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Metronidazole resistance and nim genes in anaerobes: A review
Accepted Manuscript Metronidazole resistance and nim genes in anaerobes: a review Corentine Alauzet, Alain Lozniewski, Hélène Marchandin PII:
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DOI:
10.1016/j.anaerobe.2018.10.004
Reference:
YANAE 1955
To appear in:
Anaerobe
Received Date: 3 January 2018 Revised Date:
1 October 2018
Accepted Date: 5 October 2018
Please cite this article as: Corentine Alauzet, Alain Lozniewski, Hélène Marchandin, Metronidazole resistance and nim genes in anaerobes: a review, Anaerobe (2018), doi: 10.1016/ j.anaerobe.2018.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Metronidazole resistance and nim genes in anaerobes: a review
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Corentine Alauzet 1,2, Alain Lozniewski 1,2, Hélène Marchandin 3,4*
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France
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Laboratory of Bacteriology, Nancy University Hospital, F-54000 Nancy, France
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HydroSciences Montpellier, CNRS, IRD, Univ Montpellier, CHU de Nîmes, Montpellier,
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France
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Department of Microbiology, Nîmes University Hospital, Nîmes, France
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Université de Lorraine, EA7300 Stress Immunité Pathogènes (SIMPA), F-54000 Nancy,
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* Corresponding author: Prof. Hélène Marchandin, UMR 5569 HydroSciences Montpellier,
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Equipe Pathogènes Hydriques Santé Environnements, U.F.R. des Sciences Pharmaceutiques
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et Biologiques, Université de Montpellier, 15, avenue Charles Flahault, BP 14491, 34093
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Montpellier Cedex 5, France
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[email protected]
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Declarations of interest: none.
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ACCEPTED MANUSCRIPT Abstract
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Acquired resistance to metronidazole, a 5-nitroimidazole drug largely used worldwide in the
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empirical treatment of infections caused by anaerobes, is worrisome, especially since such
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resistance has been described in multidrug-resistant anaerobic bacteria. In anaerobes, acquired
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resistance to metronidazole may be due to a combination of various and complex
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mechanisms. Among them, nim genes, possibly located on mobile genetic elements, encode
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nitro-imidazole-reductases responsible for drug inactivation. Since the first description of
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Nim proteins about 25 years ago, more nim genes have been identified; currently 11 nim
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genes are known (nimA to nimK). Mostly reported in Bacteroides fragilis group isolates, nim
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genes are now described in a variety of anaerobic genera encompassing the 4 main groups of
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Gram-negative and Gram-positive bacilli and cocci, with variable expression ranging from
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phenotypically silent to low-level or high-level resistance to metronidazole.
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This review describes the trends of metronidazole resistance rates among anaerobes over the
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past 15 years and summarizes current knowledge on mechanisms involved in this resistance.
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It also provides an update on the phylogenetic and geographical distribution of nim genes, the
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mechanisms involved in their expression and regulation, and their role in metronidazole
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resistance.
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Keywords: metronidazole, resistance, nim gene, anaerobes
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ACCEPTED MANUSCRIPT 1. Metronidazole, a prodrug used as first-line treatment in the management of a variety
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of infectious diseases
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Metronidazole and other 5-nitroimidazoles (5-Ni) are antimicrobial agents that are remarkable
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regarding their activities against both parasites and microaerophilic and anaerobic bacteria
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[1]. Metronidazole is indicated for use in a variety of infections involving anaerobic bacteria
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including bacterial vaginosis and Clostridium difficile infections, but also in other indications
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encompassing symptomatic intestinal and extraintestinal amebiasis, trichomoniasis, giardiasis,
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and Helicobacter pylori infections. This synthetic derivative of azomycin, a nitroimidazole
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compound isolated from Streptomyces spp., was first used in the late 50s to treat infections
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caused by parasites [1]. Its antibacterial activity was discovered in 1962 when it was observed
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that metronidazole was able to cure a patient from both trichomoniasis and bacterial gingivitis
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[2]. Subsequent studies demonstrated the clinical efficacy of metronidazole for the treatment
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of infections caused by anaerobes which led to its extensive use for the treatment of these
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infections from the 1970s until today [3].
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5-Ni are prodrugs, which penetrate bacterial cells via passive diffusion and are further
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intracellularly activated before exhibiting their bactericidal activity (Figure 1) [4]. Although
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the mechanism of action of 5-Ni is not entirely clear, it is believed that these drugs are
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activated by the reduction of the nitro group. As reduction proceeds, a favorable
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transmembrane concentration gradient is created which further enhances intracellular
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diffusion [4]. The absence of electron-transport proteins with sufficient negative redox in
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aerobic bacteria explains their intrinsic resistance to 5-Ni [3]. The reductive activation of 5-Ni
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is assumed to occur in a series of steps beginning with the formation of an anionic nitro-
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radical anion (R-NO2•−) followed by the formation of a nitrous derivative (R-NO), a nitrous
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free radical (R-NO−) and finally a hydroxylamine derivative (R-NHOH). It is assumed that R-
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NO2•− is one of the more active molecules able to degrade cellular macromolecules, and
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ACCEPTED MANUSCRIPT particularly DNA, via oxidation [4]. Oxidation of DNA in turn causes strand breaks and
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subsequent cell death. In anaerobes, reduction occurs when metronidazole receives an
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electron from ferredoxin or flavodoxin that was reduced by the pyruvate-
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ferredoxin/flavodoxin oxidoreductase (PFOR) system. Metronidazole acts as an electron trap
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that captures electrons that would normally be donated to hydrogen ions to form molecular
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hydrogen. This system is involved in the decarboxylation of pyruvate to acetyl-CoA. It is
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noteworthy that pyruvate may also be converted to lactate by lactate dehydrogenase (LDH).
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Some strict and facultative anaerobes, such as Cutibacterium spp., Propionibacterium spp.,
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Lactobacillus spp. and most bifibobacteria, Eubacterium spp. and Actinomyces spp. display
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intrinsically reduced susceptibility or resistance to metronidazole that could be linked to a
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lack of the PFOR system or to the use of another way to catabolize pyruvate involving the
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NADH-producing pyruvate dehydrogenase [3,5]. These taxa apart, other anaerobes remain
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highly susceptible to metronidazole.
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This excellent activity against anaerobes, its low cost, its favorable pharmacokinetic and
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pharmacodynamics properties, as well as minor adverse effects make metronidazole still the
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cornerstone for the management of anaerobic infections, despite the emergence of strains with
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acquired resistance [3,6].
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2. Resistance to metronidazole in anaerobes is complex, still uncommon but worrisome
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2.1 A low but steady and geographically diverse increase in prevalence
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The first report of acquired resistance to metronidazole described a B. fragilis isolate
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recovered in 1978 from a patient with Crohn’s disease who had been extensively treated with
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metronidazole [7]. Since then, the prevalence of acquired resistance among anaerobes has
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remained overall relatively low. Most studies about metronidazole resistance are focused on
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Bacteroides (Table 1). These studies reported low level (<3%) of metronidazole resistance in
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ACCEPTED MANUSCRIPT most part of the world [8–33], except higher resistance rates reported in Spain (4.8%), in
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South Africa (8.7%) and in Pakistan (16%) [34–36]. Regarding other Gram-negative
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anaerobes (Table 2), resistance to metronidazole has until now only rarely been detected
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among Fusobacterium isolates. Indeed, except for the studies by Katsandri et al. [14] (17
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strains isolated in Greece, 11.7% of reduced susceptibility to metronidazole ) and Wang et al.
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[31] (48 strains isolated in Taiwan, 4.2% of reduced susceptibility to metronidazole), isolates
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included in all other surveillance studies were found susceptible to metronidazole
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[9,10,12,17,19,25,29,30,32]. In contrast, reduced susceptibility to metronidazole is more
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frequent among Prevotella and to a lesser extent among Veillonella isolates depending on the
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geographical area [9,10,12,14,16,17,22,24,25,29–31,33,37–39]. In several countries, reduced
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susceptibility of Prevotella to metronidazole has been found in more than 10% of the isolates
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tested [9,14,16,29]. In two studies, which included a large number of Prevotella, clinical
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strains recovered in The Netherlands (2011-2013) and in the USA (2010-2012),
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metronidazole resistance rates of 2 and 3% were observed, respectively [10,30]. Regarding
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Gram-positive anaerobes, reduced susceptibility to metronidazole has so far only been rarely
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detected in surveillance studies among Gram-positive cocci including Parvimonas micra,
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Finegoldia magna and Peptostreptococcus sp. isolates, as well as among clostridia including
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Clostridium perfringens, Clostridium ramosum and Clostridium bifermentans
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[9,10,12,17,22,29,31–33,40,41] (Table 3). However, Koch et al. [42] described 8.1% of
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metronidazole resistance in Peptostreptococcus spp. and Ng et al. [19] described 21.5% of
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resistance in Clostridium spp. It is noteworthy that for most surveillance studies performed in
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European countries, breakpoints interpretation was based on European Committee on
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Antimicrobial Susceptibility Testing (EUCAST) recommendations
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(http://www.eucast.org/clinical_breakpoints/) while for studies emanating from other
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countries, Clinical and Laboratory Standards Institute (CLSI) breakpoints [43] were used.
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within these two groups of countries, it cannot be ruled out that some of the differences
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observed around the world are due to differences in the number of strains and species
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included, testing methods and breakpoints used. This underlines the importance of
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standardizing study methods on a global scale.
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While the overall metronidazole resistance rate is still relatively low, it is noticeable that
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isolates exhibiting simultaneous resistance to metronidazole and to other antibiotics have been
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increasingly reported during the last twenty years. The associated resistances concern a wide
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range of antibiotics routinely used to treat anaerobic infections, including carbapenems. Such
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multidrug-resistant (MDR) isolates, mainly belonging to the B. fragilis group, have been
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involved in human infections and, in many cases, have been considered as being responsible
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for therapeutic failure [11,18,25,44–55].
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2.2 Acquired metronidazole resistance mechanisms in anaerobes Several molecular mechanisms have been associated with metronidazole resistance, mainly
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described in B. fragilis (Figure 1). Resistance related to drug inactivation by nitroimidazole
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reductase encoded by nim genes is the mechanism that has been most studied and detected in
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a wide range of anaerobic species [56–58]. This mechanism is detailed in part 3.1.
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Metronidazole resistance could also be related to impaired intracellular activation as observed
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for the first described B. fragilis isolate (NCTC 11295) that exhibited high-level resistance to
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metronidazole [7]. The resistance was non-transferable by conjugation [57], and, by
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comparing this strain with susceptible ones, Narikawa et al. [59] showed that its
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metronidazole resistance was related to a decreased activity of PFOR associated with a high
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activity of LDH. The impact of impaired enzymatic activity of the PFOR complex on
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metronidazole resistance in B. fragilis has been further confirmed by Diniz et al. [60].
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Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were
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metronidazole [61].
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Metronidazole resistance in B. fragilis may also be due to other mechanisms such as drug
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extrusion by multidrug efflux pumps, increased DNA repair capacity and activation of
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antioxidant defense systems [58,62–65]. It has also been shown that the deficiency of the
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ferrous iron transporter FeoAB is linked with metronidazole resistance in B. fragilis
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suggesting an impact of intracellular iron homeostasis on metronidazole activity [66]. Patel et
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al. [67] demonstrated the relationship between overexpression of the rhamnose catabolism
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regulatory protein RhaR and metronidazole resistance in Bacteroides thetaiotaomicron by a
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yet unknown mechanism. In C. difficile, it has also been proposed that, in addition to impaired
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intracellular activation, other mechanisms such as increased DNA repair capacity, altered iron
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metabolism and biofilm formation, may contribute to metronidazole resistance [61,68]. Other
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studies reported the mutagenic activity of metronidazole in diverse bacterial species including
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B. fragilis and mutations in the nitroreductase-encoding gene rdxA have been associated with
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the induction of metronidazole resistance in H. pylori [69]. Despite such an association
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between mutagenicity and resistance inducibility has not been formally demonstrated in
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anaerobic species, such mutations should therefore also be considered as another potential
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source for metronidazole resistance in anaerobes.
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Finally, it is interesting to note that induced resistance to metronidazole has been reported in
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several studies, highlighting the importance of careful susceptibility testing of anaerobes in
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order to not overlook inducible homogeneous or heterogeneous metronidazole resistance after
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prolonged exposure to this antibiotic. Indeed, some works reported the growth of small
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colonies inside the inhibition zone of the disk or of the Etest strip after prolonged incubation
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of 72 to 120 hours (Table 4), and showed that slowly growing sub-populations displayed
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stable enhanced metronidazole MICs ranging from 8 to >256 mg/L [70,71]. Other studies
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ACCEPTED MANUSCRIPT reported that metronidazole resistance could also be induced in anaerobes by successive
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culture in presence of raising subinhibitory concentrations or high doses of metronidazole
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[60,70–75]. A recent study exploring the global mechanisms of bacterial survival upon
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metronidazole exposure in the absence of nim gene using a RNA-seq transcriptomic approach
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confirmed the complexity of metronidazole resistance [76]. In this study, de Freitas et al.
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compared a metronidazole-susceptible B. fragilis strain to 4 derivative metronidazole-resistant
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strains that were induced by subcultures with subinhibitory antibiotic concentrations. They
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showed that several metabolic pathways associated with metronidazole response, such as
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impaired drug activation (via PFOR complex or LDH), high expression level of multidrug
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efflux pumps or DNA repair systems, were altered in the resistant strains. These authors also
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found that changes in the whole gene expression patterns were maintained even when the
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metronidazole selection pressure was stopped, suggesting that drug exposure led to drastic
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persistent gene expression changes and that such persistent alterations may be involved in the
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emergence of resistant strains. Such results are in accordance with the stability of induced
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metronidazole resistance that was described in most of the studies dealing with this subject
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[70,71,73,75].
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However, none of the mechanisms that may be involved in metronidazole resistance have
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been shown to be responsible per se for high-level metronidazole resistance. Diniz et al. [60]
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reported that none of the B. fragilis mutants lacking the genes for flavodoxin and/or pyruvate-
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ferredoxin oxidoreductase was as resistant as the spontaneous metronidazole resistant strain
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derived from B. fragilis ATCC 25285T after selection by metronidazole exposure. Pumbwe et
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al. showed that MICs of metronidazole were only moderately increased in B. fragilis mutants
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overexpressing bmeRABC5, which encodes a RND-family efflux pump that confers
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metronidazole resistance [63]. Similar results were obtained when an inhibitor of the
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multidrug efflux pumps bme was used, thereby showing that metronidazole efflux systems
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ACCEPTED MANUSCRIPT alone did not cause high levels of resistance [77]. Similarly, only low to moderate levels of
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metronidazole resistance were found in feoAB-deficient mutants generated in B. fragilis and
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in B. thetaiotaomicron mutants overexpressing RhaR [66,67]. Regarding nim genes (details in
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part 3.4), several studies have also demonstrated that their presence is not per se sufficient to
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confer high-level metronidazole resistance [71,78–80]. Thus, there is nowadays unambiguous
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evidence that metronidazole resistance is a complex phenomenon that is likely to be
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multifactorial and to include probably yet unknown mechanisms [6].
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3. nim genes and metronidazole resistance
3.1. From nimA to nimK: from the pioneering research at the Pasteur Institute to
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published works based on whole genome sequencing
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3.1.1. Description of nimA to nimD genes
The presence of specific transferable 5-Ni resistance determinants was first reported in 1989
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by Breuil et al. [81] in a Bacteroides vulgatus strain, BV17. This strain carries four plasmids,
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one of which (pIP417) was responsible for low-level metronidazole resistance after
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introduction in a susceptible strain, B. fragilis 638R. The resistance of BV17, as well as that
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of B. thetaiotaomicron BT13 (pIP419-positive) and of B. fragilis BF8, a plasmid-free strain,
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have been shown to be transferable by a conjugal-like process to susceptible strains with a
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frequency ranging from 10−3 to 10−7 per donor [81–84]. The associated genetic determinants
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were identified in 1994 by the team of Madeleine Sebald at the Pasteur Institute of Paris, with
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the description of nimA and nimB genes [85] followed by that of nimC and nimD genes [86].
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Two years later, Carlier et al. explored the enzymatic activity of the nim gene products by
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comparing the metabolism of a 5-Ni-susceptible B. fragilis strain with that of the same strain
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harboring a nimA-carrying plasmid [56]. While the classic reduction of 5-Ni drugs to its nitro
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radical anion was observed in the susceptible strain, the nimA-positive strain seemed to
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ACCEPTED MANUSCRIPT mainly reduce 5-Ni to its amine derivative, thereby avoiding the formation of toxic nitroso
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radicals that are essential for antimicrobial activity. These findings led the authors to conclude
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that Nim proteins exhibited a nitroimidazole reductase activity, which causes the reduction of
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the nitro group of 4- or 5-Ni to an amino group leading to a 5-aminoimidazole inactive
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compound [56]. The crystal structure of NimA from Deinococcus radiodurans (NimA Dr)
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was elucidated seven years later [87]. This protein is weakly related (less than 26% identity)
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to the Nim proteins described in Bacteroides and Prevotella (Table 5). Leiros et al. [87–89]
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characterized specific and conserved residues either in the active site (His-71) or in the 5-Ni-
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binding site (Pro-56, Tyr-111) of Nim proteins. From structure analysis, the following
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mechanism has been proposed: i) native NimA structure with hydrogen-binding of pyruvate
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from the PFOR complex to the His-71, ii) modification of link between His-71 and pyruvate
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that became covalent upon metronidazole binding, iii) oxidation of His-71 and pyruvate into a
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His-71-Pyr residue associated with the release of two electrons and one proton, iv) reduction
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of metronidazole by the released electrons into a non toxic compound [87–89].
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3.1.2. Detection of nim genes by specific targeted PCR approach or whole
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genome sequencing
Considering the impact of an eventual dissemination of such genes among anaerobes, Trinh &
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Reysset proposed in 1996 a PCR method based on universal primers NIM3 (5’-
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ATGTTCAGAGAAATGCGGCGTAAGCG-3’) and NIM5 (5’-
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GCTTCCTTGCCTGTCATGTGCTC-3’) that is able to detect all subtypes of nim genes by
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targeting conserved sequences [90]. Since then, this method, which has been widely used to
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evaluate the presence of nim genes among anaerobes, has led to the discovery of five new
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variants. nimE was first described in B. fragilis, B. thetaiotaomicron and Bacteroides ovatus
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[91], nimF in B. vulgatus [73], nimG and nimH in B. fragilis [71,92], and nimI in Prevotella
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baroniae [70]. A tenth nim gene was identified by Husain et al. after whole genome
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ACCEPTED MANUSCRIPT sequencing (WGS) of two B. fragilis strains [78]. In this study, the annotation of their
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genomes using the RAST annotation server (http://rast.nmpdr.org) revealed a gene coding for
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a protein of the pyridoxamine 5′-phosphate oxidase-related protein family, which includes
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Nim proteins. This gene, named nimJ, was not detected by the universal primers NIM3-NIM5
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due to distinct nucleotide sequences at the sites of primers annealing; however, the predicted
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amino acid sequence of NimJ was identical to that of the universal NIM3 forward primer
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(MFREMRRK) and differed by one amino acid from that of the universal NIM5 reverse
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primer (EHMTGKEEHLTGKE) [54,77,78]. By demonstrating that at least one nim variant
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could not be detected by the universal primers used so far, Husain et al. highlighted that this
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might be the case with other unknown nim alleles and that this could account at least in part
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for some nim-negative metronidazole resistant strains observed in several epidemiological
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studies [78]. The latest variant described is nimK, recently detected by WGS in three
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metronidazole-resistant Prevotella bivia clinical isolates [93]. The sequence of nimK shows,
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however, that this gene could have been amplified by using NIM3-NIM5 primers. Beside
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detection of new nim genes, WGS led to identify known nim genes associated to molecular
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markers of resistance to other antibiotics. For example, Ank et al. described nimE together
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with cfiA, ermF and tetQ genes in a multidrug-resistant B. fragilis isolate exhibiting
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piperacillin-tazobactam, carbapenem, metronidazole, clindamycin and tetracycline resistance
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[44]. Similarly, nimD was found in the complete genome sequence of a metronidazole-
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resistant Bacteroides stercoris isolate from a polymicrobial intra-abdominal abscess [74]. In
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the latter WGS-based studies, nim genes were mostly detected by using a database
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specifically designed for the identification of acquired antibiotic resistance genes in totally or
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partially sequenced bacterial isolates, i.e., ResFinder
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(http://cge.cbs.dtu.dk/services/ResFinder/) [94] or on the basis of functional annotation.
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3.1.3. Current diversity of nim genes
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ACCEPTED MANUSCRIPT To date, eleven nim genes (nimA to nimK) sharing between 57.6 and 89.8% nucleotide
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sequence identities (corresponding amino acid sequence identities range: 54 and 90%) have
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been described (Table 5). The highest degree of amino acid identity was observed between
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NimD and NimG. The phylogenetic relationships between Nim proteins are shown in Figure
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2. Two clusters of proteins are demonstrated within the Nim protein family, the NimA-H/K
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clade grouping NimA to NimH and NimK proteins displaying more than 59% of sequence
271
identity and the NimI-J clade including NimI and NimJ proteins that display 74% of sequence
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identity (Table 5).
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The advent of WGS led to improve the exploration of nim genes diversity as this approach
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permit the discovery of new variants not detected by universal primers. Two putative new nim
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genes (protein accession No: WP_005782870 and WP_005811941) were thereby detected in
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the metronidazole-resistant Bacteroides strain UW, sharing respectively the higher amino acid
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sequence identity with NimE (33%) and NimF (40%) [95]. Similarly, WGS of a multidrug
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resistant B. thetaiotaomicron clinical isolate revealed the presence of nimD gene (99.2% of
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sequence identity with B. fragilis nimD (X76949)) [50]. In this strain, a second nim-like
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element, distantly related to known Nim proteins (26.8% of nucleotide sequence identity with
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nimE), and sharing 97.5% identity with a B. thetaiotaomicron VPI 5482 gene encoding an
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uncharacterized 5-Ni antibiotic resistance protein (AAO78184), was also detected. The latter
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observations highlighted the presence of several nim determinants within a genome. Although
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multiple nim copies of a same gene were previously described in B. fragilis (2 copies of nimJ
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[78], 2 copies of nimA [Marchandin et al., personal communication]), this is to our knowledge
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the first observations of different nim determinants in the same genome.
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3.2. nim genes through the phyla, hosts and continents
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nim genes were initially described in several cultivated Bacteroides species, mainly belonging
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to the B. fragilis group, as well as in the reclassified Parabacteroides distasonis (Table 6).
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splanchnicus, Porphyromonas sp. and in various species of Prevotella. Indeed, within the last
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decade, one nimA-positive Prevotella bivia as well as two nimB-positive strains (Prevotella
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dentalis and Prevotella denticola), one nimC-positive Prevotella oralis and two nimE-positive
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isolates (Prevotella oralis and Prevotella buccalis) were detected [14,96,97]. It is interesting
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to note that nimI seems to be intrinsic to the species Prevotella baroniae as it has only been
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recovered in nine P. baroniae French clinical isolates as well as in the British type strain
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DSM 16972T whereas it was not detected in 33 type strains belonging to other Prevotella
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species [70]. nim genes were also described in anaerobes belonging to other phyla (Table 6).
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Indeed, they were detected in Actinobacteria (Actinomyces odontolyticus, Cutibacterium
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acnes and Propionibacterium sp.), in Firmicutes (Gram-positive anaerobic cocci, Clostridium
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bifermentans, and Veillonella sp.) as well as in Fusobacteria (Fusobacterium sp.)
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[14,80,96,98].
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Interestingly, only half of the nim subtypes present a G+C content that is compatible with
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their main host’s genome (i.e. 39-49 mol% for Bacteroides sp. and 40-52 mol% for Prevotella
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sp.) [78,99,100]. The G+C content of nimA, nimC, nimD, nimF and nimI is >50 mol% and the
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newly described nimK has a very low G+C content of 37.2 mol% (Table 6). These
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observations, associated with the presence of nim genes in a wide range of bacterial phyla,
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suggest that nim genes could have resulted from a transfer from an unknown common
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ancestor followed by independent evolution. In addition, the increasing availability of
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bacterial genome sequences leads to the detection of Nim-related proteins, for which the
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function is still unknown, in phylogenetically distant species including aerobic genera (Ex:
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Deinococcus radiodurans and Streptomyces avermitilis, with respective accession numbers
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AAF10419 and Q827C5) and Archaea (Ex: Methanosarcina mazei, accession number
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Q8PT76). These observations suggested that the nim gene family is ancient and widespread in
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ACCEPTED MANUSCRIPT bacteria [78,87,101]. Although the reservoir is still unknown, it could be hypothesized that the
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intestinal microbiota could represented a very favorable environment and that, once
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introduced and adapted in Bacteroides, a genus particularly abundant in the gut and actively
318
involved in horizontal gene transfer, nim genes could easily spread under selective pressure
319
[78,99,102].
320
Studies that have evaluated the prevalence of nim genes among anaerobes are scarce and
321
showed overall relatively low level of these genes varying from 0.5 to 2.8% among
322
Bacteroides spp. [14,21,27,29,32] and from 0 to 5.3% or 5.9% for Prevotella spp. or
323
Fusobacterium spp., respectively [14,29] (Tables 1 to 3). Although still infrequent, nim-
324
positive strains are described in most of the continents. The majority of studies examining the
325
distribution of nim genes in anaerobes result from European groups [27,32,44,54,70,71,73–
326
75,78,79,85,89–92,98,103–108] but nim-positive isolates have also been reported in Brazil,
327
China, India, Kuwait, South Africa or USA [29,49–51,77,78,80,96,97,107,109–114]. In these
328
studies, nim-positive strains were isolated from various clinical specimens, mainly from
329
abdominal specimens (abscess, empyema, pus, appendectomy fluid, pancreatic pseudocyst,
330
…) but also from blood cultures as well as from periodontal, osteoarticular and, more rarely,
331
respiratory tract specimens. Few strains were recovered from fecal samples of healthy
332
volunteers [85,105,109,111,115] and one B. fragilis carrying a nimB gene was isolated from
333
polluted aquatic environment [114].
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3.3. Genetic environment and regulation of nim gene expression
335
Early descriptions of nim genes described their location on a variety of small mobilizable nim-
336
positive plasmids (pIP417, pIP419 and pIP421) [81,84–86]. It was also shown that nimB was
337
located on the chromosome of B. fragilis BF8 [82]. Since then, it has been shown that nimA,
338
nimC and nimD genes could be either plasmidic or chromosomal (Table 6). nimB and nimI
339
seem to be exclusively chromosomal whereas nimE gene was recovered on plasmid. The
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ACCEPTED MANUSCRIPT locations of nimF and nimG have not been elucidated and an exclusive chromosomal location
341
of nimH, nimJ and nimK needs to be further confirmed on other isolates.
342
It has been assumed that the transcription of these genes may be activated by insertion
343
sequence (IS) elements. Indeed, Husain et al. introduced nimE and nimJ, cloned into the
344
strong promoter expression vector pMCL140, in B. fragilis 638R and observed higher
345
metronidazole MICs for the recipient strain despite not reaching those of the original clinical
346
isolates [78]. nim transcription levels were also 2- to 4-fold lower in strain 638R than in the
347
original clinical isolates. The fact that nim genes were cloned into pMCL140 without their
348
associated IS element could explain the discrepant result as compared to the study of Soki et
349
al. who transferred the nimE-positive plasmid pBF388c by mating into B. fragilis 638R and
350
observed an increased metronidazole MIC of the transconjugant, similar to that of the nimE
351
donor strain (16 mg/L) [107]. Several transposases belonging to the IS4 or IS5 families have
352
been described upstream of the nim genes but they are not consistently present and not always
353
dedicated specifically to a variant of nim (Table 6) [107,116,117]. These IS were either
354
identical or similar to those described in imipenem-resistant strains [86,92,107]. When
355
present, IS1168 (isoform of IS1186) was the most frequently transposase associated with
356
nimA and nimB. IS1169 was the only transposase recovered in association with nimD and was
357
also rarely detected upstream of nimA. IS1170 and ISBf6 have only been described associated
358
to nimC and nimE, respectively. In B. fragilis, one case of association between IS612 and
359
nimB was observed by Soki et al. [107] and the new transposase ISBf13 was detected by
360
WGS upstream of nimA by Sydenham et al. [108]. Finally, IS614B, a putative mosaic or
361
hybrid of IS612, IS614 and IS942 was found in association with nimH and nimJ [78,92].
362
Open reading frame (ORF) of these IS are in the opposite orientation of the nim ORF and they
363
usually carry on their right end outward-oriented promoters that could activate nim
364
transcription [86,92,107]. For example, based on sequences homologous to consensus
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ACCEPTED MANUSCRIPT sequences of B. fragilis promotors, TAnnTTTG in the −7 region and TnTG in the −33 region,
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a putative promotor sequence of nimH was described within the IS614B [92,118].
367
Additionally, IS elements have been assigned a role in the expression of other resistance
368
genes in Bacteroides spp. such as those linked with resistance to macrolides (erm), cefoxitin
369
(cepA), cephalosporin (cfxA) or carbapenems (cfiA), and have been proved to be
370
interchangeable between these genes [3,107,119]. Among all the studies recovered in the
371
literature, the presence of an IS upstream of a nim gene is not always associated with
372
metronidazole resistance [47,71,73,85,90,107] and, conversely, a nim gene without an
373
upstream identified IS can be associated to metronidazole resistance [47,71,73,74,95,107,108]
374
(Table 6). In the latter case, the resistance could be explained by another nim-independent
375
mechanism (see part 2.2) or by the presence of a new IS not detected by the methods used.
376
For example, Sydenham et al. [108] described the presence of ISBf13 upstream of nimA but
377
on the complement strand. The action of such IS promoters upstream of nim genes remains
378
controversial and need to be further explored as no experimental evidence has confirmed their
379
role.
380
The presence of IS elements in the upstream region of nim genes could not only potentially
381
influence their expression but also their dissemination. Indeed, IS are small mobile genetic
382
elements involved in plasticity and adaptability of prokaryote genomes [116,117]; They are
383
widespread in all bacterial phyla and can occur in a wide range of copy numbers in a genome.
384
They can move within a chromosome, participating in modulation of neighboring gene
385
expression. They can also be involved in transfer of genetic material from chromosome to
386
other mobile genetic elements vectors such as phages and plasmids, thus participating to gene
387
dissemination [116,117]. It has been shown that nim gene-positive plasmid transfers have
388
occurred in vitro but it was also hypothesized that they have occurred in vivo, as plasmids
389
similar or identical to pIP417 or pIP419 have been detected in different clinical B. fragilis
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ACCEPTED MANUSCRIPT 390
isolates [81,82,84,107,120]. Regarding chromosomal nim genes, it can also be suggested that
391
the presence of IS could permit dissemination.
392
3.4. A variably expressed and inducible resistance Studies based on WGS showed that the presence of nim genes (A, D, E, J, new nim genes)
394
correlated with phenotypically resistant strains (metronidazole MICs ranging from 6 to >256
395
mg/L) [44,50,73,77,93]. However, several other studies showed that the presence of nim gene
396
does not always lead to resistance and a wide range of metronidazole MICs are observed in
397
nim-positive isolates ranging from 0.125 to >256 mg/L. For example, Gal et al., studying 50
398
Bacteroides spp. strains with positive detection of nim genes by specific PCR, found that
399
about half of them only presented MICs ranging from 16 to >32 mg/L and thus above the
400
resistance breakpoint [71]. Except for nimI, that seemed to be a ‘silent’ gene as it has never
401
been associated with metronidazole resistance in all P. baroniae strains tested [102], and for
402
nimF and nimG, that were described in unique metronidazole-susceptible (respective MICs of
403
1 and 2 mg/L) [71,73], no correlation were observed between levels of metronidazole
404
resistance and the type of nim gene identified (Table 6). To further assess the role of nim
405
genes in the development of metronidazole resistance, Leitsh et al. studied their expression at
406
the protein level [72]. They used 2D gel electrophoresis to identify and quantify Nim (A, B, D
407
and E) proteins and they did not observe a correlation between Nim levels and metronidazole
408
resistance levels. This study challenged the implication of Nim proteins in the inactivation of
409
metronidazole while reinforcing the complexity of metronidazole resistance and the fact that
410
nim genes per se are not sufficient to confer high-level metronidazole resistance.
411
nim-gene positive strains may also display induced and stable heterogeneous or homogeneous
412
resistance to metronidazole after in vitro exposure to the drug [70,71], similarly to nim-
413
negative strains (see part 2.2). A prolonged exposure to metronidazole has also been reported
414
as inducing 5-Ni resistance in vivo in a clinical B. fragilis isolate carrying a silent nimA,
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ACCEPTED MANUSCRIPT 415
leading to treatment failure [104]. However, despite these observations were limited to
416
isolates harboring nim genes in some studies [71–73], similar findings were observed in nim
417
gene-negative isolates in other studies (Table 4) [60,70,75,76,121].
418
3.5. nim genes as part of the genetic content of multidrug resistant anaerobes The emergence of MDR metronidazole-resistant isolates may be explained by the
420
accumulation of mutations within single strains under selective antibiotic pressure but also by
421
the spread of mobile genetic elements such as transposable elements and plasmids carrying
422
nim genes as well as other resistance genes (Table 6) [44,50,77,78,95,99,106,108]. In most
423
cases, multidrug resistance has been observed in B. fragilis clinical isolates harboring known
424
or putative nim genes and displaying at least resistance to piperacillin-tazobactam,
425
clindamycin, carbapenems and metronidazole [44,78,95,106]. The diffusion of such strains is
426
worrisome and an international cluster of MDR B. fragilis isolates harboring nimB and cfiA
427
genes, has been recently recognized [106]. Multidrug resistance (resistant to piperacillin-
428
tazobactam, metronidazole, clindamycin, ertapenem, and meropenem) was also reported in a
429
B. thetaiotaomicron isolate harboring nimD and a second putative nim gene beside two β-
430
lactamase genes, two tetX genes, tetQ, ermF, two cat genes, and several genes encoding
431
efflux pumps [50].
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4. nim genes and cultivation-independent studies
434
Two studies conducted by Koukos and colleagues specifically searched for nim genes in the
435
oral microbiota during health and disease according to the procedure of Trinh et al. [90]. In
436
the first one, they aimed at comparing the prevalence of antibiotic resistance genes in subjects
437
with successful (n=20) and failing (n=20) dental implants [122]. In the second one, they
438
investigated the presence of nim genes in 343 oral samples from 154 adult subjects who did
439
not received metronidazole in the last 12 months and distributed as follows, 50 periodontally
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ACCEPTED MANUSCRIPT healthy patients, 52 cases of gingivitis and 52 cases of chronic periodontitis [122]. In both
441
studies, nim genes were not detected supporting the continuing use of metronidazole alone or
442
in combination in the management of oral infections.
443
Another particularly interesting investigation performed by Rani et al. in the New Dehli area
444
highlighted the impact of selective pressure exerted by metronidazole on the emergence of
445
bacteria harbouring nim genes in the gut of individuals receiving metronidazole [123]. These
446
authors reported that the presence of a nim gene (nimE) was frequently detected (11 out of 19
447
patients) in fecal samples from patients with amebic liver abscesses treated with
448
metronidazole whereas no nim gene could be detected in fecal samples obtained in individuals
449
living in the same area (19 healthy individuals and 11 asymptomatic Entamoeba histolytica
450
carriers) who had not received antibiotics for 3 months prior to sampling. In this study, it was
451
also shown that a three-day course of metronidazole given to 11 individuals (8 healthy
452
volunteers and 3 patients suffering from irritable bowel syndrome) resulted in an increase of
453
the frequency of nim detection in fecal samples (72.7 % [8/11] after metronidazole exposure
454
versus 18.8% [2/11] before metronidazole exposure). Importantly, this study showed that nim
455
genes may rapidly emerged among bacterial communities after metronidazole treatment
456
concomitantly to dysbiosis. In the specific case of amebiasis, difference in metronidazole
457
efficiency was observed against ameba according to location in intestinal tissue or lumen and
458
this was thought to be related, at least in part, to anaerobes that co-exist with the ameba and
459
may responsible for metronidazole inactivation [124].
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461
5. Concluding remarks
462
Metronidazole resistance is complex and multifactorial, and the mechanisms and drivers of
463
resistance are still not fully elucidated. While nim genes appears to be associated with
464
metronidazole resistance, dissemination and/or selection of metronidazole resistant strains in
19
ACCEPTED MANUSCRIPT some studies, a strong correlation between nim genes and metronidazole resistance cannot be
466
clearly and definitely established at this time.
467
Recently, the use of WGS to predict antimicrobial susceptibility pattern has been increasingly
468
investigated and discussed. Despite some studies showed high levels (over 90%) of
469
concordance between WGS and antimicrobial susceptibility testing (AST) in some aerobes
470
[94,125–127], the EUCAST Subcommittee recently stated that “for most bacterial species
471
there is currently insufficient evidence to support the use of WGS-inferred AST to guide
472
clinical decision making” [128]. In anaerobes, phenotypic characterization of metronidazole
473
resistance is still essential, as WGS can’t be totally accurate in predicting metronidazole
474
resistance due to the multiplicity of metronidazole resistance mechanisms and the complexity
475
of nim genes expression [101]. In addition, the issue of the incompleteness of antibiotic
476
resistance genes (ARG) databases available for the identification of nim genes in whole
477
genome sequences among metagenomic data need to be addressed.
478
nim gene-mediated resistance has however to be under surveillance because numerous risk
479
factors are combined for a predicted emergence of metronidazole resistance. The most
480
important are the location of nim genes on mobile genetic elements and a high selective
481
pressure coming from indication of metronidazole administration as a first-line drug in
482
diverse infections, including the highly prevalent C. difficile infections, as well as H. pylori
483
infections and amebiasis. This is reinforced by the description of nim genes in emergent
484
multidrug resistant anaerobes, for which associated resistances to other important anti-
485
anaerobes like piperacillin-tazobactam, clindamycin and carbapenems make the choice for the
486
most effective treatment challenging [129]. Such observations should also prompt the
487
development of new 5-nitroimidazole or anti-anaerobic drugs effective in case of
488
metronidazole resistance.
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Washington State, J. Clin. Microbiol. 42 (2004) 4127–4129. doi:10.1128/JCM.42.9.41274129.2004. J.M.B.D. Vieira, R.F. Boente, K.R. Miranda, K.E.S. Avelar, R.M.C.P. Domingues, M.C. de S. Ferreira, Decreased susceptibility to nitroimidazoles among Bacteroides species in Brazil, Curr. Microbiol. 52 (2006) 27–32. doi:10.1007/s00284-005-0068-0. A.A. Kangaba, F.Y. Saglam, H.B. Tokman, M. Torun, M.M. Torun, The prevalence of enterotoxin and antibiotic resistance genes in clinical and intestinal Bacteroides fragilis group isolates in Turkey, Anaerobe. 35 (2015) 72–76. doi:10.1016/j.anaerobe.2015.07.008. J. Mahillon, M. Chandler, Insertion sequences, Microbiol. Mol. Biol. Rev. MMBR. 62 (1998) 725–774. J. Vandecraen, M. Chandler, A. Aertsen, R. Van Houdt, The impact of insertion sequences on bacterial genome plasticity and adaptability, Crit. Rev. Microbiol. 43 (2017) 709–730. doi:10.1080/1040841X.2017.1303661. D.P. Bayley, E.R. Rocha, C.J. Smith, Analysis of cepA and other Bacteroides fragilis genes reveals a unique promoter structure, FEMS Microbiol. Lett. 193 (2000) 149–154. doi:10.1111/j.1574-6968.2000.tb09417.x. N. García, G. Gutiérrez, M. Lorenzo, J.E. García, S. Píriz, A. Quesada, Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections, J. Antimicrob. Chemother. 62 (2008) 942–947. doi:10.1093/jac/dkn347. S. Trinh, A. Haggoud, G. Reysset, Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region., J. Bacteriol. 178 (1996) 6671–6676. F. Mory, J.-P. Carlier, C. Alauzet, M. Thouvenin, H. Schuhmacher, A. Lozniewski, Bacteremia caused by a metronidazole-resistant Prevotella sp. strain, J. Clin. Microbiol. 43 (2005) 5380– 5383. doi:10.1128/JCM.43.10.5380-5383.2005. G. Koukos, A. Konstantinidis, L. Tsalikis, M. Arsenakis, T. Slini, D. Sakellari, Prevalence of βlactam (blaTEM) and metronidazole (nim) resistance genes in the oral cavity of Greek subjects, Open Dent. J. 10 (2016) 89–98. doi:10.2174/1874210601610010089. R. Rani, R.S. Murthy, S. Bhattacharya, V. Ahuja, M.A. Rizvi, J. Paul, Changes in bacterial profile during amebiasis: demonstration of anaerobic bacteria in ALA pus samples, Am J Trop Med Hyg. 75 (2006) 880–885. M. Müller, Mode of action of metronidazole on anaerobic bacteria and protozoa, Surgery. 93 (1983) 165–171. N.C. Gordon, J.R. Price, K. Cole, R. Everitt, M. Morgan, J. Finney, A.M. Kearns, B. Pichon, B. Young, D.J. Wilson, M.J. Llewelyn, J. Paul, T.E.A. Peto, D.W. Crook, A.S. Walker, T. Golubchik, Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing, J. Clin. Microbiol. 52 (2014) 1182–1191. doi:10.1128/JCM.03117-13. V.N. Kos, M. Déraspe, R.E. McLaughlin, J.D. Whiteaker, P.H. Roy, R.A. Alm, J. Corbeil, H. Gardner, The resistome of Pseudomonas aeruginosa in relationship to phenotypic susceptibility, Antimicrob. Agents Chemother. 59 (2015) 427–436. doi:10.1128/AAC.03954-14. C.U. Köser, M.J. Ellington, S.J. Peacock, Whole-genome sequencing to control antimicrobial resistance, Trends Genet. TIG. 30 (2014) 401–407. doi:10.1016/j.tig.2014.07.003. M.J. Ellington, O. Ekelund, F.M. Aarestrup, R. Canton, M. Doumith, C. Giske, H. Grundman, H. Hasman, M.T.G. Holden, K.L. Hopkins, J. Iredell, G. Kahlmeter, C.U. Köser, A. MacGowan, D. Mevius, M. Mulvey, T. Naas, T. Peto, J.-M. Rolain, Ø. Samuelsen, N. Woodford, The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee, Clin. Microbiol. Infect. 23 (2017) 2–22. doi:10.1016/j.cmi.2016.11.012. M. Gajdács, G. Spengler, E. Urbán, Identification and antimicrobial susceptibility testing of anaerobic bacteria: Rubik’s cube of clinical microbiology?, Antibiotics. 6 (2017) 25. doi:10.3390/antibiotics6040025.
AC C
844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895
28
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AC C
EP
TE D
M AN U
SC
RI PT
896
29
ACCEPTED MANUSCRIPT 897
Legends to figures
898
Figure 1. Summarized metronidazole mode of action and main mechanisms involved in
900
resistance. , nitroimidazole reductase activity encoded by nim genes that reduce the nitro
901
group of 4- or 5-Ni to an amino group leading to a 5-aminoimidazole inactive compound,
902
metabolic shift away to the pathway related to conversion of pyruvate to lactate via lactate
903
dehydrogenase (LDH) associated with high LDH activity that compensates decreased
904
pyruvate ferredoxin oxidoreductase (PFOR) activity and thus with decreased 5-Ni activation,
905
increased efflux of the antibiotic (via overexpression of RND-family efflux pump encoding
906
genes of such as bmeRABC5), increased DNA repair capacity (via overexpression of DNA
907
repair proteins such as RecA), activation of antioxidant defense systems (via antioxidant
908
stress-related enzymes such as superoxide dismutase), deficiency of the ferrous iron
909
transporter FeoAB, overexpression of the rhamnose catabolism regulatory protein RhaR.
TE D
M AN U
SC
RI PT
899
910
Figure 2. Maximum likelihood (computed by PHYML, model WAG) tree based on partial
912
derived Nim amino acid sequences (150 AA). Numbers at nodes indicate percentages of
913
bootstrap support when > 70%, based on analysis of 100 replicates. Nodes indicated with
914
stars were conserved by using a Neighbor-Joining analysis from a Dayhoff DNADIST F84
915
matrix. Accession numbers of corresponding nucleotide sequences are given in brackets. Bar,
916
0.05 substitutions per site.
AC C
EP
911
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Table 1. Resistance to metronidazole and prevalence of nim genes in Bacteroides spp. MIC range (µg/ml) 0.064-4
RI PT
≤0.12-32 0.032->256
SC
% of I+R 0 0 0.3 2.9 1.8 0 1 0 0.5 1.2 2 0 1 0 1.3 1 1.6 0.3 1 0 0 0 0 1.6 16 0 0 2.9 1 2.9 0 0 8.7 4.8 0 0.7 0 0
M AN U
Breakpoints EUCAST EUCAST CLSI EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST CLSI CLSI EUCAST EUCAST EUCAST EUCAST CLSI CLSI CLSI EUCAST CLSI EUCAST EUCAST EUCAST CLSI CLSI EUCAST CLSI CLSI CLSI EUCAST CLSI EUCAST EUCAST
TE D
Testing method Etest Agar dilution Broth micro-dilution Etest Agar dilution Agar dilution Disk screening/Etest Disk screening/Etest Agar dilution Agar dilution Agar dilution Agar dilution Broth micro-dilution Agar dilution Etest Etest Disk screening/Etest Agar dilution Agar dilution Agar dilution Broth micro-dilution Etest Agar dilution Etest Agar dilution Etest Agar dilution Etest Etest Etest Etest Agar dilution Etest Etest Agar dilution Agar dilution Etest Agar dilution
EP
Study period 2011-2012 2008-2009 2010-2011 2013 2008-2009 2008-2009 2010-2015 2002-2009 2010-2016 2008-2009 2008-2009 2008-2009 2007 2008-2009 2002-2004 2003-2005 2015 2014-2016 2008-2009 2008-2009 2014-2015 2013-2014 2012 2009-2013 2010-2011 2013-2015 2010-2013 2004-2014 2009-2011 2008-2010 2015 1995-1996 2003-2005 2006-2010 2008-2009 2008-2012 2011-2013 2008-2009
AC C
Country (no of isolates) Belgium (180) Belgium (103a) Canada (387 a) Croatia (35) Croatia (56 a) Czech Republic (91 a) Denmark (203 a) Denmark (114 a) Europe c (2451) Finland (85 a) France (51 a) Germany (72 a) Germany (72) Greece (75 a) Greece (82 a) Greece (191 a) Hong Kong (741 a) Hungary (400 a) Hungary (100 a) Italy (23 a) Japan (113 a) Japan (25) Korea (147 a) Norway (122) Pakistan (39 b) Poland (74 a) Romania (53 a) Russia (67) Singapore (68 a) Singapore (69) Slovenia (869 a) South Africa (44a) South Africa (23a) Spain (792 a) Sweden (97 a) Taiwan (256) The Netherlands (283 a) The Netherlands (32 a)
0.5-32 0.125-16 ≤2->32 0.064-16
0.25-4
≤0.125-256 0.06-64 0.016-0.5 0.125-1 0.25->256 ≤0.01->256 0.016-4 ≤0.125-8 0.047-≥256 0.016->256 0.12-16 <0.016-1.5
% of nim genes 2.8 NR NR NR NR NR NR NR NR NR NR NR NR NR 1 0.5 NR NR NR NR NR NR NR NR NR NR 2 NR 1.5 d NR NR NR 0 NR NR NR NR NR
Reference [32] [18] [13] [20] [18] [18] [8] [8] [22] [18] [18] [18] [24] [18] [21] [14] [11] [23] [18] [18] [28] [33] [16] [9] [35] [15] [27] [25] [29] [19] [12] [42] [34] [36] [18] [31] [30] [18]
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AC C
EP
TE D
M AN U
SC
RI PT
Turkey (34 a) 2008-2009 Agar dilution EUCAST 0 NR [18] USA (779 a) 2010-2012 Agar dilution CLSI 0 ≤1-2 NR [26] USA (3981 a) 2010-2012 Agar dilution CLSI 2 NR [10] USA (332) 2010-2011 Etest CLSI 0.6 (R) <0.015->256 NR [17] USA (1580 a) 2007-2009 Agar dilution CLSI 0 NR [10] a Bacteroides fragilis group; b Bacteroides fragilis; c data of the european Tigecycline Evaluation and Surveillance Trial (participating countries: France, Germany, Czech Republic, Hungary, Spain, Belgium, Italy, Sweden, United Kingdom) [22]; d nim-targeting PCR only on metronidazole-resistant strains [74]. I, intermediate; R, resistant. NR, not researched. EUCAST, European Committee on Antimicrobial Susceptibility Testing. EUCAST breakpoints for metronidazole: ≤4, susceptible; >4, resistant (http://www.eucast.org/clinical_breakpoints/). CLSI, Clinical and Laboratory Standards Institute. CLSI breakpoints for metronidazole: ≤8, susceptible; ≥32, resistant [43].
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Country (no of isolates)
Study period
Testing method
Breakpoints
% of I+R
Prevotella spp.
Bulgaria (192) Europe* (1046) France (84) Germany (21) Greece (57) Japan (46) Korea (16) Norway (13) Romania (33) Russia (42) Singapore (7) Slovenia (373) Taiwan (28) The Netherlands (123) USA (571) USA (60) USA (173)
2003-2009 2010-2016 2007-2016 2007 2003-2005 2013-2014 2012 2009-2013 2011-2012 2004-2014 2009-2011 2015 2008-2012 2011-2013 2010-2012 2010-2011 2007-2009
Modified agar dilution Agar dilution Etest Broth microdilution Etest Etest Agar dilution Etest Etest Etest Etest Etest Agar dilution Etest Agar dilution Etest Agar dilution
CLSI EUCAST EUCAST EUCAST CLSI CLSI CLSI EUCAST CLSI/EUCAST EUCAST CLSI EUCAST CLSI EUCAST CLSI CLSI CLSI
0 1 1.2 0 14 4 19 15.4 0 7.1 29 0 0 2 3 0 0
Belgium (21) Greece (17)
2011-2012 2003-2005
Etest Etest
EUCAST CLSI
0 11.7
Norway (13) Russia (13) Singapore (9) Singapore (9) Slovenia (178) Taiwan (48) The Netherlands (39) USA (27) USA (33) USA (44)
2009-2013 2004-2014 2008-2010 2009-2011 2015 2008-2012 2011-2013 2010-2012 2010-2011 2007-2009
Etest Etest Etest Etest Etest Agar dilution Etest Agar dilution Etest Agar dilution
EUCAST EUCAST CLSI CLSI EUCAST CLSI EUCAST CLSI CLSI CLSI
France (116) Korea (12) Singapore (5) Slovenia (83) Taiwan (26)
1999-2001 2012 2009-2011 2015 2008-2012
Agar dilution Agar dilution Etest Etest Agar dilution
CLSI CLSI CLSI EUCAST CLSI
Veillonella spp.
SC
M AN U
EP
AC C
Fusobacterium spp.
% of nim genes
Reference
NR NR NR NR 5.3 NR NR NR NR NR 0 NR NR NR NR NR NR
[39] [22] [37] [24] [14] [33] [16] [9] [38] [25] [29] [12] [31] [30] [10] [17] [10]
<0.016-0.25 ≤2->32
NR 5.9
[32] [14]
0 0 0 0 0 4.2 0 0 0 0
≤0.01-0.25 0.015-1
NR NR NR 0 NR NR NR NR NR NR
[9] [25] [19] [29] [12] [31] [30] [10] [17] [10]
0 8 0 0 7.6
0.25-8 ≤0.125-16 1-2 0.032-4 0.5->128
0.9 NR 0 NR NR
[97] [16] [29] [12] [31]
RI PT
Genus
TE D
Table 2. Resistance to metronidazole and prevalence of nim genes in Prevotella spp., Fusobacterium spp. and Veillonella spp. MIC range (µg/ml)
0.016-8 0.5-4 ≤2->32 0.25-16 0.12-32 0.016-1 0.03-16 0.125-≥256 0.016-4 0.03-4 <0.016-24 ≤0.015-4
≤0.01-0.5 0.016-2 0.03->128 <0.016-0.25 ≤0.015-0.25
1
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AC C
EP
TE D
M AN U
SC
RI PT
The Netherlands (19) 2011-2013 Etest EUCAST 0 <0.016-3 NR [30] USA (32) 2010-2012 Agar dilution CLSI 3 NR [10] USA (9) 2010-2011 Etest CLSI 0 1-4 NR [17] USA (28) 2007-2009 Agar dilution CLSI 14 NR [10] * data of the european Tigecycline Evaluation and Surveillance Trial (participating countries: France, Germany, Czech Republic, Hungary, Spain, Belgium, Italy, Sweden, United Kingdom) [22] I, intermediate; R, resistant. NR, not researched. EUCAST, European Committee on Antimicrobial Susceptibility Testing. EUCAST breakpoints for metronidazole: ≤4, susceptible; >4, resistant (http://www.eucast.org/clinical_breakpoints/). CLSI, Clinical and Laboratory Standards Institute. CLSI breakpoints for metronidazole: ≤8, susceptible; ≥32, resistant [43].
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Table 3. Resistance to metronidazole and prevalence of nim genes in Gram-positive anaerobic cocci (GPACs) and Clostridium spp. Testing method Etest Etest Agar dilution Etest Agar dilution Etest Agar dilution Etest Agar dilution Etest Agar dilution Etest Etest Agar dilution Etest Agar dilution
% of I+R 1 28.6 0.4 0 5.9 0 0 1.2 8.1 0 8 0 0.8 4 5.7(R) 2
MIC range (µg/ml) <0.016->256 0.25->256
≤0.125->32 ≤0.01-2 0.03-4 0.03-≥32 ≤0.06->128 0.016-4 0.06-128 <0.016-2 0.023->256 ≤0.015->256
% of nim genes NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
Reference [32] [20] [22] [33] [16] [9] [35] [40] [42] [12] [31] [30] [41] [10] [17] [10]
Belgium (38) 2011-2012 Etest EUCAST 0 <0.016-4 NR [32] Croatia (4) 2013 Etest EUCAST 0 1-2 NR [20] Europe* (1584) 2010-2016 Agar dilution EUCAST 0.8 NR [22] Japan (25) 2013-2014 Etest CLSI 0 NR [33] Korea (26) 2012 Agar dilution CLSI 0 ≤0.125-4 NR [16] Norway (32) 2009-2013 Etest EUCAST 0 0.032-4 NR [9] Pakistan (32) 2010-2011 Agar dilution CLSI 3 0.015-32 NR [35] Singapore (26) 2009-2011 Etest CLSI 19 ≤0.01-≥256 0 [29] Singapore (28) 2008-2010 Etest CLSI 21.5 NR [19] Slovenia (176) 2015 Etest EUCAST 0 0.016-4 NR [12] Taiwan (93) 2008-2012 Agar dilution CLSI 0 0.03-8 NR [31] The Netherlands (62) 2011-2013 Etest EUCAST 0 <0.016-4 NR [30] USA (614) 2010-2012 Agar dilution CLSI 0.8 NR [10] USA (228) 2010-2011 Etest CLSI 0 ≤0.015-8 NR [17] USA (116) 2007-2009 Agar dilution CLSI 0 NR [10] * data of the european Tigecycline Evaluation and Surveillance Trial (participating countries: France, Germany, Czech Republic, Hungary, Spain, Belgium, Italy, Sweden, United Kingdom) [22] I, intermediate; R, resistant. NR, not researched. EUCAST, European Committee on Antimicrobial Susceptibility Testing. EUCAST breakpoints for metronidazole: ≤4, susceptible; >4, resistant (http://www.eucast.org/clinical_breakpoints/). CLSI, Clinical and Laboratory Standards Institute. CLSI breakpoints for metronidazole: ≤8, susceptible; ≥32, resistant [43].
AC C
EP
TE D
Clostridium spp.
Breakpoints EUCAST EUCAST EUCAST CLSI CLSI EUCAST CLSI Eucast CLSI EUCAST CLSI EUCAST EUCAST CLSI CLSI CLSI
RI PT
Study period 2011-2012 2013 2010-2016 2013-2014 2012 2009-2013 2010-2011 2004-2014 1995-1996 2015 2008-2012 2011-2013 2002-2004 2010-2012 2010-2011 2007-2009
SC
Country (no. of isolates) Belgium (72) Croatia (14) Europe* (1741) Japan (26) Korea (34) Norway (15) Pakistan (14) Russia (81) South Africa Slovenia (589) Taiwan (50) The Netherlands (249) The Netherlands (115) USA (611) USA (176) USA (168)
M AN U
Microorganisms GPACs
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Table 4. Heterogeneity, inducibility and stability of resistance to metronidazole in clinical Prevotella isolates, P. baroniae DSM 16972T, and B. fragilis ATCC 25285T [adapted from [69] and from personal data].
Original
SGCb
P. baroniae DSM 16972T
nimI
2
NAc
P. baroniae LBN 427
nimI
4
NA
P. baroniae LBN 430
nimI
4
NA
P. baroniae LBN 432
nimI
2
NA
P. baroniae LBN 475
nimI
4
P. baroniae LBP19
nimI
1
P. bivia LBN 332
−
4
P. bivia LBN 371
−
4
P. bivia LBN 467
−
16
P. buccae LBN 465
−
0.5
P. nanceiensis LBN 293b
−
2
P. nanceiensis LBN 410
− −
B. fragilis ATCC 25285 a
Post-induction Post-stability 16
128
128
128
128
64
64
NA
256
256
NA
128
128
32
>256
>256
32
>256
>256
32
>256
>256
NA
128
64
32
256
256
1
32
256
256
0.5
NA
256
128
EP
TE D
M AN U
SC
16
AC C
T
RI PT
MICs determined by agar dilution (µg/ml)
Presence of nim gene
Straina
LBN/LBP, clincal strains from collection of Laboratoire de Bactériologie de Nancy and Laboratoire de Bactériologie de Poitiers, SGC, slowly growing colonies within the inhibition zone of the Etest strip, c NA, not applicable (absence of slowly growing colonies). b
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Nim protein nim gene
NimA
NimB
NimC
NimD
NimE
NimF
Accession CAA50581 CAA50578 CAA54269 CAA54273 CAB82516 CAD56147 No
RI PT
Table 5. Percentage of amino acid (upper diagonal) and nucleotide (lower diagonal) sequence identities between NimA to NimK as well as compare to the nitroimidazole reductase of Deinococcus radiodurans (NimA Dr) obtained by using the BioEdit Sequence Alignment Editor. NimG *
NimH
NimI
ACR56004 FJ940883
NimJ
NimK
NimA Dr
WP_005812 AXA20009 AAF10419 825
X71444
-
77.3%
68.6%
78.6%
78.0%
74.0%
79.3%
78.0%
58.0%
55.3%
64,6%
25,5%
nimB
X71443
71.1%
-
73.3%
85.3%
82.0%
78.6%
90.0%
82.6%
63.3%
58.0%
67,3%
24,4%
nimC
X76948
71.8%
69.8%
-
74.6%
72.6%
71.3%
75.3%
74.6%
60.6%
54.6%
59,3%
22,0%
nimD
X76949
73.3%
82.5%
72.4%
-
78.6%
78.6%
89.3%
85.3%
62.6%
56.6%
64,0%
22,0%
nimE
AJ244018
70.4%
75.1%
68.2%
73.5%
-
74.6%
80.0%
80.0%
63.3%
59.3%
66,0%
24,4%
nimF
AJ515145
71.5%
75.7%
71.1%
78.1%
70.0%
-
82.0%
80.6%
62.0%
56.6%
66,6%
23,8%
nimG
*
72.4%
89.8%
70.7%
85.2%
73.3%
77.9%
-
86.6%
64.0%
56.6%
66,6%
23,8%
nimH
KX576455
73.1%
81.4%
72.2%
82.3%
73.5%
75.5%
83.9%
-
64.0%
59.3%
66,0%
23,2%
nimI
FJ940883
60.1%
59.7%
62.1%
62.8%
61.7%
63.2%
62.5%
63.8%
-
74.0%
58,6%
24,4%
nimJ
KJ816753
57.6%
61.7%
58.3%
60.6%
59.3%
57.8%
59.8%
60.2%
68.2%
-
54,0%
22,0%
nimK
MG827401
63,8%
66,9%
61,2%
65,6%
66,0%
65,8%
66,0%
66,5%
57,3%
57,5%
-
22,6%
nimA Dr AE000513
40.2%
38.1%
36.5%
39.0%
39.6%
40.0%
38.1%
35.0%
M AN U
TE D
EP
AC C 40.9%
40.9%
SC
nimA
-
* nimG sequence was not available in GenBank. The nucleotidic sequence published by Gal & Brazier as well as its deduced amino acid sequence were used [71].
1
ACCEPTED MANUSCRIPT
Table 6. Characteristics and diversity of nim genes described in the literature.
(0.75−>128 µg/mL)
variable nimBa
C
(0.125−>25 6 µg/mL) variable
nimC
P or C
57.27
IS1170
(1−>32 µg/mL) variable
P or C
50.22
IS1169
(0.25−≥64 µg/mL) variable
nimE
P
e
41.19
ISBf6
B. fragilis, B. thetaiotaomicron, B. vulgatus, Bacteroides stercoris, Fusobacterium sp., Odoribacter splanchnicus
AC C
nimD
(1.5−>256 µg/mL)
Associated in vitro antimicrobial resistance
RI PT
42.73
IS1168 IS612
variable
B. fragilis, B. thetaiotaomicron, B. vulgatus, Bacteroides ovatus, Bacteroides uniformis, P. distasonis, Prevotella bivia, Actinomyces odontolyticus, Cutibacterium acnes, Propionibacterium sp., Clostridium bifermentans B. fragilis, B. thetaiotaomicron, B. vulgatus, P. distasonis, Prevotella denticola, Prevotella dentalis, Finegoldia magna, Peptostreptococcus anaerobius, Anaerococcus prevotii, Parvimonas micra B. fragilis, B. thetaiotaomicron, P. distasonis, Prevotella oralis, Porphyromonas sp.
SC
54.63
IS1168 IS1169 ISBf13
Bacterial species or genera
M AN U
P or C
G+C (mol%)
TE D
nimA
Genetic location
EP
Nim gene
Association IS present with upstream resistance of nim to MTZ (MICs)
B. fragilis, B. thetaiotaomicron, B. vulgatus, B. ovatus, Parabacteroides merdae, Prevotella buccalis, Prevotella oralis, Veillonella sp.
Associated resistance genes
References
cepA, blaOXAAMX, AMC, PIP, , cfiA, ermF, TZP, FOX, CAR, 37 linAn2, mefE, ERY, CLI, TET tetQ, bexB
[14,32,49,53,7 1,73,79,85,90, 91,96,103,104, 107,108,110,1 13]
AMP, PIP, TZP, FOX, CAR, CLI, TET, RIF
cfxA, cfiA, ermF, tetQ
[27,71,73,80,8 5,90,91,96,97, 106,107,109,1 14]
TET
cfiA, tetQ
[14,71,73,79,8 6,91,107]
cepA, cfxA, cfiA, ermF, AMP, TZP, CAR, linAn2, mefE, TET, CLI tetQ, cat, bexB-liked AMP, AMX, cepA, cfxA, AMC, PIP, TZP, blaTEM, blaCMY, FOX, CAZ, CAR, blaOxA-1, cfiA, ERY, CLI, TET, ermF, tetQ, TGC, CM, CIP aac(6’)Ib-cr
[14,32,50,71,7 3,74,86,91,107 –109]
[14,44,49,51,7 1,73,78,79,91, 98,107,108,11 0,112]
1
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ND
51.88
noneb
no (1 µg/mL)
B. vulgatus
ND
ND
[73]
nimG
ND
47.14
ND
no (2 µg/mL)
B. fragilis
ND
ND
[71]
nimH
C
48.46
IS614B
AMX
NDf
[92]
nimI
C
58.64
nonec
ND
ND
[70]
AMP, AMX, AMC, TZP, FOX, CFP, CAR, ERY, CLI, TET
cfiA, ermR, tetQ, bexBliked, overexpression of bmeABC5 genes
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45.14
IS614 IS614B IS4 family
P. baroniae
yes (>32 µg/mL)
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B. fragilis
B. fragilis
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nimJ
yes (8 µg/mL) no (1−4 µg/mL)
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nimF
[54,77,78,108]
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yes efflux SMR [93] C 37.20 (6−12 P. bivia AMX, CLI nimK transporter µg/mL) P, plasmid; C, chromosomal; ND, not determined; MTZ, metronidazole; MIC, minimal inhibitory concentrations; Other antibiotics’ abbreviations: AMP, ampicillin; AMX, amoxicillin, AMC, amoxicillin/clavulanic acid; PIP, piperacillin; TZP, piperacillin/tazobactam; FOX, cefoxitin; CAZ, ceftazidime; CFP, cefoperazone; CAR, carbapenems; ERY, erythromycin; CLI, clindamycin; TET, tetracycline; TGC, tigecycline; RIF, rifampicin; CM, chloramphenicol; CIP, ciprofloxacin; a a nimB-like element sharing 84% identity with nimB, was also described in Peptoniphilus asaccharolyticus (sequence non available in GenBank) [80]; b among IS1168, IS1169 and IS1170; c among IS1168, IS1169, IS1170, ISBf6 and IS612; d putative bexB gene (91% sequence identity) [108]; e data obtained by Sydenham et al. from WGS could suggest a chromosomal location of nimE [108]. f absence of cfiA gene, other resistance genes not determined [92]. IS1380 family
2
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Figure 1
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Figure 2
ACCEPTED MANUSCRIPT Highlights
- Metronidazole is a drug of choice for treatment of infections caused by anaerobes. - MTZ acquired resistance may associate several mechanisms including Nim proteins.
- nim genes are present in a wide range of bacterial phyla.
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- 10 nim genes of variable expression (silentvarious resistance levels) are described.
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- nim genes are part of the genetic content of multidrug resistant anaerobes.
S1075-9964(18)30176-8
DOI:
10.1016/j.anaerobe.2018.10.004
Reference:
YANAE 1955
To appear in:
Anaerobe
Received Date: 3 January 2018 Revised Date:
1 October 2018
Accepted Date: 5 October 2018
Please cite this article as: Corentine Alauzet, Alain Lozniewski, Hélène Marchandin, Metronidazole resistance and nim genes in anaerobes: a review, Anaerobe (2018), doi: 10.1016/ j.anaerobe.2018.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Metronidazole resistance and nim genes in anaerobes: a review
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Corentine Alauzet 1,2, Alain Lozniewski 1,2, Hélène Marchandin 3,4*
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France
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Laboratory of Bacteriology, Nancy University Hospital, F-54000 Nancy, France
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HydroSciences Montpellier, CNRS, IRD, Univ Montpellier, CHU de Nîmes, Montpellier,
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France
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Department of Microbiology, Nîmes University Hospital, Nîmes, France
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Université de Lorraine, EA7300 Stress Immunité Pathogènes (SIMPA), F-54000 Nancy,
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* Corresponding author: Prof. Hélène Marchandin, UMR 5569 HydroSciences Montpellier,
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Equipe Pathogènes Hydriques Santé Environnements, U.F.R. des Sciences Pharmaceutiques
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et Biologiques, Université de Montpellier, 15, avenue Charles Flahault, BP 14491, 34093
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Montpellier Cedex 5, France
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[email protected]
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Declarations of interest: none.
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ACCEPTED MANUSCRIPT Abstract
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Acquired resistance to metronidazole, a 5-nitroimidazole drug largely used worldwide in the
22
empirical treatment of infections caused by anaerobes, is worrisome, especially since such
23
resistance has been described in multidrug-resistant anaerobic bacteria. In anaerobes, acquired
24
resistance to metronidazole may be due to a combination of various and complex
25
mechanisms. Among them, nim genes, possibly located on mobile genetic elements, encode
26
nitro-imidazole-reductases responsible for drug inactivation. Since the first description of
27
Nim proteins about 25 years ago, more nim genes have been identified; currently 11 nim
28
genes are known (nimA to nimK). Mostly reported in Bacteroides fragilis group isolates, nim
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genes are now described in a variety of anaerobic genera encompassing the 4 main groups of
30
Gram-negative and Gram-positive bacilli and cocci, with variable expression ranging from
31
phenotypically silent to low-level or high-level resistance to metronidazole.
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This review describes the trends of metronidazole resistance rates among anaerobes over the
33
past 15 years and summarizes current knowledge on mechanisms involved in this resistance.
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It also provides an update on the phylogenetic and geographical distribution of nim genes, the
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mechanisms involved in their expression and regulation, and their role in metronidazole
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resistance.
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Keywords: metronidazole, resistance, nim gene, anaerobes
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ACCEPTED MANUSCRIPT 1. Metronidazole, a prodrug used as first-line treatment in the management of a variety
41
of infectious diseases
42
Metronidazole and other 5-nitroimidazoles (5-Ni) are antimicrobial agents that are remarkable
43
regarding their activities against both parasites and microaerophilic and anaerobic bacteria
44
[1]. Metronidazole is indicated for use in a variety of infections involving anaerobic bacteria
45
including bacterial vaginosis and Clostridium difficile infections, but also in other indications
46
encompassing symptomatic intestinal and extraintestinal amebiasis, trichomoniasis, giardiasis,
47
and Helicobacter pylori infections. This synthetic derivative of azomycin, a nitroimidazole
48
compound isolated from Streptomyces spp., was first used in the late 50s to treat infections
49
caused by parasites [1]. Its antibacterial activity was discovered in 1962 when it was observed
50
that metronidazole was able to cure a patient from both trichomoniasis and bacterial gingivitis
51
[2]. Subsequent studies demonstrated the clinical efficacy of metronidazole for the treatment
52
of infections caused by anaerobes which led to its extensive use for the treatment of these
53
infections from the 1970s until today [3].
54
5-Ni are prodrugs, which penetrate bacterial cells via passive diffusion and are further
55
intracellularly activated before exhibiting their bactericidal activity (Figure 1) [4]. Although
56
the mechanism of action of 5-Ni is not entirely clear, it is believed that these drugs are
57
activated by the reduction of the nitro group. As reduction proceeds, a favorable
58
transmembrane concentration gradient is created which further enhances intracellular
59
diffusion [4]. The absence of electron-transport proteins with sufficient negative redox in
60
aerobic bacteria explains their intrinsic resistance to 5-Ni [3]. The reductive activation of 5-Ni
61
is assumed to occur in a series of steps beginning with the formation of an anionic nitro-
62
radical anion (R-NO2•−) followed by the formation of a nitrous derivative (R-NO), a nitrous
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free radical (R-NO−) and finally a hydroxylamine derivative (R-NHOH). It is assumed that R-
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NO2•− is one of the more active molecules able to degrade cellular macromolecules, and
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ACCEPTED MANUSCRIPT particularly DNA, via oxidation [4]. Oxidation of DNA in turn causes strand breaks and
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subsequent cell death. In anaerobes, reduction occurs when metronidazole receives an
67
electron from ferredoxin or flavodoxin that was reduced by the pyruvate-
68
ferredoxin/flavodoxin oxidoreductase (PFOR) system. Metronidazole acts as an electron trap
69
that captures electrons that would normally be donated to hydrogen ions to form molecular
70
hydrogen. This system is involved in the decarboxylation of pyruvate to acetyl-CoA. It is
71
noteworthy that pyruvate may also be converted to lactate by lactate dehydrogenase (LDH).
72
Some strict and facultative anaerobes, such as Cutibacterium spp., Propionibacterium spp.,
73
Lactobacillus spp. and most bifibobacteria, Eubacterium spp. and Actinomyces spp. display
74
intrinsically reduced susceptibility or resistance to metronidazole that could be linked to a
75
lack of the PFOR system or to the use of another way to catabolize pyruvate involving the
76
NADH-producing pyruvate dehydrogenase [3,5]. These taxa apart, other anaerobes remain
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highly susceptible to metronidazole.
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This excellent activity against anaerobes, its low cost, its favorable pharmacokinetic and
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pharmacodynamics properties, as well as minor adverse effects make metronidazole still the
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cornerstone for the management of anaerobic infections, despite the emergence of strains with
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acquired resistance [3,6].
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2. Resistance to metronidazole in anaerobes is complex, still uncommon but worrisome
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2.1 A low but steady and geographically diverse increase in prevalence
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The first report of acquired resistance to metronidazole described a B. fragilis isolate
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recovered in 1978 from a patient with Crohn’s disease who had been extensively treated with
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metronidazole [7]. Since then, the prevalence of acquired resistance among anaerobes has
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remained overall relatively low. Most studies about metronidazole resistance are focused on
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Bacteroides (Table 1). These studies reported low level (<3%) of metronidazole resistance in
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ACCEPTED MANUSCRIPT most part of the world [8–33], except higher resistance rates reported in Spain (4.8%), in
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South Africa (8.7%) and in Pakistan (16%) [34–36]. Regarding other Gram-negative
92
anaerobes (Table 2), resistance to metronidazole has until now only rarely been detected
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among Fusobacterium isolates. Indeed, except for the studies by Katsandri et al. [14] (17
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strains isolated in Greece, 11.7% of reduced susceptibility to metronidazole ) and Wang et al.
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[31] (48 strains isolated in Taiwan, 4.2% of reduced susceptibility to metronidazole), isolates
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included in all other surveillance studies were found susceptible to metronidazole
97
[9,10,12,17,19,25,29,30,32]. In contrast, reduced susceptibility to metronidazole is more
98
frequent among Prevotella and to a lesser extent among Veillonella isolates depending on the
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geographical area [9,10,12,14,16,17,22,24,25,29–31,33,37–39]. In several countries, reduced
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susceptibility of Prevotella to metronidazole has been found in more than 10% of the isolates
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tested [9,14,16,29]. In two studies, which included a large number of Prevotella, clinical
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strains recovered in The Netherlands (2011-2013) and in the USA (2010-2012),
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metronidazole resistance rates of 2 and 3% were observed, respectively [10,30]. Regarding
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Gram-positive anaerobes, reduced susceptibility to metronidazole has so far only been rarely
105
detected in surveillance studies among Gram-positive cocci including Parvimonas micra,
106
Finegoldia magna and Peptostreptococcus sp. isolates, as well as among clostridia including
107
Clostridium perfringens, Clostridium ramosum and Clostridium bifermentans
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[9,10,12,17,22,29,31–33,40,41] (Table 3). However, Koch et al. [42] described 8.1% of
109
metronidazole resistance in Peptostreptococcus spp. and Ng et al. [19] described 21.5% of
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resistance in Clostridium spp. It is noteworthy that for most surveillance studies performed in
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European countries, breakpoints interpretation was based on European Committee on
112
Antimicrobial Susceptibility Testing (EUCAST) recommendations
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(http://www.eucast.org/clinical_breakpoints/) while for studies emanating from other
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countries, Clinical and Laboratory Standards Institute (CLSI) breakpoints [43] were used.
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ACCEPTED MANUSCRIPT Even though results obtained in these studies give an overall idea of metronidazole resistance
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within these two groups of countries, it cannot be ruled out that some of the differences
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observed around the world are due to differences in the number of strains and species
118
included, testing methods and breakpoints used. This underlines the importance of
119
standardizing study methods on a global scale.
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While the overall metronidazole resistance rate is still relatively low, it is noticeable that
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isolates exhibiting simultaneous resistance to metronidazole and to other antibiotics have been
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increasingly reported during the last twenty years. The associated resistances concern a wide
123
range of antibiotics routinely used to treat anaerobic infections, including carbapenems. Such
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multidrug-resistant (MDR) isolates, mainly belonging to the B. fragilis group, have been
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involved in human infections and, in many cases, have been considered as being responsible
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for therapeutic failure [11,18,25,44–55].
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2.2 Acquired metronidazole resistance mechanisms in anaerobes Several molecular mechanisms have been associated with metronidazole resistance, mainly
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described in B. fragilis (Figure 1). Resistance related to drug inactivation by nitroimidazole
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reductase encoded by nim genes is the mechanism that has been most studied and detected in
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a wide range of anaerobic species [56–58]. This mechanism is detailed in part 3.1.
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Metronidazole resistance could also be related to impaired intracellular activation as observed
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for the first described B. fragilis isolate (NCTC 11295) that exhibited high-level resistance to
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metronidazole [7]. The resistance was non-transferable by conjugation [57], and, by
135
comparing this strain with susceptible ones, Narikawa et al. [59] showed that its
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metronidazole resistance was related to a decreased activity of PFOR associated with a high
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activity of LDH. The impact of impaired enzymatic activity of the PFOR complex on
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metronidazole resistance in B. fragilis has been further confirmed by Diniz et al. [60].
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Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were
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metronidazole [61].
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Metronidazole resistance in B. fragilis may also be due to other mechanisms such as drug
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extrusion by multidrug efflux pumps, increased DNA repair capacity and activation of
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antioxidant defense systems [58,62–65]. It has also been shown that the deficiency of the
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ferrous iron transporter FeoAB is linked with metronidazole resistance in B. fragilis
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suggesting an impact of intracellular iron homeostasis on metronidazole activity [66]. Patel et
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al. [67] demonstrated the relationship between overexpression of the rhamnose catabolism
148
regulatory protein RhaR and metronidazole resistance in Bacteroides thetaiotaomicron by a
149
yet unknown mechanism. In C. difficile, it has also been proposed that, in addition to impaired
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intracellular activation, other mechanisms such as increased DNA repair capacity, altered iron
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metabolism and biofilm formation, may contribute to metronidazole resistance [61,68]. Other
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studies reported the mutagenic activity of metronidazole in diverse bacterial species including
153
B. fragilis and mutations in the nitroreductase-encoding gene rdxA have been associated with
154
the induction of metronidazole resistance in H. pylori [69]. Despite such an association
155
between mutagenicity and resistance inducibility has not been formally demonstrated in
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anaerobic species, such mutations should therefore also be considered as another potential
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source for metronidazole resistance in anaerobes.
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Finally, it is interesting to note that induced resistance to metronidazole has been reported in
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several studies, highlighting the importance of careful susceptibility testing of anaerobes in
160
order to not overlook inducible homogeneous or heterogeneous metronidazole resistance after
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prolonged exposure to this antibiotic. Indeed, some works reported the growth of small
162
colonies inside the inhibition zone of the disk or of the Etest strip after prolonged incubation
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of 72 to 120 hours (Table 4), and showed that slowly growing sub-populations displayed
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stable enhanced metronidazole MICs ranging from 8 to >256 mg/L [70,71]. Other studies
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ACCEPTED MANUSCRIPT reported that metronidazole resistance could also be induced in anaerobes by successive
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culture in presence of raising subinhibitory concentrations or high doses of metronidazole
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[60,70–75]. A recent study exploring the global mechanisms of bacterial survival upon
168
metronidazole exposure in the absence of nim gene using a RNA-seq transcriptomic approach
169
confirmed the complexity of metronidazole resistance [76]. In this study, de Freitas et al.
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compared a metronidazole-susceptible B. fragilis strain to 4 derivative metronidazole-resistant
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strains that were induced by subcultures with subinhibitory antibiotic concentrations. They
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showed that several metabolic pathways associated with metronidazole response, such as
173
impaired drug activation (via PFOR complex or LDH), high expression level of multidrug
174
efflux pumps or DNA repair systems, were altered in the resistant strains. These authors also
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found that changes in the whole gene expression patterns were maintained even when the
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metronidazole selection pressure was stopped, suggesting that drug exposure led to drastic
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persistent gene expression changes and that such persistent alterations may be involved in the
178
emergence of resistant strains. Such results are in accordance with the stability of induced
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metronidazole resistance that was described in most of the studies dealing with this subject
180
[70,71,73,75].
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However, none of the mechanisms that may be involved in metronidazole resistance have
182
been shown to be responsible per se for high-level metronidazole resistance. Diniz et al. [60]
183
reported that none of the B. fragilis mutants lacking the genes for flavodoxin and/or pyruvate-
184
ferredoxin oxidoreductase was as resistant as the spontaneous metronidazole resistant strain
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derived from B. fragilis ATCC 25285T after selection by metronidazole exposure. Pumbwe et
186
al. showed that MICs of metronidazole were only moderately increased in B. fragilis mutants
187
overexpressing bmeRABC5, which encodes a RND-family efflux pump that confers
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metronidazole resistance [63]. Similar results were obtained when an inhibitor of the
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multidrug efflux pumps bme was used, thereby showing that metronidazole efflux systems
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ACCEPTED MANUSCRIPT alone did not cause high levels of resistance [77]. Similarly, only low to moderate levels of
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metronidazole resistance were found in feoAB-deficient mutants generated in B. fragilis and
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in B. thetaiotaomicron mutants overexpressing RhaR [66,67]. Regarding nim genes (details in
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part 3.4), several studies have also demonstrated that their presence is not per se sufficient to
194
confer high-level metronidazole resistance [71,78–80]. Thus, there is nowadays unambiguous
195
evidence that metronidazole resistance is a complex phenomenon that is likely to be
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multifactorial and to include probably yet unknown mechanisms [6].
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3. nim genes and metronidazole resistance
3.1. From nimA to nimK: from the pioneering research at the Pasteur Institute to
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published works based on whole genome sequencing
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3.1.1. Description of nimA to nimD genes
The presence of specific transferable 5-Ni resistance determinants was first reported in 1989
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by Breuil et al. [81] in a Bacteroides vulgatus strain, BV17. This strain carries four plasmids,
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one of which (pIP417) was responsible for low-level metronidazole resistance after
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introduction in a susceptible strain, B. fragilis 638R. The resistance of BV17, as well as that
206
of B. thetaiotaomicron BT13 (pIP419-positive) and of B. fragilis BF8, a plasmid-free strain,
207
have been shown to be transferable by a conjugal-like process to susceptible strains with a
208
frequency ranging from 10−3 to 10−7 per donor [81–84]. The associated genetic determinants
209
were identified in 1994 by the team of Madeleine Sebald at the Pasteur Institute of Paris, with
210
the description of nimA and nimB genes [85] followed by that of nimC and nimD genes [86].
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Two years later, Carlier et al. explored the enzymatic activity of the nim gene products by
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comparing the metabolism of a 5-Ni-susceptible B. fragilis strain with that of the same strain
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harboring a nimA-carrying plasmid [56]. While the classic reduction of 5-Ni drugs to its nitro
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radical anion was observed in the susceptible strain, the nimA-positive strain seemed to
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radicals that are essential for antimicrobial activity. These findings led the authors to conclude
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that Nim proteins exhibited a nitroimidazole reductase activity, which causes the reduction of
218
the nitro group of 4- or 5-Ni to an amino group leading to a 5-aminoimidazole inactive
219
compound [56]. The crystal structure of NimA from Deinococcus radiodurans (NimA Dr)
220
was elucidated seven years later [87]. This protein is weakly related (less than 26% identity)
221
to the Nim proteins described in Bacteroides and Prevotella (Table 5). Leiros et al. [87–89]
222
characterized specific and conserved residues either in the active site (His-71) or in the 5-Ni-
223
binding site (Pro-56, Tyr-111) of Nim proteins. From structure analysis, the following
224
mechanism has been proposed: i) native NimA structure with hydrogen-binding of pyruvate
225
from the PFOR complex to the His-71, ii) modification of link between His-71 and pyruvate
226
that became covalent upon metronidazole binding, iii) oxidation of His-71 and pyruvate into a
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His-71-Pyr residue associated with the release of two electrons and one proton, iv) reduction
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of metronidazole by the released electrons into a non toxic compound [87–89].
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3.1.2. Detection of nim genes by specific targeted PCR approach or whole
230
genome sequencing
Considering the impact of an eventual dissemination of such genes among anaerobes, Trinh &
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Reysset proposed in 1996 a PCR method based on universal primers NIM3 (5’-
233
ATGTTCAGAGAAATGCGGCGTAAGCG-3’) and NIM5 (5’-
234
GCTTCCTTGCCTGTCATGTGCTC-3’) that is able to detect all subtypes of nim genes by
235
targeting conserved sequences [90]. Since then, this method, which has been widely used to
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evaluate the presence of nim genes among anaerobes, has led to the discovery of five new
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variants. nimE was first described in B. fragilis, B. thetaiotaomicron and Bacteroides ovatus
238
[91], nimF in B. vulgatus [73], nimG and nimH in B. fragilis [71,92], and nimI in Prevotella
239
baroniae [70]. A tenth nim gene was identified by Husain et al. after whole genome
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ACCEPTED MANUSCRIPT sequencing (WGS) of two B. fragilis strains [78]. In this study, the annotation of their
241
genomes using the RAST annotation server (http://rast.nmpdr.org) revealed a gene coding for
242
a protein of the pyridoxamine 5′-phosphate oxidase-related protein family, which includes
243
Nim proteins. This gene, named nimJ, was not detected by the universal primers NIM3-NIM5
244
due to distinct nucleotide sequences at the sites of primers annealing; however, the predicted
245
amino acid sequence of NimJ was identical to that of the universal NIM3 forward primer
246
(MFREMRRK) and differed by one amino acid from that of the universal NIM5 reverse
247
primer (EHMTGKEEHLTGKE) [54,77,78]. By demonstrating that at least one nim variant
248
could not be detected by the universal primers used so far, Husain et al. highlighted that this
249
might be the case with other unknown nim alleles and that this could account at least in part
250
for some nim-negative metronidazole resistant strains observed in several epidemiological
251
studies [78]. The latest variant described is nimK, recently detected by WGS in three
252
metronidazole-resistant Prevotella bivia clinical isolates [93]. The sequence of nimK shows,
253
however, that this gene could have been amplified by using NIM3-NIM5 primers. Beside
254
detection of new nim genes, WGS led to identify known nim genes associated to molecular
255
markers of resistance to other antibiotics. For example, Ank et al. described nimE together
256
with cfiA, ermF and tetQ genes in a multidrug-resistant B. fragilis isolate exhibiting
257
piperacillin-tazobactam, carbapenem, metronidazole, clindamycin and tetracycline resistance
258
[44]. Similarly, nimD was found in the complete genome sequence of a metronidazole-
259
resistant Bacteroides stercoris isolate from a polymicrobial intra-abdominal abscess [74]. In
260
the latter WGS-based studies, nim genes were mostly detected by using a database
261
specifically designed for the identification of acquired antibiotic resistance genes in totally or
262
partially sequenced bacterial isolates, i.e., ResFinder
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(http://cge.cbs.dtu.dk/services/ResFinder/) [94] or on the basis of functional annotation.
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3.1.3. Current diversity of nim genes
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ACCEPTED MANUSCRIPT To date, eleven nim genes (nimA to nimK) sharing between 57.6 and 89.8% nucleotide
266
sequence identities (corresponding amino acid sequence identities range: 54 and 90%) have
267
been described (Table 5). The highest degree of amino acid identity was observed between
268
NimD and NimG. The phylogenetic relationships between Nim proteins are shown in Figure
269
2. Two clusters of proteins are demonstrated within the Nim protein family, the NimA-H/K
270
clade grouping NimA to NimH and NimK proteins displaying more than 59% of sequence
271
identity and the NimI-J clade including NimI and NimJ proteins that display 74% of sequence
272
identity (Table 5).
273
The advent of WGS led to improve the exploration of nim genes diversity as this approach
274
permit the discovery of new variants not detected by universal primers. Two putative new nim
275
genes (protein accession No: WP_005782870 and WP_005811941) were thereby detected in
276
the metronidazole-resistant Bacteroides strain UW, sharing respectively the higher amino acid
277
sequence identity with NimE (33%) and NimF (40%) [95]. Similarly, WGS of a multidrug
278
resistant B. thetaiotaomicron clinical isolate revealed the presence of nimD gene (99.2% of
279
sequence identity with B. fragilis nimD (X76949)) [50]. In this strain, a second nim-like
280
element, distantly related to known Nim proteins (26.8% of nucleotide sequence identity with
281
nimE), and sharing 97.5% identity with a B. thetaiotaomicron VPI 5482 gene encoding an
282
uncharacterized 5-Ni antibiotic resistance protein (AAO78184), was also detected. The latter
283
observations highlighted the presence of several nim determinants within a genome. Although
284
multiple nim copies of a same gene were previously described in B. fragilis (2 copies of nimJ
285
[78], 2 copies of nimA [Marchandin et al., personal communication]), this is to our knowledge
286
the first observations of different nim determinants in the same genome.
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3.2. nim genes through the phyla, hosts and continents
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nim genes were initially described in several cultivated Bacteroides species, mainly belonging
289
to the B. fragilis group, as well as in the reclassified Parabacteroides distasonis (Table 6).
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291
splanchnicus, Porphyromonas sp. and in various species of Prevotella. Indeed, within the last
292
decade, one nimA-positive Prevotella bivia as well as two nimB-positive strains (Prevotella
293
dentalis and Prevotella denticola), one nimC-positive Prevotella oralis and two nimE-positive
294
isolates (Prevotella oralis and Prevotella buccalis) were detected [14,96,97]. It is interesting
295
to note that nimI seems to be intrinsic to the species Prevotella baroniae as it has only been
296
recovered in nine P. baroniae French clinical isolates as well as in the British type strain
297
DSM 16972T whereas it was not detected in 33 type strains belonging to other Prevotella
298
species [70]. nim genes were also described in anaerobes belonging to other phyla (Table 6).
299
Indeed, they were detected in Actinobacteria (Actinomyces odontolyticus, Cutibacterium
300
acnes and Propionibacterium sp.), in Firmicutes (Gram-positive anaerobic cocci, Clostridium
301
bifermentans, and Veillonella sp.) as well as in Fusobacteria (Fusobacterium sp.)
302
[14,80,96,98].
303
Interestingly, only half of the nim subtypes present a G+C content that is compatible with
304
their main host’s genome (i.e. 39-49 mol% for Bacteroides sp. and 40-52 mol% for Prevotella
305
sp.) [78,99,100]. The G+C content of nimA, nimC, nimD, nimF and nimI is >50 mol% and the
306
newly described nimK has a very low G+C content of 37.2 mol% (Table 6). These
307
observations, associated with the presence of nim genes in a wide range of bacterial phyla,
308
suggest that nim genes could have resulted from a transfer from an unknown common
309
ancestor followed by independent evolution. In addition, the increasing availability of
310
bacterial genome sequences leads to the detection of Nim-related proteins, for which the
311
function is still unknown, in phylogenetically distant species including aerobic genera (Ex:
312
Deinococcus radiodurans and Streptomyces avermitilis, with respective accession numbers
313
AAF10419 and Q827C5) and Archaea (Ex: Methanosarcina mazei, accession number
314
Q8PT76). These observations suggested that the nim gene family is ancient and widespread in
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316
intestinal microbiota could represented a very favorable environment and that, once
317
introduced and adapted in Bacteroides, a genus particularly abundant in the gut and actively
318
involved in horizontal gene transfer, nim genes could easily spread under selective pressure
319
[78,99,102].
320
Studies that have evaluated the prevalence of nim genes among anaerobes are scarce and
321
showed overall relatively low level of these genes varying from 0.5 to 2.8% among
322
Bacteroides spp. [14,21,27,29,32] and from 0 to 5.3% or 5.9% for Prevotella spp. or
323
Fusobacterium spp., respectively [14,29] (Tables 1 to 3). Although still infrequent, nim-
324
positive strains are described in most of the continents. The majority of studies examining the
325
distribution of nim genes in anaerobes result from European groups [27,32,44,54,70,71,73–
326
75,78,79,85,89–92,98,103–108] but nim-positive isolates have also been reported in Brazil,
327
China, India, Kuwait, South Africa or USA [29,49–51,77,78,80,96,97,107,109–114]. In these
328
studies, nim-positive strains were isolated from various clinical specimens, mainly from
329
abdominal specimens (abscess, empyema, pus, appendectomy fluid, pancreatic pseudocyst,
330
…) but also from blood cultures as well as from periodontal, osteoarticular and, more rarely,
331
respiratory tract specimens. Few strains were recovered from fecal samples of healthy
332
volunteers [85,105,109,111,115] and one B. fragilis carrying a nimB gene was isolated from
333
polluted aquatic environment [114].
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3.3. Genetic environment and regulation of nim gene expression
335
Early descriptions of nim genes described their location on a variety of small mobilizable nim-
336
positive plasmids (pIP417, pIP419 and pIP421) [81,84–86]. It was also shown that nimB was
337
located on the chromosome of B. fragilis BF8 [82]. Since then, it has been shown that nimA,
338
nimC and nimD genes could be either plasmidic or chromosomal (Table 6). nimB and nimI
339
seem to be exclusively chromosomal whereas nimE gene was recovered on plasmid. The
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ACCEPTED MANUSCRIPT locations of nimF and nimG have not been elucidated and an exclusive chromosomal location
341
of nimH, nimJ and nimK needs to be further confirmed on other isolates.
342
It has been assumed that the transcription of these genes may be activated by insertion
343
sequence (IS) elements. Indeed, Husain et al. introduced nimE and nimJ, cloned into the
344
strong promoter expression vector pMCL140, in B. fragilis 638R and observed higher
345
metronidazole MICs for the recipient strain despite not reaching those of the original clinical
346
isolates [78]. nim transcription levels were also 2- to 4-fold lower in strain 638R than in the
347
original clinical isolates. The fact that nim genes were cloned into pMCL140 without their
348
associated IS element could explain the discrepant result as compared to the study of Soki et
349
al. who transferred the nimE-positive plasmid pBF388c by mating into B. fragilis 638R and
350
observed an increased metronidazole MIC of the transconjugant, similar to that of the nimE
351
donor strain (16 mg/L) [107]. Several transposases belonging to the IS4 or IS5 families have
352
been described upstream of the nim genes but they are not consistently present and not always
353
dedicated specifically to a variant of nim (Table 6) [107,116,117]. These IS were either
354
identical or similar to those described in imipenem-resistant strains [86,92,107]. When
355
present, IS1168 (isoform of IS1186) was the most frequently transposase associated with
356
nimA and nimB. IS1169 was the only transposase recovered in association with nimD and was
357
also rarely detected upstream of nimA. IS1170 and ISBf6 have only been described associated
358
to nimC and nimE, respectively. In B. fragilis, one case of association between IS612 and
359
nimB was observed by Soki et al. [107] and the new transposase ISBf13 was detected by
360
WGS upstream of nimA by Sydenham et al. [108]. Finally, IS614B, a putative mosaic or
361
hybrid of IS612, IS614 and IS942 was found in association with nimH and nimJ [78,92].
362
Open reading frame (ORF) of these IS are in the opposite orientation of the nim ORF and they
363
usually carry on their right end outward-oriented promoters that could activate nim
364
transcription [86,92,107]. For example, based on sequences homologous to consensus
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ACCEPTED MANUSCRIPT sequences of B. fragilis promotors, TAnnTTTG in the −7 region and TnTG in the −33 region,
366
a putative promotor sequence of nimH was described within the IS614B [92,118].
367
Additionally, IS elements have been assigned a role in the expression of other resistance
368
genes in Bacteroides spp. such as those linked with resistance to macrolides (erm), cefoxitin
369
(cepA), cephalosporin (cfxA) or carbapenems (cfiA), and have been proved to be
370
interchangeable between these genes [3,107,119]. Among all the studies recovered in the
371
literature, the presence of an IS upstream of a nim gene is not always associated with
372
metronidazole resistance [47,71,73,85,90,107] and, conversely, a nim gene without an
373
upstream identified IS can be associated to metronidazole resistance [47,71,73,74,95,107,108]
374
(Table 6). In the latter case, the resistance could be explained by another nim-independent
375
mechanism (see part 2.2) or by the presence of a new IS not detected by the methods used.
376
For example, Sydenham et al. [108] described the presence of ISBf13 upstream of nimA but
377
on the complement strand. The action of such IS promoters upstream of nim genes remains
378
controversial and need to be further explored as no experimental evidence has confirmed their
379
role.
380
The presence of IS elements in the upstream region of nim genes could not only potentially
381
influence their expression but also their dissemination. Indeed, IS are small mobile genetic
382
elements involved in plasticity and adaptability of prokaryote genomes [116,117]; They are
383
widespread in all bacterial phyla and can occur in a wide range of copy numbers in a genome.
384
They can move within a chromosome, participating in modulation of neighboring gene
385
expression. They can also be involved in transfer of genetic material from chromosome to
386
other mobile genetic elements vectors such as phages and plasmids, thus participating to gene
387
dissemination [116,117]. It has been shown that nim gene-positive plasmid transfers have
388
occurred in vitro but it was also hypothesized that they have occurred in vivo, as plasmids
389
similar or identical to pIP417 or pIP419 have been detected in different clinical B. fragilis
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ACCEPTED MANUSCRIPT 390
isolates [81,82,84,107,120]. Regarding chromosomal nim genes, it can also be suggested that
391
the presence of IS could permit dissemination.
392
3.4. A variably expressed and inducible resistance Studies based on WGS showed that the presence of nim genes (A, D, E, J, new nim genes)
394
correlated with phenotypically resistant strains (metronidazole MICs ranging from 6 to >256
395
mg/L) [44,50,73,77,93]. However, several other studies showed that the presence of nim gene
396
does not always lead to resistance and a wide range of metronidazole MICs are observed in
397
nim-positive isolates ranging from 0.125 to >256 mg/L. For example, Gal et al., studying 50
398
Bacteroides spp. strains with positive detection of nim genes by specific PCR, found that
399
about half of them only presented MICs ranging from 16 to >32 mg/L and thus above the
400
resistance breakpoint [71]. Except for nimI, that seemed to be a ‘silent’ gene as it has never
401
been associated with metronidazole resistance in all P. baroniae strains tested [102], and for
402
nimF and nimG, that were described in unique metronidazole-susceptible (respective MICs of
403
1 and 2 mg/L) [71,73], no correlation were observed between levels of metronidazole
404
resistance and the type of nim gene identified (Table 6). To further assess the role of nim
405
genes in the development of metronidazole resistance, Leitsh et al. studied their expression at
406
the protein level [72]. They used 2D gel electrophoresis to identify and quantify Nim (A, B, D
407
and E) proteins and they did not observe a correlation between Nim levels and metronidazole
408
resistance levels. This study challenged the implication of Nim proteins in the inactivation of
409
metronidazole while reinforcing the complexity of metronidazole resistance and the fact that
410
nim genes per se are not sufficient to confer high-level metronidazole resistance.
411
nim-gene positive strains may also display induced and stable heterogeneous or homogeneous
412
resistance to metronidazole after in vitro exposure to the drug [70,71], similarly to nim-
413
negative strains (see part 2.2). A prolonged exposure to metronidazole has also been reported
414
as inducing 5-Ni resistance in vivo in a clinical B. fragilis isolate carrying a silent nimA,
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ACCEPTED MANUSCRIPT 415
leading to treatment failure [104]. However, despite these observations were limited to
416
isolates harboring nim genes in some studies [71–73], similar findings were observed in nim
417
gene-negative isolates in other studies (Table 4) [60,70,75,76,121].
418
3.5. nim genes as part of the genetic content of multidrug resistant anaerobes The emergence of MDR metronidazole-resistant isolates may be explained by the
420
accumulation of mutations within single strains under selective antibiotic pressure but also by
421
the spread of mobile genetic elements such as transposable elements and plasmids carrying
422
nim genes as well as other resistance genes (Table 6) [44,50,77,78,95,99,106,108]. In most
423
cases, multidrug resistance has been observed in B. fragilis clinical isolates harboring known
424
or putative nim genes and displaying at least resistance to piperacillin-tazobactam,
425
clindamycin, carbapenems and metronidazole [44,78,95,106]. The diffusion of such strains is
426
worrisome and an international cluster of MDR B. fragilis isolates harboring nimB and cfiA
427
genes, has been recently recognized [106]. Multidrug resistance (resistant to piperacillin-
428
tazobactam, metronidazole, clindamycin, ertapenem, and meropenem) was also reported in a
429
B. thetaiotaomicron isolate harboring nimD and a second putative nim gene beside two β-
430
lactamase genes, two tetX genes, tetQ, ermF, two cat genes, and several genes encoding
431
efflux pumps [50].
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4. nim genes and cultivation-independent studies
434
Two studies conducted by Koukos and colleagues specifically searched for nim genes in the
435
oral microbiota during health and disease according to the procedure of Trinh et al. [90]. In
436
the first one, they aimed at comparing the prevalence of antibiotic resistance genes in subjects
437
with successful (n=20) and failing (n=20) dental implants [122]. In the second one, they
438
investigated the presence of nim genes in 343 oral samples from 154 adult subjects who did
439
not received metronidazole in the last 12 months and distributed as follows, 50 periodontally
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441
studies, nim genes were not detected supporting the continuing use of metronidazole alone or
442
in combination in the management of oral infections.
443
Another particularly interesting investigation performed by Rani et al. in the New Dehli area
444
highlighted the impact of selective pressure exerted by metronidazole on the emergence of
445
bacteria harbouring nim genes in the gut of individuals receiving metronidazole [123]. These
446
authors reported that the presence of a nim gene (nimE) was frequently detected (11 out of 19
447
patients) in fecal samples from patients with amebic liver abscesses treated with
448
metronidazole whereas no nim gene could be detected in fecal samples obtained in individuals
449
living in the same area (19 healthy individuals and 11 asymptomatic Entamoeba histolytica
450
carriers) who had not received antibiotics for 3 months prior to sampling. In this study, it was
451
also shown that a three-day course of metronidazole given to 11 individuals (8 healthy
452
volunteers and 3 patients suffering from irritable bowel syndrome) resulted in an increase of
453
the frequency of nim detection in fecal samples (72.7 % [8/11] after metronidazole exposure
454
versus 18.8% [2/11] before metronidazole exposure). Importantly, this study showed that nim
455
genes may rapidly emerged among bacterial communities after metronidazole treatment
456
concomitantly to dysbiosis. In the specific case of amebiasis, difference in metronidazole
457
efficiency was observed against ameba according to location in intestinal tissue or lumen and
458
this was thought to be related, at least in part, to anaerobes that co-exist with the ameba and
459
may responsible for metronidazole inactivation [124].
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461
5. Concluding remarks
462
Metronidazole resistance is complex and multifactorial, and the mechanisms and drivers of
463
resistance are still not fully elucidated. While nim genes appears to be associated with
464
metronidazole resistance, dissemination and/or selection of metronidazole resistant strains in
19
ACCEPTED MANUSCRIPT some studies, a strong correlation between nim genes and metronidazole resistance cannot be
466
clearly and definitely established at this time.
467
Recently, the use of WGS to predict antimicrobial susceptibility pattern has been increasingly
468
investigated and discussed. Despite some studies showed high levels (over 90%) of
469
concordance between WGS and antimicrobial susceptibility testing (AST) in some aerobes
470
[94,125–127], the EUCAST Subcommittee recently stated that “for most bacterial species
471
there is currently insufficient evidence to support the use of WGS-inferred AST to guide
472
clinical decision making” [128]. In anaerobes, phenotypic characterization of metronidazole
473
resistance is still essential, as WGS can’t be totally accurate in predicting metronidazole
474
resistance due to the multiplicity of metronidazole resistance mechanisms and the complexity
475
of nim genes expression [101]. In addition, the issue of the incompleteness of antibiotic
476
resistance genes (ARG) databases available for the identification of nim genes in whole
477
genome sequences among metagenomic data need to be addressed.
478
nim gene-mediated resistance has however to be under surveillance because numerous risk
479
factors are combined for a predicted emergence of metronidazole resistance. The most
480
important are the location of nim genes on mobile genetic elements and a high selective
481
pressure coming from indication of metronidazole administration as a first-line drug in
482
diverse infections, including the highly prevalent C. difficile infections, as well as H. pylori
483
infections and amebiasis. This is reinforced by the description of nim genes in emergent
484
multidrug resistant anaerobes, for which associated resistances to other important anti-
485
anaerobes like piperacillin-tazobactam, clindamycin and carbapenems make the choice for the
486
most effective treatment challenging [129]. Such observations should also prompt the
487
development of new 5-nitroimidazole or anti-anaerobic drugs effective in case of
488
metronidazole resistance.
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Washington State, J. Clin. Microbiol. 42 (2004) 4127–4129. doi:10.1128/JCM.42.9.41274129.2004. J.M.B.D. Vieira, R.F. Boente, K.R. Miranda, K.E.S. Avelar, R.M.C.P. Domingues, M.C. de S. Ferreira, Decreased susceptibility to nitroimidazoles among Bacteroides species in Brazil, Curr. Microbiol. 52 (2006) 27–32. doi:10.1007/s00284-005-0068-0. A.A. Kangaba, F.Y. Saglam, H.B. Tokman, M. Torun, M.M. Torun, The prevalence of enterotoxin and antibiotic resistance genes in clinical and intestinal Bacteroides fragilis group isolates in Turkey, Anaerobe. 35 (2015) 72–76. doi:10.1016/j.anaerobe.2015.07.008. J. Mahillon, M. Chandler, Insertion sequences, Microbiol. Mol. Biol. Rev. MMBR. 62 (1998) 725–774. J. Vandecraen, M. Chandler, A. Aertsen, R. Van Houdt, The impact of insertion sequences on bacterial genome plasticity and adaptability, Crit. Rev. Microbiol. 43 (2017) 709–730. doi:10.1080/1040841X.2017.1303661. D.P. Bayley, E.R. Rocha, C.J. Smith, Analysis of cepA and other Bacteroides fragilis genes reveals a unique promoter structure, FEMS Microbiol. Lett. 193 (2000) 149–154. doi:10.1111/j.1574-6968.2000.tb09417.x. N. García, G. Gutiérrez, M. Lorenzo, J.E. García, S. Píriz, A. Quesada, Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections, J. Antimicrob. Chemother. 62 (2008) 942–947. doi:10.1093/jac/dkn347. S. Trinh, A. Haggoud, G. Reysset, Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region., J. Bacteriol. 178 (1996) 6671–6676. F. Mory, J.-P. Carlier, C. Alauzet, M. Thouvenin, H. Schuhmacher, A. Lozniewski, Bacteremia caused by a metronidazole-resistant Prevotella sp. strain, J. Clin. Microbiol. 43 (2005) 5380– 5383. doi:10.1128/JCM.43.10.5380-5383.2005. G. Koukos, A. Konstantinidis, L. Tsalikis, M. Arsenakis, T. Slini, D. Sakellari, Prevalence of βlactam (blaTEM) and metronidazole (nim) resistance genes in the oral cavity of Greek subjects, Open Dent. J. 10 (2016) 89–98. doi:10.2174/1874210601610010089. R. Rani, R.S. Murthy, S. Bhattacharya, V. Ahuja, M.A. Rizvi, J. Paul, Changes in bacterial profile during amebiasis: demonstration of anaerobic bacteria in ALA pus samples, Am J Trop Med Hyg. 75 (2006) 880–885. M. Müller, Mode of action of metronidazole on anaerobic bacteria and protozoa, Surgery. 93 (1983) 165–171. N.C. Gordon, J.R. Price, K. Cole, R. Everitt, M. Morgan, J. Finney, A.M. Kearns, B. Pichon, B. Young, D.J. Wilson, M.J. Llewelyn, J. Paul, T.E.A. Peto, D.W. Crook, A.S. Walker, T. Golubchik, Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing, J. Clin. Microbiol. 52 (2014) 1182–1191. doi:10.1128/JCM.03117-13. V.N. Kos, M. Déraspe, R.E. McLaughlin, J.D. Whiteaker, P.H. Roy, R.A. Alm, J. Corbeil, H. Gardner, The resistome of Pseudomonas aeruginosa in relationship to phenotypic susceptibility, Antimicrob. Agents Chemother. 59 (2015) 427–436. doi:10.1128/AAC.03954-14. C.U. Köser, M.J. Ellington, S.J. Peacock, Whole-genome sequencing to control antimicrobial resistance, Trends Genet. TIG. 30 (2014) 401–407. doi:10.1016/j.tig.2014.07.003. M.J. Ellington, O. Ekelund, F.M. Aarestrup, R. Canton, M. Doumith, C. Giske, H. Grundman, H. Hasman, M.T.G. Holden, K.L. Hopkins, J. Iredell, G. Kahlmeter, C.U. Köser, A. MacGowan, D. Mevius, M. Mulvey, T. Naas, T. Peto, J.-M. Rolain, Ø. Samuelsen, N. Woodford, The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee, Clin. Microbiol. Infect. 23 (2017) 2–22. doi:10.1016/j.cmi.2016.11.012. M. Gajdács, G. Spengler, E. Urbán, Identification and antimicrobial susceptibility testing of anaerobic bacteria: Rubik’s cube of clinical microbiology?, Antibiotics. 6 (2017) 25. doi:10.3390/antibiotics6040025.
AC C
844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895
28
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AC C
EP
TE D
M AN U
SC
RI PT
896
29
ACCEPTED MANUSCRIPT 897
Legends to figures
898
Figure 1. Summarized metronidazole mode of action and main mechanisms involved in
900
resistance. , nitroimidazole reductase activity encoded by nim genes that reduce the nitro
901
group of 4- or 5-Ni to an amino group leading to a 5-aminoimidazole inactive compound,
902
metabolic shift away to the pathway related to conversion of pyruvate to lactate via lactate
903
dehydrogenase (LDH) associated with high LDH activity that compensates decreased
904
pyruvate ferredoxin oxidoreductase (PFOR) activity and thus with decreased 5-Ni activation,
905
increased efflux of the antibiotic (via overexpression of RND-family efflux pump encoding
906
genes of such as bmeRABC5), increased DNA repair capacity (via overexpression of DNA
907
repair proteins such as RecA), activation of antioxidant defense systems (via antioxidant
908
stress-related enzymes such as superoxide dismutase), deficiency of the ferrous iron
909
transporter FeoAB, overexpression of the rhamnose catabolism regulatory protein RhaR.
TE D
M AN U
SC
RI PT
899
910
Figure 2. Maximum likelihood (computed by PHYML, model WAG) tree based on partial
912
derived Nim amino acid sequences (150 AA). Numbers at nodes indicate percentages of
913
bootstrap support when > 70%, based on analysis of 100 replicates. Nodes indicated with
914
stars were conserved by using a Neighbor-Joining analysis from a Dayhoff DNADIST F84
915
matrix. Accession numbers of corresponding nucleotide sequences are given in brackets. Bar,
916
0.05 substitutions per site.
AC C
EP
911
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Table 1. Resistance to metronidazole and prevalence of nim genes in Bacteroides spp. MIC range (µg/ml) 0.064-4
RI PT
≤0.12-32 0.032->256
SC
% of I+R 0 0 0.3 2.9 1.8 0 1 0 0.5 1.2 2 0 1 0 1.3 1 1.6 0.3 1 0 0 0 0 1.6 16 0 0 2.9 1 2.9 0 0 8.7 4.8 0 0.7 0 0
M AN U
Breakpoints EUCAST EUCAST CLSI EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST EUCAST CLSI CLSI EUCAST EUCAST EUCAST EUCAST CLSI CLSI CLSI EUCAST CLSI EUCAST EUCAST EUCAST CLSI CLSI EUCAST CLSI CLSI CLSI EUCAST CLSI EUCAST EUCAST
TE D
Testing method Etest Agar dilution Broth micro-dilution Etest Agar dilution Agar dilution Disk screening/Etest Disk screening/Etest Agar dilution Agar dilution Agar dilution Agar dilution Broth micro-dilution Agar dilution Etest Etest Disk screening/Etest Agar dilution Agar dilution Agar dilution Broth micro-dilution Etest Agar dilution Etest Agar dilution Etest Agar dilution Etest Etest Etest Etest Agar dilution Etest Etest Agar dilution Agar dilution Etest Agar dilution
EP
Study period 2011-2012 2008-2009 2010-2011 2013 2008-2009 2008-2009 2010-2015 2002-2009 2010-2016 2008-2009 2008-2009 2008-2009 2007 2008-2009 2002-2004 2003-2005 2015 2014-2016 2008-2009 2008-2009 2014-2015 2013-2014 2012 2009-2013 2010-2011 2013-2015 2010-2013 2004-2014 2009-2011 2008-2010 2015 1995-1996 2003-2005 2006-2010 2008-2009 2008-2012 2011-2013 2008-2009
AC C
Country (no of isolates) Belgium (180) Belgium (103a) Canada (387 a) Croatia (35) Croatia (56 a) Czech Republic (91 a) Denmark (203 a) Denmark (114 a) Europe c (2451) Finland (85 a) France (51 a) Germany (72 a) Germany (72) Greece (75 a) Greece (82 a) Greece (191 a) Hong Kong (741 a) Hungary (400 a) Hungary (100 a) Italy (23 a) Japan (113 a) Japan (25) Korea (147 a) Norway (122) Pakistan (39 b) Poland (74 a) Romania (53 a) Russia (67) Singapore (68 a) Singapore (69) Slovenia (869 a) South Africa (44a) South Africa (23a) Spain (792 a) Sweden (97 a) Taiwan (256) The Netherlands (283 a) The Netherlands (32 a)
0.5-32 0.125-16 ≤2->32 0.064-16
0.25-4
≤0.125-256 0.06-64 0.016-0.5 0.125-1 0.25->256 ≤0.01->256 0.016-4 ≤0.125-8 0.047-≥256 0.016->256 0.12-16 <0.016-1.5
% of nim genes 2.8 NR NR NR NR NR NR NR NR NR NR NR NR NR 1 0.5 NR NR NR NR NR NR NR NR NR NR 2 NR 1.5 d NR NR NR 0 NR NR NR NR NR
Reference [32] [18] [13] [20] [18] [18] [8] [8] [22] [18] [18] [18] [24] [18] [21] [14] [11] [23] [18] [18] [28] [33] [16] [9] [35] [15] [27] [25] [29] [19] [12] [42] [34] [36] [18] [31] [30] [18]
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AC C
EP
TE D
M AN U
SC
RI PT
Turkey (34 a) 2008-2009 Agar dilution EUCAST 0 NR [18] USA (779 a) 2010-2012 Agar dilution CLSI 0 ≤1-2 NR [26] USA (3981 a) 2010-2012 Agar dilution CLSI 2 NR [10] USA (332) 2010-2011 Etest CLSI 0.6 (R) <0.015->256 NR [17] USA (1580 a) 2007-2009 Agar dilution CLSI 0 NR [10] a Bacteroides fragilis group; b Bacteroides fragilis; c data of the european Tigecycline Evaluation and Surveillance Trial (participating countries: France, Germany, Czech Republic, Hungary, Spain, Belgium, Italy, Sweden, United Kingdom) [22]; d nim-targeting PCR only on metronidazole-resistant strains [74]. I, intermediate; R, resistant. NR, not researched. EUCAST, European Committee on Antimicrobial Susceptibility Testing. EUCAST breakpoints for metronidazole: ≤4, susceptible; >4, resistant (http://www.eucast.org/clinical_breakpoints/). CLSI, Clinical and Laboratory Standards Institute. CLSI breakpoints for metronidazole: ≤8, susceptible; ≥32, resistant [43].
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Country (no of isolates)
Study period
Testing method
Breakpoints
% of I+R
Prevotella spp.
Bulgaria (192) Europe* (1046) France (84) Germany (21) Greece (57) Japan (46) Korea (16) Norway (13) Romania (33) Russia (42) Singapore (7) Slovenia (373) Taiwan (28) The Netherlands (123) USA (571) USA (60) USA (173)
2003-2009 2010-2016 2007-2016 2007 2003-2005 2013-2014 2012 2009-2013 2011-2012 2004-2014 2009-2011 2015 2008-2012 2011-2013 2010-2012 2010-2011 2007-2009
Modified agar dilution Agar dilution Etest Broth microdilution Etest Etest Agar dilution Etest Etest Etest Etest Etest Agar dilution Etest Agar dilution Etest Agar dilution
CLSI EUCAST EUCAST EUCAST CLSI CLSI CLSI EUCAST CLSI/EUCAST EUCAST CLSI EUCAST CLSI EUCAST CLSI CLSI CLSI
0 1 1.2 0 14 4 19 15.4 0 7.1 29 0 0 2 3 0 0
Belgium (21) Greece (17)
2011-2012 2003-2005
Etest Etest
EUCAST CLSI
0 11.7
Norway (13) Russia (13) Singapore (9) Singapore (9) Slovenia (178) Taiwan (48) The Netherlands (39) USA (27) USA (33) USA (44)
2009-2013 2004-2014 2008-2010 2009-2011 2015 2008-2012 2011-2013 2010-2012 2010-2011 2007-2009
Etest Etest Etest Etest Etest Agar dilution Etest Agar dilution Etest Agar dilution
EUCAST EUCAST CLSI CLSI EUCAST CLSI EUCAST CLSI CLSI CLSI
France (116) Korea (12) Singapore (5) Slovenia (83) Taiwan (26)
1999-2001 2012 2009-2011 2015 2008-2012
Agar dilution Agar dilution Etest Etest Agar dilution
CLSI CLSI CLSI EUCAST CLSI
Veillonella spp.
SC
M AN U
EP
AC C
Fusobacterium spp.
% of nim genes
Reference
NR NR NR NR 5.3 NR NR NR NR NR 0 NR NR NR NR NR NR
[39] [22] [37] [24] [14] [33] [16] [9] [38] [25] [29] [12] [31] [30] [10] [17] [10]
<0.016-0.25 ≤2->32
NR 5.9
[32] [14]
0 0 0 0 0 4.2 0 0 0 0
≤0.01-0.25 0.015-1
NR NR NR 0 NR NR NR NR NR NR
[9] [25] [19] [29] [12] [31] [30] [10] [17] [10]
0 8 0 0 7.6
0.25-8 ≤0.125-16 1-2 0.032-4 0.5->128
0.9 NR 0 NR NR
[97] [16] [29] [12] [31]
RI PT
Genus
TE D
Table 2. Resistance to metronidazole and prevalence of nim genes in Prevotella spp., Fusobacterium spp. and Veillonella spp. MIC range (µg/ml)
0.016-8 0.5-4 ≤2->32 0.25-16 0.12-32 0.016-1 0.03-16 0.125-≥256 0.016-4 0.03-4 <0.016-24 ≤0.015-4
≤0.01-0.5 0.016-2 0.03->128 <0.016-0.25 ≤0.015-0.25
1
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AC C
EP
TE D
M AN U
SC
RI PT
The Netherlands (19) 2011-2013 Etest EUCAST 0 <0.016-3 NR [30] USA (32) 2010-2012 Agar dilution CLSI 3 NR [10] USA (9) 2010-2011 Etest CLSI 0 1-4 NR [17] USA (28) 2007-2009 Agar dilution CLSI 14 NR [10] * data of the european Tigecycline Evaluation and Surveillance Trial (participating countries: France, Germany, Czech Republic, Hungary, Spain, Belgium, Italy, Sweden, United Kingdom) [22] I, intermediate; R, resistant. NR, not researched. EUCAST, European Committee on Antimicrobial Susceptibility Testing. EUCAST breakpoints for metronidazole: ≤4, susceptible; >4, resistant (http://www.eucast.org/clinical_breakpoints/). CLSI, Clinical and Laboratory Standards Institute. CLSI breakpoints for metronidazole: ≤8, susceptible; ≥32, resistant [43].
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Table 3. Resistance to metronidazole and prevalence of nim genes in Gram-positive anaerobic cocci (GPACs) and Clostridium spp. Testing method Etest Etest Agar dilution Etest Agar dilution Etest Agar dilution Etest Agar dilution Etest Agar dilution Etest Etest Agar dilution Etest Agar dilution
% of I+R 1 28.6 0.4 0 5.9 0 0 1.2 8.1 0 8 0 0.8 4 5.7(R) 2
MIC range (µg/ml) <0.016->256 0.25->256
≤0.125->32 ≤0.01-2 0.03-4 0.03-≥32 ≤0.06->128 0.016-4 0.06-128 <0.016-2 0.023->256 ≤0.015->256
% of nim genes NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR
Reference [32] [20] [22] [33] [16] [9] [35] [40] [42] [12] [31] [30] [41] [10] [17] [10]
Belgium (38) 2011-2012 Etest EUCAST 0 <0.016-4 NR [32] Croatia (4) 2013 Etest EUCAST 0 1-2 NR [20] Europe* (1584) 2010-2016 Agar dilution EUCAST 0.8 NR [22] Japan (25) 2013-2014 Etest CLSI 0 NR [33] Korea (26) 2012 Agar dilution CLSI 0 ≤0.125-4 NR [16] Norway (32) 2009-2013 Etest EUCAST 0 0.032-4 NR [9] Pakistan (32) 2010-2011 Agar dilution CLSI 3 0.015-32 NR [35] Singapore (26) 2009-2011 Etest CLSI 19 ≤0.01-≥256 0 [29] Singapore (28) 2008-2010 Etest CLSI 21.5 NR [19] Slovenia (176) 2015 Etest EUCAST 0 0.016-4 NR [12] Taiwan (93) 2008-2012 Agar dilution CLSI 0 0.03-8 NR [31] The Netherlands (62) 2011-2013 Etest EUCAST 0 <0.016-4 NR [30] USA (614) 2010-2012 Agar dilution CLSI 0.8 NR [10] USA (228) 2010-2011 Etest CLSI 0 ≤0.015-8 NR [17] USA (116) 2007-2009 Agar dilution CLSI 0 NR [10] * data of the european Tigecycline Evaluation and Surveillance Trial (participating countries: France, Germany, Czech Republic, Hungary, Spain, Belgium, Italy, Sweden, United Kingdom) [22] I, intermediate; R, resistant. NR, not researched. EUCAST, European Committee on Antimicrobial Susceptibility Testing. EUCAST breakpoints for metronidazole: ≤4, susceptible; >4, resistant (http://www.eucast.org/clinical_breakpoints/). CLSI, Clinical and Laboratory Standards Institute. CLSI breakpoints for metronidazole: ≤8, susceptible; ≥32, resistant [43].
AC C
EP
TE D
Clostridium spp.
Breakpoints EUCAST EUCAST EUCAST CLSI CLSI EUCAST CLSI Eucast CLSI EUCAST CLSI EUCAST EUCAST CLSI CLSI CLSI
RI PT
Study period 2011-2012 2013 2010-2016 2013-2014 2012 2009-2013 2010-2011 2004-2014 1995-1996 2015 2008-2012 2011-2013 2002-2004 2010-2012 2010-2011 2007-2009
SC
Country (no. of isolates) Belgium (72) Croatia (14) Europe* (1741) Japan (26) Korea (34) Norway (15) Pakistan (14) Russia (81) South Africa Slovenia (589) Taiwan (50) The Netherlands (249) The Netherlands (115) USA (611) USA (176) USA (168)
M AN U
Microorganisms GPACs
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Table 4. Heterogeneity, inducibility and stability of resistance to metronidazole in clinical Prevotella isolates, P. baroniae DSM 16972T, and B. fragilis ATCC 25285T [adapted from [69] and from personal data].
Original
SGCb
P. baroniae DSM 16972T
nimI
2
NAc
P. baroniae LBN 427
nimI
4
NA
P. baroniae LBN 430
nimI
4
NA
P. baroniae LBN 432
nimI
2
NA
P. baroniae LBN 475
nimI
4
P. baroniae LBP19
nimI
1
P. bivia LBN 332
−
4
P. bivia LBN 371
−
4
P. bivia LBN 467
−
16
P. buccae LBN 465
−
0.5
P. nanceiensis LBN 293b
−
2
P. nanceiensis LBN 410
− −
B. fragilis ATCC 25285 a
Post-induction Post-stability 16
128
128
128
128
64
64
NA
256
256
NA
128
128
32
>256
>256
32
>256
>256
32
>256
>256
NA
128
64
32
256
256
1
32
256
256
0.5
NA
256
128
EP
TE D
M AN U
SC
16
AC C
T
RI PT
MICs determined by agar dilution (µg/ml)
Presence of nim gene
Straina
LBN/LBP, clincal strains from collection of Laboratoire de Bactériologie de Nancy and Laboratoire de Bactériologie de Poitiers, SGC, slowly growing colonies within the inhibition zone of the Etest strip, c NA, not applicable (absence of slowly growing colonies). b
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Nim protein nim gene
NimA
NimB
NimC
NimD
NimE
NimF
Accession CAA50581 CAA50578 CAA54269 CAA54273 CAB82516 CAD56147 No
RI PT
Table 5. Percentage of amino acid (upper diagonal) and nucleotide (lower diagonal) sequence identities between NimA to NimK as well as compare to the nitroimidazole reductase of Deinococcus radiodurans (NimA Dr) obtained by using the BioEdit Sequence Alignment Editor. NimG *
NimH
NimI
ACR56004 FJ940883
NimJ
NimK
NimA Dr
WP_005812 AXA20009 AAF10419 825
X71444
-
77.3%
68.6%
78.6%
78.0%
74.0%
79.3%
78.0%
58.0%
55.3%
64,6%
25,5%
nimB
X71443
71.1%
-
73.3%
85.3%
82.0%
78.6%
90.0%
82.6%
63.3%
58.0%
67,3%
24,4%
nimC
X76948
71.8%
69.8%
-
74.6%
72.6%
71.3%
75.3%
74.6%
60.6%
54.6%
59,3%
22,0%
nimD
X76949
73.3%
82.5%
72.4%
-
78.6%
78.6%
89.3%
85.3%
62.6%
56.6%
64,0%
22,0%
nimE
AJ244018
70.4%
75.1%
68.2%
73.5%
-
74.6%
80.0%
80.0%
63.3%
59.3%
66,0%
24,4%
nimF
AJ515145
71.5%
75.7%
71.1%
78.1%
70.0%
-
82.0%
80.6%
62.0%
56.6%
66,6%
23,8%
nimG
*
72.4%
89.8%
70.7%
85.2%
73.3%
77.9%
-
86.6%
64.0%
56.6%
66,6%
23,8%
nimH
KX576455
73.1%
81.4%
72.2%
82.3%
73.5%
75.5%
83.9%
-
64.0%
59.3%
66,0%
23,2%
nimI
FJ940883
60.1%
59.7%
62.1%
62.8%
61.7%
63.2%
62.5%
63.8%
-
74.0%
58,6%
24,4%
nimJ
KJ816753
57.6%
61.7%
58.3%
60.6%
59.3%
57.8%
59.8%
60.2%
68.2%
-
54,0%
22,0%
nimK
MG827401
63,8%
66,9%
61,2%
65,6%
66,0%
65,8%
66,0%
66,5%
57,3%
57,5%
-
22,6%
nimA Dr AE000513
40.2%
38.1%
36.5%
39.0%
39.6%
40.0%
38.1%
35.0%
M AN U
TE D
EP
AC C 40.9%
40.9%
SC
nimA
-
* nimG sequence was not available in GenBank. The nucleotidic sequence published by Gal & Brazier as well as its deduced amino acid sequence were used [71].
1
ACCEPTED MANUSCRIPT
Table 6. Characteristics and diversity of nim genes described in the literature.
(0.75−>128 µg/mL)
variable nimBa
C
(0.125−>25 6 µg/mL) variable
nimC
P or C
57.27
IS1170
(1−>32 µg/mL) variable
P or C
50.22
IS1169
(0.25−≥64 µg/mL) variable
nimE
P
e
41.19
ISBf6
B. fragilis, B. thetaiotaomicron, B. vulgatus, Bacteroides stercoris, Fusobacterium sp., Odoribacter splanchnicus
AC C
nimD
(1.5−>256 µg/mL)
Associated in vitro antimicrobial resistance
RI PT
42.73
IS1168 IS612
variable
B. fragilis, B. thetaiotaomicron, B. vulgatus, Bacteroides ovatus, Bacteroides uniformis, P. distasonis, Prevotella bivia, Actinomyces odontolyticus, Cutibacterium acnes, Propionibacterium sp., Clostridium bifermentans B. fragilis, B. thetaiotaomicron, B. vulgatus, P. distasonis, Prevotella denticola, Prevotella dentalis, Finegoldia magna, Peptostreptococcus anaerobius, Anaerococcus prevotii, Parvimonas micra B. fragilis, B. thetaiotaomicron, P. distasonis, Prevotella oralis, Porphyromonas sp.
SC
54.63
IS1168 IS1169 ISBf13
Bacterial species or genera
M AN U
P or C
G+C (mol%)
TE D
nimA
Genetic location
EP
Nim gene
Association IS present with upstream resistance of nim to MTZ (MICs)
B. fragilis, B. thetaiotaomicron, B. vulgatus, B. ovatus, Parabacteroides merdae, Prevotella buccalis, Prevotella oralis, Veillonella sp.
Associated resistance genes
References
cepA, blaOXAAMX, AMC, PIP, , cfiA, ermF, TZP, FOX, CAR, 37 linAn2, mefE, ERY, CLI, TET tetQ, bexB
[14,32,49,53,7 1,73,79,85,90, 91,96,103,104, 107,108,110,1 13]
AMP, PIP, TZP, FOX, CAR, CLI, TET, RIF
cfxA, cfiA, ermF, tetQ
[27,71,73,80,8 5,90,91,96,97, 106,107,109,1 14]
TET
cfiA, tetQ
[14,71,73,79,8 6,91,107]
cepA, cfxA, cfiA, ermF, AMP, TZP, CAR, linAn2, mefE, TET, CLI tetQ, cat, bexB-liked AMP, AMX, cepA, cfxA, AMC, PIP, TZP, blaTEM, blaCMY, FOX, CAZ, CAR, blaOxA-1, cfiA, ERY, CLI, TET, ermF, tetQ, TGC, CM, CIP aac(6’)Ib-cr
[14,32,50,71,7 3,74,86,91,107 –109]
[14,44,49,51,7 1,73,78,79,91, 98,107,108,11 0,112]
1
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ND
51.88
noneb
no (1 µg/mL)
B. vulgatus
ND
ND
[73]
nimG
ND
47.14
ND
no (2 µg/mL)
B. fragilis
ND
ND
[71]
nimH
C
48.46
IS614B
AMX
NDf
[92]
nimI
C
58.64
nonec
ND
ND
[70]
AMP, AMX, AMC, TZP, FOX, CFP, CAR, ERY, CLI, TET
cfiA, ermR, tetQ, bexBliked, overexpression of bmeABC5 genes
C
45.14
IS614 IS614B IS4 family
P. baroniae
yes (>32 µg/mL)
SC
B. fragilis
B. fragilis
M AN U
nimJ
yes (8 µg/mL) no (1−4 µg/mL)
RI PT
nimF
[54,77,78,108]
AC C
EP
TE D
yes efflux SMR [93] C 37.20 (6−12 P. bivia AMX, CLI nimK transporter µg/mL) P, plasmid; C, chromosomal; ND, not determined; MTZ, metronidazole; MIC, minimal inhibitory concentrations; Other antibiotics’ abbreviations: AMP, ampicillin; AMX, amoxicillin, AMC, amoxicillin/clavulanic acid; PIP, piperacillin; TZP, piperacillin/tazobactam; FOX, cefoxitin; CAZ, ceftazidime; CFP, cefoperazone; CAR, carbapenems; ERY, erythromycin; CLI, clindamycin; TET, tetracycline; TGC, tigecycline; RIF, rifampicin; CM, chloramphenicol; CIP, ciprofloxacin; a a nimB-like element sharing 84% identity with nimB, was also described in Peptoniphilus asaccharolyticus (sequence non available in GenBank) [80]; b among IS1168, IS1169 and IS1170; c among IS1168, IS1169, IS1170, ISBf6 and IS612; d putative bexB gene (91% sequence identity) [108]; e data obtained by Sydenham et al. from WGS could suggest a chromosomal location of nimE [108]. f absence of cfiA gene, other resistance genes not determined [92]. IS1380 family
2
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 1
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Figure 2
ACCEPTED MANUSCRIPT Highlights
- Metronidazole is a drug of choice for treatment of infections caused by anaerobes. - MTZ acquired resistance may associate several mechanisms including Nim proteins.
- nim genes are present in a wide range of bacterial phyla.
RI PT
- 10 nim genes of variable expression (silentvarious resistance levels) are described.
AC C
EP
TE D
M AN U
SC
- nim genes are part of the genetic content of multidrug resistant anaerobes.