Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades

Anaerobe xxx (2014) 1e7

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Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades Lyudmila Boyanova a, *, Rossen Kolarov b, Ivan Mitov a a b

Department of Medical Microbiology, Medical University of Sofia, Zdrave Street 2, 1431 Sofia, Bulgaria University Hospital of Maxillofacial Surgery, Sofia, Bulgaria

a b s t r a c t Keywords: Antibiotic Resistance Anaerobes Anaerobic Factors Increase

Evolution of antibiotic resistance in the anaerobes was reviewed using recent data covering 2000e2013 as compared to previous years. All studies reported growing moxifloxacin resistance in Bacteroides/ Parabacteroides spp. in Europe and USA and in Clostridium difficile in Europe. In half or more studies, the resistance rates in Bacteroides/Parabacteroides spp. to amoxicillineclavulanate or ampicillinesulbactam and clindamycin rose. In some studies, an increase in resistance was found in Bacteroides/Parabacteroides spp. to cefoxitin/cefotetan and carbapenems, in Prevotella spp. to penicillins, in anaerobic cocci to clindamycin and in Bacteroides/Parabacteroides spp. and C. difficile to metronidazole. Decreasing resistance was also observed, e.g. in Bacteroides/Parabacteroides spp. to cephalosporins, in Prevotella spp. and C. difficile to tetracyclines and in C. difficile to rifampin. No resistance changes were found to tigecycline, in Bacteroides/Parabacteroides spp. to chloramphenicol and in C. difficile to vancomycin. Factors influencing the resistance were the species, ribotype, country, hospital centre, antibiotic consumption and specimen type. In conclusion, the antibiotic resistance changes in the anaerobes are diverse and dynamic. Regular national surveys of resistance and both anaerobic microbiology and susceptibility testing of the isolates become more and more valuable. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction As yet, not many laboratories perform anaerobic microbiology and test the susceptibility of anaerobic isolates. In the USA, 89% of the laboratories performed anaerobic culture but only 21% tested in-house the susceptibility of the isolates; another 20% of the laboratories contacted the reference centers for this purpose [1,2]. In Scotland, UK, 19% of the laboratories did not identify anaerobes routinely [3]. Quality control for diagnostic oral microbiology laboratories in European countries revealed that only half of the laboratories isolated and identified the anaerobes from patients with periodontitis [4]. For this reason, the treatment of anaerobic

Abbreviations: ADM, agar dilution method; BMM, broth microdilution method; CA-SFM, Antibiogram Committee of the French Society of Microbiology; CLSI, Clinical and Laboratory Standards Institute; ET, E test; EUCAST, European Committee on Antimicrobial Susceptibility Testing; SGE, spiral gradient endpoint analysis. * Corresponding author. Tel.: þ359 2 91 72 730. E-mail addresses: [email protected], [email protected] (L. Boyanova).

infections in many hospital centers not only starts but remains empirical based on published surveys. However, both the global and local changes in resistance rates over time should be considered to provide optimal treatment of the patients. Susceptibility testing of anaerobes is crucial in serious infections such as bloodstream infection and those isolates from normally sterile body sites as well as in those not responsive to empirical therapy. It is strongly recommended that anaerobes that should be considered for susceptibility testing are the highly virulent pathogens and those with unpredictable susceptibility patterns including the Gram-negative species Bacteroides spp., Prevotella spp., Fusobacterium spp., Bilophila wadsworthia and Sutterella wadsworthensis as well as the Gram-positive Clostridium spp. [5]. Current susceptibility testing methods for anaerobes include agar dilution technique with supplemented Brucella agar (the reference method), broth microdilution with supplemented Brucella broth (for Bacteroides and Parabacteroides spp.,) E test, b-lactamase test (with limited usefulness) and the spiral gradient endpoint system [2,5]. Importantly, the disk diffusion method is unsuitable for anaerobes. Different antibiotic breakpoints are used, mostly those of Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing

http://dx.doi.org/10.1016/j.anaerobe.2014.05.004 1075-9964/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Boyanova L, et al., Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.004

Agenta Penicillins Ampicillin (HLR) Penicillin

Cephalosporins Cefoxitin

Cefotetan

Ampicillinsulbactam Piperacillintazobactam

Carbapenems Imipenem

Meropenem Lincosamides Clindamycin

Anaerobic speciesb

Country/ region

No. of strains (specimen typec)

Methodd

Bacteroides/Parabacteroides spp. B. fragilis Prevotella spp. P. intermedia/P. nigrescens Prevotella spp. Anaerobic cocci

Europe Kuwait Bulgaria Switzerland Taiwan Belgium

2113 (V) 831 (V) 133 (O) 326 (O) 248 (V) 119 (V)

ADM ET BSTM ET ADM ET

16.0 100.0 15.4 14.0 NS 62.0 15.0 NS

(1988e89) (2002e04) (2003e04) (1991e94) (2002) (1987)

44.5 100.0 60.6 14.7 NS 94.0 12.0 NS

Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides B. fragilis B. thetaiotaomicron

spp. spp. spp. spp. spp. spp.

Canada Belgium Europe Spain Spain USA Korea Korea

735 (V) 418 (V) 2113 (V) 286 (V) 830 (V) 4369 (V) 276 (V) 106 (V)

BMM ET ADM ADM ET ADM ADM ADM

26.0 38.0 NS 3.0 3.4 37.6e38.0 NS 4e8.0 15.0 100.0

(1992) (2004) (1988e89) (1999) (2006e07) (1981e89) (1997) (1997)

Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Fusobacterium spp. Anaerobic cocci GPAC B. fragilis

spp. spp. spp. spp.

Canada Europe Spain Spain Kuwait Belgium Kuwait Taiwan

735 (V) 2113 (V) 1343 (V) 830 (V) 75 (V) 119 (V) 496 (V) 1605 (V)

BMM ADM ADM ET ET ET ET ADM

0.8 1.0 5.0e8.9 5.9e14.8 NS 0.0 2.0 NS 0.0 <30.0 NS

Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides B. fragilis

spp. spp. spp. spp.

USA Canada Europe Spain Kuwait

5220 (V) 735 (V) 2108 (V) 830 (V) 831 (V)

ADM BMM ADM ET ET

Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Prevotella spp. Fusobacterium spp. B. fragilis

spp. spp. spp. spp. spp.

Europe USA Canada Spain Spain Kuwait Kuwait Kuwait

2113 (V) 4369 (V) 735 (V) 1343 (V) 830 (V) 532 (V) 75 (V) 96 (V)

Canada Europe France Kuwait Spain Spain USA Korea Bulgaria Belgium UK Belgium Bulgaria

Bacteroides/Parabacteroides spp. Bacteroides/Parabacteroides spp. Bacteroides/Parabacteroides spp. B. fragilis Bacteroides/Parabacteroides spp. B. fragilis Bacteroides/Parabacteroides spp. B. thetaiotaomicron Prevotella spp. Prevotella, Porphyromonas, Campylobacter and Sutterella spp. C. difficile Anaerobic cocci Anaerobic cocci

Resistance rate % (years)e

Resistance trend

Breakpoint MIC (mg/L)f

Reference

(2008e09) (2005e07) (2007-09) (2001e04) (2006) (2011e12)

Increase None Increase None Increase None

64 CLSI CLSI CLSI CLSI EUCAST

[19] [20] [9] [13] [10] [14,16]

15.2 44.0 NS 17.2 27.1 22.1e36.5 NS 9.0 29.0 27.0

(2010e11) (2011e12) (2008e09) (2006) (2008e10) (2000e07) (2004) (2004)

Decrease None Increase Increase None None Increase Decrease

CLSI CLSI CLSI CLSI CLSI CLSI CLSI CLSI

[18] [16] [19] [7] [22] [21] [17] [17]

(1992) (1988e89) (1997e99) (2006e07) (2002e04) (1987) (2002e04) (2000e03)

6.2 10.4 2.2e20.9 7.4e13.8 NS 0.0 3.0 NS 0.0 48.0 NS

(2010e11) (2008e09) (2000e06) (2008e10) (2005e07) (2011e12) (2005e07) (2007)

Increase Increase Slight increase None None None None Increase

CLSI CLSI CLSI CLSI CLSI EUCAST CLSI CLSI

[18] [19] [7] [22] [20] [14,16] [20] [10]

<1 0.0 <1.0 10.0e11.9 NS 0.0

(1990e99) (1992) (1999e01) (2006e07) (2002e04)

<1 0.5 3.1 4.4e8.1 NS 0.0

(2000e07) (2010e11) (2008e09) (2008e10) (2005e07)

None None None None None

CLSI CLSI CLSI CLSI CLSI

[21] [18] [19] [22] [20]

ADM ADM BMM ADM ET ET ET ET

0.0 <1.0 0.0 1.5 0e1.0 NS 0.0 0.0 1.0

(1988e89) (1981e89) (1992) (1997) (2006e07) (2002e04) (2002e04) (1999)

1.2 <1.0 0.5 1.5 1.2e4.2 NS 0.0 0.0 7.9

(2008e09) (2000e07) (2010e11) (2006) (2008e10) (2005-07) (2005e07) (2007)

None None None None Slight increase None None Increase

CLSI CLSI CLSI CLSI CLSI CLSI CLSI CLSI

[19] [21] [18] [7] [22] [20] [20] [20]

735 (V) 2113 (V) 1347 (V) 299 (V) 358 (V) 830 (V) 4369 (V) 106 (V) 131 (O) 91 (V)

BMM ADM NAh ET ADM ET ADM ADM BSTM ET

8.9 9.0 17.0 43.0 33.5 47.2e54.0 NS 5.0e6.0 67.0 7.7 8.0 NS

(1992) (1988e89) (1992e96) (2002) (1997) (2006e07) (1981e89) (1997) (2003e04) (1987)

34.1 32.4 30.4 60.0 47.9 37.1e47.5 NS 31.0->35.0 91.0 13.0 31.0 NS

(2010e11) (2008e09) (2000e03) (2007) (2006) (2009e10) (2000e07) (2004) (2007e09) (2011e12)

Increase Increase Increase Increase Increase None Increase Increase None Increase

CLSI CLSI CLSI CLSI CLSI CLSI CLSI CLSI CLSI EUCAST

[18] [19] [8] [20] [7] [22] [21] [17] [9] [14e16]

179 119 (V) 78 (V)

ADM ET BSTM

91.8 (1996e04) 17.0 NS (2011e12) 30.2 (2011e12)

None None Increase

CLSI EUCAST CLSI

[24] [14,16] [23],Bg

97.1 (1979e86) 6.0 NS (1987) 8.6 (1996e99)

L. Boyanova et al. / Anaerobe xxx (2014) 1e7

BL-BLI Amoxicillinclavulanate

2

Please cite this article in press as: Boyanova L, et al., Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.004

Table 1 Examples of evolution of antibiotic resistance in anaerobes according to data from publications in the last six years.

Fluoroquinolones Moxifloxacin

Trovafloxacin Fluoroquinolones Tetracyclines Tetracycline

Glycylcyclines Tigecycline

Amphenicols Chloramphenicol

Glycopeptides Vancomycin Teicoplanin Other Rifampin Fusidic acid

0.0 0.1 RS 0.8 0.0 0.0 0.0 0.0 4.0 NS 1.5 0.0 0.0 0.0 0.0 0 RSe 6.0 NS 8.6

(1988e89) (1988e89) (1992e96) (1997) (1981e89) (1992) (1997) (2006e07) (2002) (2002e04) (2002e04) (1993e99) (1979e86) (1995e2001) (1987) (1996e99)

<1.0 2.7 RS 2.2 0.0 0.06 0.3 0.0 3.9e4.0 NS 1.0 0.0 0.0 0.0 0.0 24.4 RS 1.0 NS 9.3

ADM ADM ADM ET ET ADM ET, gyr mutation assay

9.0 6.0 0 4.0 12.0 NS 16 10.0

(1999e2001) (1997) (1979e86) (2004) (2004) (1990e99) (1985e2001)

13.6 25.0 10.2 23.0 34.0 NS >40 56.0

112 (O) 326 (O) 179 (F)

BSTM ET ADM

43.3 (2003e04) 12.6 NS (1991e94) 13.0 (1979e86)

20.7 (2007e09) 12.9 NS (2001e04) 2.0 (1996e04)

USA USA Sweden

1021 (V) 136 (V) 205 (F)

ADM ADM ET

4.7 (2006e07) 0 (2006e07) 0.0 (2004)

Bacteroides/Parabacteroides spp. Bacteroides/Parabacteroides spp. Bacteroides/Parabacteroides spp.

USA Spain Korea

4369 (V) 1343 (V) 466 (V)

ADM ADM ADM

C. difficile C. difficile C. difficile

Sweden UK Sweden

606 (F) 179 (F) 401 (F)

ET ADM ET

C. difficile BI/NAP1 clone C. difficile C. difficile

USA Sweden Sweden

205 (F) 606 (F) 606 (F)

ET ET ET

Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides B. fragilis Prevotella Fusobacterium spp. C. difficile C. difficile C. difficile ribotype 001 Anaerobic cocci Anaerobic cocci

spp. spp. spp. spp. spp. spp. spp. spp.

Europe Europe France Korea USA Canada Spain Spain Kuwait Kuwait Kuwait Sweden UK UK Belgium Bulgaria

2113 (V) 2113 (V) 1347 (V) 466 (V) 4369 (V) 735 (V) 1343 (V) 830 (V) 299 (F) 532 (V) 75 (V) 606 (F) 179 (F) 158 (F) 119 (V) 78 (V)

ADM ADM NA ADM ADM BMM ADM ET ET ET ET ET ADM SGE, ET ET BSTM

Bacteroides/Parabacteroides spp. Bacteroides/Parabacteroides spp. C. difficile C. difficile Clostridium spp. Bacteroides/Parabacteroides spp. C. difficile

Europe Spain UK Sweden Belgium USA Italy

2108 (V) 358 (V) 179 (F) 606 (F) 95 (V) 5220 (V) 147 (F)

Prevotella P. intermedia/P. nigrescens C. difficile

Bulgaria Switzerland UK

B. fragilis B. vulgatus C. difficile

(2008e09) (2008e09) (2000e03) (2004) (2000e07) (2010e11) (2006) (2009e10) (2007) (2005e07) (2005e07) (2002e07) (1996e04) (2005e06) (2011e12) (2011e12)

None None Increase None None None None None None None None None None Increase None None

CLSI 4 EUCAST CLSI CLSI CLSI CLSI CLSI CLSI CLSI CLSI EUCAST CLSI CLSI EUCAST CLSI

[19] [19] [8] [17] [21] [18] [7] [22] [20] [20] [20] [28] [24] [25] [14,16] [23],B

(2008e09) (2006) (1996e2004) (2007) (2011e12) (2000e07) (2002e08)

Increase Increase Increase Increase Increase Increase Increase

CLSI CLSI CLSI >32 CLSI CLSI CLSI

[19] [7] [24] [28] [14-16] [21] [27]

Decrease None Decrease

CLSI CLSI CLSI

[9] [13] [24]

5.4 (2008e09) 4.3 (2008e09) 0.0 (2007)

None Slight increase None

FDA FDA >0.25

[29] [29] [28]

0 (1981e89) 0 (1997e99) 0.0 (1997)

0 (2000e07) 0 (2000e06) 0.0 (2004)

None None None

CLSI CLSI CLSI

[21] [7] [17]

0.5 (1993e97) 0 (1979e86) 0 (1993e99)

0 (2004e07) 0 (1996e04) 0 (2002)

None None None

EUCAST CLSI >2

[28] [24] [28]

96.0 (2001e02) 29.8 (1993e99) 0 (1993e97)

67.9 (2005) 11.4 (2002e07) 1.5 (2004e07)

Decrease Decrease None

>32 >32 >4

[30] [28] [28]

L. Boyanova et al. / Anaerobe xxx (2014) 1e7

Please cite this article in press as: Boyanova L, et al., Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.004

Nitroimidazoles Metronidazole

Agent: HLR- high-level resistance, BL-BLI-b-lactam/b-lactamase inhibitors. Anaerobic species: GPAC- Gram-positive anaerobic cocci. c Specimen type: V-various, O-oral or oral/head and neck, F-fecal. d Methods: ADM-agar dilution method, BMM-broth microdilution method, BSTM-breakpoint susceptibility testing method, ET-E test. e Resistance rate: RS-reduced susceptibility. f Breakpoint: CA-SFM- Antibiogram committee of the French society of microbiology, CLSI- Clinical and Laboratory Standards Institute, EUCAST- European Committee on Antimicrobial Susceptibility Testing, FDA- Food and Drug Administration, SGE- spiral gradient endpoint analysis. g References: B-Boyanova (unpublished data). h Other: NA-non-available. a

b

3

4

L. Boyanova et al. / Anaerobe xxx (2014) 1e7

(EUCAST), which affects to some extent the comparison of the results. The aim of the study was to reveal the recent changes in antibiotic resistance rates in anaerobic bacteria over time, especially over the last decade. 2. Material and methods In this review we assessed evolution of antibiotic resistance in the anaerobes by recent articles. Appropriate publications were identified in Medline, PubMed and Google Scholar with the keywords or word combinations: “anaerobic bacteria”, “anaerobes”, “Bacteroides”, “Prevotella” or “Clostridium” and “antibiotic resistance” or “multidrug resistance” as well as “evolution”, “resistance trends” or “over years”. Studies written in English or French that were published in 2008e2013 were included. Publications reporting resistance trends in fewer than 70 strains as well as those based on susceptibility testing by disk diffusion method were excluded. The studies with small sample sizes were excluded as larger samples usually increase the reliability of the results. Overall, there were 112 antibiotic-organism combinations from 20 studies about the resistance of a certain species/group to a particular antibiotic over time. Of them 61 were obtained by E test (ET), 37 by agar dilution method (ADM), six by broth microdilution method (BMM), five by a breakpoint susceptibility testing method, a modified ADM, using several consecutive concentrations per antibiotic [6], one by E test and spiral gradient endpoint analysis (SGE) and for two combinations, no data were available (Table 1). Antibiotic-organism combinations from studies from America (Canada and USA, 17), Europe (Belgium, Bulgaria, France, Germany, Italy, Spain, Sweden, Switzerland, the Netherlands and UK, 75) and Asia (Kuwait, Korea and Taiwan, 20) were included. Most (52) antibiotic-organism combinations from the studies were about Bacteroides and Parabacteroides spp., 17 included Clostridium difficile, one for other clostridia, 13 for Prevotella spp., 11 for anaerobic cocci, 10 for Fusobacterium spp., six for Porphyromonas gingivalis and two for Prevotella, Porphyromonas and other Gramnegative species. Non-fragilis Bacteroidales species like those in the genus Parabacteroides were sometimes reported as members of Bacteroides fragilis group. For susceptibility and resistance breakpoint, most (71) antibiotic-organism combinations were based on CLSI criteria and the others were according to EUCAST (9) and Food and Drug Administration (FDA, 2) and other/arbitrary endpoints (9). Minimal inhibitory concentration (MIC) values over time were compared in 21 of the combinations. Antibiotic-organism combinations were available for susceptibility to penicillins (11), cephalosporins (8), b-lactam/b-lactamase inhibitors (18), carbapenems (10), clindamycin (17), metronidazole (20), fluoroquinolones (8), tetracycline (7), tigecycline (3), chloramphenicol (3), glycopeptides (3), rifampin (3) and fusidic acid (1). For some antibiotic-organism combinations in several publications [7,8], the p values were calculated. Differences between groups were evaluated by chi-square test. Criteria used to assess the risk factors for resistance were statistical significance of factors associated with the resistance or discussions concerning individual antibiotics or antibiotic class. 3. Results of comparison of antibiotic resistance data published in the literature

years), while no such evolution was observed in Belgium, Switzerland (for non-susceptibility) and the Netherlands [9e16]. Growing cefoxitin/cefotetan resistance in Bacteroides and Parabacteroides spp. was reported in four studies from Europe (5.7-fold over 21 years), including Spain (8-fold increase from 1999 to 2006, p ¼ 0.0001) and Korea (1.9-fold increase in B. fragilis over seven years). Conversely, a decrease in resistance rates was observed in Canada (1.7-fold decrease over 19 years) and in Bacteroides thetaiotaomicron in Korea (3.7-fold drop over seven years) [7,17e19]. Ten studies (five of them about Bacteroides and Parabacteroides spp.) evaluating the evolution of the resistance to amoxicillineclavulanate or ampicillinesulbactam are discussed. Bacteroides and Parabacteroides spp. showed rising resistance rates to amoxicillineclavulanate in Canada (7.8-fold increase over 19 years), Europe (10.4-fold increase over 20 years) and Taiwan (>1.6-fold increase in non-susceptibility rates over seven years) [10,18,19]. Conversely, no increase in piperacillinetazobactam resistance was found in anaerobes in Europe, USA, Canada and Kuwait [18e22]. No increase in resistance or non-susceptibility to beta-lactam/betalactamase inhibitor combinations were reported among non-Bacteroides/Parabacteroides anaerobic genera [11,12,14,16,20]. Carbapenem resistance was rare in the anaerobes, including Bacteroides and Parabacteroides spp., Prevotella spp. and Fusobacterium spp. Increase in resistance was found only in B. fragilis to meropenem in Kuwait (7.9-fold increase over eight years) [20] and a slight increase in Bacteroides/Parabacteroides resistance to both imipenem and ertapenem has been reported recently in Spain [22]. No increase was observed in another Spanish study and in USA and Canada [7,18,21]. 3.2. Resistance to clindamycin Increase in Bacteroides/Parabacteroides resistance to clindamycin over years was found in most studies from Europe, including France (p ¼ 0.001) and Spain (one of two studies, p ¼ 0.006) as well as in the USA, Canada, Kuwait, and for B. thetaiotaomicron, in Korea [7,8,17e22]. In a study from the USA, the clindamycin resistance rate was about 6-fold higher in 2000e2007 compared with that in 1981e1989 [21]. Among non-Bacteroides/Parabacteroides anaerobes, the clindamycin resistance increased in anaerobic cocci in Bulgaria (3.5-fold increase over 16 years), whereas no evolution was detected in the Netherlands and Scotland [[11,12,23,24], Boyanova, unpublished data]. In Belgium, the clindamycin nonsusceptibility rate (acc. to EUCAST breakpoint) in the group of Prevotella spp., Porphyromonas spp., Campylobacter rectus and S. wadsworthensis increased continuously from 8% in 1987 to 18% in 2004 and to 31% in 2011e12 [14e16]. 3.3. Resistance to metronidazole Within 16 studies from Europe, USA, Canada and Kuwait, no evolution in metronidazole resistance or reduced susceptibility was observed in the anaerobes except for those in C. difficile ribotype 001 in UK (24.4-fold increase in the reduced susceptibility rate over 11 years) and Bacteroides and Parabacteroides spp. in France (2.8fold increase in the resistance rate over 11 years, p ¼ 0.049) [8,25]. In addition, the metronidazole MIC90 against oral Prevotella intermedia/Prevotella nigrescens in the Netherlands slightly rose (7.8-fold over seven years) [11,12]. 3.4. Resistance to fluoroquinolones

3.1. Resistance to beta-lactams Increase in Prevotella resistance to penicillins was found in Bulgaria (3.9-fold over six years) and Taiwan (1.5-fold over four

Growing fluoroquinolone resistance was observed in Bacteroides and Parabacteroides spp. in 3/3 studies in USA and Europe and in C. difficile in 4/4 studies in Europe [7,19,21,24,26e28]. In Spain,

Please cite this article in press as: Boyanova L, et al., Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.004

L. Boyanova et al. / Anaerobe xxx (2014) 1e7

5

Bacteroides/Parabacteroides resistance rate to moxifloxacin in 2006 was 4.2-fold higher (25.0%) than that in 1997 (6.0%) [7]. Among C. difficile strains, moxifloxacin resistance increased in Scotland (10.2-fold increase in resistance rates over 25 years), Italy (5.6-fold increase over 23 years, p < 0.001), Sweden (5.8-fold increase over three years) and Germany (32-fold increase in MIC90 over 18 years) [24,26e28]. Similarly, a sharp decline in susceptibility rates of non-difficile Clostridium spp. to moxifloxacin (acc. to CLSI breakpoint, 88% in 2004 vs. 66% in 2011e2012, p ¼ 0.019) was reported in Belgium [14e16].

No evolution in vancomycin/teicoplanin resistance in C. difficile was reported in two studies from Europe [24,28]. In Sweden, the proportion of C. difficile strains resistant to >4 mg/L fusidic acid remained low (0e2.9%) over 14 years [28]. In the USA, there was a 1.4-fold decrease in rifampin MICs >32 mg/L in C. difficile BI/NAP1 clone over four years [30]. In Sweden, the number of C. difficile strains resistant to >32 mg/L rifampin dropped 2.6-fold from 1993 to 99 to 2002e07 [28]. Antibiotic resistance of the anaerobes can vary according to a number of factors, such as species or ribotype, country, hospital centre, antibiotic consumption and specimen (Table 2).

3.5. Resistance to tetracycline

4. Discussion

In non-Bacteroides/Parabacteroides anaerobic bacteria in Europe, no increase (in the Netherlands) or decrease in tetracycline resistance rates (in two studies) was found [9,11e13,24]. The decrease was 6.5-fold in C. difficile in Scotland over 25 years and 2.1-fold in Prevotella spp. in Bulgaria over six years [9,24]. In the Netherlands, the MICs90 of the periodontal pathogen P. gingivalis increased >10fold over seven years, although the strains remained susceptible to the agent [11,12].

Main mechanisms for antibiotic resistance in the anaerobes are evaluated. b -lactam resistance is associated with the presence of blactamase genes, changes in penicillin-binding proteins and changes in membrane permeability such as loss of porin [8]. In European Bacteroides/Parabacteroides strains, cepA gene was often linked to ampicillin resistance [31]. Chromosomal cephalosporinase hyperproduction in Bacteroides and Parabacteroides spp. combined with a loss of porins results in b-lactam/b lactamase inhibitor resistance [8]. Class B metallo-b-lactamase encoded by cfiA gene (if expressed by insertion of a sequence upstream of the gene), causes the still rare resistance to carbapenems [22]. For instance, while 9.4% of the European B. fragilis strains harbored cfiA gene, <10% of them exhibited imipenem resistance [31]. Clindamycin resistance is associated with the acquisition of erm genes [32,33]. Metronidazole resistance is linked to metronidazole (nim) genes in Bacteroides/Parabacteroides spp. and Prevotella spp., [34]. In Belgium, only 2.8% of Bacteroides/Parabacteroides spp. strains had nim genes in 2011e2012 [16]. Quinolone resistance in

3.6. Resistance to other antibiotics In the USA, tigecycline resistance rates of B. fragilis did not increase [29]. However, tigecycline resistance was absent in Bacteroides vulgatus strains isolated in the US in 2006e2007, but emerged in 4.3% of the isolates from 2008 to 2009 [29]. All Swedish C. difficile strains were tigecycline susceptible [28]. Chloramphenicol resistance in Bacteroides and Parabacteroides spp. was absent in all the three studies from USA, Spain and Korea [7,17,21].

Table 2 Proposed factors associated with the resistance according to the authors of the publications. Agent

Species/group

Country

Specimen type

Resistance trend

Resistance associated with

Reference

Penicillin Penicillin and tetracycline Amoxicillin-clavulanate Amoxicillin-clavulanate and piperacillintazobactam Cefoxitin

Prevotella spp. P. intermedia/P. nigrescens Bacteroides/Parabacteroides spp. Bacteroides/Parabacteroides spp.

Bulgaria Switzerland Europe Spain

Oral Oral Various Various

Increase None Increase None

Antibiotic use (increased outpatient use of penicillins) Antibiotic use (low) Species (non-fragilis isolates), country Species (Parabacteroides distasonis)

[9] [13] [19] [22]

Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides

Canada Europe Spain USA USA Europe

Various Various Various Various Various Various

Decrease Increase None None None None

Bacteroides species, country Species (P. distasonis, B. thetaiotaomicron and B. ovatus) Species (B. ovatus) Species (B. vulgatus) Species (B. ovatus) For reduced susceptibility: strains, specimen (abscesses/wounds, intraabdominal and blood) Species (P. distasonis)

[18] [19] [22] [21] [21] [19]

Antibiotic consumption (relatively rare) Species and health care center Species, country Species (B. ovatus) Ribotype 001 (reduced susceptibility) Ciprofloxacin use (in the 1980s) Ribotype 001 Species (B. vulgatus), country Species (B. ovatus and B. uniformis), health care center Fluoroquinolone use (increased), ribotype (PCR ribotype 126 or 018) Tetracycline consumption (decreased outpatient use) Prior therapy (long-term vancomycin therapy) Prior therapy by fusidic acid Epidemic clone, prior rifamycin use Ribotype (012)

[9] [21] [19] [22] [25] [24] [26] [19] [21] [27]

Piperacillin-tazobactam Imipenem

spp. spp. spp. spp. spp. spp.

Ertapenem and Imipenem

Bacteroides/Parabacteroides spp.

Spain

Various

Clindamycin

Fluoroquinolones

Prevotella spp. Bacteroides/Parabacteroides Bacteroides/Parabacteroides Bacteroides/Parabacteroides C. difficile C. difficile C. difficile Bacteroides/Parabacteroides Bacteroides/Parabacteroides C. difficile

Bulgaria USA Europe Spain UK UK Germany Europe USA Italy

Oral Various Various Various Fecal Fecal Fecal Various Various Fecal

Slight increase None Increase Increase None Increase Increase Increase Increase Increase Increase

Tetracycline Vancomycin Fusidic acid Rifampin Rifampin

Prevotella spp. C. difficile C. difficile C. difficile C. difficile

Bulgaria Sweden Sweden USA Sweden

Oral Fecal Fecal Fecal Fecal

Decrease None None Decrease Decrease

Metronidazole Moxifloxacin

spp. spp. spp.

spp. spp.

[22]

[9] [28] [28] [30] [28]

Please cite this article in press as: Boyanova L, et al., Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.004

6

L. Boyanova et al. / Anaerobe xxx (2014) 1e7

the anaerobic bacteria can result from mutations in gyrA and parC genes and efflux mechanisms such as bexA gene encoded efflux pump in Bacteroides spp., [31,32]. Importantly, different resistance breakpoints of CLSI and EUCAST for several antibiotics such as metronidazole, penicillin and piperacillinetazobactam can influence the interpretation of the MIC results. The resistance breakpoints of EUCAST and CLSI are >0.5 and 2 mg/L for penicillin, >16/4 and 128/4 for piperacillinetazobactam, and >4 (>2 for C. difficile) and 32 mg/L for metronidazole, respectively. In the study of Nagy et al. [19], higher resistance rates (10.3%) were found in Bacteroides and Parabacteroides spp. according to EUCAST breakpoints compared with those (3.1%) acc. to CLSI criteria. In addition, for some antibiotics such as tigecycline, rifampin and fusidic acid, no EUCAST or CLSI breakpoints are available; thus, FDA breakpoints for tigecycline or arbitrary breakpoints are used [28e30]. Another possible limitation is that including non-fragilis Bacteroidales species, e.g., Odoribacter splanchnicus (ex-Bacteroides splanchnicus) as members of B. fragilis group in some studies may lead to underestimation of the resistance rates in the group. In the publications from the last six years, several important traits of the evolution of the antibiotic resistance in the anaerobes can be highlighted. Importantly, increase in resistance rates in Bacteroides and Parabacteroides spp. to amoxicillineclavulanate or ampicillinesulbactam and clindamycin was common and was reported in 1/2 of the studies from different countries/regions. Another topic of concern is the change in fluoroquinolone resistance in the anaerobes as all studies detected rising moxifloxacin resistance in Bacteroides and Parabacteroides spp. as well as in C. difficile. In some studies, an increase in resistance rates was observed in Bacteroides and Parabacteroides spp. to cefoxitin/cefotetan and carbapenems, in Prevotella spp. to penicillins, in anaerobic cocci to clindamycin and in Bacteroides/Parabacteroides spp. and C. difficile to metronidazole. In several studies, a decrease in antibiotic resistance in anaerobes was also found, e.g. in Bacteroides/Parabacteroides spp. to cephalosporins, in Prevotella spp. and C. difficile to tetracyclines and in C. difficile to rifampins. During the last years, no evolution in the resistance was reported to tigecycline, in Bacteroides/Parabacteroides spp. to chloramphenicol and in C. difficile to vancomycin and teicoplanin. The most dynamic changes were found in Bacteroides/Parabacteroides spp. and C. difficile. High (10-fold) increase in resistance rates was found in the amoxicillineclavulanate resistance of Bacteroides/Parabacteroides spp. in Europe over 21 years, in metronidazole reduced susceptibility of C. difficile ribotype 001 in UK over 11 years and moxifloxacin resistance of C. difficile in Scotland over 25 years [19,24,25]. Possible factors associated with the resistance were the species/ ribotype, country, hospital centre, antibiotic consumption and specimen. For example, non-fragilis Bacteroides/Parabacteroides spp., e.g., Parabacteroides distasonis, B. thetaiotaomicron, B. vulgatus and Bacteroides ovatus showed higher resistance rate to antibiotics compared with those of B. fragilis [19e22]. In the USA, Bacteroides/ Parabacteroides resistance to clindamycin was 3.4-fold lower in P. distasonis (14.3%) compared with that in Bacteroides uniformis (49.2%) [21]. Some ribotypes of C. difficile were associated with resistance to metronidazole, fluoroquinolones or multidrug resistance [24e27]. For instance, in Scotland, most C. difficile ribotype 012 strains were resistant to clindamycin (88.9%), ceftriaxone (66.7%) and tetracycline (88.9%) [24]. Growing clindamycin resistance was found in anaerobic cocci but not in Prevotella spp. in Bulgaria [[9,23], Boyanova, unpublished data]. National antibiotic consumption is an important factor for the evolution of the resistance in the anaerobes. Growing antibiotic

consumption was associated with increased penicillin resistance in Prevotella spp. in Bulgaria and high fluoroquinolone consumption was linked to the increasing moxifloxacin resistance in C. difficile in Scotland and Italy [9,24,27]. Conversely, decreasing or low antibiotic consumption was associated with lack of evolution in phenoxymethylpenicillin and tetracycline resistance rates in P. intermedia/P. nigrescens in Switzerland and with decreasing tetracycline resistance in Prevotella spp. in Bulgaria [9,13]. The country of the study is important for the evolution of the resistance and in many cases can be associated with the antibiotic consumption. For instance, cefoxitin resistance rates in Bacteroides and Parabacteroides spp. increased in Europe and declined in Canada [18,19]. In the USA, Bacteroides/Parabacteroides resistance rates to clindamycin and moxifloxacin varied widely by health care center [21]. Patients evaluated, country of residence and year of the study can influence the results in the same country. Additionally, differences in the antibiotic resistance rates can be associated with epidemics by resistant strains, different specimens evaluated and different treatment regimens used. For example, long-term intravenous vancomycin therapy led to appearance of vancomycin resistant C. difficile strains in Sweden [28]. Epidemic clones were associated with rifampin resistance rates in the USA and Sweden [28,30]. Type of the specimens is linked to different resistance rates in the anaerobic isolates. In Europe, Bacteroides/Parabacteroides strains with reduced susceptibility to imipenem were isolated from abscesses, wounds, intraabdominal specimens and blood [19]. Multidrug resistance can be defined as resistance to 3 antibiotics of different classes. It is concerning that multidrug resistant anaerobes “gain” new territories. In Afganistan, a multidrug resistant B. fragilis strain from wound specimens was detected in 2009 [35]. The strain was resistant to ampicillinesulbactam, piperacillinetazobactam, clindamycin, metronidazole, cefoxitin, imipenem, meropenem, tigecycline and chloramphenicol. The strain was susceptible only to moxifloxacin and linezolid [35]. Multidrug resistant B. fragilis strains were detected, although infrequently in Europe; however, in 2012, such a strain was reported for the first time in Scandinavia [32]. The strain was resistant to the main antibiotics for anaerobic infections, e.g., metronidazole, meropenem, imipenem, piperacillinetazobactam and clindamycin and was susceptible only to tigecycline [32]. Unfortunately, many patients infected by multidrug resistant Bacteroides strains have fatal outcomes. Presence of resistance to commonly used anti-anaerobic agents such as penicillin, clindamycin, cefoxitin and amoxicillineclavulanate [36] and drugs for mixed aerobic-anaerobic infections such as moxifloxacin [37] strongly indicates the need for regular monitoring of the antibiotic resistance patterns of the clinically important anaerobic bacteria at national or regional level in order to guide the choice of the empirical therapy. Identification to species level of Bacteroides strains can be clinically important as different species can exhibit important differences in resistance rates to different antibiotics. 5. Conclusions In conclusion, during the past years, Bacteroides and C. difficile resistance to moxifloxacin increased globally. Bacteroides, Prevotella, C. difficile and anaerobic cocci showed resistance changes. Increasing moxifloxacin resistance in Bacteroides/Parabacteroides spp. and C. difficile were found in all studies. Rising resistance patterns were detected in Bacteroides and Parabacteroides spp. to cefoxitin/cefotetan, b-lactam/b-lactamase inhibitor combinations and clindamycin, in non-difficile Clostridium spp. to moxifloxacin, in

Please cite this article in press as: Boyanova L, et al., Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.004

L. Boyanova et al. / Anaerobe xxx (2014) 1e7

non-Bacteroides/Parabacteroides Gram-negative anaerobes to clindamycin as well as in Prevotella spp. to penicillins. Declining antibiotic resistance in some anaerobic species was also detected in several studies. Multidrug resistant B. fragilis strains were reported. Five main factors were found to influence the resistance rates. Anaerobic bacteria exhibited recent changes in antibiotic resistance. Thus the importance of regular monitoring of resistance patterns of the anaerobes and both anaerobic microbiology and susceptibility testing of the isolates increases over time. References [1] Goldstein EJ, Citron DM, Goldman PJ, Goldman RJ. National hospital survey of anaerobic culture and susceptibility methods: III. Anaerobe 2008;14:68e72. [2] Goldstein EJC, Citron DM. Resistance trends in antimicrobial susceptibility of anaerobic bacteria, part I. Clin Microbiol Newsl 2011;33:1e8. [3] Smith AJ, Lockhart DE, Tyers A, Poxton IR. A survey of the identification and susceptibility testing of anaerobes in diagnostic microbiology laboratories in Scotland, UK. J Antimicrob Chemother 2010;65:805. [4] Rautemaa-Richardson R, der Reijden Wa WA, Dahlen G, Smith AJ. Quality control for diagnostic oral microbiology laboratories in European countries. J Oral Microbiol 2011;3. [5] Brook I, Wexler HM, Goldstein EJ. Antianaerobic antimicrobials: spectrum and susceptibility testing. Clin Microbiol Rev 2013;26:526e46. [6] Boyanova L, Kolarov R, Gergova G, Deliverska E, Madjarov J, Marinov M, et al. Anaerobic bacteria in 118 patients with deep-space head and neck infections from the University Hospital of Maxillofacial Surgery, Sofia, Bulgaria. J Med Microbiol 2006;55:1285e9.
mez M, Lo
pez F, Rodríguez-Avial I, Picazo JJ. Resistance [7] Betriu C, Culebras E, Go trends of the Bacteroides fragilis group over a 10-year period, 1997 to 2006, in Madrid, Spain. Antimicrob Agents Chemother 2008;52:2686e90. [8] Dubreuil L, Odou MF. Anaerobic bacteria and antibiotics: what kind of unexpected resistance could I find in my laboratory tomorrow? Anaerobe 2010;16: 555e9. [9] Boyanova L, Kolarov R, Gergova G, Dimitrova L, Mitov I. Trends in antibiotic resistance in Prevotella species from patients of the University Hospital of Maxillofacial Surgery, Sofia, Bulgaria, in 2003-2009. Anaerobe 2010;16: 489e92. [10] Liu CY, Huang YT, Liao CH, Yen LC, Lin HY, Hsueh PR. Increasing trends in antimicrobial resistance among clinically important anaerobes and Bacteroides fragilis isolates causing nosocomial infections: emerging resistance to carbapenems. Antimicrob Agents Chemother 2008;52:3161e8. [11] Veloo AC, Seme K, Raangs E, Rurenga P, Singadji Z, Wekema-Mulder G, et al. Antibiotic susceptibility profiles of oral pathogens. Int J Antimicrob Agents 2012;40:450e4. [12] van Winkelhoff AJ, Herrera D, Oteo A, Sanz M. Antimicrobial profiles of periodontal pathogens isolated from periodontitis patients in The Netherlands and Spain. J Clin Periodontol 2005;32:893e8. [13] Kulik EM, Lenkeit K, Chenaux S, Meyer J. Antimicrobial susceptibility of periodontopathogenic bacteria. J Antimicrob Chemother 2008;61:1087e91. [14] Pierard D, De Meyer A, Rosseel P, Glupczynski Y, Struelens MJ, Delmee M, et al. In vitro activity of amoxycillin plus clavulanic acid and ticarcillin plus clavulanic acid compared with that of other antibiotics against anaerobic bacteria. Acta Clin Belg 1989;44:228e36. [15] Pierard D, De Meyer A, Rosseel P, Van Cauwenbergh M, Struelens MJ, Delmee M, et al. In vitro activity of amoxycillin/clavulanate and ticarcillin/ clavulanate compared with that of other antibiotics against anaerobic bacteria: comparison with the results of the 1987 survey. Acta Clin Belg 1996;51: 70e9. [16] Wybo I, Van den Bossche D, Soetens O, Vekens E, Vandoorslaer K, Claeys G, et al. Fourth Belgian multicentre survey of antibiotic susceptibility of anaerobic bacteria. J Antimicrob Chemother 2014;69:155e61. [17] Roh KH, Kim S, Kim CK, Yum JH, Kim MS, Yong D, et al. Resistance trends of Bacteroides fragilis group over an 8-year period, 1997-2004, in Korea. Korean J Lab Med 2009;29:293e8.

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[18] Karlowsky JA, Walkty AJ, Adam HJ, Baxter MR, Hoban DJ, Zhanel GG. Prevalence of antimicrobial resistance among clinical isolates of Bacteroides fragilis group in Canada in 2010-2011: CANWARD surveillance study. Antimicrob Agents Chemother 2012;56:1247e52. [19] Nagy E, Urb
an E, Nord CE, ESCMID Study Group on Antimicrobial Resistance in Anaerobic Bacteria. Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe: 20 years of experience. Clin Microbiol Infect 2011;17: 371e9. [20] Jamal W, Shahin M, Rotimi VO. Surveillance and trends of antimicrobial resistance among clinical isolates of anaerobes in Kuwait hospitals from 2002 to 2007. Anaerobe 2010;16:1e5. [21] Snydman DR, Jacobus NV, McDermott LA, Golan Y, Hecht DW, Goldstein EJ, et al. Lessons learned from the anaerobe survey: historical perspective and review of the most recent data (2005-2007). Clin Infect Dis 2010;50(Suppl. 1): S26e33. ~ o M, Areses P, Pen ~ alver MD, Cortizo S, Pardo F, del Molino ML, et al. [22] Trevin Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF. Anaerobe 2012;18:37e43. [23] Boyanova L, Osmanliev D, Petrov D, Mitov I, Usunova I, Petrov S, et al. Anaerobic cocci and their resistance patterns to penicillin, cefoxitin, clindamycin and metronidazole: a Bulgarian study. Clin Microbiol Infect 2000;6: 623e4. [24] Taori SK, Hall V, Poxton IR. Changes in antibiotic susceptibility and ribotypes in Clostridium difficile isolates from southern Scotland, 1979-2004. J Med Microbiol 2010;59:338e44. [25] Baines SD, O'Connor R, Freeman J, Fawley WN, Harmanus C, Mastrantonio P, et al. Emergence of reduced susceptibility to metronidazole in Clostridium difficile. J Antimicrob Chemother 2008;62:1046e52. [26] Ilchmann C, Zaiss NH, Speicher A, Christner M, Ackermann G, Rohde H. Comparison of resistance against erythromycin and moxifloxacin, presence of binary toxin gene and PCR ribotypes in Clostridium difficile isolates from 1990 and 2008. Eur J Clin Microbiol Infect Dis 2010;29:1571e3. [27] Spigaglia P, Barbanti F, Dionisi AM, Mastrantonio P. Clostridium difficile isolates resistant to fluoroquinolones in Italy: emergence of PCR ribotype 018. J Clin Microbiol 2010;48:2892e6.
n T, Alriksson I, Akerlund T, Burman LG, Unemo M. In vitro susceptibility [28] Nore to 17 antimicrobials of clinical Clostridium difficile isolates collected in 19932007 in Sweden. Clin Microbiol Infect 2010;16:1104e10. [29] Snydman DR, Jacobus NV, McDermott LA, Golan Y, Goldstein EJ, Harrell L, et al. Update on resistance of Bacteroides fragilis group and related species with special attention to carbapenems 2006-2009. Anaerobe 2011;17: 147e51. [30] Curry SR, Marsh JW, Shutt KA, Muto CA, O'Leary MM, Saul MI, et al. High frequency of rifampin resistance identified in an epidemic Clostridium difficile clone from a large teaching hospital. Clin Infect Dis 2009;48: 425e9.
ki J, Urb
[31] Eitel Z, So an E, Nagy E, ESCMID Study Group on Anaerobic Infection. The prevalence of antibiotic resistance genes in Bacteroides fragilis group strains isolated in different European countries. Anaerobe 2013;21:43e9.
ki J, Nagy E, Justesen US. Multidrug-resistant Bacteroides [32] Hartmeyer GN, So fragilis group on the rise in Europe? J Med Microbiol 2012;61:1784e8. [33] Kirchner M, Mafura M, Hunt T, Card R, Anjum MF. Antibiotic resistance gene profiling of faecal and oral anaerobes collected during an antibiotic challenge trial. Anaerobe 2013;23:20e2. [34] Alauzet C, Mory F, Teyssier C, Hallage H, Carlier JP, Grollier G, et al. Metronidazole resistance in Prevotella spp. and description of a new nim gene in Prevotella baroniae. Antimicrob Agents Chemother 2010;54:60e4. [35] Sherwood JE, Fraser S, Citron DM, Wexler H, Blakely G, Jobling K, et al. Multidrug resistant Bacteroides fragilis recovered from blood and severe leg wounds caused by an improvised explosive device (IED) in Afghanistan. Anaerobe 2011;17:152e5. [36] Brook I. Antimicrobial treatment of anaerobic infections. Expert Opin Pharmacother 2011;12:1691e707. [37] Cheadle W, Lee JT, Napolitano LM, Nichols RL. Clinical update on the use of moxifloxacin in the treatment of community-acquired complicated intraabdominal infections. Surg Infect (Larchmt) 2010;11:487e94.

Please cite this article in press as: Boyanova L, et al., Recent evolution of antibiotic resistance in the anaerobes as compared to previous decades, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.05.004