Co-resistance: an opportunity for the bacteria and resistance genes

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Co-resistance: an opportunity for the bacteria and resistance genes Rafael Canto´n1,2 and Patricia Ruiz-Garbajosa1 Co-resistance involves transfer of several genes into the same bacteria and/or the acquisition of mutations in different genetic loci affecting different antimicrobials whereas pleiotropic resistance implies the same genetic event affecting several antimicrobials. There is an increasing prevalence of isolates with co-resistance which are over-represented within the socalled high-risk clones. Compensatory events avoid fitness cost of co-resistance, even in the absence of antimicrobials. Nevertheless, they might be selected by different antimicrobials and a single agent might select co-resistant isolates. This process, named as co-selection, is not avoided with cycling or mixing strategies of antimicrobial use. Coresistance and co-selection processes increase the opportunity for persistence of the bacteria and resistance genes and should be considered when designing strategies for decreasing antimicrobial resistance. Addresses 1 Servicio de Microbiologı´a and CIBER en Epidemiologı´a y Salud Pu´blica (CIBERESP), Hospital Universitario Ramo´n y Cajal and Instituto Ramo´n y Cajal de Investigacio´n Sanitaria (IRYCIS), Carretera Colmenar Km 9.1. 28034 Madrid, Spain 2 Unidad de Resistencia a Antibio´ticos y Virulencia Bacteriana Asociada al Consejo Superior de Investigaciones Cientı´ficas (CSIC), Hospital Universitario Ramo´n y Cajal, Carretera Colmenar Km 9.1. 28034 Madrid, Spain Corresponding author: Canto´n, Rafael ([email protected])

Current Opinion in Pharmacology 2011, 11:477–485 This review comes from a themed issue on Anti-infectives Edited by U. Theuretzbacher and J.W. Mouton Available online 11th August 2011 1471-4892/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2011.07.007

Introduction Antimicrobial agents are natural compounds that are able to kill bacteria or inhibit their growth. They achieve an antimicrobial action when administered to a patient infected with pathogenic bacteria but they might also produce a collateral effect over the normal patient’s microbiota [1]. Moreover, these compounds also act as selective forces for bacterial evolution. They can select variants over the natural susceptible bacterial population with decreased antimicrobial susceptibility owing to the presence of mutations or by the acquisition of the so www.sciencedirect.com

called ‘resistance genes’ [2]. These mutations or resistance genes can be accumulated in certain pathogenic bacterial species leading to complex phenotypes that are increasingly recognized all over the world [3,4]. They are in general named as multidrug-resistant bacteria and because of this particularity they can survive under the action of different antimicrobial compounds, increasing the opportunity for the spread [5]. This review highlights the current knowledge on multidrug-resistance from the clinical microbiological perspective and novel findings on this topic. Different usage patterns at the institutional level might play a role in the emergence and persistence of these multidrug-resistant isolates and subsequent coselection processes [6].

Multiresistance, co-resistance, crossresistance and pleiotropic resistance Different definitions have been used for multidrug-resistant bacteria. A joint initiative by the European Center for Disease Prevention and Control (ECDC) and the Center for Disease Control and Prevention (CDC) recently redefined ‘multidrug-resistant’ (MDR), ‘extensively drug-resistant’ (XDR) and ‘pandrug resistant’ (PDR) bacteria [7] (see Table 1 for specific definitions) to ascertain complex patterns of resistance in microorganisms commonly found in health-care associated infections, such as infection caused by Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii [8]. These definitions are established at phenotypic level but do not consider genetic aspects of the involved resistance mechanisms. Other terms have also been used in the scientific literature to refer to multidrug-resistant phenotypes. They include ‘co-resistance’, ‘cross-resistance’ and ‘pleiotropic resistance’ (Table 1). Co-resistance involves transfer of several genetic elements into the same bacterial isolate and/or the acquisition of mutations in different genetic loci affecting different antibacterial drugs, whereas crossresistance is produced by mutations or by the acquisition of resistance genes affecting antimicrobials agents from the same class. An excellent example to address these definitions is the methicillin resistant S. aureus (MRSA). This organism is considered multidrug-resistant owing to the accumulation of resistance mechanisms (co-resistance) that include, among others, beta-lactams, macrolides, aminoglycosides or fluoroquinolones. Moreover, it exhibits cross-resistance within specific antibiotic classes, for example, cross-resistance to all fluoroquinolones owing to the mutations in the topoisomerases genes ( parC Current Opinion in Pharmacology 2011, 11:477–485

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Table 1 Definition of different terms of multi-drug resistance Term

Definition

Multidrug resistant (MDR)

Non-susceptibility to at least one agent in three or more antimicrobial categoriesa

Extensive, extensively or extremely drug resistant (XDR)

Non-susceptibility to at least one agent in all but two or fewer antimicrobial categoriesa Non-susceptibility to all agents in all antimicrobial categories

Pan-drug resistance (PDR)

Co-resistance

Cross-resistance

Pleiotropic resistance

a

Presence of different resistance mechanisms encoded by mutated or acquired genes Presence of mutated or acquired resistance genes affecting antimicrobials agents from the same class. Presence of a resistance mechanism affecting several antimicrobial classes owing to the same genetic event such as mutation or acquisition of a resistance gene.

Comment Resistant to multiple antimicrobial agents (generally three or more), classes or subclasses of antimicrobial agents. This is the most general term that includes most of the situations defined below (XDR, PDR, etc.). Initially created for Mycobacterium tuberculosis, Streptococcus pneumoniae and Staphylococcus aureus, it was later used for Gramnegative bacteria involved in the nosocomial infections. It is based on in vitro susceptibility data with the aim to alert clinicians when treating patients and also for infection control purposes Resistant to all, or almost all, approved antimicrobial agents. Initially created for Mycobacterium tuberculosis, it was later used for Gramnegative bacteria involved in the nosocomial infections. It applies for organisms susceptible to only one or two categories Different definitions have been used in publications including organisms resistant to almost all commercially available antimicrobials, resistant to all antimicrobials routinely tested and resistant to all antimicrobial antibiotics available for empirical treatment. From a practical point of view no agent tested as susceptible for that organism Complex multidrug resistance phenotypes affecting different antimicrobial classes The resistance mechanism affects different antimicrobials from the same categories The resistance mechanism affects several antimicrobials from different categories

Therapeutic categories (i.e. aminoglycosides, fluoroquinolones, cephalosporins, carbapenems, etc.).

and/or gyrA) or macrolides owing to the presence of different erm, mef or mrs genes [9]. Finally, pleiotropic resistance affects several antimicrobial classes owing to the same genetic event such as mutation or acquisition of a resistance gene. Mutations producing upregulation of efflux pump regulatory genes in P. aeruginosa give a pleiotropic resistance phenotype affecting some of the beta-lactam compounds, fluoroquinolones and aminoglycosides [10]. Moreover, acquisition of the cfr gene, responsible for the production of a methylase that affects the ribosome, determines resistance not only to linezolid in S. aureus and coagulase negative staphylococci but also to cloramphenicol, lincosamides, pleuromutilins and streptogramin A [11]. Beyond its clinical interest, these definitions are also important from an evolutionary point of view and for predictions of antimicrobial resistance. Emergence of multidrug-resistant pathogens and their later selection and spread should also be considered under the scope of the genetic linkage in addition to the antimicrobial use.

Selection and co-selection processes Within a susceptible bacterial population there is always, at a variable frequency, a resistant subpopulation that can be selected under antibiotic exposure. This can be Current Opinion in Pharmacology 2011, 11:477–485

produced between a range of concentrations where the lower boundary is the concentration at which the susceptible population is killed or inhibited (the MIC or minimum inhibitory concentration) and the upper limit, the concentration at which the resistant subpopulation is killed or inhibited. During therapeutic administration of antimicrobials, fluctuating concentrations owing to the natural pharmacokinetic process in the infection compartment occur. The importance of the antibiotics gradients in the selection of resistant bacteria was established during the nineties with organism-antibacterial models involving mutational events in the resistance development but it can also be applied to organisms harboring acquired resistance genes [12,13]. With this perspective and that of the pharmacokinetic and pharmacodynamic one, adequate antibacterial concentrations should be achieved at the infection site during treatment to avoid the resistance development [14]. It is obvious that when an isolate is resistant to an antimicrobial agent owing to a selection process, a new challenge with an antimicrobial of a different class can select a new resistant variant thus accumulating different resistance phenotypes. In environments where exposure to different selective antimicrobial drugs is frequent (such as the hospital settings), the organisms harboring more resistant traits have higher opportunities of being www.sciencedirect.com

Selection of antimicrobial resistance and co-resistance Canto´n and Ruiz-Garbajosa 479

Figure 1

(a) Sequential acquisition of resistance genes

(b)

Co-selection process

* * *

susceptible isolate resistance genes

* isolate with co-resistance antimicrobials Current Opinion in Pharmacology

Emergence and selection of multidrug-resistance: (a) Sequential acquisition of resistance genes (mutations or gene transfer). The sequential exposure to different antimicrobials might accumulate resistance genes in bacteria; (b) The use of different antimicrobials might select different patterns of coresistant bacteria owing to the presence of different resistance genes. Eventually, exposure to a single antimicrobial produce the same selective effect than exposure to different antimicrobials (co-selection).

selected. This concept was coined as ‘genetic capitalism’; the rich (resistant) tend to become richer (more resistant) [5,15]. In this scenario, a single antibiotic might select different multidrug-resistant isolates and different antimicrobials might select a multidrug-resistant isolate. These processes can be named as co-selection and are illustrated in Figure 1. The increasing prevalence of multidrug-resistant isolates is currently driven by coselection processes as well as their persistence, even in the absence of antibiotic selection pressure [16].

Co-resistance and resistance genes as units for selection The presence of different resistance genes (co-resistance) undoubtedly gives advantages to the bacteria under antibiotic selective forces. It is in general recognized that the emergence of drug resistance is a direct consequence of the use of the antibiotics and that resistance is produced in a Darwinian way by mutational events [2]. Nevertheless, selection does not only involve mutations but also resistance genes transmitted by lateral transfer [13]. The pre-existence of the so called ‘resistance genes’ in nature that can be captured and inserted in the genome of the bacterial pathogens leading resistance phenotypes is well known [17]. These ‘resistance genes’ participate in ordinary functions in environmental bacteria, including those www.sciencedirect.com

producing antimicrobials, but they determine resistance traits when present in microorganisms different from those naturally producing these genes. Both mutated and acquired resistance genes are now considered as part of the ‘resistome’ with increasing importance in the clinics [17]. Recent studies using mutant library screens, microarray technologies and mutation frequency analysis have identified large collections of resistance genes not only in human bacteria but also in those recovered from animals and insect vectors [18–20]. These findings depict not only the importance of these genetic determinants as units of potential selection but also the bacteria harboring the resistance genes. The importance of different units of antimicrobial resistance (genes, genetic platforms harboring resistance genes, clones, clonal complexes, species) (Table 2) in selection processes has been stressed and should be considered when designing strategies for controlling antimicrobial resistance. Genetic linkage of resistance genes is relevant for their expression and maintenance. During the past years we have learned that certain insertion sequences, such as IS26 or ISCR1, have participated in both the mobilization of the resistance genes and in their phenotypic expression. This Current Opinion in Pharmacology 2011, 11:477–485

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Table 2 Different examples of resistance units as levels of selection Resistance units Gene

Example gyrA mecA

Insertion sequence

IS26 ISCR1

Integrons

Class-1 integrons

Transposons

Tn1546

Comment Mutations in the quinolone resistance determining region (QRDR) of topoisomerases (i.e. gyrA) affect fluoroquinolones activity An acquired resistance gene encoding PBP2a in staphylococci determining resistance to all beta-lactams An insertion sequence involved in the mobilization of different resistance genes, including blaESBLs An insertion sequence involved in the mobilization and expression of different resistance genes, including blaESBLs

Plasmids

IncFII

Genetic structure found in Gram-negative organism able to recruit different resistance genes such as carbapanemases and aminoglycoside modifying enzymes A transposon harboring vancomycin resistance genes (vanA and regulatory genes) in enterococci A well disperse incompatibility plasmid group associated with dissemination of blaCTX-M-15

Sequence type

ST131 of Escherichia coli ST258 of Klebsiella pneumoniae

A high-risk clone involved in the spread of the CTX-M-15 extended-spectrum beta-lactamase A high-risk clone involved in the spread of KPC (Klebsiella pneumoniae carbapenemase) carbapenemases

Clonal complex

CC17 of Enterococcus faecium

A cluster of different ST associated with dissemination of ampicillin and/or vancomycin in Enterococcus faecium

has been well established with extended-spectum betalactamase (ESBL) genes and more recently with other beta-lactamases, including plasmid AmpC enzymes and new carbapenemases [21,22]. Other genetic machineries that are relevant for the maintenance of the resistance genes are the so-called integrons, which are particularly important in Gram-negatives. Integrons are inherited genetic structures able to capture and integrate several resistance genes (named cassettes) [23]. They were initially associated with transposons and/or plasmids but have been also detected in the chromosome (named chromosomal-integrons or superintegrons) with large arrangements of cassette genes. Although chromosomal-integrons were found in environmental isolates, it has been shown that they can exchange genetic material with pathogenic organisms. Recent studies in fresh water biofilms have demonstrated a dynamic exchange of qac gene cassettes from class-1 integrons with other integrons in environmental bacteria. These cassettes lead to resistance to ammonium quaternary compounds [24]. Not surprisingly, integrons and insertion sequences are increasingly present in pathogenic bacteria and commensal organisms from human and animal origins [25,26]. A recent study in Australia revealed that class-1 integrons, the most prevalent one among these platforms, were present in 10% of Escherichia coli isolates isolated from animals and in 23% from healthy and ill humans [27]. IS26 is highly prevalent in isolates with TEM-1, the most ubiquitous beta-lactamase in Gram-negatives, linked with genetic platforms participating in lateral gene transfer such as composite transposons and conjugative plasmids [25]. These platforms also harbor other resistance Current Opinion in Pharmacology 2011, 11:477–485

genes including methylases and aminoglycoside modifying enzymes affecting aminoglycosides, plasmid mediated fluoroquinolone or sulfonamide resistance genes [26,28]. All these traits might facilitate selection under antimicrobial use, even at subtherapeutical level or in combination as recently demonstrated in beef cattle when administered chlortetracycline alone or in combination with sulfametazine [29].

Co-resistance and clones and clonal complexes as units for selection Current knowledge on resistance has focused attention not only on genes and genetic platforms facilitating lateral transfer but also on other supra-levels of selection (Table 2), including clones and clonal complexes (CC). Even, the concept of ‘high-risk clones’ has been used to address the importance of specific populations within species [30,31,32]. These high-risk clones exhibit co-resistance and are spread over the world. The popularization of the multi-locus sequence typing (MLST) schemes, which include sequencing of short regions (around 400– 450 bp) of 5–7 housekeeping genes and bioinformatics analysis, has facilitated identification of these clones. They are classified in sequence types (ST) which can be clustered into CC. Several high-risk clones exemplify the importance of this concept as their manteinace in nature increases the opportunity of accumulating resistance genes. Within Gram-negatives, the ST131 E. coli was recognized all over the world as harboring the blaCTX-M-15 gene, which encodes the most successful ESBL. Nowadays, it has also been shown that this clone produces carbapenemases, www.sciencedirect.com

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including the new NDM-1 metallo-beta-lactamase, and other resistance genes [33,34]. Moreover, within Klebsiella pneumoniae, the ST258 clone was recognized as a KPC (K. pneumoniae carbapenemase) producer [35]. Once this high-risk clone emerges in an institution, it can be persistent over years and difficult to eliminate [36]. This can be explained partly owing to the multidrug-resistance phenotype exhibited by this clone and co-selection processes. Interestingly, in a large multicenter study in Greece with 40 hospitals, it was observed that persistence of the ST258 K. pneumoniae clone gives opportunity for transmission of the resistant trait (KPC gene) to other STs and also for the acquisition of other resistance genes, including other carbapenemase genes such as blaVIM [36]. In A. baumannii the population structure is dominated by three international clonal lineages, CC1 (European clone I comprising ST1, ST7, ST8, ST19 and ST20), CC2 (European clone II comprising ST2, ST45 and ST47) and CC3 (European clone III comprising ST3 and ST14), the majority of them exhibiting a multidrug-resistant phenotype [32]. Moreover, in recent studies in the Mediterranean area, it was shown that few P. aeruginosa clones (ST111, ST175 and ST235) are well-represented within the multidrug-resistant isolates despite a high genetic diversity of the general population [30,37]. In Gram-positives, the best examples can be found in genogroup CC17 Enterococcus faecium and CC2 and CC9 Enterococus faecalis. Both are multidrug-resistant to several antibiotics, including vancomycin, and have been found well-adapted in the hospital environment and also in farm animals [4]. In E. faecium, the worldwide emergence of vancomycin resistant isolates in the nosocomial setting has been associated with the expansion of ST belonging to genogroup CC17. Also in E. faecalis, vancomycin resistance is linked with particular CCs, such as CC2 and CC9 that have been identified in different European and American hospitals [4,38]. Contrary to enterococci, high-risk clones of S. aureus are also emerging in the community although different from those traditionally encountered in the hospital setting [31,39]. In contrast to North America, where the USA300 clone (ST8-IV) predominates, community-associated MRSA in Europe is characterized by clonal diversity. The most common is the European clone (ST80-IV), although reports of USA300 are increasing.

Co-resistance and antimicrobial use. The ecological perspective In practice, the discovery or the introduction of an antibiotic into clinical use frequently reveals the presence of resistance mechanisms affecting this new antibiotic class. The increase in prevalence of the corresponding resistance trait is a matter of time clearly associated with the antimicrobial use. This is well exemplified with some www.sciencedirect.com

pathogens, such as S. aureus, that have sequentially acquired resistance mechanisms in parallel to the introduction of antimicrobial agents into clinical use. Even, the intensive use of some of the antimicrobials over the years has depicted a nearly universal resistance trait of this pathogen as can be seen with MRSA and the resistance to fluoroquinolones, which rapidly emerged after the introduction of ciprofloxacin in clinical practice. This was owing to the selection of isolates with topoisomerase mutations and also with the concomitant spread of MRSA fluoroquinolone resistant clones. These findings were well illustrated during the nineties and at the beginning of the current century but recent publications have also shown a dynamic process of replacement of the preexisting clones with new ones, but with no decrease in the fluoroquinolone resistance phenotype [40]. Recent studies have demonstrated the persistence of high frequency of fluoroquinolone resistance in MRSA isolates, particularly in those from hospital origin [41–43]. Even, the new ST398 MRSA clone from animal origin, which was initially susceptible to fluoroquinolones, has recently acquired resistance to these compounds [44]. Interestingly, ST398 MRSA clone has also recruited new resistance genes such as the new cfr gene leading to resistance to linezolid, cloramphenicol, lincosamides pleuromutilins, and streptogramin A or vga(A) or vga(C) genes leading to resistance to lincosamides, pleuromutilins and streptogramin A [44,45]. As in humans, persistence of this clone in animals has been associated with antimicrobial use [46]. Other examples, but in Gram-negatives can be illustrated with ESBL and carbapenemases. Although not clearly demonstrated at a global scale, it has been observed in different institutions that the increasing use of carbapenems owing to the increasing prevalence of infections with ESBL producing organisms determines in parallel the increasing prevalence of carbapenemases [47]. Risk factors analysis also links fluoroquinolone, cephalosporin and carbapenem use with infections owing to carbapenemase producing Enterobacteriaceae [48]. Moreover, prior exposure to more than three different classes of antibiotics as well as carbapenems is also associated with blood stream infection with VIM-1 producing K. pneumoniae isolates [49]. This is not surprising as carbapenemase producing Enterobacteriaceae are frequently resistant to several antimicrobial agents, including fluoroquinolones and aminoglycosides [50].

The fitness cost, a contra-selective process? Fitness is defined as the capability of a genotype or individual bacterial cell to survive and reproduce [51]. Antimicrobials disrupt bacterial functions affecting their viability. When resistant mutants emerge, either by mutation or lateral gene transfer, the bacteria suffer a fitness cost which might affect the selection processes. In theory, the presence of different resistance genes might Current Opinion in Pharmacology 2011, 11:477–485

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increase the fitness cost of the bacteria. Nevertheless, the bacteria might undergo compensatory events to decrease this cost enabling the resistant trait to persist. It has recently been demonstrated that accumulation of several topoisomerase mutations affecting fluoroquinolone activity might even increase fitness of the bacteria. This breaks the general assumption of antimicrobial resistance and fitness cost [52]. Moreover, epistatic effects (interactions between genes such as the effect of one gene being modified by one or several other genes) might occur even in the absence of antibiotics, thus decreasing fitness cost [53]. This process can also be produced with bacterial gene amplification [54]. In addition, mutator bacteria have shown better compensatory adaptation in the fitness cost of resistance, which is a situation that can be produced in specific environments, including biofilms [55,56]. All these data show that the fitness cost is not a problem for resistance and does not preclude survival of multidrug resistant isolates.

Antimicrobial use affecting acquisition of resistance traits As previously stated, antimicrobial use affects selection and co-selection processes and increases opportunities for multidrug resistant bacteria. Reducing the selection pressure exerted by antimicrobials has been promoted to curtail multidrug resistant bacteria [14]. Nevertheless and to a certain extent, this is not rigorously possible in the hospital setting, particularly in intensive care units (ICUs), where antimicrobials are part of the benefits produced by healthcare.

two units were randomized to a predominant beta-lactam antibiotic regimen (weekly cycling of ceftriaxone, amoxicillin-clavulanic acid and fluoroquinolones) or a fluoroquinolone regimen for three months, with cross-over for another three months. The influence on the acquisition of third-generation cephalosporin-resistant or fluoroquinolone-resistant Enterobacteriaceae was monitored with microbiological surveillance cultures [59]. Reduction of beta-lactam exposure (up to 40%) was not associated with reduced acquisition of third-generation cephalosporinresistant Enterobacteriaceae, whereas an increase of fluoroquinolone use (nearly 250%) increased acquisition of fluoroquinolone-resistant Enterobacteriaceae. In another study, exposure to quinolones and antipseudomonal cephalosporins was not associated with the acquisition of resistance in P. aeruginosa, whereas it was linked with usage of all other agents [60]. Previously it was shown that in critical ill patients, a strategy of monthly rotation of anti-pseudomonal beta-lactams and ciprofloxacin may perform better than a strategy of mixing in the acquisition of P. aeruginosa resistant to beta-lactams. These results underscore the need for further studies not only monitoring resistance in clinical samples but also in surveillance cultures. Also, further studies should not only monitor resistance units (i.e. resistance genes and genetic platforms harboring resistance genes) but also resistant bacteria. Accumulation of resistance genes over cycling and mixing strategies has been considered one of the most important drawbacks of these strategies. Finally, combination therapy has traditionally been proposed to avoid the emergence of resistance. This strategy can be effective in preventing mutational events or even acquisition of resistance genes. Nevertheless, when multidrug-resistant bacteria have emerged, the use of combination therapy does not assure a decrease in bacterial resistance owing to co-selection processes (see above). This has been traditionally observed with Mycobacterium tuberculosis and more recently in patients with AIDS. Moreover, in vitro studies have shown the beneficial effect of combination therapy in preventing emergence of resistance in P. aeruginosa, including hypermutator isolates [61,62]. Nevertheless, it can be differently affected depending on the antimicrobial combination, as recently demonstrated in P. aeruginosa [63]. While the combination of fosfomycin with tobramycin prevents the emergence of resistance to both antibiotics when administered together, the combination of fosfomycin plus imipenem does not avoid the appearance of mutants resistant to both antibiotics.

Different interventions have promoted the reduction of the selection pressure, including cycling and mixing strategies [6]. Cycling, also known as rotation, consists of the subsequent use at ward level of different antimicrobial classes possessing a comparable spectrum of activity and preferably a different mechanism of action during identical predefined periods of time. Once the first antibiotic period ends, the compound is withdrawn and substituted by a second one, and then by a third and a forth one. Finally, the cycle restarts with the first compound. Mixing strategies are used at the level of individual patients (e.g. consecutive patients get a carbapenem, a quinolone, a beta-lactambeta-lactamase inhibitor combination, a cephalosporin, and so on in a cycling manner). Both strategies, grouped as antibiotic heterogeneity when referring to protocols of antimicrobial use, aim to prevent development of resistance. They are contrary to homogeneity which in each patient implies the strictly use of recommended guidelines that usually have been developed in response to bacterial resistance that has already occurred.

Concluding remarks

Benefit of antibiotic heterogeneity has been questioned. Even conflicting results are obtained when using mathematical models [57,58]. From a practical point of view,

During the past years, increasing prevalence of isolates with co-resistance has been recognized in surveillance studies. These multidrug resistant bacteria have emerged owing to several selection processes involving mutations

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under antimicrobial pressure but also by acquisition of different resistance genes. They can be selected not only by a single agent but also by different antimicrobials exerting co-selection processes. Moreover, fitness cost does not hamper persistence of these multidrug resistant bacteria owing to compensatory events, even in the absence of antimicrobials. Isolates with co-resistance are well represented by the so-called high-risk clones and can be maintained owing to co-selection processes. This fact also favors persistence of resistance genes and increases the opportunity for transmission. Different models of antimicrobial use in the clinical setting, such as cycling or mixing, might not prevent but even facilitate co-resistance and co-selection. Interventions on antimicrobial use should consider co-resistance and co-selection processes to avoid opportunities to the multidrug-resistant bacteria and the corresponding resistance genes.

Conflict of interest None to declare.

Acknowledgements Scientific background of the manuscript was partially obtained through research EU founded projects (LSHM-CT-2003-503335 and HEALTH-F32008-223031). We thank Dr. Fernando Baquero for continuous inspiration and discussion in the laboratory. We also thank Ana Moreno-Bofarull for substantial administrative support and Mary Harper for helping with the manuscript production.

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38. Ruiz-Garbajosa P, Bonten MJ, Robinson DA, Top J, Nallapareddy SR, Torres C, Coque TM, Canto´n R, Baquero F, Murray BE et al.: Multilocus sequence typing scheme for Enterococcus faecalis reveals hospital-adapted genetic complexes in a background of high rates of recombination. J Clin Microbiol 2006, 44:2220-2228. 39. Otter JA, French GL: Molecular epidemiology of communityassociated meticillin-resistant Staphylococcus aureus in Europe. Lancet Infect Dis 2010, 10:227-239.

25. Bailey JK, Pinyon JL, Anantham S, Hall RM: Distribution of the blaTEM gene and blaTEM-containing transposons in commensal Escherichia coli. J Antimicrob Chemother 2011, 66:7457-7551.

40. Armand-Lefevre L, Buke C, Ruppe E, Barbier F, Lolom I, Andremont A, Ruimy R, Lucet JC: Secular trends and dynamics of hospital associated methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect 2010, 16:1435-1441.

26. Deng Y, Zeng Z, Chen S, He L, Liu Y, Wu C, Chen Z, Yao Q, Hou J, Yang T, Liu JH: Dissemination of IncFII plasmids carrying rmtB and qepA in Escherichia coli from pigs, farm workers and the environment. Clin Microbiol Infect 2011. Jan 24, doi:10.1111/ j.1469-0691.2011.03472.x [Epub ahead of print].

41. Vindel A, Cuevas O, Cercenado E, Marcos C, Bautista V, Castellares C, Trincado P, Boquete T, Pe´rez-Va´zquez M, Marı´n M et al.: Methicillin-resistant Staphylococcus aureus in Spain: molecular epidemiology and utility of different typing methods. J Clin Microbiol 2009, 47:1620-1627.

27. Dawes FE, Kuzevski A, Bettelheim KA, Hornitzky MA, Djordjevic SP, Walker MJ: Distribution of class 1 integrons with IS26-mediated deletions in their 30 -conserved segments in Escherichia coli of human and animal origin. PLoS ONE 2010, 5:e12754.

42. Song JH, Hsueh PR, Chung DR, Ko KS, Kang CI, Peck KR, Yeom JS, Kim SW, Chang HH, Kim YS et al.: Spread of methicillin-resistant Staphylococcus aureus between the community and the hospitals in Asian countries: an ANSORP study. J Antimicrob Chemother 2011, 66:1061-1069.

28. Curiao T, Canto´n R, Garcilla´n-Barcia MP, de la Cruz F, Baquero F, Coque TM: Association of composite IS26-sul3 elements with highly transmissible IncI1 plasmids in extended-spectrumbeta-lactamase-producing Escherichia coli clones from humans. Antimicrob Agents Chemother 2011, 55:2451-2457. 29. Wu RB, Alexander TW, Li JQ, Munns K, Sharma R, McAllister TA: Prevalence and diversity of class 1 integrons and resistance genes in antimicrobial-resistant Escherichia coli originating from beef cattle administered subtherapeutic antimicrobials. J Appl Microbiol 2011, 111:511-523 [Epub ahead of print].

43. Nichol KA, Adam HJ, Hussain Z, Mulvey MR, McCracken M, Mataseje LF, Thompson K, Kost S, Lagace´-Wiens PR, Hoban DJ et al.: Comparison of community-associated and health careassociated methicillin-resistant Staphylococcus aureus in Canada: results of the CANWARD 2007–2009 study. Diagn Microbiol Infect Dis 2011, 69:320-325.

30. Garcı´a-Castillo M, Del Campo R, Morosini MI, Riera E, Cabot G, Willems R, van Mansfeld R, Oliver A, Canto´n R: Wide dispersion of ST175 clone despite a high genetic diversity of carbapenem-non-susceptible Pseudomonas aeruginosa clinical strains from 16 Spanish hospitals. J Clin Microbiol 2011, 49:2905-2910 [Epub ahead of print].

44. Argudı´n MA, Tenhagen BA, Fetsch A, Sachsenro¨der J,  Ka¨sbohrer A, Schroeter A, Hammerl JA, Hertwig S, Helmuth R, Bra¨unig J et al.: Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources. Appl Environ Microbiol 2011, 77:3052-3060. An excellent example of the emergence of a new resistance clone able to acquire different mutations and several resistance genes.

31. Willems RJ, Hanage WP, Bessen DE, Feil EJ: Population biology  of Gram-positive pathogens: high-risk clones for dissemination of antibiotic resistance. FEMS Microbiol Rev 2011, 35:872-900 [Epub ahead of print]. Excellent review of Gram-positive high-risk clones.

45. Kadlec K, Pomba CF, Couto N, Schwarz S: Small plasmids carrying vga(A) or vga(C) genes mediate resistance to lincosamides, pleuromutilins and streptogramin A antibiotics in methicillin-resistant Staphylococcus aureus ST398 from swine. J Antimicrob Chemother 2010, 65:2692-2693.

32. Woodford N, Turton JF, Livermore DM: Multiresistant Gram negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev 2011, 35:736-755 [Epub ahead of print]. A detailed catalogue of Gram-negative high-risk clones.

46. Graveland H, Wagenaar JA, Heesterbeek H, Mevius D, van Duijkeren E, Heederik D: Methicillin resistant Staphylococcus aureus ST398 in veal calf farming: human MRSA carriage related with animal antimicrobial usage and farm hygiene. PLoS ONE 2010, 5:e10990.

33. Peirano G, Schreckenberger PC, Pitout JD: Characteristics of NDM-1-producing Escherichia coli isolates that belong to the successful and virulent clone ST131. Antimicrob Agents Chemother 2011, 55:2986-2988.

47. Meyer E, Schwab F, Schroeren-Boersch B, Gastmeier P: Dramatic increase of third-generation cephalosporinresistant E. coli in German intensive care units: secular trends in antibiotic drug use and bacterial resistance, 2001 to 2008. Crit Care 2010, 14:R113.

34. Mantengoli E, Luzzaro F, Pecile P, Cecconi D, Cavallo A, Attala L, Bartoloni A, Rossolini GM: Escherichia coli ST131 producing extended-spectrum b-lactamases plus VIM-1 carbapenemase: further narrowing of treatment options. Clin Infect Dis 2011, 52:690-691. 35. Samuelsen Ø, Naseer U, Tofteland S, Skutlaberg DH, Onken A, Hjetland R, Sundsfjord A, Giske CG: Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing plasmid-mediated KPC carbapenemase in Norway and Sweden. J Antimicrob Chemother 2009, 63:654-658. 36. Giakkoupi P, Papagiannitsis CC, Miriagou V, Pappa O, Polemis M,  Tryfinopoulou K, Tzouvelekis LS, Vatopoulos AC: An update of the evolving epidemic of blaKPC-2-carrying Klebsiella pneumoniae in Greece (2009–10). J Antimicrob Chemother 2011, 66:1510-1513. An example of the persistence and dominance of a high-risk-clone. 37. Cholley P, Thouverez M, Hocquet D, van der Mee-Marquet N, Talon D, Bertrand X: Most Multidrug-resistant Pseudomonas aeruginosa isolates from hospitals in Eastern France belong to a few clonal types. J Clin Microbiol 2011, 49:2578-2583. Current Opinion in Pharmacology 2011, 11:477–485

48. Hussein K, Sprecher H, Mashiach T, Oren I, Kassis I, Finkelstein R: Carbapenem resistance among Klebsiella pneumoniae isolates: risk factors, molecular characteristics, and susceptibility patterns. Infect Control Hosp Epidemiol 2009, 30:666-671. 49. Daikos GL, Vryonis E, Psichogiou M, Tzouvelekis LS, Liatis S, Petrikkos P, Kosmidis C, Tassios PT, Bamias G, Skoutelis A: Risk factors for bloodstream infection with Klebsiella pneumoniae producing VIM-1 metallo-beta-lactamase. J Antimicrob Chemother 2010, 65:784-788. 50. Livermore DM, Warner M, Mushtaq S, Doumith M, Zhang J, Woodford N: What remains against carbapenem-resistant Enterobacteriaceae? Evaluation of chloramphenicol, ciprofloxacin, colistin, fosfomycin, minocycline, nitrofurantoin, temocillin and tigecycline. Int J Antimicrob Agents 2011, 37:415-419. 51. Andersson DI, Hughes D: Antibiotic resistance and its cost: is it possible to reverse resistance? Nat Rev Microbiol 2010, 8:260-271. www.sciencedirect.com

Selection of antimicrobial resistance and co-resistance Canto´n and Ruiz-Garbajosa 485

52. Marcusson LL, Frimodt-Møller N, Hughes D: Interplay in the  selection of fluoroquinolone resistance and bacterial fitness. PLoS Pathog 2009, 5:e1000541. A paper showing that increasing the number of mutations leading to higher resistance levels does not necessarily increase the fitness cost. 53. Ward H, Perron GG, Maclean RC: The cost of multiple drug resistance in Pseudomonas aeruginosa. J Evol Biol 2009, 22:997-1003. 54. Soo VW, Hanson-Manful P, Patrick WM: Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli. Proc Natl Acad Sci USA 2011, 108:1484-1489. 55. Garcı´a-Castillo M, del Campo R, Baquero F, Morosini MI, Turrientes MC, Zamora J, Canto´n R: Stationary biofilm growth normalizes mutation frequencies and mutant prevention concentrations in Pseudomonas aeruginosa from cystic fibrosis patients. Clin Microbiol Infect 2011, 17:704-711. 56. Perron GG, Hall AR, Buckling A: Hypermutability and  compensatory adaptation in antibiotic-resistant bacteria. Am Nat 2010, 176:303-311. Hypermutation leads to antimicrobial resistance but can also facilitate events to compensate fitness cost. 57. Beardmore RE, Pena-Miller R: Antibiotic cycling versus mixing:  the difficulty of using mathematical models to definitively quantify their relative merits. Math Biosci Eng 2010, 7:923-933. The use of mathematical model to address different ways of antimicrobial use.

www.sciencedirect.com

58. Bergstrom CT, Lo M, Lipsitch M: Ecological theory suggests that antimicrobial cycling will not reduce antimicrobial resistance in hospitals. Proc Natl Acad Sci USA 2004, 101:13285-13290. 59. Nijssen S, Fluit A, van de Vijver D, Top J, Willems R, Bonten MJ: Effects of reducing beta-lactam antibiotic pressure on intestinal colonization of antibiotic-resistant Gram-negative bacteria. Intensive Care Med 2010, 36:512-519. 60. Martı´nez JA, Delgado E, Martı´ S, Marco F, Vila J, Mensa J, Torres A, Codina C, Trilla A, Soriano A et al.: Influence of antipseudomonal agents on Pseudomonas aeruginosa colonization and acquisition of resistance in critically ill medical patients. Intensive Care Med 2009, 35:439-447. 61. Louie A, Grasso C, Bahniuk N, Van Scoy B, Brown DL, Kulawy R, Drusano GL: The combination of meropenem and levofloxacin is synergistic with respect to both Pseudomonas aeruginosa kill rate and resistance suppression. Antimicrob Agents Chemother 2010, 54:2646-2654. 62. Plasencia V, Borrell N, Macia´ MD, Moya B, Pe´rez JL, Oliver A: Influence of high mutation rates on the mechanisms and dynamics of in vitro and in vivo resistance development to single or combined antipseudomonal agents. Antimicrob Agents Chemother 2007, 51: pp. 2574–1581. 63. Rodrı´guez-Rojas A, Macia´ MD, Couce A, Go´mez C, Castan˜edaGarcı´a A, Oliver A, Bla´zquez J: Assessing the emergence of resistance: the absence of biological cost in vivo may compromise fosfomycin treatments for P. aeruginosa infections. PLoS ONE 2010, 5:e10193.

Current Opinion in Pharmacology 2011, 11:477–485