Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 Mechanisms of Bacterial Resistance to Antimicrobial Agents ENGELINE VAN DUIJKEREN,1 ANNE-KATHRIN SCHINK,2 MARILYN C. ROBERTS,3 YANG WANG,4 and STEFAN SCHWARZ2 1 Center for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands; 2 Institute of Microbiology and Epizootics, Centre of Infection Medicine, Department of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany; 3 Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98195-7234; 4 Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China ABSTRACT During the past decades resistance to virtually all antimicrobial agents has been observed in bacteria of animal origin. This chapter describes in detail the mechanisms so far encountered for the various classes of antimicrobial agents. The main mechanisms include enzymatic inactivation by either disintegration or chemical modification of antimicrobial agents, reduced intracellular accumulation by either decreased influx or increased efflux of antimicrobial agents, and modifications at the cellular target sites (i.e., mutational changes, chemical modification, protection, or even replacement of the target sites). Often several mechanisms interact to enhance bacterial resistance to antimicrobial agents. This is a completely revised version of the corresponding chapter in the book Antimicrobial Resistance in Bacteria of Animal Origin published in 2006. New sections have been added for oxazolidinones, polypeptides, mupirocin, ansamycins, fosfomycin, fusidic acid, and streptomycins, and the chapters for the remaining classes of antimicrobial agents have been completely updated to cover the advances in knowledge gained since 2006. INTRODUCTION With regard to their structures and functions, antimicrobial agents represent a highly diverse group of low-molecular-weight substances which interfere with bacterial growth, resulting in either a timely limited growth inhibition (bacteriostatic effect) or the killing of the bacteria (bactericidal effect). For more than 60 years, antimicrobial agents have been used to control bacterial infections in humans, animals, and plants. Nowadays, antimicrobial agents are among the most frequently used therapeutics in human and veterinary medicine (1, 2). In the early days of antimicrobial chemotherapy, antimicrobial resistance was not considered as an important problem, since the numbers of resistant strains were low and a large number of new highly effective antimicrobial agents of different classes were detected. These early antimicrobial agents represented products of the metabolic pathways of soil bacteria (e.g., Streptomyces, Bacillus) or fungi (e.g., Penicillium, Cephalosporium, Pleurotus) (Table 1) and provided their producers with a selective advantage in the fight for resources and the colonization of ecological niches (3). This in turn forced the susceptible bacteria living in close contact with the antimicrobial producers to develop and/or refine mechanisms to circumvent the inhibitory effects of antimiReceived: 24 February 2017, Accepted: 6 November 2017, Published: 11 January 2018 Editors: Frank Møller Aarestrup, Technical University of Denmark, Lyngby, Denmark; Stefan Schwarz, Freie Universität Berlin, Berlin, Germany; Jianzhong Shen, China Agricultural University, Beijing, China, and Lina Cavaco, Statens Serum Institut, Copenhagen, Denmark Citation: van Duijkeren E, Schink AK, Roberts MC, Wang Y, Schwarz S. 2017. Mechanisms of bacterial resistance to antimicrobial agents. Microbiol Spectrum 6(1):ARBA-0019-2017. doi:10.1128 /microbiolspec.ARBA-0019-2017. Correspondence: Stefan Schwarz, stefan.schwarz@fu-berlin.de © 2017 American Society for Microbiology. All rights reserved. ASMscience.org/MicrobiolSpectrum 1 Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 crobial agents. As a consequence, the origins of bacterial resistance to antimicrobial agents can be assumed to be in a time long before the clinical use of these substances. With the elucidation of the chemical structure of the antimicrobial agents, which commonly followed soon after their detection, it was possible not only to produce antimicrobial agents synthetically in larger amounts at lower costs, but also to introduce modifications that altered the pharmacological properties of these substances and occasionally also extended their spectrum of activity. The increased selective pressure imposed by the widespread use of antimicrobial agents since the 1950s has distinctly accelerated the development and the spread of bacterial resistance to antimicrobial agents. In most cases, it took not longer than three to five years after the introduction of an antimicrobial agent into clinical use until the first resistant target bacteria occurred (1). This is particularly true for broad-spectrum antimicrobial agents, such as tetracyclines, aminoglycosides, macrolides, and β-lactams, which have been used for multiple purposes in human and veterinary medicine, horticulture, and/or aquaculture. In contrast, this time span was extended to ≥15 years for narrow-spectrum agents, such as glycopeptides, which were used at distinctly lower quantities and only for specific applications. Multiple studies have also revealed that resistance to completely synthetic antimicrobial agents, such as sulfonamides, trimethoprim, fluoroquinolones, and oxazolidinones, can develop quickly (4–7). These observations underline the enormous flexibility of the bacteria to cope with less favorable environmental conditions by constantly exploring new ways to survive in the presence of antimicrobial agents. TABLE 1 Origins of antimicrobial agents Class Antimicrobial agent Producing organisms Year(s) of isolation/ description β-Lactam antibiotics Natural penicillins Penicillium notatum, Penicillium chrysogenum 1929, 1940 Cephalosporin C Cephalosporium acremonium 1945, 1953 Imipenem Streptomyces cattleya 1976 Aztreonam Gluconobacter spp., Chromobacterium violaceum 1981 Glycopeptides Vancomycin Amycolatopsis orientalis mid-1950s Teicoplanin, avoparcin Amycolatopsis coloradensis subsp. labeda 1975 Macrolides Erythromycin Streptomyces erythreus 1952 Spiramycin Streptomyces ambofaciens 1955 Lincosamides Lincomycin Streptomyces lincolnensis 1963 Streptogramins Streptogramin A+B Streptomyces diastaticus 1953 Virginiamycin A+B Streptomyces virginiae 1955 Tetracyclines Chlortetracycline Streptomyces aureofaciens 1948 Oxytetracycline Streptomyces rimosus 1950 Phenicols Chloramphenicol Streptomyces venezuelae 1947 Aminoglycosides Streptomycin Streptomyces griseus 1943 Neomycin Streptomyces fradiae 1949 Kanamycin Streptomyces kanamyceticus 1957 Gentamicin Micromonospora purpura 1963 Tobramycin Streptomyces tenebrarius 1967 Aminocyclitols Spectinomycin Streptomyces spectabilis 1961 Pleuromutilins Pleuromutilin, Tiamulin Pleurotus spp.; synthetic 1951, 1976 Polypeptide antibiotics Polymyxin B Bacillus polymyxa (aerosporus) 1947 Polymyxin E (colistin) B. polymyxa var. colistinus 1949 Bacitracin Bacillus licheniformis 1943 Epoxide antibiotics Fosfomycin Streptomyces fradiae, Streptomyces wedmorensis, Pseudomonas syringae 1969 Pseudomonic acid antibiotics Mupirocin Pseudomonas fluorescens 1971 Steroid antibiotics Fusidic acid Fusidium coccineum 1960 Streptothricins Nourseothricin Streptomyces noursei 1963 Sulfonamides Prontosil, sulfamethoxazole, etc. Synthetic 1935 Trimethoprim Trimethoprim Synthetic 1956 Quinolones Nalidixic acid Synthetic 1962 Fluoroquinolones Flumequine, enrofloxacin, etc. Synthetic 1973 Oxazolidinones Linezolid Synthetic 1987, 1996 2 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 This chapter summarizes the latest information on resistance mechanisms and the mobile elements involved. It is a completely revised and updated version of the chapter that was published in 2006 (8). RESISTANCE TO ANTIMICROBIAL AGENTS Resistance to antimicrobial agents can be divided into two basic types, intrinsic resistance and acquired resistance (1, 3, 8, 9). Intrinsic resistance, also known as primary or innate resistance, describes a status of general insensitivity of bacteria to a specific antimicrobial agent or class of agents. This is commonly due to the lack or the inaccessibility of target structures for certain antimicrobial agents, e.g., resistance to β-lactam antibiotics and glycopeptides in cell wall-free bacteria, such as Mycoplasma spp., or vancomycin resistance in Gramnegative bacteria due to the inability of vancomycin to penetrate the outer membrane. It can also be due to the presence of export systems or the production of speciesspecific inactivating enzymes in certain bacteria, e.g., the AcrAB-TolC system or the production of AmpC β-lactamase in certain Enterobacteriaceae. In addition, some bacteria, such as enterococci, can use exogenous folates and are thus not dependent on a functional folate synthesis pathway. As a consequence, they are intrinsically resistant to folate pathway inhibitors, such as trimethoprim and sulfonamides (9). Intrinsic resistance is a genus- or species-specific property of bacteria. In contrast, acquired resistance is a strain-specific property which can be due to the acquisition of foreign resistance genes or mutational modification of chromosomal target genes. Mutations that upregulate the expression of multidrug transporter systems may also fall into this category. Three different basic types of resistance mechanisms can be differentiated: (i) enzymatic inactivation by either disintegration or chemical modification of the antimicrobials (Table 2), (ii) reduced intracellular accumulation by decreased influx and/or increased efflux of antimicrobials (Table 3), and (iii) modification of the cellular target sites by mutation, chemical modification, or protection of the target sites, but also overexpression of sensitive targets or the replacement of sensitive target structures by alternative resistant ones (Table 4) (1, 3, 8, 9). The following subsections illustrate that bacterial resistance to antimicrobial agents varies depending on the agents, the bacteria, and the resistance mechanism. Resistance to the same antimicrobial agent can be mediated by different mechanisms. In some cases, the same resistance gene/mechanism is found in a wide variety of bacteria, whereas in other cases, resistance genes or mechanisms appear to be limited to certain bacterial species or genera. The data presented in the following subsections do not focus exclusively on resistance genes and mechanisms so far detected in bacteria of animal origin but also include resistance genes and mechanisms identified in bacteria from humans. For the best possible overview of the mechanisms and genes accounting for resistance to a specific class of antimicrobial agents, all data are presented under the names of the classes of antimicrobial agents. Resistance to β-Lactam Antibiotics A number of penicillins, alone or in combination with a β-lactamase inhibitor, as well as first- to fourthgeneration cephalosporins, are licensed for use in veterinary medicine. No carbapenems or monobactams are currently approved for use in animals. Resistance to β-lactam antibiotics is mainly due to inactivation by β-lactamases (10) and decreased ability to bind to penicillin-binding proteins (PBPs) (11) in both Grampositive and Gram-negative bacteria, but may also be based on decreased uptake of β-lactams due to permeability barriers or increased efflux via multidrug transporter systems (12, 13). Inactivation via β-lactamases is most commonly seen, with a wide range of β-lactamases involved. The evolution of β-lactamases which differ distinctly in their substrate spectra is believed to have occurred in response to the selective pressure imposed by the various β-lactam antibiotics that have been introduced into clinical use during the past decades (14). Enzymatic inactivation of β-lactam antibiotics is based on the cleavage of the amino bond in the β-lactam ring by β-lactamases (10, 15, 16). At present, more than 1,000 β-lactamases have been described, most of which are variants of known β-lactamases that differ in their substrate spectra or their enzyme stability. Two classification schemes are currently in use. The initial classification scheme was based on the similarities in the amino acid sequences and divided the β-lactamases into two molecular classes, A and B (17), which were later expanded to four molecular classes, A to D (18). The second functional classification of β-lactamases was updated in 2010 by Bush and Jacoby (18) and is done on the basis of their substrate spectra and their susceptibility to β-lactamase inhibitors such as clavulanic acid (18). This system subdivides the β-lactamases into three groups, 1 to 3, with group 1 currently comprising 2 subgroups, group 2 comprising 12 subgroups, and group 3 comprising 2 subgroups. ASMscience.org/MicrobiolSpectrum 3 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 TABLE 3 Examples of resistance to antimicrobials by enzymatic inactivationa Resistance mechanism Resistance gene(s) Gene product Resistance phenotype Bacteria involved Location of the resistance geneb Hydrolytic degradation bla β-Lactamases β-Lactam antibiotics Various Gram+, Gram–, aerobic, anaerobic bacteria P, T, GC, C ere(A), ere(B) Esterase Macrolides Gram+, Gram– bacteria P, GC vgb(A), vgb(B) Lactone hydrolases Streptogramin B antibiotics Staphylococcus, Enterococcus P Chemical modification aac, aad (ant), aph Acetyl-, adenyl-, phosphotransferases Aminoglycosides Gram+, Gram–, aerobic bacteria T, GC, P, C aad (ant) Adenyltransferases Aminoglycosides/ aminocyclitols Gram+, Gram–, aerobic bacteria T, GC, P, C catA, catB Acetyltransferases Chloramphenicol Gram+, Gram–, aerobic, anaerobic bacteria P, T, GC, C vat(A-G) Acetyltransferases Streptogramin A antibiotics Bacteroides, Staphylococcus, Enterococcus, Lactobacillus, Yersinia P, C mph(A-G) phosphotransferases Macrolides Gram+, Gram– bacteria P, T, C lnu(A-P) nucleotidyltransferases Lincosamides Gram+, Gram– bacteria P tet(X), tet(37), tet(56) oxidoreductases Tetracyclines Gram– bacteria, unknown, Legionella T, P aModified from reference 8. bAbbreviations: P, plasmid; T, transposon; GC, gene cassette; C, chromosomal DNA. TABLE 2 Examples of resistance to antimicrobials by decreased intracellular drug accumulationa,b Resistance mechanism Resistance gene(s) Gene product Resistance phenotype Bacteria involved Location of the resistance gene Efflux via specific exporters mef(A) Efflux system of the major facilitator superfamily 14-, 15-Membered macrolides Streptococcus, other Gram+ and Gram–bacteria T, P, C tet(A-E, G, H, I, J, K, L, Z), tetA(P), tet(30) 12-, 14-TMS efflux system of the MFS Tetracyclines Various Gram+ and Gram– bacteria P, T, C pp-flo, floR, floRV 12-TMS efflux system of the MFS Chloramphenicol, florfenicol Photobacterium, Vibrio, Salmonella, Escherichia, Klebsiella, Pasteurella T, P, C cmlA 12-TMS efflux system of the MFS Chloramphenicol Pseudomonas, Salmonella, E. coli T, P, C fexA 14-TMS efflux system of the MFS Chloramphenicol, florfenicol Staphylococcus T, P, C fexB 14-TMS efflux system of the MFS Chloramphenicol, florfenicol Enterococcus P vga(A), vga(C), vga(E) Efflux system of the ABC transporter family Lincosamides, pleuromutilins, streptogramin A compounds Staphylococcus P, T, C msr(A) Efflux system of the ABC transporter family Macrolides, streptogramin B compounds Staphylococcus P optrA Efflux system of the ABC transporter family Oxazolidinones, phenicols Enterococcus, Staphylococcus P, C Efflux via multidrug transporters emrE 4-TMS multidrug efflux protein Tetracyclines, nucleic acid binding compounds E. coli C blt, norA 12-TMS multidrug efflux protein of the MFS Chloramphenicol, fluoroquinolones, nucleic acid binding compounds Bacillus, Staphylococcus C mexB-mexA-oprM, acrA-acrB-tolC Multidrug efflux in combination with specific OMPs Chloramphenicol, β-lactams, macrolides, fluoroquinolones, tetracyclines, etc. Pseudomonas, E. coli, Salmonella C aModified from reference 8. bP, plasmid; T, transposon; GC = gene cassette; C, chromosomal DNA; MFS, major facilitator superfamily; TMS, transmembrane segments. 4 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 Group 1 β-lactamases (molecular class C), for example, AmpC, CMY, ACT, DHA, FOX, and MIR, are cephalosporinases that are more active on cephalosporins than benzylpenicillin and are usually not inhibited by clavulanic acid. They are widespread among Gram-negative bacteria. The ampC genes are commonly located on the chromosome but may also be found on plasmids. Some of these ampC genes are expressed inducibly; others are expressed constitutively (18). Point mutations in the promotor or attenuator region may increase β-lactamase production. Subgroup 1e enzymes are group 1 variants with greater activity against ceftazidime and other oxyimino-β-lactams as a result of amino acid substitutions, insertions, or deletions and include GC1 in Enterobacter cloacae and plasmidmediated CMY-10, CMY-19, and CMY-37. They have been named extended-spectrum AmpC β-lactamases (18). Group 2 β-lactamases (molecular classes A and D) represent diverse enzymes, most of which are sensitive to inhibition by clavulanic acid. Subgroup 2a (molecular class A) includes enzymes such as BlaZ from staphylococci, which can inactivate only penicillins. Subgroup 2b (molecular class A) comprises broad-spectrum βlactamases, such as TEM-1, TEM-2, SHV-1, and ROB- 1, which can hydrolize penicillins and broad-spectrum cephalosporins. Subgroup 2be represents extendedspectrum β-lactamases (ESBLs; e.g., variants of TEM and SHV families and CTX-M type enzymes), which can also inactivate oxyimino cephalosporins and monobactams. Subgroups 2a, 2b, and 2be enzymes are sensitive to inhibition by clavulanic acid. Due to their wide spectrum of activity, ESBLs are a serious cause of concern (19). Most currently known ESBLs belong to the TEM, SHV, CTX-M, or OXA families of β-lactamases. Less common ESBLs include BEL-1, BES-1, SFO-1, TLA-1, TLA-2, and members of the PER and VEB enzyme families. Details about the structure and function of these ESBLs, their location on mobile elements, their dissemination among bacteria of different species and TABLE 4 Examples of resistance to antimicrobials by target modificationa Resistance mechanism Resistance gene(s) Gene product Resistance phenotype Bacteria involved Location of the resistance geneb Methylation of the target site erm rRNA methylase MLSB Various Gram+ bacteria, Escherichia, Bacteroides P, T, C Protection of the target site tet(M, O, P, Q, S, T) Ribosome protective proteins Tetracyclines Various Gram+ and Gram– bacteria T, P, C Replacement of a sensitive target by an alternative drug-resistant target sul1, sul2, sul3 Sulfonamide-resistant dihydropteroate synthase Sulfonamides Various Gram– bacteria P, I dfrA, dfrB, dfrD, dfrG, dfrK Trimethoprim-resistant dihydrofolate reductase Trimethoprim Various Gram+ and Gram– bacteria P, GC, T, C mecA, mecC Penicillin-binding proteins with altered substrate specificity Penicillins, cephalosporins, carbapenems, monobactams Staphylococcus C vanA-E Alternative D-Ala-D-Lac or D-Ala-D-Ser peptidoglycan precursors Glycopeptides Enterococcus, Staphylococcus T, P, C Alteration of the LPS mcr-1, mcr-2, mcr-3, mcr-4, mcr-5 Phosphoethanolamine transferase Colistin Enterobacteriaceae T, P, C Mutational modification of the target site Mutation in the gene coding for ribosomal protein S12 Streptomycin Several Gram+ and Gram– bacteria C Mutation in the 16S rRNA Streptomycin Mycobacterium C Mutation in the 23S rRNA Macrolides Mycobacterium C Mutation in the 16S rRNA Tetracyclines Propionibacterium C Mutations in the genes for DNA gyrase and topoisomerase Fluoroquinolones Various Gram+ and Gram– bacteria C Mutation in the gene for the ribosomal protein L3 Tiamulin E. coli C Mutational modification of regulatory elements Mutations in the marRAB soxR or acrR genes Fluoroquinolones E. coli C aModified from reference 8. bP, plasmid; T, transposon; GC, gene cassette; C, chromosomal DNA; I, integron. ASMscience.org/MicrobiolSpectrum 5 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 genera, and information on ESBL detection methods can be found in several reviews (19–21). Moreover, a continuosly updated database which lists the known ESBLs and inhibitor-resistant β-lactamases including TEM, SHV, OXA, CTX-M, CMY, IMP, and VIM types can be found at https://www.ncbi.nlm.nih.gov/pathogens/beta -lactamase-data-resources/. The enzymes of subgroup 2br (molecular class A) are also broad-spectrum βlactamases, such as TEM-30, TEM-31, and SHV-10, which however, are not inhibited by clavulanic acid. Analysis of the β-lactamases of subgroups 2b, 2be, and 2br—in particular, those of the TEM and SHV types— revealed the presence of mutations which either extended the substrate spectrum or affected the enzyme stability (10, 14, 19). TEM enzymes that exhibit an extended spectrum and increased resistance to clavulanic acid inhibition are organized in subgroup 2ber and are called complex mutant TEM (CMT); these include TEM-50 (CMT-1) and TEM-158 (CMT-9) (18). Subgroup 2c (molecular class A) includes inhibitor-sensitive carbenicillinases such as CARB-3, PSE-1, and RTG, whereas the extended-spectrum carbenicillinase RTG-4 in subgroup 2ce shows enhanced activity against cefepime and cefpirome. The β-lactamases of subgroup 2d (molecular class D) (e.g., OXA-type enzymes) exhibit variable sensitivity to inhibitors and can hydrolyze oxacillin or cloxacillin. The extended spectrum of the enzymes in subgroup 2de (e.g., OXA-11 and OXA-15) is defined by their ability to hydrolize oxacillin or cloxacillin as well as oxyimino β-lactams with a preference for ceftazidime. The subgroup 2df assembles OXA enzymes which are not inhibited by clavulanic acid and show carbapenem-hydrolyzing activities. The genes have been detected on plasmids and in the chromosome of Gram-negative bacteria (18). The β-lactamases of subgroups 2e and 2f represent cephalosporinases (e.g., CepA) or serine-carbapenemases (e.g., SME-1, IMI-1, KPC-2), which are sometimes inhibited by clavulanic acid (18). While the β-lactamases of groups 1 and 2 have a serine residue in the catalytic center, the β-lactamases of group 3 (molecular class B) hydrolyze β-lactams by divalent cations (Zn2+ ) and are referred to as metallo-βlactamases (e.g., IMP-type, VIM-type, and NDM-type enzymes). Subgroup 3a consists of plasmid-encoded metallo-β-lactamases, which require two bound zinc ions for their activity. These enzymes can inactivate all β-lactams except monobactams and are insensitive to clavulanic acid but are inhibited by metal ion chelators such as EDTA. The metallo-β-lactamases in subgroup 3b preferentially hydrolyze carbapenems, especially if only one zinc-binding side is occupied (18). The location of many of the β-lactamase genes (bla) on either plasmids, transposons, or gene cassettes favors their dissemination (20–22). Altered PBPs are often associated with resistance due to decreased binding of β-lactam antibiotics (11). PBPs are transpeptidases which play an important role in cell wall synthesis. They are present in most cell wallcontaining bacteria, but they vary from species to species in number, size, amount, and affinity to β-lactam antibiotics (11). The acquisition of a novel PBP, such as the mecA-encoded PBP2a, which replaces the original β-lactam-sensitive PBP, is the cause of methicillin resistance in Staphylococcus aureus, Staphylococcus pseudintermedius, and coagulase-negative staphylococci (23, 24). Methicillin-resistant staphylococci are resistant to virtually all β-lactam antibiotics except cefpirome. The mecA gene, which codes for the alternative PBP2a, is part of a genetic element, designated Staphylococcus cassette chromosome mec (SCCmec) (25). So far, 11 SCCmec types have been described (26). In 2011, a new mecA homologue, mecC (formerly called mecLGA251), which is part of a distinct SCCmec type (SCCmec XI), was identified in methicillin-resistant S. aureus (MRSA) (27, 28). The majority of known MRSA-carrying mecC belong to clonal complex (CC) 130, but other CCs (CC425, CC1943, CC599, CC49) have also been found to harbor mecC. In addition, mecC is not restricted to S. aureus but has been found in several staphylococcal species including Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus stepanovicii, and Staphylococcus xylosus (29–31). In addition to PBP2a, the Fem proteins are involved in expression of methicillin resistance. The FemAB proteins contribute to the formation of the pentaglycine crossbridge, which is a unique staphylococcal cell wall component (32). Inactivation of femAB has been found to completely restore susceptibility to β-lactams and other antimicrobial agents in MRSA strains (33). PBPs with low affinity for β-lactams have also been detected in streptococci and enterococci (11). Homologous recombinations in the genes coding for PBPs 1a, 2a, and 2b are assumed to result in mosaic proteins with decreased affinity to β-lactams in Streptococcus pneumoniae and Neisseria spp.. PBPs, which have a low affinity to β-lactams, have been reported to be overproduced in resistant strains of Enterococcus faecium and Enterococcus faecalis. It is noteworthy that alterations in PBPs do not necessarily result in complete resistance to all β-lactams but can also lead to elevated MICs of selected β-lactam antibiotics (11). 6 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 Reduced uptake of β-lactams is due to decreased outer membrane permeability and/or the lack of certain outer membrane proteins, which serve as entries for β-lactams to the bacterial cell, and has been described in various Enterobacteriaceae, Pseudomonas aeruginosa, and other bacteria (34–36). In Escherichia coli and Klebsiella pneumoniae, β-lactam resistance can be based on the decreased expression or the structural alteration of the porins OmpF (37) and OmpK36 (38), by which β-lactams cross the outer membrane. In P. aeruginosa, resistance to imipenem has been shown to be based on the loss of the porin OprD (39). Several multidrug transporters such as the MexAB/ OprM and the MexCD/OprJ systems in P. aeruginosa, the SmeAB/SmeC system in Stenotrophomonas maltophilia, and the AcrAB/TolC system in Salmonella enterica and E. coli (40, 41) are known to mediate the export of β-lactam antibiotics. Resistance to Tetracyclines Among this family of antimicrobial agents, oxytetracycline, chlortetracycline, and tetracycline have been used in veterinary medicine since the 1950s. More recently, doxycycline has been approved for dogs, cats, and pigs. Up to now, minocycline and glycylcyclines have not been licensed for use in animals. Based on aggregated data from a survey on sales of veterinary antimicrobial agents in 25 European countries, the sales of tetracyclines accounted for 37% of the total sales of veterinary antimicrobial agents in 2011 (42). Thus, it is not surprising that tetracycline resistance has become widespread among bacteria of veterinary importance, including in aquaculture (43–45). Tetracycline resistance is usually due to the acquisition of new genes (46). There are 33 efflux genes, which code for energy-dependent efflux of tetracyclines, 12 ribosomal protection genes, which code for a protein that protects bacterial ribosomes, 13 genes which code for enzymes that modify and inactivate the tetracycline molecule, and 1 gene [tet(U)] which specifies tetracycline resistance by an unknown mechanism. The products of different tet genes share ≤79% amino acid identity (47). An updated database listing the currently known tet genes and their occurrence in various bacteria is available at http://faculty.washington.edu/marilynr/. This website is updated twice each year. New tet gene names are approved by Dr. Stuart B. Levy, Tufts University, Boston. Antibiotic resistance genes are not randomly distributed among bacteria. This has been well documented with the distribution of tet genes (47–50). The energy-dependent efflux of tetracyclines is mediated by membrane-associated proteins which exchange a proton for a tetracycline-cation complex (46, 51). These tetracycline resistance efflux proteins are part of the major facilitator superfamily and share amino acid and protein structure similarities with other efflux proteins (12). Of the 33 efflux genes, 14 [tet(A) to tet(E), tet(G), tet(H), tet(J), tet(Y), tet(30), tet(31), tet(35), tet(41), tet(57)] are found only in Gram-negative genera. The remaining are found in both Gram-negative and Grampositive genera or in bacteria of unknown source. The tet(B) gene has been identified in 33 Gram-negative genera, while the tet(L) gene has been identified in a total of 46 genera, including 24 Gram-negative and 22 Grampositive genera (http://faculty.washington.edu/marilynr/). The Tn10-associated tet(B) gene codes for a unique efflux protein, which confers resistance to both tetracycline and minocycline but not to the new glycylcyclines (46). All the 32 other efflux proteins confer resistance to tetracycline but not to minocycline or glycylcyclines. Laboratoryderived mutations in the tet(A), tet(B), tet(K), and tet(L) genes have led to glycylcycline resistance, suggesting that bacterial resistance to tigecycline may develop over time and with clinical use (46, 52, 53). The tet efflux genes code for an approximately 46-kDa membrane-bound efflux protein. The tetracycline efflux proteins present in Gramnegative bacteria commonly exhibit 12 transmembrane segments (TMSs), and upstream of the structural gene and read in the opposite direction is a specific tet repressor gene. Induction of the structural gene is based on the binding of a tetracycline-Mg2+ complex to the tet repressor protein which, in the absence of tetracycline, blocks transcription of the tet structural gene (54). The tet(A) (n = 25), tet(B) (n = 33), tet(C) (n = 16), tet(D) (n = 22), tet(G) (n = 16), tet(H) (n = 12), and tet(L) (n = 22) genes are most widespread among Gram-negative bacteria of human and veterinary origin, while tet(D) and tet(E) are often associated with aquaculture environments and fish (44). Their location on either transposons, such as Tn1721 [tet(A)] (55), Tn10 [tet(B)] (56, 57), or Tn5706 [tet(H)] (58), and plasmids facilitates their spread within the Gram-negative gene pool. The Grampositive tet(K) and tet(L) efflux genes are not regulated by repressors and confer resistance to tetracyclines, but not to minocycline. They code for proteins with 14 TMSs and are regulated by translational attenuation, which requires the presence of tetracyclines as inducers for the translation of the tet gene transcripts (59). These genes are generally found on small transmissible plasmids, which on occasion become integrated into the chromosome and occasionally may undergo interplasmidic recombination with other resistance plasmids (54, 59–61). ASMscience.org/MicrobiolSpectrum 7 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 The ribosomal protection genes code for cytoplasmic proteins which protect the ribosomes from the action of tetracycline both in vitro and in vivo and confer resistance to tetracycline, doxycycline, and minocycline (46, 62). These proteins have sequence similarity to the ribosomal elongation factors EF-G and EF-Tu and are grouped in the translation factor superfamily of GTPases (63). Their interaction with the ribosome causes an allosteric disruption of the primary tetracycline binding site(s), which then leads to the release of the tetracycline from the ribosome. This allows the ribosome to return to its functional normal posttranslocational conformational state, which was altered by the binding of tetracycline. A detailed review of the various experiments conducted to elucidate the mode of action of these proteins can be found in Connell et al. (63). The ribosomal protection genes are of Gram-positive origin and are found extensively among Gram-positive cocci. However, they have also been found in a number of Gramnegative genera. The first gene of this group, the tet(M) gene, has the widest host range of all tet genes, with 79 genera, of which 41 are Gram-positive and 38 are Gramnegative (http://faculty.washington.edu/marilynr/). This gene is located on conjugative transposons or integrative and conjugative elements, such as Tn916 (64, 65). The other commonly found genes of both human and veterinary origin are tet(O) (20 Gram-positive, 18 Gramnegative genera), tet(Q) (eight Gram-positive, 11 Gramnegative genera), and tet(W) (11 Gram-positive, 22 Gram-negative genera). Recent work suggests that mutations within the tet(M) gene may confer increased resistance to tigecycline and thus may over time increase resistance to tigecycline in nature (52). Enzymatic inactivation of tetracycline is mediated by 13 genes found in Gram-negative bacteria, nine of which [tet(47) to tet(55)] have recently been identified by soil functional metagenomic studies (66). The first inactivating gene described was the tet(X) gene (67) (which encodes an NADP-requiring oxidoreductase), which modifies and inactivates the tetracycline molecule in the presence of oxygen but was originally found only in a strict anaerobe, Bacteroides, where oxygen is excluded. The tet(X) gene has now been identified in 13 Gramnegative genera (http://faculty.washington.edu/marilynr/). This gene confers weak intrinsic resistance to tigecycline. The tigecycline activity can be improved by at least four different amino acid substitutions in the Tet(X) protein to obtain clinically relevant tigecycline resistance levels without loss of activity to other tetracyclines and was thought to be alarming for the future of tigecycline therapy (52). The gene tet(37) has been identified from the oral cavity of humans but is unrelated to the tet(X) or other genes in this class, and the function of the corresponding enzyme depends on oxygen (68). No bacterial host has been identified which carries tet(37). A third gene, tet(34), with similarities to the xanthine-guanine phosphoribosyl transferase gene of Vibrio cholerae, has also been identified in four Gram-negative genera (69). The recently described gene tet(56) has been identified in one Gram-negative genus (66), while the genes tet(47) to tet(55) have been isolated from grasslands and agricultural soils by functional genomics, where the genes were cloned into E. coli and shown to inactiviate tetracycline (66). With this recent study, the number of new genes coding for inactivating enzymes from the environment has greatly increased and may also be found in the future in bacteria of veterinary importance. The tet(U) gene has been identified in the three Grampositive genera: Enterococcus, Staphylococcus, and Streptococcus. However, it is still not clear if the gene confers tetracycline resistance in any of the bacteria it has been identified in. A mutation in the 16S rRNA consisting of a single base exchange (1058G → 1058C) has been identified in tetracycline-resistant Propionibacterium acnes (70). Position 1058 is located in a region which plays an important role in the termination of peptide chain elongation as well as in the accuracy of translation. Mutations which alter the permeability of the outer membrane porins and/or LPSs in the outer membrane can also affect resistance to tetracycline. A permeability barrier due to the reduced production of the OmpF porin, by which tetracyclines cross the outer membrane, has been described in E. coli. Mutations in the marRAB operon, which also regulates OmpF expression, may play a role in this type of tetracycline resistance (13). Different types of multidrug transporters mediating resistance to tetracycline in addition to resistance to a number of structurally unrelated compounds have been described, for instance, in E. coli (EmrE), S. enterica (AcrAB/TolC), and P. aeruginosa (MexAB/OprM, MexCD/OprJ) (12, 40, 41). Resistance to Macrolides, Lincosamides, and Streptogramins (MLS) Several macrolide antibiotics, such as erythromycin, spiramycin, tylosin and tilmicosin, tulathromycin, gamithromycin, and tildipirosin, as well as lincosamide antibiotics, such as clindamycin, lincomycin, and pirlimycin, are approved for use in animals. Since the ban of growth promotors in the European Union, no streptogramin antibiotics are licensed for veterinary use in the 8 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 European Union, but they may be used in other countries. The 16-membered macrolide antibiotics tylosin and spiramycin were previously used as feed additives for animal growth promotion but remain as therapeutics for veterinary use for the control of bacterial dysentery, respiratory disease, and mastitis. Erythromycin, the first macrolide, was introduced into clinical use over 60 years ago and has good activity against Gram-positive cocci and other Gram-positive bacteria and activity against some Gram-negative bacteria such as Campylobacter spp.. Enterobacteriaceae and Pseudomonas spp. have been considered to be innately nonsusceptible to erythromycin due to multidrug transporters which have 14-membered macrolides as substrates (71). A number of Gram-negative aerobic, facultatively aerobic, and anaerobic genera carry a variety of acquired macrolide-lincosamide and/or streptogramin resistance genes (http://faculty.washington.edu /marilynr/). The data over the past 20 years clearly show that both Gram-positive and Gram-negative bacteria may become MLS resistant by acquisition of new genes normally associated with mobile elements. Acquired resistance mechanisms include specific efflux pumps, rRNA methylases that reduce binding of the antibiotic to the 50S subunit of the ribosome, or a variety of genes that inactivate the antibiotics (72–77). MLS antibiotics, though chemically distinct, are usually considered together because they share overlapping binding sites on the 50S ribosomal subunit, and a number of resistance genes confer resistance to more than one class of these antibiotics (72–74). Target site modification occurs by rRNA methylases, which are encoded by erm genes. The erm genes were the first acquired genes that were identified to confer resistance to macrolides, lincosamides, and streptogramin B (MLSB) antibiotics (74, 75). These genes are found in Gram-positive, Gram-negative, aerobic, and anaerobic genera. Currently, 43 rRNA methylases have been characterized. Each of these enzymes adds one or two methyl groups to a single adenine (A2058 in E. coli) in the 23S rRNA moiety which prevents binding of the antibiotic to the target site and thus confers MLSB resistance to the host bacterium (http://faculty.washington.edu/marilynr; 74–76). The erm genes may be expressed all the time (constitutively) or inducibly via translational attenuation (77). This means that the gene is turned on in the presence of low doses of specific antibiotics (74–77); the type of expression depends on a regulatory region upstream of the erm gene and on which the antibiotic is able to cause induction (75, 77). In staphylococci, erythromycin and other 14- and 15-membered macrolides are able to induce erm gene expression, whereas 16membered macrolides, lincosamides, and streptogramin B antibiotics are considered noninducers (77). Laboratory selection of S. aureus produced mutants that had structural alterations in the translational attenuator region due to deletions, tandem duplications, point mutations, and the insertion of IS256 (78–80). Similar mutations have also been detected in naturally occurring strains carrying the erm genes (81). Efflux genes include 21 ATP transporters and 5 major facilitator superfamily transporters. These genes confer a variety of resistance patterns including resistance to carbomycin, erythromycin, lincomycin, oleandomycin, spiramycin, tylosin, streptogramin A, streptogramin B, and pleuromutilins, alone or in varying combinations (http://faculty.washington.edu/marilynr; 71–74, 82). The vga(A) and vga(B) genes share 59% identical amino acids and have G+C contents of 29 to 36%. The msr(A) gene confers inducible resistance to 14- and 15-membered macrolides and streptogramin B (MSB) and is found in staphylococci. The hydrophilic protein made from the msr(A) gene contains two ATP-binding motifs characteristic of the ABC transporters (74, 83, 84). The msr(A) gene confers lower levels of erythromycin resistance than the rRNA methylases (85). There are two groups of major facilitator superfamily transporters: one group encompasses lmr(A) and lmr(B), which code for lincomycin-specific efflux pumps, and the second group includes mef(A), mef(B) and mef(C) genes, which code for specific efflux pumps for 14- and 15-membered macrolides. The mef(A) gene was first described in the 1990s from Streptococcus spp. (86), but more recently it has been shown to be present in old isolates of pathogenic Neisseria spp. (87) and is now found in 30 different genera. It was the most common acquired macrolide resistance gene in a collection of 176 randomly collected commensal Gram-negative bacteria (88). Downstream of the mef(A) gene is a gene for an ABC transporter that has now been shown to independently confer macrolide resistance and has been named msr(D) (89). In contrast, the mef(B) gene is found in Escherichia spp. and the mef (C) gene in Photobacterium spp. and Vibrio spp.. The 28 inactivating enzymes identified so far encode three esterases, two lyases, 16 transferases, and seven phosphorylases (http://faculty.washington.edu/marilynr; 74, 82). The esterases [Ere(A), Ere(B), and Ere(D)] hydrolyze the lactone ring of the macrolides. The esterases have been found in both Gram-negative and Grampositive bacteria, and their genes are often associated with plasmids, though the ere(A) gene has been associated with both class 1 and class 2 integrons (90). The lyase gene, vgb(A), has been identified in the genera Enterococcus and Staphylococcus, while the vgb(B) gene ASMscience.org/MicrobiolSpectrum 9 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 has been identified in Staphylococcus. These enzymes inactivate quinupristin by opening the lactone ring (91). The newest inactivating enzymes have been identified as transferases which confer resistance by adding an acetyl group to streptogramin A, thereby inactivating the antibiotic. Sixteen genes have been found in both Grampositive and/or Gram-negative genera as described below (http://faculty.washington.edu/marilynr; 74, 82). The nine lincosamide nucleotidyltransferases [lnu genes] confer resistance to lincosamides but not to macrolides by modification and inactivation of the antibiotic. The lnu(A) gene has been identified in five Gram-positive genera, and the lnu(B) gene in four Gram-positive genera. The gene lnu(C) was identified in Streptococcus and Haemophilus. The gene lnu(E) was found in Streptococcus and Enterocococcus, while lnu(F) was identified in Aeromonas, Comamonas, Desulfobacterium, Escherichia, Leclercia, Morganella, Proteus, and Salmonella. The gene lnu(D) is associated with Streptococcus, lnu(G) with Enterococcus, and lnu(H) with Riemerella. Furthermore, lnu(P) has been identified in Clostridium. Seven virginiamycin O-acetyltransferases (vat genes) have been identified, six of which are associated with mobile elements in Enterococcus, Lactobacillus, Staphylococcus, and/or Bacteroides. Each gene was found in only one or two of these genera. In contrast, vat(F) is chromosomally encoded in Yersinia enterocolitica. There are seven enzymes, encoded by mph genes, which confer resistance by phosphorylation of erythromycin. The mph(A) gene is unqiue because it confers resistance to azithromycin, while mph(B) and mph(C) confer resistance to spiramycin (92). Six phosphorylases, encoded by the genes mph(A), mph(B), mph(D), mph(E), mph(F), and mph(G), have been found exclusively in Gram-negative species. To date, the gene mph(A) is found in 11 genera, while mph(D) and mph(E) are each found in six genera. The mph(B) gene is present in four genera, while mph(F) is found in Pseudomonas. The mph(G) gene has been found in the fish pathogens Photobacterium and Vibrio spp. The mph(C) gene, which was originally characterized in Staphylococcus spp., has now been identified in a clinical S. maltophilia isolate (http://faculty.washington.edu/marilynr/). Usually, mutational changes that affect the 23S RNA, ribosomal proteins, and/or innate efflux pumps may lead to moderate changes in susceptibility (76, 82). Various mutations have been identified in the 23S rRNA (93). Originally, mutations at either the A2058 or A2059 position (E. coli numbering) were found in pathogens that had one or two copies of the 23S rRNA, such as Mycobacterium or Helicobacter (94). Resistance to tylosin, erythromycin, and clindamycin in Brachyspira hyodysenteriae was also associated with an A → T substitution at the nucleotide position homologous with position 2058 of the E. coli 23S rRNA gene (95). Variations at positions 2058 and 2059 in the 23S rRNA have also been described in erythromycin-resistant Streptococcus pyogenes, S. pneumoniae, Campylobacter coli, Campylobacter jejuni, and Haemophilus influenzae (96, 97). An A → G substitution at position 2075 of the 23S rRNA was detected in C. coli from poultry and pigs which exhibited high-level erythromycin resistance (97). Mutations in ribosomal proteins L4 and/or L22 have been identified which confer elevated MICs of the newer agent telithromycin and/or of other members of the MLS group. Clinical Gram-positive bacteria have been found with the same mutations as mutants created in laboratories. Missense mutations, deletions, and/or insertions may alter the expression of innate pumps which then may alter resistance to the MLS antibiotics. A detailed discussion can be found in reference 71. Resistance to Aminoglycosides and Aminocyclitols Various aminoglycoside antibiotics, including gentamicin, kanamycin, amikacin, neomycin, (dihydro)streptomycin, paromomycin and framycetin, are licensed for use in both human and veterinary medicine. Among the aminocyclitol antibiotics, spectinomycin is approved for use in humans and animals, whereas apramycin is used exclusively in veterinary medicine. The main mechanism of resistance to aminoglycosides and aminocyclitols is enzymatic inactivation (98–101). In addition, reduction of the intracellular concentrations of aminoglycosides and modification of the molecular target can also result in resistance to aminoglycosides (102). Decreased intracellular concentration can result from either reduced drug uptake or from active efflux mechanisms. Chromosomal mutations conferring high-level resistance to streptomycin have also been described (13) and are the main resistance mechanism in mycobacteria. Enzymatic inactivation of aminoglycosides and aminocyclitols is conferred by any of the three types of modifying enzymes: N-acetyltransferases (AACs), O-nucleotidyltransferases (also referred to as Oadenyltransferases [ANTs]), or O-phosphotransferases (APHs) (98–101). Acetyl-coenzyme A serves as a donor of acetyl groups in acetylation reactions at amino groups, while ATP is used for the adenylation and phosphorylation reactions at hydroxyl groups. For each of these three classes of aminoglycoside-modifying enzymes, numerous members are known which differ more or less 10 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 extensively in their structure. Most modifying enzymes exhibit a narrow substrate spectrum. Several reviews have listed the known enzymes involved in modification of aminoglycosides/aminocyclitols and their molecular relationships (49, 98–101). However, new genes for aminoglycoside/aminocyclitol-inactivating enzymes or variants of already known ones are constantly being reported. Unfortunately, a continuously updated database for the currently known aminoglycoside/aminocyclitolinactivating enzymes is not available. Another problem is the lack of an unambiguous nomenclature. There are at least two alternatively used designations for genes coding for the same modifying enzyme: one designation, for example, aph(3″)-Ib, refers to the type of modification (aph) and the position where the modification is introduced (3″) and lists the subtype of the gene (Ib); the other designation, for example, strA, is easier to handle, refers only to the corresponding resistance phenotype (str for streptomycin resistance), and indicates the subtype (A). So far, four classes of AACs are known which acetylate the amino groups at positions 1, 3, 2′, and 6′ (98–102). To date, at least 80 AACs have been identified, most of which vary in their substrate spectra. The vast majority of the AAC enzymes were identified in Gram-negative bacteria. Combined resistance to apramycin and gentamicin is due to the enzyme AAC(3)-IV; the corresponding gene emerged after the introduction of apramycin into veterinary use. It was first detected in E. coli and Salmonella from animals (103) and was found later in E. coli from humans as well (104–106). A gene for a bifunctional enzyme, which displays acetyltransferase AAC(6′) and phosphotransferase APH(2″) activities, is usually found on Tn4001-like transposons, which are widely spread among staphylococci, streptococci, and enterococci (107–110). To date, five classes of ANTs are categorized depending on the position of adenylation (6, 9, 4′, 2″, and 3″) on the AG molecule (98–101). The ANT(2″) and ANT(3″) enzymes are more frequent among Gram-negative bacteria, whereas the ANT(4′), ANT(6), and ANT(9) enzymes are usually found in Gram-positive bacteria (100). The different ANT enzymes also vary considerably in their substrate spectra. Among the seven phosphotransferases [APH(2″), APH(3′), APH(3″), APH(4), APH(6), APH(7″), and APH(9)] which modify the aminoglycosides at positions 2″, 3′, 3″, 4, 6, 7″, and 9 (100), numerous variants have been identified which confer distinctly different resistance phenotypes. Most aac, ant, and aph genes are located on mobile genetic elements, such as plasmids, transposons, and gene cassettes (98–102, 111, 112). The gene apmA codes for an acetyltransferase, which confers resistance to apramycin and decreased susceptibility to gentamicin. It has been detected on plasmids of variable sizes in MRSA ST398 (113–115). In staphylococci, spectinomycin resistance is mediated by the adenyltransferase genes spc, spd, and spw (116–120). Multidrug efflux systems, such as MexXY in P. aeruginosa and AmrAB in Burkholderia pseudomallei (40), or the multidrug transporter AcrD in E. coli (121) can export aminoglycosides. The transporter MdfA from E. coli (122) has also been reported to mediate the efflux of the aminoglycosides kanamycin, neomycin, and hygromycin A. Decreased uptake of aminoglycosides may be based on a mutation in lipopolysaccharide (LPS) phosphates or on a change in the charge of the LPS in E. coli and P. aeruginosa, respectively (123). Since the entry of aminoglycosides across the cytoplasmic membrane is mainly based on the electron transport system, anaerobic bacteria and facultative anaerobic bacteria exhibit relatively high insensitivity to aminoglycosides (13). Methylation of the ribosomal target (16S rRNA) is responsible for high-level aminoglycoside resistance. It is also an emerging mechanism of great concern in clinically relevant Gram-negative bacteria. The first plasmidmediated gene identified was the 16S rRNA methylase armA (124). To date, nine additional genes that encode methylases have been reported: rmtA, rmtB, rmtC, rmtD, rtmD2, rmtE, rmtF, rmtG, and npmA (125). The rmt genes confer resistance to gentamicin and amikacin, whereas npmA confers resistance to gentamicin, neomycin, amikacin, and apramycin, but not to streptomycin (126). Mutations in the gene rpsL for the ribosomal protein S12 have been shown to result in high-level streptomycin resistance (127). Single base-pair substitutions at different positions in the gene rrs, which encodes 16S rRNA in mycobacteria, have been described to be involved in either streptomycin resistance (128) or resistance to amikacin, kanamycin, gentamicin, tobramycin, and neomycin, but not to streptomycin (129). In Mycobacterium tuberculosis, mutations in the gene rpsL, which encodes the ribosomal protein S12, can cause high-level streptomycin resistance. Overexpression of the acetyltransferase-encoding gene, eis, has mainly been associated with resistance to kanamycin. Mutations in the gidB gene, which encodes a 7-methylguanosine methyltransferase, are also associated with resistance to aminoglycosides in mycobacteria. It has been suggested that loss of function of this gene confers resistance (129). ASMscience.org/MicrobiolSpectrum 11 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 Resistance to Sulfonamides and Trimethoprim Various sulfonamides, trimethoprim, and combinations of sulfonamides and trimethoprim are licensed for use in humans and animals. There are no restrictions on the use of any of these compounds in food animals. Sulfonamides and trimethoprim are competitive inhibitors of different enzymatic steps in folate metabolism. In this regard, sulfonamides represent structural analogs of p-aminobenzoic acid and inhibit the enzyme dihydropteroic acid synthase (DHPS), whereas trimethoprim inhibits the enzyme dihydrofolate reductase (DHFR). Various mechanisms of intrinsic and acquired resistance to sulfonamides and trimethoprim have been described in bacteria (130–134). Permeability barriers and efflux pumps play a relevant role by either preventing the influx or promoting the efflux of both compounds. Intrinsic resistance to both compounds in P. aeruginosa was initially thought to be based on outer membrane impermeability. However, the multidrug exporter system MexAB/OprM was found to be mainly responsible for resistance to sulfonamides and trimethoprim in P. aeruginosa (135). For other bacteria, such as K. pneumoniae and Serratia marcescens, impaired membrane permeability is still considered to play a role in sulfonamide and trimethoprim resistance (131, 132). Naturally insensitive DHFR enzymes and folate auxotrophy play an important role in intrinsic resistance to sulfonamides and trimethoprim. DHFR enzymes which exhibit low affinity for trimethoprim and thus render their hosts intrinsically resistant to trimethoprim are known to occur in several bacterial genera including Clostridium, Neisseria, Brucella, Bacteroides, and Moraxella (13). Bacteria such as enterococci and lactobacilli which can utilize exogenous folates also show intrinsic resistance to trimethoprim and sulfonamides. Mutational or recombinational changes in the target enzymes have been observed in a wide variety of bacteria. Mutations in the chromosomal dhps gene that lead to sulfonamide resistance by single amino acid substitutions can be generated under in vitro conditions but also occur in vivo. Such mutations have been identified in E. coli, S. aureus, Staphylococcus haemolyticus, C. jejuni, and Helicobacter pylori (131). In S. pneumoniae, two amino acid duplications which change the tertiary structure of the DHPS have been found to be responsible for sulfonamide resistance (136). Recombinational events between the naturally occurring gene coding for a susceptible DHPS and that of a horizontally acquired resistant DHPS are believed to account for sulfonamide resistance in Neisseria meningitidis (131). Trimethoprim resistance has also been shown to be due to a single amino acid substitution in the DHFR protein in S. aureus (137) and S. pneumoniae (138). Mutations in the promoter region of chromosomal dhfr genes have been described to occur in E. coli and resulted in overexpression of the trimethoprim-susceptible DHFR (131). Mutations in both the promoter region and the dhfr gene have been identified in trimethoprim-resistant H. influenzae (139). The replacement of sensitive enzymes by resistant enzymes usually causes high-level resistance (130–134). To date, three types of resistant DHPS enzymes encoded by the genes sul1, sul2, and sul3 have been described to occur in Gram-negative bacteria (140–143). The gene sul1 is part of class 1 integrons and thus is often associated with other resistance genes. As part of transposons, such as Tn21, and conjugative plasmids, it is spread into various Gram-negative species and genera (140, 142). The sul2 gene often occurs together with the Tn5393associated streptomycin resistance genes strA-strB on conjugative or nonconjugative plasmids (141, 142). The gene sul3 was originally found on a conjugative plasmid from porcine E. coli, where it was flanked by copies of the insertion sequence IS15Δ/26 (143). Meanwhile, it has also been identified in E. coli from humans and animals other than pigs, as well as in S. enterica from animal and food sources (134–146). More than 40 DHFR (dfr, formerly also referred to as dhfr) genes have been identified. The genes occurring in Gram-negative bacteria are subdivided on the basis of their structure into two major groups, dfrA and dfrB (147). The 33 dfrA genes code for DHFR enzymes of 152 to 189 amino acids (aa), whereas the eight dfrBencoded DHFR enzymes consist of only 78 aa. The dfrA genes have been detected more frequently than the dfrB genes. Additionally, there are dfr gene groups in Grampositive bacteria that currently consist of only one gene each. The gene dfrG codes for an enzyme of 165 aa and has been detected in the chromosome of S. aureus (148). In S. haemolyticus and Listeria monocytogenes, the gene dfrD, which codes for an enzyme of 162 aa, has been identified on plasmids (149, 150). The gene for the 163aa DHFR DfrK was first detected on plasmids in S. aureus and linked to the tet(L) gene (151). Since then, dfrK has been found as part of transposon Tn559 in the chromosomal DNA of staphylococci and enterococci (152, 153) and on small plasmids from Staphylococcus hyicus that confer only trimethoprim resistance (114). In staphylococci, the composite transposon Tn4003 has been identified on various multiresistance plasmids. Tn4003 is composed of a central dfrA gene (also known as dfrS1) bracketed by copies of the insertion sequence IS257 (154). 12 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 Transferable trimethoprim resistance genes have been identified in a wide variety of Gram-negative bacteria; several of these genes are part of plasmids, transposons, or gene cassettes (111, 132, 134, 155) and thus are easily disseminated across species and genus borders. Several studies showed the relationships between the dfr genes (132, 134, 155). Resistance to Quinolones and Fluoroquinolones Quinolones and fluoroquinolones are potent inhibitors of bacterial DNA replication. While early quinolones such as nalidixic acid and pipemidic acid have not been used in veterinary medicine, oxolinic acid and flumequine (the first fluorinated quinolone) have been used in food-producing animals, including fish, worldwide (1, 156). Since the first of the newer fluoroquinolones, enrofloxacin, was licensed for use in animals in the late 1980s (157), several other fluoroquinolones have been approved for veterinary use in recent years, including marbofloxacin, orbifloxacin, difloxacin, ibafloxacin, danofloxacin, and pradofloxacin. Two major mechanisms account for resistance to fluoroquinolones: mutations in the genes for DNA topoisomerases and decreased intracellular drug accumulation (158–162). In addition, plasmid-mediated (fluoro)quinolone resistance genes have been described in the past decade (162). Mutational alteration of the target genes gyrA and gyrB (coding for the A and B subunits of the DNA gyrase) as well as parC and parE (coding for A and B subunits of the DNA topoisomerase IV) is frequently seen in (fluoro)quinolone-resistant bacteria. Both enzymes are tetramers consisting of two A and B units. The mutations in gyrA are commonly located within a region of ca. 130 bp which is referred to as the “quinolone resistance-determining region” (163). Mutations resulting in changes of Ser-83 (to Tyr, Phe, or Ala) and Asp-87 (to Gly, Asn, or Tyr) have been detected most frequently. In addition, double mutations at both positions and various other mutations have been described in Gram-positive and Gram-negative bacteria of human and veterinary importance (156, 159–161, 164). Stepwise mutations in gyrA and parC can result in an incremental increase in resistance to quinolones (156). Moreover, various mutations may also have different effects on resistance to the various fluoroquinolones (165). The complex interplay between individual mechanisms may also have different effects on fluoroquinolone resistance (166). Multidrug efflux systems also conferring fluoroquinolone resistance have been identified in various Gram-positive and Gram-negative bacteria, such as P. aeruginosa (MexAB/OprM, MexCD/OprJ), S. aureus (NorA), S. pneumoniae (PmrA), Bacillus subtilis (Blt), E. coli, and S. enterica (AcrAB/TolC); for reviews see references 161, 167, and 168. Since the basal level of expression of these efflux systems is low, upregulation of their expression is required to confer resistance to fluoroquinolones and other antimicrobials. In E. coli the level of production of the AcrAB-TolC efflux system is under the control of several regulatory genes, in particular the global regulatory systems marRAB and soxRS, but also acrR (167–170). Mutations in these regulatory systems may lead to overproduction of the AcrAB-TolC efflux pump and expression of the multidrug resistance phenotype (171, 172). Besides overproduction of the AcrAB-TolC efflux pump, it has been recently shown using macroarrays that E. coli strains constitutively expressing marA showed altered expression of more than 60 chromosomal genes (173). Interplay between several resistance mechanisms may lead to high-level resistance to quinolones and to other antibiotics when multidrug efflux pumps and decreased outer membrane permeability are involved (166, 174). For in vitro selected quinolone-resistant E. coli mutants, it has been shown that first-step quinolone-resistant mutants acquire a gyrA mutation. Second-step mutants reproducibly acquire a multidrug resistance phenotype and show enhanced fluoroquinolone efflux. In some third-step mutants, fluoroquinolone efflux is further enhanced and additional topoisomerase mutations are acquired. In clinical E. coli isolates from humans and animals, the situation appears to be the same, where high-level fluoroquinolone resistance is reached when mutations at several chromosomal loci are acquired (166, 174). It is noteworthy that inactivation of the AcrAB efflux pump renders resistant E. coli strains, including those with target gene mutations, hypersusceptible to fluoroquinolones and certain other unrelated drugs (174). Thus, in the absence of the AcrAB efflux pump, gyrase mutations fail to produce clinically relevant levels of fluoroquinolone resistance (175). The same observation has been made for P. aeruginosa, in which deletion of the MexAB-OprM efflux pump, which is the homolog of the AcrAB-TolC efflux pump in this species, resulted in a significant decrease in resistance to fluoroquinolones even for strains carrying target gene mutations (176). In high-level fluoroquinolone-resistant S. enterica serovar Typhimurium DT204 strains, carrying multiple target gene mutations in gyrA, gyrB, and parC, inactivation of AcrB or TolC resulted in a 16- to 32-fold decrease of resistance levels to fluoroquinolones (177, 178). ASMscience.org/MicrobiolSpectrum 13 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 Decreased drug uptake in Gram-negative bacteria is due to the marRAB-mediated downregulation of OmpF porin production. OmpF is an important porin for the entry of quinolones and fluoroquinolones into the bacterial cell (179, 180). Moreover, mutations in different gene loci (cfxB, norB, nfxB, norC, or nalB) are also associated with decreased permeability (181, 182). Plasmid-mediated quinolone resistance mechanisms have been described in recent years in addition to the aforementioned chromosomal resistance mechanisms (183–185). These plasmid-mediated quinolone resistance mechanisms usually confer low-level (fluoro) quinolone resistance by (i) target protection, (ii) acetylation, and (iii) efflux pumps. The protection of the DNA gyrase is mediated by qnr genes. Six qnr gene families (qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC) with multiple alleles per gene have been described and are organized in a database (www.lahey.org/qnrStudies). The qnr genes are associated with several mobile genetic elements and located on plasmids of varying sizes and different incompatibility groups (184, 185). The gene aac(6′)-Ib-cr codes for an aminoglycoside acetyltransferase, which is able to acetylate the amino nitrogen on the piperazinyl ring of quinolones such as ciprofloxacin and norfloxacin (184, 185). Efflux pumps are encoded by the genes qepA and oqxAB. The OqxAB efflux pump has a wide substrate specificity and is found not only on plasmids but also in the chromosomal DNA. These plasmid-mediated resistance genes have been found in several Gramnegative bacteria. The plasmid-located gene for an efflux pump qacBIII is able to confer decreased susceptibility to ciprofloxacin and norfloxacin in S. aureus and is the first plasmid-mediated quinolone resistance gene in Gram-positive bacteria (184, 185). Resistance to Phenicols Two members of the phenicols, chloramphenicol and its fluorinated derivative florfenicol, are currently approved for use in animals. The predominant mechanism of chloramphenicol resistance in Gram-positive and Gramnegative bacteria is enzymatic inactivation (186–188). In addition, efflux systems that mediate either resistance to only chloramphenicol or combined resistance to chloramphenicol and florfenicol have also been identified (187). Furthermore, permeability barriers and multidrug transporters play a role in certain Gram-negative bacteria (8, 12, 187, 189). Detailed reviews on the different genes and mechanisms accounting for bacterial resistance to chloramphenicol and florfenicol have been published (48, 187). Enzymatic inactivation of chloramphenicol is commonly achieved by chloramphenicol acetyltransferases (CATs) which transfer acetyl groups from acetyl-CoA to the C3 position of the chloramphenicol molecule. Subsequent transfer of the acetyl group to the C1 position and transfer of a second acetyl group to C3 results in mono- or diacetylated chloramphenicol derivatives, both of which are unable to inhibit bacterial protein biosynthesis (186–188). Two distinct types of CAT enzymes, which differ in their structures, are known: the classical CATs (type A) and a novel type of CAT (type B) (186, 187). All type A and type B CATs have a trimeric structure composed of three identical monomers. The cat gene codes for a CAT monomer, the size of which varies between 207 and 238 aa (type A CATs) and 209 and 212 aa (type B CATs) (187). Using the cutoff as set for the classification of tetracycline and MLS resistance genes (47, 74), 16 classes of catA determinants and at least another five classes of catB determinants can be differentiated (48). Among the catA genes, those formerly referred to as catI, catII, and catIII are most widespread among Gram-negative bacteria (48, 190– 192). They are associated with either nonconjugative transposons such as Tn9 or plasmids. Expression of these catA genes is constitutive. Various catA genes, indistinguishable from or closely related to those present on the S. aureus plasmids pC221, pC223/pSCS7, and pC194 (48, 192–195), have been detected in coagulasepositive and -negative staphylococci, but also in members of the genera Streptococcus, Bacillus, and Listeria, respectively. Expression of these mostly plasmid-borne catA genes is inducible by chloramphenicol via translational attenuation (196), whereas the Tn4451-borne catA genes of Clostridium spp. are expressed constitutively (197). The catB genes—also referred to as xat (xenobiotic acetyltransferase) genes—differ distinctly from the catA genes but are related to acetyltransferase genes, such as vat(A-E), involved in streptogramin resistance (186). Some of the catB genes have been found exclusively on the chromosome of either Agrobacterium tumefaciens, P. aeruginosa, or V. cholerae, whereas others proved to be part of transposons (Tn2424, Tn840) or plasmid-borne integrons. Studies of the level of catBmediated chloramphenicol resistance revealed a distinctly lower level of chloramphenicol resistance compared to that conferred by type A CATs (186). In addition to inactivation via CATs, enzymatic inactivation of chloramphenicol can also occur by O-phosphorylation or by hydrolytic degradation to p-nitrophenylserinol (48). Since these mechanisms have so far only been seen in the chloramphenicol producer 14 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 Streptomyces venezuelae and in a soil metagenome library, they are believed to play a role as self-defense mechanisms (48, 187). A total of 11 classes of specific exporters which mediate either chloramphenicol or chloramphenicol/ florfenicol resistance have been identified (48, 187). Among them, seven classes are represented by 10- to 12-TMS chloramphenicol exporters of soil bacteria of the genera Streptomyces, Rhodococcus, and Corynebacterium or of bacteria of unknown origin, whereas four classes of 12-TMS exporters were found among Gram-negative bacteria of medical importance (187, 198). Among these latter classes, one class represents the cmlA subgroup, and the others represent the floR subgroup. The gene cmlA, which codes for a chloramphenicol exporter, is a Tn1696-associated cassetteborne gene which, however, is inducibly expressed via translational attenuation (199). Genes related to cmlA are mainly found in Enterobacteriaceae and Pseudomonas. Genes related to floR have been identified in Photobacterium, Vibrio, Klebsiella, E. coli, and various S. enterica serovars and in Pasteurella multocida as part of the chromosomally located ICEPmu1 (48, 200– 207). In Vibrio and Salmonella, the gene floR has been detected as part of chromosomal multiresistance gene clusters (206, 208), and in E. coli, as part of conjugative and nonconjugative multiresistance plasmids (200, 201). In S. maltophilia of porcine origin, a novel floR variant, floRV, has been identified as part of a chromosomal genomic island (209). Another class of phenicol exporters is represented by FexA, the first specific chloramphenicol/florfenicol exporter of Gram-positive bacteria (210). The gene fexA, located on the transposon Tn558 (211) from Staphylococcus lentus, codes for a 14-TMS exporter of the major facilitator superfamily and is expressed inducibly via translational attenuation. A second phenicol exporter, FexB, which exhibited 56.1% amino acid identity with the FexA protein, has been exclusively identified in enterococci (212). Multidrug transporter systems that export chloramphenicol have been described to occur in several Gramnegative bacteria, including the systems MexAB/OprM and MexCD/OprJ in P. aeruginosa, AcrAB/TolC in E. coli and S. enterica, CeoAB/OpcM in Burkholderia cepacia, and ArpAB/ArpC and TtgAB/TtgC in Pseudomonas putida (40, 180). Permeability barriers based on the reduced expression of the OmpF porin in S. enterica serovar Typhi or a major outer membrane protein in H. influenzae (187) have also been described to confer chloramphenicol resistance. The mar locus which is found in various Enterobacteriaceae can contribute to chloramphenicol resistance in two ways: on one hand, it can activate the AcrAB/TolC efflux system, leading to increased efflux of chloramphenicol, and on the other hand, MarA can activate the gene micF, whose transcripts represent an antisense RNA that effectively inhibits translation of ompF transcripts, which results in a decreased influx of chloramphenicol (179, 180). Mutations in the major ribosomal protein clusters of E. coli and B. subtilis, but also mutations in the 23S rRNA of E. coli, have been described to mediate chloramphenicol resistance (213). Resistance to Oxazolidinones Oxazolidinones are a class of synthetic antibiotics that are highly active against Gram-positive bacteria. Currently, two oxazolidinones, linezolid and tedizolid, are exclusively approved for use in human medicine and are considered last-resort antimicrobial agents for the treatment of infections caused by MRSA, vancomycinresistant enterococci, and penicillin-resistant S. pneu- moniae. Initially, point mutations within either the 23S rRNA and/or the genes coding for the ribosomal proteins L3 (rplC), L4 (rplD), and L22 (rplV) were recognized as the main mechanisms of reduced oxazolidinone susceptibility (214–216). Mutations in clinical staphylococcal and enterococcal isolates, including G2247T, T2500A, A2503G, T2504C, G2505A, and G2576T, usually were found in the vicinity of the peptidyltransferase center (216). Mutations in the gene rplC, including F147L and A157R, resulted in at least 2-fold increases of the oxazolidinone MICs of laboratory and clinical staphylococci (217). The mutations in the rplD gene, which resulted in amino acid exchanges K68N or K68Q, and the insertions 71GGR72, 65WR66, and 68KG69 lead to oxazolidinone resistance in S. pneumoniae (218). Little is known about the effects of L22 mutations on linezolid resistance; whether the amino acid exchange A29V in L22 detected in two linezolid-resistant MRSA isolates is responsible for the linezolid resistance in these isolates remains to be investigated (219). The gene cfr from S. sciuri was the first transferable oxazolidinone resistance gene (220). Initially described as a novel chloramphenicol/florfenicol resistance gene, the elucidation of the resistance mechanism revealed that cfr codes for an rRNA methylase, which confers resistance not only to phenicols but also to lincosamides, oxazolidinones, pleuromutilins, and streptogramin A by methylating the adenine residue at position 2503 in 23S rRNA, which is located in the overlapping ribosomal ASMscience.org/MicrobiolSpectrum 15 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 binding site of these antibiotics (221, 222). The gene cfr is mainly located on plasmids in staphylococci and has been distributed across species and genus boundaries. In recent years, it has been detected in both Grampositive and Gram-negative genera, including Bacillus, Enterococcus, Escherichia, Jeotgalicoccus, Macrococcus, Staphylococcus, Streptococcus, and Proteus (48, 223, 224). Recently, variants of the cfr gene have been identified, including cfr(B) in E. faecium and Clostridium difficile (225, 226) and cfr(C) in Campylobacter and Clostridium (227). An ABC transporter, which is able to mediate resistance to chloramphenicol and florfenicol as well as the oxazolidinones linezolid and tedizolid, is encoded by the gene optrA, which has been identified on a conjugative plasmid in E. faecalis (228). The insertion sequence IS1216E and the transposon Tn558 have been identified in the optrA flanking regions on plasmids and on the chromosome of enterococci from humans, pigs, and chickens (229). Moreover, the gene optrA has also been detected on plasmids and in the chromosomal DNA of porcine S. sciuri (230, 231). Since then, this gene has been identified in genomes of clinical isolates of staphylococci, enterococci, and streptococci (232). Resistance to Glycopeptides Since the ban of the growth promotor avoparcin in 1996, no glycopeptide antibiotics are approved for use in animals. Glycopeptide antibiotics, such as vancomycin and teicoplanin, act by binding to the D-alanine-D-alanine termini of peptidoglycan precursors, thereby preventing transglycosylation and transpeptidation of the bacterial cell wall (233, 234). Modification of the target site is the common mechanism of bacterial resistance to glycopeptides. So far, four D-Ala-D-Lac operons (vanA, vanB, vanD, and vanM) and five D-Ala-D-Ser operons (vanC, vanE, vanG, vanL, and vanN) have been described in enterococci (235). They differ in their levels of resistance to vancomycin and teicoplanin (234). In the D-Ala-D-Lac operons, the terminal dipeptide D-alanine-D-alanine is replaced by D-alanine-D-lactate, whereas in the D-Ala-DSer operons, it is replaced by D-alanine-D-serine. These replacements reduce the ability of glycopeptides to bind to the peptidoglycan precursors and result in the case of D-lactate in high-level and in the case of D-serine in low-level glycopeptide resistance. The VanC operon is responsible for the intrinsic resistance of Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens to glycopeptides (234). Similar to VanC, VanD and VanE, both from E. faecalis, have been reported not to be transferable (234). In contrast, the vanA and vanB operons are associated with transposons which can be located on conjugative and nonconjugative plasmids in enterococci. The VanA phenotype is associated with the nonconjugative transposon Tn1546, which contains a total of nine reading frames, five of which are essential for highlevel glycopeptide resistance (236). Among these, the two genes vanR and vanS code for a response regulator protein and a sensor protein, respectively, involved in regulatory processes. Three genes are directly involved in resistance: vanH, vanA, and vanX. The gene vanH codes for a cytoplasmatic dehydrogenase that produces D-lactate from pyruvate, whereas the gene vanX codes for a D,D-dipeptidase which cleaves the D-alanine-D-alanine, and the gene vanA codes for a ligase that joins the remaining D-alanine with D-lactate. While glycopeptide resistance is often found in enterococci (237, 238), transfer studies showed that conjugative transfer of VanA-mediated vancomycin resistance from E. faecalis to S. aureus is possible under in vitro conditions (239). In 2002, the first patients infected with vanA-carrying high-level vancomycin-resistant S. aureus isolates were detected in the United States (240). Genes homologous to enterococcal glycopeptide resistance genes vanA and vanB have also been detected among members of the genera Paenibacillus and Rhodococcus (241). Moreover, a new glycopeptide resistance operon, vanOHX, has recently been identified in Rhodococcus equi (235). Impaired membrane permeability renders Gramnegative bacteria intrinsically resistant to glycopeptides, large molecules which can cross the outer membrane only poorly, if at all (234). Resistance to Pleuromutilins The pleuromutilins tiamulin and valnemulin are mainly used in veterinary medicine for the control and specific therapy of gastrointestinal and respiratory tract infections in swine and to a lesser extent in poultry and rabbits. Retapamulin is used as an ointment to treat bacterial skin infections in humans. Products for systemic use in humans with infections caused by multidrug-resistant bacteria are currently being developed. The main target bacteria in veterinary medicine are B. hyodysenteriae, Brachyspira pilosicoli, Lawsonia intracellularis, and Mycoplasma spp. Resistance derives from chromosomal mutations in the 23S rRNA and rplC genes or mobile resistance genes located on plasmids or transposons, such as the cfr genes and certain vga, lsa, and sal genes (242, 243). The mechanism of resistance varies among bacterial species. 16 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 In B. hyodysenteriae, reduced susceptibility to tiamulin has been associated with point mutations in the V domain of the 23S rRNA gene (positions 2032, 2055, 2447, 2499, 2504, and 2572 in E. coli numbering) and/ or the ribosomal protein L3 gene (244, 245). Mutation at nucleotide position 2032 appears to be related to pleuromutilin resistance and to decreased susceptibility to lincosamides (245). Tiamulin resistance in B. hyodysenteriae develops in a stepwise manner both in vitro and in vivo, suggesting that multiple mutations are needed to achieve high levels of resistance. The MICs of valnemulin are generally a few dilution steps lower than those of tiamulin (246). To date, data on the resistance mechanisms of B. pilosicoli and L. intracellularis are lacking, and data on resistance mechanisms of mycoplasmata are limited. A single mutation of the 23S rRNA gene caused elevated tiamulin and valnemulin MICs in Mycoplasma gallisepticum, but combinations of two or three mutations were necessary to produce high levels of resistance to these drugs (247). Resistance in staphylococci can be due to point mutations in the V domain of 23S rRNA or in the rplC gene, encoding the ribosomal protein L3 (248). Transferable resistance in staphylococci can be caused by vga genes, encoding ABC transporters, resulting in resistance to pleuromutilins, streptogramin A, and lincosamides. There are several vga genes which confer pleuromutilin resistance in addition to lincosamide and streptogramin A resistance: vga(A) and its variants, vga(C), and vga(E) and its variant. All these genes have been found on plasmids and transposons of staphylococci (242). Transferable resistance to five classes of antimicrobials (phenicols, lincosamides, oxazolidinones, pleuromutilins, streptogramin A) in staphylococci is mediated by the gene cfr. The sal(A) genes from S. sciuri have been shown to mediate combined resistance to lincosamides, pleuromutilins, and streptogramin A antibiotics (243). E. faecalis is intrinsically resistant to pleuromutilins, streptogramin A antibiotics, and lincosamides by the production of the ABC transporter Lsa(A). In E. faecium, acquired resistance to the above-mentioned antimicrobials is mediated by the ABC transporter gene eat(A)V (249). The enterococcal ABC transporter gene lsa(E) has been detected in methicillin-susceptible S. aureus and in MRSA of human and animal origin (250). Resistance to Polypeptide Antibiotics There are three polypeptide antibiotics that are used in human and/or veterinary medicine: bacitracin, polymyxin B, and colistin (polymyxin E). Bacitracin inhibits cell wall synthesis and is active against Gram-positive bacteria. It used to be used as a growth promoter (251). Since the ban of antimicrobial growth promoters in 2006 in the European Union, it is no longer approved for veterinary use as growth promotor in the EU. In China, colistin has also been banned from use as growth promoter in food animals, as of April 2017. However, it is used for that purpose in other countries, and it is still approved for therapeutic purposes in the European Union and China. Polymyxin B and colistin (polymyxin E) disrupt the outer bacterial cell membrane of certain Gram-negative bacteria, such as most Enterobacteriaceae, P. aeruginosa, and Acinetobacter baumannii, whereas other Gram-negative bacteria, including Proteus spp., Providencia spp., Morganella morganii, Serratia spp., Edwardsiella tarda, and bacteria of the B. cepacia complex exhibit intrinsic resistance to these polypeptide antibiotics. Recently, colistin was included in the WHO list of critically important antibiotics (252). In human medicine, colistin is used as a last-line drug in the treatment of severe infections caused by multiresistant Gram-negative bacteria, whereas it is used in veterinary medicine for the treatment of enteric diseases, mainly in swine and poultry (253). So far, several mechanisms of resistance to polymyxins (polymyxin B and colistin) have been described (254). These include a variety of LPS modifications, such as modifications of lipid A with phosphoethanolamine and 4-amino-4-deoxy-Larabinose, efflux pumps, the formation of capsules, and overexpression of the outer membrane protein OprH (254). Such resistance is chromosomally encoded, and hence spread entailed either de novo emergence or clonal expansion of resistant isolates. The alteration of the LPS on its lipid A moiety is the primary mechanism of resistance to polycationic polymyxins. 4-Amino-4-deoxy-L-arabinose (L-Ara4N) or phosphoethanolamine is added to lipid A by enzymes such as ArnT and EptA, resulting in a decrease of the negative charge of the LPS, thereby lowering the affinity of the positively charged polymyxins to the outer membrane. The genes arnT (part of the operon arnBCADTEF) and eptA, which code for those enzymes, are controlled by chromosomally encoded twocomponent regulatory systems, such as PmrAB and PhoPQ. Mutations in the operons pmrAB and phoPQ may lead to an upregulation of arnT and eptA expression, resulting in polymyxin resistance. This has been described in E. coli, K. pneumoniae, S. enterica, and P. aeruginosa (254). Furthermore, mutations in the gene for the negative feedback regulator of PhoPQ, mgrB, can activate the arnBCADTEF operon in K. pneumoniae ASMscience.org/MicrobiolSpectrum 17 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 (255). Other genes coding for phosphoethanolamine transferases are eptB in E. coli, eptC in C. jejuni, and lptA in N. meningitidis. Those phosphoethanolamine transferases add phosphoethanolamine to different positions on lipid A. The main polymyxin resistance mechanism in A. baumannii is mutations in the genes of the PmrAB system and the resulting overexpression of pmrC, which codes for an EptaA-like phosphoethanolamine transferase. In P. aeruginosa five two-component regulatory systems (PhoPQ, PmrAB, ParRS, CprRS, ColRS) have been identified, and mutations in the genes for those systems play a role in overexpression of the arnBCADTEF-ugd operon (255). In addition, plasmid-mediated resistance to polymyxins was reported in 2016 (256). The gene mcr-1 has been identified on a conjugative plasmid in E. coli of animal and human origin and codes for a phosphoethanolamine transferase. The mcr-1 gene was first identified among isolates from China, but since then it has been detected in isolates of various Enterobacteriaceae from five continents (253). Furthermore, the gene mcr-2 has been described in porcine and bovine E. coli isolates from Belgium (257). Most recently, another three mcr genes, designated mcr-3 (258), mcr-4 (259), and mcr-5 (260), have been identified in E. coli and/or S. enterica. Several variants of mcr-1 have been detected in Enterobacteriaceae (258), while mcr-2 variants have been found in Moraxella spp. (261). Variants of the gene mcr-3 have been identified in Aeromonas spp. (262, 263). Polymyxin resistance due to the complete loss of LPS in A. baumannii is based on the inactivation of genes such as lpxA, lpxC, lpxD, and lpsB, the products of which are involved in LPS biosynthesis (264). A variety of efflux pumps in several bacterial species have been described to be involved in polymyxin resistance in Gram-negative bacteria. Sensitive antimicrobial peptide proteins, encoded by the sapABCDF operon, and the resistance-nodulation-cell division transporter AcrAB-TolC seem to play a role in the susceptibility to polymyxins in E. coli, S. enterica, and Proteus mirabilis (265). The resistance-nodulation-cell division transporter VexB is involved in polymyxin resistance in V. cholerae (266). The efflux pump KpnEF, which has been described in isolates of K. pneumoniae, belongs to the small multidrug resistance protein family and is part of the Cpx regulon, which regulates capsule synthesis. Resistance to several antibiotics such as colistin, rifampicin, erythromycin, and ceftriaxone is influenced by KpnEF (266). Other efflux pumps of the small multidrug resistance protein family involved in polymyxin resistance have been identified in B. subtilis (EbrAB) and in A. baumannii (AbeS) (265). Capsule formation is another mechanism of polymyxin resistance. Capsule polysaccharides limit the interaction of polymyxins with their target sites, thus playing an important role in polymyxin resistance, not only in intrinsically resistant bacteria such as N. meningitidis and C. jejuni, but also in K. pneumoniae, E. coli, and P. aeruginosa (267, 268). Moreover, anionic bacterial capsule polysaccharides have been shown to neutralize the bactericidal activity of cationic polypeptide antibiotics and antimicrobial peptides (268). Overexpression of the outer membrane protein OprH contributes to polymyxin resistance in P. aeruginosa. OprH is a basic protein that binds to divalent cationbinding sites of LPSs, making these sites unavailable for polymyxins (269). Resistance to Mupirocin Mupirocin is a topical antibiotic used mainly for decolonization of MRSA and methicillin-susceptible S. aureus in patients and in health care personnel, but also for treatment of local skin and soft tissue infections caused by S. aureus and streptococci (270). It is not approved for veterinary use. Mupirocin prevents bacterial protein synthesis by inhibiting the bacterial isoleucyl-tRNA synthetase. Low-level resistance against mupirocin results from point mutations in the native isoleucyl-tRNA synthetase gene, whereas the acquisition of genes coding for alternative isoleucyl-tRNA synthetases leads to high-level resistance. The gene mupA (also referred to as ileS2) has been identified on conjugative plasmids (271–273), while the gene mupB has been found on nonconjugative plasmids in staphylococci (273, 274). Resistance to Ansamycins Certain ansamycins, such as rifampicin and rifamycin, are used in veterinary medicine for the treatment of infections of horses caused by R. equi. Rifampicin inhibits the bacterial RNA polymerase by interacting with the β-subunit, which is encoded by the gene rpoB. Resistance to rifampicin and related compounds is mainly due to point mutations that cause amino acid substitutions in at least one of three rifampicin resistance-determining regions within the rpoB gene. It is noteworthy that not all amino acid substitutions have the same effect. In R. equi, mutations at different positions within the rpoB gene have been shown to correlate with different rifampicin MIC values (275, 276). Mutations of rpoB have been described in several bacteria, 18 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 such as R. equi (275, 276), E. coli (277), Mycobacterium spp. (278), B. subtilis (279), S. aureus (280), and S. pseudintermedius (281). Furthermore, RNA polymerase binding proteins, such as RbpA in Streptomyces coelicolor and DnaA in E. coli, play a role in increased insensitivity to rifampicin (282). Arr enzymes are ADP-ribosyltransferases, which are able to modify rifampicin by ADP-ribosylation and thus inactivate it. M. smegmatis carries the arr gene in its chromosomal DNA (283), homologues of which have been identified in the genomes of bacteria such as S. maltophilia, Burkholderia cenocepacia, and other environmental bacteria (283). The gene arr-2 has been identified in chromosomal DNA and on various plasmids as part of class 1 integrons or composite transposons in Gram-negative bacteria such as P. aeruginosa, K. pneumoniae, and E. coli (282). An arr-3 gene was detected in a class 1 integron of Aeromonas hydrophila from a koi carp (284). Other modification mechanisms are glucosylation and phosphorylation in Nocardia spp. and phosphorylation in Bacillus spp. (282). Resistance to Fosfomycin Fosfomycin interferes with the peptidoglycan synthesis of bacteria by inhibiting the enzyme MurA. The irreversible inhibition is due to alkylation of the catalytic cysteine of MurA (285, 286). The exchange of cystein for asparagine is a target alteration, leading to intrinsic fosfomycin resistance in bacteria such as M. tuberculosis, Chlamydia trachomatis, and Borrelia burgdorferi (285, 286). Fosfomycin reaches its target site through GlpT, a glycerol-3-phosphate transporter, or via UhpT, a glucose-6-phosphate transporter. Both substrates induce the expression of their transporter, which is regulated by cAMP. Mutations in the genes coding for GlpT and UhpT or their regulators may lead to defective or inactive transporters, resulting in fosfomycin resistance (285, 286). Enzymatic inactivation of fosfomycin can be achieved by several fosfomycin-modifying enzymes. The main enzymes described are three types of metalloenzymes (FosA, FosB, and FosX) and two kinases (FomA and FomB). FosA and FosB are thiol transferases, while FosX is a hydrolase. The metalloenzymes open the oxirane ring of fosfomycin and thus render it inactive. FosA enzymes are glutathione-S-transferases which use Mn2+ and K+ as metal cofactors. They add glutathione to the oxirane ring, thereby opening the ring and inactivating fosfomycin (280). Various fosA genes have been identified on plasmids or in the chromosomal DNA of Gram-negative bacteria such as S. marcescens, E. coli, K. pneumoniae, E. cloacae, and P. aeruginosa (285, 286). They have been described as parts of transposons such as Tn2921 or flanked by copies of IS26 on plasmid pFOS18 (287). The gene fosC2, which also codes for a glutathione-S-transferase, has been identified on a gene cassette in a class 1 integron on a conjugative plasmid in E. coli (288). Other fos genes have been described in numerous bacteria, including fosC in Achromobacter denitrificans and fosK in Acinetobacter soli (289). FosB enzymes are bacillithiol-S-transferases that use Mg2+ as a cofactor. Several fosB gene variants have been detected in the chromosomal DNA of Gram-positive bacteria, such as Staphylococcus epidermidis (290), B. subtilis, Bacillus anthracis, and S. aureus (285, 286). The gene fosB3 has been identified on a conjugative plasmid in E. faecium (291), while the genes fosB1, fosB5, and fosB6 were located on small plasmids in S. aureus (292). The gene fosD is related to fosB and has been found in avian Staphylococcus rostri (293). FosX enzymes are Mn2+ -dependent epoxide hydrolases, which use water to break the oxirane ring (285, 286). Variants of the gene fosX have been detected in the chromosomal DNA of Clostridium botulinum, L. monocytogenes, and Brucella melitensis (286). The gene fosXCC was detected as part of a multidrug-resistance genomic island in C. coli (294). FomA and FomB are kinases that originate from the fosfomycin producer Streptomyces wedmorensis. They sequentially add phosphates to the phosphonate moiety of fosfomycin by using Mg2+ as a cofactor (285). Most likely, these enzymes represent part of the self-defense system of the fosfomycin producer (285). Another such kinase, originally called FosC and found in another fosfomycin producer, Pseudomonas syringae, is an ortholog of FomA (295). Resistance to Fusidic Acid Fusidic acid is a steroidal compound which was isolated from Fusidium coccineum. It exhibits antimicrobial activitiy against Gram-positive bacteria, such as staphylococci and Corynebacterium spp., as well as the Gramnegative Neisseria gonorrhoeae, N. meningitidis, and Moraxella catarrhalis (296). Fusidic acid is mainly used topically to treat skin infections caused by staphylococci, but it can also be administered systemically. Fusidic acid binds to elongation factor G and thus prevents polypeptide chain elongation during protein synthesis (297). Elongation factor G is encoded by the gene fusA. Several mutations in the gene fusA that cause resistance to ASMscience.org/MicrobiolSpectrum 19 Mechanisms of Bacterial Resistance to Antimicrobial Agents Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 fusidic acid have been described in S. aureus, with L461K being the most prevalent (297). Protection of the target site and subsequent fusid acid resistance is conferred by the protein FusB, which prevents the interaction of fusidic acid with elongation factor G (298). The gene fusB has been identified on the widespread plasmid pUB101 in S. aureus (299). A fusB-related gene, fusF, has recently been identified in Staphylococcus cohnii (300). In contrast, the genes fusC and fusD, which also confer fusidic acid resistance by target protection, have been detected as part of a chimeric SCCmecIV-SCC476 element in the chromosomal DNA of S. aureus (301) and in the chromosomal DNA of S. saprophyticus (302), respectively. Resistance to Streptothricins Streptothricins are antibiotics that consist of a streptolidine ring, a glucosamine, and a polylysine side chain. One of them, nourseothricin, was used as an antimicrobial feed additive in industrial animal farming in the former East Germany (303). Several sat genes have been identified which mediate streptothricin resistance by enzymatic inactivation via acetylation. In Gram-negative bacteria, particularly in Enterobacteriaceae, sat or sat2 genes are usually located on gene cassettes in class 1 or class 2 integrons (304, 305). In staphylococci, the sat4 gene is part of Tn5405 and, as such, is commonly detected in staphylococci that also harbor aphA3 and aadE. Furthermore, this gene has been detected in canine and feline S. pseudintermedius (306–308) and in MRSA of CC8 (ST254) from horses in Germany (309). Resistance to Substances with Antimicrobial Activity Formerly Used as Growth Promoters A number of substances with antimicrobial activity have been licensed as growth promoters for livestock. In the European Union, all growth promoters with antimicrobial activity were banned or withdrawn by 2006. The mechanisms of resistance to the macrolides tylosin and spiramycin, the streptogramin virginiamycin, and the glycopeptide avoparcin were described in the sections “Resistance to Macrolides, Lincosamides, and Streptogramins (MLS)” and “Resistance to Glycopeptides.” Hence, a brief summary of resistance to the remaining classes of growth promoters is given below. Two reviews (251, 310) are recommended for detailed insight into the various aspects of the use of growth promoters. Bacitracin resistance was first described in the producer organism Bacillus licheniformis, in which an ABC transporter system, BcrABC, acts as a self-defense system by exporting the antibiotic from the producer cell (311). In B. subtilis, two independent but complementaryacting resistance mechanisms have been detected: an ABC transporter, YtsCD, that mediates the efflux of bacitracin, and a protein designated YwoA, which is believed to compete with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (312). In E. coli, the gene bacA, which codes for an undecaprenyl pyrophosphate phosphatase, may account for bacitracin resistance (313). An ABC transporter, also termed BcrAB, that mediates bacitracin resistance was identified on a conjugative plasmid in E. faecalis (314). Avilamycin resistance in the producer organism Streptomyces viridochromogenes Tü57 is based on the activity of an ABC transporter and two rRNA methyltransferases (315). In E. faecalis and E. faecium, resistance to avilamycin was initially described to be due to variations in the ribosomal protein L16 (316). Later, an rRNA methyltransferase, EmtA, which confers highlevel resistance to avilamycin and evernimicin, was identified (317). Another two methylases—AviRa, which methylates 23S rRNA at the guanosine 2535 base, and AviRb, which methylates the uridine 2479 ribose— have been shown to confer avilamycin resistance (318). In addition, mutations at specific positions in the 23S rRNA also give rise to avilamycin resistance (319). Flavophospholipol (also known as flavomycin or bambermycin) has been reported to have a “plasmid-curing effect” on multiresistant E. coli under experimental conditions in vitro and in vivo (320). Cross-resistance to other antimicrobials has not been observed (251). Moreover, no genes or mutations conferring flavophospholipol resistance have been observed, to date. Ionophores, such as salinomycin-Na and monensinNa, are mainly used for the prevention of infections with parasites, such as Eimeria spp. (coccidiosis), Plasmodium spp., and Giardia spp. (251). Resistance or decreased susceptibility has been described in S. hyicus, coagulase-negative staphylococci from cattle and from E. faecium and E. faecalis from poultry and pigs. Genes or mutations accounting for acquired resistance to ionophores have not yet been described (251). Resistance to quinoxalines, such as carbadox and olaquindox, has been reported. An early study identified carbadox resistance to be associated with a conjugative multiresistance plasmid in E. coli (321). More than 20 years later, the genes oqxA and oqxB, which are responsible for olaqindox resistance, were cloned from a conjugative plasmid in E. coli (322). The corresponding gene products are homologous to several resistance-nodulation- 20 ASMscience.org/MicrobiolSpectrum van Duijkeren et al. Downloaded from www.asmscience.org by IP: 130.60.98.21 On: Mon, 05 Feb 2018 09:28:49 cell-division family efflux systems and use TolC as the outer membrane component. Interestingly, the OqxABTolC system also mediates resistance to chloramphenicol and ethidium bromide (322). CONCLUSION The development of antimicrobial resistance—by either mutations, development of new resistance genes, or the acquisition of resistance genes already present in other bacteria—is a complex process that involves various mechanisms. Numerous resistance genes specifying different resistance mechanisms have been identified in various bacteria. The speed of resistance development differs with regard to the bacteria involved, the selective pressure imposed by the use of antimicrobial agents, and the availability and transferability of resistance genes in the gene pools accessible to the bacteria. These basic facts apply to resistance development in bacteria from humans as well as in bacteria from animals. The loss of acquired resistance properties is often a cumbersome process which is influenced mainly by selective pressure, but also by the colocation of the resistance genes on multiresistance plasmids or in the chromosomal DNA and the organization of the resistance genes in multiresistance gene clusters or integron structures. When organized in resistance gene clusters or integrons, loss of resistance genes may not be expected even in the absence of direct selective pressure. Because we know that the use of every antimicrobial substance can select for resistant bacteria, prudent use of antimicrobial agents is strongly recommended in both human and veterinary medicine, but particularly in food animal production to retain the efficacy of antimicrobial agents for the control of bacterial infections in animals. REFERENCES 1. Schwarz S, Chaslus-Dancla E. 2001. Use of antimicrobials in veterinary medicine and mechanisms of resistance. 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