Mechanisms of Antibacterial Resistance

SECTION 7 Anti-infective Therapy

138 

Mechanisms of Antibacterial Resistance GIAN MARIA ROSSOLINI  |  FABIO ARENA  |  TOMMASO GIANI

KEY CONCEPTS • Unlike other drugs, antibiotics tend to lose their efficacy over time due to the emergence of microbial drug resistance. • No antibiotic has escaped resistance, which has often emerged soon after the introduction of the antibiotic in clinical practice. • Several biochemical mechanisms can be responsible for antibiotic resistance, including drug inactivation, target modification or by-pass, and reduced drug uptake. • Resistance can be a feature typical of a bacterial species (intrinsic resistance) or acquired by individual strains of a species that are naturally susceptible (acquired resistance). • Acquired resistance can emerge due to chromosomal mutations or acquisition of resistance genes by horizontal gene transfer mechanisms. • Resistance to multiple antibiotics can be acquired by individual strains, resulting in multidrug-resistant (MDR) phenotypes. • The acquisition of multiple resistance determinants can eventually result in strains that remain susceptible to only a few antibiotics, designated as extremely drug-resistant (XDR) strains.

Introduction The activity of antibiotics against bacterial pathogens is a prerequisite for clinical efficacy. To this purpose, the activity of antibiotics against bacterial pathogens is normally measured by standardized laboratory methods for determining susceptibility and resistance. Infections caused by bacterial strains that are categorized as susceptible to an antibiotic can be treated with that drug with a high likelihood of clinical success (although additional factors can contribute to determine the eventual outcome). On the other hand, infections caused by bacterial strains that are categorized as resistant to an antibiotic are likely not to respond to treatment with that drug, which should not be used for treatment.1 Categorization of bacterial strains as susceptible or resistant following susceptibility testing is based on comparison of results with reference values of minimal inhibitory concentrations (or of zone inhibition diameters) indicated as clinical breakpoints. The biochemical mechanisms by which bacteria resist the inhibitory action of antibiotics include: • the presence of an enzyme that inactivates the antibiotic; • modification of the antibiotic target by mutation or by posttranslational mechanisms which reduce binding of the antibiotic to the target; • by-pass of the function dependent on the antibiotic target by an alternative enzyme that is not inhibited by the antibiotic; and • reduced uptake of the antibiotic inside the cell, due to reduced permeability of the cell envelopes or to active efflux. When a resistance mechanism is present and functional in most or all strains of a bacterial species, the species is categorized as intrinsically resistant to the antibiotic(s) affected by that mechanism and resistance can be directly predicted from bacterial identification (Table 138-1). On the other hand, acquired resistance occurs when strains of a



susceptible species acquire one or more resistance mechanisms. Acquired resistance is not predictable from species identification, and the purpose of in vitro susceptibility testing in the laboratory is to identify acquired resistance among infecting pathogens isolated from clinical specimens, to guide the choice of definitive antimicrobial therapy. Acquired resistance can be due to mutations of chromosomal genes or to the acquisition of resistance determinants by horizontal gene transfer mechanisms. Transferable resistance genes are usually carried by plasmids or other types of mobile genetic elements (MGEs), and play a relevant contribution to the evolution of microbial drug resistance. There is a strong correlation between the presence of some resistance determinants and the outcome of antimicrobial therapy. For instance, the presence of the mecA gene in Staphylococcus aureus is highly predictive of methicillin resistance and, therefore, resistance to all conventional β-lactam antibiotics. However, the presence of a resistance gene is not equivalent to treatment failure: the gene also must be expressed in sufficient amounts to lead to phenotypic resistance.

Resistance to β-Lactam Antibiotics

β-Lactam antibiotics interfere with peptidoglycan synthesis by inhibition of enzymes, called penicillin-binding proteins (PBPs), that are responsible for the formation of the peptide bonds which cross-link the peptidoglycan chains. Penicillin is the oldest β-lactam antibiotic, and the active β-lactam ring has been exploited to obtain a broad array of β-lactam antibiotics, including penicillins, cephalosporins, monobactams and carbapenems, characterized by different antimicrobial spectra and pharmacokinetic properties. Overall, β-lactams are among the most prescribed antibiotics in clinical practice due to their efficacy, safety and versatility. Resistance to β-lactams can be caused by different mechanisms including: 1) the production of β-lactamases, which destroy the β-lactam ring; 2) the presence of altered PBPs, which have lower affinity for β-lactams; and 3) a reduced permeability of the outer membrane or active efflux of the drug from the periplasmic space, which impair the access of β-lactams to their PBP targets (in gram-negative bacteria).

β-LACTAMASE-MEDIATED RESISTANCE

Production of β-lactamase activity is a common mechanism of intrinsic and acquired resistance to β-lactams in gram-positive and gramnegative pathogens and, in the latter, is overall the most important mechanism of β-lactam resistance. The number of β-lactamases detected in pathogenic bacteria has risen steadily since the introduction of penicillin. β-Lactamases have been classified according to their functional properties, considering the substrate preference and the behavior towards some inhibitors (Table 138-2).2 From the clinical standpoint, the most challenging enzymes are: 1) the extended-spectrum β-lactamases (ESBLs), which are able to hydrolyze penicillins, cephalosporins (both narrow- and expanded-spectrum) and monobactams; 2) the carbapenemases, which are able to hydrolyze carbapenems and usually most other β-lactams. β-lactamases have also been classified according to the amino acid sequence similarity and mechanistic features into four molecular

1181

R R

Enterococcus faecium

R R R

Citrobacter freundii

Enterobacter cloacae

Klebsiella spp.

R R R R

Serratia marcescens

Pseudomonas aeruginosa

Acinetobacter baumannii

Stenotrophomonas maltophilia

Proteus mirabilis

Ampicillin

Gram-negativesb

Leuconostoc spp., Pediococcus spp.

Listeria monocytogenes

R

Enterococcus faecalis

Fusidic Acid

Streptococcus spp.

Staphylococcus aureus

Gram-positivesa

R

R

R

R

R

R

Amoxicillin– clavulanate

R

R

R

R

Ceftazidime

R

Piperacillin

R

R

R

Cefotaxime

R

R

R

Cephalosporins (Except Ceftazidime)

R

R

R

R

R

Cefoxitin

b

R

R

R

R

R

R

R

R

Polymyxins

Clindamycin

Tetracyclines/ Tigecycline

Erythromycin

Examples of Intrinsic Resistances of Some Gram-Positive and Gram-Negative Pathogens

Gram-positive bacteria are also intrinsically resistant to aztreonam, temocillin, polymyxins and nalidixic acid. Gram-negative bacteria are also intrinsically resistant to glycopeptides, lincosamides, daptomycin and linezolid. Modified from EUCAST expert rules in antimicrobial susceptibility testing, version 2 (www.eucast.org).

a

TABLE

138-1 

R

R

R

Ertapenem

R

Quinupristin– dalfopristin

R

Meropenem

R

Vancomycin

R

Trimethoprim– sulfamethoxazole

R

Teicoplanin

1182 SECTION 7  Anti-infective Therapy



Chapter 138  Mechanisms of Antibacterial Resistance

TABLE

138-2 

1183

Classification of β-Lactamases Based on Relevant Functional Properties and Molecular Class RELEVANT FUNCTIONAL CHARACTERISTICS Behavior With Inhibitors*

Functional Group (Bush–Jacoby)

Substrate Preference

1

Molecular Class

Representative Enzymes

SBLI

EDTA

Cephalosporins (including cephamycins)

R

R

C

AmpC of Pseudomonas aeruginosa, ACT-1, CMY-1, FOX-1

1e

Same as group 1, but increased hydrolysis of oxyiminocephalosporins

R

R

C

GC1 of Enterobacter cloacae CMY-19, CMY-37

2a

Penicillins

S

R

A

PC1

2b

Penicillins, narrow-spectrum cephalosporins (broad-spectrum)

S

R

A

TEM-1, TEM-2, SHV-1

2be

Same as group 2b, but including expandedspectrum cephalosporins and monobactams (extended-spectrum)

S

R

A

TEM-3, SHV-12, CTX-M-15, PER-1, VEB-1

2br

Same as group 2b but resistant to SBLI

R

R

A

TEM-30, SHV-10

2ber

Same as group 2be but resistant to SBLI

R

R

A

TEM-50

2c

Carbenicillin

S

R

A

PSE-1, CARB-3

2ce

Same as group 2c, but including oxyiminocephalosporins

S

R

A

RTG-4

2d

Penicillins (including cloxacillin)

V

R

D

OXA-1, OXA-2, OXA-10

2de

Same as group 2d, but including oxyiminocephalosporins (extendedspectrum)

V

R

D

OXA-11, OXA-15

2df

Penicillins, carbapenems

V

R

D

OXA-23, OXA-24, OXA-48

2e

Cephalosporins (excluding cephamycins)

S

R

A

CepA of Bacteroides fragilis

2f

Broad-spectrum including carbapenems

V

R

A

KPC-2, IMI-1, SME-1

3a

Broad-spectrum including carbapenems (not active on monobactams)

R

S

B (B1, B3)

IMP-1, VIM-1, NDM-1 L1 of Stenotrophomonas maltophilia

3b

Carbapenems (not active on monobactams)

R

S

B (B2)

CphA of Aeromonas hydrophila

*SBLI, mechanism-based serine β-lactamase inhibitors including clavulanate, sulbactam, tazobactam and avibactam; R, resistant; S, susceptible; V, variable susceptibility. Adapted from Bush K., Jacoby G.A. Antimicrob Agents Chemother 2010; 54(3):969–76.

classes. Enzymes of classes A, C and D have a serine residue at their active site, whereas those of class B require a zinc co-factor for activity (metallo-β-lactamases, MBLs). The relationships between structure and function are complex: members of the same molecular class exhibit some conserved functional properties (dependent on the structural features and catalytic mechanism defining the class), but can also exhibit significant functional diversity, while functional similarities can exist among enzymes of different classes (Table 138-2).2 Class A β-lactamases are found as resident chromosomally-encoded enzymes in some species (e.g. Klebsiella pneumoniae, Citrobacter koseri, Proteus vulgaris, Bacteroides fragilis) and are among the most prevalent acquired plasmid-encoded β-lactamases encountered in the clinical setting. They include, for instance, the plasmid-encoded broadspectrum TEM-1, TEM-2 and SHV-1 enzymes that have emerged and broadly disseminated in Enterobacteriaceae since the 1970s and have contributed the most common mechanism of acquired resistance to amino-penicillins in enterobacterial species such as Escherichia coli, Proteus mirabilis and Salmonella enterica. Their activity is usually inhibited by clavulanic acid, sulbactam, tazobactam and avibactam, thereby rendering penicillin derivatives active again. The broadspectrum TEM and SHV β-lactamases are not active against the expanded-spectrum cephalosporins (e.g. cefotaxime, ceftriaxone and ceftazidime). However, under the selective pressure generated by the use of the latter compounds, the TEM and SHV enzymes have shown the ability to evolve an expanded spectrum of activity through mutations at specific positions, which may lead to resistance against the

expanded-spectrum cephalosporins and monobactams.3 These TEMand SHV-type ESBL derivatives, of which a large number of variants have been described (http://www.lahey.org/studies/), have played an important role in the evolution of resistance to expanded-spectrum cephalosporins among Enterobacteriaceae since the mid-1980s. On the other hand, the TEM and SHV enzymes have also shown the ability to evolve mutations that confer resistance to β-lactamase inhibitors.4 More recently, plasmid-encoded class A ESBLs other than TEM and SHV derivatives have also emerged in Enterobacteriaceae. Of these, the CTX-M-type enzymes have been the most successful. In fact, they have largely replaced the TEM- and SHV-type ESBLs in many clinical settings, and are currently the most prevalent ESBLs in Enterobacteriaceae from several regions.5 The class A β-lactamases also include some enzymes with carbapenemase activity: the most important are the KPC-type (after Klebsiella pneumoniae carbapenemase) enzymes, which emerged in the late 1990s and have thenceforth disseminated worldwide providing a major contribution as a carbapenem resistance mechanism in carbapenem-resistant Enterobacteriaceae (CRE).6 Class C β-lactamases (also called AmpC-type enzymes) are found as resident chromosomally-encoded β-lactamases in several gramnegative bacilli including Pseudomonas aeruginosa, Acinetobacter baumannii, and some members of the family Enterobacteriaceae (e.g. Citrobacter freundii, Enterobacter cloacae, Serratia marcescens and Morganella morganii). Production of these enzymes is normally regulated and contributes to intrinsic resistance to those β-lactams that act as

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SECTION 7  Anti-infective Therapy

inducers and are hydrolyzed by the enzyme (e.g. ampicillin and narrow-spectrum cephalosporins). Escherichia coli is also provided with a chromosomally-encoded class C β-lactamase, but normally the gene is expressed only at negligible levels and is not inducible, which explains why most Escherichia coli strains remain susceptible to ampicillin and narrow-spectrum cephalosporins.7 Some genes encoding AmpC-type enzymes have been mobilized to plasmids and can disseminate by horizontal transfer. These plasmid-encoded AmpC-type β-lactamases are usually produced constitutively, and their prevalence among Enterobacteriaceae is increasing.4 Class C β-lactamases are active against penicillins and many cephalosporins (including cephamycins and some expanded-spectrum cephalosporins, but usually not cefepime), are not inhibited by clavulanic acid, sulbactam or tazobactam, but are inhibited by cloxacillin and avibactam.7 Class D β-lactamases (also called OXA-type enzymes after their efficient hydrolysis of oxacillin) are found as resident chromosomallyencoded enzymes in several bacterial species, and also as plasmidencoded enzymes. They were originally considered to be less important due to their overall lower diffusion and narrow substrate profile (including penicillins and some narrow-spectrum cephalosporins). However, the recent emergence of plasmid-encoded class D enzymes endowed with carbapenemase activity, which are spreading among major gram-negative pathogens including Acinetobacter spp. (e.g. OXA-23, OXA-24 and OXA-58) and members of the family Enterobacteriaceae (e.g. OXA-48) and which are responsible for acquired carbapenem resistance in those species,4 has remarkably increased the clinical relevance of these enzymes. Class D enzymes are usually resistant to clavulanate, sulbactam and tazobactam and, when co-produced with class A β-lactamases, can be responsible for an inhibitor-resistant phenotype. Class B β-lactamases are zinc-dependent enzymes whose catalytic mechanism is completely different from that of the serine-β-lactamases. MBLs are resistant to serine-β-lactamase inhibitors including the diaza­bicyclooctane derivatives such as avibactam, and unlike serineβ-lactamases, are inhibited by EDTA. The clinical importance of MBLs is largely related with their constant and efficient carbapenemase activity, and their spectrum often extends to most other β-lactams. MBLs are found as resident chromosomally-encoded enzymes in some environmental species of low pathogenic potential (e.g. Stenotrophomonas maltophilia, Aeromonas hydrophila, Elizabethkingia meningoseptica, Chryseobacterium indologenes), but since the mid-1990s several plasmid-encoded MBLs have emerged as acquired carbapenemases in isolates of gram-negative non-fermenters and of Enterobacteriaceae. The VIM, NDM and IMP-type enzymes are currently the most prevalent and widespread acquired MBLs encountered among clinical isolates.8

of the genetic context of the mecA gene.9 Recently, mecA-negative MRSA strains carrying a second type of mec gene, named mecC, have been detected from animal and human infections. The mecC gene is about 30% divergent from mecA and is not detected by molecular probes targeting mecA.11 Resistance to penicillin in Streptococcus pneumoniae is due to the presence of altered PBPs, encoded by genes that have undergone recombination with PBP genes from other species, to yield mosaic PBPs.12 On the other hand, in Enterococcus faecium mutations in PBP5 can be responsible for resistance to ampicillin, which is frequently detected in this species.13 β-Lactam resistance by altered PBP targets can also be encountered in some gram-negative pathogens including Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus influenzae.14

β-LACTAM RESISTANCE MEDIATED BY IMPERMEABILITY OR EFFLUX Reduced drug uptake is the third major mechanism responsible for β-lactam resistance in gram-negative bacteria, where β-lactams need to enter the periplasmic space to bind the PBP targets located in the cytoplasmic membrane. In fact, in gram-negative bacteria, the activity of β-lactams against the bacterial cell depends on the complex interplay of a number of factors (Figure 138-1), including: • the concentration of the antibiotic in the environment; • the rate of antibiotic entry through the outer membrane; • the amount of β-lactamase produced; • the catalytic efficiency of the β-lactamase for the antibiotic; and • the affinity of the PBPs for the antibiotic. Reduced drug uptake can be due either to a reduction or alteration in the porin channels used by β-lactams to cross the outer membrane, or to the presence of efflux pumps that can actively extrude β-lactams from the periplasmic space. Reduced uptake is often encountered as a β-lactam resistance mechanism in Pseudomonas aeruginosa, but also in Acinetobacter baumannii and Enterobacteriaceae. In Pseudomonas aeruginosa, mutational loss or alterations of the OprD2 porin, which is the entry channel for carbapenems, is one of the most common mechanisms of acquired resistance to these drugs, while upregulation of the resident RND-type MexAB multidrug efflux pump can contribute to acquired resistance to several β-lactams which are effluxed by the pump from the periplasmic space, including meropenem, and anti-pseudomonas cephalosporins and penicillins.15 Mode of action and resistance of β-lactam antibiotics in gram-negative bacteria

β-LACTAM RESISTANCE MEDIATED BY ALTERED PBPS

Altered PBPs are also a major cause of resistance against β-lactam antibiotics, especially among gram-positive cocci. Acquisition of a novel PBP, which takes over the functions of the resident PBPs and is not inhibited by conventional β-lactams, is responsible for methicillin resistance in staphylococci. Both methicillin-resistant Staph. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci are important causes of difficult-to-treat nosocomial infections. Moreover, community-associated and livestock-associated MRSA strains have emerged, compounding the epidemiology of MRSA infections.9,10 The modified PBP associated with methicillin resistance (PBP2a) is encoded by the mecA gene. Regulation of methicillin resistance is complex. Expression can be heterogeneous, whereby only a few cells express the phenotype. The mecA determinant apparently originated from some coagulase-negative staphylococci, and is associated with a peculiar type of MGE, named staphylococcal chromosome cassette mec (SCCmec), which is able to integrate at a specific locus (orfX gene) of the staphylococcal chromosome. Several types of SCCmec elements have been described, based on the type of ccr recombinase genes (involved in the mobilization of SCC elements) and on the structure

β-Lactam antibiotics

Porins

β-Lactamases

Outer membrane

Periplasm

Penicillin-binding proteins Cytoplasmic membrane

Figure 138-1  Mode of action and resistance of β-lactam antibiotics in gramnegative bacteria. In gram-negative bacteria, β-lactam activity depends on the complex interplay among several factors, including the concentration of the drug in the environment, the rate of entry through the outer membrane (usually across porins), the amount of β-lactamase produced and present in the periplasmic space, the catalytic efficiency of the β-lactamase for the antibiotic, and the affinity of the antibiotic for the penicillin-binding protein (PBP) targets, located in the cytoplasmic membrane.



Chapter 138  Mechanisms of Antibacterial Resistance

TABLE

138-3 

1185

Characteristics of a Selected Set of Aminoglycoside-Modifying Enzymes DISTRIBUTION

Resistance Mechanism

Name

Resistance Phenotype*

Gram-negative

N-acetyltransferases (AAC)

AAC(3)-I

Gm

AAC(3)-II AAC(3)-III AAC(3)-IV AAC(3)-VI AAC(6′)-I

Gm,Tm Gm,Tm Gm,Tm Gm Ak, Tm

AAC(6′)-II AAC(6′)-Ib-cr AAC(6′)-APH(2′′)

Gm, Tm Ak, Tm† Ak, Gm, Tm, Sm

Enterobacteriaceae, Acinetobacter baumannii, Pseudomonas aeruginosa Enterobacteriaceae Pseudomonas spp. Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae, Acinetobacter spp., Pseudomonas aeruginosa Enterobacteriaceae, Pseudomonas spp. Enterobacteriaceae

ANT(2′′)-I

Gm, Tm

ANT(3′′)-I

Sm

ANT(4′)-I

Ak, Tm

ANT(4′)-II

Ak, Tm

ANT(6)-I

Sm

ANT(9)-I

Sm

APH(3′)-III

Ak

APH(3′)-VI APH(6)-I

Ak Sm

O-nucleotydyltransferases (ANT)

O-phosphotransferases (APH)

Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii

Enterobacteriaceae, Acinetobacter baumannii Enterobacteriaceae, Pseudomonas aeruginosa

Gram-positive

Enterococcus spp.

Enterococcus spp., Staphylococcus spp.

Enterococcus spp., Staphylococcus spp. Enterococcus spp., Staphylococcus spp., Streptococcus spp. Enterococcus spp., Staphylococcus spp. Staphylococcus aureus, Enterococcus spp.

*Only the clinically relevant antibiotics are listed: Ak, amikacin; Gm, gentamicin; Tm, tobramycin; Sm, streptomycin. † Also confers decreased susceptibility to some quinolones. Data from Vakulenko S.B., Mobashery S. Clin Microbiol Rev 2003; 16(3):430–50 and Ramirez M.S., Tolmasky M.E. Drug Resist Updat 2010; 13(6):151–71.

In Enterobacteriaceae, reduced uptake by mutational loss or alteration of some porins, in combination with the overproduction of ESBLs or AmpC-type β-lactamases, can be responsible for a low-level carba­ penem resistance phenotype that can be selected during carbapenem treatment.16

Resistance to Aminoglycosides The first clinically effective aminoglycoside introduced in clinical practice was streptomycin, in the 1940s. Numerous aminoglycosides have since been isolated and synthetic derivatives were also produced. The most important aminoglycoside antibiotics for clinical practice are gentamicin, tobramycin, amikacin and streptomycin. They have an overall broad antimicrobial spectrum but are not active against anaerobes. Aminoglycosides bind to the bacterial ribosome (30S subunit) and interfere with protein synthesis exerting a bactericidal action. To reach the ribosomal target, aminoglycosides enter the cytoplasmic membrane via an energy-dependent transport mechanism which is not active in anaerobes. In gram-negative bacteria, aminoglycosides first bind to anionic sites on the cell envelope. This binding displaces magnesium ions and allows entry of the aminoglycosides across the outer membrane. Inactivation of aminoglycosides by aminoglycoside-modifying enzymes (AMEs) is the most common mechanism of acquired resistance against these antibiotics. Other resistance mechanisms include ribosomal target modification and reduced drug uptake. Aminoglycoside resistance genes encoding AMEs or rRNA methylases that modify the ribosomal target are believed to originate from genes present in aminoglycoside-producing species (e.g. Streptomyces griseus). AMEs belong to three major classes, depending on the type of modification that causes inactivation: phosphotransferases (APH),

acetyltransferases (AAC) and nucleotidyltransferases (ANT). Each class includes several enzymes that may differ by the site of modification on the substrate and by the substrate specificity (Table 138-3).17,18 Often AMEs are able to modify several structurally related aminoglycosides, and the spectrum of resistance conferred by each enzyme depends on the substrate specificity. Some AMEs are bifunctional enzymes that can modify aminoglycosides by two different mechanisms. One such enzyme is the bifunctional AAC(6′)-APH(2′′) enzyme, which is encoded by transposon Tn4001 found in Staph. aureus and in Enterococcus faecalis isolates, that apparently arose through the fusion of two genes, each encoding one of the partners. The AMEs must inactivate their targets before they reach the ribosomes and are either located inside the cell or associated with the cytoplasmic membrane. AMEs can be found as acquired resistance determinants in grampositive and gram-negative bacterial pathogens. In staphylococci, aminoglycoside resistance mediated by AMEs is well documented. In enterococci, the acquisition of AMEs such as the bifunctional enzyme AAC(6’)-Ie-APH(2”)-Ia, is of clinical relevance since it is responsible for high-level aminoglycoside resistance and the loss of synergistic action with β-lactams.17 In gram-negative bacilli, a large number of acquired AMEs has been detected, which can variably contribute to resistance to the various aminoglycosides. Some species have resident chromosomal AMEs that may contribute to the intrinsic resistance of that species versus some aminoglycosides (e.g. Serratia marcescens, which produces a chromosomally-encoded AAC(6’)-Ic enzyme that affects the activity of all aminoglycosides except streptomycin and gentamicin).18 Modification of the ribosomal binding site is another resistance mechanism to aminoglycosides. Modification can consist in methylation of the rRNA or in mutation of some ribosomal proteins. Methylation of rRNA can confer a high-level broad-spectrum aminoglycoside

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SECTION 7  Anti-infective Therapy

resistance including gentamicin, tobramycin and amikacin. Several types of plasmid-encoded 16S rRNA methylases have been discovered, spreading among gram-negative pathogens including Enterobacteriaceae and gram-negative non-fermenters. The most widespread 16S rRNA methylases are the ArmA and RmtB enzymes that have been detected worldwide in isolates from both human and animal origin and are often co-expressed with other clinically-relevant resistance determinants.19 Mutation of the S12 protein in the small ribosomal subunit can be associated with resistance to streptomycin. Reduced uptake has also been reported as a mechanism of aminoglycoside resistance. In Pseudomonas aeruginosa, in particular, mutational upregulation of the resident RND-type MexXY efflux system can be responsible for acquired resistance to multiple aminoglycosides.20

Resistance to Quinolones Quinolone antibiotics exert their antibacterial effects by inhibition of certain bacterial topoisomerase enzymes, namely DNA gyrase and topoisomerase IV. These bacterial enzymes regulate the topology of the bacterial chromosome (which is maintained in a supercoiled state) and their function is essential in chromosomal replication, segregation, transcription, recombination and repair. DNA gyrase and topoisomerase IV are heterotetrameric proteins composed of two subunits, designated A and B. The genes encoding the A and B subunits are referred to as gyrA and gyrB (DNA gyrase) or parC and parE (DNA topoisomerase IV; grlA and grlB in Staph. aureus). Quinolones bind to the quinolone-binding pocket of DNA topoisomerases while they are working on DNA by forming a ternary complex (enzyme–DNA–quinolone). This interaction blocks the enzyme activity and eventually results in DNA fragmentation and rapid killing of the bacterial cell. The affinity of quinolones for their dual topoisomerase targets can be different depending on the quinolone and on the bacterial species. In gram-negatives, DNA gyrase is the primary target for most quinolones, whereas topoisomerase IV appears to be the primary target in Staph. aureus and Strep. pneumoniae. However, different quinolones can have different primary targets in the same bacterial species and the primary target can be dependent on the bacterial species as well as on the quinolone structure. For instance, in Strep. pneumoniae topoisomerase IV is the primary target for ciprofloxacin while DNA gyrase is the primary target for sparfloxacin. Resistance to quinolones can be due to several different mechanisms including: 1) topoisomerase target modification by mutation; 2) reduced drug uptake by reduced permeability or active efflux; 3) topoisomerase target protection by specific proteins; and 4) drug inactivation. These mechanisms can variably cooperate among each other to increase stepwise the resistance level to quinolones.

QUINOLONE RESISTANCE BY TARGET MODIFICATION Alterations of the target topoisomerases by mutations that reduce the affinity for quinolones without compromising the enzyme function are overall the most common mechanism of acquired resistance to quinolones and have been reported in many bacterial species.21 The mutations associated with resistance are clustered in discrete regions of the enzyme subunits, which are called quinolone resistance determining regions (QRDRs). In most cases, the amino acid substitutions within the QRDR involve the replacement of a hydroxyl group with a bulky hydrophobic residue that alters the geometry of the quinolone-binding pocket present in the enzyme and impedes binding of the quinolone molecule.22 In Escherichia coli and other gram-negatives, DNA gyrase is usually the primary target and the first-step mutations leading to quinolone resistance usually occur in the QRDR of GyrA and also GyrB. Although quinolones are thought to interact primarily with the A subunit of DNA gyrase, there are mutations in the B subunit that also confer

quinolone resistance in some species. However, the frequency of GyrB mutations has been shown to be lower compared with the frequency of GyrA mutations. No GyrB mutations have been reported as resulting in cross-resistance between quinolones and the B subunit inhibitors coumermycin and novobiocin. This is consistent with the fact that the GyrB protein comprises two distinct domains: an N-terminal domain containing the sites for hydrolysis of adenosine triphosphate and binding of novobiocin and coumermycin, and a C-terminal domain containing the QRDR. Topoisomerase IV is usually a secondary target for quinolones in Escherichia coli and other gram-negatives. Thus, mutations in the QRDR of ParC are typically selected for in GyrA mutants (second-step mutations) and result in further decreased susceptibility. Second-step mutations that result in decreased quinolone susceptibility have also been reported in ParE, but they are overall less common in clinical isolates. In Staph. aureus and Strep. pneumoniae topoisomerase IV is usually the primary target of quinolones, and first-step mutations leading to quinolone resistance are usually found in ParC and ParE, while second-step mutations leading to further increased quinolone resistance are found in the gyrase subunits.21 In general, the nature of the primary target of each quinolone in a bacterial species can be deduced by the location of the first-step target mutations that are selected upon quinolone exposure. Combinations of multiple mutations within individual targets can also increase the resistance level. For instance, combinations of multiple mutations in the GyrA proteins were shown to be associated with higher minimum inhibitory concentration (MIC) values for ciprofloxacin than single point mutations. Similarly, combinations of single point mutations within GrlA were shown associated with higher ciprofloxacin MIC values than single mutations in Staph. aureus.23

QUINOLONE RESISTANCE BY DECREASED UPTAKE/ACTIVE EFFLUX DNA gyrase and topoisomerase IV are located in the cytoplasm of the bacterial cell. In order to reach their targets, quinolone antibiotics must enter the cell envelope. In gram-negative bacteria the fluoroquinolones must first cross the outer membrane. Changes in the outer membrane proteins of gram-negative bacteria have been associated with increased resistance to quinolones by decreased drug uptake.21,24 Active efflux as a mechanism of fluoroquinolone resistance has been reported in several bacterial species. In Staph. aureus the resident chromosomally-encoded NorA efflux pump is responsible for a low basal level of quinolone efflux, with a preference for hydrophilic fluoroquinolones, and can be responsible for increased resistance following mutations that cause overexpression of the norA gene.25 In P. aeruginosa, resistance to fluoroquinolones as well as to a number of other antimicrobial agents is often associated with mutational upregulation of resident RND-type multidrug efflux pumps, such as MexAB, MexCD, MexEF and MexXY, that can efflux fluoroquinolones.26 Escherichia coli has also been shown to possess resident efflux systems for quinolones, including EmrAB and AcrAB, that can decrease quinolone susceptibility upon mutational upregulation.26 Recently, plasmid-encoded quinolone efflux systems have also been reported in Enterobacteriaceae, namely QepA and OqxAB. QepA is a an efflux pump that belongs in the major facilitator superfamily (MFS) of transporters, and that can efflux some quinolones including nalidixic acid, ciprofloxacin and norfloxacin increasing the MICs up 2- to 64-fold.27 The qepA gene is often associated with other resistance determinants (e.g. the rmtB gene encoding a 16S ribosomal methylase conferring protection to aminoglycosides) in transferable resistance plasmids, and has been detected at high rates in China, but occasionally also in other countries.28 OqxAB is an RND-type efflux pump that was originally identified in animal isolates of Escherichia coli resistant to olaquindox, a quinoxaline derivative used in agriculture and as a growth promoter. OqxAB is a multidrug efflux pump that can extrude also chloramphenicol and some quinolones including nalidixic acid and ciprofloxacin, causing a moderate MIC increase

(8- to 16-fold) for these agents.29 Plasmids encoding OqxAB have mostly been detected in animal isolates, but also in clinical isolates of Enterobacteriaceae including Salmonella enterica and Escherichia coli. The oqxAB genes are also present in the chromosome of Klebsiella pneumoniae.30

QUINOLONE RESISTANCE BY TARGET PROTECTION Acquired quinolone resistance by protection of the topoisomerase target was discovered in the late 1990s and was the first example of plasmid-encoded transferable mechanism of quinolone resistance. Target protection is conferred by a family of small pentapeptide-repeat proteins, named Qnr proteins, that bind to the topoisomerase targets and protect them from the interaction with quinolones.30 A similar mechanism has evolved in bacteria to protect topoisomerases from microcins, which are proteins of the pentapeptide-repeat family that are produced by some bacteria as a mechanism of biological competition and can kill susceptible bacteria by inhibiting their topoisomerases. Qnr production leads to a 10- to 100-fold increase in the MIC for quinolones. Because MIC values for quinolones are often extremely low, the production of Qnr may be insufficient for MIC to reach the breakpoint for resistance (or even an intermediate level of susceptibility). Nevertheless, the MIC increase may significantly affect the mutant-prevention concentration (MPC) favoring the selection of QRDR mutants with higher levels of resistance.30 Several types of plasmid-encoded Qnr proteins, indicated by letters (e.g. QnrA, QnrB, QnrC, QnrD, QnrS) have been described, including multiple variants for some of these types, indicated by numbers (e.g. QnrA1, QnrA2 etc.).31 Acquired qnr genes have been reported worldwide, mostly in strains of Enterobacteriaceae, and this resistance mechanism is now considered of growing importance.28,30

QUINOLONE RESISTANCE BY DRUG INACTIVATION Inactivation by drug modification was the most recently described resistance mechanism to quinolones. Modification is due to acetylation, and is carried out by a plasmid-encoded AAC enzyme variant (named AAC(6’)-Ib-cr) that, in addition to aminoglycosides, has evolved (by mutations) the ability to acetylate also some quinolone molecules that have unsubstituted piperazinyl secondary amines, such as ciprofloxacin and norfloxacin (other quinolones lacking unsubstituted piperazinyl secondary amines are not affected). In presence of this mechanism, the MICs of quinolones are increased by two- to fourfold and usually remain lower than the breakpoint for susceptibility. However, as observed with Qnr proteins, the MIC increase may significantly affect the MPC and favor the selection of QRDR mutants with higher levels of resistance. Following its discovery, plasmidencoded AAC(6’)-Ib-cr has been detected worldwide, mostly in E. coli but also in other enterobacterial species.30

Resistance to Macrolides, Lincosamides and Streptogramins Macrolide, lincosamines and streptogramin (MLS) antibiotics are chemically distinct inhibitors of the protein synthesis acting by binding to the 50S subunit of the bacterial ribosome and usually resulting in a bacteriostatic effect. Macrolides are hydrophobic molecules having a central 12- to 16-membered-ring lactone attached to amino or neutral sugars. The macrolides of human importance are natural or semisynthetic 14-, 15- and 16-membered-ring molecules. Lincosamines are alkylderivatives of proline and are devoid of a lactone ring. Clindamycin is a semisynthetic derivative of 7-chloro-7-deoxy lincomycin, the firstdiscovered member of the family, and represents the only member of lincosamines currently used in the clinical practice. Streptogramins are composed of a mixture of two types of molecules: group A streptogramins and group B streptogramins. The two molecules act via a

Chapter 138  Mechanisms of Antibacterial Resistance

1187

synergistic interaction in the binding of the two antibiotics to the ribosome. Dalfopristin–quinupristin, a hydrosoluble derivative of pristinamycin, is the unique member of this class used in clinical practice. Although azithromycin has been used in the treatment of infections caused by some gram-negative bacilli such as Salmonella typhi and Shigella spp., Enterobacteriaceae and gram-negative non-fermenters are considered naturally resistant to MLS antibiotics due to resident efflux systems associated with a certain degree of impermeability of the outer membrane. Some clinically relevant enterococcal species, including Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus are intrinsically resistant to lincosamides and streptogramins. Resistance in these species is mediated by the presence of a resident lsa gene encoding an efflux pump. For some species, notably Haemophilus influenzae, the correlation between susceptibility testing and clinical outcome is weak and wild-type isolates are currently categorized as intermediate (see EUCAST clinical breakpoint v 6.0, http://www.eucast.org/clinical_breakpoints/). Acquisition of resistance to macrolides by naturally susceptible species was documented only one year after market introduction of erythromycin. In 1953, in fact, clinical isolates of macrolide-resistant staphylococci were described in reports from France, England, Japan and the USA.32 After these first cases, resistance to MLS antibiotics has become of clinical relevance in several cases. Resistance to macrolides can impair the efficacy of macrolide-including empirical regimens for the treatment of community-acquired pneumonia since, in some epidemiological settings the concurrent presence of macrolide resistance and reduced susceptibility to β-lactams is not uncommon in Strep. pneumoniae.33 Similarly, clindamycin resistance can impact on the treatment of skin, soft tissue and bone infections sustained by Staph. aureus and Streptococcus pyogenes.34 Resistance to macrolide in Strep. pyogenes can represent also a limitation in the treatment of pharyngitis in penicillin-allergic patients.35 Furthermore, from an epidemiological point of view, acquisition of resistance to MLS antibiotics by hyperepidemic clones has probably played a relevant role in the abrupt worldwide diffusion of strains of Clostridium difficile.36 Three main types of mechanisms can be responsible for acquired resistance to MLS antibiotics, including target modification, active efflux and drug inactivation by enzymatic modification.37 Table 138-4 summarizes the most clinically relevant mechanisms of acquired resistance together with the resulting phenotypes and their distribution.

RESISTANCE BY TARGET MODIFICATION Modification of the ribosomal target causing reduction of affinity for their binding site can cause resistance to MLS antibiotics. The most frequently encountered mechanism consists in a posttranscriptional modification of the 23S rRNA by methylases, usually named Erm (erythromycin resistance methylase), which add one or two methyl groups to a single adenine residue (A2058 in Escherichia coli) in the 23S rRNA moiety. Since adenine 2058 is a common binding site for macrolides, lincosamides and streptogramin B, this modification confers cross-resistance to all these drugs and the phenotype is called MLSB. After the first description in 1956, the number of Ermtype enzymes have grown and the nomenclature for these genes has varied. The nomenclature currently used, proposed by Roberts and colleagues in 1999,37 assigns two genes of ≥80% amino acid identity to the same class and same letter designation, while two genes that show ≤79% amino acid identity are given a different letter designation. An updated database of genes encoding transferable mechanisms of resistance to MLS antibiotics is available at the website http:// faculty.washington.edu/marilynr. rRNA methylase genes have been reported from a large number of gram-positive and gram-negative bacterial genera including intracellular and anaerobic species. However their clinical relevance is mostly linked to the spread in Staphylococcus spp. and Streptococcus spp. erm(A) and erm(C) genes predominate in staphylococcal species while erm(B) and erm(TR) are prevalent in streptococcal isolates (Table 138-4). Dissemination of these genes is attributed to the fact that these determinants can be transported by transposons and plasmids.

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TABLE

138-4 

SECTION 7  Anti-infective Therapy

The Most Relevant Acquired Resistance Mechanisms to MLS Antibiotics and Resulting Resistance Phenotypes DISTRIBUTION

Resistance Mechanism

Gene

Phenotype

Gram-positive

Gram-negative

erm(A)* erm(B)

Inducible or constitutive MLSB Inducible or constitutive MLSB

erm(C)

Inducible or constitutive MLSB

erm(D) erm(E) erm(F)

Inducible or constitutive MLSB Inducible or constitutive MLSB Inducible or constitutive MLSB

Staphylococcus, Enterococcus, Streptococcus Staphylococcus, Enterococcus, Streptococcus, Clostridium, Corynebacterium Staphylococcus, Enterococcus, Streptococcus, Clostridium, Corynebacterium

erm(G) erm(Q) erm(T) erm(X) erm(Y) erm(33) erm(35) erm(37) erm(38) erm(39) erm(40) erm(41) erm(43)

Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible Inducible

Haemophilus, Bacteroides Campylobacter, Escherichia, Haemophilus, Neisseria Bacteroides, Escherichia, Haemophilus, Neisseria Salmonella Bacteroides, Shigella Bacteroides, Haemophilus, Neisseria Bacteroides Bacteroides

cfr

PhLOPSA

Staphylococcus, Enterococcus

Escherichia

mef(A)‡

M

Staphylococcus, Enterococcus, Streptococcus, Clostridium, Corynebacterium

mef(B)

M

Bacteroides, Haemophilus, Neisseria, Escherichia, Salmonella Escherichia

msr(A)

Inducible MSB

msr(C) msr(D)

Inducible MSB Inducible MSB

lsa(B) lsa(C) lsa(E) vga(A) vga(B) vga(C) vga(D) vga(E) eat(A) sal(A)

LSAPh LSAPh LSAPh LSAPh LSAPh LSAPh LSAPh LSAPh LSAPh LSAPh

Staphylococcus, Enterococcus, Streptococcus, Corynebacterium Enterococcus Staphylococcus, Enterococcus, Streptococcus, Corynebacterium, Clostridium Staphylococcus Streptococcus Staphylococcus, Enterococcus Staphylococcus Enterococcus, Staphylococcus Staphylococcus Enterococcus Staphylococcus Enterococcus Staphylococcus

Esterases

ere(A) ere(B)

M M

Staphylococcus

Lyases

vgb(A) vgb(B)

SB SB

Enterococcus, Staphylococcus Staphylococcus

Transferases

lnu(A) lnu(B) lnu(C) lnu(D) lnu(E) vat(A) vat(B) vat(C) vat(D) vat(E) vat(H)

L L L L L SA SA SA SA SA SA

Staphylococcus Enterococcus, Staphylococcus, Streptococcus Streptococcus Streptococcus Streptococcus Staphylococcus Enterococcus, Staphylococcus Staphylococcus Enterococcus Enterococcus Enterococcus

Phosphorylases

mph(A) mph(B) mph(C) mph(D) mph(E)

M M M M M

TARGET MODIFICATION rRNA methylases

rRNA methyltransferase

or or or or or or or or or or or or or

constitutive constitutive constitutive constitutive constitutive constitutive constitutive constitutive constitutive constitutive constitutive constitutive constitutive

MLSB MLSB MLSB MLSB MLSB MLSB MLSB MLSB MLSB MLSB MLSB MLSB MLSB

Staphylococcus, Enterococcus, Streptococcus, Clostridium, Corynebacterium Staphylococcus Staphylococcus, Streptococcus, Clostridium Staphylococcus, Enterococcus, Streptococcus Corynebacterium Staphylococcus Staphylococcus Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Staphylococcus

spp.† spp.† spp.† spp.† spp.†

Bacteroides

EFFLUX Major Facilitator Superfamily

ATP-binding transporter

Escherichia, Neisseria, Bacteroides

INACTIVATING ENZYMES

Staphylococcus

M, macrolides; L, lincosamides; SA: streptogramins A; SB: streptogramins B; O: oxazolidinones; Ph: phenicols; P: pleuromutilins. *Includes also erm(TR). † Acid-fast bacteria, nontuberculous mycobacteria. ‡ Includes also mef(E). Data from Roberts M.C., Sutcliffe J., et al. Antimicrob Agents Chemother 1999; 43(12):2823–30.

Escherichia, Salmonella Escherichia

Clostridium Clostridium Haemophilus

Shigella, Escherichia Escherichia Escherichia Escherichia Escherichia

The expression of erm genes can be inducible or constitutive resulting in different phenotypes. When the expression is constitutive the resulting strain is resistant to all macrolides, lincosamides and streptogramin B. The synergy between streptogramin A and B is conserved, but in Staphylococcus spp. the bactericidal activity of the streptogramin combination is lost. In the inducible phenotype, the enzyme is only expressed in presence of 14- and 15-membered macrolides and the strain remains susceptible to 16-membered macrolides, lincosamides and streptogramins. The inducible expression depends on the sequence of the regulatory region upstream from the structural gene for the methylase. Regulation occurs by a translational attenuation mechanism in which the mRNA secondary structure normally prevents translation, which is released in the presence of inducing macrolides. Single nucleotide changes, deletions or duplications in the regulatory region can also convert inducibly resistant strains to constitutively resistant ones that are cross-resistant to MLSB antibiotics.32 Since selection of resistant mutants is not unusual during clindamycin therapy, a conservative approach should be suggested in the treatment of infection caused by inducible strains and the use of clindamycin should be discouraged when other therapeutic options are available. A new methyltransferase called Cfr, has recently been described in staphylococcal isolates. The target of this enzyme, differing by previously described Erm enzymes, is represented by the adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. This modification does not confer resistance to macrolides but impairs the efficacy of lincosamides, streptogramin A, oxazolidinones, pleuromutilins and phenicols.38

RESISTANCE BY EFFLUX Active efflux is another mechanism of resistance to MLS antibiotics, by pumping the antibiotic out of the cytoplasmic membrane, keeping intracellular concentrations low and avoiding the binding to the ribosomal target. Two classes of efflux pumps of the ATP-binding cassette (ABC) transporter superfamily or of the MFS have been increasingly detected in gram-positive pathogens. ABC transporters are composed of a channel with two cytoplasmic domains and two ATP-binding domains situated on the internal surface of the membrane. ABC transporters use ATP as the energy source, while MFS efflux pumps derive energy from the proton-motive force. The msrA gene, encoding a member of the ABC transporter superfamily, is a common cause of reduced susceptibility to 14- and 15-membered macrolides in staphylococcal isolates. Expression is inducible by macrolides. This determinant also confers resistance to streptogramin B, but only after induction by macrolides. However, the synergism between streptogramins A and B is conserved. Acquisition of two members of the MFS, mef(A) and mef(E) is clinically relevant in Strep. pyogenes and Strep. pneumoniae respectively. Due to the high degree of homology between the two genes they have been assigned to the same class (mef(A)). The resistance phenotype is characterized by reduced susceptibility to 14- and 15-membered macrolides. Clindamycin and 16-membered macrolide activity is preserved.

RESISTANCE BY ENZYMATIC MODIFICATION Several enzymes can act in modifying specific antibiotics. These proteins usually confer resistance to only one of the three classes (M, L, or S) or one component such as streptogramin A, but not streptogramin B. Enzymes which hydrolyze streptogramin B (encoded by vgb(A) and vgb(B) genes) or modify the antibiotic by adding an acetyl group (acetyltransferases) to streptogramin A (encoded by vat(A), vat(B) and vat(C) genes) have been described alone or in association in Enterococcus spp. and Staphylococcus spp. When present simultaneously, they confer resistance to dalfopristin–quinupristin. Nucleotidyltransferases of the lnu(A) class, encoding 3-lincomycin- and 4-clindamycin Onucleotidyltransferases, have been identified as a cause of isolated lincosamides resistance in staphylococcal strains. Similarly lnu(B) and lnu(C) genes can be responsible of resistance to lincosamides in

Chapter 138  Mechanisms of Antibacterial Resistance

1189

Streptococcus agalactiae isolates. Although mainly reported in enterobacteria, the phosphotransferase MphC and the esterase EreA can be responsible for erythromycin inactivation in Staph. aureus isolates.37

Resistance to Tetracyclines Tetracyclines are broad-spectrum bacteriostatic antibiotics that inhibit bacterial protein synthesis by binding the 30S ribosomal subunit and preventing the attachment of the aminoacyl-tRNA and eventually the elongation phase of protein synthesis. Resistance to tetracyclines can be due to several different mechanisms. From the clinical point of view the principal mechanisms of tetracycline resistance are represented by active efflux and ribosomal target protection.

RESISTANCE TO TETRACYCLINE BY RIBOSOMAL PROTECTION Acquired tetracycline resistance can result from production of elongation-factor G (EF-G)-like ribosomal protection proteins that interact with the ribosome so that protein synthesis is unaffected by the presence of the antibiotic. Several different tet determinants that confer tetracycline resistance by this mechanism have been identified (Table 138-5).39 The most studied determinants of ribosomal protection have been those encoded by the tet(M) and tet(O) genes. The ribosomal protection proteins encoded by the other classes have an amino acid sequence identity of at least 40% to Tet(M), and the mechanism of action is presumed to be similar for all ribosomal protection proteins. The Tet(M) ribosomal protection protein has amino acid sequence similarity to EF-G (which translocates the peptidyl transfer RNA during protein synthesis) and EF-Tu, has a ribosome-dependent guanosine triphosphatase activity, and seems to confer resistance by reversible binding to the ribosome. Ribosomal protection proteins interact with the ribosome at the level of the protein h34 causing the release of the tetracycline molecules. The ribosome returns to its standard conformational state and protein synthesis proceeds.39 Tet ribosomal protection proteins are encoded by different types of MGEs and are a common cause of acquired tetracycline resistance both in gram-positive and gram-negative bacteria (Table 138-5). This mechanism can confer resistance to both tetracycline and minocycline, but not to tigecycline, a new glycylcycline derivative of minocycline whose modified structure allows escaping most tetracycline resistance mechanisms including ribosomal protection and active efflux.

TETRACYCLINE EFFLUX SYSTEMS Acquired tetracycline efflux systems encoded by plasmid-encoded tet genes have been described both in gram-positive and gram-negative bacteria. These efflux pumps, generally, are membrane proteins with 12–14 transmembrane domains, member of the MFS of efflux systems, that are able to actively pump tetracyclines out of the cell preventing intracellular accumulation and consequently ribosome binding.39 The energy for the efflux is derived from the proton-motive force. Several different classes of Tet efflux pumps have been described, encoded by different types of MGEs and found as a common cause of acquired tetracycline resistance in bacteria (Table 138-5). Tet efflux pumps generally confer resistance to tetracycline but, with the exception of Tet(B), not to minocycline. Tigecycline is not affected by Tet efflux pumps. Expression of Tet efflux systems is often regulated by the presence of the antibiotic. In some cases (e.g. with Tet(A), Tet(B), Tet(C), Tet(D), Tet(E), Tet(G) and Tet(H)) expression is regulated by a repressor (TetR) which, in the absence of tetracycline, binds to the operator region upstream of the tet gene and represses the expression of the efflux pump.39 When tetracycline enters the cell, it binds the TetR repressor promoting a conformational change that results in a decreased ability to bind the operator region, thus allowing expression of the efflux pump. In other cases (e.g. with Tet(K) and Tet(L)), expression is regulated by mRNA attenuation in a similar way to that

1190

TABLE

138-5 

SECTION 7  Anti-infective Therapy

The Most Relevant Acquired Resistance Mechanisms to Tetracycline Antibiotics and Resulting Resistance Phenotypes DISTRIBUTION

Resistance Mechanism

Gene

Gram-positive

Gram-negative

RIBOSOMAL PROTECTION

tet(M)

Enterococcus, Staphylococcus, Streptococcus, Mycobacterium spp.† Enteroccoccus, Staphylococcus, Streptococcus, Mycobacterium spp.† Streptococcus Enteroccoccus, Staphylococcus, Streptococcus Enteroccoccus, Streptococcus Staphylococcus, Streptococcus Streptococcus

Enterobacteriaceae, Haemophilus, Stenotrophomonas

tet(O) tet(Q) tet(S) tet(T) tet(W) tet(32) tet(44) otr(A) EFFLUX

tet(A) tet(B) tet(C) tet(D) tet(E) tet(G) tet(H) tet(J) tet(K) tet(L) tet(V) tet(Y) tet(35) tet(39) tet(38) tet(40) tetAB(46) otr(B)

ENZYMATIC INACTIVATION

tet(X)

UNKNOWN

tet(U)

Mycobacterium spp.†

Enteroccoccus, Staphylococcus, Streptococcus, Mycobacterium spp.† Enteroccoccus, Staphylococcus, Streptococcus, Mycobacterium spp.† Mycobacterium spp.†

Staphylococcus Staphylococcus, Streptococcus Streptococcus Mycobacterium spp.†

Campylobacter, Enterobacteriaceae, Stenotrophomonas Enterobacteriaceae Stenotrophomonas Enterobacteriaceae Campylobacter Enterobacteriaceae Enterobacteriaceae, Haemophilus Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Haemophilus Enterobacteriaceae Enterobacteriaceae Stenotrophomonas Enterobacteriaceae, Stenotrophomonas

Enterobacteriaceae Enteroccoccus, Staphylococcus, Streptococcus

†

Acid-fast bacteria, nontuberculous mycobacteria. Data from Roberts M.C. FEMS Microbiol Lett 2005; 245(2):195–203.

described for gram-positive erm genes encoding rRNA methylase (see above) and cat genes encoding chloramphenicol acetyltransferases.40 Tetracyclines can also be effluxed by some resident MDR efflux systems of gram-negative bacteria. In some species (e.g. Proteus mirabilis, Pseudomonas aeruginosa), the basal-level efflux confers intrinsic resistance to tetracyclines. In other species, the basal-level efflux is not sufficient to confer intrinsic resistance, but mutations upregulating the resident efflux systems can be responsible of acquired tetracycline resistance. One of the best known examples is represented by mutations of the mar locus in E. coli, which lead to an overexpression of the transcriptional activator MarA, that in turn causes the overexpression of the resident multidrug efflux pump AcrAB causing tetracycline resistance.41 Efflux mediated by upregulation of some resident efflux systems has also been involved with acquired resistance to tigecycline in Enterobacteriaceae and Acinetobacter baumannii. In Escherichia coli and other enterobacteria, mutations of the regulatory genes ramA, marA, rarA and soxS, leading to overexpression of the resident AcrAB efflux system, have been associated with acquired tigecycline resistance.42 In Acinetobacter, decreased tigecycline susceptibility was found associated with mutations upregulating the resident AdeABC efflux system.43 In addition to point mutations, also insertion sequences can upregulate the expression of resident efflux systems.

Resistance to Chloramphenicol Chloramphenicol is a bacteriostatic antibiotic that binds to the 50S ribosomal subunit and inhibits the peptidyltransferase step in protein synthesis. Resistance to chloramphenicol is mostly due to

inactivation of the antibiotic by chloramphenicol acetyltransferase (CAT) enzymes that acetylate the antibiotic. In certain gram-negative bacteria, reduced drug uptake can also be responsible for resistance to chloramphenicol.

RESISTANCE BY DRUG INACTIVATION Chloramphenicol contains two hydroxyl groups that are acetylated in a reaction catalyzed by CAT enzymes. Monoacetylated and diacetylated derivatives are unable to bind to the 50S ribosomal subunit and to inhibit the prokaryotic peptidyltransferase. The cat genes are usually associated with MGEs and often carried on plasmids that mediate their diffusion among bacterial pathogens.44 Expression of the cat genes in gram-positive pathogens (Staph. aureus, Strep. pneumoniae and E. faecalis) is often inducible, and appears to be regulated by translational attenuation in a similar manner to the erm genes conferring resistance to macrolides (see above). In these cases the cat gene is preceded by a nine amino acid leader peptide, and the leader mRNA can form a stable stem-loop structure which masks the ribosome binding site of the cat gene. Chloramphenicol appears to cause the ribosome to stall on the leader sequence, opening the stem-loop structure, thereby exposing the cat ribosome binding site and allowing cat gene expression. In gramnegative bacteria, resistance to chloramphenicol is usually mediated by plasmid-mediated cat genes that are expressed constitutively.44

RESISTANCE BY DECREASED DRUG UPTAKE In gram-negative bacteria, resistance to chloramphenicol may also be due to reduced drug uptake mediated by chromosomal mutations or

by acquired resistance genes. In E. coli, for instance, chromosomal mutations of the mar locus can result in resistance to chloramphenicol and structurally unrelated antibiotics as part of the MAR phenotype, mediated by a reduced drug uptake mechanism (see below). Moreover, the cmlA1 gene carried on a mobile gene cassette associated with some integrons encodes a chloramphenicol efflux system that can contribute to acquired resistance to chloramphenicol in gram-negative bacteria.44

Chapter 138  Mechanisms of Antibacterial Resistance

TABLE

138-6 

van Gene Clusters Mediating Resistance to Glycopeptides in Gram-positive Cocci

Type of Target Modification

van Gene Cluster

Phenotype

Distribution

D-Ala–D-Lac

vanA

Inducible V,T

vanB

Inducible V

vanD

Constitutive V, T

vanM

V,T

Enterococcus spp., Staphylococcus aureus Enterococcus faecium, Enterococcus faecalis Enterococcus faecium, Enterococcus faecalis Enterococcus faecium

vanC

Inducible/ constitutive V

vanE

Resistance to Glycopeptides The glycopeptide antibiotics vancomycin and teicoplanin inhibit peptidoglycan synthesis in gram-positive bacteria by binding with high affinity to the terminal D-alanyl-D-alanine (D-Ala-D-Ala) group of the pentapeptide side chains of peptidoglycan precursors and blocking the transglycosylation and transpeptidation reactions required for poly­ merization of peptidoglycan. Gram-negative bacteria are intrinsically resistant to glycopeptides since these relatively large molecules cannot cross the outer membrane and reach their peptidoglycan target. Acquired resistance to glycopeptides is a major problem in enterococci, and has also been reported in staphylococci.

D-Ala–D-Ser

GLYCOPEPTIDE RESISTANCE IN ENTEROCOCCI

vanG

Inducible/ constitutive V Inducible V

Acquired resistance to glycopeptides can be relatively common, especially in Enterococcus faecium. Infections caused by glycopeptideresistant enterococci (usually named vancomycin-resistant enterococci, VRE) are difficult to treat since only few treatment options remain available.45 Resistance to glycopeptides is due to the production of low-affinity pentapeptide precursors, ending either with d-lactate (d-Lac) or d-serine (d-Ser) residues, which can be incorporated in the peptidoglycan. Production of these precursors is dependent on new biosynthetic pathways which include a new d-amino acid ligase and also enzymes that degrade the normal peptidoglycan precursors. Genes encoding these pathways (named van genes), together with regulatory genes, are usually found clustered on MGEs that, upon transfer, can confer glycopeptide resistance to the bacterial host. Several different clusters of van genes have been described, indicated by letters, that can be associated with different resistance phenotypes (Table 138-6).45

vanL

Inducible V

vanN

Constitutive V

vanA-type Resistance The vanA gene cluster is one of the most frequent glycopeptide resistance determinants encountered in enterococci and, therefore, among the most clinically relevant. It confers high-level resistance to vancomycin and teicoplanin, since its expression can be induced by both drugs. The vanA gene cluster is carried within a transposon (usually Tn1546) and is composed by genes involved in glycopeptide resistance (vanHAXYZ) and by regulatory genes (vanRS). VanA is the ligase that catalyzes the formation of d-Ala-d-Lac precursors. The vanH gene apparently encodes an enzyme that catalyzes the conversion of pyruvate, common in nature, to d-lactic acid, rarely found in nature. The VanA ligase uses this as a substrate to form the depsipeptide d-Ala-dLac, which is then incorporated into an alternative, vancomycinresistant peptidoglycan precursor (Figure 138-2). The VanX protein cleaves the d-Ala-d-Ala dipeptide, decreasing the amount of substrate that is available for the formation of the normal pentapeptide. This step is important since resistance would not be expressed in the presence of wild-type precursors which allow binding of glycopeptides. The VanY protein is a carboxypeptidase that may reduce the levels of the normal precursor already present so that the alternative precursor predominates. The genes vanR and vanS encode a two-component signal transducing regulatory system that sense the presence of glycopeptides by the VanS sensor and responds by activating the VanR transcriptional activator that, in turn, activates the transcription of the other van genes. The environmental stimulus that triggers the initial phosphorylation of VanS has not been identified, but it is probably

1191

Enterococcus gallinarum, Enterococcus casseliflavus Enterococcus faecalis Enterococcus faecalis Enterococcus faecalis Enterococcus faecium

V, vancomycin; T, teicoplanin. Adapted from Cattoir V., Leclercq R. J Antimicrob Chemother 2013; 68(4):731–42.

related to the presence of the glycopeptide and its interaction with the d-Ala-d-Ala target site, which inhibits transglycosylation and transpeptidation.

Other van-type resistances Other van gene clusters that are found as acquired resistance genes in enterococci are vanB, vanD, vanE, vanG, vanL, vanM and vanN, while the vanC gene cluster is intrinsic in Enterococcus gallinarum and Enterococcus casseliflavus (Table 138-6). Among them, vanB is the most widespread and clinically relevant. VanB-positive strains display various levels of inducible resistance to vancomycin but remain susceptible to teicoplanin that it is not an inducer. However, the emergence of mutants that express vanB constitutively and are also resistant to teicoplanin has been described.46 Resistance mediated by vanB may also be transferable. The other acquired van genes are overall less common. In some cases the genes are located chromosomally and are constitutively expressed. In some cases (e.g. vanD and vanM), transfer by conjugation has been demonstrated. The vanC resistance determinants are present on the chromosome in Enterococcus casseliflavus and Enterococcus gallinarum and are intrinsic characteristics of these species. VanC-harboring enterococci have low-level resistance to vancomycin and remain susceptible to teicoplanin. The pentapeptide that results from the action of the VanC ligase terminates in d-Ala-d-Ser.47 This substitution probably reduces vancomycin binding, albeit not to the same degree as the depsipeptide found in VanA and VanB enterococci. VanC-harboring strains with high-level resistance to glycopeptides as a result of the acquisition of the vanA gene cluster have also been isolated.

GLYCOPEPTIDE RESISTANCE IN STAPHYLOCOCCI The glycopeptides are front-line drugs for MRSA infections. Despite their abundant use, resistance to glycopeptides in Staph. aureus has remained overall uncommon and is phenotypically diverse, depending on the mechanism of resistance.48 High-level glycopeptide resistance is observed with strains that have acquired a VanA-type resistance mechanism identical to that

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SECTION 7  Anti-infective Therapy

The mechanism of peptidoglycan target modification in VanA-type resistance to glycopeptide antibiotics Glycopeptide-resistant cells

Glycopeptide-susceptible cells

Pyruvate

2 L-Ala

D-Ala–D-Ala

VanH NADH

VanX 2 D-Ala

Ala racemase 2 D-Ala

D-Lac

+D-Ala

VanA ATP

D-Ala–D-Ala

D-Ala–D-Lac

UDP–Mur–L-Ala–D-Glu–L-Lys

ligase

D-Ala–D-Ala

D-Ala–D-Ala–adding

enzyme

UDP–Mur–L-Ala–D-Glu–L-Lys

ATP

UDP–Mur–L-Ala–D-Glu–L-Lys–D-Ala–D-Lac

UDP–Mur–L-Ala–D-Glu–L-Lys–D-Ala–D-Ala Vancomycin

Vancomycin

UDP–Mur–L-Ala–D-Glu–L-Lys–D-Ala–D-Ala VanY (carboxypeptidase) UDP–Mur–L-Ala–D-Glu–L-Lys–D-Ala + D-Ala Figure 138-2  The mechanism of peptidoglycan target modification in VanA-type resistance to glycopeptide antibiotics. The various products of the vanA gene cluster are responsible for the synthesis of a modified peptidoglycan precursor and removal of the native precursors. ATP, adenosine triphosphate; Lac, lactate; UDP, uridine diphosphate. Adapted from Cattoir V., Leclercq R. J Antimicrob Chemother 2013; 68(4):731–42.

described for enterococci. These strains, indicated as vancomycin- or glycopeptide-resistant Staph. aureus (VRSA or GRSA), have been described since the early 2000s but thus far have remained unusual and do not exhibit a tendency to disseminate, possibly due to a remarkable fitness cost associated with the resistance mechanism.49 Lower level glycopeptide resistance is observed with strains that have acquired some chromosomal mutations. These mutants are also indicated as vancomycin- or glycopeptide-intermediate Staph. aureus (VISA or GISA). In some cases, named heteroresistant glycopeptideintermediate Staph. aureus (hVISA or hGISA) resistance is expressed only in a minority of the bacterial population. These strains are susceptible to vancomycin (MICs ≤2 mg/L) but with minority populations (typically 1 organism on 105 to 106 colony forming units) with higher vancomycin MIC, and their detection needs a population analysis profile. The GISA and hGISA strains exhibit a thicker cell wall which limits the access of glycopeptides to the d-Ala-d-Ala target in the peptidoglycan precursors. Furthermore, most of these strains show reduced peptidoglycan cross-linking when compared with isogenic revertants. The genetic mechanism for this cell-wall thickening is not fully understood, but seems to be related to mutation of many genes involved in the regulation of peptidoglycan metabolism.50 Several mutations associated with the GISA phenotypes have been characterized,50 and it has also been documented how stepwise mutations involving certain loci (e.g. graRS and vraSR and walKR) can lead first to a hGISA and then to a homogeneous GISA phenotype.51

amino acid and nucleotide synthesis. Sulfonamides are analogs of para-aminobenzoic acid. They competitively inhibit the enzyme dihydropteroate synthase (DHPS), which catalyzes the condensation of dihydropteridine with p-aminobenzoic acid at an early step of the folate synthesis pathway. Trimethoprim is an analog of dihydrofolic acid which competitively inhibits the enzyme dihydrofolate reductase (DHFR). DHFR catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid, the final step in tetrahydrofolic acid synthesis. Trimethoprim–sulfamethoxazole (co-trimoxazole) is a formulation of trimethoprim with a sulfonamide, which has a synergistic effect showing a broader spectrum of activity and a bactericidal action. A number of different resistance mechanisms to sulfonamides and trimethoprim have been described, including reduced drug uptake, target modification and target by-pass by resistant enzymes.

Resistance to Trimethoprim and Sulfonamides

Both high- and low-level resistance has been reported in several species. In some cases, acquired trimethoprim resistance may be due to chromosomal mutations leading to: 1) overproduction of the host DHFR caused by promoter mutation, thus requiring more

Trimethoprim and sulfonamides are synthetic agents that affect the biosynthesis of tetrahydrofolic acid, an essential metabolite used in

INTRINSIC RESISTANCE TO TRIMETHOPRIM AND SULFONAMIDES Reduced drug uptake is responsible for intrinsic resistance to trimethoprim of Pseudomonas aeruginosa. Intrinsic resistance to trimethoprim in a number of other species (e. g. Acinetobacter baumannii and Stenotrophomonas maltophilia) is due to host DHFR enzymes with low affinity for the drug. Enterococci, which unlike other species are able to use exogenous preformed folates, exhibit reduced susceptibilities to sulfonamides and trimethoprim.

ACQUIRED RESISTANCE TO TRIMETHOPRIM

trimethoprim concentration for the inhibition (described in Enterobacteriaceae); 2) mutations in the DHFR structural gene (described in streptococci, staphylococci). These two mechanisms are often associated in Enterobacteriaceae and in Haemophilus influenzae resulting in high-level resistance.52 High-level resistance to trimethoprim in enterobacteria is mostly caused by the acquisition of exogenous genes that encode a trimethoprim-resistant DHFR with an altered active site. Several different trimethoprim-resistant DHFRs have been characterized in gram-negative organisms, belonging in at least two groups, encoded by the dfrA and dfrB genes. In Enterobacteriaceae these genes are usually carried on mobile gene cassettes associated with integrons.53 The acquisition of the trimethoprim-resistant DHFR genes, dfrA, and the mutation of the chromosomal DHFR gene (dfrB) are currently considered to be key determinants of trimethoprim resistance in Staph. aureus of human origin.54

ACQUIRED RESISTANCE TO SULFONAMIDES Chromosomally-encoded sulfonamide resistance has been described and resistance seems to be due to an increased production of paraaminobenzoic acid and to alterations of DHPS that lower the enzyme affinity for sulfonamides. Acquired sulfonamide resistance can also result from the acquisition of plasmids harboring genes that encode a drug-resistant DHPS. This mechanism is typical for gram-negative bacilli and there are at least three genes involved, named sul1, sul2 and sul3. These genes code for a DHPS with low affinity for sulfonamide and confer high resistance levels.52

Resistance to Other Antibiotics Linezolid has been the first licensed oxazolidinone agent, and is mostly used to treat infections caused by vancomycin-resistant enterococci and MRSA. Linezolid is a protein synthesis inhibitor that targets the large subunit of the bacterial ribosome. Acquired resistance to linezolid remains uncommon but has been reported both in enterococci and in staphylococci, either sporadically or even in small outbreaks.55 Resistance can be due to mutations of the ribosomal target (nucleotide substitutions of 23S rRNA, such as G2505A or G2576U, or mutations of the L3 and L4 ribosomal proteins) or to target modification by methylation at specific positions of the 23S rRNA, that impede linezolid binding to the target.56 Resistance mediated by ribosomal target mutations is usually selected after a long exposure to the drug and, in the case of rRNA target mutations, resistance can be expressed at variable levels depending on the number of rDNA genes that carry the mutation. Ribosomal rRNA methylation, on the other hand, is mediated by the plasmid-encoded Cfr methyltransferase, which modifies the 23S rRNA at residue A2503. The latter mechanism, which is of remarkable concern due to the transferable nature, is responsible for cross-resistance to linezolid and other anti-ribosomal drugs including phenicols, lincosamides, pleuromutilins and streptogramin A (the so-called PhLOPSA phenotype), and its dissemination has likely been promoted by the use of florphenicol in veterinary medicine. Fusidic acid binds elongation-factor G (EF-G) preventing its release from the ribosome and blocking bacterial protein synthesis. In staphylococci, which are the main clinical target for fusidic acid, acquired resistance can be due either to mutations in the fusA gene, which encodes EF-G, or to the acquisition of resistance genes (fusB and fusC) that encode proteins able to bind the ribosome and protect it from fusidic acid.57 Reduced uptake and enzymatic inactivation have also been occasionally reported as resistance mechanisms to fusidic acid. Mupirocin inhibits bacterial protein synthesis by inhibition of isoleucyl tRNA synthetase (IleRS) and is used as a topical antibiotic for nasal decolonization of Staph. aureus (both MSSA and MRSA). Lowlevel resistance is caused by mutations in the chromosomal gene encoding the IleRS enzyme, which are not associated with high fitness cost. Acquisition of a novel mupirocin-resistant isoleucyl tRNA synthetase, encoded by the mupA gene can confer high-level resistance.

Chapter 138  Mechanisms of Antibacterial Resistance

1193

Plasmids carrying mupA have been detected in all major circulating MRSA clones. Recently a new plasmid-mediated mechanism for highlevel mupirocin resistance, mupB, was detected, but the prevalence of this mechanism remains to be determined.58 Metronidazole resistance in Helicobacter pylori usually results from mutational inactivation of the rdxA gene that encodes NADPH nitroreductase. This enzyme converts metronidazole into a metabolite that is toxic for the bacterial cell. Inactivation of other reductase-encoding genes could also be involved in metronidazole resistance.59 Polymyxins are last-resort drugs for multiresistant gram-negative pathogens. They exert bactericidal action by damaging the bacterial membrane after binding to the lipid A moiety of the bacterial lipopolysaccharide (LPS) present in the outer membrane of gram-negative bacteria. The interest for these drugs was recently increased by the dissemination of extremely drug-resistant (XDR) gram-negative pathogens for which polymyxins are among the few drugs that retain activity. Resistance to polymyxins generally arises following modification of the LPS target by decoration of lipid A with amino-arabinose or phosphoethanolamine residues, thereby reducing the negative charge of lipid A and the binding of polymyxins. A similar resistance mechanism has been detected in polymyxin-resistant clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter, and can be due to different chromosomal mutations.60 In Acinetobacter, acquired polymyxin resistance has also been associated with mutations causing a loss of the LPS target. Fosfomycin is a peptidoglycan synthesis inhibitor that acts by blocking the MurA enzyme, involved in the first steps of the peptidoglycan biosynthetic pathway. Its interest was largely confined to treatment of uncomplicated urinary tract infections, but recently it has also been reconsidered as a salvage option for infections caused by some XDR gram-negative bacteria. Resistance to fosfomycin can be due to several mechanisms including: 1) chromosomal mutations that alter the expression of the transport systems (for 1-α-glycero-3 phosphate and for hexose monophosphates) that fosfomycin uses to enter the cytoplasmic membrane; 2) chromosomal mutations altering the affinity of MurA enzyme for fosfomycin; 3) chromosomal mutations causing overexpression of the MurA enzyme; and 4) inactivation of fosfomycin by modifying enzymes.61 Several plasmid-encoded inactivating enzymes, such as FosA, FosB, FosC, FosD, FosK, FomX, FomA, and FomB have been described. FosA, the first characterized, is a glutathione S-transferase, which catalyzes the addition of glutathione to fosfomycin.

Resistance in Mycobacterium tuberculosis Despite the global fall in incidence and mortality related to tuberculosis (TB), MDR and extensively drug-resistant (XDR) TB represents an emerging challenge. The World Health Organization (WHO) estimated there were 210 000 drug-resistant TB-related deaths in 2013 worldwide.62 Since isoniazid and rifampin represent the backbone agents of combination therapy commonly used in the treatment of TB, the emergence of resistance to these agents poses a serious clinical challenge. Resistance to isoniazid was reported soon after its introduction in 1952. Isoniazid inhibits the synthesis of mycolic acid of the cell wall and triggers the production of toxic free radicals. Modification of numerous genes has been involved in the development of isoniazid resistance. The most common mechanism of resistance involves the katG gene that encodes for a catalase-peroxidase enzyme essential for the conversion of isoniazid to the active form. Mutations in this gene, causing enzyme conformational changes, usually lead to a high level of resistance. The most frequently observed mutation occurs at codon 315. Also mutations in the inhA gene, which encodes for an NADH-dependent enoyl-ACP reductase, and/or in its promoter cause low-level isoniazid resistance associated with ethionamide cross-resistance.

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SECTION 7  Anti-infective Therapy

Rifampin and related antibiotics (rifabutin and rifapentine) block transcription initiation by binding to the β subunit of the bacterial RNA polymerase. Resistance is caused by mutation in the rpoB gene that encodes this subunit. Most often these mutations are located at codons between nucleotide 507 and 533 (numbered according to rpoB coding sequence of Escherichia coli). From a clinical standpoint resistance to pyrazinamide and ethambutol also are relevant. Resistance to pyrazinamide is most commonly caused by mutations in the pncA gene, or its upstream region. The pncA product is required to convert pyrazinamide into its active form. The target of ethambutol is the arabinosyltransferase enzyme involved in mycolic acid synthesis. Acquired resistance is frequently caused by mutations in the embB gene encoding for this enzyme.63

Multidrug Resistance Bacteria are often resistant to more than one antimicrobial agent. Multidrug resistance can be conferred by three mechanisms: 1) reduced permeability affecting more than one drug; 2) active efflux affecting more than one drug; 3) presence of multiple resistance genes. Reduced permeability is generally caused by mutational alterations affecting the structure of the outer membrane of gram-negative bacteria, mostly consisting of the reduced expression of porins that are the main entry channel for several antibiotics. One of the best known examples is the reduction of the outer membrane protein OmpF in Escherichia coli, leading to a decreased uptake of antibiotics.26 Active efflux of antibiotics is a common resistance mechanism. Some efflux pumps are only able to pump out a single antibiotic and its close structural homologues (e.g. pumps dedicated to tetracycline efflux). However, more general-purpose efflux systems also exist. These pumps can handle a wide variety of different compounds, including many antibiotics, and thus contribute multidrug resistance phenotypes. Efflux systems may belong to a number of different families:26 • ATP-binding cassette (ABC); • major facilitator superfamily (MFS); • resistance-nodulation-division (RND); • small multidrug resistance (SMR); • multidrug and toxic compound extrusion (MATE). Efflux systems can be composed of either a single polypeptide or multiple polypeptide components, depending on the family (Figure 138-3). Multicomponent efflux systems are typical of gram-negative bacteria, where the compounds must be transported across both the cytoplasmic and outer membrane. The ABC transporters are dependent on ATP as an energy source for their activity, whereas the protonmotive force is used by other transporters. Many of these efflux systems are encoded by resident chromosomal genes and provide a contribution to the basal level of resistance to

various antibiotics expressed by the corresponding bacterial species. In this case, mutations can be responsible for upregulation of the efflux system resulting in increased resistance to multiple antibiotics (depending on the spectrum of the substrates recognized by the system). Well known examples of similar systems are the AcrAB pump of Escherichia coli and the MexAB pump of Pseudomonas aeruginosa, which belong to the RND family. The latter pump is responsible for efflux of several different compounds under basal conditions, and can be upregulated by mutations. Mutational upregulation can contribute to acquired multidrug resistance to several anti-pseudomonas agents including fluoroquinolones, anti-pseudomonas penicillins and cephalosporins, and meropenem, but not imipenem or aminoglycosides. Resistance to disinfectants is usually also mediated by efflux pumps.26

Genetic Bases of Acquired Antibiotic Resistance Acquired antibiotic resistance can arise by mutations of chromosomal genes or by acquisition of exogenous resistance genes following events of horizontal gene transfer between bacteria. Mutations leading to resistance can affect a single antibiotic or class of antibiotics, or can even be responsible for the emergence of MDR phenotypes. The latter occurs when mutations upregulate multidrug efflux pumps or affects regulatory systems that activate multiple resistance mechanisms. The MAR (multiple antibiotic resistance) system in Escherichia coli is one of the best studied regulatory systems that controls resistance to multiple antibiotics by different mechanisms.26 The system includes a three-gene operon containing marRAB. The MarR product acts as a negative regulator for the mar operon. The MarA product is required for resistance and acts by downregulating the expression of OmpF protein and upregulating the expression of the AcrAB efflux pump (Figure 138-4). Mutations in marR or in the promoter region of the mar operon can activate expression of marA leading to decreased susceptibility to multiple antibiotics (e.g. tetracycline, chloramphenicol, rifampin, nalidixic acid) that can be effluxed by the AcrAB pump and/or enter the cell via OmpF. Homologues of the mar locus exist in other members of the family Enterobacteriaceae as well as in other bacteria. Acquisition of resistance genes by horizontal gene transfer is an important mechanism of evolution of microbial drug resistance. Acquired resistance genes are typically associated with MGEs, such as

The MAR system of Escherichia coli and its regulation

marR

Structure of multidrug transporter families

MarR

OM

micF

marA

marB

MarA

acrR

acrA

acrB

Downregulation

CM ATP

ADP+Pi ABC

AcrR

H+

H+

H+/Na+

H+

RND

MFS

MATE

SMR

Figure 138-3  Structure of different multidrug transporter families. Different subunits are indicated by different colors. Efflux direction is indicated by a solid arrow. The energy source for efflux is indicated by a broken arrow. The cellular membrane (CM) is present in all cells, but gram-positive bacteria lack an outer membrane (OM). Some multidrug transporter families are present in both gram-positive and gram-negative bacteria as indicated by dotted lines for the OM (see text for details.)

OmpF

AcrA

AcrB

Efflux pump upregulation

Figure 138-4  The MAR system of Escherichia coli and its regulation. The MarR product acts as a negative regulator for the mar operon. The MarA product acts by downregulating the expression of OmpF protein (via upregulation of micF antisense RNA) and upregulating the expression of the AcrAB efflux pump. Production of MarA is normally repressed. Mutations in marR or in the promoter region of the mar operon can activate expression of marA leading to decreased susceptibility to multiple antibiotics that can be effluxed by the AcrAB pump and/ or enter the cell via OmpF.



Chapter 138  Mechanisms of Antibacterial Resistance

plasmids or integrative and conjugative elements (ICEs, formerly named conjugative transposons), that can be transferred between different bacterial cells by conjugation.53,64 Plasmids and ICEs can carry multiple resistance genes and, upon transfer, confer an MDR phenotype to the new host. Conjugative plasmids are circular DNA molecules that contain an origin of replication, a locus for partitioning, genes encoding plasmid maintenance and transfer functions, and accessory genes that often include one or more resistance determinants (Figure 138-5). Some plasmids are lacking transfer functions, but can be transferred if these functions are provided in trans by a conjugative plasmid simultaneously present in the cell. Transposons and integrons can be responsible for the capture of resistance genes on plasmids and ICEs and for their dissemination among these elements. Transposons are MGEs that range in size from a few to more than 150 kilobases and can move from one site to another of the same or of another replicon by a mechanism named

1195

transposition (Figure 138-6). Several different transposons have been described, and many of them carry one or more antibiotic-resistance determinants.64 Integrons are a peculiar group of genetic elements consisting of an integrase gene and a nearby recombination site at which mobile gene cassettes can be directionally inserted or excised by a site-specific recombination mechanism catalyzed by the integron integrase. The mobile gene cassettes are small units usually containing a single gene and a recombination site, which is recognized by the integron integrase (Figure 138-7). There are two groups of integrons: resistance integrons and superintegrons. Superintegrons are found in many gram-negative species and are located on the chromosome. These integrons may contain tens to hundreds of gene cassettes, which encode a large variety of different functions. Resistance integrons contain a lower number of gene cassettes which usually carry resistance determinants to antibiotics or disinfectants. The most common resistance integrons belong to

The general structure of a conjugative plasmid carrying antibiotic resistance genes

Replication, partitioning and maintenance modules

Transfer modules

Heavy metal resistance genes

Resistance modules

Others

Figure 138-5  The general structure of a conjugative plasmid carrying antibiotic resistance genes. The plasmid is a circular DNA molecule (the map is shown linearized to facilitate readability). The plasmid has a modular structure including modules for plasmid replication, partitioning and stable maintenance, and for the plasmid transfer apparatus; in addition it carries resistance genes for antibiotics and heavy metals, which are associated with transposons.

The structure of transposons carrying resistance genes res

IR Tn3 subgroup

tnpA

IR tnpR

resistance genes (bla)

DR

DR Pc

Tn402 harboring integron

IR Tn21/Tn501 subgroup

res tnpA

tnpR

IR resistance genes (mer)

DR

DR Pc

res

IR Tn5044 subgroup

resistance genes (mer)

tnpA

IR tnpR

DR

DR Pc

Figure 138-6  The structure of transposons carrying resistance genes. Transposons carry genes (tnp) encoding the enzymes (transposases and resolvases) responsible for the transposition process. The transposons are delimited by inverted repeats (IR) which are recognized by the transposases, and usually flanked by direct repeats (DR) that are generated following the transposition process. Resistance genes (to antibiotics, heavy metals, disinfectants) can be found at different positions and are mobilized together with the transposon.

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SECTION 7  Anti-infective Therapy

TABLE

The structure of integrons

138-7 

attC intI

attI

Resistance and Mechanism of Resistance for Gene Cassettes

Antimicrobial Agents

Resistance Determinants (Examples)

β-Lactams

Class A β-lactamases (blaGES; blaPSE; blaCARB) Class B β-lactamases (blaIMP-type; blaVIM-type) Class D β-lactamases(blaOXA-10; blaOXA-9)

Aminoglycosides

Aminoglycoside adenylyltransferases (ant(3’)-1a) Aminoglycoside acetyltransferases (aac(6’)-Ia) Aminoglycoside phosphotransferases (aphA15)

Chloramphenicol

Chloramphenicol acetyltransferases (catB2) Chloramphenicol exporter (cmlA, cmlB)

Trimethoprim

Class A dihydrofolate reductases (dfrA1) Class B dihydrofolate reductases (dfrB1)

Erythromycin

Erythromycin esterases (eraA1)

Lincosamides

Lincomycin nucleotidyltransferases (linF)

Rifampin (rifampicin)

ADP ribosylation (arr2)

Fosfomycin

Fosfomycin inactivating enzyme (fosA)

Antiseptics and disinfectants

SMR-type efflux pumps (smr1, qacE)

Mobile gene cassette

IntI integrase

Circular intermediate

intI

Adapted from Partridge S.R., et al. FEMS Microbiol Rev 2009; 33(4):757–84.

Cassette array (directional) Figure 138-7  The structure of integrons. The integron consists of an intI gene, encoding a DNA integrase, and a recombination site (attI), which is located upstream from the integrase gene. The integron integrase can insert and excise mobile gene cassettes at the attI recombination site via a site-specific recombination mechanism between attI and a recombination site (attC) that is present in the gene cassette. The gene cassettes are small mobile genetic units that normally contain a single gene (indicated by the red and blue arrows) and the attC recombination site. In resistance integrons most gene cassettes carry resistance genes to antibiotics and disinfectants (see text for details).

class 1, but other classes are known as well. A large number of gene cassettes have been described, including resistance genes for β-lactams, aminoglycosides, trimethoprim, chloramphenicol and antiseptics and disinfectants (Table 138-7). Generally the cassettes do not have promoters, but transcription occurs from one of two promoter sequences present upstream from the integron recombination site. Integrons are widespread in Enterobacteriaceae and also in gram-negative nonfermenters. ISCRs are another type of MGEs that can capture resistance genes, and that are often found associated with integron platforms.53,64

Conclusions Antibiotic resistance is ubiquitous and increasing. The most challenging resistant pathogens from the clinical and epidemiological standpoint are currently represented by MRSA, VRE, ESBL-producing Enterobacteriaceae, and carbapenemase-producing gram-negative bacilli. These strains usually exhibit MDR or extensively drug-resistant (XDR) phenotypes for which the treatment options may be very limited. Examples of XDR pathogens are represented by carbapenemresistant Acinetobacter baumannii (CRAb), which usually remain susceptible only to polymyxins, and by CRE, which often remain susceptible only to polymyxins, tigecycline and some aminoglycosides.65 Selection and dissemination of resistant strains following the use of antimicrobial agents is unavoidable. However, the phenomenon can be minimized by the prudent use of antibiotics and a strict implementation of infection control and prevention measures. The misuse and overuse of antibiotics and the presence of poor hygienic conditions facilitate the cross-transmission of resistant strains in healthcare settings and also in the community. References available online at expertconsult.com.

KEY REFERENCES Almeida Da Silva P.E.A., Palomino J.C.: Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs. J Antimicrob Chemother 2011; 66(7):1417-1430. Bush K., Jacoby G.A.: Updated functional classification of beta-lactamases. Antimicrob Agents Chemother 2010; 54(3):969-976. Cattoir V., Leclercq R.: Twenty-five years of shared life with vancomycin-resistant enterococci: is it time to divorce? J Antimicrob Chemother 2013; 68(4):731-742. Chambers H.F., Deleo F.R.: Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 2009; 7(9):629-641.

Jacoby G.A.: Mechanisms of resistance to quinolones. Clin Infect Dis 2005; 41(Suppl. 2):S120-S126. Macgowan A.P., BSAC Working Parties on Resistance Surveillance: Clinical implications of antimicrobial resistance for therapy. J Antimicrob Chemother 2008; 62(Suppl. 2):ii105-ii114. Munoz-Price L.S., Poirel L., Bonomo R.A., et al.: Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 2013; 13(9):785-796. Poole K.: Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 2005; 56(1):20-51.

Ramirez M.S., Tolmasky M.E.: Aminoglycoside modifying enzymes. Drug Resist Updat 2010; 13(6):151-171. Roberts M.C.: Update on acquired tetracycline resistance genes. FEMS Microbiol Lett 2005; 245(2):195-203. Roberts M.C., Sutcliffe J., Courvalin P., et al.: Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrob Agents Chemother 1999; 43(12):2823-2830. Toleman M.A., Walsh T.R.: Combinatorial events of insertion sequences and ICE in Gram-negative bacteria. FEMS Microbiol Rev 2011; 35(5):912-935.



Chapter 138  Mechanisms of Antibacterial Resistance 1196.e1

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