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recently gained prominence in Enterobacteriaceae. More than 300 natural ESBL variants have been identified since the mid-1980s but in-vitro studies suggest that ESBL evolution has certainly not come to an end; they may also help in predicting future developments.124
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ESBLs are known as extended-spectrum because they are able to hydrolyze a broader spectrum of β-lactam antibiotics than the simple parent β-lactamases from which they are derived. These β -lactamases are thus capable of hydrolyzing penicillins, broad-spectrum cephalosporins and monobactams, hence they are called extended spectrum beta-lactamases (ESBLs). They are acquired plasmid-mediated β-lactamases. They constitute a heterogenous molecular cluster (20 to
>90% identity) with great diversity in substrate preferences and susceptibility profiles. They have the ability to inactivate β-lactam antibiotics containing an oxyimino-group such as oxyimino-cephalosporins (e.g., ceftazidime, ceftriaxone, cefotaxime) as well as oxyimino-monobactam (aztreonam). They are not active against cephamycins and carbapenems. Generally, they are inhibited by ß-lactamase-inhibitors such as clavulanate and tazobactam.126,127
Mechanism of Action of Β-Lactamases and Extended Spectrum β-Lactamases The most important and widespread mechanism of resistance to β-lactam antibiotics in Gram-negative bacteria is by enzyme mediated hydrolysis. Three classes of enzymes that can hydrolyse β-lactam antibiotics are (1) the β-lactamases (2) acylases, and (3) esterases. The β-lactamase enzymes hydrolyze the β-lactam ring of the β-lactam antibiotics to acidic derivatives without antibacterial properties thus rendering the drug ineffective.119 Classes of such β-lactam antibiotics include penicillins, cephalosporins, and monobactams. With the altered configuration of
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the active serine site opening the active serine site to accommodate the large oxyimino side chain, as in ESBLs, there is increase in the substrate spectrum, hence the extended spectrum of the ESBLs. 122
The β-lactamases with relatively narrow substrate specificities are described as either penicillinases or cephalosporinases according to their substrate specificities.
Other "broad spectrum" enzymes are less discriminant and can hydrolyze a variety of β-lactam antibiotics.119
Despite the diversity of ESBL types and their different origins, most have qualitatively similar hydrolytic activity, being able to hydrolyze all cephalosporins except cephamycins. Aztreonam is a substrate too, as are all penicillins except temocillin. The carbapenems are virtually all stable to ESBL. Most ESBLs are inhibited by beta-lactamase inhibitors such as clavulanate, sulbactam, or tazobactam in vitro; but the clinical effectiveness of beta-lactam/beta-lactamase inhibitor combinations cannot be relied on consistently for therapy. This inhibition by beta-lactamase inhibitors is, however, useful for in vitro confirmation of the presence of ESBLs.128,129 Some TEM (e.g. TEM-3) and most SHV variants similarly have strong activity against cefotaxime and ceftazidime, whereas others with different substitutions have a predominantly ceftadizimase profile and are relatively less active against cefotaxime and ceftriaxone.116 In contrast, most CTX-M enzymes are more active against cefotaxime, ceftriaxone and cefepime than
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ceftazidime, which has important implications for laboratory detection. However, ceftazidimase activity and contingent resistance is much increased for some mutants such as CTX-M-15 and -19. 116,130 Depending on the particular enzyme, its quantity, and the impermeability of the host strain, some ESBL producers may (falsely) appear susceptible to oxyimino-cephalosporins. Klebsialla and E. coli remain the most frequent hosts of CTX-M and TEM / SHV ESBLs, but these enzyme types also occur widely in enterobacteriaceae. TEM, SHV and CTX-M ESBLs remain rare in non-fermenters. The stability of temocillin and the cephamycins reflects the presence of an α-methoxy group on the 6 or 7 position;
that of the carbapenems reflects their unique ring structure.116 Classification Schemes
Gram-negative bacteria produce a much greater variety of beta-lactamases than do gram-positive bacteria. This diversity has led to several classification schemes.
Several different schemes have been proposed to classify this large family of enzymes and a review by Bush (2001) reported that more than 190 unique enzymes have been described. Fortunately, the enzymes can be classified on the basis of their primary structure into four molecular classes (A through D) in the Ambler classification, 131 or on the basis of their substrate spectrum and responses to inhibitors into a larger number of functional groups in the Bush, Jacoby and Medeiros classification.132
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In the Ambler classification, beta-Lactamases are grouped into four classes: A through D. Class A beta-lactamases include the extended-spectrum β-lactamases (ESBLs) and degrade penicillins, some cephalosporins, and, in some instances, carbapenems. Class A enzymes are inhibited in vitro by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam Class B beta-lactamases are Zn2+-dependent enzymes that destroy all β-lactams except aztreonam, whereas class C beta-lactamases are active against cephalosporins. Class D includes cloxacillin-degrading enzymes and are however rare.
The functional classification scheme of β-lactamases proposed by Bush, Jacoby and Medeiros defines four groups according to their substrate and inhibitor profiles. Group 1 are cephalosporinases that are not well inhibited by clavulanic acid; group 2 penicillinases, cephalosporinases, and broad-spectrum ß-lactamases that are generally inhibited by active site-directed β-lactamase inhibitors; group 3 metallo-β-lactamases that hydrolyze penicillins, cephalosporins, and carbapenems and that are poorly inhibited by almost all β-lactam-containing molecules; group 4 penicillinases that are not well inhibited by clavulanic acid132 (See betalactamases table). While more than 600 distinct β-lactamases have been described, the Bush 2be group of ESBLs has proliferated and constitutes the largest subset of all such enzymes. ESBLs are located in two subgroups of group 2, namely subgroups 2be (extended-spectrum β-lactamases, Ambler’s class A enzymes) and 2 d (cloxacillin
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hydrolyzing β-lactamases, Ambler’s class D ESBLs) The total number of ESBLs that are characterized exceeds 200. Details of these may be found on the authoritative website on the nomenclature of ESBLs hosted by George Jacoby and Karen Bush. (http:www.lahey.org/studies/webt.htm). Although ESBLs have been described in a range of Enterobacteriaceae and Pseudomonadaceae from different parts of the world, they are most often identified in Klebsiella pneumoniae and Escherichia coli.
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Bush- Jacoby-Medeiros group1
Ambler class2
Representative enzymes (examples)
Preferred substrates
Inhibited by Organisms Location
CA3 EDTA4
1 C AmpC Cephalosporins - - Gramnegative rods Chromosome
(Plasmid)
2a A PC1 Penicillins + - Grampositive cocci (and
rods)
Plasmid
2b A TEM-1, 2
SHV-1
Penicillins cephalosporins
+ - Gramnegative rods
Gramnegative cocci
Plasmid Chromosome
2be A
D
TEM-derivates SHV-derivates CTX-M-1-26
PER GES-1 OXY-1/2 (K1) OXA-11,14,16,17
Penicillins narrow- and extended-spectrum cephalosporins
+
± -
- - -
Gramnegative rods
P.aeruginosa P.aeruginosa K.oxytoca
Acinetobacter spp P.aeruginosa
Plasmid
Chromosome
Plasmid
2br A TEM-30-36,
TRC-1, SHV-49
Penicillins ± - E.coli,
K.pneumoniae (inhibitor-resistant)
Plasmid
2c A PSE-1,3 4
BRO-1-3
Penicillins, carbenicillin
+ - P.aeruginosa
A.baumanii M.catarrhalis
Variable
2d D OXA-1-10
PSE-2
Penicillins cloxacillin
± - Enterobacteriaceae P.aeruginosa
Variable
2e A CepA
FPM-1 L2
Cephalosporins + - Bacteroides sp Proteus sp
S.maltophilia (inducible)
Variable
2f A
NMC-A Sme-1-3 Imi-1-3 KPC-1,2
Penicillins cephalosporins carbapenems
+
E.cloacae S.marcescens
K.pneumoniae
Chromosome
Plasmid
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D
GES-2 OXA-24-26,40, 51,58,72
±
P.aeruginosa A.baumanii
Plasmid Chromosome
3 B VIM, IMP, SPM,
GIM L1 CcrA
Penicillins cephalosporins carbapenems
- + P.aeruginosa
A.species Enterobacteriacae
S.maltophilia Bacteroides sp
Variable
TABLE 2.1 CLASSIFICATION OF BETA- LACTAMASES. 1 Functional classification based on substrate profile, 2Structural classification based on amino acid sequence similarities, 3CA, clavulanic acid (for inhibition of betalactamase activity), 4EDTA, metal chelator (for the inactivation of zinc-dependant enzymes) .From References 131 & 132
Epidemiology of ESBL
Extended-spectrum β-lactamases derived from the TEM-1 β-lactamase were first identified in the USA in outbreak strains of Klebsiella pneumoniae in the middle to late 1980s, together with the SHV-5 ESBL. The TEM-10, TEM-12 and TEM-26 enzymes have remained in US hospitals, but have been joined by other ESBLs that are variants of the SHV-1 broad-spectrum β-lactamase. In recent surveys from hospitals in the eastern part of the USA, the most prominent ESBLs have been the SHV-7 and SHV-12 enzymes. Surprisingly, few CTX-M ESBLs have yet been reported in the USA, in contrast to the rest of the world, where the CTX-M enzymes have become a predominant ESBL family.
In Canada, a wider variety of ESBLs has been identified, with multiple members of the TEM, SHV and CTX-M classes being represented in surveillance isolates.
SHV-type and CTX-M ESBLs have appeared in many Canadian isolates, with an outbreak of CTX-M-14-related enzymes from Calgary, but limited TEM-derived ESBLs.133
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In South American countries, the class A ESBLs so far recognised belong to the CTX-M, Pseudomonas Extended Resistance (PER), SHV and TEM families.
ESBL rates in South America are among the highest in the world, probably due to multiple factors. SHV- and TEM-type ESBLs have been frequently encountered, but CTX-M is endemic and widely dominant. Community-acquired ESBLs are starting to appear.134
The first types described were derivatives of the TEM-1, TEM-2 and SHV-1 enzymes during the 1980s in Europe, mainly in Klebsiella pneumoniae associated with nosocomial outbreaks. Nowadays, they are mostly found among Escherichia coli isolates in community-acquired infections, with an increasing occurrence of CTX-M enzymes. The prevalence of ESBLs in Europe is higher than in the USA but lower than in Asia and South America. However, important differences among European countries have been observed. Spread of mobile genetic elements, mainly epidemic plasmids, and the dispersion of specific clones have been responsible for the increase in ESBL-producing isolates, such as those with TEM-4, TEM-2TEM-4, TEM-52, SHV-12, CTX-M-9, CTX-M-1TEM-4, CTX-M-3, CTX-M-15 and CTX-M-32 enzymes.135
Asia is almost certainly a part of the world in which extended-spectrum β-lactamases (ESBLs) have emerged de novo, with some early antimicrobial resistance studies showing high levels of the ESBL phenotype, particularly among
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Klebsiella, and most notably in China, Korea, Japan and India. There is a lack of genotyping studies but work from the late 1990s suggests that SHV-5 and SHV-12 were most common then, with only very rare reports of TEM-related ESBL genes.
As in other parts of the world, quite marked differences have since been seen in the pattern of ESBL genes, particularly in relation to the CTX-M family. The early emergence of TOHO CTX-M-2 in Japan contrasted with CTX-M-3 and -14 in China and many other parts of the Far East, suggesting the separate transfer of genes from the genome of Kluyvera spp. to mobile genetic elements in human-associated Enterobacteriaceae. ESBL production rates are now very high compared with Europe. In most countries, there are mixtures of CTX-M types, with VEB appearing significantly in Vietnam and Thailand, and ESBL isolates from India being completely dominated by the presence of blaCTX-M-15 alone, with no other CTX-M types reported. With the total population of India and China being about 2.4 billion and with faecal carriage rates of, probably, 10%, these countries represent major reservoirs of blaCTX-M genes. Increasing international travel and trade will lead to the movement of many of these ESBL genes. The high prevalence of ESBL genes in Asia means that the empirical treatment of serious infections with β-lactam antibiotics, except carbapenems, is seriously compromised.136
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Several outbreaks of infections with ESBL-producing Klebsiella have been reported from South Africa, but no national surveillance figures have been published. However, it has been reported that 36.1% of Klebsiella pneumoniae isolates collected in a single South African hospital in 1998 and 1999 were ESBL producers. ESBLs have also been documented in a variety of North African countries. Outbreaks of Klebsiella infections with strains resistant to third-generation cephalosporins have been reported in Nigeria14,15,17 and Kenya without documentation of ESBL production. A novel CTX-M enzyme (CTX-M-12) has been found in Kenya. Members of the family Enterobacteriaceae producing SHV-2 have been isolated from three different African countries, namely, Tunisia, Senegal, and Egypt, while TEM-3, TEM-20, and TEM-21 have also been recovered from Tunisia. Characterization of ESBLs from South Africa has revealed TEM and SHV types (especially SHV-2 and SHV-5). A nosocomial outbreak of infections with Pseudomonas aeruginosa, expressing GES-2 has been described in South Africa.137
Risk Factors for Infection with ESBL Producers
Identified risk factors for colonization or infection with ESBL producers in out-patients include older age and previous use of quinolones and cephalosporins.138 In institutional settings, severe illness with prolonged hospital stays, prolonged presence of invasive medical devices (urinary catheters, endotracheal tubes,
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central venous lines, arterial lines), total parenteral nutrition, recent surgery, haemodialysis, presence of decubitus ulcers, poor nutritional status, and heavy antibiotic use have been implicated as risk factor. Use of a variety of other antibiotic classes has been found to be associated with subsequent infections due to ESBL-producing organisms. Conversely, prior use of β-lactam/ β -lactamase inhibitor combinations, penicillins, or carbapenems seems not to be associated with frequent infections with ESBL-producing organisms.137 Other risk factors identified include poor functional status, liver disease, and the use of histamine2 receptor antagonists.139
2.6 DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASES