Largest group of class A-lactamases was characterized on the basis of 26 strictly conserved residues and molecular comparisons helped standard numbering scheme as indicated by the label “ABL” (for class A-lactamase) [27]. Further updated list was reported for the residues that are involved in the catalytic mechanism and/ or in substrate binding by Matange et al. [28] [29].

It has been reported that, 268 sequences aligned for representative of class A-lactamases from subclasses A1 and A2, 100% was confirmed and highly conserved (between 90% and 99%) residues, such as Gly45, Ser70, Lys73, Leu81, Pro107, Ser130, Asp131, Asn132, Ala134, Gly144, Gly156, Glu166, Lys/ Arg234, Thr/Ser235, and Gly236, differentiated between subclasses. The two subclasses were distinguished as A1 [28] [29] [30] and A2 (discovered more recently) as subgroups due to their different conserved residues. PER-1 and PER-2, an alignment of subclass A2-lactamase sequences revealed the presence of several insertions [31] [32]. An examination of the overall amino acid composition of lactamases revealed that representative enzymes from subclass A2 had small numbers of arginine residues (8.2 3.9 residues on average) and large numbers of lysine residues (29.0 5.5 residues).

A great diversity of amino acid sequences has been noted between the different clusters of class A β-lactamases and further, X-ray crystallography has been able to determine the tertiary structure of these protein molecules and helped to explore the two subclasses of A1 and A2. (http://www.rcsb.org/pdb/home/home.do). Structures of Beta lactamases such as, TEM types, SHV types, CTX-M types, KPC-2, L2, NMC-A, OXY-1, PenA, PenI, PSE-4, SED-1, SME-1, Toho-1, and Francisella tularensis that are produced by Gram negative organisms have been determined with the exception of PER-1 and PER-2, belong to subclass A1 [31] [32].

Overall structures of these are found to be same with the structural features surrounding the active sites with two subdomains generating a cleft as in Figure 2 [28] [33] [34]. Of the two sub domains, the alpha subdomain is largely α-helical in contrast, to the alpha/beta subdomain that consists of a five-stranded β-sheet flanked by α-helices. Active sites are present on the cleft of the two subdomains and contain catalytic Ser70 residue and Glu166, Asn170, and Ser70 that is deacylated with water primed by interactions.

According to Ambler molecular classification scheme, which is based on the protein sequence similarity these are classified into four classes A, B, C and D. This classification also is based on conserved and variable amino acid motifs of the proteins. Class A, C, and D include the enzymes that hydrolyze their substrates by forming acyl enzymes via the active site serine, while class B (metalloenzymes) utilizes active site zinc to facilitate β-lactam hydrolysis [9] [39].

Bush-Jacoby-Medeiros functional classification scheme classify these enzymes according to the similarity in their functional (substrates and inhibitors profile) characteristics. Although the molecular classification is the easiest scheme to group these diverse enzymes, the functional classification enables the clinicians and laboratory microbiologists to correlate these enzymes with their clinical roles [9]. Table 1 reveals the detailed classification of the β-lactamases.

TYPES OF ESβL:

The agent that inhibits β-lactamase enzymes by irreversible binding to its active site leads to permanent inactivation. The first clinically used β-lactamase inhibitor was clavulanic acid isolated from S. clavuligerus which exhibited weak antimicrobial activity. But, if combined with amoxicillin, it significantly increases the antimicrobial activity of the later. β-lactamase inhibitors are most effective against class-A β-lactamase including CTX-M, TEM and SHV-ESβLs [55].

ESβLs are currently a universal problem in hospitalized patients as well as community settings. Prevalence of ESβLs among clinical isolates is variable with respect to different institutions, countries and continents [56]. Recent studies have shown a significant global increase in the ESβL rate. In North America the ESβL rate in Klebsiella sps., E. coli and P. mirabilis ranges from 4.2% - 44%, 3.3% - 4.7% and 3.1% - 9.5%, respectively. In Latin America, the ESβL rate of Klebsiella sps., E. coli and P. mirabilis lie in the ranges 40% - 47.3%, 6.7% - 25.4% and 9.5% - 35.5%, respectively. In the Far East-Western Pacific area, the ESβL rate in Klebsiella sps., E. coli, Salmonella sps. and P. mirabilis ranges between 11.3% - 51%, 7.9% - 23.6%, 3.4% and 1.4% - 1.8%, respectively [57].

The highest rate was detected in Egypt and Greece (38.5% and 27.4%, respectively) and the lowest rate was in Netherlands and Germany (2% and 2.6%, respectively) according to the Pan European Antimicrobial Resistance Local Surveillance (PEARLS) study done between the years 2001-2002 [58]. However, in comparison to other countries in United States (US) the prevalence of ESβL- encoding Enterobacteriaceae was around 3%. A study reflects that lower ESβL prevalence in Northern European countries compared to South-Eastern European countries [53] [59]. Whereas in Spain, only 1.5% among 1962 invasive E. coli isolates in 2001 were found to produce ESβL [60]. In contrary, France had 11.4% K. pneumoniae and 47.7% E. aerogenes isolates were seen which produced ESβL in a surveillance covering many medical centres during 1996 to 2000 [61]. Northern European countries still have the lowest prevalence of ESβL- producing Enterobacteriaceae ranging from <1% in the Netherlands to 3% in Sweden.

It has been observed that ESβL allele may be restricted to certain country or a certain geographical region, like blaTEM-10 that has been detected in the United States in several outbreaks for many years before the detection of this allele was found in Europe [62]. Another example demonstrated by blaTEM-3 which has not been detected in the United States but is been frequently observed in France [63]. In contrast, there are ESβL alleles which are commonly encountered worldwide like SHV-5 and CTX-M-15 [53].

Travel plays an important role in the dissemination of antibiotic resistance [50]. The global dissemination of CTX-M-14 and CTX-M-15 producing/ST131 E. coli is attributed to the colonization or infection of travellers returning from high risk area like Indian subcontinent and the Middle East Asia confirming that returning travellers are most likely to acquire the predominant ESβL-determi- nant in the visited country. Such acquisition can be achieved even without hospitalization or contact with the health care system in the visited country [64].

Several outbreaks have been reported, majority of which occurred in tertiary hospitals where the transfer of a colonized patient provide a chance for dissemination of the ESβL-producing organism [65]. In a French Hospital, SHV-5 expressing K. pneumoniae were isolated from six peripartum women and two neonates. PFGE profiles of these strains indicated that all of the strains have PFGE-patterns identical to that of a strain isolated from contaminated ultrasonography coupling gel [66]. ESβL-producing E. coli and K. pneumoniae having different PFGE-types, but carrying identical plasmid encoding TEM-10 have been isolated from many patients in different hospitals in Chicago. The occurrence of the same plasmid in the strains of different PFGE-genotypes is a clue for plasmid transfer [62] [67].

More interestingly, blaTEM-24 encoding, 180 kb, conjugative plasmid was detected in four different enterobacterial species E. coli, K. pneumoniae, E. aerogenes and P. rettigeri isolated from the same patient suggesting horizontal transfer between the normal flora of the gut [68]. The findings of bla_TEM were reported to be 75 percent among ESβL-producing K. pneumoniae isolates [69] [16].

In India, reports have shown high rates of ESβLs since 1990s [70]. ESβL producing Enterobacteriaceae are well established in the community were a study conducted showed 24% of ESβL producers and 74% and 76% had a history of prior use, at some time of a cephalosporin and quinolone, respectively [71].

Relatively very few genotyping studies have been carried out, SHV-12 in K. pneumoniae has been reported from Southern India to be linked to the qnrB plasmid-mediated quinolone resistance determinant, the first report being from S. marcescens [72] [73]. There is a report of SHV-5 produced by Salmonella Senftenberg causing an outbreak on a burns ward in Delhi [74]. The only genotype of a TEM ESβL is TEM-104, reported in ten isolates of K. pneumoniae from New Delhi in 2001-2002 [75].

CTX-M-15 β-lactamase is now widely distributed across the world, but was first described in a small number of isolates from Delhi in 2000 [76]. A very recent survey from three widely dispersed centers in India showed that 95/130 cefpodoxime-resistant E. coli and K. pneumoniae isolates obtained between 2003 and 2005 carried a bla CTX-M gene, and when genotyped confirmed as blaCTX- M-15 in all the cases [77]. The study by Ensor et al. (2006) prompted a letter reporting on isolates of Klebsiella spp. and E. coli collected in the late 1990s from six widely dispersed centers; 47 isolates were examined using PCR and DNA sequencing, and 37 were found to carry blaCTX-M-15, which was the only CTX-M genotype found [78]. CTX-M-15 was also identified from two of the K. pneumoniae isolates collected during 2002-2003 in Coimbatore, South India [72]. India, therefore, not only appears to have very high rates of ESβL production across the country, but is entirely dominated by blaCTX-M-15 gene.

Currently, the emergence and rapid dissemination of CTX-M positive ESβL producing bacteria have caused a change in ESβL epidemiology [11] [79]. Furthermore, the recent identification of ESβL producing isolates that have acquired carbapenemases has further limited the therapeutic options available for treatment of these multidrug resistant microorganisms [16] [63].

The recognition of the importance of ESβL’s as a major mechanism of β-lactam resistance throughout the region came with presentation of data from the 1998-1999 SENTRY antimicrobial surveillance programme [80]. The incidence of ESβL production (no genotyping was undertaken) among E. coli isolates in the four Chinese sites varied from 13% to 35%. In India, the rate of ESβL producers from pregnant women in E. coli isolates was 36.8% [81]. Rates >20% for the ESβL phenotype in K. pneumoniae in all participating mainland Chinese centers (one reaching 60%), in one each of three Japanese and Taiwanese centers, and in the single Singapore center and Philippines center, were confirmed. Such high rates had previously been reported only from South America, in a follow-up study (1998-2002) lower rates were found in K. pneumoniae isolates from Australia and Japan (<10%), but that in China was 30% and comparatively, low incidence rate in India have also been reported [81] [82]. The other area of Asia that is the Indian subcontinent high rates of ESβL production has been reported.

A number of other studies in India reported the incidence of ESβL producers to be 6.6% to 68%. In south India, Subha et al. (2002) [83] reported 6.6% ESβL producers whereas Babypadmini et al. (2004) [84] reported 40.3%. The ESβL production which was reported among Gram negative bacteria by Mathur et al. (2002) [85] was 68% and Singhal, et al. (2005) [86] detected ESβL in 64% isolates and Rodrigues, et al. (2004) [87] reported 53% ESβL production. Other studies in India have also reported a very high prevalence of ESβL producing Enterobacteriaceae. Accordingly, in North India about 46% of uropathogens belonging to Enterobacteriaceae were found to be ESβL producers [88].

Some reports have also described the molecular epidemiology of the ESβL producers [78]. One of the reasons contributing to the high prevalence of ESβL producers in India may be the crowded hospital conditions, including implementation of optimal hygienic practices, likely fueled by unrestricted use of antimicrobials without doctor’s prescription [89]. A study reported that ESβL producing Enterobacteriaceae families were responsible for the onset of community infections in India [71]. CTX-M-15 is known to be having a peculiar association with the community onset of E. coli and K. pneumoniae [16] [90] [91].

MβLs are a group of clinically important hydrolytic enzymes belonging to the molecular class B β-lactamases or group 3 according to the Bush-Jacoby-Medeiros functional classification [39]. These enzymes are about 250 amino acid residues in length [97] and require divalent cation(s), usually Zinc, for their hydrolysing activities [93] [95]. MβLs act on carbon nitrogen bond of β-lactam ring [97], but they are mechanically different from other β-lactamases, which have serine at the active site [93]. Some of these require only one Zinc ion per molecule, while others require two Zinc ions per molecule (a binuclear active site) [97]. The metal ion(s) at the active site enables them to hydrolyze broad spectrum β-lactam agents including carbapenems, but their ability can be inhibited by metal ion chelators, such as EDTA, 1,10-o-phenanthroline, and mercapto compounds [95].

MβLs are generally encoded by genes carried on mobile genetic elements, such as plasmids, transposons, and integrons [94] [98]. General characteristics of IMP type MβLs were capable to hydrolyze carbapenems, but not monobactam. In fact, the blaIMP genes were often mediated by integrons, where aminoglycoside acetyl transferase and dihydropteroate synthetase genes also co-exist [99] [100].

Currently there are no clinically validated inhibitors of MβLs are available. Inhibitors that covalently modify MβLs include small thiol modifying reagents, such as mercuric (II) salts, p-chloromercuribenzoate [114], iodoacetic acid and mercaptoacetic acid thiol esters [115]. Inhibitors that chelate the active site of Zinc include EDTA, 1, 10-o-phenanthroline, dipicolinic acid, two phenazines from Streptomyces sps. [116], bis (1-N-tetrazol-5-yl) amine [117] and EDTA. More promising compounds are those which reversibly block the active site by competitive inhibition, since these offers the potential for modification of the structure to improve the specificity of the inhibitor for MβLs alone. Such compounds include biphenyl tetrazoles [117], mercaptophenyl acetic acid derivatives, which do not covalently modify the enzyme (probably because the phenyl ring sterically hinders the hydrolysis of the carbonyl thiol bond) [115], trifluoro methyl alcohols and ketones [118], N-(2'-mercaptoethyl)-2-phenylacetamide [119], thiomandelic acid [120], D- and L-captopril inhibitors [121], 6-(mercaptomethyl) and penicillinases [122].

During the past decade, both the global dimension of this problem and an unanticipated diversity of enzymes have been revealed, as acquired MβLs have been detected in clinical isolates from Asia as well as from Europe, North and South America [92] [100] [109] [112] [123]. Currently, the most prevalent and widespread acquired MβLs are the IMP-type and VIM type enzymes, of which several variants are known. Other types of acquired MβLs SPM-1, GIM-1, and SIM-1 have also been identified [98] [124].

Peleg and his colleagues (2005) for the first time reported the emergence and rapid dissemination of an acquired MβL determinant in a hospital setting in Australia, a continental resistance [125]. The MβL gene involved in the outbreak was blaIMP-4, an allelic variant of blaIMP-1 gene previously identified in clinical isolates of Acinetobacter sps. and Citrobacter youngae from Hong Kong and the People’s Republic of China [109] [110]. It was likely imported to Australia from those areas via international travelers and following the first detection in P. aeruginosa, the MβL gene was found in hospital acquired isolates of Gram-negative pathogens of 5 different species, including P. aeruginosa, K. pneumoniae, Serratia marcescens, Enterobacter cloacae, and E. coli. This is the first report for a rapid emergence in a single hospital of the same acquired MβL determinant in several different species, as well as in different strains of the same species (clonal diversity was observed among the MβL positive isolates of K. pneumoniae and S. marcescens) [125]. In fact, MβL producers usually exhibit complex MDR phenotypes because of their nosocomial origin and because of the frequent links between MβL genes and other resistance genes on the mobile DNA elements that are involved in their dissemination [126].

Only a low number of MβL producing isolates appeared to be carbapenem resistant [125] [127]. It is known that, unlike P. aeruginosa and Acinetobacter species, members of Enterobacteriaceae with acquired MβL genes tend to exhibit carbapenem MICs that remain lower than the breakpoint for resistance, unless permeability is also impaired [126]. This phenomenon has major implications for the detection of similar isolates (and consequently for surveillance) and also for the selection of antimicrobial chemotherapy.