An Overview of Extended Spectrum Beta Lactamases and Metallo Beta Lactamases

Antibiotic resistant β-lactamases are diverse and complex enzymes produced by most of the Gram-negative bacteria that are mediated by number of plasmids. The impact of these enzymes has posed a major threat to the health sectors and has challenged the available treatment options for both community and hospital acquired infections. These include the uncomplicated most severe life-threatening infections. Moreover, with resistance to the cephalosporin drugs these MDR strains exhibit co-resistance patterns with different class of antibiotics which is a cause of concern that leads to narrow the limited treatment options. It is alarming situation since there is a steep rise in MDR—Beta lactamase pathogens mainly in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii and Pseudomonas aeruginosa. Currently, the clinical detection of Extended Spectrum of β-Lactamases (ESβL) and MβL producing pathogens are carried out by antibiotic sensitivity test on the guidelines of Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) since, the other methods being too expensive. The choice of antimicrobial treatment for infections should rely on the clinical data and the tests (AST) in asymptomatic and mild cases. However, this does not imply for critical infections. The last resorts of treatment for ESβL pathogens are carbapenem and nevertheless, resistances have also been reported for the same. With increasing resistance rate to the antibiotics, it’s very essential to follow the guidelines for detection, implementation of antibiotic rotation to reduce these pathogens, followed by the efficient infection control practices and strategies to avoid such outbreaks.


Introduction
Infectious diseases are the potential transmission of a pathogenic agent from one species to another and these pathogens are contagious, which are also known as communicable diseases [1]. These pathogens have shown high resistance to the antibiotics, which has become a worldwide problem with the consequences on the infectious disease's treatment. Usually antibiotics are given empirically before the laboratory results of culture are available to ensure appropriate therapy.
There is an alarming increase of antibiotic resistance in bacteria that cause either community infections or hospital acquired infections. Many of these multidrug pathogens are of particular interest such as, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus, and extensively drugs resistant Mycobacterium tuberculosis [2].
According to WHO (2018) reports, 750,000 deaths every year may shoot high due to bacteria exhibiting resistance if proper action is not taken. Hence, antibiotic resistance has become one of the greatest threats to global health and survival statistics, which may be linked to global problem. Dispersion of the successful clones of Multidrug Resistant (MDR) bacteria is found to be common with the importance of plasmids carrying MDR markers in Shigella sps., and E. coli first described in the seminal work in Japan over 57 years ago [3]. Leplae and his coworkers (2006) showed the mechanism of transmission of mosaic plasmids from one bacterium to other to give rise to the MDR phenotype [4].
β-lactam antibiotics are the most common drugs used for the treatment of Gram-negative bacteria and their continuous misuse has led to the resistance worldwide [5]. Bacterial strains have induced continuous production and mutation of β-lactamases, due to the continuous exposure Beta lactam drugs, expanding their activity even against the newly developed β-lactam antibiotics. Incidence and treatment of ESβL-producing MDR strains is a matter of scientific concern for the difficulties to resolve due to various reasons, difficulty in detecting ESβL production and inconsistencies in reporting [6]. Recently, a significant increase in the incidents of ESβL-related infections has been observed throughout the globe [7] [8].
Microorganisms that produce beta-lactamases can break and enable the beta-lactam molecules inactive thus, conferring the resistance to the pathogens.
More than 500 beta-lactamases have been reported so far (http://www.lahey.org/studies). These beta-lactamases are widespread across the world that are mostly reported in Gram negative organisms with common resistance mechanism mediated by plasmid or expressed chromosomally, specifically the CTX-M-15 family. Chromosomally located inducible expression is also common, while plasmid mediated enzymes are generally expressed constitutively in these Gram-negative organisms [9] [10].
ESβL enzymes are commonly found in the members of Enterobacteriaceae family and are of over 120 types. ESβLs are generally acquired by horizontal gene

Mechanism of Antibiotic Resistance by Beta-Lactamases
In the year 1940, β-lactam antibiotics came into clinical use and it has been observed that many strains of bacteria have emerged resistant to these drugs [18].
Resistance to these agents is in both Gram-positive and Gram-negative bacterial pathogens and occurs as a result of drug inactivation by β-lactamases, target site (i.e. PBP) alterations, diminished permeability and efflux of drugs [19]. The primary mechanism of the β-lactam resistance is by enzymatic cleavage of the β-lactam ring by β-lactamases produced by microbes [18].
In 1983, SHV-2, a plasmid borne ESβL produced by K. ozaenae was discovered in Germany. Other ESβL types, TEM and SHV were predominant until 1990. Later on, prevalence of SHV, TEM and a new CTX-M family of ESβL coproduced mainly by E. coli had emerged. CTX-M has become the predominant and CTX-M-producing E. coli has spread globally and has been involved in nosocomial outbreaks and community acquired infections [20]. In 1990 the first blaCTX-M was detected in clinically isolated E. coli in Germany [21] then on CTX-M-producing Enterobacteriaceae has been detected globally [22] [23].
Beta-lactamases producing bacteria hydrolyse beta-lactam drugs and render them inactive before it gets to the PBP (Penicillin binding proteins) target. The structural similarities with PBP makes the lactamases bind acylate and hydrolyse using water molecules and inactivate the Beta lactam drugs. These enzymes contain either serine residue (Ambler classes A, C, D) or metal ion Zn 2+ (Ambler class B) in their active site, that attack beta-lactam ring and break the amide bond in the ring Figure 1 [24] [25] [26]. Advances in Microbiology

Primary Structure/Molecular Structure
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 con-  [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).

Structure-Function Relationships of Class A Enzymes
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. 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. Figure 2. Secondary structures of the class A β-lactamase of Mycobacterium tuberculosis, with the spatial arrangements of the three catalytic center defining amino acid groupings, the α domain (left), and an α/β domain (right) [35]. The helices are represented as H1 to h11, and the strands are represented as S1 to S5. The figure was created with PyMOL (Delano Scientific).

Extended-Spectrum Beta-Lactamase (ESβL)
The term was used initially to refer to TEM and SHV enzymes that have the ability to hydrolyze oxyimino-cephalosporins. Later on, this term has been widened to include:  Enzymes derived from other sources and have resistance spectra similar to that of TEM and SHV mutants e.g. CTX-M and VEB types.  Enzymes exhibit wider resistance than their parents but do not belong to 2be group e.g. OXA and Amp C mutants with increased activity against cefepime [36].
Currently used definition for ESβL is β-lactamase that is able to render the bacterium resistant to the penicillin, first, second, and third-generation cephalosporins and aztreonam, but not cephamycins or carbapenems, by hydrolysis that could be inhibited by β-lactamase inhibitors of these antibiotics [11] [37] [38].

Classification of Beta-Lactamases
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.  [40]. TEM-1 hydrolyses ampicillin at a rate higher than that of carbenicillin, oxacillin, and cephalothin but fail to hydrolyse the extended-spectrum cephalosporins. TEM-2 has the same hydrolytic activity of TEM-1 but has more active native promotor and a different isoelectric point (5.6 instead 5.4). The plasmid mediated β-lactamase TEM-3 (an ESβL member) was detected in 1987 in K. pneumoniae which was isolated in France. It was originally named CTX-1 due to its higher activity against cefotaxime [41]. There is steep rise in the TEM novel variants that has been reported ( Figure 3) from different parts of the world that can be accessed at http://www.lahey.org/Studies/temtable.asp.

SHV
SHV-type ESβL was the most frequently detected in clinical isolates [42]. This is referred to sulfhydryl variable because it was thought that the inhibition of the enzyme activity by p-chloromercuribenzoate was substrate-dependent and variable according to the substrate used in the assay [43]. In 1983, a new SHV-βlactamase (designated SHV-2) efficiently hydrolyzes cefotaxime and to lesser extent ceftazidime had been detected in K. ozaenae in Germany [44]. SHV-2 differs from SHV-1 by only one amino acid at the 238 th position (glycine replaced by serine). This substitution (Gly238Ser) that resulted from a point mutation accounts for the activity of this enzyme against extended-spectrum cephalosporin. SHV-2 spread globally due to the selection pressure exerted by third-generation cephalosporins being detected in a wide range of Enterobacteriaceae but mainly in Klebsiella sps. [11] [45] [46].

Oxa-Beta-Lactamase
OXA name refers to the oxacillin-hydrolysing ability of these β-lactamases. They hydrolyze oxacillin and cloxacillin at a higher rate than 50% that of benzylpenicillin [39]. OXA-β-lactamases are mainly found in P. aeruginosa [51]. The most common OXA-type β-lactamase is OXA-1. It has been detected in up to 10% of E. coli isolates [52].

Other ESβL-Types
It includes PER-1 (for Pseudomonas extended resistance), PER-2, VEB-1 (for Vietnamese extended spectrum), VEB-2, GES (Guiana-extended spectrum) and SFO (Serratia fonticola) which share only 25% and 27% homology with TEM and SHV types. It was observed that, PER-1 with 86% amino acid homology and PER-2 incidence was high in Turkey and South America respectively. VEB-1 and TLA-1 were discovered in E. coli clinical isolates from Vietnamese patient hospitalized in France and Mexico respectively [53]. PER-1, PER-2, VEB-1 and TLA-1 are related to Bacteroides sps. with reference to their homology to the chromosomal β-lactamase [53]. These novel enzymes are found infrequently and the details of these enzymes are reviewed elsewhere [42] [54].

Inhibitors for 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 an-

Epidemiology of ESβLs
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].  [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 ESβLproducing 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 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)(1999)(2000)(2001)(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].

Metallo Beta Lactamases (MβL)
Metallo β-lactamase was first identified in 1966 as β-lactamase II (BcII) in Bacillus cereus, when the cephalosporinase activity was shown to be inhibited by metal chelators like EDTA [92]. The early reported MβL determinants were located on the chromosome and were produced by organisms of minor clinical relevance, e.g. Flavobacterium odoratum, B. cereus, and Legionella gormanii, that were regarded as rare curiosities [93].
The first report of transferable MβL was recorded in P. aeruginosa from Japan in 1991 [94] and then on MβLs were discovered with more clinically relevant genera such as Serratia, Bacteroides, and Pseudomonas [93]. The class B β-lactamases exhibit resistance to commercially available β lactamase inhibitors, but are inhibited by metal ion chelators, such as EDTA. This class of enzymes is of particular interest and concern owing to the ability to hydrolyze and thus provide resistance to virtually all classes of β-lactams, including the carbapenems. A number of clinical Burkholderia cepacia isolates producing an inducible metalloenzymes (PCM-I) that also shows preferential hydrolysis of carbapenems/ imipenem have also been described [95]. A limited number of B. fragilis isolates have been shown to produce a chromosomal metalloenzyme, CfiA/CcrA that provides resistance to imipenem [96].

Classification of MβLs
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 mole-K. Nagshetty et al. cule, 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.

Epidemiology of MβL
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.

Classification Based on Sequence Similarities by Hidden Markov Models (HMM) for β-Lactamase Annotation
Most widely used classification scheme for BLs is the Ambler structural classification, which is based on sequence similarity and it's still the best currently used.
It classifies the BLs into 4 classes: the classes A, C, and D of serine-β-lactamases (SBLs) and the Class B of metallo-β-lactamases (MβLs), where Class B is further divided into subclasses B1, B2, and B3, using sequence conservation data [27] [128] [129]. Even though SBLs and MBLs are able to break amide and ester bonds they do not belong to a common ancestor due to 2 distinct protein superfamilies [130]. SBL's tertiary structures are similar enough among themselves to  [131] while, the differences lie between their primary structures and catalytic mechanisms that divide them into classes A, C, and D [132]. The 3 subclasses of MBL, B1 and B2 have detectable sequence similarity and a common ancestor between them but not with B3 [133] [134]. The scheme proposed by Hall and Barlow (2005), based on structural information the former subclasses B1 and B2 were merged and renamed as class MB, whereas subclass B3 was renamed as class ME. Thus, the 5 BL classes which are considered as third classification level namely, SA, SC, SD, MB, and ME with subclasses MB1 and MB2 represent the fourth and last hierarchical level [132].
In few studies, phylogenetic and amino acid-based sequence similarities showed further division of classes SA and SD into 2 different new BL subclasses [35] [135]. Molecular classification of BLs does not represent currently to its actual sequence diversity and no precise definition for various classes and subclasses have been precised. Silveria et al. (2018) proposed new BL class with fused domains and extended action spectrum. This classification is based on a previous hierarchical scheme, profiles of Hidden Markov Models (HMM) and sequence clustering using similarity was proposed [136] that provides genomes in BL annotation and know the BLs distribution among bacteria phyla suggesting new BL subclasses.

Conclusion
Beta-lactamases producing bacteria causing nosocomial infections have resulted in a steep rise in their incidence in recent years. The scientific community reported extensively about the high morbidity-mortality rates due to these β-lactamases resistant MDR strains. Henceforth, detection of community and hospital transmission by β-lactamase pathogens becomes a paramount importance. Rapid identification of ESβL pathogens and their antibiotic resistance patterns will help the clinicians to select appropriate drug regimens like combination therapy and reduce their further spread. Since the epidemiology of ESβL and MβL producing bacteria is becoming more complex in both hospitals and community, it is important to regulate and monitor MDR among clinical isolates. "Super bug", bacteria might evolve in the near coming future for relatively all the antibiotics if we are not cautious in handling them. This alarms the development and spreading of the disease globally and incapability to cure the infection, and ultimately leading to mortality or death. In this situation, a constant monitoring, dispense of antibiotics for non-emergency cases only on the clinical reports and careful worldwide surveillance is urgently warranted.