**1. Introduction**

In 1941, the Magic Bullet Penicillin was first used clinically in an Oxfordshire constable, Albert Alexander, but within one year Rammelkamp reported the identification of isolates of *Stphylococcus aureus,* resistant to this miracle drug [1]. Within a short span of 70 years, from discovery of Penicillin to Tigecycline, some hospital strains have developed which are resistant to almost all available antimicrobials and the mankind is busy in writing obitu‐ ary for antimicrobials [2].

Infact, the rising trend of developing resistance to multiple antibiotics in microbes, leads to therapeutic failure. Presently, antimicrobial resistance (AMR) is a major threat to patient care and disease control throughout the world.

The World Health Day theme on 7th April 2011 was 'Antimicrobial resistance and its global spread'. World Health Organisation has raised the issue in 2011 that "Combat drug Resistance - No action today, No cure tomorrow" [3]. Not only the Gram positive bacteria like Methicillin Resistant *Staphylococcus aureus* (MRSA), Vancomycin Resistant Enterococci (VRE) etc. even the several Gram negative bacteria of Enterobacteriaceae family e.g*. E.coli, Klebsiella pneumoniae, Enterobacter sp. etc,* and *Pseudomonas aeruginosa, Acinetobacter baumani, Burkholderia species* etc. also develop resistance to most of the antibiotics now a days.

Actually, the pace in which bacteria develop resistance is much higher than the rate of development of newer antimicrobials. The antibiotic resistance is mainly developed due to inappropriate and irrational use of antibiotics. In 2011, two new terms were coined i.e. multidrug resistant (MDR) and Extreme drug resistant (XDR) organisms. MDR is defined as non-susceptibility to one or more antibiotics in three or more antibiotic classes and XDR is defined as non-susceptibility to all potentially effective antimicrobials respectively [4]. β – lactamases are the important mechanism of drug resistance among the Gram negative bacteria.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Though *E.coli* is a commensal of human or animal intestine, *E.coli* is the most commonly isolated bacterial species in clinical laboratories and is incriminated in infectious diseases involving every human tissue and organ system [5]. Theodare Escherich, a German pediatri‐ tian identified *Escherichia coli* in 1885 and named it '*Bacterium coli commune'* [6]. He also established the pathogenic role of *E.coli* through his studies. The nomenclature '*Bacterium coli*' was widely used until 1919 when Castellani and Chalmers described the genus Escherichia [7]. The type species is *Escherichia coli*. Some species like *E.fergusonii, E.hermanii* and *E.vulne‐ ris* are considered opportunistic pathogens and are associated with wound infection in humans [8]. *Escherichia albertii*, the sixth species associated with cases of diarrhoea in children of Bangladesh, has been proposed by Huys et al in 2003 [9].

of recurrent cystitis [13]. *E.coli* strains involved in urinary tract infections includes O groups

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49

*E.coli* can be detected in the laboratory by conventional tests [5]. *E.coli* are Gram negative rod, motile and on Mac Conkey's agar form lactose fermenting nonmucoid colonies. Routine biochemical tests done for E.coli are : Catalase test: Positive, Oxidase test: Negative, Triple sugar iron agar test: Acid slant/Acid butt with gas, Indole test: Positive, Methyl red test: Positive, Voges-Proskauer (VP) test: Negative, Citrate utilization test: Negative, Nitrate reduction test: Positive, Oxidative / Fermentative test (Hugh & Leifson): Fermentative

Intrinsic resistance is rarely seen in genus Escherichia. Susceptibility of individual strain to different antimicrobials varies greatly, thus antibiogram is used as an epidemiological marker. This resistance pattern is plasmid mediated. *E.coli* shows transferable resistance to one or multiple drugs [14]. Recently *E.coli* isolated from intestinal as well as extraintestinal specimens

Beta lactamases are enzymes produced by wide range of Gram negative and Gram positive bacteria. This enzyme is responsible for resistance to β-lactam antibiotics like Penicillins, Cephalosporins, Cephamycins and Carbapenems. The β-lactamase enzymes break the betalactam ring and thus inactivate the antibacterial properties. Abraham and Chain (1940) discovered penicillinase, first β-lactamase to be identified in *E.coli* before Penicillin was introduced for clinical use [15]. The name β-lactamase was given by Pollock in 1960 [16]. First plasmid mediated β-lactamase was ''TEM'' named after the name of first patient Temoniera

Ambler in 1980s classified β – lactamases into various groups and according to him, there are three main classes as A, C and D [19]. Metallobetalactamases belong to class B which exhibits potent hydrolyzing activity not only against Carbapenems but also to other β – lactam antibiotics. *E.coli* is one of those Gram negative bacteria that has potential to develop all the

Determination of β-lactamase production could be done by acidometric method, iodometric

Extended spectrum beta – lactamase (ESBL) producing strains are emerging pathogens causing Health care associated infections (HAI) and pose great therapeutic challenge in recent years.

1, 2, 4, 6, 7 etc.

**B. Laboratory diagnosis**

**A. Beta - lactamases**

metabolism, Lysine Decarboxylase test: Positive.

has become resistant to most of the routinely used antibiotics.

**1.1. Antibiotibiotic sensitivity pattern**

in whom the enzyme was first detected [17]. Classification of β-Lactamases can be done by -

**b.** Molecular or Ambler classification [19]

**B. Extended spectrum beta – lactamase (ESBL)**

**a.** Functional or Bush Jacoby Mederios classification [18]

major classes of β – lactamases including Metallobetalactamases.

method and Chromogenic cephalosporin method using nitrocephin [20].

About 80% cases of urinary tract infection are due to *E.coli* [8]. It is commonly involved in Gram negative sepsis and endotoxic shock. *E.coli* can cause diarrhoea, wound infections, pneumonia in hospitalized patients and meningitis in neonates.

*E.coli* is the organism that is most widely studied in its various aspects. *E.coli* is the model organism for studies on cell structure, growth and metabolism. It is considered to be good vehicle for the cloning of genes from prokaryotic and eukaryotic cells and for expression of gene products [8].

*E.coli* is susceptible to 97% ethylene trioxide (ETO) and 95% Hydrochlorofluorocarbon-ETO (HCFC-ETO) respectively [10]. Chlorine 1 ppm is capable of eliminating approximately 4 log10 of *E.coli* O157:H7 within a minute [11]. E.coli strains causing intestinal and extra intestinal infections, exhibits many virulence factors such as: Adhesins, O antigens, Hemolysin, Sidero‐ phore production / Iron sequestration / Aerobactin production, Cell surface hydrophobicity, Outer membrane proteins e.g. Porins etc. Haemagglutinin, Verotoxin, Cytotoxic necrotizing factor (CNF), Enterotoxin, Colicins, Gelatinase production, Serum resistance are other major virulence factors of *E.coli*. Complete serotyping of *E.coli* is based on three antigen detection as O, K & H i.e. O:K:H. If fimbrial virulence factor is present then serotype should be expressed in terms of O:K:H:F.

#### **A.** *E.coli* **— Role as a pathogen**

They are classified into two types [12] – (a) Enterovirulent *E.coli,* (b) Uropathogenic *E.coli*

**a. ENTEROVIRULENT***E.coli***:** Adherence is one of the most important virulence mecha‐ nisms of enterovirulent *E.coli*.

Enterovirulent *E.coli* strains are classified into

Enteropathogenic *E.coli* (EPEC), Enterotoxigenic *E.coli* (ETEC), Enteroinvasive *E.coli* (EIEC), Verocytotoxin – producing *E.coli* (VTEC), Enteroaggregative *E.coli* (EAEC), Diffusely adherent *E.coli* (DAEC) etc.

#### **b. UROPATHOGENIC***E.Coli* (UPEC)

*E.coli* is the predominant uropathogen isolated from acute community acquired urinary tract infections and is responsible for 85% of asymptomatic bacteriuria and more than 60% of recurrent cystitis [13]. *E.coli* strains involved in urinary tract infections includes O groups 1, 2, 4, 6, 7 etc.

#### **B. Laboratory diagnosis**

Though *E.coli* is a commensal of human or animal intestine, *E.coli* is the most commonly isolated bacterial species in clinical laboratories and is incriminated in infectious diseases involving every human tissue and organ system [5]. Theodare Escherich, a German pediatri‐ tian identified *Escherichia coli* in 1885 and named it '*Bacterium coli commune'* [6]. He also established the pathogenic role of *E.coli* through his studies. The nomenclature '*Bacterium coli*' was widely used until 1919 when Castellani and Chalmers described the genus Escherichia [7]. The type species is *Escherichia coli*. Some species like *E.fergusonii, E.hermanii* and *E.vulne‐ ris* are considered opportunistic pathogens and are associated with wound infection in humans [8]. *Escherichia albertii*, the sixth species associated with cases of diarrhoea in children of

About 80% cases of urinary tract infection are due to *E.coli* [8]. It is commonly involved in Gram negative sepsis and endotoxic shock. *E.coli* can cause diarrhoea, wound infections, pneumonia

*E.coli* is the organism that is most widely studied in its various aspects. *E.coli* is the model organism for studies on cell structure, growth and metabolism. It is considered to be good vehicle for the cloning of genes from prokaryotic and eukaryotic cells and for expression of

*E.coli* is susceptible to 97% ethylene trioxide (ETO) and 95% Hydrochlorofluorocarbon-ETO (HCFC-ETO) respectively [10]. Chlorine 1 ppm is capable of eliminating approximately 4 log10 of *E.coli* O157:H7 within a minute [11]. E.coli strains causing intestinal and extra intestinal infections, exhibits many virulence factors such as: Adhesins, O antigens, Hemolysin, Sidero‐ phore production / Iron sequestration / Aerobactin production, Cell surface hydrophobicity, Outer membrane proteins e.g. Porins etc. Haemagglutinin, Verotoxin, Cytotoxic necrotizing factor (CNF), Enterotoxin, Colicins, Gelatinase production, Serum resistance are other major virulence factors of *E.coli*. Complete serotyping of *E.coli* is based on three antigen detection as O, K & H i.e. O:K:H. If fimbrial virulence factor is present then serotype should be expressed

They are classified into two types [12] – (a) Enterovirulent *E.coli,* (b) Uropathogenic *E.coli*

**a. ENTEROVIRULENT***E.coli***:** Adherence is one of the most important virulence mecha‐

Enteropathogenic *E.coli* (EPEC), Enterotoxigenic *E.coli* (ETEC), Enteroinvasive *E.coli* (EIEC), Verocytotoxin – producing *E.coli* (VTEC), Enteroaggregative *E.coli* (EAEC), Diffusely adherent

*E.coli* is the predominant uropathogen isolated from acute community acquired urinary tract infections and is responsible for 85% of asymptomatic bacteriuria and more than 60%

Bangladesh, has been proposed by Huys et al in 2003 [9].

in hospitalized patients and meningitis in neonates.

gene products [8].

48 Trends in Infectious Diseases

in terms of O:K:H:F.

*E.coli* (DAEC) etc.

**A.** *E.coli* **— Role as a pathogen**

nisms of enterovirulent *E.coli*.

**b. UROPATHOGENIC***E.Coli* (UPEC)

Enterovirulent *E.coli* strains are classified into

*E.coli* can be detected in the laboratory by conventional tests [5]. *E.coli* are Gram negative rod, motile and on Mac Conkey's agar form lactose fermenting nonmucoid colonies. Routine biochemical tests done for E.coli are : Catalase test: Positive, Oxidase test: Negative, Triple sugar iron agar test: Acid slant/Acid butt with gas, Indole test: Positive, Methyl red test: Positive, Voges-Proskauer (VP) test: Negative, Citrate utilization test: Negative, Nitrate reduction test: Positive, Oxidative / Fermentative test (Hugh & Leifson): Fermentative metabolism, Lysine Decarboxylase test: Positive.

## **1.1. Antibiotibiotic sensitivity pattern**

Intrinsic resistance is rarely seen in genus Escherichia. Susceptibility of individual strain to different antimicrobials varies greatly, thus antibiogram is used as an epidemiological marker. This resistance pattern is plasmid mediated. *E.coli* shows transferable resistance to one or multiple drugs [14]. Recently *E.coli* isolated from intestinal as well as extraintestinal specimens has become resistant to most of the routinely used antibiotics.

#### **A. Beta - lactamases**

Beta lactamases are enzymes produced by wide range of Gram negative and Gram positive bacteria. This enzyme is responsible for resistance to β-lactam antibiotics like Penicillins, Cephalosporins, Cephamycins and Carbapenems. The β-lactamase enzymes break the betalactam ring and thus inactivate the antibacterial properties. Abraham and Chain (1940) discovered penicillinase, first β-lactamase to be identified in *E.coli* before Penicillin was introduced for clinical use [15]. The name β-lactamase was given by Pollock in 1960 [16]. First plasmid mediated β-lactamase was ''TEM'' named after the name of first patient Temoniera in whom the enzyme was first detected [17].

Classification of β-Lactamases can be done by -


Ambler in 1980s classified β – lactamases into various groups and according to him, there are three main classes as A, C and D [19]. Metallobetalactamases belong to class B which exhibits potent hydrolyzing activity not only against Carbapenems but also to other β – lactam antibiotics. *E.coli* is one of those Gram negative bacteria that has potential to develop all the major classes of β – lactamases including Metallobetalactamases.

Determination of β-lactamase production could be done by acidometric method, iodometric method and Chromogenic cephalosporin method using nitrocephin [20].

#### **B. Extended spectrum beta – lactamase (ESBL)**

Extended spectrum beta – lactamase (ESBL) producing strains are emerging pathogens causing Health care associated infections (HAI) and pose great therapeutic challenge in recent years. ESBLs were first detected in *Klebsiella pneumoniae* in 1983 [21, 22]. ESBL are classified under molecular AMBLer class A penicillinase having serine residue at their active site. ESBLs are responsible for resistance to one or more β-lactam antibiotics including third generation cephalosporins. Typically ESBLs are derived from genes for TEM-1, TEM-2 or SHV-1 by mutations that alter the amino acid sequence around the active site of β-lactamases [23,24]. ESBLs are inhibited by β-lactam inhibitors like Clavulanic acid, Sulbactam and Tazobactam. ESBLs are produced by Enterobacteriaceae family, *Pseudomonas aeruginosa, Acinetobacter species* etc.

**D. Carbapenemases**

These include β – lactamases which cause Carbapenem hydrolysis, with elevated Carbapenem MICs and they belonged to molecular classes A, B and D. Molecular classes A, C and D include the β – lactamases with serine at their active site, whereas class B β – lactamases are all

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**Molecular class A carbapenemase** – Class A serine carbapenemases belong to functional group 2f include chromosomally encoded NMC(Not Metalloenzyme Carbapenemase), IMI (Imipenem hydrolyzing β-lactamase) and SME(*Serratia marcenscens* enzyme) and plasmid mediated KPC (*Klebsiella pneumoniae* carbapenemase) and GES / IBC(integron borne cephalo‐ sporinase), etc [40]. All have the ability to hydrolyse Carbapenems, Cephalosporins, Penicillins and Aztreonem and all are inhibited by Clavulanic acid and Tazobactam. The chromosomal class A carbapenemase are infrequently found and can be induced by Imipenem and Cefoxitin. The KPC (*Klebsiella pneumoniae* carbapenemase) producing strains are found in *Klebsiella pneumoniae*, Enterobacter species, Salmonella species and other Enterobacteriaceae [41,42].

**Class D Serine carbapenemases** - The OXA (Oxacillin hydrolysing) β-lactamase with carba‐ penemase activity was detected by Patow et al in 1993 and the enzyme was purified from *Acinetobacter baumani [40].* They have been also found in Enterobacteriaceae and *P.aeruginosa* and were described as penicillinase capable of hydrolyzing Oxacillin and Cloxacillin [43,44]. They are not inhibited by Clavulanic acid and EDTA and were designated as ARI-1 (Acineto‐

They belong to molecular class B β – lactamases, requiring one or more divalent cations

The first MBL detected was chromosomally encoded and was detected in *Bacillus cereus* [46]. They possess a high level of resistance to all Carbapenems, Penicillins, Cephamycins, Cepha‐

On the basis of Imipenem and other β-lactam hydrolysis, MBLs are classified into different

Subgroup3a- possess broad spectrum activity; Subgroup3b – preferential avidity for carbape‐ nem; Subgroup 3c – hydrolyze carbapenems poorly compared to other β-lactam substrate.

Class B1 – possess key Zn co-ordinating residues of three histidines and one cystein eg. IMP,

metalloenzymes which require Zn++ for their activation [40].

bacter Resistant to Imipenem) and reside on large plasmid.

(Zn++) for their activation [45] and have 3 characteristics [2] –

**2.** Resistant to clinically used β – lactamase inhibitors and

**3.** Inhibited by EDTA, a metal ion chelator.

losporins and beta-lactamase inhibitor combinations.

At molecular level MBLs are classified into [48] –

**Metallobetalactamases (MBL)**

**1.** Hydrolyze carbapenems

**Classification of MBLs**

VIM, GIM and SPM – 1,

subgroups as [47] –

#### **Detection of Extended Spectrum β-lactamase (ESBL) production:**

The ESBL in *E.coli* is detected by two approaches: 1.Screening tests and 2.Confirmatory tests. Screening test detect reduced susceptibility to indicator drugs. According to Clinical Labora‐ tory Standard Institute (CLSI) guidelines indicator drugs used for *E.coli* & Klebsiella spp. are Cefotaxime, Ceftazidime, Ceftriaxone or Aztreonam [25]. Screening tests are not specific because mechanism other than ESBLs may also give positive results. Therefore, positive screening test should be followed by confirmatory tests.

Different confirmatory tests include Double disk approximation test, [26,27]. Comparison of Minimum inhibitory concentratin (MIC) or inhibitory zone around disk in presence or absence of β-lactamase inhibitor [28], Vitek ESBL test [29], Etest [30], three-dimentional test [31] etc. For identification of specific ESBL different molecular detection methods can be applied like DNA probes, PCR with oligonucleotide primers oligotyping, PCR followed by restriction fragment length polymorphism analysis, ligase chain reaction and nucleotide sequencing etc [32]. Commonly primers used for detecting bla genes are [33]:

CTX Forward – CGCTTTGCGATGTGCAG

#### Reverse - ACCGCGATATCGTTGGT

These techniques are available only in research centers and are beyond the scope of routine Clinical Microbiology Laboratories in India considering the presence of too many different types of ESBLs and the high cost.

#### **C. Amp C beta-lactamase [34]**

Amp C β-lactamases were first reported in 1988. Amp C β-lactamases are found either on chromosome (inducible) or on plasmid (non-inducible). Amp C β-lactamase producing bacteria show resistance to most of Cephalosporins including Cephamycin (Cefoxitin, Cefotetan) except Carbapenems. But they also hydrolyze Penicillins and Aztreonem. These are not inhibited by beta-lactam inhibitors such as Clavulanic acid, Tazobactam and Sulbactam.

**Detection of AMPC β-Lactamase production:** Presently, all plasmid mediated Amp C βlactamases have similar substrate profile to chromosomal Amp C β-lactamases. But the only difference is chromosomal Amp C β -lactamases are inducile where as plasmid mediated Amp C β –lactamases are uninducible [35]. Amp C β-lactamase producing strains could be detected by disc antagonism test [36], modified three dimensional test (MTDT) [37], Amp C disc test [38], double disk synergy test and disc potentiation test using 3-amino-phenyl-boronic acid (APB) etc [39].

### **D. Carbapenemases**

ESBLs were first detected in *Klebsiella pneumoniae* in 1983 [21, 22]. ESBL are classified under molecular AMBLer class A penicillinase having serine residue at their active site. ESBLs are responsible for resistance to one or more β-lactam antibiotics including third generation cephalosporins. Typically ESBLs are derived from genes for TEM-1, TEM-2 or SHV-1 by mutations that alter the amino acid sequence around the active site of β-lactamases [23,24]. ESBLs are inhibited by β-lactam inhibitors like Clavulanic acid, Sulbactam and Tazobactam. ESBLs are produced by Enterobacteriaceae family, *Pseudomonas aeruginosa, Acinetobacter*

The ESBL in *E.coli* is detected by two approaches: 1.Screening tests and 2.Confirmatory tests. Screening test detect reduced susceptibility to indicator drugs. According to Clinical Labora‐ tory Standard Institute (CLSI) guidelines indicator drugs used for *E.coli* & Klebsiella spp. are Cefotaxime, Ceftazidime, Ceftriaxone or Aztreonam [25]. Screening tests are not specific because mechanism other than ESBLs may also give positive results. Therefore, positive

Different confirmatory tests include Double disk approximation test, [26,27]. Comparison of Minimum inhibitory concentratin (MIC) or inhibitory zone around disk in presence or absence of β-lactamase inhibitor [28], Vitek ESBL test [29], Etest [30], three-dimentional test [31] etc. For identification of specific ESBL different molecular detection methods can be applied like DNA probes, PCR with oligonucleotide primers oligotyping, PCR followed by restriction fragment length polymorphism analysis, ligase chain reaction and nucleotide sequencing etc

These techniques are available only in research centers and are beyond the scope of routine Clinical Microbiology Laboratories in India considering the presence of too many different

Amp C β-lactamases were first reported in 1988. Amp C β-lactamases are found either on chromosome (inducible) or on plasmid (non-inducible). Amp C β-lactamase producing bacteria show resistance to most of Cephalosporins including Cephamycin (Cefoxitin, Cefotetan) except Carbapenems. But they also hydrolyze Penicillins and Aztreonem. These are not inhibited by beta-lactam inhibitors such as Clavulanic acid, Tazobactam and Sulbactam. **Detection of AMPC β-Lactamase production:** Presently, all plasmid mediated Amp C βlactamases have similar substrate profile to chromosomal Amp C β-lactamases. But the only difference is chromosomal Amp C β -lactamases are inducile where as plasmid mediated Amp C β –lactamases are uninducible [35]. Amp C β-lactamase producing strains could be detected by disc antagonism test [36], modified three dimensional test (MTDT) [37], Amp C disc test [38], double disk synergy test and disc potentiation test using 3-amino-phenyl-boronic acid

**Detection of Extended Spectrum β-lactamase (ESBL) production:**

screening test should be followed by confirmatory tests.

[32]. Commonly primers used for detecting bla genes are [33]:

CTX Forward – CGCTTTGCGATGTGCAG

Reverse - ACCGCGATATCGTTGGT

types of ESBLs and the high cost. **C. Amp C beta-lactamase [34]**

(APB) etc [39].

*species* etc.

50 Trends in Infectious Diseases

These include β – lactamases which cause Carbapenem hydrolysis, with elevated Carbapenem MICs and they belonged to molecular classes A, B and D. Molecular classes A, C and D include the β – lactamases with serine at their active site, whereas class B β – lactamases are all metalloenzymes which require Zn++ for their activation [40].

**Molecular class A carbapenemase** – Class A serine carbapenemases belong to functional group 2f include chromosomally encoded NMC(Not Metalloenzyme Carbapenemase), IMI (Imipenem hydrolyzing β-lactamase) and SME(*Serratia marcenscens* enzyme) and plasmid mediated KPC (*Klebsiella pneumoniae* carbapenemase) and GES / IBC(integron borne cephalo‐ sporinase), etc [40]. All have the ability to hydrolyse Carbapenems, Cephalosporins, Penicillins and Aztreonem and all are inhibited by Clavulanic acid and Tazobactam. The chromosomal class A carbapenemase are infrequently found and can be induced by Imipenem and Cefoxitin. The KPC (*Klebsiella pneumoniae* carbapenemase) producing strains are found in *Klebsiella pneumoniae*, Enterobacter species, Salmonella species and other Enterobacteriaceae [41,42].

**Class D Serine carbapenemases** - The OXA (Oxacillin hydrolysing) β-lactamase with carba‐ penemase activity was detected by Patow et al in 1993 and the enzyme was purified from *Acinetobacter baumani [40].* They have been also found in Enterobacteriaceae and *P.aeruginosa* and were described as penicillinase capable of hydrolyzing Oxacillin and Cloxacillin [43,44]. They are not inhibited by Clavulanic acid and EDTA and were designated as ARI-1 (Acineto‐ bacter Resistant to Imipenem) and reside on large plasmid.

#### **Metallobetalactamases (MBL)**

They belong to molecular class B β – lactamases, requiring one or more divalent cations (Zn++) for their activation [45] and have 3 characteristics [2] –


The first MBL detected was chromosomally encoded and was detected in *Bacillus cereus* [46]. They possess a high level of resistance to all Carbapenems, Penicillins, Cephamycins, Cepha‐ losporins and beta-lactamase inhibitor combinations.

#### **Classification of MBLs**

On the basis of Imipenem and other β-lactam hydrolysis, MBLs are classified into different subgroups as [47] –

Subgroup3a- possess broad spectrum activity; Subgroup3b – preferential avidity for carbape‐ nem; Subgroup 3c – hydrolyze carbapenems poorly compared to other β-lactam substrate.

At molecular level MBLs are classified into [48] –

Class B1 – possess key Zn co-ordinating residues of three histidines and one cystein eg. IMP, VIM, GIM and SPM – 1,

Class B2 – possess asparagine instead of histidine at first position of principal Zn binding motif, NXHXD. e.g. Aeromonas species and *Serratia fonticola* enzyme SFH – 1.

MBL detection tests involving inhibitors such as ethylene diamine tetraacetic acids (EDTA) and 2-Mercaptopropionic acids (2-MPA) have been recommended by various workers [57]. Tris/EDTA disks can also be used in combination with a Carbapenem disk to detect Carbape‐ nem - hydrolyzing enzymes and to differentiate between class A enzymes and MBLs. MBLs are inhibited by the Tris/EDTA disk. The inhibition of MBL can be enhanced by the addition of chelators. Double disk synergy test (DDST) [55] and Disk potentiation tests [58] are based on this principle. For detection of MBL many other methods used are MBL E-test using imipenem/imipenem-EDTA [59], reduction of MIC in presence of EDTA and polymerase chain

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PCR is specific for gene family IMP, VIM, etc. and hence, many other specific primers can be used for different MBL genes. The main disadvantage of PCR is that it requires tailor-made DNA primers and cannot differentiate between variants and may not detect new variants.

KPCs can be mainly detected by Combined disk method using Imipenem and Imipenem with

Recently, Carbapenem Resistant Enterobacteriaceae (CRE) pose a real threat to Medical fraternity as the increased frequency with which Enterobacteriaceae cause infection and the mortality associated with infection caused by CRE. Most of the studies reported newer βlactamases including MBL production in nonfermenters like *Pseudomonas aeruginosa*, *Acineto‐ bacter species* etc. There are very few studies that report MBL production in Enterobacteriaceae [19]. Hence, we have conducted the study to detect newer β - lactamases producing *E.coli*

A total Number of 450 *E.coli* strains isolated from different clinical specimens like urine, stool, blood, pus etc. were studied. The strains were characterized as *E.coli* according to conventional identification tests [5]. *E.coli* ATCC 25922 was used as positive control for all the conventional tests. Few recent tests were also included to identify *E.coli* which could reduce the number of biochemical tests and there by cost also e.g. Motility- Indole- Lysine (MIL) medium [62], Methylumbelliferyl- β-D- Glucuronide(MUG) MacConkey's medium [63]. All the *E.coli* strains isolated from urine samples were subcultured on Hi chrome UTI agar for direct detection of

Commonly primers used for detecting Class B metalloenzyme genes are [40]:

**E. Detection of Klebsiella pneumoniae carbapenamases (KPCs) [61]**

strains by phenotypic methods, isolated from different clinical specimens.

VIM-1 Forward – TTATGGAGCAGCAACCGATGT

Phenyl boronic acid, Molecular methods like PCR etc.

Reverse - CAAAAGTCCCGCTCCAACGA

reaction (PCR) [60].

**2. Material & methods**

*E.coli*.

Class B3 – MBL L1 unique among all β-lactamases in being functionally represented as a tetramer.

The numbering scheme has been recently updated to accommodate newly discovered MBLs.

MBLs are inhibited by EDTA (Ethylene diamino tetraacetic acid), 2 Mercaptoethanol, 5- Mercaptoacetic acid, 2 Mercapto propionic acid, Copper Chloride and Ferric Chloride. MBLs are classified mainly into two types – Chromosomally encoded and Plasmid encoded or Acquired or Transmissible type. Usually metallobetalactamase producing strains are suscep‐ tible to Colistin or Polymyxin B. MBLs do not hydrolyze aztreonam very well, which charac‐ teristic is different than ESBLs or Class A β-lactamases [48].

The acquired MBLs are further classified into different types depending on their place of origin as VIM (Italy or Greece), SPM (Brazil), GIM (Germany), SIM (Korea), DIM (Dutch), NDM/PCM (New Delhi metallobetalactamases/Plasmid coded metallobetalactamases).

**NDM – 1:** NDM – 1 was named after New Delhi, capital of India as NDM – 1 and was first described by Young et al in December 2009 in an individual who acquired infection in a Hospital in India due to Carbapenam resistant *Klebsiella pneumoniae* strain [49].

In March 2010, researchers from Mumbai found that most of Carbapenam resistant bacteria carried blaNDM–1 gene. The gene is carried on plasmids and is readily transferred between different strains of bacteria by horizontal gene transfer. All these strains were resistant to most of routinely used antibiotics like Aminoglycosides, β-lactams, Quinolones but sensitive to Tigecycline and Colistin [50]. Recently, Espinal et al identified a new variant of NDM-1 in *Acinetobacter baumannii* and designated it as NDM-2. They reported that, the clonal dissemi‐ nation of a NDM-2 producing *A. baumannii* was isolated in an Israeli rehabilitation ward [51]. Recently, a new variant of the New Delhi metallo-enzyme (NDM) carbapenemase, NDM-4 and NDM-5, was identified in *E.coli* from two patients both of them had a history of hospitalization in India [52,53].

#### **Detection of Metallo β-lactamase production**

Carbapenems often used as an antibiotic of last resort for treating serious infections caused by multi-drug resistant (MDR) organism. Reduced susceptibility to any Carbapenem can be used as a screen for carbapenemases. Positive screening tests are to be followed by a confirmatory test for MBL production.

Although a variety of phenotypic methods have been proposed for the detection of carbape‐ nemases, none have been recommended by CLSI. The classical Hodge [54], Modified Hodge test (MHT) [55] are economical approach for detection and confirmation of carbapenemase activity and Re – Modified Hodge test [56] for detection of MBL. However, the first two tests cannot differentiate between a class A carbapenemase and MBL, making a further confirma‐ tory test necessary. Imipenem is more sensitive but less specific Carbapenem for this test allowing detection of even OXA carbapenemases.

MBL detection tests involving inhibitors such as ethylene diamine tetraacetic acids (EDTA) and 2-Mercaptopropionic acids (2-MPA) have been recommended by various workers [57]. Tris/EDTA disks can also be used in combination with a Carbapenem disk to detect Carbape‐ nem - hydrolyzing enzymes and to differentiate between class A enzymes and MBLs. MBLs are inhibited by the Tris/EDTA disk. The inhibition of MBL can be enhanced by the addition of chelators. Double disk synergy test (DDST) [55] and Disk potentiation tests [58] are based on this principle. For detection of MBL many other methods used are MBL E-test using imipenem/imipenem-EDTA [59], reduction of MIC in presence of EDTA and polymerase chain reaction (PCR) [60].

Commonly primers used for detecting Class B metalloenzyme genes are [40]:

#### VIM-1 Forward – TTATGGAGCAGCAACCGATGT
