**2. Material & methods**

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 *E.coli*.

**II. MATERIAL & METHODS** 

not detect new variants.

Molecular methods like PCR etc.

chrome UTI agar for direct detection of *E.coli*.

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

tests are to be followed by a confirmatory test for MBL production.

**Photo 1 : Motility – Indole – Lysine medium Motility +VE, Indole production test +ve, Lysine decarboxylase test +ve.**

 In March 2010, researchers from Mumbai found that most of Carbapenam resistant bacteria carried blaNDM–1 gene. The gene 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, Colistin [50]. Recently, Espinal et al identified a new variant of NDM-1 in *Acinetobacter baumannii* and designated as NDM-2. They reported that, the clonal dissemination 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,

. 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

Although a variety of phenotypic methods have been proposed for the detection of carbapenemases, 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 confirmatory test necessary. Imipenem is more

 MBL detection tests involving inhibitors such as ethylene diamine tetraacetic acids (EDTA) and 2-Mercaptopropionic acids (2-MPA) have been recommended various workers [57]. Tris/EDTA disks can also be used in combination with a Carbapenem disk to detect Carbapenem - 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

 PCR is specific for gene family IMP, VIM, etc. and hence, many others 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

KPCs can be mainly detected by Combined disk method using Imipenem and Imipenem with Phenyl boronic acid,

 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 β- lactamases including MBL production in nonfermenters like *Pseudomonas aeruginosa*, *Acinetobacter 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* strains by phenotypic methods, isolated from different clinical specimens.

 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

Glucuronide(MUG) MacConkey's medium[63]. All the *E.coli* strains isolated from urine samples were subcultured on Hi

was identified in *E. coli* from two patients both of them had a history of hospitalization in India[52,53].

sensitive but less specific Carbapenem for this test allowing detection of even OXA carbapenemases.

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

**I.2.d. DETECTION OF KLEBSIELLA PNEUMONIAE CARBAPENEMASES (KPCs) [61]** :

VIM – 1 Forward – TTATGGAGCAGCAACCGATGT Reverse - CAAAAGTCCCGCTCCAACGA

imipenem/imipenem-EDTA[59], reduction of MIC in presence of EDTA and polymerase chain reaction (PCR)[60].

**Photo 2: MUG Mac Conkey's agar Typical bluish fluorescence**

**Photo 3 : Hi Chrome UTI agar :** *E.coli*

4

In ESBL E-test, lawn culture of test strain (turbidity adjusted to McFarland 0.5 standard) was done on a MH agar plate & ESBL E-test strip (AB Biomeriux) was placed. After overnight

deformation of ellipse or phantom zone present was considered positive for ESBL production.

For, detection of Amp C β –lactamase producing strains substrate inducer combination of Imipenem (10μg) / Ceftazidime(30 μg) disks and for confirmation disk potentiation test using

In Disk potentiation test, lawn culture of test strain (turbidity adjusted to McFarland 0.5 standard) was done on MH agar plate. Two ceftazidime(30μg) disks with centre to centre distance of 30mm were placed on that MH plate. 3-aminophenylboronic acid (APB) was dissolved in DMSO at a concentration of 100mg/ml. 10μl of this APB solution was added to

≥5mm around the Ceftazidime - APB disc compared to Ceftazidime disc only was recorded as

As ESBL and AMPC β – lactamase can be produced by a single strain and ESBL production is suppressed if the same strain also produces Amp C β –lactamases ,we followed the following

Lawn culture of test strain (turbidity adjusted to McFarland 0.5 standard) was done on MH agar plate. To detect the strains producing both ESBL and AMPC β – lactamases, we used one disk containing Ceftazidime and Clavulanic acid (CAC) and the other 02 disks containing Ceftazidime (CAZ) only, placed widely apart. On CAC disk 10μl of 3-aminophenyl boronic acid (3 – APB) (100mg/ml) solution was put. 3 – APB inhibit the growth of AmpC β – lactamases and ESBL genes can be expressed whereas 10 μl of 3 – APB solution was also put on one of the

CAC disk with 3 - APB compared to CAZ only was recorded as ESBL positive and increase in zone diameter of ≥5 mm around CAZ and 3 – APB disc compared to zone diameter of CAZ

All 450 *E.coli* strains were screened for Carbapenemase activity by Classical Hodge test [54] and for MBL production by Re-Modified Hodge test [56],DDST [55], DP test [58] and MBL

**Re-Modified Hodge Test (Re–MHT)** [56]: All 450 *E.coli* strains were subjected to Re-modified Hodge test for detection of carbapenemase activity. The broth culture of *Escherichia Coli* ATCC 25922 was adjusted to a turbidity of 0.5 McFarland standards and was used to put lawn culture on MH agar plates with sterile swab. After drying, a 10μg Imipenem disc (HiMedia) was placed

C, MIC ratio of ceftazidime/Ceftazidime Clavulanic acid (TZ/TZL) ≥ 8 or

C, an increase in zone size of

Newer β-Lactamases and *E.coli* — A Cause of Concern

http://dx.doi.org/10.5772/57578

55

C overnight. The zone diameter of ≥ 5 mm around

incubation at 370

methods.

ETest.

*2.2.2. Detection of AmpC β- lactamases [39]*

3 aminophenyl boronic acid (100 mg/ml) was used.

a positive result for Amp C β-lactamase production.

CAZ disk. The plates were incubated 370

*2.2.4. Detection of metallobetalactamases (MBL)*

one of the ceftazidime disk. After overnight incubation at 370

*2.2.3. Detection of both ESBL & AmpC β -lactamase producing strains [66]*

only was considered positive for AmpC β – lactamase production.
