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

4

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

 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 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

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

Antibiotic susceptibility test for all 450 strains of *E.coli* were done using Mueller Hinton(MH) agar plate with commercially available antibiotic discs (Himedia Pvt Ltd, India) by Kirby Bauer disc diffusion method [64] according to CLSI guidelines [65]. *E.coli* ATCC 25922 was used as

Lawn culture of test strain (turbidity adjusted to 0.5 Mc Farland standard) was put on MH Agar plate. The antibiotic disks were put on inoculated plate with all aseptic precaution. Antibiotic susceptibility test was done for Aminoglycosides like Amikacin (AK-30μg), Gentamicin (GEN-10μg), Cephalosporins like Ceftazidime (CAZ-30μg), Cefotaxime (CTX-30μg), Fluoroquinolones like Ciprofloxacin (CIP–5μg), Monobactams like Aztreonam (AT-30μg), Carbapenems such as Imipenem (IPM-10μg), Etrapenem (ETP-10μg) etc. For urine sample an additional disk of Nitrofurantoin (NIT-300μg) and only for MBL producing strains,

All 450 *E.coli* strains were tested for newer β-lactamases e.g. Extended Spectrum β-lactamas‐ es(ESBLs), AMPC β-lactamases Metallobetalactamases(MBLs) and Klebsiella pneumoniae producing Carbapenemases (KPCs) [28, 30, 39, 58, 59, 61]. As Metallobetalactamases are also found in carbapenem susceptible organisms., we have screened carbapenem sensitive strains

Combined disk method as per CLSI guideline and ESBL E-test were used for ESBL detection. In Combined disk method, lawn cultures of test strains (turbidity adjusted to McFarland 0.5 standard) were put on MH agar plates. Ceftazidime (CAZ) 30 μg disc and Ceftazidime plus Clavulanate (CAC) 30μg plus 10μg discs were put widely apart on that MH plate. After

C increase in zone diameter of ≥5 mm with CAC disk as compared

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].

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

not detect new variants.

Molecular methods like PCR etc.

**II. MATERIAL & METHODS** 

54 Trends in Infectious Diseases

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

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

**2.1. Antibiotic susceptibility test**

Colistin (CL-10μg) disk were used

**2.2. Detection of newer β-lactamases**

*2.2.1. Detection of ESBL production [28]*

to CAZ disk alone was considered positive for ESBL detection.

also for MBL production.

overnight incubation at 370

control.

tests are to be followed by a confirmatory test for MBL 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 3 aminophenyl boronic acid (100 mg/ml) was used.

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 one of the ceftazidime disk. After overnight incubation at 370 C, an increase in zone size of ≥5mm around the Ceftazidime - APB disc compared to Ceftazidime disc only was recorded as a positive result for Amp C β-lactamase production.

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

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 methods.

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 CAZ disk. The plates were incubated 370 C overnight. The zone diameter of ≥ 5 mm around 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 only was considered positive for AmpC β – lactamase production.

#### *2.2.4. Detection of metallobetalactamases (MBL)*

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 ETest.

**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 at the centre and10 μl of 50mM zinc sulfate solution was added to Imipenem disk. Then, a test strain of E.coli was streaked from the edge of the disk to the periphery in four different directions. The plate was incubated overnight at 37°C. The presence of a cloverleaf shaped zone of inhibition due to MBL production by the test strain was considered as positive Re - Modified Hodge test (Re - MHT).

*2.2.6. Detection of class D enzymes*

is given below ( photo 4,5,6,7,8,9,10,11,12)

22

MBL only

62

ESBL only

tamases phenotypically is given below ( photo 4,5,6,7,8,9,10,11,12)

42

AmpC only

MBL + ESBL

**Detection of MBL** 

**3. Observation**

**III. OBSERVATION** 

detection of Class D carbapenemase in our study.

Several workers have reported that Class D enzymes i.e. OXA – 48 type are the most difficult carbapenemase producers to be identified phenotypically [42,43]. Hence, we did not include

Figure 1: Incidence of MBL, ESBL & Amp C β –lactamase producing *E.coli* strains (n = 450)

223

MBL only ESBL only AmpC only MBL + ESBL MBL + AmpC MBL + ESBL + AmpC ESBL + AmpC

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

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

57

 Figure 1 shows incidence of different β – lactamases e.g. MBL, ESBL, AmpC β – lactamases producing *E.coli* strains. Out of 450 *E.coli* strains studied, 378 (84%) strains produced any of the 3 types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases, either alone or in combinations. Photographs of different methods used to detect newer β-lactamases phenotypically

**Figure 1: Incidence of MBL, ESBL & AmpC β – lactamase producing** *E.coli* **strains (n = 450)**

7 8 14

MBL + AmpC

MBL + ESBL +

AmpC

**Figure 1.** Shows incidence of different β – lactamases e.g. MBL, ESBL, AMPC β – lactamases producing *E.coli* strains. Out of 450 *E.coli* strains studied, 378 (84%) strains produced any of the 3 types of β – lactamases i.e. MBL, ESBL and Amp C β –lactamases, either alone or in combinations. Photographs of different methods used to detect newer β-lac‐

**Photo 4 : ESBL Combined disc test+ve Photo 5 : ESBL Etest :positive** 

**Photo 6 : Both ESBL & AmpC β- lactamase +ve Photo 7 : Classical Hodge test +ve** 

ESBL + AmpC

7

8

**Imipenem-EDTA double disk synergy test (DDST)** [55]: The IMP-EDTA double disk synergy test was performed for detection of Metallobetalactamases. Test strains i.e. *E.coli* (turbidity adjusted to 0.5 McFarland standard) were inoculated on to Mueller Hinton agar plate. After drying, a 10μg Imipenem disk and a blank sterile filter paper disk (6mm in diameter, Whatman filter paper no.2) were placed 10mm apart from edge to edge. 10 μl of 50mM zinc sulfate solution was added to the 10 μg Imipenem disk. Then, 10μl of 0.5 M EDTA (Sigma, USA) solution was applied to the blank filter paper disk. As disodium-EDTA is difficult to be solubilised in sterile water, we had used dipotassium-EDTA which is easily soluble in sterile water. Enhancement of the zone of inhibition towards the EDTA disk was interpreted as a positive result.

**Disk Potentiation Test (DP)** [58]: The IMP-EDTA combined disk test was performed for detection of metallobetalactamases. Test strains (turbidity adjusted to 0.5 McFarland standard ) were inoculated on to MH agar plate. Two imipenem disk (10 μg) were placed on the plate wide apart and 10 μl of 50mM zinc sulphate solution was added to each of the imipenem disks. Then 10μl of 0.5M EDTA solution was added to one of the disk and the plates were incubated at 350 C for 16-18 hrs. If the increase in inhibition zone with the Imipenem and EDTA disk was ≥7 mm than the imipenem disk alone, it was considered as MBL positive.

#### **MBL E-Test — Confirmatory test**

The MBL E-test was done and interpreted using test strains and Quality control strains according to manufacturer's instructions. Overnight broth culture of test strain (turbidity adjusted to 0.5 McFarland standard) was used to inoculate MH agar plate. The MBL E-test strip was put on that inoculated MH plate with a sterile forceps and plates were incubated at 370 C for 18-20 hrs. After incubation, MIC ratio of Imipenem /Imipenem-EDTA (IP/IPI) of ≥8 or deformations of ellipse or phantom zone indicate MBL production.

**Colistin E test:** All MBL producing *E.coli* strains were tested with Colistin E test (AB bioMer‐ ieux, Solana, Sweden). The Colistin E-test was done and interpreted using test strains and Quality control strains according to manufacturer's instructions.

#### *2.2.5. Detection of Klebsiella pneumoniae carbapenemases (KPCS)*

It was done by Combined disk method [61]. Lawn culture of test strain (turbidity adjusted to 0.5 Mc Farland) was put on MH agar plate and 2 Imipenem (10 μg) disks were put widely apart. To one Imipenem disk 10 μl Phenyl boronic acid solution (400μg/disk) was put. Then the MH agar plates were incubated at 370 C overnight. After incubation, the test should be considered positive when growth inhibitory zone around the disk containing Imipenem and Phenyl boronic acid was ≥ 5 mm compared to zone diameter of Imipenem alone.

#### *2.2.6. Detection of class D enzymes*

Several workers have reported that Class D enzymes i.e. OXA – 48 type are the most difficult carbapenemase producers to be identified phenotypically [42,43]. Hence, we did not include detection of Class D carbapenemase in our study.
