**3. Observation**

**III. OBSERVATION** 

**Detection of MBL** 

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 -

**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

**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

C for 16-18 hrs. If the increase in inhibition zone with the Imipenem and EDTA disk was

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

C for 18-20 hrs. After incubation, MIC ratio of Imipenem /Imipenem-EDTA (IP/IPI) of ≥8 or

**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

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

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.

C overnight. After incubation, the test should be

≥7 mm than the imipenem disk alone, it was considered as MBL positive.

deformations of ellipse or phantom zone indicate MBL production.

Quality control strains according to manufacturer's instructions.

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

the MH agar plates were incubated at 370

Modified Hodge test (Re - MHT).

56 Trends in Infectious Diseases

**MBL E-Test — Confirmatory test**

positive result.

at 350

370

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

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

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

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

7

8

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

#### **Detection of MBL 3.1. Detection of MBL**

for Colistin (bioMe'rieux) (Photo 12).

In photo 5 ESBL E Test positive shows MIC of Ceftazidime (TZ) 6 μg/ml and Ceftazidime Clavulanic acid (TZL) 0.25μg/ml respectively i.e. MIC ratio of TZ/TZL is 24. Out of 378 β lactamase producing E.coli strains 223(59%) produced both ESBL andAmp C β –lactamases. Out of total 51 MBL producing *E.coli* strains 14 (27.5%) strains produced all the three types of β – lactamases i.e. MBL, ESBL and AMPC β – lactamases. In Photo 11 MBL E Test positive shows MIC of Imipenem (IP) 24 μg/ml and Imipenem-EDTA (IPI) < 1μg/ml respectively i.e. MIC ratio of IP/IPI is > 24 and also presence of Phantom zone.

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

8 All 51MBL positive *E.coli* strains were sensitive to Colistin with MIC range from 0.032 to 0.25μg/ml and were detected by E test for Colistin (bioMe'rieux) (Photo 12).

**Photo 11 : MBL E test +ve Photo 12: Colistin E test : MIC 0.125 μg/ml** 

In photo 2 ESBL E Test positive shows MIC of Ceftazidime (TZ) 6 µg/ml and Ceftazidime Clavulanic acid (TZL) 0.25µg/ml respectively i.e. MIC ratio of TZ/TZL is 24. Out of 378 β - lactamase producing E.coli strains 223(59%) produced both ESBL and AmpC β- lactamases. Out of total 51 MBL producing *E.coli* strains 14 (27.5%) strains produced all the three types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases. In Photo 11 MBL E Test positive shows MIC of Imipenem (IP) 24 µg/ml and Imipenem-EDTA (IPI) < 1µg/ml respectively i.e. MIC ratio of IP/IPI is > 24 and also presence of Phantom zone.

All 51MBL positive *E.coli* strains were sensitive to Colistin with MIC range from 0.032 to 0.25µg/ml and were detected by E test

**Figure 2: Isolation of MBL producing** *E.coli* **strains from different clinical specimens (n = 51)** 

**Photo 8 : Remodified Hodge test +ve** 

**Phantom zone**

**Photo 10 : Disc potentiation test +ve** 

9

9

Out of total 450 *E.coli* strains 218 (48.4%) were isolated from urine, 92 (20.4%) from stool, 61 from pus and wound swab, 30

Figure 2 shows out of total 51 MBL positive *E.coli* strains maximum 27(53%) strains were isolated from urine followed by 11(21.6%) strains from pus and wound swab. Out of 27 MBL positive *E.coli* strains isolated from urine 12(44.4%) had history of catheterization and 2(7.4%) had history of instrumentation in urethra (e.g.dilatation, etc). Only 01 urine sample received from High Dependency Unit and that *E.coli* strain produced all 3 types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases. In our study, total 14 *E.coli* strains were positive for all 3 types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases ,and out of which 11 (78.6%) strains were isolated from urine samples which was quite alarming. No MBL producing *E.coli* strain was isolated from body fluids. Out of 92 stool samples, 5 (5.4%) were MBL producers, 15 (16.3%) were only ESBL producer

**Figure 3: Isolation of only MBL and MBL with other β - lactamase producing** *E.coli* **strains from different clinical** 

MBL only

MBL + ESBL

MBL + AmpC

MBL + ESBL + AmpC

Figure 3 shows maximum 9/51 (17.7%) MBL producing *E.coli* strains were isolated from Pediatrics ward. No MBL producing strain was isolated from Cardiovascular & Thoracic Surgery (CVTS) ward. From Medicine ward, 16 *E.coli* strains were only

from blood, 10 from body fluids and 39 from other specimens e.g. ET tube secretions, broncho-alveolar lavage etc.

Others

10

**Photo 9 : DDST test +ve** 

**IMP + EDTA** 

**EDTA**

**Photo 10 : Disc potentiation test +ve** 

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

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

59

**Photo 11 : MBL E test +ve Photo 12: Colistin E test : MIC 0.125 μg/ml** 

In photo 2 ESBL E Test positive shows MIC of Ceftazidime (TZ) 6 µg/ml and Ceftazidime Clavulanic acid (TZL) 0.25µg/ml respectively i.e. MIC ratio of TZ/TZL is 24. Out of 378 β - lactamase producing E.coli strains 223(59%) produced both ESBL and AmpC β- lactamases. Out of total 51 MBL producing *E.coli* strains 14 (27.5%) strains produced all the three types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases. In Photo 11 MBL E Test positive shows MIC of Imipenem (IP) 24 µg/ml and Imipenem-EDTA (IPI) < 1µg/ml respectively i.e. MIC ratio of IP/IPI is > 24 and also presence of Phantom zone.

All 51MBL positive *E.coli* strains were sensitive to Colistin with MIC range from 0.032 to 0.25µg/ml and were detected by E test

and 13 (14.1%) were only AmpC β – lactamase producer.

Wound swab

Out of total 450 *E.coli* strains 218 (48.4%) were isolated from urine, 92 (20.4%) from stool, 61 from pus and wound swab, 30 from blood, 10 from body fluids and 39 from other specimens

Figure 2 shows out of total 51 MBL positive *E.coli* strains maximum 27(53%) strains were isolated from urine followed by 11(21.6%) strains from pus and wound swab. Out of 27 MBL positive *E.coli* strains isolated from urine 12(44.4%) had history of catheterization and 2(7.4%) had history of instrumentation in urethra (e.g.dilatation, etc). Only 01 urine sample received from High Dependency Unit and that *E.coli* strain produced all 3 types of β – lactamases i.e. MBL, ESBL and Amp C β –lactamases In our study, total 14 *E.coli* strains were positive for all 3 types of β – lactamases i.e. MBL, ESBL and AMPC β – lactamases,and out of which 11 (78.6%) strains were isolated from urine samples which was quite alarming. No MBL producing *E.coli* strain was isolated from body fluids. Out of 92 stool samples, 5 (5.4%) were MBL producers, 15 (16.3%) were only ESBL producer and 13 (14.1%) were only AMPC β – lactamase producer.

Blood Body

fluids

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

ESBL producers and 10 were only Amp C β – lactamase producers.

**Figure 2: Isolation of MBL producing** *E.coli* **strains from different clinical specimens (n = 51)** 

Urine Stool Pus +

**Figure 2.** Isolation of MBL producing *E.coli* strains from different clinical specimens (n = 51)

e.g. e.g. Endotracheal (ET) tube secretions, broncho-alveolar lavage etc.

**Photo 8 : Remodified Hodge test +ve** 

**Phantom zone**

for Colistin (bioMe'rieux) (Photo 12).

0

1

2

3

4

5

6

**specialities (n = 51)**

**Photo 9 : DDST test +ve** 

**IMP + EDTA** 

**EDTA**

**Photo 8 : Remodified Hodge test +ve** 

8

**Photo 9 : DDST test +ve** 

**IMP + EDTA** 

**Photo 10 : Disc potentiation test +ve** 

9

58 Trends in Infectious Diseases

for Colistin (bioMe'rieux) (Photo 12).

**Detection of MBL 3.1. Detection of MBL**

**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** 

In photo 5 ESBL E Test positive shows MIC of Ceftazidime (TZ) 6 μg/ml and Ceftazidime Clavulanic acid (TZL) 0.25μg/ml respectively i.e. MIC ratio of TZ/TZL is 24. Out of 378 β lactamase producing E.coli strains 223(59%) produced both ESBL andAmp C β –lactamases. Out of total 51 MBL producing *E.coli* strains 14 (27.5%) strains produced all the three types of β – lactamases i.e. MBL, ESBL and AMPC β – lactamases. In Photo 11 MBL E Test positive shows MIC of Imipenem (IP) 24 μg/ml and Imipenem-EDTA (IPI) < 1μg/ml respectively i.e.

All 51MBL positive *E.coli* strains were sensitive to Colistin with MIC range from 0.032 to

**EDTA**

**Photo 11 : MBL E test +ve Photo 12: Colistin E test : MIC 0.125 μg/ml** 

In photo 2 ESBL E Test positive shows MIC of Ceftazidime (TZ) 6 µg/ml and Ceftazidime Clavulanic acid (TZL) 0.25µg/ml respectively i.e. MIC ratio of TZ/TZL is 24. Out of 378 β - lactamase producing E.coli strains 223(59%) produced both ESBL and AmpC β- lactamases. Out of total 51 MBL producing *E.coli* strains 14 (27.5%) strains produced all the three types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases. In Photo 11 MBL E Test positive shows MIC of Imipenem (IP) 24 µg/ml and Imipenem-EDTA (IPI) < 1µg/ml respectively i.e. MIC ratio of IP/IPI is > 24 and also presence of Phantom zone.

All 51MBL positive *E.coli* strains were sensitive to Colistin with MIC range from 0.032 to 0.25µg/ml and were detected by E test

**Figure 2: Isolation of MBL producing** *E.coli* **strains from different clinical specimens (n = 51)** 

MIC ratio of IP/IPI is > 24 and also presence of Phantom zone.

**Photo 8 : Remodified Hodge test +ve** 

**Phantom zone**

0.25μg/ml and were detected by E test for Colistin (bioMe'rieux) (Photo 12).

**Photo 10 : Disc potentiation test +ve** 

**Photo 11 : MBL E test +ve Photo 12: Colistin E test : MIC 0.125 μg/ml** 

9

MBL only

MBL + ESBL

MBL + AmpC

MBL + ESBL + AmpC

Figure 3 shows maximum 9/51 (17.7%) MBL producing *E.coli* strains were isolated from Pediatrics ward. No MBL producing strain was isolated from Cardiovascular & Thoracic Surgery (CVTS) ward. From Medicine ward, 16 *E.coli* strains were only

10

In photo 2 ESBL E Test positive shows MIC of Ceftazidime (TZ) 6 µg/ml and Ceftazidime Clavulanic acid (TZL) 0.25µg/ml

Out of total 450 *E.coli* strains 218 (48.4%) were isolated from urine, 92 (20.4%) from stool, 61 from pus and wound swab, 30 from blood, 10 from body fluids and 39 from other specimens e.g. ET tube secretions, broncho-alveolar lavage etc. **Figure 2.** Isolation of MBL producing *E.coli* strains from different clinical specimens (n = 51)

Figure 2 shows out of total 51 MBL positive *E.coli* strains maximum 27(53%) strains were isolated from urine followed by 11(21.6%) strains from pus and wound swab. Out of 27 MBL positive *E.coli* strains isolated from urine 12(44.4%) had history of catheterization and 2(7.4%) had history of instrumentation in urethra (e.g.dilatation, etc). Only 01 urine sample received from High Dependency Unit and that *E.coli* strain produced all 3 types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases. Out of total 450 *E.coli* strains 218 (48.4%) were isolated from urine, 92 (20.4%) from stool, 61 from pus and wound swab, 30 from blood, 10 from body fluids and 39 from other specimens e.g. e.g. Endotracheal (ET) tube secretions, broncho-alveolar lavage etc.

In our study, total 14 *E.coli* strains were positive for all 3 types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases ,and out of which 11 (78.6%) strains were isolated from urine samples which was quite alarming. No MBL producing *E.coli* strain was isolated from body fluids. Out of 92 stool samples, 5 (5.4%) were MBL producers, 15 (16.3%) were only ESBL producer and 13 (14.1%) were only AmpC β – lactamase producer. **Figure 3: Isolation of only MBL and MBL with other β - lactamase producing** *E.coli* **strains from different clinical specialities (n = 51)** Figure 2 shows out of total 51 MBL positive *E.coli* strains maximum 27(53%) strains were isolated from urine followed by 11(21.6%) strains from pus and wound swab. Out of 27 MBL positive *E.coli* strains isolated from urine 12(44.4%) had history of catheterization and 2(7.4%) had history of instrumentation in urethra (e.g.dilatation, etc). Only 01 urine sample received from High Dependency Unit and that *E.coli* strain produced all 3 types of β – lactamases i.e. MBL, ESBL and Amp C β –lactamases In our study, total 14 *E.coli* strains were positive for all 3 types of β – lactamases i.e. MBL, ESBL and AMPC β – lactamases,and out of which 11 (78.6%) strains were isolated from urine samples which was quite alarming. No MBL producing *E.coli* strain was isolated from body fluids. Out of 92 stool samples, 5 (5.4%) were MBL producers, 15 (16.3%) were only ESBL producer and 13 (14.1%) were only AMPC β – lactamase producer.

ESBL producers and 10 were only Amp C β – lactamase producers.

0

1

2

3

4

5

6

**specialities (n = 51)**

and 13 (14.1%) were only AmpC β – lactamase producer.

Wound swab

Blood Body fluids

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

Urine Stool Pus +

Out of total 450 *E.coli* strains 218 (48.4%) were isolated from urine, 92 (20.4%) from stool, 61 from pus and wound swab, 30

Figure 2 shows out of total 51 MBL positive *E.coli* strains maximum 27(53%) strains were isolated from urine followed by 11(21.6%) strains from pus and wound swab. Out of 27 MBL positive *E.coli* strains isolated from urine 12(44.4%) had history of catheterization and 2(7.4%) had history of instrumentation in urethra (e.g.dilatation, etc). Only 01 urine sample received from High Dependency Unit and that *E.coli* strain produced all 3 types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases. In our study, total 14 *E.coli* strains were positive for all 3 types of β – lactamases i.e. MBL, ESBL and AmpC β – lactamases ,and out of which 11 (78.6%) strains were isolated from urine samples which was quite alarming. No MBL producing *E.coli* strain was isolated from body fluids. Out of 92 stool samples, 5 (5.4%) were MBL producers, 15 (16.3%) were only ESBL producer

**Figure 3: Isolation of only MBL and MBL with other β - lactamase producing** *E.coli* **strains from different clinical** 

from blood, 10 from body fluids and 39 from other specimens e.g. ET tube secretions, broncho-alveolar lavage etc.

Others

strain was isolated from Cardiovascular & Thoracic Surgery (CVTS) ward. From Medicine ward, 16 *E.coli* strains were only ESBL producers and 10 were only Amp C β – lactamase producers. **Figure 3.** Isolation of only MBL and MBL with other β - lactamase producing *E.coli* strains from different clinical spe‐ cialities (n = 51)

Figure 3 shows maximum 9/51 (17.7%) MBL producing *E.coli* strains were isolated from Pediatrics ward. No MBL producing

 Figure 3 shows maximum 9/51 (17.7%) MBL producing *E.coli* strains were isolated from Pediatrics ward. No MBL producing strain was isolated from Cardiovascular & Thoracic Surgery (CVTS) ward. From Medicine ward, 16 *E.coli* strains were only ESBL producers and 10 were only Amp C β – lactamase producers.

**Figure 4 : Antibiotic resistance pattern of MBL producing** *E.coli* **strains (n = 51)**

10

Amikacin and only 28.2% strains were sensitive to Ciprofloxacin. Nitrofurantoin was used for urine specimen only (n = 218) and 67.9% strains were sensitive to Nitrofurantoin. Amongst the 51 MBL producing *E.coli* strain, 12 (23.5%) strains were sensitive to Imipenem and Etrapenem by disk diffusion test. Out of 12 Imipenem sensitive MBL producing *E.coli* strain, 5 (41.7%)

**Re - MHT DDST DP**

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

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

61

**45 46 51**

**401 402 399**

strains produced all 3 types of β – lactamases.

**Strains Phenotypic methods**

**False negative 6 5 0 False positive 4 2 0 Sensitivity % 88.2 90.2 100 Specificity % 99 99.5 100 Positive predictive value 91.8 95.8 100 Negative predictive value 98.5 98.8 100 Efficiency 97.8 98.5 100**

**Table 1.** Performance of different phenotypic methods compared to MBL – E test in identifying MBL + ve *E.coli*

and DP was 100%, when compared to MBL - E test as standard reference method.

Table 1 shows Sensitivity, Specificity, Positive predictive value, Negative predictive value and Efficiency calculated for Re – Modified Hodge test (Re – MHT), Double disk synergy test (DDST) and Disk potentiation (DP) test, compared to MBL – E test in identifying MBL positive E.coli strains. MBL – E test is considered as standard phenotypic reference method for detection of MBL positive strains. The sensitivity of Re - MHT was 88.2% and specificity was 99% whereas sensitivity of DDST was 90.2% and specificity was 99.5%. DP test was having sensitivity and specificity of 100%. The efficiency of Re – MHT was 97.8%, DDST was 98.5%

The emergence of antibiotic resistance occurs by a) spontaneous mutation and vertical gene transfer and b) horizontal gene transfer through transformation, transduction, conjugation, transposons (jumping genes) etc. The rapidity of development of antimicrobial resistance in organisms, leads to selection pressure of antibiotics like 3rd generation of cephalosporin- ESBL inhibitor combination, Monobactams and Carbapenems. Recently, Carbapenem resistant Enterobacteriaceae (CRE) pose a real threat to Medical fraternity as the increased frequency

**MBL + ve (n = 51) By MBL – E test**

**MBL – ve (n = 399) By MBL – E test**

**4. Discussion**

11

**Figure 4.** Antibiotic resistance pattern of MBL producing *E.coli* strains (n = 51)

**Strains Phenotypic methods** 

**MBL + ve (n = 51) By MBL – E test** 

**MBL – ve (n = 399) By MBL – E test** 

Figure 4 shows out of total 51 MBL producing *E.coli* strains, 39 (76.5%) strains were resistant to Imipenem and Etrapenem by disc diffusion method. The MBL producing strains of *E.coli* showed total resistance to Ampicillin, Gentamicin, Ciprofloxacin, Co – trimoxazole, Tetracycline, Ceftazidime, Cephotaxime and Cefoxitin. But all MBL positive *E.coli* strains (100%) were sensitive to Colistin. Out of total 450 *E.coli* strains, only 58.9% strains were sensitive to Amikacin and only 28.2% strains were sensitive to Ciprofloxacin. Nitrofurantoin was used for urine specimen only (n = 218) and 67.9% strains were sensitive to Nitrofurantoin. Amongst the 51 MBL producing *E.coli* strain, 12 (23.5%) strains were sensitive to Imipenem and Etrapenem by disk diffusion test. Out of 12 Imipenem sensitive MBL producing *E.coli* strain, 5 (41.7%) strains produced all 3 types of β – lactamases. Figure 4 shows out of total 51 MBL producing *E.coli* strains, 39 (76.5%) strains were resistant to Imipenem and Etrapenem by disc diffusion method. The MBL producing strains of *E.coli* showed total resistance to Ampicillin, Gentamicin, Ciprofloxacin, Co – trimoxazole, Tetracy‐ cline, Ceftazidime, Cephotaxime and Cefoxitin. But all MBL positive *E.coli* strains (100%) were sensitive to Colistin. Out of total 450 *E.coli* strains, only 58.9% strains were sensitive to

**Table 1: Performance of different phenotypic methods compared to MBL – E test in identifying MBL + ve** *E.coli*

**False negative 6 5 0 False positive 4 2 0 Sensitivity % 88.2 90.2 100 Specificity % 99 99.5 100 Positive predictive value 91.8 95.8 100** 

**Negative predictive value 98.5 98.8 100 Efficiency 97.8 98.5 100** 

97.8%, DDST was 98.5% and DP was 100%, when compared to MBL - E test as standard reference method.

**Re - MHT DDST DP** 

**45 46 51** 

**401 402 399** 

Table 1 shows Sensitivity, Specificity, Positive predictive value, Negative predictive value and Efficiency calculated for Re – Modified Hodge test (Re – MHT), Double disk synergy test (DDST) and Disk potentiation (DP) test, compared to MBL – E test in identifying MBL positive E.coli strains. MBL – E test is considered as standard phenotypic reference method for detection of MBL positive strains. The sensitivity of Re - MHT was 88.2% and specificity was 99% whereas sensitivity of DDST was 90.2% and specificity was 99.5%. DP test was having sensitivity and specificity of 100%. The efficiency of Re – MHT was Amikacin and only 28.2% strains were sensitive to Ciprofloxacin. Nitrofurantoin was used for urine specimen only (n = 218) and 67.9% strains were sensitive to Nitrofurantoin. Amongst the 51 MBL producing *E.coli* strain, 12 (23.5%) strains were sensitive to Imipenem and Etrapenem by disk diffusion test. Out of 12 Imipenem sensitive MBL producing *E.coli* strain, 5 (41.7%) strains produced all 3 types of β – lactamases.


**Table 1.** Performance of different phenotypic methods compared to MBL – E test in identifying MBL + ve *E.coli*

Table 1 shows Sensitivity, Specificity, Positive predictive value, Negative predictive value and Efficiency calculated for Re – Modified Hodge test (Re – MHT), Double disk synergy test (DDST) and Disk potentiation (DP) test, compared to MBL – E test in identifying MBL positive E.coli strains. MBL – E test is considered as standard phenotypic reference method for detection of MBL positive strains. The sensitivity of Re - MHT was 88.2% and specificity was 99% whereas sensitivity of DDST was 90.2% and specificity was 99.5%. DP test was having sensitivity and specificity of 100%. The efficiency of Re – MHT was 97.8%, DDST was 98.5% and DP was 100%, when compared to MBL - E test as standard reference method.
