**4. Discussion**

10

11

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

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

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

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

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.

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*

**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

Out of 12 Imipenem sensitive MBL producing *E.coli* strain, 5 (41.7%) strains produced all 3 types of β – lactamases.

0 10 20 30 40 50 60

> **9 9**

**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.

**Strains Phenotypic methods** 

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

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

Ampicillin Gentamicin Amikacin Ciprofloxacin Co‐trimoxazole Tetracycline Nitrofurantoin Imipenem Etrapenem Ceftazidime Cephotaxime Cefoxitin Aztreonam

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

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

Others

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 +

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 were only Amp C β – lactamase producers.

cialities (n = 51)

0

1

2

3

4

5

6

60 Trends in Infectious Diseases

**specialities (n = 51)**

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 **IV. DISCUSSION** 

AmpC β-lactamase producers.

**ICUs** 

**52**

**56**

**342**

Gupta V et al have reported 17(68%) *E.coli* strains to be ESBL positive[71].

Re – modified Hodge test (Re – MHT) Double Disk Synergy test(DDST)

True + ve : 45 True + ve : 46 False + ve : 4 False + ve : 2

with which Enterobacteriaceae cause infection and the mortality associated with infection caused by CRE and ESBL producing bacteria. In mid1990, CTX-M 15 was first reported as ESBL in India. Now, CTX-M 15 is established as globally dominant ESBL and primary cause of acquired resistance to 3rd generation Cephalosporins in Enterobacteriaceae. Walsh TR et al in year 2005 noted that MBL genes have spread from *Pseudomonas aeruginosa* to Enterobacteria‐ ceae and a clinical scenario for MBL appears to simulate the global spread of ESBL in recent future. bla NDM-1 gene on plasmid can be readily transferred between different strains of bacteria by horizontal genre transfer [47]. **IV. DISCUSSION**  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

In the present study, 52(11.6%), 56(12.4%) and 342 (76%) *E.coli* strains were isolated from Outpatient Departments(OPDs), Intensive Care units (ICUs) and Inpatient Departments(IPDs) respectively. Maximum 25 (44.6%) *E.coli* strains were isolated from Medicine ICU (MICU) and High dependency unit (HDU). In a previous study conducted in our laboratory in 2008, Basak et al have already reported the incidence of ESBL producing *E.coli* in our hospital as 41.3% [67] whereas 5 years after, in the present study, the incidence of ESBL producing *E.coli* were 68%, out of which only 13.8% strains produced ESBL alone and other strains produced ESBL, Amp C β-lactamases and MBL in combination. Pakzad I et al in 2011 have reported 28% of their *E.coli* strains as ESBL producers [68]. Sinha et al in 2008 had reported that 40.8% of *E.coli* strains were ESBL producers and 24% were AMPC β-lactamase producers [69]. 37.5% and 47.8% of *E.coli* strains were reported to be Amp C β –lactamases producers in the study conducted in Chennai, India and Kolkata,India respectively, whereas in our study 65.3% *E.coli* were Amp C β-lactamase producers. threat to Medical fraternity as the increased frequency with which Enterobacteriaceae cause infection and the mortality associated with infection caused by CRE and ESBL producing bacteria. In mid1990, CTX-M 15 was first reported as ESBL in India. Now, CTX-M 15 is established as globally dominant ESBL and primary cause of acquired resistance to 3rd generation Cephalosporins in Enterobacteriaceae. Walsh TR et al in year 2005 noted that MBL genes have spread from *Pseudomonas aeruginosa* to Enterobacteriaceae and a clinical scenario for MBL appears to simulate the global spread of ESBL in recent future. bla NDM-1 gene on plasmid can be readily transferred between different strains of bacteria by horizontal genre transfer [47]. In the present study, 52(11.6%), 56(12.4%) and 342 (76%) *E. coli* strains were isolated from Outpatient Departments(OPDs), Intensive Care units (ICUs) and Inpatient Departments(IPDs) respectively. Maximum 25 (44.6%) *E.coli* strains were isolated from Medicine ICU (MICU) and High dependency unit (HDU). In a previous study conducted in our laboratory in 2008, Basak et al have already reported the incidence of ESBL producing *E.coli* in our hospital as 41.3% [67] whereas 5 years after, in the present study, the incidence of ESBL producing *E.coli* were 68%, out of which only 13.8% strains produced ESBL alone and other strains produced ESBL, Amp C β-lactamases and MBL in combination. Pakzad I et al in 2011 have reported 28% of their *E.coli* strains as ESBL producers [68]. Sinha et al in 2008 had reported that 40.8% of *E.coli* strains were ESBL producers and 24% were AmpC β-lactamase producers [69]. 37.5% and 47.8% of *E.coli* strains were reported to be AmpC β-lactamase producers in the study conducted in Chennai, India and Kolkata,India respectively, whereas in our study 65.3% *E.coli* were AmpC β-lactamase producers. 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 with which Enterobacteriaceae cause infection and the mortality associated with infection caused by CRE and ESBL producing bacteria. In mid1990, CTX-M 15 was first reported as ESBL in India. Now, CTX-M 15 is established as globally dominant ESBL and primary cause of acquired resistance to 3rd generation

**Figure 5.** Isolation of *E.coli* strains from OPD, IPD and ICUs OT‐ICU

OPD

IPD

ICUs

Cephalosporins in Enterobacteriaceae. Walsh TR et al in year 2005 noted that MBL genes have spread from *Pseudomonas* 

Gupta V et al have reported 17(68%) *E.coli* strains to be ESBL positive[71].

**= 56)** 

Re – modified Hodge test (Re – MHT) Double Disk Synergy test(DDST)

True + ve : 45 True + ve : 46 False + ve : 4 False + ve : 2

**Figure 7 : Phenotypic detection of metallobetalactamase (MBL) producing** *E.coli* **strains by various methods. (n = 450)** 

12

**Figure 5 : Isolation of** *E.coli* **strains from OPD, IPD and** 

12

error [45].

**Figure 6 : Isolation of** *E.coli* **strains from different ICUs (n = 56)** 

**14**

**10**

**25**

**<sup>7</sup>** MICU

NICU

to be ESBL positive [71].

Disk Potentiation test (DP)

True + ve : 45 True + ve : 46 False + ve : 4 False + ve : 2

True + ve : 51

 **Disk Potentiation test (DP) True + ve : 51**

Re – modified Hodge test (Re – MHT) Double Disk Synergy test(DDST)

**Re ‐ MHT**

**4 45**

**<sup>0</sup> <sup>1</sup>**

potentiation (DP) test.

**V. EPIDEMIOLOGY -** 

Carbapenem resistant metallobetalactamases)[79].

Various authors have reported MBL producing *E.coli* strains from 2.9% (Pandya et al from Gujrat, India) [70], to 6.8% (Tsakris et al from Greece) [61] to 25% (Enwuru NV et al from Nigeria) [13]. In the present study 51(11.3%) MBL producing *E.coli* strains were isolated. MBL production was detected in both Imipenem resistant (39/41 i.e. 95.1%) and Imipenem sensitive 12/409 i.e. 2.9% strains also. It indicates that if only Imipenem resistant strains would have been screened, 2.9% MBL producing strains would have been missed. But no Klebsiella pneumoniae producing carbapenemases were detected in our present study. In 2011, Tsakris et al reported 15.9% KPC producing *E.coli* in their study. Tsakris et al have also reported that 19(43.2%) *E.coli* strains produced Amp C β –lactamases and ESBL and 15(34%) *E.coli* strains produced ESBL [61]. In another study in 2012, Gupta V et al have reported 17(68%) *E.coli* strains

> Walsh et al in 2002 have reported that the MBL – E test results were in 100% agreement with the results from the genotypic Polymerase chain reaction (PCR) and biochemical methods [59]. They have also reported that the E test MBL strip IP/IPI has the ability to detect MBLs both chromosomally and plasmid mediated, in aerobic and anaerobic bacteria. This novel

In the present study, we studied MBL positive *E.coli* strains by MBL E – test and compared the results of other phenotypic methods for MBL detection i.e. Re – Modified Hodge test (Re- MHT), Double disk synergy test (DDST) and Disk

In figure 7, the venndiagram showing interrelationship of Re – modified Hodge test (Re – MHT), Double disk synergy

Most common MBL found worldwide in Enterobacteriaceae were VIM (Verona integron encoded MBL) and IMP (active on imipenem). Multidrug resistant *E.coli* harboring New Delhi metallobetalactamase - 1 (NDM-1) isolated from a patient returned to Canada from India[75], was reported first in 2009. NDM -1 was also recognized among Enterobacteriaceae 32 from Mumbai, 13 from Varanasi and 3 from Guwahati in India and 25 isolates from eight different cities in Pakistan. These isolates

 In the present study when results of all three phenotypic methods were compared with MBL – E test results, it was found that 45/51 (88.2%) MBL positive strains were positive by all three phenotypic method i.e. Re – Modified Hodge test (Re-MHT), Double disk synergy test (DDST) and Disk potentiation (DP) tests. 04 and 02 were false positive by Re – MHT and DDST methods respectively. Whereas 6/51 (11.8%) and 5/51(9.8%) were false negative by Re – MHT and DDST method respectively. Amongst all three phenotypic methods, DP was best correlated with MBL – E test. By DP test 51 MBL positive *E.coli* strains were detected and no false positive and false negative result was found (Sensitivity 100% and specificity 100%).

*E.coli* are responsible for various infections like urinary tract infection, diarrhoea, pneumonia, bacteremia, upper

 The successful outcome of clinical use of 3rd generation cephalosporines unfortunately led to the increased use and emergence of ESBL producing Enterobacteriaceae. With the emergence of ESBL and Amp C β – lactamase production in *E.coli*, Klebsiella pneumoniae and other Enterobacteriaceae, Carbapenems were used as last resort to treat those infections. Because of

were from cases of bacteraemia, ventilator associated pneumonia and community acquired urinary tract infections [76].

 NDM - 1 spread largely to different countries like Australia, Japan, Brazil, Belgium, Canada, Germany etc [77]. The gene encoding NDM – 1is called blaNDM – 1, located on transmissible plasmid which may include other antibiotic resistance genes also leading to extensive drug resistant phenotypes (so called 'superbugs'). A recent report from ICU and wards of Sir Gangaram hospital Delhi, India showed 8.1% NDM – 1 positive E.coli [78]. In January 2011, the name of NDM–1 was changed to PCM (Plasmid encoding

Metallobatalactamases are also found in Carbapenem susceptible organisms. This hidden MBL gene can spread

selective pressure of Carbapenems, even carbapenemases producing Enterobacteriaceae (CRE) has emerged.

unnoticed in hospitals if isolates are reported sensitive without screening for presence of MBL[48].

 Omair et al in 2012, have reported that MBL – E test have been taken as a gold standard method for MBL detection [72]. Manoharan et al have reported that MBL - E test has taken as a phenotypic standard method for MBL detection though the test is expensive. Double disk synergy test (DDST) and Disk potentiation (DP) tests are economical and simple to perform but

**DDST**

**2**

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

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

63

method could be used by Clinical Laboratories to monitor the emergence of the MBL [59].

Walsh et al in 2002 have reported that the MBL – E test results were in 100% agreement with the results from the genotypic Polymerase chain reaction (PCR) and biochemical methods [59]. They have also reported that the E test MBL strip IP/IPI has the ability to detect MBLs both chromosomally and plasmid mediated, in aerobic and anaerobic bacteria. This novel method

**Figure 7.** Phenotypic detection of metallobetalactamase (MBL) producing *E.coli* strains by various methods. (n = 450)

**5 DP**

test (DDST) and Disk potentiation test (DP) for detection of MBL producing *E.coli*.

Omair et al in 2012, have reported that MBL – E test have been taken as a gold standard method for MBL detection [72]. Manoharan et al have reported that MBL - E test has taken as a phenotypic standard method for MBL detection though the test is expensive. Double disk synergy test (DDST) and Disk potentiation (DP) tests are economical and simple to perform but DDST is observer dependent while DP test is measurable with lesser chance of subjective

could be used by Clinical Laboratories to monitor the emergence of the MBL [59].

respiratory tract infections, wound infections, osteomyelitis and neonatal meningitis[73,74].

DDST is observer dependent while DP test is measurable with lesser chance of subjective error [45].

13

PICU

**Figure 5 : Isolation of** *E.coli* **strains from OPD, IPD and Figure 6 : Isolation of** *E.coli* **strains from different ICUs (n Figure 6.** Isolation of *E.coli* strains from different ICUs (n = 56)

Various authors have reported MBL producing *E.coli* strains from 2.9% (Pandya et al from Gujrat, India) [70], to 6.8% (Tsakris et al from Greece) [61] to 25% (Enwuru NV et al from Nigeria) [13]. In the present study 51(11.3%) MBL producing *E.coli* strains were isolated. MBL production was detected in both Imipenem resistant (39/41 i.e. 95.1%) and Imipenem sensitive 12/409 i.e. 2.9% strains also. It indicates that if only Imipenem resistant strains would have been screened, 2.9% MBL producing strains would have been missed. But no Klebsiella pneumoniae producing carbapenemases were detected in our present study. In 2011, Tsakris et al reported 15.9% KPC producing *E.coli* in their study. Tsakris et al have also reported that 19(43.2%) *E.coli* strains produced AmpC β – lactamases and ESBL and 15(34%) *E.coli* strains produced ESBL [61]. In another study in 2012,

**Figure 7 : Phenotypic detection of metallobetalactamase (MBL) producing** *E.coli* **strains by various methods. (n = 450)** 

13

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 with which Enterobacteriaceae cause infection and the mortality associated with infection caused by CRE and ESBL producing bacteria. In mid1990, CTX-M 15 was first reported as ESBL in India. Now, CTX-M 15 is established as globally dominant ESBL and primary cause of acquired resistance to 3rd generation Cephalosporins in Enterobacteriaceae. Walsh TR et al in year 2005 noted that MBL genes have spread from *Pseudomonas*  Various authors have reported MBL producing *E.coli* strains from 2.9% (Pandya et al from Gujrat, India) [70], to 6.8% (Tsakris et al from Greece) [61] to 25% (Enwuru NV et al from Nigeria) [13]. In the present study 51(11.3%) MBL producing *E.coli* strains were isolated. MBL production was detected in both Imipenem resistant (39/41 i.e. 95.1%) and Imipenem sensitive 12/409 i.e. 2.9% strains also. It indicates that if only Imipenem resistant strains would have been screened, 2.9% MBL producing strains would have been missed. But no Klebsiella pneumoniae producing carbapenemases were detected in our present study. In 2011, Tsakris et al reported 15.9% KPC producing *E.coli* in their study. Tsakris et al have also reported that 19(43.2%) *E.coli* strains produced Amp C β –lactamases and ESBL and 15(34%) *E.coli* strains produced ESBL [61]. In another study in 2012, Gupta V et al have reported 17(68%) *E.coli* strains to be ESBL positive [71].

 **Disk Potentiation test (DP) True + ve : 51** Walsh et al in 2002 have reported that the MBL – E test results were in 100% agreement with the results from the genotypic Polymerase chain reaction (PCR) and biochemical methods [59]. They have also reported that the E test MBL strip IP/IPI has the ability to detect MBLs both chromosomally and plasmid mediated, in aerobic and anaerobic bacteria. This novel Disk Potentiation test (DP) True + ve : 51 Re – modified Hodge test (Re – MHT) Double Disk Synergy test(DDST) True + ve : 45 True + ve : 46 False + ve : 4 False + ve : 2

Carbapenem resistant metallobetalactamases)[79].

12

**= 56)** 

**14**

**10**

**25**

**<sup>7</sup>** MICU

NICU

PICU

with which Enterobacteriaceae cause infection and the mortality associated with infection caused by CRE and ESBL producing bacteria. In mid1990, CTX-M 15 was first reported as ESBL in India. Now, CTX-M 15 is established as globally dominant ESBL and primary cause of acquired resistance to 3rd generation Cephalosporins in Enterobacteriaceae. Walsh TR et al in year 2005 noted that MBL genes have spread from *Pseudomonas aeruginosa* to Enterobacteria‐ ceae and a clinical scenario for MBL appears to simulate the global spread of ESBL in recent future. bla NDM-1 gene on plasmid can be readily transferred between different strains of bacteria

In the present study, 52(11.6%), 56(12.4%) and 342 (76%) *E.coli* strains were isolated from Outpatient Departments(OPDs), Intensive Care units (ICUs) and Inpatient Departments(IPDs) respectively. Maximum 25 (44.6%) *E.coli* strains were isolated from Medicine ICU (MICU) and High dependency unit (HDU). In a previous study conducted in our laboratory in 2008, Basak et al have already reported the incidence of ESBL producing *E.coli* in our hospital as 41.3% [67] whereas 5 years after, in the present study, the incidence of ESBL producing *E.coli* were 68%, out of which only 13.8% strains produced ESBL alone and other strains produced ESBL, Amp C β-lactamases and MBL in combination. Pakzad I et al in 2011 have reported 28% of their *E.coli* strains as ESBL producers [68]. Sinha et al in 2008 had reported that 40.8% of *E.coli* strains were ESBL producers and 24% were AMPC β-lactamase producers [69]. 37.5% and 47.8% of *E.coli* strains were reported to be Amp C β –lactamases producers in the study conducted in Chennai, India and Kolkata,India respectively, whereas in our study 65.3% *E.coli* were Amp

> **Figure 5 : Isolation of** *E.coli* **strains from OPD, IPD and ICUs**

> > **25**

**<sup>7</sup>** MICU

**52**

**56**

**342**

Gupta V et al have reported 17(68%) *E.coli* strains to be ESBL positive[71].

**Figure 6 : Isolation of** *E.coli* **strains from different ICUs (n = 56)** 

**14**

**10**

Re – modified Hodge test (Re – MHT) Double Disk Synergy test(DDST)

True + ve : 45 True + ve : 46 False + ve : 4 False + ve : 2

*aeruginosa* to Enterobacteriaceae and a clinical scenario for MBL appears to simulate the global spread of ESBL in recent future. bla NDM-1 gene on plasmid can be readily transferred between different strains of bacteria by horizontal genre transfer [47].

 In the present study, 52(11.6%), 56(12.4%) and 342 (76%) *E. coli* strains were isolated from Outpatient Departments(OPDs), Intensive Care units (ICUs) and Inpatient Departments(IPDs) respectively. Maximum 25 (44.6%) *E.coli* strains were isolated from Medicine ICU (MICU) and High dependency unit (HDU). In a previous study conducted in our laboratory in 2008, Basak et al have already reported the incidence of ESBL producing *E.coli* in our hospital as 41.3% [67] whereas 5 years after, in the present study, the incidence of ESBL producing *E.coli* were 68%, out of which only 13.8% strains produced ESBL alone and other strains produced ESBL, Amp C β-lactamases and MBL in combination. Pakzad I et al in 2011 have reported 28% of their *E.coli* strains as ESBL producers [68]. Sinha et al in 2008 had reported that 40.8% of *E.coli* strains were ESBL producers and 24% were AmpC β-lactamase producers [69]. 37.5% and 47.8% of *E.coli* strains were reported to be AmpC β-lactamase producers in the study conducted in Chennai, India and Kolkata,India respectively, whereas in our study 65.3% *E.coli* were

OPD

IPD

ICUs

NICU

PICU

OT‐ICU

**Figure 7 : Phenotypic detection of metallobetalactamase (MBL) producing** *E.coli* **strains by various methods. (n = 450)** 

12

by horizontal genre transfer [47].

62 Trends in Infectious Diseases

**IV. DISCUSSION** 

AmpC β-lactamase producers.

C β-lactamase producers.

OPD

IPD

ICUs

**IV. DISCUSSION** 

AmpC β-lactamase producers.

**Figure 5 : Isolation of** *E.coli* **strains from OPD, IPD and ICUs** 

**52**

**56**

**342**

Gupta V et al have reported 17(68%) *E.coli* strains to be ESBL positive[71].

Re – modified Hodge test (Re – MHT) Double Disk Synergy test(DDST)

True + ve : 45 True + ve : 46 False + ve : 4 False + ve : 2  In the present study, 52(11.6%), 56(12.4%) and 342 (76%) *E. coli* strains were isolated from Outpatient Departments(OPDs), Intensive Care units (ICUs) and Inpatient Departments(IPDs) respectively. Maximum 25 (44.6%) *E.coli* strains were isolated from Medicine ICU (MICU) and High dependency unit (HDU). In a previous study conducted in our laboratory in 2008, Basak et al have already reported the incidence of ESBL producing *E.coli* in our hospital as 41.3% [67] whereas 5 years after, in the present study, the incidence of ESBL producing *E.coli* were 68%, out of which only 13.8% strains produced ESBL alone and other strains produced ESBL, Amp C β-lactamases and MBL in combination. Pakzad I et al in 2011 have reported 28% of their *E.coli* strains as ESBL producers [68]. Sinha et al in 2008 had reported that 40.8% of *E.coli* strains were ESBL producers and 24% were AmpC β-lactamase producers [69]. 37.5% and 47.8% of *E.coli* strains were reported to be AmpC β-lactamase producers in the study conducted in Chennai, India and Kolkata,India respectively, whereas in our study 65.3% *E.coli* were

Various authors have reported MBL producing *E.coli* strains from 2.9% (Pandya et al from Gujrat, India) [70], to 6.8% (Tsakris et al from Greece) [61] to 25% (Enwuru NV et al from Nigeria) [13]. In the present study 51(11.3%) MBL producing *E.coli* strains were isolated. MBL production was detected in both Imipenem resistant (39/41 i.e. 95.1%) and Imipenem sensitive 12/409 i.e. 2.9% strains also. It indicates that if only Imipenem resistant strains would have been screened, 2.9% MBL producing strains would have been missed. But no Klebsiella pneumoniae producing carbapenemases were detected in our present study. In 2011, Tsakris et al reported 15.9% KPC producing *E.coli* in their study. Tsakris et al have also reported that 19(43.2%) *E.coli* strains produced AmpC β – lactamases and ESBL and 15(34%) *E.coli* strains produced ESBL [61]. In another study in 2012,

**Figure 6.** Isolation of *E.coli* strains from different ICUs (n = 56)

**Figure 7 : Phenotypic detection of metallobetalactamase (MBL) producing** *E.coli* **strains by various methods. (n = 450)** 

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 with which Enterobacteriaceae cause infection and the mortality associated with infection caused by CRE and ESBL producing bacteria. In mid1990, CTX-M 15 was first reported as ESBL in India. Now, CTX-M 15 is established as globally dominant ESBL and primary cause of acquired resistance to 3rd generation Cephalosporins in Enterobacteriaceae. Walsh TR et al in year 2005 noted that MBL genes have spread from *Pseudomonas aeruginosa* to Enterobacteriaceae and a clinical scenario for MBL appears to simulate the global spread of ESBL in recent future. bla NDM-1 gene on plasmid can be readily transferred between different strains of bacteria by horizontal genre transfer [47].

method could be used by Clinical Laboratories to monitor the emergence of the MBL [59]. Omair et al in 2012, have reported that MBL – E test have been taken as a gold standard method for MBL detection **Figure 7.** Phenotypic detection of metallobetalactamase (MBL) producing *E.coli* strains by various methods. (n = 450)

**Figure 6 : Isolation of** *E.coli* **strains from different ICUs (n**  Various authors have reported MBL producing *E.coli* strains from 2.9% (Pandya et al from Gujrat, India) [70], to 6.8% (Tsakris et al from Greece) [61] to 25% (Enwuru NV et al from Nigeria) [13]. In the present study 51(11.3%) MBL producing *E.coli* **Figure 5.** Isolation of *E.coli* strains from OPD, IPD and ICUs OT‐ICU [72]. Manoharan et al have reported that MBL - E test has taken as a phenotypic standard method for MBL detection though the test is expensive. Double disk synergy test (DDST) and Disk potentiation (DP) tests are economical and simple to perform but DDST is observer dependent while DP test is measurable with lesser chance of subjective error [45]. In the present study, we studied MBL positive *E.coli* strains by MBL E – test and compared the results of other phenotypic methods for MBL detection i.e. Re – Modified Hodge test (Re- MHT), Double disk synergy test (DDST) and Disk potentiation (DP) test. In figure 7, the venndiagram showing interrelationship of Re – modified Hodge test (Re – MHT), Double disk synergy Walsh et al in 2002 have reported that the MBL – E test results were in 100% agreement with the results from the genotypic Polymerase chain reaction (PCR) and biochemical methods [59]. They have also reported that the E test MBL strip IP/IPI has the ability to detect MBLs both chromosomally and plasmid mediated, in aerobic and anaerobic bacteria. This novel method could be used by Clinical Laboratories to monitor the emergence of the MBL [59].

test (DDST) and Disk potentiation test (DP) for detection of MBL producing *E.coli*.

strains were isolated. MBL production was detected in both Imipenem resistant (39/41 i.e. 95.1%) and Imipenem sensitive 12/409 i.e. 2.9% strains also. It indicates that if only Imipenem resistant strains would have been screened, 2.9% MBL producing strains would have been missed. But no Klebsiella pneumoniae producing carbapenemases were detected in our present study. In 2011, Tsakris et al reported 15.9% KPC producing *E.coli* in their study. Tsakris et al have also reported that 19(43.2%) *E.coli* strains produced AmpC β – lactamases and ESBL and 15(34%) *E.coli* strains produced ESBL [61]. In another study in 2012, In the present study when results of all three phenotypic methods were compared with MBL – E test results, it was found that 45/51 (88.2%) MBL positive strains were positive by all three phenotypic method i.e. Re – Modified Hodge test (Re-MHT), Double disk synergy test (DDST) and Disk potentiation (DP) tests. 04 and 02 were false positive by Re – MHT and DDST methods respectively. Whereas 6/51 (11.8%) and 5/51(9.8%) were false negative by Re – MHT and DDST method respectively. Amongst all three phenotypic methods, DP was best correlated with MBL – E test. By DP test 51 MBL positive *E.coli* strains were detected and no false positive and false negative result was found (Sensitivity 100% and specificity 100%). **V. EPIDEMIOLOGY -**  Omair et al in 2012, have reported that MBL – E test have been taken as a gold standard method for MBL detection [72]. Manoharan et al have reported that MBL - E test has taken as a phenotypic standard method for MBL detection though the test is expensive. Double disk synergy test (DDST) and Disk potentiation (DP) tests are economical and simple to perform but DDST is observer dependent while DP test is measurable with lesser chance of subjective error [45].

respiratory tract infections, wound infections, osteomyelitis and neonatal meningitis[73,74].

*E.coli* are responsible for various infections like urinary tract infection, diarrhoea, pneumonia, bacteremia, upper

 The successful outcome of clinical use of 3rd generation cephalosporines unfortunately led to the increased use and emergence of ESBL producing Enterobacteriaceae. With the emergence of ESBL and Amp C β – lactamase production in *E.coli*, Klebsiella pneumoniae and other Enterobacteriaceae, Carbapenems were used as last resort to treat those infections. Because of

were from cases of bacteraemia, ventilator associated pneumonia and community acquired urinary tract infections [76].

 NDM - 1 spread largely to different countries like Australia, Japan, Brazil, Belgium, Canada, Germany etc [77]. The gene encoding NDM – 1is called blaNDM – 1, located on transmissible plasmid which may include other antibiotic resistance genes also leading to extensive drug resistant phenotypes (so called 'superbugs'). A recent report from ICU and wards of Sir Gangaram hospital Delhi, India showed 8.1% NDM – 1 positive E.coli [78]. In January 2011, the name of NDM–1 was changed to PCM (Plasmid encoding

Metallobatalactamases are also found in Carbapenem susceptible organisms. This hidden MBL gene can spread

Most common MBL found worldwide in Enterobacteriaceae were VIM (Verona integron encoded MBL) and IMP (active on imipenem). Multidrug resistant *E.coli* harboring New Delhi metallobetalactamase - 1 (NDM-1) isolated from a patient returned to Canada from India[75], was reported first in 2009. NDM -1 was also recognized among Enterobacteriaceae 32 from Mumbai, 13 from Varanasi and 3 from Guwahati in India and 25 isolates from eight different cities in Pakistan. These isolates

selective pressure of Carbapenems, even carbapenemases producing Enterobacteriaceae (CRE) has emerged.

unnoticed in hospitals if isolates are reported sensitive without screening for presence of MBL[48].

In the present study, we studied MBL positive *E.coli* strains by MBL E – test and compared the results of other phenotypic methods for MBL detection i.e. Re – Modified Hodge test (Re-MHT), Double disk synergy test (DDST) and Disk potentiation (DP) test.

Metallobatalactamases are also found in Carbapenem susceptible organisms. This hidden MBL gene can spread unnoticed in hospitals if isolates are reported sensitive without screening for

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

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

65

The prevalence of ESBL and Amp C beta lactamases in a single isolate reduces effectiveness of beta – lactam and beta - lactamase inhibitor combinations while MBLs and Amp C beta– lactamases confer resistance to carbapenems and Cephamycin. Unfortunatenly thses enzymes

As *E.coli* are one of the commonest cause of both health care and community acquired infections, rapid identification of beta lactamase producing *E.coli* is crucial for appropriate treatment and timely implementation of infection control measures in Health care set-up. Indeed, delayed detection of ESBLs, Amp C β – lactamase and MBL producing strains, raise the possibility of spread of these strains into the community. These issues combined with the limited therapeutic options available to treat patient infected with these organisms, have made CRE of epidemiological importance globally [ 80]. ESBLs and Carbapenem resistant strains

Phenotypic methods can be useful for routine detection of ESBLs and carbapenemase pro‐

Screening of colonisation with multidrug-resistant organisms (MDROs) upon admission to hospitals has been advocated in patients who have already received healthcare in endemic countries. The CDC recommends, if previously unrecognized cases are identified of being infected with β-lactamase producing strains, a round of surveillance culture from high risk areas i.e. ICUs or wards from where detected, should be considered in any Health Care Setup. In addition prompt notification, must be made to infection control team members when CRE

Antimicrobial stewardship has been suggested as the most important efforts to control multidrug resistant organisms (MDROs) [81]. It has been found to be most effective, if efforts are directed towards an overall decrease in antimicrobial use rather than targeting a specific antimicrobial class. Limiting use of invasive devices is another potentially important preven‐ tive mechanism for MDROs including β- lactamase producing organisms. Health care workers (HCW) should follow hand hygiene practices while giving patient care preferably using an alcohol based hand rubs or antimicrobial soap and water if hands are visibly soiled, and also

In the present study, all MBL – E test positive *E.coli* strains (100%) were detected by Disk potentiation test also. MBL producing *E.coli* strains must be tested in both carbapenem resistant

duction, among Gram negative bacteria particularly when PCR is not available.

follow Standard precautions and Additional precautions as per the indications.

may lead to outbreaks of infection in HealthCare Set-up also.

are identified in Clinical Microbiology Laboratories.

**7. Conclusion**

presence of MBL [48].

usually co-exist in same isolate.

**6. Prevention and control**

In figure 7, the venndiagram showing interrelationship of Re – modified Hodge test (Re – MHT), Double disk synergy test (DDST) and Disk potentiation test (DP) for detection of MBL producing *E.coli*.

In the present study when results of all three phenotypic methods were compared with MBL – E test results, it was found that 45/51 (88.2%) MBL positive strains were positive by all three phenotypic method i.e. Re – Modified Hodge test (Re- MHT), Double disk synergy test (DDST) and Disk potentiation (DP) tests. 04 and 02 were false positive by Re – MHT and DDST methods respectively, whereas6/51 (11.8%) and 5/51(9.8%) were false negative by Re – MHT and DDST method respectively. Amongst all three phenotypic methods, DP was best correlated with MBL – E test. By DP test 51 MBL positive E.coli strains were detected and no false positive and false negative result was found (Sensitivity 100% and specificity 100%).
