Banu, Swaminathan, Kamath, Joshi, Ravishkhar K, and Choudhary: Retrospective study of incidence of metallo-β-lactamase-producing Non-fermenters in nosocomial infections of a tertiary care hospital, in Navi Mumbai


The increase in the rates of antibiotic resistance is a major cause for concern in both non-fermenting bacilli . β-lactams have been the mainstay of treatment for serious infections. Most active of these are the carbapenems, which are advocated for use for the treatment of infections caused by extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumonia, and non-fermenters. Among non-fermenters, Pseudomonas spp. and Acinetobacter spp. are commonly isolated.

Five different types of MBLs whose prevalence are increasing rapidly1 are IMP(Imipenamse), VIM(Verona Integron-encoded metallo-beta lactamse; Meyrol, SPM-1(Sao Paulo Metallo-Brta-lacamase, GIM(German imipenamse; and AIM, Australian Imipenamase; Among these, IMP and VIM (Verona Integron-encoded Metallo Beta lactamase are the most predominant. With the global increase in the occurrence and types of MBLs, early detection is crucial, the benefits of which include timely implementation of strict infection control practices and treatment with alternative antimicrobials. Among the simple and economic methods available for testing MBL production, the imipenem (IMP)-EDTA combined disc test is sensitive and specific. According to (IMP) 10 μg-EDTA 750 μg combined disc test has 95.7% sensitivity and 91.0% specificity for detection of metallo-beta-lactamase in MBL-producing Pseudomonas spp and Acinetobacter spp.

Acinetabacter baumanii is a typical nosocomial pathogen causing infections and a high mortality often among patients hospitalized in the Intensive care unit. 2

Pseudomonas aeruginosa has been described as the most common and seroius cause of infection. 3

Acquired metallo-β-lactamases (MBL) have recently emerged as one of the most worrisome resistance mechanisms owing to their capacity to hydrolyze all β-lactams, including carbapenems. Such strains are not susceptible to therapeutic serine β-lacatamase inhibitors (such as clavulanate and sulfones). Moreover, their genes are carried on highly mobile elements, allowing easy dissemination. Invasive infections with MBL-producing isolates are also associated with a higher morbidity and mortality.4 The occurrence of an MBL-positive isolate in a hospital environment poses not only a therapeutic problem but is also a serious concern for infection control management. Pseudomonas aeruginosa and Acinetobacter baumannii are among the most important causes of healthcare-associated infections. 5 As a result of being difficult to detect, such organisms pose significant risks, particularly due to their role in unnoticed spread within institutions and their ability to participate in horizontal MBL gene transfer with other pathogens in the hospital. In recent years, MBL genes have spread from P. aeruginosa, Acinetobacter Spp. to the members of the Enterobacteriaceae.

Our hospital, a tertiary care centre in Navi Mumbai Maharashtra, has a high prevalence of infections due to non-fermenting bacilli and Enterobacteriaceae. Carbapenems and cephalosporin inhibitor combinations are being used as the “last resort” in these infections since the last few years. This research investigation is undertaken to ascertain the prevalence of MBL-producing non-fermenting bacilli in our hospital. Although a global increase in the prevalence of MBL-producing non-fermenting bacilli has been reported limited data is available from NaviMumbai.

Materials and Methods

The study was conducted over period of 1 year in our hospital, which is a 1250 bedded teaching hospital with ICUs, trauma centre and one neonatal intensive care unit (NICU). Clinical samples like pus, blood, urine, body fluids, tissues and respiratory secretions from patients admitted in medical, surgical intensive care unit were collected using aseptic precautions. All samples were processed using standard Microbiological procedures.

Metallo-β-lactamase screening

MBL production was detected in imipenem-resistant isolates by phenotypic tests. The Imipenem (IMP)-EDTA combined disc test was used. 5

The Imipenem (IMP)-EDTA combined disk test was performed as described by Pseudomonas aeruginosa ATCC 27853 used as the control. Test organisms were inoculated on to plates of Mueller-Hinton agar as recommended by the CLSI. Two 10-μg imipenem disks (Hi-Media Laboratories, BD Diagnostics Pvt. Ltd.) were placed on the plate and appropriate amounts of 10 μL of EDTA solution was added to one of them to obtain the desired concentration (750 μg). The inhibition zones of the imipenem and Imipenem(IMP)-EDTA disks were compared after 16–18 h of incubation in air at 35°C. In the combined disc test, if the increase in inhibition zone with the imipenem and EDTA disc was ≥7 mm than the imipenem disc alone, it was considered as MBL positive.

Antimicrobial susceptibility

Antimicrobial susceptibility of all the isolates was performed by the Kirby-Bauer disc diffusion method according to the CLSI guidelines. 6

Antibiotics used are amikacin (30mcg), gentamicin (10mcg), netilmicin (30mcg), cefoperazone (75mcg), cefipime (30mcg) ceftazidime (30mcg) ceftrizoxime (30mcg) ciprofloxacin (5mcg), lomefloxacin (10mcg)


Bacterial isolates

A total of 107 gram negative non-fermenting bacteria were isolated from various clinical samples of admitted patients were included in the study.Clinical samples were all clinical branches ans also from patients with co morbid conditions like burns, cancer and diabetes.

All isolates were non-duplicate. The isolates were identified by conventional methods. Out of 107 samples,51 were from pus,18 from tracheal aspirates, 22 from urine,8 from sputum and 10 from blood culture.

Pseudomonas aeruginosa was isolated from 66(61.8%) samples and 41(38.3%) are from Acinetobacter species.19(65.5%) males and 10(34.5%) females were MBL producers, showing a male preponderance.12 out of 66(18.18%)were MBL producing Pseudomonas.aeruginosa. and 17out of 41(41.46%) were Acinetobacter species. Mean age of MBL producers were 34 years

Mean patients age of MBL producers - 34 years.

Antibiotic sensitivity of MBL positive Pseudomonas aeruginosa

Collostin (100%),Gatifloxacin (50%), Piperacillin-tazobactam (33.3%),Chloramphenicol (33.3%) and Netilmicin (16.66%)

Antibiotic senstivity of MBL positive Acinetobacter spp

Colistin (70.6%),Gatifloxacin (17.3%) and piperacillin - tazobactam (11.6%).

All 29 MBL isolates tested positive by the combined disc (Imipenem + EDTA ) method

Out of 29 MBL isolates screened positive by the combined disc method,4(13.7%) were non determinable by the E-test MBL

Out of the remaining 25 (86.2%) that tested positives by the E-test MBL, 6 showed deformation of the ellipse.

Mean hospital stay was 28 days in the MBL producers.

Infection associated with hospital related devices was 17 out of the 29,(58.6%) MBL producers. Of these 17,12 (70.5%) were Acinetobacter spp. Ans 5(29.5%) were Pseudomonas aeruginosa.

Mortality was seen only in MBL producers,15(51.7%) out of 29 succumbed to death.

Mortality is higher in Acinetobacter MBL producers,12 out of 17,(70.5%) as compared to Pseudomonas aeruginosa 3 out of 12,(20.5%).

Highest mortality is seen in infections due to hospital related devices, 6 out of 15(40%), followed by multi-organ damage 4 out of 15(26.4%), septicemia 3 out of 15(20%) and renal failure 2 out of 15(13.3%).

Table 1

Total number of isolates:










Table 0



F(2, 3)




Table 2

Sex wise distribution










Table 3

Patients Mortality rate and bacteria isolated
















Table 4

Clinical reason causing mortality in MBL producers

Device related

Multi organ Damage

Renal failure










Table 5

Age wise distribution


12-5 27




Above 75













Table 6

Other Co-morbid conditions of the MBL isolates




Infections associated with Hospital Devices










MBLs have been found in number of bacteria including Pseudomonas aeruginosa and Acinetobacter species. And there is no CLSI recommended methodology for their detection though simple techniques like combined disc test are available. Molecular typing(genotypic detection)7 would help in knowing that which alleles of the genes of MBL’s are prevalent in different parts of the world. The diagnostic laboratories worldwide need in house standardized phenotypic tests for their detection Multi-drug resistant or pan drug resistant strains are increasingly being found in hospital settings. MBL producing Pseudomonas aeruinosa are responsible for nosocomial outbreaks across the world. 8 Treatment options for these organisms are very limited which needs urgent attention by pharma companies to find new molecule.

Acinetobacter baumanii is an important cause of healthcare associated infections and are difficult to treatas they possess antimicrobial resistance. 9 Acquired metallo bete lacatamses ahev recently emerged as one of the most worrisome resistant mechanisms.10 Acinetobacter Spp playa significant role in the colonization and infection of patients admitted in the hospitals. 11

The mainstay of curbing this dreaded menace would be strict infection control practices and restricted use of antibiotics. As in critical care units worldwide,the dictum is hit high,hit hard so start off a patient in ICU with imipenem or Meropenem. But a culture profile with an antibiotic sensitivity must be sent immediately and de-escalate the antibiotic according to the report.Cost effective technique is necessary to prevent their dissemination as well as formulation and regulation of antimicrobial stewardship policy. 12 All Microbiologists, infectious disease specialists and who ever involved in infection control committee of the every hospital should undergo such antimicrobial stewardship program to gain updated knowledge to curb such menaces.A.baumannii has been enlisted as one of the top priority pathogens by World Health Organization in 2017. 13

We have used the combined disc method to be very sensitive for routine detection of MBL’s in the laboratory. It is very easy, economical and reliable. The E-test MBL on the other hand is specifically designed to detect as many clinically significant MBL as possible. The Etest MBL was reliable for detecting the IMP-1- and VIM-2-producing Pseudomonas and Acinetobacter isolates. 14 However given the cost constrains of E-test, the combined disc method is a better option for routine testing in the clinical microbiology laboratories which are Incorporated in many of the laboratories.

Hospital infection control committee and antibiotic policy 15 makers at hospital level, District, state, national 16 and international level has to come together and device a concrete policy to treat patients as per the policy to minimize the mortality.In India we should have our own country specific policy and everyone has to follow this without any compliance.


Our findings show that there are significant numbers of isolates showing MBL production along with MDR. There is a need for active surveillance to detect MBL producers. The Imipenem(IMP)-EDTA combined disc test is a simple test that can be used in district health laboratories or where molecular diagnostic techniques are not available to detect these types of resistance. There should be judicious use of carbapenems to prevent their spread and use of effective antibiotics as per the antibiotic-sensitivity report.

Source of Funding

No financial support was received for the work within this manuscript.

Conflict of Interest

The authors declare they have no conflict of interest.



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