Epidemiological Pattern of Non-Fermenting Metallo Beta Lactamase Producing Bacterial Pathogens Isolated From Clinical Specimens in a Tertiary Care Hospital in Kakinada

Research Article

J Bacteriol Mycol. 2017; 4(1): 1043.

Epidemiological Pattern of Non-Fermenting Metallo Beta Lactamase Producing Bacterial Pathogens Isolated From Clinical Specimens in a Tertiary Care Hospital in Kakinada

Usha Rani P and Vijayalakshmi P*

Department of Microbiology, Gitam Institute of Medical Sciences and Research, Gitam University, India

*Corresponding author: Payala Vijayalakshmi, Department of Microbiology, Gitam Institute of Medical Sciences and Research, Gitam University, India

Received: December 01, 2016; Accepted: February 20, 2017; Published: February 22, 2017


Background: Non-fermentative Gram negative bacilli are known causative pathogens responsible for hospital associated infection and for high morbidity and mortality rate across all genders and ages.

Objectives: This study examined the prevalence of Non-Fermentative Gram Negative Bacilli Metallo Beta-Lactamase Isolates (NFGNB-MBL) among carbapenem-resistant strains from clinical specimens and the epidemiological pattern (Age, Sex and Source of infection).

Methodology: A total of 250 clinical specimens from diverse infections were collected and analyzed, using standard microbiological methods. For MBL detection, two previously described methods were employed i.e. Imipenem- EDTA combined disc test and Imipenem-EDTA double disc synergy test (DDST).

Results: Sixty (24%) NFGNB isolates were identified, highest number from hospital associated infections (93.33%). Pseudomonas aeruginosa accounted for n=40 (66.67%) followed by Acinetobacter baumannii n=10 (16.67%), Alcaligenes faecalis n=6 (10%) and Stenotrophomonas maltophilia n=4 (6.66%) respectively. The isolates showed high sensitivity to imipenem (91.67%) and the incidence of MBL producing non-fermenters among the imipenem resistant organisms was found to be 100%. Conclusion: The prevalence of MBL producing isolates and association with hospital infection is of clinical concern and the therapeutic option in treatment and management of patient.

Keywords: Non-fermenters; Gram negative bacilli; Antibiotic sensitivity; Imipenem; MBL production


Non-fermenting Gram negative bacilli are a group of aerobic, non-sporing organisms that either do not use carbohydrates as a source of energy or degrade them through metabolic pathways other than fermentation [1]. These organisms are abundant in nature like soil, water, plants, decaying vegetation, food stuffs and normal flora of humans. Although NFGNB are considered commensals or contaminants, their pathogenic potential has been well established by their frequent isolation from clinical samples and their association with clinical disease. The out breaks of nosocomial infections, emerging antimicrobial resistance and epidemiology complexity have made NFGNB the remarkable organisms. NFGNB are innately resistant to many antibiotics and are known to produce extended spectrum beta-lactamase and metallo-beta-lactamase. They account for around 15% of all bacterial isolates from clinical samples. Most of the Non-fermenting Gram negative bacilli have emerged as important nosocomial pathogens in hospitalized patients of severe burns, urinary tract infections, wound infections, septicaemia, pneumonia, osteomyelitis, peritonitis, meningitis, cirrhosis of liver etc. Some can cause serious infections in immuno-compromised hosts [2]. The most important members are Pseudomonas followed by Acinetobacter, Stenotrophomonas, Moraxella, Alcaligenes, Flavobacterium, Achrobacterium etc. All these may be acquired nosocomially, so that rapid identification and epidemiological characterization are important in tracking hospital outbreaks. The rising incidence of Pseudomonas infection in hospital and its ubiquitous association in hospital environmental soil and water had led to an increased interest in identifying the strain by various bacteriological and biochemical properties by different research workers. Non-fermenting Gram negative bacilli were isolated from blood cultures of patients admitted to high risk units like oncology, nephrology, burns, NICU and cardiology [3]. With particular references to ICU and major superficial and systematic infections, Pseudomonas aeruginosa is the most common isolate followed by Acinetobacter and Alcaligenes. Bacteraemia due to Pseudomonas and Acinetobacter has become an important cause of morbidity and mortality in hospital association infections. Prolonged hospital stay, more invasive procedures for diagnosis and therapy, indiscriminate use of broad spectrum antibiotics and underlying host factors also contribute to morbidity and mortality.

In the present study an attempt has been made for isolation and identification of various non-fermenting gram negative bacilli and to study the various biochemical characters of isolated non-fermenting organisms from various clinical materials. As some of the nonfermenting organisms show multidrug resistance, the nosocomial outbreaks of carbapenem resistant non-fermenters is due to metallobeta- lactamase enzyme production. So this enzyme is responsible for multidrug resistance, and they possess high hydrolytic activity that leads to degradation of higher generation cephalosporins. Plasmid mediated MBL genes spread rapidly to other species of gram negative bacilli, therefore rapid detection of metallo-beta-lactamase production is necessary to modify therapy and to initiate effective infection control to prevent their dissemination [4]. So identification of metallo- beta-lactamase producing non-fermenters was also included in this study.

Materials and Methods

Different clinical samples collected from both IP and OP patients in Tertiary care hospital, Kakinada for a period of one year, constituted the material for this study. A total of 60 non-fermenters isolated from different clinical specimens including Pus, Urine, Endotracheal aspirates, Sputum, Blood, Catheter tips, Ascitic fluid, Pleural fluid and CSF from 250 patients. The cases included individuals of both sexes and all age groups. Gram staining was performed initially to study the morphological characteristics of the clinical isolates [5].

Culture and biochemical reactions

All the clinical specimens were initially processed to separate the non-fermenters from the other Gram negative bacilli.

Pus, respiratory secretions and other specimens were inoculated onto 5% blood agar, Mac-conkey agar and Nutrient agar and urine samples were inoculated into CLED agar and incubated aerobically at 37°C for 24h and then examined for growth. The wound swabs are inoculated into nutrient broth and incubated for 3-4 h at 37°C and then after noting the turbidity due to growth sub cultured onto MacConkey, 5% blood agar and incubated for 12-18 h at a temperature of 37°C. In case of Acinetobacter growth on MacConkey medium showed a faint pink tint, while no pigmentation was seen on blood agar. After observing the motility of organisms, Gram stain was done and confirmed that the organisms are gram negative.

Non fermenting organisms are provisionally identified by colonial morphology and pigment production and these were subjected to various biochemical and biological tests for confirmation [5]. The tests include carbohydrate fermentation tests with glucose, lactose, sucrose, xylose, mannitol and maltose. The organisms were also tested for Indole production, Methyl red test, Voges-proskauer test, Citrate utilization, Urease test, Oxidase test, Catalase test, Nitrate reduction test, Triple sugar iron agar test, Hughs Leifsons test (O/F test), Gelatin hydrolysis, Amino acid decarboxylation test, Growth on nutrient agar containing 6% NaCl, growth at 420C etc. In addition to the biochemical reactions, strains of Pseudomonas aeruginosa were further tested for their ability to grow on cetrimide (0.3%) agar. Pseudomonas aeruginosa was identified by its colony characters, pigment production and its characteristic odour, growth on cetrimide agar with 6% NaCl.

Antibiotic sensitivity testing of isolated Non-fermenters

Overnight broth culture having semi-confluent growth of the isolated bacterial strain was used as inoculums. Muller-Hinton agar plates were used and subjected to antibiotic susceptibility test using Disc-diffusion method of Kirby-Bauer, using the concentration of antibiotics per discs, recommended by the WHO experts committee on biological standardization. The antibiotics used were Amikacin (30μg), Gentamicin (10μg), Cefoperazone (75μg), Ceftazidime (30μg), Ceftriaxone (30μg), Ciprofloxacin (5μg), Imipenem (10μg), Piperacillin/Tazobactum (100/10μg), and Polymyxin B (300U). Results were interpreted in Zone of inhibition and comparing with the standard zone size given by Kirby-Bauer chart [5].

Detection of metallo beta lactamase (MBL) production

Screening for MBL production was done in Multidrug resistant isolates of the non-fermenters. MDR isolate was defined as resistant to 2 or more drugs or drug classes of therapeutic relevance. Among these some carbapenem resistant isolates MBL producers [6]. The following methods were used for the MBL detection in the present study which includes Imipenem-EDTA combined disc test and Imipenem-EDTA Double Disc Synergy Test (DDST). Imipenem- EDTA combined disc test, the test organisms were inoculated onto plates with Muller-Hinton agar. Two 10μg imipenem discs were placed on the plate, an appropriate amount of 10μl of EDTA solution were added to one of them to obtain the desired concentration (750μg). The inhibition zones of the imipenem-imipenem and EDTA discs were compared after 16-18h incubation at 37°C. In the combined disc test, if the increase in inhibition zone with the imipenem and EDTA was >7mm than the imipenem disc alone, it was considered as MBL positive [8]. Discs were prepared by adding EDTA solution to10μg imipenem discs to obtain a concentration of 750μg. The discs were dried immediately in an incubator and stored at 4°C or at -20°C in an airtight vial without desiccant. Test strains were adjusted to the Mc Farland 0.5 standard and were inoculated to MH agar. A 10μg imipenem discs and an imipenem + EDTA 750μg were placed on MH agar. Another disc containing only 750μg EDTA was also placed as a control. After overnight incubation, the established zone diameter difference of >7mm between imipenem discs and imipenem + EDTA was interpreted as EDTA synergy positive. These were compared with ATCC 27853 strains of Pseudomonas aeruginosa as a negative control [6,7].

The Imipenem-EDTA double disc synergy test was performed as described by Lee et al. Test organisms were inoculated onto plates with MH agar as recommended by the CLSI. An imipenem (10μg) disc was placed 20mm centre to centre from a blank disc containing 10μl of 0.5M EDTA (750μg). Enhancement of the zone of the inhibition in the area between imipenem and the EDTA disc in comparison with the zone of inhibition on the far side of the drug was interpreted as a positive result [8].


Of the 250 clinical specimens analyzed, 60 (24%) were identified as Non-fermentative Gram negative bacilli. These 60 were used for further processing. Various age groups ranging from 1 and 79 years of both sexes were included in the present study. Out of 250 patients, 160 were males and 90 were females. The analysis of these cases in different age group is represented in (Table 1). From the table it was noticed that, different types of infections in the age group 20-29, that is 58 (23.2%) and a lower number of cases in the age group 70-79, which is 10 (4%). The rate of infection was more in males 160 (64%) while comparing with females 90 (36%) who attended the hospital from various infections (Figure 1).