Prevalence of plasmid mediated multi drug resistant Enterobacteriaceae in Hospital environment at Bangalore, India

Seema Tharannum*, Nithya J, Manjula T S.

Department of Biotechnology (P.G.), PES Institute of Technology, Bangalore – 560 085, India.

*Corresponding author: seemakabir@gmail.com

Abstract- Air being a major medium allows pathogens to disseminate and cause infections. An initial prevalence survey conducted in the air samples of the hospital environment especially in the out patient unit/ department (OPD) of 2 government and 2 private hospitals in Bangalore using a centrifugal air sampler system, when subjected to different standard morphological studies, staining procedures and biochemical techniques showed existence of nosochomial infection causing organisms in government hospital environment only. The highest frequency of nosochomial infection causing organisms in government hospitals are gram negative enterobacteriaceae and was recorded to be the dominant (81%). The organisms thus isolated were subjected to multidrug antibiotic resistance test which include a group of β-lactam antibiotics that are frequently used in the treatment. Susceptibility testing revealed that the isolates from government hospitals are resistant to old β lactams penicillin (95%), ampicillin (100%) as well to the first and second generation cephalosporins (cephaloxin-40%, cephalothin-60%, cefoxitin-45%). The plasmids were isolated from these resistant strains and were observed that around 90% of the isolates had plasmid. The possible reason of antibiotic resistance in the organism being plasmid meditated. Incidences of these multidrug resistant enterobacteriaceae are serious threat to the hospital community. The organism can become endemic within the hospital setting with continued presence in the OPD environment and nosochomial spread. Hence the administration and management of the hospital must give top priority to support the hospital infection control program by establishing a committee to constantly monitor on hygienic conditions and apply the appropriate methods for preventing nosochomial infections. Key words: Air sampler, Nosocomial infections, β-lactamases, Enterobacteriaceae, plasmid, antibiotic resistance.

I. INTRODUCTION A nosochomial infection or hospital acquired infection occurs in a patient in a hospital or other health care facility in whom the infection was not present or at the time of admission. Many factor promote infection among hospitalized patients; decreased immunity among patients, the increasing variety of medical procedures and invasive techniques during surgery and one of the reason being the transmission of drug resistant bacteria among crowded

hospital populations, where poor infection control practices may facilitate transmission. Nosochomial infection occur world wide and affects hospitalized patients, out patients and hospital staff. Infections acquired in health care settings are among the major causes of death and increased morbidity among hospitalized patients. An initial prevalence survey conducted in the air of government and private health centers showed existence of nosochomial infection causing organism. The highest frequencies of nosochomial infection organism were reported in government hospital and it was found to be an enterobacteriaceae. Some of the drawbacks of nosochomial infections in patients are functional disability, emotional stress that may lead to disabling conditions that reduce the quality of life. Nosochomial infections are also one of the leading causes of death [1]. Prolonged stay due to these post infections mainly increases direct costs of the patient [2,3]. If organisms are multi resistant, they may cause significant disease in the community. The major factors influencing the development of nosochomial infections are microbial agent, the environmental factors, patients’ susceptibility and bacterial resistance Our objective was to identify the microbial agent and look for its existence in the environment and to study the resistance ability of the organisms. Among the microbial agent the cause for nosochomial infection can be different bacteria, viruses, fungi and parasites. The organism can be resistant to antimicrobial agents, intrinsic virulence and inoculum of infective material. The infection may be cross infection or due to endogenous infection or due to environmental infection. The hygienic environment and hygienic practices and antibiotics in medical practice, matters and contributes to the nosochomial infections. Previous studies have revealed that the most infections acquired in hospitals today are caused by microorganisms which are common in general population like Staphylococcus aureus, coagulase negative staphylococci, enterococci and enterobacteriaceae. Health care settings are an environment where both infected persons and persons at increased risk of infections are seen together. Patients admitted are potential source of infection for patients and staff.

2009 Second International Conference on Environmental and Computer Science

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DOI 10.1109/ICECS.2009.76

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Patients who became infected in the hospital are further source of infection. Air being a major medium allows pathogens to disseminate and cause infections. Human exposures to deadly microbes are known to cause severe infections. The diversity of microbes includes opportunistic organisms which causes infections when the immune system of the individual is low due to other infection. Crowded conditions in hospital especially the out patient department (OPD), frequent transfers of patients from one unit to another, concentration of patient susceptible to infection contribute to the development of nosochomial infections. Many patients receive antimicrobial drugs through a mechanism of selection and exchange of genetic resistance elements; antibiotics promote the emergence of multi drug resistant strains of bacteria. Most of sensitive microbes in the environment are suppressed, while a resistant strain persists and may become endemic in the hospital. As an antimicrobial agent becomes widely used, bacteria resistant to this drug eventually emerge and may spread in the health care setting. Multi resistant Klebsiella, Staphylococci, Pseudomonas are prevalent in many hospitals. This problem is said to be critical in developing countries where more expensive second line antibiotics may not be available or affordable [4]. The enterobacteriaceae are among the most important causes of serious nosochomial and community onset bacterial infections in humans and resistance to antimicrobial agents in these species has become an increasingly relevant problem of health care centers. β -lactam and fluoroquinolone antibiotics are frequently used to treat infections. Emerging resistance mechanism is due to the production of β -lactamases and plasmid mediated Amp C β-lactamases. Eg: cephamycin, extended spectrum β-lactamases eg: cefotaxime and carbapenem hydrolyzing enzymes, KPC (Klebsiella pneumoniae carbapenemase) types and metallo β – lactamase [5] The treatment of these multi drug resistant enterococcal infections continues to be a challenge for clinicians. Glycopeptide and β -lactam resistance is now a common feature of the majority of enterococcus species hospital isolates and resistance to common antibiotics further complicates the problem. New antibiotics such as tigecycline, lipoglycopeptides, and cephalosporin with activity against enterococcus may have potential activity against these resistant enterococcal strains. Thus optimal therapy for these infections is not well established [6] Our findings has revealed that the air sample isolates are dominantly enterobacteriaceae and these isolates from hospital environment have shown resistance to some common β -lactams and cephalosporin (extended spectrum β -lactamases). The organisms can become endemic within the hospital setting with continued presence in the OPD environment and nosochomial spread. Hence it becomes necessary to constantly monitor

the indoor air quality of the health care facility to improve the health of patients and as well to protect the health of visitors and staff. Programs focusing on rational use of antibiotics are mandatory for prevention and control of such infections. It is also advisable for the hospitals to incorporate local practice guidelines.

II. MATERIAL AND METHODS A. Hi media Centrifugal Air Sampler

The unit consists of a propeller or turbine that pulls a known volume of air into the unit and then propels the air outward to impact on a tangentially placed LB Agar strip set on a flexible plastic base. The Centrifugal Air sampler is designed to capture large particles which results in higher airborne counts than any other air sampler. The air sampler samples 280L/min. Media used: - LB Agar B. Staining Techniques

a) Gram Staining: Gram Staining is the differential staining used to differentiate into Gram positive and Gram negative microorganisms. In this staining technique the bacterial smear is subjected to 4 reagents namely Crystal Violet, Gram’s Iodine solution, 95% Ethyl Alcohol, Safranin. The two types of bacteria are distinguished based on the complexity of constituents of cell wall Gram positive bacteria contain high percentage of polysaccharides and low percentage of lipids. Gram negative bacteria contain low percentage of polysaccharides and high percentage of lipids.

b) Negative Staining Capsule staining is the common negative staining procedure followed to identify the capsule producing microorganisms which is a gelatinous outer layer that is secreted by the cell that surrounds and adheres to the cell wall. Indian ink is the common stain used for staining the capsules which helps in identification of capsule producing microorganisms which act as a defensive structure and helps in resistance to chemicals and other treatments. C. Biochemical tests

Enterobacteriaceae (enteric) are Gram-negative bacteria that grow in the intestinal tract of humans and other animals. The IMViC tests are frequently employed for identification of this group of microbes which includes such organisms as Klebsiella, Enterobacter, and Escherichia coli.

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The predicted positive samples from the above gram’s staining were subjected to further biochemical assays. The biochemical tests performed include Indole production, Methyl red, Vogues-Proskauer test, Citrate test, Oxidize production and catalase production. These biochemical tests were performed as per standard Microbiological methods (Cappuccino and Sherman, 2007). Analytical grade chemicals and reagents obtained from Himedia were used for all the tests.

D. Antibiotic test (Antimicrobial Susceptibility Testings)

Antimicrobial Susceptibilities were determined by means of the Kirby-Bauer disk diffusion test on Mueller-Hinton agar for the following antibiotics; penicillin, ampicillin, Chloramphenicol as well to the first and second generation cephalosporins (cephaloxin, cephalothin, cefoxitin).Interpretations were performed according to the guidelines of the National Committee for Clinical Laboratory Standards.

E. Plasmid DNA isolation

Single colonies of the isolated bacteria were inoculated into 2 ml of Luria Bertani broth and grown overnight at 37o C at 150 rpm in a shaker incubator. The cells were pelleted and resuspended in 100 ul of solution I (2mM Tris, pH-8, 10mM EDTA), 200 ul of solution II(2N NaoH, 1%SDS) and 150 ul of solution III (3M sodium acetate) and the contents were spun at 10000rpm for 15min at 4 degree C. Supernatant was discarded and pellet was dried at room temperature. The pellet was resuspended in 20microlitre of TE and the contents were mixed and run on 0.8 % EtBr Agarose gel to visualize the bands. Working ampicillin concentration is 100 microgram/ml.

III. RESULTS AND DISCUSSION

A. Air Sampling The air of the out patient department of the hospitals was sampled using himedia air sampler and the organisms trapped on the air sampling Leuria Bertani agar strips were incubated in an incubator at 37 deg C for 24 hours.

Fig 1.Air Sampler with Power pack

Fig 2. Air sampler strips showing colonies

B. Gram’s staining Analysis

All the isolates of the private hospital air sample were gram positive rods. Gram’s staining done on the isolates of government hospital sample I showed that 81% are gram negative and 75% in isolates of government hospital 2.

SAMPLE 1 (Government Health Care Centre)

SAMPLE 2 (Government Health Care Centre)

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C. Biochemical Analysis (Sample 1 and 2):

TABLE 1. RESULTS OF VARIOUS BIOCHEMICAL TEST PARAMETERS EXPRESSED IN PERCENTAGE OF CULTURES

Test parameter

Government hospital sample 1

Government hospital sample 2

Positive (%)

negative (%)

positive (%)

negative (%)

Indole test 12 88 20 80 Methyl red test

0 100 10 90

Vogues Proskauer test

100 0 85 15

Citrate test 70 30 75 25 Oxidase test

30 70 20 80

Catalase test

100 0 100 0

D. Antibiotic test: (Antimicrobial Susceptibility Testings)

Old and new generation antibiotics were tried on the lawn of hospital isolates in which it was observed that more than 90% were resistant to ampicillin, penicillin and some cephalosporins as the zone of inhibition is <15 mm , but were sensitive to chloramphenicol (Zone of inhibition is >15mm).

Fig.3. Lawn culture of the isolates with zone of inhibition

Fig.4. Antimicrobial Susceptibility Test

TABLE 2 . LIST OF ANTIBIOTICS USED IN FIG.3

Antimicrobial Agent

Zone of inhibition in mm

Ampicillin 15 Penicillin 12 Chloramphenicol 20

TABLE 3. LIST OF ANTIBIOTICS USED IN FIG 4

E.Plasmid DNA isolation: Plasmids from these resistant mutants were isolated and visualized on 0.8% agarose gel.

Fig 5. Agarose gel with ethidium bromide showing plasmid bands.

CONCLUSION Nosocomial infections are dangerous as they affect patients who have low immunity because of the primary infections and can lead to complications. Therefore the health care centers should do regular air sampling methods and adopt appropriate sterilization methods like fumigation, proper disposal of hospital wastes, incineration of used needles, linens etc. and maintain hygienic environment to reduce the threat of nosocomial infection.

Antimicrobial Agent

Zone of inhibition in mm

Ciprofloxacin < 15 Ceftazid 10 Cephaloxin 13 Cephalothin 10 Cefoxitin 12 Nalidixic acid 15

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ACKNOWLEDGEMENTS

The authors would like to acknowledge the Management of PESIT, Bangalore for their support in carrying out this work.

REFERENCES

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