Resistance Pattern acquired by nosocomial pathogens : a plasmid profile study

Article history: Received on: 13/08/2013 Revised on: 01/09/2013 Accepted on: 20/09/2013 Available online: 30/09/2013 Nosocomial (Hospital acquired) bacterial strains were isolated from the premises of V.S.S. Medical College and Hospital, Burla, Odisha. The strains were preliminarily characterized by using “Routine diagnostic methods” and were identified as Pseudomonas sp., Klebsiella sp., Bacillus sp. respectively. The resistance nature of all the nosocomial strainswere clearly evident from the “Sensitivity testing” by following Kirby-Bauer method. A plasmid profile study revealed that all the isolated and identified strains were harboured with plasmids. Provisionally a concluding statement was given for the possible role of plasmids in drug resistance acquired by nosocomial bacterial strains.


INTRODUCTION
A hospital-acquired infection, also known as a HAI or in medical literature as a nosocomial infection, is an infection whose development is favoured by a hospital environment, such as one acquired by a patient during a hospital visit or one developing among hospital staff.Such infections include fungal and bacterial infections and are aggravated by the reduced resistance of individual patients.
Bacteria recognized as nosocomial pathogens generally fall into three categories: those that grow slowly, those that are fastidious in their nutritional or atmospheric requirements and those that resemble commensals (Fraser, 1981).Because some of these organisms are fastidious, they have not always been readily cultured and identified.Other species have not been fully appreciated as nosocomial pathogens because of taxonomic uncertainties (Mayer and Zinner, 1985).Gram positive bacteria are the commonest cause of nosocomial infections with Staphylococcus aureus being the predominant pathogen (Inweregbu et al., 2005).Therefore there is a need for a solution to .

* Corresponding Author Dr (Mrs) Smaranika Pattnaik Laboratory of Medical Microbiology School of Life Sciences
Sambalpur University Jyoti Vihar, Burla 768019, Odisha combat with these nosocomial members.Keeping this quandary in mind, this study is a small piece of research , an attempt to nuke out the nature of resistance acquired by some of isolated bacteria strains from a hospital Environment.V.S.S. Medical College, Burla Odisha, which is regarded as a Primary Health Care Centre of Western Odisha and neighbouring states.The present pursuit had taken this Hospital environment to study about the nature of resistance acquired by the nosocomials.

Media taken for the study
Nutrient agar and broth were taken for the study was prepared according to HI-Media company instructions.

Bacterial strains taken for the study
The bacterial strains were obtained from Deptt. of Microbiology, V.S. S. Medical College in the form of Research material.The strains were Pseudomonas sp., Klebsiella sp. and Bacillus sp.respectively.The strains were subjected to further identification in the laboratory of Medical Microbiology, School of Life Sciences, Sambalpur University by following routine diagnostic procedures.
The bacterial cultures were subjected to streaking on Nutrient agar plates to get isolated colonies.Further isolated colonies were studied for morphology i.e. colour, odour, elevation, margin etc.The results are depicted in Table #1.Further the colonies were subjected to Gram staining as well as live cells staining to differentiate Gram positive and Gram negative as well as motile and non motile respectively.

Sensitivity test Disc diffusion method
The procured antibiotics were subjected to sensitivity test by following Kirby and Bayer method (1968).The Antibiotics were dissolved in sterile distilled water maintaining concentration as mentioned previously.The Antibiotic solution impregnated (4µl/disc) filter paper discs were put onto seeded bacterial culture Nutrient agar plates.A control plate was included for a comparison study.The plates were incubated at 37 0 C for a period of 14 to 18 hrs.The clear zone around each disc was measured and referred as sensitive, very sensitive, moderately sensitive or resistant by referring Kirby Bayer chart.

Tube dilution method
Tube Dilution method (Pattnaik et al., 2010) was followed to determine the minimum inhibitory concentration of drugs.The drugs were taken in a range starting from lower concentration, 1µg/ml (v/v) to higher concentration 50 µg/ml (v/v) in bacterial culture seeded broths.After incubation for a period of 48 hrs, the broths were observed with growth (Resistant) or no growth (Sensitive) of bacterial strains.

Plasmid profile study of bacterial strains
The three bacterial strains namely Pseudomonas, Klebsiella and Bacillus were subjected to isolation for plasmids (if any) by following method of Sambook and Russel ( 2001) in the M and M laboratory, National Institute of Technology (NIT), Rourkela, Odisha.

Identification of nosocomial bacterial strains
The bacterial strains (NCB1, NCB 2 and NCB3) grown on respective nutrient agar plates were observed to be members of three different genera namely Pseudomonas sp., Klebsiella sp. and Bacillus sp. which was evident from their colony characteristics (Table #1).Additionally the light microscopic study substantiated the observation (Table #2).

Study for sensitivity Disc diffusion method
From the results depicted in Table # 3, it was observed that the three nosocomial bacterial strains were resistant to the all the antibiotics having a minimized zone of inhibition or no zones of inhibition either.But the Bacillus stain was observed to be sensitive towards Levofloxacin and intermediately sensitive to Streptomycin respectively.Based upon this result it may be inferred that all the test strains were resistant to most of drugs.
Table .3: depicting the results obtained from Test for sensitivity (Disc diffusion) of antibacterial drugs depicting zones of inhibition in diameter (mm), no zones of inhibition: R, S: Sensitive, IS: Intermediately sensitive.

Names of bacteria NCB1
Pseudomonas sp

Tube dilution method
In an experiment regarding the sensitivity test for nosocomial bacterial strains towards the antibiotics namely, Amoxicillin, Ciprofloxacin, Gentamycin, Levofloxacin, Penicillin-G, Streptomycin and Tetracycline, it was found that the bacterial strains were resistant upto ≤ 50 µg/ml (v/v).From this observation it may be anecdotal about the resistance nature of nosocomials.

Plasmid profile study of bacterial strains
The Figure # 1 depicts the plasmid profile of the nosocomial strains.From the Agarose gel electrophoresis of plasmid DNA, it was observed that the Klebsiella sp. had maximum bands of plasmid DNA, while in Pseudomonas showed two number of plasmid DNA bands.In contrast, the Bacillus strain was having a diffused band of plasmid DNA.Plasmid profile analysis (PP) is a method of determining a number and size of plasmids in bacterial isolates (Selimovic et al, 2008).This observation could explain the drug resistance among nosocomials could be due to presence of resistance genes in their respective plasmids (Patwardhan et al, 2009).

CONCLUSION
From this piece of research work it may be concluded that the nosocomial bacterial strains isolated from Hospital environment were rigid towards a number of antibacterial drugs.Further it was observed that the strains were endowed with plasmids.And moreover, it was observed Klebsiella sp was found to be harbored with more number of bands.This is pertinent to mention here that, plasmids are notorious for horizontal gene transfer among bacterial strains.The nosocomial bacterial strains are always in course of horizontal gene transfer (hGT) conferring the strains drug resistant (Dzidic and Bedekovic, 2003).This study may be in juvenile stage, but may pave a way for investigating more about the mechanism responsible for hGT among nosocomial bacterial strains isolated and identified from a regional health hub.

Fig. 1 :
Fig. 1: depicting plasmid bands in the agarose gel.Lanes A: Bacillus with a faint plasmid band .B: Klebsiella with 5 plasmid bands.C: Pseudomonas with 2 plasmid bands.Arrows are indicative of plasmid bands.