Comparison of Phenotypic and Genotypic methods for biofilm forming Staphylococcus epidermidis from various clinical samples- A cross sectional study

Abstract

Coagulase Negative Staphylococci (CoNS), the indigenous flora of human skin and mucus membrane now represents one of the major nosocomial pathogen due to the patient and procedure related changes. Accurate species identification in clinically relevant framework helps in the diagnosis of the CoNS infection. Biofilm producing bacteria are responsible for several chronic infections. Biofilms protect microorganisms against both antibiotics used to treat infections and host immune system responses. The major virulence factor determining the pathogenicity of Staphylococcus epidermidis has now been well defined and found to be biofilm production. Objectives 1. To compare Phenotypic and Genotypic methods for biofilm forming Staphylococcus epidermidis from various clinical samples 2. To isolate and identify all Coagulase negative Staphylococci isolates(CoNS) from various clinical samples in hospitalized patients . Materials and method The study was conducted in the Dept of Microbiology, Dr Prabhakar Kore hospital and MRC, KLE University Belagavi. A total of 86 clinical isolates of CoNS were initially identified by colony morphology, Gram staining, Catalase test, Slide and Tube coagulase test and mannitol fermentation.The simple, inexpensive test were selected from scheme of Kloos and Schleifer for speciation. A total of 25 clinical isolates of S. epidermidis were characterized and subjected to biofilm detection by tissue culture plate method (TCP), modified congo red agar method (MCRA) and PCR (polymerase chain reaction) for icaA gene detection. Results: Out of the toal 86 isolates S.epidemidis is the most frequent isolate 25 ( 29.07% ), followed by S. hemolyticus 24 ( 27.9% ), S. hominis 20 ( 23.25% ), S. kohnii 5 (5.81% ), S. simulans 3 (3.49%), S. warneri 3 ( 3.49 %), S. schleiferi 3 ( 3.49 % ), S. intermedius 1 ( 1.16 % ), S. hyicus 1 (1.16 %)andS. Saprophyticus 1 (1.16%). Biofilm formation by Staphylococcus epidermidis (25 isolates) was detected by phenotypic and genotypic methods. By MCRA method 10 (40%) Staphylococcus epidermidis isolates were defined as biofilm producers through their black colonies. By TCP method 14 isolates (56%) were found to be biofilm producers with different grades: 6 (24%) strong producers and 8(32%) moderate producers.The icaA gene was detected concomitantly in 17(68%) isolates. In comparison with the data of icaA gene detection MCRA had the sensitivity of 52.9% and specificity of 87.5% while TCP method represented 70.4% sensitivity and 75% sepecificity. It was also noticed that all strong biofilm producers by TCP method were positive for icaA genes. Conclusion: In this study, out of 86 CoNS, 52 (60.47 %) were in > 40 years age group. Isolations were more prevalent in males 55 (63.95%). The need for prevalence and identification of CoNS by simple and inexpensive methodology helps in identifying pathogenic CoNS involved in nosocomial infections. For optimum evaluation of biofilm production by Staphylococcus epidermidis both genotypic and phenotypic analysis are needed. Timely diagnosis and appropriate treatment helps in faster patient cure and invariably cuts down the hospital expenses.