Conventional and molecular characterization of coagulase-negative staphylococcus in hospital isolates.

Background: During the last decade, coagulase-negative staphylococci (CoNS) have emerged as a major cause of nosocomial infections. They constitute a major component of the normal skin and mucosal microflora, and are particularly responsible for catheter- and medical device-related sepsis. They present unique problems in diagnosis and treatment of infections. Purpose: The present study has been designed to evaluate phenotypic and genotypic characteristics of CoNS among nosocomial isolates. Setting and Design: This study was carried out in a tertiary care hospital. Data from 150 samples collected from 73 hospitalized patients and 15 healthy volunteers between October 2003 and May 2005 were analyzed. Patients and Methods: A total of 100 CoNS strains responsible for sepsis or implant-associated infections and 50 saprophytic strains were studied. Invasive CoNS strains were selected on the basis of different colony morphologies, drug resistance patterns, and biofilm formation. The same criteria were used to select saprophytic isolates. Multiplex PCR was used to explore the ica, mecA, and atlE genes, which might contribute to the pathogenicity of CoNS and the formation of biofilms. Results: Most of the invasive strains that formed the biofilm were resistant to multiple antibiotics, with more than 80% resistant to methicillin. ica and mecA genes were detected significantly in pathogenic strains (chi-square test, P<0.0001) whereas atlE was ubiquitously amplified in all the strains. All those strains which had ica and mecA genes were resistant to multiple antibiotics and were positive for biofilm formation. Conclusion: These genetic markers thus appear to discriminate between potential invasive virulent and saprophytic strains of CoNS.

Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi.

Aim: Enteric fever is an ongoing problem in the developing nations. Resistance and reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. The present study was carried out with the objective of determining molecular basis of resistance to fluoroquinolone among the clinical isolates of Salmonella enterica serovar Typhi from different parts of India. Materials and Methods: A total of 60 S.Typhi clinical isolates were subjected to antimicrobial susceptibility testing and determination of minimum inhibitory concentration (MIC) to ciprofloxacin and nalidixic acid. Polymerase chain reaction (PCR) for GyrA gene followed by restriction fragment length polymorphism (RFLP) with restriction enzyme (RE) SSiI was performed to detect mutation at position Ser83. Further confirmation of mutation was done by nucleotide sequencing of GyrA gene. Results: Isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Twelve of the 60 isolates (18%) were susceptible to nalidixic acid (NASST) and the remaining 48 (82%) were resistant to nalidixic acid (NARST). Of these 48 NARST strains, 46 (97.5%) had reduced susceptibility to ciprofloxacin (MIC 0.25-1.0 μg/mL), whereas 2 strains (2.75%) were resistant to ciprofloxacin (MIC 4.0 μg/mL). In RFLP analysis, all the NASST strains showed 3 fragments, whereas all the NARST strains showed 2 fragments due to the loss of 1 restriction site as a result of mutation. All the NARST strains with reduced susceptibility to ciprofloxacin (n = 46) had a single mutation in gyrA gene (Ser 83→Tyr or Ser 83→Phe), whereas double mutations (Ser 83→Phe and Asp 87→Asn) were found in each of the 2 ciprofloxacin-resistant strains. None of the NASST strains (n = 12) revealed any mutation. Conclusion: Our study exemplifies the correlation between nalidixic acid screening test, MIC values, and the detection of mutation in GyrA gene by PCR-RFLP with a novel RE SSiI.This was further confirmed by nucleotide sequencing.

Differentiation of wild-type varicella-zoster strains from India and the Oka vaccine strain using a VZV open reading frame - 62 based PCR-RFLP technique

Since the introduction of varicella vaccination in India, surveillance of circulating VZV strains has gained significance. Differentiating wild-type VZV strains from the Oka vaccine strain can be achieved only by molecular genotyping methods. The development of PCR methods for VZV strain differentiation has been hampered by the fact that the VZV genome is highly conserved. We used VZV ORF 62 PCR-RFLP analysis to identify and differentiate wild-type VZV strains in India from the Oka vaccine strain. Digestion of VZV ORF 62 amplicons with SmaI, enabled accurate strain differentiation; the Oka strain was positive for three SmaI sites, compared to two SmaI sites in the wild-type VZV strains that we tested.