Application of tuf gene sequence analysis for the identification of species of coagulase-negative staphylococci in clinical samples and evaluation of their antimicrobial resistance pattern

Abstract

Azar Dokht Khosravi,1,2 Mitra Roointan,2 Effat Abbasi Montazeri,2 Sajad Aslani,3,4 Mohammad Hashemzadeh,1,2 Moslem Taheri Soodejani5 1Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 2Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 3Department of Microbiology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 4Student Research Committee, Kerman University of Medical Sciences, Kerman, Iran; 5Department of Biostatistics and Epidemiology, School of Health, Kerman University of Medical Sciences, Kerman, Iran Introduction: Coagulase-negative staphylococci (CoNS) are normal inhabitants of human skin and mucous membranes. However, CoNS represent one of the major nosocomial pathogens, especially in immunocompromised patients. The increasing incidence of CoNS and mainly methicillin-resistant strains underlines the need for an accurate identification of Staphylococcus isolates at the species level. Analysis of the tuf gene proved to be an accurate tool for the species identification of CoNS. The aims of this study were to identify the CoNS species by tuf gene-based polymerase chain reaction method and sequencing, and to determine the frequency of CoNS clinical isolates resistant to methicillin (MRCoNS) and other antibiotics.Methods: A total of 200 staphylococci isolates were collected from various clinical samples. Phenotyping methods were used for initial identification followed by polymerase chain reaction amplification of tuf gene with subsequent sequencing. The phylogenetic relationships among species were analyzed using the neighbor-joining method based on the partial gene sequence of tuf. Microbroth dilution test was used for screening methicillin resistance, and disk diffusion susceptibility testing was performed for evaluation of antibiotic resistance among the isolates.Results: In the present study, 125 isolates were identified as CoNS; among them, Staphylococcus epidermidis 54(43.2%) and Staphylococcus haemolyticus 50 (40.0%) were demonstrated as the most prevalent species. Resistance to methicillin was detected in 54.4% of the CoNS based on microbroth dilution method. In disk diffusion susceptibility testing, the greatest resistance of CoNS was demonstrated for cefoxitin (65.4%), cotrimethoxazole (54.4%), and clindamycin (49.6%), while daptomycin (87.2%) and linezolid (83.2%) showed the greatest effectiveness for CoNS isolates.Conclusion: Our results confirmed the predominance of S. epidermidis and S. haemolyticus among CoNS isolates. The high prevalence of MRCoNS strains is a serious concern and strongly suggests the need for control program measures in our hospitals in order to reduce MRCoNS infections, especially in immunocompromised patients. Keywords: coagulase-negative staphylococci, tuf gene, antimicrobial resistance, PCR, susceptibility testing

Keywords

• Coagulase-negative staphylococci (CoNS)
• tuf gene
• antimicrobial resistance
• PCR
• susceptibility testing