Abstract
Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health.
Highlights
Staphylococci includes coagulase-positive staphylococci (CPS), almost exclusively represented by Staphylococcus aureus, and coagulase-negative staphylococci (CNS) (Becker et al 2004)
Most studies on staphylococcal pathogenicity have focused on S. aureus, and little attention has been paid to CNS (ChajeckaWierzchowska et al 2015)
All isolates were identified as staphylococci: three were S. aureus and the remaining 52 belonged to nine CNS species (S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. hominis, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus) (Table 3)
Summary
Staphylococci includes coagulase-positive staphylococci (CPS), almost exclusively represented by Staphylococcus aureus, and coagulase-negative staphylococci (CNS) (Becker et al 2004). CNS infections are difficult to control because these bacteria produce biofilms on the surfaces of foreign materials and are resistant to multiple antibiotics (John and Harvin 2007). Wholegenome DNA–DNA hybridization analysis (Svec et al 2004) has been used for the identification of Staphylococcus species These two methods are not suitable for routine use, and their major disadvantages include sample manipulation after the polymerase chain reaction (PCR) step and the requirement for gene probes, whose preparation may be time-consuming. The divergence of the staphylococcal sodA gene is higher than that of 16S rRNA (Poyart et al 2001); this gene has been used as a target in staphylococcal species identification (Iwase et al 2007) These studies require additional processes after the PCR step, such as DNA sequencing and homology comparisons. Antimicrobial resistance and virulence factors produced by the CNS isolates were evaluated to investigate their pathogenicity
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