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Molecular Characterization and Toxicity Confirmation of LukM/F'-PV Producing Staphylococcus aureus Isolated from Bovine Mastitis Samples in Mysore, India.

ABSTRACT: The widespread status of subclinical condition of bovine mastitis is often associated with the production of leukotoxin M/F'-PV producing Staphylococcus aureus. The present study aims for the profiling of such leukotoxin producers through conventional and molecular methods in parallel to their leukotoxicity. The incidence of this particular pathogen was assessed in mastitis infected Holstein-Friesian cattle, where eight isolates of staphylococci were found to be present in 20 % of collected samples. Being intermediately resistant to vancomycin, they showed characteristic double zone hemolysis on 7 % sheep blood agar and typical type II reaction for coagulase test indicating the pathogenic attributes. Further with RAPD-PCR and 16S rDNA-RFLP, epidemiological specificity and genotypic relatedness of isolates to S. aureus was confirmed. Subsequently, the presence of leukotoxin (lukM) gene in native isolates was detected by leukotoxin gene specific PCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay evaluated for secreted leukotoxin in cell free supernatant was estimated to be 223 toxic units which had an LD50 cytotoxic activity on bovine neutrophil. Thus, the data acquired during study can be of prime diagnostic method for timely and accurate analysis of subclinical mastitis samples which goes undetected at consumer level.

SUBMITTER: Padmaja RJ

PROVIDER: S-EPMC3689397 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Molecular Characterization and Toxicity Confirmation of LukM/F'-PV Producing Staphylococcus aureus Isolated from Bovine Mastitis Samples in Mysore, India.

Padmaja R J RJ Halami P M PM

Indian journal of microbiology 20130209 3

The widespread status of subclinical condition of bovine mastitis is often associated with the production of leukotoxin M/F'-PV producing Staphylococcus aureus. The present study aims for the profiling of such leukotoxin producers through conventional and molecular methods in parallel to their leukotoxicity. The incidence of this particular pathogen was assessed in mastitis infected Holstein-Friesian cattle, where eight isolates of staphylococci were found to be present in 20 % of collected samp ...[more]

PMID: 24426122

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Project description:BackgroundBovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India.ResultsThe results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes.ConclusionThis is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains.

Project description:Many efforts have been made to understand the pathogenesis of bovine mastitis to reduce losses and promote animal welfare. Staphylococcus aureus may cause bovine clinical mastitis, but it is mainly associated with subclinical infection, which is usually persistent and can easily reoccur. Here, we conducted a comparative genomic analysis between strains of S. aureus causing subclinical infection (Sau170, 302, 1269, 1364), previously sequenced by our group, and two well-characterized strains causing clinical mastitis (N305 and RF122) to find differences that could be linked to mastitis outcome. A total of 146 virulence-associated genes were compared and no appreciable differences were found between the bacteria. However, several nonsynonymous single nucleotide polymorphisms (SNPs) were identified in genes present in the subclinical strains when compared to RF122 and N305, especially in genes encoding host immune evasion and surface proteins. The secreted and surface proteins predicted by in silico tools were compared through multidimensional scaling analysis (MDS), revealing a high degree of similarity among the strains. The comparison of orthologous genes by OrthoMCL identified a membrane transporter and a lipoprotein as exclusive of bacteria belonging to the subclinical and clinical groups, respectively. No hit was found in RF122 and N305 for the membrane transporter using BLAST algorithm. For the lipoprotein, sequences of Sau170, 302, 1269, and 1364 with identities between 68-73% were found in the MDS dataset. A conserved region found only in the lipoprotein genes of RF122 and N305 was used for primer design. Although the polymerase chain reaction (PCR) on field isolates of S. aureus did not validate the findings for the transporter, the lipoprotein was able to separate the clinical from the subclinical isolates. These results show that sequence variation among bovine S. aureus, and not only the presence/absence of virulence factors, is an important aspect to consider when comparing isolates causing different mastitis outcomes.

Dataset Information

Molecular Characterization and Toxicity Confirmation of LukM/F'-PV Producing Staphylococcus aureus Isolated from Bovine Mastitis Samples in Mysore, India.

Publications

Molecular Characterization and Toxicity Confirmation of LukM/F'-PV Producing Staphylococcus aureus Isolated from Bovine Mastitis Samples in Mysore, India.

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