Dataset Information

Comparative analysis of agr groups and virulence genes among subclinical and clinical mastitis Staphylococcus aureus isolates from sheep flocks of the Northeast of Brazil.

ABSTRACT: Staphylococcus aureus is one of the most frequent mastitis causative agents in small ruminants. The expression of most virulence genes of S. aureus is controlled by an accessory gene regulator (agr) locus. This study aimed to ascertain the prevalence of the different agr groups and to evaluate the occurrence of encoding genes for cytotoxin, adhesins and toxins with superantigen activity in S. aureus isolates from milk of ewes with clinical and subclinical mastitis in sheep flocks raised for meat production The agr groups I and II were identified in both cases of clinical and subclinical mastitis. Neither the arg groups III and IV nor negative agr were found. The presence of cflA gene was identified in 100% of the isolates. The frequency of hla and lukE-D genes was high - 77.3 and 82.8%, respectively and all isolates from clinical mastitis presented these genes. The sec gene, either associated to tst gene or not, was identified only in isolates from subclinical mastitis. None of the following genes were identified: bbp, ebpS, cna, fnbB, icaA, icaD, bap, hlg, lukM-lukF-PV and se-a-b-d-e.

SUBMITTER: de Almeida LM

PROVIDER: S-EPMC3833151 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Comparative analysis of agr groups and virulence genes among subclinical and clinical mastitis Staphylococcus aureus isolates from sheep flocks of the Northeast of Brazil.

de Almeida Lara M LM de Almeida Mayra Zilta P R B MZ de Mendonça Carla L CL Mamizuka Elsa M EM

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20131030 2

Staphylococcus aureus is one of the most frequent mastitis causative agents in small ruminants. The expression of most virulence genes of S. aureus is controlled by an accessory gene regulator (agr) locus. This study aimed to ascertain the prevalence of the different agr groups and to evaluate the occurrence of encoding genes for cytotoxin, adhesins and toxins with superantigen activity in S. aureus isolates from milk of ewes with clinical and subclinical mastitis in sheep flocks raised for meat ...[more]

PMID: 24294245

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Project description:Staphylococcus aureus is one of the most important contagious bacteria causing subclinical bovine mastitis. This bacterial infection is commonly identified by determine the pathogen in bovine milk samples through conventional technique including coagulase test. However, this test has several disadvantages as low sensitivity, risk of biohazard, cost expensive, and limited preparation especially in local area.Aim of this study was to compare and assess the screening method, Mannitol fermentation test (Mannitol salt agar [MSA]), and deoxyribonuclease (DNase) test, for S. aureus identification in milk samples.A total of 224 subclinical bovine mastitis milk samples were collected from four provinces of Thailand and determined S. aureus using conventional method and also subjected to the screening test, MSA and DNase test. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) among both tests were analyzed and compared to the tube coagulase test (TCT), as reference method. Immunological test by latex agglutination and molecular assay by determined spa gene were also used to identify and differentiate S. aureus.A total of 130 staphylococci were isolated by selective media, Gram-stain, and catalase test. The number of S. aureus which identified using TCT, MSA and DNase test were 32, 102, and 74 isolates, respectively. All TCT results were correlated to results of latex agglutination and spa gene which were 32 S. aureus. MSA showed 100% sensitivity, 28.57% specificity, 31.37% PPV, and 100% NPV, whereas DNase showed 53.13% sensitivity, 41.84% specificity, 22.97% PPV, and 73.21% NPV. DNase test showed higher specificity value than MSA but the test presented 26.79% false negative results whereas no false-negative result from MSA when comparing to TCT.MSA had a tendency to be a good preference for screening S. aureus because of its high sensitivity and NPV. The result from this study will improve a choice to use a screening test to diagnose S. aureus of veterinary field for prompt disease controlling and effective treatment.

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.

Project description:BackgroundStaphylococcus spp. are major cause of bovine mastitis (BM) worldwide leading to economic damage to dairy farms and public health threat. Recently, a newly emerged Staphylococcus argenteus has been found as a human and animal pathogen. Molecular characteristics, virulence and antibiotic resistant phenotypes of bacteria causing BM in Thailand are rare. This study aimed to investigated Staphylococcus spp. associated with subclinical bovine mastitis (SCM) in Thailand.MethodsMilk samples were collected from 224 cows of 52 dairy herds in four central and northeast provinces. Total somatic cell counts (SCC) and California mastitis test (CMT) were used to identify SCM cows. Milk samples were cultured for Staphylococcus spp. Coagulase-positive isolates were subjected to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Organisms suspected as S. argenteus were verified by detecting nonribosomal peptide synthetase gene. All isolates were checked for antibiograms and the presence of various virulence genes.ResultsFrom the 224 milk samples of 224 cows, 132 (59%) were positive for SCM by SCC and CMT and 229 staphylococcal isolates were recovered. They were 32 coagulase-positive (24 S. aureus and eight S. argenteus) and 197 coagulase-negative. PFGE of the S. aureus and S. argenteus revealed 11 clusters and a non-typeable pattern. MLST of representatives of the 11 PFGE clusters, three PFGE non-typeable S. aureus isolates from different locations and S. argenteus showed 12 sequence types. The eight S. argenteus isolates belonged to ST1223 (three isolates), ST2250 (two isolates), and ST2793 (two isolates). The antimicrobial tests identified 11 (46%) methicillin-resistant S. aureus and 25 (13%) methicillin-resistant coagulase-negative isolates, while seven S. argenteus were methicillin-susceptible and one isolate was methicillin-resistant. All of the 229 isolates were multiply resistant to other antibiotics. The most prevalent virulence genes of the 24 S. aureus isolates were clfA, coa and spa (X and IgG-binding region) (100%), hla (96%), pvl (96%) and sec (79%). Six S. argenteus isolates carried one enterotoxin gene each and other virulence genes including coa, clfA, hla/hlb, spa, tsst and pvl, indicating their pathogenic potential.Conclusion and perspectiveThis is the first report on the S. argenteus from cow milk samples with SCM. Data on the molecular characteristics, virulence genes and antibiograms of the Staphylococcus spp. obtained from the present study showed a wide spread and increasing trend of methicillin-resistance and multiple resistance to other antibiotics. This suggests that the "One Health" practice should be nurtured, not only at the dairy farm level, but also at the national or even the international levels through cooperation of different sectors (dairy farmers, veterinarians, medical and public health personnel and scientists) in order to effectively combat and control the spread of these pathogens.

Project description:The expression of most Staphylococcus aureus virulence factors is controlled by the agr locus, which encodes a two-component signaling pathway whose activating ligand is an agr-encoded autoinducing peptide (AIP). A polymorphism in the amino acid sequence of the AIP and of its corresponding receptor divides S. aureus strains into four major groups. Within a given group, each strain produces a peptide that can activate the agr response in the other member strains, whereas the AIPs belonging to different groups are usually mutually inhibitory. We investigated a possible relationship between agr groups and human S. aureus disease by studying 198 S. aureus strains isolated from 14 asymptomatic carriers, 66 patients with suppurative infection, and 114 patients with acute toxemia. The agr group and the distribution of 24 toxin genes were analyzed by PCR, and the genetic background was determined by means of amplified fragment length polymorphism (AFLP) analysis. The isolates were relatively evenly distributed among the four agrgroups, with 61 strains belonging to agr group I, 49 belonging to group II, 43 belonging to group III, and 45 belonging to group IV. Principal coordinate analysis performed on the AFLP distance matrix divided the 198 strains into three main phylogenetic groups, AF1 corresponding to strains of agr group IV, AF2 corresponding to strains of agr groups I and II, and AF3 corresponding to strains of agr group III. This indicated that the agr type was linked to the genetic background. A relationship between genetic background, agr group, and disease type was observed for several toxin-mediated diseases: for instance, agr group IV strains were associated with generalized exfoliative syndromes, and phylogenetic group AF1 strains with bullous impetigo. Among the suppurative infections, endocarditis strains mainly belonged to phylogenetic group AF2 and agr groups I and II. While these results do not show a direct role of the agr type in the type of human disease caused by S. aureus, the agr group may reflect an ancient evolutionary division of S. aureus in terms of this species' fundamental biology.

Dataset Information

Comparative analysis of agr groups and virulence genes among subclinical and clinical mastitis Staphylococcus aureus isolates from sheep flocks of the Northeast of Brazil.

Publications

Comparative analysis of agr groups and virulence genes among subclinical and clinical mastitis Staphylococcus aureus isolates from sheep flocks of the Northeast of Brazil.

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