Jolanta Karakulska

Identification and methicillin resistance of coagulase-negative staphylococci isolated from nasal cavity of healthy horses

The aim of this study was an analysis of the staphylococcal flora of the nasal cavity of 42 healthy horses from 4 farms, along with species identification of CoNS isolates and determination of resistance to 18 antimicrobial agents, particularly phenotypic and genotypic methicillin resistance. From the 81 swabs, 87 staphylococci were isolated. All isolates possessed the gap gene but the coa gene was not detected in any of these isolates. Using PCR-RFLP of the gap gene, 82.8% of CoNS were identified: S. equorum (14.9%), S. warneri (14.9%), S. sciuri (12.6%), S. vitulinus (12.6%), S. xylosus (11.5%), S. felis (5.7%), S. haemolyticus (3.4%), S. simulans (3.4%), S. capitis (1.1%), S. chromogenes (1.1%), and S. cohnii subsp. urealyticus (1.1%). To our knowledge, this was the first isolation of S. felis from a horse. The species identity of the remaining Staphylococcus spp. isolates (17.2%) could not be determined from the gap gene PCR-RFLP analysis and 16S rRNA gene sequencing data. Based on 16S–23S intergenic transcribed spacer PCR, 11 different ITS-PCR profiles were identified for the 87 analyzed isolates. Results of API Staph were consistent with molecular identification of 17 (19.5%) isolates. Resistance was detected to only 1 or 2 of the 18 antimicrobial agents tested in the 17.2% CoNS isolates, including 6.9% MRCoNS. The mecA gene was detected in each of the 5 (5.7%) phenotypically cefoxitin-resistant isolates and in 12 (13.8%) isolates susceptible to cefoxitin. In total, from 12 horses (28.6%), 17 (19.5%) MRCoNS were isolated. The highest percentage of MRCoNS was noted among S. sciuri isolates (100%).

Staphylococci isolated from ready-to-eat meat - Identification, antibiotic resistance and toxin gene profile.

The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumers specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of staphylococci could be of public health significance.

Comparative Analysis of Superantigen Genes in Staphylococcus xylosus and Staphylococcus aureus Isolates Collected from a Single Mammary Quarter of Cows with Mastitis

The purpose of this study was to analyze and compare genes encoding superantigens (SAgs) in Staphylococcus xylosus and Staphylococcus aureus isolates collected simultaneously from milk of the same cows with clinical mastitis. Genes encoding staphylococcal enterotoxins and enterotoxin-like proteins (sea-selu), toxic shock syndrome toxin 1 (tst-1) and exfoliative toxins (eta and etd) were investigated. It was found that among 30 isolates of S. xylosus, 16 (53.3%) harbored from 1 to 10 SAg genes. In total, in 16 SAg positive S. xylosus, 11 different enterotoxin genes were detected: sec, sed, seg, seh, sei, selm, seln, selo, selp, ser, selu and one etd gene encoding exfoliative toxin D. The most prevalent genes were ser, selu, and selo. Among all the positive isolates of S. xylosus, a total of 14 different SAg gene combinations were detected. One combination was repeated in 3 isolates, whereas the rest were detected only once. However, in the case of S. aureus all the 30 isolates harbored the same combination of SAg genes: seg, sei, selm, seln, selo and on the basis of PFGE analysis all belonged to the same clonal type. Also noteworthy was the observation that SAg genes detected in S. aureus have also been found in S. xylosus. The findings of this study further extend previous observations that SAg genes are present not only in S. aureus but also in coagulase-negative staphylococci, including S. xylosus. Therefore, taking into account that the SAg genes are encoded on mobile genetic elements it is possible that these genes can be transferred between different species of coexisting staphylococci.

Superantigen gene profiles, genetic relatedness and biological activity of exosecretions of Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis.

This study evaluated the superantigen gene profiles, genetic relatedness and biological activity of exosecretions of 50 Staphylococcus aureus isolates obtained from milk of cows with clinical mastitis. Genomic relatedness of S. aureus was determined by pulsed field gel electrophoresis analysis of macro‐restricted chromosomes. The presence of genes encoding superantigens was confirmed by multiplex PCR. To study the biological activity of S. aureus exosecretions, the supernatants from bacterial liquid cultures were classified into three groups: those with leukotoxin‐like properties, those with superantigen‐like properties and those with no particular activity on leukocytes cultured in vitro. It was shown that all analyzed bacterial isolates belonged to the same clonal type and harbored the same combination of superantigen genes, namely sed, selj and ser. However, 22% of all isolates produced factors with superantigen‐like and 48% of them with leukotoxin‐like activities. Finally, although there were no detectable genetic differences between the analyzed bacterial isolates, the virulence factors secreted by them differed considerably.

Influence of S. aureus exosecretions on cytokine profile in bovine leukocyte cultures in vitro

The aim of the research was to evaluate the in vitro effect of Staphylococcus aureus exosecretions on the expression of genes encoding IL-2 and IL-12 and secretion of IFN-γ and TNF-α, in bovine leukocyte cultures in vitro. The research was based on 30 S. aureus isolates collected from milk samples from cows with clinical mastitis. Supernatants prepared from the bacterial liquid cultures, which were used to treat leukocytes, were divided into three groups: one with superantigen-like properties, one with leukotoxic-like properties and the one without superantigen or leukotoxic-like properties. The MNC, PMN and MIX (consisted of MNC and PMN leukocytes) cultures were grown and treated with the supernatants. The work shows that the effect on the cytokine gene expression and cytokine secretion caused by S. aureus exosecretions is mainly due to the presence of virulence factors connected with superantigen-like activity and less with leukotoxic-like activity whereas exosecretions of other activity are not or only slightly involved in this process.

The Staphylococcal Panton-Valentine Leukocidin (PVL)

Abstract It is generally accepted that Pantone-Valentine leucocidin (PVL)-mediated pore formation in the cytoplasmic membrane of leukocytes (polymorphonuclear neutrophils—PMNs) in vitro eventually leads to cytolysis. However, this hypothesis has not been confirmed in vivo. Furthermore, the studies performed to date indicate that PVL concentration in vivo is probably insufficient to cause cell lysis. On the other hand, sublytic amounts of PVL prime neutrophil production of reactive oxygen species release pro-inflammatory molecules and cause exocytosis of granules. Although the molecular basis of the PVL-mediated PMNs priming is still unclear, infections caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates are closely related to the presence of the toxin. The PVL-producing S. aureus are often isolated from human CA-MRSA infections; however, little is still known regarding PVL-positive strains from animals. An explanation of PVL’s impact in S. aureus diseases is an important issue in the field of infectious diseases. Therefore, understanding the structure and function of PVL in S. aureus pathology is considered to be crucial for the development of new diagnostic tools, as well as drugs and vaccines. This chapter provides detailed information concerning the genetic basis for PVL synthesis and secretion, along with its chemical structure and pathogenic role.

Influence of milk, milk fractions and milk proteins on the growth and viability of mastitis-causing Staphylococcus aureus strain

Abstract The aim of this study was to investigate the impact of milk and milk fractions (cell-reduced, skim and whey) obtained from different cows on the growth rate of mastitis-causing Staphylococcus aureus strain at low inoculum density, simulating the early phase of intramammary infection. The association of the selected milk proteins, including α-lactalbumin, β-lactalbumin, lactoferrin, bovine serum albumin, γ-globulin and casein with the bacterial growth was also analysed. Twelve Polish Holstein-Friesian cows having no history of mastitis during the previous and current lactation were selected for this study. The S. aureus strain used in this study was isolated from a cow with clinical mastitis and was characterised by confirmed ability to spread among cows within a herd. Linear regression coefficients were calculated for associations between milk constituents and bacterial counts in whole milk as dependent variables. The comparison of bacterial growth between whole milk, cell reduced, skim and whey fractions was determined by a one-way analysis of variance (ANOVA). The results of the present study showed that the growth of mastitis-causing S. aureus was less stimulated by whole milk samples and their individual fractions in comparison to the nutrient microbiological medium. The strongest inhibition of bacterial growth was observed for whey fraction. Lactoferrin was the only protein causing a slight decrease in the growth of S. aureus. It was concluded that, depending on its growth medium and antimicrobial properties, milk may be among the factors of key importance for the incidence of this disease among individual cows.