The Two-Track Investigation of Fibronectin Binding Protein A of Staphylococcus aureus from Bovine Mastitis as a Potential Candidate for Immunodiagnosis: A Pilot Study.

Bovine mastitis is the most common disease affecting dairy cattle worldwide and it generates substantial losses for cattle breeders. One of the most common pathogens identified in infected milk samples is Staphylococcus aureus. Currently, there is no fast test for recognizing bacteria species on the market. The aim of this study was to bioinformatically and laboratory detect and characterize the fibronectin binding protein A (FnBPA) of S. aureus (SA) in milk samples obtained from cows diagnosed with mastitis. More than 90,000,000 amino acid sequences were subjected to bioinformatic detection in the search for a potential biomarker for bovine SA. The analysis of FnBPA included the detection of signal peptides and nonclassical proteins, antigenicity, and the prediction of epitopes. To confirm the presence of the fnbA gene in four SA isolates, amplification with specific primers was performed. FnBPA was detected by immunoblotting. The immunoreactivity and selectivity were performed with monoclonal anti-FnBPA antibodies and SA-negative serum. The bioinformatic analysis showed that FnBPA is a surface, conservative, immunoreactive, and species-specific protein with antigenic potential. Its presence was confirmed in all of the SA isolates we studied. Immunoblotting proved its immunoreactivity and specificity. Thus, it can be considered a potential biomarker in mastitis immunodiagnostics.

Activity of Patchouli and Tea Tree Essential Oils against Staphylococci Isolated from Pyoderma in Dogs and Their Synergistic Potential with Gentamicin and Enrofloxacin.

In this paper, we show the effect of some essential oils (EOs) on staphylococci, including multidrug-resistant strains isolated from pyoderma in dogs. A total of 13 Staphylococcus pseudintermedius and 8 Staphylococcus aureus strains were studied. To assess the sensitivity of each strain to the antimicrobial agents, two commercial EOs from patchouli (Pogostemon cablin; PcEO) and tea tree (Melaleuca alternifolia; MaEO) as well as two antibiotics (gentamicin and enrofloxacin) were used. The minimum inhibitory concentration (MIC) followed by checkerboards in the combination of EO-antibiotic were performed. Finally, fractional inhibitory concentrations were calculated to determine possible interactions between these antimicrobial agents. PcEO MIC ranged from 0.125 to 0.5 % v/v (1.2-4.8 mg/mL), whereas MaEO MIC was tenfold higher (0.625-5% v/v or 5.6-44.8 mg/mL). Gentamicin appeared to be highly prone to interacting with EOs. Dual synergy (38.1% of cases) and PcEO additive/MaEO synergism (53.4%) were predominantly observed. On the contrary, usually, no interactions between enrofloxacin and EOs were observed (57.1%). Both commercial EOs were characterized by natural composition without artificial adulteration. Patchouli and tea tree oils can be good alternatives for treating severe cases of pyoderma in dogs, especially when dealing with multidrug-resistant strains.

Identification, Superantigen Toxin Gene Profile and Antimicrobial Resistance of Staphylococci Isolated from Polish Primitive Sheep Breeds.

The study aimed to analyze staphylococcal microbiota of the nasal cavity of the primitive sheep breeds Polish Swiniarka and Wrzosówka kept on the same ecological farm. The research included the identification of staphylococcal species, evaluation of the prevalence of genes encoding enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, toxic shock syndrome toxin 1, and detection of antimicrobial resistance. From 61 swab samples gathered from Swiniarka (33) and Wrzosówka (28) healthy sheep, 127 coagulase-negative staphylococci (CoNS) were isolated. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V enzymes, the isolates were identified as: Staphylococcus xylosus (33.9%), S. equorum (29.1%), S. arlettae (15%), S. warneri (9.4%), S. lentus (7.9%), S. succinus (3.9%) and S. sciuri (0.8%). Three of these species, S. lentus, S. succinus, and S. sciuri, were detected only from the Swiniarka breed. It was found that 77.2% of isolates harbored from 1 to 7 out of 21 analyzed genes for superantigenic toxins. The greatest diversity of toxin genes was recorded for S. equorum (16 different genes). The most prevalent gene was ser (40.2%). The incidence and number of resistances to antimicrobials were found to be bacterial species but not sheep breed dependent. The highest percentage of resistance was found for S. sciuri. The most frequent resistance was observed to clindamycin (45.7%). The findings of this study prove that toxigenic and antimicrobial resistant CoNS can colonize the nasal cavity of healthy sheep.

Development of an Autochthonous Microbial Consortium for Enhanced Bioremediation of PAH-Contaminated Soil.

The main objectives of this study were to isolate bacteria from soil chronically contaminated with polycyclic aromatic hydrocarbons (PAHs), develop an autochthonous microbial consortium, and evaluate its ability to degrade PAHs in their native contaminated soil. Strains with the best bioremediation potential were selected during the multi-stage isolation process. Moreover, to choose bacteria with the highest bioremediation potential, the presence of PAH-degrading genes (pahE) was confirmed and the following tests were performed: tolerance to heavy metals, antagonistic behavior, phytotoxicity, and antimicrobial susceptibility. In vitro degradation of hydrocarbons led to the reduction of the total PAH content by 93.5% after the first day of incubation and by 99.22% after the eighth day. Bioremediation experiment conducted in situ in the contaminated area resulted in the average reduction of the total PAH concentration by 33.3% after 5 months and by over 72% after 13 months, compared to the concentration recorded before the intervention. Therefore, this study implicates that the development of an autochthonous microbial consortium isolated from long-term PAH-contaminated soil has the potential to enhance the bioremediation process.

Evaluation of nutritional value and microbiological safety in commercial dog food.

In addition to properly balancing nutritional value in accordance with the needs of a dog, estimating the microbiological quality of dog food is crucial in providing healthy and safe foods. The aim of this study was to examine the quality of dry food for adult dogs, with particular reference to: (1) evaluating the nutritional value and compliance with nutritional guidelines for dogs, (2) comparing the nutritional value of dog foods, with particular emphasis on the division into cereal and cereal-free foods, and (3) evaluating their microbiological safety. All thirty-six evaluated dry dog foods met the minimum European Pet Food Industry FEDIAF requirement for total protein and fat content. The total aerobic microbial count in the analyzed dry dog foods ranged from 2.7 × 102 to above 3.0 × 107 cfu/g. In five (14%) dog foods the presence of staphylococci was detected; however, coagulase positive Staphylococcus (CPS) was not found. Mold presence was reported in one cereal-free dog food and in six cereal foods. In none of the analyzed foods Enterobacteriaceae were found, including coliforms, Escherichia coli and Salmonella spp. Bacteria of the genus Listeria and Clostridium as well as yeasts were also not detected. In conclusion, the evaluated dry dog foods had varied microbiological quality. The detected number of microorganisms may have some implications for long-term consumption of contaminated food. The lack of European Commission standards regarding the permissible amounts of microorganisms in pet food may result in insufficient quality control of these products.

Glycoside hydrolase (PelAh) immobilization prevents Pseudomonas aeruginosa biofilm formation on cellulose-based wound dressing.

Bacterial cellulose (BC) is recognized as a wound dressing material well-suited for chronic wounds; however, it has no intrinsic antimicrobial activity. Further, the formation of biofilms can limit the effectiveness of the pre-saturation of BC with antimicrobial agents. Here, to hinder biofilm formation by P. aeruginosa, we immobilized the hydrolytic domain of PelA (a glycohydrolase involved in the synthesis of biofilm polysaccharide Pel) on the surface of BC. The immobilization of 32.35 ±â€¯1.05 mg PelAh per g BC membrane resulted in an eight-fold higher P. aeruginosa cell detachment from BC membrane, indicating reduced biofilm matrix stability. Further, 1D and 2D infrared spectroscopy analysis indicated systematic reduction of polysaccharide biofilm elements, confirming the specificity of immobilized PelAh. Importantly, BC-PelAh was not cytotoxic towards L929 fibroblast cells. Thus, we conclude that PelAh can be used in BC wound dressings for safe and specific protection against biofilm formation by P. aeruginosa.

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 consumer's 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 leukotoxin­like properties, those with superantigen­like 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 superantigen­like and 48% of them with leukotoxin­like 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.

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%).

Bacteria adhesion to the surface of various prosthetics materials.

Increased life span of humans and dynamic development of prosthetic treatment has caused that increased number of people are using removable dentures for longer periods of time. The materials used to make those prosthesis are becoming a potential pathogen factor for oral mucosa being in contact with this material. Prosthetic stomatopathy occurs in 20% to 70% of the patients who are using removable dentures. The present paper evaluates and compares adhesion of bacterial plague to the most common materials used for removable dentures i.e. acrylic material Vertex R.S., metal alloy used for frame prosthesis and a material which is an alternate to acrylic resin and acetal metal-resin Acetal Pressing D. Samples were made from the above mentioned materials and placed in mediums with four basic bacteria cultures and fungus Candida albicans (fungus) as the adhesion of bacterial plague to individual materials was evaluated. Such an evaluation facilitates choice of appropriate prosthetic material, allowing to make prosthetic restoration that is functional and aesthetic at the same time taking into account prosthetic stomatopathy prevention.

[Evaluation of practical usefulness of selected phenotypic and genotypic markers for pathogenicity of enterotoxic and entero-hemorrhagic strains of E.coli. ]. / Ocena praktycznej przydatnosci wybranych fenotypowych i genotypowych wyznaczników patogennosci enterotoksycznych i enterokrwotocznych paleczek E. coli.

This study included a description of enterotoxic and verocytotoxic activity of thirty strains of E. coli and their ability to produce beta-haemolysis in a ram blood medium. Enterotoxic and verocytoxic activity was determined by using RPLA test. The synthesis of enterotoxin LT was observed in 10 strains and the production of E. coli shiga toxins type 1--Stx1 (1 strain) or type 2 (2 strains) was observed in 3 strains of serotype O157: H7. The beta-haemolytic characteristics in vitro were demonstrated by 17 strains (57%) isolated from pigs. Among them 16 (94%) were found to possess the genes that determine the enterotoxins or enterotoxins and E. coli shiga toxin Stx2v synthesis. Using the PCR technique, 21 strains (70%) were found to possess the genetic determinants of enterotoxins LT, STa and/or STb synthesis or enterotoxins and E. coli shiga toxin Stx2v synthesis. Further genetic study showed that the strains possessed the genes elt and estB were predominant (33%) among the toxic strains of E. coli isolated from piglets and calves.

[Biochemical properties of enterotoxic and enterohemorrhagic E-coli strains]. / Biochemiczne wlasciwosci enterotoksycznych i enterokrwotocznych szczepów E. coli.

The purpose of this paper has been to describe the biological characteristics of thirty strains of E. coli. The E. coli strains were isolated from cases of colibacillosis in animals and from human faeces and cow milk samples. Of the thirty analyzed strains, 19 strains (63%) were found to belong to 7 serogroups: O8, O101, O138, O141, O147, O149 and O157. In 17 strains (57%) fimbriae F4 was discovered and in 1 strain (3%) the presence of fimbriae F5 and F41 was detected. Serological and biochemical researches, based on the analysis of 35 enzymatic reactions, confirmed that all strains, used in this study, belonged to the species E. coli. The strains demonstrated differences in biochemical activity for 12 substrates. It was found that strains of serotype O157: H7 had biochemical homogeneity, except in their rate of sucrose fermentation and their ability to hydrolyze arginine and sorbitol after longer incubation. On the basis of the biochemical activity, O157: H7 strains were affiliated with biotype C. In identifying serotype O157: H7, SMAC medium with sorbitol and liquid and solid media with MUG reagent were very useful.