Virulence factors, antimicrobial resistance and phylogeny of bovine mastitis-associated Streptococcus dysgalactiae.

We carried out a thorough genetic evaluation of Streptococcus dysgalactiae isolated from clinical bovine mastitis cases and performed a phylogenetic analysis to represent the evolutionary relationship between S. dysgalactiae sequences. A total of 35 S. dysgalactiae strains were isolated from cases of clinical mastitis identified at a large commercial dairy farm located near Ithaca, New York. Whole-genome sequencing identified twenty-six antibiotic resistance genes, four of which were acquired genes, in addition to fifty virulence genes. Multi-locus sequence typing detected three new sequence types (STs). We conclude that a high proportion of this microorganism carries multiple virulence determinants and resistance genes, and that this indicates its potential to cause mastitis. Eight different STs were identified, of which ST453 (n = 17) was the most prevalent and ST714, ST715, ST716 were novel STs.

Whole-genome sequencing reveals high genetic diversity of Streptococcus uberis isolated from cows with mastitis.

BACKGROUND: Bovine mastitis is an important cause of economic loss in dairy farms. Streptococcus uberis is among the most frequently isolated bacterial species isolated from cows with mastitis. The aim of this study was to perform an in-depth genetic assessment of S. uberis strains isolated from bovine clinical mastitis (CM) and to perform a phylogenetic analysis to represent the evolutionary relationship among S. uberis sequences. RESULTS: A total of 159 isolates was genetically characterized using whole genome sequencing. According to the virulence determinants, all strains harbored the hasC, leuS, perR, purH, and purN virulence genes. Thirty-four resistance genes were identified in at least one strain. In terms of acquired genes, we observed that 152 (95.6 %) strains had a resistance gene to lincosamine (lnuD), 48 (30.2 %) to tetracycline (tetM), 4 (2.51 %) to tobramicine (ant6), and 1 to lincosamide (lsa(E)). MLST detected the Sequence Type (ST)797 (n = 23), while 85.5 % of the strains did not match to known STs. CONCLUSIONS: Then, eleven distinct ST were identified after we submitted the new alleles to assign new STs. The other prevalent STs observed were ST1215 (n = 58), ST1219 (n = 35), and ST1213 (n = 15). And it was not possible to identify the MLST of four strains. Phylogenetic lineages indicated a high genomic diversity of S. uberis in our collection, confirming that most strains isolated from bovine mastitis have different reservoirs, typical of environmental pathogens.

Preliminary functional analysis of the subgingival microbiota of cats with periodontitis and feline chronic gingivostomatitis.

The subgingival microbial communities of domestic cats remain incompletely characterized and it is unknown whether their functional profiles are associated with disease. In this study, we used a shotgun metagenomic approach to explore the functional potential of subgingival microbial communities in client-owned cats, comparing findings between periodontally healthy cats and cats with naturally occurring chronic periodontitis, aggressive periodontitis, and feline chronic gingivostomatitis. Subgingival samples were subjected to shotgun sequencing and the metagenomic datasets were analyzed using the MG-RAST metagenomic analysis server and STAMP v2.1.3 (Statistical Analysis of Metagenomic Profiles) software. The microbial composition was also described to better understand the predicted features of the communities. The Respiration category in the level 1 Subsystems database varied significantly among groups. In this category, the abundance of V-Type ATP-synthase and Biogenesis of cytochrome c oxidases were significantly enriched in the diseased and in the healthy groups, respectively. Both features have been previously described in periodontal studies in people and are in consonance with the microbial composition of feline subgingival sites. In addition, the narH (nitrate reductase) gene frequency, identified using the KEGG Orthology database, was significantly increased in the healthy group. The results of this study provide preliminary functional insights of the microbial communities associated with periodontitis in domestic cats and suggest that the ATP-synthase and nitrate-nitrite-NO pathways may represent appropriate targets for the treatment of this common disease.

Development and evaluation of a new recombinant protein vaccine (YidR) against Klebsiella pneumoniae infection.

Vaccination is a promising approach to prevent Klebsiella infection; however, the high heterogeneity of strains is a limiting factor. The best antigenic target for an anti-Klebsiella vaccine should be expressed by all or most of strains. We previously found YidR protein to be highly conserved among K. pneumoniae strains independently of antigen serotype. Therefore, in the present study, we developed a recombinant YidR protein vaccine and evaluated its protective efficacy against lethal challenge with K. pneumoniae in a mouse model. The yidR gene was cloned in Escherichia coli for recombinant expression. The lethal dose (LD100) of K. pneumoniae was determined and lethal challenge was carried out after immunization with recombinant purified YidR. After immunization, the concentration of total serum IgG was significantly higher in YidR-immunized mice than in non-immunized mice, indicating strong induction of antibodies. Mice were challenged with LD100 of K. pneumoniae, and significantly lower murine sepsis and higher body weight were observed in YidR-immunized mice compared to unvaccinated controls. Moreover, ∼90% of YidR-immunized mice survived beyond 10 days of observation, whereas none of the control mice survived past 48 h. The protective effect of YidR recombinant protein vaccine was demonstrated and YidR may be a promising vaccine candidate to prevent klebsiellosis.

Methicillin-resistant Staphylococcus aureus in food and the prevalence in Brazil: a review.

Foodborne diseases (FBD) occur worldwide and affect a large part of the population, being a cause of international concern among health authorities. Staphylococcus aureus can be transmitted by contaminated food, and it is one of the pathogens that most cause foodborne outbreaks in Brazil. Currently, this organism's ability in developing resistance to antibiotics is notorious; methicillin-resistant Staphylococcus aureus-MRSA-is known for its resistance to methicillin, oxacillin, and others. MRSA is one of the leading causes of infections, becoming a major threat to human health worldwide due to the numerous toxins that can produce. At first, the transmission of MRSA occurred in clinical environments; but in recent decades, its presence has been reported in the community, outside the hospital environment, including food and food-producing animals around the world. In this review, information about MRSA was gathered to verify MRSA incidence in the world but especially in Brazil in food samples, food handlers, food-producing animals, and food processing environments. The studies show that MRSA is easily found and in certain cases with high frequency, thus representing a potential risk to public health.

Endodontic Microbiome of Fractured Non-vital Teeth in Dogs Determined by 16S rRNA Gene Sequencing.

Dental fractures resulting in pulp exposure will lead to an endodontic infection with microbes from the oral cavity. However, data on the endodontic microbial composition in veterinary dentistry is lacking. The aim of this study was to examine the microbiome of naturally occurring primary endodontic infections in client-owned dogs. The endodontic microbiome of 10 non-vital teeth with exposed pulp cavities was assessed using a 16S rRNA gene sequencing approach. The results were compared to the microbiome of the subgingival plaque of the same teeth. Analysis revealed an abundant mixed microflora of a comparable richness and diversity and with mostly the same phyla obtained from sulcal and endodontic samples. However, further analysis revealed significant differences between sulcal and endodontic samples in the relative abundance of the most abundant phyla and genera, with the relative abundance of Bacteriodetes being significantly higher in endodontic samples. Although each sample presented a particular profile regarding the genera identified, Bacteroides was the most abundant genus in the endodontic samples. Snowella was also significantly more abundant in endodontic samples, while Porphyromonas and Fusobacterium were significantly more abundant in sulcal samples. We confirmed that the microbiome of the diseased endodontic system is comparably abundant with microorganisms to the healthy subgingival plaque indicating that previous culture-based studies of primary endodontic infections in dogs underestimated the richness and diversity of the endodontic microbiota.

The subgingival microbial community of feline periodontitis and gingivostomatitis: characterization and comparison between diseased and healthy cats.

Periodontitis is a common and important health problem in domestic cats. The subgingival microbiota of cats diagnosed with chronic periodontitis (CP), aggressive periodontitis (AP), and feline chronic gingivostomatitis (FCGS) are not well characterized. Thus, the aim of the present study was to characterize and compare the periodontal microbiota of periodontally healthy cats versus cats diagnosed with CP, AP, and FCGS by using next-generation sequencing. In total, 44 domestic cats were enrolled, and 139 subgingival samples were subjected to 16S rRNA gene sequencing to investigate the microbiota composition of each periodontal group evaluated. Our results identified several key genera previously described in periodontal disease (e.g. Treponema and Filifactor) and in the oral microbiota (e.g. Moraxella and Capnocytophaga) of healthy cats. Phylogenetic beta diversity analysis showed that the microbiota of periodontally healthy cats were distinguishable from diseased cats. Even though most of the genera known to be associated with periodontal disease were also identified in healthy cats, they were present at significantly lower relative abundance. Remarkably, alpha diversity was found to be higher in the disease groups compared to healthy animals. These results suggest a pathological mechanism involving opportunistic behavior. Our findings corroborate those in the current literature regarding the complexity of the subgingival microbiota of the domestic cat and reveal both differences and similarities among periodontally healthy and diseased cats.

Phenotypic and genotypic characterization of Staphylococcus spp. isolated from mastitis milk and cheese processing: Study of adherence and biofilm formation.

The aim of this study was to identify the phenotypic and genotypic profiles of Staphylococcus spp. isolated from mastitis milk and cheese processing plant.To evaluate the biofilm production of wild-type strains on contact surfaces by testing different factors through adhered cells and biofilm quantifications, finally, these biofilms were observed by Scanning Electron Microscopy (SEM). Congo red agar (CRA) plate method was used to identify slime production by strains. Screening of genes encoding adhesion factors and biofilm formation was carried out using PCR. After strains selection, adhesion and biofilm assays were designed testing different times (12, 48, 96 h), strains (n = 13), contact surfaces (stainless steel and polypropylene), and temperatures (5 °C and 25 °C); and then, bacterial count and crystal violet staining were conducted. Relative frequencies of positive on CRA and genes presence were determined, and Friedman test was applied for bacterial counts and OD values. Additionally, significant factors (P ≤ .05) were subjected to multiple comparisons using the Nemenyi test. The slime production in CRA was observed by visual inspection in 38.7% of strains. A large distribution of genes was described among strains, implying a high variability of genotypic profiles. Moreover, relative frequencies of CRA positive and gene presence were described. The developed assay showed that the strain, temperature, contact surface, were significant for both variables. The SEM corroborated the findings, showing greater biofilm formation on stainless steel at 25 °C. Thus, it is essential to highlight the importance of temperature control and material with low superficial energy to avoid biofilm formation by staphylococci.

Molecular characterization and antibiotic resistance of Staphylococcus spp. isolated from cheese processing plants.

The aim of this research paper was to characterize coagulase-positive and coagulase-negative staphylococci from raw milk, Minas cheese, and production lines of Minas cheese processing. One hundred isolates from 3 different cheese producers were characterized using molecular approaches, such as PCR, molecular typing, and DNA sequencing. Staphylococcus aureus (88% of the isolates) was the most abundant followed by Staphylococcus epidermidis, Staphylococcus hyicus, and Staphylococcus warneri. Among the 22 enterotoxin genes tested, the most frequent was seh (62% of the isolates), followed by selx and ser. Hemolysin genes were widely distributed across isolates, and Panton-Valentine leukocidin and toxic shock syndrome toxin genes were also identified. Methicillin-resistant S. aureus were staphylococcal cassette chromosome mec III, IVa, IVd, and others nontypeable. In the phenotypic antibiotic resistance, multiresistant isolates were detected and resistance to penicillin was the most observed. Using spa typing, we identified several types and described a new one, t14969, isolated from cheese. These findings suggest that antibiotic resistance and potentially virulent strains from different sources can be found in the Brazilian dairy processing environment. Further research should be conducted with collaboration from regulatory agencies to develop programs of prevention of virulent and resistant strain dissemination in dairy products and the processing environment.

Levantamento da ocorrência de resíduos de antibióticos em leite cru produzido na região dos Campos Gerais, Paraná / Survey of the occurrence of antibiotic residues in raw milk produced in the region of Campos Gerais, Paraná

A detecção de resíduos de antibióticos em leite é indispensável para direcionar ações corretivas visando à saúde do consumidor, a qualidade do leite e dos produtos derivados. Desta forma, uma política de incentivo à qualidade da produção leiteira foi implantada em 2005 na região dos Campos Gerais, Paraná. Neste caso, o produtor é penalizado com desconto sobre a produção mensal quando sua produção de leite cru for identificada com resíduos de agentes antibacterianos. Assim, o objetivo do presente artigo foi efetuar um levantamento que permita estabelecer a ocorrência de resíduos de antibióticos em leite cru na região dos Campos Gerais no período de implantação e pós-implantação da política de pagamento quanto ao requisito ausência de inibidor. Foram analisadas amostras provenientes de 400 produtores de leite no período de 2005, ano da implantação da política, até a ano de 2010, usando o teste microbiológico comercial Delvotest® SP-NT. A análise dos dados, realizada a partir da frequência de aparecimento de resíduos, apontou redução da ocorrência de antibióticos e baixa ocorrência quando comparada a outras regiões do país. Portanto, foi possível realizar o levantamento de resíduos de antibióticos em leite cru na região dos Campos Gerais, permitindo demonstrar que a ocorrência de antibióticos está reduzindo sensivelmente desde o ano da implantação da política de pagamento. Além da política de pagamento implantada, a elevação na qualidade de leite se deve a diversas ações corretivas e preventivas, principalmente no manejo, por meio de esclarecimentos ao produtor.

The detection of antibiotic residues in milk is essential for directing corrective actions searching for the consumer's health and quality of milk and milk products. Thus, a policy to promote the quality of milk production was implemented in 2005 in the region of Campos Gerais, Paraná State. In this case, the producer is penalized with discount on the monthly production when the raw milk production is identified with residues of antibacterial agents. The objective of this paper was to perform a survey that would establish the occurrence of antibiotic residues in raw milk in Campos Gerais region during deployment and post-deployment policy of payment regarding the absence of inhibitor. The samples from 400 dairy farmers were analyzed from 2005, the year of implementation of the policy, to 2010, using the commercial microbiological inhibition test Delvotest® SP-NT. The data analysis showed a decrease of antibiotic residues in raw milk, which was lower than other regions in Brazil. It was possible to study these residues present in raw milk from Campos Gerais region, showing that its occurrence is decreasing significantly since the year of the payment policy implementation. In addition to the payment policy, the milk quality improved due to various corrective and preventive actions, particularly in handling through an educational program involving the producers.