Streptococcus pyogenes Lineage ST62/emm87: The International Spread of This Potentially Invasive Lineage.

Streptococcus pyogenes is known to be associated with a variety of infections, from pharyngitis to necrotizing fasciitis (flesh-eating disease). S. pyogenes of the ST62/emm87 lineage is recognized as one of the most frequently isolated lineages of invasive infections caused by this bacterium, which may be involved in hospital outbreaks and cluster infections. Despite this, comparative genomic and phylogenomic studies have not yet been carried out for this lineage. Thus, its virulence and antimicrobial susceptibility profiles are mostly unknown, as are the genetic relationships and evolutionary traits involving this lineage. Previously, a strain of S. pyogenes ST62/emm87 (37-97) was characterized in our lab for its ability to generate antibiotic-persistent cells, and therapeutic failure in severe invasive infections caused by this bacterial species is well-reported in the scientific literature. In this work, we analyzed genomic and phylogenomic characteristics and evaluated the virulence and resistance profiles of ST62/emm87 S. pyogenes from Brazil and international sources. Here we show that strains that form this lineage (ST62/emm87) are internationally spread, involved in invasive outbreaks, and share important virulence profiles with the most common emm types of S. pyogenes, such as emm1, emm3, emm12, and emm69, which are associated with most invasive infections caused by this bacterial species in the USA and Europe. Accordingly, the continued increase of ST62/emm87 in severe S. pyogenes diseases should not be underestimated.

Anti-Restriction Gene Homologs Are Highly Represented in Methicillin-Resistant and Multidrug-Resistant Staphylococcus aureus ST239 and ST398: Implications for Resistance Gene Acquisitions.

Multidrug resistance is commonly acquired by transferring DNA from one bacterium to another. However, the mechanisms that enhance the acquisitions of foreign genes are poorly understood, as well as the dynamics of their transmission between hosts in different environments. Here, genomic approaches were applied to evaluate the enrichment of the S. aureus chromosome with resistance traits in groups of genomes with or without anti-restriction genes and to analyze some evolutionary aspects of these acquisitions. Furthermore, the role played by an anti-restriction gene in improving multiresistance in MRSA was investigated by molecular cloning. A strong association was observed between the presence of anti-restriction gene homologs and patterns of multidrug resistance. Human isolates, mainly ST239-SCCmecIII, carry ardA-H1, and from animal sources, mainly CC398, carry ardA-H2. Increased DNA transfer was observed for clones that express the ardA-H1 allele, corroborating its role in promoting gene transfer. In addition, ardA-H1 was expressed in the dsDNA format in the BMB9393 strain. The evolution of successful multidrug-resistant MRSA lineages of the ST239 and ST398 was initiated not only by the entry of the mec cassette but also by the acquisition of anti-restriction gene homologs. Understanding the mechanisms that affect DNA transfer may provide new tools to control the spread of drug resistance.

High frequency of increased triclosan MIC among CC5 MRSA and risk of misclassification of the SCCmec into types.

BACKGROUND: Typing of staphylococcal cassette chromosome mec (SCCmec) elements is commonly used for studies on the molecular epidemiology of MRSA. OBJECTIVES: To perform an investigation centred on uncovering the reasons for misclassification of MRSA clonal complex 5 (CC5) SCCmec type II clinical isolates in our laboratory. METHODS: MRSA isolates from CC5 were subjected to WGS and SCCmec typing. RESULTS: This investigation led to the discovery that the classification failure was due to an insertion of IS1272 carrying the fabI gene on a transposable element (TnSha1) that confers increased MIC to the biocide triclosan. Genomic analysis revealed that fabI was present in 25% of the CC5 MRSA isolates sampled. The frequency of TnSha1 in our collection was much higher than that observed among publicly available genomes (0.8%; n = 24/3142 CC5 genomes). Phylogenetic analyses revealed that genomes in different CC5 clades carry TnSha1 inserted in different integration sites, suggesting that this transposon has entered CC5 MRSA genomes on multiple occasions. In at least two genotypes, ST5-SCCmecII-t539 and ST5-SCCmecII-t2666, TnSha1 seems to have entered prior to their divergence. CONCLUSIONS: Our work highlights an important misclassification problem of SCCmecII in isolates harbouring TnSha1 when Boye's method is used for typing, which could have important implications for molecular epidemiology of MRSA. The importance of increased-MIC phenotype is still a matter of controversy that deserves more study given the widespread use of triclosan in many countries. Our results suggest expanding prevalence that may indicate strong selection for this phenotype.

Cellular Growth Arrest and Efflux Pumps Are Associated With Antibiotic Persisters in Streptococcus pyogenes Induced in Biofilm-Like Environments.

Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections; however, none of them have fully elucidated this phenomenon. In this study, we show that GAS strains have the ability to form antimicrobial persisters when inoculated on abiotic surfaces to form a film of bacterial agglomerates (biofilm-like environment). Our data suggest that efflux pumps were possibly involved in this phenomenon. In fact, gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persisters arising in the presence of penicillin. Phenotypic reversion assay and whole-genome sequencing indicated that this event was due to non-inherited resistance mechanisms. The persister cells showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, the proteomic analysis revealed that susceptible cells express higher levels of ribosome proteins. It is remarkable that previous studies have reported the recovery of S. pyogenes viable cells from tissue biopsies of patients presented with GAS invasive infections and submitted to therapy with antibiotics. The persistence phenomenon described herein brings new insights into the origin of therapeutic failures in S. pyogenes infections. Multifactorial mechanisms involving protein synthesis inhibition, cell growth impairment and efflux pumps seem to play roles in the formation of antimicrobial persisters in S. pyogenes.

Peritoneopericardial diaphragmatic hernia in a healthy adult feline (Felis catus domesticus): diagnosis to surgical treatment - Case report.

Peritoneopericardial diaphragmatic hernia (PPDH) is a communication between the abdomen and the pericardial sac generated by congenital anomalies triggered during diaphragmatic and pericardial development. This report aimed to present the case of an adult, mixed-breed cat, affected by PPDH, focusing on the period from diagnosis to successful surgical correction. The patient had a capricious appetite and weight loss for about four months and started, at the end of this period, a state of apathy. On abdominal ultrasound, the gallbladder (GB) was close to the heart, suggesting diaphragmatic discontinuity. On thoracic radiography, there were changes suggestive of PPDH, pericardial efusion or cardiomegaly with probable dilated cardiomyopathy. Based on these findings, an echocardiogram was performed, highlighting the hepatic lobe and GB internally to the pericardium, causing cardiac compression, although without severe cardiac changes. During surgery, a diaphragmatic defect of 4 cm in diameter was observed with the congested right medial hepatic lobe and hyperemic GB in the pericardial sac. The defect was sutured using the sultan pattern in separate stitches and polyamide threads. The feline returned to feeding with greater interest soon after the surgery, and after 15 days it was fed with dry food and had normal behavior. PPDH can be diagnosed in healthy adult cats, even if there are no apparent respiratory, gastrointestinal, or cardiac signs. The echocardiogram is relevant in the definitive diagnosis, in addition to excluding differential diagnoses, and simple surgical treatment with polyamide thread and sultan suture is successful.

A hérnia periotônio-pericárdica diafragmática (HPPD) comunica o abdome e o saco pericárdico, é gerada por anomalias congênitas deflagradas no desenvolvimento diafragmático e pericárdico. O objetivo deste relato é apresentar o caso de uma gata adulta, mestiça, acometida por HPPD, destacando do diagnóstico à correção cirúrgica bem sucedida. A paciente exibia apetite caprichoso e emagrecimento há cerca de 4 meses, iniciando ao final deste período, apatia. Na ultrassonografia abdominal a vesícula biliar (VB) estava próxima ao coração sugerindo ruptura diafragmática. Na radiografia torácica evidenciou-se alterações sugestivas de HPPD ou cardiomegalia com provável miocardiopatia dilatada. Devido tais achados realizou-se ecocardiograma destacando o lobo hepático e a VB no pericárdio comprimindo o coração sem comprometer sua função. Na cirurgia observou-se defeito diafragmático (4 cm), passagem do lobo hepático medial direito e da VB para o pericárdio. Suturou-se o defeito com padrão sultan e fio poliamida 3.0. A felina voltou a se alimentar com maior interesse logo após a cirurgia. A HPPD pode ser diagnosticada em felinos adultos saudáveis, mesmo que não haja sinais respiratórios, gastrointestinais ou cardíacos aparentes. O ecocardiograma é relevante no diagnóstico definitivo, além de excluir diagnósticos diferenciais, sendo o simples tratamento cirúrgico com fio poliamida e sutura sultan bem sucedido.

Low lineage diversity and increased virulence of group C Streptococcus dysgalactiae subsp. equisimilis.

Introduction. In some species, the population structure of pathogenic bacteria is clonal. However, the mechanisms that determine the predominance and persistence of specific bacterial lineages of group C Streptococcus remain poorly understood. In Brazil, a previous study revealed the predominance of two main lineages of Streptococcus dysgalactiae subsp. equisimilis (SDSE).Aim. The aim of this study was to assess the virulence and fitness advantages that might explain the predominance of these SDSE lineages for a long period of time.Methodology. emm typing was determined by DNA sequencing. Adhesion and invasion tests were performed using human bronchial epithelial cells (16HBE14o-). Biofilm formation was tested on glass surfaces and the presence of virulence genes was assessed by PCR. Additionally, virulence was studied using Caenorhabditis elegans models and competitive fitness was analysed in murine models.Results. The predominant lineages A and B were mostly typed as emm stC839 and stC6979, respectively. Notably, these lineages exhibited a superior ability to adhere and invade airway cells. Furthermore, the dominant lineages were more prone to induce aversive olfactory learning and more likely to kill C. elegans. In the competitive fitness assays, they also showed increased adaptability. Consistent with the increased virulence observed in the ex vivo and in vivo models, the predominant lineages A and B showed a higher number of virulence-associated genes and a superior ability to accumulate biofilm.Conclusion. These results suggest strongly that this predominance did not occur randomly but rather was due to adaptive mechanisms that culminated in increased colonization and other bacterial properties that might confer increased bacteria-host adaptability to cause disease.

Characterisation of penicillin and tetracycline resistance in Staphylococcus aureus isolated from bovine milk samples in Minas Gerais, Brazil.

This Regional Research Communication describes the characterisation of ampicillin, penicillin and tetracycline resistance in Staphylococcus aureus isolated from bovine subclinical mastitis in Minas Gerais State, Brazil. Ninety S. aureus isolates from bovine mastitis exhibiting phenotypic resistance to ampicillin, penicillin and/or tetracycline were selected for this study. The minimum inhibitory concentration (MIC) of each antibiotic was determined using the E-Test® and the production of beta-lactamase was determined by cefinase disks. The resistance genes blaZ, tet(K), tet(L), tet(M), and tet(O) were investigated by PCR in all of the isolates. The MIC results classified 77, 83 and 71% of the isolates as resistant to ampicillin, penicillin and tetracycline, respectively. The MIC50 and MIC90 were, respectively, 1 and 2 µg/ml for ampicillin, 0·5 and 1 µg/ml for penicillin and 32 and 64 µg/ml for tetracycline. Eighty-six per cent of beta-lactamase producing isolates were detected. Of the 90 isolates investigated, 97% amplified blaZ, 84% amplified tet(K), 9% amplified tet(L), 2% amplified tet(M) and 1% amplified tet(O). Seventy-nine isolates (88%) showed blaZ together with at least one tet gene. S. aureus isolates showed high MIC50 and MIC90 values for the three antimicrobials. The blaZ and tet(K) genes were widespread in the herds studied, and most of the isolates harboured blaZ and tet(K) concomitantly.

Quality and safety of artisan cheese produced in the serrana region of Santa Catarina / Qualidade e inocuidade de queijos artesanais produzidos na região serrana em Santa Catarina

The serrano artisan cheese produced from raw milk of dairy cattle is a typical product of high-altitude farms in the states of Santa Catarina and Rio Grande do Sul. However, marketing of the cheeses occurs illegally because they lack the minimum maturation period required for cheese produced from raw milk. The production of artisan cheeses is required to follow strict hygiene standards. This study aimed to test the quality and safety of cheeses that were produced in 31 farms of the Serrana region in Santa Catarina after 14 and 28 days of maturation. Coliform count was measured at 35 °C, and presence of other microorganisms such as Escherichia coli, Staphylococcus, Listeria spp., and Salmonella spp. were also tested. Fat and protein percentages, acidity, salt content, and humidity were also evaluated. Data were subjected to statistical analyses using the SAS® software. After 14 and 28 days of maturation, 74.19% (23/31) and 64.52% (20/31) of samples, respectively, showed higher numbers of coliforms at 35 °C than those permissible by law. Higher than permissible numbers of E. coli were observed in 45.16% (14/31) and 48.39% (15/31) of the samples analyzed after 14 and 28 days of maturation, respectively. Coagulasepositive staphylococci values above 103 CFU/g were observed in 54.84% (17/31) and 51.61% (16/31) of cheese samples after 14 and 28 days of maturation, respectively. Contamination with Salmonella spp. was not detected. However, Listeria monocytogenes serovar 4b was isolated in 3.23% (1/31) and 6.45% (2/31) of samples after 14 and 28 days of maturation, respectively. The results of humidity tests classified the cheese samples into three categories: low, medium, and high humidity. Semi fat cheeses were predominant in both maturation periods, although the samples were classified in thin, semi fat, and fat cheeses. The main variations in the compositions of analyzed samples occurred for salt and acidity levels.[...](AU)

O queijo artesanal serrano, produzido a partir de leite cru de bovinos, é um produto típico dos campos de altitude dos estados de Santa Catarina e do Rio Grande do Sul. No entanto, a sua comercialização na grande maioria ocorre às margens da legislação, por ser fabricado com leite cru e não possuir um período mínimo de maturação. Tendo em vista que a produção de queijos artesanais necessita seguir normas rigorosas de higiene, o presente estudo objetivou desenvolver um diagnóstico da qualidade e inocuidade dos queijos com 14 e 28 dias de maturação produzido em 31 propriedades da Serra Catarinense. Foram realizadas contagens de coliformes a 35°C, Escherichia coli e Staphylococcus coagulase positiva; pesquisa de Salmonella sp. e Listeria monocytogenes. Os percentuais de gordura, acidez, sal, umidade e proteína também foram avaliados. Os dados foram submetidos as análises estatísticas realizadas no software SAS®. Após 14 e 28 dias de maturação, 74,19% (23/31) e 64,52% (20/31) das amostras de queijo analisadas, respectivamente, apresentaram limites acima do disposto para coliformes a 35 °C. Contagens de E. coli superiores ao aceitável foram observadas em 45,16% (14/31) e 48,39% (15/31) das amostras analisadas após 14 e 28 dias de maturação, respectivamente. Quanto à contagem de Staphylococcus coagulase positiva, 54,84% (17/31) e 51,61% (16/31) das amostras de queijos apresentaram após 14 e 28 dias de maturação valores acima de 103 UFC/g. Nenhuma das amostras avaliadas estava contaminada com Salmonella spp., porém Listeria monocytogenes sorovar 4b foi isolada em 3,23% (1/31) e em 6,45% (2/31) das amostras aos 14 e 28 dias de maturação, respectivamente. Em relação à umidade, os queijos foram classificados como de baixa, média e alta umidade. Em relação aos teores de gordura houve a predominância de queijos semigordos nos dois períodos de maturação, embora as amostras tenham sido enquadradas como queijos magros, semigordos e gordos. [...](AU)

Quality and safety of artisan cheese produced in the serrana region of Santa Catarina / Qualidade e inocuidade de queijos artesanais produzidos na região serrana em Santa Catarina

The serrano artisan cheese produced from raw milk of dairy cattle is a typical product of high-altitude farms in the states of Santa Catarina and Rio Grande do Sul. However, marketing of the cheeses occurs illegally because they lack the minimum maturation period required for cheese produced from raw milk. The production of artisan cheeses is required to follow strict hygiene standards. This study aimed to test the quality and safety of cheeses that were produced in 31 farms of the Serrana region in Santa Catarina after 14 and 28 days of maturation. Coliform count was measured at 35 °C, and presence of other microorganisms such as Escherichia coli, Staphylococcus, Listeria spp., and Salmonella spp. were also tested. Fat and protein percentages, acidity, salt content, and humidity were also evaluated. Data were subjected to statistical analyses using the SAS® software. After 14 and 28 days of maturation, 74.19% (23/31) and 64.52% (20/31) of samples, respectively, showed higher numbers of coliforms at 35 °C than those permissible by law. Higher than permissible numbers of E. coli were observed in 45.16% (14/31) and 48.39% (15/31) of the samples analyzed after 14 and 28 days of maturation, respectively. Coagulasepositive staphylococci values above 103 CFU/g were observed in 54.84% (17/31) and 51.61% (16/31) of cheese samples after 14 and 28 days of maturation, respectively. Contamination with Salmonella spp. was not detected. However, Listeria monocytogenes serovar 4b was isolated in 3.23% (1/31) and 6.45% (2/31) of samples after 14 and 28 days of maturation, respectively. The results of humidity tests classified the cheese samples into three categories: low, medium, and high humidity. Semi fat cheeses were predominant in both maturation periods, although the samples were classified in thin, semi fat, and fat cheeses. The main variations in the compositions of analyzed samples occurred for salt and acidity levels.[...]

O queijo artesanal serrano, produzido a partir de leite cru de bovinos, é um produto típico dos campos de altitude dos estados de Santa Catarina e do Rio Grande do Sul. No entanto, a sua comercialização na grande maioria ocorre às margens da legislação, por ser fabricado com leite cru e não possuir um período mínimo de maturação. Tendo em vista que a produção de queijos artesanais necessita seguir normas rigorosas de higiene, o presente estudo objetivou desenvolver um diagnóstico da qualidade e inocuidade dos queijos com 14 e 28 dias de maturação produzido em 31 propriedades da Serra Catarinense. Foram realizadas contagens de coliformes a 35°C, Escherichia coli e Staphylococcus coagulase positiva; pesquisa de Salmonella sp. e Listeria monocytogenes. Os percentuais de gordura, acidez, sal, umidade e proteína também foram avaliados. Os dados foram submetidos as análises estatísticas realizadas no software SAS®. Após 14 e 28 dias de maturação, 74,19% (23/31) e 64,52% (20/31) das amostras de queijo analisadas, respectivamente, apresentaram limites acima do disposto para coliformes a 35 °C. Contagens de E. coli superiores ao aceitável foram observadas em 45,16% (14/31) e 48,39% (15/31) das amostras analisadas após 14 e 28 dias de maturação, respectivamente. Quanto à contagem de Staphylococcus coagulase positiva, 54,84% (17/31) e 51,61% (16/31) das amostras de queijos apresentaram após 14 e 28 dias de maturação valores acima de 103 UFC/g. Nenhuma das amostras avaliadas estava contaminada com Salmonella spp., porém Listeria monocytogenes sorovar 4b foi isolada em 3,23% (1/31) e em 6,45% (2/31) das amostras aos 14 e 28 dias de maturação, respectivamente. Em relação à umidade, os queijos foram classificados como de baixa, média e alta umidade. Em relação aos teores de gordura houve a predominância de queijos semigordos nos dois períodos de maturação, embora as amostras tenham sido enquadradas como queijos magros, semigordos e gordos. [...]