Antimicrobial resistance of Enterobacteriaceae and Staphylococcus spp. isolated from raw cow's milk from healthy, clinical and subclinical mastitis udders.

Mastitis is the most common disease of dairy cattle and can be manifested in clinical and subclinical forms. The overuse of antimicrobials in the treatment and prevention of mastitis favours antimicrobial resistance and milk can be a potential route of dissemination. This study aimed to evaluate the biological quality of bulk tank milk (BTM) and the microbiological quality and signs of mastitis of freshly milked raw milk. In addition, to evaluate antimicrobial resistance in Enterobacteriaceae and Staphylococcus spp. isolated from freshly milked raw milk. None of the farms were within the official Brazilian biological quality limits for BTM. Freshly milked raw milk with signs of clinical (CMM), subclinical (SCMM) and no signs (MFM) of mastitis were detected in 6.67%, 27.62% and 65.71% samples, respectively. Most samples of freshly milked raw milk showed acceptable microbiological quality, when evaluating the indicators total coliforms (78.10%), Escherichia coli (88.57%) and Staphylococcus aureus (100%). Klebsiella oxytoca and S. aureus were the most prevalent microorganisms in SCMM and MFM samples. Antimicrobial resistance and multidrug resistance (MDR) were observed in 65.12% and 13.95% of Enterobacteriaceae and 84.31% and 5.88% of Staphylococcus spp., respectively, isolated from both SCMM and MFM samples. Enterobacteriaceae resistant to third-generation cephalosporin (3GCR) (6.98%) and carbapenems (CRE) (6.98%) and methicillin-resistant S. aureus (MRSA) (4.88%) were observed. Antimicrobial-resistant bacteria can spread resistance genes to previously susceptible bacteria. This is a problem that affects animal, human and environmental health and should be evaluated within the one-health concept.

Kombucha: A review of substrates, regulations, composition, and biological properties.

Kombucha has been gaining prominence around the world and becoming popular due to its good health benefits. This beverage is historically obtained by the tea fermentation of Camellia sinensis and by a biofilm of cellulose containing the symbiotic culture of bacteria and yeast (SCOBY). The other substrates added to the C. sinensis tea have also been reported to help kombucha production. The type as well as the amount of sugar substrate, which is the origin of SCOBY, in addition to time and temperature of fermentation influence the content of organic acids, vitamins, total phenolics, and alcoholic content of kombucha. The route involved in the metabolite biotransformation identified in kombucha so far and the microorganisms involved in the process need to be further studied. Some nutritional properties and benefits related to the beverage have already been reported. Antioxidant and antimicrobial activities and antidiabetic and anticarcinogenic effects are some of the beneficial effects attributed to kombucha. Nevertheless, scientific literature needs clinical studies to evaluate these benefits in human beings. The toxic effects associated with the consumption of kombucha are still unclear, but due to the possibility of adverse reactions occurring, its consumption is contraindicated in infants and pregnant women, children under 4-years-old, patients with kidney failure, and patients with HIV. The regulations in place for kombucha address a number of criteria, mainly for the pH and alcohol content, in order to guarantee the quality and safety of the beverage as well as to ensure transparency of information for consumers.

Antimicrobial, modulatory, and antibiofilm activity of tt-farnesol on bacterial and fungal strains of importance to human health.

In this study, we analyzed the antimicrobial, antibiofilm, and modulatory activities of trans-trans-farnesol (tt-farnesol). The minimum inhibitory concentration (MIC) of this sesquiterpene was evaluated against 31 Gram-positive and Gram-negative bacterial strains and 4 species of the genus Candida. Furthermore, we examined its inhibitory action on biofilm production as well as antibiotic modulation. Only Gram-positive species presented susceptibility to tt-farnesol (MIC ranging from 8 µg/mL to 128 µg/mL). No synergistic or antagonistic effects were observed between tt-farnesol (1/4 and 1/8 of MIC) and first-choice antibiotics against multidrug resistant strains. However, the modulatory action of tt-farnesol (1/2 and 1/4 of the MIC) decreased 8 × MIC of non-inhibitory ß-lactam antibiotic against a Methicillin-resistant strain. In the antibiofilm assay, tt-farnesol inhibited biofilm formation, especially in Methicillin-resistant Staphylococcus aureus (MRSA) strains, at concentrations ranging from 2 µg/mL to 128 µg/mL. Additionally, in the molecular docking study, the tt-farnesol molecule demonstrated a remarkable binding affinity with important proteins involved in the biofilm production, such as IcaA and Srt proteins. The antimicrobial action of tt-farnesol on Streptococcus pyogenes and Streptococcus agalactiae strains was evaluated for the first time, presenting an MIC of 16 µg/mL for both strains. Our findings reveal the antibacterial, antibiofilm, and modulatory potential of tt-farnesol to aid in the fight against infectious processes.

Methicillin-resistant Staphylococcus aureus in minas frescal cheese: evaluation of classic enterotoxin genes, antimicrobial resistance and clonal diversity.

This study aimed to investigate classical enterotoxin (sea to see) and mecA genes, by polymerase chain reaction and anitimicrobial susceptibility, by disk diffusion test of Staphylococcus aureus isolated from minas frescal cheese (MFC). All methicillin-resistant S. aureus (MRSA) isolates were investigated for the presence of Panton-Valentine leukocidin (PVL) genes and clonal diversity. Thirty-one S. aureus were isolated from four MFC samples. Seven (22.6%) S. aureus carried mecA gene and two of them carried enterotoxin genes seb/sec and sea/seb. Five (16.1%) S. aureus isolates showed induced resistance to clindamycin and nine (29%) were resistant to multiple -antibiotics (MDR), among these, six were MRSA. No MRSA isolates presented the PVL genes. Four MRSA were grouped into three clones and three isolates were not typable by pulsed field gel electrophoresis. MRSA isolates showed, by multilocus sequence typing, sequence types ST1, ST5, ST72 and ST4304 (new ST) and S. aureus protein A (spa type) t127, t568 and t2703. These data suggest that MFC may constitute a risk to the consumer because of its potential for staphylococcal food poisoning; however it might, also, become one of MRSA and MDR strains disseminator, including clones usually found in the hospital environment.

Balneotherapy is a potential risk factor for Pseudomonas aeruginosa colonization

ABSTRACT The practice of immersion in burn patient has been abandoned in many parts of the world but in Brazil it is still common. The aim of this study was to ascertain if balneotherapy is a risk factor for Pseudomonas aeruginosa colonization in thermally injured patients. Eighteen patients from a Burn Center were studied for 14 weeks for Pseudomonas aeruginosa. Samples were collected by swabbing the exudate of wounds, before and after giving bath to the patients and from balneotherapy table. Pulsed-field gel electrophoresis was used to determine bacterial genetic relatedness. Thirty-seven P. aeruginosa isolates were detected from 292 swabs collected from patients' burn surface area and from the balneotherapy table. Profile analysis of P. aeruginosa DNA fragmentation showed 10 clones among the 37 strains analyzed. Type A is the most prevalent clone, with 23 strains distributed into eight subtypes. These were present in the swabs collected, before and after the patients' bath, from the surface of the bath table, suggesting that there was cross-contamination between the patients in different ways. This work demonstrates that balneotherapy is a risk factor in the Burn Center studied, because the same clone was found among P. aeruginosa isolates collected at various points and times.

RESUMO A prática de balneotarapia em paciente queimado foi abandonada em muitas partes do mundo, mas no Brasil ainda é comum. O objetivo deste estudo foi verificar se a balneoterapia é um fator de risco para a colonização por Pseudomonas aeruginosa em pacientes queimados. Dezoito pacientes internados em um Centro de Queimadura (CQ) foram acompanhados por 14 semanas. Amostras foram coletadas do exsudato de feridas, antes e depois do banho dos pacientes e também da mesa onde a balneoterapia foi realizada. A relação genética entre as cepas de P. aeruginosa foi determinada pela electroforese em gel de campo pulsado. Trinta e sete cepas foram detectadas a partir de 292 swabs coletados de área de superfície das feridas dos pacientes e da mesa de balneoterapia. Análise de fragmentação do DNA das 37 P. aeruginosa mostrou a existência de 10 clones. O tipo A foi o clone mais prevalente, com 23 cepas distribuídas em oito subtipos. Estas estavam presentes nas lesões dos pacientes antes e após o banho e na mesa onde o banho foi realizado, sugerindo contaminação cruzada inter e intra-pacientes e pacientes e mesa de banho. Este trabalho mostra que a balneoterapia é um fator de risco para colonização por P. aeruginosa, no CQ estudado, pois um mesmo clone da bactéria foi encontrado nos isolados coletados em vários pontos e épocas diferentes.

Formação de biofilmes de escherichia coli produtora de toxina Shiga sorotipos O153: H25, O113:H21 e O111: H8 em superfície de aço inoxidável e eficácia de sanitizante / Biofilm formation of shiga toxin-producing escherichia coli O153: H25, O113:H21 and O111: H8 on the stainless steel surface and the effectiveness of sanitizer

Neste estudo as cepas de Escherichia coli produtora de toxina Shiga (STEC) O153:H25, O113:H21 e O111:H8, isoladas de rebanhos do país, foram avaliadas quanto à capacidade de formar biofilmes em superfície de aço inoxidável utilizada na indústria de alimentos, bem como a eficácia de diferentes concentrações de hipoclorito de sódio na inativação desses biofilmes. A capacidade de formação de biofilme foi detectada em todas as cepas de E. coli produtoras de toxina Shiga. Na avaliação da eficáciado sanitizante hipoclorito de sódio, nas concentrações de 100 mg.L-1 e 200 mg.L-1, observou-se a redução a <1 log UFC/cm2 em todas as cepas e nos tempos avaliados. Estes dados reforçam aimportância do correto procedimento de higienização, uma vez que biofilmes podem tornar-se importantes fontes de contaminação no ambiente de produção de alimentos.

This study aimed at evaluating the biofilm formation of Shiga toxin-producing Escherichia coli O153:H25, O113:H21 and O111:H8 (STEC non-O157), isolated from Brazilian cattle, on the stainless steel surface, and also the efficacy of different s concentrations of sodium hypochlorite for inactivating these biofilms. The ability to form biofilm was demonstrated in all of Shiga toxin-producing E. coli strains. In assessing the effectiveness of sodium hypochlorite sanitizer, a reduction to <1 log CFU/cm2 was observed in all of the evaluated strains and times. These data strengthen the relevance of the correct cleaning procedure, considering that biofilms formations might be an important source of food contamination.

Viabilidade de Escherichia coli O153:H25, O113:H21 e O111:H8 (STEC não-O157) produtoras de toxina Shiga em queijo minas frescal / Viability of Escherichia coli O153:H25, O113:H21 e O111:H8 (STEC non-O157) Shiga toxin-producing in minas frescal cheese

A existência de um reservatório animal é de grande importância na transmissão de Escherichia coli, produtora de toxina shiga (STEC) aos humanos. Epidemiologicamente, o sorotipo O157:H7 tem sido o mais envolvido em surtos de doença humana causada por STEC, porém surtos envolvendo STEC não pertencentes ao sorogrupo O157 (STEC não-O157) têm sido descritos. Inúmeros trabalhos constatam uma elevada ocorrência destes microrganismos em fezes de bovinos no Brasil, entretanto, pouco se sabe sobre a transmissão destes aos produtos de origem animal em nosso país. Neste trabalho, foi avaliada a viabilidade de E.coli O153:H25; O113:H21 e O111:H8 em Queijo Minas Frescal (QMF), produzido com inóculos de STEC não O157: H7 e armazenados a 8ºC. Realizaram-se contagens de E. coli e psicrotróficos totais após o processamento do queijo e com intervalos de sete e quinze dias. Foi observado aumento nas contagens de E. coli STEC não O157: H7 e psicrotróficos totais logo após o processamento do QMF, bem como durante o armazenamento a 8ºC, temperatura máxima recomendada pela legislação brasileira. Demonstra-se que, caso haja contaminação da matéria-prima com STEC não O157: H7 (deste estudo), o processamento do QMF não elimina os microrganismos e a temperatura máxima recomendada pela legislação não inibe a multiplicação bacteriana, mantendo-se o risco à população. Reforça-se, portanto, a atenção à qualidade da matéria-prima, das ferramentas de qualidade no campo e na indústria de alimentos para garantir a inocuidade do produto final.

The existence of an animal reservoir is of great importance in the transmission of shiga toxin-producing E. coli (STEC) to humans. Epidemiologically serotype O157:H7 has been the most involved in human disease outbreaks caused by STEC, however STEC not belonging to serogroup O157 (STEC non-O157) have been described in outbreaks. Studies have revealed a risk in occurrence of these organisms in feces of cattle in Brazil, but little is known about the transmission to animal's products origin in our country. This study evaluated the viability of E. coli O153:H25, O113:H21 and O111:H8 in Minas Frescal Cheese (MFC), produced with non- STEC O157:H7 and stored at 8ºC. Counts of E. coli and total psychrotrophic were performed after processing and at intervals seven and fifteen days. There were an increase in the counts of E. coli and total psychrotrophic just after MFC processing as well as during storage at 8°C. The results demonstrated that, if the raw material (milk) is contaminated with STEC non O157:H7 (this study), the MFC processing does not eliminate the microorganisms and the maximum temperature recommended does not eliminate bacterial growth, keeping the risk to the population. The results reinforces, the attention to the quality of the raw material, the quality tools in the field and in the food industry to ensure the safety of final products.

Molecular investigation of tRNA genes integrity and its relation to pathogenicity islands in Shiga toxin-producing Escherichia coli (STEC) strains

tRNA genes are known target sites for the integration of pathogenicity islands (PAI) and other genetic elements, such as bacteriophages, into bacterial genome. In most STEC (Shiga toxin-producing Escherichia coli), the PAI called LEE (locus of enterocyte effacement) is related to bacterial virulence and is mostly associated to the tRNA genes selC and pheU. In this work, we first investigated the relationship of LEE with tRNA genes selC and pheU in 43 STEC strains. We found that 28 strains (65 percent) had a disrupted selC and/or pheU. Three of these strains (637/1, 650/5 and 654/3) were chosen to be submitted to a RAPD-PCR technique modified by the introduction of specific primers (corresponding to the 5'end of genes selC and pheU) into the reaction, which we called "anchored RAPD-PCR". The PCR fragments obtained were transferred onto membranes, and those fragments which hybridized to selC and pheU probes were isolated. One of these fragments from strain 637/1 was partially sequenced. An 85-nucleotide sequence was found to be similar to the cfxA2 gene that encodes a beta-lactamase and is part of transposon Tn4555, a pathogenicity island originally integrated into the Bacteroides genome.

Genetic analysis of Escherichia coli strains carrying enteropathogenic Escherichia coli (EPEC) markers, isolated from children in Rio de Janeiro city, Brazil

No presente estudo, 47 amostras enteropatogênicas de Escherichia coli, previamente caracterizadas pelo sorotipo, fenótipo de aderência, habilidade de induzir a formação da lesão histopatológica e presença das seqüências genéticas eae, bfp e EAF, foram analisadas de acordo com o perfil de fragmentação do DNA cromossômico pela técnica de eletroforese em campo pulsado (PFGE), as variantes isoenzimáticas através da eletroforese de isoenzimas (MLEE) e a presença de seqüências específicas da região LEE (eae, espA, espB, tir) e respectivos alelos. A amplificação destas seqüências mostrou a presença de 18 padrões genéticos distintos. A tipagem do gene eae revelou que a maior parte das amostras apresentou intimina não-tipável (42%) seguida dos tipos alélicos b (35%), g e a (12% cada). A fragmentação do DNA cromossômico detectou um elevado polimorfismo genético entre as amostras estudadas e não foi observada uma correlação com os marcadores de virulência investigados. Por outro lado, a análise das variantes isoenzimáticas sugeriu uma distribuição clonal específica de variantes genéticas do locus eae, o que nos leva a indicar a sua utilização como um marcador promissor para definir as relações genéticas neste grupo de microrganismos.

Genetic analysis of Escherichia coli strains carrying enteropathogenic Escherichia coli (EPEC) markers, isolated from children in Rio de Janeiro city, Brazil

In the present study, 47 enteropathogenic Escherichia coli strains identified according to serotyping, presence of eae, bfp and EAF sequences, adherence phenotype and ability to induce attaching-effacing lesions were analyzed by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MLEE), and the presence of LEE genes (eae, espA, espB, tir) as well as the respective alleles. Amplification of LEE genes subtypes revealed 18 different pathotypes. Typing of the eae gene showed that most strains contained nontypable intimin (42%) followed by beta (35%), gamma and alpha genes (12% each). PFGE analysis revealed a variable degree of polymorphism among isolates and, in general, no clear correlation was observed among PFGE profiles and the virulence markers identified. Otherwise, grouping based on MLEE analysis showed a close association between eae allele and clonal cluster distribution leading us to indicate the eae profile as a promising marker to establish relatedness among such microorganisms.

No presente estudo, 47 amostras enteropatogênicas de Escherichia coli, previamente caracterizadas pelo sorotipo, fenótipo de aderência, habilidade de induzir a formação da lesão histopatológica e presença das seqüências genéticas eae, bfp e EAF, foram analisadas de acordo com o perfil de fragmentação do DNA cromossômico pela técnica de eletroforese em campo pulsado (PFGE), as variantes isoenzimáticas através da eletroforese de isoenzimas (MLEE) e a presença de seqüências específicas da região LEE (eae, espA, espB, tir) e respectivos alelos. A amplificação destas seqüências mostrou a presença de 18 padrões genéticos distintos. A tipagem do gene eae revelou que a maior parte das amostras apresentou intimina não-tipável (42%) seguida dos tipos alélicos beta (35%), gama e alfa (12% cada). A fragmentação do DNA cromossômico detectou um elevado polimorfismo genético entre as amostras estudadas e não foi observada uma correlação com os marcadores de virulência investigados. Por outro lado, a análise das variantes isoenzimáticas sugeriu uma distribuição clonal específica de variantes genéticas do locus eae, o que nos leva a indicar a sua utilização como um marcador promissor para definir as relações genéticas neste grupo de microrganismos.