ABSTRACT: Enterococci have been used as sentinel organisms for monitoring antimicrobial resistance in food, humans, and other animals. In this sense, the present study evaluated the antimicrobial susceptibility profile and the presence of genes associated with resistance to erythromycin (msrC and ermB) and tetracycline [tet(M) and/or tet(L)] in enterococci isolated from raw sheep’s milk and cheeses (colonial, feta-, and pecorino-type) from South region of Brazil. A total of 156 enterococci were isolated from milk (n=80) and cheese (n=76) samples, identified by MALDI-TOF. Enterococcus faecalis (50.6%; n=79) was the most frequent species isolated from both samples. According to in vitro susceptibility tests, enterococci strains were not susceptible to the most commonly antimicrobial agents used in human and veterinary medicine. The frequency of MDR strains in enterococci isolated from milk (53.7%) was higher than those from cheese (24.2%). The tet(M) gene was the most commonly detected among tetracycline not-susceptible strains. The present study provided the first evidence of antimicrobial not-susceptible enterococci in raw sheep’s milk and cheeses in South Brazil. Drug-resistant strains, particularly those that are MDR, constitute a One Health issue.

RESUMO: Os enterococos têm sido usados como organismos sentinela para monitorar o padrão de suscetibilidade a antimicrobianos em alimentos, humanos e outros animais. Neste sentido, o presente estudo objetivou avaliar o perfil de susceptibilidade a antimicrobianos e os genes associados com a resistência a eritromicina (msrC and ermB) e à tetraciclina [tet(M) and/or tet(L)] em enterococos isolados de leite cru de ovelha e queijos (colonial, tipo-feta e tipo-pecorino) do Sul do Brasil. Um total de 156 enterococos foram isolados de leite (n=80) e queijo (n=76), identificados por MALDI-TOF. Enterococcus faecalis (50,6%; n=79) foi a espécie mais frequentemente isolada de ambas as amostras. De acordo com o teste de suscetibilidade in vitro, as cepas de enterococos não foram susceptíveis aos agentes antimicrobianos mais comumente utilizados na clínica humana e veterinária. A frequência de cepas de enterococos MDR isoladas do leite (53,7%) foi superior à do queijo (24,2%). O gene tet(M) foi o mais comumente detectado entre as cepas não susceptíveis à tetraciclina. O presente estudo fornece as primeiras evidências de enterococos não susceptíveis aos antimicrobianos em leite cru de ovelha e queijos no Sul do Brasil. Cepas resistentes a drogas, particularmente as que são MDR, representam uma preocupação de Saúde Única.

Abstract Antimicrobial resistance has been attributed to the overuse of antibiotics. To control the use of antibiotics, Brazil adopted the RDC 20/2011. A comparison the antibiotic-resistance profile of bacterial has provided important insights into resistance evolution. Enterococci are ubiquitous bacteria recommended to be used as a sentinel organism, in national surveillance systems, for tracking antimicrobial resistance through the food chain. The present study aimed to evaluate the diversity and antimicrobial resistance of enterococci collected from food in South Brazil in 2017 (pos-RDC 20/11) for comparison with isolated in 2007 (pre-RDC 20/11). A total of 310 enterococci were isolated from vegetables and products of animal origin, identified by PCR and MALDI-TOF, tested for antimicrobial susceptibility and screened for resistance genes. Enterococcus casseliflavus was dominant in vegetables and E. faecalis in products of animal origin. Enterococcal isolates in 2017 were mostly sensitive to ampicillin, gentamicin, chloramphenicol, and vancomycin when compared to isolated collected in 2007. While resistance levels to most compounds remained relatively stable, multidrug resistance decreased by 24% during this period. Our results suggest that RDC 20/11 had a positive outcome in controlling the spread of antimicrobial resistance. This study provides baseline data to measure future changes in the prevalence of resistant enterococci.

Abstract The purpose of this study was to compare the composition and stability of bacteria and fungi communities during the propagation of sourdoughs prepared with organic or conventional whole wheat (Triticum aestivum) flours from South Brazil. Sourdoughs were prepared and samples were collected during different fermentation times (0 to 216 h). Total DNA of sourdough samples were extracted and the 16S rRNA gene and Internal Transcribed Spacer region were sequenced by MiSeq-Illumina. A total of 43 and 56 OTUs were identified and defined as core taxa in the bacterial and fungal communities, respectively. The analysis revealed increases in the relative abundances of the lactic acid (Pediococcus pentosaceus, Weissella hellenica and Limosilactobacillus pontis) and acetic acid bacteria (Gluconobacter frateurii and Acetobacter tropicalis) during the sourdough propagation. The filaments fungi, Alternaria tenuissima, Fusarium culmorum, Fusarium petersiae and Microdochium seminicola remained more stable in organic than conventional during propagation cycles. After 216 h of fermentation, both sourdoughs were dominated by acid- and salt-tolerant yeast Issatchenkia orientalis (syn Pichia kudriavzevii, and Candida glycerinogenes). In conclusion, there were no significant differences in microbial communities among the sourdough samples. This study revealed that both flours contain autochthonous LAB, AAB, and yeasts with biotechnological applications in sourdough bread-making.

RESUMO: Nos últimos anos, compostos com propriedades biológicas produzidas por plantas têm recebido atenção como alternativa de controle de micro-organismos. Óleos essenciais extraídos de folhas verdes de Eucalyptus sp. têm demonstrado atividades antimicrobianas. No entanto, até o momento não há nenhum relato de atividade antimicrobiana de óleos essenciais extraídos de folhas secas de Eucalyptus staigeriana. O objetivo deste estudo foi determinar a composição química dos óleos essenciais obtidos de folhas secas de E. staigeriana e avaliar in vitro a sua atividade antimicrobiana e de antibiofilme contra gram-positivas e gram-negativas e também resistentes e multirresistentes de Enterococcus faecalis isolados de amostras de alimentos e clínicas. A caracterização de E. staigeriana foi realizada por CG-EM. Para este estudo foram utilizadas 26 cepas bacterianas, que incluíram 11 cepas referência e 15 cepas de E. faecalis resistentes a antibióticos. A atividade antimicrobiana de E. staigeriana contra gram-positivas e gram-negativas foi determinada utilizando o método de disco-difusão. Os valores da concentração inibitória mínima foram avaliados pela técnica de microdiluição. Os efeitos de antibiofilme foram avaliados pelo método de placa de microtitulação. Como resultado, 21 compostos foram identificados, sendo monoterpenos oxigenados (69,58%) a grande família química. E. staigeriana mostrou apenas atividade antimicrobiana contra cepas gram-positivas. Cepas de E. faecalis resistentes e multirresistentes mostraram a menor concentração inibitória mínima (3,12 para 6,25%) quando comparado com a cepa referência de E. faecalis. E. staigeriana apresentou a capacidade de inibir a formação de biofilme, mas pouca ou nenhuma capacidade de inibir o biofilme pré-formado. Este estudo demonstra que o óleo essencial obtido de folhas secas de E. staigeriana é uma alternativa promissora para controle importante de bactérias gram-positivas resistentes de origem alimentar e clínicas.

ABSTRACT: In recent years, compounds with biological properties produced by plants have received attention as an alternative to control microorganisms. Essential oils extracted from green leaves of Eucalyptus sp. have been demonstrated to have antimicrobial activities, but so far there are no reports of antimicrobial activity of essential oils extracted from dried leaves of Eucalyptus staigeriana. So, the objectives of this study were to determine the chemical composition of the essential oils obtained from dried leaves of E. staigeriana (EOdlES) and to evaluate in vitro antimicrobial and antibiofilm activities of EOdlES against gram-positive and gram-negative, resistance and multiresistant Enterococcus faecalis isolated from food and clinical samples. The characterization of EOdlES was performed by gas chromatography-mass spectrometry (GC/MS). For this study, 26 bacterial strains were used, which included 11 reference strains and 15 antibiotic resistant and multiresistant E. faecalis strains. Antimicrobial activities of EOdlES against gram-positive and gram-negative were determined using the disc diffusion method. The minimum inhibitory concentration (MIC) value was evaluated by a microbroth dilution technique. The antibiofilm effects were assessed by microtiter plate method. As a result, 21 compounds were identified, being oxygenated monoterpenes (69.58%) the major chemical family. EOdlES showed only antimicrobial activity against gram-positive strains. E. faecalis resistant and multiresistant strains show the lowest MIC (3.12 to 6.25%), when compared with reference E. faecalis strain. EOdlES has the ability to inhibit the biofilm formation, but little or none ability to inhibit the preformed biofilm. This study demonstrates that EOdlES is a promising alternative to control important foodborne and clinic gram-positive resistant bacteria.

ABSTRACTINTRODUCTION:Exposure to subinhibitory concentrations (SICs) of antimicrobials may alter the bacterial transcriptome.METHODS: Here, we evaluated the expression of nine virulence-related genes in vancomycin-resistant enterococci (VRE) urinary tract infection isolates grown at SICs of vancomycin.RESULTS:A Subinhibitory concentrations of vancomycin interferes with gene modulation, but does not affect the phenotype of a VRE strain in vitro .CONCLUSIONS:Subinhibitory concentrations of vancomycin may regulate the expression of virulence factors in vivo or contribute to the selection of vancomycin-resistant strains.

Sanitary conditions are essential for the production of meals and control of the presence of pathogensis important to guarantee the health of customers. The aim of this study was to evaluate the sanitary quality of food services by checking the presence of thermotolerant coliforms, Staphylococcus sp. and evaluate the toxigenic potential from the latter. The analysis was performed on water, surfaces, equipment, ready-to-eat foods, hands and nasal cavity of handlers in seven food services. The water used in food services proved to be suitable for the production of meals. Most food, equipment and surfaces showed poor sanitary conditions due to the presence of thermotolerant coliforms (60.6%). Twenty-six Staphylococcus species were identified from the 121 Staphylococcus isolates tested. Staphylococci coagulase-negative species were predominant in the foods, equipment and surfaces. In food handlers and foods, the predominant species was Staphylococcus epidermidis. Twelve different genotypes were found after PCR for the classical enterotoxin genes. The seb gene (19.8%) was the most prevalent among all Staphylococcus sp. Both coagulase-positive and coagulase-negative Staphylococci showed some of the genes of the enterotoxins tested. We conclude that there are hygienic and sanitary deficiencies in the food services analyzed. Although coagulase-positive Staphylococci have not been present in foods there is a wide dispersion of enterotoxigenic coagulase-negative Staphylococci in the environment and in the foods analyzed, indicating a risk to consumer health.

The Firmicutes bacteria participate extensively in virulence and pathological processes. Enterococcus faecalis is a commensal microorganism; however, it is also a pathogenic bacterium mainly associated with nosocomial infections in immunocompromised patients. Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups involved in diverse biological processes, whose in vivo formation requires several specific protein machineries. Escherichia coli is one of the most frequently studied microorganisms regarding [Fe-S] cluster biogenesis and encodes the iron-sulfur cluster and sulfur assimilation systems. In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis. The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions. Quantitative real-time polymerase chain reaction demonstrated, for the first time, that Gram-positive bacteria possess an OxyR component responsive to oxidative stress conditions, as fully described for E. coli models. Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

A dinâmica da microbiota no trato gastrointestinal (TG) de animais pode ser afetada por patógenos, tais como Eimeria spp. Os enterococos são bactérias saprófitas que colonizam o TG de mamíferos e aves. A influência sobre a microbiota intestinal está relacionada com a capacidade de adaptação das bactérias em se aderir às células hospedeiras e de colonizar as células das mucosas. O objetivo deste estudo foi analisar a frequência de genes de virulência ace, agg e operon do bopABCD em Enterococcus faecalis isolados de swabs cloacais de frangos de corte desafiados com Eimeria spp e alimentados com dietas padrões suplementadas ou não com anticoccidiano (monesina) e também avaliar a capacidade dessas cepas em formar biofilmes sob condições in vitro. Um total de 70 E. faecalis foram selecionadas e o gene agg foi mais freqüente em cepas isoladas de frangos de corte alimentados com anticoccidiano (92,3%) quando comparado ao grupo que não recebeu anticoccidiano (70,5%). Por outro lado, os genes ace e do operon bopABCD não demostraram nenhuma diferença significativa entre os dois grupos de frangos (P>0,005). Os E. faecalis isolados de frangos de corte alimentados com anticoccidiano demostraram uma maior frequência de fortes aderentes quando crescendo em meio suplementado com glicose (92,3-88,5%) e urina (77%), quando comparado com enterococos isolados de frangos que não receberam anticoccidiano. Observou-se que E. faecalis isolados de frangos tratados com anticoccidiano mostraram uma maior frequêencia dos genes dos fatores de virulência e de perfil de fortes formadores de biofilme, o que indica uma melhor adaptação dos isolados em ambiente intestinal saudável.

The microbiota dynamics in the gastrointestinal tract (GT) of animals can be disrupted by pathogens, such as Eimeria spp. Enterococci are saprophytic bacteria that colonize the GT of mammals and birds. The influence on the intestinal microbiota is related to the adaptive capacity of bacteria to adhere to host cells and colonize the mucosal cells. The aim of this study was to analyze the frequency of virulence genes ace, agg and bopABCD operon in Enterococcus faecalis isolated from cloacal swabs of broilers challenged with Eimeria spp. and fed with a standard diet supplemented or not with anticoccidial (monensin), and, also to evaluate for the ability of these strains to form biofilms under in vitro conditions. A total of 70 E. faecalis were selected and the agg gene was more frequent in strains isolated from the broilers treated with anticoccidial (92.3%) when compared to the group that not received anticoccidial (70.5%). On the other hand, the ace and bopABCD operon genes showed no significant difference between the two groups of broilers (P>0.005). The E. faecalis isolated from the broilers treated with anticoccidial showed a higher frequency of strong biofilm formation when growing in medium supplemented with glucose (92.3-88.5%) and urine (77%) when compared with enterococci isolated from broilers that not received anticoccidial. It was observed that E. faecalis isolated from broilers treated with anticoccidial showed a higher frequency of virulence factors genes and stronger biofilms formation, indicating better adaptation of the isolates in healthy intestinal environment.

Here we report the presence and expression levels of the vanC 1 and vanC 2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC 1 and vanC 2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC 1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.

INTRODUÇÃO: Estafilococos são patógenos responsáveis por surtos de doenças transmitidas por alimentos. O estudo investigou a prevalência de genes de enterotoxinas e o perfil de resistência aos antimicrobianos em estafilococos coagulase-negativo (CoNS) e estafilococos coagulase-positivo (CoPS) isolados de morcilhas no sul do Brasil. MÉTODOS: Duzentas colônias típicas e atípicas do ágar Baird-Parker foram inoculadas em ágar sal-manitol. Oitenta e dois estafilococos manitol-positivos foram submetidos a testes bioquímicos e perfil de susceptibilidade antimicrobiana. A presença dos genes da coagulase (coa) e enterotoxinas (se) foi investigada por reação em cadeia da polimerase (PCR). RESULTADOS: Os isolados foram divididos em dois grupos: 75,6% (62/82) CoNS e 24,4% (20/82) CoPS. Através dos testes bioquímicos, 9 espécies foram determinadas, Staphylococcus saprophyticus (37,8%) e Staphylococcus carnosus (15,9%) foram as mais prevalentes. Testes de susceptibilidade demostraram fenótipos de resistência aos antibióticos administrados em humanos, como gentamicina, tetraciclina, cloranfenicol e eritromicina. O gene coa foi detectado em 19,5% (16/82) das cepas e quatro fragmentos de DNA polimórficos foram observados. Cinco CoNS contendo o gene coa foram submetidos ao sequenciamento do 16S rRNA e três mostraram similaridade com CoNS. Quarenta amostras foram positivas para pelo menos um gene se, os mais frequentes foram sea (28,6%) e seb (27,5%). CONCLUSÕES: A presença de resistência aos antimicrobianos e de genes se nos isolados de morcilha indicou que este alimento pode representar um risco potencial à saúde, já que a presença nos alimentos pode causar doenças de origem alimentar ou ser uma possível rota de transferência de estafilococos resistentes aos humanos.

INTRODUCTION: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. METHODS: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. RESULTS: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). CONCLUSIONS: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

INTRODUÇÃO: Listeria monocytogenes é um microrganismo que se encontra disseminado na natureza, sendo responsável por causar listeriose, uma doença infecciosa causada pelo consumo de alimentos contaminados. MÉTODOS: A análise molecular de 19 linhagens de Listeria monocytogenes, sorovares 1/2a, 1/2b, 4b, 4c, isoladas de produtos lácteos do Rio Grande do Sul, Brasil. As técnicas moleculares aplicadas foram: Amplificação Randômica do DNA Polimórfico e Análise por Enzimas de Restrição. Além da análise molecular foi realizado o perfil de resistência antimicrobiana. RESULTADOS: As linhagens estudadas mostraram baixo grau de diversidade, em relação ao perfil de resistência antimicrobiana desses microrganismos das amostras analisadas todas foram susceptíveis aos antimicrobianos testados. CONCLUSÕES: As técnicas moleculares estudadas apresentaram um bom poder de discriminação para as linhagens estudadas. Além disso, todas as amostras analisadas foram susceptíveis aos antimicrobianos analisados.

INTRODUCTION: Listeria monocytogenes is a ubiquitous microorganism in nature and is responsible for listeriosis, an infectious disease caused by consumption of contaminated food. METHODS: Molecular characterization was performed on 19 strains of Listeria monocytogenes (serovars 1/2a, 1/2b, 4b and 4c), isolated from dairy products in Rio Grande do Sul, Brazil. The molecular techniques applied were random amplification of polymorphic DNA and restriction enzyme analysis. In addition to the molecular analysis, the antimicrobial resistance profile was determined. RESULTS: The strains studied showed a low degree of diversity. In relation to the antimicrobial resistance profile of those microorganisms from the samples analyzed, all of them were susceptible to the antimicrobials tested. CONCLUSIONS: The molecular techniques that were used presented good discriminatory power for the strains studied. Furthermore, all of the samples that were analyzed were susceptible to the antimicrobials tested.

INTRODUÇÃO: O objetivo deste estudo foi confirmar a identificação de amostras clínicas e alimentos de Enterococcus gallinarum e Enterococcus casseliflavus por PCR-RFLP. MÉTODOS: Cinquenta e duas cepas identificadas por exames bioquímicos convencionais foram submetidos a amplificação por PCR e digestão com HinfI. Apenas 20 (38,5%) das 52 amostras apresentaram um padrão de DNA esperado E. gallinarum e E. casseliflavus. RESULTADOS: Analise dos resultados deste estudo demonstraram que, algumas vezes E. gallinarum e E. casseliflavus são erroneamente identificados e confirmaram a potencial aplicação da análise do 16S rDNA para identificação exata destas espécies. CONCLUSÕES: A correta identificação é importante a fim de distinguir entre resistência intrínseca e adquirida à vancomicina.

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

Cinqüenta e seis cepas de Enterococcus spp. foram isoladas de alimentos no Sul do Brasil, confirmados por PCR e classificadas como Enterococcus faecalis (27), Enterococcus faecium (23) e Enterococcus spp. (6). Testes de susceptibilidade aos antimicrobianos demonstraram fenótipos de resistência a uma gama de antibióticos administrados em humanos, como gentamicina, estreptomicina, ampicilina e vancomicina.

Fifty-six Enterococcus spp. strains were isolated from foods in Southern Brazil, confirmed by PCR and classified as Enterococcus faecalis (27), Enterococcus faecium (23) and Enterococcus spp(6). Antimicrobial susceptibility tests showed resistance phenotypes to a range of antibiotics widely administrated in humans such as gentamycin, streptomycin, ampicillin and vancomycin.

A seqüência da região polimórfica do gene iap foi analisada em 25 cepas de Listeria monocytogenes isoladas de queijo no Estado do Rio Grande do Sul e comparadas com cepas referências. Esta investigação distinguiu L. monocytogenes em dois grupos: I (20 cepas) e II (5 cepas).

The polymorphic region sequences in the iap gene were analyzed in 25 strains of Listeria monocytogenes isolated from cheeses in the state of Rio Grande do Sul, and compared with reference strains. This investigation distinguished two clusters of L. monocytogenes: I (20 strains) and II (5 strains).