A watershed impacted by anthropogenic activities: Microbial community alterations and reservoir of antimicrobial resistance genes.

Water is the main resource for maintaining life. Anthropic activities influence the microbial epidemiological chain in watersheds, which can act as ways of disseminating microorganisms resistant to antimicrobial drugs, with impacts on human, animal, and environmental health. Here, we characterized aquatic microbial communities and their resistomes in samples collected along Rio das Ostras watershed during two seasons. Surface water samples were collected at eleven sites from the Jundiá, Iriry, and Rio das Ostras rivers in two seasons (dry and wet season). Microbial DNA was extracted, high-throughput sequenced and screened for antimicrobial resistance genetic (ARG) markers. The physicochemical characteristics and the microbiota data confirmed that Rio das Ostras watershed can be divided into three well defined portions: rural, urban, and marine. Rural areas were enriched by bacteria typically found in limnic environments and Patescibacteria phyla. The urban portion was characterized by sites with low pH and groups associated with iron oxidation. Some genera of clinical relevance were also identified, though in relatively low abundance. The marine site was enriched mainly by Cyanobacteria and bacteria that showed strong correlation with conductivity, salinity, and chloride. Twenty-six ARG markers were identified on the resistome, being found most frequently in the urban area, despite being present in rural sites. Among them were some related to classes of great clinical concern, such as genes coding for extended-spectrum beta-lactamase (blaCTX-M and blaTEM), resistance to carbapenems (blaKPC) and to methicillin by Staphylococcus aureus (mecA). These results broaden our understanding of the microbial community of a watershed impacted by anthropogenic actions. The large number of ARGs detected along the Rio das Ostras watershed contrasts with the small number of microorganisms of clinical relevance observed, suggesting that antimicrobial resistance has arisen from non-clinical environments and microbes. Our results corroborate that freshwater acts as a reservoir of antimicrobial resistance genes.

Aplicação de ferramentas de segurança alimentar como proposta de incentivo ao aprimoramento da produção de produtos lácteos caprinos / Application of food safety tools as a proposal of incentive for the enhancement of dairy goat production

O leite é um alimento nutritivo e perecível, tornando imprescindível a utilização de Boas Práticas de Fabricação (BPF) para sua produção segura. O objetivo do trabalho foi propor melhorias das BPF à um pequeno laticínio da Mesorregião Centro Fluminense que processa leite cabra. A partir de visitas técnicas, foram coletadas amostras de alimentos para realização de análises físico-químicas e microbiológicas bem como foi feito um diagnóstico das condições de produção por meio de uma lista de verificação. Diante das inadequações encontradas que incluíram desvios na estrutura predial, organização do laticínio, higiene e controle de qualidade da matéria-prima, produto final e seu transporte até os pontos de venda, foi elaborado um plano de ação para melhoria das BPF baseado no grau de importância e custo. Foi elaborado ainda alguns Procedimentos Padrão de Higiene Operacional e revisão do Manual de BPF do estabelecimento. Esta ação contribuiu para conscientizar o laticínio sobre a importância de avaliação constante e melhoria das BPF dos produtos lácteos caprinos.

Frequency of zoonotic bacteria among illegally traded wild birds in Rio de Janeiro

Abstract The illegal wildlife trade may increase the risk of infectious disease transmission, and it may not only cause disease outbreaks in humans but also threaten livestock, native wild populations, and ecosystems' health. Bird species may act as carriers in the transmission of enteric pathogens. However, epidemiological studies on zoonotic bacteria in wild birds are rare in Brazil. From March 2011 to March 2012, we investigated the frequency of Enterobacteriaceae in cloacal swab samples from 109 birds of the passerine and Psittacidae families. These birds were recovered from illegal trade in Rio de Janeiro, Brazil, and sent to a rehabilitation center. Gram-negative bacteria were isolated from 86 wild birds (78.9%). A mean (±SD) of 1.68 (±1.30) different bacterial species were isolated per bird, with a maximum of five bacterial species from three bird species. The most frequently isolated bacteria were Escherichia coli, followed by Enterobacter spp., Klebsiella pneumoniae and other enteric bacteria. Salmonella ser. Typhimurium was isolated from a Temminck's seedeater (Sporophila falcirostris), and two Salmonella ser. Panama were isolated from two specimens of chestnut-capped blackbird (Chrysomus ruficapillus). Of the 70 selected bacterial isolates, 60 exhibited antibiotic resistance. The resistance patterns varied from one to nine of the antibiotics tested. Resistance to ceftiofur was the most prevalent, followed by ampicillin and ceftriaxone. The dissemination potential of resistant strains in situations typically seen in the management of captive birds may become a problem for the conservation of natural bird populations and for public health.

Frequency of zoonotic bacteria among illegally traded wild birds in Rio de Janeiro.

The illegal wildlife trade may increase the risk of infectious disease transmission, and it may not only cause disease outbreaks in humans but also threaten livestock, native wild populations, and ecosystems' health. Bird species may act as carriers in the transmission of enteric pathogens. However, epidemiological studies on zoonotic bacteria in wild birds are rare in Brazil. From March 2011 to March 2012, we investigated the frequency of Enterobacteriaceae in cloacal swab samples from 109 birds of the passerine and Psittacidae families. These birds were recovered from illegal trade in Rio de Janeiro, Brazil, and sent to a rehabilitation center. Gram-negative bacteria were isolated from 86 wild birds (78.9%). A mean (±SD) of 1.68 (±1.30) different bacterial species were isolated per bird, with a maximum of five bacterial species from three bird species. The most frequently isolated bacteria were Escherichia coli, followed by Enterobacter spp., Klebsiella pneumoniae and other enteric bacteria. Salmonella ser. Typhimurium was isolated from a Temminck's seedeater (Sporophila falcirostris), and two Salmonella ser. Panama were isolated from two specimens of chestnut-capped blackbird (Chrysomus ruficapillus). Of the 70 selected bacterial isolates, 60 exhibited antibiotic resistance. The resistance patterns varied from one to nine of the antibiotics tested. Resistance to ceftiofur was the most prevalent, followed by ampicillin and ceftriaxone. The dissemination potential of resistant strains in situations typically seen in the management of captive birds may become a problem for the conservation of natural bird populations and for public health.

Characteristics of Salmonella spp. Isolated from Wild Birds Confiscated in Illegal Trade Markets, Rio de Janeiro, Brazil.

The prevalence of Salmonella spp. was investigated in 109 wild birds poached in the illegal wildlife trade in Rio de Janeiro; most of them are passerines from Thraupidae family and three from Psittacidae. One strain of Salmonella ser. Typhimurium and two strains of Salmonella ser. Panama were isolated from passerine species and all of them showed resistance to multiple antimicrobial drugs, like ampicillin, ceftriaxone, ceftiofur, tetracycline, gentamicin, nalidixic acid, ciprofloxacin, and enrofloxacin. PFGE showed 100% similarity among the Salmonella ser. Typhimurium strain isolated from a Temminck's seedeater (Sporophila falcirostris) and the strains isolated from a human outbreak, in southern Brazil. The two Salmonella ser. Panama strains isolated from two chestnut-capped blackbirds (Chrysomus ruficapillus) present in the same catch showed the same clonal origin and have never been associated with epizooties and human outbreaks. Potential for dissemination of resistant Salmonella through situations offered by captive management and the isolation of the same strain from wild birds and human sources may become a problem for the conservation of natural populations and to public health.

Caracterização fenotípica da resistência a antimicrobianos e detecção do gene mecA em Staphylococcus spp. coagulase-negativos isolados de amostras animais e humanas / Phenotypic characterization of antimicrobial resistance and detection of the mecA gene in coagulase-negative Staphylococcus spp. isolates from animal and human samples

Os estafilococos coagulase-negativos (ECN) fazem parte da microbiota normal da pele e, apesar de terem sido considerados saprófitas por muito tempo, o seu significado clínico como agente etiológico tem aumentado com o passar dos anos. Neste estudo, foram obtidos 72 isolados de ECN a partir de amostras do conduto auditivo de cães, de mastite bovina e de infecções humanas. Staphylococcus xylosus foi o microrganismo mais isolado, nas amostras animais, e S. cohnii subsp. cohnii em humanos. Os isolados foram avaliados de modo a traçar o perfil fenotípico de sua resistência aos antimicrobianos mais indicados no tratamento de infecções estafilocócicas. Foi detectado um elevado nível de resistência à penicilina e ampicilina. A gentamicina, a vancomicina e a associação ampicilina+sulbactam foram eficientes frente aos isolados testados. A resistência à oxacilina foi avaliada por meio dos testes de difusão em disco modificada, ágar screen, microdiluição em caldo e diluição em ágar para constatar, se à semelhança do que ocorre com os estafilococos coagulase-positivo, esta pode ser mediada pelo gene mecA e apresentada de forma heterogênea. A presença do gene mecA foi determinada pelo método da Reação em Cadeia de Polimerase (PCR), sendo 5,6 por cento dos isolados mecA positivos.

Coagulase-negative staphylococci (SCN) make part of the normal microbiota skin and although they have been considered saprophytics for years, nowadays their clinical significance as an etiologic agent has increased. In this study, 72 SCN isolates obtained from external ear canals of dogs, bovine mastitis and human nosocomial infections were evaluated. Staphylococcus xylosus was the most prevalent microorganism in animal samples and S. cohnii subsp. cohnii in human samples. SCN isolates were evaluated in order to establish a phenotypical resistance pattern towards the most indicated antibiotics for staphyloccocal infections. A high level of resistance to penicillin and ampicillin was detected. The most efficient antibiotics evaluated were gentamicin, vancomicin and the association between ampicillin and sulbactam. To certify the heterogeneous resistance pattern, oxacillin resistance was phenotypically detected by a modified-disc-diffusion test, agar screen, broth micro-dilution and agar dilution. The presence of the mecA gene was detected in 5.6 percent of the SCN isolates by Polimerase Chain Reaction (PCR).

Mapeamento do perfil de resistência e detecção do gene mecA em Staphylococcus aureus e Staphylococcus intermedius oxacilina-resistentes isolados de espécies humanas e animais / Resistance pattern and detection of mecA gene in oxacillin-resistant isolates of Staphylococcus aureus and Staphylococcus intermedius from animal and human samples

Espécies de Staphylococcus spp resistentes a antimicrobianos representam um problema cosmopolita, sendo o controle de sua disseminação um importante desafio. O perfil de resistência a antimicrobianos de isolados de Staphylococcus aureus e Staphylococcus intermedius em amostras clínicas humanas e animais foi avaliado através do método de difusão em disco, no qual foi possível detectar um elevado nível de resistência à ampicilina e à penicilina. A avaliação da resistência à oxacilina, devido à heterogeneidade de resposta do gênero estudado, foi desenvolvida também através das seguintes técnicas: difusão em ágar modificado, ágar screen e microdiluição em caldo, e posteriormente correlacionada com a detecção do gene mecA, pela técnica de PCR, nas amostras consideradas resistentes em pelo menos um dos testes utilizados. A correlação entre os resultados obtidos nos testes fenotípicos com a presença do gene de resistência, considerado um método de referência, foi utilizada para validar a sensibilidade destes. De um total de 80 amostras avaliadas, 28 apresentaram resistência à oxacilina, sendo possível detectar a presença do gene de resistência mecA em 12 destas amostras. Os testes de suscetibilidade à oxacilina apresentaram sensibilidade superior a 50,0 por cento, sendo a difusão em disco simples e o ágar screen considerados mais sensíveis, e a difusão em disco modificada, o de menor sensibilidade.

Antimicrobial resistant Staphylococcus species represent an important cosmopolitan problem, and its spreading control is a significative challenge. Resistance pattern of Staphylococcus aureus and Staphylococcus intermedius species isolated from animals and humans clinical samples to different antibiotics was evaluated through disk diffusion method, where ampicillin and penicillin presented the highest level of resistance. The evaluation of the resistance to oxacillin, due to the heterogeneity of the response of the studied genus was carried out through the following tests: modified agar diffusion, agar screen and microdilution, and further correlation with the detection of mecA gene in samples that showed resistance in at least one of the susceptibility tests used. The correlation between the results obtained from phenotypic methods and the detection of resistance gene, considered as a reference method, was used in order to validate its sensitivity. Eighty clinical staphylococcal isolates (29 human and 51 animal isolates) were evaluated, 28 were oxacillin-resistant, mecA gene being detected in 12 samples. Susceptibility assessment tests to oxacillin presented above 50 percent of specificity, disk diffusion and agar screen being the most sensitive one, while modified disk diffusion presented the lowest sensibility rate. Ampicillin and penicillin presented the highest level of resistance.

Isolamento de agentes microbianos a partir de amostras de sangue e umbigo de bezerros mestiços neonatos / Microorganisms isolated from blood samples and umbilical structures of newborns calves

Infecção das estruturas umbilicais pode resultar em bacteremia, septicemia e morte em neonatos com falha na imunidade passiva sendo os microrganismos usuais de onfalites isolados freqüentemente em animais com bacteremia. Um estudo foi desenvolvido entre setembro 2002 e setembro 2003 utilizando-se 44 bezerros, visando verificar a freqüência de bacteremia em bezerros neonatos e a correlação entre os agentes isolados a partir de amostras de sangue de estruturas umbilicais. Amostras de sangue foram obtidas por punção da jugular e submetidas à cultura para isolamento de agentes microbianos, sendo 24 obtidas entre 48 e 72 horas e 20 entre o terceiro e o quinto dia após o nascimento. Através de “Swabs” procedeu-se à coleta de material das estruturas umbilicais para a mesma finalidade. Obteve-se crescimento microbiano em 17 (38,67%) amostras de sangue e 100% das amostras de estruturas umbilicais. Os microorganismos mais freqüentes em amostras de sangue foram Staphylococcus sp., E. coli, Bacillus spp, Pseudomonas sp. e Streptococcus. Todos os animais com bacteremia apresentaram manifestações de enfermidade focal ou sistêmica. O sinal clínico mais freqüentemente relacionado com cultura de sangue positiva foi o espessamento das estruturas umbilicais. Bacillus spp, Enterobacter spp, Micrococcus spp. e E. coli foram os microrganismos isolados das estruturas umbilicais. Os dados confirmam uma flora bacteriana mista nos casos de onfalite e sugerem uma prevalência alta de bacteremia em bezerros neonatos, sobretudo aqueles com onfalite, evidenciando a importância de boa transmissão de imunoglobulinas através do colostro.

Umbilical structures infections can be followed by bacteremic, septicemic infections and death in newborns with passive immunity deficiency. Microorganisms isolated in omphalitis have been also isolated from animals with bacteremia. From september/2002 to september/2003, a research was developed using 44 calves, in order to evaluate bacteremia frequency in newborns and to correlate microorganisms isolated from blood samples and umbilical structures. Blood samples were collected fram jugular vein for microorganisms isolation. Twenty four samples were collected in a period of 24 to 48 hours, the other 20 samples from thirty to fifty days after birth. Umbilical structures materials were collected though swabs. Microbial growth occurred in 17 (38,67%) blood samples and in 100% of umbilical samples. Staprylococcus spp., E. coli, Bacillus spp, Pseudomonas spp. and Streptococcus spp were the major isolated microorganisms from blood. All bacteremic animals presented systemic or localized clinical manifestations. The most reported clinical sign was thickness of umbilical structures. Bacillus spp, Enterobacter spp, Mierococcus spp. and E. coli were isolated from umbilical structures. Data confirm a mixed bacterial environment in omphalitis, and suggest a high prevalence of bacteremic infections in newborns calves, pointing out the need of passive immunity transfer through colostrum.