Smelly shark, smelly ray: what is infecting you?

AIMS: Although elasmobranchs are consumed worldwide, bacteriological assessments for this group are still sorely lacking. In this context, this study assessed bacteria of sharks and rays from one of the most important landing ports along the Rio de Janeiro coast. METHODS AND RESULTS: Bacteria were isolated from the cloacal swabs of the sampled elasmobranchs. They were cultured, and Vibrio, Aeromonas, and Enterobacterales were isolated and identified. The isolated bacteria were then biochemically identified and antimicrobial susceptibility assays were performed. Antigenic characterizations were performed for Salmonella spp. and Polymerase Chain Reaction (PCR) assays were performed to identify Escherichia coli pathotypes. Several bacteria of interest in the One Health context were detected. The most prevalent Enterobacterales were Morganella morganii and Citrobacter freundii, while Vibrio harveyi and Vibrio fluvialis were the most prevalent among Vibrio spp. and Aeromonas allosacharophila and Aeromonas veronii bv. veronii were the most frequent among Aeromonas spp. Several bacteria also displayed antimicrobial resistance, indicative of Public Health concerns. A total of 10% of Vibrio strains were resistant to trimethoprim-sulfamethoxazole and 40% displayed intermediate resistance to cefoxitin. Salmonella enterica strains displayed intermediate resistance to ciprofloxacin, nalidixic acid and streptomycin. All V. cholerae strains were identified as non-O1/non-O139. The detected E. coli strains did not exhibit pathogenicity genes. This is the first study to perform serology assessments for S. enterica subsp. enterica isolated from elasmobranchs, identifying the zoonotic Typhimurium serovar. Salmonella serology evaluations are, therefore, paramount to identify the importance of elasmobranchs in the epidemiological salmonellosis chain. CONCLUSIONS: The detection of several pathogenic and antibiotic-resistant bacteria may pose significant Public Health risks in Brazil, due to high elasmobranch consumption rates, indicating the urgent need for further bacteriological assessments in this group.

Detection of Escherichia coli and other Enterobacteriales members in seabirds sampled along the Brazilian coast.

Escherichia coli, an Enterobacterales member, is a normal representative of the microbiota of homeothermic animals. Most strains are commensal, but several pathotypes can cause disease, and numerous antimicrobial resistance factors have been identified. These bacteria have spread rapidly in recent years, highlighting the importance of screening the environment and non-human reservoirs for virulent strains and/or those presenting resistance factors, in addition to other microorganisms of public health importance. In this context, this study aimed to survey Enterobacteriales present in seabirds sampled along the Brazilian coast, comparing findings between migratory and resident birds, as well as between wrecked and non-wrecked animals. Escherichia coli pathotypes were also characterized through rapid seroagglutination and polymerase chain reaction techniques and antimicrobial resistance profiles were investigated through the disc agar diffusion method. Cloacal, ocular, oral, tracheal, and skin lesion swabs, as well as fresh feces, were collected from 122 seabirds. The findings indicate these animals as important hosts for opportunistic human pathogens. Escherichia coli strains were identified in 70 % of the analyzed seabirds, 62 % of which displaying resistant or intermediate profiles to at least one antimicrobial, while 7% were multiresistant. Resistance to tetracycline (22 %), nalidixic acid (15 %), trimethoprim-sulfamethozaxol (14 %) and ampicillin (12 %) were the most prevalent. Resistance to cefoxitin, a critically important antimicrobial for human medicine, was also detected. Virulence genes for one of the EAEC, ETEC or EPEC pathotypes were detected in 30 % of the identified strains, the first two described in seabirds for the first time. The EAEC gene was detected in 25 % of the sampled seabirds, all resident, 8 % of which exhibited a multidrug-resistant profile. Thus, seabirds comprise important reservoirs for this pathotype. Escherichia coli was proven an ubiquitous and well-distributed bacterium, present in all evaluated bird species and sampling sites (except Marajó Island). According to the chi-square test, no significant differences between E. coli prevalences or antimicrobial resistance profiles between migratory and resident and between wrecked and non-wrecked seabirds were observed. Thus, migratory birds do not seem to contribute significantly to E. coli frequencies, pathotypes or antimicrobial resistance rates on the Brazilian coast.

Virulence-Associated Genes and Antimicrobial Resistance of Aeromonas hydrophila Isolates from Animal, Food, and Human Sources in Brazil.

Aeromonads are natural inhabitants of aquatic environments and may be associated with various human or animal diseases. Its pathogenicity is complex and multifactorial and is associated with many virulence factors. In this study, 110 selected Aeromonas hydrophila isolates isolated from food, animals, and human clinical material from 2010 to 2015 were analyzed. Antimicrobial susceptibility testing was performed by the disk diffusion method, and polymerase chain reaction was conducted to investigate the virulence genes hemolysin (hlyA), cytotoxic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), aerolysin (aerA), and DNase-nuclease (exu). At least 92.7% of the isolates had one of the investigated virulence genes. Twenty different virulence profiles among the isolates were recognized, and the five investigated virulence genes were observed in four isolates. Human source isolates showed greater diversity than food and animal sources. Antimicrobial resistance was observed in 46.4% of the isolates, and multidrug resistance was detected in 3.6% of the isolates. Among the 120 isolates, 45% were resistant to cefoxitin; 23.5% to nalidixic acid; 16.6% to tetracycline; 13.7% to cefotaxime and imipenem; 11.8% to ceftazidime; 5.9% to amikacin, gentamicin, and sulfamethoxazole-trimethoprim; and 3.9% to ciprofloxacin and nitrofurantoin. Overall, the findings of our study indicated the presence of virulence genes and that antimicrobial resistance in A. hydrophila isolates in this study is compatible with potentially pathogenic bacteria. This information will allow us to recognize the potential risk through circulating isolates in animal health and public health and the spread through the food chain offering subsidies for appropriate sanitary actions.

Detection of multidrug-resistant Enterobacteriaceae isolated from river waters flowing to the Guanabara Bay and from clinical samples of hospitals in Rio de Janeiro, Brazil / Detección de enterobacterias multirresistentes aisladas en aguas de los ríos que desembocan en la bahía de Guanabara y en muestras de hospitales de Río de Janeiro, Brasil

Abstract Introduction: The use of antibiotics in humans, animal husbandry and veterinary activities induces selective pressure leading to the colonization and infection by resistant strains. Objective: We evaluated water samples collected from rivers of the Guanabara Bay, which have suffered minor and major environmental degradation, and clinical samples of hospital origin to detect evidence of the presence of resistance genes to aminoglycosides, beta-lactam antibiotics and fluoroquinolones in strains of Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and Escherichia coli. Materials and methods: For isolation of the water strains we employed culture media containing 32 μg/ml cephalotin and 8 μg/ml gentamicin. The strains from clinical materials were selected using culture media containing 8 μg/ml gentamicin. The strains were identified and subjected to antimicrobial susceptibility testing (AST), plasmid DNA extraction and polymerase chain reaction (PCR) to detect genes encoding enzymes modifying aminoglycosides (EMA), extended-spectrum beta-lactamases (ESBL) and plasmid mechanisms of quinolone resistance (PMQR). Results: The AST of the isolates recovered from water samples showed multidrug-resistance profiles similar to those found in isolates recovered from clinical materials. All isolates from water samples and 90% of the isolates from clinical samples showed at least one plasmid band. In the PCR assays, 7.4% of the isolates recovered from water samples and 20% of those from clinical materials showed amplification products for the three antimicrobial classes. Conclusion: We believe that the detection of microorganisms presenting genetic elements in environments such as water is necessary for the prevention and control of their dissemination with potential to infect humans and other animals in eventual contact with these environments.

Resumen Introducción. El uso de antibióticos en seres humanos, en la industria pecuaria y en las actividades veterinarias induce una presión selectiva que resulta en la colonización e infección con cepas resistentes. Objetivo. Determinar la presencia de genes de resistencia a aminoglucósidos, betalactámicos y fluoroquinolonas en cepas de Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae y Escherichia coli, obtenidas de muestras de agua de los ríos que desembocan en la bahía de Guanabara y de muestras clínicas de hospitales de Río de Janeiro. Materiales y métodos. En la selección de las cepas resistentes obtenidas de las muestras de agua de los ríos, se emplearon medios de cultivo que contenían 32 μg/ml de cefalotina y 8 μg/ ml de gentamicina. En el caso de las muestras de especímenes clínicos, se usaron medios de cultivo que contenían 8 μg/ml de gentamicina. Las cepas se identificaron y se sometieron a pruebas de sensibilidad antimicrobiana, extracción de ADN plasmídico y pruebas de reacción en cadena de la polimerasa (PCR) para detectar los genes que codifican aquellas enzimas que modifican los aminoglucósidos, las betalactamasas de espectro extendido (BLEE) y los mecanismos de resistencia a las quinolonas mediados por plásmidos. Resultados. Se encontraron perfiles de resistencia a los antimicrobianos similares en los dos grupos. En todas las bacterias obtenidas de las muestras de agua y en 90 % de las muestras clínicas, se evidenciaron bandas de plásmidos asociados con la transferencia de genes de resistencia. En las pruebas de PCR, se obtuvieron productos de amplificación de los genes de resistencia para las tres clases de antimicrobianos analizados, en el 7,4 % de las bacterias recuperadas de las muestras de agua y en el 20 % de aquellas recuperadas de las muestras clínicas. Conclusión. La detección de microorganismos con elementos genéticos que confieren resistencia a los antibióticos en ambientes como el agua, es una estrategia necesaria para prevenir y controlar la diseminación de estos agentes patógenos con potencial para infectar a humanos y a otros animales en dichos ambientes.

Microbiological Quality and Prevalence of ß-Lactam Antibiotic Resistance Genes in Oysters ( Crassostrea rhizophorae ).

The microbiological quality of oysters reflects the microbiological quality of their habitats because they are filter feeders. The objective of this study was to assess the bacterial composition of the edible oyster Crassostrea rhizophorae in urban and preserved estuaries. Particularly, we assessed the presence of pathogenic bacteria, investigated antibiotic susceptibility in bacterial isolates, and quantified ß-lactam antibiotic resistance genes (blaTEM, blaSHV, and blaKPC) via quantitative PCR of oyster DNA. Our results detected total coliforms, Escherichia coli , and enterobacteria in the oysters from urban estuaries, which is indicative of poor water quality. In addition, our detection of the eaeA and stxA2 virulence genes in 16.7% of E. coli isolates from oysters from this region suggests the presence of multiantibiotic-resistant enteropathogenic and enterohemorrhagic E. coli strains. During periods of low precipitation, increased contamination by E. coli (in winter) and Vibrio parahaemolyticus (in autumn) was observed. In contrast, cultivated oysters inhabiting monitored farms in preserved areas had low levels of bacterial contamination, emphasizing that oyster culture monitoring enhances food quality and makes oysters fit for human consumption. Distinct antibiotic resistance profiles were observed in bacteria isolated from oysters collected from different areas, including resistance to ß-lactam antibiotics. The presence of the blaTEM gene in 91.3% of oyster samples indicated that microorganisms in estuarine water conferred the capability to produce ß-lactamase. To our knowledge, this is the first study to directly quantify and detect ß-lactam antibiotic resistance genes in oysters. We believe our study provides baseline data for bacterial dynamics in estuarine oysters; such knowledge contributes to developing risk assessments to determine the associated hazards and consequences of consuming oysters from aquatic environments containing pathogenic bacteria that may possess antibiotic resistance genes.

Estudo fenotípico e molecular de resistência aos antimicrobianos em amostras clínicas e ambientais de enterobactérias / Molecular and phenotypic study of antimicrobial resistance in clinical and environmental samples of enterobacteriaceae

Buscamos detectar evidências da presença de genes envolvidos na produção de Enzimas Modificadoras de Aminoglicosídeos (EMAs), Beta-lactamases de espectro estendido (ESBLs) e Mecanismos Plasmidiais de Resistência a Quinolonas (PMQRs) em cepas de K. pneumoniae, K. ozaenae e E. coli isoladas de amostras de água de rios afluentes da Baía de Guanabara e de materiais clínicos de origem hospitalar, além de avaliar o "status sanitário" dos corpos aquáticos abordados no tocante à contaminação fecal recente e indicações de contaminação hospitalar e por outros ambientes de alta seletividade. As cepas de materiais clínicos foram selecionadas entre Maio e Julho de 2010, a partir da semeadura em meio de cultura contendo 8 ug/mL de gentamicina. As amostras de água foram coletadas em Abril e em Julho de 2009. Realizamos testes de colimetria, empregando para tal, a metodologia convencional e outra, na qual adicionamos 32 ug/mL de cefalotina e 8 ug/mL de gentamicina aos caldos Lactosado e Escherichia coli (caldo EC), a fim de detectar e quantificar coliformes resistentes. Para o isolamento das cepas empregamos meios de cultura contendo 32 ug/mL de cefalotina e 8 ug/mL de gentamicina. As cepas foram identificadas e submetidas a testes de susceptibilidade aos antimicrobianos (TSA), testes presuntivos para presença de ESBLs, extração de DNA plasmidial e ensaios de Reação em Cadeia de Polimerase (PCR) para a detecção dos genes. A utilização de agentes antimicrobianos nos testes de colimetria nos permitiu detectar a presença e quantificar coliformes totais e fecais resistentes nas amostras de água analisadas nos diferentes pontos. O TSA das cepas isoladas de amostras de água exibiu perfis de multirresistência, compatíveis com o de bactérias de origem hospitalar, semelhante ao encontrado nas cepas isoladas de materiais clínicos. Todas as cepas isoladas de amostras de água e 90% das cepas de materiais clínicos apresentaram pelo menos uma banda plasmidial. Os ensaios de PCR...

We seek to detect evidence of the presence of genes involved in the production of Aminoglycoside Modifying Enzymes (AME), Extended-spectrum beta-lactamases (ESBLs) and Plasmid Mechanisms of Resistance to Quinolones (PMQRs) in strains of K. pneumoniae, K. ozaenae and E. coli isolated from water samples from rivers of Guanabara Bay and clinical samples of hospital origin, and to evaluate the "health status" of water bodies addressed in relation to recent fecal contamination and signs of hospital contamination and other environments with high selectivity. The strains from clinical materials were selected between May and July 2010, using culture media containing 8 ug/mL gentamicin. Water samples were collected in April and July 2009. Colimetric assays were performed, using the conventional methodology and other which we added 32 ug/mL cephalothin and 8 ug/mL of gentamicin at Lactose and Escherichia coli broth (EC broth), in order to detect and to count resistant coliforms. For isolation of the strains we employed culture media containing 32 ug/mL cephalothin and 8 ug/mL gentamicin. The strains were identified and submitted to tests for antimicrobial susceptibility (TSA), presumptive tests for the presence of ESBLs, plasmid DNA extraction and tests of the Polymerase Chain Reaction (PCR). The use of antimicrobial agents in colimetric assays allowed us to detect and to count the resistant total and fecal coliforms in the water samples analyzed at different points. The TSA of the isolates recovered from water samples showed multidrug-resistance profiles, compatible with that of nosocomial bacteria, similar to that found in isolates recovered from clinical materials. All isolates from water samples and 90% of the isolates of clinical samples showed at least one plasmid band. PCR assays demonstrated the presence of amplification products to AME, ESBLs and PMQRs, and 7.4% of the isolates recovered of samples of water and 20% of the isolates of clinical materials...

Ecological aspects of the antimicrobial resistance in bacteria of importance to human infections

In view of the intimate relationship of humans with coastal lagoons (used for recreation, tourism, water supply, etc.), the discharge of domestic effluents may lead to the establishment of routes of dissemination of pathogenic microorganisms, including microorganisms carrying genes for resistance to antimicrobials, through the surrounding human communities. The objective of the present investigation was to relate the presence of antimicrobial-resistant bacteria to the environmental characteristics of three coastal lagoons, comparing the results with those from hospital sewage. Of the lagoons evaluated, two (Geriba and Imboassica) receive domestic sewage discharge, and the other (Cabiunas) is still in a natural state. We isolated in a culture medium containing 32 1/4ìg/ml of Cephalothin, fecal coliforms (E. coli), non-fecal coliforms (Klebsiella, Enterobacter, Serratia, and Citrobacter), non-glucose-fermenting Gram-negative bacilli, and Aeromonas sp. In cultures from the hospital drain we found strains showing numerous markers for resistance to most of the 11 antimicrobials tested. On the other hand, in cultures from Cabiunas and Imboassica lagoons, we found strains showing resistance only to antibiotics frequently observed in non-selective situations (considered as "common" markers). The capacity for dilution in the ecosystem, and salinity appeared related with the occurrence of multi-resistant bacterial strains. The intensity of recent fecal contamination was not shown to be associated with the numbers and types of markers found.