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.

Avaliação microbiológica de superfícies em terapia intensiva: reflexões sobre as estratégias preventivas de infecções nosocomiais / Microbiological evaluation of surfaces in intensive care: thinking about nosocomial infection prevention strategies / Evaluación microbiológica de superficies en cuidados intensivos: reflexiones sobre las estrategias preventivas de infecciones nosocomiales

Objetivo: determinar o perfil microbiológico de bactérias isoladas e identificadas nos leitos e bombas infusoras na unidade de terapia intensiva de um hospital universitário do Estado do Rio de Janeiro. Método: foram coletadas nove amostras de grades das camas dos pacientes e oito de teclados de bomba infusora de uma unidade de terapia intensiva, em outubro de 2014, delimitando-se uma área de 100cm² como parâmetro para ambas as coletas. As amostras foram coletadas através de swabs estéreis que foram umedecidos e transportados em Carry & Blair. Os microrganismos foram isolados, classificados e depois testados em relação à resistência antimicrobiana. Resultados: o gênero Staphylococcus coagulase negativa foi o mais prevalente. Os testes de suscetibilidade a antimicrobianos apontaram alguns destes Staphylococci como multirresistentes. Conclusão: chama-se atenção para a necessidade de ampliação do debate multiprofissional sobre questões de segurança hospitalar, apresentando a educação permanente como um possível caminho de sucesso no controle das infecções.

Objective: to determine the microbiological profile of bacteria isolated and identified from beds and infusion pumps in the intensive care unit of a university hospital in Rio de Janeiro state. Method: nine samples were collected from patients' bed side rails and eight from infusion pump keypads in an intensive care unit in October 2014. An area of 100cm² was delimited as the sampling parameter. Samples were collected using sterile swabs, which were wetted and transported with Cary-Blair. The microorganisms were isolated, classified, and then tested for antimicrobial resistance. Results: coagulase-negative Staphylococcus was the most prevalent type. Antimicrobial susceptibility testing indicated some of these Staphylococci were multi-drug resistant. Conclusion: multi-professional discussion of hospital safety issues must be expanded, and continuing professional development emerges as one possible pathway to success in nosocomial infection control.

Objetivo: determinar el perfil microbiológico de bacterias aisladas e identificadas en las camas y las bombas de infusión en la unidad de terapia intensiva de un hospital universitario de Rio de Janeiro. Método: se recolectaron nueve muestras de rejas de camas de pacientes y ocho de paneles de las bombas de infusión de una unidad de terapia intensiva, en octubre de 2014, delimitandose un área de 100 cm2 como parámetro para ambas recolecciones. Se recolectaron las muestras a través de swabs estériles que fueron humedecidos y transportados en Carry y Blair. Los microorganismos fueron aislados, clasificados y después probados repecto a la resistencia antimicrobiana. Resultados: el género Staphylococcus coagulasa negativo fue el más prevalente. Las pruebas de susceptibilidad a antimicrobianos mostraron algunos Staphylococci como resistentes a múltiples fármacos. Conclusión: se señala la necesidad de ampliación del debate entre los profesionales de la salud, sobre cuestiones de seguridad hospitalaria, presentando la educación permanente como un posible camino de éxito en el control de las infecciones.

Sphygmomanometers and thermometers as potential fomites of Staphylococcus haemolyticus: biofilm formation in the presence of antibiotics

BACKGROUND The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO3^2-) is toxic to most microorganisms, TeO3^2--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO3^2--resistance (Te^R) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative Te^R determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO3^2- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegansand the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the Te^R and pathogenic potential of C. diphtheriae.

β-lactamase producing enterobacteria isolated from surveillance swabs of patients in a cardiac intensive care unit in Rio de Janeiro, Brazil

There is a high incidence of infections caused by betalactamase-producing Gram-negative microorganisms in Brazil. These organisms are of clinical and epidemiological importance, since their mobile genetic elements facilitate cross-infection. The present study was conducted in sentinel rectal swabs from patients admitted to a cardiac surgery hospital in Rio de Janeiro, from January through December 2007, in a consecutive manner. The aim of the study was to characterize the genotype and phenotype of these isolates from colonized patients. Biochemical tests, antimicrobial susceptibility tests, a confirmatory test for the expression of extended spectrum betalactamase (ESBL) production and polymerase chain reaction for the blaTEM, blaSHV, CTX-M1, Toho-1 and AmpC genes were performed at the University Hospital of Universidade do Estado do Rio de Janeiro (UERJ). The most frequently isolated bacteria were Escherichia coli 9/41 (21.95 percent) and Klebsiella pneumoniae 14/41 (34.1 percent). In 24/41 (58 percent), the ESBL genotype was confirmed. The most prevalent genes in samples that expressed ESBL were blaTEM 13/24 (54 percent), AmpC 12/24 (50 percent), blaSHV 6/24 (25 percent), CTX-M1 7/24 (29 percent), and Toho-1 6/24 (25 percent). Of these, 14/24 (58 percent) presented more than one genotype for the tested primers. In nine (37 percent) samples other than E. coli, K. pneumoniae or Proteus spp., the phenotype for ESBL was found and confirmed by PCR. The most sensitive substrate in the approximation test in ESBL positive samples was ceftriaxone (83 percent). Fifty percent of the samples expressed AmpC were associated with other genes. Intermediate susceptibility to ertapenem was found in 2/41 (5 percent).

Resistência aos antimicrobianos em estafilococos coagulase-negativa isolados de hemocultura / Antimicrobial resistance in coagulase-negative staphylococci isolated of hemocultura

Os estafilococos coagulase-negativa (ECNs) estão entre os microganismos que constituem a microbiota nor al da pele. O seu significado clínico como agente causal de várias infecções em pacientes com materiais implantados e outras infecções hospitalares tem aumentado nas últimas décadas. No presente estudo avaliou-se a resistência de 36 amostras de ECNs, isoladas de hemoculturas de pacientes internados em hospital público da cidade do Rio de Janeiro. As amostras revelaram um perfil de alta resistência às drogas do grupo betalactâmico, e o emprego da cefoxitina, em substituição à oxacilina, para indicação da presença do gene mecA e detecção da resistência a estas drogas, mostrou-se adequado. O perfil de alta resistência observado entre as amostras recomenda maior rigor e critério na seleção de drogas para o tratamento das infecções po ECNs.

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.

Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenothypical gentamicin resistance amplification (frequencies of 10-3 to 10-5, compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromossomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy

Biotipagem e resistotipagem para o traçado epidemiológico da origem fecal de klebsiella pneumoniae em infecçöes urinárias / Biotyping and resistotyping for epidemiological tracing of the fecal origin of klebsiella pneumoniae in urinary infections

Em 24(82,7%) de 29 pacientes com infecçäo urinária por Klebsiella pneumoniae isolamos a mesma espécie a partir de amostras fecais. Estudamos 24 cepas urinárias e 219 cepas fecais encontrando 50 biótipos distintos (em média quatro biótipos por amostra fecal). Em dez (34,4%) dos 29 pacientes o biótipo de uma ou mais cepas fecais corresponderam ao biotipo da cepa urinária - sem encontrarmos associaçäo entre a simultaneidade e a prévia cateterizaçäo vesical (p>0,05). Na resistotipagem - utilizando quatro substâncias químicas previamente escolhidas entre 34 produtos testados (verde brilhante, verde malaquita, telurito de potássio e cloreto mercúrico) encontramos 16 resistotipos distintos. Em 14 (58,3%), dos 24 casos houve detecçäo do mesmo resistotipo em cepa(s) fecal(is) e urinária do mesmo paciente, entretanto, só em cinco (20,9%) dos casos houve concordância com a biotipagem na indicaçäo de simultaneidade. A concordância de resultados quanto à ausência ou presença de biotipos ou resistotipos simultâneos, na urina e nas fezes, foi de somente 54,2%. A presença de resistência aos íons telurito e mercúrio, entre cepas fecais e cepas urinárias, no mesmo paciente, estava significativamente associada (p<0,001)

Resistência a antibióticos e íons inorgânicos em Klebsiella pneumoniae de pacientes hospitalizados e näo hospitalizados / Resistance to antibiotics and inorganic ions in Klebsiella pneumoniae of hospitalized and non-hospitalized patients

No estudo da presença de marcadores de resistência a um conjunto de antimicrobianos de uso terapêutico (gentamicina, canamicina, trimetroprim-sulfametoxazol, tetraciclina e cloranfenicol), aos íons mercúrio e telurito, em Klebsiella pneumoniae, näo foram encontradas diferenças significativas decorrentes da origem dos pacientes, isto é, se internados ou se de atendimento ambulatorial. Nos dois grupos, o número de marcadores de resistência foi reduzido devido, provavelmente, ao uso restrito de antimicrobianos, no período prévio à avaliaçäo