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.

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.

ABSTRACT 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.

RESUMEN 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.

RESUMO 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.

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, 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 (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813 gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- 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 elegans and 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 TeR and pathogenic potential of C. diphtheriae.

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%) and Klebsiella pneumoniae 14/41 (34.1%). In 24/41 (58%), the ESBL genotype was confirmed. The most prevalent genes in samples that expressed ESBL were blaTEM 13/24 (54%), AmpC 12/24 (50%), blaSHV 6/24 (25%), CTX-M1 7/24 (29%), and Toho-1 6/24 (25%). Of these, 14/24 (58%) presented more than one genotype for the tested primers. In nine (37%) 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%). Fifty percent of the samples expressed AmpC were associated with other genes. Intermediate susceptibility to ertapenem was found in 2/41 (5%).

Tendo em vista a íntima relação entre os seres humanos e as lagoas costeiras (usadas para recreação, turismo, fornecimento de água, etc.), o lançamento de efluentes domésticos e hospitalares podem contribuir para o estabelecimento de rotas de disseminação de microorganismos patogênicos, incluindo aqueles carreadores de genes de resistência a antimicrobianos. O objetivo da presente investigação foi o de relacionar a presença de bactérias resistentes a antimicrobianos com características ambientais de três lagoas costeiras, comparando os resultados com os obtidos em esgoto hospitalar. Das lagoas estudadas, duas (Geribá e Imboassica) recebem efluentes domésticos e outra (Cabiúnas), se encontra em estado natural. Utilizando cultura contendo 32 µg/ml de Cefalotina, isolamos coliformes fecais (E. coli), coliformes não-fecais (Klebsiella, Enterobacter, Serratia e Citrobacter), bacilos gram-negativos não fermentadores de glicose e Aeromonas sp. Nas culturas de esgoto hospitalar foram isoladas cepas contendo marcadores de resistência para a maioria dos 11 antibióticos testados. No outro extremo, foram isoladas apenas cepas apresentando resistências a antibióticos freqüentemente observadas em situações não-seletivas (considerados aqui como marcadores "comuns") nas lagoas Cabiúnas e Imboassica. A capacidade de diluição do corpo aquático e a salinidade mostraram-se relacionados com a ocorrência de cepas multirresistentes, enquanto a intensidade de contaminação fecal recente não se mostrou associada ao número e tipos de marcadores detectados.

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 (Geribá and Imboassica) receive domestic sewage discharge, and the other (Cabiúnas) is still in a natural state. We isolated in a culture medium containing 32 ¼ µg/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 Cabiúnas 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.

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.

Nosocomial infections are a relevant factor in complicating the recovery of patients interned for even minor causes. In attempt to determine their origin it is crucial to consider that their origin is of an endogenous nature. Looking for an acessible expression of intestinal colonization we analyzed fecal samples from 3 separate groups of hospital patients collected after different lenghts of time. For practical reasons one group was studied prospectively and two other groups (patients hospitalized for up to 7 days and patients hospitalized for more than 7 days) were compared to one another. We looked for the emergence of tellurite resistance among Enterobacteriaceae using a selective medium. MacConkey potassium tellurite (MCPT). The frequence of prospectively studied patients with tellurite resistant strains was significantly greater after 7 days of hospitalization. For the two other groups, patients with more than 7 days of hospitalization showed a significant increase of bacterial species and of strains with new antimicrobial resistance markers. High molecular weigth plasmids were detected in some of these strains. These data show that the MCPT medium is a useful tool for the investigation of bowel colonization in hospitalized patients by drug-resistant Enterobacteriaceae.

Em 24 (82,7%) de 29 pacientes com infecção 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 associação entre a simultaneidade e a prévia cateterização 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 detecção 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 indicação 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).

Twenty-four (82.7%) out of 29 patients suffering from hospital acquired urinary infections by Klebsiella pneumoniae had the same species in their faeces. Biotyping of 24 urinary and 219 fecal strains of K. pneumoniae resulted in 50 different biotypes - an average of four biotypes per fecal sample. Ten patients (34.4%) had the same biotype in urine and faeces without any correlation with previous vesical catheterization (p>0.05). Using resistotyping to four chemical compounds selected among 34 tested substances (brilliant green, malachite green, potassium tellurite and mercuric chloride) 16 different resistotypes were found. Fourteen patients (58.3%) presented the same resistotype in urine and faeces but only in five patients was there correlation with simultaneous biotyping identity. Simultaneous occurrence of identical biotypes or resistotypes in faeces and urine occurred in only 54.2% of cases. However, there was a significant association between resistance ot mercuric and tellurite ions in fecal and urinary strains isolated from the same patient (p<0.001).

São relatadas observações acumuladas nos períodos pré-epidêmico e epidêmico (1973-1975) do atual surto de meningoencefalite meningocócica na área do Grande Rio, e realizados estudos bacteriológicos baseados em 1.000 casos suspeitos de meningoencefalite e submetidos à punção lombar, no Hospital Estadual São Sebastião. É proposto e discutido esquema simples e eficaz para processamento bacteriológico dos LCR suspeitos, a partir da colheita e pronta semeadura do material. É também discutida a real contribuição da bacterioscopia no diagnóstico presuntivo das meningoencefalites, definindo-se as limitações da técnica. Foi obtido elevado grau de isolamento de microorganismos, variando de 3%, para líquores entre zero e 10 células/mm' e 72%, para líquores acima de 1000 células/mm³. No decorrer do estudo, foram isoladas e caracterizadas 356 amostras bacterianas, assim discriminadas: N. meningitidis, 281; Haemophilus sp., 22; Enterobacteriaceae, 15; D. pneumoniae, 26; bastonetes gram negativos oxidattvos, 3; estreptococo beta hemolitico, 1 e enterococo, 1. As amostras de meningococos eram, em 15% dos casos, do grupo sorológico A, em 2% do grupo B e em 14%, do grupo sorológico C. Os testes de sensibilidade, em disco, aos agentes antimicrobianos principalmente utilizados na quimioprofilaxia e tratamento da doença meningocócica revelaram alto grau de sensibilidade das amostras ensaiadas a todos os agentes testados. A resistência à sulfadiazina sódica, em testes realizados segundo as normas preconizadas pela Food and Drug Administration (F.D.A.), revelaram elevado grau de resistência, particularmente dos meningococos do grupo C.

Data from the pre-epidemic period and from the present epidemic of meningococcal meningoencephalitis in the Great Rio de Janeiro area are presented. A thousand suspected cases were bacteriologically studied after lumbar spinal puncture at the Hospital Estadual São Sebastião, in Rio de Janeiro. Aiming at a rapid and simple diagnostic procedure to be applied to spinal fluids errors and hazards of some techniques, based on stained microscopic slides and limited choice of culture media, are discussed. Isolation of etiologic agents varied from three per cent in specimens with less than 10 white cells/mm³ to 72 per cent with fluids containing more than a thousand cells/mm³. The distribution of 356 isolated strains correspended to: Neisseria meningitidis (281); Haemophilus sp. (22); Enterobacteriaceae (15); pneumococci (26); Gram-negative oxidative rods (3); beta hemolytic streptococci (1) and enterococci (1). Serologically the meningococci corresponded to 51 per cent group A, 2 per cent group B and 14 per cent group C strains. The remaining strains were not grouped with the mentioned available diagnostic sera or demonstrated spontaneous agglutination. Very few cases of resistance to antibioties were found by using the diffusion sensitivity tests with discs of the common antimeningococcal drugs. Sulphadiazine resistence was frequent, however, particularly within the group C meningococcal isolations, when the dilution tests in solid media, according with the Food and Drug Administration (U.S.AJ instructions were employed.