Abstract Identification of nonfermenting Gram-negative bacteria (NFGNB) of cystic fibrosis patients is hard and misidentification could affect clinical outcome. This study aimed to propose a scheme using polymerase chain reaction to identify NFGNB. This scheme leads to reliable identification within 3 days in an economically viable manner when compared to other methods.

RESUMO: Salmonella Gallinarum (S. Gallinarum) e Salmonella Pullorum (S. Pullorum) são patógenos hospedeiro-específico de aves, agentes do tifo aviário e pulorose, respectivamente. Estes biovares causam infecções septicêmicas, resultando em alta mortalidade. Surtos são frequentemente relatados em diversos países, causando prejuízos devido à eliminação de lotes infectados e tratamentos. Agentes antimicrobianos são utilizados frequentemente em granjas avícolas para prevenir ou tratar infecções gastrointestinais. No presente trabalho, foi avaliada a susceptibilidade antimicrobiana de 50 isolados de S. Gallinarum e S. Pullorum, obtidos durante surtos que ocorreram entre 1987 a 1991 e entre 2006 a 2013. A comparação dos perfis de sensibilidade mostrou que todas as amostras são sensíveis aos β-lactâmicos. Todos os isolados de 1987-1991 foram sensíveis a todos os antibióticos testados, exceto NAL e CIP (78%). O perfil de susceptibilidade de S. Gallinarum (surtos de 2006 a 2013) foi NAL (58%), CIP (63%), ENR (67%), TET (92%), FFC (96%) e SXT (96%). Isolados de S. Pullorum (surtos de 2006 a 2013) apresentaram as seguintes taxas de sensibilidade: NAL (65%), CIP (71%), ENR (94%) e TET (94%). Todas as amostras foram sensíveis ao β-lactâmicos testados, no entanto, a resistência às quinolonas e fluoroquinolonas aumentou ao longo do tempo. Além disso, baixos níveis de resistência a outros antibióticos foram encontrados em isolados recentes, tais como as tetraciclinas.

ABSTRACT: Salmonella Gallinarum (S. Gallinarum) and Salmonella Pullorum (S. Pullorum) are poultry host-specific, agents of fowl typhoid and pullorum disease, respectively. These biovars cause septicemic infections, resulting in high mortality. Outbreaks are frequently reported worldwide, causing losses due to the elimination of infected flocks and treatments. The use of antimicrobial agents is frequent in poultry farms to prevent or treat gastrointestinal infections. In the present research it was evaluated the antimicrobial susceptibility of 50 S. Gallinarum and S. Pullorum isolates, from outbreaks that occurred between 1987 to 1991 and 2006 to 2013. The comparison of the susceptibility profiles showed that all isolates were susceptible to β-lactams. All isolates from 1987-1991 were susceptible to all antibiotics tested except NAL and CIP (78%). The susceptibility profile of S. Gallinarum (2006 - 2013 period) was the following NAL (58%), CIP (63%), ENR (67%), TET (92%), FFC (96%) and SXT (96%). S. Pullorum isolates (2006 - 2013 period) showed the following susceptibility rates to NAL (65%), CIP (71%), ENR (94%) and TET (94%). All isolates were susceptible to β-lactams tested, however, resistance to quinolones and fluoroquinolones increased over time. Furthermore, low levels of resistance to other antibiotics were found in recent isolates, such as tetracyclines.

This study evaluated the relationship between previous colonization of the oropharynx and development of ventilator-associated pneumonia through the classification of genomic fingerprint pattern by pulsed-field gel electrophoresis of both oxacillin-resistant and oxacillin-susceptible Staphylococcus aureus isolates obtained from hospitalized patients in an intensive care unit.

Background: The carrier state of methicillin-resistant Staphylococcus aureus is pointed as an infection predictor and a factor for environmental and person-to-person dissemination, including health service workers. These, when colonized are commonly associated to outbreaks. Objective: Analyze the prevalence of Staphylococcus aureus in saliva of workers at a university hospital. Methodology: Epidemiologic longitudinal study carried out in Curitiba, Paraná, Brazil, with 486 workers between April 2006 and June 2008. Three saliva samples were collected and a data collection instrument was applied. Staphylococcus aureus were isolated from the clinical specimen and characterized by phenotypes. The data from the instrument and the laboratory results were organized and processed with EPI-Info software and analyzed via descriptive statistics. Results: Among the healthcare workers studied, 60.9% were colonized by Staphylococcus aureus in saliva; of those, 67.9% were transitory carriers and 32.1% were persistent carriers; the prevalence of meticillin-resistant Staphylococcus aureus (MRSA) among the isolated cases was 15.7%. The average prevalence of MRSA was 12.7% and higher among nurses’ aides (21.4%) and cleaning aides (20.6%) and lower among nurses (4.5%) and doctors (5.9%). Conclusions: Healthcare workers presented high prevalence of Staphylococcus aureus in saliva, indicating the mouth as an important body site to investigate colonization by MRSA and a potential source to its dissemination.

Introdução: O estado de portador de Staphylococcus aureus resistente à meticilina é apontado como preditor de infecção e fator para a disseminação ambiental e de pessoa a pessoa, incluindo trabalhadores de serviço de saúde. Estes quando colonizados são freqüentemente associados a surtos. Objetivo: Analisar a prevalência de Staphylococcus aureus na saliva de trabalhadores de hospital universitário. Metodologia: Estudo epidemiológico longitudinal realizado em Curitiba, Paraná, Brasil, com 486 trabalhadores no período de abril de 2006 a junho de 2008 compreendeu a coleta de três amostras de saliva e aplicação de instrumento de coleta de dados. Staphylococcus aureus foram isolados dos espécimes clínicos e caracterizados fenotipicamente. Os dados foram organizados e processados no Programa EPI-Info e analisados por meio de estatística descritiva. Resultados:Entre os trabalhadores investigados, 60,9% estavam colonizados por Staphylococcus aureus na saliva, sendo 67.9% carreadores transitórios e 32.1% carreadores persistentes; a prevalência de Staphylococcus aureus resistente à meticilina (MRSA) entre os isolados foi de 15.7%. A prevalência média de MRSA foi de 12.7% sendo maior entre técnicos em enfermagem (21.4%) e auxiliares de limpeza (20.6%) e menor entre enfermeiros (4.5%) e médicos (5.9%). Conclusões: Os trabalhadores apresentaram alta prevalência de Staphylococcus aureus na saliva, indicando a boca como importante sítio corporal para a investigação da colonização por MRSA e potencial fonte para sua disseminação.

In this study we report the first isolation of VanA-type vancomycin-resistant Enterococcus faecalis strains from two different patients hospitalized in the same intensive care unit at the hospital of Universidade Federal do Triângulo Mineiro (UFTM), Uberaba, Minas Gerais, Brazil.

Three Enterococcus faecalis and one Enterococcus faecium strains were characterized by plasmid profile, pulsed-field gel electrophoresis (PFGE) and determination of antimicrobial minimal inhibitory concentrations. VanA elements were characterized by Long PCR, overlapping PCR and DNA sequencing. Enterococcal strains showed resistance to vancomycin and harbored the vanA gene, and three these were teicoplanin susceptible while one showed intermediate resistance to teicoplanin. Two E. faecalis strains showed indistinguishable PFGE profile while the third was unrelated. E. faecalis strains showed a deletion in the right terminal region of the Tn1546-like element. The E. faecium strain showed an insertion element in the vanXY intergenic region. Mutations in VanA elements were not found. Rearrangements in the VanA element could be responsible for incongruities in genotype and phenotype in these strains.

We report the first case of vancomycin-resistant Enterococcus faecalis with VanA phenotype and vanA genotype isolated from a surgical patient in a general Hospital in Uberlândia, state of Minas Gerais, Brazil.

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopetide resistance by conjugative transfer of VanA elements.

Foram estudados os fatores de virulência de trinta e duas amostras de Enterococcus faecalis, isolados de casos clínicos. Vinte e quatro amostras (75%) produziram hemolisina em ágar sangue preparado com hemácias de carneiro. No sobrenadante da cultura em BHI nenhuma amostra produziu hemolisina, no entanto quando cultivadas em meio BHI suplementado com 1% de glucose e 0,03% de L-arginina (BHI-GA), 100% das amostras lisaram hemácias de carneiro. Vinte e quatro (75%) amostras produziram caseinase e 23 (71,9%) lipase, mas nenhuma amostra produziu gelatinase. Dezesseis (46,9%) causaram arredondamento e alteração na membrana das células, com formação de vesículas e, em seguida, a morte das células HeLa e HEp-2. Treze amostras (40,6) aglutinaram eritrócitos de coelhos, mas não aglutinaram outros eritrócitos na presença ou na ausência de 1% de D-manose. Dezesseis (50%) aderiram em células HeLa e 13 (40,6%) em células HEp-2, mas todas as amostras de E. faecalis aderiram na microplaca de polipropileno, indicando que E. faecalis isolados de casos clínicos possuem capacidade de formar biofilme "in vitro". Todos os isolados mostraram-se resistentes à ação bactericida do soro normal e não produziram aerobactina. Esses resultados sugerem que, inicialmente, a colonização ou infecção por E. faecalis ocorre pela aderência e formação de biofilme nas células epiteliais e a produção de hemolisina, lípase e caseinase pode atuar como fatores de virulência na infecção por E. faecalis.

Thirty-two clinical isolates of Enterococcus faecalis were screened for virulence factors. Twenty-four (75%) isolates produced hemolysin on Mueller-Hinton blood agar plates with sheep erythrocytes. However, the cell free heat-stable hemolysin was detected in all isolates (100%) of E. faecalis when grown in BHI-GA (BHI medium supplemented with 1% glucose and 0.03% L-arginine), but not in BHI broth alone. Twenty-four isolates (75%) produced caseinase and 23 (71.9%) lipase, but none of the isolates produced gelatinase. Fifteen (46.9%) culture filtrates caused rounding and membrane alterations with blebbing formation followed by death in HeLa and HEp-2 cells, but not in Vero cells. Thirteen isolates (40.6%) agglutinated rabbit erythrocytes, but did not produce hemagglutination in other bloods, containing or not 1% D-manose. Sixteen (50%) E. faecalis isolates adhered to HeLa cells and thirteen (40.6%) to HEp-2 cells, but all isolates adhered to polypropylene microtiter plates, indicating that clinical E. faecalis possess the ability to form biofilm in vitro. All the isolates were resistant to the bactericidal action of normal serum and did not produce aerobactin. These findings suggest that adherence and consequently biofilm formation on ephitelial host cells are the first steps in the E. faecalis virulence and that hemolysin, lipase, caseinase and other virulence factors act as causative of human epithelial cell damages.

No Brasil, enterococos resistente à vancomicina (VRE) têm sido descritos como patógenos hospitalares, desde 1998. Durante um monitoramento de VRE em um hospital, foram detectadas duas cepas de Enterococcus faecalis com genótipo vanA, e sensibilidade à teicoplanina. Este é o primeiro relato do isolamento de enterococo fenótipo VanB e genótipo vanA de amostra clínica no Brasil.

In Brazil, vancomycin-resistant enterococci (VRE) have been reported as nosocomial pathogens since 1998. Recently, in a VRE surveillance in a hospital, we detected two Enterococcus faecalis isolates with vanA genotype and susceptible to teicoplanin. This is the first report of VanB phenotype-vanA genotype enterococci isolated from humans in Brazil.

Staphylococcus aureus e Estafilococos coagulase-negativa (ECN) estão entre os patógenos hospitalares mais importantes em pacientes de unidades de terapia intensiva neonatal, principalmente em infecções da corrente sanguínea. O principal objetivo deste estudo foi determinar a ocorrência de infecções hospitalares por estes microrganismos usando dois sistemas de vigilância (laboratorial e "National Nosocomial Infection Surveillance" - NNIS) e determinar os fatores de risco mais importantes durante o período de dois anos (2001-2002). Dois surtos por ambos S. aureus suscetível a meticilina (1.5%) e ECN resistente à meticilina (1.0%) foram observados, de janeiro a fevereiro/02 e agosto a setembro/02. Taxas de incidência endêmica de 3.77% e 5.16% para S.aureus e ECN foram detectadas respectivamente. Fatores de risco incluíram idade <=7 dias, hospitalização > ou = 7 dias e utilização de cateter vascular central (CVC) de polietileno através de dissecação de veia (flebotomia), mas, nenhum desses fatores independentes foram confirmados pela análise multivariada. Por outro lado, ECN resistente à oxacilina prevaleceu (66.0%) nos episódios epidêmicos. Análise molecular através de gel de eletroforese em campo pulsátil mostrou a natureza policlonal das amostras de S. aureus. Em conclusão, nós identificamos dois surtos de etiologia mista por S. aureus suscetível à meticilina e ECN resistente à meticilina associados à falta de material adequado (cateter vascular central) para neonatos, relacionados a procedimento invasivo. Os dois surtos foram controlados com a substituição de CVC de polietileno pelo cateter central de inserção periférica.

Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are among the most important nosocomial pathogens in patients from neonatal intensive care units, mainly in bloodstream infections. The main objective of this study is to determine the occurrence of nosocomial infections by these microorganisms using two surveillance systems (Laboratorial Surveillance and National Nosocomial Infection Surveillance System) and to determine the most important risk factors during a two-year period (2001-2002). Two outbreaks by both methicillin susceptible S. aureus (MSSA) (1.5%) and methicillin resistant CoNS (MRCoNS) (1.0%) were observed, from January to February/02 and August to September/02. Endemic incidence rates of 3.77% and 5.16% of S. aureus and CoNS, respectively were detected. Risk factors included age <=7 days, hospitalization > or = 7 days and utilization of polietilene central vascular catheter (CVC) through vein dissection (phlebotomy), but none of these independent factors were confirmed by the multivariate analysis. However, oxacillin resistant CoNS prevailed (66.0%) in the epidemic episodes. Molecular analysis by pulsed field gel electrophoresis showed the polyclonal nature of S. aureus isolates. In conclusion, two outbreaks were identified of mixed etiology by MSSA and MRCoNS associated to the lack of an adequate material (central venous catheter) for neonates, related invasive procedure. The outbreaks were controlled with the substitution of polietilene CVC for peripherally inserted central catheter.

We studied an outbreak of two multi-drug resistant clones of Acinetobacter baumannii in the Neonatal Intensive Care Unit of the Uberlândia Federal University Hospital in Minas Gerais state, Brazil, and we analyzed the contribution of cross-transmission in the rise in infection rates. Eleven neonates who developed multi-drug resistant A. baumannii nosocomial infection were matched to 22 neonates who were admitted to the same unit and did not develop an infection during the outbreak period, in order to identify risk factors for infection. Three out of the 11 neonates died. Epidemiological investigation included molecular typing, using pulsed field gel electrophoresis. Prior to the outbreak, from December 2001 to March 2002, no case of infection by this microorganism was diagnosed. Environmental and healthcare worker hand cultures were negative. Nine isolates had similar pulsed field gel electrophoresis patterns and two had another clone. The first clone was brought into the unit by an infected patient who was transferred from another hospital without a history of antibiotic use. The second clone did have its origin clearly defined. Both infected groups led us to conclude that several factors contributed to infection with A. baumannii. These factors were: exposure to antibiotics and invasive devices, birth weight < 1500g, age < 7 days and duration of hospitalization > 7 days. Based on logistic regression, infected neonates were more exposed to carbapenem and mechanical ventilation than the control group. Cross transmission between infants contributed to the rise in the rates of multi-drug resistant A. baumannii infection.

This study reports the results about antimicrobial resistance of Enterococcus spp. isolated from intestinal tract of patients from a university hospital in Brazil. The identification of strains at species level was performed by conventional biochemical tests, API 20 Strep (bioMérieux), and polymerase chain reaction assay. The specie distribution was E. faecium (34%), followed by E. faecalis (33%), E. gallinarum (23.7%), E. casseliflavus (5.2%), E. avium (1%), and E. hirae (1%). Intrinsic resistance to vancomycin characterized by presence of vanC genes was found in E. gallinarum and E. casseliflavus. The high prevalence of VanC phenotype enterococci is very important because these species have been reported as causing a wide variety of infections. Vancomycin-resistant E. faecium or E. faecalis were not found and no one isolate of these species was a beta-lactamase producer. Thirteen clinical isolates of enterococci (13.4%) showed multiresistance patterns, which were defined by resistance to three classes of antibiotics plus resistance to at least one aminoglycoside (gentamicin and/or streptomycin). The resistance to several antimicrobials shown by enterococcal strains obtained in this study is of concern because of the decrease in the therapeutic options for treatment of infections caused by enterococci.

O objetivo foi relatar a ocorrência de dois surtos subseqüentes na UTIN do HC-UFU, por amostras epidêmicas de P. aeruginosa e A. baumannii multirresistentes nos períodos de Mar - Set/01 e Out - Mar/02, respectivamente. O surto por P. aeruginosa incluiu sete neonatos com conjuntivite e três com bacteremia e um estudo caso-controle foi realizado no surto por A. baumannii com 11 e 22 neonatos respectivamente. Os isolados de A. baumannii foram resistentes a gentamicina e ciprofloxacina e os de P. aeruginosa a ampicilina/sulbactam além de gentamicina e ciprofloxacina. As culturas ambientais e das mãos dos profissionais de saúde foram negativas. O surto por P. aeruginosa resultou no aumento do uso de imipenem o que pode ter favorecido a emergência do surto por A. baumannii que embora suscetível a este beta-lactâmico apresentou uma multiresistência importante. A análise univariada relacionou os fatores de risco predisponentes para infecção por A. baumannii: peso < 1500g, idade < 7 dias, hospitalização > 7 dias e uso de carbapenema. O término dos surtos foi alcançado através medidas de controle de infecção e isolamento de coorte dos neonatos infectados, não sendo possível definir o reservatório e a via de transmissão.

The study documents the occurrence of two subsequent outbreaks in the NICU of HC-UFU, caused by epidemic strains of multiresistant Pseudomonas aeruginosa and Acinetobacter baumannii, that occurred between March/01 and September and between October/01 and March/02, respectively. The P. aeruginosa outbreak included seven neonates with conjunctivitis and three with bacteremia. A case-control study was conducted for the A. baumannii outbreak, with 11 and 22 neonates, respectively. The isolates of A. baumannii were resistant to gentamacin and ciprofloxacin. P. aeruginosa isolates were resistant to ampicillin/sulbactam gentamicin and ciprofloxacin. The hands of healthcare workers and environmental cultures were negative. The outbreak of P. aeruginosa resulted in the increase of use of imipenem, which could have favoured the emergence of a A. baumannii epidemic strain, despite of its susceptibility to this antibiotic. The risk factors for A. baumannii infection were: weight <1500g, age < 7 days, hospitalization > 7 days and use of carbapenems. Containment of the two outbreaks was achieved by introduction of strict hygiene measures and careful nursing care of the infected infants. The reservatory and the route of transmission were not found.

Nesse estudo, para detecção de S. aureus resistente à meticilina (MRSA), a amplificação do gene mecA baseada no multiplex PCR foi comparada com os testes de difusão com disco para 1 µg/mL de oxacilina, detecção da concentração inibitória mínima (CIM), meio de triagem com 4% de NaCl e 6 µg/mL de oxacilina. Na investigação de 24 isolados obtidos de sangue, o genótipo mecA foi detectado em apenas 16 (66,7%) dos isolados pelo multiplex PCR. A CIM apresentou valores variando de 0,19 a 512 µg/mL entre os isolados de MRSA. Os dados obtidos pelos testes de triagem e diluição em ágar apresentaram sensibilidades de 80,0% e 72,8% respectivamente, quando comparados com a presença do gene mecA (multiplex). Todos os isolados, incluindo os negativos, quando reavaliados com a técnica de PCR exclusivo para este gene, o resultado foi positivo. A produção de beta-lactamase foi positiva em 20/25 (80,0%) dos isolados. Cerca de ¼ dos pacientes evoluiu para óbito apesar da maioria (83,3%) ter sido tratada adequadamente. A identificação simultânea da bactéria e sua susceptibilidade aos antibióticos é necessária para a escolha de uma terapia adequada para casos de sepse estafilocócica, mas é importante considerar a sensibilidade, especificidade e validação dos kits disponíveis.

In this study, for detection of methicillin-resistant S. aureus (MRSA), a mecA multiplex PCR-based amplification was compared with the 1 µg oxacillin disk diffusion test, detection of minimal inhibitory concentration (MIC), and screening in agar with 4% NaCl and 6 µg/mL oxacillin. Among 24 isolates obtained from blood, mecA gene was detected in only 16 (66.7%) isolates by multiplex PCR. The MIC test showed a range of resistance to oxacillin from 0.19 to 512 µg/mL, among these isolates. Data obtained by screening and dilution tests showed that sensitivity to methicillin was 80.0% and 72.8%, respectively, when compared with the presence of mecA gene (multiplex). All isolates, including the negatives, when revaluated for mecA gene by PCR were positive. beta-lactamase production was positive for 20/25 isolates (80.0%). About ¼ of patients died dispite most of them (83.3%) were adequately treated. The simultaneous identification of the bacteria and determination of this susceptibility to antibiotics are necessary for the choice of empiric antibiotic therapy in suspected staphylococcal sepse, but is important to considering the sensibility, specificity and validation of the available kits.