Resistencia a la meticilina y producción de biopelícula en aislamientos clínicos de Staphylococcus aureus y Staphylococcus coagulasa negativa en México / Methicillin resistance and biofilm production in clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus in México

Resumen Introducción. Las infecciones por Staphylococcus aureus y Staphylococcus coagulasa negativa multirresistentes a los antibióticos y asociadas con la atención en salud tienen un gran impacto epidemiológico por su alta morbimortalidad; además, se han relacionado con la formación de biopelículas, lo cual también se asocia con la resistencia a los antimicrobianos. Objetivo. Determinar la resistencia a la meticilina y cuantificar la producción de biopelículas para establecer su posible relación con los aislamientos clínicos de S. aureus y Staphylococcus coagulasa negativa. Materiales y métodos. Se estudiaron 11 cepas de S. aureus y 12 de Staphylococcus coagulasa negativa. La resistencia a la meticilina se determinó con discos de cefoxitina tomando como valores de referencia los estándares del Clinical Laboratory Standards Institute (CLSI) de 2018. La producción de biopelícula se cuantificó con cristal violeta. Los genes mecA e icaADBC se identificaron mediante reacción en cadena de la polimerasa (PCR), y se hizo un análisis bivariado con la prueba de ji al cuadrado y el coeficiente V de Cramér, utilizando el programa SPSS™, versión 20.0. Resultados. Nueve cepas de S. aureus fueron resistentes a la meticilina (SARM) y dos fueron sensibles. Ocho cepas de Staphylococcus coagulasa negativa fueron resistentes y cuatro fueron sensibles. El genotipo mecA se encontró en ocho de las nueve cepas de S. aureus y en seis de las ocho de Staphylococcus coagulasa negativa resistentes a meticilina. Todas las cepas formaron biopelícula. Diez cepas de S. aureus y 11 de Staphylococcus coagulasa negativa presentaron el genotipo icaADCB. No se encontró asociación entre la resistencia a meticilina y la formación de biopelícula. Conclusiones. La cefoxitina es suficiente para determinar el fenotipo resistente a meticilina y se asoció con el genotipo mecA. Las cepas resistentes a la meticilina y poseedoras del gen mecA pueden presentar un mecanismo de resistencia alterno. Los dos grupos de cepas formadoras de biopelícula se relacionaron con la presencia del operón icaADCB. La formación de biopelícula y la resistencia a la meticilina se expresaron como características independientes en los dos grupos de cepas.

Abstract Introduction: Infections associated with health care caused by S. aureus and coagulase- negative Staphylococci multi-resistant to antibiotics cause a high epidemiological impact due to their high morbidity and mortality. Biofilm formation, which has been associated with antimicrobial resistance, can also occur. Objectives: To determine methicillin resistance and to quantify the biofilm production to establish if there is a relationship in clinical isolates of S. aureus and coagulase-negative Staphylococci. Material and methods: A total of 11 strains of S. aureus and 12 of coagulase-negative Staphylococci were studied. Methicillin resistance was determined with cefoxitin discs and the Clinical Laboratory Standards Institute (CSLI), 2018 reference values. Biofilm production was quantified by the crystal violet method. The mecA and icaADBC genes were identified by PCR. A bivariate analysis was performed with chi-square (c2) and Cramér's V statistical tests, using SPSS™, version 20.0 software. Results: Nine S. aureus strains were methicillin-resistant and two were sensitive. Eight coagulase-negative Staphylococci strains were resistant and four were sensitive. The mecA genotype was found in eight of the nine S. aureus resistant strains and six of eight resistant coagulase-negative Staphylococci. All strains formed biofilms. Ten strains of S. aureus and 11 of coagulase-negative Staphylococci presented the icaADCB genotype. No association was found between methicillin-resistance and biofilm formation. Conclusions: Cefoxitin is enough to define the resistance phenotype and is associated with the mecA genotype. All strains formed biofilms and were related to the presence of the icaADCB operon. Biofilm formation and methicillin resistance were independent features in both groups of strains.

Detecção de beta-lactamase por Staphylococcus coagulase-negativo isolados de queijo mussarela fatiados e fatiadores de frios / Beta-lactamase detection in coagulase-negative Staphylococci isolated from sliced mozzarella cheese and cold-cut slicers

A resistência de bactérias a antimicrobianos é considerada um problema de saúde pública, sendo a resistência aos beta-lactâmicos uma das mais importantes. O objetivo do presente estudo foi detectar a produção da enzima β-lactamase por isolados de Staphylococcus coagulase-negativo (SCN), provenientes de queijos Mussarela fatiados e equipamentos de fatiamento de frios. Os testes foram realizados utilizando discos impregnados com cefalosporina cromógena para detecção da β-lactamase. Dos 103 isolados de Staphylococcus spp. analisados, 55 (53%) produziram β-lactamase e 48 (47%) não produziram. Portanto, é possível inferir que SCN isolados neste estudo, podem inativar antimicrobianos β-lactâmicos e assim, exercer influência negativa na saúde pública, devido ao potencial em transferir genes de resistência antimicrobiana para outras bactérias.

Matrix-assisted laser desorption ionization-time of flight: a promising alternative method of identifying the major coagulase-negative Staphylococci species

Abstract INTRODUCTION. This study aimed to evaluate different methods for differentiation of species of coagulase-negative staphylococci (CoNS) that caused infections in hospitalized immunocompromised patients. METHODS. A total of 134 CoNS strains were characterized using four different methods. RESULTS. The results of matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analysis were in complete agreement with those of tuf gene sequencing (kappa index = 1.00). The kappa index of Vitek 2® Compact analysis was 0.85 (very good) and that of the conventional method was 0.63 (moderate). CONCLUSIONS . MALDI-TOF MS provided rapid and accurate results for the identification of CoNS (134; 100%).

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity

Abstract: INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Tiempo de crecimiento bacteriano en hemocultivos en neonatos / Time bacterial growth in blood cultures in neonates Abstract

Introducción: La sepsis es causa importante de morbimortalidad neonatal. Objetivos: Detectar el tiempo en que la curva de crecimiento bacteriano es evidenciada en la muestra de sangre inoculada en los hemocultivos y comparar estos tiempos de crecimiento bacteriano entre bacterias gramnegativas y grampositivas, entre los tipos de sepsis neonatal y determinar las bacterias más frecuentemente aisladas entre neonatos prematuros y de término. Pacientes y método: Estudio descriptivo de recién nacidos en riesgo de sepsis o con sospecha de sepsis por manifestaciones clínicas o de laboratorio, en que se evaluaron 114 hemocultivos positivos entre 1.932 hemocultivos tomados entre mayo de 2010 y mayo de 2014. Los datos se analizaron con Stata® 11.0. Resultados: El 5,9% de los hemocultivos tuvieron crecimiento bacteriano. La mediana y rango intercuartílico de tiempos de crecimiento bacteriano para gramnegativos fue 11 h (10-13 h), para grampositivos diferentes a Staphylococcus coagulasa negativo (SCoN) 12 h (12-18 h) y para SCoN 42h (36-44h). El 95,8% de las bacterias grampositivas y el 96% de las gramnegativas tuvieron tiempos de crecimiento bacteriano ≤ 24 h de incubación, mientras que en los SCoN el 100% de los hemocultivos fue positivo en ≤ 62 h de incubación. Conclusión: El 100% de sepsis por bacterias gramnegativas, grampositivas no SCoN y 90% de las ocasionadas por SCoN, son identificadas en los hemocultivos en las primeras 48 h, por lo cual podemos concluir que para descartar una sepsis, un período de incubación en hemocultivos de 48 h es suficiente.

Introduction: Sepsis is a major cause of neonatal morbidity and mortality. Objectives: To detect the time when the bacterial growth curve is evidenced in the blood sample inoculated blood cultures and comparing the times of bacterial growth between Gram negative and Gram positive bacteria, among the types of neonatal sepsis and identifying microorganisms more often isolated from preterm and term. Patients and method: A descriptive study. 114 positive blood cultures from 1,932 blood cultures taken from 01-May-2010 and 31-May-2014 were evaluated. Data were analyzed with Stata® 11.0. Results: 5.9% of blood cultures had bacterial growth. The median and interquartile range of Gram negative times of bacterial growth was 11 h (10-13 h), for Gram positive coagulase-negative Staphylococcus different (CoNS) 12h (12-18h) and CoNS 42h (36-44h). 95.8% of Gram positive and 96% of Gram negative, were the times of bacterial growth ≤ 24 h incubation, whereas the 100% CoNS was positive ≤ 62 h of incubation. Conclusion: 100% of sepsis by Gram negative and Gram positive no CoNS and 90% of those caused by CoNS are identified in blood cultures in 48 h, so we can conclude that to rule out sepsis, an incubation period of 48 h in blood cultures is sufficient.

Eventos estressores na família e indicativos de problemas de saúde mental em crianças com idade escolar / Stressor events in the family environment that are indicative of mental health problems in children of school age

Resumo O objetivo deste artigo é avaliar a relação entre eventos estressores ocorridos no último ano na família de crianças e adolescentes com indicativos de problemas de saúde mental em uma amostra de estudantes de duas escolas de uma cidade no sul do Brasil. Estudo transversal com 1.075 estudantes matriculados em duas escolas públicas de ensino fundamental (uma estadual e outra municipal). Foi utilizado o Strengths and Difficulties Questionnaire para avaliação de fatores emocionais e comportamentais da criança, e a Escala de Avaliação de Reajustamento Social de Holmes e Rahe (1967) para avaliar os eventos estressores. Foram utilizados o teste qui-quadrado e a regressão de Poisson, com ajuste robusto para variância, expressando os resultados em razão de prevalências (RP) e intervalos de confiança de 95%. As chances de apresentar problemas de hiperatividade foram 1,42 (IC 95% 1,10-1,83) vezes maiores no tercil intermediário e 1,37 (IC 95% 1,06-1,78) no tercil superior, quando comparados ao tercil inferior. Quanto aos problemas de relacionamento, as chances foram de 1,49 (IC 95% 1,15-1,93) vezes maiores no tercil superior ao serem comparados com o tercil inferior. Os resultados sugerem que fatores ambientais podem ser fortemente relacionados à etiologia dos transtornos mentais na infância e adolescência.

Abstract The scope of this article is to evaluate the relationship between stressor events that occurred last year in the family of children and adolescents that are indicative of mental health problems in a sample of students from two schools in a city in southern Brazil. It involved a cross-sectional study with 1,075 students enrolled in two public elementary schools. The Strengths and Difficulties Questionnaire was used to assess emotional and behavioral factors of the child and the Social Readjustment Rating Scale (SRRS) of Holmes and Rahe (1967) to assess stressor events. The chi-square and Poisson regression test with robust variance adjustment for expressing the results in the prevalence ratio (PR) and confidence intervals of 95% were used. The chances of presenting problems of hyperactivity were 1.42 (95% CI 1.10 to 1.83) times higher in the intermediate tercile and 1.37 (95% CI 1.06-1.78) in the higher tercile compared with the lower tercile. With respect to relationship problems the chances were of 1.49 (95% CI 1.15 to 1.93) times higher in the higher tercile when compared with the lower tercile. The results suggest that environmental factors may be strongly related to the etiology of mental disorders in childhood and adolescence.

Utilization of coconut oil mill waste as a substrate for optimized lipase production, oil biodegradation and enzyme purification studies in Staphylococcus pasteuri

Background Oil and grease laden wastewaters pose hindrance to the treatment units and further threaten the receiving water bodies. Lipase-producing microbial strains are increasingly being exploited for the remediation of such effluents. Results When bacterial strains isolated from oil mill effluent were screened for their lipolytic activity, two isolates, COM-4A and COM-6B showed significant extracellular lipase activity. They were identified to be Staphylococcus pasteuri and Bacillus subtilis, respectively. S. pasteuri COM-4A was cultivated in nutrient media based on coconut oil mill waste (CMW), in which it showed good growth at concentrations up to 20 g/L. While growing in such media, it was capable of producing lipase and other important extracellular hydrolytic enzymes. Furthermore, the isolate was able to effectively biodegrade the CMW supplemented in the medium. Applying the Box Behnken Design of Response Surface Methodology, lead to a 1.4-fold increase in both lipase production and oil removal by the isolate. The lipase was purified 9.02-fold and the molecular weight of the monomeric enzyme was deduced to be around 56 kDa. Characterization of the enzyme revealed it to be alkaliphilic and moderately thermophilic in nature, with pH and temperature optima of 9.0 and 50°C, respectively. The enzyme was also quite stable in the presence of water-miscible organic solvents. Conclusion Hence, the COM-4A lipase could be considered to be suitable for a variety of industrial applications such as in detergent formulations and in biodiesel production as well, apart from the possibility of applying it for bioremediation of fat and oil contaminants.

Blood Culture Proven Early Onset Sepsis and Late Onset Sepsis in Very-Low-Birth-Weight Infants in Korea

Neonatal sepsis remains one of the most important causes of death and co-morbidity in very-low-birth-weight (VLBW) infants. The aim of this study was to determine the current incidences of early-onset sepsis (EOS) and late-onset sepsis (LOS), the distribution of pathogens, and the impact of infection on co-morbidities in VLBW infants. We analyzed the data including sepsis episode from 2,386 VLBW infants enrolled in Korean Neonatal Network from January 2013 to June 2014. We defined EOS as a positive blood culture occurring between birth and 7 days of life and LOS after 7 days of life. Sepsis was found in 21.1% of VLBW infants. The risk of sepsis was inversely related to birth weight and gestational age. EOS was found in only 3.6% of VLBW infants, however the mortality rate was as high as 34.1%. EOS was associated with the increased odds for bronchopulmonary dysplasia and intraventricular hemorrhage. The vast majority of EOS was caused by Gram-positive organisms, particularly coagulase-negative staphylococci (30.6%). LOS developed in 19.4% of VLBW infants with a 16.1% mortality rate. Pathogens in LOS were dominated by coagulase-negative staphylococci (38.3%). Twenty-five percent and fifty percent of first LOS episode occurred after 12 days and 20 days from birth, respectively. Younger and smaller VLBW infants showed the earlier occurrence day for the 25% of first LOS episode. This study provides a recent nationwide epidemiology of sepsis in VLBW infants in Korea. Based on this study, successful strategies to reduce infections would improve survival and reduce morbidity.

Evaluation of fuur different dna extraction metods in coagulase-negative staphhylococci clinical isolates / Avaliacao de quatro metodos diferentes de extracao de DNA em isolados clinicos de estafilococos coagulase negativos (SCoN)

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

Atualmente, para extrair o DNA bacteriano, existem diversos métodos baseados em diferentes princípios. Entretanto, a quantidade e qualidade do DNA obtido por cada um destes métodos é variável e depende do tipo de micro-organismo em questão; os estafilococos coagulase-negativos (CoNS), por exemplo, possuem parede celular espessa difícil de lisar. O objetivo deste estudo foi comparar a quantidade e a qualidade do DNA extraído de isolados clínicos de CoNS utilizando quatro metodologias diferentes: dois protocolos caseiros e dois kits comerciais. A determinação da quantidade e da qualidade do DNA foi realizada por espectrofotometria. O DNA extraído também foi analisado em eletroforese em gel de agarose e por PCR. A concentração média de DNA foi mais alta no método de lise térmica (). Entretanto, com relação à qualidade do DNA, o kit comercial que utiliza um método de extração baseado em uma coluna de separação apresentou melhor resultado (média da relação A260/280 = 1,95). As quatro técnicas testadas forneceram DNA passível de amplificação por PCR, porém com diferentes rendimentos. A qualidade do DNA extraído de bactérias é importante, pois possibilita a realização de maior número de técnicas de biologia molecular e também armazenamento do material por maior período de tempo. Neste sentido, a técnica de extração por coluna de separação apresentou melhor desempenho frente aos CoNS.

Methicillin-resistant coagulase-negative staphylococci from healthy dogs in Nsukka, Nigeria

The occurrence, resistance phenotype and molecular mechanisms of resistance of methicillin-resistant staphylococci from groin swabs of 109 clinically healthy dogs in Nsukka, Nigeria were investigated. The groin swab samples were cultured on mannitol salt agar supplemented with 10 µgof cloxacillin. Sixteen methicillin-resistant coagulase negative staphylococci (MRCoNS), all harbouring the mecA gene were isolated from 14 (12.8%) of the 109 dogs studied. The MRCoNS isolated were: S. sciuri subspecies rodentium, S. lentus, S. haemolyticus, and S. simulans with S. sciuri subspecies rodentium (62.5%) being the predominant species. Thirteen (81.3%) of the MRCoNS were resistant to tetracycline while 12 (75%) and 10 (62.5%) were resistant to kanamycin and trimthoprim-sulphamethoxazole respectively. None of the isolates was resistant to fusidic acid, linezolid and vancomycin. Thirteen (81.3%) of the MRCoNS were multi-drug resistance (MDR). Other antimicrobial genes detected were: blaZ, tet(K), tet(M), tet(L), erm(B), lnu(A), aacA-aphD, aphA3, str, dfr(G), cat pC221,and cat pC223. Methicillin-resistant staphylococci are common colonizers of healthy dogs in Nigeria with a major species detected being S. sciuri subsp. rodentium.

Staphylococcus coagulasa-negativa clínicamente significativos: Especies más frecuentes y factores de virulencia / Clinically significant coagulase-negative staphylococci: Most frequent species and virulence factors

Coagulase-negative staphylococci have emerged as responsible for a large number of infections. However, it is often difficult to assess its pathogenic role or to discard it as a contaminant. Aim: The goal of this study was to identify clinically significant coagulase-negative staphylococci to the species level and their virulence factors. Isolates came from patients consulting at the San Roque Laboratory from 2009 to 2011. Material and Methods: Species identification was performed by De Paulis et al simplified method. Production of biofilm, hemolysins, lipases, lecithinases and DNase were determined by conventional methods; methicillin-resistance by diffusion method and mecA and Panton-Valentine genes, by multiplex PCR. Results: Out of 64 isolates, 40.6% were S. epidermidis; 20.3%, S. haemolyticus, and 15.6%, S. lugdunensis. Biofilm production was detected in 73.1% of S. epidermidis, 53.8% of S. haemolyticus and 40% of S. lugdunensis. mecA gene was identified in 69.2% of S. epidermidis, 92.3% of S. haemolyticus and none of S. lugdunensis. 83% of mecA (+) S. epidermidis isolates were biofilm producers as compared to 50% of the mecA (-). Conclusion: The frequency of S. lugdunensis, the most virulent coagulase-negative staphylococci species, was relatively high. The main virulence factor in S. epidermidis was biofilm production, being higher in those resistant to methicillin.

Staphylococcus coagulasa-negativa ha emergido como responsable de un gran número de infecciones. No obstante, con frecuencia es difícil asegurar su rol patógeno o descartarlo como contaminante. Objetivo: Estudiar a nivel de especies Staphylococcus coagulasa-negativa clínicamente significativos y sus factores de virulencia, de aislados provenientes de pacientes del Laboratorio San Roque de Asunción, Paraguay entre los años 2009 y 2011. Material y Métodos: Para la identificación de especies fue utilizado el método simplificado de De Paulis y cols. La producción de biopelícula, hemolisinas, lipasas, lecitinasas, AD-Nasa, fue determinada por métodos convencionales; la resistencia a meticilina por difusión y los genes mecA y Panton-Valentine por RPC múltiple. Resultados: De 64 aislados, 40,6% correspondió a S. epidermidis, 20,3% S. haemolyticus y 15,6% S. lugdunensis. La producción de biopelícula fue detectada en S. epidermidis en 73,1%, S. haemolyticus 53,8% y S. lugdunensis 40%. El gen mecA fue identificado en 69,2% de S. epidermidis, 92,3% de S. haemolyticus y en ninguno de S. lugdunensis. El 83% de S. epidermidis mecA (+) fue productor de biopelícula en comparación a 50% de los mecA (-). Conclusión: La frecuencia de S. lugdunensis, una de las especies más virulentas de Staphylococcus coagulasa-negativa fue relativamente alta; y el principal factor de virulencia en S. epidermidis fue la producción de biopelícula, siendo mayor en los resistentes a meticilina.

Infecções relacionadas à assistência à saúde por Staphylococcus coagulase negativa em unidade de terapia intensiva neonatal / Healthcare associated infections caused by coagulase-negative Staphylococci in a neonatal intensive care unit

OBJETIVO: Avaliar as infecções relacionadas à assistência à saúde, em unidade de terapia intensiva neonatal, causadas pelo Staphylococcus coagulase negativa, verificando o perfil de sensibilidade antimicrobiana e possíveis esquemas antibióticos eficazes. MÉTODOS: Estudo descritivo retrospectivo de uma série de casos de infecções relacionadas à assistência à saúde tardias de origem hospitalar atribuída ao Staphylococcus coagulase negativa, avaliando o perfil de sensibilidade antimicrobiana. Foram estudados os recém-nascidos internados entre 1º de janeiro de 2010 a 30 de junho de 2012 em uma unidade de terapia intensiva neonatal da cidade do Rio de Janeiro, sendo todos os pacientes oriundos de outras unidades. RESULTADOS: Foram admitidos 765 pacientes, totalizando 3.051 pacientes-dia e uma densidade de incidência de infecção geral de 18,9 por 1.000 pacientes-dia. A taxa de utilização de cateteres venosos centrais foi de 71,6% e a positividade das culturas de todos os sítios para todas as infecções relacionadas à assistência à saúde foi de 68,4%. O Staphylococcus coagulase negativa foi implicado em 11 (19,2%) das 57 infecções relacionadas à assistência à saúde e Klebsiela pneumoniae produtor de betalactamase de espectro estendido e Candida sp em 5 ocasiões cada. Das 11 infecções, 10 (90,9%) foram atribuídas a infecções primárias de corrente sanguínea. A sensibilidade dos isolados de Staphylococcus coagulase negativa em relação à vancomicina, clindamicina, ciprofloxacin, oxacilina e gentamicina foi de 100%, 81,8%, 72,7%, 27,2%, 22,2%, respectivamente. Não houve óbito atribuído diretamente à infecção por Staphylococcus coagulase negativa. CONCLUSÃO: O Staphylococcus ...

OBJECTIVE: This study sought to evaluate infections related to health care caused by coagulase-negative Staphylococci in a neonatal intensive care unit by assessing antimicrobial susceptibility profiles and potentially effective antibiotic regimens. METHODS: This was a retrospective descriptive study performed on a case series of healthcare-associated infections, and the antimicrobial susceptibility profiles were evaluated. Newborns from other hospitals who were admitted to a neonatal intensive care unit in Rio de Janeiro between January 1, 2010, and June 30, 2012, were studied. RESULTS: In total, 765 patients were admitted, totaling 3,051 patient-days, and the incidence density of general infection was 18.9 per 1,000 patient-days. The rate of central venous catheter use was 71.6%, and the positive culture rate for all sites and all infections related to health care were 68.4%. Coagulase-negative Staphylococci were identified in 11 (19.2%) of 57 health care-related infections, and infections with extended-spectrum beta-lactamase producing Klebsiella pneumoniae and Candida sp. constituted 5 cases each. Of the 11 cases of coagulase-negative Staphylococci, 10 (90.9%) were primary bloodstream infections. The sensitivity of the coagulase-negative Staphylococci isolates to vancomycin, clindamycin, ciprofloxacin, oxacillin and gentamycin was 100%, 81.8%, 72.7%, 27.2% and 22.2%, respectively. There were no deaths directly attributed to coagulase-negative Staphylococci infection. CONCLUSION: Coagulase-negative Staphylococci was the main agent identified in healthcare-associated infections, with low rates of infections related to central venous catheter. In hospitals with a high oxacillin resistance profile, similar to those included in this study, vancomycin may be used as an initial therapy, although clindamycin represents a viable alternative. .

Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding / Prevalência de genes codificadores de enterotoxinas e resistência antimicrobiana em estafilococos coagulase-negativo e coagulase-positivo isolados de morcilhas no sul do Brasil

INTRODUCTION: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. METHODS: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. RESULTS: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). CONCLUSIONS: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

INTRODUÇÃO: Estafilococos são patógenos responsáveis por surtos de doenças transmitidas por alimentos. O estudo investigou a prevalência de genes de enterotoxinas e o perfil de resistência aos antimicrobianos em estafilococos coagulase-negativo (CoNS) e estafilococos coagulase-positivo (CoPS) isolados de morcilhas no sul do Brasil. MÉTODOS: Duzentas colônias típicas e atípicas do ágar Baird-Parker foram inoculadas em ágar sal-manitol. Oitenta e dois estafilococos manitol-positivos foram submetidos a testes bioquímicos e perfil de susceptibilidade antimicrobiana. A presença dos genes da coagulase (coa) e enterotoxinas (se) foi investigada por reação em cadeia da polimerase (PCR). RESULTADOS: Os isolados foram divididos em dois grupos: 75,6% (62/82) CoNS e 24,4% (20/82) CoPS. Através dos testes bioquímicos, 9 espécies foram determinadas, Staphylococcus saprophyticus (37,8%) e Staphylococcus carnosus (15,9%) foram as mais prevalentes. Testes de susceptibilidade demostraram fenótipos de resistência aos antibióticos administrados em humanos, como gentamicina, tetraciclina, cloranfenicol e eritromicina. O gene coa foi detectado em 19,5% (16/82) das cepas e quatro fragmentos de DNA polimórficos foram observados. Cinco CoNS contendo o gene coa foram submetidos ao sequenciamento do 16S rRNA e três mostraram similaridade com CoNS. Quarenta amostras foram positivas para pelo menos um gene se, os mais frequentes foram sea (28,6%) e seb (27,5%). CONCLUSÕES: A presença de resistência aos antimicrobianos e de genes se nos isolados de morcilha indicou que este alimento pode representar um risco potencial à saúde, já que a presença nos alimentos pode causar doenças de origem alimentar ou ser uma possível rota de transferência de estafilococos resistentes aos humanos.

Conventional and molecular characterization of coagulase-negative staphylococcus in hospital isolates.

Background: During the last decade, coagulase-negative staphylococci (CoNS) have emerged as a major cause of nosocomial infections. They constitute a major component of the normal skin and mucosal microflora, and are particularly responsible for catheter- and medical device-related sepsis. They present unique problems in diagnosis and treatment of infections. Purpose: The present study has been designed to evaluate phenotypic and genotypic characteristics of CoNS among nosocomial isolates. Setting and Design: This study was carried out in a tertiary care hospital. Data from 150 samples collected from 73 hospitalized patients and 15 healthy volunteers between October 2003 and May 2005 were analyzed. Patients and Methods: A total of 100 CoNS strains responsible for sepsis or implant-associated infections and 50 saprophytic strains were studied. Invasive CoNS strains were selected on the basis of different colony morphologies, drug resistance patterns, and biofilm formation. The same criteria were used to select saprophytic isolates. Multiplex PCR was used to explore the ica, mecA, and atlE genes, which might contribute to the pathogenicity of CoNS and the formation of biofilms. Results: Most of the invasive strains that formed the biofilm were resistant to multiple antibiotics, with more than 80% resistant to methicillin. ica and mecA genes were detected significantly in pathogenic strains (chi-square test, P<0.0001) whereas atlE was ubiquitously amplified in all the strains. All those strains which had ica and mecA genes were resistant to multiple antibiotics and were positive for biofilm formation. Conclusion: These genetic markers thus appear to discriminate between potential invasive virulent and saprophytic strains of CoNS.

Bacteremias por Staphylococcus coagulase negativos oxacilina resistentes em um hospital escola na cidade de Santa Maria, Estado do Rio Grande do Sul / Oxacillin-resistant coagulase-negative Staphylococci bacteremia at a teaching hospital in Santa Maria, State of Rio Grande do Sul, Brazil

INTRODUÇÃO: Neste estudo, objetivou-se caracterizar a prevalência e o perfil de suscetibilidade de cepas de Staphylococcus coagulase negatives resistentes à oxacilina isoladas de culturas de sangue, em um hospital escola, localizado na Cidade de Santa Maria. Além disso, buscou-se comparar ao teste genotípico de referência, diferentes metodologias fenotípicas para a caracterização da resistência mediada pelo gene mecA. MÉTODOS: Após identificação (MicroScan® - Siemens), os isolados foram submetidos a testes de sensibilidade antimicrobiana a partir da difusão do disco e automação (MicroScan® - Siemens). A presença do gene mecA foi evidenciada através da técnica molecular de reação em cadeia da polimerase. RESULTADOS: A espécie prevalente foi Staphylococcus epidermidis (67 por cento). O gene mecA foi detectado em 90 por cento das cepas e conforme análise dos perfis de sensibilidade, observou-se um índice elevado de resistência a várias classes de antimicrobianos. Contudo, todos os isolados mostraram-se uniformemente sensíveis à vancomicina e tigeciclina. O disco de cefoxitina foi a metodologia fenotípica que melhor correlacionou-se com o padrão ouro. CONCLUSÕES: A análise da significância clínica de SCN isolados de hemoculturas e a detecção precisa da resistência à oxacilina representam fatores decisivos para a instituição correta da antibioticoterapia. Apesar da vancomicina constituir o tratamento usual na maioria dos hospitais brasileiros, tem a redução de seu emprego recomendada.

INTRODUCTION: This study aimed to characterize the prevalence and susceptibility profile to oxacillin-resistant Coagulase-negative Staphylococci strains isolated from blood cultures in a teaching hospital, located in Santa Maria, RS. In addition, different methodologies for phenotypic characterization of mecA-mediated oxacillin resistance were compared with genotypic reference testing. METHODS: After identification (MicroScan® - Siemens), the isolates were tested for antimicrobial sensitivity using disk diffusion and automation (MicroScan® - Siemens). The presence of mecA gene was identified by the polymerase chain reaction molecular technique. RESULTS: The most common species was Staphylococcus epidermidis (n=40, 67 percent). The mecA gene was detected in 54 (90 percent) strains, while analysis of the sensitivity profiles revealed a high rate of resistance to multiple classes of antimicrobial drugs. However, all isolates were uniformly sensitive to vancomycin and tigecycline. The cefoxitin disk was the phenotypic method that best correlated with the gold standard. CONCLUSIONS: Analysis of the clinical significance of CoNS isolated from hemocultures and the precise detection of oxacillin resistance represent decisive factors for the correct choice of antibiotic therapy. Although vancomycin constitutes the normal treatment in most Brazilian hospitals, reduction in its use is recommended.

Distribution of erm genes and low prevalence of inducible resistance to clindamycin among staphylococci isolates

INTRODUCTION: Resistance to macrolides, lincosamides and streptogramins B (MLS B antibiotics) in staphylococci may be due to modification in ribosomal target methylase encoded by erm genes. The expression of MLS B resistance lead to three phenotypes, namely constitutive resistance (cMLS B), inducible resistance (iMLS B), and resistance only to macrolides and streptogramins B (MS B). The iMLS B resistance is the most difficult to detect in the clinical laboratory. OBJECTIVE: This study investigated the expression of MLS B resistance and the prevalence of the erm genes among 152 clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus (CNS) from Hospital de Clínicas de Porto Alegre. METHODS: Primary MLS B resistance was detected by the disk diffusion method. Isolates with iMLS B phenotype were tested by double-disk induction method. All isolates were tested by a genotypic assay, PCR with specific primers. RESULTS: A total of 46.7 percent of staphylococci were positive for cMLS B; 3.3 percent for iMLS B and 3.3 percent for MS B. One or more erm genes were present in 50.1 percent of isolates. The gene ermA was detected in 49 isolates, ermC in 29 and ermB in 3. CONCLUSION: The prevalence of the ermA, ermB and ermC genes were 29.6 percent, 17.1 percent and 0.66 percent respectively, and constitutive resistance was the most frequent as compared to the other two phenotypes.

Thermostable lipase from a newly isolated staphylococcus xylosus strain; process optimization and characterization using RSM and ANN

Normal feed forward back-propagation artificial neural network (ANN) and cubic backward elimination response surface methodology (RSM) were used to build a predictive model of the combined effects and optimization of culture parameters for the lipase production of a newly isolated staphylococcus xylosus. The results demonstrated a high predictive accuracy of artificial neural network compared to response surface methodology. The optimum operating condition obtained from the ANN model was found to be at 30ºC incubation temperature, pH 7.5, 60 hrs incubation period, 1.8 percent inoculum size and 60 rpm agitation. The lipase production increased 3.5 fold for optimal medium. The produced enzyme was characterized biochemically and this is the first report about a mesophilic staphylococci bacterium with a high thermostable lipase which is able to retain 50 percent of its activity at 70ºC after 90 min and at 60ºC after 120 min. This lipase is also acidic and alkaline resistant which remains active after 24 hrs in a broad range of pH (4-11).

Detecção do gene mecA em estafilococos coagulase negativa resistentes à oxacilina isolados da saliva de profissionais da enfermagem / Detection of mecA gene in oxacillin-resistant coagulase-negative staphylococci isolated from the saliva of nursing professionals

Estafilococos coagulase negativa estão frequentemente associados às infecções nosocomiais e os profissionais da saúde podem ser reservatório e dissemina-los no hospital e comunidade. O objetivo desse estudo foi identificar espécies de estafilococos coagulase negativa isolados da saliva de profissionais da enfermagem, determinar o perfil de resistência e detectar o gene mecA. Foram selecionados 100 estafilococos coagulase negativa, sendo 41 identificados como Staphylococcus epidermidis, 25 Staphylococcus saprophyticus, 18 Staphylococcus haemolyticus, 8 Staphylococcus cohnii, 4 Staphylococcus lugdunenses, 3 Staphylococcus capitis, e 1 Staphylococcus Simulans. Desses, 32 por cento apresentaram resistência à oxacilina, 84,4 por cento à mupirocina, 32 por cento à cefoxitina, e todos sensíveis a vancomicina. Dos estafilococos coagulase negativa resistentes à oxacilina, 93,7 por cento desenvolveram-se no agar oxacilina (6µg/ml) e o gene mecA foi detectado em 75 por cento. Os resultados sinalizam que maiores investimentos devem ser direcionados a identificação das espécies de estafilococos coagulase negativa nas instituições de saúde e na comunidade.

Coagulase-negative staphylococci are frequently associated with nosocomial infections, and healthcare professionals can be reservoirs and spread them in hospitals and in the community. The aim of this study was to identify species of coagulase-negative staphylococci isolated from the saliva of nursing professionals, determine the resistance profile and detect the mecA gene. One hundred coagulase-negative staphylococci were selected: 41 were identified as Staphylococcus epidermidis, 25 as Staphylococcus saprophyticus, 18 as Staphylococcus haemolyticus, eight as Staphylococcus cohnii, four as Staphylococcus lugdunenses, three as Staphylococcus capitis and one as Staphylococcus simulans. Of these, 32 percent presented oxacillin resistance, 84.4 percent mupirocin resistance and 32 percent cefoxitin resistance, and all were vancomycin sensitive. Among the oxacillin-resistant coagulase-negative staphylococci, 93.7 percent developed in oxacillin agar (6µg/ml) and the mecA gene was detected in 75 percent. The results indicate that higher investments should be directed towards identifying coagulase-negative staphylococcus species in healthcare institutions and in the community.

Identification and detection of methicillin resistance in Non-Epidermidis coagulase-negative staphylococci

The NCCLS (2004) presented a new methodology to detect, by disk-diffusion agar, oxacillin-resistance using a cefoxitin disk. We identified coagulase-negative staphylococci (SCoN) to the species level and compared the use of cefoxitin disks (30 µg) with oxacillin disks (1 µg), agar dilution (minimum inhibitory concentration of oxacillin) and mecA gene detection in isolates of coagulase-negative bacteria other than Staphylococcus epidermidis (SCoNne). A total of 238 SCoNne was evaluated; oxacillin-resistance (the mecA gene) was detected in 71 percent of the isolates. All methods gave 100 percent sensitivity, based on presence of the mecA gene. The specificity of the cefoxitin disk was 100 percent, while the oxacillin disk gave a specificity of 91 percent and agar dilution oxacillin gave a specificity of 88 percent. We conclude that the cefoxitin disk is an efficient test, and it is an easy method for use in clinical laboratories to detect oxacillin-resistance in staphylococci.

Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestations / Vírus influenza e bactéria proteolítica co-infectantes em trato respiratório de indivíduos com manifestações respiratórias

A role for proteolytic bacteria in the exacerbation of influenza virus has been shown in natural hosts such as pigs and humans. Four hundred seven samples were collected from the respiratory tract of individuals presenting clinical manifestations, during influenza season (2003-2005) in São Paulo City. The aim of this study was to evaluate the incidence of determined bacteria co-infecting virus in human respiratory tract. Tests, such as bacteriological, immunofluorescence (IF), RT/PCR and hemagglutination (HA) were used for bacterial and viral investigation. Thirty seven (9.09 percent) positive for influenza virus were screened by IF. The RT/PCR confirmed the presence of influenza virus in these samples. Bacterial and agar casein tests demonstrated that 18 (48.64 percent) individuals were infected with proteolytic bacteria such as Staphylococcus spp., Streptococcus spp. and Pseudomonas spp. Among these samples, 13 (35.13 percent) were co-infected with influenza A virus. Influenza type B, co-infecting bacteria were found in five (13.51 percent) samples. In vitro the S. aureus protease increased the influenza HA titer after contact for 30 min at 25 ºC. Results revealed the occurrence of co-infection with proteolytic bacteria and influenza in the evaluated individuals. This finding corroborates that virus versus bacteria synergism could be able to potentiate respiratory infection, increasing damage to hosts.

O papel da bactéria proteolítica na exacerbação do vírus influenza tem sido demonstrado em hospedeiros naturais como porcos e humanos. Foram coletadas 407 amostras do trato respiratório de indivíduos apresentando manifestações clínicas, durante a estação da influenza (2003-2005) na cidade de São Paulo. Este trabalho teve como objetivo avaliar a incidência de determinadas bactérias que junto com vírus co-infectarem o trato respiratório humano. Testes bacteriológicos, e virológicos como imunofluorescência (IF), RT/PCR e hemaglutinação (HA) foram usados nas investigações viral e bacteriana. Pelo teste de IF foram selecionadas trinta e sete (9,09 por cento) amostras positivas para o vírus influenza. A presença do vírus influenza foi confirmada pela técnica de RT/PCR. Pelos testes bacteriológicos e do agar caseina, verificou-se que 18 (48,64 por cento) dos indivíduos foram infectados com bactérias proteolíticas tais como Staphylococcus spp., Streptococcus spp. e Pseudomonas spp. Destas amostras, 13 (35,13 por cento) foram co-infectadas com vírus influenza tipo A, e 5 (13,51 por cento) com influenza tipo B. No experimento in vitro com influenza e S. aureus, detectou-se aumento do título hemaglutinante deste vírus, após contacto de 30 min a 25 ºC. Os resultados obtidos revelaram a ocorrência de co-infecção com bactéria proteolítica e vírus influenza nos indivíduos avaliados. Estes achados corroboram com a investigação do sinergismo, entre bactéria e vírus, que poderia ser capaz de potencializar infecção respiratória, aumentando os riscos aos hospedeiros.