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.

Occurrence of the vanA gene in Staphylococcus epidermidis from nasopharyngeal secretion of Health-Care Workers, Recife, Brazil

Abstract INTRODUCTION: The increasing reports of vancomycin-resistant Staphylococcus strains (VRS) haves caused concern worldwide, from the laboratory detection to patient management. This study aimed to identify the occurrence of VRS strains among healthcare professionals from a university hospital. METHODS: A total of 102 Staphylococcus sp. isolates from healthcare professionals, obtained in a previous study were evaluated according to standard techniques for VRS detection. RESULTS: After screening inoculation of plates containing 6µg/ml of vancomycin, 19 resistant isolates were identified. The susceptibility profile to other antimicrobials revealed 18 multidrug resistant isolates. The minimum inhibitory concentration (MIC) was determined by E-test and broth microdilution. According to E-tests, of 19 isolates grown in BHI-V6, four isolates presented MIC ≥ 128 µg/ml, seven with MIC ranging from 4 to 8 µg/ml, and eight with MIC ≤ 2µg/ml. By broth microdilution, 14 isolates presented MIC ≤ 2 µg/ml and five with MIC ≥ 16µg/ml. The presence of the gene vanA was determined by PCR in the five resistant isolates, and this gene was detected in one of the strains. Furthermore, among the 19 strains, the gene mecA was found in 13 (39,4%) isolates, including the strain carrying the gene vanA. CONCLUSIONS: Based on these results, we highlight the presence of one strain carrying both vanA and the mecA genes, as well as multidrug-resistant strains colonizing healthcare professionals, and their importance as potential vectors to spread strains carrying resistance genes in the hospital environment.

Characterization of biofilm formation, antimicrobial resistance, and staphylococcal cassette chromosome mec analysis of methicillin resistant Staphylococcus hominis from blood cultures of children

Abstract INTRODUCTION: Methicillin resistant Staphylococcus hominis (MRSHo) has been recognized as an important human pathogen, particularly in immunocompromised patients. METHODS: A total of 19 S. hominis isolates were collected from children at the Children's Medical Centre, Tehran, Iran, from March 2012 to February 2013. MRSHo susceptibility against 13 antimicrobial and 3 antiseptic agents was determined using disk diffusion (DAD) and minimum inhibitory concentration (MIC), respectively. All isolates were subjected to polymerase chain reaction (PCR) assay for 15 distinct resistance genes, staphylococcal cassette chromosome mec (SCCmec), and arginine catabolic mobile elements (ACMEs). Biofilm production of the isolates was determined using a colorimetric microtiter plate assay. RESULTS: Of the 19 isolates, 16 were resistant to oxacillin and harbored mecA. High resistance was also observed against trimethoprim/sulfamethoxazole (81.2%). All MRSHo isolates were susceptible to the three disinfectants tested (Septicidine-PC, Septi turbo, and Sayacept-HP). In total, 15 (78.9%) isolates produced biofilms. Three isolates had SCCmec types (V and VIII), 13 were untypable (UT), and 5 had ACME type II. CONCLUSIONS: The results indicate that MRSHo with high antibiotic resistance and unknown SCCmec might become a serious problem in the future for the treatment of patients such as children.

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL-1, including 15.7% with an MIC of 8 μg mL-1 and 2% with an MIC of 16 μg mL-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.

Isolation, pathogenicity and disinfection of Staphylococcus aureus carried by insects in two public hospitals of Vitória da Conquista, Bahia, Brazil

Currently, hospital infection is a serious public health problem, and several factors may influence the occurrence of these infections, including the presence of insects, which are carriers of multidrug-resistant bacterial species. The aim of this study was to isolate staphylococci carried by insects in two public hospitals of Vitoria da Conquista, Bahia and to identify the resistance profile, pathogenicity and efficacy of disinfection of the premises. A total of 91 insects were collected in 21 strategic points of these hospitals, and 32 isolated strains ofStaphylococcus aureus were isolated. Based on antibiogram and Minimum Inhibitory Concentration results, 95% of these strains were susceptible to oxacillin. These strains were also evaluated for the presence of resistance genes encoding resistance to oxacillin/methicillin by polymerase chain reaction, but the sample was negative for this gene. Pathogenicity tests were performed in vitro biofilm formation induced by glucose, where it was found that eight (27.58%) strains were classified as biofilm producers and 21 (72.4%) as stronger producers. In addition, we performed PCR for their virulence genes: Sea (enterotoxin A), SEB (B), Sec (C), PVL (Panton-Valentine Leukocidin), ClfA (clumping factor A) and Spa (protein A). Of these, Sea, Spa PVL were positive in 7 (21.8%), 2 (6.3%) and 1 (3.1%) samples, respectively. The analysis of cytokine induction in the inflammatory response of J774 macrophages by isolates from the two hospitals did not show statistical difference at the levels of IL-6, TNF-α, IL-1 and IL-10 production. In addition, we verified the antimicrobial activity of disinfecting agents on these strains, quaternary ammonium, 0.5% sodium hypochlorite, 1% sodium hypochlorite, 2% sodium hypochlorite, 2% glutaraldehyde, Lysoform®, 70% alcohol solution of chlorhexidine digluconate, 2% peracetic acid, and 100% vinegar. Resistance was seen in only for the following two disinfectants: 70% alcohol in 31 (96.8%) samples tested and vinegar in 30 (93.8%) samples. The study demonstrated the presence of resistant and pathogenic organisms conveyed by insects, thus suggesting improvement in efforts to control these vectors.

Cambios a lo largo del tiempo en la distribución de los complejos de clones dominantes de Staphylococcus aureus resistente a la meticilina en Medellín, Colombia / Changes over time in the distribution of dominant clonal complexes of methicillin-resistant Staphylococcus aureus in Medellín, Colombia

Introducción. Parte del éxito de Staphylococcus aureus resistente a la meticilina (SARM) como patógeno se debe a la rápida diseminación de linajes pandémicos con perfiles variables de virulencia y sensibilidad antimicrobiana. En Colombia se han identificado clones asociados al hospital como el pediátrico (CC5-ST5-SCC mec IV), el brasilero (CC8-ST239-SCC mec III) y el chileno/cordobés (CC5-ST5-SCC mec I). Asimismo, se describió el USA300 (CC8-ST8-SCC mec IV), tradicionalmente asociado a la comunidad, causante de infecciones hospitalarias . Objetivo. Describir el comportamiento en el tiempo de los clones de SARM provenientes de un hospital universitario de Medellín en aislamientos recolectados con una década de diferencia. Materiales y métodos. Se analizaron 398 aislamientos de SARM, 67 recolectados en 1994 y 331 recolectados entre 2008 y 2010. La identificación y la sensibilidad a la meticilina se confirmaron mediante los genes nuc y mec A. La caracterización molecular incluyó la tipificación de spa , SCC mec , la electroforesis en gel de campo pulsado ( Pulsed Field Gel Electrophoresis, PFGE), y la tipificación por secuenciación de locus múltiples ( Multilocus Sequence Typing , MLST). Resultados. Al analizar los aislamientos de SARM de 1994 se encontró que pertenecían a un único linaje, el CC5-SCC mec IV, mientras que los aislamientos de 2008 a 2010 presentaron dos linajes dominantes: el CC8-SCC mec IVc, con cepas de los tipos spa t008 y t1610, estrechamente relacionadas con el clon USA 300, y el CC5-SCC mec I, con las de tipo spa t149, relacionadas con el clon chileno; no se detectaron cepas del linaje encontrado en 1994. Conclusiones. En este estudio se demuestra una dinámica en el tiempo de las cepas de S. aureus , y se señala la importancia de la vigilancia local y la difusión de los resultados, sobre todo en países como el nuestro, donde SARM es prevalente y la comprensión de su epidemiología es limitada.

Introduction: Part of the success of methicillin-resistant Staphylococcus aureus (MRSA) as a pathogen responds to the rapid spread of pandemic lineages with diverse virulence and antimicrobial susceptibility profiles. In Colombia, several healthcare-associated MRSA (HA-MRSA) clones have been found, including the pediatric clone (CC5-ST5-SCC mec IV), the Brazilian clone (CC8-ST239-SCC mec III), and the Chilean/Cordobés clone (CC5-ST5-SCC mec I). Moreover, the community-associated MRSA (CA-MRSA) clone USA300 has been reported as causing hospital-acquired infections. Objective: To describe the changes over time in the distribution of MRSA clones from a university hospital in Medellín collected at two time points a decade apart. Materials and methods: A total of 398 MRSA strains were analyzed. Of these, 67 strains were collected in 1994, while the remaining 331 strains were collected between 2008 and 2010. Species identification and methicillin resistance were confirmed by detection of nuc and mec A genes, respectively. Molecular characterization included spa typing, SCC mec typing, PFGE and MLST. Results: Analysis of the MRSA strains collected in 1994 revealed that they belonged to a single clone, the CC5-SCC mec IV, whereas among the isolates from 2008-2010, two dominant clones were identified: CC8-SCC mec IVc, which included spa types t008 and t1610 and is closely related to the USA 300 clone, and CC5-SCC mec I ( spa type t149), related to the Chilean clone. The ST5-SCC mec IV clone from 1994 was not detected. Conclusions: This study identifies temporal dynamics in MRSA clone diversity, and highlights the importance of local surveillance and dissemination of results, especially in countries like Colombia where MRSA is prevalent and knowledge regarding its epidemiology is still insufficient.

Aislamientos de Staphylococcus aureus sensibles a meticilina relacionados genéticamente con el clon USA300, ¿origen de los aislamientos SARM de genotipo comunitario en Colombia? / Methicillin-sensitive Staphylococcus aureus isolates related to USA300 clone: Origin of community-genotype MRSA in Colombia?

Introducción. USA300 es un linaje genético que se encuentra en aislamientos de Staphylococcus aureus sensibles (SASM) y resistentes a meticilina (SARM). Actualmente, en Colombia las infecciones por SARM en hospitales y en la comunidad son causadas principalmente por un clon con genotipo comunitario (SARM-GC) relacionado genéticamente con el clon USA300. El origen de esta variante es aún desconocido. Objetivo. Identificar y caracterizar aislamientos de S. aureus resistentes y sensibles a meticilina con el fin de aportar información para establecer un posible origen de los aislamientos SARM-GC en Colombia. Materiales y métodos. Se realizó una caracterización de aislamientos SASM relacionados con el clon USA300 detectados a partir de un análisis de 184 aislamientos de S. aureus (90 SARM y 94 SASM) causantes de infecciones. La relación genética de los aislamientos se determinó por electroforesis en gel de campo pulsado (PFGE), tipificación de secuencias multilocus (MLST) y tipificación del gen de la proteína A ( spa ). Resultados. De los 184 aislamientos, 27 (14,7 %) presentaron características moleculares y relación genética con el clon USA300, y de ellos, 18 fueron SARM y nueve fueron SASM. Todos los aislamientos SARM relacionados con este clon albergaban un casete estafilocócico cromosómico mec (SCC mec ) IVc (3.1.2). En ningún aislamiento SASM se detectaron secuencias remanentes de SCC mec o una duplicación del sitio att B que evidenciaran la pérdida del casete. Conclusión. El origen de los aislamientos SARM-GC en Colombia probablemente se encuentre en la diseminación de clones SASM relacionados con el clon USA300 que adquirieron el SCC mec IVc posteriormente.

Introduction: USA300 is a genetic lineage found both in methicillin-resistant (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) isolates. In Colombia, hospital and community MRSA infections are caused by a USA300-related community genotype MRSA (CG-MRSA) clone. The genetic origin of this clone is unknown yet. Objective: To identify and characterize methicillin-resistant (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates in order to improve the information about the origin of the CG-MRSA isolates in Colombia. Materials and methods: USA300-related MSSA isolates were detected and characterized from a study of 184 S. aureus isolates (90 MRSA and 94 MSSA) recovered from infections. The genetic relatedness of the isolates was established by means of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and protein A gene typification ( spa typing). Results: Among 184 isolates, 27 (14.7%) showed molecular characteristics and genetic relationship with the USA300 clone, of which 18 were MRSA and nine were MSSA. All USA300-related MRSA harbored Staphylococcal cassette chromosome mec (SCC mec ) IVc (3.1.2). In the MSSA isolates, SCC mec remnants or att B duplicate sites were not detected. Conclusions: In Colombia, the CG-MRSA isolates probably originated in the dissemination of an USA300-related MSSA clone which later acquired SCC mec IVc.

Methicillin-resistant Staphylococcus aureus causes both c ommunity-associated and health care-associated infections in children at the Hospital Universitario de Santander / Staphylococcus aureus resistente a meticilina causante de infecciones comunitarias y de infecciones asociadas a la atención en salud en pacientes pediátricos del Hospital Universitario de Santander

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of infection in the pediatric population. Initially, MRSA was restricted to hospitals; however, outbreaks in the community among people without health care-related risk factors have been reported worldwide. Currently, MRSA is a frequent cause of both hospital and community-associated infections. Objective: To describe the relationships between the molecular characteristics of MRSA isolates (staphylococcal cassette chromosome mec (SCCmec) type and Panton-Valentine leukocidin (PVL) carriage) and the characteristics of infection (the origin and localization of infection) in pediatric patients at the Hospital Universitario de Santander in Bucaramanga, Colombia. Materials and methods: A total of 43 MRSA isolates were obtained from hospitalized pediatric patients. SCCmec typing (I-V), SCCmec IV subtyping and PVL carriage were determined and related to the clinical characteristics. Results: Among the MRSA isolates studied, SCCmec IVc was present in 77%, followed by 16% for SCCmec I and 2% for SCCmec IVa. Two isolates were not typeable (NT). PVL genes were carried by 88% of the MRSA isolates, including the SCCmec IVc/IVa and SCCmec I isolates. SCCmec IV caused both community-acquired infection (CAI) (47%) and nosocomial infection (HAI) (53%). SCCmec IV, PVL-positive MRSA was associated with both CAI (47%) and HAI (53%) and caused mostly SSTI and osteoarticular infection. Conclusions: These findings suggest that the presence of community-associated MRSA (CA-MRSA) (SCCmec IV and PVL positive) causes both health care-associated infection (HCAI) and nosocomial infection (HAI) in pediatric patients in Colombia.

Introducción. Staphylococcus aureus resistente a la meticilina (SARM) es un agente frecuente de infección en la población pediátrica. Aunque inicialmente las cepas de SARM estaban restringidas a los hospitales, se han reportado a nivel mundial brotes de infección por SARM en individuos sin factores de riesgo y, actualmente, SARM es una causa frecuente de infecciones hospitalarias y comunitarias. Objetivo. Describir la relación entre las características moleculares de aislamientos de SARM (casete cromosómico estafilocócico mec SCCmec y leucocidina Panton-Valentine) y el origen de la infección y su presentación clínica en pacientes pediátricos del Hospital Universitario de Santander en Bucaramanga, Colombia. Materiales y métodos. Se incluyeron 43 aislamientos de SARM obtenidos de niños hospitalizados. La clasificación del SCCmec (I-V) y la subclasificación del SCCmec-IV se realizaron en todos los aislamientos. Además, los genes de la leucocidina Panton-Valentine se detectaron mediante amplificación por PCR. Las características moleculares fueron asociadas con las características clínicas de cada paciente. Resultados. Entre los 43 SARM tipificados, el SCCmec-IVc fue el más frecuente con 77 %, seguido por el SCCmec-I con 16 % y el SCCmec-IVa con 2 %. Tres aislamientos no pudieron ser tipificados. Los genes de la leucocidina Panton Valentine se detectaron en 88 % de los SARM en aislamientos portadores del SCCmec-IVc/IVa y el SCCmec-I. Los SARM SCCmec-IV positivos para la leucocidina Panton-Valentine se asociaron con infecciones adquiridas en la comunidad (47 %) y en el hospital (53 %) con compromiso de piel y tejidos blandos, y en los casos más graves, con compromiso osteoarticular. Conclusiones. Estos resultados sugieren la presencia de cepas SARM-CO (SCCmec-IV positiva para PVL) causantes de infecciones adquiridas en la comunidad y en el medio hospitalario en pacientes pediátricos en Colombia.

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.

Subtyping of Chilean Methicillin-Resistant Staphylococcus aureus strains carrying the staphylococcal cassette chromosome mec type I

The cassette chromosome mec (SCCmec) present in methicillin-resistant Staphylococcus aureus (MRSA) has two essential components, the ccr gene complex and the mec gene complex. Additionally, SCCmec has non-essential components called J regions which are used for MRSA subtyping. This study was performed to determine subtypes MRSA strains carrying SCCmec type I based on polymorphism of regions located downstream of the mecA gene. A total of 98 MRSA strains carrying SCCmec type I isolated from patients hospitalized at the County Hospital of Valdivia (Chile) between May 2007 and May 2008, were analyzed by multiplex PCR designed to amplify the mecA gene and 7 DNA hypervariable regions located around the mecA gene. MRSA strains were classified into seventeen genotypes accordingly to amplification patterns of DNA hypervariable regions. Five genotypes showed amplification patterns previously described. The remaining twelve genotypes showed new amplification patterns. Genotypes 18 and Genotype 19 were the most frequently detected. Regions HVR, Ins117 and pI258 stand out as being present in more than 60% of tested isolates. The acquisition of hypervariable regions by MRSA is a continuous horizontal transfer process through which the SCCmec have been preserved intact, or even may give rise to new types and subtypes of SCCmec. Therefore it is possible to infer that most MRSA strains isolated at the County Hospital of Valdivia (Chile) were originated from two local clones which correspond to Genotype 18 and Genotype 19.

Frecuencia de portadores nasales de Staphylococcus aureus resistente a meticilina en personal de salud de hospitales de Nicaragua / Frequency of nasal carriers of methicillin-resistant Staphylococcus aureus among health workers in Nicaraguan hospitals

Objetivo. Conocer la frecuencia de portadores nasales de cepas de Staphylococcus aureus resistentes a meticilina (SARM) y el patrón de resistencia antimicrobiana de esas cepas obtenidas de trabajadores de la salud de cuatro hospitales de Nicaragua. Métodos. Se realizó un estudio descriptivo, transversal, en el período del 1 de junio de 2009 al 30 de septiembre de 2010. Los hisopados nasales de los trabajadores de la salud que aceptaron voluntariamente participar en el estudio fueron cultivados en medio agar base de detección de resistencia a oxacilina (ORSAB). La identificación de los aislados de S. aureus se realizó por métodos cotidianos y la resistencia a meticilina se determinó por la presencia del gen mecA con la técnica de reacción en cadena de polimerasa. El patrón de resistencia antimicrobiana se detectó por difusión en disco. Cada participante firmó un consentimiento informado con anterioridad a la toma de la muestra. Resultados. Participaron en el estudio 569 trabajadores de la salud, de los cuales 208 eran del hospital de León, 155 de dos hospitales de Chinandega y 206 del de Managua. La frecuencia de portadores nasales de SARM fue de 9,6% en León, 11,6% en Chinandega y 6,7% en Managua. El perfil de resistencia de las cepas SARM fue similar en los cuatro hospitales y todas las cepas fueron sensibles a vancomicina. Del total de cepas SARM aisladas, 15% fueron multirresistentes. El porcentaje de resistencia a eritromicina fue el más alto, seguido del de clindamicina. Conclusiones. Los resultados del estudio se pueden considerar una advertencia sobre la circulación de cepas SARM entre el personal de salud de los hospitales participantes y aportan información relevante en relación al perfil de resistencia de las cepas SARM.

Objective. To determine the frequency of nasal carriers of strains of methicillinresistant Staphylococcus aureus (MRSA) and the antimicrobial resistance pattern of these strains, obtained from health workers from four hospitals in Nicaragua. Methods. A descriptive cross-sectional study was conducted between 1 June 2009 and 30 September 2010. Nasal swabs were taken from health workers who voluntarily agreed to participate in the study, and were cultured on an oxacillin-resistant screening agar base (ORSAB) medium. The S. aureus isolates were identified using ordinary methods, and methicillin resistance was confirmed based on the presence of the mecA gene using the polymerase chain reaction technique. The antimicrobial resistance pattern was detected by the disk diffusion method. Each participant signed an informed consent form before the samples were taken. Results. A total of 569 health workers participated in the study: 208 from one hospital in León, 155 from two hospitals in Chinandega, and 206 from one hospital in Managua. The frequency of nasal MRSA carriers was 9.6% in León, 11.6% in Chinandega, and 6.7% in Managua. The MRSA resistance profile was similar in the four hospitals, and all the strains were sensitive to vancomycin. Of the total MRSA strains isolated, 15% were multi-drug resistant. Erythromycin had the highest percentage of resistance, followed by clindamycin. Conclusions. The results of the study may be regarded as a warning that MRSA strains are circulating among health workers in the participating hospitals. The study also contributes important information regarding the resistance profile of MRSA strains.

Vancomycin resistance among methicillin resistant Staphylococcus aureus isolates from intensive care units of tertiary care hospitals in Hyderabad.

Background & objectives: Multidrug resistant methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial and community acquired infections and is on the rise. The glycopeptide vancomycin has been proposed as the drug of choice for treating such infections. The present study aimed at identifying the vancomycin resistance both phenotypically and genotypically among the MRSA isolates from two tertiary care hospitals in Hyderabad, south India. Methods: MRSA were isolated and identified from different clinical samples collected from ICUs of tertiary care hospitals in Hyderabad using conventional methods. Antibiogram of the isolates and vancomycin MIC were determined following CLSI guidelines. vanA was amplified by PCR using standard primers. Results: All vancomycin resistant S. aureus (VRSA) isolates were MRSA. The VRSA isolates were positive for vanA gene, except one which was negative. All VRSA had a vancomycin MIC in the range of 16-64 mg/l. Interpretation & conclusions: The increase in vancomycin resistance among MRSA and excessive use of antimicrobial agents have worsened the sensitivity. Larger studies need to be done in various geographical regions of the country to better define the epidemiology, mechanism of vancomycin resistance in S. aureus and its clinical implications.

Methicillin-resistant Staphylococcus lugdunensis carrying SCCmec type V misidentified as MRSA

Staphylococcus lugdunensis is a rare cause of severe infections and clinical manifestations are similar to those related to S. aureus infection. We describe a hospital-acquired bacteremia due to methicillin-resistant Staphylococcus lugdunensis, misidentified as methicillin-resistant S. aureus. The oxacillin MIC was 16 µg/mL and the mecA gene and SCCmec type V were determined by PCR. Although treatment had been appropriated, the patient died after rapid progressive respiratory failure and another nosocomial sepsis. It is important not only to identify S. lugdunensis in view of its clinical course, but also to determine its susceptibility to oxacillin by detecting the mecA gene or its product.

Prevalence of phenotypic resistance of Staphylococcus aureus isolates to macrolide, lincosamide, streptogramin B, ketolid and linezolid antibiotics in Turkey

The incidence of drug-resistant pathogens differs greatly between countries according to differences in the usage of antibiotics. The purpose of this study was to investigate the phenotypic resistance of 321 methicillin resistance Staphylococcus aureus (MRSA) and 195 methicillin susceptible S. aureus (MSSA) in a total of 516 S. aureus strains to macrolide, lincosamide, streptogramin B (MLS B), ketolid, and linezolid. Disk diffusion method was applied to determine MLS B phenotype and susceptibility to different antibiotic agents. It was found that 54.6 percent of the isolates were resistant to erythromycin (ERSA), 48 percent to clindamycin, 55 percent to azithromycin, 58.7 percent to spiramycin, 34.7 percent to telithromycin, and 0.4 percent to quinupristin-dalfopristin, respectively. No strain resistant to linezolid was found. The prevalence of constitutive (cMLS B), inducible (IMLS B), and macrolides and type B streptogramins (M/MS B) among ERSA isolates (237 MRSA, 45 MSSA) was 69.6 percent, 18.2 percent, and 12.2 percent in MRSA and 28.9 percent, 40 percent, and 31.1 percent in MSSA, respectively. In conclusions, the prevalence of cMLS B was predominant in MRSA; while in MSSA strains, iMLS B and M/MS B phenotype were more higher than cMLS B phenotype resistance. The resistance to quinupristindalfopristin was very low, and linezolid was considered as the most effective antibiotic against all S.aureus strains.

Methicillin/Oxacillin-resistant Staphylococcus aureus as a hospital and public health threat in Brazil

Methicillin-resistant Staphylococcus aureus is an established nosocomial pathogen (HA-MRSA, hospital acquired MRSA), but has recently begun to appear in the community (CA-MRSA, community acquired MRSA). The cause of resistance to methicillin and all other â-lactam antibiotics is the mecA gene, which is situated on a mobile genetic element, the Staphylococcal Cassette Chromosome mec (SCCmec). Seven major variants of SCCmec, type I to VII are distinguished. HA-MRSA disseminated worldwide and causes the majority of S. aureus nosocomial infections with a limited number of clones disseminated including the Brazilian Epidemic Clone (BEC, ST239-MRSA-III). CA-MRSA isolates are susceptible to non-â-lactam antibiotics, usually isolated from healthy individuals which do not possess any unknown risk factors for MRSA infection and are associated with a larger clonal diversity compared with HA-MRSA. However, during recent years distinction between HA-MRSA and CA-MRSA is beginning to fade. Actually, knowledge about MRSA disseminating clones is required to implement any strategies to control the transmission of MRSA either within hospitals or in community. For this reason, rapid identification of strains is an important issue. The rate of HA-MRSA can be reduced substantially through the implementation of interventions strategies, even in settings where MRSA is endemic as in most Brazilian hospitals. However, these policies could be quite complicated in the light of an increasing CA-MRSA prevalence in healthcare facilities, considering that distinction between HA-MRSA and CA-MRSA has started to disappear.

Molecular identification of methicillin-resistant staphylococcus aureus in Khartoum teaching hospital

Antimicrobial resistance has become a great public health problem worldwide and multi-drug resistant Staphylococcus aureus has been widely reported. The presence or absence of methicillin resistance gene [mecA] in 48 clinical wound isolates of S. aureus was examined by the polymerase chain reaction [PCR]. The results were analyzed in parallel to the disk diffusion method by oxacillin [1 microg]. Polymerase chain reaction was amplified at a sequence of mecA gene at 1319 bp. Nine [18.75%] out of the 48 isolates and were found to be identical to those of disk diffusion test. All strains were studied for their susceptibility to traditionally used antibiotics. The results revealed that multi-drug resistance was common among MRSA strains. The drug of choice for the treatment of MRSA and MSSA was vancomycin. The study concluded that multiple antibiotic resistance was common, and the PCR assay can be used to confirm MRSA infection

Caracterización fenotípica y genotípica de la resistencia a meticilina de cepas de Staphylococcus aureus aisladas en Antofagasta / Phenotypic and genotypic characterization of methicillin resistance in strains of Staphylococcus aureus isolated in Antofagasta

Staphylococcus aureus resistant to methicillin (SARM) has been associated with nosocomial infections due to its capacity to develop resistance to multiple antibiotics. There is little information about the SARM which are found in the hospital services of Antofagasta. We studied the phenotypic and genotypic characteristics of methicillin resistance in 38 strains of S. aureus isolated in Antofagasta, identified by coagulase and API Staph tests and by a biochemical test (Ph-system). The susceptibility to antibiotics was studied using the agar dilution technique, identifying SARM strains with discs of oxacillin. Beta-lactamase with nitrocephine, and the gene mecA by means of PCR. Eighty nine percent (34 strains) were SARM with a high resistance to ampicillin, penicillin, erythromycin, claritromycin. gentiamycin, amikacine and ciprofloxacine. All isolates were susceptible to vancomycin and rifampicin. Beta-lactamase was demonstrated in 79% of the SARM strains. Strain typing and resistance patterns revealed a great diversity of PhP-types and antibiotypes in the isolates. Ninety seven percent of the SARM strains had the gene mecA. One PhP-type (C6) was dominant (5 SARM strains) all had the mecA gene, produced beta lactamase and had the same pattern of antibiotic resistance. We conclude that the dominant phenotypes of SARM strains which have the mecA gene and multiple resistance to antibiotics are present in the hospitals of Antofagasta, and sound the alert on the risk of nosocomial transmission of epidemic clones of SARM.

Staphylococcus aureus resistentes a meticilina (SARM) han sido asociados con infecciones nosocomiales por su capacidad para desarrollar resistencia a múltiple antibióticos, existiendo escasa información acerca de SARM que están circulando en los servicios hospitalarios de Antofagasta. Se estudió características fenotIpicas y genotípicas de la resistencia a meticilina en 38 cepas de S. aureus aisladas en Antofagasta, identificadas por tests de coagulasa y API Staph y por tipificación bioquímica (Ph-Sistem). La susceptibilidad a antibióticos se realizo por técnica de dilución en agar, las cepas SARM fueron identificadas con discos de oxacilina, beta-lactamasa por nitrocefina y gen mecA fue detectado pot PCR. El 89% (34 cepas), fueron SARM con una alta resistencia a ampicilina, penicilina, eritromicina, gentamicina, amikacina y ciprofloxacino. Todos los aislados fueron susceptibles a vancomocina y rifampicina. Beta lactamasa fue demostrada en 79% de las cepas SARM. La tipificación y los patrones de resistencia revelaron una alta diversidad de PhP tipos y antibioticos en los aislamientos. El 97% de las cepas SARM albergaban el gen mecA. Un PhP tipo (C6) fue dominante. (5 cepas SARM), todos presentando el gen mecA, produciendo beta lactamasa y mostrando el mismo patrón de resistencia antibiótica. Se concluye que los fenotipos dominantes de cepas SARM que albergan el gen mecA y resistencia múltiples alos antibióticos están circulando en los hospitales de Antofagasta, alertando sobre el riesgo de transmisión intranosocomial de clones epidémicos de SARM.

Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in two metropolitan areas of São Paulo State, southeast Brazil

One hundred and fifty-one methicillin-resistant z (MRSA) strains have been isolated from patients admitted in tertiary care hospitals in two metropolitan areas (Campinas City and Ribeirão Preto City) in the southeast region of Brazil and analyzed through PCR-based techniques [(PCR amplification of spa, coa, and housekeeping genes (arcC, aroE, gmk, pta, tpi, yqiL)] and further restriction fragment typing of coa and of housekeeping genes. The heterogeneity of spa gene was determined directly by agarose gel electrophoresis migration. The results obtained indicate the existence of three (A, B, C) main clusters. Since the strain distribution in these three clusters is much characteristic, it denotes the existence of three main clones. All strains isolated in Campinas were grouped in clusters A and B, while most of the strains isolated in Ribeirão Preto were grouped in cluster C. This distribution denotes the existence of different founder strains that undergo independent genetic variability. The strains considered representative of the Brazilian Epidemic Clone (BEC) were categorized as cluster A. These results indicate a possible higher variability among Brazilian MRSA strains than currently described and indicate that the techniques herein used can be used as an alternative to Pulsed Field Gel Electrophoresis (PFGE).

Epidemiological trend of methicillin-resistant staphylococcus aureus and emerging challenges for control

Following its first recognition in early 1960s, the increasing incidence of nosocomial and community - acquired methicillin-resistant Staphylococcus aureus [MRSA] infections has become a global problem. The emergence of multiple-drug resistant MRSA strains and dissemination of epidemic antibiotic clones including presence of wide spectrum of virulence and predisposing risk factors complicate diagnosis, chemotherapy and control causing significant morbidity and mortality. Resistance to methicillin is mediated by altered penicillin-binding protein 2a [PBP-2a], encoded by mec gene determinant [i.e. I-V types with different genomes, antibiograms and toxin profile] embedded in staphylococcal cassette chromosome [SCCmec]. It is also partly due to hyper beta-lactamase production by the organism. Detection of MRSA strains in domestic animals and associated persons including a wide spread protozoan has widened the organism's epidemiologic characters and may influence infection control policies. Routine and regular surveillance [uncommon in poor-resourced developing areas], good hospital practices and personal hygiene, development of effective therapeutic agents and rational administration of antibiotics based on reliable test results may cause limit in the spread of MRSA infections

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus.

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.