Tipificación del cassette cromosómico estafilocócico de Staphylococcus aureus resistentes al meticilino en el estado de Aragua, Venezuela / Typification of the staphylococcal chromosome cassette of methicillin-resistant Staphylococcus aureus in the state of Aragua, Venezuela

RESUMEN Objetivos: Tipificar el casette SCCmec en cepas de Staphylococcus aureus resistentes a meticilino (SARM) en aislados clínicos de centros de salud del Estado Aragua-Venezuela y comparar la presencia de los genotipos SCCmec entre los centros de salud del estado y según el tipo de infección. Materiales y métodos: Durante enero y agosto de 2015 se estudiaron 81 cepas SARM de cuatro centros de salud del estado de Aragua en Venezuela. La resistencia al meticilino se midió con el método de Kirby-Bauer con discos de oxacilina (1 µgr) y cefoxitina (30 µgr). El gen mecA y el SCCmec se analizaron por la técnica de reacción en cadena de polimerasa múltiple. Resultados: 55 aislados (67,9%) amplificaron el gen mecA, y 24 cepas (43,6%) amplificaron el SCCmec. El SCCmec I fue el más frecuente, seguido de SCCmecIV y SCCmec III, representaron el 62,5%, 25% y 12,5%, respectivamente. El SCCmec I fue predominante en el centro de salud A (80%), mientras que el SCCmec IV se encontró en el centro de salud B (60%) y C (100%). En el centro de salud D, 50% resultó ser SCCmec I y 50% SCCmec IVd. Se encontró relación entre el SCCmec y el centro de salud con significancia estadística. En infecciones de piel y tejidos blandos y en las respiratorias predominó el SCCmec I con 63,2% y 50% respectivamente. Conclusiones: La frecuencia de SCCmec I y IV permitirá establecer nuevas medidas en el uso y control de la resistencia a los antibióticos.

ABSTRACT Objective: Typify the SCCmec cassette in methicillin-resistant strains of Staphylococcus aureus in clinical isolates from health centers in the State of Aragua-Venezuela and compare the presence of SCCmec genotypes among the state health centers and according to the type of infection. Materials and methods: 81 MRSA strains from four health centers of the Aragua-Venezuela State were studied. Methicillin resistance was performed with the Kirby-Bauer method with oxacillin (1 µg) and cefoxitin (30 µg) disks. The mecA gene and SCCmec were analyzed by the multiple PCR technique. Results: Only 55 isolates (67.9%) amplified the mecA gene, and 24 strains (43.6%) amplified SCCmec. SCCmec type I was the most frequency, followed by SCCmec IV and SCCmec III, representing 62.5%, 25% and 12.5%, respectively. SCCmec I was predominant in health center A (80%), while in B and C 60% and 100% respectively were SCCmec IV. At health center D, 50% turned out to be SCCmec I and 50% SCCmec IVd. A relationship was found between the SCCmec and the health center with statistical significance. SCCmec I predominated in skin and soft tissue and respiratory infections with 63.2% and 50%, respectively. There was no association between genotype and type of infection with a p value greater than 0.05. Conclusions: The prevalence of SCCmec I and IV will allow establishing new measures in the use of antibiotics and epidemiological control.

Comparison of Latex Agglutination Test with Polymerase Chain Reaction for Detection of mecA Gene in Methicillin Resistant Staphylococcus aureus

Background: Antimicrobial resistance is a devastating question that threatens health globally. The extensive, indiscriminate and unnecessary consumption of antibiotics for humans, as well as wildlife and in agriculture; lead to the development of notoriously resistant Staphylococcus aureus; through possession of mecA gene, encoded by modified Penicillin binding protein (PBP2a); being labeled “Methicillin resistant Staphylococcus aureus”. Conventional phenotypic techniques for MRSA detection rely on standardization of cultural characteristics. The latex agglutination method can be adopted as an accurate strategy for rapid detection of MRSA. Methodology: A total of 713 consecutive, non-duplicate isolates of Staphylococcus aureus were processed. Methicillin resistance was determined using cefoxitin (30 µg) by Kirby-Bauer method following Clinical and Laboratory Standards Institute (CLSI) guideline, latex agglutination method; and PCR for mecA gene. Results: The results showed that out of 713 Staphylococcus aureus isolates, 12.90% isolates were detected as MRSA due to resistance to cefoxitin. By latex agglutination method, 87 (94.50%) were positive for PBP2a; while on PCR mecA gene was detected only in 82 (89.10%) MRSA isolates. When assessed with PCR (gold standard) the sensitivity and diagnostic accuracy of latex agglutination was 100% and 94.57%, respectively. Conclusion: Latex agglutination test can be used as a prompt and reliable diagnostic technique for mecA gene detection in MRSA isolates, where molecular methods are limited. This can effectively minimize the misdiagnosis of resistant strains, and over/misuse of antibiotics.

Detection of mecA gene and drug-resistance analysis / 中华老年医学杂志

Objective To determine whether coagulase-negative non-epidermal staphylococcus is methicillin-resistant coagulase-negative staphylococcus by mecA gene test,when the minimal inhibitory concentration(MIC)of oxacillin is between 0.5-2.0 mg/L.Methods The mecA gene was detected and analyzed by the cefoxitin disk diffusion,E-test,VITEK-2 Compact and polymerase chain reaction (PCR)purification.Results A total of 300 strains of coagulase-negative staphylococci were screened from 4032 patients(7.4％),of which 45 strains of Staphylococcus saprophyticus and 80 strains of Staphylococcus hemolyticus were identified by Compact VITEK-2.There was a statistically significant difference in the positive rate of mecA gene detection between Staphylococcus saprophyticus and Staphylococcus hemolyticus(P ＜0.05).The results of detection of cefoxitin disk diffusion(inhibitory zone diameter ≥ 25 mm),E-test(MIC of oxacillin between 0.5-2.0 mg/L)and Compact VITEK-2 (MIC of oxacillin between 0.5-2.0 mg/L)showed that there were 81 strains of coagulase-negative non-Staphylococci,of which 10 strains with positive mecA gene were confirmed by PCR.Conclusions When the minimal inhibitory concentration (MIC)of oxacillin against coagulase-negative non-Staphylococci stains is between 0.5-2.0 mg/L,the guidelines of the American clinical laboratory standardization institute(CLSI)should be strictly implemented in clinical microbiology laboratory and the mecA gene must be tested.Based on the wide dissemination of the mecA gene in Staphylococcus aureus population,if the mecA gene test is negative,it is reported as methicillin-susceptible coagulase-negative Staphylococcus(MSCNS),and the reverse result is reported as methicillin-resistant coagulase-negative staphylococcus(MRCNS).

Ceftaroline resistance in Staphylococcus pseudintermedius gene mecA carriers / Resistência à ceftarolina em Staphylococcus pseudintermedius portadores do gene mecA

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) being a constant concern, ceftaroline fosamil has been recently approved as a new cephalosporin, active against MRSA, for use in humans; only rare cases of resistance have been reported till date. There is no report of resistance to ceftaroline in Staphylococcus pseudintermedius, which is the main bacterium causing dermatitis and otitis in dogs. To evaluate staphylococcal resistance to ceftaroline, 35 isolates of methicillin-resistant S. pseudintermedius (MRSP), carrying the mecA gene, from 26 dogs with folliculitis and nine dogs with external otitis, underwent disk diffusion test with cefoxitin, oxacillin, and ceftaroline. Tests with cefoxitin and oxacillin showed > 90% sensitivity in methicillin resistance detection. In the disk diffusion test, 97.14% (34/35) were resistant to cefoxitin, 94.29% (33/35) to oxacillin, and 31.43% (11/35) to ceftaroline. Of the ceftaroline-resistant strains, 27.27% (3/11) were obtained from the ears of dogs while the rest (8/11) were from the skin. The current report is the first description of MRSP resistance to ceftaroline.(AU)

Infecções causadas por Staphylococcus aureus resistente à meticilina (MRSA) são uma preocupação médica constante. A ceftarolina fosamila é uma nova cefalosporina ativa contra Staphylococcus aureus resistente à meticilina recentemente aprovada para uso em humanos e raros casos de resistência relatados até agora. Não há relatos de resistência à ceftarolina em Staphylococcus pseudintermedius, principal bactéria causadora de dermatite e otite em cães. Com o objetivo de avaliar a resistência estafilocócica à ceftarolina, 35 amostras de S. pseudintermedius resistentes à meticilina (MRSP), portadoras do gene mecA, provenientes de 26 cães com foliculite e 9 com otite externa foram submetidos ao teste de disco-difusão com cefoxitina, oxacilina e ceftarolina. Os testes realizados com cefoxitina e oxacilina mostraram mais de 90% de sensibilidade na detecção da resistência à meticilina em ambas. No teste da disco-difusão, 97,14% (1/35) foram resistentes à cefoxitina, 94,29% (3/35) à oxacilina e 31,43% (11/35) à ceftarolina. Das cepas resistentes às ceftarolina, 27,27 (3/11) foram provenientes de ouvido de cães e as demais (8/11), provenientes da pele, sendo essa primeira descrição de resistência de MRSP à ceftarolina na literatura atual.(AU)

Comparación de métodos fenotípico y genotípico en la detección de Staphylococcus aureus meticilino resistente en centros hospitalarios de Pereira / Comparison between phenotype and genotype methods in the detection of Methicillin-Resistant Staphylococcus aureus in Center Hospital of Pereira

Resumen Introducción: Staphylococcus aureus es un importante patógeno, puede causar infecciones leves de piel, hasta enfermedades con compromiso vital. La aparición de Staphylococcus aureus meticilino-resistentes, MRSA; ha aumentado su resistencia antimicrobiana, especialmente a β-lactámicos; dificultando el manejo de las infecciones, aumentando las tasas de morbi-mortalidad, convirtiéndose en un problema de salud pública. La expresión fenotípica de la resistencia suele ser heterogénea, dificultando su detección en el laboratorio por métodos convencionales; lo cual, incrementa los costos en la atención hospitalaria de infecciones por MRSA. Objetivo: Comparar métodos fenotípico y genotípico para la identificación de aislamientos hospitalarios de MRSA en centros hospitalarios de Pereira. Métodos: A partir de aislamientos de S. aureus obtenidos de tres instituciones de salud de alta complejidad clasificadas como A, B y C; se determinó la resistencia a meticilina por concentración mínima inhibitoria en sistemas automatizados y el gen mecA por PCR múltiple. Resultados: La prevalencia fenotípica de MRSA fue 44,4%, la institución A presentó la mayor tasa con 48,65%. La prevalencia genotípica fue 57,4%; en las instituciones A, B y C fue 55,2%, 41,7% y 75%, respectivamente, con diferencia estadísticamente significativa (p<0.05). La sensibilidad y especificidad del método fenotípico fue 99,0% y 94,7%, respectivamente, frente al método gold estándar de la PCR. El índice Kappa fue 0,942 indicando un nivel de concordancia muy bueno entre métodos. Conclusión: La prevalencia de aislamientos MRSA en las instituciones de Pereira fue alta. Los índices de concordancia de los métodos fenotípicos demostraron que son confiables para el diagnóstico de infecciones por MRSA.

Abstract Introduction: Staphylococcus aureus is an important pathogen, can cause mild skin infections, to diseases with compromise vital. The appearance of Methicillin-Resistant Staphylococcus aureus MRSA; It has increased its antimicrobial resistance, especially to β-lactam; hampering the handling of them infections, increasing the rates of morbidity-mortality, becoming a health public problem. The phenotype expression of the resistance tend to be heterogeneous, hindering its detection in the laboratory by conventional methods; which increases costs in the hospital care of MRSA infections. Objective: To compare the phenotypes and genotypes methods for identification of hospital isolates MRSA in Pereira. Methods: From isolates of S. aureus obtained of three high complexity institutions of health classified as A, B and C; determined resistance to Methicillin by minimum inhibitory concentration in automated systems and mecA gene by multiplex PCR. Results: The phenotype prevalence of MRSA was 44.4%, the institution A presented the highest rate with 48.65%. The genotype prevalence was 57.4%; in the institutions A, B and C was 55.2%, 41.7% and 75%, respectively, with difference statistically significant (p < 0.05). The sensitivity and specificity of the phenotype method were 99.0% and 94.7%, respectively, against the gold standard of the PCR method. The Kappa index was 0,942 indicating a very good level of concordance between methods. Conclusion: The prevalence of isolates MRSA in the institutions of Pereira was high. The concordance index of phenotype methods showed that they are reliable for the diagnosis of MRSA infections.

Detection of the mecA gene and identification of Staphylococcus directly from blood culture bottles by multiplex polymerase chain reaction

ABSTRACT Introduction: Staphylococcus spp. - both S. aureus, including methicillin-resistant strains (MRSA) and coagulase negative staphylococci (CoNS) - are relevant agents of healthcare-associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant CoNS from blood stream infections is critically important for patient management. It is worth noting that inappropriate empiric therapy has been associated with higher in-hospital mortality. Material and methods: In this study we evaluated a multiplex polymerase chain reaction (multiplex PCR) standardized to detect Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin resistance directly from blood culture bottles. A total of 371 blood cultures with Gram-positive microorganisms confirmed by Gram-stain were analyzed. Results from multiplex PCR were compared to phenotypic characterization of isolates. Results: Staphylococcus aureus was detected in 85 (23.0%) blood cultures and CoNS in 286 (77.0%). There was 100% agreement between phenotypic and multiplex PCR identification. Forty-three (50.6%) of the 85 S. aureus carried the mecA gene and among the 286 CoNS, 225 (78.7%) were positive for the mecA gene. Conclusions: The multiplex PCR assay developed here was found to be sensitive, specific, rapid, and showed good agreement with the phenotypic results besides being less expensive. This PCR method could be used in clinical laboratories for rapid identification and initiation of specific and effective treatment, reducing patient mortality and morbidity. Furthermore, this method may reduce misuse of antimicrobial classes that are more expensive and toxic, thus contributing to the selection of antibiotic-resistant Staphylococcus spp.

Characterization, prevalence and antibiogram study of Staphylococcus aureus in poultry

Objective To reveal the presence of methicillin resistant Staphylococcus aureus (S. aureus) (MRSA) in poultry samples and to determine the antibiogram pattern against five antibiotics. Methods Samples from different poultry farm of Chittagong city, Bangladesh were examined for S. aureus by different biochemical tests and confirmed as MRSA by identifying the presence of mecA gene using PCR. Antibiotic resistance pattern in S. aureus was determined by antibiotic disk diffusion method. Results In this study, a total of 60 samples (30 from nasal swabs and 30 from cloacal swabs) were used, of which 54 were confirmed as S. aureus by different biochemical tests. Among these, 12 were confirmed as MRSA by detecting mecA gene using PCR. During antibiogram study, both nasal and cloacal samples showed the highest resistance against penicillin-G and the lowest resistance was observed against neomycin. Conclusions Based on the present study, it can be said that different antibiotics are used extensively in poultry that leads to MRSA and is alarming for human health.

A Review of Staphylococcal Cassette Chromosome mec (SCCmec) Types in Coagulase-Negative Staphylococci (CoNS) Species

Coagulase-negative staphylococci (CoNS) are considered low pathogenic organisms. However, they are progressively causing more serious infections with time because they have adapted well to various antibiotics owing to their ability to form biofilms. Few studies have been conducted on CoNS in both, hospital and community-acquired settings, especially in Malaysia. Thus, it is important to study their species and gene distributions. A mobile genetic element, staphylococcal cassette chromosome mec (SCCmec), plays an important role in staphylococci pathogenesis. Among CoNS, SCCmec has been studied less frequently than Staphylococcus aureus (coagulase-positive staphylococci). A recent study (8) conducted in Malaysia successfully detected SCCmec type I to VIII as well as several new combination patterns in CoNS species, particularly Staphylococcus epidermidis. However, data are still limited, and further research is warranted. This paper provides a review on SCCmec types among CoNS species.

Occurrence and genetic characterization of Staphylococcus aureus in milk samples of cattle with mastitis, and in the Veterinary Hospital personnel and dairy workers / Ocorrência e caracterização genética de Staphylococcus aureus em amostras de leite de gado com mastite, na equipe de um Hospital Veterinário e em pessoas que trabalham com o gado leiteiro

Staphylococcus aureus is one of the most common microorganisms responsible for high morbidity and mortality in humans and animals. Methicillin-resistant S. aureus are responsible for several outbreaks worldwide and therapeutic arsenal has become scarce. The present investigation verified the epidemiological profile of S. aureus strains isolated from the veterinary hospital staff, from dairy cattle workers and also from milk samples of dairy cattle presenting mastitis. Samples were characterized phenotypically by antibiogram, catalase, and coagulase tests, and also by Voges-Proskauer test. The isolated strains were characterized genotypically by specific Polymerase Chain Reaction and Amplified Ribosomal DNA Restriction Analysis (ARDRA). From the 218 isolated strains, 27 were identified as S. aureus (12%), four of them were resistant to oxacillin and two of them were classified as MRSA (Methicillin-resistant S. aureus). The prevalence of isolated strains among animal personnel care was low (2%) but all MRSA isolates were found among the clinical staff. Results of ARDRA pointed out that S. aureus strains isolated from different animal care personnel were grouped in the same cluster when HindIII and HinfII restriction enzymes were used. When ARDRA was performed with HaeIII enzyme, the formation of two clusters was observed, but the isolated strains were not correlated. The prevalence of S. aureus strains isolated was higher in clinical staff and the biochemical and molecular assays of them presented 100% of correlation.(AU)

Staphylococcus aureus está entre os microrganismos que apresentam as maiores taxas de morbidade e mortalidade em seres humanos e animais. Linhagens de S. aureus resistentes a meticilina podem causar surtos de infecção em todo o mundo, o que contribui para a escassez de arsenal terapêutico. Este trabalho analisou o perfil epidemiológico de estirpes de S. aureus isoladas de pessoas que trabalham em contato com animais em um hospital veterinário com gado leiteiro e também em amostras de leite de vacas acometidas por mastite. As estirpes de S. aureus isoladas foram caracterizadas fenotipicamente por meio de antibiograma, testes de catalase e coagulase, e pelo teste de Voges-Proskauer. As amostras também foram caracterizadas genotipicamente pela técnica de Análise de Restrição de DNA Ribossômico Amplificado (ARDRA-PCR). Das 218 estirpes isoladas, 27 foram identificadas como S. aureus (12%). Entre essas, quatro estirpes foram resistentes à oxacilina e duas classificadas como SARM (S. aureus resistente à meticilina). A ocorrência de estirpes de S.aureus isoladas entre o pessoal que trabalha em contato com os animais foi baixa (2%), mas estirpes identificadas como SARM foram encontradas na equipe clínica. As análises de ARDRA realizadas com as enzimas de restrição HindIII e HinfII demonstraram que S. aureus isolados de diferentes indivíduos que trabalhavam com animais foram agrupados no mesmo cluster. Quando a ARDRA foi realizada com HaeIII foi observada formação de dois grupos, mas os isolados não se correlacionaram. Conclusão: a ocorrência de estirpes de S. aureus isoladas foi maior na equipe clínica, apresentando também correlação de 100% nos ensaios bioquímicos e moleculares.(AU)

Characterization, prevalence and antibiogram study of Staphylococcus aureus in poultry

Objective: To reveal the presence of methicillin resistant Staphylococcus aureus (S. aureus) (MRSA) in poultry samples and to determine the antibiogram pattern against five antibiotics. Methods: Samples from different poultry farm of Chittagong city, Bangladesh were examined for S. aureus by different biochemical tests and confirmed as MRSA by identifying the presence of mecA gene using PCR. Antibiotic resistance pattern in S. aureus was determined by antibiotic disk diffusion method. Results: In this study, a total of 60 samples (30 from nasal swabs and 30 from cloacal swabs) were used, of which 54 were confirmed as S. aureus by different biochemical tests. Among these, 12 were confirmed as MRSA by detecting mecA gene using PCR. During antibiogram study, both nasal and cloacal samples showed the highest resistance against penicillin-G and the lowest resistance was observed against neomycin. Conclusions: Based on the present study, it can be said that different antibiotics are used extensively in poultry that leads to MRSA and is alarming for human health.

Detección del gen mecA en cepas de Staphylococcus coagulasa positiva aisladas desde gatos / Detection of the mecA gene in coagulase positive Staphylococcus isolated from cats

Introduction: Bacterial resistance is a global concern for public health. Reports of antimicrobial resistance, including that against methicillin, have increased in strains of coagulase positive Staphylococcus (CPS) isolated from pets, however in Chile this information is limited. Objectives: To determine the antimicrobial susceptibility profiles and to detect the mecA gene in CPS strains isolated from cats in Chile. Materials and Methods : 134 samples were obtained from healthy cats and cats with skin lesions. These strains were characterized in their coagulase production and identified by BBL Crystal kit. The antimicrobial susceptibility was determined by Kirby Bauer method against 12 antimicrobials, including oxacillin. All strains were subjected to PCR to detect the mecA gene. Results: 72 CPS strains were isolated, including S. aureus and S. intermedius. Antimicrobial resistance against at least one drug was detected in strains from both healthy cats (75%) and from cats with skin lesions (87.5%). The mecA gene was detected in eight methicillin-resistant strains and also in three sensitive strains, being in general multi-resistant. Discussion: These results highlight the role of pets as reservoirs of bacterial resistance, and their potential impact on national public health.

Introducción: La resistencia bacteriana constituye un tema de preocupación para la salud pública mundial. Últimamente han aumentado los reportes de resistencia a antimicrobianos, incluida meticilina, en cepas de Staphylococcus coagulasa positiva (SCP) aisladas desde mascotas. Sin embargo, en Chile esta información es escasa. Objetivos: Determinar el perfil de susceptibilidad antimicrobiana y detectar el gen mecA en cepas de SCP aisladas desde gatos en Chile. Materiales y Métodos: Se obtuvieron 134 muestras desde gatos sanos y con lesiones dermatológicas. Las cepas fueron caracterizadas en su producción de coagulasa e identificadas mediante kit BBL Crystal. La susceptibilidad antimicrobiana se determinó mediante el método de Kirby Bauer ante 12 antimicrobianos, incluida oxacilina. Todas las cepas fueron sometidas a RPC para la detección del gen mecA. Resultados: 72 cepas de SCP fueron aisladas, incluyendo S. aureus y S. intermedius. Se detectó resistencia antimicrobiana a al menos un antimicrobiano en cepas de gatos sanos (75%) y de gatos con lesiones cutáneas (87,5%). El gen mecA fue detectado en ocho cepas resistentes a meticilina y en tres cepas sensibles, siendo en general multi-resistentes. Discusión: Estos resultados destacan el rol de las mascotas como reservorios de resistencia bacteriana y su potencial impacto en la salud pública.

Staphylococcus aureus con resistencia múltiple a los antibióticos (MDR) en un Hospital de Maracaibo- Venezuela / Multidrug-resistant (MDR) Staphylococcus aureus in a Maracaibo’s hospital, Venezuela

Staphylococcus aureus resistente a oxacilina (SAOR) continúa siendo una causa importante de infecciones nosocomiales en todo el mundo. Se determinó la resistencia a los antibióticos de cepas intrahospitalarias, clasificándolas en multidrogo-resistentes, extensamente drogo-resistentes o pandrogo-resistentes. Las muestras biológicas fueron recolectadas entre septiembre 2013-febrero 2014 y procesadas de acuerdo a técnicas de bacteriología convencional. La resistencia a los antibióticos se determinó mediante el método de difusión con discos en agar y el gen mecA se detectó mediante reacción en cadena de la polimerasa. Se observó baja prevalencia de SAOR intrahospitalario (13,86%). La mayor resistencia fue a eritromicina (66,07%), mientras que la resistencia frente a aminoglucósidos, fluoroquinolonas, clindamicina y tetraciclina fue inferior al 25%; la resistencia frente a trimetoprim/sulfametoxazol fue muy baja y el 100% de las cepas mostraron sensibilidad a rifampicina, linezolid, vancomicina y teicoplanina. El fenotipo de resistencia a MLSB más frecuente fue el de resistencia a eritromicina y susceptibilidad a clindamicina (33,93%, fenotipo MSB). Las cepas SAOR aisladas presentaron 25 antibiotipos diferentes, siendo la mayoría de los aislamientos multidrogo-resistentes (55,36%). No se observó resistencia extensa a los antibióticos ni pandrogo-resistencia y la presencia del gen mecA se demostró en todos los aislamientos resistentes a oxacilina.

Oxacillin-resistant Staphylococcus aureus (ORSA) has remained a major cause of nosocomial infections worldwide. The antibiotic resistance of isolations was determined and we classify them in multidrug-resistant, extensively drug-resistant or pandrug-resistant. The biological samples of patients from a Maracaibo’s Hospital, during September 2013 to February 2014, were processed according to conventional techniques of bacteriology. Antibiotic resistance was determined by disk diffusion method in agar and the mecA gene was detected by polymerase chain reaction. It was observed a low prevalence of nosocomial ORSA (13.86%). The higher antibiotic resistance was observed against erythromycin (66.07%) and a resistance lower than 25% to aminoglycosides, fluoroquinolones, tetracycline and clindamycin. The isolates showed a very low resistance to trimethoprim/sulfamethoxazole and all isolates were susceptible to rifampicin, linezolid, vancomycin and teicoplanin.The majority of isolates had a MSB phenotype (33.93%), with erythromycin resistance and susceptibility to clindamycin. The ORSA isolates in this study had 25 different antibiotypes and the majority of them were multidrug-resistant (55.36%). There was not both extensively drug-resistant and pandrug-resistant isolates and the presence of the mecA gene was demonstrated in all isolates of ORSA.

Cloning,prokaryotic expression,purification and identification of the transpeptidase domain of penicillin binding protein 2a of methicillin-resistant Staphylococcus aureus / 国际检验医学杂志

Objective To construct the prokaryontic expression vector of the gene fragment which encodes the transpeptidase domain of penicillin binding protein 2a(PBP2a) of methicillin‐resistant Staphylococcus aureus(MRSA) ,and to express ,purify and i‐dentify the objective protein .Methods Strains of MRSA were isolated and identified from clinical samples ,according to the se‐quence of mecA gene recorded in GenBank ,the primers of mecA fragment which encoded the transpeptidase domain of PBP2a was designed .The gene fragment from MRSA was amplified by using polymerase chain reaction(PCR) and cloned into pET28a(+ ) plasmid .After being identified by enzyme digestion and sequencing ,the recombinant plasmid was transformed into the strain of Escherichia coli BL21(DE3)plysS .The expression of transpeptidase domain of PBP2a was induced by 0 .7 mmol/L IPTG ,the ex‐pressed products were purified by using Ni afinity chromatography ,then were analyzed by using Western blot .Results The recom‐binant expression vector was digested by BamHⅠ and EcoRⅠ ,and the products were at the expected size .The result of sequencing showed two bases undergoing mutation ,while there were no frameshift mutations .The expressed protein was identified by using SDS‐PAGE and Western blot ,a new protein band was visible at the relative molecular mass of 38 × 103 .Conclusion The corre‐sponding prokaryotic expression vector is successfully constructed ,and the transpeptidase domain of PBP2a is successfully ex‐pressed and purified .

Preliminary study of biofilm formation properties and antibiotic susceptibility pattern of MRSA and MSSA isolates obtained in Yogyakarta, Indonesia

Aims: Nowadays, Staphylococcus aureus, especially methicillin resistant Staphylococcus aureus (MRSA), emerged as a major pathogenic agent of nosocomial infection and sepsis worldwide. Infections caused by these bacteria are often difficult to treat because of the development of antibiotic resistance. Biofilm formation is an important factor in the pathogenicity of staphylococcal infections and one of the reason of antibiotic treatment failure. In this study, the relationship between biofilm formation properties, the presence of mecA, icaA/D genes and antimicrobial resistance pattern were investigated in 10 methicillin sensitive Staphylococcus aureus (MSSA) and 10 MRSA clinical isolates. Methodology and results: Staphylococcal strains were identified by conventional microbiological methods, while determination of methicillin susceptibility was distinguished by the presence of mecA gene. To investigate biofilm production, congo red agar and microtiter plate test were performed. PCR was done to detect the presence of icaA/D genes, which responsible for biofilm production. Antibiotic sensitivity was carried out by agar diffusion method. The majority of MRSA isolates (90%) were not able to form biofilm, only one isolate (10%) showed capability of weak biofilm producer. Meanwhile, fully established biofilms were formed by all of MSSA isolates (100%). In addition, all MRSA and almost MSSA isolates (90%) harboured both icaA/D genes in their chromosomes. Antibiotic resistance profile of MRSA was more dominant than MSSA isolates. Conclusion, significance, and impact of study: Biofilm production of staphylococci showed difference regulation with regard to methicillin susceptibility. Antibiotic resistance profile was more dominant in MRSA, however biofilm production was found mostly in MSSA isolates.

Differences of Nuc-mecA Gene Carriage in the Noses among Different Altitudes in High Humidity District / 现代检验医学杂志

Objective To investigate the carriage of nuc-mecA gene among different altitudes in high humidity district,provid-ed guiding data for prevention of staphylococcus aureus and drug-resistant bacteria,standardizing the usage for antibiotics. Methods The nose swabs were collected in different altitudes:1 000 m,1 200 m and 1 400 m,nuc-mecAgene was confirmed by multi-channel real-time PCR.Results The carrier of nuc gene in the noses were 4.878%,2.899% and 7.143%,in 1 000 m,1 200 m and 1 400 m respectively,and there were no statistical significant among the altitudes (P>0.05).The carrier of mecA gene were 14.634%,31.884% and 41.837% in the 1 000 m,1 200 m and 1 400 m respectively,the difference showed statistical significe (P0.05).Conclusion The carrier of mecA gene in noses was increased with the increasing of the altitude. The residents who living at higher altitude should keep the colonization sites of pathogens clean,and needed timely medical when got sick,shouldn’t abuse the antibiotics without authorization.Medical staff should rational use of antibiotic drugs,a-voided overusing of antibiotics and overtreatment.

Rapid monitoring and prevention of methicillin-resistant Staphylococcus aureus in ICU / 国际检验医学杂志

Objective To understand the colonization ,distribution and prevention of methicillin‐resistant Staphylococcus aureus (MRSA) in ICU .Methods The nasal vestibule swabs were immediately taken from new patients in ICU and the sputum suction catheter specimens were collected from the patients conforming to respiratory tract infection at 72 h after entering ICU .The fluores‐cence PCR was adopted to rapidly detect mecA ,nuc gene .The related environment specimens such as nasal cavity and hands in med‐ical staffs were performed the regular tracking monitoring ,prevention and control .Results The MRSA‐detection rates were 9 .9%for nasal vestibule swabs ,2 .7% for sputum suction catheter ;7 .1% for nasal vestibule swabs of medical staffs ,4 .0% for hands and 4 .4% for cuffs;the medical staffs and patient′s nasal cavity with MRSA‐positive adopted mupirocin for scrubing ,MRSA detection rate in hands after reinforce disinfection was 0 .0% ,but nasal cavity MRSA in medical staff was still detected out every quarter . Conclusion The PCR method has significantly higher positive detection rate compared with the conventional culture method .Nasal vestibule is a major colonization site of M RSA and the main infection source of infection ,the hands are the important route of trans‐mission ,monitoring nasal vestibule in ICU patients and medical staffs and hands in medical staffs is important to control MRSA nosocomial infection .

Portación de Staphylococcus aureus multiresistentes a antimicrobianos en cavidad bucal de niños que concurren para un tratamiento en una clínica odontológica, Paraguay / Carriage of Multidrug-resistant Staphylococcus aureus in the Oral Cavities of Children Presenting for Treatment at a Dental Clinic in Paraguay

Introducción: Staphylococcus aureus es un microorganismo que posee características particulares de virulencia y resistencia a los antibióticos para uso clínico cuya diseminación es de gran importancia en salud pública. Objetivo: Identificar S. aureus sensibles y resistentes a antimicrobianos, colonizantes en cavidad bucal de niños que concurrieron para un tratamiento a una Clínica Odontológica en Asunción. Materiales y Métodos: En este estudio descriptivo, observacional realizado de agosto a septiembre de 2013, se incluyeron niños de 2 a 15 años de edad. Hisopados de cavidad bucal fueron colectados y cultivados en agar-manitol-sal. Los aislados de S. aureus fueron caracterizados respecto a la susceptibilidad a 11 antibióticos y por métodos moleculares se buscó la presencia del gen mecA y del factor de virulencia pvl. Se empleó un cuestionario sobre datos socio-demográficos y factores de riesgo asociados a infección con S. aureus. Resultados: Se incluyeron 112 niños. Se aisló S. aureus en 37 (33%) hisopados y 35 (94,6%) aislados presentaban resistencia a al menos un antibiótico. Resultaron resistentes a: Penicilina (89%), Cloranfenicol (16,2%), Oxacilina (10,8%), Eritromicina (8%), Clindamicina (8%), Gentamicina (5,4%) y un aislado con resistencia intermedia a Ciprofloxacina (2,7%). Cuatro aislados de S. aureus presentaban resistencia a múltiples drogas, los mismos portaban el gen mecA. No se detectó portación del factor de virulencia PVL. Conclusión: La elevada portación en cavidad oral de S. aureus multiresistentes en niños sin factores de riesgo como hospitalización previa o consumo de antibióticos, implica un alto riesgo para posibles infecciones endógenas, además del potencial de transferencia de determinantes de resistencia entre gérmenes de flora bucal normal .

Introduction: Staphylococcus aureus isa bacterium with special characteristicsof virulence and resistance to theantibiotics used in clinical practice, andwhose spread presents a significantpublic health challenge...

Impairments of mecA gene detection in bovine Staphylococcus spp.

Staphylococcus aureus antimicrobial resistance, especially to beta-lactams, favors treatment failures and its persistence in herd environment. This work aimed to develop a more specific primer for mecA gene detection based on the comparison of the conserved regions from distinct host origins and also investigated the presence of homologue mecA LGA251 in bovine strains. A total of 43 Staphylococcus spp. were included in this study, comprising 38 bovine S. aureus, two human and three equine coagulase-negative staphylococci (CNS). Phenotypical methicillin-resistance detection was performed through oxacillin agar-screening and cefoxitin disk-diffusion test. None isolate tested positive for mecA LGA251 gene. For mecA gene PCR, new primers were designed based on the sequences of human S. aureus (HE681097) and bovine S. sciuri (AY820253) mecA. The new primers based on the S. aureus mecA sequence amplified fragments of human and equine CNS and the ones based on S. sciuri mecA sequence only yielded fragments for S. aureus bovine strains. Multiples alignments of mecA gene sequences from bovine, human and equine revealed punctual but significant differences in bovine strains that can lead to the mecA gene detection impairment. The observed divergences of mecA gene sequences are not a matter of animal or human origin, it is a specificity of bovine samples.

PCR-based identification of methicillin-resistant Staphylococcus aureus strains and their antibiotic resistance profiles

<p><b>OBJECTIVE</b>To evaluated the PCR for mecA gene compared with the conventional oxacillin disk diffusion method for methicillin-resistant Staphylococcus aureus (S. aureus) identification.</p><p><b>METHODS</b>A total of 292 S. aureus strains were isolated from various clinical specimens obtained from hospitalized patients. Susceptibility test to several antimicrobial agents was performed by disk diffusion agar according to Clinical and Laboratory Standards Institute guidelines. The PCR amplification of the mecA gene was carried out in all the clinical isolates.</p><p><b>RESULTS</b>Among antibiotics used in our study, penicillin showed the least anti-staphylococcal activity and vancomycin was the most effective. The rate of methicillin-resistant S. aureus prevalence determined by oxacillin disk diffusion method was 47.6%; whereas, 45.1% of S. aureus isolates were mecA- positive in the PCR assay.</p><p><b>CONCLUSIONS</b>This study is suggestive that the PCR for detection of mecA gene is a fast, accurate and valuable diagnostic tool, particularly in hospitals in areas where methicillin-resistant S. aureus is endemic.</p>

PCR-based identification of methicillin-resistant Staphylococcus aureus strains and their antibiotic resistance profiles

Objective: To evaluated the PCR for mecA gene compared with the conventional oxacillin disk diffusion method for methicillin-resistant Staphylococcus aureus (S. aureus) identification. Methods: A total of 292 S. aureus strains were isolated from various clinical specimens obtained from hospitalized patients. Susceptibility test to several antimicrobial agents was performed by disk diffusion agar according to Clinical and Laboratory Standards Institute guidelines. The PCR amplification of the mecA gene was carried out in all the clinical isolates.Results:activity and vancomycin was the most effective. The rate of methicillin-resistant S. aureus prevalence determined by oxacillin disk diffusion method was 47.6%; whereas, 45.1% of S. aureus isolates were mecA- positive in the PCR assay. Among antibiotics used in our study, penicillin showed the least anti-staphylococcal Conclusions: This study is suggestive that the PCR for detection of mecA gene is a fast, accurate and valuable diagnostic tool, particularly in hospitals in areas where methicillin-resistant S. aureus is endemic.