The inhibition effect of Chlorpromazine against the β-lactam resistance of MRSA

OBJECTIVE@#To investigate the gene related to β-lactam resistance and to confirm the mechanism about a synergy effect between CPZ and β-lactam antibiotics.@*METHODS@#To measure antibacterial activity, we performed a minimum inhibitory concentration (MIC) and synergy test. Transmission electron microscopy (TEM) was used in morphological analysis. To analyze gene expression, we conducted reverse transcriptase polymerase chain reaction (PCR).@*RESULTS@#We confirmed a synergy effect between CPZ and β-lactam antibiotics. Furthermore, we observed that CPZ affect the cell envelope of MRSA by using TEM. At the gene level, CPZ reduced the expression of resistance genes.@*CONCLUSIONS@#Through this result, we hypothesize that a decrease of resistance factor expressions was caused by CPZ because it disrupts the activity of a sensor protein located in the cell membrane.

The inhibition effect of Chlorpromazine against the β-lactam resistance of MRSA

Objective To investigate the gene related to β-lactam resistance and to confirm the mechanism about a synergy effect between CPZ and β-lactam antibiotics. Methods To measure antibacterial activity, we performed a minimum inhibitory concentration (MIC) and synergy test. Transmission electron microscopy (TEM) was used in morphological analysis. To analyze gene expression, we conducted reverse transcriptase polymerase chain reaction (PCR). Results We confirmed a synergy effect between CPZ and β-lactam antibiotics. Furthermore, we observed that CPZ affect the cell envelope of MRSA by using TEM. At the gene level, CPZ reduced the expression of resistance genes. Conclusions Through this result, we hypothesize that a decrease of resistance factor expressions was caused by CPZ because it disrupts the activity of a sensor protein located in the cell membrane.

Evaluation of different methods for detecting methicillin resistance in staphylococcus aureus isolates in a university hospital located in the northeast of Brazil

Many methods have been described for the detection of methicillin-resistant Staphylococcus aureus (MRSA), but the heterogeneous expression of methicillin resistance affects the reliability of these methods. The aim of the present study was to evaluate some methods for detecting methicillin resistance in Staphylococcus aureus isolates in a university hospital located in the Northeast of Brazil. Among the isolates, 15 were methicillin-susceptible and 45 were methicillin-resistant, including low-level heterogeneous resistance strains. Both the 30 µg-cefoxitin disk and PBP2a test had 100 percent sensibility/specificity and appear to be good options for the detection of MRSA in the clinical laboratory.

Different Responses of MSSA and MRSA to Oxacillin of Their Respective MICs

All of the methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit resistance to oxacillin by producing PBP2a encoded by mecA, whereas methicllin-susceptible Staphylococcus aureus (MSSA) strains do not. To investigate phenotypic differences other than oxacillin resistance level in responses to oxacillin between MSSA and MRSA, we compared alterations of viability and ultrastructure of MSSA by oxacillin treatment with those of MRSA. When MSSA and MRSA strains were exposed to oxacillin of their respective MICs, and then were assayed for viability and observed by transmission electron microscope, increase in thickness of cell wall was more prominent in MRSA strains than in MSSA strains, while decrease in number of surviving cells was more evident and change in morphology of growing cross wall was greater in MSSA strains than in MRSA strains. It is assumed that these different responses to oxacillin between MSSA and MRSA strains may be due to activation of some PBP2a unbound to oxacillin. In conclusion, MSSA and MRSA showed different functional and morphological responses to oxacillin, although they were treated with oxacillin of concentrations that respectively inhibit their proliferation.

Caracterización fenotípica de cepas de Staphylococcus coagulasa negativa aisladas de una unidad de alto riesgo neonatal / Phenotypic characterization of coagulase-negative Staphylococcus strains isolated from a high risk neonatal unit

Las infecciones nosocomiales constituyen un problema de salud pública, debido a las altas tasas de morbimortalidad que ocasiona y por los altos costos económicos que generan. Las unidades de cuidados intensivos son una de las principales áreas donde se registra una alta incidencia de infección nosocomial, siendo la sepsis la principal infección en la cual se involucran una gran variedad de microorganismos. El grupo de Staphylococcus coagulasa negativa (SCN), es uno de los agentes etiológicos más frecuentemente aislados. De ahí nuestro interés en realizar la caracterización de 32 cepas de SCN aisladas de neonatos con infección nosocomial en la Unidad de Alto Riesgo Neonatal (UARN) del Instituto Autónomo Hospital Universitario de Los Andes (IAHULA), Mérida, Venezuela; durante el período diciembre 1997-Abril 1999. Los resultados muestran que el aislamiento de SCN fue de 47,37 por ciento. El 78,1 por ciento de las cepas estudiadas se aislaron de neonatos con bacteremia. Las especies más frecuentes fueron S. epidermidis (46,9 por ciento) y S. warneri (34,4 por ciento). Todas las cepas evaluadas mostraron resistencia a la penicilina y en un 18,8 por ciento de ellas mediada por la producción de B-lactamasa. El 68,8 por ciento de las cepas fueron resistentes a oxacilina y el 78,1 por ciento a gentamicina. La mayoría de las cepas resistentes a oxacilina mostraron valores de CIM g/mL, y se detectó la presencia de la PBP2a. Ninguna de las cepas fueron hiperproductoras de B-lactamasa. Se observó una excelente actividad de la vancomicina y quinupristin-dalfopristin sobre todas las cepas SCN evaluadas. Debido al papel que tienen los SCN en la UARN del IAHULA, es necesario extremar las medidas de asepsia durante los procedimientos de diagnóstico y terapéuticos invasivos, con el propósito de evitar las infecciones causadas por este grupo de microorganismos.

Nosocomial infections constitute a public health problem due to a high level of morbidity and mortality, generating high health-care costs in hospitals. Intensive care units are the principal areas where a high incidence of nosocomial infections is reported. Bacterimia is the principal infection involving a large variety of microorganisms; coagulase-negative Staphylococcus (CNS) is one of the most frequently isolated pathogens. Therefore, the purpose of this study was to characterize the 32 strains of CNS isolated from neonates with nosocomial infections in the High Risk Neonatal Unit (HRNU) at the University of the Andes Hospital Autonomous Institute (UAHAI), Mérida, Venezuela, from December 1997 to April 1999. Results showed that the isolation of CNS was 47.37 percent; 78.1 percent of the species were isolated from neonates with bacteremia. S. epidermidis (46.9 percent), and S. warneri (34.4 percent) were the species most frequently found. All pathogens showed resistance to penicillin and 18.8 percent of them produced ß-lactamase; 68.8 percent were resistant to oxacillin and 78.1 percent to gentamicin. Most of the oxacillin-resistant strains showed MIC values above 0.5 mg/mL and the presence of PBP2a was detected. None of the strains were hyper-producers of ß-lactamase. Vancomicin and quinuprintin/dalfopristin showed excellent activity against these CNS. Due to the role of CNS as a pathogen in the HRNU of UAHAI, strong asepsis measures during diagnosis and therapeutic invasive procedures must be taken to prevent infections caused by this group of microorganisms.

Estafilococos oxacilino resistentes en queso blanco fabricado en el estado Anzoátegui, Venezuela / Oxacyllin resistant Staphylococcus in white cheese manufactured at the Anzoategui State, Venezuela

El objetivo de esta investigación fue detectar especies estafilocócicas oxacilino resistentes en quesos blanco duro y blando, fabricados artesanalmente en diversos municipios de la zona norte del estado Anzoátegui. Las cepas de Staphylococcus fueron aisladas e identificadas empleando métodos convencionales y se confirmaron las especies coagulasa negativas mediante galerías API 32 Staph (BioMérieux); la susceptibilidad a oxacilina y otros antibióticos por el método de difusión en disco y la presencia del gen mecA, por prueba de látex MRSA Slidex (BioMérieux) que pone de manifiesto la proteína PBP2a. De 130 quesos evaluados, se aislaron 171 cepas estafilocócicas, 26 (15,2%) resistentes a oxacilina (61,5% con presencia del gen), distribuidas como sigue: 14 cepas de S. epidermidis (8,2%), 6 de S. capitis (3,4%), 2 de S. aureus (1,2%), 2 de S. hominis (1,2%), 1 de S. warneri (0,6%) y 1 de S. saprophyticus (0,6%). La presencia del gen que codifica para la proteína PBP2a se detectó tanto en cepas con homorresistencia (76,9%) como con heterorresistencia (23,1%). S. haemolitycus fue 100% sensible a oxacilina. La presencia de cepas estafilocócicas oxacilino resistentes en el queso blanco, puede representar un riesgo para salud pública ya que podría servir como fuente de diseminación de éstas y acrecentar el problema de la resistencia.

The purpose of this study was to detect oxacyllin resistant Staphylococcus species in hard and soft white cheese manufactured by artisans at various municipalities of the northern area of Anzoátegui State. The Staphylococcus strains were isolated and identified using conventional methods and coagulase negative species were confirmed through API 32 Staph (BioMérieux) galleries; susceptibility to oxacyllin and other antibiotics was determined by the disk diffusion method and presence of the mecA gene with the MRSA Slidex (BioMérieux) latex test that identifies PBP2a protein. From 130 cheeses evaluated, we isolated 171 Staphylococcus strains, 26 of which (15,2%) were oxacyllin-resistant (61,5% with presence of the gene), distributed as follows: 14, S. epidermidis strains (8,2%); 6, S. capitis (3,4%); 2, S. aureus (1,2%); 2, S. hominis (1,2%); 1, S. warneri (0.6%); and 1, S. saprophyticus (0.6%). Presence of the protein PBP2a codifying gene was detected in both homoresistant (76.9%) and heteroresistant (23.1%) strains. S. haemolitycus was 100% oxacyllin sensitive. Presence of oxacyllin resistant strains in white cheese can represent a public health risk since it could serve as a dissemination source, increasing the resistance problem.

Asociación de los genes implicados en la codificaciónde proteina de unión a la penicilina 2a (pbp2a) conla expresión fenotípica de resistencia a la meticilinaen cepas De staphylococcus spp / Association of genes involved in coding binding protein penicillin 2a (PBP2a) with phenotypic expression of methicillin resistance in strains of Staphylococcus spp

Objetivo: Determinar si existe asociación entre genes implicados en la codificación de PBP2a con la expresión fenotípica de resistencia a meticilina en cepas de Staphylococcus spp. Diseño: Descriptivo Transversal. Metodologias: e determinó la resistencia y sensibilidad de 67 aislamientos, mediante pruebas fenotípicas (difusión en disco, concentración inhibitoriamínima CIM, producción de PBP2a y pruebas genotípicas para detectar los genes mecA y sus reguladores mecR1 y mecI por Reacción em Cadena de la Polimerasa (PCR). Resultados: De 9 cepas de S. aureus resistentes por difusión en disco solo 1 fue sensible por CIM. De 7 cepas resistentes por CIM, fueron sensibles por difusión en disco. Por el contrario las 7 cepas de Staphylococcus coagulasa negativo sensibles por difusión en disco fueron resistentespor CIM.En cuanto a la prueba de producción de PBP2a, los resultados fueron discordantes con la prueba de difusión en disco en 20..

Objective: Determining the association between genes involved in the codifi cation of Penicillin Binding Proteins 2A (PBP2A) with the phenotypic expression of methicillin resistance in Staphylococcus spp. Strains Design: Descriptive cross sectional Methodology: The sensitivity of 67 isolates was determined by means of a phenotypic test (disk diffusion, minimum inhibitory concentration CIM, production of PBP2a) and genotype tests to detect the mecA gene and its regulatory mecR1 and mecI by Polymerase Chain Reaction (PCR). Results: From 9 S. aureus resistant strains by disk diffusion 1 was sensitive by CIM, 7 CIM resistant strains were sensitive by disk diffusion. The 7 coagulase negative (CNS) sensitive strains by disk diffusion were resistant by CIM. By production of PBP2a, the results were discordant with the disk diffusion test in 20% and 34%with CIM. The genotype, reveals that, from 60 S.aureus strains 10(17%), and 7 S. coagulase negative strains 4 (57%) carry the mecA gene. From 10 S. aureus mecA positive strains, 5 carry the mecR1 gene and 7 carry the mecI gene. Of the 4 strains of S.coagulase negative mecA positive 2 carry the mecR1 and 2 carry the mecI gene. Conclusion: There is no association between genotype and phenotype in Staphylococcus spp. methicillin resistant strains, since, the resistance is due to many factors that the classical phenotypic test does not include.

Evaluation of the humoral immune response in BALB/c mice immunized with a naked DNA vaccine anti-methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is the major pathogen involved in nosocomial infections, leading to high rates of morbidity and mortality in hospitals worldwide. The methicillin resistance occurs due to the presence of an additional penicillin-binding protein, PBP2a, which has low affinity for b-lactam antibiotics. In the past few years, vancomycin has been the only antibiotic option for treatment of infections caused by multiresistant MRSA; however, reports of vancomycin-resistant strains have generated great concerns regarding the treatment to overcome these infections. In the present study, we report preliminary results regarding the humoral immune response generated in BALB/c mice by two different doses of naked DNA vaccine containing an internal region, comprising the serine-protease domain, of the PBP2a of MRSA. The immunization procedure consisted of four immunizations given intramuscularly within 15-day intervals. Blood was collect weekly and anti-PBP2a-specific antibodies were screened by ELISA. BALB/c mice immunized with DNA vaccine anti-PBP2a have shown higher antibody titers mainly after the fourth immunization, and intriguingly, no correlation between the humoral immune response and DNA dose was observed. Our results suggest that the DNA vaccine anti-PBP2a induced an immune response by production of specific antibodies anti-MRSA in a non-dose-dependent manner, and it could represent a new and valuable approach to produce specific antibodies for passive immunization to overcome MRSA infections.

Evaluation of Various Methods for Detection of Methicillin Resistance Staphylococcus aureus (MRSA) / 대한임상미생물학회지

BACKGROUND: Traditional antimicrobial susceptibility test methods for detection of methicillin resistant Staphylococcus aureus(MRSA) require 24 hours to perform. In addition, accuracies of these methods can be influenced by prevalence of strains that express heterogeneous resistance. The mechanism of methicillin resistance in S. aureus is based on the production of an additional lowaffinity penicillin binding protein (PBP 2a), which is encoded by mecA gene. Therefore, PCR for mecA gene and immunological methods for PBP 2a could be used to determine resistance, but most clinical laboratories do not have resources to efficiently perform these technique on routine basis. Recently, slide latex agglutination test using latex particles sensitized with a monoclonal antibody against PBP 2a for the direct detection of PBP 2a was developed. In this study, we evaluated this new latex agglutination test, and compared to oxacillin disk diffusion test and PCR detection of mecA gene for detection of MRSA. METHODS: A total 151 clinical isolates of coagulase positive S. aureus were selected. All isolates were subjected to "blinded"testing with oxacillin disk diffusion, PBP 2a latex agglutination, and mecA PCR for detection of MRSA. RESULTS: Of 151 S. aureus, 116 (76.8%) strains were MRSA. The sensitivities and specificities of disk diffusion, latex agglutination and PCR were 94.0 and 91.4%, 97.4 and 100%, 98.3 and 100%, respectively. CONCLUSIONS: PCR for detection of mecA gene and latex agglutination test for PBP 2a are more sensitive and specific methods for detection of MRSA than oxacillin disk diffusion test. Latex agglutination test is rapid, simple, and easier to perform than PCR. In conclusion, PBP 2a detection with latex agglutination test has the potential to be used for routine applications in the microbiology laboratory where mecA gene detection with PCR is not readily available.

Detection of MRCNS and analysis of their drug resistance / 重庆医科大学学报

0.5).The resistance rates of MRCNS to PEN,GEN,SXT,CIP,TET,ALL and ERY,are all above 50%.The resistant rates of MSCNS to other antibiotics are below about 50% except for PEN(80.4%)and ERY(57.1%).MRS are more than 70% and no VAN resistant in CNS.Conclusion:All the 5 methods are suitable for clinical detection of MRCNS ,and latex agglutination is the most convenient and rapid.MRCNS had a serious cross-resistance in aminoglycosides,guinolones,tetracycline,macrolides,sulfanilamide and,so the best choice for therapy is VAN.GEN,SXT,CIP,TET and CLI could be used for MSCNS,except VAN.