Antibacterial effect of isoeugenol against Pseudomonas aeruginosa

Abatsract Pseudomonas aeruginosa is an important nosocomial pathogen and its clinical importance is mainly related to nosocomial infections. Increased rates of bacterial resistance in recent years has led WHO to publish a global priority list to guide research and discovery of new antibiotics, where P. aeruginosa is among the group of bacteria for which there is a critical level of priority for new drugs to be discovered. In this context, isoeugenol appears as an interesting alternative and the objective of this study was to investigate its action against P. aeruginosa. Isoeugenol presented significant antibacterial activity, with minimum inhibitory concentration (MIC) of 64µg/mL and minimum bactericidal concentration (MBC) of 128µg/mL, and was considered bactericidal against this species. Molecular docking revealed interactions that suggest that isoeugenol may bind to the enzyme Penicillin-Binding Protein 3 and interfere with the bacterial cell wall synthesis process. This study reinforces the antibacterial potential of this compound and emphasizes that more studies are needed in order to better investigate its mechanism of antibacterial action.

Coagulase-negative staphylococci: a 20-year study on the antimicrobial resistance profile of blood culture isolates from a teaching hospital

ABSTRACT The increasing rates of nosocomial infection associated with coagulase-negative staphylococci (CoNS) were the rationale for this study, aiming to categorize oxacillin-resistant CoNS species recovered from blood culture specimens of inpatients at the UNESP Hospital das Clínicas in Botucatu, Brazil, over a 20-year period, and determine their sensitivity to other antimicrobial agents. The mecA gene was detected in 222 (74%) CoNS samples, and the four types of staphylococcal chromosomal cassette mec (SCCmec) were characterized in 19.4%, 3.6%, 54.5%, and 14.4% of specimens, respectively, for types I, II, III, and IV. Minimal inhibitory concentration (MIC) values to inhibit 50% (MIC50) and 90% (MIC90) of specimens were, respectively, 2 and >256 µL/mL for oxacillin, 1.5 and 2 µL/mL for vancomycin, 0.25 and 0.5 µL/mL for linezolid, 0.094 and 0.19 µL/mL for daptomycin, 0.19 and 0.5 µL/mL for quinupristin/dalfopristin, and 0.125 and 0.38 µL/mL for tigecycline. Resistance to oxacillin and tigecycline and intermediate resistance to quinupristin/dalfopristin were observed. Eight (2.7%) of all 300 CoNS specimens studied showed reduced susceptibility to vancomycin. Results from this study show high resistance rates of CoNS to antimicrobial agents, reflecting the necessity of using these drugs judiciously and controlling nosocomial dissemination of these pathogens.

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.

First Case in Korea of Group B Streptococcus With Reduced Penicillin Susceptibility Harboring Amino Acid Substitutions in Penicillin-Binding Protein 2X

No abstract available.

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.

Bases moleculares de la resistencia a meticilina en Staphylococcus aureus / Molecular basis of methicillin-resistance in Staphylococcus aureus

Resumen Desde el inicio de la era antimicrobiana se han ido seleccionando gradualmente cepas de Staphylococcus aureus resistentes a antimicrobianos de amplio uso clínico. Es así como en 1960 se describen en Inglaterra las primeras cepas resistentes a meticilina, y algunos años después son informadas en hospitales de Chile. Actualmente, S. aureus resistente a penicilinas antiestafilocóccicas es endémico en los hospitales de nuestro país y del mundo, siendo responsable de una alta morbimortalidad. La resistencia es mediada habitualmente por la síntesis de una nueva transpeptidasa, denominada PBP2a o PBP2' que posee menos afinidad por el β-lactámico, y es la que mantiene la síntesis de peptidoglicano en presencia del antimicrobiano. Esta nueva enzima se encuentra codificada en el gen mecA, a su vez inserto en un cassette cromosomal con estructura de isla genómica, de los cuales existen varios tipos y subtipos. La resistencia a meticilina se encuentra regulada, principalmente, por un mecanismo de inducción de la expresión del gen en presencia del β-lactámico, a través de un receptor de membrana y un represor de la expresión. Si bien se han descrito mecanismos generadores de resistencia a meticilina mec independientes, son categóricamente menos frecuentes.

Staphylococcus aureus isolates resistant to several antimicrobials have been gradually emerged since the beginning of the antibiotic era. Consequently, the first isolation of methicillin-resistant S. aureus occurred in 1960, which was described a few years later in Chile. Currently, S. aureus resistant to antistaphylococcal penicillins is endemic in Chilean hospitals and worldwide, being responsible for a high burden of morbidity and mortality. This resistance is mediated by the expression of a new transpeptidase, named PBP2a or PBP2', which possesses lower affinity for the β-lactam antibiotics, allowing the synthesis of peptidoglycan even in presence of these antimicrobial agents. This new enzyme is encoded by the mecA gene, itself embedded in a chromosomal cassette displaying a genomic island structure, of which there are several types and subtypes. Methicillin resistance is mainly regulated by an induction mechanism activated in the presence of β-lactams, through a membrane receptor and a repressor of the gene expression. Although mec-independent methicillin resistance mechanisms have been described, they are clearly infrequent.

Portación nasal deStaphylococcus aureus y sus perfiles de resistencia a antibióticos en niños que viven en zonas de gran altitud del sudoeste de China / Staphylococcus aureus nasal carriage and its antibiotic resistance profiles in children in high altitude areas of Southwestern China

Antecedentes/Objetivo. Describir el perfil epidemiológico de la portación nasal de cepas de Staphylococcus aureus (S. aureus), su resistencia a antibióticos y la presencia de los genes de leucocidina de Panton-Valentine (LPV) y mecA en niños en edad escolar que viven en zonas de gran altitud del sudoeste de China. Métodos. En el estudio transversal, se analizaron hisopados nasales de estudiantes a fin de detectar S. aureus. Se realizó la prueba de la reacción en cadena de la polimerasa (RCP) para identificar los genes de LPV y mecA. Resultados. Del total de 314 niños, se detectó S. aureus en el 5,10% (16/314). La resistencia de las cepas aisladas a la penicilina, eritromicina, clindamicina, rifampicina y cefoxitina fue del 100%, 81,3%, 81,3%, 0,0% y 6,3%, respectivamente. Ninguna de las cepas mostró resistencia a la vancomicina. Se detectó la expresión del gen mecA en 3 cepas aisladas, y 10 cepas aisladas dieron resultado positivo para el gen de LPV. Conclusión. Se detectó S. Aureus en el 5,10% (16/314) de la población del estudio; el 0,96% (3 /314) presentó S. Aureus resistente a la meticilina (SARM). Además, se detectó la expresión de los genes de LPV y mecA en 10 y 3 cepas aisladas, respectivamente.

Background/Aim. To describe the epidemiological profile of nasal carriage of Staphylococcus aureus (S. aureus) strains, its antibiotic resistance and mecA and Panton Valentine leukocidin (PVL) genes presence, in school children residing in high altitude areas of Southwestern China. Methods. The cross sectional study screened nasal swabs taken from students for S. aureus. PCR was performed to identify mecA and PVL genes. Results. Of the total 314 children 5.10% (16/314) was detected S. aureus. The resistance of isolated strains to penicillin, erythromycin, clindamycin, rifampicin and cefoxitin was 100%, 81.3%, 81.3%, 0.0%, and 6.3% respectively. No strains demonstrated resistance to vancomycin; expression of mecA gene was detected in 3 isolates and 10 isolates were PVL-positive. Conclusion. S. aureus was detected in 5.10% (16/314) of the study population; 0.96% (3/314) had methicillin resistant S. aureus (MRSA); expression of the mecA and PVL genes were detected in 3 and 10 isolates respectively.

Staphylococcus aureus adquiridos en la comunidad: caracterización clínica, fenotípica y genotípica de aislados en niños paraguayos / Community-acquired Staphylococcus aureus isolated from Paraguayan children: clinical, phenotypic and genotypic characterization

Introduction: The prevalence of Staphylococcus aureus in the community has increased, being the pediatric population the most affected. This fact highlights the need for epidemiological surveillance. Aim: To characterize clinical, phenotypic and genotypic isolates of S. aureus children’s samples with community-acquired infections, collected in hospitals of Asuncion and the Central Department, between November 2009 and December 2010. Materials and Methods: Descriptive and transverse analysis with analytical component. Clinical data collected by medical records, antibiotic susceptibility according to CLSI criteria and detection of mecA (encoding methicillin resistance) and luk-PV genes (encoding Panton Valentine leucocidin) by PCR using specific oligonucleotides. Results: 123 isolates of S. aureus, 76% came from skin and soft tissue infections and 20% from sepsis. 18.7% (n = 23) were resistant to methicillin (MRSA). The presence of the mecA gene, a variant there and the PVL was detected in 12.2 and 48 isolates respectively. 43% of MRSA (n = 10) was carrying luk-PV. The clinical and demographic differences between patients infected with MRSA or MSSA were not statistically significant. Discussion: This study constitutes the first phenotypic and genotypic characterization of S. aureus associated with pediatric patients in Paraguay.

Introducción: La prevalencia de infecciones por Staphylococcus aureus en la comunidad ha aumentado, siendo la población pediátrica la más afectada; poniendo de relieve la necesidad de una vigilancia epidemiológica. Objetivo: Caracterizar clínica, fenotípica y genotípicamente aislados de S. aureus de muestras de niños con infecciones adquiridas en la comunidad, recolectadas en hospitales de Asunción y el Departamento Central, entre noviembre de 2009 y diciembre de 2010. Materiales y Métodos: Estudio descriptivo de corte trasverso. Datos clínicos fueron recabados de fichas, la susceptibilidad a antimicrobianos se hizo según criterio del CLSI y la detección de genes mecA y luk-PV se realizó por RPC empleando oligonucleótidos específicos. Resultados: De 123 aislados de S. aureus, 76% provenían de infecciones de piel y tejidos blandos y 20% de pacientes con bacteriemias. 18,7% (n: 23) fueron resistentes a meticilina (SARM). Se detectó la presencia de genes mecA, una variante del mismo y luk-PV en 9,8%, 1,6 y 39% de los aislados, respectivamente. El 43% de los SARM (n: 10) fue portador de luk-PV. Las diferencias clínicas y demográficas entre pacientes infectados por SARM o SASM no fueron estadísticamente significativas. Discusión: Este estudio constituye la primera caracterización clínica, fenotípica y genotípica de S. aureus asociados a la comunidad en población pediátrica realizada en Paraguay.

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.

Synergistic Anti-bacterial Effects of Phellinus baumii Ethyl Acetate Extracts and beta-Lactam Antimicrobial Agents Against Methicillin-Resistant Staphylococcus aureus

BACKGROUND: The development of new drugs or alternative therapies effective against methicillin-resistant Staphylococcus aureus (MRSA) is of great importance, and various natural anti-MRSA products are good candidates for combination therapies. We evaluated the antibacterial activities of a Phellinus baumii ethyl acetate extract (PBEAE) and its synergistic effects with beta-lactams against MRSA. METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the PBEAE. The PBEAE synergistic effects were determined by evaluating the MICs of anti-staphylococcal antibiotic mixtures, with or without PBEAE. Anti-MRSA synergistic bactericidal effects of the PBEAE and beta-lactams were assessed by time-killing assay. An ELISA was used to determine the effect of the PBEAE on penicillin binding protein (PBP)2a production. RESULTS: The MICs and MBCs of PBEAE against MRSA were 256-512 and 1,024-2,048 microg/mL, respectively. The PBEAE significantly reduced MICs of all beta-lactams tested, including oxacillin, cefazolin, cefepime, and penicillin. However, the PBEAE had little or no effect on the activity of non-beta-lactams. Time-killing assays showed that the synergistic effects of two beta-lactams (oxacillin and cefazolin) with the PBEAE were bactericidal in nature (Deltalog10 colony forming unit/mL at 24 hr: 2.34-2.87 and 2.10-3.04, respectively). The PBEAE induced a dose-dependent decrease in PBP2a production by MRSA, suggesting that the inhibition of PBP2a production was a major synergistic mechanism between the beta-lactams and the PBEAE. CONCLUSIONS: PBEAE can enhance the efficacy of beta-lactams for combined therapy in patients infected with MRSA.

Characterization of mannitol-fermenting methicillin-resistant staphylococci isolated from pigs in Nigeria

This study was conducted to determine the species distribution, antimicrobial resistance pheno- and genotypes and virulence traits of mannitol-positive methicillin-resistant staphylococci (MRS) isolated from pigs in Nsukka agricultural zone, Nigeria. Twenty mannitol-positive methicillin-resistant coagulase-negative staphylococcal (MRCoNS) strains harboring the mecA gene were detected among the 64 Staphylococcus isolates from 291 pigs. A total of 4 species were identified among the MRCoNS isolates, namely, Staphylococcus sciuri (10 strains), Staphylococcus lentus (6 strains), Staphylococcus cohnii (3 strains) and Staphylococcus haemolyticus (one strain). All MRCoNS isolates were multidrug-resistant. In addition to β-lactams, the strains were resistant to fusidic acid (85%), tetracycline (75%), streptomycin (65%), ciprofloxacin (65%), and trimethoprim/sulphamethoxazole (60%). In addition to the mecA and blaZ genes, other antimicrobial resistance genes detected were tet(K), tet(M), tet(L), erm(B), erm(C), aacA-aphD, aphA3, str, dfrK, dfrG, cat_pC221, and cat_pC223. Thirteen isolates were found to be ciprofloxacin-resistant, and all harbored a Ser84Leu mutation within the QRDR of the GyrA protein, with 3 isolates showing 2 extra substitutions, Ser98Ile and Arg100Lys (one strain) and Glu88Asp and Asp96Thr (2 strains). A phylogenetic tree of the QRDR nucleotide sequences in the gyrA gene revealed a high nucleotide diversity, with several major clusters not associated with the bacterial species. Our study highlights the possibility of transfer of mecA ...

Evaluation of penicillin expandase mutants and complex substrate inhibition characteristics at high concentrations of penicillin G / 生物工程学报

Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application.

Epidemiological study on nasal carriage in hospitalized children infected with Staphylococcus aureus / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the relationship between nasal carriage and Staphylococcus aureus (S. aureus) infection in hospitalized children.</p><p><b>METHODS</b>Fifty-six hospitalized children infected with S. aureus were recruited in this study. Nasal swabs were collected and cultured, and the nasal carriage rate of S. aureus was examined. PVL virulence gene and mecA resistance gene were both detected in clinical strains and nasal carriage strains by PCR.</p><p><b>RESULTS</b>Twenty-two (39%) of the 56 children had nasal carriage of S. aureus, and most of them (18 cases) were younger than one year. Among these 22 children, 11 (50%) had previous hospitalization over the past year. In the infected strains, the rate of methicillin-resistant S. aureus (MRSA) was 29% (16/56), while it was 32% (7/22) in carriage strains. The mecA positive results in clinical strains were consistent with the results in nasal carriage strains. Among 5 PVL-positive nasal carriage strains, 4 (90%) could be matched with their clinical strains, all of which were MRSA.</p><p><b>CONCLUSIONS</b>Nasal carriage is a potential risk factor for S. aureus infection. Nosocomial transmission may lead to nasal carriage, which can cause S. aureus infection. The isolation rate of MRSA is high in hospitalized children infected with S. aureus, which implies that more attention is needed for this situation. The isolates from noses may be clonally identical to the isolates from clinical secretions, and the homology between them needs to be confirmed by multi-locus sequence typing.</p>

Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae / Identificação de Haemonchus placei e Haemonchus contortus por PCR e por análise morfológica de parasitas adultos e larvas infectantes

Molecular and morphological methods were evaluated to distinguish between Haemonchus contortus and Haemonchus placei species. A total of 141 H. contortus and 89 H. placei male adult specimens collected from artificially infected lambs were identified individually by PCR analysis, using a species-specific primer pair. These PCR results were used as gold standard for Haemonchus spp. identification. Haemonchus placei presented higher mean spicule and barb lengths than H. contortus (P<0.05). However, some measurements overlapped. For this reason, a discriminate function did not allow the correct identification of 13 H. contortus and one H. placei specimen. The sheath tail length of the third stage larvae (L3), which comprises the distance between the tip of the larval tail and the end of the sheath tail, were measured. Only three of the 485 H. placei larvae (0.619%) had a sheath tail shorter than 85 µm, while only four of the 500 H. contortus larvae (0.8%) presented a sheath tail longer than 85 µm. The results indicated that 6.09% of the male adult specimens would be misclassified based on the discriminate function, while only 0.71% of infective larvae would be misclassified. Therefore, identification of L3 can be used as the first method to indicate the presence of H. placei and/or H. contortus in a population of domestic ruminants.

Métodos moleculares e morfológicos foram avaliados para a identificação de Haemonchus contortus e Haemonchus placei. No total, 141 H. contortus e 89 H. placei machos adultos, obtidos de cordeiros artificialmente infectados, foram identificados individualmente por PCR com o emprego de um par de “primers” espécie-específico. Esses resultados da análise por PCR foram considerados como padrão para a identificação das espécies de Haemonchus. Haemonchus placei apresentou valores médios de espículos e ganchos superiores aos de H. contortus (P<0,05). Entretanto, houve sobreposição de alguns valores. Por essa razão, a função discriminante não permitiu a identificação correta de 13 exemplares de H. contortus e de um, de H. placei. Foi medida a cauda da bainha de larvas infectantes (L3), que compreende a distância entre a ponta da cauda da larva e a ponta da cauda da bainha. Apenas três das 485 L3 de H. placei (0,619%) apresentaram a cauda da bainha com medida inferior a 85 µm e somente em quatro das 500 L3 de H. contortus (0,8%) essa medida foi superior a 85 µm. Os resultados demonstraram que 6,09% dos machos adultos seriam identificados erroneamente com base na função discriminante, enquanto a identificação incorreta de L3 seria de apenas 0,71%. Portanto, a identificação de L3 pode ser utilizada como método inicial para indicar a presença de H. placei e/ou H. contortus em uma população de ruminantes domésticos.

Genetic analysis of mecA gene and detection of homologue pbpD in Stahylococcus sciuri group

Oxacillin/methicillin-resistance is related to the mecA and its regulatory genes mecR1 and mecI. Its origin is still unknown, although evidences support that it is related to CNS, once mecA and a homologue gene, pbpD, were both detected in Staphylococcus sciuri species group. The present work evaluated 210 samples of skin and ear swabs from rodents and 60 nasal swabs from equines of Army Biologic Institute, Rio de Janeiro. Pheno- and genotypic characterization provided 59.52% (25/42) and 78.57% (11/14) S. lentus and S. sciuri, respectively. It was observed that although all S. sciuri isolates tested positive for pbpD, there was no correlation with oxacillin-resistance. On the other hand, isolates tested positive for mecA gene also presented phenotypic oxacillin-resistance in at least one assay. The alignment of the mecA gene showed that the nucleotide sequences were sorted into 2 different groups, one comprising the bovine strains and the other containing human and equine strains.

The multifaceted resources and microevolution of the successful human and animal pathogen methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important bacterial pathogens based on its incidence and the severity of its associated infections. In addition, severe MRSA infections can occur in hospitalised patients or healthy individuals from the community. Studies have shown the infiltration of MRSA isolates of community origin into hospitals and variants of hospital-associated MRSA have caused infections in the community. These rapid epidemiological changes represent a challenge for the molecular characterisation of such bacteria as a hospital or community-acquired pathogen. To efficiently control the spread of MRSA, it is important to promptly detect the mecA gene, which is the determinant of methicillin resistance, using a polymerase chain reaction-based test or other rapidly and accurate methods that detect the mecA product penicillin-binding protein (PBP)2a or PBP2’. The recent emergence of MRSA isolates that harbour a mecA allotype, i.e., the mecC gene, infecting animals and humans has raised an additional and significant issue regarding MRSA laboratory detection. Antimicrobial drugs for MRSA therapy are becoming depleted and vancomycin is still the main choice in many cases. In this review, we present an overview of MRSA infections in community and healthcare settings with focus on recent changes in the global epidemiology, with special reference to the MRSA picture in Brazil.

Analysis of Gene Mutations Associated with Antibiotic Resistance in Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.

Analysis of Gene Mutations Associated with Antibiotic Resistance in Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.

Development of a rapid multi-residue assay for detecting β-lactams using penicillin binding protein 2x* / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a rapid multi-residue assay for detecting 16 demanded by the European Union (EU).</p><p><b>METHODS</b>A recombinant penicillin-binding protein (PBP) 2x* from Streptococcus pneumoniae R6 was expressed in vitro and six β-lactams were conjugated to HRP by four methods. A rapid multi-residue assay for β-lactams was established with PBP2x* and HRP-conjugate.</p><p><b>RESULTS</b>PBP2x* was expressed and purified successfully and the ideal HRP-conjugate was identified. The multi-residue assay was developed. After optimization, penicillin G, ampicillin, amoxicillin, cloxacillin, dicloxacillin, oxacillin, nafcillin, cephalexin, ceftiofur, cefalonium, cefquinome, cefazolin, cefoperazone, cephacetrile, and cephapirin can be detected at levels below MRL in milk with simple pretreatment.</p><p><b>CONCLUSION</b>This assay developed can detect all 16 β-lactams demanded by the European Union (EU). The whole procedure takes only 45 min and can detect 42 samples and the standards with duplicate analysis.</p>

Substitutions in Penicillin-Binding Protein 1 in Amoxicillin-Resistant Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: A worldwide increase in amoxicillin resistance in Helicobacter pylori is having an adverse effect on eradication therapy. In this study, we investigated the mechanism of the amoxicillin resistance of H. pylori in terms of amino acid substitutions in penicillin-binding protein 1 (PBP1). METHODS: In total, 150 H. pylori strains were isolated from 144 patients with chronic gastritis, peptic ulcers, or stomach cancer. The minimum inhibitory concentrations (MICs) of the strains were determined with a serial 2-fold agar dilution method. The resistance breakpoint for amoxicillin was defined as >0.5 microg/mL. RESULTS: Nine of 150 H. pylori strains showed amoxicillin resistance (6%). The MIC values of the resistant strains ranged from 1 to 4 microg/mL. A PBP1 sequence analysis of the resistant strains revealed multiple amino acid substitutions: Val16-->Ile, Val45-->Ile, Ser414-->Arg, Asn562-->Tyr, Thr593-->Ala, Gly595-->Ser, and Ala599-->Thr. The natural transformation of these mutated genes into amoxicillin-sensitive strains was performed in two separate pbp1 gene segments. A moderate increase in the amoxicillin MIC was observed in the segment that contained the penicillin-binding motif of the C-terminal portion, the transpeptidase domain. CONCLUSIONS: pbp1 mutation affects the amoxicillin resistance of H. pylori through the transfer of the penicillin-binding motif.