Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Time-kill and synergism studies of ceftobiprole against Enterococcus faecalis, including beta-lactamase-producing and vancomycin-resistant isolates.

Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of Enterococcus faecalis (including 16 beta-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10(7) CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two beta-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 microg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of

Characterization of the beta-lactamase gene from {\it Enterococcus faecalis\/} strain HH22

The plasmid-encoded, constitutively produced $\beta$-lactamase gene from Enterococcus faecalis strain HH22 was genetically characterized. A restriction endonuclease map of the 5.1 kb EcoRI fragment encoding the enterococcal $\beta$-lactamase was prepared and compared with the restriction map of a cloned staphylococcal $\beta$-lactamase gene (from the naturally-occurring staphylococcal $\beta$-lactamase plasmid pI258). Comparison and hybridization studies showed that there were identical restriction sites in the region of the $\beta$-lactamase structural gene but not in the region surrounding this gene. Also the enterococcal $\beta$-lactamase plasmid did not encode resistance to mercury or cadmium which is encoded by the small, transducible staphylococcal $\beta$-lactamase plasmids. The nucleotide sequence of the enterococcal gene was shown to be identical to the published sequences of three of four staphylococcal type A $\beta$-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred-forty nucleotides upstream of the $\beta$-lactamase start codon were also determined for the inducible staphylococcal $\beta$-lactamase gene on pI258; this sequence was identical to that of the constitutively expressed enterococcal gene indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The gene for the enterococcal $\beta$-lactamase and an inducible staphylococcal $\beta$-lactamase were each cloned into a shuttle vector and then transformed into enterococcal and staphylococcal recipients. The major difference between the two host backgrounds was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene but no qualitative difference was seen between the two genera. Also a difference in the level of resistance to ampicillin was seen between the two backgrounds with the cloned enzymes by MIC and time-kill studies. The location of the enzyme was found to be host dependent since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell bound enzyme in the enterococcus. Based on the identity of the enterococcal $\beta$-lactamase and several staphylococcal $\beta$-lactamases, these data suggest recent spread of $\beta$-lactamase to enterococci and also suggest loss of a functional repressor. ^

Prevalencia de bacterias productoras de beta-lactamasas en el Hospital Vicente Corral Moscoso periodo enero-diciembre del 2014

Antecedentes: la adaptación de las bacterias a los tratamientos antibióticos ha ido mejorando, hasta el punto de llegar a presentar resistencia a los tratamientos más agresivos, esto se debe a la evolución constante que han sufrido estas con la aparición de nuevas especies, por mecanismos como la conjugación o trasmisión de plásmidos, con la producción de diferentes tipos de beta-lactamasas, estas características nuevas les han permitido mejorar su capacidad de evadir los mecanismos de acción farmacológicos. Objetivo general: establecer la prevalencia de bacterias productoras de beta-lactamasas en el Hospital Vicente Corral Moscoso, durante el periodo de enero a diciembre del 2014, Cuenca - Ecuador. Metodología: Tipo de estudio: se realizó un estudio de tipo descriptivo, observacional; Universo y muestra: historias clínicas de todos los pacientes atendidos en el Hospital Vicente Corral Moscoso, a los que se había realizado cultivo y antibiograma con reporte de producción de beta-lactamasas, durante el periodo enero a diciembre del 2014; Método de recolección de datos: observación y revisión de historias clínicas que fueron registrados en el formulario de recolección de datos; Tabulación y análisis de los resultados: Todos los datos fueron tabulados y procesados en el programa SPSS Versión 15.0, elaborando tablas simples, compuestas. Resultados: de un total de 160 bacterias aisladas, la prevalencia de bacterias productoras de betalactamasas fue 13,1%, 74,4%, 12,5% para BLEA, BLEE y carbapenemasas respectivamente. El sexo femenino fue el más afectado por las bacterias productoras de BLEA, y carbapenemasas, pero el sexo masculino fue el más afectado por bacterias productoras de BLEE. La E. coli representa el 74,79% de bacterias productoras de BLEE, representando la Klebsiella pneumoniae el 50% de todas las bacterias productoras de carbapenemasas. Al analizar la mortalidad se observa que al incrementar la resistencia incrementa el riesgo de mortalidad: BLEA 5%, BLEE 15% y Carbapenemasa 25%

Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060

Clavulanic acid (CA) is a potent inhibitor of beta-lactamases, produced by some resistant pathogenic microorganisms, which allows efficient treatment of infectious diseases. The kinetic and thermodynamic parameters of CA production by a new isolate of Streptomyces DAUFPE 3060 and its degradation were evaluated. The effect of temperature on the system was investigated in the range 24-40 degrees C adopting an overall model accounting for (a) the Arrhenius-type formation of CA by fermentation, (b) the hypothetical reversible unfolding of the enzyme limiting the overall metabolism, and (c) the irreversible first-order degradation of CA. The higher rates of CA formation (k(CA) = 0,107 h(-1)) and degradation (k(d) = 0.062 h(-1)) were observed at 32 and 40 degrees C, respectively. The main thermodynamic parameters of the three above hypothesized events were estimated. In particular, the activation parameters of degradation (activation energy = 39.0 kJ/mol; Delta H(d)* = 36.5 kJ/mol; Delta S(d)* = -219.7 J/(mol K); Delta G(d)* = 103.5 kJ/mol) compare reasonably well with those reported in the literature for similar system without taking into account the other two events. (C) 2009 Elsevier Inc. All rights reserved.

Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach

Clavulanic acid (CA) is a beta-lactam antibiotic that alone exhibits only weak antibacterial activity, but is a potent inhibitor of beta-lactamases enzymes. For this reason it is used as a therapeutic in conjunction with penicillins and cephalosporins. However, it is a well-known fact that it is unstable not only during its production phase, but also during downstream processing. Therefore, the main objective of this study was the evaluation of CA long-term stability under different conditions of pH and temperature, in the presence of variable levels of different salts, so as to suggest the best conditions to perform its simultaneous production and recovery by two-phase polymer/salt liquid-liquid extractive fermentation. To this purpose, the CA stability was investigated at different values of pH (4.0-8.0) and temperature (20-45 degrees C), and the best conditions were met at a pH 6.0-7.2 and 20 degrees C. Its stability was also investigated at 30 degrees C in the presence of NaCl, Na(2)SO(4), CaCl(2) and MgSO(4) at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 6.5). All salts led to increased CA instability with respect to the buffer alone, and this effect decreased in following sequence: Na(2)SO(4) > MgSO(4) > CaCl(2) > NaCl. Kinetic and thermodynamic parameters of CA degradation were calculated adopting a new model that took into consideration the equilibrium between the active and a reversibly inactivated form of CA after long-time degradation. (C) 2009 Elsevier B.V. All rights reserved.

Clonal transmission of ESBL-producing Klebsiella spp. at a university hospital in Brazil

The present study was designed to determine the prevalence and extended-spectrum beta-lactamase (ESBL) types in clinical isolates of Klebsiella spp. at a university hospital located in the Brazilian southern region (Ribeirao Preto, Sao Paulo) as well as their antibiotic susceptibility and genetic profiles. This study included 147 non-repeat Klebsiella spp. isolates collected from January to June 2000, of which 23 K. pneumoniae and 8 K. oxytoca were selected as ESBL producers by using the Oxoid combination disk method and Etest ESBL strip. beta-lactamases were characterized by IEF, PCR and sequencing assays using primers for ESBL genes. Antibiotic susceptibility was evaluated by MicroScan system. Dissemination of two major clones of ESBL-producing Klebsiella spp. occurred in the hospital. According to the results obtained in this study there was a clonal spread of CTX-M-producing K. oxytoca in five clinics and dissemination of ESBL-producing K. pneumoniae in the nursery and pediatrics wards.

CTX-M-producing Klebsiella spp. in a Brazilian hospital: what has changed in 6 years?

CTX-M-encoding genes from Klebsiella spp. strains isolated in 2000 and 2006 were characterized as well as their genetic environment. CTX-M-2 variants were predominant in Klebsiella pneumoniae strains, which showed a greater variability in bla(CTX-M) genes, integrons, and plasmids in 2006 when compared to strains collected in 2000. CTX-M-9-producing Klebsiella oxytoca was identified in 2000 as clonal dissemination. (C) 2010 Elsevier Inc. All rights reserved.

The characteristics of NDM-producing Klebsiella pneumoniae from Canada

After recent hospitalization in India (New Delhi and Mumbai), 2 patients, on their return to Canada, presented with lower urinary tract infections due to multiresistant Klebsiella pneumoniae that produced New Delhi metall-beta-lactamase and CTX-M-15. The organisms belonged to clones ST147 and ST340, and were positive for aac(6`)-Ib-cr, as well as for the ccdAB and vagCD addiction systems. The bla(NDM) plasmid was located on the IncFIIA and IncA/C replicon groups of plasmids. Clones ST147 and ST340 are also responsible for harbouring bla(KPC), and it is possible that they played an important role in the intercontinental spread of antimicrobial resistance. (C) 2011 Elsevier Inc. All rights reserved.

Eficácia e segurança de Sultamicilina (Ampicilina/Sulbactam) e Amoxacilina/Clavulanato no tratamento das infecções de via aéreas superiores em adultos: um estudo multicêntrico, aberto e randomizado

As IVAS em crianças e adultos são os motivos mais freqüentes de consulta médica e os que mais demandam o uso de antibióticos. A crescente resistência bacteriana causada pela produção das beta-lactamases constitui um dos mais sérios problemas atuais. A Sultamicilina é uma pró-droga dupla da ampicilina e do sulbactam, um potente inibidor de beta-lactamases que pode fazer frente a estas dificuldades. OBJETIVO: avaliar a eficácia, segurança e tolerabilidade da Ampicilina/Sulbactan comparada à Amoxacilina/Ácido Clavulânico no tratamento de IVAS, em adultos. METODOLOGIA: 102 pacientes com diagnóstico de IVAS foram randomizados em dois grupos recebendo Ampicilina/Sulbactan ou Amoxacilina/Clavulanato por 10 dias. Foram avaliados 10 e 30 dias após para análise da resposta terapêutica. RESULTADOS: Não houve diferença entre os grupos com relação à proporção de pacientes curados ao final do tratamento (visita 2) ou do estudo (visita 3). No grupo que recebeu Amoxacilina/Clavulanato, as proporções de cura foram de 61.7% e 93.2% nas visitas 2 e 3, comparadas a 64.4% e 97.4%, respectivamente, no grupo que recebeu Ampicilina/Sulbactan. A proporção de pacientes que experimentou pelo menos um evento adverso foi semelhante nos dois grupos (p = 0.940). A diarréia foi significativamente mais freqüente no grupo Amoxacilina-Clavulanato (70.6%) do que no grupo Ampicilina/Sulbactan (29.4%), (p=0.0164). CONCLUSÕES: A Ampicilina/Sulbactan é tão segura e eficaz quanto a Amoxacilina/Clavulanato no tratamento empírico de IVAS em adultos. A ocorrência significativamente menor de quadros de diarréia no grupo recebendo Ampicilina/Sulbactan necessita confirmação em estudos posteriores.

Prevalência de resistência a antimicrobianos em isolados ambientais de Escherichia coli e enterococos

A água é um recurso escasso e indispensável à vida, podendo ser um importante veículo de microrganismos patogénicos com origem fecal. A matéria fecal é também uma fonte de microrganismos resistentes a antimicrobianos e contribui para a sua disseminação e dos seus genes de resistência no ambiente e entre as comunidades microbianas comensais e microrganismos patogénicos humanos e animais. A qualidade microbiológica da água é monitorizada recorrendo à utilização de bioindicadores como Escherichia coli, enterococos e microrganismos totais. O presente estudo apresentou como principal objetivo determinar a prevalência de ESBLs e AmpCs e ainda avaliar a prevalência de estirpes de enterococos com resistência à vancomicina (VRE) em águas do rio Douro e da orla costeira da cidade do Porto. As amostragens de água foram realizadas em quatro locais localizados no estuário do rio Douro e orla costeira da cidade do Porto entre o mês de Abril e Julho. A deteção e quantificação dos bioindicadores foram realizadas pelo método de filtração por membrana. A suscetibilidade das estirpes de E. coli e enterococos foi testada pelo método de difusão em disco relativamente a várias classes de antimicrobianos. As contagens microbianas mais elevadas foram determinadas entre Abril e Junho e em amostras de água doce. Foram isoladas 62 estirpes de E. coli e 49 estirpes de enterococos que apresentaram prevalências de resistência a antimicrobianos de 90,3% (56/62) e 83,7% (41/49), respetivamente. As estirpes de E. coli apresentaram altas frequências de resistência à ampicilina (74,2%) e tetraciclina (61,3%). Nestas estirpes verificou-se ainda fenótipos associados a multirresistência a um mínimo de três classes de antimicrobianos em 56,5% (35/62) dos isolados. Verificou-se que as estirpes de enterococos apresentaram altos níveis de resistência à rifampicina (34,7%) e azitromicina (40,8%), detetando-se ainda a manifestação de fenótipo de resistência à vancomicina em 26,5% das estirpes. Observou-se uma prevalência de 36,7% (18/49) de estirpes de enterococos associadas a fenómenos de multirresistência antimicrobiana. Ana Martins vi Os resultados obtidos sugerem que o rio Douro e orla costeira, bem como os ambientes aquáticos, constituem reservatórios de bactérias e genes de resistência a antimicrobianos e possuem um papel preponderante na sua disseminação.

SUSCEPTIBILITY TO ANTIBIOTICS IN URINARY TRACT INFECTIONS IN A SECONDARY CARE SETTING FROM 2005-2006 AND 2010-2011, IN SÃO PAULO, BRAZIL: DATA FROM 11,943 URINE CULTURES

Introduction: Urinary tract infection (UTI) has a high incidence and recurrence, therefore, treatment is empirical in the majority of cases. Objectives: The aim of this study was to analyze the urine cultures performed at a secondary hospital, during two periods, 2005-2006 and 2010-2011, and to estimate the microbial resistance. Patients and methods: We analyzed 11,943 aerobic urine cultures according to basic demographic data and susceptibility to antibiotics in accordance with the Clinical and Laboratory Standards Institute (CLSI) for Vitek 1 and 2. Results: Most of our cohort consisted of young adult females that were seen at the Emergency Department. E. coli was the most frequent (70.2%) among the 75 species isolated. Resistance of all isolates was ≥ 20% for trimethoprim/sulfamethoxazole (TMP/SMX), norfloxacin, nitrofurantoin, cefazolin and nalidixic acid. Although E. coli was more susceptible (resistance ≥ 20% for TMP/SMX and nalidixic acid) among all of the isolates, when classified by the number and percentage of antibiotic resistance. Global resistance to fluoroquinolones was approximately 12%. Risk factors for E. coli were female gender and an age less than 65 years. Men and patients older than 65 years of age, presented more resistant isolates. Extended spectrum beta-lactamases (ESBL) were identified in 173 out of 5,722 Gram-negative isolates (3.0%) between 2010 and 2011. Conclusion: E. coli was the most frequent microbe isolated in the urine cultures analyzed in this study. There was a significant evolution of bacterial resistance between the two periods studied. In particular, the rise of bacterial resistance to fluoroquinolones was concerning.

Enterobacteriaceae ISOLATES FROM THE ORAL CAVITY OF WORKERS IN A BRAZILIAN ONCOLOGY HOSPITAL

The evaluation of workers as potential reservoirs and disseminators of pathogenic bacteria has been described as a strategy for the prevention and control of healthcare-associated infections (HAIs). The aim of this study was to evaluate the presence of Enterobacteriaceae in the oral cavity of workers at an oncology hospital in the Midwest region of Brazil, as well as to characterize the phenotypic profile of the isolates. Saliva samples of 294 workers from the hospital’s healthcare and support teams were collected. Microbiological procedures were performed according to standard techniques. Among the participants, 55 (18.7%) were colonized by Enterobacteriaceae in the oral cavity. A total of 64 bacteria were isolated, including potentially pathogenic species. The most prevalent species was Enterobacter gergoviae (17.2%). The highest rates of resistance were observed for β-lactams, and 48.4% of the isolates were considered multiresistant. Regarding the enterobacteria isolated, the production of ESBL and KPC was negative. Nevertheless, among the 43 isolates of the CESP group, 51.2% were considered AmpC β-lactamase producers by induction, and 48.8% were hyper-producing mutants. The significant prevalence of carriers of Enterobacteriaceae and the phenotypic profile of the isolates represents a concern, especially due to the multiresistance and production of AmpC β-lactamases.

Resistência a β-lactâmicos em Acinetobacter spp isolados de efluente hospitalar no sul do Brasil

Acinetobacter spp é um importante patógeno causador de infecções nosocomiais que acomete pacientes imunocomprometidos e capaz de adquirir resistência a antimicrobianos com facilidade. Os esgotos hospitalares são importantes disseminadores de genes de resistência a antimicrobianos para a microbiota ambiental. Neste contexto, 30 cepas de Acinetobacter spp provenientes de efluente de um hospital em Porto Alegre, RS, foram analisados quanto ao perfil de susceptibilidade a β-lactamases, quinolonas e aminoglicosídeos através de antibiograma e testes de triagem para metalo beta-lactamases e β-lactamases de espectro estendido. O perfil encontrado revela cepas multi-resistentes e que mecanismos de resistência como a produção de β-lactamases de espectro estendido e bombas de efluxo podem estar presentes nesses isolados.

Prevalence and clinical outcomes of episodes of ventilator-associated pneumonia caused by SPM-1-producing and non-producing imipenem-resistant Pseudomonas aeruginosa

INTRODUCTION: Pseudomonas aeruginosa is a leading cause of ventilator-associated pneumonia (VAP) and exhibits high rates of resistance to several antimicrobial drugs. The carbapenens are usually the drugs of choice against this microorganism. However, the carbapenem resistance has increased among these strains worldwide. The presence of metallo-β-lactamases (MBL) has been pointed out as a major mechanism of resistance among these strains. No previous study addressed outcomes of respiratory infections caused by these strains. METHODS: Our group sought to analyze the epidemiology and clinical outcomes of patients with VAP caused by imipenem-resistant P. aeruginosa. A total of 29 clinical isolates of carbapenem-resistant Pseudomonas aeruginosa were screened for metallo-β-lactamase (MBL) genes. RESULTS: Demographic and clinical variables were similar between the SPM-1-producing and non-SPM-1-producing group. Five (17.2%) isolates were positive for blaSPM-1. No other MBL gene was found. All patients were treated with polymyxin B. The infection-related mortality was 40% and 54.2% for SPM-1-producing and -non-producing isolates, respectively. CONCLUSIONS: There were no differences in epidemiological and clinical outcomes between the two groups.