B-lactamase-mediated resistance to antimicrobials : the relationship between genotype and phenotype

This thesis examined the ability to predict the emergence of bacteria resistant to antibiotics using genetic markers in the bacteria. Bacteria containing the genetic markers were able to become resistant to antibiotics, whereas bacteria that did not have the genetic markers remained susceptible. Existing techniques can identify the presence of resistance by looking at the characteristics of the bacteria during growth. However, having the ability to predict antibiotic resistance before it emerges could improve the preservation of currently available antibiotics and minimise treatment failure.

Influência da seleção do grupo controle na análise de fatores de risco para infecções hospitalares por Klebsiella pneumoniae produtora de B-lactamase de espectro estendido

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Conformational studies of phynyl and isoxazolyl penicillins

Conformational energy calculations have been carried out on a few representative b-lactamase resistant and susceptible, phenyl and isoxazolyl penicillins. These studies, in agreement with those of earlier workers, show that the 6b-side chains of resistants penicillins are highly rigid as compared to those of susceptible penicillins. The present studies also suggest that the degree of resistant to b-lactamases depends not only on the rigidity of the side chain but also on the nature and orientation of the substituent, beyond the amide carbonyl group in the side chain. The overall shapes of these penicillins correlate well with their antibacterial properties.

Perfil microbiológico, prevalencia de resistencia antibiótica y patrones de susceptibilidad en infección urinaria en una población pediátrica

Introducción: El incremento de la resistencia antibiótica se considera un problema de salud pública con consecuencias clínicas y económicas, por lo tanto se determinará la prevalencia de resistencia antibiótica en Infección del Tracto Urinario (ITU, el perfil microbiológico y los patrones de susceptibilidad en una población pediátrica atendida en la Fundación Cardioinfantil. Materiales y métodos: Estudio observacional de corte transversal, retrospectivo, entre 1 mes a 18 años de edad, con diagnóstico de ITU comunitaria atendidos entre Enero de 2011 y Diciembre de 2013. Se excluyeron pacientes con dispositivos en la vía urinaria, instrumentación quirúrgica previa, trayectos fistulosos entre la vía urinaria y sistema digestivo, ITU luego de 48 horas de hospitalización y recaída clínica en tratamiento. Se estableció la prevalencia de ITU resistente y se realizó un análisis descriptivo de la información. Resultados: Se evaluaron 385 registros clínicos, con una mediana de 1.08 años (RIQ 0.8 – 4.08), el 73.5% eran niñas. La fiebre predominó (76.5%), seguido de emesis (32.0%), disuria (23.7%) y dolor abdominal (23.1%). El uropatógeno más frecuente fue E.coli (75%), seguido de Proteus mirabilis (8.5%) y Klebsiella spp. (8.3%). La Ampicilina, el Trimetropim sulfametoxazol, la Ampicilina sulbactam y el ácido nalidixico tuvieron mayor tasa de resistencia. La prevalencia de BLEE fue 5.2% y AmpC 3.9%. La prevalencia de resistencia antimicrobiana fue de 11.9%. Conclusiones: La E.coli es el uropatogeno más frecuentemente aislado en ITU, con resistencia a la ampicilina en 60.2%, cefalosporinas de primera generación en 15.5%, trimetropin sulfametoxazol en 43.9%, cefepime 4.8%. La prevalencia de resistencia antimicrobiana fue de 11.9%.

Influence of free amino acids on clavulanic acid production by Streptomyces clavuligerus in synthetic medium

The effect of nitrogen source on clavulanic acid production was investigated in shake flasks. Media containing asparagine plus one of several different amino acids or a combination of two amino acids was tested. The best result, ca. 180 mg/L clavulanic acid, CA, in 60 h, was obtained with the lysine-tyrosine pair In an aerated and agitated fermentor this medium led to CA concentrations of ca. 210 mg/L, a remarkable production for synthetic medium utilization. Amino acids analysis during cultivation indicated that, while asparagine was consumed rapidly, lysine and tyrosine were metabolized slowly, promoting CA production.

A putative twin-arginine translocation system in the phytopathogenic bacterium Xylella fastidiosa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detection of bla(CTX-M)-type genes in complex class 1 integrons carried by Enterobacteriaceae isolated from retail chicken meat in Brazil

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

Características y perfil microbiológico de pacientes con colecistitis moderada y severa en Mederi de 2014 a 2016

Introducción: Las guías de Tokyo de 2013 lograron un consenso respecto al manejo antibiótico de la infección biliar. Sus recomendaciones están sustentadas en estudios internacionales de la epidemiología bacteriana, pero también recalcan la importancia de conocer la microbiología local para ajustar las guías de manejo. Materiales y métodos: Se diseñó un estudio descriptivo tipo serie de casos de pacientes tratados por colecistitis aguda moderada y severa en Méderi Hospital Universitario Mayor (HUM), describiendo los aislamientos microbiológicos y perfiles de resistencia de los cultivos de bilis tomados durante la cirugía. Resultados: Se analizaron 131 pacientes con una edad promedio de 63 años, la mayoría sin comorbilidades médicas. Se encontró un 48% de positividad en los cultivos, predominantemente enterobacterias siendo la más frecuente Escherichia coli, seguida de especies de Klebsiella y de Enterococcus. Los perfiles de resistencia evidenciaron un 93% de multisensibilidad antibiótica y se aislaron 4 microorganismos multirresistentes. No se encontraron diferencias en comorbilidades, alteraciones paraclínicas, presencia de síndrome biliar obstrutivo, pancreatitis o instrumentación previa de la vía biliar entre los pacientes con cultivo positivo y negativo. Conclusiones: Los resultados concuerdan con los reportes internacionales en cuanto a la flora bacteriana aislada, pero los perfiles de resistencia evidenciados en esta serie son diferentes a los que sustentan las guías de manejo de Tokio revisadas en 2013. Este hallazgo obliga a ajustar las guías de manejo institucionales con base en la epidemiología local.

Perfil epidemiológico y análisis de la formulación antibiótica en pacientes con infección urinaria nosocomial en la Fundación Cardioinfantil en el año 2014

Introducción: La IVU es muy frecuenten en la (FCI - IC), Alrededor el 60% de los pacientes con diagnóstico de IVU nosocomial corresponden a gérmenes resistente, Desde el año 2010 el CLSI disminuyó los puntos de corte de sensibilidad en las enterobacteriaceae y removió la necesidad de tamizaje y confirmación de (BLEE), en el presente trabajo se pretende determinar el perfil epidemiológico de la formulación antibiótica en pacientes con IVU nosocomial. Diseño: Se realizó un estudio observacional analítico de corte transversal. Métodos: Se realizó un análisis univariado, bivariado y multivariado. El análisis bivariado y multivariado se realizó para determinar la medida de asociación teniendo en cuenta la formulación de Carbapenemico la variable dependiente, evaluándose mediante chi cuadrado. Resultados: Se revisaron 131 urocultivos, se incluyeron 116. Los aislamientos microbiológicos más frecuentemente encontrados fueron E. Coli y K. Pneumoniae, el 43.4% de los aislamientos, presentaron expresión de BLEE, 90% de los aislamientos fueron sensibles a Cefepime. La mayoría de los modelos obtenidos mostraron una fuerte asociación entre el reporte de BLEE en antibiograma con la formulación de carbapenémicos como terapia final OR 33,12 IC 95% (2,90 – 337,4). Conclusión: La epidemiologia de la IVU nosocomial en la FCI-IC no difiere de las referencias internacionales, no hay adherencia a las guías de manejo intrahospitalario y el reporte de la palabra BLEE en el antibiograma predice la formulación de antibiótico carbapenémico por el médico que lee el urocultivo

The molecular basis for beta-lactamase gene expression in B. anthracis, B. cereus and B. thuringiensis

The susceptibility of most Bacillus anthracis strains to β-lactam antibiotics is intriguing considering that the B. anthracis genome harbors two β-lactamase genes, bla1 and bla2, and closely-related species, Bacillus cereus and Bacillus thuringiensis, typically produce β-lactamases. This work demonstrates that B. anthracis bla expression is affected by two genes, sigP and rsp, predicted to encode an extracytoplasmic function sigma factor and an antisigma factor, respectively. Deletion of the sigP/rsp locus abolished bla expression in a penicillin-resistant clinical isolate and had no effect on bla expression in a prototypical penicillin-susceptible strain. Complementation with sigP/rsp from the penicillin-resistant strain, but not the penicillin-susceptible strain, conferred β-lactamase activity upon both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsp in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsp homologues are required for inducible penicillin resistance in those species. Expression of the B. cereus or B. thuringiensis sigP and rsp genes in a B. anthracis sigP/rsp-null mutant confers resistance to β-lactam antibiotics, suggesting that while B. anthracis contains the genes necessary for sensing β-lactam antibiotics, the B. anthracis sigP/rsp gene products are insufficient for bla induction. ^ Because alternative sigma factors recognize unique promoter sequence, direct targets can be elucidated by comparing transcriptional profiling results with an in silico search using the sigma factor binding sequence. Potential σP -10 and -35 promoter elements were identified upstream from bla1 bla2 and sigP. Results obtained from searching the B. anthracis genome with the conserved sequences were evaluated against transcriptional profiling results comparing B. anthracis 32 and an isogenic sigP/rsp -null strain. Results from these analyses indicate that while the absence of the sigP gene significantly affects the transcript levels of 16 genes, only bla1, bla2 and sigP are directly regulated by σP. The genomes of B. cereus and B. thuringiensis strains were also analyzed for the potential σP binding elements. The sequence was located upstream from the sigP and bla genes, and previously unidentified genes predicted to encode a penicillin-binding protein (PBP) and a D-alanyl-D-alanine carboxypeptidase, indicating that the σ P regulon in these species responds to cell-wall stress caused by β-lactam antibiotics. ^ β-lactam antibiotics prevent attachment of new peptidoglycan to the cell wall by blocking the active site of PBPs. A B. cereus and B. thuringiensis pbp-encoding gene located near bla1 contains a potential σP recognition sequence upstream from the annotated translational start. Deletion of this gene abolished β-lactam resistance in both strains. Mutations in the active site of the PBP were detrimental to β-lactam resistance in B. cereus, but not B. thuringiensis, indicating that the transpeptidase activity is only important in B. cereus. I also found that transcript levels of the PBP-encoding gene are not significantly affected by the presence of β-lactam antibiotic. Based on these data I hypothesize that the gene product acts a sensor of β-lactam antibiotic. ^

Hydrolysis of Organophosphate Esters: Phosphotriesterase Activity of Metallo-beta-lactamase and Its Functional Mimics

The phosphotriesterase (PTE) activity of a series of binuclear and mononuclear zinc(II) complexes and metallo-beta-lactamase (m beta 1) from Bacillus cereus was studied. The binuclear complex 1, which exhibits good m beta 1 activity, shows poor PTE activity. In contrast,complex 2, a poor mimic of m beta 1, exhibits much higher activity than 1 The replacement of Cl- ligands by OH- is important for the high PTE activity of complex 2 because this complex does not show any catalytic activity in methanol. The natural enzyme m beta 1 from B. cereus is also found to be an inefficient catalyst in the hydrolysis of phosphotriesters. These observations indicate that the binding of beta-lactam substrates at the binuclear zinc(II) center is different from that of phosphotriesters. Furthermore, phosphodiesters, the products from the hydrolysis of triesters, significantly inhibit the PTE activity of m beta 1 and its functional mimics. Although the mononuclear complexes 3 and 4 exhibited significant m beta 1 activity, these complexes are found to be almost inactive in the hydrolysis of phosphotriesters. These observations indicate that the elimination of phosphodiesters from the reaction site is important for the PTE activity of zinc(II) complexes.

Metallo-beta-lactamase and phosphotriesterase activities of some zinc(II) complexes

Metallo-beta-lactamases (m beta l) and phosphotriesterase (PTE) are zinc(II) enzymes, which hydrolyze the beta-lactam antibiotics and toxic organophosphotriesters, respectively. In the present work, we have synthesized a few asymmetric phenolate-based ligands by sequential Mannich reaction and their corresponding zinc(II) complexes. These zinc(II) complexes were studied for their m beta l and PTE activities. It is shown that the zinc(II) complexes can hydrolyze oxacillin, the beta-lactam antibiotic, at much higher rates as compared to the hydrolysis of p-nitrophenyl diphenylphosphate (PNPDPP), the phosphotriester. Among the complexes studied, the binuclear asymmetric complex 1 having a water molecule coordinated to one of the zinc(II) ions exhibits much better mbl activity than the mononuclear complexes. However, the mononuclear zinc(II) complexes having labile chloride ions exhibit significant PTE activity, which can be ascribed to the replacement of chloride ions by hydroxide ions during hydrolysis reactions. (C) 2011 Elsevier B.V. All rights reserved.

Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii

This study aims in identifying MBLs particularly Zn requiring Molecular Class B enzymes produced by Pseudomonas aeruginosa and Acinetobacter baumannii. The resistance by these organisms are in a rise against all antibiotics including carbapenems and no prescribed CLSI guidelines is available for detecting them. Clinical isolates antibiotic susceptibility was determined by number of phenotypic tests by addition of 50mM of 10 mu l zinc as cofactor for metallo beta lactamase production along with 0.5M ETDA of 5 mu l (930 mu g per disk) plain disks. Increase in zone size of the meropenem -EDTA disk compared to the meropenem disk without EDTA was recorded positive. For Zn requiring MBLs zone towards both disks of EDTA and Zn along with meropenem is detected by DDST.

Balanoposthitis caused by Pseudomonas aeruginosa co-producing metallo-beta-lactamase and 16S rRNA methylase in children with hematological malignancies

Balanoposthitis is defined as the inflammation of the glans penis and its foreskin. In the presence of other underlying medical conditions, this localized infection may spread systemically, serving as a source of fever and bacteremia in neutropenic males. Two rare cases of balanoposthitis caused by a clonally related Pseudomonas aeruginosa isolate co-producing the SPM-1 metallo-beta-lactamase and the novel 16S rRNA methylase RmtD are described. Four multidrug-resistant (MDR) P. aeruginosa isolates were successively recovered from glans/foreskin swabs and urine cultures from two uncircumcised pediatric patients, one with Burkitt`s non-Hodgkin`s lymphoma and one with acute lymphoblastic leukemia. Clinically, preputial colonization by MDR P. aeruginosa evolved to severe balanoposthitis with glans/foreskin lesions as a source of fever. Combination therapy of ciprofloxacin and/or aztreonam (systemic) plus polymyxin B (topical) was effective once reversion of the neutropenic condition was achieved. Although P. aeruginosa remains an unusual cause of balanoposthitis, these cases should alert the physician to the potential pathogenicity of this bacterium. Furthermore, co-production of metallo-beta-lactamase and 16S rRNA methylase has a potential impact on the empirical management of complicated infections caused by P. aeruginosa. Crown Copyright (C) 2009 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. All rights reserved.

Risk factors for colonisation of newborn infants during an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in an intermediate-risk neonatal unit

We describe a cross-sectional, survey to identify risk factors for colonisation of neonates by extended-spectrum P-Lactamase (ESBL)-producing Klebsiella pneumoniae. This occurred following exposure to a colonised healthcare worker during an outbreak in an intermediate-risk neonatal. unit. In total, 120 neonates admitted consecutively during a three-month period were screened for ESBL-producing K. pneumoniae by rectal swabbing and 27 were identified as colonised. Multivariate analysis showed colonisation to be independently associated with use of antibiotics and absence of breastfeeding. Previous use of antibiotics presented an odds ratio (OR) of 12.3 [95% confidence interval. (Cl): 3.66-41.2, P < 0.001]. The most commonly used antibiotics were penicillin and amikacin. Breastfeeding was associated with reduced risk for colonisation (OR: 0.22; 95% Cl: 0.05-0.99; P = 0.049). Nine isotates recovered during the first stage of the outbreak and 27 isolates from surveillance cultures were typed thereafter by pulsed-field gel electrophoresis, revealing six different profiles (A-F). Clones A, C, and E were implicated in the first stage of the outbreak, whereas among the 27 strains recovered from surveillance cultures, all six clones were identified. Clone A was also found on the hand of a nursing auxiliary with onychomycosis. We concluded that prior antimicrobial use predisposed to colonisation. The possible role of breastfeeding as a protective factor needs to be further elucidated. Detection of different genotypes of ESBL-producing K. pneumonioe suggests that dissemination of mobile genetic elements bearing the ESBL gene may have been superimposed on the simple dissemination of a clone during the outbreak. (c) 2008 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.