Klebsiella variicola and Klebsiella quasipneumoniae with capacity to adapt to clinical and plant settings / Klebsiella variicola y Klebsiella quasipneumoniae con capacidad para adaptarse al ambiente clínico y a las plantas

Abstract: Objective: To compare the genetic determinants involved in plant colonization or virulence in the reported genomes of K. variicola, K. quasipneumoniae and K. pneumoniae. Materials and methods: In silico comparisons and Jaccard analysis of genomic data were used. Fimbrial genes were detected by PCR. Biological assays were performed with plant and clinical isolates. Results: Plant colonization genes such as cellulases, catalases and hemagglutinins were mainly present in K. variicola genomes. Chromosomal β-lactamases were characteristic of this species and had been previously misclassified. K. variicola and K. pneumoniae isolates produced plant hormones. Conclusions: A mosaic distribution of different virulence- and plant-associated genes was found in K. variicola and in K. quasipneumoniae genomes. Some plant colonizing genes were found mainly in K. variicola genomes. The term plantanosis is proposed for plant-borne human infections.

Resumen: Objetivo: Comparar genes de colonización de plantas o de virulencia en los genomas reportados de K. variicola, K. quasipneumoniae y K. pneumoniae. Material y métodos: Se utilizaron análisis in silico y de Jaccard. Por PCR se detectaron genes de fimbrias. Se realizaron ensayos biológicos con aislados de plantas y clínicos. Resultados: Los genes de colonización de plantas como celulasas, catalasas y hemaglutininas se encontraron principalmente en genomas de K. variicola. Las β-lactamasas cromosómicas son características de la especie y en algunos casos estaban mal clasificadas. K. variicola y K. pneumoniae producen hormonas vegetales. Conclusiones: Se encontró una distribución en mosaico de los genes de asociación con plantas y de virulencia en K. variicola y K. quasipneumoniae. Principalmente en K. variicola se encontraron algunos genes involucrados en la colonización de plantas. Se propone el término plantanosis para las infecciones humanas de origen vegetal.

Perfil de sensibilidad antimicrobiana de microorganismos causantes de infecciones urinarias adquiridas en la comunidad en pacientes con diabetes mellitus en Colombia / Antimicrobial susceptibility profile in urinary pathogens causing community-acquired infections in diabetic patients in Colombia

Resumen Introducción. La infección de las vías urinarias es la más frecuente en pacientes diabéticos, y es un factor determinante de la morbilidad y la mortalidad en este grupo de pacientes. El aumento de la resistencia de los microorganismos adquiridos en la comunidad a los antibióticos comúnmente utilizados para combatirla es alarmante. Objetivo. Determinar el perfil de sensibilidad a los antibióticos de los microorganismos responsables de infecciones urinarias adquiridas en la comunidad en pacientes diabéticos atendidos en algunos hospitales de Colombia. Materiales y métodos. Se hizo un estudio descriptivo de un subgrupo de pacientes diabéticos en el marco de una investigación en adultos con infección de origen comunitario de las vías urinarias. Durante un año, se recolectaron aislamientos de Escherichia coli, Klebsiella spp. y Proteus mirabilis en nueve hospitales de Colombia y se determinó su perfil de sensibilidad mediante métodos microbiológicos y moleculares, para establecer la presencia de betalactamasas de espectro extendido del tipo AmpC y de carbapenemasas del tipo KPC. Resultados. Se recolectaron 68 aislamientos (58 de E. coli, nueve de Klebsiella spp. y uno de P. mirabilis). Cuatro (6,9 %) de los aislamientos de E. coli expresaron dichas betalactamasas, en dos (3,4 %) de ellos, pertenecientes al grupo filogenético B2 y al clon ST131, se detectaron las betalactamasas TEM-1 y CTM-X-15. En otros cuatro (6,9 %) aislamientos de E. coli se encontró el fenotipo AmpC, y en tres de ellos se produjeron las betalactamasas TEM-1 y CMY-2. Un aislamiento de K. pneumoniae expresó la carbapenemasa KPC-3. Conclusión. Se confirmó la presencia de cepas productoras de betalactamasas de espectro extendido y carbapenemasas en microorganismos responsables de infección urinaria adquirida en la comunidad en pacientes diabéticos.

Abstract Introduction: Urinary tract infection is the most common pathology in diabetic patients, and an important determinant of morbidity and mortality among them. The increasing resistance of uropathogens acquired in the community to commonly used antibiotics is alarming. Objective: To identify the profile of antibiotic susceptibility of uropathogens responsible for community-acquired infections among diabetic patients in hospitals in Colombia. Materials and methods: We conducted a descriptive study in a subgroup of diabetic patients in the framework of a larger study in adults with urinary tract infection acquired in the community. Over one year, we collected Escherichia coli, Klebsiella spp. and Proteus mirabilis isolates from nine hospitals in Colombia. Their susceptibility profile was determined using microbiological and molecular methods to establish the presence of extended-spectrum AmpC betalactamases and KPC carbapenemases. Results: We collected 68 isolates (58 E. coli, nineKlebsiella spp. and oneProteus mirabilis). Four (6.9%) of the E. coli isolates expressed extended spectrum betalactamases,two (3.4%) of thembelonged to the phylogenetic group B2 andto ST131 clone and expressed the TEM-1 and CTM-X-15 betalactamases. The AmpC phenotype was found in four(6.9%) of the E. coli isolates, three of which producedTEM-1 and CMY-2 betalactamases. One K. pneumoniaeisolate expressed the KPC-3 carbapenemase. Conclusion: The presence of extended spectrum betalactamases and carbapenemases in uropathogens responsible for community-acquired infection was confirmed in diabetic patients.

CTX-M extended-spectrum ß-lactamase-producing Klebsiella spp, Salmonella spp, Shigella spp and Escherichia coli isolates in Iranian hospitals

Abstract This study was conducted in Iran in order to assess the distribution of CTX-M type ESBLs producing Enterobacteriaceae. From January 2012 to December 2013, totally 198 E. coli, 139 Klebsiella spp, 54 Salmonella spp and 52 Shigella spp from seven hospitals of six provinces in Iran were screened for resistance to extended-spectrum cephalosporins. After identification and susceptibility testing, isolates presenting multiple-drug resistance (MDR) were evaluated for ESBL production by the disk combination method and by Etest using (cefotaxime and cefotaxime plus clavulanic acid). All isolates were also screened for bla CTX-M using conventional PCR. A total of 42.92%, 33.81%, 14.81% and 7.69% of the E. coli, Klebsiella spp, Salmonella spp and Shigella spp isolates were MDR, respectively. The presence of CTX-M enzyme among ESBL-producing isolates was 85.18%, 77.7%, 50%, and 66.7%, in E. coli, Klebsiella spp, Salmonella spp and Shigella spp respectively. The overall presence of CTX-M genes in Enterobacteriaceae was 15.4% and among the resistant isolates was 47.6%. This study indicated that resistance to β-lactams mediated by CTX-M enzymes in Iran had similar pattern as in other parts of the world. In order to control the spread of resistance, comprehensive studies and programs are needed.

Mortalidad por bacteriemia causada por Escherichia coli y Klebsiella spp. productoras de beta lactamasas de espectro extendido: cohorte retrospectiva en un hospital de Lima, Perú / Mortality caused by bacteremia Escherichia coli and Klebsiella spp. extended-spectrum beta-lactamase- producers: a retrospective cohort from a hospital in Lima, Peru

Objetivos. Evaluar los factores asociados a la mortalidad causada por bacteriemias por Escherichia coli y Klebsiella spp. productoras de beta lactamasas de espectro extendido (BLEE). Materiales y Métodos. Se realizó un estudio de cohortes retrospectivo, que incluyó 85 pacientes mayores de 16 años con diagnóstico de bacteriemia por Escherichia coli o Klebsiella spp. hospitalizados entre 2006 y 2008 en el Hospital Nacional Cayetano Heredia. Las cohortes se clasificaron de acuerdo a la producción de BLEE según los resultados de los hemocultivos. Se evaluaron los factores asociados a la mortalidad cruda y atribuible empleando regresión de Poisson en un modelo multivariado, con lo que se obtuvo riesgos relativos ajustados (RRa). Además, se construyeron curvas de mortalidad. Resultados. Se encontró que el 35,3% de las bacteriemias fueron debidas a cepas productoras de BLEE. El análisis de la mortalidad cruda mostró una mayor mortalidad en el grupo de cepas productoras de BLEE (63,3%). El RRa fue de 1,5 (IC95%: 1,02-2,3). En el caso de mortalidad atribuible, la proporción también fue mayor (63,3%), el RRa fue de 1,9 (IC95%: 1,2-2,9). El uso de catéter venoso central fue otro factor asociado tanto a la mortalidad cruda (RRa= 2,4; IC95%: 1,2- 4,8) como a la mortalidad atribuible (RRa= 3,8; IC95%: 1,6-8,8). Conclusiones. La producción de BLEE es un factor de riesgo independiente para mortalidad por bacteriemia causada por E. coli y Klebsiella spp. Su presencia debe evaluarse tras la sospecha diagnóstica y la elaboración de la terapéutica inicial, lo que podría disminuir la mortalidad por esta causa.

Objectives. To evaluate the factors associated to mortality caused by bacteremia due to Escherichia coli and Klebsiella spp. producers of extended-spectrum beta-lactamase (ESBL). Materials and methods. We performed a retrospective cohort study, including 85 patients older than 16 and diagnosed with bacteremia by Escherichia coli or Klebsiella spp., hospitalized between 2006 and 2008 in Cayetano Heredia National Hospital. Cohorts were classified according to the ESBL production following blood culture results. Factors associated to gross and attributable mortality were evaluated using the Poisson regression in a multivariate model, through which adjusted relative risks (ARRs) were obtained. Mortality curves were also built. Results. 35.3% of bacteremia cases were caused by ESBL-producing strains. The analysis of gross mortality showed a higher mortality rate in the group with ESBL producing strains (63.3%), ARR being 1.5 (CI 95%: 1.02-2.3). In the case of the attributable mortality, the proportion was also higher (63.3%), ARR being 1.9 (CI 95%: 1.2-2.9). The use of a central venous catheter was another factor associated to both gross mortality (ARR= 2.4; CI 95%: 1.2-4.8) and attributable mortality (ARR= 3.8; CI 95%: 1.6-8.8). Conclusions. The production of ESBL is an independent risk factor for bacteremia mortality caused by E. coli and Klebsiella spp. Its presence should be evaluated following diagnosis consideration and initial therapy elaboration, which could in turn decrease the mortality by this cause.

Phenotypic & molecular characterization of AmpC β-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. from five Indian Medical Centers.

Background & objectives: AmpC β-lactamases which are often plasmid mediated hydrolyze all β-lactam antibiotics except cefepime and carbapenems. We evaluated the presence of AmpC β-lactamases among Enterobacteriaceae strains recovered prospectively from patients at five Indian tertiary care centres. Methods: The study included 909 consecutive Gram-negative isolates recovered from clinically significant specimens during June 2007 - May 2008 as part of an ICMR-ESBL study. Among the study isolates, 312 were found to be cefoxitin resistant by disc diffusion test (DDT). Minimum inhibitory concentration (MIC) determination by E test was done against amikacin, levofloxacin, impinem, meropenem, ertapenem, tigecycline and piperacillin-tazobactam. Combined DDT using phenyl boronic acid as inhibitor with cefoxitin was used for phenotypic confirmation of AmpC phenotype. The common Amp C genotypes ACC, FOX, MOX, DHA, CIT and EBC were detected by multiplex PCR. Results: Plasmid mediated Amp C phenotype was confirmed in 114 of the 312 (36.5%) cefoxitin resistant isolates with 255 (81.7%) showing multidrug resistance. Susceptibility to tigecycline was highest (99%) followed by imipenem, meropenem (97%), ertapenem (89%), amikacin (85%), and piperacillin-tazobactam (74.6%). Levofloxacin resistance was 82 per cent. ESBL co carriage was observed among 92 per cent of Amp C producers. Among 114 Amp C producers, 48 could be assigned a genotype, this included CIT- FOX (n=25), EBC (n=10), FOX (n = 4), CIT (n=3), EBC-ACC (n=2) and one each of DHA, EBC-DHA, FOX -DHA and FOX-EBC-DHA. Interpretation & Conclusions: Overall, AmpC phenotypes were found in 12.5 per cent isolates, multidrug resistance and ESBL co-carriage among them was high suggesting plasmid mediated spread. The study results have implications in rational antimicrobial therapy and continued surveillance of mechanisms of resistance among nosocomial pathogens.

Nosocomial and community infections due to class A extended-spectrum β-lactamase (ESBLA)-producing Escherichia coli and Klebsiella spp. in southern Brazil

OBJECTIVES: To determine the prevalence of class A extended spectrum β-lactamases (ESBL)-producing Escherichia coli and Klebsiella spp., and to investigate clonality among ESBL-producing isolates of nosocomial and community infections. METHODS: The study involved 354 nosocomial infections samples and 992 community infections samples, obtained between 2003 and 2006 at Caxias do Sul, RS. The detection of ESBL was performed by the disk-diffusion test. Presence of blaCTX-M, blaSHV and blaTEM β-lactamase genes was evaluated by PCR, and genomic typing was determined by pulsed-field gel electrophoresis analysis. RESULTS: Higher frequency of ESBL-producing isolates were detected among nosocomial samples of E. coli (6.7 percent) and Klebsiella (43.7 percent), than those obtained from community infections (0.4 percent and 2.6 percent). blaTEM and blaCTX were the most prevalent ESBL gene families in both E. coli and Klebsiella isolates. Different pulsotypes were obtained among ESBL-producing E. coli and 11 clones for Klebsiella spp., which occurred over the years and in different hospital wards. Among ESBL-producing K. pneumoniae, 74.3 percent transferred ESBL genes by conjugation and exhibited concomitant decreased aminoglycosides susceptibility. CONCLUSION: ESBL-producing E. coli, and especially K. pneumoniae are essentially a nosocomial problem, and their dissemination to the community is relatively limited. The great genetic variability observed among ESBL-producing bacteria indicates polyclonal spread and high transference of ESBL genes between bacteria in the hospital environment. This information is of paramount importance for nosocomial infection control.

Detección de cepas productoras de b-lactamasas de espectro extendido por el sistema DIRAMIC: Comparación con los métodos doble difusión con discos y E-test / Detection of expanded-espectrum-betalactamases by DIRAMIC system: Comparison with the double-disk synergy test and E-test

The capacity of the DIRAMIC system to detect strains producing extended-spectrum-betalactamase (ESBL) was evaluated through the comparison with two phenotypic confirmatory tests: double-disk synergy test and E-test. Ninety seven clinical isolates of Escherichia coli y Klebsiella spp. previously characterized; not repeated and suspected of being ESBL producers were studied by the three methods. In comparison with E-test and double-disk synergy test, DIRAMIC system showed a sensitivity of 85.7 percent and 92.7 percent as well as specificity of 100 percent and 92.9 percent; respectively. The values found have a very high degree of concordance (kappa index > 0.80). The results obtained vouch for the utility of the DIRAMIC as a rapid method to alert about the presence of strains producing ESBL enzymes.

Se evaluó la capacidad del sistema DIRAMIC-Cuba para detectar cepas productoras de p-lactamasas de espectro extendido (BLEE), mediante la comparación con dos métodos fenotípicos confirmatorios: doble difusión con discos y E-test. Noventa y siete aislados clínicos de Escherichia coli y Klebsieüa spp previamente caracterizados, no repetidos, sospechosos de producir BLEE se estudiaron por los tres procedimientos para determinar sensibilidad, especificidad y concordancia entre los resultados. Para el sistema DIRAMIC se encontró una sensibilidad de 85,7 y 92,7 por ciento y una especificidad de 100 y 92,9 por ciento en comparación con E-test y doble difusión con discos, respectivamente. Los valores de concordancia encontrados fueron muy altos (índice kappa > 0,80). Los resultados obtenidos avalan la utilidad del sistema DIRAMIC como vía rápida, para alertar al médico acerca de la presencia de cepas productoras de BLEE, aunque es necesario profundizar y ampliar el estudio a modo de emitir resultados más precisos.

Prevalência das famílias TEM, SHV e CTX-M de -lactamases de espectro estendido em Escherichia coli e Klebsiella spp no Hospital Universitário de Santa Maria, Estado do Rio Grande do Sul / Prevalence of the TEM, SHV and CTX-M families of extended-spectrum -lactamases in Escherichia coli and Klebsiella spp at the University Hospital of Santa Maria, State of Rio Grande do Sul

Neste estudo estimou-se a distribuição e prevalência de β-lactamases de espectro estendido pertencentes às famílias TEM, SHV e CTX-M entre amostras de Escherichia coli e Klebsiella spp. no Hospital Universitário de Santa Maria, Rio Grande do Sul. Durante 14 meses, 90 microrganismos foram selecionados como prováveis produtores de ESBL. Os isolados foram submetidos a testes fenotípicos confirmatórios para a presença de ESBL. A seguir, os tipos de ESBLs presentes em cada microrganismo foram determinados através da pesquisa dos respectivos genes através da reação em cadeia da polimerase. Empregando-se o método do disco combinado, a presença de ESBLs foi confirmada em 55 (61,1 por cento) amostras; quando o método do duplo disco foi utilizado, 57 (63,3 por cento) amostras foramprodutoras de ESBLs. Com base na PCR, as ESBLs do tipo TEM e SHV foram mais presentes em Klebsiella pneumoniae enquanto que ESBL do tipo CTX-M foram mais presentes em Klebsiella oxytoca.

In this study, the distribution and prevalence of extended-spectrum β-lactamases belonging to the TEM, SHV and CTX-M families were estimated among samples of Escherichia coli and Klebsiella spp. at the university hospital of Santa Maria, Rio Grande do Sul. Over a 14-month period, 90 microorganisms were selected as likely ESBL producers. The isolates were subjected to confirmatory phenotype tests for the presence of ESBL. Through investigating the respective genes using the polymerase chain reaction, the ESBL types present in each microorganism were then determined. Fifty-five samples (61.1 percent) were confirmed as ESBL-positive by means of the combined disc method, and 57 (63.3 percent) were found to be ESBL producers by means of the double disc method. From the polymerase chain reaction, ESBLs of TEM and SHV types were more frequently present in Klebsiella pneumoniae, while ESBL of CTX-M type was more frequently present in Klebsiella oxytoca.

Glucosyltransferase production by Klebsiella sp. K18 and conversion of sucrose to palatinose using immobilized cells / Produção de glicosiltransferase por Klebsiella sp. K18 e conversão de sacarose em palatinose utilizando células imobilizadas

The strain Klebsiella sp. K18 produces the enzyme glucosyltransferase and catalyses the conversion of sucrose to palatinose, an alternative sugar that presents low cariogenicity. Response Surface Methodology was successfully employed to determine the optimal concentration of culture medium components. Maximum glucosyltransferase production (21.78 U mL-1) was achieved using the optimized medium composed by sugar cane molasses (80 g L-1), bacteriological peptone (7 g L-1) and yeast extract (20 g L-1), after 8 hours of fermentation at 28ºC. The conversion of sucrose to palatinose was studied utilizing immobilized cells in calcium alginate. The effects of the alginate concentration (2-4%), cell mass concentration (20-40%) and substrate concentration (25-45%) were evaluated and the yield of palatinose was approximately 62.5%.

A linhagem Klebsiella sp. K18 produz a enzima glicosiltransferase que catalisa a conversão de sacarose em palatinose, um açúcar alternativo que apresenta baixa cariogenicidade. Metodologia de Superfície de Resposta foi empregada com sucesso para determinar a concentração ótima dos componentes do meio de cultivo. A máxima produção deglicosiltransferase (21,78 U mL-1) foi obtida utilizando o meio de cultivo otimizado composto por melaço de cana de açúcar (80 g L-1), peptona bacteriológica (7 g L-1) e extrato de levedura (20 g L-1), após 8 horas de fermentação a 28ºC. A conversão desacarose em palatinose foi estudada utilizando células imobilizadas em alginato de cálcio. Os efeitos da concentração de alginato (2-4%), concentração de massa celular (20-40%) e concentração de substrato (25-45%) foram avaliados e a porcentagem de palatinose foi de aproximadamente 62,5%.

Evaluation of the MicroScan NegCombo Panel Type 44 for Detection of Extended-Spectrum beta-Lactamase among Clinical Isolates of Escherichia coli, Klebsiella species, and Proteus mirabilis / 대한진단검사의학회지

BACKGROUND: Accurate and rapid detection of extended-spectrum beta-lactamases (ESBLs) is important in guiding proper antimicrobial therapy for infected patients. We evaluated the performance of MicroScan NegCombo Type 44 panel (Dade Behring, USA), which was developed to confirm ESBL-producing Enterobacteriaceae using ceftazidime/clavulanate and cefotaxime/clavulanate. METHODS: From August 30 to September 20, 2007, 206 non-duplicate clinical isolates, including 106 Escherichia coli, 81 Klebsiella pneumoniae, 11 Klebsiella oxytoca, and 8 Proteus mirabilis were subcultured and tested with Type 32 and Type 44 panels. The results were compared with those of the CLSI phenotypic confirmatory test (CLSI-PCT) and disk approximation test (DAT). Isolates not susceptible to cefotetan or flagged as "Possible ESBL, unable to interpret confirm test (Possible ESBL)" on Type 44 panel were tested with boronic acid disks to confirm AmpC beta-lactamases (AmpC) production. RESULTS: Of the 206 isolates tested, 44 (21.4%) produced ESBL by CLSI-PCT or DAT, including 27 E. coli, 14 K. pneumoniae, 2 K. oxytoca, and 1 P. mirabilis. Thirty-eight isolates flagged as "Confirmed ESBL" on Type 44 panel were all confirmed as ESBL-producers. Of 14 K. pneumoniae flagged as "Possible ESBL", 6 were confirmed as ESBL and AmpC co-producers and 8 as AmpC-producers. CONCLUSIONS: Type 44 panel showed an excellent performance in detecting ESBL-producing E. coli, Klebsiella spp., and P. mirabilis. When flagged as "Confirmed ESBL", no other confirmatory test was necessary to report as ESBL; however, "Possible ESBL" required a differential test for AmpC production.

Evaluation of the Phoenix Automated Microbiology System for Detecting Extended-Spectrum beta-Lactamase in Escherichia coli, Klebsiella species and Proteus mirabilis / 대한진단검사의학회지

BACKGROUND: The aim of this study was to compare the BD Phoenix (Beckton Dickinson Diagnostic Systems, USA) extended-spectrum beta-lactamase (ESBL) test with the Clinical and Laboratory Standards Institute (CLSI) ESBL phenotypic confirmatory test by disk diffusion (CLSI ESBL test) in Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Proteus mirabilis. METHODS: We tested 224 clinical isolates of E. coli, K. pneumoniae, K. oxytoca and P. mirabilis during May 2006 to March 2007. These isolates were examined by the Phoenix and the CLSI ESBL tests simultaneously. For the isolates showing discordant results between the two tests, boronic acid disk test was performed to differentiate AmpC beta-lactamase and ESBL. RESULTS: Among the 224 clinical isolates, 75 and 79 isolates were positive for ESBL by CLSI ESBL test and Phoenix test, respectively. Having detected 4 more isolates as ESBL-producers, Phoenix test showed a 98.2% agreement with a 100% sensitivity and 97.3% specificity compared with CLSI ESBL test. Among the four false positive isolates, three were AmpC-positive but ESBL-negative. CONCLUSIONS: The BD Phoenix ESBL test was sensitive and specific, and can be used as a rapid and reliable method to detect ESBL production in E. coli, Klebsiella species, and P. mirabilis.

SHV-type extended-spectrum beta-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico

OBJECTIVE: In this work we report the molecular characterization of beta-lactam antibiotics resistance conferred by genes contained in plasmids from enterobacteria producing extended-spectrum beta-lactamases (ESBL). MATERIAL AND METHODS: Fourteen enterobacterial clinical isolates selected from a group of strains obtained from seven different hospitals in Mexico during 1990-1992 and 1996-1998 were analyzed at the Bacterial Resistance Laboratory (National Institute Public Health, Cuernavaca). Molecular characterization included PFGE, IEF of beta-lactamases, bacterial conjugation, PCR amplification and DNA sequencing, plasmid extraction and restriction. RESULTS: Isolates were genetically unrelated. ESBL identified were SHV-2 (5/14) and SHV-5 (9/14) type. Cephalosporin-resistance was transferable in 9 of 14 (64 percent) clinical isolates with only one conjugative plasmid, DNA finger printing showed a similar band pattern in plasmids. CONCLUSIONS: The dissemination of cephalosporin resistance was due to related plasmids carrying the ESBL genes.

OBJETIVO: En este trabajo se reporta la caracterización molecular de la resistencia a antibiótico beta-lactámicos conferida por genes contenidos en plásmidos de enterobacterias productoras de beta-lactamasas de espectro extendido (BLEEs). MATERIAL Y MÉTODOS: Catorce aislamientos clínicos de enterobacterias fueron seleccionados por conveniencia de un banco de cepas obtenidas de siete diferentes hospitales de México durante los periodos 1990-1992 y 1996-1998 y fueron procesados en el Laboratorio de Resistencia Bacteriana (Instituto Nacional de Salud Pública, Cuernavaca). En la caracterización se empleó PFGE, IEF para beta-lactamasas, conjugación bacteriana, amplificación por PCR y secuenciación de DNA, extracción y restricción de plásmidos. RESULTADOS: Las 14 cepas fueron no relacionadas genéticamente. Se identificaron BLEEs tipo SHV-2 (5/14) y SHV-5 (9/14). La resistencia a cefalosporinas fue transferida por conjugación en 9 de 14 (64 por ciento) aislamientos clínicos mediante un plásmido que mostró un patrón de restricción similar entre ellos. CONCLUSIÓN: Se sugiere que la diseminación de la resistencia a cefalosporinas fue debida a plásmidos relacionados que contienen los genes que codifican BLEEs.

Occurrence of TEM & SHV gene in extended spectrum beta-lactamases (ESBLs) producing Klebsiella sp. isolated from a tertiary care hospital.

BACKGROUND & OBJECTIVE: Extended-spectrum beta-lactamases (ESBLs) are rapidly evolving group of beta-lactamase enzymes produced by the Gram negative bacteria. These enzymes have been derived from TEM and SHV genes by mutations and have been well described in Klebsiella pneumoniae. Information on molecular types of ESBL positive Klebsiella sp. is lacking from India. We therefore undertook this study to look for the TEM and SHV genes in ESBL positive Klebsiella sp. isolated from the patients admitted to a tertiary care hospital in north India. METHODS: A total of 204 multidrug-resistant isolates of Klebsiellae obtained from clinical samples; blood (n=108), urine (n=15), pus (n=2) and sputum (n=79) were obtained and screened for resistance to 3rd generation cephalosporins (3GC). The ESBL status was determined by double disk diffusion test (DDDT) and further by ESBL E-test. Multiplex PCR specific for TEM and SHV genes was performed to distinguish four different genotypes: TEM-positive, SHV-positive, TEM- and SHV-positive and non-TEM non-SHV ESBL types. RESULTS: Eighty six per cent (175 of 204) of the isolates were found to be resistant to at least one of the 3GCs, of which 97.1 per cent (170) of Klebsiella sp. isolates were confirmed to be positive for ESBL. Of these 170 isolates, 95 were randomly selected for PCR of TEM and SHV genes. Isolates having both TEM and SHV genes were common (67.3%) whereas only 20 per cent isolates possessed TEM gene and 8.4 per cent SHV gene alone. INTERPRETATION & CONCLUSION: Our findings showed that the majority of the ESBL positive clinical isolates of Klebsiella sp. carried both TEM and SHV genes followed by TEM alone. Such studies need to be done in various geographical regions of the country to know about the prevalent genotypes for better management of infection.

Clinical outcome and risk factors related to extended-spectrum beta-lactamase-producing Klebsiella spp. infection among hospitalized patients

Over the past two decades, nosocomial infections caused by extended-spectrum beta-lactamase (ESBL)-producing Klebsiella spp. have become a major problem all around the world. This situation is of concern because there are limited antimicrobial options to treat patients infected with these pathogens, and also because this kind of resistance can spread to a wide variety of Gram-negative bacilli. Our objectives wereto evaluate among in-patients at a publicuniversity tertiary-care hospital with documented infection due to Klebsiella spp., which were the risk factors (cross-sectional analysis) and the clinical impact (prospective cohort) associated with an ESBL-producing strain. Study subjects were all patients admitted at the study hospital between April 2002 and October 2003, with a clinically and microbiologically confirmed infection caused by Klebsiella spp. at any body site, except infections restricted to the urinary tract. Of the 104 patients studied, 47 were infected with an ESBL-producing strain and 57 with a non-ESBL-producing strain. Independent risk factors associated with infection with an ESBL-producing strain were young age, exposure to mechanical ventilation, central venous catheter, use of any antimicrobial agent, and particularly use of a 4th generation cephalosporin or a quinolone. Length of stay was significant longer for patients infected with ESBL-producing strains than for those infected with non-ESBL-producing strains, although fatality rate was not significantly affected by ESBL-production in this cohort. In fact, mechanical ventilation and bacteremia were the only variables withindependent association withdeath detected in this investigation.

Production of extracellular proteases and amylases by some acidophilic and alkalophilic bacteria.

Twelve bacteria were isolated from two effluent sources of Shaw-Wallace Gelatins, Jabalpur. Six bacteria from dicalcium phosphate plant effluent (pH-5) and six from main drain of the factory (pH-10) were isolated. Two facultatively acidophilic and two facultatively alkalophilic bacteria were selected and tentatively identified as Plesiomonas shigelloides, Aeromonas hydrophilla, Klebsiella pneumoniae and Staphylococcus saprophyticus respectively. Acidic amylases were produced in higher amounts on 4th day of incubation by Plesiomonas shigelloides and on 6th day by Aeromonas hydrophilla. Alkaline amylases were produced in higher amounts on 4th day of incubation by Klebsiella pneumoniae and on 8th day by Staphylococcus saprophyticus in vitro.

Phytase from Klebsiella Sp. No. PG-2: purification and properties.

A phytase (EC 3.1.3.8) was extracted from rat intestinal bacterium, Klebsiella Sp. No. PG.-2, and purified 50-fold by ammonium sulphate fractionation, ion-exchange chromatography and gel filtration. The enzyme is inducible in nature. The pH optimum was at 6.0 for all the inositol phosphates studied and this characterized the enzyme as an acid phosphohydrolase. Of a range of potential substrates tested, only p-nitrophenyl phosphate alongwith the inositol phosphates was hydrolyzed. It exhibits a Km of 2.0 mM; temperature optimum of 37 degrees C and energy of activation 9,120 cal/mole for all the inositol phosphates studied. The activity was inhibited by Ag2+, Hg2+, Cu2+, fluoride and high substrate concentration.

Purification and properties of alpha-galactosidase from Klebsiella Sp. No. PG-2.

alpha-Galactosidase has been purified from Klebsiella Sp. No. PG-2, a bacterium isolated from rat small intestine, using calcium phosphate gel, DEAE-cellulose column chromatography and gel filtration technique. About 130-fold increase in specific activity was observed, the pH optimum of 6.5-7.0 characterizes the enzyme as neutral alpha-galactosidase. The optimum temperature was 37 degrees C and the energy of activation was 11,856 cal/mole. Km values obtained for raffinose, mellibose, stachyose and p-nitrophenyl-alpha-D-galactopyranoside were 20.0, 6.6 33.3 and 4.0 mM respectively. The activity was inhibited by p-CMB; iodoacetate, Ag2+, Hg2+, Cu2+, Pb2+ and galactose. Examination of the enzyme activity indicated that the enzyme is cytosolic and is inducible in nature.