Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina / Detección molecular de cepas patógenas de Escherichia coli aisladas de terneros neonatos de tambo en la provincia de Córdoba, Argentina

The aim of this study was to perform a current molecular characterization of bovine pathogenic Escherichia coli strains isolated from random samplings in Argentinean dairy farms. Rectal swabs were obtained from 395 (63.7 %) healthy and 225 (36.3 %) diarrheic calves, belonging to 45 dairy farms in Cordoba Province, Argentina. E. coli isolates were examined for virulence genes (f5, f41, f17, sta, stb, lt, eae, vt) using PCR and the prevalence of E. coli virulence profiles was spatially described in terms of spatial distribution. A total of 30.1 % isolates were found to be positive for at least one of the virulence genes. Depending on the different gene combinations present, 11 virulence profiles were found. Most of the isolates analyzed had a single gene, and no combination of fimbrial and enterotoxin gene was predominant. There was no association between the frequency and distribution of E. coli virulence genes and calf health status. Most of the virulence profiles were compatible with ETEC strains and showed a homogeneous distribution over the sampled area. A clustering pattern for E. coli virulence profiles could not be recognized. This work provides updated information on the molecular characterization of pathogenic E. coli strains from dairy herds in Cordoba, Argentina. These findings would be important to formulate prevention programs and effective therapies for diarrhea in calves caused by E. coli.

El objetivo de este trabajo fue realizar una caracterización molecular actualizada de cepas patógenas bovinas de Escherichia coli aisladas de un muestreo aleatorio en tambos de una de las principales zonas lecheras de Argentina. Se obtuvieron hisopados rectales de 395 terneros neonatos sanos (63,7 %) y 225 diarreicos (36,3 %) pertenecientes a 45 tambos de la provincia de Córdoba, Argentina. Los genes de virulencia f5, f41, f17, sta, stb, lt, eae y vt se analizaron mediante PCR y se investigó la prevalencia de los perfiles de virulencia en función de la distribución geográfica. La prevalencia de aislamientos de E. coli patogénicos con al menos un gen de virulencia fue del 30,1 %. Once perfiles de virulencia fueron identificados, dependiendo de la combinación de genes presentes. La mayor parte de las muestras presentó un solo gen de virulencia, y no predominó ninguna combinación de genes de fimbrias y toxinas. No hubo asociación entre la frecuencia y la distribución de los genes de virulencia y el estado de salud de los terneros. La mayoría de los perfiles de virulencia fueron compatibles con cepas ECET y se distribuyeron cubriendo toda el área geográfica muestreada. No se reconoció ningún patrón de agrupamiento espacial para dichos perfiles. Este trabajo provee información actualizada sobre la caracterización molecular de E. coli patógena en rodeos lecheros de Córdoba, Argentina. Estos resultados serían importantes para formular programas preventivos y terapias eficaces contra la diarrea bovina causada por E. coli.

stg fimbrial operon from S. Typhi STH2370 contributes to association and cell disruption of epithelial and macrophage-like cells

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) stg operon, encoding a chaperone/usher fimbria (CU), contributes to an increased adherence to human epithelial cells. However, one report suggests that the presence of the Stg fimbria impairs the monocyte-bacteria association, as deduced by the lower level of invasion to macrophage-like cells observed when the stg fimbrial cluster was overexpressed. Nevertheless, since other CU fimbrial structures increase the entry of S. Typhi into macrophages, and considering that transcriptomic analyses revealed that stg operon is indeed expressed in macrophages, we reassessed the role of the stg operon in the interaction between S. Typhi strain STH2370 and human cells, including macrophage-like cells and mononuclear cells directly taken from human peripheral blood. RESULTS: We compared S. Typhi STH2370 WT, a Chilean clinical strain, and the S. Typhi STH2370 Astg mutant with respect to association and invasion using epithelial and macrophage-like cells. We observed that deletion of stg operon reduced the association and invasion of S. Typhi, in both cellular types. The presence of the cloned stg operon restored the WT phenotype in all the cases. Moreover, we compared Salmonella enterica sv. Typhimurium 14028s (S. Typhimurium, a serovar lacking stg operon) and S. Typhimurium heterologously expressing S. Typhi stg. We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells. CONCLUSIONS: S. Typhi stg operon encodes a functional adhesin that participates in the interaction bacteria-eukary-otic cells, including epithelial cells and macrophages-like cells. The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.

Prevalence of papC and usp Virulence Factors in Uropathogenic Escherichia coli Causing Asymptomatic Urinary Tract Infections in Adolescents.

Aims: To determine the prevalence of two virulence genes associated with uropathogenic Escherichia coli; papC gene of the P fimbriae for adherence to uro-epithelial cells and usp (uropathogen-specific protein) gene, a Vibrio cholerae toxin gene homologue. Study Design: Cross sectional. Place and Duration of Study: Department of Biochemistry and Biotechnology and the Clinical Analysis Laboratory, Kwame Nkrumah University of Science and Technology, Kumasi, between October 2011 and February 2012. Methodology: Escherichia coli isolates (n= 149) from an adolescent population of ages 13- 18 years (from a total sampled population of 85 males and 64 females) were screened for papC and usp, using specific primers for the two genes in polymerase chain reactions. Results: The usp gene was the most prevalent (72.48%), followed by papC (51.00%) and papC+usp (24.16%). Significant difference (P = .002) was observed between papC and usp and also papC and papC+usp (P < .0001). usp Gene prevalence was also significantly different from that of papC+usp (P < .0001). Conclusion: This study suggests that a higher proportion of strains of uropathogenic Escherichia coli implicated in UTI in the studied population possess the usp gene whose protein product potentially serves to reduce competing microbes in the urinary tract.

Virulence factors in Escherichia coli isolated from calves with diarrhea in Vietnam

This study was conducted to determine the prevalence and characteristics of pathogenic Escherichia (E.) coli strains from diarrheic calves in Vietnam. A total of 345 E. coli isolates obtained from 322 diarrheic calves were subjected to PCR and multiplex PCR for detection of the f5, f41, f17, eae, sta, lt, stx1, and stx2 genes. Of the 345 isolates, 108 (31.3%) carried at least one fimbrial gene. Of these 108 isolates, 50 carried genes for Shiga toxin and one possessed genes for both enterotoxin and Shiga toxin. The eae gene was found in 34 isolates (9.8%), 23 of which also carried stx genes. The Shiga toxin genes were detected in 177 isolates (51.3%) and the number of strains that carried stx1, stx2 and stx1/stx2 were 46, 73 and 58, respectively. Among 177 Shiga toxin-producing E. coli isolates, 89 carried the ehxA gene and 87 possessed the saa gene. Further characterization of the stx subtypes showed that among 104 stx1-positive isolates, 58 were the stx1c variant and 46 were the stx1 variant. Of the 131 stx2-positive strains, 48 were stx2, 48 were stx2c, 11 were stx2d, 17 were stx2g, and seven were stx2c/stx2g subtypes. The serogroups most prevalent among the 345 isolates were O15, O20, O103 and O157.

Genes involucrados en la biogénesis de fimbrias afectan la formación de biopelículas por parte de Klebsiella pneumoniae / Genes involved in fimbrial biogenesis affect biofilm formation in Klebsiella pneumoniae

Introducción. Klebsiella pneumoniae es un patógeno oportunista comúnmente asociado con infecciones hospitalarias. La persistencia y patogénesis de este microorganismo pueden estar asociadas con su capacidad para formar biopelículas. Entre los factores implicados en la formación de biopelículas en diversos microorganismos están las fimbrias o pili. K. pneumoniae expresa tanto fimbria tipo 1 como fimbria tipo 3, estructuras proteicas importantes en la mediación de la adhesión a células epiteliales y la virulencia. Objetivo. Identificar genes importantes en la formación de biopelículas de K. pneumoniae. Materiales y métodos. K. pneumoniae MZ2098 se mutagenizó con el transposón miniTn10Km y subsecuentemente se tamizó para deficiencias en la formación de biopelículas en placas de 96 pozos usando medio BHI-MOPS. Los mutantes seleccionados se analizaron bajo diversas condiciones variando los medios de cultivo y las superficies utilizadas. Los genes interrumpidos por el transposón se identificaron mediante reacción en cadena de la polimerasa arbitraria y secuenciación. Resultados. De un banco de 9.300 inserciones en K. pneumoniae se obtuvieron 37 mutantes deficientes en su capacidad para formar biopelículas. Se identificaron tres mutantes con inserciones en genes para fimbria con fenotipos notables por su diferencia en cuanto a la capacidad para adherirse a superficies in vitro. Conclusión. Los resultados indicaron que las fimbrias tipo 1 y 3, ésta última ya implicada en este fenómeno en K. pneumoniae, son factores importantes para la adhesión y la formación de agregados multicelulares.

Introduction. Klebsiella pneumoniae is an opportunistic pathogen commonly associated with nosocomial infections. The persistence and pathogenesis of this microorganism is associated with its capacity to form biofilms. Pili or fimbriae are among the factors implicated in biofilm formation in diverse microorganisms. Klebsiella pneumoniae expresses both type 1 and type 3 fimbriae—proteinacious structures that mediate adhesion to epithelial cells and are important for virulence. Objective. To identify genes involved in biofilm formation in K. pneumoniae. Materials and methods. Klebsiella pneumoniae MZ2098 was subjected to mutagenesis with the miniTn10Km transposon and screened for defects in ability to form biofilms. The bacteria were curltured in 96-well plates using BHI-MOPS medium. Selected mutants were analyzed under diverse conditions by varying culture conditions and growth surfaces. Genes interrupted by the transposon were identified by arbitrary polymerase chain reaction and sequencing. Results. Thirty-seven mutants deficient in biofilm formation were obtained by screening 9,300 transposon-insertion mutants in K. pneumoniae. Three of these mutants had insertions in genes that affected fimbrial formation, and their phenotypes showed severe defects in the capacity to adhere to surfaces in vitro. Conclusion. Type 1 and type 3 fimbriae are important factors for adhesion and formation of multicellular aggregates of K. pneumoniae.

Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil / Fatores de virulência de Escherichia coli uropatogênicas provenientes de um Hospital Universitário em Ribeirão Preto, São Paulo, Brasil

The aim of the study was to determine the occurrence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of a tertiary-care teaching hospital, between July and August 2000, showing clinical and laboratory signs of urinary tract infection (UTI). The presence of genes (pap, afa, sfa) for fimbriae expression was assayed using specific primers in a polymerase chain reaction. Among the isolates studied, the prevalence of the virulence factors was 96.0 percent, 76.0 percent, 24.0 percent, for hemolysin, aerobactin and colicin, respectively; the prevalence of genes coding for fimbrial adhesive systems was 32.0 percent, 19.0 percent and 11.0 percent for pap, sfa and afa respectively. The strains isolated from the outpatients displayed a greater number of virulence factors compared to those from hospitalized subjects, emphasizing the difference between these two kinds of patients.

O objetivo do trabalho foi determinar a ocorrência de fatores de virulência, tais como, a expressão de fímbrias, produção de hemolisina, colicina e aerobactina em 100 cepas de Escherichia coli isoladas de pacientes ambulatoriais e hospitalizados de um hospital universitário de nível de atendimento terciário, entre os meses de julho e agosto de 2000, que apresentavam sinais clínicos e laboratoriais de infecção do trato urinário (ITU). Foram pesquisados os genes pap, afa e sfa responsáveis pela expressão de fímbrias através da técnica de PCR. A freqüência dos fatores de virulência entre as cepas estudadas foi de 96,0 por cento, 76,0 por cento e 24,0 por cento para hemolisina, aerobactina e colicina respectivamente, e a prevalência dos genes para os sistemas de adesinas fimbriais foi de 32,0 por cento, 19,0 por cento e 11,0 por cento para os genes pap, sfa e afa respectivamente. As cepas isoladas dos pacientes ambulatoriais exibiram um número maior de fatores de virulência quando comparadas com aquelas provenientes de indivíduos hospitalizados.

Validity of mechanism of gene transfer in the process called conjugation in bacteria.

We have attempted a new evaluation of the process of conjugation in bacteria, because of some basic dissimilarities observed between this and that of eukaryotes, or plants and animals. Reference donor and recipient strains, widely used to prove conjugation in bacteria, were chosen; addition of DNase during the conjugation process, led to an unexpected but highly reproducible increase in the transconjugant colony counts (TCC; ca. > or = 1 log), when compared with that of the controls without DNase. Transconjugants were also obtained when the same live donors were substituted with the UV-killed ones although the TCC was very low initially. Contrarily, donors treated with DNA-intercalating agents, e.g. acridine orange or ethidium bromide, resulted in a complete failure to produce transconjugants. There was a quantitative relationship between the DNase used on donors and levels of DNA sugars/nucleotides/DNA, which possibly resulted from interaction between the DNase and DNA being present/produced on the donor surface. This may be indicative of what may actually happen in the donor-recipient mixtures in the conjugation test proper, where the recipient DNase may activate a donor DNA production cycle. The evidences presented did not suggest that the donor DNA in the conjugation process is actually vestibuled through any intercellular conjugation passages, and is susceptible to the action of DNase or the intercalating dyes.