Correlation between virulence markers of Helicobacter pylori in the oral cavity and gastric biopsies / Correlação entre marcadores de virulência de Helicobacter pylori na cavidade oral e em biópsias gástricas

ABSTRACT BACKGROUND: The clinical outcome of Helicobacter pylori infection has been associated with virulence factors. The presence of these factors is useful as molecular markers in the identification of the high risk for developing severe gastric pathologies. OBJECTIVE: To correlate the presence of virulence markers cagA and bab2A of H. pylori in oral and gastric biopsy samples. METHODS: An observational, prospective, descriptive, and cross-sectional study was carried out between September 2011 and September 2012. Patients suffering dyspepsia with indication for upper gastrointestinal video endoscopy who attended the Gastroenterology Service of the Hospital Dr. Julio C. Perrando were included. Epidemiological investigation was completed. To detect the bacteria and their virulence genes, samples of saliva, dental plaque and gastric biopsy were taken and processed by PCR. RESULTS: Sixty-one patients were selected for this study (30 women and 31 men). H. pylori was detected in 31 gastric biopsies and 31 oral samples. Significant difference between oral and gastric samples was found in cagA genotype. Agreement between oral and gastric genotypes was found in 38.7% of samples from the same patient. CONCLUSION: This study is the first in provide information about the genotypes of the Argentinean Northeast H. pylori strains. Despite the high prevalence of H. pylori infection, the most of patients had less virulent genotypes in oral cavity and gastric tissue. The cagA / babA2 combination was not frequent in the samples studied. There was not a statistical correlation between the virulence genes and gastroduodenal or oral diseases. Although in some patients the same genotype was found both in oral and gastric samples, it cannot be ensure that they corresponding to the same strain because a DNA sequencing was not performed.

RESUMO CONTEXTO: O resultado clínico da infecção por Helicobacter pylori tem sido associado com fatores de virulência. A presença desses fatores como marcadores moleculares é útil na identificação do risco elevado para o desenvolvimento de graves patologias gástricas. OBJETIVOS: Correlacionar a presença de marcadores de virulência cagA e bab2A do H. pylori em amostras de biópsias gástricas e orais. MÉTODOS: Um estudo observacional, prospectivo, descritivo e transversal foi realizado entre setembro de 2011 e setembro de 2012. Foram incluídos pacientes com sintomas de dispepsia com indicação de endoscopia gastrointestinal que compareceram ao Serviço de Gastroenterologia do Hospital Dr. Julio C. Perrando . Investigação epidemiológica foi concluída. Para detectar a bactéria e seus genes de virulência, amostras de saliva, placa dentária e biópsia gástrica foram tomadas e processadas pelo PCR. RESULTADOS: Sessenta e um pacientes foram selecionados para este estudo (30 mulheres e 31 homens). H. pylori foi detectado em 31 biópsias gástricas e 31 amostras orais. Foi encontrada diferença significativa entre as amostras orais e gástricas no genótipo cagA . A ocorrência simultânea entre genótipos orais e gástricos do mesmo paciente foi encontrada em 38,7% das amostras. CONCLUSÃO: Este é o primeiro estudo a fornecer informações sobre os genótipos das cepas do H. pylori no Nordeste Argentino. Apesar da alta prevalência da infecção pelo H. pylori , a maioria dos pacientes tinha genótipos menos virulentos na cavidade oral e tecido gástrico. A combinação cagA / babA2 não foi frequente nas amostras estudadas. Não houve correlação estatística entre os genes de virulência e doenças gastroduodenais ou orais. Embora em alguns pacientes o mesmo genótipo tenha sido encontrado tanto nas amostras orais quanto gástricas, não se pode garantir que correspondam à mesma variação, pois um sequenciamento de DNA não foi realizado.

Genotipificación de los genes rgpA y kgp que codifican para las gingipaínas de Porphyromonas gingivalis / Genotyping of rgpA and kgp genes encoding Pophyromonas gingivalis gingipains

Porphyromonas gingivalis es un microorganismo fuertemente asociado con la etiología de la periodontitis. Esta bacteria posee varios factores de virulencia, dentro de los que destacan las gingipaínas, debido a sus múltiples acciones relacionadas con la destrucción de la matriz extracelular del tejido conectivo periodontal, la modulación del sistema inmune del hospedero y la estimulación de la expresión de citoquinas pro-inflamatorias. Estas proteinasas tienen afinidades específicas siendo Arg-gingipaínas (RgpA y RgpB, codificadas por los genes rgpA y rgpB, respectivamente) y Lys-gingipaínas (Kgp, codificada por el gen kgp). Se ha descrito que existen polimorfismos en los genes que codifican para esta proteinasas. El objetivo del presente estudio fue describir la frecuencia de los genotipos identificados para los genes rgpA y kgp en aislados clínicos de P. gingivalis, obtenidos desde pacientes con periodontitis. Para ello se utilizó amplificación por PCR de los genes rgpA y kgp, seguido de análisis de restricción. De un total de 47 aislados provenientes de 4 individuos con periodontitis crónica y 2 con periodontitis agresiva, se genotipificaron 38 aislados para el gen rgpA, exhibiendo la totalidad de éstos el patrón electroforético A (100 por ciento). Para el gen kgp se genotipificaron 43 aislados, presentando 28 de ellos (65.2 por ciento) el perfil electroforético kgp-I y 15 aislados (34.8 por ciento) el perfil kgp-II. En los aislados provenientes de un individuo fue posible apreciar ambos genotipos descritos para el gen kgp. Los resultados indican un predominio del patrón electroforético A (rgpA) y que el genotipo kgp-I fue el más frecuentemente encontrado de los genotipos kgp.

Porphyromonas gingivalis is a microorganism strongly associated with the etiology of periodontitis. This periodontal bacterium possesses an array of virulence factors, among which gingipains have a key importance, being involved with extracellular matrix destruction of periodontal tissues, modulation of host immune response and stimulation in the production of pro-inflammatory cytokines by different types of cells. These proteinases have specific affinities, being Arg-gingipains (RgpA and RgpB, encoded by rgpA and rgpB genes, respectively) and Lys-gingipains (Kgp, encoded by the kgp gene). It has been described that there are polymorphisms in the genes encoding for gingipains. Therefore, the aim of the present study was to describe the frequency of rgpA and kgp genotypes in clinical isolates of P. gingivalis obtained from periodontitis patients. For determining the rgpA and kgp genotypes, we used PCR amplification and restriction analysis. From 47 isolates obtained from 4 individuals with chronic periodontitis and 2 subjects with aggressive periodontitis, 38 were typified for rgpA gene and all exhibited the electrophoretic pattern A (100 percent). For kgp gene, we characterized 43 isolates, 28 of them (65.2 percent) with the kgp-I electrophoretic profile and 15 isolates (34.8 percent) with the kgp-II profile. In the isolates belonging to one individual, we found both genotypes of kgp gene. The results indicate a clear predominance of the electrophoretic pattern A (for rgpA gene) and kgp-I genotype was the most frequently found of the kgp genotypes.

Application of fnbA gene as new target for the species-specific and quantitative detection of Staphylococcus aureus directly from lower respiratory tract specimens by real time PCR.

Staphylococcus aureus is a significant cause of hospital-acquired pneumonia (HAP), particularly in mechanically ventilated patients. We used the fibronectin-binding protein A gene (fnbA) for the species-specific and quantitative detection of S. aureus directly from lower respiratory tract (LRT) specimens by a Taq Man real time PCR. For this reason, a total of 269 lower respiratory tract (LRT) specimens collected from patients with hospital-acquired pneumonia were assayed. Amplification of fnbA in serial dilutions ranged from 10 9 CFU/ ml to 10 2 CFU/ml. Standard curve of triplicate every dilution had slope 3.34 ± 0.1 and R 2 > 0.99 with SD 0.1. Based on these data, the sensitivity and specificity of the newly developed real time PCR targeting the fnbA gene were both 100%. The Cohen's Kappa test showed the Kappa value of 1.0. The fnbA gene is a potential marker for the species-specific detection of S. aureus and can be used to detect this bacterium in any clinical specimens by real time PCR. Moreover, this method reduces the time needed for quantitative detection of Staphylococcus aureus from LRT specimens to nearly 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.

Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

The Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein, a toll-like receptor 4 agonist, enhances dendritic cell-based cancer vaccine potency

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.

Identification & characterization of Shiga toxin-producing Escherichia coli isolates from patients with diarrhoea in Iran.

Background & objectives: Verotoxigenic Escherichia coli are important serotypes of enterohaemorrhagic E. coli (EHEC) subgroup that cause attaching and effacing lesions in enterocytes by producing verotoxins or shiga-like toxins resulting in haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). The aim of this study was to detect these serotypes specially E. coli O157:H7 in stool samples of patients with diarrhoea and identification of virulence genes (STX1, STX2, Hly and EAE) in Shahrekord-Iran area using PCR technique. Methods: Two hundred diarrhoeal stool samples of patients were collected through 2007-2008. Microbiological and biochemical examinations were done to detect the E. coli. Serological tests carried out to identify the O157 or O157:H7 serotypes. Results: Of the 58 E. coli isolates, 16 (27.6%) were detected as STX1 carrying E. coli, four (6.9%) carrying STX2, eight (13.8%) carrying both STX1 and STX2, and 12 (20.7%) were Hly carrying E. coli, but none of the isolates contained EAE gene. None of the isolates were E. coli O157 or O157:H7 serotypes. Interpretation & conclusions: Our results revealed that verotoxigenic E. coli isolates other than O157 serotype were involved in causing diarrhoea in Shahrekord-Iran.

Detección histológica y molecular de Helicobacter pylori y genotipificación con base en los genes de virulencia babA2 e iceA en pacientes con patología gástrica benigna / Molecular and histological detection of Helicobacter pylori and genotyping based on babA2 and iceA in patients with benign gastric pathologies

Objective: To investigate the presence of Helicobacter pylori in a Colombian population. Material and Methods: Gastric biopsies from 60 patients with benign gastric pathologies were submitted to histopathological examination and genotyped by PCR through amplification of babA2 and iceA genes. Results: The presence of H. pylori was demonstrated in 78.3 percent, 63.3 percent and 66.7 percent of the samples after Giemsa staining, andH. pylori 16S DNAr and iceA gene amplification, respectively. In addition, H. pylori babA2 positive was found infecting 7 patients. Among the iceA positive samples, 35 percent were identified as iceAl, 47.5 percent as iceA2 and 17.5 percent contained iceAl and iceA2 or different iceA2 types. This work allowed detection and genotyping of H. pylori, demonstrating a low proportion of patients carrying strains babA2 positive and an iceA genotype distribution according to previous reports. No association was found between the presence of babA2 and iceA genes and ulcer disease.

Objetivo: El presente estudio investiga la presencia de Helicobacter pylori en pacientes de una población colombiana con enfermedad gastro-duodenal benigna y realiza su genotipificación usando como blanco los genes babA2 e iceA. Pacientes y Métodos: Se analizaron biopsias gástricas de 60 pacientes usando histopatologíay RPC. Resultados: La presencia de H. pylori se demostró en 78,3 por ciento, 63,3 por ciento y 66,7 por ciento de los pacientes, mediante tinción de Giemsa, amplificación del gen 16S ADNr y del locus iceA, respectivamente. Helicobacter pylori babA2 positivo se encontró infectando 7 pacientes y de las 40 muestras positivas para la presencia del locus iceA, 35 por ciento eran iceAl, 47.5 por ciento iceA2 y en 17,5 por ciento se evidenció infección múltiple. Se aportan datos sobre la prevalencia de H. pylori encontrando una baja proporción de casos babA2 positivos y una distribución de los genotipos iceA acorde con datos reportados previamente. La presencia de los genes babA2 e iceA no se encontró asociada con la presentación de úlcera.

Maternal and neonatal screening for Group B streptococci by SCP B gene based PCR: a preliminary study.

OBJECTIVE: To detect the magnitude of group B streptococcal (GBS) colonization and disease among a sample of pregnant women and their infants in Egypt. STUDY DESIGN: Prospective observational study. PARTICIPANTS: The study included 95 pregnant females, 35-37 weeks of gestational age, attending the antenatal outpatient clinic at AlFayom University Hospital between September 2006 and June 2007. All participants were screened with vaginorectal swabs by a conventional GBS PCR assay. Participants were grouped into group A (GBS present, 17 patients) and group B (GBS absent, 78 patients). Details with regard to labor and delivery were recorded and placental pathology was examined to detect histological chorioamnionitis. Ninety-five infant data were also recorded. All neonates of group A (17 out of 95 with known positive maternal GBS) underwent collection of simultaneous specimens from surface sites for PCR before their first bath and within four hours of birth. RESULTS: GBS carriage rate in the study sample was 17.89%. Chorioamnionitis confirmed in three patients by placental pathology (one was in group A and two in group B) was statistically not significant. Twenty-two women had rupture of membranes (< 12 hours) before delivery (four from group A and 18 from group B) that was not statistically significant. There were three infants out of 17 in group A who had GBS colonized at one or more sites by PCR which was statistically significant. However, only one infant was admitted to neonatal intensive care unit (NICU) that was not statistically significant. CONCLUSION: Maternal GBS carriage is associated with a significant increase in neonatal infection rate but is not associated with an increase in neonatal intensive care admission. An accurate evaluation of colonization rate (using a larger sample) is desired to evaluate neonatal invasive disease and determine the cost effectiveness of PCR to select an appropriate preventive strategy in Egypt.

Diagnóstico de Mycoplasma genitalium por amplificación de los genes MgPa y ARN ribosomal 16S / Diagnosis of Mycoplasma genitalium by MgPa and rRNA 16S gene amplification

OBJETIVO: El microorganismo Mycoplasma genitalium se ha relacionado con la uretritis no gonocócica (UNG). La técnica de PCR se ha convertido en el principal método de detección de este patógeno. En consecuencia, debe aplicarse un método de diagnóstico mediante la amplificación de fragmentos de ADN por la técnica PCR. MATERIAL Y MÉTODOS: Se seleccionaron los cebadores MGF-MGR y MgPaF-MgPaR, complementarios de los genes de ARNr 16S y MgPa de M. genitalium, respectivamente. Se efectuaron ensayos de especificidad y sensibilidad y se estudiaron muestras clínicas. RESULTADOS: La PCR con cada grupo de cebadores utilizado fue específica sólo para M. genitalium y la sensibilidad fue mayor con el grupo de cebadores MGF-MGR. En el estudio de 34 muestras clínicas, 18.5 por ciento fue positivo a M. genitalium y se encontró un mayor número de muestras positivas al utilizar los cebadores MgPaF-MgPaR. CONCLUSIONES: Debe aplicarse en la práctica clínica el diagnóstico de M. genitalium mediante la amplificación del ADN por PCR en los pacientes con UNG.

OBJECTIVE: Mycoplasma genitalium has been associated with nongonococcal urethritis (NGU). Diagnosis by PCR has become the primary detection method for this organism. Thus, diagnosis by DNA amplification using the PCR technique should be utilized. MATERIAL AND METHODS: GMF/GMR and MgpF/MgpR primer pairs, complementary to the M. genitalium 16S rRNA and MgPa genes, respectively, were selected. Specificity and sensibility assays were conducted and clinical samples were studied. RESULTS: The PCR with each primer pair was specific only for M. genitalium, and the sensibility was higher with the GMF/GMR primers. In the study of 34 clinical samples, 18,5 percent were positive for M. genitalium, with more positive samples when the MgpF/MgpR primers were used. CONCLUSIONS: DNA amplification by PCR should be applied in clinical practice to the diagnosis of M. genitalium in patients with NGU should using.

Association of ompU gene in Vibrio cholerae from patients and environment with bile resistance.

The objective of this study was to determine whether Vibrio cholerae, possessing ompU isolated from patients and the environment, conferred bile resistance and whether other virulence genes were also related to bile resistance. Fifty-two V cholerae O1 and non-O1 isolates were examined by PCR for the presence of the virulence-associated and regulatory genes, ctxA, tcpA, zot, ace, ompU, toxR, hlyA and stn/sto. V. cholerae possessing ompU resistant to equal or greater than 10% sodium deoxycholate were found in 93% of isolates but only in 9% of V. cholerae isolates not possessing ompU. The effects of other virulence genes on bile resistance could not be ascertained in this study. Thus V cholerae non-O1 with ompU and possibly other virulence genes isolated from the environment have the potential of affecting public health.

Detection of shiga-toxigenic Escherichia coli (STEC) in diarrhoeagenic stool & meat samples in Mangalore, India.

BACKGROUND & OBJECTIVE: Shiga-toxigenic Escherichia coli (STEC) are causative agents of bloody diarrhoea, haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Humans acquire infections primarily through contaminated beef. In India, STEC has not been implicated as a major cause of diarrhoea. Hence, isolation of STEC from diarrhoeagenic stool samples of patients and beef samples marketed through retail outlets was attempted in Mangalore, India. METHODS: Diarrhoeagenic stool samples (n = 192) and meat samples (n = 103) were screened for STEC, using conventional culture methods and polymerase chain reaction (PCR) from December 2003 to 2006 in the department of Microbiology, Kasturba Medical College, Mangalore. All the E. coli isolates were subjected to antibiotic susceptibility testing and serotyping. RESULTS: Of the 40 eae positive E. coli isolates from meat sample, one was positive for all the STEC genes, namely stx1, stx2, rfb O157 and EHEC hlyA. This isolate belonged to O157 serogroup. Of the 110 eae positive E. coli isolated from stool samples, two were positive for EHEC hlyA and belonged to serogroup O8 and one was positive for bfp gene and found to be of O6 serogroup. Among the 192 stool enrichment broths tested, 160 were positive for eae gene, of which two were EHEC hlyA positive and one was bfp gene positive. Among the 103 meat enrichment cultures, 90 were positive for eae gene and one among them was positive for all the STEC genes. INTERPRETATION & CONCLUSION: Our results showed a low incidence of STEC and high prevalence of eae positive E. coli other than STEC in stool and meat samples. A low positivity was observed for PCR performed directly on stool and meat samples. However, PCR on enrichment cultures gave better results. Since E. coli O157 was isolated and detected by PCR in one of the meat samples, this organism may be of public health significance. A study on a large sample may provide some answer.

Aislamiento, caracterización y subtipificación de cepas de Escherichia coli O157:H7 a partir de productos cárnicos y leche / Isolation, characterization and typing of Escherichia coli O157:H7 strains from beef products and milk

Escherichia coli productor de toxina Shiga (Stx) (STEC) O157:H7 es un patógeno asociado a enfermedades transmitidas por alimentos, fundamentalmente de origen animal. Se investigó la presencia de E. coli O157 en 250 muestras de carne picada y hamburguesas obtenidas de comercios de las ciudades de Santa Fe y Santo Tomé (Pcia. de Santa Fe) y en 150 muestras de leche provenientes de tanques de enfriado de tambos de la región, utilizando enriquecimiento selectivo y separación inmunomagnética. A partir de 3 muestras de carne (1,2%) se aislaron cepas E. coli O157:H7 stx2, eae, y ehxA positivas, que pudieron ser diferenciadas mediante electroforesis de campo pulsado, fagotipificación y genotipificación de stx. No se aislaron cepas STEC O157:H7 a partir de las muestras de leche. Estos hallazgos confirman la participación de los alimentos de origen animal en la epidemiología de las enfermedades producidas por E. coli O157:H7.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is an emergent pathogen associated with foodborne diseases, especially foodstuffs of animal origin. A total of 250 beef samples (ground beef and hamburgers) obtained from retail outlets in Santa Fe and Santo Tomé cities, and 150 milk samples from bulk tank milk from dairy barns of the region were analyzed by selective enrichment and immunomagnetic separation. Escherichia coli O157:H7 stx2, eae and ehxA positive strains were isolated from three (1.2%) beef samples. The strains could be differentiated by pulsed-field gel electrophoresis, phagetyping and genotyping of stx. The milk samples were negative for STEC O157. These findings confirm the role of food of animal origin in the epidemiology of E. coli O157:H7 - associated diseases.

Genotipificación de aislados clínicos de Helicobacter pylori en base a genes asociados a virulencia cagA, vacA y babA2: Primer aislamiento de una cepa babA2 positiva en pacientes chilenos / Genotypying of clinical isolates of Helicobacter pylori by cagA, vacA and babA2 virulence associated genes: First detection of a babA2 positive strain in Chilean patients

Background: Helicobacter pylori-associated gastroduodenal diseases depends on host characteristics, environmental conditions and bacterial virulence factors, such as cagA, vacA y babA2 gene products. Moreover, peptic ulcer disease has been related with cagA+, vacAs1m1 strains, while metaplasia and gastric cancer has been associated to cagA+, vacAs1 and babA2+ H pylori strains. Gene babA2 has not yet been described in clinical isolates from Chilean patients. Aim: To investigate the presence of cagA, vacA (s and m) and babA2 genes in clinical isolates of H pylori from Chilean patients. Material and Methods: Sixty six isolates from 41 patients were genotyped by PCR, using primers for s1a, s1b, s2, m1, m2, cagA and babA2 genes as previously described. Results: cagA gene was detected in 16 isolates (24.2 percent) while vacAs1a, vacAs1b, vacAs2, vacAm1 and vacAm2 were detected in 28 (42.4 percent), 14 (21.2 percent), 17 (25.8 percent), 21 (31.8 percent) and 29 isolates (43.9 percent), respectively. One isolate (1.5 percent) was babA2 positive, being the first isolate with this genotype described in Chile. Besides the babA2+ genotype this clinical isolate also presented cagA+ and vacAs1a which has been related with metaplasia or gastric cancer. Five isolates showed an ulcerogenic profile cagA+, vacAs1m1. Conclusions: The results presented indicate the prevalence of vacAs1m1 genotype among the clinical isolates analyzed, and a low frequency of babA2 genotype.

Utilidad de la reacción de polimerasa en cadena para la detección de Mycoplasma pneumoniae en adultos mayores con neumonía adquirida en la comunidad / Diagnostic utility of the polymerase chain reaction for the diagnosis of Mycoplasma pneumoniae in elderly patients with community-acquired pneumonia

Mycoplasma pneumoniae es una causa frecuente de neumonía adquirida en la comunidad (NAC) en niños y adultos jóvenes, existiendo escasa información de su frecuencia en el adulto mayor. Se analizó la reacción de polimerasa en cadena (RPC) con dos pares de partidores, gen de la adhesina P1 y gen 16S rRNA, para la detección de M. pneumoniae en lavado faríngeo de 84 pacientes de 60-96 años con diagnóstico clínico de NAC, desde septiembre de 2002 hasta agosto de 2004. Los resultados de la RPC fueron comparados con los de la serología mediante inmunofluorescencia indirecta (IFI). Se detectó infección por M. pneumoniae mediante serología o RPC en 11 de 84 pacientes (13,1%). La serología fue positiva en 8 (72,7%) y la RPC en 7 (63,6%) muestras. La serología en la muestra de suero en fase aguda fue positiva en 5 de 11 pacientes (45,4%), en 4 de ellos por la presencia de IgM. En 4 pacientes con serología positiva la RPC fue negativa. En 3 pacientes con serología negativa la RPC fue positiva. Las dos RPC mostraron 100% de correlación y la sensibilidad fue la misma; no se detectaron muestras con efecto inhibitorio de la reacción. En conclusión, M. pneumoniae debería ser considerado en la etiología de la NAC en adultos mayores. La detección de este microorganismo debe basarse en el uso combinado de serología y RPC.

Isolation and identification of Escherichia coli O157:H7 using different detection methods and molecular determination by multiplex PCR and RAPD

Escherichia coli O157:H7 is recognized as a significant food-borne pathogen, so rapid identification is important for food hygiene management and prompt epidemiological investigations. The limited prevalence data on Shiga toxin-producing E. coli (STEC) and E. coli O157:H7 in foods and animals in Korea made an assessment of the risks difficult, and the options for management and control unclear. The prevalence of the organisms was examined by newly developed kit-E. coli O157:H7 Rapid kit. For the isolation of E. coli O157:H7, conventional culture, immunomagnetic separation, and E. coli O157:H7 Rapid kit were applied, and multiplex PCR and randomly amplified polymorphic DNA (RAPD) were performed for the molecular determination. There was high molecular relatedness among 11 Korean isolates and 17 U.S. strains at 63% level. Additionally, distinct differentiation between pig and cattle isolates was determined. It implied that RAPD had a capacity to distinguish strains with different sources, however it could not discriminate among isolates according to their differences in the degree of virulence. In antimicrobial susceptibility tests, 45.5% of isolates showed antibiotic resistance to two or more antibiotics. Unlike the isolates from other countries, domestic isolates of E. coli O157:H7 was mainly resistant to ampicillin and tetracylines. In summary, the application of E. coli O157:H7 Rapid kit may be useful to detect E. coli O157:H7 due to its sensitivity and convenience. Moreover, combinational analysis of multiplex PCR together with RAPD can aid to survey the characteristics of isolates.

Clinical diagnosis of group B streptococci by scpB gene based PCR.

BACKGROUND & OBJECTIVES: The goal of the present study was to improve and simplify the diagnosis of Streptococcus agalactiae (group B Streptococcus, GBS) infection for routine clinical practice. METHODS: A total of 71 clinical samples were tested by microbiologic culture, counter immunoelectrophoresis (CIE) and PCR described in the literature. Southern hybridization was accomplished with the Enzo(TM) "DNA Labeling and Detection Kit", Roche (Germany). The computer techniques were used for selection of the specific primers and for analysis of the sizes of PCR products. RESULTS: The primers for the regions around the 51 bp deletion in C5a peptidase gene (scpB) of GBS were selected. PCR analysis revealed the 255 bp amplification fragment in GBS, 306 bp fragment in groups A and G streptococci (GAS, GGS) and did not reveal any fragments in other bacterial species. Among 71 urine and serum clinical samples tested, none were found to be GBS positive by microbiologic culture, 16 samples by CIE, 36 by PCR. The specificity of amplification was confirmed by Southern hybridization. INTERPRETATION & CONCLUSION: The 51 bp deletion in scpB gene in comparison with scpA and scpG genes can be used as a diagnostic tool for identification of GBS. The 51 bp deletion based PCR proved to be faster and more reliable test than microbiologic culture or CIE.

Distribution of virulence genes in Salmonella serovars isolated from man & animals.

BACKGROUND & OBJECTIVES: Pathogenesis of Salmonellosis depends upon a large number of factors controlled by an array of genes that synergise into the actual virulence of Salmonella. A study was undertaken to observe the distribution of three such genes, namely, Salmonella enterotoxin (stn), Salmonella Enteritidis fimbrial (sef and plasmid encoded fimbrial (pef genes, among different serovars of Salmonella enterica isolated from man and animals. METHODS: A total of 95 isolates belonging to S. Typhimurium (51), S. Enteritidis (36), S. Bareilly (3), and S. Paratyphi B (5) serovars were subjected to polymerase chain reaction (PCR) assay for the detection of stnl ssf and pef genes using their specific primers and the PCR products were analysed by 1 per cent agarose gel electrophoresis for the presence of the respective genes. RESULTS: Varying distribution pattern of these genes was observed amongst the isolates. While, stn was found in all the 95 strains, sef was found only among the S. Enteritidis isolates. The pef gene was found to be absent in 10 isolates including the three S. Bareilly isolates. INTERPRETATION & CONCLUSION: Findings indicated that the stn gene is widely distributed among Salmonella irrespective of the serovars and source of isolation. However, the sef gene appears to be serovar specific. Since the stn gene is found in all the isolates, it can be a viable target gene to explore the possibility of direct detection of Salmonella from samples from biological sources.

Molecular Relatedness between Isolates Yersinia pseudotuberculosis from a Patient and an Isolate from Mountain Spring Water

A 40-yr-old buddhist monk was admitted to the hospital with abdominal pain, fever, and confusion. He had a history of drinking untreated mountain spring water in his temple, and experienced the above symptoms for several days before admission. In past medical history, he had suffered from hepatic cirrhosis. Yersinia pseudotuberculosis was isolated from his blood and ascitic fluid. The mountain spring water that he had ingested was cultivated and Y. pseudotuberculosis was also isolated. For identification of pathogenic Y. pseudotuberculosis, each isolate from the three sources (blood, ascitic fluid, and drinking water) was also analysed for the inv gene for Y. pseudotuberculosis and the virF gene for virulent plasmid by PCR. All strains were positive for both the virF and the inv genes and also positive for autoagglutination test. For relationship study, each isolate from the three sources was also analysed with serotyping and restriction endonuclease analysis of virulence plasmid DNA (REAP) using BamHI. All belonged to the serotype 4b and REAP pattern D. Thus, all these findings supported that the mountain spring water was the source of the Y. pseudotuberculosis infection in this case.

Frequency of pap and pil operons in Escherichia coli strains associated with urinary infections

Strains of E. coli isolated from patients with urinary tract infection were examined for P and type 1 adhesin production by colony hybridization with pap and pil operons. The P pili probe detected 45 (46.4 per cent) of the total of 97 strains studied and the type 1 pili probe detected 83 (85.6 per cent). The pap operon was detected in 39 (53.4 per cent) of 73 strains isolated from urine of patients with urinary disease and in 6 (25.0 per cent) of 24 strains isolated from feces of healthy individuals employed as controls (P = 0.029), and the pil operon was detected in 67 (91.8 per cent) of the urinary strains and in 16 (66.6 per cent) of the fecal strains (P = 0.007). Our data did not show significant differences in frequency of P pili among isolates from pyelonephritis (78.5 per cent), cystitis (45.8 per cent) and asymptomatic bacteriuria (54.5 per cent). Type 1 pili were not associated with the different types of infection; the frequency of these pili was 100 per cent in pyelonephritis and in asymptomatic bacteriuria, and 87.5 per cent in cystitis. The incidence of pap operon in strains isolated from pyelonephritis and from asymptomatic bacteriuria was higher in 11-to 40-year old women. These data show a high frequency of pap and pil operons among uropathogenic strains of E. coli, which seems to be an important factor in the development of urinary infection.