Prevalence of Porphyromonas gingivalis fimA II genotype in generalized aggressive periodontitis patients

Purpose: The objective of this study was to evaluate the prevalence of Porphyromonas gingivalis (Pg) and its filmA II genotype in a sample of Brazilian patients with generalized aggressive periodontitis (GAgP) and to correlate the presence of each pathogen/genotype eith clinical parameters. Methods: We used polymerase chain reaction (PCR) to evaluate the presence of Pg and filmA II genotype in subgingival plaque samples collected from the deepest site of 45 Brazilian patients aged 15-40 years with GAgP and correlated findings with age and clinical parameters (plaque index, gingival bleeding index, probing depth and clinical attachment loss). Results: Pg was identified in 64.4% patients. FilmA II genotype was present in 82.6% of Pg-positive patients. The presence of Pg and filmA II genotype was significantly associated with greater clinical attachment loss at the sampled periodontal site. Pg-positive patients were slightly older than Pg-negative patients. Conclusions: Pg and filmA II genotype were highly prevalente in Brazilian patients with GAgP. Pg was more commonly observed in slightly older individuals and in sites with more clinical attachment loss. (AU)

Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells

Abstract This study was undertaken in order to assess the involvement of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer in the M. tuberculosis-epithelial cell interaction. A MTP-deficient strain of M. tuberculosis demonstrated a significant reduction of 69.39% (p = 0.047) and 56.20% (p = 0.033) in its ability to adhere to and invade A549 pulmonary epithelial cells, respectively, in comparison with the wild-type strain. Complementation of the MTP-deficient mutant restored its adhesion and invasion capacity back to the wild-type levels. Overall, it was found that similar concentrations of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFN-γ, MCP-1, and TNF-α were induced in A549 cells infected with the MTP-proficient and MTP-deficient strains. However, at 48 h post-infection, the MTP-deficient mutant induced significantly lower levels of TNF-α than the wild-type strain (p = 0.033). Furthermore, at 72 h post-infection, the mutant induced significantly higher levels of IL-8 than the wild-type (p = 0.005). We conclude that MTP is an adhesin/invasin of epithelial cells and, while playing a role in M. tuberculosis entry, they do not appear to largely influence the epithelial cell cytokine response.

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.

Sodium houttuyfonate inhibits virulence related motility of Pseudomonas aeruginosa / 中国中药杂志

Sodium houttuyfonate (SH) is a derivative of effective component of a Chinese material medica, Houttuynia cordata, which is applied in anti-infection of microorganism. But, the antimicrobial mechanisms of SH still remain unclear. Here, we firstly discovered that SH effectively inhibits the three types of virulence related motility of.Pseudomonas aeruginosa, i.e., swimming, twitching and swarming. The plate assay results showed that the inhibitory action of SH against swimming and twitching in 24 h and swarming in 48 h is dose-dependent; and bacteria nearly lost all of the motile activities under the concentration of 1 x minimum inhibitory concentration (MIC) (512 mg x L(-1) same as azithromycin positive group (1 x MIC, 16 mg x L(-1)). Furthermore, we found that the expression of structural gene flgB and pilG is down-regulated by SH, which implies that inhibitory mechanism of SH against motility of P. aeruginosa may be due to the inhibition of flagella and pili bioformation of P. aeruginosa by SR Therefore, our presented results firstly demonstrate that SH effectively inhibits the motility activities of P. aeruginosa, and suggest that SH could be a promising antipseudomonas agents in clinic.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

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.

Efficacy of bacterin-, outer membrane protein- and fimbriae extract-based vaccines for the control of Salmonella Enteritidis experimental infection in chickens / Eficácia de bactéria inativada (bacterina), proteína da membrana externa e extrato de fimbrias no controle de infecção experimental por Salmonella Enteritidis (SE) em galinhas

The efficacy of three vaccines was evaluated in chickens for the control of experimental infection with Salmonella Enteritidis (SE) phage type 4. The vaccines were produced with bacterin, outer membrane proteins (OMP) and fimbriae crude extract (FE). The chickens were vaccinated intramuscularly with two doses of each vaccine at 12 and 15 weeks of age. The chickens were then orally challenged with 10(9) CFU/chicken Salmonella Enteritidis phage type 4 at 18 weeks of age. Fecal swabs were performed for the recovery of shedding SE, and SE was recovered from the liver and spleen. Additionally, antibody titers were measured in the serum by micro-agglutination test. The results indicated that the vaccine produced with bacterin yielded better results and resulted in reduction of fecal shedding and organ invasion by SE after oral challenge, although no vaccine was 100% effective for the control of SE experimental infection.

A eficácia de três vacinas de Salmonella Enteritidis fagotipo 4, produzidas na forma de bacterina, proteínas de membrana externa (OMP) e extrato bruto de fímbrias (FE) foi avaliada para proteção de aves infectadas experimentalmente. As aves foram vacinadas por via intramuscular com duas doses de cada vacina as 12 e 15 semanas de idade e desafiadas com 10(9) UFCs de Salmonella Enteritidis fagotipo 4 às 18 semanas de idade, por via oral. A eficácia foi determinada através do reisolamento da bactéria nas fezes e no fígado e baço, e os anticorpos foram mensurados no soro. Os resultados demonstraram que a vacina produzida com a bacterina foi mais eficaz em comparação às outras vacinas examinadas, para reduzir a excreção fecal e a invasão de órgãos após o desafio por SE.

Virulencia y variabilidad de Porphyromonas gingivalis y Aggregatibacter actinomycetemcomitans y su asociación a la periodontitis / Virulence and variability on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans and their association to periodontitis

Las periodontitis son un conjunto de patologías de naturaleza inflamatoria y etiología infecciosa producidas por el biofilm patogénico subgingival. Porphyromonas gingivalis y Aggregatibacter actinomycetemcomitans son bacterias periodonto-patógenas que pueden causar daño directo a las estructuras periodontales a través de los diversos factores de virulencia que expresan. Sobre la base de estos factores de virulencia, distintos genotipos y serotipos bacterianos se han descrito, cada uno de ellos con una potencial variable patogenicidad. En esta revisión bibliográfica se describen diferentes factores de virulencia de P. gingivalis y A. actinomycetemcomitans y se discute la variable inmunogenicidad y patogenicidad de los distintos genotipos y serotipos descritos para ellos. Tanto P. gingivalis como A. actinomycetemcomitans poseen diversos factores de virulencia asociados al inicio, progresión y severidad de las periodontitis. En P. gingivalis, los factores de virulencia para los cuales se describen distintos genotipos y/o serotipos son fimbria, LPS y cápsula bacteriana, y en A. actinomycetemcomitans son leucotoxina A, Cdt y LPS. Cada uno de estos distintos genotipos y serotipos induce una respuesta inmuno-inflamatoria diferente en el hospedero y, por lo tanto, se podrían asociar a una variable patogenicidad y podrían determinar las características clínicas de la enfermedad.

Periodontitis represents a heterogenic group of periodontal infections elicited by bacteria residing at the subgingival biofilm. Although this biofilm is constituted by a broad variety of bacterial species, only a limited number has been associated with the periodontitis aetiology, among them Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Both P. gingivalis and A. actinomycetemcomitans express a number of virulence factors that contribute to direct tissue damage and, based on them, distinct genotypes and serotypes have been described, each one with a potential variable pathogenicity. This review aimed to analyze the different virulence factors described for P. gingivalis and A. actinomycetemcomitans and to discuss the variable immunogenicity and pathogenicity of their serotypes and genotypes. P. gingivalis and A. actinomycetemcomitans express different virulence factors and they determine the initiation, progression, and severity of periodontitis. In P. gingivalis, distinct serotypes and/or genotypes are described based on fimbriae, LPS, and capsule. Additionally, in A. actinomycetemcomitans distinct serotypes and/or genotypes are described based on leucotoxin A, Cdt, and LPS. These distinct serotypes and genotypes induce a differential immunoinflammatory response and, thus, could be associated with variations in pathogenicity and reflected in clinic characteristics of the disease.

Genótipos fimA de porphyromonas gingivalis e periodontite / Porphyromonas gingivalis fimA genotypes and periodontitis

Porphyromonas gingivalis é um dos principais patógenos envolvidos com o início e progressão da doença periodontal. Este microorganismo exibe uma diversidade genotípica e fenotípica que pode resultar em diferenças na capacidade dos clones induzirem destruição periodontal. Variações no potencial patogênico dos distintos genótipos do gene fimA de P. gingivalis foram relacionadas a diferentes doenças periodontais em diversos estudos. Esta revisão teve o objetivo de analisar criticamente os estudos que relacionaram as condições periodontais e os diferentes genótipos fimA de P. gingivalis. Foram incluídas publicações na língua inglesa de estudos clínicos em humanos, que avaliaram a relação entre as condições periodontais e os diferentes genótipos fimA de P. gingivalis. Doze estudos foram considerados válidos para a realização desta revisão. Pôde-se concluir que os genótipos fimA II e IV de P. gingivalis encontram-se frequentemente associados à periodontite crônica

Porphyromonas gingivalis is a major pathogen involved in the initiation and progression of periodontal disease. This microorganism exhibits a genotypic and phenotypic diversity which may result in differences in the ability to induce periodontal destruction of the clones. Variations in the pathogenic potential of different genotypes of fimA gene of P. gingivalis were related to different periodontal diseases in several studies. This systematic review aimed to critically analyze the studies that have linked periodontal conditions and the different fimA genotypes of P. gingivalis. We included English language publications of human clinical studies that evaluated the relationship between periodontal conditions and the different fimA genotypes of P. gingivalis. Twelve studies were considered valid for the completion of this review. It was concluded that fimA genotypes II and IV of P. gingivalis are often associated with chronic periodontitis.

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.

Virulence factors, antimicrobial resistance, and plasmid content of Escherichia coli isolated in swine commercial farms / Fatores de virulência, resistência aos antimicrobianos, presença de plasmídeos em Escherichia coli isoladas de amostras clínicas e ambientais de suínos

Virulence factors and antimicrobial resistance patterns of Escherichia coli isolates were evaluated. A total of 80 E. coli isolates were evaluated, being 64 from clinical samples (intestinal content and fragments of organs from diarrheic piglets), seven from feces of clinically healthy piglets and sows, and nine environmental samples (five from facilities, two from feed, one from insect, and one from waste). Molecular characterization was performed by PCR detection of fimbriae and toxin genes and plasmid content determination. The isolates were also characterized according to their resistance or sensitivity to the following drugs: ampicillin, trimethoprim:sulfamethoxazole, tetracycline, amikacine, colistin, norfloxacin, florfenicol, enrofloxacin, cefalexin, trimethoprim, neomycin, chloramphenicol, and gentamicin. From 80 E. coli isolates, 53.8 percent were classified as enterotoxigenic E. coli (ETEC), 2.5 percent were shiga toxin-producing E. coli (STEC), and 43.8 percent showed a non specific pattern and were unclassified. One fecal isolate from non-diarrheic piglet was classified as ETEC by PCR. Clinical isolates showed resistance mainly for tetracycline and trimethoprim:sulfamethoxazole. Plasmidial DNA was observed in 70 isolates, being 78.5 percent of clinical isolates, 8.57 percent of non-diarrheic feces, and 12.8 percent of environment.

Os fatores de virulência e a resistência aos antimicrobianos foram avaliados em Escherichia coli. Um total de 80 isolados de E. coli, sendo 64 de amostras clínicas (conteúdo intestinal e fragmentos de órgãos de leitões diarreicos), sete das fezes de porcas e leitões saudáveis e nove de amostras ambientais (cinco de instalações, dois de alimentos, um de inseto e um de esterqueira). A caracterização molecular feita pela PCR objetivou detectar fimbrias e toxinas, bem como a determinação do conteúdo de plasmídeos. Os isolados foram caracterizados quanto à resistência ou sensibilidade às seguintes drogas: ampicilina, sulfazotrim, tetraciclina, amikacina, colistina, norfloxacina, florfenicol, enrofloxacina, cefalexina, trimetoprim, neomicina, cloranfenicol e gentamicina. Dos 80 isolados, 53,8 por cento foram classificados como E. coli enterotoxigênica (ETEC), 2,5 por cento como E. coli produtora de shiga toxina (STEC) e 43,8 por cento, por não apresentarem padrão específico, não foram classificadas. Pela PCR, um isolado de fezes de suíno sem diarreia foi classificado como ETEC. Os isolados das amostras clínicas foram principalmente resistentes à tetraciclina e à sulfazotrim. Em 70 isolados, observaram-se DNA plasmidial, destes 78,5 por cento foram obtidos de amostras clínicas, 8,57 por cento de leitões sadios e 12,8 por cento de amostras ambientais.

Bacterial biofilm formation, pathogenicity, diagnostics and control: An overview.

Bacterial biofilms are complex, mono- or poly-microbialn communities adhering to biotic or abiotic surfaces. This adaptation has been implicated as a survival strategy. The formation of biofilms is mediated by mechanical, biochemical and genetical factors. The biofilms enhance the virulence of the pathogen and have their potential role in various infections, such as dental caries, cystic fibrosis, osteonecrosis, urinary tract infection and eye infections. A number of diagnostic techniques, viz., bright-field microscopy, epifluorescence microscopy, scanning electron microscopy, confocal laser scanning microscopy and amplicon length heterogeneity polymerase chain reaction, have been employed for detection of these communities. Researchers have worked on applications of catheter lock solutions, a fish protein coating, acid shock treatment, susceptibility to bacteriophages, etc., for biofilm control. However, we need to rearrange our strategies to have thorough insight and concentrate on priority basis to develop new accurate, precise and rapid diagnostic protocols for detection and evaluation of biofilm. Above all, the strict compliance to these techniques is required for accurate diagnosis and control.

[Molecular study of phase variation of type 1 fimbriae in uropathogenic escherichia coli 044 serotypes during touching with solid surfaces]

Type 1 fimbriae is the most common adhesion factor in urine tract infection. In this Study, presence of virulence genes in isolated strains of uropathogenic E. Coli, O serotyping and molecular detection of phase variation of type 1 fimbriae were assessed during solid surfaces exposure. Isolated E. coli from urine samples of patients were serotyped by using serologic methods. Phenotypic estimation of phase variation was applied by mannose - resistant hemagglutination [MRHA]. Fim opron phase variation was studied by HinFI digestion and PCR reaction. For all 158 E. coli strains, the most occurrences belonged to O44. Forty nine percent of the isolates were mannose-sensitive and expressed fim operon in agar medium. While, 51% of strains were resistant to mannose in the same position. In Broth medium 68% of isolates were mannose-sensitive and 32% were mannose-resistant. PCR products with 359bp and 200bp long fragment demonstrated ON position and those with 489bp and 70bp long fragment indicated ON and OFF positions. Uropathologic E. coli strains posses few number of O serotypes. Environmental factors play an important role in regulation of fimberiae operon expression. Strains recovered from these urine samples, however, were shown capable to switch the fim operon to the ON position after culture in broth medium. Type 1 fimbrial expression and flagella motility are probably representative of an essential dynamic interplay between bacterial adhesion and motility. The strains present in urine samples but nonattached to the epithelium are inactive for type 1 fimbriae expression

Ultrastructure and types of fimbriae in uropathogenic E-coli among bilharzial patients

The aim of this work was to study the association of different types of fimbriae of urinary E coli isolates with different disease entities. We collected a total of 57 urinary E. coli isolates from 3 groups of bilharrzial patients: group 1: with cystitis [21 isolates]; group 2: with pyelonephritis [18 isolates] and group 3: with urinary bladder carcinoma [18 isolates]. Each isolate was studied for: I. Fimbrial expression and type determination by haemagglutination [HA] of human and guinea pig erythrocytes. II. Electron microscopic [E/M] structure using negative staining, standard transmission and scanning electron microscopy. It was found that infection with mannose sensitive type 1 fimbriated E. coli dominated in group 1 and 3 [80.95% and 77.78% respectively]. In group2 [55.56%] were caused by mannose resistant P fimbriated E. coli. Although there was a perfect correlation between HA and the presence of fimbiriae by E/M [P< 0.01], yet E/M detected other types of fimibriae which could-have been missed by HA alone. Negative staining was the best technique in electron microscopy. We concluded that detection of P flimbriae in urinay E. coli strains may justify a vigorous antibiotic treatment to prevent development of pyelonephritis. Although type 1 fimbriae was associated with simple cystitis, yet follow up and complete investigations are recommended to detect an associated carcinoma

Identification of the 987P protein receptors for enterotoxigenic Escherichia coli / 生物工程学报

The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediates adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by Ligand blot, protein bands with a set of 32-35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC(RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequences of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The above results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins.

Patogénesis molecular, epidemiología y diagnóstico de Escherichia coli enteropatógena / Molecular pathogenesis, epidemiology and diagnosis of enteropathogenic Escherichia coli

Escherichia coli enteropatógena (EPEC) es una de las principales causas de diarrea en niños menores de dos años en países en vías de desarrollo. La principal característica histopatológica de la infección es una lesión que induce la EPEC en el intestino conocida como la lesión A/E (adherencia y eliminación). Las bacterias se adhieren a los enterocitos y permiten la acumulación de la actina del citoesqueleto en la región apical de la célula, hasta formar una estructura de tipo "pedestal" y causar la eliminación de las microvellosidades intestinales. A pesar de que se conoce de modo detallado el proceso de formación de los pedestales de actina, aún no se ha esclarecido el mecanismo global de la diarrea que induce EPEC. La diarrea se ha vinculado con: a) la destrucción de las microvellosidades del enterocito, b) la salida masiva de iones hacia la luz intestinal y c) la secreción de alguna enterotoxina. En estudios realizados en países en vías de desarrollo se ha demostrado que EPEC es uno de los principales agentes participantes en la diarrea infantil, con elevadas tasas de morbilidad y mortalidad. El diagnóstico microbiológico de la infección se realiza con metodologías adicionales a las utilizadas con regularidad en el laboratorio de microbiología clínica, entre ellas las siguientes: a) serotipificación, b) ensayo de adherencia, c) prueba de FAS (tinción fluorescente para actina) y d) detección específica de genes que codifican a proteínas incluidas en la patogénesis, como el bfpA y eae. Un objetivo de esta revisión es actualizar los avances observados en la patogénesis molecular de la infección por EPEC, las metodologías para el diagnóstico microbiológico y la epidemiología en México y otros países en vías de desarrollo.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea in infants less than two years of age in developing countries. To induce diarrhea EPEC uses several virulence factors acting on a still unknown and mysterious mechanism. The hallmark of EPEC infection is a histological intestinal alteration known as the attaching and effacing (A/E) lesion. The bacterium attaches intimately to the enterocyte and induces assembly of cytoskeleton intracellular actin on the cellular surface. Rearrangements of the actin cytoskeleton form a pedestal-like structure where bacterium tightly cups the cells, leading to degeneration of brush border microvilli. Although the mechanism of EPEC-induced pedestal formation has been dissected in detail, the overall mechanism of diarrhea is still obscure. It is believed that EPEC-mediated secretory diarrhea is related to a) intestinal microvilli effacement, b) massive loss of intracellular ions into the intestinal milieu and c) secretion of an EPEC enterotoxin. Epidemiological studies conducted in developing countries have shown that EPEC is one of the main bacteria frequently isolated from children with diarrhea, causing high morbidity and mortality rates. The microbiological diagnosis of EPEC-induced disease is performed with analytic methodologies different from those used by the standard microbiology laboratory, the most relevant being: a) serotypification, b) the adherence assay, c) FAS test, and d) the specific detection of virulence-involved genes (bfpA and eae genes) using molecular biology techniques. The purpose of this review is to update the most recent findings regarding the molecular pathogenesis of EPEC, its epidemiology in Mexico as well as other developing countries, and also the developed methodology for the diagnosis of EPEC infection.

In vitro expression of Lgeionella pneumophila pilE gene and its immunogenicity / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant plasmid containing Lgeionella pneumophila pilE gene, detect its expression in NIH3T3 cells and evaluate its immunogenicity.</p><p><b>METHODS</b>PilE gene (LP) was amplified from Legionella pneumophila DNA by PCR and inserted into pcDNA3.1(+) vector to construct the recombinant plasmid pcDNA3.1-pilE, which as verified by restriction endonuclease digestion, PCR and DNA sequencing analysis. NIH3T3 cells were transfected with the recombinant plasmid with Lipofection strategy. Transient and stable pilE gene products were detected by immunofluorescence and Western blotting, respectively. To evaluate the immunogenicity of pcDNA3.1-pilE, the recombinant plasmid was used as a DNA vaccine to immunize female BALB/c mice intramuscularly and the specific antibodies, lymphocyte proliferation response, interferon (IFN)-gamma production and cytotoxic T-lymphocyte response of the immunized mice were detected and evaluated.</p><p><b>RESULTS</b>The pilE gene of 429 bp in length was amplified. After transfection of NIH3T3 cells with the recombinant plasmid, strong green fluorescence was observed on the cell membrane and inside the cell. A protein with relative molecular mass of 15.7 kD was detected in the transfected cells with Western blotting, suggesting successful protein expression of pilE gene. pcDNA3.1-pilE resulted in much stronger immune response in the immunized mice than pcDNA3.1(+) (P<0.01).</p><p><b>CONCLUSION</b>The recombinant plasmid containing Lgeionella pneumophila pilE gene constructed in this study is capable of expression in NIH3T3 cells, and can induce specific humoral and cellular immune responses in mice.</p>

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.