Presencia de genes fimH y afa en aislamientos urinarios de Escherichia coli productora de betalactamasas de espectro extendido en Lima, Perú / Presence of fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases producing Escherichia coli in Lima, Peru

RESUMEN Con el objetivo de determinar la presencia de los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de betalactamasas de espectro extendido (BLEE), se realizó un estudio descriptivo, con aislamientos del cepario del proyecto TO-06/09 del Instituto Nacional de Salud del Niño en Lima, Perú. Se incluyeron 75 aislamientos urinarios de Escherichia coli. La identificación de genes se realizó por reacción en cadena de la polimerasa. De los 75 aislamientos, 74 (98,7%) fueron positivos para el gen fimH y 6 (8,0%) fueron positivos para el gen afa. Se evidenció la presencia de los factores de virulencia producidos por los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de BLEE.

ABSTRACT Descriptive study conducted in order to determine the presence of the fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli. Isolates from project TO-06/09 of the Instituto Nacional de Salud del Niño in Lima, Peru were used. A total of 75 urinary isolates of Escherichia coli were included. Gene identification was performed by polymerase chain reaction. From the 75 isolates, 74 (98.7%) were positive for the fimH gene and 6 (8.0%) were positive for the afa gene. Virulence factors produced by the fimH and afa genes were evident in urinary isolates of ESBL producing Escherichia coli.

Analysis of porphyromonas gingivalis fimA genotypes in severe periodontitis patients

Abstract The aim of this study was to i) evaluate the prevalence of P. gingivalis and the genotypes fim A I, Ib, II, III, IV, and V in Brazilian patients with periodontitis stage III and IV, grades B and C, ii) compare periodontitis grades B and C with regard to the prevalence of P. gingivalis and fim A genotypes, and iii) correlate the presence of these pathogens with clinical periodontal variables. Two samples of subgingival biofilm were collected from the interproximal sites with the greatest clinical attachment loss (CAL) of each patient (grade B = 38; grade C = 54) and submitted to polymerase chain reaction (PCR) for the identification of P. gingivalis and fim A genotypes. The collected periodontal clinical parameters included gingival index, plaque index, probing depth (PD), bleeding on probing (BoP) and CAL. P. gingivalis was present in 61.96% of the samples, but more prevalent in patients with grade C periodontitis (p = 0.048) and higher CAL (p < 0.001), PD (p < 0.001), and BoP (p = 0.01) values, and at sites with high CAL values (p = 0.01). The fim A II genotype was more prevalent in patients with greater mean PD (p = 0.04) and a higher proportion of bleeding sites (p = 0.006). Thus, in this sample of Brazilian periodontitis patients, the presence of P. gingivalis was associated with grade C periodontitis and periodontal destruction, while the fim A II genotype was associated with increased PD and BoP, supporting the notion that P. gingivalis fim A II is an important virulence factor in periodontal tissues.

Frequency of Porphyromonas gingivalis fimA in smokers and nonsmokers after periodontal therapy

Abstract Porphyromonas gingivalis is one of the most important Gram-negative anaerobe bacteria involved in the pathogenesis of periodontitis. P. gingivalis has an arsenal of specialized virulence factors that contribute to its pathogenicity. Among them, fimbriae play a role in the initial attachment and organization of biofilms. Different genotypes of fimA have been related to length of fimbriae and pathogenicity of the bacterium. Objectives The aim of this study was to identify 5 types of fimA genotype strains in smokers and nonsmokers with periodontitis, before and after periodontal therapy. Material and Methods Thirty-one patients with periodontitis harboring P. gingivalis were selected: 16 nonsmokers (NS) and 15 smokers (SM). Clinical and microbiological parameters were evaluated at baseline and 3 months after periodontal treatment, namely: plaque index, bleeding on probe, probing depth, gingival recession and clinical attachment level. The frequency of P. gingivalis and fimA genotype strains were determined by polymerase chain reaction. Results Type I fimA was detected in the majority of SM and NS at baseline, and the frequency did not diminish after 3 months of treatment. The frequency of type II genotype was higher in SM than NS at baseline. After 3 months, statistical reduction was observed only for types II and V fimA genotypes in SM. The highest association was found between types I and II at baseline for NS (37.5%) and SM (53.3%). Conclusion The most prevalent P. gingivalis fimA genotypes detected in periodontal and smoker patients were genotypes I and II. However, the presence of fimA genotype II was higher in SM. Periodontal treatment was effective in controlling periodontal disease and reducing type II and V P. gingivalis fimA.

Distribución gen fimA de Porphyromonas gingivalis en pacientes chilenos con periodontitis crónica / Distribution of fimA genotypes of Porphyromonas gingivalis in Chilean patients with chronic periodontitis

RESUMEN: Antecedentes: Gen fimA de Porphyromonas gingivalis es un importante factor de virulencia asociado al desarrollo y la progresión de periodontitis. Objetivo: Cuantificar los niveles de P. gingivalis y la prevalencia de genotipos fimA en pacientes chilenos con diferentes grados de severidad de periodontitis crónica. Metodología: Se analizaron 135 muestras subgingivales de 45 adultos (15 con leve, 15 con moderada y 15 con periodontitis severa) mediante qPCR para P. gingivalis y genotipos fimA (I-V and Ib). Resultados: Se detectó P. gingivalis en el 73,3% de los pacientes con periodontitis crónica (46,6%, 73,3% y 100% para las formas leve, moderada y severa, respectivamente). El gen fimA se detectó en el 66% de los sujetos positivos para P. gingivalis, siendo el fimA IV y I los genotipos más prevalentes. Además, se detectó fimA IV en el 75% y fimA I en el 62,5% de los casos severos y moderados de periodontitis, respectivamente. Los niveles aumentados de fimA IV se asociaron con periodontitis crónica severa. Conclusiones: Los resultados sugieren una alta prevalencia de P. gingivalis y de sus genotipos fimA IV y I en pacientes con periodontitis crónica. Además fimA IV fue asociado con formas más severas de periodontitis crónica en esta población chilena.

ABSTRACT: Background: Porphyromonas gingivalis fimA gene is a key virulence factor and has been associated with development and progression of periodontal diseases. Aim: To quantify the levels of P. gingivalis and the prevalence of fimA genotypes in Chilean patients with different severity of chronic periodontitis. Methodology: One hundred and thirty five subgingival samples from 45 adults (15 with slight, 15 with moderate and 15 with severe chronic periodontitis, respectively) were analyzed by qPCR for P. gingivalis and fimA genotypes (I-V and Ib). Results: P. gingivalis was detected in 73.3% of patients (46.6%, 73.3% and 100% of patients with slight, moderate and severe chronic periodontitis, respectively). The genotype fimA was detected in 66% of positive subjects for P. gingivalis, whereas fimA IV and I were the most prevalent genotypes. In addition, fimA IV was detected in 75% and fimA I in 62.5% of severe and moderate cases, respectively. Increased levels of fimA IV were associated with severe chronic periodontitis. Conclusions: These findings suggest that there is a high prevalence of P. gingivalis and its fimA IV and I genotypes in chronic periodontitis patients. Furthermore, fimA IV was associated with severe chronic periodontitis in this Chilean population.

Prevalencia de los genotipos fimA II y fimA IV de Porphyromonas gingivalis en un grupo de mujeres mexicanas con diabetes gestacional en la región centro de México / Prevalence of fimA II and fimA IV Porphyromonas gingivalis genotypes in a group of Mexican women with gestational diabetes in the central region of México

La diabetes gestacional (DG) es una de las complicaciones médicas que más frecuentemente afectan a las mujeres embarazadas; algunos autores reportan una prevalencia entre el 9,7 y el 13,9%. La DG puede ser causa de efectos adversos como: nacimiento pretérmino, macrosomia, nacimiento por cesárea, hiperbilirrubinemia, hipertensión gestacional, así como la predisposición de desarrollar posteriormente diabetes mellitus tipo 2 y síndrome metabólico. La literatura señala la asociación entre los microorganismos presentes en el biofilm subgingival, etiológicos de la inflamación de los tejidos de soporte dentarios y diabetes mellitus. Uno de estos microorganismos, Porphyromonas gingivalis, expresa, entre otros factores de virulencia, una proteína llamada fimbrilina, la cual presenta variaciones genotípicas relacionadas con su capacidad de inducción en la expresión de mediadores inflamatorios; los genotipos fimA II y fimA IV se consideran con mayor capacidad de virulencia y su presencia se ha asociado con la resistencia a la insulina. En este estudio analizamos la prevalencia de los genotipos fimA II y fimA IV en un grupo de mujeres mexicanas de la región central de México con DG, en mujeres con embarazo sin diabetes y mujeres sin embarazo y sin diabetes. Los resultados encontrados muestran una elevada presencia del genotipo fimA II en mujeres con DG (p < 0,05)

Gestational diabetes (GD) is one of the most common complications in pregnant women, with some authors reporting prevalence between 9.7% and 13.9%. GD can lead to the following adverse effects: preterm birth, macrosomia, cesarean birth, hyperbilirubinemia, gestational hypertension, and predisposition to later develop diabetes mellitus type 2 and metabolic syndrome. The literature shows an association between microorganisms in the subgingival biofilm, which produces inflammation of the dental support tissue, and diabetes mellitus. Porphyromonasgingivalis is one of these microorganisms, and among other virulence factors, it expresses a protein called fimbrilin which has genotypic variations related to its ability to induce expression of inflammatory mediators. Genotypes fimA II and fimA IV are considered to have a greater virulence and their presence has been associated with insulin resistance. An analysis is made on the prevalence of genotypes fimA II and fimA IV in a group of women in central region of Mexico with GD, pregnant women without diabetes, and non-pregnant women without diabetes. The results show an elevated presence of genotype fimA II in women with GD (P <.05)

Regulatory T-cell vaccination independent of auto-antigen

To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25+ Treg cells are stimulated, but for arthritis CD39+ Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-beta and interleukin-10.

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.

Determination of adhesion virulence factors of enteropathogenic escherichia coli [eaeA,bfpA] isolates from asymptomatic individuals compared to those with diarrhea

The aim of the this study was to investigate the prevalence of toxB, paa, Ipf and iha adhesion genes in enteropathogenic Escherichia coli [EPEC] isolates lacking in two important adhesion factors, the eaeA and bfpA genes. We examined a total of 70 serologically confirmed EPEC [eaeA, bfpA] isolates. DNA from the isolates was extracted by the phenol-chloroform method. toxB, paa, Ipf and iha adhesion genes in the EPEC isolates were detected by polymerase chain reaction. Data were analyzed by SPSS software and statistical analysis using the chi square test. P-values less than 0.05 were considered significant. PCR was positive for the toxB gene in 2 [2.85%], paa in 3 [4.28%], Ipf in 32 [45.71%] and iha in 15 [21.42%] of the 70 strains. Statistically, none of the toxB.paa, and Ipf genes were associated with diarrhea. However, the iha gene showed a weak significant relation to diarrhea [P=0.11]. The main mechanism of pathogenicity for EPEC is attachment and effacement. Therefore, EPEC [eaeA, bfpA] should have another adhesin factor, which should be investigated. EPEC strains [eaeA-, bfpA-] that possess the Ipf gene are common. Further investigations of the virulence properties of these strains are necessary in order to elucidate the role of these virulence factors in diarrhea among Iranian children

Diarrhoeagenic E. coli pathotypes in children with and without diarrhoea in an Iranian referral paediatrics centre

Diarrhoeagenic Escherichia coli can be considered as the most important etiologic agents of diarrhoea in the Islamic Republic of Iran, particularly in children. This study determined the frequency of diarrhoeagenic E. coliisolates collected from children with acute diarrhoea [n= 50] and a control group [n= 50] at an Iranian referral paediatric centre during a 1-year period. Using multiplex PCR, diarrhoeagenic E. coli was identified in 90% of the case group and 20% of controls. Enterotoxigenic E. coli was the most frequently identified pathotype in both groups [26% in cases; 10% in controls]. Shiga toxin-producing E. coli was the second most isolated pathotype [17%], followed by enteroaggregative E. coli [12%]. No enteroinvasive E. coliand enteropathogenic E. colistrains were recovered. More than 80% of isolates harboured the fimHgene. This high proportion of diarrhoeagenic E. coli and diversity of E. coli types highlights the need for enhanced surveillance of gastroenteritis agents in children in this country

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

Construction of recombinant plasmid with Porphyromonas gingivalis FimA deficiency / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct the recombinant plasmid pPHU281_A_Spec_B, which knock out Porphyrmonas gingivalis (Pg) FimA gene.</p><p><b>METHODS</b>Genomic DNA was extracted from PgATCC33277 which was cultured in anaerobic condition. The upstream and downstream gene of FimA was cloned from Pg genenomic DNA with specific restriction sites by polymerase chain reaction. Suicide vector pPHU281 was inserted by three fragments: upstream, downstream of FimA gene and spectinomycin resistance gene. The recombinant plasmid was confirmed by electrophoresis and sequenced after amplification in compentent cells DH-5α.</p><p><b>RESULTS</b>The gene sequence was identified by DNA sequencing analysis. The recombinant plasmid pPHU281_A_Spec_B was successfully constructed.</p><p><b>CONCLUSIONS</b>The recombinant plasmid pPHU281_A_Spec_B was constructed, which may be used for the constructon of FimA deficient Pg.</p>

Comparative study on the ability of adhesion and invasion of different fimA genetypes of Porphyromonas gingivalis to oral epithelial cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To compare the ability of adhesion and invasion to epithelial cells by Porphyromonas gingivalis (Pg) strains with different fimA separated from Chinese.</p><p><b>METHODS</b>Cultured method and antibiotic protection method were used to determine the adhesive and invasive ability of Pg with different fimA genetypes. The adhesion was observed by scanning electron microscope.</p><p><b>RESULTS</b>All the strains adhered and invaded to KB cells, and the adhesion rate ranged from 0.523% to 37.125% and invasive rate from 0.017% to 3.750%.The adhesive and invasive ability among different fimA genotypes showed no significant difference (P > 0.05).</p><p><b>CONCLUSIONS</b>There is no significant correlation between fimA genotype and ability in adhesion and invasion to KB cells.</p>

Survival properties of ETEC surface-displayed K88ac-LT(B) on Lactobacillus casei / 生物工程学报

K88ac-LT(B) gene derived from pQE30-K88ac-LT(B) was cloned into the expression vector pLA and then the recombinant vector was transformed into the competent cells Lactobacillus casei 525. The recombinant bacteria were grown at 37 degrees C, in MRS broth. Western blotting analysis with rabbit-anti-K88ac-LT(B) polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 71.2 kD. The K88ac-LT(B) fusion protein on the cell surface was confirmed by immunofluorescence mciroscopy and flow cytometric analysis. In addition, the survival of recombinant Lactobacillus casei 525 was studied in imitative gastrointestinal environments such as artificial gastro fluid (pH 1.5-5.5), artificial intestinal fluid, bile(0.3-3.0 g/L). The results indicated that the recombinant strain survived well in artificial gastric fluids at pH 2.5-4.5 in 5 h. The recombinant Lactobacillus casei 525 could slowly grow in the artificial intestinal fluid for different time, and could survive in 0.3% bile.

Matrix metalloproteinase 8 and 9 regulations of polymorphonuclear leukocytes stimulated by Porphyromonas gingivalis with different fimA genotypes / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the pathogenicity of matrix metalloproteinase 8, 9 (MMP-8, MMP-9) regulations of polymorphonuclear leukocytes (PMNs) by challenge of Porphyromonas gingivalis (P. gingivalis) with different fimA genotypes.</p><p><b>METHODS</b>The studies mainly adopt the isopycnic sedimentation separation to separate the PMNs from human peripheral blood. P. gingivalis ATCC 33277 (type I), WCSP 115 (type II), WCSP 1.5 (type III), W83 (type IV), WCSP 559 (type IV) were assessed for their inductions of MMP-8, MMP-9 expression in PMNs. MMP-8, MMP-9 protein levels in culture supernatant were determined by ELISA at different time intervals (5 min, 30 min, 1 h, 2 h) following continuous co-culture of bacteria with PMNs.</p><p><b>RESULTS</b>MMP-8 and MMP-9 protein levels produced by PMNs co-culture with the I fimA-IV fimA P. gingivalis were significantly stronger than unsimulated group. The velocity and quantity of MMP-8 produced by PMNs co-culture with the II fimA P. gingivalis and IV fimA P. gingivalis were more than III fimA, IVfimA P. gingivalis. The MMP-9 protein levels produced by PMNs co-culture with the I fimA, II fimA, IV fimA P. gingivalis was significantly stronger than III fimA and IV fimA P. gingivalis.</p><p><b>CONCLUSION</b>II fimA and IV fimA P. gingivalis have stronger pathogenicity relatively, which indicate that fimA genotype is associated with pathogenesis of P. gingivalis.</p>

Cloning of the fimA gene of Porphyromonas gingivalis and its expression and purification in Escherichia coli / 华西口腔医学杂志

<p><b>UNLABELLED</b>OBJECTIVE; To clone the fimA gene of Porphyromonas gingivalis (P. gingivalis) and detect its expression in Escherichia coli (E. coli).</p><p><b>METHODS</b>The fimA gene was obtained by PCR from the genome of P. gingivalis to construct a prokaryotic expression plasmid pT-BAD/fimA. pT-BAD/fimA was transformed into E. coli BL21 (DE3) competent cells and the recombination protein was characterized by means of matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. The bound protein was eluted with different concentrations of imidazole (250, 200, 150, 100, 50 micromol x L(-1)) respectively.</p><p><b>RESULTS</b>DNA sequencing showed that the fragment was 99.9% consistent with that of the published. After induction with L-arabinose, a new 3.8 x 10(4) protein appeared on SDS-PAGE gel. The protein was further identified by MALDI-TOF-MS. Purity of 95% of the target protein was purified by Ni-NTA Purification System after eluted with 100 micromol x L(-1) imidazole.</p><p><b>CONCLUSION</b>The fimA gene of P. gingivalis was cloned successfully and its protein was expressed correctly in E. coli. A high purity of protein FimA was obtained and it could be applied for follow-up researches.</p>

Matrix metalloproteinases regulations of human gingival fibroblasts by Porphyromonas gingivalis with different fimA genotypes / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of matrix metalloproteinases (MMP) regulations of human gingival fibroblasts (HGF) by challenge of Porphyromonas gingivalis (Pg) with different fimA genotypes.</p><p><b>METHODS</b>Pg ATCC 33277 (type I), WCSP115 (type II), WCSP1.5 (type III), W83 (type IV) were assessed for their inductions of MMP-1 and MMP-2 expression in HGF. MMP mRNA levels of HGF were determined by real-time RT-PCR and MMP protein levels in culture supernatant were determined by ELISA at different time intervals (1, 3, 6 and 12 h) following continuous co-culture of bacteria with HGF.</p><p><b>RESULTS</b>When co-cultured with Pg, the MMP-1 and MMP-2 mRNA and protein expression of HGF significantly increased compared with the negative control group (P < 0.01). The group of type II showed greater up-regulated than other fimA genotypes in the mRNA and protein expressions of MMP-1 and MMP-2, MMP-1 mRNA [(28.88 +/- 3.12) - (231.01 +/- 24.99)] and protein [(1.35 +/- 0.17) - (3.08 +/- 1.20)] microg/L; MMP-2 mRNA [(20.42 +/- 2.21) - (188.34 +/- 37.37)] and protein [(2.57 +/- 0.76) - (18.08 +/- 1.15)] microg/L for different time periods; While the group of type III was weaker than other fimA genotypes, the level of MMP-1 mRNA was [(5.11 +/- 0.55) - (72.84 +/- 8.84)] and protein [(0.68 +/- 0.13) - (1.46 +/- 0.94)] microg/L, MMP-2 mRNA [(4.55 +/- 0.55) - (25.75 +/- 3.12)] and protein [(2.28 +/- 0.93) - (11.22 +/- 2.46)] microg/L (P < 0.05).</p><p><b>CONCLUSIONS</b>Pg could induce HGF to over-express MMP, and fimA genotypes of Pg may be related to this pathogenicity, which might indicate fimA genotype is associated with pathogenesis of Pg.</p>

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.

[Distribution of sefA gene among Salmonella enteritidis isolates from poultry sources and potential as diagnostic and epidemiological tools]

This study was conducted to detect the presence of sefA gene among Salmonella Enteritidis isolates from poultry sources by polymerase chain reaction [PCR] and evaluate its potential as diagnostic and epidemiological tools. Thirty Salmonella isolates from poultry sources: broilers, broiler breeders, layers, hatcheries, and poultry abattoirs were investigated. Upper and forward primers were constructed based on the published sequence of the sefA gene that encodes the SEF14 fimbrial subunit [fimbrin]. The size of target product was 526 bp. To confirm the specificity, the PCR products were digested with BamHI restriction enzyme that divides the product to two segments of 186 and 340 bp. The PCR reaction was set up as described in the previous literature. All Salmonella Enteritidis isolates showed the presence of 526 bp product. None of isolates belonging to serogroups B and C were positive for the 526 bp fragment. The restriction enzyme BamHl divided each 526 bp product into two fragments of 186 and 340 bp. This pattern was demonstrated for all Salmonella Enteritidis isolates. The results of the present study showed that the sefA gene carries a high potential to be used as a diagnostic and an epidemiological tool for Salmonella Enteritidis

Monocyte chemoattractant protein-1 regulations of human gingival fibroblasts by Porphyromonas gingivalis with different fimA genotypes / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of monocyte chemoattractant protein-1 (MCP-1) regulations of human gingival fibroblasts (HGF) by challenge of Porphyromonas gingivalis (Pg) with different fimA genotypes.</p><p><b>METHODS</b>Pg ATCC 33277 (type I), WCSP115 (type II), WCSP1.5 (type III), W83 (type IV) were assessed for their inductions of MCP-1 expression in HGF. MCP-1 mRNA levels of HGF were determined by real-time RT-PCR and MCP-1 protein levels in culture supernatant by ELISA at different time intervals (1 h, 3h, 6h and 12h) following continuous co-culture of bacteria with HGF.</p><p><b>RESULTS</b>MCP-1 mRNA and protein levels were both up-regulated when HGF co-cultured with different Pg fimA genotypes. Type II was stronger than other fimA genotypes, HGF expressed significantly great amount of MCP-1 mRNA [(25.75 +/- 3.12)-(326.69 +/- 35.35)] and protein [(178.20 +/- 46.20)-(443.46 82.19) ng/L] for different time periods; While Type III was weaker than other fimA genotypes, and the level of MCP-1 mRNA was [ (4.16 +/- 0.82)-(94.17 +/- 18.56)] and protein [(86.95 +/- 23.90)-(264.01 +/- 28.59) ng/L](P < 0.05).</p><p><b>CONCLUSIONS</b>fimA genotypes of Pg are related with the inductions of MCP-1, which might indicate fimA genotype is associated with pathogenesis of Pg.</p>

Cloning, expression and immunity of pilA gene and ompC gene from avian pathogenic Escherichia coli / 生物工程学报

In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.