临床分离肺炎链球菌的毒力基因及血清型分析

目的 了解肺炎链球菌的5种常见毒力基因(psaA、pspA、nanA、ply、lytA)在临床分离株中的分布情况.方法 以2006-2008年从临床分离133株肺炎链球菌为研究对象,采用PCR法对5种常见的毒力基因进行检测.结果 lytA、psaA、nanA、pspA、ply这5种基因在133株菌中的检测阳性率分别为94.7%、85.0%、84.2%、60.2%、82.7%;5种毒力基因在87株常见血清型菌中的阳性率分别为100%、87.4%、89.7%、67.8%、86.2%.结论 5种毒力基因在本地区肺炎链球菌中的阳性检测率均较高,在常见血清型菌株中的阳性检测率较其他血清型高.这5种基因是肺炎链球菌致病的主要毒力基因,可能作为新刑肺炎蛋白疫苗的候选基因.     

Enteroinvasive Escherichia coli: a cause of bacteremia in patients with AIDS.

A strain of enteroinvasive Escherichia coli was isolated from the blood of a patient with advanced human immunodeficiency virus disease on repeated occasions, associated with severe diarrheal illness. The isolate was killed in vitro by control sera but not by sera collected from the patient before or after his bacterial illnesses.     

Ongoing horizontal and vertical transmission of virulence genes and papA alleles among Escherichia coli blood isolates from patients with diverse-source bacteremia

The phylogenetic distributions of multiple putative virulence factors (VFs) and papA (P fimbrial structural subunit) alleles among 182 Escherichia coli blood isolates from patients with diverse-source bacteremia were defined. Phylogenetic correspondence among these strains, the E. coli Reference (ECOR) collection, and other collections of extraintestinal pathogenic E. coli (ExPEC) was assessed. Although among the 182 bacteremia isolates phylogenetic group B2 predominated, exhibited the greatest concentration of individual VFs, and contained the largest number of familiar virulent clones, other phylogenetic groups exhibited greater concentrations of certain VFs than did group B2 and included several additional virulent clones. Certain of the newly detected VF genes, e.g., fyuA (yersiniabactin; 76%) and focG (F1C fimbriae; 25%), were as prevalent or more prevalent than their more familiar traditional counterparts, e.g., iut (aerobactin; 57%) and sfaS (S fimbriae; 14%), thus possibly offering additional useful targets for preventive interventions. Considerable diversity of VF profiles was observed at every level within the phylogenetic tree, including even within individual lineages. This suggested that many different pathways can lead to extraintestinal virulence in E. coli and that the evolution of ExPEC, which involves extensive horizontal transmission of VFs and continuous remodeling of pathogenicity-associated islands, is a highly active, ongoing process.     

Clinical Escherichia coli strains carrying stx genes: stx variants and stx-positive virulence profiles

Altogether, 173 Shiga toxin-producing Escherichia coli (STEC) serotype O157 (n = 111) and non-O157 (n = 62) isolates from 170 subjects were screened by PCR-restriction fragment length polymorphism for eight different stx genes. The results were compiled according to serotypes, phage types of O157, production of Stx toxin and enterohemolysin, and the presence of eae. The stx genes occurred in 11 combinations; the most common were stx(2) with stx(2c) (42%), stx(2) alone (21%), and stx(1) alone (16%). Of the O157 strains, 64% carried stx(2) with stx(2c) versus 2% of the non-O157 strains (P < 0.001). In the non-O157 strains, the prevailing gene was stx(1) (99% versus 1% in O157 strains; P < 0.001). In addition, one strain (O Rough:H4:stx(2c)) which has not previously been described as associated with hemolytic-uremic syndrome (HUS) was found. Ten stx-positive virulence profiles were responsible for 71% of all STEC infections. Of these profiles, five accounted for 71% of the 21 strains isolated from 20 patients with HUS or thrombotic thrombocytopenic purpura (TTP). The strains having the virulence profile that caused mainly HUS or TTP or bloody diarrhea produced Stx with titers of >/=1:128 (90%) more commonly than did other strains (51%; P < 0.001). These strains were also more commonly enterohemolytic (98% versus 68% for other strains; P < 0.001) and possessed the eae gene (100%) more commonly than did other strains (74%; P < 0.001). A particular virulence profile, O157:H7:PT2:stx(2):stx(2c):eae:Ehly, was significantly more frequently associated with HUS and bloody diarrhea than were other profiles (P = 0.02) and also caused the deaths of two children. In this study, the risk factors for severe symptoms were an age of <5 years and infection by the strain of O157:H7:PT2 mentioned above.     

Molecular characterization of Escherichia coli strains that cause symptomatic and asymptomatic urinary tract infections

The differences between Escherichia coli strains associated with symptomatic and asymptomatic urinary tract infections (UTIs) remain to be properly determined. Here we examined the prevalence of plasmid types and bacteriocins, as well as genetic relatedness, in a defined collection of E. coli strains that cause UTIs. Comparative analysis identified a subgroup of strains with a high number of virulence genes (VGs) and microcins M/H47. We also identified associations between microcin genes, VGs, and specific plasmid types.     

Virulence determinants in nontoxinogenic Escherichia coli O157 strains that cause infantile diarrhea.

Ten sorbitol-fermenting Escherichia coli O157 strains that cause infantile diarrhea and are positive in the fluorescence actin staining test were determined to be negative for Shiga-like toxin (SLT) genes. We amplified their complete eae genes, contrasting them with those of SLT-producing E. coli O157 by restriction fragment length polymorphism analysis and nucleotide sequence analysis of a 400-bp stretch of the 3' end of eae. The data substantiated the presence of two eae genotypes within serogroup O157, one resembling eae of enteropathogenic E. coli (EPEC) strain E2348/69, found in nontoxinogenic E. coli O157 strains, and the other resembling eae of EHEC strain EDL 933, found in toxinogenic E. coli O157 strains. Another EPEC-specific virulence determinant was also shown to be large plasmids harboring EPEC adherence factor sequences. The SLT-negative E. coli O157 strains described here fall under the heading of EPEC, which serves as an explanation for their virulence in infants, and represent a third pathogroup within serogroup O157.     

Prevalence of adhesive genes among uropathogenic Escherichia coli strains isolated from patients with urinary tract infection in Mangalore

The study was carried out to detect the adhesive genes pap (pyelonephritis associated pili), sfa (S fimbrial adhesin) and afa (afimbrial adhesin) from Escherichia coli strains isolated in patients diagnosed with urinary tract infection (UTI). A total of 23% of the isolates were positive for pap, sfa and afa genes with a prevalence of 60.87% (14/23), 39.1% (9/23) and 39.1% (9/23), respectively. Prevalence of multiple adhesive genes was 8.7% (2/23) for pap and afa, 30.43% (7/23) for pap and sfa. Significant numbers of isolates were positive for Congo red binding (80%) and haemolysin production 60%. The prevalence of multiple adhesive genes indicate the potential to adhere and subsequently cause a systemic infection among UTI patients.     

Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea.

The genetic relationships among 1,300 isolates of Escherichia coli representing 16 serotypes associated with enteric disease, including O157:H7 strains recovered from patients with hemorrhagic colitis and hemolytic uremic syndrome and O26:H11, O55:H6, O55:H7, O111:H2, and O128:H2 strains, many of which were isolated originally from infants with diarrhea, were estimated from allelic variation among 20 enzyme-encoding genes detected by multilocus enzyme electrophoresis. Multiple electrophoretic types were observed among isolates of each serotype, with isolates of the same O serogroup differing on average at 28% of the enzyme loci. Comparisons of the multilocus enzyme profiles revealed that 72% of the isolates belong to 15 major electrophoretic types, each of which corresponds to a bacterial clone with a wide geographic distribution. Genetically, the O157:H7 clone is most closely related to a clone of O55:H7 strains that has long been associated with worldwide outbreaks of infantile diarrhea. We propose that the new pathogen emerged when an O55:H7-like progenitor, already possessing a mechanism for adherence to intestinal cells, acquired secondary virulence factors (Shiga-like cytotoxins and plasmid-encoded adhesins) via horizontal transfer and recombination.     

Differentiation in virulence patterns of Escherichia coli possessing eae genes

In this study 98 Escherichia coli strains which belonged to traditional enteropathogenic (EPEC) serotypes and 82 enterohemorrhagic E. coli (EHEC) strains were screened by polymerase chain reaction (PCR) for the presence of E. coli -attaching and -effacing (eae) genes. These strains were also hybridized with the enteropathogenic adherence factor (EAF) probe and examined in the fluorescence actin staining (FAS) test. The results obtained from the individual strains demonstrated that all 26 class I EPEC with localized adherence to HEp-2 cells carried EAF and eae genes. In contrast, of 72 EPEC strains with no or diffuse adherence only 1 strain was EAF positive and 6 strains had eae. Of 82 EHEC strains a total of 75 carried eae sequences. Of considerable interest, 15 of 21 E. coli strains that lost their slt genes during subcultivation were found to be eae positive. As controls a total of 53 enterotoxigenic and enteroinvasive E. coli, and 125 E. coli strains from the normal flora were investigated and all displayed negative results in the eae-PCR. From the 201 strains comprising classical EPEC serotypes, EHEC and E. coli with lost slt genes, a total of 126 displayed a positive FAS test and 122 reacted in the eae-PCR. Only 4 strains were FAS test positive but eae-PCR negative. Our data indicate that E. coli strains possessing the eae genes are heterogenous with respect to their virulence determinants. Loss of virulence plasmids and phage-encoded slt genes either in the host or during storage may contribute to this heterogeneity. The eae-PCR detected all class I EPEC and 91.5% of the EHEC. For diagnostic purposes we, therefore, recommend the combination of eae- and slt-specific gene probes which allowed a 100% detection of the class I EPEC and EHEC strains investigated here.     

Multiplex PCRs for identification of Escherichia coli virulence genes

PCRs were developed to detect 11 Escherichia coli virulence genes. Primers amplified the respective genes without cross-reaction with other genes. Specificity was maintained in multiplex reactions; excellent amplification of target genes was possible with a minimum of four multiplex reactions. These reactions successfully identified genes in E. coli from the feces of four dogs.     

A uropathogenicity island contributes to the pathogenicity of Escherichia coli strains that cause neonatal meningitis

We report that the archetypal Escherichia coli strain C5 causing neonatal meningitis harbors a pathogenicity island (PAI) designated PAI I(C5) that is similar to the PAI II(J96) of uropathogenic E. coli J96 inserted in the leuX-tRNA gene. PAI-negative C5 mutants had a lower capacity than C5 to induce high-level bacteremia in a neonatal rat model. However, no change in their resistance to the bactericidal effect of serum and their capacity to cross the blood-brain barrier was observed.     

Prevalence of diarrheagenic Escherichia coli strains detected by PCR in patients with travelers' diarrhea

Objective: To determine the prevalence of the different categories of diarrheagenic Escherichia coli , enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), verotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC), and enteropathogenic E. coli (EPEC), associated with travelers' diarrhea.
Methods: Stool specimens from 350 patients with travelers' diarrhea were collected between 1994 and 1996. The virulence factors of the diarrheagenic E. coli isolated were detected by PCR technique, in subcultures of single colonies of all morphotypes of E. coli observed in culture on MacConkey agar.
Results: ETEC (15.7%), EAggEC (13.4%) and DAEC (9.14%) are significantly more prevalent than EIEC (3.4%), EPEC (2.86%) and VTEC (0.86%) (p < 0.05; z -test). The prevalence of ETEC and EAggEC was similar in all geographic areas visited.
Conclusions: PCR is a rapid and specific technique to use in the identification of the different categories of diarrheagenic E. coli and greatly increases the yield of potential enteropathogens from cases of travelers' diarrhea. Not only ETEC but also EAggEC and DAEC strains play a major role in the etiology of travelers' diarrhea, whereas EIEC, EPEC, and VTEC strains play a minor role, leading to the question of whether it is necessary to search routinely for these three types of E. coli in diagnostic laboratories.     

Genetic relatedness and virulence gene profiles of Escherichia coli strains isolated from septicaemic and uroseptic patients

We investigated the relationship between clonality and virulence factors (VFs) of a collection of Escherichia coli strains isolated from septicaemic and uroseptic patients with respect to their origin of translocation. Forty septicaemic and 30 uroseptic strains of E. coli were tested for their phylogenetic groupings, genetic relatedness using randomly amplified polymorphic DNA (RAPD), biochemical fingerprinting method (biochemical phenotypes [BPTs]), adherence to HT-29 cells and the presence of 56 E. coli VF genes. Strains belonging to phylogenetic groups B2 and D constituted 93% of all strains. Fifty-four (77%) strains belonged to two major BPT/RAPD clusters (A and B), with cluster A carrying significantly (P = 0.0099) more uroseptic strains. The degree of adhesion to HT-29 cells of uroseptic strains was significantly (P = 0.0012) greater than that of septicaemic strains. Of the 56 VF genes tested, pap genes was the only group that were found significantly (P < 0.0001) more often among uroseptic isolates. Phylogenetic group B2 contained a significantly higher number of strains carrying pap genes than those in group D. We conclude that uroseptic E. coli are clonally different from septicaemic strains, carry more pap genes and predominantly adhere more to the HT-29 cell model of the gut.     

Genetic profiling of enterohemorrhagic Escherichia coli strains in relation to clonality and clinical signs of infection

Sixty-seven human strains of enterohemorrhagic Escherichia coli (EHEC) (from patients with more or less severe symptoms) were serogrouped and arranged according to pulsed-field gel electrophoresis (PFGE) patterns. We used PCR to investigate the strains according to known or putative virulence factors, and associations with disease were studied. All EHEC strains with the same PFGE pattern belonged to the same serogroup. On the contrary, two serogroups (O157 and O8) included strains with different PFGE patterns. We found several different combinations of chromosomal and plasmid-borne determinants, encoding the putative virulence factors, among the strains. As judged from clinical symptoms, there was no marked difference in pathogenicity among the strains and their combinations of virulence traits. All strains of O157 had the genes coding for verocytotoxin (VT) 2, intimin (eaeA), E. coli hemolysin (E-hly), and secreted serine protease (espP). Among EHEC non-O157 strains, the genes coding for VT1 and VT2 were equally dispersed. EaeA positivity was just as common among VT1- as VT2-positive strains. Among the plasmid-borne determinants, E-hly and espP were the most common and E-hly might be a pathogenicity marker among EHEC non-O157 strains. The conclusion is that PFGE is a very useful tool in epidemiological studies. The EHEC plasmids are heterogeneous in their gene composition, with the four plasmid-borne determinants found in many combinations. There was no reliable correlation between chromosomal and plasmid-borne virulence factors and human disease.     

Clonal relationships and variation in virulence among Escherichia coli strains of avian origin

Average genetic relatedness among 44 Escherichia coli strains of serotypes O1, O2, and O78 isolated mainly from birds with colibacillosis or swollen-head syndrome from France or Saudi Arabia was estimated based on allelic variation detected by multilocus enzyme electrophoresis. For 20 enzyme-encoding loci, we resolved 2.8 alleles per locus and distinguished 17 electrophoretic types (ETs) that were used to mark naturally occurring cell lineages or clones. On average, ETs differed at 37% of their loci. Forty-eight percent of the isolates represent three ETs, two of which belong to previously defined complexes of clones identified in avian disease in North America and Europe. Virulence of strains, assessed in experimental infections of day-old chicks, showed little variation among isolates of a clone, but was significantly variable among isolates of different clone complexes. These findings add support to the evidence that a majority of avian isolates that cause colibacillosis belong to a few cosmopolitan pathogenic clones and indicate a substantial between-clone component of pathogenicity.     

Occurrence of hlyA and sheA genes in extraintestinal Escherichia coli strains

The association of a hemolytic phenotype with the carriage of the alpha-hemolysin gene (hlyA) and/or the silent hemolysin gene (sheA or clyA) among 540 extraintestinal clinical isolates of Escherichia coli and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal E. coli infection, the role of SheA is not completely clarified. Two hemolytic sheA+ E. coli strains that lacked hlyA and possessed no other hemolysin genes were identified. No hlyA+ sheA+ strains were identified, suggesting that there is possible incompatibility between hlyA and sheA in the chromosome of E. coli.     

Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains

The ability of 15 Escherichia coli strains to form biofilms on polystirene plates was studied. The strains were serotyped, and their phenotypic expression of surface virulence factors (VFs), and antibiotic susceptibility was also determined. Moreover, 30 VFs-associated genes were analysed, including 15 adhesins (papC, papG and its three alleles, sfa/focDE, sfaS, focG, afa/draBC, iha, bmaE, gafD, nfaE, fimH, fimAvMT78, agn43, F9 fimbriae and type 3 fimbriae-encoding gene clusters), four toxins (hlyA, cnf1, sat and tsh), four siderophore (iron, fyuA, iutA and iucD), five proctetins/invasion-encoding genes (kpsM II, kpsMT III, K1 kps variant- neuC, traT and ibeA), and the pathogenicity island malX and cvaC. Morphological appearance and thickness of biofilms of two strong and three weak biofilm producers were also studied by confocal laser scanning microscopy (CLSM). Seven strains were classified as strong biofilm producers and the remaining eight strains were regarded as weak biofilm producers. Mannose-resistant haemagglutination was the only phenotypically expressed surface virulence factor more frequently found in the strong biofilm group. Five virulence-associated genes were more common (p<0.05) in strong biofilm producers: papC and papG alleles, sfa/focDE, focG, hlyA and cnf1. CLSM images showed irregular biofilms with projections at the top mainly in strong biofilm.     

Presence and characterization of extraintestinal pathogenic Escherichia coli virulence genes in F165-positive E. coli strains isolated from diseased calves and pigs

The virulence genotype profile and presence of a pathogenicity island(s) (PAI) were studied in 18 strains of F165-positive Escherichia coli originally isolated from diseased calves or piglets. On the basis of their adhesion phenotypes and genotypes, these extraintestinal pathogenic strains were classified into three groups. The F165 fimbrial complex consists of at least two serologically and genetically distinct fimbriae: F165(1) and F165(2). F165(1) is encoded by the foo operon (pap-like), and F165(2) is encoded by fot (sfa related). Strains in group 1 were foo and fot positive, strains in group 2 were foo and afa positive, and strains in group 3 were foo positive only. The strains were tested for the presence of virulence genes found mainly in extraintestinal pathogenic E. coli (ExPEC) strains. Although all the strains were positive for the papA variant encoding F11 fimbriae incD, traT, and papC, the prevalence of virulence genes commonly found in PAIs associated with ExPEC strains was highly variable, with strains of group 2 harboring most of the virulence genes tested. papG allele III was detected in all strains in group 1 and in one strain in group 3. All other strains were negative for the known alleles encoding PapG adhesins. The association of virulence genes with tRNA genes was characterized in these strains by using pulsed-field gel electrophoresis and DNA hybridization. The insertion site of the foo operon was found at the pheU tRNA locus in 16 of the 18 strains and at the selC tRNA locus in the other 2 strains. Furthermore, 8 of the 18 strains harbored a high-pathogenicity island which was inserted in either the asnT or the asnV/U tRNA locus. These results suggest the presence of one or more PAIs in septicemic strains from animals and the association of the foo operon with at least one of these islands. F165-positive strains share certain virulence traits with ExPEC, and most of them are pathogenic in piglets, as tested in experimental infections.     

Aerobactin and other virulence factor genes among strains of Escherichia coli causing urosepsis: association with patient characteristics.

To assess the role of aerobactin as a virulence factor among uropathogenic Escherichia coli, we determined the prevalence, location, and phenotypic expression of aerobactin determinants among 58 E. coli strains causing bacteremic urinary tract infections. We correlated the presence of the aerobactin system with antimicrobial-agent resistance, the presence and phenotypic expression of other uropathogenic virulence factor determinants (P fimbriae, hemolysin, and type 1 fimbriae), and characteristics of patients. Colony and Southern hybridization of total and plasmid DNA with DNA probes for each virulence factor showed that aerobactin determinants were present in 78% of the strains and were plasmid associated in 21%, whereas P fimbria, hemolysin, and type 1 fimbria determinants were present in 74, 43, and 98% of the strains, respectively, and were always chromosomal. Chromosomal aerobactin, P fimbria, and hemolysin determinants occurred together on the chromosome more often in strains from patients without predisposing urological or medical conditions (P = 0.04). Strains with plasmid-encoded aerobactin lacked determinants for P fimbriae (P = 0.004) and hemolysin (P = 0.0004), were resistant to multiple antimicrobial agents (P = 0.0001), and were found only in compromised patients. Mating experiments demonstrated that some aerobactin plasmids also encoded antimicrobial-agent resistance. These findings suggest that the determinants for aerobactin, P fimbriae, and hemolysin are conserved on the chromosome of the antimicrobial-agent-susceptible uropathogenic strains of E. coli which invade noncompromised patients. In contrast, these chromosomal virulence factors are often absent from E. coli strains causing urosepsis in compromised hosts; these strains may acquire plasmid aerobactin in conjunction with antimicrobial-agent resistance genes.