Genetic markers of pathogenicity of conditionally pathogenic enterobacteria isolated from children with diarrheal diseases

In the process of examination of 89 children from different age groups with diarrheal disease caused by bacteria from Enterobacteriaceae family 89 microorganisms were isolated including Klebsiella spp. (37 isolates), Citrobacter spp. (9 isolates), Enterobacter spp. (17 isolates), Hafnia alvei (1 isolate), Morganella morganii (11 isolates), Proteus spp. (14 isolates). Presence of genes associated with pathogenicity islands (PAIS): hlyA, hlyB (hemolysin), sfaG (fimbria antigen type S), cnf1 (cytotoxic necrotizing factor 1), estB (heat-stable enterotoxin B)were studied in these cultures by PCR. It was found that 32.6% of examined isolates had fragments of PAIS's genes--hlyA was detected in 9 cases (10.1%), hlyB--in 10 cases (11.2%), sfaG --in 8 cases (9%), cnf1--in 9 cases (10.1%), and estB--in 3 cases (3.4%). Positive correlation between genetic determinants hlyB and cnf1 as well as hlyA and sfaG was found while estB was not associated with other genes. Weak positive correlation between presence of sfaG and resistance to tetracycline and chloramphenicol was detected. Factors coded by revealed determinants of PAIS can play a role in the development of diarrheal syndrome.     

Genetic determinants of Escherichia coli pathogenicity isolated from urine and feces of children with different clinical variants of urinary system infection

In the process of examination of 156 children of different age groups 176 E. coli cultures were isolated; of these, 98 cultures were isolated from acute cystitis and pyelonephritis patients, 28--from urine in cases of aysmptomatic bacteriuria, 30--from feces in cases of asymtomatc bacteriuria and intestinal dysbacteriosis, while 20 cultures--from feces of healthy children. In these bacteria the presence of genes associated with pathogenicity islets (PI) hlyA, hlyB, cnf-1, papC, sfaG and gene irp-2 (iron-regulated protein) was established with PCR. The detection rate of PI determinants in uropathogenic E. coli (UPEC) was shown to depend on the variants of the clinical manifestation of urinary tract infection. The total detection rate of PI gene fragments in UPEC cultures of different origin was indicative of their definitely less frequent occurrence in asymptomatic bacteriuria, observed simultaneously with intestinal dysbacteriosis, in comparison with acute urological infection. Practically the same detection rate of PI determinants in E. coli, isolated in asymptomatic bacteriuria in children, reflected high probability of genetic exchange in the above-mentioned fragments and made it possible to presume the existence of DNA sites, characteristic mainly of pathogenic clones. The established heterogeneity of the detection rate of PI determinants in E. coli clinical isolates requires further study.     

The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae

A pathogenicity island termed high-pathogenicity island (HPI) is present in pathogenic Yersinia. This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin. Recently, the HPI was also detected in various strains of Escherichia coli. In this study, the distribution of the HPI in the family Enterobacteriaceae was investigated. Among the 67 isolates pertaining to 18 genera and 52 species tested, nine (13.4%) harbored the island. These isolates were three E. coli, one Citrobacter diversus and five Klebsiella of various species (Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Klebsiella planticola, and Klebsiella oxytoca). As in Yersinia sp., all nine isolates synthesized the HPI-encoded iron-repressible proteins HMWP1 and HMWP2. In the K. oxytoca strain, the right-end portion of the HPI was deleted, whereas the entire core region of the island was present in the eight other enterobacteria strains analyzed. In most of these isolates, the HPI was bordered by an asn tRNA locus, as in Yersinia sp. This report thus demonstrates the spread of the HPI among various members of the family Enterobacteriaceae.     

Simple and reliable multiplex PCR assay for detection of blaTEM,bla(SHV) and blaOXA-1 genes in Enterobacteriaceae

Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type beta-lactamases in Enterobacteriaceae. TEM-type and OXA-1 enzymes are the major plasmid-borne beta-lactamases implicated in amoxicillin-clavulanic acid resistance in Escherichia coli isolates. We have developed a rapid and simple multiplex polymerase chain reaction (PCR) which discriminates bla(TEM), bla(SHV) and bla(OXA-1) genes by generating fragments of 516, 392 and 619 bp respectively. Multiplex PCR analysis of 51 amoxicillin-clavulanate resistant E. coli isolates detected bla(TEM) and bla(SHV) genes in 45 and two strains, respectively, and only one strain harboured a bla(OXA-1) gene. Twenty-three of the 40 cefotaxime-resistant Enterobacteriaceae isolates produced amplicons with a size compatible with the presence of bla(TEM) (13 strains), bla(SHV) (six strains) genes or the association of both genes (four strains). These results were verified by colony hybridisation. Therefore, multiplex PCR is a suitable tool for initial rapid screening of bla genes in Enterobacteriaceae.     

Virulence characteristics of Escherichia coli isolates from children with urinary tract infections

The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common infecting agent in children and adults of both sexes. In an attempt to evaluate the intrinsic virulence of E. coli uroisolates from children, 54 strains were assessed by using PCR for the presence of five representative genetic determinants coding for adherence systems (pap, sfa/foc, afa), and toxins (hly and cnf). The prevalence of pap, sfa/foc and afa genes was 55%, 54%, and 44%, respectively. Hemolysin-encoding gene hly was detected in 55% strains, while cnf was exhibited by 35% of the screened E. coli isolates. Among the 39 PCR positive strains isolated from children's urine cultures the co-occurrence of the various targeted virulence genes was detected in 30 strains, the virulence profiles identified suggesting the presence of their localization on chromosomal regions known as pathogencity-associated islands. The rapid and reliable detection of the intrinsic virulence potential by this molecular approach could be very useful when evaluating the importance of microorganism pathogenicity versus host's susceptibility for developing an overt symptomatology of infection.     

The<Emphasis Type="Italic">Escherichia fergusonii iucABCD iutA</Emphasis> genes are located within a larger chromosomal region similar to pathogenicity islands

Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues. The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E. coli. Aerobactin molecules synthesized by E. fergusonii and E. coli strains stimulated growth of aerobactin indicator strains harboring either E. coli or E. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins from E. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E. fergusonii strains tested. These data, together with the genetic organization of this region, suggest that E. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.     

Occurrence of P.fimbrii in strains from selected genera of Enterobacteriaceae]

This study was aimed at recognition of frequency of occurrence of P fimbriae in strains of Escherichia coli isolated from samples of feces of children with symptoms of diarrhoea and at search of these adhesions in strains representing other than Escherichia genera of Enterobacteriaceae strains. One hundred forty laboratory strains were investigated. They belonged to genus Citrobacter, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Salmonella, Shigella, and Yersinia. Also were tested 1277 colonies of enteric rods isolated from the MacConkey's medium inoculated with samples of feces from 163 children with symptoms of diarrhoea. Mannose-resistant active hemagglutination test was performed with human group O erythrocytes and guinea pig erythrocytes stabilized with glutaraldehyde. Presence of P fimbriae was detected by the slide latex test with latex covered by P1 glycoprotein. Among 140 laboratory strains of Enterobacteriaceae in 21 strains (3-E. cloaceae, 2-Hafnia, 13-K. pneumoniae, 2-P. rettgeri and in one strains of Providencia) presence of MRHA adhesins was demonstrated. Nine of these strains (2-Hafnia, 5-K. pneumoniae and 2-P. rettgeri) reacted specifically in the latex test. Among 1142 colonies of E. coli isolated from children with symptoms of diarrhoea, 326 colonies belonged to 13 EPEC serotypes. With 118 (36.2%) of EPEC colonies a positive result of MRHA reaction was found with human erythrocytes and 34 (10.4%) with guinea pig erythrocytes. Positive latex test was obtained with 77 (23.6%) colonies. All these colonies possessed MRHA adhesins. Remaining 816 colonies of E. coli strains did not represent microorganisms belonging to serotypes accepted as enteropathogenic. From 112 (13.7%) colonies out of 816 not belonging to EPEC, positive results was obtained in the MRHA test with human erythrocytes and this was the case also with 41 (5.0%) colonies in MRHA reaction with application of guinea pig erythrocytes. The latex test was positive in 65 (7.9%) colonies of E. coli. From remaining 135 colonies other than E. coli, positive result of latex test of presence of P fimbriae was obtained with 54 (40.0%) colonies, including 14 colonies of E. cloacae, 23-K. pneumoniae and 17-K. oxytoca. In all these strains presence of MRHA adhesins was demonstrated. These investigations demonstrated that among EPEC strains significantly more frequently, than not belonging to these serotypes of E. coli, MRHA adhesins, including P fimbriae was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild-type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemolytic activity were attained similarly by the E. coli post-translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram-negative human and animal pathogens.     

Conservation of transfer ribonucleic acid and 5S ribonucleic acid cistrons in Enterobacteriaceae.

The genes for tranfer ribonucleic acid (tDNA) and 5S ribonucleic acid (5SDNA) were isolated from the total deoxyribonucleic acid (DNA) of Escherichia coli. The relatedness of tDNA and 5S from E. coli and other species of Enterobacteriaceae was determined by reassociation of the isolated genes labeled with 32PO4 to unlabeled, unfractionated DNA. Double-stranded DNA was separated from unreacted DNA by hydroxyapatite chromatography. Thermal elution profiles were done to determine the amount of unpaired bases present in related DNA sequences. Relative to total DNA, both 5S DNA and tDNA were highly conserved throughout the Enterobacteriaceae, including the genera Yersinia and Proteus.     

Analysis of genome plasticity in pathogenic and commensal Escherichia coli isolates by use of DNA arrays

Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA "pathoarray" developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.     

Homology of the Gene Coding for Outer Membrane Lipoprotein Within Various Gram-Negative Bacteria

The mRNA for a major outer membrane lipoprotein from Escherichia coli was found to hybridize specifically with one of the EcoRI and one of the HindIII restriction endonuclease-generated fragments of total DNA from nine bacteria in the family Enterobacteriaceae: E. coli, Shigella dysenteriae, Salmonella typhimurium, Citrobacter freundii, Klebsiella aerogenes, Enterobacter aerogenes, Edwardsiella tarda, Serratia marcescens, and Erwinia amylovora. However, among the Enterobacteriaceae, DNA from two species of Proteus (P. mirabilis and P. morganii) did not contain any restriction endonuclease fragments that hybridized with the E. coli lipoprotein mRNA. Furthermore, no hybrid bands were detected in four other gram-negative bacteria outside the family Enterobacteriaceae: Pseudomonas aeruginosa, Acinetobacter sp. HO1-N, Caulobacter crescentus, and Myxococcus xanthus. Envelope fractions from all bacteria in the family Enterobacteriaceae tested above cross-reacted with antiserum against the purified E. coli free-form lipoprotein in the Ouchterlony immunodiffusion test. Both species of Proteus, however, gave considerably weaker precipitation lines, in comparison with the intense lines produced by the other members of the family. All of the above four bacteria outside the family Enterobacteriaceae did not cross-react with anti-E. coli lipoprotein serum. From these results, the rate of evolutionary changes in the lipoprotein gene seems to be closely related to that observed for various soluble enzymes of the Enterobacteriaceae.     

Chromosomal replication origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae are functional in Escherichia coli.

The chromosomal DNA replication origins (oriC) from two members of the family Enterobacteriaceae, Enterobacter aerogenes and Klebsiella pneumoniae, have been isolated as functional replication origins in Escherichia coli. The origins in the SalI restriction fragments of 17.5 and 10.2 kilobase pairs, cloned from E. aerogenes and K. pneumoniae, respectively, were found to be between the asnA and uncB genes, as are the origins of the E. coli and Salmonella typhimurium chromosomes. Plasmids containing oriC from E aerogenes, K. pneumoniae, and S. typhimurium replicate in the E. coli cell-free enzyme system (Fuller, et al., Proc. Natl. Acad. Sci. U.S.A. 78:7370--7374, 1981), and this replication is dependent on dnaA protein activity. These SalI fragments from E. aerogenes and K. pneumoniae carry a region which is lethal to E. coli when many copies are present. We show that this region is also carried on the E. coli 9.0-kilobase-pair EcoRI restriction fragment containing oriC. The F0 genes of the atp or unc operon, when linked to the unc operon promoter, are apparently responsible for the lethality.     

Comparative analysis of virulence genes, genetic diversity, and phylogeny of commensal and enterotoxigenic Escherichia coli isolates from weaned pigs

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (alpha-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.     

Common evolutionary origin of chromosomal beta-lactamase genes in enterobacteria

A 32P-labeled fragment of DNA, encoding the major part of the chromosomal ampC beta-lactamase gene of Escherichia coli K-12, was used as a hybridization probe for homologous DNA sequences in colonies of Neisseria gonorrhoeae, Pseudomonas aeruginosa, and different enterobacterial species. The ampC probe detected the presence of homologous DNA sequences in clinical isolates of E. coli, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, Salmonella typhimurium, Serratia marcescens, and P. aeruginosa. No hybridization was found with N. gonorrhoeae colonies. In Southern blotting experiments the ampC probe hybridized to chromosomal DNA fragments of the same size in all enterobacterial species tested. However, the degree of hybridization differed with DNA from different species. DNA from the Shigella species strongly hybridized to the ampC probe. Furthermore, antibodies raised against purified E. coli K-12 ampC beta-lactamase precipitated beta-lactamases from the Shigella species, suggesting extensive sequence similarities between the ampC genes of these genera. The production of chromosomal beta-lactamase in S. sonnei increased with increasing growth rate similar to E. coli K-12. This growth rate response was abolished in two beta-lactamase-hyperproducing S. sonnei mutants, which thus seem similar to E. coli K-12 attenuator mutants. We propose that both the structure and regulation of the chromosomal beta-lactamase genes are very similar in E. coli and in S. sonnei.     

Amikacin resistance of clinical strains of Enterobacteriaceae and Pseudomonas]

Amikacin resistance was studied in 380 bacterial strains of Enterobacter, Klebsiella, Serratia, Pseudomonas and E. coli isolated in clinics of the Moscow Region. It was shown that 69 isolates were resistant to amikacin. Plasmid DNA was detected in 10 amikacin resistant isolates. Three of them belonging to Klebsiella and 3 belonging to E. coli contained plasmids controlling resistance to amikacin. The plasmids isolated from the strains of Klebsiella determined as well resistance to kanamycin and streptomycin but did not control resistance to sisomicin, tobramycin and gentamicin while the plasmids isolated from the strains of E. coli determined resistance to amikacin, kanamycin, gentamicin, tobramycin and sisomicin.     

A novel integrative and conjugative element (ICE) of Escherichia coli: the putative progenitor of the Yersinia high-pathogenicity island

Diversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high-pathogenicity island (HPI) carrying genes involved in siderophore-mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA-processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4-like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.     

Characteristics of E.coli O157 strains isolated in Poland from clinical material and food samples

E. coli belonging to the O157 serological group are among the organisms isolated most frequently out of all the so called entero-hemorrhagic E. coli strains (EHEC). Since several years they have been isolated also in Poland. The purpose of the present study was determination on selected phenotypic and genotypic properties of E. coli O157 strains isolated in our country from clinical material samples and from food. The serotype of the strains was determined, together with the following properties regarded as pathogenicity markers of verotoxic E. coli strains such as absence of beta-glucuronidase activity and sorbitol fermentation ability, as well as production of verotoxins SLT I and/or SLT II and entero-hemolysin. Besides that, by the PCR method the fragments of the genes coding for verotoxins, intimin and enterohaemolysin were amplified. The products of PCR were analysed by the restriction enzyme analysis (RFLP). All verotoxic E. coli O157 strains isolated in Poland were analysed by the pulsed field gel electrophoresis of genomic DNA (PFGE). The studied group comprised E. coli O157 strains, among them 40 strains were isolated from human faeces and 5 from food. The remaining strains were the reference E. coli O157:H7 EDL 933 and G 5244 strains and strains from NIH collection. The obtained results showed that the tested strains were a very varying population. 21 of them (all isolated from food, 11 from faeces and 5 reference strains) belonged to serotype O157:H7, five were not peritrichous O157:NM and the remaining ones had other ciliary antigen than H7. All strains isolated from food, reference strains and only 3 O157:NM strains isolated from humans were verotoxic. The strains from food and two reference strains produced only SLT II, 2 of 3 strains isolated from humans and one reference strain also produced only SLT II and the other produced both verotoxins. Apart from these 13 verotoxic strains all remaining strains caused sorbitol fermentation.     

Genetic and phenotypic diversity of plant growth promoting rhizobacteria isolated from sugarcane plants growing in pakistan

Bacteria were isolated from roots of sugarcane varieties grown in the fields of Punjab. They were identified by using API20E/NE bacterial identification kits and from sequences of 16S rRNA and amplicons of the cpn60 gene. The majority of bacteria were found to belong to the genera of Enterobacter, Pseudomonas, and Klebsiella, but members of genera Azospirillum, Rhizobium, Rahnella, Delftia, Caulobacter, Pannonibacter, Xanthomonas, and Stenotrophomonas were also found. The community, however, was dominated by members of the Pseudomonadaceae and Enterobacteriaceae, as representatives of these genera were found in samples from every variety and location examined. All isolates were tested for the presence of five enzymes and seven factors known to be associated with plant growth promotion. Ten isolates showed lipase activity and eight were positive for protease activity. Cellulase, chitinase, and pectinase were not detected in any strain. Nine strains showed nitrogen fixing ability (acetylene reduction assay) and 26 were capable of solubilizing phosphate. In the presence of 100 mg/l tryptophan, all strains except one produced indole acetic acid in the growth medium. All isolates were positive for ACC deaminase activity. Six strains produced homoserine lactones and three produced HCN and hexamate type siderophores. One isolate was capable of inhibiting the growth of 24 pathogenic fungal strains of Colletotrichum, Fusarium, Pythium, and Rhizoctonia spp. In tests of their abilities to grow under a range of temperature, pH, and NaCl concentrations, all isolates grew well on plates with 3% NaCl and most of them grew well at 4 to 41degrees C and at pH 11.     

Occurrence of Escherichia coli O157 in feces of healthy persons, from selected groups, in the country

The purpose of the study was determination of the occurrence of E. coli O157 in faeces samples of healthy subjects and characterization of the isolated strains with respect to their potential pathogenicity. The study was carried out in two stages. In the first one in 5 sanitary-epidemiological stations samples were tested from healthy subjects after inoculation onto McConkey (MC) or/and McConkey with sorbitol (SMC) media and isolating from each culture 10 lactose-positive (on MC medium) or sorbitol-negative (on SMC) colonies. Then latex test was done with each isolate for E. coli O157 presence. In all, 1005 samples were studied, including 260 taken from children aged 0-2 years, 180 samples from children aged 3-10 years, and 565 samples from older children and adults. E. coli O157 rods were cultured from 6 adults (0.6%). In the second stage carried out at the Laboratory of Enterobacteriaceae, Bacteriology Department, National Institute of Hygiene strains obtained from territorial laboratories were studied determining their phenotypic and genotypic traits regarded as virulence markers of verotoxic E. coli O157 strains, such as inability to ferment sorbitol and MUG breakdown, and production of verotoxins and enterohaemolysin. By the PCR method fragments were sought of genes coding for production of verotoxins, intimin and enterohaemolysin. The results showed that no E. coli O157 strain obtained from healthy individuals produced verotoxins, but three studied strains contained the eae gene determining intimin production and they were regarded as enteropathogenic.