Stb Genes Research Articles

Antibiotic resistance of enterotoxigenic and entroaggrigative Escherichia coli isolated from gastroenteritis cases

Background: Toxigenic strains of Escherichia coli are common enteric pathogens of human. The aim of this study was to detect virulence genes and antibiotic resistance pattern of the enterotoxigenic and enteroaggrigative E. coli isolated from diarhoeal stool samples. Methods: Totally, 234 diarrhoeal stool samples were collected. Microbiological examinations were done to detect the E. coli. PCR was used to identify Lt, Sta, Stb and East1 genes. Antibiotic resistance test was performed using the Disk diffusion method. Results: Out of 114 isolated E. coli, 15(13.5%) harbored Stb, 52(45.61%) East1, 30 (26.31%) Lt gene. The Sta gene was not detected in tested samples. The lowest resistance was for gentamicin (0%) while the highest resistance was for trimethoprim (79.8%). Resistance of E. coli isolates to chloramphenicol, cephotaxime, sulphametoxazole, ciprofloxacin, ampicillin and tetracycline were 3.5%, 7.01%, 71.05%, 10.5%, 52.63% and 3.5%, respectively. Conclusions: Toxigenic E. coli strains perticipated in diarrhoea in Shahrekord- Iran. The high presences of antibiotic resistance have been shown to trimethoprim, sulphametoxazole and ampicillin.

Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic Escherichia coli strain that expresses the same adhesion and enterotoxins

Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88+E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LTR192G) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LTR192G-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88+/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12–24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin.

The Distribution ofEscherichia coliSerovars, Virulence Genes, Gene Association and Combinations and Virulence Genes Encoding Serotypes in PathogenicE. coliRecovered from Diarrhoeic Calves, Sheep and Goat

Ruminants, especially cattle, have been implicated as a principal reservoir of one of the enterovirulent Escherichia coli pathotypes. The detection of the virulence genes in diarrhoeic calves and small ruminants has not been studied in Egypt. To determine the occurrence, serotypes and the virulence gene markers, stx1, stx2, hylA, Flic(h7) , stb, F41, K99, sta, F17, LT-I, LT-II and eae, rectal swabs were taken from diarrhoeic calves, sheep and goats and subjected to bacterial culture and PCR. The E.coli prevalence rate in the diarrhoeic animals was 63.6% in calves, 27.3% in goat and 9.1% in sheep. The 102 E.coli strains isolated from the calves, goat and sheep were 100% haemolytic non-verotoxic and fitted into the Eagg group. The isolates belonged to seven O serogroups (O25, O78, O86, O119, O158, O164 and O157). The eae gene was detected in six of the strains isolated from the calves. The 102 bovine, ovine and caprine E.coli strains isolated in this study were negative for stx1, stx2, F41, LT-I and Flic(h7) genes. The highest gene combinations were found to occur in the form of 24/102 isolates (23.5%) that carried the F17 gene predominantly associated with eaeA, hylA, K99 and Stb genes in the calves, while the hylA, K99 and Sta were the only genes found to be in conjunction in both calves and goats (6/102; 5.9% each). Our data show that in Egypt, large and small ruminants could be a potential source of infection in humans.

Genetic analysis of resistance to septoria tritici blotch in the French winter wheat cultivars Balance and Apache

The ascomycete Mycosphaerella graminicola is the causal agent of septoria tritici blotch (STB), one of the most destructive foliar diseases of bread and durum wheat globally, particularly in temperate humid areas. A screening of the French bread wheat cultivars Apache and Balance with 30 M. graminicola isolates revealed a pattern of resistant responses that suggested the presence of new genes for STB resistance. Quantitative trait loci (QTL) analysis of a doubled haploid (DH) population with five M. graminicola isolates in the seedling stage identified four QTLs on chromosomes 3AS, 1BS, 6DS and 7DS, and occasionally on 7DL. The QTL on chromosome 6DS flanked by SSR markers Xgpw5176 and Xgpw3087 is a novel QTL that now can be designated as Stb18. The QTLs on chromosomes 3AS and 1BS most likely represent Stb6 and Stb11, respectively, and the QTLs on chromosome 7DS are most probably identical with Stb4 and Stb5. However, the QTL identified on chromosome 7DL is expected to be a new Stb gene that still needs further characterization. Multiple isolates were used and show that not all isolates identify all QTLs, which clearly demonstrates the specificity in the M. graminicola–wheat pathosystem. QTL analyses were performed with various disease parameters. The development of asexual fructifications (pycnidia) in the characteristic necrotic blotches of STB, designated as parameter P, identified the maximum number of QTLs. All other parameters identified fewer but not different QTLs. The segregation of multiple QTLs in the Apache/Balance DH population enabled the identification of DH lines with single QTLs and multiple QTL combinations. Analyses of the marker data of these DH lines clearly demonstrated the positive effect of pyramiding QTLs to broaden resistance spectra as well as epistatic and additive interactions between these QTLs. Phenotyping of the Apache/Balance DH population in the field confirmed the presence of the QTLs that were identified in the seedling stage, but Stb18 was inconsistently expressed and might be particularly effective in young plants. In contrast, an additional QTL for STB resistance was identified on chromosome 2DS that is exclusively and consistently expressed in mature plants over locations and time, but it was also strongly related with earliness, tallness as well as resistance to Fusarium head blight. Although to date no Stb gene has been reported on chromosome 2D, the data provide evidence that this QTL is only indirectly related to STB resistance. This study shows that detailed genetic analysis of contemporary commercial bread wheat cultivars can unveil novel Stb genes that can be readily applied in marker-assisted breeding programs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-011-1623-7) contains supplementary material, which is available to authorized users.

Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs

This experiment was conducted to study the prevalence of Salmonella and Escherichia coli ( E. coli). from the surface of egg shells, egg shell membranes or pores, and internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Egg shell swabs, shell crush and egg internal contents (yolk and albumen) of an individual egg were processed for bacteriological examination. Salmonella spp. were not detected from any of the egg shell surfaces, egg shell crush or egg internal contents. Thirty five E. coli isolates were isolated from the egg shell surface. Ten E. coli strains were also isolated from shell crush. However, the internal contents of eggs appeared to be sterile. Polymerase chain reaction was performed on forty-five E. coli isolates using primers for heat stable enterotoxin genes A and B ( STa and STb) and also for colicin V gene ( cvaC). STa gene was detected in four E. coli isolates isolated from egg shell surfaces. All the E. coli isolates were negative for STb and cvaC genes. These data provide useful information regarding the prevalence of virulent E. coli and Salmonella spp. on and in unwashed eggs collected from layer farms. These data also suggest that unwashed eggs collected from caged layer farms are unlikely to be sources of Salmonella outbreaks. Egg shell translucency could be due to changes in the mammillary layer and mamillary cores during the early phases of egg shell formation and has the potential to increase the incidence of microcracks in egg shells, and hence, may be responsible for bacterial penetration. There was a significant correlation between egg shell translucency and egg shell penetration by Salmonella Infantis and E coli. Both strains of bacteria were able to penetrate the translucent egg shells even at very low doses. The penetration, however, was hindered in both translucent and non translucent eggs at 4 °C, as compared with room temperature which highlights the importance of storage of eggs at refrigerated temperatures.

Comparison of the effects of different nutrient media on production of heat-stable enterotoxin-b by Escherichia coli

Trypticase soy broth (TSB) has been commonly used to culture Escherichia coli strains for detection or harvest of heat-stable enterotoxin-b (STb); however, in our experience, the yields have been low. In this study, we compared STb yields resulting from growth in brain heart infusion broth (BHI) supplemented with 2% casamino acids (BHI-CA) with that from TSB. Since strains may concurrently express heat-labile enterotoxin (LT) and lincomycin has been reported to cause increased expression of LT, we also compared its effects on STb production. STb + clones had significantly higher production of STb when grown in BHI-CA compared to TSB, based on enzyme-linked immunosorbent assay (ELISA) conducted on cell-free supernatants. The superiority of BHI-CA to TSB was further tested on 17 porcine enterotoxigenic E. coli (ETEC) isolates, all positive for the STb gene ( estB) by PCR. Cell-free supernatants of 100% of the ETEC isolates were detectably positive for STb by ELISA when grown in BHI-CA, in contrast to only 10 strains positive (59%) when TSB supernatants were analyzed. For all the samples, the amounts of STb expressed in BHI-CA based on ELISA were significantly higher than those from TSB ( p ≤ 0.03). Incorporation of lincomycin at a concentration optimal for LT production into these media caused a significant decrease in STb levels, and use of both lincomycin and polymyxin B at these concentrations abolished detectable STb production altogether. We conclude that BHI-CA is a better medium for supporting STb production than is TSB. Culture in BHI-CA followed by testing by STb-ELISA is a useful combination for detecting STb production.

Prevalence of enterotoxigenic Escherichia coli virulence genes from scouring piglets in Zimbabwe

World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe. We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened in toxin positive isolates. Toxin positive strains lacking tested fimbriae genes were characterized using transmission electron microscopy, agglutination and agglutination inhibition tests. Approximately 32% of the 1,984 isolates tested positive for STa, STb, LT or Stx-2e genes. Of these, approximately 81% had F4, F5, F6, F18 or F41 fimbriae genes. The remaining toxin positive strains lacked tested fimbriae genes and appeared to either express F1-like fimbriae, or lacked fimbriae. The data constitute an important framework for implementation of prevention measures, such as using relevant fimbriae-based vaccines against ETEC induced diarrhea or VTEC-induced edema.

Detection of LT and ST genes in the Escherichia coli isolated from the dogs, sheep and poultry

This study was designed to identify if the heat-labile enterotoxin (LT)-, heat-stable enterotoxin (ST)a- and STb-encoding genes are detectable in faecal samples from different healthy hosts and also on which part of the genome (chromosome or plasmid) they are located. Seventy-five samples of Escherichia coli were isolated from dog, sheep and poultry faecal samples (25 from each host). Plasmid and chromosomal DNA were extracted and polymerase chain reaction performed on all plasmid and chromosomal DNA using LT, STa and STb primers. Additionally, the plasmid profile of all E. coli isolates was defined using gel electrophoresis. The results showed that 36% of the E. coli isolates possessed genes for the production of LT toxin. All samples were negative for STa and STb genes. The plasmid profile of different hosts showed that all samples harboured plasmids. The results of this study indicate that enterotoxigenic E. coli are present in the faeces of different hosts. Since the genes encoding for LT and ST toxins reside on plasmids, or occasionally on transposons, they may transfer among the Gram-negative bacteria especially to the enterobacteriaceae family including E. coli which then may infect other hosts, for example, humans.

P1139 Presence of STa, STb and LT enterotoxin genes in Klebsiella strains isolated in different hospitals from gastrointestinal tracts

P1139 Presence of STa, STb and LT enterotoxin genes in Klebsiella strains isolated in different hospitals from gastrointestinal tracts

Prevalence of virulence genes in Escherichia coli strains recently isolated from young pigs with diarrhea in the US

Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning diarrhea (PWD) is economically one of the most important diseases for the swine industry. Porcine ETEC strains typically express K88 or F18 fimbria and heat-labile (LT) and/or heat-stable (STa, STb) enterotoxins. However, recent studies indicate that EAST1 toxin, adhesin involved in diffuse adherence (AIDA-I) and porcine attaching and effacing-associated factor (paa) may also be expressed by ETEC strains associated with diarrhea. To better understand the virulence factors of E. coli strains that cause PWD, we applied PCR to screen for K88, F18, F41, 987P and K99 fimbrial genes; LT, STa, STb, Stx2e and EAST1 toxic genes; and AIDA-I, paa and EAE adhesin genes in E. coli strains recently isolated from young pigs with PWD in the US. Of 304 E. coli isolates from diarrheic pigs submitted for testing, 175 (57.6%) strains possessed fimbrial genes: K88 (64.6%), F18 (34.3%), F41 (0.57%), K99 (0.57%), 987P (0); toxin genes: LT (57.7%), STb (72.6%), STa (27.4%), STx2e (17.4%), EAST1 (35%); and adhesin genes: AIDA-I (26.9%), paa (60%), EAE (1.1%). All toxin genes except the EAST1 toxin gene, were almost exclusively associated with K88+ or F18+ isolates, and most of these isolates carried multiple toxin genes. The non-fimbrial adhesin paa was found present in over half of the K88+ isolates. A total of 129 (42%) isolates carried no fimbrial genes, including 66 (21.7%) isolates that did not have any of the above virulence genes. These results suggest a broad array of virulence genes associated with PWD in pigs.

Detection of a plasmid-encoded pathogenicity island in F18+ enterotoxigenic and verotoxigenic Escherichia coli from weaned pigs

AbstractMost virulence genes of enterotoxigenic Escherichia coli (ETEC) are located on plasmids. The gene for heat-stable enterotoxin I (sta) is part of the transposon Tn1681, and the heat-stable enterotoxin II (stb) gene was described to be part of the transposon Tn4521. In the studies presented here, we describe the linkage of the sta and stb genes on an approximately 10-kb fragment designated as toxin-specific locus (TSL). The TSL has been isolated from the 120-kb virulence plasmid pTC of the porcine ETEC strain 2173 that produces F18 fimbriae. The nucleotide sequence of the TSL fragment was determined. Sequences in the flanking regions of the sta gene indicated the presence of Tn1681, but – unexpectedly – flanking sequences of the neighbouring stb gene were completely different from those of Tn4521. The 10-kb TSL is part of a 40-kb fragment that contains the replication origin of pTC. This 40-kb fragment was mobilised into plasmid pACYC177 and the nucleotide sequence of the bordering fragments was determined. The 40-kb fragment was flanked by IS10 elements at both ends, indicating the existence of a new 40-kb pathogenicity island (PAI) in strain 2173. Several F4(K88)+ETEC and F18+ETEC as well as F18+E. coli strains producing enterotoxins and verotoxin-2 (ETEC/VTEC) from weaned pigs of different geographical origin were tested for the flanking regions by PCR to see if they belong to the “Tn4521-like” or the “pTC-like”stb type. It turned out that the Tn4521-like stb-type was characteristic of F4(K88)+ETEC, while the pTC-like stb type was present in most F18+ETEC and F18+ETEC/VTEC, although polymorphism was observed both in the K88 and F18 groups. These results suggest the existence and worldwide spread of a new plasmid-encoded pathogenicity island in porcine post weaning ETEC and ETEC/VTEC strains producing F18 fimbriae.

Expression of heat-stable enterotoxin STb by adherent Escherichia coli is not sufficient to cause severe diarrhea in neonatal pigs.

The role of Escherichia coli heat-stable enterotoxin B (STb) in neonatal porcine diarrhea caused by enterotoxigenic E. coli was examined by comparing adherent isogenic strains with or without STb. The cloned STb gene (in the plasmid pRAS1) was electroporated into a nonenterotoxigenic strain (226M) which expresses the F41 adhesin. Strain 226M pRAS1 adhered and expressed STb in vivo, causing fluid secretion in ligated ileal loops in neonatal pigs. Although strain 226M pRAS1 caused very mild diarrhea in some orally inoculated neonatal pigs, the weight loss in these pigs was similar to that caused by the parental strain without STb. We conclude that STb does not significantly contribute to diarrhea caused by enterotoxigenic E. coli in neonatal pigs.

Identification of the Saccharomyces cerevisiae genes STB1-STB5 encoding Sin3p binding proteins.

The yeast SIN3 gene functions as a transcriptional repressor, despite the fact that Sin3p does not bind DNA directly. We have conducted a two-hybrid screen to look for proteins that interact with Sin3p, using the PAH2 domain of Sin3p as bait. Five new genes, STB1-STB5 were identified, as well as the STB6 gene, which is similar to STB2. STB1, STB2, STB3, and STB6 are novel genes, and STB4 and STB5 encode C6 zinc cluster DNA-binding proteins. None of these genes is essential for viability, and several of these genes may encode transcriptional activators. Several special problems were encountered in using a transcriptional repressor in a two-hybrid screen. For example, the STB genes will interact with a LexA-Sin3(PAH2) fusion protein containing a region of Sin3p, but a LexA-Sin3p fusion protein containing full-length Sin3p, along with a STB clone, does not produce two-hybrid activation of a transcriptional reporter. In addition, a sin3 mutation reduces the transcriptional activation by two-hybrid partners, suggesting that a sin3 mutation reduces the transcriptional efficiency of the Gal4p and VP16 activation domains. We have shown previously that Sin3p is part of a large multiprotein complex, and we show here that Stb1p and Stb2p are present in this complex.

Characterization of Escherichia coli isolated from foals

Serotype, biotype, antibiogram, hemolysin production, fimbrial hemagglutinins, select toxin genes (STb, STaP, LT, slt1 and slt2) and the attaching effacing ( eae) gene were determined for 99 foal strains of E. coli. E. coli from diarrheic and normal foals could not be distinguished by serotype, biotype, or antibiogram. Differences ( P ≤ 0.05) were observed in hemolysin production (11.5% vs. 0%) and the expression of mannose-resistant hemagglutinins (23% vs. 13%) among E. coli from diarrheic and healthy foals, respectively. Three of the E. coli strains from diarrheic foals were positive with probes for slt genes and one was positive for STb and LT genes. One strain from a healthy foal possessed the STb gene. As determined by the polymerase chain reaction, 8 strains possessed the eae gene. Seven of the 8 strains were from diarrheic foals and one eae-positive strain was from a healthy foal. The slt-positive strains did not possess eae genes and the eae-positive strains did not possess slt genes. These results indicate that enterotoxigenic strains of E. coli are not implicated in any substantial degree in sporadic foal diarrhea. However, the identification of slt-positive and eae-positive strains in foal feces indicate the presence of potentially virulent strains among foals.

The detection of K88, K99 fimbrial antigen and enterotoxin genes of Escherichia coli isolated from piglets and calves with diarrhoea in Indonesia

Enterotoxigenic Escherichia coli (ETEC) strains cause diarrhoeal disease in piglets and calves in Indonesia. These strains possess two virulence factors namely attachment and enterotoxin antigens . These factors could be detected phenotypically and genetically. Haemolytic Escherichia coli (E coli) isolates possessing K88 fimbrial antigen associated with 0-group 108 and 149. They were positive for K88 gene and demonstrated their ability to produce heat labile enterotoxin (LT) and genetically were all positive for LT gene . Seventeen isolates ofE coli K88 which associated with 0-group 149 were positive forSTb gene, other O-serotypes were negative . Ten isolates of Ecoli K88 which associated with 0-group 108 possessed K88, K99, LT and STa genes, but negative for STb gene . However, phenotypically the K99 antigen and STa toxin were not expressed under laboratory conditions, the reason was not well understood . E. coli K99 strains isolated from calves wit h diarrhoea were all associated with 0-group 9 and produced STa toxin when tested by suckling mousse bioassay. The E. coli K99 calf isolates were all hybridized with K99 and STa gene only . It is likely that K99 gene is associated with STa gene . The DNA hybridization technique is more convenience to be used for confirmation diagnosis of colibacillosis, however, not all veterinary laboratories could perform these tests . Keywords: Excherichia coli, probe, gene detection, virulence determinant

Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity

The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59. Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity.

Evaluation of three new techniques for the detection of STb-positive Escherichia coli strains

A study was conducted to compare different techniques for the detection of heat-stable enterotoxin b (STb)-positive E. coli strains. Antisera against purified STb was used to develop an enzyme-linked immunosorbent assay (ELISA). STb-positive strains identified by ELISA were tested for bioactivity in rat jejunal loops. Our ELISA was as sensitive as, but less specific than, the bioassay for detection of STb-positive strains. A non-radioactive DNA probe to detect the gene coding for STb was also developed by incorporating digoxigenin-11-dUTP into DNA by the random primed labelling technique. The non-radioactive digoxigenin-labelled DNA probe demonstrated a similar detectability to the radioactive probe and was more convenient to manipulate but was less sensitive and specific than the bioassay and the radioactive probe. In addition, the polymerase chain reaction (PCR) was used to amplify a specific portion of the gene coding for STb. The PCR was a highly specific and practical technique for the detection of STb-positive strains. All E. coli strains tested containing the STb gene produced the STb toxin.

Detection of genes for fimbrial antigens and enterotoxins associated with Escherichia coli serogroups isolated from pigs with diarrhea

A total of 1,226 Escherichia coli strains isolated from 1979 to 1989 from pigs with diarrhea were examined for serogroup and fimbrial antigen F4 (K88) production. Four main patterns of isolation of the various serogroups were observed, depending on the ages of the pigs from which isolates were obtained and the production of F4. In pattern I, serogroups O8:K"S16", O9:K35, O9/O101:K30, O9/O101:K103, O9 (group), O20:K101, and O64:K"V142" were predominant in pigs aged 0 to 6 days (41.9% of isolates) and were less frequent in pigs aged 7 to 27 days (24.6% of isolates) but were rarely found in pigs aged 28 to 60 days (4.0% of isolates). In pattern II, the F4-associated serogroups O8:K"4627", O157:K"V17", O149:K91, and O147:K89 were predominant in pigs aged 7 to 27 days (29.8% of isolates) and in pigs aged 28 to 60 days (35.0% of isolates). In pattern III, serogroups O8 (group), O115:K"V165", and O147:K89 were rarely isolated from pigs aged 0 to 6 days but were equally distributed in pigs aged 7 to 27 days (10.1% of isolates) and in pigs aged 28 to 60 days (10.9% of isolates). In pattern IV, serogroups O138:K81, O139:K82, O141:K85ac, O45:K"E65", and O26:K60 were most frequently isolated in pigs aged 28 to 60 days (19.3% isolates). Over the period from 1979 to 1989, the proportion of isolates belonging to serogroups of pattern II and the proportion of F4 isolates within the serogroup O157:K"V17" declined, whereas the proportion of isolates of serogroups O147:K89, O8:K"S16", and O9:K35 increased. For 228 isolates selected from the most important serogroups, good agreement was observed between the results of gene probes and immunofluorescence for the detection of fimbrial antigens F4 (K88), F5 (K99), F6 (987P), and F41 and between the results of gene probes and biological assays for the detection of heat-labile enterotoxin (LT) and heat-stable enterotoxins a and b (STa and STb). The STa gene was mostly associated with isolates of pattern I serogroups, which had the F5, F6, and F41 genes alone or in various combinations. The LT and/or STb genes, with the F4 gene, mostly were observed in isolates of pattern II serogroups. The STb gene alone was observed mostly in isolates of pattern III serogroups, although isolates were negative for all fimbrial antigen genes. Similarly, isolates of pattern IV serogroups were negative for all fimbrial antigen genes and rarely positive for the enterotoxin genes. However, verotoxin production was associated with isolates of serogroups O138:K81 and O139:K82. The most important pathotypes among enterotoxigenic isolates in this study were F4:LT:STb, F5:STa, STb, F5:F41:STa, F4:STb, F6, STa, and LT.

Expression of the cloned gene for enterotoxin STb of Escherichia coli

This study involved the construction of hybrid plasmids to produce heat-stable enterotoxin type II of Escherichia coli (STb). The translation of the open reading frame for the STb gene estA was demonstrated in several ways. Studies using in vivo labeling with [35S]cysteine demonstrated a radiolabeled protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expected molecular weight of 5,000 for toxin STb. Insertion of translational or transcriptional termination signals into the BglII site of the estA gene blocked the expression of estA. The estA gene was cloned into high-expression vector pKC30 downstream from the strong pL promoter. Northern (RNA) blotting assays revealed a 10- to 20-fold increase in mRNA produced by strain C600F(pKC30STb) over other STb-producing strains, compared with little or no increase in enterotoxin activity demonstrated by bioassay. The estA gene, with its own promoter and Shine-Delgarno region and a portion of the sequence for the signal peptide deleted, was also inserted under the control of the tac promoter. Even after induction of the tac promoter by addition of isopropyl-beta-D-thiogalactopyranoside, no biologic enterotoxin activity could be identified. Neutralizing antibodies to STb were produced in rabbits by using either a purified OmpF-STb-beta-galactosidase fusion protein or a 19-amino-acid synthetic STb peptide coupled to keyhole limpet hemocyanin.

Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli serotype O8:KX105 and O8:K"2829" strains isolated from piglets with diarrhea.

Twelve pathogenic and seven nonpathogenic enterotoxigenic Escherichia coli strains which were previously identified as belonging to serogroup O8:KX105 (A. Broes, J. M. Fairbrother, S. Larivière, M. Jacques, and W. M. Johnson, Infect. Immun. 56:241-246, 1988) were further examined for their phenotypic and genotypic properties. Only the 12 pathogenic strains were confirmed to possess the capsular antigen KX105. The seven nonpathogenic strains did not possess this antigen and thus were incorrectly assigned to have capsular antigen KX105. All seven nonpathogenic strains apparently possessed a previously unrecognized capsular antigen which has been designated K"2829". Studies with antisera prepared against F1 (type 1) fimbriae from three E. coli strains suggested that at least three antigenic subtypes of F1 fimbriae were represented among the O8:KX105 strains examined. By using serotyping, biotyping, and outer membrane protein profile analyses, the O8:KX105 strains were divided into at least two distinct clusters, whereas the O8:K"2829" strains were grouped into a single unique cluster. Most of the strains of the same cluster were further differentiated by testing for antibiotic resistance and colicin production and resistance and by analysis of plasmid content. With the exception of one strain which lost its enterotoxicity during storage, all of the O8:KX105 strains hybridized with the gene probes for the heat-labile (LT) and heat-stable (STb) enterotoxins. For each O8:KX105 strain, a single plasmid ranging in size from 61 to 77 megadaltons carried the LT and STb genes. All of the enterotoxigenic O8: KX105 strains fermented sorbose, whereas the nonenterotoxigenic strain did not. All of the O8:K "2829" strains hybridized with the STb probe only. For each O8:K "2829" strain, the STb genes were located on a single plasmid of 61 or 22 megadaltons. None of the strains demonstrated homology with the genes encoding the F4 (K88), F5 (K99), F6 (987P), and F41 fimbrial antigens and STaP and STaH.