Development of a rapid immunochromatographic test to identify enteropathogenic and enterohemorrhagic Escherichia coli by detecting EspB.

A rapid immunochromatographic (IC) test to identify enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) was developed to detect EspB secreted by the type III secretion system of these bacteria. The detection limit of the test system was 4 ng/mL. All 33 of 34 strains harboring the eae gene encoding intimin were positive in the IC test, and all 40 of the eae-negative strains were negative. The results showed that the sensitivity was 96.9% and specificity was 100%. The IC test also detected EspB in a stool sample artificially supplemented with 60 ng EspB/mL. The IC test for the detection of EspB may be a practical method to define EPEC or EHEC both in clinical laboratories and the field.

The long polar fimbriae genes identified in Shiga toxin-producing Escherichia coli are present in other diarrheagenic E. coli and in the standard E. coli collection of reference (ECOR) strains.

Long polar fimbriae (LPF) are related to type I fimbriae in genetic organization and were first identified in Salmonella enterica serovar Typhimurium. Four lpfA genetic variants designated lpfA(O157/OI-141), lpfA(O157/OI-154), lpfA(O26) and lpfA(O113) have been identified in Shiga toxin-producing Escherichia coli (STEC). In this study, PCR was employed to determine the distribution of STEC-lpfAs in enteropathogenic, enteroaggregative, enterotoxigenic and enteroinvasive E. coli (EPEC, EAEC, ETEC and EIEC) and in the standard E. coli collection of reference (ECOR). Among the 97 diarrheagenic strains from our collection, only 2 EPEC strains of serotypes O55:H7 and O119:NM were positive for both lpfA(O157/OI-141) and lpfA(O157/OI-154). lpfA(O157/OI-141) was also positive in 1 of 25 ETEC strains. lpfA(O113) was present in 51 of 97 strains and lpfA(O26) in 13 of 97 strains of diverse diarrheagenic categories. STEC-lpfAs were also present in non-pathogenic ECOR strains of all phylogenetic groups. This study showed that the lpfA genes identified in the genome of STEC strains are not specific to this category. Our results suggest that there is a relationship between the lpfA variant and the phylogenetic group.

Characterization of Salmonella isolated in Okinawa, Japan.

Salmonella enterica strains isolated in Okinawa between 1995 and 2005 were analyzed with respect to their serovars and antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) was used to examine their digestion patterns. A total of 1,071 isolates, including 610 from humans, 358 from animal rectal swabs and 103 from meat obtained at grocery stores, were examined. The first 3 most frequent serovars in human isolates were Enteritidis, Weltevreden and Bareilly, together accounting for 65% of the isolates. In isolates from the rectal swabs of laying hens, the predominant serovars were Albany, Saintpaul and Aarhus, accounting for 82% of the isolates. In broilers, 123 of 124 isolates belonged to serovar Infantis, which reflected the high ratio of this serovar in the chicken sold at grocery stores. An antibiogram of human isolates was different from that of broilers and chicken. Chromosomal DNAs of S. Infantis isolated from humans and from the rectal swab of broilers and chickens were examined by PFGE using the restriction enzymes XbaI and BlnI. The digestion patterns of human isolates were not coincident with those of the isolates from the rectal swab of broilers and chicken-meat samples.

Short report: isolation of Escherichia coli O157:H7 from fecal samples of cows in Vietnam.

Enterohemorrhagic Escherichia coli O157:H7 was isolated for the first time in Vietnam. Shiga toxin-producing E. coli were isolated from 8 of 100 cows examined. The two strains showing serotype O157:H7 carried the eae, ehxA, and stx2c genes, but the other six were negative for the eae gene.

Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method.

Here we report a loop-mediated isothermal amplification (LAMP) method for detecting Shigella and enteroinvasive Escherichia coli. The target for this LAMP method is the ipaH gene which is carried by both of the pathogens. The LAMP method efficiently detected the gene within 2 h at a minimal amount of bacteria (8 CFU) per reaction.

Pathogenicity of Shigella in healthy carriers: a study in Vientiane, Lao People's Democratic Republic.

Shigella spp. isolated from diarrheal patients and non-diarrheal carriers were examined by PCR for the presence of two pathogenic genes, chromosomal ipaH and invasive plasmid encoded ial. Shigella spp. were detected in 7 of 72 diarrheal cases examined (9.7%), and 9 of 145 non-diarrheal cases (6.2%). All isolates from diarrheal cases harbored both ipaH and ial, while all isolates from non-diarrheal cases were positive for ipaH but not ial. These results suggested that Shigella spp. in healthy carriers were basically non-pathogenic.

The relationship between O-antigens and pathogenic genes of diarrhea-associated Escherichia coli.

To evaluate the serogrouping-based diagnosis of diarrheagenic Escherichia coli, a total of 1,130 strains of E. coli isolated in several countries were studied. The strains were regarded as enterovirulent on the basis of their O-antigens determined using a commercially available kit containing 43 antisera, and the presence of diarrhea-associated genes (eae, stx, aggR, est, elt, ipaH) was evaluated by PCR. Two hundred sixty-three strains of 1,130 (23.3%) were identified as diarrheagenic based on the presence of at least one pathogenic gene. The probability that E. coli identified as diarrheagenic on the basis of serogrouping actually possessed some pathogenic gene was highest for serogroup O119 (78.4%); other serogroups with a positive rate for pathogenic genes higher than 60% were O111 and O126. No target genes were detected among the strains belonging to serogroups O1, O29, O112ac, O143, O158 and O168. Our results suggest that, in practice, serogrouping is useful for the identification of diarrheagenic E. coli in a very limited number of serogroups.

[Symptoms of food-borne diseases and gastroenteritis in Kyushu, Japan].

In this study we analyzed the symptoms of gastroenteritis or food-borne disease caused by the 10 most prevalent pathogens: Norovirus, Salmonella, Vibrio parahaemolyticus, Campylobacter jejuni, Clostridium perfringens, Shiga toxin-producing Escherichia coli (STEC), enterotoxigenic E. coli (ETEC), Shigella sonnei/flexneri (Shigella), Staphylococcus aureus, and emetic-type Bacillus cereus. The symptoms diarrhea, vomiting, fever, abdominal pain, and headache, and the incubation period in 646 cases in 10 districts of Kyushu between January 2000 and December 2004 were recorded. The pathogen with the shortest mean incubation period was B. cereus (0.8 h), and was followed by S. aureus (3.3 h), C. perfringens (10.7 h) and V. parahaemolyticus (16.4 h). All the patients infected with B. cereus and S. aureus developed symptoms within 6 hours, and those infected with V. parahaemolyticus and C. perfringens developed symptoms within 24 hours. Bloody diarrhea was associated with STEC and Shigella, but rare with other pathogens. Vomiting was associated with almost all cases of S. aureus and B. cereus infection, and occurred in 71.5% of the Norovirus cases and 56.1% of the V. parahaemolyticus cases. Vomiting was less common in the C. perfringens (22.0%) and the ETEC and STEC (both about 5%). Bloody diarrhea, abdominal pain, and vomiting were statistically significantly more common with STEC 0157 infection than with STEC non-0157 infection. Since the cases analyzed in this study included all degrees of illness, mild to severe, and a wide range of ages, the information obtained will serve as a good reference material for administrative and laboratory work when an outbreak takes place.

Detection of EspB using reversed passive latex agglutination: application to determination of enteropathogenic Escherichia coli.

We developed a new practical method to identify enteropathogenic Escherichia coli (EPEC) by detecting the pathogenic factor, EspB. E. coli were cultured in Dulbecco's Modification of Eagle's Medium (DMEM), and EspB was detected in the culture supernatant by reversed passive latex agglutination (RPLA). All 63 E. coli strains harboring the eaeA gene encoding intimin were positive for RPLA, and all 25 strains without the eaeA gene were negative. Among these 63 eaeA-positive strains, 38 Shiga toxin-producing E. coli (STEC) produced Shiga toxin (Stx) under the same culture conditions (DMEM). Subtypes of EspB alpha, beta and gamma were antigenically cross-reactive to each other as determined by RPLA and Western blotting. A kit for Stx detection (RPLA) is commercially available and therefore this RPLA for detection of EspB could be a practical method to define EPEC in both clinical laboratories and the field.

Purification and characterization of a novel metalloprotease isolated from Aeromonas caviae.

A novel protease produced by Aeromonas caviae was purified and characterized. The molecular weight of the protease (AP19) was estimated as 19 kDa on SDS-polyacrylamide gel electrophoresis. The protease activity was not inhibited completely by heating at 100 degrees C for 15 min. The proteolytic activities were inhibited by metalloprotease inhibitor. The N-terminal amino acid sequence of AP19 was VTASVSFSGRCTN. AP19 did not activate Aeromonas proaerolysin, and did not show fluid accumulation in the rabbit intestinal loop test. A high concentration of the protease showed cytotoxic activity against Vero cells.

Aerolysin is activated by metalloprotease in Aeromonas veronii biovar sobria.

Aeromonas veronii is an opportunistic human pathogen that causes diarrhoea and extraintestinal infections, such as wound infection and septicaemia. An A. veronii protease (AVP) from a biovar sobria strain, AeG1, was partially purified and characterized. Mature AVP hydrolysed casein but not elastin, and protease activity was inhibited by metalloprotease inhibitors. Nucleotide sequence analysis showed that AVP belongs to the thermolysin family of proteases. An AVP-deficient mutant was constructed to investigate the role of AVP in aerolysin activation. Western blot analysis using anti-aerolysin antisera revealed that proaerolysin (52 kDa) in the AVP-deficient mutant was not completely activated to mature aerolysin (47 kDa) as seen in the wild-type strain. The AVP-deficient mutant showed lower cytotoxic and haemolytic activities than wild type. AVP and proaerolysin had no haemolytic activity; however, activity appeared after incubating both proteins. Taken together, these results suggested that AVP plays an indirect role in virulence through activating aerolysin, which is an essential step for cytotoxic activity.

Molecular analysis of VcfQ protein involved in Vibrio cholerae type IV pilus biogenesis.

The nucleotide sequence of an ORF (vcfQ) within the type IV pilus gene cluster of Vibrio cholerae O34 strain NAGV14 was determined, thereby completing the sequence analysis of the structural operon. The vcfQ gene showed homology to the mshQ gene of the mannose-sensitive haemagglutinin pilus gene cluster. The vcfQ was 651 bp larger than mshQ, and the G+C content of the extra 651 bp portion (35.6 mol%) was lower than that of the overall vcfQ gene (42.5 mol%). Except for the first 270 aa residues, the deduced amino acid sequence of VcfQ showed high homology to the MshQ protein. There was immunological cross-reaction between VcfQ and MshQ by Western blotting. Cell fractionation studies showed that VcfQ is located in both the inner and the outer membranes. Mutational analysis showed that vcfQ-deficient mutant expressed detectable levels of major pilin (VcfA), but failed to assemble them into pili, indicating that VcfQ is essential for pilus assembly. Colony-blotting analyses showed that the N-terminal region of vcfQ is variable in V. cholerae strains.

Minor pilin subunits are conserved in Vibrio cholerae type IV pili.

The nucleotide sequences of five open reading frames within the Vibrio cholerae NAGV14 type IV pilus gene cluster were determined. The genes showed high homology to the mannose-sensitive hemagglutinin (MSHA) pilus genes mshB, mshC, mshD, mshO and mshP. PCR analysis showed that a MSHA-like gene cluster is highly conserved among different V. cholerae strains, with the exception of the previously reported major pilin subunit. Recombinant MshB and MshO proteins were purified and specific antiserum was raised to each of them. Western blotting analyses showed that these antisera reacted with purified NAGV14 and MSHA pili. The results suggested that MshB and MshO are minor components of the pilus fiber. Although there was no cross-reaction between the major pilin subunits of NAGV14 and MSHA pili, minor components seemed to be highly homologous and immunologically cross-reactive.

Comparison of antimicrobial susceptibility between invasive and non-invasive Shigella organisms.

Antimicrobial susceptibility was determined for ten strains of Shigella spp. comparing invasive (invasion plasmid containing) and non-invasive members of each strain. The activity of the antimicrobial agents could be classified into three types from the differences between the minimum inhibitory concentration (MIC) for the invasive and non-invasive shigellae. For type 1, there was no difference between the MIC (an MIC gap) for invasive and non-invasive organisms. For type 2, the MIC for the invasive organisms of a strain was higher than that of non-invasive organisms of the strain. In the third type, macrolides taken in by shigellae through the type III secretion apparatus, more effectively inhibited the growth of invasive than non-invasive organisms.

The incidence of Escherichia coli having pathogenic genes for diarrhea: a study in the People's Democratic Republic of Lao.

The incidence of Escherichia coli having pathogenic genes for diarrhea was studied in Laos in 2002. A total of 525 E. coli strains from 278 patients (basically, two E. coli isolates from each patient) were examined by PCR to detect the known pathogenic genes (stx, eae, elt, est, ipaH, and aggR). These genes were detected in 23 strains from 16 patients (16/278: 5.8%). In 10 cases of the 16, one of the two isolates from each individual was negative for the gene, and in the other six cases, both isolates had the gene (same gene in four cases). E. coli having eae but no stx (enteropathogenic E. coli [EPEC]) was found in two cases out of 278 (0.7%). Nevertheless, Class I classical EPEC (serogroup-based) was found in 77 cases (28%). Enterotoxigenic E. coli, enteroaggregative E. coli, and enterohemorrhagic E. coli were found in 9, 4, and 1 cases, respectively. Enteroinvasive E. coli was not detected. This study suggested that the incidence of diarrhea due to E. coli is not as high as has been previously thought.

A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing.

Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.

Oxalate-degrading Providencia rettgeri isolated from human stools.

BACKGROUND: Oxalate-degrading bacteria are thought to metabolize intestinal oxalate and thus decrease the urinary excretion of oxalate by reducing its intestinal absorption. METHODS: We have isolated several novel oxalate-degrading bacteria from human stools. Oxalate degrading bacteria were investigated to characterize their protein profiles with antibodies against oxalyl-coenzyme A decarboxylase (65 kDa) and formyl-coenzyme A transferase (48 kDa) purified from Oxalobacter formigenes. RESULTS: One of these isolates was identified as Providencia rettgeri, which showed two proteins (65 kDa and 48 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) that were not found in non-oxalate-degrading P. rettgeri. Antibodies reacted with the 65 and 48 kDa proteins from the P. rettgeri strain on Western blotting. An Oxalobacter formigenes formyl-coenzyme A transferase gene probe reacted with chromosomal DNA from P. rettgeri on Southern blotting under high stringency conditions, while an Oxalobacter formigenes oxalyl-coenzyme A decarboxylase gene probe did not react under the same conditions. CONCLUSIONS: The mechamism of oxalate degradation by P. rettgeri appears to be similar to that of Oxalobacter formigenes. This is the first report of a facultative oxalate-degrading organism that is one of the Enterobacteriaceae.

Characterization of the outermembrane proteins of Vibrio cholerae expressed in in vivo culture.

Two outer membrane proteins (Omps) of Vibrio cholerae O1, expressed in the intestine (in vivo) but not in culture media (in vitro), were investigated. The molecular masses of those proteins were 116 kDa and 15 kDa, and they were not associated with iron-regulated proteins. Convalescent cholera patients' sera reacted with the 15 kDa protein but not with the 116 kDa protein. The N-terminal amino acid sequence of the 15 kDa protein was homologous to V. cholerae OmpT. Anti-serum to the 15 kDa protein caused agglutination of the organisms grown in the intestine, but not the organisms in in vitro culture. The anti-serum was bactericidal, but it did not inhibit the adhesion of the organisms to the intestine and HEp-2 cells. These findings suggest the possibility that the 15 kDa protein could be involved in post-infection immunity.

An Aeromonas veronii biovar sobria infection with disseminated intravascular gas production.

We report a case of Aeromonas veronii biovar sobria infection with disseminated intravascular gas production. The patient was an afebrile 15-year-old girl who had been quite healthy until the onset of the illness. She came to the hospital because of a 6-h history of increasing pain and swelling in her left thigh. On admission, no infection was suspected, and a tentative diagnosis of a ruptured left gracilis muscle was made. Because the pain increased continuously, the treatment concentrated on pain control. Unexpectedly, abrupt death occurred 23 h after her admission. Postmortem computed tomographic (CT) scans showed an abundance of gas in the blood vessels of the entire body. Postmortem investigation revealed disseminated intravascular gas production, marked intravascular hemolysis, and numerous intravascular Gram-negative bacilli in all organs examined. The organisms were identified as A. veronii biovar sobria, and were highly susceptible to third-generation cephalosporins. Regarding therapeutic problems, the early administration of these antibiotics should reduce the fatality rate in such infections. It is critical to keep the possibility of such an infection in mind when a patient complains of severe muscle pain.

Aeromonas trota strains, which agglutinate with Vibrio cholerae O139 Bengal antiserum, possess a serologically distinct fimbrial colonization factor.

Pili of Aeromonas trota strain 1220, which agglutinates with Vibrio cholerae O139 Bengal antiserum, were purified and characterized. The molecular mass of the subunit protein was estimated to be 20 kDa and the pl was 5 center dot 4. The pili were immunologically unrelated to the other Aeromonas pili reported so far. However, the N-terminal amino acid sequence of the subunit pilin was similar to those of the pilins from other Aeromonas pili reported previously. Neither A. trota cells nor pili purified from strain 1220 agglutinated human and rabbit erythrocytes, but both adhered to the rabbit intestine. Bacterial cells pretreated with antipilus antibody (Fab portion) failed to adhere to the rabbit intestine. Moreover, bacteria did not adhere to the rabbit intestine pretreated with the purified pili. This pilus antigen was not detected in V. cholerae O139 Bengal and other Aeromonas spp. These findings suggest that the pilus of the A. trota strain is a novel colonization factor of Aeromonas spp.