Association of cytolethal distending toxin-II gene-positive Escherichia coli with Escherichia albertii, an emerging enteropathogen.

Cytolethal distending toxin (CDT)-producing Escherichia coli have been isolated from patients with diarrhea, sepsis and urinary tract infection. CDT of E. coli is divided into five types (CDT-I through CDT-V) based on differences in amino acid sequences and its genomic location. However, in our recent studies, a few strains of cdt-II gene-positive bacteria, initially identified as atypical E. coli, were re-identified as Escherichia albertii, an emerging enteropathogen, by extensive characterization including multilocus sequence (MLS) analysis and sugar utilization tests. This finding prompted us to investigate if bacteria previously identified as cdt-II gene-positive E. coli might be E. albertii. In the present study, we therefore re-examined the identity of 20 cdt-II gene-positive bacteria isolated from children with diarrhea, which were initially identified as atypical E. coli. By extensive sugar utilization tests, these bacteria showed a closer relatedness to E. albertii than E. coli, because they did not ferment any of the tested sugars including dulcitol, lactose, d-melibiose, l-rhamnose and d-xylose. Further, both phylogenetic analyses based on nucleotide sequences of 7 housekeeping genes (MLS analysis) and rpoB gene showed that all the cdt-II gene-positive bacteria belonged to a distinct lineage of E. albertii from those of E. coli and Shigella boydii. They were also positive by an E. albertii-specific PCR. Taken together, these data suggest that cdt-II gene-positive bacteria previously identified as E. coli are actually E. albertii. Therefore, we suggest a new definition for cdt-II gene-positive E. coli as E. albertii with the inclusion of CDT-II in E. albertii CDT.

Molecular characterization of cytolethal distending toxin gene-positive Escherichia coli from healthy cattle and swine in Nara, Japan.

BACKGROUND: Cytolethal distending toxin (CDT)-producing Escherichia coli (CTEC) has been isolated from patients with gastrointestinal or urinary tract infection, and sepsis. However, the source of human infection remains unknown. In this study, we attempted to detect and isolate CTEC strains from fecal specimens of healthy farm animals and characterized them phenotypically and genotypically. RESULTS: By PCR analysis, the cdtB gene was detected in 90 and 14 out of 102 and 45 stool specimens of healthy cattle and swine, respectively, and none from 45 chicken samples. Subtypes of the cdtB genes (I to V) were further examined by restriction fragment length polymorphism analysis of the amplicons and by type-specific PCRs for the cdt-III and cdt-V genes. Of the 90 cdtB gene-positive cattle samples, 2 cdt-I, 25 cdt-III, 1 cdt-IV, 52 cdt-V and 1 both cdt-III and cdt-V gene-positive strains were isolated while 1 cdt-II and 6 cdt-V gene-positive were isolated from 14 cdtB positive swine samples. Serotypes of some isolates were identical to those of human isolates. Interestingly, a cdt-II gene-positive strain isolated from swine was for the first time identified as Escherichia albertii. Phylogenetic analysis grouped 87 E. coli strains into 77 phylogroup B1, 6 B2, and 4 D, respectively. Most of the B1 strains harbored both lpfAO113 and ehaA. Three and twenty-two cdt-V gene-positive strains harbored eaeA and stx genes, respectively, and seven possessed cdt-V, stx and subAB genes. The cnf2 gene, normally present in cdt-III gene-positive strains, was also detected in cdt-V gene-positive strains. CONCLUSIONS: Our results suggest that healthy cattle and swine could be the reservoir of CTEC, and they could be a potential source of human infections.

Molecular characterizations of cytolethal distending toxin produced by Providencia alcalifaciens strains isolated from patients with diarrhea.

Cytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides--CdtA, CdtB, and CdtC--encoded by the genes cdtA, cdtB, and cdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles of cdtB (Cdt-I through Cdt-V) in Escherichia coli and used the assay to investigate the prevalence and characteristic of CDT-producing E. coli in children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206-215, 2009). In these assays, two untypable cdtB genes were detected and the organisms harboring the cdtB gene were identified as Providencia alcalifaciens (strains AH-31 and AS-1). Nucleotide sequence analysis of the cdt gene cluster revealed that the cdtA, cdtB, and cdtC genes of P. alcalifaciens are of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of the cdt genes among closely related species, the presence of cdt genes was screened in various Providencia spp. by colony hybridization assay, and the cdt gene cluster was found in only limited strains of P. alcalifaciens. Genome walking revealed that the cdt gene cluster of P. alcalifaciens is located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT of P. alcalifaciens (PaCDT) showed some homology with the CDT of Shigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells, E. coli CDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G(2)/M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinical P. alcalifaciens strains could produce variants of the CDTs compared.

Distribution of virulence genes related to adhesins and toxins in shiga toxin-producing Escherichia coli strains isolated from healthy cattle and diarrheal patients in Japan.

Shiga toxin-producing Escherichia coli (STEC) isolated from Japan were investigated for the distribution of virulence genes. A total of 232 STEC strains including 171 from cattle and 61 from human were examined for the occurrence of genes responsible for bacterial adhesions to intestine, e.g., eae (intimin, E. coli attaching and effacing), saa (STEC autoagglutinating adhesin), iha (irgA homologue adhesin), efa1 (E. coli factor for adherence), lpfA(O113) (long polar fimbriae), and ehaA (EHEC autotransporter) by colony hybridization assay. Similarly, the presence of toxigenic cdt (cytolethal distending toxin), and subAB (subtilase cytotoxin) genes were also checked. Among cattle isolates, 170, 163, 161, 155, 112 and 84 were positive for lpfA(O113) (99%), ehaA (95%), iha (94%), saa (91%), subAB (65%), and cdt-V (49%), respectively, while 2 were positive for eae (1.2%) and efa1 (1.2%) each. In case of human isolates, 60, 59, 58 and 58 were positive for ehaA (98%), iha (97%), efa1 (95%), and eae (95%), respectively, while 11, 2, 2, and 1 were positive for lpfA(O113) (18%), saa (3.3%), cdt-V (3.3%), and subAB (1.6%), respectively. Therefore, in human STEC isolates efa1 and eae whereas in cattle isolates saa, lpfA(O113), cdt-V and subAB were prevalent. These data indicate differential occurrence of some pathogenic genes in human and cattle originated STEC strains in Japan.

A collaborative study on a method to detect Salmonella in food.

Fourteen laboratories with expertise in Salmonella detection in food joined in a collaborative study. The laboratories performed qualitative analyses of ground pork samples using the proposed detection method. Salmonella Typhimurium (hydrogen sulfide-producing strain) and Salmonella Senftenberg (hydrogen sulfide-nonproducing strain) were used for inoculation. Three levels of Salmonella contamination were used for the study (0, 1-10, and 11-100 cfu/25 g). We evaluated the presence of Salmonella in each sample and the serological O group. Unmarked samples delivered to the laboratories were accurately judged to be inoculated or not inoculated with Salmonella at a 99.8% (419/420) detection rate in this collaborative study. The proposed method is suitable as a standard method to detect Salmonella in food.

Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis.

A multiplex PCR assay has been developed for the identification of the six common Campylobacter taxa associated with human gastroenteritis and/or septicaemia, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the five Campylobacter species and the one subspecies, and each of the PCR products was sufficiently distinguished by a difference in size by agarose gel electrophoresis. On evaluation of efficacy with 142 Campylobacter strains, the assay correctly identified all strains as 1 of the 6 Campylobacter taxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the six Campylobacter taxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.

Isolation and Characterization of an Escherichia albertii Strain Producing Three Different Toxins from a Child with Diarrhea.

Here, we report a bacterium-isolated as the sole pathogen from a child with diarrhea-harboring eae and 2 different cytolethal distending toxin genes (cdt) that are homologous to Escherichia coli cdt-I and cdt-II. The bacterium was originally identified as atypical E. coli by conventional biochemical testing, but was finally identified as E. albertii by multilocus sequence analysis, which is the only method that can currently differentiate E. albertii from E. coli. The Shiga toxin 2f (stx2f) genes were also detected in the strain. Production of these 3 toxins was confirmed by western blotting and/or a cytotoxicity assay using eukaryotic cell lines. This is the first report showing the biological activity of CDT-I, CDT-II, and Stx2f in E. albertii.

Isolation and characterization of a cytotoxin produced by Plesiomonas shigelloides P-1 strain.

In order to clarify the enteropathogenicity of Plesiomonas shigelloides, we investigated a cytotoxin produced by the P-1 strain isolated from patients suffering from diarrhea. The cytotoxicity of the culture filtrate of the strain reached a maximum in culture at 37 degrees C after 12 h shaken in BHI medium. The cytotoxin in the cultures was purified by (NH4)2SO4 precipitation, and Sephacryl S-100, Mono Q HR, and Superdex 200 HR column chromatographies. An approximate 340-fold purification was achieved, with a recovery of about 1.4%, from the culture supernatant. The cytotoxin is heat-stable, and is a complex of three major proteins (LPS-binding proteins with molecular weights of 32, 40, and 48 kDa), with lipopolysaccharide (LPS) giving a total a molecular weight of more than 600 kDa. The ratio of protein to LPS in the cytotoxin was 6-5. The cytotoxic activity was reduced by about 80% by proteinase K treatment or when incubated with anti-cholera toxin antibody (Anti-CT). Western blotting of the cytotoxin with Anti-CT demonstrated the presence of two anti-cholera toxin-reactive protein (ACRP) bands with molecular weights of 40 kDa (a major single protein band) and 48 kDa. The N-terminal amino acid sequence (20 residues) of the 40 kDa protein was 75% identical to Pasteurella multocida cell membrane proteins. The cytotoxin gave a positive reaction in the suckling mouse assay whereas LPS alone hardly exhibited any cytotoxic or enterotoxigenic activity. In conclusion, P. shigelloides produces a cytotoxin that consists of a complex of protein and LPS with the former component exhibiting both cytotoxicity and enteropathogenicity. This cytotoxin has the potential to have an important role in the enteropathogenicity of P. shigelloides.

Photobacterium phosphoreum caused a histamine fish poisoning incident.

An incident of histamine fish poisoning (HFP) occurred due to the consumption of iwashi maruboshi (dried sardine) in Osaka, Japan in March 2002. A histamine-producing bacterial strain, YS4-7, was isolated from iwashi maruboshi that contained 1700 mg of histamine per kilogram. This strain was identified as Photobacterium phosphoreum by biochemical examinations and partial sequencing of 16S rDNA. P. phosphoreum YS4-7 showed greater capability as a histamine producer at 4 and 12 degrees C than Morganella morganii JCM 1672. Strain YS4-7 produced 546 mg of histamine per kilogram in a sardine homogenate stored for 12 h at 20 degrees C. M. morganii, Raoultella planticola and Hafnia alvei have been isolated from fish implicated in HFP incidents, whereas this is the first report of P. phosphoreum being the causative bacterium in a sporadic case of histamine food poisoning.

Long-term excretion of Shiga toxin-producing Escherichia coli (STEC) and experimental infection of a sheep with O157.

To investigate a long-term shedding of Shiga toxin (Stx)-producing Escherichia coli (STEC) from sheep, a fifteen-month study for STEC isolation from a sheep, which had yielded STEC before, was attempted. The sheep continued to shed STEC and 39 STEC were isolated. The number of STEC in the feces was estimated at 1.7 x 10(3) per gram. In addition, although Stx1-negative O157 and stx2-encoding bacteriophage were experimentally infected to the sheep, Stx-positive O157 or Stx2- producing bacterial cells were not detected. The genetical and biochemical characterization of those 39 STEC strains showed that all STEC strains produced Shiga toxin 1 (Stx1) and were divided into three classes (I to III). From phylogenetic analysis of their amino acid sequences, class-I STEC was classified as group 1 comprising mainly human STEC, and classes II/III were as group 2 comprising sheep STEC. Our results suggest that STEC easily colonized in sheep and that the sheep continued to shed STEC, showing that sheep might be an important reservoir for human STEC infection.

[The serotype and Shiga toxin type of Shiga toxin-producing Escherichia coli from humans and various animals].

To clear the route of STEC (Shiga toxin-producing Escherichia coli) infection to humans, we examined the serotype. Shiga toxin genotype and eae gene of STEC strains from humans and various animals. The most predominant serotype originated from humans was O157:H7, followed by O26:H11, and other serotypes were O91:H21, O103:H2, O111:NM, O121:H19, etc. The eae gene was found in 79 of 93 strains from human origin. The serotypes of STEC from cattle were significant by similar to that of STEC from humans. The eae gene was found in 44 of 87 strains from cattle. Shiga toxin genotypes possessed O157 strains from humans and cattle, were divided into six groups, stx1, stx2, stx2c, stx1 + stx2, stx1 + stx2c and stx2 + stx2c. Moreover, frequency rates of Shiga toxin genotypes of O157 were also similar to both human and cattle origins. The serotypes of STEC from sheep were also a little similar to that of STEC from humans. Seven of 8 strains from deer possessed stx2d gene that the strains from humans seldom possessed, and none of the strains possessed eae gene. In STEC originated from swine, 15 of 25 strains were O139:H1 that Shiga toxin genotype was stx2e. It was thought that the sources of STEC infection to human are cattle and sheep, and deer and swine had little possibility to human STEC infection.

Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan.

In the present study, we examined the prevalence and characteristics of CTEC among diarrheal children in Japan during a year-long surveillance study. A PCR-RFLP assay for the detection and differentiation of five types of E. coli cdtB gene (types I through V) was developed, and 362 stool specimens collected from patients reporting to pediatric departments in two hospitals were analyzed. Of the 35 samples (9.7%) that were positive for the cdtB gene, 21 were positive for cdt-I, three for cdt-II, four for cdt-III, three for cdt-IV and four samples were positive for cdt-V, as determined by different molecular techniques. The recovery of CTEC having cdt alleles was a little less, which included 19 with cdt-I, one cdt-II, three cdt-III, three cdt-IV and four with cdt-V. Among 30 CTEC strains isolated, the majority of them (43%) belonged to serogroup 02. The other virulence genes such as astA, cnfl, eaeA, cnf2 and bfpA genes were detected in 14 (47%), 11 (37%), four (13%), three (10%) and one (3.3%) strains of CTEC, respectively. However, the other common virulence-associated genes specific for DEC were not detected in these strains. Interestingly, an untypable cdt gene was detected by PCR-RFLP in Providencia alcalifaciens. Our data indicate that CTEC may be associated with diarrheal children in Japan and most of them do not belong to a conventional enteropathogenic pathovar and thus differ from strains isolated in developing countries.

Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury.

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.

Evaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses.

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT-LAMP assay and compared with the results of routine RT-PCR. The results of the RT-LAMP assay corresponded well to that of RT-PCR. These findings suggest the practical application of the RT-LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT-LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT-LAMP system is able to detect NVs in clinical specimens within a wide range.

Cytolethal distending toxin (Cdt)-producing escherichia coli isolated from a child with bloody diarrhea in Japan.

In a retrospective analysis by PCR, the cdtI gene encoding the cytolethal distending toxin (Cdt) was detected in Escherichia coli O2:H12 strain isolated from the bloody diarrheal stool specimen of a child. To our knowledge, this is the first report showing the possible association of Cdt-producing E. coli in Japan, particularly in a child with bloody diarrhea.