Na⁺/H⁺ exchangers (NHEs) have been shown to be involved in regulating cell volume and maintaining fluid and electrolyte homeostasis. Pooled evidences have suggested that loss of Na⁺/H⁺ exchanger isoform 8 (NHE8) impairs intestinal mucosa. Whether NHE8 participates in the pathology of infectious colitis is still unknown. Our previous study demonstrated that somatostatin (SST) could stimulate the expression of intestinal NHE8 so as to facilitate Na⁺ absorption under normal condition. This study further explored whether NHE8 participates in the pathological processes of infectious colitis and the effects of SST on intestinal NHE8 expression in the setting of infectious colitis. Our data showed that NHE8 expression was reduced in Citrobacter rodentium (CR) infected mice. Up-regulation of NHE8 improved diarrhea symptom and mucosal damage induced by CR. In vitro, a similar observation was also seen in Enteropathogenic E. coli (EPEC) infected Caco-2 cells. Seglitide, a SST receptor (SSTR) 2 agonist, partly reversed the inhibiting action of EPEC on NHE8 expression, but SSTR5 agonist (L-817,818) had no effect on the expression of NHE8. Moreover, SST blocked the phosphorylation of p38 in EPEC-infected Caco-2 cells. Taken together, these results suggest that enhancement of intestinal NHE8 expression by SST could ameliorate the symptoms of mice with infectious colitis.
Absorption ; Animals ; Anti-Inflammatory Agents ; Caco-2 Cells ; Cell Size ; Citrobacter rodentium ; Colitis* ; Diarrhea* ; Enteropathogenic Escherichia coli ; Homeostasis ; Humans ; In Vitro Techniques ; Intestinal Mucosa ; Mice* ; Pathologic Processes ; Pathology ; Phosphorylation ; Somatostatin* ; Up-Regulation*

BACKGROUND: Although Escherichia coli is a common cause of bacterial enteritis in Korea, reports on community-acquired E. coli enteritis in Korean children are scarce. This study aimed to determine the clinical characteristics and pathotype distribution of community-acquired E. coli enteritis diagnosed by a multiplex polymerase chain reaction (PCR) assay in Korean children. MATERIALS AND METHODS: The medical records of children aged 18 years or less who were diagnosed with acute gastroenteritis by the attending physician between 2013 and 2016 were retrospectively reviewed. The clinical characteristics of children diagnosed with E. coli enteritis were investigated and compared with those diagnosed with Salmonella enteritis. E. coli and Salmonella infections were diagnosed by a stool PCR assay. RESULTS: Among 279 children, in whom PCR assays for E. coli and Salmonella spp. were performed, Salmonella enteritis and E. coli enteritis were diagnosed in 43 (15.4%) and 39 (14.0%) children, respectively. Among the 39 children with E. coli enteritis, enteropathogenic E. coli (n=21, 53.8%) and enteroaggregative E. coli (n=15, 38.4%) were the most common causative agents. Empirical antibiotics were administered to 33 (84.6%) children. A total of 31 (79.5%) children developed fever, and 25 (80.6%) of them had the fever for 3 days or less, which resolved a median of 1 day (range 0-3 days) after hospitalization. The most frequent gastrointestinal symptom was diarrhea (n=36, 92.3%). Significantly more children with E. coli enteritis were aged 2 years or less as compared with those with Salmonella enteritis (41.0% vs. 21.9%, P = 0.021). Children with Salmonella enteritis more frequently complained of fever (97.7% vs. 79.5%, P = 0.012), abdominal pain (90.7% vs. 64.1%, P = 0.004), and hematochezia (46.5% vs. 10.3%, P < 0.001) than those with E. coli enteritis. Erythrocyte sedimentation rate and C-reactive protein levels were significantly higher in children with Salmonella enteritis than those with E. coli enteritis (P < 0.001). CONCLUSION: Enteropathogenic E. coli was the most frequent pathotype in Korean children with E. coli enteritis that caused mild clinical symptoms. A stool PCR assay for E. coli may be useful for epidemiological purpose and for an early diagnosis of E. coli enteritis.
Abdominal Pain ; Anti-Bacterial Agents ; Blood Sedimentation ; C-Reactive Protein ; Child* ; Diarrhea ; Early Diagnosis ; Enteritis* ; Enteropathogenic Escherichia coli ; Escherichia coli* ; Escherichia* ; Fever ; Gastroenteritis ; Gastrointestinal Hemorrhage ; Hospitalization ; Humans ; Korea ; Medical Records ; Multiplex Polymerase Chain Reaction ; Polymerase Chain Reaction* ; Retrospective Studies ; Salmonella ; Salmonella Infections

OBJECTIVE: The study aimed to determine the basic histomorphologic effects of Bacillus clausii (B. clausii) spores in enteropathogenic Escherichia coli (E. coli) O127:H21-infected mice by evaluating the spleen, mesenteric lymph nodes, and intestinal mucosa.

METHODS: The study involved 46 apparently healthy Balb/c mice (Mus musculus) which were acclimatized for 19 days prior to any intervention. Sixteen mice were used to determine the sublethal dose of E. coli, which was performed by administering serially-diluted solutions and subsequent generation of a standard curve. From the remaining 30 mice, ten served as normal controls while the remaining 20 were randomized to receive either B. clausii or placebo of sterile water for a week. All mice were then challenged with E. coli for another week and euthanized, and the spleen, mesenteric lymph nodes, and small intestine harvested and examined microscopically. All study personnel were blinded of the treatment assignments.

RESULTS: Histologic evaluation of the small intestine in E. coli only-fed mice exhibited prominent attachment effacement lesions, with severely denuded mucosa, lymphocytic infiltration, and debris in the intestinal lumen. However, mice given B. clausii prior to E. coli infection displayed only minimal mucosal damage with less sloughing of villus tips, plus increased mucus-secreting goblet cells. In the spleen, E. coli only-fed mice showed moderate to severe lymphoid hyperplasia with blurred boundaries between red and white pulp. In contrast, mice which received B. clausii prior to E. coli infection had only mild degrees of lymphoid hyperplasia. Similar findings were seen in the mesenteric lymph nodes where E. coli only-fed mice showed moderate to severe lymphoid hyperplasia while those given B. clausii prior to E. coli infection merely had mild lymphoid hyperplasia.

CONCLUSION: B. clausii exerts a potential protective and immunomodulatory action in E. coli O127:H21-infected mice based on histomorphologic effects. However, additional studies are needed to fully characterize these mechanisms.mice based on histomorphologic effects. 

Animal ; Enteropathogenic Escherichia Coli ; Goblet Cells ; Mice, Inbred Balb C ; Spleen ; Bacillus Clausii ; Hyperplasia ; Escherichia Coli Infections ; Intestinal Mucosa ; Lymph Nodes

OBJECTIVETo investigate the molecular typing feature of enteropathogenic Escherichia coli (EPEC) strains isolated from different reservoirs in eight provinces of China from 2006 to 2014.

METHODSAccording to the time, place, reservoir, and PFGE pattern of the EPEC strains isolated from stools of humans with diarrhea, animal feces, and foods in eight provinces of China between 2006 and 2014, 149 EPEC strains were selected and characterized by multilocus sequence typing (MLST) using seven housekeeping genes provided by E.coli MLST database. Strain analysis demonstrated 56 different sequence types (STs). SeqMan II, MEGA 5.05, and eBURST V3 were applied to analyze the genetic relationships of domestic and forein existing 392 strains (243 EPEC strains included in the E.coli MLST database and 149 EPEC strains comprised in the present study).

RESULTSAmong the 56 different STs, the prevalent ST was ST-40, which included 19 (19/149, 12.8%) isolates. Nineteen new STs were identified. Eleven new alleles were detected in six house-keeping genes (adk, fumC, gyrB, icd, mdh, and purA). Six STs were simultaneously detected among EPEC strains isolated from patients with diarrhea and animals. And these EPEC strains were all aEPEC strains. Two STs were simultaneously identified among EPEC strains isolated from patients with diarrhea and foods. Also, these EPEC strains were all aEPEC strains. 33 out of 173 STs were divided into five major clone complexes by eBURST, STC-29, STC-10, STC-20, STC-28, and STC-517. The remaining EPEC strains included in the other 140 STs were part of the other clone complexes or just were singletons.

CONCLUSIONA high degree of phylogenetic heterogeneity was observed among the EPEC strains isolated in eight provinces of China. The EPEC strains with same STs of human isolates isolated from animal feces and foods were all aEPEC strains.

Animals ; China ; Diarrhea ; Enteropathogenic Escherichia coli ; Escherichia coli ; Escherichia coli Proteins ; Feces ; Humans ; Multilocus Sequence Typing ; Phylogeny

OBJECTIVETo understand the distribution of virulence gene and the epidemiological characteristics of diarrheagenic Escherichia(E.) coli (DEC) from diarrheal patients in Beijing.

METHODSStool specimens from diarrheal patients were cultured which were collected from the hospitals under sentinel surveillance program, during 2012-2013. DNA was examined by real-time PCR.

RESULTS253 out of 6 370 specimens were positive for DEC detection with the rate as 4.0%. A total number of 262 DEC strains were isolated. Two different pathotypes of DEC strains with mixed infection, were isolated from 9 specimens. Different pathotypes would show the following profiles: 42.8% for enteropathogenic E. coli (EPEC) including 42.0% atypical and 0.8% typical; 38.9% for enterotoxigenic E. coli (ETEC) including 24.8% st positive, 9.9% lt positive and 4.2% st and lt both positive;15.3% for enteroaggregative E. coli(EAEC);2.7% for enteroinvasive E. coli (EIEC); one strain STEC with serotype O26:K60. ETEC had obvious characteristics on age. All kinds of DEC were isolated throughout the year with seasonal fluctuation.

CONCLUSIONDEC isolates from diarrheal patients in Beijing were dominated by EPEC and ETEC, with atypical ones accounted for the majority of EPEC. One specimen was found under mixed infection. Pathotypes DEC were found to have different age and seasonal distributions.

China ; epidemiology ; Diarrhea ; microbiology ; Enteropathogenic Escherichia coli ; genetics ; isolation & purification ; Enterotoxigenic Escherichia coli ; genetics ; isolation & purification ; Epidemics ; Escherichia coli ; genetics ; isolation & purification ; Escherichia coli Infections ; epidemiology ; microbiology ; Genotype ; Humans ; Virulence

BACKGROUND: We investigated the prevalence of various pathogens (13 enteric bacteria and 5 viruses) which cause diarrhea using multiplex PCR of stool specimens and compared two multiplex PCR methods for detecting diarrheagenic Escherichia coli. METHODS: A total of 405 stool specimens submitted between November 2010 to February 2011 for routine culture of enteric pathogens were included and screened for five viruses (astrovirus, Group A rotavirus, enteric adenovirus, norovirus G1/G2) and eight bacteria (Salmonella spp., Shigella spp., Campylobacter spp., Vibrio spp., C. difficile Toxin B, C. perfringens, Y. enterolytica, Aeromonas spp.) using the Seeplex(R) Diarrhea ACE detection kit (Seegene). In addition, virulence-associated genes of enteropathogenic E. coli, (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli, (EIEC), enterotoxigenic E. coli (ETEC), and enteroaggressive E. coli (EAEC) were detected using 16-plex PCR and a commercial diarrheagenic E. coli detection (DEC) PCR kit (SSI Diagnostica). RESULTS: Overall, 138 (34.1%) of 405 samples was positive for pathogen. The positive rate for virus was 18.5%. norovirus G2, Group A rotavirus, enteric adenovirus, astrovirus and norovirus G1 were detected in 40, 23, 8, 3 and 1 samples, respectively. The positive rate for bacteria was 24.4% (99/405). C. difficile toxin B was the most frequently detected, followed by C. perfringens, EPEC, and EAEC. The agreements of the two multiplex PCR methods for detecting EPEC and EHEC were 99.3% and 100%, respectively. CONCLUSION: The detection rate was high (34.1%) including various diarrheagenic E. coli (6.2%) and C. perfringens (5.2%). Multiplex PCR is thus useful for detecting various pathogens.
Adenoviridae ; Aeromonas ; Bacteria ; Campylobacter ; Diarrhea ; Enterobacteriaceae ; Enterohemorrhagic Escherichia coli ; Enteropathogenic Escherichia coli ; Enterotoxigenic Escherichia coli ; Escherichia ; Escherichia coli ; Multiplex Polymerase Chain Reaction ; Norovirus ; Polymerase Chain Reaction ; Prevalence ; Rotavirus ; Shigella ; Vibrio ; Viruses

In infants, urinary tract infections (UTIs) are quite common and primarily caused by bacterial pathogens. However, little research has been conducted regarding the relationship between uropathogenic bacteria, virulent genes, and uropathogenic viruses that might induce UTIs in infants. In this study, we evaluated infants with UTIs to determine the influence of bacterial virulent genes and type of viral infections on clinical aspects. First, we detected 44 cases of bacterial UTI from 600 suspected cases in infants and children. We detected E. coli urovirulence genes (kps, usp, pap, ireA, and cnf), two enteropathogenic E. coli genes (bfpA, and eae) and four S. aureus and S. epidermidis genes (mecA, pvl, bbp, and icaA) in urine samples from infant UTI cases. We also simultaneously detected hematuria-related adenovirus type 11, 21, and BK virus (BKV) in urine samples by PCR. As a result, E. coli was the most prevalent bacteria and in Dimercaptosuccinic acid (DMSA)-positive UTI cases, the uropathogenic E. coli virulence factor pap was significantly high. We found that BKV detection was significantly higher in DMSA-positive UTI infants (89%) compared with 50% of non-UTI (no bacteria detected) cases. These results are indicative of combined multiple bacterial and viral infections and show severe infant pyelonephritis.
Adenoviridae ; Bacteria ; Benzophenones ; BK Virus ; Boronic Acids ; Child ; Enteropathogenic Escherichia coli ; Escherichia ; Escherichia coli ; Humans ; Infant ; Polymerase Chain Reaction ; Pyelonephritis ; Succimer ; Urinary Tract ; Urinary Tract Infections

In order to obtain an attenuated vaccine candidate for enteropathogenic Escherichia coli (EPEC) O45, a ler deletion mutant of pig enteropathogenic E. coli (PEPEC) O45 was constructed by using the suicide vector pCVD442, termed as PEPEC O45(deltaler). The culture supernatant of PEPEC O45(deltaler) deletion mutant was inoculated in vero cell culture. PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the PEPEC O45(deltaler) deletion mutant and the virulent strain O45 respectively. Mice were observed daily for clinical signs and weight changes. Test group of mice inoculated with PEPEC O45(deltaler) gained weight normally and experienced no clinical signs. In contrast, control group of mice inoculated with virulent strain O45 exhibited weight loss and all died in four days. In another experiment, pregnant mice and pig were orally vaccinated by PEPEC O45(deltaler) twice at interval of 14 days respectively. Subsequently, the suckling mice and pig were orally challenged with O45 at 7 days of age respectively. The results showed that 80% of the sucking mice born by vaccinated mice and 75% of the sucking pig born by vaccinated pig were survival; 15% of the sucking mice born by non-vaccinated mice and 10% of the sucking pig born by non-vaccinated pig were survival. This study demonstrated that PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell and to be safety to mice and pig. Oral immunization can induce specific immune responses in mice and pig, and this mutant strain could be used as an attenuated vaccine candidate against PEPEC O45.
Animals ; Enteropathogenic Escherichia coli ; genetics ; immunology ; Escherichia coli Infections ; microbiology ; prevention & control ; Escherichia coli Proteins ; genetics ; Escherichia coli Vaccines ; biosynthesis ; genetics ; immunology ; Gene Deletion ; Mice ; Mutagenesis, Site-Directed ; Swine ; microbiology ; Swine Diseases ; microbiology ; prevention & control ; Trans-Activators ; genetics ; Vaccines, Attenuated ; biosynthesis ; genetics ; immunology

Shiga toxin-producing Escherichia coli (STEC) can cause a broad spectrum of human illness ranging from symptom-free to hemolytic uremic syndrom (HUS). Associations between known or putative virulence factors of STEC and diseases in human were investigated. PCR analyses showed that 33 (78.6%) isolates carried an ehxA enterohemolysin gene and 6 (14.3%) isolates possessed an saa autoaggutinating adhesin gene, and 31 (73.8%) isolates carried an eae intimin gene (7 isolates with type beta, 16 with type gamma, and 3 with type epsilon). Twenty-nine (69%) isolates from patients carried eae+, ehxA+, saa- (genotype A) and 68 (86%) isolates from asymptomatic outbreaks and 4 (36%) isolates from bovine possessed eae-, ehxA+, saa+ (genotype C). Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. In HEp-2 cell adherence assay, isolates carrying eae gene exhibited a localized adherence phenotype, the other isolates carrying saa showed LC (loose clusters of bacteria) and IS (isolated bacteria). In conclusion, most STEC isolated from cattle feces in Gwangju, Korea showed characteristics different from those isolated from patients. But these results may be useful information for pathogenesis judgement of STEC.
Animals ; Cattle ; Diarrhea ; Disease Outbreaks ; Enteropathogenic Escherichia coli ; Escherichia coli Proteins ; Feces ; Hemolysin Proteins ; Humans ; Korea ; Lifting ; Molecular Biology ; Phenotype ; Plasmids ; Polymerase Chain Reaction ; Shiga-Toxigenic Escherichia coli ; Virulence Factors

A pair primer was designed by Oligo 6.0 according to the pilA gene sequence of E. coli isolated from human in GenBank. The pilA Gene was obtained by PCR with the enteropathogenic E. coli isolated from ducks as template and cloned into pMD18-T vector. It was identified by PCR, restriction endonuclease analysis, DNA sequencing and then subcloned into BamH I/Hind III site of prokaryotic expression vector pET-32a(+) and recombinant expression plasmid pET-32a-pilA was constructed successfully. The plasmid was transformed into Eschericha coli BL21 (DE3) and 36kD pilA recombinant protein was expressed be induced with IPTG. The protein was purified by Ni-agarose affinity chromatograghy and was prepared as vaccine with Freund' s adjuvant. The ducklings were immunized with the vaccine at 1 and 8-day-old respectively. Two weeks after last immunized, the antibody titer of duck serum was detected by ELISA and the ducklings were challenged with 10(9) PFU enteropathogenic E. coli GH1.2 virulent strain. The immunoprotection effect of pilA recombinant protein vaccine was evaluated according to the mortality, re-isolated rate of E. coli, and grades of pathological changes. The results show that the antibody titer are 1:12800, but 1:200 were detected from ducklings immunized with homologous whole cells E. coli inactivated vaccine. The mortality, re-isolated rate of E. coli, degree of pathological changes of immunized ducklings is lower than that of the control ducklings and showed significant or extremely significant differences (P < 0.01 or P < 0.05), but non-significant difference compared to the ducklings which immunized with homologous whole cells E. coli inactivated vaccine (P > 0.05). The results show that pilA recombinant protein has some immunoprotection effect with the challenging of virulent strains of E. coli GH1.2.
Animals ; Animals, Newborn ; Antibodies, Bacterial ; blood ; immunology ; Ducks ; microbiology ; Electrophoresis, Polyacrylamide Gel ; Enteropathogenic Escherichia coli ; genetics ; immunology ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli Proteins ; genetics ; immunology ; metabolism ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Humans ; Immunization ; Polymerase Chain Reaction ; Poultry Diseases ; immunology ; mortality ; prevention & control ; Recombinant Proteins ; administration & dosage ; immunology ; metabolism ; Survival Rate ; Virulence