Genetic and Phenotypic Variation of Campylobacter jejuni NCTC11168 Caused by flhA Mutation during Laboratory Passage

Objective Campylobacter jejuni NCTC11168 is commonly used as a standard strain for flagellar biosynthesis research.In this report,two distinguished phenotypic isolates(CJ1Z,flhA mutant strain,lawn;CJ2S,flhA complemented strain,normal colony)appeared during laboratory passages for NCTC11168.Methods Phenotypic assessments,including motility plates,transmission electron microscopy,biofilm formation assay,autoagglutination assay,and genome re-sequencing for these two isolates(CJ1Z,flhA mutant strain;CJ2S,flhA complemented strain)were carried out in this study.Results Transmission electron microscopy revealed that the flagellum was lost in CJ1Z.Phenotypic assessments and genome sequencing of the two isolates were performed in this study.The capacity for biofilm formation,colony auto-agglutination,and isolate motility was reduced in the mutant CJ1Z.Comparative genomic analysis indicated a unique native nucleotide insertion in flhA(nt,2154)that caused the I719Y and I720Y mutations and early truncation in flhA.Conclusion FlhA has been found to influence the expression of flagella in C.jejuni.To the best of our knowledge,this is the first study to describe the function of the C-terminal of this protein.

In Vitro Protein Expression Profile of Campylobacter jejuni Strain NCTC11168 by Two-dimensional Gel Electrophoresis and Mass Spectrometry

Objective To investigate the protein expression profiles of the major food‐borne pathogen Campylobacter jejuni NCTC11168.Methods Membrane and soluble cellular proteins were extracted from the genome‐sequenced C.jejuni strain NCTC11168.Protein expression profiles were determined using two‐dimensional gel electrophoresis（2‐DE）.All the detected spots on the 2‐DE map were subjected to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry（MALDI‐TOF/TOF） analysis.Results A total of 537 and 333 spots were detected from the whole cell and membrane‐associated proteins of C.jejuni NCTC11168 cultured on Columbia agar medium at 42 ℃ by 2‐DE and Coomassie Brilliant Blue staining,respectively.Analyses of whole cell and membrane‐associated proteins included 399 and 133 spots,respectively,which included 182 and 53 functional proteins identified by MALDI‐TOF/TOF analysis.Conclusion The comprehensive expression protein profiles of C.jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation.

Can Campylobacter jejuni play a role in development of celiac disease? A hypothesis

Celiac disease （CD） is an entropathy with malabsortive condition in which an allergic reaction to the cereal grain-protein （gluten） causes small intestine rnucosal injury. CD is a multifactorial disorder in which both genetic and environmental factors contribute to the disease development. Mechanisms have been described to explain the pathology of CD. T cells specific for multiple gluten peptides are found in virtually all patients. Generation of such a broad T cell response may be a prerequisite for disease development. CD is associated with multiple extraintestinal presentations, including neurological deficits. Recent studies have shown a significant correlation between anti-ganglioside antibodies and neurological disorders in patients with underlying CD. Gangliosides are glycosphingolipids which are abundant in nervous system and in other tissues including gastrointestinal tract. It is not known what triggers the release of anti-ganglioside antibodies in people with gluten sensitivity. But, the mechanism is likely to involve the intestinal immune system response to ingested gliadin, a component of wheat gluten. Studies showed that mechanisms different from gluten exposure may be implicated in antibody formation, and other environmental factors may also exist. In addition, considering the fact that genetic predisposition dysregulating mucosal immune responses in the presence of certain environmental triggers like gastrointestinal infections may be strong etiological factors for developing chronic intestinal inflammation including CD, the hypothesis raised in our mind that antiganglioside antibody formation in CD may play a role not only in development of neurological complications in celiac patients, but also in development of CD itself. As presence of Campylobacter jejuni in other diseases with antigangliosides antibody formation has been established, we propose the possible role of Campylobacter jejuni in development of CD in association with other genetic and environmental factors by the mechanism that molecular mimicry of gangliosides-like epitopes common to both lipo-polysacharide coats of certain strains of Campylobacter jejuni and gangliosides in cell structure of gastrointestinal mucosa may cause an autoimmune response and consequently lead to atrophy and degeneration of mucosa possibly by apoptosis.

Disruption of colonic barrier function and induction of mediator release by strains of Campylobacter jejuni that invade epithelial cells

AIM: To study the mechanisms by which Campylobacter jejuni (C. jejuni) causes inflammation and diarrhea. In particular, direct interactions with intestinal epithelial cells and effects on barrier function are poorly under- stood. METHODS: To model the initial pathogenic effects of C. jejuni on intestinal epithelium, polarized human colonic HCA-7 monolayers were grown on permeabilized filters and infected apically with clinical isolates of C. jejuni. Integrity of the monolayer was monitored by changes in monolayer resistance, release of lactate dehydrogenase, mannitol fluxes and electron microscopy. Invasion of HCA-7 cells was assessed by a modified gentamicin protection assay, translocation by counting colony forming units in the basal chamber, stimulation of mediator release by immunoassays and secretory responses in monolayers stimulated by bradykinin in an Ussing chamber. RESULTS: All strains translocated across monolayers but only a minority invaded HCA-7 cells. Strains that invaded HCA-7 cells destroyed monolayer resistance over 6 h, accompanied by increased release of lactate dehydrogenase, a four-fold increase in permeability to [3H] mannitol, and ultrastructural disruption of tight junctions, with rounding and lifting of cells off the filter membrane. Synthesis of interleukin (IL)-8 and prostaglandin E2 was increased with strains that invaded the monolayer but not with those that did not. CONCLUSION: These data demonstrate two distinct effects of C. jejuni on colonic epithelial cells and provide an informative model for further investigation of initial host cell responses to C. jejuni.

Genetic Characteristics of Lipooligosaccharide and Capsular Polysaccharide of Campylobacter jejuni from Different Sources in China

Objective To determine the distribution of two important virulence factors[lipooligosaccharide(LOS)and capsular polysaccharide(CPS)]in Campylobacter jejuni(C.jejuni)isolated from different sources in China and to develop a rapid screening method for Guillain–Barrésyndrome(GBS)-associated strains.Methods Whole-genome sequencing was carried out for 494 C.jejuni strains.The Ortho MCL software was used to define the LOS/CPS gene clusters.CPS genotyping was performed with serotype-specific sequence alignment using the BLAST software.Real-time Polymerase chain reaction(PCR)was developed with the unique sequences of specific CPS types.Results Nine novel and 29 previously confirmed LOS classes were identified.LOS classes A,B,and C were the most common(48.2%,238/494)among the 494 strains.Twenty-six capsular types were identified in 448 strains.HS2,HS4c,HS5/31,HS19,and HS8/17 were the most frequent CPS genotypes(58.7%,263/448).Strains of 17 CPS genotypes(strain number>5)had one or two prevalent LOS classes(P<0.05).Multiplex real-time PCR for rapid identification of HS2,HS19,and HS41 was developed and validated with strains of known serotypes.Conclusion Our results describe the genetic characteristics of the important virulence factors in C.jejuni strains in China.The multiplex real-time PCR developed in this study will facilitate enhanced surveillance of GBS-associated strains in China.

Modification and Evaluation of Brucella Broth Based Campylobacter jejuni Transport Medium

Reliable transport of Campylobocter jejuni isolates is critical to microbial epidemiology research, especially in developing countries without a good temperature control mailing system. Various factors, including oxygen, temperature, transport medium composition, could affect the survival of C jejunL In this study, the protective effects of different ingredients in C. jejuni transport media at 4 ℃ and 25 ℃ and under aerobic condition were quantitatively evaluated respectively. The results showed that enriched medium, supplementation with 5% blood and being kept at 4 ℃ could improve the viability of different C. jejuni strains during transport. In addition, supplementation with 25 mmol/L L-fucose in Wang＇s transport medium could significantly improve the survival of C.

Synthesis of a conjugable hexasaccharide corresponding to the capsular polysaccharide of Campylobacter jejuni strain BH0142

The first assembly of a conjugation-ready hexasaccharide from the capsular glycan of C.jejuni.strain BH0142 has been accomplished.The synthesis features the efficient preparation of 6-deoxy-D-idoheptopyranosyl fluoride donors proceeding from allylα-D-C-glucopyranoside by a C1-to-C5 switch strategy with radical dehydroxymethylative fluorination as a key step,stereocontrolled construction of 1,2-trans-α-D-ido-heptopyranosidic bonds and of 1,2-cis-α-D-galactopyranosidic linkages.The obtained target oligosaccharide sets a solid foundation for making structurally-defined multivalent glycoconjugate vaccine candidates against C.jejuni.infections.

Genetic and Antibiotic Resistance Characteristics ofCampylobacterjejuni Isolated from Diarrheal Patients,Poultry and Cattle in Shenzhen

Objective To investigate genetic and antibiotic resistance characteristics of Campylobacter jejuni(C. jejuni) isolated from Shenzhen. Methods Multilocs sequence typing and agar dilution methods were used to define the genotype and antibiotic resistance of C. jejuni, respectively. Results In total, 126 C. jejuni strains were isolated. The prevalence of C. jejuni was 5.3% in diarrheal patients. The prevalence in poultry meat(36.5%) was higher than that in cattle meat(1.1%). However, the prevalence in poultry cloacal swabs(27.0%) was lower than that in cattle stool(57.3%). Sixty-two sequence types were obtained, among which 27 of the STs and 10 alleles were previously unreported. The most frequently observed clonal complexes were ST-21(11.9%), ST-22(10.3%), and ST-403(7.1%). ST-21, ST-45, ST-354, ST-403, and ST-443 complexes overlapped between isolates from patients and cattle, whereas ST-45 and ST-574 complexes overlapped between isolates from patients and poultry. All C. jejuni were resistant to at least one antibiotic. The highest resistance rate was toward ciprofloxacin(89.7%), followed by tetracycline(74.6%), and nalidixic acid(69.0%). Conclusion This is the first report of the genotypes and antibiotic resistance of C. jejuni in Shenzhen. Overlapping clonal complexes were found between isolates from patients and cattle, and between patients and poultry.

GM1 ANTIBODY IN GUILLAIN-BARRE SYNDROME AFTER CAMPYLOBACTER JEJUNI INFECTION

Fecal culture of Campylobacter jejuni was prepared by the method of Skirrow, and serum class specific antibodies (IgG.IgM and IgA)to Campylobacter jejuni and class specific antibodies(IgG IgM)to GMI were prepared with solid phase enzyme linked immunasorbent assay in 16 cases

Risk factors and clinical implication of superimposed Campylobacter jejuni infection in patients with underlying ulcerative colitis

Background and aims:Superimposed Campylobacter jejuni infection(CJI)has been described in patients with ulcerative colitis(UC).Its risk factors and impact on the disease course of UC are not known.Our aims were to evaluate the risk factors for CJI in UC patients and the impact of the bacterial infection on outcomes of UC.Methods:Out of a total of 918 UC patients tested,21(2.3%)of patients were found to be positive for CJI(the study group).The control group comprised 84 age-matched UC patients who had tested negative for CJI.Risk factors for CJI and UC-related outcomes at 1 year after diagnosis of CJI were compared between the two groups.Results:Ten patients(47.6%)with CJI required hospital admission at the time of diagnosis,including eight for the management of“UC flare”.Treatment with antibiotics resulted in improvement in symptoms in 13 patients(61.9%).On multivariate analysis,hospital admission in the preceding year was found to be an independent risk factor for CJI[odds ratio(OR):3.9;95%confidence interval(CI):1.1–14.1]and there was a trend for chronic liver disease as a strong risk factor(OR:5.0;95%CI:0.9–28.3).At 1-year follow up,there was a trend for higher rates of UC-related colectomy(28.8% vs.14.3%;P=0.11),and mortality(9.5%vs.1.2%;P=0.096)in the study group.Conclusion:Recent hospitalization within 1 year was found to be associated with increased risk for CJI in UC patients.There was a trend for worse clinical outcomes of UC with in patients with superimposed CJI,which was frequently associated with UC flare requiring hospital admission.

A Campylobacteriosis Outbreak Caused by One Asymptomatic Food Handler Carrier

In August 2021,three students with diarrhea from the same school visited a local hospital in the S district of Beijing.An epidemic investigation showed that there were more students with diarrhea in the same school and they had one meal together.Campylobacter jejuni was isolated from both patients with diarrhea and asymptomatic food handlers;however,the latter also carried Campylobacter coli.Phylogenomic analysis showed that there was a campylobacteriosis outbreak among the students,and the asymptomatic food handler may have been the source of the infection.Routine inspection and surveillance for Campylobacter is needed for the food producing staff,particularly those cooking in the cafeteria in schools or other public food services.

A Case-control Study on Children with Guillain-barre Syndrome in North China

To explore the risk factors for Guillain-barre syndrome. Methods Case-control study design was used in 51 cases of Guillain-barre syndrome,and 51 matched controls.All of the 51 cases in this study had been examined by electrophysiology. Serum IgG antibodies specific for C. jejuni were determined in all the subjects by ELISA. Each case and control were interviewed using an ad hoc questionnaire, including his/her demographic information,onset of the illness, their personal hygiene and so on. Results The study showed that Guillain-barre syndrome was associated with a few factors, such as polio vaccine immunization before onset of illness (OR=7.27), no hand washing after defecation and before meals (OR=6.15). Infection of C. jejuni was strongly associated with the illness (OR=9.5,P<0.001). Conclusion It is suggested that occurrence of Guillain-barre syndrome may correlate to infection of C. jejuni and poor personal hygiene in children.

HLA alleles in patients with Guillain Barre syndrome

To determine whether genetic factors may work in concert with autoimmune factors in the pathogenesis of Guillain Barre syndrome (GBS) Methods We determined the differences in the distribution of HLA alleles between GBS patients and normal controls HLA DQA, DQB and DRB alleles were typed by the sequence specific primer polymerase chain reaction (PCR SSP) methods in 47 GBS, 7 patients with Campylobacter jejuni (CJ) enteritis and 50 normal controls Results There were no differences in the frequency of HLA DQA, DQB and DRB among GBS group, CJ group, normal controls and GBS subgroups with respect to recent CJ infection, GM1 IgG and GM1 IgM antibodies ( P >0 05) There was an increasing tendency of DQA1*0301 ( P =0 056, RR =2 991) in the subgroup with GM1 IgG antibodies and DQA1 *0302 ( P =0 087, RR =3 587) in the subgroup with recent CJ infection No difference was found among GBS, CJ enteritis patients and normal controls Conclusions The increasing tendency of the two alleles suggests that there may be some relation between genetic factors and immunological factors, but a definite conclusion waits for more cases