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.

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.

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.

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.

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.

Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction

Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.

Genetic Characteristics and Antimicrobial Susceptibility of Arcobacter butzleri Isolates from Raw Chicken Meat and Patients with Diarrhea in China

Arcobacter is an emerging foodborne pathogen worldwide.In this study,the prevalence,antimicrobial susceptibility and genetic characteristics of Arcobacter from different sources were investigated.Eighteen A.butzleri isolates were obtained from 60 raw chicken meat samples(16/60,27%)and 150 patients with diarrhea(2/150,1.3%).The resistance ratios to nalidixic acid,ciprofloxacin,clindamycin,chloramphenicol,and florfenicol were 83.33%(15/18),38.89%(7/18),38.89%(7/18),33.33%(6/18)and 33.33%(6/18),respectively.We performed whole genome sequencing of the 18 isolates,and we predicted antibiotic resistance genes and virulence factors by using assembled genomes through blastx analysis.Two resistance genes,blaOXA-464 and tet(H),and the C254T mutation in gyrA,were identified in the genomes of some resistant isolates.Furthermore,virulence genes,such as flgG,flhA,flhB,fliI,fliP,motA,cadF,cjl349,ciaB,mviN,pldA and tlyA,were found in all strains,whereas hecA,hecB and iroE were found in only some strains.Phylogenetic tree analysis of A.butzleri isolates on the basis of the core-genome single nucleotide polymorphisms showed that two isolates from patients with diarrhea clustered together,separately from the isolates from raw chicken and the chicken strains.This study is the first comprehensive analysis of Arcobacter isolated in Beijing.

Comparison of the Pathogenicity of Neisseria meningitidis Isolates of Hyperinvasive Sequence Type 7 Belonging to Serogroups A,B,C,and X

Objective To compare the pathogenicity of isolates of sequence type 7(ST-7)Neisseria meningitidis(N.meningitidis)belonging to four different serogroups(A,B,C,and X).Methods Four ST-7 N.meningitidis isolates serogrouped as A,B,C,and X and characterized by different capsule structures,were examined for their adhesion and invasion properties,and their ability to induce cytokine release and apoptosis in the host cell(the A549 cell line).Results Among the four ST-7 N.meningitidis isolates,the serogroup A isolate possessed the strongest adhesion and invasion ability.This isolate also induced the release of the highest levels of the proinflammatory mediators interleukin-6,interleukin-1β,and interferon,and the highest apoptosis rate in the host cells.However,there was no significant difference in interleukin-8 and tumor necrosis factor-αsecretion between the four isolates.Based on the findings,the serogroup X N.meningitidis isolate had the weakest pathogenicity,whereas there was almost no difference in the pathogenicity of the isolates from serogroups B and C.Conclusions The differences in the capsular structure of the four isolates of ST-7 N.meningitidis affected their pathogenic capacities.The findings also imply that the hyperinvasive ST-7 N.meningitidis lineage may include hypoinvasive isolates.

Comparative Genomic Analysis of Gene Variations of Two Chinese Yersinia pestis Isolates from Vaccine Strain EV76

Objective To investigate genomic variations of two Chinese Yersinia pestis isolates that were isolated from different plague foci obtained from vaccine strain EV76 from the Yunnan province of China. Methods A microarray containing 12 000 probes covering the entire genome of seven Yersinie pestis and two Yersinia pseudotuberculosis strains, was used. PCR assays were performed to confirm microarray results. Results The gene variations detected included the absence of five genes related to the synthesis of betaine in both EV76 and another sequenced attenuated strain, KIM D27. Several genes related to phage-related membrane proteins were found to be absent in the Antiqua biovar Yunnan strain, 485, which was isolated from a rodent plague foci. Conclusion These findings provide initial insight into the distinct strains isolated from natural foci, within their genomic context, including Yunnan Y. pestis strains. This information will be used therefore to establish subsequent comparisons of these sequences with published complete genomes of other strains.