Prediction of superantigen active sites and clonal expression of staphylococcal enterotoxin-like W / 中华流行病学杂志

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.

Genetic and Phenotypic Variation of Campylobacter jejuni NCTC11168 Caused by flhA Mutation during Laboratory Passage / 生物医学与环境科学（英文）

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.

Comparison of the Pathogenicity of Isolates of Hyperinvasive Sequence Type 7 Belonging to Serogroups A, B, C, and X / 生物医学与环境科学（英文）

Objective@#To compare the pathogenicity of isolates of sequence type 7 (ST-7) ( ) belonging to four different serogroups (A, B, C, and X).@*Methods@#Four ST-7 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 isolates, the serogroup A isolate possessed the strongest adhesion and invasion ability. This isolate also induced the release of the highest levels of the pro-inflammatory 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-α secretion between the four isolates. Based on the findings, the serogroup X 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 affected their pathogenic capacities. The findings also imply that the hyperinvasive ST-7 lineage may include hypoinvasive isolates.

Genetic and Antibiotic Resistance Characteristics of Campylobacter jejuni Isolated from Diarrheal Patients, Poultry and Cattle in Shenzhen / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate genetic and antibiotic resistance characteristics of Campylobacter jejuni (C. jejuni) isolated from Shenzhen.</p><p><b>METHODS</b>Multilocs sequence typing and agar dilution methods were used to define the genotype and antibiotic resistance of C. jejuni, respectively.</p><p><b>RESULTS</b>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%).</p><p><b>CONCLUSION</b>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.</p>

Antibiotics susceptibility and genetic characteristics analysis for quinolone resistant Campylobacter jejuni isolated from China / 中国人兽共患病学报

According to CLSI,agar dilution method was used to analyze the minimum inhibitory concentrations (MIC) of the nalidixic acid and ciprofloxacin for the isolates from different sources.Mutations in the quinolone resistant determining region (QRDR) of gyrA and gyrB were examined by DNA sequencing of 102 resistant C.jejuni isolates and 27 sensitive isolates.The results showed that 218 isolates(93.16％) were resistant to nalidixic acid among the entire tested 234 isolates.Among these,the resistant rates of the isolates from chicken feaces,duck feaces,human feaces,food animal and cow feaces were 100.00％,100.00％,97.96％,97.83％ and 77.97％,respectively.The 211 isolates(90.17％) were resistant to ciprofloxa-cin.Among these,the resistant rates of the isolates from chicken feaces,duck feaces,human feaces,food animal and cow feaces were 100.00％,100.00％,91.84％,95.65％ and 77.97％,respectively.The differences were both statistically significant.All of the resistant isolates on the QRDR of gyrA had Thr-86-Ile mutation.However,the point substitutions in gyrB gene were synonymous mutations.The results indicated that the C.jejuni isolates in this study showed highly resistant to nalidixic acid and ciprofloxacin.The Thr-86-Ile mutation on the QRDR of gyrA can cause highly resistant to quinolone and fluoroquinolone for C.jejuni.

In vitro protein expression profile of Campylobacter jejuni strain NCTC11168 by two-dimensional gel electrophoresis and mass spectrometry / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the protein expression profiles of the major food-borne pathogen Campylobacter jejuni NCTC11168.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 °C 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.</p><p><b>CONCLUSION</b>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.</p>

Comparative genomic analysis of gene variations of two Chinese Yersinia pestis isolates from vaccine strain EV76 / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>A microarray containing 12 000 probes covering the entire genome of seven Yersinia pestis and two Yersinia pseudotuberculosis strains, was used. PCR assays were performed to confirm microarray results.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Polymorphism of Helicobacter pylori vacA, isolated in China / 中华流行病学杂志

Objective To understand the polymorphism of Helicobacter pylori (H. pylori) vacA alleles in China. Methods A total of 119 H. pylori strains were isolated from different gastroduodenal diseases in 7 different geographic regions in China. vacA and its alleles were identified according to the length of PCR products with DNA electrophoresis. The distributions of vacA alleles were statistically analyzed. The core fragment of vacA was sequentially analyzed by software MEGA4.0. Results The alleles in vacA dominantly belonged to sla, m2 and il in the tested strains.The distribution appeared to be 97.5%(116/119) ,68.9%(82/119) and 91.6%(109/119),respectively.The mlb allele appeared to be 26.1% (31/119). slb and mla were not found. The major vacA recombination was between slaim2/il and 62.2% , followed by sla/mlb/il (25.2% , 30/119). No association was found between the distribution of sla allele and the clinical outcome, as well as the geographical regions (P>0.05). However, the distribution of m alleles showed significant difference both among the types of disease and the geographic regions (P<0.01), The present of i alleles did not show significant differences among disease patterns, but had significant differences between different geographic groups (P<0.01). Three clusters were identified among these 119 isolates according to the DNA sequence of vacA. Conclusion sla/m2/il appeared to be the main allele in H. pylori vacA isolates from China in this study. The distribution of m alleles in vacA was correlated both to the regions and the disease patterns. The presence of i allele was associated to the regions but not the disease patterns.

Multi-PCR identification and virulence genes detection of Campylobacter jejuni isolated from China / 中华流行病学杂志

<p><b>OBJECTIVE</b>This study was to simultaneously identify Campylobacter jejuni and Campylobacter coli isolates in China by Multi-PCR assay and to study the prevalence of six virulence and toxin genes on them.</p><p><b>METHODS</b>A multi-PCR method with three sets of primers specifically designed for application of a 16S rRNA as a universal control, mapA, ceuE based on the specific sequence of C. jejuni and C. coli, was applied to detect 65 Campylobacter isolates from China. Another two separately PCR Primers were directed towards the hippuricase gene (hipO) characteristic of C.jejuni and glyA gene characteristic of C. coli were performed for further confirmation. The presence of the cadF, virB11, flaA, cdtA, cdtB, cdtC genes among these 65 strains were investigated by PCR.</p><p><b>RESULTS</b>From multi-PCR detection, 42 isolates belonged to C. jejuni, other 23 isolates belong to C. coli. Data showing the identification were 100% in concordance with the separated PCR for hipO and glyA amplification. The efficiency (100%) of identification by these three primers multi-PCR method was higher than the biochemical test (83.1%). The cadF and flaA genes were detected from 100% (65/65) of the isolates and the PCR product of each gene were identical with each isolate. Only 10.8% (7/65) of the isolates were positive for virB11. The cdtA gene was found in 92% (60/65) of the isolates. 97.6% (41/42) of C. jejuni had cdtB gene, whereas no PCR product with this primers for all the C. coli isolates. cdtC was presented in all the isolates but the lengths of PCR products were different. For C. jejuni, it was 555 bp, for C. coli, it was about 465 bp.</p><p><b>CONCLUSION</b>This three primers simultaneous multi-PCR method seemed to be useful for the identification of C. jejuni and C. coli isolates from China since cadF and flaA genes were widely spread in Campylobacter isolates in this country. The present report on virB11 was similar to previous reports from other countries, but the distribution of cdt gene cluster in Campylobacter species isolated from China might be different.</p>

Distribution of cagA 3' region, iceA, vacA and HP0519 on Helicobacter pylori isolated from China / 中华流行病学杂志

<p><b>OBJECTIVE</b>This study was aimed to characterize the Helicobacter pylori strains isolated from different geographic regions in China and different ethnic groups in Yunnan province in terms of cagA, iceA, vacA and HP0519 genes which were proposed to be related to the pathogenesis.</p><p><b>METHODS</b>150 Helicobacter pylori strains were collected from Yunnan province, Fujian province and Beijing. Chromosome DNA was extracted and polymerase chain reaction (PCR) was carried out to determine the 3' region of cagA, iceA, vacA and HP0519 status with specific primers. PCR results were analyzed statistically according to their isolated original and clinical outcomes.</p><p><b>RESULTS</b>For cagA 3' region, 93% (139/150) of the Chinese Helicobacter pylori strains belonged to East Asian type according to the specific primer of TF/JR. Among the 150 strains, 75% (113/150) belonged to iceA1, and 19% (29/150) to iceA2. The dissemination of iceA was not associated with any of the geographic regions, different ethnic groups or different clinical outcomes. 96% (144/150) of the vacA s region belonged to s1. In the vacA middle region, m2, m1b, m1b-m2 were 57% (85/150), 27% (41/150) and 11% (16/150) respectively. However, m1a was only observed in two strains from Fujian. Neither vacA s1 nor m2 showed significant difference between Yunnan, Fujian and Beijing. However, the distribution of mlb-m2 in Yunnan was higher than that in Fujian and Beijing. In Yunnan province, the distribution of vacA s1 was not associated with different ethnic groups but m2 from Bai group was less than other two ethnic groups. The ratio of m1b in Bai group was higher than that in other groups. Both vacA' s region and m region alleles had no significant relationship with the clinical outcomes. With the 15 bp and 24 bp DNA insertion and deletion primers test, 93% (140/150) of the strains were positive. The distributions of the 15 bp and 24 bp DNA insertion or deletion were different according to the different ethnic groups.</p><p><b>CONCLUSION</b>By JF/TR primer, 93% of the Chinese strains cagA's 3' region belonged to East Asian type. Most of the Chinese strains vacA's allele was s1. The distribution of vacA s1 had no relationship with the clinical outcome of the isolates. From different geographic regions and ethnic groups, the distribution of vacA m region allele was different. 93% of the Chinese strains HP0519 genes had 24 bp or 15 bp insertion or deletion character. The biological meaning of the polymorphism of HP0519 needs advanced investigation.</p>

Development of a Multiplex PCR-Microarray Method for Detection of Important Enteropathogen / 中国生物工程杂志

Objective: To establish a multiplex PCR-microarray method for detecting important enteropahogens.Methods: Uniplex and multiplex PCR were performed to obtain the best primer sets for identifying the target bacteria at species and multi-species level.Fluorescent dyes were mixed into PCR reaction to determine whether it can affect the efficiency of amplification.To improve the efficiency of microarray,a 35 pairs primer-labeling system was optimized based on the hybridization results to find the best combination to avoid false negative results.Results: Specific PCR products were all obtained using species-specific primer sets.More preferential amplification may happen when more primer pairs were added to the reaction.The hybridization results showed a positive association between the efficiency of multiplex-PCR and signal intensity.Conventional PCR yielded more products than fluorescent dyes labeled PCR.Thirty-five primers were divided into three different combinations to label target respectively,hybridization results showed a high specificity.Conclusion: Mixing fluorescent dyes into PCR may reduce the efficiency of amplification and hybridization,but may have no effect on the analysis of hybridization results.The hybridization efficiency of microarray depends on the amplification efficiency of multiplex PCR.For microarray target labeling,three primer sets could be used to avoid negative hybridization led by preferential amplification of multiplex-PCR.It indicates that the multiplex PCR-microarray method is an attractive diagnosis tool for the high-throughput identification of enteropathogenic organisms especially for multiple causative agents and epidemiological investigations.