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.

Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection

AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.

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.

Reaction of antibodies to Campylobacter jejuni and cytolethal distending toxin B with tissues and food antigens

BACKGROUND The bacteria Campylobacter jejuni(C. jejuni) is commonly associated with GuillaneBarré syndrome(GBS) and irritable bowel syndrome(IBS), but studies have also linked it with Miller Fisher syndrome, reactive arthritis and other disorders, some of which are autoimmune. It is possible that C. jejuni and its toxins may be crossreactive with some human tissues and food antigens, potentially leading to autoimmune responses.AIM To measure the immune reactivity of C. jejuni and C. jejuni cytolethal distending toxin(Cdt) antibodies with tissue and food antigens to examine their role in autoimmunities.METHODS Using enzyme-linked immunosorbent assay(ELISA) methodology, specific antibodies made against C. jejuni and C. jejuni Cdt were applied to a variety of microwell plates coated with 45 tissues and 180 food antigens. The resulting immunoreactivities were compared to reactions with control wells coated with human serum albumin(HSA) which were used as negative controls and with wells coated with C. jejuni lysate or C. jejuni Cdt which served as positive controls.RESULTS At 3 SD above the mean of control wells coated with HSA or 0.41 OD, the mouse monoclonal antibody made against C. jejuni showed moderate to high reactions with zonulin, somatotropin, acetylcholine receptor, β-amyloid and presenilin.This immune reaction was low with an additional 25 tissue antigens including asialoganglioside, and the same antibody did not react at all with another 15 tissue antigens. Examining the reaction between C. jejuni antibody and 180 food antigens, we found insignificant reactions with 163 foods but low to high immune reactions with 17 food antigens. Similarly, we examined the reaction of C. jejuni Cdt with the same tissues and food antigens. The strongest reactions were observed with zonulin, intrinsic factor and somatotropin. The reaction was moderate with 9 different tissue antigens including thyroid peroxidase, and reaction was low with another 10 different antigens, including neuronal antigens.The reaction of C. jejuni Cdt antibody with an additional 23 tissue antigens was insignificant. Regarding the reaction of C. jejuni Cdt antibody with different food antigens, 160 out of 180 foods showed insignificant reactions, while 20 foods showed reactions ranging from low to high.CONCLUSION Our findings indicate that C. jejuni and its Cdt may play a role in inflammation and autoimmunities beyond the gut.

Polyphosphate and associated enzymes as global regulators of stress response and virulence in Campylobacter jejuni

Campylobacter jejuni(C. jejuni),a Gram-negative microaerophilic bacterium,is a predominant cause of bacterial foodborne gastroenteritis in humans worldwide. Despite its importance as a major foodborne pathogen,our understanding of the molecular mechanisms underlying C. jejuni stress survival and pathogenesis is limited. Inorganic polyphosphate(poly P) has been shown to play significant roles in bacterial resistance to stress and virulence in many pathogenic bacteria. C. jejuni contains the complete repertoire of enzymes required for poly P metabolism. Recent work in our laboratory and others have demonstrated that poly P controls a plethora of C. jejuni properties that impact its ability to survive in the environment as well as to colonize/infect mammalian hosts. This review article summarizes the current literature on the role of poly P in C. jejuni stress survival and virulence and discusses on how poly P-related enzymes can be exploited for therapeutic/prevention purposes. Additionally,the review article identifies potential areas for future investigation that would enhance our understanding of the role of poly P in C. jejuni and other bacteria,which ultimately would facilitate design of effective therapeutic/preventive strategies to reduce not only the burden of C. jejuni-caused foodborne infections but also of other bacterial infections in humans.

Antibiotic susceptibility profiling and virulence potential of Campylobacter jejuni isolates from different sources in Pakistan

Objective:To determine antibiotic resistance patterns and virulence potential of Campylobacter jejuni(C.jejuni) isolates from clinical human diarrheal infections,cattle and healthy broilers.Methods:Antibiotic sensitivity patterns of C.jejuni isolates were determined by Kirby Bauer Disc Diffusion assay.These isolates were then subjected to virulence profiling for the detection of map A(membrane-associated protein).cadF(fibronectin binding protein).wlaN(beta-1.3-galaclosyltransferase) and neu AB(sialic acid biosynthesis gene).Further C.jejuni isolates were grouped by random amplification of polymorphic DNA(RAPD) profiling.Results:A total of436 samples from poultry(n=88).cattle(n=216) and humans(n=132) from different locations were collected.Results revealed percentage of C.jejuni isolates were 35.2%(31/88).25.0%(54/216) and 11.3%(15/132) among poultry,cattle and clinical human samples respectively.Antibiotic susceptibility results showed that similar resistance patterns to cephalothin was ie.87.0%,87.1%and 89%among humans,poultry and cattle respectively,followed by sulfamethoxazolc+trimcthoprim 40.0%,38.7%and 31.0%in humans,poultry and cattle and Ampicillin 40%,32%and 20%in humans,poultry and cattle respectively.Beta-lactamase activity was detected in 40.00%humans.20.37%cattle and 32.25%in poultry C.jejuni isolates.CadF and mapA were present in all poultry,cattle and human C jejuni isolates.wlauN was not detected in any isolate and neu AB was found in 9/31(36%) poultry isolates.RAPD profiling results suggested high diversity of C.jejuni isolates.Conclusions:Detection of multidrug resistant C.jejuni strains from poultry and cattle is alarming as they can be potential hazard to humans.Moreover,predominant association of virulence factors,cadF and map A(100%each) in C.jejuni isolates from all sources and neuAB(36%) with poultry isolates suggest the potential source of transmission of diverse types of C.jejuni to humans.

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.

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.

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.

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.

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.

Antibiotic resistance profile and RAPD analysis of Campylobacter jejuni isolated from vegetables farms and retail markets

Objective:To investigate antibiotic resistance profile and characterize Campylobacter jejuni(C.jejuni) isolates using random amplified polymorphic DNA(RAPD) analysis.Methods:Ninety eight C.jejuni isolates from farms and retail outlets were screened against 10 antibiotics commonly used clinically and agriculturally by using disk diffusion method.RAPD analysis was done to characterize 98 C.jejuni isolates.Results:Fifty-one percent of the isolates had multiple antibiotic resistance index 0.2 and below.This indicated that the isolates in the vegetables were not from the high risk environment or extensive farming practices.C.jejuni isolates found resistant towards penicillin G(93%),vancomycin(86%),ampicillin(35%),erythromycin(28%),gentamycin(4%),amikacin(3%),enrofloxacin(1%),norfloxacin(1%) and no resistance towards ciprofloxacin.RAPD clustering analysis showed that the contamination of C.jejuni in vegetables was likely due to cross contamination at retail markets.Conclusions:C.jejuni contamination in vegetables at retail markets was due to cross contamination.Current finding proved that C.jejuni in small scale vegetables production was less expose towards antibiotic abuse.

Effect of Stress-Adaptation on Antibiotic Sensitivity Profiles of <i>Campylobacter jejuni</i>

Campylobacter jejuni is one of the leading causes of human gastroenteritis. Campylobacter jejuni requires special conditions and media in the laboratory for its growth. In nature, however, this organism is able to survive in very diverse and hostile environments and produce disease in humans and animals. The different mechanisms by which C. jejuni survives stressful conditions in the environment still remain unclear. Stress-adaptation may be one of the factors helping this organism to survive stresses. Some C. jejuni strains have been found to have increased antibiotic resistance in last several years. To determine the effect of acid adaptation on the antibiotic sensitivity profile of C. jejuni, 4 different isolates of C. jejuni (a human isolate and 3 poultry isolates) were exposed to an acid pH of 5.5 and then rechallenged with different stresses. The antibiotic sensitivity profiles of C. jejuni after stress-adaptation were compared with antibiotic sensitivity profiles of non-stressed C. jejuni using the Kirby Bauer agar disc diffusion assay. The antibiotic sensitivity profiles of the C. jejuni isolates used in this study were found to change when the acidadapted bacteria were subjected to further stresses such as an acidic pH of 4.5, aerobic atmosphere and starvation. In the majority of the cases, antibiotic-resistant C. jejuni isolates were found to be more sensitive to antibiotics after stress-adaptation, but in a few cases C. jejuni showed increased resistance. These results indicate that increasing various stresses in a sequential pattern may, in some cases, reduce antibiotic resistance of C. jejuni isolates.

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.

Public Health Significance of <i>Campylobacter jejuni</i>

Campylobacter jejuni is a prominent bacterial cause of human gastroenteritis. Campylobacteriosis outbreaks reported were related to the ingestion of the contaminated food. Meat are reported to be frequently infected with C. jejuni. It is well recognized that C. jejuni is one of the main causes of gastroenteritis in humans, and poultry meat is reported to be the main source. A number of studies in several countries have shown the occurrence of C. jejuni in animal farms, slaughterhouses, and meat. This review simply describes the occurrence, spreading, and public health significance of C. jejuni.

一株空肠弯曲菌噬菌体vB_Cj_QDYZ的生物学特性和全基因组分析

空肠弯曲菌是一种重要的人畜共患病原菌,能引起人类细菌性腹痛腹泻。随着空肠弯曲菌耐药性日益严重,亟需筛选能高效裂解空肠弯曲菌的噬菌体,为应用噬菌体防治空肠弯曲菌污染提供理论基础。该研究分离一株能高效裂解空肠弯曲菌的噬菌体,将其命名为vB_Cj_QDYZ。电镜结果显示该噬菌体有一个正二十面体的头部(直径约100.81 nm)和一条可伸缩的尾部(长度约65.90 nm)。噬菌体vB_Cj_QDYZ的温度耐受范围为30~60℃,在pH值为3~12时效价较稳定。噬菌体的最佳感染复数为0.01,一步生长曲线表明,噬菌体的潜伏期是60 min,裂解期是150 min,爆发量是45 PFU/cell。噬菌体的基因组全长为130627 bp,G+C含量为26%,共有165个开放阅读框,3个tRNA。基因组中不含毒力基因、抗生素耐药基因及过敏原基因。比较基因组学表明噬菌体vB_Cj_QDYZ可能是来自Eucampyvirinae亚科,Fletchervirus属的新成员。噬菌体vB_Cj_QDYZ是一株裂解能力较强、热稳定性和pH稳定性良好的空肠弯曲菌噬菌体,具有开发为新型空肠弯曲菌抗菌剂的潜力。

2016-2022年北京市人源空肠弯曲菌多重耐药及流行特征分析

目的 对2016-2022年北京地区人源空肠弯曲菌流行特征和多重耐药进行分析。方法 对2016-2022年肠道门诊的感染性腹泻患者粪便样本进行滤膜驱动法分离培养空肠弯曲菌并采用琼脂稀释法(MIC)进行耐药实验。结果 2016-2022年共检出人源空肠弯曲菌614株,检出月份以4-10月为主,占87.46%(537/614),发病年龄集中在20~<40岁,占51.63%(317/614),男、女性别比为1.52∶1。耐药结果显示,614株菌以萘啶酸耐药率最高,为78.99%(485/614),红霉素耐药率最低,为4.89%(30/614)。614株菌有116种耐药谱型,耐三重及以上耐药菌株占65.64%(403/614),耐药谱以耐萘啶酸、环丙沙星、四环素谱型最多,占23.62%(145/614)。结论 空肠弯曲菌引起的腹泻逐年增多,多重耐药严重且谱型复杂。

苏州市空肠弯曲菌耐药与分子特征分析

目的了解苏州市空肠弯曲菌的耐药情况和分子特征。方法对2018年-2022年苏州市分离的空肠弯曲菌进行全基因组测序分析了解毒力基因、耐药基因、多位点序列分型(Multi-locus sequence typing,MLST)等分子特征,通过琼脂稀释法了解其耐药表型。结果61株空肠弯曲菌被分为42个ST型别,以ST464占比最高(9.8%),包含12个CC群,以CC21占比最高(19.7%)。61株菌共检出18种耐药基因,97种毒力基因。药敏试验耐药率排前3位的抗生素分别是萘啶酸,四环素和环丙沙星,耐药率分别为90.2%,88.5%和73.8%,对红霉素100.0%敏感,多重耐药率达到41.0%。结论苏州市空肠弯曲菌多重耐药率高,携带耐药基因和毒力基因较多,克隆群以CC21为主,但ST型别较为分散,呈现较高的遗传多样性。

2020-2022年广东省儿童弯曲菌的耐药性和分子流行特征分析

目的了解2020-2022年广东省儿童感染空肠弯曲菌和结肠弯曲菌的检出率、耐药特征、毒力特征及多位点序列分型等分子流行特征和病原学特征。方法在广东省广州市2家医院采集2020-2022年疑似感染儿童病例的肛拭子或粪便样本,过滤法分离培养弯曲菌,用微生物质谱系统进行鉴定;采用琼脂稀释法进行抗生素耐药水平分析;提取细菌基因组核酸,开展全基因组测序,基于全基因组测序结果分析弯曲菌耐药基因、毒力基因、多位点序列分型和基于全基因组单核苷酸多态性的系统发育分析。结果本研究的持续性常规监测检出弯曲菌53株,弯曲菌的阳性检出率为2.94%,其中,空肠弯曲菌占81.13%(43/53),结肠弯曲菌占18.87%(10/53)。集中采样处理的多病原监测筛查出弯曲菌16株,其中,空肠弯曲菌11株,结肠弯曲菌5株。对其中46株弯曲菌完成了耐药实验和全基因组测序,包括33株空肠弯曲菌和13株结肠弯曲菌。弯曲菌对临床治疗广泛使用的红霉素的耐药率为21.73%(10/46),对四环素的耐药率为80.43%(37/46),对喹诺酮类抗生素萘啶酸和环丙沙星的耐药率为76.08%(35/46)和71.73%(33/46),对氯霉素的耐药率最低,为2.17%(1/46)。结肠弯曲菌的耐药率普遍高于空肠弯曲菌,在红霉素、庆大霉素、链霉素、泰利霉素和克林霉素指标上的差异具有统计学意义。共检出30株多重耐药菌,有9种多重耐药表型。基于全基因组序列分析,46株弯曲菌携带喹诺酮类、四环素类、β-内酰胺类、氨基糖苷类等抗生素耐药基因;携带5类共128种毒力因子基因;所有46株弯曲菌比对出35种ST型,系统发育分析显示无明显优势ST型,结肠弯曲菌较空肠弯曲菌的SNPs更多。结论2020-2022年广东省弯曲菌的阳性检出趋于稳定,空肠弯曲菌的检出率高于结肠弯曲菌。弯曲菌分离株对四环素和喹诺酮类抗生素耐药严重,多重耐药谱广泛,其中结肠弯曲菌耐药更为严重;耐药基因和耐药表型有较强的关联,具有一定的预测意义。空肠弯曲菌的毒力基因较结肠弯曲菌更为丰富,可能与更高的检出率和致病性有关。系统发育分支明确且序列呈现较高的遗传多样性,无明显优势克隆群。

大肠杆菌肠菌素对空肠弯曲菌的促生长作用研究

[目的]调查鸡肠道内大肠杆菌分泌肠菌素的水平,以及其对空肠弯曲菌的促生长作用。[方法]利用间接竞争ELISA(ic-ELISA)方法检测临床分离的30株鸡肠道大肠杆菌分离株的肠菌素产量;利用λRed同源重组系统构建大肠杆菌entB基因缺失突变株,并用ic-ELISA方法测定大肠杆菌亲本株和突变株的肠菌素产量;检测亲本株和突变株在不同限铁培养基中的生长曲线;将大肠杆菌亲本株和突变株培养上清接种空肠弯曲菌,探究大肠杆菌肠菌素对空肠弯曲菌生长的影响。[结果]30株鸡肠道大肠杆菌分离株分泌肠菌素水平介于11~126μmol/L之间。利用λRed同源重组系统成功构建2株大肠杆菌entB基因缺失突变株。定性结果显示,大肠杆菌亲本株在CAS培养基中产生黄色晕圈,而entB基因缺失株不产生晕圈。ic-ELISA检测结果显示,亲本株分泌肠菌素浓度为100~150μmol/L,而基因缺失株未检测出肠菌素。大肠杆菌亲本株和entB基因缺失株在富铁培养基中生长无显著差异(P>0.05),在限铁培养基中达到平台期的D 600 nm值分别为0.6和0.3。促生长试验结果显示,大肠杆菌亲本株培养上清接种空肠弯曲菌24和36 h,菌落数分别为7.8和8.2 lg CFU/mL,而entB基因缺失株培养上清接种后菌落数分别为7.0和7.3 lg CFU/mL。[结论]大肠杆菌具有分泌肠菌素的能力,且其在限铁环境下能促进空肠弯曲菌的生长,试验结果为深入研究肠菌素介导的肠道细菌之间的相互作用奠定基础。