基于全基因组序列的弯曲菌特征分析

空肠弯曲菌(Campylobacter jejuni)和结肠弯曲菌(Campylobacter coli)是引起人类腹泻的主要致病菌。传统生化方法在鉴定弯曲菌时存在步骤多、耗时长、通量低等问题。本研究通过利用生物信息学方法对弯曲菌全基因组进行序列、基因注释、耐药基因、多位点序列分型以及CRISPR-Cas系统等分析,挖掘能够有效区分空肠弯曲菌和结肠弯曲菌的高分辨力特征。实验结果表明,空肠弯曲菌和结肠弯曲菌在基因组序列长度、GC含量、基因数量、多位点序列分型以及CRISPR-Cas系统等方面存在显著差异。同时,研究还发现了一段在空肠弯曲菌基因组中广泛存在的高分辨力CRISPR重复序列。这些特征可用于构建能够准确鉴别空肠弯曲菌和结肠弯曲菌的生物信息学方法。     

空肠弯曲菌增菌方法的研究与血清学分型

本文对空肠弯曲菌增菌培养基作了改进,以增菌法与直接分离法(直接法)对比进行实验。从379份腹泻成人粪便中分离出29株空肠弯曲菌,其中增菌法分离出29株(100%),直接法15株(51.72%)。增菌法提高检出率48.28%,P<0.001,两法差异非常显著,符合率为96.3%,说明增菌法优于直接法。另外,用烛缸培养法从鸡和猪粪便中分离出空肠弯曲菌分别为50和32株。对人、鸡和猪粪便中检出的110株菌作了血清学和生物学分型。从分出的15个血清型谱中发现不同来源菌株有共同性血清型,多集中于45型     

空肠弯曲菌生物学分型的研究

用马尿酸水解,28℃中生长及10种试剂抗性试验,研究了空肠弯曲菌的生物学分型,每株菌按3种方法,4组实验进行计数,我们对86株弯曲菌进行了生物学分型,其中空肠弯曲菌79株,分为17种类型,结肠弯曲菌5株为5种类型,海鸥弯曲菌2株为2种类型。这对菌株在流行病学上分析有一定意义。     

肉及肉制品中空肠弯曲菌的污染情况调查

我们对辽宁地区肉及肉制品进行弯曲杆菌属检查,发现12.9%(23/177)的样品为弯曲杆菌属阳性.经API Campy鉴定系统鉴定,其中26.1%(6/23)为空肠弯曲菌(C.jejuni)阳性,同时发现1株大肠弯曲菌.该调查证实,辽宁地区进出口的肉及肉制品中,存在空肠弯曲菌的污染,如果销售不当或再加工卫生不良,会对消费者的健康构成潜在威胁.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

江苏部分地区猪源结肠弯曲菌耐药性分析及多位点序列分型

【背景】弯曲菌(Campylobacter)是重要的人畜共患肠道病原菌,可通过食物链传播,引起人类腹泻性肠炎。【目的】了解猪源弯曲菌耐药特征和分子遗传特征,对江苏省10个规模化猪场进行弯曲菌分离和耐药性检测,并研究分离株的分子分型。【方法】采用琼脂平板稀释法进行最低抑菌浓度(minimal inhibitory concentration,MIC)测定,PCR方法扩增耐药基因,以弯曲菌7个管家基因(aspA、glnA、gltA、glyA、pgm、tkt和uncA)为目的基因进行多位点序列分型(multilocus sequence typing,MLST)研究。【结果】100份样品共分离出结肠弯曲菌22株,分离率为22%,弯曲菌检出情况与养殖规模和日龄无关(P>0.05)。耐药性试验结果显示,20株分离株为多重耐药菌株(81.82%,20/22),猪源结肠弯曲菌分离株对10种抗生素耐药程度不一,分别为:庆大霉素36.36%,链霉素50%,克林霉素27.27%,氯霉素13.64%,四环素40.91%,环丙沙星18.18%,萘啶酸63.63%,泰利霉素59.09%,红霉素100%,阿...     

从腹泻患儿粪便中检出空肠弯曲菌

近年来,空肠弯曲菌在世界各国已成为引起腹泻的重要病原菌之一。1982年在我国上海首次报道后,各地已相继有报道。为了解空肠弯曲菌在我市儿童中发病情况和探索其病原特点,我院肠道门诊于1986年9月～1987年8月从549例腹泻患儿粪便中分离出12株空肠弯曲菌,其阳性     

广西地区空肠弯曲菌的血清型

本文以加拿大Lior博士提供的标准抗血清,采用快速玻片凝集法对广西地区人、畜、禽所携带的38株空肠/结肠弯曲菌进行血清学分型鉴定,结果(见表1)是人源菌2株均为血清第8型;鸡源菌16株分别为血清第1、4、8、11、17、21型,不能分型3株;鸭源菌11株,分别为血清第5、8、20、21、46、53型,不能分型2株;豚鼠源菌7株,分别为血清第4、6、7,8、21型;鸽子源菌2株分别为血清第5型和21型,各源菌株中,与人源血清型相同(第8型)的鸡源株占25％;鸭源株占18.2％;豚鼠源株占42.8％,以上研究说明这些动物与人感染空肠/结肠弯曲菌有密切关系,家禽是人感染的重要贮存宿主之一。     

124株鹌鹑空肠弯曲菌生物学性状的观察

由鹌鹑分出的１２４株空肠弯曲菌,经鉴定,其形态学特征、培养特性以及生化反应、生长与抑制生长试验的特点等均与人源空肠弯曲菌基本一致,但半数以上菌株具有ａ型溶血性能。按Ｌｉｏｎ生物学分型,主要为Ⅳ型（占７５．８％）,其次是Ⅲ型、Ⅱ型（分别占１７．７％、６．５％）,互型缺如。研究证实,氯化镉对全部菌株均能抑制生长,而０／１２９无此作用。药敏试验表明,鹌鹑空肠弯曲菌依次对氟哌酸、麦迪霉素、氯霉素、呋喃妥因、丁胺卡那霉素、红霉素和链霉素呈现高敏,但均有少数菌株例外;对青霉素类和菌必治则大多数菌株表现耐药。     

弯曲菌染色体DNA限制性内切酶带型研究

从鸡、羊、鹅、鸭粪便中分离出的空肠弯曲菌,提取染色体DNA做酶切分析,得到几乎相同的带型;与从比利时引进的结肠弯曲菌及海鸥弯曲菌比较,带型完全不同;同时,从人胃粘膜中分离出的幽门螺旋菌,做染色体DNA酶切分析,得到与前者完全不同的带型。     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle.

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

DNA base compositions and base sequence relatedness of atypical Campylobacter jejuni strains from clinical material

Abstract The relationships of nitrate-negative campylobacters (NNC) resembling Campylobacter jejuni were investigated by DNA base composition estimation (T _m method) and DNA-DNA hybridization (S1 endonuclease assay). The 8 NNC strains which were from clinical material formed a homogeneous DNA group with a high level of relatedness (approx. 70%) to typical (nitrate-positive) C. jejuni , but were less similar (approx. 35%) to Campylobacter coli , and only slightly related (≥ 10%) to Campylobacter fetus, Campylobacter laridis and Campylobacter sputorum . The NNC strains showed small but consistent genome differences from typical C. jejuni . As these differences can be correlated with several bacteriological test differences, we conclude that the NNC strains constitute a distinct subspecies within C. jejuni .     

The epidemiology of antibiotic resistance in Campylobacter

Antibiotic resistance, particularly with the fluoroquinolones and macrolide antibiotics, has now emerged globally with thermophilic campylobacters, including Campylobacter jejuni and C. coli, giving rise to concerns about how these organisms have acquired such resistance characteristics, as well as consequences for human and animal treatment. This review examines (i) the clinical epidemiology of antibiotic resistance in human and animal thermophilic campylobacters, (ii) an update on resistance rates globally, (iii) surveillance of antimicrobial resistance in campylobacters originating from animals, particularly poultry, (iv) the role of the environment in the acquisition and transmission of antibiotic-resistant campylobacters, as well as (v) issues of biocide resistance in campylobacters.     

Validation of a polymerase chain reaction/restriction enzyme analysis method for species identification of thermophilic campylobacters isolated from domestic and wild animals

AIMS: To compare and evaluate a polymerase chain reaction/restriction enzyme analysis (PCR/REA) method with standard phenotypic tests for the identification and differentiation of the thermophilic campylobacters Campylobacter jejuni, C. coli, C. lari and C. upsaliensis. METHODS AND RESULTS: One hundred and eighty-two presumptive thermophilic campylobacters from 12 different animal species were tested by a recently published PCR/REA and standard phenotypic tests. By PCR/REA, 95% of the isolates were clearly identified as either one of the four thermophilic Campylobacter species or as not belonging to this group of organisms at all. By standard phenotyping, 174 of the 182 isolates were initially identified as either C. jejuni, C. coli, C. lari or C. upsaliensis. Additional genotypic tests and phenotyping showed that 52 of these identifications were either incorrect or unreliable. Of the C. jejuni isolates, 19% were identified as C. coli by initial phenotyping and 27 sheep isolates phenotyped as C. coli or C. lari were, in fact, arcobacters. CONCLUSIONS: The PCR/REA was more reliable than standard phenotyping for the identification of thermophilic campylobacters from different animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Routinely used phenotypic tests often resulted in unreliable identifications, requiring additional testing. The PCR/REA, however, gave unequivocal results and was considered useful for the routine identification of thermophilic campylobacters from different animals.     

Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples

Three different enrichment broths and two selective growth media were compared for isolating thermophilic campylobacters by combined membrane filtration and enrichment techniques from surface waters of different physical, chemical and bacteriological characteristics. Fifty-two strains of campylobacters were isolated from total of 1668 cultures. The various broth/medium combinations did not affect the dominance of C. jejuni over C. coli (total 49 C. jejuni and three C. coli). The most efficient combinations of enrichment broth and growth media were either Oosterom broth/blood-free charcoal-cefoperazone-deoxycholate agar (CCDA) medium or blood-free charcoal-cefoperazone-deoxycholate (CCD) broth/CCDA medium. Modified Preston broth (sheep blood instead of horse blood) with either of the growth media gave significantly lower yields although it suppressed efficiently the growth of contaminants. Skirrow medium had lower selectivity than CCDA medium and gave slightly lower isolation rate. Enrichment time (24 or 48 h) did not affect the isolation frequency of campylobacters but longer enrichment time increased the growth of contaminants. Prefiltration through membranes of pore sizes 5.0 and 1.2 microns decreased the growth of contaminants. However, these membranes retain campylobacters and must be cultured to avoid underestimation. From more polluted waters campylobacters were isolated most frequently with CCD broth and CCDA medium.     

Ribotypes and AP-PCR fingerprints of thermophilic campylobacters from marine recreational waters

Thirty-two strains of thermophilic campylobacters isolated from marine recreational waters and seven reference strains were biotyped and analysed by chromosomal DNA Hae III ribopatterns and AP-PCR profiles based on a random 10-mer primer (5'-CAA TCG CCG T-3'). The majority of seawater isolates (90%) were Campylobacter coli , and three strains were Camp. jejuni. Southern blot hybridization analysis showed differences between the strains, and in a numerical analysis three main clusters were formed at the 45% similarity level, that corresponded to Camp. jejuni subsp. jejuni, Camp. coli , and a combination of Camp. coli and Camp. jejuni subsp. doylei. AP-PCR profiles also differentiated between the species but were less discriminatory than ribotyping because six strains (17%) could not be typed by this method. Numerical analysis gave four main clusters at the 45% similarity level, corresponding to Camp. jejuni subsp. jejuni, Camp. coli (two clusters) and Camp. lari. The study shows that strains within each species are diverse genomically. Both molecular methods were highly discriminatory, although some strains with identical ribotypes could be distinguished by AP-PCR, and they are valuable new alternatives to traditional typing in epidemiological studies of environmental campylobacters.     

Ribosomal RNA gene restriction fragment diversity amongst Lior biotypes and Penner serotypes of Campylobacter jejuni and Campylobacter coli

Diversity based on ribosomal RNA gene-restriction endonuclease digest patterns was detected amongst 42 strains of Campylobacter jejuni and 18 strains of C. coli including representatives of 53 different Penner serotypes. HaeIII ribopatterns were coded for numerical analysis which showed that all except two were different including those of several strains of the same serotype (P2 and P20). At the 30% similarity level, four groupings were formed in the analysis of which three corresponded to C. jejuni, C. coli and C. lari phenotypes respectively. Eight strains (13%) were atypical as their phenotypic and ribopattern associations did not correspond. Ribopattern fragments of 3.0, 5.0 and 9.3 kb were characteristic of the majority of C. jejuni, whereas 1.5, 2.2-, 2.3- and 4.7-kb fragments were commonly present in C. coli. These fragments provided novel species-specific markers. We conclude that HaeIII ribotyping was as discriminatory as Penner serotyping of C. jejuni and C. coli and may even provide a basis for distinguishing between strains of the same serotype and for identifying new groups within the thermophilic campylobacters.     

The isolation and characterization of Campylobacter jejuni subsp. jejuni from domestic geese (Anser anser)

AIMS: The objectives of this study were to determine the presence of thermophilic Campylobacter spp. in free range domestic geese, and to characterize isolated strains using phenotyping criteria and SDS-PAGE of whole-cell proteins. METHODS AND RESULTS: Forty cloacal swabs from two different flocks of domestic geese were examined. All Camp. jejuni strains isolated from geese were biotyped using the Lior biotyping scheme. Twelve Camp. jejuni isolates were also tested for their susceptibility to 17 different antibacterial agents by a disc diffusion METHOD: Fourteen of the isolates were also subjected to SDS-PAGE. All of the geese examined were found to harbour Camp. jejuni. Six geese carried more than one species of Campylobacter. All strains examined were susceptible to various antibiotics but resistant to penicillin G and cephalothin. Eleven strains (92%) were resistant to sodium cefuroxime, and eight (67%) were resistant to cloxacillin, ampicillin and colistin sulphate. Three strains (25%) were resistant to tetracycline, and one strain was resistant to sulfamethoxazole/trimethoprim and kanamycin. Nine strains were subtyped as Camp. jejuni subsp. jejuni biotype II and the remaining ones as biotype I. There were 96% and 100% similarities between all the strains examined by SDS-PAGE. CONCLUSION: This study showed that Camp. jejuni were common in the intestinal tract of domestic geese. SIGNIFICANCE AND IMPACT OF THE STUDY: Geese should be considered as potential reservoirs for human and animal campylobacteriosis. The antibiotic resistance data from this study also showed that fluoroquinolone resistance, which appears to be a problem in poultry isolates in some countries, is not yet a problem in these geese.     

Reduction of Campylobacter jejuni colonization of chicks by cecum-colonizing bacteria producing anti-C. jejuni metabolites.

Cecum-colonizing bacteria were isolated from Campylobacter jejuni-free White Leghorn (Gallus domesticus) laying hens and screened for the ability to produce anti-C. jejuni metabolites. Nine isolates were obtained that possessed this characteristic. The peroral administration of the nine isolates as a mixture (ca. 10(9) per chick) to 1-day-old chicks was followed 1 week later by peroral inoculation of Campylobacter jejuni (ca. 10(9) per chick) to determine if the cecal isolates could protect chicks from colonization by campylobacters. The nine-strain mixture of cecal bacteria provided from 41 to 85% protection from C. jejuni colonization. The protective bacteria were reduced to a mixture of three strains on the basis of their ability to utilize mucin as a sole substrate for growth. These strains included Klebsiella pneumoniae 23, Citrobacter diversus 22, and Escherichia coli (O13:H-) 25. Four feeding trials with this three-strain mixture provided from 43 to 100% (average, 78%) protection from C. jejuni colonization. The dominant cecal bacterium of chicks treated with the three-strain mixture was consistently E. coli O13:H-. Similarly, three trials with only E. coli 25 used as the protective bacterium resulted in 49 to 72% (average, 59%) protection from C. jejuni colonization, with E. coli O13:H- being the dominant cecal bacterium in all cases. Although not completely effective, E. coli 25 substantially reduced the incidence of C. jejuni colonization of chicks. For all trials, fewer C. jejuni were present in the ceca of colonized chicks receiving the protective bacteria before exposure to C. jejuni than in chicks receiving only C. jejuni.(ABSTRACT TRUNCATED AT 250 WORDS)