多位点序列分型分析空肠弯曲菌华东动物源分离株

【目的】研究空肠弯曲菌菌株间的分子特征,对不同宿主来源的空肠弯曲菌进行分子分型研究。【方法】选择空肠弯曲菌的7个看家基因gltA、aspA、glnA、glyA、pgm、tkt和uncA作为目的基因,对2006-2008年间华东地区分离的42株空肠弯曲菌样本进行PCR扩增后测序。将测序结果软件分析并上传到数据库进行比对,将结果制作多位点序列分型(multilocus sequence typing,MLST)遗传进化树并进行分析。【结果】与数据库已有类型比对,发现了24个新的ST型,通过进化树得到其遗传关系。【结论】MLST方法对于研究空肠弯曲菌的菌株群体基因差异与进化趋势具有重要意义。     

Multilocus sequence typing

Numerous computer-based statistical packages have been developed in recent years and it has become easier to analyze nucleotide sequence data and gather subsequent information that would not normally be available. Multilocus sequence typing (MLST) is used for characterizing isolates of bacterial and fungal species and uses nucleotide sequences of internal fragments of housekeeping genes. This method is finding a place in clinical microbiology and public health by providing data for epidemiological surveillance and development of vaccine policy. It adds greatly to our knowledge of the genetic variation that can occur within a species and has therefore been used for studies of population biology. Analysis requires the detailed interpretation of nucleotide sequence data obtained from housekeeping and nonhousekeeping genes. This is due to the amount of data generated from nucleotide sequencing and the information generated from an array of analytical tools improves our understanding of bacterial pathogens. This can benefit public health interventions and the development of enhanced therapies and vaccines. This review concentrates on the analytical tools used in MLST and their use in the clinical microbiology and public health fields.     

两株炭疽芽胞杆菌MLST 新序列型(ST)

【目的】2株炭疽芽胞杆菌(Bacillus anthracis)17003-14和17003-32的多位点序列分型(Multilocussequence typing,MLST)研究。【方法】选取B.anthracis基因组7个常见管家基因位点glpF、gmk、ilvD、pta、pur、pycA和tpi进行PCR扩增、测序,与MLST数据库中的等位基因序列进行比对,确定菌株的序列型(sequence type,ST)。【结果】B.anthracis 17003-14和17003-32的等位基因编号分别为113、31、1、43、1、53、7和113、31、1、43、1、53、37,比对结果显示这2株细菌的等位基因编号组合未见报道。【结论】17003-14和17003-32为新ST菌株,已被MLST数据库确认,注册号(pubMLST id)分别为id-1053和id-1054。     

安徽部分地区动物源弯曲菌的分离鉴定及多位点序列分型

【背景】弯曲菌是一种重要的食源性人兽共患病原菌,革兰氏阴性、微需氧、弯曲螺旋状。【目的】为了解安徽地区弯曲菌流行状况和分子遗传特征,对安徽6个不同地区动物源的弯曲菌进行分离鉴定,并研究分离株分子分型。【方法】通过形态学及培养特性观察、生化试验、PCR方法对菌株进行鉴定。以弯曲菌7个管家基因asp A、gln A、glt A、gly A、pgm、tkt和unc A为目的基因对分离株进行多位点序列分型,并制成遗传进化树。【结果】共分离到42株弯曲菌菌株,源自6个地区的分离株具有较为一致的形态特性和相似的生化特性。多位点序列分型结果显示,本研究中共获得32种ST型,共发现9种新的ST型(8190、8222、8223、8831、8833、8841、8832、8834和8843)和6个新的等位基因(gln A606、gln A607、glt A518、gly A680、pgm863和unc A541)。进化树结果显示,空肠弯曲菌与结肠弯曲菌遗传关系相差甚远,聚集归为两个大群,分别有5个分支和3个分支。【结论】安徽6个地区不同来源的空肠弯曲菌与结肠弯曲菌均有丰富的基因型,且没有明显优势的基因型。从遗传变异的角度来看,空肠弯曲菌复杂多样,结肠弯曲菌相对保守。     

Multilocus sequence typing of Campylobacter jejuni, and the correlation between clonal complex and pulsed-field gel electrophoresis macrorestriction profile

The characterization of Campylobacter jejuni has been significantly improved by the use of multilocus sequence typing (MLST), which allows the relationship between isolates to be determined. The sequence types (STs) of 261 isolates of C. jejuni from New Zealand were determined. Isolates were obtained from a range of sources including chicken meat, cattle, pigs, duck, sheep, water and human infections. Thirty-two new alleles and 44 new STs were identified. Comparison of the MLST data and pulsed-field gel electrophoresis macrorestriction profiles showed that the macrorestriction profiles were good predictors of the clonal complex (CC) but not ST. All the major CCs identified elsewhere in the world were found in New Zealand as well as the association of certain CCs with particular animal niches. The majority of new STs identified were from river water isolates.     

Multilocus sequence typing performed on Campylobacter coli isolates from humans, broilers, pigs and cattle originating in Denmark

AIMS: To assess whether Campylobacter coli isolated from different sources in Denmark constitute separate populations. METHODS AND RESULTS: Multilocus sequence typing (MLST) was applied to 160 C. coli isolates from animal origin, food products and human cases of gastroenteritis. The isolates were collected in Denmark over a 2-year period. In total, 84 sequence types (STs) were obtained and 57 of these STs were novel to this study. Ten per cent of the isolates possessed STs that were found in both human, poultry and pig isolates. Only 10% of the isolates from pigs shared ST with isolates from humans, and these shared STs were found in poultry isolates as well. CONCLUSIONS: Great genetic diversity was seen within the Danish C. coli population. Furthermore, we found that the C. coli types isolated from Danish pigs constitute a small fraction of the C. coli causing human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: C. coli isolates from pigs is presumably not a significant source of human campylobacteriosis in Denmark. The Danish C. coli isolates include 68% STs novel to this study, showing a great diversity compared with studies from other countries.     

47株空肠弯曲菌湖北禽源株的多位点序列分型

【目的】为了解湖北地区家禽空肠弯曲菌的流行状况及其分子特征,应用多位点序列分型方法对2013–2014年的47株禽源空肠弯曲菌湖北分离株进行分子分型研究。【方法】以空肠弯曲菌的7个管家基因aspA、glnA、gltA、glyA、pgm、tkt和uncA为目的基因,提取样本基因组后PCR扩增,测序和分析。将测序结果上传数据库进行比对,制作成多位点序列分型(Multilocus sequence typing,MLST)遗传进化树。【结果】分离株共有38个ST型,10个克隆群,其中最多的克隆群为ST-353CC和ST-464CC,发现2个新的等位基因编号和25个新的ST型。遗传进化树显示,不同家禽宿主中空肠弯曲菌序列型存在一定的差异,不同地区和来源的空肠弯曲菌呈现出遗传多样性。【结论】本研究对湖北分离的47株禽源空肠弯曲菌进行了MLST分析,其结果显示菌株多样性较为丰富,将为我国家禽空肠弯曲菌的流行病学调查提供科学的数据。     

Molecular typing of Campylobacter jejuni and C. coli from chickens and patients with gastritis or Guillain‐Barré syndrome based on multilocus sequence types and pulsed‐field gel electrophoresis patterns

Campylobacter jejuni has recently been noted as the most common cause of bacterial foodborne diseases in Japan. In the present study, we determined ST types of C. jejuni and Campylobacter coli isolated from chickens and patients with enteritis or GBS in Japan and Thailand. C. jejuni from chickens, enteritis, and GBS exhibited divergent ST types and included several novel types in addition to worldwide common types. C. coli from enteritis was also divergent. Novel ST types may represent unidentified native clones in each country. Pulsed‐field gel electrophoresis confirmed the above typing and demonstrated long‐term persistence and transmission.     

MLST clustering of Campylobacter jejuni isolates from patients with gastroenteritis,reactive arthritis and Guillain–Barré syndrome

Aims: To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain–Barré syndrome (GBS) and reactive arthritis (RA). Methods and Results: Multilocus sequence typing (MLST) was used to characterize 122 isolates, including 18 from patients with RA and 8 from patients with GBS. The GI and RA isolates were collected in Denmark during 2002–2003 and the GBS isolates were obtained from other countries. In overall, 51 sequence types (STs) were identified within 18 clonal complexes (CCs). Of these three CCs, ST‐21, ST‐45 and ST‐22 clonal complexes accounted for 64 percent of all isolates. The GBS isolates in this study significantly grouped into the ST‐22 clonal complex, consistent with the PubMLST database isolates. There was no significant clustering of the RA isolates. Conclusions: Isolates from Denmark were found to be highly genetically diverse. GBS isolates grouped significantly with clonal complex ST‐22, but the absence of clustering of RA isolates indicated that the phylogenetic background for this sequela could not be reconstructed using variation in MLST loci. Possibly, putative RA‐associated genes may vary, by recombination or expression differences, independent of MLST loci. Significance and Impact of the Study: MLST typing of C. jejuni isolates from Danish patients with gastroenteritis confirmed that the diversity of clones in Denmark is comparable to that in other European countries. Furthermore, a verification of the grouping of GBS isolates compared to RA isolates provides information about evolution of the bacterial population resulting in this important sequela.     

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.     

Genotyping of Campylobacter coli isolated from humans and retail meats using multilocus sequence typing and pulsed-field gel electrophoresis

Aims:  To determine the antimicrobial resistant profiles and clonality of Campylobacter coli isolated from clinically ill humans and retail meats.
Methods and Results:  A total of 98 C. coli isolates (20 from humans and 78 from retail meats) were phenotypically characterized. Antimicrobial susceptibility testing was done using agar dilution method for ciprofloxacin, gentamicin, erythromycin and doxycycline. Seventy C. coli isolates including humans ( n  = 20) and retail meats ( n  = 50) were genotyped by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Resistance to ciprofloxacin was found in 29% and 15% of isolates from retail meats and humans. We observed 61 PFGE profiles using two enzymes ( Sma I, Kpn I) with an Index of discrimination of 0·99, whereas MLST generated 37 sequence types. Two clonal complexes were identified with 58 (82%) C. coli isolates clustered in the ST-828 complex.
Conclusions:  Resistance to ciprofloxacin and erythromycin was identified in C. coli obtained from retail meats and ill humans. PFGE typing of C. coli isolates was more discriminatory than MLST. Grouping of C. coli isolates (82%) by MLST in ST-828 clonal complex indicates a common ancestry.
Significance and Impact of the Study:  A high frequency of resistance found to ciprofloxacin and erythromycin is concerning from food safety perspective. PFGE using single or double restriction enzymes was found to be more discriminatory than MLST for genotyping C. coli . Overall, the C. coli populations recovered from humans and retail meats were genotypically diverse.     

Genotypic comparison of five isolates of Rickettsia prowazekii by multilocus sequence typing

Genetic traits of five Rickettsia prowazekii isolates, including the first from Africa and North America, and representatives from human and flying squirrels were compared using multilocus sequence typing. Four rickettsial genes encoding 17 kDa genus-common antigen (17 kDa gene), citrate synthase (gltA), OmpB immunodominant antigen (ompB) and 120 kDa cytoplasmic antigen (sca4) were examined. Sequence identities of 17 kDa gene and gltA were 100% among the isolates. Limited sequence diversity of ompB (0.02-0.11%) and sca4 (0.03-0.20%) was enough to distinguish the isolates, and evaluation of the combined four genes provided a method to easily differentiate R. prowazekii from other rickettsiae.     

Ribosomal operon intergenic sequence region (ISR) heterogeneity in Campylobacter coli and Campylobacter jejuni

Aims: The intergenic sequence regions (ISR) between the 16S and 23S genes of Campylobacter jejuni and Campylobacter coli are markedly different for each species. However, in the genomic sequence for Camp. coli RM2228 , two rRNA operons have an ISR that is characteristic of Camp. coli, and the third operon is characteristic of Camp. jejuni. The aim of this study was to determine the prevalence of ISR heterogeneity in these organisms. Methods and Results: PCR primers were designed to yield a 327‐base pair (bp) product for Camp. coli and 166‐bp product for Camp. jejuni. A strain like Camp. coli RM2228 should yield products of both sizes. DNA from a panel of Camp. coli (n = 133) and Camp. jejuni (n = 134) isolates were tested. All of the isolates yielded products of the predicted size for the species. To verify the data for Camp. coli RM2228 , each ribosomal operon from the isolate was individually amplified by PCR and tested with the ISR primer pair. Products of both sizes were produced as predicted. Conclusions: The cross‐species heterogeneity of the ISR seen in Camp. coli RM2228 is uncommon. Significance and Impact of the Study: The heterogeneity must have been caused by horizontal gene transfer at a frequency lower than predicted from housekeeping gene data. Thus, it can be expected that species identification based on the ISR can be confused in rare isolates.     

Multilocus sequence typing for comparison of veterinary and human isolates of Campylobacter jejuni

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.     

Enterotoxigenicity and frequency of Campylobacter jejuni, C. coli and C. laridis in human and animal stool isolates from different countries

Campylobacter jejuni and C. coli strains were collected during three different years from adult patients with enterocolitis in Sweden (n = 372) from 49 patients in Kuwait, and Campylobacter strains from hens from Mexico, Pakistan and Sweden (n = 107) and Swedish pigs (n = 47). C. jejuni was the predominant species in human and hen isolates, and C. coli in pigs C. coli was significantly more common in human isolates from Sweden, and more common in hen isolates from Pakistan, than in hens from Sweden and Mexico. C. laridis was only isolated from pigs (17%) and was in no case enterotoxigenic. Both in human and hen isolates, C. jejuni strains were more enterotoxigenic than C. coli strains. C. jejuni strains from Swedish hens were less enterotoxigenic than those from Pakistan and Mexico (P less than 0.001), and strains from pigs were less enterotoxigenic than those from hens (P less than 0.001). We conclude that C. jejuni are more often enterotoxigenic and possibly more virulent than c. coli and C. laridis. The relative frequency of C. jejuni and C. coli in humans and animals differs from one country to another.     

Evaluation of cytotoxic activity in fecal filtrates from patients with Campylobacter jejuni or Campylobacter coli enteritis

We sought to determine the prevalence of cytotoxic activity in fecal filtrates from persons with C. jejuni or C. coli enteritis. Stool specimens were collected from 20 persons with C. jejuni or C. coli enteritis, 20 persons with acute diarrheal illnesses of other causes, and 9 healthy, asymptomatic persons. Fecal filtrates were then incubated with Chinese hamster ovary (CHO) or HeLa cells. The fecal filtrate from 1 of the 20 (5%) persons with Campylobacter enteritis was cytotoxic for HeLa cells at a titer of 1:40, and 10 (50%) were cytotoxic for CHO cells at maximum titers of 1:20. Cytotoxic activity for CHO cells at a median titer of 1:20 was also present in 40% of the fecal filtrates from persons with diarrhea due to causes other than Campylobacter enteritis, and in 33% of filtrates from healthy, asymptomatic persons. The observed low level of cytotoxicity in fecal filtrates from all patient groups studied likely resulted from non-specific factors, unrelated to the pathogenesis of Campylobacter enteritis.     

Genotyping of Campylobacter jejuni strains from Danish broiler chickens by restriction fragment length polymorphism of the LPS gene cluster

AIMS: To apply and evaluate LG (LPS genes) genotyping, which is a genotyping method based on a cluster of genes involved in the synthesis of surface lipopolysaccharides (LPS) in Campylobacter species, for typing of Campylobacter jejuni isolates obtained from Danish broiler chickens. Furthermore, the LG genotyping method was used to study the genetic stability of four C. jejuni strains after gastrointestinal passage through experimentally infected chickens. METHODS AND RESULTS: In the present study, the LG genotyping method was modified with respect to the restriction enzymes used. To validate the method, 63 Penner serotype reference strains and 107 C. jejuni chicken isolates, representing the most common Penner serotypes of C. jejuni in Danish poultry, were selected for typing. The method was successfully used for typing all isolates and the LG genotype profiles were reproducible. There were no changes in the LG genotype of the C. jejuni strains obtained after experimental passage through chickens. CONCLUSIONS: All C. jejuni strains obtained from broiler chickens were typeable by the LG genotyping method. Application of the RsaI restriction enzyme improved the method in terms of ease and consistency of analyses and increase of discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The LG genotyping method is a valuable tool for typing C. jejuni isolates obtained from poultry. However, the association between Penner serotyping based on passive haemagglutination of heat-stable antigens and LG genotyping was low when applied to poultry isolates. This is in contrast to previous studies on isolates of human origin that reported a high correlation between results obtained by the two typing methods (Shi et al. 2002).     

Reference isolates for the clonal complexes of Campylobacter jejuni

AIMS: To identify and make available through the National Collection of Type Cultures (NCTC) a set of reference isolates for the clonal complexes of Campylobacter jejuni. METHODS AND RESULTS: The development of a multilocus sequence typing scheme for C. jejuni enabled the genetic characterization of a large number of isolates (n = 814) from cases of human disease, animals, birds and their food products. The nucleotide sequence data were used to assign each isolate an allelic profile or sequence type (ST) and examine the C. jejuni population structure in terms of clonal complexes. The clonal complexes consisted of an abundant central or founder genotype (ST), after which the complex was named, together with very closely related, generally less abundant genotypes differing from the founder at one, two or three loci. The clonal complex is an informative unit for the study C. jejuni epidemiology. It provides data which enabled the choice of 13 C. jejuni founder isolates for submission to the NCTC as a representative cross-section of the C. jejuni population. CONCLUSIONS: These 13 isolates provide a defined resource for further research into aspects of C. jejuni biology such as genomic diversity, virulence and adaptation to particular hosts or environmental survival. SIGNIFICANCE AND IMPACT OF STUDY: This isolate collection is available through the NCTC and provides a resource for further research.     

Diversities and similarities in PFGE profiles of Campylobacter jejuni isolated from migrating birds and humans

AIMS: To genetically sub-type Campylobacter jejuni strains isolated from migratory birds, and to compare these with clinical strains collected in the same area and corresponding time period, with the aim to increase our knowledge on sub-types occurring among wild birds and their possible impact on human disease. METHODS AND RESULTS: We sub-typed C. jejuni strains from migrating birds (n = 89) and humans (n = 47), using macrorestriction profiling by pulsed-field gel electrophoresis. Isolates from migrant birds often exhibited sub-types with higher levels of similarity to isolates from birds of the same species or feeding guild, than to isolates from other groups of birds. Likewise, could the vast majority of sub-types found among the migrant bird isolates not be identified among sub-types from human cases. Only two bird strains, one from a starling (Sturnus vulgaris) and one from a blackbird (Turdus merula), had sub-types that were similar to some of the human strain sub-types. CONCLUSIONS: Isolates from one bird species, or feeding guild, often exhibited high similarities, indicating a common transmission source for individuals, or an association between certain sub-types of C. jejuni and certain ecological guilds or phylogenetic groups of birds. Sub-types occurring among wild birds were in general distinctively different from those observed in patients. The two bird isolates that were similar to human strains were isolated from bird species that often live in close associations with human settlements. SIGNIFICANCE AND IMPACT OF STUDY: Wild birds have often been mentioned as a potential route for transmission of C. jejuni to humans. Our study demonstrates that strains isolated from birds most often are different from clinical strains, but that some strain similarities occur, notably in birds strongly associated with human activities.