Association of Campylobacter jejuni metabolic traits with multilocus sequence types.
ABSTRACT: In this study, we describe the association of three Campylobacter jejuni metabolism-related traits, ?-glutamyl-transpeptidase (GGT), fucose permease (fucP), and secreted L-asparaginase [ansB(s)], with multilocus sequence types (STs). A total of 710 C. jejuni isolates with known STs were selected and originated from humans, poultry, bovines, and the environment. Among these isolates, we found 31.1% to produce GGT and 49.3% and 30.3% to be positive for ansB(s) and fucP, respectively. The combination of GGT production, the presence of ansB(s), and the absence of fucP was associated with ST-22, ST-586, and the ST-45 and ST-283 clonal complexes (CCs), which were the main STs and CCs found among the human and chicken isolates. The ST-21 CC was associated with the presence of fucP and was the major CC among the bovine isolates. Although the ST-61 CC was the second major CC among the bovine isolates, these isolates did not have any of the markers studied, making the role of fucP in bovine gut colonization questionable. The ST-45 CC was subdivided into three groups that were attributed solely to ST-45. One group showed a marker combination described previously, another group was found to be positive for ansB(s) only, and the third group did not have any of the markers studied. These results suggest that the host association of these markers seems to be indirect and may arise as a consequence of host-ST and -CC associations. Thus, a representative collection of STs should be tested to draw sensible conclusions in similar studies.
Project description:In this study, multilocus sequence typing (MLST) was combined with the genetic detection of six genetic markers, ansB, dmsA, ggt, cj1585c, cjj81176-1367/71 (cj1365c), and the two-gene marker tlp7 (cj0951c plus cj0952c), to assess if their presence correlated with different C. jejuni clonal groups. Using a collection of 266 C. jejuni isolates from (in decreasing order of sample size) humans, chickens, cattle, and turkeys, it was further investigated whether the resulting genotypes correlated with the isolation source. We found combinations of the six marker genes to be mutually exclusive, and their patterns of presence or absence correlated to some degree with animal source. Together with MLST results, the obtained genotypes could be segregated into six groups. An association was identified for ansB, dmsA, and ggt with the MLST-clonal complexes (MLST-CC) 22, 42, 45, and 283, which formed the most prominent group, in which chickens were the most prevalent animal source. Two other groups, characterized by the presence of cj1585c, cjj81176-1367/71, and the two-gene marker tlp7, associated with either MLST-CC 21 or 61, were overrepresented in isolates of bovine origin. Mutually exclusive marker gene combinations were observed for ansB, dmsA, and ggt, typically found in CC 45 and the related CC 22, 42, and 283, whereas the other three marker genes were found mostly in CC 21, 48, and 206. The presence of the two-gene marker tlp7, which is typical for MLST 21 and 53 as well as for MLST-CC 61, strongly correlates with a bovine host; this is interpreted as an example of host adaptation. In cases of C. jejuni outbreaks, these genetic markers could be helpful for more effective source tracking.
Project description:Campylobacter jejuni is a leading cause of human gastroenteritis worldwide. This study aimed at a better understanding of the genetic diversity of this pathogen disseminated in Japan. We performed multilocus sequence typing (MLST) of Campylobacter jejuni isolated from different sources (100 human, 61 poultry, and 51 cattle isolates) in Japan between 2005 and 2006. This approach identified 62 sequence types (STs) and 19 clonal complexes (CCs), including 11 novel STs. These 62 STs were phylogenetically divided into 6 clusters, partially exhibiting host association. We identified a novel ST (ST-4526) that has never been reported in other countries; a phylogenetic analysis showed that ST-4526 and related STs showed distant lineage from the founder ST, ST-21 within CC-21. Comparative genome analysis was performed to investigate which properties could be responsible for the successful dissemination of ST-4526 in Japan. Results revealed that three representative ST-4526 isolates contained a putative island comprising the region from Cj0737 to Cj0744, which differed between the ST-4526 isolates and the reference strain NCTC11168 (ST-43/CC-21). Amino acid sequence alignment analyses showed that two of three ST-4526 isolates expressed 693aa- filamentous hemagglutination domain protein (FHA), while most of other C. jejuni strains whose genome were sequenced exhibited its truncation. Correspondingly, host cell binding of FHA-positive C. jejuni was greater than that of FHA-truncated strains, and exogenous administration of rFHA protein reduced cell adhesion of FHA-positive bacteria. Biochemical assays showed that this putative protein exhibited a dose-dependent binding affinity to heparan sulfate, indicating its adhesin activity. Moreover, ST-4526 showed increased antibiotic-resistance (nalidixic acid and fluoroquinolones) and a reduced ability for DNA uptake. Taken together, our data suggested that these combined features contributed to the clonal thriving of ST-4526 in Japan.
Project description:<b>Objectives:</b> <i>Campylobacter jejuni</i> is responsible for 80% of <i>Campylobacter</i> infections in Israel, a country with a high incidence reaching 91/100,000 population. We studied the phylogeny, diversity and prevalence of virulence factors using whole genome sequencing (WGS) of a national sample of <i>C. jejuni</i> clinical, food, and animal isolates collected over a 10-year period (2003-2012). <b>Methods:</b> <i>C. jejuni</i> isolates (<i>n</i> = 263) were subject to WGS using Illumina sequencing (PE 250bpx2). Raw reads and <i>de novo</i> assemblies were analyzed with the BioNumerics whole genome MLST (wgMLST) pipeline. Reads were screened for 71 virulence genes by the SRST2 script. Allelic profiles were analyzed to create minimum spanning trees and allelic core distances were investigated to determine a reliable cutoff for strain determination. <b>Results:</b> wgMLST analysis of 263 <i>C. jejuni</i> isolates indicated significant diversity among the prevalent clonal complexes (CCs) with CC-21 and CC-353 being the most diverse, and CC-574 the most clonal. Within CC-21, sequence type (ST)-1359 created a separate clade. Human, poultry and bovine isolates clustered together across the different STs. Forty four percent of studied isolates were assigned to 29 genetic clusters. Temporal and geographical relatedness were found among the minority of clusters, while most phylogenetically associated cases appeared diffuse and unassociated epidemiologically. The majority of virulence factors were highly prevalent across the dataset and not associated with genotype, source of isolation or invasiveness. Conversely, all 13 genes associated with type VI secretion system (T6SS) were lineage-related and identified in only 18% of the isolates. T6SS was detected in 95.2% of ST-1359, a common type in Israel. <b>Conclusions:</b> wgMLST supported the assessment that poultry and cattle are likely food sources of infection in Israel. Substantial genetic clustering among <i>C. jejuni</i> isolates suggested multiple point source and diffuse outbreaks that were previously unreported in Israel. The high prevalence of T6SS among ST-1359 isolates is unique to Israel, and requires further investigation. This study exemplifies the importance of studying foodborne pathogens using advanced genomic approaches across the entire spectrum of One Health.
Project description:Campylobacter species are zoonotic pathogens and the leading cause of bacterial enteritis worldwide. With the increase of antimicrobial resistance to fluoroquinolones and macrolides, they have been identified by the World Health Organization (WHO) as high-priority antimicrobial-resistant pathogens. There is currently little known about the prevalence and antimicrobial resistance characteristics of Campylobacter species in Beijing. In this study, we performed a 2-year surveillance of Campylobacter in Beijing, China. We used multilocus sequence typing (MLST) and antimicrobial susceptibility testing to analyze 236 Campylobacter isolates recovered from 230 clinical infectious cases in Beijing between 2017 and 2018. The Campylobacter isolation rate in diarrhea patients was 7.81%, with higher isolation rates in male patients than female patients and in autumn compared with other seasons. We identified 125 sequence types (STs) of 23 cloning complexes (CCs) among the 236 isolates, including four new alleles and 19 new STs. The most commonly isolated STs of Campylobacter jejuni were ST-22 and ST-760 (4.50%), and the most commonly isolated ST of Campylobacter coli was ST-9227 (16.67%). We also compared our isolates with clinical Campylobacter isolates from other countries in Asia, CC-353 of Campylobacter coli was found in eight countries, CC-1034 and CC-1287 of Campylobacter coli were found only in China. All C. jejuni isolates were resistant to at least one antimicrobial. C. jejuni showed the highest rate of resistance toward ciprofloxacin (94.50%), followed by tetracycline (93.50%), and nalidixic acid (92.00%), while C. coli showed highest resistance toward ciprofloxacin (94.44%) and tetracycline (94.44%) followed by nalidixic acid (88.89%). The most commonly observed MDR combination of C. jejuni were quinolone, phenicol and tetracycline (11.50%), while the most commonly observed MDR combination of C. coli were macrolide, quinolone, phenicol, tetracycline and lincosamide (30.56%). Surveillance of molecular characterization will provide important information for prevention of Campylobacter infection. This study enhances insight into Campylobacter infections in diarrheal patients, with relevance for treatment regimens in Beijing.
Project description:<h4>Background</h4>Campylobacter jejuni, the most common bacterial pathogen causing gastroenteritis, shows a wide genetic diversity. Previously, we demonstrated by the combination of multi locus sequence typing (MLST)-based UPGMA-clustering and analysis of 16 genetic markers that twelve different C. jejuni subgroups can be distinguished. Among these are two prominent subgroups. The first subgroup contains the majority of hyperinvasive strains and is characterized by a dimeric form of the chemotaxis-receptor Tlp7(m+c). The second has an extended amino acid metabolism and is characterized by the presence of a periplasmic asparaginase (ansB) and gamma-glutamyl-transpeptidase (ggt).<h4>Results</h4>Phyloproteomic principal component analysis (PCA) hierarchical clustering of MALDI-TOF based intact cell mass spectrometry (ICMS) spectra was able to group particular C. jejuni subgroups of phylogenetic related isolates in distinct clusters. Especially the aforementioned Tlp7(m+c)(+) and ansB+/ ggt+ subgroups could be discriminated by PCA. Overlay of ICMS spectra of all isolates led to the identification of characteristic biomarker ions for these specific C. jejuni subgroups. Thus, mass peak shifts can be used to identify the C. jejuni subgroup with an extended amino acid metabolism.<h4>Conclusions</h4>Although the PCA hierarchical clustering of ICMS-spectra groups the tested isolates into a different order as compared to MLST-based UPGMA-clustering, the isolates of the indicator-groups form predominantly coherent clusters. These clusters reflect phenotypic aspects better than phylogenetic clustering, indicating that the genes corresponding to the biomarker ions are phylogenetically coupled to the tested marker genes. Thus, PCA clustering could be an additional tool for analyzing the relatedness of bacterial isolates.
Project description:Campylobacter is a zoonotic pathogen that causes foodborne diarrheal illness globally. To better understand health risks in Southeastern China, Campylobacter spp. were surveyed in humans and representative poultry products over 3 years. One hundred and ninety-five representative isolates (n = 148, Campylobacter jejuni; n = 45, Campylobacter coli; n = 2 Campylobacter hyointestinalis) were examined for genetic relatedness and antimicrobial susceptibility. Nearly all Campylobacter isolates (99.0%, 193/195) were resistant to at least one class of antimicrobials, and 45.6% (89/195) of the isolates exhibited multidrug resistance. Genotypic analysis revealed high diversity among tested strains. Multilocus sequence typing (MLST) displayed 120 sequence types (STs) including 42 novel STs being added to the PubMLST international database. Sixty-two STs belonged to 16 previously characterized clonal complexes (CCs), of which CC-21, CC-45, CC-464, CC-574, CC-353, and CC-828 were most frequently identified. In addition, pulsed-field gel electrophoresis (PFGE) fingerprinting resulted in 66 PFGE SmaI patterns among the 125 isolates, with eight patterns shared between human and poultry sources. Subtyping data did not correlate with antimicrobial resistance phenotypes. Taken together, this large-scale surveillance study highlights high antimicrobial resistance and molecular features of Campylobacter isolates in Southeastern China.
Project description:Source attribution using molecular subtypes has implicated cattle and sheep as sources of human Campylobacter infection. Whether the Campylobacter subtypes associated with cattle and sheep vary spatiotemporally remains poorly known, especially at national levels. Here we describe spatiotemporal patterns of prevalence, bacterial enumeration, and subtype composition in Campylobacter isolates from cattle and sheep feces from northeastern (63 farms, 414 samples) and southwestern (71 farms, 449 samples) Scotland during 2005 to 2006. Isolates (201) were categorized as sequence type (ST), as clonal complex (CC), and as Campylobacter jejuni or Campylobacter coli using multilocus sequence typing (MLST). No significant difference in average prevalence (cattle, 22%; sheep, 25%) or average enumeration (cattle, 2.7 x 10(4) CFU/g; sheep, 2.0 x 10(5) CFU/g) was found between hosts or regions. The four most common STs (C. jejuni ST-19, ST-42, and ST-61 and C. coli ST-827) occurred in both hosts, whereas STs of the C. coli ST-828 clonal complex were more common in sheep. Neither host yielded evidence for regional differences in ST, CC, or MLST allele composition. Isolates from the two hosts combined, categorized as ST or CC, were more similar within than between farms but showed no further spatiotemporal trends up to 330 km and 50 weeks between farm samples. In contrast, both regions yielded evidence for significant differences in ST, CC, and allele composition between hosts, such that 65% of isolates could be attributed to a known host. These results suggest that cattle and sheep within the spatiotemporal scales analyzed are each capable of contributing homogeneous Campylobacter strains to human infections.
Project description:A total of 95 human Campylobacter jejuni isolates acquired from domestic infections and collected from three districts in Finland during the seasonal peak (June to September) in 2012 were analyzed by PCR-based multilocus sequence typing (MLST) and by whole-genome sequencing (WGS). Four predominant sequence types (STs) were detected among the isolates: ST-45 (21%) and ST-230 (14%, ST-45 clonal complex [CC]), ST-267 (21%, ST-283 CC), and ST-677 (19%, ST-677 CC). In districts 1 and 3, most of the infections occurred from early July to the middle of August, with a peak at weeks 29 to 31, but in district 2, the infections were dispersed more evenly throughout 3 months (June to August). WGS data were used for further whole-genome MLST (wgMLST) analyses of the isolates representing the four common STs. Shared loci of the isolates within each ST were analyzed as distance matrices of allelic profiles by the neighbor-net algorithm. The highest allelic variations (>400 different alleles) were detected between different clusters of ST-45 isolates (1,121 shared loci), while ST-230 (1,264 shared loci), ST-677 (1,169 shared loci), and ST-267 isolates (1,217 shared loci) were less diverse with the clusters differing by <40 alleles. Closely related isolates showing no allelic variation (subclusters) were detected among all four major STs. In some cases, they originated from different districts, suggesting that isolates can be epidemiologically connected and may have the same infection source despite being originally identified as sporadic infections.
Project description:An intensive study of 443 isolates of Campylobacter jejuni and Campylobacter coli from 2031 fecal samples excreted by animal sources including cattle, sheep, and pigs, a range of wild and domesticated avian species and pets is described. The prevalence found in the majority of animal sources ranged from 22% to 28% with poultry being highest at 41% and cats and dogs lowest (<5%). The average count excreted for each animal source was found not to be significantly different ranging from approximately 10(2) to 10(5) cfu/g. Multilocus sequence typing (MLST) identified phylogenies that exhibited host specificity. A number of clonal complexes (CCs) and sequence types (STs) were characteristic of particular hosts (e.g., CC-179, ST-637, and ST-1341 found only in pigeons and gulls). Analysis of genetic distance demonstrated numerous significant differences in the distribution of MLST types (CC, ST, and allele) between animal sources. Host association was quantified using structure that correctly assigned the nine animal sources with accuracies of 28%, 24%, and 55% at the CC, ST, and allele levels, respectively. This is substantially higher than would be expected by random allocation (11%) but farmyard poultry had the lowest assignment accuracy (13%, 13%, and 21%) suggesting that isolates were shared with a wide range of other animals. This study demonstrates the link between MLST type and host and provides data that can be used in risk assessment and food attribution models. Further, it demonstrates the applicability of MLST to characterize Campylobacter strains from a broad range of environmental sources.
Project description:Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels.