Utility of multilocus sequence typing as an epidemiological tool for investigation of outbreaks of gastroenteritis caused by Campylobacter jejuni.
ABSTRACT: Multilocus sequence typing (MLST) has been proven useful for the study of the global population structure of Campylobacter jejuni; however, its usefulness for the investigation of outbreaks of disease caused by C. jejuni has not been proven. In this study, MLST plus sequencing of the flaA short variable region (SVR) were applied to 47 isolates from 12 outbreaks of C. jejuni infection whose relatedness has been determined previously, and the results were compared to those of serotyping and pulsed-field gel electrophoresis (PFGE). Isolates implicated in an outbreak were indistinguishable by all four subtyping methods, with sporadic isolates being distinguished from outbreak isolates. Two sporadic isolates from one outbreak were resistant to SmaI digestion and therefore nontypeable by PFGE but were differentiated from the outbreak strain by the other methods. PFGE and flaA SVR typing were the most discriminatory methods, with discriminatory indices (DI) of 0.930 and 0.923, respectively. However, an epidemic strain from one outbreak was distinguished from the other outbreak isolates by flaA SVR typing; its flaA allele was different at five nucleotides, suggesting that this change was possibly mediated by recombination. MLST was less discriminatory than PFGE and flaA SVR typing (DI = 0.859), and many of the epidemic strains possessed common sequence types (STs) including ST-8, -21, -22, and -42. However, further discrimination within STs was achieved by flaA SVR typing or PFGE. The results from this study demonstrate that a combined approach of MLST plus flaA SVR typing provides a level of discrimination equivalent to PFGE for outbreak investigations.
Project description:Campylobacter is genetically highly diverse and undergoes frequent intraspecific recombination. Turkeys have been identified as an important reservoir for Campylobacter jejuni which is of public health significance. The assessment of the genetic diversity among Campylobacter population is critical for our understanding of the epidemiology of this bacterium. The genetic profiles were different according to the molecular typing methods used. The performance of established flaA genotyping, multilocus sequencing typing (MLST) and DNA microarray assay based on the ArrayTube™ technology was evaluated using 14 Campylobacter jejuni isolated from a commercial turkey flock. The flaA typing was performed using PCR-RFLP with restriction enzymes Sau3AI, AluI, a 'composite' flaA analysis of AluI and Sau3AI and DdeI. The 14 isolates were differentiated into 3, 5, 7 and 9 genotypes, respectively. Entire flaA gene and short variable region (SVR) sequences were analysed. Sequencing of the entire flaA provided 11 different genotypes. flaA-SVR sequence analysis detected 8 flaA alleles and 4 flaA peptides. One new flaA allele type (528) was identified. MLST analysis represented 10 different sequence types (STs) and 5 clonal complexes (CCs). The microarray assay recognised 14 different genotypes. The discriminatory indices were 0.560, 0.802, 0.857, and 0.912 for flaA-RFLP depending on the used enzymes, 0.890 for flaA-SVR, 0.967 for entire flaA sequencing, 0.945 for MLST and 1.00 for the DNA microarray assay. The flaA gene was genetically stable over 20 passages on blood agar. In conclusion, the different typing tools demonstrated a high level of genetic heterogeneity of Campylobacter jejuni in a turkey flock, indicating that a single flock can be infected by multiple genotypes within one rearing cycle. DNA microarray-based assays had the highest discriminatory power when compared with other genotyping tools.
Project description:In October 1998, the Centers for Disease Control and Prevention (CDC) assisted in an investigation of an outbreak of campylobacteriosis at a school in Salina, Kansas. Twenty-two isolates were submitted from the Kansas state public health laboratory to CDC, 9 associated with the outbreak and 13 epidemiologically unrelated sporadic isolates. Pulsed-field gel electrophoresis (PFGE) using SmaI and SalI was initially used to validate the epidemiologic data. We then tested the ability of other subtyping techniques to distinguish the outbreak-associated isolates from unrelated sporadic isolates. The methods employed were somatic O serotyping, PCR-restriction fragment length polymorphism (RFLP) analysis of flaA, DNA sequence analysis of 582 bp of flaA that included the short variable region (SVR), and sequencing of the entire flaA gene. PFGE was the most discriminatory technique, yielding 11 SmaI and 10 SalI restriction profiles. All outbreak isolates were indistinguishable by PFGE, somatic O serotyping, and sequencing of the 582-bp region of the flaA gene. fla typing by PCR-RFLP grouped one sporadic isolate with the outbreak strain. Analysis of the DNA sequence of a 582-bp segment of flaA produced strain groupings similar to that generated by PCR-RFLP but further differentiated two flaA PCR-RFLP types (with a 1-bp difference in the 582-bp region). Two sporadic strains were distinct by flaA PCR-RFLP but differed only by a single base substitution in the 582-bp region. The entire flaA gene was sequenced from strains differing by a single base pair in the 582-bp region, and the data revealed that additional discrimination may in some cases be obtained by sequencing outside the SVR. PFGE was superior to all other typing methods tested for strain discrimination; it was crucial for understanding the Kansas outbreak and, when SmaI was used, provided adequate discrimination between unrelated isolates.
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.
Project description:To compare the two Acinetobacter baumannii multi-locus sequence typing (MLST) schemes and to assess their suitability to aid in outbreak analysis we investigated the molecular epidemiology of 99 Acinetobacter baumannii isolates representing outbreak-related and sporadic isolates from 24 hospitals in four different countries (Germany, Poland, Sweden, and Turkey). Pulsed-field gel electrophoresis (PFGE) was used as the reference method to determine the epidemiologic relatedness of isolates and compared to MLST using both the Oxford and Pasteur scheme. Rep-PCR was used to define international clonal lineages (IC). We identified 26 unique outbreak strains and 21 sporadic strains. The majority of outbreaks were associated with carbapenem-resistant A. baumannii harbouring oxacillinase OXA-23-like and corresponding to IC 2. Sequence types (STs) obtained from the Oxford scheme correlate well with PFGE patterns, while the STs of the Pasteur scheme are more in accordance with rep-PCR grouping, but neither one is mirroring completely the results of the comparator. On two occasions the Oxford scheme identified two different STs within a single outbreak where PFGE patterns had only one band difference. The CCs of both MLST schemes were able to define clonal clusters that were concordant with the ICs determined by rep-PCR. IC4 corresponds to the previously described CC15 Pasteur (= CC103 Oxford). It can be concluded that both MLST schemes are valuable tools for population-based studies. In addition, the higher discriminatory power of the Oxford scheme that compares with the resolution obtained with PFGE can often aid in outbreak analysis.
Project description:Campylobacteriosis is the most commonly reported form of human bacterial gastroenteritis in the world. Sound identification of infectious sources requires subtyping, but the most widely used methods have turnaround times measured in days and require specialist equipment and skills. A multiplex ligation-dependent probe amplification-binary typing (MBiT) assay was developed for subtyping Campylobacter jejuni and Campylobacter coli. It was tested on 245 isolates, including recent isolates from Belgium and New Zealand, and compared to multilocus sequence typing (MLST). When used in an outbreak setting, MBiT identified the predominant genotype and possible additional cases days before pulsed-field gel electrophoresis (PFGE) results were available. MBiT was more discriminatory than MLST and, being a single assay with results produced within 6 h, was more rapid and cost-effective than both MLST and PFGE. In addition, MBiT requires only basic molecular biology equipment and skills.
Project description:We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost- and time-efficient routine monitoring.
Project description:To evaluate the impact of pig farm management on the genetic diversity and on the virulence of Campylobacter coli, we characterized isolates from 19 organic pig farms (62 isolates) and from 24 conventional pig farms (58 isolates). The 120 C. coli isolates were typed using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and the presence of nine virulence genes was screened using real-time PCR. The capacity of adhesion and invasion of 61 isolates (32 from organic and 29 from conventional farms) were then tested on human intestinal Caco-2 cells. A total of 59 PFGE types and of 50 sequence types (STs) were identified. Twelve PFGE types and nine STs, accounting for 34 and 41.6% of the isolates, respectively, were common between the two production systems with ST854 dominating (18.3% of the isolates). Twenty-nine PFGE types and 25 STs were only found in isolates from organic farms, and 18 PFGE types and 16 STs from conventional farms. No significant differences were found in diversity despite the differences in rearing systems, except at the locus level for the glnA, gltA, and uncA genes. All isolates, regardless of their origin, carried the ceuE, iam, ciaB, and flaA genes and more than 95% of the isolates carried the cadF and cdtABC genes. No significant differences were found in pathogenicity between the two farming systems. The pathogenicity of the C. coli isolates was low compared to C. jejuni control strains tested. The plasmid gene virb11 was detected in only 13 isolates from organic farms; these isolates showed greater invasion capacity than those without this gene. Our study indicates that pig farm management does not significantly affect the diversity and the virulence of Campylobacter coli isolated from pigs. The common genotypes between conventional and organic farms may indicate that some genotypes are adapted to pigs.
Project description:At present, the most used methods for Klebsiella pneumoniae subtyping are multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). However, the discriminatory power of MLST could not meet the need for distinguishing outbreak and non-outbreak isolates and the PFGE is time-consuming and labor-intensive. A core genome multilocus sequence typing (cgMLST) scheme for whole-genome sequence-based typing of K. pneumoniae was developed for solving the disadvantages of these traditional molecular subtyping methods. Firstly, we used the complete genome of K. pneumoniae strain HKUOPLC as the reference genome and 907 genomes of K. pneumoniae download from NCBI database as original genome dataset to determine cgMLST target genes. A total of 1,143 genes were retained as cgMLST target genes. Secondly, we used 26 K. pneumoniae strains from a nosocomial infection outbreak to evaluate the cgMLST scheme. cgMLST enabled clustering of outbreak strains with <10 alleles difference and unambiguous separation from unrelated outgroup strains. Moreover, cgMLST revealed that there may be several sub-clones of epidemic ST11 clone. In conclusion, the novel cgMLST scheme not only showed higher discriminatory power compared with PFGE and MLST in outbreak investigations but also showed ability to reveal more population structure characteristics than MLST.
Project description:Historically, a number of typing methods have been evaluated for Staphylococcus aureus strain characterization. The emergence of contemporary strains of community-associated S. aureus, and the ensuing epidemic with a predominant strain type (USA300), necessitates re-evaluation of the discriminatory power of these typing methods for discerning molecular epidemiology and transmission dynamics, essential to investigations of hospital and community outbreaks. We compared the discriminatory index of 5 typing methods for contemporary S. aureus strain characterization. Children presenting to St. Louis Children's Hospital and community pediatric practices in St. Louis, Missouri (MO), with community-associated S. aureus infections were enrolled. Repetitive sequence-based PCR (repPCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa), and staphylococcal cassette chromosome (SCC) mec typing were performed on 200 S. aureus isolates. The discriminatory index of each method was calculated using the standard formula for this metric, where a value of 1 is highly discriminatory and a value of 0 is not discriminatory. Overall, we identified 26 distinct strain types by repPCR, 17 strain types by PFGE, 30 strain types by MLST, 68 strain types by spa typing, and 5 strain types by SCCmec typing. RepPCR had the highest discriminatory index (D) of all methods (D = 0.88), followed by spa typing (D = 0.87), MLST (D = 0.84), PFGE (D = 0.76), and SCCmec typing (D = 0.60). The method with the highest D among MRSA isolates was repPCR (D = 0.64) followed by spa typing (D = 0.45) and MLST (D = 0.44). The method with the highest D among MSSA isolates was spa typing (D = 0.98), followed by MLST (D = 0.93), repPCR (D = 0.92), and PFGE (D = 0.89). Among isolates designated USA300 by PFGE, repPCR was most discriminatory, with 10 distinct strain types identified (D = 0.63). We identified 45 MRSA isolates which were classified as identical by PFGE, MLST, spa typing, and SCCmec typing (USA300, ST8, t008, SCCmec IV, respectively); within this collection, there were 5 distinct strain types identified by repPCR. The typing methods yielded comparable discriminatory power for S. aureus characterization overall; when discriminating among USA300 isolates, repPCR retained the highest discriminatory power. This property is advantageous for investigations conducted in the era of contemporary S. aureus infections.
Project description:Campylobacter jejuni is one of the most common bacterial causes of human gastroenteritis, and recent findings suggest that turkeys are an important reservoir for this organism. In this study, 80 C. jejuni isolates from eastern North Carolina were characterized for resistance to nine antimicrobials, and strain types were determined by fla typing, pulsed-field gel electrophoresis (PFGE) with SmaI and KpnI, and (for 41 isolates) multilocus sequence typing (MLST). PFGE analysis suggested that many of the isolates (37/40 [ca. 93%]) in a major genomic cluster had DNA that was partially methylated at SmaI sites. Furthermore, 12/40 (30%) of the isolates in this cluster were completely resistant to digestion by KpnI, suggesting methylation at KpnI sites. MLST of 41 isolates identified 10 sequence types (STs), of which 4 were new. Three STs (ST-1839, ST-2132 and the new ST-2934) were predominant and were detected among isolates from different farms. The majority of the isolates (74%) were resistant to three or more antimicrobials, and resistance to ciprofloxacin was common (64%), whereas resistance to the other drug of choice for treatment of human campylobacteriosis, erythromycin, was never encountered. Most (33/34) of the kanamycin-resistant isolates were also resistant to tetracycline; however, only ca. 50% of the tetracycline-resistant isolates were also kanamycin resistant. Isolates with certain antimicrobial resistance profiles had identical or closely related strain types. Overall, the findings suggest dissemination of certain clonal groups of C. jejuni isolates in the turkey production industry of this region.