Multilocus Sequence Typing Alleles Research Articles

Development and validation of a random forest algorithm for source attribution of animal and human Salmonella Typhimurium and monophasic variants of S. Typhimurium isolates in England and Wales utilising whole genome sequencing data.

Source attribution has traditionally involved combining epidemiological data with different pathogen characterisation methods, including 7-gene multi locus sequence typing (MLST) or serotyping, however, these approaches have limited resolution. In contrast, whole genome sequencing data provide an overview of the whole genome that can be used by attribution algorithms. Here, we applied a random forest (RF) algorithm to predict the primary sources of human clinical Salmonella Typhimurium (S. Typhimurium) and monophasic variants (monophasic S. Typhimurium) isolates. To this end, we utilised single nucleotide polymorphism diversity in the core genome MLST alleles obtained from 1,061 laboratory-confirmed human and animal S. Typhimurium and monophasic S. Typhimurium isolates as inputs into a RF model. The algorithm was used for supervised learning to classify 399 animal S. Typhimurium and monophasic S. Typhimurium isolates into one of eight distinct primary source classes comprising common livestock and pet animal species: cattle, pigs, sheep, other mammals (pets: mostly dogs and horses), broilers, layers, turkeys, and game birds (pheasants, quail, and pigeons). When applied to the training set animal isolates, model accuracy was 0.929 and kappa 0.905, whereas for the test set animal isolates, for which the primary source class information was withheld from the model, the accuracy was 0.779 and kappa 0.700. Subsequently, the model was applied to assign 662 human clinical cases to the eight primary source classes. In the dataset, 60/399 (15.0%) of the animal and 141/662 (21.3%) of the human isolates were associated with a known outbreak of S. Typhimurium definitive type (DT) 104. All but two of the 141 DT104 outbreak linked human isolates were correctly attributed by the model to the primary source classes identified as the origin of the DT104 outbreak. A model that was run without the clonal DT104 animal isolates produced largely congruent outputs (training set accuracy 0.989 and kappa 0.985; test set accuracy 0.781 and kappa 0.663). Overall, our results show that RF offers considerable promise as a suitable methodology for epidemiological tracking and source attribution for foodborne pathogens.

A multi-provincial Salmonella Typhimurium outbreak in Canada associated with exposure to pet hedgehogs, 2017-2020.

In October 2020, an investigation began in Canada on an outbreak of Salmonella Typhimurium infections of the same strain as a concomitant outbreak in the United States (US) that was linked to pet hedgehogs. The objective of this article is to identify the source of the outbreak, determine if there was a link between the Canadian and US outbreaks and identify risk factors for infection to inform public health interventions. Cases were identified through whole genome sequencing of S. Typhimurium isolates. Information was collected on case exposures, including animal contact. Hedgehog and environmental specimens were tested for S. Typhimurium and a trace back investigation was conducted. There were 31 cases in six provinces, with illness onset dates from June 1, 2017, to October 15, 2020. Median case age was 20 years and 52% were female. Isolates grouped together between 0-46 whole genome multi locus sequence typing allele differences. Of 23 cases with available exposure information, 19 (83%) reported contact with hedgehogs in the seven days prior to symptoms; 15/18 (83%) reported direct contact and 3/18 (17%) reported indirect contact. Trace back investigation did not identify a common source of hedgehogs but uncovered an industry with a complex distribution network. The outbreak strain was detected in samples collected from a hedgehog in one case's home and from a hedgehog in a Québec zoo. Direct and indirect contact with hedgehogs was identified as the source of this S. Typhimurium outbreak. Public health communications aimed to increase awareness about the risks of zoonoses from hedgehogs and shared key hygienic practices to reduce disease transmission.

Molecular Characterisation of Corynebacterium diphtheriae Isolates of Faucial Diphtheria Cases from Assam: A Cross-sectional Study

Introduction: Diphtheria caused by toxigenic Corynebacterium diphtheriae is an acute respiratory infection characterised by pseudomembrane formation in the throat. Although diphtheria is often reported sporadically across India, molecular characterisation of C.diphtheriae strains could play a crucial role in epidemiological investigations. Aim: The present study aimed at molecular characterisation of Corynebacterium diphtheriae isolates of faucial diphtheria cases from Assam, North-East India utilising Multi-Locus Sequence Typing (MLST). Materials and Methods: In this cross-sectional study, three isolates were obtained from three different cluster of cases that occurred across two districts of Assam, India for a period from November 2019 to June 2020. MLST of these three C.diphtheriae isolates were performed in Multidisciplinary Research Unit of Assam Medical College and Hospital by using the 7-gene MLST alleles of C.diphtheriae to determine the Sequence Types (ST). Results: The MLST of the three isolates of this study revealed two distinct STs, ST-588 and ST-576 with multi-clonal lineages with ST-466, reported in C.diphtheriae strains of North Kerala and ST-301 related strains of Bangladesh respectively. Conclusion: In spite of frequent reports of diphtheria cases from Assam, molecular typing of the C.diphtheriae strains found in North-Eastern region of India has not yet been performed. This is one of the initial investigations from this region of India to use MLST for typing C.diphtheriae strains. Therefore, studies related to molecular characterisation of C.diphtheriae strains found throughout India could immensely help in determining the clonality and diversity existing among these strains.

Genotyping Diversity of Pseudomonas aeruginosa Isolates, Isolated from Baquba City

This research aims to assess the similarities and variances between MDR Pseudomonas aeruginosa clinicalisolates from different infections using MLST molecular typing technique in Baquba city. One hundredeighty clinical samples were collected from urinary tract, wounds and burns infections, included (110)urine, (50) wounds and (20) burns. Twenty isolates (11.11%) of Pseudomonas aeruginosa were diagnosedbased on microscopic, morphological, biochemical tests and confirmed by VITEK-2 system. Antibioticsusceptibility test for the twenty isolates was performed against six antibiotics using disk diffusion method,the results showed (100%) resistance for Cephalothin, Ceftazdim (60%), Colistin (35%), Amikacin (25%),Ciprofloxacin (20%), while (0%) resistance for Imipenem. The results also showed that 16 (80%) of isolateswere produced extended spectrum beta-lactamase (ES?L) and did not show their production for Metallobeta-lactamase (M?Ls). Biofilm was detected by Micro-Titer Plate method and the result showed that all theisolates (100%) were biofilm producers. Multi-locus sequence typing (MLST) was used because broadnessand ease of access of MLST database. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA,andtrpE) were sequenced and the results were compared with MLST database and allele profile for eachisolate was used to determine sequence types (STs).New Iraqi bacterial strain has been identified andrecorded in database of MLST (PubMLST) under the name (AS-85U).

1256. New Acquisitions of ET-12 Burkholderia cenocepacia in Adults With Cystic Fibrosis: Role of Whole Genome Sequencing in Outbreak Investigation

BackgroundTransmission of Burkholderia cenocepacia ET-12 strain (ET-12Bc) can cause epidemics in the cystic fibrosis (CF) population. The Toronto Adult CF center currently follows 500 patients; 20% have infection with B. cepacia complex (BCC), including 48 patients infected with ET-12Bc. The center adheres to the 2013 infection prevention and control guidelines and patients are also segregated by clinics. Despite this, there have been 11 new acquisitions of ET-12Bc since 2008. The objective of this study was to describe the investigation of an ET-12Bc outbreak in CF patients, using whole-genome sequencing (WGS).MethodsInvestigations included multilocus sequence typing (MLST) and WGS of 34 isolates (11 new ET-12Bc acquisitions, 18 isolates of known ET-12Bc patients (including all patients with hospital admissions that overlapped with new acquisitions), four isolates from CF patients in the USA and the J2315 reference strain). Each of the seven MLST alleles from ET12Bc strain J2315 was downloaded from PubMLST and used to “Blast” each of the 16 WGS databases. WGS was done using 150 bp paired-end runs on an Illumina HiSeq4000. Single nucleotide polymorphisms (SNPs) between the newly sequenced strains and J2315 were profiled.ResultsTen patients had a hospital admission within the 2 months preceding their first ET-12Bc positive sputum culture, except for one in whom ET-12Bc was detected 12 months following hospital admission. In all isolates, the seven alleles (atpD, gltB, gyrB, recA, lepA, phaC and trpB) matched 100% to sequence type 28 and clonal complex 31, and were identical to J2315. WGS SNP analysis confirmed that transmission occurred from known cases on the unit in 10/11 (90.9%) patients. To date, 8/11 patients with new acquisitions have died (median survival of 95 days).ConclusionOur investigations found epidemiological evidence suggestive of ET-12Bc transmission on the CF unit, which was confirmed by MLST and WGS SNP analysis. Compared with MLST, WGS SNP analysis had better discriminatory power and was well correlated with the identified epidemiological links between patients. Recognition of ET-12Bc transmission with associated high mortality rates has led to a change in our hospital policy. ET-12Bc positive patients will no longer be cared for on the same unit as ET-12Bc negative patients with CF.Disclosures All authors: No reported disclosures.

Rapid and precise alignment of raw reads against redundant databases with KMA

BackgroundAs the cost of sequencing has declined, clinical diagnostics based on next generation sequencing (NGS) have become reality. Diagnostics based on sequencing will require rapid and precise mapping against redundant databases because some of the most important determinants, such as antimicrobial resistance and core genome multilocus sequence typing (MLST) alleles, are highly similar to one another.In order to facilitate this, a novel mapping method, KMA (k-mer alignment), was designed. KMA is able to map raw reads directly against redundant databases, it also scales well for large redundant databases. KMA uses k-mer seeding to speed up mapping and the Needleman-Wunsch algorithm to accurately align extensions from k-mer seeds. Multi-mapping reads are resolved using a novel sorting scheme (ConClave scheme), ensuring an accurate selection of templates.ResultsThe functionality of KMA was compared with SRST2, MGmapper, BWA-MEM, Bowtie2, Minimap2 and Salmon, using both simulated data and a dataset of Escherichia coli mapped against resistance genes and core genome MLST alleles. KMA outperforms current methods with respect to both accuracy and speed, while using a comparable amount of memory.ConclusionWith KMA, it was possible map raw reads directly against redundant databases with high accuracy, speed and memory efficiency.

Prevalence and molecular characterization of Staphylococcus aureus isolated from goats in Chongqing, China

BackgroundStaphylococcus aureus is an important zoonotic pathogen which not only causes significant economic loss in livestock production but also poses a potential threat to public health. Compared with bovine and swine, the information on the colonization of S. aureus in goats is very limited. To understand the prevalence and characteristics of S. aureus in goats, we used the nasal swabs collected from apparently healthy goats to isolate S. aureus, and tested their antimicrobial susceptibility, virulence gene carrying levels, and multilocus sequence typing (MLST).ResultsIn 74 nasal swabs of apparently healthy goats, 32 (43.24%) S. aureus strains were isolated and identified, most of which were susceptible to many antibiotics, except for trimethoprim, furazolidone, amoxicillin, lincomycin and roxithromycin, and the resistance incidence of which were 50%, 40.63%, 37.5%, 28.13%, and 21.88% respectively. All the isolates were methicillin-susceptible S. aureus (MSSA) and mecA-negative. Enterotoxin genes were found in 53.13% of the strains. Of which, sej was the most prevalent (21.88%), followed by seb, sec, and see with the same level (18.75%). The most prevalent combination were seb + see and seb + tst. None of the S. aureus isolates harbored sea, sed, seh, eta and etb. Multilocus sequence typing (MLST) revealed 6 new alleles (aroe-552, aroe-553, glpf-500, pta-440, yqil-482 and yqil-496) and 5 new sequence types (STs) (3431,3440,3444,3445 and 3461). Using eBURST, the 5 STs were assigned to clonal complex 522 (CC522) and a further CC with no predicted ancestor. Phylogenetic analysis of seven concatenated MLST alleles revealed that the 5 STs were grouped into cluster I composed of S. aureus mainly from goats and sheep.ConclusionWe provide the data for prevalence of S. aureus in goats in Chongqing municipality and their characterization which will help in tracking evolution of epidemic strains and their control methods.

Molecular diversity of Wolbachia in Lepidoptera: Prevalent allelic content and high recombination of MLST genes

Wolbachia are common endosymbiotic bacteria of Arthropoda and Nematoda that are ordinarily transmitted vertically in host lineages through the egg cytoplasm. Despite the great interest in the Wolbachia symbiont, many issues of its biology remain unclear, including its evolutionary history, routes of transfer among species, and the molecular mechanisms underlying the symbiont's effect on its host. In this report, we present data relating to Wolbachia infection in 120 species of 13 Lepidoptera families, mostly butterflies, from West Siberian localities based on Multilocus sequence typing (MLST) and the wsp locus and perform a comprehensive survey of the distribution of Wolbachia and its genetic diversity in Lepidoptera worldwide. We observed a high infection incidence in the studied region; this finding is probably also true for other temperate latitude regions because many studied species have broad Palearctic and even Holarctic distribution. Although 40 new MLST alleles and 31 new STs were described, there was no noticeable difference in the MLST allele content in butterflies and probably also in moths worldwide. A genetic analysis of Wolbachia strains revealed the MLST allele core in lepidopteran hosts worldwide, viz. the ST-41 allele content. The key finding of our study was the detection of rampant recombination among MLST haplotypes. High rates of homologous recombination between Wolbachia strains indicate a substantial contribution of genetic exchanges to the generation of new STs. This finding should be considered when discussing issues related to the reconstruction of Wolbachia evolution, divergence time, and the routes of Wolbachia transmission across arthropod hosts.

Whole genome multilocus sequence typing as an epidemiologic tool for Yersinia pestis

Human plague is a severe and often fatal zoonotic disease caused by Yersinia pestis. For public health investigations of human cases, nonintensive whole genome molecular typing tools, capable of defining epidemiologic relationships, are advantageous. Whole genome multilocus sequence typing (wgMLST) is a recently developed methodology that simplifies genomic analyses by transforming millions of base pairs of sequence into character data for each gene. We sequenced 13 US Y. pestis isolates with known epidemiologic relationships. Sequences were assembled de novo, and multilocus sequence typing alleles were assigned by comparison against 3979 open reading frames from the reference strain CO92. Allele-based cluster analysis accurately grouped the 13 isolates, as well as 9 publicly available Y. pestis isolates, by their epidemiologic relationships. Our findings indicate wgMLST is a simplified, sensitive, and scalable tool for epidemiologic analysis of Y. pestis strains.

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison.

Comparison of multilocus sequence typing, RAPD, and MALDI-TOF mass spectrometry for typing of β-lactam–resistant Klebsiella pneumoniae strains

Extended spectrum of β-lactam (ESBL) resistance of Klebsiella pneumoniae has become an increasing problem in hospital infections. Typing of isolates is important to establish the intrahospital surveillance of resistant clones. In this study, the discriminatory potential of randomly amplified polymorphic DNA and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analyses were compared with multilocus sequence typing (MLST) by using 17 β-lactam–resistant K. pneumoniae isolates of different genotypes. MLST alleles were distributed in 8 sequence types (STs). Among ESBL strains of the same ST, the presence of different β-lactamase genes was common. RAPD band patterns also revealed 8 types that corresponded to MLST-defined genotypes in 15 out of 17 cases. MALDI-TOF analysis could differentiate 5 clusters of strains. The results of this work show that RAPD may be usable as a rapid screening method for the intrahospital surveillance of K. pneumoniae, allowing a discrimination of clonally related strains. MALDI-TOF-based typing was not strongly corresponding to genotyping and warrants further investigation.

Population Genetic Structure of Streptococcus pneumoniae in Kilifi, Kenya, Prior to the Introduction of Pneumococcal Conjugate Vaccine

BackgroundThe 10-valent pneumococcal conjugate vaccine (PCV10) was introduced in Kenya in 2011. Introduction of any PCV will perturb the existing pneumococcal population structure, thus the aim was to genotype pneumococci collected in Kilifi before PCV10. Methods and FindingsUsing multilocus sequence typing (MLST), we genotyped >1100 invasive and carriage pneumococci from children, the largest collection genotyped from a single resource-poor country and reported to date. Serotype 1 was the most common serotype causing invasive disease and was rarely detected in carriage; all serotype 1 isolates were members of clonal complex (CC) 217. There were temporal fluctuations in the major circulating sequence types (STs); and although 1-3 major serotype 1, 14 or 23F STs co-circulated annually, the two major serotype 5 STs mainly circulated independently. Major STs/CCs also included isolates of serotypes 3, 12F, 18C and 19A and each shared ≤2 MLST alleles with STs that circulate widely elsewhere. Major CCs associated with non-PCV10 serotypes were predominantly represented by carriage isolates, although serotype 19A and 12F CCs were largely invasive and a serotype 10A CC was equally represented by invasive and carriage isolates. ConclusionsUnderstanding the pre-PCV10 population genetic structure in Kilifi will allow for the detection of changes in prevalence of the circulating genotypes and evidence for capsular switching post-vaccine implementation.

Extensive Diversity of Streptococcus pyogenes in a Remote Human Population Reflects Global-Scale Transmission Rather than Localised Diversification

The Indigenous population of the Northern Territory of Australia (NT) suffers from a very high burden of Streptococcus pyogenes disease, including cardiac and renal sequelae. The aim of this study was to determine if S. pyogenes isolated from this population represent NT endemic strains, or conversely reflect strains with global distribution. emm sequence typing data were used to select 460 S. pyogenes isolates representing NT S. pyogenes diversity from 1987–2008. These isolates were genotyped using either multilocus sequence typing (MLST) or a high resolution melting-based MLST surrogate (Minim typing). These data were combined with MLST data from other studies on NT S. pyogenes to yield a set of 731 MLST or Minim typed isolates for analysis. goeBURST analysis of MLST allelic profiles and neighbour-joining trees of the MLST allele sequences revealed that a large proportion of the known global S. pyogenes MLST-defined diversity has now been found in the NT. Specifically, fully sequence typed NT isolates encompass 19% of known S. pyogenes STs and 43% of known S. pyogenes MLST alleles. These analyses provided no evidence for major NT-endemic strains, with many STs and MLST alleles shared between the NT and the rest of the world. The relationship between the number of known Minim types, and the probability that a Minim type identified in a calendar year would be novel was determined. This revealed that Minim types typically persist in the NT for >1 year, and indicate that the majority of NT Minim types have been identified. This study revealed that many diverse S. pyogenes strains exhibit global scale mobility that extends to isolated populations. The burden of S. pyogenes disease in the NT is unlikely to be due to the nature of NT S. pyogenes strains, but is rather a function of social and living conditions.

Global distribution and diversity of ovine-associated Staphylococcus aureus

Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe (n=153), Africa (n=28), South America (n=14) and Australia (n=1); 172 bovine, 68 caprine and 433 human S. aureus profiles. Overall there were 59 STs and 87 spa types in the 293 ovine isolates; in the 97 new ovine isolates there were 22 STs and 37 spa types, including three novel MLST alleles, four novel STs and eight novel spa types. Three main CCs (CC133, CC522 and CC700) were detected in sheep and these contained 61% of all isolates. Four spa types (t002, t1534, t2678 and t3576) contained 31% of all isolates and were associated with CC5, CC522, CC133 and CC522 respectively. spa types were consistent with MLST CCs, only one spa type (t1403) was present in multiple CCs. The three main ovine CCs have different but overlapping patterns of geographical dissemination that appear to match the location and timing of sheep domestication and selection for meat and wool production. CC133, CC522 and CC700 remained ovine-associated following the inclusion of additional host species. Ovine isolates clustered separately from human and bovine isolates and those from sheep cheeses, but closely with caprine isolates. As with cattle isolates, patterns of clonal diversification of sheep isolates differ from humans, indicative of their relatively recent host-jump.

Prominence of an O75 Clonal Group (Clonal Complex 14) among Non-ST131 Fluoroquinolone-Resistant Escherichia coli Causing Extraintestinal Infections in Humans and Dogs in Australia

Fluoroquinolone (FQ)-resistant extraintestinal pathogenic Escherichia coli (FQ(r) ExPEC) strains from phylogenetic group B2 are undergoing epidemic spread. Isolates belonging to phylogenetic group B2 are generally more virulent than other E. coli isolates; therefore, resistance to FQs among group B2 isolates is concerning. Although clonal expansion of sequence type 131 (ST131) is a major factor, the contribution of additional clonal groups has not been quantified. Group B2 FQ(r) ExPEC isolates from humans (n = 250) and dogs (n = 12) in Australia were screened for ST131, a recently recognized and rapidly emerging multidrug-resistant and virulent clonal group that is important in both human and companion animal medicine. Non-ST131 isolates underwent virulence genotyping, PCR-based O typing, partial multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and FQ resistance mechanism analysis. Of 49 non-ST131 isolates (45 human, 4 canine), 49% (24 human, 2 canine) represented O-type O75 and exhibited conserved virulence genotypes (F10 papA allele, iha, fimH, sat, vat, fyuA, iutA, kpsMII, usp, ompT, malX, K1/K5 capsule) and MLST allele profiles corresponding with clonal complex CC14. Two clusters, each containing canine and human isolates, were identified by PFGE (differentiated by K1 and K5 capsules). Australian FQ(r) O75 isolates exhibited commonality with an historical FQ-susceptible O75 urosepsis isolate (also CC14). The isolation from humans and dogs of highly similar FQ(r) derivatives of the classic O75:K1/K5 (CC14) ExPEC lineage suggests recent acquisition of FQ resistance and potential cross-host-species transfer. This lineage should be targeted with ST131 in future epidemiological investigations of FQ(r) ExPEC.

Concurrent Serotyping and Genotyping of Pneumococci by Use of PCR and Electrospray Ionization Mass Spectrometry

A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ionization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65- to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single- and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates.

Multilocus Sequence Typing of Total-Genome-Sequenced Bacteria

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

Characterisation of novel strains of multiply antibiotic-resistant Salmonella recovered from poultry in Southern Senegal

Non-typhoidal Salmonella (NTS) contamination in poultry and poultry products is a major cause of food-borne disease in humans. This study presents the molecular epidemiology of NTS isolated from poultry in Senegal. A total of 261 NTS recovered from broiler farms, chicken carcasses and street vendors were characterized using random amplification of polymorphic DNA (RAPD) and multilocus sequence typing (MLST) techniques. We observed 20 distinct RAPD profiles corresponding to 18 different serotypes. Strains from each of these 20 groups were further analysed using MLST. Consequently, 12 new MLST alleles and 17 new sequence types were discovered. Three sequence types (S. Kentucky ST198, S. Agona ST13 and S. Istanbul ST33) have previously been described in Senegal and other countries, suggesting that these clones are geographically widely distributed and are circulating in a wide range of hosts. Nine clones showed multi-resistance to the most commonly used antibiotics in both humans and animals. However, a novel multi-resistant clone of S. Kentucky ST832 was found. This study gives new insights into the genetic diversity of NTS in Senegal. Molecular tools remain essential to improve our understanding of the epidemiology of NTS by tracking the sources of infection and/or contamination.

Allele Intersection Analysis: A Novel Tool for Multi Locus Sequence Assignment in Multiply Infected Hosts

Wolbachia are wide-spread, endogenous α-Proteobacteria of arthropods and filarial nematodes. 15–75% of all insect species are infected with these endosymbionts that alter their host's reproduction to facilitate their spread. In recent years, many insect species infected with multiple Wolbachia strains have been identified. As the endosymbionts are not cultivable outside living cells, strain typing relies on molecular methods. A Multi Locus Sequence Typing (MLST) system was established for standardizing Wolbachia strain identification. However, MLST requires hosts to harbour individual and not multiple strains of supergroups without recombination. This study revisits the applicability of the current MLST protocols and introduces Allele Intersection Analysis (AIA) as a novel approach. AIA utilizes natural variations in infection patterns and allows correct strain assignment of MLST alleles in multiply infected host species without the need of artificial strain segregation. AIA identifies pairs of multiply infected individuals that share Wolbachia and differ in only one strain. In such pairs, the shared MLST sequences can be used to assign alleles to distinct strains. Furthermore, AIA is a powerful tool to detect recombination events. The underlying principle of AIA may easily be adopted for MLST approaches in other uncultivable bacterial genera that occur as multiple strain infections and the concept may find application in metagenomic high-throughput parallel sequencing projects.

High-Resolution Melting System to Perform Multilocus Sequence Typing of Campylobacter jejuni

Multi-locus sequence typing (MLST) has emerged as the state-of-the-art method for resolving bacterial population genetics but it is expensive and time consuming. We evaluated the potential of high resolution melting (HRM) to identify known MLST alleles of Campylobacter jejuni at reduced cost and time. Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM. The approach was investigated using 47 C. jejuni isolates, previously characterized by classical MLST, representing isolates from diverse environmental, animal and clinical sources and including the six most prevalent sequence types (ST) and the most frequent alleles. HRM was then applied to a validation set of 84 additional C. jejuni isolates from chickens; 92% of the alleles were resolved in 35 hours of laboratory time and the cost of reagents per isolate was $20 compared with $100 for sequence-based typing. HRM has the potential to complement sequence-based methods for resolving SNPs and to facilitate a wide range of genotyping studies.