Multilocus Typing Research Articles

Multilocus fragment analysis of Cryptosporidium parvum from pre-weaned calves in Colombia

The intra-species genetic diversity of Cryptosporidium parvum in dairy cattle farms in the central area of Colombia was investigated using a multilocus fragment typing approach with nine variable-number tandem-repeat (VNTR) loci and the gp60 gene. Genomic DNA of 70 C. parvum isolates from pre-weaned calves in 32 farms was analysed. Most markers showed two (ML1, MSB, CP47, and MSC6-7) or three alleles (5B12, Cgd2_3850, and Cgd6_5400), although they exhibited a major allele accounting for more than 69% of specimens, which explains their low discriminatory index. The TP14 microsatellite was monomorphic while a total of six alleles were found at the ML2 microsatellite. The two novel allelic variants (219bp, 245bp) exhibited by more than 36% of specimens at the latter locus were a remarkable finding. The 10-markers typing tool provided a Hunter-Gaston discriminatory value of 0.940 (95% CI, 0.918 – 0.961) and differentiated 22 multilocus subtypes (MLTs). Nevertheless, the combination of the three most informative markers (ML2, gp60, and Cgd2_3850) differentiated 68% of MLTs and hardly impaired the discriminatory index. The fact that many MLTs (13/22) were distinctive for individual farms provides evidence for the endemic nature of the infection and the major role played by transmission within farms. The eBURST algorithm suggested a low degree of genetic divergence. All but three MLTs were clustered in a clonal complex with a star-like topology typical of clonal expansion, however linkage analysis did not find evidence of linkage disequilibrium. Bayesian analysis also identified a genetic structure with K = 3 being the best estimation of ancestral clusters, although a large proportion of isolates (35%) could not be allocated to a single population, which indicates their mixed origin. The results confirm the genetic distinctiveness of C. parvum in cattle farms in this geographical area. This is the first multilocus analysis on the intra-specific variability of Cryptosporidium from calves in South America.

Phylogenetics of the Spiroplasma ixodetis endosymbiont reveals past transfers between ticks and other arthropods

The bacterium Spiroplasma ixodetis is a maternally inherited endosymbiont primarily described from ticks but also found widespread across other arthropods. While it has been identified as a male-killing agent in some insect species, the consequences of infection with S. ixodetis in ticks are entirely unknown, and it is unclear how this endosymbiont spreads across tick species. Here, we have investigated this aspect through the examination of the diversity and evolutionary history of S. ixodetis infections in 12 tick species and 12 other arthropod species. Using a multi-locus typing approach, we identified that ticks harbor a substantial diversity of divergent S. ixodetis strains. Phylogenetic investigations revealed that these S. ixodetis strains do not cluster within a tick-specific subclade but rather exhibit distinct evolutionary origins. In their past, these strains have undergone repeated horizontal transfers between ticks and other arthropods, including aphids and flies. This diversity pattern strongly suggests that maternal inheritance and horizontal transfers are key drivers of S. ixodetis spread, dictating global incidence of infections across tick communities. We do not, however, detect evidence of S. ixodetis-based male-killing since we observed that infections were widely present in both males and females across populations of the African blue tick Rhipicephalus decoloratus.

Cutibacterium acnes in primary reverse shoulder arthroplasty: from skin to deep layers

The aim of this study was to determine the presence of Cutibacterium acnes (formerly Propionibacterium acnes) on the skin and in deep tissue in a real clinical scenario of primary reverse shoulder arthroplasty. This prospective study included 90 primary reverse shoulder arthroplasties, and 12 cultures were obtained from each patient. Each sample was homogenized and used to inoculate PolyVitex (bioMérieux, Marcy-l'Etoile, France) agar and Schaedler (bioMérieux) agar plates. The same procedure was also followed with a thioglycolate broth. Culture was considered positive for C acnes when 2 or more colonies were observed. Total DNA from C acnes isolates was extracted using the InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA, USA) method. The phylotype was determined, and single-locus sequence typing was done on all isolates. We obtained 1080 tissue cultures from the 90 patients included, and 62 of those tissue cultures (5.7%) were positive for C acnes. There were 22 C acnes-positive tissue cultures before prosthesis implantation and 40 after implantation. C acnes was isolated in 17 patients (18.8%). We sent 38 positive samples for blinded phylotyping, single-locus sequence typing, and multi-locus sequence typing type determination. Many of the clusters isolated belonged to phylotype IB and clonal complex (CC) 36 or phylotype II and CC53. In the real scenario of patients undergoing primary reverse shoulder arthroplasty using antibiotic prophylaxis and standard preoperative skin preparation with chlorhexidine, C acnes was isolated in the deep layers of 18.8% of the patients. The C acnes K1 and K2 subtypes (belonging to phylotype II and CC53), reported to be commonly involved in prosthetic joint infection, were usually isolated.

Fast and flexible bacterial genomic epidemiology with PopPUNK.

The routine use of genomics for disease surveillance provides the opportunity for high-resolution bacterial epidemiology. Current whole-genome clustering and multilocus typing approaches do not fully exploit core and accessory genomic variation, and they cannot both automatically identify, and subsequently expand, clusters of significantly similar isolates in large data sets spanning entire species. Here, we describe PopPUNK (Population Partitioning Using Nucleotide K-mers), a software implementing scalable and expandable annotation- and alignment-free methods for population analysis and clustering. Variable-length k-mer comparisons are used to distinguish isolates’ divergence in shared sequence and gene content, which we demonstrate to be accurate over multiple orders of magnitude using data from both simulations and genomic collections representing 10 taxonomically widespread species. Connections between closely related isolates of the same strain are robustly identified, despite interspecies variation in the pairwise distance distributions that reflects species’ diverse evolutionary patterns. PopPUNK can process 103–104 genomes in a single batch, with minimal memory use and runtimes up to 200-fold faster than existing model-based methods. Clusters of strains remain consistent as new batches of genomes are added, which is achieved without needing to reanalyze all genomes de novo. This facilitates real-time surveillance with consistent cluster naming between studies and allows for outbreak detection using hundreds of genomes in minutes. Interactive visualization and online publication is streamlined through the automatic output of results to multiple platforms. PopPUNK has been designed as a flexible platform that addresses important issues with currently used whole-genome clustering and typing methods, and has potential uses across bacterial genetics and public health research.

Differential identification of Anaplasma in cattle and potential of cattle to serve as reservoirs of Anaplasma capra, an emerging tick-borne zoonotic pathogen

Bovine anaplasmosis is a tick-borne, infectious, non-contagious disease caused by Anaplasma marginale, A. centrale, A. bovis, and zoonotic A. phagocytophilum. Recently, Anaplasma capra detected in goats was identified as a novel zoonotic pathogen. To determine whether A. capra can infect bovines, we used PCR to differentially diagnose Anaplasma spp. in 1219 South Korean cattle by performing multilocus gene typing and restriction fragment length polymorphism (RFLP). Nucleotide sequencing and phylogenetic analysis detected the 16S rRNA gene of A. bovis and four genes from A. capra in 12 (1.0%) and five (0.4%) cattle, respectively. Supplementary discrimination between A. bovis and A. capra was accomplished by RFLP. The 16S rRNA, msp4, groEL, and gltA genes of A. capra identified in this study had much lower degrees of identity to those in A. centrale and other Anaplasma spp. A. phagocytophilum was not detected in any of the tested cattle. Although the prevalence was low, this study suggests the potential of cattle to serve as reservoirs of A. capra. Thus, further studies are needed to clarify the pathogenesis of A. capra in cattle and its possible involvement in transmission to humans.

Sand bedding as a reservoir for Lactococcus garvieae dissemination in dairy farms.

Lactococcus garvieae is now recognized as a species with clinical significance for human and veterinary medicine. The aim of this study was to evaluate the presence of this pathogen in sand bedding and milk samples. Two farms in Minnesota with problems of clinical and subclinical mastitis due to streptococci-like organisms were selected. Twenty-four Lactococcus garvieae isolates from sand bedding and 18 isolates from quarter milk were comparatively studied using a genotypic approach. RAPD (random amplification of polymorphic DNA) PCR and REP (repetitive element palindromic) PCR experiments highlighted a similar electrophoretic profile. When genes belonging to the core genome of L. garvieae were tested through a MLRT (multilocus restriction typing), we again observed that all L. garvieae isolates coming from sand bedding and milk shared a common profile, distinguishable from previously studied representative L. garvieae strains. These data indicate that the L. garvieae isolated from sand bedding and milk originated from a few strains adapted to persist in the same habitat. This supports the hypothesis that sand bedding can represent a reservoir of L. garvieae strains and be a potential vehicle for their dissemination in dairy farms.

Pertussis Outbreak in a Primary School in China: Infection and Transmission of the Macrolide-resistant Bordetella pertussis.

A pertussis outbreak was studied in a primary school in Xi'an, China, in March 2016. The school consisted of 536 pupils 6-12 years of age who were divided into 12 classes of 6 grades (2 classes for each grade). The identified index case was an 11-year-old girl at class 2 of grade 5. Interview was conducted and nasopharyngeal swabs were taken from all pupils (N = 94) in the 2 classes of grade 5. Nasopharyngeal swabs were tested by both culture and polymerase chain reaction (PCR). Four culture- and 17 PCR-positive cases were identified in 94 pupils. Infection rate was significantly higher in class 2 compared with that in class 1 [37.0% (17/46) vs. 14.6% (7/48), χ(2) = 4.26, P < 0.05]. All Bordetella pertussis isolates were macrolide-resistant, harbored prn1/ptxP1/fim3-1 as previously reported and belonged to multilocus variable tandem repeat analysis type MLVA 195. Of the 17 DNAs positive for diagnostic PCR, 12 were also positive for 23S ribosomal RNA PCR. All the 12 DNAs had the A2047G mutation of 23S rRNA gene of B. pertussis. This study described a pertussis outbreak caused by macrolide-resistant B. pertussis in a primary school and indicated that close contact of index case causes the bacterial transmission.

Multi-locus phylogenetics of the Midichloria endosymbionts reveals variable specificity of association with ticks.

Candidatus Midichloria mitochondrii is a maternally inherited bacterium of ticks with a unique intra-mitochondrial lifestyle. Here, we investigate on the evolutionary history of these associations and the degree of Midichloria-tick specificity. While previous surveys used the 16S rRNA gene as an exclusive molecular marker, we rather developed a multi-locus typing method based on four more variable housekeeping genes (groEL, rpoB, dnaK and ftsZ) and on one flagellum gene (fliC) present in Midichloria genomes. Using this method, multi-locus phylogenetic analyses revealed the structuring of a wide Midichloria genetic diversity into three distinct lineages associated with ticks. Overall, two distinct evolutionary strategies are obvious depending on lineage: two Midichloria lineages are generalists with infections acquired through horizontal transfers between distantly related tick species but one other Midichloria lineage rather show a high specificity degree to the Ixodes tick genus. This pattern suggests a capacity of certain Midichloria strains to maintain infections in only limited range of related tick species. These different infection strategies of Midichloria highlight an unexpected variability in their dependency to their tick hosts. We further conjecture that this pattern is also likely to indicate variability in their effects on ticks.

Epidemiology and Molecular Identification and Characterization of Mycoplasma pneumoniae, South Africa, 2012-2015.

During 2012–2015, we tested respiratory specimens from patients with severe respiratory illness (SRI), patients with influenza-like illness (ILI), and controls in South Africa by real-time PCR for Mycoplasma pneumoniae, followed by culture and molecular characterization of positive samples. M. pneumoniae prevalence was 1.6% among SRI patients, 0.7% among ILI patients, and 0.2% among controls (p<0.001). Age <5 years (adjusted odd ratio 7.1; 95% CI 1.7–28.7) and HIV infection (adjusted odds ratio 23.8; 95% CI 4.1–138.2) among M. pneumonia–positive persons were associated with severe disease. The detection rate attributable to illness was 93.9% (95% CI 74.4%–98.5%) in SRI patients and 80.7% (95% CI 16.7%–95.6%) in ILI patients. The hospitalization rate was 28 cases/100,000 population. We observed the macrolide-susceptible M. pneumoniae genotype in all cases and found P1 types 1, 2, and a type 2 variant with multilocus variable number tandem repeat types 3/6/6/2, 3/5/6/2, and 4/5/7/2.

Comparative analysis of core genome MLST and SNP typing within a European Salmonella serovar Enteritidis outbreak

Multi-country outbreaks of foodborne bacterial disease present challenges in their detection, tracking, and notification. As food is increasingly distributed across borders, such outbreaks are becoming more common. This increases the need for high-resolution, accessible, and replicable isolate typing schemes. Here we evaluate a core genome multilocus typing (cgMLST) scheme for the high-resolution reproducible typing of Salmonella enterica (S. enterica) isolates, by its application to a large European outbreak of S. enterica serovar Enteritidis. This outbreak had been extensively characterised using single nucleotide polymorphism (SNP)-based approaches. The cgMLST analysis was congruent with the original SNP-based analysis, the epidemiological data, and whole genome MLST (wgMLST) analysis. Combination of the cgMLST and epidemiological data confirmed that the genetic diversity among the isolates predated the outbreak, and was likely present at the infection source. There was consequently no link between country of isolation and genetic diversity, but the cgMLST clusters were congruent with date of isolation. Furthermore, comparison with publicly available Enteritidis isolate data demonstrated that the cgMLST scheme presented is highly scalable, enabling outbreaks to be contextualised within the Salmonella genus. The cgMLST scheme is therefore shown to be a standardised and scalable typing method, which allows Salmonella outbreaks to be analysed and compared across laboratories and jurisdictions.

Multilocus typing for characterization of ‘Candidatus Phytoplasma asteris’-related strains in several ornamental species in Italy

Multilocus typing for characterization of ‘<i>Candidatus</i> Phytoplasma asteris’-related strains in several ornamental species in Italy

Epidemiological studies based on multi-locus sequence typing genotype of methicillin susceptible Staphylococcus aureus isolated from camel's milk.

One hundred milk samples were collected from camel’s milk for the isolation of Staphylococcus aureus. Thirty-one isolates were S. aureus, 45 were other forms of staphylococci and 24 represented other bacteria. Five isolates from S. aureus were methicillin resistant S. aureus (MRSA) and 26 samples were methicillin susceptible S. aureus (MSSA). The whole genome sequence of S. aureus was annotated and visualised by rapid annotation using subsystem technology (RAST) which is a fully-automated service for annotating complete or nearly complete bacterial genomes. Four isolates from MSSA strains were subjected to multi-locus sequence typing (MLST). Three multi-locus sequences types or sequence types (MLST/ST) were found, namely ST15, ST1153 and ST130. The phylogenetic analysis of the concatenated sequences of the seven genes forming the MLST profile of S. aureus classification revealed a high degree of similarity and close relationship between the ST15 and ST1153 while the third ST (ST130) was located in a different cluster.

Estandarización del análisis multi-locus de número variable de repeticiones en tándem para el estudio de Staphylococcus aureus resistentes a meticilina aislados de la comunidad en Paraguay

Staphylococcus aureus es un patógeno capaz de causar infecciones con amplio rango de severidad y adaptarse a diferentes tejidos. Su epidemiología es compleja, por circulación de cientos de clones a nivel mundial, lo que requiere de métodos moleculares reproducibles y de alto poder discriminatorio para la identificación de los mismos. El presente estudio tuvo como objetivo principal la estandarización del análisis multi-locus de número variable de repeticiones en tándem (MLVA) para análisis de variabilidad genética de aislados de S. aureus previamente tipificados por electroforesis en gel de campo pulsado (PFGE), gold standard para tipificación de aislados. La MLVA se realizó por amplificación de 7 locus VNTR (clfA, clfB, sdrC, sdrD, sdrE, sspA y spA) por PCR. Se alcanzó un alto nivel de reproducibilidad. El empleo de cepas previamente tipificadas por análisis de secuencias multi-locus (MLST), PFGE, locus spa y cassette SCCmec, permitió validar de forma comparativa el agrupamiento generado por MLVA. Los aislados que fueron agrupados como idénticos por MLVA presentaron resultados congruentes con la totalidad de las otras técnicas moleculares y ésta demostró ser más sensible que PFGE para distinguir entre aislados que presentaron patrones PFGE idénticos. La MLVA cumple todos los criterios de un método de tipificación útil.

Development and evaluation of a core genome multilocus typing scheme for whole-genome sequence-based typing of Acinetobacter baumannii.

We have employed whole genome sequencing to define and evaluate a core genome multilocus sequence typing (cgMLST) scheme for Acinetobacter baumannii. To define a core genome we downloaded a total of 1,573 putative A. baumannii genomes from NCBI as well as representative isolates belonging to the eight previously described international A. baumannii clonal lineages. The core genome was then employed against a total of fifty-three carbapenem-resistant A. baumannii isolates that were previously typed by PFGE and linked to hospital outbreaks in eight German cities. We defined a core genome of 2,390 genes of which an average 98.4% were called successfully from 1,339 A. baumannii genomes, while Acinetobacter nosocomialis, Acinetobacter pittii, and Acinetobacter calcoaceticus resulted in 71.2%, 33.3%, and 23.2% good targets, respectively. When tested against the previously identified outbreak strains, we found good correlation between PFGE and cgMLST clustering, with 0–8 allelic differences within a pulsotype, and 40–2,166 differences between pulsotypes. The highest number of allelic differences was between the isolates representing the international clones. This typing scheme was highly discriminatory and identified separate A. baumannii outbreaks. Moreover, because a standardised cgMLST nomenclature is used, the system will allow inter-laboratory exchange of data.

A Novel Pan-Genome Reverse Vaccinology Approach Employing a Negative-Selection Strategy for Screening Surface-Exposed Antigens against leptospirosis.

Reverse vaccinology (RV) has been widely used for screening of surface-exposed proteins (PSEs) of important pathogens, including outer membrane proteins (OMPs), and extracellular proteins (ECPs) as potential vaccine candidates. In this study, we applied a novel RV negative strategy and a pan-genome analysis for screening of PSEs from 17 L. interrogans strains covering 11 predominately epidemic serovars and 17 multilocus typing (MLST) sequence types (STs) worldwide. Our results showed, for instance, out of a total of 633 predicted PSEs in strain 56601, 92.8% were OMPs or ECPs (588/633). Among the 17 strains, 190 core PSEs, 913 dispensable PSEs and 861 unique PSEs were identified. Of the 190 PSEs, 121 were further predicted to be highly antigenic and thus may serve as potential vaccine candidates against leptospirosis. With the exception of LipL45, OmpL1, and LigB, the majority of the 121 PSEs were newly identified antigens. For example, hypothetical proteins BatC, LipL71, and the OmpA family proteins sharing many common features, such as surface-exposed localization, universal conservation, and eliciting strong antibody responses in patients, are regarded as the most promising vaccine antigens. Additionally, a wide array of potential virulence factors among the predicted PSEs including TonB-dependent receptor, sphingomyelinase 2, leucine-rich repeat protein, and 4 neighboring hypothetical proteins were identified as potential antigenicity, and deserve further investigation. Our results can contribute to the prediction of suitable antigens as potential vaccine candidates against leptospirosis and also provide further insights into mechanisms of leptospiral pathogenicity. In addition, our novel negative-screening strategy combined with pan-genome analysis can be a routine RV method applied to numerous other pathogens.

MICA Gene Deletion in 3411 DNA Samples from Five Distinct Populations in Mainland China and Lack of Association with Nasopharyngeal Carcinoma (NPC) in a Southern Chinese Han population.

Deletion of major histocompatibility complex class I chain-related genes A (MICA*Del) was investigated in 3,411 DNA samples from two southern Chinese Han populations (Hunan Han, HNH; Guangdong Han, GDH), two northern Chinese populations (Inner Mongolia Han, IMH; Inner Mongolia Mongol, IMM) and one southeastern Chinese Han population (Fujian Han, FJH) using an in-house polymerase chain reaction-sequence specific priming (PCR-SSP) assay, which enables direct discrimination between heterozygote and homozygote for MICA*Del. MICA*Del showed a frequency ranging from 0.8% in FJH to 5.7% in IMM (Pcorrected < 0.05), indicating northward increase in frequency of MICA*Del in Chinese populations. In contrast to the association reported recently in a Taiwan Chinese population and aMalaysian Chinese cohort, MICA*Del distribution did not differ between 1,120 patients with nasopharyngeal carcinoma (NPC) and 1,483 normal controls in the HNH population (1.03% in NPC cases vs 1.18% in the controls, OR (95% CI) = 0.87 (0.51-1.47), p = 0.69). Further gender-stratified analysis also failed to disclose any male-specific association reported in a Taiwan Chinese population. Multi-locus typing of the 94 samples carrying MICA*Del revealed two new haplotypes, HLA-A*11:01-B*13:01-MICA*Del-MICB*009N-DRB1*04:06 and HLA-B*35:01-MICA*Del-MICB*009N-DRB1*15:01, in addition to HLA-B*48-MICA*Del. Unexpectedly, two samples with MICA*Del in the HNH population were each consistently found to have two distinct MICA alleles, indicating the existence of two MICA gene copies on certain HLA haplotypes. Based on the results from a sizeable case-control study, our data suggest that there is no association between MICA*Del and NPC in the southern Chinese Han population.

Multilocus typing of Cryptosporidium spp. in young calves with diarrhea in Korea

We assessed the prevalence and performed molecular analysis of Cryptosporidium spp. in diarrheal feces of calves in Korea. Diarrheal fecal samples were collected from 951 young calves (<3months) on 425 farms. Cryptosporidium prevalence was assessed by PCR and ELISA, and molecular characterization was performed by targeting the 18S rRNA, heat-shock protein 70 (hsp70), and glycoprotein 60 (gp60) genes. Data were analyzed according to the sex, type of cattle, region, season, and type of diarrhea. PCR analysis revealed Cryptosporidium spp. in 9.9% (94/951) of diarrheal fecal samples. C. parvum and C. bovis/ryanae were present in 6.1% (58/951) and 4.1% (39/951) of diarrheal fecal samples, respectively. In addition, ELISA showed positive results for C. parvum in 9.7% (92/951) samples. Statistical analysis of the PCR and ELISA results revealed a lower prevalence of C. parvum in the hemorrhagic diarrheal samples (P<0.05). For C. bovis/ryanae, seasonality and high prevalence in hemorrhagic diarrhea were observed (P<0.05). Of the 951 samples tested for C. parvum, 903 samples showed agreement with a κ value of 0.65, indicating good agreement between the two tests. Although C. bovis and C. ryanae share highly similar 18S rRNA sequences, PCR based on hsp70 successfully distinguished C. bovis from C. ryanae. Sequence analysis of gp60 revealed that C. parvum belonged to the IIa families and was further subtyped as IIaA18G3R1 and IIaA16G3R1, which have not been previously reported in Asia. These findings indicate that Cryptosporidium spp. play an important role in diarrhea in young calves in Korea. Considering the zoonotic significance of C. parvum IIa subtype and dense rearing system of cattle in Korea, prevention and continuous monitoring of Cryptosporidium are required.

MetaMLST: multi-locus strain-level bacterial typing from metagenomic samples.

Metagenomic characterization of microbial communities has the potential to become a tool to identify pathogens in human samples. However, software tools able to extract strain-level typing information from metagenomic data are needed. Low-throughput molecular typing schema such as Multilocus Sequence Typing (MLST) are still widely used and provide a wealth of strain-level information that is currently not exploited by metagenomic methods. We introduce MetaMLST, a software tool that reconstructs the MLST loci of microorganisms present in microbial communities from metagenomic data. Tested on synthetic and spiked-in real metagenomes, the pipeline was able to reconstruct the MLST sequences with >98.5% accuracy at coverages as low as 1×. On real samples, the pipeline showed higher sensitivity than assembly-based approaches and it proved successful in identifying strains in epidemic outbreaks as well as in intestinal, skin and gastrointestinal microbiome samples.

Whole genome sequencing of Salmonella Typhimurium illuminates distinct outbreaks caused by an endemic multi-locus variable number tandem repeat analysis type in Australia, 2014.

BackgroundSalmonella Typhimurium (STM) is an important cause of foodborne outbreaks worldwide. Subtyping of STM remains critical to outbreak investigation, yet current techniques (e.g. multilocus variable number tandem repeat analysis, MLVA) may provide insufficient discrimination. Whole genome sequencing (WGS) offers potentially greater discriminatory power to support infectious disease surveillance.MethodsWe performed WGS on 62 STM isolates of a single, endemic MLVA type associated with two epidemiologically independent, food-borne outbreaks along with sporadic cases in New South Wales, Australia, during 2014. Genomes of case and environmental isolates were sequenced using HiSeq (Illumina) and the genetic distance between them was assessed by single nucleotide polymorphism (SNP) analysis. SNP analysis was compared to the epidemiological context.ResultsThe WGS analysis supported epidemiological evidence and genomes of within-outbreak isolates were nearly identical. Sporadic cases differed from outbreak cases by a small number of SNPs, although their close relationship to outbreak cases may represent an unidentified common food source that may warrant further public health follow up. Previously unrecognised mini-clusters were detected.ConclusionsWGS of STM can discriminate foodborne community outbreaks within a single endemic MLVA clone. Our findings support the translation of WGS into public health laboratory surveillance of salmonellosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0831-3) contains supplementary material, which is available to authorized users.

Discovery of new variable number tandem repeat loci in multiple Cryptosporidium parvum genomes for the surveillance and investigation of outbreaks of cryptosporidiosis

Cryptosporidium parvum is a protozoan parasite causing gastro-intestinal disease (cryptosporidiosis) in humans and animals. The ability to investigate sources of contamination and routes of transmission by characterization and comparison of isolates in a cost- and time-efficient manner will help surveillance and epidemiological investigations, but as yet there is no standardised multi-locus typing scheme. To systematically identify variable number tandem repeat (VNTR) loci, which have been shown to provide differentiation in moderately conserved species, we interrogated the reference C. parvum Iowa II genome and seven other C. parvum genomes using a tandem repeat finder software. We identified 28 loci that met criteria defined previously for robust typing schemes for inter-laboratory surveillance, that had potential for generating PCR amplicons analysable on most fragment sizing platforms: repeats ≥6 bp, occurring in tandem in a single repeat region, and providing a total amplicon size of <300 bp including 50 bp for the location of the forward and reverse primers. The qualifying loci will be further investigated in vitro for consideration as preferred loci in the development of a robust VNTR scheme.