Multilocus Sequence Typing Assay Research Articles

Development of molecular assays for the analysis of genetic relationships of Mycoplasma iowae

Mycoplasma iowae is a worldwide spread and economically important avian pathogen that mostly infects turkeys. Currently, multi-locus sequence typing (MLST) serves as the gold standard method for strain identification in M. iowae. However, additional robust genotyping methods are required to effectively monitor M. iowae infections and conduct epidemiological investigations. The first aim of this study was to develop genotyping assays with high resolution, that specifically target M. iowae, namely a multiple-locus variable number of tandem-repeats analysis (MLVA) and a core genome multi-locus sequence typing (cgMLST) schema. The second aim was the determination of relationships among a diverse selection of M. iowae strains and clinical isolates with a previous and the newly developed assays. The MLVA was designed based on the analyses of tandem-repeat (TR) regions in the six serotype reference strains (I, J, K, N, Q and R). The cgMLST schema was developed based on the coding sequences (CDSs) common in 95% of the examined 99 isolates. The samples were submitted for a previously published MLST assay for comparison with the developed methods. Out of 94 TR regions identified, 17 alleles were selected for further evaluation by PCR. Finally, seven alleles were chosen to establish the MLVA assay. Additionally, whole genome sequence analyses identified a total of 676 CDSs shared by 95% of the isolates, all of which were included into the developed cgMLST schema. The MLVA discriminated 19 distinct genotypes (GT), while with the cgMLST assay 79 sequence types (ST) could be determined with Simpson’s diversity indices of 0.810 (MLVA) and 0.989 (cgMLST). The applied assays consistently identified the same main clusters among the diverse selection of isolates, thereby demonstrating their suitability for various genetic analyses and their ability to yield congruent results.

Discrimination of Bacillus subtilis from Other Bacillus Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes.

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

Detection of NDM-5 and MCR-1 antibiotic resistance encoding genes in Enterobacterales in long-distance migratory bird species Ciconia ciconia, Algeria

β-lactams and colistin resistance in Enterobacterales is a global public health issue. In this study we aimed to investigate the occurrence and genetic determinants of Extended-Spectrum β-lactamases, carbapenemases and mcr-encoding-genes in Enterobacterales isolates recovered from the migratory bird species Ciconia ciconia in an Algerian city. A total of 62 faecal samples from white storks were collected. Samples were then subjected to selective isolation of β-lactams and colistin-resistant-Enterobacterales. The representative colonies were identified using Matrix-Assisted Laser Desorption-Ionisation Time-of-Flight Mass Spectrometry. Susceptibility testing was performed using the disk-diffusion method. ESBL, carbapenemases, and colistin resistance determinants were searched for by PCR and sequencing. The clonality relationships of the obtained isolates were investigated by multilocus sequence typing assays. Mating experiments were carried out to evaluate the transferability of the carbapenemase and mcr-genes. Forty-two isolates were identified as follows: Escherichia coli (n = 33), Klebsiella pneumoniae (n = 4), Proteus mirabilis (n = 4) and Citrobacter freundii (n = 1). Molecular analysis showed that twelve isolates carried the blaESBL genes alone, fifteen E. coli isolates were positive for the blaOXA-48 gene, six isolates were NDM-5-carriers (two P. mirabilis, two K. pneumoniae and two E. coli) and eight E. coli strains were positive for the mcr-1 gene. MLST results showed a high clonal diversity, where NDM-5-producing strains were assigned to two sequence types (ST167 for E. coli and ST198 for K. pneumoniae), whereas the mcr-1 positive E. coli isolates belonged to ST58, ST224, ST453, ST1286, ST2973, ST5542, ST9815 and the international high-risk resistant lineage ST101. To the best of our knowledge, this is the first report of blaNDM-5 gene in white storks and also the first describing the mcr-1 gene in white storks in Algeria. This study underlines the important role of migratory white storks as carriers of high-level drug-resistant bacteria, allowing their possible implication as indicators and sentinels for antimicrobial resistance surveillance.

Preponderance of bla KPC-Carrying Carbapenem-Resistant Enterobacterales Among Fecal Isolates From Community Food Handlers in Kuwait.

Carbapenem-resistant Enterobacterales (CRE) are pathogens that have been found in several countries, with a significant public health concern. Characterizing the mode of resistance and determining the prevailing clones are vital to the epidemiology of CRE in our community. This study was conducted to characterize the molecular mode of resistance and to determine the clonality of the CRE fecal isolates among community food handlers (FHs) vs. infected control patients (ICPs) in Kuwait. Fecal CRE isolates obtained from FHs and ICPs from September 2016 to September 2018 were analyzed for their resistance genes. Gene characterization was carried out by polymerase chain reaction (PCR) assays and sequencing. Clonality of isolates was established by multilocus sequence typing (MLST). Of the 681 and 95 isolates of the family Enterobacterales isolated from FHs and ICPs, 425 (62.4%) and 16 (16.8%) were Escherichia coli, and 18 (2.6%) and 69 (72.6%) were Klebsiella pneumoniae, respectively. A total of 36 isolates were CRE with a prevalence of 5.3% among FH isolates and 87 (91.6%) among the ICPs. Of these, carbapenemase genes were detected in 22 (61.1%) and 65 (74.7%) isolates, respectively (p < 0.05). The detected specific genes among FHs and ICPs were positive for blaKPC 19 (86.4%) and 35 (40.2%), and blaOXA 10 (45.5%) and 59 (67.8%), in addition to blaNDM 2 (9.1%) and 32 (36.8%), respectively. MLST assays of the E. coli and K. pneumoniae isolates revealed considerable genetic diversity and polyclonality as well as demonstrated multiple known ST types and eight novel sequence types. The study revealed a relatively high number of CRE harboring predominantly blaKPC-mediated CRE among the community FH isolates vs. predominant blaOXA genes among the ICPs. Those heterogeneous CRE isolates raise concerns and mandate more efforts toward molecular surveillance. A multinational study is recommended to monitor the spread of genes mediating CRE in the community of Arabian Peninsula countries.

Establishment and application of a multilocus sequence typing assay for Corynebacterium striatum

Objective: To establish a multilocus sequence typing (MLST) assay for Corynebacterium (C.) striatum, explore the population structure and evolution relationship of clinical isolates of C. striatum. Methods: Seven housekeeping genes (gyrA, gyrB, hsp65, sodA, secA1, rpoB, 16S rRNA) were amplified with PCR by using self-designed specific primers and sequenced. Then, the sequences were assembled with software SeqMan. The gene diversity and gene recombination characteristics were evaluated by using software DnaSP 5.10.01 and Splits tree 4.14.2. The phylogenetic tree and the minimum spanning tree were constructed based on the sequence types (ST) characteristics by using software MEGA 7.0.14 and BioNumerics, respectively. In addition, the genetic evolutionary relationship among STs were analyzed by using software eBURST 3.0. Results: The expected amplification products of seven sites selected in all the test strains were obtained. Splits tree showed that the clustering of all C. striatum strains was consistent, suggesting that gene recombination is the potential driving force for the evolution of C. striatum. All of the 344 C.striatum strains were divided into 72 STs by MLST and 85.7% of the strains formed clonal complexes. CC19 was the predominant clonal complex, whereas ST16 in the clonal complex was detected in the most strains. ST had a certain geographic clustering and a certain correlation with the isolation time. Conclusions: C. striatum showed high genetic diversity in China and CC19 was the predominant clonal complex. The MLST assay established in this study can be used for the typing of C. striatum, but further improvement is needed.

DEVELOPMENT OF MULTILOCUS SEQUENCE TYPING (MLST) OF ACTINOBACILLUS PLEUROPNEUMNIAE

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae (AP) infection leads to considerable financial loss in the swine industry worldwide. Multilocus sequence typing (MLST), a definitive molecular typing technique that is highly reproducible and is capable of comparing data across laboratories, has not been studied on AP. Therefore, the aim of this work is to develop a MLST assay to characterize AP isolates collected from Taiwan. A total of 85 AP isolates collected from pleuropneumonia of diseased pigs with respiratory symptoms and seven reference isolates from other countries were included for comparison. Seven housekeeping genes (recF, gly, rho, tpi, pyk, recN, and rpo) were selected for sequencing. Capsule types that determine serotypes and Apx toxins were analyzed by the polymerase chain reaction (PCR). The sequencing results showed that the 7 housekeeping genes differentiated the 92 isolates into 14 sequence types that belonged to three major clonal complexes and five singletons by eBURST. The 85 isolates assigned to detection of capsule types and Apx toxins showed that serotype 1 with Apx I, II, IV (55.3%) followed by serotype 5 with Apx I, II, IV (29.4%) were the most prevalent in Taiwan. Notably, serotype 15 was identified for the first time. Clonal complexes based on ST profiles from MLST analysis were highly associated with the distribution of capsule types, suggesting that MLST scheme was sufficient to identify DNA samples directly from AP. Therefore, MLST might be a useful tool for identification and further epidemiological assay of AP.

Multilocus sequence typing of the goose pathogen Mycoplasma anserisalpingitidis

Mycoplasma anserisalpingitidis infection is associated with the inflammation of the genital tract and cloaca, embryo lethality, and decreased egg production in geese, leading to serious economic losses. M. anserisalpingitidis has been detected mainly in Central and Eastern Europe, especially in Hungary, but the pathogen was identified recently in China, predicting it’s worldwide occurrence. In this study, a novel multilocus sequence typing (MLST) scheme was developed to analyse phylogenetic relationships between M. anserisalpingitidis field isolates and clinical specimens originating from different geographical locations.Five loci (atpG, fusA, pgiB, plsY, and uvrA) were selected for the final MLST study. The examined 89 M. anserisalpingitidis samples yielded 76 unique sequence types with a 0.994 Simpson’s index of diversity. The samples were originated from Hungary, Poland, Ukraine, China, and Vietnam. Phylogenetic analysis revealed the existence of three distinct clades (A–C) and six subclades within clade C. Generally, samples originating from the same geographical locations or livestock integration clustered together. Isolates in clade A showed the closest relationships to the M. anatis outgroup due to sequence similarity of the plsY locus. The highest genetic distance was observed in 5C among the subclades of clade C, containing the Asian and some Hungarian field isolates.The developed MLST assay revealed high diversity of the investigated M. anserisalpingitidis samples. The method proved to be a valuable and cost-effective tool for sequence typing of this waterfowl Mycoplasma species, enabling the better understanding of its phylogeny and providing a robust assay for future molecular epidemiological investigations.

Development of a New High-Throughput Multilocus Sequence Typing Method to Monitor Causative Agents of Nosocomial Infections

Background: Nosocomial infections cause 4%–56% mortality in newborns. Several epidemiological studies have shown that transmission of opportunistic pathogens from the sink to the patient, including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Serratia marcescens are associated with nosocomial infections in neonatal intensive care units (NICUs). In this project, we aimed to develop fast, accurate, and high-throughput multilocus sequence typing assays (HiMLST-Illumina) to detect opportunistic pathogens to assess their distribution in the sink environment of NICUs and their transfer to patients. Methods: Genome sequences of P. aeruginosa (n = 45), S. maltophilia (n = 23) and S. marcescens (n = 34) strains were retrieved from public genome databases to build their pangenomes, using the open-source PGAdb-builder server. The core genome was identified for each opportunistic pathogen and was searched for genes displaying the highest polymorphism. The minimal number of loci to include in a HiMLST-Illumina assay was determined by comparing topology of phylogenetic trees of concatenated loci based on genome similarity, computed as the average nucleotide identity (ANI) score. The primers used for HiMLST-Illumina schemes were designed in silico on a conserved domain and were tested on reference strains of each species. Results: Bioinformatics analyses showed that 3–4 loci (&lt;300 base pairs per locus) distinguished strains with the same performances than ANI scores. The assays were tested using opportunistic pathogen isolates and environmental DNA originating from NICU sinks. The HiMLST-Illumina analysis of environmental DNA revealed the presence of at least 1 of the 3 studied opportunistic pathogens in 50% of sampled drains (n = 20). In a previous sampling, P. aeruginosa was isolated on selective culture media before and 48 hours after disinfection of a sink drain with chlorine. S. marcescens was also isolated from another sink 2 weeks after disinfection. Identification of the isolates was confirmed by HiMLST-Illumina analyses and will be typed to compare with clinical isolates. Conclusions: Initial in silico tests predict a high discriminating power of the HiMLST-Illumina method, suggesting that it would be possible to quickly identify strains of interest in a large number of samples. The power of this method is also in the possibility for molecular typing without a need for cultivation. Preliminary results suggest that sinks are readily colonized by opportunistic pathogens. This HiMLST-Illumina scheme will be applied in a 2-year intensive survey of NICUs in 3 hospitals in Montreal to evaluate the performance of new sink designs in limiting bioaerosol production and transmission of opportunistic pathogens to patients.Funding: NoneDisclosures: None

Genotyping Mycoplasma hyorhinis by multi-locus sequence typing and multiple-locus variable-number tandem-repeat analysis

Mycoplasma hyorhinis is a swine pathogen bacterium, which causes significant economic losses. The infection spreads through direct contact between the animals. Powerful genotyping methods like PCR based multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA) are necessary to monitor the infections and to conduct epidemiological investigations; hence supporting the control of the disease. The aims of the present study were to examine M. hyorhinis isolates originating mainly from Hungary with MLST and MLVA developed in the study, and to compare the results of the two typing methods.To characterize 39 M. hyorhinis isolates and the type strain (NCTC 10,130), six house-keeping genes were selected for MLST and six tandem-repeat regions were chosen for MLVA. We were able to differentiate 31 sequence types and 37 genotypes within the 40 analyzed isolates by the MLST and the MLVA, respectively. With the combination of the two newly developed assays all examined isolates were distinguished with the exception of the ones originating from the same animal. The developed MLST assay provided a robust and high resolution phylogenetic tree, while the MLVA system is suitable for the differentiation of closely related isolates from the same farm, hence the assay is appropriate for epidemiologic studies.

Risk factors for the carriage of Streptococcus infantarius subspecies infantarius isolated from African fermented dairy products.

Streptococcus infantarius subsp. infantarius (Sii) has been identified as predominant lactic acid bacteria in spontaneously fermented dairy products (FDPs) in sub-Saharan Africa including Côte d'Ivoire. However, Sii belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC). Most SBSEC members are assumed to be involved as opportunistic pathogens in serious diseases in both humans and animals. A population-based cross-sectional survey, including 385 participants was conducted in Korhogo, northern Côte d'Ivoire, to identify risk factors for Sii fecal carriage, including consumption of local FDPs. A structured questionnaire was used to gather participant's socio-demographic and economic characteristics, their relation to livestock and dietary habits. In addition, fresh stool and milk samples were collected. The identification of Sii was done using a SBSEC-specific PCR assay targeting 16S rRNA and groEL genes. The overall prevalence of SBSEC and Sii carriage was 23.2% (confidence interval CI 95% = 18.9–27.5) and 12.0% (CI 95% = 8.4–15.5) for stool, respectively. Prevalence of Sii was significantly higher in consumers of artisanal butter compared with non-consumers (57.1% vs 10.1%, odds ratio OR: 11.9, 95% CI: 3.9–36.6), as well as in persons handling livestock (OR = 3.9; 95% CI = 1.6–9.3) and livestock primary products (OR = 5.7; 95% CI = 2.3–14.3). The closer contact with livestock was a risk factor for Sii fecal carriage. Sii strains were isolated from fresh and fermented milk products with a prevalence of 30.4% and 45.4%, respectively. Analysis of Sii population structure through the SBSEC multi locus sequence typing assay revealed a close relationship across human and dairy isolates, possibly linked to a Kenyan human isolate. All these outcomes underline the interest of in-depth investigations on the ecology, potential reservoirs and pathways of contamination by Sii at the human-animal-environment interface in comparison to yet to be collected data from Europe, Asia and the Americas to further elucidate the various roles of Sii.

Development of a Multilocus Sequence Typing Assay for Mycoplasma gallisepticum.

Mycoplasma gallisepticum (MG) is the most pathogenic avian mycoplasma species. It affects commercial and noncommercial poultry and wild birds. Current MG sequence typing methods rely on the partial sequence of one or more surface antigen genes. Multilocus sequence typing (MLST), a widely used typing method for many human and animal pathogens, relies on conserved housekeeping genes. Recently, MLST assays have been developed for Mycoplasma synoviae (MS) and Mycoplasma iowae. Additionally, a whole genome-based core genome MLST (cgMLST) assay has been developed for MG and MS. However, cgMLST can be implemented only on pure isolates and cannot be applied to clinical samples. Here, we have developed a seven-locus-based MLST scheme for MG that can be applied directly on clinical samples without the need for isolation. The seven loci were selected from 425 genes recently used for the cgMLST assay. A total of 101 diverse MG samples, including isolates and clinical samples, were typed with the newly developed seven-locus MLST. The phylogeny and discriminatory power of the seven-locus MLST were evaluated and compared with the cgMLST and gene-targeted sequencing methods currently used for MG sequence typing. The seven-locus MLST provided optimum discriminatory power and congruent phylogeny to cgMLST. Additionally, a database for MG MLST was created and is currently available for public use online. This assay will increase accessibility to MG sequence typing and provide a stable and expandable nomenclature compatible with cgMLST. The seven-locus MLST assay represents an important tool for epidemiologic investigation of MG that can contribute to better control and eradication efforts.

Direct culture-independent sequence typing of Taylorella equigenitalis obtained from genital swabs and frozen semen samples from South African horses.

We report herein the use of crude extracts obtained from samples of Taylorella equigenitalis-infected horses for the purpose of multi-locus sequence typing (MLST). Samples (n = 36) were collected from horses in South Africa from 1996 to 2017: 34 from genital swabs (stored at -20°C for 2-3 y) and 2 from cryopreserved raw semen aliquots (stored at -70°C for 18 y) prior to assay. The MLST assay showed a single sequence type (ST), designated ST4, that supported a point introduction and thus a common source for the South African outbreak of contagious equine metritis.

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates.

Mycoplasma gallisepticum is among the most economically significant mycoplasmas causing production losses in poultry. Seven melt-curve and agarose gel-based mismatch amplification mutation assays (MAMAs) and one PCR are provided in the present study to distinguish the M. gallisepticum vaccine strains and field isolates based on mutations in the crmA, gapA, lpd, plpA, potC, glpK, and hlp2 genes. A total of 239 samples (M. gallisepticum vaccine and type strains, pure cultures, and clinical samples) originating from 16 countries and from at least eight avian species were submitted to the presented assays for validation or in blind tests. A comparison of the data from 126 samples (including sequences available at GenBank) examined by the developed assays and a recently developed multilocus sequence typing assay showed congruent typing results. The sensitivity of the melt-MAMA assays varied between 101 and 104M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 103 to 105 template copies/reaction, and no cross-reactions occurred with other Mycoplasma species colonizing birds. The presented assays are also suitable for discriminating multiple strains in a single sample. The developed assays enable the differentiation of live vaccine strains by targeting two or three markers/vaccine strain; however, considering the high variability of the species, the combined use of all assays is recommended. The suggested combination provides a reliable tool for routine diagnostics due to the sensitivity and specificity of the assays, and they can be performed directly on clinical samples and in laboratories with basic PCR equipment.

Genotyping Mycoplasma gallisepticum by multilocus sequence typing

Mycoplasma gallisepticum causes chronic respiratory disease and reproductive disorders in many bird species, resulting in considerable economic losses to the poultry industry. Maintenance of M. gallisepticum-free flocks is the most adequate method to control infection. To this end, monitoring systems and vaccination programs with live vaccine strains are applied worldwide. There is strong demand for efficient epidemiological investigation tools to distinguish M. gallisepticum strains in order to control disease. Up to now, multilocus sequence typing (MLST) has been regarded as gold standard for genotyping bacteria due to its good reproducibility and high discriminatory power.The aim of this study was to develop an MLST assay which can determine phylogenetic distances between M. gallisepticum strains. After analysing more than 30 housekeeping genes, six loci (atpG, dnaA, fusA, rpoB, ruvB, uvrA) were selected for the MLST assay due to their genomic location and high diversity.Examination of 130 M. gallisepticum strains with this MLST method yielded 57 unique sequence types (STs) with a 0.96 Simpson’s index of diversity.Considering the large number of STs and high diversity index, this MLST method was found to be appropriate to discriminate M. gallisepticum strains. In addition, the developed method was shown to be suitable for epidemiological investigations, as it confirmed linkage between related strains from outbreaks in different farms. Besides, MLST also suggested high impact of extensive international trade on the spread of different M. gallisepticum strains. Furthermore this method can be used for differentiation among vaccine and field strains.

Genotyping Mycoplasma synoviae: Development of a multi-locus variable number of tandem-repeats analysis and comparison with current molecular typing methods

Control of one of the most important avian mycoplasmas, Mycoplasma synoviae, and tracing the spread of the infection can be challenging as the pathogen is transmissible by both horizontal and vertical routes, and it can be disseminated through long distances via the hatching eggs, day-old chicks or pullets during intensive international trade. Genetic information provided by molecular typing methods support control programmes and epizootiologic studies. The aims of the present study were to develop a multi-locus variable number of tandem-repeats analysis (MLVA) method for the typing of M. synoviae isolates and to evaluate the currently used molecular typing methods which are applicable directly on clinical samples.Tandem repeat (TR) regions were selected from the whole genome sequence of the M. synoviae type strain (WVU1853) to characterise the genetic diversity of 86 M. synoviae strains originating from 15 countries. The strains were also submitted to multi-locus sequence typing (MLST) assays, vlhA gene sequence analysis and to assays designed to differentiate live vaccine strains from field strains.The developed MLVA involves the examination of seven TR regions and provides similar genetic resolution as the tested MLST assays by identifying 35 genotypes among the tested strains. Differentiation of the live vaccine strains from field strains was also successful with the developed assay.The provided MLVA method proved to be a highly discriminative, rapid and cost-effective alternative typing technique for the genetic characterisation of M. synoviae and it is also suitable for the complementation of live vaccine strain differentiating assays in ambiguous cases.

An Extended Multilocus Sequence Typing (MLST) Scheme for Rapid Direct Typing of Leptospira from Clinical Samples.

BackgroundRapid typing of Leptospira is currently impaired by requiring time consuming culture of leptospires. The objective of this study was to develop an assay that provides multilocus sequence typing (MLST) data direct from patient specimens while minimising costs for subsequent sequencing.Methodology and FindingsAn existing PCR based MLST scheme was modified by designing nested primers including anchors for facilitated subsequent sequencing. The assay was applied to various specimen types from patients diagnosed with leptospirosis between 2014 and 2015 in the United Kingdom (UK) and the Lao Peoples Democratic Republic (Lao PDR). Of 44 clinical samples (23 serum, 6 whole blood, 3 buffy coat, 12 urine) PCR positive for pathogenic Leptospira spp. at least one allele was amplified in 22 samples (50%) and used for phylogenetic inference. Full allelic profiles were obtained from ten specimens, representing all sample types (23%). No nonspecific amplicons were observed in any of the samples. Of twelve PCR positive urine specimens three gave full allelic profiles (25%) and two a partial profile. Phylogenetic analysis allowed for species assignment. The predominant species detected was L. interrogans (10/14 and 7/8 from UK and Lao PDR, respectively). All other species were detected in samples from only one country (Lao PDR: L. borgpetersenii [1/8]; UK: L. kirschneri [1/14], L. santarosai [1/14], L. weilii [2/14]).ConclusionTyping information of pathogenic Leptospira spp. was obtained directly from a variety of clinical samples using a modified MLST assay. This assay negates the need for time-consuming culture of Leptospira prior to typing and will be of use both in surveillance, as single alleles enable species determination, and outbreaks for the rapid identification of clusters.

Development of multilocus sequence typing (MLST) assay for Mycoplasma iowae

Mycoplasma iowae (MI) infection is an economically and commercially important disease of turkeys. There are no sequence typing assays available for MI strain identification, the only available molecular tools for this purpose, are DNA fingerprinting assays. In addition to their low reproducibility, fingerprinting assays require isolation of the microorganism in pure culture, which is difficult for avian mycoplasma. Therefore, we propose a multilocus sequence typing (MLST) assay as the first genotyping assay for identification of MI. Based on the two available MI genomes on GenBank, 26 loci of housekeeping genes were identified and studied in a diverse sample set. Finally, six genes were selected for the newly developed MLST assay. The final sequence analysis of the six loci (total of 5019bp) (dppC, ulaA, valS, rpoC, leuS, kdpA) allowed the differentiation of 47 MI samples into 23 unique sequence types. Moreover, when only 4 loci were used to type the same set of samples, they resulted in 20 unique sequence types. Analysis of phylogenetic trees and clonal groups generated by MLST displayed a high degree of agreement with geographical and temporal information of the tested samples. MLST is a highly reproducible molecular epidemiology assay that can be used to identify positive clinical cases directly from DNA samples. Therefore, it provides a useful tool allowing for better identification, control and eradication efforts.

Prevalence, Distribution, and Development of an Ecological Niche Model of Dermacentor variabilis Ticks Positive for Rickettsia montanensis.

Rickettsia montanensis has long been considered a nonpathogenic member of the spotted fever group rickettsiae. However, the infection potential of R. montanensis is being revisited in light of its recent association with a case of human infection in the United States and the possibility that additional cases may have been misdiagnosed as Rocky Mountain spotted fever. To this end, DNA was extracted from American dog ticks (Dermacentor variabilis) removed from Department of Defense (DoD) personnel and their dependents at DoD medical treatment facilities (MTFs) during 2002-2012 (n = 4792). These 4792 samples were analyzed for the presence of R. montanensis (n = 36; 2.84%) and all vector DNA was confirmed to be of D. variabilis origin using a novel Dermacentor genus-specific quantitative real-time polymerase chain reaction procedure, Derm, and a novel Dermacentor species multilocus sequence typing assay. To assess the risk of R. montanensis infection, the positive and negative samples were geographically mapped utilizing MTF site locations. Tick localities were imported into a geographical information systems (GIS) program, ArcGIS, for mapping and analysis. The ecological niche modeling (ENM) program, Maxent, was used to estimate the probability of tick presence in eastern United States using locations of both R. montanensis-positive and -negative ticks, climate, and elevation data. The ENM for R. montanensis-positive D. variabilis estimated high probabilities of the positive ticks occurring in two main areas, including the northern Midwest and mid-Atlantic portions of the northeastern regions of United States, whereas the R. montanensis-negative D. variabilis tick model showed a wider estimated range. The results suggest that R. montanensis-positive and -negative D. variabilis have different ranges where humans may be at risk and are influenced by similar and different factors.

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA.

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.

Analysis of the genetic distribution among members of Clostridium botulinum group I using a novel multilocus sequence typing (MLST) assay

Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partial sequencing of the adk, atpH, gyrB, proC, rpoD and spo0A genes from 51 various C. botulinum/sporogenes isolates was performed, resulting in 37 different sequence types (STs). Analysis of the sequence data revealed a genetic distribution in five larger clusters with a loose correlation to the BoNT serotypes. The developed MLST assay had a slightly lower resolution ability when compared to the MLVA (multilocus variable number of tandem repeat analysis), but the two methods resulted in similar subclusters of the strains possessing the BoNT serotypes A, B and F. The current work presents the development of a novel MLST assay useful for genotyping C. botulinum related to basic phylogenetic research and trace-back analysis in microbial forensic studies.