Variable Number Tandem Repeat Loci Research Articles

An Outbreak of Pseudomonas Aeruginosa Infection Linked to a "Black Friday" Piercing Event.

IntroductionOutbreaks linked to cosmetic piercing are rare, but can cause significant illness. We report the investigation and management of a point-source outbreak that occurred during a Black Friday promotional event in North West England.MethodsOutbreak investigation was led by Public Health England, and included active case finding among individuals pierced at a piercing premises between 25/11/2016 (Black Friday) and 7/12/2016. Detailed epidemiological, environmental (including inspection and sampling), and microbiological investigation was undertaken.ResultsDuring the Black Friday event (25/11/2016), 45 people were pierced (13 by a newly-appointed practitioner). Eleven cases were identified (7 microbiologically-confirmed, 2 probable, and 2 possible). All cases had clinical signs of infection around piercing sites, and five required surgical intervention, with varying degrees of post-operative disfigurement. All confirmed and probable cases had a scaffold piercing placed with a guide bar by the newly-appointed practitioner. Pseudomonas aeruginosa, indistinguishable at nine-locus variable-number tandem repeat loci, was isolated from four of the confirmed cases, and from pre- and post-flush samples from five separate water taps (three sinks) in the premises. Water samples taken after remedial plumbing work confirmed elimination of Pseudomonas contamination.DiscussionAlthough high levels of Pseudomonas water contamination and some poor infection control procedures were identified, infection appeared to require additional exposure to an inexperienced practitioner, and the more invasive scaffold piercing. A proactive collaborative approach between piercers and health and environmental officials is required to reduce outbreak risk, particularly when unusually large events are planned

Evolution of Variable Number Tandem Repeats and Its Relationship with Genomic Diversity in Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is the most common Salmonella serovar causing human infections in Australia and many other countries. A total of 12,112 S. Typhimurium isolates from New South Wales were analyzed by multi-locus variable number of tandem repeat (VNTR) analysis (MLVA) using five VNTRs from 2007 to 2014. We found that mid ranges of repeat units of 8–14 in VNTR locus STTR5, 6–13 in STTR6, and 9–12 in STTR10 were always predominant in the population (>50%). In vitro passaging experiments using MLVA type carrying extreme length alleles found that the majority of long length alleles mutated to short ones and short length alleles mutated to longer ones. Both data suggest directional mutability of VNTRs toward mid-range repeats. Sequencing of 28 isolates from a newly emerged MLVA type and its five single locus variants revealed that single nucleotide variation between isolates with up to two MLVA differences ranged from 0 to 12 single nucleotide polymorphisms (SNPs). However, there was no relationship between SNP and VNTR differences. A population genetic model of the joint distribution of VNTRs and SNPs variations was used to estimate the mutation rates of the two markers, yielding a ratio of 1 VNTR change to 6.9 SNP changes. When only one VNTR repeat difference was considered, the majority of pairwise SNP difference between isolates were 4 SNPs or fewer. Based on this observation and our previous findings of SNP differences of outbreak isolates, we suggest that investigation of S. Typhimurium community outbreaks should include cases of 1 repeat difference to increase sensitivity. This study offers new insights into the short-term VNTR evolution of S. Typhimurium and its application for epidemiological typing.

The Majority of Genotypes of the Virulence Gene inlA Are Intact among Natural Watershed Isolates of Listeria monocytogenes from the Central California Coast.

Internalin A is an essential virulence gene involved in the uptake of the foodborne pathogen Listeria monocytogenes into host cells. It is intact in clinical strains and often truncated due to Premature Stop Codons (PMSCs) in isolates from processed foods and processing facilities. Less information is known about environmental isolates. We sequenced the inlA alleles and did Multi Locus Variable Number Tandem Repeat Analysis (MLVA) on 112 L. monocytogenes isolates from a 3-year period from naturally contaminated watersheds near a leafy green growing area in Central California. The collection contained 14 serotype 1/2a, 12 serotype 1/2b, and 86 serotype 4b strains. Twenty-seven different inlA alleles were found. Twenty-three of the alleles are predicted to encode intact copies of InlA, while three contain PMSCs. Another allele has a 9-nucleotide deletion, previously described for a clinical strain, indicating that it is still functional. Intact inlA genes were found in 101 isolates, and 8 isolates contained the allele predicted to contain the 3-amino acid deletion. Both allele types were found throughout the 3-year sampling period. Three strains contained inlA alleles with PMSCs, and these were found only during the first 3 months of the study. SNP analysis of the intact alleles indicated clustering of alleles based on serotype and lineage with serotypes 1/2b and 4b (lineage I strains) clustering together, and serotype 1/2a (lineage II strains) clustering separately. The combination of serotype, MLVA types, and inlA allele types indicate that the 112 isolates reflect at least 49 different strains of L. monocytogenes. The finding that 90% of environmental L. monocytogenes isolates contain intact inlA alleles varies significantly from isolates found in processing plants. This information is important to public health labs and growers as to the varieties of L. monocytogenes that could potentially contaminate fresh produce in the field by various means.

Longitudinal study of Salmonella 1,4,[5],12:i:- shedding in five Australian pig herds

The shedding patterns of Salmonella spp. and MLVA profiles of Salmonella enterica subspecies enterica (I) serotype 1,4,[5],12:i:- were monitored in a 12-month longitudinal observational study of five pig herds to inform management; provide indications of potential hazard load at slaughter; and assist evaluation of MLVA for use by animal and public health practitioners. Twenty pooled faecal samples, stratified by age group, were collected quarterly. When Salmonella was cultured, multiple colonies were characterized by serotyping and where S. Typhimurium-like serovars were confirmed, isolates were further characterized by phage typing and multiple locus variable number tandem repeat analysis (MLVA). Salmonella was detected in 43% of samples. Salmonella 1,4,[5],12:i- was one of several serovars that persisted within the herds and was found among colonies from each production stage. Virtually all Salmonella 1,4,[5],12:i:- isolates were phage type 193, but exhibited 12 different, closely-related MLVA profiles. Salmonella 1,4,[5],12:i:- diversity within herds was low and MLVA profiles were stable indicating colonization throughout the herds and suggesting each farm had an endemic strain. High prevalence of S. 1,4,[5],12:i:- specific shedding among terminal animals indicated high hazard load at slaughter, suggesting that primary production may be an important pathway of S. 1,4,[5],12:i:- into the human food chain, this has implications for on-farm management and the application and targeting control measures and further evidence of the need for effective process control procedures to be in place during slaughter and in pork boning rooms. These findings have implications for animal health and food safety risk mitigation and risk management.

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.

Genotyping of Klebsiella Pneumonia Strains Isolated from Eldly Inpatients by Multiple-locus Variable-number Tandem-repeat Analysis.

Objective To investigate the genotype of klebsiella pneumonia strains isolated from eldly inpatients by multiple-locus variable-number tandem-repeat analysis. Methods Totally 184 klebsiella pneumonia strains,isolated from eldly inpatients,were collected,and their genome DNA were extracted. The polymorphism of 7 variable-number tandem-repeat locus in the DNA samples was analyzed by multiple primers polymerase chain reaction and capillary electrophoresis. The clustering analysis of genotyping was carried out with the BioNumerics 5.1 software. Results A total of 139 genotypes were identified in 184 klebsiella pneumonia clinical strains,showing obvious genetic polymorphisms. With clustering analysis of genotypes,all the strains were categorized into three gene clusters (genogroups 1,2,and 3). The genogroup 1 was the biggest cluster,containing 93.06% of the isolated strains. Conclusion There was a predominant cluster in the klebsiella pneumonia strains isolated from eldly inpatients in our center,and the major source of klebsiella pneumonia infection remained the nosocomial infection.

Feasibility of Using Multiple-Locus Variable-Number Tandem-Repeat Analysis for Epidemiology Study of Vibrio parahaemolyticus Infections

Vibrio parahaemolyticus causes foodborne gastroenteritis, which is often associated with the consumption of raw or undercooked shellfish. Molecular typing can provide critical information for detecting outbreaks and for source attribution. In this study, we describe the development and evaluation of an optimized multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) for the characterization of V. parahaemolyticus isolates. The discriminatory power of MLVA was compared to that of pulsed-field gel electrophoresis (PFGE) by typing 73 sporadic isolates. Epidemiologic concordance was evaluated by typing 23 isolates from five epidemiologically well-characterized outbreaks. The optimized MLVA was applied in early warning, epidemiological surveillance, and source tracking for V. parahaemolyticus infections. There was no significant difference in the discriminatory power of PFGE and MLVA with six or eight VNTR loci for the sporadic isolates. All isolates within an outbreak were indistinguishable by MLVA with six loci, except for one outbreak. Typically, the epidemiological survey could be initiated according to PFGE clusters. We applied MLVA with six loci on 22 isolates in two PFGE clusters. Isolates in one PFGE cluster were distinguished by MLVA. Although a follow-up investigation showed that both clusters had no epidemiological concordance, MLVA decreased the frequency of initiation of epidemiological surveys, thereby reducing labor costs. The ability of MLVA to trace the source of infection was evaluated by isolates from two outbreaks and shrimp samples. The isolates from one of outbreaks and a shrimp had the same MLVA type, suggesting that an epidemiological survey was initiated. Data from the epidemiological investigation subsequently indicated that contaminated shrimp from a nearby city (Dongguan) might be the source of the outbreak. In conclusion, these results indicate that the optimized MLVA may be a promising tool for early warning and epidemiological surveillance of V. parahaemolyticus infections.

State of Chimerism as a Promising Tool for Follow up of Hematopoietic Stem Cell Transplantation in Egyptian Patients

Follow up of patients after HSCT by chimerism detection can detect the outcome either engraftment, relapse or rejection. Many variables affect the result of chimerism either increasing or decreasing chimerism. Lineage specific chimerism is specific and sensitive than chimerism detection by whole blood. Here in this study the importance of these variables on the outcome of chimerism detection is studied. Subject and method: This study was conducted at Ain Shams University Hospital during the periodbetween January 2011and January 2015. 46 patients were included (16 child and 30 adult). They weretransplanted for malignant and non malignant hematological diseases from totally matched siblings.Themedian duration of follow-up was one year. Chimerism status was detected for all patients by polymerase chain reaction based on amplification of variable number tandem repeat (VNTR) markers. Six VNTR loci were detected in every subject pre-transplant in order to detect an informative locus to be used in follow uppost-transplant. Also lineage specific chimerism and dilution experiment were conducted on some patients. Results: One patient showed no informative locus pre-transplantation, 37 patients showed complete donor chimersm, two patients retained recipient pattern post-transplantation and six patients showed MC. Lineage cell specific chimerism and dilution experiment were conducted for patients who showed failure or mixed chimerism. Conclusion: Chimerism detection by VNTR is dependable method to follow up patients after BMT however,some cases need some modifications as repeated analysis, short duration, lineage specific in comparison to whole blood analysis, dilution experiments in order to predict the outcome.

Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks.

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.

A Simple Variable Number of Tandem Repeat-Based Genotyping Strategy for the Detection of Handling Errors and Validation of Sample Identity in Biobanks.

Biobanking biological samples involve multiple handling, processing, and labeling steps. Each step may be a source of error, which if unnoticed or uncorrected may have consequences for research. We aimed to develop a simple and inexpensive genotyping method that would be valuable to detect such errors and confirm sample identity. For this purpose, seven variable number of tandem repeat (VNTR) loci were selected, analyzed by polymerase chain reaction (PCR) amplification, and organized in a PCR-based DNA profiling algorithm that proved useful to minimize the number of steps required for the procedure. Match probability calculations suggest that this method/algorithm has the potential to discriminate every participant of a biobank. As a proof of concept, the algorithm was applied on samples taken from the PROCURE Prostate Cancer Biobank. It was applied on 403 DNA samples from 101 randomly chosen patients who provided prostate tissues at surgery and blood at two to three different time points over a period of up to 7 years. A unique DNA profile requiring the analysis of no more than four VNTR loci (D16S83, D17S5, D1S80, D19S20) was successfully obtained for each of the 101 cases studied and led to the identification of two mismatches among the 403 samples evaluated (0.5% error rate). Further investigations using the same genotyping method revealed that one of the errors was due to tissue mishandling and that the other was due to tissue mislabeling. These errors, typical to the complex biobanking process, highlight the importance to implement a routine genotyping method as part of quality assurance in biobanking.

Identification of the Rare, Four Repeat Allele of IL-4 Intron-3 VNTR Polymorphism in Indian Populations.

Cytokines are cell signaling molecules which upon release by cells facilitate the recruitment of immune-modulatory cells towards the sites of inflammation. Genetic variations in cytokine genes are shown to regulate their production and affect the risk of infectious as well as autoimmune diseases. Intron-3 of interleukin-4 gene (IL-4) harbors 70-bp variable number of tandem repeats (VNTR) that may alter the expression level of IL-4 gene. To determine the distribution of IL-4 70-bp VNTR polymorphism in seven genetically heterogeneous populations of Chhattisgarh, India and their comparison with the finding of other Indian and world populations. A total of 371 healthy unrelated individuals from 5 caste and 2 tribal populations were included in the present study. The IL-4 70-bp VNTR genotyping was carried out using PCR and electrophoresis. Overall, 3 alleles of IL-4 70-bp VNTR (a2, a3 and a4) were detected. The results demonstrated the variability of the IL-4 70-bp VNTR polymorphism in Chhattisgarh populations. Allele a3 was the most common allele at the 70-bp VNTR locus in all populations followed by a2 allele. This study reports the presence four repeat allele a4 at a low frequency in the majority of the Chhattisgarh populations studied. Further, the frequency of the minor allele (a2) in Chhattisgarh populations showed similarity with the frequencies of European populations but not with the East Asian populations where the a2 allele is a major allele. Our study provides a baseline for future research into the role of the IL-4 locus in diseases linked to inflammation in Indian populations.

Development of a novel multi-locus variable-number tandem repeat analysis (MLVA) combined with high resolution melting analysis (HRMA) for Listeria innocua typing and its application in a food processing plant

Listeria monocytogenes is the causative bacteria of listeriosis, a type of food poisoning with high mortality, which mainly infects people through the consumption of ready-to-eat food. Most cases of L. monocytogenes contamination have occurred at food processing plants. Therefore, food companies must carefully monitor the proliferation and spread of this bacterium. Listeria innocua has often been reported to exist in food processing plants when the presence of the genus Listeria is investigated. In addition, because L. innocua and L. monocytogenes are known to act together, L. innocua is an important hygiene indicator for food companies. Multilocus variable-number tandem repeat analysis (MLVA) is a method used to discriminate between strains, based on the discrepancies in the number of repeating units within the tandem repeats (TRs). In many cases, MLVA uses capillary electrophoresis (CE) or base sequence analysis. However, the equipment used for these analyses is expensive, and requires a certain level of knowledge and skill for operation. Therefore, it is only used at research facilities. However, in order to rapidly trace the sources of contamination in food processing plants, it is necessary to easily discriminate between strains within the working areas. Therefore, in this study, we established a simpler MLVA method, using high resolution melting analysis (HRMA) to analyze the diversity of variable-number of tandem repeats (VNTRs). We investigated three VNTR loci (used in a previous MLVA-CE analysis) using MLVA-HRMA. The MLVA-HRMA of 23 L. innocua strains showed that this method differentiates between strains at a more or less equal level with MLVA, which uses base sequence analysis. HRMA is also used to investigate gene polymorphisms based on the Tm value of the PCR products. Therefore, a different peak pattern is observed depending on the internal SNP even if the number of repeating units are the same. We then evaluated the efficacy of MLVA-HRMA in differentiating between 96 strains isolated at food processing plants. This analysis demonstrated the usefulness of the MLVA-HRMA method and its discriminatory ability in tracing the sources of contamination in food processing plants. Since this method is simpler, and cheaper than the existing MLVA methods, it could see widespread use in food processing plants as a rapid investigative tool.

Molecular Characterization of Shigella sonnei: An Increasingly Prevalent Etiologic Agent of Shigellosis in Guizhou Province, Southwest of China.

BackgroundShigellosis is a serious problem in Guizhou and Shigella sonnei is an increasingly prevalent etiologic agent of local shigellosis cases. No data, however, are available about the molecular characterization of the local isolates of S. sonnei. We have conducted this study to molecularly characterize the clinical isolates of S. sonnei in Guizhou Province.Results76 S. sonnei isolates, including four isolates from 1974–1982 and 72 isolates from 2008–2010, were used for analysis in this study. Pulsed-field gel electrophoresis (PFGE) based on XbaI digestion divided the 76 isolates into 38 PFGE patterns (PT) and 15 PTs were represented by more than one isolates with PT31 (N = 8) containing the most number of isolates, followed with PT2 (N = 6). Multiple-Locus Variable number tandem repeat (VNTR) Analysis (MLVA) based on seven VNTR loci discriminated them into 19 different MLVA types (MTs), and four MTs were represented by more than one isolate with MT4 (N = 39) containing the most number of isolates, followed with MT12 (N = 18). 15 Multilocus sequence typing (MLST) base on 15 loci differentiated the isolates into six sequence types (STs), among which four STs were novel. The most common STs are ST76 (N = 43) and ST116 (N = 25), accounting for 92.1%. Correlation between genetic relationships and geographical origins or isolation years was observed among the isolates studied. Majority of isolates were clustered in accordance with the origin of isolation years based on the genetic data, which were also from similar geographical origins.ConclusionsOur results revealed the molecular characteristics including the specific genotypes such as four novel STs, clonal relationship, and genetic changes of local isolates from different years, which enhances our understanding of molecular characteristics of S. sonnei and contributes to the prevention and control of shigellosis in Guizhou Province.

Intra-Species Genetic Diversity and Clonal Structure of Cryptosporidium parvum in Sheep Farms in a Confined Geographical Area in Northeastern Spain.

A multilocus fragment typing approach including eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to investigate the intra-farm and intra-host genetic diversity of Cryptosporidium parvum in sheep farms in a confined area in northeastern Spain. Genomic DNA samples of 113 C. parvum isolates from diarrheic pre-weaned lambs collected in 49 meat-type sheep farms were analyzed. Loci exhibited various degrees of polymorphism, the finding of 7–9 alleles in the four most variable and discriminatory markers (ML2, Cgd6_5400, Cgd6_3940, and GP60) being remarkable. The combination of alleles at the twelve loci identified a total of 74 multilocus subtypes (MLTs) and provided a Hunter-Gaston discriminatory index of 0.988 (95% CI, 0.979−0.996). The finding that most MLTs (n = 64) were unique to individual farms evidenced that cryptosporidial infection is mainly transmitted within sheep flocks, with herd-to-herd transmission playing a secondary role. Limited intra- host variability was found, since only five isolates were genotypically mixed. In contrast, a significant intra-farm genetic diversity was seen, with the presence of multiple MLTs on more than a half of the farms (28/46), suggesting frequent mutations or genetic exchange through recombination. Comparison with a previous study in calves in northern Spain using the same 12-loci typing approach showed differences in the identity of major alleles at most loci, with a single MLT being shared between lambs and calves. Analysis of evolutionary descent by the algorithm eBURST indicated a high degree of genetic divergence, with over 41% MLTs appearing as singletons along with a high number of clonal complexes, most of them linking only two MLTs. Bayesian Structure analysis and F statistics also revealed the genetic remoteness of most C. parvum isolates and no ancestral population size was chosen. Linkage analysis evidenced a prevalent pattern of clonality within the parasite population.

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

Multiple-locus variable-number tandem repeat analysis for strain discrimination of non-O157 Shiga toxin-producing Escherichia coli

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of growing concern worldwide that have been associated with several recent multistate and multinational outbreaks of foodborne illness. Rapid and sensitive molecular-based bacterial strain discrimination methods are critical for timely outbreak identification and contaminated food source traceback. One such method, multiple-locus variable-number tandem repeat analysis (MLVA), is being used with increasing frequency in foodborne illness outbreak investigations to augment the current gold standard bacterial subtyping technique, pulsed-field gel electrophoresis (PFGE). The objective of this study was to develop a MLVA assay for intra- and inter-serogroup discrimination of six major non-O157 STEC serogroups—O26, O111, O103, O121, O45, and O145—and perform a preliminary internal validation of the method on a limited number of clinical isolates. The resultant MLVA scheme consists of ten variable number tandem repeat (VNTR) loci amplified in three multiplex PCR reactions. Sixty-five unique MLVA types were obtained among 84 clinical non-O157 STEC strains comprised of geographically diverse sporadic and outbreak related isolates. Compared to PFGE, the developed MLVA scheme allowed similar discrimination among serogroups O26, O111, O103, and O121 but not among O145 and O45. To more fully compare the discriminatory power of this preliminary MLVA method to PFGE and to determine its epidemiological congruence, a thorough internal and external validation needs to be performed on a carefully selected large panel of strains, including multiple isolates from single outbreaks.

Single nucleotide polymorphisms and risk factors predictive of pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease progressing asymptomatically until death within months after diagnosis. Defining at-risk populations should promote an early diagnosis and efficient follow-up, therefore avoiding its development. Single nucleotide polymorphisms, or SNPs, constitute the most abundant form of genetic variation in the human genome. SNPs are markers of diverse populations or individuals, yet also can be associated with differences in susceptibility or severity of certain diseases and/or individual responses to drugs. Many SNPs have previously been identified in studies of healthy subjects and patients with different alleles of a given gene. To date, around forty SNPs from the human genome have been correlated with predisposition to PDAC by pan-genomic studies or genome wide association studies (GWAS). However, parts of the human genome located within the GC-rich repeated domain of chromosomes are unsuitable for GWAS. Unfortunately, of those forty SNPs none are currently used in routine clinical protocols as potential biomarkers for PDAC. Exon 11 of the bile salt-dependent lipase gene ( BSDL ) plays a key role in pancreatic disease and encodes variable number of tandem repeat (VNTR) sequences, therefore Martinez et al . [Oncotarget. 2015; 6: 39855-39864.] hypothesized that a genetic link exists between mutations in VNTR loci and predisposition to pancreatic cancer (PC). Authors reported that the c.1719C>T transition (SNP rs488087) present in BSDL VNTR may be a useful marker for defining a population at risk of developing PC (occurrence: 63.90% in the PC group versus 27.30% in the control group). Notably, the odds ratio (OR) of 4.7 for the T allele was larger than those already determined for other SNPs suspected to be predictive of PC. Further studies on tumor pancreatic tissue suggested that the T allele may favor Kras G12R/G12D somatic mutations which represent negative prognostic factors associated with reduced survival. Furthermore, a robust method using probes for droplet digital (dd)-PCR was designed to specifically discriminate the C/C major from C/T or T/T minor genotypes. Altogether, Martinez et al . propose that detection of the T allele in rs488087 SNP should lead to an in-depth follow-up of these patients, particularly if associated with other potential risk factors of PC.

Potential Sources and Transmission of Salmonella and Antimicrobial Resistance in Kampala, Uganda.

In sub‒Saharan Africa, non‒typhoidal Salmonellae (NTS) cause invasive disease particularly in children and HIV infected adults, but the disease epidemiology is poorly understood. Between 2012 and 2013, we investigated NTS sources and transmission in Kampala. We detected Salmonella in 60% of the influent and 60% of the effluent samples from a wastewater treatment plant and 53.3% of the influent and 10% of the effluent samples from waste stabilization ponds that serve the human population; 40.9% of flush‒water samples from ruminant slaughterhouses, 6.6% of the poultry fecal samples from live bird markets and 4% of the fecal samples from swine at slaughter; and in 54.2% of the water samples from a channel that drains storm–water and effluents from the city. We obtained 775 Salmonella isolates, identified 32 serovars, and determined resistance to 15 antimicrobials. We genotyped common serovars using multiple‒locus variable number tandem repeats analysis or pulsed‒field gel electrophoresis. In addition, we analyzed 49 archived NTS isolates from asymptomatic livestock and human clinical cases. Salmonella from ruminant and swine sources were mostly pan‒susceptible (95%) while poultry isolates were generally more resistant. Salmonella Kentucky isolated from poultry exhibited extensive drug resistance characterized by resistance to 10 antimicrobials. Interestingly, similar genotypes of S. Kentucky but with less antimicrobial resistance (AMR) were found in poultry, human and environmental sources. The observed AMR patterns could be attributed to host or management factors associated with production. Alternatively, S. Kentucky may be prone to acquiring AMR. The factors driving AMR remain poorly understood and should be elucidated. Overall, shared genotypes and AMR phenotypes were found in NTS from human, livestock and environmental sources, suggesting zoonotic and environmental transmissions most likely occur. Information from this study could be used to control NTS transmission.

Large Diversity of Porcine Yersinia enterocolitica 4/O:3 in Eight European Countries Assessed by Multiple-Locus Variable-Number Tandem-Repeat Analysis.

A total of 253 multiple-locus variable-number tandem-repeat analysis (MLVA) types among 634 isolates were discovered while studying the genetic diversity of porcine Yersinia enterocolitica 4/O:3 isolates from eight different European countries. Six variable-number tandem-repeat (VNTR) loci V2A, V4, V5, V6, V7, and V9 were used to study the isolates from 82 farms in Belgium (n = 93, 7 farms), England (n = 41, 8 farms), Estonia (n = 106, 12 farms), Finland (n = 70, 13 farms), Italy (n = 111, 20 farms), Latvia (n = 66, 3 farms), Russia (n = 60, 10 farms), and Spain (n = 87, 9 farms). Cluster analysis revealed mainly country-specific clusters, and only one MLVA type consisting of two isolates was found from two countries: Russia and Italy. Also, farm-specific clusters were discovered, but same MLVA types could also be found from different farms. Analysis of multiple isolates originating either from the same tonsils (n = 4) or from the same farm, but 6 months apart, revealed both identical and different MLVA types. MLVA showed a very good discriminatory ability with a Simpson's discriminatory index (DI) of 0.989. DIs for VNTR loci V2A, V4, V5, V6, V7, and V9 were 0.916, 0.791, 0.901, 0.877, 0.912, and 0.785, respectively, when studying all isolates together, but variation was evident between isolates originating from different countries. Locus V4 in the Spanish isolates and locus V9 in the Latvian isolates did not differentiate (DI 0.000), and locus V9 in the English isolates showed very low discriminatory power (DI 0.049). The porcine Y. enterocolitica 4/O:3 isolates were diverse, but the variation in DI demonstrates that the well discriminating loci V2A, V5, V6, and V7 should be included in MLVA protocol when maximal discriminatory power is needed.

Repeat-based Sequence Typing of Carnobacterium maltaromaticum

Carnobacterium maltaromaticum is a Lactic Acid Bacterium (LAB) of technological interest for the food industry, especially the dairy as bioprotection and ripening flora. The industrial use of this LAB requires accurate and resolutive typing tools. A new typing method for C. maltaromaticum inspired from MLVA analysis and called Repeat-based Sequence Typing (RST) is described. Rather than electrophoresis analysis, our RST method is based on sequence analysis of multiple loci containing Variable-Number Tandem-Repeats (VNTRs). The method described here for C. maltaromaticum relies on the analysis of three VNTR loci, and was applied to a collection of 24 strains. For each strain, a PCR product corresponding to the amplification of each VNTR loci was sequenced. Sequence analysis allowed delineating 11, 11, and 12 alleles for loci VNTR-A, VNTR-B, and VNTR-C, respectively. Considering the allele combination exhibited by each strain allowed defining 15 genotypes, ending in a discriminatory index of 0.94. Comparison with MLST revealed that both methods were complementary for strain typing in C. maltaromaticum.