Variable Number Of Tandem Repeat Markers Research Articles

Development of a diagnostic variable number tandem repeat marker and dual TaqMan genotyping assay to distinguish Lophophora species.

The Lophophora genus of the Cactaceae family includes Lophophora diffusa and Lophophora williamsii, which has traditionally been used as a natural analgesic; however, its use is now under strict regulation worldwide as it contains mescaline, a unique psychotropic agent. Recently, non-medical and illegal distribution and abuse of L. williamsii have increased worldwide; thus, effective species identification methods are urgently needed. Here, we identified a new variable number tandem repeat (VNTR) marker in the trnL intron region to identify and characterize species in forensic analyses. The VNTR marker has a unique structure of tandem repeats, each with 13 nucleotides; one repeat unit was found in L. williamsii and two in L. diffusa. Phylogenetic and length polymorphism analyses confirmed that this novel VNTR marker could distinguish between Lophophora species. Furthermore, our newly developed TaqMan genotyping assay utilizes two probes; the color and position of dots on the discrimination plot differ according to the tandem repeat count within the VNTR marker. The limits of detection of the assay were 0.000063ng (LW-VNTR probe-1) and 0.000066ng (LW-VNTR probe-2), indicating high sensitivity. Moreover, when crime scene samples of 16 presumed L. williamsii species were analyzed, the results coincided with those of gas chromatography-mass spectrometry, confirming the applicability of our marker for Lophophora species identification. Thus, the tandem repeats within the trnL intron region can be exploited as a VNTR marker to identify L. williamsii and L. diffusa. Our dual TaqMan genotyping assay based on a novel marker demonstrates potential for forensic applications.

Novel strategy of multiple-locus variable number tandem repeats analysis for genetic fingerprinting of human.

The variable number of tandem repeat (VNTR) analyses are methods based on the detection of repeated sequences within the human genome. In order to perform DNA typing at the personal laboratory, it is necessary to improve the VNTR analysis. The VNTR markers were difficult to popularize because PCR amplification was difficult due to its GC-rich and long nucleotide sequence. The aim of this study was to select the multiple VNTR markers that could only be identified by PCR amplification and electrophoresis. We genotyped each of the 15 VNTR markers using genomic DNA from 260 unrelated individuals by PCR amplification. Differences in the fragment length of PCR products are visualized by agarose gel electrophoresis. To confirm their usefulness as a DNA fingerprint these 15 markers were simultaneously analyzed with the DNA of 213 individuals and verified the statistical significance. In addition, to investigate the usefulness of each of the 15 VNTR markers as paternity markers, Mendelian segregation by meiotic division within a family consisting of two or three generations was confirmed. Fifteen VNTR loci selected in this study could be easily amplified by PCR and analyzed by electrophoresis, and were newly named DTM1 ~ 15. The number of total alleles in each VNTR showed from 4 to 16, and 100 to 1600bp in length, and their heterozygosity ranged from 0.2341 to 0.7915. In simultaneous analysis of 15 markers from 213 DNAs, the probability of chance appearing the same genotype in different individuals was less than 4.09E-12, indicating its usefulness as a DNA fingerprint. These loci were transmitted through meiosis by Mendelian inheritance in families. Fifteen VNTR markers have been found to be useful as DNA fingerprints for personal identification and kinship analysis that can be used at the personal laboratory level.

Diversidade genética de populações de Xanthomonas phaseoli pv. manihotis em mandioca por meio de marcadores rep-PCR e VNTRs

Abstract The objective of this work was to evaluate the genetic diversity of Xanthomonas phaseoli p v. manihotis (Xpm) from eight populations from five cassava producing states in Brazil, through the rep-PCR (BOX-PCR and ERIC-PCR) and variable number of tandem repeat (VNTR) markers. Cassava leaves with symptoms of cassava bacterial blight were collected in eight municipalities, and the Xpm isolates were identified by amplification with primers specific for these isolates. The identity of the Xpm isolates was confirmed with the BOX-PCR, ERIC-PCR, and VNTR markers. The observed selection pressure, together with the mode of reproduction and the mechanisms that increase genetic variability, allows of the pathogen populations to adapt according to microclimate variation, contributing to a differentiated reproductive success. ERIC-PCR and VNTRs are the best markers for evaluating the genetic variability in the eight studied Xpm populations. However, ERIC-PCR is the marker that best separated the groups by population and presented a higher similarity between the isolates of the same population. The study of the genetic diversity of Xpm is key to improve disease monitoring and management strategies in cassava crops.

Validation of a multilocus genotyping scheme for subtyping Cryptosporidium parvum for epidemiological purposes

Subtyping Cryptosporidium parvum for outbreak investigations or epidemiological surveillance usually relies on DNA sequence analysis of a gene coding for a 60 KDa glycoprotein (gp60). Although gp60 can be useful for allelic discrimination and to help investigate sources and routes of transmission, the presence of common subtypes and recombination during the parasite's sexual life-cycle demand a multilocus-based method for more discriminatory genotyping. While whole genome sequencing would provide the ultimate approach, it is a time consuming and expensive option for faecal parasites such as Cryptosporidium that occur at low density and are difficult to propagate routinely. In this study, we selected and evaluated a panel of previously identified variable-number tandem-repeat (VNTR) markers, to establish a multilocus genotyping scheme based on fragment sizing, appropriate for inter-laboratory surveillance and outbreak investigations. Seven VNTR markers were validated in vitro and demonstrated typeability of 0.85 and discriminatory power of 0.99. The discriminatory power was much greater than the currently used gp60 sequencing (0.74), which identified 26 subtypes, compared to 100 different MLVA profiles within the same sample set. The assay was robust, with repeatable results and reproducibility across three laboratories demonstrating the scheme was suitable for inter-laboratory comparison of C. parvum subtypes. As the majority of genotypes (79%) were unique among epidemiologically unrelated samples, there was efficiency to infer linkage, and epidemiological concordance was observed in historical outbreaks. We propose that the multilocus variable number of tandem repeats analysis scheme is suitable to assist outbreak investigations.

Characterization of Tularemia Cases in Slovenia with Multiple-Locus Variable-Number Tandem Repeat Analysis.

Francisella tularensis is the etiologic agent of tularemia, a bacterial zoonotic disease. The genome of F. tularensis shows a recent evolutionary change, especially in reservoirs. Variable number of tandem repeats (VNTR) is described as a high-speed molecular clock and can thus be used as a high-resolution typing system. The main objective of our study was to investigate the molecular diversity of F. tularensis strains and reveal possible sources of infection. Using real-time PCR targeting the ISFtu2 region, we successfully amplified targeted DNA in 13/31 Slovenian patients with a clinical diagnosis of tularemia, and with PCR targeting the fopA gene, we obtained 11/13 PCR products. Sequencing revealed that all samples were identified as F. tularensis subsp. holarctica. We successfully obtained one F. tularensis isolate from a lymph node aspirate by culture on chocolate agar. Our isolate was clustered into major clade B12 (subclade B43). We optimized VNTR typing to be used directly on clinical samples. Multiple-locus VNTR analysis (MLVA) revealed five unique MLVA types; 45.5% samples had the same MLVA type, another 27.3% shared a different MLVA type, and each of the remaining had a unique MLVA type. Most samples differed at only two VNTR markers (Ft-M03 and Ft-M06). Additionally, we investigated samples from small mammals (n = 532) and Ixodes ricinus ticks (n = 232) captured in the same geographical area in which patients with tularemia were found. No F. tularensis DNA was detected in samples of small mammals or I. ricinus ticks. The diversity of MLVA types in Slovenia was high, despite the small region, but most of the samples from the same region shared the same MLVA type. Our results suggest that MLVA is a useful tool for quick molecular characterization of F. tularensis directly from patient samples, especially when investigating geographically localized outbreaks.

Genetic Traces of the Francisella tularensis Colonization of Spain, 1998-2020.

More than 1000 humans have acquired the febrile disease tularemia in Spain since the first notification of human cases in 1997. We here aimed to study the recent molecular evolution of the causative bacterium Francisella tularensis during disease establishment in Spain. Single-nucleotide polymorphisms (SNPs) and variable-number tandem repeats (VNTRs) were analyzed in whole-genome sequences (WGS) of F. tularensis. Short-read WGS data for 20 F. tularensis strains from humans infected in the periods 2014–2015 and 2018–2020 in Spain were generated. These data were combined with WGS data of 25 Spanish strains from 1998 to 2008 and two reference strains. Capillary electrophoresis data of VNTR genetic regions were generated and compared with the WGS data for the 11 strains from 2014 to 2015. Evolutionary relationships among strains were analyzed by phylogenetic methods. We identified 117 informative SNPs in a 1,577,289-nucleotide WGS alignment of 47 F. tularensis genomes. Forty-five strains from Spain formed a star-like SNP phylogeny with six branches emerging from a basal common node. The most recently evolved genomes formed four additional star-like structures that were derived from four branches of the basal common node. VNTR copy number variation was detected in two out of 10 VNTR regions examined. Genetic clustering of strains by VNTRs agreed with the clustering by SNPs. The SNP data provided higher resolution among strains than the VNTRs data in all but one cases. There was an excellent correlation between VNTR marker sizing by capillary electrophoresis and prediction from WGS data. The genetic data strongly support that tularemia, indeed, emerged recently in Spain. Distinct genetic patterns of local F. tularensis population expansions imply that the pathogen has colonized a previously disease-free geographical area. We also found that genome-wide SNPs provide higher genetic resolution among F. tularensis genomes than the use of VNTRs, and that VNTR copy numbers can be accurately predicted using short-read WGS data.

A review of recent research on Theileria parva: Implications for the infection and treatment vaccination method for control of East Coast fever.

The infection and treatment (ITM) live vaccination method for control of Theileria parva infection in cattle is increasingly being adopted, particularly in Maasai pastoralist systems. Several studies indicate positive impacts on human livelihoods. Importantly, the first detailed protocol for live vaccine production at scale has recently been published. However, quality control and delivery issues constrain vaccination sustainability and deployment. There is evidence that the distribution of T.parva is spreading from endemic areas in East Africa, North into Southern Sudan and West into Cameroon, probably as a result of anthropogenic movement of cattle. It has also recently been demonstrated that in Kenya, T.parva derived from cape buffalo can 'breakthrough' the immunity induced by ITM. However, in Tanzania, breakthrough has not been reported in areas where cattle co-graze with buffalo. It has been confirmed that buffalo in northern Uganda national parks are not infected with T.parva and R.appendiculatus appears to be absent, raising issues regarding vector distribution. Recently, there have been multiple field population genetic studies using variable number tandem repeat (VNTR) sequences and sequencing of antigen genes encoding targets of CD8+ T-cell responses. The VNTR markers generally reveal high levels of diversity. The antigen gene sequences present within the trivalent Muguga cocktail are relatively conserved among cattle transmissible T.parva populations. By contrast, greater genetic diversity is present in antigen genes from T.parva of buffalo origin. There is also evidence from several studies for transmission of components of stocks present within the Muguga cocktail, into field ticks and cattle following induction of a carrier state by immunization. In the short term, this may increase live vaccine effectiveness, through a more homogeneous challenge, but the long-term consequences are unknown.

Tandem repeat analysis for typing of Pseudomonas aeruginosa carrying resistance genes in southern Iran

Pseudomonas aeruginosa is known to be one of the most important causes of nosocomial infections, with high antimicrobial resistance. Thus, the current study was conducted to evaluate usefulness of multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA) plan for epidemiological studies and clustering of P. aeruginosa isolated in different wards of general hospital in Bandar Abbas, south of Iran, as well as assessment of presence of integrons and resistance genes in these strains. A total of 72 clinical isolates of P. aeruginosa were isolated from the patients hospitalized in the hospital from December 2017 to June 2018 in Bandar Abbas, southern Iran. MLVA of seven VNTR markers was performed for differentiating and genetic relatedness of isolates. PCR amplification was conducted for detection of bla HSV, bla VEB , int1 and int2 genes. MLVA assay with seven loci resolved 36 different genotypes. Nearly all the isolates were classified into 6 different clonal complexes and 15 singletons. High-level resistance rate was observed in ceftazidime (44; 61%). Nine (12.5%) of these isolates showed multiple drug resistance (MDR) phenotypes. Seven (9.7%), 5 (6.9%), 46 (63.8%) and 6 (8.3%) of isolates carried bla HSV, bla VEB , int1 and int2 genes, respectively. Seven VNTR markers used in this study provided a moderate discriminatory power and differentiated 36 genotypes among 72 isolates. In addition, presence of class 1 and class 2 integrons, bla HSV and bla VEB in different clones indicated a high risk of spreading of MDR isolates among these clones. Results obtained from specification of different tandem repeats in the current study can be helpful to be used for MLVA in epidemiological surveillance of P. aeruginosa isolates in different regions.

Evaluation of Multilocus Variable-Number Tandem-Repeat (MLVA-8Orsay) for Typing of Carbapenem-Resistant Acinetobacter baumannii Isolated from Patients in Tehran, Iran

Objectives: Acinetobacter baumannii has emerged worldwide as a major nosocomial pathogen. The aim of this study was to evaluate the MLVA-8Orsay scheme suitability for clustering and differentiating A. baumannii strains isolated from two hospitals in Tehran. Methods: A panel of eight variable-number tandem-repeat (VNTR) markers was used for typing analysis (MLVA-8Orsay) of 89 carbapenem-resistant A. baumannii strains isolated from two hospitals in Tehran. Results: Abaum0845 (S-VNTR marker) was excluded from the MLVA scheme because of its great allelic diversity at this locus. The modified MLVA-7 scheme provided excellent discriminatory power and detected 82 MLVA types among the 83 isolates. Additionally, the great diversity associated with MLVA markers did not permit to apply the previously proposed criteria for clustering the isolates. However, six MLVA complexes were defined assuming a 71% genetic similarity cut-off, corresponding to the existence of identically sized alleles for at least five VNTR markers. The blaOXA-23-like gene (93.2%) was the most prevalent gene in resistant isolates collected from the two hospitals. Conclusions: MLVA-7 discriminatory power was found to be excellent. This method was also appeared to be useful and reliable in epidemiological investigations of A. baumannii strains. MLVA-7 analysis revealed that carbapenemase-carrying A. baumannii isolates might be polyclonal and provided evidence for gene transfer among different strains collected from the two hospitals. Overall, the study provided evidence supporting the use of MLVA-7 scheme as an alternative or complementary method for typing of A. baumannii isolates.

Genetic diversity of Fusarium graminearum isolated from weeds

Fusarium head blight (FHB) is a devastating disease of cereals worldwide. The primary hosts of this pathogen are wheat and barley, but it also can survive and spread in segetal plants. Data on Fusarium graminearum subpopulations in weeds is lacking. We collected isolates of F. graminearum sensu stricto from segetal plants growing in six separate crop fields and used variable number of tandem repeats (VNTR) markers to gain insights into the diversity of its subpopulations. Ten primer pairs of VNTR markers were tested on 57 F. graminearum isolates obtained from 415 sampled plants representing 52 species. High genetic diversity was found, even in this relatively small number of isolates.

MOLECULAR CHARACTERIZATION OF TUNISIAN STRAINS OF ERWINIA AMYLOVORA

The present study focused on the molecular characterization of a collection of Erwinia amylovora isolates recovered from different outbreaks in Tunisia between 2012 and 2014. Analysis of 54 isolates, including the reference type strain CFBP 1430T revealed that all Tunisian isolates produced the expected amplicons with diverse primer pairs routinely used for molecular diagnostics of E. amylovora. We also evaluated the genetic variability of these isolates by PCR fingerprinting, using specific primers for clustered regularly interspaced short palindromic repeats (CRISPRs) and for variable number of tandem repeats (VNTR) sequences. For the first method, our results revealed that all the primers used, except those for CRISPR3, which produced an identical amplicon for all isolates, showed some variability among Tunisian isolates. For the second method, forty-nine isolates showed the same fingerprint patterns as the reference type strain CFBP 1430T with all the primers used, whereas four of the isolates showed very divergent patterns. These results suggest that there has been a main introduction of European-type isolates in Tunisia, and possibly a few mutations or other few independent introductions of the pathogen. Additionally, these results indicate that PCR fingerprinting using VNTR markers is a most useful tool for discriminating among E. amylovora strains and for their identification in epidemiological studies.

Acquisition of Tn6018-3′ CS regions increases colistin MICs against Acinetobacter baumannii isolates harboring new variants of AbaRs

Colistin is the last hope to treat extensively drug resistance (XDR) Acinetobacter baumannii (A. baumannii) infections, but resistance to colistin is currently reported in clinical centers all over the world. Here, we studied two colistin-resistant A. baumannii isolates with a difference in minimum inhibitory concentrations (MICs) that were isolated from a single burn patient during treatment in the hospitalization period. The international clonal (IC) lineage, multilocus sequence typing (MLST), and multiple loci variable number tandem repeat (VNTR) analysis (MLVA) typing were used to characterize the relatedness of A. baumannii isolates. Lipopolysaccharides (LPS) and PmrAB system analysis by PCR sequencing, polyacrylamide gel electrophoresis (PAGE), and real-time PCR were performed to determine the intactness and probable modifications of the LPS as the main resistance mechanisms to colistin. A combination of PCR, sequencing, and restriction fragment length polymorphism (RFLP) was used for A. baumannii resistance islands (AbaR) mapping as resistance-determinant reservoirs. Two isolates were identical at all MLST and VNTR marker loci that indicated the isolates were the same strain. In comparison to colistin-heteroresistant A. baumannii strain TEH267 (MIC=1.5mg/L), colistin-resistant A. baumannii strain TEH273 (MIC ≥256mg/L) acquired two genomic regions including Tn6018-topA sequence and topA sequence-3' CS in its AbaR structure containing ispA and cadA genes which, it would appear, could be associated with eightfold increase in colistin MIC. Both isolates had new variants of AbaR-like structures which could be derivatives of the typical AbaR3. According to the results of this study, AbaRs could be associated with an increase in MIC to colistin.

Inter- and intra-strain variability of tandem repeats in Mycoplasma pneumoniae based on next-generation sequencing data.

To characterize inter- and intra-strain variability of variable-number tandem repeats (VNTRs) in Mycoplasma pneumoniae to determine the optimal multilocus VNTR analysis scheme for improved strain typing. Whole genome assemblies and next-generation sequencing data from diverse M. pneumoniae isolates were used to characterize VNTRs and their variability, and to compare the strain discriminability of new VNTR and existing markers. We identified 13 VNTRs including five reported previously. These VNTRs displayed different levels of inter- and intra-strain copy number variations. All new markers showed similar or higher discriminability compared with existing VNTR markers and the P1 typing system. Our study provides novel insights into VNTR variations and potential new multilocus VNTR analysis schemes for improved genotyping of M. pneumoniae.

Genetic Diversity of Indonesian Bacterial Leaf Blight Isolate (Xanthomonas oryzae pv. oryzae) Core Collection based on the VNTR and avrXa7 Molecular Markers

Bacterial leaf blight ( BLB) is one of the major diseases in rice caused by Xanthomonas oryzae pv. oryzae . This study aimed to identify and analyze the genetic diversity of 18 BLB isolates that consist of 7 races and 11 haplotypes from various locations in Indonesia. The genetic diversity analysis was conducted on the basis of the VNTR (Variable Number of Tandem Repeat) markers and the avrxa7 gene marker . The b anding pattern of the amplification product was made into binary data as input for the construction of a dendogram. Based on the dendogram , three X. oryzae pv. oryzae genotype groups with different virulence levels were formed . The VII ( IXO80_021) race of X. oryzae pv. oryzae genotype group I and the VIII - A ( IXO 80_024 ) race of genotype group II were avirulent, whereas the races and haplotypes of genotype group III were virulent.

Development and evaluation of a multiple-locus variable-number tandem-repeats analysis assay for subtyping Salmonella typhi strains from Sub-Saharan Africa

Background: Typhoid fever a major public health problem in developing countries. Epidemiological investigations of Salmonella enterica serovar Typhi (Salmonella Typhi) infections using molecular sub-typing methods are challenging due to the highly homologous nature of Salmonella Typhi strains. In recent years, several approaches in using multiple-locus variable-number tandem-repeats analysis (MLVA) for molecular sub-typing of Salmonella Typhi have been made. To date, a standardized set of variable-number tandem-repeats (VNTR) loci for the typing of homologous Salmonella Typhi strains has not been established. The aim of our study was to develop and evaluate a MLVA assay consisting of 5 VNTR markers to analyse Salmonella Typhi strains from Sub-Saharan Africa (SSA). Methods & Materials: To develop and evaluate the MLVA assay, 50 Salmonella Typhi strains from humans were selected from the culture collection at the Centre for Enteric Diseases (CED) at the National Institute for Communicable Diseases (NICD) from a potential 1080 isolates. This strain panel represented the diverse Salmonella Typhi pulsed-field gel electrophoresis (PFGE) profiles in the CED database, specimen collection dates and geographic areas within the SSA region. The 50 Salmonella Typhi strains were used to evaluate 12 polymorphic VNTR loci that were previously published. These include TR1, TR2, TR3, TR4, TR5, Sal02, Sal06, Sal10, Sal16, Sal20, TR4500 and TR4600. The assay included PCR amplification of VNTR loci using fluorescently labelled primers and size determination of PCR products by capillary electrophoresis. Results: Of the 12 VNTR loci that were evaluated, 5 (TR3, TR4, TR5, Sal06, Sal10 and TR4500) were found unsuitable as they showed poor allele variation (between 1 and 3 alleles). The remaining 6 VNTR loci showed good allele variation and good diversity indices; with Sal20, Sal 16 and TR1 having 6, 9 and 11 alleles respectively; and Sal02, TR4699 and TR2 having 15, 20 and 23 alleles respectively. Conclusion: In summary, we have identified 6 polymorphic VNTR loci suitable for MLVA analysis of Salmonella Typhi strains. The five most diverse VNTR loci will be selected for the MLVA assay and will be used to analyse Salmonella Typhi strains from SSA. This work will assist in rapidly identifying strain relatedness and assist outbreak detection in typhoid fever outbreaks.

Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites

Erwinia amylovora causes a major disease of pome fruit trees worldwide, and is regulated as a quarantine organism in many countries. While some diversity of isolates has been observed, molecular epidemiology of this bacterium is hindered by a lack of simple molecular typing techniques with sufficiently high resolution. We report a molecular typing system of E. amylovora based on variable number of tandem repeats (VNTR) analysis. Repeats in the E. amylovora genome were identified with comparative genomic tools, and VNTR markers were developed and validated. A Multiple-Locus VNTR Analysis (MLVA) was applied to E. amylovora isolates from bacterial collections representing global and regional distribution of the pathogen. Based on six repeats, MLVA allowed the distinction of 227 haplotypes among a collection of 833 isolates of worldwide origin. Three geographically separated groups were recognized among global isolates using Bayesian clustering methods. Analysis of regional outbreaks confirmed presence of diverse haplotypes but also high representation of certain haplotypes during outbreaks. MLVA analysis is a practical method for epidemiological studies of E. amylovora, identifying previously unresolved population structure within outbreaks. Knowledge of such structure can increase our understanding on how plant diseases emerge and spread over a given geographical region.

Diversity among strains of Pseudomonas aeruginosa from manure and soil, evaluated by multiple locus variable number tandem repeat analysis and antibiotic resistance profiles

The results of a multiple locus variable number of tandem repeat (VNTR) analysis (MLVA)-based study designed to understand the genetic diversity of soil and manure-borne Pseudomonas aeruginosa isolates, and the relationship between these isolates and a set of clinical and environmental isolates, are hereby reported. Fifteen described VNTR markers were first selected, and 62 isolates recovered from agricultural and industrial soils in France and Burkina Faso, and from cattle and horse manure, along with 26 snake-related isolates and 17 environmental and clinical isolates from international collections, were genotyped. Following a comparison with previously published 9-marker MLVA schemes, an optimal 13-marker MLVA scheme (MLVA13-Lyon) was identified that was found to be the most efficient, as it showed high typability (90%) and high discriminatory power (0.987). A comparison of MLVA with PFGE for typing of the snake-related isolates confirmed the MLVA13-Lyon scheme to be a robust method for quickly discriminating and inferring genetic relatedness among environmental isolates. The 62 isolates displayed wide diversity, since 41 MLVA types (i.e. MTs) were observed, with 26 MTs clustered in 10 MLVA clonal complexes (MCs). Three and eight MCs were found among soil and manure isolates, respectively. Only one MC contained both soil and manure-borne isolates. No common MC was observed between soil and manure-borne isolates and the snake-related or environmental and clinical isolates. Antibiotic resistance profiles were performed to determine a potential link between resistance properties and the selective pressure that might be present in the various habitats. Except for four soil and manure isolates resistant to ticarcillin and ticarcillin/clavulanic acid and one isolate from a hydrocarbon-contaminated soil resistant to imipenem, all environmental isolates showed wild-type antibiotic profiles.

Identification and typing of Francisella tularensis with a highly automated genotyping assay

A PCR assay was developed to genotypically characterize Francisella tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella samples. Sample DNA was amplified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR amplicons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay.

Simple and Highly Discriminatory VNTR-Based Multiplex PCR for Tracing Sources of Aspergillus flavus Isolates

Aspergillus flavus is second only to A. fumigatus in causing invasive aspergillosis and it is the major agent responsible for fungal sinusitis, keratitis and endophthalmitis in many countries in the Middle East, Africa and Southeast Asia. Despite the growing challenge due to A. flavus, data on the molecular epidemiology of this fungus remain scarce. The objective of the present study was to develop a new typing method based on the detection of VNTR (Variable number tandem repeat) markers. Eight VNTR markers located on 6 different chromosomes (1, 2, 3, 5, 7 and 8) of A. flavus were selected, combined by pairs for multiplex amplifications and tested on 30 unrelated isolates and six reference strains. The Simpson index for individual markers ranged from 0.398 to 0.818. A combined loci index calculated with all the markers yielded an index of 0.998. The MLVA (Multiple Locus VNTR Analysis) technique proved to be specific and reproducible. In a second time, a total of 55 isolates from Chinese avian farms and from a Tunisian hospital have been evaluated. One major cluster of genotypes could be defined by using the graphing algorithm termed Minimum Spanning Tree. This cluster comprised most of the isolates collected in an avian farm in southern China. The MLVA technique should be considered as an excellent and cost-effective typing method that could be used in many laboratories without the need for sophisticated equipment.

Molecular Characterization of Fusarium Head Blight Pathogens Sampled from a Naturally Infected Disease Nursery Used for Wheat Breeding Programs in China.

Fusarium head blight (FHB) is an important disease of wheat and barley worldwide. The disease is primarily caused by members of the Fusarium graminearum species complex, consisting of at least 14 phylogenetically distinct species. To determine the population structure of the FHB pathogens in a naturally infected disease nursery located at Jianyang, Fujian province, China, 160 isolates of the F. graminearum complex were recovered from symptomatic wheat spike samples collected in two consecutive years (2008 and 2009) and characterized using species- and chemotype-specific polymerase chain reaction as well as variable number tandem repeat (VNTR) markers. All isolates analyzed were identified as F. asiaticum except for one isolate, which was identified as F. avenaceum. Among the 159 F. asiaticum isolates, 126 (79%) isolates were of the nivalenol (NIV) type while 29 (18%) isolates were of the 15-acetyl deoxynivalenol type and only 4 (3%) isolates were of the 3-acetyl deoxynivalenol type. The 10 VNTR markers revealed 124 distinct haplotypes and 76 polymorphic alleles across the whole population. The two subpopulations (FA-08 and FA-09) grouped based on the year of collection exhibited low genetic differentiation (Fst = 0.032) and high gene flow (Nm = 15.13). However, a significant genetic differentiation was found within the NIV-type isolates as revealed by the Structure software. The pairwise linkage disequilibrium tests did not support the hypothesis of random mating in the population because half (48.8%) of the locus pairs showed a linkage disequilibrium (P > 0.05). Our results suggest that FHB in this nursery was caused by a genetically homogenous and non-random mating population of F. asiaticum in 2008 and 2009, which consisted of all three trichothecene types with various levels of aggressiveness.