VNTR Research Articles

Molecular epidemiological analysis of Salmonella Schwarzengrund isolated in Japan by newly developed multi-locus variable-number tandem repeat analysis method

To establish molecular epidemiological analysis method for Salmonella Schwarzengrund and investigate its origin and genetic relationships among isolates in Japan, S. Schwarzengrund strains were examined by the pulsed-field gel electrophoresis (PFGE), the multi-locus sequencing typing (MLST), and the multi-locus variable-number tandem repeat analysis (MLVA) developed in this study. By the PFGE and MLST methods, the strains were typed into two clades (A and B) and two sequence types (STs) (96 and 241), respectively. Minimum spanning tree showed that the strains belonging ST241 genetically close to ST96, suggesting that ST241 originates from ST96. For MLVA, new primers for variable-number tandem repeat (VNTR) loci were designed, and five VNTR loci finally selected. In the MLVA method, the strains were typed into 11 sequence types. The MLVA method was able to differentiate strains belonging to the same clade of PFGE and MLST methods. These results indicate that the MLVA developed in this study shows a higher discriminatory power for S. Schwarzengrund compared to the PFGE and MLST methods and that it appears to be a useful method for the molecular epidemiological investigation of S. Schwarzengrund despite the low genetic diversity of this serotype in Japan.

High hemolytic activity of the Staphylococcus aureus spa t1081 among clonal complex 45 in Taiwan

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 45 was first reported in Taiwan in 2006. Since then, the prevalence of ST45 MRSA in clinical isolates has increased. This study was carried out to understand the changes in the proportions, evolutionary relationships, and infection advantages of ST45 and its related clones. Materials and methods: S. aureus including MRSA and MSSA (methicillin-sensitive S. aureus), and clonal complex (CC) 45 blood isolates were collected in 2000, 2005, and from January 2010 to August 2014. Molecular typing, multiple-locus variable-number tandem repeat analysis (MLVA) and single nucleotide polymorphism (SNP)-based phylogenetic analysis were performed. Fitness and virulence analyses were used to understand the infection advantages of the isolates. ResultsAmong the 67 CC45 isolates, only MSSA ST508 isolates were found in 2000 and 2005. Since 2010, the prevalence of MRSA has increased, t1081/ST45 has become dominant, and MRSA ST508 has been found. Phylogenetic analysis indicated that most of the ST45 isolates were located in a cluster distinct from those of ST508 and ST929. However, the t026 isolates clustered with the ST508 isolates rather than with the other ST45 isolates. Moreover, fitness and virulence analyses revealed that the t1081 isolates had higher hemolytic activity than the t026 and ST508 isolates did. ConclusionOur findings indicated that the increased prevalence of ST45 MRSA isolates from blood cultures in Taiwan was due to the t1081 isolates, and their high hemolytic activity may provide an infection advantage.

Molecular typing methods to characterize Brucella spp. from animals: A review.

Brucellosis is an infectious disease of animals that can infect humans. The disease causes significant economic losses and threatens human health. A timely and accurate disease diagnosis plays a vital role in the identification of brucellosis. In addition to traditional diagnostic methods, molecular methods allow diagnosis and typing of the causative agent of brucellosis. This review will discuss various methods, such as Bruce-ladder, Suiladder, high-resolution melt analysis, restriction fragment length polymorphism, multilocus sequence typing, multilocus variable-number tandem repeat analysis, and whole-genome sequencing single-nucleotide polymorphism, for the molecular typing of Brucella and discuss their advantages and disadvantages.

Association of CTLA-4 (AT)n Variants in Basal Cell Carcinoma and Squamous Cell Carcinoma Patients from Western Mexico.

The incidence of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) is constantly increasing, becoming a significant health problem. CTLA-4 is a critical immune checkpoint, and it has been suggested that a variant of variable-number tandem repeat in the 3'-UTR of its gene, known as (AT)n, may be associated with a higher susceptibility to some cancers; however, little is known about genetic variants of the CTLA-4 gene in NMSC. To establish the association of this genetic variant in the CTLA-4 gene with the susceptibility of NMSC carcinogenesis in the Western Mexican population, samples from 150 BCC patients, 150 SCC patients, and 150 healthy individuals as the reference group (RG) were analyzed by endpoint PCR, followed by electrophoresis to genotype the samples. We found that the short-repeat 104/104 bp genotype may be a risk factor for BBC carcinogens (OR = 2.92, p = 0.03), whereas the long-repeat 106/106 bp genotype may be a protective factor for both BCC (OR = 0.13, p = 0.01) and SCC (OR = 0.32, p = 0.01) susceptibility. Our results show that in the Western Mexican population, long-repeat (AT)n variants in the CTLA-4 gene are associated with a protective factor in BCC and SCC. In contrast, short repeats are associated with a risk factor.

Bacillus anthracis in South Africa, 1975–2013: are some lineages vanishing?

The anthrax-causing bacterium Bacillus anthracis comprises the genetic clades A, B, and C. In the northernmost part (Pafuri) of Kruger National Park (KNP), South Africa, both the common A and rare B strains clades occur. The B clade strains were reported to be dominant in Pafuri before 1991, while A clade strains occurred towards the central parts of KNP. The prevalence of B clade strains is currently much lower as only A clade strains have been isolated from 1992 onwards in KNP. In this study 319 B. anthracis strains were characterized with 31-loci multiple-locus variable-number tandem repeat analysis (MLVA-31). B clade strains from soil (n = 9) and a Tragelaphus strepsiceros carcass (n = 1) were further characterised by whole genome sequencing and compared to publicly available genomes. The KNP strains clustered in the B clade before 1991 into two dominant genotypes. South African strains cluster into a dominant genotype A.Br.005/006 consisting of KNP as well as the other anthrax endemic region, Northern Cape Province (NCP), South Africa. A few A.Br.001/002 strains from both endemic areas were also identified. Subclade A.Br.101 belonging to the A.Br.Aust94 lineage was reported in the NCP. The B-clade strains seems to be vanishing, while outbreaks in South Africa are caused mainly by the A.Br.005/006 genotypes as well as a few minor clades such as A.Br.001/002 and A.Br.101 present in NCP. This work confirmed the existence of the rare and vanishing B-clade strains that group in B.Br.001 branch with KrugerB and A0991 KNP strains.

The evaluation of IL-4 VNTR intron 3 and TNF-α (rs1799964) gene polymorphisms in Egyptian patients with alopecia areata: a case–control study

BackgroundAlopecia areata (AA) is a non-scarring hair loss condition that usually affects the scalp. The exact pathogenesis is poorly understood; however, multiple factors like genetics, environmental, psychological, and immunological factors may have a role. The purpose of this study was to look into possible links between the functional interleukin-4 (IL-4) gene intron 3 variable number of tandem repeats (VNTR) and TNF-(rs1799964) gene polymorphism and AA susceptibility. This case–control study consisted of 79 unrelated patients and 156 age- and sex-matched healthy individuals as a control group. The Severity of Alopecia Tool was used to assess the extent of hair loss from the scalp. Polymerase chain reaction (PCR) with specific primers was used to determine IL-4 gene 70-bp VNTR polymorphism while polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) was used to investigate TNF-α (rs1799964) gene polymorphism.ResultsNone of the selected polymorphisms for both genotypes and alleles had statistical significance when patients and controls were compared with each other (p-values for IL-4 VNTR were 0.11, 0.74, 0.052 and 0.27 and for TNF-α polymorphism was 0.71, 0.43, 0.65 and 0.55, respectively, for codominant, dominant, recessive and overdominant models of inheritance, respectively). Furthermore, the same results were retrieved when the genotypes were compared with the patient’s clinical and demographic data (p-value > 0.05).ConclusionThe findings indicate that IL-4 VNTR intron 3 and TNF-α (rs1799964) gene polymorphisms are not linked to the development of AA in the Egyptian population.

Genetic Diversity and Virulence of Phytopathogenic Burkholderia glumae Strains Isolated from Rice Cultivars in Valleys of the High Jungle of Perú.

Burkholderia glumae causes bacterial leaf blight in rice, and its global spread has been exacerbated by climate change. To understand the genetic diversity and virulence of B. glumae strains isolated from rice cultivars in Perú, 47 isolates were obtained from infected rice fields, all belonging to B. glumae, and confirmed by recA and toxB sequences. The BOX-PCR typing group has 38 genomic profiles, and these turn into seven variable number tandem repeats (VNTR) haplotypes. There was no correlation between clustering and geographical origin. Nineteen strains were selected for phenotypic characterization and virulence, using both the maceration level of the onion bulb proxy and inoculation of seeds of two rice cultivars. Several strains produced pigments other than toxoflavin, which correlated with onion bulb maceration. In terms of virulence at the seed level, all strains produced inhibition at the root and coleoptile level, but the severity of symptoms varied significantly between strains, revealing significant differences in pathogenicity. There is no correlation between maceration and virulence scores, probably reflecting different virulence mechanisms depending on the host infection stage. This is the first study to evaluate the VNTR diversity and virulence of Peruvian strains of B. glumae in two commercial cultivars.

Exploring Gene Polymorphisms Associated with Otitis Media through Variable Number Tandem Repeats (VNTR) Region

Background: Otitis is an inflammation and infection of the inner lining of the middle ear. The problem known as suppurative otitis media if there are droplets and holes in the tympanic membrane due to this inflammation and infection of the mucous membrane. The present study focused on specific genes suspected to be associated with otitis media in 60 subjects clinically diagnosed with OM The study focuses on two genes, MUC5B and IL-1RN, whose genes mutations by variable number of tandem repeat (VNTR) regionsMethods: Variable numbers of tandem repeats (VNTR) regions were used to genotype 60 patients with otitis media and 30 healthy individuals for genotypic polymorphisms for the MUC5B and IL-1RN genes by number of tandem repeats (VNTR) regions on a variable basis Blood samples were collected from participants, and three milliliters were placed directly into EDTA tubes. Subsequently, PCR products were detected by agarose gel electrophoresis and visualized by ethidium bromide staining. The size of amplified DNA fragments was determined by comparison with a 100-bp DNA ladder molecule size markerResults: About the same, the paper represents the association of gene polymorphisms with otitis media by genotyping a total of 60 patients as OM, and, on the other hand, 30 individuals were noted to be healthy. This was to determine genotypic polymorphisms for the two genes, that is, MUC5B and IL1RN, within the VNTR region. The results showed that the carriers of allele 2 in the genotypic position had a risk of nearly twofold getting otitis media.Conclusion: Polymorphism of the MUC5B and IL-1RN genes can be responsible for the severity of the inflammatory process in the middle ear, complicated by otitis media or hearing loss.Keywords: Otitis media; Gene polymorphism; MUC5B; IL-1RN; VNTR

An Evaluation of the Lineage of Brucella Isolates in Turkey by a Whole-Genome Single-Nucleotide Polymorphism Analysis.

Brucellosis is a disease caused by the Brucella (B.) species. It is a zoonotic disease that affects farm animals and causes economic losses in many countries worldwide. Brucella has the ability to persist in the environment and infect the host at low doses. Thus, it is more important to trace brucellosis outbreaks, identify their sources of infection, and interrupt their transmission. Some countries already have initial data, but most of these data are based on a Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA), which is completely unsuitable for studying the Brucella genome. Since brucellosis is an endemic disease in Turkey, this study aimed to examine the genome of Turkish Brucella isolates collected between 2018 and 2020, except for one isolate, which was from 2012. A total of 28 strains of B. melitensis (n = 15) and B. abortus (n = 13) were analyzed using a core-genome single-nucleotide polymorphism (cgSNP) analysis. A potential connection between the Turkish isolates and entries from Sweden, Israel, Syria, Austria, and India for B. melitensis was detected. For B. abortus, there may be potential associations with entries from China. This explains the tight ties found between Brucella strains from neighboring countries and isolates from Turkey. Therefore, it is recommended that strict measures be taken and the possible effects of uncontrolled animal introduction are emphasized.

The influence of PER3 VNTR genotypes on the age of onset in a group of bipolar I disorder patients: an exploratory study

BackgroundPER3 is a circadian gene that contains a variable number of tandem repeats (VNTR) which codifies for three genotypes: 4/4; 4/5; and 5/5 and is involved in non-visual response to light, a critical process associated with bipolar disorder onset. Benedetti et al. (Neurosci Lett 445(2):184–7) related this VNTR with bipolar disorder age of onset and linked genotype 5/5 with an earlier onset. In this study, we aimed to investigate these associations of PER3 VNTR genotypes with age of onset in a homogenous sample of German patients with bipolar I disorder through Kaplan-Meier curves.Methods45 patients were enrolled and divided into three groups according to PER3 VNTR genotypes. Recognizing common biological features, we built a combined group of -5 allele carriers (4/5 + 5/5). As a primary outcome, Kaplan-Meier analysis was conducted to delineate the three genotypes’ influence on age of onset. The secondary Kaplan-Meier analysis aimed to evaluate the relation between the 4/4 homozygotes group and the combined group (4/5 + 5/5) with age of onset. Finally, we proceeded to compare groups through a Log Rank Test and performed an analysis of covariance (ANCOVA).ResultsThe Kaplan-Meier analysis with three separate genotypes didn’t replicate the findings of Benedetti’s study. The analysis comparing genotype 4/4 with the combined group showed the influence of PER3 VNTR variants on the age of onset and relates genotype 4/4 to an earlier onset. ANCOVA between the combined and the 4/4 genotype groups, correlated genotype 4/4 with an increased number of depressive episodes.ConclusionThis study showed no significant effect of PER3 VNTR genotypes on the age of onset and in linking genotype 5/5 with an earlier onset age. Contrasting results may arise from intrinsic differences between the two studies but also shed light on hypothetically different levels of functioning of PER3 VNTR genotypes in the context of bipolar pathology. Further studies will require bigger and more homogeneous clinical samples.

Structural and genetic diversity in the secreted mucins MUC5AC and MUC5B

The secreted mucins MUC5AC and MUC5B are large glycoproteins that play critical defensive roles in pathogen entrapment and mucociliary clearance. Their respective genes contain polymorphic and degenerate protein-coding variable number tandem repeats (VNTRs) that make the loci difficult to investigate with short reads. We characterize the structural diversity of MUC5AC and MUC5B by long-read sequencing and assembly of 206 human and 20 nonhuman primate (NHP) haplotypes. We find that human MUC5B is largely invariant (5,761-5,762 amino acids [aa]); however, seven haplotypes have expanded VNTRs (6,291-7,019 aa). In contrast, 30 allelic variants of MUC5AC encode 16 distinct proteins (5,249-6,325 aa) with cysteine-rich domain and VNTR copy-number variation. We group MUC5AC alleles into three phylogenetic clades: H1 (46%, ∼5,654 aa), H2 (33%, ∼5,742 aa), and H3 (7%, ∼6,325 aa). The two most common human MUC5AC variants are smaller than NHP gene models, suggesting a reduction in protein length during recent human evolution. Linkage disequilibrium and Tajima's D analyses reveal that East Asians carry exceptionally large blocks with an excess of rare variation (p<0.05) at MUC5AC. To validate this result, we use Locityper for genotyping MUC5AC haplogroups in 2,600 unrelated samples from the 1000 Genomes Project. We observe a signature of positive selection in H1 among East Asians and a depletion of the likely ancestral haplogroup (H3). In Europeans, H3 alleles show an excess of common variation and deviate from Hardy-Weinberg equilibrium (p<0.05), consistent with heterozygote advantage and balancing selection. This study provides a generalizable strategy to characterize complex protein-coding VNTRs for improved disease associations.

Multiple locus variable number tandem repeat analysis genotype polymorphism of Bacillus anthracis in China

Objective: To analyze the multiple locus variable number tandem repeat analysis (MLVA) genotype polymorphism of Bacillus (B.) anthracis and establish a MLVA genotype database of B. anthracis in China. Methods: B. anthracis strains isolated from different sources in China since 1947 were collected. Genotype identification was carried out using the MLVA15 genotyping protocol based on 15 variable number tandem repeat loci. The genotypes were uniformly numbered and named. The distribution characteristics of the MLVA genotypes of strains were analyzed. Software Bionumerics was used to construct clustering diagrams to analyze the genetic relationships. Results: The MLVA15 clustering analysis subdivided the isolates into 4 major groups and 91 genotypes, 54 of which were unique to China. The genotypes from MLVA15-CHN1 to MLVA15-CHN6 were widely distributed throughout China and in all eras, while other genotypes were restricted to certain regions or eras. Conclusions: This study established a MLVA genotype database of B. anthracis, which provides basis for the understanding of MLVA genetic polymorphisms and the control and molecular source tracing of the anthrax outbreaks in China.

Molecular epidemiology and genomic features of Bordetella parapertussis in Shanghai, China, 2017-2022.

Pertussis is a highly contagious respiratory illness mainly caused by Bordetella pertussis (BP). Bordetella parapertussis (BPP) can induce symptoms compatible with pertussis, but has been underdiagnosed and underreported. The current pertussis vaccines offer low protection against BPP. Herein, we aim to reveal the epidemiology and genomic evolution of BPP in Shanghai, China. Children diagnosed with BPP infection from January 2017 to December 2022 in Shanghai, China were enrolled. We performed antimicrobial susceptibility testing (AST), multiple locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing (WGS) analysis. A total of 260 international BPP genomes were chosen for comparison to investigate the genomic diversity and phylogenetic characteristics of Chinese strains within a global context. Sixty patients were diagnosed with BPP infection by culture, with the positive ratio of 3.5‰ (60/17337) for BPP in nasopharyngeal swap samples. The average age of patients was 4.5 ± 0.3 years. BPPs contained four MLVA types including MT6 (65.0%), MT4 (26.7%), untype-1 (6.7%) and MT5 (1.7%), and none of strains showed resistance to macrolides. All strains carried virulence genotype of ptxP37/ptxA13/ptxB3/ptxC3/ptxD3/ptxE3/fim2-2/fim3-10. MT4 and MT5 strains carried prn54, whereas MT6 and untype-1 BPPs expressed prn101. We identified two outbreaks after 2020 caused by MT4 and MT6 strains, each corresponding to distinct WGS-based phylogenetic lineages. The MT4-lineage is estimated to have originated around 1991 and has since spread globally, being introduced to China between 2005 and 2010. In contrast, the MT6-lineage was exclusively identified in China and is inferred to have originated around 2002. We revealed the genomic diversity of BPPs circulating in Shanghai, China, and reported the outbreaks of MT6 and MT4 BPPs after 2020. This is the first report on the emergence and regional outbreak of MT6 BPPs in the world, indicating that continuous surveillance on BPPs are thus required.

A study of association of the VNTR MIR-137 rs58335419 with schizophrenia

The MIR137 gene encodes microRNA-137 (miR-137), which is a brain-enriched miR that is highly expressed in various brain regions. miR-137 has been identified as a modulator of processes involved in the pathogenesis of neuropsychiatric disorders. Functional polymorphism of variable number of tandem repeats (VNTR) rs58335419 was found in the regulatory region of the MIR137 gene. It is associated with a change in the expression of miR-137 and, as a result, with an increased risk of developing psychopathologies, including schizophrenia. In this study, we for the first time have analyzed the distribution of frequencies of alleles and genotypes of VNTR MIR137 in a large sample from the Russian population. The association of VNTR with the risk of schizophrenia has been studied. It was found that the presence of VNTR alleles with more than three repeats, as well as a genotype homozygous for such alleles, is associated with an increased risk of developing schizophrenia (OR = 1.4, 95% CI: 1.01-1.95).

Association of NLRP3 and IL-4 VNTR polymorphisms and genetic susceptibility to preeclampsia: A case-control study

IntroductionAbnormalities in the maternal immune system and insufficient gestational immune tolerance may significantly contribute to the development of preeclampsia (PE). The NLR family pyrin domain containing 3 (NLRP3) functions as a pattern recognition receptor that identifies pathogen-associated molecular patterns. Interleukin-4 (IL-4) is a potent anti-inflammatory cytokine that modulates the immune response. Therefore, this study aims to elucidate the impact of NLRP3 and IL-4 variable number of tandem repeats (VNTR) polymorphisms on susceptibility to PE. Materials and MethodsA total of 1,018 patients with PE and 1,007 normal pregnant women were recruited as the case group and the control group, respectively. Peripheral blood DNA was extracted, and NLRP3 and IL-4 VNTR polymorphisms were genotyped using polymerase chain reaction and gel electrophoresis. Genotypes and allele frequencies of pregnant women were assessed in both cohorts. ResultsThe NLRP3 VNTR 9–7 genotype in the PE group was significantly lower than that in the control group, but 9 and 14 allele frequencies were significantly higher in patients with PE. Individuals with IL-4 VNTR genotypes 1–2 had a lower risk of PE than controls, and the IL-4 VNTR 2 allele frequency was significantly lower in patients with PE. ConclusionsThis study, the first of its kind in the literature, evaluates the impact of NLRP3 VNTR and IL-4 VNTR polymorphisms on PE, revealing a significant correlation with PE susceptibility. This investigation contributes to understanding the pathogenesis of PE and provides a reference point for developing strategies to prevent and treat the disease in the future.

The role of immune checkpoint inhibitors: Variable number of tandem repeat (VNTR) polymorphism in the second exon of the P-selectin glycoprotein ligand-1 (PSGL-1) gene polymorphism in multiple myeloma.

Somatic mutations and polymorphisms may play a role in multiple myeloma (MM) susceptibility and survival. One of the immune checkpoint inhibitors is P-selectin glycoprotein ligand-1 (PSGL-1); the majority of tumor-infiltrating leukocytes express PSGL-1, causing T cell and immune inhibition via PSGL-1 mediator molecules. We aimed to investigate the effect of variable number of tandem repeat (VNTR) polymorphism in the second exon of the PSGL-1 gene on MM susceptibility, response to treatment and survival in our patient group. A total of 238 patients diagnosed with MM between January 2010 and January 2021 and 162 healthy individuals as a control group were included in this cross-sectional study. The genotypes of the VNTR polymorphism in the second exon of the PSGL-1 gene were statistically compared between patients and healthy controls; the statistically significant effects of the genotypes on response to first-line treatment and survival were examined. The AC genotype was significantly higher in healthy controls compared to patients diagnosed with MM (p < 0.001). The median PFS in patients with AA/AB/AC was 56 months, while it was 100 months in patients with BB/CC. The hazard ratio of 1.34 for PFS was found to be clinically significant and having the BB/CC genotype could provide a longer PFS compared to others, but it was not statistically significant due to the sample size. Our study results will shed light on new study plans in terms of immune checkpoint target therapies among conventional treatment preferences in MM.

Rhesus Macaques: VNTR Polymorphism of the FCGRT Gene.

Variable number tandem repeat (VNTR) polymorphisms of the human neonatal IgG Fc receptor α-chain gene (FCGRT) are known to influence the expression levels of FCGRT and IgG in serum. Monkeys are considered to be a relevant biological model for studying the effects of immunobiological drugs. The study determined the functional VNTR polymorphisms of the FCGRT gene in 109 male rhesus macaques from the nursery of the Kurchatov Complex of Medical Primatology. PCR amplification of samples was carried out followed by electrophoretic separation of DNA fragments in a 2% agarose gel. Individual DNA amplification products were sequenced (according to Sanger system) in forward and reverse directions to confirm the specificity. The genotyping showed that the VNTR polymorphism of the FCGRT gene in the studied population of rhesus macaques is presented by 9 variants. The frequency of the VNTR5 allele associated with lower IgG levels was 14.2%, and the most common one was the VNTR7 allele (25.2%). We also identified alleles that have not been previously reported: VNTR3, VNTR4, VNTR6, VNTR8, and VNTR9. The study allows to consider rhesus macaques as a potential model for studying the immunological response depending on the genetic VNTR variant of FCGRT.

Mechanistic Characterization of De Novo Generation of Variable Number Tandem Repeats in Circular Plasmids during Site-Directed Mutagenesis and Optimization for Coding Gene Application.

Site-directed mutagenesis for creating point mutations, sometimes, gives rise to plasmids carrying variable number tandem repeats (VNTRs) locally, which are arbitrarily regarded as polymerase chain reaction (PCR) related artifacts. Here, the alternative end-joining mechanism is reported rather than PCR artifacts accounts largely for that VNTRs formation and expansion. During generating a point mutation on GPLD1 gene, an unexpected formation of VNTRs employing the 31 bp mutagenesis primers is observed as the repeat unit in the pcDNA3.1-GPLD1 plasmid. The 31 bp VNTRs are formed in 24.75% of the resulting clones with copy number varied from 2 to 13. All repeat units are aligned with the same orientation as GPLD1 gene. 43.54% of the repeat junctions harbor nucleotide mutations while the rest don't. Their demonstrated short primers spanning the 3' part of the mutagenesis primers are essential for initial creation of the 2-copy tandem repeats (TRs) in circular plasmids. The dimerization of mutagenesis primers by the alternative end-joining in a correct orientation is required for further expansion of the 2-copy TRs. Lastly, a half-double priming strategy is established, verified the findings and offered a simple method for VNTRs creation on coding genes in circular plasmids without junction mutations.

Evaluation of a Multilocus Variable-Number Tandem-Repeat Analysis Scheme for Typing Ochrobactrum anthropi.

Ochrobactrum anthropi (O. anthropi) is found in water, soil, plants and animals. Even though it has low virulence, it has increasingly been found to cause a number of infectious diseases in people with low immunity. The identification of O. anthropi mainly uses biochemical methods, such as the API 20NE or Vitek-2. The typing studies of O. anthropi have mainly utilized PFGE, rep-PCR, AFLP, 16s rDNA sequencing, RecA-PCR RFLP, and MALDI-TOF MS. This study aims to evaluate the polymorphisms of variable-number tandem-repeats (VNTRs) within genomic DNA of O. anthropi strains. The tandem repeats (TRs) in genomic DNA are discovered using Tandem Repeat Finder software (version 4.09). Twelve different VNTRs are designated and assigned to the nomenclature. The primers for PCR of 12 loci are designed. The PCR product size is converted to the number of tandem repeats in every locus. The relatedness of 65 O. anthropi strains from geographically different countries are analyzed by means of 12-variable-number tandem-repeat analysis(MLVA-12). A total of 51 different genotypes are found in 65 O. anthropi strains. These strains, which were collected from the same environmental samples, hospitals, and countries, are clustered within the same or closely genotypes. The MLVA-12 assay has a good discriminatory power for species determination, typing of O. anthropi, and inferring the origin of bacteria.

Virulent properties and genomic diversity of Vibrio vulnificus isolated from environment, human, diseased fish.

The incidence of Vibrio vulnificus infections, with high mortality rates in humans and aquatic animals, has escalated, highlighting a significant public health challenge. Currently, reliable markers to identify strains with high virulence potential are lacking, and the understanding of evolutionary drivers behind the emergence of pathogenic strains is limited. In this study, we analyzed the distribution of virulent genotypes and phenotypes to discern the infectious potential of V. vulnificus strains isolated from three distinct sources. Most isolates, traditionally classified as biotype 1, possessed the virulence-correlated gene-C type. Environmental isolates predominantly exhibited YJ-like alleles, while clinical and diseased fish isolates were significantly associated with the nanA gene and pathogenicity region XII. Hemolytic activity was primarily observed in the culture supernatants of clinical and diseased fish isolates. Genetic relationships, as determined by multiple-locus variable-number tandem repeat analysis, suggested that strains originating from the same source tended to cluster together. However, multilocus sequence typing revealed considerable genetic diversity across clusters and sources. A phylogenetic analysis using single nucleotide polymorphisms of diseased fish strains alongside publicly available genomes demonstrated a high degree of evolutionary relatedness within and across different isolation sources. Notably, our findings reveal no direct correlation between phylogenetic patterns, isolation sources, and virulence capabilities. This underscores the necessity for proactive risk management strategies to address pathogenic V. vulnificus strains emerging from environmental reservoirs.IMPORTANCEAs the global incidence of Vibrio vulnificus infections rises, impacting human health and marine aquacultures, understanding the pathogenicity of environmental strains remains critical yet underexplored. This study addresses this gap by evaluating the virulence potential and genetic relatedness of V. vulnificus strains, focusing on environmental origins. We conduct an extensive genotypic analysis and phenotypic assessment, including virulence testing in a wax moth model. Our findings aim to uncover genetic and evolutionary factors that drive pathogenic strain emergence in the environment. This research advances our ability to identify reliable virulence markers and understand the distribution of pathogenic strains, offering significant insights for public health and environmental risk management.