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Background/objectives: Pseudomonas aeruginosa can cause community-acquired infections affecting various body sites. The present retrospective study investigated the genetic diversity of 173 isolates (166 clinical, 7 environmental) of P. aeruginosa collected from clinical pathology laboratories in Abidjan, Côte d'Ivoire (2001–2011). Methods: Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) using 13 loci was applied to all isolates and compared to published MLVA data. The antibiotics status of the isolates was compiled when available and compared to published profiles. Results: Among 95 isolates analyzed for their antibiotics status, 14 displayed concerning resistance profiles: five multidrug-resistant (MDR) and nine extensively drug-resistant (XDR). MLVA typing revealed a high genetic diversity (>130 genotypes), with many genotypes represented by a single strain. Notably, thirteen clusters (≥4 related isolates) were observed. Some clusters displayed close genetic relatedness to isolates from France, Korea, and well-studied strains (ST560, LES and PA14). Comparative analysis suggested the presence of international high-risk MDR clones (CC233, CC111) in Côte d'Ivoire. Importantly, MLVA clustering revealed a close relationship of CC235-MDR strains with a locally identified cluster (group 9). Conclusions: These findings support MLVA as a reliable and cost-effective tool for low-resource settings, allowing the selection of relevant strains for future whole genome sequence analyses. This approach can improve outbreak investigations and public health interventions aimed at curbing MDR P. aeruginosa transmission within hospitals and at the national level. [ABSTRACT FROM AUTHOR]

Understanding the epidemiology and evolution of Staphylococcus aureus (SA) is crucial for implementing effective infection prevention and control measures. In this study, a short-term (4-month) and local molecular epidemiology of SA in the nosocomial setting was investigated using multiple-locus variable number tandem repeat (VNTR) analysis (MLVA). The study included 31 clinical SA isolates. The analysis was based on two complimentary panels encompassing 14 VNTR loci wherein a first-line assay comprised a subset of 10 loci (Panel 1), followed by a second subset of four loci (Panel 2). Thirty MLVA profiles were generated. Small amplicons were obtained for three out of the four loci included in Panel 2 for more than half of the isolates, making the calculation of the number of repeats impossible. Therefore, phylogenetic clustering was based on the analysis of basic loci included in Panel 1 (which identified two major genetic clusters). Cluster I included 26 (84%) isolates, among which 14 were classified into five sub-clusters (cutoff value ≥65%). Only one pair of the isolates shared an identical MLVA pattern. Our study indicates the usefulness of a MLVA tool comprised of a primary subset of 10 loci to track the possibility of SA intra-hospital transmission. We report a high genetic diversity of the isolates and suggest a limited degree of intra- and inter-ward SA transmission. Further genetic studies are necessary to investigate the nature of the small alleles yielded in Panel, as their subset can provide even higher resolution of the assay.

Tularemia is an acute febrile disease caused by the Gram-negative bacillus Francisella tularensis. Based on genetic and phenotypic characteristics, three subspecies are distinguished: tularensis, holarctica, and mediasiatica. F. tularensis subsp. mediasiatica remains the least studied subspecies. Over the past decade, new foci of distribution of F. tularensis subsp. mediasiatica have been discovered in Russia (Siberia), expanding the possible distribution area by thousands of kilometers. This article provides whole genome single nucleotide polymorphism (wgSNP) and polymorphic tandem repeats (MLVA) analyses of 28 mediasiatica strains isolated between 1965 and 2004 in Kazakhstan. Despite high genetic homogeneity, MLVA with eleven loci (MLVA11) demonstrates a high discriminatory ability (diversity index, 0.9497). The topological structure of the trees based on wgSNP and MLVA is not comparable; however, clustering remains congruent for most outbreaks, with the exception of two strains from one outbreak that are identical in terms of wgSNP but differ at three tandem repeat loci. Based on wgSNP, the strains are assigned to one of the three currently known mediasiatica sublineages, lineage M.I, together with other historical strains maintained in collections in Russia and Sweden. wgSNP shows limited previously unknown genetic diversity, with the M.I lineage size being only 118 SNPs. The wgSNP genotype is not strongly correlated with year and place of isolation. [ABSTRACT FROM AUTHOR]

Background/objectives: Pseudomonas aeruginosa can cause community-acquired infections affecting various body sites. The present retrospective study investigated the genetic diversity of 173 isolates (166 clinical, 7 environmental) of P. aeruginosa collected from clinical pathology laboratories in Abidjan, Côte d’Ivoire (2001–2011). Methods: Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) using 13 loci was applied to all isolates and compared to published MLVA data. The antibiotics status of the isolates was compiled when available and compared to published profiles. Results: Among 95 isolates analyzed for their antibiotics status, 14 displayed concerning resistance profiles: five multidrug-resistant (MDR) and nine extensively drug-resistant (XDR). MLVA typing revealed a high genetic diversity (>130 genotypes), with many genotypes represented by a single strain. Notably, thirteen clusters (≥4 related isolates) were observed. Some clusters displayed close genetic relatedness to isolates from France, Korea, and well-studied strains (ST560, LES and PA14). Comparative analysis suggested the presence of international high-risk MDR clones (CC233, CC111) in Côte d’Ivoire. Importantly, MLVA clustering revealed a close relationship of CC235-MDR strains with a locally identified cluster (group 9). Conclusions: These findings support MLVA as a reliable and cost-effective tool for low-resource settings, allowing the selection of relevant strains for future whole genome sequence analyses. This approach can improve outbreak investigations and public health interventions aimed at curbing MDR P. aeruginosa transmission within hospitals and at the national level.

Strains of Bacillus genus were isolated from soil samples in the permafrost region (Yakutia, Russia). The phenotypic characteristics of the strains are given. The analysis of the obtained data made it possible to assign them to the Bacillus cereus complex. PCR analysis made it possible to determine the profile of B. cereus toxin synthesis genes in the genomes of the studied strains. Genetic characterization was obtained by RAPD genotyping and using MLVA loci used for genotyping of the anthrax pathogen. The results of genotyping at different levels of resolution made it possible to differentiate the studied strains from the B. anthracis species and to show their intraspecific genetic differences and the degree of relationship. Whole genome sequencing was carried out, based on the data of which MLST genotyping was carried out, which revealed two known sequence types and one new one that is described for the first time in this work. The results we obtained are of practical importance and are extremely interesting from the point of view of the evolution and phylogeography of the B. cereus group, since the fact that strains were isolated from permafrost suggests that their age may be much older than expected. [ABSTRACT FROM AUTHOR]

Tularemia is an acute febrile disease caused by the Gram-negative bacillus Francisella tularensis. Based on genetic and phenotypic characteristics, three subspecies are distinguished: tularensis, holarctica, and mediasiatica. F. tularensis subsp. mediasiatica remains the least studied subspecies. Over the past decade, new foci of distribution of F. tularensis subsp. mediasiatica have been discovered in Russia (Siberia), expanding the possible distribution area by thousands of kilometers. This article provides whole genome single nucleotide polymorphism (wgSNP) and polymorphic tandem repeats (MLVA) analyses of 28 mediasiatica strains isolated between 1965 and 2004 in Kazakhstan. Despite high genetic homogeneity, MLVA with eleven loci (MLVA11) demonstrates a high discriminatory ability (diversity index, 0.9497). The topological structure of the trees based on wgSNP and MLVA is not comparable; however, clustering remains congruent for most outbreaks, with the exception of two strains from one outbreak that are identical in terms of wgSNP but differ at three tandem repeat loci. Based on wgSNP, the strains are assigned to one of the three currently known mediasiatica sublineages, lineage M.I, together with other historical strains maintained in collections in Russia and Sweden. wgSNP shows limited previously unknown genetic diversity, with the M.I lineage size being only 118 SNPs. The wgSNP genotype is not strongly correlated with year and place of isolation.

The aim of the present study was to characterize (phenotypically and genotypically) two strains of Brucella abortus identified as belonging to biovar 4 isolated from cattle in Brazil. The strains were isolated from cervical bursitis from cattle in the states of Pará and Rio Grande do Sul, respectively. In the phenotypic identification, the isolates were positive in CO2 requirement, produced H2S, were resistant to basic fuchsin (20 µg / mL) and sensitive to thionin (20 µg / mL and 40 µg / mL) and presented M surface antigen, but A surface antigen is absent. The isolates were positive in the PCR for the bcsp31 gene (genus-specific) and in the AMOS-enhanced PCR, both isolates showed a band profile consistent with B. abortus biovar 1, 2 or 4. Moreover, both isolates also showed restriction patterns identical to the reference strain when tested by the omp2b PCR-RFLP. In genotyping using Multiple Locus Variable Number of Tandem Repeat (VNTR) Analysis - MLVA (MLVA16), the isolates showed differences in several loci (Bruce42, Bruce19, Bruce04, Bruce16 and Bruce30); by Multiple Locus Sequence Typing (MLST), they also exhibited differences in sequence type (ST), strain 16/02 ST1 (2-1-1-2-1-3-1-1-1) and strain 128/11 ST (22-1-1 -8-9-3-1-1-1). The extensive typing of B. abortus strains isolated from cattle in Brazil using different approaches confirmed the occurrence of rare B. abortus biovar 4 in the country. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease in humans and animals that may be asymptomatic long-term and quickly develop to pneumonia and septicemia in immunocompromised state and due to predisposing factors. Melioidosis is endemic in countries with tropical and subtropical climates, where B. pseudomallei is a part of the soil and water microbiota of stagnant water bodies, as well as the plant rhizosphere. The recording of imported melioidosis cases in countries with temperate climate zone, along with the continued threat of using this pathogen as a bioterrorism agent, indicate the relevance of research aimed at developing modern methods for its diagnosing and typing. Current research in the intraspecific differentiation of pathogenic strains of infectious diseases tends to use two or more types of molecular markers. A promising direction for the B. pseudomallei strains genotyping is based on using a combination of VNTR loci, which provides a high discriminating ability of the method of multi-locus variable tandem repeat number analysis (MLVA), and slowly evolving single nucleotide polymorphisms (SNPs). The aim of this work was to identify new VNTR and SNP loci suitable for use as genetic markers in molecular typing of the melioidosis causative agent. 20 strains of B. pseudomallei from the collection of the Volgograd Plague Control Research Institute and whole genome sequences of 85 B. pseudomallei strains from the GenBank NCBI database were analyzed. While typing melioidosis causative agent strains from the GenBank NCBI database for 4 VNTR loci, 74 genotypes were identified, of which 64 were unique (HunterGaston index 0.997). It was found that the high allelic polymorphism of VNTR loci limited a potential to determine the geographical and phylogenetic relationships of B. pseudomallei isolates by using the MLVA-4 scheme, and the identified null alleles increased the risk of homoplasia. In this regard, the MLVA-4 scheme was supplemented with a VNTR marker in the BPSS1974 locus encoding a collagen-like protein as well as with SNP markers in the genes of lipid A biosynthesis lauroyl acyltransferase, RNA polymerase sigma factor RpoH, and glutamine amidotransferase. For amplification of the select loci, primers and probes were designed as part of the developed scheme, which were tested in the typing of B. pseudomallei collection strains. According to the results of a cluster analysis for 105 strains of the melioidosis causative agent, an integrated approach turned out to be optimal, allowing differentiation of strains within SNP groups based on VNTR profiles. The methodological approach developed for B. pseudomallei genetic typing, based on a comprehensive analysis of 4 SNP- and 5 MLVA-markers in our modification, allowed to differentiate strains according to the geographical regions of their origin and establish the clonal origin of isolates upon revealing cases of melioidosis.

A Multi‐Locus Variable number of tandem repeat Analysis (MLVA) genotyping scheme was developed for the epidemiological study of Moritella viscosa, which causes 'winter ulcer' predominantly in sea‐reared Atlantic salmon (Salmo salar L.). The assay involves multiplex PCR amplification of six Variable Number of Tandem Repeat (VNTR) loci, followed by capillary electrophoresis and data interpretation. A collection of 747 spatiotemporally diverse M. viscosa isolates from nine fish species was analysed, the majority from farmed Norwegian salmon. MLVA distributed 76% of the isolates across three major clonal complexes (CC1, CC2 and CC3), with the remaining forming minor clusters and singletons. While 90% of the salmon isolates belong to either CC1, CC2 or CC3, only 20% of the isolates recovered from other fish species do so, indicating a considerable degree of host specificity. We further highlight a series of 'clonal shifts' amongst Norwegian salmon isolates over the 35‐year sampling period, with CC1 showing exclusive predominance prior to the emergence of CC2, which was later supplanted by CC3, before the recent re‐emergence of CC1. Apparently, these shifts have rapidly swept the entire Norwegian coastline and conceivably, as suggested by typing of a small number of non‐Norwegian isolates, the Northeast Atlantic region as a whole. [ABSTRACT FROM AUTHOR]

Aims The ability to distinguish between Klebsiella pneumoniae strains is critical for outbreak investigations. A new typing method, intergenic region polymorphism analysis (IRPA), was developed, validated, and the discriminatory power was determined by comparison with multiple-locus variable-number tandem repeat analysis (MLVA) in this study. Methods and results This method is based on the idea that every IRPA locus (polymorphic fragment of intergenic regions present in one strain but not in other strains or different fragment sizes in other strains) could divide strains into different genotypes. A 9-loci IRPA scheme was designed to type 64  K. pneumoniae isolates. Five IRPA loci were identified that conferred the same level of discrimination as the 9-loci initially examined. Among these K. pneumoniae isolates, 7.81% (5/64), 6.25% (4/64), 4.96% (3/64), 9.38% (6/64), and 1.56% (1/64) were capsular serotypes K1, K2, K5, K20, and K54, respectively. The discriminatory power of the IRPA method was better than that of MLVA expressed in Simpson's index of diversity (SI) at 0.997 and 0.988, respectively. The congruent analysis of the IRPA method and MLVA showed moderate congruence between the two methods (AR = 0.378). The AW indicated that if IRPA data are availabl, one can accurately predict the MLVA cluster. Conclusion The IRPA method was found to have higher discriminatory power than MLVA and allowed for simpler band profile interpretation. The IRPA method is a rapid, simple, and high-resolution technique for molecular typing of K. pneumoniae. [ABSTRACT FROM AUTHOR]

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. • PFGE and MLST largely typed Japanese strains into two clades. • S. Schwarzengrund ST241 was predominant ST in Japan. • ST241 was genetically close to ST96, suggesting that ST241 originates from ST96. • The developed MLVA classified Japanese strains into 11 types. • The strains of ST241 were typed as single locus variants by the developed MLVA. [ABSTRACT FROM AUTHOR]

Although we live in the genomic era, the accessibility of the complete genome sequence of Coxiella burnetii , the etiological agent of Q fever, has increased knowledge in the field of genomic diversity of this agent However, it is still somewhat of a "question" microorganism. The epidemiology of Q fever is intricate due to its global distribution, repository and vector variety, as well as absence of surveys defining the dynamic interaction among these factors. Moreover, C. burnetii is a microbial agent that can be utilized as a bioterror weapon. Therefore, typing techniques used to recognize the strains can also be used to trace infections back to their source which is of great significance. In this paper, the latest and current typing techniques of C. burnetii spp. are reviewed illustrating their advantages and constraints. Recently developed multi locus VNTR analysis (MLVA) and single-nucleotide polymorphism (SNP) typing methods are promising in improving diagnostic capacity and enhancing the application of genotyping techniques for molecular epidemiologic surveys of the challenging pathogen. However, most of these studies did not differentiate between C. burnetii and Coxiella -like endosymbionts making it difficult to estimate the potential role that ticks play in the epidemiology of Q fever. Therefore, it is necessary to analyze the vector competence of different tick species to transmit C. burnetii. Knowledge of the vector and reservoir competence of ticks is important for taking adequate preventive measures to limit infection risks. The significant prevalence observed for the IS1111 gene underscores its substantial presence, while other genes display comparatively lower prevalence rates. Methodological variations, particularly between commercial and non-commercial kit-based methods, result in different prevalence outcomes. Variations in sample processing procedures also lead to significant differences in prevalence rates between mechanical and non-mechanical techniques. • Coxiella burnetii is a Gram-negative bacterium causing the "Q (query) fever disease". • Diagnostics by Sequencing, PCR-RFLP, Multiple Locus Variable-number Tandem Repeat Analysis and Multi Locus Sequence Typing • Strain differentiation is crucial for disease diagnosis and treatment, outbreak investigations, and epidemiological surveys. [ABSTRACT FROM AUTHOR]

Abstract Background The correlation between various factors (geographical region, clinical incidence, and host type) and the genomic heterogeneity has been shown in several bacterial strains including Chlamydia abortus. Methods The aim of this study was to survey the predominant types of C. abortus strains isolated from ruminants in Iran by the multiple loci variable number of tandem repeats (VNTR) analysis (MLVA) method. C. abortus infection was evaluated in a total of 117 aborted fetuses by real-time PCR. The isolation was done via the inoculation of the positive samples in chicken embryo and the L929 cell line. Genotyping was carried out by MLVA typing technique. Results Forty samples (34.2%) were detected with C. abortus infection; however, chlamydial infection in ruminants of Charmahal/Bakhtiari (3 bovines and 35 sheep) was higher than that of Khuzestan (2 sheep). All MLVA types (MT1-MT8) were detected in the collected samples from Charmahal/Bakhtiari but only 2 types (MT1 and MT3) were reported in samples from Khuzestan. The main MT type was MT1 (32% of aborted fetuses). Although in this study only 9 cow samples were investigated, they possessed similar clusters to those obtained from sheep (MT1 and MT6). Variation of type in sheep samples (MT1 to MT8) was more than that of bovine samples (MT1, and MT6). Conclusion By this research revealed that C.abortus was responsible for a significant percentage of ruminant abortion in two studied regions. The main MT type was MT1 (32% of aborted fetuses) and also 7 different genotypes were involved in infections. So it is concluded that diversity in C.abortus genotyping is high in two regions.

ABSTRACT: Mycoplasma hyopneumoniae is one of the most challenging respiratory pathogens involved with swine pneumonia worldwide, responsible for a chronic infection with high morbidity, which predisposes secondary bacterial infections in growing and finishing pigs. Advances in diagnostic techniques allowed identification of genetic characteristics associated with high antigenic and proteomic variability among bacterial strains. This study aimed to evaluate the genetic diversity of M. hyopneumoniae strains in lungs with pneumonic lesions obtained from 52 pig farms located in Minas Gerais, one of the largest swine production states in Brazil. Genotyping was performed using multilocus variable number of tandem repeat (VNTR) analysis (MLVA), targeting two loci encoding P97 and P146 adhesins VNTR. The results showed that this agent is widely disseminated in pig farms and there is a high polymorphism of M. hyopneumoniae variants circulating in the state of Minas Gerais. Different M. hyopneumoniae genotypes are randomly distributed in several regions of the state, with no specific geographic population structure pattern. M. hyopneumoniae association with viral agents was sporadic (3.17% with Influenza A and 1.9% with PCV2).

Brucellosis is one of the most common neglected zoonotic diseases globally, with a public health significance and a high economic loss in the livestock industry caused by the bacteria of the genus Brucella. In this study, 136 Egyptian Brucella melitensis strains isolated from animals and humans between 2001 and 2020 were analysed by examining the whole‐core‐genome single‐nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA‐16). Almost all Egyptian isolates were belonging to the West Mediterranean clade, except two isolates from buffalo and camel were belonging to the American and East Mediterranean clades, respectively. A significant correlation between the human case of brucellosis and the possible source of infection from animals was found. It seems that several outbreak strains already existing for many years have been spread over long distances and between many governorates. The cgSNP analysis, in combination with epidemiological metadata, allows a better differentiation than the MLVA‐16 genotyping method and, hence, the source definition and tracking of outbreak strains. The MLVA based on the currently used 16 markers is not suitable for this task. Our results revealed 99 different cgSNP genotypes with many different outbreak strains, both older and widely distributed ones and rather newly introduced ones as well. This indicates several different incidents and sources of infections, probably by imported animals from other countries to Egypt. Comparing our panel of isolates to public databases by cgSNP analysis, the results revealed near relatives from Italy. Moreover, near relatives from the United States, France, Austria and India were found by in silico MLVA. [ABSTRACT FROM AUTHOR]

Abstract Background Coxiella burnetii is the etiological agent of Q fever, a zoonosis affecting many animal species including sheep and goats. The aims of this study were to evaluate the shedding of Coxiella burnetii in small ruminant herds and to identify the pathogen’s genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods. Results Overall, 165 samples from 43 herds of goats and 9 flocks of sheep were collected including bulk tank milk (BTM), individual milk samples, vaginal swabs, tissue sections from stillborn kids, feces and placentas. These were tested by real-time PCR targeting the IS1111 element. C. burnetii infection was confirmed in 51.16% of the herds of goats and 22.2% of the flocks of sheep. Six out of nine samples originating from goats were successfully genotyped using the MLVA method. The presence was confirmed of two widely distributed MLVA genotypes (I and J) and genotype PL1 previously reported only in cattle. Only one sequence type (ST61) was identified; however, the majority of specimens represented partial STs and some of them may belong to ST61. Other partial STs could possibly be ST74. Conclusion This study confirmed the relatively common occurrence of Coxiella burnetii in small ruminant herds in Poland. Interestingly, all genotyped samples represent cattle-associated MLVA genotypes.

Abstract Background Prevalence of brucellosis and MLVA genotyping in animals and humans, isolated from different regions of Pakistan was performed. Animals having history of brucellosis from the field and local farms of Bannu, Mardan, Peshawar, Swat, Lahore and Islamabad were selected for blood collection. Humans that work with them were also selected for sampling in this study. Total of 600 samples were taken from cattle and humans and subjected to Rose Bengal plate Test (RBPT) for the initial screening of positive samples. Designed primers of B.abortus for cattle and B.melitensis for humans were utilised to perform PCR. Culturing and isolation was carried to further to perform MLVA genotyping assay through the selection of two panels of primer markers. Results RBPT showed more number of cases of brucellosis in animals and humans compared to the PCR findings. Genotyping findings based upon MLVA‐15 set of markers demonstrated that the isolated strains of B.abortus fall in the same clade with strain A1, P8 and A2 from Pakistan and also similar with BCCN#02‐45 strain from India. On the other hand, B.melitensis isolated from different districts of Pakistan shared the same clade with BwIM‐AFG 63, BwIM‐AFG 44 strains from Afghanistan and BwIM IRN 37 strain from Iran. Selected VNTR alleles were sequenced for calibration purposes. Conclusion It is concluded that Brucella is prevalent in animals and humans in studied districts of Pakistan. Moreover, A1, P8, BwIM‐AFG 63, BwIM‐AFG 44 and A2 were found the common genotypes in Pakistan.

ABSTRACT: The aim of the present study was to characterize (phenotypically and genotypically) two strains of Brucella abortus identified as belonging to biovar 4 isolated from cattle in Brazil. The strains were isolated from cervical bursitis from cattle in the states of Pará and Rio Grande do Sul, respectively. In the phenotypic identification, the isolates were positive in CO2 requirement, produced H2S, were resistant to basic fuchsin (20 µg / mL) and sensitive to thionin (20 µg / mL and 40 µg / mL) and presented M surface antigen, but A surface antigen is absent. The isolates were positive in the PCR for the bcsp31 gene (genus-specific) and in the AMOS-enhanced PCR, both isolates showed a band profile consistent with B. abortus biovar 1, 2 or 4. Moreover, both isolates also showed restriction patterns identical to the reference strain when tested by the omp2b PCR-RFLP. In genotyping using Multiple Locus Variable Number of Tandem Repeat (VNTR) Analysis - MLVA (MLVA16), the isolates showed differences in several loci (Bruce42, Bruce19, Bruce04, Bruce16 and Bruce30); by Multiple Locus Sequence Typing (MLST), they also exhibited differences in sequence type (ST), strain 16/02 ST1 (2-1-1-2-1-3-1-1-1) and strain 128/11 ST (22-1-1 -8-9-3-1-1-1). The extensive typing of B. abortus strains isolated from cattle in Brazil using different approaches confirmed the occurrence of rare B. abortus biovar 4 in the country.

Background: The correlation between various factors (geographical region, clinical incidence, and host type) and the genomic heterogeneity has been shown in several bacterial strains including Chlamydia abortus. Methods: The aim of this study was to survey the predominant types of C. abortus strains isolated from ruminants in Iran by the multiple loci variable number of tandem repeats (VNTR) analysis (MLVA) method. C. abortus infection was evaluated in a total of 117 aborted fetuses by real-time PCR. The isolation was done via the inoculation of the positive samples in chicken embryo and the L929 cell line. Genotyping was carried out by MLVA typing technique. Results: Forty samples (34.2%) were detected with C. abortus infection; however, chlamydial infection in ruminants of Charmahal/Bakhtiari (3 bovines and 35 sheep) was higher than that of Khuzestan (2 sheep). All MLVA types (MT1-MT8) were detected in the collected samples from Charmahal/Bakhtiari but only 2 types (MT1 and MT3) were reported in samples from Khuzestan. The main MT type was MT1 (32% of aborted fetuses). Although in this study only 9 cow samples were investigated, they possessed similar clusters to those obtained from sheep (MT1 and MT6). Variation of type in sheep samples (MT1 to MT8) was more than that of bovine samples (MT1, and MT6). Conclusion: By this research revealed that C.abortus was responsible for a significant percentage of ruminant abortion in two studied regions. The main MT type was MT1 (32% of aborted fetuses) and also 7 different genotypes were involved in infections. So it is concluded that diversity in C.abortus genotyping is high in two regions. [ABSTRACT FROM AUTHOR]

Aims: This study aimed to identify the genotypic fingerprinting of Brucellamelitensis biovar 3 isolates from ruminants in Kafr El‐Sheikh, Egypt, to compare with other peers globally and to highlight the epidemiology and potential causes of brucellosis control failure. Methods and Results: A multilocus variable‐number tandem‐repeat analysis (MLVA 16) was carried out on 41 B. melitensis bv3 isolates, 31 from the preferential hosts (28 sheep and three goats) and 10 from atypical hosts (nine cattle and one buffalo), identified by bacteriological and molecular techniques. MLVA‐16 analysis revealed 19 genotypes with nine as singletons. The most prevalent genotypes were M3_K.E (3,5,3,13,1,1,3,3,7,43,8,7,6,7,5,3), M13_K.E (3,5,3,13,1,1,3,3,7,43,8,5,8,7,7,3) and M5_K.E (3,5,3,13,1,1,3,3,7,43,8,4,8,7,11,3) circulating between different animal species. The B. melitensis isolation from aborted cows in farms that had never reared small ruminants indicates the likelihood of cow to cow B. melitensis transmission. Different genotypes of B. melitensis could be isolated from the same animal. The local geographic distribution of genotypes showed a very close genetic relatedness with genotypes reported outside the study area. Worldwide, our genotypes were mostly related to the Western Mediterranean lineage and less likely to the America's clonal lineage. Conclusion: There is a high genetic similarity of B. melitensis bv3 genotypes among different ruminant species, and the same animal could be infected with different genotypes. There is a high probability of spreading of B. melitensis among atypical hosts in the absence of the original hosts. The genetic relatedness of B. melitensis bv3 genotypes in the study area with other different geographic areas highlighted the national and international ruminants movement role as a potential factor for maintaining B. melitensis infection. Significance and Impact of the Study: Further investigations are required to understand the impact of the presence of more than one genotype of B. melitensis in the same animal on the efficacy of brucellosis control strategies. [ABSTRACT FROM AUTHOR]

Brucellosis is an important zoonosis that is emerging in some regions of the world, gaining increased relevance with the inclusion of the causing agent Brucella spp. in the class B bioterrorism group. Until now, multi-locus VNTR Analysis (MLVA) based on 16 loci has been considered as the gold standard for Brucella typing. However, this methodology is laborious, and, with the rampant release of Brucella genomes, the transition from the traditional MLVA to whole genome sequencing (WGS)-based typing is on course. Nevertheless, in order to avoid a disruptive transition with the loss of massive genetic data obtained throughout the last decade and considering that the transition timings will vary considerably among different countries, it is important to determine WGS-based MLVA alleles of the nowadays sequenced genomes. On this regard, we aimed to evaluate the performance of a Python script that had been previously developed for the rapid in silico extraction of the MLVA alleles, by comparing it to the PCR-based MLVA procedure over 83 strains from different Brucella species. The WGS-based MLVA approach detected 95.3% of all possible 1,328 hits (83 strains×16 loci) and showed an agreement rate with the PCR-based MLVA procedure of 96.4% for MLVA-16. According to our dataset, we suggest the use of a minimal depth of coverage of ~50x and a maximum number of ~200 contigs as guiding "boundaries" for the future application of the script. In conclusion, the evaluated script seems to be a very useful and robust tool for the in silico determination of MLVA profiles of Brucella strains, allowing retrospective and prospective molecular epidemiological studies, which are important for maintaining an active epidemiological surveillance of brucellosis. [ABSTRACT FROM AUTHOR]

Background: Coxiella burnetii is the etiological agent of Q fever, a zoonosis affecting many animal species including sheep and goats. The aims of this study were to evaluate the shedding of Coxiella burnetii in small ruminant herds and to identify the pathogen's genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods. Results: Overall, 165 samples from 43 herds of goats and 9 flocks of sheep were collected including bulk tank milk (BTM), individual milk samples, vaginal swabs, tissue sections from stillborn kids, feces and placentas. These were tested by real-time PCR targeting the IS1111 element. C. burnetii infection was confirmed in 51.16% of the herds of goats and 22.2% of the flocks of sheep. Six out of nine samples originating from goats were successfully genotyped using the MLVA method. The presence was confirmed of two widely distributed MLVA genotypes (I and J) and genotype PL1 previously reported only in cattle. Only one sequence type (ST61) was identified; however, the majority of specimens represented partial STs and some of them may belong to ST61. Other partial STs could possibly be ST74. Conclusion: This study confirmed the relatively common occurrence of Coxiella burnetii in small ruminant herds in Poland. Interestingly, all genotyped samples represent cattle-associated MLVA genotypes. [ABSTRACT FROM AUTHOR]

Background: Prevalence of brucellosis and MLVA genotyping in animals and humans, isolated from different regions of Pakistan was performed. Animals having history of brucellosis from the field and local farms of Bannu, Mardan, Peshawar, Swat, Lahore and Islamabad were selected for blood collection. Humans that work with them were also selected for sampling in this study. Total of 600 samples were taken from cattle and humans and subjected to Rose Bengal plate Test (RBPT) for the initial screening of positive samples. Designed primers of B.abortus for cattle and B.melitensis for humans were utilised to perform PCR. Culturing and isolation was carried to further to perform MLVA genotyping assay through the selection of two panels of primer markers. Results: RBPT showed more number of cases of brucellosis in animals and humans compared to the PCR findings. Genotyping findings based upon MLVA‐15 set of markers demonstrated that the isolated strains of B.abortus fall in the same clade with strain A1, P8 and A2 from Pakistan and also similar with BCCN#02‐45 strain from India. On the other hand, B.melitensis isolated from different districts of Pakistan shared the same clade with BwIM‐AFG 63, BwIM‐AFG 44 strains from Afghanistan and BwIM IRN 37 strain from Iran. Selected VNTR alleles were sequenced for calibration purposes. Conclusion: It is concluded that Brucella is prevalent in animals and humans in studied districts of Pakistan. Moreover, A1, P8, BwIM‐AFG 63, BwIM‐AFG 44 and A2 were found the common genotypes in Pakistan. [ABSTRACT FROM AUTHOR]

Brucellosis is an important zoonosis that is emerging in some regions of the world, gaining increased relevance with the inclusion of the causing agent Brucella spp. in the class B bioterrorism group. Until now, multi-locus VNTR Analysis (MLVA) based on 16 loci has been considered as the gold standard for Brucella typing. However, this methodology is laborious, and, with the rampant release of Brucella genomes, the transition from the traditional MLVA to whole genome sequencing (WGS)-based typing is on course. Nevertheless, in order to avoid a disruptive transition with the loss of massive genetic data obtained throughout the last decade and considering that the transition timings will vary considerably among different countries, it is important to determine WGS-based MLVA alleles of the nowadays sequenced genomes. On this regard, we aimed to evaluate the performance of a Python script that had been previously developed for the rapid in silico extraction of the MLVA alleles, by comparing it to the PCR-based MLVA procedure over 83 strains from different Brucella species. The WGS-based MLVA approach detected 95.3% of all possible 1,328 hits (83 strains×16 loci) and showed an agreement rate with the PCR-based MLVA procedure of 96.4% for MLVA-16. According to our dataset, we suggest the use of a minimal depth of coverage of ~50x and a maximum number of ~200 contigs as guiding “boundaries” for the future application of the script. In conclusion, the evaluated script seems to be a very useful and robust tool for the in silico determination of MLVA profiles of Brucella strains, allowing retrospective and prospective molecular epidemiological studies, which are important for maintaining an active epidemiological surveillance of brucellosis.

The aim was to develop a short MLVA-typing scheme of the causative agent of glanders and to assess the possibility of its use for differentiation of Burkholderia mallei strains and study their genetic polymorphism.Materials and methods. The study was carried out on 14 B. mallei strains from the collection of the Volgograd Research Institute for Plague Control and 12 whole genome sequences of the B. mallei strains presented in GenBank NCBI. A set of 32 loci proposed for differentiation of the melioidosis pathogen was used to select VNTR-loci for typing the causative agent of glanders. Polyacrylamide gel electrophoresis, sequencing, and fragment analysis were applied to detect the size of the VNTR fragments.Results. VNTR loci 993, 3145, 3652, 20, 2862, and 1217, which were selected as the most variable among the causative agent of glanders, were included in the final MLVA typing scheme. The parameters of setting and detecting the results of MLVA typing have been optimized.Conclusion. Analisys of the typing results of 26 B. mallei strains showed a high discriminating power of the developed method of intraspecies differentiation of glanders pathogen based on 6-loci MLVA-scheme and the prospects of its use for epidemiological investigation to determine the source of the glanders outbreak.

Abstract Background The rapid identification of lineage remains a challenge in the genotyping of clinical isolates of recombinogenic pathogens. The chromosome of Mycobacterium avium subsp. hominissuis (MAH), an agent of Mycobacterium avium complex (MAC) lung disease, is often mosaic and is composed of chromosomal segments originating from different lineages. This makes it difficult to infer the MAH lineage in a simple experimental set-up. To overcome this difficulty, we sought to identify chromosomal marker genes containing lineage-specific alleles by genome data mining. Results We conducted genetic population structure analysis, phylogenetic analysis, and a survey of historical recombination using data from 125 global MAH isolates. Six MAH lineages (EA1, EA2, SC1, SC2, SC3, and SC4) were identified in the current dataset. One P-450 gene (locus_tag MAH_0788/MAV_0940) in the recombination-cold region was found to have multiple alleles that could discriminate five lineages. By combining the information about allele type from one additional gene, the six MAH lineages as well as other M. avium subspecies were distinguishable. A recombination-cold region of 116 kb contains an insertion hotspot and is flanked by a mammalian cell-entry protein operon where allelic variants have previously been reported to occur. Hence, we speculate that the acquisition of lineage- or strain-specific insertions has introduced homology breaks in the chromosome, thereby reducing the chance of interlineage recombination. Conclusions The allele types of the newly identified marker genes can be used to predict major lineages of M. avium. The single nucleotide polymorphism typing approach targeting multiallelic loci in recombination-cold regions will facilitate the epidemiological study of MAC, and may also be useful for equivalent studies of other nontuberculous mycobacteria potentially carrying mosaic genomes.

Abortion in ruminants is an important problem in Turkey and it leads to serious economic losses for farmers. The aim of this study is to diagnose Chlamydia abortus in ruminant abortions in the Marmara Region of Turkey and to determine the genotypes of the agent and obtain the first national epidemiological data in this sense. For this purpose, a total of 730 abortion materials (fetal tissue, fetal stomach contents, placentas, cotyledons, vaginal swabs) belonging to 267 cattle, 380 sheep, 70 goats and 13 water buffaloes were examined for C. abortus with species specific real-time PCR. DNAs of positive samples were genotyped by the Multilocus Variable Number Tandem Repeat Analysis (MLVA) method. From 730 materials, 87 (11.9%) were found positive for C. abortus. Positivity rates were 21.4% in goats, 16.6% in sheep, 7.7% in water buffaloes and 3% in cattle. The dominant genotype was found to be MLVA genotype 2 (93.1%), and 4 different genotypes including genotypes 3, 4, and 5 were involved in infections. So it is concluded that genotypic diversity of C. abortus is high in the Marmara region. It was also revealed that C. abortus was responsible for a significant proportion of small ruminant abortions in this region. [ABSTRACT FROM AUTHOR]

Nationwide increases in Mycoplasma pneumoniae pneumonia cases in Japan were reported in 2011, 2012, 2015, and 2016. In this study, we isolated 554 M. pneumoniae strains in 4 areas in Japan (Kanagawa, Okayama, Osaka, and Saitama) between 2006 and 2019, and performed genotyping analysis. More than 80% of the strains isolated in 2011 and 2012 harbored type 1 p1 adhesin gene; however, strains harboring type 2 or its variant p1 gene increased in 2015 and 2016 and dominated after 2017. These findings suggested that a shift in the prevalent genotype of M. pneumoniae clinical strains occurred recently in Japan. More than 90% of the type 1 strains isolated after 2010 harbored macrolide-resistance mutations in their 23S rRNA gene, whereas most type 2 lineage strains had no such mutations. Consequently, the increase in type 2 lineage strains in Japan has reduced the macrolide resistance rate of clinical M. pneumoniae strains. During this analysis, we also identified M. pneumoniae strains carrying a novel variant type 1 p1 gene, and we classified it as type 1b. We then sequenced the genomes of 81 selected M. pneumoniae strains that we collected between 1976 and 2017 in Japan, and compared them with 156 M. pneumoniae genomes deposited in public databases to provide insights into the interpretation of M. pneumoniae genotyping methods, including p1 typing, multiple-locus variable-number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST), and typing by 8 single-nucleotide polymorphism markers (SNP-8). As expected, p1 typing, MLST, and SNP-8 results exhibited good correlation with whole-genome SNP analysis results in terms of phylogenetic relationships; however, MLVA typing results were less comparable to those of the other methods. MLVA may be useful for the discrimination of strains derived from a single outbreak within a limited area; however, is not reliable for classification of strains collected from distantly separated areas at different time points. This study showed the usefulness of genome-based comparison of M. pneumoniae for molecular epidemiology. Genome sequencing of more strains will improve our understanding of global propagation routes of this pathogen and evolutionary aspects of M. pneumoniae strains.

Salmon gill poxvirus (SGPV) can cause serious gill disease in Atlantic salmon (Salmo salar L.) and represents a significant problem to aquaculture industries in Northern Europe. Here, a single-tube multi-locus variable-number tandem-repeat (VNTR) analysis (MLVA) genotyping assay, targeting eight VNTR loci, was developed for studying the epizootiology of SGPV. Through MLVA typing of SGPV positive samples from 180 farmed and wild Atlantic salmon in Northern Europe, the first molecular population study of this virus was undertaken. Comparison of resulting MLVA profiles by cluster analysis revealed considerable micro-diversity, while only a limited degree of specific clustering by country of origin could be observed, and no clustering relating to the severity of disease outbreaks. Phylogenetic analysis, based on genomic data from six SGPV specimens (three Norwegian, one Scottish, one Faroese and one Canadian), complemented and corroborated MLVA by pointing to a marked transatlantic divide in the species, with one main, relatively conserved, SGPV lineage as predominant in Europe. Within certain fjord systems and individual freshwater salmon smolt farms in Norway, however, discrete MLVA clustering patterns that prevailed over time were observed, likely reflecting local predominance of specific SGPV sub-lineages. MLVA typing was also used to refute two suspected instances of vertical SGPV transmission from salmon broodstock to offspring, and to confirm a failed disinfection attempt in one farm. These novel insights into the previously undocumented population structure of SGPV provide important clues, e.g., regarding the mechanisms underlying spread and recurrence of the virus amongst wild and farmed salmon populations, but so far no indications of more or less virulent SGPV sub-lineages have been found. The MLVA scheme represents a highly sensitive genotyping tool particularly well suited for illuminating SGPV infection routes, and adds to the relatively low number of MLVA protocols that have so far been published for viral species. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed within a single working day. Resulting MLVA profiles can be readily shared and compared across laboratories, facilitating rapid placement of samples in an international ezpizootiological context.

Abstract Background As the epidemiology of human Q Fever generally reflects the spread of Coxiella burnetii in ruminant livestock, molecular characterization of strains is essential to prevent human outbreaks. In this study we report the genetic diversity of C. burnetii in central Italy accomplished by MST and MLVA-6 on biological samples from 20 goat, sheep and cow farms. Results Five MST and ten MLVA profiles emerged from the analysis establishing a part of C. burnetii strain world atlas. In particular, ST32 occurred on 12 farms (60%), prevalently in goat specimens, while ST12 (25%) was detected on 4 sheep and 1 goat samples. ST8 and a variant of this genotype were described on 2 different sheep farms, whereas ST55 was observed on a goat farm. Five complete MLVA profiles different from any other published genotypes were described in this study in addition to 15 MLVA incomplete panels. Despite this, polymorphic markers Ms23, Ms24 and Ms33 enabled the identification of samples sharing the same MST profile. Conclusions Integration of such data in international databases can be of further help in the attempt of building a global phylogeny and epidemiology of Q fever in animals, with a “One Health” perspective.

BACKGROUND AND OBJECTIVE: Pseudomonas aeruginosa is considered as one of the important causes of urinary infections in hospitals. The aim of the current study is the genetic typing for the number of bacterial strains isolated from patients using MLVA technique. METHODS: In this study, 70 isolates were collected from different hospitals located in Tehran city. First, DNA extraction was conducted for genotyping analysis by MLVA method. Subsequently, VNTR sequences located in several genes of bacterial genomes such as MS-214, MS-215, MS-217, MS-222, MS-223, MS-142 and MS-173 were amplified by specific primers using PCR technique. After confirming the PCR amplification using electrophoresis and visualization of their bonds on agarose gel, relationship evolutionary graph for the different strains was constructed based on MLVA technique. FINDINGS: After the electrophoresis and determination of VNTR copy-numbers, 70 strains were classified as 36 types. The genetic evolutionary tree was also constructed based on VNTRs Data. According to the results, PCR products of 70 strains visualized by electrophoresis are divided to 39 types based on VNTR analysis. Evolution tree and MSR algorithm was also constructed for all 70 clinical isolates. According to the MST algorithm, 70 clinical strains divided into 11 clonal complexes which these criteria is interpreted as genetic distance based on the difference of VNTR copy numbers for each group CONCLUSION: The present study showed that MLVA could be helpful for typing clinical strains of P. aeruginosa. The results also showed that this method had great potential to differentiate those strains with high phenotypic similarity.

Nationwide increases in Mycoplasma pneumoniae pneumonia cases in Japan were reported in 2011, 2012, 2015, and 2016. In this study, we isolated 554 M. pneumoniae strains in 4 areas in Japan (Kanagawa, Okayama, Osaka, and Saitama) between 2006 and 2019, and performed genotyping analysis. More than 80% of the strains isolated in 2011 and 2012 harbored type 1 p1 adhesin gene; however, strains harboring type 2 or its variant p1 gene increased in 2015 and 2016 and dominated after 2017. These findings suggested that a shift in the prevalent genotype of M. pneumoniae clinical strains occurred recently in Japan. More than 90% of the type 1 strains isolated after 2010 harbored macrolide-resistance mutations in their 23S rRNA gene, whereas most type 2 lineage strains had no such mutations. Consequently, the increase in type 2 lineage strains in Japan has reduced the macrolide resistance rate of clinical M. pneumoniae strains. During this analysis, we also identified M. pneumoniae strains carrying a novel variant type 1 p1 gene, and we classified it as type 1b. We then sequenced the genomes of 81 selected M. pneumoniae strains that we collected between 1976 and 2017 in Japan, and compared them with 156 M. pneumoniae genomes deposited in public databases to provide insights into the interpretation of M. pneumoniae genotyping methods, including p1 typing, multiple-locus variable-number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST), and typing by 8 single-nucleotide polymorphism markers (SNP-8). As expected, p1 typing, MLST, and SNP-8 results exhibited good correlation with whole-genome SNP analysis results in terms of phylogenetic relationships; however, MLVA typing results were less comparable to those of the other methods. MLVA may be useful for the discrimination of strains derived from a single outbreak within a limited area; however, is not reliable for classification of strains collected from distantly separated areas at different time points. This study showed the usefulness of genome-based comparison of M. pneumoniae for molecular epidemiology. Genome sequencing of more strains will improve our understanding of global propagation routes of this pathogen and evolutionary aspects of M. pneumoniae strains. [ABSTRACT FROM AUTHOR]

Salmon gill poxvirus (SGPV) can cause serious gill disease in Atlantic salmon (Salmo salar L.) and represents a significant problem to aquaculture industries in Northern Europe. Here, a single-tube multi-locus variable-number tandem-repeat (VNTR) analysis (MLVA) genotyping assay, targeting eight VNTR loci, was developed for studying the epizootiology of SGPV. Through MLVA typing of SGPV positive samples from 180 farmed and wild Atlantic salmon in Northern Europe, the first molecular population study of this virus was undertaken. Comparison of resulting MLVA profiles by cluster analysis revealed considerable micro-diversity, while only a limited degree of specific clustering by country of origin could be observed, and no clustering relating to the severity of disease outbreaks. Phylogenetic analysis, based on genomic data from six SGPV specimens (three Norwegian, one Scottish, one Faroese and one Canadian), complemented and corroborated MLVA by pointing to a marked transatlantic divide in the species, with one main, relatively conserved, SGPV lineage as predominant in Europe. Within certain fjord systems and individual freshwater salmon smolt farms in Norway, however, discrete MLVA clustering patterns that prevailed over time were observed, likely reflecting local predominance of specific SGPV sub-lineages. MLVA typing was also used to refute two suspected instances of vertical SGPV transmission from salmon broodstock to offspring, and to confirm a failed disinfection attempt in one farm. These novel insights into the previously undocumented population structure of SGPV provide important clues, e.g., regarding the mechanisms underlying spread and recurrence of the virus amongst wild and farmed salmon populations, but so far no indications of more or less virulent SGPV sub-lineages have been found. The MLVA scheme represents a highly sensitive genotyping tool particularly well suited for illuminating SGPV infection routes, and adds to the relatively low number of MLVA protocols that have so far been published for viral species. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed within a single working day. Resulting MLVA profiles can be readily shared and compared across laboratories, facilitating rapid placement of samples in an international ezpizootiological context. [ABSTRACT FROM AUTHOR]

Brucellosis, caused by the bacteria of the genus Brucella, is one of the most neglected common zoonotic diseases globally with a public health significance and a high economic loss among the livestock industry worldwide. Since little is known about the distribution of B. abortus in Egypt, a total of 46 B. abortus isolates recovered between 2012–2020, plus one animal isolate from 2006, were analyzed by examining the whole core genome single nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA). Both cgSNP analysis and MLVA revealed three clusters and one isolate only was distantly related to the others. One cluster identified a rather widely distributed outbreak strain which is repeatedly occurring for at least 16 years with marginal deviations in cgSNP analysis. The other cluster of isolates represents a rather newly introduced outbreak strain. A separate cluster comprised RB51 vaccine related strains, isolated from aborted material. The comparison with MLVA data sets from public databases reveals one near relative from Argentina to the oldest outbreak strain and a related strain from Spain to a newly introduced outbreak strain in Egypt. The distantly related isolate matches with a strain from Portugal in the MLVA profile. Based on cgSNP analysis the oldest outbreak strain clusters with strains from the UK. Compared to the in silico analysis of MLVA, cgSNP analysis using WGS data provides a much higher resolution of genotypes and, when correlated to the associated epidemiological metadata, cgSNP analysis allows the differentiation of outbreaks by defining different outbreak strains. In this respect, MLVA data are error-prone and can lead to incorrect interpretations of outbreak events.

Abstract The pig is one of the main reservoirs of Yersinia enterocolitica strains pathogenic to humans. A description of the Y. enterocolitica population in this reservoir, and accurate discriminatory techniques for typing isolates are needed for prevention, outbreak investigation, and surveillance. This study investigates the genetic diversity of pathogenic Y. enterocolitica isolates obtained from pig tonsils in a French pig slaughterhouse in 2009 (S1) and 2010 (S2). The use of Pulsed‐Field Gel Electrophoresis (PFGE) and MLVA as typing techniques was also compared and evaluated. First, a total of 167 isolates (12 of biotype 3 recovered during S1, and 155 of biotype 4 recovered during S1 and S2) were typed by PFGE using the XbaI enzyme. MLVA was then tested on all the biotype 3 isolates in addition to 70 selected biotype 4 isolates recovered over the 2 years. PFGE generated two specific XbaI‐PFGE profiles for biotype 3 isolates. Nine XbaI profiles were obtained for biotype 4, with a higher diversity (ID = 0.599) than biotype 3 (ID = 0.167). Two out of the nine XbaI profiles were reported during both surveys and at different months. MLVA improved the differentiation between isolates; the index of diversity reached 0.621 and 0.958, respectively, for biotype 3 (three MLVA types) and biotype 4 (32 MLVA types). The MLVA types for biotype 4 differed over the two surveys, but some isolates with different MLVA types were genetically closely related. This study provides an initial evaluation of the genetic diversity of Y. enterocolitica strains isolated from pigs in France. We show that some PFGE profiles are maintained in the pig production sector, and, through MLVA, that part of the Y. enterocolitica population remained genetically close over the two years. MLVA proved its effectiveness as a tool for investigating pathogenic Y. enterocolitica strains isolated from pigs.

Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. Several distinct genetic lineages or genomic groups have been shown to exist, with evidence for different virulence potential of these lineages. Multispacer Sequence Typing (MST) and Multiple-Locus Variable number tandem repeat Analysis (MLVA) are being used to genotype strains. However, it is unclear how these typing schemes correlate with each other or with the classification into different genomic groups. Here, we created extensive databases for published MLVA and MST genotypes of C. burnetii and analysed the associated metadata, revealing associations between animal host and human disease type. We established a new classification scheme that assigns both MST and MLVA genotypes to a genomic group and which revealed additional sub-lineages in two genomic groups. Finally, we report a novel, rapid genomotyping method for assigning an isolate into a genomic group based on the Cox51 spacer sequence. We conclude that by pooling and streamlining existing datasets, associations between genotype and clinical outcome or host source were identified, which in combination with our novel genomotyping method, should enable an estimation of the disease potential of new C. burnetii isolates.

• Genotypic properties of P.aeruginosa strains caused UTI were assessed using MLVA. • Based on MLVA method, 61 various genotypes of P. aeruginosa were identified. • MLVA is a cost effective, easy, and high resolution technique. • To detect the presence of TEM and SHV resistance genes, conventional PCR was performed. • Approximately 80% and 70% of strains carried the TEM and SHV resistance genes, respectively. Numerous nosocomial infections including urinary tract infection (UTI) have been reported to be linked to Pseudomonas aeruginosa (P. aeruginosa). This bacterium is one of the most common pathogen colonized in the urinary tract. The main purpose of this study was to evaluated the presence of antibiotic resistance genes and also the most frequent genotype patterns of P. aeruginosa in the patients with UTI hospitalized in different wards of hospitals. In this study, 70 strains of P. aeruginosa isolated of urine samples from the patients with UTI were assessed. The isolated strains were genotyped using Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) method. We have also analyzed the presence of TEM and SHV resistant genes in the isolates. A total of 70 P. aeruginosa strains was isolated from the UTI patients. Based on MLVA method, 61 various genotypes of P. aeruginosa were identified which grouped into two main clusters and 4 sub-clusters. Moreover, approximately 80% and 70% of isolated strains carried the TEM and SHV resistance genes, respectively. Our findings showed that the majority of patients hospitalized in different wards of hospitals have experienced the urinary tract infection caused by P. aeruginosa. According to the genotyping results, a high diversity of the P. aeruginosa population was observed in the patients with UTI. Our results can provide a better understanding of the P. aeruginosa genotype distribution and epidemiology of infection, which can be applied as basic data for future antibiotic therapies. [ABSTRACT FROM AUTHOR]

Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.

Mastitis in goats is mainly caused by coagulase-negative Staphylococcus (CNS). The identification methods for this group are based on evaluating the expression of phenotypic characteristics such as the ability to metabolize various substrates; however, this is disadvantageous as these methods are dependent on gene expression. In recent years, genotyping methods such as the Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) and gene identification have been useful for epidemiological study of several bacterial species. To develop a genotyping method, the genome sequence of Staphylococcus chromogenes MU970 was analysed. The analysis showed nine virulence genes described in Staphylococcus aureus. The MLVA was developed using four loci identified in the genome of S. chromogenes MU970. This genotyping method was examined in 23 strains of CNS isolated from goat mastitis. The rate of discrimination for MLVA was 0.8893, and the highest rates of discrimination per the index of Simpson and Hunter-Gaston were 0.926 and 0.968 for the locus 346_06, respectively. The virulence genes were present in all strains of S. chromogenes but not in other CNS. The genotyping method presented in this paper is a viable and easy method for typifying CNS isolates from mastitis cases in different regions and is an ideal mean of tracking this disease.

Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets. [ABSTRACT FROM AUTHOR]

Currently, although the prevalence of brucellosis in Kazakhstan remains high, there are limited data available on the genetic diversity of circulating Brucella strains. Here, MLVA was employed to genotype a panel of 102 Brucella isolates collected from eight Kazakh regions and neighboring countries (Russia, Kyrgyzstan) during the period 1935–2017. MLVA-11 analysis classified 64 B . abortus strains into genotypes 72, 82, 331, 71, 341 and 69, while one genotype was novel, having no correspondence within the MLVA international database. MLVA-11 analysis of 37 B . melitensis strains showed 100% identity with genotypes 116, 114 and 11. One B . suis strain was classified into genotype 33. Phylogeography based on MLVA-15 demonstrated that all B . abortus and B . melitensis strains belonged to “Abortus C” and “East Mediterranean” lineages, respectively. B . abortus strains from Kazakhstan and Russia resulted genetically related to Portuguese, Brazilian and US isolates, suggesting ancient spread of these lineages from Europe westwards to South America and eastwards to Turkey, Russia and Asia. Most of Kazakh B . melitensis isolates were related to strains circulating in China, likely due to long-term trading partnerships between the two countries. In fine-scale MLVA-15 analysis, 17 B . abortus and 12 B . melitensis genotypes were identified; among them 12 are novel. Interestingly, epidemiological information supporting molecular data were retrieved for two clusters within the B . abortus group, thus proving that MLVA is an appropriate tool for effective traceback analyses. Our findings suggest that molecular genotyping should be applied systematically to support control plans for eradication of brucellosis in Kazakhstan. [ABSTRACT FROM AUTHOR]

Bacillus cereus sensu lato group, which includes the causative agent of anthrax Bacillus anthracis, has a high degree of genetic similarity. However, in recent years, the development of molecular genetics has made it possible to find some molecular markers for identifying Bacillus anthracis in order to differentiate it from the closely related bacilli, get the clustering of the collection of strains, and describe the phylogenetic relationships between the clusters. This article deals with up-to-date methods of genotyping of Bacillus anthracis; it also evaluates its discrimination ability, informational value, validity of the results, efficiency, simplicity, and ease of use. In addition, international online resources and the possibility of their use for comparing collections of the B. anthracis strains isolated from various parts of the world are discussed. [ABSTRACT FROM AUTHOR]

Estimated has been efficacy of MLVA25 method, used for differentiation and clusterization of Yersinia pestis strains, in reference to Yersinia pseudotuberculosis strains, as well as the search of Y. pseudotuberculosis strains mostly closely related to Y. pestis . Applied was the reduced version of the technique deploying 14 out of 25 VNTR loci. Comparative study of 71 Y. pseudotuberculosis isolates and five Y. pestis strains by means of MLVA25 and MLVA14 variants revealed 75 and 54 genotypes, respectively. Y. pseudotuberculosis strains of certain serovars corresponded to certain clusters of MLVA types. According to MLVA14 typing of 221 Y. pseudotuberculosis isolates the clusters including the strains belonging to MLST types ST19 (serovar O:3) and ST43 (serovar O:1b) were the most closely related to Y. pestis .

Background: Cephalosporin resistance in clinical E. coli isolates is increasing internationally. The increase has been caused by virulent and often multidrug-resistant clones, especially the extended spectrum β-lactamase (ESBL) producing E. coli clone O25b-ST131. Methods: In Norway, recommended empirical treatment of sepsis consists of gentamicin and penicillin combined, or a broad-spectrum cephalosporin. To investigate if increased gentamicin and cephalosporins resistance rates in our hospital could be caused by specific clones, we conducted a retrospective study on E. coli blood culture isolates from 2011 through 2015. All E. coli isolates non-susceptible to gentamicin and/or third-generation cephalosporins were genotyped using multiple-locus variable-number of tandem repeat analysis (MLVA) and compared with antibiotic susceptible isolates. The frequency of the most common genes causing ESBL production (blaCTX-M, blaampC) was examined by Real-Time PCR. Results: A total of 158 cephalosporin and/or gentamicin resistant and 97 control isolates were differentiated into 126 unique MLVA types. Of these, 31% of the isolates belonged to a major MLVA cluster consisting of 41% of the gentamicin resistant and 35% of the cephalosporin resistant isolates. The majority (65/80 isolates) of this MLVA cluster contained MLVA types associated with the E. coli O25b-ST131 clone. Genes encoding CTX-M enzyme phylogroups 1 and 9 occurred in 65% and 19% of cephalosporin resistant isolates, respectively, whereas blaampC-CIT was identified in 3%. Conclusion: No local E. coli bacteraemia clone was identified. Antibiotic resistance was dispersed over a variety of genotypes. However, association with the international E. coli O25b-ST131 clone was frequent and may be an important driver behind increased resistance rates. Monitoring and preventing dissemination of these resistant clones are important for continued optimal treatment. [ABSTRACT FROM AUTHOR]