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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]

Background: Brucellosis is the most important public health problem worldwide, and the annual incidence of the disease in humans is 2.1 million. The Brucella genome is highly conserved, with over 90% similarity among species. The aim of this study was to perform species-level identification of Brucella spp. strains isolated from humans diagnosed with brucellosis and to further investigate the phylogenetic relationships using multiple locus variable number of tandem repeats analysis (MLVA)-16 and 16S rRNA sequencing analysis. Materials and Methods:Brucella spp. was isolated from the blood cultures of 54 patients who tested positive for brucellosis through serological examinations. Real-time PCR was used to identify the isolates in species, and the genus level of Brucella was confirmed with 16S rRNA. All isolates were subjected to phylogenetic analysis using variable number of tandem repeat analysis with multiple loci. Results: Subsequent analysis via real-time PCR confirmed these isolates to be of the Brucella melitensis species. The 16S rRNA sequence analysis showed 100% homogeneity among the isolates. MLVA revealed the formation of five different genotypic groups. While two groups were formed based on the 16S rRNA sequence analysis, five groups were formed in the MLVA. Conclusions: The study concluded that 16S rRNA sequence analysis alone did not provide sufficient discrimination for phylogenetic analysis but served as a supportive method for identification. MLVA exhibited higher phylogenetic power. The widespread isolation of B. melitensis from human brucellosis cases highlights the importance of controlling brucellosis in small ruminants to prevent human infections. [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. [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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: Human brucellosis causes serious public health concerns in Ningxia, China. Methods: This study employed epidemiological, bacteriological, and multiplelocus variable-number tandem repeat analysis (MLVA) methods to conduct an epidemiological investigation, which is necessary for devising tailored control strategies. Results: Between 1958 and 2022, 29,892 cases were reported, with an average annual number of cases and incidence of 467 and 7.1/100,000, respectively. The epidemic situation gradually worsened, with cases escalating from 26 cases in 2005 to 6,292 in 2022, with the incidence rate rising from 0.441 in 2005 to 86.83 in 2022. Geographically, the disease spread from a single affected county in 2004 to encompass all 22 counties in 2022. Yanchi County had the highest incidence, followed by the Hongsibao and Tongxin counties. These data suggest that Brucella infection has become a rampant regional concern in human brucellosis. Between 1958 and 2019, a total of 230 Brucella strains were identified across four studied hosts. These strains comprised four species with 12 biovars, including B. melitensis bv. 1, bv. 2, bv. 3, B. abortus bv. 1, bv. 3, bv. 4, bv. 5, bv. 6, bv. 7, B. suis bv. 1 and bv. 3, and B. canis. These data highlight the high species/biovars and host diversity of the Brucella population, posing a substantial challenge to brucellosis surveillance. There was an apparent transition from multiple species/biovars historically to the current dominance of a single species, B. melitensis, emphasizing the requirement for strengthening surveillance of B. melitensis. Genotypes 42 and 116, constituting 96.2% of the total number of genotypes, predominated in panel 1 and MLVA-11, indicating that all strains belong to the East Mediterranean lineage. MLVA cluster analysis revealed persistent transmission of dominant circulating genotypes, presenting an epidemic pattern characterized primarily by epidemiologically related cases with a few sporadic cases. Strains in this study exhibited high genetic homogeneity with strains from the Northwest, and those from Kazakhstan and Mongolia. Conclusion: The epidemic situation of human brucellosis has gradually worsened; the rampant epidemic of the disease has become a regional concern. The present study highlights that implementing the of targeted surveillance and intervention strategies is urge. [ABSTRACT FROM AUTHOR]

Legionella is an important waterborne pathogen that causes legionellosis. Public baths are considered the primary cause of legionellosis infection in Japan. We investigated the prevalence and genetic distribution of 338 Legionella spp. isolates from 81 public bath facilities, includ- ing 35 hot springs and 46 other facilities, through annual periodic surveillance in Kobe, Japan, from 2016 to 2021. In addition, the genotypes of nine clinical strains of unknown infectious source from the same period were compared to those of bathwater isolates. We elucidated the differences in the distribution of Legionella species, serogroups, and genotypes between hot springs and other public baths. Legionella israe- lensis, L. londiniensis, and L. micdadei colonized hot springs along with L. pneumophila. The minimum spanning tree analysis based on multiple-locus variable number tandem repeat analysis (MLVA) also identified four major clonal complexes (CCs) in L. pneumophila SG1 and found that CC1 of the four CCs is a specific novel genotype with the lag-1 gene in hot springs. The same MLVA genotypes and sequence types as those of the clinical strains were not present among the strains isolated from bath water. Thus, our surveillance is useful for estimat- ing the sources of legionellosis infection in Japan and developing prevention strategies. [ABSTRACT FROM AUTHOR]

In the present research, we aimed to determine the characteristics of E. faecalis strains collected from an Iranian Children's Hospital for four years. Sixty-seven E. faecalis isolates with virulence genes detection, variablenumber tandem repeat (VNTR), and multiple-locus variable-number tandem repeat analysis (MLVA) typing were investigated. A high frequency of virulence genes belonged to gelatinase (73%) and Enterococcus faecalis (62%). The MLVA of 67 E. faecalis isolates revealed 52 VNTR patterns and 38 MLVA types (MTs). Furthermore, genetic diversities with the majority of the MT1 as a major MT in different Wards of the Children's Hospital were found. [ABSTRACT FROM AUTHOR]

Since 2010, the cases and incidences of human brucellosis have been increasing annually in Qinghai (QH) Province. Molecular epidemiology and phylogenetic analyses of strains from this region are crucial to better understand the transmission of the disease and the evolutionary patterns of Brucella strains. In this study, classical bio-typing assay, multilocus variable-number tandem repeat analysis, and the whole-genome sequencing–single-nucleotide polymorphism approach were used to illustrate the epidemiological and evolutionary patterns of Brucella melitensis. A total of 54 B. melitensis bv. 3 strains were isolated and molecularly characterized, with all strains belonging to the East Mediterranean lineages. Cross-regional transmission events (i.e., between counties) were caused by common sources of infection, suggesting that predominant circulating genotypes are endemic in different regions. Strengthening surveillance in animal brucellosis and controlling infected animals’ cross-border movement are necessary. Two strains isolated from humans and marmots were clustered in the same sub-clade, implying the possible existence of direct and/or indirect contact between sheep (and goats) and wildlife (marmots), but this needs to be verified by further investigations. The global-scale phylogenetic analysis indicated that 54 strains sorted into six subclades, four of which formed independent lineages, suggesting that the increase in the incidence rate of human brucellosis may be caused by local circulating lineages. Further strengthening the serology and pathogen surveillance of animals (wildlife) and humans will contribute to an indepth understanding of the transmission chain of human brucellosis in this region. [ABSTRACT FROM AUTHOR]

Escherichia coli is an important microorganism for cattle breeding. The aim of this study was to investigate the presence of phylogenetic groups, virulence factors, genotyping with multi-locus variable tandem repeat analysis (MLVA), and susceptibility to commonly used antimicrobial agents in E. coli strains isolated from aborted bovine fetal samples. In this study, phylogrouping and various virulence genes were analyzed by PCR in E. coli strains isolated from 637 bovine fetal tissue samples. Consequently, E. coli was isolated and identified in 24 samples in culture. Of the 24 isolates identified as positive, 12.5% were defined as group A, 83.3% as B1, and 4.2% as group B2. Of the E. coli isolates, virulence factor fimH was identified in eight (33.3%), traT in 15 (62.5%), ompT in five (20.8%), CNF1 in one (4.16%), and CNF2 in six (25%). Seven genotypic groups were determined as a result of the analysis with the MLVA 10 method. According to the antimicrobial susceptibility test results, high resistance was determined against amoxicillin/clavulanic acid and oxytetracycline. In conclusion, strains of E. coli containing CNF1, CNF2, fimH, traT , and ompT virulence factors can be associated with bovine abortions. It is noteworthy that the dominant phylogenetic group B1 has been observed in cases of cattle abortions. [ABSTRACT FROM AUTHOR]

Brucella melitensis primarily affects sheep, goats and is associated with brucellosis in humans, which is one of the world's most widespread neglected zoonotic disease. The current study attempted the determination of genetic diversity through comparative genome analysis of B. melitensis strains reported from India with other countries. The study also reports the isolation and identification of B. melitensis BMNDDB8664 from a cow with a history of abortion, whole-genome sequencing (WGS), determination of virulence factors, genotyping, and comparative genome analysis. Multilocus sequence typing, Multiple locus variable number of tandem repeats analysis (MLVA), and WGS based phylogeny revealed the predominance of ST-8 and genotypes (116 and II respectively) that clustered to the East Mediterranean lineage. Identification of hitherto unreported genotypes by MLVA also indicated the existence and circulation of West Mediterranean and American lineages in India. Though the AMOS-PCR results suggest the BMNDDB8664 isolate as Brucella abortus, the outcomes from multiplex PCR, ribosomal multilocus sequence typing, and WGS analysis confirmed it as B. melitensis. The analysis revealed the presence of adeF gene (aids conferring resistance to fluoro-quinolone and tetracyclines). The isolate lacked two important T4SS genes virB2 and virB7 genes (roles in infection and rifampicin resistance respectively) and also lacked the Brucella suis mprF gene that aids intracellular survival. Further, BMNDDB8664 lacked some of the genes associated with LPS synthesis (wbkB, wbkC) and transport (wzm, wzt) and hence, is most likely a rough strain. WGS-based phylogenetic analysis revealed close genetic relatedness of this BMNDDB8664 with a sheep isolate and two human isolates. The results prompt systematic, broad-based epidemiological studies on brucella infection at the species level. For effective control of human brucellosis, a concerted One Health approach with studies encircling the identification of aetiology at species, strain level to find their prevalence, spread, and inter-host transmission patterns need to be understood, for better design and implementation of effective control strategies in India and other endemic regions. [ABSTRACT FROM AUTHOR]

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]

Objective: Brucellosis caused by bacteria of the Brucella genus is a globally important zoonotic disease. B. abortus, which usually causes abortion and infertility in cows, can cross-infect ruminants and also cause chronic disease in humans. Controlling outbreaks in ruminants is essential to prevent economic losses and human disease due to bovine brucellosis. Since classical phenotypic methods alone are insufficient in monitoring epidemic strains of B. abortus, genotypic surveillance has become crucial today. We aimed to determine the role of B. abortus strains in the abortion etiology of domestic ruminants in the Cukurova region, Türkiye, reveal the phylogenetic relationships of the isolated strains using molecular methods and examine the availability of these methods in this field. Methods: One hundred eighteen abortion samples were included in the study. Following the isolation and identification of B. abortus, the clonal relationship between these isolates was investigated by Pulse-Field Gel Electrophoresis (PFGE) and Multi-Locus Variable Number Tandem Repeat Analysis (MLVA) methods. Results: B. abortus was detected in 17 (14.4%) of 118 abortion cases examined. The distribution of B. abortus isolates among cattle, goats and sheep was 72.2% (13/18), 5.9% (3/51) and 2.0% (1/49), respectively. The 14 (82.4%) of B. abortus isolates were biovar 3. A single cluster and four pulsotypes (HGDI=0.5662) were determined by the PFGE, five types (HGDI=0.6838) by the MLVA-16, and six genotypes (HGDI=0.7132) by evaluating these two methods together. Genotype 1, which consists of nine (52.9%) isolates, six obtained from cattle and three from goats, was predominant. Conclusion: Although the discriminative powers of the PFGE and MLVA methods were not high enough when a small number of samples were examined, the discriminatory power of MLVA-16 was determined to be higher than PFGE in determining the regionally predominant B. abortus genotypes. Moreover, the use of both together was even more effective. Since using the two methods together in epidemiological studies requires additional costs and effort, it was concluded that the predominant genotypes could be determined, especially during epidemic periods and evaluated in vaccine studies. Additional examination of human clinical specimens in epidemic areas will further strengthen the results to be obtained. Thus, our study will provide essential data for the studies to be performed in order to control brucellosis outbreaks, which constitute a serious public health problem. [ABSTRACT FROM AUTHOR]

The epidemiology of Salmonella Infantis is complex in terms of its distribution and transmission. The continuous collection and analysis of updated data on the prevalence and antimicrobic resistance are essential. The present work aimed to investigate the antimicrobial resistance and the correlation among S. Infantis isolates from different sources through the multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA). A total of 562 Salmonella strains isolated from 2018 to 2020 from poultry, humans, swine, water buffalo, mussels, cattle, and wild boar were serotyped, and 185 S. Infantis strains (32.92%) were identified. S. Infantis was commonly isolated in poultry and, to a lesser extent, in other sources. The isolates were tested against 12 antimicrobials, and a high prevalence of resistant strains was recorded. S. Infantis showed high resistance against fluoroquinolones, ampicillin, and tetracycline, which are commonly used in human and veterinary medicine. From all S. Infantis isolates, five VNTR loci were amplified. The use of MLVA was not sufficient to understand the complexity of the epidemiological relationships between S. Infantis strains. In conclusion, an alternative methodology to investigate genetic similarities and differences among S. Infantis strains is needed. [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]

The genotyping of the multidrug-resistant Klebsiella pneumoniae species complex is essential to identify outbreaks and to track their source and spread. The aim of this study was to improve and extend the typeability, availability, cost and time efficiency of an existing multi-locus VNTR analysis (MLVA). A modified scheme (MLVA8+) was adopted and validated for strain-level differentiation of the three Klebsiella species involved in human pathology. A diverse set of 465 K. pneumoniae clinical isolates from 22 hospitals and 3 outpatient laboratories in Bulgaria were studied, where 315 were carbapenem-resistant. The MLVA8+ typeability was significantly improved and the typing data were validated against 158 isolates which were previously typed by WGS. The MLVA8+ results were highly concordant with the classic 7-locus MLST and the novel K. variicola MLST, but had greater congruency coefficients (adjusted Wallace). A major advantage was the differentiation of the hybrid cluster ST258 into its corresponding clades. Furthermore, the applicability of MLVA8+ was demonstrated by conducting a retrospective investigation of the intra-hospital spread of blaKPC-, blaNDM- and blaOXA-48-like producers. The MLVA8+ has improved utility and extended typing scope to K. variicola and K. quasipneumoniae, while its cost and time-to-result were reduced. [ABSTRACT FROM AUTHOR]

In this study, Listeria isolates (214) were characterized as follows: L. innocua (77.10%), L. monocytogenes (11.21%), L. welshimeri (5.61%), L. grayi (1.40%), L. seeligeri (0.93%), and L. species (3.73%) that were not identified at the species level, from beef and beef based products from retail and farms in Mpumalanga and North West provinces of South Africa. MLVA was further used to type Listeria innocua isolates (165) and Listeria monocytogenes isolates (24). The L. monocytogenes isolates were also serogrouped using PCR. The MLVA protocol for L. monocytogenes typing included six tandem repeat primer sets, and the MLVA protocol for L. innocua included the use of three tandem repeats primer sets. The L. monocytogenes serogroups were determined as follows: 4b-4d-4e (IVb) (37.50%), 1/2a-3a (IIa) (29.16%), 1/2b-3b (IIb) (12.50%), 1/2c-3c (IIc) (8.33%), and IVb-1 (4.16%). MLVA could cluster isolates belonging to each specie, L. monocytogenes, and L. innocua isolates, into MLVA-related strains. There were 34 and 10 MLVA types obtained from the MLVA typing of L. innocua and L. monocytogenes, respectively. MLVA clustered the L. monocytogenes isolates irrespective of sample category, serogroups, and geographical origin. Similarly, the L. innocua isolates clustered irrespective of meat category and geographical origin. MLVA was able to cluster isolates based on MLVA relatedness. The clustering of isolates from farms and retailers indicates transmission of Listeria spp. MLVA is an affordable, simple, and discriminatory method that can be used routinely to type L. monocytogenes and L. innocua isolates. [ABSTRACT FROM AUTHOR]

Escherichia coli ST131 is a pandemic clone with high antibiotic resistance, and it is a major causative agent of urinary tract infection (UTI) and bloodstream infections. This study evaluated the distribution and expression of virulence genes and genotyping of E. coli O25b/ST131 by Multi-locus variable number tandem repeat analysis (MLVA) method among UTI in patients at Tehran hospitals, Iran. A total of 107 E. coli isolates were collected from UTI patients. Polymerase chain reaction (PCR) amplification of the pabB gene was used to identify E. coli O25b/ST131 and the prevalence of sat and hlyA virulence genes was also analyzed. The microtiter method quantified biofilm formation ability in E. coli O25b/ST131. The Real-Time PCR (qRT-PCR) was performed to evaluate the expression of sat and hlyA genes. Finally, MLVA was performed for E. coli O25b/ST131 genotyping by targeting seven tandem repeats. SPSS-16 software was used for statistical analysis. Molecular study showed that 71% of isolates carried the pabB gene and were considered E. coli O25b/ST131 strains. Also, 45.8% and 17.8% of isolates carried sat and hlyA genes, respectively. The 57.9% isolates had biofilm formation ability. Expression of the studied virulence genes showed an increase in strong biofilm producing E. coli O25b/ST131 strains. A total of 76 (100%) E. coli O25b/ST131 strains were typed by the MLVA method. High prevalence of E. coli O25b/ST131 isolates in UTI patients can be a serious warning to the treatment due to the high antibiotic resistance rate, expression of virulence genes, and biofilm formation. [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]

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]

Climate change brings new risks of emergence of especially dangerous diseases. The paper reports the possibility of assessing the pathogenic potential of bacteria as demonstrated by studying the allelic polymorphism of anthrax bacterium pathogenicity factor genes, which is a prerequisite for assessing the associated microbiological risks. The allelic polymorphism of the capBCADE operon (capB, capC, capA, capD, and capE genes) encoding the capsule biosynthesis proteins of Bacillus anthracis, and the acpA and acpB genes encoding the expression regulators of this operon have been studied for the first time. A number of single nucleotide polymorphisms (SNPs) were described in the strains of the studied sample, including 5 SNPs in the capB gene, 3 in capC, 4 in capA, 14 in capD, 2 in capE, and 15 in acpB, as well as 7 SNPs and one insertion in the acpA gene. As a result, the sample has been divided into sequence types for each gene and 17 genotypes, which are combinations of the identified sequence types. In silico translation of the detected alleles of the studied genes revealed three isoforms of the CapB and CapA proteins, two isoforms of the CapC and CapE proteins, six isoforms of the CapD protein, five isoforms of the AcpA protein, and four isoforms in the AcpB protein. It has been demonstrated that the SNP in the 351A → G position of capC is a marker of A.Br.Aust94 group strains. Based on the results, A.Br.Vollum group strains were divided into two subgroups. The strains in the evolutionary lines B and C differed from the line A strains by the presence of an 853G → A SNP in the acpA gene. In addition, a previously unknown variable number tandem repeat (VNTR), has been found in the acpA gene and the possibility of using it for differentiating and genotyping of B. anthracis strains has been demonstrated. [ABSTRACT FROM AUTHOR]

Background: Coxiella burnetii (Cb) is the causative agent of the zoonotic disease Q fever which is distributed worldwide. Molecular typing of Cb strains is essential to find out the infectious source and prevent Q fever outbreaks, but there has been a lack of typing data for Cb strains in China. The aim of this study was to investigate the genotypes of Cb strains in wild rats in Yunnan Province, China. Results: Eighty-six wild rats (Rattus flavipectus) were collected in Yunnan Province and 8 of the 86 liver samples from the wild rats were positive in Cb-specific quantitative PCR (qPCR). The Cb strains from the 8 rats were then typed into 3 genotypes using 10-spacer multispacer sequence typing (MST), and 2 of the 3 genotypes were recognized as novel ones. Moreover, the Cb strains in the wild rats were all identified as genotype 1 using 6-loci multilocus variable number of tandem repeat analysis (MLVA). Conclusions: This is the first report of genotypic diversity of Cb strains from wild rats in China. Further studies are needed to explore the presence of more genotypes and to associate the genotypes circulating in the wildlife-livestock interaction with those causing human disease to further expand on the epidemiological aspects of the pathogen. [ABSTRACT FROM AUTHOR]

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]

Objective(s): Human gastrointestinal tract harbors a variety of bacteria with vital roles in human health. Bacteroides fragilis is considered one of the dominant constituents of gut microflora which can act as an opportunistic pathogen leading to various diseases, including colon cancer, diarrhea, uterine and intrathecal abscesses, septicemia, and pelvic inflammation. In this study, multiple locus variable number of tandem repeats analysis (MLVA) was performed to genetically differentiate 50 B. fragilis isolates.Materials and Methods: Eight suitable tandem repeats (TRs) were selected by bioinformatics tools and were then subjected to PCR amplification using specific primers. Finally, MLVA profiles were clustered using BioNumerics 7.6 software package. Results: All VNTR loci were detected in all isolates using the PCR method. Overall, B. fragilis isolates were differentiated into 27 distinct MLVA types. The highest diversity index was allocated to TR1, TR2, TR5, TR6, and TR8; with this taken into account, strain type 14 was the most prevalent with 12 strains belonging to this type. Clustering revealed three major clusters of A, B, and C. With regards to the pathogenicity of B. fragilis and the outcomes of infections related to this microorganism, it is imperative to study this microorganism isolated from both patients and healthy individuals.Conclusion: This study aimed at evaluating the efficiency of MLVA for the genetic differentiation of B. fragilis. The results of this study indicate the promising efficiency of MLVA typing for cluster detection of this bacterium.

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]

Rhipicephalus microplus , a vector that can transmit many pathogens to humans and domestic animals, is widely distributed in Yunnan province, China. However, few reports on the prevalence of tick-borne pathogens (TBPs) in Rh. microplus in Yunnan are available. The aim of this study was to detect TBPs in Rh. microplus in Yunnan and to analyze the phylogenetic characterization of TBPs detected in these ticks. The adult Rh. microplus (n = 516) feeding on cattle were collected. The pooled DNA samples of these ticks were evaluated using metagenomic next-generation sequencing (mNGS) and then TBPs in individual ticks were identified using genus- or group-specific nested polymerase chain reaction (PCR) combined with DNA sequencing assay. As a result, Candidatus Rickettsia jingxinensis (24.61%, 127/516), Anaplasma marginale (13.18%, 68/516), Coxiella burnetii (3.10%, 16/516), and Coxiella- like endosymbiont (CLE) (8.33%, 43/516) were detected. The dual coinfection with Ca. R. jingxinensis and A. marginale and the triple coinfection with Ca. R. jingxinensis , A. marginale , and CLE were most frequent and detected in 3.68% (19/516) and 3.10% (16/516) of these ticks, respectively. The results provide insight into the diversity of TBPs and their coinfections in Rh. microplus in Yunnan province of China, reporting for the first time that C. burnetii had been found in Rh. microplus in China. Multilocus variable number tandem repeat analysis with 6 loci (MLVA-6) discriminated the C. burnetii detected in Rh. microplus in Yunnan into MLVA genotype 1, which is closely related to previously described genotypes found primarily in tick and human samples from different regions of the globe, indicating a potential public health threat posed by C. burnetii in Rh. microplus in Yunnan. [ABSTRACT FROM AUTHOR]

aim of this study was Clostridioides difficile outbreak investigation due to the emergence of rifampicin resistant ribotype 027 (RT 027) fecal isolates from patients of Polish tertiary care hospital between X. 2017 and II. 2018 using multilocus variable tandem repeat analysis (MLVA). Materials and Methods: Twenty-nine C. difficile fecal isolates from patients of tertiary care hospital in Southern Poland were ribotyped and analyzed by MLVA. Multiplex PCR (mPCR) for genes encoding GDH (gluD), toxins A (tcdA)/ B (tcdB), 16S rDNA and binary toxin genes (ctdA and ctdB) was performed. The antibiotic susceptibility profile was determined by E-test. Results: The A, B and binary toxins encoding genes were detected in all 29 C. difficile strains which were sensitive to metronidazole, vancomycin and were resistant to erythromycin, clindamycin, and moxifloxacin; resistance to imipenem demonstrated 97%, to rifampicin – 45% isolates. C. difficile strains could be grouped by MLVA into 5 distinct clusters, and the largest cluster II contains 16 strains. The comparison of rifampicin GM MIC of cluster II (n=16 strains) with all others (n=13) showed that strains from clusters I, III, IV and V possessed significantly (p < 0.005) higher GM MIC and were more resistant to rifampicin. Conclusion: MLVA analysis proved transmission and recognized outbreak due to multidrug-resistant RT 027 C. difficile among patients of tertiary care hospital in Southern Poland. The reason for this is probably the widespread occurrence of spores in the hospital environment, which includes, among others, neglect of hygienic procedures and epidemic supervision. High resistance to imipenem (97%) and to rifampicin (45%) among C. difficile RT 027 Silesian isolates is threatening and requires further studies to elucidate this phenomenon. [ABSTRACT FROM AUTHOR]

Background: The natural hosts of Shigella are typically humans and other primates, but it has been shown that the host range of Shigella has expanded to many animals. Although Shigella is becoming a major threat to animals, there is limited information on the genetic background of local strains. The purpose of this study was to assess the presence of virulence factors and the molecular characteristics of S. flexneri isolated from calves with diarrhea. Results: Fifty-four S. flexneri isolates from Gansun, Shanxi, Qinghai, Xinjiang and Tibet obtained during 2014 to 2016 possessed four typical biochemical characteristics of Shigella. The prevalences of ipaH, virA, ipaBCD, ial, sen, set1A, set1B and stx were 100 %, 100 %, 77.78 %, 79.63 %, 48.15 %, 48.15 and 0 %, respectively. Multilocus variable number tandem repeat analysis (MLVA) based on 8 variable number of tandem repeat (VNTR) loci discriminated the isolates into 39 different MLVA types (MTs), pulsed field gel electrophoresis (PFGE) based on NotI digestion divided the 54 isolates into 31 PFGE types (PTs), and multilocus sequence typing (MLST) based on 15 housekeeping genes differentiated the isolates into 7 MLST sequence types (STs). Conclusions: The findings from this study enrich our knowledge of the molecular characteristics of S. flexneri collected from calves with diarrhea, which will be important for addressing clinical and epidemiological issues regarding shigellosis. [ABSTRACT FROM AUTHOR]

Background: Cholera has been present and recurring in Zambia since 1977. However, there is a paucity of data on genetic relatedness and diversity of the Vibrio cholerae isolates responsible for these outbreaks. Understanding whether the outbreaks are seeded from existing local isolates or if the outbreaks represent separate transmission events can inform public health decisions.Results: Seventy-two V. cholerae isolates from outbreaks in 2009/2010, 2016, and 2017/2018 in Zambia were characterized using multilocus variable number tandem repeat analysis (MLVA) and whole genome sequencing (WGS). The isolates had eight distinct MLVA genotypes that clustered into three MLVA clonal complexes (CCs). Each CC contained isolates from only one outbreak. The results from WGS revealed both clustered and dispersed single nucleotide variants. The genetic relatedness of isolates based on WGS was consistent with the MLVA, each CC was a distinct genetic lineage and had nearest neighbors from other East African countries. In Lusaka, isolates from the same outbreak were more closely related to themselves and isolates from other countries than to isolates from other outbreaks in other years.Conclusions: Our observations are consistent with i) the presence of random mutation and alternative mechanisms of nucleotide variation, and ii) three separate transmission events of V. cholerae into Lusaka, Zambia. We suggest that locally, case-area targeted invention strategies and regionally, well-coordinated plans be in place to effectively control future cholera outbreaks. [ABSTRACT FROM AUTHOR]

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

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]

Q fever is a zoonotic disease caused by the bacteria Coxiella burnetii. Domestic ruminants are the primary source for human infection, and the identification of likely contamination routes from the reservoir animals the critical point to implement control programs. This study shows that Q fever is detected in Belgium in abortion of cattle, goat and sheep at a different degree of apparent prevalence (1.93%, 9.19%, and 5.50%, respectively). In addition, and for the first time, it is detected in abortion of alpaca (Vicugna pacos), raising questions on the role of these animals as reservoirs. To determine the relationship between animal and human strains, Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) (n=146), Single-Nucleotide Polymorphism (SNP) (n=92) and Whole Genome Sequencing (WGS) (n=4) methods were used to characterize samples/strains during 2009-2019. Three MLVA clusters (A, B, C) subdivided in 23 subclusters (A1-A12, B1-B8, C1-C3) and 3 SNP types (SNP1, SNP2, SNP6) were identified. The SNP2 type/MLVA cluster A was the most abundant and dispersed genotype over the entire territory, but it seemed not responsible for human cases, as it was only present in animal samples. The SNP1/MLVA B and SNP6/MLVA C clusters were mostly found in small ruminant and human samples, with the rare possibility of spillovers in cattle. SNP1/MLVA B cluster was present in all Belgian areas, while the SNP6/MLVA C cluster appeared more concentrated in the Western provinces. A broad analysis of European MLVA profiles confirmed the host-species distribution described for Belgian samples. In silico genotyping (WGS) further identified the spacer types and the genomic groups of C. burnetii Belgian strains: cattle and goat SNP2/MLVA A isolates belonged to ST61 and genomic group III, while the goat SNP1/MLVA B strain was classified as ST33 and genomic group II. In conclusion, Q fever is widespread in all Belgian domestic ruminants and in alpaca. We determined that the public health risk in Belgium is likely linked to specific genomic groups (SNP1/MLVA B and SNP6/MLVA C) mostly found in small ruminant strains. Considering the concordance between Belgian and European results, these considerations could be extended to other European countries. [ABSTRACT FROM AUTHOR]

Salmonella enterica subspecies enterica causes salmonellosis in humans and animals and is an important cause of food infections worldwide. In recent years, the multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA), a fast molecular typing method with strong epidemiological discrimination, has facilitated the effective control of diverse infections. This study aimed at the typing of 28 human origined Salmonella enteritidis, Salmonella infantis, and Salmonella typhimurium strains by using a single MLVA protocol. Previously these strains have been identified by pulsed field gel electrophoresis (PFGE) method and it has been shown that each strain produced a distinct PFGE banding profile. One MLVA protocol was tested on 3 serotypes simultaneously and it produced three banding patterns specific to each of the three common Salmonella serotypes. MLVA also constitute a relatively more cost-effective and faster method than PFGE. [ABSTRACT FROM AUTHOR]

Pseudomonas savastanoi is a bacterial species included in the Pseudomonas syringae complex. It is further sub-typed in pathovars which are the causal agents of a group of diseases of woody plants, such as the "knot disease" on olive and oleander and the bacterial canker on ash. Given its long-established presence in the Mediterranean area, the pathogen causing the afore-mentioned diseases can be considered endemic. Here, an MLVA approach was developed to assess the genetic relationships among and within those pathovars, with a specific focus on P. savastanoi pv. savastanoi. By analyzing the genome of the P.savastanoi pv. savastanoi strain NCPPB 3335 (accession n° CP008742), 14 Tandem Repeat (TR) loci were identified and the corresponding primers were designed and used for the amplification of genomic DNAs from 84 strains belonging to Pseudomonas savastanoi pathovars. Data were analyzed using different approaches, such as hierarchical clustering, STRUCTURE, and k-means clustering with DAPC to evaluate the effectiveness of the assay in describing pathovars and population structure of the pathogen. Results reveal a very complex articulation of genetic relationships, as expected from a long-time evolving pathogen, while providing the possibility to discriminate the pathovars between each other. At intra-pathovar level, the MLVA assay clusters isolates mainly according to their hosts and geographic origin. This resulted particularly useful in the identification and tracking of P. savastanoi populations at local level. [ABSTRACT FROM AUTHOR]

Objective(s): Human gastrointestinal tract harbors a variety of bacteria with vital roles in human health. Bacteroides fragilis is considered one of the dominant constituents of gut microflora which can act as an opportunistic pathogen leading to various diseases, including colon cancer, diarrhea, uterine and intrathecal abscesses, septicemia, and pelvic inflammation. In this study, multiple locus variable number of tandem repeats analysis (MLVA) was performed to genetically differentiate 50 B. fragilis isolates. Materials and Methods: Eight suitable tandem repeats (TRs) were selected by bioinformatics tools and were then subjected to PCR amplification using specific primers. Finally, MLVA profiles were clustered using BioNumerics 7.6 software package. Results: All VNTR loci were detected in all isolates using the PCR method. Overall, B. fragilis isolates were differentiated into 27 distinct MLVA types. The highest diversity index was allocated to TR1, TR2, TR5, TR6, and TR8; with this taken into account, strain type 14 was the most prevalent with 12 strains belonging to this type. Clustering revealed three major clusters of A, B, and C. With regards to the pathogenicity of B. fragilis and the outcomes of infections related to this microorganism, it is imperative to study this microorganism isolated from both patients and healthy individuals. Conclusion: This study aimed at evaluating the efficiency of MLVA for the genetic differentiation of B. fragilis. The results of this study indicate the promising efficiency of MLVA typing for cluster detection of this bacterium. [ABSTRACT FROM AUTHOR]

Background/purpose: To date, molecular typing studies on Mycoplasma pneumoniae are limited. We evaluated the molecular types of Mycoplasma pneumoniae in pediatric patients in Taiwan in 2016.Methods: We used real-time quantitative PCR on respiratory specimens to identify M. pneumoniae in children with community-acquired pneumonia. The domain V of their 23S rRNA were sequenced for detection of macrolide-resistant point mutations. Molecular typing with multiple locus variable-number tandem repeat analysis (MLVA) was done for both macrolide-susceptible and -resistance M. pneumoniae samples.Results: M. pneumoniae was detected in 22% (180/826) respiratory samples during the study period. Among all M. pneumoniae-positive samples, 24% (43/180) had harbored macrolide-resistant genotypes, and 86% (37/43) of them were A2063G mutation. Forty-two macrolide-resistant strains and 20 randomly selected macrolide-susceptible strains underwent MLVA profiling. MLVA 4-5-7-2 was the most frequent type (32/62, 52%), followed by 4-5-7-3 (17/62, 27%) and 1-5-6-2 (9/62, 15%). There was a strong association between MLVA 4-5-7-2 and macrolide resistance (p < 0.001). In contrast, M 4-5-7-3 and 1-5-6-2 were related to macrolide susceptibility (p < 0.001, and p = 0.025, respectively).Conclusion: Macrolide resistance was relatively low (24%) in this age group in 2016 in Taiwan, and A2063G was the dominant point mutation. MLVA 4-5-7-2 was associated with macrolide resistance. [ABSTRACT FROM AUTHOR]

Background: Brucellosis is considered a main health concern in humans and animals. Neither familiar molecular methods nor the classical biotyping techniques are acceptable for subtyping Brucella spp. Loci containing variable number tandem repeats (VNTRs) have recently demonstrated their practicality in typing isolates from human and animal origin despite the excessive genetic homogeneity in the genus Brucella. Methods: The genotypic characteristics of sixty-six Brucella melitensis and thirty-four Brucella abortus isolates from veterinary samples and human brucellosis cases in Iran during 2014 - 2018. They were analyzed using multiple- locus variable-number tandem-repeat analysis (MLVA) which consisted of sixteen primer pairs and designed and classified as belonging to one of the three panels: panel 1 (MLVA-8: eight loci including Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, and Bruce55), panel 2A (three loci including Bruce18, Bruce19, and Bruce21), and panel 2B (five loci including Bruce04, Bruce07, Bruce09, Bruce16, and Bruce30); MLVA-11 (panels 1 and 2A), and MLVA-16 (panels 1, 2A, and 2B) using BioNumerics software (Version 7.6). Results: Using panel 1, 2A, and 2B (MLVA-16), 59 genotypes with a genetic similarity coefficient ranging from 91 to 100% were obtained from the 100 Brucella spp. isolates. For all isolates, only genotype 36 and genotype 26 were obtained using panels 1 and 2A, respectively. The B. abortus isolates showed variations at 9 different genotypes, while B. melitensis isolates have been dispersed in 50 different genotypes. Bruce16 and Bruce4 showed the highest discriminatory power. Conclusions: The MLVA-16 assay appeared to be a useful and important molecular genotyping tool that is capable of proving epidemiological linkages in outbreak and trace-back investigations and is helpful in improving the effectiveness of brucellosis control programs. [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. [ABSTRACT FROM AUTHOR]

Objective: Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of Leptospira spp., and peridomiciliary rodents are the most important reservoir animals for human infection. Dogs are known to be the reservoir animal of L. interrogans serovar Canicola, but the importance of dogs in zoonotic transmission of other Leptospira serotypes/genotypes remains unclear. This study reports the isolation of L. interrogans serogroup Autumnalis from two human patients in Japan and describes the genetic comparison between canine and mouse isolates using multiple-locus variable-number tandem repeat analysis (MLVA). Results: MLVA revealed that 8 out of the 11 loci compared were identical between the two human isolates. The human isolates clustered with the dog but not the mouse isolates. Moreover, the profile of one of the human isolates was identical to that of one of the dog isolates. [ABSTRACT FROM AUTHOR]

(A. baumannii) is a pathogen responsible for nosocomial infections among the hospitalized patients. The aim of this study was to investigate genotyping and molecular characterization and to examine the biofilm formation ability of A. baumannii isolates. In total, 70 A. baumannii isolates were collected from patients admitted to Imam Khomeini Hospital in Ahvaz, Southwestern Iran. Minimum inhibitory concentrations (MIC) test was performed using Vitek 2 system. The presence of genes encoding metallo-β-lactamases, oxacillinases, and integrase and the biofilm formation ability were then evaluated. Multiple locus variable-number tandem repeat (VNTR) analysis (MLVA) typing and multiplex PCR were performed to determine the genetic relationships. The blaOXA-23-like gene had the highest prevalence. The frequency of genes encoding blaSPM, blaIMP, and blaVIM among MDR A. baumannii isolates were 12 (17.1%), 18 (25.7%), and 22 (31.4%), respectively. Moreover, 46 isolates (75.4%) harbored class I integron and 10 isolates (16.39%) carried class II integron. The number of weak, moderate and strong biofilm-producing isolates were 3 (4.3%), 7 (10%), and 55 (78.5%), respectively. The results showed that 70 A. baumannii isolates were grouped into 12 distinct MLVA types with five clusters and four singleton genotypes. In addition, 25 (35.7%) isolates were assigned to international clone (IC) variants, 37 (52.8%) isolates belonged to group 1 (IC II), and 8 (11.4%) isolates belonged to group 2 (IC I). Our findings revealed that the population structure of the A. baumannii isolates was genetically diverse. More focus on genetic variation and antibiotic resistance of A. baumannii isolates are recommended. [ABSTRACT FROM AUTHOR]

Melioidosis is a common infectious disease in Southeast Asia and Northern Australia. In Hainan, several cases have been reported, but no systematic study has yet been done on the molecular epidemiology profiles of the organism. An investigation of the molecular epidemiology links and population structure of Burkholderia pseudomallei would help to better understand the clonally of the isolates and differences among them. In this study, multilocus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) were applied to examine the epidemiological relatedness and population structure of 166 B. pseudomallei isolates obtained during 2002–2014 in Hainan, China. Both the MLVA_4 and MLST approaches had high discriminatory power for this population, with diversity indices of 0.9899 and 0.9457, respectively. However, the MLVA_4 assay showed a higher discriminatory power than the MLST approach, and a variable-number tandem repeat (VNTR3 933) found by the MLVA approach was the most useful in discriminating strains from this province. A total of 166 strains yielded 99 MLVA_4 genotypes, of which 34 genotypes were shared by 101 isolates, for a clustering rate of 60.8% (101/166), which suggested that some cases may have a common source. Additionally, 65 isolates showed distinct genotypes, indicating that more than 39.2% (65/166) of melioidosis cases in Hainan had epidemiologically unrelated or sporadic characteristics. The 166 isolates were resolved into 48 STs, of which five STs (ST55, -70, -46, -50, and -58) were here found to be predominant. Phylogenetic analysis of 116 isolates conducted using the eBURST v3 segregated the 48 STs into eight groups with ST50 as predicted founder, and 21 STs were found to be singletons, which suggest that the strains in the Hainan region represent a high diversity of ST clones, indicating that many B. pseudomallei clone groups are endemic to this region. Moreover, ST50 had 5 SLV, 7 DLV, 6 TLV, and 29 satellite STs and formed a radial expansion pattern, suggesting that the melioidosis epidemic in this study was mainly caused by the clonal expansion of ST 50. Phylogenetic analysis on global scale suggests that China's isolates are closely related to isolates from Southeast Asia, particularly from Thailand and Malaysia. [ABSTRACT FROM AUTHOR]

Background: Anthrax is an endemic disease that persists in the rural regions of China. The global genetic population structure of B.anthracis has also been defined by the canonical single-nucleotide polymorphisms (canSNP) and multiple-locus variable-number tandem repeat analysis (MLVA). Five canSNP lineages were found in China, and the A.Br.Ames lineage has been the second predominant group in recent years in China. The objective of this study was to reveal genetic diversity of the Ames lineage strains by MLVA.Methods: Two molecular typing methods, canSNP and MLVA with 15markers were used to study the genetic relationship among the Ames lineage strains. The outbreak information associated with these strains was also collected and investigated.Results: From 2007 to 2018, a total of 21 human anthrax infection outbreaks (68 patients) associated with B. anthracis Ames lineage strains were reported in China. Ames lineage strain-associated human anthrax is mainly distributed in the northern part of China, including the provinces of Inner Mongolia, Liaoning, Gansu, and Xinjiang. In the study, a total of 30 Ames lineage strains were included and 10 MLVA15 genotypes were identified. These strains were mainly found in northeast China, Inner Mongolia and Liaoning. In recent years, the Ames lineage strains were isolated in the two provinces every year. The 18 Ames lineage strains isolated from Inner Mongolia were divided into eight MLVA15 genotypes. From 2010 to 2015, there were continuous reports of outbreaks in Keyouzhongqi County, Inner Mongolia, and the strains that were isolated annually in succession belonged to the MLVA15-30 genotype.Conclusions: The Ames lineage strains are widely distributed in northern China. Their genetic diversity can be illustrated by the results of the MLVA. The genetic characteristics of the Ames lineage strains from outbreaks in different provinces varied. In some areas, human anthrax outbreaks occurred annually in succession, and these related strains grouped together. These observations indicate that the local environment was persistently contaminated with B. anthracis spores, vaccination of livestock should become the fundamental control measure in the areas. [ABSTRACT FROM AUTHOR]

Background: Entero-invasive E. coli (EIEC) is one of the causes of bacillary dysentery in adults and children. The ability of EIEC to invade and colonize the surface of epithelial cells is influenced by many virulence factors. This study aimed to investigate the distribution of virulence factor genes in EIEC strains isolated from patients with diarrhea in Ahvaz, Iran, as well as the genetic diversity between these isolates by Multilocus variable-number tandem repeat analysis (MLVA). Materials and Methods: A total of 581 diarrheic stool samples were collected from patients with diarrhea attending two hospitals, in Ahvaz, Iran. The E. coli strains were identified by biochemical methods. Subsequently, all E. coli isolates were identified as EIEC by polymerase chain reaction (PCR) for the ipaH gene. The EIEC isolates evaluated by PCR for the presence of 8 virulence genes (ial, sen, virF, invE, sat, sigA, pic, and sepA). All EIEC strains were genotyped by the MLVA typing method. Results: A total of 13 EIEC isolates were identified. The presence of ial, virF, invE, sen, sigA, pic, and sat genes was confirmed among 92.3%, 84.6%, 84.6%, 76.9%, 69.2%, and 15.3% of EIEC isolates, respectively. On the other hand, none of the isolates were positive for the sepA gene. The EIEC isolates were divided into 11 MLVA types. Conclusion: Our results showed a high distribution of virulence genes among EIEC isolates in our region. This study showed that MLVA is a promising typing technique for epidemiological studies. MLVA can supply data in the form of codes that can be saved in the database and easily shared among laboratories, research institutes, and even hospitals. [ABSTRACT FROM AUTHOR]

Background: Human brucellosis has become a severe public health problem in China's Guangxi Province, and there has been higher prevalence of brucellosis in this region after 2010. Both multiple locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) assay schedules were used to genotype isolates and determine relationships among isolates. Results: A total of 40 isolates of Brucella were obtained from humans, pigs, and dogs from 1961 to 2016. There were at least three species of Brucella detected in Guangxi Province, Brucella melitensis, Brucella suis, and Brucella canis, with 16, 17, and 7 isolates, respectively. Of which B. suis biovar 3 was the predominant species resulting in pig brucellosis in the area examined before 2000s. Moreover, B. melitensis biovar 3 was found to be mainly responsible for human brucellosis during 2012–2016. All B. melitensis isolates in this study belonged to East Mediterranean lineage. MLVA-11 genotype 116 was the dominant genotype and represented 81.2% of the isolates. MLVA cluster analysis showed there to be 44% (7/16) brucellosis cases caused by B. melitensis with a profile of outbreak epidemic from 2012 to 2016. However, nearly 83.3% (20/24) of brucellosis cases resulting from both B. suis and B. canis showed no epidemiological links or sporadic characteristics. MLVA-16 analysis confirmed extensive genotype-sharing events between B. melitensis isolates from Guangxi and other northern provinces within China. These data revealed that there are potential epidemiology links among these strains. B. suis strains of this study showed a unique genetic lineage at the global level and may have existed historically in this area. However, present B. canis isolates were closely related to previously reported isolates in Korea, where they may have originated. MLST typing showed that the population structure of Brucella strains had changed considerably in this province; ST17 and ST21, two previously predominant populations appeared to have been replaced by recently emerging ST8 group. Conclusions: Our investigation data have inspired the hypothesis that Guangxi Province had been subject to an imported human brucellosis epidemic. Our data suggest that strains found in Northern regions of China are the principal source of infections in recent cases of human brucellosis in Guangxi Province. Comparative genomic analysis from more strains is necessary to confirm this hypothesis. This work will facilitate better understanding of the epidemiology and improve the effectiveness of control and prevention of brucellosis in this region. [ABSTRACT FROM AUTHOR]

Clostridioides difficile has become the leading nosocomial Gram-positive pathogen in the developed countries. In Lithuania, the national surveillance program for C. difficile started in 2017. Enzyme immunoassay, the real-time PCR system, and culture are used for laboratory confirmation of C. difficile infection in Lithuanian clinical laboratories. No reference laboratory for C. difficile is present in Lithuania. Fifty-eight isolates of C. difficile were collected in 2016 and 2017 in two hospitals using real-time PCR and culture methods. Agarose gel–based PCR ribotyping, multilocus variable number tandem repeats analysis (MLVA), and multilocus sequence typing (MLST) were used for the genotypic characterization of 28 isolates. PCR ribotyping and MLST showed that 78.6% of the tested toxigenic isolates belong to the ribotype RT027/ST1. Using MLVA, 95.5% of RT027 isolates were genetically related. MLVA revealed three clonal complexes in RT027. Six non-RT027 isolates showed four different electrophoretic profiles in PCR ribotyping and were assigned to the MLST sequence types ST2, ST13, ST54, and ST63. The highest discriminatory power showed the genotyping by MLVA. In total, 20 MLVA profiles were identified. This genotyping technique allowed to identify four groups of RT027/ST1 isolates that were indistinguishable by PCR ribotyping and MLST. Our study is the first genotypic characterization of C. difficile isolates in Lithuania. We observed a high prevalence of presumptive RT027 that suggests unfavorable epidemiological situation in Lithuania. Our results stress for implementation of genotyping of C. difficile isolates in Lithuanian surveillance. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to characterize antimicrobial resistance (AMR), molecular determinants of AMR and virulence, as well as clonal relationship of Streptococcus agalactiae isolates from women at 35-37 weeks of gestation in the Chaoshan metropolitan area of southern China. Methodology: Bacterial strains isolated from vaginal swabs were identified and antimicrobial susceptibility tests were performed by using a Vitek 2 Compact system (BioMérieux, France). Resistance and virulence genes were detected by polymerase chain reaction (PCR) and the clonal relationship was analysed by multiple locus variable number tandem repeat analysis (MLVA). Statistical analysis was carried out by using SPSS software, version 19.0. Results: All GBS were susceptible to benzylpenicillin, ampicillin, quinupristin/dalfopristin, tigecycline, linezolid and vancomycin, but a considerable proportion was resistant to clindamycin (29.67%), erythromycin (46.15%), azithromycin (63.74%), tetracycline (84.62%) and quinolones (25.27%). The carrier rates of ermB (69.04%) and mefA/E (64.28%) were detected in these GBS strains resistant to erythromycin. In terms of MLVA detection, 91 GBS strains were categorized into 43 genotypes and 6 clusters. All GBS harboured hylB and cylE genes, most of which carried a combination of PI-1 and PI-2a genes as a common virulence gene profile. Conclusions: The high level of resistance conferred by some corresponding resistance genes to macrolides, lincosamides and quinolones of GBS isolates from pregnant women in southern China, has reinforced the necessity for monitoring GBS strain resistance to the above agents. Comparative genetic studies of GBS isolates, especially efforts to understand the relationship between pilus islands and genotype, were essential for conducting infection control and epidemiological comparisons between countries. [ABSTRACT FROM AUTHOR]