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Carbapenem-Resistant Acinetobacter baumannii (CRAB) outbreak in intensive care units (ICUs) is a significant problem for healthcare facilities. In this study, we aimed to investigate the occurrence of CRAB isolates among ICU-admitted patients during the three waves of the COVID-19 pandemic in Iran using Multiple-Locus Variable Number Tandem-Repeat Analysis (MLVA). We obtained 50 (A) baumannii isolates from tracheal aspirate and blood culture samples. In the disc diffusion method, all isolates were cefotaxime, ceftriaxone, and cefepime-resistant, while 98% (49/50) of isolates were resistant to piperacillin, piperacillin-tazobactam, ceftazidime, and ciprofloxacin. Levofloxacin and tobramycin resistance was found in 76% (38/50) of isolates. In the microbroth dilution test all isolates were resistant to imipenem, 98% (49/50) to meropenem, 68% (34/50) to colistin, and 20% (10/50) to polymyxin (B) Based on the PCR findings, all isolates harbored blaOXA−40, ISAba-1, and int-2 genes. There were no isolates found that have the blaOXA−58, blaOXA−143, blaVEB−1, blaVIM, and int-3 genes. Among Extended-spectrum beta-lactamases (ESBL) genes, blaCTX−M, blaTEM, blaSHV, blaGES, and blaPER−1 have a prevalence of 42% (21/50), 84% (42/50), 58% (29/50), 78% (39/50), and 54% (27/50), respectively. 74% (37/50) of the isolates had the blaOXA−23 gene, while all of the isolates carried the blaOXA−40 gene. Among MBL genes, blaIPM, blaGIM, blaSIM, and blaNDM−1 have a prevalence of 20% (10/50), 8% (4/50), 22% (11/50), and 60% (30/50), respectively. The prevalence of int-1 was documented as 74% (37/50). Accordingly, all isolates were identified as CRAB. The co-existence of blaOXA−23/int-2 and blaOXA−23/isaba-1 was 74% (37/50). The co-existence of blaNDM−1/ISAba-1 was observed in 30 (60%) isolates. Using an 80% similarity threshold on the dendrogram constructed through MLVA typing, all isolates were grouped into two clusters: cluster A with 9 isolates from wave 3, and cluster B with 41 isolates from waves 3, 4, and 5. Our study confirms a clonal transmission of CRAB during the study period and suggests using molecular typing methods like MLVA in healthcare settings to identify dominant clones, antibiotic resistance patterns, and transmission routes. This will help to better manage the emergence and spread of antibiotic-resistant strains in future outbreaks. [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. [ABSTRACT FROM AUTHOR]

Cassava bacterial blight (CBB), caused by Xanthomonas phaseoli pv. manihotis (Xpm), poses a significant threat to cassava production in Burkina Faso. Prior to this study, the geographical distribution, incidence and origin of CBB introductions were poorly understood. In this investigation, we assessed the incidence of CBB in major cassava‐cropping regions and analysed the genetic diversity of 344 Xpm strains collected from 19 fields between 2015 and 2016, using a multilocus variable number of tandem repeat analysis (MLVA) scheme targeting 14 microsatellite markers. Our results reveal the presence of CBB in three out of four cropping regions, with incidences ranging from 0% to 100% at locality and variety scale. Burkina Faso's Xpm strains exhibited high genetic diversity at multiple levels, including country scale (He = 0.6), cropping region (0.43 to 0.57), locality (0.25 to 0.50) and field (0.25 to 0.52). Pairwise comparisons among populations from different cropping areas indicated a strong genetic differentiation. The categorical minimum spanning tree reveals that the Cascades region has the higher diversity indices and consequently could be the origin site of dispersion of Xpm in the other cropping regions of cassava. Human activities play a major role in the dissemination of Xpm through cassava cuttings. The importance of the sanitary state of cuttings must be emphasized in order to avoid CBB and ensure good cassava production. [ABSTRACT FROM AUTHOR]

Abstract Carbapenem-Resistant Acinetobacter baumannii (CRAB) outbreak in intensive care units (ICUs) is a significant problem for healthcare facilities. In this study, we aimed to investigate the occurrence of CRAB isolates among ICU-admitted patients during the three waves of the COVID-19 pandemic in Iran using Multiple-Locus Variable Number Tandem-Repeat Analysis (MLVA). We obtained 50 (A) baumannii isolates from tracheal aspirate and blood culture samples. In the disc diffusion method, all isolates were cefotaxime, ceftriaxone, and cefepime-resistant, while 98% (49/50) of isolates were resistant to piperacillin, piperacillin-tazobactam, ceftazidime, and ciprofloxacin. Levofloxacin and tobramycin resistance was found in 76% (38/50) of isolates. In the microbroth dilution test all isolates were resistant to imipenem, 98% (49/50) to meropenem, 68% (34/50) to colistin, and 20% (10/50) to polymyxin (B) Based on the PCR findings, all isolates harbored bla OXA−40 , ISAba-1, and int-2 genes. There were no isolates found that have the bla OXA−58 , bla OXA−143 , bla VEB−1 , bla VIM , and int-3 genes. Among Extended-spectrum beta-lactamases (ESBL) genes, bla CTX−M , bla TEM , bla SHV , bla GES , and bla PER−1 have a prevalence of 42% (21/50), 84% (42/50), 58% (29/50), 78% (39/50), and 54% (27/50), respectively. 74% (37/50) of the isolates had the bla OXA−23 gene, while all of the isolates carried the bla OXA−40 gene. Among MBL genes, bla IPM , bla GIM , bla SIM , and bla NDM−1 have a prevalence of 20% (10/50), 8% (4/50), 22% (11/50), and 60% (30/50), respectively. The prevalence of int-1 was documented as 74% (37/50). Accordingly, all isolates were identified as CRAB. The co-existence of bla OXA−23 /int-2 and bla OXA−23 /isaba-1 was 74% (37/50). The co-existence of bla NDM−1 /ISAba-1 was observed in 30 (60%) isolates. Using an 80% similarity threshold on the dendrogram constructed through MLVA typing, all isolates were grouped into two clusters: cluster A with 9 isolates from wave 3, and cluster B with 41 isolates from waves 3, 4, and 5. Our study confirms a clonal transmission of CRAB during the study period and suggests using molecular typing methods like MLVA in healthcare settings to identify dominant clones, antibiotic resistance patterns, and transmission routes. This will help to better manage the emergence and spread of antibiotic-resistant strains in future outbreaks.

Abstract Brucella spp. are facultative intracellular pathogens that cause zoonosis- brucellosis worldwide. There has been a trend of the re-emergence of brucellosis worldwide in recent years. The epidemic situation of brucellosis is serious in Xinjiang. To analyze the epidemic situation of Brucella spp. in Xinjiang among humans and animals, this study identified 144 Brucella isolates from Xinjiang using classical identification and 16 S rRNA sequencing. MLVA, drug resistance testing, and wgSNP detection were also performed. At the same time, analysis was conducted based on the published data of Brucella isolates worldwide. The results showed that the dominant species was B. melitensis biovar 3, which belonged to GT42 (MLVA-8 typing) and the East Mediterranean lineage. The correlation among isolates was high both in humans or animals. The isolates in Xinjiang exhibited higher polymorphism compared to other locations in China, with polymorphism increasing each year since 2010. No amikacin/kanamycin-resistant strains were detected, but six rifampicin-intermediate isolates were identified without rpoB gene variation. The NJ tree of the wgSNP results indicated that there were three main complexes of the B. melitensis epidemic in Xinjiang. Based on the results of this study, the prevention and control of brucellosis in Xinjiang should focus on B. melitensis, particularly strains belonging to B. melitensis bv.3 GT42 (MLVA-8 typing) and East Mediterranean lineage. Additionally, the rifampicin- and trimethoprim-sulfamethoxazole- resistance of isolates in Xinjiang should be closely monitored to avoid compromising the therapeutic efficacy and causing greater losses. These results provide essential data for the prevention and control of brucellosis in Xinjiang and China. Although the isolates from Xinjiang have significant characteristics among Chinese isolates and can reflect the epidemiological situation of brucellosis in China to some extent, this study cannot represent the characteristics of isolates from other regions.

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.

Brucella spp. are facultative intracellular pathogens that cause zoonosis- brucellosis worldwide. There has been a trend of the re-emergence of brucellosis worldwide in recent years. The epidemic situation of brucellosis is serious in Xinjiang. To analyze the epidemic situation of Brucella spp. in Xinjiang among humans and animals, this study identified 144 Brucella isolates from Xinjiang using classical identification and 16 S rRNA sequencing. MLVA, drug resistance testing, and wgSNP detection were also performed. At the same time, analysis was conducted based on the published data of Brucella isolates worldwide. The results showed that the dominant species was B. melitensis biovar 3, which belonged to GT42 (MLVA-8 typing) and the East Mediterranean lineage. The correlation among isolates was high both in humans or animals. The isolates in Xinjiang exhibited higher polymorphism compared to other locations in China, with polymorphism increasing each year since 2010. No amikacin/kanamycin-resistant strains were detected, but six rifampicin-intermediate isolates were identified without rpoB gene variation. The NJ tree of the wgSNP results indicated that there were three main complexes of the B. melitensis epidemic in Xinjiang. Based on the results of this study, the prevention and control of brucellosis in Xinjiang should focus on B. melitensis, particularly strains belonging to B. melitensis bv.3 GT42 (MLVA-8 typing) and East Mediterranean lineage. Additionally, the rifampicin- and trimethoprim-sulfamethoxazole- resistance of isolates in Xinjiang should be closely monitored to avoid compromising the therapeutic efficacy and causing greater losses. These results provide essential data for the prevention and control of brucellosis in Xinjiang and China. Although the isolates from Xinjiang have significant characteristics among Chinese isolates and can reflect the epidemiological situation of brucellosis in China to some extent, this study cannot represent the characteristics of isolates from other regions. [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]

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]

Xanthomonas hortorum pv. carotae is a pathogen responsible for the bacterial blight of carrot. Although it infects carrot fields worldwide, its diversity remains unknown. Here, we validated by PCR the identification as X. hortorum pv. carotae of most of the strains isolated from symptomatic carrots. We studied their diversity by sequencing seven housekeeping genes. The analysis confirmed the identity of most of the strains previously identified as X. hortorum pv. carotae and highlighted the presence of two clusters inside the pathovar carotae. The 18 non-X. hortorum pv. carotae have been identified as Xanthomonas from other species. As the X. hortorum pv. carotae strains clustered only in two groups by this analysis, a new multilocus variable number of tandem repeats analysis scheme was developed. It allowed us to validate the subclustering in two groups and observe diversity among them. At the same time, based on this study and data from the literature, we proposed the strain CFBP 7900 as the neopathotype strain of the pathovar. The strain should replace the holopathotype strain CFBP 4997, as it is no longer available in international collections. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The aim of the present study was to evaluate the effectiveness of analysis of the high resolution melting curves obtained after amplification of VNTR loci for the identification and differentiation of Brucella strains.Materials and methods. 16 strains of Brucella species – B. canis (n=1), B. abortus (n=9), B. melitensis (n=2), B. suis (n=4) – of different geographical origin were used as objects of the research. The MLVA-typing was performed using conventional PCR followed by separation of amplicons in agarose gel and real-time PCR with post-amplification analysis of the curves of VNTR loci melting in the presence of intercalating dye SybrGreen. Bioinformatics analysis was conducted with the help of Vector NTI 9.1, Mega 11 software (MUSCLE algorithm). Phylogenetic analysis was carried out applying UPGMA method using the Mega 11 program.Results and discussion. MLVA approach based on the analysis of the melting point curves of the obtained after amplification of VNTR-loci PCR fragments has shown that each of the 16 strains of Brucella is characterized by a unique melting temperature profile. PCR followed by electrophoresis has demonstrated that despite the high variability of the used VNTR sequences (h=0.48…0.74), only post-amplification melting curves of the Bru7, Bru9, Bru18, Bru21 loci had sufficient information content to determine the genetic polymorphism of the studied Brucella strains. Based on the results of phylogenetic analysis of the Bru7, Bru9, Bru18, Bru21 sequences, it has been found that the majority of the studied Brucella strains are distributed in the dendrogram in accordance with their taxonomic and geographical position. Thus, HRM analysis of melting curves obtained after amplification of the Bru7, Bru9, Bru18, Bru21 loci has the potential to be used for differentiating Brucella strains.

Hongmei Xue,1 Jiquan Li,1 Li Ma,1 Xuxin Yang,1 Lingling Ren,1 Zhijun Zhao,1 Jianling Wang,1 Yuanbo Zhao,1 Zhongzhi Zhao,1 Xuefei Zhang,1 Zhiguo Liu,2 Zhenjun Li2 1Department of Brucellosis Prevention and Control, Qinghai Institute for Endemic Disease Prevention and Control, Xining, Qinghai, People’s Republic of China; 2National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of ChinaCorrespondence: Zhiguo Liu; Zhenjun Li, Email wlcblzg@126.com; lizhenjun@icdc.cnObjective: Brucellosis is a serious public health issue in Qinghai (QH), China. Surveying the seroprevalence and isolation of B. abortus strains from marmots is key to understanding the role of wildlife in the maintenance and spread of brucellosis.Methods: In this study, a set of methods, including a serology survey, bacteriology, antibiotic susceptibility, molecular genotyping (MLST and MLVA), and genome sequencing, were employed to characterize the two B. abortus strains.Results: The seroprevalence of brucellosis in marmots was 7.0% (80/1146) by serum tube agglutination test (SAT); one Brucella strain was recovered from these positive samples, and another Brucella strain from a human. Two strains were identified as B. abortus bv. 1 and were susceptible to all eight drugs examined. The distribution patterns of the accessory genes, virulence associated genes, and resistance genes of the two strains were consistent, and there was excellent collinearity between the two strains on chromosome I, but they had significant SVs in chromosome II, including inversions and translocations. MLST genotyping identified two B. abortus strains as ST2, and MLVA-16 analysis showed that the two strains clustered with strains from northern China. WGS-SNP phylogenetic analysis showed that the strains were genetically homogeneous with strains from the northern region, implying that strains from a common lineage were spread continuously in different regions and hosts.Conclusion: Seroprevalence and molecular clues demonstrated frequent direct or indirect contact between sheep/goats, cattle, and marmots, implying that wildlife plays a vital role in the maintenance and spread of B. abortus in the Qinghai–Tibet Plateau.Keywords: Brucella abortus, marmots, isolated, MLST, MLVA, WGS-SNP, QH

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.

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]

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]

The genetic diversity of Brucella strains has not been fully understood. To investigate this, the genetic characteristics of 64 isolates of Brucella melitensis from sheep and goats' milk were studied using random fragment length polymorphism (RFLP) and multiple locus variable-number tandem repeat analysis (MLVA-16) methods developed in Orsay, France (MLVA-16Orsay). The RFLP analysis revealed that all 64 isolates were of biovar one. The MLVA-typing showed that one sample was simultaneously infected with two strains of B. melitensis and the genotype of 65 isolate was analyzed. Four genotypes (47, 42, 43, and 63) were identified using MLVA-8 (panel 1), whereas six genotypes (138, 125, 116,108, and two unknown genotypes) were identified using MLVA11 (panels 1 and 2A). From the review of MLVA-16 (panels 1, 2A, and 2B), panel 2B showed a very high discriminatory power. Two loci of Bruc04 and Bruc30 from this panel had diversity index values higher than 0.71 and the average diversity index was 0.619. So MLVA-16Orsay 34 showed the genotype indicating a low genetic homogeneity among the isolates. The findings of MLVA genotyping of the isolates suggest that strains of B. melitensis isolated from the milk of small ruminants in Iran are most closely related to the isolates from neighboring countries of the Eastern Mediterranean group. To the best of our knowledge, this is the first study to indicate the potential use of MLVA genotyping for simultaneous detection of specimen contamination using two different B. melitensis biovars. [ABSTRACT FROM AUTHOR]

Mammary-like vulvar adenocarcinoma (MLVA) is an exceedingly rare subtype of vulvar adenocarcinoma that shares features with mammary gland tissue. Due to its rarity and lack of consensus, MLVA presents diagnostic challenges to pathologists. We present the case of a 59-year-old female with an ulcerated mass on the right side of the external genitalia, diagnosed as MLVA. Comprehensive immunohistochemistry (IHC) and gene sequencing studies were performed to characterize the tumor. IHC analysis revealed triple expression of hormonal receptors (estrogen receptor, progesterone receptor, and HER2), supporting the mammary gland origin of the tumor. Gene sequencing identified unique genetic mutations associated with the expression of hormonal markers. One fusion gene (ERBB2-NAGLU) has not been reported in any tumors, and other mutations with unique mutation types have not been previously reported in MLVA. Our findings shed light on the molecular characteristics of MLV and may help improve the diagnosis and treatment of this rare type of vulvar adenocarcinoma.

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, including 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 israelensis, 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 estimating the sources of legionellosis infection in Japan and developing prevention strategies. HIGHLIGHTS We elucidated differences in the distribution of Legionella species, serogroups, and genotypes between hot springs and other public baths.; 10 STs (ST-KB1-KB10) identified as unique types in this study had novel allelic profiles.; No strains from bath water in our samples exhibited the same MLVA genotypes or STs as the clinical strains collected through surveillance during the same period.; Our study will be useful for estimating the sources of infection in Japan and developing prevention strategies.;

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.

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.

Introduction: Mycobacterium avium complex (MAC) bacteria are the most prominent etiological agents of lymphadenitis in pigs. M. avium subspecies hominissuis (MAH) is a member of MAC and has been reported in many parts of the world to be the most prevalent non-tuberculous mycobacteria (NTM) to cause mycobacteriosis in humans, mainly in children. Thus, the economic and zoonotic impact of MAC species are increasingly being recognized. In South Africa, little is known about the distribution of NTM and the molecular epidemiology of M. avium in pigs. Materials and methods: In this study, lymph nodes including mandibular, mesenteric, submandibular, and retropharyngeal, with tuberculosis-like lesions were collected during routine meat inspection of slaughter pigs with no disease symptoms (n = 132), between 1991 and 2002. These pigs were slaughtered at 44 abattoirs distributed across seven of the nine South African provinces. Mycobacterial culture, polymerase chain reaction (PCR), and sequencing of the Mycobacterium specific 577 bp 16S rRNA gene fragment were performed for species and subspecies identification. Results: The majority of the isolates (each per sample); 114 (86.4%) were identified as MAH, 8 (6%) as MAA/M. avium subsp. silvaticum, 4 (3%) were Mycobacterium tuberculosis, 2 (1.5%) as Mycobacterium intracellulare, and 1 (0.75%) as Mycobacterium bovis. The other isolates were identified as Mycobacterium lentiflavum (0.75%), Mycobacterium novocastrense (0.75%), and a Micrococcus spp. (0.75%). Using an eight-marker MLVA typing tool, we deciphered at least nine MIRU VNTR INMV types of MAH and MAA. Discussion: Identification of known zoonotic mycobacteria, including MAH, MAA, M. intracellulare, M. bovis, and M. tuberculosis, from slaughter pigs has a potential public health impact and also strengthens recognition of the potential economic impact of MAC. This study has also for the first time in South Africa, revealed MAC MIRU VNTR INMV genotypes which will aid in the future epidemiological investigation of MAC in South Africa. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae causing nosocomial infections is increasingly multi-drug-resistant. Rapid and efficient typing tools are required for monitoring. To assess a simple, rapid (<5 h) multiplex polymerase chain reaction method based on multi-locus variable number tandem repeat analysis (MLVA) as a screening tool to determine whether or not K. pneumoniae strains are related. The global discriminatory power of the method was assessed on 72 unrelated K. pneumoniae isolates, including community carriage isolates, highly virulent strains causing liver abscess, and extended-spectrum beta-lactamase- and carbapenemase-producing strains. Suspected related strains from a suspected outbreak and a relapsed meningitis case were also studied. MLVA results were compared with whole-genome sequencing (WGS) analysis and multi-locus sequence typing (MLST). MLVA and MLST had similar discriminatory power, each distinguishing 54 profiles among the 72 unrelated isolates (Hunter–Gaston index 0.989). Each strain belonging to one sequence type (ST) or ST complex had its own MLVA type, with few exceptions. Two strains of ST268 and ST1119 shared the same MLVA profile, and two unrelated strains of ST307, ST86, ST45 and ST37 exhibited two different MLVA types each. Moreover, investigation of seven grouped cases of K. pneumoniae neonatal sepsis pointed to strong suspicion of a common source for five isolates, while two isolates with a different MLVA profile were excluded from this cluster. The MLVA approach is a useful, rapid and reliable tool for epidemiological investigation requiring only basic molecular biology equipment, and permits identification of sporadic isolates that are not part of an outbreak. However, analysis of strains sharing the same MLVA type by a highly discriminatory technique, such as WGS, remains necessary. [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]

Objective To conduct a molecular epidemiological tracing and analysis of Yersinia pestis strains isolated from two human plague outbreaks with unknown sources in Gansu Province, China. The results of this analysis would provide a basis for isolating and controlling the sources of Yersinia pestis. Methods The strains of Yersinia pestis isolated from two human plague outbreaks occurring on December 12, 2017, and September 27, 2019 were genotyped by the different region (DFR) and the clustered regularly interspaced short palindromic repeats (CRISPR). The repeat numbers of the variable number tandem repeat (VNTR) loci in the tested strains of Yersinia pestis were calculated by the multiple variable number tandem repeats analysis (MLVA), and the location of the phylogenetic tree of the tested strains was determined with the method of minimum spanning tree (MST) by the software BioNumerics 6.6. Results The strain of 20171212 lacked DFR01, DFR02, DFR03, DFR04, DFR13, DFR23, and the DFR type was identified as type 8. The space sequence of YPa was a1'-a2-a3-a4-a5-a6- a7-a35, the space sequence of YPb was b1-b2-b3-b4, the space sequence of YPc was c1-c2-c3, the gene cluster of CRISPR was Ca35', the genotype of CRISPR was 26'. MLVA clustering analysis showed that the strain clustered within in the cluster of Yuerhong pasture in Subei County and formed an independent branch. On the other hand, the strain of 20190927 lacked DFR01, DFR13 and DFR23, with the DFR type identified as type 1b. The space sequence of YPa was a1-a2-a3-a4- a5-a6-a7, the space sequence of YPb was b1-b2-b3-b4, the space sequence of YPc was c1-c2-c3, the gene cluster of CRSIPR was Ca7, the genotype of CRSIPR was 22 MLVA clustering analysis showed that the strain was located close to the cluster of Dangjinshan in Akesai County, and relatively distant from the cluster of Yuerhong pasture in Subei County. Conclusions The genotypes of strain 20171212 by DFR and CRISPR were consistent with the main genotypes of Y. pestis from Himalayana Marmota foci in Subei County, which confirmed that the human plague cases were naturally occurring locally. However, the strain gathered the cluster of Yuerhong pasture in Subei County, which indicated that the source of infection was not in Yanchiwan Town, but in the surrounding area of the Yuerhong pasture. The genotypes of strain 20190927 by DFR and CRISPR were in accordance with the main genotype of Y. pestis from Himalayana Marmota foci in Akesai County and were closer to the cluster of Dangjinshan in Aksai County than to [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]

Abstract Background Pseudomonas aeruginosa is a common co-infecting pathogen recognized among COVID-19 patients. We aimed to investigate the antimicrobial resistance patterns and molecular typing of Pseudomonas aeruginosa isolates among Coronavirus disease-19 patients. Methods Between December 2020 and July 2021, 15 Pseudomonas aeruginosa were isolated from COVID-19 patients in the intensive care unit at Sina Hospital in Hamadan, west of Iran. The antimicrobial resistance of the isolates was determined by disk diffusion and broth microdilution methods. The double-disk synergy method, Modified Hodge test, and polymerase chain reaction were utilized to detect Pseudomonas aeruginosa extended spectrum beta-lactamase and carbapenemase producers. Microtiter plate assay was performed to evaluate the biofilm formation ability of the isolates. The isolates phylogenetic relatedness was revealed using the multilocus variable-number tandem-repeat analysis method. Results The results showed Pseudomonas aeruginosa isolates had the most elevated resistance to imipenem (93.3%), trimethoprim-sulfamethoxazole (93.3%), ceftriaxone (80%), ceftazidime (80%), gentamicin (60%), levofloxacin (60%), ciprofloxacin (60%), and cefepime (60%). In the broth microdilution method, 100%, 100%, 20%, and 13.3% of isolates showed resistance to imipenem, meropenem, polymyxin B, and colistin, respectively. Ten (66.6%) isolates were identified as multiple drug resistance. Carbapenemase enzymes and extended spectrum beta-lactamases were identified in 66.6% and 20% of the isolates, respectively and the biofilm formation was detected in 100% of the isolates. The bla OXA-48, bla TEM, bla IMP, bla SPM, bla PER, bla VEB, bla NDM, bla SHV, and bla CTX-M genes were detected in 100%, 86.6%, 86.6%, 40%, 20%, 20%, 13.3%, 6.6%, and 6.6% of the isolates, respectively. The bla VIM , bla GIM , bla GES , and bla MCR-1 genes were not identified in any of the isolates. The MLVA typing technique showed 11 types and seven main clusters and most isolates belong to cluster I, V and VII. Conclusion Due to the high rate of antimicrobial resistance, as well as the genetic diversity of Pseudomonas aeruginosa isolates from COVID-19 patients, it is indispensable to monitor the antimicrobial resistance pattern and epidemiology of the isolates on a regular basis.

IntroductionMycobacterium avium complex (MAC) bacteria are the most prominent etiological agents of lymphadenitis in pigs. M. avium subspecies hominissuis (MAH) is a member of MAC and has been reported in many parts of the world to be the most prevalent non-tuberculous mycobacteria (NTM) to cause mycobacteriosis in humans, mainly in children. Thus, the economic and zoonotic impact of MAC species are increasingly being recognized. In South Africa, little is known about the distribution of NTM and the molecular epidemiology of M. avium in pigs.Materials and methodsIn this study, lymph nodes including mandibular, mesenteric, submandibular, and retropharyngeal, with tuberculosis-like lesions were collected during routine meat inspection of slaughter pigs with no disease symptoms (n = 132), between 1991 and 2002. These pigs were slaughtered at 44 abattoirs distributed across seven of the nine South African provinces. Mycobacterial culture, polymerase chain reaction (PCR), and sequencing of the Mycobacterium specific 577 bp 16S rRNA gene fragment were performed for species and subspecies identification.ResultsThe majority of the isolates (each per sample); 114 (86.4%) were identified as MAH, 8 (6%) as MAA/M. avium subsp. silvaticum, 4 (3%) were Mycobacterium tuberculosis, 2 (1.5%) as Mycobacterium intracellulare, and 1 (0.75%) as Mycobacterium bovis. The other isolates were identified as Mycobacterium lentiflavum (0.75%), Mycobacterium novocastrense (0.75%), and a Micrococcus spp. (0.75%). Using an eight-marker MLVA typing tool, we deciphered at least nine MIRU VNTR INMV types of MAH and MAA.DiscussionIdentification of known zoonotic mycobacteria, including MAH, MAA, M. intracellulare, M. bovis, and M. tuberculosis, from slaughter pigs has a potential public health impact and also strengthens recognition of the potential economic impact of MAC. This study has also for the first time in South Africa, revealed MAC MIRU VNTR INMV genotypes which will aid in the future epidemiological investigation of MAC in South Africa.

Listeria monocytogenes is a serious human pathogen and an enduring challenge to control for the ready-to-eat food processing industry. Cost-effective tools that can be deployed by commercial or in-house laboratories to rapidly investigate and resolve contamination events in the built food processing environment are of value to the food industry. Multilocus variable number tandem-repeat analysis (MLVA) is a molecular subtyping method, which along with other same-generation methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) is being superseded in disease tracking and outbreak investigations by whole-genome sequencing (WGS). In this paper, it is demonstrated that MLVA can continue to play a valuable role as a valid, fast, simple, and cost-effective method to identify and track Listeria monocytogenes subtypes in factory environments, with the method being highly congruent with MLST. Although MLVA does not have the discriminatory power of WGS to identify truly persistent clones, with careful interpretation of results alongside isolate metadata, it remains a powerful tool in situations and locations where WGS may not be readily available to food business operators.

Introduction: Brucellosis was made statutorily notifiable in 1955, in China, while in Guizhou Province, the pathogen of human brucellosis was isolated for the first time in 2011. However, currently, the brucellosis epidemic is becoming more and more severe in Guizhou Province. The type distribution and genetic characteristics of Brucella in Guizhou Province, as well as its evolutionary relationship with domestic and foreign strains, are still unclear. Methods: MLST, MLVA, and rpoB typing techniques were used for the molecular epidemiological study of the 83 Brucella isolates in Guizhou province. Results: Among the 83 Brucella strains, MLST identified three ST genotypes, of which ST39 is a newly reported type in China. MLVA-16 generated 49 genotypes, and MLVA-11 generated 5 known genotypes and 2 unreported genotypes. Six genotypes were identified by rpoB technology. Discussion: MLVA has a high resolution, but differences at the Bruce 04 and 16 loci cannot exclude associations between epidemics, and combining MLST and rpoB typing methods for epidemiologic tracing can avoid erroneous judgments. Moreover, through the combined analysis of the three typing techniques, the possible origin of the new Brucella can be reasonably inferred, which is also conducive to promoting the subsequent research of the novel Brucella. [ABSTRACT FROM AUTHOR]

This review aims to provide a comprehensive overview of the significant Clostridioides difficile molecular typing techniques currently employed in research and medical communities. The main objectives of this review are to describe the key molecular typing methods utilized in C. difficile studies and to highlight the epidemiological characteristics of the most prevalent strains on a global scale. Geographically distinct regions exhibit distinct strain types of C. difficile, with notable concordance observed among various typing methodologies. The advantages that next-generation sequencing (NGS) offers has changed epidemiology research, enabling high-resolution genomic analyses of this pathogen. NGS platforms offer an unprecedented opportunity to explore the genetic intricacies and evolutionary trajectories of C. difficile strains. It is relevant to acknowledge that novel routes of transmission are continually being unveiled and warrant further investigation, particularly in the context of zoonotic implications and environmental contamination. [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.

Background & Objective: Brucellosis is an endemic disease with a high prevalence in Iran whose highest frequency is in the western region of the country. Genetic diversity investigation is an important method to determine the epidemiological relationship of Brucella isolates in different geographical areas. Therefore, the present study aimed to investigate the genetic diversity of human Brucella melitensis (B. melitensis) strains using the Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) Typing method in the west of the country. Materials & Methods: In this study, 20 strains of isolated B. melitensis were collected from the human serum samples of suspected Brucellosis in the west of the country and were analyzed by MLVA-16 method. Results: The results showed that 3 genotype numbers 42, 43 and 47 were identified using MLVA-8 method and using MLVA-11 method genotypes 125, 138 and 111 were recognized. Also, 16 different genotypes were detected from the analysis of the isolates by MLVA-16 method which shows a high degree of polymorphism among the isolates due to the high genetic diversity of the isolates in Panel 2B loci. Conclusion: The results showed the high genetic diversity of B. melitensis isolates in the west of the country and their genetic relationship with the known strains in the neighboring countries of the Eastern Mediterranean area, as well as the importance of the MLVA method in identifying the source of infection.

Background: Several studies have highlighted the incidence of Clostridioides difficile infections (CDIs) in Taiwan and certain ribotypes have been related to severe clinical diseases. A study was conducted to investigate the polymerase chain reaction (PCR) ribotypes and genetic relatedness of clinical C. difficile strains collected from January 2009 to December 2015 at a hospital in northeastern Taiwan. Material and methods: A modified two-step typing algorithm for C. difficile was used by combining a modified 8-plex and 3′-truncated tcdA screening PCR. In addition, MLVA typing was adopted for investigation of bacterial clonality and transmission. Results: Among a total of 86 strains, 24 (28%) were nontoxigenic and 62 (72%) had both tcdA and tcdB (A + B+). No tcdA-negative and tcdB-positive (A−B+) strains were identified. Binary toxin (CDT)-producing (cdtA+/cdtB+) strains were started to be identified in 2013. The 21 (34%) A+B+ clinical strains with binary toxin and tcdC deletion were identified as RT127 strains, which contained both RT078-lineage markers and fluoroquinolone (FQ)-resistant mutations (Thr82Ile in gyrA). Multiple loci variable-number tandem repeat analysis (MLVA) for phylogenetic relatedness of RT127 strains indicated that 20 of 21 strains belonged to a clonal complex that was identical to a clinical strain collected from southern Taiwan in 2011, suggestive of a clonal expansion in Taiwan. Conclusion: A two-step typing method could rapidly confirm species identification and define the toxin gene profile of C. difficile isolates. The clonal expansion of RT127 strains in Taiwan indicates monitoring and surveillance of toxigenic C. difficile isolates from human, animal, and environment are critical to develop One Health prevention strategies.

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]

Brucellosis is a widely prevalent zoonotic disease of major public health significance. A collection of Brucella melitensis and Brucella abortus field isolates of animal and human origin were subjected to MLVA-15 typing followed by phylogeography studies. The MLVA-15 analysis of B. melitensis (n = 65) field isolates resulted in 48 different profiles. The panel I marker bruce45 was found to be most conserved, while the rest of the panel I markers showed low to moderate length polymorphism. Among the panel II markers, bruce04, bruce16 and bruce30 showed a high discriminatory index. The MLVA-15 typing of 13 B. abortus field isolates revealed 13 different genotypes with panel II markers showing higher discriminatory ability vis-à-vis panel I. The minimum spanning tree analysis (MST) in comparison with isolates from the international database revealed that all B. melitensis and B. abortus isolates from this study belonged to the 'Eastern Mediterranean' and the 'abortus C' lineage, respectively. The MLVA-15 typing could differentiate field isolates of B. abortus and B. melitensis originating from different regions, reaffirming the technique's potential of high resolution and suitability for local epidemiological studies. The MLVA scheme also has the advantage of comparison of local isolates with a worldwide database, allowing for phylogeographical studies. [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]

Background: Pseudomonas aeruginosa is a common co-infecting pathogen recognized among COVID-19 patients. We aimed to investigate the antimicrobial resistance patterns and molecular typing of Pseudomonas aeruginosa isolates among Coronavirus disease-19 patients. Methods: Between December 2020 and July 2021, 15 Pseudomonas aeruginosa were isolated from COVID-19 patients in the intensive care unit at Sina Hospital in Hamadan, west of Iran. The antimicrobial resistance of the isolates was determined by disk diffusion and broth microdilution methods. The double-disk synergy method, Modified Hodge test, and polymerase chain reaction were utilized to detect Pseudomonas aeruginosa extended spectrum beta-lactamase and carbapenemase producers. Microtiter plate assay was performed to evaluate the biofilm formation ability of the isolates. The isolates phylogenetic relatedness was revealed using the multilocus variable-number tandem-repeat analysis method. Results: The results showed Pseudomonas aeruginosa isolates had the most elevated resistance to imipenem (93.3%), trimethoprim-sulfamethoxazole (93.3%), ceftriaxone (80%), ceftazidime (80%), gentamicin (60%), levofloxacin (60%), ciprofloxacin (60%), and cefepime (60%). In the broth microdilution method, 100%, 100%, 20%, and 13.3% of isolates showed resistance to imipenem, meropenem, polymyxin B, and colistin, respectively. Ten (66.6%) isolates were identified as multiple drug resistance. Carbapenemase enzymes and extended spectrum beta-lactamases were identified in 66.6% and 20% of the isolates, respectively and the biofilm formation was detected in 100% of the isolates. The blaOXA-48, blaTEM, blaIMP, blaSPM, blaPER, blaVEB, blaNDM, blaSHV, and blaCTX-M genes were detected in 100%, 86.6%, 86.6%, 40%, 20%, 20%, 13.3%, 6.6%, and 6.6% of the isolates, respectively. The blaVIM, blaGIM, blaGES, and blaMCR-1 genes were not identified in any of the isolates. The MLVA typing technique showed 11 types and seven main clusters and most isolates belong to cluster I, V and VII. Conclusion: Due to the high rate of antimicrobial resistance, as well as the genetic diversity of Pseudomonas aeruginosa isolates from COVID-19 patients, it is indispensable to monitor the antimicrobial resistance pattern and epidemiology of the isolates on a regular basis. [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]

IntroductionBrucellosis, a serious public health issue affecting animals and humans, is neglected in West Africa (WA).MethodsIn the present study, bio-typing, multi-locus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing single-nucleotide polymorphism (WGS-SNP) analysis were used to characterize the Brucella abortus (B. abortus) strains from WA.ResultsAll of the 309 strains analyzed in this study were extracted and downloaded from the international MLVA bank and were from 10 hosts (cattle, humans, ovine, buffalo, dromedaries, horse, sheep, zebu, dog, and cat) distributed in 17 countries in WA. Based on the bio-typing, three biovars, dominated by B. abortus bv.3, were observed and reported across seven decades (1958–2019). With MLST, 129 B. abortus strains from the present study were sorted into 14 STs, with ST34 as the predicted founder. These 14 STs clustered into the global MLST data into three clone complexes (C I–C III) with the majority of strains clustering in C I, while C II forms an independent branch, and C III harbors three STs shared by different continents. These data revealed that most cases were caused by strains from native lineages. According to the MLVA-11 comparison, 309 strains were divided into 22 MLVA-11 genotypes, 15 of which were unique to WA and the remaining seven had a global distribution. MLVA-16 analysis showed that there were no epidemiological links among these strains. Based on the MLVA data, B. abortus strains from WA have high genetic diversity, and predominated genotypes were descended from a native lineage. While the MLVA-16 globally highlights that the dominant native and few introduced lineages (from Brazil, the USA, South Korea, Argentina, India, Italy, Portugal, the UK, Costa Rica, and China) co-driving the B. abortus ongoing prevalence in WA. The high-resolution SNP analysis implied the existence of introduced B. abortus lineages, which may be reasonably explained by the movement and trade of dominant hosts (cattle) and/or their products.DiscussionOur results indicated that B. abortus strains in WA consist of native and introduced strains that necessitate control such as vaccination, testing, slaughtering, and movement control by the relevant country authorities to reduce brucellosis in livestock.

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]

Paratuberculosis is a worldwide chronic enteric disease of ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). While MAP has been widely investigated all around the world, little is known about the different strains that circulate in each country. This study describes the genetic diversity of MAP isolates from different bovine and deer herds from Argentina, analyzed by Multiple-Locus Variable number tandem repeat Analysis (MLVA), as well as the phylogenetic relatedness between geographically distant isolates through Whole Genome Sequencing (WGS) and core-genome analysis. A total of 90 MAP isolates were analyzed. The results showed seven different MLVA genotypes, with almost 75% of them belonging to pattern INMV 1, described in all the herds studied. WGS results suggested the presence of a common INMV 1 strain circulating throughout the country. Our results allow confirming the coexistence of different strains in time and space and the mixed infections identified in some animals. These observations suggest the absence of animal monitoring prior to introduction to the herds and the need for a control program in the country. This study represents the first to report WGS of MAP strains in Argentina. [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]