MLVA Research Articles

Brucella ceti in Common Dolphins (Delphinus delphis) in Portugal—Characterization of First Isolates

This study investigates Brucella ceti infection in marine mammals stranded along the Lisbon and Tagus Valley coast between 2022 and mid-2024, marking the first report of Brucella presence in Portuguese waters. Out of 59 examined marine mammals, B. ceti was isolated in three common dolphins (5.1%), a prevalence rate consistent with previous studies from other coastlines. PCR-based detection indicated a higher infection rate (23.7%), suggesting an underestimation of the prevalence of B. ceti infection in this population. Multi-locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) revealed distinct genetic profiles and close relationships to B. ceti strains from the Atlantic, supporting the hypothesis of specific host-adapted lineages in dolphins. Virulence genes, including those for host interaction (bspE, btpB) and intracellular survival (virB7, vceA), were consistent across isolates, highlighting the pathogenic potential. Additionally, antimicrobial resistance (AMR) genes, such as mprF and efflux proteins (bepC-G), were also identified. These findings underscore the need for further research and surveillance to understand B. ceti transmission, host range, and impacts on Atlantic cetaceans, as well as to develop effective diagnostic and management strategies to mitigate infection risks in marine environments.

Genetic structure and diversity of Mycoplasma hyopneumoniae based on a MLVA typing scheme.

Several epidemiological studies have been carried out using Multiple-Locus Variable-number tandem repeat Analysis (MLVA) for M. hyopneumoniae typing. However, a global perspective on the implications of the genetic diversity of this pathogen is lacking. This study aimed to determine and to analyze the genetic structure of M. hyopneumoniae based on the p97R1-p146R3 MLVA typing scheme and to characterize, analyze and compare MLVA types among countries where the information was publicly available. A set of 797 publicly available data of M. hyopneumoniae p97R1-p146R3 MLVA genetic types from six different countries were analyzed using Genalex 6.41 software to characterize loci polymorphism and using Structure 2.3.4 software in order to identify the genetic structure. A total of 185 MLVA types were identified among the analyzed data. For the p97R1 and p146R3 loci, most of the molecular variation in M. hyopneumoniae populations was identified within countries. Three genetic clusters and their recombinations were identified globally. M. hyopneumoniae is a genetically diverse pathogen with limited clonality and three well-defined clusters and their combinations were identified in this investigation. The greatest genetic variation of M. hyopneumoniae was observed within countries.

Genetic diversity atlas of Brucella melitensis strains from Sichuan Province, China

Human brucellosis is a re-emerging disease in Sichuan Province, China. In this study, bacteriology, conventional bio-typing, multi-locus sequence typing (MLST), and multiple locus variable-number tandem repeat analysis (MLVA) were applied to preliminarily characterize the strains in terms of genetic diversity and epidemiological links. A total of 101 Brucella strains were isolated from 16 cities (autonomous prefectures) from 2014 to 2021, and all of the strains were identified as Brucella melitensis bv. 3, suggesting that surveillance should focus on ruminants. MLST analysis identified four STs, namely, ST8 (n = 93), ST39 (n = 6), ST101 (n = 1), and ST118 (n = 1). The latter were new STs, indicating that strains displayed high population diversity. Six MLVA-8, namely, 42, 43, 45, 63, 83, and 114, and eight MLVA-11, namely, 111, 115, 116, 125, 180, 291, 298, and 342, genotypes were identified, demonstrating that all of the strains were from the Eastern Mediterranean lineage, and these strains exhibited a high genotype diversity. MLVA-16 analysis revealed that there was a co-existing transmission pattern, where sporadic cases and multiple outbreak events had a common origin. The dominant STs and MLVA genotypes of strains were epidemic in Northern, China, and 36 MLVA-16 genotypes were shared among strains (n = 51, 50.4%, 51/101) from Sichuan and strains from 22 other provinces. The findings imply that infected animals were introduced from outside the province. The surveillance and control of the disease have become public health challenges. Animal quarantines should be strengthened to prevent the spread of B. melitensis species among adjacent regions.

Unravelling Antimicrobial Resistance in Mycoplasma hyopneumoniae: Genetic Mechanisms and Future Directions.

Antimicrobial resistance (AMR) in Mycoplasma hyopneumoniae, the causative agent of Enzootic Pneumonia in swine, poses a significant challenge to the swine industry. This review focuses on the genetic foundations of AMR in M. hyopneumoniae, highlighting the complexity of resistance mechanisms, including mutations, horizontal gene transfer, and adaptive evolutionary processes. Techniques such as Whole Genome Sequencing (WGS) and multiple-locus variable number tandem repeats analysis (MLVA) have provided insights into the genetic diversity and resistance mechanisms of M. hyopneumoniae. The study underscores the role of selective pressures from antimicrobial use in driving genomic variations that enhance resistance. Additionally, bioinformatic tools utilizing machine learning algorithms, such as CARD and PATRIC, can predict resistance traits, with PATRIC predicting 7 to 12 AMR genes and CARD predicting 0 to 3 AMR genes in 24 whole genome sequences available on NCBI. The review advocates for a multidisciplinary approach integrating genomic, phenotypic, and bioinformatics data to combat AMR effectively. It also elaborates on the need for refining genotyping methods, enhancing resistance prediction accuracy, and developing standardized antimicrobial susceptibility testing procedures specific to M. hyopneumoniae as a fastidious microorganism. By leveraging contemporary genomic technologies and bioinformatics resources, the scientific community can better manage AMR in M. hyopneumoniae, ultimately safeguarding animal health and agricultural productivity. This comprehensive understanding of AMR mechanisms will be beneficial in the adaptation of more effective treatment and management strategies for Enzootic Pneumonia in swine.

A single-center analysis of clonal transmission of carbapenem-resistant Acinetobacter baumannii among intensive care unit patients during the COVID-19 pandemic

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.

Molecular epidemiology of brucellosis in Asia: insights from genotyping analyses.

Brucellosis infects humans and animals worldwide but is particularly prevalent in Asia. In many Asian countries, molecular diagnostic tools for accurate molecular diagnostics and molecular epidemiology are lacking. Nonetheless, some countries have conducted in-depth molecular epidemiological studies. The objective of this study was to reveal the genetic relationships, geographic origins, and distributions of Brucella strains across Asia for two primary species, B. abortus and B. melitensis. For this, we systematically searched genotyping data from published studies on the molecular epidemiology of Brucella species for both humans and livestock in Asia. We used data from multilocus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing analysis of Brucella strains. We also analyzed the MLVA genotypes of 129 B. abortus isolates and 242 B. melitensis isolates with known origins in Asia from an online MLVA database using MLVA-11 data in minimum spanning trees and MLVA-16 data in neighbor-joining trees. We found that the B. melitensis East Mediterranean lineage is predominant across the continent, with only a small number of samples from the Africa and Americas lineages, and none from the West Mediterranean lineage. The "abortus C" genotype was the most common group of B. abortus in Asia, with limited genetic variation for this species. Several studies also reported that Near Eastern countries frequently encounter human brucellosis cases of B. abortus from genotypes 42 and 43. Our study highlights the inconsistent collection of genetic data for Brucella species across Asia and a need for more extensive sampling in most countries. Finally, a consistent nomenclature is necessary to define various groupings of strains within a lineage (i.e., clade) so uniform terminology should denote particular genetic groups that are understood by all researchers.

Multiple-Locus Variable-Number Tandem Repeat Analysis of Helicobacter pylori Strains Isolated from Biopsy Samples

Background: Multiple-locus variable-number tandem-repeat analysis (MLVA) is a reliable, fast, and useful method for conducting phylogenetic and genotyping analyses. However, its application in Helicobacter pylori typing is limited. Objectives: This study aimed to determine the genetic diversity of H. pylori strains isolated from biopsy samples using six variable loci in MLVA typing. Methods: Over one year, we collected 70 non-duplicative biopsy samples from patients at Rohani Hospital, Babol, in northern Iran. After DNA extraction, H. pylori strains were confirmed through the amplification of the glmM gene in the PCR test. Multiple-locus variable-number tandem-repeat analysis typing utilized six VNTR markers: VNTR-180, VNTR-263, VNTR-614, VNTR-607, VNTR-2181, and VNTR-2457. The PHYLOViZE software was employed to construct the phylogenetic tree. Results: Out of the 70 specimens, 43 tested positive for H. pylori. The phylogenetic tree revealed some strains with completely similar MLVA patterns. Our findings indicated that type 2 is the dominant type in this region, with most patients infected with this type exhibiting gastritis. Conclusions: The high allelic variability observed at VNTR-614, -607, and -2457 indicates high intraspecific variability for these markers in all isolates from our region. Consequently, these VNTRs can be considered ideal sites for typing.

Microbial Forensics: Comparison of MLVA Results According to NGS Methods, and Forensic DNA Analysis Using MLVA

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled <i>in silico</i> anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were <i>B. anthracis</i> through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of <i>B. anthracis</i> were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of <i>B. anthracis</i>, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as <i>B. anthracis</i> strain A4564 which is associated with injectional anthrax isolates in heroin users.

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

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

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

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

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

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

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

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

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

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

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

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

The Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) of Staphylococuss aureus clinical isolates recovered from the Provincial Specialist Hospital in Lublin, Poland

Abstract 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.

Multiple-Locus Variable-Number Tandem Repeat Analysis Genotyping of Biofilm-Producing Pseudomonas aeruginosa Clinical Isolates

Background: Pseudomonas aeruginosa significantly contributes to hospital-acquired infections. Objectives: This study aimed to investigate the genetic diversity of P. aeruginosa strains using multiple-locus variable-number tandem repeat analysis (MLVA) and to explore the relationship between biofilm production and antibiotic resistance. Methods: In this cross-sectional study, 79 P. aeruginosa isolates were collected. Antibiotic sensitivity was tested using the Kirby-Bauer method, and biofilm production capability was assessed through the microtiter plate method. Genetic diversity was evaluated by MLVA, analyzing eight variable-number tandem repeat (VNTR) loci: MS-213, MS-214, MS-207, MS-217, MS-222, MS-209, MS-77, and MS-172. Phylogenetic relationships were delineated using PHYLOViZ 2.0 software. Results: The patient cohort comprised 51.9% males, with the majority of samples (35.4%) obtained from urine. Ceftazidime (CAZ 30µg) showed the highest resistance rate at 77.2%. Notably, 92.4% of isolates were capable of forming biofilms, categorized as 22.7% weak, 28.7% moderate, and 46.5% strong. Phylogenetic analysis demonstrated variability across one or more VNTR loci. Simpson’s index (0.906) and Shannon-Weiner diversity indices (H: 3.466, J: 0.910, Hmax: 3.807, Hmin: 1.242) identified MS77 as the most informative marker for genetic diversity among the isolates. Conclusions: The study highlights an alarming trend in antibiotic resistance, underscoring the necessity of regular monitoring. The findings confirm that MLVA is a straightforward, rapid genotyping method suitable for assessing the genetic diversity of P. aeruginosa.

Occurrence, antimicrobial resistance, and molecular characteristics of Salmonella enterica serovar 1,4,[5],12:i:- from food and patients in China

Salmonella enterica serovar Typhimurium monophasic variant 1,4,[5],12:i:- is increasingly responsible for human infections worldwide. However, studies assessing the prevalence and epidemiological characteristics of this emerging serovar are lacking in China. Here, a collection of 125 S. Typhimurium-like strains derived from food and patients in China between 2011 and 2019 were analyzed to investigate the occurrence ratio of S. 1,4,[5],12:i:-. Among them, 23.2% (22/95) of food-related isolates and 70.0% (21/30) of human isolates were assigned as S. 1,4,[5],12:i:-. Only two strains were sequence type (ST) 19; the rest all belonged to ST34. These isolates were further divided into 29 multiple locus variable-number tandem repeat analysis (MLVA) profiles, with the same dominant profile present in both food-source and human isolates. More than 90.0% of S. 1,4,[5],12:i:- isolates were multidrug-resistant. The high prevalence of blaCTX-M (33.3%, 7/21) in extended-spectrum β-lactamase-producing S. 1,4,[5],12:i:- isolates from patients identified here will raise clinical concern over human salmonellosis treatment. Twelve different deletion types of the fljAB operon region associated with the loss of phase 2 flagellar antigen were identified. These results suggested that the Chinese S. 1,4,[5],12:i:- strains consist of multiple distinct clones and their high rates of prevalence and multidrug resistance could constitute potential risks to public health.

Molecular typing of clinical multidrug-resistant Klebsiella pneumoniae isolates.

Klebsiella pneumoniae is an opportunistic pathogen which is an important cause of hospital-acquired and antibiotic resistance infections. Therefore, this study aimed to determine the frequency of resistance to antibiotics, as well as the molecular typing of the associated isolates, and compare multiple-locus VNTR analysis (MLVA) and Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) methods to specify the degree to which distinctions can be separated from each other. One hundred K. pneumoniae isolates were obtained from different sources of infections from patients admitted to hospitals. Antibiotic susceptibility testing was then performed by applying the Kirby-Bauer disk diffusion method. Typing of K. pneumoniae was done by utilizing MLVA and ERIC-PCR methods. Eighty-six multidrug-resistant (MDR) K. pneumoniae isolates were identified, which resistance to ampicillin, trimethoprim/sulfamethoxazole, and ceftriaxone was the most frequent in the considered isolates (100, 93, and 93%, respectively). A total of 50 different antibiotic susceptibility patterns were observed among the MDR K. pneumonia, with the most frequent pattern being resistance to all antibiotics (12.79%) and resistance to all antibiotics except amikacin (10.47%). The isolates were then divided into 37 different MLVA types and seven clonal complexes were obtained from the minimum spanning tree analysis. Finally, the isolates were assigned to 38 different ERIC types. The discriminatory power of MLVA and ERIC methods also showed a value of 0.958, and 0.974. Both PCR-typing methods with phenotypic patterns can be useful for the epidemiological typing of K. pneumoniae isolates with the highest performance in discriminating isolates.

Epidemiological changes and molecular characteristics of Brucella strains in Ningxia, China.

Human brucellosis causes serious public health concerns in Ningxia, China. This study employed epidemiological, bacteriological, and multiple-locus variable-number tandem repeat analysis (MLVA) methods to conduct an epidemiological investigation, which is necessary for devising tailored control strategies. 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. 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.

Molecular Analysis of Enterococcus Faecalis Isolates in a 4-year Period.

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, variable-number 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.