Variable-number Tandem Repeat Analysis Research Articles

Unravelling Chlamydia trachomatis diversity in Amhara, Ethiopia: MLVA-ompA sequencing as a molecular typing tool for trachoma.

Trachoma is the leading infectious cause of blindness worldwide and is now largely confined to around 40 low- and middle-income countries. It is caused by Chlamydia trachomatis (Ct), a contagious intracellular bacterium. The World Health Organization recommends mass drug administration (MDA) with azithromycin for treatment and control of ocular Ct infections, alongside improving facial cleanliness and environmental conditions to reduce transmission. To understand the molecular epidemiology of trachoma, especially in the context of MDA and transmission dynamics, the identification of Ct genotypes could be useful. While many studies have used the Ct major outer membrane protein gene (ompA) for genotyping, it has limitations. Our study applies a typing system novel to trachoma, Multiple Loci Variable Number Tandem Repeat Analysis combined with ompA (MLVA-ompA). Ocular swabs were collected post-MDA from four trachoma-endemic zones in Ethiopia between 2011-2017. DNA from 300 children with high Ct polymerase chain reaction (PCR) loads was typed using MLVA-ompA, utilizing 3 variable number tandem repeat (VNTR) loci within the Ct genome. Results show that MLVA-ompA exhibited high discriminatory power (0.981) surpassing the recommended threshold for epidemiological studies. We identified 87 MLVA-ompA variants across 26 districts. No significant associations were found between variants and clinical signs or chlamydial load. Notably, overall Ct diversity significantly decreased after additional MDA rounds, with a higher proportion of serovar A post-MDA. Despite challenges in sequencing one VNTR locus (CT1299), MLVA-ompA demonstrated cost-effectiveness and efficiency relative to whole genome sequencing, providing valuable information for trachoma control programs on local epidemiology. The findings suggest the potential of MLVA-ompA as a reliable tool for typing ocular Ct and understanding transmission dynamics, aiding in the development of targeted interventions for trachoma control.

Correlation of DNA load, genotyping, and clinical phenotype of Mycoplasma pneumoniae infection in children.

This study aimed to investigate the correlation between Mycoplasma pneumoniae (MP)-DNA load in the bronchoalveolar lavage fluid (BALF) of children with MP pneumonia (MPP) and its subtypes, relevant laboratory data, imaging, extrapulmonary complications in infected children, and its clinical significance in evaluating the disease. Children hospitalized with MPP at Tianjin Children's Hospital between December 2017 and December 2020 were selected for the study, excluding those with mixed viral, bacterial, and fungal infections. Children were divided into low- and high-load groups according to the MP DNA load in BALF using real-time quantitative fluorescence polymerase chain reaction (PCR). After a successful MP culture, positive specimens were subjected to PCR-Restriction fragment length polymorphism and Multiple-locus variable number tandem repeat analysis typing. Basic data, clinical information, laboratory data, and radiological results were collected from all children included in the study. The PI-I type dominated the different load groups. Children in the low-load group had more wheezing and shortness of breath; however, children in the high-load group had a higher length of hospitalization, maximum fever temperature, higher chills/chilliness, incidence of abdominal pain, and higher C-reactive protein (CRP), procalcitonin (PCT) and aspartate aminotransferase (AST) levels. Children in the high-load group were more likely to have imaging changes such as pleural effusion, and the incidence of respiratory infections and extrapulmonary complications was higher than that of those in the low-load group. We applied Spearman's correlation analysis to clarify the relationship between MP DNA load and the clinical severity of MPP. We found that MP DNA load was positively correlated with length of hospitalization, maximum fever temperature, CRP, PCT, Interleukin-6 (IL-6), and AST levels, and negatively correlated with fever and cough durations, white blood cell count (WBC), and proportion of monocytes (MONO). The degree of correlation was as follows: length of hospitalization > IL-6 > cough duration > AST > fever duration > PCT > WBC > proportion of MONO > maximum fever temperature > CRP levels. MP DNA load was not correlated with MP typing but was significantly correlated with the children's clinical phenotype. Therefore, the MP DNA load helps in the early diagnosis of infection and can better predict disease regression.

Klebsiella pneumoniae sequence type 147: a high-risk clone increasingly associated with plasmids carrying both resistance and virulence elements.

Introduction. The first hybrid resistance/virulence plasmid, combining elements from virulence plasmids described in hypervirulent types of Klebsiella pneumoniae with those from conjugative resistance plasmids, was described in an isolate of sequence type (ST) 147 from 2016. Subsequently, this type has been increasingly associated with these plasmids.Hypothesis or gap statement. The extent of carriage of hybrid virulence/resistance plasmids in nosocomial isolates of K. pneumoniae requires further investigation.Aim. To describe the occurrence of virulence/resistance plasmids among isolates of K. pneumoniae received by the UK reference laboratory, particularly among representatives of ST147, and to compare their sequences.Methodology. Isolates received by the laboratory during 2022 and the first half of 2023 (n=1278) were screened for virulence plasmids by PCR detection of rmpA/rmpA2 and typed by variable-number tandem repeat analysis. Twenty-nine representatives of ST147 (including a single-locus variant) from seven hospital laboratories were subjected to long-read nanopore sequencing using high-accuracy q20 chemistry to provide complete assemblies.Results. rmpA/rmpA2 were detected in 110 isolates, of which 59 belonged to hypervirulent K1-ST23, K2-ST86 and K2-ST65/375. Of the remainder, representatives of ST147 formed the largest group, with 22 rmpA/rmpA2-positive representatives (out of 47 isolates). Representatives were from 19 hospital laboratories, with rmpA/rmpA2-positive isolates from 10. Nanopore sequencing of 29 representatives of ST147 divided them into those with no virulence plasmid (n=12), those with non-New Delhi metallo-β-lactamase (NDM) virulence plasmids (n=6) and those carrying bla NDM-5 (n=9) or bla NDM-1 (n=2) virulence plasmids. These plasmids were of IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) replicon types. Most of the non-NDM virulence plasmids were highly similar to the originally described KpvST147L_NDM plasmid. Those carrying bla NDM-5 were highly similar to one another and to previously described plasmids in ST383 and carried an extensive array of resistance genes. Comparison of the fully assembled chromosomes indicated multiple introductions of ST147 in UK hospitals.Conclusion. This study highlights the high proportion of representatives of ST147 that carry IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) hybrid resistance virulence plasmids. It is important to be aware of the high probability that representatives of this type carry these plasmids combining resistance and virulence determinants and of the consequent increased risk to patients.

Genotyping and phylogenetic analysis of Coxiella burnetii in domestic ruminant and clinical samples in Iran: insights into Q fever epidemiology

Coxiella burnetii, a zoonotic pathogen, is the causative agent of Q fever, an endemic disease in Iran. However, there is currently a lack of available data on the genotypes of C. burnetii in the country. Here, we typed 26 C. burnetii isolates detected in milk, abortion, cotylodon, and cardiac valve samples from various geographical areas and hosts (7 cattle, 8 goats, 10 sheep, and 1 human) using Multilocus Variable Number Tandem Repeat Analysis (MLVA/VNTR) with five loci:ms24, ms27, ms28, ms33, and ms34. As IS1111 was observed to be spontaneously inserted in locus ms23 across all of our examined C. burnetii samples, five loci were employed for MLVA/VNTR genotyping. Among the 26 C. burnetii strains, 22 distinct genotypes (A–V) were identified in the discriminative loci. In silico analysis categorized Iranian C. burnetii strains into five genomic groups along with seven singletons, representing 11 exiting clonal complexes worldwide. Clusters 10 and 11 exclusively consisted of Iranian samples. These findings revealed high genotyping diversity among C. burnetii isolates in Iran. The genotypes circulating in Iran differed significantly from those found in other regions worldwide. To gain a comprehensive understanding of Q fever epidemiology in Iran, it is crucial to conduct large-scale studies that assess the distribution of C. burnetii genotypes across different geographical areas, hosts, and sources.

Development of molecular assays for the analysis of genetic relationships of Mycoplasma iowae

Mycoplasma iowae is a worldwide spread and economically important avian pathogen that mostly infects turkeys. Currently, multi-locus sequence typing (MLST) serves as the gold standard method for strain identification in M. iowae. However, additional robust genotyping methods are required to effectively monitor M. iowae infections and conduct epidemiological investigations. The first aim of this study was to develop genotyping assays with high resolution, that specifically target M. iowae, namely a multiple-locus variable number of tandem-repeats analysis (MLVA) and a core genome multi-locus sequence typing (cgMLST) schema. The second aim was the determination of relationships among a diverse selection of M. iowae strains and clinical isolates with a previous and the newly developed assays. The MLVA was designed based on the analyses of tandem-repeat (TR) regions in the six serotype reference strains (I, J, K, N, Q and R). The cgMLST schema was developed based on the coding sequences (CDSs) common in 95% of the examined 99 isolates. The samples were submitted for a previously published MLST assay for comparison with the developed methods. Out of 94 TR regions identified, 17 alleles were selected for further evaluation by PCR. Finally, seven alleles were chosen to establish the MLVA assay. Additionally, whole genome sequence analyses identified a total of 676 CDSs shared by 95% of the isolates, all of which were included into the developed cgMLST schema. The MLVA discriminated 19 distinct genotypes (GT), while with the cgMLST assay 79 sequence types (ST) could be determined with Simpson’s diversity indices of 0.810 (MLVA) and 0.989 (cgMLST). The applied assays consistently identified the same main clusters among the diverse selection of isolates, thereby demonstrating their suitability for various genetic analyses and their ability to yield congruent results.

Prevalence and genetic distribution of Legionella spp. in public bath facilities in Kobe City, Japan.

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.

Molecular typing and antimicrobial resistance of group B Streptococcus clinical isolates in Saudi Arabia

Group B Streptococcus (GBS) has emerged as an important cause of severe infections in adults. However, limited data are available regarding the epidemiology of GBS in Saudi Arabia. Isolates were collected over a period of eight months from colonized (n=104) and infected adults (n=95). Serotypes and virulence determinants were detected by polymerase chain reactions (PCRs). Genetic relatedness was assessed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Antimicrobial susceptibilities were determined by disk diffusion. Serotypes III and V (25% each) were the most prevalent, followed by serotypes II (16.18%), Ia (13.24%), VI (9.31%), and Ib (8.82%), while five isolates remained non-typeable (2.45%). Hypervirulent serotype III/CC17 clone (n=21) accounted for 41.18% of the serotype III isolates. Most isolates (53.92%) harboured pilus island (PI) 1 and 2a types, while PI-2b was predominantly detected in the hypervirulent clone. Isolates were variably resistant to tetracycline (76.47%), erythromycin (36.76%), clindamycin (25.49%), and levofloxacin (6.37%), but remained susceptible to penicillin. Macrolide resistant isolates exhibited constitutive (55.42%) and inducible macrolide-lincosamide-streptogramin B resistance phenotypes (33.74%), while a few had L (9.64%) or M (1.2%) phenotypes. MLVA patterns of dominant serotypes III and V revealed 40 different types divided into 12 clusters and 28 singletons. Interestingly, macrolide resistance was significantly associated with two major MLVA types. GBS isolates belonged predominantly to serotypes III and V, but there were no clear associations between serotypes and patient groups. The studied isolates exhibited high levels of resistance to erythromycin and clindamycin that need further surveillance.

Optimization of MLVA loci combination using metaheuristic methods

Multilocus variable number tandem repeat analysis (MLVA) utilizes short DNA repeat polymorphism in genomes, which is termed variable number tandem repeat (VNTR), to differentiate closely related organisms. One research challenge is to find an optimal set of VNTR to distinguish different members accurately. An intuitive method is to use an exhaustive search method. However, this method is not an efficient way to find optimal solutions from a dataset comprising many attributes (loci) due to the curse of dimensionality. In this study, metaheuristic methods are proposed to find an optimal set of loci combination. Basic genetic algorithm (BGA) and modified genetic algorithm (MGA) were proposed in our previous work for this purpose. However, they require prior knowledge from an experienced user to specify the minimum number of loci for achieving good results. To impose no such expertise requirement for parameter setting, a GA with Duplicates (GAD), which allows the inclusion of duplicated loci in a chromosome (potential solution) during the search process, is developed. The study also investigates the search performance of a hybrid metaheuristic method, namely quantum-inspired differential evolution (QDE). Hunter-Gaston Discriminatory Index (HGDI) is used to indicate the discriminatory power of a loci combination. Two Mycobacterium tuberculosis MLVA datasets obtained from a public portal and a local laboratory respectively, are used. The results obtained by using exhaustive search and metaheuristic methods are first compared, followed by a performance comparison among BGA, MGA, GAD, and QDE by a statistical approach. The best-performing GA method (i.e., GAD) and QDE are selected for a performance comparison with several recent metaheuristic methods using both MLVA datasets by a statistical approach. The statistical results show that both GAD and QDE could achieve higher HGDI than the recent methods using a small but informative set of loci combination.

Spatial and phylogenetic patterns reveal hidden infection sources of Bacillus anthracis in an anthrax outbreak in Son La province, Vietnam.

Bacillus anthracis, the bacterial cause of anthrax, is a zoonosis affecting livestock and wildlife often spilling over into humans. In Vietnam, anthrax has been nationally reportable since 2015 with cases occurring annually, mostly in the northern provinces. In April 2022, an outbreak was reported in Son La province following the butchering of a water buffalo, Bubalus bubalis. A total of 137 humans from three villages were likely exposed to contaminated meat from the animal. Early epidemiological investigations suggested a single animal was involved in all exposures. Five B. anthracis isolates were recovered from human clinical cases along with one from the buffalo hide, another from associated maggots, and one from soil at the carcass site. The isolates were whole genome sequenced, allowing global, regional, and local molecular epidemiological analyses of the outbreak strains. All recovered B. anthracis belong to the A.Br.001/002 lineage based on canonical single nucleotide polymorphism analysis (canSNP). Although not previously identified in Vietnam, this lineage has been identified in the nearby countries of China, India, Indonesia, Thailand, as well as Australia. A twenty-five marker multi-locus variable number tandem repeat analysis (MLVA-25) was used to investigate the relationship between human, soil, and buffalo strains. Locally, four MLVA-25 genotypes were identified from the eight isolates. This level of genetic diversity is unusual for the limited geography and timing of cases and differs from past literature using MLVA-25. The coupled spatial and phylogenetic data suggest this outbreak originated from multiple, likely undetected, animal sources. These findings were further supported by local news reports that identified at least two additional buffalo deaths beyond the initial animal sampled in response to the human cases. Future outbreak response should include intensive surveillance for additional animal cases and additional molecular epidemiological traceback to identify pathogen sources.

Molecular characterization of MRSA collected during national surveillance between 2008 and 2019 in the Netherlands

Background.Although the Netherlands is a country with a low endemic level, methicillin-resistant Staphylococcus aureus (MRSA) poses a significant health care problem. Therefore, high coverage national MRSA surveillance has been in place since 1989. To monitor possible changes in the type-distribution and emergence of resistance and virulence, MRSA isolates are molecularly characterized.Methods.All 43,321 isolates from 36,520 persons, collected 2008–2019, were typed by multiple-locus variable number tandem repeats analysis (MLVA) with simultaneous PCR detection of the mecA, mecC and lukF-PV genes, indicative for PVL. Next-generation sequencing data of 4991 isolates from 4798 persons were used for whole genome multi-locus sequence typing (wgMLST) and identification of resistance and virulence genes.Results.We show temporal change in the molecular characteristics of the MRSA population with the proportion of PVL-positive isolates increasing from 15% in 2008–2010 to 25% in 2017–2019. In livestock-associated MRSA obtained from humans, PVL-positivity increases to 6% in 2017–2019 with isolates predominantly from regions with few pig farms. wgMLST reveals the presence of 35 genogroups with distinct resistance, virulence gene profiles and specimen origin. Typing shows prolonged persistent MRSA carriage with a mean carriage period of 407 days. There is a clear spatial and a weak temporal relationship between isolates that clustered in wgMLST, indicative for regional spread of MRSA strains.Conclusions.Using molecular characterization, this exceptionally large study shows genomic changes in the MRSA population at the national level. It reveals waxing and waning of types and genogroups and an increasing proportion of PVL-positive MRSA.

Investigation of virulence factors, phylogenetic grouping, multiple-locus variable number tandem repeat analysis, and antimicrobial susceptibility of E. coli isolated from aborted bovine fetal tissue.

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.

Validating the Utility of Multilocus Variable Number Tandem-repeat Analysis (MLVA) as a Subtyping Strategy to Monitor Listeria monocytogenes In-built Food Processing Environments

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.

A minisatellite-based MLVA for deciphering the global epidemiology of the bacterial cassava pathogen Xanthomonas phaseoli pv. manihotis.

Cassava Bacterial Blight (CBB) is a destructive disease widely distributed in the different areas where this crop is grown. Populations studies have been performed at local and national scales revealing a geographical genetic structure with temporal variations. A global epidemiology analysis of its causal agent Xanthomonas phaseoli pv. manihotis (Xpm) is needed to better understand the expansion of the disease for improving the monitoring of CBB. We targeted new tandem repeat (TR) loci with large repeat units, i.e. minisatellites, that we multiplexed in a scheme of Multi-Locus Variable number of TR Analysis (MLVA-8). This genotyping scheme separated 31 multilocus haplotypes in three clusters of single-locus variants and a singleton within a worldwide collection of 93 Xpm strains isolated over a period of fifty years. The major MLVA-8 cluster 1 grouped strains originating from all countries, except the unique Chinese strain. On the contrary, all the Xpm strains genotyped using the previously developed MLVA-14 microsatellite scheme were separated as unique haplotypes. We further propose an MLVA-12 scheme which takes advantage of combining TR loci with different mutation rates: the eight minisatellites and four faster evolving microsatellite markers, for global epidemiological surveillance. This MLVA-12 scheme identified 78 haplotypes and separated most of the strains in groups of double-locus variants (DLV) supporting some phylogenetic relationships. DLV groups were subdivided into closely related clusters of strains most often sharing the same geographical origin and isolated over a short period, supporting epidemiological relationships. The main MLVA-12 DLV group#1 was composed by strains from South America and all the African strains. The MLVA-12 scheme combining both minisatellite and microsatellite loci with different discriminatory power is expected to increase the accuracy of the phylogenetic signal and to minimize the homoplasy effects. Further investigation of the global epidemiology of Xpm will be helpful for a better control of CBB worldwide.

Novel strategy of multiple-locus variable number tandem repeats analysis for genetic fingerprinting of human.

The variable number of tandem repeat (VNTR) analyses are methods based on the detection of repeated sequences within the human genome. In order to perform DNA typing at the personal laboratory, it is necessary to improve the VNTR analysis. The VNTR markers were difficult to popularize because PCR amplification was difficult due to its GC-rich and long nucleotide sequence. The aim of this study was to select the multiple VNTR markers that could only be identified by PCR amplification and electrophoresis. We genotyped each of the 15 VNTR markers using genomic DNA from 260 unrelated individuals by PCR amplification. Differences in the fragment length of PCR products are visualized by agarose gel electrophoresis. To confirm their usefulness as a DNA fingerprint these 15 markers were simultaneously analyzed with the DNA of 213 individuals and verified the statistical significance. In addition, to investigate the usefulness of each of the 15 VNTR markers as paternity markers, Mendelian segregation by meiotic division within a family consisting of two or three generations was confirmed. Fifteen VNTR loci selected in this study could be easily amplified by PCR and analyzed by electrophoresis, and were newly named DTM1 ~ 15. The number of total alleles in each VNTR showed from 4 to 16, and 100 to 1600bp in length, and their heterozygosity ranged from 0.2341 to 0.7915. In simultaneous analysis of 15 markers from 213 DNAs, the probability of chance appearing the same genotype in different individuals was less than 4.09E-12, indicating its usefulness as a DNA fingerprint. These loci were transmitted through meiosis by Mendelian inheritance in families. Fifteen VNTR markers have been found to be useful as DNA fingerprints for personal identification and kinship analysis that can be used at the personal laboratory level.

The first report of Brucella melitensis biovar 2 strain isolated from cattle in Turkey

Bovine brucellosis is an infectious zoonotic disease of great impact on animal welfare and has significant economic implications on livestock farm worldwide. The disease is caused primarily by Brucella abortus (B. abortus), while B. melitensis is less common, and B. suis infection is rare. B. melitensis is the most common causative agent of brucellosis in small ruminants and humans. Although the main host of B. melitensis is considered to be small ruminants, this bacterium is also present in large ruminants. Despite brucellosis has been eradicated in many European countries, it is still endemic in Mediterranean countries and Turkey. The most prevalent Brucella species in the Mediterranean basin and Turkey is B. melitensis biovar (bv) 3. Previous studies have reported that B. melitensis bv2 is quite low in Turkey. This is the first study to isolate B. melitensis bv2 from cattle in Turkey. The strains were characterized using classical biotyping methods and then were molecularly confirmed. Multilocus variable number tandem repeat analysis (MLVA-16) typing of the strains revealed a novel genotype (1-5-3-13-3-2-3-2-4-41-8-5-4-3-3-7), which matches the Multilocus sequence typing (MLST) profiles in the database of ST8 (3-2-3-2-1-5-3-2-8). These results indicate that B. melitensis bv2 can easily infect cattle and this has to be considered in the epidemiology and control of bovine brucellosis. Circulating the highly pathogenic B. melitensis bv2 in cattle farms is of public health concern.

A pseudo-outbreak of MRSA due to laboratory contamination related to MRSA carriage of a laboratory staff member

BackgroundMethicillin resistant Staphylococcus aureus (MRSA) is a major burden for hospitals globally. However, in the Netherlands, the MRSA prevalence is relatively low due to the ‘search and destroy’ policy. Routine multiple-locus variable-number of tandem repeat analysis (MLVA) of MRSA isolates supports outbreak detection. However, whole genome multiple locus sequence typing (wgMLST) is superior to MLVA in identifying (pseudo-)outbreaks with MRSA. The present study describes a pseudo-outbreak of MRSA at the bacteriology laboratory of a large Dutch teaching hospital.MethodsAll staff members of the bacteriology laboratory of the Elisabeth-TweeSteden hospital were screened for MRSA carriage, after a laboratory contamination with MRSA was suspected. Clonal relatedness between the index isolate and the MRSA isolates from laboratory staff members and all previous MRSA isolates from the Elisabeth-TweeSteden hospital with the same MLVA-type as the index case was examined based on wgMLST using whole genome sequencing.ResultsOne of the staff members was identified as the probable source of the laboratory contamination, because of carriage of a MRSA possessing the same MLVA-type as the index case. Eleven other isolates with the same molecular characteristics were found in the database, of which seven were retrospectively suspected of contamination. Clonal relatedness was found between ten isolates, including the isolate found in the staff member and the MRSA found in the index patient with a maximum of eleven alleles difference. All isolates were epidemiologically linked through the laboratory staff member, who had worked on all these cultures.ConclusionsThe present study describes a MRSA pseudo-outbreak over a 2.5-year period due to laboratory contamination caused by a MRSA carrying laboratory staff member involving nine patients. In case of unexpected bacteriological findings, the possibility of a laboratory contamination should be considered.

Application of a new multi-locus variable number tandem repeat analysis (MLVA) scheme for the seasonal investigation of Cryptosporidium parvum cases in Wales and the northwest of England, spring 2022

The protozoan Cryptosporidium parvum is an important cause of gastroenteritis in humans and livestock, and cryptosporidiosis outbreaks are common. However, a multi-locus genotyping scheme is not widely adopted. We describe the further development and application of a seven-locus multi-locus variable number of tandem repeats analysis (MLVA) scheme. From 28th March to 31st July 2022, confirmed C. parvum stools (n = 213) from cryptosporidiosis patients (cases) in Wales (n = 95) and the north west of England (n = 118) were tested by MLVA. Typability (defined as alleles identified at all seven loci in a sample) was 81.2% and discriminatory power estimated by Hunter Gaston Discriminatory Index was 0.99. A MLVA profile was constructed from the alleles, expressed in chromosomal order. Profiles were defined as simple (single allele at each locus) or mixed (more than one allele at any locus). A total of 161 MLVA profiles were identified; 13 were mixed, an additional 38 simple profiles contained null records, and 110 were complete simple profiles. A minimum spanning tree was constructed of simple MLVA profiles and those identical at all seven loci defined genetic clusters of cases (here, null records were considered as an allele); 77 cases formed 25 clusters, ranging from two to nine (mode = two) cases. The largest cluster, following epidemiological investigation, signalled a newly-identified outbreak. Two other cases with mixed profiles that contained the outbreak alleles were included in the outbreak investigation. In another epidemiologically-identified outbreak of six initial cases, MLVA detected two additional cases. In a third, small outbreak of three cases, identical MLVA profiles strengthened the microbiological evidence. Review of the performance characteristics of the individual loci and of the seven-locus scheme suggested that two loci might be candidates for review, but a larger dataset over a wider geographical area and longer timeframe will help inform decision-making about the scheme by user laboratories and stakeholders (such as public health agencies). This MLVA scheme is straightforward in use, fast and cheap compared to sequence-based methods, identifies mixed infections, provides an important tool for C. parvum surveillance, and can enhance outbreak investigations and public health action.

Genotypic properties of collection plague microbes strains from the natural plague foci of Kazakhstan

Background. Epidemiological and epizootological monitoring of natural plague foci requires an integrated approach to solving problems, taking into account the phenotypic and genetic variability of Y. pestis and zoning of natural plague foci. The introduction of a new molecular genetic methodology aimed at studying the genomic polymorphism of the plague pathogen provides reliable results for the differentiation of not only groups, but also individual strains.The aim. To determine the genotypes of the plague microbe from different autonomous foci of the Republic of Kazakhstan.Materials and methods. 105 strains of Y. pestis isolated from various natural plague foci of Kazakhstan in 1951–2015 were studied. The phenotypic properties of the strains were studied using standard microbiological methods. A polymerase chain reaction (PCR) was used to detect fragments of the cafl, pst and YPO2088 genes. Multilocus variable number tandem repeat (VNTR) analysis (MLVA) was performed for 25 VNTR loci.Results. The phenotypic properties of the strains were preliminarily studied and the strains of the plague microbe were tested for specificity using the Pest-Quest test system (Kazakhstan). The PCR study confirmed the species-specific affiliation of Y. pestis strains. A variety of strains with typical phenotypic characteristics was revealed. MLVA for 25 key loci (MLVA25) revealed that the studied strains of the plague microbe are phylogenetically closest to the Mediaevalis biovar representatives. A phylogenetic tree of the studied strains has been obtained. It was found that 9 genotypes circulate on the territory of Kazakhstan, and their distribution in certain natural plague foci was determined.Conclusions. The resulting clustering indicates the relationship between the strain groups obtained on the dendrogram by the MLVA25 method and the territories of certain natural plague foci.

MLVA and com1 genotyping of Coxiella burnetii in farmed ruminants in Great Britain

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, has been shown to be endemic in Great Britain, but information on the prevailing genomic lineages or Genomic Groups (GGs) of Coxiella burnetii is limited. The aim of this study was to genotype C. burnetii isolates from infected farmed ruminants by Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) and identify their associated Genomic Group. A total of 51 Coxiella-containing abortion samples from farmed ruminants (sheep, goats, and cattle), which were collected in Great Britain during 2013–2018, were included in the study, 34 of which returned a C. burnetii MLVA genotype. All bovine samples (n = 18), 5/7 of the ovine samples, and 3/9 of the caprine samples belonged to an MLVA cluster which we could link to the MST20 genotype of GG III, whereas 6/9 of the caprine samples and 2/7 of the ovine samples belonged to MLVA clusters which we could link to the MST33 or MST32 genotypes of GG II (7 vs 1 sample(s), respectively). We also noted that the Coxiella-specific com1 gene contained unique mutations that could genomotype isolates, i.e. assign them to a Genomic Group. In conclusion, both goats and sheep in Great Britain (from 2014 onward) were found to carry the same MLVA genotypes (MST33-like; GG II) that were linked to a human Q fever outbreak in the Netherlands. This knowledge in combination with the usage of genotyping/genomotyping methods should prove useful in future surveillance programs and in the management of outbreaks.

Distribution and expression of virulence genes (hlyA, sat) and genotyping of Escherichia coli O25b/ST131 by multi-locus variable number tandem repeat analysis in Tehran, Iran.

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.