Multilocus Variable Number Tandem Repeat Research Articles

Prediction of Listeria monocytogenes Clonal Complexes from Multilocus Variable Number Tandem Repeat Analysis Patterns Using a Machine Learning Approach.

Multilocus variable number tandem repeat analysis (MLVA) is a molecular subtyping technique that remains useful for those without the resources to access whole genome sequencing for the tracking and tracing of bacterial contaminants. Unlike techniques such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis, MLVA did not emerge as a standardized subtyping method for Listeria monocytogenes, and as a result, there is no reference database of virulent or food-associated MLVA subtypes as there is for MLST-based clonal complexes (CCs). Having previously shown the close congruence of a 5-loci MLVA scheme with MLST, a predictive model was created using the XGBoost machine learning (ML) technique, which enabled the prediction of CCs from MLVA patterns with ∼85% (±4%) accuracy. As well as validating the model on existing data, a straightforward update protocol was simulated for if and when previously unseen subtypes might arise. This article illustrates how ML techniques can be applied with elementary coding skills to add value to previous-generation molecular subtyping data in-built food processing environments.

Strain typing and antimicrobial susceptibility of Salmonella enterica Albany isolates from duck farms in South Korea

Salmonella enterica is distributed worldwide and is a common cause of bacterial food poisoning in humans and a serious public health problem. Although duck meat consumption has recently increased in Korea, studies on the epidemiological relationship between S. enterica contamination in duck farms are scarce. Salmonella enterica serovar Albany isolates recovered from duck farms were analyzed using two typing methods — IR Biotyper® (IRBT) and multilocus variable-number tandem repeat analysis (MLVA). The clustering results were compared with the epidemiological survey findings and the antimicrobial resistance profiles. From April 2019 to October 2020, 20 individual feces per farm from 5–6-week-old ducks were collected repeatedly from 105 duck farms. Salmonella spp. isolated from duck feces were identified using PCR and multilocus sequence typing to investigate the prevalence and distribution of the Salmonella serovars. The prevalence of S. enterica was 19%, and S. enterica Albany was the predominantly recovered isolate. The S. enterica Albany isolates underwent antimicrobial susceptibility testing to determine the minimum inhibitory concentration. MLVA and IRBT methods established relatedness and diversity among the S. enterica Albany isolates. Multidrug-resistant S. enterica Albany was distributed in all the farms. Antimicrobial resistance profiles reflected the duck farm characteristics and isolates recovered from the same farm showed an identical profile. Isolates repeatedly recovered from the same farm also showed identical IRBT clusters and MLVA groups. These findings suggest that the isolates remained on the duck farm and re-infected new duck flocks. Thus, proper cleaning and disinfection is required before the farms are repopulated.

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.

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.

Native circulating Brucella melitensis lineages causing a brucellosis epidemic in Qinghai, China.

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 in-depth understanding of the transmission chain of human brucellosis in this region.

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.

Whole genome sequencing of Ethiopian Brucella abortus isolates expands the known diversity of an early branching sub-Saharan African lineage.

Brucellosis remains one of the most significant zoonotic diseases globally, responsible for both considerable human morbidity and economic losses due to its impacts on livestock productivity. Despite this, there remain significant evidence gaps in many low- and middle-income countries, including those of sub-Saharan Africa. Here we report the first molecular characterisation of Brucella sp. from Ethiopia. Fifteen Brucella sp. isolates from an outbreak in cattle from a herd in central Ethiopia were identified as Brucella abortus, using bacterial culture and molecular methods. Sequencing of the Ethiopian B. abortus isolates allowed their phylogenetic comparison with 411 B. abortus strains of diverse geographical origins, using whole genome single nucleotide polymorphisms (wgSNP). The Ethiopian isolates belonged to an early-branching lineage (Lineage A) previously only represented by data from two strains, both of sub-Saharan African origin (Kenya and Mozambique). A second B. abortus lineage (Lineage B), also comprised solely of strains originating from sub-Saharan Africa, was identified. The majority of strains belonged to one of two lineages of strains originating from a much broader geographical range. Further analyses based on multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA) expanded the number of B. abortus strains available for comparison with the Ethiopian isolates and were consistent with the findings from wgSNP analysis. MLST profiles of the Ethiopian isolates expanded the sequence type (ST) diversity of the early branching lineage of B. abortus, equivalent to wgSNP Lineage A. A more diverse cluster of STs, equivalent to wgSNP Lineage B, was comprised solely of strains originating from sub-Saharan Africa. Similarly, analysis of B. abortus MLVA profiles (n = 1891) confirmed that the Ethiopian isolates formed a unique cluster, similar to only two existing strains, and distinct from the majority of other strains of sub-Saharan African origin. These findings expand the known diversity of an under-represented lineage of B. abortus and suggest a potential evolutionary origin for the species in East Africa. In addition to providing information concerning Brucella species extant within Ethiopia this work serves as the basis for further studies on the global population structure and evolutionary history of a major zoonotic pathogen.

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.

Another advantage of multi-locus variable-number tandem repeat analysis that can putatively subdivide enterohemorrhagic Escherichia coli O157 strains into clades by maximum a posteriori estimation.

Enterohemorrhagic Escherichia coli O157 (O157) strains can be subdivided into clades based on their single-nucleotide polymorphisms, but such analysis using conventional methods requires intense effort by laboratories. Although multi-locus variable-number tandem repeat analysis (MLVA), which can be performed with low laboratory burden, has been used as a molecular epidemiological tool, it has not been evaluated whether MLVA can be used the clade subdivision of O157 strains like it can for that of other pathogenic bacteria. This study aimed to establish a method for subdividing O157 strains into clades using MLVA data. The standardized index of association, ISA, for O157 strains isolated in Chiba prefecture, Japan (Chiba isolates) revealed the presence of unique tandem repeat patterns in each major clade (clades 2, 3, 7, 8, and 12). A likelihood database of tandem repeats for these clades was then constructed using the Chiba isolates, and a formula for maximum a posteriori (MAP) estimation was constructed. The ratio of the number of O157 strains putatively subdivided into a clade by MAP estimation from MLVA data relative to the number of O157 strains subdivided using single-nucleotide polymorphism analysis (designated as the concordance ratio [CR]) was calculated using the Chiba isolates and O157 strains isolated in Yamagata prefecture (Yamagata isolates). The CRs for the major Chiba and Yamagata isolate clades, other than clade 2, were 89%-100%. Although the CR for clade 2 Chiba isolates was >95%, that of the Yamagata isolates was only 78.9%. However, these clade 2 CRs were not significantly different from one another, indicating that clade 2 strains can be subdivided correctly by MAP estimation. In conclusion, this study expands the utility of MLVA, previously applied predominantly for molecular epidemiological analysis, into a low-laboratory-burden tool for subdividing O157 strains into phylogenetic groups.

Identification of Listeria species and Multilocus Variable-Number Tandem Repeat Analysis (MLVA) Typing of Listeria innocua and Listeria monocytogenes Isolates from Cattle Farms and Beef and Beef-Based Products from Retail Outlets in Mpumalanga and North West Provinces, South Africa.

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.

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.

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.

THE USE OF MOLECULAR GENETIC METHODS BASED ON MLVA ANALYSIS TO CONFIRM THE UNIQUENESS OF COLLECTION STRAINS OF BRUCELLA

Collections of pathogenic bacterial cultures are necessary for the storage of commercially valuable and typical strains for the purpose of patenting, production and testing of diagnostic and preventive drugs, as well as epidemiological strains isolated during the diagnosis of infectious diseases for detailed study and monitoring of changes in sanitary and epidemiological well-being. Collections are replenished with new unique strains, so it is very important that the characteristics of these strains should be carried out using highly discriminatory molecular genetic methods. This article presents the results of the evaluation of the genetic diversity of 36 collection strains of Brucella abortus and 57 strains of Brucella melitensis by the Multilocus variable number tandem repeat (VNTR) analysis (MLVA-16). Generic identification and absence of contamination by other bacterial species was confirmed by sequencing of the 16S rRNA gene. MLVA analysis allowed to establish contamination of 4 strains with different genotypes of brucella and to reveal that the deposited 36 strains isolated in 14 outbreaks of brucellosis are identical genotypes of the pathogen. Our studies have confirmed the presence of identical brucella strains in the collection. The use of MLVA-16 in testing isolated bacterial cultures from a single outbreak will allow differentiating unique strains for their subsequent deposition. Verification of deposited strains with MLVA characteristics with a detailed analysis of metadata about deposited strains will optimize the collection, in terms of preserving only unique strains, which will reduce economic costs and risks to ensure biological safety.

Urban rats as carriers of invasive Salmonella Typhimurium sequence type 313, Kisangani, Democratic Republic of Congo.

Invasive non-typhoidal Salmonella (iNTS-mainly serotypes Enteritidis and Typhimurium) are major causes of bloodstream infections in children in sub-Saharan Africa, but their reservoir remains unknown. We assessed iNTS carriage in rats in an urban setting endemic for iNTS carriage and compared genetic profiles of iNTS from rats with those isolated from humans. From April 2016 to December 2018, rats were trapped in five marketplaces and a slaughterhouse in Kisangani, Democratic Republic of the Congo. After euthanasia, blood, liver, spleen, and rectal content were cultured for Salmonella. Genetic relatedness between iNTS from rats and humans-obtained from blood cultures at Kisangani University Hospital-was assessed with multilocus variable-number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing (MLST) and core-genome MLST (cgMLST). 1650 live-capture traps yielded 566 (34.3%) rats (95.6% Rattus norvegicus, 4.4% Rattus rattus); 46 (8.1%) of them carried Salmonella, of which 13 had more than one serotype. The most common serotypes were II.42:r:- (n = 18 rats), Kapemba (n = 12), Weltevreden and Typhimurium (n = 10, each), and Dublin (n = 8). Salmonella Typhimurium belonged to MLST ST19 (n = 7 rats) and the invasive ST313 (n = 3, isolated from deep organs but not from rectal content). Sixteen human S. Typhimurium isolates (all ST313) were available for comparison: MLVA and cgMLST revealed two distinct rat-human clusters involving both six human isolates, respectively, i.e. in total 12/16 human ST313 isolates. All ST313 Typhimurium isolates from rats and humans clustered with the ST313 Lineage 2 isolates and most were multidrug resistant; the remaining isolates from rats including S. Typhimurium ST19 were pan-susceptible. The present study provides evidence of urban rats as potential reservoirs of S. Typhimurium ST313 in an iNTS endemic area in sub-Saharan Africa.

Molecular Tools for Typing Mycoplasma pneumoniae and Mycoplasma genitalium

Mycoplasma pneumoniae and Mycoplasma genitalium are cell wall-less bacteria with strongly reduced genome content and close phylogenetic relatedness. In humans, the only known natural host, the microorganisms colonize the respiratory or genitourinary mucosa and may cause a broad range of clinical presentations. Besides fundamental differences in their tissue specificity, transmission route, and ability to cause prevalence peaks, both species share similarities such as the occurrence of asymptomatic carriers, preferred populations for infection, and problems with high rates of antimicrobial resistance. To further understand the epidemiology of these practically challenging bacteria, typing of strains is necessary. Since the cultivation of both pathogens is difficult and not performed outside of specialized laboratories, molecular typing methods with adequate discriminatory power, stability, and reproducibility have been developed. These include the characterization of genes containing repetitive sequences, of variable genome regions without the presence of repetitive sequences, determination of single and multi-locus variable-number tandem repeats, and detection of single nucleotide polymorphisms in different genes, respectively. The current repertoire of procedures allows reliable differentiation of strains circulating in different populations and in different time periods as well as comparison of strains occurring subsequently in individual patients. In this review, the methods for typing M. pneumoniae and M. genitalium, including the results of their application in different studies, are summarized and current knowledge regarding the association of typing data with the clinical characteristics of infections is presented.

Genetic Profiling of Salmonella enteritidis Strains Isolated in the Stavropol Region in 2016–2019

Introduction: Salmonella enteritidis is the most common etiologic agent of salmonellosis in humans in the Stavropol Region, which is in line with the global trend. In this regard, the definition of a serovar provides little information in the course of an epidemiological investigation and necessitates subspecific typing of S. enteritidis isolates. Objective: MLVA typing of S. enteritidis strains isolated in the Stavropol Region in 2016–2019 and the analysis of the genetic structure of the local Salmonella population. Materials and methods: We studied 122 strains of S. enteritidis isolated in 2016–2019 from stool samples of patients with acute intestinal infections in the city of Stavropol and the Caucasian Mineral Waters (CMW) of the Stavropol Region. Multi Locus Variable Number Tandem Repeat (MLVA) typing was performed based on five variable loci. The size of amplified loci was determined by capillary electrophoresis. Results: The studied strains were distinguished by high genetic heterogeneity and belonged to 25 MLVA genotypes. S. enteritidis strains belonging to 24 MLVA types were identified on the territory of Stavropol. The dominant genetic variant in the city was 3-10-5-4-1, which included 40 Salmonella strains (44.4%) isolated in 2016–2019. In some years, the proportion of other genetic variants increased. In the CMW area, strains belonging to seven MLVA genotypes were isolated, most of which belonged to the profile 3-10-5-4-1, while a change in the dominant variant of S. Enteritidis was registered annually. Prevalent regional MLVA types are widespread in the world and have a significant epidemic potential. Conclusions: We obtained new data on S. enteritidis MLVA genotypes in the Stavropol Region, established dominant genetic variants, and noted differences in the ratio of circulating MLVA genotypes of S. enteritidis in 2016–2019. Our findings necessitate constant molecular genetic monitoring to assess real-time dynamic changes in the genetic structure of the Salmonella population. They can also be used in investigations of possible cases (outbreaks) of salmonellosis to determine their source and routes of transmission.

Investigation of OXA-23, OXA-24, OXA-40, OXA-51, and OXA-58 Genes in Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae Isolates from Patients with Urinary Tract Infections

Background: Escherichia coli and Klebsiella pneumoniae are frequently responsible for urinary tract infections (UTIs). The high rate of carbapenem resistance in Enterobacteriaceae has become a global therapeutic concern. Objectives: The study investigated OXA-23, OXA-24, OXA-40, OXA-51, and OXA-58 genes in uropathogenic E. coli and K. pneumoniae isolates. Methods: We isolated 500 uropathogenic isolates of E. coli and K. pneumoniae from patients at Milad Hospital, Tehran, Iran. Antibiotic susceptibility testing was performed using a strip-test method, and the carbapenem-nonsusceptoble isolates were confirmed with an automated antibiotic sensitivity testing system. The OXA genes were determined by multiplex PCR. Molecular typing was performed by multilocus variable-number tandem repeat (VNTR) analysis (MLVA). Results: Out of 500 isolates, 40 (8%) were detected as carbapenem-resistant, including 13 E. coli and 27 K. pneumoniae. All carbapenem-resistant isolates were ESBL-producing and resistant to ceftriaxone, ciprofloxacin, meropenem, ceftazidime, and amoxicillin-clavulanate. Moreover, 46.1% and 26% of carbapenem-insensitive E. coli and K. pneumoniae isolates carried a beta-lactamase-producing gene associated with the OXA-23-like group. Finally, E. coli and K. pneumoniae isolates were divided into two and three MLVA patterns, respectively. Conclusions: This is the first report of OXA-51, 58, and 24 carbapenemases in clinical isolates of E. coli and K. pneumoniae from UTI patients in Iran. Significant differences were seen in OXA-51, 58, and 24 genes between carbapenem-insensitive and carbapenem-sensitive E. coli and K. pneumoniae isolates. Molecular typing suggested the vertical transmission of resistance genes.

Tracking the diversity and Mediterranean lineage of Brucella melitensis isolates from different animal species in Turkey using MLVA-16 genotyping

Brucellosis is a zoonotic disease with a high prevalence in humans and farm animals in Turkey. However, data on the genetic diversity of Brucella spp. circulating in Turkey and parts of the Mediterranean region are limited. In the present study, the genetic diversity of 50 B. melitensis isolates from seven regions of Turkey was investigated using multi-locus variable number tandem repeats analysis (MLVA-16). The profiles were compared with 163 B. melitensis isolates recovered from the Mediterranean basin. B. melitensis strains from Turkey contain 46 different genotypes and consist of two main clusters. B. melitensis isolates from Turkey were closely related to isolates from Greece and some Portuguese strains. The same genotypes isolated from different sites show the spread between sites. Therefore, uncontrolled animal movements and the trade of imported animals can be important factors for the spread of brucellosis. The endemic occurrence of B. melitensis in the Mediterranean basin is a result of socio-historical links between Mediterranean countries. Turkish strains belong to the Eastern Mediterranean line. Eradicating brucellosis in countries of the Mediterranean basin with high prevalence is a demanding need to reduce trade barriers and, more importantly, prevent human suffering