Multilocus Variable-number Tandem-repeat Analysis Research Articles

Genetic diversity and incidence of cassava bacterial blight (CBB) caused by Xanthomonas phaseoli pv. manihotis in Burkina Faso

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

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.

Molecular epidemiological analysis of Salmonella Schwarzengrund isolated in Japan by newly developed multi-locus variable-number tandem repeat analysis method

To establish molecular epidemiological analysis method for Salmonella Schwarzengrund and investigate its origin and genetic relationships among isolates in Japan, S. Schwarzengrund strains were examined by the pulsed-field gel electrophoresis (PFGE), the multi-locus sequencing typing (MLST), and the multi-locus variable-number tandem repeat analysis (MLVA) developed in this study. By the PFGE and MLST methods, the strains were typed into two clades (A and B) and two sequence types (STs) (96 and 241), respectively. Minimum spanning tree showed that the strains belonging ST241 genetically close to ST96, suggesting that ST241 originates from ST96. For MLVA, new primers for variable-number tandem repeat (VNTR) loci were designed, and five VNTR loci finally selected. In the MLVA method, the strains were typed into 11 sequence types. The MLVA method was able to differentiate strains belonging to the same clade of PFGE and MLST methods. These results indicate that the MLVA developed in this study shows a higher discriminatory power for S. Schwarzengrund compared to the PFGE and MLST methods and that it appears to be a useful method for the molecular epidemiological investigation of S. Schwarzengrund despite the low genetic diversity of this serotype in Japan.

Molecular typing methods to characterize Brucella spp. from animals: A review.

Brucellosis is an infectious disease of animals that can infect humans. The disease causes significant economic losses and threatens human health. A timely and accurate disease diagnosis plays a vital role in the identification of brucellosis. In addition to traditional diagnostic methods, molecular methods allow diagnosis and typing of the causative agent of brucellosis. This review will discuss various methods, such as Bruce-ladder, Suiladder, high-resolution melt analysis, restriction fragment length polymorphism, multilocus sequence typing, multilocus variable-number tandem repeat analysis, and whole-genome sequencing single-nucleotide polymorphism, for the molecular typing of Brucella and discuss their advantages and disadvantages.

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.

Whole-Genome sequencing in routine Mycobacterium bovis epidemiology - scoping the potential.

Mycobacterium bovis the main agent of bovine tuberculosis (bTB), presents as a series of spatially-localised micro-epidemics across landscapes. Classical molecular typing methods applied to these micro-epidemics, based on genotyping a few variable loci, have significantly improved our understanding of potential epidemiological links between outbreaks. However, they have limited utility owing to low resolution. Conversely, whole-genome sequencing (WGS) provides the highest resolution data available for molecular epidemiology, producing richer outbreak tracing, insights into phylogeography and epidemic evolutionary history. We illustrate these advantages by focusing on a common single lineage of M. bovis (1.140) from Northern Ireland. Specifically, we investigate the spatial sub-structure of 20 years of herd-level multi locus VNTR analysis (MLVA) surveillance data and WGS data from a down sampled subset of isolates of this MLVA type over the same time frame. We mapped 2108 isolate locations of MLVA type 1.140 over the years 2000-2022. We also mapped the locations of 148 contemporary WGS isolates from this lineage, over a similar geographic range, stratifying by single nucleotide polymorphism (SNP) relatedness cut-offs of 15 SNPs. We determined a putative core range for the 1.140 MLVA type and SNP-defined sequence clusters using a 50 % kernel density estimate, using cattle movement data to inform on likely sources of WGS isolates found outside of core ranges. Finally, we applied Bayesian phylogenetic methods to investigate past population history and reproductive number of the 1.140 M. bovis lineage. We demonstrate that WGS SNP-defined clusters exhibit smaller core ranges than the established MLVA type - facilitating superior disease tracing. We also demonstrate the superior functionality of WGS data in determining how this lineage was disseminated across the landscape, likely via cattle movement and to infer how its effective population size and reproductive number has been in flux since its emergence. These initial findings highlight the potential of WGS data for routine monitoring of bTB outbreaks.

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.

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.

Genetic Structure and TALome Analysis Highlight a High Level of Diversity in Burkinabe Xanthomonas Oryzae pv. oryzae Populations

Bacterial Leaf Blight of rice (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major threat for food security in many rice growing countries including Burkina Faso, where the disease was first reported in the 1980’s. In line with the intensification of rice cultivation in West-Africa, BLB incidence has been rising for the last 15 years. West-African strains of Xoo differ from their Asian counterparts as they (i) are genetically distant, (ii) belong to new races and, (iii) contain reduced repertoires of Transcription Activator Like (TAL) effector genes. In order to investigate the evolutionary dynamics of Xoo populations in Burkina Faso, 177 strains were collected from 2003 to 2018 in three regions where BLB is occurring. Multilocus VNTR Analysis (MLVA-14) targeting 10 polymorphic loci discriminated 24 haplotypes and showed that Xoo populations were structured according to their geographical localization and year of collection. Considering their major role in Xoo pathogenicity, we assessed the TAL effector repertoires of the 177 strains upon RFLP-based profiling. Surprisingly, an important diversity was revealed with up to eight different RFLP patterns. Finally, comparing neutral vs. tal effector gene diversity allowed to suggest scenarios underlying the evolutionary dynamics of Xoo populations in Burkina Faso, which is key to rationally guide the deployment of durably resistant rice varieties against BLB in the country.

Controlled Experimental Infection in Pigs with a Strain of Yersinia enterocolitica Harboring Genetic Markers for Human Pathogenicity: Colonization and Stability.

Yersinia enterocolitica (Ye) is one of the major causes of foodborne zoonosis. The BT4/O:3 bioserotype is most commonly isolated in human infections. Pigs are considered the main reservoir of Ye, and hence, understanding the dynamics of infection by this pathogen at the individual and group levels is crucial. In the present study, an experimental model was validated in Large White pigs infected with a BT4/O:3 strain. This study showed that Ye contamination in pigs may occur via the introduction of the bacteria not only by mouth but also by snout, with a colonization process consisting of three periods corresponding to three contamination statuses of pigs: P1, corresponding to the 24 h following ingestion or inhalation of Ye with the appearance of bacteria in tonsils or in feces; P2, from 2 days postinoculation (dpi), corresponding to expansion of Ye and colonization of the digestive system and extraintestinal organs associated with an IgG serological response; and P3, after 21 dpi, corresponding to regression of colonization with intermittent Ye detection in tonsils and feces. Although the inoculated strain persisted up to 56 dpi in all pigs, genetic variations with the loss of the gene yadA (a gene involved in human infection) and the emergence of two new multilocus variable-number tandem-repeat analysis (MLVA) profiles were observed in 33% of the 30 isolates studied. This experimental infection model of pigs by Ye provides new insights into the colonization steps in pigs in terms of bacterial distribution over time and bacterial genetic stability.

The clinical significance of macrolide resistance in pediatric Mycoplasma pneumoniae infection during COVID-19 pandemic.

Mycoplasma pneumoniae (MP) is a commonly occurring pathogen causing community-acquired pneumonia (CAP) in children. The global prevalence of macrolide-resistant MP (MRMP) infection, especially in Asian regions, is increasing rapidly. However, the prevalence of MRMP and its clinical significance during the COVID-19 pandemic is not clear. This study enrolled children with molecularly confirmed macrolide-susceptible MP (MSMP) and MRMP CAP from Beijing Children's Hospital Baoding Hospital, Capital Medical University between August 2021 and July 2022. The clinical characteristics, laboratory findings, chest imaging presentations, and strain genotypes were compared between patients with MSMP and MRMP CAP. A total of 520 hospitalized children with MP-CAP were enrolled in the study, with a macrolide resistance rate of 92.7%. Patients with MRMP infection exhibited more severe clinical manifestations (such as dyspnea and pleural effusion) and had a longer hospital stay than the MSMP group. Furthermore, abnormal blood test results (including increased LDH and D-dimer) were more common in the MRMP group (P<0.05). Multilocus variable-number tandem-repeat analysis (MLVA) was performed on 304 samples based on four loci (Mpn13-16), and M3562 and M4572 were the major types, accounting for 74.0% and 16.8% of the strains, respectively. The macrolide resistance rate of M3562 strains was up to 95.1%. The prevalence of MRMP strains in hospitalized CAP patients was extremely high in the Baoding area, and patients infected with MRMP strains exhibited more severe clinical features and increased LDH and D-dimer. M3562 was the predominant resistant clone.

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.

Multilocus VNTR analysis-ompA typing of Chlamydia trachomatis isolates in Tehran, Iran

IntroductionThis study is the first to describe the genetic diversity of C. trachomatis strains derived from patients with signs and symptoms of genitourinary infections admitted to Tehran health centers and hospitals using the high-resolution genotyping method, multilocus variable-number tandem-repeat analysis with ompA sequencing (MLVA)-ompA. MethodsOne hundred and sixty-seven urogenital specimens were collected from October 2019 to July 2020. Specimens were inoculated to cell culture and examined for the presence of C. trachomatis isolates by microscopic valuation. Out of 167 samples, 19 (11.3%) viable C. trachomatis organisms were isolated in cell culture. Eighteen isolates were successfully genotyped by MLVA-ompA analysis. ResultsThe most prevalent ompA genotypes were E, D, F and G, comprising 42%, 26.3% and 21% and 10.5% of isolates, respectively. Other genotypes were not detected from any of the samples. Out of the 18 fully genotyped isolates, 10 different MLVA-ompA genotypes were obtained. The most prevalent MLVA-ompA genotypes were 8.6.1-E (33.3%) and 8.5.2-D (16.6%). Genotype 8.6.1-E was common in both females and males. ConclusionsOur results showed that MLVA-ompA analysis was more discriminatory than ompA typing alone and, therefore, a suitable complement to ompA. Using this method, dominant genotypes in the community and transmission patterns in sexual networks could be identified. The high diversity of C. trachomatis strains in Tehran may be due to the low level of public health and awareness, and future studies are needed.

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.

Investigation of antimicrobial resistance patterns and molecular typing of Pseudomonas aeruginosa isolates among Coronavirus disease-19 patients

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

MLVA genotyping of Moritella viscosa reveals serial emergence of novel, host-specific clonal complexes in Norwegian salmon farming.

A Multi-Locus Variable number of tandem repeat Analysis (MLVA) genotyping scheme was developed for the epidemiological study of Moritella viscosa, which causes 'winter ulcer' predominantly in sea-reared Atlantic salmon (Salmo salar L.). The assay involves multiplex PCR amplification of six Variable Number of Tandem Repeat (VNTR) loci, followed by capillary electrophoresis and data interpretation. A collection of 747 spatiotemporally diverse M. viscosa isolates from nine fish species was analysed, the majority from farmed Norwegian salmon. MLVA distributed 76% of the isolates across three major clonal complexes (CC1, CC2 and CC3), with the remaining forming minor clusters and singletons. While 90% of the salmon isolates belong to either CC1, CC2 or CC3, only 20% of the isolates recovered from other fish species do so, indicating a considerable degree of host specificity. We further highlight a series of 'clonal shifts' amongst Norwegian salmon isolates over the 35-year sampling period, with CC1 showing exclusive predominance prior to the emergence of CC2, which was later supplanted by CC3, before the recent re-emergence of CC1. Apparently, these shifts have rapidly swept the entire Norwegian coastline and conceivably, as suggested by typing of a small number of non-Norwegian isolates, the Northeast Atlantic region as a whole.

Improvement and Validation of a Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA8+) for Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae

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

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.

The Association between Mycoplasma pneumoniae Genotype and Cutaneous Disease.

Mycoplasma pneumoniae (Mp) can cause several extrapulmonary manifestations, most frequently dermatological ones. It is largely unknown whether Mp genotype determines Mp-induced cutaneous disease. The aim of our study was to assess the association between Mp genotype and this clinical outcome. We performed a retrospective study of children referred with signs of acute Mp infection from 1 January 2014 to 31 December 2014. We compared the characteristics of children presenting as cutaneous disease, upper (URTI) and lower respiratory tract infection (LRTI). In addition, we separately analyzed the data of patients presenting with Mp-induced cutaneous disease. We evaluated data from 435 patients (mean age 7.3 years, SD 3.4 years; 52.0% boys) who had Mp PCR-positive pharyngeal swab, P1 genotype and/or multilocus variable-number tandem-repeat analysis (MLVA) genotype defined and no viral co-detection, presenting as cutaneous disease (38/435), URTI (46/435) or LRTI (351/435). The majority of patients had urticarial (55%, 21/38) or maculopapular eruptions (37%, 14/38). We found no association between Mp genotype and clinical outcome of cutaneous disease, nor any specific dermatological presentation. In the group with cutaneous disease, 18% (7/38) required hospital admission because of rash. We found that infection with MLVA-3,6,6,2 strains was more common in admitted patients than in outpatients (40% vs. 4%, p = 0.017) and significantly affected the likelihood of hospital admission in a logistic regression model. The results of our cohort study suggest that Mp genotype does not determine Mp-induced cutaneous disease or a specific dermatological presentation. Nevertheless, infections with certain MLVA strains could induce more severe cutaneous disease requiring hospitalization.