Multilocus Variable-number Tandem-repeat Analysis Research Articles

Genomic epizootiology of a Brucella abortus outbreak in Northern Ireland (1997–2012)

BackgroundIn the recent past (1997–2012), Northern Ireland in the United Kingdom suffered an outbreak of Brucella abortus, which at its height affected over 200 cattle herds. Initially, isolates were characterized using multi-locus variable number tandem repeats analysis (MLVA). While informative in this setting, hyper-variability in some loci limited the resolution necessary to infer fine-scale disease transmission networks. Consequently, we applied whole-genome sequencing to isolates from this outbreak to evaluate higher resolution markers for disease epizootiology. ResultsPhylogenetic analysis revealed that the B. abortus outbreak in Northern Ireland was caused by two distinct pathogen lineages. One contained isolates consistent with the 1997–2012 outbreak being linked to a previous endemic infection thought eradicated. The dominant second lineage exhibited little genetic diversity throughout the recrudescent outbreak, with limited population sub-structure evident. This finding was inconsistent with prior MLVA molecular characterizations that suggested the presence of seven clonal complexes. Spatio-temporal modeling revealed a significant association of pairwise SNP differences between isolates and geographic distances. However, effect sizes were very small due to reduced pathogen diversity. ConclusionsGenome sequence data suggested that hyper-variability in some MLVA loci contributed to an overestimate of pathogen diversity in the most recent outbreak. The low diversity observed in our genomic dataset made it inappropriate to apply phylodynamic methods to these data. We conclude that maintaining data repositories of genome sequence data will be invaluable for source attribution/epizootiological inference should recrudescence ever re-occur. However genomic epizootiological methods may have limited utility in some settings, such as when applied to recrudescent/re-emergent infections of slowly-evolving bacterial pathogens.

Evaluation of genetic diversity among aquatic and fecal isolates of Escherichia coli using multilocus variable number of tandem repeat analysis.

In developing countries like India, fecal pollution of surface waters is a major threat to public and environmental health. The aim of the study was to assess serological, phylogenetic and molecular diversity among aquatic Escherichia coli isolates from Yamuna river and their comparison with the animal fecal isolates. A total of 97 E. coli isolates from Yamuna river and domesticated animals were characterized by multilocus variable number tandem repeat analysis (MLVA) using four VNTR loci. The pathogenicity of these strains by serological and phylogenetic analysis was also determined. E. coli strains were differentiated into 53 distinct MLVA types with high discriminatory power, Simpson's index of 0.95 (95% CI 0.923-0.978). Cluster analysis and population modeling using minimum spanning tree suggested a possible epidemiological linkage among aquatic and fecal isolates. The study also reported the presence of highly diverse and pathogenic serotypes belonging to STEC and EPEC strains, particularly O157 and high prevalence of pathogenic phylogroups (phylogroup, B2 and D). The presence of such a high molecular heterogeneity among aquatic and fecal E. coli isolates emphasizes upon the need to develop proper fecal pollution abatement strategies for Indian natural bodies.

Distribution of Genes Encoding Virulence Factors and the Genetic Diversity of Enteroinvasive Escherichia coli (EIEC) Isolates from Patients with Diarrhea in Ahvaz, Iran.

BackgroundEntero-invasive E. coli (EIEC) is one of the causes of bacillary dysentery in adults and children. The ability of EIEC to invade and colonize the surface of epithelial cells is influenced by many virulence factors. This study aimed to investigate the distribution of virulence factor genes in EIEC strains isolated from patients with diarrhea in Ahvaz, Iran, as well as the genetic diversity between these isolates by Multilocus variable-number tandem repeat analysis (MLVA).Materials and MethodsA total of 581 diarrheic stool samples were collected from patients with diarrhea attending two hospitals, in Ahvaz, Iran. The E. coli strains were identified by biochemical methods. Subsequently, all E. coli isolates were identified as EIEC by polymerase chain reaction (PCR) for the ipaH gene. The EIEC isolates evaluated by PCR for the presence of 8 virulence genes (ial, sen, virF, invE, sat, sigA, pic, and sepA). All EIEC strains were genotyped by the MLVA typing method.ResultsA total of 13 EIEC isolates were identified. The presence of ial, virF, invE, sen, sigA, pic, and sat genes was confirmed among 92.3%, 84.6%, 84.6%, 76.9%, 69.2%, and 15.3% of EIEC isolates, respectively. On the other hand, none of the isolates were positive for the sepA gene. The EIEC isolates were divided into 11 MLVA types.ConclusionOur results showed a high distribution of virulence genes among EIEC isolates in our region. This study showed that MLVA is a promising typing technique for epidemiological studies. MLVA can supply data in the form of codes that can be saved in the database and easily shared among laboratories, research institutes, and even hospitals.

Current advances in molecular subtyping using multilocus variable number of tandem repeat analysis of Salmonella Enteritidis and Salmonella Typhimurium in Egyptian chickens.

Aim:This study aimed to characterize the genetic diversity, evolutionary level, and prevalence of genotypes of common isolates of Salmonella (Salmonella Enteritidis and Salmonella Typhimurium). Using one of the most advanced molecular recognition techniques, multilocus variable number of tandem repeat analysis (MLVA), we characterized the genotype and prevalence of S. Enteritidis and S. Typhimurium.Materials and Methods:One hundred and twenty-five internal organ samples were collected from the major chicken slaughterhouses in Egypt, and Salmonella species were isolated. PCR was utilized to amplify the IE-1 and Flic-C genes to identify S. Enteritidis and S. Typhimurium DNA, respectively, from Salmonella isolates. MLVA was applied on nine samples of S. Enteritidis DNA and three samples of S. Typhimurium DNA. Six variable number tandem repeat (VNTR) loci (Sal02, Sal04, Sal06, Sal10, Sal20, and Sal23) were amplified.Results:Of the examined samples (n=125), a total of 12 isolates (9.6%) were either identified as Enteritidis or Typhimurium. PCR-mediated amplification of IE-1 and Flic-C revealed that 75% (n=9) of the 12 Salmonella isolates were S. Enteritidis and 25% (n=3) were S. Typhimurium. The six loci amplified through MLVA had allelic diversity. The most discriminatory heterogenic locus for S. Enteritidis was Sal20. Sal04 and Sal23 were the most discriminatory heterogenic loci for S. Typhimurium. VNTR allelic profile analysis revealed nine unique genotypes for S. Enteritidis and three for S. Typhimurium.Conclusion:This study was the first to use MLVA analysis to identify S. Enteritidis and S. Typhimurium strains isolated from chickens in Egypt. The molecular typing data reported herein allowed us to characterize the genotypes of S. Enteritidis and S. Typhimurium that are most prevalent in Egyptian chickens. Moreover, this epidemiological information provides valuable insight on how to prevent disease transmission. Moreover, our methods provide an alternative to traditional serotyping techniques that may produce inaccurate strain identifications for organisms with rough lipopolysaccharide structures.

Frequency and molecular epidemiology of class A ESBLs producing Enteroinvasive Escherichia coli (EIEC) isolates among patients with diarrhea.

This study aimed to investigate the frequency and molecular epidemiology of class A ESBLs producing Enteroinvasive Escherichia coli (EIEC) isolates among patients with diarrhea. Antibiotic resistance is widespread among diarrheagenic Escherichia coli (DEC) in developing countries. Information regarding Extended-Spectrum β-Lactamases (ESBLs) in diarrheagenic pathogens should be considered in clinical management when an optimal treatment is required. A total of 581 stool samples were collected from patients with diarrhea in Ahvaz, Iran. PCR was used for the presence of the ipaH gene to confirm EIEC strains. The antibiotic resistance pattern of all EIEC isolates was determined by the disk diffusion method. EIEC isolates were screened for class A β-lactamase genes. Genotyping of harboring β-lactamase genes was performed by Multi-Locus VNTR Analysis (MLVA). Among 13 EIEC isolates, 9 isolates (69.2%) were found ESBL positive by double-disk synergy test (DDST) and PCR. Furthermore, bla CTX-M-15 and bla CTX-M-1 genes were detected in 77.8% (n=7) and 44.5% (n=4) of the bla CTX-M-1 group. On the other hand, the bla TEM-1 gene was detected in 66.6% (n=6). None of the isolates had bla SHV-1, bla KPC, or bla GES genes. Six MLVA genotypes were identified. The current study revealed that the presence of ESBLs genes mediates the resistance of EIEC isolates to the majority of antibiotics in this region. The presence of ESBLs genes in different MLVA types showed that one specific clone was not responsible for spreading the EIEC isolates.

Diversity in Genotype Distribution of Mycoplasma pneumoniae Obtained from Children and Adults.

The aim of this study was to explore whether there was any specific genotype responsible for the high prevalence of Mycoplasma pneumoniae infection in children. A total of 247 M. pneumoniae-DNA positive clinical specimens including 200 from children and 47 from adults, collected in Beijing, China, during the same period, were analyzed. We performed P1-restriction fragment length polymorphism analysis (RFLP), multi-locus variable number tandem repeat analysis (MLVA) and detected the macrolide resistance-associated mutations in 23S rRNA of the clinical specimens. In the present study, we observed P1 genotype 1 and MLVA type M4-5-7-2 accounted for the majority of the cases across all ages in Beijing. Macrolide resistance-associated mutants of M. pneumoniae were also at a high level with 90.5% (181/200) in children and 76.6% (36/47) in adults. However, more diverse genotypes and a higher prevalence of macrolide resistance-associated mutations were found in the pediatric specimens. Further investigations are warranted to help to explain the difference of morbidity and molecular characteristics across the demographic spectrum.

New Genotypes of Coxiella burnetii Circulating in Brazil and Argentina.

Coxiella burnetii, the zoonotic agent of Q fever, has a worldwide distribution. Despite the vast information about the circulating genotypes in Europe and North America, there is a lack of data regarding C. burnetii strains in South America. Here, we show the presence of novel multispacer sequence typing (MST) genotypes of C. burnetii in two clusters detected in Brazil and Argentina that seem to be distant in parenthood. Argentinian strains isolated from a tick belongs to a new phylogenetic branch of C. burnetii, and the Brazilians strains may be related to MST 20 and 61. Multilocus variable number tandem repeats analysis (MLVA) typing provided a deeper resolution that may be related to host clusters of bovines, caprine, ovine, and ticks. Our results corroborate with the reports of geotypes of C. burnetii. Thus, we highlight the need for more genotyping studies to understand the genetic diversity of C. burnetii in South America and to confirm the hypothesis of host-related genotypes. We also emphasize the importance of virulence studies for a better understanding of Q fever in the region, which may help in surveillance and disease prevention programs.

Phylogenetic Placement of Isolates Within the Trans-Eurasian Clade A.Br.008/009 of Bacillus anthracis.

The largest phylogenetic lineage known to date of the anthrax pathogen Bacillus anthracis is the wide-spread, so-called Trans-Eurasian clade systematically categorized as the A.Br.008/009 group sharing two defining canonical single-nucleotide polymorphisms (canSNP). In this study, we genome-sequenced a collection of 35 B. anthracis strains of this clade, derived from human infections, animal outbreaks or soil, mostly from European countries isolated between 1936 and 2008. The new data were subjected to comparative chromosomal analysis, together with 75 B. anthracis genomes available in public databases, and the relative placements of these isolates were determined within the global phylogeny of the A.Br.008/009 canSNP group. From this analysis, we have detected 3754 chromosomal SNPs, allowing the assignation of the new chromosomal sequences to established sub-clades, to define new sub-clades, such as two new Spanish, one Bulgarian or one German group(s), or to introduce orphan lineages. SNP-based results were compared with that of a multilocus variable number of tandem repeat analysis (MLVA). This analysis indicated that MLVA typing might provide additional information in cases when genomics yields identical genotypes or shows only minor differences. Introducing the delayed mismatch amplification assay (DMAA) PCR-analysis, we developed a cost-effective method to interrogate for a set of ten phylogenetically informative SNPs within genomes of A.Br.008/009 canSNP clade strains of B. anthracis. By this approach, additional 32 strains could be assigned to five of ten defined clades.

High Prevalence of Macrolide-Resistant Bordetella pertussis and ptxP1 Genotype, Mainland China, 2014-2016.

According to the government of China, reported cases of pertussis have increased remarkably and are still increasing. To determine the genetic relatedness of Bordetella pertussis strains, we compared multilocus variable-number tandem-repeat analysis (MLVA) results for isolates from China with those from Western countries. Among 335 isolates from China, the most common virulence-associated genotype was ptxA1/ptxC1/ptxP1/prn1/fim2–1/fim3A/tcfA2, which was more frequent among isolates from northern than southern China. Isolates of this genotype were highly resistant to erythromycin. We identified 36 ptxP3 strains mainly harboring ptxA1 and prn2 (35/36); ptxP3 strains were sensitive to erythromycin and were less frequently from northern China. For all isolates, the sulfamethoxazole/trimethoprim MIC was low, indicating that this drug should be recommended for patients infected with erythromycin-resistant B. pertussis. MLVA of 150 clinical isolates identified 13 MLVA types, including 3 predominant types. Our results show that isolates circulating in China differ from those in Western countries.

Longitudinal dynamics of herd-level Mycobacterium bovis MLVA type surveillance in cattle in Northern Ireland 2003–2016

Investigating genetically-structured diversity in pathogen populations over time is important to better understand disease maintenance and spread. Herd-level surveillance of Mycobacterium bovis genotypes (multi-locus VNTR analysis types, MLVA types) from all culture-confirmed bovine tuberculosis (TB) herd cases was undertaken in Northern Ireland (NI), generating an unparalleled, longitudinal, population-level 14-year survey for this pathogen.Across this population, 295 genetically-distinct M. bovis MLVA types were identified in the 19,717 M. bovis isolates surveyed. Of these, the most frequent was MLVA type 002 (23.0%); 151 MLVA types were represented more than once, in groups ranging from 2 to 4438 isolates. Only 23 MLVA types were isolated in all 14 years. Investigating inter-annual frequency of M. bovis MLVA types, examples of statistically-significant expansions (MLVA types 002, 004, 006, 009 and 027), contractions (MLVA types 001, 007 and 011) and maintenance (MLVA types 003 and 005) were disclosed, during a period of fluctuating bovine TB herd-level incidence at the NI scale.The fixed period frequency distribution of MLVA types remained highly right-skewed. Novel VNTR copy number variant MLVA types (N = 242; an average of 17 per annum) were identified throughout the survey. The MLVA type distribution in the landscape was not random; MLVA types showed statistically-significant geographical localization and strong spatial associations with Divisional Veterinary Office (DVO) regions. There was also evidence of differential risk of particular MLVA types across breeds (Holstein/Friesian vs. other), age-class, and sex and some evidence of an association between the number of animals testing positive for bovine TB during the disclosing test and particular MLVA types, although there was substantial variation.

Investigation of the molecular epizootiological characteristics and tracking of the geographical origins of Brucella canis strains in China.

Brucellosis is a global pandemic infectious zoonosis. Brucella canis is a rare source of human brucellosis in China, and its public health significance remains under debate. Moreover, data pertaining to the epizootiological characteristics and geographical origin of B.canis on a nationwide scale are limited, and the risk to public safety posed by B.canis infections is unknown. The MLVA (multilocus variable-number tandem repeat analysis) assay can be helpful to analyse epidemiological correlations among Brucella isolates and to track their geographic origins. To accomplish this task, MLVA-16 was used to analyse the epidemiological links of 63 isolates obtained from dogs and humans. Sixty-three B.canis strains were sorted into three large clusters (A, B and C) and 50 different genotypes (GT1-50), and 43 unique genotypes were represented by single isolates, suggesting that these strains had no obvious epidemiological links and that canine brucellosis is predominantly sporadic in China. The other seven shared genotypes (among a total of 20 isolates) were each represented by two to eight isolates, indicating that strains from each shared genotype were epidemiologically correlated. Five of the shared genotypes were from 16 strains obtained from Beijing, indicating that canine brucellosis in Beijing originates from multipoint outbreaks with multiple sources of infection. Based on comprehensive case analysis of clinical B.canis infection, we preliminarily suggest that human B.canis infections are associated with Mycoplasma pneumoniae infection that results in decreased patient immunity. B.canis may have limited epidemiological significance for the healthy population, but it remains a significant threat to the canine breeding industry and to humans who come into close contact with dogs. Based on MLVA-11 data, B.canis strains were clustered into 16 genotypes and divided into five evolutionary branches; these data confirm that this population covers an extensive geographic area and exhibits characteristics of the origin and evolution of co-existing introduced and locally native lineages. We believe this study will contribute to strengthening efforts to prevent and control canine brucellosis and to improve public understanding of the health risks posed by B.canis.

Multilocus variable number tandem repeat analysis (MLVA)-typing of Brucella abortus isolates of India reveals limited genetic diversity.

Multilocus variable number tandem repeat analysis (MLVA) technique has wide applications in studying phylogenies and short-term epidemiology of pathogens. The technique has been extensively used worldwide in molecular epidemiology of Brucella genus. Only one study on this aspect is reported from India despite its economic and public health significance in country. The present study isolated B. abortus from domesticated bovines of Jammu region of Jammu and Kashmir state, India, and applied MLVA for 16 loci (MLVA-16). MLVA results were compared with the results of a previous study and with MLVA data of Indian isolates present in http://microbesgenotyping.i2bc.paris-saclay.fr/database. In the study, 136 samples from bovines (cattle and buffaloes) of 47 farms of Jammu region were processed for isolation. Eleven isolates of B. abortus biovar 1 from 6 farms were obtained. In MLVA-16 analysis, although the isolates were classified in a single cluster, 5 genotypes were obtained with a specific genotype being prevalent on each farm. The study identifies that MLVA-16 is capable to differentiate B. abortus strains in an area having high genetic similarity among isolates. On comparing the results with previous study and database, the isolates were found to have high genetic similarity indicating that the genetic diversity of B. abortus in India is very limited. It probably indicates that India is contaminated recently with B. abortus. To test this hypothesis, analysis of whole genome sequencing data of diverse collection of Indian B. abortus strains is required.

Enhancing genomics-based outbreak detection of endemic Salmonella enterica serovar Typhimurium using dynamic thresholds.

Salmonella enterica serovar Typhimurium is the leading cause of salmonellosis in Australia, and the ability to identify outbreaks and their sources is vital to public health. Here, we examined the utility of whole-genome sequencing (WGS), including complete genome sequencing with Oxford Nanopore technologies, in examining 105 isolates from an endemic multi-locus variable number tandem repeat analysis (MLVA) type over 5 years. The MLVA type was very homogeneous, with 90 % of the isolates falling into groups with a five SNP cut-off. We developed a new two-step approach for outbreak detection using WGS. The first clustering at a zero single nucleotide polymorphism (SNP) cut-off was used to detect outbreak clusters that each occurred within a 4 week window and then a second clustering with dynamically increased SNP cut-offs were used to generate outbreak investigation clusters capable of identifying all outbreak cases. This approach offered optimal specificity and sensitivity for outbreak detection and investigation, in particular of those caused by endemic MLVA types or clones with low genetic diversity. We further showed that inclusion of complete genome sequences detected no additional mutational events for genomic outbreak surveillance. Phylogenetic analysis found that the MLVA type was likely to have been derived recently from a single source that persisted over 5 years, and seeded numerous sporadic infections and outbreaks. Our findings suggest that SNP cut-offs for outbreak cluster detection and public-health surveillance should be based on the local diversity of the relevant strains over time. These findings have general applicability to outbreak detection of bacterial pathogens.

Molecular characterization of Mycoplasma pneumoniae infections in Moscow from 2015 to 2018

The aim of this study was to assess which Mycoplasma pneumoniae genotypes were present in Moscow during the years 2015-2018 and whether the proportion between detected genotypes changed over time. We were also interested in the presence of macrolide resistance (MR)Mycoplasma pneumoniae. We performed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), SNP typing, and mutation typing in the 23S rRNA gene from 117 M. pneumoniae clinical isolates. Our analysis suggests two major MLVA types: 4572 and 3562. In 2017-2018, MLVA type 4572 gradually became predominant. In general, the SNP type range is the same as described earlier for European countries. The analysis of MR mutations showed that 7% of the isolates had an A2063G mutation in the 23S rRNA gene with no isolates carrying an A2064G mutation. In 2017-2018, MLVA type 4572 (SNP type 1) begins to spread in Moscow, which was widespread globally, especially in Asian countries. SNP typing of our sample showed higher discriminatory power than MLVA typing.

MAC-INMV-SSR: a web application dedicated to genotyping members of Mycobacterium avium complex (MAC) including Mycobacterium avium subsp. paratuberculosis strains

Genotyping of Mycobacterium avium subsp. paratuberculosis (Map) is an indispensable tool for surveillance of this significant veterinary pathogen. For Map, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRUs) and other variable number variable-number tandem repeats (VNTRs) was established using 8 markers. In the recent past this standard, portable, reproducible and discriminatory typing method has been frequently applied alone or in combinations with multi-locus short-sequence-repeat (MLSSR) sequencing. With the widespread use of these genotyping methods, standardization between laboratories needs to be managed, and knowledge of existing profiles and newly defined genotypes should be indexed and shared. To meet this need, a web application called “MAC-INMV-SSR database” was developed. This freely accessible service allows users to compare MLVA and MLSSR subtype data of their strains with those of existing reference strains analyzed with the same genotyping methods.

Supporting evidence for a human reservoir of invasive non-Typhoidal Salmonella from household samples in Burkina Faso.

BackgroundSalmonella Typhimurium and Enteritidis are major causes of bloodstream infection in children in sub-Saharan Africa. This study assessed evidence for their zoonotic versus human reservoir.MethodsIndex patients were children with blood culture confirmed Salmonella infection recruited during a microbiological surveillance study in Nanoro, rural Burkina between May 2013 and August 2014. After consent, their households were visited. Stool from household members and livestock (pooled samples per species) as well as drinking water were cultured for Salmonella. Isolates with identical serotype obtained from index patient and any household sample were defined as “paired isolates” and assessed for genetic relatedness by multilocus variable number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS).ResultsTwenty-nine households were visited for 32/42 (76.2%) eligible index patients: two households comprised two index patients each, and in a third household the index patient had a recurrent infection. Among the 32 index patients, serotypes were Salmonella Typhimurium (n = 26), Salmonella Enteritidis (n = 5) and Salmonella Freetown (n = 1). All Typhimurium isolates were sequence type (ST)313. Median delay between blood culture sampling and household visits was 13 days (range 6–26). Salmonella was obtained from 16/186 (8.6%) livestock samples (13 serotypes) and 18/290 (6.2%) household members (9 serotypes). None of the water samples yielded Salmonella. Paired Salmonella Typhimurium isolates were obtained from three households representing four index patients. MLVA types were identical in two pairs and similar in the third (consisting of two index patients and one household member). WGS showed a strong genetic relatedness with 0 to 2 core genome SNPs difference between pairs on a household level. Livestock samples did not yield any Salmonella Typhimurium or Salmonella Enteritidis, and the latter was exclusively obtained from blood culture. Other serotypes shared by human and/or livestock carriers in the same household were Salmonella Derby, Drac, Tennessee and Muenster.Conclusions/SignificanceThe current study provides further evidence of a human reservoir for invasive non-Typhoidal Salmonella (iNTS) in sub-Saharan Africa.

In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness

Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.

Linked seasonal outbreaks of Salmonella Typhimurium among passerine birds, domestic cats and humans, Sweden, 2009 to 2016.

In 2016, an outbreak of Salmonella Typhimurium (STm) with multilocus variable-number tandem repeat analysis (MLVA) profiles historically associated with passerine birds (2-[11-15]-[3-4]-NA-212) occurred among passerines, cats and humans in Sweden. Our retrospective observational study investigated the outbreak and revisited historical data from 2009–16 to identify seasonality, phylogeography and other characteristics of this STm variant. Outbreak isolates were analysed by whole-genome single nucleotide polymorphism (SNP) typing. The number of notified cases of passerine-associated STm among passerines, cats and humans per month and county, and their MLVA profiles, were compared to birdwatchers’ counts of passerines. Seasonal trend decomposition and correlation analysis was performed. Outbreak isolates did not cluster by host on SNP level. Passerine-associated STm was seasonal for birds, cats and humans, with a peak in March. Cases and counts of passerines at bird feeders varied between years. The incidence of passerine-associated STm infections in humans was higher in the boreal north compared with the southern and capital regions, consistent with passerine population densities. Seasonal mass migration of passerines appears to cause STm outbreaks among cats certain years in Sweden, most likely via predation on weakened birds. Outbreaks among humans can follow, presumably caused by contact with cats or environmental contamination.

A two-step approach for the investigation of a Clostridium difficile outbreak by molecular methods

Abstract The recent review “How to: molecular investigation of a hospital outbreak” written by Nutman and Marchaim published in Clinical Microbiology and Infection journal summarized data on typing methods used for a hospital outbreak investigation. Regarding Clostridium difficile infection (CDI), a whole genome sequencing (WGS) was recommended as the initial and a single molecular test for characterisation of C. difficile isolates in a suspected outbreak situation. According to results from our survey on typing capacity for C. difficile in Europe, whole genome sequencing was available only in five National reference laboratories for C. difficile in 2017 in Europe. Because of limited availability of WGS, we suggest a two-step approach that includes the typing of C. difficile isolates with a high discriminatory method, capillary electrophoresis ribotyping and the subtyping of C. difficile isolates by Multilocus Variable-number Tandem-repeats Analysis (MLVA) and/or WGS when a common cluster of CDIs is confirmed. This two-step approach would provide an epidemiological relevant timeframe for confirmation of a suspected CDI outbreak as well as being cost-effective by selecting highly related C. difficile isolates for further investigation.

Prevalence and Dynamics of Pathogenic Yersinia enterocolitica 4/O:3 Among Finnish Piglets, Fattening Pigs, and Sows.

Pigs are considered the main reservoir of Yersinia enterocolitica, and hence, understanding the ecology of this foodborne pathogen at the farm level is crucial. We calculated Bayesian estimates for the ability of a commercial enzyme-linked immunosorbent assay (ELISA) diagnostic test kit to detect antibodies against pathogenic Yersinia in pigs. The sensitivity and specificity of the test were 75.4% and 98.1%, respectively. We also studied the dynamics of Y. enterocolitica infection in 3 farrow-to-finish pig farms by following the same 30 pens of pigs through their lifetime from farrowing unit to slaughterhouse. Each farm was sampled 4 times, and 864 fecal and 730 serum samples were collected altogether. Pathogenic Y. enterocolitica 4/O:3 was isolated from 31.6% of the fecal samples by culturing, and Yersinia antibodies were detected in 38.2% of the serum samples with the commercial ELISA test. The pathogen was not isolated from farrowing units or all-in/all-out weaning units. However, in the weaning and fattening units using continuous management systems, the pathogen was isolated from every pen at some point of the study. After the pigs were transported into slaughterhouse, 150 tonsils were collected and 96.7% were positive by culturing. Among the strains isolated from feces and tonsils, 56 different genotypes of pathogenic Y. enterocolitica 4/O:3 were found by multilocus variable-number tandem-repeat analysis (MLVA). Finally, we collected tonsils of 266 sows from 115 farrowing farms, and Y. enterocolitica 4/O:3 was detected in 6.0% of the samples by the culture method, whereas 77.1% of the tonsils were serologically positive; the estimate for true seroprevalence was 95.8%. In conclusion, sows may not be the main source of Y. enterocolitica for piglets, although sows may still play a role in maintaining Y. enterocolitica in pig farms. Instead, pigs appear to get this foodborne pathogen mainly during the fattening period, especially if continuous management is applied.