Multilocus Variable-number Tandem-repeat Analysis Research Articles

Rapid and simple SNP genotyping for Bordetella pertussis epidemic strain MT27 based on a multiplexed single-base extension assay

Multilocus variable-number tandem repeat analysis (MLVA) is widely used for genotyping of Bordetella pertussis, the causative bacteria for pertussis. However, MLVA genotyping is losing its discriminate power because prevalence of the epidemic MT27 strain (MLVA-27) is increasing worldwide. To address this, we developed a single nucleotide polymorphism (SNP) genotyping method for MT27 based on multiplexed single-base extension (SBE) assay. A total of 237 MT27 isolates collected in Japan during 1999–2018 were genotyped and classified into ten SNP genotypes (SG1 to SG10) with a Simpson’s diversity index (DI) of 0.79 (95% CI 0.76–0.82). Temporal trends showed a marked increase in the genotypic diversity in the 2010s: Simpson’s DI was zero in 1999–2004, 0.16 in 2005–2009, 0.83 in 2010–2014, and 0.76 in 2015–2018. This indicates that the SNP genotyping is applicable to the recently circulating MT27 strain. Additionally, almost all outbreak-associated MT27 isolates were classified into the same SNP genotypes for each outbreak. Multiplexed SBE assay allows for rapid and simple genotyping, indicating that the SNP genotyping can potentially be a useful tool for subtyping the B. pertussis MT27 strain in routine surveillance and outbreak investigations.

Improved evaluation of the genetic variability of Brazilian strains of Erwinia psidii with newly developed microsatellite markers

AbstractErwinia psidii is the causal agent of bacterial blight of guava, and wilt and dieback of eucalypt in Brazil. Previous studies using repetitive sequence‐based PCR (rep‐PCR) revealed limited genetic diversity in E. psidii populations. Here, the draft genome sequence of the type strain IBSBF435 was probed for variable number of tandem repeats (VNTRs) that could be used to study E. psidii genetic diversity by multilocus variable number of tandem repeat analysis (MLVA). Eleven selected VNTRs were used to assess genetic relatedness among 108 E. psidii strains from guava (36) and eucalypt (72), of different geographic origins and years of collection. A total of 79 haplotypes were detected in the strain collection. The number of alleles per locus ranged from 3 to 15. A minimum spanning tree indicated the occurrence of nine clonal complexes, of which two contained only guava strains, five only eucalypt strains, and two contained guava and eucalypt strains. The three oldest strains collected from guava in the 1980s (IBSBF435, IBSBF454, and IBSBF493) exhibited distinct haplotypes. IBSBF435 was genetically more closely related to some eucalypt strains than to guava strains, which supports previously reported lack of host specificity. IBSBF454 grouped with guava strains more recently collected from different states, suggesting that it could have been disseminated to different Brazilian areas. The implication of the lack of differentiation between guava and eucalypt strains is discussed in relation to the detection of E. psidii affecting eucalypt more than 25 years after the first description of guava bacterial blight.

First report of macrolide-resistant and -susceptible Mycoplasma pneumoniae clinical strains isolated from a single case

ObjectivesThe aim of this study was to investigate the biological characteristics and effect of antibiotic treatment for different Mycoplasma pneumoniae isolates co-infecting the same patient. MethodsTwo throat swab specimens from a single patient, on the day of admission (Sp01) and discharge (Sp13), were liquid cultured and subcultured on agar medium to obtain M. pneumoniae monoclones. The 23S rRNA gene of 50 monoclones from specimens Sp01 and Sp13 were analysed. Real-time PCR assay was used for detection of mutations and genotyping. Two typical monoclones were isolated for antimicrobial susceptibility testing. ResultsGenotype 1 monoclones accounted for 70.8% (34/48) in Sp01 and 95.7% (44/46) in Sp13. All genotype 1 monoclones were of the 4-5-7-2 multilocus variable-number tandem-repeat analysis (MLVA) type, while all genotype 2 monoclones were 3-5-6-2 MLVA type. The genotype 1 monoclone, which harboured the A2063G mutation in 23S rRNA gene, was resistant to erythromycin and azithromycin in vitro, whilst genotype 2, which did not carry the mutation, was susceptible to macrolides. The proportion of macrolide-resistant M. pneumoniae monoclones in the specimen cultures collected rose from 70.8% to 95.7% at the time of discharge. ConclusionThis is the first report on the isolation of macrolide-resistant and -susceptible strains of M. pneumoniae from the same patient. After treatment, the proportion of macrolide-resistant M. pneumoniae increased, but the patient still carried viable macrolide-susceptible strains, meaning that the macrolide-susceptible strains did not disappear completely.

Retrospective Analysis of the Relationship between Two Anthrax Outbreaks in Kazakhstan Based on Genomic Data.

We present a retrospective analysis of strains from two anthrax outbreaks in western Kazakhstan in 2009. The outbreaks occurred during the same period and in the same area located close to main roads, favoring a single source of infection. However, multilocus variable-number tandem-repeat analysis (MLVA), canonical single-nucleotide polymorphism (CanSNP) analysis, and genome-wide analysis demonstrated that the outbreaks were not connected.

Contrasting genetic diversity and structure among Malagasy Ralstonia pseudosolanacearum phylotype I populations inferred from an optimized Multilocus Variable Number of Tandem Repeat Analysis scheme.

The Ralstonia solanacearum species complex (RSSC), composed of three species and four phylotypes, are globally distributed soil-borne bacteria with a very broad host range. In 2009, a devastating potato bacterial wilt outbreak was declared in the central highlands of Madagascar, which reduced the production of vegetable crops including potato, eggplant, tomato and pepper. A molecular epidemiology study of Malagasy RSSC strains carried out between 2013 and 2017 identified R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II). A previously published population biology analysis of phylotypes II and III using two MultiLocus Variable Number of Tandem Repeats Analysis (MLVA) schemes revealed an emergent epidemic phylotype II (sequevar 1) group and endemic phylotype III isolates. We developed an optimized MLVA scheme (RS1-MLVA14) to characterize phylotype I strains in Madagascar to understand their genetic diversity and structure. The collection included isolates from 16 fields of different Solanaceae species sampled in Analamanga and Itasy regions (highlands) in 2013 (123 strains) and in Atsinanana region (lowlands) in 2006 (25 strains). Thirty-one haplotypes were identified, two of them being particularly prevalent: MT007 (30.14%) and MT004 (16.44%) (sequevar 18). Genetic diversity analysis revealed a significant contrasting level of diversity according to elevation and sampling region. More diverse at low altitude than at high altitude, the Malagasy phylotype I isolates were structured in two clusters, probably resulting from different historical introductions. Interestingly, the most prevalent Malagasy phylotype I isolates were genetically distant from regional and worldwide isolates. In this work, we demonstrated that the RS1-MLVA14 scheme can resolve differences from regional to field scales and is thus suited for deciphering the epidemiology of phylotype I populations.

Multilocus Variable-Number Tandem-Repeat Analysis for Genotyping of Escherichia coli Strains Isolated from Hospital Wastewater, Tehran, Iran

Background:Escherichia coli is one of the most frequent causes of many common bacterial infections. As a potential reservoir, hospital wastewater is considered for the dissemination of bacterial pathogens such as E. coli. Therefore, research on hospital waste’s bacteria by low-cost, rapid and easy molecular typing methods such as multilocus variable-number tandem-repeat analysis (MLVA) can be helpful for the study of epidemics.Methods:E. coli strains were isolated from hospital wastewater sources in Tehran, Iran, over a 24-month sampling period (Jun 2014- Jun 2016) and identified by standard bacteriological methods. The diversity of repeated sequences of seven variable-number tandem-repeat (VNTR) loci was studied by MLVA method base on polymerase chain reaction (PCR).Results:Overall, 80 E. coli isolates were discriminated into 51 different genotypes. Analysis of the MLVA profiles using a minimum spanning tree (MST) algorithm showed two clonal complexes with 71 isolates and only nine isolates were stayed out of clonal complexes in the form of a singleton. High genotypic diversity was seen among E. coli strains isolated from hospital wastewaters; however, a large number of isolates showed a close genetic relationship.Conclusion:MLVA showed to be a rapid, inexpensive and useful tool for the analysis of the phylogenetic relationships between E. coli strains under the study.

Biogeography and Environmental Drivers of Legionella pneumophila Abundance and Genotype Composition Across the West Bank: Relevance of a Genotype-Based Ecology for Understanding Legionella Occurrence.

The West Bank can be considered as a high-risk area for Legionella prevalence in drinking water due to high ambient temperature, intermittent water supply, frequent pressure loss, and storage of drinking water in roof containers. To assess occurrence of Legionella species, especially L. pneumophila, in the drinking water of the West Bank, the drinking water distribution systems of eight hospitals were sampled over a period of 2.3 years covering the seasonal cycle and the major geographic regions. To gain insight into potential environmental drivers, a set of physico-chemical and microbiological parameters was recorded. Sampling included drinking water and biofilm analyzed by culture and PCR-based methods. Cultivation led to the isolation of 180 strains of L. pneumophila that were genotyped by Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). Surprisingly, the abundance of culturable L. pneumophila was low in drinking water of the sampling sites, with only three out of eight sites where Legionella was observed at all (range: 30–500 CFU/Liter). By contrast, biofilm and PCR-based analyses showed a higher prevalence. Statistical analyses with physico-chemical parameters revealed a decrease of L. pneumophila abundance for water and biofilm with increasing magnesium concentrations (>30 mg/L). MLVA-genotype analysis of the L. pneumophila isolates and their spatial distribution indicated three niches characterized by distinct physico-chemical parameters and inhabited by specific consortia of genotypes. This study provides novel insights into mechanisms shaping L. pneumophila populations and triggering their abundance leading to an understanding of their genotype-specific niches and ecology in support of improved prevention measures.

An Outbreak of Clostridium (Clostridioides) difficile Infections within an Acute and Long-Term Care Wards Due to Moxifloxacin-Resistant PCR Ribotype 176 Genotyped as PCR Ribotype 027 by a Commercial Assay.

We aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert® C. difficile/Epi assay by polymerase chain reaction (PCR) ribotyping, toxin genes’ detection and multi-locus variable number tandem repeats analysis (MLVA). The susceptibility to metronidazole, vancomycin and moxifloxacin was determined by agar dilution. In addition, the presence of Thr82Ile in the GyrA and a single nucleotide deletion at position (Δ117) in the tcdC gene were investigated. Between January 1 and June 30, 2016, of 114 CDIs, 75 cases were genotyped as presumptive PCR ribotype (RT) 027 infections using a commercial assay. C. difficile isolates cultured from presumptive RT027 stool samples belonged to RT176. These isolates carried genes for toxin A (tcdA), B (tcdB), binary (cdtA/B) and had Δ117 in the tcdC gene. Using MLVA, the 71/75 isolates clustered into two clonal complexes (CCs). Of these, 39 isolates (54.9%) were from patients hospitalized in acute care and 32 isolates (45.1%) were isolated from patients hospitalized in the long-term care department. All isolates were susceptible to metronidazole and vancomycin, and 105 isolates were resistant to moxifloxacin (92%) carrying Thr83Ile in the GyrA. An outbreak of RT176 CDIs, suspected as RT027, was recognized in a Slovakian hospital. In order to monitor the emergence and spread of RT027-variants, the identification of a presumptive RT027 CDI should be confirmed at a strain level by PCR ribotyping.

Genotyping of Equine Lawsonia intracellularis Sampled in Japan by Using Multilocus Variable-Number Tandem Repeat Analysis

The incidence of equine proliferative enteropathy, caused by Lawsonia intracellularis, is increasing around the world. To investigate the relationships of variable-number tandem repeat (VNTR) patterns with host species and clinical status in horses, multilocus VNTR analysis (MLVA) was applied to 98 L. intracellularis samples collected from horses, seven from pigs, seven from wildlife, one vaccine strain, and 17 public strains. The VNTR patterns were highly diverse: a total of 130 samples identified 99 distinct patterns, and the 98 horses were classified into 71 different patterns. A phylogenetic tree based on the MLVA showed three clusters: porcine, equine, and miscellaneous cluster. The equine cluster contained 46 horse samples, of which 42 (91.3%) were collected from two sampling areas. The MLVA could discriminate horse samples from pig, but the horse samples in the miscellaneous cluster could not be distinguished from wildlife samples. As for clinical data of the horses, the VNTR patterns were unrelated to horse age, clinical signs, and clinical outcomes. This study shows that VNTR patterns had no clear connection with equine clinical status, but the MLVA could be useful to investigate its epidemiological relationships, and interspecific transmission of L. intracellularis between horse and wildlife cannot be ruled out.

Molecular Characterization of Mycoplasma pneumoniae Isolates in the United States from 2012 to 2018

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae We collected 446 M. pneumoniae-positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required.

A molecular and epidemiological study of Vibrio cholerae isolates from cholera outbreaks in southern Ghana.

Cholera remains a major global public health threat and continuous emergence of new Vibrio cholerae strains is of major concern. We conducted a molecular epidemiological study to detect virulence markers and antimicrobial resistance patterns of V. cholerae isolates obtained from the 2012–2015 cholera outbreaks in Ghana. Archived clinical isolates obtained from the 2012, 2014 and 2015 cholera outbreaks in Ghana were revived by culture and subjected to microscopy, biochemical identification, serotyping, antibiotic susceptibility testing, molecular detection of distinct virulence factors and Multi-Locus Variable-Number of Tandem-Repeat Analysis (MLVA). Of 277 isolates analysed, 168 (60.6%) were confirmed to be V. cholerae and 109 (39.4%) isolates constituted other bacteria (Escherichia coli, Aeromonas sobria, Pseudomonas aeruginosa, Enterobacter cloacae and Enterococci faecalis). Serotyping the V. cholerae isolates identified 151 (89.9%) as Ogawa, 3 (1.8%) as Inaba and 14 (8.3%) as non-O1/O139 serogroup. The O1 serogroup isolates (154/168, 91.7%) carried the cholera toxin ctxB gene as detected by PCR. Additional virulence genes detected include zot, tcpA, ace, rtxC, toxR, rtxA, tcpP, hlyA and tagA. The most common and rare virulence factors detected among the isolates were rtxC (165 isolates) and tcpP (50 isolates) respectively. All isolates from 2014 and 2015 were multidrug resistant against the selected antibiotics. MLVA differentiated the isolates into 2 large unique clones A and B, with each predominating in a particular year. Spatial analysis showed clustering of most isolates at Ablekuma sub-district. Identification of several virulence genes among the two different genotypes of V. cholerae isolates and resistance to first- and second-line antibiotics, calls for scaleup of preventive strategies to reduce transmission, and strengthening of public health laboratories for rapid antimicrobial susceptibility testing to guide accurate treatment. Our findings support the current WHO licensed cholera vaccines which include both O1 Inaba and Ogawa serotypes.

Genetic diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000–2013

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants).Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59.SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates.

Genotyping of Salmon Gill Poxvirus Reveals One Main Predominant Lineage in Europe, Featuring Fjord- and Fish Farm-Specific Sub-Lineages.

Salmon gill poxvirus (SGPV) can cause serious gill disease in Atlantic salmon (Salmo salar L.) and represents a significant problem to aquaculture industries in Northern Europe. Here, a single-tube multi-locus variable-number tandem-repeat (VNTR) analysis (MLVA) genotyping assay, targeting eight VNTR loci, was developed for studying the epizootiology of SGPV. Through MLVA typing of SGPV positive samples from 180 farmed and wild Atlantic salmon in Northern Europe, the first molecular population study of this virus was undertaken. Comparison of resulting MLVA profiles by cluster analysis revealed considerable micro-diversity, while only a limited degree of specific clustering by country of origin could be observed, and no clustering relating to the severity of disease outbreaks. Phylogenetic analysis, based on genomic data from six SGPV specimens (three Norwegian, one Scottish, one Faroese and one Canadian), complemented and corroborated MLVA by pointing to a marked transatlantic divide in the species, with one main, relatively conserved, SGPV lineage as predominant in Europe. Within certain fjord systems and individual freshwater salmon smolt farms in Norway, however, discrete MLVA clustering patterns that prevailed over time were observed, likely reflecting local predominance of specific SGPV sub-lineages. MLVA typing was also used to refute two suspected instances of vertical SGPV transmission from salmon broodstock to offspring, and to confirm a failed disinfection attempt in one farm. These novel insights into the previously undocumented population structure of SGPV provide important clues, e.g., regarding the mechanisms underlying spread and recurrence of the virus amongst wild and farmed salmon populations, but so far no indications of more or less virulent SGPV sub-lineages have been found. The MLVA scheme represents a highly sensitive genotyping tool particularly well suited for illuminating SGPV infection routes, and adds to the relatively low number of MLVA protocols that have so far been published for viral species. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed within a single working day. Resulting MLVA profiles can be readily shared and compared across laboratories, facilitating rapid placement of samples in an international ezpizootiological context.

Molecular Characteristics of Burkholderia pseudomallei Collected From Humans in Hainan, China.

Melioidosis is a common infectious disease in Southeast Asia and Northern Australia. In Hainan, several cases have been reported, but no systematic study has yet been done on the molecular epidemiology profiles of the organism. An investigation of the molecular epidemiology links and population structure of Burkholderia pseudomallei would help to better understand the clonally of the isolates and differences among them. In this study, multilocus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) were applied to examine the epidemiological relatedness and population structure of 166 B. pseudomallei isolates obtained during 2002–2014 in Hainan, China. Both the MLVA_4 and MLST approaches had high discriminatory power for this population, with diversity indices of 0.9899 and 0.9457, respectively. However, the MLVA_4 assay showed a higher discriminatory power than the MLST approach, and a variable-number tandem repeat (VNTR3 933) found by the MLVA approach was the most useful in discriminating strains from this province. A total of 166 strains yielded 99 MLVA_4 genotypes, of which 34 genotypes were shared by 101 isolates, for a clustering rate of 60.8% (101/166), which suggested that some cases may have a common source. Additionally, 65 isolates showed distinct genotypes, indicating that more than 39.2% (65/166) of melioidosis cases in Hainan had epidemiologically unrelated or sporadic characteristics. The 166 isolates were resolved into 48 STs, of which five STs (ST55, -70, -46, -50, and -58) were here found to be predominant. Phylogenetic analysis of 116 isolates conducted using the eBURST v3 segregated the 48 STs into eight groups with ST50 as predicted founder, and 21 STs were found to be singletons, which suggest that the strains in the Hainan region represent a high diversity of ST clones, indicating that many B. pseudomallei clone groups are endemic to this region. Moreover, ST50 had 5 SLV, 7 DLV, 6 TLV, and 29 satellite STs and formed a radial expansion pattern, suggesting that the melioidosis epidemic in this study was mainly caused by the clonal expansion of ST 50. Phylogenetic analysis on global scale suggests that China’s isolates are closely related to isolates from Southeast Asia, particularly from Thailand and Malaysia.

MLVA fingerprinting of Brucella melitensis circulating among livestock and cases of sporadic human illness in Egypt.

Brucella melitensis is a serious public health threat, with human infection exhibiting acute febrile illness and chronic health problems. The present study investigated the genetic diversity and epidemiological links of the important zoonotic bacterium B.melitensis in Egypt using multilocus variable-number tandem repeat analysis (MLVA-16) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. A total of 118 isolates were identified as B.melitensis biovar 3 by classical biotyping and Bruce-ladder assay. Although B.melitensis is primarily associated with infection in sheep and goats, most of B.melitensis isolates in this study were obtained from secondary hosts (cattle, buffaloes, humans and a camel) suggesting cross-species adaptation of B.melitensis to large ruminants in Egypt. The MLVA-16 scheme competently discriminated 70 genotypes, with 51 genotypes represented by single isolates, and the remaining 19 genotypes were shared among 67 isolates, suggesting both sporadic and epidemiologically related characteristics of B.melitensis infection. Matching of local genotypes with representatives of global genotypes revealed that the majority of Egyptian isolates analysed had a West Mediterranean descendance. As this study represents the first comprehensive genotyping and genetic analysis of B.melitensis from different sources in Egypt, the information generated from this study will augment knowledge about the main epidemiological links associated with this bacterium and will allow a better understanding of the current epidemiological situation of brucellosis in Egypt. Ultimately, this will help to adopt effective brucellosis intervention strategies in Egypt and other developing nations.

Molecular detection of Coxiella burnetii in raw meat intended for pet consumption.

The discovery of antibodies against Coxiella burnetii in cattery-confined breeding cats indicating prior or current exposure (Shapiro etal., 2015) prompted an investigation into possible sources of infection. One hypothesis was that raw meat diets containing reservoir species may provide a source of C. burnetii transmission. The aim of this pilot study was to determine whether C. burnetii DNA was present in raw meat sold exclusively for companion animal consumption. The sample population consisted of raw meat packages (n=58) of primarily kangaroo origin, with three to four aliquots (50-120mg) randomly selected from each package. Genomic DNA was extracted from whole tissue in each of these aliquots using a modified protocol. Three quantitative PCR assays were used for the detection of C.burnetii targeting the IS1111 gene, the heat shock operon htpAB and the C.burnetii outer membrane protein-coding gene, com1. Coxiella burnetii DNA was detected in 25/58 samples (43%) using the IS1111, htpAB and/or com1 PCR assays and confirmed by DNA sequencing. All samples amplifying a product in the com1 assay also amplified a product in the htpAB and IS1111 assays. A total of 17/58 (29%) packets were positive with all three genes, 4/58 (7%) were positive with two genes (IS1111 and htpAB) and 4/58 (7%) were positive with the IS1111 gene only. Coxiella burnetii DNA was five times more likely to be found in offal than skeletal muscle meat samples. All meat samples in which C.burnetii DNA was found were from kangaroo tissues, while samples labelled as non-kangaroo meat (n=4) were negative. Multi-locus variable number of tandem repeat analysis (MLVA) identified three different genotypes of C.burnetii that have all been identified previously from Australian human clinical Q fever cases. Further investigations are required to determine the potential role of certain raw meats in the transmission of C.burnetii to cats and humans.

Molecular characterization of Brucella ovis in Argentina

Brucellosis in rams is caused by Brucella ovis or Brucella melitensis and it is considered one of the most important infectious diseases of males in sheep-raising countries. Molecular characterization of Brucella spp. achieved by multi-locus variable number of tandem repeats analysis (MLVA) is a powerful tool to genotype Brucella spp. However, data regarding B. ovis genotyping is scarce. Thus, the aim of this study was to characterize the molecular diversity of B. ovis field-strains in Argentina. A total of 115 isolates of B. ovis from Argentina and Uruguay were genotyped using MLVA-16 and analyzed altogether with 14 publicly available B. ovis genotypes from Brazil. The Discriminatory Power (D) was 0.996 for MLVA-16 and 0.0998 for MLVA-8 and MLVA-11. Analysis of MLVA-16 revealed 100 different genotypes, all of them novel, including 90 unique ones. There was no correlation between geographical distribution and genotype and results showed a higher diversity within provinces than between provinces. Clustering analysis of the strains from Argentina, Uruguay and Brazil revealed that the 129 isolates were grouped into two clades. Whole Genome Sequencing analysis of the 19 B. ovis genomes available in public databases, and including some of the Argentinian strains used in this study, revealed clustering of the Argentinian isolates and closer relationship with B. ovis from New Zealand and Australia. This work adds new data to the poorly understood distribution map of genotypes regionally and worldwide for B. ovis and it constitutes the largest study of B. ovis molecular genotyping until now.

Brucella melitensis in Asian Badgers, Northwestern China

Abstract We isolated Brucella melitensis biovar 3 from the spleen of an Asian badger (Meles leucurus) in Nilka County, northwestern China. Our investigation showed that this isolate had a common multilocus variable-number tandem-repeat analysis 16 genotype, similar to bacterial isolates from local aborted sheep fetuses.

Brucella melitensis in Asian Badgers, Northwestern China.

We isolated Brucella melitensis biovar 3 from the spleen of an Asian badger (Meles leucurus) in Nilka County, northwestern China. Our investigation showed that this isolate had a common multilocus variable-number tandem-repeat analysis 16 genotype, similar to bacterial isolates from local aborted sheep fetuses.

Erratum for Cadel-Six et al., "Draft Genome Sequences of Salmonella enterica subsp. enterica Serovar Dublin Strains from Raw Milk Cheeses Characterized by Multilocus Variable-Number Tandem-Repeat Analysis Profiles Associated with Two Outbreaks in France".

Volume 8, no. 1, e01361-18, 2019, [https://doi.org/10.1128/MRA.01361-18][1]. Page 1: The article title should read as given above. Page 1, abstract, line 2: “St. Nectaire and Morbier cheeses” should read “two raw milk cheeses.” Page 1, abstract, lines 3 and 4: “fatal outbreaks” should