MLVA Profiles Research Articles

Validation of a multilocus genotyping scheme for subtyping Cryptosporidium parvum for epidemiological purposes

Subtyping Cryptosporidium parvum for outbreak investigations or epidemiological surveillance usually relies on DNA sequence analysis of a gene coding for a 60 KDa glycoprotein (gp60). Although gp60 can be useful for allelic discrimination and to help investigate sources and routes of transmission, the presence of common subtypes and recombination during the parasite's sexual life-cycle demand a multilocus-based method for more discriminatory genotyping. While whole genome sequencing would provide the ultimate approach, it is a time consuming and expensive option for faecal parasites such as Cryptosporidium that occur at low density and are difficult to propagate routinely. In this study, we selected and evaluated a panel of previously identified variable-number tandem-repeat (VNTR) markers, to establish a multilocus genotyping scheme based on fragment sizing, appropriate for inter-laboratory surveillance and outbreak investigations. Seven VNTR markers were validated in vitro and demonstrated typeability of 0.85 and discriminatory power of 0.99. The discriminatory power was much greater than the currently used gp60 sequencing (0.74), which identified 26 subtypes, compared to 100 different MLVA profiles within the same sample set. The assay was robust, with repeatable results and reproducibility across three laboratories demonstrating the scheme was suitable for inter-laboratory comparison of C. parvum subtypes. As the majority of genotypes (79%) were unique among epidemiologically unrelated samples, there was efficiency to infer linkage, and epidemiological concordance was observed in historical outbreaks. We propose that the multilocus variable number of tandem repeats analysis scheme is suitable to assist outbreak investigations.

Identification and molecular characterization of Brucella abortus and Brucella melitensis isolated from milk in cattle in Azerbaijan

BackgroundAzerbaijan currently ranks thirteenth in global incidence of human brucellosis, with an estimated annual incidence through 2000 at over 50 cases per million. Brucella melitensis has been isolated from patients and is thought to have been acquired through contact with small ruminants or as a foodborne infection. To reduce the burden of human brucellosis, the Azerbaijani government began in 2002, a nationwide vaccination control campaign in small ruminants. There is serological evidence of bovine brucellosis (presumably due to Brucella abortus) in Azerbaijan, but no prevalence estimates were available when this study started in March 2017. The aim of this study was to isolate and identify Brucella spp. from cow milk in the Ganja region, where brucellosis takes a heavy toll on humans and livestock.ResultsBlood and milk samples were collected from cows (n = 1075) in early lactation (up to 90-days) in farms that had a history of previous positive serological results and abortions. Twenty-two out of 57 milk samples collected from seropositive cows, showed growth on Farrell’s media, when incubated with 5% CO2. Eight additional milk samples showed growth in the absence of CO2. The classical biotyping classified them as Brucella abortus (22) and Brucella melitensis (8). RT-PCR confirmed that strains belonged to the genus Brucella. MLVA profiles were obtained for DNA extracted from two B. abortus and six B. melitensis strains. While the B. abortus genetic profile was described in the MLVA database, matching the profile of B. abortus strains isolated in East Europe, Central Asia and China, we found a new genotype for the B. melitensis strains isolated in Azerbaijan, clustering with strains belonging to the American clade, rarely identified in the region.ConclusionDespite the implementation of the vaccination program in small ruminants, our results suggest that spill-over events of B. melitensis from small ruminants to cattle have occurred. However, cattle are likely to be primarily infected with B. abortus, which warranted the implementation of a bovine brucellosis program. Such a program started in fall 2017. In the Ganja region, cattle should be considered as a potential source of B. abortus and B. melitensis for humans.

MLVA GENOTYPING OF B. ANTHRACIS IN THE FORMATION OF A COLLECTION OF REPRESENTATIVE STRAINS

Anthrax is an infectious disease that affects many species of mammals and humans. Bacteriological isolation of the pathogen and its identification by phenotypic and PCR methods remains the gold standard. However, these methods do not allow determining the cause of the outbreak, namely, their origin from one or more sources. In addition, the causative agents of especially dangerous infections are deposited in the National Collections, and genotyping with the selection of a single representative strain is a prerequisite to prevent the loss of strains with unique properties, as well as the deposition of identical strains. In this study, we performed MLVA genotyping of 10 isolates from 5 outbreaks of anthrax in animals in 2021. The obtained MLVA profiles allowed differentiation of isolates in different outbreaks and confirmed the identity of genotypes in each outbreak. MLVA genotyping of B. anthracis can be recommended as the primary method for genotyping and selection of unique strains for replenishment of the collection.

Molecular characterization of Vibrio cholerae O1 isolates obtained from outbreaks in the Philippines, 2015-2016.

Introduction. The Philippines, comprising three island groups, namely, Luzon, Visayas and Mindanao, experienced an increase in cholera outbreaks in 2016. Previous studies have shown that Vibrio cholerae isolates obtained from the Philippines are novel hybrid El Tor strains that have evolved in the country and are clearly distinct from those found in Mozambique and Cameroon.Gap statement. The characterization of the strains isolated from outbreaks has been limited to phenotypic characteristics, such as biochemical and serological characteristics, in most previous studies.Aim. We performed multilocus variable-number tandem repeat (VNTR) analysis (MLVA) for V. cholerae isolates obtained from 2015 to 2016 to further characterize and understand the emergence and dissemination of the strains in the Philippines.Methodology. A total of 139 V. cholerae O1 Ogawa biotype El Tor isolates were obtained from the Philippines during diarrhoeal outbreaks in 18 provinces between 2015 and 2016. VNTR data were analysed to classify the MLVA profiles where the large-chromosome types (LCTs) were applied for grouping.Results. We identified 50 MLVA types among 139 isolates originating from 18 provinces, and 14 LCTs. The distribution of the LCTs was variable, and a few were located in specific areas or even in specific provinces. Based on eBURST analysis, 99 isolates with 7 LCTs and 32 MLVA types belonged to 1 group, suggesting that they were related to each other. LCT A was predominant (n=67) and was isolated from Luzon and Visayas. LCT A had 14 MLVA types; however, it mostly emerged during a single quarter of a year. Eight clusters were identified, each of which involved specific MLVA type(s). The largest cluster involved 23 isolates showing 3 MLVA types, 21 of which were MLVA type A-14 isolated from Negros Occidental during quarter 4 of 2016. Comparative analysis showed that almost all isolates from the Philippines were distinct from those in other countries.Conclusions. The genotypic relationship of the V. cholerae isolates obtained during outbreaks in the Philippines was studied, and their emergence and dissemination were elucidated. MLVA revealed the short-term dynamics of V. cholerae genotypes in the Philippines.

Evaluation of the genetic relatedness of Bacteroides fragilis isolates by TRs analysis.

Objective(s):Human gastrointestinal tract harbors a variety of bacteria with vital roles in human health. Bacteroides fragilis is considered one of the dominant constituents of gut microflora which can act as an opportunistic pathogen leading to various diseases, including colon cancer, diarrhea, uterine and intrathecal abscesses, septicemia, and pelvic inflammation. In this study, multiple locus variable number of tandem repeats analysis (MLVA) was performed to genetically differentiate 50 B. fragilis isolates.Materials and Methods:Eight suitable tandem repeats (TRs) were selected by bioinformatics tools and were then subjected to PCR amplification using specific primers. Finally, MLVA profiles were clustered using BioNumerics 7.6 software package. Results:All VNTR loci were detected in all isolates using the PCR method. Overall, B. fragilis isolates were differentiated into 27 distinct MLVA types. The highest diversity index was allocated to TR1, TR2, TR5, TR6, and TR8; with this taken into account, strain type 14 was the most prevalent with 12 strains belonging to this type. Clustering revealed three major clusters of A, B, and C. With regards to the pathogenicity of B. fragilis and the outcomes of infections related to this microorganism, it is imperative to study this microorganism isolated from both patients and healthy individuals.Conclusion:This study aimed at evaluating the efficiency of MLVA for the genetic differentiation of B. fragilis. The results of this study indicate the promising efficiency of MLVA typing for cluster detection of this bacterium.

Epidemiological Investigation and Etiological Analysis of a Cutaneous Anthrax Epidemic Caused by Butchering Sick Cattle in Guizhou, China.

A suspected human cutaneous anthrax epidemic caused by butchering sick cattle occurred in Zhijin County of Guizhou Province, Southwest of China, in 2016. Epidemiological investigation and etiological analysis were performed to provide a scientific basis for the source tracking of the epidemic. The epidemic was epidemiologically investigated, and skin blister samples collected from patients and soil samples collected from the butchering spots were used for Bacillus anthracis isolation. The suspicious B. anthracis isolates were identified using conventional methods and PCR, followed by genotyping using multiple-locus variable-number tandem repeats (VNTRs) analysis (MLVA-15) and canonical single-nucleotide polymorphism (canSNP). The genetic relationship of epidemic strains and isolates collected from other regions was analyzed. Epidemiological investigation results showed that the patients may be infected by B. anthracis during butchering sick cattle. Two suspected B. anthracis strains were isolated from blood samples and blister fluids, respectively. Conventional methods identified the two suspected isolates as B. anthracis, while PCR results showed that anti-protective antigen (PA) and capsule (CAP) gene were positive in the two isolates. MLVA-15 showed that the MLVA profiles of the two isolates were 9-20-12-53-16-2-8-8-8-4-4-4-4-10-4, which is different from the MLVA profiles of representative strains from other regions. CanSNP analysis showed that the two strains belonged to cluster A.Br.001/002. Clustering analysis and minimum spanning tree (MST) demonstrated that the two isolates were clustered with strains previously isolated from Guizhou Province. The results indicated that B. anthracis was the pathogen for this epidemic, and the patients were infected during butchering the sick. The genetic characteristics and the relationship of the B. anthracis isolates to strains from other regions indicated that the epidemic was a local occurrence.

Backward compatibility of whole genome sequencing data with MLVA typing using a new MLVAtype shiny application for Vibrio cholerae.

Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is widely used by laboratory-based surveillance networks for subtyping pathogens causing foodborne and water-borne disease outbreaks. However, Whole Genome Sequencing (WGS) has recently emerged as the new more powerful reference for pathogen subtyping, making a data conversion method necessary which enables the users to compare the MLVA identified by either method. The MLVAType shiny application was designed to extract MLVA profiles of Vibrio cholerae isolates from WGS data while ensuring backward compatibility with traditional MLVA typing methods. To test and validate the MLVAType algorithm, WGS-derived MLVA profiles of nineteen Vibrio cholerae isolates from Democratic Republic of the Congo (n = 9) and Uganda (n = 10) were compared to MLVA profiles generated by an in silico PCR approach and Sanger sequencing, the latter being used as the reference method. Results obtained by Sanger sequencing and MLVAType were totally concordant. However, the latter were affected by censored estimations whose percentage was inversely proportional to the k-mer parameter used during genome assembly. With a k-mer of 127, less than 15% estimation of V. cholerae VNTR was censored. Preventing censored estimation was only achievable when using a longer k-mer size (i.e. 175), which is not proposed in the SPAdes v.3.13.0 software. As NGS read lengths and qualities tend to increase with time, one may expect the increase of k-mer size in a near future. Using MLVAType application with a longer k-mer size will then efficiently retrieve MLVA profiles from WGS data while avoiding censored estimation.

Genetic Diversity of Vibrio cholerae О1 El Tor during Epidemic Complications in the Siberian and Far East Regions

The epidemic of cholera in Siberia and the Russian Far East during the seventh pandemic was recorded as sporadic cases of introduction of the infection into the territory or severe local outbreaks associated with the infection’s importation in the 1970s and 1990s. The genetic diversity and relationship of 83 V. cholerae O1 El Tor strains isolated from the region with epidemic complications were studied on the basis of the multilocus analysis of the variable number tandem repeats at five loci: VcA, VcB, VcC, VcD, and VcG. The VcB locus was characterized by the maximum polymorphism in the sample of the studied strains, while the VcC locus displayed the minimum polymorphism. Cluster analysis revealed the differentiation of V. cholerae strains during epidemic complications into seven clusters depending on the structure of the pathogenic determinants (typical and atypical El Tor vibrios), as well as on the time and direction of the pathogen importation. In some outbreaks in the Far East in 1999, the heterogeneity of the etiological agent populations was shown (five genotypes were detected among the outbreak strains from Yuzhno-Sakhalinsk, and six genotypes were determined among the strains from Primorskii krai). At the same time, the predominant MLVA profiles and their single locus variants were identified. The absence of a strict connection between the territory and the genotypes of V. cholerae outbreak strains (one strain from Yuzhno-Sakhalinsk showed the identity of the allele profile with the isolates from Vladivostok), as well as no correspondence of the V. cholerae genotype to a specific infection focus, was shown. According to the data of the MLVA typing, the V. cholerae O1 El Tor strains isolated from the Siberian and Far Eastern regions (Russia) with epidemic complications were characterized by the heterogeneity of their genetic organization, which agrees with the period and direction of their importation to the territory. During sporadic outbreaks of cholera, the clonality of the etiological agent was revealed. Closely related subclones, the emergence of which could result from the presence of the causative agent in the environment or passaging through a susceptible organism, were identified.

Bioburden and transmission of pathogenic bacteria through elevator channel during endoscopic retrograde cholangiopancreatography: application of multiple-locus variable-number tandem-repeat analysis for characterization of clonal strains

ABSTRACTObjectives: Endogenous and exogenous infection of the biliary tract could occur during endoscopic retrograde cholangiopancreatography.Methods: Bile samples of patients with hepatobiliary diseases, and swab samples of elevator channel samples of duodenoscope and washing instruments were prepared simultaneously and cultured aerobically and anaerobically. Antimicrobial susceptibility of the most common characterized bacterial species was tested, and their genetic relatedness was analyzed by multiple locus variable number of tandem repeats method.Results: Contamination with Pseudomonas aeruginosa was detected in 38.2% of the elevator channels’ and 26.6% of the bile samples. Staphylococcus aureus, Enterococcus spp., Staphylococcus epidermidis, Escherichia coli, Enterobacter spp., and Clostridium perfringenes were among other bacterial isolates in the elevator channel swab samples. Highest antimicrobial resistance rate among P. aeruginosa isolates from the bile and swab samples were detected against gentamicin (100% and 73%, respectively), while the lowest one was measured to piperacillin-tazobactam (25% and 0%, respectively). Out of the 27 distinct MLVA profiles, relatedness of P. aeruginosa strains in the bile samples compared with those from the elevators was shown in three series of the samples.Conclusion: Identity of P. aeruginosa strains among the bile and elevator channel samples showed possibility of cross-contamination among patients even at distinct time intervals.Expert opinion: Bacterial infection is considered as main complications of ERCP. Entry of bacteria into the biliary tract via contaminated device and its related instruments and their proliferation in this tissue could cause serious infections. To prevent this side effect, reprocessing of medical equipment via standard cleaning and disinfection procedures are needed. Our results showed incompliance of methods used for endoscope cleaning and disinfection procedure. Although host risk factors, such as sphincterotomy, could increase rate of infection with different types of bacteria, their ability for formation of biofilm and spore, which could help them to resist disinfectants and washing procedures seems to be main cause of persistent colonization and transmission among different patients. New standards for disinfection compared with currently used methods and use of materials to eliminate formation of bacterial microcolonies seem to be necessary to prevent cross-contamination.

Genotyping of Coxiella burnetii in sheep and goat abortion samples

BackgroundQ fever, caused by Coxiella burnetii, is a zoonosis that presents a worldwide distribution and affects both humans and animals. The route of dispersal of the pathogen by ruminants into the environment usually involves stages of abortion and parturition, nevertheless the agent can, also, be detected in other animal samples. Therefore it is considered as important in terms of proper diagnosis, as well as, for epidemiology and surveillance purposes, to genotype the pathogen. The aim of the current study was to investigate the presence of different genotypes of the agent in animals that had suffered from abortion during a two-year survey in Greece.ResultsSixty nine tissue samples (37 stomach contents, 11 liver samples, 21 cotyledons) were collected from 59 abortion cases in sheep (N = 45) and goats (N = 14) from 65 farms at eight different areas of Greece. Samples were screened by qPCR and positive ones were further genotyped using a 10-locus multiple loci (ms 1, 3, 7, 12, 20, 21, 22, 26, 30 and 36) variable number of tandem repeat analysis (MLVA) method.Three genotypes were identified in sheep (A, B, C). Samples representing each of the obtained MLVA profile were further used for MST genotyping. Ten spacers (Cox 2, 5, 6, 18, 20, 22, 37, 51, 56 and 57) were amplified. A close relatedness among the identified MLVA genotypes was confirmed since they all belonged to MST group 32.ConclusionsThe current study introduces into the aspect of genotyping of C. burnetii in Greece. Further studies are needed to explore the presence of more genotypes, to associate the genotypes circulating in the animal and tick population with those causing human disease in order to further expand on the epidemiological aspects of the pathogen.

The role of domestic reservoirs in domestically acquired Salmonella infections in Norway: epidemiology of salmonellosis, 2000-2015, and results of a national prospective case-control study, 2010-2012.

In Norway, incidence of sporadic domestically acquired salmonellosis is low, and most frequently due to Salmonalla Typhimurium. We investigated the risk factors for sporadic Salmonella infections in Norway to improve control and prevention measures. Surveillance data for all Salmonella infections from 2000 to 2015 were analysed for seasonality and proportion associated with domestic reservoirs, hedgehogs and wild birds. A prospective case-control study was conducted from 2010 to 2012 by recruiting cases from the Norwegian Surveillance System for Communicable Diseases and controls from the Norwegian Population Registry (389 cases and 1500 controls). Univariable analyses using logistic regression were conducted and a multivariable model was developed using regularised/penalised logistic regression. In univariable analysis, eating snow, dirt, sand or playing in a sandbox (aOR 4.14; CI 2.15-7.97) was associated with salmonellosis. This was also the only exposure significantly associated with illness in the multivariable model. Since 2004, 34.2% (n = 354) of S. Typhimuirum cases had an MLVA profile linked to a domestic reservoir. A seasonal trend with a peak in August for all Salmonella types and in February for S. Typhimurium was observed. Indirect exposure to domestic reservoirs remains a source of salmonellosis in Norway, particularly for children. Information to the public about avoiding environmental exposure should be strengthened and initiatives to combat salmonellosis in the food chain should be reinforced.

Investigating Nano particles in Bioinformatic Analysis of the Brucella Genome for Indication and Differentation by qPCR

Based on the identified genes, the design of specific primers and probes for qPCR was made, which makes it possible to differentiate B. melitensis and B. abortus in a single reaction. For the differentiation of Brucella strains of the same species, the PCR method of multilocus VNTR analysis, optimized for uniform temperature conditions, is described. In addition, MLVA profiles for B. abortus and B. melitensis genomes are presented. All methods of indication and differentiation of Brucella species and strains are described in detail, which allows them to be used in the everyday work of any PCR laboratory dealing with brucellosis. Control of amplification in the indication of bacteria of the genus Brucella and determination of their belonging to B. abortus and B. melitensis species is recommended to be carried out using the plasmid DNA designed in this work.

Multilocus Variable-Number Tandem-Repeat Analysis of Yersinia ruckeri Confirms the Existence of Host Specificity, Geographic Endemism, and Anthropogenic Dissemination of Virulent Clones.

A multilocus variable-number tandem-repeat analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen Yersinia ruckeri, which causes yersiniosis in salmonids. The assay involves amplification of 10 variable-number tandem-repeat (VNTR) loci in two five-plex PCRs, followed by capillary electrophoresis. A collection of 484 Y. ruckeri isolates, originating from various biological sources and collected from four continents over 7 decades, was analyzed. Minimum-spanning-tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin, and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, subclustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout, Y. ruckeri strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific subclustering further indicates persistent colonization of freshwater farms in Norway. Identification of genetically diverse Y. ruckeri isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less-virulent or avirulent strains.IMPORTANCE This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context.

Genetic diversity of Coxiella burnetii in domestic ruminants in central Italy

BackgroundAs the epidemiology of human Q Fever generally reflects the spread of Coxiella burnetii in ruminant livestock, molecular characterization of strains is essential to prevent human outbreaks. In this study we report the genetic diversity of C. burnetii in central Italy accomplished by MST and MLVA-6 on biological samples from 20 goat, sheep and cow farms.ResultsFive MST and ten MLVA profiles emerged from the analysis establishing a part of C. burnetii strain world atlas. In particular, ST32 occurred on 12 farms (60%), prevalently in goat specimens, while ST12 (25%) was detected on 4 sheep and 1 goat samples. ST8 and a variant of this genotype were described on 2 different sheep farms, whereas ST55 was observed on a goat farm. Five complete MLVA profiles different from any other published genotypes were described in this study in addition to 15 MLVA incomplete panels. Despite this, polymorphic markers Ms23, Ms24 and Ms33 enabled the identification of samples sharing the same MST profile.ConclusionsIntegration of such data in international databases can be of further help in the attempt of building a global phylogeny and epidemiology of Q fever in animals, with a “One Health” perspective.

Molecular epidemiology of Neisseria gonorrhoeae strains circulating in Indonesia using multi-locus variable number tandem repeat analysis (MLVA) and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) techniques

BackgroundControl of gonorrhea in resource-limited countries, such as Indonesia, is mostly unsuccessful. Examining Neisseria gonorrhoeae (Ng) transmission networks using strain typing might help prioritizing public health interventions.MethodsIn 2014, urogenital Ng strains were isolated from clients of sexually transmitted infection clinics in three Indonesian cities. Strains were typed using Multiple-Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) and Ng Multi-Antigen Sequence Typing (NG-MAST) at the Public Health Service, Amsterdam, the Netherlands, and compared to Dutch strains collected from 2012 to 2015. Minimum spanning trees (MSTs) were constructed using MLVA profiles incorporating demographics and NG-MAST genogroups. A cluster was defined as ≥5 strains differing in ≤1 VNTR locus. The concordance between MLVA and NG-MAST was examined with the adjusted Wallace coefficients (AW).ResultsWe collected a total of 78 Indonesian strains from Yogyakarta (n = 44), Jakarta (n = 25), and Denpasar (n = 9). Seven MLVA clusters and 16 non-clustered strains were identified. No cluster was specific for any geographic area, risk group or age group.Combined with 119 contemporary Dutch strains, 8 MLVA clusters were identified, being four clusters of Indonesian strains, two of Dutch strains, and two of both Indonesian and Dutch strains. Indonesian strains (79.5%) were more often clustered compared to Dutch strains (24.3%).The most prevalent NG-MAST genogroups among Indonesian strains was G1407 (51.3%) and among Dutch strains was G2992 (19.3%). In Indonesian strains, the AW [95% confidence interval] for MLVA to NG-MAST was 0.07 [0.00–0.27] and for NG-MAST to MLVA was 0.03 [0.00–0.12].ConclusionIndonesian Ng strains are more often clustered than Dutch strains, but show no relation with geographical area, risk group, or age group, suggesting a more clonal Ng epidemic in Indonesia. Some similar Ng strains circulate in both Indonesia and the Netherlands.

Annual report of the Scientific Network on Microbiological Risk Assessment 2017

Annual report of the Scientific Network on Microbiological Risk Assessment 2017

Whole genome sequencing of Brucella melitensis isolated from 57 patients in Germany reveals high diversity in strains from Middle East.

Brucellosis, a worldwide common bacterial zoonotic disease, has become quite rare in Northern and Western Europe. However, since 2014 a significant increase of imported infections caused by Brucella (B.) melitensis has been noticed in Germany. Patients predominantly originated from Middle East including Turkey and Syria. These circumstances afforded an opportunity to gain insights into the population structure of Brucella strains. Brucella-isolates from 57 patients were recovered between January 2014 and June 2016 with culture confirmed brucellosis by the National Consultant Laboratory for Brucella. Their whole genome sequences were generated using the Illumina MiSeq platform. A whole genome-based SNP typing assay was developed in order to resolve geographically attributed genetic clusters. Results were compared to MLVA typing results, the current gold-standard of Brucella typing. In addition, sequences were examined for possible genetic variation within target regions of molecular diagnostic assays. Phylogenetic analyses revealed spatial clustering and distinguished strains from different patients in either case, whereas multiple isolates from a single patient or technical replicates showed identical SNP and MLVA profiles. By including WGS data from the NCBI database, five major genotypes were identified. Notably, strains originating from Turkey showed a high diversity and grouped into seven subclusters of genotype II. MLVA analysis congruently clustered all isolates and predominantly matched the East Mediterranean genetic clade. This study confirms whole-genome based SNP-analysis as a powerful tool for accurate typing of B. melitensis. Furthermore it allows special allocation and therefore provides useful information on the geographic origin for trace-back analysis. However, the lack of reliable metadata in public databases often prevents a resolution below geographic regions or country levels and corresponding precise trace-back analysis. Once this obstacle is resolved, WGS-derived bacterial typing adds an important method to complement epidemiological surveys during outbreak investigations. This is the first report of a detailed genetic investigation of an extensive collection of B. melitensis strains isolated from human cases in Germany.

Quantification of hygiene indicators and Salmonella in the tonsils, oral cavity and rectal content samples of pigs during slaughter

The tonsils, oral cavity and faeces of 94 pigs at slaughter were sampled to assess the numbers of total aerobic bacteria, Enterobacteriaceae and Escherichia coli in the rectal content, tonsils and oral cavity of pigs at time of evisceration. Moreover, the prevalence, numbers and types of Salmonella spp. were determined. Mean numbers of Enterobacteriaceae in tonsils and the oral cavity differed between slaughterhouses. The proportion of Enterobacteriaceae relative to total aerobic bacteria differed between the different tissues, though large variations were observed between animals. Salmonella spp. were mostly detected in oral cavity swabs (n = 51, 54%), of which six samples were contaminated in numbers over 2.0 log CFU/100 cm2. Salmonella spp. were also recovered from 17 tonsillar tissue samples (18%) and 12 tonsillar swabs (13%). Out of the 29 rectal content samples from which Salmonella was recovered (31%), most were lowly contaminated, in the range between −1 and 0 log CFU/g. The predominant serotypes were S. Typhimurium and its monophasic variant, which were recovered from 33 and 13 pigs, respectively. In most cases, the same serotypes and MLVA profiles were found in pigs slaughtered during the same day, thus suggesting a common source of contamination.

Yersinia enterocolitica, a Neglected Cause of Human Enteric Infections in Côte d’Ivoire

BackgroundEnteropathogenic Yersinia circulate in the pig reservoir and are the third bacterial cause of human gastrointestinal infections in Europe. In West Africa, reports of human yersiniosis are rare. This study was conducted to determine whether pathogenic Yersinia are circulating in pig farms and are responsible for human infections in the Abidjan District.Methodology/Principal findingsFrom June 2012 to December 2013, pig feces were collected monthly in 41 swine farms of the Abidjan district. Of the 781 samples collected, 19 Yersinia strains were isolated in 3 farms: 7 non-pathogenic Yersinia intermedia and 12 pathogenic Yersinia enterocolitica bioserotype 4/O:3. Farm animals other than pigs and wild animals were not found infected. Furthermore, 2 Y. enterocolitica 4/O:3 strains were isolated from 426 fecal samples of patients with digestive disorders. All 14 Y. enterocolitica strains shared the same PFGE and MLVA profile, indicating their close genetic relationship. However, while 6 of them displayed the usual phage type VIII, the other 8 had the highly infrequent phage type XI. Whole genome sequencing and SNP analysis of individual colonies revealed that phage type XI strains had unusually high rates of mutations. These strains displayed a hypermutator phenotype that was attributable to a large deletion in the mutS gene involved in DNA mismatch repair.Conclusions/SignificanceThis study demonstrates that pathogenic Y. enterocolitica circulate in the pig reservoir in Côte d'Ivoire and cause human infections with a prevalence comparable to that of many developed countries. The paucity of reports of yersiniosis in West Africa is most likely attributable to a lack of active detection rather than to an absence of the microorganism. The identification of hypermutator strains in pigs and humans is of concern as these strains can rapidly acquire selective advantages that may increase their fitness, pathogenicity or resistance to commonly used treatments.

Tolerance to quaternary ammonium compound disinfectants may enhance growth of Listeria monocytogenes in the food industry

The antibacterial effect of disinfectants is crucial for the control of Listeria monocytogenes in food processing environments. Tolerance of L. monocytogenes to sublethal levels of disinfectants based on quaternary ammonium compounds (QAC) is conferred by the resistance determinants qacH and bcrABC. The presence and distribution of these genes have been anticipated to have a role in the survival and growth of L. monocytogenes in food processing environments where QAC based disinfectants are in common use. In this study, a panel of 680 L. monocytogenes from nine Norwegian meat- and salmon processing plants were grouped into 36 MLVA profiles. The presence of qacH and bcrABC was determined in 101 isolates from the 26 most common MLVA profiles. Five MLVA profiles contained qacH and two contained bcrABC. Isolates with qacH and bcrABC showed increased tolerance to the QAC Benzalkonium chloride (BC), with minimal inhibitory concentrations (MICs) of 5–12, 10–13 and <5ppm for strains with qacH (two allele variants observed), bcrABC, and neither gene, respectively. Isolates with qacH or bcrABC were not more tolerant to BC in bactericidal tests in suspension or in biofilms compared with isolates lacking the genes. Water residue samples collected from surfaces in meat processing plants after QAC disinfection had bactericidal effect against L. monocytogenes when the sample BC levels were high (>100ppm). A sample with lower BC concentrations (14ppm of chain length C-12 and 2.7ppm of chain length C-14) inhibited growth of L. monocytogenes not containing bcrABC or qacH, compared to strains with these genes.The study has shown that L. monocytogenes harbouring the QAC resistance genes qacH and bcrABC are prevalent in the food industry and that residuals of QAC may be present in concentrations after sanitation in the industry that result in a growth advantage for bacteria with such resistance genes.