MLVA Research Articles

Genome-based population structure analysis of the strawberry plant pathogen Xanthomonas fragariae reveals two distinct groups that evolved independently before its species description.

Xanthomonas fragariae is a quarantine organism in Europe, causing angular leaf spots on strawberry plants. It is spreading worldwide in strawberry-producing regions due to import of plant material through trade and human activities. In order to resolve the population structure at the strain level, we have employed high-resolution molecular typing tools on a comprehensive strain collection representing global and temporal distribution of the pathogen. Clustered regularly interspaced short palindromic repeat regions (CRISPRs) and variable number of tandem repeats (VNTRs) were identified within the reference genome of X. fragariae LMG 25863 as a potential source of variation. Strains from our collection were whole-genome sequenced and used in order to identify variable spacers and repeats for discriminative purpose. CRISPR spacer analysis and multiple-locus VNTR analysis (MLVA) displayed a congruent population structure, in which two major groups and a total of four subgroups were revealed. The two main groups were genetically separated before the first X. fragariae isolate was described and are potentially responsible for the worldwide expansion of the bacterial disease. Three primer sets were designed for discriminating CRISPR-associated markers in order to streamline group determination of novel isolates. Overall, this study describes typing methods to discriminate strains and monitor the pathogen population structure, more especially in the view of a new outbreak of the pathogen.

Multiple-locus variable-number tandem repeat analysis using multiplex polymerase chain reaction and next-generation sequencing - A novel high-throughput method for subtyping Listeria strains

Listeria monocytogenes is significantly associated with listeriosis infection. Several species of Listeria other than L. monocytogenes directly affect the food industry. In the present study we developed a novel subtyping method for tracking the source of L. monocytogenes and L. innocua in large-scale investigations using multiple-locus variable-number tandem repeat analysis (MLVA) coupled with next-generation sequencing (NGS). Forty-eight L. monocytogenes and L. innocua strains isolated from a food processing plant and the environment were used for the amplification of 15 specific variable number tandem repeat (VNTR) loci. The results exhibited 100% concordance for amplicon detection using both the subtyping methods. Coupling of MLVA with capillary electrophoresis (CE) or NGS detected 38 and 89 different alleles, respectively. MLVA coupled with NGS showed significantly higher discriminating power than MLVA coupled with CE. In MLVA coupled with NGS method, locus JLR1 of L. monocytogenes had 54% discrimination while locus TR3 had 100% discrimination solely in L. innocua. In addition, MLVA coupled with NGS method had significantly higher discrimination power (DI = 1.0 with 48 MLVA patterns) than that of MLVA coupled with CE (DI = 0.79 with 15 MLVA patterns). These results indicate the advantage of MLVA coupled with NGS method in detecting not only the length but also sequence polymorphism in a single read run. MLVA coupled with NGS led to significant reduction in labor and cost than CE and the traditional Sanger sequencing. Thus, MLVA coupled with NGS is an effective and feasible technique for the molecular typing of Listeria species in food industries.

Antimicrobial susceptibility and MLVA analysis of S. Typhimurium strains isolated from human and poultry samples in Tunisia.

Salmonella enterica infections are a significant public health concern worldwide, being Salmonella Typhimurium one of the most prevalent serovars. Human salmonellosis is typically associated with the consumption of contaminated foods, such as poultry, eggs and processed meat. The extensive use of antimicrobials in humans and animals has led to an increase in multidrug resistance among Salmonella strains, becoming multidrug-resistant (MDR) strains a major public health concern. This study was designed to investigate the antimicrobial susceptibility and the genotypic diversity of Salmonella Typhimurium strains isolated in Tunisia from human and poultry sources from 2009 to 2015. Fortyfive strains were analyzed by disk-diffusion test to determine the antimicrobial susceptibility. The presence of antimicrobial resistance genes was tested by PCR, and genotyping was performed using multiple-locus variable-number tandem repeats analysis (MLVA). About 50% of the strains were resistant to at least 3 antibiotics (multidrug-resistant strains, MDR). The most frequent resistance profile in clinical strains was AMP-TIC-TET-MIN-SXT (n = 7) and TET-MIN in poultry origin strains (n = 7). The MLVA typing grouped the strains in 2 main clusters. Cluster I was mostly formed by human isolates, whereas in cluster II both human and poultry isolates were grouped. Simpson's diversity index was 0.870 and 0.989 for antimicrobial resistance profiles and MLVA, respectively. Multiresistance is common in Salmonella Typhimurium isolated from human and poultry sources in Tunisia. The genotyping results suggest that some strains isolated from both sources may descend from a common subtype.

Genotyping of pathogenic leptospira by Multiple Locus Variable-number Tandem Repeat Analysis (MLVA)

ObjectiveTo introduce the method of molecular genotyping (MLVA) to determine the genotype of field isolates of leptospira.IntroductionLeptospirosis (ictherohemoglobinuria, Leptospirosis biliousness) is a natural focal and zoonotic infectious disease dangerous for humans and farm animals. It is important to identify specific leptospira strains isolated from rodents or sick and suspicious animals by the serotype or genotype. In comparison with serotyping using micro agglutination test (MAT), molecular genotyping makes it possible to accurately identify a specific pathogen strain. The genetic classification now becomes more significant than the phenotypic classification.MethodsSpecific oligonucleotide primers, which flank fragments of the genome locus of pathogenic leptospira varies in terms of the number of tandem repeats VNTR-4, -7, -10 specific for L.interrogans, L.kirschneri, and L.borgpetersenii were used. The amplification products were detected using agar gel electrophoresis with the following identification of the fragment length with a molecular weight marker and comparison with the collection of VNTR profiles of the strains described in the literature.ResultsIt was established that the method of leptospira molecular genotyping by determining the number of variable tandem repeats of a locus (VNTR-variable number tandem repeats analysis) is suitable for molecular epizootology studies in Ukraine. The advantages of the method are the simplicity of performance and availability for diagnostic and research laboratories in Ukraine compared to other pathogen genome sequencing based genotyping methods, in particular Multilocus sequence typing (MLST) or Multispacer Sequence Typing (MST), which require complex equipment and operating conditions. The reference strain of Leptospira M20 serotype Copengageni serogroup Icterohaemorrhagiae from the NAAS IVM collection of was studied and its VNTR profile was identified with the genotype of the strain Fiocruz L1-130 that is described in the literature as a serotype of Copengageni serogroup Icterohaemorrhagiae. The genotype of the leptospira field isolate obtained from a rat in Lviv Oblast of Ukraine was specified and its identity was established in the aforementioned genotype. The obtained data support the prospects of using MLVA genotyping method to study the distribution of different genotypes of leptospira. The research will continue to study the specificities of molecular epizootology of leptospirosis in Ukraine.ConclusionsThe method of leptospira molecular genotyping by multilocus analysis of the number of variable tandem repeats has been tested in the Leptospirosis Research Laboratory in collaboration with the Museum of Microorganisms at the National Academy of Sciences, the Ukraine Institute of Veterinary Medicine, the ELISA and PCR Research Laboratory, and the Bila Tserkva National Agrarian University. The genotype of the reference strain has been correlated with its serological profile; identification of the genotype of the field isolate pathogenic leptospira has been completed. The tested method is planned to be implemented in surveillance and control over leptospirosis spreading in Ukraine, and aimed to help in development and improvement of leptospirosis vaccine formulations. Additionally, method of Multiple-Locus Variable number tandem repeat Analysis will be used for molecular epidemiology research in Ukraine.ReferencesSalaün L, Mérien F, Gurianova S, Baranton G, Picardeau M. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol. 2006;44(11):3954-3962. doi:10.1128/JCM.00336-06.Caimi K, Repetto SA, Varni V, Ruybal P. Infection , Genetics and Evolution Leptospira species molecular epidemiology in the genomic era. Infect Genet Evol. 2017;54(July):478-485. doi:10.1016/j.meegid.2017.08.013.Ayral F, Zilber AL, Bicout DJ, Kodjo A, Artois M, Djelouadji Z. Distribution of leptospira interrogans by multispacer sequence typing in urban Norway rats (Rattus norvegicus): A survey in France in 2011-2013. PLoS One. 2015;10(10):1-14. doi:10.1371/journal.pone.0139604.

Resurgence of Pertussis and Emergence of the Ptxp3 Toxin Promoter Allele in South Italy.

Despite universal immunization programs, pertussis remains a major public health concern. This study aimed to describe the pertussis epidemiology in the Puglia region in 2006-2015 and to identify recent polymorphisms in Bordetella pertussis virulence-associated genes. The pertussis cases in 2006-2015 were identified from the National Hospital Discharge Database and the Information System of Infectious Diseases. Samples of pertussis cases in 2014-2016 that were confirmed by the Regional Reference Laboratory were subjected to ptxA, ptxP and prn gene sequencing and, in 10 cases, multiple-locus variable-number tandem repeat analysis. In Puglia in 2006-2015, the pertussis incidence rose from an average of 1.39/100,000 inhabitants in 2006-2013 to 2.56-2.54/100,000 in 2014-2015. In infants <1 year of age, the incidence rose from an average of 60.4/100,000 infants in 2006-2013 to 149.9/100,000 in 2015. Of the 661 cases recorded in 2006-2015, 80.3% required hospitalization; of these, 45.4% were <1 year of age. Of the 80 sequenced samples, the allelic profile ptxA1-ptxP3-prn2 was detected in 74. This variant was detected in both vaccinated and unvaccinated people. Six Bordetella pertussis samples were prn deficient. The multiple-locus variable-number tandem repeat analysis cases exhibited multiple-locus variable-number tandem repeat analysis-type 27. The pertussis incidence in Puglia has risen. The hypervirulent strain was also found in vaccinated people. This suggests bacterial adaptation to the vaccine and raises questions about acellular vaccine effectiveness. Prevention of infant pertussis cases is best achieved by immunizing the pregnant mother. Enhanced surveillance and systematic laboratory confirmation of pertussis should be improved in Italy.

Characterisation of Streptococcus pneumoniae isolates from invasive disease in adults following the introduction of PCV10 in Brazil.

Purpose. Invasive pneumococcal disease (IPD) in the elderly is an important public health issue due to the increased proportion of this population in many countries including Brazil. We aimed to characterise pneumococci isolates in adults >50 years with IPD, following the introduction of the 10-valent pneumococcal conjugate vaccine (PCV10) as part of the National Childhood Immunisation Program for children ≤2 years in March 2010.Methodology. Between 2013 and 2015, pneumococcal isolates were collected and serotypes were determined using multiplex PCR and/or Quellung reaction. Antimicrobial susceptibility was defined by E-test (bioMérieux); genetic diversity was determined using Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and, in selected isolates, Multi Locus Sequence Typing (MLST) was performed.Results/Key findings. Among 102 pneumococcal isolates, the most frequent serotypes were 19A, 13 of 102 (12.7 %) and 22F, 10 of 102 (9.8 %). Ninety-eight isolates were tested for antimicrobial susceptibility. Intermediate resistance to penicillin was present in 2/98 (2.0 %), ceftriaxone in 7/98 (7.1 %) and meropenem in 7/95 (7.4 %) of the isolates (non-meningitis breakpoint: 4 µg ml-1/2 µg ml-1/0.5 µg ml-1, respectively). Resistance to penicillin (meningitis breakpoint ≥0.12 µg ml-1) was observed in 31/98 (31.6 %) of the isolates. Genetic analysis presented two relevant clonal groups, belonging to non-PCV10 serotypes: 19A (ST320, linked to non-susceptibility) and 22F (ST6403).Conclusion. Our data suggest a predominance of non-PCV10 serotypes among IPD in the elderly population in circulating strains ca. 3 to 5 years after the introduction of PCV10 in Brazil.

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia.

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients’ data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia.

Enzootic situation and molecular epidemiology of Brucella in livestock from 2011 to 2015 in Qingyang, China

A large-scale survey was conducted in domestic animal populations from 2011 to 2015 in Qingyang, China. A total of 448,398 animals from different districts of Qingyang were tested for the presence of Brucella-specific antibodies using the Rose Bengal Plate Test (RBPT) and the Standard Agglutination Test (SAT). From 2011 to 2015, the yearly average positive rates were between 0.04 and 4.75% in the eight counties tested. In addition, the prevalence rates were between 0 and 9.96% in these eight counties. Sheep was the dominant host of Brucella in Qingyang, and the prevalence rate in sheep (2.74%) was higher than those in the other animals tested. Identification of 10 Brucella isolates from sheep confirmed that the epidemic strains were B. melitensis biovar 3 (n = 9) and B. melitensis biovar 1 (n = 1). MLVA-11 (multiple-locus variable-number tandem repeat analysis) analysis of the 10 isolates showed three genotypes: genotype 116 (n = 8), genotype 115 (n = 1) and genotype 136 (n = 1). Furthermore, analysis of the whole-genome sequences of the representative B. melitensis strain QY1 indicated that this isolate was closely related to isolates from China and India. The results of serum epidemiology confirmed that the region of northern Qingyang was a critical Brucella epidemic area and that the disease showed a rising trend, especially from 2013 to 2015. An analysis of the isolate genotypes suggested that sheep brucellosis mainly resulted from conventional B. melitensis (East Mediterranean group), although the external strain (American group) also occurred in Qingyang.

Genotyping and Phylogenetic Analysis of Group B Streptococcus by Multiple Locus Variable Number Tandem Repeat Analysis in Iran

Background: Group B streptococcus (GBS), also known as Streptococcus agalactiae, is well known as a causative agent for neonatal invasive diseases; it is also a major pathogen in adults. Analytic epidemiology is required to monitor the clinical isolates of GBS. However, there is insufficient information on the genetic background of GBS in Iran, and this information is needed to guide and develop a GBS vaccine. Materials and Methods: In total, 90 well-characterized GBS isolates were collected from April 2014 to August 2015. In this study, molecular typing was used to disclose a relationship between the multiple-locus variable number tandem repeat analysis (MLVA) types, serotyping, and pilus islands. The isolates were characterized by the types of capsular polysaccharides and pilus islands and were examined by MLVA to study the epidemiological relationship of isolates. Results: The results indicate that there is a significant relationship between the distribution of serotypes and pilus island genes; GBS isolates were differentiated into 12 types by capsular polysaccharides and pilus islands analysis. The discriminatory power of an MLVA analysis was high based on the five most variable numbers of tandem repeat loci and 44 MLVA types that were identified. Conclusions: This study has provided useful insights into the genetic heterogeneity of GBS isolates in Tehran and Alborz, Iran. The extensive distribution of pilus islands in various serotypes and MLVA types throughout the GBS population refers to the advancement of the pilus-based GBS vaccines. [GMJ.2018;7:e1121]

Genotyping and Phylogenetic Analysis of Group B Streptococcus by Multiple Locus Variable Number Tandem Repeat Analysis in Iran

Background: Group B streptococcus (GBS), also known as Streptococcus agalactiae, is well known as a causative agent for neonatal invasive diseases; it is also a major pathogen in adults. Analytic epidemiology is required to monitor the clinical isolates of GBS. However, there is insufficient information on the genetic background of GBS in Iran, and this information is needed to guide and develop a GBS vaccine. Materials and Methods: In total, 90 well-characterized GBS isolates were collected from April 2014 to August 2015. In this study, molecular typing was used to disclose a relationship between the multiple-locus variable number tandem repeat analysis (MLVA) types, serotyping, and pilus islands. The isolates were characterized by the types of capsular polysaccharides and pilus islands and were examined by MLVA to study the epidemiological relationship of isolates. Results: The results indicate that there is a significant relationship between the distribution of serotypes and pilus island genes; GBS isolates were differentiated into 12 types by capsular polysaccharides and pilus islands analysis. The discriminatory power of an MLVA analysis was high based on the five most variable numbers of tandem repeat loci and 44 MLVA types that were identified. Conclusions: This study has provided useful insights into the genetic heterogeneity of GBS isolates in Tehran and Alborz, Iran. The extensive distribution of pilus islands in various serotypes and MLVA types throughout the GBS population refers to the advancement of the pilus-based GBS vaccines. [GMJ.2018;7:e1121]

Genotyping and Phylogenetic Analysis of Group B Streptococcus by Multiple Locus Variable Number Tandem Repeat Analysis in Iran.

Background: Group B streptococcus (GBS), also known as Streptococcus agalactiae, is well known as a causative agent for neonatal invasive diseases; it is also a major pathogen in adults. Analytic epidemiology is required to monitor the clinical isolates of GBS. However, there is insufficient information on the genetic background of GBS in Iran, and this information is needed to guide and develop a GBS vaccine. Materials and Methods: In total, 90 well-char - acterized GBS isolates were collected from April 2014 to August 2015. In this study, molecular typing was used to disclose a relationship between the multiple-locus variable number tandem repeat analysis (MLVA) types, serotyping, and pilus islands. The isolates were characterized by the types of capsular polysaccharides and pilus islands and were examined by MLVA to study the epidemiological relationship of isolates. Results: The results indicate that there is a significant relationship between the distribution of serotypes and pilus island genes; GBS isolates were differentiated into 12 types by capsular polysaccharides and pilus islands analysis. The discriminatory power of an MLVA analysis was high based on the five most variable numbers of tandem repeat loci and 44 MLVA types that were identified. Conclusion: This study has provided useful insights into the genetic heterogeneity of GBS isolates in Tehran and Alborz, Iran. The extensive distribution of pilus islands in various serotypes and MLVA types throughout the GBS population refers to the advancement of the pilus-based GBS vaccines.

Molecular epidemiology of Shiga toxin-producing O113:H21 isolates from cattle and meat.

The serotype O113:H21 is considered one of the relevant non-O157 STEC serotypes associated with severe human infections. Due to the increased detection of O113 strains and their relationship with clinical cases, which emphasizes the importance of this serogroup as an emerging pathogen, our aim was to determine the characteristics of STEC O113:H21 strains circulating in bovine cattle and retail meat from Argentina. For this purpose, we determined the presence and combinations of various virulence genes (and their variants) related to adhesion and toxicity in a collection of 34 isolates. Their genetic relatedness using multiple-locus variable-number tandem repeat analysis (MLVA) was also studied. Subtyping of stx genes indicated that O113:H21 strains circulating in Argentina mainly present stx2a alone or together with stx2c or, less frequent, with stx2d , all of which are subtypes associated with human disease. We found plasmid markers, such as saa, ehxA and subA, in a higher proportion than previous studies, and five variants of saa, two of which were novel ones. In relation to MLVA subtyping, we detected a limited diversity among the isolates considering that several loci were not discriminative and, that in some farms, the same clone seemed to remain circulating throughout the year. The O113:H21 strains studied harbour several toxin and adhesion genes (saa, espP, fimCD, ehaA, iha, hcpA, elfA, lpfO113, ecpA, subA, cdt-V) and Stx subtypes associated with human disease. Results also highlighted that subtyping of stx and saa is useful to discriminate O113:H21 strains that share virulence genes. In conclusion, this study shows that a number of O113:H21 strains that occur in foods and bovines could be pathogenic for humans. This situation calls for further attention in the prevention and control of foodborne disease caused by these strains.

Molecular characterization, toxin detection and resistance testing of human clinical Clostridium difficile isolates from Lebanon

Clostridium (Clostridioides) difficile is the main cause for nosocomial diarrhoea in industrialised nations. Epidemiologic data on the pathogen’s occurrence in other world regions are still scarce. In this context we characterized with phenotypic and molecular genetic methods C. difficile isolates stemming from hospitalised patients with diarrhoea in Lebanon.From 129 stool samples of symptomatic patients at a tertiary care University hospital in Lebanon, a total of 107 C. difficile strains were cultivated and underwent ribotyping, toxin gene detection and antibiotic resistance testing.Ribotype 014 (RT014, 16.8%) predominated, followed by RT002 (9.3%), RT106 (8.4%) and RT070 (6.5%). Binary toxin gene-positive isolates (RT023, RT078 and RT126) were rarely detected and RT027 was absent. Interestingly, within one isolate only the toxin A gene (tcdA) was detected. Multiple-locus variable-number tandem repeat analysis (MLVA) revealed strong strain diversity in most RTs. The isolates were sensitive to metronidazole and vancomycin, and only a small proportion of strains displayed resistance against moxifloxacin, rifampicin, and clarithromycin (5.6%, 1.9%, and 2.8%), respectively.The data indicate that the genetic strain composition of Lebanese strains differs markedly from the situation seen in Europe and North America. Especially the epidemic RTs seen in the latter regions were almost absent in Lebanon. Interestingly, most strains showed almost no resistance to commonly used antibiotics that are suspected to play a major role in the development of C. difficile infection, despite frequent use of these antibiotics in Lebanon. Thus, the role of antimicrobial resistance as a major driving force for infection development remains uncertain in this area.

Molecular Investigation of the Transmission Dynamics of Brucellosis Observed Among Children in the Province of South - East Anatolia, Turkey

Background: Brucellosis is a zoonotic disease caused by Brucella species. Although brucellosis is considered as an occupational disease in adults, recently it has become an infectious disease affecting all age groups, including children. Molecular epidemiological studies are crucial for control and treatment of disease in children. Objectives: This study aimed at identifying Brucella species, to detect antibiotic susceptibilities and define transmission dynamics between the Brucella isolates in children. Methods: A total of 77 Brucella isolates were identified by conventional and polymerase chain reaction methods. Anti - biotic susceptibilities were investigated by E - test strips. The isolates were genotyped by using multiple locus variable number tandem repeat analysis (MLVA) (MLVA - 16 Orsay), including 8 mini - satellite (panel 1) and 8 microsatellite (panel 2A and 2B) markers. Results: The mean age was 9.14 ± 3.4 years. All patients had been consuming unpasteurized milk. All isolates were Brucella melitensis biovar 3. Only 2 isolates were resistant to ceftriaxone, while the other isolates were susceptible to other antimicrobial agents. The MLVA - 16 typing revealed 42 MLVA profiles. Eighteen profiles included 2 or more isolates, indicating a clustering rate of 66.7%. Twenty - four isolates showed a unique profile. Single locus, double locus, and 3 locus variants were detected in 32, 26, and 15 isolates, respectively. Bruce 30, Bruce 16, Bruce 9, Bruce 7, and Bruce 4 were highly discriminatory loci, respectively. All strains were defined as genotype 122, according to MLVA - 11, and genotype 43 according to MLVA - 8, and were in the Eastern Mediterranean genotype. Conclusions: High clustering rate revealed that brucellosis among the children mainly resulted from common sources. Controlling animal movements and avoiding contaminated milk products have an importance to interrupt spread of brucellosis in children.

Relationship between macrolide resistance mutations in Mycoplasma pneumoniae and its genotype

Objective To analyze the relationship between macrolide resistance mutations in Mycoplasma pneumoniae (Mp) and its genotype by multiple-locus variable-number tandem-repeat analysis (MLVA). Methods One hundred and forty-three Mp-positive specimens were collected in Beijing (54 collected at the Affiliated Children′s Hospital of the Capital Institute of Pediatrics), the United States (59 collected at four different geographical locations: Kansas City, Missouri; Seattle, Washington; New York, New York; Chicago, Illinois) and Australia (30 provided by the diagnostic laboratory at the Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Sydney). Nested PCR was used to detect mutations in 23S rRNA. A capillary electrophoresis-based single tube multiplex PCR (mPCR-CE) was used to analyze the MLVA types of Mp in those samples. Results A2063G mutation was identified in 57 specimens including 49 from Beijing, seven from the United States and one from Australia. The 143 Mp-positive specimens were typed into 10 distinct MLVA types. Fifty-four specimens collected in Beijing belonged to four MLVA types, which were M4-5-7-2 (44/54, 81.5%), M3-5-6-2 (7/54, 13.0%), M4-5-6-2 (2/54, 3.70%) and M4-5-5-2 (1/54, 1.85%). Fifty-nine specimens collected in the United States belonged to six MLVA types including M4-5-7-2 (27/59, 45.8%), M3-5-6-2 (18/59, 30.5%), M3-6-6-2 (11/59, 18.6%), M3-5-6-1 (1/59, 1.69%), M4-5-7-3 (1/59, 1.69%) and M5-5-7-2 (1/59, 1.69%). Thirty specimens of Mp from Australia were grouped to five types with M3-5-6-2 (12/30, 40.0%) and M4-5-7-2 (10/30, 33.3%) and M3-5-7-2 (5/30, 16.7%) being the predominant types. Macrolide resistance mutations were detected in 57 out of 143 specimens (49 from Beijing, seven from the United States and one from Sydney) and 50 of them were MLVA type of M4-5-7-2. Conclusion The MLVA type of M4-5-7-2 is associated with macrolide resistance in Mp. Key words: Mycoplasma pneumoniae; Macrolide resistance; 23S rRNA; MLVA genotype

The role of transportation in the spread of Brachyspira hyodysenteriae in fattening farms

BackgroundDirect and indirect contact among animals and holdings are important in the spread of Brachyspira hyodysenteriae. The objective of this study was to investigate the role of slaughterhouse vehicles in spreading B. hyodysenteriae between unconnected farms.ResultsMultilocus sequence typing (MLST) and Multiple Locus Variable number tandem repeat Analysis (MLVA) were used to characterize B. hyodysenteriae strains isolated from trucks. Before cleaning, 976 batches of finishing pigs transported by 174 trucks from 540 herds were sampled. After cleaning, 763 of the 976 batches were also sampled. Sixty-one of 976 and 4 of 763 environmental swabs collected from trucks before and after cleaning and disinfection operations, respectively, were positive for B. hyodysenteriae. The 65 isolates in this study originated from 48 farms. Trucks were classified into five categories based on the number of visited farms as follows: category 1: 1–5 farms, category 2: 6–10 farms, category 3: 11–15 farms, category 4: 16–20 farms, category 5: >21 farms. Although the largest number of vehicles examined belonged to category 1, the highest percentage of vehicles positive for B. hyodysenteriae was observed in categories 3, 4 and 5. Specifically, 90.9% of trucks belonging to category 5 were positive for B. hyodysenteriae, followed by categories 4 and 3 with 85.7% and 83.3%, respectively. The results of MLST and MLVA suggest that trucks transporting pigs from a high number of farms also play a critical role in spreading different B. hyodysenteriae genetic profiles. STVT 83–3, which seems to be the current dominant type in Italy, was identified in 56.25% of genotyped isolates. The genetic diversity of isolated strains from trucks was high, particularly, in truck categories 3, 4 and 5. This result confirmed that MLST and MLVA can support the study of epidemiological links between different B. hyodysenteriae farm strains.ConclusionsThis study highlights the potential role of shipments in B. hyodysenteriae spread. Moreover, it emphasizes the importance of strict vehicle hygiene practices for biosecurity programmes.

Tracing Back the Source of an Outbreak of Salmonella Typhimurium; National Outbreak Linked to the Consumption of Raw and Undercooked Beef Products, the Netherlands, October to December 2015.

Introduction. On 23 October 2015, six related cases with gastroenteritis called the Netherlands Food and Consumer Product Safety Authority. They suspected filet américain, a raw beef spread, to be the source of infection. Leftovers and stool samples tested positive for Salmonella Typhimurium. Multiple locus variable-number of tandem repeat analysis (MLVA) revealed a MLVA pattern (02-23-08-08-212), which had not been detected in the Netherlands before. Concomitantly, an increase of this MLVA type was observed in the national Salmonella surveillance, amounting to 46 cases between 26 October and 9 December.Methods. To investigate whether filet américain or an alternative (related) source could be linked to surveillance-reported cases, cases (n=38) were invited to complete a questionnaire and upstream source tracing to map the food supply chain was initiated.Results. Rapid interdisciplinary action resulted in identification of a contaminated 46-ton batch of beef distributed via a Dutch deboning plant as the likely source of infection. In total, 24/29 respondents (83%) could be linked to the incriminated batch of beef products (predominantly filet américain and minced beef).Discussion. Repeated identification of raw meat products as a source of infection emphasizes the importance of awareness of the risk of infection when handling or consuming these products. Improved measures and procedures on product labelling, pre-treatment or product testing should be considered.

MLVA and LPS Characteristics of Brucella canis Isolated from Humans and Dogs in Zhejiang, China.

Brucella canis is a pathogenic bacterium that causes brucellosis in dogs, and its zoonotic potential has been increasing in recent years. B. canis is a rare source of human brucellosis in China, where Brucella melitensis has been the major pathogen associated with human brucellosis outbreaks. In late 2011, a case of a B. canis infection was detected in a human patient in Zhejiang Province, China. To compare the genotypes between strains of B. canis isolated from the patient and from dogs, a multiple-locus variable-number tandem-repeat analysis (MLVA-16) was performed. In addition, the lipopolysaccharide-synthesis-related genes were analyzed with the B. canis reference strain RM6/66. 32 B. canis strains were divided into 26 genotypes using MLVA-16 [Hunter-Gaston Diversity Index (HGDI) = 0.976]. The HGDI indexes for various loci ranged between 0.000 and 0.865. All four Hangzhou isolates were indistinguishable using panel 1 (genotype 3) and panel 2A (genotype 28). However, these strains were distinctly different from other isolates from Beijing, Jiangsu, Liaoning, and Inner Mongolia at Bruce 09. The emergence of a human B. canis infection was limited to an area. Comparative analysis indicated B. canis from canines and humans have no differences in lipopolysaccharide-synthesis locus. The comprehensive approaches have been used to analyze human and canine B. canis isolates, including molecular epidemiological and LPS genetic characteristics. Further detailed analysis of the whole genomic sequencing will contribute to understanding of the pathogenicity of B. canis in humans.

Characterization of Pathogenic Vibrio parahaemolyticus from the Chesapeake Bay, Maryland.

Vibrio parahaemolyticus is the leading cause of bacterial gastroenteritis associated with seafood consumption in the United States. Here we investigated the presence of virulence factors and genetic diversity of V. parahaemolyticus isolated from water, oyster, and sediment samples from the Chesapeake Bay, Maryland. Of more than 2,350 presumptive Vibrio collected, more than half were confirmed through PCR as V. parahaemolyticus, with 10 encoding both tdh and trh and 6 encoding only trh. Potentially pathogenic V. parahaemolyticus were then serotyped with O1:KUT and O3:KUT predominant. Furthermore, pulsed-field gel electrophoresis was performed and the constructed dendrogram displayed high diversity, as did results from multiple-locus VNTR analysis. Vibrio parahaemolyticus was readily isolated from Chesapeake Bay waters but was less frequently isolated from oyster and sediment samples collected during this study. Potentially pathogenic V. parahaemolyticus was isolated in fewer numbers and the isolates displayed expansive diversity. Although characteristics of the pathogenic V. parahaemolyticus were highly variable and the percent of pathogenic V. parahaemolyticus detected was low, it is important to note that, pathogenic V. parahaemolyticus are present in the Chesapeake Bay, warranting seafood monitoring to minimize risk of disease for the public, and to reduce the economic burden of V. parahaemolyticus related illness.

In vitro invasion capacity of Salmonella Typhimurium DT9 isolates sourced from humans and layer hen environments.

In Australia, Salmonella Typhimurium definitive type 9 is frequently isolated during foodborne outbreaks of salmonellosis. Multiple-locus variable number tandem repeat analysis (MLVA) trace back investigations frequently identify isolate distribution patterns that may be epidemiologically linked to disease outbreaks. In this study, the in vitro virulence potential of S. Typhimurium DT9 isolates possessing different MLVA patterns (03 15 07 11 550, 03 24 11 10 523, 03 15 08 11 550 and 03 14 08 11 550) isolated from either humans or layer hens was assessed using a human colon carcinoma cell line. Four strains per MLVA from each host for a total of 32 isolates were included in these experiments. Bacteria were grown to stationary phase and added to cells at a multiplicity of infection of 100. Across all isolates, mean percent recovery ranged from 7.1±1.1 to 33.3±7.1%. The layer hen isolate, KC900 (MLVA profile 03 15 08 11 550), exhibited the greatest invasion with a mean percent recovery of 33.3±7.1%. Overall, layer hen isolates of S. Typhimurium DT9 had significantly higher invasion into Caco2 cells than human isolates (p=.0021). RAPD and enterobacterial repetitive intergenic consensus genomic fingerprinting was also performed. Irrespective of source, the SalmonellaDT9 isolates included in this study exhibited similar fingerprint patterns.