MLVA Types Research Articles

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.

Prevalence, genetic relatedness and antibiotic resistance of hospital-acquired clostridium difficile PCR ribotype 018 strains

Clostridium difficile infection (CDI) is a major healthcare-associated infection. The aim of this study was to investigate the genetic relatedness of the endemic C. difficile PCR ribotype 018 strains in an institution and changes to their characteristics during a five-year period.A total of 207 isolates from inpatients at Hanyang University Hospital from 2009 to 2013 were analysed using multilocus variable-number tandem-repeat analysis (MLVA). Minimum inhibitory concentrations (MICs) of several antibiotics were determined.In total, 204 (98.6%) were genetically related, with a summed tandem-repeat distance (STRD) ≤ 10. Minimum-spanning-tree analysis identified 78 MLVA types, categorized into six clonal complexes (CCs). The largest cluster, CC-I, included 51 MLVA types from 148 isolates (71.5%) and the second largest cluster, CC-II, included 10 MLVA types from 36 isolates (17.4%). Resistance rates for antibiotics were: clindamycin (CLI), 97.6%; moxifloxacin (MXF), 98.6%; vancomycin (VAN), 1.4%; and rifaximin (RFX), 8.2%. All isolates were susceptible to piperacillin/tazobactam (TZP) and metronidazole (MTZ). Comparing the MICs of antibiotics for the isolates each year from 2009 to 2013, MICs of antibiotics that promote CDI, such as CLI, MXF, TZP and RFX, increased over the five-year period (P–value by Kruskal-Wallis test: < 0.0001, <0.0001, <0.0001, and <0.0001 respectively); however, MICs of VAN or MTZ, antibiotics for treatment of CDI, did not increase or decreased over the same time period (P–value by Kruskal-Wallis test: 0.166, <0.0001).C. difficile RT018 isolates in a tertiary hospital over a five-year period presented a close clonal relationship. MICs of antibiotics promoting CDI increased with this clonal expansion.

Molecular epidemiology of Bordetella pertussis in Greece, 2010–2015

PurposeTo determine the predominant strains of Bordetella pertussis in Greece during 2010–2015.MethodologyInfants and children (n=1150) (15 days to 14 years) of Greek, Roma and immigrant origin with different vaccination statuses were hospitalized in Athens, Greece with suspected pertussis infection. IS481/IS1001 real-time PCR confirmed Bordetella spp./B. pertussis infection in 300 samples. A subset of samples (n=153) were analysed by multi-locus variable number tandem repeat analysis (MLVA) and (n=25) by sequence-based typing of the toxin promotor region (ptxP) on DNA extracted from clinical specimens.Results/Key findingsA complete MLVA profile was determined in 66 out of 153 samples; the B. pertussis MLVA type 27 (n=55) was the dominant genotype and all tested samples (n=25) expressed the ptxP3 genotype. The vaccine coverage in the Greek population was 90 %; however, the study population expressed complete coverage in 2 out of 264 infants (0–11 months) and in 20 out of 36 children (1–14 years). Roma and immigrant minorities represent 7 % of the Greek population, but make up 50 % of the study population, indicating a low vaccine coverage among these groups.ConclusionsThe B. pertussis MT27 and ptxP3 genotype is dominant in Greek, Roma and immigrant infants and children hospitalized in Greece. Thus, the predominant MLVA genotype in Greece is similar to other countries using acellular vaccines.

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

Multi‐country outbreak of Salmonella Enteritidis infections linked to Polish eggs

EFSA Supporting PublicationsVolume 14, Issue 12 1353E Technical reportOpen Access Multi-country outbreak of Salmonella Enteritidis infections linked to Polish eggs European Food Safety Authority, European Food Safety AuthoritySearch for more papers by this authorEuropean Centre for Disease Prevention and Control, European Centre for Disease Prevention and ControlSearch for more papers by this author European Food Safety Authority, European Food Safety AuthoritySearch for more papers by this authorEuropean Centre for Disease Prevention and Control, European Centre for Disease Prevention and ControlSearch for more papers by this author First published: 15 December 2017 https://doi.org/10.2903/sp.efsa.2017.EN-1353Citations: 7 Requestor: European Commission Question number: EFSA-Q-2017-00720 This article was originally published on the EFSA website www.efsa.europa.eu on 12 December 2017 as part of EFSA's urgent publication procedures. AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 European Centre for Disease Prevention and Control and European Food Safety Authority. Multicountry outbreak of Salmonella Enteritidis phage type 8, MLVA type 2-9-7-3-2 and 2-9-6-3-2 infections, 7 March 2017. Available from: https://ecdc.europa.eu/sites/portal/files/media/en/publications/Publications/rapid-outbreak-assessment-Salmonella-Enteritidis-7-mar-2017.pdf. 2 REGULATION (EC) No 2160/2003 of the European Parliament and of the Council of 17 November 2003 on the control of salmonella and other specified food-borne zoonotic agents.2003; OJL, L325:[15 p.]. Available from: http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32003R2160&rid=2. 3 European Centre for Disease Prevention and Control. The European Surveillance System (TESSy). 2016. 4 European Centre for Disease Prevention and Control. Laboratory standard operating procedure for multiplelocus variable-number tandem repeat analysis of Salmonella enterica serotype Enteritidis. Stockholm: ECDC; 2016. Available from: http://ecdc.europa.eu/en/publications/Publications/Salmonella-Enteritidis-Laboratory-standard-operating-procedure.pdf. 5 European Food Safety Authority and European Centre for Disease Prevention and Control. The European Union summary report on trends and sources of zoonoses, zoonotic agents, and food-borne outbreaks in 2016. EFSA Journal 2017; 15(12):5077[228 p.]. Available from: https://doi.org/10.2903/j.efsa.2017.5077 6 Official Journal of the European Union. Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents, amending Council Decision90/424/EEC and repealing Council Directive 92/117/EEC. 2003 12.12.2003. Report No. 7 Ashton PM, Nair S, Peters TM, Bale JA, Powell DG, Painset A, et al. Identification of Salmonella for public health surveillance using whole genome sequencing. PeerJ. 2016; 4:e1752. 8Inns T, Ashton PM, Herrera-Leon S, Lighthill J, Foulkes S, Jombart T, et al. Prospective use of whole genome sequencing (WGS) detected a multi-country outbreak of Salmonella Enteritidis. Epidemiology and infection. 2016 Oct 26:1-10. 9Ashton P., Nair S., Peters T., Tewolde R., Day M., Doumith M., et al. Revolutionising Public Health Reference Microbiology using Whole Genome Sequencing: Salmonella as an exemplar. bioRxiv. 2015. Citing Literature Volume14, Issue12December 20171353E ReferencesRelatedInformation

Salmonella in breeding pigs: Shedding pattern, transmission of infection and the role of environmental contamination in Irish commercial farrow-to-finish herds.

This study aimed to provide new insights into the epidemiology of Salmonella in pig production, focusing on potential shedding patterns in breeding pigs throughout a full production cycle and the risk of transmission of infection from the sow to her offspring. A longitudinal study was conducted on five farrow-to-finish commercial pig farms. In each herd, shedding of Salmonella in faeces was monitored in breeders through service, gestation and lactation. Swabs of the farrowing room floor and pools of faeces from piglets were collected on two occasions during lactation. Environmental pen swabs were also taken in the weaning and finisher houses. Salmonella isolates were serotyped, tested for antimicrobial resistance (AMR) and typed by Multiple-Locus Variable number tandem repeat Analysis (MLVA). Shedding by breeding pigs was low in all stages of the production cycle; 5% of sows shed at service, the production stage with highest risk of shedding (p<.01), 1.6% shed during gestation and 2.5% after farrowing. Salmonella was detected in 4% of piglet faecal pools in the second week post-farrowing and 5% in the fourth week. Serotyping and AMR profiles of Salmonella isolates revealed that strains in sows and gilts were mostly different from strains isolated in weaner and finisher facilities. MLVA typing confirmed that the source of infection in piglets was in most instances the contaminated environment rather than their dam. Based on the typing results, it appears that sows do not pose a major risk in the maintenance and transmission of Salmonella to their progeny but instead the contaminated pen environment is more significant in the perpetuation of the organism on farm.

Persistence of the same genetic type of Mycoplasma hyopneumoniae in a closed herd for at least two years

Two cross-sectional studies were carried out in 2013 and 2015 monitoring for Mycoplasma hyopneumoniae presence in a swine farm. In these studies, the genetic diversity of M. hyopneumoniae was assessed in clinical specimens using a Multiple Locus Variable-number tandem repeat Analysis (MLVA) targeting P97 R1, P146 R3 and H4 loci. The samples from August 2015 showed the MLVA profile prevalent in June 2013, therefore it can be concluded that a same genetic type of M. hyopneumoniae can persist for at least two years in a closed herd. In addition, the nested PCR reactions implemented in this study showed to be useful for MLVA typing in non-invasive clinical samples.

Species identification and molecular typing of human Brucella isolates from Kuwait

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90–99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

Molecular epidemiology of Bordetella pertussis in Cambodia determined by direct genotyping of clinical specimens

ObjectivesThis study sought to determine the genotypes of circulating Bordetella pertussis, the causative agent of pertussis, in Cambodia by direct molecular typing of clinical specimens. MethodsDNA extracts from nasopharyngeal swabs obtained from 82 pertussis patients in 2008–2016 were analyzed by multilocus variable-number tandem repeat analysis (MLVA). B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter ptxP were also investigated by DNA sequence-based typing. ResultsForty-four DNA extracts (54%) yielded a complete MLVA profile, and these were sorted into 8 MLVA types (MT18, MT26, MT27, MT29, MT43, MT72, MT95, and MT200). MT27 and MT29, which are common in developed countries, were the predominant strain types (total 73%). The predominant profile of virulence-associated allelic genes was the combination of ptxP3/ptxA1/prn2/fim3A (48%). MT27 strains were detected during the entire study period, whereas MT29 strains were only found in 2014–2016. ConclusionsThe B. pertussis population in Cambodia, where a whole-cell pertussis vaccine (WCV) has been continuously used, resembled those observed previously in developed countries where acellular pertussis vaccines are used. Circulating B. pertussis strains in Cambodia were distinct from those in other countries using WCVs.

Pyrazinamide resistance-conferring mutations in pncA and the transmission of multidrug resistant TB in Georgia

BackgroundThe ongoing epidemic of multidrug-resistant tuberculosis (MDR-TB) in Georgia highlights the need for more effective control strategies. A new regimen to treat MDR-TB that includes pyrazinamide (PZA) is currently being evaluated and PZA resistance status will largely influence the success of current and future treatment strategies. PZA susceptibility testing was not routinely performed at the National Reference Laboratory (NRL) in Tbilisi between 2010 and September 2015. We here provide a first insight into the prevalence of PZA resistant TB in this region.MethodsPhenotypic susceptibility to PZA was determined in a convenience collection of well-characterised TB patient isolates collected at the NRL in Tbilisi between 2012 and 2013. In addition, the pncA gene was sequenced and whole genome sequencing was performed on two isolates.ResultsOut of 57 isolates tested 33 (57.9%) showed phenotypic drug resistance to PZA and had a single pncA mutation. All of these 33 isolates were MDR-TB strains. pncA mutations were absent in all but one of the 24 PZA susceptible isolate. In total we found 18 polymorphisms in the pncA gene. From the two major MDR-TB clusters represented (94–32 and 100–32), 10 of 15, 67.0% and 13 of 14, 93.0% strains, respectively were PZA resistant. We also identified a member of the potentially highly transmissive clade A strain carrying the characteristic I6L substitution in PncA. Another strain with the same MLVA type as the clade A strain acquired a different mutation in pncA and was genetically more distantly related suggesting that different branches of this particular lineage have been introduced into this region.ConclusionIn this high MDR-TB setting more than half of the tested MDR-TB isolates were resistant to PZA. As PZA is part of current and planned MDR-TB treatment regimens this is alarming and deserves the attention of health authorities. Based on our typing and sequence analysis results we conclude that PZA resistance is the result of primary transmission as well as acquisition within the patient and recommend prospective genotyping and PZA resistance testing in high MDR-TB settings. This is of utmost importance in order to preserve bacterial susceptibility to PZA to help protect (new) second line drugs in PZA containing regimens.

Epidemiology and Molecular Characteristics of Mycoplasma pneumoniae During an Outbreak of M. pneumoniae-associated Stevens-Johnson Syndrome.

An increase in Mycoplasma pneumoniae-associated Stevens-Johnson syndrome (SJS) cases at a Colorado pediatric hospital led to an outbreak investigation. We describe the epidemiologic and molecular characteristics of M. pneumoniae among SJS case-patients and surrounding community members during the outbreak. M. pneumoniae polymerase chain reaction-positive respiratory specimens from 5 Colorado hospitals and 4 referral laboratories underwent confirmatory polymerase chain reaction testing; positive specimens then underwent multilocus variable-number tandem-repeat analysis (MLVA) and macrolide resistance testing. Three SJS-M. pneumoniae case-patient households were surveyed using a standardized questionnaire, and nasopharyngeal/oropharyngeal swabs were obtained from all consenting/assenting household contacts. International Classification of Diseases, 9th revision codes were used to identify pneumonia cases among Colorado patients 5-21 years of age from January 2009 to March 2014. Three different M. pneumoniae MLVA types were identified among the 5 SJS case-patients with confirmed infection; MLVA type 3-X-6-2 was seen more commonly in SJS case-patients (60%) than in 69 non-SJS community specimens (29%). Macrolide resistance was identified in 7% of community specimens but not among SJS case-patients. Of 15 household contacts, 5 (33%) were M. pneumoniae positive; all MLVA types were identical to those of the corresponding SJS case-patient, although the specimen from 1 contact was macrolide resistant. Overall pneumonia cases as well as those caused by M. pneumoniae specifically peaked in October 2013, coinciding with the SJS outbreak. The outbreak of M. pneumoniae-associated SJS may have been associated with a community outbreak of M. pneumoniae; clinicians should be aware of the M. pneumoniae-SJS relationship. Household transmission of M. pneumoniae was common within the households investigated.

Multi-locus variable-number tandem repeat analysis of Bordetella pertussis isolates circulating in Poland in the period 1959-2013.

Despite the long history of pertussis vaccination and high vaccination coverage in Poland and many other developed countries, pertussis incidence rates have increased substantially, making whooping cough one of the most prevalent vaccine-preventable diseases. Among the factors potentially involved in pertussis resurgence, the adaptation of the Bordetella pertussis population to country-specific vaccine-induced immunity through selection of non-vaccine-type strains still needs detailed studies. Multi-locus variable-number tandem repeat analysis (MLVA), also linked to MLST and PFGE profiling, was applied to trace the genetic changes in the B. pertussis population circulating in Poland in the period 1959-2013 versus country-specific vaccine strains. Generally, among 174 B. pertussis isolates, 31 MLVA types were detected, of which 11 were not described previously. The predominant MLVA types of recent isolates in Poland were different from those of the typical isolates circulating in other European countries. The MT27 type, currently predominant in Europe, was rarely seen and detected in only five isolates among all studied. The features of the vaccine strains used for production of the pertussis component of a national whole-cell diphtheria-tetanus-pertussis (DTP) vaccine, as studied by MLVA and MLST tools, were found to not match those observed in the currently circulating B. pertussis isolates in Poland. Differences traced by MLVA in relation to the MLST and PFGE profiling confirmed that the B. pertussis strain types currently observed elsewhere in Europe, even if appearing in Poland, were not able to successfully disseminate within a human population in Poland that has been vaccinated with a whole-cell pertussis vaccine not used in other countries.

Subtyping of Canadian isolates of Salmonella Enteritidis using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) alone and in combination with Pulsed-Field Gel Electrophoresis (PFGE) and phage typing

Salmonella enterica subspecies enterica serovar Enteritidis (SE) is one of the most common causes of human salmonellosis and in Canada currently accounts for over 40% of human cases. Reliable subtyping of isolates is required for outbreak detection and source attribution. However, Pulsed-Field Gel Electrophoresis (PFGE), the current standard subtyping method for Salmonella spp., is compromised by the high genetic homogeneity of SE. Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) was introduced to supplement PFGE, although there is a lack of data on the ability of MLVA to subtype Canadian isolates of SE. Three subtyping methods, PFGE, MLVA and phage typing were compared for their discriminatory power when applied to three panels of Canadian SE isolates: Panel 1: 70 isolates representing the diversity of phage types (PTs) and PFGE subtypes within these PTs; Panel 2: 214 apparently unrelated SE isolates of the most common PTs; and Panel 3: 27 isolates from 10 groups of epidemiologically related strains.For Panel 2 isolates, four MLVA subtypes were shared among 74% of unrelated isolates and in Panel 3 isolates, one MLVA subtype accounted for 62% of the isolates. For all panels, combining results from PFGE, MLVA and PT gave the best discrimination, except in Panel 1, where the combination of PT and PFGE was equally as high, due to the selection criteria for this panel. However, none of these methods is sufficiently discriminatory alone for reliable outbreak detection or source attribution, and must be applied together to achieve sufficient discrimination for practical purposes. Even then, some large clusters were not differentiated adequately. More discriminatory methods are required for reliable subtyping of this genetically highly homogeneous serovar. This need will likely be met by whole genome sequence analysis given the recent promising reports and as more laboratories implement this tool for outbreak response and surveillance.

Within-Flock Population Dynamics of Dichelobacter nodosus.

Footrot causes 70–90% of lameness in sheep in Great Britain. With approximately 5% of 18 million adult sheep lame at any one time, it costs the UK sheep industry £24–84 million per year. The Gram-negative anaerobe Dichelobacter nodosus is the causative agent, with disease severity influenced by bacterial load, virulence, and climate. The aim of the current study was to characterize strains of D. nodosus isolated by culture of swabs from healthy and diseased feet of 99 ewes kept as a closed flock over a 10-month period and investigate persistence and transmission of strains within feet, sheep, and the flock. Overall 268 isolates were characterized into strains by serogroup, proline–glycine repeat (pgr) status, and multi-locus variable number tandem repeat analysis (MLVA). The culture collection contained 87 unique MLVA profiles and two major MLVA complexes that persisted over time. A subset of 189 isolates tested for the virulence marker aprV2 were all positive. The two MLVA complexes (76 and 114) comprised 62 and 22 MLVA types and 237 and 28 isolates, respectively. Serogroups B, and I, and pgrB were associated with MLVA complex 76, whereas serogroups D and H were associated with MLVA complex 114. We conclude that within-flock D. nodosus evolution appeared to be driven by clonal diversification. There was no association (P > 0.05) between serogroup, pgr, or MLVA type and disease state of feet. Strains of D. nodosus clustered within sheep and were transmitted between ewes over time. D. nodosus was isolated at more than one time point from 21 feet, including 5 feet where the same strain was isolated on two occasions at an interval of 1–33 weeks. Collectively, our results indicate that D. nodosus strains persisted in the flock, spread between sheep, and possibly persisted on feet over time.

Multi‐country outbreak of Salmonella Enteritidis phage type 8, MLVA profile 2‐9‐7‐3‐2 and 2‐9‐6‐3‐2 infections

EFSA Supporting PublicationsVolume 14, Issue 3 1188E Technical reportOpen Access Multi-country outbreak of Salmonella Enteritidis phage type 8, MLVA profile 2-9-7-3-2 and 2-9-6-3-2 infections European Food Safety Authority, European Food Safety AuthoritySearch for more papers by this authorEuropean Centre for Disease Prevention and Control, European Centre for Disease Prevention and ControlSearch for more papers by this author European Food Safety Authority, European Food Safety AuthoritySearch for more papers by this authorEuropean Centre for Disease Prevention and Control, European Centre for Disease Prevention and ControlSearch for more papers by this author First published: 10 March 2017 https://doi.org/10.2903/sp.efsa.2017.EN-1188Citations: 9 Requestor: European Commission Question number: EFSA-Q-2017-00109 This article was originally published on the EFSA website www.efsa.europa.eu on 7 March 2017 as part of EFSA's urgent publication procedures. AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 European Centre for Disease Prevention and Control (ECDC) and European Food Safety Authority (EFSA). Multi-country outbreak of Salmonella Enteritidis phage type 8 MLVA type 2-9-7-3-2 and 2-9-6-3-2 infections, 27 October 2016. Stockholm: ECDC; 2016. Available from: http://ecdc.europa.eu/en/publications/Publications/ROA-ECDC-EFSA-Salmonella%20Enteritidis.pdf. 2 Regulation (EC) No 2160/2003 of the European Parliament and of the Council of 17 November 2003 on the control of salmonella and other specified food-borne zoonotic agents. 2003; OJL, L325:[15 p.]. Available from: http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32003R2160&rid=2. 3 European Centre for Disease Prevention and Control. The European Surveillance System (TESSy) [Database]. ECDC: Stockholm; 2016. 4 European Centre for Disease Prevention and Control. Laboratory standard operating procedure for multiplelocus variable-number tandem repeat analysis of Salmonella enterica serotype Enteritidis. Stockholm: ECDC; 2016. Available from: http://ecdc.europa.eu/en/publications/Publications/Salmonella-Enteritidis-Laboratorystandard-operating-procedure.pdf. 5 EFSA (European Food Safety Authority), ECDC (European Centre for Disease Prevention and Control). The European Union summary report on trends and sources of zoonoses, zoonotic agents, and food-borne outbreaks in 2015. EFSA Journal. 2016; 14(12):[231 p.]. Available from: http://ecdc.europa.eu/en/publications/Publications/EU-summary-report-trends-sources-zoonoses-2015.pdf. 6 Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents, amending Council Decision 90/424/EEC and repealing Council Directive 92/117/EEC. 2003. 7Ashton PM, Nair S, Peters TM, Bale JA, Powell DG, Painset A, et al. Identification of Salmonella for public health surveillance using whole genome sequencing. PeerJ. 2016 Apr 5;4:e1752. 8Inns T, Ashton PM, Herrera-Leon S, Lighthill J, Foulkes S, Jombart T, et al. Prospective use of whole genome sequencing (WGS) detected a multi-country outbreak of Salmonella Enteritidis. Epidemiol Infect. 2017 Jan; 145(2): 289– 298. 9Ashton P, Nair S, Peters T, Tewolde R, Day M, Doumith M, et al. Revolutionising public health reference microbiology using whole genome sequencing: Salmonella as an exemplar. bioRxiv 033225; doi: https://doi.org/10.1101/033225. Available from: http://biorxiv.org/content/biorxiv/early/2015/11/29/033225.full.pdf Citing Literature Volume14, Issue3March 20171188E ReferencesRelatedInformation

Population and evolutionary dynamics of Shiga-toxin producing Escherichia coli O157 in a beef herd: A longitudinal study.

Shiga toxin producing Escherichia coli O157:H7 (STEC O157) is naturally found in the gastrointestinal tract of cattle and can cause severe disease in humans. There is limited understanding of the population dynamics and microevolution of STEC O157 at herd level. In this study, isolates from a closed beef herd of 23 cows were used to examine the population turnover in the herd. Of the nine STEC O157 clades previously described, clade 7 was found in 162 of the 169 isolates typed. Multiple locus variable number tandem repeat analysis (MLVA) differentiated 169 isolates into 33 unique MLVA types. Five predominant MLVA types were evident with most of the remaining types containing only a single isolate. MLVA data suggest that over time clonal replacement occurred within the herd. Genome sequencing of 18 selected isolates found that the isolates were divided into four lineages, representing four different 'clones' in the herd. Genome data confirmed clonal replacement over time and provided evidence of cross transmission of strains between cows. The findings enhanced our understanding of the population dynamics of STEC O157 in its natural host that will help developing effective control measures to prevent the spread of the pathogen to the human population.

Dynamics of Salmonella Shedding and Welfare of Hens in Free-Range Egg Production Systems.

The current study investigated the effect of environmental stressors (i.e., weather changes) on Salmonella shedding in free-range production systems and the correlations with behavioral and physiological measures (i.e., fecal glucocorticoid metabolites). This involved longitudinal and point-in-time surveys of Salmonella shedding and environmental contamination on four commercial free-range layer farms. The shedding of Salmonella was variable across free-range farms and in different seasons. There was no significant effect of season on the Salmonella prevalence during this investigation. In this study, the combined Salmonella most probable number (MPN) counts in environmental (including feces, egg belt, dust, nest box, and ramp) samples were highest in samples collected during the summer season (4th sampling, performed in February). The predominant serovars isolated during this study were Salmonella enterica serovar Mbandaka and Salmonella enterica serovar Typhimurium phage types 135 and 135a. These two phage types were involved in several egg product-related Salmonella outbreaks in humans. Multilocus variable-number tandem-repeat analysis (MLVA) results indicated that MLVA types detected from human food poisoning cases exhibited MLVA patterns similar to the strains isolated during this study. All Salmonella isolates (n = 209) were tested for 15 different genes involved in adhesion, invasion, and survival of Salmonella spp. We also observed variations for sopA, ironA, and misL There were no positive correlations between fecal corticosterone metabolite (FCM) and Salmonella prevalence and/or shedding in feces. Also, there were no positive correlations between Salmonella prevalence and Salmonella count (log MPN) and any of the other welfare parameters.IMPORTANCE In this study, the welfare of laying hens and Salmonella shedding were compared over a prolonged period of time in field conditions. This study investigated the long-term shedding of Salmonella serovars in a free-range egg production system. Given that there is increasing demand for free-range eggs, it is essential to understand the risks associated with such a production system.

Molecular typing of Salmonella paratyphi A strains isolated in Hangzhou area from 2002 to 2013

Objective To analyze the molecular characteristics of Salmonella paratyphi A (S.paratyphi A) strains prevailing in Hangzhou area in recent years. Methods Pulse field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) were performed for molecular typing and epidemiological analysis of 72 S. paratyphi A strains isolated in Hangzhou area during 2002 to 2013. Results The 72 S. paratyphi A strains were divided into 11 PFGE (by using restriction enzymes of Xba Ⅰ and Bln Ⅰ) and 6 MLVA types. Among the selected 34 variable number tandem repeat (VNTR) sites, 4 sites (1188K, 2075K, 2201K and 4346K) showed high polymorphism, in which PFGE displayed a higher resolution than MLVA. Except for the 5 PFGE types of X4B5, X7B7, X8B8, X9B9 and X10B10, the other 6 PFGE types belonged to a same clone sharing a similarity of greater than 95%, and the S. Paratyphi A strains in that clone accounted for 93.1% of the total strains isolated in Hangzhou. Conclusion The occurrence of paratyphoid A in Hangzhou area from 2002 to 2013 was mainly caused by S. paratyphi A strains belonging to the same clone. Combination of PFGE with MLVA was conducive to epidemiological investigation of paratyphoid A. Key words: Salmonella paratyphi A; Pulse field gel electrophoresis; Multiple-locus variable-number tandem repeat analysis