Multiple-locus Variable-number Tandem Repeat Research Articles

Antimicrobial Susceptibility Evaluation and Multiple-Locus Variable Number Tandem Repeat Analysis of Neisseria gonorrhoeae Isolates in China in 2012.

This study aimed to gain information on the antimicrobial susceptibility and molecular epidemiological typing of Neisseria gonorrhoeae (NG) isolates in China in 2012. A total of 244 NG isolates were consecutively recovered from patients with uncomplicated gonorrhea attending sexually transmitted disease clinics in 3 Chinese cities-Guangzhou, Nanjing, and Tianjin-in 2012. Neisseria gonorrhoeae susceptibilities to penicillin and tetracycline were examined by detecting penicillinase-producing NG (PPNG) and high-level tetracycline-resistant NG, and NG susceptibilities to ciprofloxacin, spectinomycin, ceftriaxone, and cefixime were determined using an agar dilution method. Neisseria gonorrhoeae isolates were typed by multiple-locus variable number tandem repeat analysis. We conducted a χ analysis to compare clusters with Bonferroni correction and Kruskal-Wallis test. Neisseria gonorrhoeae isolates gathered from the 3 cities differed significantly in the prevalence of tetracycline-resistant NG (P < 0.001) and NG treated with ceftriaxone with a minimum inhibitory concentration of 0.125 mg/L or higher (P < 0.001). The analysis of the combination of the 7 variable number of tandem repeats loci for all of the 244 isolates yielded 110 multiple-locus variable number tandem repeat analysis types falling into 5 clusters. Cluster III was associated with PPNG, whereas cluster II was associated with non-PPNG (P < 0.05) and NG treated with ceftriaxone with a minimum inhibitory concentration of 0.125 mg/L or higher (P < 0.05). Antimicrobials that can be used with confidence to treat NG infection currently in China include ceftriaxone and spectinomycin, but not penicillin, tetracycline, ciprofloxacin, and cefixime. Moreover, some of the resulting clusters were associated with PPNG and NG with decreased ceftriaxone susceptibility.

Foodborne Salmonellosis in Italy: Characterization of Salmonella enterica Serovar Typhimurium and Monophasic Variant 4,[5],12:i− Isolated from Salami and Human Patients

Salmonella enterica serovar Typhimurium (STm) and its monophasic variant 4,[5],12:i:- (VMSTm) have been responsible for an increased number of foodborne infections in humans in Europe in recent years. The aim of this study was to investigate the origin of three foodborne salmonellosis outbreaks that occurred in Pavia Province (Lombardy region, northern Italy) in 2010. Phenotypic and genetic characteristics of the STm and VMSTm isolates from patients and from food that were recovered in the framework of the three outbreaks were evaluated through serotyping, phage typing, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multiple-locus variable-number tandem repeat analysis (MLVA). Salami from three artisan producers, which had all purchased meat from the same slaughterhouse, was the food source of infection in outbreak I. STm isolates were recovered from salami and patients with symptoms of gastroenteritis. These isolates had the same PFGE type and the same rare MLVA profile (3-18-9-NA-211). The same molecular profiles were found in an STm isolate from a salami, which likely was the source of another family outbreak (II). A VMSTm strain with common phenotypic and molecular profiles was isolated from three hospitalized patients and identified as the cause of another putative outbreak (III). During the following 3 years (2011 through 2013), 360 salami produced in Pavia Province were monitored for the presence of S. enterica . In 2011, no STm and VMSTm isolates were recovered from 159 salami tested. During 2012 and 2013, 13.9% of 201 tested salami harbored S. enterica , and half of the isolates were VMSTm, mainly in salami from those artisan producers involved in the previous outbreaks. These isolates were genetically variable, especially in terms of MLVA profiles. The data collected suggest that from 2012, VMSTm has replaced STm in the environments of the salami producers monitored in this study, and these data confirm the dominance of this emergent serovar along the pork supply chain.

Prevalence, antimicrobial resistance and multiple-locus variable-number tandem-repeat analysis profiles of diarrheagenic Escherichia coli isolated from different retail foods

Diarrheagenic E. coli (DEC) isolates were recovered from local retail markets and the Osaka Municipal Central Wholesale Market in Japan. Retail food samples were collected for analysis in Osaka Japan from 2005 to 2008 and consisted of 32 beef, 28 pork, 20 poultry, 136 fish, 66 fruits and vegetables and 51 ready–to-eat (RTE) food samples. A total of 82 DEC strains were recovered from 64 (19%) food samples with the highest prevalence in poultry (100%, 20/20), followed by pork (54%, 15/28), beef (28%, 9/32), fruits and vegetables (12%, 8/66), fish (6.6%, 9/136) and RTE foods (5.9%, 3/51). Most of the strains belonged to E. coli possessing the enteroaggregative E. coli (EAEC) heat-stable enterotoxin 1 (EAST1) gene (EAST1EC; n=62, P<0.0001) and enteropathogenic E. coli (EPEC; n=16, P<0.01), whereas only 1 strain belonged to Shiga toxin-producing E. coli (STEC), 1 to EAEC and 2 to enterotoxigenic E. coli (ETEC) strains. Of the 82 DEC isolates, 22 O and 13H serogroups were detected, including some specific serogroups (O91, O103, O115, O119, O126, and O157) which have been associated with human diarrheal infections. Phylogenetic group A and B1 were predominant among the DEC isolates. Antimicrobial resistance to tetracycline was most common (49%), followed by nalidixic acid (28%), ampicillin (24%), sulfamethoxazole/trimethoprim (20%), and cephalothin (18%). All isolates were susceptible to aztreonam. Of the resistant strains, 44% (22/50) demonstrated resistance to >3 antimicrobial agents. Isolates resistant to >5 antimicrobials were only found in the meat samples, while isolates from the fruits and vegetables as well as RTE foods showed resistance to only 1 or 2 antimicrobial agents. Sixty one percent of EAST1EC, 56% of EPEC and all of the EAEC and ETEC were resistant to at least 1 antimicrobial agent. Multiple-locus variable-number tandem repeat analysis (MLVA) was used in this study for genotyping of DEC. The 82 isolates collected for this study showed 77 distinct MLVA profiles located among 3 branches. The Simpson's Index of Diversity (D) was 99.9% at its highest. The high diversity of these food strains would suggest their originating from a variety of sources and environments. In conclusion, retail food samples in Japan were contaminated with DEC; EAST1EC, a putative DEC, were detected at high rates in poultry, pork and beef. Isolates resistant to >3 antimicrobials were found only in raw meat and fish. Food animals may act as the reservoir for multi-resistant bacteria. Due to the finding that nearly 1/3 of EAST1EC strains were resistant to >3 antimicrobials, additional surveillance for EAST1EC should be initiated.

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

MLVA for Salmonella enterica subsp. enterica Serovar Dublin: Development of a Method Suitable for Inter-Laboratory Surveillance and Application in the Context of a Raw Milk Cheese Outbreak in France in 2012.

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) figures among the most frequently isolated Salmonella strains in humans in France. This serovar may affect production and animal health mainly in cattle herds with corresponding high economic losses. Given that the current gold standard method, pulsed-field gel electrophoresis (PFGE), provides insufficient discrimination for epidemiological investigations, we propose a standard operating procedure in this study for multiple-locus variable number tandem repeat analysis (MLVA) of S. Dublin, suitable for inter-laboratory surveillance. An in silico analysis on the genome of S. Dublin strains CT_02021853 was performed to identify appropriate microsatellite regions. Of 21 VNTR loci screened, six were selected and 401 epidemiologically unrelated and related strains, isolated from humans, food and animals were analyzed to assess performance criteria such as typeability, discriminatory power and epidemiological concordance. The MLVA scheme developed was applied to an outbreak involving Saint-Nectaire cheese for which investigations were conducted in France in 2012, making it possible to discriminate between epidemiologically related strains and sporadic case strains, while PFGE assigned only a single profile. The six loci selected were sequenced on a large set of strains to determine the sequence of the repeated units and flanking regions, and their stability was evaluated in vivo through the analysis of the strains investigated from humans, food and the farm environment during the outbreak. The six VNTR selected were found to be stable and the discriminatory power of the MLVA method developed was calculated to be 0.954 compared with that for PFGE, which was only 0.625. Twenty-four reference strains were selected from the 401 examined strains in order to represent most of the allele diversity observed for each locus. This reference set can be used to harmonize MLVA results and allow data exchange between laboratories. This original MLVA protocol could be used easily and routinely for monitoring of serovar Dublin isolates and for conducting outbreak investigations.

Molecular Epidemiology of Leptospira Serogroup Pomona Infections Among Wild and Domestic Animals in Spain.

Strains of Leptospira serogroup Pomona are known to cause widespread animal infections in many parts of the world. Forty-three isolates retrieved from domestic animals and wild small mammals suggest that serogroup Pomona is epidemiologically relevant in Spain. This is supported by the high prevalence of serovar Pomona antibodies in livestock and wild animals. In this study, the strains were serologically and genetically characterized in an attempt to elucidate their epidemiology. Serological typing was based on the microscopic agglutination test but molecular typing involved species-specific polymerase chain reaction, restriction endonuclease analysis, and multiple-locus variable-number tandem repeat analysis. The study revealed that the infections are caused by two serovars, namely Pomona and Mozdok. Serovar Pomona was derived only from farm animals and may be adapted to pigs, which are recognized as the maintenance host. The results demonstrated that serovar Pomona is genetically heterogeneous and three different types were recognized. This heterogeneity was correlated with different geographical distributions of the isolates. All strains derived from small wild mammals were identified as serovar Mozdok. Some isolates of this serovar retrieved from cattle confirm that this serovar may also be the cause of infections in food-producing animals for which these wild species may be source of infection.

Linezolid-resistant enterococci in Polish hospitals: species, clonality and determinants of linezolid resistance

The significant increase of the linezolid-resistant enterococci (LRE) has been observed in Polish hospitals since 2012 and our study aimed at elucidating the possible reasons for this phenomenon. Polish LRE isolates were analysed by multilocus-sequence typing (MLST) and multiple locus variable-number tandem repeat (VNTR) analysis (MLVA), polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) to establish clonal relatedness and mechanism of linezolid resistance, respectively. Fifty analysed LRE (2008–2015) included mostly Enterococcus faecium (82%) and Enterococcus faecalis (16%). Enterococcus faecium belonged to the hospital-adapted lineages 17/18 and 78, while E. faecalis isolates represented ST6, a hospital-associated type, and ST116, found in both humans and food-production animals. The G2576T 23S rRNA mutation was the most frequent (94%) mechanism of linezolid/tedizolid resistance of LRE. None of the isolates carried the plasmid-associated gene of Cfr methyltransferase, whereas optrA, encoding the ABC-type drug transporter, was identified in two E. faecalis isolates. In these isolates, optrA was located on a plasmid, transferable to both E. faecium and E. faecalis, whose partial (36.3 kb) sequence was 100% identical to the pE394 plasmid, identified previously in China in both clinical and farm animal isolates. The optrA–E. faecium transconjugant displayed a significant growth deficiency, in contrast to the optrA–E. faecalis. Our study indicates the role of mutation acquisition by hospital-adapted clones of enterococci as a major driver of increasing resistance to linezolid and tedizolid. Transferability and apparent lack of a biological cost of resistance suggest that E. faecalis may be a natural reservoir of optrA, an emerging mechanism of oxazolidinone resistance.

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

Improvement of infection control management by routine molecular evaluation of pathogen clusters

BackgroundUndetected pathogen clusters can often be a source of spreading in-hospital infections. Unfortunately, detection of clusters can be problematic because epidemiological connection is not always easily established. Infection prevention and control (IPC) measures, however, are most effective when applied at the earliest possible stage. AimThe goal of our study was to evaluate the benefits of routine use of molecular typing techniques for IPC management in a large University teaching hospital. MethodsWe implemented daily routine molecular typing of pathogen clusters using cost-effective standard methods such as random amplified polymorphic DNA PCR, multiple-locus variable number tandem repeat analysis, and spa-typing over a 4-year study period (2012–2015). FindingsFour pathogen clusters were evaluated: (I) 14 cases of Clostridium difficile in a peripheral ward, (II) 17 cases of methicillin-resistant Staphylococcus aureus (MRSA) in two intensive-care units (ICUs), (III) 21 cases of multidrug-resistant Klebsiella pneumoniae within one department, and (IV) 6 cases of vancomycin-resistant Enterococcus faecium in an interdisciplinary ICU. Typing revealed that cluster I was not caused by an outbreak strain but was likely due to different endogenous infections. Clusters III and IV showed a classical space–time clustering of point source outbreaks. Cluster II represented a prolonged temporal cluster, which would have gone undetected without molecular typing because of large intercase intervals. ConclusionImplementing daily routine molecular typing is effective for detecting and analyzing pathogen clusters. Falsely suspected outbreaks can be quickly resolved, whereas actual outbreaks can be identified faster, so that targeted IPC measures can be applied earlier.

Marine Bivalve Mollusks As Possible Indicators of Multidrug-Resistant Escherichia coli and Other Species of the Enterobacteriaceae Family

The mechanisms for the development and spread of antibacterial resistance (ABR) in bacteria residing in environmental compartments, including the marine environment, are far from understood. The objective of this study was to examine the ABR rates in Escherichia coli and other Enterobacteriaceae isolates obtained from marine bivalve mollusks collected along the Norwegian coast during a period from October 2014 to November 2015. A total of 549 bivalve samples were examined by a five times three tube most probable number method for enumeration of E. coli in bivalves resulting in 199 isolates from the positive samples. These isolates were identified by biochemical reactions and matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry, showing that 90% were E. coli, while the remaining were species within the genera Klebsiella, Citrobacter, and Enterobacter. All 199 isolates recovered were susceptibility tested following the European Committee on Antimicrobial Susceptibility Testing disk diffusion method. In total, 75 of 199 (38%) isolates showed resistance to at least one antibacterial agent, while multidrug-resistance were seen in 9 (5%) isolates. One isolate conferred resistance toward 15 antibacterial agents. Among the 75 resistant isolates, resistance toward extended-spectrum penicillins (83%), aminoglycosides (16%), trimethoprim (13%), sulfonamides (11%), tetracyclines (8%), third-generation cephalosporins (7%), amphenicols (5%), nitrofurans (5%), and quinolones (5%), were observed. Whole-genome sequencing on a selection of 10 E. coli isolates identified the genes responsible for resistance, including blaCTX-M genes. To indicate the potential for horizontal gene transfer, conjugation experiments were performed on the same selected isolates. Conjugative transfer of resistance was observed for six of the 10 E. coli isolates. In order to compare E. coli isolates from bivalves with clinical strains, multiple-locus variable number tandem repeats analysis (MLVA) was applied on a selection of 30 resistant E. coli isolates. The MLVA-profiles were associated with community-acquired E. coli strains causing bacteremia. Our study indicates that bivalves represent an important tool for monitoring antibacterial resistant E. coli and other members of the Enterobacteriaceae family in the coastal environment.

Impact of diversity of Mycoplasma hyopneumoniae strains on lung lesions in slaughter pigs

The importance of diversity of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is not yet fully known. This study investigated the genetic diversity of M. hyopneumoniae strains in ten pig herds, and assessed associations between the presence of different strains of M. hyopneumoniae and lung lesions at slaughter. Within each herd, three batches of slaughter pigs were investigated. At slaughter, from each batch, 20 post mortem bronchoalveolar lavage fluid samples were collected for multiple locus variable-number tandem repeat analysis (MLVA), and lung lesions (Mycoplasma-like lesions, fissures) were examined. Multivariable analyses including potential risk factors for respiratory disease were performed to assess associations between the number of different strains per batch (three categories: one strain, two–six strains, ≥seven strains), and the lung lesions as outcome variables. In total, 135 different M. hyopneumoniae strains were found. The mean (min.–max.) number of different strains per batch were 7 (1–13). Batches with two–six strains or more than six strains had more severe Mycoplasma-like lesions (P = 0.064 and P = 0.012, respectively), a higher prevalence of pneumonia [odds ratio (OR): 1.30, P = 0.33 and OR: 2.08, P = 0.012, respectively], and fissures (OR = 1.35, P = 0.094 and OR = 1.70, P = 0.007, respectively) compared to batches with only one strain. In conclusion, many different M. hyopneumoniae strains were found, and batches of slaughter pigs with different M. hyopneumoniae strains had a higher prevalence and severity of Mycoplasma-like lung lesions at slaughter, implying that reducing the number of different strains may lead to less lung lesions at slaughter and better respiratory health of the pigs.

Genetic source tracking of an anthrax outbreak in Shaanxi province, China

BackgroundAnthrax is an acute zoonotic infectious disease caused by the bacterium known as Bacillus anthracis. From 26 July to 8 August 2015, an outbreak with 20 suspected cutaneous anthrax cases was reported in Ganquan County, Shaanxi province in China. The genetic source tracking analysis of the anthrax outbreak was performed by molecular epidemiological methods in this study.MethodsThree molecular typing methods, namely canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA), and single nucleotide repeat (SNR) analysis, were used to investigate the possible source of transmission and identify the genetic relationship among the strains isolated from human cases and diseased animals during the outbreak.ResultsFive strains isolated from diseased mules were clustered together with patients’ isolates using canSNP typing and MLVA. The causative B. anthracis lineages in this outbreak belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-31 genotype (the 31 genotype in MLVA15 scheme). Because nine isolates from another four provinces in China were clustered together with outbreak-related strains by the canSNP (A.Br.001/002 subgroup) and MLVA15 method (MLVA15-31 genotype), still another SNR analysis (CL10, CL12, CL33, and CL35) was used to source track the outbreak, and the results suggesting that these patients in the anthrax outbreak were probably infected by the same pathogen clone.ConclusionsIt was deduced that the anthrax outbreak occurred in Shaanxi province, China in 2015 was a local occurrence.

Evaluation of MLVA for epidemiological typing and outbreak detection of ESBL-producing Escherichia coli in Sweden

BackgroundTo identify the spread of nosocomial infections and halt outbreak development caused by Escherichia coli that carry multiple antibiotic resistance factors, such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases, is becoming demanding challenges due to the rapid global increase and constant and increasing influx of these bacteria from the community to the hospital setting. Our aim was to assess a reliable and rapid typing protocol for ESBL-E. coli, with the primary focus to screen for possible clonal relatedness between isolates. All clinical ESBL-E. coli isolates, collected from hospitals (n = 63) and the community (n = 41), within a single geographical region over a 6 months period, were included, as well as clinical isolates from a polyclonal outbreak (ST131, n = 9, and ST1444, n = 3). The sporadic cases represented 36 STs, of which eight STs dominated i.e. ST131 (n = 33 isolates), ST648 (n = 10), ST38 (n = 9), ST12 and 69 (each n = 4), ST 167, 405 and 372 (each n = 3). The efficacy of multiple-locus variable number tandem repeat analysis (MLVA) was evaluated using three, seven or ten loci, in comparison with that of pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST).ResultsMLVA detected 39, 55 and 60 distinct types, respectively, using three (GECM-3), seven (GECM-7) or ten (GECM-10) loci. For GECM-7 and −10, 26 STs included one type and eleven STs each included several types, the corresponding numbers for GECM-3 were 29 and 8. The highest numbers were seen for ST131 (7,7 and 8 types, respectively), ST38 (5,5,8) and ST648 (4,5,5). Good concordance was observed with PFGE and GECM-7 and −10, despite fewer types being identified with MLVA; 78 as compared to 55 and 60 types. The lower discriminatory power of MLVA was primarily seen within the O25b-ST131 lineage (n = 34) and its H30-Rx subclone (n = 21). Epidemiologically unrelated O25b-ST131 isolates were clustered with O25b-ST131 outbreak isolates by MLVA, whereas the ST1444 outbreak isolates were accurately distinguished from unrelated isolates.ConclusionMLVA, even when using only three loci, represents an easy initial typing tool for epidemiological screening of ESBL-E. coli. For the ST131-O25b linage, complementary methods may be needed to obtain sufficient resolution.

Molecular epidemiology and antimicrobial resistance phenotypes of Acinetobacter baumannii isolated from patients in three hospitals in southern Vietnam.

Multidrug resistance in the nosocomial pathogen Acinetobacter baumannii limits therapeutic options and impacts on clinical care. Resistance against carbapenems, a group of last-resort antimicrobials for treating multidrug-resistant (MDR) A. baumannii infections, is associated with the expression (and over-expression) of carbapenemases encoded by the blaOXA genes. The aim of this study was to determine the prevalence of antimicrobial-resistant A. baumannii associated with infection in three hospitals in southern Vietnam and to characterize the genetic determinants associated with resistance against carbapenems. We recovered a total of 160 A. baumannii isolates from clinical samples collected in three hospitals in southern Vietnam from 2012 to 2014. Antimicrobial resistance was common; 119/160 (74 %) of isolates were both MDR and extensively drug resistant (XDR). High-level imipenem resistance (>32 µg ml-1) was determined for 109/117 (91.6 %) of the XDR imipenem-nonsusceptible organisms, of which the majority (86.7 %) harboured the blaOXA-51 and blaOXA-23 genes associated with an ISAba1 element. Multiple-locus variable number tandem repeat analysis segregated the 160 A. baumannii into 107 different multiple-locus variable number tandem repeat analysis types, which described five major clusters. The biggest cluster was a clonal complex composed mainly of imipenem-resistant organisms that were isolated from all three of the study hospitals. Our study indicates a very high prevalence of MDR/XDR A. baumannii causing clinically significant infections in hospitals in southern Vietnam. These organisms commonly harboured the blaOXA-23 gene with ISAba1 and were carbapenem resistant; this resistance phenotype may explain their continued selection and ongoing transmission within the Vietnamese healthcare system.

Molecular Subtyping of Salmonella enterica Serovar Typhi by Pulsed-Field Gel Electrophoresis and Multiple-Locus Variable-Number Tandem-Repeat Analysis in India: Their Association with Antimicrobial Resistance Profiles.

Molecular subtyping and DNA sequencing-based methods, which are commonly used for discriminating Salmonella enterica serovar Typhi (S. Typhi) isolates, lead to improved molecular epidemiological investigations for prevention and control of typhoid fever. We obtained S. Typhi blood isolates (n = 66) from India during 2007-14 for molecular subtyping by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) in association with antibiotic resistance profiles. Genotypic diversity was observed more by MLVA (Simpson's index of diversity, D value = 0.997) than PFGE (D value = 0.864). Two prevalent pulsotypes containing nalidixic acid-resistant (NALR) and NALR-ciprofloxacin-resistant (CIPR) S. Typhi isolates circulated in India. Multidrug-resistant (MDR), NALR-CIPR, and most NALR isolates were found to be clonal by PFGE. MLVA could differentiate the clonal isolates. Most of the MDR and NALR-CIPR isolates showed variation in single or double VNTR loci, whereas NALR isolates varied in more than 2 loci, reflecting higher genetic diversity among the NALR isolates. Of the 6 VNTR loci, TR4,699 (D value = 0.838) and Sal02 (D value = 0.890) loci played important roles as MLVA cluster-supporting alleles. The rapid turnaround time and high-level discriminatory power of MLVA may be useful for tracking and controlling the transmission of S. Typhi isolates during epidemiological investigations.

Shiga Toxin (Verotoxin)-producing Escherichia coli and Foodborne Disease: A Review.

Shiga toxin (verotoxin)-producing Escherichia coli (STEC) is an important cause of foodborne disease. Since outcomes of the infections with STEC have a broad range of manifestation from asymptomatic infection or mild intestinal discomfort, to bloody diarrhea, hemolytic uremic syndrome (HUS), end-stage renal disease (ESRD), and death, the disease is a serious burden in public health and classified as a notifiable infectious disease in many countries. Cattle and other ruminants are considered to be the major reservoirs of STEC though isolation of STEC from other animals have been reported. Hence, the source of contamination extends to a wide range of foods, not only beef products but also fresh produce, water, and environment contaminated by excretes from the animals, mainly cattle. A low- infectious dose of STEC makes the disease relatively contagious, and causes outbreaks with unknown contamination sources and, therefore, as a preventive measure against STEC infection, it is important to obtain characteristics of prevailing STEC isolates in the region through robust surveillance. Analysis of the isolates by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) could help finding unrecognized foodborne outbreaks due to consumption of respective contaminated sources. However, though the results of molecular analysis of the isolates could indicate linkage of sporadic cases of STEC infection, it is hardly concluded that the cases are related via contaminated food source if it were not for epidemiological information. Therefore, it is essential to combine the results of strain analysis and epidemiological investigation rapidly to detect rapidly foodborne outbreaks caused by bacteria. This article reviews STEC infection as foodborne disease and further discusses key characteristics of STEC including pathogenesis, clinical manifestation, prevention and control of STEC infection. We also present the recent situation of the disease in Japan based on the surveillance of STEC infection.

Multiple-Locus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Isolates between 2004 and 2014 in Yamagata, Japan: Change in Molecular Characteristics during an 11-year Period.

Multiple-locus variable-number tandem-repeat analysis (MLVA) typing was performed for Mycoplasma pneumoniae strains isolated between 2004 and 2014 in Yamagata, Japan. The results were examined by considering the combination of the P1 type and prevalence of macrolide resistance-associated mutations. Four-locus (Mpn13-16) MLVA classified 347 strains into 9 MLVA types, including 3 major types: 3-5-6-2, 4-5-7-2, and 4-5-7-3. All type 3-5-6-2 strains (77 strains) were P1 type 2 variants (2a or 2c), while types 4-5-7-2 (181 strains) and 4-5-7-3 (75 strains) were P1 type 1. MLVA type 4-5-7-2 strains circulated and were dominant until 2010, accounting for 88.4% of the 121 strains isolated between 2004 and 2010. The prevalence of types 4-5-7-3 and 3-5-6-2 strains increased rapidly in 2011 and 2012, respectively, resulting in cocirculation of 3 MLVA types, including type 4-5-7-2, between 2011 and 2013. The prevalence of macrolide resistance-associated mutations in MLVA types 4-5-7-2, 4-5-7-3, and 3-5-6-2 strains was 59.7% (108/181), 25.3% (19/75), and 0% (0/77), respectively. Because the prevalence of macrolide resistance-associated mutations differed by current MLVA types in Yamagata, continued surveillance combined with molecular typing and identification of macrolide resistance-associated mutations is necessary.

Clonal structure in Ichthyobacterium seriolicida, the causative agent of bacterial haemolytic jaundice in yellowtail, Seriola quinqueradiata, inferred from molecular epidemiological analysis.

Bacterial haemolytic jaundice caused by Ichthyobacterium seriolicida has been responsible for mortality in farmed yellowtail, Seriola quinqueradiata, in western Japan since the 1980s. In this study, polymorphic analysis of I.seriolicida was performed using three molecular methods: amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Twenty-eight isolates were analysed using AFLP, while 31 isolates were examined by MLST and MLVA. No polymorphisms were identified by AFLP analysis using EcoRI and MseI, or by MLST of internal fragments of eight housekeeping genes. However, MLVA revealed variation in repeat numbers of three elements, allowing separation of the isolates into 16 sequence types. The unweighted pair group method using arithmetic averages cluster analysis of the MLVA data identified four major clusters, and all isolates belonged to clonal complexes. It is likely that I.seriolicida populations share a common ancestor, which may be a recently introduced strain.

MRSA in persons not living or working on a farm in a livestock-dense area: prevalence and risk factors.

MRSA emerged in livestock and persons in contact with livestock is referred to as livestock-associated MRSA (LA-MRSA). We assessed the prevalence and risk factors for MRSA carriage in persons not living or working on a farm. A cross-sectional study was performed among 2492 adults living in close proximity of livestock farms. Persons working and/or living on farms were excluded. Nasal swabs were cultured using selective media. Participants completed questionnaires and the distance from the residential address to the nearest farm was calculated. The Mann-Whitney U -test was used to compare median distances. Risk factors were explored with logistic regression. Fourteen persons carried MRSA (0.56%; 95% CI 0.32%-0.92%), 10 of which carried LA-MRSA of multiple-locus variable-number tandem repeat analysis complex (MC) 398 (0.40%; 95% CI 0.20%-0.71%). MRSA MC 398 carriers lived significantly closer to the nearest farm than non-carriers (median: 184 versus 402 m; P < 0.01). In bivariate analyses correcting for contact with livestock, this difference remained significant. Although the prevalence was low, living near farms increased the risk of MRSA MC 398 carriage for persons not living or working on a farm. Further research is necessary to identify the transmission routes.

Investigation of a national outbreak of STEC Escherichia coli O157 using online consumer panel control methods: Great Britain, October 2014.

In October 2014, Public Health England (PHE) identified cases of Shiga toxin-producing Escherichia coli (STEC) serogroup O157 sharing a multiple locus variable-number tandem repeat analysis (MLVA) profile. We conducted a case-control study using multivariable logistic regression to calculate adjusted odds ratios (aOR) and 95% confidence intervals (CI) testing a range of exposures. Cases were defined as laboratory-confirmed STEC O157 with the implicated MLVA profile, were UK residents aged ⩾18 years with symptom onset between 25 September and 30 October 2014, and had no history of travel abroad within 5 days of symptom onset. One hundred and two cases were identified. Cases were mostly female (65%; median age 49, range 2-92 years). It was the second largest outbreak seen in England, to date, and a case-control study was conducted using market research panel controls and online survey methods. These methods were instrumental in the rapid data collection and analysis necessary to allow traceback investigations for short shelf-life products. This is a new method of control recruitment and this is the first in which it was a standalone recruitment method. The case-control study suggested a strong association between consumption of a ready-to-eat food and disease (aOR 28, 95% CI 5·0-157) from one retailer. No reactive microbiological testing of food items during the outbreak was possible due to the short shelf-life of the product. Collaboration with industrial bodies is needed to ensure timely traceback exercises to identify contamination events and initiate appropriate and focused microbiological testing and implement control measures.