MLVA Types Research Articles

Use of multiple locus variable number tandem repeats analysis in systematics of the causative agent of brucellosis

Methods that allow one to carry out molecular genetic differentiation of strains of the same species are acquiring increasing importance in the modern system of struggle with brucellosis. The multiple locus variable number tandem repeats analysis (MLVA) method was selected for molecular genetic differentiation up until the strain level and simultaneous establishment of genetic relationship of the studied strains. The aim of the study was typing (by the MLVA method) of strains of three pathogenic to humans Brucella species with the analysis of stability of selected loci, identification of differences between them and conformity of the data, obtained by this method and by phenotypic method of Brucella differentiation, for use of the MLVA method in systematics of the causative agent of brucellosis. Materials and methods. Twenty-six Brucella strains, which are reference (n = 15); vaccine (n = 2); field strains of three pathogenic Brucella species, including B. melitensis (n = 3), B. abortus (n = 2), and B. suis (n = 2); and isolates (n = 2) of unknown taxonomic position were typed by the MLVA method with nine pairs of primers for known variable loci for the Brucella genome. Analysis of the stability of selected loci, their differences according to the Hunter-Gaston discrimination index (HGDI), and conformity of the data obtained with phenotypic methods of identification was conducted. Results. The MLVA method confirms the results of phenotypic methods of identification and the stability of selected loci in most reference and vaccine strains with a high variability HGDI index (0.9969) for all loci. A dendrogram based on the data of the MLVA typing allowed us to allocate the Brucella strains into related clusters according to their taxonomic species and biovar position and development of 25 genotypes. The B. melitensis strains generated a cluster related to the B. melitensis 63/9 biovar 2 reference strain. Australian Brucella 83-4 and Brucella 83-6 isolates from mouse like rodents generated a cluster distant from other Brucella strains. Conclusions. MLVA is a promising method of Brucella strain differentiation with known and undetermined taxonomic status for their systematics and creation of the MLVA genotype catalogue, which will favor a qualitative improvement of the system of epidemiological surveillance for this infection in Russia.

Improving resolution of public health surveillance for human Salmonella entericaserovar Typhimurium infection: 3 years of prospective multiple-locus variable-number tandem-repeat analysis (MLVA)

BackgroundProspective typing of Salmonella enterica serovar Typhimurium (STM) by multiple-locus variable-number tandem-repeat analysis (MLVA) can assist in identifying clusters of STM cases that might otherwise have gone unrecognised, as well as sources of sporadic and outbreak cases. This paper describes the dynamics of human STM infection in a prospective study of STM MLVA typing for public health surveillance.MethodsDuring a three-year period between August 2007 and September 2010 all confirmed STM isolates were fingerprinted using MLVA as part of the New South Wales (NSW) state public health surveillance program.ResultsA total of 4,920 STM isolates were typed and a subset of 4,377 human isolates was included in the analysis. The STM spectrum was dominated by a small number of phage types, including DT170 (44.6% of all isolates), DT135 (13.9%), DT9 (10.8%), DT44 (4.5%) and DT126 (4.5%). There was a difference in the discriminatory power of MLVA types within endemic phage types: Simpson's index of diversity ranged from 0.109 and 0.113 for DTs 9 and 135 to 0.172 and 0.269 for DTs 170 and 44, respectively. 66 distinct STM clusters were observed ranging in size from 5 to 180 cases and in duration from 4 weeks to 25 weeks. 43 clusters had novel MLVA types and 23 represented recurrences of previously recorded MLVA types. The diversity of the STM population remained relatively constant over time. The gradual increase in the number of STM cases during the study was not related to significant changes in the number of clusters or their size. 667 different MLVA types or patterns were observed.ConclusionsProspective MLVA typing of STM allows the detection of community outbreaks and demonstrates the sustained level of STM diversity that accompanies the increasing incidence of human STM infections. The monitoring of novel and persistent MLVA types offers a new benchmark for STM surveillance.A part of this study was presented at the MEEGID × (Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases) Conference, 3-5 November 2010, Amsterdam, The Netherlands

Evaluation of Fourier transform infrared (FT-IR) spectroscopy and chemometrics as a rapid approach for sub-typing Escherichia coli O157:H7 isolates

The importance of tracking outbreaks of foodborne illness and the emergence of new virulent subtypes of foodborne pathogens have created the need for rapid and reliable sub-typing methods for Escherichia coli O157:H7. Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate statistical analyses was used for sub-typing 30 strains of E. coli O157:H7 that had previously been typed by multilocus variable number tandem repeat analysis (MLVA) and pulsed field gel electrophoresis (PFGE). Hierarchical cluster analysis (HCA) and canonical variate analysis (CVA) of the FT-IR spectra resulted in the clustering of the same or similar MLVA types and separation of different MLVA types of E. coli O157:H7. The developed FT-IR method showed better discriminatory power than PFGE in sub-typing E. coli O157:H7. Results also indicated the spectral relatedness between different outbreak strains. However, the grouping of some strains was not in complete agreement with the clustering based on PFGE and MLVA. Additionally, HCA of the spectra differentiated the strains into 30 sub-clusters, indicating the high specificity and suitability of the method for strain level identification. Strains were also classified (97% correct) based on the type of Shiga toxin present using CVA of the spectra. This study demonstrated that FT-IR spectroscopy is suitable for rapid (≤16 h) and economical sub-typing of E. coli O157:H7 with comparable accuracy to MLVA typing. This is the first report of using an FT-IR-based method for sub-typing E. coli O157:H7.

Prevalence and Genetic Characterization of Pertactin-Deficient Bordetella pertussis in Japan

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn−) B. pertussis was observed in Japan. The Prn− isolate was first discovered in 1997, and 33 (27%) Prn− isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn− isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn− isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn− isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn− isolates belong to MLVA-186, and 6 and 3 Prn− isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn− clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn− isolates have a higher growth potential than the Prn+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn− strains have arisen and that Prn expression is not essential for fitness under these conditions.

Clonal Spread ofMycoplasma pneumoniaein Primary School, Bordeaux, France

Clonal Spread of<i>Mycoplasma pneumoniae</i>in Primary School, Bordeaux, France

Clonal outbreak of ST17 multidrug-resistant Enterococcus faecium harbouring an Inc18-like::Tn1546 plasmid in a haemo-oncology ward of a Spanish hospital

To report a clonal outbreak of ST17 vancomycin-resistant Enterococcus faecium (VREfm) carrying Tn1546 (vanA) in a haemo-oncology ward of a tertiary teaching hospital in the south of Spain (January-September 2009). Twenty-two VREfm strains from 13 patients were characterized by PFGE, multiple-locus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). Genes encoding antibiotic resistance and putative virulence traits and the Tn1546 backbone were investigated by PCR. Plasmid characterization included determination of size (S1-PFGE) and replication modules (PCR, hybridization and sequencing). Patient clinical records were analysed retrospectively. A single ST17 E. faecium clone (MT-7 MLVA type) carrying esp and hyl plus a 30 kb Inc18-like::Tn1546 (IS1216) plasmid was identified. Ampicillin resistance was linked to PBP5 showing mutations at positions 24, 27, 34, 66, 68, 85, 100, 144, 172, 177, 204, 216, 324, 462, 466', 470, 485, 496, 499, 525, 546, 558, 582, 586, 629, 632, 642 and 667. Other resistance genes identified were erm(B), ant(6')-Ia and aph(3')-IIIa. Fluoroquinolone resistance was attributable to ParC (Arg-61 → Gly and Ser-80 → Arg) and GyrA (Ser-83 → Arg) mutations. A nosocomial outbreak caused by an ST17 (CC17) E. faecium clone harbouring Esp and Hyl and a 30 kb Inc18-like::Tn1546 plasmid among haemo-oncology patients is reported. The failure of early infection control practices indicates an undetected reservoir and the ability of this strain to persist over long periods. The potential spread of epidemic clones and broad host plasmids carrying vancomycin resistance in Spain is of concern since it might contribute towards a higher rate of VREfm infection.

Characterization of Salmonella enterica serovar Enteritidis isolates recovered from blood and stool specimens in Thailand

BackgroundBacteremia due to Salmonella spp. is a life-threatening condition and is commonly associated with immune compromise. A 2009 observational study estimated risk factors for the ten most common non-typhoidal Salmonella (NTS) serovars isolated from Thai patients between 2002–2007. In this study, 60.8% of Salmonella enterica serovar Enteritidis isolates (n = 1517) were recovered from blood specimens and infection with Salmonella serovar Enteritidis was a statistically significant risk factor for bacteremia when compared to other NTS serovars. Based on this information, we characterized a subset of isolates collected in 2008 to determine if specific clones were recovered from blood or stool specimens at a higher rate. Twenty blood isolates and 20 stool isolates were selected for antimicrobial resistance testing (MIC), phage typing, PFGE, and MLVA.ResultEight antibiogrammes, seven MLVA types, 14 XbaI/BlnI PFGE pattern combinations, and 11 phage types were observed indicating considerable diversity among the 40 isolates characterized. Composite analysis based on PFGE and MLVA data revealed 22 genotypes. Seven of the genotypes containing two or more isolates were from both stool and blood specimens originating from various months and zones. Additionally, those genotypes were all further discriminated by phage type and/or antibiogramme. Ninety percent of the isolates were ciprofloxacin resistant.ConclusionsThe increased percentage of bloodstream infections as described in the 2009 observational study could not be attributed to a single clone. Future efforts should focus on assessing the immune status of bacteriaemic patients and identifying prevention and control measures, including attribution studies characterizing non-clinical (animal, food, and environmental) isolates.

MLVA genotyping of Chinese human Brucella melitensisbiovar 1, 2 and 3 isolates

BackgroundSince 1950, Brucella melitensis has been the predominant strain associated with human brucellosis in China. In this study we investigated the genotypic characteristics of B. melitensis isolates from China using a multiple-locus variable-number tandem-repeat analysis (MLVA) and evaluated the utility of MLVA with regards to epidemiological trace-back investigation.ResultsA total of 105 B. melitensis strains isolated from throughout China were divided into 69 MLVA types using MLVA-16. Nei's genetic diversity indices for the various loci ranged between 0.00 - 0.84. 12 out 16 loci were the low diversity with values < 0.2 and the most discriminatory markers were bruce16 and bruce30 with a diversity index of > 0.75 and containing 8 and 7 alleles, respectively. Many isolates were single-locus or double-locus variants of closely related B. melitensis isolates from different regions, including the north and south of China. Using panel 1, the majority of strains (84/105) were genotype 42 clustering to the 'East Mediterranean' B. melitensis group. Chinese B. melitensis are classified in limited number of closely related genotypes showing variation mainly at the panel 2B loci.ConclusionThe MLVA-16 assay can be useful to reveal the predominant genotypes and strain relatedness in endemic or non-endemic regions of brucellosis. However it is not suitable for biovar differentiation of B. melitensis. Genotype 42 is widely distributed throughout China during a long time. Bruce 16 and bruce 30 in panel 2B markers are most useful for typing Chinese isolates.

Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping ofEhrlichia ruminantiumand its application to infectedAmblyomma variegatumcollected in heartwater endemic areas in Uganda

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost- effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.

Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks.

P4-S1.08 Performance comparison of Neisseria gonorrhoeae (NG) sequence typing methods NG-MAST and NG-MLVA on a defined panel of isolates

BackgroundThe increase in N gonorrhoeae (NG) prevalence since the mid-1990s, increasing antibiotic resistance, and the absence of an effective vaccine, demand more efficient national and international surveillance of the NG...

Emergence of Clostridium difficile Ribotype 027 in Korea

BackgroundClostridium difficile infection (CDI) has markedly risen and is associated with hypervirulent ribotype 027 outbreaks in North America and Europe since 2003. The aims of this study were to determine the prevalence of ribotype 027 among C. difficile isolates in Korea, to characterize the ribotype 027 isolates, and to determine the clinical severity of CDI in patients infected with these isolates.MethodsA total of 1,251 isolates of C. difficile recovered from stool specimens of suspected CDI patients at two tertiary-care hospitals and one commercial laboratory between 2002 and 2009. Genes for toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA and cdtB) were detected by PCR. Mutation in the tcdC gene was detected by sequencing after PCR amplification. For molecular genotyping, we performed PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat analysis (MLVA). Minimum inhibitory concentrations of moxifloxacin were determined using Etest strips (AB bioMérieux, Sweden).ResultsWe identified 7 isolates as ribotype 027. These isolates had the same tcdC mutation as the epidemic strain, and 6 of them were resistant to moxifloxacin. The isolates were categorized into 3 different PFGE types and 7 different MLVA types. All the 7 cases had occurred sporadically.ConclusionsC. difficile ribotype 027 is uncommon, but it has emerged in Korea. The spread of this ribotype should be closely monitored in order to avoid an outbreak of CDI in Korea.

Culture-independent multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae

A PCR approach for multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae directly from respiratory samples was developed and evaluated on 54 specimens from M. pneumoniae-positive pneumonia patients. The method resulted in a clearly identifiable MLVA type in all samples tested and can be used for MLVA without laborious isolation of the pathogen.

Genetic characterization of Brucella melitensis isolates from Mongolia, Russia, and Azerbaijan

The goal of this work was to provide comparative genetic characterization of the human and animal Brucella melitensis isolates from Mongolia, Russia and Azerbaijan using current molecular-genetic typing methods. Twenty eight Mongolian (n = 18), Russian (n = 6), and Azerbaijan (n = 4) human and animal Brucella melitensis isolates were studied using 2 molecular typing methods based on PCR for differential species and biovar specific ORF (open reading frames) molecular targets and MLVA (multiple locus variable number tandem repeats analysis) using primers to 12 known loci. The PCR was used for differential molecular targets (all B. melitensis isolates) were characterized as the B. melitensis biovar 2. The MLVA revealed 7 identical and 5 variable MLVA loci. All the isolates were classified into 25 genotypes using the dendrogram on the data of 12 loci and the cluster related to reference strain B. melitensis 63/9 biovar 2. The B. melitensis isolates having related MLVA genotypes were connected to Mongolian, Russian and Azerbaijan regions. The circulation for two B. melitensis isolates to not typical hosts as camel and yak was demonstrated using molecular typing methods. The genetic characterization of twenty eight B. melitensis isolates from different geographical regions in Mongolia, Russia, and Azerbaijan recognized genetic relationships. On the other hand, the MLVA has high discrimination power with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.9841 revealing genetic diversity for the isolates forming of 25 MLVA genotypes. To improve the system of the brucellosis surveillance in Russia MLVA typing of B. melitensis isolates are necessary to investigate from the Siberian (Republics Tuva, Buryatia, and Irkutsk region) and South (Republics Dagestan, Kalmykia, and Stavropol region) Districts having frontier areas with Mongolia and Azerbaijan.

Multiple-Locus Variable Number Tandem Repeat Analysis for Streptococcus pneumoniae: Comparison with PFGE and MLST

In the era of pneumococcal conjugate vaccines, surveillance of pneumococcal disease and carriage remains of utmost importance as important changes may occur in the population. To monitor these alterations reliable genotyping methods are required for large-scale applications. We introduced a high throughput multiple-locus variable number tandem repeat analysis (MLVA) and compared this method with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLVA described here is based on 8 BOX loci that are amplified in two multiplex PCRs. The labeled PCR products are sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The composite of the number of repeats of the BOX loci makes up a numerical profile that is used for identification and clustering. In this study, MLVA was performed on 263 carriage isolates that were previously characterized by MLST and PFGE. MLVA, MLST and PFGE (cut-off of 80%) yielded 164, 120, and 87 types, respectively. The three typing methods had Simpson's diversity indices of 98.5% or higher. Congruence between MLST and MLVA was high. The Wallace of MLVA to MLST was 0.874, meaning that if two strains had the same MLVA type they had an 88% chance of having the same MLST type. Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%. For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup. Overall, MLVA is a promising genotyping method that is easy to perform and a relatively cheap alternative to PFGE and MLST. In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in the Netherlands before the introduction of the 7-valent conjugate vaccine.

Population Structure of Invasive Streptococcus pneumoniae in the Netherlands in the Pre-Vaccination Era Assessed by MLVA and Capsular Sequence Typing

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA).To assess the serotype of each isolate CST was used. Based on the determination of the partial sequence of the capsular wzh gene, this method assigns a capsular type of an isolate within a single PCR reaction using multiple primersets. The genetic background of pneumococcal isolates was assessed by MLVA. MLVA and CST were used to create a snapshot of the Dutch pneumococcal population causing invasive disease before the introduction of the 7-valent pneumococcal conjugate vaccine in the Netherlands in 2006. A total of 1154 clinical isolates collected and serotyped by the Netherlands Reference Laboratory for Bacterial Meningitis were included in the snapshot. The CST was successful in discriminating most serotypes present in our collection. MLVA demonstrated that isolates belonging to some serotypes had a relatively high genetic diversity whilst other serotypes had a very homogeneous genetic background. MLVA and CST appear to be valuable tools to determine the population structure of pneumococcal isolates and are useful in monitoring the effects of pneumococcal vaccination.

Molecular Epidemiological Investigation of a Diffuse Outbreak Caused bySalmonella entericaSerotype Montevideo Isolates in Osaka Prefecture, Japan

In Osaka Prefecture, Japan, three foodborne outbreaks were caused by Salmonella enterica serotype Montevideo in rapid succession between September 2007 and May 2008. Further, Salmonella Montevideo was also isolated from several sporadic diarrhea patients and asymptomatic carriers examined during approximately the identical period. To investigate the relatedness of the isolates, we performed antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) analysis, and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) for 29 Salmonella Montevideo isolates obtained in this region between 1991 and 2008. Although antimicrobial susceptibility tests had low discriminatory power, PFGE patterns revealed 17 unique types with <90% similarity in combined analyses involving XbaI and BlnI. Moreover, we detected three VNTR loci that were useful to genotype Salmonella Montevideo isolates, with our method ultimately classifying the isolates into 11 MLVA types based on differences in repeat unit number in each examined locus. Six isolates obtained from patients of two separate foodborne disease outbreaks, one sporadic patient, and three different carriers between 2007 and 2008 had nearly identical PFGE patterns and were classified into the identical MLVA type; further, the isolates with this PFGE and MLVA pattern appeared only at that time between 1991 and 2008. These data strongly suggest that genetically identical Salmonella Montevideo strains may have caused the 2007 and 2008 outbreaks in Osaka Prefecture. Our results demonstrate that PFGE using XbaI and BlnI is useful for discriminating between Salmonella Montevideo isolates, even within a limited area, and reconfirm that continuous epidemiological surveillance for bacterial intestinal infections such as salmonellosis may be useful to not only monitor changes in the genetic diversity of isolates, but to also detect diffuse outbreaks.

Defining criteria to interpret multilocus variable-number tandem repeat analysis to aid Clostridium difficile outbreak investigation

PFGE is currently the North American standard for surveillance for Clostridium difficile but lacks discriminatory power to aid outbreak investigation. A further limitation to PFGE is the high baseline rate of the epidemic North American pulsotype (NAP) 1 strain in hospitals. Multilocus variable-number tandem repeat analysis (MLVA) appears to have superior discriminatory power but criteria to define clonality have not been set. We conducted surveillance for toxin-positive C. difficile infection (CDI) at a single academic health sciences centre between September 2009 and April 2010. Seventy-four patient specimens resulting in 86 discrete CDI episodes were subjected to PFGE and MLVA. Results were analysed using Bionumerics software to generate phylogenetic trees and coupled to patient demographic data. Amongst the NAP1 strains, two distinct clusters were identified by MLVA using 90 % similarity as a cut-off by Manhattan distance-based clustering, four clusters using 95 % and seven clusters using 97 %. Population analysis conducted on multiple colonies (n = 25) demonstrated that 1-3 % difference in MLVA types was typical for a single individual. Typing was also conducted in the context of institutional outbreaks (n = 42, three outbreaks) in order to determine clusters within the NAP1 strain. By combining longitudinal surveillance with epidemiological information, single specimen population analysis and typing in the context of institutional outbreaks, we conclude that the use of the Manhattan distance-based clustering with a cut-off of 95-97 % is capable of distinguishing outbreak clones from sporadic isolates.

MLVA is a valuable tool in epidemiological investigations of Escherichia coli and for disclosing multiple carriage

Escherichia coli is a common cause of healthcare-associated urinary tract infection, and is frequently present in the urine of elderly people. Transmission of E. coli between individuals has been suggested, and individuals can be concurrently colonized with several types. Efficient typing methods are required to investigate these epidemiological relationships, and we have examined the applicability of multiple-locus variable number tandem repeat analysis (MLVA). Up to 20 E. coli isolates were sampled per individual from 30 elderly residents at 2 long-term care facilities, and typed using MLVA, pulsed-field gel electrophoresis (PFGE) and PhenePlate (PhP). Thirty-one E. coli types were identified using MLVA, compared to 38 and 32 using PFGE and PhenePlate, respectively. All isolates were typeable using MLVA and PhenePlate, whereas PFGE failed to type isolates from 2 individuals. The Wallace 1 coefficient indicated a high probability that isolates of the same PFGE type were also of the same type according to the other 2 methods. However, the Wallace 2 coefficient indicated a low probability that isolates of the same PhP type would be classified as the same type by PFGE. Twenty-four of the MLVA types were uniquely restricted to single individuals, whilst 7 MLVA types were found in more than 1 individual. Colonization with more than 1 MLVA type was seen in 8 individuals. There was no evidence of specific institutional types at either of the 2 long-term care facilities. MLVA displays a high discriminatory power, and shows substantial potential with respect to epidemiological studies and infection control issues.

Multilocus variable-number tandem repeat analysis: a helpful tool for subtyping French Clostridium difficile PCR ribotype 027 isolates

The objective of this study was to evaluate the usefulness of multilocus variable-number tandem repeat analysis (MLVA) for typing and subtyping of Clostridium difficile. Sixty-eight strains were studied, including strains from PCR ribotypes 027, 078/126, 014/020/077, 017 and 023. The stability of variable-number tandem repeat (VNTR) loci was tested by comparing the MLVA results of two strains subcultured 11 times. After DNA extraction, seven tandem repeat loci (A6, B7, C6, E7, F3, G8, H9) from published MLVA schemes were amplified by PCR and sequenced. The distance between two strains was determined by calculating the summed tandem repeat difference. Genomic diversity was evaluated by using the minimum spanning tree (Bionumerics 5.1 software program; Applied Maths). Among the 68 C. difficile isolates examined, 65 unique MLVA types were identified, suggesting a high discriminatory power. An overall good agreement was observed between MLVA types and PCR ribotypes. The stability of VNTR loci was good. MLVA could separate isolates of the hypervirulent PCR ribotype 027 clone in several clusters; all 027 strains isolated within a hospital were grouped in a specific cluster or were placed very close to each other. Results of MLVA confirmed that strains from PCR ribotypes 078 and 126 were closely related although some were located in different branches of the tree. Similar results were observed for most strains from PCR ribotypes 014, 020 and 077. This highly discriminatory method is time-consuming and expensive, but is a valuable tool for subtyping of C. difficile, especially of 027 strains.