Multiple-locus Variable-number Tandem Repeat Analysis Patterns Research Articles

Multiple-Locus Variable-Number Tandem Repeat Analysis of Helicobacter Pylori Strains Isolated from Biopsy Samples

Background: Multiple-locus variable-number tandem-repeat analysis (MLVA) is a reliable, fast, and useful method for conducting phylogenetic and genotyping analyses. However, its application in Helicobacter pylori typing is limited. Objectives: This study aimed to determine the genetic diversity of H. pylori strains isolated from biopsy samples using six variable loci in MLVA typing. Methods: Over one year, we collected 70 non-duplicative biopsy samples from patients at Rohani Hospital, Babol, in northern Iran. After DNA extraction, H. pylori strains were confirmed through the amplification of the glmM gene in the PCR test. Multiple-locus variable-number tandem-repeat analysis typing utilized six VNTR markers: VNTR-180, VNTR-263, VNTR-614, VNTR-607, VNTR-2181, and VNTR-2457. The PHYLOViZE software was employed to construct the phylogenetic tree. Results: Out of the 70 specimens, 43 tested positive for H. pylori. The phylogenetic tree revealed some strains with completely similar MLVA patterns. Our findings indicated that type 2 is the dominant type in this region, with most patients infected with this type exhibiting gastritis. Conclusions: The high allelic variability observed at VNTR-614, -607, and -2457 indicates high intraspecific variability for these markers in all isolates from our region. Consequently, these VNTRs can be considered ideal sites for typing.

The serological and genetic diversity of the Leptospira interrogans Icterohaemorrhagiae serogroup circulating in the UK.

Strains of Leptospira interrogans belonging to two very closely related serovars, Icterohaemorrhagiae and Copenhageni, have been associated with disease in mammalian species and are the most frequently reported agents of human leptospirosis. They are considered the most pathogenic serovars and represent more than half of the leptospires encountered in severe human infections. Nineteen such isolates from the United Kingdom - human, domestic and wildlife species - were typed using three monoclonal antibodies (F12 C3, F70 C14 and F70 C24) in an attempt to elucidate their epidemiology. They were further examined by restriction endonuclease analysis (REA), multiple-locus variable-number tandem repeat analysis (MLVA) and lic12008 gene sequence analysis. Monoclonal antibody F12 C3, which is highly specific for Icterohaemorrhagiae and Copenhageni, confirmed that all the strains belonged to these two serovars. Sixteen strains were identified as Copenhageni and three as Icterohaemorrhagiae serovar. Only one restriction pattern type was identified, thus confirming that REA is not able to discriminate between the Icterohaemorrhagiae and Copenhageni serovars. Variable-number tandem-repeat analysis found three loci with differences in the repeat number, indicating genetic diversity between British isolates. Sequences of the lic12008 gene showed that all isolates identified as the Icterohaemorrhagiae serotype have a single base insertion, in contrast to the same sequences of the Copenhageni serotype. Copenhageni is the predominant serovar in the Icterohaemorrhagiae serogroup isolated in British Isles. There is a genetic diversity of MLVA patterns of the isolates but no genetic tool used in the study was able to determine serovars.

Molecular typing and antimicrobial resistance of group B Streptococcus clinical isolates in Saudi Arabia

Group B Streptococcus (GBS) has emerged as an important cause of severe infections in adults. However, limited data are available regarding the epidemiology of GBS in Saudi Arabia. Isolates were collected over a period of eight months from colonized (n=104) and infected adults (n=95). Serotypes and virulence determinants were detected by polymerase chain reactions (PCRs). Genetic relatedness was assessed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Antimicrobial susceptibilities were determined by disk diffusion. Serotypes III and V (25% each) were the most prevalent, followed by serotypes II (16.18%), Ia (13.24%), VI (9.31%), and Ib (8.82%), while five isolates remained non-typeable (2.45%). Hypervirulent serotype III/CC17 clone (n=21) accounted for 41.18% of the serotype III isolates. Most isolates (53.92%) harboured pilus island (PI) 1 and 2a types, while PI-2b was predominantly detected in the hypervirulent clone. Isolates were variably resistant to tetracycline (76.47%), erythromycin (36.76%), clindamycin (25.49%), and levofloxacin (6.37%), but remained susceptible to penicillin. Macrolide resistant isolates exhibited constitutive (55.42%) and inducible macrolide-lincosamide-streptogramin B resistance phenotypes (33.74%), while a few had L (9.64%) or M (1.2%) phenotypes. MLVA patterns of dominant serotypes III and V revealed 40 different types divided into 12 clusters and 28 singletons. Interestingly, macrolide resistance was significantly associated with two major MLVA types. GBS isolates belonged predominantly to serotypes III and V, but there were no clear associations between serotypes and patient groups. The studied isolates exhibited high levels of resistance to erythromycin and clindamycin that need further surveillance.

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

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

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

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

Colonization of toxigenic Clostridium difficile among ICU patients: a prospective study.

BackgroundA prospective study was performed to investigate the prevalence of colonization among ICU patients and to examine whether asymptomatic carriers were the source of subsequent C. difficile infection (CDI) and acquisition of toxigenic C. difficile.MethodsRectal swabs were collected from adult patients on admission to and at discharge from a 50-bed medical ICU of a major referral hospital in western China, from August to November 2014. Stools were collected from patients who developed ICU-onset diarrhea. Both swabs and stools were screened for tcdB (toxin B gene) by PCR. Samples positive to tcdB were cultured for C. difficile and isolates recovered were screened for tcdB and the binary toxin genes by PCR. Strain typing was performed using multilocus sequence typing and isolates belonging to the same sequence type (ST) were further typed using multiple-locus variable number tandem repeat analysis (MLVA).ResultsDuring the 4-month period, rectal swabs were collected from 360 (90.9 %) out of 396 patients who were admitted to the ICU. Among the 360 patients, 314 had stayed in the ICU more than 3 days, of which 213 (73.6 %) had a rectal swab collected within the 3 days prior to discharge from ICU. The prevalence of toxigenic C. difficile colonization was 1.7 % (6 cases) and 4.3 % (10 cases) on admission and discharge, respectively. Only four (1.1 %) out of 360 patients had CDI, corresponding to 10.7 cases per 10,000 ICU days. None of the four cases had toxigenic C. difficile either on admission or at discharge. Toxigenic C. difficile isolates were recovered from all swabs and stool samples positive for tcdB by PCR and belonged to 7 STs (ST2, 3, 6, 37, 54, 103 and 129). None of the isolates belonging to the same ST had identical MLVA patterns. Binary toxin genes were detected in one ST103 isolate that caused colonization.ConclusionThe prevalence of colonization with toxigenic C. difficile among patients on admission to ICU was low in our setting. ICU-acquired toxigenic C. difficile were not linked to those detected on admission. Active screening for toxigenic C. difficile may not be a resource-efficient measure in settings with a low prevalence of colonization.

Introduction of quinolone resistant Escherichia coli to Swedish broiler population by imported breeding animals

During recent years a rapid increase of quinolone resistant Escherichia coli have been noted in the Swedish broiler population, despite the lack of a known selective pressure. The current study wanted to investigate if imported breeding birds could be a source for the quinolone resistant E. coli. The occurrence of quinolone resistant E. coli was investigated, using selective cultivation with nalidixic acid, in grand-parent birds on arrival to Sweden and their progeny. In addition, sampling in hatcheries and empty cleaned poultry houses was performed. Clonality of isolates was investigated using a 10-loci multiple-locus variable number tandem repeat analysis (MLVA). To identify the genetic basis for the resistance isolates were also analysed for occurrence of plasmid-mediated quinolone resistance (PMQR) determinants and characterization of chromosomal mutations. E. coli resistant to nalidixic acid occurred in grandparent birds imported to Sweden for breeding purposes. Four predominant MLVA types were identified in isolates from grandparent birds, parent birds and broilers. However, resistant E. coli with identical MLVA patterns were also present in hatcheries and poultry houses suggesting that the environment plays a role in the occurrence. Nalidixic acid resistance was due to a mutation in the gyrA gene and no PMQR could be identified. The occurrence of identical clones in all levels of the production pyramid points to that quinolone resistant E. coli can be introduced through imported breeding birds and spread by vertical transmission to all levels of the broiler production pyramid.

Genetic Types, Gene Repertoire, and Evolution of Isolates of the Salmonella enterica Serovar 4,5,12:i:− Spanish Clone Assigned to Different Phage Types

Salmonella enterica subsp. enterica 4,[5],12:i:- is one of the most prevalent serovars associated with human infections worldwide. Two multidrug-resistant clones, designated Spanish and European clones, are recognized as having importance for public health and are subject to control measures in the European Union. In this study, 23 clinical isolates belonging to the Spanish clone were characterized by multilocus sequence typing, multiple-locus variable number tandem repeat analysis (MLVA), PCR amplification and sequencing, and a DNA microarray targeting 263 genes, in order to provide new insights into their origins and further evolution. The derived data were compared with information available from other studies for S. 4,[5],12:i:- isolates of both the Spanish and the European clones, to identify differential molecular markers which could be potentially used as surveillance tools in the control of dissemination of this serovar. The isolates analyzed were assigned to sequence type 19 and to 17 MLVA patterns, with 3-13-16-NA-311 being the most prevalent. Highly similar virulence, metabolic, and prophage-associated gene profiles were identified, but DNA mobility markers distinguished five genotypes. Two types of deletions, caused by insertion of IS26, presumably donated by pUO-STmR/RV1-like plasmids typically found in the Spanish clone, affected the fljAB operon and surrounding DNA. The Spanish and European clones differ in sequence type, MLVA patterns, gene repertoire, and fljAB deletion type. The observed variability supports an independent evolution of the two successful monophasic clones from different Salmonella enterica serovar Typhimurium ancestors and can be taken into consideration for epidemiological surveillance.

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

Multiple-locus variable-number tandem repeat analysis of reference strains used for the diagnosis of leptospirosis in Argentina.

Leptospirosis is a worldwide zoonosis caused by a spirochete that belongs to the genus Leptospira. In the last years, new methods, such as the PCR-based multiple-locus variable-number tandem repeat analysis (MLVA), have been developed for the genotyping of leptospires. In the present work, the MLVA patterns for all reference strains used in Argentina for bovine, ovine, porcine, equine, caprine and canine leptospirosis diagnosis, as well as in human and wild animal diagnosis, were obtained. MLVA results are presented in such a way that they can be readily used for the identification of these strains by the simple and direct comparison of agarose gels. Making the use and interpretation of the MLVA for leptospires typing easier will help increase the use of this method as a routine procedure for human and animal diagnosis, for epidemiological studies, vaccine control and other applications.

Outbreak of Shiga-Toxigenic Escherichia coli O157:H7 Infections Associated with Rodeo Attendance, Utah and Idaho, 2009

In summer 2009, the Utah Department of Health investigated an outbreak of Shiga-toxigenic Escherichia coli (STEC) O157:H7 (O157) illness associated with attendance at multiple rodeos. Patients were interviewed regarding exposures during the week before illness onset. A ground beef traceback investigation was performed. Ground beef samples from patient homes and a grocery store were tested for STEC O157. Rodeo managers were interviewed regarding food vendors present and cattle used at the rodeos. Environmental samples were collected from rodeo grounds. Two-enzyme pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) were performed on isolates. Fourteen patients with primary STEC O157 illness were reported in this outbreak. Isolates from all patients were indistinguishable by PFGE. Isolates from nine patients had identical MLVA patterns (main outbreak strain), and five had minor differences. Thirteen (93%) patients reported ground beef consumption during the week before illness onset. Results of the ground beef traceback investigation and ground beef sampling were negative. Of 12 primary patients asked specifically about rodeo attendance, all reported having attended a rodeo during the week before illness onset; four rodeos were mentioned. All four rodeos had used bulls from the same cattle supplier. An isolate of STEC O157 identified from a dirt sample collected from the bullpens of one of the attended rodeos was indistinguishable by PFGE and MLVA from the main outbreak strain. Recommendations were provided to rodeo management to keep livestock and manure separate from rodeo attendees. This is the first reported STEC O157 outbreak associated with attendance at multiple rodeos. Public health officials should be aware of the potential for rodeo-associated STEC illness.

Genotyping of Leptospira interrogans strains from Argentina by Multiple-Locus Variable-number tandem repeat Analysis (MLVA)

Leptospirosis outbreaks occur regularly in Argentina and other South American countries, but little is known about their epidemiological relationships. Application of new molecular tools, such as the Multiple-Locus Variable-number tandem repeat Analysis (MLVA) is limited by scant available data on regional strains. We have analyzed the genetic diversity of a collection of 31 strains of Leptospira interrogans isolated in Argentina during the past five decades from humans and animals, including a strain from an environmental source and another isolated from an opossum. Genotyping was performed by MLVA using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45]. Clustering analysis revealed eight distinct MLVA genotypes, with a dominant one, genotype A. Strains with this genotype were consistently isolated since 1960, representing 55% of the total strains and spanning an extensive geographical distribution. Other seven genotypes were less frequent, and only genotypes A and Hond Utrecht IV were isolated during the last decade. Different kinds of repeat blocks for each VNTR locus were identified by sequence analysis. VNTR copy number differences among genotypes always involved only one of these blocks. MLVA patterns obtained reveal the genetic diversity and relationships between strains, and constitute the framework for the genotyping of leptospires in the region.

Methicillin‐Resistant Coagulase‐Negative Staphylococci in the Community: High Homology of SCCmec IVa betweenStaphylococcus epidermidisand Major Clones of Methicillin‐ResistantStaphylococcus aureus

Data on community spread of methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are scarce. We assessed their potential role as a reservoir of staphylococcal cassette chromosome mec (SCCmec) IVa, the leading SCCmec subtype in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Nasal carriage of MR-CoNS was prospectively investigated in 291 adults at hospital admission. MR-CoNS were characterized by SCCmec typing, long-range polymerase chain reaction (PCR) for SCCmec IV, and multiple-locus variable-number tandem repeat analysis (MLVA) for Staphylococcus epidermidis (MRSE) strains. Three SCCmec IVa elements were fully sequenced. The carriage rate of MR-CoNS was 19.2% (25.9% and 16.5% in patients with and patients without previous exposure to the health care system, respectively; P = .09). MR-CoNS strains (n = 83, including 58 MRSE strains with highly heterogeneous MLVA patterns) carried SCCmec type IVa (n = 9, all MRSE), other SCCmec IV subtypes (n = 9, including 7 MRSE), other SCCmec types (n = 15), and nontypeable SCCmec (n = 50). Long-range PCR indicated structural homology between SCCmec IV in MRSE and that in MRSA. Complete sequences of SCCmec IVa from 3 MRSE strains were highly homologous to those available for CA-MRSA, including major clones USA300 and USA400. MR-CoNS are probably disseminated in the community, notably in subjects without previous exposure to the health care system. MRSE, the most prevalent species, may act as a reservoir of SCCmec IVa for CA-MRSA.

Multiple-locus variable-number tandem repeat analysis (MLVA) of Leptospira interrogans serovar Pomona from Argentina reveals four new genotypes

Outbreaks of leptospirosis occur regularly in Argentina, but little is known about their epidemiological relationships. We have analyzed the genetic diversity of a collection of 16 strains of Leptospira interrogans serovar Pomona isolated from animals and humans in Argentina during the past 45 years. Genotyping was performed by multiple-locus variable-number tandem repeat analysis (MLVA) using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45]. Clustering analysis revealed four new distinct MLVA genotypes, with a dominant one. Strains with this genotype were consistently isolated since 1960 to the present, mainly from cows and pigs, but also from humans, representing 75% of the total strains studied. These strains coexisted temporally and geographically with isolates presenting the other new genotypes. VNTR4 locus, with four different alleles, presented the highest diversity between the VNTR loci analyzed. MLVA patterns obtained will be useful for future diagnostic and epidemiological tracing analysis.

Comparison of Multiple-Locus Variable-Number Tandem Repeat Analysis, Pulsed-Field Gel Electrophoresis, and Phage Typing for Subtype Analysis of Salmonella enterica Serotype Enteritidis

Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of Salmonella serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonella serotype Enteritidis.