scholarly journals Rapid and Simple UniversalEscherichia coliGenotyping Method Based on Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Single-Tube Multiplex PCR and Standard Gel Electrophoresis

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
François Caméléna ◽  
André Birgy ◽  
Yasmine Smail ◽  
Céline Courroux ◽  
Patricia Mariani-Kurkdjian ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Tandem Repeat ◽  
Gel Electrophoresis ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Discriminatory Power ◽  
Content Type ◽  
E Coli ◽  
High Discriminatory Power ◽  
St131 Clone

ABSTRACTWe developed a multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing ofEscherichia coliandShigellaisolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: theE. colireference (ECOR) collection (n = 72), O1:K1 isolates causing neonatal meningitis (n = 38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone (n = 38), Shiga toxin-producingE. coli(STEC) isolates of serogroups O157:H7 (n = 21) and O26 (n = 16, 8 of which belonged to an outbreak), 27Shigellaisolates (22Shigella sonneiisolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27Shigellaisolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing ofE. coli/Shigellaisolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE.IMPORTANCEFast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of allE. coliisolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates).

scholarly journals Comparison of Multilocus Variable-Number Tandem-Repeat Analysis with Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis for Enterococcus faecalis

2011 ◽  
Vol 60 (4) ◽  
pp. 335-339 ◽  
Author(s):  
EWA SADOWY ◽  
ALEKSANDRA SIEŃKO ◽  
WALERIA HRYNIEWICZ
Keyword(s):  
Enterococcus Faecalis ◽  
Gel Electrophoresis ◽  
Tandem Repeats ◽  
Diversity Index ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Pulsed Field Gel Electrophoresis ◽  
Discriminatory Power ◽  
Pulsed Field ◽  
High Discriminatory Power

Enterococcus faecalis represents recently an important etiological agent of health care-associated infections (HAIs) and there is a need for evaluation and comparison of typing methods available for this microorganism. We tested multilocus VNTR (variable-number tandem repeats) analysis (MLVA) on a well-characterized collection of 153 clinical isolates of E. faecalis, corresponding to 52 multilocus sequence types and 67 pulsed-field gel electrophoresis (PFGE) profiles. MLVA showed high discriminatory power, discerning 111 different types (diversity index equal 98.9%). The concordance MLVA/MLST and MLVA/PFGE was 0.95 and 0.74, respectively. High discriminatory power of MLVA indicates its utility for local epidemiology such as outbreak investigation, and for differentiation of clones defined by other methods.

scholarly journals Development of a Multiple-Locus Variable-Number Tandem-Repeat Typing Scheme for Genetic Fingerprinting of Burkholderia cenocepacia and Application to Nationwide Epidemiological Analysis

2014 ◽  
Vol 53 (2) ◽  
pp. 398-409 ◽  
Author(s):  
Christine Segonds ◽  
Michelle Thouverez ◽  
Antoine Barthe ◽  
Nadège Bossuet-Greif ◽  
Lenka Tisseyre ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Burkholderia Cepacia ◽  
Genetic Relatedness ◽  
Variable Number Tandem Repeat ◽  
Cost Effective ◽  
Variable Number ◽  
Discriminatory Power ◽  
Burkholderia Cenocepacia ◽  
Content Type ◽  
Burkholderia Multivorans

Organisms of theBurkholderia cepaciacomplex are especially important pathogens in cystic fibrosis (CF), with a propensity for patient-to-patient spread and long-term respiratory colonization.B. cenocepaciaandBurkholderia multivoransaccount for the majority of infections in CF, and major epidemic clones have been recognized throughout the world. The aim of the present study was to develop and evaluate a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme forB. cenocepacia. Potential VNTR loci were identified upon analysis of the annotated genome sequences ofB. cenocepaciastrains AU1054, J2315, and MCO-3, and 10 of them were selected on the basis of polymorphisms and size. A collection of 100B. cenocepaciastrains, including epidemiologically related and unrelated strains, as well as representatives of the major epidemic lineages, was used to evaluate typeability, epidemiological concordance, and the discriminatory power of MLVA-10 compared with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Longitudinal stability was assessed by testing 39 successive isolates from 14 patients. Typeability ranged from 0.91 to 1, except for that of one marker, which was not amplified in 53% of theB. cenocepaciaIIIA strains. The MLVA types were shown to be stable in chronically colonized patients and within outbreak-related strains, with excellent epidemiological concordance. Epidemic and/or globally distributed lineages (epidemic Edinburgh-Toronto electrophoretic type 12 [ET-12], sequence type 32 [ST-32], ST-122, ST-234, and ST-241) were successfully identified. Conversely, the discriminatory power of MLVA was lower than that of PFGE or MLST, although PFGE variations within the epidemic lineages sometimes masked their genetic relatedness. In conclusion, MLVA represents a promising cost-effective first-line tool inB. cenocepaciasurveillance.

Escherichia coli O157:H7 Outbreak Associated with Restaurant Beef Grinding

2015 ◽  
Vol 78 (7) ◽  
pp. 1272-1279 ◽  
Author(s):  
LAUREN M. TORSO ◽  
RONALD E. VOORHEES ◽  
STEPHEN A. FOREST ◽  
ANDREW Z. GORDON ◽  
SHARON A. SILVESTRI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Tandem Repeat ◽  
Gel Electrophoresis ◽  
Variable Number Tandem Repeat ◽  
Ground Beef ◽  
Variable Number ◽  
Pulsed Field Gel Electrophoresis ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Repeat Analysis

Escherichia coli O157:H7 is a common cause of foodborne illness in the United States. Beef ground at establishments regulated by the U.S. Department of Agriculture, Food Safety and Inspection Service is routinely tested for E. coli O157:H7. Prior to December 2013, boxed beef product (wholesale cuts of beef, such as beef loin, packaged into bags and boxed for shipping) was not always tested for this pathogen. Downstream processors or retailers may grind the product; and, if the ground beef is not cooked to the recommended temperature, pathogens on the exterior of the beef introduced to the interior through grinding may survive. On 18 October 2013, the Allegheny County Health Department identified two E. coli O157:H7 cases, both of whom were food handlers at restaurant A, a restaurant that ground locally produced boxed beef for hamburgers on site. Case finding was conducted through public messaging, employee surveys, and disease surveillance. All potential cases were interviewed using a standard questionnaire. A confirmed case was defined as laboratory-confirmed E. coli O157:H7 with exposure to restaurant A. A probable case was defined as a patient with compatible symptoms and exposure to restaurant A but without laboratory confirmation. All human and food isolates were characterized by pulsed-field gel electrophoresis and multilocus variable-number tandem repeat analysis. The analysis identified 14 confirmed and 10 probable cases of E. coli; 18 nonintact ground beef samples tested positive for E. coli O157:H7. Nine confirmed cases were restaurant A employees. All confirmed cases recalled eating a restaurant A hamburger in the 10 days before illness onset; most cases reported consuming medium to rare hamburgers. Multiple pulsed-field gel electrophoresis and multilocus variable-number tandem repeat analysis patterns were identified among both the human and ground beef isolates, and the patient isolates matched those found in ground beef samples. Restaurant A voluntarily closed for 1.5 days, changed beef suppliers, ceased grinding beef in-house, and has had no new cases since reopening.

Prevalence and Molecular Characterization of Escherichia coli O157:H7 by Multiple-Locus Variable-Number Tandem Repeat Analysis and Pulsed-Field Gel Electrophoresis in Three Sheep Farming Operations in California

2011 ◽  
Vol 74 (9) ◽  
pp. 1413-1421 ◽  
Author(s):  
CHRISTOPHER KILONZO ◽  
EDWARD R. ATWILL ◽  
ROBERT MANDRELL ◽  
MELISSA GARRICK ◽  
VERONICA VILLANUEVA ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Gel Electrophoresis ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Pulsed Field Gel Electrophoresis ◽  
Escherichia Coli O157 ◽  
Fecal Samples ◽  
Multiple Locus ◽  
E Coli ◽  
Repeat Analysis

A year-long study was conducted to determine the fecal prevalence of Escherichia coli O157:H7 in three sheep ranches. Strain diversity and persistence were compared with multiple-locus variable-number tandem repeat analysis and pulsed-field gel electrophoresis. Ranch C, a feedlot, consisted of young sheep raised predominantly on a high-grain diet. The other two sites consisted of sheep raised on native pasture and a combination of native and irrigated pasture. Forty fecal samples were collected every month from each ranch. Samples were examined for E. coli O157:H7 by immunomagnetic separation and culture of the magnetic beads onto selective media. Detection of virulence markers in positive isolates was determined by PCR. E. coli O157:H7 was isolated from 100 (22.7%) of 440 fecal samples collected from ranch C. On ranch B, 9 (1.9%) of the 480 fecal samples were positive for the pathogen, while none of the samples from ranch A were positive. On ranch C, the odds of detecting E. coli O157:H7 was 3.2 times greater during the warmer months compared with the cooler months of the year. There was no association between days spent in the feedlot and fecal prevalence of the pathogen (P = 0.62). Most multiple-locus variable-number tandem repeat analysis types were isolated only once from ranch C (14 of 23), but several strains were isolated over 4 to 6 months, often in many intervening negative months. This study revealed that the prevalence of E. coli O157:H7 can be high in some sheep ranches in California, especially in feedlots where young sheep are fed predominantly high-grain rations.

scholarly journals Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks

2011 ◽  
Vol 77 (14) ◽  
pp. 4949-4958 ◽  
Author(s):  
C. Sekse ◽  
M. Sunde ◽  
B.-A. Lindstedt ◽  
P. Hopp ◽  
T. Bruheim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Human Pathogens ◽  
Content Type ◽  
Representative Sampling ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Close Relationship ◽  
Large Survey

ABSTRACTA national survey ofEscherichia coliO26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, andE. coliO26 was detected in 17.9% of the flocks. One hundred forty-twoE. coliO26 isolates were examined for flagellar antigens (H typing) and four virulence genes, includingstxandeae, to identify possible Shiga toxin-producingE. coli(STEC) and enteropathogenicE. coli(EPEC). Most isolates (129 out of 142) were identified asE. coliO26:H11. They possessedeaeand may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between theE. coliO26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all theE. coliO26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing onE. coliO26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.

scholarly journals Prediction of Local Transmission of Mycobacterium tuberculosis Isolates of a Predominantly Beijing Lineage by Use of a Variable-Number Tandem-Repeat Typing Method Incorporating a Consensus Set of Hypervariable Loci

2017 ◽  
Vol 56 (1) ◽  
Author(s):  
Yoshiro Murase ◽  
Kiyohiko Izumi ◽  
Akihiro Ohkado ◽  
Akio Aono ◽  
Kinuyo Chikamatsu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Trend Test ◽  
Typing Method ◽  
Content Type ◽  
Tuberculosis Transmission ◽  
High Prevalence ◽  
Hypervariable Loci

ABSTRACT Strain genotyping based on the variable-number tandem repeat (VNTR) is widely applied for identifying the transmission of Mycobacterium tuberculosis. A consensus set of four hypervariable loci (1982, 3232, 3820, and 4120) has been proposed to improve the discrimination of Beijing lineage strains. Herein, we evaluated the utility of these four hypervariable loci for tracing local tuberculosis transmission in 981 cases over a 14-month period in Japan (2010 to 2011). We used six different VNTR systems, with or without the four hypervariable loci. Patient ages and weighted standard distances (a measure of the dispersion of genotype-clustered cases) were used as proxies for estimating local tuberculosis transmission. The highest levels of isolate discrimination were achieved with VNTR systems that incorporated the four hypervariable loci (i.e., the Japan Anti-Tuberculosis Association [JATA]18-VNTR, mycobacterial interspersed repetitive unit [MIRU]28-VNTR, and 24Beijing-VNTR). The clustering rates by JATA12-VNTR, MIRU15-VNTR, JATA15-VNTR, JATA18-VNTR, MIRU28-VNTR, and 24Beijing-VNTR systems were 52.2%, 51.0%, 39.0%, 24.1%, 23.1%, and 22.0%, respectively. As the discriminative power increased, the median weighted standard distances of the clusters tended to decrease (from 311 to 80 km, P < 0.001, Jonckheere-Terpstra trend test). Concurrently, the median ages of patients in the clusters tended to decrease (from 68 to 60 years, P < 0.001, Jonckheere-Terpstra trend test). These findings suggest that strain typing using the four hypervariable loci improves the prediction of active local tuberculosis transmission. The four-locus set can therefore contribute to the targeted control of tuberculosis in settings with high prevalence of Beijing lineage strains.

Microbiological Effects of a Routine Treatment for Decontaminating Hide-On Carcasses at a Large Beef Packing Plant

2015 ◽  
Vol 78 (2) ◽  
pp. 256-263 ◽  
Author(s):  
XIANQIN YANG ◽  
MADHU BADONI ◽  
FRANCES TRAN ◽  
COLIN O. GILL
Keyword(s):  
Escherichia Coli ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Routine Treatment ◽  
Multiple Locus ◽  
E Coli ◽  
Repeat Analysis ◽  
Before And After

To investigate the microbiological effects of a hide-on carcass decontaminating treatment recently implemented at a beef packing plant, carcasses undergoing routine processing at the plant were sampled during successive periods in January/February, April/May, and September/October. During each period, samples were collected from carcasses before and after the decontamination of hide-on carcasses, after skinning, before decontamination of the skinned carcasses, and at the end of the carcass dressing process. At each stage of processing during each period, samples were obtained by swabbing an area of 1,000 cm2 on each of 25 carcasses. Aerobes, coliforms, and Escherichia coli were enumerated. In most samples, coliforms were predominantly E. coli. In all three periods, the log mean numbers of aerobes and E. coli recovered from hides before decontamination were between 6.6 and 6.8 and between 5.3 and 5.9 log CFU/1,000 cm2, respectively. The log mean numbers of aerobes recovered from decontaminated hides were 6.6 log CFU/1,000 cm2 in January/February and April/May but 5.4 log CFU/1,000 cm2 in September/October. The log total numbers of E. coli recovered from decontaminated hides in January/February and April/May were 2.4 and 3.8 log CFU/25,000 cm2, respectively, but no E. coli was recovered from such carcasses in September/October. Log total numbers of aerobes and E. coli recovered from skinned or dressed carcasses were mostly &gt;4 and between 1 and 2 log CFU/25,000 cm2, respectively. Typing of 480 E. coli isolates by multiple-locus variable-number tandem repeat analysis (MLVA) identified 218 MLVA types. Most isolates recovered from carcasses in different periods or at different stages of processing were of different MLVA types. However, small numbers of MLVA types were recovered in more than one period or from both hides before and after decontamination and skinned or dressed carcasses. The findings show that the hide-decontaminating treatment disrupted the usual transfer of E. coli from hides to meat surfaces during carcass skinning.

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