Novel multiple-locus variable-number tandem-repeat analysis method for rapid molecular typing of human Staphylococcus aureus

We evaluated the use of a novel multiple-locus variable-number tandem-repeat analysis (MLVA) method for typing of human Staphylococcus aureus. For a total of 150 clinical isolates, MLVA demonstrated the highest discriminatory power. MLVA correctly assigned isolates to outbreaks or identified isolates as unlinked. MLVA is a rapid and simple method for the epidemiological typing of S. aureus.     

New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates

The PCR-based methodology applied to multiple-locus variable numbers of tandem repeat (VNTR) analysis was recently shown to be a useful technique for the molecular typing of clinical isolates of several bacterial species. We have adopted this method for the molecular typing of methicillin-resistant Staphylococcus aureus. Five staphylococcal VNTR loci (sdr, clfA, clfB, ssp, and spa) were subjected to analysis, and it was shown that the method allows typing of S. aureus strains with the discriminatory power and reproducibility of pulsed-field gel electrophoresis while at the same time being rapid and applicable to analysis of large numbers of isolates.     

Microfluidic-chip-based multiple-locus variable-number tandem-repeat fingerprinting with new primer sets for methicillin-resistant Staphylococcus aureus

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.     

Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates

Multiple-locus variable-number tandem-repeat analysis (MLVA), a new PCR-based method of typing Staphylococcus aureus, was compared to pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) on a group of 59 S. aureus (mostly methicillin-resistant) clinical isolates. The aim of the study was to establish possible criteria of clustering MLVA patterns and to check concordance levels between the results produced by MLVA and the three other typing methods. As in our earlier study, MLVA turned out to have discriminatory power similar to that of PFGE. Comparison of data obtained by the two approaches allowed us to propose a 70% or ca. 80% cutoff value of the similarity between two MLVA patterns, depending on a cutoff level applied to interpret the PFGE results, 75% or ca. 90%, respectively. The cutoff values corresponded to the difference of up to six or four bands, respectively, among maximum 14 bands in total produced by two isolates in the analysis. The MLVA clusters matched well those obtained by PFGE, and they were also consistent in general with clusters generated by spa typing and MLST, these latter methods characterized lower resolution. Our results suggest that MLVA may be reliable in shorter-term S. aureus epidemiological studies, including analyses of outbreaks and hospital-to-hospital strain transmission events. Well-known advantages of typing methods based on PCR (low cost, short time, and easiness of performance) make MLVA a method that may be useful in a variety of laboratories, including those performing routine microbiological analyses within medical centers.     

Multiple-locus variable-number tandem repeat analysis for typing of Staphylococcus epidermidis

We applied a high-resolution PCR-based typing method, multiple-locus variable-number tandem repeat analysis (MLVA), for discrimination of 30 multidrug-resistant clinical isolates of Staphylococcus epidermidis. The results of MLVA were congruent with results obtained by pulsed-field gel electrophoresis (PFGE). MLVA generated discrete character data, and its discriminatory capacity was comparable to that of PFGE.     

Typing and subtyping of Clostridium difficile isolates by using multiple-locus variable-number tandem-repeat analysis

Using the genomic sequence of Clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (MLVA) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for C. difficile. All reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of PCR ribotype 001, could be discriminated from each other. Application of MLVA to 28 isolates from 7 outbreaks due to the emerging hypervirulent PCR ribotype 027-pulsed-field gel electrophoresis type NAP1 resulted in recognition of 13 clusters. Additionally, 29 toxin A-negative, toxin B-positive isolates belonging to PCR ribotype 017 from eight different countries revealed eight country-specific clusters. MLVA is a highly discriminatory genotyping method and a new tool for subtyping of newly emerging variants of C. difficile.     

Comparison of multiple-locus variable-number tandem-repeat analysis with other PCR-based methods for typing Brucella suis isolates

Multiple-locus variable-number tandem-repeat analysis (MLVA), multiplex PCR, and PCR-restriction fragment length polymorphism analysis were compared for typing Brucella suis isolates. A perfect concordance was obtained among these molecular assays. However, MLVA was the only method to demonstrate brucellosis outbreaks and to confirm that wildlife is a reservoir for zoonotic brucellosis.     

A chromogenic method for rapid identification of Staphylococcus aureus

Staphylococcus aureus is responsible for septicaemia and serious nosocomial infections. A rapid and specific identification of this species is of great importance in clinical microbiology. Current methods for S. aureus identification require a 18 to 24 h-incubation. We describe a two hour-identification method based on the detection of the staphylocoagulase, using human prothrombin and a chromogenic substrate. 242 staphylococcal strains (160 S. aureus, 82 coagulase-negative staphylococci (CNS)) were collected from 4 French hospitals. They have been identified by the following methods: (i) clotting of citrated rabbit plasma, which is considered as reference method; (ii) biochemical tests (Rapidec Staph and Api Staph or ID 32 Staph); (iii) and agglutination test (Pastorex Staph or Pastorex Staph-plus). A strain of S. intermedius was provided by the Collection of the Pasteur Institute (Paris). An adapted culture medium is inoculated with staphylococci and adjusted to 2 Mac Farland unities. This medium is then mixed to an equal volume with a human prothrombin solution and the chromogenic substrate. After 1 to 2 hours incubation at 37 degrees C, the strength of the yellow colour of the mixture is observed to the naked eye, or measured at 405 nm with a spectrophotometer. Fifteen chromogenic tripeptides having a thrombin-like affinity and paranitroanilin as leaving group were compared. With the substrate which has the higher hydrolysis velocity and enzymatic affinity (SQ149), all S. aureus strains gave a positive result: 94.7% of the methicillin-susceptible S. aureus were detected after 1 hour incubation, but only 52.3% of the methicillin-resistant S. aureus. 98.4% of the methicillin-resistant S. aureus were detected after 2 hours. No false positive result was observed for the 82 CNS strains. The chromogenic method shows good within-run and day-to-day precision tests. It doesn't need any complementary test. The sensitivity and the specificity are 99.4% and 100% respectively.     

Strain typing of Borrelia burgdorferi,Borrelia afzelii,and Borrelia garinii by using multiple-locus variable-number tandem repeat analysis

Human Lyme borreliosis (LB) is the most prevalent arthropod-borne infection in temperate climate zones around the world and is caused by Borrelia spirochetes. We have identified 10 variable-number tandem repeat (VNTR) loci present within the genome of Borrelia burgdorferi and subsequently developed a multiple-locus VNTR analysis (MLVA) typing system for this disease agent. We report here the successful application of MLVA for strain discrimination among a group of 41 globally diverse Borrelia isolates including B. burgdorferi, B. afzelii, and B. garinii. PCR assays displayed diversity at these loci, with total allele numbers ranging from two to nine and Nei's diversity (D) values ranging from 0.10 to 0.87. The average D value was 0.53 across all VNTR loci. A clear correlation exists between the repeat copy number and the D value (r = 0.62) or the number of alleles (r = 0.93) observed across diverse strains. Cluster analysis by the unweighted pair-group method with arithmetic means resolved the 30 observed unique Borrelia genotypes into five distinct groups. B. burgdorferi, B. afzelii, and B. garinii clustered into distinct affiliations, consistent with current 16S rRNA phylogeny studies. Genetic similarity and diversity suggest that B. afzelii and B. garinii are close relatives and were perhaps recently derived from B. burgdorferi. MLVA provides both phylogenetic relationships and additional resolution to discriminate among strains of Borrelia species. This new level of strain identification and discrimination will allow more detailed epidemiological and phylogenetic analysis in future studies.     

Modified multiple-locus variable-number tandem-repeat analysis for rapid identification and typing of Clostridium difficile during institutional outbreaks

A modified multiple-locus variable-number tandem-repeat analysis (MMLVA) method was validated on Clostridium difficile-infected stool specimens from institutional outbreaks. The method allows simultaneous detection of toxin genes, deletions, and tandem repeats from cultured isolates or stool specimens. Results were used to aid institutional outbreak investigation by identifying clusters of NAP1/027.     

黄疸出血群钩端螺旋体MLVA分型研究

目的 初步探讨MLVA(multiple-locus variable-number tandem-repeat analysis)技术在黄疸出血群钩端螺旋体基因分型中的应用.方法 选取7个VNTR位点,对我国致病性钩端螺旋体黄疸出血群菌株提取基因组DNA,采用PCR扩增和琼脂糖凝胶电泳技术检测,应用BioNumerics(Ver-sion4.0)软件进行聚类分析.结果 共对117株钩端螺旋体的7个VNTR位点进行了检测,聚类分析分为3个群(A群、B群、C群)28种基因型,其中A群占11.97%(14/117)、B群占0.85%(1/117)、c群占87.18%(102/117);多态性指数介于0.0831与0.8005之间;MLVA基因型存在明显的地域性.结论 MLVA分型技术可初步对钩端螺旋体进行遗传学分类鉴定,应用该技术,将在钩体病分子流行病学研究中发挥重要的作用.     

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.     

Rapid genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays

This study evaluated a DNA oligonucleotide array that recognised 38 different Staphylococcus aureus targets, including all relevant resistance determinants and some toxins and species-specific controls. A new method for labelling sample DNA, based on a linear multiplex amplification that incorporated biotin-labelled dUTP into the amplicon, was established, and allowed detection of hybridisation of the amplicons to the array with an enzymic precipitation reaction. The whole assay was validated by hybridisations with a panel of reference strains and cloned specific PCR products of all targets. To evaluate performance under routine conditions, the assay was used to test 100 methicillin-resistant S. aureus (MRSA) isolates collected from a university hospital in Saxony, Germany. The results showed a high correlation with conventional susceptibility data. The ermA and ermC macrolide resistance genes were found in 40% and 32% of the isolates, respectively. The most prevalent aminoglycoside resistance gene was aphA3 (57% of the isolates), followed by aacA-aphD (29%) and aadD (29%); tet genes, mupR and dfrA were rare. There were no isolates with van genes or genes involved in resistance to quinupristin-dalfopristin. Enterotoxins were detected in 27% of the isolates. Genes encoding Panton-Valentine leukocidin, toxic shock syndrome toxin and exfoliative toxins were not found. The DNA array facilitated rapid and reliable detection of resistance determinants and toxins under conditions used in a routine laboratory and has the potential to be used for array-based high-throughput screening.     

Multiplex PCR for rapid detection of Staphylococcus aureus isolates suspected to represent community-acquired strains

The continuous spread of community-acquired methicillin-resistant Staphylococcus aureus (caMRSA) and the introduction of these highly virulent isolates into hospitals represent increasing threats. The timely recognition of caMRSA strains is crucial for infection control purposes. Thus, we developed a PCR-based assay for the easy and rapid determination of those caMRSA clones that currently are the most prevalent in Germany and Central Europe. This assay was able to correctly identify the majority of the isolates as caMRSA of sequence type 80 (ST80), clonal complex 1 (USA400), and ST8 (USA300). In combination with spa typing-BURP (based upon repeat pattern) analysis and resistance typing, it provides a means for the extensive characterization of suspicious isolates. Thus, this assay represents a reliable tool for monitoring the emergence and spread of different caMRSA clones. The resulting information, in combination with careful interpretation of the epidemiological records, might help to prevent the further spread of those highly virulent caMRSA clones.     

A reliable,high-resolution and high-throughput genotyping method for HLA-DRB1

Human leukocyte antigen (HLA) DNA typing is an essential part to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow. Thus, fetching quickly high-resolution genotype is very important at present. However, current HLA DNA typing methods especially for HLA-DRB1 generally yielded ambiguous typing results because of high degree of heterozygosity on exome region and primer pairs design limitations, which generally amplified only exon2 of HLA-DRB1 and the position of its primer pairs is on exome region. To solve this problem, we developed a reliable, high-resolution and high-throughput (RHH) sequence based typing approach for HLA-DRB1 through massively parallel paired-end sequencing. Several primer pairs were designed to amplify three exon regions, which are part of exon1, the whole region of exon2 and exon3 of HLA-DRB1, to genotyping for HLA-DRB1 by synthesis. 94 samples were included to analyze and the highly successful genotyping ratio (98.94%) and no ambiguous genotyping result demonstrated the accurate performance of our method for HLA-DRB1 genotyping.     

Use of Staphylococcus aureus for rapid radioimmunoassay of influenza A virus haemagglutinin.

In a rapid method for the radioimmunoassay (RIA) of influenza A virus haemagglutinin, Staphylococcus aureus (strain Cowan I, Czechoslovak State Collection No Mau 55/64) was used for separation of bound and free antigens. With rabbit and human immune sera, the binding of antigen-antibody complexes to heat-killed, formalin-fixed staphylocci was comparable to the double antibody technique. The time required for the completion of binding reaction was about 10 min compared to 18--24 hr required for double antibody precipitation. S. aureus did not bind directly (i.e. in the absence of specific antibody) a significant amount of radiolabelled antigen.     

Evaluation of an isothermal signal amplification method for rapid detection of methicillin-resistant Staphylococcus aureus from patient-screening swabs

A new molecular assay (CytAMP) utilizing isothermal signal-mediated amplification of RNA was evaluated for rapid detection of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). The assay targeted the coa (coagulase) and mecA genes, thereby simultaneously identifying S. aureus and methicillin (oxacillin) resistance. Results were obtained in approximately 3.5 h as a color signal in 96-well microtiter plates. The detection limit was between 2 x 10(5) and 10(6) CFU/assay, equivalent to 4 x 10(6) to 2 x 10(7) CFU/ml in an overnight broth. This level of growth was obtained with an initial inoculum of 10 to 50 CFU. The CytAMP assay and a mecA-femB PCR assay both detected 113 MRSA strains among 396 clinical isolates of bacteria (CytAMP sensitivity and specificity were both 100% relative to those of PCR). Conventional culture detected 109 MRSA strains, but with 19 false-positive and 23 false-negative results relative to both molecular methods. Discrepancies were also observed for 100 enrichment broths containing MRSA screening swabs, with 11 broths culture negative but PCR positive. CytAMP and PCR were more in agreement, but six broths were CytAMP negative and PCR positive. Five of these contained 10(2) to 10(5) CFU/assay (below the CytAMP detection limit of 2 x 10(5) CFU/assay), and the sixth contained 10(6) CFU/assay. Overall, culture and CytAMP had similar sensitivities and specificities relative to those of PCR, but the CytAMP assay enabled swabs to be analyzed as a batch following overnight incubation in enrichment broth, with results reported before 12 noon the next day.     

Screening method for rapid detection of methicillin-resistant (heteroresistant) Staphylococcus aureus.

A broth screening method was developed to detect methicillin-resistant (heteroresistant) strains of Staphylococcus aureus from primary plates. Lyophilized vials containing cation-supplemented Mueller-Hinton broth, 2% NaCl, and 10 micrograms of methicillin and 6 micrograms of oxacillin per ml were hydrated and tested with 129 methicillin-resistant and 35 methicillin-susceptible strains of S. aureus. With the addition of a tetrazolium indicator after 5 h of incubation, 96.9% of resistant strains were detected. No false-positive results occurred.     

Screening for methicillin-resistant Staphylococcus aureus in clinical swabs using a high-throughput real-time PCR-based method.

The presence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and the community is a serious problem. Accordingly, a comprehensive plan has been implemented in the County of Vejle, Denmark, to identify colonised and/or infected individuals and to control the spread of MRSA. Since 2005, all patients and healthcare personnel have been screened for MRSA colonisation, involving analysis of 300-400 samples daily. To deal with this number of samples, a PCR-based method customised for high-throughput analysis and a system for fast reporting of MRSA carrier status were developed. Swab samples were incubated overnight in a selective tryptone soya broth and were analysed by PCR the following day. Using this strategy, non-colonised individuals were identified within 24 h, while MRSA-positive samples were analysed further by traditional microbiological methods to determine the resistance pattern. This is a cost-effective approach, as the greatest expense in hospitals involves the isolation of patients of unknown MRSA status. The method was evaluated by testing 2194 clinical samples, with a sensitivity and specificity of 100% and 94%, respectively. The analytical sensitivity was 97%, with 161 of 166 different MRSA strains and isolates generating positive results according to PCR analysis. Using four control strains, the inter-assay variation was revealed to be a maximum of 2.6%, indicating good reproducibility.