Molecular Strain Typing Using Repetitive Sequence–Based PCR

Frye, Stacie R.; Healy, Mimi

doi:10.1007/0-387-32892-0_26

Cited by 10 publications

(6 citation statements)

References 154 publications

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“…An alternative solution to RAPD-PCR, characterized by both high differential strength and satisfactory reproducibility, is the amplification of repetitive regions dispersed in bacterial genomes [48,49]. In our research, we used ERIC, (GTG) 5 and BOXA1R primers (Table S1) for this purpose.…”

Section: Rep-pcrmentioning

confidence: 99%

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level

Jarocki

Komoń-Janczara

Glibowska

et al. 2020

IJMS

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The genus Lactobacillus includes, among others, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus, species that are collectively referred to as the Lactobacillus casei group. Many studies have shown that strains belonging to this group may decrease lactose intolerance, the effects of inflammatory bowel disease, diarrhea, constipation, food allergies and even colon cancer. Moreover, evidences exists of positive effects of these bacteria on mucosal immunity and blood cholesterol level. Because of their beneficial influence on human health, many of them are used as food additives and probiotic pharmaceuticals. It should be stressed that health-promoting properties are not attributed at the species level, but to specific strains. Therefore, procedures are necessary to allow specific identification at each phylogenetic level—genus, species and strain. In this paper we present a practical overview of molecular methods for the identification and differentiation of L. casei bacteria. The research included 30 bacterial strains belonging to three species: L.casei, L. paracasei and L. rhamnosus. Among the tested procedures were genus- and species-specific PCR, multiplex-PCR, Real-Time HRM analysis, RFLP-PCR, rep-PCR, RAPD-PCR, AFLP-PCR, and proteomic methods such as MALDI-TOF MS typing and SDS-PAGE fingerprinting. The obtained results showed that multiplex-PCR and MALDI-TOF MS turned out to be the most useful methods to identify the tested bacteria at the species level. At the strain level, the AFLP-PCR method showed the highest discriminatory power. We hope that the presented results will allow for the easy selection of an appropriate procedure, depending on the experiment conducted and the equipment capabilities of any given laboratory.

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Section: Rep-pcrmentioning

confidence: 99%

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level

Jarocki

Komoń-Janczara

Glibowska

et al. 2020

IJMS

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“…To analyze the phylogenetic relatedness of the isolates used in this study, repetitive-sequence PCR (rep-PCR) was performed as previously described (29,30). The banding patterns obtained on a virtual gel were compared to determine the similarity index.…”

Section: Bacterial Isolatesmentioning

confidence: 99%

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group

Burnham

Westblade

et al. 2016

J Clin Microbiol

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Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection of mecA-or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/ mecC PCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered. Staphylococcus pseudintermedius is a coagulase-positive, hemolytic species that colonizes the nares and anal mucosa of healthy dogs and cats (1-4). Clinically, S. pseudintermedius is the most common cause of canine pyoderma and occasionally causes urinary tract and joint infections in dogs and cats (5-8). Phenotypically, S. pseudintermedius is difficult to differentiate from Staphylococcus intermedius and Staphylococcus delphini, which are also coagulase-positive veterinary staphylococci. Collectively, these three species are referred to as the S. intermedius group (SIG). Differentiation of the members of this group requires molecular analysis (9), although matrix-assisted laser desorption ionization-time of flig...

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“…The PCR products were loaded into two different DiversiLab DNA chips (bioMérieux; Durham, NC) according to the manufacturer's specifications and resolved using the 2100 Bioanalyzer instrument. The resultant banding patterns were analyzed using the DiversiLab Bacterial Barcodes software program, which uses curve-based Pearson coefficients for pairwise similarity scores (14). The data were organized into a dendrogram, and a similarity index (SI) for each pair was calculated (Fig.…”

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confidence: 99%

Development and Evaluation of a Novel, Semiautomated Clostridium difficile Typing Platform

et al. 2013

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dWe describe a novel, semiautomated Clostridium difficile typing platform that is based on PCR-ribotyping in conjunction with a semiautomated molecular typing system. The platform is reproducible with minimal intra-or interassay variability. This method exhibited a discriminatory index of 0.954 and is therefore comparable to more arduous typing systems, such as pulsedfield gel electrophoresis. C lostridium difficile, the etiological agent of C. difficile infection (CDI), is an important cause of both hospital-and community-acquired infectious diarrhea (1, 2). The emergence of hypervirulent C. difficile isolates and in particular the NAP1/BI/027 isolate has altered the epidemiology of C. difficile infections in many health care institutions, resulting in increased severity and duration of disease, with concomitant increases to the length and cost of hospitalization (1). Therefore, typing methods that can discriminate NAP1/BI/027 and other emerging hypervirulent C. difficile isolates may be important for understanding the transmission dynamics of the organism. In addition, it has recently been demonstrated that the analytical sensitivity and specificity of C. difficile diagnostic assays may be dependent on the C. difficile strain type (3). In addition, recent reports suggest that the relapse rate following treatment with certain novel antianaerobic and C. difficile-specific antimicrobials could correlate with C. difficile strain type (4, 5); thus, an appreciation of the isolate type may play a role in patient management in the future. As such, C. difficile typing may have the potential to improve the management of CDI beyond clinical surveillance, especially in the hospital setting, and it may be important for clinical laboratorians and infection control specialists to have a baseline understanding of the different C. difficile isolates circulating in their institutions.Pulsed-field gel electrophoresis (PFGE) is the principal reference method employed for C. difficile typing in North America (6). While PFGE affords acceptable discriminatory power (6), it does suffer from some important limitations, in particular the labor intensity, technical expertise, turnaround time, and necessity for control strains to be processed alongside isolates of epidemiological interest. In Europe, the predominant method for C. difficile typing is PCR-ribotyping, which involves the PCR amplification of the intergenic space region between the 16S and 23S rRNA genes (7,8,9). For many years, epidemiologic studies for C. difficile have relied on PFGE and PCR-ribotyping to determine strain relatedness; multiple-locus variable-number tandem-repeat analysis (MLVA) for C. difficile typing has also been described for recent studies (6,10,11, 12). Although this method does appear to be reproducible and discriminatory, it requires access to a genetic analyzer/DNA sequencer, which is cost prohibitive to many laboratories. The objective of this study was to develop and validate a semiautomated PCR-ribotyping platform that would further reduce labor...

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Molecular Strain Typing Using Repetitive Sequence–Based PCR

Cited by 10 publications

References 154 publications

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group

Development and Evaluation of a Novel, Semiautomated Clostridium difficile Typing Platform

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