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Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus.

ABSTRACT: Fifty-nine Staphylococcus aureus isolates and 1 isolate of Staphylococcus intermedius were typed by investigators at eight institutions by using either antibiograms, bacteriophage typing, biotyping, immunoblotting, insertion sequence typing with IS257/431, multilocus enzyme electrophoresis, restriction analysis of plasmid DNA, pulsed-field or field inversion gel electrophoresis, restriction analysis of PCR-amplified coagulase gene sequences, restriction fragment length polymorphism typing by using four staphylococcal genes as probes, or ribotyping. Isolates from four well-characterized outbreaks (n = 29) and a collection of organisms from two nursing homes were mixed with epidemiologically unrelated stock strains from the Centers for Disease Control and Prevention. Several isolates were included multiple times either within or between the sets of isolates to analyze the reproducibilities of the typing systems. Overall, the DNA-based techniques and immunoblotting were most effective in grouping outbreak-related strains, recognizing 27 to 29 of the 29 outbreak-related strains; however, they also tended to include 3 to 8 epidemiologically unrelated isolates in the same strain type. Restriction fragment length polymorphism methods with mec gene-associated loci were less useful than other techniques for typing oxacillin-susceptible isolates. Phage typing, plasmid DNA restriction analysis, and antibiogram analysis, the techniques most readily available to clinical laboratories, identified 23 to 26 of 29 outbreak-related isolates and assigned 0 to 6 unrelated isolates to outbreak strain types. No single technique was clearly superior to the others; however, biotyping, because it produced so many subtypes, did not effectively group outbreak-related strains of S. aureus.

SUBMITTER: Tenover FC

PROVIDER: S-EPMC263045 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus.

Tenover F C FC Arbeit R R Archer G G Biddle J J Byrne S S Goering R R Hancock G G Hébert G A GA Hill B B Hollis R R

Journal of clinical microbiology 19940201 2

Fifty-nine Staphylococcus aureus isolates and 1 isolate of Staphylococcus intermedius were typed by investigators at eight institutions by using either antibiograms, bacteriophage typing, biotyping, immunoblotting, insertion sequence typing with IS257/431, multilocus enzyme electrophoresis, restriction analysis of plasmid DNA, pulsed-field or field inversion gel electrophoresis, restriction analysis of PCR-amplified coagulase gene sequences, restriction fragment length polymorphism typing by usi ...[more]

PMID: 7908673

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Project description:BackgroundStaphylococcus aureus causes a variety of severe infections such as bacteremia and sepsis. At present, 60-80% of S. aureus isolates from Taiwan are methicillin resistant (MRSA). It has been shown that certain MRSA clones circulate worldwide. The goals of this study were to identify MRSA clones in Taiwan and to correlate the molecular types of isolates with their phenotypes.MethodsA total of 157 MRSA isolates from bacteremic patients were collected from nine medical centers. They were typed based on polymorphisms in agr, SCCmec, MLST, spa, and dru. Phenotypes characterized included Panton-Valentine leucocidin (pvl), inducible macrolide-lincosamide-streptogramin B resistance (MLSBi), vancomycin (VA) and daptomycin (DAP) minimal inhibitory concentrations (MIC), and superantigenic toxin gene profiles. Difference between two consecutive samples was determined by Mann-Whitney-U test, and difference between two categorical variables was determined by Fisher's exact test.ResultsFour major MRSA clone complexes CC1, CC5, CC8, and CC59 were found, including 4 CC1, 9 CC5, 111 CC8, and 28 CC59 isolates. These clones had the following molecular types: CC1: SCCmecIV and ST573; CC5: SCCmecII and ST5; CC8: SCCmecIII, ST239, and ST241, and CC59: SCCmecIV, SCCmecV(T), ST59, and ST338. The toxin gene profiles of these clones were CC1: sec-seg-(sei)-sell-selm-(seln)-selo; CC5: sec-seg-sei-sell-selm-(seln)-selp-tst1; CC8: sea-selk-selq, and CC59: seb-selk-selq. Most isolates with SCCmecV(T), ST59, spat437, and dru11 types were pvl(+) (13 isolates), while multidrug resistance (?4 antimicrobials) were associated with SCCmecIII, ST239, spa t037, agrI, and dru14 (119 isolates) (p<0.001). One hundred and twenty four isolates with the following molecular types had higher VA MIC: SCCmecII and SCCmecIII; ST5, ST239, and ST241; spa t002, t037, and t421; dru4, dru10, dru12, dru13, and dru14 (p<0.05). No particular molecular types were found to be associated with MLSBi phenotype.ConclusionsFour major MRSA clone complexes were found in Taiwan. Further studies are needed to delineate the evolution of MRSA isolates.

Project description:Staphylococcus aureus is a major bacterial human pathogen that causes a wide variety of clinical manifestations. The main aim of the presented study was to determine and optimize a novel sequencing independent approach that enables molecular typing of S. aureus isolates and elucidates the transmission of emergent clones between patients. In total, 987 S. aureus isolates including both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates were used to evaluate the novel typing approach combining high-resolution melting (HRM) analysis of multilocus sequence typing (MLST) genes (mini-MLST) and spa gene (spa-HRM). The novel approach's discriminatory ability was evaluated by whole-genome sequencing (WGS). The clonal relatedness of tested isolates was set by the BURP and BURST approach using spa and MLST data, respectively. Mini-MLST classified the S. aureus isolates into 38 clusters, followed by spa-HRM classifying the isolates into 101 clusters. The WGS proved HRM-based methods to effectively differentiate between related S. aureus isolates. Visualizing evolutionary relationships among different spa-types provided by the BURP algorithm showed comparable results to MLST/mini-MLST clonal clusters. We proved that the combination of mini-MLST and spa-HRM is rapid, reproducible, and cost-efficient. In addition to high discriminatory ability, the correlation between spa evolutionary relationships and mini-MLST clustering allows the variability in population structure to be monitored. IMPORTANCE Rapid and cost-effective molecular typing tools for Staphylococcus aureus epidemiological applications such as transmission tracking, source attribution and outbreak investigations are highly desirable. High-resolution melting based methods are effective alternative to those based on sequencing. Their good reproducibility and easy performance allow prospective typing of large set of isolates while reaching great discriminatory power. In this study, we established a new epidemiological approach to S. aureus typing. This scheme has the potential to greatly improve epidemiological investigations of S. aureus.

Project description:Background Staphylococcus aureus is one of the most important foodborne pathogens in the world and the main cause of dairy cow mastitis. Few studies have investigated the epidemic pedigree of S. aureus of bovine origin in Hunan, China. Therefore, we aimed to analyze the capsular polysaccharides (CP), molecular typing, and antibiotic resistance characteristics of S. aureus isolated from raw milk of dairy farms in Hunan Province. Methods Between 2018 and 2022, 681 raw milk samples were collected from dairy cows from farms in Changsha, Changde, Shaoyang, Yongzhou, and Chenzhou in Hunan Province. S. aureus was isolated from these samples, and the isolates were subjected to molecular typing, CP typing, and determination of antibiotic resistance through broth dilution and polymerase chain reaction (PCR). Results From 681 raw milk samples, 76 strains of S. aureus were isolated. The pathogenicity of 76 isolates was determined preliminarily by detecting cp5 and cp8 CP genes. Eighteen types of antimicrobial resistance phenotypes of 76 S. aureus strains were detected by the broth dilution method, and 11 kinds of related resistance genes were amplified by PCR. The S. aureus isolates had CP5 (42.10%) and CP8 (57.89%). S. aureus had a multiple antimicrobial resistance rate of 26.75%. The isolated strains had the highest resistance rate to penicillin (82.89%) and showed varying degrees of resistance to other drugs, but no isolate showed resistance to doxycycline. The 76 isolates all carried two or more antibiotic resistance genes, with a maximum of eight antibiotics resistance genes. FemB was detected in all isolates, but none of isolates carried vanA, ermA, or glrA. The 76 isolates were divided into 22 sequence types (ST) and 20 spa types by MLST and spa typing, and the number of t796-ST7 (n = 15) isolates was the highest, which may be the major epidemic strain of multidrug-resistant S. aureus. Conclusion The present findings indicate the need to increase production of the CP8 S. aureus vaccine in Hunan Province and strengthen resistance monitoring of t796-ST7 isolates with the prevalent molecular type of multi-drug resistant strains. The use of β-lactam, macrolides, and lincosamides should be reduced; doxycycline, sulfonamides, and glycopeptides could be appropriately added to veterinary antibiotics to treat infectious diseases in dairy cows.

Project description:In a point-prevalence study performed in 145 Spanish hospitals in 2006, we collected 463 isolates of Staphylococcus aureus in a single day. Of these, 135 (29.2%) were methicillin (meticillin)-resistant S. aureus (MRSA) isolates. Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE) after SmaI digestion, staphylococcal chromosomal cassette mec (SCCmec) typing, agr typing, spa typing with BURP (based-upon-repeat-pattern) analysis, and multilocus sequence typing (MLST). The 135 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (72.6%), gentamicin (20.0%), erythromycin (66.7%), and clindamycin (39.3%). Among the isolates resistant to erythromycin, 27.4% showed the M phenotype. All of the isolates were susceptible to glycopeptides. Twelve resistance patterns were found, of which four accounted for 65% of the isolates. PFGE revealed 36 different patterns, with 13 major clones (including 2 predominant clones with various antibiotypes that accounted for 52.5% of the MRSA isolates) and 23 sporadic profiles. Two genotypes were observed for the first time in Spain. SCCmec type IV accounted for 6.7% of the isolates (70.1% were type IVa, 23.9% were type IVc, 0.9% were type IVd, and 5.1% were type IVh), and SCCmec type I and SCCmec type II accounted for 7.4% and 5.2% of the isolates, respectively. One isolate was nontypeable. Only one of the isolates produced the Panton-Valentine leukocidin. The isolates presented agr type 2 (82.2%), type 1 (14.8%), and type 3 (3.0%). spa typing revealed 32 different types, the predominant ones being t067 (48.9%) and t002 (14.8%), as well as clonal complex 067 (78%) by BURP analysis. The MRSA clone of sequence type 125 and SCCmec type IV was the most prevalent throughout Spain. In our experience, PFGE, spa typing, SCCmec typing, and MLST presented good correlations for the majority of the MRSA strains; we suggest the use of spa typing and PFGE typing for epidemiological surveillance, since this combination is useful for both long-term and short-term studies.

Dataset Information

Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus.

Publications

Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus.

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