CRISPR Typing Increases the Discriminatory Power of Streptococcus agalactiae Typing Methods.
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ABSTRACT:
SUBMITTER: Beauruelle C
PROVIDER: S-EPMC8328194 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.
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Project description:Historically, a number of typing methods have been evaluated for Staphylococcus aureus strain characterization. The emergence of contemporary strains of community-associated S. aureus, and the ensuing epidemic with a predominant strain type (USA300), necessitates re-evaluation of the discriminatory power of these typing methods for discerning molecular epidemiology and transmission dynamics, essential to investigations of hospital and community outbreaks. We compared the discriminatory index of 5 typing methods for contemporary S. aureus strain characterization. Children presenting to St. Louis Children's Hospital and community pediatric practices in St. Louis, Missouri (MO), with community-associated S. aureus infections were enrolled. Repetitive sequence-based PCR (repPCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa), and staphylococcal cassette chromosome (SCC) mec typing were performed on 200 S. aureus isolates. The discriminatory index of each method was calculated using the standard formula for this metric, where a value of 1 is highly discriminatory and a value of 0 is not discriminatory. Overall, we identified 26 distinct strain types by repPCR, 17 strain types by PFGE, 30 strain types by MLST, 68 strain types by spa typing, and 5 strain types by SCCmec typing. RepPCR had the highest discriminatory index (D) of all methods (D?=?0.88), followed by spa typing (D?=?0.87), MLST (D?=?0.84), PFGE (D?=?0.76), and SCCmec typing (D?=?0.60). The method with the highest D among MRSA isolates was repPCR (D?=?0.64) followed by spa typing (D?=?0.45) and MLST (D?=?0.44). The method with the highest D among MSSA isolates was spa typing (D?=?0.98), followed by MLST (D?=?0.93), repPCR (D?=?0.92), and PFGE (D?=?0.89). Among isolates designated USA300 by PFGE, repPCR was most discriminatory, with 10 distinct strain types identified (D?=?0.63). We identified 45 MRSA isolates which were classified as identical by PFGE, MLST, spa typing, and SCCmec typing (USA300, ST8, t008, SCCmec IV, respectively); within this collection, there were 5 distinct strain types identified by repPCR. The typing methods yielded comparable discriminatory power for S. aureus characterization overall; when discriminating among USA300 isolates, repPCR retained the highest discriminatory power. This property is advantageous for investigations conducted in the era of contemporary S. aureus infections.
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Project description:Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Epidemiological studies of GBS infections require comprehensive typing systems that provide information about variable characteristics, such as antigenic type, virulence, or antibiotic resistance, as well as the "backbone" structure or the genetic lineage of isolates. We have previously described a 3-set genotyping system that identifies the molecular serotype (MS) or molecular serosubtype (msst), the protein gene profile, and the presence of several mobile genetic elements (F. Kong, D. Martin, G. James, and G. L. Gilbert, J. Med. Microbiol. 52:337-344, 2003). In this study, 83 clinical GBS isolates which had been previously studied by multilocus sequence typing (MLST) (N. Jones, J. F. Bohnsack, S. Takahashi, K. A. Oliver, M. S. Chan, F. Kunst, P. Glaser, C. Rusniok, D. W. Crook, R. M. Harding, N. Bisharat, and B. G. Spratt, J. Clin. Microbiol. 41:2530-2536, 2003) were examined by using the 3-set genotyping system. Genotypes were assigned to five isolates that were nontypeable by conventional serotyping. There were 27 "3-set" genotypes, 24 multilocus sequence types (STs), and 35 unique combinations (or strains), of which the 4 most common, msst III-2 (ST-17), msst III-1 (ST-19), Ia-1 (ST-23), and V-1 (ST-1), accounted for more than 60% of isolates. The 83 isolates were grouped into seven clusters, with a good correlation between the multilocus STs and the genotypes. The combination of 3-set genotyping and MLST adds discriminatory power to strain typing of GBS, which will be useful for future studies of the epidemiology and pathogenesis of GBS disease.
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Project description:We investigated the antibiotic and heavy-metal resistance mechanisms, virulence genes and clonal relationships of macrolide- and/or lincosamide-resistant (M+/-LR) Streptococcus agalactiae (group B Streptococcus, GBS) isolates from pregnant women in La Rioja in Northern Spain, a region with a significant immigrant population. In total 375 GBS isolates were recovered during 2011. About three-quarters of isolates were from European nationals and the remainder distributed among 23 other nationalities. Seventy-five (20%) were classified as M+/-LR strains and 28 (37%) of these were resistant to ⩾3 classes of antibiotics. Capsular serotypes III (29·3%), V (21·3%) and II (12%) were the most frequent. A wide variety of antibiotic resistance genes were detected in M+/-LR strains; notably, 5·3% harboured the lsa(C) gene associated with cross-resistance, and tet(W) was identified in a single strain. We report, for the first time, the detection of cadmium and copper resistance encoded by tcrB + cadA + cadC genes in 20 M+/-LR strains, which raises the possibility of co-selection of antibiotic and heavy-metal resistance disseminated through mobile genetic elements. The M+/-LR strains were highly diverse by DNA macrorestriction profiles (65 patterns) and 16 multilocus sequence types (STs) distributed among six clonal complexes; the most frequent were ST1, ST19, and ST12, and two strains were novel (ST586 and ST601). In conclusion, a wide diversity of genetic lineages of macrolide, lincosamide and heavy-metal- resistant GBS strains was observed in an ethnically diverse maternal population.
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Project description:Surveillance of circulating microbial populations is critical for monitoring the performance of a molecular diagnostic test. In this study, we characterized 31 isolates of Streptococcus agalactiae (group B Streptococcus [GBS]) from several geographic locations in the United States and Ireland that contain deletions in or adjacent to the region of the chromosome that encodes the hemolysin gene cfb, the region targeted by the Xpert GBS and GBS LB assays. PCR-negative, culture-positive isolates were recognized during verification studies of the Xpert GBS assay in 12 laboratories between 2012 and 2018. Whole-genome sequencing of 15 GBS isolates from 11 laboratories revealed four unique deletions of chromosomal DNA ranging from 181 bp to 49 kb. Prospective surveillance studies demonstrated that the prevalence of GBS isolates containing deletions in the convenience sample was <1% in three geographic locations but 7% in a fourth location. Among the 15 isolates with chromosomal deletions, multiple pulsed-field gel electrophoresis types were identified, one of which appears to be broadly dispersed across the United States.
| S-EPMC6440789 | biostudies-literature
Project description:Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is increasingly recognized as a major cause of soft tissue and invasive diseases in the elderly and diabetic populations. Antibiotics like penicillin are used with great frequency to treat these infections, although antimicrobial resistance is increasing among GBS strains and underlines a need for alternative methods not reliant on traditional antibiotics. GBS granadaene pigment is related to the hemolysin/cytolysin of GBS, which is critical for the pathogenesis of GBS diseases. Here, we show that photobleaching granadaene dampens the hemolytic activity of GBS. Furthermore, photobleaching of this antioxidant was found to increase GBS susceptibility to killing by reactive oxygen species like hydrogen peroxide. Treatment with light was also shown to affect GBS membrane permeability and contribute to increased susceptibility to the cell membrane-targeting antibiotic daptomycin. Overall, our study demonstrates dual effects of photobleaching on the virulence and antimicrobial susceptibility of GBS and suggests a novel approach for the treatment of GBS infection.
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