Sequencing and staphylococci identification.
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ABSTRACT:
SUBMITTER: Mellmann A
PROVIDER: S-EPMC3373113 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Coagulase-negative staphylococci (CNS) make up a diverse bacterial group, appearing in a myriad of ecosystems. To unravel the composition of staphylococcal communities in these microbial ecosystems, a reliable species-level identification is crucial. The present study aimed to design a primer set for high-throughput amplicon sequencing, amplifying a region of the tuf gene with enough discriminatory power to distinguish different CNS species. Based on 2566 tuf gene sequences present in the public European Nucleotide Archive database and saved as a custom tuf gene database in-house, three different primer sets were designed, which were able to amplify a specific region of the tuf gene for 36 strains of 18 different CNS species. In silico analysis revealed that species-level identification of closely related species was only reliable if a 100% identity cut-off was applied for matches between the amplicon sequence variants and the custom tuf gene database. From the three primer sets designed, one set (Tuf387/765) outperformed the two other primer sets for studying Staphylococcus-rich microbial communities using amplicon sequencing, as it resulted in no false positives and precise species-level identification. The method developed offers interesting potential for a rapid and robust analysis of complex staphylococcal communities in a variety of microbial ecosystems.
| S-EPMC7356217 | biostudies-literature
Project description:Staphylococcal species heterogeneity in the nasal microbiome during antibiotic prophylaxis
| PRJNA324352 | ENA
Project description:Staphylococci are among the commonly isolated bacteria from intramammary infections in bovines, where Staphylococcus aureus is the most studied species. This species carries a variety of virulence genes, contributing to bacterial survival and spread. Less is known about non-aureus staphylococci (NAS) and their range of virulence genes and mechanisms, but they are the most frequently isolated bacteria from bovine milk. Staphylococci can also carry a range of antimicrobial resistance genes, complicating treatment of the infections they cause. We used Illumina sequencing to whole genome sequence 93 staphylococcal isolates selected from a collection of staphylococcal isolates; 45 S. aureus isolates and 48 NAS isolates from 16 different species, determining their content of antimicrobial resistance genes and virulence genes. Antimicrobial resistance genes were frequently observed in the NAS species as a group compared to S. aureus. However, the lincosamide resistance gene lnuA and penicillin resistance gene blaZ were frequently identified in NAS, as well as a small number of S. aureus. The erm genes conferring macrolide resistance were also identified in several NAS isolates and in a small number of S. aureus isolates. In most S. aureus isolates, no antimicrobial resistance genes were detected, but in five S. aureus isolates three to six resistance genes were identified and all five of these carried the mecA gene. Virulence genes were more frequently identified in S. aureus, which contained on average five times more virulence genes compared to NAS. Among the NAS species there were also differences in content of virulence genes, such as S. chromogenes with a higher average number of virulence genes. By determining the content of a large selection of virulence genes and antimicrobial resistance genes in S. aureus and 16 different NAS species our results contribute with knowledge regarding the genetic basis for virulence and antimicrobial resistance in bovine staphylococci, especially the less studied NAS. The results can create a broader basis for further research into the virulence mechanisms of this important group of bacteria in bovine intramammary infections.
| S-EPMC8721127 | biostudies-literature
Project description:To screen isolates and to identify mecA alleles, published mecA sequences were analyzed, and a microarray for the rapid discrimination of mecA alleles was designed. A GenBank analysis yielded 135 full-length gene sequences annotated as mecA. These sequences clustered into 32 different alleles corresponding to 28 unique amino acid sequences and to 15 distinct hybridization patterns on this microarray. A collection of 78 clinical and veterinary isolates of Staphylococcus spp. was characterized using this assay. Nine of the 15 expected patterns, as well as one as-yet-unknown pattern, were identified. These patterns were detected in various epidemic methicillin-resistant Staphylococcus aureus strains, in S. pseudintermedius, and in coagulase-negative species such as S. epidermidis, S. fleurettii, or S. haemolyticus. There was no correlation between the different mecA hybridization patterns and the SCCmec type. Determination of MICs showed that mecA alleles corresponding to only four of these nine patterns were associated with ?-lactam resistance. The mecA alleles that did not confer ?-lactam resistance were largely restricted to coagulase-negative staphylococci of animal origin, such as S. sciuri and S. vitulinus. Because of the diversity of sequences and the different impact on ?-lactam susceptibility, the existence of different mecA alleles needs to be taken into account when designing diagnostic assays for the detection of mecA.
| S-EPMC3486549 | biostudies-literature
Project description:In veterinary medicine, coagulase-positive staphylococci (CoPS) other than Staphylococcus aureus have frequently been misidentified as being S. aureus strains, as they have several phenotypic traits in common. There has been no reliable method to distinguish among CoPS species in veterinary clinical laboratories. In the present study, we sequenced the thermonuclease (nuc) genes of staphylococcal species and devised a multiplex-PCR (M-PCR) method for species identification of CoPS by targeting the nuc gene locus. To evaluate sensitivity and specificity, we used this M-PCR method on 374 staphylococcal strains that had been previously identified to the species level by an hsp60 sequencing approach. We could successfully distinguish between S. aureus, S. hyicus, S. schleiferi, S. intermedius, S. pseudintermedius, and S. delphini groups A and B. The present method was both sensitive (99.8%) and specific (100%). Our M-PCR assay will allow the routine species identification of CoPS isolates from various animal species for clinical veterinary diagnosis.
| S-EPMC2832457 | biostudies-literature
Project description:Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health.
| S-EPMC5758691 | biostudies-literature
Project description:UNLABELLED:Linezolid resistance is uncommon among staphylococci, but approximately 2% of clinical isolates of coagulase-negative staphylococci (CoNS) may exhibit resistance to linezolid (MIC, ?8 µg/ml). We performed whole-genome sequencing (WGS) to characterize the resistance mechanisms and genetic backgrounds of 28 linezolid-resistant CoNS (21 Staphylococcus epidermidis isolates and 7 Staphylococcus haemolyticus isolates) obtained from blood cultures at a large teaching health system in California between 2007 and 2012. The following well-characterized mutations associated with linezolid resistance were identified in the 23S rRNA: G2576U, G2447U, and U2504A, along with the mutation C2534U. Mutations in the L3 and L4 riboproteins, at sites previously associated with linezolid resistance, were also identified in 20 isolates. The majority of isolates harbored more than one mutation in the 23S rRNA and L3 and L4 genes. In addition, the cfr methylase gene was found in almost half (48%) of S. epidermidis isolates. cfr had been only rarely identified in staphylococci in the United States prior to this study. Isolates of the same sequence type were identified with unique mutations associated with linezolid resistance, suggesting independent acquisition of linezolid resistance in each isolate. IMPORTANCE:Linezolid is one of a limited number of antimicrobials available to treat drug-resistant Gram-positive bacteria, but resistance has begun to emerge. We evaluated the genomes of 28 linezolid-resistant staphylococci isolated from patients. Multiple mutations in the rRNA and associated proteins previously associated with linezolid resistance were found in the isolates investigated, underscoring the multifocal nature of resistance to linezolid in Staphylococcus. Importantly, almost half the S. epidermidis isolates studied harbored a plasmid-borne cfr RNA methylase gene, suggesting that the incidence of cfr may be higher in the United States than previously documented. This finding has important implications for infection control practices in the United States. Further, cfr is commonly detected in bacteria isolated from livestock, where the use of phenicols, lincosamides, and pleuromutilins in veterinary medicine may provide selective pressure and lead to maintenance of this gene in animal bacteria.
| S-EPMC4030478 | biostudies-literature
Project description:A home-made array for the detection of antimicrobial resistance genes in gram-positive bacteria
| PRJNA868571 | ENA
Project description:Coagulase-negative staphylococcus isolates were identified using Sensititre GPID plates and API strips (n = 156). For selected isolates, partial sequencing of the 16S rRNA, sodA, and tuf genes was performed. The Sensititre plates correctly identified 68.9% of isolates, with a concordance of 86% for Staphylococcus haemolyticus and 73% for Staphylococcus epidermidis.
| S-EPMC2832405 | biostudies-literature
Project description:BackgroundStaphylococcus aureus and S. pseudintermedius are the two most common coagulase positive staphylococci (CPS). S. aureus is more prevalent among humans, whereas S. pseudintermedius is more commonly isolated from dogs, however, both can cause various community and hospital acquired diseases in humans.MethodsIn the current study we screened 102 dogs and 84 owners in Hungary. We tested the antibiotic susceptibility of the strains and in order to get a better picture of the clonal relationship of the strains, we used pulsed-field gel electrophoresis. In addition, three pairs of isolates with identical PFGE patterns were whole genome sequenced, MLST and spa types were established.ResultsCarriage rate of S. aureus was 23.8% in humans and 4.9% in dogs and two cases of co-carriage were found among dogs and owners. S. pseudintermedius carriage rate was 2.4% and 34.3%, respectively, with only one co-carriage. The isolates were generally rather susceptible to the tested antibiotics, but high tetracycline resistance of S. pseudintermedius strains was noted. The co-carried isolates shared almost the same resistance genes (including tet(K), bla(Z), norA, mepR, lmrS, fosB) and virulence gene pattern. Apart from the common staphylococcal enzymes and cytotoxins, we found enterotoxins and exfoliative toxins as well. The two S. aureus pairs belonged to ST45-t630, ST45-t671 and ST15-t084, ST15-t084, respectively. The co-carried S. pseudintermedius isolates shared the same housekeeping gene alleles determining a novel sequence type ST1685.ConclusionsBased on the genomic data, dog-owner co-carried strains displayed only insignificant differences therefore provided evidence for potential human-to-dog and dog-to-human transmission.
| S-EPMC7799803 | biostudies-literature