Project description:Nineteen linezolid-resistant Staphylococcus epidermidis and two Staphylococcus aureus isolates recovered from two medical institutions in northeast Ohio and an S. aureus cfr index strain previously collected in the same facilities during the 2007 SENTRY Antimicrobial Surveillance Program were investigated for the genetic basis of oxazolidinone resistance and the location of cfr. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST). The location of cfr was determined by Southern blotting and hybridization. Plasmid sequencing was performed using the 454 Life Sciences (Roche) GS-FLX DNA platform. The two S. aureus isolates showed unique PFGE patterns but were multilocus sequence type 5 (ST5) and spa type t002, whereas the S. aureus index strain was ST239 and t037. Southern blot and hybridization experiments showed that cfr was plasmid located and that the S. epidermidis isolates, one of the S. aureus isolates, and the S. aureus index strain shared an identical cfr-carrying plasmid (39.3 kb). Sequencing results confirmed these findings. A 10-kb fragment containing cfr showed the highest identity (99.9%) to a 9.5-kb fragment of plasmid pSCFS3 from a bovine Staphylococcus lentus isolate from Germany. In addition, these 39.3-kb plasmids from human S. epidermidis and S. aureus exhibited BglII restriction profiles very similar to that observed for plasmid pSCFS3. The cfr-carrying plasmid detected in the remaining S. aureus isolate (7.9 kb) was distinct and showed the highest identity to the chromosomal cfr integrate found in the chromosomal DNA of a Proteus vulgaris isolate from a pig in China.
Project description:Background:Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid. Methods:Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction. Results:Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use. Conclusions:The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.
Project description:The clinical Staphylococcus epidermidis isolate 426-3147L exhibits an unusually high resistance to linezolid that exceeds 256 ?g/ml. The presence of the cfr gene, encoding the RNA methyltransferase targeting an rRNA nucleotide located in the linezolid binding site, accounts for a significant fraction of resistance. The association of cfr with a multicopy plasmid is one of the factors that contribute to its elevated expression. Mapping of the cfr transcription start sites identified the native cfr promoter. Furthermore, analysis of the cfr transcripts in Staphylococcus epidermidis 426-3147L showed that some of them originate from the upstream plasmid-derived promoters whose activity contributes to efficient cfr transcription. The genetic environment of the cfr gene and its idiosyncratic transcription pattern result in increased activity of Cfr methyltransferase, leading to a high fraction of the ribosomes being methylated at A2503 of the 23S rRNA. Curing of the Staphylococcus epidermidis 426-3147L isolate from the cfr-containing plasmid reduced the linezolid MIC to 64 ?g/ml, indicating that other determinants contribute to resistance. Nucleotide sequence analysis revealed the presence of the C2534T mutation in two of the six 23S rRNA gene alleles as well as the presence of mutations in the genes of ribosomal proteins L3 and L4, which were previously implicated in linezolid resistance. Thus, the combination of resistance mechanisms operating through alteration of the drug target site appears to cause an unusually high level of linezolid resistance in the isolate.
Project description:Staphylococcus epidermidis causes a large number of catheter-related sepsis infections annually in the United States. We present the 2.54-Mbp complete genome assembly of reference strain S. epidermidis AmMS 205, including a single 37.7-kbp plasmid. The annotated assembly is available in GenBank under accession numbers CP009046 and CP009047.
Project description:Whole genome sequencing is a useful tool to monitor the spread of resistance mechanisms in bacteria. In this retrospective study, we investigated genetic resistance mechanisms, sequence types (ST) and respective phenotypes of linezolid-resistant <i>Staphylococcus epidermidis</i> (LRSE, <i>n</i> = 129) recovered from a cohort of patients receiving or not receiving linezolid within a tertiary hospital in Innsbruck, Austria. Hereby, the point mutation G2603U in the 23S rRNA (<i>n</i> = 91) was the major resistance mechanism followed by the presence of plasmid-derived <i>cfr</i> (<i>n</i> = 30). The majority of LRSE isolates were ST2 strains, followed by ST5. LRSE isolates expressed a high resistance level to linezolid with a minimal inhibitory concentration of ≥256 mg/L (<i>n</i> = 83) in most isolates, particularly in strains carrying the <i>cfr</i> gene (<i>p</i> < 0.001). Linezolid usage was the most prominent (but not the only) trigger for the development of linezolid resistance. However, administration of linezolid was not associated with a specific resistance mechanism. Restriction of linezolid usage and the monitoring of plasmid-derived <i>cfr</i> in LRSE are potential key steps to reduce linezolid resistance and its transmission to more pathogenic Gram-positive bacteria.
Project description:The heterogeneous course, severity, and treatment responses among patients with atopic dermatitis (AD; eczema) highlight the complexity of this multifactorial disease. Prior studies have used traditional typing methods on cultivated isolates or sequenced a bacterial marker gene to study the skin microbial communities of AD patients. Shotgun metagenomic sequence analysis provides much greater resolution, elucidating multiple levels of microbial community assembly ranging from kingdom to species and strain-level diversification. We analyzed microbial temporal dynamics from a cohort of pediatric AD patients sampled throughout the disease course. Species-level investigation of AD flares showed greater Staphylococcus aureus predominance in patients with more severe disease and Staphylococcus epidermidis predominance in patients with less severe disease. At the strain level, metagenomic sequencing analyses demonstrated clonal S. aureus strains in more severe patients and heterogeneous S. epidermidis strain communities in all patients. To investigate strain-level biological effects of S. aureus, we topically colonized mice with human strains isolated from AD patients and controls. This cutaneous colonization model demonstrated S. aureus strain-specific differences in eliciting skin inflammation and immune signatures characteristic of AD patients. Specifically, S. aureus isolates from AD patients with more severe flares induced epidermal thickening and expansion of cutaneous T helper 2 (TH2) and TH17 cells. Integrating high-resolution sequencing, culturing, and animal models demonstrated how functional differences of staphylococcal strains may contribute to the complexity of AD disease.
Project description:The skin colonizing coagulase-negative Staphylococcus epidermidis causes nosocomial infections and is an important opportunistic and highly adaptable pathogen. To gain more insight into this species, we sequenced the genome of the biofilm positive, methicillin susceptible S. epidermidis O47 strain (hereafter O47). This strain belongs to the most frequently isolated sequence type 2. In comparison to the RP62A strain, O47 can be transformed, which makes it a preferred strain for molecular studies. S. epidermidis O47's genome has a single chromosome of about 2.5 million base pairs and no plasmid. Its oriC sequence has the same directionality as S. epidermidis RP62A, S. carnosus, S. haemolyticus, S. saprophyticus and is inverted in comparison to Staphylococcus aureus and S. epidermidis ATCC 12228. A phylogenetic analysis based on all S. epidermidis genomes currently available at GenBank revealed that O47 is closest related to DAR1907. The genome of O47 contains genes for the typical global regulatory systems known in staphylococci. In addition, it contains most of the genes encoding for the typical virulence factors for S. epidermidis but not for S. aureus with the exception of a putative hemolysin III. O47 has the typical S. epidermidis genetic islands and several mobile genetic elements, which include staphylococcal cassette chromosome (SCC) of about 54 kb length and two prophages ?O47A and ?O47B. However, its genome has no transposons and the smallest number of insertion sequence (IS) elements compared to the other known S. epidermidis genomes. By sequencing and analyzing the genome of O47, we provide the basis for its utilization in genetic and molecular studies of biofilm formation.
Project description:Staphylococcus epidermidis ATCC 12228 was sequenced using a long-read method to generate a complete genome sequence, including some plasmid sequences. Some differences from the previously generated short-read sequence of this nonpathogenic and non-biofilm-forming strain were noted. The assembly size was 2,570,371 bp with a total G+C% content of 32.08%.
Project description:<i>Staphylococcus epidermidis</i> is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical <i>S. epidermidis</i> strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical <i>S. epidermidis</i> isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical <i>S. epidermidis</i> isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in <i>Escherichia coli</i> DC10B containing a ?<i>dcm</i> mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of <i>S. epidermidis</i> and hence improve our understanding of their pathogenesis in human infections.<b>IMPORTANCE</b> <i>Staphylococcus epidermidis</i> is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of <i>S. epidermidis</i> are poorly understood. Genetic manipulations of clinically relevant strains of <i>S. epidermidis</i> are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant <i>S. epidermidis</i> isolates has now become possible, thus improving our understanding of <i>S. epidermidis</i> as a pathogen.
Project description:Staphylococcus epidermidis 1457 is a frequently utilized strain that is amenable to genetic manipulation and has been widely used for biofilm-related research. We report here the whole-genome sequence of this strain, which encodes 2,277 protein-coding genes and 81 RNAs within its 2.4-Mb genome and plasmid.