Co-culturing with Streptococcus anginosus alters Staphylococcus aureus transcriptome when exposed to tonsillar cells.

Introduction: Improved understanding of Staphylococcus aureus throat colonization in the presence of other co-existing microbes is important for mapping S. aureus adaptation to the human throat, and recurrence of infection. Here, we explore the responses triggered by the encounter between two common throat bacteria, S. aureus and Streptococcus anginosus, to identify genes in S. aureus that are important for colonization in the presence of human tonsillar epithelial cells and S. anginosus, and further compare this transcriptome with the genes expressed in S. aureus as only bacterium. Methods: We performed an in vitro co-culture experiment followed by RNA sequencing to identify interaction-induced transcriptional alterations and differentially expressed genes (DEGs), followed by gene enrichment analysis. Results and discussion: A total of 332 and 279 significantly differentially expressed genes with p-value < 0.05 and log2 FoldChange (log2FC) ≥ |2| were identified in S. aureus after 1 h and 3 h co-culturing, respectively. Alterations in expression of various S. aureus survival factors were observed when co-cultured with S. anginosus and tonsillar cells. The serine-aspartate repeat-containing protein D (sdrD) involved in adhesion, was for example highly upregulated in S. aureus during co-culturing with S. anginosus compared to S. aureus grown in the absence of S. anginosus, especially at 3 h. Several virulence genes encoding secreted proteins were also highly upregulated only when S. aureus was co-cultured with S. anginosus and tonsillar cells, and iron does not appear to be a limiting factor in this environment. These findings may be useful for the development of interventions against S. aureus throat colonization and could be further investigated to decipher the roles of the identified genes in the host immune response in context of a throat commensal landscape.

Exploring differentially expressed genes of Staphylococcus aureus exposed to human tonsillar cells using RNA sequencing.

BACKGROUND: The nose and the throat are the most predominant colonizing sites of Staphylococcus aureus, and colonization is a risk factor for infection. Nasal colonization is well described; however, we have limited knowledge about S. aureus throat colonization. The main objective of this study was to explore differentially expressed genes (DEGs) in S. aureus throat isolate TR145 exposed to human tonsil epithelial cells (HTEpiC) by using RNA sequencing (RNA-seq) and pathway analysis. DEGs in S. aureus at 1 or 3 hours (h) interaction with its host were explored. RESULTS: S. aureus was co-cultured in absence and presence of tonsillar cells at 1 or 3 h. Over the 3 h time frame, the bacteria multiplied, but still caused only minor cytotoxicity. Upon exposure to tonsillar cell line, S. aureus changed its transcriptomic profile. A total of 508 DEGs were identified including unique (1 h, 160 DEGs and 3 h, 78 DEGs) and commonly shared genes (1 and 3 h, 270 DEGs). Among the DEGs, were genes encoding proteins involved in adhesion and immune evasion, as well as iron acquisition and transport. Reverse transcription qPCR was done on selected genes, and the results correlated with the RNA-seq data. CONCLUSION: We have shown the suitability of using HTEpiC as an in vitro model for investigating key determinants in S. aureus during co-incubation with host cells. Several DEGs were unique after 1 or 3 h exposure to host cells, while others were commonly expressed at both time points. As their expression is induced upon meeting with the host, they might be explored further for future targets for intervention to prevent either colonization or infection in the throat.

Social network analysis of Staphylococcus aureus carriage in a general youth population.

OBJECTIVES: Staphylococcus aureus carriage increases the risk of infection. We used social network analysis to evaluate whether contacts have the same S. aureus genotype indicating direct transmission or whether contagiousness is an indirect effect of contacts sharing the same lifestyle or characteristics. METHODS: The Fit Futures 1 study collected data on social contact among 1038 high school students. S. aureus carriage was determined from two nasal swab cultures and the genotype was determined by spa-typing of positive throat swabs. RESULTS: S. aureus carriage and spa-type were transmitted in the social network (P < 0.001). The probability of carriage increased by 5% for each S. aureus positive contact. Male sex was associated with a 15% lower risk of transmission compared to the female sex, although the carriage prevalence was higher for men (36% vs 24%). Students with medium physical activity levels, medium/high alcohol use, or normal weight had a higher number of contacts and an increased risk of transmission (P < 0.002). CONCLUSION: We demonstrated the direct social transmission of S. aureus. Lifestyle factors are associated with the risk of transmission, suggesting the effects of indirect social groups on S. aureus carriage, such as friends having more similar environmental exposures. The male predominance in the carriage is determined by sex-specific predisposing host characteristics as the social transmission is less frequent in males than females. Information on social networks may add to a better understanding of S. aureus epidemiology.

Shotgun-metagenomics based prediction of antibiotic resistance and virulence determinants in Staphylococcus aureus from periprosthetic tissue on blood culture bottles.

Shotgun-metagenomics may give valuable clinical information beyond the detection of potential pathogen(s). Identification of antimicrobial resistance (AMR), virulence genes and typing directly from clinical samples has been limited due to challenges arising from incomplete genome coverage. We assessed the performance of shotgun-metagenomics on positive blood culture bottles (n = 19) with periprosthetic tissue for typing and prediction of AMR and virulence profiles in Staphylococcus aureus. We used different approaches to determine if sequence data from reads provides more information than from assembled contigs. Only 0.18% of total reads was derived from human DNA. Shotgun-metagenomics results and conventional method results were consistent in detecting S. aureus in all samples. AMR and known periprosthetic joint infection virulence genes were predicted from S. aureus. Mean coverage depth, when predicting AMR genes was 209 ×. Resistance phenotypes could be explained by genes predicted in the sample in most of the cases. The choice of bioinformatic data analysis approach clearly influenced the results, i.e. read-based analysis was more accurate for pathogen identification, while contigs seemed better for AMR profiling. Our study demonstrates high genome coverage and potential for typing and prediction of AMR and virulence profiles in S. aureus from shotgun-metagenomics data.

Shotgun-Metagenomics on Positive Blood Culture Bottles Inoculated With Prosthetic Joint Tissue: A Proof of Concept Study.

Clinical metagenomics is actively moving from research to clinical laboratories. It has the potential to change the microbial diagnosis of infectious diseases, especially when detection and identification of pathogens can be challenging, such as in prosthetic joint infection (PJI). The application of metagenomic sequencing to periprosthetic joint tissue (PJT) specimens is often challenged by low bacterial load in addition to high level of inhibitor and contaminant host DNA, limiting pathogen recovery. Shotgun-metagenomics (SMg) performed directly on positive blood culture bottles (BCBs) inoculated with PJT may be a convenient approach to overcome these obstacles. The aim was to test if it is possible to perform SMg on PJT inoculated into BCBs for pathogen identification in PJI diagnosis. Our study was conducted as a laboratory method development. For this purpose, spiked samples (positive controls), negative control and clinical tissue samples (positive BCBs) were included to get a comprehensive overview. We developed a method for preparation of bacterial DNA directly from PJT inoculated in BCBs. Samples were processed using MolYsis5 kit for removal of human DNA and DNA extracted with BiOstic kit. High DNA quantity/quality was obtained, and no inhibition was observed during the library preparation, allowing further sequencing process. DNA sequencing reads obtained from the BCBs, presented a low proportion of human reads (<1%) improving the sensitivity of bacterial detection. We detected a 19-fold increase in the number of reads mapping to human in a sample untreated with MolYsis5. Taxonomic classification of clinical samples identified a median of 96.08% (IQR, 93.85-97.07%; range 85.7-98.6%) bacterial reads. Shotgun-metagenomics results were consistent with the results from a conventional BCB culture method, validating our approach. Overall, we demonstrated a proof of concept that it is possible to perform SMg directly on BCBs inoculated with PJT, with potential of pathogen identification in PJI diagnosis. We consider this a first step in research efforts needed to face the challenges presented in PJI diagnoses.

Culturing periprosthetic tissue in BacT/Alert® Virtuo blood culture system leads to improved and faster detection of prosthetic joint infections.

BACKGROUND: Blood culture bottles (BCBs) provide a semiautomated method for culturing periprosthetic tissue specimens. A study evaluating BCBs for culturing clinical samples other than body fluids is needed before implementation into clinical practice. Our objective was to evaluate use of the BacT/Alert® Virtuo blood culture system for culturing periprosthetic tissue specimens. METHODS: The study was performed through the analysis of spiked (n = 36) and clinical (n = 158) periprosthetic tissue samples. Clinical samples were analyzed by the BCB method and the results were compared to the conventional microbiological culture-based method for time to detection and microorganisms identified. RESULTS: The BacT/Alert® Virtuo blood culture system detected relevant bacteria for prosthetic joint infection in both spiked and clinical samples. The BCB method was found to be as sensitive (79%) as the conventional method (76%) (p = 0.844) during the analyses of clinical samples. The BCB method yielded positive results much faster than the conventional method: 89% against 27% detection within 24 h, respectively. The median detection time was 11.1 h for the BCB method (12 h and 11 h for the aerobic and the anaerobic BCBs, correspondingly). CONCLUSION: We recommend using the BacT/Alert® Virtuo blood culture system for analyzing prosthetic joint tissue, since this detect efficiently and more rapidly a wider range of bacteria than the conventional microbiological method.

Genetic variability in the sdrD gene in Staphylococcus aureus from healthy nasal carriers.

BACKGROUND: Staphylococcus aureus cell wall anchored Serine Aspartate repeat containing protein D (SdrD) is a member of the microbial surface component recognising adhesive matrix molecules (MSCRAMMs). It is involved in the bacterial adhesion and virulence. However the extent of genetic variation in S. aureus sdrD gene within isolates from healthy carriers are not known. The aim of this study was to evaluate allelic variation of the sdrD gene among S. aureus from healthy nasal carriers. RESULTS: The sdrD A region from 48 S. aureus isolates from healthy carriers were analysed and classified into seven variants. Variations in the sdrD A region were concentrated in the N2 and N3 subdomains. Sequence analysis of the entire sdrD gene of representative isolates revealed variations in the SD repeat and the EF motifs of the B repeat. In silico structural modelling indicates that there are no differences in the SdrD structure of the 7 variants. Variable amino acid residues mapped onto the 3D structure revealed that the variations are surface located, exist within the groove between the N2-N3 subdomains and distributed mainly on the N3 subdomain. Comparison of adhesion to keratinocytes in an in vitro cell adhesion assay, using NCTC 8325-4∆sdrD strains expressing the various sdrD gene variants, indicated a significant difference between only two complements while others showed no major difference in their adhesion. CONCLUSIONS: This study provides evidence of sequence variations across the different domains of SdrD from S. aureus isolated from healthy nasal carriers. Proper understanding of these variations is necessary in the study of S. aureus pathogenesis.

Localization of Staphylococcus aureus in tissue from the nasal vestibule in healthy carriers.

BACKGROUND: Colonization of the body is an important step in Staphylococcus aureus infection. S. aureus colonizes skin and mucous membranes in humans and several animal species. One important ecological niche of S. aureus is the anterior nares. More than 60% of the S. aureus in the nose are found in vestibulum nasi. Our aim was to describe the localization of S. aureus in nasal tissue from healthy carriers. METHODS: Punch skin biopsies were taken from vestibulum nasi from healthy volunteers (S. aureus carriers and non-/intermittent carriers, n = 39) attending the population-based Tromsø 6 study. The tissue samples were processed as frozen sections before immunostaining with a specific S. aureus antibody, and finally evaluated by a confocal laser-scanning microscope. RESULTS: Our results suggest that S. aureus colonize both the upper and lower layers of the epidermis within the nasal epithelium of healthy individuals. The number of S. aureus in epidermis was surprisingly low. Intracellular localization of S. aureus in nasal tissue from healthy individuals was also detected. CONCLUSIONS: Knowledge of the exact localization of S. aureus in nasal tissue is important for the understanding of the host responses against S. aureus. Our results may have consequences for the eradication strategy of S. aureus in carriers, and further work can provide us with tools for targeted prevention of S. aureus colonisation and infection.

The interaction between Staphylococcus aureus SdrD and desmoglein 1 is important for adhesion to host cells.

Staphylococcus aureus is known as a frequent colonizer of the skin and mucosa. Among bacterial factors involved in colonization are adhesins such as the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Serine aspartate repeat containing protein D (SdrD) is involved in adhesion to human squamous cells isolated from the nose. Here, we identify Desmoglein 1 (Dsg1) as a novel interaction partner for SdrD. Genetic deletion of sdrD in S. aureus NCTC8325-4 through allelic replacement resulted in decreased bacterial adherence to Dsg1- expressing HaCaT cells in vitro. Complementary gain-of-function was demonstrated by heterologous expression of SdrD in Lactococcus lactis, which increased adherence to HaCaT cells. Also ectopic expression of Dsg1 in HEK293 cells resulted in increased adherence of S. aureus NCTC8325-4 in vitro. Increased adherence of NCTC8325-4, compared to NCTC8325-4ΔsdrD, to the recombinant immobilized Dsg1 demonstrated direct interaction between SdrD and Dsg1. Specificity of SdrD interaction with Dsg1 was further verified using flow cytometry and confirmed binding of recombinant SdrD to HaCaT cells expressing Dsg1 on their surface. These data demonstrate that Dsg1 is a host ligand for SdrD.

A Look into the Melting Pot: The mecC-Harboring Region Is a Recombination Hot Spot in Staphylococcus stepanovicii.

INTRODUCTION: Horizontal gene transfer (HGT) is an important driver for resistance- and virulence factor accumulation in pathogenic bacteria such as Staphylococcus aureus. METHODS: Here, we have investigated the downstream region of the bacterial chromosomal attachment site (attB) for the staphylococcal cassette chromosome mec (SCCmec) element of a commensal mecC-positive Staphylococcus stepanovicii strain (IMT28705; ODD4) with respect to genetic composition and indications of HGT. S. stepanovicii IMT28705 was isolated from a fecal sample of a trapped wild bank vole (Myodes glareolus) during a screening study (National Network on "Rodent-Borne Pathogens") in Germany. Whole genome sequencing (WGS) of IMT28705 together with the mecC-negative type strain CM7717 was conducted in order to comparatively investigate the genomic region downstream of attB (GenBank accession no. KR732654 and KR732653). RESULTS: The bank vole isolate (IMT28705) harbors a mecC gene which shares 99.2% nucleotide (and 98.5% amino acid) sequence identity with mecC of MRSA_LGA251. In addition, the mecC-encoding region harbors the typical blaZ-mecC-mecR1-mecI structure, corresponding with the class E mec complex. While the sequences downstream of attB in both S. stepanovicii isolates (IMT28705 and CM7717) are partitioned by 15 bp direct repeats, further comparison revealed a remarkable low concordance of gene content, indicating a chromosomal "hot spot" for foreign DNA integration and exchange. CONCLUSION: Our data highlight the necessity for further research on transmission routes of resistance encoding factors from the environmental and wildlife resistome.

Staphylococcus aureus nasal isolates from healthy individuals cause highly variable host cell responses in vitro: the Tromsø Staph and Skin Study.

Studies on Staphylococcus aureus populations colonizing the nasal cavity reveal that some bacterial strains are more common, while others are rarely found. This study included five isolates with the most common spa types and five isolates with rare spa types from healthy population. Selected phenotypic traits and genomic content among nasal S. aureus isolates were compared. Besides the rather similar growth rates, our data revealed a high diversity among isolates; that is, in biofilm formation, the ability to attach to and be internalized in keratinocytes as well as ability to induce pro- and anti-inflammatory cytokines. The results showed that S. aureus isolates from healthy hosts are phenotypically diverse and cause highly variable host cell responses. Therefore, generalizing the results from one S. aureus isolate to all is highly questionable.

Host- and microbe determinants that may influence the success of S. aureus colonization.

Staphylococcus aureus may cause serious skin and soft tissue infections, deep abscesses, endocarditis, osteomyelitis, pneumonia, and sepsis. S. aureus persistently colonizes 25-30% of the adult human population, and S. aureus carriers have an increased risk for infections caused by the bacterium. The major site of colonization is the nose, i.e., the vestibulum nasi, which is covered with ordinary skin and hair follicles. Several host and microbe determinants are assumed to be associated with colonization. These include the presence and expression level of bacterial adhesins, which can adhere to various proteins in the extracellular matrix or on the cellular surface of human skin. The host expresses several antimicrobial peptides and lipids. The level of ß-defensin 3, free sphingosine, and cis-6-hexadecenoic acid are found to be associated with nasal carriage of S. aureus. Other host factors are certain polymorphisms in Toll-like receptor 2, mannose-binding lectin, C-reactive protein, glucocorticoid-, and vitamin D receptor. Additional putative determinants for carriage include genetic variation and expression of microbial surface components recognizing adhesive matrix molecules and their interaction partners, as well as variation among humans in the ability of recognizing and responding appropriately to the bacteria. Moreover, the available microflora may influence the success of S. aureus colonization. In conclusion, colonization is a complex interplay between the bacteria and its host. Several bacterial and host factors are involved, and an increased molecular understanding of these are needed.

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis.

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes.

The AmpC phenotype in Norwegian clinical isolates of Escherichia coli is associated with an acquired ISEcp1-like ampC element or hyperproduction of the endogenous AmpC.

OBJECTIVES: The aim of the study was to examine resistance mechanisms associated with an AmpC phenotype in Norwegian clinical isolates of Escherichia coli. METHODS: Clinical E. coli isolates (n = 106) with reduced susceptibility to third-generation cephalosporins without clavulanic acid synergy were collected from 12 Norwegian laboratories from 2003 to 2005. Twenty-two isolates with an AmpC phenotype were selected for further characterization by PFGE, isoelectric focusing, different PCR-based techniques, DNA sequencing, AmpC qRT-PCR, transfer studies and plasmid analyses. RESULTS: The 22 isolates were not clonally related by the PFGE analysis. All isolates expressed a beta-lactamase with a pI of 9.0-9.2. Ten isolates contained a bla(CMY) gene, which was linked to an ISEcp1-like element in all cases. Twelve isolates had mutations or insertions in the promoter or the attenuator regions, leading to increased expression of the chromosomal ampC gene. One of these isolates had an ISEc10 element inserted upstream of the chromosomal ampC gene. CONCLUSIONS: This is the first molecular study of Norwegian clinical E. coli isolates with an AmpC phenotype. Resistance was mediated either by expression of bla(CMY) from acquired ISEcp1-like-bla(CMY) elements, or by mutations or insertions in the chromosomal ampC gene control region leading to hyperproduction of the endogenous AmpC enzyme. There was no correlation between the level of ampC mRNA and the MICs of cephalosporins.

Methicillin-resistant Staphylococcus aureus (MRSA) isolated from small and exotic animals at a university hospital during routine microbiological examinations.

Clinical specimens of small animals (n=869) were screened for the occurrence of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA; MRSA) during routine microbiological examinations, and results were confirmed by a multiplex PCR strategy. The genetic relatedness of all mecA-positive S. aureus isolates was further investigated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR for Panton-Valentine leukocidine genes (PVL) and staphylococcal cassette chromosome mec-typing (SCCmec). A total of 61 S. aureus isolates were found during a 20-month period of investigation, 27 (44.3%) of them harbouring the mecA gene for methicillin-resistance. The majority of MRSA were isolated in specimens from dogs (n=18) and cats (n=4). One guinea pig and one rabbit were found to be positive for an MRSA infected site. Similarly, three exotic animals, a turtle, a bat and a parrot, were found to be infected with MRSA. PFGE and MLST analysis revealed a certain genotype ("A" and "A-1") dominating the isolate collection (23 of 27). Furthermore, one isolate showed homologous PFGE pattern to the German epidemic strain Barnim ("BE") and another one ("BE-1") was considered to be closely related. A third genotype ("B") was detected in two cases. Two different sequence types (ST) were identified among the 27 MRSA isolates. PFGE type "A" and both strains related to the Barnim epidemic strain were assigned to ST22, whereas ST239 was associated to PFGE profile "B". The present data show that certain MRSA genotypes are capable of infecting a wide spectrum of small and exotic animals, especially in clinical facilities.

Multiple staphylococcal cassette chromosomes and allelic variants of cassette chromosome recombinases in Staphylococcus aureus and coagulase-negative staphylococci from Norway.

We investigated the nature of the staphylococcal cassette chromosome mec (SCCmec) elements and cognate insertion sites in a collection of 42 clinical staphylococcal isolates of various species from Norway. The ccr and mec genes and the attachment sites (attL/attR) were identified by PCR, Southern blot hybridization, and DNA sequencing. We found 10 possibly new SCCmec types and one previously unreported variant of SCCmec type III (mec complex A, ccrAB3, and ccrC7) in Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis. Eleven of 42 strains contained multiple copies of ccr, suggesting the presence of mosaic structures composed of multiple SCC elements. S. haemolyticus contained ccrAB2 genes identical to those in S. aureus SCCmec type IV but lacked IS1272 and mec regulators. Two new allelic ccr variants, ccrC6 and ccrC7, were identified. Also, the presumed functional version of ccrB1 was found in a mecA-positive S. hominis strain and in mecA-negative S. epidermidis and S. hominis strains. Only minor differences in direct repeats in the left and right boundaries (attR/attL) were observed, while there was more variation in the inverted repeats. Coagulase-negative staphylococci (CoNS) contained several representatives of different ccr complexes and thus seemed to harbor multiple or composite new types of SCCmec. The enormous diversity observed in the SCCmec elements implies a large SCCmec reservoir in CoNS.

SCCmec in staphylococci: genes on the move.

Staphylococcal cassette chromosome (SCC) elements are, so far, the only vectors described for the mecA gene encoding methicillin resistance in staphylococci. SCCmec elements are classified according to the type of recombinase they carry and their general genetic composition. SCCmec types I-V have been described, and SCC elements lacking mecA have also been reported. In this review, we summarize the current knowledge about SCC structure and distribution, including genetic variants and rudiments of the elements. Its origin is still unknown, but one assumes that staphylococcal cassette chromosome is transferred between staphylococci, and mecA-positive coagulase-negative staphylococci may be a potential reservoir for these elements. Staphylococcal genomes seem to change continuously as genetic elements move in and out, but no mechanism of transfer has been found responsible for moving SCC elements between different staphylococcal species. Observations suggesting de novo production of methicillin-resistant staphylococci and horizontal gene transfer of SCCmec will be discussed.

Dissemination of community-acquired methicillin-resistant Staphylococcus aureus clones in northern Norway: sequence types 8 and 80 predominate.

Increasing frequencies of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain isolation have been reported from many countries. The overall prevalence of MRSA in Norway is still very low. MRSA isolates (n = 67) detected between 1995 and 2003 in northern Norway were analyzed by pulsed-field gel electrophoresis, multilocus sequence typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Sixty-seven isolates were associated with 13 different sequence types. Two successful MRSA clones predominated. Sequence type 8 (ST8) (40%) and ST80 (19%) containing SCCmec type IV were detected in hospitals and communities in different geographic regions during a 7-year period. In general, there was a low level of antimicrobial resistance. Only 26% of the isolates were multiresistant. International epidemic clones were detected. The frequent findings of SCCmec type IV (91%) along with heterogeneous genetic backgrounds suggest a horizontal spread of SCCmec type IV among staphylococcal strains in parallel with the clonal spread of successful MRSA strains.

Local variants of Staphylococcal cassette chromosome mec in sporadic methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococci: evidence of horizontal gene transfer?

The mecA gene in Staphylococcus aureus is located on the genetic element staphylococcal cassette chromosome (SCC). Different SCCmecs have been classified according to their putative recombinase genes (ccrA and ccrB) and overall genetic composition. Clinical isolates of coagulase-negative staphylococci (CoNS; n = 39) and S. aureus (n = 20) from Norway, India, Italy, Finland, the United States, and the United Kingdom were analyzed by pulsed-field gel electrophoresis, which showed that most isolates were genetically unrelated. Cluster analyses of 16S rRNA gene and pta sequences confirmed the traditional biochemical species identification. The mecI, mecR1, mecA, and ccrAB genes were detected by PCRs, identifying 19 out of 20 S. aureus and 17 out of 39 CoNS isolates as carriers of one of the three published ccrAB pairs. New variants of SCCmec were identified, as well as CoNS isolates containing ccrAB genes without the mec locus. ccrAB and mec PCRs were verified by hybridization. Sequence alignments of ccrAB genes showed a high level of diversity between the ccrAB alleles from different isolates, i.e., 94 to 100% and 95 to 100% homology for ccrAB1 and ccrAB2, respectively. All of the ccrAB3 genes identified were identical. Genetically unique and sporadic methicillin-resistant S. aureus (MRSA) contained local variants of ccrAB gene pairs identical to those found in MR-CoNS but different from those in MRSA from other regions. Allelic variants of ccrAB in isolates from the same geographic region showed sequence conservation independent of species. The species-independent sequence conservation found suggests that there is a closer genetic relationship between ccrAB2 in Norwegian staphylococci than between ccrAB2 sequences in international MRSA and Norwegian MRSA. This might indicate that different staphylococcal species acquire these genes locally by horizontal gene transfer.