Anne-Merethe Hanssen

Local Variants of Staphylococcal Cassette Chromosome mec in Sporadic Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Coagulase-Negative Staphylococci: Evidence of Horizontal Gene Transfer?

ABSTRACT 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.

Dissemination of Community-Acquired Methicillin-Resistant Staphylococcus aureus Clones in Northern Norway: Sequence Types 8 and 80 Predominate

ABSTRACT Increasing frequencies of community-acquired methicillin-resistant Staphylcoccus 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.

Multiple Staphylococcal Cassette Chromosomes and Allelic Variants of Cassette Chromosome Recombinases in Staphylococcus aureus and Coagulase-Negative Staphylococci from Norway

ABSTRACT 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.

Staphylococcus aureus: Determinants of human carriage

Staphylococcus aureus is a common human commensal but carriage varies between e.g. geographic location, age, gender, ethnicity and body niche. The nares, throat and perineum are the most prevalent sites for carriage in the general adult population. Other sites of the skin and the intestine are also frequently colonised. Thus, a successful establishment is dependent on multiple factors. This review describes results from observational studies of S. aureus carriage and the influence bacterial, host and environmental/modifiable factors might have on the relationship.

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 β-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.

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.

Clinical isolates of Staphylococcus aureus from the Arkhangelsk region, Russia: antimicrobial susceptibility, molecular epidemiology, and distribution of Panton‐Valentine leucocidin genes

A total of 91 consecutive clinical isolates of Staphylococcus aureus were collected at the Regional Hospital of Arkhangelsk, Russia, from May to December 2004, and examined for antimicrobial susceptibility, methicillin resistance and presence of Panton‐Valentine leucocidin (PVL) genes. Epidemiological typing was performed by pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Methicillin‐resistant S. aureus (MRSA) isolates were examined by staphylococcal cassette chromosome mec (SCCmec) typing. High‐to‐moderate rates of resistance to penicillin (β‐lactamase production; 93%), tetracycline (40%), erythromycin and clindamycin (32%) were observed. Forty out of ninety‐one (44%) isolates were positive for PVL genes. Thirty‐six (40%) PVL‐positive methicillin‐susceptible S. aureus (MSSA) strains were shown by PFGE and MLST typing (ST121, ST681, ST837) to be part of a nosocomial outbreak caused by clonal complex (CC) 121. PFGE, MLST and SCCmec typing revealed three MRSA clones. Sequence type (ST) 239‐III (n=11), ST1097‐III (n=1) and ST8‐IV (n=3) belong to CC8 of epidemic multiresistant MRSA, whereas ST426‐MRSA‐IV/CC395 (n=1) has not been reported previously. All MRSA strains were PVL negative. The overall results underline the necessity of microbiological sampling, antimicrobial susceptibility testing, and epidemiological typing as a rational basis for antimicrobial treatment of S. aureus infections, and infection control measures to limit the spread of multiresistant MRSA and epidemic MSSA clones.

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 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.