Novel pseudo-staphylococcal cassette chromosome mec element (ψSCCmec57395) in methicillin-resistant Staphylococcus pseudintermedius CC45

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to β-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (ΨSCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ΨSCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ΨSCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ΨSCCmec57395 element amplified by long-range PCR revealed the presence of ΨSCCmec57395 in the 33 additional isolates of MRSP CC45. The ΨSCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.

Multilocus sequence typing for characterization of Staphylococcus pseudintermedius

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.

Daptomycin versus vancomycin for osteoarticular infections due to methicillin-resistant Staphylococcus aureus (MRSA): a nested case-control study

Vancomycin is the standard antibiotic for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. While daptomycin is approved for MRSA bacteremia, its effectiveness in osteoarticular infections (OAIs) has not been established. A 1:2 nested case-control study of adult patients with MRSA OAIs admitted to an academic center from 2005 to 2010 was carried out. Clinical outcomes and drug toxicity in patients treated with daptomycin versus vancomycin were compared. Twenty patients with MRSA OAIs treated with daptomycin were matched to 40 patients treated with vancomycin. The median age of the patients was 52 years (range, 25-90), and 40 (67 %) were male. Most patients had osteomyelitis (82 %), predominantly from a contiguous source (87 %). Forty percent were diabetics. Diabetic patients were more likely to receive vancomycin than daptomycin [20 (50 %) vs. 4 (20 %); p = 0.03]. Vancomycin was more often combined with antibiotics other than daptomycin [22 (55 %) vs. 5 (25 %); p = 0.03]. The median total antibiotic treatment duration was 48 (daptomycin) vs. 46 days (vancomycin) (p = 0.5). Ninety percent of daptomycin-treated patients had previously received vancomycin for a median of 14.5 days (range, 2-36). Clinical success rates were similar between daptomycin and vancomycin at 3 months [15 (75 %) vs. 27 (68 %); p = 0.8] and 6 months [14 (70 %) vs. 23 (58 %); p = 0.5], even after propensity score-based adjustment for antibiotic assignment. The frequency of adverse events was similar between treatment groups [1 (5 %) vs. 7 (18 %); p = 0.2]. Daptomycin and vancomycin achieved similar rates of clinical success and drug tolerability. Daptomycin is a reasonable alternative for treating MRSA OAIs, particularly in patients where therapy with vancomycin has not been well tolerated.

Host-pathogen interactions of secreted and surface Staphylococcus aureus factors

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, the amino-terminus, is composed of three immunoglobulin-folded subdomains (N1, N2, and N3) that contain ligand-binding activity. Clumping factors A and B (ClfA and ClfB) and SdrG are Sdr proteins that bind to fibrinogen (Fg), a large, plasma glycoprotein that is activated during the clotting cascade to form fibrin. In addition to recognizing fibrinogen, ClfA and ClfB can bind to other host ligands. Analysis of S. aureus strains that cause osteomyelitis led to the discovery of the bone-sialoprotein-binding protein (Bbp), an Sdr protein. Because several MSCRAMMs target more than one molecule, I hypothesized that Bbp may recognize other host proteins. A ligand screen revealed that the recombinant construct BbpN2N3 specifically recognizes human Fg. Surface plasmon resonance was used to determine the affinity of BbpN2N3 for Fg, and a dissociation constant of 540 nM was determined. Binding experiments performed with recombinant Fg chains were used to map the binding of BbpN2N3 to the Fg Aalpha chain. Additionally, Bbp expressed on the surface of Lactococcus lactis and S. aureus Newman bald mediated attachment of these bacteria to Fg Aalpha. To further characterize the interaction between the two proteins, isothermal titration calorimetry and inhibition assays were conducted with synthetic Fg Aalpha peptides. To determine the physiological implications of Bbp binding to Fg, the effect of Bbp on fibrinogen clotting was studied. Results show that Bbp binding to Fg inhibits the formation of fibrin. The consequences of this interaction are currently under investigation. Together, these data demonstrate that human Fg is a novel ligand for Bbp. This study indicates that the MSCRAMM Bbp may aid in staphylococcal attachment by targeting both an extracellular matrix and a blood plasma protein. The implications of these novel findings are discussed.

Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America.

BACKGROUND: Methicillin-resistant Staphylococus aureus (MRSA) is an important nosocomial and community-associated (CA) pathogen. Recently, a variant of the MRSA USA300 clone emerged and disseminated in South America, causing important clinical problems. METHODS: S. aureus isolates were prospectively collected (2006-2008) from 32 tertiary hospitals in Colombia, Ecuador, Peru, and Venezuela. MRSA isolates were subjected to antimicrobial susceptibility testing and pulsed-field gel electrophoresis and were categorized as health care-associated (HA)-like or CA-like clones on the basis of genotypic characteristics and detection of genes encoding Panton-Valentine leukocidin and staphylococcal cassette chromosome (SCC) mec IV. In addition, multilocus sequence typing of representative isolates of each major CA-MRSA pulsotype was performed, and the presence of USA300-associated toxins and the arcA gene was investigated for all isolates categorized as CA-MRSA. RESULTS: A total of 1570 S. aureus were included; 651 were MRSA (41%)--with the highest rate of MRSA isolation in Peru (62%) and the lowest in Venezuela (26%)--and 71%, 27%, and 2% were classified as HA-like, CA-like, and non-CA/HA-like clones, respectively. Only 9 MRSA isolates were confirmed to have reduced susceptibility to glycopeptides (glycopeptide-intermediate S. aureus phenotype). The most common pulsotype (designated ComA) among the CA-like MRSA strains was found in 96% of isolates, with the majority (81%) having a < or =6-band difference with the USA300-0114 strain. Representative isolates of this clone were sequence type 8; however, unlike the USA300-0114 strain, they harbored a different SCCmec IV subtype and lacked arcA (an indicator of the arginine catabolic mobile element). CONCLUSION: A variant CA-MRSA USA300 clone has become established in South America and, in some countries, is endemic in hospital settings.

Determination of an inoculum effect with various cephalosporins among clinical isolates of methicillin-susceptible Staphylococcus aureus.

Using 98 clinical methicillin-susceptible Staphylococcus aureus isolates of known beta-lactamase (Bla) type, we found a pronounced inoculum effect for cephalexin (mostly Bla type A and C strains), a mild inoculum effect for cephalothin (especially types B and C), and no inoculum effects for ceftriaxone and cefuroxime. Ceftobiprole showed the lowest MICs at a high inoculum but with a slight increase for Bla-positive versus Bla-negative strains. Since a potential therapeutic effect associated with a cephalosporin inoculum effect has been described, further studies are warranted.

Inoculum effect with cefazolin among clinical isolates of methicillin-susceptible Staphylococcus aureus: frequency and possible cause of cefazolin treatment failure.

Methicillin (meticillin)-susceptible Staphylococcus aureus (MSSA) strains producing large amounts of type A beta-lactamase (Bla) have been associated with cefazolin failures, but the frequency and impact of these strains have not been well studied. Here we examined 98 MSSA clinical isolates and found that 26% produced type A Bla, 15% type B, 46% type C, and none type D and that 13% lacked blaZ. The cefazolin MIC(90) was 2 microg/ml for a standard inoculum and 32 microg/ml for a high inoculum, with 19% of isolates displaying a pronounced inoculum effect (MICs of >or=16 microg/ml with 10(7) CFU/ml) (9 type A and 10 type C Bla producers). At the high inoculum, type A producers displayed higher cefazolin MICs than type B or C producers, while type B and C producers displayed higher cefamandole MICs. Among isolates from hemodialysis patients with MSSA bacteremia, three from the six patients who experienced cefazolin failure showed a cefazolin inoculum effect, while none from the six patients successfully treated with cefazolin showed an inoculum effect, suggesting an association between these strains and cefazolin failure (P = 0.09 by Fisher's exact test). In summary, 19% of MSSA clinical isolates showed a pronounced inoculum effect with cefazolin, a phenomenon that could explain the cases of cefazolin failure previously reported for hemodialysis patients with MSSA bacteremia. These results suggest that for serious MSSA infections, the presence of a significant inoculum effect with cefazolin could be associated with clinical failure in patients treated with this cephalosporin, particularly when it is used at low doses.

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Small colony variant of methicillin-resistant Staphylococcus pseudintermedius ST71 presenting as a sticky phenotype

We first observed the phenomenon of small colony variants (SCVs) in a Staphylococcus pseudintermedius sequence type 71 (ST71) strain, isolated from a non-pet owner. Although we found that small-sized colonies share main features with Staphylococcus aureus SCVs, they nevertheless show a novel, particular, and sticky phenotype, whose expression was extremely stable, even after subcultivation.

Genetic Relatedness, Antimicrobial and Biocide Susceptibility Comparative Analysis of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius from Portugal

Forty methicillin-resistant and -susceptible Staphylococcus pseudintermedius (MRSP and MSSP, respectively) from colonization and infection in dogs and cats were characterized for clonality, antimicrobial, and biocide susceptibility. MSSP were genetically more diverse than MRSP by multi-locus sequence typing and pulsed-field gel electrophoresis. Three different spa types (t06, t02, t05) and two SCCmec types (II-III and V) were detected in the MRSP isolates. All MRSP and two MSSP strains were multidrug-resistant. Several antibiotic resistance genes (mecA, blaZ, tet(M), tet(K), aac(6')-Ie-aph(2')-Ia, aph(3')-III, ant(6)-Ia, sat4, erm(B), lnu(A), dfr(G), and catpC221) were identified by microarray and double mutations in the gyrA and grlA genes and a single mutation in the rpoB gene were detected by sequence analysis. No differences were detected between MSSP and MRSP in the chlorhexidine acetate (CHA) minimum inhibitory concentrations (MICs). However, two MSSP had elevated MIC to triclosan (TCL) and one to benzalkonium chloride and ethidium bromide. One MSSP isolate harboured a qacA gene, while in another a qacB gene was detected. None of the isolates harboured the sh-fabI gene. Three of the biocide products studied had high bactericidal activity (Otodine(®), Clorexyderm Spot Gel(®), Dermocanis Piocure-M(®)), while Skingel(®) failed to achieve a five log reduction in the bacterial counting. S. pseudintermedius have become a serious therapeutic challenge in particular if methicillin- resistance and/or multidrug-resistance are involved. Biocides, like CHA and TCL, seem to be clinically effective and safe topical therapeutic options.

New MLSB resistance gene erm(43) in Staphylococcus lentus

The search for a specific rRNA methylase motif led to the identification of the new macrolide, lincosamide, and streptogramin B resistance gene erm(43) in Staphylococcus lentus. An inducible resistance phenotype was demonstrated by cloning and expressing erm(43) and its regulatory region in Staphylococcus aureus. The erm(43) gene was detected in two different DNA fragments, of 6,230 bp and 1,559 bp, that were each integrated at the same location in the chromosome in several S. lentus isolates of human, dog, and chicken origin.

Emergence of methicillin-resistant Staphylococcus pseudintermedius in Switzerland: three cases of urinary tract infections in cats

Methicillin resistance has emerged in clinical isolates of Staphylococcus pseudintermedius from cats in Switzerland. Three cats suffering from urinary tract infections were infected with methicillin-resistant S. pseudintermedius (MRSP). Phenotypic and genotypic characterization of the resistance profile showed that the isolates displayed resistance to all beta-lactams and cephalosporins (blaZ, mecA), fluoroquinolones, tetracyclines [tet(K)], macrolides, lincosamides and streprogramins B [erm(B)], chloramphenicol (catpC221), trimethoprim [dfr(G)] and the aminoglycosides gentamicin [aac(6')-Ie-aph(2')-Ia], kanamycin and neomycin [aph(3')-III] and streptomycin [ant(6)-Ia]. They also harbor the leukocidin gene lukS-I. MRSP represents a new challenge for antibiotic therapy and this zoonotic bacteria may rapidly spread to animals and humans.

Characterization of new staphylococcal cassette chromosome mec (SCCmec) and topoisomerase genes in fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius

Fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius isolates harbor two new staphylococcal cassette chromosome mec (SCCmec) elements that belong to class A, allotype 3 (SCCmec II-III), and to the new allotype 5 (SCCmec VII). Analysis of the complete nucleotide sequences of the topoisomerase loci gyrB/gyrA and grlB/grlA revealed mutations involved in fluoroquinolone resistance.

Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk

Staphylococcus aureus genotype B (GTB) is a contagious mastitis pathogen in cattle, occurring in up to 87% of individuals. Because treatment is generally insufficient, culling is often required, leading to large economic loss in the Swiss dairy industry. As the detection of this pathogen in bulk tank milk (BTM) would greatly facilitate its control, a novel real-time quantitative PCR-based assay for BTM has previously been developed and is now being evaluated for its diagnostic properties at the herd level. Herds were initially classified as to their Staph. aureus GTB status by a reference method. Using BTM and herd pools of single-quarter and 4-quarter milk, the herds were then grouped by the novel assay, and the resulting classifications were compared. A total of 54 dairy herds were evaluated. Using the reference method, 21 herds were found to be GTB positive, whereas 33 were found to be negative. Considering the novel assay using both herd pools, all herds were grouped correctly, resulting in maximal diagnostic sensitivities (100%) and specificities (100%). For BTM samples, diagnostic sensitivities and specificities were 90 and 100%, respectively. Two herds were false negative in BTM, because cows with clinical signs of mastitis were not milked into the tank. Besides its excellent diagnostic properties, the assay is characterized by its low detection level, high efficiency, and its suitability for automation. Using the novel knowledge and assay, eradication of Staph. aureus GTB from a dairy herd may be considered as a realistic goal.