Importance of early detection of vancomycin-resistant subpopulations in apparently susceptible meticillin-resistant Staphylococcus aureus clinical isolates.

This study aimed to investigate the presence of vancomycin-non-susceptible subpopulations in apparently susceptible meticillin-resistant Staphylococcus aureus (MRSA) and the ability of these isolates to develop into homogeneously resistant strains. Vancomycin MICs of 200 clinical MRSA isolates were determined using agar dilution (AD) and spiral gradient endpoint (SGE) technique. Isolates with an MIC≤2mg/L but displaying subpopulations with an MIC>2mg/L by SGE were re-tested by Etest and PAP-AUC and were incubated with 2mg/L vancomycin for 2 weeks. MIC testing was repeated weekly by AD, Etest and SGE to observe progression to non-susceptibility. A total of 17.5% and 16.0% of isolates were non-susceptible to vancomycin (MIC>2mg/L) by SGE and AD, respectively. Eight isolates (4%) displayed a resistant subpopulation; five met the definition of hVISA by PAP-AUC. The initial Etest MIC for these isolates was 2mg/L, but resistant subpopulations were observed in only three isolates on prolonged incubation. MICs of all eight isolates increased rapidly in the presence of vancomycin, reaching ≥3.0mg/L by Day 7 and ≥4mg/L after 14 days by all three methods. The prevalence of vancomycin-non-susceptible MRSA was high, and non-susceptibility developed rapidly in seemingly susceptible isolates with covert subpopulations. These were effectively detected by SGE. With increasing reports of vancomycin clinical failure, early detection of potentially non-susceptible isolates before or early in vancomycin therapy is essential to avoid further resistance development and poor clinical outcomes. SGE offers a novel and cost-effective technique for detection of potentially non-susceptible strains.

Occupational exposure to raw meat: a newly-recognized risk factor for Staphylococcus aureus nasal colonization amongst food handlers.

Staphylococcus aureus contaminating raw meat may increase nasal colonization risk for occupationally-exposed food handlers. Food handlers from six catering establishments were nasally sampled for S. aureus and completed a questionnaire on carriage risk factors. Isolates were characterized for antibiotic susceptibility, spa type and, for methicillin-resistant strains, SCCmec type. Of 434 food handlers, 99 (22.8%) were colonized with S. aureus. Five isolates were methicillin-resistant belonging to SCCmec IV (2) and V (3). Resistance to tetracycline (20%), and erythromycin (16%) was high, but <10% to other antibiotics. Spa typing revealed 17% of isolates as t189, with 8% each t127 and t1081. Food handlers ever handling raw meat had a significantly higher colonization risk (OR=2.7; 95% CI: 1.7-4.5), increasing to 3.7 (95% CI: 2.0-6.8) for those always exposed. This is the first report of increased colonization risk in food handlers exposed to raw meat. This occupational hazard may increase infection risk, so improved compliance with workplace hygiene may be required.

Role of stop codons in development and loss of vancomycin non-susceptibility in methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Problems of vancomycin non-susceptible Staphylococcus aureus (VISA) and subsequent treatment failure are increasing. This study aimed to observe development and loss of vancomycin non-susceptibility, determine exposure time needed for resistance development, and follow mutations in the VraSR and GraSR two-component systems during these processes. METHODS: Sequences of vraS, graR and rpoB, proposed as critical sites of mutation associated with non-susceptibility development, were compared in susceptible clinical methicillin-resistant S. aureus isolates both initially and following vancomycin induction and its withdrawal, to identify mutations. Mutations were correlated with exposure time, increase in vancomycin MIC and phenotypic changes. RESULTS: Both time required for heterogeneous VISA and VISA development, and maximum MIC attained (6-20 mg/L) varied between strains. Sequence analysis revealed the presence of stop codons in an initial strain with delayed non-susceptibility development. Other changes in vraS and graR occurred during VISA development in all isolates. After removal of vancomycin pressure, most strains reverted to susceptibility accompanied by emergence of stop codons in both vraS and graR. One strain not displaying stop codons remained resistant in the absence of vancomycin pressure. A substitution in GraR (D148Q) appeared to be associated with an elevated MIC (20 mg/L). No rpoB mutations were observed throughout VISA development. CONCLUSIONS: Vancomycin non-susceptibility developed in all strains tested. Mutations in vraS and graR appeared to be essential for VISA development, with stop codons playing an important role in delaying non-susceptibility development and reversion. Absence of mutations in rpoB suggests that these are not essential for vancomycin resistance. Further work is required to confirm consistent changes involved in non-susceptibility development.

Prevalence of antiseptic-resistance genes in Staphylococcus aureus and coagulase-negative staphylococci colonising nurses and the general population in Hong Kong.

Reduced biocide susceptibility in staphylococci is associated with quaternary ammonium compound (qac) gene-encoding efflux proteins. This study compared the prevalence of antiseptic-resistance genes (qacA/B, smr) in staphylococci colonising nurses and non-healthcare workers. Staphylococcus aureus and coagulase-negative staphylococci (CoNS) isolated from 249 nurses were compared for qacA/B and smr positivity with carriage isolates from non-healthcare workers. Associations between qac genes and antibiotic resistance were investigated and minimum inhibitory concentrations (MICs)/minimum bactericidal concentrations (MBCs) to benzalkonium chloride and chlorhexidine determined. Both genes had higher prevalence in CoNS from nurses (OR: 8.4; 95% CI: 5.4-13.2) and qacA/B was more common in nurses'S. aureus isolates than those of the general population (OR: 5.5; 95% CI: 2.7-11.2). Meticillin-resistant S. aureus (MRSA) carriage was low (3.2% nurses; 0.5% general population). The risk of harbouring qacA/B and smr was associated with presence of mecA (OR: 2.9; 95% CI: 1.8-4.8) and contact with MRSA-infected patients (OR: 2.0; 95% CI: 1.0-3.9) in S. aureus and CoNS. S. aureus with qac genes displayed significantly more antibiotic resistance and all gene-positive isolates had higher MICs and MBCs to antiseptics. Increased prevalence of antiseptic-resistance genes in staphylococci from nurses indicates that the hospital environment could exert selective pressure for carriage of these strains. The increased proportion of qac genes in meticillin-resistant strains suggests co-selection of these genes, as does the increased carriage of gene-positive strains by those in contact with MRSA-positive patients. Reduced antiseptic susceptibility may allow persistence of organisms in the presence of low level residues and contribute to survival of MRSA.

Tracking changes in the vraSR and graSR two component regulatory systems during the development and loss of vancomycin non-susceptibility in a clinical isolate.

We investigated changes in regulatory genes, vraS and graR, during development of vancomycin non-susceptibility in a patient with methicillin-resistant Staphylococcus aureus who failed therapy and following in-vitro vancomycin exposure and a subsequent drug-free growth period. Minimum Inhibitory Concentration (MICs) were determined and genes sequenced at each stage. After 30 days of vancomycin exposure, the strain attained maximum MIC (20 mg/L) and was resistant to all antibiotics. Reversion to vancomycin susceptibility occurred 21 days after removal. We observed mutations in vraS and graR during non-susceptibly development and novel stop codons in the reverted strain. Mutations in graR appear important for development of intermediate susceptibility to vancomycin. The results suggest that monitoring of vancomycin therapy could allow earlier change to appropriate agents.

Prevalence of Staphylococcus aureus carriage among dogs and their owners.

Case reports have indicated transmission of Staphylococcus aureus between humans and pets. We investigated associations between level of contact between dog and owner, and S. aureus colonization. In a cross-sectional study, nasal carriage and antibiotic susceptibility of S. aureus was determined for 830 dogs and 736 owners. Relatedness of isolates was investigated using antibiograms and pulsed-field gel electrophoresis (PFGE). Associations between carriage and demographics or amount of contact between owners and dogs were documented. S. aureus was isolated in 24% of humans and 8.8% of dogs. Antibiotic resistance was significantly more common in canine isolates. Of 17 owner/dog colonized pairs, six were indistinguishable by PFGE. Colonization of dogs was not associated with close human contact, but was strongly associated with health-care occupations (OR 3.29, 95% CI 1.49-7.26, P=0.002). In outbreak situations health-care workers' pets should be considered as a source of S. aureus. High rates of resistance indicate increased monitoring of antibiotic use in veterinary practice is needed.

Characterisation of methicillin-resistant Staphylococcus aureus isolates from dogs and their owners.

Ten methicillin-resistant Staphylococcus aureus (MRSA) isolates from healthy owners and their pets were characterised by susceptibility testing, staphylococcal chromosome cassette (SCC)mec and agr typing, and detection of the Panton-Valentine leukocidin (PVL) genes. Two human and three dog isolates harbouring SCCmec type III appeared to be of hospital origin. The five remaining isolates carried SCCmec type IV, with three being multidrug-resistant. One type IV isolate was PVL-positive and a prototypic agr type 3, typified by strain MW2. This is the first report of this type in association with nasal carriage. Drug resistance may be increasing among community isolates of MRSA.

The prevalence and source of methicillin-resistant Staphylococcus aureus (MRSA) in the community in Hong Kong.

Although reports of isolation of methicillin-resistant Staphylococcus aureus (MRSA) from patients admitted from the community have increased, few studies have investigated colonization of healthy subjects. This study aimed to determine community levels of MRSA in Hong Kong. Nasal swabs from a cross section of young adults and family units were cultured for MRSA. Antibiotic sensitivities and risk factors for carriage were determined and clonal relationships were investigated by pulsed-field gel electrophoresis (PFGE). Overall carriage was low (1.4%), and associated with health-care exposures (OR 13.56, 95% CI 1.11-165.21). Subjects working in health care yielded multi-resistant MRSA strains, but isolates from non-hospital-exposed subjects were methicillin-resistant only. Strains indistinguishable by PFGE were carried by subjects working together, and some spread to other contacts was observed. MRSA colonization in the community is rare in Hong Kong and is largely associated with working in health care. Community-acquired staphylococcal infections may be treated with agents effective against methicillin-sensitive strains.

Penicillin and vancomycin tolerance among clinical isolates of Streptococcus pneumoniae in Hong Kong.

OBJECTIVE: To determine the prevalence of penicillin and vancomycin tolerance in clinical isolates of Streptococcus pneumoniae. DESIGN: Laboratory testing of 50 consecutive clinical isolates. SETTING: District hospital, Hong Kong. PATIENTS: Fifty patients with pneumonia or meningitis who were admitted to a district hospital in Hong Kong between August and October 2002. MAIN OUTCOME MEASURES: Lysis, as assessed by loss of optical density at 4 hours at 540 nm, and reduction in viable count expressed as the log of the number killed ('log kill') after exposure of cultures to 10 times the minimum inhibitory concentration of penicillin and vancomycin. RESULTS: Of the 50 isolates, five (10%) were tolerant to penicillin and one (2%) was tolerant to vancomycin. CONCLUSION: This is the first report of vancomycin tolerance in Streptococcus pneumoniae in Hong Kong. Because vancomycin tolerance is associated with clinical failure in the treatment of pneumococcal meningitis, increased monitoring for resistant strains of organisms is suggested.

Prevalence of carriage of antimicrobial resistant strains of Streptococcus pneumoniae in primary school children in Hong Kong.

A cross-sectional survey was conducted to determine prevalence and assess risk factors for carriage of antibiotic resistant strains of Streptococcus pneumoniae in healthy school children in Hong Kong. Throat swabs were collected from 1455 subjects and written questionnaires providing demographic data and medical history were completed by parents. The overall carriage rate of Streptococcus pneumoniae was 3.5%, of which 49% were penicillin resistant. High levels of resistance to tetracycline (73%), erythromycin (52%), trimethoprim (66%) and ciprofloxacin (57%) were observed. Carriage was associated with presence of a younger sibling (OR = 1.79) and use of antibiotics (OR = 2.31). High use of day care and small size of housing units did not result in a high rate of carriage. The low rate of carriage may be linked to high use of antibiotics, geographical factors or ethnicity. High rates of antibiotic resistance reflect heavy use of antibiotics by general practitioners.