Margaret O'Donoghue

Colonization of Butchers with Livestock-Associated Methicillin-Resistant Staphylococcus aureus

Reports have documented colonization of swine in Europe, North America and more recently in China with livestock‐associated methicillin‐resistant Staphylococcus aureus (LA‐MRSA). Contamination of pig farmers, veterinarians and abattoir workers with these strains has been observed. However, although contamination levels of 10% of retail pork were reported from the Netherlands and Canada, there are limited data of contamination rates of workers handling raw meat. We investigated the rates of MRSA contamination of local butchers working in wet markets, where recently slaughtered pigs are cut up. Nasal swabs collected from 300 pork butchers at markets throughout Hong Kong were enriched in brain heart infusion broth with 5% salt and cultured on MRSASelect®. Isolates were confirmed as Staphylococcus aureus and susceptibility testing performed. The presence of mecA was confirmed, SCCmec and spa type determined and relatedness investigated by PFGE. Subjects completed a questionnaire on MRSA carriage risk factors. Seventeen samples (5.6%) yielded MRSA, 15 harbouring SCCmec IVb. Ten strains were t899 (CC9), previously reported from local pig carcasses. Five strains were healthcare associated: SCCmec type II, t701(CC6), colonizing two subjects at the same establishment, and single isolates of t008 (CC8), t002 (CC5) and t123 (CC45). The remaining isolates were t359 (CC97), previously reported from buffaloes, and t375 (CC5), reported from bovine milk. None of these butchers reported recent hospitalization or a healthcare worker in the family. Two had recently received antibiotics, one for a skin infection. Four reported wound infections within the last year. All were exposed to meat for >9 h per day. Carriage of MRSA was higher in butchers than in the general community. Although five strains were probably of healthcare origin, the high incidence of t899 (CC9) suggests that cross‐contamination from pork occurs frequently. Washing of hands after touching raw pork is advised.

Isolation of Methicillin-Resistant Staphylococcus aureus (MRSA) from Retail Meats in Hong Kong

OBJECTIVES The presence of methicillin-resistant Staphylococcus aureus (MRSA) on meat purchased from retail outlets may allow its spread to households and represents a risk for colonization and possibly infection of consumers. Improved isolation methods have indicated that more than 10% of samples are positive. We aimed to determine rates of MRSA contamination of meat samples, including comparison of fresh and frozen samples. We characterized isolates and determined their antibiotic susceptibility. METHODS Samples of raw meats commonly consumed in Hong Kong were investigated for MRSA contamination using a double-enrichment isolation method. Isolates were characterized by antibiotic susceptibility testing, presence of mecA, SCCmec type, staphylococcal enterotoxins, Panton-Valentin leukocidin (PVL), and spa type. Differences in rates of MRSA contamination between meat types, rearing method, locations, sources, and fresh or frozen storage were compared. RESULTS MRSA was recovered from 21.9% of pork samples (78/355), 6.8% chicken (31/455), and 4.4% of beef (17/380). Isolation was considerably higher from fresh pork (47%) than frozen (0.6%), whereas contamination rates in fresh (6%) and frozen (7%) chicken were similar. All strains were multidrug resistant. All contaminated fresh pork and most frozen chicken originated from China. Most isolates belonged to CC9, being SCCmec IVb and spa type t899 or closely related spa types, but one chicken sample yielded ST398. Five strains carried spa types associated with human isolates. The egc enterotoxin group was present in the majority of isolates, but PVL in only three from chicken. CONCLUSIONS The predominance of t899 in isolates indicates that the primary source of contamination may be pig carcasses, previously demonstrated to frequently harbor CC9-positive MRSA in Hong Kong and China. The high rates of meat contamination suggest that improvements in food safety and personal hygiene guidelines may be advisable to reduce risk of spread of these MRSA strains in the community.

Characterization of methicillin-resistant Staphylococcus aureus isolates from pig carcasses in Hong Kong.

This study describes the isolation and characterization of methicillin‐resistant Staphylococcus aureus (MRSA) from slaughtered pigs sampled from local markets in Hong Kong. The nares of 400 slaughtered pigs were cultured and MRSA isolates characterized for the presence of antibiotic‐resistance determinants, toxins and SCCmec and spa types using PCR. Clonality was investigated using PFGE and MLST. The prevalence of MRSA colonization of slaughter pigs was 39.3%, the majority (92%) harbouring SCCmec type IVb. Of the 157 samples yielding MRSA, 13 had two distinct MRSA strains present. Spa type t899 was predominant, with only 5/170 isolates displaying closely related types (t4474, t1939, t2922 and t5390). PFGE with sma1 and MLST confirmed the strains as ST9. Most isolates were multidrug resistant. Tetracycline resistance (97%) was mainly attributable to tet(K) with only 3% of isolates additionally harbouring tet(M). Resistance to erythromycin (89%) and chloramphenicol (71%) was associated with the presence of erm(C), and fex(A), respectively. No strains carried cfr and there was no resistance to linezolid, although minimum inhibitory concentration (MICs) were close to the resistance break point. Resistance to clindamycin (99%), ciprofloxacin(78%), quinopristin–dalfopristin (44%) and cotrimoxazole (32%) was common, but remained low for fusidic acid (4%) and rifampicin (2%). All strains were negative for PVL, exfoliative, and enterotoxins. This survey confirmed the uniformity of MRSA isolates in pigs from several regions of China, in contrast to more diversified characteristics reported in European studies. Colonization rates were higher than previously reported. Isolates were resistant to a wide range of antibiotics, but resistance was not detected to linezolid, nitrofurantoin, vancomycin or tigecycline. Although the clinical importance of ST9 in humans is uncertain, continued surveillance, in particular of those occupationally‐exposed, is recommended.

Sustainable reduction of nasal colonization and hand contamination with Staphylococcus aureus in food handlers, 2002-2011.

A longitudinal study of nasal colonization and hand contamination of food handlers with Staphylococcus aureus commenced in 2002 prior to the outbreak of severe acute respiratory syndrome. In the follow-up in 2003 when hygiene measures were strictly implemented, significant reductions in carriage were observed. To investigate if this change was sustained, nasal and hand carriage rates were compared between the earlier studies and a further sampling in 2011. The initial nasal carriage rate was 35% and hand contamination 41·2%, decreasing to 23·5% and 11·6%, respectively in 2003 (P < 0·001). In 2011, nasal carriage was similar to 2003 (22·9%), while hand contamination dropped further to 3·7% (P < 0·001). Spa-typing revealed 39 types in 2002 and 42 in 2011. This study reveals that the marked reduction in colonization had been sustained. This may be attributed to reduced opportunities for spread due to enhanced hygiene and reinforces its importance for control of disease.

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.

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.

Rapid detection of vancomycin-non-susceptible Staphylococcus aureus using the spiral gradient endpoint technique

OBJECTIVES Several methods have been introduced for detection of vancomycin-non-susceptible Staphylococcus aureus [heterogeneous vancomycin-intermediate S. aureus (hVISA) and vancomycin-intermediate S. aureus (VISA)]. However, the limitations of these methods can delay appropriate therapy for the patient. This study evaluated the spiral gradient endpoint (SGE) technique for detection of hVISA/VISA. METHODS The SGE method was evaluated for intra-batch, inter-batch and inter-observer reproducibility in comparison with MICs determined by agar dilution. Three media, Mueller-Hinton agar, brain heart infusion agar and brain heart infusion agar with 5% glucose, were evaluated. The SGE method was compared with agar dilution for correlation of MIC and susceptibility category using control strains, clinical isolates and induced vancomycin-non-susceptible strains. RESULTS The SGE method had good reproducibility and there was excellent correlation of MICs generated by SGE using brain heart infusion agar with those by agar dilution (r(2) =0.950), with no difference in resistance categories generated by the two methods. All VISA isolates were correctly identified and the method allowed easy identification of hVISA by means of the trailing endpoint. CONCLUSIONS SGE offers a simple, rapid and cost-effective alternative method for the detection of hVISA/VISA for the routine laboratory. Early recognition of vancomycin-non-susceptible strains can allow the change to appropriate antibiotics, resulting in potentially better patient outcomes.

Spiral gradient endpoint susceptibility testing: a fresh look at a neglected technique

OBJECTIVES Increasing antibiotic resistance and interest in matching antibiotic therapy with pharmacokinetic/pharmacodynamic characteristics of isolates has led to increasing demands for determination of MICs. This can lead to increased costs for the laboratory. The spiral gradient endpoint (SGE) technique, a low-cost method of MIC determination, was developed some years ago. Although the technique showed good correlation with reference methods, it was not widely employed, mainly due to the introduction of alternative methods. We have revisited this technique and evaluated it for the determination of MICs for fastidious organisms. METHODS The SGE method was first optimized for fastidious organisms using Haemophilus influenzae. Intra-batch and inter-batch reproducibility was determined for H. influenzae, Streptococcus pneumoniae, Moraxella catarrhalis and Neisseria gonorrhoeae. The method was then evaluated by comparison of MICs for clinical isolates of these organisms determined by SGE with those determined with the reference method. RESULTS Optimization of the technique resulted in a method with excellent reproducibility for all organisms tested [SD 0.10-0.337; coefficient of variation (CV) 8.59%-18.66%]. These SDs/CVs were lower than those of the reference methods (0.27-2.34; 31.0%-63.8%). There was excellent correlation of the MICs with the reference methods (0.908-0.930) and insignificant differences in numbers of strains in each resistance category, with no tendency for SGE to produce higher or lower MICs than the reference method (P > 0.05). CONCLUSIONS SGE was shown to be reproducible and produced results that correlated well with standard techniques for fastidious organisms. The method offers a rapid, flexible, cost-effective alternative for smaller laboratories and for routine use in developing countries.

Tracking sources of Staphylococcus aureus hand contamination in food handlers by spa typing.

We aimed to identify the source of Staphylococcus aureus contaminating hands of food handlers. Nasal samples and direct fingertip imprints were collected on 2 occasions from food handlers and characterized to determine likely sources of hand contamination. Most hand contamination was attributable to nasal isolates of persistently colonized coworkers who had presumably contaminated the environment. Regular handwashing should be supplemented by effective environmental disinfection.

High Levels of Staphylococcus aureus Contamination in Chinese-Style Roast Pork

Chinese roasted pork has been implicated as a major source of food poisoning caused by Staphylococcus aureus. Establishing the source, either as contaminants from raw meat or from food handlers, could facilitate drafting more appropriate guidelines for better prevention of food poisoning. To determine the rate and source of staphylococcal contamination, roasted pork purchased from 50 sui-mei shops in Hong Kong was sampled for presence of S. aureus by enrichment and subsequent culture. Isolates were characterized for methicillin sensitivity, spa type, and presence of Panton-Valentine leukocidin (PVL) and staphylococcal enterotoxins (SEs). Methicillin-resistant isolates were confirmed by presence of mecA and SCCmec type and sensitivity to vancomycin investigated. S. aureus was isolated from 25 (50%) samples, with 3 yielding two colony types. Of the 28 isolates, 3 were resistant to cefoxitin, but only 2 were mecA positive and belonged to SCCmec type V. The mecA negative isolate also lacked mecC, but had a penicillin minimum inhibitory concentration of 10 mg/L. A livestock-associated spa type (t034) was only observed in one methicillin-sensitive strain, all other isolates appearing to be of human origin, with 30% belonging to t189. One isolate was PVL positive and five carried genes for classical SEs. The high rate of staphylococcal contamination observed was probably associated with food handlers, as the strains belonged to spa types previously reported in clinical and nasal carriage isolates. The presence of enterotoxins in 18% of isolates confirms the risk of food poisoning associated with this product and emphasizes the need for improved guidelines for handling after preparation. Use of refrigerated display areas should be considered.