>>>>>>> Discussion <<<<<<<

This study showed the presence of S. aureus on almost 50% of samples of roasted pork and MRSA in two samples. Five samples (10%) yielded toxin producing strains.

Such contamination allows bacterial growth and production of SE causing Staphylococcal Food Poisoning if the food is not well stored – it is usually displayed at ambient temperature.

Wound infections or colonization of hands and other body sites such as nasal cavity may occur leading to spread of the bacteria in the community if contaminated pork is handled without gloves5,6 (see Fig. 7)

Two samples yielded more than one S. aureus strain and one sample yielded both S. aureusand MRSA. S. aureus may become MRSA through exchange of genetic materials.

Contamination may be from more than one source (see Fig. 7). 1) Food handler – nasal cavity, hand, apron (see Fig. 8) 2) Cross contamination by raw meat7

Fig. 7. Transmission route of S. aureus and MRSA Fig. 8. Poor hygiene of food handler’s apron

One S. aureus strain was PVL gene-positive and five carried SE genes. Presence of these virulence factors increase possible infection and food poisoning risks from roasted pork.

As roasted pork has been associated with food poisoning and this study has shown contamination with SE positive S. aureus, improvement in food hygiene in line with food safety guidelines should be implemented.

Further investigation: A larger sample including specimens from all parts of Hong Kong, complete antibiotic profiling, investigation of more virulence factors, spa and MLST typing should be performed.

Isolation and Characterization of Staphylococcus aureus Strains from Roasted Pork in Hong KongChi-Pong Young, Maureen Boost , Margaret O’Donoghue

Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR

>>>>>> Introduction <<<<<<Staphylococcus aureus is a major cause of food poisoning and in Hong Kong the majority of such outbreaks have been ascribed to intake of roasted pork although there appear to have been no studies to determine rates of contamination of this popular local food. Staphylococcal food poisoning is caused by enterotoxins (SE). Recently, livestock associated methicillin-resistant S. aureus (LA-MRSA) has been isolated from locally slaughtered pork.

>>>>>>Materials and methods <<<<<<1. Samples:

Portions of roasted pork (see Fig. 1a) were collected from 50 retail outlets (siu-mei stores)in Hong Kong that prepare roast pork on the premises, on three separate days during January and February 2011.Several small pieces totaling 25g were aseptically cut from the meat and homogenised in Trptone Soy Broth (TSB). 5ml of the homogenate was enriched in 6% NaCl TSB for 24h.

2. Isolation of S. aureus / MRSA:

a. S. aureus: After enrichment, plated on ChromID SA –green round colonies (Fig 1b).

b. MRSA: An aliquot from the TSB enrichment was transferredto TSB + 5 mg/L ceftizoxime and 75 mg/L aztreonam ,incubated overnight and then plated onto ChromID MRSA– Green round colonies (Fig 1c).

c. Confirmation by Gram stain and latex agglutination test

3. Antibiotic susceptibility testing for methicillin resistance: 1 μg oxacillin and 30 μg cefoxitin disks were placed on Mueller Hinton Agar (MHA) previously inoculated with a bacterial suspension at a turbidity of 0.5 McFarland . Results were determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.Susceptibility to both antibiotics = S. aureus; Resistance to both antibiotics = MRSA

4. Detection of MIC of vancomycin: The Spiral gradient endpoint method was used to create a concentration gradient of vancomycin across Brain Heart Infusion agar. A 0.5 McFarland bacterial suspension was streaked across the gradient. The endpoint of the growth was measured after 24 h and the MIC calculated using SpiralBiotech software. Susceptibility was determined according to the CLSI guidelines.

5. Characterization

Positive samples

Number of isolates

β-lactamasehyperproducer

Vancomycin susceptibility

PVL gene positive

SE genes mecA gene positive

SCCmec

S. aureus

24(48%)

26 1* N/A 1 sea gene: 1sec gene: 3see gene: 2

[Total 6]

N/A N/A

MRSA 2(4%)

2 N/A All susceptible

0 0 2 IVb: 1V: 1

Fig. 4 shows the positive results of seb-see gene - PVL gene - sea gene - sec gene

- see gene - mecA gene - SCCmec

Roasted pork

Community

FarmersPigs

Raw meat

Food handlers

>>>>>> Conclusion <<<<<<This is the first report of the presence of S. aureus and MRSA on roasted pork in Hong Kong. It demonstrates that MRSA can be transferred by contaminated food.

>>>>>> Acknowledgements <<<<<<

The study was funded by the Dept of Health Technology & Informatics, The Hong Kong Polytechnic University. We would like to thank Jeff Ho, Vijaya C. Doddangoudar and Carol Zhang for technical assistance.

PVL SE mecA SCCmec

Increasing virulence of skin and soft tissue infections 2 Causing SFP1 Leading to ᵦ-lactam antibiotic resistance3 Carrying antibiotic resistance genes4

>>>>>>> Aims of this study<<<<<<<(i) To isolate S. aureus and MRSA from roasted pork in Hong Kong;

(ii) To characterize isolates for presence of Panton Valentin Leukocidin (PVL) gene, SE genes and antibiotic profiling;(iii) To confirm MRSA by presence of mecA and characterization of staphylococcal cassette chromosome mec (SCCmec)

genotype.

Table 1. Functions of the genes

Fig. 1a. Portion of roasted pork

Fig 1b. S. aureus colony on ChromID SA

Fig 1c. MRSA on ChromID MRSA

DNA Extraction

MRSA only:mecA and SCCmec typing

PERFORM PCR

S. aureus and MRSA:PVL + sea-see genes

If mecA negative, MIC of benzylpenicillin was determined

by Standard Agar Dilution

>>>>>> Results <<<<<<

*This strain did not have the mecA gene but was resistant to cefoxitin, oxacillin and benzylpenicillin

>>>>>> References <<<<<<1. Department of Health. Scientific Committee on Enteric Infections and Foodborne Diseases. Review of Staphylococcal Food Poisoning in Hong Kong. 2010 [Accessed 2011 Jan 15]. Available from

http://www.chp.gov.hk/files/pdf/Review of Staphylococcal Food Poisoning in Hong Kong.pdf2. Maltezou HC, Giamarellou H. Community-acquired methicillin-resistant Staphylococcus aureus infections. International Journal of Antimicrobial Agents 2006; 27(2):87-96. 3. Smith TL, Jarvis WR. Antimicrobial resistance in Staphylococcus aureus. Microbes and Infection 1999; 1(10):795-805.4. Malachowa N, DeLeo FR. Mobile genetic elements of Staphylococcus aureus. Cellular and Molecular Life Sciences 2010; 67(18):3057-3071.5. Paciorek ML, Kochman M, Piekarska K, Grochowska A, Wingdyga B. The distribution of enterotoxin and enterotoxin-like genes in Staphylococcus aureus strains isolated from nasal carriers and food samples. International Journal of Food Microbiology 2007; 117(3):319-323.6. Elston DM. Community-acquired methicillin-resistant Staphylococcus aureus. Journal of the American Academy of Dermatology 2007; 56(1):1-16. 7. Baird RM, Lee WH. Media used in the detection and enumeration of Staphylococcus aureus. International Journal of Food Microbiology 1995; 26(1):15-24.

Fig 2 PVL gene

>>>>>> Results (2) <<<<<<

Fig 3 sea gene

Fig 5 mecA Fig 6 SCCmec IVb and V