Panton Valentine Leukocidin (PVL) Toxin Positive MRSA

Panton valentine leukocidin (PVL) toxin positive MRSA

strains isolated from companion animals

Shelley Rankin8'*, Scott Roberts13, Kathleen O'Shea0, Donna Maloney3,

Marianne Lorenzo'1, Charles E. Benson0

"Matthew J. Ryan Veterinary Hospital, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104, USA

University a/Pennsylvania, School of Veterinary Medicine. 3850 Spruce Street, Philadelphia, PA 19104, USA

c University of Pennsylvania, New Bolton Center, 382 West St Rd, Kennett Sq., PA 19148. USA

Received 19 November 2004; received in revised form 17 February 2005; accepted 18 February 2005

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a highly pathogenic multiple-drug resistant (MDR) microorganism

that has recently become more prevalent in the community. It has been found that MRSA strains can also contain genes that

encode the panton valentine leukocidin toxin (PVL). The PVL toxin has been shown to be responsible for many of the severe

clinical symptoms of infection with MRSA, such as furunculosis, severe necrotizing pneumonia, and necrotic lesions of the skin

and soft tissues. The aim of this study was to determine the presence of the S. aureus PVL toxin genes (/^S-PVand lukF-PV) in

MRSA strains isolated from companion animals. Eleven MRSA isolates, from a total of 23 tested, were shown to possess the

mecA gene and the PVL toxin genes. Pulsed-field gel electrophoresis showed that the 11 PVL toxin positive MRSA strains were

highly clonal.

Keywords: Panton valentine leukocidin (PVL); Methicillin-resistant Staphylococcus aureus (MRSA)

* Corresponding author. Tel.: +1 215 573 1189; c fax: +1 215 898 0503. \

1. Introduction

Methicillin-resistant Staphylococcus aureus (MRSA), traditionally regarded as a pathogen of humans, causes a wide range of disease states; specifically, septicemia, endocarditis, pneumonia; and wound, bone, joint and dermatological infections (Fell et al., 2003). MRSA is an emerging pathogen in companion animals where it has been shown to cause numerous conditions ranging from soft tissue infection, such as chronic pyoderma, post-surgical wound infection, joint invasion or even death (Seguin et al., 1999; Pak et al., 1999; Gortel et al., 1999; van Duijkeren et al., 2003; Guardabassi et al., 2004; van Duijkeren et al., 2004a). Several case reports suggest that MRSA infection can be transferred between humans and animals thus the isolation of MRSA from companion animals may present a potential human health risk (Cefai et al., 1994; Manian, 2003; van Duijkeren et al., 2004b).

The panton valentine leukocidin (PVL) is a cytotoxin that causes leukocyte destruction and tissue necrosis. In 1999, Lina et al. howed that the PVL toxin is produced by fewer than 5of5'. aureus strains. More recently, the PVL toxin has been associated with community-acquired methicillin-resistant S. aureus strains (CA-MRSA) (Gillet et al., 2002). The aim of this study was to determine the presence of the S. aureus PVL toxin gene in MRSA strains isolated from companion animals.

2. Experimental methods

Twenty-three MRSA strains obtained from clinical samples submitted to the Mathew J. Ryan Veterinary Hospital at the University of Pennsylvania (MJR-VHUP) were included in the study. Strains were identified by a MicroScan Walkaway 40 (Dade Behring, Sacramento, CA) PC20 Gram Positive combo-panel; antimicrobial susceptibility to a further 22 antibiotics was determined using the same MicroScan panel. MRSA strains were stored at —70 °C in Microbank tubes (ProLab Diagnostics, Austin, TX). All 23 isolates were tube coagulase positive and slide coagulase (Staphaurex, Remel, Lenexa, KS) positive. S. aureus strain ATCC 49775 was used as a positive control.

2.1. DNA extraction and real-time PCR detection of the PVL toxin genes. DNA was extracted from 23 MRSA strains using a MasterPure DNA Extraction Kit (Epicentre Technologies, Madison, WI) and 5 |xl was used as a template for PCR amplification. Real-time PCR using primers, PVLSC-F, GCTCAGGAGATACAAG and PVLSC-R, GGATAGCAAAAGCAATG was carried out on a SmartCycler™ (Cepheid, Sunnyvale, CA) using a LightCycler® FastStart DNA Master SYBR Green I kit (Roche Applied Bioscience, Indianapolis, IN), as described previously (Roberts et al., 2005).

In addition, the presence of the mecA gene, that confers resistance to oxacillin, was determined by PCR using primers and conditions, as described previously (Sakoulas et al., 2001).

2.2. Pulsed-field gel electrophoresis (PFGE)

PFGE was performed on 11 PVL-positive MRSA isolates as described previously (Roberts et al., 2005). Using BioNumerics software ver 3.0 (Applied Maths, Kortrijk, Belgium) percent similarities were identified on a dendrogram derived from the unweighted pair group method using arithmetic averages and based on Dice coefficients. A similarity coefficient of 80 was selected to define pulsed-field profile (PFP) clusters as described for S. aureus (McDougal et al., 2003).

3. Results

Table 1 shows details of 11 MRSA isolates that were positive for the PVL toxin genes. All isolates were obtained from animals admitted to MJR-VHUP in 2002 and 2003. Eight isolates were from dogs and one isolate each was obtained from a cat, a rabbit and an African grey parrot. The strains presented in Table 1 are in chronological order. Each animal had a unique diagnosis and analysis of their medical records did not suggest that nosocomial transmission had occurred while at MJR-VHUP.

Fig. 1 shows the PFGE profiles obtained from 11 PVL-positive MRSA isolates. Six unique PFGE profiles were identified and with the exception of strain 1950 all isolates formed a cluster that showed >80 similarity. Antibiotic resistance profiles are also shown in Table 1 and there was no correlation between resistance phenotype and PFGE profiles.

Dice (Opl.0 50) (To! 1,5. •8.5) (MtO.d'.i S»0.0i SO.O'A-IOO.O'1';)

Sma 1 Sma1

Fig. 1. A graphical representation of the PFGE profiles obtained from 11 PVL-positive MRSA strain. Three pulsed-field clusters were evident and with the exception of isolate 1950 all PFGE profiles were >80 similar suggesting that these strains were highly clonal.

Table 1
Source, PFGE profile, PVL and mecA gene PCR results and resistance profile of strains in this study
Isolate / Animal / Diagnosis / PFGE profile / lukS-PV/lukF-PV / mecA / Resistance profile
5567 / Canine / Infiltrative lipoma / 1 / Positive / Positive / Ox, Cd, Cp, E
1866 / Canine / Wound infection / 1 / Positive / Positive / Ox, Cd, Cp, E
1950 / Parrot / Chronic sinusitis / 6 / Positive / Positive / Ox, Cp
3196 / Canine / Carpal joint infection / 3 / Positive / Positive / Ox, Cd, Cp, E
3542 / Canine / Prostatitis / 4 / Positive / Positive / Ox, Cd, Cp, E, Te
3759 / Canine / Sepsis / 2 / Positive / Positive / Ox, Cd, Cp, E, Te
3779 / Canine / Pneumonia / 2 / Positive / Positive / Ox, Cd, Cp, E
4342 / Canine / Joint infection / 5 / Positive / Positive / Ox.Te
4691 / Rabbit / Ear infection / 3 / Positive / Positive / Ox, Cd, Cp, E, Te
4711 / Feline / Tooth abscess / 1 / Positive / Positive / Ox, Cd, Cp, E, Te
4714 / Canine / CNS infection / 3______/ Positive / Positive / Ox, Cd, Cp, E

4. Discussion

This is the first study to demonstrate the presence of the PVL toxin genes in MRSA strains isolated from companion animals. The results are noteworthy in that MRSA strains that produce the PVL toxin have been shown in many studies to cause pneumonia, necrotizing dermatitis, and other primary diseases in humans and these conditions were mirrored here in animals. In 2003, Vandenesch et al. described the worldwide emergence of CA-MRSA strains that produce PVL toxin. Genetic comparison of CA-MRSA strains from the United States and Australia indicated that these strains belonged to five different clonal complexes, thus suggesting diverse genetic backgrounds rather than the global spread of a single clone. The 11 strains in this study are highly related and appear to belong to a single clone. It is not known at this time whether these strains belong to a CA-MRSA clone or a healthcare-associated MRSA clone but we would welcome comparison. Several recent studies have indicated that it is possible to transmit MRSA between humans and companion animals (Seguin et al., 1999; Manian, 2003; van Duijkeren et al., 2004b) and it will undoubtedly be inferred from this study that companion animals infected by PVL-positive MRSA strains may act as a reservoir of infection for humans. However, the alternative hypothesis that humans may serve as a reservoir for infection in companion animals seems equally feasible.

Now that we have shown that the PVL toxin is present in MRSA strains from companion animals, further research into the clinical aspects, risk factors and epidemiology of MRSA infections in veterinary medicine should be a priority.

Acknowledgements

Scott Roberts was supported by the Merck/NIH summer research program under NIH training grant RR07065 and a grant from the Merck Foundation.

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