Download Veterinary Microbiology 108 (2005) 145-148

Veterinary Microbiology 108 (2005) 145-148
Panton valentine leukocidin (PVL) toxin positive MRSA
strains isolated from companion animals
Shelley Rankin a,*, Scott Roberts b, Kathleen O' Shea °, Donna Maloney a,
Marianne Lorenzo a, Charles E. Benson
"Mathew J. Ryan Veterinary Hospital, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104, USA
b University of Pennsylvania, School of Veterinary Medicine, 3850 Spruce Street, Philadelphia, PA 19104, USA
'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 (lukS-PV and 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.
© 2005 Elsevier B.V. All rights reserved.
E-mail address: [email protected] (S. Rankin).
Keywords: Panton valentine leukocidin (PVL); Methicillin-resistant Staphylococcus aureus (MRSA)
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 (Feil et a1., 2003). MRSA is an emerging pathogen in companion
animals where it has been shown to cause numerous conditions ranging from soft tissue infec tion, 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. showed that the PVL toxin is produced by fewer than 5% of S. 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 (MJRVHUP) were included in the study. Strains were
identified by a MicroScan Walkaway 40 (bade 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). A1123 isolates were tube
coagulase positive and slide coagulase (Staphaurex, Remel, Lenexa, KS) positive. S. aureus strain ATCC
49775 was used as a positive control.
21. 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 µl 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
SmartCyclerTM (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 l shows details of l l 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 I
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.
Table I
Source, PFGE profile, PVL and m e c A 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
I
Positive
Positive
Ox, Cd, Cp, E
1866
1950
3196
3542
3759
3779
4342
4691
4711
4714
Canine
Parrot
Canine
Canine
Canine
Canine
Canine
Rabbit
Feline
Canine
Wound infection
Chronic sinusitis
Carpal joint infection
Prostatitis
Sepsis
Pneumonia
Joint infection
Ear infection
Tooth abscess
CNS infection
I
6
3
4
2
2
5
3
I
3
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Ox, Cd, Cp, E
Ox, Cp
Ox, Cd, Cp, E
Ox, Cd, Cp, E, Te
Ox, Cd, Cp, E, Te
Ox, Cd, Cp, E
Ox, Te
Ox, Cd, Cp, E, Te
Ox, Cd, Cp, E, Te
Ox, Cd, Cp, E
CNS: central nervous system; Ox: oxacillin; Cd: clindamycin; Cp: ciproftoxacin; E: erythromycin and Te: tetracycline.
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 I and there
was no correlation between resistance phenotype and PFGE profiles.
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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* Corresponding author. Tel.: +1 215 573 1189;
fax: +1 215 898 0503.