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Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy
Accepted Manuscript Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy A. Traversa, G.R. Gariano, S. Gallina, D.M. Bianchi, R. Orusa, L. Domenis, P. Cavallerio, L. Fossati, R. Serra, L. Decastelli PII:
S0740-0020(15)00145-8
DOI:
10.1016/j.fm.2015.07.012
Reference:
YFMIC 2430
To appear in:
Food Microbiology
Received Date: 29 April 2014 Revised Date:
12 May 2015
Accepted Date: 20 July 2015
Please cite this article as: Traversa, A., Gariano, G., Gallina, S., Bianchi, D., Orusa, R., Domenis, L., Cavallerio, P., Fossati, L., Serra, R., Decastelli, L., Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy, Food Microbiology (2015), doi: 10.1016/ j.fm.2015.07.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy A. Traversa a,b, G R. Gariano a,b, S. Gallina a,b, D M. Bianchi a,b, R. Orusa a,c, L. Domenis a,c, P. Cavallerio d, L. Fossati d, R. Serra d, L. Decastelli a,b Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta - IZSPLV
b
Struttura Complessa Controllo Alimenti e Igiene delle Produzioni – Turin, Italy
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Centro di Referenza Nazionale per le Malattie degli Animali Selvatici – Aosta, Italy
d
AOU San Giovanni Battista, Laboratorio di Microbiologia, Turin, Italy
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Corresponding author: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta – IZSPLV, Struttura Complessa Controllo Alimenti e Igiene delle Produzioni – Turin, Italy. Tel: +39 (0)11 2686233; Fax: +39 (0)11
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2473450. E-mail address: [email protected]. (A. Traversa)
ACCEPTED MANUSCRIPT 1. Introduction
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Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of hospital-
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associated infections in humans; treatment has become increasingly difficult owing to the
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development of resistance to many commonly used antimicrobials (EFSA and ECDC, 2012).
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Methicillin resistance is mediated by the mecA gene, chromosomally located in the
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staphylococcal cassette chromosome mec (SCCmec), which encodes penicillin-binding
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protein (PBP) 2a with a low affinity for beta-lactams (EFSA, 2009).
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In addition to hospital-acquired MRSA infections, community-associated infections caused
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by MRSA (CA-MRSA) have become a growing public health problem (Witte et al., 2004).
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CA-MRSA clones have evolved outside hospital settings and may carry the gene for Panton-
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Valentine leukocidin (PVL) cytotoxin, a potential virulence factor (EFSA, 2009). New
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MRSA, known as livestock-associated (LA)-MRSA, has been detected in food-producing
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animals as well, including pigs, cattle, chickens (De Neeling et al., 2007; Lee, 2006), horses,
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and companion animals (Cuny et al., 2010). LA-MRSA and the new clone ST398 (MRSA
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lineage multilocus sequence type 398), belonging to the clonal complex (CC)398 may also be
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harboured by humans, especially where there is occupational contact with affected livestock
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(EFSA and ECDC, 2014). Food-producing animals and their products are considered a
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potential source of MRSA infection in humans (ECDC, EFSA and EMEA, 2009).
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Foods and food chains are an important and potential route of transmission of
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microorganisms between food-producing animals, including wildlife, and humans and vice
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versa. Surveillance and monitoring of MRSA in humans and food-producing animals is
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recommended in all European Member States; further work should be performed to
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harmonize the methods for the detection of MRSA as a contaminant of food (ECDC, EFSA
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and EMEA 2009). Data on MRSA in food-producing animals, especially in pig holdings,
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have been reported worldwide, yet the currently available data about MRSA in foods are still
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ACCEPTED MANUSCRIPT few. The occurrence of MRSA in wild animals has been sporadically reported to date
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(Wardyn et al., 2012; Porrero et al., 2013). Northwest Italy is rich in animal wildlife where
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many species are hunted as game for human consumption. Wildlife data may be useful to
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assess MRSA circulation in wild species and the potential route of transmission to domestic
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animals and, ultimately, humans.
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The present study was undertaken to investigate the occurrence of methicillin resistance in
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Staphylococcus aureus strains isolated from food and from wild animal carcasses. The aims
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were: 1) to assess the level of contamination of MRSA in foods analysed for the detection of
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S. aureus according to national official control programs and regional monitoring activities;
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and 2) to evaluate the occurrence of MRSA strains in wild species collected by the National
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Reference Center for Wild Animal Diseases (Ce.R.M.A.S.); and 3) to characterize MRSA
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isolates by using staphylococcal cassette chromosome mec typing, spa typing, PVL encoding
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gene detection, and antimicrobial susceptibility testing.
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2. Materials and Methods
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2.1 Sampling
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Food. A total of 2162 samples of food were analysed by the Food Control Laboratory of the
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IZS PLV in Turin in 2008 for the enumeration of coagulase-positive staphylococci (CPS) and
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the detection of S. aureus according to national official monitoring activities and regional
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agreements. Details of the analyses are given in Table 1.
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Wildlife. In the period 2003-2009, the carcasses of 242 alpine wild ruminants (Rupicapra
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rupicapra, Capra ibex, and Capreolus capreolus), 276 foxes (Vulpes vulpes), 134 mustelids
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(Martes foina, Meles meles, Martes martes), and 16 rodents (Marmota marmota, Sciurus
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vulgaris) were autopsied at the Ce.R.M.A.S for establishing the cause of death or the
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examination of lesions in game animals killed by hunters; the submandibular lymph nodes of
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ACCEPTED MANUSCRIPT 697 wild boars (Sus scrofa) were subjected to anatomopathological examination within a
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monitoring plan for wild animal tuberculosis. All animals came from the northwestern Alps
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(Aosta Valley and North Piedmont). Bacteriological examination of tissue samples of lesion
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was generally performed using blood agar (Microbiol Diagnostici) and Mc Conkey agar
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incubated for 24 h at 37° C, aerobically and/or anaerobically depending on the suspect
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microorganism.
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2.2 Enumeration of coagulase-positive staphylococci and identification of S. aureus
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Food. For the enumeration of CPS from food samples, accredited methods according to
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standard microbiological procedures (ISO 6888-2:1999/Amend1: 2003) were used. S. aureus
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isolates were identified using the ID 32 Staph System (bioMérieux, France) or GP-Vitek2
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Cards (bioMérieux). All S. aureus isolates were stored at -20° C in Microbank (Mast
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Diagnostic, Merseyside, UK) until further analysis.
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Proportions of S. aureus positive samples in each type of food were compared by chi-square
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or Fisher’s exact tests. A p-value < 0.05 was considered statistically significant. Analyses
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were carried out using GraphPad Prism software.
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Wildlife. All S. aureus isolates were confirmed by API STAPH (bioMérieux) and
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agglutination tests.
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2.3 Phenotypic methicillin resistance detection methods
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Stocked cultures of S. aureus isolates were subcultured onto blood agar plates (Microbiol
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Diagnostici) and RPF-Baird-Parker agar plates (Biolife italiana). After incubation for 24 h at
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37° C, each strain was inoculated onto Brilliance MRSA medium (Oxoid) then incubated for
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18 h at 37° C: presumptive MRSA strains showed colonies with denim blue coloration. At
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the same time, each strain was tested for susceptibility to cefoxitin 30 µg using the disc
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diffusion method on Mueller–Hinton agar according to the Clinical Laboratory Standards
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Institute (CLSI M100-S19, 2009) guidelines.
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ACCEPTED MANUSCRIPT MRSA ATCC 49775™ was included as positive control strain for both tests.
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2.4 SCCmec typing, spa-typing and detection of PVL gene of MRSA
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The DNA of presumptive MRSA strains was extracted using EZ1 (Qiagen, Hilden, Germany)
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and tested for staphylococcal cassette chromosome mec (SCCmec) typing performed by
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multiplex PCR (Zhang et al., 2005). The polymorphic X region of the protein A gene (spa)
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was amplified by PCR using specific primers (www.ridom.de, spa-server). All sequencing
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reactions were carried out using an ABI 3130 genetic analyzer (Applied Biosystems, Foster
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City, CA).
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SCCmec type IV or V strains were further tested by PCR for the PVL encoding gene (Lina et
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al., 1999).
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2.5 Antimicrobial susceptibility test
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Minimal inhibitory concentration (MIC) of a panel of 20 antimicrobials was determined
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against MRSA isolates using Microscan Walk Away (Siemens). The antimicrobial
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breakpoints used for interpretation of susceptibility tests refer to Clinical Laboratory
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Standards Institute (CLSI, 2009) guidelines.
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3. Results
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3.1 Occurrence of S. aureus in food
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The prevalence of S. aureus in food was 17.1% (370/2162). The associations between the
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presence of S. aureus strains and bulk tank milk samples, as well as cow cheese samples,
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were found to be statistically significant (P<0.0001).
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Among the positive samples (Table 2), 42 samples of cheese had CPS >100,000 CFU/g, 2 of
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these, collected during foodborne outbreaks, resulted positive for staphylococcal enterotoxins
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(SEs) on ELFA-VIDAS or ELISA according to the ANSES EU-CRL screening method for
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CPS.
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S. aureus was found in 2.0% of wild animal carcasses and in 3.2% of wild boar lymph nodes.
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In particular, 6.25% (1/16) of rodents samples (Martes foina), 4% (10/242) of wild ruminants
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(8 Rupicapra rupicapra and 2 Capra ibex), 0.8% (1/134) of mustelids (Marmota marmota),
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and 0.4% (1/276) of Vulpes vulpes were positive for S. aureus.
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3.3 Phenotypic methicillin resistance detection methods
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Food. S. aureus strains isolated from food samples were tested for both MRSA-Brilliance
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(Oxoid) growth and susceptibility to cefoxitin 30 µg using the disc agar diffusion method on
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Mueller–Hinton agar: 8 strains grew on MRSA Brilliance with typical colonies, but only 1
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exhibited resistance to cefoxitin (strain 1). One strain, which did not grow on the medium,
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was resistant to cefoxitin on disc diffusion testing (strain 2).
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Wildlife. None of the 35 S. aureus isolates tested by both phenotypic methods grew on Oxoid
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Brilliance MRSA or showed resistance to cefoxitin. As no phenotypic methicillin resistance
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was reported, these isolates were not tested with molecular methods, in accordance with our
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protocol.
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3.4 SCCmec typing, spa-typing and detection of PVL gene of MRSA strains
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SCCmec typing and spa-typing of the 9 strains that resulted methicillin resistant according to
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the results of at least one of the phenotypic methods showed that 2 out of the 9 strains, both
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from bulk tank milk, carried spa-type t899 and SCCmec type IV (strain 1) and V (strain 2)
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but were negative for the detection of the PVL gene.
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3.5 Antimicrobial susceptibility test
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MRSA isolates were resistant to beta-lactams and tetracycline but were susceptible to
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glycopeptides and linezolid. In addition, strain 1 showed resistance against erythromycin,
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clarithromycin, clindamycin and trimethoprim/sulfamethoxazole, and strain 2 only against
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gentamicin.
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4. Discussion
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In this survey, the occurrence of S. aureus strains in food samples was 17.1% (370/2162). In
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accordance with national official monitoring programs and regional agreements, the search
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for S. aureus was conducted in foods more likely to be contaminated with CPS and S. aureus.
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The prevalence of S. aureus in bulk tank milk and cow cheese was 41.0% (107/261) and
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32.6% (155/476), respectively; these foodstuffs were observed to be significantly associated
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(P<0.05) with the presence of S. aureus. The occurrence of this microorganism in dairy
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products or other foodstuff varies across studies: Haran et al. (2012) reported a 62% S. aureus
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prevalence in bulk tank milk collected from Minnesota dairy farms, whereas Shanehbandi et
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al. (2014) found a prevalence of S. aureus about 16% in traditional cheese samples.
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In our survey, MRSA phenotypic prevalence differed depending on whether the Oxoid
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MRSA Brilliance medium or susceptibility to cefoxitin 30 µg by the agar diffusion method
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was used: 2.2% versus 0.5% in the tested strains (8/2162 or 0.37% and 2/2162 or 0.09% in
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the food samples), respectively. Molecular methods confirmed MRSA phenotypic prevalence
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obtained with the disc agar diffusion method. Oxoid MRSA Brilliance showed high
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specificity (0.98) and negative predictive value (1.00) but very low sensitivity (0.5) and
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positive predictive value (0.13). The data reported in other studies are discordant: Lee et al.
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(2013) found that Brilliance MRSA Agar had high specificity and low sensitivity, whereas
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Malhotra-Kumar et al. (2010) reported high sensitivity but low specificity.
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In the opinion of these authors, because positive results on screening methods need further
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confirmation steps, chromogenic media may afford advantages in time and costs;
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nonetheless, it is our opinion that choosing media with better performance would be
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advisable. In addition, the recent finding of the new mecA homologue, mecALGA251, also
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known as mecC, has brought the routine testing for mecA as a confirmatory test for MRSA
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ACCEPTED MANUSCRIPT into question. MecC produces a negative result in conventional PCRs for mecA but shows
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phenotypically resistance to beta-lactams (Paterson et al., 2014). Therefore, the use of a good
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method to detect phenotypic methicillin resistance is needed to screen for both mecA and
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mecC positive strains.
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We observed a low occurrence of MRSA in food of animal origin (0.09%): two strains, both
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from bulk tank milk, were resistant to cefoxitin on disc diffusion testing and were found to
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harbour the mecA gene by PCR assays. MRSA prevalence in the bulk tank milk samples was
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0.77% (2/261), previous reports by Haran et al. (2012), Paterson et al. (2012), and
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Kreausukon et al. (2012) reported values ranging between 1.3% and 4.4%. The most
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prevalent spa-type among MRSA isolates from bovine sources is t011 (Haran et al., 2012;
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Paterson et al., 2012; Kreausukon et al., 2012). In our study, the two MRSA strains isolated
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from bulk tank milk harbored spa-type t899 and SCCmec type IV and V, respectively. Spa-
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type t899, like t011 and many others, has been associated with ST938, the LA-MRSA clone
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emerging in Europe (EFSA, 2009; Fessler et al., 2011; Monaco et al., 2013). LA-MRSA has
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been shown to be generally resistant against tetracycline (Spohr et al., 2011) and often
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susceptible to fluoroquinolones (EFSA, 2009), as shown by the two MRSA isolates. Spa-type
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t899 has been reported in pork, chicken, turkey and fowl meat (de Boer et al., 2009; Fessler et
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al., 2011; Boost et al., 2013), in the pork food chain (van Duijkeren et al., 2008; Pan et al.,
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2009; Battisti et al., 2010; Monaco et al., 2013), and in bulk tank milk, but only in Italy
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(Benedetti, et al., 2010). Moreover, the two MRSA strain isolated from bulk tank milk
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harbored the SCCmec type IV and V, as generally expected in LA-MRSA. In our study, none
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of the MRSA isolates was positive for PVL; however, PVL-positive MRSA has been
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detected in bovine milk in Korea, in raw milk in China (Wang et al., 2014), and from bulk
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tank milk samples in the United States (Haran et al., 2012) and in Italy (Benedetti et al.,
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2010).
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ACCEPTED MANUSCRIPT In our survey, none of the S. aureus strains isolated from the wild animal carcasses was
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methicillin resistant. Reports on MRSA infections and carriage in wildlife are scarce
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worldwide. Wardyn et al. (2012) found three samples positive for MRSA (2.6%), two of
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which were positive to the PVL gene, and one PVL-positive MSSA strain from a European
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beaver (Castor fiber). Porrero et al. (2013) reported a 0.37% prevalence of MRSA in nasal
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and skin samples of wild species in Spain.
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5. Conclusions
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No MRSA were found in the S. aureus strains isolated from wild animal carcasses. While this
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finding may be reassuring for the moment, wildlife can carry pathogenic genes (PVL) and
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MRSA (Wardyn et al., 2012; Porrero et al., 2013). Further studies are therefore needed to
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assess the risk among a broader range of game species, the potential transmission to domestic
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animals and, ultimately, humans, and to assess MRSA circulation if we are to understand the
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mechanisms of transmission of MRSA strains in wildlife.
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The low prevalence of MRSA observed in this study suggests that there is a limited risk of
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MRSA transmission to humans via food. However, the detection of LA-MRSA in bulk tank
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milk suggests that healthy animals, or cows with subclinical mastitis in lactation, could play a
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role in the spread of MRSA among animals, workers, and the farm environment. Raw milk
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and raw milk products are listed in the food categories recommended to be collected for
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MRSA monitoring in Europe (EFSA, 2012).
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Since LA-MRSA t899 strains have been reported in bulk tank milk only in Italy (Benedetti et
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al., 2010), wider sampling is needed to determine the spread of this spa-type in Italian milk
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productions. Furthermore, greater attention should be paid to the cattle food chain, which
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may be a possible route of transmission of LA-MRSA to humans through raw dairy products.
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This is especially critical in Italy, where a variety of raw milk cheeses is produced and raw
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milk sold in vending machines is becoming increasingly popular.
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Acknowledgements
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This study was partially funded by the Italian Ministry of Health (IZS PLV/RC/02/07).
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Porrero, M.C., Mentaberre, G., Sanchez, S., Fernandez-Llario, P., Gómez-Barrero, S., Navarro-Gonzalez, N., Serrano, E., Casas-Díaz, E., Marco, I., Fernández-Garayzabal, J.F., Mateos, A., Vidal, D., Lavín, S., Domínguez, L., 2013. Methicillin resistant
297
Staphylococcus aureus (MRSA) carriage in different free-living wild animal species
298
in Spain.Vet. J. 2013;198(1):127-30.
299
Shanehbandi, D., Baradaran, B., Sadigh-Eteghad, S., Zarredar, H., 2014. Occurrence of
300
Methicillin Resistant and Enterotoxigenic Staphylococcus aureus in Traditional
12
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Cheeses in the North West of Iran. ISRN Microbiol. Volume 2014, Article ID
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129580, 5 pages.
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Spohr, M., Rau, J., Friedrich, A., Klittich, G., Fetsch, A., Guerra, B., Hammerl, J.A., Tenhagen, B.A., 2011. Methicillin-resistant Staphylococcus aureus (MRSA) in three
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dairy herds in southwest Germany. Zoonoses Public Health 58, 252-61.
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van Duijkeren, E., Ikawaty, R., Broekhuizen-Stins, M.J., Jansen, M.D., Spalburg, E.C., de
307
Neeling, A.J., Allaart, J.G., van Nes, A., Wagenaar, J.A., Fluit, A.C., 2008.
308
Transmission of methicillin-resistant Staphylococcus aureus between different kinds
309
of pig farms. Vet. Microbiol. 126, 383-9.
Wang, X., Li, G., Xia, X., Yang, B., Xi, M., Meng, J., 2014. Antimicrobial Susceptibility and
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Molecular Typing of Methicillin-Resistant Staphylococcus aureus in Retail Foods in
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Shaanxi, China. Foodborne Pathog Dis. 11(4):281-6.
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Wardyn, S.E., Kauffman, L.K., Smith, T.C., 2012. Methicillin-resistant Staphylococcus aureus in Central Iowa Wildlife. Journal of Wildlife Disease, 48, 4, 1069-1073. J.
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Wildl. Dis.;48(4):1069-73.
318 319 320 321
community-acquired MRSA in Germany. Euro Surveill.;9(1):16-8.
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Witte, W., Cuny, C., Strommenger, B., Braulke, C., Heuck, D., 2004. Emergence of a new
Zhang, K., McClure, J.A., Elsayed, S., Louie, T., Conly, J.M., 2005. Novel Multiplex PCR
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Assay for Characterization and Concomitant Subtyping of Staphylococcal Cassette Chromosome mec Types I to V in Methicillin-Resistant Staphylococcus aureus. J. Clin. Microbiol. 43 (10), 5026-5033.
13
Food matrix
ACCEPTED Samples (%)
Cow’s cheese Raw cow’s milk for vending machine Organs - slaughter activities – no lesions Bovine Equine Wild boar Swine Rabbit Caprine Goat’s and sheep’s milk Bulk tank milk Meat preparations Fresh meat (not poultry)
476 453 363 (342) (6) (6) (3) (3) (3) 283 261 100 76
MANUSCRIPT Food matrix
(22.0) (21.0) (16.8)
Samples (%)
Pasta, rice Meat products Fruit and vegetables Sauces and flavouring Poultry fresh meat Ready to eat – not meat Butter and animal fat Ice cream Egg products Crustaceans and molluscs Fish threads and slices Canned fish Pastry
(1.8) (1.5) (0.7) (0.6) (0.6) (0.5) (0.3) (0.2) (0.2) (0.2) (0.1) (0.1) (0.1)
2162
(100)
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(13.1) (12.1) (4.6) (3.5)
38 32 16 13 12 11 7 5 4 4 3 3 2
Total
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Table 1. Total number and type of foodstuff samples collected and tested for both the enumeration of coagulase positive staphylococci and the search for S. aureus in the period January-December 2008.
Samples positive for S. aureus (%)
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Cow’s cheese Bulk tank milk Raw cow’s milk for vending machine Goat’s and sheep’s milk Organs - slaughter activities – no lesions Bovine Caprine Meat preparations Pasta, rice Fresh meat (not poultry) Meat products Pastry Total
155 107 46 38 9 (7) (2) 6 4 2 2 1 370
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Food matrix
(41.9) (28.9) (12.4) (10.3) (2.4)
(1.6) (1.1) (0.5) (0.5) (0.3) (100)
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Table 2. Total number and type of foodstuff samples positive for S. aureus.
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Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy
Highlights: 1. MRSA occurrence is low: the risk of transmission to humans via food is limited 2. 2 MRSA strains (0.77%), both spa-type t899, were isolated from bulk tank milk 3. MRSA t899, associated with ST398, was detected in bulk tank milk only in Italy 4. The cattle food chain may be a possible route of transmission of LA-MRSA to humans
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5. Wild species had no MRSA but further studies on MRSA spreading are needed
S0740-0020(15)00145-8
DOI:
10.1016/j.fm.2015.07.012
Reference:
YFMIC 2430
To appear in:
Food Microbiology
Received Date: 29 April 2014 Revised Date:
12 May 2015
Accepted Date: 20 July 2015
Please cite this article as: Traversa, A., Gariano, G., Gallina, S., Bianchi, D., Orusa, R., Domenis, L., Cavallerio, P., Fossati, L., Serra, R., Decastelli, L., Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy, Food Microbiology (2015), doi: 10.1016/ j.fm.2015.07.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy A. Traversa a,b, G R. Gariano a,b, S. Gallina a,b, D M. Bianchi a,b, R. Orusa a,c, L. Domenis a,c, P. Cavallerio d, L. Fossati d, R. Serra d, L. Decastelli a,b Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta - IZSPLV
b
Struttura Complessa Controllo Alimenti e Igiene delle Produzioni – Turin, Italy
c
Centro di Referenza Nazionale per le Malattie degli Animali Selvatici – Aosta, Italy
d
AOU San Giovanni Battista, Laboratorio di Microbiologia, Turin, Italy
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Corresponding author: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta – IZSPLV, Struttura Complessa Controllo Alimenti e Igiene delle Produzioni – Turin, Italy. Tel: +39 (0)11 2686233; Fax: +39 (0)11
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2473450. E-mail address: [email protected]. (A. Traversa)
ACCEPTED MANUSCRIPT 1. Introduction
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Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of hospital-
3
associated infections in humans; treatment has become increasingly difficult owing to the
4
development of resistance to many commonly used antimicrobials (EFSA and ECDC, 2012).
5
Methicillin resistance is mediated by the mecA gene, chromosomally located in the
6
staphylococcal cassette chromosome mec (SCCmec), which encodes penicillin-binding
7
protein (PBP) 2a with a low affinity for beta-lactams (EFSA, 2009).
8
In addition to hospital-acquired MRSA infections, community-associated infections caused
9
by MRSA (CA-MRSA) have become a growing public health problem (Witte et al., 2004).
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CA-MRSA clones have evolved outside hospital settings and may carry the gene for Panton-
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Valentine leukocidin (PVL) cytotoxin, a potential virulence factor (EFSA, 2009). New
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MRSA, known as livestock-associated (LA)-MRSA, has been detected in food-producing
13
animals as well, including pigs, cattle, chickens (De Neeling et al., 2007; Lee, 2006), horses,
14
and companion animals (Cuny et al., 2010). LA-MRSA and the new clone ST398 (MRSA
15
lineage multilocus sequence type 398), belonging to the clonal complex (CC)398 may also be
16
harboured by humans, especially where there is occupational contact with affected livestock
17
(EFSA and ECDC, 2014). Food-producing animals and their products are considered a
18
potential source of MRSA infection in humans (ECDC, EFSA and EMEA, 2009).
19
Foods and food chains are an important and potential route of transmission of
20
microorganisms between food-producing animals, including wildlife, and humans and vice
21
versa. Surveillance and monitoring of MRSA in humans and food-producing animals is
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recommended in all European Member States; further work should be performed to
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harmonize the methods for the detection of MRSA as a contaminant of food (ECDC, EFSA
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and EMEA 2009). Data on MRSA in food-producing animals, especially in pig holdings,
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have been reported worldwide, yet the currently available data about MRSA in foods are still
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ACCEPTED MANUSCRIPT few. The occurrence of MRSA in wild animals has been sporadically reported to date
27
(Wardyn et al., 2012; Porrero et al., 2013). Northwest Italy is rich in animal wildlife where
28
many species are hunted as game for human consumption. Wildlife data may be useful to
29
assess MRSA circulation in wild species and the potential route of transmission to domestic
30
animals and, ultimately, humans.
31
The present study was undertaken to investigate the occurrence of methicillin resistance in
32
Staphylococcus aureus strains isolated from food and from wild animal carcasses. The aims
33
were: 1) to assess the level of contamination of MRSA in foods analysed for the detection of
34
S. aureus according to national official control programs and regional monitoring activities;
35
and 2) to evaluate the occurrence of MRSA strains in wild species collected by the National
36
Reference Center for Wild Animal Diseases (Ce.R.M.A.S.); and 3) to characterize MRSA
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isolates by using staphylococcal cassette chromosome mec typing, spa typing, PVL encoding
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gene detection, and antimicrobial susceptibility testing.
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2. Materials and Methods
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2.1 Sampling
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Food. A total of 2162 samples of food were analysed by the Food Control Laboratory of the
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IZS PLV in Turin in 2008 for the enumeration of coagulase-positive staphylococci (CPS) and
44
the detection of S. aureus according to national official monitoring activities and regional
45
agreements. Details of the analyses are given in Table 1.
46
Wildlife. In the period 2003-2009, the carcasses of 242 alpine wild ruminants (Rupicapra
47
rupicapra, Capra ibex, and Capreolus capreolus), 276 foxes (Vulpes vulpes), 134 mustelids
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(Martes foina, Meles meles, Martes martes), and 16 rodents (Marmota marmota, Sciurus
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vulgaris) were autopsied at the Ce.R.M.A.S for establishing the cause of death or the
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examination of lesions in game animals killed by hunters; the submandibular lymph nodes of
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ACCEPTED MANUSCRIPT 697 wild boars (Sus scrofa) were subjected to anatomopathological examination within a
52
monitoring plan for wild animal tuberculosis. All animals came from the northwestern Alps
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(Aosta Valley and North Piedmont). Bacteriological examination of tissue samples of lesion
54
was generally performed using blood agar (Microbiol Diagnostici) and Mc Conkey agar
55
incubated for 24 h at 37° C, aerobically and/or anaerobically depending on the suspect
56
microorganism.
57
2.2 Enumeration of coagulase-positive staphylococci and identification of S. aureus
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Food. For the enumeration of CPS from food samples, accredited methods according to
59
standard microbiological procedures (ISO 6888-2:1999/Amend1: 2003) were used. S. aureus
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isolates were identified using the ID 32 Staph System (bioMérieux, France) or GP-Vitek2
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Cards (bioMérieux). All S. aureus isolates were stored at -20° C in Microbank (Mast
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Diagnostic, Merseyside, UK) until further analysis.
63
Proportions of S. aureus positive samples in each type of food were compared by chi-square
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or Fisher’s exact tests. A p-value < 0.05 was considered statistically significant. Analyses
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were carried out using GraphPad Prism software.
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Wildlife. All S. aureus isolates were confirmed by API STAPH (bioMérieux) and
67
agglutination tests.
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2.3 Phenotypic methicillin resistance detection methods
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Stocked cultures of S. aureus isolates were subcultured onto blood agar plates (Microbiol
70
Diagnostici) and RPF-Baird-Parker agar plates (Biolife italiana). After incubation for 24 h at
71
37° C, each strain was inoculated onto Brilliance MRSA medium (Oxoid) then incubated for
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18 h at 37° C: presumptive MRSA strains showed colonies with denim blue coloration. At
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the same time, each strain was tested for susceptibility to cefoxitin 30 µg using the disc
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diffusion method on Mueller–Hinton agar according to the Clinical Laboratory Standards
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Institute (CLSI M100-S19, 2009) guidelines.
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2.4 SCCmec typing, spa-typing and detection of PVL gene of MRSA
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The DNA of presumptive MRSA strains was extracted using EZ1 (Qiagen, Hilden, Germany)
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and tested for staphylococcal cassette chromosome mec (SCCmec) typing performed by
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multiplex PCR (Zhang et al., 2005). The polymorphic X region of the protein A gene (spa)
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was amplified by PCR using specific primers (www.ridom.de, spa-server). All sequencing
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reactions were carried out using an ABI 3130 genetic analyzer (Applied Biosystems, Foster
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City, CA).
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SCCmec type IV or V strains were further tested by PCR for the PVL encoding gene (Lina et
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al., 1999).
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2.5 Antimicrobial susceptibility test
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Minimal inhibitory concentration (MIC) of a panel of 20 antimicrobials was determined
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against MRSA isolates using Microscan Walk Away (Siemens). The antimicrobial
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breakpoints used for interpretation of susceptibility tests refer to Clinical Laboratory
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Standards Institute (CLSI, 2009) guidelines.
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3. Results
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3.1 Occurrence of S. aureus in food
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The prevalence of S. aureus in food was 17.1% (370/2162). The associations between the
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presence of S. aureus strains and bulk tank milk samples, as well as cow cheese samples,
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were found to be statistically significant (P<0.0001).
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Among the positive samples (Table 2), 42 samples of cheese had CPS >100,000 CFU/g, 2 of
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these, collected during foodborne outbreaks, resulted positive for staphylococcal enterotoxins
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(SEs) on ELFA-VIDAS or ELISA according to the ANSES EU-CRL screening method for
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CPS.
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S. aureus was found in 2.0% of wild animal carcasses and in 3.2% of wild boar lymph nodes.
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In particular, 6.25% (1/16) of rodents samples (Martes foina), 4% (10/242) of wild ruminants
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(8 Rupicapra rupicapra and 2 Capra ibex), 0.8% (1/134) of mustelids (Marmota marmota),
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and 0.4% (1/276) of Vulpes vulpes were positive for S. aureus.
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3.3 Phenotypic methicillin resistance detection methods
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Food. S. aureus strains isolated from food samples were tested for both MRSA-Brilliance
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(Oxoid) growth and susceptibility to cefoxitin 30 µg using the disc agar diffusion method on
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Mueller–Hinton agar: 8 strains grew on MRSA Brilliance with typical colonies, but only 1
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exhibited resistance to cefoxitin (strain 1). One strain, which did not grow on the medium,
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was resistant to cefoxitin on disc diffusion testing (strain 2).
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Wildlife. None of the 35 S. aureus isolates tested by both phenotypic methods grew on Oxoid
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Brilliance MRSA or showed resistance to cefoxitin. As no phenotypic methicillin resistance
114
was reported, these isolates were not tested with molecular methods, in accordance with our
115
protocol.
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3.4 SCCmec typing, spa-typing and detection of PVL gene of MRSA strains
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SCCmec typing and spa-typing of the 9 strains that resulted methicillin resistant according to
118
the results of at least one of the phenotypic methods showed that 2 out of the 9 strains, both
119
from bulk tank milk, carried spa-type t899 and SCCmec type IV (strain 1) and V (strain 2)
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but were negative for the detection of the PVL gene.
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3.5 Antimicrobial susceptibility test
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MRSA isolates were resistant to beta-lactams and tetracycline but were susceptible to
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glycopeptides and linezolid. In addition, strain 1 showed resistance against erythromycin,
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clarithromycin, clindamycin and trimethoprim/sulfamethoxazole, and strain 2 only against
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gentamicin.
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4. Discussion
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In this survey, the occurrence of S. aureus strains in food samples was 17.1% (370/2162). In
129
accordance with national official monitoring programs and regional agreements, the search
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for S. aureus was conducted in foods more likely to be contaminated with CPS and S. aureus.
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The prevalence of S. aureus in bulk tank milk and cow cheese was 41.0% (107/261) and
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32.6% (155/476), respectively; these foodstuffs were observed to be significantly associated
133
(P<0.05) with the presence of S. aureus. The occurrence of this microorganism in dairy
134
products or other foodstuff varies across studies: Haran et al. (2012) reported a 62% S. aureus
135
prevalence in bulk tank milk collected from Minnesota dairy farms, whereas Shanehbandi et
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al. (2014) found a prevalence of S. aureus about 16% in traditional cheese samples.
137
In our survey, MRSA phenotypic prevalence differed depending on whether the Oxoid
138
MRSA Brilliance medium or susceptibility to cefoxitin 30 µg by the agar diffusion method
139
was used: 2.2% versus 0.5% in the tested strains (8/2162 or 0.37% and 2/2162 or 0.09% in
140
the food samples), respectively. Molecular methods confirmed MRSA phenotypic prevalence
141
obtained with the disc agar diffusion method. Oxoid MRSA Brilliance showed high
142
specificity (0.98) and negative predictive value (1.00) but very low sensitivity (0.5) and
143
positive predictive value (0.13). The data reported in other studies are discordant: Lee et al.
144
(2013) found that Brilliance MRSA Agar had high specificity and low sensitivity, whereas
145
Malhotra-Kumar et al. (2010) reported high sensitivity but low specificity.
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In the opinion of these authors, because positive results on screening methods need further
147
confirmation steps, chromogenic media may afford advantages in time and costs;
148
nonetheless, it is our opinion that choosing media with better performance would be
149
advisable. In addition, the recent finding of the new mecA homologue, mecALGA251, also
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known as mecC, has brought the routine testing for mecA as a confirmatory test for MRSA
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ACCEPTED MANUSCRIPT into question. MecC produces a negative result in conventional PCRs for mecA but shows
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phenotypically resistance to beta-lactams (Paterson et al., 2014). Therefore, the use of a good
153
method to detect phenotypic methicillin resistance is needed to screen for both mecA and
154
mecC positive strains.
155
We observed a low occurrence of MRSA in food of animal origin (0.09%): two strains, both
156
from bulk tank milk, were resistant to cefoxitin on disc diffusion testing and were found to
157
harbour the mecA gene by PCR assays. MRSA prevalence in the bulk tank milk samples was
158
0.77% (2/261), previous reports by Haran et al. (2012), Paterson et al. (2012), and
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Kreausukon et al. (2012) reported values ranging between 1.3% and 4.4%. The most
160
prevalent spa-type among MRSA isolates from bovine sources is t011 (Haran et al., 2012;
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Paterson et al., 2012; Kreausukon et al., 2012). In our study, the two MRSA strains isolated
162
from bulk tank milk harbored spa-type t899 and SCCmec type IV and V, respectively. Spa-
163
type t899, like t011 and many others, has been associated with ST938, the LA-MRSA clone
164
emerging in Europe (EFSA, 2009; Fessler et al., 2011; Monaco et al., 2013). LA-MRSA has
165
been shown to be generally resistant against tetracycline (Spohr et al., 2011) and often
166
susceptible to fluoroquinolones (EFSA, 2009), as shown by the two MRSA isolates. Spa-type
167
t899 has been reported in pork, chicken, turkey and fowl meat (de Boer et al., 2009; Fessler et
168
al., 2011; Boost et al., 2013), in the pork food chain (van Duijkeren et al., 2008; Pan et al.,
169
2009; Battisti et al., 2010; Monaco et al., 2013), and in bulk tank milk, but only in Italy
170
(Benedetti, et al., 2010). Moreover, the two MRSA strain isolated from bulk tank milk
171
harbored the SCCmec type IV and V, as generally expected in LA-MRSA. In our study, none
172
of the MRSA isolates was positive for PVL; however, PVL-positive MRSA has been
173
detected in bovine milk in Korea, in raw milk in China (Wang et al., 2014), and from bulk
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tank milk samples in the United States (Haran et al., 2012) and in Italy (Benedetti et al.,
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2010).
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methicillin resistant. Reports on MRSA infections and carriage in wildlife are scarce
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worldwide. Wardyn et al. (2012) found three samples positive for MRSA (2.6%), two of
179
which were positive to the PVL gene, and one PVL-positive MSSA strain from a European
180
beaver (Castor fiber). Porrero et al. (2013) reported a 0.37% prevalence of MRSA in nasal
181
and skin samples of wild species in Spain.
182
5. Conclusions
183
No MRSA were found in the S. aureus strains isolated from wild animal carcasses. While this
184
finding may be reassuring for the moment, wildlife can carry pathogenic genes (PVL) and
185
MRSA (Wardyn et al., 2012; Porrero et al., 2013). Further studies are therefore needed to
186
assess the risk among a broader range of game species, the potential transmission to domestic
187
animals and, ultimately, humans, and to assess MRSA circulation if we are to understand the
188
mechanisms of transmission of MRSA strains in wildlife.
189
The low prevalence of MRSA observed in this study suggests that there is a limited risk of
190
MRSA transmission to humans via food. However, the detection of LA-MRSA in bulk tank
191
milk suggests that healthy animals, or cows with subclinical mastitis in lactation, could play a
192
role in the spread of MRSA among animals, workers, and the farm environment. Raw milk
193
and raw milk products are listed in the food categories recommended to be collected for
194
MRSA monitoring in Europe (EFSA, 2012).
195
Since LA-MRSA t899 strains have been reported in bulk tank milk only in Italy (Benedetti et
196
al., 2010), wider sampling is needed to determine the spread of this spa-type in Italian milk
197
productions. Furthermore, greater attention should be paid to the cattle food chain, which
198
may be a possible route of transmission of LA-MRSA to humans through raw dairy products.
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This is especially critical in Italy, where a variety of raw milk cheeses is produced and raw
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milk sold in vending machines is becoming increasingly popular.
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Acknowledgements
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This study was partially funded by the Italian Ministry of Health (IZS PLV/RC/02/07).
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Food matrix
ACCEPTED Samples (%)
Cow’s cheese Raw cow’s milk for vending machine Organs - slaughter activities – no lesions Bovine Equine Wild boar Swine Rabbit Caprine Goat’s and sheep’s milk Bulk tank milk Meat preparations Fresh meat (not poultry)
476 453 363 (342) (6) (6) (3) (3) (3) 283 261 100 76
MANUSCRIPT Food matrix
(22.0) (21.0) (16.8)
Samples (%)
Pasta, rice Meat products Fruit and vegetables Sauces and flavouring Poultry fresh meat Ready to eat – not meat Butter and animal fat Ice cream Egg products Crustaceans and molluscs Fish threads and slices Canned fish Pastry
(1.8) (1.5) (0.7) (0.6) (0.6) (0.5) (0.3) (0.2) (0.2) (0.2) (0.1) (0.1) (0.1)
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(100)
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(13.1) (12.1) (4.6) (3.5)
38 32 16 13 12 11 7 5 4 4 3 3 2
Total
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Table 1. Total number and type of foodstuff samples collected and tested for both the enumeration of coagulase positive staphylococci and the search for S. aureus in the period January-December 2008.
Samples positive for S. aureus (%)
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Cow’s cheese Bulk tank milk Raw cow’s milk for vending machine Goat’s and sheep’s milk Organs - slaughter activities – no lesions Bovine Caprine Meat preparations Pasta, rice Fresh meat (not poultry) Meat products Pastry Total
155 107 46 38 9 (7) (2) 6 4 2 2 1 370
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Food matrix
(41.9) (28.9) (12.4) (10.3) (2.4)
(1.6) (1.1) (0.5) (0.5) (0.3) (100)
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Table 2. Total number and type of foodstuff samples positive for S. aureus.
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Methicillin resistance in Staphylococcus aureus strains isolated from food and wild animal carcasses in Italy
Highlights: 1. MRSA occurrence is low: the risk of transmission to humans via food is limited 2. 2 MRSA strains (0.77%), both spa-type t899, were isolated from bulk tank milk 3. MRSA t899, associated with ST398, was detected in bulk tank milk only in Italy 4. The cattle food chain may be a possible route of transmission of LA-MRSA to humans
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5. Wild species had no MRSA but further studies on MRSA spreading are needed