Human health risks associated with antimicrobial-resistant enterococci and Staphylococcus aureus on poultry meat

Accepted Manuscript Human health risks associated with antimicrobial-resistant enterococci and Staphylococcus aureus on poultry meat. Valeria Bortolaia, Carmen Espinosa-Gongora, Luca Guardabassi PII:

S1198-743X(15)01029-0

DOI:

10.1016/j.cmi.2015.12.003

Reference:

CMI 461

To appear in:

Clinical Microbiology and Infection

Received Date: 6 October 2015 Revised Date:

1 December 2015

Accepted Date: 1 December 2015

Please cite this article as: Bortolaia V, Espinosa-Gongora C, Guardabassi L, Human health risks associated with antimicrobial-resistant enterococci and Staphylococcus aureus on poultry meat., Clinical Microbiology and Infection (2016), doi: 10.1016/j.cmi.2015.12.003. 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 1

Human health risks associated with antimicrobial-resistant enterococci and

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Staphylococcus aureus on poultry meat.

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Valeria Bortolaia1a, Carmen Espinosa-Gongora1a, Luca Guardabassi1,2*

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The authors contributed equally to the work.

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Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences,

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University of Copenhagen, Stigbøjlen 4, 1870 Frederiksberg C, Denmark

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Kitts, West Indies

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Department of Biomedical Sciences, Ross University School of Veterinary Medicine, St

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Keywords: Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, chicken,

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broiler, turkey, foodborne transmission

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Running title: Antimicrobial resistant enterococci and S. aureus on poultry meat

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*Corresponding author:

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Tel: +1869 4654161 Ext. 401-1329

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Enterococci and staphylococci are frequent contaminants on poultry meat. Enterococcus

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faecalis, Enterococcus faecium and Staphylococcus aureus are also well known aetiological

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agents of a wide variety of infections resulting in major healthcare costs. This review

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provides an overview of the human health risks associated to the occurrence of these

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opportunistic human pathogens on poultry meat with particular focus on the risk of foodborne

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transmission of antimicrobial resistance. In the lack of conclusive evidence of transmission,

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this risk was inferred using data from scientific articles and national reports on prevalence,

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bacterial load, antimicrobial resistance and clonal distribution of these three species on

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poultry meat. The risks associated to ingestion of antimicrobial-resistant enterococci of

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poultry origin comprise horizontal transfer of resistance genes and transmission of multidrug-

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resistant E. faecalis lineages such as sequence type ST16. E. faecium lineages occurring in

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poultry meat products are distantly related from those causing hospital-acquired infections

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but may act as donors of quinupristin/dalfopristin resistance and other resistance determinants

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of clinical interest to the human gut microbiota.

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Ingestion of poultry meat contaminated with S. aureus may lead to food poisoning. However,

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antimicrobial resistance in the toxin-producing strains does not have clinical implications

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since food poisoning is not managed by antimicrobial therapy. Recently methicillin-resistant

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S. aureus (MRSA) of livestock origin has been reported on poultry meat. In theory handling

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or ingestion of contaminated meat is a potential risk factor for colonization by MRSA.

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However, this risk is presently regarded as negligible by public health authorities.

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ACCEPTED MANUSCRIPT Introduction

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Enterococci and staphylococci are frequent contaminants on poultry meat and well

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established opportunistic pathogens in human medicine. Enterococcus faecalis, Enterococcus

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faecium and Staphylococcus aureus are leading causes of human infections that may affect

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virtually any body site and range in severity from uncomplicated wound infections to fatal

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endocarditis [1, 2]. Taken together, E. faecalis and E. faecium are ranked as the third cause of

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bacteraemia in Europe and in America accounting for approximately 11-13% of all

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bacteraemia cases [3, 4]. S. aureus is the most common cause of skin and soft tissue

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infections (SSTI) and nosocomial bacteraemia in America and Europe [2]. All these Gram-

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positive cocci are also normal commensals in poultry and other domestic animals. Strains of

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animal origin may be transmitted to humans through direct contact with animals or via

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exposure to contaminated food. In industrialized countries, the risk of zoonotic transmission

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by direct contact with poultry is limited to farmers, veterinarians and slaughterhouse workers,

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which represent a small fraction of the general human population. Foodborne transmission

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may affect a larger part of the population through consumption and handling of contaminated

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poultry meat and other food items that may be cross-contaminated in the kitchen. The

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consequences of exposure to contaminated poultry meat differ between enterococci and S.

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aureus. Enterococci can colonise the consumer’s gut, mainly the colon, where they represent

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a small proportion (less than 1%) of the culturable microbiota [5], and subsequently may act

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as opportunistic pathogens and donors of antimicrobial resistance determinants to the

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indigenous microbiota. Ingestion of S. aureus strains may lead to food poisoning if the strains

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present on meat have the ability to produce enterotoxins [6]. In addition, it has been

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hypothesized that handling or consumption of poultry meat may result in colonisation of skin

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and mucosae (e.g. nasal and oral mucosae) [7, 8], which is considered an important risk

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factor for S. aureus infections [9]. Foodborne transmission of livestock-associated

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ACCEPTED MANUSCRIPT methicillin-resistant S. aureus (MRSA) is of particular concern in view of the recent

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emergence of these multidrug-resistant bacteria in meat products, including poultry meat [10,

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11].

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Zoonotic transmission of enterococci and S. aureus from poultry has been studied almost

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exclusively in relation to antimicrobial resistance, primarily vancomycin and methicillin

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resistance, respectively. Experimental data on colonization of the human gut by resistant

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strains of poultry origin are scarce and limited to enterococci. Hence frequency and load of

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these bacteria on poultry meat and comparative analysis of antimicrobial resistance patterns

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and clonal types among poultry and human clinical isolates are the only scientific elements

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available to assess the risk of foodborne transmission.

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The present review provides state-of-the-art knowledge of the human health risks associated

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to the occurrence of these opportunistic pathogens on poultry meat with particular focus on

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the contribution of antimicrobial-resistant strains of poultry origin to human infections.

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PubMed was searched systematically for articles published in the last five years on

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prevalence, antimicrobial resistance and host specificity of enterococci and S. aureus on

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poultry meat as well as for older articles documenting health risks derived from ingestion of

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strains of poultry origin. National surveillance reports were used to complement such

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information. Epidemiological links between poultry meat and human infections were inferred

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based on similarities in the patterns of antimicrobial resistance and clonal distribution

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between poultry and human clinical isolates.

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Prevalence and loads of enterococci and S. aureus on poultry meat

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Poultry meat may get contaminated with E. faecalis, E. faecium and S. aureus at slaughter or

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through food handling. For enterococci, it is assumed that most bacteria present on meat

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derive from animals because gut bacteria from food handlers should not reach food products

ACCEPTED MANUSCRIPT by following good hygiene standards. It should be noted that enterococci are normal

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commensals in the gut of poultry and contamination of carcasses by faecal bacteria is more

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common in poultry than in other food-producing animal species, mainly due to lower

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hygienic standards in poultry slaughtering compared to pig and cattle slaughtering.

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Consequently, the prevalence of poultry meat samples contaminated by enterococci is

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generally high, even though it varies greatly depending on the sample types and the isolation

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methods employed by different studies as well as by the hygiene conditions of the

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slaughterhouse(s) under study (Table 1). Based on the available scientific literature, the

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prevalence of raw poultry meat products contaminated with E. faecalis and E. faecium may

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reach 96% (Table 1). The load of enterococci on poultry meat ranges from 101 to 103 colony

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forming units (CFU) per gram of raw chicken or turkey meat [12, 13].

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S. aureus in retail poultry meat products can have either animal or human origin as a

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consequence of possible contamination by meat handlers. Both S. aureus and MRSA have

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been shown to be more frequent in turkey meat (19.4-77% and 32-50% respectively) than in

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chicken meat (17.8-68% and 0.3-25% respectively) (Table 2) [10, 11, 14-30]. This reflects

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the higher prevalence in turkey at the farm level [16, 19]. The frequency of S. aureus/MRSA

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isolation from poultry meat is generally similar to that of pork but higher than that in beef

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[10, 21-23, 26]. Based on the few data available in the scientific literature, the load of S.

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aureus in poultry raw meat is generally below 102 CFU/g [31-33] with the exception of

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minced meat, where S. aureus concentrations as high as 104 CFU/g have been reported in one

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study [33]. In regard to the load of MRSA, a Canadian study estimated very low level of

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contamination (10-30 CFU/g) on raw chicken meat [28]. Experimental infection of healthy

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and traumatized skin was successfully reproduced in 50% of skin sites (ID50) using an

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inoculum of 103 and 102 CFU/cm2, respectively, although occlusion was always necessary

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[34]. Based on these data MRSA loads on raw chicken meat appear to be too low for causing

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infection by contact with healthy intact skin.

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Antimicrobial resistance in enterococci and S. aureus isolated from poultry meat

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Enterococci are intrinsically resistant to various antimicrobial classes and there are

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remarkable differences in the patterns and to a lesser extend in the genetic bases of acquired

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resistance between E. faecium and E. faecalis [1]. A wide variety of resistance genes has been

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detected in S. aureus of animal origin conferring resistance to virtually all antimicrobials

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classes used in veterinary medicine [35]. Data on antimicrobial resistance in enterococci

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isolated from meat products are provided by numerous studies [36-45] and surveillance

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programmes [30, 46-50] that have employed these microorganisms as indicators of

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antimicrobial resistance. On the contrary, data on antimicrobial resistance in S. aureus of

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meat origin are scarce and largely biased to MRSA. Prevalence of antimicrobial resistance

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changes over time and varies depending on host species and country of origin (Figure 1) [51],

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most likely as a consequence of differences in antimicrobial usage.

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At present, the resistance phenotypes of clinical relevance that may be linked to poultry meat

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comprise resistance to ampicillin, gentamicin, quinupristin-dalfopristin and vancomycin in E.

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faecium and resistance to gentamicin in E. faecalis. Resistance to ampicillin and vancomycin

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in E. faecalis and resistance to other clinically relevant drugs such as linezolid, daptomycin

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and tigecycline in both species are rare or even not detected among poultry isolates [51]. On

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the contrary, enterococci of poultry origin often exhibit resistance to tetracyclines but such

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phenotype has limited importance from a human clinical perspective [51]. Methicillin

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resistance is by far the most important resistance phenotype in S. aureus isolated from poultry

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meat in consideration of the importance of β-lactams in the therapy of staphylococcal

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infections and of the emergence of livestock-associated MRSA in poultry farming [2, 16, 20].

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ACCEPTED MANUSCRIPT Other antimicrobial classes used in the management of S. aureus infections in clinical

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practice and to which resistance is observed among poultry isolates include fluoroquinolones,

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aminoglycosides and lincosamides. Even though tetracyclines, macrolides and trimethoprim-

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sulfamethoxazole are rarely used for management of staphylococcal infections, these

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antimicrobial agents are used in poultry production and resistance can be used as marker to

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track zoonotic transmission [51, 52].

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Poultry reservoirs of antimicrobial-resistant enterococci and S. aureus may be identified by

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comparing patterns of antimicrobial resistance in isolates from poultry meat and diseased

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humans within defined geographical regions. National surveillance programmes represent an

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optimal source of data representative of large populations and comparable across countries

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and over time. This explains why the data on occurrence of antimicrobial resistance in

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enterococci presented in this review are largely based on national surveillance reports

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including data for both human and poultry isolates (Figure 1). Ampicillin resistance is

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relatively common in E. faecium compared to E. faecalis. The prevalence of ampicillin-

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resistant E. faecium (AREF) in poultry meat products varies significantly depending on

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geographical region and poultry species (Figure 1A). For example, in Denmark AREF

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accounts for 3% and 16% of E. faecium isolates from domestic and imported broiler meat,

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respectively [50], whereas in the USA they represent 10% and 54% of the isolates from

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chicken and turkey meat, respectively [48].

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Ampicillin resistance occurs at high levels (> 90%) in human clinical isolates independent of

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geographical origin (Figure 1), which is an indirect indication that poultry meat does not

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contribute significantly to occurrence of ampicillin resistance in human pathogenic E.

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faecium. This is further supported by the population genetics of human pathogenic AREF that

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are to a certain degree distinct from poultry strains (see the next section and references

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therein) and by the fact that ampicillin resistance mainly spreads in E. faecium by vertical

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ACCEPTED MANUSCRIPT transmission of chromosomal mutations whereas conjugative transfer of ampicillin resistance

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has been reported sporadically to date [53, 54]. Thus the risk that AREF possibly ingested via

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food may transfer ampicillin resistance determinants horizontally to human pathogenic E.

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faecium is very limited based on current knowledge [1].

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The patterns of gentamicin resistance in E. faecium also vary significantly between countries.

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In Denmark, high level gentamicin resistance (HLGR) is absent in poultry meat isolates

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despite being high (72%) among clinical isolates (Figure 1A.a), while in the USA HLGR

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occurs less frequently (13%) among clinical isolates but more frequently among poultry meat

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isolates (7% and 8% in broiler and turkey meat, respectively) (Figure 1A). Similar patterns

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are also observed for HLGR in E. faecalis (Figure 1B). These data suggest that the potential

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risk of transfer of HLGR from poultry to human enterococci is higher in the USA than in

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Denmark, although comparative studies of the mobile genetic elements (MGEs) encoding

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gentamicin resistance in poultry and clinical strains are needed to assess this zoonotic risk.

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Similarly, the patterns of vancomycin-resistant E. faecium (VREF) appear to be unconnected

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between humans and poultry within defined geographical areas. In Denmark, prevalence of

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VREF has never been higher than 4.5% among clinical isolates, while it ranged from 80% in

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1995 to undetectable levels in 2013 in E. faecium isolated from poultry and poultry meat

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[50]. On the contrary, VREF is widespread in hospitals in the USA (up to 76% of clinical

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isolates), while it is virtually absent among isolates from poultry [48] (Figure 1A.e). These

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prevalence data clearly mirror the patterns of glycopeptide use in human medicine in the two

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countries since vancomycin is rarely used in Denmark while it has been extensively used

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since two decades in the USA [1, 50]. Transfer of vancomycin resistance from poultry to

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clinical enterococci has been hypothesized mainly for the vanA type since other resistance

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determinants such as vanB and vanN have been detected in poultry meat isolates only

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sporadically [44, 55]. This hypothesis was supported by finding of a poultry-specific

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ACCEPTED MANUSCRIPT nucleotide polymorphism in the vanX allele (G at position 8234) in the vanA-carrying

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transposable element (Tn1546)in clinical isolates [56, 57]. However, this zoonotic risk has

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diminished considerably after the ban of avoparcin use and the consequent decrease of VREF

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in poultry flocks [58], even though a high frequency (50%) of VREF-positive broilers flocks

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at slaughter has been reported in Denmark 15 years after the ban of avoparcin [59].

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Quinupristin/dalfopristin resistance occurs at variable frequency (28-73%) among E. faecium

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isolates from poultry meat both in Europe and in the USA (Figure 1A), suggesting that it is

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not directly linked to use of the quinuprisitin/dalfopristin analogue virginiamycin, which was

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discontinued in European poultry production in 1999 but is still used in the USA. Co-

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selection of quinopristin/dalfopristin-resistant strains in poultry may be enhanced by genetic

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linkages to genes conferring resistance to other antimicrobial classes (e.g. macrolides) [60].

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Information on occurrence of quinopristin/dalfopristin resistance in human isolates is limited.

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Different authors suggested that poultry may represent a reservoir of this resistance, but the

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hypothesis has not been corroborated by molecular epidemiology data on strains of poultry

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and clinical origin [61, 62].

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Also tetracycline resistance occurs at variable frequency in enterococci isolates from poultry

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meat of different geographical origin (10-75% in E. faecium and 35-87% in E. faecalis) but in

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this case there is a plausible link between occurrence of tetracycline resistance and the

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widespread use of tetracyclines in poultry production [30, 46-50]. Information on tetracycline

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resistance in clinical isolates is generally unavailable since it is not relevant from a clinical

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perspective. Nevertheless, occurrence of tetracycline resistance genes may play a role in co-

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selection of genes conferring resistance to clinically relevant antimicrobials as hypothesized

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for glycopeptides [63].

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The few data available on antimicrobial resistance in S. aureus isolates from poultry originate

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from research articles with limited numbers of isolates and the antimicrobials tested often do

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ACCEPTED MANUSCRIPT not correspond with those tested for human clinical isolates. An extensive dataset including S.

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aureus isolates from poultry meat and human infections in the USA indicates significantly

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higher prevalence of resistance to aminoglycosides, fluoroquinolones, macrolides,

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quinupristin-dalfopristin and trimethoprim-sulfamethoxazole among human clinical isolates

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and higher prevalence of tetracycline resistance among poultry meat isolates (Figure 1C) [21-

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23, 29, 64, 65]. High prevalence of tetracycline resistance (as high as 100%) has also been

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reported in poultry meat isolates in Germany [66], Poland [17], Hong Kong [26] and Korea

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[25]. Overall these data suggest that poultry isolates are less often resistant to antimicrobials

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with the exception of tetracyclines, an antimicrobial class that is widely used in production of

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poultry and other livestock but has limited importance in the management of human S. aureus

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infections. The recent emergence of MRSA in poultry meat is a reason for concern due to the

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importance of methicillin resistance in clinical settings [67]. However, there is no evidence

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that poultry meat may act as a source of MRSA colonization or invasive infection in humans

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[7]. Furthermore, methicillin resistance and other resistance phenotypes of clinical relevance

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have no implications for S. aureus food poisoning since this gastrointestinal illness is not

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managed by antimicrobial therapy [67]. Altogether the human health risks associated with the

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presence of antimicrobial-resistant S. aureus on poultry meat appear to be limited on the basis

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of the current scientific literature.

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Host-specificity of enterococcal and S. aureus lineages on poultry meat

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Zoonotic transmission of enterococci from poultry has been studied almost exclusively in

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relation to antimicrobial resistance and in particular vancomycin resistance. Antimicrobial-

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resistant enterococci of poultry origin ingested via food have been shown both to colonise the

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digestive tract of healthy humans for a variable time up to 14 days after ingestion and to

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exchange antimicrobial resistance genes with the indigenous microbiota [68, 69]. It is

ACCEPTED MANUSCRIPT unknown to what extent these events happen in non-experimental conditions and contribute

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to human infections. Recent studies on population structure of E. faecalis and E. faecium of

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human and animal origins provide useful information to limit this knowledge gap.

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Specific E. faecalis lineages such as clonal complex CC2, CC16 and CC87 (as defined by

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multilocus sequence typing, MLST) are enriched among hospital-derived strains, which is

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largely attributable to acquisition of virulence factors and antimicrobial resistance genes via

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horizontal gene transfer [70]. However, in the E. faecalis population structure there is no

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categorical evolutionary distinction between clinical, commensal and animal strains, as

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shown by Bayesian-based modelling of population structure (BAPS) [71]. This implies that

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E. faecalis occurring on poultry meat, and especially multidrug-resistant strains belonging to

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sequence type ST16, may retain a zoonotic potential mainly linked to the lack of host

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specificity. This generalist lifestyle may allow foodborne strains to colonise the human gut

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after ingestion of contaminated food and cause infection if pathogenic conditions arise.

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Differently from E. faecalis, the majority of E. faecium infections in humans is caused by

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strains clustering in clade A1, as defined by single nucleotide polymorphism (SNP)-based

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phylogenetic analysis of WGS data from strains collected across Europe and the USA. Clade

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A1 strains, which include strains belonging to the hospital-associated lineages 17

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(comprising, among others, ST16 and ST17), 18 (ST18) and 78 (ST78 and ST192) differ

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considerably from human commensal isolates and, to a smaller extent, from animal isolates

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[72]. In particular, ST78 isolates have putative evolutionary origin in common with pets

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(dogs and cats) and poultry isolates, and diversified mainly through recombination and

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acquisition or loss of MGEs, which eventually led to adaptation to different ecological niches

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[71-75]. Although this ecological distinction is not absolute, it appears that the main zoonotic

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risk linked to E. faecium isolates is represented by transfer of MGEs harbouring antimicrobial

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resistance genes.

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ACCEPTED MANUSCRIPT Whereas living poultry is colonized by typically poultry-associated S. aureus (e.g. ST5, spa

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types t002, t306 and t13620) [17, 18, 76] or livestock-associated MRSA lineages (e.g. ST9

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and ST398) [15, 18-20], strains isolated from poultry meat comprise a combination of

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animal- and human-associated lineages [11, 17, 20, 23, 27, 77] (Figure 2). These data suggest

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persistence of poultry lineages through the meat production chain [20] but most importantly,

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provide a strong indication that slaughtering and meat handling represent critical points for

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meat contamination with strains of human origin [17, 21, 28]. Frequent S. aureus and MRSA

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lineages in poultry such as ST5 (CC5) and ST398 (CC398) are also quite common among

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human clinical isolates (Figure 2) [78, 79]. It has been proposed that the S. aureus ST5 sub-

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lineage associated to poultry has evolved by a single human-to-poultry host jump [80], as

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previously hypothesized for livestock-associated MRSA ST398 in pigs [81]. However,

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poultry ST5 isolates can be discerned from human clinical isolates by highly discriminatory

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typing methods such as single nucleotide polymorphysm (SNP), revealing a high degree of

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adaptation [80]. The role of poultry as a source of MRSA CC398 human infections cannot be

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assessed as it is not possible to discern between strains originating from poultry and other

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animal species. However, the overall impact of this livestock-associated lineage appears to be

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limited, especially in countries with high MRSA prevalence in the human population [82].

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Other MRSA lineages that are possibly epidemiologically linked to poultry include ST239

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(CC8), which has been reported among healthy chickens in Belgium [16] and is a worldwide

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disseminated hospital-acquired MRSA [79] but to the best of our knowledge has never been

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reported on poultry meat. MRSA lineages that have been reported sporadically on poultry

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meat products but not in live poultry such as PVL-positive ST8 (USA300) and ST45 are well

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known human-adapted lineages and their occurrence on chicken meat [11, 20, 23] is most

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likely attributable to contamination.

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ACCEPTED MANUSCRIPT Transmission of S. aureus from livestock is strongly associated to direct contact to living

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animals [15] and secondary human-to-human propagation from farm settings to the

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community has been considered limited until recently, when proportions of 11 and 38% of

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MRSA CC398 colonized people report no contact with pigs, veal calves or broilers [50, 83].

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The majority of these cases with unknown CC398 origin are reported in areas of high

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livestock production [84]. It has been suggested that MRSA ST398 cases in people without

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contact to livestock could be due to meat handling [85] and consumption of poultry has been

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identified as a risk factor for MRSA carriage [86]. However, the risk of MRSA colonization

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via exposure to contaminated meat was estimated to be low for professional meat handlers in

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Europe and even lower for the general population [87], although the risk appears higher in

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other parts of the world [88]. In countries like Denmark and The Netherlands, where the

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population is highly concentrated in a few urban areas, the vast majority of MRSA ST398

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cases occur in rural areas with high pig population densities [89, 90]. The low frequency of

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infections in urban areas provides indirect evidence that food is not an important source for

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transmission of livestock-associated MRSA. As concluded by Wendlandt et al. [67], the

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occurrence of MRSA in meat products does not equate to MRSA being considered a

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foodborne pathogen. Even though colonization by ingestion cannot be neglected [28],

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considering the low loads of both S. aureus and MRSA on contaminated poultry meat, the

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risk is likely extremely low if meat is consumed after proper cooking. Accordingly, the

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European Food Safety Authority (EFSA) reached the conclusion that the the risk of infection

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to slaughterhouse workers and persons handling meat appears to be low based on the

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published literature [91].

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Conclusions

ACCEPTED MANUSCRIPT Although chicken and to a greater extent turkey meat are often contaminated with E. faecalis,

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E. faecium and S. aureus, the bacterial loads are generally low and the risk that humans may

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become colonized after ingestion or handling of contaminated poultry meat can be prevented

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by proper cooking and kitchen hygiene. Foodborne colonization of the human gut is more

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plausible for enterococci than for S. aureus but the risk that multidrug-resistant enterococci of

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poultry origin may cause infections concerns mainly E. faecalis. The magnitude of this risk

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remains unknown and appears to be limited to specific lineages such as ST16. Colonization

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by MRSA and other multidrug-resistant S. aureus after ingestion of contaminated meat is not

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supported by scientific evidence. Even though in theory meat handling is a possible

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transmission route and there is still a substantial uncertainty with respect to the prevalence of

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infections caused by MRSA outside the hospital, epidemiological data from countries where

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MRSA is notifiable and more information is available on the MRSA types causing

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community-acquired infections indicate that this risk is low for the general population [92].

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Transmission of antimicrobial resistance by horizontal gene transfer represents the main risk

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posed by resistant enterococci on poultry meat. Transfer of gentamicin resistance in E.

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faecium and E. faecalis and of quinupristin/dalfopristin resistance in E. faecium are the major

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potential risks with remarkable differences between geographical regions. Antimicrobial

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resistance is of limited importance in S. aureus strains causing food poisoning as treatment

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does not involve antimicrobials. Moreover, there is no evidence supporting a flow of

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resistance genes of clinical relevance from poultry to human S. aureus.

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Current knowledge on the impact of foodborne E. faecium, E. faecalis and S. aureus on

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poultry meat is limited and often based on indirect evidence. Data on the minimum dose of

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resistant strains of poultry origin required to colonize the human gut and a deeper insight into

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strain and host factors that mitigate for or against colonization and transfer of antimicrobial

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ACCEPTED MANUSCRIPT 336

resistance to the commensal microbiota are needed to assess the actual health risks to

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consumers.

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Transparency declaration

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The authors declare no conflict of interest.

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Authorship/contribution

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VB and CEG collected relevant literature on enterococci and S. aureus, respectively. LG

344

coordinated the study. All authors contributed to critical discussion of the available literature

345

and writing of the manuscript.

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ACCEPTED MANUSCRIPT Figure legends

633

Figure 1. Prevalence of antimicrobial resistance in E. faecium (A), E. faecalis (B) and S.

634

aureus (C) isolated from poultry meat and human invasive infections within defined

635

geographical areas. Figure 1A-B: data were retrieved from surveillance reports [46-48, 50] if

636

available; otherwise data from EFSA (for meat samples) [30] and EARS-net/ResistanceMap

637

(for invasive infections) [49, 65] were used. Figure 1C: data were retrieved from [21-23, 29,

638

64, 65]. AMP: ampicillin; BSB: β-lactamase-susceptible β-lactam; BRB: β-lactamase-

639

resistant β-lactam; DAP: daptomycin; FQ: fluoroquinolones; GEN: gentamicin; KAN:

640

kanamycin; LZD: linezolid; MLS: macrolide/lincosamide/streptogramin; Q/D:

641

quinupristin/dalfopristin; RIF: rifampin; TET: tetracycline; TMP/SMX: trimethoprim-

642

sulfamethoxazole; VAN, vancomycin. Numbers in brackets represent the total number of

643

isolates tested. *Prevalence of Q/D resistance in Danish E. faecium isolates is 54% according

644

to the EFSA report in which a cut-off value of > 1 µg/ml is used.

M AN U

SC

RI PT

632

TE D

645

Figure 2. Proportions of S. aureus lineages in live poultry and poultry raw meat (modified

647

from 10, 11, 15-28, 77).

AC C

EP

646

ACCEPTED MANUSCRIPT Table 1. Prevalence (%) of E. faecalis and E. faecium in raw poultry meat as determined by selective enrichment (SE) and direct plating (DP) on selective media. E. faecalis

E. faecium

No. of samples

Meat type

Greece

0

0

19

C

Greece

9

12

300

C

Italy

13

11

238

C/T

Tunisia

63

33

51

C/T

Canada

>94

4

206

Canada

>94

0

91

USA

42

44

USA

85

10

USA

nd

30

Colombia

81

13

Denmark

48

Denmark

74

Sweden

78

Method Reference (36)

RI PT

DP

(37)

DP

(38)

DPa

(39)

SC

DP

SE

(40)

T

SE

(40)

C

SE

(48)

478

T

SE

(48)

343

C

SE

(41)

200

C

SE

(42)

TE D

M AN U

C

480

96

150

Cb

SE

(50)

81

167

Cc

SE

(50)

10

100

C

SE

(46)

EP

Country

nd, not determined; C, chicken; T, turkey; C/T, chicken and turkey. Direct plating preceded by culture in non-selective broth

b

Broiler meat produced in Denmark.

c

Broiler meat imported to Denmark.

AC C

a

ACCEPTED MANUSCRIPT Table 2. Prevalence (%) of S. aureus and methicillin-resistant S. aureus (MRSA) in raw poultry meat as determined by selective enrichment (SE) and direct plating (DP) on selective media. S. aureus MRSA

Meat type

No. of isolates

Method

Reference

SE

(28)

Canada

1

C

250

USA

0

C

222

DP

(29)

SE

(22)

SE

(22)

57

nd

(23)

76

nd

(23)

18

0

C

45

USA

19

0

T

36

USA

25

2

T

USA

25

4

C

USA

41

0

USA

77

4

35

M AN U

The Netherlands

C

46

SE

(21)

T

26

SE

(21)

Ca

520

SE

(10)

Ta

116

SE

(10)

TE D

16

Germany Germany

24

C

443

SE

(30)

25

Ca

24

SE

(24)

32

T

460

SE

(20)

50

Ta

22

SE

(24)

Denmark

EP

Germany

SC

USA

The Netherlands

RI PT

Country

0

Cb

121

SE

(11)

Denmark

18

Cc

193

SE

(11)

68

1

C

125

SE

(17)

41

2

C

264

SE

(27)

18

1

C

209

SE

(14)

7

Ca

255

SE

(26)

0,5

C

913

nd

(25)

Poland China

AC C

Germany

Pakistan Hong Kong Korea

43

ACCEPTED MANUSCRIPT nd, not determined; C, chicken; T, turkey. including imported meat.

b

Broiler meat produced in Denmark.

c

Broiler meat imported to Denmark.

AC C

EP

TE D

M AN U

SC

RI PT

a

100 90 80 70 60 50 40 30 20 10 0

Figure 1A

RI PT

b) Sweden

0

0

0 0,7

AMP (10/548)

GEN (10/351)

VAN (10/456)

100 90 80 70 60 50 40 30 20 10 0

d) Slovenia

M AN U 0 0 GEN (72/211)

Q/D (72/0)

e) United States of America

VAN (72/436)

AC C

Prevalence 8%) of resistance

VAN (96/81/715)

c) The Netherlands

AMP (72/436) 100 90 80 70 60 50 40 30 20 10 0

Q/D (96/81/0)

0 0

TE D

Prevalence (%) of resistance

AMP GEN (96/81/714) (96/81/41)

*

100 90 80 70 60 50 40 30 20 10 0

SC

0

Prevalence (%) of resistance

a) Denmark

Prevalence (%) of resistance

100 90 80 70 60 50 40 30 20 10 0

EP

Prevalence (%) of resistance

ACCEPTED MANUSCRIPT

0 0 AMP GEN Q/D VAN (213/48/710) (213/48/640) (213/48/0) (213/48/733)

0 AMP (16/115)

GEN (16/113)

0 Q/D (16/0)

0 VAN (16/115)

0,2

0 GEN (224/279)

e) Hungary

0 1,7 AMP (35/658)

0

0

0

AMP (78/1036)

60 55 50 45 40 35 30 25 20 15 10 5 0

VAN (224/481)

0 GEN (35/659)

RI PT

VAN (48/74/517)

AC C

Prevalence (%) of resistance

GEN (48/74/60)

0 0,2

c) The Netherlands

AMP (224/529)

60 55 50 45 40 35 30 25 20 15 10 5 0

0

VAN (35/657)

60 55 50 45 40 35 30 25 20 15 10 5 0

Figure 1B

b) Sweden

SC

60 55 50 45 40 35 30 25 20 15 10 5 0

0

60 55 50 45 40 35 30 25 20 15 10 5 0

1

0

0

GEN (78/673)

VAN (78/894)

d) Slovenia

M AN U

Prevalence (%) of resistance

AMP (48/74/586)

0

Prevalence (%) of resistance

0 0,7

TE D

0

Prevalence 8%) of resistance

a) Denmark

Prevalence (%) of resistance

60 55 50 45 40 35 30 25 20 15 10 5 0

EP

Prevalence (%) of resistance

ACCEPTED MANUSCRIPT

0

0

0

0

AMP (77/120)

GEN (77/119)

VAN (77/120)

f) United States of America

0

0

1

AMP (202/407/1110)

0 GEN (202/407/1020)

0

VAN (202/407/1121)

ACCEPTED MANUSCRIPT 100

Figure 1C

United States of America

90

RI PT

70 60 50

SC

40 30

M AN U

20 10 0 1

0 0

0

0

1 0 0

0 0

0 0 0

0 0 0

Broiler meat – imported (only applicable to Denmark)

EP

Broiler meat (in Denmark, domestically produced)

TE D

BSB BRB MLS GEN/KAN TET FQ TMP/SMX Q/D VAN RIF LZD DAP (61/36/1257) (61/36/4581) (61/36/1634) (56/35/924) (36/36/451) (61/36/3595) (31/35/1660) (31/35/662) (56/35/4684) (31/35/3771) (31/35/3719) (31/35/2848)

AC C

Prevalence (%) of resistance

80

Turkey meat

Human infections

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT