Antimicrobial susceptibility and oxymino-β-lactam resistance mechanisms in Salmonella enterica and Escherichia coli isolates from different animal sources

Accepted Manuscript Antimicrobial susceptibility and oxymino-β-lactam resistance mechanisms in Salmonella enterica and Escherichia coli isolates from different animal sources Lurdes Clemente, Vera Manageiro, Daniela Jones-Dias, Ivone Correia, Patricia Themudo, Teresa Albuquerque, Margarida Geraldes, Filipa Matos, Cláudia Almendra, Eugénia Ferreira, Manuela Caniça PII:

S0923-2508(15)00097-2

DOI:

10.1016/j.resmic.2015.05.007

Reference:

RESMIC 3413

To appear in:

Research in Microbiology

Received Date: 1 December 2014 Revised Date:

15 May 2015

Accepted Date: 22 May 2015

Please cite this article as: L. Clemente, V. Manageiro, D. Jones-Dias, I. Correia, P. Themudo, T. Albuquerque, M. Geraldes, F. Matos, C. Almendra, E. Ferreira, M. Caniça, Antimicrobial susceptibility and oxymino-β-lactam resistance mechanisms in Salmonella enterica and Escherichia coli isolates from different animal sources, Research in Microbiologoy (2015), doi: 10.1016/j.resmic.2015.05.007. 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.

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ACCEPTED MANUSCRIPT

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Lurdes Clemente a, Vera Manageiro b,c, Daniela Jones-Dias b,c, Ivone Correia a, Patricia Themudo a, Teresa Albuquerque a, Margarida Geraldes d, Filipa Matos d, Cláudia Almendra d, Eugénia Ferreira b,c, Manuela Caniça b,c* a

[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] “*Correspondence and reprints” [email protected]

Abstract

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INIAV – National Institute for Agrarian and Veterinary Research. Microbiology and Mycology Laboratory. Estrada de Benfica, 701, 1549-011, Lisbon, Portugal b National Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections, Department of Infectious Diseases, NIH, National Institute of Health Dr. Ricardo Jorge. Av. Padre Cruz, 1649-016 Lisbon, Portugal c Center for the Studies of Animal Science, Institute of Agrarian and Agri-Food Sciences and Technologies, University of Oporto, Oporto, Portugal d INIAV – National Institute for Agrarian and Veterinary Research. Microbiology and Mycology Laboratory. Rua dos Lagidos, 4485-655, Vairão, Portugal

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Antimicrobial susceptibility and oxymino-β-lactam resistance mechanisms in Salmonella enterica and Escherichia coli isolates from different animal sources

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The impact of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC

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β-lactamases (PMAβs) of animal origin has been a public health concern. In this study, 562

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Salmonella enterica and 598 Escherichia coli isolates recovered from different animal species

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and food products were tested for antimicrobial resistance. Detection of ESBL-, PMAβ-,

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plasmid-mediated quinolone resistance (PMQR)-encoding genes and integrons was performed

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in isolates showing non-wild-type phenotypes.

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Susceptibility profiles of Salmonella spp. isolates differed according to serotype and

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origin of the isolates. The occurrence of cefotaxime non-wild-type isolates was higher in pets

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than in other groups. In nine Salmonella isolates, blaCTX-M (n=4), blaSHV-12 (n=1), blaTEM-1

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(n=2) and blaCMY-2 (n=2) were identified. No PMQR-encoding genes were found. In 47 E.

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coli isolates, blaCTX-M (n=15), blaSHV-12 (n=2), blaCMY-2 (n=6), blaTEM-type (n=28) and PMQR-

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encoding genes qnrB (n=2), qnrS (n=1) and aac(6')-Ib-cr (n=6) were detected. To the best of

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our knowledge, this study is the first to describe the presence of blaCMY-2 (n=2) and blaSHV-12

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(n=1) genes among S. enterica from broilers in Portugal.

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This study highlights the fact that animals may act as important reservoirs of isolates

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carrying ESBL-, PMAβ- and PMQR-encoding genes that might be transferred to humans

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through direct contact or via the food chain.

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Keywords: Antimicrobial resistance; Salmonella enterica; Escherichia coli; ESBL; PMAβ;

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PMQR

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1.

Introduction

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Salmonella is a widely distributed foodborne pathogen and one of the most common

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causes of bacterial foodborne illnesses, with tens of millions of human cases occurring

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worldwide every year (http://www.who.int). In the European Union it is the second most

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reported zoonotic disease in humans, with a total of 92,916 cases; most infections are caused

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by serovars Enteritidis, Typhimurium and Typhimurium monophasic 1,4,[5], 12:i:- [1].

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Escherichia coli is the most prevalent commensal of the human and animal

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gastrointestinal tract and remains

one of the most frequent causes of several bacterial

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infections in both humans and animals [2].

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Antimicrobial resistance in Enterobacteriaceae, namely in non-typhoidal Salmonella

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serotypes and E. coli, is an expanding problem [3,4]. Wide uncontrolled use of antimicrobial

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compounds in human and veterinary practices, animal production, agriculture and industrial

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technology and an increase in population mobility and in the circulation of food and raw

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materials for food production across

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emergence and dissemination of resistant and multiresistant bacterial strains that constitute a

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risk for

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associated with treatment of infections [4,5].

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different countries are factors responsible for the

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human and animal health due to an increase in morbidity, mortality and the cost

β-lactams and fluoroquinolones are two important classes of antimicrobials used to treat

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severe infections in humans and in animals [4]. Resistance to third generation cephalosporins

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is generally due to

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mediated AmpC β-lactamases (PMAβs). Various β-lactamase-encoding genes have been

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detected in diverse serotypes located in plasmids or in integrons, facilitating its transmission

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between serotypes and other bacteria [6].

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production of extended-spectrum β-lactamases (ESBLs) and plasmid-

Animals have the potential to act as reservoirs for a number of zoonotic infections,

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including those caused by pathogenic and commensal E. coli ESBL producers, which might

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be transmitted to humans through direct contact or via the food chain [5,6].

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In Europe, a wide range of ESBL genotypes have been reported from animals [6,7],

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some of which are also found in humans [8]. Companion animals, including horses, dogs and

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cats, also constitute a potential reservoir of ESBL-encoding genes, as they often live in close

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contact with their owners, facilitating

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living in the wilderness or in captivity may also represent a source of ESBL-producing E.

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coli isolates to the ecosystems [12,13], stressing the importance of the environment on

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dissemination of resistance genes and

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between zoo animals, zoo keepers and visitors [14].

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occurrence of transmission

[9-11]. Wild animals

potential zoonotic transmission due to

contact

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In this study, we present updated data on antimicrobial resistance in Salmonella

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recovered from animals, with particular emphasis on food-producing animals, poultry feed

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and food of animal origin, as well as in E. coli isolates collected from food-producing

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animals, pets and zoo animals. The presence of antimicrobial resistance mechanisms in

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isolates with reduced susceptibility to third-generation cephalosporins and/or cephamycins

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was also evaluated.

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2.

Materials and methods

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2.1. Bacterial isolates

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This study included 562 Salmonella spp. isolates representing 50 different serotypes

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(Table 1), recovered from breeders (n=23), broilers (n=193), layers (n=73), turkeys (n=17),

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animal feed (n=52), other animal species (n=22) and food products of animal origin (n=182),

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over the period of 2012-2013.

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In poultry farms, samples were collected from feces and environment using sterile

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boots/sock swabs. Food products consisted of: uncooked fresh products like minced meat,

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hamburgers, meat cuts, sausages and table eggs, randomly collected at various retail stores.

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Samples from other animal species (pigeons, partridges, ducks, pets and exotic animals)

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consisted of hemoculture and organs (lung, liver, spleen, kidneys and intestine) collected

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during post-mortem examination for bacteriological examination.

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All samples were examined according to ISO norm 6579:2002 applied to Salmonella

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detection in food and animal feeding stuffs [15]. After biochemical confirmation, Salmonella

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isolates were sent to the Salmonella National Reference Laboratory (INIAV-Lisbon) in triple

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sugar iron slopes or SMID plates for serotype characterization.

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Also included in this study were 598 E. coli isolates (Table 1) collected over the period

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of 2009-2013 from food-producing animals [(bovine, swine and poultry), (n=215), pets (dogs,

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cats, horses and cage birds), (n=113) and zoo animals (terrestrial and aquatic mammals, birds

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and reptiles), (n=270)]. Samples consisted of swabs from organic fluids and cavities, fecal

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samples, urine samples, hemocultures and organs collected during post-mortem examination

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and submitted for bacteriological analysis. Suspected E. coli colonies obtained in MacConkey

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agar were confirmed by means of API 20E strips.

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Salmonella spp. and E. coli isolates were preserved in cryovials containing tryptose

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soya broth and glycerol at -70ºC for further antimicrobial susceptibility tests and molecular

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assays.

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2.2.

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Serotyping of Salmonella isolates

Salmonella isolates were serotyped by the slide agglutination method for their O and H antigens using the method of Kauffmann-White scheme [16].

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2.3. Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MICs) were determined by agar dilution following

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standard recommendations [17], using a panel of nine antimicrobial compounds: ampicillin

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(A),

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chloramphenicol (C), tetracycline (T), sulfamethoxazole (Su) and trimethoprim (Tm) (Table

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2).

(Ct),

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cefotaxime

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nalidixic

acid

(Na),

ciprofloxacin

To assess non-wild-type strains, interpretation of

(Cp),

gentamicin

(G),

results was done according to the

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epidemiological cut-off values recommended by the European Committee on Antimicrobial

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Susceptibility Testing (EUCAST, http://mic.eucast.org/Eucast2/). For Salmonella spp., the

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cut-off value used for sulfamethoxazole was that for sulfonamides from Clinical Standards

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Laboratory Institute [18]. E. coli ATCC 25922 was used as the quality control strain.

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Rates of resistance to antimicrobials important in humans (ampicillin, cefotaxime,

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ciprofloxacin and gentamicin) were calculated according to clinical breakpoints established

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by EUCAST for Enterobacteriaceae [19]. Isolates were considered multidrug-resistant

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(MDR) if they presented diminished susceptibility or non-wild-type phenotypes against three

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or more antimicrobials not structurally related (Tables 2 and 3).

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2.3. Screening and identification of ESBLs and PMAβs

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Salmonella spp. and E. coli isolates exhibiting MIC > 0.5µg/mL and > 0.25µg/mL,

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respectively, were considered non-wild-type to cefotaxime and tested for the production of

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ESBL by a combined disk test using cefotaxime, ceftazidime and cefpodoxime as single

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drugs and in combination with clavulanic acid.

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In addition, a cefoxitin disk (30 µg) was added to this test to detect presumptive PMAβ

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producers. All isolates classified as intermediate or resistant using CLSI criteria (≤17mm) to

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cefoxitin [18] were suspected to be PMAβ producers and also included in this study.

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2.4. Molecular characterization of resistance

In order to detect the presence of β-lactamase-encoding genes in the isolates showing

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non-wild-type phenotypes to cefotaxime and/or cefoxitin, different PCR reactions were

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performed according to the phenotypes found. The blaESBL (blaTEM, blaSHV, blaOXA-1-type,

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blaCTX-M) and blaPMAβ (blaCMY, blaMOX, blaFOX, blaLAT, blaACT, blaMIR, blaDHA, blaMOR,

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blaACC) genes were detected by PCR, as previously described [20]. Positive and negative

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controls were used in all PCR reactions. PCR products were purified and all amplicons were

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further sequenced directly on both strands using automatic sequencer ABI3100 (Applied

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Biosystems).

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Additionally, the isolates were subjected to the detection of class 1 and 2 integrons

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through amplification of integrase-encoding genes, as reported elsewhere [21].

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Salmonella spp. and E. coli isolates evidencing a non-wild phenotype to cefotaxime

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and, simultaneously, non-susceptibility to quinolones, were screened for the following PMQR

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encoding genes: qnrA, qnrB, qnrC, qnrD, qnrS, aac(6´)-Ib-cr and qepA [22-25].

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173 174 3. Results

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3.1. Serotypes of Salmonella spp.

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Wide serotype diversity (n=50) was seen among the 562 Salmonella isolates. Overall, in

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poultry, particularly in broilers, S. Havana and S. Enteritidis were the two main serotypes

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identified; S. Mbandaka was the most common serotype in layers (Table 1).

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S. Enteritidis is one of the most commonly reported serotypes in humans and it was found in poultry food products (100%, 10/10),

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13/57) and in poultry feed (15.8%, 9/57).

broilers (54.4%, 31/57),

layers (22.8%,

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S. I 4,[5],12:i:- and S. Typhimurium were the most common serotypes recovered from

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food products of swine and poultry origin. Since serovar I 4,[5],12:i:- is considered an

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emerging serotype in humans, it was identified in 61.7% (29/47) of isolates recovered from

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swine and 34% (16/47) from poultry products; in live birds, 37.5% (6/16) and 31.3% (5/16)

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were from broilers and turkeys, respectively (Table 1).

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3.2. Antimicrobial susceptibility phenotype of Salmonella spp. and E. coli isolates Susceptibility profiles of Salmonella spp. differed according to the origin (Table 2) and serotypes of the isolates (Table 3).

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Although clinical resistance to ciprofloxacin was absent (Table 3), the frequency of

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non-wild-type isolates to this antimicrobial was higher in poultry and poultry food products

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(both 30.1%). Therefore, isolates recovered from bovine and swine food products showed

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higher

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sulfamethoxazole (60% and 67.6%, respectively) and ampicillin (20% and 50%, respectively)

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(Table 2).

non-wild-type

phenotypes

for

tetracycline

(64%

and

73%,

respectively),

Regarding cefotaxime, nine isolates of S. enterica (1.6%, 9/562) (four serotype Havana

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and one serotype Enteritidis recovered from broilers, two belonging to serotype I 4,[5],12:i:

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obtained from a partridge and pork sausage, one serotype Heidelberg from broiler neck skin

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and one

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cefotaxime (MIC ranging from 1 to ≥ 8µg/mL) (Table 4).

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serotype London from a pork hamburger) presented non-wild-type MICs for

MDR was observed in five isolates from turkeys (29.4%), 26 isolates from broilers

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(13.5%), two isolates from layers (2.8%), six isolates from other animal species (27.3%), 44

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isolates from food of swine origin (59.5%), 22 isolates from food of poultry origin (26.5%), 5

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isolates from food of bovine origin (20.0%) and two isolates from animal feed (3.9%). No

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MDR isolates were detected in broiler breeders. The higher frequency of MDR isolates was

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observed in S. I 4,[5],12:i:- and S. Typhimurium serovars (Table 3).

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With regard to susceptibility of E. coli isolates (Table 2), the frequency of non-wild-

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type isolates to all antimicrobials tested was higher when compared with Salmonella isolates,

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particularly to ciprofloxacin, where clinical resistance was high, namely in food-producing

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animals (24.2%) and pets (21.1%) (Table 2). Eight isolates of E. coli from food-producing

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animals (3.7%, 8/215), twelve from pets (10.6%, 12/113) and seven from zoo animals (2.6%,

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7/270) showed a non-wild-type to cefotaxime (MIC ranging 0.5 to ≥8mg/L) (Table 4).

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MDR was recorded in 115 isolates from food-producing animals (53.7%), 39 isolates from pets (34.5%) and 81 isolates from zoo animals (30%) (Table 2).

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3.3. Characterization of ESBL, PMAβ, PMQR and integrons

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ESBL and PMAβ phenotypes were detected in five and two isolates, respectively (Table

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4). In two MDR Salmonella Havana (ACtSuT, ACtCpSuTm), one Salmonella Heidelberg

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(ACtSuTm) and one Salmonella London (ACt) isolate, blaCTX-M-type, blaCTX-M-1 and blaCTX-M-

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14

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Havana isolates showing the ACt phenotype. The blaSHV-12 gene was detected in one MDR S.

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Enteritidis isolate (showing ACtNaCpCSuT) recovered from a broiler flock. Two S. I

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4,[5],12:i:- isolates (with ACtSuT and ACtNaCpGT phenotypes)

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sausage and a partridge presented the blaTEM-1 gene.

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genes were detected, respectively. Additionally, blaCMY-2 gene was encountered in two S.

pork

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recovered from

Although three isolates, including two S. Havana and one S. I 4,[5],12:i:-, showed a

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non-wild-type MIC for ciprofloxacin (0.25 and 1 µg/mL, respectively), no PMQR-encoding

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genes were detected (Table 4).

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One isolate of each serotype Havana, Enteritidis and Heidelberg harbored class 1 integrons and one isolate belonging to serotype I 4,[5],12:i:- carried a class 2 integron. The ESBL/PMAβ-encoding genes identified in E. coli from food-producing animals

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were: blaCTX-M-1 (n=4), blaSHV-12 (n=2), blaCMY-2 (n=2) and blaCTX-M-32 (n=1); in pets, they

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were blaCMY-2 (n=4), blaCTX-M-15 (n=2) and blaCTX-M-14 (n=1); and in zoo animals, blaCTX-M-15

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(n=5), blaCTX-M-1 (n=1) and blaCTX-M-14 (n=1) (Table 4).

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Although the frequency of non-wild-type isolates to ciprofloxacine was lower in zoo

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animals than in pets and food-producing animals (Table 2), PMQR-encoding genes were

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more frequently detected in isolates recovered from these animals, with aac(6')-Ib-cr being

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most prevalent (Table 4).

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Six out of nine ESBL/PMAβ isolates from food-producing animals (66.7%), three out

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of seven isolates from pets (42.9%) and seven out of seven isolates from zoo animals (100%)

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were MDR. No ESBL/PMAβ-encoding genes were detected in two and three isolates

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recovered from food-producing animals and pets, respectively, showing resistance to

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cefoxitin, and none of these enzymes were produced by E. coli isolates with intermediate

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susceptibility to this antibiotic.

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Most of the E. coli isolates harbored class 1 integrons. Nevertheless, class 2 was also found in one isolate recovered from a dog and in one isolate from a turkey.

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4. Discussion

In the last six years, in addition to S. Enteritidis, serovars Havana and Mbandaka were

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most frequently isolated from broilers, layers and poultry feed in Portugal, suggesting that

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they are well adapted to the poultry population [26,27]. It is likely that poultry feed containing

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cereal grain imported from certain countries is one of the main sources of the high frequency

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of occurrence of these serotypes in live birds. S. Havana has been previously detected in

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Danish

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(http://unsafefood.eu/notification). Although, in European countries, the prevalence of

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serovars Mbandaka and Havana in Gallus gallus is low (5.48% and 0.21%, respectively) [4],

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it is a matter of concern, since they are considered potentially pathogenic for humans [28,29].

and

rapeseed

meals

imported

from

other

countries

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With regard to food products, Salmonella I 4,[5],12:i:- and S. Typhimurium were the

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most common serotypes recovered from food products, followed by S. Enteritidis. Food

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products and animals seem to be important reservoirs for human infection. As previously

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reported, monophasic S. Typhimurium was in third place in the top 10 list of the most

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commonly reported serovars in human cases

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importance in many countries, having caused a substantial number of infections in both

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humans and animals bred for food [4].

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in 2012 and appears to be of increasing

In this study, important differences (≥3-fold dilutions) between MIC50 and MIC90 were noted among some species, particularly

E. coli isolates for ampicillin, nalidixic acid,

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ciprofloxacin, sulfamethoxazole, tetracycline and trimethoprim, and also

Salmonella spp.,

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for ampicillin, nalidixic acid, sulfamethoxazole and tetracycline. For each antimicrobial,

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MIC50 and MIC90 distributions indicate that at least two bacterial subpopulations may exist

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(wild and non-wild-type) [30], which corroborates other findings [27,31]. Regarding E. coli isolates, the frequency of non-wild-type phenotypes to all

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antimicrobials tested (except for cefotaxime) was higher in food-producing animals, followed

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by companion and zoo animals, which might be due to the high consumption of veterinary

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antimicrobials, particularly tetracyclines and fluoroquinolones [32]. Currently, nearly 25% of

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isolates from food-producing animals are clinically resistant to ciprofloxacin, leading to the

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increasing use of third-generation cephalosporins in food animals. Use of these antimicrobial

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is reported to be an important factor in the emergence of extended-spectrum cephalosporin-

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resistant E. coli [33]. Although the consumption reported for these antimicrobials is not high

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(0.2 mg/PCU) [32], it might be underestimated since, in companion animal practice, human

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cephalosporins are frequently prescribed.

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A comparison between results obtained in this study with those from a previous work

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[26] show that there was an increase in the frequency of non-wild-type MICs for cefotaxime

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in Salmonella spp. isolates from broilers, and from food products at the national level, from

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0.44% to 1.6%, as reported in other studies [34]. In our study, CTX-M-1, CTX-M-14, TEM-1 and SHV-12 β-lactamases were found in

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Salmonella,

in agreement with findings from other European and non-European countries

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[6,35,36]. The low frequency of TEM-1 and SHV-12 follows the current situation in Europe

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[6]. However, to our knowledge, SHV-12 was first described in one isolate of S. Enteritidis

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from broilers in Portugal.

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While blaCMY-2 is the most frequently reported PMAβ-encoding gene in other countries

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and in different serovars [6,34,37], we report its occurrence here for the first time in two

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isolates of S. Havana from broilers resistant only to β-lactams. The spread of blaCMY-2

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harboring Salmonella through the food chain also has important public health implications;

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like ESBLs, it encodes resistance to third-generation cephalosporins,

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antibiotics used to treat complicated human infections, including salmonellosis [2].

an important class of

In all 47 E. coli non-wild-type isolates to cefotaxime and/or cefoxitin, the ubiquitous

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ampC gene was detected and frequently associated with blaTEM-type. It should be noted that E.

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coli possess a chromosomal ampC gene that is normally repressed or only weakly expressed.

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Alterations in ampC gene promoter regions increase the production of AmpC and confer

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variable resistance levels to penicillins and cephalosporins, including cephamycins and

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oxyimino-cephalosporins, suggesting that this resistance mechanism might have been

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triggered among our isolates [38].

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Overall, CTX-M-group enzyme isolates were detected in 15 out of 28 (53.6%) isolates

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exhibiting a non-wild phenotype to cefotaxime. CTX-M-1 was the major ESBL enzyme found

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in food-producing (n=4) and also in zoo animals (n=1). It has been disseminated in several

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countries in food-producing animals and in wildlife [12,13,35], although not commonly

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observed in humans. Therefore, it has been previously reported as a possible cross-

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contamination between humans, avian hosts and meat, highlighting the importance of its

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possible transmission to humans [8]. Currently, CTX-M-15 is the most common ESBL CTX-

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M variant detected worldwide in clinically important human pathogens [6]. In our work, it

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was detected in two isolates from companion animals (dogs) and in five isolates from

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dolphins. Similarly, CTX-M-15 has also been reported in E. coli isolates from companion [9-

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11], wild [12] and zoo animals [14,39]. Due to the potentially frequent contact between pets

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and owners and zoo animals, zookeepers and visitors, bacteria containing such genes might

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spread among these different reservoirs.

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PMAβ enzymes were also found in 6 E. coli isolates, from food-producing (n=2) and

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companion animals (n=4), confirming findings in other studies [10,34].

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The presence of PMQR, which have been increasingly reported in animals [14,40,41],

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may have an additional effect on chromosomal quinolone resistance mechanisms, which

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might explain

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ciprofloxacin, respectively, observed in some of our isolates. Indeed, resistance to quinolones

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in Enterobacteriaceae is mostly linked to chromosomal mutations in the quinolone resistance-

325

determining region (QRDR) [40,42].

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the high MIC values of >512 mg/l and ≥8 mg/l to nalidixic acid and

In Europe, qnrS and qnrB variants were the most frequently detected in different

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Salmonella serotypes from animals and food [43]; however, no PMQR-encoding genes were

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detected among such isolates in our study. In contrast, qnrS1 and qnrB19 genes were detected

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in E. coli isolates from food-producing animals co-expressing blaCMY-2 in one of the isolates,

330

which increases the threat of antimicrobial resistance, as they are plasmid-mediated [43]. In

331

pets and zoo animals, aac(6')-Ib-cr was the most frequently PMQR detected, which is

332

common in human E. coli spreading worldwide [43,44]. The spread of MDR isolates

333

producing ESBL or PMAβ is a matter of concern, especially when they carry other resistant

334

traits conferring resistance to aminoglycosides, fluoroquinolones or mobile genetic elements

335

like integrons. [44].

EP

TE D

M AN U

SC

326

Among the nine Salmonella non-wild-type isolates for cefotaxime found in our study,

337

six were MDR (66.7%); additional resistance was also detected for sulfamethoxazole,

338

tetracycline, trimethoprim and gentamicin. Since these drugs are used in animal production,

339

co-selection may have played a role in the onset of ESBL-producing isolates compared with

340

previous studies [26].

AC C

336

341

Thirty-five (35/47, 74.5%) E. coli isolates were MDR, comprising a significant

342

contribution by the food-producing animals group. Twenty-seven (77.1%, 27/35) carried

343

integrons. Similarly, among six MDR Salmonella isolates, four also carried integrons

14

ACCEPTED MANUSCRIPT 344

(66.7%). This supports the hypothesis of an association between the presence of emerging

345

MDR isolates and integrons, as well as other mobile genetic elements, contributing to the

346

spreading of antimicrobial resistance determinants [43]. In conclusion, we report for the first time the detection, in Portugal, of the blaCMY-2

348

gene in two isolates of S. Havana from broilers, and of blaSHV-12 in one isolate of S. Enteritidis

349

also from broilers.

350

Overall,

RI PT

347

results presented in this study indicate that animals should be considered

potential reservoirs for ESBL-, PMAβ- and PMQR-producing isolates. Prudent use of

352

antimicrobials in animals should be strongly encouraged, as well as

353

antimicrobial resistance genes, in order to monitor future trends in the occurrence of

354

resistance to oxymino-β-lactams and fluoroquinolones.

355 356

358

Conflict of interest

The authors declare no conflict of interests.

359 Acknowledgements

EP

360

TE D

357

characterization of

M AN U

SC

351

V. Manageiro and D. Jones-Dias were supported by grants SFRH/BPD/77486/2011 and

362

SFRH/BD/80001/2011, respectively, from the Fundação para a Ciência e Tecnologia, Lisbon,

363

Portugal. These results were partially presented at the 3rd ASM Conference on Antimicrobial

364

Resistance in Zoonotic Bacteria and Foodborne Pathogens in Animals, Humans and the

365

Environment, 26-29 June 2012, Aix-la-Provence and at TEMPH (Trends in Environmental

366

Microbiology for Public Health, 18-21 September 2014, Lisbon.

367 368

AC C

361

The authors are grateful to all staff involved for their technical assistance.

15

ACCEPTED MANUSCRIPT 369 References

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public health risks of enterobacterial isolates producing extended-spectrum β-lactamases

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in Salmonella enterica and Escherichia coli isolated from animals, humans, food and the

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of class 1 integrons among Salmonella enterica serovar Enteritidis isolated from humans and

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poultry. FEMS Immunol Med Microbiol 2012;64:237-243.

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503

clonal group. Clin Microbiol Rev 2014;27:543-574.

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ACCEPTED MANUSCRIPT

21

505 506

a

1 4 4 2 2 8 21

0 6 1 5 0 4 16

0 0 10 10

3 12 26 41

2 29 16 47

0 102 1 0 8 0 111 0 0 2 2

Other Total serotypesa

SC

1 31 13 1 9 2 57

Important serotypes in national poultry population S. S. Havana Mbandaka 0 5 13 0 7 0 25

21 45 41 9 26 8 150

23 193 73 17 52 22 380

0 0 2 2

20 33 27 80

25 74 83 182

M AN U

Animal species Breeders Broilers Layers Turkeys Animal feed Other species Total Food Bovine Swine Poultry Total

Emergent reported serotype in humans S. 4,[5],12,i:-

TE D

Samples

Most common reported serotypes in humans S. S. Enteritidis Typhimurium

Escherichia coli

RI PT

Salmonella spp.

Samples

Isolates (n)

Animal species Pets Zoo Food-producing

113 270 215

Total

598

EP

Serotypes: Agona, Altona, Anatum, Bardo, Bovismorbificans, Brandenburg, Bredeney, Cerro, Cubana, Derby, Duesseldorf, Give, Heidelberg, Hadar, Indiana, Kentucky, Kingston, Kottbus, Javiana, Landau, Lexington, Llandoff, London, Muenchen, Newport, Reading, Redba, Rissen, Saintpaul, Schwarzengrund, Seftenberg, Stanleyville, Taksony, Tennessee, Tomegbe, Virchow, I 3,19 :-:-z27, I:17:b:?, II 48:z10:[1,5], II 6,7:z6:?, II 42:b:e,n,x,z15, I 9,46:?:?, II 4, [5],12:?, 4,12:eh:?, I 6,7,?:?

AC C

507 508 509 510 511 512 513

Table 1. Salmonella spp. (n=562) and Escherichia coli (n=598) isolates

22

ACCEPTED MANUSCRIPT

516 517 518 519 520 521 522

Table 2. MIC50 and MIC90 for Salmonella spp. (n=562) and E. coli (n=598) isolates Salmonella spp. Breeders Broilers Layers Turkeys

Escherichia coli

Animal Other Food feed species Bovine Swine Poultry n=52 n=22 n=25 n=74 n=83

n=23

n=193

n=73

n=17

A MIC50 MIC90 % DS a

2 2 8.7

2 >64 16.1

1 2 4.2

2 >64 35.3

2 4 3.8

2 >64 27.3

2 >64 20

2 >64 50

Ct MIC50 MIC90 % DS a % Rb

≤0.06 0.125 0 0

≤0.06 0.25 3.1 2.6

≤0.06 0.125 0 0

0.125 0.125 0 0

≤0.06 0.125 0 0

≤0.06 0.125 4.5 0

0.125 0.125 0 0

≤0.06 0.125 2.7 1.4

Na MIC50 MIC90 % DS a

4 4 8.7

8 512 26

4 256 11.1

4 256 17.6

4 4 0

4 >512 18.2

4 4 0

4 8 4.1

Cp MIC50 MIC90 % DS a %Rb

0.015 0.03 8.7 0

0.03 0.5 30.1 0

0.015 0.125 9.7 0

0.03 0.125 17.6 0

0.015 0.03 0 0

0.03 1 22.7 0

0.03 0.03 0 0

C MIC50 MIC90 % DS a

4 8 0

8 16 3.1

4 8 0

8 8 5.9

8 8 0

4 16 9.1

G MIC50 MIC90 % DS a

0.5 0.5 0

≤0.25 0.5 1

0.5 0.5 0

0.5 0.5 0

0.5 0.5 0

Su MIC50 MIC90 % DS a

32 64 4.3

64 >1024 27.5

64 128 19.4

64 >1024 47.1

T MIC50 MIC90 % DS a

1 2 0

Tm MIC50 MIC90 % DS a MDR % DS

c

8 >64 43.9

8 >64 38.9

≤0.06 1 10.6 8

≤0.06 0.125 3 3

4 >512 28.9

4 >512 38.6

4 >512 23.7

4 >512 14.1

0.03 0.03 4.1 0

0.03 0.5 30.1 1.2

0.03 >8 41.9 24.2

0.015 >8 27.2 21.1

0.015 8 17.4 11.1

4 256 12

8 >256 20.3

4 8 3.6

8 128 21.4

4 8 7

4 8 5.5

0.5 8 13.6

0.5 1 0

0.5 2 9.5

0.5 1 2.4

0.5 8 11.6

0.5 1 5.3

0.5 1 5.2

64 64 5.8

32 >1024 13.6

>1024 >1024 60

>1024 >1024 67.6

64 >1024 27.7

128 >1024 47

32 >1024 25.4

32 >1024 24.7

16 >64 52.9

2 4 1.9

2 >64 27.3

64 >64 64

>64 >64 73

2 >64 31.3

64 >64 62.3

2 >64 30.7

2 >64 31.7

M AN U

SC

≤0.06 0.125 3.7 3.7

TE D 1 2 1.4

8 >64 47.4

≤0.06 0.125 1.2 1.2

EP 2 64 10.4

2 >64 30.1

Food Zoo Pets animals animals n=215 n=113 n=270

RI PT

Antimicrobials

AC C

514 515

≤0.25 ≤0.25

≤0.25 0.5

≤0.25 0.5

≤0.25 ≤0.25

≤0.25 0.5

≤0.25 0.5

≤0.25 ≤0.25

≤0.25 >32

≤0.25 0.5

0.5 >32

0.5 >32

0.5 >32

0

9.8

1.4

0

1.9

45.5

0

14.9

8.4

36.7

22

19.6

0

13.5

2.8

29.4

3.9

27.3

20

59.5

26.5

53.7

34.5

30

A, ampicillin; Ct, cefotaxime; Na, nalidixic acid; Cp, ciprofloxacin; C, chloramphenicol; G, gentamicin; Su, sulfamethoxazole; T, tetracycline; Tm, trimethoprim a Decreased susceptibility - EUCAST epidemiological breakpoints b Resistance - EUCAST clinical breakpoints c Multidrug resistance

525 526 527 528 529 530 531

532

23 Table 3. MIC50 and MIC90 among the most important Salmonella serotypes

>64 >64 66.7 66.7

2 4 7.7 7.7

S. 4, [5], 12, i:Food Broilers Turkeys Swine Poultry (n=6) (n=5) (n=29) (n=16) >64 >64 83.3 83.3

RI PT

Antimicrobials Broilers Layers Animal feed Food Poultry (n=31) (n=13) (n=9) (n=10) A MIC50 2 2 2 2 MIC90 2 2 2 4 % DS a 6.5 0 0 0 %Rb 6.5 0 0 0

S. Typhimurium Food Swine Poultry (n=12) (n=26)

S. Havana Broilers Animal feed (n=102) (n=16)

>64 >64 100 100

>64 >64 79.3 79.3

>64 >64 56.3 56.3

4 >64 18.6 18.6

2 2 0 0

SC

S. Enteritidis

0.125 0.125 3.2 3.2

≤0.06 0.125 0 0

≤0.06 0.125 0 0

0.125 0.125 0 0

0.125 0.125 0 0

≤0.06 0.125 0 0

≤0.06 ≤0.06 0 0

≤0.06 0.125 0 0

≤0.06 0.125 3.4 0

≤0.06 0.125 0 0

0.25 0.25 3.9 3.9

0.125 0.125 0 0

Cp MIC50 MIC90 % DS a %Rb

0.25 0.25 80.6 0

0.125 0.25 53.8 0

0.03 0.03 0 0

0.25 0.25 90 0

0.03 0.03 0 0

0.03 0.25 15.4 0

0.03 0.03 16.7 0

0.03 0.03 0 0

0.03 0.03 34.5 0

0.03 0.03 0 0

0.03 0.5 26.5 0

0.03 0.03 0 0

≤0.25 0.5 0

0.5 0.5 0

≤0.25 0.5 0

≤0.25 0.5 0

0.5 0.5 0

0.5 1 0

≤0.25 0.5 0

0.5 1 0

≤0.25 1 6.9

0.5 0.5 0

≤0.25 0.5 2

0.5 0.5 0

0

0

0

0

0

0

0

0

6.9

0

2

0

3.2

0

0

66.7

3.8

83.3

80

82.8

50

13.7

0

% MDR c

TE D

EP

G MIC50 MIC90 % DS a %Rb

0

M AN U

Ct MIC50 MIC90 % DS a %Rb

AC C

523 524

ACCEPTED MANUSCRIPT

A, ampicillin; Ct, cefotaxime; Cp, ciprofloxacin; G, gentamicin a Decreased susceptibility - EUCAST epidemiological breakpoints b Resistance - EUCAST clinical breakpoints c Multidrug resistance

24 Table 4. Characteristics of Salmonella spp. and E. coli isolates displaying non-wild-type phenotypes to cefotaxime and or cefoxitin

Pets EC30 EC50 EC175 EC200 EC274 EC315

E. coli E. coli E. coli E. coli E. coli E. coli

dog cat dog dog cat dog

a

Ct

Cp

a

>8 0.25 >8 0.03 >8 0.25 8 0.03 8 0.06 1 1 ≤0.06 8 ≤0.06 0.125 0.25 >8 0.125 0.015 4 2 ≤0.06 0.03 >8 >8 ≤0.06 0.03 0.125 8 0.25 >8 ≤0.06 ≤0.008 >8 >8 >8 0.06 >8 0.015 >8 >8 0.125 8 0.125 0.25 0.125 4 4 >8 4 >8

FOX

Genetic profile

b

c

bla genes

PMQRd genes

RI PT

A, Ct, Na, Cp, C, Su, T A, Ct, Su, T A, Ct, Cp, Su, Tm A, Ct A, Ct A, Ct, Na, Cp, G, T Na, Cp, Su, G, T A, Na, Cp, Su, T A, Na, Cp, Su, C, T A, Su, C, T A, Ct, Na, Cp A A, Ct, Na, Cp, C, Su, T, Tm, A A, Na, Cp, C, Su, T, Tm A, Na, Cp, G, Su, T, Te, C A, Su, T, Tm A, Ct, Na, Cp, C, Su, T, Tm, A, Ct, Na, C, Su, T, Tm, A, Ct, Su A, Ct, Na, Cp, Su, Tm A, Na, Cp, G, T A, Cp, T A, Na, Cp, Su, A, Ct, Na, Cp, C, Su, T A, Ct, Na, Cp, C, Su, T

Decreased susceptibility

S S S R R S I R R I R I S R I I I S S S S I I I S S

blaSHV-12 bla CTX-M-1 bla CTX-M-type bla CMY-2 bla CMY-2 bla TEM-1 bla TEM-type, blaAMP-C bla TEM-type , blaAMP-C bla TEM-type, blaAMP-C blaAMP-C, bla OXA-type bla TEM-type , blaAMP-C, bla CMY-2 blaAMP-C blaAMP-C, bla CTX-M-32 bla TEM-type, blaAMP-C, bla CMY-2 bla TEM-type, blaAMP-C bla TEM-type, blaAMP-C bla TEM-type , blaAMP-C bla TEM-type , blaAMP-C, bla CTX-M-1 blaAMP-C, bla CTX-M-1 bla TEM-type , blaAMP-C, bla CTX-M-1 blaAMP-C, bla CTX-M-1 bla TEM-type, blaAMP-C bla TEM-type, blaAMP-C bla TEM-type, blaAMP-C bla TEM-type, blaAMP-C, blaSHV-12 bla TEM-type, blaAMP-C, blaSHV-12

A, Na, Cp, Ct, C, Su, TE, Su, Tm >8 A, Ct 1 A, Ct >8 A, Te ≤0.06 A, Ct, Na, Cp, G, Su, T, Tm >8 A, Ct, Su, T, Tm >8

0.25 0.015 0.015 0.015 >8 0.015

I R R R R S

bla TEM-type, blaAMP-C blaAMP-C blaAMP-C, bla CMY-2 bla TEM-type, blaAMP-C blaAMP-C bla TEM-type, blaAMP-C, bla CTX-M-14

Integrons

Class 1 Class 1

SC

broiler broiler broiler broiler broiler partridge turkey swine turkey swine broiler bovine swine broiler chicks turkey bovine calf swine bovine chicks swine bovine broiler chicks broiler chicks rabbits bovine bovine

Antimicrobial resistance phenotype

M AN U

S. Enteritidis S. Havana S. Havana S. Havana S. Havana S. 4,[5],12:i:E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli

Animal species

TE D

Food-producing animals SA12434 SA22067 SA34303 SA6423 SA6427 SA18281 EC32 EC230 EC235 EC241 EC261 EC269 EC276 EC296 EC350 EC382 EC383 EC421 EC427 EC439 LC64 LC215 LC217 LC219 EC492 EC505

Species identification

EP

Sample Isolate

AC C

533 534

ACCEPTED MANUSCRIPT

Class 2 Class 1; Class 2 Class 1 Class 1 Class 1 qnrS1 Class 1 Class 1 Class 1 Class 1 Class 1 Class 1 Class 1 Class 1 qnrB 19 Class 1 Class 1 Class 1

Class 1

Class 1 Class 1

25 E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli

cat dog dog cat dog dog mandarim parrot

EC92

E. coli

dolphin

EC126 EC128 EC163 EC212 EC248 EC325 EC337 EC338 EC361 LC218 EC456 EC536

E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli

turtledove lemur frog dolphin tiger dolphin dolphin dolphin dolphin bear otter Cariama cristata

A, Ct A, Ct, Na, Cp A, Na, Cp, Su, T, Tm A, Ct, Na, Cp A, Ct, An, Cp, G, Su, Tm A, Ct, Na, Cp, Tm A, Ct A, Ct, Na, Cp, Su

0.5 >8 0.25 8 >8 >8 8 2

0.015 >8 8 >8 >8 >8 0.015 >8

I S I R S R R S

>8

>8

S

≤0.06 ≤0.06 ≤0.06 >8 >8 >8 0.125 >8 >8 0.125 0.125 >8

0.25 0.25 0.015 >8 0.015 >8 >8 >8 >8 8 0.03 >8

I I I I S S I S S I I S

bla TEM-type, blaAMP-C, bla OXA-type; bla CTX-M-15 bla TEM-type, blaAMP-C bla TEM-type, blaAMP-C blaAMP-C blaAMP-C, bla OXA-type; bla CTX-M-15 bla TEM-type, blaAMP-C, bla CTX-M-1 blaAMP-C, bla OXA-type; bla CTX-M-15 bla TEM-type, blaAMP-C blaAMP-C, bla OXA-type; bla CTX-M-15 blaAMP-C, bla OXA-type; bla CTX-M-15 bla TEM-type, blaAMP-C bla TEM-type, blaAMP-C blaAMP-C, bla CTX-M-14

>8 2 >8

0.03 0.03 0.03

S S S

bla CTX-M-1 bla TEM-1 bla CTX-M-14

Food

a

EP

SA4810 S. Heidelberg broiler carcass A, Ct, Su, Tm SA31501 S. 4,[5],12:i:pork sausage A, Ct, Su, T SA31511 S. London hamburger swine A, Ct

TE D

A, Na, Cp, C, Su, T, Tm A, Na, Cp, C, Su, T, Tm A A, Ct, Na, Cp, G, T A, Ct, S, T, Tm A, Ct, Na, Cp, G, T A, Na, Cp, C, Su, T, Tm A, Ct, Na, Cp, G, T A, Ct, Na, Cp, G, T A, Na, Cp, T A A, Ct, An, Cp, T

M AN U

A, Ct, An, Cp, G, T

SC

Zoo animals

blaAMP-C blaAMP-C, CTX-M-15 bla TEM-type, blaAMP-C blaAMP-C, bla CMY-2 blaAMP-C, CTX-M-15; bla TEM-type, blaAMP-C, bla CMY-2 bla TEM-type, blaAMP-C, bla CMY-2 blaAMP-C

RI PT

EC321 EC356 EC385 EC425 EC433 EC443 EC459 EC498

Class 1 aac(6')-Ib-cr

Class 1 Class 2

aac(6')-Ib-cr

Class 1 Class 1 Class 1

aac(6')-Ib-cr Class 1 aac(6')-Ib-cr Class 1 aac(6')-Ib-cr aac(6')-Ib-cr qnrB 19

Minimum inhibitory concentration (MIC) Disk diffusion (Kirby-Bauer method) A, ampicillin; Ct, cefotaxime; Na, nalidixic acid; Cp, ciprofloxacin; FOX, cefoxitin; C, chloramphenicol; G, gentamicin; Su, sulfamethoxazole; T, tetracycline; Tm, trimethoprim S, susceptible; I, intermediate; R, resistant c β-Lactame genes d Plasmid-mediated quinolone resistance b

AC C

535 536 537 538 539 540 541 542

ACCEPTED MANUSCRIPT

Class 1

Class 1