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Prevalence and fluoroquinolones resistance of Campylobacter and Salmonella isolates from poultry carcasses in Rio de Janeiro, Brazil
Accepted Manuscript Prevalence and fluoroquinolones resistance of Campylobacter and Salmonella isolates from poultry carcasses in Rio de Janeiro, Brazil Pedro Henrique Nunes Panzenhagen, Waldemir Silva Aguiar, Beatriz da Silva Frasão, Virginia Léo de Almeida Pereira, Dayse Lima da Costa Abreu, Dalia dos Prazeres Rodrigues, Elmiro Rosendo do Nascimento, Maria Helena Cosendey de Aquino PII:
S0956-7135(15)30224-3
DOI:
10.1016/j.foodcont.2015.10.002
Reference:
JFCO 4680
To appear in:
Food Control
Received Date: 12 July 2015 Revised Date:
2 October 2015
Accepted Date: 6 October 2015
Please cite this article as: Panzenhagen P.H.N., Aguiar W.S., da Silva Frasão B., de Almeida Pereira V.L., da Costa Abreu D.L., dos Prazeres Rodrigues D., do Nascimento E.R. & de Aquino M.H.C., Prevalence and fluoroquinolones resistance of Campylobacter and Salmonella isolates from poultry carcasses in Rio de Janeiro, Brazil, Food Control (2015), doi: 10.1016/j.foodcont.2015.10.002. 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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Prevalence and fluoroquinolones resistance of Campylobacter and
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Salmonella isolates from poultry carcasses in Rio de Janeiro, Brazil
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Pedro Henrique Nunes Panzenhagena*, Waldemir Silva Aguiara, Beatriz da Silva Frasãoa,
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Virginia Léo de Almeida Pereiraa, Dayse Lima da Costa Abreua, Dalia dos Prazeres
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Rodriguesb, Elmiro Rosendo do Nascimentoa, Maria Helena Cosendey de Aquinoa
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a
Department of Veterinary Public Health and Public Health, Federal Fluminense University (UFF), 24230-
340, 64 Vital Brazil Filho Street, Niterói, RJ, Brazil. b
National Reference Laboratory Diagnosis of Enteric Bacteria, Oswaldo Cruz Institute, Oswaldo Cruz
Foundation (FIOCRUZ), 21045-900, 4365 Brazil Avenue, Rio de Janeiro, RJ, Brazil
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* Corresponding Author at: Analytical Laboratory Center, Department of Food Technology, Faculty of
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Veterinary, Fluminense Federal University, 64, Vital Brazil Filho, Niterói - Rio de Janeiro - Brazil Zip Code:
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24230-340. E-mail: [email protected] / Phone: +55-21-99567-9533 / +55-21-2268-5367
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13 Abstract
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To investigate the prevalence of Campylobacter and Salmonella in poultry carcasses in state of Rio de
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Janeiro, Brazil, 60 samples from 6 slaughterhouses were collected over a period of 6 months. A total
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of 82 Campylobacter isolates were obtained from twenty seven (45%) positive chicken carcasses,
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including 44 isolates (53.66%) of Campylobacter jejuni and 38 (46.34%) of Campylobacter coli. The
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identification of all strains was confirmed by PCR. Salmonella was isolated from 4 (6.67%) carcasses
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by conventional method and was detected in 5 (8.33%) of 60 chicken carcasses by PCR. Two
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Salmonella Albany and two Salmonella Typhimurium were identified. Antimicrobial susceptibility
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testing was primarily done by the disk diffusion method and later by assessing minimum inhibitory
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concentrations (MICs) against all the isolates. All the Campylobacter isolates were resistant to
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ciprofloxacin and enrofloxacin. It was observed high MIC values for enrofloxacin (64 µg/mL) in one
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C. jejuni and two C. coli strains, and for ciprofloxacin (≥ 128 µg/mL) in one C. jejuni and three C.
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coli strains. No Salmonella isolate was resistant to these antibiotics by both methods. These findings
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reveal a broad extent of fluoroquinolone resistance in Campylobacter isolates from chicken carcasses
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in Brazil and underline the need for prudent use of these antibiotics in poultry production to minimize
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the spread of fluoroquinolone resistant Campylobacter.
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Keywords: Campylobacter, Salmonella, Enrofloxacin, Ciprofloxacin, Chicken carcasses, Brazil
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1. Introduction
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Salmonellosis and Campylobacteriosis are among the most frequently reported foodborne
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diseases worldwide. While numerous potential vehicles of transmission exist, commercial chicken
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meat has been identified as one of the most important food vehicles for these organisms (FAO/WHO, 1
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2009). Although The Center for Disease Control and Prevention (CDC) reports Salmonella as leading
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causes of hospitalization by foodborne illness in United States (Scallan et al., 2011), interestingly,
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Campylobacter remains the most commonly reported gastrointestinal bacterial pathogen in humans
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since 2005 within European Union population (EFSA, 2013). In developing countries, outbreak information is frequently incomplete because health
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authorities lack the capabilities or resources for detection, or presumably, because diarrheal diseases
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are highly endemic and outbreaks may be less common or obvious than in industrialized countries
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(Zaidi et al., 2008). Despite this incomplete outbreak information in Brazil there are several reports of
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Salmonella prevalence in chicken carcasses ranging from 5.9% to 86.7% (Cardoso & Tessari, 2008;
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Duarte et al., 2009; Fuzihara, Fernandes, & Franco, 2000; Matheus, Rudge, & Gomes, 2003; Oliveira
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et al., 2006).
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Campylobacter prevalence around the world is very variable and range from 0.29% to 96.7%
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in chicken carcass (Aquino, Pacheco, Ferreira, & Tibana, 2002; Garin et al., 2012; Wang, Guo, & Li,
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2013). In Brazil, the Campylobacter presence is not investigated in most cases of bacterial
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gastroenteritis because the methodologies of isolation and characterization are different from those
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used in the research of common enteropathogenic bacteria, such as Salmonella, Shigella and the E.
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coli group. However, some authors such as Aquino et al. (2002) and Hungaro et al. (2015) found 60%
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and 16.8% of Campylobacter prevalence in chicken carcasses respectively.
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Fluoroquinolones, such as ciprofloxacin and enrofloxacin, have an extensive application both
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in human and veterinary medicine with spectrum of action over Gram-negative and Gram-positive
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bacteria (Ruiz, 2003). Enrofloxacin, a quinolone developed exclusively for use in animals, has a wide
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antibacterial activity and is commonly used in poultry production in Brazil. Ciprofloxacin, a
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metabolite of enrofloxacin (Idowu, Peggins, Cullison, & Bredow, 2010), besides its use in poultry
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production, is also used for the treatment of human Salmonellosis and Campylobacteriosis (Agunos et
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al., 2013). Once fluoroquinolones residues could persists in the animal body and may result in the
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development of bacteria resistant strains, several studies have linked the therapeutically and
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prophylactically use with the emergence and spread of resistance from these pathogens (Cheng et al.,
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2012; Finley et al., 2013; Yan, Wang, Qin, Liu, & Du, 2011).
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Antimicrobial resistance is an increasing worldwide concern and has been developed over the
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past 30 years regarding the emergence of multidrug-resistant phenotypes among Salmonella and
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Campylobacter (Hur, Jawale, & Lee, 2012). Alarmed by the rise in multidrug-resistant Salmonella in
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the 1960s, the United Kingdom’s Swann Report of 1969 recognized the possibility that AGPs
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(Antimicrobial Growth Promoters) were contributing largely to the problem of drug-resistant
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infections. These reports concluded that animal growth promotion with antibiotics used for human
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therapy should be banned. However, this practice has continued in many countries although with
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antibiotics that are not used therapeutically in humans (Marshall & Levy, 2011). 2
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We notice that little is known with regards to the simultaneous occurrence of Campylobacter
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and Salmonella on chicken carcasses in Brazil, and their resistance to fluoroquinolones. Therefore, the
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aim of this study was to investigate their prevalence and pattern of enrofloxacin and ciprofloxacin
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resistance in carcasses of slaughtered chicken in Rio de Janeiro State, Brazil.
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2. Materials and methods
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2.1. Sample collection
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During 6 months in 2013, 60 chicken carcasses were collected from 6 slaughterhouses in state
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of Rio de Janeiro, Brazil. From each slaughterhouse, 10 chicken carcasses were randomly collected
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from the chiller tank and transported on ice in sterilized plastic bags to the laboratory. Microbiological
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analyses were carried out within at least 3 hours after collection. The slaughterhouses were also
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randomly selected and its identification were preserved changing their names by the letters A to F. 2.2. Salmonella examination method
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Skin samples of neck, breast and cloacal (25g) were homogenized in a stomacher (Stomacher
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80 Laboratory Blender Seward) for 2 minutes and pre enriched with 225 mL of buffered peptone water
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(BPW) at 37°C for 24h. After incubation, isolation of Salmonella was performed in general
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accordance with U.S.FDA Bacteriological Analytical Manual (Hammack, Andrews, & Jacobson,
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2014). Isolates were subjected to Salmonella Poly O and Poly H antibody assays (Probac do Brazil®). At the same time, Salmonella detection was performed by PCR. The analyses were carried out
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using 1.0 mL of 24h pre-enrichment incubated buffered peptone water (BPW) at 37°C. DNA
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extraction and amplifications were performed in accordance with Myint, Johnson, Tablante, &
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Heckert (2006). The primer set of ST 11 (AGC CAA CCA TTG CTA AAT TGG CGC A) and ST 15
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(GGT AGA AAT TCC CAG CGG GTA CTG), originally designed by Aabo, Rasmussen, Roseen,
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Sørensen, & Olsen (1993), is highly specific for Salmonella species and defines an amplified fragment
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of 429 bp. Salmonella isolates were sent to the National Reference Center, Institute Oswaldo Cruz,
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Rio de Janeiro, Brazil for serotyping.
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2.3. Campylobacter examination method The chicken carcass were rinsed with 250 mL of 0.1% buffered peptone water and massaged
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in sterile plastic bag. Loopfuls were used for Campylobacter isolation according to Stern, Patton,
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Doyle, Park, & Mccardell (1992) and 3 to 5 suspected colonies per each plate were picked and
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identificated by PCR in accordance with Harmon, Ransom, & Wesley (1997). DNA was extracted
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with the commercial extraction kit 'Wizard® Genomic DNA Purification Kit' (PROMEGA®). Primers
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used were pg3/pg50 that amplify a conserved region in the two species (C. jejuni and C. coli) related
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to flagellin gene and primers C-1/C-4 which amplify a specific region of the species C. jejuni strains.
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The amplification reaction was performed with a final volume of 50 mL containing 5µL of the sample 3
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dGTP and dTTP, 0.4 uM of each primer pg3 and pg50, 0.2 µM of each primer C1 and C4, 2.5 U Taq
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polymerase and 5.5 mM/L MgCl2. The initial denaturation was performed at 94°C for four minutes
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followed by 25 amplification cycles consisting of one minute at 94°C, one minute at 55°C, one minute
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at 72°C and extension at 72°C for seven minutes. Verification of amplicons was performed in a
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horizontal electrophoresis tank 'Electrophoresis Cell (BioAmérica) with 0.5x TBE, with Pac Power
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source 300 (Bio-Rad) in 1.5% agarose gel stained with GelRed.
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2.4. Antimicrobial susceptibility test
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Salmonella and Campylobacter isolates were tested for enrofloxacin (5µg) and ciprofloxacin (5µg)
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(Cefar Brazil) susceptibilities. The susceptibility testing was primarily done by the disk diffusion
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method and later by assessing minimum inhibitory concentrations (MICs) against all the resistant
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isolates detected by disk diffusion method. The minimal inhibitory concentration (MIC) was
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determined by the agar dilution method containing ciprofloxacin and enrofloxacin (Sigma-Aldrich®)
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on the following concentrations: 128 µg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL, 8 µg/mL, 4 µg/mL, 2
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µg/mL, 1 µg/mL, 0.5 µg/mL (NCCLS, 2003). The MIC were decided based on visible growth and
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breakpoint of ≥ 4 µg/mL for ciprofloxacin (CLSI, 2008) and enrofloxacin (Chen et al., 2010) were
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used. C. jejuni ATCC 33560 and C. Coli ATCC 33559 were included on every plate as a quality
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control.
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2.5. Statistical analysis
Statistical analyses for Salmonella detection methods followed procedures described
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previously (Thrusfield, 2007). The program InStat, version 3.1 (GraphPad, 2009) was used for the
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calculations. The Chi-square test and Fisher’s exact two-tailed test were used for statistical analysis. A
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P value < 0.05 was used for statistical significance.
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3. Results and Discussion
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Salmonella was isolated from 4 (6.67%) of 60 samples. Two S. Albany and one S. Typhimurium
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were isolated from the slaughterhouse “B” and one S. Typhimurium was isolated from the
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slaughterhouse “F” (Table 1). Salmonella was detected in 5 (8.33%) of 60 chicken carcasses by PCR,
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but only four Salmonella positive carcasses by isolation matched the PCR results (Table 1). No
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significant statistical differences were found between conventional isolation and PCR detection by the
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Fisher Exact Test (p > 0.05).
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Relating to other previous investigations conducted in Brazil, our results have shown a slightly
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lower overall Salmonella contamination of chicken carcasses by both detection techniques when
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compared to Fuzihara et al. (2000) (41%) and Oliveira et al. (2006) (11.8%). However, similar results
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were described by Matheus et al. (2003) (5.9%) and Duarte et al. (2009) (9.6%). Salmonella
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prevalence observed in this study (6.67%) is also much lower than the observed in other developing
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countries including Iran (33%) (Dallal et al., 2010), Cambodia (88.2%) (Lay, Vuthy, Song, Phol, & 4
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Sarthou, 2011) Vietnam (45.9%) (Ta et al., 2012), and Colombia (27%) (Donado-Godoy et al., 2012).
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It is well known that laborers training, good manufacturing practices and HACCP influence the
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contamination level during the slaughter process. Most of visited slaughterhouses had good
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manufacturing practices what could explain the low prevalence described in this study. Although the PCR technique has detected 5 positive samples versus 4 positive samples
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obtained by isolation, no statistical difference was found between both methods. Salmonella
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Typhimurium and S. Albany were the only serotypes identified in this research. Besides S. Enteritidis,
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S. Typhimurium head the two top serotypes responsible for human salmonellosis in the world
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(Hendriksen et al., 2011). Although S. Albany is an uncommon serotype on chicken carcass around
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the world, this was not the first time that this serotype was isolated from chicken carcasses in Brazil
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(Fuzihara et al., 2000). Ta et al. (2014), also found S. Albany serotype on Chicken carcasses from
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Retail Markets in Vietnam.
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Unlike Salmonella, Campylobacter was isolated from all slaughterhouses investigated in this
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study, and 82 Campylobacter isolates were obtained from 27 (45%) chicken carcasses contaminated
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with this pathogen. Forty four isolates (53.66%) were identified as Campylobacter jejuni and 38
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(46.34%) as Campylobacter coli. (Table 2). Campylobacter jejuni was predominant and these data are
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similar to reports from other countries as in Senegal, Cameroon, New Caledonia, Madagascar,
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Vietnam (Garin et al., 2012) and China (Huang, Zong, Zhao, Zhu, & Jiao, 2015).
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We found a prevalence of Campylobacter in carcasses ranging from 10% to 80% depending on
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the slaughterhouse. In two slaughterhouses (B and F) was observed a higher prevalence of both
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Campylobacter and Salmonella, what could be explained by the poor conditions of the evisceration
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process. On the other hand, no Salmonella and a lower Campylobacter prevalence were found in
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slaughterhouses D and E. The occurrence of both Salmonella and Campylobacter in the same broiler
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samples was also reported before (Franz, van der Fels-Klerx, Thissen, & van Asselt, 2012) and their
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presence in this product is resultant of the contamination level and hygienic processing in poultry
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raising sites, layers, hatcheries, chicken farms and slaughterhouses. In this study, the investigated
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slaughterhouses were registered in State Inspection Service. In Brazil, there is a Pathogen Reduction
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Program of Agriculture Ministry for detection of Salmonella sp. in broiler and turkey carcasses in
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natura, involving all slaughterhouses registered in Federal Inspection Service, however, there is no
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requirement for Campylobacter detection.
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We found in this study, Campylobacter more prevalent than Salmonella. Many previous
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studies have found that chicken is more contaminated with Campylobacter compared to Salmonella
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(Berghaus et al., 2013; Huang et al., 2015; Madden, Moran, Scates, McBride, & Kelly, 2011). The
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variable prevalence of Campylobacter and Salmonella on poultry carcasses around the world can be
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resultant of the type and number of samples, different methods of collection of samples, transport
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conditions, laboratory methods, and different sanitary conditions on poultry farms and
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slaughterhouses. 5
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Campylobacter isolates were resistant to these antibiotics by Disk Diffusion Method and Minimal
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Inhibitory Concentration (MIC). A high-level resistance, substantiated by the values obtained at CIM,
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ranging from ≥ 8 µg.mL-1 to ≤ 64 µg.mL-1 for enrofloxacin and from ≥ 16 µg.mL-1 to ≤ 128 µg.mL-1
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for ciprofloxacin were observed. One C. jejuni and two C. coli strains had MIC = 64 µg/mL to
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enrofloxacin, and one C. jejuni and three C. coli strains had MIC = 128 µg/mL to ciprofloxacin by the
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agar dilution method (Table 3).
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Salmonella isolates in this study were sensitive to fluoroquinolones and although several
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studies around the world have reported high prevalence of Salmonella resistant strains to
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fluoroquinolones, no resistance was also observed in studies in Iran (Dallal et al., 2010), Spain
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(Álvarez-Fernández, Alonso-Calleja, García-Fernández, & Capita, 2012) and USA (Berrang et al.,
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2009). On the other hand, our findings revealed a high prevalence (100%) of fluoroquinolone-resistant
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Campylobacter in chicken carcasses. Results from other studies performed in Brazil, also showed a
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high C. jejuni - resistance rate of 100% and 95% to ciprofloxacin (de Moura et al., 2013; Hungaro et
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al., 2015), respectively. In other countries, similar results were found such as in Korea (88%) (Kang et
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al., 2006), South Africa (91%) (Bester & Essack, 2008), China (98%) (Chen et al., 2010), and Italy
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(79%) (Nobile, Costantino, Bianco, Pileggi, & Pavia, 2013). Our study also revealed a high level of
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resistance to the fluoroquinolones, since 77.27% of C. jejuni and 76.32% of C. coli had ciprofloxacin
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MIC’s ≤ 64 µg.mL-1 and four strains (4.87%) had MIC’s ≤ 128 µg.mL-1. A lower level of resistance to
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enrofloxacin was observed when compared to ciprofloxacin, since only one strain of C. jejuni (2.27%)
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and two strains of C. coli (5.26%) had enrofloxacin MIC’s = 64 µg.mL-1.
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Accordingly to Mayrhofer, Paulsen, Smulders, & Hilbert (2004), the lack of resistance by
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Salmonella strains might originate from the function of the main target of the drugs, the DNA gyrase.
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It might well be that in aerobically growing bacteria as Salmonella and E. coli, DNA gyrase has a
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more important role to stabilize the helix structure, as the helix is less supercoiled than in
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anaerobically or micro aerobically growing bacteria and that could explain why it is most common to
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find high quinolones resistance in Campylobacter than in Salmonella. Furthermore, fluoroquinolone
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treatment in Campylobacter rapidly selects high levels of resistance and a single point mutation in the
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gyrA gene is sufficient to confer high level of fluoroquinolone resistance (McDermott et al., 2002).
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This appears to be a characteristic of Campylobacter, since in the results reported by van Boven,
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Veldman, Jong, & Mevius (2003) the treatment with fluoroquinolones could reduce the presence of
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Escherichia coli below the level of detection and do not induced resistance in this organism.
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Although fluoroquinolone-resistant Campylobacter has already been well reported, the high
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level and prevalence of resistance found in this study appear to be among the most alarming. This is a
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concerning situation once fluoquinolone-resistant Campylobacter become dominant in poultry even in
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the absence of selection pressure (Luo et al., 2005) and only ban the use of it in poultry production
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would be inefficient. This enhances the potential role of raw chicken carcasses in the circulation of 6
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resistant Campylobacter strains in humans in Brazil, and demands more careful attention on antibiotics
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use in the animal production. In countries where fluoroquinolones are not accepted to use in poultry
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production, no ciprofloxacin and enrofloxacin resistant Campylobacter were isolated from poultry,
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such as in Australia and Norway (Miflin, Templeton, & Blackall, 2007; Norström et al., 2006). Fluoroquinolones are not labelled for use as growth promotion in Brazil. However,
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enrofloxacin is routinely used in poultry production for preventive and therapeutic purposes and might
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represent one potential source of pollution to the environment, enhancing fluoroquinolone resistance.
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Leal, Figueira, Tornisielo, & Regitano (2012) evaluated sorption–desorption and occurrence of
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commonly used fluoroquinolones in poultry litter and soil samples from São Paulo state in Brazil and
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found enrofloxacin as the most often detected compound, present in 30% of poultry litters and in 27%
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of soils at the highest mean concentrations.
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4. Conclusions
Besides the presence of Salmonella, this study reveals a significant contamination of chicken
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carcasses with fluoroquinolone-resistant Campylobacter in Rio de Janeiro state. These data may draw
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the attention about the widespread use of fluoroquinolones at poultry production in Brazil and alert
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veterinary services about these enteric bacteria in chicken products in order to avoid their occurrence
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at the poultry farming and to improve the hygiene practices at the slaughter.
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Acknowledgments
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We wish to thank Dr. Dalia Rodrigues (FIOCRUZ) for Salmonella serotyping. This study was supported by a grant from the CAPES for post-graduation program in Brazil.
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ACCEPTED MANUSCRIPT Table 1 Total prevalence, PCR detection and serotype identification of Salmonella isolated from chicken carcasses slaughtered in State of Rio de Janeiro, Brazil, 2013. Sample identification
Serotype
PCR detection
A
-
Not isolated
Negative
11
S. Typhimurium
12
S Albany
14
S Albany
C
-
Not isolated
D
-
Not isolated
Negative
E
-
Not isolated
Negative
51
F
52
Salmonella spp. Salmonella spp. Negative
Not isolated
Salmonella spp.
S. Typhimurium
Salmonella spp.
4 (6.67)
5 (8.33)
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Table 2
Salmonella spp.
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Campylobacter prevalence and distribution among chicken carcasses slaughtered in State of Rio de Janeiro, Brazil, 2013. Contaminated chicken carcass (%)
C. jejuni (%)
C. coli (%)
Total
A
3 (30%)
11 (13.41%)
Not isolated
11
7 (70%)
06 (7.32%)
17 (20.73%)
23
6 (60%)
01 (1.22%)
18 (21.95%)
19
D
2 (20%)
03 (3.66%)
Not isolated
03
E
1 (10%)
01 (1.22%)
01 (1.22%)
02
F
8 (80%)
22 (26.83%)
02 (2.44%)
24
Total
27 (45%)
44 (53.66%)
38 (46.34%)
82
B
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ACCEPTED MANUSCRIPT Table 3 Enrofloxacin and ciprofloxacin resistance level of Campylobacter isolates by Minimal Inhibitory Concentration (MIC) tested. Enrofloxacin
64 µg/mL
32 µg/mL
16 µg/mL
C. jejuni 44 (100%) C. coli 38 (100%)
01 (2.27%)
01 (2.27%)
15 (34.09%)
02 (5.26%)
05 (13.16%)
18 (47.37%)
8 µg/mL
4 µg/mL
41 (93.18%)
44 (100%)
32 (84.21%)
38 (100%)
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Species (n) (%)
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Dilutions
Ciprofloxacin
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Dilutions 128 µg/mL
64 µg/mL
32 µg/mL
16 µg/mL
8 µg/mL
C. jejuni 44 (100%) C. coli 38 (100%)
01 (2.27%)
34 (77.27%)
42 (95.45%)
42 (95.45%)
44 (100%)
03 (7.89%)
29 (76.32%)
30 (78.95%)
32 (84.21%)
38 (100%)
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Species (n) (%)
2
S0956-7135(15)30224-3
DOI:
10.1016/j.foodcont.2015.10.002
Reference:
JFCO 4680
To appear in:
Food Control
Received Date: 12 July 2015 Revised Date:
2 October 2015
Accepted Date: 6 October 2015
Please cite this article as: Panzenhagen P.H.N., Aguiar W.S., da Silva Frasão B., de Almeida Pereira V.L., da Costa Abreu D.L., dos Prazeres Rodrigues D., do Nascimento E.R. & de Aquino M.H.C., Prevalence and fluoroquinolones resistance of Campylobacter and Salmonella isolates from poultry carcasses in Rio de Janeiro, Brazil, Food Control (2015), doi: 10.1016/j.foodcont.2015.10.002. 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
Prevalence and fluoroquinolones resistance of Campylobacter and
2
Salmonella isolates from poultry carcasses in Rio de Janeiro, Brazil
3
Pedro Henrique Nunes Panzenhagena*, Waldemir Silva Aguiara, Beatriz da Silva Frasãoa,
4
Virginia Léo de Almeida Pereiraa, Dayse Lima da Costa Abreua, Dalia dos Prazeres
5
Rodriguesb, Elmiro Rosendo do Nascimentoa, Maria Helena Cosendey de Aquinoa
7 8 9
a
Department of Veterinary Public Health and Public Health, Federal Fluminense University (UFF), 24230-
340, 64 Vital Brazil Filho Street, Niterói, RJ, Brazil. b
National Reference Laboratory Diagnosis of Enteric Bacteria, Oswaldo Cruz Institute, Oswaldo Cruz
Foundation (FIOCRUZ), 21045-900, 4365 Brazil Avenue, Rio de Janeiro, RJ, Brazil
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* Corresponding Author at: Analytical Laboratory Center, Department of Food Technology, Faculty of
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Veterinary, Fluminense Federal University, 64, Vital Brazil Filho, Niterói - Rio de Janeiro - Brazil Zip Code:
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24230-340. E-mail: [email protected] / Phone: +55-21-99567-9533 / +55-21-2268-5367
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13 Abstract
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To investigate the prevalence of Campylobacter and Salmonella in poultry carcasses in state of Rio de
16
Janeiro, Brazil, 60 samples from 6 slaughterhouses were collected over a period of 6 months. A total
17
of 82 Campylobacter isolates were obtained from twenty seven (45%) positive chicken carcasses,
18
including 44 isolates (53.66%) of Campylobacter jejuni and 38 (46.34%) of Campylobacter coli. The
19
identification of all strains was confirmed by PCR. Salmonella was isolated from 4 (6.67%) carcasses
20
by conventional method and was detected in 5 (8.33%) of 60 chicken carcasses by PCR. Two
21
Salmonella Albany and two Salmonella Typhimurium were identified. Antimicrobial susceptibility
22
testing was primarily done by the disk diffusion method and later by assessing minimum inhibitory
23
concentrations (MICs) against all the isolates. All the Campylobacter isolates were resistant to
24
ciprofloxacin and enrofloxacin. It was observed high MIC values for enrofloxacin (64 µg/mL) in one
25
C. jejuni and two C. coli strains, and for ciprofloxacin (≥ 128 µg/mL) in one C. jejuni and three C.
26
coli strains. No Salmonella isolate was resistant to these antibiotics by both methods. These findings
27
reveal a broad extent of fluoroquinolone resistance in Campylobacter isolates from chicken carcasses
28
in Brazil and underline the need for prudent use of these antibiotics in poultry production to minimize
29
the spread of fluoroquinolone resistant Campylobacter.
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Keywords: Campylobacter, Salmonella, Enrofloxacin, Ciprofloxacin, Chicken carcasses, Brazil
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1. Introduction
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Salmonellosis and Campylobacteriosis are among the most frequently reported foodborne
34
diseases worldwide. While numerous potential vehicles of transmission exist, commercial chicken
35
meat has been identified as one of the most important food vehicles for these organisms (FAO/WHO, 1
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2009). Although The Center for Disease Control and Prevention (CDC) reports Salmonella as leading
37
causes of hospitalization by foodborne illness in United States (Scallan et al., 2011), interestingly,
38
Campylobacter remains the most commonly reported gastrointestinal bacterial pathogen in humans
39
since 2005 within European Union population (EFSA, 2013). In developing countries, outbreak information is frequently incomplete because health
41
authorities lack the capabilities or resources for detection, or presumably, because diarrheal diseases
42
are highly endemic and outbreaks may be less common or obvious than in industrialized countries
43
(Zaidi et al., 2008). Despite this incomplete outbreak information in Brazil there are several reports of
44
Salmonella prevalence in chicken carcasses ranging from 5.9% to 86.7% (Cardoso & Tessari, 2008;
45
Duarte et al., 2009; Fuzihara, Fernandes, & Franco, 2000; Matheus, Rudge, & Gomes, 2003; Oliveira
46
et al., 2006).
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Campylobacter prevalence around the world is very variable and range from 0.29% to 96.7%
48
in chicken carcass (Aquino, Pacheco, Ferreira, & Tibana, 2002; Garin et al., 2012; Wang, Guo, & Li,
49
2013). In Brazil, the Campylobacter presence is not investigated in most cases of bacterial
50
gastroenteritis because the methodologies of isolation and characterization are different from those
51
used in the research of common enteropathogenic bacteria, such as Salmonella, Shigella and the E.
52
coli group. However, some authors such as Aquino et al. (2002) and Hungaro et al. (2015) found 60%
53
and 16.8% of Campylobacter prevalence in chicken carcasses respectively.
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Fluoroquinolones, such as ciprofloxacin and enrofloxacin, have an extensive application both
55
in human and veterinary medicine with spectrum of action over Gram-negative and Gram-positive
56
bacteria (Ruiz, 2003). Enrofloxacin, a quinolone developed exclusively for use in animals, has a wide
57
antibacterial activity and is commonly used in poultry production in Brazil. Ciprofloxacin, a
58
metabolite of enrofloxacin (Idowu, Peggins, Cullison, & Bredow, 2010), besides its use in poultry
59
production, is also used for the treatment of human Salmonellosis and Campylobacteriosis (Agunos et
60
al., 2013). Once fluoroquinolones residues could persists in the animal body and may result in the
61
development of bacteria resistant strains, several studies have linked the therapeutically and
62
prophylactically use with the emergence and spread of resistance from these pathogens (Cheng et al.,
63
2012; Finley et al., 2013; Yan, Wang, Qin, Liu, & Du, 2011).
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Antimicrobial resistance is an increasing worldwide concern and has been developed over the
65
past 30 years regarding the emergence of multidrug-resistant phenotypes among Salmonella and
66
Campylobacter (Hur, Jawale, & Lee, 2012). Alarmed by the rise in multidrug-resistant Salmonella in
67
the 1960s, the United Kingdom’s Swann Report of 1969 recognized the possibility that AGPs
68
(Antimicrobial Growth Promoters) were contributing largely to the problem of drug-resistant
69
infections. These reports concluded that animal growth promotion with antibiotics used for human
70
therapy should be banned. However, this practice has continued in many countries although with
71
antibiotics that are not used therapeutically in humans (Marshall & Levy, 2011). 2
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We notice that little is known with regards to the simultaneous occurrence of Campylobacter
73
and Salmonella on chicken carcasses in Brazil, and their resistance to fluoroquinolones. Therefore, the
74
aim of this study was to investigate their prevalence and pattern of enrofloxacin and ciprofloxacin
75
resistance in carcasses of slaughtered chicken in Rio de Janeiro State, Brazil.
77
2. Materials and methods
78
2.1. Sample collection
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During 6 months in 2013, 60 chicken carcasses were collected from 6 slaughterhouses in state
80
of Rio de Janeiro, Brazil. From each slaughterhouse, 10 chicken carcasses were randomly collected
81
from the chiller tank and transported on ice in sterilized plastic bags to the laboratory. Microbiological
82
analyses were carried out within at least 3 hours after collection. The slaughterhouses were also
83
randomly selected and its identification were preserved changing their names by the letters A to F. 2.2. Salmonella examination method
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Skin samples of neck, breast and cloacal (25g) were homogenized in a stomacher (Stomacher
86
80 Laboratory Blender Seward) for 2 minutes and pre enriched with 225 mL of buffered peptone water
87
(BPW) at 37°C for 24h. After incubation, isolation of Salmonella was performed in general
88
accordance with U.S.FDA Bacteriological Analytical Manual (Hammack, Andrews, & Jacobson,
89
2014). Isolates were subjected to Salmonella Poly O and Poly H antibody assays (Probac do Brazil®). At the same time, Salmonella detection was performed by PCR. The analyses were carried out
91
using 1.0 mL of 24h pre-enrichment incubated buffered peptone water (BPW) at 37°C. DNA
92
extraction and amplifications were performed in accordance with Myint, Johnson, Tablante, &
93
Heckert (2006). The primer set of ST 11 (AGC CAA CCA TTG CTA AAT TGG CGC A) and ST 15
94
(GGT AGA AAT TCC CAG CGG GTA CTG), originally designed by Aabo, Rasmussen, Roseen,
95
Sørensen, & Olsen (1993), is highly specific for Salmonella species and defines an amplified fragment
96
of 429 bp. Salmonella isolates were sent to the National Reference Center, Institute Oswaldo Cruz,
97
Rio de Janeiro, Brazil for serotyping.
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2.3. Campylobacter examination method The chicken carcass were rinsed with 250 mL of 0.1% buffered peptone water and massaged
100
in sterile plastic bag. Loopfuls were used for Campylobacter isolation according to Stern, Patton,
101
Doyle, Park, & Mccardell (1992) and 3 to 5 suspected colonies per each plate were picked and
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identificated by PCR in accordance with Harmon, Ransom, & Wesley (1997). DNA was extracted
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with the commercial extraction kit 'Wizard® Genomic DNA Purification Kit' (PROMEGA®). Primers
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used were pg3/pg50 that amplify a conserved region in the two species (C. jejuni and C. coli) related
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to flagellin gene and primers C-1/C-4 which amplify a specific region of the species C. jejuni strains.
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The amplification reaction was performed with a final volume of 50 mL containing 5µL of the sample 3
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dGTP and dTTP, 0.4 uM of each primer pg3 and pg50, 0.2 µM of each primer C1 and C4, 2.5 U Taq
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polymerase and 5.5 mM/L MgCl2. The initial denaturation was performed at 94°C for four minutes
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followed by 25 amplification cycles consisting of one minute at 94°C, one minute at 55°C, one minute
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at 72°C and extension at 72°C for seven minutes. Verification of amplicons was performed in a
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horizontal electrophoresis tank 'Electrophoresis Cell (BioAmérica) with 0.5x TBE, with Pac Power
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source 300 (Bio-Rad) in 1.5% agarose gel stained with GelRed.
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2.4. Antimicrobial susceptibility test
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Salmonella and Campylobacter isolates were tested for enrofloxacin (5µg) and ciprofloxacin (5µg)
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(Cefar Brazil) susceptibilities. The susceptibility testing was primarily done by the disk diffusion
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method and later by assessing minimum inhibitory concentrations (MICs) against all the resistant
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isolates detected by disk diffusion method. The minimal inhibitory concentration (MIC) was
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determined by the agar dilution method containing ciprofloxacin and enrofloxacin (Sigma-Aldrich®)
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on the following concentrations: 128 µg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL, 8 µg/mL, 4 µg/mL, 2
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µg/mL, 1 µg/mL, 0.5 µg/mL (NCCLS, 2003). The MIC were decided based on visible growth and
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breakpoint of ≥ 4 µg/mL for ciprofloxacin (CLSI, 2008) and enrofloxacin (Chen et al., 2010) were
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used. C. jejuni ATCC 33560 and C. Coli ATCC 33559 were included on every plate as a quality
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control.
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2.5. Statistical analysis
Statistical analyses for Salmonella detection methods followed procedures described
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previously (Thrusfield, 2007). The program InStat, version 3.1 (GraphPad, 2009) was used for the
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calculations. The Chi-square test and Fisher’s exact two-tailed test were used for statistical analysis. A
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P value < 0.05 was used for statistical significance.
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3. Results and Discussion
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Salmonella was isolated from 4 (6.67%) of 60 samples. Two S. Albany and one S. Typhimurium
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were isolated from the slaughterhouse “B” and one S. Typhimurium was isolated from the
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slaughterhouse “F” (Table 1). Salmonella was detected in 5 (8.33%) of 60 chicken carcasses by PCR,
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but only four Salmonella positive carcasses by isolation matched the PCR results (Table 1). No
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significant statistical differences were found between conventional isolation and PCR detection by the
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Fisher Exact Test (p > 0.05).
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Relating to other previous investigations conducted in Brazil, our results have shown a slightly
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lower overall Salmonella contamination of chicken carcasses by both detection techniques when
139
compared to Fuzihara et al. (2000) (41%) and Oliveira et al. (2006) (11.8%). However, similar results
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were described by Matheus et al. (2003) (5.9%) and Duarte et al. (2009) (9.6%). Salmonella
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prevalence observed in this study (6.67%) is also much lower than the observed in other developing
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countries including Iran (33%) (Dallal et al., 2010), Cambodia (88.2%) (Lay, Vuthy, Song, Phol, & 4
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Sarthou, 2011) Vietnam (45.9%) (Ta et al., 2012), and Colombia (27%) (Donado-Godoy et al., 2012).
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It is well known that laborers training, good manufacturing practices and HACCP influence the
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contamination level during the slaughter process. Most of visited slaughterhouses had good
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manufacturing practices what could explain the low prevalence described in this study. Although the PCR technique has detected 5 positive samples versus 4 positive samples
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obtained by isolation, no statistical difference was found between both methods. Salmonella
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Typhimurium and S. Albany were the only serotypes identified in this research. Besides S. Enteritidis,
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S. Typhimurium head the two top serotypes responsible for human salmonellosis in the world
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(Hendriksen et al., 2011). Although S. Albany is an uncommon serotype on chicken carcass around
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the world, this was not the first time that this serotype was isolated from chicken carcasses in Brazil
153
(Fuzihara et al., 2000). Ta et al. (2014), also found S. Albany serotype on Chicken carcasses from
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Retail Markets in Vietnam.
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Unlike Salmonella, Campylobacter was isolated from all slaughterhouses investigated in this
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study, and 82 Campylobacter isolates were obtained from 27 (45%) chicken carcasses contaminated
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with this pathogen. Forty four isolates (53.66%) were identified as Campylobacter jejuni and 38
158
(46.34%) as Campylobacter coli. (Table 2). Campylobacter jejuni was predominant and these data are
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similar to reports from other countries as in Senegal, Cameroon, New Caledonia, Madagascar,
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Vietnam (Garin et al., 2012) and China (Huang, Zong, Zhao, Zhu, & Jiao, 2015).
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We found a prevalence of Campylobacter in carcasses ranging from 10% to 80% depending on
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the slaughterhouse. In two slaughterhouses (B and F) was observed a higher prevalence of both
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Campylobacter and Salmonella, what could be explained by the poor conditions of the evisceration
164
process. On the other hand, no Salmonella and a lower Campylobacter prevalence were found in
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slaughterhouses D and E. The occurrence of both Salmonella and Campylobacter in the same broiler
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samples was also reported before (Franz, van der Fels-Klerx, Thissen, & van Asselt, 2012) and their
167
presence in this product is resultant of the contamination level and hygienic processing in poultry
168
raising sites, layers, hatcheries, chicken farms and slaughterhouses. In this study, the investigated
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slaughterhouses were registered in State Inspection Service. In Brazil, there is a Pathogen Reduction
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Program of Agriculture Ministry for detection of Salmonella sp. in broiler and turkey carcasses in
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natura, involving all slaughterhouses registered in Federal Inspection Service, however, there is no
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requirement for Campylobacter detection.
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We found in this study, Campylobacter more prevalent than Salmonella. Many previous
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studies have found that chicken is more contaminated with Campylobacter compared to Salmonella
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(Berghaus et al., 2013; Huang et al., 2015; Madden, Moran, Scates, McBride, & Kelly, 2011). The
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variable prevalence of Campylobacter and Salmonella on poultry carcasses around the world can be
177
resultant of the type and number of samples, different methods of collection of samples, transport
178
conditions, laboratory methods, and different sanitary conditions on poultry farms and
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slaughterhouses. 5
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Campylobacter isolates were resistant to these antibiotics by Disk Diffusion Method and Minimal
182
Inhibitory Concentration (MIC). A high-level resistance, substantiated by the values obtained at CIM,
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ranging from ≥ 8 µg.mL-1 to ≤ 64 µg.mL-1 for enrofloxacin and from ≥ 16 µg.mL-1 to ≤ 128 µg.mL-1
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for ciprofloxacin were observed. One C. jejuni and two C. coli strains had MIC = 64 µg/mL to
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enrofloxacin, and one C. jejuni and three C. coli strains had MIC = 128 µg/mL to ciprofloxacin by the
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agar dilution method (Table 3).
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Salmonella isolates in this study were sensitive to fluoroquinolones and although several
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studies around the world have reported high prevalence of Salmonella resistant strains to
189
fluoroquinolones, no resistance was also observed in studies in Iran (Dallal et al., 2010), Spain
190
(Álvarez-Fernández, Alonso-Calleja, García-Fernández, & Capita, 2012) and USA (Berrang et al.,
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2009). On the other hand, our findings revealed a high prevalence (100%) of fluoroquinolone-resistant
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Campylobacter in chicken carcasses. Results from other studies performed in Brazil, also showed a
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high C. jejuni - resistance rate of 100% and 95% to ciprofloxacin (de Moura et al., 2013; Hungaro et
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al., 2015), respectively. In other countries, similar results were found such as in Korea (88%) (Kang et
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al., 2006), South Africa (91%) (Bester & Essack, 2008), China (98%) (Chen et al., 2010), and Italy
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(79%) (Nobile, Costantino, Bianco, Pileggi, & Pavia, 2013). Our study also revealed a high level of
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resistance to the fluoroquinolones, since 77.27% of C. jejuni and 76.32% of C. coli had ciprofloxacin
198
MIC’s ≤ 64 µg.mL-1 and four strains (4.87%) had MIC’s ≤ 128 µg.mL-1. A lower level of resistance to
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enrofloxacin was observed when compared to ciprofloxacin, since only one strain of C. jejuni (2.27%)
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and two strains of C. coli (5.26%) had enrofloxacin MIC’s = 64 µg.mL-1.
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Accordingly to Mayrhofer, Paulsen, Smulders, & Hilbert (2004), the lack of resistance by
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Salmonella strains might originate from the function of the main target of the drugs, the DNA gyrase.
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It might well be that in aerobically growing bacteria as Salmonella and E. coli, DNA gyrase has a
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more important role to stabilize the helix structure, as the helix is less supercoiled than in
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anaerobically or micro aerobically growing bacteria and that could explain why it is most common to
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find high quinolones resistance in Campylobacter than in Salmonella. Furthermore, fluoroquinolone
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treatment in Campylobacter rapidly selects high levels of resistance and a single point mutation in the
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gyrA gene is sufficient to confer high level of fluoroquinolone resistance (McDermott et al., 2002).
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This appears to be a characteristic of Campylobacter, since in the results reported by van Boven,
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Veldman, Jong, & Mevius (2003) the treatment with fluoroquinolones could reduce the presence of
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Escherichia coli below the level of detection and do not induced resistance in this organism.
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Although fluoroquinolone-resistant Campylobacter has already been well reported, the high
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level and prevalence of resistance found in this study appear to be among the most alarming. This is a
214
concerning situation once fluoquinolone-resistant Campylobacter become dominant in poultry even in
215
the absence of selection pressure (Luo et al., 2005) and only ban the use of it in poultry production
216
would be inefficient. This enhances the potential role of raw chicken carcasses in the circulation of 6
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resistant Campylobacter strains in humans in Brazil, and demands more careful attention on antibiotics
218
use in the animal production. In countries where fluoroquinolones are not accepted to use in poultry
219
production, no ciprofloxacin and enrofloxacin resistant Campylobacter were isolated from poultry,
220
such as in Australia and Norway (Miflin, Templeton, & Blackall, 2007; Norström et al., 2006). Fluoroquinolones are not labelled for use as growth promotion in Brazil. However,
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enrofloxacin is routinely used in poultry production for preventive and therapeutic purposes and might
223
represent one potential source of pollution to the environment, enhancing fluoroquinolone resistance.
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Leal, Figueira, Tornisielo, & Regitano (2012) evaluated sorption–desorption and occurrence of
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commonly used fluoroquinolones in poultry litter and soil samples from São Paulo state in Brazil and
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found enrofloxacin as the most often detected compound, present in 30% of poultry litters and in 27%
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of soils at the highest mean concentrations.
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4. Conclusions
Besides the presence of Salmonella, this study reveals a significant contamination of chicken
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carcasses with fluoroquinolone-resistant Campylobacter in Rio de Janeiro state. These data may draw
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the attention about the widespread use of fluoroquinolones at poultry production in Brazil and alert
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veterinary services about these enteric bacteria in chicken products in order to avoid their occurrence
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at the poultry farming and to improve the hygiene practices at the slaughter.
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Acknowledgments
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We wish to thank Dr. Dalia Rodrigues (FIOCRUZ) for Salmonella serotyping. This study was supported by a grant from the CAPES for post-graduation program in Brazil.
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ACCEPTED MANUSCRIPT Table 1 Total prevalence, PCR detection and serotype identification of Salmonella isolated from chicken carcasses slaughtered in State of Rio de Janeiro, Brazil, 2013. Sample identification
Serotype
PCR detection
A
-
Not isolated
Negative
11
S. Typhimurium
12
S Albany
14
S Albany
C
-
Not isolated
D
-
Not isolated
Negative
E
-
Not isolated
Negative
51
F
52
Salmonella spp. Salmonella spp. Negative
Not isolated
Salmonella spp.
S. Typhimurium
Salmonella spp.
4 (6.67)
5 (8.33)
TE D
Total (%)
Table 2
Salmonella spp.
SC
M AN U
B
RI PT
Slaughterhouse
Campylobacter prevalence and distribution among chicken carcasses slaughtered in State of Rio de Janeiro, Brazil, 2013. Contaminated chicken carcass (%)
C. jejuni (%)
C. coli (%)
Total
A
3 (30%)
11 (13.41%)
Not isolated
11
7 (70%)
06 (7.32%)
17 (20.73%)
23
6 (60%)
01 (1.22%)
18 (21.95%)
19
D
2 (20%)
03 (3.66%)
Not isolated
03
E
1 (10%)
01 (1.22%)
01 (1.22%)
02
F
8 (80%)
22 (26.83%)
02 (2.44%)
24
Total
27 (45%)
44 (53.66%)
38 (46.34%)
82
B
AC C
C
EP
Slaughterhouse
1
ACCEPTED MANUSCRIPT Table 3 Enrofloxacin and ciprofloxacin resistance level of Campylobacter isolates by Minimal Inhibitory Concentration (MIC) tested. Enrofloxacin
64 µg/mL
32 µg/mL
16 µg/mL
C. jejuni 44 (100%) C. coli 38 (100%)
01 (2.27%)
01 (2.27%)
15 (34.09%)
02 (5.26%)
05 (13.16%)
18 (47.37%)
8 µg/mL
4 µg/mL
41 (93.18%)
44 (100%)
32 (84.21%)
38 (100%)
SC
Species (n) (%)
RI PT
Dilutions
Ciprofloxacin
M AN U
Dilutions 128 µg/mL
64 µg/mL
32 µg/mL
16 µg/mL
8 µg/mL
C. jejuni 44 (100%) C. coli 38 (100%)
01 (2.27%)
34 (77.27%)
42 (95.45%)
42 (95.45%)
44 (100%)
03 (7.89%)
29 (76.32%)
30 (78.95%)
32 (84.21%)
38 (100%)
AC C
EP
TE D
Species (n) (%)
2