Clonal relatedness and resistance patterns of Salmonella Corvallis from poultry carcasses in a Brazilian slaughterhouse

Introduction: Salmonella is a major cause of foodborne disease, and poultry products are important contributors to the transmission of this zoonotic pathogen. Although considered to be rare in most countries, Salmonella Corvallis has been reported in specific geographic areas isolated from both human and non-human sources. The aim of this study was to report the occurrence, the antimicrobial resistance profiles including the extended-spectrum beta-lactamase (ESBL) production, and the clonal relatedness of S. Corvallis strains. Methodology: A total of 132 fragments of poultry carcasses from a slaughterhouse in São Paulo State, Brazil, were collected at different stages of the manufacturing process (post-bleeding, post-plucking, and post-chilling) and analyzed for the presence of Salmonella. Antimicrobial resistance was determined by disc diffusion method and Etest. Clonal relatedness was determined by pulsed-field gel electrophoresis (PFGE). Results: Among the 272 Salmonella strains recovered, fourteen were S. Corvallis. Ten (71.4%) showed ESBL production and resistance to at least three antimicrobial agents. Nalidixic acid resistance and reduced ciprofloxacin susceptibility was verified in four (28.6%) strains. PFGE analyses showed that all the S. Corvallis strains belonged to the same pulsotype. Conclusion: This study identified genetically related S. Corvallis strains exhibiting ESBL production and reduced susceptibility to quinolone. The results suggest the need to improve the sanitary conditions in the slaughterhouse. Moreover, from a public health perspective, continuous surveillance on Salmonella is needed to control the dissemination of this important zoonotic pathogen and its resistance determinants.


Introduction
Foodborne diseases have serious economic and health implications.In the United States alone, more than 300,000 hospitalizations and 3,000 deaths were estimated to be attributed to foodborne infections in 2011 [1].Salmonella is a major agent of foodborne illness [1] and contaminated eggs, poultry meat, and by-products are considered the main sources of nontyphoidal salmonellosis [2].Although 2,610 serovars of Salmonella have been identified [3,4], the majority of human infections are caused by a limited number of serovars.S. enterica Typhimurium and S. enterica Enteritidis are the most prevalent serovars isolated from human infections, although other serovars have been recognized in specific regions [5].

S.
Corvallis is considered to be rare in most countries; however, in recent years, a higher incidence has been reported in specific geographic areas such as Bulgaria, Denmark, Tunisia and Japan [6][7][8].This serovar has been isolated from poultry, food products of animal origin, human infections, and even in outbreaks of foodborne diseases [6][7][8].
Cephalosporins and fluoroquinolones are the drugs of choice for treating patients with complicated Salmonella infections [9].However, the emergence of extended-spectrum β-lactamase enzymes and decreased susceptibility to ciprofloxacin in Salmonella strains has drastically reduced the treatment options, which may lead to unfavorable outcomes [9,10].In veterinary medicine, the use of antimicrobial drugs for therapeutic purposes and as growth promoters may stimulate the selection of antimicrobial-resistant Salmonella strains [11,12], causing a major public health concern.
Extended-spectrum β-lactamase (ESBL) production in Salmonella is not common.However, the reports of ESBL-producing Salmonella serovars are increasing worldwide over the last decades [13,14].ESBL-producing Salmonella strains have been detected in poultry and poultry products in different countries, including Brazil [14][15][16].
Limited information is available regarding the occurrence of ESBL-producing strains among the Salmonella strains recovered from the Brazilian poultry industry.In this study, we report the occurrence, antimicrobial resistance, and genetic relatedness of S. Corvallis strains isolated from poultry carcasses in the state of São Paulo, Brazil.

Salmonella isolation and identification
A total of 132 poultry fragments (breast, right and left legs quarter) from carcasses were collected in a slaughterhouse located in the Central Western area of the State of São Paulo, Brazil during 2011-2012.Samples were collected at three different manufacturing stages in the industrial plant: postbleeding, post-plucking, and post-chilling.Salmonella isolates were recovered using standard procedures consisting of a pre-enrichment with buffered peptone water 1%, enrichment with Rappaport Vassiliadis broth and Tetrathionate broth, and isolation onto Xylose Lysine Desoxycholate agar and Bismuth Sulfite agar plates [17].The presumptive isolates were confirmed to belong to the Salmonella genus based on a subset of conventional biochemical tests [18].Salmonella isolates were serotyped by agglutination tests using the somatic O, phase 1 and phase 2 of the H flagellar antisera according to the White-Kauffmann-Le Minor scheme [3].
ESBL production was confirmed by the doubledisk diffusion test when the key-hole effect was observed between the ceftazidime, cefepime, cefotaxime, or ceftriaxone and amoxicillin/clavulanate disks, indicating the partial or total restoration of the activity of cephalosporin by clavulanic acid [20].Escherichia coli ATCC25922, E. coli ATCC35218, and Pseudomonas aeruginosa ATCC27853 were used as reference strains for the antimicrobial susceptibility testing.

Pulsed field gel electrophoresis (PFGE)
PFGE was performed according to the PulseNet protocol [21] developed by the Centers for Disease Control and Prevention (CDC).Briefly, cell lysis was followed by proteinase K treatment and DNA restriction with XbaI (New England Biolabs, Ipswich, MA).Electrophoresis was performed in a CHEF-DR III system (Biorad Laboratories Inc., Hercules, CA) with the following running parameters: 6 V/cm; temperature, 14°C; initial switch, 2.2 s; final switch, 63.8 s; and length, 18 h.The Lambda Ladder PFG Marker (New England Biolabs, Ipswich, USA) was employed for gel normalization.TIFF images were analyzed with BioNumerics 5.0 software (Applied Maths), and a dendrogram was generated by the unweighted pair group method with arithmetic mean (UPGMA) and the Dice coefficient for band matching with 1.5% tolerance and 1.5% optimization.A S. Typhimurium strain was included as an experimental control.

Discussion
We report the occurrence of genetic related S. Corvallis presenting ESBL enzymes and reduced susceptibility to ciprofloxacin.S. Corvallis was originally described in 1949 [22] after being isolated from poultry, and its importance has been increasing in recent years due to its potential for causing local or global outbreaks [6,7,23,24].Detection of ESBL-producing Salmonella strains has emerged over the years [13,14].One of the largest studies on S. Corvallis strains showed that different types of ESBL were occasionally simultaneously present in strains recovered from human sources and food products in Bulgaria, Thailand, and Denmark [6].The emergence of ESBL in Salmonella isolated from animal production may be, at least in part, attributed to the selective pressure in this reservoir when antimicrobial agents are widely applied to promote growth [13,15,25].Evidence of similar cassette arrays found in pork and human Salmonella isolates suggests the potential transmission of multidrug resistance between food of animal origin and human isolates [26].
In addition to the beta-lactam resistance, the presence of S. Corvallis strains presenting resistance to nalidixic acid and reduced susceptibility to ciprofloxacin was noteworthy.Nalidixic acid resistance is predictive of fluoroquinolone resistance, as observed in this study: all of the nalidixic acidresistant strains presented reduced ciprofloxacin susceptibility.Fluoroquinolone resistance is an important concern that is less frequently reported in Salmonella than in other food-borne pathogens [27]; however, Salmonella with reduced susceptibility to fluoroquinolone has been reported worldwide [28,29] and in Brazil [30,31].In fact, to the best of our knowledge, this study is the first to report ESBLproducing strains of S. Corvallis with reduced ciprofloxacin susceptibility in Brazil.Detection of S. Corvallis strains resistant to both cephalosporins and quinolones antimicrobials in this poultry slaughterhouse may have an impact on public health, since animals can represent important reservoirs and potential vehicles for antimicrobial resistance dissemination through the food chain [16,26].
PFGE has been demonstrated to be sufficiently discriminatory for subtyping different Salmonella serovars, including S. Corvallis [6,32,33].Our finding that the genetic patterns of S. Corvallis recovered from different areas in the poultry processing plant were indistinguishable suggests that this strain is well adapted to the conditions at this plant.This adaptation may be attributed to the use of inadequate hygienic techniques [34] and/or to the ability of the bacteria to adhere to the materials used in the poultry industry [35].In addition, strains of S. Corvallis with the same pulsotype presented different resistance patterns and ESBL production, suggesting that the mechanisms involved in these phenotypes are associated with mobile genetic elements, such as plasmids.

Conclusion
This study identified genetically related S. Corvallis strains presenting reduced susceptibility to quinolone.To our knowledge, this is the first report of S. Corvallis, ESBL-producing isolates from slaughter plants in Brazil.Isolation of genetically related strains on different days of collection indicates the ability of this bacterium to survive in the environment.The well-recognized role of Salmonella as a zoonotic agent highlights the need for continuous surveillance to control the dissemination of this important pathogen.

Figure 1 .
Figure 1.Pulsed field gel electrophoresis profiles, extended-spectrum beta-lactamase (ESBL) status, isolation source, and antimicrobial resistance patterns of Salmonella Corvallis isolated from a poultry slaughterhouse in Brazil.