Plasmid-mediated quinolone resistance in Salmonella serotypes isolated from chicken carcasses in Turkey

Quinolones have been extensively used for treatment of a variety of invasive and systemic infections of salmonellosis. Widespread use of these agents has been associated with the emergence and dissemination of quinolone-resistant pathogens. The quinolone resistance and plasmid-mediated quinolone resistance determinants (qnrA, qnrB, qnrS and aac(6’)-Ib-cr) of 85 Salmonella isolates from chicken carcasses were investigated in this study. Isolates were serotyped according to the Kauffman-White-Le Minor scheme, and broth microdilution method was used to determine quinolone resistance. Plasmid-mediated quinolone resistance genes were investigated by real-time PCR and positive results were confirmed by sequencing. Among the Salmonella isolates, 30/85 (35%) and 18/85 (21%) were found to be resistant to enrofloxacin (MIC ≥ 2 mg/ml), and danofloxacin (MIC ≥ 2 mg/ml), respectively. All the isolates were negative for qnrA, qnrB and aac(6’)-Ib-cr genes, nevertheless 2% (S. Brandenburg and S. Dabou) were positive for qnrS (qnrS1 determinant). This study is the first and unique investigating the plasmidmediated quinolone resistance determinants of Salmonella isolated from chicken carcasses in Turkey. Quinolone susceptibility, qnr, aac(6’)-Ib-cr, real-time PCR, minimal inhibition concentration Salmonellae are among the most common Gram negative pathogens implicated in foodborne illnesses and also a major public health concern worldwide (Guner et al. 2012). Chicken meat, fresh and processed meat and eggs have been the most commonly incriminated foodstuffs in several outbreaks of salmonellosis (Tauxe 1991; Benenson and Chin 1995; Mead et al. 1999). Quinolone resistant Salmonella isolates have been documented in numerous locations with variable prevalence including Japan (Taguchi et al. 2009) and Brasil (Ferrar i et al. 2011). Molbak et al. (2002) reported that quinolone resistance in Salmonella spp. isolated in Denmark had increased almost 10 fold in five years. Unfortunately, these results indicate that widespread fluoroquinolone use for the treatment of infections both in human and in veterinary medicine has led to increasing numbers of quinolone resistant Salmonella isolates. Several reports in Turkey have evaluated the plasmid-mediated quinolone resistant (PMQR) in animal isolates of Escherichia coli strains (Cengiz et al. 2012; Mustak et al. 2012). However, this study is the first and unique one investigating the PMQR determinants of Salmonella isolated from chicken carcasses in Turkey. The aim of this study was to investigate the presence of PMQR determinants of qnrA, qnrB, qnrS, aac(b)-1b-cr in Salmonella strains isolated from chicken carcasses between 2005 and 2009 in the Bursa and Ankara provinces of Turkey. ACTA VET. BRNO 2014, 83: 281–286; doi:10.2754/avb201483040281 Address for correspondence: Dr. Artun Yibar Department of Food Hygiene and Technology Faculty of Veterinary Medicine, Uludag University 16059, Gorukle Campus, Bursa, Turkey Phone: +90 224 294 1338 Fax: +90 224 294 1202 E-mail: artunyibar@hotmail.com http://actavet.vfu.cz/ Materials and Methods


Quinolone susceptibility, qnr, aac(6')-Ib-cr, real-time PCR, minimal inhibition concentration
Salmonellae are among the most common Gram negative pathogens implicated in foodborne illnesses and also a major public health concern worldwide (Guner et al. 2012).Chicken meat, fresh and processed meat and eggs have been the most commonly incriminated foodstuffs in several outbreaks of salmonellosis (Tauxe 1991;Benenson and Chin 1995;Mead et al. 1999).Quinolone resistant Salmonella isolates have been documented in numerous locations with variable prevalence including Japan (Taguchi et al. 2009) and Brasil (Ferrari et al. 2011).Molbak et al. (2002) reported that quinolone resistance in Salmonella spp.isolated in Denmark had increased almost 10 fold in five years.Unfortunately, these results indicate that widespread fluoroquinolone use for the treatment of infections both in human and in veterinary medicine has led to increasing numbers of quinolone resistant Salmonella isolates.
Several reports in Turkey have evaluated the plasmid-mediated quinolone resistant (PMQR) in animal isolates of Escherichia coli strains (Cengiz et al. 2012;Mustak et al. 2012).However, this study is the first and unique one investigating the PMQR determinants of Salmonella isolated from chicken carcasses in Turkey.The aim of this study was to investigate the presence of PMQR determinants of qnrA, qnrB, qnrS, aac(b)-1b-cr in Salmonella strains isolated from chicken carcasses between 2005 and 2009 in the Bursa and Ankara provinces of Turkey.

Bacterial strains
A total of 85 Salmonella spp.strains which were previously isolated from chicken carcasses in Bursa and Ankara provinces of Turkey between 2005 and 2009 were obtained from Uludag University, Faculty of Veterinary Medicine Department of Food Hygiene and Technology laboratory.All presumptive Salmonella-positive isolates were confirmed by biochemical tests according to ISO 6579:2002 and serotyped according to the Kauffman-White-Le Minor scheme (ISO 2002;Grimont and Weill 2007).

Antimicrobial susceptibility testing
Enrofloxacin and danofloxacin MICs were determined by broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2012a).Enrofloxacin and danofloxacin analytical standards were obtained from Fluka (Sleinheim, Germany) and dissolved in 20:80 methanol/phosphate buffered saline (v/v).Salmonella cultures were prepared in Mueller-Hinton Broth at 37 °C for 16-20 h.Freshly prepared stock solution was sterilized using 0.20 µm single-use filter units (Minisart ® , Sartorius Stendim Biotech, Göttingen, Germany).Enrofloxacin and danofloxacin dilutions ranging from 0.008 to 256 mg/l were prepared in Mueller-Hinton Broth.Inoculants with a density equivalent to 0.5 McFarland turbidity standard were prepared and each of enrofloxacin and danofloxacin dilutions were added into 96 well plates (Thermo Scientific, Epsom, UK).After incubating at 37 °C for 16-20 h, the MICs were defined as the minimum concentration of enrofloxacin and danofloxacin inhibiting growth of bacteria.The optical densities (ODs) of the cultures were measured at a wave length of 595 nm (iMark™, Bio-Rad, Hercules, CA, USA).Escherichia coli ATCC 25922 was used as control for antimicrobial susceptibility testing.Susceptibility to enrofloxacin and danofloxacin were interpreted according to CLSI (CLSI 2012b).

Detection of PMQR genes
Plasmids were extracted from Salmonella isolates with a DNA-spin™ Plasmid DNA Purification Kit (iNtRON Biotechnology, Seoul, Republic of Korea) according to manufacturer's recommendations.Presence of PMQR was investigated with qnrA, qnrB, qnrS, aac(6')-Ib-cr specific primers designed for this study by real-time PCR using Melting Curve Analysis.The PMQR gene sequences were acquired from NCBI Data Bank (NCBI Data Bank) and sequence analyses were made by ClustalW programme (ClustalW2).Primers aiming secured sequences were designed manually and tested with Primer-Blast (NCBI/Primer-BLAST).
A total of 85 isolates were screened by qnr determinants using specific primers for qnrA (5′-CCG AGT TTG GCC AGA TAG AC-3′ [forward] and 5′ CTG CTC CAG CAA ATC CTG TT-3′ [reverse]), qnrB (5′-CAG TAC ACC GGC CAG AAA GT-3′ [forward] and 5′-TTG AAA TGG CAC ATG CTG AT-3′ [reverse]), and qnrS (5′-ACG ACA TTC GTC AAC TGC AA-3′ [forward] and 5′-CGA AGA TCT GCG ACA TCA AA-3′ [reverse]) and for aac(6')-Ib-cr (5′-TGA CCT TGC GAT GCT CTA TG-3′ [forward] and 5′ TGG TCT ATT CCG CGT ACT CC-3′ [reverse]).Amplification was carried out in a 50 µl volume containing 0.2 µM each of dNTP and the primer pairs at concentrations each of 2.5 pM and 2.5 U of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania), 2 µM MgCl 2 and 1 × EvaGreen.Isolated DNA (3 µl) was used as a template.The PCR was performed using an ABI 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) under the following conditions: initial denaturation for 10 min at 95 °C; 35 cycles of denaturation for 10 s at 94 °C, annealing for 15 s at 55 °C and extension for 10 s at 72 °C; and final extension for 5 min at 72 °C.The temperature had been raised to 95 °C from 50 °C by the speed of 0.5 °C/s in the melting curve analysis, meanwhile the specification of PCR products had been tested by reading the fluorescent constantly.
PCR products were purified and then sequenced with 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) by Sanger method.The sequences were compared and confirmed with the GenBank nucleotide database using the BLAST (Basic Local Alignment Search Tool) program (NCBI/BLAST Home).

Discussion
Quinolone resistance in Salmonella has been observed all over the world (Angulo et al. 2000;Helmuth 2000).In the present study, 11 (13%) and 8 (9%) Salmonella isolates were found to be resistant to enrofloxacin and danofloxacin, respectively.The results have also shown that Salmonella strains were highly resistant to quinolones.High resistances (≥ 256 mg/ml) to enrofloxacin and danofloxacin in two S. Hadar, one S. Kentucky and one S. Typhimurium may be due to mutations in quinolone resistance determining regions.Resistance to quinolones in Salmonella spp.have been reported by previous studies in chickens (Wang et al. 2006;De Jong et al. 2014).
The PMQR determinants are distributed among Enterobacteriaceae, including Salmonella, worldwide (Cattoir and Nordmann 2009).The qnrA, qnrB, qnrS, and aac(6')-Ib-cr genes are the most commonly identified resistance determinants in Turkey (Nazik et al. 2009;Ozgumus et al. 2009).The prevalence of qnr genes among the E. coli isolates obtained from animals in the EU and Turkey ranged between 3% and 15% (Veldman et al. 2011;Cengiz et al. 2012).In another study from Turkey, Nazik et al. (2009) reported qnr genes in Enterobacter spp., E. coli and K. pneumoniae.In agreement with previous studies (Kehrenberg et al. 2006;Avsaroglu et al. 2007), our results confirmed the presence of qnrS gene associated with fluoroquinolone resistance in two different Salmonella isolates from Turkey.In contrast, Yu et al. (2011), reported that all Salmonella isolates were negative for qnr genes, whereas a high prevalence (37.1%) of aac(6')-Ib-cr was found.
The presence of PMQR determinants has been reported in clinical isolates of S. Enteritidis (Cheung et al. 2005).In contrast, PMQR genes were not detected in Salmonella strains isolated from ready to eat foods and chicken meat as reported by Kim et al. (2011).Low prevalence (2%) of qnr genes in Salmonella isolates in comparison with other studies was observed.These genes were located on plasmids which are frequently associated with Enterobacteriaceae isolated from various foods and humans (Hopkins et al. 2007;Ferrari et al. 2011).
Even though the topic is widely investigated by several researchers, to our knowledge, there are only limited numbers of studies handling the subject in Turkey.By the present study, PMQR determinants in Salmonella isolated from chicken carcasses were investigated for the first time.This finding has indicated that chicken may be an important vector for the spread of quinolone-resistant Salmonella among animals, as well as from animals to humans through the food chain.
The PMQR determinants in Salmonella isolated from food-producing animals such as chickens are an important public health issue.Preventing the spread of quinolone-resistant Salmonella strains will require the establishment of continuous surveillance program for all resistance determinants at a national level.Furthermore, our results can provide useful information prompting further studies on the prevalence of quinolone-resistant Salmonella spp.

Table 1 .
Determined MIC values for Salmonella serotypes to enrofloxacin and danofloxacin.

Table 2 .
Number of Salmonella strains susceptible, intermediate susceptible, and/or resistant to enrofloxacin and danofloxacin.
a S/I/R, susceptible/intermediate susceptible/resistant
ND = not detected