Prevalence and characterisation of quinolone resistance mechanisms in Salmonella spp.

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VETMIC-6503; No. of Pages 8 Veterinary Microbiology xxx (2014) xxx–xxx

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Prevalence and characterisation of quinolone resistance mechanisms in Salmonella spp. Dariusz Wasyl *, Andrzej Hoszowski, Magdalena Zaja˛c Department of Microbiology, National Reference Laboratory for Salmonellosis and Antimicrobial Resistance, National Veterinary Research Institute, Partyzanto´w 57, 24-100 Puławy, Poland

A R T I C L E I N F O

Keywords: Quinolone resistance Epidemiology QRDR PMQR Qnr Salmonella Newport

A B S T R A C T

The study was focused on characterisation of quinolone resistance mechanisms in Salmonella isolated from animals, food, and feed between 2008 and 2011. Testing of Minimal Inhibitory Concentrations revealed 6.4% of 2680 isolates conferring ciprofloxacin resistance. Simultaneously 37.7% and 40.8% were accounted for, respectively, nalidixic acid and ciprofloxacin Non Wild-Type populations. Amplification and sequencing of quinolone resistance determining region of topoisomerases genes in 44 isolates identified multiple amino-acid substitutions in gyrA at positions Ser83 (N = 22; !Leu, !Phe, !Tyr), Asp87 (N = 22; !Asn, !Gly, !Tyr) and parC (Thr57Ser, N = 23; Ala141Ser, N = 1). No relevant mutations were identified in gyrB and parE. Twelve patterns combining one or two substitutions were related to neither serovar nor ciprofloxacin MIC. In 92 isolates suspected for plasmid mediated quinolone resistance two qnr alleles were found: qnrS1 (or qnrS3; N = 50) and qnrB19 (or qnrB10; N = 24). Additionally, two isolates with chromosomally encoded mechanisms carried qnrS1 and qnrS2. All tested isolates were negative for qnrA, qnrC, qnrD, qepA, aac(60 )-Ib-cr. Both chromosomal and plasmid mediated quinolone resistance determinants were found in several Salmonella serovars and Pulsed Field Gel Electrophoresis was used to assess phylogenetic similarity of selected isolates (N = 82). Salmonella Newport was found to accumulate quinolone resistance determinants and the serovar was spreading clonally with either variable gyrA mutations, qnrS1/S3, or qnrB10/B19. Alternatively, various determinants are dispersed among related S. Enteritidis isolates. Antimicrobial selection pressure, multiple resistance determinants and scenarios for their acquisition and spread make extremely difficult to combat quinolone resistance. ß 2014 Elsevier B.V. All rights reserved.

1. Introduction Fifty years have just passed since the introduction of quinolones into medical practice and decline in their therapeutic efficacy as well as emergence and spread of various resistance mechanisms are evoked (Poirel et al., 2012). The primal enthusiasm on perfect efficacy of fully

* Corresponding author. E-mail address: [email protected] (D. Wasyl).

synthetic bactericidal antimicrobials have been trimmed by discovery of chromosomal mechanisms involving spontaneous mutations (Hopkins et al., 2005; Jeong et al., 2011). Further limitations came along with the accumulation of genetic rearrangements leading to clinical resistance and the detection of transferable resistance mechanisms in late 1990-ties (Poirel et al., 2012). Due to efficacy of low dosage and favourable pharmacokinetics, fluoroquinolones are widely used in human and animals since 1980-ties (Hopkins et al., 2005; Fabrega et al., 2009). Being excreted during treatment mostly as active

http://dx.doi.org/10.1016/j.vetmic.2014.01.040 0378-1135/ß 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Wasyl, D., et al., Prevalence and characterisation of quinolone resistance mechanisms in Salmonella spp.. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.01.040

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compounds their biological action is not limited to therapeutic site, but it is moved further as resistance selection pressure into environment (Kaplan et al., 2013). Since all (fluoro)quinolones exert their antibacterial effect by inhibition of topoisomerases, the same mechanisms confer resistance to all clinically relevant molecules. Spontaneous point mutations leading to amino acid substitutions in quinolone resistance determining region (QRDR) of topoisomerases II (gyrase) and IV genes alter the quinolone binding site and result in failure of their antibacterial activity (Hopkins et al., 2005; Fabrega et al., 2009). As a step-wise process in Salmonella, single mutation in gyrA, gyrB, parC, or parE reduces susceptibility to fluoroquinolones and multiple substitutions are needed for clinical resistance (Hopkins et al., 2005; Yang et al., 2012). That scheme might be also interfered with nonspecific mechanisms including outer membrane permeability or efflux pumps (Hopkins et al., 2005; Karczmarczyk et al., 2010; Akiyama and Khan, 2012; Rushdy et al., 2013). Plasmid mediated quinolone resistance (PMQR) confer lower susceptibility to fluoroquinolones and might be considered a background for selection of chromosomeencoded resistance. Three major mechanisms are involved in PMQR: qnr peptides protect topoisomerases from antimicrobial action, variant of aminoglycoside acetyltransferase (aac(60 )-Ib-cr) modifies ciprofloxacin molecule, and QepA protein modulates quinolone efflux pump (Poirel et al., 2012). The number of discovered qnr genes is currently approaching 100 alleles divided into 5 categories (qnrA, qnrB, qnrC, qnrD, qnrS) of which qnrB accounts up to 73 variants (http://www.lahey.org/qnrStudies) (Poirel et al., 2012). Clinical salmonellosis in human is usually self-limiting food-borne disease that might be tracked back mainly to food obtained from subclinically infected animals. Neither animal carriers nor patients require antimicrobial therapy unless systemic infection occurs (Jeong et al., 2011). For that reasons antimicrobial resistance in Salmonella might be considered a side effect of fluoroquinolone selection pressure due to the other clinical indications and further spread of resistant strains along the food chain. Dissemination of quinolone resistant Salmonella clones among domestic animals, wildlife, and humans was described in Spain (Palomo et al., 2013). Previously we reported emergence of high level ciprofloxacin resistant Salmonella enterica subspecies enterica serovar (S.) Kentucky in turkeys and pet reptiles (Wasyl and Hoszowski, 2012; Zaja˛c et al., 2013) and occurrence of S. Stanley outbreak strain in Poland (Wasyl et al., 2013a). Transmission of resistant Salmonella via food (Cavaco et al., 2008b; Akiyama and Khan, 2012; Yang et al., 2012) or foreign travels (Weill et al., 2006; Hassing et al., 2011; Le Hello et al., 2013) may lead to serious clinical consequences (Jeong et al., 2011). The burden of quinolone resistant Salmonella is not limited to food-borne intoxications, but also confronted with human adopted serovars responsible for typhoid or paratyphoid fever and being often empirically treated with fluoroquinolones as drug of choice (Hassing et al., 2011). For the above mentioned reasons the knowledge on epidemiology of resistant Salmonella, the genetic mechan-

isms behind as well as public awareness are needed. Therefore, based on a broad selection of Salmonella from animals, feeds, and foods available from antimicrobial resistance monitoring the current study aimed at the detection and characterisation of quinolone resistance mechanisms. Furthermore, epidemiological links between subset of those isolates were elucidated. 2. Material and methods Minimal Inhibitory Concentration (MIC) of Nalidixic acid (Nal, dilution range 4–64 mg/L) and Ciprofloxacin (Cip, 0.008–8 mg/L) were tested in 2680 Salmonella. They were picked up from 9670 isolates from animals, food, and feed available between 2008 and 2011 according to resistance monitoring rules: no attention was paid for serovar, but one isolate per serovar and animal flock/herd or batch of food/feed, duplicates were excluded. They represented 136 Salmonella serovars and serological forms, ten of which contributed to 80.0% of study group: Enteritidis (N = 1010), Infantis (N = 266), Typhimurium (N = 247, including 21 monophasic variants), Mbandaka (N = 153), Newport (N = 126), Virchow (N = 114), Kentucky (N = 79), Hadar (N = 55), Agona (N = 54), Saintpaul (N = 39). Antimicrobial susceptibility testing was performed with Sensititre1 EUMVS2 plate (Trek Diagnostic Systems, UK). For the purpose of current study MIC were interpreted according to European Committee on Antimicrobial Susceptibility Testing criteria (Table 1, Fig. 1), both epidemiological cut-offs and clinical breakpoint (not applicable for Nal). Based on MIC distribution, a subset of isolates was selected for identification of quinolone resistance mechanisms. Forty-four Salmonella isolated in 2011 representing ciprofloxacin MIC ranging from 0.125 mg/L to 4 mg/L (Table 2) were used for identification of chromosomal mutations in QRDR of gyrA, gyrB, parC, and parE. Those isolates as well as all Salmonella with MICNal ranging from 4 to 32 mg/L and MICCip 0.125 mg/L (Table 1) were tested for PMQR mechanisms: qnrA, qnrB, qnrC, qnrD, qnrS, qepA, aac(60 )-Ib-cr. PCR assays were run in 25 ml amplification mixture composed of 12.5 ml of Maxima1 Hot Start PCR Master Mix (2X) (Fermentas Life Sciences, Lithuania), 0.1 ml of each primer (Table S1), 1 ml of DNA template (boiling lysate of whole-cell suspension), and 11.4 ml of water. The conditions were: initial denaturation (95 8C for 5 min), 30 cycles of 30 s at 95 8C, 30 s in annealing temperature, elongation for 90 s at 72 8C, and final extension at 72 8C for 10 min. Duplex-PCR was applied for simultaneous detection of qnrB and qnrS genes, whereas all other assays focused on single targets. The obtained amplicons were analysed according to product size in 2% agarose gel (9 V/cm, 80 min). S. Typhimurium ATCC 14028 was used as quality control strain in assays targeting chromosomal genes whereas PMQR positive isolates or DNA were obtained from EURL-Antimicrobial Resistance (DTU, Kgs. Lyngby, Denmark). qnrB19-positive S. Newport and qnrS1-positive S. Saintpaul originated from previously described studies (Veldman et al., 2011). Relevant amplicons were sequenced (Oligo, Institute of Biochemistry and Biophysics, Polish Academy of Sciences,


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Table 1 Ciprofloxacin and Nalidixic acid MIC distribution in tested Salmonella isolates (N = 2680). Epidemiological and clinical interpretations and prevalence of non-wild type populations (Nal and Cip) and ciprofloxacin resistant isolates. Shadowed zone designates isolates suspected for presence of plasmid mediated mechanisms (N = 92).

MIC (mg/L) ≤4 8 16 Nalidixic 32 acid 64 >64 total epidemiological interpretation clinical interpretation

≤0.008 23

0.016 592 6

0.032 0.064 0.125 893 23 5 39 5 1 1 1 1 27 1 2 75 24 598 936 29 108 WT ≤ 0.064 mg/L N = 1587 (59.2%) S ≤ 0.5 mg/L N = 2509 (93.6%)

Ciprofloxacin 0.25 0.5 1 2 4 8 >8 10 1 1 32 10 1 1 19 6 1 1 2 1 1 13 611 113 77 11 4 43 26 668 146 86 12 4 43 26 NWT N = 1093 (40.8%) R > 1 mg/L N = 171 (6.4%)

Poland) and resulting reverse and forward nucleotide sequences aligned with SeqMan Pro (DNAStar Lasergene). Aligned sequences were analysed with MEGA5 software (Center for Evolutionary Medicine and Informatics). Obtained sequences were deposited in GeneBank (Table S1). XbaI Pulsed Field Gel Electrophoresis was performed according to PulseNet protocol as described elsewhere (Wasyl and Hoszowski, 2012) to assess phylogenetic similarity of 82 isolates representing 10 Salmonella serovars and carrying various quinolone resistance mechanisms. 3. Results Distribution of MIC values among tested isolates represented full range of Cip and Nal dilutions (Table 1).

total 1548 93 29 5 42 963 2680

epidemiological interpretation WT ≤ 16 mg/L N = 1670 (62.3%) NWT N = 1010 (37.7%)

Non Wild-Type category was assigned to 37.7% (N = 1010; Nal) and 40.8% (N = 1093; Cip) of the isolates. Ciprofloxacin clinical resistance was noted in 6.4% (N = 171) of them. Sixty-nine isolates showing high level clinical resistance to ciprofloxacin (MICCip 8 mg/L) belonging exclusively to serovar Kentucky were excluded from current study since clonal spread of ST198 clone was described elsewhere (Wasyl and Hoszowski, 2012; Zaja˛c et al., 2013). The remaining 2411 isolates showing MICCip ranging from 0.008 mg/L to 4 mg/L were processed in further analyses. MICs suggesting presence of PMQR mechanisms were observed in 92 (3.4%) isolates (Table 1). Chromosomal substitutions in QRDR regions were found in gyrA (Ser83, N = 22; Asp87, N = 22) and parC (Thr57, N = 23; Ala141, N = 1). Remaining genes were either unaltered (parE) or harboured substitutions irrelevant for quinolone resistance (gyrB). Twelve patterns combining

Fig. 1. Phylogenetic similarity of S. Newport (N = 26) with quinolone resistance mechanisms: PMQR – Plasmid Mediated Quinolone Resistance, QRDR – amino-acids substitutions in Quinolone Resistance Determining Region of topoisomerases encoding genes. NWT MICs – antimicrobial abbreviations and minimal inhibitory concentration epidemiological cut-off values (EUCAST; non-wild type > mg/L): Amp – ampicillin (8 mg/L), Chl – chloramphenicol (16 mg/L), Cip – ciprofloxacin (0.064 mg/L), Flr – florfenicol (16 mg/L), Kan – kanamycin (4 mg/L), Nal – nalidixic acid (16 mg/L), Str – streptomycin (16 mg/L), Smx – sulfamethoxazole (256 mg/L), Tet – tetracycline (8 mg/L); WT – wild type gene.


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Table 2 Gyrase (gyrA, gyrB) and topoisomerase VI (parC) genes substitutions within quinolone resistance determining region by ciprofloxacin MIC values and Salmonella serovar: SA – Adelaide, SAg – Agona, SE – Enteritidis, SH – Hadar, SI – Infantis, SL – Lexington, SM – Mbandaka, SN – Newport, SS – Saintpaul, SSt – Stanley, STe – Tennessee, ST – Typhimurium, SV – Virchow. Plasmid-mediated quinolone resistance (qnrS1/3, qnrS2) presence was indicated. No relevant substitutions were found in parE. gyrA Ser83

Asp87

gyrB

parC

Leu470

Thr57

Ala141

– – – – Sere Sere Sere – Sere Sere Sere Sere

– – – – – – – – – – Serh –

Asna – – – Tyrb – c Phe – – d Tyr – – a – Asn – – Glyf – Tyrb – – Leug Asna – Phec – – d Tyr – – – – – d Tyr – (Met)i No. of tested isolates a b c d e f g h i

Number of isolates by MICCip(mg/L) (Salmonella serovar codes) 0.125 2 (SE, SV)

1 1 1 1

(SH) (SN) (SSt) (SV)

0.25 4 5 1 1 1

0.5

(ST 3, SS 1) (SE 2, SV 2, SS 1) (SV) (SE) (SH)

2

4

1 (SE, qnrS2) 1 (SV) 3 (SE)

2 (SE)

3 (SAg 1, SI 2)

3 (SI 2, SN 1)

1 (SN)

1 (SN, qnrS1/S3)

1 (SI) 8

5

2

1

5 (SH, SA 2, STe 2) 3 (SN 2, SM) 1 (SN) 1 (SL)

6

Total

1

22

4 8 4 4 2 1 6 1 3 9 1 1 44

AAC. TAC. TTC. TAC. AGC. GGC. TTG. TCG. ATG.

Salmonella without and with QRDR mutations (CI95%: 7.33–10.88% and 1.26–15.14%, respectively; N.S.). Qnrpositive isolates belonged mostly to S. Newport (55.3%) and S. Enteritidis (16.0%). Thirteen PFGE-XbaI profiles showing 41.6% similarity have been identified among 26 S. Newport isolates originating from various sources, including feed, poultry and poultry meat and exotic pet snake (Fig. 1). Most profiles have been clustered (79% similarity) and gathered mostly qnrS1/S3 positive isolates, qnrB10/B19 isolates or isolates with various gyrA substitutions at position Ser83 (!Tyr, !Phe) or Asp87Gly. All isolates

one or two substitutions were dispersed among tested isolates and correlation with neither serovar nor MICCip value were noted (Table 2). PMQR mechanisms in eleven Salmonella serovars (Table 3) were identified as qnrS1 or qnrS3 (qnrS1/S3; N = 50) and qnrB10 or qnrB19 (qnrB10/B19; N = 24). The remaining isolates were either negative for all tested resistance determinants (N = 12) or not tested (N = 6) qnrS1. Additionally, qnrS1/S3 and qnrS2 were identified in two isolates with chromosomal mutations. None of the tested isolates carried qnrA, qnrC, qnrD, or qepA, aac(60 )-Ibcr. There were no difference in the prevalence of PMQRs in

Table 3 Plasmid mediated quinolone resistance genes. Salmonella

PMQR (N = 92) Negative

Not tested

1 5

1a 5a

1

1 3

2a

8

37

2 1a

1 9

3

qnrB10/B19 Agona Enteritidis Indiana Infantis Mbandaka Montevideo Newport Oranienburg Saintpaul Typhimurium Virchow Total a b c

QRDR

3 3

qnrS1/S3

qnrS1/S3

1

6

1

1a 24

50c

12

Percentage of PMQR-positive isolates within serovar (95% CI)

2 15 3 3 3 1 52 1 4 9 1

2.1% 16.0% 3.2% 3.2% 3.2% 1.1% 55.3% 1.1% 4.3% 9.6% 1.1%

3.7% (1.0–12.5%) 1.5% (0.7–2.2%) 10.7% (3.7–27.2%) 1.1% (0.0–2.4%) 2.0% (0.0–4.2%) n.a.b 41.3% (32.7–49.9%) 5.6% (1.0–25.8%) 10.3% (4.1–23.6%) 3.6% (1.3–6.0%) 0.9% (0.0–2.6%)

94

100.0%

qnrS2 1

1 4

Total (%)

1

Including single isolates with borderline ciprofloxacin MIC = 0.125 mg/L and MICNal 4 mg/L. Not analysed (3 isolates tested). Eight isolates previously reported in Veldman et al. (2011).


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Fig. 2. Phylogenetic similarity of S. Enteritidis (N = 17) with quinolone resistance mechanisms: PMQR – Plasmid Mediated Quinolone Resistance, QRDR – amino-acids substitutions in Quinolone Resistance Determining Region of topoisomerases encoding genes. NWT MICs antimicrobial abbreviations and interpretation criteria as given in Fig. 1; Col – colistin (2 mg/L); WT – wild type gene.

tested for QRDR mutations (N = 7) showed Thr57Ser parC substitution. One of the mutants simultaneously carried qnrS1/S3 gene (Fig. 1, Table 2). Seventeen S. Enteritidis isolates revealed eight PFGEXbaI profiles (79.2% similarity). Only gyrA substitutions were noted, both at Ser83 (!Tyr or !Phe) and Asp87Tyr. PMQR-positive (qnrS1/S3, qnrS2, and qnrB10/B19) isolates were scattered over various PFGE-XbaI profiles. One of the profiles gathered 7 S. Enteritidis isolated between 2008 and 2011 and featuring either qnrS1 gene or three different substitutions in gyrA (Fig. 2, Table 2). Phylogenetic analyses of the remaining serovars were presented in Supplementary Materials. Both tested S. Adelaide reptilian isolates showed diverse PFGE-XbaI profiles but the same gyrA substitution (Fig. S1, Table 2). Two PFGE-XbaI profiles and two gyrA Asp87 substitutions (!Tyr or !Asn) were observed in three S. Hadar isolates (Fig. S2). Five S. Infantis shared indistinguishable DNA profile and gyrA (Ser83Tyr) and parC (Thr57Ser) substitutions (Fig. S3). S. Mbandaka gyrA mutant and qnrS1/S3 positive isolate shared indistinguishable PFGE-XbaI profile, whereas another profile was noted in the second qnrS1/S3 carrying isolate (Fig. S4). Four profiles with 43.2% of similarity were observed in S. Saintpaul with various resistance mechanisms (qnrS1/S3, qnrB10/B19, gyrA Asp87 ! Tyr or !Asn) (Fig. S5). Both S. Tennessee shared DNA profile and gyrA and parC mutations (Fig. S6). All nine qnrB10/B19 carrying were clustered in three similar (79.5%) profiles of S. Typhimurium, whereas two others gathered gyrA Asp87Asn mutants (Fig. S7). Finally, six S. Virchow with various gyrA substitutions, including double mutants (Ser83Leu, Asp87Asn) were clustered in three related (84.8%) PFGE-XbaI profiles (Fig. S8). 4. Discussion Current results sign in worldwide studies on characterisation of quinolone resistance mechanisms and assessment of frequency of their occurrence in Salmonella. Indeed, finding of 40.8% isolates showing NWT ciprofloxacin MIC as well as 6.4% of clinical resistance compared to susceptibility of all isolates tested in middle 1990-ties (Hoszowski et al., 1998) confirmed the increase of

quinolone resistance in Salmonella over the recent years. Although studies on human isolates indicate that clinical resistance is rare (Jeong et al., 2011; Kozoderovic et al., 2012; Rushdy et al., 2013) the reports on resistance mechanisms found in Salmonella isolated from the environment, animals and foods (Chiu et al., 2002; Cavaco et al., 2008b; Akiyama and Khan, 2012; Kim et al., 2013; Palomo et al., 2013) have to be considered a public health warning. Monitoring data show the scope of occurrence of quinolone resistance mechanisms and their association with Salmonella serovars (EFSA and ECDC, 2013). For that reason we excluded S. Kentucky from current study to avoid bias due to its clonal spread (Wasyl and Hoszowski, 2012; Le Hello et al., 2013; Zaja˛c et al., 2013). Our previous analysis has proved stable frequency of occurrence of ciprofloxacin and nalidixic acid NWT isolates within the most prevalent Salmonella serovars. The only exception was S. Enteritidis showing significant increase in isolates from broilers, but not from laying hens (Wasyl et al., 2013b). Quinolone resistance in that serovar has been recently reported from Serbia (Kozoderovic et al., 2012). The phenomenon was described in S. Schwarzengrund (Akiyama and Khan, 2012), S. Saintpaul (Beutlich et al., 2010) as well as S. Stanley outbreak strain present in several European countries (Wasyl et al., 2013a). Although numerous substitutions have been described (Hopkins et al., 2005) current study on random isolates identified substitutions only in two positions of gyrA and parC genes. Interestingly, three different amino-acids replaced Ser83 and Asp87 of gyrA in isolates representing 13 Salmonella serovars, but serine always gave parC replacement, both at Thr57 or Ala141. Unlike some other studies (Eaves et al., 2004) neither single substitutions nor substitution patterns were related to Salmonella serovar or ciprofloxacin MIC value (Karczmarczyk et al., 2010). Furthermore, Thr57Ser parC substitution was considered not or doubtfully associated to resistance phenotypes (Akiyama and Khan, 2012; Yang et al., 2012; Palomo et al., 2013). The limited number of observed substitutions and equal contribution of both gyrA mutations is quite different from Korean study where multiple QRDR alternations and prevalent Asp87 substitutions were reported (Jeong et al.,


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2011). Although Chinese results were similar for gyrA, the observed substitutions in parC varied enormously (Yang et al., 2012). In Colombia Thr57Ser parC substitution was the most prevalent but any other except Asp87Tyr in gyrA were observed (Karczmarczyk et al., 2010). Based on the reports from other parts of the world (Whichard et al., 2007; Beutlich et al., 2010; Le Hello et al., 2013; Rushdy et al., 2013) it is deemed that some differences in aminoacid substitutions and the frequency of their occurrence might depend on geographical origin of the isolates (Karczmarczyk et al., 2010; Hassing et al., 2011; Kim et al., 2013). Plasmid-mediated qnr genes showed definitely less variability than QRDR mutations. Since only three alleles of qnr genes were noted in almost a hundred isolates representing eight serovars (Table 3), it might be assumed that the genes were present in the environment and Salmonella acquired them due to plasmid exchange (Herrera-Leon et al., 2011; Dolejska et al., 2013; Kim et al., 2013). The most frequently observed qnrS1 and qnrB19 genes have already been reported from the initial part of current study, both in Salmonella and commensal Escherichia coli (Veldman et al., 2011) as well as clinical human isolates (Jeong et al., 2011). Interestingly, both genes ware reported in E. coli isolated in Poland from wild birds (Dolejska et al., 2013) and food animals (unpublished) whereas qnrB5 was identified in Salmonella food isolates (Cavaco et al., 2008b). PMQR genes found in current study have been located on different plasmids found in various serovars (Garcia-Fernandez et al., 2009; Kim et al., 2013). Karczmarczyk et al. (2010) described qnrB19 associated with small ColE plasmid found in several isolates representing various serovars. The homologous plasmid carrying qnrB19 gene was described in S. Typhimurium isolated in the Netherlands (Hammerl et al., 2010). Noteworthy, the same gene was found in all nine PMQR positive S. Typhimurium tested in current study (Table 3). Further studies are needed to characterise the plasmids and trace back their original host bacterium. Until recently qnr-positive Salmonella were relatively infrequent (Herrera-Leon et al., 2011). The survey run in several European countries defined their prevalence at 0.04% (Veldman et al., 2011). Current finding of PMQR in 3.5% of tested isolates indicated a significant (P 0.001) increase in the occurrence of transferable quinolone resistance, similar to the one observed in various bacteria (Yue et al., 2008; Kim et al., 2009; Jeong et al., 2011). Furthermore, those genes have been found in two isolates conferring chromosomally encoded quinolone resistance with the highest of the observed MICCip values (Table 2). As far as we are concerned, such MIC might not result only from accumulation of the two resistance mechanisms, but presumably some undefined resistance background was involved (Hopkins et al., 2005; Karczmarczyk et al., 2010; Akiyama and Khan, 2012; Rushdy et al., 2013). Quinolone resistant Salmonella with parC mutation and qnrS1 gene were reported from Korea and as such it might be of clinical relevance (Jeong et al., 2011). Our results proved that PMQR genes spread independently from QRDR mutations and chromosomal mechanisms did not prevent from acquiring of transferable ones. Multiple studies point

out the correlation of plasmid mediated quinolone and cephalosporin resistance (Cattoir et al., 2007; Whichard et al., 2007; Hassing et al., 2011; Herrera-Leon et al., 2011; Poirel et al., 2012). Our results did not conform to that rule since only few isolates (data not shown) conferred cephalosporin resistance (Wasyl and Hoszowski, 2012). Finding of 55.4% of PMQR-suspected isolates within S. Newport and 41.3% of that serovar carrying PMQR is another intriguing issue. Based on the diversity PFGE-XbaI profiles it might be assumed that several clones contribute to the S. Newport spread mostly in poultry and poultry meat. The clusters of similar profiles observed in isolates sharing common features like presence of different qnr gens, QRDR mutation patterns and NWT MIC profiles proves independent genetic events leading to acquisition of quinolone resistance mechanisms and further clonal spread (Fig. 1). Another clone carrying qnrB was reported from Denmark in imported turkey meat (Cavaco et al., 2008b). Similar epidemic of cephalosporin-resistant but not to quinolones S. Newport was drafted in the US (Whichard et al., 2007). The reason why different clones of S. Newport tend to collect various resistance mechanisms remains unclear (Fabrega et al., 2009). S. Enteritidis provided another scenario of spread of quinolone resistance (Fig. 2). Single clone of highly related PFGE-XbaI profiles embracing animal and food isolates with diverse gyrA substitutions and all qnr genes noted in current study indicated that those mechanisms emerged or entered an on-going epidemic of the most prevalent serovar (Palomo et al., 2013). Both scenarios were verified with the phylogenetic analysis of the other tested serovars. These includes indistinguishable S. Infantis or S. Tennessee isolates with the same mutations, diverse PFGE-XbaI profiles of the qnrS1/S3- positive S. Mbandaka isolates or diverse gyrA mutations in related S. Virchow. Interestingly, indistinguishable profile was noted in three S. Virchow with various mutations, including double gyrA mutant and still reduced ciprofloxacin susceptibility. Although few isolates tested, current result confirmed both vast genetic diversity of S. Saintpaul (Wasyl et al., 2012) and quinolone resistance mechanisms present in the isolates. The observed PFGE-XbaI profiles were indistinguishable from the ones noted in several countries (Cavaco et al., 2008b; Beutlich et al., 2010; Wasyl et al., 2012) and thus confirmed acquisition of resistance mechanisms along the infection cycle. Lastly, S. Typhimurium clones – one built of gyrA mutants and the other included exclusively qnrB10/B19positive isolates – confirmed clonal spread of as described elsewhere (Garcia-Fernandez et al., 2009; Hammerl et al., 2010). 5. Conclusions Up to our knowledge this is the first report of quinolone resistance mechanisms identification in a wide selection of Salmonella isolated in Poland. The study confirmed that in the era of increasing quinolone resistance due to antimicrobial selection pressure, multiple mechanisms might be involved in acquisition and spread of resistance mechanisms. Several amino-acid substitutions were identified, but those targeting gyrA gene were of major


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relevance. Various Salmonella serovars might harbour qnr genes which were disseminated independently from chromosomal mutations. Different scenarios might contribute to spread of quinolone resistance. Some serovars i.e. S. Enteritidis or S. Saintpaul might incidentally develop chromosomal mutation or acquire plasmids encoding for Qnr proteins during infectious cycle. In the others a successful epidemiological spread might be related with quinolone resistance mechanisms present in a given clone. Several knowledge gaps have been identified: why S. Newport is more prone to harbour various quinolone resistance mechanisms than other serovars, is it only clonal spread contributing to geographical differences in QRDR mutations of topoisomerases genes, what are environmental reservoirs of plasmid-born quinolone resistance mechanisms. Finally, to what extend the unknown resistance mechanisms that might accumulate with QRDR and PMQR causing clinical resistance and animal and public health threat. Conflict of interest statement Nothing to declare. Acknowledgements The study was supported by governmental founding of the multi-annual research project Protection of Animal and Human Health (Ministry of Council Resolution 244/2008 of October 28, 2008).

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