Ciprofloxacin and nalidixic acid resistance of Salmonella spp. isolated from retail food in Poland

Accepted Manuscript Ciprofloxacin and nalidixic acid resistance of Salmonella spp. isolated from retail food in Poland

Łukasz Mąka, Elżbieta Maćkiw, Monika Stasiak, Tomasz Wołkowicz, Joanna Kowalska, Jacek Postupolski, Magdalena Popowska PII: DOI: Reference:

S0168-1605(18)30088-6 doi:10.1016/j.ijfoodmicro.2018.03.012 FOOD 7844

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

26 October 2017 12 March 2018 16 March 2018

Please cite this article as: Łukasz Mąka, Elżbieta Maćkiw, Monika Stasiak, Tomasz Wołkowicz, Joanna Kowalska, Jacek Postupolski, Magdalena Popowska , Ciprofloxacin and nalidixic acid resistance of Salmonella spp. isolated from retail food in Poland. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Food(2017), doi:10.1016/j.ijfoodmicro.2018.03.012

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ACCEPTED MANUSCRIPT Ciprofloxacin and nalidixic acid resistance of Salmonella spp. isolated from retail food in Poland.

Łukasz Mąkaa*, Elżbieta Maćkiwa, Monika Stasiaka, Tomasz Wołkowiczb, Joanna Kowalskaa,

Department of Food Safety, National Institute of Public Health – National Institute of Hygiene,

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a

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Jacek Postupolskia, Magdalena Popowskac

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Warsaw, Poland

Department of Bacteriology, National Institute of Public Health – National Institute of Hygiene,

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c

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Warsaw, Poland

Department of Bacterial Physiology, Institute of Microbiology, Faculty of Biology, Warsaw University,

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Warsaw, Poland

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* Correspondence: Łukasz Mąka, Department of Food Safety, National Institute of Public

ABSTRACT

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[email protected]

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Health – National Institute of Hygiene, Chocimska 24, 00-791, Warsaw, Poland

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Distribution of amino acid substitutions in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, parE and determinants of plasmid-mediated quinolone resistance (PMQR) were investigated among quinolone-resistant Salmonella spp. strains isolated from retail food in Poland in the years 2008–2013. Ten different amino acid substitutions were identified in QRDRs. Five different amino acid substitutions were identified in gyrA: Ser83Tyr, Ser83Phe, Asp87Tyr, Asp87Asn, Asp87Gly, two amino acid substitutions in parC: Thr57Ser, Ser80Ile and in parE: Leu445Phe,

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ACCEPTED MANUSCRIPT Arg511Ser. One substitution - Ser464Phe - was detected within gyrB. In gyrA a single substitution (Ser83Tyr) was identified the most frequently – 34.8% (63/181). Second most frequently identified variant (21.0% – 38/181) was a co-existence of two single substitutions in gyrA: Ser83Tyr and parC: Thr57Ser. In four isolates co-existed three substitutions in three different genes: gyrA: Ser83Tyr + parC: Thr57Ser + parE: Leu445Phe (two isolates), gyrA:

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Ser83Phe + parC: Thr57Ser + parE: Leu445Phe, and gyrA: Ser83Tyr + parC: Thr57Ser + parE:

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Arg511Ser. In the two isolates four substitutions were identified – in gyrA: Ser83Phe +

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Asp87Tyr and in parC: Thr57Ser + Ser80Ile.

Among resistant isolates, MIC values varied between 32 and 2048 mg/L (nalidixic acid)

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and between 0.125 and 16 mg/L (ciprofloxacin).

MIC values of two isolates harboring qnrS1without any substitutions were 32 mg/L

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(NA) and 0.5–1.0 mg/L (CIP). The highest MIC values for NA and CIP were observed in two isolates of Salmonella spp. carrying double substitutions in gyrA: Ser83Phe + Asp87Tyr and

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parC: Thr57Ser + Ser80Ile. MIC value for NA was 2048 mg/L while for CIP – 16 mg/L.

Keywords: Salmonella spp., quinolones, QRDR mutations, PMQR, antimicrobial resistance,

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1. Introduction

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food.

Antimicrobial resistance of bacteria isolated from food is a problem of growing importance. One of the greatest challenges is resistance to quinolones and fluoroquinolones. In many reports, quinolone resistance is associated with point mutations in the quinolone resistancedetermining region (QRDR). In 1998, Martinez-Martinez et al. reported about plasmidmediated quinolone resistance (PMQR) mechanism to nalidixic acid and fluoroquinolones.

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ACCEPTED MANUSCRIPT They described a qnrA gene located on plasmid pMG252 in clinical isolate Klebsiella pneumoniae. Since then, other authors reported about qnrS (Hata et al., 2005), qnrB (Jacoby et al., 2006), qnrC (Wang et al., 2009) and qnrD (Cavaco et al., 2009). Many qnr variants have been reported (Jacoby et al., 2008). However, qnrS are not the only PMQR mechanisms. Resistance to quinolones may be also associated with QepA efflux pump (Yamane et al., 2007;

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Yamane et al., and 2008) and OqxAB multidrug resistance pumps (Zhao et al., 2010).

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Moreover, in 2006, Robicsek et al. reported that aminoglycoside acetyltransferase (aac(6’)-Ib)

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variant – aac(6’)-Ib-cr, has the ability to acetylate ciprofloxacin, which results in reduced susceptibility to this antimicrobial.

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In Poland, Salmonella spp. is one of the most frequently reported food-borne bacterial pathogens. According to epidemiological reports prepared by the Department of Epidemiology

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of the National Institute of Public Health – National Institute of Hygiene (NIPH-NIH) and Department for Communicable Disease and Infection Prevention and Control of Chief Sanitary

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Inspector, in 2015 8630 cases of salmonellosis were reported, compared with 8392 cases in

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2014 (Czarkowski et al., 2014; epidemiological reports, 2017). In the European Union (EU) Salmonella is the second most commonly reported gastrointestinal bacterial pathogen. In 2015,

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a total of 94625 confirmed cases of salmonellosis were reported (EFSA and ECDC, 2016a).

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2. Materials and methods

2.1. Selection of resistant Salmonella spp. isolates Salmonella spp. strains were isolated from retail food samples in the frame of Official Control and Monitoring Program by Sanitary and Epidemiological Stations, in accordance with the method PN-EN ISO 6579:2003/A1:2007 “Horizontal method for the detection of Salmonella

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ACCEPTED MANUSCRIPT spp.”. The tested samples included food of animal and plant origin, both ready-to-eat products and products requiring further processing. The isolates were tested using disc diffusion method and dilution method, and selected in compliance with the recommendation of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST, 2013). Because of absence of EUCAST criteria

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for nalidixic acid, authors used the criteria recommended by the Clinical Laboratory and

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Standard Institute (CLSI) (CLSI, 2012).

A total of 181 quinolone-resistant Salmonella spp. were collected from single food samples

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from all over the country in years 2008 – 2013 by Sanitary and Epidemiological Stations.

2.2. Serotyping

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Serotyping was performed by the Bacteriology Department of NIPH-NIH according to White-

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2.3. DNA isolation

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Kauffmann-Le Minor scheme (Grimont and Weill, 2007).

2.4. PCR

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DNA was isolated using the boiling technique, as described before (Mąka et al., 2015b).

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Screening for the presence of PMQR genes and gyrA, gyrB, parC and parE was performed by PCR amplification, using primers described in supplementary tables 1–4. Reaction conditions for gyrA: 95°C – 5 min, 30x (95°C – 30 s, 54°C – 30 s, 72°C – 30 s), 72°C – 5 min. For gyrB: 95°C – 5 min, 30x (94°C – 1 min, 58°C – 1 min, 72°C – 1 min), 72°C – 10 min. For parC, parE 95°C – 5 min, 30x (94°C – 1 min, 52°C – 1 min, 72°C – 1 min), 72°C – 10min. The final 25µl of PCR mixture contained 1x concentrated Dream Taq buffer,

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ACCEPTED MANUSCRIPT 2mM MgCl2, 0.2mM dNTPs, 0.3µM of each gyrA primers or 0.25 µM of each gyrB, parC, parE primers, 1U Dream Taq polymerase, 1 µl of template DNA and sterileMilli-Q water. Reaction conditions for detection of qnrA, qnrB, qnrC and qnrS fragments: 94°C – 5 min, 30x (94°C – 30 s, 67°C – 35 s, 72°C – 1 min), 72°C – 10 min. The final 25µl of PCR mixture contained 1x concentrated Dream Taq buffer, 2mM MgCl2, 0.2mM dNTPs, 1 µM of

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each primers, 1U Dream Taq polymerase, 1 µl of template DNA and sterileMilli-Q water.

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Reaction conditions for detection of the qnrD fragment: 95°C – 5 min, 30x (94°C – 30s,

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56°C – 30s, 72°C – 1 min 30 s), 72°C – 10 min. The final 25µl of PCR mixture contained 1x concentrated Dream Taq buffer, 2mM MgCl2, 0.2mM dNTPs, 0.4µM of each primers, 1U

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Dream Taq polymerase, 1 µl of template DNA and sterileMilli-Q water. Reaction conditions for detection of qepA and aac(6’)-Ib fragments: 94°C – 6 min, 30x

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(94°C – 30 s, 54°C – 35 s, 72°C – 1 min), 72°C – 10 min. The final 25µl of PCR mixture contained 1x concentrated Dream Taq buffer, 2mM MgCl2, 0.2mM dNTPs, 1 µM of each

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primers, 1U Dream Taq polymerase, 1 µl of template DNA and sterileMilli-Q water.

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All PCR products for aac(6’)-Ib were digested with BtsCI (Thermo Scientific) restriction enzyme. The digestion resulted in two products of the sizes of 210 bp and 272 bp.

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Lack of enzyme digestion site reveals presence of aac(6’)-Ib-cr. Digestion was performed

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according to the conditions recommended by the producer manufacturer.

2.5. Sequencing and bioinformatic analysis of sequences All of the PCR products for qnr, gyrA, gyrB, parC and parE were sent to Genomed (Warsaw, Poland) to sequence and read nucleotide sequences. The final nucleotide sequence was analyzed using Chromas LITE ver. 2.1.1. The sequences were compared with sequences available in GenBank. GenBank accession numbers: gyrA: AAL21173.1, gyrB: Y07916.1, parC: AAA27180.1, parE: L05544.1.

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ACCEPTED MANUSCRIPT Sequence alignment and analysis were performed online using BLAST software (http://www.ncbi.nlm.nih.gov) and CLC Sequence Viewer v.6.8.1. (CLC Inc., Aarhus, Denmark).

3. Results

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Twenty different serotypes (Enteritidis, Infantis, Virchow, Newport, Hadar,

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Typhimurium, Mbandaka, Saintpaul, Bredeney, Derby, Duisburg, Eko, Glostrup, Kentucky,

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Kottbus, Sandiego, Schwarzengrund, Tennessee, Tshiongwe and Salmonella enterica subsp. enterica) were identified among quinolone-resistant Salmonella spp. ( Table 5).

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(Supplementary table 5).

None of the tested isolates contained genes qepA or aac(6’)Ib-cr. The presence of qnr

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genes were confirmed in 10 isolates (5.5%) – 5 of them contained qnrB19 and 5 qnrS1. All of the tested isolates were negative for qnrA, qnrC and qnrD. All of the qnr-positive isolates had

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been originated from meat samples and belonged to one of the following serotypes: Infantis,

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Kottbus, Newport, Schwarzengrund and Typhimurium (Table 5) (Supplementary table 5). The digestion PCR products for aac(6’)-Ib gene resulted in two products of the sizes of

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210 bp and 272 bp. Lack of enzyme digestion site reveals presence of aac(6’)-Ib-cr. None of the tested isolates contained genes qepA or aac(6’)Ib-cr.

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Obtained sequences of fragments of gyrA, gyrB, parC and parE genes were deposited in GenBank database under accession numbers MG995855 – MG995999 and MH013512 – MH013948. DNA sequencing of gyrA, gyrB, parC and parE revealed point mutations resulting in 10 different amino acid substitutions in all tested genes. Five different amino acid substitutions were identified in gyrA: Ser83Tyr, Ser83Phe, Asp87Tyr, Asp87Asn, Asp87Gly, two amino acid substitutions both in parC: Thr57Ser, Ser80Ile and in parE: Leu445Phe, Arg511Ser. One substitution, i.e. Ser464Phe, was detected within gyrB (Table 1) (Table 6).

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ACCEPTED MANUSCRIPT Among the tested isolates 20 different variants of substitutions and co-existence of substitutions with qnr genes were detected. A single substitution in gyrA (Ser83Tyr) was identified the most frequently – 34.8% (63/181). Second most frequently identified variant (21.0% – 38/181) was the co-existence of two single substitutions in gyrA: Ser83Tyr and parC: Thr57Ser (Table 1) (Table 6).

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Apart from the identification of the single and double substitutions, In four of the

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isolates, three substitutions co-existed in three different genes: In four isolates co-existed three

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substitutions in three different genes: gyrA: Ser83Tyr + parC: Thr57Ser + parE: Leu445Phe (two isolates), gyrA: Ser83Phe + parC: Thr57Ser + parE: Leu445Phe (one isolate), and gyrA:

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Ser83Tyr + parC: Thr57Ser + parE: Arg511Ser (one isolate). Moreover, in two isolates four substitutions were identified in two isolates – in gyrA: Ser83Phe + Asp87Tyr and in parC:

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Thr57Ser + Ser80Ile (Table 1) (Table 6).

Among the resistant isolates, MIC values varied between 32 and 2048 mg/L (nalidixic

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acid) and between 0.125 and 16 mg/L (ciprofloxacin). MIC values of two isolates harboring

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qnrS1without any substitutions were 32 mg/L (NA) and 0.5–1.0 mg/L (CIP). MIC values of isolates containing single substitution in gyrA varied from 512 to 2048 mg/L (NA) and from

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0.125 to 1 mg/L (CIP).

Among the four isolates containing single substitution within parC: Thr57Ser, three

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additionally harbored qnrB19. The harboring of the genes has not influenced the nalidixic acid MIC value (64 mg/L) but it has affected the ciprofloxacin MIC value (0.5 mg/L compared to 0.125 mg/L). Forty-two isolates carried two substitutions: gyrA: Ser83Tyr + parC: Thr57Ser, and among them two isolates additionally harboured qnrB19 and other two qnrS1. MIC values of the 38 isolates containing only two substitutions ranged from 512 to 2048 mg/L (NA) and from 0.25 to 1 mg/L (CIP). For isolates with qnrB19, the MICs were 1024 mg/L (NA)

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ACCEPTED MANUSCRIPT and 0.5–2 mg/L (CIP), while for qnrS1 – positive MIC values were 1024 mg/L (NA) and 4 mg/L. The highest MIC values for NA and CIP were observed in two isolates of Salmonella spp. carrying double substitutions in gyrA: Ser83Phe + Asp87Tyr and parC: Thr57Ser +

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Ser80Ile. The MIC value for NA was 2048 mg/L while for CIP – 16 mg/L.

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4. Discussion

This study is a continuation of previously published research regarding the antimicrobial

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susceptibility of Salmonella strains isolated from retail products in Poland (Mąka et al. 2014,

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2015a and 2015b).

In this research, among quinolone-resistant Salmonella spp., ten different amino acid

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substitutions were identified in four genes: five different substitutions in gyrA, two substitutions in parC, two in parE and one substitution in gyrB. Most frequently amino acid substitutions

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occurred within genes gyrA and parC. The total of 256 substitutions were detected: 176 in gyrA,

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75 in parC, 4 in parE and 1 in gyrB. These results are in compliance with those reported by other authors. Furthermore in our study it was shown that the resistance to quinolones and

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fluoroquinolones among Salmonella spp. isolates is mostly associated with point mutations in QRDR. Amino acids substitutions result in the increase of MIC values.

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Yang et al. (2012) reported that 68 point mutations were identified in gyrA, parC and parE among 30 multiresistant Salmonella isolated from retail meat products in China in 20072008, Mutations within gyrB were not identified. Generally, mutations in genes gyrA (gyrase) and parC (topoisomerase IV) are reported more often in comparison with parE, while mutations in gyrB are sporadic (Ling et al., 2003; Eaves et al., 2004; Chen et al., 2007; Jeong et al., 2011; Kim et al., 2011; Yang et al., 2012). Most of the mutations appear in gyrA and parC, however,

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ACCEPTED MANUSCRIPT there are occasional reports about mutations in gyrB and parE (Ling et al., 2003; Eaves et al., 2004; Fabrega et al., 2008). The presence of qnr genes was confirmed in 10 isolates (5.5%) of which 5 contained qnrB19 and 5 qnrS1. Compared to other authors, it is a relatively low number of isolates harboring these determinants of plasmid-mediated quinolone resistance. Among nalidixic acid-

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resistant Salmonella spp. strains isolated from meat products in Columbia, 30.8% were qnrB19

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– positive. All of them were resistant to ciprofloxacin and did not carry any of the other genetic

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determinants of resistance (aac(6’)-Ib-cr, qepA, qnrA or qnrS) (Karczmarczyk et al., 2010). PMQR-positive Salmonella isolates of human and animal origin in Europe were confirmed in

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collaborative studies including by 13 National Reference Laboratories for Antimicrobial Resistance (NRL-AR). The results show that among Salmonella enterica isolated from different

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sources (environment, human, food, pigs, fowls, sheep, turkeys, reptiles), 59% (288/485) were PMQR-positive. Six among the food-originated isolates contained qnrS1 (one isolate was

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(Veldman et al., 2011).

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additionally aac(6’)-1b-cr – positive), two were qnrB19-positive and one harbored qnrD

Plasmid-mediated quinolone resistance plays an important role in the acquisition of

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high-level fluoroquinolone-resistance by the selection of isolates containing substitutions within gyrA and parC (Hooper, 1999).

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Among all the tested strains, Salmonella Kentucky isolates need further discussion. According to EFSA and ECDC report (2016b), Salmonella belonging to this serovar exhibited a high-level resistance to ciprofloxacin. It is an important public health concern because ciprofloxacin is a common first-line treatment for invasive salmonellosis in humans. In humanoriginated strains 84.0% of S. Kentucky was resistant to ciprofloxacin, which is consistent with the dissemination of the ciprofloxacin-resistant S. Kentucky ST198 strain in Europe and elsewhere since 2010 (Le Hello et al., 2011; 2013). Among the Salmonella spp. isolated from

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ACCEPTED MANUSCRIPT broiler meat, resistance to ciprofloxacin (42.6%) and nalidixic acid (39.7%) was observed. S. Kentucky with a high-level ciprofloxacin resistance is likely presumably to belong to the ST198 clone. S. Kentucky with a high-level ciprofloxacin resistance is associated with the ST198 clone. In our research two strains of S. Kentucky had been isolated from turkey meat samples

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as part of an Official Control. After comparing our data with the data of other authors, it seems

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it indicates appears that both isolates of S. Kentucky are presumably the clones of the epidemic

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strain ST198.

Besides the high level of resistance to ciprofloxacin (MIC=16 mg/L), our isolates carried blaTEM

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and coexisting substitutions: Asp87Tyr in gyrA and Ser80Ile in parC. Our isolates were also resistant to amoxicillin/clavulanic acid, ampicillin, gentamicin, sulfonamides, streptomycin and

et al., and 2013; Le Hello et al., 2013).

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tetracycline, which is also consistent with the data of other authors (Barua et al., 2012; Barua

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Reports about S. Kentucky were previously referred to strains isolated from turkey and

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pet reptiles (Wasyl and Hoszowski, 2012; Le Hello et al., 2013; Zając et al., 2013). This article reports about Salmonella Kentucky isolated from retail food, i.e. from food widely available to

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5. Conclusion

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consumers.

The results indicate a common prevalence of amino acid substitutions among nalidixic-resistant Salmonella spp. isolated from retail food in Poland, especially among S. Enteritidis. This mechanism of resistance cannot be disseminated via horizontal gene transfer (HGT), however point mutations may accumulate and persist in bacterial population. It was confirmed that 5.5% of isolates resistant to quinolones were qnr-positive, but what is interesting is that all of them were isolated after the year of 2011, and among 10 isolates 6 were

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ACCEPTED MANUSCRIPT isolated in 2013, which may indicate the beginning of qnr genes dissemination among Salmonella spp. isolated from retail food. However, the number of qnr-positive isolates was too small to substantiate such a conclusion. Nevertheless, it indicates the need for further monitoring in this area. The results of these studies are important in two aspects. First of all, because the results are

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related to strains isolated from food from retail, i.e. food available to consumers. Second of all,

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it is an important public health concern because ciprofloxacin is a common first-line treatment

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for invasive salmonellosis in humans. Our research indicates that quinolone-resistant isolates may disseminate via food chain, therefore there is the need for further monitoring and research

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in this field.

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Acknowledgments

This work was supported by a grant from the National Center of Science awarded on the basis

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Table 1 . Number of isolates containing amino acid substitutions, plasmid-mediated quinolone resistance (PMQR) and minimum inhibitory concentrations (MICs) of Salmonella spp. Amino acid substitutions

PMQR

Number of isolates

-

qnrS1

MIC range (mg/L) NA

CIP

2

32

0,5-1

-

63

1024-2048

0,25-1

-

29

512-2048

0,125-1

gyrA: Asp87Asn

-

5

512-1024

0,25-0,5

gyrA: Asp87Gly

-

2

512-1024

0,125-0,25

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gyrA: Ser83Tyr gyrA: Asp87Tyr

-

7

1024-2048

0,25-1

-

1

64

0,125

parC: Thr57Ser

qnrB19

3

64

0,5

gyrA: Asp87Asn + parC: Thr57Ser

-

6

1024-2048

0,25-1

gyrA: Ser83Phe + parC: Thr57Ser

-

8

512-2048

0,25-1

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gyrA: Ser83Phe parC: Thr57Ser

-

38

512-2048

0,25-1

gyrA: Ser83Tyr + parC: Thr57Ser

qnrB19

2

1024

0,5-2

gyrA: Ser83Tyr + parC: Thr57Ser

qnrS1

2

1024

4

-

2

512

0,125-0,25

gyrA: Asp87Tyr + parC: Thr57Ser gyrA: Asp87Gly + parC: Thr57Ser

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gyrA: Ser83Tyr + parC: Thr57Ser

-

4

256-512

0,125-0,25

qnrS1

1

64

2

gyrA: Ser83Tyr + parC: Thr57Ser + parE: Leu445Phe

-

2

1024-2048

0,25-0,5

gyrA: Ser83Phe + parC: Thr57Ser + parE: Leu445Phe

-

1

2048

0,5

gyrA: Ser83Tyr + parC: Thr57Ser + parE: Arg511Ser

-

1

2048

0,5

gyrA: Ser83Phe + Asp87Tyr + parC: Thr57Ser + Ser80Ile

-

2

2048

16

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gyrB: Ser464Phe + parC: Thr57Ser

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HIGHLIGHTS

Distribution of amino acid substitutions in QRDRs and qnr genes were investigated.



Ten different amino acid substitutions were identified in QRDRs.



Among ten qnr-positive isolates (5.5%) 5 contained qnrB19 and 5 qnrS1.



Ciprofloxacin MICs varied between 0.125 and 16 mg/L.



Co-existence of three and four substitutions in single strains were identified.

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

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