Characterization of nonpathogenic Listeria species isolated from food and food processing environment

    Characterization of nonpathogenic Listeria species isolated from food and food processing environment Dorota Korsak, Magdalena Szuplewska PII: DOI: Reference:

S0168-1605(16)30435-4 doi: 10.1016/j.ijfoodmicro.2016.08.032 FOOD 7357

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

27 May 2016 19 August 2016 23 August 2016

Please cite this article as: Korsak, Dorota, Szuplewska, Magdalena, Characterization of nonpathogenic Listeria species isolated from food and food processing environment, International Journal of Food Microbiology (2016), doi: 10.1016/j.ijfoodmicro.2016.08.032

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ACCEPTED MANUSCRIPT Characterization of nonpathogenic Listeria species isolated from food and food processing

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environment

Department of Applied Microbiology, Faculty of Biology, University of Warsaw,

Miecznikowa 1, 02-096 Warsaw, Poland

Department of Bacterial Genetics, Faculty of Biology, University of Warsaw,

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Dorota Korsak1*, Magdalena Szuplewska2

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Miecznikowa 1, 02-096 Warsaw, Poland

*Author for correspondence. Tel + 48 22 5541326, Fax: + 48 22 5541402 E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract A total of 127 Listeria isolates from food and food processing environments, including 75 L.

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innocua, 49 L. welshimeri, 2 L. seeligeri and 1 L. grayi were tested for susceptibility to eight

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antimicrobials, benzalkonium chloride (BC), cadmium and arsenic. The isolates were also

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screened for the presence of extrachromosomal genetic elements  plasmids, and their restriction pattern types were determined. All strains were susceptible to ampicillin, ciprofloxacin, erythromycin, gentamicin, rifampicin, trimethoprim and vancomycin. Two of

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the L. innocua isolates showed resistance to tetracycline and minocycline. The resistance was

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determined by the presence of chromosomal localization of tet(M) gene, which was not integrated in the transposon Tn916-Tn1545 family. Of analyzed isolates, 18.11% and 55.91% isolates were resistant to BC and cadmium, respectively, but all were susceptible to arsenic.

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Resistance to BC was correlated with resistance to cadmium  all BC resistant isolates were also resistant to cadmium. On the other hand, 67.61% of cadmium-resistant isolates were

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susceptible to BC, suggesting that cadmium and BC resistance were not always concurrent in Listeria species. 48.03% of isolates contained plasmids. The size of most of the identified

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replicons was in the range of 50-90 kb. All plasmids were classified into 12 groups with identical restriction pattern (I-XII). Interestingly, plasmids belonging to the same group were determined in isolates of the same species. Only in one case, plasmids with I-type profile were identified in L. innocua and L. welshimeri. There was a association between resistance to BC and plasmid DNA presence: all resistant isolates carried a plasmid. A correlation between resistance to cadmium and plasmid carriage was also observed in L. innocua and L. seeligeri isolates, but among resistant L. welshimeri, 23.08% of isolates did not have plasmids. This may suggest that resistance is associated with determinants located within the chromosome.

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ACCEPTED MANUSCRIPT To elucidate the adaptation strategies and ecology of Listeria spp., it is important to have a better understanding of its resistance to antimicrobials and environmental toxicants such as

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heavy metals and disinfectants.

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Keywords

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resistance, plasmids identification

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nonpathogenic Listeria spp., tet(M) gene, benzalkonium chloride resistance, cadmium

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ACCEPTED MANUSCRIPT 1 Introduction The genus Listeria (phylum Firmicutes), covering a group of Gram-positive, non-spore-

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forming, rod-shaped bacteria, , contains 17 species. Phylogenetic analysis showed the

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existence of four well-supported clades within the genus comprising: (i) L. monocytogenes,

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L. marthii, L. innocua, L. welshimeri, L. seeligeri and L. ivanovii, which we refer to as Listeria sensu stricto, (ii) L. fleischmannii, L. aquatica and L. floridensis, (iii) L. rocourtiae, L. weihenstephanensis, L. cornellensis, L. grandensis, L. riparia, L. borriae, L. newyorkensis

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and (iv) L. grayi (den Bakker et al., 2014; Weller et al., 2015).

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Listeria spp. are widely distributed in many different environments, including soil, surface water, vegetation, sewage, animal feed, farm environments, food processing environments, urban and suburban environments. Two species, L. monocytogenes and L. ivanovii, are

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facultative intracellular pathogens, the etiological agents of listeriosis - a food-borne infection. The other fifteen species are harmless environmental saprophytes (Bertsch et al.,

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2013b; den Bakker et al., 2014; Graves et al., 2010; Halter et al., 2013; Leclercq et al., 2010; Weller et al., 2015). Nonetheless, some of them, including L. ivanovii, L. seeligeri, L.

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innocua, L. welshimeri, and L. grayi, have been occasionally implicated in human clinical cases reports, mainly in individuals with suppressed immune functions and/or underlying illnesses (Liu, 2013).

Apart from L. grayi, listeriae are naturally susceptible to various antimicrobial agents such as

glycopeptides,

tetracyclines,

trimethoprim, penicillins,

carbapenems,

rifampicin,

macrolides, lincosamides, chloramphenicol and naturally resistant to most cephalosporins, they also show reduced susceptibility to fluoroquinolones (Troxler et al., 2000). Quaternary ammonium compounds (QACs), such as benzalkonium chloride (BC) are among the most commonly used disinfectants. They possess antimicrobial effect against a broad range of microorganisms e.g. bacteria, yeast, molds, algae, viruses and play a critical

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ACCEPTED MANUSCRIPT role in controlling the spread of environmentally transmitted pathogens in healthcare and food-processing environments, as well as in the home (Gerba, 2015). They exhibit greater

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activity against Gram-positive bacteria than against Gram-negative ones (Jennings et al.,

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2015). Bacteria are regularly exposed to sublethal concentrations of disinfectants, and this can

initially susceptible bacteria (Hegstad et al., 2010).

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lead to a selective pressure for acquisition of resistance determinants or for adoptation of

Agricultural and industrial practices have a crucial role in polluting the environment with

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heavy metal ions. Microbial populations in this habitat adapt to different concentrations of

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heavy metals and become resistant (Hobman and Crossman, 2014). Resistance to antibiotics, disinfectants and heavy metals is important for bacterial survival in contaminated environments.

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Plasmids, the natural vectors of horizontal gene transfer (HTG), play a key role in the dissemination. of genes of adaptive value (including virulence factors). Their transfer may

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lead to creation of highly virulent, multiresistant strains as well as new emerging pathogens (Schmidt and Hensel, 2004). Listeria spp. are important model for gene transfer studies since

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these bacteria are characterized by high conservation of chromosome genomes, which is a unique feature among the phylum Firmicutes. Relatively small amount of exogenous DNA in the chromosomes suggests that plasmids are major factors in determining the variability of the host strains (den Bakker et. al., 2010). A lot of studies have focused on resistance of L. monocytogens strains isolated from different sources to antimicrobial agents, disinfectants, and heavy metals. However, not to much is known about the resistance to these compounds among Listeria spp. strains, especially isolated from the food processing plant environment. Therefore, in this study we conducted a comprehensive characterization of collection of nonpathogenic Listeria spp, including L. innocua, L. welshimeri, L. seeligeri and L. grayi derived from food and food

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ACCEPTED MANUSCRIPT processing environments in Poland. We determined the susceptibility of these strains to different antimicrobials, arsenic, cadmium and benzalkonium chloride (quaternary ammonium

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compound commonly used as a disinfectant) and assessed a correlation between resistance to

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these compounds and identified extrachromosomal genetic elements  plasmids.

2 Materials and methods

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2.1 Sample collection and bacterial isolation

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All samples were collected from large retail outlets, smaller retail stores and foodproducing factories between 2001 and 2010. The samples were transported to the laboratory

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in portable insulated cold boxes and the swabs were transported in sterile tubes. The samples

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were immediately subjected to microbiological analysis. The strains were isolated according to the standard procedure ISO PN-EN ISO 11290-

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1:1999/A1:2005 as described in detail elsewhere (Korsak et al., 2012). The isolates were preidentified by beta-hemolysis reaction, acid production from rhamnose and xylose and CAMP

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test, followed by the multiplex PCR method described by Huang et al. (2007). The following references strains were used: L. monocytogenes ATCC 13932, L. grayi ATCC 25401, L. welshimeri ATCC 35987, L. seeligeri ATCC 35967, L. innocua PZH 5/04 and L. ivanovii PZH 7/04. L. innocua PZH 5/04 and L. ivanovii PZH 7/04 were obtained from the collections of the National Institute of Public Health  National Institute of Hygiene (Warsaw, Poland). Cultures were maintained in brain-heart infusion agar (BHI; Oxoid, Basingstoke, Hampshire, United Kingdom) at 4 oC throughout the study period and stored at -80 oC in BHI broth containing 20% glycerol.

2.2 Determination of susceptibility

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ACCEPTED MANUSCRIPT 2.2.1 Determination of antimicrobial susceptibility

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The resistance of Listeria spp. isolates to 8 antimicrobial agents: ampicillin (MIC range

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0.125-2 g/ml), ciprofloxacin (0.063-8 g/ml), erythromycin (0.032-2 g/ml), gentamicin

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(0.032-2 g/ml), rifampicin (0.016-2 g/ml), trimethoprim (0.032-2 g/ml), vancomycin (0.125-8 g/ml), and tetracycline (0.063-64 g/ml) was determined using broth microdilution method (antimicrobials supplied as powders by Sigma-Aldrich, St. Louis, USA) according to

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the approved CLSI guidelines M45-A2 for L. monocytogenes (CLSI, 2010). Classification of

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strains as susceptible, intermediate and resistant was based on a protocol from previous susceptibility study with different Listeria species (Troxler et al., 2000). Minimal inhibitory

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concentration (MIC) values of the antimicrobials against the isolates classified as resistant

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were retested three times. In the case of strains resistant to tetracycline, the MICs for

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minocycline were also determined.

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2.2.2 Determination of BC and heavy metal susceptibility

BC and heavy metals susceptibility of Listeria spp. was assessed as described previously by Mullapudi et al. (2008) with minor modifications. Briefly, inoculum was prepared by selecting Listeria colonies from BHI agar incubated for 24 h, and re-suspending them in sterile saline solution to obtain turbidity of 0.5 McFarland units (Densitometer II, PilavaLachema Diagnostika, Czech Republic), which corresponds to approximately 1.5 x 10 8 cfu/ml. To determine susceptibility to BC, a 3 l volume of bacterial suspension was dropped in duplicate on cation adjusted Mueller Hinton 1.2% agar plates (Becton, Dickinson and Company) with 1.2% defibrinated sheep blood supplemented with variable concentrations of BC: 0, 2.5, 5, 10, 20 and 40 g/ml. To determine susceptibility to cadmium and arsen, 3 μl of

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ACCEPTED MANUSCRIPT cell suspension was dropped in duplicate onto Iso-Sensitest agar (ISA) (Oxoid), supplemented with 35, 70, 140, 200 μg/ml anhydrous cadmium chloride (Sigma) or 25, 50, 100, 200, 300,

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500 μg/ml sodium arsenite (Sigma).

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Each plate contained the panel of test isolates, as well as the designated negative control

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strains. The plates were incubated at 37 °C for 48 h, and the quantity of growth on the test plates was compared with that on control ISA or Mueller Hinton plates. Strains were considered resistant to BC, cadmium and arsenic if they yielded confluent

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growth on agar supplemented with 10 g/ml of BC, 70 g/ml cadmium chloride and 500

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μg/ml sodium arsenite, respectively (the criteria for L. monocytogens were adopted,

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Mullapudi et al. 2008). MICs were determined in at least two independent replicates.

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2.3 DNA isolation

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2.3.1 Genomic DNA isolation

Total genomic DNA was extracted using Gene MATRIX Bacterial and Yeast Genomic

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DNA Purification Kit as recommended by the supplier (EurX, Gdańsk, Poland) or Chelex-100 (Bio-Rad, Hercules, USA) resin-based technique. Three to five colonies from BHI plate were suspended in 50 l of 5% Chelex-100. The suspensions were mixed and briefly centrifuged. After incubation for 20 minutes at 95 °C the samples were cooled on ice for 5 min and centrifuged at 2.400 x g for 3 min (Eppendorf MiniSpin Plus Centrifuge). The resulting supernatants were used as DNA templates in the PCR mixture.

2.3.2 Plasmid isolation

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ACCEPTED MANUSCRIPT All Listeria spp. isolates were grown overnight in BHI broth at 37 oC. A 4 ml volume of the culture was harvested by centrifugation (4 °C, 3 min, 2.400 x g, Eppendorf MiniSpin Plus

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Centrifuge). The pelleted cells were used immediately for plasmid DNA preparation or frozen

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at -70 °C for use on the following day. Plasmids were isolated according to the method

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described by Anderson and McKay (1983).

2.4 Genetic basis of resistance mechanisms

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2.4.1 PCR of tetracycline resistance genes and integrase gene

Based on antimicrobial susceptibility results, tetracycline and minocycline resistant Listeria isolates were further characterized by detecting the presence of tet(M), tet(K), tet(L),

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tet(S), tet(T) and int-Tn (Tn916-Tn1545) genes as described previously by Bertrand et al. (2005). PCR products were analyzed by electrophoresis in 0.8% agarose gel and, if

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necessary, purified using Gel-Out Kit (A&A Biotechnology) and cloned into pGEMT-Easy

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vector (Promega) or used as a probe in DNA-DNA hybridization.

2.4.2 DNA-DNA hybridization

Copy number and genomic localization of tet(M) gene were analyzed by DNA-DNA hybridization (Sambrook and Russel, 2001). The specific probes were prepared by PCR amplification of a selected region containing tet(M) gene, using genomic DNA of 10/01 strain as template, and specific oligonucleotide primers as described previously by Bertrand et al. (2005). The amplified DNA fragments were gel-purified and labeled with digoxigenin (DIG, Roche). Total genomic DNA or plasmid DNA of the analyzed Listeria strains (10/01 and 56/06) was digested using appropriate restriction enzymes HindIII or EcoRI and, after DNA

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ACCEPTED MANUSCRIPT electrophoresis, transferred onto nylon membrane. Hybridization and visualization of bound DIG-labelled probes were carried out as recommended by the supplier (Roche). The number

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of DNA bands binding with tet(M) gene probe was equivalent to the minimum number of

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copies of a given element within the genome.

2.4.3 Introduction of plasmid DNA into bacterial cells

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the method of described by Kushner (1978).

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DNA was introduced into Escherichia coli TG1 by chemical transformation according to

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2.4.4 DNA sequencing

The nucleotide sequence of a fragment of tet(M) gene was determined in the Laboratory

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of DNA Sequencing and Oligonucleotide Synthesis (oligo.pl) at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland. Plasmid for DNA sequencing was

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constructed by cloning purified tet(M) gene PCR fragment into pGEM-TEasy vector (Promega). Standard primer pair 21M13 (5'-TGTAAAACGACGGCCAGT-3') and M13Rev (5'-CAGGAAACAGCTATGACC-3') was used to verify the nucleotide sequence of cloned DNA fragment.

2.5 Plasmid profile analysis, restriction fragment length polymorphism (RFLP)

Plasmid DNA was digested with BamHI or EcoRI (Thermo Scientific) and resulting fragments were separated by electrophoresis in 0.8% agarose gel. The size of the plasmids was defined by migration in agarose gel in comparison to reference sequenced plasmids:

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ACCEPTED MANUSCRIPT pK8P1 (39 184 bp; Pseudomonas sp. ANT_K8), pJ2P1 (59 265 bp; Pseudomonas sp. ANT_J2) and pLM19O1 (78 679 bp; Ochrobactrum sp. LM19, Dziewit et. al., 2015).

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2.6 Statistical analysis

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The prevalence of resistance to BC and cadmium and co-resistance to these compounds among Listeria spp, and the prevalence of plasmids among species was compared statistically using chi-square analysis, with significance at P  0.05 defined with Statistica version 6

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software (Statsoft, Inc., Tulsa, USA).

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3 Results

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In this study a total of 127 isolates, including 75 L. innocua, 49 L. welshimeri, 2 L. seeligeri and 1 L. grayi were obtained from different types of food and food-related sources.

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This number includes 55 isolates from ready-to-eat products (sausages, cold cuts; 23 L. innocua, 32 L. welshimeri), 21 isolates from raw beef, pork and beef-pork minced meat (11 L.

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innocua, 10 L. welshimeri), 4 from fresh vegetables (2 L. innocua, 2 L. seeligeri), 3 from poultry carcasses (L. innocua), 2 from cheese (L. innocua), 2 from fresh fish (1 L. innocua, 1 L. welshimeri), 1 from french fries (L. welshimeri) and 39 directly from processing environments (food-contact surfaces and instruments, and nonfood-contact surfaces such as floor , 33 L. innocua, 5 L. welshimeri, 1 L. grayi (Supplementary material).

3.2 Susceptibility profiles of Listeria isolates 3.2.1 Antimicrobial susceptibility

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ACCEPTED MANUSCRIPT All isolates were susceptible to ampicillin, ciprofloxacin, erythromycin, gentamicin, rifampicin, trimethoprim and vancomycin. Only two of the tested isolates (1.57%) showed

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phenotypic resistance: L. innocua 10/01 isolated from raw minced beef meat and L. innocua

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56/06 from processing plant were resistant to both tetracycline compounds (tetracycline and

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minocycline) (Table 1). The MIC value of tetracycline was 48 g/ml and 32 g/ml for L. innocua 10/01 and 56/06 isolates, respectively, whereas MIC of minocycline was 24 g/ml

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3.2.2 Genetic basis of tetracycline resistance

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and 8 g/ml.

In resistant isolates of L. innocua (10/01 and 56/06) the presence of five tetracycline

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resistance genes [tet(M), tet(S), tet(L), tet(O), tet(K)] was investigated. In both strains, only tet(M) gene was detected. The tet(M) gene is often associated with the conjugative

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transposons Tn916 or Tn1545 but, in this study, tet(M)-positive isolates did not contain the integrase (int) gene typical for mobile elements of the Tn916-Tn1545 family. Copy number

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and localization of tet(M) in the genome of 10/01 and 56/06 isolates were analyzed using DNA hybridization. The tet(M) gene was present in a single copy in the chromosomes of both resistant strains.

3.2.3 The prevalence of resistance to BC, cadmium and arsenic among Listeria isolates

Of the 127 Listeria isolates examined, 23 (18.11%) and 71 (55.91%) were resistant to BC and cadmium, respectively (Table 1 and Supplementary material). In contrast, all the isolates were susceptible to arsenic. The prevalence of resistance to BC and cadmium varies between the species. Isolates with BC resistance were significantly more frequent among L. welshimeri (30.61%, 15 out 49) than L. innocua (9.33%, 7 out 75) (P = 0.0024). In the case of cadmium, 12

ACCEPTED MANUSCRIPT the prevalence of resistant isolates was significantly higher among L. innocua (69.33%, 52 out of 75) than L. welshimeri (36.73%, 18 out of 49) (P = 0.0003). Twenty-three isolates

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(18.11%) resistant to BC were also resistant to cadmium. It is noteworthy that the prevalence

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of co-resistant isolates was higher among L. welshimeri (30.61%, 15 out of 49) than among L.

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innocua (9.33%, 7 out of 75).

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3.3 Plasmid profile analysis

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Out of 127 isolates of Listeria spp., 61 strains (48.03%) contained plasmids: L. innocua (41 isolates), L. welshimeri (18), L. grayi (1) and L. seeligeri (1) (Table 2 and Supplementary material). Most of the analyzed strains had only one plasmid, one isolate (L. innocua 24/04)

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contained more than one (Fig. 1A). There were no significant differences between L. welshimeri and L. innocua in the prevalence of plasmids. The size of most of the identified

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plasmids was in the range of 50-90 kb. Plasmid DNA was digested with selected restriction enzymes in order to distinguish strains with identical plasmids. The plasmids were grouped

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based on the results of restriction analysis, enabling the identification of 12 replicon groups with identical restriction pattern - I-XII (Fig. 1B; Supplementary material).

3.4 Relationship between dissemination of plasmids among Listeria isolates and resistance to BC and cadmium

The relationship between resistance to BC and cadmium and detection of plasmids for 127 isolates of Listeria spp. is shown in Table 3. An association between resistance to BC and plasmid DNA carriage was observed: all BC resistant isolates of L. welshimeri, L. innocua, and L. seeligeri carried a plasmid. A correlation between resistance to cadmium and plasmid

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ACCEPTED MANUSCRIPT carriage was also observed in L. innocua and L. seeligeri isolates. In the case of L. welshimeri, 40 out of 52 isolates resistant to cadmium carried plasmids.

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4 Discussion

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Since the detection of the first antibiotic resistant and multiresistant L. monocytogenes

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strains (Poyart-Salmeron et al., 1990), new resistant strains from this genus have been reported. The levels of resistance are varied and influenced by antimicrobial use in humans and animals as well as by geographical factors (Aras and Ardiç 2015; Charpentier et al., 1995;

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Chen et al., 2010; Davis and Jackson, 2009; da Rocha et al., 2012; Gómez et al., 2014; Jamali

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et al., 2014; Jamali et al., 2015; Kovačević et al., 2012; Li et al., 2007; Morenzo et al., 2014; Pesavento et al., 2010; Rahimi et al., 2012; Walsh et al., 2001). Our previous study, including the collection of L. monocytogenes strains, indicated that

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the occurrence of resistance to antibiotics was very low (Korsak et al., 2012). In this study we obtained similar results for nonpathogenic Listeria spp. isolated from food and food-related

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sources, with only two L. innocua isolates resistant to tetracycline and minocycline. The first two tetracycline resistant strains were identified in a Canadian study that

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examined the susceptibility of 26 strains, recovered from milk, to nine antibiotics (Slade and Collins-Thompson, 1990). Since then the incidence of tetracycline resistance has been increasing in strains of Listeria spp. (Bertsch et al., 2014; Charpentier et al., 1995; Chen et al., 2010; da Rocha et al., 2012; Gómez et al., 2014; Jamali et al., 2015; Pesavento et al., 2010; Rahimi et al., 2012; Walsh et al., 2001). Tetracycline resistance is often determined by the acquisition of genes encoding energydependent efflux or a protein that protects bacterial ribosomes from the action of antibiotic. Many of these genes are associated with self-transmissible plasmids or conjugative transposons (Chopra and Roberts, 2001). In the present study we screened the tetracycline resistant strains for the presence of five known tetracycline-resistance genes described in

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ACCEPTED MANUSCRIPT Listeria spp. The determinants tet(K) and tet(L) confer the resistance to tetracycline only, while the other three genes tet(M), tet(T), and tet(S) can confer both tetracycline and

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minocycline resistance by ribosomal protection (Bertrand et al., 2005). According to most

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studies, the major genotype for tetracycline resistance in Listeria strains is the tet(M) gene

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(Bertrand et al., 2005; Bertsch et al., 2014; Charpentier et al., 1995; Chen et al. 2010; Davis and Jackson, 2009; Facinelli et al., 1993; Li et al., 2007), which is found most frequently in the chromosomes and often associated with a large, broad-host-range conjugative transposons

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of the Tn916-Tn1545 family acquired from Enterococcus-Streptococcus (Marra et al., 1999).

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In resistant L. innocua strains analyzed in this study tet(M) gene was located in the chromosome. However, none of these strains carried the integrase (int) gene of Tn916Tn1545, which greatly reduces the ability to transfer of tet(M) gene to other species.

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In this study we also identified and characterized a pool of plasmids, which were present. in 61 Listeria spp. isolates (48.03%). The size of the analyzed plasmids was in the range of

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50-90 kb typical for plasmids identified so far in this group of bacteria (Bertsch et al., 2013a; Canchaya et al., 2010; den Bakker et al., 2012; Gilmour et al., 2010; Kuenne et al., 2010;

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Kuenne et al., 2013; Nelson et al., 2004). We identified twelve different plasmid restriction patterns. Remarkably, plasmids belonging to the same group were determined in the isolates of the same species. Only in one case, plasmid of the same restriction pattern was isolated from L. innocua (36/06, 58/06, 62/06, 64/06, 75/06, 79/06) and L. welshimeri (75/06), which may indicate the conjugal transfer of this replicon. In addition, many plasmid profile were similar, suggesting the relatedness of the plasmid. Presuambly they have a conserved core genome and differ only by the presence of exogenous DNA, acquired by HTG. The plasmid restriction patterns were compared with the in silico determined patterns of other Listeria spp. plasmids, whose complete nucleotide sequences were available in the NCBI database. The analysis revealed that several plasmids identified in this study (III-type) have a similar pattern

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ACCEPTED MANUSCRIPT to pLM80 replicon of L. monocytogenes 4b strain H7858 (Nelson et al., 2004). This plasmid harbored a gene cassette conferring resistance to BC and other quaternary ammonium

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disinfectants as well as to cadmium resistance, which is a characteristic feature of many L.

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monocytogenes plasmids (Elhanafi et al., 2010; Kuenne et al., 2010; McLauchlin et al., 1997;

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Nelson et. al., 2004). All Listeria isolates analyzed in this study, which carried type III plasmids, were also resistant to BC and cadmium.The incidence of resistance to BC and heavy metals in Listeria species other than L. monocytogenes and possible correlations

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between heavy metal resistance and disinfectant resistance has not yet been detected. We

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showed that only 18.11% of nonpathogenic Listeria isolates were resistant to BC, but as many as 55.91% were resistant to cadmium. In L. innocua, L. welshimeri and L. seeligeri isolates resistance to the quaternary ammonium disinfectant BC was correlated with resistance to

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cadmium. Without exception, all isolates found to be BC resistant were also resistant to this metal. On the other hand, 67.61% of cadmium resistant strains were susceptible to BC,

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suggesting that cadmium and BC resistance are not always linked. Within the co-resistant Listeria isolates we identified plasmids, which leads to the speculation that BC resistance

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determinants were acquired by plasmids that already harbored cadmium resistance genes. Noteworthy, our findings indicate that among 71 isolates resistant to cadmium, 12 L. innocua did not have any plasmids. This may suggest that resistance is associated with determinants located within the chromosome. The processing environment may present many opportunities for nonpathogenic Listeria spp. to interact with L. monocytogenes. Katharios-Lanwermeyer et al. (2012) demonstrated the conjugative transfer of BC and cadmium resistance determinants from L. innocua and L. welshimeri to L. monocytogenes and between these two nonpathogenic Listeria species. Nonpathogenic Listeria spp. have the potential to serve as reservoirs for resistance genes to and transfer them within mobile genetic elements among themselves, as well as to L.

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ACCEPTED MANUSCRIPT monocytogenes inhabiting the same environment. Mobile DNA seems to play a significant role in the variability and evolution of Listeria genomes.

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In conclusion, presented results obtained from a collection of nonpathogenic Listeria

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isolates from many different types of food and food-related sources in Poland indicate that the

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occurrence of resistance to antibiotics in these species is very low. Our findings also demonstrate the correlation between the prevalence of resistance to disinfectants such as BC, and to heavy metal ions, such as cadmium, and plasmid carriage.

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Further studies are needed to determine the resistance mechanisms to BC and cadmium in

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these species and the possible role of different resistant determinants in the ecology and adaptation of listeriae that contaminate food and food-processing environments. Acknowledgments

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This work was partially supported financially by the Ministry of Science and Higher

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Education (Grant N N312 255335).

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Fig. 1. A) Plasmid profile of Listeria spp. Plasmid DNA was extracted using the method described by Anderson and McKay (1983). Plasmid size standards are isolated from Pseudomonas sp. ANT_K8 (pK8P1 – 39 184 bp), Pseudomonas sp. ANT_J2 (pJ2P1 – 59 265 bp), Ochrobactrum sp. LM19 (pLM19O1 – 78 679 bp; Dziewit et al., 2015). The black arrow indicates the position of chromosomal DNA. The species abbreviations: (L.i.) – L. innocua, (L.w.) – L. welshimeri, (L.s.) – L. seeligeri, (L.g.) – L. grayi. B) Plasmid restriction patterns. Plasmid DNA was digested with EcoRI restriction enzyme in order to enable the identification of 12 replicon groups with identical restriction pattern

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No. (%) resistant 7 (9.33) 15 (30.61) 1 (50) 0 (0) 52 (69.33) 18 (36.73) 1 (50) 0 (0) 2 (2.67) 0 (0) 0 (0) 0 (0)

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Table 1 Antimicrobial susceptibility of Listeria spp. isolates. MIC* MIC90 No. (%)** range Antimicrobial Species susceptible (g /ml) (g /ml) 2.5-40 Benzalkonium chloride 5 68 (90.67) L. innocua 40 34 (69.39) L. welshimeri L. seeligeri 5 1 (50) L. grayi 5 1 (100) 35-200 Cadmium 200 23 (30.67) L. innocua 200 31 (63.27) L. welshimeri L. seeligeri 70 1 (50) L. grayi 35 1 (100) 0.063-64 Tetracycline 1 73 (97.3) L. innocua 1 49 (100) L. welshimeri L. seeligeri 1 2 (100) L. grayi 1 1 (100)

The table provides results only for those antimicrobial agents and heavy metals for which the resistance was observed.

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*BC and heavy metals susceptibility was assessed as described previously by Mullapudi et al. (2008).

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**Classification of strains as susceptible and resistant was based on a study described by Troxler et al. (2000) (antimicrobial agents) and Mullapudi et al. (2008) (BC and heavy metals).

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Strains L. innocua L. welshimeri L. seeligeri L. grayi Total

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34 (90.93)/7 34/15 3 (16.67)/15 1/1 (17.07) (83.33) 23/52 1 (2.44)/40 31/18 0 (0)/18 1/1 (97.56) (100) 22/7 0 (0)/7 31/15 0 (0)/15 0/1 (100) (100)

0 (0)/1 (100) 0 (0)/1 (100) 0 (0)/1 (100)

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Table 3 Resistance to benzalkonium chloride and cadmium and presence of plasmid DNA in Listeria spp. isolates. No. of isolates L. innocua L. L. seeligeri L. grayi welshimeri Plasmid Plasmid Plasmid Plasmid Total DNA Total DNA Total DNA Total DNA S/R* detected S/R detected S/R detected S/R detected S/R (%) S/R (%) S/R (%) S/R (%)

* S – sensitive; R – resistant.

1/0 (0) 0/1 0/0

1 (100)/0 (0) 0 (0)/1 (100) 0 (0)/0 (0)

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In Poland the prevalence of resistance to antimicrobials among nonpathogenic Listeria isolates is low Two from among 127 isolates showed resistance to tetracycline and minocycline

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18.11% isolates were resistant to benzalkonium chloride while 55.91% to cadmium

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All the isolates were susceptible to arsenic

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12 different restriction patterns were identified among 61 isolates harboring plasmids

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There was an association between resistance to BC, cadmium and plasmid carriage

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