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Antimicrobial resistance profiles of Listeria monocytogenes isolated from ready-to-eat products in Poland in 2007–2011
Accepted Manuscript Antimicrobial resistance profiles of Listeria monocytogenes isolated from ready - to eat products in Poland in 2007- 2011 Elżbieta Maćkiw, Magdalena Modzelewska, Łukasz Mąka, Halina Ścieżyńska, Kamila Pawłowska, Jacek Postupolski, Dorota Korsak PII:
S0956-7135(15)30005-0
DOI:
10.1016/j.foodcont.2015.05.011
Reference:
JFCO 4445
To appear in:
Food Control
Received Date: 25 February 2015 Revised Date:
30 April 2015
Accepted Date: 10 May 2015
Please cite this article as: Maćkiw E., Modzelewska M., Mąka Ł., Ścieżyńska H., Pawłowska K., Postupolski J. & Korsak D., Antimicrobial resistance profiles of Listeria monocytogenes isolated from ready - to - eat products in Poland in 2007- 2011, Food Control (2015), doi: 10.1016/ j.foodcont.2015.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Antimicrobial resistance profiles of Listeria monocytogenes isolated from ready - to - eat products in Poland in 2007- 2011
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Abstract
The aim of the study was to characterize strains of L. monocytogenes isolated from ready to eat (RTE) products collected as part of official food control and monitoring in Poland. A total
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of 105 L. monocytogenes isolates from RTE products: 54- cakes and 51 - delicatessen products were examined. The presence L. monocytogenes in cakes and delicatessen products
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was 0.4 % and 0.7 % respectively suggesting the level of contamination of RTE products with
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L. monocytogenes is very low.
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Elżbieta Maćkiw1*, Magdalena Modzelewska1, Łukasz Mąka1, Halina Ścieżyńska1, Kamila
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Pawłowska1, Jacek Postupolski1, Dorota Korsak2
3 4 Antimicrobial resistance profiles of Listeria monocytogenes
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isolated from ready - to - eat products in Poland in 2007-2011
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National Institute of Public Health - National Institute of Hygiene, Department of Food
Safety, 24 Chocimska str , 00-791 Warszawa, Poland
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Miecznikowa str, 02-096 Warsaw, Poland
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University of Warsaw, Faculty of Biology, Department of Applied Microbiology, 1
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* corresponding author
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Elżbieta Maćkiw
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[email protected]
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Tel. + 48 22 54 21 381, Fax. +48 22 54 21 392
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1. Introduction
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Listeria monocytogenes, a ubiquitous gram-positive microaerophilic bacterium has
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been recognized as an important foodborne pathogen, causing severe listeriosis infections in
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humans. (encephalitis, meningitis and septicaemia) and animals (mastitis, diarrhea and 1
ACCEPTED MANUSCRIPT gastroenteritis). Clinical manifestations of invasive listeriosis are usually severe and include
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abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile
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gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate
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species, including birds (Vázquez-Boland et al., 2001). Groups at highest risk are pregnant
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women, neonates, adults with underlying disease (cancer, AIDS, diabetes, chronic hepatic
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disorder, transplant recipients), the elderly (>65 years old), and other immunocompromised
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individuals (McLauchlin, Mitchell, Smerdon, & Jewell, 2004; Liu, 2006; Martins & Leal
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Germano, 2011). In 2013, 27 Member States of the European Union reported 1 763 confirmed
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human cases of listeriosis, which was an 7.3 % increase compared with 2012. The EU
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notification rate was 0.44 cases per 100,000, the highest specific notification rate being
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observed in Finland (1.12) and the lowest in Bulgaria (0.04). The notification rate in Poland
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was 0.15 (EFSA 2014).
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Listeriosis is a sporadic disease which is often associated with consumption of
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contaminated milk, soft cheese, under-cooked meat, fish, eggs and egg products, ready - to -
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eat products (RTE), unwashed raw vegetables and cabbage (Aureli et al., 2003; Gibbons,
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Adesiun, Seepersadsingh, & Rahaman, 2006; De Santis et al., 2007; Arslan & Ozdemir, 2008;
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Filiousis, Johansson, Frey, & Perreten, 2009; Garrido, Vitas, & Gracia-Jalon, 2009; Harakeh
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et al., 2009; Rahimi, Ameri & Momtaz, 2010; Jacobsen, Heggebø, Sunde, & Skjervheim,
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2011; Alonso-Hernando et al., 2012; Bouayad & Hamdi, 2012; Cokal, Dagdelen, Cenet, &
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Gunsen, 2012). Control of L. monocytogenes contamination represents a significant problem
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for the food industries, public health agencies and government bodies.
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L. monocytogenes has been differentiated into 13 serotypes 1/2a, 1/2b, 1/2c, 3a, 3b,
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3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 (Liu, 2006; Jamali, Radmehr & Thong, 2013). Molecular
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serogroup-related PCR typing of L. monocytogenes is based on the detection by PCR of
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serogroup-specific regions, leading to five genoserogroups: IIa (serotype 1/2a-3a), IIb 2
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(serotypes 1/2b-3b-7), IIc (serotypes 1/2c-3c), IVa (serotype 4a-4c), IVb (serotypes 4ab-4b,
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4d-4e) (Borucki & Call, 2003; Doumith et al., 2004). Of the serotypes of L. monocytogenes which can cause illness, only four account for
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95% of human isolates: 1/2a, 1/2b, 1/2c and 4b (Kathariou, 2002; Uhitil at al., 2004; Huang et
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al, 2011; Martins & Leal Germano, 2011). In the US, in 2012 serotype 4b was the most
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commonly identified serotype, accounting for 54% of isolates and serotype 1/2a was second
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with 28% (CDC, 2014). The most frequently isolated serotype from clinical samples in Brazil
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was 4b (60.3%), followed by 1/2a (29.0%) (Hofer et al., 2006).
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Serotype 1/2a has frequently been detected in different food matrices (Martins & Leal
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Germano, 2011; Jamali, Radmehr & Thonga, 2013). The most common serotypes in 2013
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were 1/2a (57.5 %) and 4b (34.3 %), followed by 1/2b (6.4 %), 1/2c (1.4 %), 3a and 3b (both
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0.2 %). The most common PCR serogroup was IIa (44.7 %, corresponding to conventional
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serotypes 1/2a and 3a), followed by IVb (44.6 %, corresponding to serotypes 4b, 4d, and 4e),
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IIb (7.8 %, corresponding to serotypes 1/2b, 3b and 7) and IIc (2.9 %, corresponding to
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serotypes 1/2c and 3c) (EFSA 2014).
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L. monocytogenes has generally been considered susceptible to antibiotics active
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against Gram-positive bacteria. The effective treatment of choice for listeriosis is a β-lactam
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antibiotic (e.g. penicillin, ampicillin), alone or in combination with an aminoglycoside (e.g.
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gentamicin) in the case of immunocompromised patients. The second treatment of choice is
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the combination of trimethoprim and a sulfonamide (e.g. sulfamethoxazole), especially for
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patients allergic to β-lactams (Conter et al., 2009). Vancomycin and erythromycin are also
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used, respectively, to treat bacteremia and pregnant women diagnosed with listeriosis. Other
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antibiotics used in cases of listeriosis are rifampicin, tetracycline, chloramphenicol and the
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fluoroquinolones (Alonso-Hernando et al., 2012; Wang et al., 2013).
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ACCEPTED MANUSCRIPT Antimicrobials are increasingly used in feeds for the control and treatment of diseases
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in animals. Treatment of infections due to opportunistic bacteria may become a problem
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(Arlsan & Özdemir, 2008). The European Union in 2006 prohibits the use of antimicrobials
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which are consumed as human medicine in animal foods, as growth promoting agents of
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animals (Regulation (EC) No, 1831/2003). However, an increasing number of strains are
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becoming resistant to most of the known antibiotics including those used for treating
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listeriosis. Some multidrug resistant L. monocytogenes strains have been isolated from foods
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and human listeriosis cases. The levels of resistance are varied and influenced by
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antimicrobial use in humans and animals, as well as geographical differences (Korsak et al.,
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2012).
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Little information is available on the antimicrobial susceptibility of L. monocytogenes
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isolated from cakes and delicatessen products in Poland. In the first study made in Poland,
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Korsak and co-workers (2012) examined 471 L. monocytogenes isolates from different types
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of food and food-related sources (including 43 ready-to-eat products and 15 from cakes). The
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study showed low prevalence of antimicrobial resistance among L. monocytogenes isolates.
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The aim of this study was to characterize strains estimate the prevalence of L.
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monocytogenes isolated from foods cakes and delicatessen collected under official control and
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monitoring in Poland and to determine the susceptibility of L. monocytogenes isolates to
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various antimicrobial agents. Moreover, the aim was also to identify the serotypes of the
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isolates, which served to determine the diversity of the test collection of L. monocytogenes
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isolates.
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2. Materials and methods
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2.1. Food samples
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presence of L. monocytogenes in food samples. A total of 144 555 food samples were
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collected from large retail outlets and smaller units, including 27 175 samples of cakes and 20
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304 samples of delicatessen products, excluding meat. The samples were tested for the
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presence of L. monocytogenes by the laboratories of Provincial Sanitary and Epidemiological
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Stations, pursuant to the procedure described in the standard PN-EN ISO 11290-
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1:1999+A1:2005. L. monocytogenes bacteria were isolated from 257 RTE samples, including
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from 112 of the cakes and from 145 of delicatessen products. To our laboratory, i.e. the
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National Reference Laboratory for L. monocytogenes, 105 strains (51 from cakes and 54 from
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delicatessen) were sent for confirmation. These strains were subjected to further studies.
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2.2. DNA isolation
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DNA isolation was performed using kit Genomic mini (A&A BIOTECHNOLOGY, Poland).
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2.3. Serotyping of L. monocytogenes
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The multiplex PCR assay was carried according to the procedure in-house method:
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Listeria monocytogenes molecular serotyping described by the European Union Reference
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Laboratory for L. monocytogenes ANSES (Maison-Alfort, France). In brief, 25 µl of reaction
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mixture contained 1U Hot Start polymerase (KAPA Taq, Biosystem), 1× fast-start buffer, 2
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mM MgCl2, 0.2 mM dNTPs, 0.4 µM each of the following primers: LMO0737-1 and
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LMO0737-2; LMO1118-1 and LMO1118-2; ORF2110-1 and ORF2110-2; ORF2819-1 and
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ORF2819-2 and 0.1 µM of the other primers PRS-1 and PRS-2 and 0.2 µM LIP-1 and LIP-2
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(Genomed, Warsaw, Poland) and bacterial DNA. The cycling program consisted of initial
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denaturation for 3 min at 94 °C, followed by 35 cycles of denaturation at 94 °C (40 s),
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annealing at 53 °C (45 s), extension at 72 °C (1 min 15 s) and a final extension at 72 °C (7
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min). A second PCR assay was performed to detect the presence of the flaA gene. The
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ACCEPTED MANUSCRIPT amplification mix of 25 µl, contained 1U Hot Start polymerase (KAPA Taq, Biosystem); 1×
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fast-start buffer, 4 mM MgCl2, 0.2 mM dNTPs, 0.8 µM flaA-F and flaA-R primers and DNA.
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The PCR conditions were as follows: initial denaturation for 3 min at 94 °C, followed by 40
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cycles of denaturation at 94 °C (30 s), annealing at 61 °C (40 s), extension at 72 °C (1 min);
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and a final extension at 72 °C (7 min). The sequences of primers are listed in Table 1.
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Amplified PCR products were visualized by electrophoresis in 1.5% agarose gel stained with
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Midori green. The molecular group IIa (serotype 1/2a-3a) includes isolates in which genes:
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lmo1118, lmo0737, prs, prfA, flaA have been identified; molecular group IIc (serotypes 1/2c-
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3c): lmo1118, lmo0737, prs, prfA; molecular group IIb (serotypes 1/2b-3b-7): orf2819, prs,
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prfA; molecular group IVa (serotype 4a-4c): prs, prfA, flaA
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(serotypes 4ab-4b, 4d-4e): orf2110, orf2819, prs, prfA. The multiplex PCR profiles do not
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distinguish serovar 1/2a from 3a, 1/2b from 3b and 7, 1/2c from 3c or 4ab from 4b and 4d, 4e
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within L. monocytogenes.
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2.4. Antimicrobial susceptibility testing
and molecular group IVb
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Antibiotic susceptibility was assessed using the disk diffusion method on Mueller-
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Hinton agar plate containing 5% sheep blood (Oxoid) and incubated at 37 °C for 18 to 20 h.
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The following discs (Oxoid) were used: gentamicin (10 μg), meropenem (10 μg), ampicillin
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(10 μg), sulfamethoxazole-trimethoprim (23.75/1.25 μg), amoxycillin/clavulanic acid - 2:1
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(20/10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg). The inhibition
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zones were measured and scored as sensitive, intermediate susceptibility and resistant. E. coli
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ATCC 35218 and Staphylococcus aureus ATCC 25923 were used as reference strains for
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antibiotic disc control. Tests and results were performed following recommendations for
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Staphylococcus sp. (CLSI, 2012).
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3. Results and discussion The results of the study carried within the scope of official food controls indicate that
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the level of contamination with L. monocytogenes of RTE products collected in Poland from
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27 175 samples of cakes and 20 304 samples of delicatessen is very low. The presence of L.
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monocytogenes in cakes and delicatessen products was 0.4 % and 0.7 % respectively. Little
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information is available regarding the quality of cakes, delicatessen and the contamination of
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these products by L. monocytogenes in other countries. A study carried out in Croatia also
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showed a low level of contamination, which, however, was higher than in Poland. Eighteen
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samples of cakes, collected at hotels, restaurants and pastry shops throughout Croatia, were
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found to be positive for Listeria spp. (6.36%), where L. monocytogenes was isolated from 12
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samples (4.27%) (Uhitil at al., 2004). Similar results were presented by Mengesha et al.
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(2009). In this study, L. monocytogenes was isolated in ready-to-eat food items consisting of
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ice cream (11.7%), cakes (6.5%), and soft cheese (3.9%). The prevalence of L.
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monocytogenes was in agreement with a study in Serbia (Gusman et al., 2014). 912 samples
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were analyzed for the presence of L. monocytogenes and the contamination rate was found to
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be 5.6% for cream cheese and 5.6% for cakes.
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The serogroup distribution for the 105 L. monocytogenes strains from RTE was
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determined (Table 2). Our data showed higher percentages of isolates belonging to molecular
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group IVb (serotypes 4ab-4b, 4d-4e) which comprised 33 strains (31.4%) in our study.
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However, the isolation rate of molecular group IIa (serotype 1/2a-3a) was at a similar level as
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molecular group IIb (serotypes 1/2b-3b-7), respectively 21.9% and 24.8%. The molecular
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group IIc (serotypes 1/2c-3c) was relatively rare in RTE (2.9%). The presence of these
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important serotypes among such food items indicates that these foods may pose a potential
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public health risk. There were no representatives of molecular group IVa (serotype 4a-4c).
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The serotypes of L. monocytogenes present in food were diverse and the presence of potential
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number one serotype associated with human listeriosis (Zhang et al., 2007). Serovar 4b is not
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the most common subtype isolated from food, affirming that this discrepancy between the
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occurrence in foods and listeriosis cases might suggest that serotype 4b is more virulent than
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the other serotypes. The absence of a correlation between the serotype present in the foods
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and those identified in listeriosis is due to the survival characteristics of certain serotypes in
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foods (Martins & Leal Germano, 2011). The high prevalence of serotype 4ab- 4b, 4d- 4e in
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our study is similar to the results obtained by Mengesha et al. (2009). In this study, 711
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samples were randomly collected and from among them 34 isolates of L. monocytogenes
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serotypes 4b/4e (n = 32), 4c, and 4e were identified.
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However, in other studies serotype 1/2a occurred more frequently. The most
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commonly isolated from RTE products serotype of L. monocytogenes in Wales was 1/2a
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(Meldrum et al., 2010). The results obtained by Guerra at al. 2001 showed that among the
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strains isolated serogroup 1/2a was the most frequently recovered from RTE in Portugal.
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Serotype identification by PCR of the strains isolated from RTE foods in Belgian revealed
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that most belonged to the 1/2a serotype group (Van Coillie et al., 2004). In the study of
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Korsak et al. (2012) the majority of 43 isolates obtained from RTE products belonged to
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serotypes 1/2a and 3a (30.2%) and 1/2c and 3c (32.6%). The serogroup distribution for the 32
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L. monocytogenes from RTE products in Malaysia was determined to be: 1/2a- 3a (65.6%),
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1/2c-3c (21.9%) and 4b- 4d-4e (12.5%) (Jamali & Tong, 2014). The L. monocytogenes
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isolates form RTE products in China belonged to serotypes 1/2a and 4b (Wang et al., 2013).
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L. monocytogenes strains were tested for their susceptibility to antimicrobials. The
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results of this study suggest that the overall incidence of antibiotic resistance in L.
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monocytogenes is relatively very low. Ten of the tested strains (9.5%) were resistant to
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ampicillin; 5 strains isolated from delicatessen products and 5 strains isolated from cakes
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ACCEPTED MANUSCRIPT (Table 3). These results are lower than reported by Rahimi, Ameri and Momtaz (2010) who
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found that 26.3 % of L. monocytogenes isolates from milk and dairy products in Iran were
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resistant to ampicillin. Also strains isolated from ready-to-eat food in Italy showed a similar
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level of resistance to ampicillin and methicillin (Pesavento et al., 2010). Twenty percent of L.
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monocytogenes strains were determined by the disk diffusion method to be resistant to
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ampicillin and 22.5% to methicillin. It is of concern that this expanding range now includes a
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number of antibiotics used to treat listeriosis, e.g. penicillin, ampicillin. Results from the
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present study show that all of the L. monocytogenes isolates were sensitive to antibiotics
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commonly used as a second-choice therapy to treat listeriosis (ciprofloxacin, tetracycline,
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chloramphenicol, and the combination sulfamethoxazole-trimethoprim). In the study of
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Rodas-Suárez et al., (2006) no incidence of resistance to antimicrobial agents tested:
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gentamicin,
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streptomycin, meropenem, cefotaxime and amoxycillin/clavulanic acid was observed.
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ampicillin,
sulfamethoxazole-trimethoprim
erythromycin,
tetracycline,
Our data show that L. monocytogenes contamination occurred in RTE: these isolates
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were resistant to ampicillin and they belonged to serotypes 4ab-4b-4d-4e and 1/2a-3a which
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are associated with human listeriosis, suggesting that this pathogen may represent a potential
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danger to public health. Results from the present study show the sensitivity all of the L.
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monocytogenes isolates to antibiotics commonly used as second-choice therapy to treat
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listeriosis. Resistant to ampicillin bacterial strains, like those observed in this study, could be
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transmitted to people through contact or food consumption. This may further complicate the
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clinical management of disease caused by the pathogen.
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This work was supported financially by NIPH-NIH (6/ZŚ/2013-5/ZŚ/2014).
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Huang, B., Fang, N., Dimovski, K., Wang, X., Hogg, G., & Bates, J. (2011). Observation of a
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new pattern in serogroup-related PCR typing of Listeria monocytogenes 4b isolates.
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Jamali, H., Radmehr, B., & Thong, K. L. (2013). Prevalence, characterization, and
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Jamali, H., & Thong, K. L. (2014). Genotypic characterization and antimicrobial resistance of Listeria monocytogenes from ready-to-eat foods. Food Control, 44, 1-6
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Kathariou, S. (2002). Listeria monocytogenes virulence and pathogenicity, a food safety perspective. Journal of Food Protection, 65(11), 1811-1829.
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Liu, D. (2006). Identification, subtyping and virulence determination of Listeria
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monocytogenes, an important foodborne pathogen. Journal of Medical Microbiology,
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Martins, E. A., & Leal Germano, P. M. (2011). Listeria monocytogenes in ready-to-eat,
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sliced, cooked ham and salami products, marketed in the city of São Paulo, Brazil:
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Occurrence, quantification, and serotyping. Food Control, 22(2), 297–302.
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McLauchlin, J., Mitchell, R. T., Smerdon, W. J., & Jewell, K. (2004). Listeria monocytogenes
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and listeriosis: a review of hazard characterisation for use in microbiological risk
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assessment of foods. International Journal of Food Microbiology, 92(1), 15–33.
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Meldrum, R.J., Ellis, P.W., Mannion, P.T., Halstead, D., Garside, J. (2010). Prevalence of
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Listeria monocytogenes in ready-to-eat foods sampled from the point of sale in Wales,
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United Kingdom. Journal of Food Protection. 73(8), 1515-1518.
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Mengesha, D., Zewde, B.M., Toquin, M.T., Kleer, J., Hildebrandt, G., & Gebreyes, W.A. (2009).
Occurrence
and
distribution
of
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monocytogenes and 13
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other Listeria species in ready-to-eat and raw meat products. Berliner und Münchener
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tierärztliche Wochenschrift.122 (1-2): 20-24.
Pesavento, G., Ducci, B., Nieri, D., Comodo, N., & Lo Nostro, A. (2010). Prevalence and
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Control, 21(5), 708–713.
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Rahimi, E., Ameri, M., & Momtaz, H. (2010). Prevalence and antimicrobial resistance of
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Listeria species isolated from milk and dairy products in Iran. Food Control, 21(11),
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1448–1452.
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Rodas-Suárez, O.R., Flores-Pedroche, J.F., Betancourt-Rule, J.M., Quiñones-Ramírez, E.I.,&
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Vázquez-Salinas, C., (2006). Occurrence and antibiotic sensitivity of Listeria
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monocytogenes strains isolated from oysters, fish, and estuarine water. Applied and
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Environmental Microbiology, 72, 7410–7412.
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Uhitil, S., Jakšić, S., Petrak, T., Medić, H., & Gumhalter-Karolyi, L. (2004). Prevalence of
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Listeria monocytogenes and the other Listeria spp. in cakes in Croatia. Food Control,
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15(3), 213–216.
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Van Coillie, E., Werbrouck, H., Heyndrickx, M., Herman, L., & Rijpens, N. (2004).
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Prevalence and typing of Listeria monocytogenes in ready-to-eat food products on the
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Belgian market. Journal of Food Protection, 67(11), 2480-2487.
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Vázquez-Boland, J.A., Kuhn, M.,
Berche, P., Chakraborty, T., Domínguez-Bernal, G.,
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Goebel, W., et al. (2001). Listeria pathogenesis and molecular virulence determinants.
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Clinical Microbiology Reviews, 14(3), 584–640.
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Wang, X.-M., Lü, X.-F., Yin, L., Liu, H.-F., Zhang, W.-J., Si, W., Yu, S.-Y., Shao, M.-L.
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Liu,
S.-G.
(2013).
Occurrence
and
antimicrobial
susceptibility
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monocytogenes isolates from retail raw foods. Food Control, 32, 153-158.
of
Listeria
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Zhang, Y., Yeh, E., Hall, G., Cripe, J., Bhagwat, A. a, & Meng, J. (2007). Characterization of
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Listeria monocytogenes isolated from retail foods. International Journal of Food
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Microbiology, 113(1), 47–53.
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Table 1. List of primers for PCR serotyping and expected amplicon sizes.
prs
PRS 1 PRS 2
orf 2819 orf 2110 fla A
15 16
LMO1118-1 LMO1118-2
370
GCT GAA GAG ATT GCG AAA GAA G CAA AGA AAC CTT GGA TTT GCG G
274
GAT ACA GAA ACA TCG GTT GGC GTG TAA TCT TGA TGC CAT CAG G
691
AGG GCT TCA AGG ACT TAC CC ACG ATT TCT GCT TGC CAT TC
1/2a-3a, 1/2c-3c
ORF2819- 1 ORF2819-2
471
AGC AAA ATG CCA AAA CTC GT CAT CAC TAA AGC CTC CCA TTG
4ab-4d-4e-7
ORF2110-1 ORF2110-2
597
AGT GGA CAA TTG ATT GGT GAA CAT CCA TCC CTT ACT TTG GAC
4ab-4b-4d-4e
538
TTA CTA GAT CAA ACT GCT CC AAG AAA AGC CCC TCG TCC
4 5 6
D’Agostino et al., 2004
7
Doumith et al. 2004
8
Doumith et al. 2004
1/2c-3c
9 10
Doumith et al. 2004
1/2a-3a-4a-4c
3
Doumith et al. 2004
L. monocytogenes
AGG GGT CTT AAA TCC TGG AA CGG CTT GTT CGG CAT ACT TA
flaA-F flaA-R
Reference
Listeria spp.
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lmo 1118
LMO0737-1 LMO0737-2
Specific to
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lmo0737
LIP 1 LIP 2
Sequence (5’-3’)
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prfA
Size (in bp)
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Primer
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Gene targeted
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2
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Doumith et al. 2004
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Borucki and Call 2003
13 14
17
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Table 2. Molecular serogroups of L. monocytogenes isolated from ready- to- eat products in Poland in 2007–2011.
IIb
10 (19.5%)
14 (27.5%)
3 (5.9%)
0 (0%)
20 (39.2%)
Not classified 4 (7.8%)
243 (24.0%)
12 (22.2%)
0 (0%)
0 (0%)
13 (24.2%)
16 (29.6%)
23 (21.9%)
26 (24.8%)
3 (2.9%)
0 (0%)
33 (31.4%)
20 (19.0%)
19 20
25 26 27
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IVb
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IIa
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Delicatessen (excluding meat products) (N=51) Cakes (N=54) Ready-to-eat products Total (N=105)
Molecular serogroups IIc IVa
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28
2
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Table 3. Antimicrobial resistance of L. monocytogenes isolates from ready- to- eat products in Poland in 2007–2011
30
CIP (5 μg)
AMP (10 μg)
Delicatessen (excluding meat products) (N=51) Cakes (N= 54)
0/51
0/51
0/51
5/51 (9.8%)
0/54
0/54
0/54
5/54 (9.3%)
Ready-to-eat products Total (N=105)
0/105
0/105
0/105
10/105 (9.5%)
MEM (10 μg)
SXT (23.75/ 1.25 μg) 0/51
AMC (20/ 10 μg) 0/51
TET (30 μg)
0/54
0/54
0/54
54/0
0/105
0/105
0/105
0/105
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CN (10 μg)
0/51
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0/51
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C: chloramphenicol, CN: gentamycin, CIP: ciprofloxacin, AMP: ampicillin, MEM: meropenem, SXT: sulphamethoxazole-trimethoprim, amoxycillin/clavulanic acid (2:1), TET: tetracycline.
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31 32 33
C (30 μg)
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Number of strains resistant / tested (%)
3
*Highlights (for review)
ACCEPTED MANUSCRIPT Monitoring study of L. monocytogenes in RTE products on retail in Poland Low level of contamination of RTE products with L. monocytogenes High incidence of serotype 4ab-4b-4d-4e (molecular group: IVb) of L. monocytogenes Nine and a half percent of L. monocytogenes isolates were resistant to ampicillin
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All L. monocytogenes isolates were sensitive to other examined antibiotics
S0956-7135(15)30005-0
DOI:
10.1016/j.foodcont.2015.05.011
Reference:
JFCO 4445
To appear in:
Food Control
Received Date: 25 February 2015 Revised Date:
30 April 2015
Accepted Date: 10 May 2015
Please cite this article as: Maćkiw E., Modzelewska M., Mąka Ł., Ścieżyńska H., Pawłowska K., Postupolski J. & Korsak D., Antimicrobial resistance profiles of Listeria monocytogenes isolated from ready - to - eat products in Poland in 2007- 2011, Food Control (2015), doi: 10.1016/ j.foodcont.2015.05.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Antimicrobial resistance profiles of Listeria monocytogenes isolated from ready - to - eat products in Poland in 2007- 2011
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Abstract
The aim of the study was to characterize strains of L. monocytogenes isolated from ready to eat (RTE) products collected as part of official food control and monitoring in Poland. A total
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of 105 L. monocytogenes isolates from RTE products: 54- cakes and 51 - delicatessen products were examined. The presence L. monocytogenes in cakes and delicatessen products
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was 0.4 % and 0.7 % respectively suggesting the level of contamination of RTE products with
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L. monocytogenes is very low.
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Elżbieta Maćkiw1*, Magdalena Modzelewska1, Łukasz Mąka1, Halina Ścieżyńska1, Kamila
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Pawłowska1, Jacek Postupolski1, Dorota Korsak2
3 4 Antimicrobial resistance profiles of Listeria monocytogenes
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isolated from ready - to - eat products in Poland in 2007-2011
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1
National Institute of Public Health - National Institute of Hygiene, Department of Food
Safety, 24 Chocimska str , 00-791 Warszawa, Poland
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2
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Miecznikowa str, 02-096 Warsaw, Poland
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University of Warsaw, Faculty of Biology, Department of Applied Microbiology, 1
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* corresponding author
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Elżbieta Maćkiw
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[email protected]
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Tel. + 48 22 54 21 381, Fax. +48 22 54 21 392
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1. Introduction
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Listeria monocytogenes, a ubiquitous gram-positive microaerophilic bacterium has
22
been recognized as an important foodborne pathogen, causing severe listeriosis infections in
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humans. (encephalitis, meningitis and septicaemia) and animals (mastitis, diarrhea and 1
ACCEPTED MANUSCRIPT gastroenteritis). Clinical manifestations of invasive listeriosis are usually severe and include
25
abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile
26
gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate
27
species, including birds (Vázquez-Boland et al., 2001). Groups at highest risk are pregnant
28
women, neonates, adults with underlying disease (cancer, AIDS, diabetes, chronic hepatic
29
disorder, transplant recipients), the elderly (>65 years old), and other immunocompromised
30
individuals (McLauchlin, Mitchell, Smerdon, & Jewell, 2004; Liu, 2006; Martins & Leal
31
Germano, 2011). In 2013, 27 Member States of the European Union reported 1 763 confirmed
32
human cases of listeriosis, which was an 7.3 % increase compared with 2012. The EU
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notification rate was 0.44 cases per 100,000, the highest specific notification rate being
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observed in Finland (1.12) and the lowest in Bulgaria (0.04). The notification rate in Poland
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was 0.15 (EFSA 2014).
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Listeriosis is a sporadic disease which is often associated with consumption of
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contaminated milk, soft cheese, under-cooked meat, fish, eggs and egg products, ready - to -
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eat products (RTE), unwashed raw vegetables and cabbage (Aureli et al., 2003; Gibbons,
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Adesiun, Seepersadsingh, & Rahaman, 2006; De Santis et al., 2007; Arslan & Ozdemir, 2008;
40
Filiousis, Johansson, Frey, & Perreten, 2009; Garrido, Vitas, & Gracia-Jalon, 2009; Harakeh
41
et al., 2009; Rahimi, Ameri & Momtaz, 2010; Jacobsen, Heggebø, Sunde, & Skjervheim,
42
2011; Alonso-Hernando et al., 2012; Bouayad & Hamdi, 2012; Cokal, Dagdelen, Cenet, &
43
Gunsen, 2012). Control of L. monocytogenes contamination represents a significant problem
44
for the food industries, public health agencies and government bodies.
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L. monocytogenes has been differentiated into 13 serotypes 1/2a, 1/2b, 1/2c, 3a, 3b,
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3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 (Liu, 2006; Jamali, Radmehr & Thong, 2013). Molecular
47
serogroup-related PCR typing of L. monocytogenes is based on the detection by PCR of
48
serogroup-specific regions, leading to five genoserogroups: IIa (serotype 1/2a-3a), IIb 2
ACCEPTED MANUSCRIPT 49
(serotypes 1/2b-3b-7), IIc (serotypes 1/2c-3c), IVa (serotype 4a-4c), IVb (serotypes 4ab-4b,
50
4d-4e) (Borucki & Call, 2003; Doumith et al., 2004). Of the serotypes of L. monocytogenes which can cause illness, only four account for
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95% of human isolates: 1/2a, 1/2b, 1/2c and 4b (Kathariou, 2002; Uhitil at al., 2004; Huang et
53
al, 2011; Martins & Leal Germano, 2011). In the US, in 2012 serotype 4b was the most
54
commonly identified serotype, accounting for 54% of isolates and serotype 1/2a was second
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with 28% (CDC, 2014). The most frequently isolated serotype from clinical samples in Brazil
56
was 4b (60.3%), followed by 1/2a (29.0%) (Hofer et al., 2006).
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Serotype 1/2a has frequently been detected in different food matrices (Martins & Leal
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Germano, 2011; Jamali, Radmehr & Thonga, 2013). The most common serotypes in 2013
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were 1/2a (57.5 %) and 4b (34.3 %), followed by 1/2b (6.4 %), 1/2c (1.4 %), 3a and 3b (both
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0.2 %). The most common PCR serogroup was IIa (44.7 %, corresponding to conventional
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serotypes 1/2a and 3a), followed by IVb (44.6 %, corresponding to serotypes 4b, 4d, and 4e),
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IIb (7.8 %, corresponding to serotypes 1/2b, 3b and 7) and IIc (2.9 %, corresponding to
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serotypes 1/2c and 3c) (EFSA 2014).
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L. monocytogenes has generally been considered susceptible to antibiotics active
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against Gram-positive bacteria. The effective treatment of choice for listeriosis is a β-lactam
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antibiotic (e.g. penicillin, ampicillin), alone or in combination with an aminoglycoside (e.g.
67
gentamicin) in the case of immunocompromised patients. The second treatment of choice is
68
the combination of trimethoprim and a sulfonamide (e.g. sulfamethoxazole), especially for
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patients allergic to β-lactams (Conter et al., 2009). Vancomycin and erythromycin are also
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used, respectively, to treat bacteremia and pregnant women diagnosed with listeriosis. Other
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antibiotics used in cases of listeriosis are rifampicin, tetracycline, chloramphenicol and the
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fluoroquinolones (Alonso-Hernando et al., 2012; Wang et al., 2013).
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3
ACCEPTED MANUSCRIPT Antimicrobials are increasingly used in feeds for the control and treatment of diseases
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in animals. Treatment of infections due to opportunistic bacteria may become a problem
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(Arlsan & Özdemir, 2008). The European Union in 2006 prohibits the use of antimicrobials
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which are consumed as human medicine in animal foods, as growth promoting agents of
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animals (Regulation (EC) No, 1831/2003). However, an increasing number of strains are
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becoming resistant to most of the known antibiotics including those used for treating
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listeriosis. Some multidrug resistant L. monocytogenes strains have been isolated from foods
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and human listeriosis cases. The levels of resistance are varied and influenced by
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antimicrobial use in humans and animals, as well as geographical differences (Korsak et al.,
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2012).
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Little information is available on the antimicrobial susceptibility of L. monocytogenes
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isolated from cakes and delicatessen products in Poland. In the first study made in Poland,
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Korsak and co-workers (2012) examined 471 L. monocytogenes isolates from different types
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of food and food-related sources (including 43 ready-to-eat products and 15 from cakes). The
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study showed low prevalence of antimicrobial resistance among L. monocytogenes isolates.
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The aim of this study was to characterize strains estimate the prevalence of L.
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monocytogenes isolated from foods cakes and delicatessen collected under official control and
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monitoring in Poland and to determine the susceptibility of L. monocytogenes isolates to
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various antimicrobial agents. Moreover, the aim was also to identify the serotypes of the
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isolates, which served to determine the diversity of the test collection of L. monocytogenes
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isolates.
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2. Materials and methods
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2.1. Food samples
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presence of L. monocytogenes in food samples. A total of 144 555 food samples were
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collected from large retail outlets and smaller units, including 27 175 samples of cakes and 20
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304 samples of delicatessen products, excluding meat. The samples were tested for the
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presence of L. monocytogenes by the laboratories of Provincial Sanitary and Epidemiological
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Stations, pursuant to the procedure described in the standard PN-EN ISO 11290-
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1:1999+A1:2005. L. monocytogenes bacteria were isolated from 257 RTE samples, including
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from 112 of the cakes and from 145 of delicatessen products. To our laboratory, i.e. the
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National Reference Laboratory for L. monocytogenes, 105 strains (51 from cakes and 54 from
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delicatessen) were sent for confirmation. These strains were subjected to further studies.
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2.2. DNA isolation
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DNA isolation was performed using kit Genomic mini (A&A BIOTECHNOLOGY, Poland).
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2.3. Serotyping of L. monocytogenes
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The multiplex PCR assay was carried according to the procedure in-house method:
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Listeria monocytogenes molecular serotyping described by the European Union Reference
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Laboratory for L. monocytogenes ANSES (Maison-Alfort, France). In brief, 25 µl of reaction
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mixture contained 1U Hot Start polymerase (KAPA Taq, Biosystem), 1× fast-start buffer, 2
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mM MgCl2, 0.2 mM dNTPs, 0.4 µM each of the following primers: LMO0737-1 and
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LMO0737-2; LMO1118-1 and LMO1118-2; ORF2110-1 and ORF2110-2; ORF2819-1 and
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ORF2819-2 and 0.1 µM of the other primers PRS-1 and PRS-2 and 0.2 µM LIP-1 and LIP-2
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(Genomed, Warsaw, Poland) and bacterial DNA. The cycling program consisted of initial
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denaturation for 3 min at 94 °C, followed by 35 cycles of denaturation at 94 °C (40 s),
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annealing at 53 °C (45 s), extension at 72 °C (1 min 15 s) and a final extension at 72 °C (7
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min). A second PCR assay was performed to detect the presence of the flaA gene. The
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ACCEPTED MANUSCRIPT amplification mix of 25 µl, contained 1U Hot Start polymerase (KAPA Taq, Biosystem); 1×
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fast-start buffer, 4 mM MgCl2, 0.2 mM dNTPs, 0.8 µM flaA-F and flaA-R primers and DNA.
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The PCR conditions were as follows: initial denaturation for 3 min at 94 °C, followed by 40
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cycles of denaturation at 94 °C (30 s), annealing at 61 °C (40 s), extension at 72 °C (1 min);
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and a final extension at 72 °C (7 min). The sequences of primers are listed in Table 1.
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Amplified PCR products were visualized by electrophoresis in 1.5% agarose gel stained with
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Midori green. The molecular group IIa (serotype 1/2a-3a) includes isolates in which genes:
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lmo1118, lmo0737, prs, prfA, flaA have been identified; molecular group IIc (serotypes 1/2c-
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3c): lmo1118, lmo0737, prs, prfA; molecular group IIb (serotypes 1/2b-3b-7): orf2819, prs,
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prfA; molecular group IVa (serotype 4a-4c): prs, prfA, flaA
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(serotypes 4ab-4b, 4d-4e): orf2110, orf2819, prs, prfA. The multiplex PCR profiles do not
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distinguish serovar 1/2a from 3a, 1/2b from 3b and 7, 1/2c from 3c or 4ab from 4b and 4d, 4e
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within L. monocytogenes.
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2.4. Antimicrobial susceptibility testing
and molecular group IVb
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Antibiotic susceptibility was assessed using the disk diffusion method on Mueller-
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Hinton agar plate containing 5% sheep blood (Oxoid) and incubated at 37 °C for 18 to 20 h.
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The following discs (Oxoid) were used: gentamicin (10 μg), meropenem (10 μg), ampicillin
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(10 μg), sulfamethoxazole-trimethoprim (23.75/1.25 μg), amoxycillin/clavulanic acid - 2:1
140
(20/10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg). The inhibition
141
zones were measured and scored as sensitive, intermediate susceptibility and resistant. E. coli
142
ATCC 35218 and Staphylococcus aureus ATCC 25923 were used as reference strains for
143
antibiotic disc control. Tests and results were performed following recommendations for
144
Staphylococcus sp. (CLSI, 2012).
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3. Results and discussion The results of the study carried within the scope of official food controls indicate that
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the level of contamination with L. monocytogenes of RTE products collected in Poland from
150
27 175 samples of cakes and 20 304 samples of delicatessen is very low. The presence of L.
151
monocytogenes in cakes and delicatessen products was 0.4 % and 0.7 % respectively. Little
152
information is available regarding the quality of cakes, delicatessen and the contamination of
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these products by L. monocytogenes in other countries. A study carried out in Croatia also
154
showed a low level of contamination, which, however, was higher than in Poland. Eighteen
155
samples of cakes, collected at hotels, restaurants and pastry shops throughout Croatia, were
156
found to be positive for Listeria spp. (6.36%), where L. monocytogenes was isolated from 12
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samples (4.27%) (Uhitil at al., 2004). Similar results were presented by Mengesha et al.
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(2009). In this study, L. monocytogenes was isolated in ready-to-eat food items consisting of
159
ice cream (11.7%), cakes (6.5%), and soft cheese (3.9%). The prevalence of L.
160
monocytogenes was in agreement with a study in Serbia (Gusman et al., 2014). 912 samples
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were analyzed for the presence of L. monocytogenes and the contamination rate was found to
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be 5.6% for cream cheese and 5.6% for cakes.
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The serogroup distribution for the 105 L. monocytogenes strains from RTE was
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determined (Table 2). Our data showed higher percentages of isolates belonging to molecular
165
group IVb (serotypes 4ab-4b, 4d-4e) which comprised 33 strains (31.4%) in our study.
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However, the isolation rate of molecular group IIa (serotype 1/2a-3a) was at a similar level as
167
molecular group IIb (serotypes 1/2b-3b-7), respectively 21.9% and 24.8%. The molecular
168
group IIc (serotypes 1/2c-3c) was relatively rare in RTE (2.9%). The presence of these
169
important serotypes among such food items indicates that these foods may pose a potential
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public health risk. There were no representatives of molecular group IVa (serotype 4a-4c).
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The serotypes of L. monocytogenes present in food were diverse and the presence of potential
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ACCEPTED MANUSCRIPT serotype 4b in all food categories raised a public health concern as serotype 4b has been the
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number one serotype associated with human listeriosis (Zhang et al., 2007). Serovar 4b is not
174
the most common subtype isolated from food, affirming that this discrepancy between the
175
occurrence in foods and listeriosis cases might suggest that serotype 4b is more virulent than
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the other serotypes. The absence of a correlation between the serotype present in the foods
177
and those identified in listeriosis is due to the survival characteristics of certain serotypes in
178
foods (Martins & Leal Germano, 2011). The high prevalence of serotype 4ab- 4b, 4d- 4e in
179
our study is similar to the results obtained by Mengesha et al. (2009). In this study, 711
180
samples were randomly collected and from among them 34 isolates of L. monocytogenes
181
serotypes 4b/4e (n = 32), 4c, and 4e were identified.
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However, in other studies serotype 1/2a occurred more frequently. The most
183
commonly isolated from RTE products serotype of L. monocytogenes in Wales was 1/2a
184
(Meldrum et al., 2010). The results obtained by Guerra at al. 2001 showed that among the
185
strains isolated serogroup 1/2a was the most frequently recovered from RTE in Portugal.
186
Serotype identification by PCR of the strains isolated from RTE foods in Belgian revealed
187
that most belonged to the 1/2a serotype group (Van Coillie et al., 2004). In the study of
188
Korsak et al. (2012) the majority of 43 isolates obtained from RTE products belonged to
189
serotypes 1/2a and 3a (30.2%) and 1/2c and 3c (32.6%). The serogroup distribution for the 32
190
L. monocytogenes from RTE products in Malaysia was determined to be: 1/2a- 3a (65.6%),
191
1/2c-3c (21.9%) and 4b- 4d-4e (12.5%) (Jamali & Tong, 2014). The L. monocytogenes
192
isolates form RTE products in China belonged to serotypes 1/2a and 4b (Wang et al., 2013).
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L. monocytogenes strains were tested for their susceptibility to antimicrobials. The
194
results of this study suggest that the overall incidence of antibiotic resistance in L.
195
monocytogenes is relatively very low. Ten of the tested strains (9.5%) were resistant to
196
ampicillin; 5 strains isolated from delicatessen products and 5 strains isolated from cakes
8
ACCEPTED MANUSCRIPT (Table 3). These results are lower than reported by Rahimi, Ameri and Momtaz (2010) who
198
found that 26.3 % of L. monocytogenes isolates from milk and dairy products in Iran were
199
resistant to ampicillin. Also strains isolated from ready-to-eat food in Italy showed a similar
200
level of resistance to ampicillin and methicillin (Pesavento et al., 2010). Twenty percent of L.
201
monocytogenes strains were determined by the disk diffusion method to be resistant to
202
ampicillin and 22.5% to methicillin. It is of concern that this expanding range now includes a
203
number of antibiotics used to treat listeriosis, e.g. penicillin, ampicillin. Results from the
204
present study show that all of the L. monocytogenes isolates were sensitive to antibiotics
205
commonly used as a second-choice therapy to treat listeriosis (ciprofloxacin, tetracycline,
206
chloramphenicol, and the combination sulfamethoxazole-trimethoprim). In the study of
207
Rodas-Suárez et al., (2006) no incidence of resistance to antimicrobial agents tested:
208
gentamicin,
209
streptomycin, meropenem, cefotaxime and amoxycillin/clavulanic acid was observed.
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ampicillin,
sulfamethoxazole-trimethoprim
erythromycin,
tetracycline,
Our data show that L. monocytogenes contamination occurred in RTE: these isolates
211
were resistant to ampicillin and they belonged to serotypes 4ab-4b-4d-4e and 1/2a-3a which
212
are associated with human listeriosis, suggesting that this pathogen may represent a potential
213
danger to public health. Results from the present study show the sensitivity all of the L.
214
monocytogenes isolates to antibiotics commonly used as second-choice therapy to treat
215
listeriosis. Resistant to ampicillin bacterial strains, like those observed in this study, could be
216
transmitted to people through contact or food consumption. This may further complicate the
217
clinical management of disease caused by the pathogen.
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This work was supported financially by NIPH-NIH (6/ZŚ/2013-5/ZŚ/2014).
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Table 1. List of primers for PCR serotyping and expected amplicon sizes.
prs
PRS 1 PRS 2
orf 2819 orf 2110 fla A
15 16
LMO1118-1 LMO1118-2
370
GCT GAA GAG ATT GCG AAA GAA G CAA AGA AAC CTT GGA TTT GCG G
274
GAT ACA GAA ACA TCG GTT GGC GTG TAA TCT TGA TGC CAT CAG G
691
AGG GCT TCA AGG ACT TAC CC ACG ATT TCT GCT TGC CAT TC
1/2a-3a, 1/2c-3c
ORF2819- 1 ORF2819-2
471
AGC AAA ATG CCA AAA CTC GT CAT CAC TAA AGC CTC CCA TTG
4ab-4d-4e-7
ORF2110-1 ORF2110-2
597
AGT GGA CAA TTG ATT GGT GAA CAT CCA TCC CTT ACT TTG GAC
4ab-4b-4d-4e
538
TTA CTA GAT CAA ACT GCT CC AAG AAA AGC CCC TCG TCC
4 5 6
D’Agostino et al., 2004
7
Doumith et al. 2004
8
Doumith et al. 2004
1/2c-3c
9 10
Doumith et al. 2004
1/2a-3a-4a-4c
3
Doumith et al. 2004
L. monocytogenes
AGG GGT CTT AAA TCC TGG AA CGG CTT GTT CGG CAT ACT TA
flaA-F flaA-R
Reference
Listeria spp.
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lmo 1118
LMO0737-1 LMO0737-2
Specific to
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lmo0737
LIP 1 LIP 2
Sequence (5’-3’)
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prfA
Size (in bp)
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Primer
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Gene targeted
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2
11
Doumith et al. 2004
12
Borucki and Call 2003
13 14
17
1
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Table 2. Molecular serogroups of L. monocytogenes isolated from ready- to- eat products in Poland in 2007–2011.
IIb
10 (19.5%)
14 (27.5%)
3 (5.9%)
0 (0%)
20 (39.2%)
Not classified 4 (7.8%)
243 (24.0%)
12 (22.2%)
0 (0%)
0 (0%)
13 (24.2%)
16 (29.6%)
23 (21.9%)
26 (24.8%)
3 (2.9%)
0 (0%)
33 (31.4%)
20 (19.0%)
19 20
25 26 27
EP
24
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23
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21 22
IVb
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IIa
SC
Delicatessen (excluding meat products) (N=51) Cakes (N=54) Ready-to-eat products Total (N=105)
Molecular serogroups IIc IVa
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28
2
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Table 3. Antimicrobial resistance of L. monocytogenes isolates from ready- to- eat products in Poland in 2007–2011
30
CIP (5 μg)
AMP (10 μg)
Delicatessen (excluding meat products) (N=51) Cakes (N= 54)
0/51
0/51
0/51
5/51 (9.8%)
0/54
0/54
0/54
5/54 (9.3%)
Ready-to-eat products Total (N=105)
0/105
0/105
0/105
10/105 (9.5%)
MEM (10 μg)
SXT (23.75/ 1.25 μg) 0/51
AMC (20/ 10 μg) 0/51
TET (30 μg)
0/54
0/54
0/54
54/0
0/105
0/105
0/105
0/105
SC
CN (10 μg)
0/51
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0/51
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C: chloramphenicol, CN: gentamycin, CIP: ciprofloxacin, AMP: ampicillin, MEM: meropenem, SXT: sulphamethoxazole-trimethoprim, amoxycillin/clavulanic acid (2:1), TET: tetracycline.
AC C
31 32 33
C (30 μg)
RI PT
Number of strains resistant / tested (%)
3
*Highlights (for review)
ACCEPTED MANUSCRIPT Monitoring study of L. monocytogenes in RTE products on retail in Poland Low level of contamination of RTE products with L. monocytogenes High incidence of serotype 4ab-4b-4d-4e (molecular group: IVb) of L. monocytogenes Nine and a half percent of L. monocytogenes isolates were resistant to ampicillin
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All L. monocytogenes isolates were sensitive to other examined antibiotics