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The prevalence of major foodborne pathogens in ready-to-eat chicken meat samples sold in retail markets in Turkey and the molecular characterization of the recovered isolates
LWT - Food Science and Technology 81 (2017) 202e209
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The prevalence of major foodborne pathogens in ready-to-eat chicken meat samples sold in retail markets in Turkey and the molecular characterization of the recovered isolates Seçil Abay a, *, Reyhan Irkin b, Fuat Aydin a, Hamit Kaan Müs¸tak c, Kadir Serdar Diker c a b c
Erciyes University, Faculty of Veterinary Medicine, Department of Microbiology, Kayseri, Turkey Balikesir University, Engineering and Architecture Faculty, Food Engineering Department, Balikesir, Turkey Ankara University, Faculty of Veterinary Medicine, Department of Microbiology, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 November 2016 Received in revised form 18 March 2017 Accepted 30 March 2017 Available online 31 March 2017
The aims of the present study were to evaluate the prevalence of Arcobacter spp., Campylobacter spp., Listeria spp., and Salmonella spp. in heat-processed ready-to-eat (RTE) chicken products manufactured by various companies using bacterial culture methods and to perform virulence gene analysis, serotyping, genotyping, and antibacterial susceptibility tests on the isolated strains. For this purpose, 50 packages of chicken convenience products were used as the study material. Phenotypic tests and a molecular method (Polymerase Chain Reaction, PCR) were used to identify the isolated bacteria. All samples examined were negative for Arcobacter spp., Campylobacter spp., and Salmonella species. Listeria species were isolated from 12 (24%) of the examined samples. Among the Listeria species isolated, 9 were identified as L. monocytogenes, 2 were identified as L. innocua, and one was identified as L. welshimeri. All isolates were susceptible to the antibiotics tested. A detailed molecular analysis of the Listeria spp. revealed that the examined food products posed a significant public health hazard. Considering the presence of different genotypes of L. monocytogenes in RTE food production facilities, all the steps of food production must be reviewed in terms of conformity with sanitation and hygiene rules, and necessary measures must be set in place. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Arcobacter spp. Campylobacter spp. Listeria spp. Molecular characterization Ready to eat chicken meat Salmonella spp.
1. Introduction Campylobacter spp., Listeria spp. and Salmonella spp. are the most prevalent and serious foodborne pathogens (Mor-Mur & Yuste, 2010). Additionally, some infections originating from Arcobacter spp. in water and food (chicken, turkey, beef, sheep and pork) have been reported (Ho, Lipman, & Gaastra, 2006; Kayman et al., 2012; Patyal, Rathore, Mohan, Dhama, & Kumar, 2011). These bacteria are frequently found as commensals in the intestinal tracts of numerous animals, including poultry. Arcobacter spp. and Campylobacter spp. are closely related genera in the family (Vandamme, 2000). They are Gram-negative, spiral-shaped, motile organisms and cause a variety of diseases in humans and animals. Chicken meats contaminated with Campylobacter spp. constitute the largest potential source of human
* Corresponding author. E-mail addresses: [email protected], [email protected] (S. Abay). http://dx.doi.org/10.1016/j.lwt.2017.03.052 0023-6438/© 2017 Elsevier Ltd. All rights reserved.
infections by far (Butzler, 2004; Collado & Figueras, 2011). Foodborne listeriosis in humans is rare but severe. Listeria species are found in soil, water, effluents, a large variety of foods, and humans and animal feces (Barbuddhe & Chakraborty, 2009). The genus Listeria currently includes 17 recognized species (L. monocytogenes, L. seeligeri, L. ivanovii, L. welshimeri, L. marthii, L. innocua, L. grayi, L. fleischmannii, L. floridensis, L. aquatica, L. newyorkensis, L. cornellensis, L. rocourtiae, L. weihenstephanensis, L. grandensis, L. riparia and L. booriae) (Orsi & Wiedmann, 2016). These bacteria are Gram-positive, facultative anaerobes that are motile at 10e25 C, non-spore forming and are able to multiply even at high salt concentrations and in acidic conditions (McLauchlin, Catherine, & Christine, 2014). Listeria spp. can be endemic in food processing environments. Unlike most bacteria, Listeria can grow and multiply in some foods in the refrigerator. Therefore, their presence may be indicative of poor hygiene or cross-contamination, which is a possible source of Listeria contamination in processed meat (Nyenje, Odjadjare, Tanih, Green, & Ndip, 2012).
S. Abay et al. / LWT - Food Science and Technology 81 (2017) 202e209
More than 2500 Salmonella serovars are considered potential pathogens in animals and humans. S. enterica has 6 subspecies (S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S. enterica subsp. Indica). The principle sources of these microorganisms include poultry, eggs, raw meat and raw milk (Anonym, 2005). Ready-to-eat (RTE) foods are consumed without further treatment, such as cooking, that would eliminate or reduce the microbial load. A variety of RTE foods, such as cooked meat and poultry, are commonly consumed; recently, the consumption of this type of food has rapidly increased due to their good taste and simple preparation. Therefore, the safety of these products has been a source of concern to consumers, especially in relation to their microbiological contamination (Yang et al., 2016). A variety of RTE foods originate from animals, including meat, poultry, seafood and dairy products. The microbiological risks to the consumer from these products have also increased (Hwang & Huang, 2010). Improperly heated-up and/or stored RTE-type meat products (especially poultry products) can be a cause of food poisoning by bacteria such as Salmonella enteritidis, Listeria monocytogenes and Campylobacter jejuni (Pietrzak, Cegielka, Fonberg-Broczek, & Ziarno, 2011). Ready-to-eat foods are important vehicle in the transport of Listeria spp. to humans. One study that investigated 384 food samples in Ethiopia reported a 25% prevalence of L. monocytogenes, among which some isolates were multi-drug resistant (penicillin, nalidixic acid, tetracycline and chloramphenicol), indicating the need for the application of hygienic practices in the food processing industries (Dhama et al., 2015). Ready-to-eat cooked chicken products can easily be contaminated with L. monocytogenes in the post-processing steps. The consumption of contaminated RTE cooked chicken foods results in severe health problems, including listeriosis, with a high death rate (Goh et al., 2014). Similarly, humans can be subjected to Campylobacter when consuming improperly processed poultry products. However, the most important route of food-borne disease is related to the consumption of foods that are cross-contaminated with Campylobacter during food preparation of a meal with poultry products (Signorini et al., 2013). The common contamination of poultry products with Arcobacter has frequently been reported (Smet, Zutter, Hende, & Houf, 2010). Generally, Arcobacter spp. are found in foods of animal origin, such as chicken, pork, beef, lamb, and raw milk. The highest prevalence was shown for poultry, followed by pork and beef meat (Girbau, Guerra, Martinez-Malaxetxebarria, Alonso, & Astorga, 2015). Although several studies showed that Salmonella spp. prevailed in RTE poultry products (Karadal, Ertas, Hizlisoy, Abay, & Al, 2013), there are no prevalent studies concerning Arcobacter spp., Campylobacter spp., and Listeria spp. contaminations in our country. Our aims were i: to determine the prevalence of Arcobacter spp., Campylobacter spp. and Listeria spp. in commercial RTE poultry products using culture methods, ii: to perform virulence gene analysis, serotyping, and genotyping and antibacterial susceptibility tests on the isolated strains. 2. Materials and methods 2.1. Ready-to-eat poultry meat samples Various processed chicken meat samples were purchased from four local retail markets and poultry shops in the cities of Kayseri and Balıkesir, Turkey. A total of 50 samples, including meatballs (n ¼ 10), nuggets (n ¼ 8), sausages (n ¼ 8), burgers (n ¼ 8), Doner kebabs (n ¼ 8) and kebabs (n ¼ 8) (see Table 1 for details), were examined. The samples were kept cool and examined within 1 h of
203
purchase. 2.2. Isolation and identification of Arcobacter spp. Arcobacter spp. isolation was performed according to the method of Aydin, Gümüs¸soy, Atabay, Iça, and Abay (2007). 2.3. Isolation and identification of Campylobacter spp. For the isolation of Campylobacter spp. from the samples, Aydin et al. (2007)’s method was used; however, in the enrichment step, the samples were incubated at 37 C. Isolates identified with the phenotypic tests as Campylobacter spp. were confirmed using molecular analysis (mPCR) (Wang et al., 2002). 2.4. Isolation and identification of Listeria spp. Isolation and phenotypic identification of Listeria spp. from samples were performed according to the method of Abay, Aydin, and Sumerkan (2012). Isolates were confirmed at the species level by molecular methods, including hly gene-specific PCR (Pourjafar et al., 2010) and 16S rRNA sequence analysis (Lane, 1991). 2.5. Isolation and identification of Salmonella spp. The procedure was performed according to ISO 6579e2002. Positive colonies (2e3) were confirmed by the API 20E kit (Biomerieux, France). 2.6. DNA extraction DNA was extracted from each isolate using the UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories, 12224e250) following the manufacturer's instructions. 2.7. PCR amplification of the 16S rDNA gene The universal primers 27F AGAGTTTGATCMTGGCTCAG and 1492R GGTTACCTTGTTACGACTT were used to amplify the 16S rDNA gene (Lane, 1991). Amplified products were resolved by 1.5% agarose (Prona) gel electrophoresis and visualized under a UV transilluminator (G:BOX Chemi XRQ, Syngene). 2.8. Sequencing and phylogenetic analysis The amplified PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), and the Big Dye Direct Cycle Sequencing Kit (Applied Biosystems) was used in the sequence analysis; both kits were used according to the manufacturer's instructions. After cycle sequencing, the amplicons were purified with Sephadex G-50 (Sigma-Aldrich) and sequenced on the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems). All sequences were analyzed with the CLC Main Workbench 6 and compared with reference sequences in the National Center for Biotechnology Information website with BLASTN. The evolutionary history was inferred using the neighborjoining method (Saitou & Nei, 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was determined (Felsenstein, 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JukesCantor method (Jukes & Cantor, 1969) and were in the units of the number of base substitutions per site. The analysis involved 15 nucleotide sequences, including the 3 reference strains
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Table 1 Sample types and numbers (Listeria spp. positive samples). Company
Types of Samples nugget
A B C D E F G H I K Total
2n 1
2 2
Total meatballs
1* -
1 -
1
-
8
2
1 2
2
3 2 10
sausages
burgers
Donerkebabs
2
2
1 1 6
2
1
1 1 2 2
-
8
1
1 2 1
1 -
2 2
-
8
1
kebab
1
-
2 1
-
2
-
1
-
1 1
1 -
2
-
2 1 8
1
1 1 8
1 1
5 5 2 4 3 7 7 4 9 4 50
1 2 1 1 4 1 2 12
n
: Number of samples. *: Listeria spp. positive samples. -: negative samples.
L. monocytogenes (JF967620.1), L. innocua (NR_116805.1) and L. welshimeri (NR_074998.1) obtained from GenBank. All positions containing gaps and missing data were eliminated. The evolutionary analyses were conducted in MEGA6 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). 2.9. Nucleotide accession numbers The 16S rDNA sequences of the 12 isolates were deposited in GenBank under accession numbers from KX427175.1 to KX427186.1. 2.10. Enterobacterial repetitive intergenic consensus sequencebased PCR (ERIC-PCR) The ERIC motifs 1R (50 -ATG TAA GCT CCT GGG GAT TCA C-30 ) and 2 (50 -AAG TAA GTG ACT GGG GTG AGC G-30 ) were used for PCRmediated molecular subtyping of the Listeria spp. isolates (Aydin, Gümüs¸soy, Atabay, 2007). The ERIC-PCR gel image was evaluated using the GelCompar II 6.6.11 Gel Electrophoresis Software (Applied Maths) to construct the evolutionary tree of the Listeria species. 2.11. Serotyping of L. monocytogenes using molecular methods and serological tests Serotyping of the L. monocytogenes isolates was performed using molecular methods (Doumith, Buchrieser, Glaser, Jacquet, & Martin, 2004). Discrimination of serotypes obtained by mPCR from each other, a serological method based on the somatic and flagellar antigens was performed using Listeria O and H antisera (Denka Seiken 214362, Tokyo, Japan) as previously described (Karadal & Yıldırım, 2014). 2.12. Virulence gene analysis of Listeria spp. by PCR All Listeria spp. isolates were analyzed for the presence of the inlA, inlC (Wieczorek, Dmowska, & Osek, 2012), iap (Swetha, Madhava Rao, Krishnaiah, & Kumar, 2012), inlB, prfA, actA, hly, plcA, plcB, mpl, lin0558 and lin1068 (Johnson et al., 2004) virulence genes. Each virulence gene was investigated separately by PCR. The PCR and amplification conditions were performed according to Johnson et al., 2004. The expected band sizes were 800 bp, 517 bp, 131 bp, 1107 bp, 479 bp, 1994 bp, 496 bp, 798 bp, 320 bp, 674 bp, 866 bp and 613 bp for the inlA, inlC, iap, inlB, prfA, actA, hly, plcA, plcB, mpl, lin0558 and
lin1068 genes, respectively. 2.13. Antibacterial susceptibility testing The antibiotic susceptibilities of Listeria spp. to amoxicillin and clavulanic acid (AMC, 30 mg), ampicillin (AM, 10 mg), ciprofloxacin (CIP, 5 mg), enrofloxacin (ENR, 5 mg), gentamicin (CN, 10 mg), penicillin (P, 10 mg) and vancomycin (VA, 30 mg) were determined using the disk diffusion test (Bauer, Kirby, Sherris, & Turek, 1966). The antimicrobial disks were purchased from Oxoid, UK. The disk diffusion test results were interpreted using the criteria published by the Clinical and Laboratory Standards Institute (CLSI, 2008). Currently, there are no interpretative criteria provided by CLSI for Listeria, with the exception of susceptibility breakpoints for ampicillin and penicillin. Therefore, the CLSI criteria for staphylococci were applied in this study. Staphylococcus aureus ATCC 25923 was used as the control strain. 2.14. Standard strains The Arcobacter butzleri LMG 10828 (LMG bacterial collection, Ghent University), Campylobacter coli DCC2, Campylobacter jejuni NCTC 11168, L. monocytogenes serotype 1/2a (RSSK:472) and Salmonella enteritidis (from Erciyes University, Faculty of Veterinary Medicine, Dept. of Microbiology culture collection) reference strains were used throughout the study. 3. Results 3.1. Arcobacter spp., Campylobacter spp., and Salmonella spp. isolation and identification tests All of the examined samples tested negative for Arcobacter spp., Campylobacter spp., and Salmonella species. 3.2. Listeria spp. isolation and identification results Of the examined samples, twelve (24%) were positive for Listeria species. Among the isolated Listeria species, 9 were identified as L. monocytogenes, 2 were identified as L. innocua, and one was identified as L. welshimeri using phenotypic identification tests. The Listeria spp. isolates were numbered from the RTECM (ready-to-eat chicken meat) 1 to RTECM 12 samples; the isolates were referred to with these numbers when presenting the following results in subheadings. All L. monocytogenes isolates tested were positive in
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the PCR performed with primers specifically designed for the hly gene (840 bp). The results of this identification test were confirmed by the 16S rRNA sequence analysis (Fig. 1). The sources of the samples that tested positive for Listeria spp., the isolated species, and their distributions according to the companies are presented in Table 1. Accordingly, 2 out of 8 nugget samples tested positive for Listeria spp.; of these isolates, one was identified as L. welshimeri and the other was identified as L. monocytogenes. Among the 6 Listeria species isolated from the meatballs, 5 were identified as L. monocytogenes and 1 was one identified as L. innocua. One L. innocua isolate was recovered from the sausage samples, and one L. monocytogenes isolate was recovered from each of the burger, doner and kebab samples. The highest rate of listeria contamination was observed in the products of the company signified by the letter “F”.
3.3. Genotyping of the Listeria spp. isolates All isolates exhibited amplification of the 16S rDNA operon, resulting in a band of approximately 1350 bp (there were a total of 1347 positions in the final dataset). These 16S rDNA sequence data showed great similarity to the reference sequences of the strains in GenBank (between 99% and 100% at the species level). We confirmed that the 16S rDNA sequence analysis was a sufficient tool for identifying of Listeria spp. at the species level (Soni & Dubey, 2014). On the other hand, the Listeria spp. isolates recovered from RTE could not be discriminated according to their isolation sources (nugget, meatballs, sausages, burgers, Doner-kebabs and kebab) by the 16S rDNA sequence analysis (Fig. 1). The ERIC-PCR results of the 12 Listeria spp. are presented in Fig. 2. Remarkable genetic heterogeneity was observed between the isolates, as shown in Fig. 2. In particular, there was prominent genetic heterogeneity between the isolates recovered from the products of the companies signified by the letters “F” and “B” (Table 2). Likewise, there was significant genetic diversity between the L. innocua isolates.
205
3.4. Serotyping of L. monocytogenes isolates using molecular methods and serological tests Serotyping of the 9 L. monocytogenes isolates using mPCR showed that 7 isolates were serotype 1/2a, 3a and 2 were serotype 1/2c, 3c (Fig. 3A) and then the identification test results at the molecular level were confirmed by the serological tests (Listeria O and H antisera) as 7 isolates were serotype 1/2a and 2 were serotype 1/2c (Table 2). 3.5. Virulence gene analysis of Listeria spp. by PCR The virulence gene analysis results are presented in Table 3 and Fig. 3B. All L. monocytogenes isolates tested positive for the 9 virulence genes examined. Of these L. monocytogenes isolates, only 4 were positive for the actA gene. Only isolate number 1 tested positive for the lin1068 gene, and none of the isolates were positive for the lin0558 gene. The L. welshimeri isolate was negative for all of the examined virulence genes. 3.6. Antibacterial susceptibility testing All isolates were susceptible to amoxicillin and clavulanic acid, ampicillin, ciprofloxacin, enrofloxacin, gentamicin, penicillin, and vancomycin. 4. Discussion Generally, raw chicken meat and its products are highly contaminated (approximately 100%) with Campylobacter jejuni (Aydin et al., 2007; Yıldız & Diker, 1992), and the majority of human cases of campylobacteriosis (Kayman, Abay, & Hizlisoy, 2013) are associated with the consumption of improperly cooked contaminated food products or the cross-contamination of other products with Campylobacter species (Humphrey, Martin, Slander, & Durham, 2001). Poultry and meat products are also subjected to
Fig. 1. Evolutionary relationships of related Listeria spp. according to their 16S rDNA sequences by the neighbor-joining method. The optimal tree with the sum of branch lengths ¼ 0.00670292 is shown. The percentage of replicated trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. GenBank accession numbers are indicated for each sequence in parenthesis.
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Fig. 2. Dendrogram for the 12 Listeria isolates analyzed according to the ERIC-PCR gel electrophoresis band profiles. The dendrogram was constructed using the GelCompar II 6.6.11 (Applied Maths) software with UPGMA clustering based on the Dice correlation coefficient.
Table 2 Isolate numbers (company codes), species and serotypes of the Listeria spp. Company
Isolate Noa
Listeria species distribution
Isolate serotype (mPCR)
Isolate serotype (O and H Antisera)
A B
RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM
L. L. L. L. L. L. L. L. L. L. L. L.
1/2c, 3c 1/2c, 3c e 1/2a, 3a 1/2a, 3a 1/2a, 3a 1/2a, 3a 1/2a, 3a 1/2a, 3a 1/2a, 3a
1/2c 1/2c e 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a
C D F
I K a
9 11 12 1 2 4 7 8 10 6 3 5
welshimeri monocytogenes monocytogenes innocua monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes innocua monocytogenes monocytogenes
Number of Listeria isolates.
heat processing prior to presentation to the consumer. Generally, heat-processing treatment (55 C and higher) of these products inactivates Campylobacter species (Lake, Hudson, Cressey, & Gilbert, 2007). Therefore, these products are not considered to pose a significant risk to consumers. A very limited number of studies have evaluated the load of Campylobacter species in heat-processed ~ ones-Ramírez, poultry meat products (llida & Faridah, 2012; Quin V azquez-Salinas, Rodas-Su arez, Ramos-Flores, & Rodríguez-Mon~ o, 2000; Joint Food Safety and Standards Group, 1996; Bohaytan chuk et al., 2006; Federighi et al., 1999). Ilida & Faridah (2012) found that 22 heat-processed chicken products were negative for C. jejuni, whereas 758 heat-processed chicken products were negative for C. jejuni in the study by the Joint Food Safety and Standards Group (1996). Additionally, cooked poultry sausage samples were free from thermotolerant Campylobacter in a study conducted by Federighi et al. (1999). Similarly, all samples tested in the present study were negative for Campylobacter species. In contrast, Quinones-Raminez et al. (2000) found contamination with Campylobacter species in 27 out of 100 heat-processed chicken taco samples. The researchers noted that mishandling was the source of contamination and highlighted that these products posed a potential risk to public health. Arcobacter species are water- and food-borne pathogens that are important for humans and are abundantly present in raw poultry
and meat products (Aydin, Gümüs¸soy, Atabay, 2007; Mor-Mur & Yuste, 2010). However, no study has evaluated the prevalence of these microorganisms in heat-processed ready-to-eat chicken meat products. All samples tested in the present study were negative for Arcobacter species. This finding can be explained by the inactivation of Arcobacter species during the heat processing treatment. Arcobacter species are in the same family as Campylobacter species and show substantially identical morphological/physiological characteristics. Although Salmonella has largely been found in raw poultry products, all of the samples evaluated in the present study tested negative for Arcobacter species, Campylobacter species and Salmonella species. Salmonella species would be inactivated when the ready-to-eat chicken meats were subjected to the heat processing treatment. Likewise, other researchers did not report Salmonella species in heat-processed chicken meat products (Bohaychuk et al., 2006; Karadal et al., 2013). The most important finding of the present study in terms of isolation and identification was that Listeria species were isolated from 24% (12/50) of the samples (Table 1). Among the Listeria species isolated, 9 (75%) were identified as L. monocytogenes. The presence of Listeria species in heat-processed ready-to-eat chicken meat and chicken products poses a significant public health hazard. The isolation rates of Listeria spp. from RTE chicken meat
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Fig. 3. Results of L. monocytogenes serotyping and virulence genes analysis. A. Agarose gel electrophoresis of mPCR products for the identification of L. monocyogenes serotypes. Lanes 1e12 (RTECM 1e12): Listeria spp. isolates recovered from processed chicken meat. Lanes 1 and 6: L. innocua, lane 9: L. welshimeri, lanes 2, 3, 4, 5, 7, 8 and 10: L. monocytogenes serovar 1/2a, lanes 11 and 12: L. monocytogenes serovar 1/2c, C1: Control strain of L. innocua, C2: Control strain of L. monocytogenes serovar 1/2a, C3: Control strain of L. monocytogenes serovar 4b and C4: Control strain of L. monocytogenes serovar 1/2c, M: Marker, 100 bp plus. B. Representative agarose gel electrophoresis image of the L. monocytogenes, L. innocua and L. welshimeri virulence genes. Lane 1: iap gene, 131 bp; Lane 2: prfA gene, 479 bp; Lane 3: mpl, 674 bp; Lane 4: hly gene, 496 bp; Lane 5: actA, 1994 bp; Lane 6: plcA gene, 798 bp; Lane 7: plcB, 320 bp; Lane 8: lin1068, 613 bp; Lane 9: inlA, 800 bp; Lane 10: inlB, 1107 bp; Lane 11: inlC, 517 bp; M: Marker, 100 bp plus (Thermo Scientific).
Table 3 Occurrence and distribution of 12 virulence genes analyzed in Listeria spp. isolates. Isolates numbers
inlA
inlB
inlC
prfA
actA
hly
plcA
plcB
mpl
iap
lin1068
lin0558
RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ e þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ
1 Li 2 Lm 3 Lm 4 Lm 5 Lm 6 Li 7 Lm 8 Lm 9 Lw 10 Lm 11 Lm 12 Lm
þ: positive for virulence gene, : negative for virulence gene. Li: L. innocua Lm: L. monocytogenes Lw: L. welshimeri.
and products show variability. The Listeria spp. rates found in RTE chicken meat and products in different countries were 2.9% by , & Man ~ es, (2001), 3% by Bohaychuk et al. Soriano, Rico, Molto (2006), 0.02% by Kanarat, Jitnupong, and Sukhapesna (2011) and 10% by Awadallah and Suelam (2014). The results of the present study suggest higher rates than those reported by the researchers in the studies mentioned above. This difference may be related to the isolation and identification methods employed, insufficient hygiene practices, and the bacterial
loads of the products (Keeratipibul & Lekroengsin, 2008, 2009). Heat-processed ready-to-eat chicken meat and chicken products must be free from L. monocytogenes as per the relevant regulations in Turkey (Türk Gıda Kodeksi Mikrobiyolojik Kriterler € netmelig i, o 2011, http://www.mevzuat.gov.tr/MevzuatMetin/ yonetmelik/7.5.15690-ek.pdf, Republic of Turkey Ministry of Food, Agriculture and Livestock). The high isolation rate for Listeria species in the tested products in the present study suggests poor sanitation and insufficient hygiene practices in these
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establishments. Adherence to sanitation and hygiene principles during the preparation of heat-processed chicken meat and chicken products significantly reduces the Listeria species load (Keeratipibul & Lekroengsin, 2008, 2009). In the present study, products of 7 out of 10 companies tested positive, and 3 tested negative for Listeria species. The highest contamination rate was observed in the products of the company signified by the letter “F” (Table 1). The L. monocytogenes isolates recovered in the present study were not well characterized at the molecular level. All of the isolates possessed all 9 virulence genes tested (Fig. 3B and Table 3), with 7 isolates serotype 1/2a and 2 isolates serotype 1/2c (Fig. 3A and Table 2). Additionally, a phylogenetic analysis of the three different Listeria species isolated in the present study was performed (Figs. 1 and 2). The sequence analysis results allowed the identification of the isolates at the species level but failed to detect genetic diversity within a particular species. However, ERIC-PCR was a sensitive tool for the detection of genetic diversity within a species. There was remarkable diversity in the band patterns of the L. monocytogenes isolates recovered from the products of the companies signified by the letters “F” and “B” (Table 2 and Fig. 2.). This finding indicates the presence of multiple contamination sources in these establishments. L. monocytogenes has the highest mortality rate (20e40%) in humans among other food-borne pathogens. The virulence genes possessed by the bacteria are known to have a significant effect on this finding. Because the 1/2a, 1/2b and 4b serotypes of L. monocytogenes account for 98% of the listeriosis cases (Borucki & Call, 2003; Nho, Abdelhamed, Reddy, Karsi, & Lawrence, 2015; Swaminathan & Gerner-Smidt, 2007), the ready-to-eat chicken meat from which L. monocytogenes was isolated in the present study constitutes a potential hazard to human consumers. Although a number of studies have reported resistance to various antibiotics in L. monocytogenes isolates (Facinelli, Giovanetti, Varaldo, Casolari, & Fabio, 1991; Roberts, Facinelli, € Giovanetti, & Varaldo, 1996; Yücel, Cıtak, & Onder, 2005), all of the Listeria isolates tested in the present study were susceptible to the seven antibiotics tested. 5. Conclusions Fifty ready-to-eat chicken meat products tested negative for Arcobacter spp., Campylobacter spp., and Salmonella species, but 12 samples (24%) were found to be contaminated with Listeria species. A detailed molecular analysis such as identification, virulence genes screening and genotyping of the Listeria spp. revealed that the food products posed a significant public health hazard. Considering the presence of different L. monocytogenes genotypes in ready-to-eat food production facilities, all steps of food production must be reviewed in terms of conformity with the principles of sanitation and hygiene practice, and necessary measures must be set in place. References Abay, S., Aydin, F., & Sumerkan, A. B. (2012). Molecular typing of Listeria spp. isolated from different sources. Ankara Universitesi Veteriner Fakultesi Dergisi, 59, 183e190. Anonym. (2005). Salmonellosis paratyphoid, non-typhoidal salmonellosis the center for food security and public health. Iowa State University. http://www.cfsph. iastate.edu/Factsheets/pdfs/nontyphoidal_salmonellosis.pdf. Awadallah, M. A. I., & Suelam, I. I. A. (2014). Characterization of virulent Listeria monocytogenes isolates recovered from ready-to-eat meat products and consumers in Cairo, Egypt. Veterinary World, 7, 788e793. Aydin, F., Gümüs¸soy, K. S., Atabay, H. I., Iça, T., & Abay, S. (2007). Prevalence and distribution of Arcobacter species in various sources in Turkey and molecular analysis of isolated strains by ERIC-PCR. Journal of Applied Microbiology, 103, 27e35. Aydin, F., Gumussoy, K. S., Ica, T., Sumerkan, B., Es¸el, D., Akan, M., et al. (2007). The prevalence of Campylobacter jejuni in various sources in Kayseri, Turkey, and
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
The prevalence of major foodborne pathogens in ready-to-eat chicken meat samples sold in retail markets in Turkey and the molecular characterization of the recovered isolates Seçil Abay a, *, Reyhan Irkin b, Fuat Aydin a, Hamit Kaan Müs¸tak c, Kadir Serdar Diker c a b c
Erciyes University, Faculty of Veterinary Medicine, Department of Microbiology, Kayseri, Turkey Balikesir University, Engineering and Architecture Faculty, Food Engineering Department, Balikesir, Turkey Ankara University, Faculty of Veterinary Medicine, Department of Microbiology, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 November 2016 Received in revised form 18 March 2017 Accepted 30 March 2017 Available online 31 March 2017
The aims of the present study were to evaluate the prevalence of Arcobacter spp., Campylobacter spp., Listeria spp., and Salmonella spp. in heat-processed ready-to-eat (RTE) chicken products manufactured by various companies using bacterial culture methods and to perform virulence gene analysis, serotyping, genotyping, and antibacterial susceptibility tests on the isolated strains. For this purpose, 50 packages of chicken convenience products were used as the study material. Phenotypic tests and a molecular method (Polymerase Chain Reaction, PCR) were used to identify the isolated bacteria. All samples examined were negative for Arcobacter spp., Campylobacter spp., and Salmonella species. Listeria species were isolated from 12 (24%) of the examined samples. Among the Listeria species isolated, 9 were identified as L. monocytogenes, 2 were identified as L. innocua, and one was identified as L. welshimeri. All isolates were susceptible to the antibiotics tested. A detailed molecular analysis of the Listeria spp. revealed that the examined food products posed a significant public health hazard. Considering the presence of different genotypes of L. monocytogenes in RTE food production facilities, all the steps of food production must be reviewed in terms of conformity with sanitation and hygiene rules, and necessary measures must be set in place. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Arcobacter spp. Campylobacter spp. Listeria spp. Molecular characterization Ready to eat chicken meat Salmonella spp.
1. Introduction Campylobacter spp., Listeria spp. and Salmonella spp. are the most prevalent and serious foodborne pathogens (Mor-Mur & Yuste, 2010). Additionally, some infections originating from Arcobacter spp. in water and food (chicken, turkey, beef, sheep and pork) have been reported (Ho, Lipman, & Gaastra, 2006; Kayman et al., 2012; Patyal, Rathore, Mohan, Dhama, & Kumar, 2011). These bacteria are frequently found as commensals in the intestinal tracts of numerous animals, including poultry. Arcobacter spp. and Campylobacter spp. are closely related genera in the family (Vandamme, 2000). They are Gram-negative, spiral-shaped, motile organisms and cause a variety of diseases in humans and animals. Chicken meats contaminated with Campylobacter spp. constitute the largest potential source of human
* Corresponding author. E-mail addresses: [email protected], [email protected] (S. Abay). http://dx.doi.org/10.1016/j.lwt.2017.03.052 0023-6438/© 2017 Elsevier Ltd. All rights reserved.
infections by far (Butzler, 2004; Collado & Figueras, 2011). Foodborne listeriosis in humans is rare but severe. Listeria species are found in soil, water, effluents, a large variety of foods, and humans and animal feces (Barbuddhe & Chakraborty, 2009). The genus Listeria currently includes 17 recognized species (L. monocytogenes, L. seeligeri, L. ivanovii, L. welshimeri, L. marthii, L. innocua, L. grayi, L. fleischmannii, L. floridensis, L. aquatica, L. newyorkensis, L. cornellensis, L. rocourtiae, L. weihenstephanensis, L. grandensis, L. riparia and L. booriae) (Orsi & Wiedmann, 2016). These bacteria are Gram-positive, facultative anaerobes that are motile at 10e25 C, non-spore forming and are able to multiply even at high salt concentrations and in acidic conditions (McLauchlin, Catherine, & Christine, 2014). Listeria spp. can be endemic in food processing environments. Unlike most bacteria, Listeria can grow and multiply in some foods in the refrigerator. Therefore, their presence may be indicative of poor hygiene or cross-contamination, which is a possible source of Listeria contamination in processed meat (Nyenje, Odjadjare, Tanih, Green, & Ndip, 2012).
S. Abay et al. / LWT - Food Science and Technology 81 (2017) 202e209
More than 2500 Salmonella serovars are considered potential pathogens in animals and humans. S. enterica has 6 subspecies (S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S. enterica subsp. Indica). The principle sources of these microorganisms include poultry, eggs, raw meat and raw milk (Anonym, 2005). Ready-to-eat (RTE) foods are consumed without further treatment, such as cooking, that would eliminate or reduce the microbial load. A variety of RTE foods, such as cooked meat and poultry, are commonly consumed; recently, the consumption of this type of food has rapidly increased due to their good taste and simple preparation. Therefore, the safety of these products has been a source of concern to consumers, especially in relation to their microbiological contamination (Yang et al., 2016). A variety of RTE foods originate from animals, including meat, poultry, seafood and dairy products. The microbiological risks to the consumer from these products have also increased (Hwang & Huang, 2010). Improperly heated-up and/or stored RTE-type meat products (especially poultry products) can be a cause of food poisoning by bacteria such as Salmonella enteritidis, Listeria monocytogenes and Campylobacter jejuni (Pietrzak, Cegielka, Fonberg-Broczek, & Ziarno, 2011). Ready-to-eat foods are important vehicle in the transport of Listeria spp. to humans. One study that investigated 384 food samples in Ethiopia reported a 25% prevalence of L. monocytogenes, among which some isolates were multi-drug resistant (penicillin, nalidixic acid, tetracycline and chloramphenicol), indicating the need for the application of hygienic practices in the food processing industries (Dhama et al., 2015). Ready-to-eat cooked chicken products can easily be contaminated with L. monocytogenes in the post-processing steps. The consumption of contaminated RTE cooked chicken foods results in severe health problems, including listeriosis, with a high death rate (Goh et al., 2014). Similarly, humans can be subjected to Campylobacter when consuming improperly processed poultry products. However, the most important route of food-borne disease is related to the consumption of foods that are cross-contaminated with Campylobacter during food preparation of a meal with poultry products (Signorini et al., 2013). The common contamination of poultry products with Arcobacter has frequently been reported (Smet, Zutter, Hende, & Houf, 2010). Generally, Arcobacter spp. are found in foods of animal origin, such as chicken, pork, beef, lamb, and raw milk. The highest prevalence was shown for poultry, followed by pork and beef meat (Girbau, Guerra, Martinez-Malaxetxebarria, Alonso, & Astorga, 2015). Although several studies showed that Salmonella spp. prevailed in RTE poultry products (Karadal, Ertas, Hizlisoy, Abay, & Al, 2013), there are no prevalent studies concerning Arcobacter spp., Campylobacter spp., and Listeria spp. contaminations in our country. Our aims were i: to determine the prevalence of Arcobacter spp., Campylobacter spp. and Listeria spp. in commercial RTE poultry products using culture methods, ii: to perform virulence gene analysis, serotyping, and genotyping and antibacterial susceptibility tests on the isolated strains. 2. Materials and methods 2.1. Ready-to-eat poultry meat samples Various processed chicken meat samples were purchased from four local retail markets and poultry shops in the cities of Kayseri and Balıkesir, Turkey. A total of 50 samples, including meatballs (n ¼ 10), nuggets (n ¼ 8), sausages (n ¼ 8), burgers (n ¼ 8), Doner kebabs (n ¼ 8) and kebabs (n ¼ 8) (see Table 1 for details), were examined. The samples were kept cool and examined within 1 h of
203
purchase. 2.2. Isolation and identification of Arcobacter spp. Arcobacter spp. isolation was performed according to the method of Aydin, Gümüs¸soy, Atabay, Iça, and Abay (2007). 2.3. Isolation and identification of Campylobacter spp. For the isolation of Campylobacter spp. from the samples, Aydin et al. (2007)’s method was used; however, in the enrichment step, the samples were incubated at 37 C. Isolates identified with the phenotypic tests as Campylobacter spp. were confirmed using molecular analysis (mPCR) (Wang et al., 2002). 2.4. Isolation and identification of Listeria spp. Isolation and phenotypic identification of Listeria spp. from samples were performed according to the method of Abay, Aydin, and Sumerkan (2012). Isolates were confirmed at the species level by molecular methods, including hly gene-specific PCR (Pourjafar et al., 2010) and 16S rRNA sequence analysis (Lane, 1991). 2.5. Isolation and identification of Salmonella spp. The procedure was performed according to ISO 6579e2002. Positive colonies (2e3) were confirmed by the API 20E kit (Biomerieux, France). 2.6. DNA extraction DNA was extracted from each isolate using the UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories, 12224e250) following the manufacturer's instructions. 2.7. PCR amplification of the 16S rDNA gene The universal primers 27F AGAGTTTGATCMTGGCTCAG and 1492R GGTTACCTTGTTACGACTT were used to amplify the 16S rDNA gene (Lane, 1991). Amplified products were resolved by 1.5% agarose (Prona) gel electrophoresis and visualized under a UV transilluminator (G:BOX Chemi XRQ, Syngene). 2.8. Sequencing and phylogenetic analysis The amplified PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), and the Big Dye Direct Cycle Sequencing Kit (Applied Biosystems) was used in the sequence analysis; both kits were used according to the manufacturer's instructions. After cycle sequencing, the amplicons were purified with Sephadex G-50 (Sigma-Aldrich) and sequenced on the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems). All sequences were analyzed with the CLC Main Workbench 6 and compared with reference sequences in the National Center for Biotechnology Information website with BLASTN. The evolutionary history was inferred using the neighborjoining method (Saitou & Nei, 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was determined (Felsenstein, 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JukesCantor method (Jukes & Cantor, 1969) and were in the units of the number of base substitutions per site. The analysis involved 15 nucleotide sequences, including the 3 reference strains
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Table 1 Sample types and numbers (Listeria spp. positive samples). Company
Types of Samples nugget
A B C D E F G H I K Total
2n 1
2 2
Total meatballs
1* -
1 -
1
-
8
2
1 2
2
3 2 10
sausages
burgers
Donerkebabs
2
2
1 1 6
2
1
1 1 2 2
-
8
1
1 2 1
1 -
2 2
-
8
1
kebab
1
-
2 1
-
2
-
1
-
1 1
1 -
2
-
2 1 8
1
1 1 8
1 1
5 5 2 4 3 7 7 4 9 4 50
1 2 1 1 4 1 2 12
n
: Number of samples. *: Listeria spp. positive samples. -: negative samples.
L. monocytogenes (JF967620.1), L. innocua (NR_116805.1) and L. welshimeri (NR_074998.1) obtained from GenBank. All positions containing gaps and missing data were eliminated. The evolutionary analyses were conducted in MEGA6 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). 2.9. Nucleotide accession numbers The 16S rDNA sequences of the 12 isolates were deposited in GenBank under accession numbers from KX427175.1 to KX427186.1. 2.10. Enterobacterial repetitive intergenic consensus sequencebased PCR (ERIC-PCR) The ERIC motifs 1R (50 -ATG TAA GCT CCT GGG GAT TCA C-30 ) and 2 (50 -AAG TAA GTG ACT GGG GTG AGC G-30 ) were used for PCRmediated molecular subtyping of the Listeria spp. isolates (Aydin, Gümüs¸soy, Atabay, 2007). The ERIC-PCR gel image was evaluated using the GelCompar II 6.6.11 Gel Electrophoresis Software (Applied Maths) to construct the evolutionary tree of the Listeria species. 2.11. Serotyping of L. monocytogenes using molecular methods and serological tests Serotyping of the L. monocytogenes isolates was performed using molecular methods (Doumith, Buchrieser, Glaser, Jacquet, & Martin, 2004). Discrimination of serotypes obtained by mPCR from each other, a serological method based on the somatic and flagellar antigens was performed using Listeria O and H antisera (Denka Seiken 214362, Tokyo, Japan) as previously described (Karadal & Yıldırım, 2014). 2.12. Virulence gene analysis of Listeria spp. by PCR All Listeria spp. isolates were analyzed for the presence of the inlA, inlC (Wieczorek, Dmowska, & Osek, 2012), iap (Swetha, Madhava Rao, Krishnaiah, & Kumar, 2012), inlB, prfA, actA, hly, plcA, plcB, mpl, lin0558 and lin1068 (Johnson et al., 2004) virulence genes. Each virulence gene was investigated separately by PCR. The PCR and amplification conditions were performed according to Johnson et al., 2004. The expected band sizes were 800 bp, 517 bp, 131 bp, 1107 bp, 479 bp, 1994 bp, 496 bp, 798 bp, 320 bp, 674 bp, 866 bp and 613 bp for the inlA, inlC, iap, inlB, prfA, actA, hly, plcA, plcB, mpl, lin0558 and
lin1068 genes, respectively. 2.13. Antibacterial susceptibility testing The antibiotic susceptibilities of Listeria spp. to amoxicillin and clavulanic acid (AMC, 30 mg), ampicillin (AM, 10 mg), ciprofloxacin (CIP, 5 mg), enrofloxacin (ENR, 5 mg), gentamicin (CN, 10 mg), penicillin (P, 10 mg) and vancomycin (VA, 30 mg) were determined using the disk diffusion test (Bauer, Kirby, Sherris, & Turek, 1966). The antimicrobial disks were purchased from Oxoid, UK. The disk diffusion test results were interpreted using the criteria published by the Clinical and Laboratory Standards Institute (CLSI, 2008). Currently, there are no interpretative criteria provided by CLSI for Listeria, with the exception of susceptibility breakpoints for ampicillin and penicillin. Therefore, the CLSI criteria for staphylococci were applied in this study. Staphylococcus aureus ATCC 25923 was used as the control strain. 2.14. Standard strains The Arcobacter butzleri LMG 10828 (LMG bacterial collection, Ghent University), Campylobacter coli DCC2, Campylobacter jejuni NCTC 11168, L. monocytogenes serotype 1/2a (RSSK:472) and Salmonella enteritidis (from Erciyes University, Faculty of Veterinary Medicine, Dept. of Microbiology culture collection) reference strains were used throughout the study. 3. Results 3.1. Arcobacter spp., Campylobacter spp., and Salmonella spp. isolation and identification tests All of the examined samples tested negative for Arcobacter spp., Campylobacter spp., and Salmonella species. 3.2. Listeria spp. isolation and identification results Of the examined samples, twelve (24%) were positive for Listeria species. Among the isolated Listeria species, 9 were identified as L. monocytogenes, 2 were identified as L. innocua, and one was identified as L. welshimeri using phenotypic identification tests. The Listeria spp. isolates were numbered from the RTECM (ready-to-eat chicken meat) 1 to RTECM 12 samples; the isolates were referred to with these numbers when presenting the following results in subheadings. All L. monocytogenes isolates tested were positive in
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the PCR performed with primers specifically designed for the hly gene (840 bp). The results of this identification test were confirmed by the 16S rRNA sequence analysis (Fig. 1). The sources of the samples that tested positive for Listeria spp., the isolated species, and their distributions according to the companies are presented in Table 1. Accordingly, 2 out of 8 nugget samples tested positive for Listeria spp.; of these isolates, one was identified as L. welshimeri and the other was identified as L. monocytogenes. Among the 6 Listeria species isolated from the meatballs, 5 were identified as L. monocytogenes and 1 was one identified as L. innocua. One L. innocua isolate was recovered from the sausage samples, and one L. monocytogenes isolate was recovered from each of the burger, doner and kebab samples. The highest rate of listeria contamination was observed in the products of the company signified by the letter “F”.
3.3. Genotyping of the Listeria spp. isolates All isolates exhibited amplification of the 16S rDNA operon, resulting in a band of approximately 1350 bp (there were a total of 1347 positions in the final dataset). These 16S rDNA sequence data showed great similarity to the reference sequences of the strains in GenBank (between 99% and 100% at the species level). We confirmed that the 16S rDNA sequence analysis was a sufficient tool for identifying of Listeria spp. at the species level (Soni & Dubey, 2014). On the other hand, the Listeria spp. isolates recovered from RTE could not be discriminated according to their isolation sources (nugget, meatballs, sausages, burgers, Doner-kebabs and kebab) by the 16S rDNA sequence analysis (Fig. 1). The ERIC-PCR results of the 12 Listeria spp. are presented in Fig. 2. Remarkable genetic heterogeneity was observed between the isolates, as shown in Fig. 2. In particular, there was prominent genetic heterogeneity between the isolates recovered from the products of the companies signified by the letters “F” and “B” (Table 2). Likewise, there was significant genetic diversity between the L. innocua isolates.
205
3.4. Serotyping of L. monocytogenes isolates using molecular methods and serological tests Serotyping of the 9 L. monocytogenes isolates using mPCR showed that 7 isolates were serotype 1/2a, 3a and 2 were serotype 1/2c, 3c (Fig. 3A) and then the identification test results at the molecular level were confirmed by the serological tests (Listeria O and H antisera) as 7 isolates were serotype 1/2a and 2 were serotype 1/2c (Table 2). 3.5. Virulence gene analysis of Listeria spp. by PCR The virulence gene analysis results are presented in Table 3 and Fig. 3B. All L. monocytogenes isolates tested positive for the 9 virulence genes examined. Of these L. monocytogenes isolates, only 4 were positive for the actA gene. Only isolate number 1 tested positive for the lin1068 gene, and none of the isolates were positive for the lin0558 gene. The L. welshimeri isolate was negative for all of the examined virulence genes. 3.6. Antibacterial susceptibility testing All isolates were susceptible to amoxicillin and clavulanic acid, ampicillin, ciprofloxacin, enrofloxacin, gentamicin, penicillin, and vancomycin. 4. Discussion Generally, raw chicken meat and its products are highly contaminated (approximately 100%) with Campylobacter jejuni (Aydin et al., 2007; Yıldız & Diker, 1992), and the majority of human cases of campylobacteriosis (Kayman, Abay, & Hizlisoy, 2013) are associated with the consumption of improperly cooked contaminated food products or the cross-contamination of other products with Campylobacter species (Humphrey, Martin, Slander, & Durham, 2001). Poultry and meat products are also subjected to
Fig. 1. Evolutionary relationships of related Listeria spp. according to their 16S rDNA sequences by the neighbor-joining method. The optimal tree with the sum of branch lengths ¼ 0.00670292 is shown. The percentage of replicated trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. GenBank accession numbers are indicated for each sequence in parenthesis.
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Fig. 2. Dendrogram for the 12 Listeria isolates analyzed according to the ERIC-PCR gel electrophoresis band profiles. The dendrogram was constructed using the GelCompar II 6.6.11 (Applied Maths) software with UPGMA clustering based on the Dice correlation coefficient.
Table 2 Isolate numbers (company codes), species and serotypes of the Listeria spp. Company
Isolate Noa
Listeria species distribution
Isolate serotype (mPCR)
Isolate serotype (O and H Antisera)
A B
RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM
L. L. L. L. L. L. L. L. L. L. L. L.
1/2c, 3c 1/2c, 3c e 1/2a, 3a 1/2a, 3a 1/2a, 3a 1/2a, 3a 1/2a, 3a 1/2a, 3a 1/2a, 3a
1/2c 1/2c e 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a
C D F
I K a
9 11 12 1 2 4 7 8 10 6 3 5
welshimeri monocytogenes monocytogenes innocua monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes innocua monocytogenes monocytogenes
Number of Listeria isolates.
heat processing prior to presentation to the consumer. Generally, heat-processing treatment (55 C and higher) of these products inactivates Campylobacter species (Lake, Hudson, Cressey, & Gilbert, 2007). Therefore, these products are not considered to pose a significant risk to consumers. A very limited number of studies have evaluated the load of Campylobacter species in heat-processed ~ ones-Ramírez, poultry meat products (llida & Faridah, 2012; Quin V azquez-Salinas, Rodas-Su arez, Ramos-Flores, & Rodríguez-Mon~ o, 2000; Joint Food Safety and Standards Group, 1996; Bohaytan chuk et al., 2006; Federighi et al., 1999). Ilida & Faridah (2012) found that 22 heat-processed chicken products were negative for C. jejuni, whereas 758 heat-processed chicken products were negative for C. jejuni in the study by the Joint Food Safety and Standards Group (1996). Additionally, cooked poultry sausage samples were free from thermotolerant Campylobacter in a study conducted by Federighi et al. (1999). Similarly, all samples tested in the present study were negative for Campylobacter species. In contrast, Quinones-Raminez et al. (2000) found contamination with Campylobacter species in 27 out of 100 heat-processed chicken taco samples. The researchers noted that mishandling was the source of contamination and highlighted that these products posed a potential risk to public health. Arcobacter species are water- and food-borne pathogens that are important for humans and are abundantly present in raw poultry
and meat products (Aydin, Gümüs¸soy, Atabay, 2007; Mor-Mur & Yuste, 2010). However, no study has evaluated the prevalence of these microorganisms in heat-processed ready-to-eat chicken meat products. All samples tested in the present study were negative for Arcobacter species. This finding can be explained by the inactivation of Arcobacter species during the heat processing treatment. Arcobacter species are in the same family as Campylobacter species and show substantially identical morphological/physiological characteristics. Although Salmonella has largely been found in raw poultry products, all of the samples evaluated in the present study tested negative for Arcobacter species, Campylobacter species and Salmonella species. Salmonella species would be inactivated when the ready-to-eat chicken meats were subjected to the heat processing treatment. Likewise, other researchers did not report Salmonella species in heat-processed chicken meat products (Bohaychuk et al., 2006; Karadal et al., 2013). The most important finding of the present study in terms of isolation and identification was that Listeria species were isolated from 24% (12/50) of the samples (Table 1). Among the Listeria species isolated, 9 (75%) were identified as L. monocytogenes. The presence of Listeria species in heat-processed ready-to-eat chicken meat and chicken products poses a significant public health hazard. The isolation rates of Listeria spp. from RTE chicken meat
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Fig. 3. Results of L. monocytogenes serotyping and virulence genes analysis. A. Agarose gel electrophoresis of mPCR products for the identification of L. monocyogenes serotypes. Lanes 1e12 (RTECM 1e12): Listeria spp. isolates recovered from processed chicken meat. Lanes 1 and 6: L. innocua, lane 9: L. welshimeri, lanes 2, 3, 4, 5, 7, 8 and 10: L. monocytogenes serovar 1/2a, lanes 11 and 12: L. monocytogenes serovar 1/2c, C1: Control strain of L. innocua, C2: Control strain of L. monocytogenes serovar 1/2a, C3: Control strain of L. monocytogenes serovar 4b and C4: Control strain of L. monocytogenes serovar 1/2c, M: Marker, 100 bp plus. B. Representative agarose gel electrophoresis image of the L. monocytogenes, L. innocua and L. welshimeri virulence genes. Lane 1: iap gene, 131 bp; Lane 2: prfA gene, 479 bp; Lane 3: mpl, 674 bp; Lane 4: hly gene, 496 bp; Lane 5: actA, 1994 bp; Lane 6: plcA gene, 798 bp; Lane 7: plcB, 320 bp; Lane 8: lin1068, 613 bp; Lane 9: inlA, 800 bp; Lane 10: inlB, 1107 bp; Lane 11: inlC, 517 bp; M: Marker, 100 bp plus (Thermo Scientific).
Table 3 Occurrence and distribution of 12 virulence genes analyzed in Listeria spp. isolates. Isolates numbers
inlA
inlB
inlC
prfA
actA
hly
plcA
plcB
mpl
iap
lin1068
lin0558
RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM RTECM
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ e þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ
1 Li 2 Lm 3 Lm 4 Lm 5 Lm 6 Li 7 Lm 8 Lm 9 Lw 10 Lm 11 Lm 12 Lm
þ: positive for virulence gene, : negative for virulence gene. Li: L. innocua Lm: L. monocytogenes Lw: L. welshimeri.
and products show variability. The Listeria spp. rates found in RTE chicken meat and products in different countries were 2.9% by , & Man ~ es, (2001), 3% by Bohaychuk et al. Soriano, Rico, Molto (2006), 0.02% by Kanarat, Jitnupong, and Sukhapesna (2011) and 10% by Awadallah and Suelam (2014). The results of the present study suggest higher rates than those reported by the researchers in the studies mentioned above. This difference may be related to the isolation and identification methods employed, insufficient hygiene practices, and the bacterial
loads of the products (Keeratipibul & Lekroengsin, 2008, 2009). Heat-processed ready-to-eat chicken meat and chicken products must be free from L. monocytogenes as per the relevant regulations in Turkey (Türk Gıda Kodeksi Mikrobiyolojik Kriterler € netmelig i, o 2011, http://www.mevzuat.gov.tr/MevzuatMetin/ yonetmelik/7.5.15690-ek.pdf, Republic of Turkey Ministry of Food, Agriculture and Livestock). The high isolation rate for Listeria species in the tested products in the present study suggests poor sanitation and insufficient hygiene practices in these
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establishments. Adherence to sanitation and hygiene principles during the preparation of heat-processed chicken meat and chicken products significantly reduces the Listeria species load (Keeratipibul & Lekroengsin, 2008, 2009). In the present study, products of 7 out of 10 companies tested positive, and 3 tested negative for Listeria species. The highest contamination rate was observed in the products of the company signified by the letter “F” (Table 1). The L. monocytogenes isolates recovered in the present study were not well characterized at the molecular level. All of the isolates possessed all 9 virulence genes tested (Fig. 3B and Table 3), with 7 isolates serotype 1/2a and 2 isolates serotype 1/2c (Fig. 3A and Table 2). Additionally, a phylogenetic analysis of the three different Listeria species isolated in the present study was performed (Figs. 1 and 2). The sequence analysis results allowed the identification of the isolates at the species level but failed to detect genetic diversity within a particular species. However, ERIC-PCR was a sensitive tool for the detection of genetic diversity within a species. There was remarkable diversity in the band patterns of the L. monocytogenes isolates recovered from the products of the companies signified by the letters “F” and “B” (Table 2 and Fig. 2.). This finding indicates the presence of multiple contamination sources in these establishments. L. monocytogenes has the highest mortality rate (20e40%) in humans among other food-borne pathogens. The virulence genes possessed by the bacteria are known to have a significant effect on this finding. Because the 1/2a, 1/2b and 4b serotypes of L. monocytogenes account for 98% of the listeriosis cases (Borucki & Call, 2003; Nho, Abdelhamed, Reddy, Karsi, & Lawrence, 2015; Swaminathan & Gerner-Smidt, 2007), the ready-to-eat chicken meat from which L. monocytogenes was isolated in the present study constitutes a potential hazard to human consumers. Although a number of studies have reported resistance to various antibiotics in L. monocytogenes isolates (Facinelli, Giovanetti, Varaldo, Casolari, & Fabio, 1991; Roberts, Facinelli, € Giovanetti, & Varaldo, 1996; Yücel, Cıtak, & Onder, 2005), all of the Listeria isolates tested in the present study were susceptible to the seven antibiotics tested. 5. Conclusions Fifty ready-to-eat chicken meat products tested negative for Arcobacter spp., Campylobacter spp., and Salmonella species, but 12 samples (24%) were found to be contaminated with Listeria species. A detailed molecular analysis such as identification, virulence genes screening and genotyping of the Listeria spp. revealed that the food products posed a significant public health hazard. Considering the presence of different L. monocytogenes genotypes in ready-to-eat food production facilities, all steps of food production must be reviewed in terms of conformity with the principles of sanitation and hygiene practice, and necessary measures must be set in place. References Abay, S., Aydin, F., & Sumerkan, A. B. (2012). Molecular typing of Listeria spp. isolated from different sources. Ankara Universitesi Veteriner Fakultesi Dergisi, 59, 183e190. Anonym. (2005). Salmonellosis paratyphoid, non-typhoidal salmonellosis the center for food security and public health. Iowa State University. http://www.cfsph. iastate.edu/Factsheets/pdfs/nontyphoidal_salmonellosis.pdf. Awadallah, M. A. I., & Suelam, I. I. A. (2014). Characterization of virulent Listeria monocytogenes isolates recovered from ready-to-eat meat products and consumers in Cairo, Egypt. Veterinary World, 7, 788e793. Aydin, F., Gümüs¸soy, K. S., Atabay, H. I., Iça, T., & Abay, S. (2007). Prevalence and distribution of Arcobacter species in various sources in Turkey and molecular analysis of isolated strains by ERIC-PCR. Journal of Applied Microbiology, 103, 27e35. Aydin, F., Gumussoy, K. S., Ica, T., Sumerkan, B., Es¸el, D., Akan, M., et al. (2007). The prevalence of Campylobacter jejuni in various sources in Kayseri, Turkey, and
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