Food Control 27 (2012) 64e72

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Food Control

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Tracking sources of contamination in a cooked chicken meat factory by PCR-RAPD-based DNA fingerprinting

Suwimon Keeratipibul*, Punnida Techaruwichit

Department of Food Technology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand article info abstract

Article history: As a means to reduce the risk of Listeria spp. contamination in cooked frozen chicken meat process this Received 29 August 2011 study investigated the sources and the routes of infection using PCR-RAPD-based molecular typing. A Received in revised form total of 12,833 samples of final products (865), intermediate stage precut and packaged meat (4325) and 20 February 2012 environmental surfaces (7643) were screened for the presence of Listeria spp. Of the 401 positive isolates Accepted 25 February 2012 from the processing environment, the species were comprised of (82.3%), Listeria wel- shimeri (11.2%), Listeria seeligeri (5.5%) and (1%). Twelve positive isolates of Keywords: L. innocua and one each of L. welshimeri and L. seeligeri were found in the finished product. A total of 415 Chicken meat fi Listeria Listeria contaminated samples were further subjected to RAPD (randomly ampli ed polymorphic DNA) fi Transmission route analysis to evaluate the relationship of the contaminants in the nal product and those in the envi- Molecular typing ronment. L. innocua type LI 1.1, L. welshimeri type LW 1.5 and L. seeligeri type LS 1 were the only isolates RAPD found in the finished product, whilst L. innocua type LI 1.1 was persistently found in the surfaces of the food processing plant throughout the sampling period. The surfaces from which Listeria spp. were most frequently recovered were the liquid N2 chiller exhaust pipe, the metal detector conveyor belt and the freezer drain. Therefore, the cleaning and sanitizing procedures were revised and strictly implemented to reduce and eliminate the real sources of Listeria contamination in the cooked frozen chicken meat process. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Listeria innocua, Listeria welshimeri, Listeria seeligeri, Listeria grayi, , Listeria rocourtiae (Leclercq et al., 2009) and Listeria Cooked chicken meat products are economically important to marthii (Graves et al., 2010). L. monocytogenes is the species most several countries, spanning the developed to the developing widely associated with human disease (listeriosis), where this countries, and include the Thai food processing industry. The pathogen most often affects those with severe underlying condi- annual export value for Thailand of 1e1.7 billion U.S. dollars (Thai tions (such as immunosuppressive therapy, AIDS, and chronic Broiler Processing Exporters Association, 2010) makes Thailand conditions, such as cirrhosis), pregnant women, unborn or newly the fourth largest cooked chicken meat exporting country in the delivered infants and the elderly. All strains of L. monocytogenes world, after the United States of America, Brazil and the European appear to be pathogenic and infections can be life threatening, with Union (EU). Japan and the EU are the main importers of Thai fatality rates of 20e30% (WHO/FAO, 2004). Despite the fact that chicken exports (Department of Foreign Trade, 2009). However, a wide variety of foods may be contaminated with L. mono- despite the significant production and distribution of Thai cooked cytogenes, outbreaks and sporadic cases of listeriosis are predomi- chicken meat products, tracking the relevant pathogens to the nately associated with ready-to-eat foods (Pradhan et al., 2009; source of contamination in these products remains poorly studied WHO/FAO, 2004). An important factor in foodborne listeriosis in Thailand. is that the pathogen can grow to significant numbers at refrigera- The genus Listeria, which are gram-positive, non-spore forming tion temperatures when given sufficient time (ICMSF, 1996; , are comprised of eight species: Listeria monocytogenes, MacGowan, Bowker, & Mclauchlin, 1994; WHO/FAO, 2004). Hence, L. monocytogenes directly affects the chilled and frozen ready-to-eat food industries. Although the regulatory standards of the Department of * Corresponding author. Tel.: þ66 2 2185515 6, þ66 2 2185519; fax: þ66 2 2544314. Livestock Development of Thailand, and various countries E-mail address: [email protected] (S. Keeratipibul). including Japan and the EU for ready-to-eat meat and poultry

0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.02.026 S. Keeratipibul, P. Techaruwichit / Food Control 27 (2012) 64e72 65 products require a zero tolerance (negative in 25 g sample) for Steaming L. monocytogenes (Commission Regulation (EC) No. 2073/2005; (Product core temperature of 75 oC for not Department of Livestock Development, 2010), Listeria spp. occur- rence, and not only L. monocytogenes, in these products is unac- less than 1 min) ceptable to both the exporters and importers (Interview data from QA manager of the cooked chicken meat factory in Thailand). Many studies support that the presence of any Listeria species in Pre-chilling fi a speci c environment can indicate the presence of the others, in a liquid N2 chiller including L. monocytogenes (Barros et al., 2007; Slade, 1992). Thus, (Product core temperature of 50 – 60 oC) fairly extensive research by worldwide food producers and researchers related to Listeria elimination and prevention has been conducted. Many authors have also demonstrated a high Chilling prevalence of L. monocytogenes, and other Listeria spp., in meat and poultry product processing environments; for example: in in an air blast spiral chiller o chilling and cutting rooms (Van de Elen & Snijders, 1993), (Product core temperature of 10 C) workers’ hands (Kerr, Kite, Heritage, & Hawkey, 1995), conveyor belt rollers (Tompkin, 2002) and processing equipment (Lawrence & Gilmour, 1995), strongly suggesting that the processing envi- Slicing or dicing ronment represents a significant source of these organisms in by man or machine finished products. While processed meat and poultry products are cooked to destroy Listeria, these bacteria can recontaminate the product while it is being handled, packaged or distributed (Lekroengsin, Keeratipibul, & Trakoonlerswilai, 2007; Tompkin, Individual quick freezing (IQF) o Scott, Bernard, Sveum, & Gombas, 1999). (Product core temperature of -18 C) Early studies of transmission routes depended solely on isolating and counting the organism at different places along the processing line (Eklund et al., 1995; Lekroengsin et al., 2007). When Filling and packing the organism was found on any environmental surface, cleaning and sanitizing was then implemented on the contaminated Fig. 1. The schematic flow diagram of the cooked chicken meat processing line. surfaces. However, the organism found on those surfaces might not be the organism that contaminated the product since by using conventional species identification, different strains of the same chicken meat was cooked by steaming to bring the product core species could not be identified. Therefore, there has been a limita- temperature to 75 C for not less than 1 min. After that the meat tion to find the real source of product contamination. As a conse- was pre-chilled in a liquid nitrogen (N2) chiller until the product quence, the control and prevention strategies implemented might core temperature reached around 50e60 C and further cooled not correct the contamination source. Thus, recent studies have down to 10 C in an air blast spiral chiller. Then, the meat was diced been greatly facilitated by the use of molecular-typing methods or sliced by hand or dicing machine, frozen in an individual quick with high discriminatory power, including randomly amplified freezer (IQF) until the product core temperature was lower polymorphic DNA (RAPD) profile analysis (Boerlin, Bannerman, than 18 C and packed in a plastic bag. Ischer, Rocourt, & Bille, 1995; Byun, Jung, & Yoo, 2001; Chambel The ambient temperature in the plant was controlled at 10 C. et al., 2007; Fonnesbech Vogel, Jørgensen, Ojeniyi, Huss, & Gram, The different processing operations of the cooked meat took place 2001). Molecular studies on the ecology of Listeria species strains in a big hall with separated sections and with a continuous flow present in the food processing environment provide crucial infor- process. A strict procedure regarding personal hygiene was mation for the development of better control and prevention instructed and followed. All employees wore gloves which were strategies for this important foodborne pathogen (Norton et al., sanitized every half an hour and changed at least twice a shift. Daily 2001). However, despite its importance, at present there are only cleaning and sanitizing was carried out at the middle and the end of a few studies on the Listeria profile and its control in the Thai each production shift (day shift: 12:00e13:00, 17:00e18:00; night chicken meat processing plants, and there is no report of the shift: 24:00e1:00, 5:00e6:00). A thorough cleaning was carried molecular typing of Listeria to track the source of contamination to out on Sunday when there is no production. implement a better understanding and control of the route of food contamination by this organism in Thailand. Therefore, the purpose 2.2. Sampling of this study was to investigate the prevalence and the transmission routes of Listeria species to cooked (steamed) chicken meat prod- The processing environmental surfaces and the different inter- ucts in a representative Thai cooked frozen chicken meat process- mediate stages of chicken meat processing and the finished ing plant using PCR-RAPD molecular detection. product, were sampled over a 16-week period between Oct 2009 and Jan 2010. Sampling was performed three days per week 2. Materials and methods (Tuesday, Thursday and Friday) at six times per, day. That is for the day shift at 7:00 (start of the day-shift production), 10:00 and 2.1. Processing plant and product manufacturing 16:00 h; and for the night shift at 19:00 (start of the night shift), 22:00 and 4:00 h. A total of 11,968 samples were collected and This study was performed in a cooked (steamed) chicken meat screened for Listeria spp. processing plant in Thailand. The steam production line has a pro- Samples of chicken meat were immediately taken after each cessing capacity of 23 metric tons per day, using raw chicken meat processing step, i.e. after the cooking, pre-chilling, chilling, dicing, obtained from a certified slaughter house. The flow diagram of the and freezing steps. In all cases, 500 g of each product sample was cooked chicken meat processing line is presented in Fig. 1. The raw taken and sealed in a sterile plastic bag and transported in a cooler 66 S. Keeratipibul, P. Techaruwichit / Food Control 27 (2012) 64e72 to the laboratory for further analysis. A bag (5 kg e packaged) of the (Niederhauser et al., 1994), PB1, PB4 (Byun et al., 2001), HLWL 74, finished product was also sampled before exporting to the HLWL 85, OMP-01 (Aguado, Vitas, & Garcia-Jalon, 2004), CsM 13, inl customers and transported in a cooler to the laboratory. AF and pH (Chambel et al., 2007), was performed with five strains of Environmental sampling was performed in the different pro- L. innocua, three strains of L. welshimeri and two strains of cessing plant areas. Sampling locations were chosen to represent L. seeligeri isolated from the environmental surfaces of the pro- those most likely to harbor Listeria. Sampling sites were divided cessing plant. Based upon the PCR-RAPD banding profiles obtained, into three zones, based on the contact and proximity to the product. four of these 16 primers were selected for RAPD typing. Amplifi- Zone 1 is the product-contact surfaces, zone 2 is the non-product cation was performed in a thermocycler (Corbett Research), using contact surfaces in close proximity to the product and zone 3 is a total volume of 20 ml solution which included 10 reaction buffer, the non-product contact surfaces that are further away from the 1 U of Taq DNA polymerase (Invitrogen), 10 mM of each deoxy- product. The area of environmental sampling varied depending on nucleoside triphosphate (Fermentas), 50 mM Mg2Cl, 10 mMof the sampling location. All sampling sites in the production envi- primer and 1 ml of DNA extract. A concentration of 5 ng/reaction ronment were swabbed with sterile cotton swabs moistened with DNA template was used in the RAPD reaction. The same volume 0.85% NaCL (w/v). After sampling, the swabs were soaked in 10 ml (1 ml) of double deionized water was used to replace the bacterial of Dey/Engley(D/E) Neutralizing Broth and kept in a cooler during DNA extract as a negative control, while a template DNA of transport to the laboratory. L. innocua was used as a positive control. The primers selected were the universal forward sequencing Ò 2.3. Listeria species identification by VIDAS method primer (UFS: 50TTATGTAAAACGACGGCCAGT30), HLWL 74 (50ACGTATCTGC30), HLWL 85 (50ACAACTGCTC30) and OMP-01 Upon arrival at the laboratory, 25 g chicken samples were each (50GTTGGTGGCT30). The PCR cycling conditions used for these homogenized for 1 min in 225 ml Half-Fraser (HF) broth (bio- primers were as follows. For the UFS primer, 94 C for 3 min Mérieux) in a stomacher, and incubated at 30 1 C for 20e26 h as followed by 4 cycles of 94 C for 45 s, 26 C for 2 min and 72 C for a pre-enrichment step. One ml of the suspension was transferred to 2 min; 30 cycles of 94 C for 45 s, 36 C for 1 min and 72 C for tubes containing 10 ml of Fraser Broth and incubated at 30 1 C 2 min; plus one additional cycle at 72 C for 5 min. For the HLWL for 20e26 h. Then, 1 ml of the Fraser Broth was transferred to tubes 74, HLWL 85 and OMP-01 primer, the cycling program was 45 and heated at 95e100 C for 15 1 min. After heating, the tubes cycles of 94 C for 4 min, 39 C for 45 s, 72 C for 1 min; plus 1 were cooled and mixed, and then 0.5 ml of the boiled broth was additional cycle of 72 C for 10 min. To differentiate the strains, Ò transferred into the sample well on VIDAS strip. Vidas Assay pattern analysis was performed. One ml of the amplification (Vidas LIS/48 min) for Listeria spp. detection was then performed. products from each of the four selected primer reactions was For the environmental samples, the swabs were resuspended in mixed and loaded on an Agilent DNA 7500 kit and the pattern was 90 ml HF broth and incubated at 30 1 C for 24e26 h. One ml of examined by using Agilent 2100 Bioanalyzer. the suspension was then transferred to tubes and heated at 95e100 C for 15 1 min. Then, 0.5 ml of the boiled broth was 3. Results Ò transferred into the sample well on VIDAS strip. Vidas Assay (Vidas LSX/70 min) for Listeria spp. detection was then performed 3.1. Prevalence of Listeria species (bioMérieux, Durham, France). One loop of all positive samples were streaked on Listeria selective agar (Oxford; OXOID) and From a total of 4325 intermediate chicken meat samples, taken Ottaviani Agosti agar (OAA) plates (bioMérieux), incubated at from five processing steps, none of the samples were positive for 37 1 C for 48 2 h and then observed for the presence of typical Listeria spp. However, from a total of 865 finished product samples Listeria colonies according to ISO 11290-1. From each plate, three that were analyzed, 14 (approximately 1.6%) were found to be colonies with morphological characteristics of Listeria were picked positive for Listeria spp., comprised of 12 samples being positive for off, streaked onto TSAYE (Tryptone Soy Agar; OXOID) with 0.6% (w/ L. innocua and one each for L. welshimeri and L. seeligeri, with no v) Yeast Extract (Merck) plates and incubated at 37 1 C for samples being co-infected with multiple species of Listeria spp. 18e24 h. Colonies presumptive for Listeria spp. on TSAYE were A total of 2954 environmental samples from zone 1 surfaces, selected and subjected to Gram staining, catalase test and 3645 samples from zone 2 and 1044 samples from zone 3 were motility at 25 1 C for 48 h. The API Listeria System incubated at swabbed and revealed the prevalence of Listeria in 1.1, 5.8 and 13.4% 35 1 C for 18e24 h was used to confirm the identified species of the samples, respectively. The species compositions of these 401 (bioMérieux S.A.). contaminated samples were L. innocua (82.3%), L. welshimeri (11.2%), L. seeligeri (6%) and L. monocytogenes (1.0%). 2.4. Listeria strain identification by RAPD The prevalence (% positive samples) of Listeria spp. on the surfaces at the start of day-shift production (7:00 h) ranged from 2.4.1. DNA preparation low, but not zero (0.8%) at zone 1 up to high, (12.1%) for zone 3 and A single isolated colony of Listeria on TSAYE was picked up and typically increased by the start of night-shift production (19:00 h), cultured in TSBYE (Tryptic Soy Broth with 0.6% (w/v) Yeast Extract; especially in zone 3 (Fig. 2). Importantly, Listeria spp. were present Merck) overnight at 37 C. The cells from 3 ml of the overnight on the surfaces in zones 1, 2 and 3 at all production times, but culture were recovered by centrifugation (10,000 g for 10 min), whilst the prevalence of Listeria spp. on surfaces in zones 1 and 2 from which the total DNA was extracted using the Genomic DNA were broadly consistent in most production times, that on the zone Extraction Kit (YGB, RBC Bioscience). The concentration of extrac- 3 surfaces increased rapidly and continuously from 10:00 (minima) ted DNA was determined by measuring the absorbance at O.D.260. until the end of the production time of the night shift (04.00 h).

2.4.2. RAPD analysis 3.2. Development and optimization of RAPD assay For single primed PCR-RAPD fingerprinting, an initial screening of the 16 different primers; the universal forward (UFS), Tn21 Sixteen primers, which have been used in other studies for and ampC gene sequencing primers (MacGowan et al., 1993); RAPD analyses of L. monocytogenes and other species in this genus UBC155, UBC156, UBC127 (Farber & Addison, 1994), HR4, ECO2 of different origin, were tested for their discriminatory abilities S. Keeratipibul, P. Techaruwichit / Food Control 27 (2012) 64e72 67

(4.1%), LI 1.6 (3.5%), LI 2.1 (1.8%), LI 3 (1.8%) and LI 1.5 (0.3%). Only strain LI 1.1 was found in the finished product. This strain was widely distributed on the environmental surfaces of all zones of every processing step along the processing line throughout the sampling period. The high frequency of contamination (58.6%) was at the exhaust pipe of the in-feed liquid N2 chiller where the product was fed into the chiller. On this surface, a greater diversity of L. innocua strains were found, being LI 1.2, LI 1.6, LI 2.1 and LI 2.2, than on any other environmental surface (Table 1). For L. welshimeri, three main different RAPD profiles (strains LW 1, LW 2 and LW 3) were obtained from the 46 isolates, with strain LW 1 being further divided into five substrains (LW 1.1, LW 1.5, LW 1.6, LW 1.7 and LW 1.8), as shown in Fig. 3. L. welshimeri strain LW 1.7 was the dominant strain (37.5%) of L. welshimeri found in the processing environment. The other L. welshimeri strains contami- nated in the processing environment were LW 1.1 (25%), LW 2 Fig. 2. Trend of Listeria spp. prevalence as that recovered from all environmental surface swabs with respect to each zone and the daily production time. (10%), LW 3 (10%), LW 1.6 (7.5%), LW 1.8 (7.5%) and LW 1.5 (5%). The only L. welshimeri found in finished product was strain LW 1.5. The prevalence of the various L. welshimeri strains on the against five isolates of L. innocua, three of L. welshimeri and two of environmental surfaces in the processing plant are shown in L. seeligeri isolates from the environmental surfaces of the pro- Table 2. The main sources of L. welshimeri contamination were in cessing plant. The fingerprints produced by eight of the primers (Tn the late process stages and in zone 3, followed by zone 2 and then 21, PB 1, PB 4, CsM 13, inl AF, pH, HR4 and ECO2), could not zone 1. The main sources in zone 1 (direct contact) were the differentiate the strains that showed different fingerprints when workers’ gloves in quality control/packing (LW 1.7 and LW3) and the other primers were used. However, four of the primers (OMP- the freezer (LW1.6). 01, HLWL 74, HLWL 85 and UFS), provided the highest discrimi- For L. seeligeri, one sample of the finished product was nating power among the 16 tested primers. Primer OMP-01 could contaminated, whilst 21 contaminated areas were found in the differentiate all five L. innocua isolates, primers HLWL 85 and HLWL processing environment. All 22 isolates belonged to a single RAPD 74 could differentiate all three L. welshimeri isolates, and primers strain (LS 1, Fig. 3). A high prevalence was found in the late process OMP-01, HLWL 85, HLWL 74 and UFS could differentiate both stages on the floor of the packing area and the freezer area surfaces, L. seeligeri isolates. However, to enhance the discriminatory which are the same areas as L. welshimeri LW 1.7, except that one potential, the four primers (OMP-01, HLWL 85, HLWL 74 and UFS) LS1 sample was also isolated on the floor of dicing area. were used for every isolate regardless of which species it was. For L. monocytogenes, one main RAPD profile based strain, LM 1, was obtained from the four L. monocytogenes isolates, which was 3.3. RAPD profiling and prevalence of Listeria species and strains further divided into substrain LM 1.1 and LM 1.2 (Fig. 3). No L. monocytogenes isolate was found in the finished product. Two Reproducible distinct electrophoretic patterns were obtained isolates belonging to substrain LM 1.1 were found on floor of the using the combined amplicons from the separate RAPD-PCR packing area and gloves of the quality control worker at the packing amplifications of all four primers (OMP-01, HLWL 74, HLWL 85 step, whilst the other two LM 1.2 isolates being found on the wire- and UFS). Three main RAPD patterns (strains LI 1, LI 2 and LI 3) were mesh tray which was connected to the out-feed conveyor belt of obtained for the 342 L. innocua isolates examined with strain LI 1 the freezer. being further divided into four substrains (LI 1.1, LI 1.2, LI 1.5 and LI 1.6), as shown schematically in Fig. 3, with an example of the strain and substrain classification shown in Fig. 4. Strain LI 2 was divided 3.4. Correlation of Listeria RAPD types in cooked chicken meat into two different substrains (LI 2.1 and LI 2.2) whilst strain LI 3 did and in the processing environment not have a different RAPD substrain. L. innocua strain LI 1.1 was the dominant strain, represented by As mentioned in Section 3.1, a total of 14 Listeria isolates,12 from 77.1% of all the L. innocua samples. The other L. innocua strains L. innocua LI 1.1 and one each from L. welshimeri LW 1.5 and presented in the processing environment were LI 2.2 (11.5%), LI 1.2 L. seeligeri LS 1,were obtained from the finished chicken product.

Fig. 3. Representative RAPD patterns of Listeria species strains/substrains from a cooked chicken meat processing plant generated with primer OMP-01, HLWL 74, HLWL 85 and UFS. L, ladder. Lanes 1 and 2, L. monocytogenes strains (LM 1.1 and LM 1.2, respectively). Lanes 3e9, L. innocua strains (LI 1.1, LI 1.2, LI 1.5, LI 1.6, LI 2.1, LI 2.2 and LI 3, respectively). Lanes 10e15, L. welshimeri strains (LW 1.1, LW 1.5, LW 1.6, LW 1.7, LW 2, and LW 3, respectively). Lane 16, L. seeligeri strain (LS 1). 68 S. Keeratipibul, P. Techaruwichit / Food Control 27 (2012) 64e72

Fig. 4. Examples of electropherograms of L. innocua RAPD subtypes among 363 isolates. The seven strains of L. innocua shown are (A) LI 1.1, (B) LI 1.2, (C) LI 1.5, (D) LI 1.6, (E) LI 2.1, (F) LI 2.2 and (G) LI 3. The electropherograms were obtained from an Agilent 2100 Bioanalyzer. The vertical axis is the concentration of the RAPD products (nmol/l). The horizontal axis is the elution time (s) of the RAPD products. The first and last peaks represent the lower and higher mass standard markers, respectively.

Examination of the environmental surfaces in the processing packing area (zone 3) and the draining pipe of the freezer (zone 3) area that were contaminated with L. innocua LI 1.1 on the same or were positive for L. seeligeri LS 1. a nearby day that the finished products were contaminated reveals that L. innocua LI 1.1 was mainly found in the exhaust pipe of in- 4. Discussion feed liquid N2 chiller as well (Tables 3 and 4). Moreover, L. innocua LI 1.1 was also found on conveyor belts of the in-feed Detectable levels of Listeria spp. were isolated from samples of liquid N2 chiller, dicer, metal detector and heat sealer, as well as the finished meat product and environmental surface swabs in the the control monitor of the dicer, tray supporter under the liquid N2 processing plant, but none in the intermediate product. Thus, either chiller, floor of the dicing area, draining pipe of the freezer, floor of Listeria spp. contamination occurs in the processing, or else pre- the packing area and the gloves of the worker who carried equip- existing contaminations in the meat are at a low rate and concen- ment to be cleaned. tration or are distributed unevenly in the product making it difficult For L. welshimeri, substrain LW 1.5 was found in the finished to detect Listeria spp. Given the proportion of Listeria spp. found in product on 14/11/09 yet no environmental surface in the processing the finished product and environmental surfaces of the processing line was found to be contaminated with L. welshimeri LW 1.5 on or plant, compared to the number of negative intermediate meat close to that day. One month later on 18/12/09, L. welshimeri LW 1.5 samples, it is likely that contamination of Listeria spp. in the was found once again in the draining pipe of the freezer. The source finished product came, at least in part if not totally, from the of contamination of substrain LW 1.5 in the finished product on 14/ environmental surface. 11/09 has not been revealed yet. Certainly, Listeria spp. contamination was found to occur on at Only one finished product sample was contaminated with least some environmental surfaces in every zone throughout both L. seeligeri LS 1, but no environmental surface was contaminated the production shifts, suggesting that, the cleaning and sanitizing at with L. seeligeri LS 1 at that time. However, 4 days before the the end of each production shift could not completely eliminate product was contaminated, the gloves of packing worker (zone 1 Listeria spp. on all the surfaces of each zone, in addition to any new surface) were contaminated with L. seeligeri LS 1. In addition, two contamination brought in by the workers each day. The increasing days after the date the product was contaminated the floor of the prevalence of Listeria spp. on zone 3 surfaces during the two S. Keeratipibul, P. Techaruwichit / Food Control 27 (2012) 64e72 69

Table 1 Prevalence (% positive from 7643 screened samples) of L. innocua strains on the environmental surfaces in the three zones (see Methods) of the processing line.

Zone Swab surfaces L. innocua strains/substrains

LI 1.1 LI 1.2 LI 1.5 LI 1.6 LI 2.1 LI 2.2 LI 3 1 Out-feed conveyor belt of cooker eee0.6 eee

In-feed conveyor belt of liq. N2 chiller 2.3 eeee0.6 e In-feed conveyor belt of chiller 0.6 eeee0.6 e Out-feed conveyor belt of chiller 0.6 0.6 eeeee Conveyor belt of dicer 1.7 eeeeee Gloves of QC worker after dicing 1.1 eeeeee In-feed conveyor belt of freezer 0.6 eeeeee Out-feed conveyor belt of freezer 1.1 0.6 eeeee Gloves of worker at freezer 1.1 eeeeee Gloves of packing worker 0.6 0.6 eeeee

2 Frame of the exit of cooker 0.6 eeeeee Tray supporter under conveyor belt of the cooker 0.6 eeeee

Exhaust pipe of liquid N2 chiller 58.6 0.6 e 2.9 2.3 9.2 e Tray supporter under conveyor belt of liquid N2 chiller 4.0 eeeeee Monitor of dicer controller 0.6 eeeeee External area of the dicer 0.6 eeeee1.1 Controller box of dicer 1.1 eeee0.6 e Packing table 1.1 eeeeee Conveyor belt of metal detector and heat sealer 7.5 ee1.7 ee0.6 Belt for transferring equipment 0.6 eeeeee

3 Gloves of worker carrying used equipment 0.6 eeeee Floor of dicing room 9.8 1.1 e 1.1 e 2.3 0.6 Wall of freezer eeeee0.6 Draining pipe of freezer 22.4 e 0.6 eee0.6 Floor at packing area 14.4 0.6 ee0.6 0.6 e

For the finished product, only isolate L1.1 was found in 12/865 samples. production shifts reflects that more attention was paid to surfaces The development of a better understanding of the route of in zones 1 and 2 during the mid-shift cleaning and sanitizing, contamination with Listeria species in food processing plants, in including the frequent brief sanitizing with 70% (v/v) ethanol combination with rapid, standardized detection and typing during production which can reduce the prevalence of Listeria spp. systems, will provide critical tools and knowledge for the devel- However, even with respect to zones 1 and 2, the cleaning and opment and verification of improved control strategies (Norton sanitizing procedures do not appear to be able to totally eliminate et al., 2001). Among various molecular fingerprinting methods, Listeria spp. PCR-RAPD has been shown to be a rapid, reproducible and powerful In this study site four of the eight Listeria species were recov- genomic typing method for L. monocytogenes (Boerlin et al., 1995; ered, with L. grayi, L. ivanovii, L. rocourtiae and L. marthii not being Cocolin et al., 2005; Farber & Addison, 1994; Fonnesbech Vogel found. Moreover, only four of the positive samples were found to be et al., 2001; Kerr et al., 1995). Comparing with other subtyping isolates of the pathogenic L. monocytogenes (1%). Instead, L. innocua methods, Kerouanton et al. (1998) compared serotyping, electro- (82.3%) was the most prevalent species. This result is in accordance phoretic typing of esterases (zymotyping), restriction fragment with other authors, especially the predominance of L. innocua over length polymorphism of ribosomal DNA (ribotyping), pulsed-field L. monocytogenes and other species (Barros et al., 2007; Capita, gel electrophoresis (PFGE) and RAPD (with 1 primer) in a study Alonso-Calleja, Moreno, & Garcia-Fernandez, 2001). However, designed to adapt a strategy for epidemiologically typing of L. the presence of Listeria spp. is useful in assessing the potential monocytogenes strains. Five serotypes, eight zymotypes, ten ribo- presence of L. monocytogenes in the process plant environment types, 12 PFGE patterns and 13 RAPD patterns were identified (Tompkin, 2002). Rather strong and prompt action should be taken among their 35 strains. Giovannacci et al. (1999) determined the following the isolation of any Listeria spp. origin of pork cuts contamination by L. monocytogenes using RAPD

Table 2 Prevalence (% positive from 7643 screened samples) of L. welshimeri strains on the environmental surfaces in the three zones of the processing line.

Zone Swab surfaces L. welshimeri strains/substrains

LW 1.1 LW 1.5 LW 1.6 LW 1.7 LW 1.8 LW 2 LW 3 1 Gloves of worker at the freezer ee0.6 eeee Gloves of packing worker and QC worker at packing step eee0.6 ee0.6

2 Exhaust pipe of liquid N2 chiller eeeeee0.6 Monitor of dicer controller 0.6 ee0.6 eee Conveyor belt of metal detector and heat sealer 2.3 e 0.6 0.6 0.6 ee

3 Floor at cooking area eeeee1.1 e Wall of the freezer 1.1 ee2.9 0.6 0.6 0.6 Draining pipe of the freezer 1.7 0.6 e 2.9 0.6 e 0.6 Floor at packing area eee0.6 e 1.1 e

For the finished product, only isolate LW 1.5 was found, and in 2/865 cases. 70 S. Keeratipibul, P. Techaruwichit / Food Control 27 (2012) 64e72

Table 3 The contamination of L. innocua LI 1.1 on different environmental surfaces on the day, or close by it, that the finished product contamination was found.

Source of contamination Zone 15/11/09 20/11/09 21/11/09 14/12/09 04/01/10 13/01/10 16/01/10 (4a) (1) (1) (1) (3) (1) (1)

Conveyor belt of in-feed liquid N2 chiller 1 eeeee1 e Exhaust pipe on in-feed liquid N2 chiller 2 1 4 4 4 6 5 4 Tray supporter under the conveyor belt of liquid N2 chiller 2 eeeee1 e Floor of dicing room 3 e 12e 1 e 1 Monitor of dicing controller 2 eeeee1 e Conveyor belt of dicer 1 eeeee1 e Draining pipe of freezer 3 eeee124 Floor at packing area 3 eeeeee1 Conveyor belt of heat sealer 2 eee1 eee Conveyor belt of metal detector 2 eee11e 1 Gloves of worker carrying equipment 1 eeee1 ee

a Number of contaminated finished product found on the day. with five different primers, PFGE and a PCR-restriction enzyme spp. In addition and importantly, no Listeria spp. were detected in analysis (PCR-REA) based on the polymorphism existing within the the product taken after the cooking step. This result is in accor- inlA and inlB genes. Results obtained from RAPD and PFGE were dance with Lundén, Autio, and Korkeala (2002), who reported that closely related and distinguished respectively 17 RAPD types and 17 the sources of contamination came from the processing lines i.e. PFGE types among the 287 isolates, whereas the PCR-REA analysis dicing machine, not from the raw material. Autio, Keto-Timonen, only yielded two profile. Therefore, RAPD method was chosen as Lunden, Björkroth, and Korkeala (2003) and Miettinen, Björkroth, a tool for Listeria investigation in this study. In general, it has been and Korkeala (1999) also reported that they did not find persis- recommended that at least three informative (polymorphic) tent L. monocytogenes (pulsed-field gel electrophoresis typing) in primers be used if RAPD is the sole molecular-typing method raw materials. Miettinen et al. (1999) showed that the packing employed (Kerr et al., 1995). In this study the four informative machine sustained the contamination, and Autio et al. (2003) (polymorphic) primers (OMP-01, HLWL 74, HLWL 85 and UFS) that observed that L. monocytogenes contamination was associated were selected and used in this study have already shown their with processing machines, particularly dicing machines. discriminatory power in other studies (Aguado et al., 2004; Although many potential sources of contamination of L. innocua MacGowan et al., 1993). LI 1.1 were found in this study, the surface that was the most The L. innocua strains in this study contained PCR-RAPD profiles frequently infected was the exhaust pipe of the in-feed liquid N2 that were comprised of largely similar product sizes, but there were chiller (used for cooling the product of 75 C down to about some different bands allowing discriminatory designation of 40e50 C), and this correlated to product contamination. Although different substrains. Of the isolates that were placed in the same located in zone 2, and so is not a product-contact surface, it was substrain, they were analyzed at different times and on different quite near the product and the employee traffic flow around there days, suggesting the reproducibility of this system. In addition to was always congested. Thus, the cross contamination potential was environmentally induced mutations, the genomic heterogeneity of very high. Moreover, the condensate from the exhaust pipe of the Listeria might result from differential selection from environmental liquid N2 chiller frequently dropped on the floor, where it might be stresses, such as the use of sanitizers and/or the change of cleaning carried and distributed to other environmental surfaces by and sanitizing agents, upon a mixed population, or mixed colo- employees working around that area. Although the environmental nizers brought in, for example, by the workers. Buchrieser, Cossart, surfaces in zone 1 (product-contact surface) are the direct route for Kunst, Glaser, and Rusniok (2003) and Lou and Yousef (1997) product contamination, any persistent contamination in zone 2 can indicated that acid sanitizers and ethanol might cause adaptation also easily cause product contamination through zone 1 (e.g. of the organism to endure and survive in the environment. Adap- conveyor belt of in-feed liquid N2 chiller) and workers. Therefore, tive responses may also occur in response to heat and acid stresses more attention should be paid to the non-product contact surfaces factors that are frequently involved in cleaning and sanitizing (zones 2 and 3) as well. treatments (Hill, Driscoll, & Booth, 1995). That L. innocua occurred on the exhaust pipe at in-feed side L. innocua LI 1.1 was found in the cooked chicken meat pro- (product entering side) and not on the out-feed side (product cessing plant at all sampling periods with a persistent contamina- leaving side) of the liquid N2 chiller is likely explained by the tion of the processing line. Continuous contamination of processing difference in their temperatures. The in-feed side (6e10 C) being lines by incoming raw material does not seem probable since the compatible with Listeria growth in contrast to the colder out-feed cooking temperature and time is sufficient to destroy the Listeria side (91 C). The liquid nitrogen chiller exhaust vent is of a non- universal design. The in-feed pipe is very long and installed high up through the ceiling to exhaust the N2 gas out of the factory. Table 4 Additionally, the vent pipe is not straight but is doglegged, bending The occurrence of L. innocua on the exhaust pipe at in-feed side (product incoming at 45 to the perpendicular for about 2.5 m before reverting back to side) and out-feed side (product leaving side) of liquid N chiller. 2 vertical and venting for a considerable height out of the building. In Month Number of contaminated samples/number of analyzed samples addition, some parts cannot be disassembled and so prevents The exhaust pipe The exhaust pipe a considerable difficulty in thoroughly cleaning and sanitizing. Due at in-feed side at out-feed side to the high-speed flow of the exhausted N2 gas, chicken meat January 0/5 1/1 residues accumulated inside the pipe. Since Listeria can grow and February 9/10 0/10 multiply at temperatures as low as 0.4 C(Walker, Archer, & March 3/5 0/5 Banks, 1990), the in-feed side provides ideal conditions for Lis- April 4/5 0/5 teria growth and it is a perfect harborage site (residence) of Listeria. May 7/11 0/5 Although this is not a general design, but a specific problem to this S. Keeratipibul, P. Techaruwichit / Food Control 27 (2012) 64e72 71 meat plant, the general principal is nevertheless important. That is Boerlin, P., Bannerman, E., Ischer, F., Rocourt, J., & Bille, J. (1995). Typing Listeria fi that the design of all aspects of a processing plant, and not just monocytogenes: a comparison of random ampli cation of polymorphic DNA with 5 other methods. Research in Microbiology, 146,35e49. those in zone 1 but also those in zones 2 and 3, should allow for Buchrieser, C., Cossart, P., Kunst, F., Glaser, P., & Rusniok, C. (2003). Comparison of ease of complete sanitization to prevent the development of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for harborage sites. evolution and pathogenicity. FEMS Immunology and Medical Microbiology, 35, 207e213. Considering the contamination of L. welshimeri, substrain LW 1.7 Byun, S. K., Jung, S. C., & Yoo, H. S. (2001). Random amplification of polymorphic was found to be the most prevalent L. welshimeri in the processing DNA typing of Listeria monocytogenes isolated from meat. International Journal environment, but no analyzed product sample was contaminated of Food Microbiology, 69,227e235. Capita, R., Alonso-Calleja, C., Moreno, B., & Garcia-Fernandez, M. C. (2001). Occur- with substrain LW 1.7. Indeed, in all the sampling in this study, only rence of Listeria species in retail poultry meat and comparison of a cultural/ one sample of the finished product was contaminated with immunoassay for their detection. International Journal of Food Microbiology, 65, L. welshimeri, and this was with substrain LW 1.5. Thus, although 75e82. Chambel, L., Manuela, S., Fernandes, I., Barbosa, M., Zilhão, I., Barata, B., et al. (2007). investigation of contamination at molecular level can help reveal Occurrence and persistence of Listeria spp. in the environment of ewe and cow’s the real source of product contamination, and so help in reducing milk cheese dairies in Portugal unveiled by an integrated analysis of identifi- the contamination of the cooked chicken meat, in this study the cation, typing and spatialetemporal mapping along production cycle. Interna- e real source of L. welshimeri LW 1.5 contamination was not found tional Journal of Food Microbiology, 116,52 63. Cocolin, L., Stella, S., Nappi, R., Bozzetta, E., Cantoni, C., & Comi, G. (2005). Analysis of and further investigation is needed. PCR-based methods for characterization of Listeria monocytogenes strains iso- After investigating the potential sources of Listeria contamina- lated from different sources. International Journal of Food Microbiology, 103, e tion in the product, various corrective and preventive actions were 167 178. Commission Regulation (EC) No. 2073/2005. Available from http://ec.europa.eu/ implemented to eradicate the contamination. These included food/food/biosafety/hygienelegislation/legisl_en.htm. dissembling all equipments at the end of the shift, revision of Department of Foreign Trade. (2009). Available from http://www.depthai.go.th/dep/ cleaning and sanitizing procedures (types of chemical and doc/ 53/53003610.doc. Department of Livestock Development. (2010). Microbiological criteria of livestock frequency of cleaning and sanitizing), and for those parts of products for export [online]. Available from http://www.dld.go.th/certify/th/index. equipment which could not be dissembled, then subjected to steam php? option¼com_content&view¼article&id¼609:law&catid¼118:docuqmentary. heating at 75 C for 15 min. After implementing the new procedure, Eklund, M. W., Poysky, F. T., Paranjpye, R. N., Lashbrook, L. C., Peterson, M. E., & fi Pelroy, G. A. (1995). Incidence and sources of Listeria monocytogenes in cold- the prevalence of Listeria spp. in the nished product and pro- smoked fishery products and processing plants. Journal of Food Protection, 58, cessing environment were decreased. The contamination of Listeria 502e508. spp. in the finished product decreased some eight-fold (from 1.6% Farber, J. M., & Addison, C. J. (1994). RAPD typing for distinguishing species and fi strains in the genus Listeria. Journal of Applied Bacteriology, 77, 242e250. down to 0.2%). However, the Listeria strain found in the nal Fonnesbech Vogel, B., Jørgensen, L. V., Ojeniyi, B., Huss, H. 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