2022 Volume 28 Issue 2 Pages 141-149
In this study, the occurrence, serogroups, virulence genes, antibiotic susceptibility profiles, and diversity were estimated for Listeria monocytogenes isolated from Pangasius fish in two processing facilities in Vietnam. L. monocytogenes was most frequently detected in wash water samples at 20.8% (15 out of 72), and in Pangasius fish it was 16.6% (15 out of 90 fish samples). Serotypes 1/2b were dominant, and internalin genes (inlA, inlC, and inlJ) were found in all the L. monocytogenes isolates. Most of the isolates were resistant to cefoxitin, oxacillin, and fosfomycin. Genotyping of L. monocytogenes by random amplified polymorphic DNA analysis revealed three clusters of the isolates specific to the facilities to some extent. This study suggests a high risk of L. monocytogenes contamination from wash water to fish. Hence, maintaining the quality of water and designing sanitation procedures to reduce L. monocytogenes contamination are required to ensure food safety at Pangasius fish processing plants.
Listeria monocytogenes is a gram-positive facultative anaerobic bacterium that inhabits a wide range of environments, including food materials and food contact surfaces (Lungu et al., 2009). L. monocytogenes causes listeriosis, which is a serious disease in humans with exceptionally high risks for fetuses, immunocompromised patients, and the elderly (Hernandez-Milian and Payeras-Cifre, 2014). Infection from L. monocytogenes often occurs through the ingestion of contaminated foods such as milk products, meat products, raw (undercooked) fish, and vegetables (Ramaswamy et al., 2007; Sant'Ana et al., 2014). In general, fish is usually contaminated with L. monocytogenes by spreading from the intestinal contents to fish muscles and/or cross-contamination due to the unhygienic handling of fish (Jami et al., 2014). Inadequate cleaning and disinfection procedures promote a high risk of L. monocytogenes contamination from the processing environment to fish products. Hence, one prevention measure to control L. monocytogenes is to revise the facility environment to make it less bacteria friendly.
Pangasius production in Vietnam has been estimated at approximately 1.4 million tons, making Vietnam the largest producer and exporter of Pangasius in the global marketi). The Mekong Delta is the leading cultural region of Pangasius, accounting for over 75% of the total national productionii). During the grow-out stage, large amounts of antimicrobial agents are used to control diseases and increase production (Rico et al., 2013). So far, there are more than 20 antimicrobials, mainly β-lactams, phenicols, quinolones, and sulfonamides, authorized for use in Pangasius aquaculture in Vietnam (Phu et al., 2015). Treatment failure can occur when farmers use their own medicated feed, which can result in antimicrobial resistance (Pham et al., 2015). A previous study investigated antibiotic resistance of pathogens such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella at Pangasius farms and in frozen Pangasius products (Boss et al., 2016). Despite L. monocytogenes being the most frequently isolated pathogen, there are no studies characterizing L. monocytogenes strains isolated from Vietnamese Pangasius products throughout the processing system. Hence, in this study, we estimated the occurrence, antibacterial resistance, pathogenicity, and diversity of L. monocytogenes strains from fish and environmental samples in Pangasius processing plants.
Media for L. monocytogenes isolation Demi-Fraser broth (Merck) and Fraser broth (Merck, Darmstadt, Germany) were prepared by adding the Fraser Listeria selective supplement (1.00093.0010; Merck) and Fraser Listeria Ammonium iron (III) (1.00092.0010; Merck). Listeria agar (base) acc. OTTAVIANI and AGOSTI (Merck) was mixed with Chromocult Listeria agar selective supplement (1.00432.0010; Merck) and Chromocult Listeria agar enrichment supplement (1.00439.0010; Merck).
Sample collection All samples were collected from two Pangasius fish processing facilities in the Vietnamese Mekong Delta (VMD) from September 2018 to January 2019. A flow diagram of the processing line and sampling locations is shown in Fig. 1. Within each processing line, different types of samples were collected at 16 sampling locations in 3 consecutive weeks (n = 16 × 3). Overall, a total of 288 samples including Pangasius fish (90 samples), water (72 samples), food contact surfaces (72 samples), and hand/gloves (54 samples) were collected at the two facilities. The raw fish and fillets were randomly selected with sterile tweezers and placed in separate sterile Stomacher® bags (Interscience, Île-de-France, France). Environmental samples, including food contact surfaces and hands/gloves, were collected by swabbing vertically, horizontally, and diagonally on a 100 cm2 surface with sterile-premoistened swabs (Merck), and the swab was then placed in 5 mL Demi-Fraser broth. Approximately 500 mL of potable water that was used to wash the fillets were collected from the washing tanks in separate sterile Stomacher® bags. All samples were stored in a cool box with ice and frozen gel packs and transported to the Laboratory of Microbiology and Biotechnology (Can Tho University, Can Tho, Vietnam) for enrichment and isolation of L. monocytogenes within 24 h of sampling. Strains were analyzed at the Laboratory of Food Hygienic Chemistry at Kyushu University (Fukuoka, Japan).
The fish processing system and sampling locations at two Pangasius plants, A and B. The processing steps are shown in italics for plant B.
Isolation and identification of L. monocytogenes Fish samples (25 g) were aseptically removed from different parts of each fillet using sterile scalpels and tweezers and placed in a sterile Stomacher® bag containing 225 mL of Demi-Fraser broth. For water samples, 1 mL was transferred to 4 mL of Demi-Fraser broth in sterile tubes. The fish, water, and swab samples in Demi-Fraser broth were pre-enriched by incubation for 24 h at 30 °C. Afterward, 0.1 mL was then inoculated into 10 mL of Fraser broth and incubated for 48 h at 37 °C. This culture was then streaked on Listeria agar and incubated for 48 h at 37 °C. A typical colony of L. monocytogenes on Listeria agar showed a green-blue color, surrounded by an opaque halo. The presumptive colonies of L. monocytogenes were selected and tested using PCR. The primer pair used was LM1 and LM2 to amplify a 702 bp DNA fragment of the hlyA gene (Aznar and Alarcón, 2003). L. monocytogenes 687 (1/2a, No. 174) and 689 (4b, No. 185) were used as reference strains in this study (Liu et al., 2012). The strains were named as follows: name of the processing plant - identification number - time of sampling (V1: visit 1, V2: visit 2, or V3: visit 3, respectively).
Serotyping of L. monocytogenes The strains were serotyped using L. monocytogenes serotyping antisera (Denka Seiken, Tokyo, Japan) according to the manufacturer's instructions.
Detection of virulence genes in L. monocytogenes Multiplex PCR was performed to detect L. monocytogenes internalin genes (inl), namely inlA, inlC, and inlJ, as previously described by Liu et al. (2007). Standard PCR was conducted to detect the inlB gene in all strains according to the methods of Jamali and Thong (2014).
Antibiotic susceptibility tests The L. monocytogenes strains from fish processing plants were subjected to antimicrobial susceptibility tests following the broth microdilution and disc diffusion method as described by CLSI (2017). For the broth microdilution method, 96-well DP 32 dry plates (Eiken Chemical Co., Tokyo, Japan) were used to test susceptibility to oxacillin, ampicillin, cefazolin, cefmetazole, flomoxef, imipenem, gentamycin, arbekacin, minocycline, cefoxitin, erythromycin, clindamycin, vancomycin, teicoplanin, linezolid, fosfomycin, sulfamethoxazole/trimethoprim, and levofloxacin. The minimum inhibitory concentrations (MICs) for 50% and 90% (MIC50 and MIC90) of the L. monocytogenes strains were calculated. The results were interpreted according to CLSI (2017) as shown in Table 2. For the disc diffusion method, the sensitivities of L. monocytogenes strains to tetracycline (30 µg), chloramphenicol (30 µg), and ciprofloxacin (5 µg) were investigated, as they are widely used for the treatment of diseases in fish and pond water. The diameter of the inhibitory zone was measured and interpreted according to the zone diameter breakpoints for Staphylococcus species. Against tetracycline, chloramphenicol, and ciprofloxacin, zone diameters of ≥ 19, 18, and 21 mm were classified as susceptible, 15–18, 13–17, and 16–20 mm were intermediate, and ≤ 14, 12, and 15 mm were resistant, respectively (CLSI, 2017).
Genotyping of L. monocytogenes strains using Random Amplified of Polymorphic DNA (RAPD) RAPD typing was performed on L. monocytogenes strains using the primer HLW85 (5′-ACAACTGCTC-3′) (Hansen et al., 2006). Genomic DNA was extracted from the strains using Cica Geneus DNA Extraction Reagent (Kanto Chemical Co., Inc., Tokyo, Japan) according to the manufacturer's instructions. The quality of the DNA extract was determined using a Nanodrop ND 1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). To be considered as high-quality genomic DNA, the ratio of the absorbance at 260 and 280 nm is approximately 1.8. Afterward, the PCR program consisted of an initial denaturation step of 94 °C for 2 min, followed by 45 amplification cycles of 1 min at 95 °C, 2 min at 35 °C, and 1 min at 72 °C, and a final extension of 72 °C for 10 min. The PCR products were electrophoresed and visualized by staining with the Midori Green Advance DNA stain (Nippon Genetics Europe GmbH, Germany). Dendrograms were drawn with Bionumerics software (Applied Maths, Sint-Martens-Latem, Belgium) by means of Dice correlation and cluster analysis using the unweighted pair group method (UPGM).
The occurrence of L. monocytogenes in Pangasius fish and environmental samples The results in Table 1 show that the incidence of L. monocytogenes was estimated at 15.6% (45 out of 288) among the 288 samples of Pangasius fish and environmental samples from two processing plants, A and B. Concerning the type of sample, L. monocytogenes was most frequently detected in 20.8% of wash water samples (15 out of 72 samples). Regarding each facility, 24 out of 144 samples (16.6%) were positive for L. monocytogenes in plant A, while 21 out of 144 samples (14.5%) were positive in plant B. Concerning sampling time, L. monocytogenes was found at various sampling locations over the three visits in plant A. However, this pathogen was highly detected at visit 1 in plant B but decreased at visits 2 and 3 after the trimming process. Our results showed that contamination of the raw material was maintained at a low percentage, whereas the incidence in end products was greatly higher. This reveals that these end products were contaminated with L. monocytogenes during the processing. The contamination seems to have occurred during the washing process, since L. monocytogenes was most frequently detected from wash water samples. These samples were taken from the washing tanks containing potable water and/or chlorine water at a certain concentration at steps of bleeding, washing 1, washing 2, and glazing.
| SL | Type of sample | Processing step | No. of samples | No. of positive samples | Total | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Visit 1 | Visit 2 | Visit 3 | Visit 1 | Visit 2 | Visit 3 | No. of samples | No. of positive samples | % positive samples | |||||||||
| A | B | A | B | A | B | A | B | A | B | A | B | ||||||
| SL1 | Fish | Raw | 3 | 3 | 3 | 3 | 3 | 3 | 1 | 90 | 15 | 16.6% | |||||
| SL3 | Filleting | 3 | 3 | 3 | 3 | 3 | 3 | ||||||||||
| SL7 | Trimming | 3 | 3 | 3 | 3 | 3 | 3 | 2 | 1 | 1 | |||||||
| SL12 | Cooling | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 2 | 1 | |||||||
| SL14 | Packaging | 3 | 3 | 3 | 3 | 3 | 3 | 1 | 2 | 1 | |||||||
| SL4 | Hands/gloves | Filleting | 3 | 3 | 3 | 3 | 3 | 3 | 1 | 54 | 6 | 11.1% | |||||
| SL8 | Trimming | 3 | 3 | 3 | 3 | 3 | 3 | 2 | 1 | ||||||||
| SL15 | Packaging | 3 | 3 | 3 | 3 | 3 | 3 | 2 | |||||||||
| SL6 | Food contact surfaces | Skinning | 3 | 3 | 3 | 3 | 3 | 3 | 72 | 9 | 12.5% | ||||||
| SL9 | Trimming | 3 | 3 | 3 | 3 | 3 | 3 | 1 | 2 | ||||||||
| SL11 | Washing 2 | 3 | 3 | 3 | 3 | 3 | 3 | 1 | 1 | 3 | |||||||
| SL16 | Packaging | 3 | 3 | 3 | 3 | 3 | 3 | 1 | |||||||||
| SL2 | Water | Bleeding | 3 | 3 | 3 | 3 | 3 | 3 | 2 | 1 | 72 | 15 | 20.8% | ||||
| SL5 | Washing 1 | 3 | 3 | 3 | 3 | 3 | 3 | 1 | |||||||||
| SL10 | Washing 2 | 3 | 3 | 3 | 3 | 3 | 3 | 2 | 3 | 1 | 3 | ||||||
| SL13 | Glazing | 3 | 3 | 3 | 3 | 3 | 3 | 1 | 1 | ||||||||
| Total | 48 | 48 | 48 | 48 | 48 | 48 | 7 | 13 | 6 | 4 | 11 | 4 | 288 | 45 | 15.6% | ||
| Antimicrobial agents | MIC range (µg/ml) | MIC Breakpoints (µg/ml) | MIC50 (µg/ml) | MIC90(µg/ml) | ||||
|---|---|---|---|---|---|---|---|---|
| S | I | R | A | B | A | B | ||
| Oxacillin | 0.12–4 | ≤ 2 | - | ≥ 4 | 4 | 4 | 4 | 4 |
| Ampicillin | 0.12–16 | ≤ 4 | - | ≥ 16 | 0.5 | 0.5 | 0.5 | 0.5 |
| Cefazolin | 0.5–16 | ≤ 8 | - | ≥ 32 | 2 | 4 | 4 | 4 |
| Cefmetazole | 1–32 | ≤16 | 32 | ≥ 64 | 16 | 32 | 32 | 32 |
| Flomoxef | 0.5–16 | ≤ 8 | > 8 | 8 | 8 | 8 | 8 | |
| Imipenem | 0.25–8 | ≤ 4 | 8 | ≥ 16 | 0.25 | 0.25 | 0.25 | 0.25 |
| Gentamycin | 0.25–8 | ≤ 4 | 8 | ≥ 16 | 0.25 | 0.25 | 0.25 | 0.25 |
| Arbekacin | 0.25–8 | - | - | ≥ 4 | 0.25 | 0.25 | 0.25 | 0.25 |
| Minocycline | 2–8 | ≤ 4 | 8 | ≥ 16 | 2 | 2 | 2 | 2 |
| Cefoxitin | 4–16 | ≤ 4 | - | ≥ 8 | >16 | >16 | >16 | >16 |
| Erythomycin | 0.12–4 | ≤ 0.5 | 1–4 | ≥ 8 | 0.12 | 0.12 | 0.12 | 0.12 |
| Clindamycin | 0.06–2 | ≤ 0.5 | 1–2 | ≥ 4 | 1 | 1 | 1 | 1 |
| Vancomycin | 0.5–16 | ≤ 2 | 4–8 | ≥ 16 | 0.5 | 0.5 | 0.5 | 0.5 |
| Teicoplanin | 0.5–16 | ≤ 8 | 16 | ≥ 32 | 0.5 | 0.5 | 0.5 | 0.5 |
| Linezolid | 0.25–8 | ≤ 4 | - | ≥ 8 | 2 | 2 | 2 | 2 |
| Fosfomycin | 32–128 | ≤ 32 | - | ≥ 32 | >128 | >128 | >128 | >128 |
| Sulfomethoxazole-Trimethoprim | 9.5/0.5–38/2 | ≤ 9.5/0.5 | 19/1 – 38/2 | 76/4 | 0.5 | 0.5 | 0.5 | 0.5 |
| Levofloxacin | 0.25–4 | ≤ 1 | 2 | ≥ 4 | 1 | 1 | 1 | 1 |
S, Susceptible
I, Intermediate
R, Resistant
Bleeding is a step at which the raw fish is manually cut at the main artery from the heart to the gills, then dipped in a potable water tank for 30 min before being transferred to the subsequent stages of processing. Washing 1 and washing 2 are steps at which fillets are dipped in an aerated chlorine water tank (with 50 mg/L available chlorine) for about 2–3 min at 22 °C and 16 °C, respectively. Glazing is a step at which the frozen fillets are dipped into a potable water tank at 1–5 °C for 5–10 s. The wash water is renewed every 2–3 h (after washing 4–5 tons of fillets), and the washing tanks are cleaned after 18–24 h. During the washing steps, the levels of bacterial contamination in the wash water gradually increase as a function of time due to the accumulation of bacteria and large amounts of nutrients from suspended organic matter (Ragaert et al., 2007). In this way, there is a high probability of transmission of microorganisms from wash water into products (Allende et al., 2008; Chen et al., 2010), despite having a water monitoring program at these processing facilities for water source reception, distribution, and storage within the factory to ensure compliance with standards for potable water. Moreover, the effectiveness of decontamination in Pangasius fish facilities was not expected due to the improper use of chlorine; chlorine was only added at the beginning of the batch without readjustment of available chlorine concentration (Tong Thi et al., 2015). As a consequence, the end products pose a high risk because of cross-contamination of pathogens, such as L. monocytogenes. Similar studies also indicated that 15% and 40% of Pangasius fish were contaminated with L. monocytogenes in two Pangasius processing facilities in VMD (Noseda et al., 2013; Ngoc et al., 2020). Although Vietnamese regulations (TCVN) require the absence of L. monocytogenes in 25 g of Pangasius fish (TCVN, 2010), the ongoing situation is still alarming. So far, the plants have improved their food safety management system to control and ensure the safety of output. In the two plants, controlling L. monocytogenes in the wash water is an important factor in reducing the hazard of cross-contamination.
Characterization of L. monocytogenes Fig. 3 shows a dendrogram generated based on RAPD analysis of L. monocytogenes isolates from two Pangasius processing plants, A and B, and the source, processing step, serotype, virulence genes, and resistance profile of the isolates. All of the 45 L. monocytogenes isolates were positive for 3 out of 4 genes related to internalin (inlA, inlC, and inlJ), suggesting that these strains could be virulent (Fig. 3). Similar observations in previous studies showed the presence of the tested internalin genes in almost all of the L. monocytogenes strains from an open-air fish market (Jamali et al., 2015), clinical samples, and different kinds of foods (Pournajaf et al., 2016). Among these internalin genes, inlJ in particular has emerged as an excellent target for determining L. monocytogenes virulence (Liu, 2006). As shown in Fig. 3, the predominant serotypes of L. monocytogenes strains from the two facilities were 1/2b (40%), 3b (15.6%), 4b (22.2%), and 4c (13.4%), which is consistent with results observed for Turkish seafood products (Siriken et al., 2013) and fresh aquatic products in China (Chen et al., 2018). There is no information on the serogroups of L. monocytogenes from sporadic cases of listeriosis; hence, it is difficult to determine which serogroups are linked to listeriosis in Vietnam. Further investigations should be conducted to better understand the specificity of serogroups of L. monocytogenes strains in different types of food in Vietnam.
Antibiotic susceptibility profile The MIC values for L. monocytogenes strains from plants A and B are listed in Table 2. The antimicrobial susceptibility of 45 strains to 21 antibacterial agents is shown in Fig. 2. All of the isolates were susceptible to 13 antimicrobials, including ampicillin, cefazolin, imipenem, gentamycin, arbekacin, minocycline, erythromycin, teicoplanin, linezolid, sulfamethoxazole-trimethoprim, tetracycline, chloramphenicol, and ciprofloxacin. The MIC50 and MIC90 values of antimicrobials determined for the strains from plant A were similar to those for the strains from plant B (Table 2). As shown in Fig. 2, cefoxitin resistance was detected in 100% of L. monocytogenes strains from both plants A and B. The strains from plant A were resistant to fosfomycin (96%), oxacillin (83%), and flomoxef (8%), and the strains from plant B showed resistance to oxacillin (100%), fosfomycin (100%), flomoxef (33%), and levofloxacin (5%). Most of the L. monocytogenes isolates from both plants were resistant to cefoxitin, oxacillin, and fosfomycin. In fact, it has been shown that L. monocytogenes is intrinsically resistant to these antibiotics (Troxler et al., 2000; Krawczyk-Balska and Markiewicz, 2016). These antibiotics act by inhibiting the synthesis of the peptidoglycan that forms the cell walls of bacteria (Bennett et al., 2015). The mechanisms of antimicrobial resistance include: (1) the production of enzymes inactivating the antibiotics, (2) alteration of existing proteins targets, and (3) the reduction of cell membrane permeability to antibiotics (King et al., 2014). In Pangasius fish farming, a high resistance level to tetracycline (60.9%), ampicillin (69.6%), trimethoprim-sulfamethoxazole (60.9%), and chloramphenicol (32.6%) has been observed for gram-negative bacteria (Sarter et al., 2007). So far, there is a lack of available information on the antibiotic-resistance profiles of L. monocytogenes or other gram-positive bacteria isolated from the aquaculture regions of Pangasius fish.
Antibiotic resistance profile including, susceptibility ( □ ), intermediate ( ■ ), and resistance ( ■ ) of Listeria monocytogenes isolates from Pangasius fish processing plants A, and B to antimicrobials. MPIPC, Oxacillin; ABPC, Ampicillin; CEZ, Cefazolin; CMZ, Cefmetazole; FMOX, Flomoxef; IPM, Imipenem; GM, Gentamycin; ABK, Arbekacin; MINO, Minocycline; CFX, Cefoxitin; EM, Erythromycin; CLDM, Clindamycin; VCM, Vancomycin; TEIC, Teicoplanin; LZD, Linezolid; FOM, Fosfomycin; ST, Sulfomethoxazole-Trimethoprim; LVFX, Levofloxacin; CIP, Ciprofloxacin; TET, Tetracycline; CHL, Chloramphenicol.
Regarding multiple drug resistance, 82.2% (37 out of 45 strains) of L. monocytogenes isolates in the present study were multidrug-resistant strains (Fig. 3). Antimicrobial resistance profiles revealed that the strains showed five resistance patterns in total: (1) cefoxitin; (2) cefoxitin and fosfomycin; (3) oxacillin, cefoxitin, and fosfomycin; (4) oxacillin, flomoxef, cefoxitin, and fosfomycin; and (5) oxacillin, cefoxitin, fosfomycin, and levofloxacin (Fig. 3). The ratios appear to be similar to those of strains from chickens in Japan (Maung et al., 2019) and livestock and poultry meat in China (Chen et al., 2015). However, the multidrug resistance rate of L. monocytogenes strains from Pangasius in the VMD is lower than that of other kinds of food in some countries, e.g., 100% resistance in strains from salad vegetables in Nigeria (Ieren et al., 2013) and 100% from milk and soft cheese in Egypt (Alashmawy et al., 2014). Even though L. monocytogenes is known to be susceptible to various antibiotics, the presence of multidrug-resistant strains is worrisome. Therefore, monitoring and reducing the use of antimicrobial agents is crucial to better control antibiotic resistance.
Genetic diversity The results of the RAPD analysis revealing the genetic correlation among the strains are shown in Fig. 3. The strains were divided into three different clusters. The majority of strains isolated from plant B were present in cluster I, while most of the strains from plant A were in cluster III, suggesting the settlement of specific L. monocytogenes strains for each facility. The isolates not classified into clusters I and III were classified into cluster II, suggesting a certain genetic similarity among these strains. Moreover, the results showed that despite the different times of sampling, the strains were grouped in the same cluster by RAPD. These antimicrobial-resistance profiles were widely distributed in clusters I, II, and III. Additionally, the strains within the same cluster did not always show the same serotype or antimicrobial-resistance profile. Interestingly, in cluster II, two strains from different plants, A-65-V3 and B-111-V2, also showed the same patterns of serogroup, inl genes, antimicrobial-resistance profiles, and the same fingerprint, indicating the spread of some L. monocytogenes strains through VMD. Considering the location, plants A and B are located on the two main branches of the Mekong River in the VMD at a distance of 70 km. A dense network of canal rivers originating from the two central rivers could facilitate mutual contamination. In cluster I, two strains, B-77-V1 and B-70-V1, showed the same pattern, suggesting genetic similarity between the two strains. However, these strains, which were isolated from very close processing steps, had different antimicrobial-resistance profiles. The result might be key for elucidating the genetic diversity of L. monocytogenes, and additional studies should be performed to clarify the genetic diversity of L. monocytogenes strains isolated from the facilities and in VMD.
In conclusion, in addition to the presence of L. monocytogenes in the final product, potential virulent and epidemiologically important serogroups from these strains could also indicate trouble for human health. How to control the quality of water as well as to avoid cross-contamination during processing are fundamental aspects that the Pangasius facilities have to cope with to ensure the safety of their products. Moreover, further studies are vital to identify the spreading route of L. monocytogenes in fish processing plants so as to implement effective hygiene plans.
A dendrogram generated based on random amplification of polymorphic DNA (RAPD) analysis of 45 L. monocytogenes isolates from two Pangasius processing plants A and B, and source, processing step, serotype, virulence genes, and resistant profile of the isolates. Virulence genes: inlA - C - J, inlA – inlC – inlJ; inl A - B - C - J, inlA – inl B – inlC – inlJ. Antimicrobials: CFX, Cefoxitin; FMOX, Flomoxef; FOM, Fosfomycin; LVFX, Levofloxacin; MPIPC, Oxacillin.
Acknowledgements This work was supported by the Can Tho University Improvement Project VN14-P6, and supported by a Japanese ODA loan.
Conflict of interest There are no conflicts of interest to declare.
