Prevalence and Antibiotic Resistance of Listeria Monocytogenes in Chicken Meat Retailers in Yazd, Iran

ORIGINAL ARTICLE Introduction: Listeria monocytogenes is a pathogen bacteria transmitted by food stuffs. Due to the lack of information about contamination of retail chicken meat to Listeria monocytogenes in Iran, this study aimed to determine the prevalence and antibiotic resistance of this bacterium in retail chicken meat in Yazd, Iran. Materials and Methods: This cross-sectional descriptive study was conducted from January 2018 to June 2018 on 811 randomly selected samples from four districts located in Yazd city, Iran. Isolation of Listeria monocytogenes was performed using enrichment and selective culture media as well as biochemical tests. The positive samples were confirmed by PCR assay. Results: In total, 247 samples (30.5%) were infected with at least one of the Listeria spp. Among the 247 samples, the isolates were 68 (27%) L. monocytogenes, 155 (63%) L. innocua, 5 (2%) L. seeligeri, 19 (8%) L. ivanovii. In current study, the antibiotic resistance of positive samples was also evaluated that especially Listeria monocytogenes were resistant to tetracycline and penicillin. Conclusions: The presence of this pathogenic microorganism in chicken meat can be a health risk, especially for pregnant women, the elderly, and those with immune deficiency. Considering the amount of contamination with L. monocytogenes in chicken and the high mortality rate caused by it, monitoring of the health principles and standards during the production, transportation, and storage as well as training of employees in this industry are necessary. Article History: Received: 16 September 2019 Accepted: 20 November 2019


Introduction
Listeria is a genus of bacteria with different strains found widely throughout the natural environment, responsible for the contamination of numerous food products. Among these, L. monocytogenes as gram-positive intracellular zoonotic pathogen present in the food industry, can cause listeriosis 1 in human. The highest mortality rate resulting by the foodborne pathogens (about 17%) has been observed in relation to this bacterial infection 2 . The bacteria can exist and survive in all conditions, including low temperatures, different pH values, high concentrations of salt or bile, and oxidative stress. Regarding previous studies, some of foodborne diseases caused by contaminated meat and meat products are related to L. monocytogenes [3][4] . Accordingly, one of the major problems in the food chain is due to poor management of this widely distributed bacterium which is compatible with various conditions 5 .
It is obvious that use of poultry meat and its products have been accounted for many human dietary programs around the world because of highquality nutrients such as protein, vitamins, and minerals 6 . Hence, these foods are highly desirable, palatable and digestible, as well as inexpensive rather than other types of meat like beef and mutton. Moreover, the consumption of beef, sheep and poultry meat per Capita has increased nowadays 7 . The poultry industry is a common place that Listeria spp. can infect the chicken-meat and products. Listeria contamination of poultry carcasses can occur in poultry slaughterhouses and processing plants during various stages, including improper cleaning and disinfecting of environment and equipment, as well as unsanitary transportation of the resulting products 8 . The environment, workers, surfaces, and equipment are possible resources to infect prepared chicken products after cooking 9 .
Along with the mentioned problems, globally antimicrobial and multidrug resistance among the pathogens is other serious troublemakers for human beings. The major reason for the current study is the abuse and overuse of antimicrobial agents that may result in the spread of antimicrobial-resistant pathogens, including Listeria spp., despite the effect of these agents to control the infections. Public health consequences in human population can be happened due to the spread of bacterial resistant to antimicrobial agents in contaminated food products 10 . According to our knowledge, no investigations have been conducted on Listeria spp. contamination in retail poultry meat concerning the prevalence and antibiotic susceptibility in the city of Yazd, Iran. Hence, the main purposes of the current study were to isolate and identify Listeria spp. from the chicken as well as to determine antibiotic resistance patterns of the isolates.

Sample collection
This cross-sectional descriptive study was conducted from January to June 2018 on 811 randomly selected samples in Yazd city among the most popular and the busiest stores. The samples were taken separately from the stainless steel table (n = 30), poultry moving carts (n = 100), sliced carcasses (n = 170, 85 thighs and 85 chests), packed chicken (n = 100), gloves (n = 60), workers' hands (n = 30), apron (n = 30), knife (n = 45), chicken liver (n = 100), gizzard (n = 90), refrigerator with temperature of 0-4°C (n = 56). All swab specimens were stored in Falcon tubes containing 1 ml of 0.1% peptone water, as well as the meat, liver and viscera samples (25 g) in sterile containers. All samples were transported to the laboratory in the cold box.

Isolation and identification of Listeria spp.
Guidelines proposed by the United States Department of Agriculture (USDA) were applied for isolation, so that 25 g of the sample was aseptically added to 225 ml of University of Vermont-Modified Listeria enrichment broth (UVM I enrichment medium), mixed with a stomacher device, and incubated at 37°C for 24 hours. After 24 hours, 1 ml of the initial enrichment medium was transferred to 9 ml of UVM II medium and incubated at 37°C for 24 hours, and then cultured on palcam agar medium and incubated at 25°C for 48 hours. Three suspicious colonies (black and deep) were cultured on tryptone soy agar with 0.6% yeast extract and incubated at 37°C for 24 hours [11][12] . The isolated Listeria species were identified through biochemical tests, including Gram staining, catalase, mobility at 25°C and 37°C, fermentation of glucose, maltose, rhamnose, xylose and mannitol, esculin reduction, nitrate reduction, beta hemolysis, MR/VP, and CAMP test 13 .

PCR assays
The isolated bacteria were cultured in Brain Heart Infusion (BHI) medium and incubated at 37°C for 18 hours 14 . The extraction was performed by the boiling method, and then the DNA was extracted. The primers for Listeria genus were related to the prs gene to identify all Listeria species, including forwarding 01 (5′-GCTGAAGAGATTGCGAAAGAAG-3′) and reverse 02 (5′-CAAAGAAACCTTGGATTTGC GG-3′) to amplify 370-pb DNA fragment. The oligonucleotide primers released from hly gene for Listeria monocytogenes consisted of DG69 (5'-GTGCCGCCAAGAAAAGGTTA-3') and DG74 (5'-CGCCACACTTGAGATAT-3') to amplify 636-pb DNA fragment. The PCR mixture was used for each sample after optimization of its component concentrations, including the primers, distilled water, Master Mix and Template DNA in 25 μl for each PCR reaction. The mixture was placed in a thermal cycle for gene amplification. Subsequently, the PCR products were electrophoresed on 1.5% agarose gel [15][16] .
Then, each Listeria monocytogenesis strain was cultured as scratch method and the disks were placed onto each plate (5 antibiotic disks per plate) and incubated at 35°C for 24 hours. The inhibition zone diameter was measured. The findings were interpreted according to the criteria provided by the CLSI (Clinical and Laboratory Standards  Institute) table.
Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922 were used as quality control organisms in the determination of antibiotic susceptibility.

Statistical analysis
The data were analyzed by SPSS 18 software using descriptive statistics and Chi-square test to compare the prevalence of contamination and bacterial resistance.
In current study, all Listeria isolates were tested for resistance to 12 different antibiotics by the disk diffusion method. Most isolates were resistant to tetracycline and penicillin. The bacterial resistance to various antibiotics is shown in Table 2. The results obtained from the present study indicated high resistance of L. monocytogenes and L. innocua to tetracycline and penicillin. Among all samples, 73 samples (29.5%) were resistant to one antibiotic, 76 samples (30.7%) to two antibiotics and 80 samples (32.3%) to more than two antibiotics. Among all samples, 10 (4%) and 15 (6%) samples had the minimum resistance to vancomycin and erythromycin, respectively.

Discussion
Poultry meat centers are environments with suitable temperature and humidity for the growth of Listeria bacteria. Therefore, the bacterium can remain in the environment for few months or even years, which leads to food contamination. Sustainability of Listeria in the environment is due to its ability to create a biofilm on different levels. The first comprehensive study on the prevalence of listeria in Iran was reported by Jalali and Abedi in Isfahan. In their study, from the total 617 samples achieved from different foods, 4.6% had Listeria. The infection rate in fresh poultry meat was 4.5% and all positive samples were related to Listeria innocua; none of the samples were contaminated with Listeria monocytogenes 19 . Fallah et al. reported a prevalence of 34% for raw poultry meat at the supply level in center of Iran 20 . The amounts of contamination in current study for Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri were 14.1%, 16.1%, 2.51%, and 2.01%, respectively. Jamali et al. also reported that the prevalence of Listeria in the chicken retail centers of Tehran was 16.1%. The infection rates related to L. ivanovii, L. monocytogenes, L. innocua, and L. seeligeri were respectively 9.8%, 3.6%, 2.1%, and 0.7%. In another study conducted in 5 large cities of Iran 21 , the mean prevalence of Listeria in meat retail centers was 15.8%. In present research, the prevalence of Listeria in Yazd was 17.7%. The prevalence of L. monocytogenes, L. innocua, and L. welshimeri was reported as 2.1%, 14.6%, and 1%, respectively. The prevalence of Listeria achieved from the current study was higher than the previous studies carried out in Iran, but it was consistent with the results reported from different countries; 34% in Canada 22 , 28% in Japan 23 , and 32.7% in Turkey 24 .
In order to compare the results of the present study with those of other studies, the country, the separation technique, and the investigated type of tissue should be considered. The actual incidence of L. monocytogenes is generally higher than the reported results through the traditional isolation method, which relies on selective enrichment medium (UVM) and Fraser broth to purify L. monocytogenes. At temperatures below 40 ° C, L. innocua grows faster than L. monocytogenes and this is one of the reasons for higher prevalence of L. innocua than L. monocytogenes which was reported in the current study because the conventional enrichment method is more in favor of L. innocua spp. growth. In previous studies, L. innocua was more prevalent than the other species [20][21][25][26][27] . The prevalence of L. monocytogenes in the northern European countries was 22% 28 , in Italy 21.8% 26 , and in Sri Lanka was 34% 29 that is compatible with the current results.
In Loura et al. research, the chicken liver samples were investigated and 10% and 20% of infections were reported by L. innocua and L. grayi, respectively. None of the samples were contaminated with L. monocytogenes 30 . A study conducted in Bulgaria showed that from 112 samples of goose liver, 35.7% were contaminated with L. monocytogenes 31 . The prevalence of L. monocytogenes in the chicken liver in Iran was 21.6 20 , in Brazil 23.3% 32 , in Malaysia 25% 33 , which was consistent with the results achieved form the current study. Chicken's liver and gizzard are not contaminated during slaughter; their most important source of contamination is after slaughter and in processing by cross-contamination between other parts of the poultry and table, equipment, and the work environment [33][34] .
Laura et al. found that the knife used to cut chicken was 80% and 50% contaminated with L. monocytogenes and L. innocua, respectively, while no contamination with L. monocytogenes was observed in workers' gloves 30 . Barbalho et al. 35 reported that contamination of workers' hands and gloves by L. monocytogenes and L. innocua, were 11.8% and 40.5%, respectively which are consistent with the current study. Kerr 36 and Yuewei 37 found 11.8% of contamination with L. monocytogenes in workers' hands and gloves that were in line with the current results. High contamination in equipment shows that conventional disinfection methods are inefficient 30 .
Studies represent that the antibiotic resistance of L. monocytogenes isolated from foods is increasing  Vol (4), Issue (4), December 2019, 895-902 Jehsd.ssu.ac.ir 900 worldwide 38 . In a study on chicken meat offered in Ankara, Turkey 27 , L. monocytogenes, L. welshimeri, and L. innocua species were very sensitive to chloramphenicol (88-100%), but they showed resistance to ampicillin (66-100%). In present study, the bacteria were most resistance to tetracycline and penicillin, while the least rate of resistance was to vencomycin and erythromycin. Antibiotic resistance results in present study showed high resistance of L. monocytogenes to ampicillin, tetracycline, chloramphenicol, and penicillin antibiotics. Antibiotic resistance of Listeria sp. is due to the acquisition of genetic elements such as moving plasmids and conjugate transposons 39 .
Osaili et al. 25 , in Jordan, observed that from 10 antibiotic tests of chicken meat samples, the highest resistance was related to tetracycline. Furthermore, Fallah et al. 20 , studied poultry products in the central part of Iran and reported that the highest antibiotic resistance was for ampicillin, penicillin, and tetracycline that are consistent with the results of the present study. Walsh et al. 40 , showed that the highest antibiotic resistance of L. monocytogenes is related to tetracycline and penicillin antibiotics with 6.7% and 3.7% of prevalence, respectively. The antibiotic resistance of L. innocua in current study was more than that of L. monocytogenes. Although the genetic basis for these differences is not known, the presence of plasmids encoded antibiotic resistance was reported in L. innocua 40 . In the current study, L. innocua had the highest resistance to antibiotics spectrum, which is consistent with the above study. There are different factors causing the antibiotic resistance of various bacteria, such as excessive consumption of antibiotics in the poultry industry. The conducted studies in the field of antibiotic resistance of Listeria bacteria have indicated that this bacterium has a growing resistance to different antibiotics, which can increase the risk of infection for children, pregnant women, the elderly, and those with immune deficiency 41 .

Conclusion
The results of present study provide information about the contamination state of raw poultry meat and retailers' equipment in Yazd, Iran. The presence of this pathogenic microorganism in chicken meat can be a health risk, especially for pregnant women, the elderly, and those with immune deficiency. Considering the amount of contamination with L. monocytogenes in chicken and the high mortality rate caused by it, monitoring of the health principles and standards during the production, transportation, and storage as well as training of employees in this industry are necessary.