Prevalence and Antimicrobial Susceptibility of Listeria monocytogenes in Fresh Poultry Products in Bandung, Indonesia. Yoni Darmawan Sugiri1*, Josef Kleer2, Greta Gölz2, Tongkorn Meeyam3, Warangkhana Chaisowwong3, and Thomas Alter2 Joint Master Course in Veterinary Public Health, Freie Universität Berlin and Chiang Mai University, Thailand, and Balai Pengujian dan Penyidikan Penyakit Hewan dan Kesmavet (BP3HK), Dinas Peternakan Provinsi Jawa Barat, West Java, Indonesia. 2 Institute of Food Hygiene, Department of Veterinary Medicine, Freie Universität Berlin, Germany. 3 Department of Veterinary Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Thailand. 1

*Corresponding author; Emails: [email protected] & [email protected]

Key words: Listeria monocytogenes, Poultry products, Antimicrobial susceptibility, Serotyping, Multiplex PCR.

Abstract The purpose of this study was to determine the prevalence and the number of Listeria monocytogenes in 184 samples of fresh poultry products sold in traditional markets and supermarkets in Bandung, West Java, Indonesia, and also to determine the antimicrobial resistance pattern and molecular characterization of the isolated L. monocytogenes strains. The samples were analyzed following ISO 11290-1: 1996 and ISO 11290-2: 1998. Disc diffusion method was applied for antimicrobial susceptibility test and multiplex polymerase chain reaction was applied for molecular serotyping of the isolated L. monocytogenes. Overall L. monocytogenes was detected in 15.8 % of the fresh poultry products, with prevalence of 15.2 % and 16.3 % for traditional markets and supermarkets samples respectively. There was no significant difference between traditional markets and supermarkets regarding the prevalence and the count number of L. monocytogenes. The average L. monocytogenes count in poultry products sold in traditional markets was 1.08 log cfu/g, and 1.03 log cfu/g for supermarkets. The contamination level of L. monocytogenes was < 10 cfu/g in 87.5 %, between 10 and 100 cfu/g in 9.8 %, between 100 and 1000 cfu/g in 2.2 %, and > 1000 cfu/g in 0.5 % of the samples. All 29 isolates in this study belong to the molecular serogroup IIb comprising the serovars 1/2b, 3b and 7. 27.6 % (8/29) isolates were resistant to at least one of ten antimicrobials tested, and 72.4 % (21/29) of the isolates were susceptible to all antimicrobials tested. Among 29 isolates, 17.2 %, 6.9 %, 6.9 % and 3.4 % were resistant to penicillin, ampicillin, erythromycin, and a combination of ampicillin and penicillin respectively. The results of this study reveal that

there is wide spread cross contamination and emerging antibiotics resistant of L. monocytogenes isolated from fresh poultry products sold in study area. There are strong needs to improve this condition by improving good hygienic practices and prudent use of antimicrobial drugs at all level of poultry production chain.

Introduction Listeria monocytogenes has been largely studied in the last decades due to its importance as food borne human pathogen with high mortality rate on susceptible hosts (1). L. monocytogenes is a psychrotrophic pathogen and can grow at refrigerator temperatures. Minimum pH for growth on foods is 4.39 and it can grow under aerobic, microaerobic, anaerobic as well as under vacuum condition (2). L. monocytogenes can be found on soil, water, fresh vegetables, and intestinal contents of birds, fish, insect and other animals (3). L. monocytogenes can also be found in the processing environments because of poor hygiene practices in the premises (4). Listeriosis is a severe foodborne disease caused by L. monocytogenes. This disease has been studied and documented in the developed or industrialized countries such as in Europe and North America, and sometimes sporadic cases and irregular outbreaks of human listeriosis have been detected in developing countries (5). More than 80 % of human listeriosis cases are caused by serovars 4b, 1/2a, 1/2b and 1/2c (6). The main risk groups for listeriosis are pregnant women and their fetuses, new born children, immune-compromised, and old people (7). There are no data or information concerning the prevalence, the quantitative load, antimicrobial

resistance pattern and serotypes of L. monocytogenes isolated from fresh poultry products in Indonesia, and only limited information regarding this disease in humans neither in animals. Therefore this study was conducted to determine the prevalence, the quantitative load, the antimicrobial resistance pattern and to characterize the L. monocytogenes, and also to compare the prevalence and quantitative load between traditional markets and supermarkets.

and breast) were homogenized with half-Fraser broth without the addition of selective agents, in order to revitalize stressed listeriae, the homogenate was allowed to rest for 1 hour at room temperature before plating on the agar. 1 ml of homogenate was spread onto Chromocult® Listeria selective agar and PALCAM agar, and incubated aerobically at 37°C for 24-48 h. Suspected L. monocytogenes colonies were counted and 3-5 colonies per plate were sub cultured on Tryptone Soya Yeast Extract agar plates for confirmation by biochemical and CAMP tests.

Material and Method

Antimicrobial Susceptibility test

Sample collection

All isolated L. monocytogenes strains were tested by the standard disc diffusion method described by the Clinical and Laboratory Standards Institute 2012 (8) on Mueller Hinton agar (Merck, Germany) incubated at 37°C for 24 h. The antibiotic discs that were used in this study were: ampicillin [10 µg], neomycin [10 µg], erythromycin [15 µg], penicillin [10 µg], ciprofloxacin [10 µg], streptomycin [10 µg], sulphametoxazole-trimetophrim [23.75-1.25 µg], kanamycin [30 µg] tetracycline [30 µg] and gentamycin [10 µg] (OXOID®, UK). Staphylococcus aureus ATCC 25923 was used as control strain.

A total of 184 fresh poultry products (chicken) composed of 92 samples from 12 traditional markets and 92 samples from 12 supermarkets were collected in Bandung, Indonesia between November 2012 and February 2013. All samples were taken by convenient random sampling, and transported to the laboratory aseptically in cool boxes. The samples were analyzed immediately, If the samples were not analyzed on the day of arrival at the lab, they were kept in a refrigerator (26°C) for not more than 24 hours before analysis.

Identification of L. monocytogenes The isolation and identification of L. monocytogenes was done according to ISO 11290-1: 1996. Ten gram of each sample (chicken skin from neck and breast) were enriched in 90 ml half Fraser broth (Merck, Germany), and incubated aerobically at 30°C for 24 h. 100 µl of enriched sample were transferred to 10 ml Fraser broth, and incubated aerobically at 37°C for 48 h. The enriched samples in Fraser broth then were streaked onto Chromocult® Listeria selective agar (Merck, Germany) and PALCAM agar (Merck, Germany), and incubated aerobically at 37°C for 24-48 h. Suspected L. monocytogenes colonies were subcultured on Tryptone Soya Yeast Extract agar plates (Merck, Germany) for confirmation by biochemical and CAMP tests. All L. monocytogenes strains isolated in this study were stored in brain heart infusion broth with 20 % glycerol and kept at -70°C for further studies.

Enumeration of L. monocytogenes The enumeration of L. monocytogenes was done according to ISO 11290-2: 1998. Ten gram of each sample (chicken skin from neck

Molecular serotyping of L. monocytogenes isolates Strains were recovered by streaking on 5 % sheep blood agar and Trypticase Soy Yeast Extract agar and were grown overnight at 37°C. The colonies were scraped and suspended in 500 µl sterile 1x Tris-EDTA mixed and boiled for 10 minutes to extract the DNA (9). Then the samples were centrifuged and the supernatant was divided into aliquots and stored at -20°C until used for PCR. The serotyping by molecular serogroup was carried out using two multiplex PCRs according to Doumith et al., 2004 (10) and Kerouanton et al., 2010 (11) with some modification. For the multiplex PCR a total of 25 µl reaction mixture were composed of 2.5 µl 10×buffer, 0.5 µl MgCl2 [25mM], 0.5 µl dNTPs [10mM each], 0.1µl Taq-DNA Polymerase (Faststart, Roche, Mannheim, Germany) [5U/µl], template DNA 0.5 µl, 2.4 µl of following primer pairs (Table 1): 0.4 µl LMO0737 [50 pmol/µl], 0.8 µl LMO1118 [50 pmol/µl], 0.4 µl ORF2819 [50 pmol/µl], 0.4 µl ORF21110 [50 pmol/µl], 0.2 µl PRS [50 pmol/µl] and 0.2 µl LIP [50 pmol/µl], and 18.5 µl sterile distilled water with the

amplification conditions: initial denaturation at 95°C for 4 min. followed by 35 cycles with 94°C for 30 s, annealing at 58°C for 50 s, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. For the flaA PCR, a total of 25 µl reaction mixture were composed of 2.5 µl 10×buffer, 2.5 µl MgCl 2 [25mM], 0.5 µl dNTPs [10mM each], 0.1µl Taq DNA Polymerase (Faststart) [5U/µl], template DNA 0.8 µl, 0.82 µl of following primers pairs (Table 1): 0.8 µl flaA [50pmol/µl] and 0.02 µl PRS [50pmol/µl], and 17.78 µl sterile distilled water with the amplification conditions: initial denaturation at 95°C for 3 min followed by 40 cycles with 94°C for 30 s, annealing at 61°C for 40 s, extension at 72°C for 1 min, and a final extension at 72°C for 7 min.

prevalence of L. monocytogenes on the fresh poultry products. L. monocytogenes was detected and isolated from 14 out of 92 samples from traditional markets (15.2 %; 95 % Confidence Interval (CI): 7.88 – 22.6), while 15 out of 92 samples from supermarkets were positive for L. monocytogenes (16.3 %; 95 % CI: 8.76 – 23.85). The prevalence for all the samples was 15.8 % (29/184) at 95 % CI (10.49 – 21.02) as shown in Table 2. Table 2 Prevalence of L. monocytogenes in traditional and supermarkets in Bandung, Indonesia

Table 1 Primer pairs used in this study

Enumeration of L. monocytogenes The average L. monocytogenes count of traditional markets was 1.08 log cfu/g, and the average L. monocytogenes count of supermarkets was 1.03 log cfu/g (Table 3). There was no statistically significant difference (p > 0.05) between the L. monocytogenes count of traditional markets and supermarkets.

Data analyses Data analysis was done using “R” statistical and data analysis free software. Descriptive statistics were used to describe the result of prevalence and enumeration analysis. The prevalence of L. monocytogenes between traditional markets and supermarkets were compared using chi square statistical test, and for L. monocytogenes enumeration data were compared using two samples independent t-test. For the antimicrobial susceptibility test, it was counted how many isolates are resistant against the antibiotics or antimicrobials used in the study. All isolated L. monocytogenes strains were serotyped and serogrouped based on the results of mPCR.

Result Identification of L. monocytogenes There was no statistically significant difference (p > 0.05) between traditional markets and supermarkets regarding the

Table 3. Descriptive statistic of L. monocytogenes counts in Bandung, Indonesia.

aSD=

Standard Deviation

There were 87.5 % (161/184) of the samples had L. monocytogenes counts less than 10 cfu/g, 9.8 % (18/184) had L. monocytogenes counts between 10 and 100 cfu/g, 2.2 % (4/184) had L. monocytogenes counts between 100 and 1,000 cfu/g and 0.5 % (1/184) had L. monocytogenes counts higher than 1,000 cfu/g. The distribution of L. monocytogenes enumeration data from all samples (184 samples) are presented in Table 4.

Table 4. Enumeration data of L. monocytogenes, in Bandung, Indonesia.

monocytogenes isolates from this study belong to the molecular serogroup IIb, comprising the serotypes 1/2b- 3b- and 7.

Antimicrobial susceptibility test From a total of 29 isolates of L. monocytogenes analyzed, eight (27.6 %, 95 % CI: 11.3 – 43.9) were resistant to at least one of ten antimicrobials tested, and 21 (72.4 %, 95 % CI: 56.1 – 88.7) were susceptible to all antimicrobials tested. Five out of 29 isolates (17.2 %, 95 % CI: 3.5 – 31.0) were resistant to penicillin, 2 (6.9 %, 95 % CI: 1.2 – 20.2) were resistant to ampicillin, and 2 (6.9 %, 95 % CI: 1.2 – 20.2) were resistant to erythromycin, while one out of 29 isolates (3.4 %, 95 % CI: 0.2 – 15.3) was resistant to penicillin and ampicillin (Table 5).

Figure 1. First multiplex PCR results. M: DNA marker 100 bp, Lane 1-4: positive control (serotype 3a, 1/2b, 4b, 1/2a), lane 5-9: L monocytogenes isolates from the study, A: lmo0737 (691 bp), B: orf2110 (597 bp), C: orf2819 (471 bp), D: prs (370 bp), E: lip (274 bp).

Table 5 Result of antimicrobials susceptibility test of L. monocytogenes.

Figure 2. Second multiplex PCR (flaA PCR) results. M: DNA marker 100 bp, lane 1: negative control, lane 2-4: positive control (serotype 3a, 1/2b, 1/2a), lane 5-9: L monocytogenes isolates from the study, A: flaA (538 bp), B: prs (370 bp).

Discussion

n: number of isolate, AMP: ampicillin, N: neomycin, E: erythromycin, P: penicillin, CIP: ciprofloxacin, S: streptomycin, SXT: sulphamethoxazole/ trimethoprim, K: kanamycin, TE: tetracycline, CN: gentamycin.

Molecular serotyping of L. monocytogenes isolates In all 29 isolated L. monocytogenes strains the prs, prfA and orf2819 gene was detected by first PCR (Figure. 1). In none of all 29 isolates the flaA gene was detected by the second PCR (Figure. 2). From the results of the first and second mPCR assay, all L.

The prevalence of L. monocytogenes from fresh poultry products in this study was relatively similar to the prevalences described in Thailand (13.9 %), Pakistan (17.5 %), and France (15.5 %) (12, 13, 14). There are many factors which can contribute to the contamination of fresh poultry products. L. monocytogenes can be found easily in the environment. It is resistant to various environmental conditions which allow longer survival under adverse conditions than most other non-spore forming food borne bacteria (15). Several studies show that L. monocytogenes can be found easily in poultry slaughterhouse or processing plants (16). Even though the

prevalence and enumeration results of L. monocytogenes between these two types of markets showed no statistically significant difference, the contamination level of L. monocytogenes in traditional markets is slightly higher than in supermarkets. There were five samples from traditional markets with level of contamination higher than 100 cfu/g, whereas the samples from supermarkets showed level of contamination lower than 100 cfu/g. In susceptible peoples, the infective dose for L. monocytogenes is maybe less than 1,000 cells (15). The results of this study indicate that the fresh poultry products which were contaminated by L. monocytogenes higher than 100 cfu/g which is derived from the traditional markets have the potential to infect humans, especially those in high risk groups such as young children, the elderly, pregnant women, and those with immune problems (YOPI). These results show that the hygienic practices are one of many factors which can contribute to the level of L. monocytogenes contamination in the fresh poultry products at the selling point (16). Therefore need to increase public awareness to always cook all fresh poultry products properly before consumption, to decrease the chance of getting infected by food borne pathogen such as L. monocytogenes. Although the incidence of antibiotic resistance of L. monocytogenes isolates in this study is still relatively low, it presents an evidence of the emergence of multi-resistant L. monocytogenes strains which can be a threat to public and animal health. We found that among 29 isolates, 17.2 %, 6.9 %, 6.9 % and 3.4 % were resistant to penicillin, ampicillin, erythromycin, and a combination of ampicillin and penicillin respectively. The resistance of L. monocytogenes strains against penicillin, ampicillin and erythromycin was also reported in the others studies, these studies showed that the resistance rate were 2.3 %, 9.2 % and 1.9 % (17), 64.3 %, 32.1 %, 57.1 % (18), 22.5 %, 23.7 %, 12.1 % (19) and 3.73 %, 1.98 %, 0.4 % (20) against penicillin, ampicillin, and erythromycin respectively. The antimicrobial resistance of L. monocytogenes strains isolated from fresh poultry products in this study may caused by uncontrolled and imprudent use of antimicrobial agents by farmers and their

excessive use in poultry farms to control diseases in Indonesia, especially in West Java (21). Resistance of L. monocytogenes against antibiotics is influenced by many factors. Several studies described that conjugation of enterococcal and streptococcal plasmids and transposons which are carrying antibiotic resistance genes can be transferred to Listeria sp. and between species of Listeria, it is known that L. monocytogenes is frequently found in the digestive tract of humans and animals where many species of enterococci and streptococci harboring conjugative plasmids and transposons are found in very high numbers (22). The most common serotype isolated from foods and food processing environments is 1/2a followed by 4b, 1/2b, 4c and 1/2c (23). All strains isolated in this study belong to serogroup IIb comprising serotype 1/2b-3b-7. By means of genetic ribotyping classification methods, L. monocytogenes was divided into three lineages: lineage I consists of serotypes 1/2b, 3b, 4b, 4d, and 4e, lineage II is composed of serotypes 1/2a, 1/2c, 3a, and 3c, and lineage III of serotypes 4a and 4c. Lineage I has the highest pathogenic potential and is mainly involved in epidemic outbreaks. Lineage II has intermediate pathogenic potential and is possibly involved in sporadic outbreaks, while Lineage III has low pathogenic risk and rarely causes human infection (7).

Conclusion The results of this study reveal that there is wide spread cross contamination of L. monocytogenes in fresh poultry products sold in Bandung, Indonesia. Although the contamination level of L. monocytogenes on fresh poultry products was relatively low, the existence of this human pathogen must be considered as having considerable public health impact especially for those people who belong to the high risk group for listerial infection such as young, old, pregnant and immuno-compromised people (YOPI). This study also found that all isolates belong to the molecular serogroup IIb comprising serotype 1/2b-3b-7 which are

categorized as highly pathogenic and involved in most of epidemic outbreaks. In addition to the public health impact, some of those isolates show antimicrobial resistance against antimicrobial drugs commonly used in human and veterinary medicine. There are strong needs to improve this condition by improving good manufacturing practices, good hygienic practices, good retailing practices and prudent use of antimicrobial drugs at all level of poultry production chain. These problems also deserve more attention from all stakeholders including the government as regulator.

Acknowledgment This study was conducted as part of Master of Veterinary Public Health thesis and was financially supported by Germany Government through DAAD and Royal Thai Government through VPHCAP Chiang Mai University. The Authors also would like to acknowledge the support provided by the Balai Pengujian dan Penyidikan Penyakit Hewan dan Kesmavet (BP3HK), Dinas Peternakan Provinsi Jawa Barat, West Java, Indonesia, for the facilities, media and chemical reagents used in this study.

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