Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 12, Number 2, October 2023 | Pages: 607-610 | DOI: 10.14421/biomedich.2023.122.607-610 ISSN 2540-9328 (online) Prevalence of Enterobacteriaceae Producing Extended Spectrum Beta-Lactamase (ESBL) in Broiler Meat (Gallus domesticus) Sold in Regional Markets of West Surabaya Kadek Rio Risnanda1, Masfufatun2,*, Agusniar Furkani Listyawati3, Kuntaman Kuntaman3, Akhmad Sudibya3 1Faculty of Medicine; 2Department of Biochemistry; 3Department of Microbiology, Faculty of Medicine, Universitas Wijaya Kusuma Surabaya Jl. Dukuh Kupang XXV No. 54, Dukuh Kupang, Kec. Dukuh Pakis, Kota Surabaya, Jawa Timur, Indonesia. Corresponding author* masfufatun@uwks.ac.id Manuscript received: 23 August, 2023. Revision accepted: 03 November, 2023. Published: 09 November, 2023. Abstract Broiler meat is a foodstuff that is easily damaged by bacterial growth. Several pathogenic bacteria from the Enterobacteriaceae family have also been found as contaminants in chicken meat. While Extended Spectrum Beta-Lactamases (ESBL) are enzymes that have the ability to hydrolyze antibiotics of the penicillin class, first, second, and third generation cephalosporins as well as the monobactam group. Based on this phenomenon, the researchers wanted to examine the prevalence of the ESBL-producing Enterobacteriaceae family in broiler chicken (Gallus domesticus) meat sold in the West Surabaya market. This research is a descriptive observational type using a cross-sectional approach. The population in this study was broiler chicken meat taken from broiler traders in several markets in West Surabaya which had met the inclusion and exclusion criteria with a large sample of 50 samples. Then the data were analyzed using the chi-square test. The results of the analysis showed that 13 samples produced ESBL. The prevalence of Enterobacteriaceae bacteria producing ESBL in broiler chicken (Gallus domesticus) meat in several markets in West Surabaya is 26% of the 50 samples used. The results of the chi-square test showed that there was no significant difference between locations where ESBL was found. Keywords: Enterobacteriaceae; Extended Spectrum Beta-Lactamase; Broiler Chicken Meat. Abbreviations: ESBL Extended Spectrum Beta-Lactamase; MDR: Multiple Drug Resistance. INTRODUCTION Poultry meat, especially chicken meat, is a source of animal protein that is in great demand in Indonesia (Region, 2019). Most of Indonesia's demand for meat comes from poultry, especially chickens. Based on 2018 data, broiler production increased by 480,309 heads. According to statistical data for 2019, the consumption of chicken meat is 4.94 kg per person per year (Direktorat Jenderal Peternakan dan Kesehatan Hewan, 2019). Broiler meat is a food ingredient that is easily damaged by bacterial growth. Marketed chicken meat can be a breeding ground for microbes that cause food poisoning. Several pathogenic bacteria from the Enterobacteriaceae family have also been found as contaminants in chicken meat, including Escherichia coli, Staphylococcus aureus, Salmonella sp., Pseudomonas sp., Clostridium perfringens and Shigella flexneri (Ray, 2014). Enterobacteriaceae contamination of food occurs through soil, air, water, dust, digestive tract, human hand contact, and the influence of animals such as cockroaches and flies (Mu’arofah et al. 2020). Laying the carcass or body parts of chickens directly on the ground after slaughtering a chicken that has been washed can result in contamination with carcass remnants, such as blood, feathers, excrement, and its own stomach contents (Ramadhani et al., 2020). A study by Mashak revealed that 16.25% of chicken meat from Alborz, Iran, was E.coli positive. Previous research also stated that several samples of chicken meat from local markets in Indonesia presented microbial contamination, which included S. aureus (6.7%), Salmonella sp. (85%), and E. coli (90.03%). Contamination of poultry products including raw broiler meat by pathogenic microorganisms, especially bacteria, has become one of the most challenging problems in the food industry worldwide (Wardhana et al. 2021). Extended Spectrum Beta-Lactamases are enzymes that have the ability to hydrolyze penicillin class antibiotics, first, second and third generation cephalosporins as well as monobactam groups and cause resistance to all of these antibiotics (Biutifasari, 2018). https://doi.org/10.14421/biomedich.2023.122.607-610 mailto:masfufatun@uwks.ac.id 608 Biology, Medicine, & Natural Product Chemistry 12 (2), 2023: 607-610 Epidemiologically, the prevalence of ESBL varies in various countries. The prevalence of ESBL produced by Enterobacteriaceae families such as E. coli and Klebsiella pneumoniae varies, ranging from 42.7% in Latin America, 5.8% in North America, 2% to 31% in Europe, and 4.8% to 12% in isolates. in Asia (Yessy, 2021). Research in five hospitals in Indonesia in 2013 showed the prevalence of ESBL-producing bacteria was 32-68% (Kuntaman, 2013). The results of the 2016 AMR study by the Department of Health Control on Antimicrobial Resistance in 8 hospitals with ESBL indicator bacteria E. coli and K. pneumoniae showed an average prevalence of 60%. Beta-lactams are used against gram-positive and gram-negative bacteria. Bacterial resistance to lactams are using three pathways: Disruption of the antibiotic lactamase enzyme, alteration of the target of the antibiotic and decreased cellular uptake of the antibiotic. All of these pathways play an important role in antibiotic resistance. However, lactam-producing and lactam- destroying bacteria are the main causes of resistance (Masruroh et al. 2016). Antibiotics are chemical substances produced by fungi or bacteria that have the ability to kill or prevent the growth of pathogenic microorganisms, while their toxicity to humans is relatively low. Irrational use of antibiotics causes negative effects, such as the immunity of microorganisms to some antibiotics, increased drug side effects and even death. Bacterial resistance to antibiotics is a global health problem that threatens the treatment of infectious diseases and causes serious disability and death in the healthcare community. Bacteria that naturally carry antibiotic resistance genes can transfer these genes to other bacteria. In addition, bacteria can produce enzymes that inhibit the action of antibiotics (Aidara-Kane et al., 2013). One of the antibiotics used is a beta-lactam class of antibiotics which works by inhibiting the cell wall. Improper use of antibiotics, including selection, dosage, and patient non- adherence to treatment, play a role in the development of antibiotic resistance. One of the resistance mechanisms of Gram-negative bacteria belonging to the Enterobacteriaceae family is the production of ESBL. According to the results of a study on 781 hospital patients throughout Indonesia, 81% of Escherichia coli were found to be resistant to various antibiotics, namely ampicillin (73%), cotrimoxazole (56%), chloramphenicol (43%), ciprofloxacin (22%), and gentamicin (18%) (Kementerian Kesehatan Republik Indonesia, 2013). Based on the data and research results obtained above, the researchers wanted to examine the prevalence of Enterobacteriaceae family bacteria such as E. coli producing ESBL in broiler chicken meat (Gallus Domesticus) sold in West Surabaya market in the hope of knowing whether the consumption of broiler chickens contaminated with bacteria can affect the percentage of Indonesian people who are resistant to antibiotics due to the high E. coli bacteria in broiler chicken meat. MATERIALS AND METHODS Research Design This research is a descriptive observational type using a cross-sectional approach. Population and Research Sample The population in this study was broiler chicken meat taken from broiler traders in several markets in West Surabaya with a sample size of 50 samples that met the inclusion and exclusion criteria. This sampling used reference to the Basic and Clinical Biostatistics book. Data Analysis Techniques The method used in this study was univariate analysis to determine the prevalence rate of ESBL-producing bacteria in meat sold in markets in West Surabaya and bivariate analysis using the chi-square test. RESULTS AND DISCUSSION Results Table 1. Identification Results of Enterobacteriaceae Producing Extended Spectrum Beta-Lactamase (ESBL) in Broiler Chicken (Gallus domesticus) Meat in Regional Markets of West Surabaya. Location Number of Samples Extended Spectrum Beta- Lactamase (ESBL) I 10 2 II 10 4 III 10 2 IV 10 2 V 10 3 Total 50 13 (26%) Notes: I: Darmo Market Location, II: Darmo Market Location, III: Kupang Dukuh Market Location. IV: Kupang Gunung Market Location, V: Simo Gunung Market Location Figure 1. Lactose fermenter bacteria isolated from broiler chicken meat. Risnanda et al. – Prevalence of Enterobacteriaceae Producing Extended Spectrum … 609 Based on the table and figure above, it can be seen that from 50 samples of broiler chickens in several markets in West Surabaya, 19 samples were positive for Enterobacteriaceae and 13 of them produced Extended Spectrum Beta-Lactamase (ESBL). At location I there were 2 positive samples for ESBL. Of the 10 samples at location II, 5 samples were positive for Enterobacteriaceae and 4 of them produced ESBL. Of the 10 samples at location III, there were 4 positive samples for Enterobacteriaceae and 3 of them produce ESBL. At location IV, 5 positive samples were found for Enterobacteriaceae and 2 of them produced ESBL and at location V, out of the 10 samples used, 3 samples were found positive for Enterobacteriaceae and all of them ESBL. Based on Table 1, it can be calculated the prevalence of Enterobacteriaceae bacteria producing ESBL in broiler chicken (Gallus domesticus) meat in several markets in West Surabaya, namely 26% of the 50 samples used. Table 2. Crosstab Places with Enterobacteriaceae Bacteria. Location Enterobacteriaceae Total Positif Negatif Pasar Darmo Permai 2 (4%) 8 (16%) 10 (20%) Pasar Darmo 5 (10%) 5 (10%) 10 (20%) Dukuh Kupang 4 (8%) 6 (12%) 10 (20%) Pasar Kupang Gunung 5 (10%) 5 (10%) 10 (20%) Pasar Simo Gunung 3 (6%) 7 (14%) 10 (20%) Total 19 (38%) 31 (62%) 50 (100%) Based on Table 2, it shows that most of the Enterobacteriaceae were found in the Darmo market and Kupang Gunung market, namely each (10%). Table 3. Relationship of Place with Extended Spectrum Beta-Lactamase (ESBL) Bacteria. Location ESBL Total p-value Positif Negatif Darmo 6 (30%) 14 (70%) 20 (100%) 0,599 Kupang 7 (23%) 23 (76%) 30 (100%) Total 13 (26%) 37 (74%) 50 (100%) The results of the chi-square test showed that there was no significant difference between locations where Extended Spectrum Beta-Lactamase (ESBL) was found. Discussion The results of the analysis showed that from 50 samples of broiler chickens in several markets in West Surabaya, 19 samples were positive for Enterobacteriaceae and 13 of them produced Extended Spectrum Beta-Lactamase (ESBL). The prevalence of Enterobacteriaceae bacteria producing Extended Spectrum Beta-Lactamase (ESBL) in broiler chicken (Gallus domesticus) meat in several markets in West Surabaya is 26% of the 50 samples used. Research by Puspandari, Sunarno, et al. was conducted in four markets and two slaughterhouses in the Jakarta area. Research with a total of 240 chicken meat samples showed that ESBL-producing E. coli was isolated from 161 (67.1%) of 240 broiler samples, 161 (84.3%) of 191 suspected colonies on MacConkey agar supplemented with cefotaxime 0 media .4% confirmed as ESBL producer E. coli (Puspandari et al., 2021). The results of this study provide information about ESBL-producing Enterobacteriaceae associated with antibiotic resistance found in broiler chickens in several markets in West Surabaya. The problem of Multiple- Drug Resistance (MDR) is exacerbated by the ability of bacteria to transfer genetic material carrying resistance properties from one bacterium to another vertically through genetic mutation and horizontally through conjugation, transduction and transformation. MDR occurs when more and more antibiotics are used, the greater the selective pressure on the process of evolution and proliferation of resistant bacterial strains to defend themselves so that vertical resistance arises from genetic mutations and horizontal resistance from the exchange of resistant gene material against various types of different antibiotic resistance mechanisms. (Gregova et al. 2012). Multidrug resistance is a common occurrence in ESBL producing bacteria. Aminoglycoside modifying enzyme (AME) and ESBL are genes that encode resistance enzymes commonly found in bacterial plasmids. That is, gene transfer occurs in genetic factors such as transposons, integrons, and plasmids (Aloccati et al., 2013). The ESBL gene that appears in E. coli is caused by a plasmid-mediated genetic mutation, especially the ESBL gene with TEM and SHV types, then a new ESBL group appears, namely CTXM (Hasibuan, 2017). This study is in accordance with research conducted (Wibisono et al. 2020) the results obtained from 185 samples of cloacal swabs which tested positive for ESBL-producing E. coli contained 7.03% ESBL (DDTS). Food products of animal origin contaminated with the Enterobacteriaceae family such as ESBL- producing E. coli can pose a health risk, although the level of risk is difficult to measure. Broiler chickens can serve as a reservoir for ESBL E. coli. ESBL bacteria are transmitted in several ways, namely: consuming contaminated meat, faecal contaminated environment containing ESBL-producing E. coli, exposure to patients or people infected with ESBL and transmitted to humans via animals and possibly causing zoonoses. Contamination of the broiler chickens under study can also come from the water used to wash the chickens, the equipment used by traders to cut the chickens and other equipment that has direct contact with the meat, as well as the hygiene of the chicken slaughterhouse itself. 610 Biology, Medicine, & Natural Product Chemistry 12 (2), 2023: 607-610 Placing chicken meat directly on the sales counter without the need for a special place and handling such as packaging or temperature control which is also commonly found in the market can affect the level of bacterial contamination, one of which is E. coli bacteria. Flies can infect meat as vectors for E. coli. In addition to flies, the hands of buyers and sellers who are not clean can cause the meat to be exposed to E. coli bacteria. It is known that E. coli is harmful to health because it produces a toxin (shiga toxin) (Reich et al., 2013). CONCLUSIONS The results of the analysis showed that out of 50 samples of broiler chickens in several markets in West Surabaya, 13 samples produced Extended Spectrum Beta- Lactamase (ESBL). The prevalence of Enterobacteriaceae bacteria producing Extended Spectrum Beta-Lactamase (ESBL) in broiler chicken (Gallus domesticus) meat in several markets in West Surabaya, namely 26% of the 50 samples used, most of the Enterobacteriaceae were found in Darmo market and Kupang Gunung market, respectively each by (10%). The results of the chi square test showed that there was no significant difference between locations where Extended Spectrum Beta-Lactamase (ESBL) was found. Acknowledgements: This research is supported by Prof. Kuntaman, dr., MS., Sp.MK(K), and also Institute Research and Community Service that has provided research funding assistance. Authors’ Contributions: Kadek Rio Risnanda & Kuntaman designed the study. Kadek Rio Risnanda, Masfufatun, and Agusniar Furkani Listyawati carried out the laboratory work. Kadek Rio Risnanda analyzed the data and wrote the manuscript. All authors read and approved the final version of the manuscript. Competing Interests: The authors declare that there are no competing interests. Funding: Funding for this research is Institute of Research and Community Service REFERENCES Aidara-Kane, A., Andremont, A., Collignon, P. (2013) Antimicrobial resistance in the food chain and the AGISAR initiative. J Infect Pub Hea. 6. 162-165. Biutifasari, V. (2018). Extended Spectrum Beta-Lactamase (ESBL). 1(1), 1–11. Direktorat Jenderal Peternakan dan Kesehatan Hewan. (2019). statistik peternakan dan kesehatan hewan. Jakarta: Direktorat Jenderal Peternakan dan Kesehatan Hewan Kementrian Pertanian RI. 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