Vol. 8 No. 3 September–December 2020 Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Available online at IJTID Website: https://e-journal.unair.ac.id/IJTID/ Research Report The Epidemiological Pattern and Risk Factor of ESBL (Extended Spectrum Β-Lactamase) Producing Enterobacteriaceae in Gut Bacterial Flora of Dairy Cows and People Surrounding in Rural Area, Indonesia Agusta Reny Soekoyo1, Sulistiawati2, Wahyu Setyorini3, K. Kuntaman3,4,5 1 Master Program of Basic Medical Sciences, Faculty of Medicine, Universitas Airlangga 2 Department of Public Health, Faculty of Medicine, Universitas Airlangga 3 Institute of Tropical Disease, Universitas Airlangga 4 Department of Medical Microbiology, Faculty of Medicine, Universitas Airlangga 5 Dr Soetomo Academic Hospital, Surabaya, Indonesia Received: 23rd January 2020; Revised: 28th January 2020; Accepted: 6th October 2020 ABSTRACT Livestock would be a risk factor of resistant bacteria that impact on human health. Rural area with farms as major economic source has become a risk of the spread of the ESBL producing Enterobacteriaceae The aim of the study was to explore the distribution and risk factor of ESBL (extended-spectrum β-lactamase) producing Enterobacteriaceae in the gut bacterial fl ora of dairy cows and people surrounding farming area. Total of 204 fecal swab samples were collected, 102 from dairy cows and 102 from farmers. Samples were sub-cultured by streaking on MacConkey agar supplemented with 2 mg/L cefotaxime. The growing colonies were confi rmed of the ESBL producer by Modifi ed Double Disk Test (M-DDST) and identifi cation of Enterobacteriaceae by biochemical test. ESBL genes were identifi ed by PCR. ESBL producing bacteria were found 13.7% in dairy cows and 34.3% in farmers. ESBL producing Enterobacteriaceae in dairy cows were 6.9% and in farmers of 33.3%. Statistical analysis showed: Distribution of ESBL producing Enterobacteriaceae strain were insignifi cant among dairy cows and farmers while blaTEM distribution was signifi cantly diff erent (p= 0,035) and use of antibiotic was identifi ed as a risk factor of colonization of ESBL producing Enterobacteriaceae in farmers (p= 0,007). Farmers had suspected as the source of ESBL producing Enterobacteriaceae based on higher prevalence. Further education of appropriate use of antibiotic need to enhance to control risk factor and prevent the colonization of ESBL producing Enterobacteriaceae. Keywords: Enterobacteriaceae, ESBL, gut fl ora, dairy cow, farmer, rural ABSTRAK Hewan ternak diduga sebagai faktor risiko kejadian bakteri resisten yang berdampak terhadap kesehatan manusia. Area rural dengan potensi ekonomi di sektor peternakan merupakan area yang berisiko terhadap penyebaran Enterobacteriaceae penghasil ESBL. Penelitian bertujuan mengeksplorasi pola distribusi dan faktor risiko Enterobacteriaceae penghasil ESBL pada bakteri fl ora usus sapi perah dan penduduk sekitarnya. Total 204 sampel swab feses, terdiri dari 102 swab feses sapi perah dan 102 swab feses peternak. Swab feses ditanam pada media MacConkey yang ditambahkan 2 mg/L cefotaxime. Koloni yang tumbuh dikonfi rmasi sebagai penghasil ESBL dengan metode Modifi ed Double Disk Test (M-DDST) and diidentifi kasi dengan uji biokimia. Identifi kasi gen ESBL menggunakan metode PCR. Prevalensi bakteri penghasil ESBL di sapi perah sebesar 13.7% dan di peternak sebesar 34.3%. Distribusi Enterobacteriaceae penghasil ESBL pada sapi perah 6.9% dan pada peternak 33.3%. Analisis statistik menunjukkan: Tidak ada perbedaan signifi kan antara distribusi bakteri Enterobacteriaceae penghasil ESBL pada sapi perah dan peternak, distribusi blaTEM pada sapi perah dan peternak berbeda signifi kan (p = 0,035), dan penggunaan antibiotik sebagai faktor risiko kolonisasi Enterobacteriaceae penghasil ESBL pada peternak (p= 0,007). Peternak diduga sebagai sumber Enterobacteriaceae penghasil ESBL. Penyuluhan * Corresponding Author: kuntaman@fk.unair.ac.id 145Agusta Reny Soekoyo, et al.: The Epidemiological Pattern and Risk Factor of ESBL Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 tentang penggunaan antibiotik secara tepat perlu ditingkatkan untuk mengendalikan faktor risiko dan mencegah kolonisasi Enterobacteriaceae penghasil ESBL. Kata kunci: Enterobacteriaceae, ESBL, fl ora usus, sapi perah, peternak, rural How to Cite: The Epidemiological Pattern and Risk Factor of ESBL (Extended Spectrum Β-Lactamase) Producing Enterobacteriaceae in Gut Bacterial Flora of Dairy Cows and People Surrounding in Rural Area, Indonesia. Soekoyo, AR. Sulistiawati, Setyorini, W. Kuntaman, K. Indonesian Journal of Tropical and Infectious Disease, 8(3), 144–151. INTRODUCTION The inappropriate use of antibiotics in human and animal health is a major cause of pathogenic bacterial resistance.1 Resistant p a t h o g e n i c b a c t e r i a t h a t h a v e i n c r e a s e d significantly over the past few decades are ESBL-producing bacteria (extended-spectrum β-lactamase).2 ESBL mainly distributed among gram-negative bacilli of the Enterobacteriaceae group.3 The use of antibiotics as growth promotion and the prevention of disease in veterinary 4 was correlated with the increase of ESBL producing Gram-negative bacteria.5 It thus, the livestock are identifi ed as a risk factor for ESBL producing Enterobacteriaceae (ESBL-E).6 In 2018, East Java Province was identifi ed has the highest population of dairy cows in Indonesia, about 283,311 cows.7 Most of the dairy farms in East Java Province are located in rural areas to empowering the community's economy. Kalipucang Village in District of Pasuruan, East Java was established as the fi rst center of public dairy farming in Indonesia at 2016.8 E S B L p r o d u c i n g E n t e ro b a c t e r i a c e a e (ESBL-E) bacteria cause various infections in humans, such as: bacteremia, gastroenteritis, respiratory infections, urinary tract infections, and infections of the central nervous system.9 In dairy cows, Escherichia coli and Klebsiella spp are identifi ed as an agent that causing infl ammation of mammary gland and udder tissue (mastitis) which impact on decreasing quantity and quality of milk production, increasing the rejected prematurely, and death.10 ESBL-E becomes a serious challenge in therapy for infection includes prolong of diagnosis and expensive, a longer duration of treatment, limited antibiotic choices that impact on higher cost of therapy for an infection, as well as increased morbidity and mortality.11 Multiple resistance to fl uoroquinolones, aminoglycosides, and trimethoprim are commonly found in ESBL- E.3 It also causes carrier in both humans12 and livestock.6 Since ESBL-E has been identifi ed as one of the causes of mastitis in dairy cows in 2000,6 dairy farming was suspected to be at risk as a source of ESBL-E transmission. It thus the epidemiological profi le of ESBL-E in farm needs to be explored. This study is the fi rst study to analyze the epidemiological patterns of ESBL producing Enterobacteriaceae in livestock and humans in rural areas in Indonesia. The aim of the study was to identify and analyzed the distribution and risk factor of ESBL producing Enterobacteriaceae in gut bacterial fl ora of dairy cows and people/farmer who have close contact with dairy cows. MATERIALS AND METHODS Design This study was conducted a cross-sectional design. This study was approved by Research Ethics Committee in Faculty of Medicine of Universitas Airlangga, no: 82/EC/KEPK/ FKUA/2019. Samples Collection Fecal samples were collected from April until July 2019 from dairy cows and farmers. Samples collected using Amies transport medium (Deltalab, Spanyol). The total dairy farming in the District of Pasuruan, East Java, are 648 clusters, of which as many as 102 were randomly included as the samples in this study, consisting 146 Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Indonesian Journal of Tropical and Infectious Disease, Vol. 8 No. 3 September–December 2020: 144–151 of 102 samples from dairy cows and 102 from humans that living around and have close contact with dairy cows. These clusters were located in Kalipucang village. The samples transportation were using a cool box and ice pack (4-8˚C). Samples were processed within 24 hours after taken from the sample source. Bacterial and ESBL Identifi cation The isolation, confi rmation, and identifi cation of ESBL-E were conducted in The Clinical Microbiology Laboratory of Dr. Soetomo Hospital, Surabaya. Amies swab was streaked on MacConkey selective medium supplemented by cefotaxime 2 mg/L and incubated for 18-24 h at 37˚C. The growing colonies were ESBL confi rmed by Modifi ed Double Disk Synergy Test (M-DDST). Colonies which grow were inoculated in Mueller Hinton medium (0,5 MacFarland) with five (5) antibiotics disk : amoxicillin/ clavulanic (AMC) 30 ug, ceftazidime (CAZ) 30 ug, cefotaxime (CTX) 30 ug, ceftriaxone (CRO) 30 ug, and aztreonam (ATM) 30 ug which placed within 15 mm of distance from edge to edge of AMC disk.13 Incubated for 18-24 hours at 37˚C. The inhibition zone which show synergy zone between one of cephalosporin disk or aztreonam disk with amoxicillin/clavulanic disk was confi rmed as ESBL producer. ESBL positive strain were bacteriologically identifi ed using the biochemical test: Triple Sugar Iron (TSI) test, Indol test, Methyl Red (MR) test, Voges Proskauer (VP) test, Urease test, and Motility test. All bacterial isolates were then stored in deep freeze minus 80˚C. Genotypic Examination Genotypic examination was held in Institute of Tropical Diseases, Universitas Airlangga, Surabaya. DNA Extraction DNA extraction was conducted by boiling method. The identifi ed ESBL producing bacteria were re-cultured on Mueller Hinton medium, incubated at 37˚C for 18 – 24 h. Four to fi ve colonies were taken and suspended in sterile distilled water in 1,5 ml Eppendorf tube. The suspension was homogenized with vortex for 15 seconds and immersed in a thermostat at 95˚C for 10 minutes, then centrifuged at 14.000 rpm for 1 minutes. The supernatant was used as DNA template in PCR and stored in -20˚C.14 DNA Amplifi cation Three ESBL gene primers are used to amplify and identify the ESBL gene, as follow (Table 1) : [15] PCR reaction was run in volume of 20 μl: 10 ul of GoTaq Green Master Mix 2x (Promega),1 ul for each of forward and reverse primers, 3 μl nuclease free water, and 5 μl DNA template. PCR was run as follow: for blaCTX-M: denaturation on 94ºC for 7 minutes and the following 35 cycles on 94ºC for 50 seconds, annealing on 50ºC for 40 seconds, extension on 72ºC for 1 minute, and fi nal extension on 72ºC for 5 minutes; for blaSHV: denaturation on 96ºC for 5 minutes and the following 35 cycles on 96ºC for 1 minute, annealing on 60ºC for 1 minute, extension on 72ºC for 1 minute, and fi nal extension on 72ºC Table 1. Primers of ESBL Genes Gen Sekuens Primer (5’-3’) Amplicon size (bp/base pair) blaCTX-M F : 5’ ATGTGCAGYACCAGTAARGT 3’ R : 5’ TGGGTRAARTARCTSACCAGA 3’ 593 blaSHV F : 5’ GGTTATGCGTTATATTCGCC 3’ R : 5’ TTAGGTTGCCAGTGCTC 3’ 867 blaTEM F : 5’ ATGAGTATTCAACATTTCCG 3’ R : 5’ CTGACAGTTACCAATGCTTA 3’ 867 147Agusta Reny Soekoyo, et al.: The Epidemiological Pattern and Risk Factor of ESBL Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 for 10 minutes; and for blaTEM: denaturation on 96ºC for 5 minutes and the following 35 cycles on 96ºC for 1 minute, annealing on 58ºC for1 minute, extension on 72ºC for 1 minute, and fi nal extension on 72ºC for 10 minutes. PCR amplicon were visualized in 2% gel electrophoresis. Questionnaire to Find Risk Factors Information about risk factor of ESBL producing Enterobacteriaceae in dairy cows and farmers were obtained through interview and questionnaires. Enterobacteriaceae strain and genotype distribution among dairy cows and farmers and risk factors were analyzed by Chi Square/Fisher Exact Test on SPSS 22 version program. RESULTS D i s t r i b u t i o n o f E S B L P r o d u c i n g Enterobacteriaceae in Dairy Cows and Farmers Prevalence of ESBL producing bacteria in dairy cows was 13.7% (14/102) and in farmers 34.3% (35/102). ESBL producing Enterobacteriaceae in dairy cows were 6.9% (7/102) and in farmers 33.3% (34/102) (Table 2). The ESBL producing Enterobacteriaceae in dairy cows were mostly: Escherichia coli 85.7% (6/7) and Enterobacter spp 14.3% (1/7), whereas among 34 ESBL-E in human were Escherichia coli 82.4% (28/34), Enterobacter spp 14.3% (3/34), Klebsiella pneumoniae 5.9% (2/34), and Klebsiella oxytoca 2.9% (1/34). There were no signifi cant diff erences in distribution of ESBL producing Enterobacteriaceae strain in dairy cows and in farmers (Table 2) and Fig.1. Escherichia coli were identifi ed as dominant strain of ESBL producing Enterobacteriaceae in dairy cows and farmers (85.7% vs. 82.4%). Distribution of ESBL Gene Among Dairy Cows and Human (Farmers) A m o n g s e v e n E S B L p r o d u c i n g Enterobacteriaceae in dairy cows, six isolates were harbored blaCTX-M (85.7%) and one an unidentifi ed gene (14.3%). Among 34 isolates of ESBL producer in farmers, 26 isolates harbored blaCTX-M (76.5%), 15 isolates blaTEM, and three Table 2. Distribution of ESBL producing Enterobacteriaceae strain in dairy cows and farmers ESBL Producer Dairy Cows (n=102) Farmers (n=102) p value ESBL producing bacteria 14 (13.7%) 35 (34.3%) Non- Enterobacteriaceae 7 (6.9%) 1 (0.9%) Enterobacteriaceae 7 (6.9%) 34 (33.3%) Escherichia coli 6 (85.7) 28 (82.4) p = 1,000 Enterobacter spp 1 (14.3) 3 (8.8) p = 0,542 Klebsiella pneumoniae 0 / 0 2 (5.9) p = 1,000 Klebsiella oxytoca 0 / 0 1 (2.9) p = 1,000 Note: ESBL-E: ESBL producing Enterobacteriaceae Figure 1. The Double Disk Synergy Test (DDST) for identifying ESBL producer bacteria. Note: The increasing of inhibition zone in area between cephalosporin disk and clavulanic acid disk was marked as positive ESBL producer. Table 3. ESBL genes distribution of ESBL producing Enterobacteriaceae in dairy cows and farmers ESBL Gene Dairy Cows (n=7) Farmers (n=34) p value blaCTX-M 6 (85.7) 26 (76.5) p = 1,000 blaSHV 0 (0) 3 (8.8) p = 1,000 blaTEM 0 (0) 15 (44.1) p = 0,035 Unidentifi ed gene 1(14.3) 0 (0) p = 0,171 blaCTX-M, blaTEM 0 (0) 8 (23.5) - blaCTX-M, blaSHV, blaTEM 0 (0) 1 (2.9) - Note: Unidentifi ed gene: gene of ESBL producing Enterobacteriaceae which couldn’t detected with specifi c primer used in this study 148 Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Indonesian Journal of Tropical and Infectious Disease, Vol. 8 No. 3 September–December 2020: 144–151 isolates blaSHV (8.8%), respectively. There was a signifi cant diff erence of blaTEM distribution in dairy cows and in farmers (p = 0, 035) (Table 3). Combination of two and three of ESBL genes were found in Enterobacteriaceae producing ESBL isolates in farmers. Eight isolates harbored blaCTX-M dan blaTEM (23.5%) and one isolate harbored blaCTX-M, blaSHV, blaTEM (2.9%) (Table 3). Risk Factor for Colonization of ESBL Producing Enterobacteriaceae Age, origin of dairy cows, history of illness during the last 3 months, history of drug use, type of drug given last 3 month, and type of feed were not risk factors for colonization of ESBL-producing Enterobacteriaceae in dairy cows. Risk factor for ESBL producing Enterobacteriaceae colonization in farmers was the use of antibiotics (p = 0.007). Gender, age, education level, household, hygiene sanitation of environment (location of dairy cow shed and type of toilet), personal hygiene sanitation (frequency and how to wash hands), and frequency of going out of the city during the last 3 months were not a risk factor. DISCUSSION C o l o n i z a t i o n o f E S B L p r o d u c i n g Enterobacteriaceae in dairy cows by 6,9% in this study is similar with study in healthy ruminant (cows and buff aloes) in rural areas in Cambodia by 7%16 and lower than in cattle farm in German by 54.5%.6 Colonization of ESBL producing Enterobacteriaceae in farmers by 33,33% lower than the colonization of ESBL-producing bacteria in healthy individuals in rural areas in Thailand by 65.7%,17 in China by 73.9%,18 and workers in cattle farms in Germany: 12.5%. 6 Human and animal gut were the natural habitat of many bacterial especially Enterobacteriaceae and become a reservoir of various infections.12 Non-appropriate and overuse of antibiotic caused selective pressure that supports the growth of resistance bacteria.9 Colonization of resistance bacteria in human and animal gut causing transmission of resistance genes in gut flora bacterial through horizontal gene transfer by conjugative plasmid.12 ESBL mostly encoded by genes in plasmids.19 Enterobacteriaceae was identifi ed as having plasmid carrying resistant genes. IncFII plasmid group known as a plasmid group that encoded ESBL genes and it widely distributed in Enterobacteriaceae. It called epidemic resistant plasmid group.20 This study identifi ed Escherichia coli as the dominant ESBL producing bacteria in dairy cows (85.7%) and farmers (82.4%). Distribution of Escherichia coli as an ESBL producer in dairy cows in this study was 85.7%, higher than in cattle farms in Mecklenburg-Western Pomerania, Germany by 54.5%.6 At farmers, distribution of ESBL producing Escherichia coli by 82.4% is lower than the distribution of Escherichia coli in healthy individuals in rural areas in Thailand by 85.4% 17 and in China 88%,18 but higher than workers in cattle farms in Germany: 12.5%. 6 Escherichia coli is the main organism that produces ESBL in communities21 and associated with urinary tract infections (UTI). It is related to their role as gut bacterial fl ora and are pathogenic to humans and animals.12 The resistance of commensal Escherichia coli to antimicrobial agents has been found in healthy individuals.22 This bacterium also acts as an indicator of 'acquired antibiotic resistance genes’ in the community.23 Distribution of blaCTX-M in the ESBL producing Enterobacteriaceae in dairy cows by 85.7% is higher than the distribution of blaCTX-M in cattle farms in Germany 80%.6 At farmers, Figure 2. Electrophoresis of amplifi ed gene of blaCTX-M (593 bp) 149Agusta Reny Soekoyo, et al.: The Epidemiological Pattern and Risk Factor of ESBL Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 distribution of blaCTX-M in the ESBL-producing Enterobacteriaceae by 76.5% higher than in healthy individuals in rural areas in China by 68.1%18 and in Thailand by 65.7%.17 blaCTX-M that mostly integrated with conjugative plasmid, and conjoint with the other resistant gene, has a higher transferability among bacteria, and impact on higher prevalence epidemiologically.24 blaCTX-M, blaSHV, and blaTEM are dominant ESBL genes in various regions worldwide and found from isolates of humans, animals and the environment. blaTEM and blaSHV mainly found in Escherichia coli and Klebsiella pneumoniae.25 The study identifi ed blaTEM in Escherichia coli and Enterobacter spp isolates and blaSHV in Klebsiella pneumoniae and Escherichia coli isolates. blaCTX-M is the dominant ESBL gene worldwide, especially in community and has increased in incidence since 2000.12 It were identifi ed in livestock and pets with Escherichia coli as the main producing bacteria.26 Our study showed that blaCTX-M was identifi ed in Escherichia coli, Enterobacter spp, and Klebsiella oxytoca. Dissemination of blaCTX-M occurs rapidly, extensive, and significantly. Plasmids are known to carry genes which encode resistance for antibiotic.27 Conjugative plasmids play an important role in facilitating the horizontal dissemination of blaCTX-M among bacteria.28 blaCTX-M was identified in various epidemic resistant plasmid groups, including: groups IncF, IncN, IncI1, IncL / M, and IncHI2.24 These plasmid group are able to capture and transfer resistant genes among bacteria.29 The IncFII Group is the largest plasmid group encoding blaCTX-M and widely found in Enterobacteriaceae24 and isolates from human and animal.20 ISEcp1 is the genetic element which associated with all variants of blaCTX-M, play a role incoding transposase and inducing blaCTX-M expression. Transposase is an enzyme that mobilizes blaCTX-M in certain plasmids.28 Other types of IS include: ISCR1 plays a role in blaCTX-M group 2 and 9 expression, IS10 in blaCTX-M group 8 expression,24 and IS26 in blaCTX-M group 1 and 9 expression. ISEcp1 and ISCR1 play a role in the mobilization of class 1 integron that encodes various types of resistant genes (MDR cassettes).28 Clones of Escherichia coli were identifi ed having a signifi cant role in the dissemination of blaCTX-M, among others, such as ST131, ST38, ST393, ST405. ST131 serotype 025: H4 phylogenetic group B2 is an extra-intestinal pathogenic E. coli strain and was mainly involved in blaCTX-M dissemination especially blaCTX-M- 15 in worldwide.24 It identifi ed having IncFII plasmid group and found in isolates originated from the animal, environment, and especially human.28 Combination of two or three ESBL genes in one bacterial isolate is due to integron and plasmid that carry several resistant genes. Enterobacteriaceae would be harboring of 5 to 6 plasmids in one isolate.30 Class 1 integron which related to blaCTX-M were identified encoding several types of resistant genes (MDR cassettes).28 The unidentifi ed gene is thought to be an ESBL gene in addition to blaCTX-M (group 1), blaSHV, and blaTEM. The fi nding of antibiotic use as risk factor of ESBL producing Enterobacteriaceae in farmers in this study (p= 0,007) related according to the study of Luvsansharav et al,17 which identifi ed the use of antibiotics in the last 3 months (OR 1,883; 95% CI 1,221-2,903) as a risk factor for colonization of ESBL producing bacteria in healthy individuals in rural area in Thailand and Zang et al18 that identifi ed antibotic use in the previous 6 months (OR 1,892; 95% CI 1,242–2,903; p = 0.034) as a risk factor for colonization of ESBL-producing bacteria in healthy individuals in rural area in China. In dairy cows in this study, there was not any antibiotic use detected based on data on questionnaires. The total of 52% of dairy cows were given anthelmintic every three months. Risk factors for colonization of ESBL producing Enterobacteriaceae in dairy cows was not identifi ed. Dissemination of ESBL producing bacteria occurs from animals to humans or vice versa.6 ESBL producing Gram negative in dairy cows have the potential as a zoonotic risk,31 especially through close contact during daily care.6 150 Copyright © 2020, IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Indonesian Journal of Tropical and Infectious Disease, Vol. 8 No. 3 September–December 2020: 144–151 The results of the study showed that the rural community could act as a reservoir of ESBL- producing Enterobacteriaceae. The fi nding of Escherichia coli and blaCTX-M as the dominant strain and ESBL gene epidemiologically indicated alarming sign. E. coli ST131 consider as virulent strain,24 multiple resistance, easily colonize and spread between humans, animals and environment isolates.27 It contributes to the spread of blaCTX-M globally through horizontal gene transfer.24 ESBL-producing E. coli and blaCTX-M have driven the spread of ESBL gene in the community. Colonization of ESBL-producing Enterobacteriaceae is a risk factor for infection of ESBL-E.3 The colonization of ESBL-producing Enterobacteriaceae in community were predicted increasingby about 5% annually.24 This certainly becomes a challenge in therapy of infectious disease. CONCLUSION Farmers had suspected as the source of ESBL producing Enterobacteriaceae based on higher prevalence. The use of antibiotic in human, was identifi ed as risk factor for colonization of ESBL producing Enterobacteriaceae while not identifi ed in dairy cows. CONFLICT OF INTEREST There is no confl ict of interest of this study. ACKNOWLEDGEMENT We thank staff and cadre of Sumberpitu Public Health Centre for assistance of collecting samples, Health Offi ce of District of Pasuruan for the study support. Microbiology Laboratory of Dr. Soetomo and Institute Tropical Diseases, Surabaya for processing samples. Thank you to Tahir professorship grant Universitas Airlangga No. 1149/UN3/2018, that supported this study. REFERENCES 1. Health Ministry of The Republic of Indonesia. Antimicrobial Resistance Control Becomes World Attention. News Release. 2018. [cited 30 January 2019]. Available from:. 2. Shaikh S, Fatima J, Shakil S, Rizvi SMD, Kamal MA. Antibiotic Resistance And Extended Spectrum β-Lactamase : Types, Epidemiology And Treatment, Saudi J Bio Sci. 2015 ;22(1) ;90-101. doi: http://dx.doi. org/10.1016/j.sjbs.2014.08.002 3. B r o l u n d A . O v e r v i e w o f E S B L - p r o d u c i n g Enterobacteriaceae From A Nordic Perspective. 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