Escherichia Coli Isolated from Raw Milk

p-ISSN 2615-787X e-ISSN 2615-790X ANSHARIETA ET AL. / Tropical Animal ScienceTropical Journal Animal 44(1):10-15 Science Journal, March 2021, 44(1):10-15 Accredited by Directorate General of Strengthening for Research DOI: https://doi.org/10.5398/tasj.2021.44.1.10 and Development No: 30/E/KPT/2018 Available online at http://journal.ipb.ac.id/index.php/tasj Genetic Identification of Shiga Toxin Encoding Gene from Cases of Multidrug Resistance (MDR) Escherichia coli Isolated from Raw Milk R. Ansharietaa, M. H. Effendib,*, & H. Plumeriastutic aMaster Program in Animal Diseases and Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, bDepartment of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, cDepartment of Veterinary Pathology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, 60115, Indonesia *Corresponding author: [email protected] (Received 29-03-2020; Revised 08-07-2020; Accepted 22-07-2020) ABSTRACT Escherichia coli is one of bacteria which have resistant to three or more classes of antimicrobial agents. E. coli having resistant to three or more classes of antimicrobial drugs can be defined as multidrug-resistant (MDR) bacteria. The aim of the study was to evaluate the expression of Shiga toxin gen in MDR E. coli. A total of 250 raw milks samples were taken from dairy farms in Kediri, Probolinggo, Pasuruan, Blitar, and Batu Region, East Java Province, Indonesia. Each sample was cultured into enrichment media Brilliant Green Bile Lactose Broth and Eosin Methyllen blue agar, then identified with TSIA agar and IMVIC biochemistry test. Antibiotic sensitivity testing was done using Kirby- Bauer disc diffusion assay on medium Mueller-Hinton agar (Oxoid, CM0337). Antibiotics disks used were 30 µg of Tetracycline (Oxoid, CT0054), 10 µg of Streptomycin (Oxoid, CT0047), 30 µg of Chloramphenicol (Oxoid, CT0013), 5 µg of Trimethoprim (Oxoid, CT0057), and 30 µg of Aztreonam (Oxoid, CT0264). Isolate showing resistance to at least 3 antibiotics disk were then continued with PCR assay to identify Shiga toxin E. coli (STEC) encoding stx2 gene. The study was designed to evaluate the nucleotide analysis of STEC gene. The result showed that 6.25% (1/16) of STEC encoding gene was found in MDR E. coli. This report of molecular identification on the presence of STEC gene in MDR E. coli confirmed a wider spread of MDR E. coli that can threaten animal health and human health. Keywords: stx2 gene; MDR; Escherichia coli; raw milk; public health INTRODUCTION human and animal health (Ventola, 2015). Irrational treatment and administration of antibiotics greater Escherichia coli which produces Shiga toxin (STEC), than the recommended dosage in agriculture increase is defined as a group of E. coli which can produce a toxin the incidence of antimicrobial resistance in daily life, called Shiga toxin (stx). There are two main types of and this condition has a great impact on the social and Shiga toxins found in this STEC group. The two toxins economic aspects (Barriere, 2014). Antibiotic resistance are stx1 and stx2 (Hunt, 2010). According to several makes health workers lose many antibiotic choices reports of findings in the field, stx2 type toxin is more in the treatment of bacterial diseases, causing the common and more prevalent than stx1 toxin in E. coli high potential for treatment failure in cases of clinical isolates obtained from livestock feces (Tahamtan et al., complications (Llor & Bjerrum, 2014). The applications 2010). Cattle are believed to be the main reservoir of of good hygiene and sanitation practices in animal the STEC bacteria (Hussein & Sakuma, 2005). STEC is farms are something that should be emphasized to often identified with foodborne outbreaks throughout prevent the distribution of MDR microbes in the the world with mild symptoms such as mild diarrhea to populations and the transfer of MDR microbes from severe symptoms such as hemolytic uremic syndrome animals to humans. (HUS) (Noris et al., 2012). Foods related to STEC infec- Although antibiotic treatment is not the first choice tion are foods without cooking processes, such as raw in cases of infections caused by STEC, multidrug- meat, cheese, non-pasteurized milk or raw milk, fruit resistant (MDR) STEC is a public health problem that and vegetable juices, and other natural ingredients is quite dangerous because the strain acts as a reservoir (Mohammadi et al., 2013). of resistant genes (Cointe et al., 2018). MDR STEC Antimicrobial resistance is a global problem, bacteria can easily transfer the resistant genes to the in which scientific research has been carried out and other Enterobacteriaceae family bacteria in the body of indicates that this problem has a negative impact on living organisms or in the environment. Some bacteria 10 March 2021 ANSHARIETA ET AL. / Tropical Animal Science Journal 44(1):10-15 in the gut of living organisms are friends and have the ducted by measuring the diameter of the inhibitory zone potential to transmit resistance genes to one another formed, based on Clinical and Laboratory Standards (Colavecchio et al., 2017). A number of researches have Institutions, and the resistance to Aztreonam that is the been conducted on STEC isolates from humans, but only monobactam can be referred as a presumptive extended a few have been identified from foodstuffs of animal spectrum beta lactamase (ESBL) (CLSI, 2018). origins. Report on the status of STEC bacterial resistance Isolate which shown resistance to at least 3 antibi- to antibiotics is also limited. Therefore, this study was otics disk was then taken from the LB Broth and trans- conducted to identify the STEC gene, stx2, which is ferred onto Nutrient Agar (Merck, 105450). The whole a prevalent form of MDR E. coli bacteria isolated from cell suspensions were made by mixing approximately several dairy farms in East Java, Indonesia. 8-10 colonies from Nutrient Agar to 0.3 mL Tris-HCl containing EDTA (TE) buffer. The cell lysate was made MATERIALS AND METHODS by heating the suspension for 10 min with the boiling method. The lysates were centrifuged for 10 min at 8000 The sample size in this study was 250 raw milk rpm to shed the cellular debris. A volume of 5 µL of the samples taken from the cow bulk milk of each dairy supernatant was used as a template for amplification farm located in Kediri (K), Probolinggo (A), Pasuruan by PCR. The specific primer used in this study was stx2 (G), Blitar (S), and Batu (H), East Java, Indonesia, according to Brenjchi et al. (2011), F: 5′- CCA TGA CAA during the period of September-December 2019. The CGG ACA GCA GTT-3′ and R: 5′- CCT GTC AAC TGA study was conducted by purposive sampling with GCA CTT TG-3′. sample criteria of poor enclosure sanitation and dirty Each PCR reaction was performed in a 25 µL am- environment (Dairy, 2019). The number of farms plification mixture consisting of 2.5 µL 10 X PCR buffer calculated on average reached 1500-1800 lactation cows (500 mM KCl, 200 mM Tris HCl), 0.5 µL dNTPs (10 per location. Samples taken at each dairy farm location mM), 1 µL MgCl2 (50 mM), 1.25 µL of each primer (0.5 were 50 samples (cow bulk milk). A total of 25 mL of µM), 0.2 µL of Taq DNA polymerase (5 unit/µL), and 2 milk samples from each milk can be taken and placed in µL of template. The thermocycler (Bio Rad, Hercules, sterile plastics. Milk samples were put into a cool box at CA) program was started with initial incubation at 94°C 4ᵒC for transportation (Arya et al., 2012; Kristianingtiyas for 5 min, followed by 35 cycles of denaturation at 94°C et al., 2020; Putra et al., 2019). for 60 sec, annealing at 52°C for 30 sec and elongation at Each sample was cultured into enrichment media 72°C for 60 sec, and a final extension at 72°C for 10 min. of Brilliant Green Bile Lactose Broth (Merck, 105454) The PCR products were separated by electrophoresis in and incubated at 37ᵒC for 18-24 hours. The positive 1.5% agarose gel at 100 V for 40 min in the tris-acetate results were characterized by the change of media from buffer, visualized by ethidium bromide staining, illumi- green to cloudy green color and the presence of gas in nated by UV-transilluminator, and documented by a gel Durham tube (Effendi et al., 2019). The positive samples documentation apparatus. DNA ladder with 100 bp was were then cultured in Eosin Methylene Blue (EMB) used as a marker for m-PCR assay. The expected size of (Merck, 101347) and incubated at 37°C for 18-24 hours products for stx2 is 779 bp (Brenjchi et al., 2011). (Putra et al., 2019). The colonies showing metallic green colors were purified and continued to the identification RESULTS steps with Triple Sugar Iron Agar (TSIA) and Indol- Motility, Methyl Red, Voges Proskauer, and Citrate A total of 250 samples were taken from sev- (IMViC) tests. The positive samples were then stored in eral dairy farms in East Java Province, Indonesia. E. LB Broth (Himedia, M1245) for further use in the next coli isolates were found in 176 samples of fresh cow’s testing. milk. The E. coli isolates were then tested using Kirby Antibiotic sensitivity testing was done us- Bauer disc diffusion assay with several antibiotics, ing Kirby-Bauer disc diffusion assay on Mueller- namely Tetracycline, Streptomycin, Trimethoprim, Hinton agar medium (Oxoid, CM0337). Antibiotics Chloramphenicol, and Aztreonam. The number of iso- disks used were 30 µg of Tetracycline (Oxoid, lates that were resistant to Tetracycline, Streptomycin, CT0054), 10 µg of Streptomycin (Oxoid, CT0047), Trimethoprim, Chloramphenicol, and Aztreonam was 30 µg of Chloramphenicol (Oxoid, CT0013), 5 µg of 17.05% (30/176), 14.2% (25/176), 9.66% (17/176), 7.95% Trimethoprim (Oxoid, CT0057), and 30 µg of Aztreonam (14/176), and 1.7% (3/176), respectively (Table 1).

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