Resistance Profile of Extended Spectrum Beta Lactamase-Producing Escherichia coli Bacteria using Vitek 2 Compact Method

Doctoral Program in Veterinary Science, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia Department of Microbiology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia Department of Veterinary Public Health, Faculty of Veterinary Medicine, Airlangga University, Surabaya, 60115, Indonesia


Introduction
Disease in commercial chicken is one of constraints causing the decline in economic value (Wiedosari and Wahyuwardani, 2015). Pathogenic microorganisms cause infectious diseases, which is one of a leading cause of death in animals or even human. The high prevalence of infectious disease is associated with the high use of antibiotic in health care (Noor and Peoleonga, 2005). A negative economic consequence in commercial chicken production can be caused by Escherichia coli (E.coli) infection. The existence of Extended Spectrum Beta Lactamase (ESBL)producing E.coli on commercial chicken is highly associated with E.coli, a normal microflora on gastrointestinal tract of commercial chicken. Feces and animal housing environment can facilitate the existence of E.coli, although, chicken show no symptoms at all of any diseases caused by the bacteria (Wibisono et al., 2018).
Commercial chicken farm is one of sources of antibiotic resistance. The uncontrolled use of antibiotic causes the emergence of antibiotic resistance. Beta lactam antibiotic is the type of antibiotic commonly used in commercial chicken production. Resistance to beta lactam antibiotic can occur in ESBL-producing E.coli (Santos et al., 2013;Hammerum et al., 2014). Extended Spectrum Beta Lactamase is an enzyme that Buletin Peternakan 44 (2): 48-53, May 2020 Bulletin of Animal Science ISSN-0126-4400/E-ISSN-2407-876X Accredited: 36a/E/KPT/2016 http://buletinpeternakan.fapet.ugm.ac.id/ causes resistance to wider spectrum on third generation of cephalosporins and mobobactams; thus, the antiobiotic becomes ineffective. ESBLproducing bacteria can also be resistant to wide array of antibiotic class: aminoglycoside, tetracycline chloramphenicol, and sulfamethoxazole-trimethoprim (Brower et al., 2017;Sudarwanto et al., 2017). Incidence on multidrug resistance to third generation cephalosporins and other antibiotic classes are often found in ESBL-producing bacteria (Masruroh et al., 2016). Multidrug resistance is a resistance to three or more different antibiotic classes (Handayani et al., 2017).
Identification of ESBL bacteria is a problem in confirming a diagnosis at the laboratory, either in therapeutic approach or in the field to prevent its spread. Clinical and Laboratory Standards Institute (CLSI) states that ESBL examination must be performed routinely. According to CLSI recommendation, ESBL detection consists of two steps. The first is an initial screen test, a filter test to reduce the susceptibility to more than one cephalosporin indicators (cefotaxime, ceftriaxone, ceftazidime, cefpodoxime) and aztreonam. The susceptibility reduction from cephalosporins shows positive results. A positive result from screening test is followed by the second step, ESBL confirmation test. The second step aims to detect the hydrolytic potency of ESBL against antibiotic used in the screening test. The ESBL confirmation test deliver a picture of collaboration action between ceftazidime or cefotaxime, and clavulanate acid (Amelia et al., 2016;CLSI, 2017;Biutifasari, 2018).
Methods of identification, antibiotic sensitivity test, and ESBL-producing bacteria confirmation test are categorized into phenotype and genotype examination methods, with their advantages and limitations. Both phenotypic bacteria identification and antibiotic sensitivity is relatively easier than genotypic examination. Phenotypic examination is performed by using agar testing method or fully automated automatic method as in VITEK ® 2 compact automated system. The VITEK ® 2 compact automated system from Biomerieux works on calorimetric principle for identification, through biochemical and antibiotic sensitivity tests. The accuracy of VITEK ® 2 compact automated system ranges from 97.8% (O'Hara, 2005) to 98.02% (Duggal et al., 2012). This study aimed to determine resistance profile and the nature of multidrug resistance in ESBL-producing E.coli against several classes of antibiotic. Antibiotic resistance is a problem occurring across the globe, including Indonesia, both in human and animal health.

Materials and Methods
This study used commercial chicken cloaca swab sample, collected from commercial chicken farms in Blitar Regency. 22 positive ESBL-producing E.coli isolate were identified using IMBIC test and confirmed as ESBL-producing E.coli using Double Disc Synergy (DDST) test (CLSI, 2017;Effendi et al., 2018). Identification of ESBL-producing E.coli isolate was performed using VITEK ® 2 GN card, while VITEK ® 2 AST cards were used to determine the resistance profile. 3 ml of sterile saline solution (0.45 -0.50%; pH 4.5 -7.0) was put into plastic tube aseptically to make a bacterial suspension. E. coli isolate was isolated on MacConkey Agar (MCA) medium, incubated at 35-37˚C for 20-24 hours. E. coli isolate was put int saline solution using sterile swab to create bacterial suspension. Bacterial suspensions were homogenized and 0.50 -0.63 McFarland bacterial turbidity were made using VITEK ® 2 DensiCHEK. These bacterial suspensions were inoculated to VITEK ® 2 card no more than after 30 minutes. Bacterial suspension tubes and VITEK ® 2 cards (GN and AST) were placed into special rack or cassette. Racks containing bacterial suspensions and cards were placed into vacuum chamber station for 30-60 seconds. Bacterial suspension was then transferred into wells. Transfer tubes were cut automatically. Cards were transferred to incubator room after 15 minutes, for approximately 8 hours at 35˚C. Cards were then analyzed automatically. Antibiotic sensitivity test on ESBL-producing E.coli was carried out using VITEK ® 2 compact, that consists of 18 antibiotics: amoxicillin, ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, cefazolin, cefotaxime, ceftazidime, ceftriaxone, cefepime, aztreonam, ertapenem, meropenem, amikacin, gentamicin, ciprofloxacin, tigecycline, nitrofurantoin, dan trimethoprim/sulfamethoxazole. The results were automatically analyzed by system and interpreted as sensitive, intermediate, or resistant (Sugiartha, 2016;Biomerieux, 2017). ESBL-producing E.coli multidrug resistance in this study used six different antibiotic classes: beta lactam, aminoglycoside, quinolone, tetracycline, nitrofuran, and sulfonamide-potential.

Results and Discussion
The resistance test results of ESBLproducing E.coli from commercial chicken cloaca swabs against several classes of antibiotic ( Figure  1) shows high level of resistance. The highest percentage of resistance was observed in amoxicillin, ampicillin, cefazolin, cefotaxime, and ceftriaxone, by 100%. This result is according to resistance study on ESBL-producing E.coli at RPHR of Bogor City that showed 100% level of resistance on penicillin and amoxicillin (Normaliska et al., 2019). Amoxicillin, ampicillin, cefazolin, cefotaxime, and ceftriaxone are categorized as beta lactam class antibiotic, amoxicillin and ampicillin belong to penicillin, cefazolin belongs to first generation cephalosporins, cefotaxime and ceftriaxone is third generation cephalosporins class. E.coli which is often referred as ESBL-producing bacteria has ability to produce beta lactamase enzyme, an enzyme that able to inhibit beta lactam (Paterson and Bonomo, 2005). Beta lactam resistance commonly occurs in gram negative bacteria, including E.coli that can be isolated form animal products. Poultry is known as ESBL-producing E.coli reservoir (Schmid et al., 2013;Hammerum et al., 2014). The high resistance level of ESBLproducing E.coli from commercial chicken cloaca swab on beta lactam class antibiotic is caused by the presence of ESBL enzyme in E.coli, which is not limited on digestive tract, but also in the cage, feces, and surrounding environment (Schroeder et al., 2004). The enzyme is not only able to hydrolyze penicillin, but also third generation cephalosporins and monobactam (Paterson and Bonomo, 2005;Nuangmek et al., 2018).
The susceptibility result shows a nearly similar pattern for all isolates (Table 1). The most resistance pattern seen on ESBL-producing E.coli was AM/AMP/KZ/CTX/CRO/ATM/GM/CIP pattern (22.73% or 5/22), and AM/AMP/KZ/CTX/CRO/ATM/GM/CIP/SXT (18.2% or 4/22). The result of t multiclass antibiotic resistance shows 86.36% (19/22) had multidrug resistance (Table 2). Multidrug resistance is certain bacterial resistance to three or more different antibacterial class (Kurniawati et al., 2015). Multidrug resistance is a problem that hard to resolve in disease treatment. This condition is caused by the practice of the antibiotic use as disease preventive measures in commercial chicken production. Antibiotic has been used to control the level of morbidity, mortality, and infection of E.coli. While antibiotics are mostly used as therapeutic, several antibiotic classes are used for sub therapeutic purpose to prevent the emergence of disease in animal production (Niasono et al., 2019). The use of antibiotic at sub therapeutic level can lead to the emergence of multidrug resistance (Wang et al., 2015).
The highest level of multidrug resistance patter of ESBL-producing E.coli was observed on BL/AG/Q/SP pattern (50% or 11/22). Other multidrug resistance patterns found in this study were BL/AG/Q/SP (18.20% or 4/22), BL/AG/Q/SP (13.64% or 3/22), and BL/AG/Q (4.55% or 1/22). This study demonstrates that the high incidence of multidrug resistance can indicate the high risk of transmission on other chicken at the farm, to cause a transmission of antibiotic resistance on multi class antibiotics. The condition can result in the high potential failure of antibiotic treatment    during the existence of infectious disease at the farm. The different resistance pattern arises due to the varied antibiotic combinations used by farmers, as stated by Bywater et al. (2014) in which various type of antibiotics, geographic conditions, and production system are factors causing the different resistance pattern.