Phenotypic and Genotypic Study of Antibiotics Resistance Profile in Escherichia coli Isolated from Broilers Farm in Cianjur, Indonesia Kajian Fenotip dan Genotip Profil Resistensi Antibiotik pada Escherichia coli yang Diisolasi dari Peternakan Ayam Broiler di Cianjur, Indonesia

This study aimed to investigate the phenotypic and genotypic of antibiotics resistance profile in Escherichia coli. The 30 samples come from cloacal swab of broilers in Cianjur, Indonesia. Isolation and identification of E. coli was performed by culturing in McConkey agar, eosin methylene blue agar, Gram staining and five essential biochemical tests (indole, methyl red, Voges-Proskauer, and citrate). In this study, 10 isolates (33.3%) were confirmed E. coli positive. Phenotypic profile was performed by screening all isolates with 8 antibiotics of 6 antibiotic groups. The screening was carried by Kirby-Bauer disk diffusion method based on the standard of CLSI. For genotypic profile, each resistant isolate was detected antibiotic resistance-encoding gene. The result showed all isolates (100%) resistant against tetracyclin, oxytetracycline and erythromycin. Nine isolates (90%) detected nalidixic acid and enrofloxacin-resistant. The ciprofloxacin and gentamicin-resistant isolates were 70% and 40%, respectively. There was no resistant isolate for chloramphenicol. Multi drug-resistant was detected on 90% isolates. Only gyrA (100%) and tetA (80%) genes were detected. This study showed high rate of occurrence of antibiotic resistance in E. coli. Not all resistant isolates were detected to have target genes encoding antibiotic resistance in this study.


INTRODUCTION
Antibiotic resistance has become an increasingly uncontrollable health problem in recent years. Global awareness of the emergence of multi drugresistant bacteria (MDR) has increased but new MDR strains have also increased (Solomon & Oliver, 2014). Therefore, the health threat due to MDR bacterial infection is increasing (Mohammad et al., 2015) Antibiotic resistance can lead to ineffectiveness of standard treatment, persistent infection, and can spread to other individuals (WHO, 2019). Patients with these infections need a more complicated treatment plan because of the ability of bacterial cells to fight off antibiotics, resulting in higher morbidity and mortality rates (Bonomo, 2000). The United States Centers for Disease Control and Prevention (CDC) estimated in 2013 that at least 2 million people acquire antibiotic-resistant bacterial infections each year and more than 23 thousand people die from these infections (Mohammad et al., 2015).
Improper use of antibiotics in livestock, farms, domestic animals, plants and humans causes antibiotic resistance in agricultural and clinical biomes. Antibiotic resistance to pathogenic bacteria is caused by the widespread and uncontrolled use of antibiotics in agricultural and medical (Cameron & McAllister, 2016). Many countries in the world have made substantial efforts to reduce the use of antibiotics in food-producing animals. These efforts include establishing national reduction targets, implementing a ban on antibiotics in animal feed for food-producing animals, comparing the use of antibiotics at the farm level, and encouraging antibiotic stewardship such as requiring susceptibility testing prior to the use of high priority antibiotics. This kind of intervention is expected to reduce the rate of antibiotic resistance in animals, the environment, and in humans (Karen et al., 2017).
Escherichia coli, including the family Enterobacteriaceae, acts as commensal bacteria and pathogens. They can be found in the environment, food, animal intestines, and human intestines (Markey et al., 2013). It is known that E. coli is a bacterium that has antibiotic resistance properties (Kurnia et al., 2018). According to WHO (2017), E. coli is the main bacterial cause of the spread of antibiotic resistance. It expresses the MDR mechanism by multiple and varied mechanisms i.e. changing the target site to prevent antibiotic binding, stopping the active pump of the antibiotic entry pathway into cells and impaired absorption of antibiotics due to reduced outer membrane permeability (Tilak, 2011). Expression of antibiotic resistance mechanisms encoded by certain genes. Bacteria that are resistant to more than one antibiotic class also have more than one resistance coding gene. Even bacteria that are resistant to one antibiotic can have more than one resistance coding gene. Tetracycline-resistant E. coli was detected to have the tetA and tetB genes; quinolone resistance was detected to have the qnrA, qnrB, and qnrS genes (Kurnia et al., 2018).
Among Gram negative bacteria, E. coli has the highest resistance pattern (Sukhumungoon et al., 2011). Antibiotic resistance testing is important to understand and monitor the pattern of antibiotic resistance conditions in E. coli, especially those from production animals. Therefore, the main objective of this study was to investigate the phenotype and genotype phenotypic and genotypic profile of antibiotic resistance in E. coli isolated from cloacal swab samples of broilers.

Samples Collection
Thirty cloacal swab samples were collected from broilers at a farm in Cianjur, West Java, Indonesia. Each sample was placed into tube containing 5 ml of 0.1% buffered peptone water (Oxoid CM1049, Thermo Scientific, England), and labelled with the identity of the sample. All of samples were stored in a cool box with a temperature of 4-10 ºC and immediately taken to the Medical Microbiology Laboratory, Faculty of Veterinary Medicine, IPB University.

Isolation and Identification of Escherichia coli
The samples were cultured into MacConkey agar (Oxoid CM0007, Thermo Scientific, England) and incubated at 37 ºC for 18-24 hours. The pink colonies surrounded by pink on the media because of acidic conditions resulting from lactose fermentation were transferred into eosin methylene blue agar (Levine) (L-EMB) (Oxoid CM0069, Thermo Scientific. England) and incubated at 37 ºC for 18-24 hours. The expected E. coli colonies were shown as methalic sheen color. A suspicious colony from each eosin methylene blue agar (Levine) (L-EMB) (Oxoid CM0069, England) plate inoculated to tryptone soya agar (Oxoid CM0131, England) slant and incubated for 18-24 hours at 37 ºC for further test. Gram staining of suspicious colonies was carried out to determine bacterial cell morphology. The five essential biochemical tests were carried out for confirmation consisting of indole, methyl red, Voges-Proskauer, and citrate (IMViC). All cultures that produced IMViC patterns of ++--were considered to be E. coli (Markey et al., 2013).

Preparation DNA Template
Escherichia coli isolates were inoculated into tryptone soya broth (Oxoid CM0129, Thermo Scientific England)and incubated for 18-24 hours at 37 ºC. Bacterial suspension were centrifuged for 1 minute 14.000-16.000 × g and the pellets were taken for DNA extraction. DNA extraction was using Presto TM Mini gDNA bacteria kit (GBB300/301, Geneaid, Taiwan) and stored at -20 ºC for further use.

Detection Antibiotics Resistance Genes
Detection tetA, aac3-IV, gyrA, and ermB as antibiotics resistace genes in E. coli isolates used polymerase chain reaction (PCR) method (Kurnia et al., 2018). This reaction was carried out by MyTaq TM HS Red Mix (Bioline BIO-25048, England) according manufacturer instruction. Annealing temperature was set according to each of the target genes (Table  1). Electrophoresis for PCR products on 1% agarose gel and Tris-Acetate-EDTA (TAE) buffer (1×) (T9650, Sigma-Aldrich, Germany) was carried at 70 Volt 50 minutes by electrophoresis power supply Bio-Rad Power Pac 300 (15008 C24, Bio-Rad, California). DNA staining used 2 μl FloroSafe DNA Stain (BIO-5130, 1st BASE, Malaysia). 100 bp DNA ladder (Invitrogen 15628019, Thermo Scientific, USA) was used as standard. The gene bands formed were adjusted to DNA marker to determine the approximate base length. The size of the DNA bands that have estimated base lengths were matched with references in Table 1.The detection antibiotics resistance genes were only for resistant isolates.

Data Analysis
Data were presented in tables and analyzed descriptively.

DISCUSSION
The emergence of antibiotics resistance agents had become a public health threat. Caution was necessary when there were few or no effective alternative antibiotic agents for the treatment of infections caused by E. coli. Although most strains of E. coli were harmless and commonly found in the intestines of humans and warm-blooded animals, some strains could cause severe foodborne disease in humans.
In this study, we studied the phenotype and genotype profile of antibiotic resistance in E. coli from cloacal swabs of broilers in Cianjur, Indonesia. The 33.3% prevalence of E. coli isolates from samples collected in this study was lower than some previous studies (Masruroh et al., 2016;Niasono et al., 2019; Azad et al., 2019). However, the results of isolation and identification of E. coli in our study were not much different Andriyani et al. (2020) that is 26.7%. Antibiotic resistance test results showed that the number of isolates resistant to three or more antibiotic classes was quite high. Multi drug-resistant of tetracycline, quinolone, fluoroquinolone and macrolides was most frequently observed. The presence of resistant E. coli on samples was a matter of concern. In general, when antibiotics were used, they would eliminate the susceptible bacteria strains and remain resistant bacteria. Then the resistant bacteria multiplied and become a dominant population which could transfer resistance genes (either horizontally or vertically) to other bacteria (Madigan et al., 2014). Resistance traits could be obtained from mutations, gene transfer by conjugation or transformation, transposons, integron, and bacteriophages (Cogliani et al., 2011). In fact, E. coli was the main bacteria that causes the spread of antibiotic resistance (WHO, 2017).
Tetracycline resistance was frequently reported from chicken samples (Kurnia et al., 2018;Al Azad et al., 2019). In this study, 100% of the isolates showed phenotypic resistance to tetracyclines and oxytetracyclines. These results were consistent with previous studies which reported tetracycline resistance in 97.3% of isolates (Niasono et al., 2019). Tetracycline resistance occured in chicken isolates because it was widely used as growth promoters in poultry (Kolar et al., 2002).
The tetA and tetB is the most common gene responsible for resistance to tetracyclines (Skockova et al., 2012). In this study, we performed the detection of tetA gene. As many as 80% of isolates that were phenotypically resistant showed the presence of tetA gene. Although the remaining 20% were also phenotypically resistant but did not carry tetA gene. This resistance could be caused by other mechanisms such as enzymatic inactivation or target modification (Chopra & Roberts, 2001).
All isolates (100%) showed erythromycin resistant. Similarly, (Talebiyan et al., 2014) reported erythromycin resistant in E. coli was 71.7%. The high resistance of erythromycin was related to the impact of Mycoplasma treatment. Erythromycin was Notes : TE: tetracycline; OT: oxytetracycline; CN: gentamicin; NA: nalidixic acid; CIP: ciprofloxacin; E: erythromycin; C: chloramphenicol; tetA for tetracycline and oxytetracycline resistant; aac(3)-IV gentamicin resistant; gyrA for nalidixic acid, ciprofloxacin or enrofloxacin resistant; and ermB for erythromycin resistant; S: susceptible; I: intermediate; R: resistant; "+":antibiotics resistance-encoding gene was present in sample; "₋": antibiotics resistance-encoding gene was not present in sample; "⁎": not performed detection antibiotics resistanceencoding genes because it was not resistant highly used in breeders against Mycoplasma synoviae and Mycoplasma gallisepticum. Erythromycin-resistant E. coli was screened for the ermB gene. Although phenotypically resistant to erythromycin, genotypically no ermB gene was detected in all E. coli isolates. These results differ from detection of the ermB gene by Kappell et al. (2015), there were 38.9% of the ermB gene detected in E. coli isolates from urban waterways. It is possible that another erythromycin resistance gene encodes this phenotype.
The prevalence of gentamicin resistance phenotype in E. coli isolates was 40%. The result was similar to the study conducted by Al Azad et al. (2019) showed 49% E. coli resistance to gentamicin. Gentamicin was used widely in the poultry industry with or without a combination of other antibiotics.
Genotypically, all gentamicin resistant isolates were not detected by the aac(3)-IV gene. Nevertheless, the study conducted by Choi et al. (2011) showed 46.2% and 50.7% of the aac(3)-IV genes in E. coli indicator and E. coli pathogenic isolates, respectively. Zarate et al. (2018) stated that there were other genes capable of coding resistance to gentamicin and other aminoglycoside antibiotics, i.e. aac(6)-I, aac (6)

-II, aac(3)-I, aac(3)-II, aac(3)-III and aac(3)-VII.
The resistance test for quinolone and fluoroquinolones in this study was carried out against nalidixic acid, enrofloxacin, and ciprofloxacin. Phenotypically, the resistance of this group is quite high (70-90%). According to the study Al Azad et al. (2019), E. coli isolates from broilers experienced high resistance (100%) to ciprofloxacin and enrofloxacin. However, different report from Niasono et al. (2019) showed lower resistance to nalidixic acid, enrofloxacin and ciprofloxacin (40.5-64.9%). The diversity in the use of antibiotics, geographic differences, and different poultry production systems gave different patterns of resistance (Bywater et al., 2004).
The gyrA gene represented quinolone and fluoroquinolone resistance coding genes. All resistant isolates of this group were phenotypically (100%) detected by the gyrA gene. Quinolone and fluoroquinolone resistance usually occurs due to gyrA gene mutation. Kotb et al. (2019) showed that 100% of the gyrA gene had a mutation in codon 83 (serine → leucine) in 71.3% of the quinolone and fluoroquinolone-resistant E. coli. In addition, the gyrA gene in the high resistance phenotype underwent a codon mutation in 87 (Asp87Asn) (4%).
There were no isolates detected chloramphenicol resistance. Similar to the finding of Hailu & Tefera (2016) had reported no resistant E. coli isolates to chloramphenicol. The current study finding was distantly related to the finding of Shecho et al. (2017) who found only 3.85% of healthy broilers resistant to chloramphenicol. The different finding was reported by Halfaoui et al. (2017), the chloramphenicol resistance was medium level (39.22%) in E. coli from broiler chickens in Algeria. Chloramphenicol was prohibited in breeding. The medium level resistance might due to the persistence of previous resistances or exposes illegal usage.
In conclusion, occurrence of resistant antibiotics in E. coli isolated from broiler chickens in Cianjur, Indonesia, was high. Multi drug-resistant was found among isolates. The high level of multi drugresistant among bacterial isolates indicates the indiscriminate use of antimicrobials in poultry sectors. In this study, not all resistant phenotypes were detected their resistance genes according to the target primers. For future studies, it was advisable to use more varied target primers.
"All authors declare that there are no conflicts of Interest".