Identification of Antibiotic Resistance Pattern, and Detection of Virulence Genes iss, and ompT in Avian Pathogenic Escherichia coli from Broiler Chickens in Chitwan, Nepal

This study aimed to identify, evaluate the antibiotic resistance pattern and detect virulence genes iss, and ompT in avian pathogenic Escherichia coli (APEC) from broiler chickens in central Nepal. To determine the antibiotic resistance pattern of the obtained isolates, the Kirby-Bauer disc Original Research Article Sankhi et al.; JPRI, 32(23): 1-12, 2020; Article no.JPRI.60969 2 diffusion method was used with six different commercial antibiotic discs: Amikacin, Gentamycin, Ciprofloxacin, Doxycycline, Chloramphenicol and Levofloxacin. A polymerase chain reaction (PCR) assay was used for the selected isolates (n=40) to screen the presence of the iss and ompT genes after the extraction of DNA using the boiling method Out of 60 suspected Colibacillosis liver samples, 40 were confirmed as E. coli positive The antibiogram profile revealed maximum resistance to Doxycycline (87.5%), followed by Levofloxacin (72.5%), Ciprofloxacin (67.5%), Chloramphenicol (40.0%), Gentamycin (32.5%) and Amikacin (10.0%).. The presence of the iss and ompT genes was found to be 100.0% and 90.0%, respectively. APEC was found to be highly resistant to most of the antibiotics. Virulence-associated genes iss and ompT were obtained at high percentages from Colibacillosis suspected broiler chickens in Chitwan, Nepal. These finding suggests that the judicial use of antimicrobials is compulsory to check antibiotic resistance and Colibacillosis outbreaks in poultry farms.


INTRODUCTION
Escherichia coli (E. coli), mostly colonized in the gastrointestinal tract of warm blooded animals and birds, is a gram-negative, rod shaped, flagellated, non-sporulating, and facultative anaerobic bacteria belonging to the Enterobacteriaceae family of class Gammaprotobacteria [1,2]. Most E. coli strains are nonpathogenic; however, some are pathogenic. Some pathogenic strains of E. coli invade various organs of birds, which leads to pericarditis, air sacculitis, perihepatitis, peritonitis, and other extraintestinal diseases, collectively termed Colibacillosis [3,4]. Avian pathogenic E. coli (APEC) strains that fall under the category of extraintestinal pathogenic E. coli (ExPEC) are highly associated with diverse diseases and are responsible for great economic losses in the avian industry [5].
Antibiotic therapy is one of the prime measures for controlling the morbidity as well as mortality caused by APEC infection. Many studies have suggested the use of antibiotics in chicken broilers as a disease preventive measure and a growth promoter [6,7]. Since the haphazard use of antibiotics leads to the occurrence and development of resistant isolates, they need to be used prudently to restore their therapeutic usefulness in both humans and animals [8]. European countries along with others have reported on the widespread use of large amounts of antibiotics without any professional consultation or supervision [7,9]. The utilization of antibiotics in food-producing animals is associated with several adverse effects, such as modification of the intestinal flora, appearance of antibiotic residues in food particles, impacts on the public environment, and emergence of antibiotic resistance in microorganisms [10,11]. Such microbes have challenged the cure of zoonotic diseases, and their transmission from animals to humans has threatened the public health care sectors [12].
The pathogenic ability of the APEC strain is determined by a broad range of virulence factors that are coded by virulence-associated genes such as iutA, papC, iroN, iss, ompT, cva/cvi, tsh, hylF, astA, iucD, and irp-2. Of the eleven genes reported worldwide, iss and ompT genes are considered the most widespread virulence genes in APEC strains [13]. The increased serum survival (iss) gene plays a major role in resistance against serum complement. E. coli resists complement by producing Iss proteins. This protein inhibits classical and alternate pathways of the complement system. It causes a 20-fold increase in complement resistance and a 60-fold increase in virulence towards poultry [14]. As the iss gene has been found more frequently among pathogenic than nonpathogenic strains, a strong association is suggested between the iss gene and pathogenicity of APEC of various serogroups but not with fecal E. coli isolates from apparently healthy birds [15]. Another virulence gene called the ompT gene encodes the OmpT protein, which is an aspartyl protease found on the outer membrane of E. coli. This ompT gene is also found on some gram-negative species of bacteria that hydrolyse peptide materials that are harmful to the bacterium and hence increase the virulence of that gene [16].
In Nepal, poultry farming has been growing tremendously over a few decades. It shares a total of 8% of AGDP and 4% of GDP [17]. The Chitwan district of Nepal is the major broiler producing district containing more than 1,365 broiler farms [18]. Studies on the molecular characterization of virulence-associated genes, such as iss and ompT, responsible for Colibacillosis are scarce in Nepal. Most of the studies have only concentrated on prevalence studies. PCR, a confirmatory test, highlights the importance of molecular detection of virulence genes and is easier to apply than other methods [13,14,19]. This type of study could provide further evidence and platform for other researchers to conduct research on candidate vaccines that might be proven successful in eliminating Colibacillosis infections in broiler chickens of Nepal [19]. Hence, the study aimed to identify, evaluate the antibiotic resistance pattern, and detect virulence genes iss and ompT in APEC from broiler chickens in central Nepal.

Study Duration and Site
This study was conducted from September 2018 to February 2019 in Chitwan District, which is located in the southwest part of Baghmati Province. The samples were obtained from the National Avian Disease Investigation Laboratory (NADIL), Bharatpur, Chitwan, where dead birds from different areas of Chitwan are taken for postmortem and disease diagnosis. Identification, antibiotic resistance patterns, and gene detection were performed at the Veterinary Microbiology Lab, Faculty of Animal Science, Veterinary Science and Fisheries (FAVF), Agriculture and Forestry University (AFU), Chitwan.

Sample Collection
Purposive sampling was performed. A total of 60 liver samples of broilers from NADIL were collected aseptically in a sterile vial containing 1 ml of sterile peptone water. It was then immediately transported on a cold chain box with ice packs to the microbiology laboratory of FAVF, Chitwan. All liver samples were taken from the broilers that were non-vaccinated, between 4-8 weeks of age and with no history of antibiotic therapy after one week of age.

Identification of E. coli
Streaking was performed directly on MacConkey agar (MAC) plates and left for incubation at 37°C aerobically for 24 hours. Afterwards, a single isolated colony from MAC agar was subcultured onto the EMB agar plate and incubated at 37°C aerobically for 24 hrs. Green metallic sheen with dark centered colonies was observed on EMB agar plates. Various morphological characteristics, such as size, shape, surface texture, edge and elevation, opacity, color, etc. of the suspected colonies on different agar media developed within 18-24 hrs of incubation was carefully studied and recorded. Suspected purified colonies were smeared, fixed and stained with Gram's staining. As E. coli is a gram-negative bacterium, the bacterial smear should appear pinkish with the shape of bacilli.

Identification Tests
Biochemical tests, such as motility, indole, methyl red, Voges-Proskauer, simmon citrate, triple sugar iron (TSI), motility indole ornithinase (MIO) test, catalase, and oxidase test, were performed. The criteria was followed for various biochemical tests to confirm E.coli, as depicted in Table 1.

Antimicrobial Susceptibility Testing
The antibiotic susceptibility test was performed using the modified Kirby-Bauer disk diffusion method as suggested by the Clinical and Laboratory Standards Institute (CLSI) [20]. All 40 APEC isolates were tested for six different antimicrobial agents on Muller Hinton agar (HiMedia). Isolates of E. coli were inoculated in brain heart infusion broth (BHIB) and incubated at 37°C for 6 hours. The turbidity of the sample was adjusted to a 0.5 McFarland standard by dilution. Instantly, after dilution, a sterile swab was dipped into the inoculum and streaked over the entire surface of the Muller Hinton agar three times. Six suitable antibiotic discs were selected. Disks of Amikacin (30 mcg), Gentamycin (10 mcg), Ciprofloxacin (5 mcg), Levofloxacin, Chloramphenicol (30 mcg), and Doxycycline (25 mcg) were used.

DNA Extraction, Quantification and Primers
The rapid boiling method was performed for deoxyribose nucleic acid (DNA) extraction [21]. Quantification and purity detection of DNA extracted from E. coli culture was performed spectrophotometrically at 260 nm and 280 nm using a Nanodrop Spectrophotometer (Quawell, UV-Vis Spectrophotometer Q5000). Forward and reverse primers of both the iss and ompT genes were used, as shown in Table 2 [22]. The polymerase chain reaction (PCR) primers used in the study were 323 bp for the iss gene and 496 bp for the ompT gene.

Statistical Analysis
Data entry and analysis were performed using Microsoft Excel 2010. .

RESULTS
Out of 60 diseased birds clinically diagnosed with Colibacillosis, 40 isolates were biochemically identified as E. coli with a prevalence rate of 66.67%.
Among the 60 liver samples obtained from Broiler chickens, 40 were confirmed to be E. coli positive through cultural and biochemical tests, as predicted in Table 3.

. Antimicrobial susceptibility test of liver samples of broiler chickens
The presence of virulence-associated genes in the E. coli isolates was detected using PCR. Out of 40 total confirmed E. coli isolates, the iss gene was present in all with the highest prevalence of 100.0%, while the ompT gene was harbored in 36 isolates, with a prevalence of 90.0%, as illustrated in Figs. 4 and 5.

DISCUSSION
Our study identified, evaluated the antibiotic resistance pattern and detected virulence genes iss and ompT in avian pathogenic Escherichia coli from broiler chickens in central Nepal over six months. The study revealed that the 40 isolates were biochemically identified as E. coli with a prevalence rate of 66.67%. This was comparatively higher than a study conducted in a similar setting in Egypt, where the prevalence rate was 34.5% [23]. The higher prevalence rate of E. coli in our study may be due to differences in the cultural/serological diagnostic tests, lower sample sizes, and persistence of unhygienic farm surroundings, unsanitized water and infections after respiratory diseases in chickens.
Antibiotics are considered an effective measure for the control of Colibacillosis. However, the emergence of multidrug-resistant strains has evolved challenges in the risk reduction of APEC infections. Our study showed a high rate of resistance to the majority of the examined antibiotics. Of the six antibiotics tested, none of  [4,19,24,25]. Alternatively, aminoglycoside amikacin was found to be most effective against 82.5% of the E. coli strain, which is slightly lower than that of a study conducted in central Nepal [19]. The antibiotic resistance pattern detected in this current research demonstrates a devastating situation of antibiotic-resistant E. coli strains in the Chitwan district of Nepal. Irrational use of antibiotics for nontherapeutic purposes even without consulting professionals may be the reason for the higher drug resistance seen in broiler chickens in Chitwan, Nepal. Virulence associated genes The frequency of two genes, iss and ompT, and their role in pathogenicity were analyzed among APEC strains. The iss gene was detected in all isolates, showing 100% prevalence, which was consistent with two other previous studies [19,26]. Conversely, the iss gene was detected in only 38.5% of the isolates in a study conducted in Egypt [23]. Likewise, our findings showed a 90% detection rate of the ompT gene. This is quite similar to the finding reported from Brazil and central Nepal, wherein the detection rate was 100% for the ompT gene [3,19]. Conversely, the detection rate of the ompT gene was comparatively lower in other studies conducted in China (57.6% and 67%, respectively) [26,27]. The difference in the detection rate of the iss and ompT genes may be due to differences in the base pair of primers, DNA extraction technique (kit method/direct boiling method), sample difference (liver/trachea/faeces/heart), differences in poultry breeds, geographical differences, environmental factors, etc.

CONCLUSION
Our study revealed APEC to be more resistant to most antibiotics. Likewise, virulence-associated genes iss and ompT were obtained at high percentages from Colibacillosis-suspected broiler chickens. Further study on the pathogenicity of the iss and ompT genes by taking larger sample sizes may provide ideas for the production of therapeutic vaccines. These findings highlight the essence of the judicial use of antibiotics at an optimum dose to ensure effective treatment of Colibacillosis in broiler chickens. It also urges the need for collaborative efforts from poultry farm owners, veterinarians and government agencies for the prevention and control of avian Colibacillosis in Nepal.

LIMITATION AND STRENGTH
There are some limitations to this study. Sample collection was limited to single centre of Chitwan district of Nepal, which along with small sample size shrinks the reliability of the study. Likewise, only two virulence genes were detected in the study. Therefore, the generalizability of the findings remains to be explored. More gene detection from poultry could have strengthen the findings on the status of virulence genes and necessity of novel vaccine production against the Colibacillosis disease in poultry. However, the use of confirmatory biochemical test for E. coli detection and molecular technique for gene detection have strengthen this study thus preventing from probable false positive result.

CONSENT
lt is not applicable.

ETHICAL APPROVAL
The Research Ethics Committee of Agricultural and Forestry University, Rampur, Chitwan, Nepal granted ethical approval for the study. Animal samples were collected and processed by following animal research ethical guidelines approved by the Ethics Committee of this University.

AVAILABILITY OF DATA AND MATERIALS
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.