ANTI-BACTERIAL RESISTANCE OF COMMENSAL ESCHERICHIA COLI STRAINS OF MECONIUM ORIGIN IN APPARENTLY HEALTHY CHICKS

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INTRODUCTION
Although Escherichia coli is a typical component of poultry intestinal microbial flora, it may also be a pathogen related to severe illnesses, such as colibacillosis.Furthermore, Escherichia coli may be the origin of resistance genes that can be transferred to humans, creating a hazard to the public's health (Shang et al., 2018).
Following the acquisition of virulence genes and pathogenicity enhancement, commensal strains of E. coli may be the ancestors of pathogenic strains via random point mutations or acquisition of chromosomal virulence operons (Duriez et al., 2001).
Antimicrobial resistance among bacteria has become a difficulty due to antimicrobials increasingly being used as growth stimulants, preventing agents and medicinal substances in the chicken business over the past few decades (Nhung et al., 2017).The administration of multiple antimicrobials during chicken rearing may cause the spread of antibiotic resistance, and the widespread use of antibiotics in chickens has raised the risk of bacterial resistance, particularly in gram-negative microbes like E. coli.(Simoneit et al., 2015).Multiple-drug resistance (MDR) has been linked to selective pressure brought on by the use of antimicrobials in the poultry sector to treat both pathogenic and commensal E. coli.(Gyles, 2008).This occurrence is caused not just by bacterial innate ability to survive and reproduce in large numbers, but by horizontal gene transfer via plasmid as well (Apata 2009).Numerous studies have discovered a connection between the use of antibiotics in livestock agriculture and the development of antibiotic resistance in human illnesses (Silbergeld et al., 2018).
Integrons have been linked to multidrug resistance in enteric organisms, such as E. coli.(Deng et al., 2015).
Integrons are a unique type of recombination system that can capture and express resistance to disinfectants and antimicrobials in gene cassettes (Hall, 2012).
Numerous researches have been conducted in recent years to identify the existence and the type of resistance cassettes, the integrons' structure, and the relationship between the presence of integrons and MDR in pathogenic and commensal E. coli isolated from animal and human samples (Kaushik et al., 2018).Both in people and animals, gram-negative bacteria typically carry Class 1 integrons.The majority of integrons in clinical isolates belong to this class also known as clinical integrons (Gillings 2017).In order to ascertain the prevalence of MDR in commensal Escherichia coli bacteria of meconium origin in seemingly healthy chicks, as well as the distribution of class 1 integron-integrase gene (intI1) in these isolates, this study was set out to identify these two factors, in addition to examine AMR in commensal E. coli in chicks and decrease the antibiotics use in farming by identifying efficient substitute therapies, utilizing more resistant breeds, and enhancing animal welfare.

Samples
Samples were taken from 30 batches (10 batches from farms, 10 from imported chicks and 10 from hatcheries), each batch has 30 chicks.Chicks were apparently healthy.The samples were taken from meconium and internal organs (liver, heart, and lung).So, the examined samples are 900 samples (300 from each group).

E. coli isolation and identification
According to (Nolan et al., 2020), Escherichia coli was isolated and identified.All collected samples underwent a preenrichment step in buffered peptone water (Lab M, UK) and were then incubated aerobically for 24 hours at 37°C.On MacConkey agar (Neogen, US) and eosin methylene blue agar (Lab M) plates, a loopful of the broth culture was used as an inoculum.The plates were then incubated at 37°C for 24 hours.Utilizing urea, Simmons' citrate agar, peptone water, and oxidase strips from Oxoid in the UK, Kovacs reagent from HiMedia in India, and triple sugar iron agar from Lab M, the isolated colonies were identified morphologically and biochemically.

Molecular assessment
Polymerase chain reaction (PCR) was used to perform additional testing on the 10 chosen E. coli isolates to determine whether the class 1 integron was present.

DNA extraction.
Adapting the manufacturer's instructions, the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) was used to extract DNA from samples.Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K, and then 200 µl of 100% ethanol was added to the lysate after incubation.After that, the sample was cleaned and centrifuged, following the manufacturer's instructions.The kit's 100 µl of elution buffer was used to elute the nucleic acid.

Priming oligonucleotide.
The primers used are indicated in Table (1) and were provided by Metabion (Germany).

PCR amplification.
A 25 µl reaction containing 12.5 µl of DreamTaq Green PCR Master Mix (2X) (Thermo Scientific), 1 µl of each primer at a concentration of 20 pmol, 5.5 µl of DNAas free water, and 5 µl of DNA template was used to test the primers.Thermal cycler 2720 from Applied Biosystems was used to carry out the process.

Examining the PCR products.
The PCR products were separated by electrophoresis on 1% agarose gel in 1x TBE buffer using gradients of 5V/cm (Applichem, Germany, GmbH).For gel analysis, 20 µl of the PCR products were loaded in each gel slot.Generuler 100 bp DNA ladder (Fermentas, Sigma) was used to determine the fragment sizes.The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

Isolation and identification of Escherichia coli
A commensal strain of E. coli was examined in 30 batches (each batch contains 30 chicks), so the number of examined chicks was 900.Pooled internal organs (heart, lungs, liver) and meconium were obtained from each batch.Commensal E. coli strains were found in all examined meconium samples with a percentage of (100%).The number of final examined samples was 30 samples (10 samples from each group).Each sample represented one batch.
E. coli colonies on Macconkey agar were pink.and violet on violet red bile agar, Eosin methylene blue agar were used for differentiation between pathogenic E.coli and commensal E.coli.Pathogenic E.coli were shin metallic colonies On EMB. Biochemical tests were applied for confirmation.From Table ( 5) we explain that all the isolated strains showed multidrug resistance.

Class 1 integron detection among isolates with multiple drug resistance
Conventional PCR was applied on ten MDR E. coli isolates for the detection of the integrase gene.
Nine out of the ten MDR isolates had the integrase gene, with a percentage of (90%) giving characteristic bands at 280 bp (Fig. 1).

DISCUSSION
Despite all the examined chicks were not administered antibiotics before the examination of the samples, all the isolated E. coli strains were found to be MDR.The results are in line with the most current report from the European Food Safety Authority (EFSA).(EFSA and ECDC2020).This might be determined by a vertical or horizontal acquisition of resistance from breeders.(Osman et al., 2018 andMarin et al., 2020) or the environment (Montoro et al., 2020 andOikarainen et al., 2019), respectively.These results show the importance of MDR acquired from the environment of breeding, hatching, or transportation (Poulsen et al., 2017;Dame et al., 2019).It has been observed that breeders' microbiota can directly vertically infiltrate one-day-old chicks (Nilsson et al., 2014) or by the bacteria that are resistant and persist in the hatchery or on delivery surfaces (Oikarainen et al., 2019;Projahn et al., 2017;Projahn et al., 2018).To reduce the selective AMR/MDR impact on breeders, hatcheries, and farm environments, they must be managed strictly in the early phases (Aarestrup 2015, Dierikx et al., 2013).In our study, high drug resistance was reported against Penicillin, Ampicillin, Amoxicillin clavulinic acid, Cephradine, Cephalexin, and Cephalothin, and the percentage was 100%, 96.6% against Cefotaxime, 86.6% against Oxillinc acid, Tobramycin, Erythromycin, Ceftriaxone, Cephradine for each, 83.3% against Doxycycline, and Oxytetracycline, and 76.6% against Amoxicillin and Streptomycin.A low percentage of drug resistance was reported against Colistin sulphate 56.6% Trimethoprime-sulfamethoxazole 50% and Norfloxacin 36.6%.
According to the outcomes listed by the (EFSA 2020) and (Martins da Costa et al., 2009), who identified resistant bacteria from one-day-old chicks to ampicillin, cephalothin, tetracycline, streptomycin, gentamicin, and enrofloxacin but discovered no E. coli resistant to chloramphenicol.Since no antimicrobial drugs had previously been administered to the chicks used in this investigation, vertical transmission of resistant strains from parent flocks is possible.(Giovanardi et al., 2005) or contamination in the environment of the hatchery (Dierikx et al., 2013) possibly be the primary factors.(Bortolaia et al., 2010).They concluded that E. coli resistance to -lactams and fluoroquinolones was caused by vertical transmission through parent hens in the same framework.Baron et al., (2014) suggested that E. coli resistance may be introduced to the hatchery facilities, either through true vertical transmission when parent poultry stocks are contaminated or through very early contamination in the hatchery itself, or during transport when the immature digestive flora is probably very receptive to early colonization.Even yet, it is impossible to exclude further lateroccurring contamination incidents on the production farm.

Table 1 :
Target genes, sequences of primers, amplicon sizes and cycling conditions.

Table 2 :
Interpretative criteria of antimicrobial drugs of commensal E.coli isolated from oneday-old native chicks (farms).

Table 3 :
Interpretative criteria of antimicrobial drugs of commensal E.coli in one-day-old Imported chicks.

Table 4 :
Interpretative criteria of antimicrobial drugs of commensal E.coli in new Hatched chicks.

Table 5 :
Commensal E.coli antimicrobial profile of resistance isolates collected from native chicks, imported chicks and newly hatched chicks: