Molecular study of resistance genes in Escherichia coli isolated from chronic respiratory disease cases in broilers

1. The role of coli in the occurrence of CRD
2. Determine the resistance of coli to the famous antibiotics.
3. The presence of resistance genes in isolated coli

Introduction

Avian pathogenic E. coli is the main pathogen in poultry. It causes many infections and is accompanied by viral and other infections (1,2). Colibacillosis has economic importance in poultry through its effect on reducing bird productivity, increased mortality, contamination of infected carcasses at slaughter, and the cost of prevention and treatment (3,4). To control and protect against infection in poultry, antimicrobials are used in feed during risky durations of bacterial infections, as prophylaxis, or as a growth stimulator (5). Bacterial resistance to antibiotics is increasing, and its relationship with the wrong use of antibiotics in controlling diseases in humans and animals has been noted (6,7(. There must be vectors that transfer antibiotic-resistant bacteria isolated from chicken meat to humans after consuming poultry meat and animal products (8). Beta-lactam antibiotics have remained the prime selection for treating E. coli infections (9,10). The E. coli resistance to cephalosporins through its weapons enzymes (ESBLs) limits therapeutic options against E. coli infections (11). Beta-lactamases are enzymes produced by E. coli that cause hydrolyzing of beta-lactam ring in penicillin and cephalosporin; thus, these antibiotics lose their therapeutic ability to give bacteria the character of resistance (12,13). One of the beta-lactamase enzymes includes extended-spectrum beta-lactamases (ESBLs) like TEM, SHV, CTX-M, and OXA (14,15). The blaCTX−M genes are swiftly pervasion and were recorded in E. coli (16,17). Plasmids encode these enzymes and are transmitted to consumers through the nourishment chain and animal contact (18,19). The goal of the existing study was to isolate Escherichia coli strains from the CRD in broiler chickens and determine their responsible genes for antibiotic resistance.

Materials and methods

Ethical approve

The endorsement certificate with the number UM.VET.2021.075 on 18/8/2021was granted by the Commission of scientific morals, which also provided the moral consent to carry out this methodical activity in the College of Veterinary Medicine.

Sampling

Seventy-four swabs from internal organs (air sacs, heart, lung, liver) were collected from broiler chickens with severe respiratory signs in Mosul City from September 2021 to March 2022. Specimens were placed in a sterile screw-capped. Specimens were conveyed to the microbiology laboratory of the College of Veterinary Medicine, University of Mosul.

Isolation and identification of E. coli

Swabs were placed on MacConkey agar containing foxime 500mg (20,21) and incubated at 37ºC for 24h. Pink fermented colonies were chosen and re-cultured on BHI (brain heart infusion) agar for purification. Colonies were classified biochemically using standard bacteriological methods (22).

Antimicrobial susceptibility testing

The disc diffusion method was used to test the sensitivity of the isolates to eleven antimicrobial agents (23). The isolates that exhibited multi-drug resistance by the disc diffusion method were selected for antimicrobial resistance gene amplification using PCR.

Extraction of DNA

Sixty-one isolates of cefotaxime-resistance E. coli underwent DNA extraction using DNA preparation Cat NO. PP-206 (Jena Bioscience, Germany). The extraction was done by following the manufacturer's instructions and depending on (24,25). Keep the extracted DNA at -20°C till use.

Amplification of DNA

The primers and their information are explained in table 1. A confirmatory genetic assay was conducted to diagnose E. coli using a specific primer for the 16S rRNA gene (ECOL200-F and ECOL400-R) (26). whereas detection of the CTX-M, TEM, and SHV genes (24-28).

Table 1: Sequence of primers

Results

Escherichia coli isolates were isolated from chronic respiratory disease infections in poultry with a percentage of 82.4% (61 out of 74 samples). All isolates showed absolute resistance to the tetracyclines, sulphamethoxazole, chloramphenicol, and levofloxacin, while all isolates were sensitive to imipenem. They also showed resistance in varying proportions to azithromycin, tobramycin, gentamycin, streptomycin, and ciprofloxacin (Table 2). All 61 isolates were molecularly detected as E. coli (232bp) (Figure 1). CTX-M gene was found in all isolates of E. coli 100%, which appeared at 550bp (Figure 2), while 52 (85.25%) isolates appeared positive for the TEM gene with the band at 822bp (Figure 3). Finally, only 9 (14.75%) isolates showed the SHV gene with an expected band size of 753bp (Figure 4).

Table 2: Antibacterial resistance of E. coli isolated from chronic respiratory disease

Figure 1: Molecular confirmation of E. coli isolates according to the sequence of 16S rRNA (M: Ladder, 1-7 positive for E. coli at 232 bp).

 Figure 2: Affirmation of CTX-M gene in E. coli isolates. M: ladder, lanes 1-7 positive CTX-M gene (550 bp)

Figure 3: Detection of TEM gene. M: Ladder, lanes 1-5 positive TEM gene (822 bp), lanes 6- 7 are negative.

 Figure 4: PCR products of SHV gene. M: Ladder, lane 7 positive SHV gene (753 bp), lanes 1-6 are negative.

Discussion

Although chicken meat is considered a primary food source in many countries, the broiler is a persistent source of bacterial spreading that causes many diseases between humans and animals. Raising chickens and keeping them in contact with humans facilitates the transmission of bacteria that cause diseases of animal origin to humans (29). Chicken meat contains some E. coli strains considered more pathogenic than others and responsible for most deaths recognized as avian pathogenic E. coli (APEC) live outside the intestines and is endemic to the respiratory system and some organs, and causes systemic diseases and major fatalities (30). The present study recorded that the isolation rate of APEC reached 82.4% from 74 samples suffering from different clinical cases such as septicemia, peritonitis, and airsacculitis. This result is close to what was obtained by researchers in India, where they recorded a rate of isolation of 67% (31), in addition to the results of other researchers in Egypt, where the isolation rate was 75.4% from cases of chronic respiratory infections (32). The isolated bacteria appeared highly resistant to azithromycin, levofloxacin, gentamicin, tetracycline, and chloramphenicol. This has been confirmed in many articles and studies (31,33). This resistance may be attributed to the wrong utilization of antibiotics without testing the sensitivity of isolates to antibiotics, as well most poultry breeders’ resort to adding antibiotics to stimulate growth and obtain high production (34). Also, one of the causes of antibiotic resistance like tetracycline is having genes that code for excluding this antibiotic from the cell leading to reduced concentration and the protection of E. coli ribosomes (35). One of the most plasmid mechanisms is the transfer and spreading of resistance genes among the bacterial population (36). Resistance was also recorded in the β-lactam group in the study due to β-lactamase production by E. coli (37). All isolated E. coli (61) was carried out with the CTX-M gene encoding ESBL (100%). This result was consistent with recent studies with similar proportions, and the gene was prevalent in isolates taken from the broilers' farm (38). The CTX enzymes have hydrolytic effects on cephalosporins (39). The plasmids carry the ESBL genes and are also easily transmitted between commensal and pathogenic bacteria in poultry (40). The animal-human transference of ESBL genes leads to the difficulty and failure of treatment in both cases and the economic losses they cause (41). In the existing results, the TEM gene, responsible for releasing an enzyme that awards resistance to β-lactam, has appeared in 52 E. coli isolates. This gene can cause ESBL by mutation altering the sequence of one amino acid nearby the active site of β-lactamases (42,43). The CTX-M gene was the most predominant in isolates of E. coli, after the TEM and SHV genes. It was consistent with what was obtained by researchers in poultry farms in America, where the highest percentage was 33.6% of gene CTX-M followed by a low frequency of TEM and SHV genes (18,42).

Finally, the current results spotlight the serious problem in poultry farms that showed the excessive resistance of E. coli isolates that are prevalent in broilers and appeared proprietor for an arsenal of resistance genes to the most usable antibacterial in the treatment of bacterial infections in these farms, which means the demand for introducing more novel antibiotics in the cure of poultry health problems.

Conclusion

The current results spotlight the serious problem in poultry farms that showed the excessive resistance of E. coli isolates that are prevalent in broilers and appeared proprietor for an arsenal of resistance genes to the most usable antibacterial in the treatment of bacterial infections in these farms, which means the demand for introducing more novel antibiotics in the cure of poultry health problems.

Acknowledgments

The authors are immensely thankful for the efforts and support of the Department of Microbiology and Deanship of the College of Veterinary Medicine, University of Mosul.

Conflict of interest

The authors declare there are no conflicts of interest or financial ties to any government institutions and that no outside funding was used to carry out this research. We declare that all identified writers have read, reviewed, and approved the work. We have also all agreed on the order in which the authors are listed.