Characterization of multiple antimicrobial-resistant patterns of E. coli isolated from broilers in Ismailia farms and markets

Characterization of multiple antimicrobial-resistant patterns of E. coli isolated from broilers in Ismailia farms and markets.

Fadwa F. Mahmoud¹, Wafaa A. A. Ibrahim², Heba M. Hassan³

¹ Food Hygiene and Microbiology, Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Ismailia 41511, Egypt.

² Biotechnology department, Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Ismailia 41511, Egypt.

³ Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Giza, Egypt.

Abstract

Colibacillosis is a septicemic illness that affects poultry economically and usually results in numerous lesions in broiler chicken flocks. Previously published studies revealed increased E. coli resistance against a wide range of clinically used antimicrobials. This study was carried out to isolate and identify E. coli isolated from diseased broiler chicken farms and markets in Ismailia governorate, detect that E. coli isolates susceptibility to commonly utilized antibiotics, such as tetracycline, , ampicillin, amoxicillin, gentamicin, streptomycin, colistin,  and co-trimoxazole, etc. as well as detect the aminoglycosides resistant genes aacC (Aminoglycoside acetyltransferase)  and Aada2  (Aminoglycoside adenyltransferases)  by PCR in E. coli-resistant isolates.

From 70 diseased broilers chicken farms (5-7 birds/farm) aged (4-38 days) were received at the Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP) - Ismailia branch during the period from September 2019 to October 2022  and 70 chicken meat samples collected from Ismailia markets. The collected samples subjected to various examinations including isolation, phenotypic identification, antimicrobial drugs susceptibility and resistance assessments, and the aminoglycosides resistant genes aacC and Aada2 identification by PCR.

Results: Showed that E. coli was isolated and phenotypically recognized from 12 out of 70 diseased broiler chicken farms with a per cent ratio of 17.14 % and from 8 out of 70 chicken breast meat samples collected from markets in Ismailia with a per cent ratio of 11.43 %. All of the isolates showed multidrug resistance and antimicrobial resistance genes aacC and Aada2 genes were recorded in 13 out of 15 and 14 out of 15 of isolated phenotypically recognized E. coli isolates with a per cent ratio of 93.33% and 87.67%, respectively. Both aacC and Aada2 genes were Aminoglycoside resistance genes against gentamicin and streptomycin, respectively.

Conclusion: Broiler chicken may constitute an important source of multi antimicrobial drugs resistant E. coli, which can be considered a potential threat to public health through the transmission of resistant bacteria via the food chain.

Keywords: Broilers, E. coli, misuse of antibiotics, multi antimicrobial drug resistance, and Antibiogram.

Introduction:

In the poultry production field, the control of infectious diseases causing significant financial losses is recognized as one of the primary challenges (McKissick, 2006). Colibacillosis is brought about by the avian pathogenic bacterial organism (E. coli) (Barnes et al., 2008). They belong to the Enterobacteriaceae family and are responsible for both extra-intestinal and enteric infections in both humans and animals (Percival and Williams, 2014). The avian pathogenic E. coli are one of the pathogenic and lethal bacteria in the majority of poultry products (broiler chickens, layers, breeding flocks, ducks, and geese). It is Gram-negative road bacteria. It displayed colibacillosis and colisepticemia symptoms such as swelling head syndrome, air vasculitis, cellulitis, omphalitis, pericarditis, and perihepatitis. On the other hand, they pose a risk to public health by storing and spreading antibiotic resistance genes globally (Nolan et al., 2013). All ages of chickens are vulnerable to colibacillosis; however, young birds are more frequently infected than older hens (Barnes et al., 2003). The colisepticemia is considered the most prevalent form of colibacillosis and causes significant financial losses in poultry production in various regions all over the world (Saif, 2003).

The global production of chicken is significantly impacted financially by colibacillosis. The majorities of financial losses were caused by the impacted birds' deaths and decreased productivity. It is a prevalent illness in flocks of chicken, particularly in intensive breeding systems (Otaki, 1995). Birds suffering from colibacillosis can exhibit a variety of symptoms, such as abrupt death or an abnormal coloration with their necks dragged into their bodies (Matin et. al., 2017). Antimicrobials are frequently utilized in the production of animals to cure infectious illnesses and enhance growth. The considerable resistance to antimicrobial agents in the normal flora of poultry and pathogenic microorganisms is largely caused by the use of antimicrobials in poultry production industries for growth promotion (Romanus et al., 2012). In practice, the employing of antimicrobials in the feed may alter the intestinal flora by exerting a selective pressure in favor of resistant bacteria populations such as resistant E. coli which may enter into the environment and food chain (Furtula et al., 2010). An important public health concern is the use of antibiotics in animals raised for food and how they contribute to bacterial resistance. E. coli is one of the microorganisms that are frequently resistant to antibiotics as a result of its widespread presence in humans and animals as well as its function as a pathogenic and commensal organism (Zhao et al., 2012).

Bacteria become resistant to both single and multiple antimicrobials with repeated use over time, making it difficult to treat some infections (Moustafa and Mourad, 2015). Antimicrobial resistance, particularly multidrug resistance, has increased dramatically in recent years in clinical isolates, including E. coli isolates from animals (Elsabet, 2011). It is a worldwide issue, and it has now been recognized as a global public health phenomenon (Kaye et al., 2004). Due to the risk of spreading these resistant bacteria to humans, antimicrobial resistance among E. coli in food animals like chicken is becoming a bigger issue (Odwaret al., 2014). In veterinary practice, the antimicrobial sensitivity testing of pathogenic microorganisms in vitro is considered the best way for the veterinarian to select the suitable treatment (Radwan et al., 2016). Additionally, it helps identify the isolates that are multidrug resistant. As a result, the right antibiotic should be chosen based on the sensitivity that can be determined through laboratory testing. Poultry vets are concerned about the widespread resistance of E. coli species to antibiotics. Much attention has been paid to this growing resistance both in Egypt and globally. According to Radwan et al., (2020) Plasmids are the main vector used to disperse resistance genes across the bacterial community. Antimicrobial resistance genes in E. coli isolates can be found with PCR, and there is a large range of multidrug resistance E. coli.

Therefore, this study aimed to isolate and identify E. coli from diseased broiler chickens collected from 70 diseased broiler chicken farms received to the Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP) - Ismailia branch during the period from September 2019 to October 2022, and 70 chicken meat samples collected from Ismailia markets. Additionally, the E. coli isolates susceptibility to commonly utilized antibiotics, such as tetracycline, kanamycin, ampicillin, amoxicillin, gentamicin, and co-trimoxazole was tested. Moreover, the detection of aacC and Aada2 genes by PCR in E. coli-resistant isolates was carried out.

Materials and Methods:

1- Sample collection:

The tested samples consisted of 70 diseased broiler chicken farms received to the Reference Laboratory for Veterinary Quality Control on poultry production (RLQP) - Ismailia branch during the period from September 2019 to October 2022 and 70 chicken meat samples were collected from Ismailia markets. The samples collected from 5 to 7 chickens per farm and the age of birds varied from 4 to 38 days) suffered from depression, ruffled feathers, diarrhea and loss of appetite were subjected to post-mortem examination under septic conditions, and the internal organs (liver, lung, spleen, and heart) were collected from birds showing colisepticemia, air vasculitis, perihepatitis, and pericarditis then pooled together for bacterial screening and isolation. Twenty-five grams (±0.5) composite chicken meat samples (represented by breast) were aseptically excised and transferred into a high-duty sterile stomacher bag with mesh containing 225 ml 0.1% (w/v) sterile buffered peptone water, BPW (Oxoid) whereas homogenized using a lab blender for 2 minutes to obtain a homogenate fluid.

2-Isolation and identification:

The samples were incubated aerobically into buffer peptone water at 37° C for 24 h. A loopful from each incubated sample was streaked onto MacConkey’s agar (HiMedia) and Eosin Methylene Blue agar (EMBA) (Lab M) plates were then incubated at 37° C for 24 hours. The suspected colonies were 1–2 mm in diameter and appeared as a pink color colony on MacConkey and metallic sheen colonies on EMBA. Suspected E. coli colonies were subjected to morphological and biochemical identification, including oxidase, urease, indole production, methyl red, Voges–Proskauer, hydrogen sulfide, and citrate tests along with glucose, lactose, sorbitol, sucrose, and mannitol fermentation (Nolan et al., 2013, Islam et al., 2014).

3-Antimicrobial susceptibility pattern of the isolated E. coli:

Antimicrobial Sensitivity Test (AST) was performed for all isolates against the most commonly used antibiotics by poultry farms in Ismailia. A pattern of 14 antibiotics discs (Oxoid) was used, which include, Tetracycline (Tetracycline 30 µg disk), Ampicillin (Ampicillin 10 µg disk), Amoxicillin (Amoxicillin 30 µg  disk), Gentamicin (Gentamicin 10 µg disk), Kanamycin (Kanamycin 30 µg disk), Co-trimoxazole (Trimethoprim+Sulfamethoxazole 2.25/23.75 µg disk), Streptomycin (Streptomycin 10 µg disk), Ceftazidim (Ceftazidime 30 µg disk), Colistin (Colistin 10 µg disk), Levofloxacin (Levofloxacin 5 µg disk), Lincomycin (Lincomycin 5 µg disk), rifampicin (Rifampicin 5 µg disk), ofloxacin (Ofloxacin 5 µg disk) and Naldixic acid (Nalidixic acid 100 µg disk), using disk diffusion method as previously described (WHO, CDC, 2013). A bacte­rial suspension of 0.5 McFarland was prepared and streaked on Mueller-Hinton agar (Oxoid) plates using cotton swabs. Finally, antibiotic disks were placed on the surface of the plates followed by incubation at 37°C for 24 h. After incubation, the inhibition zones were measured (in millimeters) using a ruler and interpreted according to the guidelines of Clinical and Laboratory Standards Institute (CLSI, 2021).

4- Detection of aacC and Aada2 genes by PCR in E. coli-resistant isolates:

Fifteen E. coli were tested for aacC (aminoglycoside acetyltransferase) and Aada2 (aminoglycoside adenyltransferases) genes by PCR. Both genes are aminoglycoside resistance genes against gentamicin and streptomycin, respectively (Lynne et al., 2008) and (Walker   et al., 2001).

DNA extraction:

DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56^OC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

Oligonucleotide Primer:

Primers used were supplied from Metabion (Germany) are listed in table (1).

Table (1): Primers sequences, aacC and Aada2 target genes, amplicon sizes and cycling conditions.

PCR amplification:

Primers were utilized in a 25- µl reaction containing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentrations, 5.5 µl of water, and 5 µl of DNA template. The reaction was performed in an applied biosystem 2720 thermal cycler.

Analysis of the PCR Products:

The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 15 µl of the products were loaded in each gel slot. A general 100 bp ladder (Fermentas, thermos, Germany) 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.

Results:

Prevalence of E. coli Isolates in Broiler Chicken Samples from Diseased Farms and Markets in Ismailia, Egypt:

In a comprehensive study conducted in Ismailia, Egypt, the prevalence rates of E. coli isolates were investigated in broiler chicken samples collected from both diseased broiler chicken farms and markets. The findings revealed varying rates of E. coli occurrence in the two settings. Out of 70 samples collected from diseased broiler chicken farms, 12 isolates of E. coli were recovered, resulting in a prevalence rate of 17.14 %. In comparison, samples obtained from markets exhibited a lower prevalence, with 8 isolates of E. coli recovered out of the 70 samples, corresponding to a prevalence rate of 11.43 %.

Antibiogram study of E. coli isolated from pooled internal organ samples of diseased broiler chickens collected from broiler farms and breast muscle samples collected from different markets in Ismailia:

In a comprehensive antibiogram study, 20 biochemically confirmed E. coli isolates from pooled internal organ samples of diseased broiler chickens from farms and breast muscle samples from various markets in Ismailia were evaluated for their resistance patterns against a range of selected antibacterial agents (N=14).

The results revealed a concerning trend of multidrug resistance among the tested isolates.

The antimicrobial resistance profile of the 20 biochemically confirmed E. coli isolates from broiler chicken samples in Ismailia revealed noteworthy patterns. Firstly, all isolates displayed 100% resistance against amoxicillin, rifampicin, lincomycin and nalidixic acid, indicating high resistance rates. Secondly, the isolates exhibited elevated resistance against ampicillin (95%), streptomycin (90%) and kanamycin (71.43%) representing the second-highest resistance rates. Conversely, ofloxacin (45.45%), and gentamicin (30%) recorded the lowest resistance rates. Notably, colistin demonstrated sensitivity across all tested isolates. Furthermore, the concerning observation of multidrug resistance patterns emerged, as all isolates exhibited resistance to three or more antimicrobial agents from different classes, underscoring the urgent need for comprehensive strategies to address the pervasive issue of multidrug-resistant E. coli strains in poultry samples.

Detection of aacC and Aada2 resistance genes by PCR in E. coli-resistant isolates:

The antimicrobial resistance genes aacC and Aada2 genes were recorded in 13 out of 15 and 14 out of 15 isolated phenotypically and biochemically recognized E. coli isolates with a per cent ratio of 93.33% and 87.67%, respectively. Both aacC and Aada2 genes were Aminoglycoside resistance genes against gentamicin and streptomycin, respectively.

Discussion:

The study involved the examination of 70 pooled internal organ samples from diseased broiler chicken farms in Ismailia, revealing that 12 of these samples (17.14%) were positive for E. coli. Additionally, 8 out of 70 samples of breast muscles from various markets in Ismailia were found to contain E. coli, resulting in an incidence of 11.43%. These findings underscore significant differences in the prevalence rates of E. coli isolates between broiler chicken samples from farms and those from markets. The higher prevalence in farm samples suggests a comparatively higher risk of E. coli contamination in these environments. Conversely, the higher prevalence in market samples indicates an increased potential for E. coli contamination in retail settings. Possible factors contributing to this difference include variations in hygiene practices, storage conditions, and transportation methods between the two sources. The study emphasizes the critical need for ongoing monitoring and targeted measures to improve food safety standards in both broiler chicken farms and marketplaces. Identifying and addressing specific risk factors associated with E. coli contamination is essential to ensure the overall safety of poultry products and safeguard public health. Continuous surveillance and collaboration between poultry producers, market stakeholders, and regulatory authorities are essential components for mitigating potential risks linked to E. coli contamination within the food supply chain.

These findings seemed somewhat compatible with Moawad et al., ( 2018) and Shecho et al., (2017) who recorded E. coli isolation from avian farms in both Egypt and Ethiopia at a very low rate (13.4% and 11%), respectively. In the same context, colibacillosis incidence was proven to be 0, 84% and 0, 8% in broiler chickens and layers (Matin et al., 2017).

On the other hand, laboratory investigation of 350 collected samples of poultry origin revealed that 132 samples were ensured to have E coli isolates with an incidence of 37.7%. These E coli isolates were segregated from chickens' internal organs with an incidence of 53.4% (Ibrahim et al., 2019).  Furthermore, out of 270 examined whole chicken carcass samples, 216 isolates of E coli were segregated with an incidence of 80% (Eltai, et al., 2020).

In chicken farms, antibiotics are utilized for a variety of purposes, including prophylaxis, growth promotion, and medicinal uses (Almofti et al., 2016 Mohamed-Noor et al., 2012). They include a large number of compounds of different types that can be given in chicken feed or drinking water. However, due to the existence of antibiotic residues and bacteria that are resistant to antibiotics, including E. coli, the careless administration of these medications may have unfavorable effects (Singer et al., 2006 Almofti et al., 2016). Furthermore, several scientific studies have shown a connection between the use of antibiotics in animals raised for food production and the development and evolution of bacteria resistant to antibiotics (Singer et al., 2006, Mohamed-Noor et al., 2012and Almofti et al., 2016).

Recent studies in Egypt and worldwide have reported antimicrobial residues and antibiotic-resistant bacteria in food animal products such as chicken meat suggesting large-scale unregulated use of antibiotics by the poultry industry (Samy et al., 2022, Brower et al., 2017, Mohamed-Noor et al., 2012, and Eckburg et al., 2005).

These seemed compatible with our findings in this study which revealed a marked predominance of antibiotic resistance among E. coli isolates obtained from different diseased broiler chicken farms and markets in Ismailia. Regarding the rising rate of E. coli isolates antimicrobial resistance in this study; these results were somewhat comparable to those published in other Egyptian publications (Amer et al., 2018; El-Seedy et al., 2019; Qurani 2019). Furthermore, numerous reports from all around the world have confirmed this finding, including those from Dou et al., (2016) in China, Rahman et al., (2017) in Bangladesh, (Danachi et al., 2018) in Lebanon, and   Subedi et al.,  ( 2018) in Nepal. These findings point to clear evidence of the indiscriminate and abusive use of certain antibiotics for infection prevention or control. These multidrug-resistant bacteria eventually take the place of the drug-sensitive ones in an environment that is saturated with antibiotics (Van den Bogaard et al., 2001).

In this study, our obtained findings revealed that all isolates of E coli showed 100% résistance against amoxicillin, rifampicin, lincomycin and Nalidixic acid. The second-highest resistance rate was recorded against ampicillin (95%), streptomycin (90%) and kanamycin (71.43%). These results are nearly similar to that of Abdel-Rahman et al., ( 2023) who recorded that most E. coli isolates from diseased cases in broiler Egyptian farms showed the highest resistance percentage to ampicillin and nalidixic acid, Samy et al., (2022) who reported highest resistance against amoxicillin was found among E coli isolates from poultry samples with percentages of 83.3% and Hamed et al., (2021) who detected high resistance of E. coli isolates against ampicillin, tetracycline and nalidixic acid.

Meanwhile, the lowest resistance rate was recorded against ofloxacin (45.45%). This recorded result was somewhat in agreement with Hamed et al., (2021) who detected that E. coli isolates from some Egyptian poultry farms showed less resistance to ciprofloxacin and Moawad et al., (2018) who reported that E. coli isolates from healthy broilers in Egypt showed a low rate of resistance to fluoroquinolones ciprofloxacin (21.4%) and levofloxacin (14.3%). However, in the current study, none of the tested isolates exhibited resistance to colistin. Previous studies conducted in Egypt have found significant differences in E. coli isolates resistance to colistin. Badr et al., (2022) noted that E. coli isolates from broilers chicken farms in three Egyptian governorates displayed a low incidence rate of resistance (41%), Awad et al., (2020) found that 54 flocks of broilers in two North Delta governorates had a high incidence of 92.31%. However, 48 broiler farms spread across five governorates in northern Egypt expressed a very low incidence (7.9%) according to Moawad et al., (2018).

In this study, all isolates showed resistance against 3 or more investigated antimicrobial agents of different class (multidrug resistance) patterns (100%). These obtained results were consistent with Radwan et al., (2020) who recorded that all E. coli isolates from broiler chickens in Beni-Suef, EL-Minia, El-Fayoum, Assiut and Sohag Governorates were 100% multidrug-resistant (MDR), Hamed et al., (2021) who found that all of E. coli isolates from some Egyptian poultry farms expressed resistance to at least three or more antimicrobials and Abdel-Rahman et al., (2023) who reported that all isolates of E. coli investigated for their sensitivity using the disk diffusion method against 18 antibiotics were described as multidrug-resistant strains.

Antimicrobial resistance (AMR) acquisition and spread are linked to several genetic pathways. Numerous mobile and mobilizable genetic components, such as integrons, transposons, insertion sequences, and plasmids, are included in the E. coli mobilome (Gillings, 2014).

It is commonly recognized that integrons have a significant role in the spread of antibiotic resistance in Gram-negative bacteria. Integrons are genetic structures that can transcribe, remove, and express genes. These genes are often found in mobile elements like plasmids permit their bacterial spread (Fluit and Schmitz, 2004). Integrons are genetic constructs that have been found in several studies to contain AMR genes in their variable region (as gene cassettes) in chicken farms (Pérez-Etayo et al., 2018 and Kalantari et al., 2021). The polymerase chain reaction (PCR) is considered one of the most important molecular methods and has been extensively employed in recent years to investigate antibiotic resistance genes.

In the current study, the antimicrobial resistance genes aacC and Aada2 genes were recorded by using PCR in 13 out of 15 and 14 out of 15 isolated phenotypically and biochemically recognized E. coli isolates with a per cent ratio of 93.33% and 87.67%, respectively. Both aacC and Aada2 genes were Aminoglycoside resistance genes against gentamicin and streptomycin, respectively. This result seemed in the same context as Radwan et al., (2018) who stated that antimicrobial resistance genes Aada2 and aacC genes were the most prevalent found in all E coli isolates (100%). Abd Elatiff et al., (2019) by using the Aada2-specific primers, PCR screening for antibiotic resistance genes in E. coli revealed that 12 serogroup isolates were positive.

Conclusion:

In conclusion, this study focus on the significant prevalence of antibiotic-resistant E. coli in broiler chickens, both in internal organ samples collected from diseased farms and in meat samples from markets in Ismailia. The high resistance rates observed against commonly used antibiotics, such as amoxicillin, rifampicin, and lincomycin, raise concerns about the indiscriminate use of these drugs in poultry farming. The detection of aacC and Aada2 genes in the majority of the isolates highlights the genetic basis for resistance against aminoglycosides, further emphasizing the need for responsible antibiotic use in the poultry industry. The study reinforces the importance of ongoing surveillance efforts to monitor and address the emergence of multidrug-resistant bacteria, safeguarding both animal and public health.

Table (2): Results of the antibiotic sensitivity test of E. coli isolates

Table (3): Detection of Aminoglycoside antimicrobial resistance genes aacC and Aada2 against gentamicin and streptomycin

Figure 1. Amplification of Aada2 resistance gene. All samples produced a band at 622 bp (positive to the Aada2 gene) except in lane 14 (negative to the Aada2 gene). Lane M: 1Kb DNA Ladder

Figure 2.Amplification of aacC resistance gene. All samples produced a band at 448 bp (positive to aacC gene) samples, except in lanes 5, and 8 (negative to aacC gene). Lane M: 1Kb DNA Ladder.