Identification of Antibiotic Resistance genes (blaCTX-M, sul1, gyrA, aac(3)-II) and Virulence genes: (tsh, iss, hlyF) of Avian Pathogenic Escherichia coli isolated from respiratory and enteric infections of broiler chickens

Introduction

Avian colibacillosis, caused by Escherichia coli, is a critical bacterial disease affecting poultry worldwide. The disease manifests as septicemia, airsacculitis, pneumonia, enteritis, and systemic organ damage, leading to significant economic losses in the poultry industry (Aworh et al., 2021) [9] . The emergence of multidrug-resistant (MDR) strains is a growing concern, particularly strains producing extended-spectrum β-lactamases (ESBLs), which complicate treatment and pose potential public health risks (Bonvegna, 2022) [11] . Resistance genes such as blaCTX-M, sul1, gyrA, and aac(3)-II are frequently associated with MDR E. coli, while virulence-associated genes, including tsh, iss, and hlyF, enhance bacterial adhesion, serum survival, and pathogenicity (Delicato et al., 2002; Mellata et al., 2009) [15,35] . Molecular typing using Multi-Locus Sequence Typing (MLST) allows characterization of genetic lineages, helping trace epidemiological patterns and potential zoonotic transmission (Vinodhkumar et al., 2024) [57] . Given the extensive use of antibiotics in poultry farming, surveillance of antimicrobial resistance, virulence determinants, and genetic diversity in local poultry populations is crucial. This study aims to provide comprehensive data on MDR E. coli in broilers in Al-Diwaniyah, Iraq, combining bacteriological, molecular, and histopathological approaches.

Materials and Methods Bacterial Isolation and Sample Collection

A total Fifty swab samples were aseptically collected from the lungs, intestines, and air sacs of broiler and layer chickens from poultry farms in Al-Diwaniyah Province, Iraq, between September and December 2025. Samples were divided into two groups: Group 1 consisted of tissue specimens fixed in 10% neutral buffered formalin for histopathological examination, while Group 2 included swab samples used for bacteriological and molecular analyses (Figures 1 and2). Sampling and handling procedures were performed according to standard veterinary microbiological protocols to ensure accurate pathogen recovery (Abd El-Tawab et al., 2020) [1] .

Bacteriological identification

Swabs were cultured on nutrient agar, MacConkey agar, and Eosin Methylene Blue (EMB) agar and incubated aerobically at 37°C for 24 hours. Lactose-fermenting colonies showing pink coloration on MacConkey agar and a metallic green sheen on EMB agar were presumptively identified as Escherichia coli, consistent with conventional diagnostic criteria (Ibrahim et al., 2019) [23] . Confirmed identification was carried out using the VITEK® 2 automated system (bioMérieux), which has demonstrated high diagnostic accuracy for Enterobacteriaceae in recent evaluations (Moges et al., 2021) [38] .

Antibiotic sensitivity testing

Ten confirmed E. coli isolates were tested for antimicrobial susceptibility using the VITEK® 2 system against ampicillin/sulbactam, cefotaxime, ciprofloxacin, colistin, gentamicin, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole. Interpretation of resistance profiles followed the standards of the Clinical and Laboratory Standards Institute (CLSI, 2024), which provides internationally accepted breakpoints for antibiotic testing.

Molecular Detection of Resistance and Virulence Genes

Genomic DNA was extracted using a commercial kit according to the manufacturer's instructions. Conventional PCR was performed to detect resistance genes (blaCTX-M, sul1, gyrA, aac(3)-II) and virulence genes (tsh, iss, hlyF), which are commonly associated with avian pathogenic E. coli (APEC) strains (Paixão et al., 2021) [43] . PCR amplification conditions and gel electrophoresis analysis were performed following established molecular diagnostic protocols (Borges et al., 2019) [12] .

Histopathological Examination

Formalin-fixed tissues were processed routinely, embedded in paraffin, sectioned at 4-5 µm, and stained with hematoxylin and eosin (H&E). Microscopic lesions were evaluated according to standard veterinary histopathological criteria (Mesquita et al., 2022) [37] .

Results s Bacteriological Identification of E. coli

All lung, intestinal, and air sac swabs were cultured on nutrient agar at 37°C for 24 hours. Suspected colonies were subcultured on MacConkey agar and EMB agar.

All confirmed E. coli isolates demonstrated:Green metallic sheen colonies on EMB agar figure 3, Rose-pink colonies on MacConkey agar (figure .4).

Antibiotic sensitivity test results

Ten confirmed E. coli isolates were tested against seven antimicrobial agents (Fig. 5) High resistance was observed to ampicillin/sulbactam and trimethoprim/sulfamethoxazole (100%). Moderate resistance was observed to gentamicin and ciprofloxacin (80%). Lower resistance was observed to cefotaxime and colistin (40%).

Molecular Detection of Antimicrobial Resistance Genes

The molecular analysis of antimicrobial resistance genes revealed a high prevalence of resistance determinants among the examined Escherichia coli isolates. All ten isolates (100%) demonstrated successful amplification of the blaCTX-M gene at 395 bp, indicating the widespread presence of extended-spectrum β-lactamase (ESBL)-associated resistance (Figure 6A). Similarly, the sul1 gene was detected in all isolates (100%) at 530 bp (Figure 6B), confirming the genetic basis of sulfonamide resistance observed phenotypically. Furthermore, the gyrA gene was amplified in 8 out of 10 isolates (80%) at 291 bp (Figure 6C), suggesting mutations associated with quinolone resistance. The aac(3)-II gene, responsible for aminoglycoside resistance, was also detected in 8 isolates (80%) with an expected product size of 451 bp (Figure 6D). Collectively, these findings indicate the coexistence of multiple antimicrobial resistance genes among the studied isolates.

Molecular Detection of Virulence Genes

The distribution of virulence-associated genes demonstrated a high pathogenic potential among the isolates. The tsh gene was detected in all isolates (100%) with an amplicon size of 420 bp (Figure 7A). Likewise, all isolates (100%) were positive for the iss gene at 323 bp (Figure 7B), highlighting their enhanced serum survival capability.

In addition, the hlyF gene was identified in 9 out of 10 isolates (90%) at 599 bp (Figure 7C), indicating the presence of hemolysin-associated virulence factors. The high prevalence of these virulence determinants supports the classification of the isolates as avian pathogenic E. coli (APEC) strains.

Lung

Histopathological evaluation of lung tissues revealed lesions compatible with serous to purulent pneumonia. There was extensive inflammatory infiltration, mainly composed of lymphocytes, surrounding and infiltrating the alveolar structures. Marked alveolar necrosis and structural disruption were evident, along with vascular dilation and congestion within pulmonary vessels. In some areas, fibrosis was observed, suggesting chronic inflammatory progression. These findings indicate severe pulmonary involvement in response to E. coli infection (Figure9).

Air Sacs

Microscopic examination of air sac tissues showed characteristic lesions of airsacculitis. The sections revealed marked inflammatory cell infiltration, predominantly lymphocytes, along with hyperplasia of the epithelial lining. Areas of necrosis were observed in association with fibrinous exudate deposition. Prominent vascular dilation, congestion, and early fibrotic changes were also evident, reflecting both acute and chronic inflammatory processes. These pathological findings confirm severe air sac involvement in infected chickens (Figure10).

Discussion

The present study revealed a high prevalence of multidrugresistant (MDR) Escherichia coli isolates recovered from poultry farms in Al-Diwaniyah Province. The observed 100% resistance to ampicillin/sulbactam and trimethoprim/sulfamethoxazole is consistent with previous global findings reporting elevated resistance rates to βlactams and sulfonamides in poultry-associated E. coli isolates (Xexaki et al., 2023; Soodan, 2022; Nasrollahian et al., 2024) [40] . The extensive use of these antimicrobial agents in poultry production, either for therapeutic or prophylactic purposes, is considered a major selective pressure driving this resistance (Islam et al., 2023) [24] . The 80% resistance observed to gentamicin and ciprofloxacin further supports the presence of MDR phenotypes in poultry environments. Similar patterns of increasing resistance to fluoroquinolones and aminoglycosides have been documented in poultry isolates worldwide (Tigabie et al., 2023; Shariati et al., 2022; Veloo et al., 2025) [48,56] . Although lower resistance rates were detected for cefotaxime and colistin (40%), the emergence of resistance to colistin is particularly alarming because it is considered a last-resort antibiotic in human medicine (Amegayibor et al., 2025) [8] . The spread of mobile colistin resistance genes such as mcr has been widely reported and represents a serious One Health concern (Mousavi et al., 2025). Molecular analysis demonstrated that all isolates (100%) harbored the blaCTX-M gene. This finding indicates widespread dissemination of ESBL-producing strains in poultry farms, similar to reports from Indonesia (Faridah et al., 2023), Senegal (Cissé et al., 2024) [13] , Pakistan (Saeed et al., 2023) [20] , and Iran (Moradzadeh et al., 2025) [39] . The dominance of CTX-M-type ESBL genes in animal production systems has been widely documented (Bonvegna, 2022) [11] , confirming their global expansion across different hosts and ecological niches. [44,45,49] . The gyrA gene was detected in 80% of isolates, confirming the molecular basis of ciprofloxacin resistance. Mutations in the quinolone resistance-determining region (QRDR) of gyrA are widely recognized as key mechanisms of fluoroquinolone resistance (Song et al., 2020) [51] . High prevalence of ciprofloxacin-resistant E. coli in broiler farms has been reported in Bangladesh (Das et al., 2023), although regional variations have been observed (Das et al., 2020; Masood et al., 2019). Local Iraqi studies have also documented gyrAmediated resistance among clinical isolates (Al-Fayyadh et al., 2017) [6] . The aac(3)-II gene, detected in 80% of isolates, explains the phenotypic resistance to gentamicin. Aminoglycosidemodifying enzymes such as AAC (3)-II reduce antibiotic binding to ribosomes, thereby impairing protein synthesis inhibition (Cirit et al., 2019). The co-occurrence of aminoglycoside resistance genes with other antimicrobial resistance determinants in poultry isolates has been reported in genomic studies (Aworh et al., 2021; Mak et al., 2022) [9,33] . Regarding virulence factors, the universal detection of tsh and iss genes (100%) and the high prevalence of hlyF (90%) indicate that these isolates possess significant pathogenic potential. The tsh gene plays an important role in adhesion and colonization of the avian respiratory tract (Dozois et al., 2000; Delicato et al., 2002) [15,17] . High prevalence of tsh among APEC isolates has been reported in recent studies (Abdelhady et al., 2025; Hasanet et al., 2023; Shah et al., 2025) [2,22,47] . The iss gene enhances serum survival and resistance to complement-mediated killing (Subedi et al., 2018) [52] , and its high prevalence in poultry isolates has been documented in [16,34] . Detection of hlyF in 90% of isolates suggests the presence of ColV-like virulence plasmids, which frequently carry multiple virulence determinants (Mellata et al., 2009; Zhuge et al., 2018) [35,63] . High prevalence of hlyF in poultry isolates has been reported in Brazil and South Korea (Jeong et al., 2021; Lúcio et al., 2025) [26] , and its dissemination may reflect selective pressure in intensive poultry systems (Alanazi, 2016) [5] . Histopathological findings in intestines, lungs, and air sacs confirmed severe inflammatory and necrotic changes. Hemorrhagic enteritis and mucosal erosion observed in the present study are consistent with intestinal barrier disruption mechanisms described previously (Olkowski et al., 2005; Koynarski et al., 2010) [41] . Inflammatory cell infiltration reflects activation of host immune defense mechanisms (Kogut et al., 2018) [29] , while tissue necrosis represents advanced pathological progression (Kapakin et al., 2015). Pulmonary lesions including serous/purulent pneumonia and alveolar necrosis correspond with previously described pathological stages of bacterial pneumonia (Song et al., 2022; Li et al., 2023; Abdelhamid et al., 2024; Jahan, 2023) [3,25,50] . Air sac lesions characterized by congestion, fibrinous exudate, and fibrosis are typical manifestations of colibacillosis in poultry (Dutta et al., 2013; Srinivasan et al., 2014; Govindhasamy et al., 2023; Zaki et al., 2025) [18,21,62] . From an immunological perspective, bacterial virulence factors interact with host immune regulatory mechanisms, potentially affecting disease severity. Recent research highlights the importance of enzymatic immune regulators in poultry antiviral and antibacterial responses, including the role of amylase-mediated pathways in immune modulation (Al-Ibadi, et al., 2025) [7] . Such interactions may influence the progression and systemic impact of bacterial infections.

Conclusion

The study revealed that Escherichia coli which have been isolated from broiler chickens in Al-Diwaniyah strong antibiotic resistance, including ampicillin and trimethoprim/sulfamethoxazole, along with the presence of multiple resistance genes (blaCTX-M, sul1, gyrA, aac(3)-II). Virulence-associated genes (tsh, iss, hlyF) were also frequently detected, indicating a high pathogenic potential. These findings underscore the importance of continuous monitoring of antibiotic use in poultry productioan and the implementation of biosecurity and infection control measures to limit the spread of resistant and pathogenic bacteria and protect both animal and human health.

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