layers, E. coli, EMB agar, PCR, antibiogram.

India is one of the world's leading producers of eggs and broiler meat, and demand is rising as the population grows, incomes rise, and dietary preferences evolve. Chickens account for over 95% of total egg production in India, with ducks and other poultry species making up the remainder. India ranks 3rd in egg output and 6th in chicken meat production globally. More output is required to meet the ICMR's recommended standards of 180 eggs and 10.8 kg of poultry meat per person per year. Many factors influence productivity; among them, infectious diseases are considered one of the most significant factors. The use of live vaccines, combined with the evolution of many pathogens, has resulted in a rise in disease incidence and resurgence, posing a threat to production. The most common respiratory pathogens, like Mycoplasma, Infectious Bronchitis Virus, Infectious Laryngotracheitis Virus, and E. coli, are the prevalent etiological agents that lead to financial and productivity losses. Avian colibacillosis is one of the major diseases caused by E. coli (Singh et al., 2011). Infected birds exhibit symptoms like cough, respiratory discomfort, poor growth, and productivity (Pang et al., 2002). E. coli, which belongs to the Enterobacteriaceae family and is considered the normal intestinal microflora of humans and birds, is typically present in the pharynx and trachea of birds. It is a gram-negative, non-acid-fast, non-sporulating, facultatively anaerobic, rod-shaped bacterium that ferments lactose, producing acid and gas. The majority of E. coli strains are non-pathogenic and are referred to as commensals. However, after acquiring certain virulence features, some of these bacteria have gained the ability to thrive in diverse organisms and are known as pathogenic E. coli, which cause clinical symptoms associated with intestinal and extraintestinal illnesses (Kaper et al., 2005). E. coli can be classified into two types: commensals and pathogenic. Pathogenic E. coli is divided into two subgroups: extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC), both of which cause gastrointestinal illnesses. Avian pathogenic Escherichia coli (APEC), a kind of extraintestinal pathogenic E. coli (ExPEC), is the cause of avian colibacillosis, an infectious disease in birds. The most common infections in chickens caused by avian pathogenic E. coli (APEC) are septicaemia, enteritis, perihepatitis, pericarditis, airsacculitis, egg peritonitis, salpingitis, coligranuloma, omphalitis, cellulitis, osteomyelitis, and decreased egg yield, quality, and hatchability in layers (Dziva et al., 2008). Layers are susceptible to APEC at any time during the growth and laying seasons, particularly during the late laying period and peak egg production, whereas broilers aged 4 to 6 weeks are more vulnerable (Dho-Moulin et al., 1999). In the presence of stressors, APEC invades the gastrointestinal and respiratory tracts via abraded tracheal and intestinal epithelium, penetrates deeply into the mucosa and submucosa before entering the bloodstream, and spreads to internal organs, resulting in septicaemia (Dho-Moulin et al., 1999; Dziva et al., 2008; Rodrigo Guabiraba et al., 2015). In most countries, poultry flocks are often raised under intensive conditions using a wide range of antimicrobials (Agunos et al., 2012; Landoni and Albarellos 2015). These antimicrobials are typically given orally with the purpose of preventing and treating disease while also boosting development and production (Page and Gautier 2012). A considerable number of these antimicrobials are regarded as important and critical for human medicine (World Health Organization et al., 2017). The indiscriminate use of antimicrobials in animals has accelerated the development of antibiotic-resistant bacteria, which can pose major public health threats (CDC: Atlanta, GA, USA et al., 2019). Furthermore, E. coli has been identified as a natural reservoir of antibiotic resistance genes (ARGs), playing a significant role in the spread of antimicrobial resistance (AMR) (CDC: Atlanta, GA, USA et al., 2019; Partridge et al., 2018), and it is frequently used as an important biomarker for monitoring AMR (Brisola et al., 2019; Poirel et al., 2018). To avoid economic and production losses, it is essential to detect organisms and antibiogram patterns early, as well as propose area-specific medication for treatment.  Regular isolation of organisms is must to know the conformation and antimicrobial pattern for selection of suitable drug. The current work sought to employ molecular approaches to detect E. coli in isolates from layers with respiratory infections and characterize their antimicrobial pattern, thereby assisting in the selection of appropriate treatments and control methods to prevent economic losses in the poultry industry.

Sample collection: Samples were collected from 12 commercial layer farms located in and around Tirupati. A total of 78 pooled oral swabs, 82 pooled tracheal swabs, 86 pooled nasal swabs, and 25 pooled infraorbital sinus exudates were collected from the ailing birds, which were showing respiratory signs and were labeled farm-wise and specimen-wise. 

Isolation and identification of Escherichia coli: After collection, the samples were inoculated into sterile test tubes containing nutrient broth and aerobically incubated at 37 °C in a bacteriological incubator for 24 hours. Following incubation, the inoculum was spread onto MacConkey agar and Eosin Methylene Blue (EMB) agar plates and incubated at 37 °C for 24 hours. Later, the inoculated plates were examined for pink-colored colonies on MCA and greenish metallic sheen colonies on EMB agar. To confirm, the colonies on EMB agar plates were subjected to gram staining and biochemical tests, viz., IMViC tests (indole production, methyl red, Voges Proskauer, citrate test), catalase test, urine production, and nitrate reduction tests, and the organisms showed the IMVC pattern of ++-- (Edwards et al., 1972).

DNA Isolation: DNA isolation was done using the boiling and snap-chilling processes. Initially, E. coli isolates that had been cultured and biochemically confirmed were incubated in nutrient broth at 37 °C overnight. After 18 hours, 1.5 mL of the enriched broth culture was centrifuged for 10 minutes at 10,000 rpm and the pellet was resuspended in 100 µL of nuclease-free water. This mixture was heated in a boiling water bath at 100 °C for 10 minutes before being rapidly chilled in an ice box at -20 °C and centrifuged at 10,000 rpm for 5 minutes. The supernatant was stored at -20 °C and utilized as a DNA template in the PCR assay. DNA concentration was determined using Nanodrop (Thermo Scientific, USA).

Polymerase Chain Reaction (PCR): All extracted DNA samples were submitted to PCR using particular primers to target the 16s rRNA gene for species-specific characterization. Table 1 lists the primers used in the investigation. The reaction used 12.5 μl of master mix, 4 μl of DNA template, 1 μl of forward and reverse primer, and 6.5 μl of nuclease-free water for a total of 25 μl. The PCR reaction was carried out in a thermal cycler with a heated cover under standardized cycling conditions, as indicated in Table 2. The amplified PCR products obtained after the reaction were run through 1.5% agarose gel electrophoresis in a horizontal electrophoresis apparatus at 5 V/cm and ethidium bromide at 0.5 μg/ml. The Alpha Innotech gel documentation system was used to examine the gel under UV transillumination in order to observe the bands. 

Table 1: Primers used for detection of E. coli (Nagendra Reddy Thopireddy, 2023).

Table 2: Cyclic conditions used for amplification of 16s rRNA gene of E. coli

Out of 271 samples, 219 were positive for E. coli, with an overall prevalence rate of 80.81%. On testing, 8 farms out of 12 were found positive for the E. coli infection. Among the 8 positive farms, 59 out of 78 (75.64% oral swabs), 72 out of 82 (87.80% tracheal swabs), 70 out of 86 (81.39% nasal swabs), and 18 out of 25 (72% infraorbital sinus exudates), totaling 219, were confirmed positive by PCR, which produced a predicted size of 585 bp in all positives. Random selection of the isolated E. coli was performed, and 32 [4 (1 from oral swabs, 1 from tracheal swabs, 1 from nasal swabs, and 1 from infraorbital sinus exudate) from each positive farm] isolates were chosen and then subjected to antimicrobial sensitivity tests. The highest resistance was observed for Cephalexin (100%) and Ceftriaxone (100%). The highest sensitivity was observed for Cotrimaxazole (65.62%), followed by Tetracycline (28.12%), Enrofloxacin (25%), Ciprofloxacin (21.87%), and Colistin (15.62%) (Table 3).

Table 3: Antibiotic susceptibility patterns of E. coli isolates from respiratory infections of commercial layers.

Fig. 1. Antibiotic sensitivity patterns of E. coli isolates.

DISCUSSION

E. coli belongs to the Enterobacteriaceae family. E. coli can be classified into two types: commensals and pathogenic. Pathogenic E. coli is divided into two subgroups: extra intestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC). ExPEC is further divided into six subpathotypes: uropathogenic E. coli (UPEC), sepsis/newborn meningitis-associated E. coli (NMEC), avian pathogenic E. coli (APEC) (Kunert Filho et al., 2015), sepsis-associated pathogenic E. coli (SePEC) (Mokady et al., 2005), mammary pathogenic E. coli (MPEC) (Shpigel et al., 2008), and endometrial pathogenic E. coli (EnPEC). DEC is divided into eight subpathotypes: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC), enteroaggregative E. coli (EAEC) (Huang et al., 2006), adherent invasive E. coli (AIEC), and Shiga-toxin-producing enteroaggregative E. coli (STEAEC).

APEC can cause both systemic and localized forms of colibacillosis, acting as a primary or secondary agent. The most prevalent localized infections caused by APEC as the primary pathogen are omphalitis, yolk sac infections, and reproductive tract infections (Landman et al., 2013). E. coli, as a secondary pathogen, contributes to respiratory colibacillosis and causes colisepticemia. Colisepticemia develops secondary to viral infections such as Newcastle disease (NDV), avian influenza (AIV), infectious bronchitis (IBV), Mycoplasma gallisepticum (MG), immunosuppressive diseases (infectious bursal disease (IBD)), and stressors (overcrowding, excessive dust, and ammonia levels) (Dho-Moulin et al., 1999; Ghunaim et al., 2014).

In all 12 poultry farms, clinical signs including ruffled feathers, nasal discharges, facial swelling, labored breathing, huddling, depression, sinus swelling, a decrease in egg production and poor egg quality, reluctance to stand or move, decreased food and water consumption, and complete or extreme prostration. Similar observations were documented by Ramasamy et al. (2008); Gowthman et al. (2013); De Carli et al. (2014); Veeraselvam et al. (2019); Thopireddyet al.(2023). The current study identified E. coli as the predominant concurrent infectious agent linked with respiratory disease complex in eight farms (80.81%), corroborating the findings of previous researchers (Gowthaman et al., 2013; Chowdhury et al., 2018; Kaore et al., 2018).

Post-mortem examination of affected birds revealed polyserositis characterized by white to yellow exudates on the liver surface (fibrinous perihepatitis), heart (fibrinous pericarditis), (Fig. 2), and intestines (peritonitis), along with blood vessel congestion and lung consolidation indicative of severe E. coli infection (Yadav et al., 2018; Surjagade et al., 2020; Halder et al., 2021). The study also identified an acute form of the disease marked by septicemia leading to death and a subacute form characterized by pericarditis, airsacculitis, and perihepatitis, as reported by Dhama et al. (2013); Dadheech et al. (2016) ; Thopireddy (2023).

Fig. 2. PM examination of the affected bird showing pericarditis and perihepatitis.

Isolation and identification of E. coli. After 24 hours of incubation at 37°C in aerobic conditions, all E. coli positive isolates on MacConkey agar exhibited lactose-fermenting pink colonies (Fig. 3), whereas E. coli colonies on EMB agar exhibited greenish metallic sheen (Fig. 4), consistent with findings by Matin et al. (2017); Veeraselvam et al. (2019); Surjagade et al.(2020); Thopireddy et al. (2023). The E. coli isolates appeared as pink-colored gram-negative bacilli, occurring singly or in pairs, (Fig. 5), aligning with observations by Amer et al. (2015); Boro et al.(2019). All E. coli isolates displayed an IMViC pattern of ++-- (Edwards et al. 1972), were urease negative, catalase positive, and oxidase negative, paralleling results reported by Amin et al. (2017); Ibrahim et al. (2019); Nagendra Reddy et al. (2023).

Fig. 3. MCA showing pink colored colonies of E. coli.

Fig. 4. EMB agar showing greenish metallic colonies of E. coli.

Fig. 5. Gram staining showing pink coccobacillary rods of E. coli.

Fig. 6. IMViC tests pattern for E. coli (1. Indole test-positive, 2. Methyl red-positive, 3. Vp test-negative, 4. Citrate test-negative).

All suspected samples (271 samples) from 12 farms were initially tested for E. coli by targeting 16sr RNA gene and the study found that 8 farms with a total of 219 samples, of which 59 (74.64% from oral swabs), 72 (87.80% from tracheal swabs), 70 (81.39% from nasal swabs), and 18 (72% from in fraorbital sinus exudates) tested positive. These results are higher than those reported by Nayak et al. (2017), who found a positivity rate of 14.2% in both tracheal and nasal swabs, and align with Banu et al. (2017), who reported a positivity rate of 57% in tracheal swabs and 57.1% in infraorbital sinus exudates. The current study observed an E. coli isolation rate of 80.8%, similar findings have been reported by other researchers, 80% by Tonu et al. (2011), 82.83% by Ievy et al. (2020) and 100% by Ibrahim et al. (2019). The high isolation rate of E. coli observed in our study, as well as in others, could be due to stringent adherence to microbiological isolation techniques, resulting in improved bacterial recovery. Such a high rate of isolation, positions E. coli as an ideal candidate for assessing antimicrobial resistance (AMR) prevalence and patterns across various production systems, including layer poultry farms (Mudenda et al., 2023).

An investigation in Andhra Pradesh identified E. coli on 12 farms, with a PCR positivity rate of 63.15% and a SYBR Green Real Time PCR positivity rate of 71.2%. On these farms, PCR detected E. coli in 92 nasal swabs (40.35%), 106 tracheal swabs (51.75%), 64 tracheal tissues (42.10%), and 72 lung tissues (47.36%). Furthermore, 118 nasal swabs (49.56%), 128 tracheal swabs (56.14%), 68 tracheal tissues (44.73%), and 77 lung tissues (50.65%) tested positive for SYBR Green Real Time PCR by Nagendra Reddy et al. (2023).

Fig. 7. Data table representing sample size.

Molecular characterization of E. coli. This work used PCR to quickly identify 16s rRNA gene of E. coli. The technique used DNA isolated from E. coli isolates and PCR primers specific to the 16s rRNA gene, which resulted in a 585 bp amplicon confirming E. coli presence. 16s rRNA was identified in 219 of 271 samples using PCR, corroborating the findings of Tonu et al. (2011); Islam et al. (2014); Matin et al. (2017).

Fig. 8. Agarose gel displaying the 585 bpE. coli 16s rRNA gene product amplified by PCR.

Antibiotic sensitivity of E. coli. E. coli includes a large number of antibiotic resistance genes, including beta-lactam resistance genes. The presence of beta-lactam antibiotic resistance genes in E. coli causes the production of beta-lactamase enzymes, which hydrolyze β-lactam antibiotics, a frequent resistance mechanism in the Enterobacteriaceae family (Pitoutet al., 1998). ESBLs are enzymes that efficiently hydrolyze third and fourth-generation cephalosporins and monobactams (e.g., aztreonam), but are inhibited by β-lactamase inhibitors such as clavulanic acid and tazobactam (Fernandes et al., 2014). Furthermore, ESBL-producing E. coli frequently exhibits resistance to multiple classes of antimicrobials, primarily fluoroquinolones, sulfonamides, aminoglycosides, chloramphenicol, trimethoprim, and tetracyclines (Bonnet et al., 2004). The major genes involved in ESBL production are the TEM (blaTEM), SHV (blaSHV), and CTX-M (blaCTX-M) genes. CTX-M-type ESBL-producing E. coli is the most common worldwide (Pfeifer et al., 2010).

Our investigation found that E. coli was resistant to Tetracycline (56.25%) and Cotrimaxazole (31.25%), which is consistent with research on laying hens in Zambia by Mudenda et al. (2023). In contrast, a study from commercial farms in Zambia's Chisamba district by Mtonga et al. (2021) reported a 100% resistance of E. coli to Tetracycline. Additionally, a high resistance rate to Colistin (71.85%) was detected, echoing the results of Hess et al., (2022). Our research indicated that the resistance of E. coli to Cephalexin was the highest (100%), with resistance to Ciprofloxacin and Enrofloxacin at 59.37% and 53.12%, respectively, corroborating Khanal et al. (2017) findings. Similarly, our study discovered E. coli to be highly resistant to Ceftriaxone (100%), which was also found in a study by Islam et al. (2023), that revealed Escherichia coli isolates demonstrating resistance to several cephalosporin antibiotics, ranging from 2.3% to 100% (95% CI ranged from 0.4% to 100%) for Ceftriaxone. 

Nagendra Reddy Thopireddy and  Surendranath Reddy Somanagari (2024). Phenotypic, Molecular Detection, and Antibiogram Patterns of E. coli isolates from Respiratory Infections of Commercial Layers in and around Tirupati. Biological Forum – An International Journal, 16(10): 48-54.