Identification of Escherichia coli and Salmonella enterica from Fecal Matter of Selected Birds and Assessment of their Susceptibility Towards Different Antibiotics

Over 10,000 birds, migrate between different countries and continents (Barrowclough et al., 2016; Walesiak et al., 2022). Wild birds are known to be a significant source of bacterial diseases which can be transmitted to both humans and animals (Montero et al., 2021; Mircea et al., 2014). These diseases also spread to aquatic environments (Zhao et al., 2017; Hird et al., 2015) and can lead to multiple abnormalities in infected humans, or cause the infected to become carriers (Lagerstrom and Hadly, 2021). Wild birds transmit these beyond local outbreaks (Rahman et al., 2021). Pakistan is home to over 650 different bird species found in three distinct zoogeographical zones: Ethiopian, Oriental, and Palearctic. This diversity makes Pakistani birds unique (Zaman et al., 2023). Wild birds can transmit over 40 different types of diseases to humans and animals (Gazzonis et al., 2021). These pathogens can also be transmitted even when the birds appear healthy and show no signs of infection (Lagerstrom and Hadly, 2021). Due to their mobility, wild birds can transport pathogens over great distances during migration, increasing the potential for disease outbreaks beyond local areas (Rahman et al., 2021). Wildlife, including wild birds, plays a vital role as a reservoir for enteric bacterial pathogens and zoonotic diseases (Gnat et al., 2021).

Enteric bacterial pathogens cause gastroenteritis resulting high morbidity and mortality which poses significant public health risks (Getie et al., 2019). Environment, water and food specimens are potential sources of such pathogens (Thompson and Seitzinger, 2019). These pathogens also include Salmonella and Escherichia. Various studies have revealed multiple antibiotic resistance (MAR) of such pathogens (Ebomah and Okoh, 2020; Igere et al., 2020). Among the zoonotic pathogens, S. enterica is particularly noteworthy for its high prevalence worldwide, and its ability to infect a wide variety of hosts (Ebani et al., 2021; Foti et al., 2018). E. coli and S. enterica are significant enteropathogens that can cause foodborne infections with a significant impact on public health and high mortality rates (Fu et al., 2022; Wuyts et al., 2015) with mortality rates and significant morbidity (Cabrera et al., 2023).

S. enterica and E. coli are the most prevalent pathogens found in wild birds with outbreaks of septicemia and deaths reported in Canada, Norway, United Kingdom, Sweden, and Switzerland (Ebani et al., 2021; Pavez-Munoz et al., 2021). Wild birds infected with S. enterica may exhibit specific symptoms such as anorexia, pneumonia, diarrhea, neurological disorders, or sudden death (Kakooza et al., 2021; Redweik et al., 2020). These pathogens are also a significant source of antimicrobial resistance (Phiri et al., 2020). S. enterica are often present in the digestive tract of animals, birds, and humans and can be spread through feces, leading to contamination of water and food sources (Ebani et al., 2021). S. enterica is a multidrug-resistant, Gram-negative, facultatively anaerobic or aerobic bacteria that belongs to the Enterobacteriaceae family. Birds can spread S. enterica through fecal shedding, direct contact, and shared environments (Sharif et al., 2020). E. coli also is a major pathogen found in the digestive tract of humans and birds, and is considered to be an important pathogen in bird microbiota (Rahman et al., 2020). E. coli is a rod-shaped, Gram-negative bacterium that belongs to the Enterobacteriaceae family. It has the ability to ferment lactose at 44°C, and produces pink colonies on MacConkey agar and dark purple colonies on EMB (eosin methylene blue) agar plates (Kavitha and Devasena, 2013).

The emergence and spread of antibiotic resistance has grown globally and it is considered a significant threat to the public health of wild birds (Cohen et al., 2020). Wild birds increase the potential for the spread of antibiotic resistance during their migration far distances (Mehmood et al., 2020; Greig et al., 2015). Prolonged use of antibiotics can lead to the development of antimicrobial resistance in microorganisms, making it difficult to treat infectious diseases caused by these microorganisms (WHO, 2018). The overuse and misuse of antibiotics is a major problem that is currently emerging globally (Ahmed et al., 2019).

The objective of this study was to examine the prevalence, susceptibility to antimicrobial agents, and occurrence of antibiotic resistance genes, of E. coli and S. enterica in eight captive and wild avian species, while considering variations in temperature and altitude.

A total of 480 mature captive birds viz. peafowls (Pavo cristatus), ring-necked pheasants (Phasianus colchicus), turkeys (Meleagris gallopavo), and pigeons (Columba livia domestica), were collected from districts in Punjab (Pakistan) including Bahawalpur, Khanewal, Okara, Kasur, Lahore, Sargodha, Chakwal, and Rawalpindi. Similarly, 480 wild birds such as sparrows (Passer domesticus), crows (Corvus splendens), mynas (Acridotheres tristis), and wild pigeons (Columba livia) were also captured from the same localities. GIS map showing sampling sites (districts), is given in Figure 1.

Fecal samples were collected aseptically from sampled birds on a monthly basis using forceps. The samples were immediately preserved in sterile conical tubes with screw caps and transported to the laboratory for further processing. The samples were serially diluted and plated on SS (Salmonella-Shigella) agar media using the streaking method. The plates were incubated at 37 °C for overnight. A single colony from this culture was selected and transferred to EMB (eosin methylene blue) agar media to obtain a pure culture of S. enterica and E. coli isolates. These isolates were saved for further use. A colony from this pure culture was then added to a falcon tube containing nutrient broth, and incubated at 37°C. 10ml of this cultured broth was then transferred to a new falcon tube containing nutrient broth and incubated at 37°C (Chandran and Mazumder, 2014). After incubation, the bacterial growth was examined using Gram’s staining to identify the presence of E. coli and S. enterica (Al-Aalim, 2017).

Freshly cultured E. coli and S. enterica colonies were subjected to morphological characterization and biochemical identification. Phenotypic characteristics of the colonies such as color, size, and edge were recorded (MacFadden, 2000).

16S rRNA gene of E. coli and S. enterica was amplified by PCR using universal primers. 1 µl of template DNA was added to a total of 25 µl reaction solution for PCR containing two primers of the 16S rRNA gene; 1 µl of forward primer (27F): AGAGTTTGATCCTGGCTCAG, 1 µl of reverse primer (1492R): CTACGGCTACCTTGTTACGA, 10 µl of water, and 12 µl of GoTaq Green Master Mix (Promega, USA) under the amplification conditions mentioned in Table I (Mohakud et al., 2019). The PCR products were electrophoresed on 1% agarose gel stained with Ethidium Bromide (EtBr) and using a standard sized molecular marker, 1Kb DNA Ladder RTU (ready-to-use, GeneDireX). The PCR products revealing the thickest bands were sequenced using Sanger’s method at BGI Hong Kong Company Limited, China. The obtained sequences were analyzed and compared for taxonomic identification using NCBI BLAST and submitted to the Genbank database. The phylogenetic relationship of E. coli and S. enterica was checked by phylogenetic tree analysis of the 16S rRNA gene of E. coli and S. enterica using the bootstrap method and MEGA 11.0 (Molecular Evolutionary Genetic Analysis) with 1000 bootstrap replications (Shah et al., 2009).

DNA was extracted from the pure culture using the Qiagen AllPrep kit (Qiagen, CA). The purity and concentration of the DNA were evaluated by gel electrophoresis on 1% agarose gel stained with Ethidium Bromide (EtBr) and using a standard sized molecular marker, 1Kb DNA Ladder RTU (Ready to use, GeneDireX) (Ramon-Laca et al., 2018). ABR genes (qnrA, blaTEM, and sul3) of E. coli and S. enterica were detected by amplifying them using PCR (Bio-Rad, USA) and species-specific primers (Macrogen, Seoul Korea) (Wu et al., 2018). A total of 25 µl PCR reaction solution containing 1 µl of template DNA, 1 µl of F (forward) primer, 1 µl of R (reverse) primer, 10 µl of water, and 12 µl of GoTaq Green Master Mix (Promega, USA) was used in PCR to detect the target ABR genes of E. coli and S. enterica under the amplification conditions mentioned in Table I. The amplified PCR products were analyzed on 1% agarose gel stained with ethidium bromide (EtBr) and using a standard sized molecular marker, 1Kb DNA Ladder RTU (Ready-to-Use, GeneDireX). PCR products revealing the thickest bands, were sequenced using Sanger’s method at BGI Hong Kong Company Limited, China.

The antibiotic susceptibility of E. coli and S. enterica was determined using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar (MHA) plates (Tenover, 2014). 14 different antibiotic discs were applied to MHA plates that had been inoculated with a suspension of E. coli and S. enterica colonies containing 1.5 x 108 cfu/ml. 6 mm discs with varying doses of antibiotics (5-30 μg) sourced from OXOID, UK were placed on the MHA plates and incubated at 37°C for 24 h. The size of the zone of inhibition around each disc was measured to classify the bacteria as resistant, moderately susceptible, or susceptible (Weinstein and Lewis, 2020).

The prevalence of E. coli and S. enterica in selected wild and captive birds was compared using descriptive statistics such as proportion and percentage. Data on prevalence and occurrence of ABR genes of E. coli and S. enterica was collected using spreadsheets (Excel 2010; Microsoft, Redmond, Washington) and analyzed using the chi-square test of independence by SPSS version 21.0 software (IBM, USA).

A total of 159 E. coli isolates (16.6%) were recovered from a sample of 960 captive and wild birds, comprising 17 turkeys, 34 captive pigeons, 26 sparrows, 19 ring-necked pheasants, 31 peafowls, 10 mynas, 13 crows, and 9 wild pigeons. Similarly, a total of 26 S. enterica isolates (2.7%) were recovered from the same sample of 960 captive and wild birds, comprising 4 turkeys, 5 captive pigeons, 2 sparrows, 5 ring-necked pheasants, 4 peafowls, 1 myna, 3 crows, and 2 wild pigeons. These findings demonstrate the prevalence of E. coli and S. enterica among captive and wild birds and provide insight into the distribution of these pathogens among different avian species.

The colonies of E. coli and S. enterica were observed on SS agar and EMB agar media plates. E. coli colonies appeared as circular, smooth and glossy, and dark purple on EMB agar media while colorless on SS agar media. E. coli were Gram-negative rods (bacilli) and typically 0.5-1.0 µm in width and 1-3 µm in length. E. coli colonies were non-spore-forming and arranged in clusters or chains. S. enterica colonies were small, circular and appeared red on SS agar while dark purple on EMB agar media. The colonies were about 2-3mm in diameter and have a smooth, glossy appearance.

E. coli colonies were characterized as gram-negative, non-spore forming, rod-shaped bacteria with peritrichous flagella. The E. coli isolates were found to be positive for lactose fermentation (indicated by the appearance of pink color), catalase fermentation, indole production, and methyl red tests. However, they were found to be negative for citrate utilization, oxidase, and Vogas-Proskauer tests. Similarly, S. enterica isolates were found to be positive for H2S production and glucose fermentation. However, they were found to be negative for citrate utilization, lactose fermentation, urease, and citrate utilization. These results provide important information on the characteristics and metabolic properties of E. coli and S. enterica isolates, which can aid in the identification and differentiation of these bacterial strains.

Captive pigeons exhibited maximum 19.1% and 3.3% occurrence of blaTEM of E. coli and S. enterica, respectively, 18.3% and 2.5% of sul3, respectively in peafowls, and 23.3% and 3.3% of qnrA, respectively in captive pigeons (Tables V and VI). Accession numbers of ABR genes are given in Table VIII.

The antibiotic resistance profiles of E. coli and S. enterica isolates were evaluated. All E. coli isolates were found to be 100% resistant to ciprofloxacin (CIP), and sulfamethoxazole (SXT) while S. enterica exhibited 75% resistance to ampicillin (AMX) and flumequine (FLU), E. coli isolates showed 75% against cefotaxime (CTX) and norfloxacin (NOR). All E. coli and S. enterica isolates were found to be 100% sensitive to doxycycline (DO), gentamicin (GM), neomycin (N), streptomycin (S), and tetracycline (T). All E. coli and S. enterica isolates were found to be 50% susceptible to chloramphenicol (C) and erythromycin (E) (Table VII).

Phylogenetic tree revealed that E. coli strains AMS081 (OP784591) and AMS082 (OP784586) isolated during this study were 100% identical to each other as well as to E. coli strains previously isolated in other studies such as FC5906 (MN661169), EIEC (MF919607), and ETEC (MF919609). Similarly, the S. enterica strains AMS0101 (OP784599), AMS0121 (OP784589), and AMS077 (OP457070) isolated in this study showed 99% similarity among them and with other S. enterica strains that have been previously isolated in other studies such as FC1857 2 (KY776580), CFSAN003959 (CP041184), Sal-1135 (CP045464), and CVM N16S321 (CP049313). This suggests that these strains are closely related and have similar characteristics to those found in previous studies. Phylogenetic tree of E. coli and S. enterica is shown in Figure 2.

The prevalence of E. coli and S. enterica were analyzed in relation to temperature, sampling sites, and elevation. The results showed that the highest incidence of E. coli and S. enterica infections were found in captive pigeons, with rates of 28.3% and 4.2%, respectively (Fig. 3).

On the other hand, the lowest infection rates were observed in wild pigeons, at 7.5% for E. coli and 1% in mynas for S. enterica (Table II). The peak of E. coli and S. enterica cases were reported in July at a temperature of 39⁰C, with infection rates of 26.7% and 5%, respectively (Table III). Overall prevalence of both E. coli and S. enterica was recorded as 16.55% and 2.7%, respectively which is depicted in Figure 1. Results of chi-squared test of independence showing P- value and chi-squared value with respect to selected parameters,are shown in Table IX.

Maximum prevalence 28.3% and 4.2% of E. coli and S. enterica respectively was recorded at elevation of 128m in isolates of birds sampled from Khanewal while minimum 5% and 0.8% of E. coli and S. enterica respectively was recorded at elevation of 508m in samples from Rawalpindi (Table IV). GIS map showing prevalence (%) of E. coli and S. enterica with respect to sampling sites (districts) and elevation level, is given in Figure 1.

In recent years, there has been an increasing interest in wildlife and natural hosts for detecting pathogens and antibiotic-resistant bacteria (Defaye et al., 2023). The potential threat of antibiotic-resistant bacterial colonization in wildlife and subsequent contamination of the environment has been widely recognized (Russo et al., 2022). Using wild animals and their natural habitats to detect antibiotic-resistant pathogens and bacteria has raised concerns in recent years (Smith et al., 2022). The possibility of antibiotic-resistant bacteria colonizing wild animals and polluting their surroundings has increased the potential risk (Liang et al., 2023).

Birds transfer pathogenic bacteria in other animals and humans of one locality to other localities (Batista et al., 2022). Pigeons have the potential to serve as carriers of zoonotic bacteria that can spread to humans, animals and other birds, thus posing a threat to the food chain (Pedersen and Clark, 2007). Among the most common zoonotic disease causing agents that can be transmitted by pigeons are E. coli and S. enterica both of which pose significant biological risks to human health (Abulreesh, 2011). While most strains of E. coli are typically harmless and are found in the digestive tract, certain strains can cause foodborne illnesses and other diseases in birds. According to the results of the current study, a majority of E. coli strains isolated were found to be pathogenic. The distribution of pathogenic and non-pathogenic strains was found to be nearly identical among the pigeons (Bhave et al., 2019). Phenotypic characterization of E. coli comes in accordance with findings of Aworh et al. (2021) and Batista et al. (2022). Lactose fermentation test helps in rapid identification E. coli and other Enterobacteriaceae (Van Belkum et al., 2012). Biochemical identification of E. coli isolates was directly in accordance with Kithar et al. (2016) and Forbes et al. (2007).

We recorded 28.3% prevalence of E. coli in captive pigeons followed by 25.8% in peafowls, 21.7% in sparrows, 15.8% in ring-necked pheasants, 14.2% in turkeys, 10.8% in crows, 8.3% in mynas and 7.5% in mynas. In a previous study by Karim et al. (2020), 40 samples were examined and they found a 52.5% prevalence of E. coli, which was higher than our recorded prevalence. Similarly, in a prior study, 69.64% prevalence of E. coli was reported in healthy pigeons out of examined 112 samples of seemingly healthy pigeons from various locations in the Mymensingh district which was very high than our recorded prevalence in pigeons (Islam et al., 2004). Another study accounted 13.89% prevalence of E. coli in pigeons. Differences in these prevalence, sample size and regional variation may have contributed to the variations among the results of various studies. The recorded colony characteristics and properties of E. coli isolates in this study were consistent with findings reported by Dutta et al. (2013). Similarly, another recent study reported overall 49.5% prevalence of E. coli in six bird species while 54.5% in crow, 61.5% in myna, 78.9% in captive pigeon, and 23.5% in sparrow in Pakistan (Saeed et al., 2023). Major factors such as bacterial strain and environmental factors, such as climate, hygiene practices, and antibiotic use, can impact the prevalence of E. coli.

Bacteria belonging to the Salmonella genus pose a significant risk to both human and animal health. These pathogens are the most common bacteria transmitted from poultry products to humans, and they are also associated with significant economic losses (Glass et al., 2019). We recorded 4.2% prevalence of S. enterica in captive pigeons and ring-necked pheasants, 3.3% in peafowls and turkeys, 2.5% in crows, 1.7% in sparrows and wild pigeons, and 0.8% in mynas. In a previous study, prevalence of 27.5% in pigeons was reported in Dhaka (Karim et al., 2020). In a previous study on pigeon diseases in Khulna Sadar and surrounding private farms, a prevalence of 20.32% salmonellosis was reported, with more cases detected among younger pigeons aged 30-90 days (Islam et al., 2004). Another study reported prevalence of 22.2%, 58.3%, and 27.5% for Salmonella spp. in cloacal swabs, footpads, and feces of pigeons from the Mymensingh district, respectively, with an overall prevalence of 35.71%. This study also reported variable prevalence of 40%, 20%, and 30% of S. enterica in pigeons in markets, farms, and villages, respectively (Hosain et al., 2012). In a recent study, overall, 0.98% prevalence of S. enterica was reported in bird species in South Korea (Wei et al., 2020). Similarly, another recent study reported overall 30.6% prevalence of S. enterica in six bird species while 22.2% in crow, 20% in myna, 36.6% in captive pigeon, and 20% in sparrow in Pakistan (Saeed et al., 2023). The big difference in the prevalence was due to different geographical location, bacterial strain, nutritional resources, and environmental factors.

In the current study, we identified three antibiotic resistance genes (qnrA, sul3, and blaTEM) in E. coli and S. enterica. Specifically, qnrA of E. coli was identified in 106 (11%) samples, sul3 in 78 (8.1%), and blaTEM in 72 (7.5%) while qnrA of S. enterica was identified in 17 (1.8%) samples, sul3 in 12 (1.2%), and blaTEM in 16 (1.7%). blaTEM and qnrA gene were also identified in pigeon (Aslantas and Govce, 2020). Similarly, these genes were also identified in E. coli (Le et al., 2020; Ben et al., 2020). In contrast to our findings, higher occurrence of three ABR genes has been reported in E. coli by (Racewicz et al., 2022). As compared to our results, very high rate of occurrence of these ABR genes of S. enterica has been reported (Zhao et al., 2017).

Our study evaluated antimicrobial resistance of E. coli and S. enterica isolates in fecal birds, and found a relatively high frequency of resistance to commonly used antibiotics bacterial infections (Assoumy et al., 2021). Using one antibiotic can cause resistance to another antibiotic through cross-resistance and co-selection (Saifullah et al., 2016). Amoxicillin and ciprofloxacin have been associated with increased resistance in E. coli, while frequent use causes higher levels of resistance (Kakooza et al., 2021). The connection between antibiotic consumption and resistance is intricate, and recent investigation has revealed both affirmative and adverse correlations of antimicrobial resistance (AMR) in E. coli and S. enterica strains extracted from captive pigeons (Ahmed et al., 2019). Almost all isolates were resistant to at least one antibiotic. Pigeons can acquire pathogens through contaminated food and water, and may contribute to the spread of antibiotic-resistant bacteria. Our findings are consistent with prior research and suggest a need for greater awareness of the human-animal-environment interface in the spread of AMR (Pouwels et al., 2018).

Captive birds have higher prevalence of E. coli and zoonotic S. enterica compared with wild bird species, which can affect human health. Misuse and frequent application of multiple antibiotics in enclosures of captive bird species cause antibiotic resistance and the emergence of ABR gene against each specific antibiotic. This ultimately enhances the rate of pathogenicity of E. coli and S. enterica, posing a major potential threat not only to captive but also to wild birds. The antibiotic resistance pattern of these bacteria causes severe infections in infected birds.

No funding was received for this study.

The current research work was approved by Departmental Board of Studies of Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Lahore, Pakistan.

Not applicable.

All the authors agreed with the content and gave explicit consent to submit and they obtained consent from the responsible authorities at the institute/organization where the work has been carried out.

These will be provided by the corresponding author on reasonable request.

The authors have declared no conflict of interest.