Prevalence of pathogenic Enterobacteriaceae in the excreta of free living wild birds in Agroecosystems

Introduction

The excreta of free-living birds harbour a variety of pathogens such as viruses, bacteria, and fungi and these pathogens can be transferred during the movements of these and result in intra-and interspecies transmission (Gogu-Bogdan et al., 2014) [1] . Although many bacteria are a part of the normal intestinal flora of birds, bird species associating with potentially contaminated environments, such as refuse dumps, sewage treatment facilities and agricultural sites are more likely to harbor pathogenic bacteria in their intestinal tracts. Most of the birds infected with zoonotic bacteria do not display clinical symptoms and are asymptomatic carriers, however, they pose a significant risk to human health (Kobuszewska, and Wysok, 2024) [2] . Of particular concern in the farm environment are the Gram-negative bacteria within the family Enterobacteriaceae that includes Salmonella species, Escherichia coli, Enterobacter aerogenes and Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirablis and Providencia alcalifaciens which are pathogenic to humans (Vilcins et al., 2002 ) [3]. Additionally, antibiotic-resistant strains of these bacteria have been detected in wild bird excreta, raising concerns about the role of wildlife in the dissemination of resistance genes (Mansoor et al., 2024) [4] . The high prevalence of Enterobacteriaceae members in the digestive tract and fecal material of birds is driven by a combination of unique avian physiology, environmental exposure, and dietary factors that favor facultative anaerobes as the birds have a shorter digestive tract, faster transit times, and a distinct anatomy where the digestive and urogenital tracts meet at the cloaca, creating a unique, oxygen-accessible environment suitable for these bacteria (Pan and Xu, 2014) [5] . The wild birds harbor antibiotic-resistant strains of Campylobacter jejuni and Escherichia coli which can be transmitted to humans through direct contact or environmental contamination and posing significant health risks. Besides this, Salmonella enteric, a multidrug-resistant, Gramnegative, facultatively anaerobic or aerobic bacteria, often found to be present in the digestive tract of birds and humans can spread through faecal material of birds and lead to contamination of water and food sources (Ebani et al., 2021) [6] . The literature reported presence of bacteria in fecal samples of birds of agroecosystems such as pigeons, house crow, myna, parakeet etc.

The excreta of free-living wild birds may act as reservoirs of pathogenic bacteria and pose a risk to public health and other wildlife. The present study was thus planned to isolate and identify the pathogenic bacteria (family Enterobacteriaceae) from the excreta of free living wild birds commonly found in agroecosystems of Ludhiana so that appropriate management practices could be followed to minimize exposure to these pathogenic bacteria.

Materials and Methods

Collection of excreta of birds

Fresh excreta of different bird species (blue rock pigeon, Columba livia; rose ringed parakeet, Psittacula krameri; common myna, Acridotheres tristis; house crow, Corvus splendens; cattle egret, Bubulcus ibis and red wattled lapwing, Vanellus indicus) was collected from their roosting, foraging and nesting sites in Punjab Agricultural University (PAU), dairy farm of Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) and residences of Ludhiana city. After collecting the excreta samples in sterile plastic bags with the help of spatula these were labeled appropriately with the source, time and date of collection and then stored at 4 °C till further processing.

Isolation and characterization of bacteria

One gram of fresh excreta sample of each species was thoroughly mixed in 9 ml of MacConkey broth and kept at 37 0 C for 24 hours for pre-enrichment of sample. Aliquots (0.1 ml) from serial logarithmic dilutions of each suspension were pipetted onto the surface of duplicate Petriplates containing MacConkey agar. The inoculum was spread evenly with a sterile T-shaped glass applicator. The Petriplates were incubated at 37 °C for 24 -48 hours. The mean colony count was determined and represented as colony forming units (cfu/g) of each sample.

Identification criteria of bacteria

The bacteria isolated were identified using Himedia Enterobacteriaceae identification kit which was provided with two sterile white opaque strips with 12 wells each for various biochemical and carbohydrate utilization tests. The tests were based on the principle of pH change and substrate utilization. On incubation the bacteria undergo metabolic changes which were indicated by a colour change in the media that was either visible spontaneously or after addition of a reagent (Table 1, Table 2).

Results and Discussion

The excreta of six bird species was tested for the presence of bacteria (Enterobacteriaceae family). Few genus of bacteria were isolated from 12 excreta samples collected from about 60 pellets of six different species i.e. blue rock pigeon(n=4), rose ringed parakeet (n=1), common myna (n=1), house crow (n=3), cattle egret (n=1) and red wattled lapwing (n=1). This may be due to the reason bacteria need a temperature of 37 ° C for their growth, but in the present studies the collection was done in fields after the elimination from the bird body where the temperature is quite different from the growth temperature of bacteria. The amount of bacteria decreased with age of feces. Birds are widely recognized as important reservoirs and disseminators of microorganisms, including several bacterial pathogens of public health concern. Due to their mobility, feeding habits, and frequent proximity to human settlements, birds can play a significant role in the environmental spread of enteric bacteria (Ebani et al., 2021) [6] . The bacterial colonies were identified by biochemical characterization. These isolates belonged to Citrobacter freundii, Enterobacter cloacae, Escherichia coli and Kleibsella pneumoniae strains based on the results of biochemical tests in the kit (Fig. 1). The members of the family Enterobacteriaceae including Salmonella species, Escherichia coli, Enterobacter aerogenes and Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirablis and Providencia alcalifaciens are pathogenic to humans (Janda and Abbott, 2021) [7] . Thus excreta of all bird species studied in present work indicated a source of infection to humans. Out of the 4 samples of pigeon excreta samples tested for Enterobacteriaceae family, 3 were positive for E. coli indicating its prevalence in 75% of the samples and C. freundii, E.cloacae and K. pneumoniae was detected in only one sample revealing 25% of the samples showing positive isolation (Table 2). The occurrence of E. cloacae and E. coli was highest (2 × 10 9 cfu/ g) followed by K. pneumoniae (1.2 × 10 9 cfu/ g) and C. freundii (8.2 × 10 8 cfu/ g) (Table 3). Earlier, 2.5% of 400 fresh fecal samples of rock pigeons found to be positive for shiga-toxin producing E. coli 0157 (Abulreesh, 2011) [8] . Shiga -toxin producing E. coli were found in fecal droppings and/or cloacal swabs of pigeons that live in urban and rural areas around the world (Kobayashi et al 2009, Kowalczyk and Wójcik-Fatla, 2025) [9,10] . The pigeons act as a possible reservoir of Shiga toxin 2f-producing E. coli associated with human disease (Wani et al., 2004, Sonntag et al., 2005) [11,12] . The excreta of rose ringed parakeet carried highest bacterial count of E. coli (7 × 10 9 cfu/ g) followed by C. freundii (6.5 × 10 9 cfu/ g), K. pneumoniae (2.7 × 10 9 cfu/ g) and E. cloacae (1.4 × 10 9 cfu/ g). E.coli (64%) was also isolated from caged parrots of dhaka zoo of Bangladesh (Akhter et al., 2010) [13] . Out of the 3 samples of house crow excreta tested for Enterobacteriaceae family, 3 were positive for K. pneumoniae indicating its prevalence in 100% of the samples E. cloacae was isolated from 2 samples only showing its prevalence in 66% of samples and C. freundii and E.coli was detected in only one sample revealing 33% of the samples showing positive isolation. The excreta of house crow carries highest bacterial count of E. cloacae (1.7 × 10 9 cfu/ g) followed by C. freundii (3.4 × 10 8 cfu/ g), E. coli (3 × 10 8 cfu/ g) and K. pneumoniae (2.3 × 10 8 cfu/ g). Earlier, 9 different species of bacteria belonging to the family Enterobacteriaceae (Citrobacter freundii, Enterobacter cloacae, Proteus mirabilis, Klebsiella pneumoniae, Kluyvera ascorbata, Salmonella arizonae, Salmonella typhi, Shigella flexneri and Shigella sonnei) in fresh fecal samples of the large-billed crow (Corvus sp.) collected from the study site at Bangsar, an urban setting in Kuala Lumpur, Malaysia (Yong et al., 2010) [14] . Crows being scavengers with very versatile feeding habits that will take food from the ground or might have fed on items which have been contaminated by infectious droppings from another infected and become infected or be carriers of these pathogens.

The excreta of common myna carries highest bacterial count of K. pneumoniae (3.5 × 10 9 cfu/ g) followed by E.coli. (2.4 × 10 9 cfu/ g), C. freundii (1.4 × 10 9 cfu/ g) and E. cloacae (8 × 10 8 cfu/ g). The excreta of cattle egret showed highest count of E. cloacae (1.6 × 10 9 cfu/ g) followed by E. coli (2 × 10 8 cfu/ g ) and C. freundii (1.7 × 10 7 cfu/ g). The excreta of red wattled lapwing carried highest population density of E. cloacae (4 × 10 9 cfu/ g) followed by E. coli (2.4 × 10 9 cfu/ g), K. pneumoniae (1 × 10 9 cfu/ g) and C. freundii (1.2 × 10 7 cfu/ g). These findings suggested that excreta of common myna, cattle egret and red-wattled lapwing act as significant carriers of pathogenic enteric bacteriaand may contribute to environmental contamination and zoonotic transmission (Cocoș et al., 2025) [15] .

In the present studies the population density of C. freundii was highest in rose ringed parakeet, E. cloacae in red wattled lapwing, E.coli in rose ringed parakeet and K. pneumoniae in common myna and 73% of excreta samples showed presence of E.coli and 64% were positive for C. freundii, E. cloacae and K. pneumoniae (Fig. 2). Escherichia coli, is a common inhabitant of the intestinal tract of numerous vertebrate species. In the past E. coli also has been reported from bird species. While most strains of E. coli are considered commensal, some strains are responsible for a variety of illnesses, including urinary tract infections(UTIs), gastroenteritis, and bacteremia but more specifically, the Shiga toxin-producing E. coli (STEC) serotype 0157:H7 has been associated with human outbreaks of hemorrhagic colitis and hemolytic uremic syndrome after the consumption of contaminated food, especially beef (Adams et al., 2016, Nemati et al., 2024) [16,17] . Enterobacter species, particularly E. cloacae and E. aerogenes, are important nosocomial pathogens responsible for various infections, including bacteremia, lower respiratory tract infections, skin and soft-tissue infections, urinary tract infections (UTIs), endocarditis, intra-abdominal infections, septic arthritis, osteomyelitis, CNS, and ophthalmic infections. Enterobacter species can also cause various community-acquired infections, including UTIs, skin and soft-tissue infections, and wound infections, among others. The bacteria, Klebsiella pneumoniae is a major cause of pneumonia (Jezzini et al., 2024) [18] , urinary tract infections (Filev et al., 2025) [19] , bloodstream infections and sepsis associated with high morbidity and mortality, particularly in cases involving multidrug-resistant strains (Shi et al., 2025) [20] . Thus, excreta of different bird species in agroecosytems harbor a wide range of pathogenic members of the Enterobacteriaceae family. As these birds are free living, so spreads of infectious agents may occur through excreta of these birds which serve as a significant source of environmental contamination and pose potential zoonotic risks to humans and emphasizing the need for proper hygiene and following of appropriate monitoring measures.

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