Gut health has become a major focus in both human and animal nutrition, as intestinal microbial communities play vital roles in digestion, nutrient absorption, immune system development, pathogen defense and overall host well-being. Probiotics are well-documented for their ability to reduce mortality, inhibit harmful bacteria, improve growth performance and support gut and immune functions (Narayanan and Subramonian, 2023; Miranda et al., 2021; Wang et al., 2023; Radhika et al., 2020; Shawky et al., 2026). They offer potential solutions to industry challenges, including high feed costs (Thirumalaisamy et al., 2016), emerging diseases (Chatterjee and Rajkumar, 2015), climate change impacts (Mottet and Tempio, 2017) and the demand for antibiotic-and residue-free production, while addressing antimicrobial resistance, food safety, animal welfare and environmental sustainability.
       
Probiotic species such as Lactobacillus, Bifidobacterium, Streptococcus, Bacillus, Aspergillus, Enterococcus, Candida and Saccharomyces have shown beneficial effects in poultry (Abreu et al., 2023). Although candidates can be sourced from diverse environments (Pino et al., 2019), strains originating from the host are generally preferred for better adaptation and colonization (Oyewole et al., 2018).
       
FAO/WHO (2002) emphasize safety as a critical criterion for probiotic use. While Lactobacillus spp. are generally regarded as safe, some strains harbor antibiotic resistance genes (Zhang and Zhang, 2019; Todorov et al., 2019), posing risks of horizontal gene transfer to pathogens or commensals (Sharma et al., 2016). Lactobacillus strains are reported to readily colonize the animal gut and potentially transfer resistance genes to bacterial pathogens (Thumu and Halami, 2019). In addition, certain strains may acquire virulence-associated traits from other bacterial species (Todorov et al., 2017). Therefore, antimicrobial resistance profiling and hemolytic activity testing are essential for comprehensive safety evaluation. In this context, the present study aimed to isolate and characterize Lactobacillus spp. from different regions of the chicken gut and assess their safety for potential probiotic application in poultry production.

Isolation of probiotic bacteria
 
The study was conducted at ICAR- Central Avian Research Institute (CARI), Izatnagar, Uttar Pradesh, India (28.4049° N, 79.4331°E). Adult broiler parent birds of the colored synthetic male line (CSML) were housed under a deep litter system with uniform management and feeding practices. Five birds were randomly selected, fasted overnight and gut samples were collected separately from the crop, proventriculus, ileum and caeca for the isolation of Lactobacillus sp.
       
Samples were pooled by gut segment into sterile PBS, homogenized and centrifuged (700×g, 5 min) to remove debris. Supernatants were enriched in MRS broth and incubated anaerobically at 37°C for 24 h. Cultures were streaked on MRS agar, incubated (37°C, 24 h, anaerobic) and forty colonies (10 per gut segment) were randomly selected based on morphology. Isolates were sub-cultured twice in MRS broth/agar to ensure purity. Purified isolates were preserved as glycerol stocks at -40°C following standard protocols. and revived in MRS broth for further characterization, including sugar fermentation assey, antibiotic susceptibility and hemolytic activity, by standard procedures.
 
Characterization of bacterial isolates
 
Phenotypic characterization of isolates was performed by Gram staining (Himedia kit K001). Catalase activity was assessed by adding 3% H₂O₂ to a loopful of culture and observing for bubble formation. Carbohydrate utilization profiles were determined using the HiCarbo™ kit (Himedia) with 24 h cultures standardized to OD₆₀₀ = 0.5, inoculated (50 µl/well), incubated at 37°C for 24 h and interpreted as per manufacturer’s instructions.
       
Genomic DNA from 40 isolates was extracted using the QIAamp® DNA Mini Kit (Cat. No: 51304) and quantified using a NanoDrop spectrophotometer (Thermo Scientific NanoDrop One). PCR amplification of the 16S-23S rRNA intergenic spacer region (ISR) was performed using primers LOLMAl-rev (5′-CTC AAA ACT AAA CAA AGT TTC-3′) and R16 (5′-CTT GTA CAC ACC GCC CGT CA-3′) (Dubernet et al., 2002). The PCR protocol consisted of initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final extension at 72°C for 7 min. Amplicons (~250 bp) were visualized on 1.5% agarose gel stained with ethidium bromide under a gel documentation system (Biozen PROCCD 516).
 
Antibiogram test
 
The strain’s antibiotic sensitivity of the probiotic bacteria was evaluated using the kirby-bauer disc method. The antibiotics used in the study included tetracycline, methicillin, cefixime, gentamicin, bacitracin, clindamycin, enrofloxacin, chlortetracycline, colistin, streptomycin, norfloxacin, penicillin, vancomycin, cotrimoxazole, kanamycin and trimethoprim. These plates were then anaerobically incubated at 37°C and growth inhibition was assessed after 24 hours. The inhibition zones 1 mm, 2-5 mm, 5-10 and more than 10 mm were classified as strains of no (-), mild (+), strong (++) and very strong (+++) inhibition, respectively.
 
Hemolytic activity test
 
The isolates were sub-cultured in MRS broth medium for 18-24 hours at 37°C. Streak plate methods were performed on sheep blood agar plates to analyze the haemolytic activity. The appearance of clear zones around the bacterial colonies was indicated as α-, β-, or γ-haemolysis (Reuben et al., 2019).

Grams staining and catalase activity
 
Forty bacterial isolates-ten each from the crop (CR 1-10), proventriculus (PR 1-10), Ileum (IL 1-10) and caeca (CE 1-10)-were obtained based on general morphological characteristics of Lactobacillus. Gram staining and light microscopy (Plate 1) revealed all isolates as gram-positive rods, occurring singly, in pairs, or in chains. All tested catalase-negative (Plate 2), distinguishing them from catalase-positive genera. Agarose gel electrophoresis (Plate 3) showed distinct amplification bands confirming the presence of conserved Lactobacillus-specific DNA sequences.






 
Sugar fermentation tests
 
The results of the sugar fermentation tests are presented in Supplementary Table 1 and Plate 4. Most of the isolates fermented maltose, fructose, dextrose, mannose, inulin, sucrose, trehalose and exhibited positive esculin hydrolysis. It was reported that the assimilation d galactose, d glucose, d lactose, d maltose, d melibiose, d raffinose and d sucrose was common for all analyzed  Lactobacillus spp (Kobierecka et al., 2017). Similarly, Kassa et al. (2024) reported that all isolates were able to ferment fructose, sucrose, galactose, maltose, mannose and mannitol. In the present study, 17 of the 40 isolates were able to ferment lactose. This finding is comparable to that of Kassa et al., (2024), who observed that four of the twelve isolates tested were unable to ferment lactose. Sugars like maltose, sucrose overlap with those fermented by our isolates, highlighting the differential saccharolytic capability of Lactobacilli.




       
In contrast, most isolates were negative for xylose, L-arabinose, sodium gluconate, glycerol, inositol, arabitol, erythritol, α-methyl-D-glucoside, α-methyl-D-mannoside, xylitol, ONPG, Cellobiose, D-arabinose, malonate, sorbose and citrate utilization. Similarly, Kassa et al., (2024) also reported that 5 out of 12 isolates tested shown no fermentation with inositiol.
       
The fermentation profiles of other sugars revealed both shared and region-specific metabolic capabilities among the studied Lactobacillus isolates (Plate 4). Lactose (17/40), melezitose (18/40) and galactose (19/40), fermentation was prevalent, with most positive isolates distributed across the crop, proventriculus and Ileum, but rarely in the caeca-indicating a moderately conserved trait that suggests differential sugar availability or adaptation. Raffinose and melibiose were widely utilized by most isolates from the crop and Ileum, but less frequently in the proventriculus and caeca. Variable fermentation of salicin, dulcitol, sorbitol, rhamnose and mannitol was observed, with higher frequencies in crop and ileum isolates, reflecting possible metabolic versatility in these regions. Rhamnose fermentation was mainly restricted to crop and proventriculus.  Adonitol was rarely utilized, with limited fermentation seen only in a few ileum isolates. This variability in sugar fermentation is consistent with previous findings that suggest adaptability of certain strains to sugar availability in their environment. In lactose-deficient conditions, some Lactobacillus strains undergo physiological adaptations to utilize readily available sugars more efficiently (Al-Naseri et al., 2013). These restrictions can be partly explained by the absence of key enzymes for fermentation in many lactic acid bacteria genomes. Instead, poultry Lactobacillus appear to specialize in the rich supply of glucose-linked sugars present in feed. The findings reveal clear differences in carbohydrate utilization pattern in Lactobacillus spp., indicating microbial specialization along the gastrointestinal tract of chickens.
 
Antibiotic sensitivity test
 
The results of the antibiotic sensitivity tests are presented in Supplementary Table 2 and Plate 5. The susceptibility of probiotics to conventional antibiotics is a desirable and health-promoting trait, as genes conferring antibiotic resistance can potentially transfer to intestinal pathogens, complicating treatment efforts (Nallala et al., 2017). The results revealed consistently high-level susceptibility to penicillin with the majority of isolates showing strong inhibitory responses (+++). However, with regard to cefixime, resistance was also observed in a few isolates showing only mild inhibition. This observation is consistent with previous reports that Lactobacillus species are typically sensitive to β-lactams like penicillin but tend to be more resistant to cephalosporins (Gueimonde et al., 2013; Ammor et al., 2008).




       
Similarly, clindamycin, chlortetracycline and enrofloxacin demonstrated high activity, with the majority of isolates showing clear sensitivity and relatively few displaying resistance or intermediate responses. However, clindamycin showed poor sensitivity or resistance in approximately 25% of the isolates, indicating a notable level of reduced efficacy. Anisimova and Yarullina (2019) demonstrated 10% of tested isolates showed resistance to clindamycin. Royan and Mortezaei (2024), also reported clindamycin resistance genes in Lactobacillus strains. Campedelli et al. (2019) identified 60 genes encoding resistance to clindamycin and observed 20% resistance to clindamycin among 182 Lactobacillus strains. In the case of enrofloxacin, although most isolates in the present study were sensitive, however, literature reports such as Dec et al. (2017) have documented a 48% resistance rate to enrofloxacin among Lactobacillus strains isolated from poultry, highlighting the potential for emerging resistance under field conditions.
       
Although some isolates exhibited reduced sensitivity, moderate to high susceptibility was noted for antibiotics such as tetracycline, norfloxacin and bacitracin. However, around 50% of the Lactobacillus isolates showed complete resistance to sulfonamides, including trimethoprim and cotrimoxazole. The variable susceptibility observed with these antibiotics may potentially be attributed to strain-specific resistance mechanisms or prior exposure to these drugs. These findings are consistent with those of Dec et al. (2017), who reported a high prevalence of resistance to tetracycline (74%) among Lactobacillus strains isolated from poultry. It was also noted that genes associated with acquired tetracycline resistance were commonly detected in probiotic products, with tetK being the most frequently identified determinant (Floris et al., 2025). Interestingly, a high level of resistance to norfloxacin was also reported by Sharma et al. (2016) in Lactobacillus species isolated from commercial probiotic preparations. Moderate to high sensitivity to bacitracin eas observed in all the isolates of Lactobacillus screened in the current study, in contrast Han et al. (2015) reported 100% resistance in 29 Lactobacillus isolates screened. Furthermore, Campedelli et al. (2019) reported 84% resistance to trimethoprim, while Klare et al. (2007) observed broad or partly species-dependent MIC profiles of LAB against trimethoprim and trimethoprim/sulfamethoxazole.
       
Widespread resistance was also observed to kanamycin, gentamicin, streptomycin and colistin, with most isolates showing minimal or no inhibition. This finding aligns with the known poor uptake of aminoglycosides under anaerobic conditions and the inherently low permeability of the Lactobacillus cell envelope to these compounds. The resistance observed is likely intrinsic rather than acquired, as reported by Anisimova and Yarullina (2019). Colistin, a cationic antibiotic that disrupts bacterial membranes, is known to be primarily effective against Gram-negative bacteria and has minimal activity against Gram-positive organisms (Bialvaei and Samadi, 2015). The isolates in this study displayed resistance to colistin, likely due to the thick peptidoglycan layer characteristic of Gram-positive Lactobacillus species.
       
Similarly, the majority of strains in this study showed resistance to methicillin and vancomycin, which may also be attributed to intrinsic mechanisms. It has been reported that, in Lactobacillus spp., the terminal D-alanine in the muramyl pentapeptide is replaced with D-lactate or D-serine, preventing vancomycin from binding effectively (Delcour et al., 1999), thereby conferring resistance.
       
Lactobacilli of poultry gut origin are reported to carry acquired antibiotic resistance genes (Saleem et al., 2018) and their transmission to other gut bacteria poses a public health concern through horizontal gene transfer (Rokon-Uz-Zaman et al., 2023). Therefore, a critical first step in evaluating the suitability of Lactobacillus isolates as probiotic feed additives is a thorough assessment of their antibiotic resistance profiles. Genetic screening for known resistance genes (e.g., those conferring resistance to commonly used antibiotics) could also enhance the selection of safe probiotic strains. These findings are essential for clinical safety assessments of probiotic formulations, as they highlight the need to monitor and verify antibiotic resistance profiles in Lactobacillus strains intended for probiotic use in poultry.
         
Hemolytic activity test
 
Hemolysis is a common virulence factor among pathogenic microorganisms, facilitating iron acquisition and potentially leading to anemia and edema in the host (Vesterlund et al., 2007). It may also result from strong hydrogen peroxide production, as observed in Streptococcus gordonii (Barnard and Stinson, 1996). Hemolytic activity was assessed for all 40 isolates on blood agar to evaluate their potential pathogenicity and interactions with host red blood cells. The results are presented in Supplementary Table 3 and Plate 6.




       
A majority of isolates (37 out of 40; 92.5%) exhibited gamma hemolysis, indicating no hemolytic activity. This is characteristic of non-pathogenic or commensal organisms, such as many Lactobacillus spp. Only three isolates (IL 7, IL 9 and IL 10) demonstrated alpha hemolysis, identified by a greenish discoloration around the colonies, suggestive of partial red blood cell degradation. Goldstein et al. (2015) reported that Lactobacillus strains are typically alpha-hemolytic. In the present study, 3 out of 40 isolates displayed alpha hemolysis, while the remaining were non-hemolytic. However, Nallala et al. (2017) observed no alpha hemolytic activity in Lactobacillus strains when cultured on human blood agar. Although reports of alpha-hemolytic Lactobacillus strains exist, they are relatively rare. Additionally, Reuben et al. (2019) reported that 3 out of 18 lactic acid bacteria (LAB) isolates were β-hemolytic, which is generally considered unsafe for probiotic use. These findings underscore the variability in hemolytic activity among Lactobacillus isolates and emphasize the importance of screening for hemolysis to ensure safety for poultry applications.

The present study was conducted to characterize Lactobacillus spp. from various parts of the chicken gastrointestinal tract and evaluated their safety profile as potential probiotic candidates. All 40 isolates exhibited typical characteristics of Lactobacillus, including gram-positive rod morphology and catalase negativity. Carbohydrate fermentation profiling revealed region-specific metabolic capabilities, indicating functional diversity among gut-associated isolates. The variability in resistance to antibiotics such as tetracycline and enrofloxacin highlights the importance of strain-specific evaluation, particularly in the context of transfer of antibiotic resistance genes.

This study was supported by the ICAR-Central Avian Research Institute, Izatnagar, Uttar Pradesh, India, through the provision of necessary facilities. The authors also wish to express their sincere gratitude to the Director, CARI, for their kind support in facilitating this research.
 
Funding
 
The work was supported by Institutional funded project of ICAR- Central Avian Research Institute, Izatnagar, Uttar Pradesh, India.
 
Author contributions
 
All authors contributed to the conception and design of the study. Dr. Simmi Tomar provided supervision and conceptual guidance. Material preparation and data collection were conducted by Dr. Hudson. G.H, Dr. Saravana Kumar. K and Dr. Vijayasarathi. M.K. The first draft of the manuscript was written by G.H. Hudson and Dr. S. Ilavarasan. All authors reviewed and provided feedback on previous versions of the manuscript. All authors read and approved the final manuscript.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Ethics statement
 
The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Central Avian Research Institute, Izatnagar and Experiment was carried out in 2024-25.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.