Probiotic Potential of Autochthonous Lactobacillus against Avian Pathogenic Escherichia coli 

Colibacillosis is a major bacterial disease in chicken caused by Escherichia coli (APEC). It affects gut of poultry causing reduced weight gains and mortality, resulting in economic loss to the poultry producers. In order to prevent and control this disease, antibiotics are frequently used, which has led to an increase in antimicrobial resistance. Probiotics are a potent substitute for antimicrobial drugs, which are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (WHO, 2002). Among a variety of probiotic organisms, Lactobacilli are the most significant bacterial group having biological, medicinal and host immune-modulating characteristics that are generally recognized as safe. Supplementing Lactobacillus species in poultry improved the birds’ resistance to infectious agents like Escherichia coli. (Jin et al., 1998).
 
An organism to be selected as a probiotic species should have the ability to attach and colonise the intestinal lining and must be able to survive the harsh conditions of bile salt and stomach acid (Mahasneh and Abbas, 2014). choosing species-specific probiotic species from the gut microbiota is beneficial since autochthonous probiotic species are more resilient and stable in the gut of host from which they originate (Soto et al., 2010). Therefore, the present research was aimed to investigate the antimicrobial ability of autochthonous poultry-specific Lactobacillus species against pathogenic E. coli in order to include them in probiotic or synbiotic formulations for poultry. 

Collection of samples
 
A total of 73 samples (50 cloacal swabs and 23 tissues) from desi and commercial chicken were collected from different areas of Krishna district, Andhra Pradesh during August 2021 to February 2022 using sterile cotton swabs. The swabs were then immediately placed in LMB (L-cystiene MRS Bromocresol green) broth and were transported to the Microbiology Laboratory of the Department of Veterinary Microbiology, NTR College of Veterinary Science, Gannavaram in anaerobic candle jar. These samples were streaked onto LMB agar plates and incubated in CO₂ incubator at 37°C for 48 h. Characteristic green centered colonies with transparent halo around colonies were tested for Gram’s staining and streaked onto MRS agar and incubated in CO₂ incubator at 37°C for 48 h.
 
Identification of Lactobacillus
 
A series of biochemical tests which included Catalase, Indole, Methyl red, Voges-Proskauer, Citrate and Sugar fermentation were performed for identification of Lactobacillus spp. and the results were interpreted according to Bergey’s Manual of determinative bacteriology. Lactobacillus spp. were further confirmed using genus specific PCR targeting 16S rRNA (Sharif et al., 2018). The primers used and the amplicon size was given in Table 1. The assay was performed in thermal cycler under standardized cycling conditions which include initial denaturation 95°C for 3 min, 1 cycle; denaturation 95°C for 30 sec, primer annealing 60°C for 1 min, extension 72°C for 1 min for 35 cycles; final extension 72°C for 7 min 1 cycle; hold/stand by 4°C for 10 min.


 
Aggregation test
 
Carried out as per the method of Jankovic et al., (2012) with some modifications. Overnight cultures of Lactobacillus were taken in MRS broth. Bacteria grown in broth were harvested by centrifugation at 3000 rotation per minute (rpm) for 5 min, then washed and resuspended in PBS to give a final optical density of 1 (about 1 x 10⁹ CFU/mL) at 600 nm. The OD values were measured for 4 h and 24 h. Percentage of aggregation was calculated according to the following equation.
 

%A= 1 - (A/A0) x 100

 
Where:
A= Absorbance after 4 h incubation and 24 h of incubation.
A0= Absorbance before incubation.
 
Cell surface hydrophobicity
 
Tested as per the method of Del et al., (2000) with some modifications. Two ml of an overnight culture of Lactobacillus cultured in MRS broth were used for the test. The cultures were centrifuged and the pellet was suspended in PBS to give a final optical density of 1 (about 1 x 10⁹ CFU/mL) at 600 nm. 500 µL of bacterial suspension was transferred into another eppendorf tube and 200 µL of xylene was added. 200 µL of n-hexadecane was added to remaining 500 µL of bacterial suspension. Hydrophobicity was calculated as the percentage decrease in the OD₆₀₀ of the bacterial suspension due to partitioning of cells into the hydrocarbon layer.
 

Where:
A₀ and A= Absorbance before and after hydrocarbon extraction respectively.
 
Acid and bile tolerance tests
 
Carried out as per the method of Torshizi et al., (2008) with some modifications. Cell suspensions of Lactobacillus were prepared in MRS at pH 2, 3 and 6.5, which were incubated for 3 h in shaker incubator at 37°C. After 3 h each sample was streaked onto MRS agar and incubated at 37°C for 48 h anaerobically to determine the presence or absence of growth, which was used to confirm livability of the strains. The OD values of the bacterial suspensions were taken at 600 nm after 3 h.

Overnight cultures of the isolates were centrifuged for 10 min at 10,000 rpm. The pellet was suspended in MRS broth with different concentrations of ox-bile (0.1%, 0.3% and 0.5%) and incubated in shaker incubator at 37°C. Test cultures were evaluated at 2, 4 and 6 h for the presence or absence of growth by streaking samples onto MRS agar and the OD values of the bacterial suspensions were taken at 600nm for 2, 4 and 6 h.
 
Agar well diffusion assay
 
Carried out as per the method of Chen et al., (2018). The MH agar plates were swabbed on the surface with pathogenic E. coli, which was isolated from an outbreak and serotyped (O101) by National Salmonella and Escherichia Centre, Central Reasearch Institute, Kasauli. Wells of 6 mm diameter were prepared and sealed with MRS agar and cellfree supernatants from isolated Lactobacilli were loaded in the wells (100 µl/well). Following 24 h incubation at 37°C, inhibition zones were recorded. DMSO was used as negative control.
 
Antibiotic susceptibility test
 
ABST was carried as per the method of (Bauer et al., 1966). The density of bacteria suspension was adjusted until the visible turbidity was equal to 0.5 McFarland standard. The inoculum was spread evenly over the entire surface of the plates. Subsequently, paper discs of 15 commonly used antibiotics were laid on the plates and incubated anaerobically at 37°C. The inhibition zone diameters were measured and the results were expressed in terms of resistance, intermediate or susceptible, according to interpretative standards (Charteris et al., 1998).
 
Sequencing
 
The 16s rRNA PCR amplified products of the different Lactobacillus isolates were sequenced using Sanger sequencing on a 3500 genetic analyser (Applied biosystems, California, USA). To identify the species of the isolate, a similarity search was performed using BLAST in the NCBI database.
 
Phylogenetic analysis
 
The evolutionary history was inferred using the UPGMA method (Sneath and Sokal, 1973). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein ,1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. This analysis involved 16 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 347 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021).
 
Haemolytic activity
 
Columbia agar plates containing 5% (w/v) sheep blood were incubated at 37°C for 48 h and evaluated based on green zones around colonies (α-haemolysis), clear zones around colonies (β-haemolysis) and no zones around colonies (γ-haemolysis) on Columbia blood agar plates (Mangia et al., 2019).
 
Gelatine liquefaction test
 
A gelatine medium containing 12% gelatine (Sigma-Aldrich, St. Louis, MO, USA) was inoculated with Lactobacillus strains at a concentration of 1% and incubated for 48 h at 37°C. Gelatine liquefaction of strains was assessed by storing medium in a refrigerator for 24 h and checking whether gelatine was hydrolyzed or not.

A total of 73 samples (50 cloacal swabs and 23 tissues) were collected from desi and commercial chicken, of which 56 (76.71%) were found positive for Lactobacillus by cultural methods. All these isolates exhibited change in colour of LMB broth from copper blue to green colour, green centered colonies with transparent halo around colonies on LMB agar and pale white/cream coloured colonies on MRS agar. These isolates were found to be Gram positive bacilli, negative for catalase test, IMViC tests and gelatin liquefaction test. These results were consistent with earlier reports that the isolated Lactobacillus spp. from the G.I. tract of chicken (Niamsup et al., 2003). All the Lactobacillus isolates which were confirmed by cultural and biochemical tests were found to be positive for 16S rRNA gene with 341 bp.

To be efficacious, a probiotic strain must be viable at the site of action and adhere to epithelial cells and mucosal surfaces (Aziz et al., 2019). We found that 37 isolates had autoaggregation ≥50%, 17 showed autoaggregation potential 20-50% whereas 2 showed little autoaggregation potential ≤20% after 24 h of incubation. Strains with ≥50% autoaggregation were considered to have high autoaggregation potential (Aziz et al., 2019).

Cell surface hydrophobicity (CSH) is amongst the most important surface attributes controlling cell adherence to abiotic and biotic surfaces and biofilm formation (Chen et al., 2018). In this study 5 isolates showed CSH values higher than 93% (strong hydrophobic) and 33 isolates showed between 66 to 93% (hydrophobic) with N-hexadecane whereas, 25 were hydrophobic and none of the isolates showed strong hydrophobicity when tested with xylene (Fig 1). Out of the 56 isolates, 22 isolates with high autoaggregation and hydrophobicity potential were selected and subjected to acid and bile tolerance tests.



Probiotic bacteria must be able to endure the acidic and bile environment of the intestine. The pH in GIT of chicken ranges from 2.6 in proventriculus to 6.3 in large intestine (Church and Pond, 1974). We found that 16 of the tested isolates withstand pH 2.5, whereas all isolates showed high viability at pH 6.5. Bile salt tolerance is required for strains to establish and survive in the chicken intestine (Du et al., 1998). In this study, all the tested 22 isolates showed viable colonies at all concentrations of ox-bile (0.1%, 0.3% and 0.5%) after 2, 4 and 6 h of incubation.

The ability of probiotic Lactobacilli to suppress the growth of pathogenic bacteria is one of their most essential characteristics (Ben et al., 2012). Sixteen isolates were selected based on viability at acidic pH and all bile concentrations and were subjected to well diffusion assay against pathogenic E. coli. All the tested isolates showed inhibitory activity against E. coli with zone of inhibition ranging from 10-18 mm (Fig 2). The inhibitory activity may be due to production of organic acids primarily lactic acid, which lowers the pH, making it unsuitable for bacterial development.



Probiotics must be tested for antibiotic sensitivity to make sure they are free of antibiotic resistance genes. (Nallala et al., 2017). All the 16 isolates showed sensitivity to chloramphenicol, ampicillin and erythromycin. Lactobacillus were sensitive to antibiotics inhibiting protein synthesis, such as clindamycin, chloramphenicol and erythromycin (Charteris et al., 1998). We found that 75% of the isolates exhibited resistance to nalidixic acid, vancomycin, tetracycline and streptomycin but the selected isolate is sensitive to tetracycline. High intrinsic resistance of Lactobacillus has been reported against streptomycin, gentamicin and vancomycin which are aminoglycosides and glycopeptide (Jose et al., 2015). Lactobacillus to be used as a feed additive must be susceptible to ampicillin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol (Marchwinska and Gwiazdowska, 2022). But, the isolates in this study exhibited resistance to vancomycin, gentamicin and streptomycin which may be chromosomally encoded and is an intrinsic feature of Lactobacillus, hence may not be transferable and such isolates may be used as a feed additive (Casarotti et al., 2017).

One of the key unfavourable metabolic activities of probiotic bacteria that contribute to increased pathogenicity is hemolytic activity and hence non-haemolytic strains should be chosen because they are considered safe (Ambalam et al., 2013). One isolate among the 16 selected Lactobacillus isolates was non-haemolytic (γ-haemolysis) and the remaining showed haemolysis (-haemolysis). Gelatinase enzyme is considered a virulence factor as it may hydrolyze collagens that initiate an inflammatory response hence, strains which are negative for gelatinase activity must be selected (Da Silva et al., 2019). All the isolates in our study were negative for gelatin hydrolysis assay.

Sequencing results revealed that the selected isolate showed 96.82% of its similarity to Lactobacillus fermentum which was the major Lactobacillus species in the gastrointestinal tracts of swine and poultry and exhibited good adherence to the intestinal epithelium, resistance to the gastric juice, bile tolerance and antagonistic effects against enteric pathogenic bacteria (Lin et al., 2007).

Phylogenetic tree revealed that the similarity of the selected isolate to different strains of Lactobacillus in descending order is as follows: 99.83% similar to Lactobacillus fermentum NR 104927.1:350-690, 99.79% to Lactobacillus fermentum NR 118978.1:258-597, 99.48% to Lactobacillus cerevisiae NR 158030.1:360-700, 99.45% to Lactobacillus garii NR 170423.1:311-652, 99.42% to Lactobacillus reuteri and Lactobacillus pantheris, 99.415% to Lactobacillus plantarum and Lactobacillus pentosus, 99.41% to Lactobacillus brevis, 99.39% to Lactobacillus vaginalis NR 041796.1:353-694, 99.32% to Lactobacillus siliginis (Fig 3).

We conclude that the autochthonous probiotic Lactobacillus fermentum species successfully suppressed pathogenic E. coli. Therefore, it may further be tested in vivo before its inclusion in probiotic/synbiotic feed additives as alternate to antibiotic growth promotor in poultry.

The authors declare that they have no conflict of interest.