Antimicrobial role of Lactobacillus species as potential probiotics against enteropathogenic bacteria in chickens

Introduction: The emergence of antimicrobial resistance among bacterial community resulted in a ban on drugs as the growth promoter in poultry feed. This situation demands to explore alternatives as food supplements with health benefit to poultry. Therefore, probiotic microorganisms, which are considered as safe and possess various health benefits can be a choice. Present study was designed to explore the probiotic potential of the isolated lactobacillus species in chickens. Methodology: Out of 220 samples, 100 Lactobacillus species were isolated from various regions of chicken intestine. They were further characterized on the basis of morphology, staining and catalase test. Species-level identification was made by amplifying Lactobacillus specific 16S rRNA gene. Out of 100 isolates, 21 were selected for sequencing on the basis of band intensity. Results: Among 21 sequences, 16 were identified as L. paracasei (n = 6), L. salivarius (n = 3), L. johnsonii (n = 3), and L. agilis, L. fermentum, L. sakei, and L. curvatus (n = 1 each). These strains were found to be significantly acid-tolerant with 81.68 85.01% survival rate at pH 2)and bile-tolerant with 81.96 -84.65% survival rate at 0.3% bile. Except three; all strains showed salt tolerance to 2% and 4% NaCl. Among 21 Lactobacillus strains, 6 showed good antimicrobial activities against S. aureus, Salmonella Typhimurium and E. coli. Conclusion: Lactobacillus species with probiotic property can be used in poultry feed formulation for their health benefit to combat gastrointestinal infections.


Introduction
Poultry industry plays a crucial role to provide protein-rich food for human consumption in terms of eggs and meat [1].With the continuous expansion of poultry industry incidence of bacterial infections are widely increased posing health concerns to human population with huge economic losses to the poultry industry from production to marketing [2].The most common causative agents of bacterial infections responsible for diarrhea and low poultry productivity are the serotypes of Salmonella and Escherichia coli [3].The transmission of Salmonella occurs through oral-fecal route via contaminated poultry products causing typhoid, food poisoning, gastroenteritis and enteric fever [4,5,6].The main reservoir of Salmonella enterica includes chickens, turkeys, ducks, parrots and coastal species.Salmonella infections are recognized as zoonotic infections transmitted through contaminated meat and processed poultry products [7,8].
To inhibit bacterial infections in poultry, antibiotics have been used for decades as feed supplements [9].Thirty different classes of antibiotics (broad and short spectrum antibiotics) were used at sub-therapeutic level as feed additives in poultry industry to enhance poultry production [10].This excessive use of antimicrobial drugs in poultry industry is one of the main causes for emerging antimicrobial resistant superbugs as well as for the hypersensitivity reactions in humans [2].Beside responsible for antimicrobial resistance, the use of antibiotics also destroys the normal microbiota of chicken making them vulnerable to various other diseases [11].
Considering the evolving problem of antimicrobial resistant bacteria, it is important to explore an alternate having growth promoting effects in animal feed.Probiotics, a name coined by Nobel laureate Élie Metchnikoff are the microorganisms that promote health benefits upon ingestion.Probiotics have a number of beneficial effects on the host; they can help to maintain intestinal microbial flora and aid in digestion and stimulate immune system.According to FAO/WHO, probiotics are live microorganisms which when administered in adequate amounts confer a health benefit for the host [12].Among the most commonly used probiotics in poultry production; lactic acid bacteria (LAB) inhibit the growth of pathogens through competitive exclusion in the gastrointestinal tract therefore enhance the health of the chickens.Out of many species of Lactobacillus, some species such as Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus reuteri, and Lactobacillus rhamnosus, have been used as probiotics [13].
The purpose of this study is to identify indigenous Lactobacilli species by molecular methods and assess their role as potential probiotic.

Samples
This research was conducted at the Poultry Research Laboratory (PRL) Department of Physiology, University of Karachi (UoK), Karachi.A total of 220 samples were collected from intestine, caecum and cloaca of chickens from various market places and poultry farms located in the vicinity of Karachi.Sterile cotton swabs were used to collect the samples and placed in the sterile vials containing De Man, Rogosa and Sharp (MRS) broth (Oxoid, Basingstoke , UK).The samples were transported aseptically to PRL for further processes.

Isolation, preliminary screening and preservation of bacterial culture
Lactobacillus-specific De Man, Ragosa and Sharpe (MRS; Oxoid, city, UK) broth and agar were used to enrich and isolate Lactobacillus species by incubating at 37 °C for 18 to 24 hours.Isolated colonies were picked and characterized on the basis of Gram staining and catalase test [14] followed by preservation in glycerol (50:50) at -80°C for further investigations.

Acid and bile salt tolerance test
Tolerance tests were carried out as described previously [15].In brief, acid and bile tolerance was carried out at pH 2.0, pH 3.0, pH 6.2 and bile salts (Oxoid, city, UK) of 0.2%, 0.3% respectively.Cell viability was calculated post 3-hrs incubation at 37°C by CFU estimation.

Effect of NaCl
Osmotic resistance to NaCl was evaluated according to Kobierecka et al., [16].Briefly fresh culture of Lactobacillus was inoculated in MRS broth containing 2.0%, 4.0% and 6.5% NaCl and incubated overnight at 37 °C.Growth was evaluated on visual inspection of turbidity and results were recorded.

Polymerase Chain Reaction (PCR) and sequencing
DNA from Gram-positive and catalase-negative isolates was extracted using Promega DNA purification kit (Promega, city, USA) as per manufacturer protocol, DNA purity and integrity was measured through UV spectrophotometer and gel electrophoresis.The extracted DNA was amplified using Lactobacillusspecific primers (Table 1) designed in this study.PCR was carried out using GoTaq Green Master Mix (Promega, city, USA) containing 50 to 100ng of genomic using conventional thermocycler (Veriti, Applied Biosystem, city, USA) under following temperature conditions; initial denaturation 95 °C for 5 minutes, 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 57 °C for 30 seconds and extension at 72 °C for 2 minutes and a final extension 72 °C for 7 minutes.The amplified PCR products were electrophoresed on 1.5 % agarose gel and visualized by using UV trans-illuminator ChemiDoc-It2 (UVP, Cambridge, UK) imager and vision works LS software (version 7.1).
For sequencing the PCR products were purified by using Gel purification kit (Bioline, UK), quantified and sequenced from Macrogen Inc. (Seoul, Rep. of Korea).Obtained sequences were assessed with Bioedit software (version 7.0).Forward and reverse sequences aligned together with ClustalW and DNA contigs were prepared.These consensus sequences of DNA were further evaluated using BLAST of the GenBank (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and species identification were confirmed after comparison of similarity with the processed DNA sequences.

Antimicrobial activity
Antimicrobial activity of Lactobacillus strains was assessed by agar well diffusion method [17].Cell free supernatant (CFS) of Lactobacillus strains was filtersterilized (0.22 µm) and evaluated for antimicrobial potential against S. aureus (NCTC-6571), Salmonella Typhimurium (ATCC-14028) and E. coli (ATCC-25922).Precisely a suspension of 10 8 cells of respective cultures was spread on Luria-Bertani (LB) agar; 6 mm diameter wells were punched and filled with 50 µL of Lactobacillus CFS.Plates were incubated at 37 °C; zone of inhibition was measured and characterized as potent (> 20 mm), moderate (10 to 20 mm) or no activity (< 10 mm).

Statistical analysis
One-way analysis of variance (ANOVA) was performed at significance level p < 0.05 for the acid and bile tolerance test SPSS (Statistics 22, IBM).Multiple comparisons of means were assessed by Tukey's HSD post hoc test at significance level p < 0.05.

Effect of NaCl concentration on survival of Lactobacillus species
In the present study, NaCl resistance were variable as all strains survived in 0.34 mol/L (2.0%NaCl) and most of the strains survived in 0.68 mol/L (4.0%NaCl).However, two strains such as L. paracasei (ZA32Cl) and L. johnsonii (ZA79Cl) did not survive n 0.68 mol/L (4.0%NaCl).None of Lactobacillus strains survived in 1.11 mol/L (6.5% NaCl) (Table 4).

Conclusion
Our results suggest that conventional culturedependent techniques are important for preliminary screening of probiotics strains however, molecularbased assessment is essential for species-level identification.Six isolated and well-characterized probiotic strain may have commercial potential to overcome the development of antibiotic resistance in poultry industry.

Table 1 .
Primers used for 16S rRNA gene of Lactobacillus species.

Table 2 .
Acid tolerance of various Lactobacillus strains at low pH.
Cfu = colony forming unit; Data represented as Mean ± SD, each in triplicate.All parameters were calculated using one-way ANOVA; P value < 0.05 taken as significant.

Table 3 .
Bile salt tolerance of various Lactobacillus strains at different bile salt concentration.
BS = Bile salt; cfu = colony forming unit; Data represented as Mean ± SD, each in triplicate; All parameters were calculated using one-way ANOVA.P value < 0.05 taken as significant.

Table 4 .
Resistance of Lactobacillus strains to NaCl + or -sign indicates growth or no growth, respectively.