Wageningen Academic P u b l i s h e r s

Beneficial Microbes, 2016; 7(1): 35-44

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Selection, characterisation and evaluation of potential probiotic Lactobacillus spp. isolated from poultry droppings S. Asghar1, M. Arif1, M. Nawaz1*, K. Muhammad1, M.A. Ali1, M.D. Ahmad2, S. Iqbal3, A.A. Anjum1, M. Khan1 and J. Nazir1 1Department of Microbiology, University of Veterinary and Animal Sciences, Out-Fall Road Lahore, 54000 Lahore, Pakistan; 2Department

of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Out-Fall Road Lahore, 54000 Lahore, Pakistan; 3Department of Food Science and Human Nutrition, University of Veterinary and Animal Sciences, Out-Fall Road Lahore, 54000 Lahore, Pakistan; [email protected] Received: 17 February 2015 / Accepted: 4 September 2015 © 2015 Wageningen Academic Publishers

RESEARCH ARTICLE Abstract Aim of the present study was to characterise and evaluate probiotic potential of lactobacilli isolated from indigenous poultry. Lactobacilli were isolated from poultry droppings and identified by genus specific polymerase chain reaction and 16S rRNA gene sequencing. Isolates were characterised in vitro by their ability to tolerate low pH and bile salts, phytase activity, antimicrobial activity, antibiotic susceptibility profile, and autoaggregation and coaggregation with poultry gut pathogens. In vivo evaluation of selected isolates was done by their effect on the body weight gain and immune response of broiler chicks. Total of 90, one-day old chicks, were randomly divided in 9 groups and given selected lactobacilli alone and in combinations (108 cfu/bird, daily) from day 7 to day 35. Body weight gain and humoral immune response to New Castle Disease Virus (NDV) vaccine were determined weekly. Three lactobacilli isolates (SMP52, SMP64 and SMP70) were selected as potentially probiotic bacteria on the basis of in vitro characterisation and identified as Lactobacillus crispatus, Lactobacillus casei and L. crispatus, respectively. Chicks supplemented with ‘SMP52’, ‘SMP64’, ‘SMP70’ and ‘SMP64+SMP70’ and a commercial probiotic product (Protexin) showed significantly higher mean weight gain per bird (1,584±35.2, 1,629±30.6, 1,668±34.7, 1,619±29.5 and 1,576±31.7 g/bird, respectively) as compared to negative control group (1,394±26.7 g/bird), on day 35. SMP 70 also showed significantly higher geometric mean titre against NDV vaccine at day 21 as compared to negative control. It is concluded that L. crispatus SMP52, L. casei SMP64 and L. crispatus SMP70 are potential probiotic candidates which alone or in different combinations may increase body weight of broilers. Keywords: Lactobacillus casei, Lactobacillus crispatus, bile tolerance, antibacterial activity

1. Introduction The poultry industry is the 2nd largest industry in Pakistan which generates around 1.5 million jobs. Its contribution to the agriculture and livestock sector is 5.76 and 10.4%, respectively. Poultry meat contributes to 26.8% of the meat requirements of the local market (Anonymous, 2014). The poultry industry faces many challenges such as mycotoxicosis, lower feed conversion rates and infectious diseases (Cegielska-Radziejewska et al., 2013). Antibiotic and toxin binders are used frequently in the poultry industry to solve these problems (Diarra and Malouin, 2014).

Probiotics provide safe and effective alternatives (Kabir, 2009). According to FAO/WHO probiotics are defined as ‘live microorganisms which when given in appropriate amounts confer health benefits on host’ (FAO/WHO, 2001). Probiotics are used for various benefits to human, poultry, ruminants and aquaculture. Benefits of probiotics in poultry include protection from bacterial pathogens, better production performance, feed conversion ratio, weight gain and meat quality, enhanced immune response, digestion and uptake of nutrients, and strengthening of gut microbiota (Gupta and Das, 2013; Kabir, 2009). Although exact mechanisms of action of probiotics are

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2015.002035

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S. Asghar et al.

still under investigations, most agreed mechanisms include production of antimicrobial substances, competitive exclusion of pathogens, production of digestive enzymes such as phytase, and neutralisation of enterotoxins and stimulation of immune response (Chaucheyras-Durand and Durand, 2010; Nava et al., 2005). Effects of probiotics depend on their ability of survival and retention in physiochemical barriers of poultry gut, and their own biochemical capabilities and genetics. Many different species and strains belonging to genus Lactobacillus, Streptococcus, Bacillus, Bifidobacterium, Enterococcus and Saccharomyces have been used as probiotics (Al-Ghazzewi and Tester, 2014; Allegretti et al., 2014). Phytate, an organic form of phosphorous, is present in plant and grain based poultry feeds. Monogastric animals, including poultry, do not have digestive enzymes for the breakdown of phytate (Hill et al., 2007). Phytate also impairs the absorption of other important nutrients by chelation. Phytase is an enzyme which breaks down the organic form of phosphorous (unavailable) into inorganic phosphorous (available) (Lee et al., 2013). Therefore addition of purified phytase enzyme in feed or phytase producing microbes in poultry gut can enhance nutrient availability (Lan et al., 2002). There are many different sources of phytase enzymes including plants, fungi and bacteria (Lee et al., 2013). Probiotic effects of microbes are specie and strain specific and it is important to explore new environments and habitats for the isolation of more efficient probiotic bacteria (Million and Raoult, 2013; Vijaya Kumar et al., 2005). One such habitat is gut of organically reared or backyard poultry which generally exhibit better immune response, health status and higher diversity of microbes as compared to broilers (Park et al., 2013), so it might be a better source of beneficial microbes. Although there are many studies throughout the world, which report phytase producing probiotics (Lan et al., 2002; Lee et al., 2013; Parvaneh et al., 2014), none is from Pakistan, where poultry is 2nd largest industry. Therefore, the present study was designed as a first step to develop phytase producing probiotics, which may cope with insufficiency of indigenous probiotics in Pakistan.

2. Materials and methods Isolation of lactobacilli Dropping samples of backyard poultry (n=20) were collected from different rural areas of Lahore, Pakistan. Samples were collected in sterilised containers, transported to Department of Microbiology, University of Veterinary and Animal Sciences (Lahore, Pakistan) at 4 °C, and stored at -20 °C until further analysis. Lactobacilli were isolated by plating serially diluted samples (10-fold) on De Man, Rogosa and Sharpe (MRS; Lab M Ltd., Bury, UK) media supplemented with nystatin (100 µg/100 ml) followed by 36

48 h incubation at 37 °C in aerobic conditions. Different colonies (one of each type) were selected, purified and stored in MRS broth supplemented with 15% glycerol at -20 °C.

Identification of lactobacilli Preliminary characterisation of isolates was done by Gram’s staining and catalase test. DNA of isolates was extracted with the TIANamp Bacterial DNA Kit (TianGen Biotech, Beijing, China P.R.) following the manufacturer’s instructions. Isolates were further identified to genera level by genus specific PCR using Lactobacillus genus specific primers XB5-F (GCCTTGTACACACCGCCCGT) and LbLMA1-R (CTCAAAACTAAACAAAGT) as described previously (Nawaz et al., 2011a). For species identification, partial 16S rRNA gene of selected isolates was amplified by universal primers 8FLP-F (AGTTTGATCCTGGCTCAG) and XB4-R (GTGTGTACAAGGCCCGGGAAC) as described previously (Nawaz et al., 2011a). Amplicons (~1,400 bp) were sequenced by using same primers by Center of Excellence In Molecular Biology, Lahore. Sequences have been submitted to NCBI GenBank.

In vitro selection of potentially probiotic lactobacilli Resistance to low pH Tolerance of Lactobacilli to low pH was determined as described by Delgado et al. (2007). Briefly, exponentially growing isolates in MRS broth were washed by centrifugation and re-suspended (~107 cfu/ml) in phosphate buffered saline (PBS) with different pH (2, 3, 4), adjusted with 0.1 N HCl, for 90 min. Tolerance to pH was determined by re-culturing 100 µl of cellular suspensions, treated with different pH, in 10 ml MRS broth for 24 h at 37 °C and optical density (OD) was measured at 600 nm. Lactobacillus rhamnosus GG (LGG) was procured from Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, maintained and grown in MRS broth and used as positive control.

Screening for phytase activity Isolates were inoculated on phytase screening medium followed by incubation at 37 °C for 4 days to screen phytase producing isolates. Phytase screening medium contained 0.3% glucose, 0.1% tryptone, 0.1% sodium phytate (SigmaAldrich, St. Louis, MO, USA), 0.0004% FeSO4, 0.05% MgSO4×7H2O, 0.03% CaCl2×2H2O, 0.004% MnCl2 pH 6.5 with 1.5% agar. Isolates surrounded by clear halo were recognised as phytase producers. Phytase enzyme activity of cell free supernatants of selected isolates was measured as described by Askelson et al. (2014). Briefly, cell free supernatants of exponentially grown isolates were used as enzyme source to liberate inorganic phosphate from Beneficial Microbes 7(1)



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sodium phytate in phytase reaction buffer (6.4 mM sodium phytate, 2 mM CaCl2, 100 mM Tris-HCl, pH 7.0) at 37 °C. The enzyme activity was defined as the micromoles of inorganic phosphate liberated in 1 min.

Tolerance to bile Tolerance of isolates to bile salts was determined as described by Bao et al. (2010). Briefly, exponentially growing cultures (1%) were inoculated in MRS broth supplemented with different concentrations of bile salts (0.3, 1.0 and 1.8%) for 24 h at 37 °C. After incubation tolerance was assessed by measuring OD at 600 nm. LGG was used as positive control.

Antimicrobial activity The antimicrobial activity of isolates against Bacillus sp. 34, Campylobacter jejuni 15, Proteus mirabilis 27, Salmonella sp. 09 (all from the Department of Microbiology, University of Veterinary and Animal Sciences), Escherichia coli ATCC25932 and Staphylococcus aureus ATCC29213 (ATCC, Manassas, VI, USA) was determined by well diffusion assay as described by Bao et al. (2010). Indicator microbes were obtained from the collection of Department of Microbiology, University of Veterinary and Animal Sciences, Lahore. Briefly, molten (43 °C) nutrient agar (Lab M) was inoculated with overnight grown indicator microbes (1%), swirled gently and poured into 90 mm petri dishes. After solidification of agar wells of appropriate size were bored in agar plates. Cell free supernatant (CFS) (100 µl) of isolates were added into wells and plates were incubated at 37 °C for 24 h. Antimicrobial activity was measured as the zone of growth inhibition around the well. To prepare CFS, overnight growth of isolates in MRS broth were filtered through a membrane filter with pore size of 0.22 µm. CFS were neutralised to pH 7.0 and boiled at 70 °C for 10 min prior to use.

Antibiotic susceptibility pattern Susceptibility of isolates to different antibiotics including penicillin (10 µg), erythromycin (15 µg), streptomycin (10 µg), nalidixic acid (5 µg), ciprofloxacin (5 µg) and moxifloxacin (5 µg) was determined using the Kirby-Bauer method (Bauer et al., 1996). Overnight grown isolates were centrifuged (5,000 rpm), resuspended in PBS and adjusted to 1 McFarland for inoculum preparation. Bacterial isolates were inoculated on MRS agar using sterile swabs to create a confluent layer. Antibiotic discs were placed on medium surface, incubated for 48 h at 37 °C followed by measuring the diameter of the zone of inhibition (mm).

Characterisation and evaluation of potential probiotics

Autoaggregation and coaggregation Autoaggregation and coaggregation of lactobacilli was determined as described by Bao et al. (2010). Briefly, for autoaggregation, overnight grown isolates were centrifuged at 6,000 rpm, resuspended in PBS and incubated at 37 °C. After different time intervals (0 min, 30 min, 1 h and 2 h) ODs were taken at 600 nm with an DEN-1 densitometer (Grant Instruments, Cambridge, UK). For coaggregation, equal volumes of resuspended (PBS) isolates and indicator microbes (S. aureus, Salmonella spp., Bacillus spp. and E. coli) were mixed and incubated 37 °C. At different time intervals (0 min, 30 min, 1 h and 2 h) ODs were measured at 600 nm.

In vivo evaluation of probiotic potential of lactobacilli in broilers Day old broiler chicks (n=90) were purchased from the local market and reared for 35 days in the experimental room of Department of Microbiology, University of Veterinary and Animal Sciences. Chicks were randomly distributed in nine groups (10 chicks per groups) and housed separately to prevent cross infection. Inoculum consisting of 3 isolates of lactobacilli were prepared according to 2 McFarland standards (108 cfu/ml) for probiotic supplementation to the broiler chicks.

Experimental design Chicks were given potentially probiotic lactobacilli (108 cfu/bird, daily) alone and in combinations (SMP52, SMP64, SMP70, SMP52+SMP64, SMP52+SMP70, SMP64+SMP70 and SMP52+SMP64+SMP70, respectively) from day 7 to day 35. The positive control group was administered with a commercial probiotic (Protexin; Hilton Pharma Ltd., Karachi, Pakistan), while the negative control group was given no probiotic. To prepare inoculums, overnight grown of isolates were centrifuged at 5,000×g, resuspended in normal saline and adjusted to OD equivalent of 1 McFarland. Birds in all groups were vaccinated against NDV using live virus ‘Lasota’ vaccine (Intervac Ltd., Sheikhupura, Pakistan) at day 6 and 16 and killed NDV at day 7.

Body weight gain Birds were weighed on the weekly basis, i.e. at day 1, 7, 14, 21, 28 and 35, while feed intake (FI) and water consumption were checked on daily basis.

Immunomodulatory effects against New Castle Disease Virus Serum samples of each bird from each group were collected at 14, 21, 28 and 35 days old and stored at the -20 °C until further processing. Serum was used to determine antibody titres against NDV by using haemagglutination inhibition

Beneficial Microbes 7(1)

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assay (HI) (Allan et al., 1978). Commercially available ‘Lasota’ vaccine of NDV was used as an antigen or viral source for the test. HI titre of every serum sample was expressed as reciprocal of serum dilution. Geometric mean titre of different groups were calculated using the ‘tube number and table/modified log 2’ method (Allan et al., 1978).

Statistical analysis Data was presented as mean ± standard deviation and compared by one-way ANOVA followed by Tukey’s multiple comparison tests (P

Selection, characterisation and evaluation of potential probiotic Lactobacillus spp. isolated from poultry droppings.

Aim of the present study was to characterise and evaluate probiotic potential of lactobacilli isolated from indigenous poultry. Lactobacilli were isol...
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