Faecal lactobacilli from common pheasants and their characterization

Common pheasants have been most often breeded for sport hunting and for culinary meat. Lactic acid bacteria (LAB) are a part of the obligate intestinal microflora. They play important role in the development of the immune system. Concerning the common pheasants, only limited information exists in relation with LAB. The aim of our work was to study faecal lactobacilli isolated of 60 common pheasants (farm in west Slovakia) to the genus, Lactobacillus were allotted two strains of Lactobacillus brevis and two to Lactobacillus plantarum (50 isolates) identified by Maldi-Tof system expressed on the basis of high score values identification; confirmed by phenotypization and polymerase chain reaction. Undesirable enzymes β-glucuronidase and β-glucosidase showed negative reaction in L. plantarum LP13, L. plantarum LP14, L. brevis LB12. Amounts of lactic acid produced by strains were in accordance with typical representants of homofermentative lactobacilli. Strains inhibited the growth of at least of two out of 16 indicators; mostly listeriae. L. plantarum LP13 was the most active strain. LB12, LP13 and LP14 tolerate very low pH and 1% bile in growth medium. Results are contribution to the limited knowledge in this area; LP13 strain was selected for the further detailed antimicrobial studies.


INTRODUCTION
Common pheasants husbandry has been most often provided for sport hunting but also for culinary meat.Therefore, healthy pheasants are of paramount importance to farmers and consumers.Immune system plays an important role in health of animals; the common intestinal microflora plays an important role in development of the immune system.Lactic acid bacteria (LAB) are a part of the obligate intestinal microflora.Only limited information can be found in the literature on the microflora of common pheasants (including LAB).Some authors assert that the dominated LAB in pheasants are bifidobacteria, lactobacilli, peptococci and streptococci (Shulin and Xiuli, 1998).LAB especially those possessing probiotic properties have shown many beneficial effect on health of animals and humans; e.g.lactobacilli are known to transform hexoses to lactic acid which has antimicrobial effect (Jahreis et al., 2002;Strompfová and Lauková, 2013).They can also tolerate lower pH or bile in the *Corresponding author.E-mail: laukova@saske.sk.Tel.+421557922964; Fax:+421 55 7287842.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Abbreviations: LAB, Lactic acid bacteria; CFU, colony forming unit; Maldi-Tof, matrix assisted laser desorption/ionization-time of flight mass spectrometry; mmo/l, millimol per liter; mm-millimeter; HSP, feed mixtures for animals; log10, logarithmus 10; nmol, nannomol; µg, microgram.
intestinal tract which is an acceptable condition for their probable application (Vinderola and Reiheimer, 2003).Moreover, some lactobacilli can cause antimicrobial effect due to the production of bacteriocin substances (De Vuyst et al., 1996;Enan, 2006).Following these properties, the aim of our work was to characterize lactobacilli detected in faeces of common pheasants; the results will contribute to limited knowledge in microflora of pheasants; promising strain with beneficial properties can be selected.

Sampling and identification
Pheasants were located in the aviaries of west Slovakia farm with free movement.They were 16-17 weeks old.Faeces from 60 pheasants were sampled.Six feacal mixture of randomly picked up faeces from 10 pheasants in each sample were provided.Pheasants were fed commercial feed mixture BZ3 (HSP, Bratislava, Slovakia) for pheasants broilers.They had access to water ad libitum.Sampling, birds handling and care were performed in relation to acceptance of Slovak Veterinary and Food Administration and farmer.Mixture samples (in each, faeces of 10 pheasants) were treated by the standard microbiological method.Briefly, 1 g of faecal mixture from each sample in Ringer solution (Merck, Germany) was treated using the Stomacher (Masticator, Spain) and then diluted in Ringer solution (ratio 1:9).To enumerate lactic acid bacteria (LAB), the appropriate dilutions were plated on MRS agar (Merck, Germany; MRS broth supplemented with 1.5% (v/w) agar, ISO 15214) and the plates were incubated at 37°C for 48 h (in CO 2 atmoshpaere).The total counts of lactic acid bacteria were enumerated as an average count of colonies grown in the highest dilution (per sample) and expressed in colony forming units per gram (CFU/g, log 10).Selected colonies were checked for their purity and prepared for the identification to the species level.The species identification was provided by the use of the Maldi-Tof BioTyper TM identification system (Bruker Daltonics, 2008) based on the analysis of bacterial proteins measured by Maldi-Tof mass spectrometry.Lysates of bacterial cells were prepared according to the instruction of producer prior to identification (Bruker Daltonics, 2008).Moreover, phenotypization using the commercial BBL Anaerobe kit (Becton and Dickinson, Cockeysville, USA) was performed.As control strain, Lactobacillus plantarum CCM 4000 (Dr.Nemcová, University of Veterinary Medicine and Pharmacy, Košice, Slovakia, deponed to Czech Culture Collection in Brno, Czech republic) was used.Bacterial DNA for the PCR reaction was extracted according to Klein-Lanhorst et al. (1991).The primers and protocol for PCR (Bethier and Ehrlich, 1998) were as follows: 5´-ATGAGGTATTCAACTTATG-3´ and 5´-GCTGGATCACCTCCTTTC-3´, initial denaturation at 95°C for 5 min, 30 cycles at 95°C for 1 min, temperature and time of annealing were 51°C 1 for min, 72°C for 1 min and elongation at 72°C for 7 min.Thermocycler Techgene KRD (the Great Britain) was used.The PCR product was separated by electrophoresis in 0.8% agarose gels (Sigma, Germany) containing ethidium bromide (1 µg/L Sigma).The molecular mass standard (100 bp DNA ladder, Invitrogen, USA) was used according to the manufacturers instruction.L. plantarum CCM 4000 (Dr.Nemcová, University of Veterinary Medicine and Pharmacy, Košice, Slovakia) was used as positive control.

Enzymes testing
The isolated strains were tested for enzyme production using the API-ZYM system (BioMérieux, France) following the manufacturers recommendations.The inocula (65 µl) of the McFarland standard 1 suspensions were pipetted into each well of the kit.Enzyme activities were evaluated after 4 h of incubation at 37°C and after the addition of Zym A and Zym B reagents.Color intensity values from 0 to 5 and their relevant value in nanomoles were assigned for each reaction according to the color chart with the kit.

Antibiotic phenotype and lactic acid production
Antibiotic phenotype of the strains was tested according to EFSA test (2012) using the antibiotic discs as follows: gentamicin (10 µg), erythromycin (15 µg), chloramphenicol (30 µg), kanamycin (30 µg), vancomycin (30 µg), rifampicin (30 µg) and tetracycline (30 µg).They were evaluated according to the recommendations of the suppliers (Becton and Dickinson, Cockeysville, USA or Lach-Ner, Czech republic) as sensitive or resistant reading of the zones of inhibition.As a control L. plantarum CCM 4000 was used (Dr.Nemcová, UVMP Košice, Slovakia).Briefly, the strains were incubated in MRS broth (Merck) up to the optical density-OD 600 1.0.Then aliquots (100 µl) were plated on MRS agar enriched with defibrinated sheep blood.The sterile discs were applied onto plates.The plates were cultivated at 37°C for 18 and/or 24 h in the atmosphaere with CO 2 .The sizes of inhibitory zones were recorded in millimeter (mm).
Lactic acid was analyzed by the validated spectrophotometric method and expressed in mmol/L.This method is based on the conversion of lactic acid to acetaldehyde by heat from sulfuric acid.Acetaldehyde reacts with 4-hydroxybiphenyl to form a color complex.Prior to the analysis, the strains were cultivated in MRS broth (Merck, Germany) at 37°C (under CO 2 atmosphaere) for 18 h.Aliquotes volumes of these cultures were then analyzed (Pryce, 1986).

Antimicrobial activity
Antimicrobial activity of the strains was tested by the qualitative method.Briefly, the plates with MRS agar (1.5%) were inoculated by testing strains of lactobacilli (as stripes) and cultivated at 37°C overnight.Indicator bacteria were grown in Trypticase soy broth (Difco, USA) at 37°C up to the optical density 1.00 (OD 600 ).Plates with grown lactobacilli were then overlaid with Trypticase soy agar (0.4%) supplemented with 200 µl of broth cultures of indicators; then they were incubated again under the same conditions.The average of the clear inhibion zones were evaluated in mm; it means the inhibition of indicator strain by tested lactobacilli.The size of zones from 5 to 10 mm was evaluated as low antimicrobial activity; zones measuring over 10 mm in average were evaluated as high antimicrobial activity.The target of indicator strains were: Enterococcus avium EA5 (our isolate from faeces of piglets-the principal indicator), Staphylococcus aureus SA5 (our isolate from mastitis milk), 12 strains of Listeria monocytogenes: (2116, P3300, P2024, P5258, P6301, P6501, 7223, P7395, P7401, P7562, Ve405, TS10811, isolates from different meat products, State Veterinary Institute, Olomouc, Czech republic), L. monocytogenes CCM4699 (clinical isolate, Czech Culture Collection-CCM, Brno, Czech republic), Listeria innocua LMG13568 (University of Brussel, Belgium).

Resistance to low pH and bile tolerance
To check resistance to low pH, the method according to Arboleya et al. (2011) was used.Lactobacilli were grown in 5 ml of MRS broth (Merck, Germany) at 37°C overnight.After centrifuging, cells were resuspended in Ringer solution (Oxoid).Bacterial suspension was added to gastric juice stimulated solution (1:9, pH 2.5 and 7) and incubated for 90 and 180 min.To check surviving of cells, samples of 0, 90 and 180 min were spread on MRS agar (Merck, Germany) to count cells (CFU/mL).The test was performed in duplicate.
To check bile tolerance, the tubes were inoculated with 0.1% of tested strain grown overnight in MRS (Merck, Germany) and enriched with 1% of bile (at 37°C).The control tubes contained MRS broth with strain and no bile).They were cultivated for 12 and 24 h and the surviving growth of strains was checked by samples plating on MRS agar (Merck, Germany).Plates were kept at 37°C for 48 h and the counts were expressed in CFU/mL.

RESULTS
The counts of lactic acid bacteria reached up to 2.72 ± 0.64 CFU/g.Among 50 randomly picked up colonies, 4 strains were taxonomically allotted to the genus Lactobacillus by the identification method used.The other strains were not lactobacilli.According to the Maldi-Tof analysis, the species detected in faecal samples of pheasants were L. brevis (2) and L. plantarum (2).This taxonomic identification was expressed on the basis of high score values (L.brevis LB11-2.401; L. brevis LB12-2.383; L. plantarum LP13-2.292; L. plantarum LP14-2.104).Moreover, phenotypic properties confirmed this identification; NH3 formation from arginine and fermentation of disacharides were in accordance with control strain reported by Vos et al. (2009).Identity of L. plantarum strains was also confirmed PCR; 256 bp band was visualized by electrophoresis.
Lactobacilli were sensitive to erythromycin, rifampicin, chloramphenicol and tetracycline.Resistance to vancomycin and kanamycin was found in the strains LB12 and LP13.In addition, LP14 strain was resistant to gentamicin.
The values of lactic acid reached from 2.82 to 4.86 mmol/L; average value was 3.73 mmol/L.L. plantarum strains showed higher lactic acid production (Table 2) as compared to L. brevis strains.
Selected strains LB12, LP13 and LP14 tolerate very well pH 2.5 (Table 3).As compared to physiological pH 7.0, the difference in enumerating counts at 90 min was 5.55 log cycle for LB12, 3.34 log cycle for LP13 and 4.26 log cycle for LP14.LP14 survived in the highest counts at incubation time of 180 min at pH 2.5 and when compared with pH 7.0, the lowest difference in counts was found at time 180 min (Table 3).
The strains were able to survive in the the presence of 1.0% (w/v) oxgall-bile with the different 1.0 log cycle as compared to the control tubes and experimental tubes after 24 h of growth; B12, 10.23:9.69log CFU/mL; LP14, 10.25:9.1 log CFU/mL.In LP13, even the same count was enumerated (10.38 log CFU/mL) in control as in experimental tubes (10.38 log CFU/mL).

DISCUSSION
Lactobacilli belong to Firmicutes, class Bacilli, order Lactobacillales, family Lactobacillaceae (Vos et al., 2009).Detection of the species L. plantarum and L. brevis presented here are in agreement with highly probable species identification of Maldi-Tof identification system (value score 2.300-3.000)and/or secure probable species identification-probable species identification (value score 2. 000-2.299;Bruker Daltonics, 2008).Only limited information exists in relation to the species of lactobacilli detected in pheasants and/or poultry, in general.For instance Heravi et al. (2011) detected two species of lactobacilli in chicken; Lactobacillus crispatus and Lactobacillus salivarius; moreover, they possessed probiotic properties.In our study, different species were identified.It seems, microbiota associated directly with pheasants are still open field for the study.However, up till now, reported studies are mostly connected with probiotic application of lactobacilli in chickens (Audisio et al., 2000).
Bacterial β-glucuronidase is known to be associated with initiation of some diseases, e. g. in human it is colon cancer (Lidbeck et al., 1992).Having in the mind probable probiotic potential of the isolated lactobacilli, it means to assess safety of the strains, the strains positive for enzymes associated with the intestinal diseases such as α-chymotrypsin, β-glucuronidase, β-glucosidase and N-acetyl-β-glucosaminidase should be eliminated for further probiotic studies.Lactobacilli presented here were mostly negative or with weak reaction with regards to the enzymes α-chymotrypsin, β-glucuronidase, β-glucosidase and N-acetyl-β-glucosaminidase.It is one of parameters which indicates their probable use as probiotic bacteria in the future.Positive strain L. brevis LB11 for former mentioned enzymes was excluded from further testing.
Different susceptibility or resistance of lactobacilli to vancomycin was described as one of the identification characteristic (Swenson et al., 1990;Hamilton-Miller and Shah, 1998).Likewise, Swenson et al. (1990) described susceptibility of vancomycin-resistant lactobacilli to other antimicrobials.Lactic acid values presented here are in accordance with typical representants of homofermentative lactobacilli (Gereková et al., 2011).
Lactobacilli showed antimicrobial activity in at least of 2 indicator bacteria from the 16 used.Antimicrobial activity in lactobacilli e.g.those due to bacteriocins have been reported as good in inhibiting the growth of listaerie which are common contaminants in feed/food industry.Those substances were for example, sakacins or plantaricin 423 (Drider et al., 2006).Therefore, we also plan to continue detail studies of antimicrobial activity of identified lactobacilli which seems done due to bioactive susbtances produced by L. plantarum LP13 and LP14; it is good that these strains should did not produce biogennic amines (Greif and Greifová, 2013).
Generally, LAB are commonly more resistant against acidic environment and low pH (Piard and Desmazeud, 1991).This fact was confirmed also by Strompfová and Lauková (2013) in lactobacilli from dogs and primates.Tolerance to low pH and bile are properties which lead to selection of strains for their application, although it can be strain-dependent.Although only 4 taxonomically allotted lactobacilli from fececes of pheasants were isolated, these results are important contribution to the microbiology of pheasants.In conclusion, L. plantarum LP13 and LP14 represent promising probiotic candidates; moreover, detail studies of their antimicrobial activity are in process.

Table 1 .
Enzymatic activities of lactobacilli from common pheasants assayed by the API ZYM system.

Table 3 .
Tolerance of lactobacilli to low pH (counts in CFU/mL).