Properties of potentially probiotic Lactobacillus isolates from poultry intestines

The most commonly used probiotic bacteria belong to the genus Lactobacillus, being regarded as beneficial for poultry health and production. However, commercial probiotics do not always ensure both expected effects. In order to improve the utility properties of new preparations, the selection of new probiotic candidates should be made on the basis of the performance of the species within the poultry digestive tract. The aim of this study was to isolate and identify lactobacilli from poultry intestines, and to select probiotic candidates for subsequent in vivo trials. Digesta from 18 poultry specimens were collected, serially diluted, plated onto Wilkins-Chalgren anaerobe agar supplemented with 30% of rumen fluid and onto De Man, Rogosa and Sharpe agar plates, and incubated at 37 °C for 48 h under anaerobic conditions. Isolated colonies were subjected to Gram staining and catalase reaction. They were then pre-identified using matrixassisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). Forty-four Lactobacillus strains belonging to 16 species were identified and subjected to evaluations of survival under simulated gastrointestinal conditions, autoaggregation and hydrophobicity. Most of the screened Lactobacillus reuteri strains as well as individual strains of L. acidophilus, L. amylovorus, L. gallinarum, L. ingluviei, L. johnsonii, L. oris, L. salivarius, L. saerimneri, and L. vaginalis showed high survival rates under gastrointestinal tract conditions and good surface properties. The results suggest their potential for further testing as probiotic candidates in in vivo trials. Lactobacillus, MALDI TOF MS, autoaggregation, hydrophobicity, probiotics Lactobacillus populations are autochthonous residents in the gastrointestinal tracts of humans and animals, including poultry (Stephenson et al. 2010). The presence of lactobacilli in the poultry intestinal ecosystem has previously been considered as beneficial to poultry health and production (Tannock 2004). Recent studies of Lactobacillus species ecology and their beneficial effects have brought new data (Wang et al. 2014; Adhikari and Kwon 2017; Duar et al. 2017b), and new probiotic candidates from this group of bacteria are being sought based on current knowledge. This renewed interest is stimulated by the urgent need to reduce the consumption of antimicrobials in the poultry industry while at the same time promoting health and improving performance indicators such as mean egg weight, body weight, and the feed conversion ratio (Angelakis and Rault 2010; Olnood et al. 2015; Shokryazdan et al. 2017). Commercial probiotic preparations containing lactobacilli do not always ensure these expected effects which, among other things, may be influenced by an inappropriate process of selecting Lactobacillus isolates (KizerwetterŚwida and Binek 2016). Moreover, commercial probiotic products commonly do not contain the probiotic strains stated on their labels or are even inappropriate for the stated target animal species (Lata et al. 2006; Šmídková and Čížek 2017). Selection criteria for new probiotic candidates should be based on actual knowledge as to the importance of particular Lactobacillus species in poultry intestines (Adhikari and Kwon 2017; Duar et al. 2017a,b) and their probiotic functional properties. In vitro selection procedures usually include survival of probiotic candidates under gastrointestinal ACTA VET. BRNO 2019, 88: 73–84; https://doi.org/10.2754/avb20198801073 Address for correspondence: Prof. MVDr. Alois Cizek, CSc. Department of Infectious Diseases and Microbiology Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Brno Palackeho tr. 1946/1, 61242 Brno, Czech Republic Phone: +420 607 652 460 E-mail: cizeka@vfu.cz http://actavet.vfu.cz/ conditions, their surface properties, capability for competitive exclusion and antibiotic susceptibility (Bujnakova et al. 2014; Kizerwetter-Świda and Binek 2016; Rajokaa et al. 2018). The objectives of the present study were to isolate and identify lactobacilli from poultry intestines and then to select probiotic candidates for subsequent in vivo trials. Materials and Methods Lactobacilli culture and growth conditions Whole caeca or jejuna from 18 randomly selected healthy chickens at 4 to 40 weeks of age were processed in an anaerobic chamber (10% CO2, 5% H2, and 85% N2 atmosphere; Concept 400, Baker Ruskinn, Sanford, Maine, USA) within 1 h after they were ethically sacrificed. Approximately 0.5 ml of caecal or jejunal digesta were collected and serially diluted in 4.5 ml of pre-reduced anaerobically sterilized dilution blanks and plated onto Wilkins-Chalgren anaerobe agar (Oxoid, Basingstoke, UK) supplemented with 30% of rumen fluid (Medvecky et al. 2018) and onto De Man, Rogosa and Sharpe (MRS) agar plates (Oxoid, Basingstoke, UK). All inoculated media were incubated at 37 °C for 48 h in an anaerobic chamber. Isolated colonies on the plates with the highest dilution were subjected to Gram staining and catalase reaction. They were then pre-identified using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS). Colonies pre-identified as lactobacilli were subcultured on Wilkins-Chalgren anaerobe agar or MRS agar plates and the pure cultures thus obtained were used for further studies. Six strains (L. amylovorus CCM4380, L. fermentum CCM7192, L. parabuchneri DSM5987, L. pentosus CCM4619, L. plantarum CCM7039, and L. salivarius subsp. salivarius CCM7274) were included as control strains in all procedures. All cultures of lactobacilli were stored in MRS broth (Oxoid, Basingstoke, UK) with 20% glycerol at −80 °C. Species identification using MALDI-TOF MS Pure cultures of Lactobacillus spp. isolates and control strains on MRS agar plates were subjected to MALDITOF MS on a Microflex LT instrument (Bruker Daltonik, Germany) as described by several authors (Duskova et al. 2012; Bujnakova et al. 2014; Dec et al. 2016). Briefly, the bacterial culture on MALDI plates was overlaid with 1 μl of matrix solution containing 10 mg/ml HCCA (a-cyano-4-hydroxycinnamic acid, SigmaAldrich, Prague, Czech Republic) dissolved in 50% acetonitrile (Sigma-Aldrich, Prague, Czech Republic) and 2.5% trifluoroacetic acid, and then air-dried. The mass spectra were processed using the MALDI Biotyper 3.0 software package (Bruker, Leipzig, Germany) containing 6903 reference spectra, including 243 for lactobacilli. Identification was performed according to the criteria recommended by the manufacturer (ID score: 1.700–1.999 probable genus identification; 2.000–2.299 secure genus identification, probable species identification; 2.300– 3.000 highly probable species identification). Tolerance to gastrointestinal conditions Tolerance of isolates to simulated gastric juice and bile salts was assayed as described by Jena et al. (2013). Briefly, for the bile tolerance test, MRS broth (Oxoid, Basingstoke, UK) was supplemented with 0.3% (w/v) bile salts (Sigma-Aldrich, Prague, Czech Republic). Simulated gastric juice contained 6.4 g NaHCO3, 0.239 g KCl, 1.28 g NaCl, and 0.1% (w/v) pepsin (Sigma-Aldrich, Prague, Czech Republic) per litre of MRS broth (pH 2.5). For both tests, 5 ml of lactobacilli culture grown overnight in MRS broth at 37 °C were pelleted and washed twice with 4 ml of phosphate buffered saline (PBS; pH 7.2). Subsequently, culture density was adjusted to McFarland turbidity standard 1.0 (Densi-La-Meter®, Erba Lachema, Brno, Czech Republic) (ca 3 × 108 cfu/ml) and inoculated into 10 ml of modified MRS broth containing bile salts or simulated gastric juice. Samples were then incubated at 37 °C for 30, 60, 90, 120, or 180 min. The number of viable cells was assessed by a serial dilution and plate count method. The results were expressed as growth rate (%) of Lactobacillus strains. Autoaggregation assay Autoaggregation capabilities were assessed according to the procedure described by Collado et al. (2007) with minor modifications. The lactobacilli culture grown overnight in MRS broth at 37 °C was pelleted, washed twice with PBS, and then resuspended in PBS. The optical density (OD) of the bacterial suspension was adjusted to McFarland turbidity standard 2.0 (Densi-La-Meter®, Erba Lachema, Brno, Czech Republic). Bacterial cell suspensions were incubated without agitation in closed measuring cuvettes (2 ml) at room temperature (22 ± 2 °C) for different time periods (0, 2, 4, 6, 24, and 48 h). At the determined time the absorbance was measured at 600 nm using a Biowave Cell Density Meter (WPA, Cambridge, UK). Autoaggregation assay was accomplished in one experiment with each isolate and was expressed as autoaggregation percentage (A% = 1 − (At/A0)/100), where At represents absorbance at the determined time and A0 the absorbance at t0. Cell surface hydrophobicity assay The degree of hydrophobicity of the cultures was specified according to procedures described previously (Rosenberg 1984; Jena et al. 2013) and which are based on affinity of bacterial cells for toluene 74


Lactobacillus, MALDI TOF MS, autoaggregation, hydrophobicity, probiotics
Lactobacillus populations are autochthonous residents in the gastrointestinal tracts of humans and animals, including poultry (Stephenson et al. 2010).The presence of lactobacilli in the poultry intestinal ecosystem has previously been considered as beneficial to poultry health and production (Tannock 2004).Recent studies of Lactobacillus species ecology and their beneficial effects have brought new data (Wang et al. 2014;Adhikari and Kwon 2017;Duar et al. 2017b), and new probiotic candidates from this group of bacteria are being sought based on current knowledge.This renewed interest is stimulated by the urgent need to reduce the consumption of antimicrobials in the poultry industry while at the same time promoting health and improving performance indicators such as mean egg weight, body weight, and the feed conversion ratio (Angelakis and Rault 2010;Olnood et al. 2015;Shokryazdan et al. 2017).Commercial probiotic preparations containing lactobacilli do not always ensure these expected effects which, among other things, may be influenced by an inappropriate process of selecting Lactobacillus isolates (Kizerwetter-Świda and Binek 2016).Moreover, commercial probiotic products commonly do not contain the probiotic strains stated on their labels or are even inappropriate for the stated target animal species (Lata et al. 2006;Šmídková and Čížek 2017).
Selection criteria for new probiotic candidates should be based on actual knowledge as to the importance of particular Lactobacillus species in poultry intestines (Adhikari and Kwon 2017;Duar et al. 2017a,b) and their probiotic functional properties.In vitro selection procedures usually include survival of probiotic candidates under gastrointestinal conditions, their surface properties, capability for competitive exclusion and antibiotic susceptibility (Bujnakova et al. 2014;Kizerwetter-Świda and Binek 2016;Rajokaa et al. 2018).
The objectives of the present study were to isolate and identify lactobacilli from poultry intestines and then to select probiotic candidates for subsequent in vivo trials.

Lactobacilli culture and growth conditions
Whole caeca or jejuna from 18 randomly selected healthy chickens at 4 to 40 weeks of age were processed in an anaerobic chamber (10% CO 2 , 5% H 2 , and 85% N 2 atmosphere; Concept 400, Baker Ruskinn, Sanford, Maine, USA) within 1 h after they were ethically sacrificed.Approximately 0.5 ml of caecal or jejunal digesta were collected and serially diluted in 4.5 ml of pre-reduced anaerobically sterilized dilution blanks and plated onto Wilkins-Chalgren anaerobe agar (Oxoid, Basingstoke, UK) supplemented with 30% of rumen fluid (Medvecky et al. 2018) and onto De Man, Rogosa and Sharpe (MRS) agar plates (Oxoid, Basingstoke, UK).All inoculated media were incubated at 37 °C for 48 h in an anaerobic chamber.Isolated colonies on the plates with the highest dilution were subjected to Gram staining and catalase reaction.They were then pre-identified using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS).Colonies pre-identified as lactobacilli were subcultured on Wilkins-Chalgren anaerobe agar or MRS agar plates and the pure cultures thus obtained were used for further studies.Six strains (L.amylovorus CCM4380, L. fermentum CCM7192, L. parabuchneri DSM5987, L. pentosus CCM4619, L. plantarum CCM7039, and L. salivarius subsp.salivarius CCM7274) were included as control strains in all procedures.All cultures of lactobacilli were stored in MRS broth (Oxoid, Basingstoke, UK) with 20% glycerol at −80 °C.

Species identification using MALDI-TOF MS
Pure cultures of Lactobacillus spp.isolates and control strains on MRS agar plates were subjected to MALDI-TOF MS on a Microflex LT instrument (Bruker Daltonik, Germany) as described by several authors (Duskova et al. 2012;Bujnakova et al. 2014;D e c et al. 2016).Briefly, the bacterial culture on MALDI plates was overlaid with 1 μl of matrix solution containing 10 mg/ml HCCA (a-cyano-4-hydroxycinnamic acid, Sigma-Aldrich, Prague, Czech Republic) dissolved in 50% acetonitrile (Sigma-Aldrich, Prague, Czech Republic) and 2.5% trifluoroacetic acid, and then air-dried.The mass spectra were processed using the MALDI Biotyper 3.0 software package (Bruker, Leipzig, Germany) containing 6903 reference spectra, including 243 for lactobacilli.Identification was performed according to the criteria recommended by the manufacturer (ID score: 1.700-1.999probable genus identification; 2.000-2.299secure genus identification, probable species identification; 2.300-3.000highly probable species identification).

Tolerance to gastrointestinal conditions
Tolerance of isolates to simulated gastric juice and bile salts was assayed as described by J e n a et al. ( 2013).Briefly, for the bile tolerance test, MRS broth (Oxoid, Basingstoke, UK) was supplemented with 0.3% (w/v) bile salts (Sigma-Aldrich, Prague, Czech Republic).Simulated gastric juice contained 6.4 g NaHCO 3 , 0.239 g KCl, 1.28 g NaCl, and 0.1% (w/v) pepsin (Sigma-Aldrich, Prague, Czech Republic) per litre of MRS broth (pH 2.5).For both tests, 5 ml of lactobacilli culture grown overnight in MRS broth at 37 °C were pelleted and washed twice with 4 ml of phosphate buffered saline (PBS; pH 7.2).Subsequently, culture density was adjusted to McFarland turbidity standard 1.0 (Densi-La-Meter ® , Erba Lachema, Brno, Czech Republic) (ca 3 × 10 8 cfu/ml) and inoculated into 10 ml of modified MRS broth containing bile salts or simulated gastric juice.Samples were then incubated at 37 °C for 30, 60, 90, 120, or 180 min.The number of viable cells was assessed by a serial dilution and plate count method.The results were expressed as growth rate (%) of Lactobacillus strains.

Autoaggregation assay
Autoaggregation capabilities were assessed according to the procedure described by Collado et al. ( 2007) with minor modifications.The lactobacilli culture grown overnight in MRS broth at 37 °C was pelleted, washed twice with PBS, and then resuspended in PBS.The optical density (OD) of the bacterial suspension was adjusted to McFarland turbidity standard 2.0 (Densi-La-Meter ® , Erba Lachema, Brno, Czech Republic).Bacterial cell suspensions were incubated without agitation in closed measuring cuvettes (2 ml) at room temperature (22 ± 2 °C) for different time periods (0, 2, 4, 6, 24, and 48 h).At the determined time the absorbance was measured at 600 nm using a Biowave Cell Density Meter (WPA, Cambridge, UK).Autoaggregation assay was accomplished in one experiment with each isolate and was expressed as autoaggregation percentage (A% = 1 − (A t /A 0 )/100), where A t represents absorbance at the determined time and A 0 the absorbance at t 0 .

Cell surface hydrophobicity assay
The degree of hydrophobicity of the cultures was specified according to procedures described previously (Rosenberg 1984  in a two-phase system.Hydrophobicity was calculated from three replicates as the percent decrease in OD of the original bacterial suspension due to cells partitioning into the hydrocarbon layer.The cell surface hydrophobicity (%) of isolate adhering to hydrocarbon solvent was calculated according to the following equation: Hydrophobicity % = OD660 before mixing − OD660 after mixing/OD660 before mixing × 100.Data processing Bacterial counts were represented as the average logarithm of colony forming units (CFU) or the average values of absorbance (OD).All data were entered into spreadsheets (Excel, Microsoft) that were used to calculate the percentage of autoaggregation, hydrophobicity, and viability of Lactobacillus strains in simulated gastrointestinal conditions.

Identification of lactobacilli
The isolates were pre-identified by Gram staining, catalase reaction, and application of MALDI TOF MS.Forty-four Lactobacillus isolates belonging to 16 species were obtained from intestinal contents of 18 poultry intestinal specimens.Table 1 summarizes the Lactobacillus species pre-identified by MALDI TOF MS and their abundance in different intestinal compartments.A dendrogram designed on cluster analysis of MALDI-Biotyper protein mass spectra of these isolates showing distinctive clusters consisting of the same Lactobacillus species along with control strains is presented as Fig. 1.Corresponding ID score values for the same set of lactobacilli are included in Table 1.The ID scores for 8 strains were between 1.589 and 1.999, and for 36 strains they ranged from 2.000 to 2.489.Despite low ID values for 2 isolates of L. oris (1.589 and 1.682), the application of MALDI TOF MS allowed identification at species level for 82% of the isolates and for 14% of the isolates provided ID values at genus level.

Tolerance to simulated gastric juice
The effect of simulated gastric juice on the growth rate of lactobacilli was evaluated for 44 isolates of poultry origin and 6 control strains.Differences were found among Lactobacillus strains in their tolerance to simulated gastric conditions (Fig. 2).After 30 min of the treatment, the following isolates lost their viability: L. agilis 358, 650, L. kitasatonis 458, L. parabuchneri DSM5987, L. pentosus CCM4619, and L. plantarum CCM7039.Generally, a low level of viability was recorded for control strains.By contrast, all strains of L. reuteri and one strain of each L. acidophilus (275), L. gasseri (197), L. oris (316), and L. vaginalis (683) showed high levels of viability through time.

Autoaggregation and hydrophobicity assay
The percentage of autoaggregation increased with time for each Lactobacillus strain.With the exceptions of strains L. gasseri 197, L. johnsonii 340, L. coleohominis 574, L. vaginalis 337, and L. fermentum CCM 7192, most of the strains (61%) were highly positive in both tests.Positive relationship between autoaggregation percentage and hydrophobicity percentage was evident in most Lactobacillus strains (91%, n = 44).Conversely, negative link was proven in the strains L. reuteri 21, L. gasseri 197, L. gallinarum 117, and L. fermentum CCM 7192.The results of autoaggregation and hydrophobicity assays are summarized in Fig. 4.    criteria, thereby confirming that these properties are strain-specific and not shared by all strains of the same species.
In conclusion, our results indicate that some Lactobacillus strains can be classified as probiotic candidates due to their tolerance for simulated gastrointestinal conditions and surface properties associated with intestinal colonization.They can therefore be included into in vivo trials for testing health-and performance-related functional properties on a poultry model.Additional in vitro studies would be then required to confirm the strains' capability for competitive exclusion and antibiotic susceptibility, as well as to assess their stability within manufacturing processes and therapeutic application forms.

Fig. 1 .
Fig. 1.Main-spectra dendrogram of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) profiles generated by the MALDI Biotyper.

Table 1 .
; J e n a et al. 2013) and which are based on affinity of bacterial cells for toluene Lactobacillus control strains and isolates of poultry origin, abundance in section of the gut (J -jejunum, C -cecum) in log