In vitro evaluation of the safety and probiotic properties of Lactobacilli isolated from chicken and calves

Anaerobe xxx (2013) 1e10

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Clinical microbiology

In vitro evaluation of the safety and probiotic properties of Lactobacilli isolated from chicken and calves Dobroslava Bujnakova, Eva Strakova*, Vladimir Kmet Institute of Animal Physiology, Slovak Academy of Sciences, Soltesovej 4/6, 040 01 Kosice, Slovak Republic

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 May 2013 Received in revised form 16 September 2013 Accepted 25 October 2013 Available online xxx

A total of 73 chicken and calves isolates were diagnosed using matrix-assisted laser desorption ionization-time-of flight mass spectrometry (Maldi-Tof MS). After a preliminary subtractive screening based on the high acid tolerance at pH 2.5 and bile resistance at 0.3% oxgall, twenty isolates belonging to the species Lactobacillus salivarius, Lactobacillus agilis, Lactobacillus reuteri, Lactobacillus murinus and Lactobacillus amylovorus were in vitro screened for the safety assessment and probiotic properties, including antibiotics susceptibility patterns, biochemical activity and potential for competitive exclusion of biofilm producing pathogens determined by crystal violet and/or quantitative Fluorescent in situ Hybridisation (FISH) assays utilizing 50 Cy 3 labelled probe Enter1432 for enteric group. Antibiotic susceptibility testing was performed according to the ISO norm 10932. The sixteen strains were susceptible to certain antimicrobial agents, except for two chicken (L. salivarius 12K, L. agilis 13K) and two calves (L. reuteri L10/1, L. murinus L9) isolates with the presence non wild-type ECOFFs (epidemiological cut-off) for gentamicin (256 mg ml1), tetracycline (128 mg ml1), kanamycin (256 mg ml1) and streptomycin (96 mg ml1). The two referenced chicken isolates gave positive aac(60 )Ie-aph(200 )Ia and tet(L) PCR results. The wild-type ECOFFs isolates were subjected to the apiZYM analysis for enzyme profile evaluation and amino acid decarboxylase activities determined by qualitative plate method and multiplex PCR for the detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via eithers ornithine decarboxylase, odc, or agmatine deiminase, agdi). From examined strains only two chicken isolates (L. reuteri 14K; L. salivarius 15K) had no harmful b-glucuronidase, b-glucosidase activities connected with detrimental effects in the gastrointestinal tract and together no amino acid decarboxylase activities and no genes associated with biogenic amines production though only chicken L. salivarius 15K whole cells and acid supernatants shown strong suppressive potential against biofilm-forming Klebsiella and Escherichia coli. Our results highlight that above-mentioned isolate L. salivarius 15K fulfils the principle requirements of a qualified probiotic and may be seen as a reliable candidate for further validation studies in chicken. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Lactobacilli Escherichia coli Klebsiella Maldi-Tof MS b-Glucuronidase Antibiotic resistance

1. Introduction Antibiotics used as growth promoters in animal feeds have been banned and nowadays, the possibility of using alternative additives instead of antibiotics is being researched. One such opportunity is probiotics. In recent years, worldwide interest in the use of probiotic bacteria for health promotion and disease prevention has increased significantly in scientific community, consumers and food producers. This interest is based on the knowledge that the targeted use of microorganisms with suitable properties may have beneficial effect on

* Corresponding author. Tel.: þ421 915 865 646. E-mail address: [email protected] (E. Strakova).

animal and human health. Bacterial strains most commonly used as probiotics are Lactobacillus, Lactococcus, Enterococcus and Bifidobacterium [1] which belong to the lactic acid bacteria. Before a screening of promising probiotic functional efficacy it is necessary to provide of preliminary in vitro screening to ensure the safety aspects of tested strains. Probiotic microorganisms should be free of undesirable traits, such as transmissible antibiotic resistance [2,3] (to avoid spreading resistance determinants in intestinal pathogenic or opportunistic bacteria), specific amino acid decarboxylase activities and thus, the possibility to synthesize biogenic amines with potential health risks to consumers [4] and harmful biochemical activities such as a-chymotrypsin, b-glucosidase, bglucuronidase, and N-acetyl-ß-glucosaminidase activities, which are often associated with intestinal diseases and involved in

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Please cite this article in press as: Bujnakova D, et al., In vitro evaluation of the safety and probiotic properties of Lactobacilli isolated from chicken and calves, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.10.009

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D. Bujnakova et al. / Anaerobe xxx (2013) 1e10

generating carcinogens and tumour promoters [5]. The next important criterion is survival under gastrointestinal condition (acid pH and bile salt). Though the safety and efficacy of probiotics needs to be demonstrated in controlled clinical trials [6], their primary selection is based on a series of well-defined in vitro tests [7,8]. With the aim of selecting new promising probiotic candidates with minimized undesirable properties, twenty chicken and calves lactobacilli isolates were subjected to comprehensive in vitro analyses to assess their key safety and functionality. 2. Materials and methods 2.1. Bacterial isolates and growth condition From 73 chicken and calves isolates identified using Maldi-Tof MS, twenty selected lactobacilli belonged to the five following species: Lactobacillus salivarius (n ¼ 7), Lactobacillus agilis (n ¼ 2), Lactobacillus reuteri (n ¼ 9) and Lactobacillus amylovorus (n ¼ 1), Lactobacillus murinus (n ¼ 1). All lactobacilli isolates were routinely grown in MRS medium (Oxoid, England) by anaerobic incubation at 37  C for 48 h. The pathogens used in the experiments were Klebsiella pneumonii KPC (carbapenemase positive strain); uropathogenic Escherichia coli K188 and E. coli DH5a/pCIB10B (ibeA positive strain), which was obtained from VA Medical Center, Minneapolis, United States. The strains of E. coli and K. pneumonii were grown in MacConkey agar (Oxoid, England) overnight at 37  C. 2.2. Bacterial identification 2.2.1. Maldi-Tof MS bacterial identification Maldi-Tof MS was performed on a Microflex LT instrument (Bruker Daltonik GmbH, Leipzig, Germany) as described Bessede et al. [9]. To identify microorganisms, the raw spectra obtained for each isolate were imported into BioTyper software, version 2.0 (Bruker Daltonik). When the obtained score was >2.00, identification was considered correct at the species level; in the range 1.7e 1.999, the identification was considered correct at the genus level; and <1.7, the identification was not sufficient. Merely lactobacilli strains with obtained scores higher than 2.3 were selected for further experiments. 2.2.2. Genotypic bacterial identification Amplification of DNA was carried out using genus-specific primers LBLMA 1-rev and R16-1. The PCR mixture and cycle parameters were set according to Dubernet et al. [10] and PCR amplification was carried out in a C-1000 Thermal Cycler (Biorad). For the species identification, lactobacilli were firstly separated by a multiplex PCR (with primers Ldel-7; LU-1; LU-3; LU-5; Lac-2) into four groups based on the nucleotide sequences of the 16Se23S rRNA intergenic spacer region and adjacent 23S rRNA gene [11] and then merely L. salivarius strains together with finally selected L. salivarius 15K and L. salivarius 12K (aac(60 )-aph(20 )-Ia positive strain) were identified with species-specific primers (For-Sal-3; Rev-Sal-1) for confirmation of our Maldi-Tof results as described Harrow et al. [12]. 2.3. Tolerance to acid and bile Tolerance to low pH and bile content was assessed as described by Anderson et al. [13], with minor modifications. Briefly, lactobacilli were grown overnight at 37  C in MRS broth in 5% CO2. Each culture was vortexed and separated 10-ml aliquots were collected by centrifugation (3800 g for 20 min). Cell pellets were suspended to an approximate cell concentration of 106 CFU ml1 in the following test solutions: MRS broth control, MRS broth adjusted to

pH 2.5, MRS broth containing 0.3% oxgall (Difco). As a control, broth without inoculation was used. For survival under the different conditions, the samples were taken at 0 h and 4 h and then 100 ml cultures were plated after appropriate dilution on MRS agar plates. Enumeration was done following 48 h incubation at 37  C. 2.4. Antibiotic susceptibility testing and MIC determinations Antibiotic susceptibility testing was performed according to the ISO 10932/IDF 223 standard [14]. The minimal inhibitory concentrations (MICs) of the lactobacilli strains towards gentamicin, kanamycin, streptomycin, neomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, penicillin, vancomycin, virginiamycin, linezolid, trimethoprim, ciprofloxacin and rifampicin was determined by the microdilution method using the microtiter VetMIC Lact-1 and Lact-2 panels for susceptibility testing of bacteria (Statens Veterinarmedicinska Anstalt, Uppsala, Sweden). In brief, strains were grown in MRS agar, colonies were resuspended in LSM medium consisted from 90% Isosensitest medium (Oxoid, England) and 10% MRS medium (Oxoid, England). The suspension was diluted 1000 times in the same medium, 100 ml (approximately 3  104 CFU/well) were then added to each well of the microdilution plate and incubated at 28  C under anaerobic conditions for 48 h. Growth within each well was determined visually after incubation for each antibiotics by comparing with the positive control as recommended ISO 10932/IDF 223 standard [14]. MIC values surpassing the microbiological breakpoints were filled and confirmed moreover by using MIC strip tests (Liofilchem, Italy). The MICs (mg ml1) were interpreted according to the recent FEEDAP document of the European Food Safety Authority (EFSA) on the update of the criteria used in the assessment of antibiotics bacterial resistance of human or veterinary importance [15] as well as epidemiological cut-off values defined by the ACE e ART Project results, ISO 10932 (2010) [14]. 2.4.1. PCR Detection of resistance genes Total DNA was extracted from bacterial cultures with the Dneasy Tissue Kit (Qiagen, France) according to manufacturer’s instructions. Purified DNA samples were then stored at 20  C. PCR-based detection of genes responsible for resistance to gentamicin and other aminoglycosides, but not streptomycin [aac(60 )-aph(20 )-Ia], tetracycline [tet(L), tet(M), tet(S)], using primers and protocols described by Refs. [16,17], kanamycin [aph(30 )-IIIa] according Vakulenko et al. [18] and streptomycin/ spectinomycin [aadA] according Clark et al. [19], was applied to strains suspected to carry acquired resistance. 2.5. Determination of ability to produce biogenic amines The biogenic amines production (tyramine, histamine and putrescine) was assessed using the decarboxylase agar previously described by Bover-Cid and Holzapfel [20]. The precursor amino acids (tyrosine, histidine and ornithine, respectively) were purchased from Sigma. Lactobacilli strains were inoculated onto the decarboxylase plates and incubated for 4 days at 37  C under aerobic and anaerobic conditions. The positive results were indicated by a change of the colour from yellow to purple in response to the pH shift caused by the production of the alkaline biogenic amines from the amino acids included in the medium. 2.5.1. The multiplex PCR for detection of genes responsible for biogenic amines production DNA was extracted from bacterial cultures as described previously. For simultaneous detection of four biogenic amines genes: histamine (histidine decarboxylase, hdc), tyramine (tyrosine

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decarboxylase, tyrdc) and putrescine (via eithers ornithine decarboxylase, odc, or agmatine deiminase, agdi) as well as a PCR internal control corresponding to the 16S rRNA coding gene was used the method described by Coton et al. [21]. 2.6. Enzyme activities The enzyme activities of the lactobacilli strains were assayed following the method of Arora et al. [22] using the apiZYM system (BioMérieux, France), which detects the following 19 enzymes: alkaline and acid phosphatases, butyrate esterase, caprylate esterase lipase, myristate lipase, leucine, valine and cystine aminopeptidases, trypsin, a-chymotrypsin, phosphoamidase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, bglucosidase, b-glucosaminidase, a-mannosidase, and a-fucosidase. Enzyme activity readings were taken after 4 h of anaerobic incubation at 37  C and after addition of Zym A and Zym B reagents. Colour intensity values from 0 to 5 and their relevant value in nanomoles (expressed as approximate nanomoles of substrate hydrolysed during 4 h of incubation from 0 to 40 nanomol) were assigned for each reaction according to a the manufacturer’s directions.

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(Fluka, Switzerland) overnight at 4  C [25] and then stored in equal volumes of PBS and 96% ethanol at 20  C. Bacterial counts were assessed using the FISH method with probe (VBC-Genomics, Austria) Enter1432 for enteric group according to Sghir et al. [26]. The hybridized samples were vacuum filtered onto 0.2-mm polycarbonate membrane filters. A microscope (BX 51; Olympus, Japan) fitted with appropriate filters for Cy3 dye was used for bacterial quantification. 2.8. Statistical analysis Experiments were performed in triplicate. Bacterial numbers were represented as the average logarithm of colony forming unit (CFU)  SEM, or average values of absorbance  SEM per well of 96well Nunc polystyrene microtitre plates. The t-test was used to compare the rate of bacterial biofilm between E. coli and Klebsiella strains incubated with or without Lactobacillus strains. Values of p < 0.05 were considered as statistically significant. 3. Results and discussion 3.1. Lactobacilli

2.7. Screening of biofilm production in Lactobacillus sp.; E. coli; K. pneumonii The strains were inoculated to the appropriate agar and incubated for 24 h at 37  C. One colony was transferred to 5 ml of broth and incubated for 24 h at 37  C. Then the culture was centrifuged (10,000  g, 10 min), and the sediment was resuspended in phosphate-buffered saline (PBS) to reach the McFarland standard 1 suspension that corresponded to 1.5e3  108 CFU ml1. A volume of 100 ml of the culture was inoculated in a well of a polystyrene microtitre plate (Nunc, Denmark) and incubated for 24 h at 37  C. A modified version of a previously described method [23] was used for crystal violet assay. The biofilm formed in the well of the microtitre plate was washed five to six times with 200 ml of PBS and dried for 30 min at 37  C in an inverted position. A volume of 50 ml of a 1% (W/V) solution of crystal violet (Merck) in ethanol was added and incubated for 15 min at 25  C. The dye solution was aspirated away, and the well was washed with 5  400 ml of distilled water. After removing water and drying for 10 min at 25  C, 200 ml of the ethanoleacetone (80:20, V/V) mixture was added. The absorbance at 570 nm of the dye solutions was measured in Synergy HT Multi-Mode Microplate Reader (BioTek, USA). When the A570 < 0.1, the biofilm production was considered as low; in the range of A570 between 0.1 and 0.2; the biofilm production was considered as moderate and if A570 > 0.2, the biofilm production was considered as strong. 2.7.1. Inhibition of biofilm formation by different lactobacilli fractions Different fractions (acid, neutralized supernatant and whole cells) from lactobacilli were prepared essentially as described previously [24] and were used to evaluate the inhibition of biofilm formation. The selected pathogens were transferred to Nunc microplates as described before, and the different Lactobacillus sp. fractions were added to each well to complete 200 ml volume. The plates were incubated for 24 h at 37  C and biofilm was evaluated by using crystal violet assay as described earlier and/or quantitative FISH methods. Pathogens or Lactobacillus sp. were included as control. All the assays were performed by triplicate. 2.7.2. The quantitative FISH assay for biofilm-forming bacteria enumeration Before enumeration, bacterial biofilms were harvested by addition of 100 ml of 0.5% Triton X-100, fixed in 4% paraformaldehyde

Lactobacilli were subjected to a subtractive system of reliable in vitro screening methods recommended by FAO/WHO committee [7]. 3.2. Bacterial identification 3.2.1. Maldi-Tof MS bacterial identification Quality assurance programmes associated with research, development, production and validation of the health benefits of probiotic Lactobacillus require their relevant identification using modern techniques. Biochemical methods, such as API 50 CH enzymatic tests may be useful to obtain a first tentative classification at the genus level, but the identification result should in any case be confirmed by molecular methods [27]. The one suitable method is considered MALDI-Tof mass spectrometry protein fingerprinting analysis. Nevertheless, DNA based technique is considered as more accurate for bacterial identification than protein fingerprinting, the available literature indicates that misidentification by Maldi-Tof MS is most probably associated with an insufficient number of reference strains available in the Maldi-Tof MS spectral database and optimization of extraction protocols for difficult-to-treat samples is undoubtedly important for increase the accuracy of identification by the Maldi-Tof MS [28]. The Maldi Tof identification has been routinely used in our lab from 2010. The methodology of samples preparation especially for lactobacilli is empirically well designed. Moreover, our database is continually actualized and currently contains 225 lactobacilli belonging to 14 species. From total of 73 animal isolates, twenty lactobacilli with obtained scores higher than 2.3 were selected for further experiments. The dendrogram generated from cluster analysis of MaldiTof mass spectra of these strains pointed out distinctive clusters consisting of the same species lactobacilli (Fig. 1). The nine L. reuteri spectra constituted the first cluster that could potentially be subdivided into four smaller groups. A distance level of 400 separated the most extreme L. reuteri spectra, whereas the sub-clustering reduced the maximal distance level in all groups to approximately 200. A second cluster regrouped L. murinus, L. amylovorus and two L. agilis spectra despite a high distance level of 500 from among the L. murinus, L. amylovorus and L. agilis. Distance level of 400 is between L. murinus and L. amylovorus, e contra distance level lesser than 100 among L. agilis. Etceteras seven strains belonging to

Please cite this article in press as: Bujnakova D, et al., In vitro evaluation of the safety and probiotic properties of Lactobacilli isolated from chicken and calves, Anaerobe (2013), http://dx.doi.org/10.1016/j.anaerobe.2013.10.009

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Fig. 1. MSP dendrogram of MALDI-TOF mass spectral profiles generated by the MALDI Biotyper; Strains clustering with distance level lower than 500 could be classified up to species level.

L. salivarius were separated to three sub-clusters and with an additional distinct spectrum of one single strain L. salivarius 15K with a maximum distance level of 300. Distance levels on horizontal axis of MSP dendrogram indicating similarity were under 500 which have been described as reliably classified bacteria [29]. Clusters under a distance level of 100 indicating a high similarity between the isolates. 3.2.2. Genotypic bacterial identification The all of tested strains gave positive results with genus-specific primers (LBLMA 1-rev and R16-1). The separation by a multiplex PCR into four groups based on the nucleotide sequences of the 16Se 23S rRNA intergenic spacer region and adjacent 23S rRNA gene showed that sixteen strains identified by Maldi-Tof MS as L. salivarius or L. reuteri belonged to group IV, such as is described in research article written by Song et al. [11]. The seven L. salivarius strains together with finally selected L. salivarius 15K and L. salivarius 12K (aac(60 )-aph(20 )-Ia positive strain) subjected to PCR with species-specific primers for L. salivarius (For-Sal-3; Rev-Sal-1) gave positive 97 bp amplicons, which confirmed results obtaining by Maldi-Tof MS. 3.3. Tolerance to acid and bile Tolerance to gastric conditions is one of the in vitro tests frequently recommended for the evaluation of the probiotic attributes of an individual strain [30]. The twenty chosen isolates expressed high acid and bile tolerance after 4-h exposure, in doing so bacterial viability varied between 48 and 86%, respectively 52%e 91%. The results are presented in Table 1. Since viability and survival of probiotic bacteria during passage through the stomach is a considerable parameter to reach the intestine and provide potentially beneficial effect, the very important step of the probiotic potential study is focused on the selection of acid and bile tolerant isolates. The pH in stomach ranges from 1, during fasting, to 4.5, after a meal, and food ingestion can take up to 3 h. Since Lactobacillus strains are known to survive at pH 4.6, lower pH values are usually examined. The studies done so far indicate that majority of examined strains were completely resistant to pH 3 after 3 h of exposure,

but most of the strains displayed loss of viability when exposed to pH 1 or 2 for 1 h. These results are in agreement with those obtained from previous similar studies, where Lactobacillus strains were able to retain their viability when exposed to pH values of 2.5e4.0, but displayed loss of viability at lower pH values [31,32]. On the other hand, it was, however, observed before that although some strains did not survive in vitro in gastric juice with pH 2 for more than 15 min, they reached the colon in viable state and exerted a beneficial effect in vivo. It is important to consider also the buffering capacity of the ingested food or using of encapsulated delivery systems which can improve acid-sensitive strain viabilities during gastric transit [33]. Actually, ingested bacteria are rarely

Table 1 Effect of acid and bile on Lactobacillus sp. viability. Strains

Viable counts of bacteria (log CFU ml1) pH 2.5

0.3% oxgall t4

t0 L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

reuteri L4 reuteri L4/1 reuteri L5 reuteri L5/1 amylovorus L6 reuteri L6/1 reuteri L7 reuteri L8 murinus L9 reuteri L10/1 agilis 4K salivarius 5K salivarius 6K salivarius 8K salivarius 9K salivarius 10K salivarius 12K agilis 13K reuteri 14K salivarius 15K

6.37 6.40 6.78 5.78 5.48 7.58 7.47 6.14 5.14 6.77 5.77 6.55 6.48 5.99 6.18 7.58 6.77 6.20 7.12 6.99

                   

0.14 0.32 0.38 0.19 0.22 0.47 0.28 0.25 0.25 0.14 0.38 0.22 0.65 0.14 0.22 0.54 0.30 0.14 0.37 0.51

5.04 5.37 5.15 4.17 3.97 5.98 5.00 4.52 4.12 3.24 3.74 4.87 4.75 4.47 4.17 5.98 4.98 5.34 5.68 5.97

t0                    

0.10 0.32 0.27 0.11 0.40 0.12 0.12 0.04 0.04 0.16 0.18 0.12 0.37 0.11 0.20 0.12 0.39 0.10 0.45 0.12

(79%) (84%) (76%) (72%) (72%) (79%) (67%) (74%) (80%) (48%) (65%) (74%) (73%) (74%) (67%) (79%) (74%) (86%) (80%) (85%)

6.33 6.35 6.21 5.55 6.54 6.48 6.21 6.54 6.00 6.33 5.33 6.05 6.56 5.75 6.52 6.33 6.48 6.93 6.98 6.85

t4                    

0.21 0.12 0.25 0.38 0.02 0.09 0.33 0.15 0.55 0.21 0.41 0.12 0.07 0.18 0.62 0.09 0.50 0.29 0.69 0.40

5.14 5.73 5.05 4.07 4.97 4.78 4.99 5.32 5.02 4.14 3.14 3.37 4.13 5.17 4.97 5.78 4.64 6.14 5.68 5.47

                   

0.17 0.22 0.25 0.10 0.39 0.12 0.48 0.24 0.44 0.47 0.17 0.22 0.25 0.10 0.20 0.12 0.12 0.17 0.42 0.22

(81%) (90%) (81%) (73%) (76%) (74%) (80%) (81%) (84%) (65%) (59%) (52%) (78%) (89%) (76%) (91%) (72%) (89%) (81%) (80%)

t0eViable counts (log CFU ml1  SEM) of each strain at 0 h. t4eViable counts (log CFU ml1  SEM) of each strain at 4 h (% viability of selected lactobacilli). in grey selected L. salivarius 15K.

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Table 2 Minimal inhibitory concentration (MIC) (in mg ml1) of selected intestinal lactobacilli strains to 16 antibiotics determined by the VETMIC Lact 1 and 2 system. Strain

L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

reuteri L4 reuteri L4/1 reuteri L5 reuteri L5/1 amylovorus L6 reuteri L6/1 reuteri L7 reuteri L8 murinus L9 reuteri L10/1 agilis 4K salivarius 5K salivarius 6K salivarius 8K salivarius 9K salivarius 10K salivarius 12K agilis 13K reuteri 14K salivarius 15K

Antibiotics Gm

Km

Sm

Nm

Tc

Em

Cl

Cm

Am

Pc

Va

Vi

Lz

Tm

Ci

Ri

2 4 2 2 2 4 2 2 256 256 2 2 2 2 2 2 256 256 4 4

8 8 8 8 16 8 4 4 256 256 16 4 2 8 8 4 256 256 4 8

8 16 16 8 8 16 16 8 96 96 32 8 8 8 8 8 96 96 8 8

1 2 2 1 0.5 0.5 0.5 1 2 4 8 2 4 0.5 0.5 1 2 0.5 0.5 4

8 8 8 4 4 8 2 8 128 128 4 1 1 1 1 1 128 128 4 2

0.25 0.25 0.5 0.5 0.25 0.25 0.25 0.25 0.25 0.5 0.25 0.5 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

0.12 0.5 0.12 0.12 0.12 0.5 0.5 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0. 5 0.12 0.12 0.12

1 2 2 1 1 2 2 2 2 2 1 1 2 1 2 1 1 1 2 2

0.12 0.12 0.5 0.12 0.25 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.25 0.25 0.25 0.25 0.25 0.12 0.12 0.12

0.5 1 0.5 1 1 0.5 0.5 0.5 0.5 1 1 0.5 0.5 0.5 0.5 0.5 0.5 1 1 1

16 128 32 16 32 16 16 16 32 16 16 2 2 2 2 2 2 64 32 2

0.5 1 0.5 1 1 1 1 1 1 0.5 1 0.5 0.5 1 0.5 0.5 1 0.5 1 1

2 2 4 2 4 2 2 2 2 2 2 4 2 2 2 4 2 2 2 2

0.12 0.12 0.25 0.5 0.5 0.12 0.12 0.12 0.25 0.25 0.12 0.12 0.12 0.12 0.12 0.12 2 2 0.25 0.25

4 8 4 4 8 8 16 16 4 4 4 4 4 8 4 8 16 32 4 4

0.5 0.5 1 1 1 0.5 0.5 1 1 1 0.5 0.5 0.5 0.5 0.5 0.5 1 1 1 0.5

Gm-gentamicin; Km-kanamycin; Sm-streptomycin; Nm-neomycin; Tc-tetracycline; Em-erythromycin; Cl-clindamycin; Cm-chloramphenicol; Am-ampicillin; Pc-penicillin; Va-vancomycin; Vi-virgniamycin; Lz-linezolid; Tm-trimethoprim; Ci-ciprofloxacin; Ri-rifampicin. In grey, MIC values surpassing the microbiological breakpoints defined by the FEEDAP Panel (European Commission 2008).

subjected to pH below 3 [34]. These previous observations were reason for selection pH value 2.5 in our lactobacilli survival capability experiment. Resistance to bile salts is generally considered as an essential property for probiotic strains to survive the conditions in the small intestine. The relevant physiological concentrations of bile range are from 0.10 to 0.30% (w/v). Thus, it is necessary that efficient probiotic bacteria should be able to grow in bile salt with these concentrations [35].

tests (Liofilchem, Italy). This small antibiotic-resistant fraction justifies an antibiotic-susceptibility assay to avoid the inclusion of resistant strains in the formulation of probiotics. Although, a phenotypic test cannot confirm the presence or absence of transferable resistance genes, some of the observed levels are compatible with common transmissible determinants. These phenotypic

3.4. Antibiotic susceptibility testing and MIC determinations Antibiotic resistance of microorganisms used as probiotic agents is an area of growing concern. It is believed that antibiotic used for food-producing animals can promote the emergence of acquired antibiotic resistance in bacteria present in the intestinal microflora. Then, the antibiotic-resistant bacteria can transfer the resistance factor to other pathogenic bacteria through the exchange of genetic material [36]. One of the safety considerations in probiotic studies is the verification that a prospective probiotic strain does not contain potentially transferable resistance genes. The sixteen isolates were susceptible to tetracycline, with minimum inhibitory concentration (MIC) values ranging from 1 to 8 mg ml1, to gentamicin (2e4 mg ml1), to erythromycin (0.25e 0.5 mg ml1), to clindamycin (0.12e0.5 mg ml1), to streptomycin (8e32 mg ml1), to chloramphenicol (1e2 mg ml1), to neomycin (0.5e8 mg ml1), to kanamycin (2e16 mg ml1), to ampicillin (0.12e 0.5 mg ml1), to penicillin (0. 5 to 1 mg ml1), to vancomycin (2e 128 mg ml1), to virginiamycin (0. 5 to 1 mg ml1), to linezolid (2e 4 mg ml1), to trimethoprim (0.12e2 mg ml1), to ciprofloxacin (4e 32 mg ml1) and to rifampicin (0. 5 to 1 mg ml1), besides two chicken (L. salivarius 12K, L. agilis 13K) and two calves (L. reuteri L10/ 1, L. murinus L9) isolates with the presence of non wild-type ECOFFs with MIC levels for gentamicin (256 mg ml1), tetracycline (128 mg ml1), kanamycin (256 mg ml1) and streptomycin (96 mg ml1) (Table 2), which are regarded as resistant according to the microbiological breakpoints given by the recent FEEDAP document of the EFSA [15]. MIC values surpassing the microbiological breakpoints were confirmed moreover by using MIC strip

Fig. 2. PCR amplification of the tet (L) gene (229 bp amplicon). The PCR products were obtained using genomic DNA of the Lactobacillus salivarius 12K (lane 1); and Lactobacillus agilis 13K (lane 2); M: Mid Range DNA Ladder 100 bp to 3 kb linear scale molecular weight marker (Jena Bioscience).

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chicken isolates, resistant to tetracycline (L. salivarius 12K, L. agilis 13K) with MIC of 128 mg ml1 were positive to efflux pump encoding tet(L)gene (Fig. 2), which is located on pL S55 plasmid as described Ammor et al. [37]. A gentamicin and a kanamycin MIC of 256 mg ml1 was found in above-mentioned chicken isolates; a kanamycin MIC of 128 mg ml1 likewise in two calves (L. reuteri L10/ 1, L. murinus L9) strains. The forenamed chicken gentamicinresistant isolate L. salivarius 12K gave positive aac(60 )Ie-aph(200 )Ia PCR result (Fig. 3) and negative ones for aph(30 )-IIIa genes, e contra the two calves kanamycin-resistant strains, which gave a negative PCR results for the both of genes. The aac(60 )Ie-aph(200 )Ia amplicon was purified, sequenced and sequence similarity was determined using BLAST in the GenBank database. This comparison showed that our sequence had 100% identity with the N-terminal region of the AAC (60 )-APH (200 ) aminoglycoside-modifying enzyme carries the acetyltransferase activity of Staphylococcus aureus (accession no. AB568461). The first report of the AAC(60 )-APH(200 ) aminoglycosidemodifying enzyme in Lactobacillus of animal origin was described by Tenorio et al. [38], who expects that it is a possibility of transfer the bifunctional gene aac(60 )Ie-aph(200 )Ia, which is a part of the composite transposon Tn4001 widely distributed among gram-positive microorganisms [39], from Enterococcus to Lactobacillus. 3.5. Determination of ability to produce biogenic amines and the multiplex PCR for detection of genes responsible for biogenic amines production

Fig. 3. PCR amplification of the aac(60 )-aph(20 )-Ia gene (348 bp amplicon). The PCR product was obtained using genomic DNA of the Lactobacillus salivarius 12K (lane 1); M: Mid Range DNA Ladder 100 bp to 3 kb linear scale molecular weight marker (Jena Bioscience).

results underline the necessity to monitor the presence of transmissible antibiotic resistance genes at the molecular level. 3.4.1. PCR Detection of resistance genes PCR-based detection of genes responsible for resistance to gentamicin [aac(60 )-aph(20 )-Ia], tetracycline [tet(L), tet(M), tet(S)], kanamycin [aph(30 )-IIIa] and streptomycin/spectinomycin [aadA] was applied to strains suspected to carry acquired resistance. Two

To ensure their safety for use in animal and their products the sixteen wild-type ECOFFs isolates were screened for potentiality to produce biogenic amines. Phenotype detection of histidine, tyrosine, ornithine decarboxylase activities determined by qualitative plate method show that four calve isolates L. reuteri (L5/1, L6/1, L7) and L. amylovorus L6 possessed histidine decarboxylase activity (Fig. 4). Though, the detection of biogenic amines producing bacteria by conventional culture techniques is often tedious and unreliable, exhibiting disadvantages such as lack of speed, appearance of false positive/negative results and low sensibility [40]. As molecular methods are fast, reliable and culture-independent, they represent an interesting alternative for the detection of bacteria producing biogenic amines. In order to rapidly screen isolates for potential biogenic amines-producing strains, a multiplex PCR method was applied to simultaneously detect four genes involved in the production of the main biogenic amines: tyramine via tyrosine decarboxylase, putrescine via ornithine decarboxylase or agmatine deiminase and histamine via histidine decarboxylase. The

Fig. 4. Phenotype detection of histidine decarboxylase activities determined by qualitative plate method-Lactobacillus reuteri L 5/1 after a) two or b) four days of incubation on decarboxylase agar supplemented with histidine and bromocresol purple as indicator dye.

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Lactobacilli, E. coli and Klebsiella biofilm-forming capacities on abiotic surfaces were evaluated. The L. salivarius 15K (A570 ¼

0 0 0 10 20 20 5 5 5 0 10 10 10 10 30 20 0 30 10 10 5 30 0 0 30 20 5 20 0 5 0 0 30 10 0 0 0 0 10 0 10 10 0 0 0 5 0 0 40 40 40 40 40 5 40 40 40 30 40 40 40 30 5 20 20 0 5 40 20 0 10 10 5 5 5 20 5 5 5 5 5 10 0 0 10 5 20 5 40 40 5 20 5 5 0 0 30 30 20 30 40 20 40 40 20 5 0 0 5 0 10 5 10 20 20 5 5 0 0 0 5 0 5 5 10 20 0 5 20 0 0 5 10 10 10

10 30 10 20 10 10 20 5 20 20 10 40 10 20 10 20 10 5 20 5

Enzyme assayeda,b

In grey L. salivarius 15K and L. reuteri 14K without harmful enzyme activities. a Enzyme activity measured as the approximate nanomoles of substrate hydrolysed during 4-h incubation. b Harmful enzyme activities.

3.7. Screening of biofilm production in Lactobacillus sp.; E. coli; K. pneumonii

Strains

The enzyme activities of twelve lactobacilli, free of acquired resistance patterns and genes involved in the main biogenic amines production, were screened. The strains displayed various enzyme profiles as summarized in Table 3. Safety assessment includes the lack of harmful activities, such as b-glucosidase, b-glucuronidase activities, which are associated with detrimental effects in the colon by releasing aglycones and deconjugating glucuronic acid-conjugated carcinogens [41,42]. The L. salivarius (6K, 8K) and L. reuteri (L4, L4/1, L8) showed moderate b-glucosidase activity (10e20 nmol of substrate hydrolysed); L. salivarius 5K, 10K and L. reuteri L5 expressed high bglucosidase activity (30 nmol of substrate hydrolysed) and moreover L. reuteri L5, L. agilis 4K and L. salivarius (5K, 10K) had slow or moderate b-glucuronidase activity (5e10 nmol of substrate hydrolysed). In contrast, the b-galactosidase released by probiotics has been implicated in the relief of lactose maldigestion symptoms [43]. The two convenient strains L. salivarius 15K and L. reuteri 14K had high b-galactosidase activities (40 nmol of substrate hydrolysed). Simultaneously mentioned strains possessed a-fucosidase activity (through to help intestine colonization) and therewithal lack of aglucosidase activity (a trait considered positive for diabetic and obese humans) [44,45].

Table 3 Enzyme activities of Lactobacillus sp. assayed by the Api-ZYM system.

3.6. Enzyme activities

reuteri L4 reuteri L4/1 reuteri L5 reuteri L8 agilis 4K salivarius 5K salivarius 6K salivarius 8K salivarius 9K salivarius 10K reuteri 14K salivarius 15K

multiplex PCR was able to detect the corresponding a 435 bp hdc amplicons in phenotypically histidine positive strains (Fig. 5). One potential hdc target gene fragments were further confirmed by sequencing simplex PCR amplification product and sequence similarity was determined using BLAST in the GenBank database and showed 99% identity on 100% query cover with the amino acid sequence of the L. reuteri DSM 20016 (accession no. YP001272408).

L. L. L. L. L. L. L. L. L. L. L. L.

Fig. 5. Genotype detection of histidine decarboxylase, 435 bp hdc amplicons; The PCR products were obtained using genomic DNA of the Lactobacillus reuteri L5/1 (lane 1); Lactobacillus reuteri L6/1 (lane 2) Lactobacillus reuteri L7 (lane 3) and Lactobacillus amylovorus L6 (lane 4); M: Mid Range DNA Ladder 100 bp to 3 kb linear scale molecular weight marker (Jena Bioscience).

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Naphtol-AS-BIAcid Esterase EsteraseLeuValCysa-Galactosidase b-Galactosidase b-Glucuronidaseb a-Glucosidase b-Glucosidaseb a-Fucosidase phosphohydrolase phosphatase (C4) lipase (C8) arylamidase arylamidase arylamidase

D. Bujnakova et al. / Anaerobe xxx (2013) 1e10

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Table 4 The potential for exclusion of biofilm producing pathogens determined by quantitative FISH. >Strains

K188 KPC DH5a/pCIB10B

Bacterial number determined by FISHa Monoculture

L. reuteri 14K

L. salivarius 15K

4.58  0.25 3.73  0.22 5.53  0.27

4.33  0.32 (5%) 3.67  0.32 (2%) 5.10  0.18 (8%)

2.78  0.37** (39%) 2.21  0.32** (41%) 3.27  0.25*** (41%)

a number of enteric groups (Enter1432 probe) are given as log10 of number of biofilm forming bacteria per well  SEM; K188, KPC, DH5a/pCIB10B: monoculture and after treatment with whole cells L. reuteri 14K or L. salivarius 15K; experiments were performed in triplicate; significantly different from monoculture at **p < 0.01, ***p < 0.001; (% of pathogens biofilm inhibition by lactobacilli).

0.395  0.015) and L. reuteri 14K (A570 ¼ 0.264  0.025) produced strong biofilms. It is well known, that selection of probiotic is primary based on good adhesion to intestinal surfaces (in vitro-mucin, cell lines etc.). The ability to adhere to abiotic surfaces is not routinely tested in probiotic strains, even though the potential role of commensal bacteria biofilm formation in host health and disease is substantial, because allow to them to colonize of gut environment, remain more resistant and play a role in the protection of the gut epithelium from adherence of pathogenic agents. Probiotics in biofilm-like communities may be essential for long-term remodelling of the healthy composition and function of the intestinal population, therefore, probiotic strategies for the prevention and treatment of disease may require the intensive search for autochthonous strains with beneficial characteristics that include the capability to form biofilm [46]. Although biofilm formation on abiotic surfaces can’t definitely replace testing of adhesion ability to biotic surfaces, in recent years, some researchers test potential probiotic for ability of biofilm formation and/or some authors have shown a statistically significant positive correlation between adhesiveness to biotic surfaces and ability to form biofilm on abiotic surfaces [47e49]. Similarly, employed E. coli K188 (A570 ¼ 0.286  0.014); E. coli DH5a/pCIB10B (A570 ¼ 0.354  0.024) and K. pneumonii (A570 ¼ 0.249  0.035) were strong biofilm producers. Indeed, many animal bacterial pathogens exist predominantly as adherent (also called biofilm or sessile) organisms within tissue and on inert surfaces and it is well known that such infections are extremely difficult treatable, regarding to their high antimicrobial resistance [50]. The tolerance towards currently available antimicrobials calls for development of alternative treatment strategies, as bacteria, in retrospect, have a unique ability to evade the actions of classic antibiotics. 3.7.1. Inhibition of biofilm formation by different lactobacilli fractions The L. salivarius 15K and L. reuteri 14K (whole cells, acid and neutralized supernatants) different fractions competence to

inhibit E. coli and Klebsiella biofilms was tested. Our results revealed that whole cell and acid supernatant of L. salivarius 15K dramatically reduced E. coli and K. pneumonii biofilmformation. In this study, we showed that co-incubation of L. salivarius 15K whole cells together with E. coli K188; DH5a/pCIB10B and K. pneumonii resulted in a strong decrease in pathogens biofilm forming capacity (39e41% inhibition), whereas L. reuteri 14K whole cells displayed only marginal biofilm competitive exclusion potential (2e8% inhibition) (Table 4). The remarkable anti-biofilm effects of L. salivarius 15K against pathogens may be related to its stronger adhesion to abiotic surfaces in comparison with L. reuteri 14K, which could induce greater competition between the probiotic bacterium and pathogens and its strong co-aggregation potential (data not shown) may be an additional interpretation for the lack of pathogens biofilm formation [51]. Another possible mechanism of inhibition pathogens biofilms formation may be related to release of biosurfactants (not confirmed in our study), which alter surface hydrophobicity and hereby inhibit the adhesion of pathogenic microorganisms [52]. Treating E. coli K188; E. coli DH5a/pCIB10B and K. pneumonii biofilms with L. salivarius 15K acid supernatants revealed significant differences (P < 0.01, P < 0.001) in optical density between pre and post treatment. Neutralization of supernatants greatly reduced inhibition pathogens biofilms formation, suggesting that the inhibitory effect was due to acids production. The obtained results are presented in Table 5. To address the possible contribution of bacteriocins in the observed inhibition of biofilms formation, lactobacilli supernatant were treated with either trypsin or proteinase K. Protease treatment of the supernatants did not reduce inhibition of biofilms formation (data not shown), suggesting that inhibition was not due to the production of a proteinaceous component. These data support the hypothesis that ability of L. salivarius 15K to inhibit biofilms formation is caused, at least in part, to the production of acids, which caused reduction of cytoplasmic pH and metabolic activities and by this way internal bacterial osmotic disorders [53,54]. The similarly results were obtained by Juarez Tomás et al. [55]. Our results indicate that L. salivarius 15K whole cells and acid supernatant is a potent antimicrobial agent against E. coli and Klebsiella biofilms and could became one of the strategies to suppress or control biofilm forming infections, but we would like to underline the necessity for further studies. 4. Conclusion Following the subtractive in vitro screening strategy, the chicken strain L. salivarius 15K was finally selected based on promising probiotic properties without harmful characteristics. This strain could be taken in further investigation in in vivo studies to elucidate its potential health benefits and its application as novel probiotic strains in the animal (poultry) feed supplements.

Table 5 The potential for inhibition of biofilm producing pathogens determined by crystal violet assay. Strains

Crystal violet assay (A570)a Monoculture

K188 KPC DH5a/pCIB10B

0.286  0.014 0.249  0.035 0.354  0.024

L. reuteri 14K

L. salivarius 15K

AS

NS

AS

NS

0.253  0.117 0.015  0.009*** 0.152  0.023

0.267  0.02 0.199  0.039 0.325  0.045

0.004  0.022*** 0.045  0.001** 0.123  0.025**

0.317  0.045 0.230  0.059 0.360  0.065

a average values of absorbance from triplicate measurements  SEM are presented, A570eabsorbance at l ¼ 570 nm; K188, KPC, DH5a/pCIB10B: monoculture and after treatment with L. reuteri 14K or L. salivarius 15K (AS: acid supernatant; NS: neutralized supernatant); significantly different from monoculture at **p < 0.01, ***p < 0.001.

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