Arch Microbiol (2013) 195:537–543 DOI 10.1007/s00203-013-0905-7
ORIGINAL PAPER
Required characteristics of Paenibacillus polymyxa JB‑0501 as potential probiotic Karim Naghmouchi · John Baah · Benoit Cudennec · Djamel Drider
Received: 5 March 2013 / Revised: 9 April 2013 / Accepted: 22 May 2013 / Published online: 12 June 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract The ability of Paenibacillus polymyxa to inhibit the growth of Escherichia coli generic ATCC 25922 (Escherichia coli ATCC 25922) and to adhere to monolayers of the enterocyte-like human cell line Caco-2 was evaluated. P. polymyxa JB-0501 (P. polymyxa JB-0501), found in a livestock feed probiotic supplement, was compared to P. polymyxa reference strain ATCC 43685 and ATCC 7070 (P. polymyxa ATCC) in terms of carbohydrate utilization and resistance to lysozyme, acid, bile salts, and hydrogen peroxide. JB-0501 grew at pH 4.5 and at H2O2 concentrations less than 7.3 μg/ml and presented a higher affinity to hexadecane and decane. Bile salts at 0.2 % inhibited the growth of all three strains. P. polymyxa JB-0501 and P. polymyxa ATCC 43865 adhered to Caco-2 cell monolayers. The percentage of cells that adhered ranged from about 0.35 to 6.5 % and was partially proportional to the number applied. Contact time (from 15 min to 1 h) had little impact on adhesion. P. polymyxa JB-0501 inhibited the growth of E. coli ATCC 25922, as proven by the diffusion
Communicated by Erko Stackebrandt. K. Naghmouchi (*) · J. Baah Lethbridge Research Center, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada e-mail:
[email protected] K. Naghmouchi Laboratoire des Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, El Manar, Tunis, Tunisia B. Cudennec · D. Drider (*) Laboratoire des Procédés Biologiques, Génie Enzymatique et Microbien (ProBioGEM), UPRES‑EA 1026, Polytech’Lille/IUTA, Université Lille Nord de France, Avenue Paul Langevin, 59655 Villeneuve d’Ascq Cedex, France e-mail: djamel.drider@univ‑lille1.fr
tests in agar. Taken together, these results suggested that P. polymyxa JB-0501 has the potential probiotic properties to justify its consideration as a livestock feed supplement. Keywords Paenibacillus polymyxa JB-0501 · Escherichia coli · Caco-2 cells · Adhesion · Pathogenic bacteria · Probiotic properties
Introduction Paenibacillus polymyxa, formerly known as Bacillus polymyxa (Ash et al. 1993), constitutes part of microbiota from soil, rhizospheres, water, diseased insect larvae, and foods (Choi et al. 2009). These species have been reported as a potential biological control agent because of its capacity to produce a cyclic lipopeptide antibiotic, which is active against a wide variety of microbes such as Pseudomonas fluorescens C7R12, Streptomyces argentelous ATCC 1109, and Fusarium acuminatum DSM 62148 (Selim et al. 2005). Paenibacillus polymyxa JB-0501 (P. polymyxa JB-0501) was isolated from a microbial-based livestock feed supplement and was shown to be active against Gram-negative bacteria such as Escherichia coli RR1, Pseudomonas fluorescens R73, Pantoea agglomerans BC1, Butyrivibrio fibrisolvens OR85, and Fibrobacter succinogenes (Naghmouchi et al. 2011). For instance, antibacterial activity of P. polymyxa JB-0501 was shown to be attributed to production of colistin A and colistin B (Naghmouchi et al. 2012), leading to possible medical applications of P. polymyxa JB-0501 or its antibacterial substances in the treatment of infections due to Gram-negative bacteria, which constitute a major clinical concern (Landman et al. 2008). The aim of this study was to determine the potential of P. polymyxa JB-0501 as a probiotic in livestock
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feed, in particular as an inhibitor of E. coli ATCC 25922 (E. coli 25922). To this end, we compared our strain P. polymyxa JB-0501 with P. polymyxa reference strains, in terms of carbohydrate utilization profile and tolerance of gastrointestinal tract conditions (lysozyme, acid, bile, and hydrogen peroxide), cell affinity for hydrocarbons, as well as adhesion to the enterocyte-like cell line Caco-2.
Materials and methods Strains and culture conditions Paenibacillus polymyxa JB-0501 and P. polymyxa reference strains ATCC (43865 and 7070) were aerobically grown for 18 h at 30 °C with agitation in tryptic soy broth (TSB) (Difco Laboratories, Detroit, MI, USA) containing 1 % of yeast extract leading to a medium named TSBYE. E. coli generic ATCC 25922 (E. coli ATCC 25922) was grown for 16 h at 37 °C in TSB (Difco). The four strains were stored at −80 °C in the corresponding medium containing 20 % glycerol (w/v) (Sigma-Aldrich, Oakville, ON, Canada). Frozen stocks were activated by re-suspending 100 μl of each conserved strain in 10 ml of growth medium, followed by incubation for 18–24 h at the corresponding temperature. Antimicrobial activity of Paenibacillus polymyxa JB‑0501 To demonstrate that inhibition of E. coli ATCC 25922 by P. polymyxa JB-0501 was attributable in part to secreted substances, culture supernatant was tested using a qualitative agar well diffusion method (Wolf and Gibbons, 1996). Briefly, TSBYE containing 0.75 % (w/v) agar (Difco) was cooled to 47 °C, seeded with overnight culture of E. coli ATCC 25922 (1 % vol), and poured into sterile Petri plates. Wells (7 mm) were cut in the solidified agar using a sterile metal cork borer and filled with 80 μl of P. polymyxa culture supernatant. The plates were incubated at 5 °C for 2 h to allow diffusion process; the incubation was continued aerobically for 18 h at 30 °C. After this period, the plates were inspected for the presence or absence of zones of inhibition. Paenibacillus polymyxa JB-0501 culture (10 ml) was centrifuged, and the cells were re-suspended in 50 μl of TSB. 20 μl of suspension were deposited into a 5-mm sterile paper disk. The paper disks impregnated with bacterial cell suspension were placed on the surface of TSBYE seeded with overnight culture of E. coli ATCC 25922 (0.25 ml in 25 ml), solidified with 0.75 % agar, and poured at 47 °C into Petri plates. The plates were incubated
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aerobically at 30 °C for 18 h and checked for zones of inhibition (clear agar) surrounding the paper disks. Tolerance of Paenibacillus polymyxa JB‑0501 to lysozyme, acid, bile, hydrogen peroxide (H2O2), and gastric acid Tolerance of the three P. polymyxa strains to lysozyme (Sigma), acidic conditions, bile salts (Sigma), and hydrogen peroxide (Sigma) was tested using sterile flat-bottom 96-well microtiter plates (Falcon, Becton–Dickinson and Company, Frankin Lakes, NJ, USA) (Gagnon et al. 2004). To TSBYE broth, we added egg-white lysozyme (Sigma, 48,000 U/mg protein) at different concentrations (0, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 μg/ml). The medium was adjusted to different pH values (7.0, 6.5, 6.0 5.5, 5.0, 4.75, 4.5, 4.25, and 4.0) using 1 M HCl. For another purpose, we added to the medium bovine bile salts (Sigma) at different concentrations (0.1, 0.2, 0.3, and 0.4 % w/v). In order to study the effect of H2O2, we added different concentrations of this compound (0, 2.5, 5, 10, 20, and 40 μg/ml) purchased from Merck (Darmstadt, Germany). Wells (in duplicate) containing 200 μl of tested broth were inoculated with 20 μl of overnight culture of P. polymyxa diluted 1/100 in TSBYE. Microplates were incubated under aerobic conditions at 30 °C for 18 h. Optical densities (OD) were read at 630 nm using a Thermomax microplate reader (Zeus, scientific Inc, Raritan, NJ). Resistance to H2O2 and lysozyme (minimum inhibitory concentration) was expressed as the lowest concentration that maintained the OD under 50 % of the OD in the “0” concentration wells. Acid resistance was expressed as the lowest pH at which the tested strain had an OD at least 40 % of its level at pH 6.5. Determination of the tolerance to a simulated gastric transit was based on the procedure described by Charteris et al. (1998). A simulated gastric juice was prepared by suspending pepsin (3 mg/ml) in sterile saline (0.5 % w/v) and adjusting the pH to 2.0 with concentrated HCl. Overnight 2 ml cultures of the three P. polymyxa variants were subjected to centrifugation in an Eppendorf centrifuge at 5,000×g for 5 min and washed two times in quarter-strength Ringer’s solution at pH 7 (Sigma). Then, 0.3 ml of the washed suspension was added to 1.5 ml of simulated gastric juice (pH 2.0) in a 2.0 ml Eppendorf tube and vortexed. In the controls, simulated gastric juice was replaced by 1.5 ml quarter-strength Ringer’s solution, and these samples were used for determination of the initial cell counts. Aliquots of 0.2 ml were removed after 15, 30, and 90 min incubation at 37 °C, and viable counts were determined by plating serial tenfold dilutions on LB agar and counting the colony numbers after anaerobic incubation at 37 °C for 48 h. Experiments were carried out in duplicate
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and were repeated two times. Results are expressed as the mean and standard deviation of three determinations. Carbohydrates utilization The abilities of P. polymyxa JB-0501 and the other P. polymyxa strains (ATCC 43865, ATCC 7070) to utilize sugars, glycogen, and pyruvate and to grow in the presence of 6.5 % NaCl as well as to tolerate polymyxin B were determined using the Vitek 2 colorimetric GN-BCL card for the identification of Gram-positive spore-forming microorganisms of the family Bacillaceae (BioMerieux, Inc. Hazelwood, MO, USA) according to the manufacturer’s instructions (Scheldeman et al. 2004). Results were reported as negative or positive. The carbohydrate utilization profiles were compared, and the homology was determined using the Vitek 2 compact system (BioMerieux, Inc. Hazelwood, MO, USA). Data are means of duplicate experiments. Determination of cell affinity for hydrocarbons Paenibacillus polymyxa JB-0501 and reference strains ATCC 43865 and ATCC 7070 were grown in TSBYE media at 30 °C to the logarithmic growth phase, harvested by centrifugation (3,000×g, 10 min), washed twice, and re-suspended at a concentration of 108 CFU/ml in 0.15 M NaCl solution. Hexadecane or decane (0.4 ml) was vortexed for 60 s, and the suspension was allowed to stand for 30 min (Doyle and Rosenberg 1995). Absorbance by the aqueous phase at 400 nm was measured using a spectrophotometer (Molecular Devices Corp., Sunnyvale, CA, USA). The percentage of cells extracted into each hydrocarbon liquid was determined by the following formula: 100 × [1 − (A/A0)], where A0 and A are the absorbances by the aqueous phase prior to and after mixing. Data are means of duplicate experiments. Caco‑2 cell culture and bacterial adhesion assay Enterocyte-like Caco-2 cells (ATCC HTB-37) obtained from the American Type Culture Collection (Rockville, MD) were cultured routinely under a 5 % (v/v) CO2 atmosphere at 37 °C as monolayer in Dulbecco-modified Eagle’s minimal essential medium (DMEM; Gibco) containing 25 mM glucose, 4 mM glutamine, and 1 mM sodium pyruvate (Gagnon et al. 2004) and then in medium supplemented with 20 % (v/v) fetal calf serum (Hyclone Laboratories, Logan, UT, USA). Wells in multiwell tissue culture plates (Falcon, Becton–Dickinson) were seeded each with 104 cells. The culture medium was replaced every 48 h, and confluent monolayers obtained after the tenth subculture were used for the adhesion assay. Trypan blue was used for
viable staining, and cells were counted by hemocytometer (Hausser scientific, Horsham, PA, USA). The following procedure of Cepeljnik et al. (2007) was followed. Caco-2 cell monolayers were washed twice with sterile phosphate-buffered saline (PBS, 100 mM, pH 7.3) (Sigma) and submerged in DMEM for 18 h, followed by one wash with PBS. Overnight culture of P. polymyxa in TSBYE broth was centrifuged, the bacterial cells were washed twice with PBS, suspended in DMEM at approximately 4 × 104 CFU/ml or 4 × 106 CFU/ml, and 2 ml was added to the wells. Adhesion to Caco-2 cells was evaluated at 15, 30, and 60 min by washing twice with sterile PBS and then adding 0.25 ml of trypsin–EDTA (0.05 % trypsin, 0.53 mM Na4-EDTA; Gibco). After 15 min of incubation at 37 °C, 0.25 ml of DMEM supplemented with 20 % FCS (heat-inactivated fetal calf serum) was added to each well to stop the trypsin reaction, and the Caco-2 layer was detached by pipetting. Serial tenfold dilutions were then plated on Bacillus cereus agar (Difco Laboratories), followed by incubation under aerobic conditions for 24 h at 37 °C. All experiments were done in duplicate.
Results Antimicrobial activity of Paenibacillus polymyxa JB‑0501 The antimicrobial substance(s) produced by P. polymyxa JB-0501 was confirmed qualitatively using the agar diffusion test (Fig. 1a) against E. coli ATCC 25922 as the indicator strain. Recently, we reported that the substances behind the anti-E. coli activity involved colistin A and colistin B (Naghmouchi et al. 2012). Using the spot test, P. polymyxa JB-0501 was found to be antagonistic toward E. coli ATCC 25922 (Fig. 1b). Paper disks loaded with P. polymyxa JB0501 produced clear zones of inhibition about 8 mm in diameter. TSB medium was tested as negative control. Lysozyme, acid, bile, and hydrogen peroxide tolerance by Paenibacillus polymyxa JB‑0501 The ability of P. polymyxa JB-0501 to grow in the presence of lysozyme, acidic pH, bile salts, and H2O2 was determined. P. polymyxa JB-0501 was able to grow in the presence of egg-white lysozyme concentrations up to 16.5 μg/ ml, whereas P. polymyxa ATCC 43865 and P. polymyxa ATCC 7070 strains were able to tolerate a high concentrations estimated to be 31.25 and 62.5 μg/ml, respectively. On the other hand, all three strains of P. polymyxa grew at pH 4.75. Nevertheless, no growth was observed for any of the strains in the presence of 0.2 % bile salts. The maximal H2O2 concentration tolerated by P. polymyxa JB-0501 was between 3.6–7.3 μg/ml.
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Fig. 1 a Agar diffusion test showing inhibition of E. coli ATCC 25922 by supernatant of 20 h culture of P. polymyxa JB-0501 (right); left = negative control (tryptic soy broth). b Paper disk spot test showing inhibition of E. coli ATCC 25922 by P. polymyxa JB-0501
100
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Log CFU/ml
Affinity (%)
80
60
40
6 4 2 0
20
0
15
30
45
60
Times (min) 0 JB0501
ATCC 43865
ATCC 7070
Fig. 2 Affinity of P. polymyxa JB-0501, P. polymyxa ATCC 43865 and P. polymyxa ATCC 7070 strains for hexadecane (white) and decane (gray). Values presented are the means of three independent measurements
Tolerance to gastric acidity and cell affinity for hydrocarbons Exposure of P. polymyxa JB-0501, P. polymyxa ATCC 43865, and P. polymyxa ATCC 7070 strains to a simulated gastric buffer containing pepsin at pH 2.0 resulted in a rapid loss of viability (Fig. 2). Viable counts of variant P. polymyxa JB-0501 strains decreased from about 6.93 to 1.61 Log CFU/ml within 15 min. P. polymyxa JB-0501 displayed higher affinity for both hexadecane and decane than the P. polymyxa ATCC 43865 and P. polymyxa ATCC 7070 (Fig. 3).
Fig. 3 Tolerance of selected P. polymyxa JB-0501 (diamond), P. polymyxa ATCC 43865 (square) and P. polymyxa ATCC 7070 (triangle) strains to a simulated gastric juice (pH 2.0). Results are means of two independent experiments, vertical bars represent standard deviations
ATCC) using API 50-CHB Gallery (bioMerieux Canada Inc.,). P. polymyxa JB-0501 can use d-galactose, d-mannose, d-trehalose, d-glucose, d-ribose, palatinose, and d-melezitose as carbohydrates. On the other hand, P. polymyxa ATCC strains can not use d-melezitose and d-tagatose as carbohydrates. All P. polymyxa strains resulted to be sensitive to polymyxin B. P. polymyxa ATCC 43865 and P. polymyxa ATCC 7070, but not P. polymyxa JB-0501, were able to grow in presence of 6.5 % NaCl. Strain P. polymyxa JB-0501 displayed a homology of about 98 %, confirming the high probability that it does indeed belong to the genus Paenibacillus. Adhesion of Paenibacillus polymyxa to Caco‑2 cells
Carbohydrate utilization and other physiological factors The carbohydrate utilization profiles, the tolerance of salt or polymyxin B, and the calculated homology were determined for the three variants of P. polymyxa (JB-0501 and
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The quantitative adhesion of P. polymyxa JB-0501 and P. polymyxa ATCC 43865 and ATCC 7070 to monolayers of enterocyte-like cells is depicted in Table 1. Starting from initial numbers of about 8.7 × 106 and 6.8 × 106
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Arch Microbiol (2013) 195:537–543 Table 1 Adhesion of P. polymyxa JB-0501, P. polymyxa ATCC 43865 and P. polymyxa ATCC 7070 to Caco-2 cells
Adherent bacteria (CFU/well)a
P. polymyxa JB-0501 P. polymyxa ATCC 43865
a
Mean ± SE of duplicate analyses
P. polymyxa ATCC 7070
Initial load
15 min
30 min
60 min
8.7 ± 0.53 × 106 8.7 ± 0.45 × 104 6.8 ± 0.45 × 106 6.8 ± 0.37 × 104 7.1 ± 0.65 × 106
3.1 ± 0.1 × 104 5.3 ± 0.22 × 103 3.2 ± 0.21 × 104 4.4 ± 0.33 × 103 3.8 ± 0.21 × 104
3.1 ± 0.31 × 104 4.4 ± 0.68 × 103 2.8 ± 0.53 × 104 2.4 ± 0.22 × 103 3.3 ± 0.13 × 104
3.4 ± 0.16 × 104 4.8 ± 0.42 × 103 4.8 ± 0.26 × 104 3.7 ± 0.32 × 103 4.8 ± 0.46 × 104
7.1 ± 0.22 × 104
2.4 ± 0.33 × 102
5.5 ± 0.58 × 102
4.1 ± 0.51 × 102
CFU/well, the number of adherent bacterial cells after 1 h of contact was about 3.4 ± 0.16 × 104 CFU/well and 4.8 ± 0.26 × 104 CFU/well, respectively, for P. polymyxa JB-0501 and P. polymyxa ATCC 43865. Starting from initial numbers 100 times smaller, the numbers of adherent bacterial cells were about ten times smaller. Contact time with the Caco-2 monolayer had little effect on the number of adherent bacterial cells counted.
Discussion Importantly, this study has confirmed the anti-E. coli activity of P. polymyxa JB-0501, which was isolated from a microbial-based livestock feed supplement (Naghmouchi et al. 2011). As a rule, E. coli was regarded as an inoffensive and common bacterium of the gastrointestinal tract of ruminants. However, the emergence of the enterohemorrhagic strain E. coli O157:H7 has become a major public concern worldwide. Indeed, this pathogen is producing potent exotoxins with severe damage to the lining of the intestine, in particular in humans, allowing the strain to invade the body and infect organs such as the kidneys, sometimes with fatal consequences. The results obtained in this study suggest some potential for P. polymyxa JB-0501 as a probiotic, based on antimicrobial activities observed using whole cells (paper disk method) and culture supernatant (agar diffusion test). P. polymyxa JB-0501 thus sheds or secretes antimicrobial substances that diffuse into agar and produce clear inhibition zones attributed at least partly to colistin A and colistin B (Naghmouchi et al. 2012). The supernatant has been shown to be effective against E. coli O157:H7, Pseudomonas aeruginosa ATCC 19442, Salmonella enterica UL, and Pseudomonas fluorescens LRC R73. Gibson and Wang (1994) reported that the antagonistic activity of several species of P. polymyxa against Gram-negative pathogens was associated with the production of polymyxin-like antimicrobial peptides. For a probiotic candidate to be effective, it must survive under gastrointestinal tract conditions, which means, resistance to salivary lysozyme, gastric acid (HCl), and bile.
The “candidate” should insure its survival and settlement within the intestinal microbiota (Fuller 1989 and Fuller 1992). In this sense, P. polymyxa JB-0501 was able to grow in the presence of 16.5 μg/ml of lysozyme; this concentration is anticipated to be higher than the physiological concentration of intestinal lysozyme (Suskovic et al. 1997). Lysozyme is known to be active against Gram-positive bacteria; it is usually used at 100 μg/ml for laboratory cell wall lysis (Suskovic et al. 1997). The growth of P. polymyxa JB0501 in TSBYE broth appeared to be unaffected at low pH as pH 4.5. Acidity is believed to be the most detrimental factor affecting the growth and viability of potential probiotic bacteria. Indeed, acidity is known to significantly affect growth when pH is below 4.5 (Lankaputhra and Shah 1995). P. polymyxa JB-0501 should therefore survive in fermented products and consequently could contribute to extending product shelf life. As stated before, resistance to bile salts stand also as a major criterion to endorse potential probiotics affiliations. Gilliland et al. (1984) used a concentration of 0.3 % bile in order to determine bile tolerance level of Lactobacillus acidophilus strains. Remarkably, none of the P. polymyxa strains used in the present work was able to survive or even tolerate a bile concentration of 0.2 %. Kristoffersen et al. (2007) studied the tolerance to bile salts in forty Bacillus cereus strains (B. cereus). Growth of all strains was observed at low bile salt concentrations, but none growth was observed on LB agar plates containing more than 0.005 % bile salts. B. cereus ATCC 14579 was grown to mid-exponential growth phase and shifted to medium containing bile salts (0.005 %). Resistance to H2O2 was also tested in order to determine whether P. polymyxa JB-0501 might be competitive with H2O2-producing organisms, in fermented food products (i.e. added as a co-ferment) or in the intestinal ecosystem. The minimum inhibitory concentrations of H2O2 for P. polymyxa JB-0501 and P. polymyxa ATCC 43865 were about 7.3–14.6 and 14.6 μg/ml, respectively; these values are slightly below the value (17 μg/ ml) usually found to be detrimental to probiotic bacteria in yogurt (Dave and Shah, 1997). The current data are somehow surprising because Paenibacillus as an aerobe is
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anticipated to contain enzymes for reducing hydrogen peroxide and superoxide. Using the human colon carcinoma cell line Caco-2 as a model of the intestinal epithelium, we established that P. polymyxa JB-0501 was slightly less adhering than the reference strain P. polymyxa ATCC 43865 after 1 h. In the case of both strains, adhesion ranged from about 0.35 to 6.5 % of the initial numbers of P. polymyxa strains contacted with the Caco-2 cells enterocytes, the proportion decreasing at the higher initial load thus suggesting that saturation of the adhesion sites was approached at the higher bacterial load tested. The results showed that adhesion was only somewhat dependent on P. polymyxa JB-0501 concentration and that contact time had even less impact. This in vitro adhesion assay (pH, well size, incubation medium, incubation time, cell lines, etc.) makes possible the comparison of our results with those reported in the literature for spore-forming or other probiotic bacteria. Andersson et al. (1998) showed in their study that B. cereus produced spores capable of adhering to Caco-2 cell monolayers and also able to germinate in an environment similar to that of the small intestine; the authors noted that the hydrophobicity of the spore was a contributing factor to adhesion. On the other hand, Angioi et al. (1995) have evaluated the capacity of B. subtilis strains to colonize the surface of Caco-2 cells, testing the bacteria both in the spore state and following stimulation of germination by exposure to low pH (as under gastric conditions) or to high temperatures. The degree of adhesion was found to vary in relation to the different physiological phases of the bacterial cells. Further studies on Bacillus spores revealed a strong affinity for hexadecane and other hydrophobic solvents (Doyle et al. 1984), whilst their treatment with strong denaturants promoted their adherence to hexadecane (Koshikawa et al. 1989). Our work is original, and this is in our opinion the first report targeting Paenibacillus species as candidate for potential probiotic grade. Based on that, we assert that P. polymyxa JB-0501 has a set of interesting probiotic characteristics including production of antimicrobials, resistance to gastrointestinal conditions (e.g. lysozyme and acidic pH), and tolerance of hydrogen peroxide, as well as adhesion to Caco-2 cells. As a future direction, further studies are needed to determine whether this inhibition is bacteriostatic or bacteriocidal and whether or not adhesion of E. coli ATCC 25922 or E. coli O157:H7 to intestinal epithelial cells is inhibited. The next undertaking would be to increase the capacity of P. polymyxa JB-0501 to tolerate gastrointestinal conditions using conventional selection procedures (e.g. in media containing bile salts) or by encapsulation in a suitable matrix for inclusion in animal feed.
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Arch Microbiol (2013) 195:537–543 Acknowledgments This research was supported by Best Environmental Technologies Inc., Edmonton, AB, Canada.
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