Folia Microbiol (2015) 60:97–101 DOI 10.1007/s12223-014-0345-z
Occurrence and characterization of Paenibacillus sp. isolated from rabbits Monika Pogány Simonová & Vladimír Kmeť & Andrea Lauková
Received: 6 March 2014 / Accepted: 4 September 2014 / Published online: 18 September 2014 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014
Introduction
Materials and methods
Paenibacillus, the representants of which previously belonged to the genus Bacillus, was re-classified as a separate genus in 1993 (Ash et al. 1993). The genus Paenibacillus includes Gram-positive, aerobic or facultative anaerobic, endospore-forming bacteria which have been isolated from a variety of environments—soil, water, rhizosphere, vegetable matter, forage and insect larvae, and clinical samples (McSpadden Gardener 2004; Ouyang et al. 2008). There is a rapidly growing interest in Paenibacillus sp. because of their production of various extracellular enzymes (polysaccharide-degrading enzymes and proteases) and antimicrobial substances against a wide spectrum of microorganisms, including pathogens (e.g. Clostridium botulinum), and the ability to enhance the immune function of animals with its polysaccharide (Li et al. 2012). Previous studies on rabbit caecal microflora revealed the great bacterial diversity of the ecosystem studied, including both the phyla Firmicutes and Bacteroides (Monteils et al. 2008). Up to now, we were more focused on Gram-positive faecal enterococci (Simonová et al. 2005). To our knowledge, there have been no previous reports of Paenibacillus sp. isolated from rabbits’ gastrointestinal tract. Because representants of the species Paenibacillus were determined in rabbits faeces, we decided to test their sensitivity to antibiotics and enterocins; it was not tested for this type of bacteriocins yet.
Isolation and identification of bacterial strains
M. P. Simonová (*) : V. Kmeť : A. Lauková Institute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovakia e-mail: [email protected]
The faecal and caecal samples from healthy rabbits (n=144, age 35–77 days, Hycole breed, various sex) were homogenized in Ringer buffer (Oxoid, UK) using stomacher, properly diluted and plated on plate count agar (Biomark, India). Plates were incubated aerobically at 37 °C for 24–48 h. The bacterial counts were expressed in log 10 of colony forming units per gram (log10 CFU/g±SD). After incubation, morphologically different colonies were picked up and re-streaked for several generations in order to isolate purified individual bacterial strains. Isolates were identified using protein “fingerprints” determined by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics MALDI Biotyper, USA) as described by Bessède et al. (2011), Gram staining and on the basis of colony characteristic. The phylogenetic trees were constructed using the neighbour-joining method with the software MEGA5 (Tamura et al. 2011) with the aim to select of suitable, not identical strain(s) with bacteriocin-like activity for further in vitro and in vivo testing. The BBL Gram-Positive ID Kit (Becton & Dickinson) was applied to determine additional physiological and biochemical features. Sensitivity of isolates to antibiotics and enterocins Antibiotic resistance of identified Paenibacillus strains (100 μL of an 18-h culture of each tested strain) was tested by the qualitative method—the agar disc diffusion method on Columbia agar (Becton & Dickinson, BD, USA) with 10 % of defibrinated sheep blood. The tests should be performed according to internationally recognized standards such as the National Committee for Clinical Laboratory Standards (NCCLS 2003). The following antibiotic discs (Oxoid, UK;
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Lach-Ner, Czech Republic) were used: amikacin (AK30 μg), amoxicillin/clavulanic acid (AMC30), ampicillin (AMP10), bacitracin (B2), chloramphenicol (C30), clindamycin (DA2), colistin (CL10), doxycycline (DOX30), erythromycin (ERY15), gentamicin (CN120), kanamycin (KAN30), linkomycin (L2), neomycin (N5), penicillin (PEN10), piperacillin (PIP100), rifampicin (RA30), streptomycin (S25), tetracycline (TE30), tobramycin (TOB10) and vancomycin (VAN30). After incubation at 37 °C for 18 h, the strains were classified as resistant or sensitive (according to the manufacturers’ instructions). The semi-purified enterocins (SPEs) were produced by strains listed in Table 1. They were isolated in our Laboratory of Animal Microbiology (Institute of Animal Physiology SAS, Košice, Slovakia) except Enterococcus haemoperoxidus
isolated in Czech Republic (provided by Dr. P. Švec, Czech Collection of Microorganisms, Masaryk University, Brno, Czech Republic). The SPEs were prepared by the following procedures: a 16-h culture (300 mL) of Enterococcus faecium CCM7420-EF2019, CCM7419-EK13, AL41, EF55, CCM4231, EF9a, EF9296 and E. haemoperoxidus EHae466 strains in MRS broth (Merck) were centrifuged for 30 min at 10,000g in order to remove the cells. After adjusting of supernatant to pH 5.0 (5.5 in the case of AL41 strain), ammonium sulphate was gently added to the supernatant to obtain 40 % (w/v) saturation, and the mixture was stirred at 4 °C for 2 h (EK13, EF9296, EF9a EHae466), for 4 h (EF2019) and for 24 h (EF55, CCM4231), at 21 °C for 1 h (AL41). After centrifugation at 10,000g for 30 min, the resulting pellet was resuspended in 10 mmol/L sodium phosphate buffer (pH 6.5).
Table 1 The susceptibility of Paenibacillus cookii, P. glucanolyticus and P. lactis isolates against semi-purified enterocins (SPEs-Ents; AU/mL) Isolates
Source
Semi-purified enterocins (SPEs) EK13
AL41
EF55
CCM4231
EF9a
EF9296
EF2019
EHae466
EA5 PC1 PL1 PL1A PL2 PL3 PL4 PL5 PL5A PL6 PL7 PL8 PL9 PL10 PL11
– Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Caecum Caecum Faeces Caecum Caecum
51,200 12,800 25,600 12,800 25,600 25,600 12,800 25,600 12,800 25,600 6400 25,600 1600 25,600 6400
3200 100 100 100 100 1600 – 6400 6400 100 200 1600 1600 200 3200
51,200 12,800 400 400 1600 25,600 200 12,800 12,800 25,600 12,800 25,600 3200 25,600 3200
100 200 100 – – – – 100 100 100 – 800 – – 100
25,600 12,800 25,600 25,600 1600 25,600 800 25,600 12,800 6400 800 25,600 200 12,800 200
25,600 12,800 12,800 800 6400 25,600 12,800 25,600 12,800 12,800 800 12,800 100 400 400
51,200 12,800 25,600 25,600 3200 25,600 12,800 25,600 12,800 1600 12,800 25,600 3200 800 1600
25,600 12,800 6400 12,800 100 6400 100 100 800 25,600 12,800 6400 3200 25,600 800
PL12 PL13 PL14 PL14A PG1 PG2 PG3 PG4 PG4A PG4B PG5 PG6
Caecum Faeces Faeces Faeces Caecum Caecum Caecum Faeces Faeces Faeces Faeces Faeces
3200 25,600 400 6400 100 25,600 25,600 25,600 25,600 12,800 3200 100
400 6400 400 200 100 – 200 100 800 800 1600 100
800 25,600 400 25,600 100 25,600 25,600 25,600 25,600 25,600 6400 400
– 100 100 – – – – – 100 100 800 –
400 25,600 100 25,600 100 25,600 25,600 25,600 25,600 25,600 6400 800
800 200 25,600 25,600 100 200 6400 25,600 25,600 12,800 3200 100
25,600 25,600 25,600 25,600 100 1600 25,600 25,600 25,600 25,600 3200 12,800
200 25,600 25,600 6400 100 25,600 25,600 25,600 25,600 25,600 6400 200
AU/mL arbitrary units per millilitre of culture medium Enterococcus avium—control indicator strain, EK13 (produced by E. faecium EK13-CCM7419), AL41 (E. faecium AL41), EF55 (E. faecium EF55), CCM4231 (E. faecium CCM4231), EF9a (E. faecium EF9a), 9296 (E. faecium 9296), EF2019 (E. faecium EF2019-CCM7420), EHae466 (E. haemoperoxidus EH466)
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The antimicrobial titer of SPE was defined as the reciprocal of the highest twofold dilution producing a distinct inhibition of the inhibitor lawn, expressed in arbitrary units per mL (AU/ mL) of culture medium. Enterococcus avium EA5 (our isolate from piglet) was used as a bacteriocin-sensitive indicator strain (the amount of 200 μL of an 18-h culture of each indicator strain) to determine bacteriocin activity levels; the bacteriocin activity of SPEs produced by different strains varied between 1600 and 25,600 AU/mL.
Results The total counts of Paenibacillus sp. in faeces of rabbits varied from 1.33 to 3.74 log CFU/mL/g (detected in 30 mixture samples); the bacterial counts in caecum were lower (from 1.39 to 1.99 log CFU/mL/g); they occurred only in several samples (18 samples). The isolates were allotted to the species Paenibacillus glucanolyticus (9; PG1-4, 4A, 4B, 5, 6), Paenibacillus lactis (17; PL1, 1A, 2–5, 5A, 6–14, 14A) and Paenibacillus cooki (PC1) by MALDI-TOF. As demonstrated in Figs. 1 and 2, the isolate pairs PG1-PG4, PL5-PL5A, PL4PL8, PL1-PL1A and PL2-PL7 formed a tight cluster on the basis of nearly identical mass spectra and could be marked as identical strains. Considering the results obtained using the BBL GramPositive ID Kit, they were in accordance with those in the Bergey’s Manual (2009); acid is produced from lactose, sucrose, mannitol (except P. cookii isolate), arabinose, glycerol (except P. lactis strains), fructose (except P. cookii isolate) and trehalose (except P. lactis isolates; Table 2). The most of isolates hydrolyzed esculin, but not urea and arginine. Fig. 1 Phylogenetic tree of Paenibacillus glucanolyticus isolates from rabbits based on the protein mass fingerprinting analysis using the neighbourjoining method
99
All isolates were resistant to colistin. The most of strains showed resistance to bacitracin, clindamycin, erythromycin, linkomycin, streptomycin (96 %), penicillin (92 %), tetracycline (88 %), ampicillin, doxycycline, kanamycin, rifampicin (81 %), chloramphenicol (77 %), amoxycillin/clavulanic acid (69 %), piperacillin (65 %), neomycin (62 %) and tobramycin (54 %). The isolates were sensitive to vancomycin (100 %), gentamicin (96 %) and amikacin (88 %). Therefore, they were submitted to test them for sensitivity to SPEs-Ents. The strains showed the highest sensitivity to SPEs-Ents EK13, EF55, EF9a, 2019, 9296 and EHae466 (in the range 100–25,600 AU/mL; Table 1); the most sensitive strain to tested SPBs-enterocins was the strain PG4A. The PG1 strain appeared as the most resistant indicator strain.
Discussion The rabbits’ gastrointestinal tract is characterized by an abundant microflora, including anaerobic and aerobic species (including both the phyla Firmicutes and Bacteroides Monteils et al. 2008). While paenibacilli are widely distributed in water, soil, vegetable matter, insect larvae, etc., there are only few studies about Paenibacillus sp. isolated from animals—honeybee (Alippi et al. 2002) and catfish (de Castro et al. 2011). Moreover, there have been no previous reports of paenibacilli from rabbits. Paenibacilli in faeces and caecum of rabbits reached lower counts than Paenibacillus sp. isolated from soil content of rhizosphere (generally in range from log 3 to log 6 cells per gram of fresh weight; McSpadden Gardener 2004). To analyze the relationship of isolates, fingerprinting by MALDI-TOF MS was performed, with the aim to exclude the identical strains for further testing. Although, five bacterial
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Fig. 2 Phylogenetic tree of Paenibacillus lactis isolated from rabbits based on the protein mass fingerprinting analysis using the neighbour-joining method
pairs (PG1-PG4, PL5-PL5A, PL4-PL8, PL1-PL1A and PL2PL7) were found as identical on the basis of phylogenetic analysis; these strains showed different sensitivity to tested enterocins (Table 1). The isolated strains were also tested to physiological and biochemical features to determine resp. confirmed the similarity between them; details are given in Table 2. According to Bergey’s Manual (Garrity 2009), the most of isolates hydrolysed esculin, but not urea and arginine (except the P. cookii isolate; this genus usually shows negative results for arginine hydrolysis). As also presented in Table 2, P. glucanolyticus and P. lactis isolates (or the most of them) showed positive Table 2 Biochemical characteristics of Paenibacillus cookii, P. glucanolyticus and P. lactis isolates from rabbits P. cookii (1)
Hydrolysis of Esculin Urea Arginine Acid from Trehalose Lactose Sucrose Mannitol Arabinose Glycerol Fructose
P. glucanolyticus (9)
P. lactis (17)
+
−
+
−
+ − +
7 3 1
2 6 8
15 4 0
2 13 17
− + + − + + −
4 8 8 7 8 7 7
5 1 1 2 1 2 2
1 16 16 17 16 1 16
16 1 1 0 1 16 1
1, 9, 17—number of isolates
results for acid production from lactose, sucrose, arabinose, mannitol, glycerol and fructose—according to Bergey’s Manual. The biochemical characteristic of the P. cookii PC1 isolate is in agreement with the data published before (Scheldeman et al. 2004; Garrity 2009). These data demonstrated and confirmed the phenotypic features to the genus level. While the majority of P. glucanolyticus and P. lactis isolates showed negative results for acid production from trehalose, P. cookii was able to produce acid; these findings are contradictory to the results presented in previous literature. Different species of Paenibacillus seem to harbour resistance to different classes of antibiotics. The present Paenibacillus strains have shown resistance to the most of antibiotics; Pednekar et al. (2010) also isolated multi-drug resistant Paenibacillus sp. from soil. In accordance with our results, some studies presented resistance to oxytetracycline/ tetracycline in many species of Paenibacillus (Evans 2003, Pednekar et al. 2010); the others showed the opposite effect (Antúnez et al. 2007). In the past, vancomycin resistance has been also demonstrated by many species of Paenibacillus (Patel et al. 2000; Pednekar et al. 2010), in contrary to our findings. The sensitivity of paenibacilli to gentamicin—as it was confirmed in our study—was reported also in the other works (Antúnez et al. 2007). The dramatic increase of antibiotic-resistant bacteria and residua in products has evoked and has supported the research on new antimicrobials (plant extracts, bacteriocins, etc.) of natural origin. While the in vitro growth-inhibitory effect of plant extracts against Paenibacillus larvae is documented (Flesar et al. 2010), the lack of knowledge exists about paenibacilli testing to bacteriocins/enterocins. In this study, the spectrum of antibacterial activity of tested enterocins (Simonová and Lauková 2007; Pogány Simonová et al.
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2010) was spread to the representants of a new genus detected in rabbits—Paenibacillus sp. Moreover, the strains were more sensitive to natural antimicrobials—enterocins than to antibiotics; it seems to be a higher effectivity of enterocins to replace antibiotics for prevention/elimination of potential pathogens. These results provide us to novel information about sensitivity to antimicrobials (both synthetic and natural) of a new, till now non-studied species of Paenibacillus sp. in rabbits—as a new source of their occurrence.
Conclusions This study constitutes the first report of its kind as to the antimicrobial resistance and sensitivity to bacteriocin produced by enterococci, reported ever in a Paenibacillus sp. of rabbit origin. The most of strains were resistant to antibiotics, but sensitive to natural antimicrobials—enterocins. Outgoing from the fact that paenibacilli are able to produce antimicrobial compounds, Paenibacillus strains isolated from rabbits’ gastrointestinal tract are now available for further studies aiming their bacteriocin production and characterization, facilitating their potential use in rabbit husbandries as a new way to prevent microbial disorders. Acknowledgments This work was supported by the projects VEGA 2/0002/11 and 2/0004/14. We are grateful to Mrs. Margita Bodnárová for her skillful technical assistance and to Dr. V. Kmeť for the phylogenetic tree construction. Conflict of interest None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
References Alippi AM, López AC, Aguilar OM (2002) Differentiation of Paenibacillus larvae subsp. larvae, the cause of American foulbrood of honeybees, by using PCR and restriction fragment analysis of genes encoding 16S rRNA. Appl Environ Microbiol 68:3655–3660 Antúnez K, Piccini C, Castro-Sowinski S, Rosado AS, Seldin L, Zumino P (2007) Phenotypic and genotypic characterization of Paenibacillus larvae isolates. Vet Microbiol 124:178–183 Ash C, Priest PG, Collins MD (1993) Molecular identification of rRNA group 3 bacilli and Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie Van Leeuwenhoek 64:253–260
101 Bessède E, Angla-gre M, Delagarde Y, Sep Hieng S, Ménard A, Mégraud F (2011) Matrix assisted laser desorption/ionization BIOTYPER: experience in the routine of a University hospital. Clin Microbiol Infect 17:533–538 de Castro ALM, Vollú RE, Peixoto RS, Grigorevski-Lima AL, Coelho RRR, Bon EPS, Rosado AS, Seldin L (2011) Cellulolytic potential of a novel strain of Paenibacillus sp. isolated from the armored catfish Parotocinclus maculicauda gut. Brazil J Microbiol 42:1608–1615 Evans JD (2003) Diverse origins of tetracycline resistance in the honeybee pathogen Paenibacillus larvae. J Invertebr Pathol 83:46–50 Flesar J, Havlik J, Kloucek P, Rada V, Titera D, Bednar M, Stropnicky M, Kokoska L (2010) In vitro growth inhibitory effect of plant-derived extracts and compounds against Paenibacillus larvae and their acute oral toxicity to adult honey bees. Vet Microbiol 145:129–133 Garrity GM (2009) Bergey’s manual of systematic bacteriology: the Firmicutes. In: Whitman et al. (Eds.), Bergey’s Manual of Systematic Bacteriology. Vol. 3, Springer, New York, pp 269–295 Li NZ, Xia T, Xu YL, Qiu RR, Xiang H, He D, Peng YY (2012) Genome sequence of Paenibacillus strain Aloe-11, an endophytic bacterium with broad antimicrobial activity and intestinal colonization ability. J Bacteriol 194(8):2017–2018 McSpadden Gardener BB (2004) Ecology of Bacillus and Paenibacillus spp. in agricultural systems. Phytopathol 94:1252–1258 Monteils V, Cauquil L, Combes S, Godon JJ, Gidenne T (2008) Potential core species and satellite species in the bacterial community within the rabbit caecum. FEMS Microbiol Ecol 66:620–629 National Committee for Clinical Laboratory Standards (2003) Approved standard M2-A8. Performance standards for antimicrobial disk susceptibility tests, 8th ed. NCCLS, Wayne, Pa Ouyang J, Pei Z, Lutwick L, Dalal S, Yang L, Cassai N, Sandhu K, Hanna B, Wieczorek RL, Bluth M, Pincus MR (2008) Case report: Paenibacillus thiaminolyticus: a new cause of human infection, inducing bacteremia in a patient on hemodialysis. Ann Clin Lab Sci 38:393–400 Patel R, Piper K, Cockerill FR III, Steckelberg JM, Yousten AA (2000) The biopesticide Paenibacillus popilliae has a vancomycin resistance gene cluster homologous to the enterococcal VanA vancomycin resistance gene cluster. Antimicrob Agents Chemother 44:705–709 Pednekar PB, Jain R, Thakur NL, Mahajan GB (2010) Isolation of multidrug resistant Paenibacillus sp. from fertile soil: an imminent menace of spreading resistance. J Life Sci 4(5):7–12 Pogány Simonová M, Lauková A, Haviarová M (2010) Pseudomonads from rabbits and their sensitivity to antibiotics and natural antimicrobials. Res Vet Sci 88:203–220 Scheldeman P, Goossens K, Rodriguez-Diaz M, Pil A, Goris J, Herman L, De Vos P, Logan NA, Heyndrickx M (2004) Paenibacillus lactis sp. nov., isolated from raw and heat-treated milk. Int J Syst Evol Microbiol 54:885–891 Simonová M, Lauková A, Štyriak I (2005) Enterococci from rabbits— potential feed additive. Czech J Anim Sci 50(9):416–421 Simonová M, Lauková A (2007) Bacteriocin activity of enterococci from rabbits. Vet Res Comm 31:143–152 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetic analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739
Occurrence and characterization of Paenibacillus sp. isolated from rabbits Monika Pogány Simonová & Vladimír Kmeť & Andrea Lauková
Received: 6 March 2014 / Accepted: 4 September 2014 / Published online: 18 September 2014 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014
Introduction
Materials and methods
Paenibacillus, the representants of which previously belonged to the genus Bacillus, was re-classified as a separate genus in 1993 (Ash et al. 1993). The genus Paenibacillus includes Gram-positive, aerobic or facultative anaerobic, endospore-forming bacteria which have been isolated from a variety of environments—soil, water, rhizosphere, vegetable matter, forage and insect larvae, and clinical samples (McSpadden Gardener 2004; Ouyang et al. 2008). There is a rapidly growing interest in Paenibacillus sp. because of their production of various extracellular enzymes (polysaccharide-degrading enzymes and proteases) and antimicrobial substances against a wide spectrum of microorganisms, including pathogens (e.g. Clostridium botulinum), and the ability to enhance the immune function of animals with its polysaccharide (Li et al. 2012). Previous studies on rabbit caecal microflora revealed the great bacterial diversity of the ecosystem studied, including both the phyla Firmicutes and Bacteroides (Monteils et al. 2008). Up to now, we were more focused on Gram-positive faecal enterococci (Simonová et al. 2005). To our knowledge, there have been no previous reports of Paenibacillus sp. isolated from rabbits’ gastrointestinal tract. Because representants of the species Paenibacillus were determined in rabbits faeces, we decided to test their sensitivity to antibiotics and enterocins; it was not tested for this type of bacteriocins yet.
Isolation and identification of bacterial strains
M. P. Simonová (*) : V. Kmeť : A. Lauková Institute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovakia e-mail: [email protected]
The faecal and caecal samples from healthy rabbits (n=144, age 35–77 days, Hycole breed, various sex) were homogenized in Ringer buffer (Oxoid, UK) using stomacher, properly diluted and plated on plate count agar (Biomark, India). Plates were incubated aerobically at 37 °C for 24–48 h. The bacterial counts were expressed in log 10 of colony forming units per gram (log10 CFU/g±SD). After incubation, morphologically different colonies were picked up and re-streaked for several generations in order to isolate purified individual bacterial strains. Isolates were identified using protein “fingerprints” determined by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics MALDI Biotyper, USA) as described by Bessède et al. (2011), Gram staining and on the basis of colony characteristic. The phylogenetic trees were constructed using the neighbour-joining method with the software MEGA5 (Tamura et al. 2011) with the aim to select of suitable, not identical strain(s) with bacteriocin-like activity for further in vitro and in vivo testing. The BBL Gram-Positive ID Kit (Becton & Dickinson) was applied to determine additional physiological and biochemical features. Sensitivity of isolates to antibiotics and enterocins Antibiotic resistance of identified Paenibacillus strains (100 μL of an 18-h culture of each tested strain) was tested by the qualitative method—the agar disc diffusion method on Columbia agar (Becton & Dickinson, BD, USA) with 10 % of defibrinated sheep blood. The tests should be performed according to internationally recognized standards such as the National Committee for Clinical Laboratory Standards (NCCLS 2003). The following antibiotic discs (Oxoid, UK;
98
Folia Microbiol (2015) 60:97–101
Lach-Ner, Czech Republic) were used: amikacin (AK30 μg), amoxicillin/clavulanic acid (AMC30), ampicillin (AMP10), bacitracin (B2), chloramphenicol (C30), clindamycin (DA2), colistin (CL10), doxycycline (DOX30), erythromycin (ERY15), gentamicin (CN120), kanamycin (KAN30), linkomycin (L2), neomycin (N5), penicillin (PEN10), piperacillin (PIP100), rifampicin (RA30), streptomycin (S25), tetracycline (TE30), tobramycin (TOB10) and vancomycin (VAN30). After incubation at 37 °C for 18 h, the strains were classified as resistant or sensitive (according to the manufacturers’ instructions). The semi-purified enterocins (SPEs) were produced by strains listed in Table 1. They were isolated in our Laboratory of Animal Microbiology (Institute of Animal Physiology SAS, Košice, Slovakia) except Enterococcus haemoperoxidus
isolated in Czech Republic (provided by Dr. P. Švec, Czech Collection of Microorganisms, Masaryk University, Brno, Czech Republic). The SPEs were prepared by the following procedures: a 16-h culture (300 mL) of Enterococcus faecium CCM7420-EF2019, CCM7419-EK13, AL41, EF55, CCM4231, EF9a, EF9296 and E. haemoperoxidus EHae466 strains in MRS broth (Merck) were centrifuged for 30 min at 10,000g in order to remove the cells. After adjusting of supernatant to pH 5.0 (5.5 in the case of AL41 strain), ammonium sulphate was gently added to the supernatant to obtain 40 % (w/v) saturation, and the mixture was stirred at 4 °C for 2 h (EK13, EF9296, EF9a EHae466), for 4 h (EF2019) and for 24 h (EF55, CCM4231), at 21 °C for 1 h (AL41). After centrifugation at 10,000g for 30 min, the resulting pellet was resuspended in 10 mmol/L sodium phosphate buffer (pH 6.5).
Table 1 The susceptibility of Paenibacillus cookii, P. glucanolyticus and P. lactis isolates against semi-purified enterocins (SPEs-Ents; AU/mL) Isolates
Source
Semi-purified enterocins (SPEs) EK13
AL41
EF55
CCM4231
EF9a
EF9296
EF2019
EHae466
EA5 PC1 PL1 PL1A PL2 PL3 PL4 PL5 PL5A PL6 PL7 PL8 PL9 PL10 PL11
– Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Caecum Caecum Faeces Caecum Caecum
51,200 12,800 25,600 12,800 25,600 25,600 12,800 25,600 12,800 25,600 6400 25,600 1600 25,600 6400
3200 100 100 100 100 1600 – 6400 6400 100 200 1600 1600 200 3200
51,200 12,800 400 400 1600 25,600 200 12,800 12,800 25,600 12,800 25,600 3200 25,600 3200
100 200 100 – – – – 100 100 100 – 800 – – 100
25,600 12,800 25,600 25,600 1600 25,600 800 25,600 12,800 6400 800 25,600 200 12,800 200
25,600 12,800 12,800 800 6400 25,600 12,800 25,600 12,800 12,800 800 12,800 100 400 400
51,200 12,800 25,600 25,600 3200 25,600 12,800 25,600 12,800 1600 12,800 25,600 3200 800 1600
25,600 12,800 6400 12,800 100 6400 100 100 800 25,600 12,800 6400 3200 25,600 800
PL12 PL13 PL14 PL14A PG1 PG2 PG3 PG4 PG4A PG4B PG5 PG6
Caecum Faeces Faeces Faeces Caecum Caecum Caecum Faeces Faeces Faeces Faeces Faeces
3200 25,600 400 6400 100 25,600 25,600 25,600 25,600 12,800 3200 100
400 6400 400 200 100 – 200 100 800 800 1600 100
800 25,600 400 25,600 100 25,600 25,600 25,600 25,600 25,600 6400 400
– 100 100 – – – – – 100 100 800 –
400 25,600 100 25,600 100 25,600 25,600 25,600 25,600 25,600 6400 800
800 200 25,600 25,600 100 200 6400 25,600 25,600 12,800 3200 100
25,600 25,600 25,600 25,600 100 1600 25,600 25,600 25,600 25,600 3200 12,800
200 25,600 25,600 6400 100 25,600 25,600 25,600 25,600 25,600 6400 200
AU/mL arbitrary units per millilitre of culture medium Enterococcus avium—control indicator strain, EK13 (produced by E. faecium EK13-CCM7419), AL41 (E. faecium AL41), EF55 (E. faecium EF55), CCM4231 (E. faecium CCM4231), EF9a (E. faecium EF9a), 9296 (E. faecium 9296), EF2019 (E. faecium EF2019-CCM7420), EHae466 (E. haemoperoxidus EH466)
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The antimicrobial titer of SPE was defined as the reciprocal of the highest twofold dilution producing a distinct inhibition of the inhibitor lawn, expressed in arbitrary units per mL (AU/ mL) of culture medium. Enterococcus avium EA5 (our isolate from piglet) was used as a bacteriocin-sensitive indicator strain (the amount of 200 μL of an 18-h culture of each indicator strain) to determine bacteriocin activity levels; the bacteriocin activity of SPEs produced by different strains varied between 1600 and 25,600 AU/mL.
Results The total counts of Paenibacillus sp. in faeces of rabbits varied from 1.33 to 3.74 log CFU/mL/g (detected in 30 mixture samples); the bacterial counts in caecum were lower (from 1.39 to 1.99 log CFU/mL/g); they occurred only in several samples (18 samples). The isolates were allotted to the species Paenibacillus glucanolyticus (9; PG1-4, 4A, 4B, 5, 6), Paenibacillus lactis (17; PL1, 1A, 2–5, 5A, 6–14, 14A) and Paenibacillus cooki (PC1) by MALDI-TOF. As demonstrated in Figs. 1 and 2, the isolate pairs PG1-PG4, PL5-PL5A, PL4PL8, PL1-PL1A and PL2-PL7 formed a tight cluster on the basis of nearly identical mass spectra and could be marked as identical strains. Considering the results obtained using the BBL GramPositive ID Kit, they were in accordance with those in the Bergey’s Manual (2009); acid is produced from lactose, sucrose, mannitol (except P. cookii isolate), arabinose, glycerol (except P. lactis strains), fructose (except P. cookii isolate) and trehalose (except P. lactis isolates; Table 2). The most of isolates hydrolyzed esculin, but not urea and arginine. Fig. 1 Phylogenetic tree of Paenibacillus glucanolyticus isolates from rabbits based on the protein mass fingerprinting analysis using the neighbourjoining method
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All isolates were resistant to colistin. The most of strains showed resistance to bacitracin, clindamycin, erythromycin, linkomycin, streptomycin (96 %), penicillin (92 %), tetracycline (88 %), ampicillin, doxycycline, kanamycin, rifampicin (81 %), chloramphenicol (77 %), amoxycillin/clavulanic acid (69 %), piperacillin (65 %), neomycin (62 %) and tobramycin (54 %). The isolates were sensitive to vancomycin (100 %), gentamicin (96 %) and amikacin (88 %). Therefore, they were submitted to test them for sensitivity to SPEs-Ents. The strains showed the highest sensitivity to SPEs-Ents EK13, EF55, EF9a, 2019, 9296 and EHae466 (in the range 100–25,600 AU/mL; Table 1); the most sensitive strain to tested SPBs-enterocins was the strain PG4A. The PG1 strain appeared as the most resistant indicator strain.
Discussion The rabbits’ gastrointestinal tract is characterized by an abundant microflora, including anaerobic and aerobic species (including both the phyla Firmicutes and Bacteroides Monteils et al. 2008). While paenibacilli are widely distributed in water, soil, vegetable matter, insect larvae, etc., there are only few studies about Paenibacillus sp. isolated from animals—honeybee (Alippi et al. 2002) and catfish (de Castro et al. 2011). Moreover, there have been no previous reports of paenibacilli from rabbits. Paenibacilli in faeces and caecum of rabbits reached lower counts than Paenibacillus sp. isolated from soil content of rhizosphere (generally in range from log 3 to log 6 cells per gram of fresh weight; McSpadden Gardener 2004). To analyze the relationship of isolates, fingerprinting by MALDI-TOF MS was performed, with the aim to exclude the identical strains for further testing. Although, five bacterial
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Fig. 2 Phylogenetic tree of Paenibacillus lactis isolated from rabbits based on the protein mass fingerprinting analysis using the neighbour-joining method
pairs (PG1-PG4, PL5-PL5A, PL4-PL8, PL1-PL1A and PL2PL7) were found as identical on the basis of phylogenetic analysis; these strains showed different sensitivity to tested enterocins (Table 1). The isolated strains were also tested to physiological and biochemical features to determine resp. confirmed the similarity between them; details are given in Table 2. According to Bergey’s Manual (Garrity 2009), the most of isolates hydrolysed esculin, but not urea and arginine (except the P. cookii isolate; this genus usually shows negative results for arginine hydrolysis). As also presented in Table 2, P. glucanolyticus and P. lactis isolates (or the most of them) showed positive Table 2 Biochemical characteristics of Paenibacillus cookii, P. glucanolyticus and P. lactis isolates from rabbits P. cookii (1)
Hydrolysis of Esculin Urea Arginine Acid from Trehalose Lactose Sucrose Mannitol Arabinose Glycerol Fructose
P. glucanolyticus (9)
P. lactis (17)
+
−
+
−
+ − +
7 3 1
2 6 8
15 4 0
2 13 17
− + + − + + −
4 8 8 7 8 7 7
5 1 1 2 1 2 2
1 16 16 17 16 1 16
16 1 1 0 1 16 1
1, 9, 17—number of isolates
results for acid production from lactose, sucrose, arabinose, mannitol, glycerol and fructose—according to Bergey’s Manual. The biochemical characteristic of the P. cookii PC1 isolate is in agreement with the data published before (Scheldeman et al. 2004; Garrity 2009). These data demonstrated and confirmed the phenotypic features to the genus level. While the majority of P. glucanolyticus and P. lactis isolates showed negative results for acid production from trehalose, P. cookii was able to produce acid; these findings are contradictory to the results presented in previous literature. Different species of Paenibacillus seem to harbour resistance to different classes of antibiotics. The present Paenibacillus strains have shown resistance to the most of antibiotics; Pednekar et al. (2010) also isolated multi-drug resistant Paenibacillus sp. from soil. In accordance with our results, some studies presented resistance to oxytetracycline/ tetracycline in many species of Paenibacillus (Evans 2003, Pednekar et al. 2010); the others showed the opposite effect (Antúnez et al. 2007). In the past, vancomycin resistance has been also demonstrated by many species of Paenibacillus (Patel et al. 2000; Pednekar et al. 2010), in contrary to our findings. The sensitivity of paenibacilli to gentamicin—as it was confirmed in our study—was reported also in the other works (Antúnez et al. 2007). The dramatic increase of antibiotic-resistant bacteria and residua in products has evoked and has supported the research on new antimicrobials (plant extracts, bacteriocins, etc.) of natural origin. While the in vitro growth-inhibitory effect of plant extracts against Paenibacillus larvae is documented (Flesar et al. 2010), the lack of knowledge exists about paenibacilli testing to bacteriocins/enterocins. In this study, the spectrum of antibacterial activity of tested enterocins (Simonová and Lauková 2007; Pogány Simonová et al.
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2010) was spread to the representants of a new genus detected in rabbits—Paenibacillus sp. Moreover, the strains were more sensitive to natural antimicrobials—enterocins than to antibiotics; it seems to be a higher effectivity of enterocins to replace antibiotics for prevention/elimination of potential pathogens. These results provide us to novel information about sensitivity to antimicrobials (both synthetic and natural) of a new, till now non-studied species of Paenibacillus sp. in rabbits—as a new source of their occurrence.
Conclusions This study constitutes the first report of its kind as to the antimicrobial resistance and sensitivity to bacteriocin produced by enterococci, reported ever in a Paenibacillus sp. of rabbit origin. The most of strains were resistant to antibiotics, but sensitive to natural antimicrobials—enterocins. Outgoing from the fact that paenibacilli are able to produce antimicrobial compounds, Paenibacillus strains isolated from rabbits’ gastrointestinal tract are now available for further studies aiming their bacteriocin production and characterization, facilitating their potential use in rabbit husbandries as a new way to prevent microbial disorders. Acknowledgments This work was supported by the projects VEGA 2/0002/11 and 2/0004/14. We are grateful to Mrs. Margita Bodnárová for her skillful technical assistance and to Dr. V. Kmeť for the phylogenetic tree construction. Conflict of interest None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
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