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- Materials and methods
Aims: Paenibacillus isolates were selected to test antimicrobial activity against bacteria, filamentous fungi and yeasts isolates, with the purpose of finding new bacterium species for microbiological control.
Methods and Results: Fifty-five strains belonging to 15 species of Paenibacillus were inoculated on trypticase soya agar, potato dextrose agar and sabouraud agar plates in order to evaluate their antimicrobial activity against 16 indicator bacteria, 14 filamentous fungi and six yeasts isolates, both reference and field strains. After these screening, culture supernatant of 17 isolates was prepared. Twenty-five Paenibacillus isolates presented antimicrobial activity, where seven species (Paenibacillus chibensis; P. koreensis; P. illinoiensis; P. validu; P. pabuli; P. brasilensis and P. peoriae) stood out inhibiting at least 13 of the 16 indicator bacteria. Only 14 of the 55 isolates exhibited antifungal activity. P. peoriae inhibited 13 among the 14 filamentous fungi and all yeasts indicator strains. Fourteen isolates produced culture supernatant with antimicrobial activity.
Conclusions: Among the 55 isolates analysed, 25 exhibited a broad inhibition spectrum against bacteria and pathogenic fungi. P. validus, P. chibensis, P. koreensis and P. peoriae isolates proved to be the subject matter for studies on the production of antimicrobial agents.
Significance and Impact of the Study: The present study revealed other two species with antimicrobial activity: P. validus and P. chibensis, and it contributed to enhance Paenibacillus biocontrolling potential, proving that it exhibit a broad action spectrum.
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- Materials and methods
The Paenibacillus genus is a Gram-positive, aerobic or facultative anaerobic rod-shaped, endospore-forming bacteria. Species of different nutritional requirements, growth conditions, metabolism and DNA structure constitute the genus. Paenibacillus strains have been detected in a variety of environments such as: soil (Berge et al. 2002), water, rhizosphere (Silveira 2003), vegetable matter, foods, tree roots, forage and insect larvae (Daane et al. 2002), as well as in hospital clinical material (Bosshard et al. 2002).
Yet, the main characteristic of the group is the secretion of extracellular enzymes in neutral and alkaline growth conditions (Meehan et al. 2001), some Paenibacillus species are also able to produce polysaccharides, amino acids and secondary metabolites such as antibiotic agents, pigments, toxins, side by side with inductors of competition and symbiosis, enzyme inhibitors, pheromones and promoters of vegetable and animal growth (Yoon et al. 2002; Dasman et al. 2003; Yoon et al. 2003).
Therefore, with the purpose of finding new bacterium species that may be used in microbiological control, the objective of this study was to characterize 55 Paenibacillus isolates, belonging to 15 different species, from environment (soil and water), for their antimicrobial activity against pathogenic filamentous fungi, yeasts and bacteria commonly found in humans, plants and animals.
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All 55 isolates of Paenibacillus were tested against 16 Gram-positive, -negative, reference strains, animal and plant pathogens, and field isolates of indicator bacteria. Out of these, 25 exhibited antibiosis, inhibiting at least one of the 16 indicator bacteria (Table 2). Ten isolates stood out inhibiting at least 13 of the 16 indicator bacteria in at least one of the experimental periods used (P. chibensis 17b and 12; P. koreensis 32M. P. illinoiensis 23M; P. validus 112,10M, and 191; P. pabuli 24 mar; P. brasilensis 16; and P. peoriae 57). Pseudomonas aeruginosa was not inhibited by any of the Paenibacillus isolates. The growth of Pseudomonas putida, Burkholderia cepacia, Ralstonia solanacearum, Citrobacter freundii and Salmonella Typhimurium was inhibited, but weakly, with average inhibition zones around 5 and 12 mm. The growth of Pectobacterium carotovorum brasiliensis, Staphylococcus aureus, Listeria monocytogenes and L. innocua was strongly inhibited, showing the average inhibition zones larger than 19 mm. Paenibacillus validus isolates had the best performance, as 10 of the 11 isolates tested inhibited the growth of the majority of the indicator bacteria at the three incubation periods used. The P. koreensis isolate (32M) presented a good antibiotic activity, inhibiting 14 of the 16 indicator bacteria used in the experiment. Paenibacillus peoriae, with one isolate only (57), was able to inhibit the growth of 15 of the 16 indicator bacteria (Table 2). Paenibacillus glucanolyticus, P. thiaminolyticus, P. borealis, P. alginolyticus and P. apiarius isolates did not present any antibiotic effect against indicator bacteria at any of the three incubation periods tested. Yet, in the light of the small number of isolates tested, this result is not to be considered statistically significant. No significant difference (P < 0,05) was observed for the three incubation periods used. This observation reinforces that a 24-h incubation period would be enough to secure a good antibiotic action, because at the end of this period the culture is at an early stationary stage, with considerable production of active metabolites.
Table 2. Antimicrobial activity of Paenibacillus isolates, which inhibit the indicator bacteria (15) in at least one incubation period
|P. validus 4IN||−#||−||−||−||−||−||−||−||−||−||−||+||−||++||−|
|Paenibacillus validus 172||−||++||+||++||+++||−||+||++||+||+++||−||++||++||+++||+++|
|P. validus 112||++||++||+||++||+||−||+||++||++||++||+++||++||++||+++||++|
|P. validus 10M||++||+++||+||++||++||+++||++||+++||++||+++||+++||+++||+++||−||+++|
|P. validus 191||++||++||++||++||+||++||++||++||++||+++||+++||++||++||+++||+++|
|P. validus 111||−||+++||++||++||++||−||++||+++||+++||+++||−||++||++||−||−|
|P. validus 168||−||+++||++||++||++||−||+++||+++||++||+++||−||+++||+++||++||−|
|P. validus 169||++||−||−||−||−||−||−||−||−||−||+++||−||−||++||+++|
|P. azotofixans 189||−||−||−||−||−||−||−||−||−||−||−||−||+||++||+|
|P. chibensis 23||−||−||−||++||++||++||−||+++||−||+++||++||++||++||+++||+++|
|P. chibensis 17b||−||+||+||++||++||++||++||++||++||++||++||++||++||+||+++|
|P. pabuli 24M||−||+||++||++||++||+++||++||++||++||++||+++||++||+++||++||++|
|P. glucanolyticus 11||−||−||−||−||−||−||−||−||−||−||−||−||−||−||++|
|P. illinoiensis 29||−||−||−||−||−||++||−||−||+||−||−||+||+||++||++|
|P. illinoiensis 23M||+||++||+||++||++||+||+||+++||+||−||++||++||++||++||++|
|P. koreensis 32M||++||++||+||++||++||+||++||++||++||++||+++||++||++||−||+++|
|P. brasiliensis 16||+||−||−||++||++||++||+||+++||++||++||+++||++||++||++||+++|
|P. validus 44||++||++||+||−||−||−||+||−||+++||+||++||−||+||−||++|
|P. validus 40||−||−||−||−||−||+++||−||++||++||++||++||+++||++||++||+|
|P. validus 4||−||−||−||−||−||++||−||−||−||+||++||+||−||+||++|
|P. chibensis 12||++||++||+||+++||++||+++||++||+++||++||++||+++||++||+++||+++||+++|
|P. azotofixans 43||−||−||−||−||−||−||−||−||−||−||−||+||−||++||+|
|P. azotofixans 34||++||+||+||−||−||−||+||−||−||+||++||−||+||+||+|
|P. azotofixans 45||++||−||−||−||−||+++||−||−||+++||−||++||++||+||+++||++|
|P. peoriae 57||++||+++||++||+++||++||+++||++||+++||+++||++||++||+++||+++||+++||+++|
Generally speaking, Gram-positive and -negative bacteria were susceptible. Xanthomonas anoxopodis was the most susceptible indicator bacterium, being inhibited by 22 of the 55 Paenibacillus isolates tested. Pectobacterium carotovorum ssp. carotovorum, Staph. aureus and Pseudomonas fluorescens were inhibited by 20 Paenibacillus isolates, followed by L. innocua and Escherichia coli, which were inhibited by 17 isolates. Among the indicator bacteria that least suffered inhibition by Paenibacillus, there were: Cit. freundii, inhibited by only 12 isolates, followed by L. monocytogenes, Enterobacter cloacae, Ps. putida and Salm. Typhimurium, inhibited by 14 isolates (Table 2).
Among the 55 isolates tested, 24 (44%) exhibited inhibitory activity against filamentous fungi, with average inhibition zones varying between 12 and 32 mm. Once more, P. validus and P. chibensis isolates stood out, as five of the nine P. validus isolates and three of the four P. chibensis isolates exhibited antibiosis to at least 13 out of the 14 indicators strains.
Paenibacillus validus (isolates 191, 10M, 112, 172 and 4). P. illinoiensis (isolate 23M) and P. brasiliensis (isolate 16) were also active against all the tested indicator fungi strains (Table 3). Only 10 (P. validus 4, 10M,168,191; P. chibensis 17b,23; P. glucanolyticus 19; P. illinoisensis 23M, P. koreensis 32M and P. peoriae 57) of the 55 isolates tested (18%) were effective against reference strains and clinical isolates of Candida spp. The strain C. guiliermondii (L 31) was only inhibited by isolate 57 (P. peoriae), which in turn inhibited the growth of all yeasts used, with average inhibition zones varying between 12 and 20 mm. The remaining 45 isolates did not inhibit any of the Candida spp. strains (Table 4).
Table 3. Inhibitory activity of Paenibacillus spp. isolates against filamentous fungi
| || ||Aspergillus niger||A. fumigatus||A. oryzae||Fusarium solani||Bipolaris oryzae||Bipolaris sorokiniana||Field strains#|
| 191||Paenibacillus validus||++*||++||+++||+++||+++||+++||+++||+++||+++||+++||+||++||+||+|
| 10 m||P. validus||++||++||+||++||+++||+++||+++||+++||+++||+++||+||+++||++||+|
| 112||P. validus||+||+||++||++||++||++||+++||+++||+++||+++||+||++||++||+|
| 172||P. validus||+++||+++||++||+++||+++||+++||+++||+++||+++||++||+||++||+||+|
| 17b||P. chibensis||+++||++||++||+++||+++||+++||+++||++||+++||+++||+||++||+||++|
| 23||P. chibensis||+||++||++||++||++||+++||+++||++||+++||+++||+||+||++||+|
| 23M||P. illinoiensis||+++||+++||++||++||+||+++||++||+++||+++||+++||+||+||+||+|
| 16||P. brasilensis||++||++||++||++||+++||+++||+++||+||+++||+++||+||+++||+||+|
| 4||P. validus||+++||+++||+++||+++||−||+++||+++||+++||+++||+++||+||++||++||++|
| 12||P. chibensis||++||+++||+++||+++||−||+++||+++||+++||+++||++||+||+||+||++|
| 57||P. peoriae||+||+||++||−||+||++||++||++||++||++||+||+||+||+|
Table 4. Inhibitory activity of Paenibacillus spp. isolates against Candida spp.
| || ||C. albicans 1||C. parapsilopsis 2||C. tropicalis 2||L30#||L31||L33|
| 191||Paenibacillus validus||++*||++||++||−||−||−|
| 10 m||P. validus||++||++||++||+||−||−|
| 168||P. validus||++||+||++||+++||−||−|
| 17b||P. chibensis||++||+||++||++||−||−|
| 23||P. chibensis||++||−||−||−||−||−|
| 19||P. glucanolyticus||++||++||++||++||−||−|
| 23 m||P. illinoiensis||+++||++||+++||−||−||−|
| 32 m||P. koreensis||++||−||−||++||−||−|
| 4||P. validus||++||+||+||−||−||+++|
| 57||P. peoriae||+||+||+||+++||+||+++|
The statistical analysis, using the nonparametric Kruskal–Wallis test, has revealed that the group formed by isolates 191, 10 m, 112, 172, 17b, 23, 23 m and 16 did not statistically differ when compared with one another. However, as a group, it exhibited a higher inhibition when compared with other isolates (P < 0·05). As regards the Paenibacillus soil isolates, isolates 4, 12 and 57, were not statistically different among one another (P < 0·05), however, as a group exhibited a greater inhibition when compared with other isolates. The nonparametric Mann–Whitney test has revealed that water Paenibacillus isolates presented higher antibiotic action when compared with soil isolates (P < 0·05).
After the triage of the isolates that produced antibiotic compounds, 17 isolates were chosen (12 water isolates and 5 soil isolates), among those that stood out inhibiting great part of the indicator micro-organisms (data not shown), to test their culture supernatant for antibiosis: eight P. validus (172, 112, 10M, 191, 111, 168, 40 and 4), four P. chibensis (17b, 23, 12 and 18), one P. koreensis (32M), one P. pabuli (24M), one P. brasiliensis (16) and the P. peoriae isolate (57).
Except for isolates 24M, 18 and 57, which did not produce active supernatants, all other isolates produced supernatants that exhibited antibiotic activity against indicator bacteria. The most sensitive indicator bacteria were L. innocua and Ps. fluorescens. The species Ps. putida, Shigella sonnei, B. cepacia, P. carotovorum and Salm. Typhimurium were inhibited by at least three supernatants. The results obtained with the filamentous fungi strains indicated an excellent inhibition action by isolate 23 (P. chibensis), in which supernatant was efficient against all fungi tested. Isolates 16, 10M and 168 exhibited some degree of antibiotic activity, inhibiting the growth of the fungi tested. The remaining Paenibacillus isolates did not produce supernatants that were active against the fungi used. None of the soil isolates that had previously inhibited filamentous fungi growth was, at this time, able to produce active supernatants against them.
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The Paenibacillus isolates tested in the present study were chosen because of their antagonistic action against a wide range of micro-organisms (Katz and Demain 1977; Nielsen and Sorensen 1997; Piuri et al. 1998; Dijksterhuis et al. 1999; Seldin et al. 1999; Beatty and Jensen 2002; von der Weid et al. 2003). Antibiotic peptides are produced by many species of micro-organisms, including Gram-positive bacteria (Emmert and Handelsman 1999). A large number of these metabolites are produced under nutritional stress, and the build-up of these substances may be observed in steady-state cultures (Martin and Demain 1980), which is the case of Paenibacillus after 24-h incubation. Previous studies have shown that antibiotic action may also bear a connection with sporulation, which starts during the steady-state growth phase (Beatty and Jensen 2002). von der Weid et al. (2003) carried out a triage study with one P. peoriae isolate NRRL BD-62 to test antibiosis against E. coli, Rals. solanacearum, Erwinia carotovora ssp. carotovora and B. cepacia among other species. The results found were similar to those here presented; that is, the bacterial strains mentioned had their growth inhibited by Paenibacillus. Girardin et al. (2002) observed a strong antibiotic action of Paenibacillus polymyxa against Clostridium botulinum isolates obtained from food samples. Seldin et al. (1999) tested a P. polymyxa strain against several micro-organisms pathogenic to humans and obtained a broad spectrum of antibiotic action. Among the organisms tested was a C. albicans strain, isolated from a patient suffering from mucocandidiasis. This strain was inhibited, which also was observed in the present study, as all Candida strains obtained from clinical isolates (C. albicans 1, C. tropicalis 2, C. parapsilopsis 1) were inhibited by Paenibacillus.
The antibiotic action exhibited by the culture supernatant is explained by the release of bacterial metabolites into the culture medium, which was observed in 14 of the 17 supernatants tested. When the cell was removed, no antibiosis was observed for the three remaining isolates, suggesting that the inhibition activity, exhibited during the initial triage, may be imputed to metabolites linked to the cell membrane or else to the production of extracellular enzymes such as chitinases, β-glycanases or proteases (Mavingui and Heulin 1994; Budi et al. 2000). It is important to mention that P. peoriae isolate (57) raised interest because of the excellent antibiotic action exhibited during the initial triage, with the cultures. Similar results were also arrived at by von der Weid et al. (2003) in a study that analysed one P. peoriae NRRL BD-62 under the same experimental conditions used in the present study. Nevertheless, the species did not exhibit the same antagonistic activity of the supernatant, as in our study. This is an indication that P. peoriae 57 may be a worthy subject matter for further enzymes purification studies, given the broad spectrum of antibiotic action against indicator micro-organisms.
Smith et al. (1993) observed that the action of Bacillus UW85 in decreasing the incidence of pumpkin rot by Phytophtora spp. was more intense when the cell was present in the culture medium. Budi et al. (2000) observed that one Paenibacillus strain and its metabolites suppressed mycelial growth, production of sporangia and elongation of the germination tube in P. parasitica, a phytopathogenic fungus. This observation is important, given that such effects interrupt the life cycle of the pathogen. Apart from this, the Paenibacillus strain also reduced the growth of other pathogenic fungi of the Fusarium genus, pointing to this genus to have a broad spectrum of antagonistic activity.
Among the 55 isolates analysed in the present study, 25 exhibited a broad inhibition spectrum against Gram-positive and -negative bacteria and pathogenic fungi. Paenibacillus validus, P. chibensis, P. koreensis and P. peoriae isolates were proved to be sound subject matter for studies on the production of antimicrobial agents. This is justified by the fact that the micro-organisms listed above inhibited 93% of the indicator bacteria and 100% of the fungi tested. Most of the work on Paenibacillus antimicrobial activity is based on P. polymyxa (Nielsen and Sorensen 1997; Piuri et al. 1998; Dijksterhuis et al. 1999; Seldin et al. 1999; Beatty and Jensen 2002) P. koreensis (Chung et al. 2000) and P. peoriae (von der Weid et al. 2003) isolates, the present study revealed other two species: P. validus and P. chibensis with this activity. Additional studies as the biochemical characterization of supernatants produced by the isolates that best inhibited growth would be the next step, because of an extensive array of compounds that are commercially interesting to the pharmaceutical industry, and that may be used as biocontrollers against field diseases.