Journal of Applied Bacteriology 1992, 73. 497-500
Range of action and genetic bacteriocin codification of Pseudomonas aeruginosa isolated from three different ecological niches C. Padilla, M. Salazar and 0. Faundez Department of Biological Sciences, University of Talca, Chile 4099/01/92: accepted 15 May 1992
c . PADILLA, M. S A L A Z A R AND 0.FAUNDEZ. 1992. Strains of Pseudomonas aeruginosa isolated from sediments in wells showed a greater bacteriocinogenic activity in those isolated from river and clinical specimens. More than half of all the strains examined had extrachromosomal DNA. Plasmid D N A was extracted from all the strains and in only 24/120, all from different origins, was the curing achieved ; all these strains coded their bacteriocins in the chromosomal DNA. INTRODUCTION
Bacteriocins are antibacterial proteins produced by bacteria (Konisky 1982). They may confer a selective advantage by killing other bacteria which are in competition with those that produce them (Farkas-Himsley 1980). Bacteriocins have been used for typing micro-organisms (De Vicente et al. 1990). In addition, they have been considered as a possible means of controlling pathogenic bacteria in the mouth as well as enteropathogens in foods and polluted water (Russell & Tagg 1981; Nielson et al. 1990; Padilla et al. 1990). One of the most important producers of bacteriocins is Pseudomonas aeruginosa, the bacteriocinogenic properties of which are widely recognized (Fyfe et al. 1984). In view of the ubiquitous nature of Ps. aeruginosa, its ability to tolerate unfavourable environmental conditions, its bacteriocinogenic capacity, and having regard to the potential use of the bacteriocins, we studied the bacteriocinogenic range of action and bactericidal potency. We also examined the genetic codification of bacteriocins in strains isolated from clinical samples, rivers and sediments of water wells. MATERIALS AND METHODS Bacterial stralns
One hundred and twenty strains of Ps..aeruginosa were isolated from clinical specimens by conventional methods. A further 88 strains were obtained from 10 different sites along the Claro River in Chile, as described by De Vicente Correspondence to :Dr C. Padilla, Department of Biological Sciences, University of Talca, Talca, Chile.
et al. (1990), and 63 were isolated from sediments of wells in the rural area in the 7th Region of Chile. In addition, several abacteriocinogenic organisms isolated in our laboratory were used as target strains: Corynebacterium xerosis, Bacillus subtilis, Streptococcus pyogenes, Staphylococcus epidermidis, enterotoxigenic Escherichia coli (CKWS), Shigella Jexneri (CT 152), Salmonella typhi (CK 098), Salm. paratyphi B and Klebsiella pneumoniae. Reference strains used to control the presence of plasmids were E. coli pBR-322 and pUC-19. Cultivation and identificationof the bacteria
The organisms from river samples were isolated by a membrane filtration technique and the strains were reisolated on MacConkey (Difco) and trypticase soy agar (Difco). The sediment samples were diluted 1 : 10 in sterile water and 0.1 ml was plated on MacConkey agar and on trypticase soy agar. These cultures were incubated at 28°C for 24 h. The same media were used for the clinical specimens. All the strains of Ps. aeruginosa were identified biochemically by conventional tests, following the identification criteria described by Elliot et al. (1981). Detectlon of inhlbitory activity
The technique used was a modification of that described by Bagliano & Hindsill (1970) and later performed according to that of Padilla et al. (1990). Obtention of piasmidial DNA
The extrachromosomal DNA was obtained according to the technique described by Birnboim et al. (1979).
498 C. PADILLA ET A L .
Table 1 Pseudornonas aeruginosa with bacteriocinogenic activity isolated from
Strains of Ps. aeruginosa Ps. aeruginosa from different origins
Total NO.
Well sediment Rivers
63 88 120
Clinical specimens
Bacteriocinogenic
Abacteriocinogenic
%
NO.
%
No.
%
100 100 100
61 41 79
96.8 46.5 65.8
2 47 41
3-1 53-4 34.1
Ellmlnatlon of extrachromosomal DNA
To ascertain if the coding genes of the bacteriocins were contained on a plasmid or were chromosomal, the extrachromosomal DNA was eliminated by a modified version of the procedure of Watanabe 81 Fukasawa (1961). Later, the method was that of Padilla et al. (1990). RESULTS AND DISCUSSION
In ecological terms, it is important that the bacteriocinogenicity in Ps. aeruginosa was observed more frequently in isolates of sediment of wells in comparison with others (Table 1). These results contrasted, in part, with those of Govan (1986) who found that the bacteriocinogenicity in strains of Ps. aeruginosa, was more frequent in clinical than in environmental strains. Table 2 shows that Ps. aeruginosa from sediments produced larger average diameter inhibition zones (haloes) of Gram-positive and Gram-negative bacteria as compared with Ps. aeruginosa strains isolated from rivers and clinical material. The table also shows that the average diameter of inhibition haloes in Gram-negative organisms are larger than these of Gram-positive bacteria. Also of interest is the variation in the susceptibility of target strains to different bacteriocins (Fig. 1). Table 3 shows the strains of Ps. aeruginosa with a capacity to inhibit more than one target strain. Nine strains from well sediments produced bacteriocins with a wide spectrum of activity and more than one bacteriocin can be manufactured by one strain. On the other
different environments
hand, five river and three clinical strains each possessed a narrow spectrum bacteriocin. Only strains P3 and P54 showed a capacity to inhibit the growth of three different target strains. It was also observed that Kl. pneumoniae was the most sensitive bacterium to different bacteriocins, whereas B. subtilis was resistant to all of them. The extraction of plasmid DNA indicated that 76% of all the microorganisms studied showed an extrachromosoma1 DNA of different sizes (Fig. 2). The plasmid DWA elimination in 12 strains from well sediments, 12 from rivers and 12 from clinical material showed that all maintained the capacity to manufacture bacteriocins. This indicates that information for bacteriocins in these micro-organisms is coded in the chromosomal DNA. I t was very interesting to observe that strains of P5. aeruginosa isolated from sediments had the most bacteriocinogenic activity. The reason could be natural selection in the habitants of an almost closed ecological niche. In the rural areas of developing countries the water for human consumption is highly contaminated with enteropathogenic bacilli (Valenzuela & Peralta 1987). The large
Table 2 Average diameter of haloes indications of
bacteriocinogenic inhibition of Pseudomonas aeruginosa isolated from different environments against Gram-negative and positive target strains
Sources of strains of Ps. aeruginosa River
Well sediment
Target strains
Inhibition zones (mm)
Gram-negative Gram-positive
12.8
21-7
9.7
16-4
Clinical
18.2 11.8
Fig. 1. Production of haloes of inhibition of Pseudomonas aeruginosa from sediment (P 18), river (R4) and clinical sample (H31), when overland with ShigellaJexneri (CT 152)
BACTERIOCIN CODIFICATION OF P S . AERUGlNOSA 499
Table 3 Pseudomonas aeruginosa from
Target strains inhibited for bacteriocins
different origins producing bacteriocins of a wide range of action
Ps. aeruginosa strain
Origin
Ec
Sf
St
Kp
p9 p2 1 PI I p25 p3 p4 p39 p54
Sediment Sediment Sediment Sediment Sediment Sediment Sediment Sediment Sediment River River River River River Clinical Clinical Clinical
o + + o o + o + o + o o + o o o +
o + + o o + + + o + + o o o o o o
+ o o + + o o o o o o o o + + o o
+ o o o + o + + o o + o + + + o +
p60
R2 1
R4 R50
R4 1 R63
H, H68
Bs o o o o o o o o o o o o o o o o o
Sp o o o o o o o o + o o o o o o + o
Se o o o o o o o o + o o + o o o o o
Cx o o o + + o o o o o o + o o o + o
activity; +, bacteriocin activity; Ec, enterotoxigenic Escherichia coli (CK 041); Sf, Shigella jexncri (CT 152); St, Salmonella typhi (CK 098); Kp, Klebsiella pneumoniae; Bs, Bacillus subtilis; Sp, Streptococcus pyogenes; Se, Staphylococcus epidermidis ; Cx, Corynebacterium xerosis.
0,No
I
2
3
4
5
6
7
8
9
10
II
number of pathogens that occur in water and sediments, along with the increasing usage of recreational waters, creates a potential health hazard (Burton et al. 1987). The inhibitory properties of bacteriocins isolated from strains of some Ps. aeruginosa obtained from sediments is an interesting phenomena and we think it is a basis for biotechnological studies in the biological decontamination of water. I n summary, we suggest that bacteriocins might be used to control pathogen microorganisms in water. Initial unpublished observations in artificial water systems indicate promising results.
REFERENCES
Fig. 2 Extraction of plasmid DNA of Pseudomonas aeruginosa
from different sources (left to right). Ps. aeruginosa from sediment: line 1 (P43), line 2 (P21), line 3 (P36), line 4 (P7); Ps. aeruginosa from river: line 5 (R24), line 7 (R24), line 8 (R31); Ps. aeruginosa from clinical : line 9 (H75), line 10 (H47), line 11 (H12); E. coli pUC-19: line 6
BIRNBOIM, H.C. & DOLY,J . (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Research 7 , 15 13- 1523. BURTON,G.A., GUNNISON, D . & L A N C E G.R. , (1987) Survival of pathogenic bacteria in various freshwater sediment. Applied and Environmental Microbiology 53, 633-638. CODINA, J . C . MARTINEZDE V I C E N T E , A., MANZANARES, M., AVILES, M., BORREGO, J.J. & ROMERO,P. (1990) Serotypes and pyocin types of Pseudomonas aeruginosa isolated from natural waters. Letters in Applied Bacteriology 10, 77-80. ELLIOT, T.B., BILARDI,B.L., H U G H , R . , WEAVER, R.E. & WELSTER, J . (1981) Identification of Glucose Non-
500 C. P A D I L L A ET A L
fermenting Gram Negative Rods. pp. 81-94. Washington DC: American Society for Microbiology. F A R K A S - H I M S L EHY. , (1980) Bacteriocins, are they broadspectrum antibiotics ? Journal of Antimicrobial Chemotherapy 6, 224-227. F Y F E ,J . M . , H A R R I SG , . M . & G O V A N J, . R . W . (1984) Revised typing methods for Pseudomonas aeruginosa. Journal of Clinical Microbiology 20,47-59. GAGLIANO V ,. & H I N D S I L LR, . (1970) Characterization of Staphylococcus aureus bacteriocin. Journal of Bacteriology 104, 117-125. G o v A N , J .R . (1986) In vivo significance of bacteriocins and bacteriocins receptors. Scandinavian Journal of Infectious Diseases 49, 31-37. K O N I S K YJ ,. (1982) Colicins and others bacteriocins with established modes of action. Annual Review of Microbiology 36, 125144. N I E L S O NJ.W., , D I C K S O NJ ,. & CROUSE, D . (1990) Use of
a bacteriocin produced by Pediococcus acidilactici to inhibit Listeria monocytogenes associated with fresh meat. Applied and Environmental Microbiology 56, 2349-2353. P A D I L L AC, . , B R E V I SP, . , S A I D ,A . & M O L I N AR, . (1990) Inhibition of the growth of enteropathogenic bacilli by bacteriocins produced by micro-organisms from the sediment of wells. Journal of Applied Bacteriology 68, 289-295. RUSSELL, C. & T A G GJ ,. R . (1981) Role of bacteriocins during plaque formation by Streptococcus salivarius and Streptococcus sanguis on a tooth in an artificial mouth. Journal of Applied Bacteriology 50, 305-3 13. V A L E N Z U E L0 A ,. & P E R A L T A . (1987) Condiciones sanitarias del abastecimiento de agua de 10s pequefios productores agricolas. Universum 2, 39-46. WATANABE, T . & F U K A S A W T. A , (1961) Episome mediate transfer of drug resistance in Enterobacteriaceae. Journal of Bacteriology 18, 67&683.
Range of action and genetic bacteriocin codification of Pseudomonas aeruginosa isolated from three different ecological niches C. Padilla, M. Salazar and 0. Faundez Department of Biological Sciences, University of Talca, Chile 4099/01/92: accepted 15 May 1992
c . PADILLA, M. S A L A Z A R AND 0.FAUNDEZ. 1992. Strains of Pseudomonas aeruginosa isolated from sediments in wells showed a greater bacteriocinogenic activity in those isolated from river and clinical specimens. More than half of all the strains examined had extrachromosomal DNA. Plasmid D N A was extracted from all the strains and in only 24/120, all from different origins, was the curing achieved ; all these strains coded their bacteriocins in the chromosomal DNA. INTRODUCTION
Bacteriocins are antibacterial proteins produced by bacteria (Konisky 1982). They may confer a selective advantage by killing other bacteria which are in competition with those that produce them (Farkas-Himsley 1980). Bacteriocins have been used for typing micro-organisms (De Vicente et al. 1990). In addition, they have been considered as a possible means of controlling pathogenic bacteria in the mouth as well as enteropathogens in foods and polluted water (Russell & Tagg 1981; Nielson et al. 1990; Padilla et al. 1990). One of the most important producers of bacteriocins is Pseudomonas aeruginosa, the bacteriocinogenic properties of which are widely recognized (Fyfe et al. 1984). In view of the ubiquitous nature of Ps. aeruginosa, its ability to tolerate unfavourable environmental conditions, its bacteriocinogenic capacity, and having regard to the potential use of the bacteriocins, we studied the bacteriocinogenic range of action and bactericidal potency. We also examined the genetic codification of bacteriocins in strains isolated from clinical samples, rivers and sediments of water wells. MATERIALS AND METHODS Bacterial stralns
One hundred and twenty strains of Ps..aeruginosa were isolated from clinical specimens by conventional methods. A further 88 strains were obtained from 10 different sites along the Claro River in Chile, as described by De Vicente Correspondence to :Dr C. Padilla, Department of Biological Sciences, University of Talca, Talca, Chile.
et al. (1990), and 63 were isolated from sediments of wells in the rural area in the 7th Region of Chile. In addition, several abacteriocinogenic organisms isolated in our laboratory were used as target strains: Corynebacterium xerosis, Bacillus subtilis, Streptococcus pyogenes, Staphylococcus epidermidis, enterotoxigenic Escherichia coli (CKWS), Shigella Jexneri (CT 152), Salmonella typhi (CK 098), Salm. paratyphi B and Klebsiella pneumoniae. Reference strains used to control the presence of plasmids were E. coli pBR-322 and pUC-19. Cultivation and identificationof the bacteria
The organisms from river samples were isolated by a membrane filtration technique and the strains were reisolated on MacConkey (Difco) and trypticase soy agar (Difco). The sediment samples were diluted 1 : 10 in sterile water and 0.1 ml was plated on MacConkey agar and on trypticase soy agar. These cultures were incubated at 28°C for 24 h. The same media were used for the clinical specimens. All the strains of Ps. aeruginosa were identified biochemically by conventional tests, following the identification criteria described by Elliot et al. (1981). Detectlon of inhlbitory activity
The technique used was a modification of that described by Bagliano & Hindsill (1970) and later performed according to that of Padilla et al. (1990). Obtention of piasmidial DNA
The extrachromosomal DNA was obtained according to the technique described by Birnboim et al. (1979).
498 C. PADILLA ET A L .
Table 1 Pseudornonas aeruginosa with bacteriocinogenic activity isolated from
Strains of Ps. aeruginosa Ps. aeruginosa from different origins
Total NO.
Well sediment Rivers
63 88 120
Clinical specimens
Bacteriocinogenic
Abacteriocinogenic
%
NO.
%
No.
%
100 100 100
61 41 79
96.8 46.5 65.8
2 47 41
3-1 53-4 34.1
Ellmlnatlon of extrachromosomal DNA
To ascertain if the coding genes of the bacteriocins were contained on a plasmid or were chromosomal, the extrachromosomal DNA was eliminated by a modified version of the procedure of Watanabe 81 Fukasawa (1961). Later, the method was that of Padilla et al. (1990). RESULTS AND DISCUSSION
In ecological terms, it is important that the bacteriocinogenicity in Ps. aeruginosa was observed more frequently in isolates of sediment of wells in comparison with others (Table 1). These results contrasted, in part, with those of Govan (1986) who found that the bacteriocinogenicity in strains of Ps. aeruginosa, was more frequent in clinical than in environmental strains. Table 2 shows that Ps. aeruginosa from sediments produced larger average diameter inhibition zones (haloes) of Gram-positive and Gram-negative bacteria as compared with Ps. aeruginosa strains isolated from rivers and clinical material. The table also shows that the average diameter of inhibition haloes in Gram-negative organisms are larger than these of Gram-positive bacteria. Also of interest is the variation in the susceptibility of target strains to different bacteriocins (Fig. 1). Table 3 shows the strains of Ps. aeruginosa with a capacity to inhibit more than one target strain. Nine strains from well sediments produced bacteriocins with a wide spectrum of activity and more than one bacteriocin can be manufactured by one strain. On the other
different environments
hand, five river and three clinical strains each possessed a narrow spectrum bacteriocin. Only strains P3 and P54 showed a capacity to inhibit the growth of three different target strains. It was also observed that Kl. pneumoniae was the most sensitive bacterium to different bacteriocins, whereas B. subtilis was resistant to all of them. The extraction of plasmid DNA indicated that 76% of all the microorganisms studied showed an extrachromosoma1 DNA of different sizes (Fig. 2). The plasmid DWA elimination in 12 strains from well sediments, 12 from rivers and 12 from clinical material showed that all maintained the capacity to manufacture bacteriocins. This indicates that information for bacteriocins in these micro-organisms is coded in the chromosomal DNA. I t was very interesting to observe that strains of P5. aeruginosa isolated from sediments had the most bacteriocinogenic activity. The reason could be natural selection in the habitants of an almost closed ecological niche. In the rural areas of developing countries the water for human consumption is highly contaminated with enteropathogenic bacilli (Valenzuela & Peralta 1987). The large
Table 2 Average diameter of haloes indications of
bacteriocinogenic inhibition of Pseudomonas aeruginosa isolated from different environments against Gram-negative and positive target strains
Sources of strains of Ps. aeruginosa River
Well sediment
Target strains
Inhibition zones (mm)
Gram-negative Gram-positive
12.8
21-7
9.7
16-4
Clinical
18.2 11.8
Fig. 1. Production of haloes of inhibition of Pseudomonas aeruginosa from sediment (P 18), river (R4) and clinical sample (H31), when overland with ShigellaJexneri (CT 152)
BACTERIOCIN CODIFICATION OF P S . AERUGlNOSA 499
Table 3 Pseudomonas aeruginosa from
Target strains inhibited for bacteriocins
different origins producing bacteriocins of a wide range of action
Ps. aeruginosa strain
Origin
Ec
Sf
St
Kp
p9 p2 1 PI I p25 p3 p4 p39 p54
Sediment Sediment Sediment Sediment Sediment Sediment Sediment Sediment Sediment River River River River River Clinical Clinical Clinical
o + + o o + o + o + o o + o o o +
o + + o o + + + o + + o o o o o o
+ o o + + o o o o o o o o + + o o
+ o o o + o + + o o + o + + + o +
p60
R2 1
R4 R50
R4 1 R63
H, H68
Bs o o o o o o o o o o o o o o o o o
Sp o o o o o o o o + o o o o o o + o
Se o o o o o o o o + o o + o o o o o
Cx o o o + + o o o o o o + o o o + o
activity; +, bacteriocin activity; Ec, enterotoxigenic Escherichia coli (CK 041); Sf, Shigella jexncri (CT 152); St, Salmonella typhi (CK 098); Kp, Klebsiella pneumoniae; Bs, Bacillus subtilis; Sp, Streptococcus pyogenes; Se, Staphylococcus epidermidis ; Cx, Corynebacterium xerosis.
0,No
I
2
3
4
5
6
7
8
9
10
II
number of pathogens that occur in water and sediments, along with the increasing usage of recreational waters, creates a potential health hazard (Burton et al. 1987). The inhibitory properties of bacteriocins isolated from strains of some Ps. aeruginosa obtained from sediments is an interesting phenomena and we think it is a basis for biotechnological studies in the biological decontamination of water. I n summary, we suggest that bacteriocins might be used to control pathogen microorganisms in water. Initial unpublished observations in artificial water systems indicate promising results.
REFERENCES
Fig. 2 Extraction of plasmid DNA of Pseudomonas aeruginosa
from different sources (left to right). Ps. aeruginosa from sediment: line 1 (P43), line 2 (P21), line 3 (P36), line 4 (P7); Ps. aeruginosa from river: line 5 (R24), line 7 (R24), line 8 (R31); Ps. aeruginosa from clinical : line 9 (H75), line 10 (H47), line 11 (H12); E. coli pUC-19: line 6
BIRNBOIM, H.C. & DOLY,J . (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Research 7 , 15 13- 1523. BURTON,G.A., GUNNISON, D . & L A N C E G.R. , (1987) Survival of pathogenic bacteria in various freshwater sediment. Applied and Environmental Microbiology 53, 633-638. CODINA, J . C . MARTINEZDE V I C E N T E , A., MANZANARES, M., AVILES, M., BORREGO, J.J. & ROMERO,P. (1990) Serotypes and pyocin types of Pseudomonas aeruginosa isolated from natural waters. Letters in Applied Bacteriology 10, 77-80. ELLIOT, T.B., BILARDI,B.L., H U G H , R . , WEAVER, R.E. & WELSTER, J . (1981) Identification of Glucose Non-
500 C. P A D I L L A ET A L
fermenting Gram Negative Rods. pp. 81-94. Washington DC: American Society for Microbiology. F A R K A S - H I M S L EHY. , (1980) Bacteriocins, are they broadspectrum antibiotics ? Journal of Antimicrobial Chemotherapy 6, 224-227. F Y F E ,J . M . , H A R R I SG , . M . & G O V A N J, . R . W . (1984) Revised typing methods for Pseudomonas aeruginosa. Journal of Clinical Microbiology 20,47-59. GAGLIANO V ,. & H I N D S I L LR, . (1970) Characterization of Staphylococcus aureus bacteriocin. Journal of Bacteriology 104, 117-125. G o v A N , J .R . (1986) In vivo significance of bacteriocins and bacteriocins receptors. Scandinavian Journal of Infectious Diseases 49, 31-37. K O N I S K YJ ,. (1982) Colicins and others bacteriocins with established modes of action. Annual Review of Microbiology 36, 125144. N I E L S O NJ.W., , D I C K S O NJ ,. & CROUSE, D . (1990) Use of
a bacteriocin produced by Pediococcus acidilactici to inhibit Listeria monocytogenes associated with fresh meat. Applied and Environmental Microbiology 56, 2349-2353. P A D I L L AC, . , B R E V I SP, . , S A I D ,A . & M O L I N AR, . (1990) Inhibition of the growth of enteropathogenic bacilli by bacteriocins produced by micro-organisms from the sediment of wells. Journal of Applied Bacteriology 68, 289-295. RUSSELL, C. & T A G GJ ,. R . (1981) Role of bacteriocins during plaque formation by Streptococcus salivarius and Streptococcus sanguis on a tooth in an artificial mouth. Journal of Applied Bacteriology 50, 305-3 13. V A L E N Z U E L0 A ,. & P E R A L T A . (1987) Condiciones sanitarias del abastecimiento de agua de 10s pequefios productores agricolas. Universum 2, 39-46. WATANABE, T . & F U K A S A W T. A , (1961) Episome mediate transfer of drug resistance in Enterobacteriaceae. Journal of Bacteriology 18, 67&683.
