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Resistance of environmental bacteria to heavy metals
Bioresource Technology 64 (1998) 7-15 © 1998 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-8524/98 $19.00 ELSEVIER
PI I:S0960-8524(97)0016
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RESISTANCE OF E N V I R O N M E N T A l . BACTERIA TO H E A V Y METALS A. Hassen"*, N. Saidi a & M. Cherif b, A. Boudabous c alnstitut National de Recherche Scientifique et Technique, URNE-Eau, B.P 15-1082, CitOMahrajbne, Tunis, Tunisie °Institut National Agronomique de Tunisie, 43 Avenue Charles Nicolle, 1082, CitOMahragbne, Tunis, Tunisie CFacultOdes Sciences de Tunis, Laboratoire de microbiologie, Campus universitaire, 1060 Tunis, Tunisie (Received 9 March 1997; revised version received 12 October 1997; accepted 22 October 1997) Jonas, 1989). The introduction of heavy metals, in various forms, in the environment, can produce considerable modifications of the microbial communities and their activities (Doelman et al., 1994; Guzzo et al., 1994; Hiroki, 1994; Starzecka and Bednarz, 1993). Heavy metals generally exert an inhibitory action on microorganisms by blocking essential functional groups, displacing essential metal ions, or modifying the active conformations of biological molecules (Doelman et al., 1994; Gadd and Griffiths, 1978; Li and Tan, 1994a,b; Wood and Wang, 1983); however, at relatively low concentration some metals are essential for micoroorganisms (e.g. Co, Cu, Zn, Ni) since they provide vital co-factors for metallo-proteins and enzymes (Eiland, 1981; Doelman et al., 1994). In naturally polluted environments, the response of microbial communities to heavy metals depends on the concentration and availability of metals and is dependent on ~the actions of complex processes, controlled by multiple factors such as the type of metal, the nature of medium and microbial species (De Rore et al., 1994; Goblenz et al., 1994; Hashemi et al., 1994; Olasupo et al., 1993; Tomioka et al., 1994). For instance, it was demonstrated that copper and silver blocked an enzyme system involved in respiration (Liebe and Stuchr, 1972; Rahn et al., 1973). The mechanism of resistance to metal takes two forms, either accumulation in the form of particular protein-metal association (Ow, 1993; Rohit and Sheela, 1994), or blockage at the level of the cell wall and the systems of membrane transportation (Ow, 1993; Tomioka et al., 1994; Wehrheim and Wettern, 1994). Heavy metal MICs (minimal inhibitory concentrations) for bacterial strains present in various natural habitats such as soil, water, sediments and sewageamended soil, have been studied (Abbas and Edwards, 1989; Chang and Broadbent, 1982; Duxbury and Bicknell, 1983; Hiroki, 1994; Nieto et al., 1989). To determine the MICs of heavy metals,
Abstract
Bacteria were isolated from different naturally polluted environments. Metal-resistant bacteria were selected and minimal inhibitory concentrations of heavy metals (MICs) for each isolate were determined. In addition, the mobility of the most important metallic cations (Cu, Zn, Cr, Cd, Co, Hg) was evaluated by comparing results obtained by two tests of toxicity in solid and liquid media. Results of the test of toxicity in solid media agreed with those in liquid, however, inhibitory concentrations in solid media were much higher than those in liquid. The range of metal concentrations tolerated in solid and liquid media yielded information on the capacity of adsorption and complexation of the metals. Mercury, and to a lesser degree copper, seemed to have a good capacity for adsorption and complexation and, consequently, had a limited diffusion in different naturally polluted environments. The presence of metals in the growth medium allowed us to maintain the tolerance of bacteria at a comparable level with that observed in naturally polluted environments. Cu and Cr were the best tolerated metals. Hg was the most toxic component for all bacteria, followed by Co and Cd. Pseudomonas aeruginosa (strain $6), with a relatively high MIC for metals and a large spectrum of antibiotic resistance appears to be a bacterial model for eco-toxicological studies. © 1998 Published by Elsevier Science Ltd. All rights reserved. Key words: heavy metals, metal-tolerant bacteria, Pseudomonas aeruginosa, toxicity, adsorption. INTRODUCTION
Numerous toxicological studies have examined the heavy metal sensitivity or resistance of bacteria isolated from different habitats (Doelman et al., 1994; Duxbury and Bicknell, 1983; Hiroki, 1992; *Author to whom correspondence should be addressed 7
8
A. Hassen et al.
most studies have used the medium that best supports the growth of the microorganism or a group of microorganisms. Frequently, media have been amended with various quantities of heavy metal salts, inoculated with the appropriate microorganism, and microbial growth measured to determine the minimal inhibitory concentration. A number of problems are associated with this approach. Metal-binding capacity of the microorganisms, chelation to various components of the media, and formation of complexes can each cause a reduction in the activities of free metals. Therefore, the activity of a free metal ion, ordinarily considered to be the toxic metal species that ultimately determines the microbial response to the metal, rarely approaches the total metal concentration added to media (Angle and Chaney, 1989). Of particular importance is the sorption or chelation of metals to unspecified organic compounds found in most biological media. For example, interactions of mercury with the constituents of Luria-Bertani broth (LB) have been studied by Chang et al. (1993) during the growth of P aeruginosa PU21 (possessing a mercuric reductase). This study showed that 30 to 40% of the mercury (Hg ~÷) formed complexes with tryptone and yeast extract of the medium within 140h of contact. Borges and Wollum (1980), in studying cadmium sensitivity of Bradyrhizobium japonicum, and using a mineral medium, showed that a significant amount of the cadmium added, bonded to uncharacterized medium components. As a consequence, it appears that MICs determined with traditional approach media cannot be related to actual metal concentrations in the habitat from which bacteria were isolated. In spite of these limits, the technique of MICs remains a valid approach to evaluate the action of heavy metals on the microbial activity in polluted habitats such as agricultural soils, sludge-amended soils, marine sediments and municipal refuse. On the other hand, the possibility of using metalresistant bacteria as bio-indicators of polluted environment, has been shown to be a sensitive and reliable tool in detecting the sub-lethal toxicity of these polluting compounds. A combination of bioassays (fish, algae, bacteria) is increasingly recommended in the framework of integrated eco-toxicological approaches, in order to gain a better insight into the potential dangers associated with the disposal of complex industrial effluent in the environment (Blaise et al., 1985; Trevors et al., 1985). The tolerance of soil bacteria to heavy metals has been proposed as an indicator of potential toxicity of metals to other forms of life (Olson and Thornton, 1982). We report here the isolation of metal-resistant bacteria from different habitats of natural and polluted environments. We have determined the heavy-metal-MICs for each bacterial isolate and have selected the most dominant group, which
comprised the most interesting bacterial species that can be used as a bacterial bio-indicator of heavymetal toxicity. We also have evaluated the mobility of these metals in terms of bioavailability to microorganisms and their binding to unspecified organic compounds found in growth media. METHODS Microorganisms and growth conditions Strains of Bacillus thuringiensis and Escherichia coil K12 were obtained from Dr H. de Barjac, Pasteur Institute of Paris. All hospital strains were isolated from the rhino-pharynx of patients of the paediatrics hospital of Siliana (Dr E1 Bour, Siliana, Tunisia). Strains from natural environments were selected as follows: suspensions of soil, sewage sludge, wastewaters and composted municipal solid waste were inoculated separately into twice concentrated nutrient broth (Oxoid, Lab-Lemco Powder, 1 g/liter; Yeast extract, 2g/liter; Peptone, 5g/liter; and sodium chloride, 5 g/liter) and incubated for at least 4 days at (37_+°C). The initial cultures were the microbial stocks used for selection. In the second phase, nutrient broth in tubes (300 x 20 mm) was supplemented with increasing amounts of metal salts, and was then inoculated with 0.5 ml of microbial stock, and incubated at (37+°C) for 72 h. The last tube with a positive culture of each series used was considered to contain metal-tolerant bacteria. These strains were then isolated in metal-free nutrient agar. All isolates were identified based on morphological features and biochemical properties (Capet, 1970; Orndoff and Colwell, 1980). Their identification was confirmed using API 20 NE and E test kits (API System SA, bioM6rienx, Marcy l'Etoile, France). Chemicals The heavy metals tested were sulfate salts: CrSOa.8H20; CuSOa.5H20; HgSO4; CdSO4.2.5H20; ZnSO4.5H20; COSO4.7H20. Stock solutions were prepared in distilled water, slightly acidified for mercury (2 to 4 drops of 6 N HC1), and were sterilized at ll0°C for 15 min. These solutions, in various concentrations according to the metal tested, were kept at 4°C for no longer than 1 month. The glassware used was leached in 2 N HNO3 and rinsed several times with distilled water before use to avoid metal contamination. Assessment of metal toxicity In order to assess quantitatively the effects of the heavy metal, a plate diffusion and a tube dilution method were tested: Plate diffusion method To each plate of nutrient agar (Oxoid), 0.5 ml of the appropriate metal salt solution was added in a
Environmental bacteria and heavy metals central well of 1 cm in diameter and 4 mm in depth. Plates were then incubated at (37+°C) for 24 h to allow diffusion of the metal into the agar. It was supposed by that time, that a concentration gradient of the metal would have been formed. On each plate, eight strains were inoculated in radial streaks and in duplicate. Plates were then incubated at (37±°C) for 48 h. After incubation, the area of growth inhibition (in mm) was measured as that from the edge of the central well to the leading edge of the growing streak. The percentage of bacterial tolerance was calculated in terms of the ratio: length of the growth in mm vs length of the total inoculated streak. The range of concentrations for heavy metals was as follows (milli-Mols): 25, 50, 100, 150, 200 and 250 for Zn, Cr and Cu; 30, 60, 80,120, 160, 180 for Cd; 5, 10, 25, 50, 75 and 100 for Co and 5, 10, 35, 70, 90 and 100 for Hg.
Tube dilution method (MIC) Concentrations of appropriate metals were prepared in tubes with a final volume of 10 ml of nutrient broth (Oxoid). Culture medium and metallic solution were sterilized separately for 15 min at ll0°C. Three tubes were prepared for each metallic concentration, then inoculated with 200/d of an 18-h-old culture of the studied bacterial strain. A positive control consisted of a metal-deficient medium inoculated with the microorganism and a negative one consisted of a metal-supplemented medium without the microorganism. Tubes were read after incubation at (37+°C) for 5 days. The lowest concentration of metal that completely prevented growth was termed the 'minimal inhibitory concentration' (MIC). Antibiotic susceptibility tests We adopted two different methods: the ATB strips (bioM6rieux, Marcy-l'Etoile, France) and the agar disc method. The ATB strips consist of 16 pairs of cupules. The first pair does not contain any antibiotic and serves as a positive growth control. The next 14 pairs contain antibiotics at one or two concentrations (c and C). The last pair is a blank, which permits the addition of an extra antibiotic when needed (unit test). The bacteria to be tested are suspended in distilled water, then transferred into the growth medium and inoculated into the strip. After 18 to 24 hours of incubation, growth may either be read visually or by the ATB instrument. The result obtained classifies the strain as Sensitive, Intermediate or Resistant.
9
heavy metals, were heterogeneous but all were Gram-negative bacteria. The dominant bacterial species belonged to the genus Pseudomonas (P. paucimobilis, P. aeruginosa, P. cepacia) and Proteus mirabilis (Table 1). Toxicity of metals in solid media The plate-diffusion method used here gave very consistent results, particularly with respect to the study of the toxicity of metals in solid media, which could be similar to polluted soil and sewage sludge. This test took into consideration the quantity of metal bound or chelated to unspecified organic components found in most biological media. The tolerance of the 25 bacterial strains listed in Table 1 was examined and only results relative to copper and mercury are reported in Figs 1 and 2. These figures report only the concentrations of metal that gave very distinct effects. While copper and mercury were, respectively, the most tolerated and the most toxic metals, chromium, zinc, cobalt and cadmium gave intermediate results (results not shown). From Fig. 1 it appears that the average inhibition of bacterial growth caused by Cu varied between 0 and 95% according to bacterial strain and metal concentration. The most tolerant bacterial strains were S18, S19, $20 and $21 of Proteus mirabilis, S12 of Pseudomonas paucimobilis, S16 of Providencia rettgeri and $25 of Klebsiella rhinoscleromatis. Fig. 2 shows that mercury reduced noticeably the growth of all strains studied. In the range of 10 to 100 of mercury, bacterial growth inhibition varied between 50 and 100%. Two strains, Streptococcus sp.1 ($3) and P. paucimobilis (Sll) showed relatively high tolerance to mercury with more than 25% of growth at the highest metal concentration (100 mM). This experiment revealed mainly a particular resistance of P. paucimobilis species to Cr, Cu, Cd and Hg, of Streptococcus sp.1 to Zn, Cr, Co and Hg and of P aeruginosa to Zn, Cu, Cr and Cd. Therefore, P aeruginosa and P. paucimobilis isolates appeared as the most metal-tolerant species and could serve as bio-indicators of heavy metals in the polluted environments. Toxicity of metals in liquid media Experiments in liquid culture were conducted to determine the precise concentrations of metals at which the species could grow. All results obtained were expressed in MIC (Table 2).
Copper toxicity RESULTS Fresh isolates Fresh isolates, selected from different natural environments and identified as strains tolerant to
Most bacterial strains studied tolerated more than 0.8mM of CuSO4.5H20 (Table 2). The most tolerant species were Providencia rettgeri ($15) and P. aeruginosa ($7) with, respectively, a MIC of 1.8 and 1.6.
A. Hassen et al.
10 Table 1. B a c t e r i a l s t r a i n s i s o l a t e d
Strain no. S1 $2 $3 $4 $5 $6 $7 $8 $9 S10 Sll S12 S13 S14 $15 $16 S17 S18 S19 $20 $21 $22 $23 $24 $25
Identification
Origin
Escherichia coli K12 (Pasteur) Bacillus thuringiensis (serotype I) Streptococcus sp. 1 (group A pyogenes) Streptococcus sp. 2 (group G) Staphylococcus aureus Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudornonas paucimobilis Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas cepacia Providencia rettgeri Providencia rettgeri Proteus mirabilis Proteus mirabilis Proteus mirabilis Proteus mirabilis Proteus mirabilis Aeromonas hydrophila Acinetobacter calcoaceticus Citrobacter freundii Klebsiella rhinoscleromatis
Institut Pasteur of Paris Institut Pasteur of Paris Rhino-pharynx of patients, paediatrics hospital of Silania, Tunisia Rhino-pharynx of patients, paediatrics hospital of Silania, Tunisia Rhino-pharynx of patients, paediatrics hospital of Silania, Tunisia Wastwater Compost of municipal solid waste, Tunis Wastwater Fresh water Municipal sewage sludge Mix of solid municipal wastewater andsewage sludge Solid waste Strain Ferchichi (ONAS Tunisia) Strain Ferchichi (ONAS Tunisia) Wastewater Compost of municipal solid waste, Tunis Wastewater Wastewater Wastewater Wastewater Wastewater Soil Municipal sewage sludge Municipal solid waste Compost of municipal solid waste, Tunis
Isolated strains were identified by their morphology and biochemical proprties. Identification was confirmed by the use of API 20 E and NE test kit (API System SA, bioM6rieux, Marcy l'Etoile, France).
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III100 mM 11200 mM r'1250 mM 1 Fig. 1. Toxicity of Copper to bacterial strains (S.) tested in agar media. Sn: see Table 1.
Environmental bacteria and heavy metals Chromium toxicity
11
aeruginosa ($6 and $7). Zinc below the MIC often
For all strains, the average level of tolerance was 1.2 mM of chromium III sulfate. Chromium has generally a moderate effect on bacterial growth. Tolerant strains of P. aeruginosa ($6 and $7), P. paucimobilis ($9) and K. rhinoscleromatis ($25) showed a respective MIC of 1.2, 1.5 and 1.8 mM of chromium III sulfate. It was observed for strains of P. aeruginosa that Cr stimulated notably pyocyanin synthesis.
stimulated the synthesis of pyoverdin in isolates of P.
aeruginosa. Mercury toxicity
Zinc toxicity
Mercury appeared as the most toxic of all metals tested (average MIC 0.05 mM of mercury salt). Its action affected simultaneously Gram-positive and Gram-negative bacteria. These results confirmed the high toxicity of mercury previously detected in solid media. It should be mentioned that most isolates showed, during the five first sub-cultures in nutrient broth, a medium tolerance that was 10 times higher than the one presented subsequently after several cultures. Indeed, the MICs decreased clearly after the fifth sub-culture. The presence of metal in the growth medium allowed the maintenance of the tolerance at a comparable level to the one observed at the isolation.
Zinc appeared toxic, sometimes at very low doses, except for some bacterial strains that were found to be relatively zinc-tolerant, such as Acinetobacter calcoaceticus ($23), Citrobacter freundii ($24) and P.
Susceptibility of strains $6 and $7 to antibiotics The two strains $6 and $7 of P. aeruginosa were studied mainly because they appeared dominant
Cobalt and cadmium toxicity Cobalt inhibited pigment synthesis in P. aeruginosa. Tolerance was often lower than 0.8mM of COSO4.7H20. Most strains tolerant to cadmium resisted average concentrations of 1.5 mM of CdSO4, 2.5H20. These strains were essentially P.
aeruginosa.
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A. Hassen et al.
12
Table 2. Minimal inhibitory concentrations of heavy metals
Strain no.
S1 $2 $3 $4 $5 $6 $7 $8 $9 $10 $11 $12 S13 $14 S15 S16 S17 S18 S19 $20 $21 $22 $23 $24 $25
MIC* of metal
Identification
Escherichia coli K12 Bacillus thuringiensis (serotype I) Streptococcus sp. 1 Streptococcus sp. 2 Staphylococcus aureus Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudoraonas paucimobilis Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas cepacia Providencia rettgeri Providencia rettgeri Proteus mirabilis Proteus mirabilis Proteus mirabilis Proteus mirabilis Proteus mirabilis Aeromonas hydrophila Acinetobacter calcoaceticus Citrobacter freundii Klebsiella rhinoscleromatis
Cu
Cr
Co
Cd
Zn
Hg
0.5 0.5 0.02 0.05 0.2 1.2 1.6 0.1 1.5 0.2 1.2 1.5 0.8 1.2 1.8 1.2 ND 1.5 1.2 1.5 1.5 1.2 ND 0.2 ND
1.5 1.0 0.8 0.5 0.2 1.2 1.5 3 1.2 1.8 1.2 1 1.2 1.2 1.2 1.5 ND 1.5 ND 1.5 1.2 1.5 ND 0.8 1.2
0.2 0.05 0.05 0.05 0.2 0.4 0.6 0.8 0.4 0.4 0.4 0.2 0.4 0.2 0.4 0.6 ND 0.2 0.2 0.2 0.2 0.6 ND 0.2 ND
0.2 1.2 0.2 0.2 0.1 1.5 1.5 0.6 0.2 0.2 1.5 0.2 0.2 0.2 0.5 0.2 ND 1.2 1.2 1.2 1.2 0.2 ND 0.2 ND
0.2 0.5 0.2 0.2 0.2 1.5 1.5 1 0.2 0.2 0.8 0.5 0.2 0.2 0.5 0.8 ND 1 0.8 1.2 1 0.2 1.2 1.5 ND
0.05 0.06 0.05 0.005 0.005 0.08 0.05 0.1 0.08 0.05 0.08 0.05 0.05 0.04 0.05 0.05 0.1 0.08 0.08 0.1 0.1 0.05 ND 0.04 ND
Minimal inhibitory concentration (MIC) expressed in mM/liter in nutrient broth (Oxoid). ND, none detected.
during the selection of bacterial strains. Table 3 indicates that $6 and $7 have a similar pattern of resistance to antibiotics. In fact, among the 42 antibiotics tested only streptomycin (10 UI), chloramphenicol (30#g), imipenem (8mg/liter) and pefloxacin (4mg/liter) showed different effects towards $6 and $7. Of particular importance is also the broad range of resistance of $6 to antibiotics. In fact, 26 antibiotics were tolerated by this strain (Table 3).
DISCUSSION
As pointed out by Trevors et al. (1985), there is a problem in defining exactly what is meant by resistance to heavy metals. Throughout this study, we have often preferred the term 'tolerance' to 'resistance'. Responses of fresh bacterial isolates to heavy metals were very heterogeneous. All isolates were Gram-negative bacteria. However, Foster (1983) has reported the resistance to heavy metals in both Gram-positive and Gram-negative bacteria. Mahler et al. (1986) have found that all isolates tolerant for cadmium and mercury were Gram-positive aerobic spore-formers, and very few of these strains were antibiotic-resistant. Duxbury (1981) reported that generally Gram-negative soil species appeared to be more tolerant than Gram-positive. This difference in
results could be explained by the conditions of each bacterial isolation and the selectivity of microbial culture techniques adopted in each study; particularly with respect to the nature and specificity of growth media. In the present study, the dominating bacterial isolates belonged to the genera Pseudomonas and Proteus. Generally, isolates identified within the same genera showed a certain heterogeneity in their biochemical characteristics and some variation in their colony morphology. This heterogeneity and variation were possibly due to the various origins of the isolates. Results of bacterial isolation have shown the abundance of Pseudomonas aeruginosa, P. paucimobilis and of Proteus rnirabilis, respectively. Strain $6 of P. aeruginosa, which had been isolated from raw wastewater of the city of Tunis, appeared as the most important isolate and could be selected as a bioindicator of toxicity. The ecological value of the sensitivity or resistance of strain $6 to metals lies in its ubiquitous presence in many habitats, in its capacity to grow in the presence of a relatively high heavy-metal concentration, and in its ability to release secondary metabolites, such as pyocines and pigments in the medium. Asparagine broth described by Highsmith and Abshire (1975) seems to be an ideal growth medium for P. aeruginosa for two reasons. First, asparagine broth has a relatively simple composition (DL-asparagine, 3 g/liter;
Environmental bacteria and heavy metals
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KzHPO4, 1g/liter; MgSO4.7H20, 0.5g/liter) as compared with other traditional media and therefore a lower metal-binding-capacity with constituents of the media. Second, asparagine often enhances the biosynthesis of pigments by P. aeruginosa. As previously stated, metals and various components of nutrient media could interact, thus complicating interpretation of the data. The test of toxicity in solid media investigated here could be useful in the evaluation of metal toxicity in sewage sludges and contaminated soil, where conditions of diffusion, complexation and availability of metals were different from those observed in liquid media. The speciation of metals was a very important determinant in understanding the quantitative aspects of metal toxicity. The plate diffusion method suggested in this study is very practical and allows us to test up to eight strains by plate• Mercury, in spite of its weak solubility and diffusion due to its strong adherence to medium components by adsorption and complexation (Behel et al., 1983), appeared as the most toxic metal. It is interesting to note that the following decreasing order of toxicity: Hg > Co > Cd > Cu-Cr > Zn, does not differ from that reported in other studies (Duxbury, 1981)• The test in liquid media was sensitive at concentrations 10 to 1000 times lower than those obtained in solid media. In spite of its limits described previously, the liquid media test allowed a good evaluation of metal toxicity in polluted environments, such as industrial effluents, incinerator residues, landfill municipal refuse and sewage sludge leachates. Statistical calculation of the ratios between 'Average tolerated concentrations in solid media' and 'Average tolerated concentrations in liquid media' for each metal and each strain tested were made (results not shown). These ratios revealed that the power of metal adsorption and complexation could be classified as follows H g > C u > C d Zn > Co-Cr. These results appear very interesting, since it is well known that mercury, and to a lesser degree copper, possess a good capacity for adsorption and complexation (Behel et al., 1983) and therefore a possibility of limited migration in natural environments, such as soil, sewage sludge or sediments, is possible• A growing body of evidence indicates that metal tolerance and antibiotic resistance are often found together in many clinical isolates and that metal and antibiotic resistance are closely associated (Timoney et al., 1987). The very broad resistance of strain $6 of P. aeruginosa to antibiotics may indicate that the latter is rich in plasmids that carry simultaneous resistance to antibiotics and metals• The net fall of MICs after the fifth sub-culture, of isolated strains, showed, however, that resistance of bacteria to heavy metals was conditioned by the
14
A. Hassen et al.
presence or the absence of the metal in the medium. Indeed, the absence of metal from the bacterial environment reduced noticeably its power of resistance (Timoney et al., 1987) and the maintenance of a metal-resistant microbial community was likely to dependent on the presence of the metal in the growth medium (Mahler et al., 1986).
ACKNOWLEDGEMENTS
This investigation was supported by grant from the National Foundation of Scientific Research (ERESMAT, B14/91). We thank Professor Hans-W. Ackermann, Department of Microbiology, Faculty of Medicine, Laval University, Quebec, Canada, for helpful discussion and critical review of the manuscript.
REFERENCES Abbas, A. & Edwards, C. (1989). Effects of metals on a range of Streptornyces specie~ Appl. Environ. Microbiol., 55, 2030-2035. Angle, J. S. &Chaney, R. L. (1989). Cadmium resistance screening in nitrilotriacetate-buffered minimal media. Appl. Environ. Microbiol., 55, 2101-2104. Behel, D., Darrel, J. R., Nelson, W. & Sommers, L. E. (1983). Assessment of heavy metal equilibria in sewage sludge-treated soil J. Environ. Qual., 12, 181-186. Blaise, C., Bermingham, N. & Van Coillie, R. (1985). La m6thode d'evaluation 6cotoxicologique int6gr6e: une contribution h la lutte contre l'6cotoxicit6. Bull. Qualit~ Eaux, 10, 58-59. Borges, A. C. & Wollum, A. G. (1980). A field study of a soil-soybean plant-Rhizobiurn system amended with cadmium. J. Environ. Qual., 9, 420-423. Capet, R. G. (1970). Aide-memoire de d~termination bact~rienne. Vigot Fr~res Ed, Paris. Chang, F. H. & Broadbent, F. E. (1982). Influence of trace metal on carbon dioxide evolution from a yolo soit Soil Sci., 132, 416-421. Chang, J. S., Hong, J., Oa, O. & Bh, O. (1993). Interaction of mercuric ions with the bacterial growth medium and its effects on enzymatic reduction of mercury. Biotechnol. Progr., 9, 526-532. De Rore, H., Top, E., Houwen, F., Mergeay, M. & Verstraete, W. (1994). Evolution of heavy metal resistant transconjugants in a soil environment with a concomitant selective pres~ FEMS Microbiol. Ecol., 14, 263 -273. Doelman, P., Jansen, E., Michels, M. & Van Til, M. (1994). Effects of heavy metals in soil on microbial diversity and activity as shown by the sensitivity-resistance index, an ecologically relevant parameter- Biol. Fertil. Soil., 17, 177-184. Duxbury, T. (1981). Toxicity of heavy metals to soil bacteria. FEMS Microbiol. Lett., 11, 217-220. Duxbury, T. & Bicknell, B. (1983). Metal-tolerant bacterial populations from natural and metal-polluted soil~ Soil Biol. Biochem., 15, 243-250. Eiland, F. (1981). The effects of application of sewage sludge on microorganisms in soil. Tidsskrift planteavl., 85, 39-46.
Foster, T. J. (1983). Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol. Rev., 47, 361-409. Gadd, G. M. & Griffiths, A. J. (1978). Microorganisms and heavy metal~ Microbiol. Ecol., 4, 303-317. Goblenz, A., Wolf, K. & Bauda, P. (1994). The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe. Metals and microorganisms: relationships and applications. FEMS Microbiol. Rev., 14, 303-308. Guzzo, A., Du Bow, M. & Bauda, P. (1994). Identification and characterization of genetically programmed responses to toxic metal exposure in Escherichia coli. Metals and microorganisms: relationships and application~ FEMS Microbiol. Rev., 14, 369-374. Hachemi, F., Leppard, G. G. & Kushner, D. J. (1994). Copper resistance in anabaena variabilis: Effects of phosphate nutrition and polyphosphate bodies. Microbial ecology, 27, 159-176. Highsmith, A. K. & Abshire, R. L. (1975). Evolution of a most probable number technique of the enumeration of Pseudomonas aeruginosa. Appl. Environ. Microbiol., 36, 596-601. Hiroki, M. (1992). Effects of heavy metal contamination on soil microbial population. Soil Sci. Plant Nutr., 38, 141-147. Hiroki, M. (1994). Populations of Cd-tolerant microorganisms in soil polluted with heavy metal~ Soil Sci. Plant Nutr., 40, 515-524. Jonas, R. B. (1989). Acute copper and cupric ion toxicity in an estuarine microbial community. Appl. Environ. Microbiol., 55, 43-49. Li, F. & Tan, T. C. (1994). Effect of heavy metal ions on the efficacy of a mixed bacilli BOD sensor- Biosens. Bioelectron., 9, 315-324. Li, F. & Tan, T. C. (1994). Monitoring BOD in the presence of heavy metal ions using a poly (4-vinylpyridine) coated microbial sensor- Biosens. Bioelectron., 9, 445-455. Liebe, D. C. & Stuchr, T. J. E. (1972). Copper II-DNA denaturation I - - concentration dependence of melting temperature and terminal relaxation time. Biopolymers, 11, 145-166. Mahler, I., Levinson, H. S., Wang, Y. & Halvorson, H. O. (1986). Cadmium and mercury-resistant Bacillus strains from a salt marsh and from Boston Harbor- Appl. Environ. Microbiol., 52, 1293-1298. Nieto, J. J., Castillo, R. F., Marquez, M. C., Ventosa, A., Quesada, E. & Ruiz Berraquero, F. (1989). Survey of metal tolerance in moderately halophilic eubacteria Water Res., 55, 2385-2390. Olasupo, N. A., Scott-Emuakpor, M. B. & Ogunshola, R. A. (1993). Resistance to heavy metals by some Nigerian yeast strain~ Folia Microbiologica, 38, 285-287. Olson, B. H. & Thornton, I. (1982). The resistance patterns to metals of bacterial populations in contaminated lancL J. Soil Sci., 33, 271-277. Orndorf, S. A. & Colwell, R. R. (1980). Distribution and characterization of kepone-resistant bacteria in the aquatic environment. Appl. Environ. Microbiol., 39, 611-622. Ow, D. (1993). Phytochelatin-mediated cadmium tolerance in Schizosaccharomyces pombe. In l~tro Cell. Develop. Biol., 29, 213-219. Rahn, R. O., Setlow, J. K. & Stout, J. E. (1973). Ultraviolet irradiation of nucleic acids complexed with heavy atoms III. Influence of Ag ÷ and Hg2 + on the sensitivity of phage and of transforming DNA to ultraviolet radiation- Photochem. Photobiol., 18, 39-41. Rohit, M. & Sheela, S. (1994). Uptake of zinc in Pseudomonas sp. strain UDG26. Appl. Environ. Microbiol., 60, 2367-2370.
Environmental bacteria and heavy metals Starzecka, A. & Bednarz, T. (1993). Comparison of development and metabolic activity of algae and bacteria in soil under the influence of short-and longterm contamination with metallurgic industrial dusts Archiv Hydrobiol., 98 Suppl., 71-88. Timoney, J. F., Port, J., Giles, J. & Spanier, J. (1987). Heavy-metal and antibiotic resistance in the bacterial flora of sediments of New York Bight. Appli. Environ. Microbiol., 36, 465-472. Tomioka, N., Uchiyama, H. & Yagi, O. (1994). Cesium accumulation and growth characteristics of Rhodococcus
15
erythropolis CS98 and Rhodococcus sp. strain CS40Z Appl. Environ. Microbiol., 60, 2227-2231. Trevors, J. T., Oddie, K. M. & Belliveau, B. H. (1985). Metal resistance in bacteria FEMS Microbiol. Rev., 32, 39-54. Wehrheim, B. & Wettern, M. (1994). Comparative studies of heavy metal uptake of whole ceils and different types of cell ills from Chlorella fusca. Biotechnol. Tech., 8, 227-232. Wood, J. M. & Wang, H. K. (1983). Microbiol resistance to heavy metals Environ. Sci. Technol., 17, 582-590.
PI I:S0960-8524(97)0016
1-2
RESISTANCE OF E N V I R O N M E N T A l . BACTERIA TO H E A V Y METALS A. Hassen"*, N. Saidi a & M. Cherif b, A. Boudabous c alnstitut National de Recherche Scientifique et Technique, URNE-Eau, B.P 15-1082, CitOMahrajbne, Tunis, Tunisie °Institut National Agronomique de Tunisie, 43 Avenue Charles Nicolle, 1082, CitOMahragbne, Tunis, Tunisie CFacultOdes Sciences de Tunis, Laboratoire de microbiologie, Campus universitaire, 1060 Tunis, Tunisie (Received 9 March 1997; revised version received 12 October 1997; accepted 22 October 1997) Jonas, 1989). The introduction of heavy metals, in various forms, in the environment, can produce considerable modifications of the microbial communities and their activities (Doelman et al., 1994; Guzzo et al., 1994; Hiroki, 1994; Starzecka and Bednarz, 1993). Heavy metals generally exert an inhibitory action on microorganisms by blocking essential functional groups, displacing essential metal ions, or modifying the active conformations of biological molecules (Doelman et al., 1994; Gadd and Griffiths, 1978; Li and Tan, 1994a,b; Wood and Wang, 1983); however, at relatively low concentration some metals are essential for micoroorganisms (e.g. Co, Cu, Zn, Ni) since they provide vital co-factors for metallo-proteins and enzymes (Eiland, 1981; Doelman et al., 1994). In naturally polluted environments, the response of microbial communities to heavy metals depends on the concentration and availability of metals and is dependent on ~the actions of complex processes, controlled by multiple factors such as the type of metal, the nature of medium and microbial species (De Rore et al., 1994; Goblenz et al., 1994; Hashemi et al., 1994; Olasupo et al., 1993; Tomioka et al., 1994). For instance, it was demonstrated that copper and silver blocked an enzyme system involved in respiration (Liebe and Stuchr, 1972; Rahn et al., 1973). The mechanism of resistance to metal takes two forms, either accumulation in the form of particular protein-metal association (Ow, 1993; Rohit and Sheela, 1994), or blockage at the level of the cell wall and the systems of membrane transportation (Ow, 1993; Tomioka et al., 1994; Wehrheim and Wettern, 1994). Heavy metal MICs (minimal inhibitory concentrations) for bacterial strains present in various natural habitats such as soil, water, sediments and sewageamended soil, have been studied (Abbas and Edwards, 1989; Chang and Broadbent, 1982; Duxbury and Bicknell, 1983; Hiroki, 1994; Nieto et al., 1989). To determine the MICs of heavy metals,
Abstract
Bacteria were isolated from different naturally polluted environments. Metal-resistant bacteria were selected and minimal inhibitory concentrations of heavy metals (MICs) for each isolate were determined. In addition, the mobility of the most important metallic cations (Cu, Zn, Cr, Cd, Co, Hg) was evaluated by comparing results obtained by two tests of toxicity in solid and liquid media. Results of the test of toxicity in solid media agreed with those in liquid, however, inhibitory concentrations in solid media were much higher than those in liquid. The range of metal concentrations tolerated in solid and liquid media yielded information on the capacity of adsorption and complexation of the metals. Mercury, and to a lesser degree copper, seemed to have a good capacity for adsorption and complexation and, consequently, had a limited diffusion in different naturally polluted environments. The presence of metals in the growth medium allowed us to maintain the tolerance of bacteria at a comparable level with that observed in naturally polluted environments. Cu and Cr were the best tolerated metals. Hg was the most toxic component for all bacteria, followed by Co and Cd. Pseudomonas aeruginosa (strain $6), with a relatively high MIC for metals and a large spectrum of antibiotic resistance appears to be a bacterial model for eco-toxicological studies. © 1998 Published by Elsevier Science Ltd. All rights reserved. Key words: heavy metals, metal-tolerant bacteria, Pseudomonas aeruginosa, toxicity, adsorption. INTRODUCTION
Numerous toxicological studies have examined the heavy metal sensitivity or resistance of bacteria isolated from different habitats (Doelman et al., 1994; Duxbury and Bicknell, 1983; Hiroki, 1992; *Author to whom correspondence should be addressed 7
8
A. Hassen et al.
most studies have used the medium that best supports the growth of the microorganism or a group of microorganisms. Frequently, media have been amended with various quantities of heavy metal salts, inoculated with the appropriate microorganism, and microbial growth measured to determine the minimal inhibitory concentration. A number of problems are associated with this approach. Metal-binding capacity of the microorganisms, chelation to various components of the media, and formation of complexes can each cause a reduction in the activities of free metals. Therefore, the activity of a free metal ion, ordinarily considered to be the toxic metal species that ultimately determines the microbial response to the metal, rarely approaches the total metal concentration added to media (Angle and Chaney, 1989). Of particular importance is the sorption or chelation of metals to unspecified organic compounds found in most biological media. For example, interactions of mercury with the constituents of Luria-Bertani broth (LB) have been studied by Chang et al. (1993) during the growth of P aeruginosa PU21 (possessing a mercuric reductase). This study showed that 30 to 40% of the mercury (Hg ~÷) formed complexes with tryptone and yeast extract of the medium within 140h of contact. Borges and Wollum (1980), in studying cadmium sensitivity of Bradyrhizobium japonicum, and using a mineral medium, showed that a significant amount of the cadmium added, bonded to uncharacterized medium components. As a consequence, it appears that MICs determined with traditional approach media cannot be related to actual metal concentrations in the habitat from which bacteria were isolated. In spite of these limits, the technique of MICs remains a valid approach to evaluate the action of heavy metals on the microbial activity in polluted habitats such as agricultural soils, sludge-amended soils, marine sediments and municipal refuse. On the other hand, the possibility of using metalresistant bacteria as bio-indicators of polluted environment, has been shown to be a sensitive and reliable tool in detecting the sub-lethal toxicity of these polluting compounds. A combination of bioassays (fish, algae, bacteria) is increasingly recommended in the framework of integrated eco-toxicological approaches, in order to gain a better insight into the potential dangers associated with the disposal of complex industrial effluent in the environment (Blaise et al., 1985; Trevors et al., 1985). The tolerance of soil bacteria to heavy metals has been proposed as an indicator of potential toxicity of metals to other forms of life (Olson and Thornton, 1982). We report here the isolation of metal-resistant bacteria from different habitats of natural and polluted environments. We have determined the heavy-metal-MICs for each bacterial isolate and have selected the most dominant group, which
comprised the most interesting bacterial species that can be used as a bacterial bio-indicator of heavymetal toxicity. We also have evaluated the mobility of these metals in terms of bioavailability to microorganisms and their binding to unspecified organic compounds found in growth media. METHODS Microorganisms and growth conditions Strains of Bacillus thuringiensis and Escherichia coil K12 were obtained from Dr H. de Barjac, Pasteur Institute of Paris. All hospital strains were isolated from the rhino-pharynx of patients of the paediatrics hospital of Siliana (Dr E1 Bour, Siliana, Tunisia). Strains from natural environments were selected as follows: suspensions of soil, sewage sludge, wastewaters and composted municipal solid waste were inoculated separately into twice concentrated nutrient broth (Oxoid, Lab-Lemco Powder, 1 g/liter; Yeast extract, 2g/liter; Peptone, 5g/liter; and sodium chloride, 5 g/liter) and incubated for at least 4 days at (37_+°C). The initial cultures were the microbial stocks used for selection. In the second phase, nutrient broth in tubes (300 x 20 mm) was supplemented with increasing amounts of metal salts, and was then inoculated with 0.5 ml of microbial stock, and incubated at (37+°C) for 72 h. The last tube with a positive culture of each series used was considered to contain metal-tolerant bacteria. These strains were then isolated in metal-free nutrient agar. All isolates were identified based on morphological features and biochemical properties (Capet, 1970; Orndoff and Colwell, 1980). Their identification was confirmed using API 20 NE and E test kits (API System SA, bioM6rienx, Marcy l'Etoile, France). Chemicals The heavy metals tested were sulfate salts: CrSOa.8H20; CuSOa.5H20; HgSO4; CdSO4.2.5H20; ZnSO4.5H20; COSO4.7H20. Stock solutions were prepared in distilled water, slightly acidified for mercury (2 to 4 drops of 6 N HC1), and were sterilized at ll0°C for 15 min. These solutions, in various concentrations according to the metal tested, were kept at 4°C for no longer than 1 month. The glassware used was leached in 2 N HNO3 and rinsed several times with distilled water before use to avoid metal contamination. Assessment of metal toxicity In order to assess quantitatively the effects of the heavy metal, a plate diffusion and a tube dilution method were tested: Plate diffusion method To each plate of nutrient agar (Oxoid), 0.5 ml of the appropriate metal salt solution was added in a
Environmental bacteria and heavy metals central well of 1 cm in diameter and 4 mm in depth. Plates were then incubated at (37+°C) for 24 h to allow diffusion of the metal into the agar. It was supposed by that time, that a concentration gradient of the metal would have been formed. On each plate, eight strains were inoculated in radial streaks and in duplicate. Plates were then incubated at (37±°C) for 48 h. After incubation, the area of growth inhibition (in mm) was measured as that from the edge of the central well to the leading edge of the growing streak. The percentage of bacterial tolerance was calculated in terms of the ratio: length of the growth in mm vs length of the total inoculated streak. The range of concentrations for heavy metals was as follows (milli-Mols): 25, 50, 100, 150, 200 and 250 for Zn, Cr and Cu; 30, 60, 80,120, 160, 180 for Cd; 5, 10, 25, 50, 75 and 100 for Co and 5, 10, 35, 70, 90 and 100 for Hg.
Tube dilution method (MIC) Concentrations of appropriate metals were prepared in tubes with a final volume of 10 ml of nutrient broth (Oxoid). Culture medium and metallic solution were sterilized separately for 15 min at ll0°C. Three tubes were prepared for each metallic concentration, then inoculated with 200/d of an 18-h-old culture of the studied bacterial strain. A positive control consisted of a metal-deficient medium inoculated with the microorganism and a negative one consisted of a metal-supplemented medium without the microorganism. Tubes were read after incubation at (37+°C) for 5 days. The lowest concentration of metal that completely prevented growth was termed the 'minimal inhibitory concentration' (MIC). Antibiotic susceptibility tests We adopted two different methods: the ATB strips (bioM6rieux, Marcy-l'Etoile, France) and the agar disc method. The ATB strips consist of 16 pairs of cupules. The first pair does not contain any antibiotic and serves as a positive growth control. The next 14 pairs contain antibiotics at one or two concentrations (c and C). The last pair is a blank, which permits the addition of an extra antibiotic when needed (unit test). The bacteria to be tested are suspended in distilled water, then transferred into the growth medium and inoculated into the strip. After 18 to 24 hours of incubation, growth may either be read visually or by the ATB instrument. The result obtained classifies the strain as Sensitive, Intermediate or Resistant.
9
heavy metals, were heterogeneous but all were Gram-negative bacteria. The dominant bacterial species belonged to the genus Pseudomonas (P. paucimobilis, P. aeruginosa, P. cepacia) and Proteus mirabilis (Table 1). Toxicity of metals in solid media The plate-diffusion method used here gave very consistent results, particularly with respect to the study of the toxicity of metals in solid media, which could be similar to polluted soil and sewage sludge. This test took into consideration the quantity of metal bound or chelated to unspecified organic components found in most biological media. The tolerance of the 25 bacterial strains listed in Table 1 was examined and only results relative to copper and mercury are reported in Figs 1 and 2. These figures report only the concentrations of metal that gave very distinct effects. While copper and mercury were, respectively, the most tolerated and the most toxic metals, chromium, zinc, cobalt and cadmium gave intermediate results (results not shown). From Fig. 1 it appears that the average inhibition of bacterial growth caused by Cu varied between 0 and 95% according to bacterial strain and metal concentration. The most tolerant bacterial strains were S18, S19, $20 and $21 of Proteus mirabilis, S12 of Pseudomonas paucimobilis, S16 of Providencia rettgeri and $25 of Klebsiella rhinoscleromatis. Fig. 2 shows that mercury reduced noticeably the growth of all strains studied. In the range of 10 to 100 of mercury, bacterial growth inhibition varied between 50 and 100%. Two strains, Streptococcus sp.1 ($3) and P. paucimobilis (Sll) showed relatively high tolerance to mercury with more than 25% of growth at the highest metal concentration (100 mM). This experiment revealed mainly a particular resistance of P. paucimobilis species to Cr, Cu, Cd and Hg, of Streptococcus sp.1 to Zn, Cr, Co and Hg and of P aeruginosa to Zn, Cu, Cr and Cd. Therefore, P aeruginosa and P. paucimobilis isolates appeared as the most metal-tolerant species and could serve as bio-indicators of heavy metals in the polluted environments. Toxicity of metals in liquid media Experiments in liquid culture were conducted to determine the precise concentrations of metals at which the species could grow. All results obtained were expressed in MIC (Table 2).
Copper toxicity RESULTS Fresh isolates Fresh isolates, selected from different natural environments and identified as strains tolerant to
Most bacterial strains studied tolerated more than 0.8mM of CuSO4.5H20 (Table 2). The most tolerant species were Providencia rettgeri ($15) and P. aeruginosa ($7) with, respectively, a MIC of 1.8 and 1.6.
A. Hassen et al.
10 Table 1. B a c t e r i a l s t r a i n s i s o l a t e d
Strain no. S1 $2 $3 $4 $5 $6 $7 $8 $9 S10 Sll S12 S13 S14 $15 $16 S17 S18 S19 $20 $21 $22 $23 $24 $25
Identification
Origin
Escherichia coli K12 (Pasteur) Bacillus thuringiensis (serotype I) Streptococcus sp. 1 (group A pyogenes) Streptococcus sp. 2 (group G) Staphylococcus aureus Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudornonas paucimobilis Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas cepacia Providencia rettgeri Providencia rettgeri Proteus mirabilis Proteus mirabilis Proteus mirabilis Proteus mirabilis Proteus mirabilis Aeromonas hydrophila Acinetobacter calcoaceticus Citrobacter freundii Klebsiella rhinoscleromatis
Institut Pasteur of Paris Institut Pasteur of Paris Rhino-pharynx of patients, paediatrics hospital of Silania, Tunisia Rhino-pharynx of patients, paediatrics hospital of Silania, Tunisia Rhino-pharynx of patients, paediatrics hospital of Silania, Tunisia Wastwater Compost of municipal solid waste, Tunis Wastwater Fresh water Municipal sewage sludge Mix of solid municipal wastewater andsewage sludge Solid waste Strain Ferchichi (ONAS Tunisia) Strain Ferchichi (ONAS Tunisia) Wastewater Compost of municipal solid waste, Tunis Wastewater Wastewater Wastewater Wastewater Wastewater Soil Municipal sewage sludge Municipal solid waste Compost of municipal solid waste, Tunis
Isolated strains were identified by their morphology and biochemical proprties. Identification was confirmed by the use of API 20 E and NE test kit (API System SA, bioM6rieux, Marcy l'Etoile, France).
a 100
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III100 mM 11200 mM r'1250 mM 1 Fig. 1. Toxicity of Copper to bacterial strains (S.) tested in agar media. Sn: see Table 1.
Environmental bacteria and heavy metals Chromium toxicity
11
aeruginosa ($6 and $7). Zinc below the MIC often
For all strains, the average level of tolerance was 1.2 mM of chromium III sulfate. Chromium has generally a moderate effect on bacterial growth. Tolerant strains of P. aeruginosa ($6 and $7), P. paucimobilis ($9) and K. rhinoscleromatis ($25) showed a respective MIC of 1.2, 1.5 and 1.8 mM of chromium III sulfate. It was observed for strains of P. aeruginosa that Cr stimulated notably pyocyanin synthesis.
stimulated the synthesis of pyoverdin in isolates of P.
aeruginosa. Mercury toxicity
Zinc toxicity
Mercury appeared as the most toxic of all metals tested (average MIC 0.05 mM of mercury salt). Its action affected simultaneously Gram-positive and Gram-negative bacteria. These results confirmed the high toxicity of mercury previously detected in solid media. It should be mentioned that most isolates showed, during the five first sub-cultures in nutrient broth, a medium tolerance that was 10 times higher than the one presented subsequently after several cultures. Indeed, the MICs decreased clearly after the fifth sub-culture. The presence of metal in the growth medium allowed the maintenance of the tolerance at a comparable level to the one observed at the isolation.
Zinc appeared toxic, sometimes at very low doses, except for some bacterial strains that were found to be relatively zinc-tolerant, such as Acinetobacter calcoaceticus ($23), Citrobacter freundii ($24) and P.
Susceptibility of strains $6 and $7 to antibiotics The two strains $6 and $7 of P. aeruginosa were studied mainly because they appeared dominant
Cobalt and cadmium toxicity Cobalt inhibited pigment synthesis in P. aeruginosa. Tolerance was often lower than 0.8mM of COSO4.7H20. Most strains tolerant to cadmium resisted average concentrations of 1.5 mM of CdSO4, 2.5H20. These strains were essentially P.
aeruginosa.
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Fig. 2. Toxicity of mercury to bacterial strains (S.) tested in agar media. S.: see Table 1.
A. Hassen et al.
12
Table 2. Minimal inhibitory concentrations of heavy metals
Strain no.
S1 $2 $3 $4 $5 $6 $7 $8 $9 $10 $11 $12 S13 $14 S15 S16 S17 S18 S19 $20 $21 $22 $23 $24 $25
MIC* of metal
Identification
Escherichia coli K12 Bacillus thuringiensis (serotype I) Streptococcus sp. 1 Streptococcus sp. 2 Staphylococcus aureus Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudoraonas paucimobilis Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas cepacia Providencia rettgeri Providencia rettgeri Proteus mirabilis Proteus mirabilis Proteus mirabilis Proteus mirabilis Proteus mirabilis Aeromonas hydrophila Acinetobacter calcoaceticus Citrobacter freundii Klebsiella rhinoscleromatis
Cu
Cr
Co
Cd
Zn
Hg
0.5 0.5 0.02 0.05 0.2 1.2 1.6 0.1 1.5 0.2 1.2 1.5 0.8 1.2 1.8 1.2 ND 1.5 1.2 1.5 1.5 1.2 ND 0.2 ND
1.5 1.0 0.8 0.5 0.2 1.2 1.5 3 1.2 1.8 1.2 1 1.2 1.2 1.2 1.5 ND 1.5 ND 1.5 1.2 1.5 ND 0.8 1.2
0.2 0.05 0.05 0.05 0.2 0.4 0.6 0.8 0.4 0.4 0.4 0.2 0.4 0.2 0.4 0.6 ND 0.2 0.2 0.2 0.2 0.6 ND 0.2 ND
0.2 1.2 0.2 0.2 0.1 1.5 1.5 0.6 0.2 0.2 1.5 0.2 0.2 0.2 0.5 0.2 ND 1.2 1.2 1.2 1.2 0.2 ND 0.2 ND
0.2 0.5 0.2 0.2 0.2 1.5 1.5 1 0.2 0.2 0.8 0.5 0.2 0.2 0.5 0.8 ND 1 0.8 1.2 1 0.2 1.2 1.5 ND
0.05 0.06 0.05 0.005 0.005 0.08 0.05 0.1 0.08 0.05 0.08 0.05 0.05 0.04 0.05 0.05 0.1 0.08 0.08 0.1 0.1 0.05 ND 0.04 ND
Minimal inhibitory concentration (MIC) expressed in mM/liter in nutrient broth (Oxoid). ND, none detected.
during the selection of bacterial strains. Table 3 indicates that $6 and $7 have a similar pattern of resistance to antibiotics. In fact, among the 42 antibiotics tested only streptomycin (10 UI), chloramphenicol (30#g), imipenem (8mg/liter) and pefloxacin (4mg/liter) showed different effects towards $6 and $7. Of particular importance is also the broad range of resistance of $6 to antibiotics. In fact, 26 antibiotics were tolerated by this strain (Table 3).
DISCUSSION
As pointed out by Trevors et al. (1985), there is a problem in defining exactly what is meant by resistance to heavy metals. Throughout this study, we have often preferred the term 'tolerance' to 'resistance'. Responses of fresh bacterial isolates to heavy metals were very heterogeneous. All isolates were Gram-negative bacteria. However, Foster (1983) has reported the resistance to heavy metals in both Gram-positive and Gram-negative bacteria. Mahler et al. (1986) have found that all isolates tolerant for cadmium and mercury were Gram-positive aerobic spore-formers, and very few of these strains were antibiotic-resistant. Duxbury (1981) reported that generally Gram-negative soil species appeared to be more tolerant than Gram-positive. This difference in
results could be explained by the conditions of each bacterial isolation and the selectivity of microbial culture techniques adopted in each study; particularly with respect to the nature and specificity of growth media. In the present study, the dominating bacterial isolates belonged to the genera Pseudomonas and Proteus. Generally, isolates identified within the same genera showed a certain heterogeneity in their biochemical characteristics and some variation in their colony morphology. This heterogeneity and variation were possibly due to the various origins of the isolates. Results of bacterial isolation have shown the abundance of Pseudomonas aeruginosa, P. paucimobilis and of Proteus rnirabilis, respectively. Strain $6 of P. aeruginosa, which had been isolated from raw wastewater of the city of Tunis, appeared as the most important isolate and could be selected as a bioindicator of toxicity. The ecological value of the sensitivity or resistance of strain $6 to metals lies in its ubiquitous presence in many habitats, in its capacity to grow in the presence of a relatively high heavy-metal concentration, and in its ability to release secondary metabolites, such as pyocines and pigments in the medium. Asparagine broth described by Highsmith and Abshire (1975) seems to be an ideal growth medium for P. aeruginosa for two reasons. First, asparagine broth has a relatively simple composition (DL-asparagine, 3 g/liter;
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13
KzHPO4, 1g/liter; MgSO4.7H20, 0.5g/liter) as compared with other traditional media and therefore a lower metal-binding-capacity with constituents of the media. Second, asparagine often enhances the biosynthesis of pigments by P. aeruginosa. As previously stated, metals and various components of nutrient media could interact, thus complicating interpretation of the data. The test of toxicity in solid media investigated here could be useful in the evaluation of metal toxicity in sewage sludges and contaminated soil, where conditions of diffusion, complexation and availability of metals were different from those observed in liquid media. The speciation of metals was a very important determinant in understanding the quantitative aspects of metal toxicity. The plate diffusion method suggested in this study is very practical and allows us to test up to eight strains by plate• Mercury, in spite of its weak solubility and diffusion due to its strong adherence to medium components by adsorption and complexation (Behel et al., 1983), appeared as the most toxic metal. It is interesting to note that the following decreasing order of toxicity: Hg > Co > Cd > Cu-Cr > Zn, does not differ from that reported in other studies (Duxbury, 1981)• The test in liquid media was sensitive at concentrations 10 to 1000 times lower than those obtained in solid media. In spite of its limits described previously, the liquid media test allowed a good evaluation of metal toxicity in polluted environments, such as industrial effluents, incinerator residues, landfill municipal refuse and sewage sludge leachates. Statistical calculation of the ratios between 'Average tolerated concentrations in solid media' and 'Average tolerated concentrations in liquid media' for each metal and each strain tested were made (results not shown). These ratios revealed that the power of metal adsorption and complexation could be classified as follows H g > C u > C d Zn > Co-Cr. These results appear very interesting, since it is well known that mercury, and to a lesser degree copper, possess a good capacity for adsorption and complexation (Behel et al., 1983) and therefore a possibility of limited migration in natural environments, such as soil, sewage sludge or sediments, is possible• A growing body of evidence indicates that metal tolerance and antibiotic resistance are often found together in many clinical isolates and that metal and antibiotic resistance are closely associated (Timoney et al., 1987). The very broad resistance of strain $6 of P. aeruginosa to antibiotics may indicate that the latter is rich in plasmids that carry simultaneous resistance to antibiotics and metals• The net fall of MICs after the fifth sub-culture, of isolated strains, showed, however, that resistance of bacteria to heavy metals was conditioned by the
14
A. Hassen et al.
presence or the absence of the metal in the medium. Indeed, the absence of metal from the bacterial environment reduced noticeably its power of resistance (Timoney et al., 1987) and the maintenance of a metal-resistant microbial community was likely to dependent on the presence of the metal in the growth medium (Mahler et al., 1986).
ACKNOWLEDGEMENTS
This investigation was supported by grant from the National Foundation of Scientific Research (ERESMAT, B14/91). We thank Professor Hans-W. Ackermann, Department of Microbiology, Faculty of Medicine, Laval University, Quebec, Canada, for helpful discussion and critical review of the manuscript.
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