Research Articles Heavy Metal Resistance

95© 2005 ecomed publishers (Verlagsgruppe Hüthig Jehle Rehm GmbH), D-86899 Landsberg and Tokyo • Mumbai • Seoul • Melbourne • ParisJSS – J Soils & Sediments 55555 (2) 95 – 100 (2005)

Research Articles

Multiple Metal Resistant Transferable Phenotypes in Bacteria as Indicators ofSoil Contamination with Heavy Metals

Robert P. Ryan, David J. Ryan and David N. Dowling*

Department of Science and Health, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland

* Corresponding author (david.dowling @itcarlow.ie)

(14%) isolates resistant to zinc, copper, nickel, arsenic and cobaltand site B had no bacteria resistant to all five of these selectedmetals. The transferability of heavy metal resistance was inves-tigated in the case of 60 multiple heavy metal resistant isolatestaken from site A and 50 multiple resistance isolates from siteB. Transfer was only detected in isolates from site A, 13% showedtransfer and expression of copper, zinc and arsenic resistancedeterminants. In most cases the transconjugants only expressedresistance to copper, zinc and arsenic, which were the metals,used for selection. This co-transfer of all three determinants sug-gests a genetic link between these resistance determinants.

Conclusion. Heavy metal resistant bacteria are present in bothsites, however, the number and presence of multiple transfer-able resistance phenotypes are confined to the isolates from theheavy metal contaminated site. The presence of high levels ofheavy metals selects these multiple resistance phenotypes. Withinthese communities there seems to be little diversity between themicroorganisms, which provides a hugely preferable environ-ment for gene transfer of such metal resistant determinants.

Recommendation and Perspective. The experiments have showna microbes ability to mobilize heavy metal determinants and arelationship between heavy metal resistance and metal contami-nation has been identified These multiple heavy metal resistantbacteria could eventually be used for detection and qualifica-tion of the level of heavy metal-polluted soil/water environments.

Keywords: Bacteria; gene transfer; heavy metal resistance; mi-crobial communities; multiple resistance; pollution; soil con-tamination

DOI: http://dx.doi.org/10.1065/jss2004.10.120

Abstract

Background, Aim and Scope. Environmental contamination byheavy metals affects microbial communities. The number of sin-gle and multiple heavy metal resistant bacteria may be an indic-tor of the level of contamination. This paper details the isola-tion and characterisation of metal resistant microorganismsisolated from rhizosphere/soil samples obtained from an aban-doned zinc, lead and copper mine and a local unaffected site.This data was compared to the level of heavy metal in the soilsto establish the effect of metals on the microbial communityand to determine the relationship between pollutant levels andresistant strains. This paper outlines the diversity of transfer-able resistance determinants between both sites and details thelevels of heavy metal resistant bacteria and those expressingtransferable multiple heavy metal tolerance.

Methods. The sample sites were located in Co. Galway, Ireland.The first sample site (site A) was a former lead, zinc and coppermine, which was closed in 1961 due to exhaustion of ore. Thesecond site (site B) was located two and a half kilometres fromthe mining site and was not affected by the mining operations.Composite soil samples were characterised for general soil ma-trix composition, organic content, pH and general chemicalparameters. The soil was also enumerated for the total viableheterotrophic counts and tested on Pseudomonas selective agar(PSA) for total Pseudomonas counts and Sucrose Asparagine(SA), which is semi-selective for fluorescent Pseudomonas.

Results and Discussion. Samples from both site A and site B wereanalysed by atomic absorption spectrophotometry for the pres-ence of heavy metals. In the case of copper, which has a Dutch listrecommended minimum permissible level of 190 µg/Kg dry weight,the levels detected at site A were 1270 µg/Kg dry weight whilesite B was detected at 36 µg/Kg. The arsenic levels detected at siteA were eight times the permissible level (416 µg/Kg) while onlyhalf the permissible level was found at site B (13 µg/Kg). Zincconcentrations were also high at site A (4460 µg/Kg) while atsite B (553 µg/Kg) they were well below the Dutchlist guidelines(720 µg/Kg). A large number of heavy metal tolerant strains wereisolated from both sites. 270 isolates (site A (170) and site B (100))were screened against 8 metals to examine the extent of multipleresistance. 82% of the strains from site A were found to be re-sistant to 5 metals. A total of 18% showed resistance to all 8metals and of those examined only 4% were resistant to onlyone metal. In contrast isolates from site B showed no multipleresistance to more than 5 metals, while 62% showed resistanceto individual metals only. Site A had a higher level of multipleheavy metal resistance strains. Stains isolated from site A had 23

Introduction

The introduction of heavy metals in various forms in theenvironment can result in considerable modifications of themicrobial communities and their activities (Nies et al. 1999).Heavy metals may exert an inhibitory action on microor-ganisms by blocking essential functional groups, displacingessential metal ions, or modifying the active confirmationsof biological molecules (Moffett et al. 2002). Numerous stud-ies have examined the heavy metal sensitivity or resistanceof bacteria isolated from different habitats and many mi-croorganisms show adaptation to the toxic metals to whichthey are exposed (Müller et al. 2001a).

In polluted environments, the response of microbial com-munities to heavy metals depends both on concentrationsand availability of metals, on biological factors such as thetype of metal, and on the nature of medium and microbe

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(Beining et al. 1996). Furthermore, the frequency of appear-ance of resistant bacteria to specific heavy metals may becorrelated with increasing loads of metals in the environ-ment. As a result heavy metal resistant bacteria may be usedas biological monitors or bio-indicators of environmentalcontamination (Ravina and Baath 2001, Richards et al.2001). Bio-indicators have been shown to be a sensitive andreliable tool in detecting the sub lethal toxicity of these pol-luting compounds (Trevors et al. 1985). A combination ofbioassays is increasingly recommended to gain an insightinto potential dangers associated with the disposal of suchtoxic compounds into the environment (Blaise et al. 1985).

In recent years, it has become obvious that chromosomaland plasmid borne determinants responsible for heavy metalresistance can transfer freely within an ecological system suchas soil (Rensing et al. 2001). For this reason bacteria in en-vironments contaminated with numerous heavy metals mayevolve or acquire a number of heavy metal resistance deter-minants, resulting in multiple resistant. Relatively little in-formation is currently available on the transfer rates associ-ated with these multiple resistance elements.

This report details the isolation of metal resistant bacteriafrom a number of different soil samples taken from an aban-doned lead, zinc and copper mine in Co. Galway, Ireland.The research was designed to observe shifts in microbialadaption in the case of a heavy metal polluted soil site whencompared to a non-polluted soil site. Although previous stud-ies have shown that heavy metal resistance is ubiquitous insoil (Clausen 2000), this paper evaluates and compares thepresence and mobility of such resistant determinants foundat both contaminated and uncontaminated sites.

This report also highlights the isolation of a significantlyincreased level of multiple metal resistant bacteria from thepolluted soil environments. The experiments were designedto monitor the evolution of similar multiple resistance pat-terns across communities after the long term exposure totoxic heavy metals. This study also examined the linkage ofheavy metal resistance determinants by investigating thetransferability patterns of metal resistances and the possiblecorrelation between the levels of toxic metals.

1 Materials and Methods

1.1 Soil analysis

The sample sites were located in the west of Ireland. The firstsample site (site A) was a former lead, zinc and copper mine,which was closed in 1961 due to exhaustion of ore. The mine

was opened in 1908 and was mined for 52 years in whichtime the copper, zinc and lead were exported for smelting.The second site (site B) was located two and a half kilometresfrom the mining site and was not affected by the mining op-erations. 250 g soil samples, down to a 10cm depth were col-lected from the experimental sites. The samples were com-posed of approximately 50% vegetation/rhizosphere and 50%soil for each sampling point. These samples were character-ised for general soil matrix composition, organic content, pHand general chemical parameters (Welch et al. 1980). The lev-els of heavy metal in the soil samples from site A and site Bwere determined using atomic absorption spectrophotometry.These concentrations were compared to the permissible levelsoutlined by European Dutchlist guidelines (Rossi 1990).

1.2 Culturable bacterial populations

10 g of each sample was added to sterile Ringers solutionand serial dilutions were plated on Luria-Bertani (LB) agarto enumerate the total viable heterotrophs, Pseudomonasselective agar (PSA) for total Pseudomonas count and Su-crose Asparagine (SA) semi-selective for fluorescent Pseu-domonas (Collins et al. 1998). Plates were incubated at 30°Cand 37°C for 24 and 48 hours respectively. The Miles andMisra drop count method (Collins et al. 1998) was used toenumerate metal resistant bacteria on selective LB agar, PSAagar and SA agar with heavy metal supplements at 30ºC(Table 1). A total of 170 isolates from site A and 100 iso-lates from site B were taken at random from the LB agarplates containing copper, zinc and arsenic supplements asrepresentative samples of heavy metal resistant bacteria.Replica plating (using an inoculum of 1 x 103 cfu/ml) wasused to screen these isolates on various concentrations ofheavy metals (see Table 1). Plates were incubated at 30ºCfor 24–48 hours (Collins et al. 1998). These were then ana-lysed and compared to determine their multiple metal re-sistance profile. Of the 170 resistant isolates from site A, 60multi-resistant strains were selected for further investiga-tion. Individual multiple resistant isolates were plated onincreasing heavy metal concentrations (see Table 1) to de-termine minimal inhibitory concentration (MIC) values. Nonmetal resistant strains E. coli JM109, B. subtilis, P. fluor-escens and P. putida were used as negative controls.

1.3 Plasmid screening

The 60 (multi-resistant (MR)) site A isolates and 50 of the(MR) site B isolates were also screened for metal resistanttransferable plasmids. Recipient strains used were E. coli

Heavy metals Plate concentrations Heavy metal MIC ranges Mercuric chloride (Hg) 250 µM 500 µM–5 mM

Nickel chloride (Ni) 2.5 mM 2 mM–5 mM

Potassium Tellurite (Tell) 2.5 mM 2 mM–5 mM

Zinc chloride (Zn) 2.5 mM 2 mM–7.5 mM

Cadmium chloride (Cd) 2.5 mM 2 mM–5 mM

Cobalt chloride (Co) 2.5 mM 500 µM–5 mM

Sodium arsenite (Ars) 10 mM 7.5 mM–11 mM

Copper chloride (Cu) 2.5 mM 500 µM–12 mM

Plate concentration = concentration of heavy metal which was frequency used as a selective agent for resistant bacteria in a particular plate

Table 1: Concentrations of heavy metals used in this study

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JM109, E. coli J53-1 (Nal 100 µg/ml) and P. putida (Amp100 µg/ml). Recipient strains were grown in LB broth, inthe presence of 50 µg ampicillin (Amp), 20 µg nalidixic acid(Nal) or high salt MacConkey. The broth cultures were in-cubated at either 30ºC or 37ºC with aeration on a rotary plat-form (100 rpm). The 110 donor strains were also cultured inLB broth containing the concentration(s) of heavy metal(s)outlined in Table 1. Conjugations were carried out using a setof liquid filter mating experiments as described by Dronen(1998). Transconjugants were selected using both the antibi-otic and heavy metal selection on LB agar plates. Transconju-gants were isolated and re-streaked on selective media plates.

2 Results

2.1 Heavy metal contamination

Both soil samples from site A and site B were deemed tohave (i) no sludge amendments, (ii) high vegetation amend-ments and (iii) high chance of heavy metal amendments.Both samples were composed of approximately 50% veg-etation/rhizosphere and 50% soil. The sample from site Ahad a soil matrix consisting of 8% clay, 50% silt and 42%sand, while the sample from site B consisted of 12% clay,50% silt/loam and 38% sand. Both soil samples were classedas sandy loam soil and had very little physical difference.Chemical analysis showed that the level of total nitrogen be-ing at site B was 0.092 ppm while at site A it was 0.075 ppm.The same was seen in the case of organic carbon with site Ahaving a lower level (0.245 ppm) while site B (0.746 ppm)was again higher. Inorganic levels of carbon were higher atsite A (2.145 ppm) than that of site B (1.895 ppm). How-ever, pH analysis showed a high acidity of the soil from siteA (pH 5.5) while the soil from site B was almost neutral(pH 7.1). Although the total nitrogen may not be statisticallydifferent, the organic carbon (0.245 vs. 0.746) and inorganiccarbon (2.145 vs 1.895) are different, this difference may notbe important in this study but would be very important whentrying to evaluate potential bioremediation applications.

Samples from both site A and site B were analysed by atomicabsorption spectrophotometry for the presence of heavymetals (Table 2). The level of each metal was compared with

the action limits outlined by the Dutchlist, which outlinesthe acceptable levels of acidity, heavy metals and oils presentin European soil and water. The sample from site A showeda high level of metal contamination well above the levelsoutlined in the Dutchlist. In the case of copper, which has apermissible level of 190 µg/l dry weight, the levels detectedat site A were 1270 µg/l dry weight while at site B was de-tected at 36 µg/l. The arsenic levels detected at site A wereeight times the permissible level (416 µg/l) while only half thepermissible level concentration was found at site B (13 µg/l).Zinc concentrations were also high at site A (4460 µg/l) whileat site B (553 µg/l) they were well below the Dutchlist guide-lines (720 µg/l). Site A also showed elevated levels of nickel,silver, tellurite, cadmium and cobalt (see Table 2). Site Bdisplayed metal levels well below those requiring actionoutlined by the Dutchlist (see Table 2).

2.2 Culturable heterotrophic aerobic bacterial populations

Total viable heterotrophic counts carried out on LB, totalPseudomonad count on PSA and total selectable fluorescentPseudomonas counts on SA were plated out for soil samplesfrom sites A and B (Table 3). In the case of site A, the totalviable count was 3.4 x 106 cfu/ml selected on LB media ofwhich 33% were Pseudomonas (1.1 x 106 cfu/ml) selectedon PSA media. Approximately 10% of the Pseudomonasfrom site A were P. fluorescens (9 x 103 cfu/ml) expressedon SA media. Counts on LB media from site B had a highernumber of organisms per gram of soil (2.1 x 107 cfu/ml)than site A. The total Pseudomonad count on PSA (1.1 x104 cfu/ml) only made up 0.05% of the culturable popula-tion found at site B. An even smaller proportion of strainsisolated from site B were fluorescent in nature when selectedon SA media with a total count of 2.5 x 103 cfu/ml.

A large number of heavy metal tolerant strains were iso-lated from both sites. The percentage of metal tolerant strainswas calculated with relation to the total viable count in thecase of each site (Fig. 1). A high number of resistance strainswere isolated from both sites with the majority of resistancedetected in strains isolated from site A. 79% of strains iso-lated from site A were resistant to either copper, zinc or

Metals Dutch list – (µg/l) Sample site A – (µg/l) Sample site B – (µg/l)

Arsenic 55 0.1 mM 416 13 mM 13 0.04 mM

Cadmium 12 0.4 mM 391 2 mM 6.6 0.02 mM

Cobalt 240 0.8 mM 106 0.4 mM 9 0.03 mM

Copper 190 0.9 mM 1270 7 mM 36 0.02 mM

Mercury 0.3 0.001 mM 4.2 0.02 mM 1 0.003 mM

Nickel 210 0.9 mM 238 1 mM 56 0.023 mM

Silver 5.6 0.001 mM 10 0.004 mM 4.4 0.001 mM

Tellurium 115 0.28 mM 171 0.45 mM 100 0.27 mM

Zinc 720 2.4 mM 4460 15 mM 553 1.9 mM

Medium Sample site A Standard Deviation Sample site B Standard Deviation TVC on LB 3.4 x 106 2.5 2.1 x 107 22.4

TVC on PSAa 1.1 x 106 25.7 1.1 x 104 14.3

TVC on SAb 9 x 103 17.2 2.5 x 103 11.9

TVC = Total Viable Count CFU/g soil (wet weight); a PSA semi-selective medium for Pseudomonads; bSA semi-selective medium for P. fluorescens group

Table 2: A comparison of the Dutchlist permissible levels of metal and those found in both soil sample sites A and B

Table 3: Aerobic heterotrophic culturable bacterial counts on different media

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arsenic (see Fig. 1). The level of metal resistance at site Branged between 30 to 35% in the case of copper, zinc andarsenic (see Fig. 1).

2.3 Multiple heavy metal resistance

270 isolates (site A (170) and site B (100)) were screenedagainst 8 metals to examine the extent of multiple resist-ance. 82% of the strains from site A were found to be resist-ant to 5 metals. A total of 18% showed resistance to all 8metals and of those examined only 4% were resistant toonly one metal. In contrast, isolates from site B showed nomultiple resistance to more than 5 metals, while 62% showedresistance to individual metals. It can be seen in Fig. 2 thatsite A has a higher level of multiple heavy metal resistancestrains. Site A having 23 (14%) isolates resistant to zinc,copper, nickel, arsenic and cobalt and site B having no bac-teria resistant to all five of these selected metals.

Further characterisation of the multiple resistant bacteria fromsite A was carried out. A random selection of 60 isolateswere identified as Gram- negative and characterised (Collinset al. 1998). These strains were isolated on 2.5 mM copper,

2.5 mM zinc and 10 mM arsenic therefore an MIC range of2.5–11 mM was examined for each metal (see Table 1). 12strains showed resistance levels to 10mM copper chloride.In many cases halos of blue deposits were seen in the sur-rounding media possibly indicating metal precipitation. 21of the isolates were resistant to zinc at levels of 5 mM wherethe MIC range for zinc chloride was 2–7.5mM. In the caseof some of the zinc resistant isolates on LB agar black de-posits were seen in the surrounding media, again possiblyindicating metal precipitation. The MIC range examined forarsenic was 7.5–15 mM and 41 of these isolates showedgrowth on a concentration of 14 mM arsenic. A total of 32isolates were resistant to low levels of cobalt (1 mM), nickel(2 mM), tellurite (2 mM), cadmium (2 mM) and mercury(500 µM) as described in Table 1. It was observed that mostof the multiple resistant isolates showed similar tolerance tocobalt, cadmium, nickel and zinc.

2.4 Multiple heavy metal resistance phenotype transfer

The genetic transfer of heavy metal resistance was investi-gated in the case of 60 multiple heavy metal resistant iso-lates taken from site A and 50 multiple resistance isolates

0

10

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40

50

60

70

80

90

100

Per

cen

tag

e m

etal

res

ista

nce

Tell -1mM

Hg -50uM

Ars -10mM

ZnCl -2.5mM

Ni -2.5mM

Co -2.5mM

Cu -2.5mM

Cd -2.5mM

Metal concentrations

site A site B

Fig. 1: Percentage of microbes displaying heavy metal resistance isolated from soil/root samples from sample sites in Co. Galway, Ireland

Fig. 2: Venn diagrams outlining the level of multiple heavy metal resistance from sample site A and sample site B

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JSS – J Soils & Sediments 55555 (2) 2005 99

from site B. These isolates were conjugated with a numberof different recipients (Table 4). Transconjugants werescreened on 2.5 mM Cu, 2.5 mM Zn and 10mM Ars. 36%of the isolates from site A showed high frequency transfer ofmetal resistance. However no transconjugants were obtainedusing isolates taken from site B. The rate of transfer from do-nors taken from site A was approximately 103 to 104 cfu/ml.The number of transconjugants isolated using site A strainswas 1 x 103 cfu/ml in the case of E. coli J53-1 and 2.6 x 103

cfu/ml in the case of E. coli JM109 and 7.1 x 104 cfu/ml whenP. putidia was used as a recipient. A total of 194 transconju-gants were isolated showing resistance to a heavy metal fromsite A yielding an average coefficient of transfer between0.02–0.1 in the case of E. coli recipients. This was a littlehigher in the case of P. putidia (0.64–1.5).

A total of 46% of recipients received resistance to Zn (2.5 mM),37% showed resistance to Cu (2.5 mM) and 22% showedresistance to Ars (10 mM). Of these heavy metal resistancerecipients, 34% showed resistance to both Zn (2.5 mM) andCu (2.5 mM). Again 12% of the isolated recipients showedresistance to both Zn (2.5 mM) and Ars (10 mM), while 19%showed resistance to both Cu (2.5 mM) and Zn (2.5 mM)and 7% showed resistance to Cu (2.5 mM) and Ars (10 mM).Only 13% of the isolated transconjugants showed resistanceto all three selected metals (Cu (2.5 mM), Zn (2.5 mM) andArs (10 mM)).

In the case of the matings plated on Zn (2.5 mM), a total of20% of the transconjugates were isolated from E. coli J53-1,18% from E. coli JM109 and 61% from P. putidia recipi-ents. A total of 66 transconjugates were isolated frommatings plated on Cu (2.5 mM) where high levels of E. coliJ53-1 (10%), E. coli JM109 (28%) and P. putidia (65%) re-cipients were found. Finally in matings plated on Ars (10 mM)a total of 39 transconjugates were isolated, 33% from E. coli

JM109, none from E. coli J53-1 and 67% from P. putidia.The three metal resistant determinants that showed high fre-quency of transfer were arsenic, copper and zinc. These metalresistant transconjugants were further screened for othersubsequent metal resistant transfer detailed in Table 4. Ofthe other metals screened it was seen that nickel and cad-mium were also seen to transfer as a result of conjugation.However, mercury and tellurite resistant determinants whichwere found in abundance in the parent donor strains showedpoor transfer to the recipients. 23 strains showed transfer ofall three metal resistance determinants copper, zinc and ar-senic, of these, 10 strains transferred the phenotype to allthree recipients. A total of 23 parent strains were identified,69% of these strains were Pseudomonad with 29% beingidentified as Pseudomonas fluorescens.

3 Discussion

The rhizophere /soil samples from both site A and site Bwere both classed as sandy loam soil similar in chemicalcomposition and physical structure. The main differencebetween both sites was the low pH (5.5) and the high levelsof metals found at site A, while site B had a neutral pH (7.1)and relatively low quantities of metals (see Table 2). Theacidity found in the soil is most likely attributed to the highlevels of metal (Ledin 2000).

Overall the soil microbial community was different betweenthe two sites reflecting a selection for heavy metal resistantbacteria at site A. Site A showed a predominance ofPseudomonads. This observation is consistent with Barkayet al. 1986, who studied the effect of metal rich sewage onbacterial communities of grasslands and observed the diver-sity of the culturable bacterial populations differing at thecontaminated site reporting a lack of bacterial diversity insamples with metal contamination.

Transconjugant Mercury 1mM Arsenic 7.5mM Cobalt 2.5mM Copper 2.5mM Tellurite 1mM Zinc 2.5mM T1 1.2 x 102 1.2 x 103 0.7 x 102 1.4 x 104 1.8 x 102 2.8 x 102 T2 ND 0.7 x 103 ND 1.5 x 102 ND 3 x 102 T3 ND 0.8 x 104 ND 2.1 x 103 ND 4.1 x 103 T4 ND 1.9 x 102 ND 3.1 x 103 ND 6.1 x 102 T5 ND 2.1 x 105 ND 6.0 x 103 ND 12 x 103 T6 ND 3.1 x 102 ND 1.2 x 105 ND 2.4 x 105 T7 1.4 x 102 4.1 x 103 3.1 x 103 9.5 x 103 1.4 x 102 1.9 x 103 T8 ND 1.1 x 103 ND 0.6 x 102 ND 1.2 x 102 T9 ND 3.9 x 103 ND 0.7 x 102 ND 1.4 x 102 T10 ND 2.6 x 103 0.7 x 102 0.2 x 103 ND 0.4 x 103 T11 ND 1.2 x 105 ND 0.4 x 103 ND 0.8 x 103 T12 ND 1.8 x 103 ND 0.8 x 102 ND 1.6 x 102 T13 ND 1.6 x 102 ND 0.7 x 103 ND 3.2 x 103 T14 ND 1.7 x 103 ND 1.6 x 103 ND 1.6 x 103 T15 1.3 x 102 2.9 x 103 1.3 x 102 9.2 x 103 ND 1.8 x 104 T16 1.1 x 102 3.2 x 103 ND 1.8 x 102 1.3 x 102 3.6 x 102 T17 ND 1.7 x 102 ND 1.5 x 103 ND 3.0 x 103 T18 ND 1.5 x 103 ND 1.6 x 103 ND 3.2 x 103 T19 ND 1.3 x 103 ND 1.9 x 104 ND 3.8 x 104 T20 ND 1.4 x 104 2.1 x 102 1.2 x 103 ND 2.4 x 103 T21 ND 2.2 x 103 ND 1.4 x 105 4.6 x 102 2.8 x 105 T22 ND 6.2 x 102 ND 3.9 x 103 ND 7.8 x 103 T23 ND 9.2 x 103 ND 4.6 x 102 ND 9.2 x 102 ND = not detected

Table 4: Frequency of transfer of metal resistant determinants Ars, Cu and Zn into E. coli JM109 from the environmental isolates

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Both sites had populations of bacteria that were resistant toheavy metals (see Fig. 1). However, the populations isolatedat site A had a greater percentage of isolates resistant to zinc(82%), copper (69%) and arsenic (79%) and this site alsohas the highest number of metal resistance isolates. Resist-ance to metals such as cobalt (33%) and cadmium (24%)were much lower in comparison. In contrast, site B had asignificantly lower percentage of heavy metal resistant iso-lates: zinc (35%), copper (34%) and arsenic (22%), cobalt(12%) and cadmium (3%).

The concentration of metal contamination appeared to correlatewith the percentage of resistant bacteria. At both sites zinc, cop-per and arsenic were the predominate contaminant metals (seeTable 2). This was reflected in the percentage of strains resistantto these metals (see Fig. 1). Less of the strains were resistant tocobalt and cadmium this may reflect the lower concentrationof these metals at both sites. This is consistent with work car-ried out by Müller et al. (2001b), where bacterial communitiessubjected to increasing levels of mercury (< 1 mM) showed anincreasing level of mercury tolerance (1 x 105 cfu/g soil).

Isolates from site A showed a higher percentage of bacterialisolates encoding multiple resistances than that of equivalentresistant isolates taken from site B. This is clearly portrayed inthe Venn diagrams showing the relationship between the fivechosen metals (zinc, nickel, copper, arsenic and cobalt) (seeFig. 2). The greater level of multiple resistances found at site Amay be attributed to the higher levels of metal contaminationin the soil. In the case of bacteria isolated from site A, heavymetal resistance determinants must have been recruited in or-der to survive in such environments (De Lorenzo et al. 1998).In the case of the three major polluting metals at site A, cop-per, zinc and arsenic, 28% of strains were resistant to all threeof these metals. Timoney et al. (1987) noted a similar responseduring work on bacterial flora of sediments where sedimentcontaminated with zinc and lead showed increased zinc andlead resistant bacterial communities. In contrast, metals withlower concentrations found at site A such as cobalt, telluriteand nickel had fewer resistant isolates. Quite the opposite wasseen in the number of multiple resistant bacteria from site Bwere low with only 3% resistant to copper, zinc and arsenic.Other evidence, of multiple resistance relating to concentra-tion of metal was seen in that the majority of isolates from siteB were only resistant to individual metals (see Fig. 2).

Over a third (36%) of the multiple resistant isolates used inthe transfer studies from site A showed transfer of metalresistances between donor and recipient. The samples takenfrom site B showed no transferable resistance phenotypesindicating that a high percentage of heavy metal resistancedeterminants found in site A were encoded by transferableelements whereas heavy metal resistances in strains at site Bwere not mobile in this experiment.

A high rate of transfer to all three recipients was observedfrom isolates acquired from site A. E. coli JM109 and E. coliJ53-1 both had very similar levels of acceptance and expres-sion of zinc and copper resistance phenotypes. E. coli JM109did receive arsenic determinants while E. coli J53-1 did not. P.putida was the best recipient for zinc resistant determinants.This may be attributed to the high percentage (33%) ofPseudomonads that made up the total population, which al-

lowed for efficient horizontal transfer of heavy metal resist-ance genes within this group. Of the total transconjugants iso-lated in these experiments, 13% showed re-transfer and ex-pression of copper, zinc and arsenic resistance determinants.In most cases the transconjugants only expressed resistance tocopper, zinc and arsenic, which were the metals, used for se-lection (see Table 4). This co-transfer of all three determinantsindicates a genetic link between these resistance determinants.

In conclusion, heavy metal contamination provides a strongpressure that selects for the recruitment multiple resistancephenotypes that encode resistance to the predominant met-als in the site and a high proportion of these are likely to beencoded by highly mobile genetic elements. In soils with highlevels of heavy metals closely related heterotrophic resistantbacteria constituted a substantial fraction of the aerobicmicroflora. Selected multiple heavy metal resistant isolatesmay eventually be used for the detection and quantificationof the levels of heavy metal-polluted soil/water environmentsand this is the focus of continuing research by the authors.

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Received: July 29th, 2004Accepted: October 21st, 2004

OnlineFirst: October 22nd, 2004