a novel nickel resistance determinant found in sewage-associated bacteria

Microb Ecol (1997) 33:230–239

q 1997 Springer-Verlag New York Inc.

A Novel Nickel Resistance Determinant Found in Sewage-Associated Bacteria

R.W. Pickup,1 H.E.H. Mallinson,1 G. Rhodes,1 L.K. Chatfield2

1 Institute of Freshwater Ecology, Windermere Laboratory, Far Sawrey, Ambleside, Cumbria, LA22 0LP, UK2 Department of Applied Biology, Univesity of Central Lancashire, Preston, Lancashire PR1 2TQ, UK

Received: 30 January 1996; Revised: 8 May 1996

A B S T R A C T

Nickel-resistant bacteria were isolated from effluent discharged from a sewage treatment outfall

over an 18-month period. One of these strains, Enterobacter cloacae FBA30, was found to harbor

a narrow host range conjugative plasmid, designated pFBA30, which confers nickel resistance on

its host. A 10.2-kb SstI restriction fragment was cloned from pFBA30 and was shown to specify

inducible nickel resistance, both in its original host and in laboratory strains of Escherichia coli.

This DNA fragment, and a 1.75-kb SmaI fragment derived from it, were used as probes to examine

other bacterial strains isolated during the study. Homologous nickel resistance genes were detected

in enteric bacteria emerging directly from the treatment plant, but not in strains isolated from

sediments downstream of the outfall, or in strains harboring well-characterized determinants such

as ncc/nre, cnr, or Klebsiella oxytoca type. Thus, this element constitutes a new nickel resistance

determinant and was named nrf.

Nickel-resistant strains were sorted into two groups, based on the sampling date and the size of

restriction fragments homologous to probes developed from pFBA30. Group A isolates all carried

a homologous 6.5-kb PvuII restriction fragment and expressed nickel resistance constitutively.

Group B strains, isolated independently from the same outfall, were different from group A in that

the nickel resistance was associated with a 4.7-kb PvuII fragment, and group B strains displayed a

slower growth rate on nickel salts media. Each group contained a range of enteric bacterial species,

including K. oxytoca, Citrobacter freundii, and Enterobacter spp. Localized genetic exchange probably

occurs within wastewater treatment effluents, and, once effluents are discharged into the environ-

ment, enteric bacteria carrying pFBA30-like determinants do not persist downstream.

Correspondence to: R.W Pickup. E-mail: [email protected].

79050E$P18 03-31-97 14:35:19

Nickel Resistance in Sewage-Associated Bacteria 231

treatment increases the proportion of bacteria that carryIntroductioneither single or multiple antibiotic resistances; between 0.1

and 1% of total coliforms in fecal material were resistant,Plasmid-encoded nickel resistance in bacteria has been char-and this reached frequencies of 10% in urban wastewater,acterized most extensively in Alcaligenes eutrophus CH3450% in river water, and 80% in potable water [16]. Bacteria[21]. This strain harbors the conjugative plasmid pMOL28that carry resistance to multiple antibiotics have also beenwhich confers resistance to a range of metals including nickelfound in both drinking water and wastewater [2, 3]. Further-and cobalt. Resistance is mediated by an inducible energy-more, chlorination of water has been shown to select fordependent specific efflux system [11, 25, 34, 35, 36, 41].multiple antibiotic resistance [23]. Resistance to heavy met-Extensive molecular characterization of the nickel efflux sys-als, linked to that of antibiotics, has been found not only intem was achieved by cloning the resistance gene (cnr) frombacteria isolated from sewage effluent [41] but also in bacteriapMOL28 [15, 37]. Most related studies have used nickel-from hospitals [19], metal-contaminated estuaries [1], andresistant strains isolated from anthropogenically pollutedsoils [18]. Bacteria affected by either water or wastewatersites, particularly those associated with the metal-reclamationtreatment are eventually released into aquatic environments.industries [20, 39], and those where nickel percolates natu-Almost all investigations of antibiotic resistance in the aquaticrally through an ecosystem [39]. Resistance levels conferredhabitat have concentrated on its incidence in bacteria of fecal

by these plasmid- or chromosomally located genes rangeorigin [5, 12–14]. However, a positive correlation was noted

from 1 to 40 mM in Tris-gluconate media [31, 33].between the occurrence of heavy metal and multiple antibiotic

There is some diversity to be found among nickel resis-resistance in a range of bacteria isolated from water distribu-

tance determinants. Some nickel resistance genes show a hightion and treatment systems [7]. The aim of this study was to

degree of homology with cnr of pMOL28 and ncc of A.develop nucleic acid probes to examine the nickel-resistant

xylosoxidans 31A [32, 33], while others have been found tobacterial community being dispersed from a wastewater treat-

carry a second resistant determinant (nre) that has only lim-ment works, and to assess whether metal resistance transfers

ited homology with pMOL28. The nre determinant expressesto the bacterial community downstream of the outfall pipe.

in E. coli and is thought to be widespread among nickel-

resistant bacteria [32]. Comparative analysis of a large num-

ber of nickel-resistant isolates enabled Stoppel and SchlegelMaterials and Methods[39] to distinguish four groups of nickel resistance. TheseBacterial Strains and Plasmidscomprised (1) those showing homology with cnr of pMOL28

[17] and ncc from pTOM (33); (2) a combination of cnr, The strains used in this study are described in Table 1. All nickel-

resistant strains were maintained on normal-strength nutrient agarncc, and nre [32]; and (3) a determinant found in K. oxytoca,(Oxoid, Basingstoke UK) supplemented with 5.0 mM nickel chlo-showing weak homology with nre [40]. These loci were notride. Escherichia coli HB101 and JM103 containing recombinantubiquitous, however, as indicated by the existence of a fourthplasmids were maintained on nutrient agar supplemented with 50

group of resistant strains exhibiting no sequence homologymg ml21 ampicillin. All other strains were maintained on nutrient

with either cnr/ncc or nre [32]. agar.The occurrence of metal resistant bacteria in anthropo-

genically polluted sites is well documented [10, 38]. Studies Sampling and Isolation of Bacteriaof bacteria emerging from a sewage treatment plant in the

Church Beck is a freshwater stream in the Coniston Fells, Cumbria,English Lake District showed the presence of a range of metal

UK and has been described previously [26, 27]. The sample siteresistances, which included nickel [26] and copper [27]. No was the sewage outfall in the lower section of Church Beck whichselection for resistance due to metal contamination was ap- feeds directly into Coniston (National Grid Reference SD 308971).

Bacteria were isolated from 11 samples of water, as described byparent at this site, since this treatment system received onlyPickup [26], except that filters were placed on nutrient agar supple-domestic inputs [26]. It has been noted previously that resis-mented with 5 mM NiCl2•6H2O (pH 7.6). Sediments were collectedtance to heavy metals occurs at a surprisingly high frequencydownstream of the outfall pipe in glass containers, vigorously shaken

in the absence of sources of direct selection [38]. in the laboratory, and allowed to settle. The supernatant was thenSewage effluent, wastewater, and water treatments have filtered and strains were isolated in the same way as the water

samples. Plates were incubated at 208C until single colonies ap-been shown to have several effects on bacteria. Wastewater

79050E$P18 03-31-97 14:35:19

232 R.W. Pickup et al.

Table 1. Bacterial strains and plasmids

Strain designationused in this study Species name and strain number Plasmid name/(content) Descriptiona Origin

FBA30 Enterobacter cloacae pFBA30/pFBA31 (2) inducible Nir This studyFBA30-1 Escherichia coli HB101 pFBA30 (1) inducible Nir This studyFBA30-2 Enterobacter cloacae NCIB 8151 pFBA30 (1) inducible Nir This studyFBA30-3 Enterobacter aerogenes pFBA30 (1) inducible Nir This studyFBA30-4 Serratia rubidae pFBA30 (1) inducible Nir This studyFBA13 Klebsiella oxytoca (group A) 0 constitutive Nir This studyFBA17B Enterobacter sakazakii (group A) not named (2) constitutive Nir This studyFBA28 Enterobacter cloacae (group A) not named (2) constitutive Nir This studyFBA1.3.21 Citrobacter freundii (group B) not named (1) inducible Nir This studyFBA1.4.2 ndb (group B) not named (1) inducible Nir This studyFBA1.5.43 Enterobacter agglomerans (group B) not named (1) inducible Nir This studyFBA30-10 Escherichia coli JM103 pFBA30-10 (1) inducible Nir This studyFBA30-11 Escherichia coli JM103 pFBA30-11 (1) inducible Nir This studyFBA30-12 Escherichia coli JM103 pFBA30-12 (1) constitutive Nir This studyNCIMBc8151 Enterobacter cloacae (0) Nis NCIMBHB101 Escherichia coli HB101 (0) Smr Nis 6JM103 Escherichia coli JM103 (0) Nis 43PaW340 Pseudomonas putida PaW340 (0) Smr trp2 42M695 Escherichia coli JM109 pSK2::KOHI4-1 Nir K. oxytoca 40M696 Escherichia coli JM109 pSK2::TBA9a Nir nre/ncc 32,33M697 Escherichia coli JM109 pHLI4 Nir cnr 17

a trp, Tryptophan; Nir, nickel resistance; Smr, streptomycin resistanceb nd, Not determinedc NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen

peared. All colonies were purified and retained. API identification ml21) were use as the selective medium. Single colonies were isolated,

purified, and examined for the presence of plasmids.strips (Biomerieux Ltd., Basingstoke, UK) were used, as directed

by the manufacturer.

DNA ManipulationsBacterial Conjugation

Plasmids from environmental isolates and transconjugants wereExogenous Plasmid Isolation. Exogenous plasmid isolation was

extracted using the method of Wheatcroft and Williams [42]. Totalmodified from the method of Bale et al. [4]. Water/effluent samples

genomic DNA was extracted using the method of Pitcher et al.of 50, 100, and 500 ml were mixed with 1 ml of recipient strain

[29], and recombinant plasmids were prepared by the method ofculture (PaW340 or HB101) and filtered through a 0.22-mm Milli-

Close and Rodriguez [8]. Plasmids used for genetic manipulationpore HC filter under vacuum. Filters were incubated on nutrient

were purified by SDS lysis/CsCl centrifugation [30].agar at 308C overnight. The accumulated growth was resuspended

Restriction endonucleases were obtained from Gibco BRL (Pais-in sterile distilled water, serially diluted, and spread onto nutrient

ley, UK). Agarose gel electrophoresis was carried out by establishedagar plates supplemented with NiCl2 (5 mM) and streptomycin (100

procedures. Reaction conditions for DNA ligation with T4 DNAmg ml21). Plates were examined for single colonies after incubation

ligase were as recommended by the supplier (Gibco BRL). Transfor-at 308C. Appropriate donor (water/effluent sample) and recipient

mation into E. coli JM103 was achieved by the procedure of Cohencontrols were performed simultaneously.

et al. [9]. DNA probes (either from purified DNA or from restriction

fragments excised from low melting temperatures gels, as directedEndogenous Plasmid Transfer. Conjugation experiments were per-

formed using a plate mating method with purified donor strains by the manufacturer) were labeled with [32P]dCTP using a Random

Primed DNA Labelling Kit (Boehringer, Lewes UK). pUC19 geneisolated from the sewage outfall, as described by Pickup [28], using

both PaW340 and HB101 as recipients. To facilitate screening a banks were screened by the method of Close and Rodrigues [9].

DNA hybridization was carried out as described previously [22].large number of isolates, streak matings were used. The recipient

was streaked vertically down the left side of the selection plate.

Donors were then streaked horizontally, in one movement, from Induction Studiesthe left to the right side through the recipient streak. The donor

streak to the left of the recipient served as a control. Nutrient agar Bacteria were cultured overnight in 50 ml normal-strength nutrient

broth and in 50 ml nutrient broth supplemented with 0.5 mMplates supplemented with NiCl2 (5 mM) and streptomycin (100 mg

79050E$P18 03-31-97 14:35:19


100 isolates, in total, were retained from sediments down-

stream of the outfall during sample trips 2 and 3. The concen-

tration of nickel-resistant isolates in the sediments was not

determined. Of the sediment isolates, 60% were confirmed

as oxidase negative.

Conjugation Experiments

An attempt was made to isolate conjugative nickel-resistant

plasmids from environmental samples by transfer to labora-

tory strains of Pseudomonas putida (PaW340) and E. coli

(HB101). No transconjugants were isolated by this method

when filtered bacteria from sewage sample 1 or either of the

sediment samples were used as donor populations, so purified

strains isolated from sample 1 were examined further by

individual streak matings. Again, no transconjugants were

observed when PaW340 was used as a recipient, but a single

isolate regularly yielded nickel-resistant transconjugants in

matings with HB101. This strain was designated FBA30. API

identification and analysis of plasmid DNA confirmed that

the colonies were true transconjugants. The donor isolate

was identified as Enterobacter cloacae (probability .99%)

using API test strips. Plasmid analysis of the donor strainFig. 1. HindIII digestion of plasmids found in FBA30 and trans-

conjugants. Lanes: 1, l (HindIII); 2, FBA30(pFBA30:pFBA31); 3, (FBA30) revealed two plasmids (designated pFBA30 andE. coli JM103(pFBA30); 4, E. cloacae NCIMB 8151(pFBA30); 5, E. pFBA31; Fig. 1). Comparison of the plasmid content ofaerogenes(pFBA30); 6, Serratia rubidae(pFBA30).

FBA30 and the transconjugants obtained by plate matings

revealed that one plasmid (pFBA30) had transferred with a

frequency of 1.9 3 1023 transconjugants per donor cell, andNiCl2 (previously determined to be a nonlethal concentration), with

was stably maintained (Fig. 1). FBA30-1 was retained asshaking (100 rpm) at 308C. The optical density at 600 nm was thenrepresentative of the transconjugants. Plate matings withmeasured (Cecil Instruments CE292 Digital UV Spectrophotome-

ter), with nutrient broth as a blank. Each culture was diluted in FBA30 and FBA30-1 showed that plasmid pFBA30 was trans-nutrient broth to an optical density of 1.0 at 600 nm. After adjust- missible between enteric bacteria (E. cloacae NCIB8151, E.ment, 1 ml of each culture was inoculated into duplicate flasks aerogenes, Serratia rubidae), but not to nonenteric strains (P.containing 50 ml nutrient both supplemented with 2.5 mM NiCl2. putida PaW340, P. fluorescens). Transconjugants harboringThese were incubated at 308C with shaking (100 rpm). The optical

pFBA30 were isolated and purified from these matings. Thesedensity of each was measured hourly for a total of 8 h after inocu-strains were designated FBA30-2, FBA30-3, and FBA30-4,lation.

respectively (Table 1). Restriction endonuclease digestion

with a range of enzymes revealed pFBA30 to be approxi-Results mately 55 kb in size (data not presented).Isolation of Nickel-Resistant Strains

Cloning of Nickel Resistance Determinant from pFBA30The sewage outfall was sampled three times (samples 1, 2,

and 3) at intervals of 6 months. Bacteria resistant to 5 mM Plasmid DNA from pFBA30 was isolated and digested with

SstI. Resultant DNA fragments were ligated into pUC19,NiCl2 were readily isolated on each occasion at a concentra-

tion of approximately 1,000 cells l21. In the first instance, and recombinant plasmids were transformed into JM103. All

clones were tested for nickel resistance, and a single, nickel-over 100 isolates were retained from sample 1. A total of 100

isolates from samples 2 and 3 were also retained for further resistant colony was obtained. The recombinant plasmid in

this strain was found to carry an inserted 10.2-kb SstI restric-analysis. All were confirmed as oxidase negative. A further

79050E$P18 03-31-97 14:35:19


tion as that of pFBA30-10, while that of pFBA30-12 (Fig. 2c)

has the reverse configuration.

When tested on nickel-supplemented media, FBA30,

JM103(pFBA30), and JM103(pFBA30-10) were resistant to

7.5 mM Ni in nutrient agar. JM103(pFBA30-11) and JM103

(pFBA30-12) were resistant to only a lower concentration (5

mM). When Tris-buffered gluconate minimal media (TMM)

was used [21], FBA30 retained its resistance profile while

those plasmids with E. coli HB101 or JM103 as a host showed

proportionally reduced levels of resistance: pFBA30-1 and

pFBA30-10 at 2.5 mM and the subclones (pFBA30-11 and

pFBA30-12) at 1 mM. This is because the nutrient agar may

act as a complexing agent enhancing nickel availability and

subsequent inhibitory effects in TMM [31, 33]. No resistance

to antibiotics, CoCl2, or other metals was observed.

Development of Probe for Nickel-Resistance Genes

A total of 500 isolates from treated sewage and sediment

were probed in hybridization experiments with the complete

pFBA30-10 plasmid bearing the 10.2-kb SstI restriction frag-

ment. Of the 400 sewage isolates, 95 showed some homology

to pFBA30-10 under conditions of low stringency, but of

these, only 32 isolates showed homology under highly strin-

gent conditions. The latter were retained. None of the isolates

from sediment showed any significant level of hybridizationFig. 2. Restriction map of the DNA insert in pUC19 associated

with nickel resistance: (a) represents pFBA30-10 with a 10.2-kb SstI to pFBA30-10, even under conditions of low stringency. Thefragment cloned into the multiple cloning site (MCS) of pUC19; subclone pFBA30-11 was developed as a probe against thethe shaded area indicates the 2.3-kb PvuII fragment subcloned in 32 isolates by digestion with PvuII and SmaI. This yielded apUC19; (b) and (c) represent pFBA30-11 and pFBA30-12 subclones

1.75-kb SmaI fragment and a 0.5-kb PvuII-SmaI fragment.of pFBA30-10 carrying a 2.6-kb PvuII fragment that is still able to

Each was used as a nucleic acid probe. The 0.5-kb fragmentconfer nickel resistance. D, DraI; H, HindIII; P, PvuII; S, SalI; Sm,did not hybridize with any of the 32 isolates showing homol-SmaI; S, SstI.

ogy to pFBA30-10. The larger probe of 1.75 kb hybridized

strongly with a number of isolates under conditions of high

stringency. However, reconfirmation of their status revealedtion fragment, which conferred resistance to NiCl2 on its E.that only 6 isolates consistently hybridized to the 1.75-kbcoli host. A comparison of restriction endonuclease fragmentsfragment. The fragments showing homology to the 1.75-kbfrom both plasmids, and hybridization to pFBA30 DNA (dataSmaI fragment were sized from a PvuII digest of total DNAnot presented) confirmed that this plasmid originated fromfrom each isolate and were shown to be divisible into twopFBA30. The fragment was examined for internal restrictionclasses, distinguishable by the sampling date. Group A com-sites, and a restriction map compiled for DraI, HindIII, PvuII,

prised isolates FBA13, FBA17B, and FBA28, each containingSalI, and SmaI (Fig. 2a). The plasmid was designated

a homologous fragment of approximately 6.5 kb. Each ofpFBA30-10. Nickel resistance was retained by subclones de-

the group B isolates (FBA1.3.21, FBA1.4.2, and FBA1.5.43)rived from pFBA30-10 by digestion with PvuII and subse-

possessed a PvuII restriction fragment of 4.7 kb that alsoquent religation. Transformation of re-ligated DNA into

showed significant homology with the 1.75-kb probe (Fig.JM103 yielded two such clones, here designated pFBA30-

3). Of the group A and B strains, only FBA13 contained no11 and pFBA30-12. Each carries a 2.3-kb PvuII restriction

observable plasmids (Table 1). Hybridization experimentsfragment which occupies opposite orientations in each plas-

mid. The insert in pFBA30-11 (Fig. 2b) has the same orienta- with plasmids from these strains suggest that the homologous

79050E$P18 03-31-97 14:35:19


sequences appeared to be located on the chromosome in

each case as hybridization could not be matched to any of

the plasmid bands (data not presented).

No homology was detected between pFBA30-11 and

pMOL154 which contains the structural cnr genes from

pMOL28 (Fig. 1). Similarly, no homology was detected with

plasmids carrying ncc/nre, cnr, or the determinant from K.

oxytoca [32, 33, 40] (data not presented).

Identification of Isolates Showing Homology to pFBA30

All nickel-resistant isolates that showed homology with

pFBA30 were found to be oxidase negative, but presented

different profiles when tested with the API identification

system. Group A strains were tentatively identified as K.

oxytoca (strain 13; probability 85%), E. sakazalai (strain 17B;

probability 97%), and E. cloacae (strain 28; probability 99%).

Group B included E. agglomerans (strain 1.5.43; probability

85%), C. freundii (strain 1.3.2; probability 85%), and strain

1.4.2 which, although not identifiable by the API test strips,

presented a different profile from the other strains. These

strains thus represent a diverse population of enteric bacteria,

but no one strain predominated either group A or B. Growth

on nickel-supplemented media showed that all strains were

resistant to 7.5 mM Ni in nutrient broth. None demonstrated

resistance to low concentrations up to 1 mM CoCl2.

Inducibility of Nir

FBA30 and laboratory E. coli strains haboring pFBA30 or a

derivative plasmid were examined for the inducibility of

nickel resistance by preculturing strains in the presence of a

nonlethal concentration of Ni (0.5 mM) prior to inoculation

into nutrient broth supplemented with 2.5 mM Ni. Control

strains, E. cloacae NCIB8151 and E. coli HB101, showed no

growth after 8 h.

The growth profile of FBA30 indicated that the nickel

resistance of this strain was inducible, since preculture in 0.5Fig. 3. Southern blot analysis of nickel-resistant isolates indicating mM Ni had a significant effect on the lag time when comparedhomology with the nickel resistance locus of pFBA30. (a) PvuII

to that of FBA30 in the absence of preculture (Fig. 4a). JM103digest of total DNA from FBA30 and environmental isolates. Lanes:

(pFBA30-10) showed identical growth characteristics (Fig.1, l (HindIII); 2, FBA13; 3, FBA17B; 4, FBA28; 5, FBA1.3.21; 6,4b). This confirms that the 10.2-kb SstI fragment of pFBA30FBA1.4.2; 7, FBA1.5.43; 8, FBA30; 9, FBA30-10. (b) Southern blot

analysis using 1.75-kb PvuII fragment (probe 1) of pFBA30-11 carried by pFBA30-10 contains the entire nickel resistanceshowing homology with 6.5-kb fragment in group A isolates and locus, and is still inducible. Both pFBA30-11 (Fig. 4c) and4.7-kb fragment in group B isolates, respectively. Lanes: as in Fig. pFBA30-12 (Fig. 4d) differed from pFBA30-10 in that each2. Please note that FBA 17B and FBA1.5.43 gave atypical signals;

showed a slower growth rate in the presence of 2.5 mM Ni.subsequent Southern blots reconfirmed the presence of the 6.5-kb

pFBA30-12 displayed a reduced level of resistance that wasand 4.7-kb fragments, respectively (data not presented). Group Anot inducible: this presumably reflects a lower rate of tran-and B isolates refer to the size of fragment homologous to the 1.75-

kb probe, which are 6.5 kb and 4.7 kb, respectively. scription of the nickel resistance locus from the vector pro-

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moter. pFBA30-11, with its insert in the same orientation as resistance systems [21, 24, 32, 37], that of pFBA30 does not

provide resistance to metals such as cobalt, zinc, mercury,that of pFBA30-10, displayed a slow growth rate and a re-

duced form of inducibility. chromate, or cadmium. Therefore, the nickel resistance

gene(s) of pFBA30 represents another, unrelated gene familyEnvironmental isolates were also examined for inducibil-

ity of Nir. Group A strains 13, 17B, and 28 demonstrated a and was designated the nrf determinant.

We also report herein that this nickel resistance was induc-common growth profile indicative of a noninducible consti-

ible, both in E. cloacae and in other enteric species. Inducibil-tutive resistance (Fig. 4e,f,g). In contrast, group B strains all

ity was retained by the 10.2-kb SstI restriction fragment ofshowed a clear inducible response (Fig. 4h,i,j). Growth rates

pFBA30-10, but not by the 2.3-kb PvuII fragment derivedfor these strains in the presence of nickel were consistently

from this plasmid. pFBA30-11 and pFBA30-12, each carryingslower than that for FBA30.

the PvuII fragment in opposite orientations, displayed consti-

tutive expression and slower growth in the presence of nickel.

This may stem from the loss of regulatory genes locatedDiscussion outside the fragment, but this has yet to be confirmed by

complementation studies. It is considered unlikely that theTransfer of nickel resistance from environmental samples to effect is due to vector promoters overriding the inducibleHB101 or PaW340 was not detected by the exogenous plas- expression of the cloned nickel resistance gene(s), since con-mid isolation procedure. Endogenous matings, again using struction of these plasmids removes the regulated plac pro-the same recipients, identified only a single strain out of 100 moter of pUC19, and the only other available promoters lieisolates that could transfer nickel resistance by conjugation. in the ori region of the plasmid.This may indicate the inability of these laboratory strains to Use of the 10.2-kb SstI restriction fragment as a hybridiza-act as recipients in matings with recently isolated bacteria tion probe against DNA extracted from environmental iso-under the conditions used here. It is significant that all nickel- lates of the Church Beck site identified 95 strains (24% ofresistant strains were enteric species, and this, in itself, may nickel-resistant isolates) sharing limited homology with thepreclude PaW340 from stably inheriting narrow host range nickel resistance locus of pFBA30. Of these, 32 showed strongplasmids from these sources. However, previous studies have homology with the probe fragment under conditions of highisolated metal resistant strains by these methods (silver resis- stringency. These 32 isolates were further challenged by hy-tant plasmids, Pickup, unpublished data), and the results bridization experiments using a 1.75-kb SmaI fragment frommay instead indicate a very low frequency of plasmid-borne pFBA30-10. Six of these demonstrated extensive homologyresistance in these bacterial populations. with this probe and were retained for further study. The

FBA30 was tentatively identified as E. cloacae and was nickel resistance of these six strains appeared to be locatedfound to contain two plasmids. One of these was designated on the bacterial chromosome of each, rather than on a plas-pFBA30 and is described as a conjugative nickel-resistance mid. The majority of previously reported nickel resistanceplasmid of approximately 55 kb. It is considered to have a genes have been shown to reside on plasmids [39], althoughnarrow host range since it was readily introduced into enteric chromosomal resistance has been reported for Alcaligenesrelatives such as E. coli, E. aerogenes, and S. rubidae, but not denitrificans 4a-2 and K. oxytoca [15, 40].into oxidase-positive strains such as P. putida or P. fluorescens. These six strains were classified into two groups, desig-The plasmid pFBA31 found in FBA30 is smaller than pFBA30 nated A and B, distinguishable by the date on which eachand was not transmissible to other strains; it was not consid- was sampled from the environment. The two groups canered further. be further distinguished by the size of chromosomal PvuII

The nickel-resistance locus was cloned on a 10.2-kb SstI restriction fragments sharing homology with the nickel resis-

restriction fragment of pFBA30 DNA, and localized to an tance gene(s) of pFBA30. Group A and B strains carry 6.5-

internal 2.3-kb PvuII fragment. Preliminary characterization and 4.7-kb PvuII fragments, respectively. which are homolo-

of the nickel resistance determinant carried by pFBA30 indi- gous with the 1.75-kb SmaI probe fragment from pFBA30.

cates that it is not related to any of the previously described All these strains are enteric species associated with sewage

nickel resistance families such as ncc/nre, cnr, or the determi- discharge. It is worth noting that one of these species (K.

nant from K. oxytoca [32, 33, 39, 40], and is apparently oxytoca; strain 13 of group A) has already been associated

with nickel resistance [39, 40]. All three group A strainsconfined to enteric bacteria. Furthermore, unlike other nickel

79050E$P18 03-31-97 14:35:19


Fig. 4. Comparison of growth of strains carrying the pFBA30-type nickel determinant in the presence of NiCl2. Each graph represents

duplicate experiments examining growth with (ln) and without (X✖) preculture in 0.5 mM Ni over a 7- to 8-h period. Graphs (a–d)represent the wild-type and recombinant strains; (e–g), group A strains; and (h–j), group B strains.

demonstrated constitutive nickel resistance, while those of but isolates from these sediments did not possess homology

group B displayed induction of resistance when precultured with the nickel resistance determinant of pFBA30. This pre-

in low levels of NiCl2. Thus, the nickel resistance genes of sumably reflects the narrow host range of this plasmid, and

groups A and B are taken as representatives of two branches a low freqency of conjugative transfer between enteric bacte-

of a divergent gene family, which differ in their regulation ria and indigenous species in this ecosystem.and flanking restriction endonuclease sites. Further studies

are underway to confirm the relationship between the group

A and group B nickel resistance loci.AcknowledgmentsNo homology with pFBA30 was detected in nickel-resis-

tant bacteria isolated from sediments downstream of theSupport from the Institute of Freshwater Ecology and Naturaloutfall pipe. The time scale of the sampling indicated thatEnvironment Research Council is gratefully acknowledged.resistant bacteria are constantly discharged from the sewageI wish to thank Prof. Dr. H.G. Schlegel for supplying plasmidstreatment plant into the river. The different nickel-resistantcarrying ncc/nre, cnr, and Klebsiella oxytoca-type resistances.populations found in each sample further indicate that theseIn addition, I would like to thank Annick Wilmotte (VITO,resistant bacteria do not constitute a stable population, but

Belgium) for supplying pMOL154, and Pamela Broadbent,rather one that changes with time in terms of its species

Nick Whalley, and Francis O’Neil, who have all contributedand genetic constitution. These bacteria represent potential

colonists of the sediments downstream of the effluent pipe, to this manuscript.

79050E$P18 03-31-97 14:35:19


Fig. 4 continued

speciation of group D streptococci from above and below a

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a novel nickel resistance determinant found in sewage-associated bacteria

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