Comparison of Two Multimetal Resistant Bacterial Strains:Enterobacter sp. YSU and Stenotrophomonas maltophilia ORO2

Andrew Holmes Æ Anubhav Vinayak Æ Cherise Benton Æ Aaron Esbenshade ÆCarlisle Heinselman Æ Daniel Frankland Æ Samatha Kulkarni ÆAdrienne Kurtanich Æ Jonathan Caguiat

Received: 16 January 2009 / Accepted: 21 July 2009 / Published online: 18 August 2009

� Springer Science+Business Media, LLC 2009

Abstract The Y-12 plant in Oak Ridge, TN, which

manufactured nuclear weapons during World War II and

the Cold War, contaminated East Fork Poplar Creek with

heavy metals. The multimetal resistant bacterial strain,

Stenotrophomonas maltophilia Oak Ridge strain O2 (S.

maltophilia O2), was isolated from East Fork Poplar Creek.

Sequence analysis of 16s rDNA suggested that our working

strain of S. maltophilia O2 was a strain of Enterobacter.

Phylogenetic tree analysis and biochemical tests confirmed

that it belonged to an Enterobacter species. This new strain

was named Enterobacter sp. YSU. Using a modified R3A

growth medium, R3A-Tris, the Hg(II), Cd(II), Zn(II),

Cu(II), Au(III), Cr(VI), Ag(I), As(III), and Se(IV) MICs for

a confirmed strain of S. maltophilia O2 were 0.24, 0.33, 5,

5, 0.25, 7, 0.03, 14, and 40 mM, respectively, compared to

0.07, 0.24, 0.8, 3, 0.05, 0.4, 0.08, 14, and 40 mM,

respectively, for Enterobacter sp. YSU. Although S.

maltophilia O2 was generally more metal resistant than

Enterobacter sp. YSU, in comparison to Escherichia coli

strain HB101, Enterobacter sp. YSU was resistant to

Hg(II), Cd(II), Zn(II), Au(III), Ag(I), As(III), and Se(IV).

By studying metal resistances in these two strains, it may

be possible to understand what makes one microorganism

more metal resistant than another microorganism. This

work also provided benchmark MICs that can be used to

evaluate the metal resistance properties of other bacterial

isolates from East Fork Poplar Creek and other metal

contaminated sites.

Introduction

The Y-12 plant in Oak Ridge, TN played an important role

in national defense of the USA during the last 60 years of

the twentieth century. It processed uranium during World

War II to make the first nuclear bomb [29]. With the

beginning of the Cold War in the 1950s, the Y-12 plant

switched to lithium processing for manufacturing hydrogen

bombs. This process required large amounts of mercury,

which was not tightly contained, and about 920,000 kg

were spilled into East Fork Poplar Creek (Poplar Creek)

and the surrounding environment [22]. Four S-3 ponds,

located near the Y-12 plant at the origin of Poplar Creek,

were used to dispose of acidic wastes that were contami-

nated with uranium and other heavy metals [4]. These

ponds were constructed without a covering or a lining to

allow the liquid wastes to evaporate or be decontaminated

as they passed through the soil. Unfortunately, most of the

wastes simply leached into the ground as well as into the

creek. In 1983, the use of these ponds was discontinued, the

remaining contents were treated, liquid was drained and the

ponds were filled and capped.

The bacterium, Stenotrophomonas maltophilia Oak

Ridge Strain ORO2 (S. maltophilia O2), was isolated from

Poplar Creek. It grew in the presence of toxic levels of

copper, platinum, mercury, gold, cadmium, lead, chro-

mium, silver, and selenium salts and detoxified them by

converting them to insoluble precipitates [21]. Sequence

analysis of 16s rDNA suggested that our working strain of

S. maltophilia O2 was related to a strain of Enterobacter.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00284-009-9471-2) contains supplementarymaterial, which is available to authorized users.

A. Holmes � A. Vinayak � C. Benton � A. Esbenshade �C. Heinselman � D. Frankland � S. Kulkarni � A. Kurtanich �J. Caguiat (&)

Department of Biological Sciences,

Youngstown State University, Youngstown, USA

e-mail: jjcaguiat@ysu.edu

123

Curr Microbiol (2009) 59:526–531

DOI 10.1007/s00284-009-9471-2

Biochemical tests confirmed these results, and the new

strain was called Enterobacter sp. YSU. The 16s rDNA

sequences, biochemical characteristics, and metal resis-

tance properties between Enterobacter sp. YSU and a

confirmed strain of S. maltophilia O2 are compared. The

high levels of metal resistances observed in S. maltophilia

O2 compared with the intermediate levels of metal resis-

tances observed in Enterobacter sp. YSU can be used as

benchmarks in studies with other bacterial isolates from

Poplar Creek and other metal contaminated sites.

Materials and Methods

Bacterial Strain Plasmids and Media

Stenotrophomonas maltophilia O2 (ATCC # 53510) was

purchased from the American Type Culture Collection

(Manassas, VA). Enterobacter sp. YSU is described here.

Escherichia coli (E. coli) strain HB101 was used as a metal

sensitive control [2].

R3A-Tris medium was a modification of R3A medium

[19] with 10 mM Tris-HCl, pH 7.5 (Fisher Scientific, Fair

Lawn, NJ) replacing phosphate. Luria–Bertani (LB) med-

ium (Fisher Scientific) was described previously [2]. When

required, R3A-Tris medium was supplemented with HgCl2,

CdCl2, ZnCl2, CuSO4, K2CrO4, Pb(NO3)2 (Fisher Scien-

tific), AgNO3, HAuCl4�3H2O, NaAsO2 (Amresco, Solon,

OH), and Na2SeO3 (MP Bio Medicals, LLC, Solon, OH).

The EnterotubeTM II Identification System [10] was pur-

chased from Becton Dickinson (Cockeysville, MD), and

other biochemical tests were described by Harley [14].

16s rDNA Sequencing

Genomic DNA was purified using an UltraCleanTM Microbial

DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad,

CA) and amplified using GoTaq polymerase (Promega,

Madison, WI), 8F primer, 1492R primer (Table 1), and

30 cycles of 95�C for 1 min, 55�C for 1 min, and 72�C for

2 min. The PCR amplicands were cloned using a Strata-

CloneTM PCR Cloning Kit (Stratagene, La Jolla, CA). The

resulting plasmids were purified using an Eppendorf� Perfect

Plasmid Mini kit (Hamburg, Germany), and sequenced using

the primers in Table 1, a Beckman Coulter (Fullerton, CA)

Genome LabTM DTCS-Quick Start Kit and a Beckman

Coulter CEQ 2000 XL DNA Analysis System.

Sequences were assembled using ContigExpress and

compared using the AlignX program from Invitrogen’s

(Carlsbad, CA) Vector NTI 10.3.0 software suite. Phylo-

genetic trees were constructed using the Molecular Evolu-

tionary Genetics Analysis version 4.0 (MEGA 4) software

suite [26]. The 16s rDNA sequences were analyzed by the

Basic Alignment Search Tool (BLAST) [1] using the

Nucleotide Collection (nr/nt) database and the ‘‘somewhat

similar sequences (blastn)’’ algorithm. Each set of sequen-

ces was aligned using the default settings of Clustal W [27]

from MEGA 4. The alignments were assembled into

Neighbor-Joining trees [23] using the Maximum Likelihood

Composite method [25] and a 2000 replicate bootstrap test

[9].

Minimal Inhibitory Concentrations (MICs)

Overnight cultures of each strain grown at 30�C in R3A-

Tris medium were diluted 1/50 into fresh medium. After

adding the desired concentration of metal, the cultures

were grown for 24 h at 30�C. Turbidity was measured

before and after the 24 h incubation period using a Klett

Colorimeter with a KS-54 filter. The average difference

between the 24 h Klett reading and the initial Klett reading

was calculated for at least 3 different trials, and the stan-

dard error for each average difference was calculated using

the student t distribution with a 95% confidence level.

Then, the percent growth at each metal concentration was

calculated by dividing the average difference of the treated

cultures by the average difference of the untreated cultures.

Percent error was calculated by dividing the error by the

average difference of the untreated cultures. A strain was

considered to be sensitive at a particular metal concentra-

tion if the percent growth was less than 10% with a percent

error extending to below 20%.

Results and Discussion

Cloned 16s rDNA fragments from the Enterobacter sp.

YSU (gi|238801115|) strain and the S. maltophilia O2

(gi|238801116|) strain were sequenced and analyzed by

BLAST [1]. All 100 hits for each strain were 99–98%

homologous with 0% gaps. After removing identical

sequence hits and sequences for uncultured strains, 25 of

the first 49 hits for Enterobacter sp. YSU and 19 of the first

24 hits for S. maltophilia O2 were selected for alignment

and tree construction.

The Neighbor-Joining [23] method in MEGA 4 placed

Enterobacter sp. YSU in a group of Enterobacter, Erwinia,

Citrobacter, and Pantoea species found in the soil and in

animals [5, 30]. From this tree, the origin and species of

Enterobacter sp. YSU were not obvious.

Stenotrophomonas maltophilia O2 was located in a

group of clinical and environmental isolates of S. malto-

philia. The clinical isolates encoded L1 and L2 b-lacta-

mases [11], while the environmental isolates, degraded

herbicides [3, 16] and encoded antifungal compounds [18].

A. Holmes et al.: Comparison of Two Multimetal Resistant Bacterial Strains 527

123

The 16s rDNA sequence did not provide enough infor-

mation to indicate an origin for S. maltophilia O2.

The EnterotubeTM II Identification System characterized

biochemical properties of Enterobacter sp. YSU. E. coli

strain HB101 (HB101), which was used as a control, tested

positive for E. coli as expected. The Enterobacter strain

tested positive for motility, glucose with gas, Voges-

Proskauer, ornithine, adonitol, lactose, arabinose, and cit-

rate and negative for the Gram stain, methyl red, lysine,

H2S, indole, sorbitol, dulcitol, phenylalanine, urea, DNase,

oxidase, and catalase. Because these biochemical pheno-

types did not completely match any previously published

phenotypes for a specific Enterobacter species [12], a

species could not be assigned to Enterobacter sp. YSU.

The EnterotubeTM II Identification System was not

designed for typing S. maltophilia, but the biochemical test

results for S. maltophilia O2 were consistent with other S.

maltophilia strains [8]. S. maltophilia O2 was positive for

the motility, glucose without gas, lysine, urea, DNase, and

catalase tests and negative for the Gram stain, methyl red,

Voges-Proskauer, ornithine, H2S, indole, adonitol, lactose,

arabinose, sorbitol, dulcitol, phenylalanine, citrate, and

oxidase tests. A positive urea test was an atypical result.

R3A medium was used to compare the levels of metal

resistances to Hg(II), Cd(II), Zn(II), Cu(II), Au(III),

Cr(VI), Ag(I), As(III), Se(IV), and Pb(II) because its

derivative medium, R2A, was capable of supporting the

growth of a wider range of bacteria than standard plate

counting medium, and R3A was used to grow and maintain

R2A isolates [20]. To avoid metal-phosphate precipitation,

phosphate was replaced with Tris-HCl, pH 7.5 to make

R3A-Tris. Overnight cultures were diluted into fresh R3A-

Tris and grown for 24 h in the presence of different metal

concentrations. Resistance of each strain was observed by

plotting the percent growth versus the metal concentration

(Fig. 1). The standard error generally became large near

each MIC because growth was variable at these borderline

concentrations.

Escherichia coli strain HB101 was used as a control in

the MIC studies because it is generally more sensitive to

metals than S. maltophilia O2 and Enterobacter sp. YSU.

In addition, the ability to genetically manipulate E. coli

makes it a good vehicle for expressing and modifying

metal resistance genes from other bacterial species.

Determining the MICs for HB101 provided future bench-

marks for measuring the levels of metal resistances

expressed by genes cloned from the Poplar Creek isolates

into E. coli.

Stenotrophomonas maltophilia O2 appeared to be much

more resistant to Hg(II), Cd(II), Zn(II), Cu(II), Au(III), and

Cr(VI) than Enterobacter sp. YSU and HB101. In addition,

Enterobacter sp. YSU was more resistant to Hg(II), Cd(II),

Zn(II), and Au(III) than HB101. The S. maltophilia O2

MIC for Hg(II) was 0.250 mM, compared to 0.07 mM for

Enterobacter sp. YSU and 0.02 mM for HB101 (Fig. 1a).

The S. maltophilia MIC for Cd(II) was 0.33 mM, com-

pared to 0.24 mM for Enterobacter sp. YSU and 0.14 mM

for HB101 (Fig. 1b). The S. maltophilia O2 MIC for Zn(II)

Table 1 Oligonucleotides used in this work

Primer Sequence Purpose

8F 50-AGAGTTTGATCCTGGCTCAG-30 Cloning S. maltophilia O2 and Enterobacter 16s rDNA fragments

1492R 50-GGTTACCTTGTTACGACTT-30 Cloning S. maltophilia O2 and Enterobacter 16s rDNA fragments

M13 F 50-GTAAAACGACGGCCAG-30 Sequencing cloned S. maltophilia O2 and Enterobacter rDNA fragments

M13 R 50-CAGGAAACAGCTATGAC-30 Sequencing cloned S. maltophilia O2 and Enterobacter rDNA fragments

SO2 F2 50-GCTCGTTGCGGGACTTAACC 30 Sequencing the cloned Enterobacter rDNA fragment

SO2 R2 50-ACACGGTCCAGACTCCTACG-30 Sequencing the cloned Enterobacter rDNA fragment

SO2 F3 50-TTGCACCCTCCGTATTACCG-30 Sequencing the cloned Enterobacter rDNA fragment

SO2 R3 50-GCGGTGAAATGCGTAGAGAT-30 Sequencing the cloned Enterobacter rDNA fragment

SO2 F4 50-CGTAGGAGTCTGGACCGTGT-30 Sequencing the cloned Enterobacter rDNA fragment

SO2 R4 50-ACCCTTATCCTTTGTTGCCA-30 Sequencing the cloned Enterobacter rDNA fragment

SO2 F5 50-AGTTCCCGAAGGCACCAATC-30 Sequencing the cloned Enterobacter rDNA fragment

ATCC-SO2 F2 50-GCTCGTTGCGGGACTTAACC-30 Sequencing the cloned S. maltophilia O2 rDNA fragment

ATCC-SO2 R2 50-AGACACGGTCCAGACTCCTA-30 Sequencing the cloned S. maltophilia O2 rDNA fragment

ATCC-SO2 F3 50-CGGTATGGCTGAATCAGGCT-30 Sequencing the cloned S. maltophilia O2 rDNA fragment

ATCC-SO2 R3 50-AAACGATGCGAACTGGATGT-30 Sequencing the cloned S. maltophilia O2 rDNA fragment

ATCC-SO2 F4 50-AGTTCTCGACATGTCAAGGC-30 Sequencing the cloned S. maltophilia O2 rDNA fragment

ATCC-SO2 F5 50-CACAACGGACTTAAACGACC-30 Sequencing the cloned S. maltophilia O2 rDNA fragment

ATCC-SO2 R4 50-CGCATACGACCTACGGGTGA-30 Sequencing the cloned S. maltophilia O2 rDNA fragment

ATCC-SO2 R5 50-AGCGTGCGTAGGTGGTCGTT-30 Sequencing the cloned S. maltophilia O2 rDNA fragment

528 A. Holmes et al.: Comparison of Two Multimetal Resistant Bacterial Strains

123

was 5 mM, compared to 0.8 mM for Enterobacter sp. YSU

and 0.5 mM for HB101 (Fig. 1c). The S. maltophilia O2

MIC was 5 mM for Cu(II), compared to 3 mM for Enter-

obacter sp. YSU and HB101 (Fig. 1d). The S. maltophilia

O2 MIC for Au(III) was 0.25 mM, compared to 0.05 mM

for Enterobacter sp. YSU and 0.03 mM for HB101

(Fig. 1e). The S. maltophilia O2 MIC for Cr(VI) was

8 mM, compared to 0.4 mM for Enterobacter sp. YSU and

HB101 (Fig. 1f).

The Enterobacter sp. YSU strain was more resistant to

Ag(I) than S. maltophilia O2 and HB101. Its MIC for Ag(I)

was 0.08 mM, compared to 0.03 mM for S. maltophilia O2

and HB101 (Fig. 1g). The Enterobacter sp. YSU and S.

maltophilia MICs for As(III) MICs were 14 mM, com-

pared to 6 mM for HB101 (Fig. 1h). The Enterobacter sp.

YSU and S. maltophilia MICs for Se(IV) were 40 mM,

compared to 70 mM for HB101 (Fig. 1i). All three pro-

duced a red precipitate which contributed to turbidity. At

10 mM selenite, S. maltophilia O2 produced a more

intense red precipitate than the other strains. Its percent

growth at this concentration was 300 ± 81%, compared to

53 ± 121% for Enterobacter sp. YSU and 77 ± 122% for

E. coli. Thus, although the E. coli strain appeared to

withstand a slightly higher concentration of selenite than

the other strains, S. maltophilia O2 precipitated the selenite

more efficiently at lower concentrations. Viable cell counts

would have provided more accurate MICs for selenite

because it would have eliminated the contribution of the

precipitate to percent growth.

The Pb(II) MICs were not accurate because Pb(II) pre-

cipitated even in the absence of phosphate. Although R3A-

Tris medium was not compatible with Pb(II), it was useful

for estimating the MICs to the other 9 metals. These metal

MICs determined for the three strains can be used as

benchmarks to test additional isolates from Poplar Creek

and other metal contaminated sites. Although S. malto-

philia O2 was more metal resistant than Enterobacter sp.

YSU, compared to HB101, Enterobacter sp. YSU was

Fig. 1 MICs for E. coli strain HB101, Enterobacter sp. YSU, and S.maltophilia O2. a HgCl2, b CdCl2, c ZnCl2, d CuSO4, eHAuCl4�3H2O, f K2CrO4, g AgNO3, h NaAsO2, i Na2SeO3. Filled

circle E. coli strain HB101, open circle Enterobacter sp. YSU, filledinverted triangle S. maltophilia O2

A. Holmes et al.: Comparison of Two Multimetal Resistant Bacterial Strains 529

123

resistant to salts of Hg(II), Cd(II), Zn(II), Au(III), Ag(I),

As(III), and Se(IV).

The reason for the higher levels of metal resistances in

S. maltophilia O2 was not known. Perhaps it simply

expressed its metal resistance genes at higher levels than

the Enterobacter sp. YSU, or maybe it used different, more

efficient resistance mechanisms.

Previous work showed that metal resistances are com-

monly found in related strains of Enterobacter cloacae (E.

cloacae) and S. maltophilia. S. maltophilia Sm777 tolerated

up to 0.05 mM Hg(II), 0.5 mM Cd(II), 5 mM Cu(II),

50 mM Se(IV), 0.02 mM Ag(I), and 5 mM Pb(II) [19].

These concentrations are similar to the estimated MICs for

S. maltophilia O2. In addition, the multimetal resistant

strain, E. cloacae BS, demonstrated MICs of 17.8 mM for

Cd(II), 7.6 mM for Zn(II), 8.7 mM for Cu(II) and 3.4 mM

for Pb(II) [15]. Two E. cloacae strains from a dental patient

grew on Mueller Hinton agar containing 1.0 mM Ag(I) [7],

and E. cloacae HO1 grew at Cr(VI) concentrations over

10 mM [28]. These MICs for E. cloacae all were all higher

than the MICs estimated for Enterobacter sp. YSU. How-

ever, since the above MICs were determined using a growth

medium other than R3A, the tested strains could respond

differently in R3A medium.

Bacteria use different metal resistance mechanisms [13,

24]. Most pump the metals out of the cells. Some oxidize or

reduce metals to less toxic forms, while others sequester

metals using metallothioneins. Many strains of bacteria

probably use a combination of these mechanisms. The

genome for S. maltophilia K279a, an opportunistic patho-

gen which was isolated from a cancer patient, was

sequenced recently [6]. It contained many different genes

which encoded proteins for resistances that may allow it to

adapt to metal contaminated environments. Although a

synopsis of the Enterobacter sp. 638 genome sequence

|gi:146309667| has not been published, a brief search for

key words in its annotated sequence revealed that it may

contain a P-type ATPase for Pb(II), Cd(II), Zn(II), and

Hg(II) transport, a ZntA protein for Zn(II) transport, and a

Cd(II)/Zn(II)/Co(II), CzcA, efflux protein. Previous work

examined nine Enterobacter isolates from a pristine site in

Brazil and found that they were all sensitive to 4 lg/ml

Hg(II) [17]. From the present study and the scan of the

Enterobacter sp. 638 genome, it appears that most Enter-

obacter species probably contain some genes that confer

resistances to low metal concentrations and acquire addi-

tional genes by horizontal gene transfer [24] when they

encounter high metal concentrations. Estimating the metal

MICs for Enterobacter sp. 638 may provide answers to this

hypothesis.

This work used 16s rDNA and biochemical studies to

identify a new strain of bacteria Enterobacter sp. YSU. It

also used a modified R3A medium, R3A-Tris, to define the

high, medium, and low metal resistance properties of S.

maltophilia O2, Enterobacter sp. YSU and E. coli strain

HB101, respectively. These properties will be used to

evaluate 2,500 other Poplar Creek bacterial colonies that

have been isolated using R2A agar medium and saved as

glycerol stocks in R3A medium. Comparing these isolates

with the strains in the present study may make it possible to

understand why one bacterial strain is more metal resistant

than another, identify novel metal resistance mechanisms

and detect gene transfer within a bacterial population.

Acknowledgments This study was funded by the National Science

Foundation through Grant numbers 0542178 and 0620240 and the

University Research Council at the Youngstown State University

(YSU) School of Graduate Studies. We thank Julio ‘‘Ed’’ Budde from

the Department of Biological Sciences at YSU for resolving our

sequencing reactions, Thomas Schmidt from Michigan State Uni-

versity (MSU) and Xiang Jia Min from YSU for help with 16s rDNA

sequencing and analysis, Jay Kerns from the Department of Mathe-

matics and Statistics at YSU for help with calculating the standard

MIC errors and Anne Summers from the University of Georgia, Julius

Jackson from Michigan State University, Xiang Jia Min and Chester

Cooper, Jr from YSU for critiquing the manuscript.

References

1. Altschul SF, Madden TL, Schaffer AA et al (1997) Gapped

BLAST and PSI-BLAST: a new generation of protein database

search programs. Nucleic Acids Res 25:3389–3402

2. Ausubel F, Brent R, Kingston RE et al (1997) Short protocols in

molecular biology, 3rd edn. Wiley, New York

3. Barreiros L, Nogales B, Manaia CM et al (2003) A novel path-

way for mineralization of the thiocarbamate herbicide molinate

by a defined bacterial mixed culture. Environ Microbiol 5:

944–953

4. Brooks SC (2001) Waste characteristics of the former S-3 ponds

and outline of uranium chemistry relevant to NABIR field

research center studies. ORNLITM-2001/27 http://www.osti.gov/

energycitations/servlets/purl/814525-OXnnBu/native/

5. Chung H, Park M, Madhaiyan M et al (2005) Isolation and

characterization of phosphate solubilizing bacteria from the

rhizosphere of crop plants of Korea. Soil Biol Biochem 37:

1970–1974

6. Crossman LC, Gould VC, Dow JM et al (2008) The complete

genome, comparative and functional analysis of Stenotropho-monas maltophilia reveals an organism heavily shielded by drug

resistance determinants. Genome Biol 9:R74

7. Davis IJ, Richards H, Mullany P (2005) Isolation of silver- and

antibiotic-resistant Enterobacter cloacae from teeth. Oral

Microbiol Immunol 20:191–194

8. Denton M, Kerr KG (1998) Microbiological and clinical aspects

of infection associated with Stenotrophomonas maltophilia. Clin

Microbiol Rev 11:57–80

9. Efron B, Halloran E, Holmes S (1996) Bootstrap confidence

levels for phylogenetic trees. Proc Natl Acad Sci USA 93:

13429–13434

10. Farmer JJIII, Davis BR, Hickman-Brenner FW et al (1985)

Biochemical identification of new species and biogroups of

Enterobacteriaceae isolated from clinical specimens. J Clin

Microbiol 21:46–76

530 A. Holmes et al.: Comparison of Two Multimetal Resistant Bacterial Strains

123

11. Gould VC, Okazaki A, Avison MB (2006) Beta-lactam resistance

and beta-lactamase expression in clinical Stenotrophomonasmaltophilia isolates having defined phylogenetic relationships. J

Antimicrob Chemother 57:199–203

12. Grimont F, Grimont P (2006) The genus Enterobacter. In:

Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt

E (eds) The prokaryotes: volume 6: proteobacteria: gamma

Subclass. Springer, New York, pp 197–214

13. Haferburg G, Kothe E (2007) Microbes and metals: interactions

in the environment. J Basic Microbiol 47:453–467

14. Harley JP (2008) Laboratory exercises in microbiology, 7th edn.

McGraw-Hill/Higher Education, Boston

15. Hu Q, Dou MN, Qi HY et al (2007) Detection, isolation, and

identification of cadmium-resistant bacteria based on PCR-

DGGE. J Environ Sci (China) 19:1114–1119

16. Laramee L, Lawrence JR, Greer CW (2000) Molecular analysis

and development of 16S rRNA oligonucleotide probes to char-

acterize a diclofop-methyl-degrading biofilm consortium. Can J

Microbiol 46:133–142

17. Lima-Bittencourt CI, Cursino L, Goncalves-Dornelas H et al

(2007) Multiple antimicrobial resistance in Enterobacteriaceaeisolates from pristine freshwater. Genet Mol Res 6:510–521

18. Minkwitz A, Berg G (2001) Comparison of antifungal activities

and 16S ribosomal DNA sequences of clinical and environmental

isolates of Stenotrophomonas maltophilia. J Clin Microbio

39:139–145

19. Pages D, Rose J, Conrod S et al (2008) Heavy metal tolerance in

Stenotrophomonas maltophilia. PLoS ONE 3:e1539

20. Reasoner DJ, Geldreich EE (1985) A new medium for the enu-

meration and subculture of bacteria from potable water. Appl

Environ Microbiol 49:1–7

21. Revis NW, Benson SB, Osborne TR (1989) Reduction of trace

elements to the elemental form by microorganisms. US patent

4826602

22. Rouse Campbell K, Ford CJ, and Levine DA (1998) Mercury

distribution in Poplar Creek, Oak Ridge, Tennessee, USA.

Environ Toxicol Chem 17:1191–1198

23. Saitou N, Nei M (1987) The neighbor-joining method: a new

method for reconstructing phylogenetic trees. Mol Biol Evol

4:406–425

24. Summers AO (2006) Genetic linkage and horizontal gene trans-

fer, the roots of the antibiotic multi-resistance problem. Anim

Biotechnol 17:125–135

25. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very

large phylogenies by using the neighbor-joining method. Proc

Natl Acad Sci USA 101:11030–11035

26. Tamura K, Dudley J, Nei M et al (2007) MEGA4: molecular

evolutionary genetics analysis (MEGA) software version 4.0.

Mol Biol Evol 24:1596–1599

27. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W:

improving the sensitivity of progressive multiple sequence align-

ment through sequence weighting, position-specific gap penalties

and weight matrix choice. Nucleic Acids Res 22:4673–4680

28. Wang PC, Mori T, Komori K et al (1989) Isolation and charac-

terization of an Enterobacter cloacae strain that reduces hexa-

valent chromium under anaerobic conditions. Appl Environ

Microbiol 55:1665–1669

29. Widner TE, Ripple SR, Buddenbaum JE (1996) Identification and

screening evaluation of key historical materials and emission

sources at the Oak Ridge Reservation. Health Phys 71:457–469

30. Wildschutte H, Lawrence JG (2007) Differential Salmonellasurvival against communities of intestinal amoebae. Microbiol-

ogy 153:1781–1789

A. Holmes et al.: Comparison of Two Multimetal Resistant Bacterial Strains 531

123