A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Section 8
Metals/Antibiotic Resistance in Bacteria 9/09/2011
Back to Myth # 4
Few people consider that metals released to sewage treatment plants will cause bacteria to develop not only resistance
to metals, but also to antibiotics. The metals are concentrated during sewage treatment, along with the bacteria used to
treat sewage, and both are spread on agricultural land as well as school grounds, parks, home lawns and gardens
where metals and bacteria continue to interact. This is not new information. What is new is that the government is now
allowing nanometals to be distributed in virtually every type of product you can think of, from cosmetics to food to
medicine. This is a new source of antibiotic resistance that has no boundary as a DNA molecule is only 1 nm wide. We
only need to review the research on heavy metals to see where this new untested science leads.
In 1975, David J. Groves and Frank E. Young, University of Rochester, reported on the “Epidemiology of Antibiotic and
Heavy Metal Resistance in Bacteria: Resistance Patterns in Staphylococci Isolated from Populations Not Known to be
Exposed to Heavy Metals.” They said, “Staphylococci were isolated from clinical specimens obtained from patients not
known to be exposed to abnormal levels of heavy metals. The antibiotic and heavy metal resistance patterns of these
strains were determined by using a disk diffusion test and computer sorting. Though not absolute, an association of
resistance to mercury and tetracycline in coagulase-negative strains was found, in contrast to resistance to copper and
penicillin in coagulase-producing strains. A high degree of correlation was observed between the resistance to phenyl
mercury and inorganic mercury, but no correlation was obtained between resistance to methylmercury and other
metals. In general, strains resistant to many agents were usually coagulase negative. A possible mechanism and
implications of these associations are considered.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC429191/
In 1975, I. Chopra, The Medical School, University of Bristol, reported on “Mechanism of Plasmid-Mediated Resistance
to Cadmium in Staphylococcus aureus.” Chopra said, “Many isolates of hospital staphylococci are resistant to heavy
metal ions, notably cadmium, mercury, and arsenic. In the majority of isolates, the genes that code for resistance to
these ions are carried by penicillinase plasmids, although in a few cultures resistance is determined by genes that,
although plasmid-located, are not linked to those coding for penicillinase production or resistance to other antibiotics.
This may imply that resistance to heavy metals gives the hospital staphylococcus a selective advantage over cultures
susceptible to these ions; indeed, there is some evidence that the use of mercury therapeutically could be a factor in
selecting resistant strains. However, there is little evidence to explain the high incidence of strains that are cadmium
resistant.” http://aac.asm.org/cgi/reprint/7/1/8.pdf
In 1977, Hideomi Nakahara, et al. The Jikei University School of Medicine at Tokyo, reported on the “Frequency of
heavy-metal resistance in bacteria from inpatients in Japan.” They said, “In many bacteria, resistance to heavy metals
is associated with a plasmid. R plasmids in Escherichia coli can determine resistance to several metallic ions such as
mercury, cobalt and nickel3, and a penicillinase plasmid mediating resistance to mercury, cadmium, arsenate, arsenite,
lead and zinc has been observed in Staphylococcus aureus. Mechanisms controlling bacterial resistances to mercury
and cadmium are quite different although both are mediated by the same penicillinase plasmid. Furthermore,
microorganisms generally detoxify mercurial compounds metabolically by the formation of volatile mercury or mercury
mercaptides. It is of interest that resistance to these heavy metals is mediated by the plasmids which determine
resistance to antibiotics. Most of these heavy metals are established or possible causes of environmental pollution;
methyl mercury causes 'Minamata disease' and cadmium causes 'Itai-Itai disease' in Japan. The role of R plasmids in
drug resistance has been widely studied, and extrachromosomal determinants are a main cause of the increase in
numbers of drug-resistant bacteria. Studies of heavy metal resistance have attempted to establish a relationship
between resistance to heavy metals and to drugs in the hospital environment. The factors selecting for these heavy-
metal-resistant bacteria have not yet been identified. We believe that heavy-metal-resistant microorganisms do not
arise by chance, but, that there must be selectional factors beyond mere drug resistance. One of these selectional
factors may be environmental contamination by heavy metals. To investigate this possibility, we studied the frequency
of drug and heavy-metal resistance in clinical isolates of E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and
S. aureus. We report here that the frequency of heavy-metal resistance in these strains was the same as, or higher
than that of antibiotic resistance. Some R plasmids in E. coli and K. pneumoniae also carried a resistance to Hg.”
http://www.nature.com/nature/journal/v266/n5598/abs/266165a0.html
In 1978, J. F. Timoney, et al., New York State College of Veterinary Medicine, Cornell University, reported on “Heavy-
Metal and Antibiotic Resistance in the Bacterial Flora of Sediments of New York Bight.” They said, “The New York Bight
extends seaward some 80 to 100 miles (ca. 129 to 161 km) from the Long Island and New Jersey shorelines to the
edge of the continental shelf. Over 14 x 106 m3 of sewage sludge, dredge spoils, acid wastes, and cellar dirt are
discharged into this area each year. Large populations of Bacillus sp. resistant to 20 µg of mercury per ml were
observed in Bight sediments contaminated by these wastes. Resistant Bacillus populations were much greater in
sediments containing high concentrations of Hg [mercury] and other heavy metals than in sediments from areas further
offshore where dumping has never been practiced and where heavy-metal concentrations were found to be low.
Ampicillin resistance due mainly to ß-lactamase production was significantly (P < 0.001) more frequent in Bacillus
strains from sediments near the sewage sludge dump site than in similar Bacillus populations from control sediments.
Bacillus strains with combined ampicillin and Hg resistances were almost six times as frequent at the sludge dump site
as in control sediments. This observation suggests that genes for Hg resistance and ß-lactamase production are
simultaneously selected for in Bacillus and that heavy-metal contamination of an ecosystem can result in a selection
pressure for antibiotic resistance in bacteria in that system. Also, Hg resistance was frequently linked with other heavy-
metal resistances and, in a substantial proportion of Bacillus strains, involved reduction to volatile metallic Hg (Hg°).”
http://aem.asm.org/cgi/content/abstract/36/3/465
In the 1980 study, “Antibiotic and metal resistance in "Escherichia coli" strains isolated from the environment and from
patients”, G. Cenci, et al., found, “The incidence and the patterns of the antibiotic and metal resistance in 106 strains
of Escherichia coli isolated from ground waters, used also as drinking water supply (sample A), was studied in
comparison with the resistance behaviour in the 104 strains of the same microorganism isolated from non hospitalized
patients (sample P). Significant differences between the percentage of resistant strains in the two examined samples
were found for some of the antibiotics and the metals tested (ampicillin, streptomycin, kanamycin, mercury and zinc)
while non statistically significant differences were found for gentamicin, tetracyclin, nalidixic acid and cadmium. From
the high percentages of the resistant strains in the environmental sample (up to 44.3% for tetracyclin) we may deduce
that also the ground waters, especially if used as drinking water, contribute to the spread of the resistant bacteria. The
patterns of the antibiotic multiresistances in the strains isolated from patients and from ground waters do not differ
greatly and this strengthens the hypothesis that resistance to antibiotics has been acquired by Escherichia coli strains
before reaching the ground waters.” http://www.ncbi.nlm.nih.gov/pubmed/7008715
In a 1984 study by the J J Calomiris, et al., Terrestrial Microbial Ecology/Biotechnology Program, U.S. Environmental
Protection Agency, “Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking
water”. They said, “Bacterial isolates from the drinking water system of an Oregon coastal community were examined to
assess the association of metal tolerance with multiple antibiotic resistance. Positive correlations between tolerance to
high levels of Cu2+ (Copper), Pb2+ (Lead), and Zn2+ (Zinc) and multiple antibiotic resistance were noted among
bacteria from distribution waters but not among bacteria from raw waters. Tolerances to higher levels of Al3+
(Aluminum) and Sn2+ (Tin) were demonstrated more often by raw water isolates which were not typically multiple
antibiotic resistant. A similar incidence of tolerance to Cd2+ (Cadmium) was demonstrated by isolates of both water
types and was not associated with multiple antibiotic resistance. These results suggest that simultaneous selection
phenomena occurred in distribution water for bacteria which exhibited unique patterns of tolerance to Cu2+, Pb2+, and
Zn2+ and antibiotic resistance.” http://aem.asm.org/cgi/content/abstract/47/6/1238
In 1985, Duncan Rouch, et al., University of Melbourne, reported on, "Inducible Plasmid-mediated Copper Resistance
in Escherichia coli." They said, “Heavy metal resistance in micro-organisms can occur by a variety of mechanisms
including physical sequestration, exclusion and/or efflux, or detoxification, which may be tandem with efflux (Summers &
Silver, 1978). For example, mercuric and organomercurial resistance involves enzymic cleavage to Hg2+ followed by
the reduction of toxic Hg2+ to the less toxic and volatile HgO (Summers & Silver, 1978; Silver, 1981). However,
cadmium resistance in Staphylococcus aureus is not due to detoxification but to reduced accumulation of Cd2+ by
plasmid-containing resistant cells as a result of an energy-dependent efflux process coded for by plasmid genes
(Tynecka et al., 1981). An energy-dependent arsenate efflux is responsible for plasmid-mediated arsenate resistance
in S. aureus and Escherichia coli (Silver & Keach, 1982). The resistance can be plasmid-, chromosomally- or
transposon-encoded and may be constitutive or inducible depending on the metal resistance studied.”
http://mic.sgmjournals.org/content/131/4/939.full.pdf
In 1989, Paul A. Rochelle, et al., University of Wales Institute of Science and Technology, reported on the "Factors
Affecting Conjugal Transfer of Plasmids Encoding Mercury Resistance from Pure Cultures and Mixed Natural
Suspensions of Epilithic Bacteria". They said, "Mercury resistance has been reported to be transferable from 5% of soil
isolates from 26% of marine bacteria (Gauthier et al., 1985), and from 9% of bacteria isolated from freshwater fish. Our
results are similar, as 24% of the pseudomonad-like isolates transferred mercury resistance to P. aeruginosa, although
transfer did not occur from random isolates (< 2%). -- The isolates included Pseudomonas spp., Alcaligenes spp.,
Acinetobacter spp., Klebsiella spp., and Citrobacter spp., all of which have been isolated previously from aquatic
sources (Nuttall, 1982; Jones et al., 1986). -- pQM3 also encoded streptomycin resistance. Both plasmids were broad
host range, transferring to a range of Pseudomonas spp. and Escherichia coli but not to Acinetobacter culcouceticus,
Proteus vulgaris or Klebsiella pneumoniae (Table 3). --The epilithic plasmids pQMl and pQM3 had maximum transfer
frequencies at 20-25°C (Fig. l), which was similar to the optimum temperatures reported for two mercury-resistance
plasmids isolated from soil (25°C; Kelly & Reanney, 1984) and for transfer of antibiotic resistance from a sewage
isolate (20-25°C; Altherr & Kasweck, 1982). Maximum frequencies for transfer of mercury resistance from a marine
pseudomonad to E. coli occurred at 30°C." http://mic.sgmjournals.org/content/135/2/409.full.pdf
In 1990, J. S. Grewal and R. P. Tiwari, Panjab University at Chandigarh, reported on “Inducible Plasmid-mediated
Copper Resistance in Escherichia coli.” They said, “Gram-negative bacteria are considered generally to be more
resistant than gram-positive bacteria to antimicrobial agents. The transfer of multiple drug resistance through R factors
has long been recognized in the enterobacteria, especially Escherichia coli. The plasmids of E. coli not only confer
resistance to antibiotics and metal ions but, in a few reports, have even been shown to confer sensitivity to mercuric
chloride and antibiotics. Specific serotypes of E. coli have been shown to carry transmissible plasmids for both
enterotoxin production (Ent +) and resistance (R) to antibiotics . The occurrence of antibiotic resistant E. coli in man,
sewage and fresh water has been reported. – Of 39 strains of Escherichia coli isolated from foodstuffs, all were
resistant to at least one of a panel of four metallic ions tested. The most common resistance (94.9%) was against
cadmium, followed by arsenate (76.9%), silver (71.8%) and mercury (61.5%). Multiple resistance to three (35.9%) or
four (38.5%) metals was seen more often than resistance to two (1 8%) or one (7.7%) metal only. The opposite trend
was seen in antibiotic resistance; resistance to one (30%) or two (49%) antibiotics was more common than to three or
more antibiotics (13%). Resistance to kanamycin correlated with resistance to silver and cadmium ions and resistance
to ampicillin or cephalothin was, with one exception, associated with resistance to cadmium ions.”
http://jmm.sgmjournals.org/content/32/4/223.full.pdf
In 1999, Amit Gupta, et al., University of Illinois at Chicago, reported on “Molecular basis for resistance to silver cations
in Salmonella.” They said, "Here we report the genetic and proposed molecular basis for silver resistance in pathogenic
microorganisms. The silver resistance determinant from a hospital burn ward Salmonella plasmid contains nine open
reading frames, arranged in three measured and divergently transcribed RNAs. The resistance determinant encodes a
periplasmic silver−specific binding protein (SilE) plus apparently two parallel efflux pumps: one, a P−type ATPase (SilP);
the other, a membrane potential−dependent three−polypeptide cation/proton antiporter (SilCBA). The sil determinant is
governed by a two−component membrane sensor and transcriptional responder comprising silS and silR, which are
co−transcribed. The availability of the sil silver−resistance determinant will be the basis for mechanistic molecular and
biochemical studies as well as molecular epidemiology of silver resistance in clinical settings in which silver is used as a
biocide." http://www.ncbi.nlm.nih.gov/pubmed/9930866
In 2003, Anne Spain, University of Oklahoma and Dr. Elizabeth Alm, Central Michigan University, reported on the
“Implications of Microbial Heavy Metal Tolerance in the Environment.” They said, “Although some heavy metals are
essential trace elements, most can be, at high concentrations, toxic to all branches of life, including microbes, by
forming complex compounds within the cell. Because heavy metals are increasingly found in microbial habitats due to
natural and industrial processes, microbes have evolved several mechanisms to tolerate the presence of heavy metals
(by either efflux, complexation, or reduction of metal ions) or to use them as terminal electron acceptors in anaerobic
respiration. Thus far, tolerance mechanisms for metals such as copper, zinc, arsenic, chromium, cadmium, and nickel
have been identified and described in detail. Most mechanisms studied involve the efflux of metal ions outside the cell,
and genes for this general type of mechanism have been found on both chromosomes and plasmids. Because the
intake and subsequent efflux of heavy metal ions by microbes usually includes a redox reaction involving the metal
(that some bacteria can even use for energy and growth), bacteria that are resistant to and grow on metals also play
an important role in the biogeochemical cycling of those metal ions. This is an important implication of microbial heavy
metal tolerance because the oxidation state of a heavy metal relates to the solubility and toxicity of the metal itself.
When looking at the microbial communities of metal-contaminated environments, it has been found that among the
bacteria present, there is more potential for unique forms of respiration. Also, since the oxidation state of a metal ion
may determine its solubility, many scientists have been trying to use microbes that are able to oxidize or reduce heavy
metals in order to remediate metal-contaminated sites. Another implication of heavy metal tolerance in the environment
is that it may contribute to the maintenance of antibiotic resistance genes by increasing the selective pressure of the
environment. Many have speculated and have even shown that a correlation exists between metal tolerance and
antibiotic resistance in bacteria because of the likelihood that resistance genes to both (antibiotics and heavy metals)
may be located closely together on the same plasmid in bacteria and are thus more likely to be transferred together in
the environment. Because of the prevalence of antibiotic resistant pathogenic bacteria, infectious diseases are
becoming more difficult and more expensive to treat; thus we need to not only be more careful of the drastic overuse of
antibiotics in our society, but also more aware of other antimicrobials, such as heavy metals, that we put into the
environment.” http://www.ruf.rice.edu/~rur/issue2_files/PDF_Final/spain.pdf
In 2003, Jennifer R. Huddleston, Texas Tech Universitym reported on “ANTIBIOTIC- AND METAL-RESISTANT
Aeromonas ISOLATED FROM ENVIRONMENTAL SOURCES.” Huddleston said, “Aeromonas is a ubiquitous aquatic
bacterium that causes serious infections in both cold- and warm-blooded animals, including humans. Clinical isolates of
the organism have shown an increasing incidence of antibiotic and antimicrobial drug resistances since the widespread
use of antibiotics began. The genes for antibiotic resistance and metal resistance are frequently carried on the same
plasmids, imparting both characteristics to the host bacterium. When there are either antibiotics or metals present in
the environment, both markers are co-selected. Two hundred eighty-three Aeromonas isolates belonging to eleven
different species were isolated from several streams and both urban and rural playa lakes in Lubbock, TX and New
Mexico. The minimal inhibitory concentrations of seven metals, six antibiotics, and two synthetic drugs were determined.
Low incidences of trimethoprim resistance, mercury resistance, and arsenite resistance were found. Antibiotic and
metal resistances were not linked in almost all of the Aeromonas isolates. Plasmids were isolated from selected strains
of the arsenite- and mercury-resistant organisms and transformed into Escherichia coli XLl-Blue MRP', showing that the
resistance genes were carried on plasmids. From the data, it was concluded that mercury and arsenite resistance
could be transferred to other organisms in natural environments.” http://etd.lib.ttu.edu/theses/available/etd-06272008-
31295017115568/unrestricted/31295017115568.pdf
In 2003, Marcus Adonai Castro-Silva, et al., Universidade do Vale do Itajaí, Itajaí, SC, Brasil, reported on “Heavy metal
resistance of microorganisms isolated from coal mining environments of Santa Catarina.” They said, “Heavy metal
resistance is a widespread attribute among microorganisms isolated from mining environments. Bacteria from
Acidiphilium and Acidocella genera are able to resist to levels as high as 1M Cd, Zn, Ni and Cu, with this resistance
being plasmid- mediated. Filamentous fungi and yeasts can also show high levels of metals and metalloids resistance,
being this resistance associated to the capacity to accumulate these elements. This work evaluated a great number of
strains of different microorganisms, and in general, the incidence of heavy metal resistance was higher among fungi.
However, except for Cd, the resistance levels were similar to those of bacteria. For fungi, the detected resistance levels
were similar to those related by Durán et al., 1999, except in the case of Zn, which was lower. Results of this study
showed that heavy metal resistance among bacteria is widespread. The strains isolated by enrichment procedures
showed an extreme tolerance (up to 100 mM) to the tested metals (except Cu), which is in agreement with results of
other studies. The high incidence of heavy metal resistance detected in this work indicates the potential of these
microorganisms as bioremediation agents.” http://www.scielo.br/scielo.php?pid=s1517-
83822003000500015&script=sci_arttext
In 2003, Vajiheh Karbasizaed, et al., Tehran University of Medical Sciences, reported on “heavy metal resistance and
plasmid profile of coliforms isolated from nosocomial infections in a hospital in Isfahan, Iran.” They said, “The
antimicrobial, heavy metal resistance patterns and plasmid profiles of Coliforms (Enterobacteriacea) isolated from
nosocomial infections and healthy human faeces were compared. Fifteen of the 25 isolates from nosocomial infections
were identified as Escherichia coli, and remaining as Kelebsiella pneumoniae. Seventy two percent of the strains
isolated from nosocomial infections possess multiple resistance to antibiotics compared to 45% of strains from healthy
human faeces. The difference between minimal inhibitory concentration (MIC) values of strains from clinical cases and
from faeces for four heavy metals (Hg, Cu, Pb, Cd) was not significant. However most strains isolated from hospital
were more tolerant to heavy metal than those from healthy persons. There was no consistent relationship between
plasmid profile group and antimicrobial resistance pattern, although a conjugative plasmid (>56.4 kb) encoding
resistance to heavy metals and antibiotics was recovered from eight of the strains isolated from nosocomial infections.
The results indicate multidrug-resistance coliforms as a potential cause of nosocomial infection in this region. – Twenty-
five coliform isolates involved in nosocomial infections and 20 isolates were from the faeces of healthy persons were
tested in this study. Fifteen of the isolates from nosocomial infections identified as Escherichia coli and the remaining
as Kelebsiella pneumoniae. The most common nosocomial infections were urinary tract (15 cases), blood stream (5
cases), respiratory tract (3 cases) and skin infection (2 cases). – In spite of the wide range of plasmids present in the
bacterial isolates from nosocomial infection, there was no consistent correlation between plasmid profiles and
antibiotic resistance pattern. This is not unexpected since the same antimicrobial resistance pattern can be encoded by
unrelated plasmids, transposons, phages and chromosomal genes.” https://tspace.library.utoronto.
ca/retrieve/2734/jb03077.pdf
In 2004, J . BERG, et al., Royal Veterinary and Agricultural University, reported on the “Copper amendment of
agricultural soil selects for bacterial antibiotic resistance in the field.” They said, “The objective of this study was to
determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance
patterns in indigenous soil bacteria. – Soil bacteria were isolated from untreated and Cu-amended field plots. Cu-
amendment significantly increased the frequency of Cu-resistant isolates. A panel of isolates were characterized by
Gram reaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics. More
than 95% of the Cu-resistant isolates were Gram-negative. Cu-resistant Gram-negative isolates had significantly higher
incidence of resistance to ampicillin, sulphanilamide and multiple (‡3) antibiotics than Cu-sensitive Gram-negative
isolates. Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher
incidence of resistance to chloramphenicol and multiple (‡2) antibiotics than corresponding isolates from control plots. –
The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also
indirectly selects for antibiotic resistance in the Cu-resistant bacteria. Hence, the widespread accumulation of Cu in
agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance.”
http://ucbiotech.org/issues_pgl/ARTICLES/BERG.PDF
In 2005, S.L. Percival, et al., reported on "Bacterial resistance to silver in wound care". They said, "Plasmid-mediated
Ag+ resistance has been identified in P. stutzeri, members of the Enterobacteriaceae and Citrobacter spp. -- A plasmid
conferring resistance to a number of antibiotics and heavy metals including Ag+ was obtained from a Salmonella
species isolated from a burns unit after causing septicaemia, death in three patients and resulting in the closure of the
burns unit at Massachusetts General Hospital. This was the first report of the genetic and molecular basis for Ag+
resistance. -- The gene cluster for AgC resistance was found to contain nine genes,23 seven of these were named and
two were classified as open reading frames (silP, ORF105, sil AB, ORF96, silC,silSR and silE). -- Laboratory studies
have provided evidence of plasmid pMG101 transfer to E. coli. In fact, Ag+ resistance conferred by plasmid pMG101
enabled the growth of E. coli in more than 0.6 mM Ag+ which is in excess of six times the tolerable concentration for
sensitive E. coli. -- It has been shown that most interactions between chemotherapeutic agents and microbial
populations occur at very low concentrations, and that low concentrations produce a substantial stress in bacterial
populations that eventually influences the rate of variation and the diversity of adaptive responses leading to high
levels of resistance." http://www.idpublications.com/journals/PDFs/JHI/JHI_MostCited_2.pdf
Kan-Jen Tsai Ph.D. gives a brief review of metal resistance in the 2006 Introduction to “Bacterial heavy metal
resistance”. He said, “Heavy metals include some toxic chemical elements and their deviated chemical compounds.
Most of these compounds are not functionally involved in any activity for life. On the other hand, these heavy metals
are frequently generating strong reactive oxygen species (ROS) and directly/indirectly causing gene mutations, and
therefore their presences are hazardous to cell. Even though their toxicities might be different, the protein damages
and their competitions of the entry for certain essential elements might be the most likely effects to cell.”
http://www.bio.sci.osaka-u.ac.jp/initiative2006/pdf/TsaiLectSum.pdf
In 2006, B. H. Belliveau, et al., reported on “Lead, tin, and multiple antibiotic resistant Pseudomonas spp. isolated from
polluted sediment.” They said. “Two Pseudomonas spp. were isolated from freshwater sediment by enrichment in
nutrient broth amended with either 1000 μg/mL lead or tin. Both organisms also displayed multiple antiobiotic
resistance. The tin-resistant pseudomonad was also resistant to 1000 μg/mL lead, whereas the Iead-resistant
Pseudomonas sp. was not resistant to 1000 μg/mL tin. Vertical and horizontal agarose gel electrophoresis of cleared
cell lysates revealed that plasmids were not present in either strain. It therefore appears that lead, tin, and antiobiotic
resistance is not plasmid encoded in these organisms.” http://onlinelibrary.wiley.com/doi/10.1002/tox.
2540020402/abstract
In 2006, Maria-Judith De Souza, et al. Reported on “Metal and antibiotic-resistance in psychrotrophic bacteria from
Antarctic Marine waters.” They said, “In the wake of the findings that Antarctic krills concentrate heavy metals at ppm
level, (Yamamoto et al., 1987), the Antarctic waters from the Indian side were examined for the incidence of metal and
antibiotic-resistant bacteria during the the austral summer (13th Indian Antarctic expedition) along the cruise track
extending from 50°S and 18°E to 65°S and 30°E. The bacterial isolates from these waters showed varying degrees of
resistance to antibiotics (Chloramphenicol, ampicillin, streptomycin, tetracycline and kanamycin) and metals (K2CrO4,
CdCl2, ZnCl2 and HgCl2) tested. Of the isolates screened, about 29% and 16% were resistant to 100ppm of cadmium
and chromium salt respectively. Tolerance to lower concentration (10ppm) of mercury (Hg) was observed in 68% of the
isolates. Depending on the antibiotics the isolates showed different percentage of resistance. Multiple drug and metal-
resistance were observed. High incidence of resistance to both antibiotics and metals were common among the
pigmented bacterial isolates. Increased resistance decreased the ability of bacteria to express enzymes. The results
reiterate previous findings by other researchers that the waters of southern ocean may not be exempt from the spread
of metal and antibiotic resistance.”
http://drs.nio.org/drs/bitstream/2264/206/1/Ecotoxicology_15_379.pdf
In 2007, Stuart A. Thompson, et al., Medical College of Georgia at Augusta, reported on “Novel Tetracycline
Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens.” They said, “As a step in
characterizing the possible indirect selection of antibiotic resistance by heavy metal contamination at the Savannah
River site, we selected an environmental bacterium [Serratia] that was resistant to mercury. It was screened for
resistance to several antibiotics and found to also be resistant to tetracycline. – Serratia marcescens is an
opportunistic gram-negative pathogen that is a significant cause of nosocomial infections, such as pneumonia, urinary
tract infections, bacteremia, meningitis, and myocarditis, including troublesome and recurrent hospital outbreaks, for
example, in neonatal intensive care units. S. marcescens infections (septicemia, pneumonia, cellulitis) are also
problematic in human immunodeficiency virus-infected persons. S. marcescens is ubiquitous in nature and can be
found in water, soil, plants, insects, and animals. – The apparent lack of plasmids in this S. marcescens strain, as well
as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were
located on the S. marcescens chromosome and may have been acquired by transduction.” http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC1855637/
In 2008, Delphine Pages, et al., Lab Ecol Microb Rhizosphere & Environ Extrem (LEMiRE), Saint-Paul-lez-Durance,
reported on “Heavy Metal Tolerance in Stenotrophomonas maltophilia,” They said, “Stenotrophomonas maltophilia is
an aerobic, non-fermentative Gram-negative bacterium widespread in the environment. S. maltophilia Sm777 exhibits
innate resistance to multiple antimicrobial agents. Furthermore, this bacterium tolerates high levels (0.1 to 50 mM) of
various toxic metals, such as Cd, Pb, Co, Zn, Hg, Ag, selenite, tellurite and uranyl. S. maltophilia Sm777 was able to
grow in the presence of 50 mM selenite and 25 mM tellurite and to reduce them to elemental selenium (Se0) and
tellurium (Te0) respectively. Transmission electron microscopy and energy dispersive X-ray analysis showed
cytoplasmic nanometer-sized electron-dense Se0 granules and Te0 crystals. Moreover, this bacterium can withstand
up to 2 mM CdCl2 [calcium chloride] and accumulate this metal up to 4% of its biomass. The analysis of soluble thiols in
response to ten different metals showed eightfold increase of the intracellular pool of cysteine only in response to
cadmium. Measurements by Cd K-edge EXAFS spectroscopy indicated the formation of Cd-S clusters in strain Sm777.
Cysteine is likely to be involved in Cd tolerance and in CdS-clusters formation. Our data suggest that besides high
tolerance to antibiotics by efflux mechanisms, S. maltophilia Sm777 has developed at least two different mechanisms to
overcome metal toxicity, reduction of oxyanions to non-toxic elemental ions and detoxification of Cd into CdS. – This
species constitutes one of the dominant rhizosphere inhabitant, frequently isolated from the rhizosphere of wheat, oat,
cucumber, maize, oilseed rape, and potato – This bacterium was also increasingly described as an important
nosocomial pathogen in debilitated and immunodeficient patients, as well as associated with a broad spectrum of
clinical syndromes, e.g. bacteraemia, endocarditis, respiratory tract infections. – S. maltophilia displays intrinsic
resistance to many antibiotics, making selection of optimal therapy difficult.” http://www.plosone.org/article/info:doi%
2F10.1371%2Fjournal.pone.0001539
In 2008, Fatih Matyar, et al., Çukurova University at Adana, reported on “Antibacterial agents and heavy metal
resistance in Gram-negative bacteria isolated from seawater, shrimp and sediment in Iskenderun Bay, Turkey.” They
said, “The aim of the present study was to determine the level of antibiotic resistance patterns and distribution of heavy
metal resistance of bacterial isolates from seawater, sediment and shrimps, and to determine if there is a relationship
between antibiotic and heavy metal resistance. We undertook studies in 2007 in the industrially polluted Iskenderun
Bay, on the south coast of Turkey. The resistance of 236 Gram-negative bacterial isolates (49 from seawater, 90 from
sediment and 97 from shrimp) to 16 different antibiotics, and to 5 heavy metals, was investigated by agar diffusion and
agar dilution methods, respectively. A total of 31 species of bacteria were isolated: the most common strains isolated
from all samples were Escherichia coli (11.4%), Aeromonas hydrophila (9.7%) and Stenotrophomonas maltophilia
(9.3%). There was a high incidence of resistance to ampicillin (93.2%), streptomycin (90.2%) and cefazolin (81.3%),
and a low incidence of resistance to imipenem (16.5%), meropenem (13.9%) and cefepime (8.0%). Some 56.8% of all
bacteria isolated from seawater, sediment and shrimp were resistant to 7 or more antibiotics. Most isolates showed
tolerance to different concentrations of heavy metals, and minimal inhibition concentrations ranged from 12.5 μg/ml to >
3200 μg/ml. The bacteria from seawater, sediment and shrimp showed high resistance to cadmium of 69.4%, 88.9%,
and 81.1% respectively, and low resistance to manganese of 2%, 6.7% and 11.3% respectively. The seawater and
sediment isolates which were metal resistant also showed a high resistance to three antibiotics: streptomycin, ampicillin
and trimethoprim-sulphamethoxazole. In contrast, the shrimp isolates which were metal resistant were resistant to four
antibiotics: cefazolin, nitrofurantoin, cefuroxime and ampicillin. Our results show that Iskenderun Bay has a significant
proportion of antibiotic and heavy metal resistant Gram-negative bacteria, and these bacteria constitute a potential risk
for public health.” http://www.sciencedirect.com/science/article/pii/S0048969708008346
In 2008, Steven L. Percival, et al., reported on the “Prevalence of Silver Resistance in Bacteria Isolated from Diabetic
Foot Ulcers and Efficacy of Silver-Containing Wound Dressings.” They said, “The micro-organisms used in this study
were routinely isolated from the chronic foot wounds of patients attending the Diabetic Foot Clinic at Tameside General
Hospital, UK who had diabetic foot ulcers exhibiting signs and symptoms of clinical infection, as well as ulcers clinically
free from infection. -- The isolates obtained for this study were coagulase negative Staphylococcus aureus (CNS — 17
strains), Staphylococcus aureus (24 strains), Enterobacter spp. (three strains), Enterobacter cloacae (four strains),
Escherichia coli (six strains), additional coliforms (two strains), Pseudomonas spp. (six strains), Pseudomonas
aeruginosa (nine strains), Enterococcus faecalis (five strains), Alcaligenes faecalis (two strains), diphtheroids (10
strains), Citrobacter spp. – The physiological, biochemical, genetic, and structural studies of the silver-resistant
determinant plasmid pMG101 established the molecular basis of silver resistance. Plasmid pMG101 is a 182 kb,
transferable plasmid26 encoding resistance to silver (nine Open Reading frames [ORFs] in three transcriptional units),
mercury, tellurite, ampicillin, chloramphenicol, tetracycline, streptomycin, and sulphonamide. It confers resistance in
bacteria at silver concentrations six or more times the concentration of what a sensitive Escherichia coli can tolerate.
Functions assigned to the genes are based on homologous systems encoding resistances to other metals. The silver-
resistance system encodes two silver efflux pumps (one an ATPase and the other chemiosmotic) and two periplasmic
Ag+-binding proteins.” http://www.o-wm.com/article/8483
In 2009, E. Eze, et al., University of Nigeria at Nsukka,reported on the “Association of metal tolerance with multidrug
resistance among bacteria isolated from sewage.” They said, “Sewage effluent from the sewage treatment plant of the
University of Nigeria, Nsukka, was analyzed for the presence of metal and non-metal ions and for the presence of metal
tolerant and drug resistant bacteria. Methods: Plasmid mediation of metal tolerance and multiple drug resistance was
demonstrated by sodium dodecyl sulphate (SDS) curing and direct cell transfer experiments. Results: Ions found
present (in mgL-1) include, among others, Mercury (50.148), Lead (41.906), Sodium (907.240), and Potassium
(700.00). Bacterial populations isolated from the effluent were members of the genera Enterobacter (n=15),
Escherichia (n=18), Achromobacter (n=18), Acinetobacter (n=25) Klebsiella (n=12), Pseudomonas (n=08), Proteus
(n=20) and Serratia (n=10). Enterobacter spp showed high percentage tolerance of 73% to Lead. Species of
Acinetobacter and Pseudomonas showed, to varying degrees, across-the-board tolerance to all the individual salts.
Also an across-the-board resistance of between 25-75% and 8.3-41.7% to the test drugs was exhibited by
Pseudomonas and Klebsiella spp respectively. Sixty per cent each of Acinetobacter and Klebsiella spp lost both metal
tolerance and drug resistance attributes simultaneously following the SDS curing protocol. Overall percentage loss of
both characteristics was 57.1%. Acquisition of metal tolerance and multidrug resistance by recipients was total (100%)
and so was the subsequent loss of these capabilities following SDS treatment of these recipients.” http://www.cabdirect.
org/abstracts/20103285316.html
In 2009, Pankaj Kumar Jain, et al., Birla Institute of Technology and Science at Pilani, reported on the "Characterization
of metal and antibiotic resistance in a bacterial population isolated from Copper mining industry." They said, "The
emergence of multiple metal/antibiotic resistance among bacterial populations poses a potential threat to human
health. The co-existence of metal/antibiotic resistance in bacterial strains suggests the role of metals as a factor which
can contribute to such a phenomenon. The objective of this study is to characterize multiple metal/antibiotic resistant
bacteria from the soil and water sample of a copper mining industry. A total of 24 strains (12 Gram-positive and 12
Gram-negative bacteria) were identified by the 16sRNA gene sequencing. Out of 24 bacterial isolates, 9 isolates show
multiple metal and antibiotic resistance. These strains were screened to find the presence of endogenous plasmid
DNA. One strain, Bacillus sp. (PRS3) was found to have plasmid DNA of 4.2kb that could replicate in both Gram
positive and Gram negative bacteria. Thus, it can be predicted that metal pollution results in selective pressure that
leads to the development of multiple metal/antibiotic resistance among bacterial populations, probable through
horizontal gene transfer. This study strongly emphasizes the need for metal laden waste treatment and safe disposal,
thus preventing the transfer of such potential pathogens to human populations."
http://www.classicrus.com/IJIB/Arch/2009/1602.pdf
In 2009, Leslie H. Wardwell, et al., reported on the “Co-selection of Mercury and Antibiotic Resistance
in Sphagnum Core Samples Dating Back 2000 Years.” They said, “Metal exposure might induce multiple drug
resistance (MDR) in bacteria in environments devoid of antibiotics via the process of co-selection, but the extent is
poorly known. Core samples from two sphagnum peat bogs in central Maine, USA, were analyzed for total Hg content
and were radiocarbon dated. Culturable bacteria isolated from various core depths were assayed for antibiotic- and Hg-
resistance and the presence of merA (mercuric reductase). Our results show that sphagnum peat bogs represent
natural ecosystems that contain ambient levels of Hg that select for indigenous bacterial strains that are not only Hg
resistant, but also possess the MDR phenotype. – Dates for deposition of sphagnum at the base of cores taken from
Round Pond bog and Hamilton Pond bog, 325BCE and 260AD (Table 2) respectively, are well before the application of
antimicrobial chemotherapy in the 1940s. Therefore, high levels of antibiotic resistance observed in bacterial
populations far below the bog surface are not a result of selection that was influenced anthropogenically, since these
bacteria have not likely been exposed to high levels of manufactured antimicrobial compounds. – MDR bacteria were
isolated from all depths of the sphagnum cores in both bogs (Tables 5 and 6). Broad maximal resistances to antibiotics
in the ß-lactam, aminoglycoside, cephem and folate pathway inhibitor families were observed. Pseudomonas spp.
demonstrated the greatest antibiotic resistance with P. putida strains RP 4 and RP 5 possessing maximal resistance to
13 of the 23 tested antibiotics. Of the gram-positive isolates, Paenibacillus sp. RP 16 exhibited the greatest antibiotic
resistance to 11 of the 19 tested antibiotics (Table 5). Only 3 gram-positive metabolically active isolates had resistance
to the fluorquinolone, ciprofloxacin (Table 5). However, endospore-forming isolates possessed ciprofloxacin
resistance (Table 6). In addition, Paenibacillus spp. RP14 and RP 16 show resistance to the macrolides erythromycin
and clarithromycin as well as the glycopeptide vancomycin (Table 5).” http://people.wcsu.
edu/gyurer/files/Wardwell2009_Coselection%20of%20mercury%20and%20AB%20resistance.pdf
In 2009, A. D. Altalhi, reported on “Plasmids profiles, antibiotic and heavy metal resistance incidence of endophytic
bacteria isolated from grapevine (Vitis vinifera L.).” Altalhi said, “Little is known about the bacterial communities
associated with the plant inhabiting desert ecosystem. In this study, the bacterial population associated with grapevine
(Vitis vinifera L.) plant, growing desert soil was analyzed using the culture dependent approach. A total of 111 bacterial
isolates were isolated from stems and leaves samples, collected from different locations and subjected to further
analyses. Based on the identification methods, the bacterial isolates were grouped into 14 genera. The main genera
are Acetobacter, Acinetobacter, Citrobacter, Enterobacter, Erwinia, Escherichia, Methylococcus, Xanthomonas, Vibrio,
Bacillus, Micrococcus, Planococcus, Staphylococcus and Streptomyces. Significant differences in the endophytic
communities were observed between plants collected from different sites and also between plant stems and leaves. All
the isolates were examined for plasmid DNA content and resistance to antibiotics (Ampicillin, Kanamycin, Tetracyclin)
and heavy metals. Minimum inhibitory concentrations (MICs) of Cu, Cd, Hg, Mn, Ni and Zn for isolates were also
determined. Resistance was most frequent to Ampicillin (57%), followed by Kanamycin (53%) and Tetracycline (26%).
The highest MICs observed were 10 µg/ml for mercury, 50 µg/ml for Cu and Cd and 200 µg/ml for other metals. On a
percentage basis, 18.48% of total strains from leaves were found to harbour plasmids, whereas, 11.83% of the roots
isolates contained plasmids.” http://www.cabdirect.org/abstracts/20093342726.html;
jsessionid=CB456F84E1242F6600C4199D06EA85C0
In 2010, Ganiyu O Oyetibo, et al., University of Lagos, Akoka, reported on “Bacteria with dual resistance to elevated
concentrations of heavy metals and antibiotics in Nigerian contaminated systems.” They said, “Samples of soil, water,
and sediments from industrial estates in Lagos were collected and analyzed for heavy metals and physicochemical
composition. Bacteria that are resistant to elevated concentrations of metals (Cd(2+), Co(2+), Ni(2+), Cr(6+), and Hg
(2+)) were isolated from the samples, and they were further screened for antibiotic sensitivity. The minimum tolerance
concentrations (MTCs) of the isolates with dual resistance to the metals were determined. The physicochemistry of all
the samples indicated were heavily polluted. Twenty-two of the 270 bacterial strains isolated showed dual resistances
to antibiotics and heavy metals. The MTCs of isolates to the metals were 14 mM for Cd(2+), 15 mM for Co(2+) and Ni
(2+), 17 mM for Cr(6+), and 10 mM for Hg(2+). Five strains (Pseudomonas aeruginosa, Actinomyces turicensis,
Acinetobacter junni, Nocardia sp., and Micrococcus sp.) resisted all the 18 antibiotics tested. Whereas Rhodococcus
sp. and Micrococcus sp. resisted 15 mM Ni(2+), P. aeruginosa resisted 10 mM Co(2+). To our knowledge, there has
not been any report of bacterial strains resisting such high doses of metals coupled with wide range of antibiotics.
Therefore, dual expressions of antibiotics and heavy-metal resistance make the isolates, potential seeds for
decommissioning of sites polluted with industrial effluents rich in heavy metals, since the bacteria will be able to
withstand in situ antibiosis that may prevail in such ecosystems.”
http://www.nextbio.com/b/search/article.nb?id=19688604
In 2010, Virender Singh, et al., HNB Garhwal University Srinagar, Uttrakhand (U.K.), reported on the “ISOLATION AND
CHARACTERIZATION OF PSEUDOMONAS RESISTANT TO HEAVY METALS CONTAMINANTS.” They said, “In the
present study total eight heavy metal resistant Pseudomonas sp were isolated from sewage of industrial effluents from
waste water treatment plant of Paonta Sahib H.P. India, against chromium, copper, nickel, cadmium. All the isolates
exhibited high resistance to heavy metals with minimum inhibitory concentration (MIC) for heavy metals ranging from
50μg/ml to 350μg/ml. All isolates showed multiple tolerances to heavy metal and were multi antibiotic resistant. Heavy
Metal Tolerance Test indicated maximum microbial tolerance of Pseudomonas sp (Ps-6) to Copper (300 μg/ml) and
lowest to Chromium (60 μg/ml).” http://www.globalresearchonline.net/journalcontents/Volume3issue2/Article%20030.pdf
In 2011, Parisa Keramati, et al., Soil Science Department, Khorasgan (Isfahan) branch, Islamic Azad University,Isfahan,
reported on the “Multi-metal resistance study of bacteria highly resistant to mercury isolated from dental clinic effluent.”
“Heavy metals pollution represents an important environmental problem. One of these metals is mercury. The aim of
this research was isolating bacteria highly resistant to mercury from dental clinic effluent to investigate their growth
potential in the presence of other heavy metals, such as Zn, Cu, Ni, Ag, Cd and Pb. Three dental wastewater samples
were selected and their mercury concentrations, pH, EC, BOD5 and COD were determined. The mercury-resistant
bacteria were found to belong to the genera of Pseudomonas, Proteus, Citrobacter, Bacillus, Corynebacterium and
Staphylococcus. In examining multi-metal resistances, the pattern of hexa-R was seen in the case of Citrobacter and
Pseudomonas genera. The highest tolerated concentration of heavy metals was 25.6 mM which is related to Ag and Cu
and tolerated by Citrobacter isolate. It is also indicated that Citrobacter was the most resistant isolates to Cd with
significant difference (P < 0.05). Results of this study demonstrate the occurrence of different groups of bacteria,
capable of high tolerance to mercury with a potential to tolerate a variety of other toxic heavy metals suggest that,
resistance to many types of toxicants may be present in the same organism; therefore, such organisms have high
potential for biotechnology purposes.” http://www.academicjournals.org/ajmr/PDF/pdf2011/4Apr/Keramati%20et%20al.
pdf
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