A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Section 4
Antibiotic Resistance in the Soil. 9/09/2011
Back to Myth # 4
For over 60 years researchers have been mining soil for Actinomycetes, Fungi and Bacteria that produce natural toxins
that we call antibiotics. They also carry the gene that makes them resistant to the toxins. Since the 1980s, we have
been re-engineering soil organisms to produce greater quantities of the toxins and to create organisms that could not
exist in nature. The re-engineered organisms include a antibiotic resistant gene to verify changes took place. Many of
these antibiotic resistant genes will end up on agricultural land. Antibiotic resistant organisms will continue to build up in
soils as pathogenic organism contaminated sewage effluent and sludge are disposed of on grazing land, cropland,
school grounds, parks, forests, home lawns and gardens as reclaimed water and biosolids. This is not new research.
What we don't know is the effect of dumping the engineered organisms on soils where they mix with the environmental
organisms or where the tipping point will be.
In 1973, Michael A. Cole, University of Illinois at Urbana, and Gerald H. Elkan, North Carolina State University at
Raleigh, reported on the “Transmissible Resistance to Penicillin G, Neomycin, and Chloramphenicol in Rhizobium
japonicum.” They said, “The genetic basis for resistance to a number of antibiotics was examined in Rhizobium
japonicum. Resistance to penicillin G, neomycin, and chloramphenicol appears to be mediated by an extrachromosomal
element similar to that found in the of toxins Enterobacteriaceae. Resistance to these antibiotics was eliminated from
cells by treatment with acridine orange, and resistance to all three antibiotics could be transferred en bloc to
Agrobacterium tumefaciens under conditions excluding transformation or transduction as possible genetic mechanisms.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC444536/pdf/aac00351-0048.pdf
In 1978, K. Bernhard, et al., Universitat Wurzburg at Rontgenring, reported on “Bacteriocin and Antibiotic Resistance
Plasmids in Bacillus cereus and Bacillus subtilis.” They said, “Sporeforming bacteria were enriched from garden soil and
identified as B. cereus or B. subtilis by standard methods. The purified strains were then tested for antibiotic resistance
and antibiotic or bacteriocin production. – In a search for Bacillus plasmids, we chose to isolate Bacillus strains, mainly
B. cereus and B. subtilis, from aerobic layers of soil, which are also populated by the main producers of antibiotics, i.e.,
Streptomyces, other Bacillus species, and fungi. – Also the described transformation rate is higher for the B. cereus
plasmid.The Tcr determined by pBC16 is relatively high, whereas pT127 renders the B. subtilis cells resistant only to low
concentrations of tetracycline. The properties of both plasmids make them potential vectors for gene cloning in B.
subtilis.” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC222102/pdf/jbacter00297-0465.pdf
In 1978, P. R. Fisher, et al., Queensland University, reported on the "Isolation and Characterization of the Pesticide-
Degrading Plasmid pJP1 from Alcaligenes paradoxus." They said, "Plasmid involvement in the degradation of camphor
was first reported by A. M. Chakrabarty and I. C. Gunsalus in 1971 (Genetics 68:S10, 1971). Subsequent investigations
have revealed plasmids encoding the degradation of other naturally occurring aromatic and aliphatic compounds such
as octane, naphthalene, salicylate, and toluate (3). Such plasmids increase the biochemical versatility of the host
bacterium, extending the range of complex organic compounds used as sole sources of carbon and energy.
Significantly, these plasmids have been isolated almost exclusively from species of the genus Pseudomonas, a group of
bacteria known to play a major role in the breakdown and recycling of naturally occurring organic molecules in both soil
and water. ... Other characteristics not associated with the presence of pJP1 were as follows. (i) The responses to
antibiotics by using Mastring-S disks were resistance to carbenicillin, penicillin G, ampicillin, cephalothin,
chloramphenicol, kanamycin, erythromycin, tetracycline, nitrofurantoin, gentamicin, streptomycin, lincomycin, and
novobiocin and sensitivity to sulfadiazine, polymyxin B, or colistin sulfate. ... Extensive, unrestricted use of synthetic
molecules such as antibiotics and pesticides has resulted in widespread development of microbial populations capable
of degrading these molecules. Unlike the development of antibiotic resistance, which is an undesirable occurrence, the
evolution and spread of pesticide-degrading genes may have a beneficial effect by removing potentially dangerous
pollutants from the environment." http://www.ncbi.nlm.nih.gov/pmc/articles/PMC222450/pdf/jbacter00292-0078.pdf
In 1979, Michael A. Cole, University of Illinois at Urbana, and Gerald H. Elkan, North Carolina State University at Raleigh,
reported on the “Multiple Antibiotic Resistance in Rhizobiumjaponicum.” They said, “A total of 48 strains of the soil
bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics. Over 60% of the
strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant
to neomycin and penicillin G, when tested by disk assay procedures. The most common grouping of resistances in
strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol,
and streptomycin (25% of all strains tested). The occurrence of multiple drug resistance in a soil bacterium that is not a
vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple
drug resistance.” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC243316/pdf/aem00209-0089.pdf
In 1981, R. H. Don and J. M. Pemberton, University of Queensland at St. Lucia, reported on the "Properties of Six
Pesticide Degradation Plasmids Isolated from Alcaligenes paradoxus and Alcaligenes eutrophus." They said,
"Biophysical and genetic properties of six independently isolated plasmids encoding the degradation of the herbicides
2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid are described. Four of the plasmids, pJP3,
pJP4, pJP5, and pJP7, had molecular masses of 51 megadaltons, belonged to the IncPl incompatibility group, and
transferred freely to strains of Escherichia coli, Rhodopseudomonas sphaeroides, Rhizobium sp., Agrobacterium
tumefaciens, Pseudomonas putida, Pseudomonas fluorescens, and Acinetobacter calcoaceticus. ... To identify
additional plasmid- encoded properties, plasmid-bearing strains were tested for their ability to utilize a variety of
compounds as sole source of carbon and energy and for resistance to antibiotics, heavy metal ions, and the
organomercurials PMA and merbromin. ... The herbicide 2,4,5-T was not utilized by any of the plasmid-bearing strains.
Other properties not associated with the presence of the plasmids were resistance to the antibiotics penicillin G,
streptomycin, tetracycline, erythromycin, kanamycin, methicillin, sulphafurazole, sulfamethoxazole, nalidixic acid,
bacitracin, trimethoprim, gentamicin, and cloxacillin and resistance to the heavy metal ions of cobalt (CoCl2), cadmium
(CdCl2), copper (CuS04), nickel (NiCl2), and zinc (ZnCl2)- ... Under natural conditions a number of routes exist for the
widespread dissemination of genetic material through bacterial populations. With antibiotic resistance genes, this spread
has been facilitated by transposable genetic elements and broad-host-range plasmids (7). The ubiquity of ,B-
lactamases has been attributed in part to the movement of transposon TnA throughout gram negative bacterial
populations on broad-hostrange plasmids such as RP4 (17). ...- Second, resistance to mercury is encoded by a variety
of antibiotic resistance plasmids, certain conjugative plasmids (28, 29), and at least one degradative plasmid (6);
therefore, it is not unusual to find that all four plasmids can convert PMA to mercury. What is interesting is that PMA is
both a fungicide and an herbicide, and as the plasmids already encode the degradation of other unrelated herbicides
(2,4-D, MCPA, and 3-chlorobenzoate), these plasmids represent the first broad-host-range, multiple-pesticide-
degrading plasmids to be reported.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC217166/pdf/jbacter00273-0017.pdf
In 1988, C. Steven McDaniel, et al., Texas A&M University, College Station, reported on the “Cloning and Sequencing of
a Plasmid-Borne Gene (opd) Encoding a Phosphotriesterase.” They said, “Synthetic organophosphorus neurotoxins are
used extensively as agricultural and domestic pesticides including insecticides, fungicides, and herbicides. Naturally
occurring bacterial isolates capable of metabolizing this class of compounds have received considerable attention since
they provide the possibility of both environmental and in situ detoxification. Pseudomonas putida MG and
Flavobacterium spp. have been shown to possess the ability to degrade an extremely broad spectrum of
organophosphorus phosphotriesters as well as thiol esters. – It is clear that many soil bacteria possess degradative,
plasmid-borne genes which could be readily transferred and expressed among a variety of bacterial and viral hosts.
This phenomenon is not limited to organophosphorus neurotoxins, since plasmid-borne genes for degradative enzymes
of herbicides have been well documented. In the case of the opd genes, a wide range of pesticides sharing a common
chemical structure are degraded, providing the potential for rapid evolution of genes to degrade a variety of pesticides
and challenging the agrochemical rationale of substituting pesticides of similar chemical structure or increasing
application rates for extended pest control. Rapid mutational adaptation in an enriched soil bacterial population could
render ineffective any subsequent applications of a similar chemical.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC211123/pdf/jbacter00183-0312.pdf
In 1990, R. B. Henschke and F. R. J. Schmidt reported on the “Screening of soil bacteria for plasmids carrying antibiotic
resistance.” They said, “To evaluate the role of plasmids in soil communities antibiotic-resistant bacteria have been
isolated from soil. Among them, 419 l-aminopeptidase positive strains (Gram-negative) and 28 l-aminopeptidase
negative strains (Gram-positive) were screened for the presence of plasmids. None of the Gram-negative organisms
contained plasmids. Among the Gram-positive bacteria plasmid-harboring strains were detected.”
http://www.springerlink.com/content/q8954667833626nh/
In 1990, Rolf B. Henschke and Friedrich R. J. Schmidt reported on "Plasmid mobilization from genetically engineered
bacteria to members of the indigenous soil microflora in situ." They said, "For study of gene transfer and expression in a
large variety of soil bacteria in situ, a gene-promoter cassette was constructed and inserted into an expression vector
that allowed expression and maintenance in a wide host range. The hybrid replicon was transformed into a mobilizer
strain that transferred its DNA to other Gram-negative bacteria at very high rates. This genetically engineered
microorganism (GEM) was introduced into nonsterile soil microcosms. Plasmid transfer from the introduced GEM to
members of the native soil flora was observed in situ."
http://www.springerlink.com/content/t51j4t6344166778/
In 1991, John L. Norwlli, et al., New York State Agricultural Experiment Station, Cornell University,
Geneva, reported on the "Homologous Streptomycin Resistance Gene Present among Diverse
Gram-Negative Bacteria in New York State Apple Orchards." They said, "The streptomycin resistance gene of
Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe
that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared
by three different DNA clones of the resistance gene. ... Resistance to streptomycin in Pseudomonas syringae pv.
papulans, the causal agent of blister spot of apple, has been observed in New York, Ohio, and Michigan (4, 7a, lla).
Blister spot of apple is predominant on, but not restricted to, the cultivar Mutsu (3, 24). Previously, growers in New York
successfully controlled the disease with streptomycin sprays. In 1985, incidents of control failure after the use of
streptomycin were reported and streptomycin-resistant strains of P. syringae pv. papulans were isolated from several
orchards (4). There was a significant correlation between the number of streptomycin sprays applied the previous
season and the detection of streptomycin-resistant strains of P. syringae pv. papulans (4). ... These results show that
DNA associated with streptomycin resistance in the plant-pathogenic bacteria P. syringae pv. papulans is part of the
gene pool of a diverse group of gram-negative bacteria in the orchard environment. There have been numerous
demonstrations of the in vitro and in planta transfer of resistance plasmids between plant pathogenic
bacteria and bacteria associated with plants and animals under laboratory or controlled experimental conditions. – DNA
homologous to SMP3 appears to be common and widespread among streptomycin-resistant plant pathogenic bacteria.
In addition to the P. syringae pv. Syringae strain reported here, SMP3 has shown homology with P. syringae strains
isolated in Michigan, Ohio, and Georgia (21b). P. syringae pv. papulans Psp36 has also been reported to contain DNA
homologous with the streptomycin resistance gene of Xanthomonas campestris pv. vesicatoria BV5-4a (21)."
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC182737/pdf/aem00055-0164.pdf
In 2001, J. C. Chee-Sanford, et al., University of Illinois at Urbana-Champaign, Urbana, found resistant bacteria in the
soil due to the “Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying
Two Swine Production Facilities.” They said, In this study, we used PCR typing methods to assess the presence of
tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying
two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P),
tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to
be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The
identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and
sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not
predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in
some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains
of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study
demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater
may be a potential source of antibiotic resistance in the food chain.” http://aem.asm.org/cgi/content/abstract/67/4/1494
In 2001, Patricia S. McManus, University of Wisconsin at Madison and Virginia O. Stockwell, Oregon State University at
Corvallis reported on "Antibiotic Use for Plant Disease Management in the United States." They said, "In the 1950s, soon
after the introduction of antibiotics in human medicine, the potential of these “miracle drugs” to work wonders on plant
diseases was explored. Nearly 40 antibiotics were screened for plant disease control (12). Of the screened compounds,
fewer than 10 were used commercially and only streptomycin had significant usage. For the control of certain bacterial
diseases of plants, streptomycin was indeed a silver bullet. Unfortunately, just as the emergence of bacterial strains
resistant to antibiotics has limited their performance in clinical settings, streptomycin resistance has destabilized plant
disease control. The antibiotic resistance crisis in medicine has been widely publicized and is recognized as a major
threat to controlling human bacterial diseases and infections worldwide (19, 20). Efforts to conserve the efficacy of
antibiotics in medicine have drawn scrutiny to all antibiotic use within and outside the medical field, including agriculture.
Concern from the general public and within the scientific community has resulted in a number of misconceptions
regarding antibiotic use on plants. We describe the current use of antibiotics on plants in the U.S., the emergence of
antibiotic-resistant plant pathogens, and the political upheaval regarding antibiotic use on plants. ... At this point,
streptomycin and oxytetracycline have been used on crop plants for the past 45 years and 25 years, respectively,
without reports of adverse effects on humans. The efficacy of these silver bullets for control of plant diseases has been
diminished in some regions due to the emergence of antibiotic-resistant strains of pathogens. However, until effective
and economic alternatives become available, antibiotics will remain important tools for the management of some of the
most devastating plant diseases." http://www.plantmanagementnetwork.org/pub/php/review/antibiotic/
In 2002, N. Esiobu, L. Armenta L and J. Ike, Florida Atlantic University, reported on “Antibiotic resistance in soil and
water environments.” They said, “Seven locations were screened for antibiotic-resistant bacteria using a modified agar
dilution technique. Isolates resistant to high levels of antibiotics were screened for r plasmids. Low-level resistance (25
micro g x ml(-1)) was widespread for ampicillin, penicillin, tetracycline, vancomycin and streptomycin but not for
kanamycin. Resistant populations dropped sharply at high antibiotic levels, suggesting that intrinsic non-emergent
mechanisms were responsible for the multiple drug resistance exhibited at low doses. Dairy farm manure contained
significantly (P < 0.01) more (%) resistant bacteria than the other sites. Bacteria isolated from a dairy water canal, a lake
by a hospital and a residential garden (fertilized by farm manure) displayed resistance frequencies of 77, 75 and 70%,
respectively. Incidence of tetracycline resistance was most prevalent at 47-89% of total bacteria. Out of 200
representative isolates analyzed, Pseudomonas, Enterococcus-like bacteria, Enterobacter and Burkholderia species
constituted the dominant reservoirs of resistance at high drug levels (50-170 micro g x ml(-1)). Plasmids were detected
in only 29% (58) of these bacteria with tetracycline resistance accounting for 65% of the plasmid pool. Overall,
resistance trends correlated to the abundance and type of bacterial species present in the habitat. Environmental
reservoirs of resistance include opportunistic pathogens and constitute some public health concern.”
http://www.ncbi.nlm.nih.gov/pubmed/12396530
Also in 2002, Nadine A. Séveno, et al., reported on the “Occurrence and reservoirs of antibiotic resistance genes in the
environment.” They said, “Antibiotic resistance genes have become highly mobile since the development of antibiotic
chemotherapy. A considerable body of evidence exists proving the link between antibiotic use and the significant
increase in drug-resistant human bacterial pathogens. The application of molecular detection and tracking techniques in
microbial ecological studies has allowed the reservoirs of antibiotic resistance genes to be investigated. It is clear that
the transfer of resistance genes has occurred on a global scale and in all natural environments. The considerable
diversity of bacteria and mobile elements in soils has meant that the spread of resistance genes has occurred by all
currently known mechanisms for bacterial gene transfer. Trans-kingdom transfers from plants to bacteria may occur in
soil. Hot spots for gene transfer in the soil/plant environment have been described and colonized niches such as the
rhizosphere and other nutrient-enriched sites, for example manured soil, have been identified as reservoirs of
resistance genes. Although exposure and selection for tolerance of antibiotics is considerable in clinical environments
there is increasing evidence that selection for resistant phenotypes is occurring in natural environments. Antibiotic-
producing bacteria are abundant in soil and there is evidence that they are actively producing antibiotics in nutrient-
enriched environments in soil. In addition there is clear evidence that the self-resistance genes found within antibiotic
gene clusters of the producers have transferred to other non-producing bacteria. Perhaps most important of all is the
use of antibiotics in agriculture as growth promotants and for treatment of disease in intensively reared farm animals.
These treatments have resulted in gut commensal and pathogenic bacteria acquiring resistance genes under selection
and then, due to the way in which farm slurries are disposed of, the spread of these genes to the soil bacterial
community. Integrons with multiple resistance gene cassettes have been selected and disseminated in this way; many of
these cassettes carry other genes such as those conferring heavy metal and disinfectant resistance which have been
co-selected in bacteria surviving in effluents and contaminated soils, further maintaining and spreading the antibiotic
resistance genes.” http://journals.lww.
com/revmedmicrobiol/Abstract/2002/01000/Occurrence_and_reservoirs_of_antibiotic_resistance.2.aspx
In 2003, Brion Duffy, et al., Swiss Federal Research Center for Fruit Production, Viticulture and Horticulture, reported on
“PATHOGEN SELF-DEFENSE: Mechanisms to Counteract Microbial Antagonism.” They said, “Natural and agricultural
ecosystems harbor a wide variety of microorganisms that play an integral role in plant health, crop productivity, and
preservation of multiple ecosystem functions. Interactions within and among microbial communities are numerous and
range from synergistic and mutualistic to antagonistic and parasitic. Antagonistic and parasitic interactions have been
exploited in the area of biological control of plant pathogenic microorganisms. To date, biocontrol is typically viewed from
the perspective of how antagonists affect pathogens. This review examines the other face of this interaction: how plant
pathogens respond to antagonists and how this can affect the efficacy of biocontrol. Just as microbial antagonists utilize
a diverse arsenal of mechanisms to dominate interactions with pathogens, pathogens have surprisingly diverse
responses to counteract antagonism. These responses include detoxification, repression of biosynthetic genes involved
in biocontrol, active efflux of antibiotics, and antibiotic resistance. Understanding pathogen self-defense mechanisms for
coping with antagonist assault provides a novel approach to improving the durability of biologically based disease
control strategies and has implications for the deployment of transgenes (microorganisms or plants).” http://www.pngg.
org/pp590_790/self%20defense.pdf
In 2003, Shahin Shafiani and Abdul Malik, reported on the “Tolerance of pesticides and antibiotic resistance in bacteria
isolated from wastewater-irrigated soil.” They said, “A total of 64 bacterial isolates (40 Pseudomonas spp., 12
Azotobacter and 12 Rhizobium spp.) were characterized on the basis of morphological, cultural and biochemical
characteristics. All the isolates were tested for their tolerance to the pesticides endosulfan, carbofuran, and malathion.
12.5% of the Pseudomonas isolates from soil tolerated concentrations of 1600 g malathion ml whereas 7.5% of isolates
tolerated the same concentration of carbofuran. However, Pseudomonas isolates demonstrated a tolerance limit to
endosulfan at a concentration of 800 g/ml. Asymbiotic N2-fixers (Azotobacter) and symbiotic N2-fixers (Rhizobium spp.)
were also able to tolerate concentrations of pesticides up to 1600 g/ml. All the isolates were further tested for their
antibiotic susceptibility against seven different antibiotics, nalidixic acid, cloxacillin, chloramphenicol, tetracycline,
amoxycillin, methicillin, doxycycline. 100% of the Pseudomonas isolates were resistant to cloxacillin and 57.5% were
resistant to methicillin. 7.5% of the isolates exhibited multiple resistance to five different antibiotics in three different
combinations whereas 25% of the isolates showed multiple resistance to four different antibiotics in seven different
combinations. Some of the resistant isolates were also screened for plasmid DNA and found to harbour a single
plasmid.” http://www.springerlink.com/content/qj65p227p041798p/
In 2003, Sören Thiele-Bruhn, University of Rostock, reported on "Pharmaceutical antibiotic compounds in soils -- a
review" Thiele-Bruhn said, "Antibiotics are highly effective, bioactive substances. As a result of their consumption,
excretion, and persistence, they are disseminated mostly via excrements and enter the soils and other environmental
compartments. Resulting residual concentrations in soils range from a few lg up to g kg±1 and correspond to those
found for pesticides. Numerous antibiotic molecules comprise of a non-polar core combined with polar functional
moieties. Many antibiotics are amphiphilic or amphoteric and ionize. However, physicochemical properties vary widely
among compounds from the various structural classes. Existing analytical methods for environmental samples often
combine an extraction with acidic buffered solvents and the use of LC-MS for determination. In soils, adsorption of
antibiotics to the organic and mineral exchange sites is mostly due to charge transfer and ion interactions and not to
hydrophobic partitioning. Sorption is strongly influenced by the pH of the medium and governs the mobility and transport
of the antibiotics. In particular for the strongly adsorbed antibiotics, fast leaching through soils by macropore or
preferential transport facilitated by dissolved soil colloids seems to be the major transport process. Antibiotics of
numerous classes are photodegraded. However, on soil surfaces this process if of minor influence. Compared to this,
biotransformation yields a more effective degradation and inactivation of antibiotics. However, some metabolites still
comprise of an antibiotic potency. Degradation of antibiotics is hampered by fixation to the soil matrix; persisting
antibiotics were already determined in soils. Effects on soil organisms are very diverse, although all antibiotics are highly
bioactive. The absence of effects might in parts be due to a lack of suitable test methods. However, dose and
persistence time related effects especially on soil microorganisms are often observed that might cause shifts of the
microbial community. Significant effects on soil fauna were only determined for anthelmintics. Due to the antibiotic
effect, resistance in soil microorganisms can be provoked by antibiotics. Additionally, the administration of antibiotics
mostly causes the formation of resistant microorganisms within the treated body. Hence, resistant microorganisms reach
directly the soils with contaminated excrements. When pathogens are resistant or acquire resistance from commensal
microorganisms via gene transfer, humans and animals are endangered to suffer from infections that cannot be treated
with pharmacotherapy. The uptake into plants even of mobile antibiotics is small. However, effects on plant growth were
determined for some species and antibiotics."
http://www.susane.info/en/ref/antibioticsinsoils.pdf
In 2003, Stuart B. Levy, Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine at
Boston, reported on "Antibiotic Resistance: Consequences of Inaction," Levy said, "In surveying the problem of
resistance, we should focus on two components of the drug resistance phenomenon: the antibiotic agent and the
resistance gene. Both are needed to produce a clinical resistance problem [2]. In 1998, some 50 million pounds of
antibiotics were produced in the United States, of which about half went to people in hospitals and homes. Of the
remainder, about 80% was given to animals for various indications. The rest was applied to other organisms—
honeybees, plants, trees, etc. In the early 1990s, I determined that 50,000 pounds of antibiotics were being used in
agriculture annually under the designation “pesticides” [3]. In the United States, pesticides include antibiotics, such as
tetracycline and streptomycin. The Environmental Protection Agency (EPA) recently reported that 300,000 pounds of
antibiotics were being sprayed onto fruit trees annually in the southern parts of the United States [4]. This practice is
also common in many parts of Central and South America. One can imagine the geographic spread of antibiotics by
means of this application, complicated further by the dilution of the drugs as rain and other natural movements disperse
them into the environment. The end result is excellent conditions for the selection of drug resistance."
http://cid.oxfordjournals.org/content/33/Supplement_3/S124.full
In 2004, Zulfiqar Ali Saqib, University of the Punjab, reported on “STUDIES ON INSECTICIDE DEGRADING BACTERIA.”
Ali Saqib said, “The soil samples were collected from agricultural fields with 5-10 years history of insecticidal spray from
different parts of Punjab, Pakistan. Fifty nine bacterial strains resistant to organochlorine insecticides, i.e. endosulfan
and heptachlor (cyclodiene group) were isolated. They were also capable of utilizing organophosphates (Anthio, Ethion,
Quinalphos), pyrethroids (Bifenthrin, Cypermethrin) and a carbamate (Carbosulfan) as a carbon source in an inorganic
M-9 agar medium. Thirty five of these bacterial strains were isolated from Lahore region and twenty four from Multan
region. The minimum inhibitory concentration (MIC) of endosulfan and heptachlor ranged from 2 to 10 mg/ml, while that
of organophosphates (OPs) from 1 to 6 mg/ml, of pyrethroids from 2 to 12 mg/ml and of carbosulfan from 1 to 4 mg/ml.
On the basis of high MICs twenty four bacterial strains were finally selected for further analysis. The optimum pH and
temperature of the selected bacterial isolates was 6.5-7.5 and 30-42°C, respectively. The growth patterns of the isolates
were studied in different media viz., LB, M9+glucose (50 mg/100 ml), M9+endosulfan (50 mg/100 ml) and M9+heptachlor
(50 mg/100ml). In LB medium typical growth patterns were observed as compared with those in M9, in which prolonged
lag or stationary phases were observed. Eight isolates, based upon the various biochemical tests, were identified as
members of the genus Bacillus, two each of the genus Kurthia and Caryophanon and one each of Sporolactobacillus,
Derxia, Streptococcus, Aureobacterium, Alcaligenes, Terrabacter, Rizhobacter, Azomonas and Pimelobacter. They were
also tested for sensitivity to eight heavy metals. The MICs of Cd2+ varied from 300-500 μg/ml, of Co2+ 1 00-200 μg/ml,
of Cr6+ 300-500 μg/ml, of Cu2+ 350-450 μg/ml, of Hg2+ 1 00-200 μg/ml,. of Ni2+ 300-550 μg/ml, of Pb2+ 1500-2500
μg/ml and of Zn2+ 300-500 μg/ml.. The antibiotic sensitivity of isolates was also checked against nine antibiotics. The
isolates were resistant to ampicillin, amoxycillin, cefixime and cefotoxim but were sensitive to minocyclin. Most of the
bacteria were sensitive to cefazoline except for CMBLI 62, 75, 76 and 87, to chloramphenicol except for CMBLI 43, 44,
46, 53, 55 and 65 and tetracycline except for CMBLI 38, 53 and 55. All isolates were found to harbour plasmids of 23 Kb
except for CMBLI 62, which had five plasmids of 23, 13, 6.5, 4.2 and 2.9 Kb size. All the isolates could be cured of their
plasmids, which made them sensitive to insecticides mixed in the growth media. The competent cells of E. coli C600 were
successfully transformed with the plasmids of isolates. The transformed E. coli could grow on M9 selective medium
containing 50 mg/100 ml of endosulfan/heptachlor. The 23 Kb plasmid was later successfully recovered from the
transformants. It was concluded that the gene for insecticide degradation was located on the plasmids. The total
proteins of some selected isolates grown in LB, M9+glucose, M9+endosulfan and M9+heptachlor (50 mg/100 ml) were
extracted in the sample buffer and were analyzed by 12% 8DS-PAGE. More protein bands were seen in isolates grown
in LB media as compared with those in M9 media. Despite the suppression of certain protein bands in isolates grown in
insecticide-containing media, protein bands of 64, 62, 59,50 and 37 Kd size were present in M9 as well as in control
media. The presence of protein bands in insecticide stress media implicate their role in detoxification of insecticides. The
isolated bacterial strains can be employed in the microbe based bioremediation of insecticide-contaminated soil and
waste water” http://eprints.hec.gov.pk/1767/1/1700.htm
In 2004, Christian S. Riesenfel, et al., University of Wisconsin at Madison, reported on “Uncultured soil bacteria are a
reservoir of new antibiotic resistance gene.” They said, “Antibiotic resistance genes are typically isolated by cloning from
cultured bacteria or by polymerase chain reaction (PCR) amplification from environmental samples. These methods do
not access the potential reservoir of undiscovered antibiotic resistance genes harboured by soil bacteria because most
soil bacteria are not cultured readily, and PCR detection of antibiotic resistance genes depends on primers that are
based on known genes. To explore this reservoir, we isolated DNA directly from soil samples, cloned the DNA and
selected for clones that expressed antibiotic resistance in Escherichia coli. We constructed four libraries that collectively
contain 4.1 gigabases of cloned soil DNA. From these and two previously reported libraries, we identified nine clones
expressing resistance to aminoglycoside antibiotics and one expressing tetracycline resistance. Based on the predicted
amino acid sequences of the resistance genes, the resistance mechanisms include efflux of tetracycline and inactivation
of aminoglycoside antibiotics by phosphorylation and acetylation. With one exception, all the sequences are
considerably different from previously reported sequences. The results indicate that soil bacteria are a reservoir of
antibiotic resistance genes with greater genetic diversity than previously accounted for, and that the diversity can be
surveyed by a culture-independent method”
http://execdeanagriculture.rutgers.edu/pdfs/goodman-092.pdf
In 2008, Gautam Dantas, et al., Harvard Medical School at Boston, reported on “Bacteria Subsisting on Antibiotics.”
They said, “Antibiotics are a crucial line of defense against bacterial infections. Nevertheless, several antibiotics are
natural products of microorganisms that have as yet poorly appreciated ecological roles in the wider environment. We
isolated hundreds of soil bacteria with the capacity to grow on antibiotics as a sole carbon source. Of 18 antibiotics
tested, representing eight major classes of natural and synthetic origin, 13 to 17 supported the growth of clonal bacteria
from each of 11 diverse soils. Bacteria subsisting on antibiotics are surprisingly phylogenetically diverse, and many are
closely related to human pathogens. Furthermore, each antibiotic-consuming isolate was resistant to multiple antibiotics
at clinically relevant concentrations. This phenomenon suggests that this unappreciated reservoir of antibiotic-
resistance determinants can contribute to the increasing levels of multiple antibiotic resistance in pathogenic bacteria.”
http://arep.med.harvard.edu/pdf/Dantas08.pdf
In 2009, Charles W. Knapp, et al., Newcastle University, and Alterra, Wageningen University, reported on the “Evidence
of Increasing Antibiotic Resistance Gene Abundances in Archived Soils since 1940.” They said, “Mass production and
use of antibiotics and antimicrobials in medicine and agriculture have existed for over 60 years, and has substantially
benefited public health and agricultural productivity throughout the world. However, there is growing evidence that
resistance to antibiotics (AR) is increasing both in benign and pathogenic bacteria, posing an emerging threat to public
and environmental health in the future. Although evidence has existed for years from clinical data of increasing AR,
almost no quantitative environmental data exist that span increased industrial antibiotic production in the 1950s to the
present; i.e., data that might delineate trends in AR potentially valuable for epidemiological studies. To address this
critical knowledge gap, we speculated that AR levels might be apparent in historic soil archives as evidenced by
antibiotic resistance gene (ARG) abundances over time. Accordingly, DNA was extracted from five long-term soil-series
from different locations in The Netherlands that spanned 1940 to 2008, and 16S rRNA gene and 18 ARG abundances
from different major antibiotic classes were quantified. Results show that ARG from all classes of antibiotics tested have
significantly increased since 1940, but especially within the tetracyclines, with some individual ARG being >15 times
more abundant now than in the 1970s. This is noteworthy because waste management procedures have broadly
improved and stricter rules on nontherapeutic antibiotic use in agriculture are being promulgated. Although these data
are local to The Netherlands, they suggest basal environmental levels of ARG still might be increasing, which has
implications to similar locations around the world.” http://pubs.acs.org/doi/abs/10.1021/es901221x
In 2010, S. Umamaheswari and M. Murali, Periyar EVR College at Tiruchirappalli, reported on the “Prevalence of
plasmid mediated pesticide resistant bacterial assemblages in crop fields.” They said, “Three crop fields namely paddy
sugarcane and tomato exposed to bavistin [Methyl (1H-benzimidazol-2-yl) carbomate], monocrotophos[Dimethyl(E)-1-
methyl-2-(methyl-carbamoyl) vinyl phosphate] and kinado plus [(EZ)-2-chloro-3-dimethoxyphosphinoyloxy-X1, X1-
diethylbut-2-enamide], respectively were chosen for the present investigation to know the bacterial population and
degradation of pesticides. The chemical nature of the soil and water samples from the pesticide contaminated fields was
analysed along with counting of the total heterotrophic bacteria (THB), Staphylococci and Enterococcci population.
Mean calcium, phosphate and biological oxygen demand were maximum in tomato field water Field water recorded
maximum phophate and silicate content, whereas, sugarcane field water elicited maximum dissolved oxygen content. On
the other hand, available phosphate and exchangeable potassium were maximum is sugarcane field soil. Significant
variations in the bacterial population were evident between the treatments in sugarcane field soil and tomato field water
exposed to monocrotophos and kinado plus, respectively In addition, significant variations between THB, Staphlyococci
and Enterococci population were also evinced in both the sugarcane and tomato fields. The dominant pesticide resistant
bacteria, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeuroginosa harboured plasmids and the
resistant trait observed were found to be plasmid borne.”
http://www.jeb.co.in/journal_issues/201011_nov10/paper_11.pdf
In 2011, R. Anjum, et al., Aligarh Muslim University, reported on the “Molecular characterization of conjugative plasmids
in pesticide tolerant and multi-resistant bacterial isolates from contaminated alluvial soil.” They said, “A total of 35
bacteria from contaminated soil (cultivated fields) near pesticide industry from Chinhat, Lucknow, (India) were isolated
and tested for their tolerance/resistance to pesticides, heavy metals and antibiotics. Bacterial isolates were identified by
16S rDNA sequencing. Gas Chromatography analysis of the soil samples revealed the presence of lindane at a
concentration of 547 ng g(-1) and α-endosulfan and β-endosulfan of 422 ng g(-1) and 421 ng g(-1) respectively. Atomic
Absorption Spectrophotometry analysis of the test sample was done and Cr, Zn, Ni, Fe, Cu and Cd were detected at
concentrations of 36.2, 42.5, 43.2, 241, 13.3 and 11.20 mg kg(-1) respectively. Minimum inhibitory concentrations of all
the isolates were determined for pesticides and heavy metals. All the multi-resistant/tolerant bacterial isolates were also
tested for the presence of incompatibility (Inc) group IncP, IncN, IncW, IncQ plasmids and for rolling circle plasmids of the
pMV158-family by PCR. Total community DNA was extracted from pesticide contaminated soil. PCR amplification of the
bacterial isolates and soil DNA revealed the presence of IncP-specific sequences (trfA2 and oriT) which was confirmed
by dot blot hybridization with RP4-derived DIG-labelled probes. Plasmids belonging to IncN, IncW and IncQ group were
neither detected in the bacterial isolates nor in total soil DNA. The presence of conjugative or mobilizable IncP plasmids
in the isolates indicate that these bacteria have gene transfer capacity with implications for dissemination of heavy metal
and antibiotic resistance genes. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant
bacteria in the contaminated soils.” http://www.ncbi.nlm.nih.gov/pubmed/21376364
Research indicates there are multiple paths for the spread of multi-resistant bacteria. The first documented path for
plasmid transformation of bacteria was in the laboratory. Later researchers discovered they could recombine bacterial
DNA to create chimera bacteria that could not exist in nature. Antibiotic resistant genes were inserted to confirm chimera
bacteria had been created. Unfortunately, until relatively recently, these antibiotic resistant chimera bacteria were
dumped into the sewers and passed through treatment plants in reclaimed water and treated sludge. Since sewage is
“treated” with either aerobic or anaerobic bacteria treatment plants became swap meets for the transfer of antibiotic
resistant genes.
Next Laboratories/Antibiotic Resistant Bacteria
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