A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Section 3
Viable, But Nonculturable to Antibiotic Resistant Synthetic Biology. 9/09/2011
Back to Myth # 4
One problem that may not stand out in the following studies that has been touched on in the sections, coliform myth
and compost myth, is that stressed bacteria not only survive under nutrient, moisture, and temperature deficient
conditions, but they are much more inclined to simply be in a stationary or dormant phase and antibiotic resistant.
Under these conditions, they generally can not be cultured by standard laboratory methods. Another point to be made
is that laboratory research is generally done with weak laboratory stock bacterial clones under nutrient rich conditions
which do not reflect real world conditions.
D. D. Woods, University of Oxford, was one of the first scientists to discuss viable but nonculturable bacteria in “The
Inaugural Marjory Stephenson Memorial Lecture” of 1953. It was accepted that bacteria exposed to irradiation,
treatment with certain chemicals, etc., lost the ability to synthesize particular essential metabolites. Not only that, but it
was a scientific fact that these traits could be induced in bacterial mutants. http://mic.sgmjournals.org/content/9/2/151.
full.pdf
Many of these bacterial mutants have been collected and continuously cloned by certain laboratories to make them
available for scientific studies by research experts. As an example, E. coli K-12 has been continuously cloned since it
was isolated at Stanford in 1922 and referred to as the wild type. It has the standard laboratory strain, but as a result of
continuous cloning, E. coli K-12 has become a weak shell of its former self, lost its virulence as well as the ability to
colonize the intestinal tract and survives poorly in the environment. Yet, it is the prototype workhorse on which all
standards are based. The clonal mutants cover the field from antibiotic resistant to radiation resistant to drug and
ethanol production. However, E. coli K-12 is not the only cultured clone that survives poorly in the envirnment.
In the following 1978 study, it is shown that cloned laboratory (American Type Culture Collection – ATCC) strains of
bacteria have much low resistance than environmental strains. L. A. Carson, et al., reported on the “Growth
characteristics of atypical mycobacteria in water and their comparative resistance to disinfectants.” They said, “With the
increasing significance of group IV atypical mycobacteria as etiological [disease causing] agents in a variety of
infections, studies were conducted to determine their growth capabilities in water and their comparative resistance to
disinfectants used to decontaminate hospital equipment. Isolates of Mycobaterium chelonei (TM strains) from peritoneal
fluids of patients and peritoneal dialysis machines were able to multiply in commercial distilled water, with generation
times at 25 degrees C ranging from 8 to 15 h. Levels of 10(5) to 10(6) cells per ml were attained, and these stationary-
phase populations declined only slightly over a 1-year period. Results of studies to determine resistance to
disinfectants showed the following. (i) TM strains of M. chelonei cultured in commercial distilled water showed survivors
in 2% aqueous formaldehyde (HCHO) solutions up to 24 h; in 8% HCHO, only a 2-log reduction in viable counts was
observed over a 2-h sampling period. Reference ATCC strains of M. chelonei and M. fortuitum were rapidly inactivated,
with no survivors after 2 h of exposure to 2% HCHO or 15 min of exposure to 8% HCHO. (ii) In 2% alkaline
glutaraldehyde, TM strains survived 60 min. whereas ATCC strains showed no survivors after 2 min of contact time. (iii)
All M. chelonei and M. fortuitum strains survived 60 min of exposure to concentrations of 0.3 and 0.7 microgram of free
chlorine per ml at pH 7” http://aem.asm.org/cgi/content/abstract/36/6/839
Once researchers were able to insert individual bits of genetic material, including antibiotic genes, into bacteria, it was
just a short step to the creation of standardized biological parts and circuits in the new field of synthetic biology. In the
2006 article, “The Promise and Perils of Synthetic Biology,” Jonathan B. Tucker and Raymond A. Zilinskas explained
the concept. They said, “Perhaps the most ambitious subfield of synthetic biology involves efforts to develop a “tool
box” of standardized genetic parts with known performance characteristics—analogous to the transistors, capacitors,
and resistors used in electronic circuits—from which bioengineers can build functional devices and, someday, synthetic
microorganisms. The Synthetic Biology Working Group at M.I.T. is attempting to turn this concept into a reality by
developing a comprehensive set of genetic building blocks, along with standards for characterizing their behavior and
the conditions that support their use. In the summer of 2004, the group established a Registry of Standard Biological
Parts. The registry is made up of components called “BioBricks,” short pieces of DNA that constitute or encode
functional genetic elements. Examples of BioBricks are a “promoter” sequence that initiates the transcription of DNA
into messenger RNA, a “terminator” sequence that halts RNA transcription, a “repressor” gene that encodes a protein
that blocks the transcription of another gene, a ribosome-binding site that initiates protein synthesis, and a “reporter”
gene that encodes a fluorescent jellyfish protein, causing cells to glow green when viewed through a fluorescence
microscope. A BioBrick must have a genetic structure that enables it to send and receive standard biochemical signals
and to be cut and pasted into a linear sequence of other BioBricks, in a manner analogous to the pieces in a Lego set.
As of early April 2006, the BioBricks registry contained 167 basic parts, including sensors, actuators, input and output
devices, and regulatory elements. Also included in the registry were 421 composite parts, and an additional 50 parts
were being synthesized or assembled. Emulating the approach employed by open-source software developers, the M.I.
T. group has placed the registry on a public website (http://parts.mit.edu/) and invited all interested researchers to
comment on and contribute to it. The ultimate goal of this effort is to develop a methodology for the assembly of
BioBricks into circuits with practical applications, while eliminating unintended or parasitic interactions that could
compromise the characterized function of the parts. To date, BioBricks have been assembled into a few simple genetic
circuits. One such circuit renders a film of bacteria sensitive to light, so that it can capture an image like a photographic
negative. In other experiments, BioBricks have been combined into devices that function as logic gates and perform
simple Boolean operations, such as AND, OR, NOT, NAND, and NOR. For example, an AND operator generates an
output signal when it gets a biochemical signal from both of its inputs; an OR operator generates a signal if it gets a
signal from either input; and a NOT operator (or inverter) converts a weak signal into a strong one, and vice versa. The
long-term goal of this work is to convert bioengineered cells into tiny programmable computers, so that it will be possible
to direct their operation by means of chemical signals or light.”
http://www.thenewatlantis.com/publications/the-promise-and-perils-of-synthetic-biology
In 2011, Nazanin Saeidi, et al., Nanyang Technological University at Singapore, reported on their work of “Engineering
microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen.”
They used engineering principles known as synthetic biology to create a new form of bacteria with off the shelf DNA
base pairs. They said, “Synthetic biology aims to engineer genetically modified biological systems that perform novel
functions that do not exist in nature, with reusable, standard interchangeable biological parts. The use of these
standard biological parts enables the exploitation of common engineering principles such as standardization,
decoupling, and abstraction for synthetic biology (Endy, 2005). With this engineering framework in place, synthetic
biology has the potential to make the construction of novel biological systems a predictable, reliable, systematic
process. While the development of most synthetic biological systems remains largely ad hoc, recent efforts to
implement an engineering framework in synthetic biology have provided long-awaited evidences that engineering
principles can facilitate the construction of novel biological systems. Synthetic biology has so far demonstrated that its
framework can be applied to a wide range of areas such as energy, environment, and health care. For example, novel
biological systems have been constructed to produce drugs (Ro et al, 2006) and biofuels (Steen et al, 2010), to
degrade containments in water (Sinha et al, 2010), and to kill cancer cells (Anderson et al, 2006). – We demonstrated
that our engineered E. coli sensed and killed planktonic P. aeruginosa, evidenced by 99% reduction in the viable cells.
Moreover, we showed that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%,
leading to much sparser and thinner biofilm matrices. These results suggest that E. coli carrying our synthetic genetic
system may provide a novel synthetic biology-driven antimicrobial strategy that could potentially be applied to fighting
P. aeruginosa and other infectious pathogens.”
http://www.nature.com/msb/journal/v7/n1/full/msb201155.html
The problem left to deal with is that one percent of the P. aeruginosa were not killed with new chimera bacteria. Were
they resistant to the chimera protein? Researchers have already created synthetic genetic sequences never before
seen in nature. The artificial proteins created from amino acids were able to sustain life in bacteria by some unknown
method.
In 2011, Hee-Sung Park, et al., Yale University, reported on “Expanding the Genetic Code of Escherichia coli with
Phosphoserine.” They said, “O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic
phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an
engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a
protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNA
synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu
to relax its quality-control function and permit Sep-tRNASep binding. To test our system, we synthesized the activated
form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally
inserted in their canonical positions (Sep218, Sep222). This system has general utility in protein engineering, molecular
biology, and disease research.”
http://www.sciencemag.org/content/333/6046/1151.abstract
Researchers are once again threading on uncharted territory without looking at the past mistakes which caused the
spread of antibiotic resistant pathogens in the community environment. It all starts and ends with the life sustaining soil.
Many soil bacteria have become human pathogens other that the notorious spore forming Bacillus anthracis and
Clostridium. One of the most interesting pathogens is the antibiotic resistant bacteria Nocardia species which requires a
treatment course of 6-12 months. It is often responsible for the accumulation of foam that occurs in activate sludge
during wastewater treatment. The major problem is that no one really knows what type of organisms are in sludge as
many organisms in sludge and soil are unculturable by standard laboratory methods and some may take days or weeks
to multiply.
Next Antibiotic Resistance in the Soil.