U.S. patent application number 14/141571 was filed with the patent office on 2014-06-26 for toxin-eating bacteria and bioremediation.
The applicant listed for this patent is PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to George M. CHURCH, Gautam DANTAS, Morten O. SOMMER.
Application Number | 20140178971 14/141571 |
Document ID | / |
Family ID | 42266688 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140178971 |
Kind Code |
A1 |
CHURCH; George M. ; et
al. |
June 26, 2014 |
Toxin-Eating Bacteria and Bioremediation
Abstract
Bacteria that can use antibiotics as a carbon source are
provided. Methods and bacteria useful for bioremediation are also
provided.
Inventors: |
CHURCH; George M.;
(Brookline, MA) ; DANTAS; Gautam; (University
City, MO) ; SOMMER; Morten O.; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PRESIDENT AND FELLOWS OF HARVARD COLLEGE |
CAMBRIDGE |
MA |
US |
|
|
Family ID: |
42266688 |
Appl. No.: |
14/141571 |
Filed: |
December 27, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12579696 |
Oct 15, 2009 |
8658416 |
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14141571 |
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61105614 |
Oct 15, 2008 |
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Current U.S.
Class: |
435/252.1 |
Current CPC
Class: |
B08B 7/00 20130101; C12R
1/01 20130101 |
Class at
Publication: |
435/252.1 |
International
Class: |
C12R 1/01 20060101
C12R001/01 |
Goverment Interests
STATEMENT OF GOVERNMENT INTERESTS
[0002] This invention was made with government support under
DE-FG02-03ER63445 (T-103693) awarded by the U.S. Department of
Energy. The government has certain rights in the invention.
Claims
1. A clonal isolate of a bacterium that can use one or more
antibiotics as a carbon source, wherein the bacterium has a 16S
nucleic acid sequence comprising a GenBank Accession Number
selected from the group consisting of EU515334, EU515335, EU515336,
EU515337, EU515338, EU515339, EU515400, EU515401, EU515402,
EU515403, EU515404, EU515405, EU515406, EU515407, EU515408,
EU515409, EU515410, EU515411, EU515412, EU515413, EU515414,
EU515415, EU515416, EU515417, EU515418, EU515419, EU515420,
EU515421, EU515422, EU515423, EU515424, EU515425, EU515426,
EU515427, EU515428, EU515429, EU515430, EU515431, EU515432,
EU515433, EU515434, EU515435, EU515436, EU515437, EU515438,
EU515439, EU515440, EU515441, EU515442, EU515443, EU515444,
EU515445, EU515446, EU515447, EU515448, EU515449, EU515450,
EU515451, EU515452, EU515453, EU515454, EU515455, EU515456,
EU515457, EU515458, EU515459, EU515460, EU515461, EU515462,
EU515463, EU515464, EU515465, EU515466, EU515467, EU515468,
EU515469 EU515470, EU515471, EU515472, EU515473, EU515474,
EU515475, EU515476, EU515477, EU515478, EU515479, EU515480,
EU515481, EU515482, EU515483, EU515484, EU515485, EU515486,
EU515487, EU515488, EU515489, EU515490, EU515491, EU515492,
EU515493, EU515494, EU515495, EU515496, EU515497, EU515498,
EU515499, EU515500, EU515501, EU515502, EU515503, EU515504,
EU515505, EU515506, EU515507, EU515508, EU515509, EU515510,
EU515511, EU515512, EU515513, EU515514, EU515515, EU515516,
EU515517, EU515518, EU515519, EU515520, EU515521, EU515522,
EU515523, EU515524, EU515525, EU515526, EU515527, EU515528,
EU515529, EU515530, EU515531, EU515532, EU515533, EU515534,
EU515535, EU515536, EU515537, EU515538, EU515539, EU515540,
EU515541, EU515542, EU515543, EU515544, EU515545, EU515546,
EU515547, EU515548, EU515549, EU515550, EU515551, EU515552,
EU515553, EU515554, EU515555, EU515556, EU515557, EU515558,
EU515559, EU515560, EU515561, EU515562, EU515563, EU515564,
EU515565, EU515566, EU515567, EU515568, EU515569, EU515570,
EU515571, EU515572, EU515573, EU515574, EU515575, EU515576,
EU515577, EU515578, EU515579, EU515580, EU515581, EU515582,
EU515583, EU515584, EU515585, EU515586, EU515587, EU515588,
EU515589, EU515590, EU515591, EU515592, EU515593, EU515594,
EU515595, EU515596, EU515597, EU515598, EU515599, EU515600,
EU515601, EU515602, EU515603, EU515604, EU515605, EU515606,
EU515607, EU515608, EU515609, EU515610, EU515611, EU515612,
EU515613, EU515614, EU515615, EU515616, EU515617, EU515618,
EU515619, EU515620, EU515621, EU515622 and EU515623.
2. The bacterium of claim 1, wherein one or more antibiotics are
from the antibiotic class selected from the group consisting of
pyrimidine derivative, sulfonamide, quinolone, glycopeptides,
beta-lactam, amphenicols, aminoglycoside and amino acid
derivative.
3. The bacterium of claim 1, wherein one or more antibiotics are
selected from the group consisting of chloramphenicol, penicillin
G, vancomycin, carbenicillin, ciprofloxacin, mafenide, kanamycin,
sisomicin, amikacin, trimethropin, D-cycloserine, gentamicin,
dicloxacillin, nalidixic acid, thiamphenicol, levofloxacin,
sulfamethizole and sulfisoxazole.
4. The bacterium of claim 1, wherein the bacterium uses the one or
more antibiotics as a sole carbon source.
Description
PRIORITY INFORMATION
[0001] This application is a divisional of U.S. application Ser.
No. 12/579,696 filed Oct. 15, 2009, which claims priority to U.S.
Provisional Patent Application No. 61/105,614, filed on Oct. 15,
2008, all of which are hereby incorporated herein by reference in
their entireties.
BACKGROUND
[0003] The seemingly unchecked spread of multiple antibiotic
resistance in clinically relevant pathogenic microbes is alarming.
Furthermore, a significant environmental reservoir of antibiotic
resistance determinants, termed the antibiotic resistome, has been
discovered (Riesenfeld et al. (2004) Environmental Microbiology
6:981; D'Costa et al. (2006) Science 311:374). The primary
microbial antibiotic resistance mechanisms include efflux pumps,
target gene-product modifications, and enzymatic inactivation of
the antibiotic compound (Walsh (2000) Nature 406:775; Alekshun and
Levy (2007) Cell 128:1037). Many of the mechanisms are common to
several species of pathogens and spread by lateral gene transfer
(Davies (1994) Science 264:375). While many bacteria growing in
extreme environments (Davies (1994) Science 264:375) and capable of
degrading toxic substrates (McAllister et al. (1996) Biodegradation
7:1) have been previously reported, only a few organisms have been
shown to subsist on a limited number of antibiotic substrates
(Kameda et al. (1961) Nature 191:1122; Johnsen (1977) Archives of
Microbiology 115:271; Abdelm et al. (1961) Nature 189:775).
SUMMARY
[0004] The present invention is based in part on the surprising
discovery that the microbiome (e.g., of soil and/or water) includes
a significant reservoir of bacteria capable of subsisting on
antibiotics. Clonal bacterial isolates were obtained from eleven
diverse soils which were capable of utilizing one of 18 different
antibiotics as the sole carbon source. The 18 antibiotics comprised
of natural, semi-synthetic and synthetic compounds and included all
major bacterial target classes.
[0005] Accordingly, in certain exemplary embodiments, a method of
reducing a level of one or more antibiotics from an
antibiotic-contaminated substance comprising culturing an organism
that can utilize the one or more antibiotics as a carbon source
(e.g., a sole carbon source) in the presence of the
antibiotic-contaminated substance for a sufficient amount of time
to reduce the level of one or more antibiotics from the
antibiotic-contaminated substance is provided. In certain aspects,
the organism is a bacterium. In other aspects, one or more
antibiotics are from the antibiotic class including one or more of
pyrimidine derivative, sulfonamide, quinolone, glycopeptides,
beta-lactam, amphenicols, aminoglycoside and amino acid derivative.
In yet other aspects, one or more antibiotics are selected from the
group including one or more of chloramphenicol, penicillin G,
vancomycin, carbenicillin, ciprofloxacin, mafenide, kanamycin,
sisomicin, amikacin, trimethropin, D-cycloserine, gentamicin,
dicloxacillin, nalidixic acid, thiamphenicol, levofloxacin,
sulfamethizole and sulfisoxazole. In still other aspects, the
antibiotic-contaminated substance is one or more of contaminated
soil, contaminated water and a contaminated work surface (e.g., in
a hospital, a clinic, a laboratory or the like).
[0006] In certain exemplary embodiments, a bacterium that can use
one or more antibiotics as a carbon source (e.g., a sole carbon
source) is provided. The bacterium has a 16S nucleic acid sequence
comprising a GenBank Accession Number selected from one or more of
EU515334, EU515335, EU515336, EU515337, EU515338, EU515339,
EU515400, EU515401, EU515402, EU515403, EU515404, EU515405,
EU515406, EU515407, EU515408, EU515409, EU515410, EU515411,
EU515412, EU515413, EU515414, EU515415, EU515416, EU515417,
EU515418, EU515419, EU515420, EU515421, EU515422, EU515423,
EU515424, EU515425, EU515426, EU515427, EU515428, EU515429,
EU515430, EU515431, EU515432, EU515433, EU515434, EU515435,
EU515436, EU515437, EU515438, EU515439, EU515440, EU515441,
EU515442, EU515443, EU515444, EU515445, EU515446, EU515447,
EU515448, EU515449, EU515450, EU515451, EU515452, EU515453,
EU515454, EU515455, EU515456, EU515457, EU515458, EU515459,
EU515460, EU515461, EU515462, EU515463, EU515464, EU515465,
EU515466, EU515467, EU515468, EU515469 EU515470, EU515471,
EU515472, EU515473, EU515474, EU515475, EU515476, EU515477,
EU515478, EU515479, EU515480, EU515481, EU515482, EU515483,
EU515484, EU515485, EU515486, EU515487, EU515488, EU515489,
EU515490, EU515491, EU515492, EU515493, EU515494, EU515495,
EU515496, EU515497, EU515498, EU515499, EU515500, EU515501,
EU515502, EU515503, EU515504, EU515505, EU515506, EU515507,
EU515508, EU515509, EU515510, EU515511, EU515512, EU515513,
EU515514, EU515515, EU515516, EU515517, EU515518, EU515519,
EU515520, EU515521, EU515522, EU515523, EU515524, EU515525,
EU515526, EU515527, EU515528, EU515529, EU515530, EU515531,
EU515532, EU515533, EU515534, EU515535, EU515536, EU515537,
EU515538, EU515539, EU515540, EU515541, EU515542, EU515543,
EU515544, EU515545, EU515546, EU515547, EU515548, EU515549,
EU515550, EU515551, EU515552, EU515553, EU515554, EU515555,
EU515556, EU515557, EU515558, EU515559, EU515560, EU515561,
EU515562, EU515563, EU515564, EU515565, EU515566, EU515567,
EU515568, EU515569, EU515570, EU515571, EU515572, EU515573,
EU515574, EU515575, EU515576, EU515577, EU515578, EU515579,
EU515580, EU515581, EU515582, EU515583, EU515584, EU515585,
EU515586, EU515587, EU515588, EU515589, EU515590, EU515591,
EU515592, EU515593, EU515594, EU515595, EU515596, EU515597,
EU515598, EU515599, EU515600, EU515601, EU515602, EU515603,
EU515604, EU515605, EU515606, EU515607, EU515608, EU515609,
EU515610, EU515611, EU515612, EU515613, EU515614, EU515615,
EU515616, EU515617, EU515618, EU515619, EU515620, EU515621,
EU515622 and EU515623. In certain aspects, one or more antibiotics
are from the antibiotic class including one or more of pyrimidine
derivative, sulfonamide, quinolone, glycopeptides, beta-lactam,
amphenicols, aminoglycoside and amino acid derivative. In other
aspects, one or more antibiotics include one or more of
chloramphenicol, penicillin G, vancomycin, carbenicillin,
ciprofloxacin, mafenide, kanamycin, sisomicin, amikacin,
trimethropin, D-cycloserine, gentamicin, dicloxacillin, nalidixic
acid, thiamphenicol, levofloxacin, sulfamethizole and
sulfisoxazole.
[0007] In certain exemplary embodiments, a method of removing one
or more antibiotics from an antibiotic-contaminated substance,
comprising culturing a bacterium described above in the presence of
the antibiotic-contaminated substance for a sufficient amount of
time to reduce the level of one or more antibiotics from the
antibiotic-contaminated substance is provided. In certain aspects,
one or more antibiotics are from the antibiotic class including one
or more of pyrimidine derivative, sulfonamide, quinolone,
glycopeptides, beta-lactam, amphenicols, aminoglycoside and amino
acid derivative. In other aspects, one or more antibiotics are
selected from the group including one or more of chloramphenicol,
penicillin G, vancomycin, carbenicillin, ciprofloxacin, mafenide,
kanamycin, sisomicin, amikacin, trimethropin, D-cycloserine,
gentamicin, dicloxacillin, nalidixic acid, thiamphenicol,
levofloxacin, sulfamethizole and sulfisoxazole. In yet other
aspects, the antibiotic-contaminated substance is one or more of
contaminated soil, contaminated water and a contaminated work
surface (e.g., in a hospital, a clinic, a laboratory or the
like.
[0008] Further features and advantages of certain embodiments of
the present invention will become more fully apparent in the
following description of the embodiments and drawings thereof, and
from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee. The foregoing and
other features and advantages of the present invention will be more
fully understood from the following detailed description of
illustrative embodiments taken in conjunction with the accompanying
drawings in which:
[0010] FIGS. 1A-1B graphically depict clonal bacterial isolates
subsisting on antibiotics. (A) Heat-map illustrating growth results
from all combinations of 11 soils by 18 antibiotics, where blue
squares represent successful isolation of bacteria from a given
soil that are able to utilize that antibiotic as sole carbon source
at 1 g/L. Soil samples labeled F1-3 were from farm soils and U1-3
were from urban soils. Soil samples P1-5 were from pristine soils,
collected from non-urban areas with minimal human exposure over the
last 100 years (Table 2). (B) High performance liquid
chromatography (HPLC) traces at 214 nm of representative penicillin
and carbenicillin catabolizing clonal isolates and corresponding
un-inoculated media controls for different time points over 20 or
28 days of growth, respectively.
[0011] FIG. 2 graphically depicts the phylogenetic distribution of
bacterial isolates subsisting on antibiotics. 16S ribosomal DNA
(rDNA) was sequenced from antibiotic catabolizing clonal isolates
using universal bacterial rDNA primers. High-quality, non-chimeric
sequences were classified using Greengenes (DeSantis et al. (2006)
Applied and Environmental Microbiology 72:5069), with consensus
annotations from RDP (Cole et al. (2007) Nucl. Acids Res. 35:D169)
and NCBI taxonomies (D. L. Wheeler et al. (2000) Nucl. Acids Res.
28:10). Phylogenetic trees were constructed using the neighbor
joining algorithm in ARB (W. Ludwig et al. (2004) Nucl. Acids Res.
32:1363) using the Greengenes aligned 16S rDNA database. Placement
in the tree was confirmed by comparing automated Greengenes
taxonomy to the annotated taxonomies of nearest neighbors of each
sequence in the aligned database. Branches of the tree are
color-coded by bacterial orders, and clonal isolates represented as
squares. Accession numbers of certain of these bacterial isolates
that have been deposited are from EU515334 to EU515623 (GenBank),
and are hereby incorporated by reference in their entirety.
[0012] FIGS. 3A-3C graphically depict antibiotic resistance
profiling of 75 clonal isolates capable of subsisting on
antibiotics. (A) Heat map illustrating the resistance profiles of a
representative subset of 75 clonal isolates capable of utilizing
antibiotics as sole carbon source (Table 3). Resistance was
determined as growth after 4 days at 22.degree. C. in Luria Broth
media containing 20 mg/liter antibiotic (top panel) and 1 g/liter
antibiotic (bottom panel). (B) Percentage of clonal isolates
resistant to each of the 18 antibiotics. Antibiotics are color
coded by class, the full height of each bar corresponds to the
percentage of clonal isolates resistant at 20 mg/liter and the
solid colored section of each bar corresponds to the percentage of
clonal isolates resistant at 1 g/liter. (C) Histogram depicting the
distribution of the number of antibiotics that the clonal isolates
were resistant to at 20 mg/liter (top panel) and 1 g/liter (bottom
panel).
[0013] FIGS. 4A-4B graphically depict the distribution of
antibiotic catabolizing bacterial isolates with respect to
antibiotics and soil. (A) Number of antibiotic catabolizing
bacteria isolated from 11 soils color-coded by antibiotic class
catabolized. (B) Percentage of soils containing antibiotic
catabolizing bacteria, color-coded by chemical origin of
antibiotic.
[0014] FIG. 5 schematically depicts the phylogenetic distribution
of bacterial isolates subsisting on antibiotics. Full set of
bacteria subsisting on antibiotics is displayed in the centre, with
branches color-coded by bacterial orders, and clonal isolates
represented as squares. Subsets comprising clonal isolates
catabolizing each antibiotic are represented as trees around the
periphery, grouped by antibiotic class. 16S ribosomal DNA (rDNA)
was sequenced from antibiotic catabolizing clonal isolates using
universal bacterial rDNA primers. High-quality, non-chimeric
sequences were classified using Greengenes (DeSantis et al. (2006)
Applied and Environmental Microbiology 72:5069), with consensus
annotations from RDP (Cole et al. (2007) Nucleic Acids Res 35:D169)
and NCBI taxonomies (Wheeler et al. (2000) Nucleic Acids Res
28:10). Phylogenetic trees were constructed using the
neighbor-joining algorithm in ARB (Ludwig et al. (2004) Nucleic
Acids Res 32:1363) using the Greengenes aligned 16S rDNA database.
Placement in the tree was confirmed by comparing automated
Greengenes taxonomy to the annotated taxonomies of nearest
neighbors of each sequence in the aligned database. The
phylogenetic distributions of species isolated from different
antibiotics as sole carbon source exhibit some interesting trends.
For instance, the fluoroquinolone antibiotics, ciprofloxacin and
levofloxacin, have similar phylogenetic distributions, as do the
aminoglycoside antibiotics, gentamycin and amikacin, but the two
sets are notably different from each other. Interestingly, the
orders of bacteria subsisting on amikacin appear more similar to
gentamycin than kanamycin despite amikacin being a semi synthetic
kanamycin derivative.
[0015] FIGS. 6A-6C depict mass spectrometry analysis of growth
media from penicillin subsisting bacterial culture. (A) Mass
spectra of day 0 growth media from penicillin culture with a major
peak at m/z of 335.10 corresponding exactly to the protonated
penicillin G molecule. (B) Mass spectra of day 4 growth media from
penicillin culture with two major peaks at m/z values 353.11 and
309.12 corresponding to protonated benzylpenicilloic acid and
benzylpenilloic acid, respectively. (C) First steps of a proposed
penicillin G degradation pathway.
[0016] FIG. 7 depicts a list of antibiotic catabolizing isolates
described in FIG. 2. AIB2: Antibiotic Box 2; S*: section; YDM-TM:
1X YDM, trace metals, pH 5.5; Extr: extraction; RT: room
temperature.
[0017] FIG. 8 depicts a list of antibiotic catabolizing isolates
described in FIG. 2. AIB3: Antibiotic Box 3; S*: section; YDM-TM:
1X YDM, trace metals, pH 5.5; Extr: extraction; RT: room
temperature.
[0018] FIG. 9 depicts a list of antibiotic catabolizing isolates
described in FIG. 2. AIB4: Antibiotic Box 4; S*: section; YDM-TM:
1X YDM, trace metals, pH 5.5; Extr: extraction; RT: room
temperature.
[0019] FIG. 10 depicts a list of antibiotic catabolizing isolates
described in FIG. 2. AIB5: Antibiotic Box 5; S*: section; YDM-TM:
1X YDM, trace metals, pH 5.5; Extr: extraction; RT: room
temperature.
[0020] FIG. 11 depicts a list of antibiotic catabolizing isolates
described in FIG. 2. AIB6: Antibiotic Box 6; S*: section; YDM-TM:
1X YDM, trace metals, pH 5.5; Extr: extraction; RT: room
temperature.
[0021] FIG. 12 depicts a list of antibiotic catabolizing isolates
described in FIG. 2. AIB7: Antibiotic Box 7; S*: section; YDM-TM:
1X YDM, trace metals, pH 5.5; Extr: extraction; RT: room
temperature.
[0022] FIG. 13 depicts a list of antibiotic catabolizing isolates
described in FIG. 2. AIB8: Antibiotic Box 8; S*: section; YDM-TM:
1X YDM, trace metals, pH 5.5; Extr: extraction; RT: room
temperature.
DETAILED DESCRIPTION
[0023] Man-made chemicals are often used to clean up contaminated
and/or toxic materials, which can be both costly and
time-consuming. Microorganisms (e.g., bacteria) are a natural,
inexpensive means for reducing and/or eliminating contamination
and/or toxicity of a substance. Accordingly, in certain exemplary
embodiments, antibiotic and/or toxin eating microorganisms (e.g.,
bacteria) that can be produced using the methods described herein
are provided. In certain aspects, a cell, cell lysate, cell
extract, cell fraction, protein(s), polypeptide(s), isolated
antibiotic(s) or any combinations thereof from one or more
antibiotic and/or toxin eating microorganisms (e.g., bacteria) are
incubated in the presence of a contaminated substance to reduce or
eliminate contamination. In another aspect, antibiotic and/or toxin
eating bacteria are used in hybrid biological/chemical
manufacturing or decontamination systems where resistance to high
levels of various chemicals is helpful in the process engineering.
A cell, cell lysate, cell extract, cell fraction, protein(s),
polypeptide(s), isolated antibiotic(s) or any combinations thereof
from one or more antibiotic and/or toxin eating microorganisms
(e.g., bacteria) can be applied to a contaminated substance or a
manufacturing system via aerosols, slurries, cleaning solutions,
animal feeds, seeds, fertilizer and the like to partially or
completely decontaminate the substance or manufacturing system.
[0024] As used herein, the terms "toxin-eating bacterium" and
"toxin-eating bacteria" refer to bacteria that can use one or more
toxins and/or contaminants as a carbon source(s) or as the sole
carbon source to support growth. As used herein, the terms
"antibiotic-eating bacterium" and "antibiotic-eating bacteria"
refer to bacteria that can use one or more antibiotics as a carbon
source(s) or as the sole carbon source to support growth.
[0025] In certain exemplary embodiments, one or more toxin-eating
bacteria described herein are used for bioremediation of one or
more contaminants from a variety of environments such as, e.g.,
earth (e.g., sand, soil, rocks, any combination thereof and the
like), water (e.g., springs, lakes, brooks, streams, rivers, bays,
estuaries, seas, oceans and the like), air, manmade surfaces (e.g.,
medical facilities, instruments, service salons, makeup counters
etc.) and the like. As used herein, the term "bioremediation"
refers to the ability of one or more bacteria described herein to
remove or reduce the levels of one or more contaminants from an
environment.
[0026] As used herein, the terms "toxic environment" and
"contaminated substance" refer to an environment or substance,
respectively, that contains one or more adverse compound(s) and/or
physical condition(s) that can inhibit growth, inhibit productivity
and/or lead to the death of one or more microorganisms exposed to
the compound(s) and/or physical condition(s). A toxic environment
includes, but is not limited to, the following: the presence of
inhibitory compounds (e.g., antibiotics, radioactive compounds,
heavy metals and the like) high or low salinity, extreme
temperatures (e.g., high temperature (e.g., in thermal vents)
and/or cold temperature (e.g., in icy conditions), water scarcity,
darkness, light, catalytic products (e.g., cell waste, alcohol and
the like) and the like. For example, a toxic environment can
include the presence of a concentration (e.g., high or low
concentrations) of a compound and/or a condition that is considered
non-toxic to the microorganism in typical concentrations and/or in
typical conditions, as well as the presence of a compound or a
physical condition that would be typically considered to be
detrimental to the organism.
[0027] In certain embodiments, the toxicity (of a toxic
environment) or contamination (of a contaminated substance) is
eliminated or reduced to non-toxic or non-contaminated levels. In
certain aspects, the toxicity and/or contamination is reduced by
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.9% or more.
[0028] In certain exemplary embodiments, DNA fragments that can be
used in a microorganism to decrease toxicity and/or contamination
of a substance are provided. In certain aspects, the identification
of useful DNA fragments occurs by introducing a diverse library of
DNA fragments into a clonal population of the production
microorganism creating a population of cells harboring different
DNA fragments. The population of microorganisms harboring the large
DNA fragment library is subjected to growth in the presence of high
concentration of the inhibitor(s) which would normally suppress
growth of the host organism. If a host cell in the population
contains a DNA fragment which encodes for resistance to, e.g., high
concentrations of inhibitor(s) (e.g., one or more antibiotics), the
cell will selectively grow and can be identified. The DNA fragment
that enabled the cell to tolerate the inhibitor can then be
isolated, characterized and subsequently introduced into the
production microorganism improving its catalytic productivity in
the presence of the inhibitor.
[0029] As used herein, the term "organism" includes, but is not
limited to, a human, a non-human primate, a cow, a horse, a sheep,
a goat, a pig, a dog, a cat, a rabbit, a mouse, a rat, a gerbil, a
frog, a toad, a fish (e.g., D. rerio) a roundworm (e.g., C.
elegans) and any transgenic species thereof. The term "organism"
further includes, but is not limited to, a yeast (e.g., S.
cerevisiae) cell, a yeast tetrad, a yeast colony, a bacterium, a
bacterial colony, a virion, virosome, virus-like particle and/or
cultures thereof, and the like.
[0030] As used herein, the terms "microorganism" and "microbe"
refer to tiny organisms. Most microorganisms and microbes are
unicellular, although some multicellular organisms are microscopic,
while some unicellular protists and bacteria (e.g., T. namibiensis)
called are visible to the naked eye. Microorganisms and microbes
include, but are not limited to, bacteria, fungi, archaea and
protists, microscopic plants, and animals (e.g., plankton, the
planarian, the amoeba) and the like.
[0031] Certain aspects of the invention pertain to vectors, such
as, for example, expression vectors, containing a nucleic acid
encoding one or more bipolar cell-specific regulatory sequences. As
used herein, the term "vector" refers to a nucleic acid sequence
capable of transporting another nucleic acid to which it has been
linked. One type of vector is a "plasmid," which refers to a
circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. By way of example, but not of limitation, a vector of the
invention can be a single-copy or multi-copy vector, including, but
not limited to, a BAC (bacterial artificial chromosome), a fosmid,
a cosmid, a plasmid, a suicide plasmid, a shuttle vector, a P1
vector, an episome, YAC (yeast artificial chromosome), a
bacteriophage or viral genome, or any other suitable vector. The
host cells can be any cells, including prokaryotic or eukaryotic
cells, in which the vector is able to replicate.
[0032] Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "expression vectors." In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" can be used interchangeably. However, the
invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses), which
serve equivalent functions.
[0033] The recombinant expression vectors of the invention comprise
a nucleic acid of interest (e.g., a nucleic acid sequence from a
microorganism) in a form suitable for expression of the nucleic
acid in a host cell, which means that the recombinant expression
vectors include one or more regulatory sequences, selected on the
basis of the host cells to be used for expression, which is
operatively linked to the nucleic acid sequence to be expressed.
Within a recombinant expression vector, "operably linked" is
intended to mean that the nucleotide sequence of interest is
present in the vector in a manner which allows for expression of
the nucleotide sequence (e.g., in an in vitro
transcription/translation system or in a host cell when the vector
is introduced into the host cell). The term "regulatory sequence"
is intended to include promoters, enhancers and other expression
control elements (e.g., polyadenylation signals). Such regulatory
sequences are described, for example, in Goeddel; Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. (1990). Regulatory sequences include those which direct
constitutive expression of a nucleotide sequence in many types of
host cells and those which direct expression of the nucleotide
sequence only in certain host cells (e.g., tissue-specific
regulatory sequences).
[0034] It will be appreciated by those skilled in the art that the
design of the expression vector can depend on such factors as the
choice of the host cell to be transformed, the level of expression
of protein desired, and the like. The expression vectors of the
invention can be introduced into host cells to thereby produce
proteins or portions thereof, including fusion proteins or portions
thereof, encoded by nucleic acids as described herein.
[0035] In certain exemplary embodiments, a nucleic acid described
herein is expressed in bacterial cells using a bacterial expression
vector such as, e.g., a fosmid. A fosmid is a cloning vector that
is based on the bacterial F-plasmid. The host bacteria will
typically only contain one fosmid molecule, although an inducible
high-copy on can be included such that a higher copy number can be
obtained (e.g., pCC1FOS.TM., pCC2FOS.TM.). Fosmid libraries are
particularly useful for constructing stable libraries from complex
genomes. Fosmids and fosmid library production kits are
commercially available (EPICENTRE.RTM. Biotechnologies, Madison,
Wis.). For other suitable expression systems for both prokaryotic
and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0036] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0037] A host cell can be any prokaryotic or eukaryotic cell. For
example, one or more bipolar cell-specific regulatory elements
and/or portion(s) thereof can be reproduced in bacterial cells such
as E. coli, viruses such as retroviruses, insect cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS
cells). Other suitable host cells are known to those skilled in the
art.
[0038] Delivery of nucleic acid sequences described herein (e.g.,
vector DNA) can be by any suitable method in the art. For example,
delivery may be by injection, gene gun, by application of the
nucleic acid in a gel, oil, or cream, by electroporation, using
lipid-based transfection reagents, or by any other suitable
transfection method.
[0039] As used herein, the terms "transformation" and
"transfection" are intended to refer to a variety of art-recognized
techniques for introducing foreign nucleic acid (e.g., DNA) into a
host cell, including calcium phosphate or calcium chloride
co-precipitation, DEAE-dextran-mediated transfection, lipofection
(e.g., using commercially available reagents such as, for example,
LIPOFECTIN.RTM. (Invitrogen Corp., San Diego, Calif.),
LIPOFECTAMINE.RTM. (Invitrogen), FUGENE.RTM. (Roche Applied
Science, Basel, Switzerland), JETPEI.TM. (Polyplus-transfection
Inc., New York, N.Y.), EFFECTENE.RTM. (Qiagen, Valencia, Calif.),
DREAMFECT.TM. (OZ Biosciences, France) and the like), or
electroporation (e.g., in vivo electroporation). Suitable methods
for transforming or transfecting host cells can be found in
Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed.,
Cold Spring harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
[0040] In certain exemplary embodiments, one or more host
microorganisms described herein are engineered with various
isolation and/or safety features such as, e.g., novel genetic
codes, broad restriction systems, extreme sensitivity to substances
common in nature (e.g., UV light), dependency on lab metabolites
uncommon in nature (e.g., diaminopimelic acid) and the like in
order to decrease the spread of antibiotic and/or toxin resistance
gene(s) from one or more host cells. A non-limiting example of a
broad restriction system would be expression in the same cell
endonucleases aimed at both the methylated and unmethylated forms
of a DNA sequence (e.g., DpnI and DpnII aimed at G-mA-T-C and
GATC). This would require the removal of all sites (GATC in the
above example) throughout the host genome.
[0041] In certain exemplary embodiments, antibiotic and/or toxin
eating microorganisms (e.g., bacteria) are used to develop novel
antibiotics. Novel antibiotics are useful for overcoming the
multi-drug resistance (MDR) that is increasingly observed among
pathogenic bacteria. In certain exemplary aspects, antibiotic
and/or toxin eating bacteria are used to manufacture novel
antibiotics either harvested metagenomically from diverse natural
microbial cells or engineered from combinatorial libraries. Even
the trace amounts need to detect biosynthesis of novel compounds
could be enough to kill the host (or put undesired pressure to be
unproductive).
[0042] Novel antibiotics can be manufactured, for example, by
metagenomic harvesting from natural microbial cells or by
engineering from combinatorial libraries. In certain exemplary
embodiments, one or more microorganisms that are resistant to one
or more compounds that typically kill and/or inhibit the growth of
the microorganism (e.g., antibiotics, toxins and the like) are used
in screening assays for identifying modulators, i.e., candidate or
test compounds or agents (e.g., antibodies, peptides, cyclic
peptides, peptidomimetics, small molecules, small organic
molecules, antibiotics or drugs) which kill or have an inhibitory
effect on the growth of one or more microorganisms are provided. In
certain aspects, such screening assays can identify novel
antibiotics as well as antibiotics that are effective in killing or
reducing the growth of one or more multiple antibiotic resistant
microorganisms.
[0043] As used herein, the term "antibiotic" refers to a
chemotherapeutic agent (e.g., an agent produced by microorganisms
and/or synthetically) that has the capacity to inhibit the growth
of and/or to kill, one or more microorganisms (e.g., bacteria,
fungi, parasites and the like) or aberrantly growing cells (e.g.,
tumor cells). As used herein, antibiotics are well-known to those
of skill in the art. Classes of antibiotics include, but are not
limited to, aminoglycosides (e.g., amikacin, gentamicin, kanamycin,
neomycin, netilmicin, streptomycin, tobramycin, paromomycin and the
like), ansamycins (e.g., geldanamycin, herbimycin and the like),
carbacephem (e.g., loracarbef), carbapenems (e.g., ertapenem,
doripenem, imipenem/cilastatin, meropenem and the like)
cephalosporins (e.g., first generation (e.g., cefadroxil,
cefazolin, cefalotin, cefalexin and the like), second generation
(e.g., cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime and
the like), third generation (e.g., cefixime, cefdinir, cefditoren,
cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,
ceftizoxime, ceftriaxone and the like), fourth generation (e.g.,
cefepime and the like) and fifth generation (e.g., ceftobiprole and
the like)), glycopeptides (e.g., teicoplanin, vancomycin and the
like), macrolides (e.g., azithromycin, clarithromycin,
dirithromycin, erythromycin, roxithromycin, troleandomycin,
telithromycin, spectinomycin and the like), monobatams (e.g.,
aztreonam and the like), penicillins (e.g., amoxicillin,
ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin,
flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin,
penicillin, piperacillin, ticacillin and the like), polypeptides
(e.g., bacitracin, colistin, polymyxin B and the like) quinolones
(e.g., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin,
lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin
and the like), sulfonamides (e.g., mafenide, prontosil,
sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine,
sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole and the
like), tetracyclines (e.g., demeclocycline, doxycycline,
minocycline, oxytetracycline, tetracycline and the like) and others
(e.g., arsphenamine, chloramphenicol, clindamycin, lincomycin,
ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid,
linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin,
pyrazinamide, quinupristin/dalfopristin, rifampin, tinidazol and
the like) (See, e.g., Robert Berkow (ed.) The Merck Manual of
Medical Information--Home Edition. Pocket (September 1999), ISBN
0-671-02727-1).
[0044] In certain exemplary embodiments, assays for screening
candidate or test compounds (e.g., antibiotics) which bind to or
modulate (e.g., kill or have an inhibitory effect on the growth of)
a microorganism are provided. The test compounds of the present
invention can be obtained using any of the numerous approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the "one-bead one-compound" library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds (Lam,
K. S. (1997) Anticancer Drug Des. 12:145).
[0045] The candidate or test compound(s) described herein can be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the nucleic
acid molecule or protein and a pharmaceutically acceptable carrier.
As used herein the language "pharmaceutically acceptable carrier"
is intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0046] In certain exemplary embodiments, a pharmaceutical
composition is formulated to be compatible with its intended route
of administration. Examples of routes of administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerin, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0047] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, CREMOPHOR EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0048] Sterile injectable solutions can be prepared by
incorporating the candidate or test compound(s) in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0049] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: A binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic, acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant: such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
[0050] In one embodiment, the candidate or test compound(s) are
prepared with carriers that will protect the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These may be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0051] Nasal compositions generally include nasal sprays and
inhalants. Nasal sprays and inhalants can contain one or more
active components and excipients such as preservatives, viscosity
modifiers, emulsifiers, buffering agents and the like. Nasal sprays
may be applied to the nasal cavity for local and/or systemic use.
Nasal sprays may be dispensed by a non-pressurized dispenser
suitable for delivery of a metered dose of the active component.
Nasal inhalants are intended for delivery to the lungs by oral
inhalation for local and/or systemic use. Nasal inhalants may be
dispensed by a closed container system for delivery of a metered
dose of one or more active components.
[0052] In one embodiment, nasal inhalants are used with an aerosol.
This is accomplished by preparing an aqueous aerosol, liposomal
preparation or solid particles containing the compound. A
non-aqueous (e.g., fluorocarbon propellant) suspension could be
used. Sonic nebulizers may be used to minimize exposing the agent
to shear, which can result in degradation of the compound.
[0053] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the agent together with
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers vary with the requirements of the
particular compound, but typically include nonionic surfactants
(Tweens, Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid, lecithin, amino
acids such as glycine, buffers, salts, sugars or sugar alcohols.
Aerosols generally are prepared from isotonic solutions.
[0054] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0055] The candidate or test compound(s) can also be prepared in
the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0056] In one embodiment, candidate or test compound(s) are
prepared with carriers that will protect them against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0057] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0058] Toxicity and therapeutic efficacy of candidate or test
compound(s) can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD50/ED50. Compounds
which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0059] Data obtained from cell culture assays and/or animal studies
can be used in formulating a range of dosage for use in humans. The
dosage typically will lie within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. For any
compound used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC50 (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0060] In certain exemplary embodiments, a method for treatment of
infection by a microorganism includes the step of administering a
therapeutically effective amount of an agent (e.g., one or more
candidate or test compounds) which modulates (e.g., kills and/or
inhibits the growth of), one or more microorganisms to a subject.
As defined herein, a therapeutically effective amount of agent
(i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg
body weight, from about 0.01 to 25 mg/kg body weight, from about
0.1 to 20 mg/kg body weight, or from about 1 to 10 mg/kg, 2 to 9
mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of an inhibitor can include a
single treatment or, in certain exemplary embodiments, can include
a series of treatments. It will also be appreciated that the
effective dosage of inhibitor used for treatment may increase or
decrease over the course of a particular treatment. Changes in
dosage may result from the results of diagnostic assays as
described herein. The pharmaceutical compositions can be included
in a container, pack, or dispenser together with instructions for
administration.
[0061] In certain embodiments, monitoring the influence of agents
(e.g., drugs, compounds) on the killing and/or inhibiting cell
growth of one or more microorganisms can be applied not only in
basic drug screening, but also in clinical trials. In certain
exemplary embodiments, a method is provided for monitoring the
effectiveness of treatment of a subject with an agent (e.g., an
agonist, antagonist, antibody, peptidomimetic, protein, peptide,
nucleic acid, small molecule, antibiotic or other drug candidate
identified by the screening assays described herein) comprising the
steps of (i) obtaining a pre-administration sample from a subject
prior to administration of the agent; (ii) detecting the level of a
microorganism in the preadministration sample; (iii) obtaining one
or more post-administration samples from the subject; (iv)
detecting the level the microorganism in the post-administration
samples; (v) comparing the level of microorganism in the
pre-administration sample with the level of microorganism in the
post-administration sample or samples; and (vi) altering the
administration of the agent to the subject accordingly. For
example, increased administration of the agent may be desirable to
increase the effectiveness of the agent. Alternatively, decreased
administration of the agent may be desirable to decrease the
effectiveness of the agent.
[0062] It is to be understood that the embodiments of the present
invention which have been described are merely illustrative of some
of the applications of the principles of the present invention.
Numerous modifications may be made by those skilled in the art
based upon the teachings presented herein without departing from
the true spirit and scope of the invention. The contents of all
references, patents and published patent applications cited
throughout this application are hereby incorporated by reference in
their entirety for all purposes.
[0063] The following examples are set forth as being representative
of the present invention. These examples are not to be construed as
limiting the scope of the invention as these and other equivalent
embodiments will be apparent in view of the present disclosure,
tables, figures, and accompanying claims.
Example I
Bacteria Subsisting on Antibiotics
[0064] 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. Hundreds of soil bacteria with the
capacity to grow on antibiotics as a sole carbon source were
isolated. Of 18 antibiotics tested, representing eight major
classes of natural and synthetic origin, 13-17 antibiotics
supported 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 is
resistant to multiple antibiotics at clinically relevant
concentrations. This phenomenon suggests this unappreciated
reservoir of antibiotic resistance determinants can contribute to
the increasing levels of multiple antibiotic resistance in
pathogenic bacteria.
[0065] Clonal bacterial isolates from 11 diverse soils (Table 2)
which were capable of utilizing one of 18 different antibiotics as
the sole carbon source were cultured. The 18 antibiotics comprised
of natural, semi-synthetic and synthetic compounds of different
ages and included all major bacterial target classes. Every
antibiotic tested was able to support bacterial growth (FIG. 1A and
FIGS. 4A-4B). Notably, 6 out of 18 antibiotics supported growth in
all 11 soils, covering 5 of the 8 classes of antibiotics tested.
Appropriate controls were performed to ensure that carbon source
contamination of the source media or carbon fixation from the air
were insignificant to this experiment (See Example II).
[0066] Clonal isolates capable of subsisting on penicillin and
carbenicillin were obtained from all the soils tested, and isolates
from 9 out of 11 soils that could subsist on dicloxacillin.
Representative isolates capable of growth on penicillin and
carbenicillin were selected for subsequent analysis by high
performance liquid chromatography (HPLC) (See Example II). Removal
of the antibiotics from the media was observed within 4 and 6 days,
respectively (FIG. 1B). Mass spectrometry analysis of penicillin
cultures is consistent with a penicillin catabolic pathway (Johnsen
(1977) Archives of Microbiology 115:271) initiated by hydrolytic
cleavage of the beta lactam ring, which is the dominant mode of
clinical resistance to penicillin and related beta lactam
antibiotics, followed by a decarboxylation step (FIGS. 6A-6C) (See
Example II).
[0067] Bacteria were isolated from all the soils tested that grew
on ciprofloxacin (FIG. 1A), a synthetic fluoroquinolone and one of
the most widely prescribed antibiotics. Clonal isolates capable of
catabolizing the other two synthetic quinolones tested,
levofloxacin and nalidixic acid, were also isolated from a majority
of the soils (FIG. 1A). Previous studies have highlighted the
strong parallels between antibiotic resistance determinants
harbored by soil dwelling microbes and human pathogens (Davies
(1994) Science 264:375; Marshall (1998) Antimicrobial Agents and
Chemotherapy 42:2215; D'Costa et al. (2007) Curr. Opin. Microbiol.
10:481). The lateral transfer of genes encoding the enzymatic
machinery responsible for subsistence on quinolone antibiotics to
human pathogens could introduce a novel resistance mechanism so far
not observed in the clinic.
[0068] Phylogenetic profiling of the clonal isolates (See Example
II) revealed a diverse set of species in the Proteobacteria (87%),
Actinobacteria (7%) and Bacteroidetes (6%) (FIG. 2 and FIG S2).
These phyla all include many clinically relevant pathogens. Of the
eleven orders represented, Burkholderiales constitute 41% of the
species isolated. The other major orders (>5%) are:
Pseudomonadales (24%), Enterobacteriales (13%), Actinomycetales
(7%), Rhizobiales (7%), and Sphingobacteriales (6%).
[0069] Without intending to be bound by scientific theory, one
explanation for the widespread catabolism of both natural and
synthetic antibiotics may relate to their organic sub-structures
which are found in nature. Metabolic mechanisms exist for
processing those sub-structures and may allow for the utilization
of the parent synthetic antibiotic molecule. It is interesting that
more than half of the bacterial isolates identified in this study
belong to the orders Burkholderiales and Pseudomonadales. Organisms
in these orders typically have large genomes of approximately 6-10
megabases, which has been suggested to be positively correlated to
their metabolic diversity and multiple antibiotic resistance
(Projan (2007) Antimicrobial Agents and Chemotherapy 51:1133).
These organisms can be thought of as scavengers, capable of
utilizing a large variety of single carbon sources as food (Parke
and Gurian-Sherman (2001) Annual Review of Phytopathology
39:225).
[0070] The magnitude of antibiotic resistance for a representative
subset of 75 clonal isolates was determined (Table 3). Each clonal
isolate was tested for resistance towards all 18 antibiotics used
in the subsistence experiments at 20 mg/L and 1 g/L in rich media
(See Example II). The clonal isolates tested on average were
resistant to 17 out of 18 antibiotics at 20 mg/L, and 14 out of 18
antibiotics at 1 g/L (FIG. 3). Furthermore, for 74 of the 75
isolates, it was determined that if a bacterial isolate was able to
subsist on an antibiotic, it was also resistant to all antibiotics
in that class at 20 mg/L.
[0071] Previous work showing that strains from the genus
Streptomyces are on average resistant to 7-8 antibiotics at 20 mg/L
has highlighted the importance of producer organisms as a reservoir
of antibiotic resistance (D'Costa et al. (2006) Science 311:374).
Here bacteria subsisting on antibiotics are described as a
substantial addition to the antibiotic resistome in terms of both
phylogenetic diversity and prevalence of resistance. The bacteria
isolated and described herein are `super resistant,` since they
tolerate concentrations of antibiotics>1 g/L which are 50-fold
higher than the antibiotic concentrations used to define the
antibiotic resistome. Id.
[0072] Greengenes (DeSantis et al. (2006) Applied and Environmental
Microbiology 72:5069) identified isolates among the bacteria
subsisting on antibiotics that are closely related to known
pathogens e.g., members of the Burkholderia cepacia complex, and
Serratia marcescens. In principle, relatedness allows for easier
transfer of genetic material, since codon usage, promoter binding
sites and other transcriptional and translational motifs are likely
to be similar. It is therefore possible that pathogenic microbes
can more readily use resistance genes originating from bacteria
subsisting on antibiotics compared to the resistance genes from
more distantly related antibiotic producer organisms.
[0073] To date, there have been no reports describing antibiotic
catabolism in pathogenic strains. However, since most sites of
serious infection in the human body are not carbon source limited
it is unlikely that pathogenic microbes would have a strong
selective advantage by catabolizing antibiotics compared to just
resisting them, so it is likely that only the resistance conferring
part of the catabolic machinery would be selected for in pathogenic
strains.
[0074] In addition to the finding that bacteria subsisting on
natural and synthetic antibiotics are widely distributed in the
environment, these results highlight an unrecognized reservoir of
multiple antibiotic resistance machinery. Bacteria subsisting on
antibiotics are phylogenetically diverse, and include many
organisms closely related to clinically relevant pathogens. It is
thus possible that pathogens could obtain antibiotic resistance
genes from environmentally distributed super-resistant microbes
subsisting on antibiotics.
REFERENCES
[0075] Riesenfeld et al. (2004) Environmental Microbiology 6:981
[0076] Walsh (2000) Nature 406:775 [0077] Alekshun and Levy (2007)
Cell 128:1037 [0078] Fredrickson et al. (2000) Applied and
Environmental Microbiology 66:2006 [0079] McAllister et al. (1996)
Biodegradation 7:1 [0080] Kameda et al. (1961) Nature 191:1122
[0081] Abd-El-Malek et al. (1961) Nature 189:775 [0082] Cole et al.
(2007) Nucleic Acids Res 35:D169 [0083] Wheeler et al. (2000)
Nucleic Acids Res 28:10 [0084] Ludwig et al. (2004) Nucleic Acids
Res 32:1363
Example II
Materials and Methods
[0085] Growth Media
[0086] All liquid media used for isolating bacteria capable of
subsisting on antibiotics was made by dissolving 1 g/L of the
relevant antibiotics (Table 1, which depicts lot purities of
antibiotics used, as reported on Certificates of Analysis from
Sigma-Aldrich) into single carbon source (SCS) media containing 5 g
(NH.sub.4).sub.2SO.sub.4, 3 g KH.sub.2PO.sub.4, 0.5 g
MgSO.sub.4.7H.sub.2O, 15 mg EDTA, 4.5 mg ZnSO.sub.4.7H.sub.2O, 4.5
mg CaCl.sub.2.2H.sub.2O, 3 mg FeSO.sub.4.7H.sub.2O, 1 mg
MnCl.sub.2.4H.sub.2O, 1 mg H.sub.3BO.sub.3, 0.4 mg
Na.sub.2MoO.sub.4.2H.sub.2O, 0.3 mg CuSO.sub.4.5H.sub.2O, 0.3 mg
CoCl.sub.2.6H.sub.2O and 0.1 mg KI per liter water. The pH was
adjusted to 5.5 using HCl, and the media was sterilized through a
0.22 .mu.m filter. Solid medium was prepared by adding 15 g agar
per liter of liquid SCS media followed by autoclaving before adding
antibiotics.
TABLE-US-00001 TABLE 1 NR, not reported. Antibiotics Lot Purity %
Ciprofloxacin 98.5 Levofloxacin 100.0 Sisomicin 99 Gentamicin NR
Kanamycin NR Amikacin 100 Penicillin G 99.7 Carbenicillin 92.9
Dicloxacillin 99.8 Chloramphenicol >99 Nalidixic acid 100
Thiamphenicol >99 Sulfisoxazole 99.7 Trimethoprim 100 Mafenide
100 Sulfamethizole 99.9 D-Cycloserine 98 Vancomycin NR
[0087] All liquid media used for resistance profiling was made by
dissolving 20 mg/L or 1 g/L of the relevant antibiotics into
autoclaved Luria broth containing 5 g Yeast Extract, 10 g NaCl and
10 g of tryptone in 1 Liter of water. The pH was adjusted to 5.5
using HCl, and the media was sterilized through a 0.22 .mu.m
filter.
[0088] Culturing of Environmental Bacteria Capable of Subsisting on
Antibiotics
[0089] Initial soil microbial inocula (soil description in Table 2,
which depicts soil information for the 11 different soils from
which bacteria capable of subsisting on antibiotics were isolated)
were prepared in minimal medium containing no carbon, and
inoculated into SCS-antibiotic media (corresponding to
approximately 125 mg of dissolved soil in 5 mL of media). To
significantly reduce the transfer of residual alternative carbon
sources present in original inocula, samples were passaged (2.5
.mu.L) into fresh SCS-antibiotic media (5 mL) two additional times
after 7 days of growth, resulting in a 5.times.10.sup.4 dilution at
each passage (resulting in a final carryover of approximately 30 ng
of soil in 5 mL of media at the third passage). Clonal isolates
from the liquid cultures were obtained by plating cultures out on
SCS-antibiotic agar medium and resulting single colonies were
picked and re-streaked on corresponding plates. Three colonies each
were then inoculated into fresh SCS-antibiotic liquid media (5 mL)
to confirm clonal phenotype. Final culture growth was recorded
after 1 month incubation without shaking at 22.degree. C. and
cultures with at least 10.sup.8 cells/mL were assayed as positive
growth.
TABLE-US-00002 TABLE 2 FIG. 1A Soil identifiers Soil type name Soil
collection location F1 Farmland S1G Corn Field with Antibiotic
Treated Manure, Great Brook Farm, Carlisle, MA F2 Farmland S1N
Alfalfa Field with Manure Treatment, Northcroft Farm, Pelican
Rapids, MN F3 Farmland S2N Alfalfa Field without Manure Treatment,
Northcroft Farm, Pelican Rapids, MN P1 Pristine S2R Raccoon Ledger,
Rockport, MA P2 Pristine S3N Prairie next to Northcroft Farm,
Pelican Rapids, MN P3 Pristine S1R Brier's Swamp, Rockport, MA P4
Pristine S1A Pristine Forest Soil, Alan Seeger Natural Area, PA P5
Pristine S2T Untreated Forested Area, Toftrees State Gameland Area,
PA U1 Urban S1T Waste Water Treated Area, Toftrees State Gameland
Area, PA U2 Urban S3F Boston Fens, MA U3 Urban S1P Boston Public
Garden, MA
[0090] Since inoculation in media lacking a carbon source (no
carbon control) did not show growth in any cases, carbon source
contamination of the source media or carbon fixation from the air
were considered insignificant to this experiment. The only other
alternative carbon substrate for growth could be impurities in the
antibiotic stocks. All antibiotics used were purchased from
Sigma-Aldrich at the highest purities available. Lot purities of
each compound used are listed in Table 1. Based on an average
carbon mass of 0.15.times.10.sup.-12 g per bacterial cell, it was
estimated that at least 15 .mu.g of carbon must be incorporated
into bacterial biomass to reach sufficient culture densities in 1
mL of culture to be rated as successful growth. Assuming 50% carbon
content of impurities, and under the most stringent assumptions of
(1) 100% incorporation of carbon impurities into biomass, and (2)
no loss of carbon as metabolic byproducts (such as CO.sub.2),
antibiotics with greater than 97% purity would have insufficient
impurities to support sole carbon source growth. Of the antibiotic
lots used in this experiment (Table 1), twelve compound stocks are
at least 99% pure, two compounds (ciprofloxacin and D-cycloserine)
have between 98 and 98.5% purity, one compound (carbenicillin) is
92.9% pure, and no purity information is available for three
compounds (kanamycin, gentamicin, and vancomycin).
Phylogenetic Profiling
[0091] The 16S ribosomal DNA (rDNA) of each of the clonal isolates
identified in this study was amplified using universal bacterial
16S primers:
TABLE-US-00003 >Bact_63f_62C (SEQ ID NO: 1) 5'-CAG GCC TAA CAC
ATG CAA GTC-3' >Bact_1389r_63C (SEQ ID NO: 2) 5'-ACG GGC GGT GTG
TAC AAG-3'
[0092] Successful 16S rDNA amplicons were sequenced for
phylogenetic profiling. High-quality, non-chimeric sequences were
classified using Greengenes (DeSantis et al. (2006) Nucleic Acids
Res 34:W394; DeSantis et al. (2006) Applied and Environmental
Microbiology 72:5069), with consensus annotations from RDP (Cole et
al. (2007) Nucleic Acids Res 35:D169) and NCBI taxonomies (Wheeler
et al. (2000) Nucleic Acids Res 28: 10). Phylogenetic trees were
constructed using the neighbor joining algorithm in ARB (Ludwig et
al. (2004) Nucleic Acids Res 32:1363) using the Greengenes aligned
16S rDNA database. Placement in the tree was confirmed by comparing
automated Greengenes taxonomy to the annotated taxonomies of
nearest neighbors of each sequence in the aligned database.
[0093] Resistance Profiling of 75 Representative Isolates Capable
of Subsisting on Antibiotics
[0094] 75 clonal isolates (Table 3, which lists strain information
for the 75 clonal isolates used for resistance profiles) were
selected to include multiple isolates capable of subsisting on each
of the 18 antibiotics and originating from each of the 11 soils
(Table 2). Bacterial cultures were inoculated into Luria Broth from
frozen glycerol stocks and were incubated at 22.degree. C. for 3
days. 500 nL of this culture was used to inoculate each of the
clonal isolates into 200 uL of Luria Broth containing one of the
eighteen different antibiotics (See Table 1) at 20 mg/L and 1 g/L.
Cultures were incubated without shaking at 22.degree. C. for 4
days. Resistance of an isolate was determined by turbidity at 600
nm using a Versamax microplate reader from Molecular Devices.
TABLE-US-00004 TABLE 3 FIG. 3A identifier Strain name Subsisting on
From soil 1 Levo-S2T-M1LLLSSL-2 Levofloxacin S2T 2
Kana-S2T-M1LLLSSL-3 Kanamycin S2T 3 Amik-S2T-M1LLLSSL-1 Amikacin
S2T 4 Carb-S2T-M1LLLSSL-2 Carbenicillin S2T 5 Chlo-S2T-M1LLLSSL-2
Chloramphenicol S2T 6 Nali-S2T-M1LLLSSL-1 Nalidixic acid S2T 7
Thia-S2T-M1LLLSSL-2 Thiamphenicol S2T 8 Trim-S2T-M1LLLSSL-1
Trimethoprim S2T 9 Mafe-S2T-M1LLLSSL-3 Mafenide S2T 10
Cycl-S2T-M1LLLSSL-3 D-Cycloserine S2T 11 Vanc-S2T-M1LLLSSL-3
Vancomycin S2T 12 Siso-S2N-M1LLLSSL-1 Sisomycin S2N 13
Gent-S2N-M1LLLSSL-2 Gentamycin S2N 14 Kana-S2N-M1LLLSSL-2 Kanamycin
S2N 15 Peni-S2N-M1LLLSSL-2 Penicillin G S2N 16 Dicl-S2N-M1LLLSSL-1
Dicloxacillin S2N 17 Trim-S2N-M1LLLSSL-1 Trimethoprim S2N 18
Vanc-S2N-M1LLLSSL-1 Vancomycin S2N 19 Dicl-S3N-M1LLLSSL-2
Dicloxacillin S3N 20 Thia-S3N-M1LLLSSL-3 Thiamphenicol S3N 21
Trim-S3N-M1LLLSSL-2 Trimethoprim S3N 22 Mafe-S3N-M1LLLSSL-2
Mafenide S3N 23 Vanc-S3N-M1LLLSSL-2 Vancomycin S3N 24
Cipr-S1P-M1LLLSSL-3 Ciprofloxacin S1P 25 Peni-S1P-M1LLLSSL-2
Penicillin G S1P 26 Chlo-S1P-M1LLLSSL-1 Chloramphenicol S1P 27
Thia-S1P-M1LLLSSL-1 Thiamphenicol S1P 28 Trim-S1P-M1LLLSSL-3
Trimethoprim S1P 29 Slfm-S1P-M1LLLSSL-2 Sulfamethizole S1P 30
Cycl-S1P-M1LLLSSL-1 D-Cycloserine S1P 31 Vanc-S1P-M1LLLSSL-3
Vancomycin S1P 32 Cipr-S1T-M1LLLSSL-2 Ciprofloxacin S1T 33
Levo-S1T-M1LLLSSL-1 Levofloxacin S1T 34 Siso-S1T-M1LLLSSL-1
Sisomycin S1T 35 Carb-S1T-M1LLLSSL-1 Carbenicillin S1T 36
Dicl-S1T-M1LLLSSL-1 Dicloxacillin S1T 37 Chlo-S1T-M1LLLSSL-1
Chloramphenicol S1T 38 Thia-S1T-M1LLLSSL-3 Thiamphenicol S1T 39
Trim-S1T-M1LLLSSL-2 Trimethoprim S1T 40 Mafe-S1T-M1LLLSSL-1
Mafenide S1T 41 Cycl-S1T-M1LLLSSL-2 D-Cycloserine S1T 42
Vanc-S1T-M1LLLSSL-1 Vancomycin S1T 43 Levo-S3F-M1LLLSSL-3
Levofloxacin S3F 44 Slfs-S3F-M1LLLSSL-3 Sulfisoxazole S3F 45
Trim-S3F-M1LLLSSL-l Trimethoprim S3F 46 Mafe-S3F-M1LLLSSL-3
Mafenide S3F 47 Slfm-S3F-M1LLLSSL-3 Sulfamethizole S3F 48
Vanc-S3F-M1LLLSSL-2 Vancomycin S3F 49 Amik-S1R-M1LLLSSL-3 Amikacin
S1R 50 Peni-S1R-M1LLLSSL-2 Penicillin G S1R 51 Mafe-S1R-M1LLLSSL-2
Mafenide S1R 52 Vanc-S1R-M1LLLSSL-2 Vancomycin S1R 53
Trim-S1N-M1LLLSSL-1 Trimethoprim S1N 54 Vanc-S1N-M1LLLSSL-1
Vancomycin S1N 55 Kana-S1A-M1LLLSSL-2 Kanamycin S1A 56
Carb-S1A-M1LLLSSL-2 Carbenicillin S1A 57 Slfs-S1A-M1LLLSSL-1
Sulfisoxazole S1A 58 Vanc-S1A-M1LLLSSL-2 Vancomycin S1A 59
Kana-S2R-M1LLLSSL-2 Kanamycin S2R 60 Amik-S2R-M1LLLSSL-3 Amikacin
S2R 61 Peni-S2R-M1LLLSSL-2 Penicillin G S2R 62 Dicl-S2R-M1LLLSSL-1
Dicloxacillin S2R 63 Mafe-S2R-M1LLLSSL-2 Mafenide S2R 64
Slfm-S2R-M1LLLSSL-1 Sulfamethizole S2R 65 Cipr-S1G-M1LLLSSL-1
Ciprofloxacin S1G 66 Levo-S1G-M1LLLSSL-1 Levofloxacin S1G 67
Gent-S1G-M1LLLSSL-3 Gentamycin S1G 68 Kana-S1G-M1LLLSSL-1 Kanamycin
S1G 69 Peni-S1G-M1LLLSSL-1 Penicillin G S1G 70 Carb-S1G-M1LLLSSL-3
Carbenicillin S1G 71 Chlo-S1G-M1LLLSSL-3 Chloramphenicol S1G 72
Nali-S1G-M1LLLSSL-2 Nalidixic acid S1G 73 Thia-S1G-M1LLLSSL-1
Thiamphenicol S1G 74 Slfs-S1G-M1LLLSSL-3 Sulfisoxazole S1G 75
Mafe-S1G-M1LLLSSL-2 Mafenide S1G
[0095] Analysis of Antibiotic Removal of Penicillin and
Carbenicillin Subsisting Bacteria
[0096] Representative isolates capable of growth on penicillin and
carbenicillin as sole carbon source were selected for analysis of
antibiotic removal from the growth media by High Performance Liquid
Chromatography (HPLC). 2 .mu.L of these cultures were re-inoculated
into fresh SCS-antibiotic medium (5 mL) and allowed to grow for 28
days. Samples of the cultures and un-inoculated media controls were
taken at regular intervals throughout the 28 day period and the
catabolism of penicillin and carbenicillin was monitored at 214 nm
by HPLC of filtered media from samples using a Hewlett Packard 1090
Liquid Chromatograph and a Vydac C-18 column. HPLC was performed at
a flow rate of 0.3 mL/min with an acetonitrile gradient going from
5% to 65% in 30 minutes in the presence of 0.1% trifluoroacetic
acid.
[0097] The HPLC chromatogram of the penicillin catabolizing culture
medium (FIG. 1B) started out with a single peak corresponding to
the penicillin peak of the un-inoculated control. This peak
disappeared at day 4 with the appearance of multiple smaller peaks
at lower elution times; by day 20 these peaks had also disappeared
in agreement with the complete catabolism of penicillin by the
culture in 20 days. In comparison, the single penicillin peak in
the un-inoculated control remained the dominant peak over the same
time course. The HPLC chromatogram of the medium from the
carbenicillin catabolizing culture (FIG. 1B) started out with a
bimodal peak corresponding to the un-inoculated carbenicillin
control, which remained stable for 2 days. At day 4, corresponding
to the appearance of visible turbidity in the inoculated culture,
the bimodal peak had almost disappeared and secondary peaks at
lower elution times were observed. These secondary peaks almost
completely disappeared by the 28.sup.th day, suggesting that
carbenicillin was almost completely catabolized within 28 days. The
bimodal carbenicillin peak remained relatively unchanged in the
un-inoculated control over the same time course.
[0098] Samples from the penicillin subsisting culture from day 0
and day 4 were prepared for LC/MS using a Waters Sep-Pak Cartridge
prior to mass spectrometry analysis using a LTQ-FT from Thermo
Electron. Mass spectra were analyzed using XCalibur 2.0.5 and the
empirically determined m/z values of all major peaks were compared
to predicted m/z values of putative penicillin degradation products
calculated using ChemDraw Ultra 9.0 (FIGS. 6A-6C).
Sequence CWU 1
1
2121DNAArtificialPCR Primer 1caggcctaac acatgcaagt c
21218DNAArtificialPCR Primer 2acgggcggtg tgtacaag 18
* * * * *