Induction of antibiotic hypersensitivity in tetracycline-resistance microorganisms

Microorganisms carrying tetracycline-resistant determinants are hypersensitized to Sm- and 2-DOS- aminoglycoside antibiotics by exposure to subinhibitory amounts of tetracycline inducer.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
Drug-resistant microorganisms pose a serious threat to effective clinical 
management of infectious diseases. It is well-settled that microorganism 
drug-resistance is afforded by both extrachromosomal resistance 
determinants and random mutation of the cells; in the presence of the 
drug, these provide a selection advantage for the survival of the 
resistant bacteria. While mechanisms of drug-resistance vary, and in many 
instances are poorly understood, it is believed that for many drugs, 
including tetracycline, resistance is a function of cellular membrane 
efflux of the drug controlled by genetic information encoded in 
extrachromosomal plasmids. The resistance trait can be transmitted from 
resistant to sensitive organisms by transferal of the genetic material 
itself via resistance transfer factors carried by the cell. It is this 
genetically-mediated transfer of bacterial drug resistance traits which 
presents the most serious clinical implications, particularly when 
multiple-drug resistance is involved. 
2. Discussion of Related Art 
The clinical treatment of infectious diseases has thus been often designed 
to obviate the development of a drug-resistant microorganism population 
insofar as possible. In particular, combinations of antibiotics are often 
favored in antibiotic therapy to suppress the emergence of mutants 
expressing resistance to an individual drug employed in the therapy, 
particularly in the treatment of infectious diseases caused by 
microorganisms which tend rapidly to mutate to resistant strains. While 
synergistic effects have been reported for some of these combinations, the 
thrust of this therapy has been to treat the infection with minimum 
inhibitory concentrations (MIC) of one or more antibiotics to inhibit the 
growth of the infecting bacteria, including singly drug-resistant mutant 
strains, and increase the spectrum of sensitive microorganisms targeted by 
the drugs. While this approach is relatively successful in combating 
drug-resistant populations arising from random mutations, it is of little 
effect in the treatment of microorganisms with constitutive resistance 
(i.e., those carrying extrachromosomal resistance determinants) to one or 
more therapeutic drugs of first choice Accordingly, the infecting 
resistant microorganisms are treated with drugs which are of less clinical 
value, usually owing to their association with adverse side effects at 
effective dosage levels. In particular, diseases caused by microorganisms 
containing tetracycline resistance determinants are commonly managed by 
treatment of the host with a drug of lesser choice, frequently an 
aminoglycoside antibiotic; at the antibiotic dosages typically employed, 
however, aminoglycoside and nephro- toxicity are serious and not uncommon 
clinical problems. It is thus desirable to improve such alternative 
therapy to decrease risk of toxicity while maintaining or improving 
efficacy. 
SUMMARY OF THE INVENTION 
The invention is directed to a method for hypersensitizing microorganisms 
which contain tetracycline-resistance determinants (Tc.sup.r) to the 
action of an aminoglycoside antibiotic, comprising exposing the Tc.sup.r 
microorganism to a subinhibitory amount of one of a class of 
tetracycline-related compounds to induce expression of the 
tetracycline-resistance determinant and sensitize the microorganism to 
antibiotic therapy. Hypersensitization according to the invention permits 
the clinical use of much lower minimum inhibitory concentrations of 
aminoglycoside antibiotic than would otherwise be required, with 
concomitant reduced risk of drug toxicity. Further, the resulting 
increased potency of the antibiotics against Tc.sup.r strains is also 
exploitable to achieve a more rapid effect, or to treat marginally 
susceptible bacteria at conventional dosage levels. Effects comparable to 
those obtained with conventional aminoglycoside therapy are thus 
obtainable at lower dosage levels, or, conversely, the antibiotic is more 
effective at conventional dosage levels. 
DETAILED DESCRIPTION OF THE INVENTION 
It has been postulated that microorganisms expressing tetracycline 
resistance are characterized by a component of the cytoplasmic membrane 
styled TET-protein, which mediates movement of tetracycline across the 
membrane and out of the cell. In addition to mediating the efflux of 
tetracycline from the cell membrane, the presence of TET-protein appears 
to be associated with microorganism hypersensitivity to a variety of 
compounds, especially lipophilic chelators such as fusaric acid (see, 
e.g., J. Bact. 143:926-933, 1980). 
It has now been discovered that a subinhibitory amount of tetracycline or a 
tetracycline-related compound is effective to hypersensitize tetracycline 
resistant microorganisms to the action of aminoglycosideantibiotics 
containing streptamine or 2-deoxystreptamine functional groups. It is 
believed that tetracycline functions to induce the expression of 
TET-resistance in the microorganism, probably by the production of 
TET-protein, which then promotes the uptake of the therapeutic antibiotic 
by the cell. Therapeutic regimens employing reduced minimum inhibitory 
concentrations (MICs) of aminoglycoside antibiotics against tetracycline 
resistant bacteria are accordingly practical. 
The microorganisms hypersensitizable according to the invention are those 
genetically resistant to tetracycline, i.e., microorganisms carrying 
extrachromosomal tetracycline resistance determinants. Clinically, the 
infecting strain of Tc.sup.r microorganism is characteristically a strain 
of bacteria, especially Gram-negative bacteria. While microorganisms vary 
in their responses to a drug, an art-accepted definition of a tetracycline 
resistant microorganism is a microorganism for which the minimum 
inhibitory concentration of tetracycline is greater than about 12.5 ug/ml, 
especially greater than about 16 ug/ml Physicians' Desk Reference: 38th 
edition, Med. Econ. Co., Orodell, New Jersey--pub. (1984) pgs. 1058, 1527, 
1941]. Typically, resistant strains according to the invention are those 
wherein the MIC is greater than about 50 ug/ml. 
The inducers are clinically employed against Tc.sup.r microorganisms in 
amounts sufficient to provide the desired hypersensitivity to the 
therapeutic antibiotic. While, as previously noted, this hypersensitivity 
is associated with induction of TET-protein production, hypersensitivity 
may also be associated with induction of abnormal or modified TET-proteins 
which do not themselves function to mediate TET-resistance in Tc.sup.r 
microorganisms, but which do function to mediate hypersensitivity to the 
aminoglycoside antibiotic. The increase in aminoglycoside sensitivity will 
vary according to several factors, including the class of Tc.sup.r 
determinant present, the type of plasmid on which the determinant is 
located, and the particular microorganism expressing the TET gene. The 
correlation between in vivo and in vitro results is high, however, and 
optimum dosages for clinical use can be readily determined by preliminary 
treatment of the pathogen with inducer and antibiotic in vitro, as 
illustrated by the Examples set forth herein. In the practice of the 
invention, the inducer is further employed in an amount providing a 
subinhibitory concentration (clinically, a subinhibitory serum 
concentration) for the infecting resistant microorganism. For 
microorganisms characterized by a resistance to tetracycline serum 
concentrations of more than about 50 ug/ml, subinhibitory serum 
concentrations of less than about 50 ug/ml of antimicrobially active 
inducer are employable. Similarly, lower subinhibitory concentrations are 
employable according to the lower resistance characteristics of the 
infecting microorganism. 
The tetracycline employed is selected from a class of tetracycline-related 
compounds herein referred to as "inducers", defined as compounds 
containing a tetracycline nucleus which do not necessarily themselves 
exhibit antibiotic properties, but which are capable of inducing Tc.sup.r 
expression, or the production of TET-protein, in the Tc.sup.r 
microorganism. The class includes compounds characterized by a 
tetracycline nucleus (I) which is unsubstituted or substituted with one or 
more groups R.sub.1 -R.sub.11, which are selected with the proviso that 
the resulting compound functions to induce Tc.sup.r expression as 
described, and is non-toxic in clinical use. 
##STR1## 
Microbially active compounds of the formula (I) include those compounds 
wherein R.sub.5, R.sub.7, R.sub.8 and R.sub.9 are OH; R.sub.4 is 
N(CH.sub.3).sub.2 ; and R.sub.6 is CONH.sub.2 ; and especially such 
compounds wherein: 
R.sub.1 is H, Cl, or N(CH.sub.3).sub.2 ; 
R.sub.2 is CH.sub.3 OH, OH, CH.sub.2, CH.sub.3, or H; 
R.sub.3 is H or OH; and 
R.sub.10 and R.sub.11 are H. 
The class further includes non-microbially active compounds of the formula 
I, such as those wherein R.sub.1 -R.sub.5 and R.sub.7 -R.sub.11 are as 
defined above and R.sub.6 is COOH, many of which are excellent inducers. 
Compounds suitable for use in the process of the invention are further 
described in the literature, especially Antimicrobial Drug Resistance, 
"Resistance to the Tetracyclines" Academic Press, Inc. (1984), pp. 
191-240, incorporated herein by reference. Compounds of the formula (I) 
which are biologically modified, such as acid-inactivated tetracycline, 
are also useful in the practice of the present invention. Inducers 
comprising tetracycline, chlortetracycline, oxytetracycline, 
demeclocycline, methacycline, doxycycline and minocycline are particularly 
contemplated. 
As previously noted, microorganisms treatable according to the present 
invention typically have a tetracycline resistance of at least about 12.5 
ug/ml, and usually at least about 16 ug/ml, and for these organisms, 
concentrations of active inducers of below about 12.5 or 16 ug/ml, 
respectively, are thus subinhibitory. Generally, for most resistant 
microorganism strains within the scope of the invention, serum 
concentrations substantially below the minimum inhibitory concentration 
are employed. Subinhibitory inducer serum concentrations of less than 
about 4 ug/ml, typically, less than about 2 ug/ml, and more typically, 
less than about 1 ug/ml, are ordinarily sufficient to hypersensitize 
Tc.sup.r strains infecting mammals, especially humans, to the selected 
aminoglycoside antibiotic, and are preferred in practice. Since serum 
levels high enough to inhibit resistant strains are not clinically 
attempted in vivo, tetracycline or related active compounds are not 
presently employed alone or in combination with other drugs to treat 
clinically pathogens which are consecutively tetracycline-resistant, 
especially highly resistant strains of the type having a resistance of 
greater than about 50 ug/ml. In applications wherein non-microbially 
active inducers are employed (i.e., those which would not inhibit growth 
of the tetracycline-resistant microorganism in any clinically contemplated 
amount), the term "subinhibitory" is defined as a non-toxic amount 
sufficient to hypersensitize the microorganism. Typically, the amounts 
employed are comparable to the subinhibitory amounts of the 
microbially-active inducers employed. 
The expression of the TET gene induced by treatment of the resistant 
pathogen with the tetracycline or tetracycline related inducer according 
to the invention mediates the potency of aminoglycoside antibiotics which 
contain either streptamine or 2-deoxystreptamine in the structure thereof: 
##STR2## 
wherein: R.sub.1 is --H, --CH.sub.3, --CH.sub.2 CH.sub.3, or guanido group 
R.sub.2 is --H, or glycosidic bond 
R.sub.3 is --H or --OH 
Exemplary therapeutic antibiotics within this class include kanamycin, 
amikacin, gentamicin, tobramycin, netilmicin, sisomicin, streptomicin, 
neomycin, paromomycin, and apramycin. Kanamycin, amikacin, gentamicin, 
tobramycin and streptomicin appear to be of particular clinical and 
veterinary interest at the present time. Since hypersensitization 
according to the invention appears to render the Tc.sup.r cells 
consistently vulnerable to compounds containing streptamine (Sm) or 
2-deoxystreptamine (2-DOS) moieties, it is contemplated that clinical 
treatment of Tc.sup.r pathogens with subinhibitory amounts of inducer 
according to the invention will hypersensitize these pathogens to a 
variety of compounds containing these streptamine or 2-deoxystreptamine 
moieties. It is particularly contemplated that the efficacy of natural 
antibiotics and other drugs such as chloramphenicol, rifampicin, or 
ampicillin, which do not contain 2-DOS- or Sm- moieties, 
and to which Tc.sup.r microorganisms are not hypersensitizable according to 
the process of the invention, can be significantly improved by the 
expedient of attaching Sm- or 2-DOS moieties to the nuclei thereof in 
conventional manner, followed by hypersensitization with a tetracycline 
inducer according to the invention Derivitization of synthetic antibiotics 
and other drugs to provide one or more Sm- or 2-DOS- functional groups on 
the molecule for use in therapeutic treatment according to the invention 
is also possible, provided that the derivatives are not toxic to the host 
in the required therapeutic amounts. Modification of compounds which are 
too toxic for use per se with 2-DOS or Sm moieties for clinical use at 
decreased dosage levels is also within the scope of the invention. The 
antibiotics are administered as well-understood in the art, in therapeutic 
amounts sufficient to inhibit growth of the infecting mircroorganism. 
While the inducer and antibiotic may be administered together, sequential 
administration over a period of time sufficient to first permit inducement 
of Tc.sup.r expression is often preferable. 
The Examples which follow demonstrate that bacteria carrying 
tetracycline-resistant determinants are more susceptible to Sm- or 2-DOS- 
glycosides after induction with an inducer according to the invention, and 
that the in vitro results correlate with in vivo studies. It is noted that 
throughout the disclosure, including the following Examples, the 
expression "ug" means "micrograms".

EXAMPLES 
METHODOLOGY: 
The antibiotic concentration that reduces bacterial plating efficiency by 
90% (the LD.sub.90) in vitro was determined as follows: Aliquots of 
exponentially growing cultures of the specified bacteria containing 
2-5.times.10.sup.3 cells were spread on L-agar plates containing a range 
of concentrations of the indicated antibiotics and after incubation at 
37.degree. C. for 24-36 hours the number of colonies was determined. 
The antibiotic concentration that reduced bacterial survival by 90% in vivo 
was determined as follows: Virulent Tc.sup.s (tetracycline-sensitive) and 
Tc.sup.r K-1 E. coli strains were grown on blood agar plates at 37.degree. 
C. The bacteria were scraped from the plates and washed by centrifugation 
through buffered saline. Aliquots containing 3.times.10.sup.6 cells in one 
ml. of buffered saline were injected into the peritoneal cavities of 
groups of 3-5 male mice, using one group for each antibiotic concentration 
to be tested. Two hours later, the specified amounts of antibiotic in one 
ml. of buffered saline were injected into the peritoneal cavities. Seven 
hours after the injection of the bacteria, the animals were killed, the 
contents of the peritoneal cavities removed, and the number of surviving 
bacteria determined by plating on L-agar. 
EXAMPLES 
EXAMPLES 1-5 
The LD.sub.90 s on kanamycin of E. coli K-12 strain HB101 containing the 
inducible tet genes carried by plasmids pSC101, pCC42, (class C tet 
genes), plasmid pJOE105 (class A tet gene), transposon Tn10 (class B tet 
gene) and plasmid pSL101 (class D tet gene) were compared with and without 
induction of the tet genes by exposure to either 0.5-1.0 ug/ml of medium 
Tc or acid-inactivated Tc (ATC), an inducer that lacks antimicrobial 
activity. These strains are resistant to Tc serum concentrations of from 
about 40-250 ug/ml, depending on which tet gene they contain. Thus, the 
inducing concentration of Tc is far below that needed for antibacterial 
activity. The strains induced with Tc (or ATC) had LD.sub.90 values that 
were approximately 25%, 50%, 25%, 40%, and 15% lower, respectively, than 
paired controls that had not been induced with Tc or ATC (Both inducers 
have been used interchangeably with comparable results). 
EXAMPLES 6-9 
The LD.sub.90 on kanamycin of Tc.sup.r clinical isolates of E. coli, 
Proteus and Klebsiella was compared with and without induction with Tc at 
1 ug/ml. Each of these strains was resistant to at least 20 ug/ml Tc. 
Therefore the inducing Tc concentration is far below that which would have 
antibacterial activity against these strains. The LD.sub.90 of the E. coli 
strain, two Proteus strains and the Klebsiella strain which had been 
induced with 1 ug/m/ tc were approximately 20%, 25%, 25% and 20% lower 
than the LD.sub.90 of the same strains which had not been induced. 
Similarly, the LD.sub.90 on amikacin of 22 isolates of E. coli were 
compared with and without induction with Tc at 1 ug/ml. Thirteen out of 
twenty-two LD.sub.90 s of the induced strains were lower than the 
LD.sub.90 s of the non-induced strains. 
EXAMPLE 10 
The LD.sub.90 on amikacin of a Tc.sup.r clinical isolate of E. coli which 
was also resistant to kanamycin was compared on amikacin with and without 
induction with 1 ug/ml Tc. The LD.sub.90 of the induced sample was 
approximately 35% lower than the LD.sub.90 of the paired control which had 
not been induced with Tc. 
EXAMPLE 11 
The LD.sub.90 on kanamycin of the virulent Tc.sup.r K-1 E. coli strain 
LA396 containing plasmid pBR322 was compared to the LD.sub.90 of the 
plasmid-free Tc.sup.s control in vivo. The LD.sub.90 of the Tc.sup.r 
strain was approximately 50% lower than the LD.sub.90 of the Tc.sup.s 
control at a dosage level of 75 ug antibiotic per animal. 
Examples 1-5demonstrate that increased susceptibility of Tc.sup.r bacteria 
to aminoglycoside antibiotics is induced when cells containing a 
representative of each of the four classes of Tc resistance genes which 
have been described in Gram-negative bacteria are exposed to subinhibitory 
concentrations of a tetracycline inducer. Examples 6-10 demonstrate that 
increased susceptibility is similarly induced when clinically isolated 
pathogens are exposed to subinhibitory concentrations of tetracycline. 
Example 11 demonstrates that the increased susceptibility is expressed in 
vivo in a mouse model of peritonitis. It is thus apparent that the process 
according to the present invention increases aminoglycoside efficacy, with 
reduced risk of toxicity.