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Antimicrobial activity of linezolid combined with minocycline against
vancomycin-resistant enterococci
Key words: linezolid; minocycline; Enterococcus; mutation
Abstract
Background. Vancomycin-resistant enterococcus(VRE) cause serious infections that
are difficult to treat. We carried out this study to 1) determine the mutant prevention
concentration (MPC) of linezolid when combined with minocycline against VRE
strains, 2) determine the mechanism of drug resistance in vitro, and 3) to provide a
theoretical basis for the rational use of drugs against VRE.
Methods. To evaluate the effect of combination therapy on linezolid-resistant
Enterococcus, we first determined the minimum inhibitory concentrations (MICs) of
linezolid and minocycline against 30 Enterococci isolates (including 20 VRE strains)
by the broth microdilution method.Drug interactions were assessed by the
checkerboard microdilution tests and confirmed by time-kill studies. Two
vancomycin-susceptible strains N27 and N40 (linezolid MIC, 2 μg/ml; minocycline
MIC, 4 μg/ml) and control strains E. faecalis ATCC 29212 and ATCC 51299 were
also tested. The MPCs of linezolid and minocycline (alone and combined) were
determined using the agar dilution method. Strains showing stable resistance were
analyzed by PCR amplification of domain V of the 23S rRNA gene.
Results. Checkerboard titration studies revealed synergistic effects of combination
therapy in 26.7% of 30 Enterococci isolates.Antagonism was not observed.
The
G2576U mutation was detected in stable linezolid-resistant strains of ATCC 29212,
N40, and N27 before and after resistance screening, and MIC values increased with
the number of G2576U mutations. The MPC of linezolid against Enterococcus
decreased dramatically when combined with minocycline, and vice versa.
Conclusions. Linezolid or minocycline alone produce resistant strains; however, their
joint use may reduce the MPC of each agent against VRE, thereby decreasing
resistant mutants and bacterial infections. Otherwise,it was difficult to close MSW in
vivo with higher MIC values of minocycline.Additional pharmacokinetic and
pharmacodynamic data are necessary in order to provide more meaningful prediction
of the in vivo in clinical practice.
INTRODUCTION
The first infections due to vancomycin-resistant enterococcus (VRE) were reported in
1988 from Great Britain[1].Since then, VRE has evolved from causing rare, sporadic
cases of infection in severely immunocompromised patients to causing widespread
outbreaks in a variety of clinical settings. It showing resistance to glycopeptides have
now been reported from many parts of the world and show heterogeneity, both
phenotypic and genotypic [2].
Emerging risk factors for a massive bacterial colonization include the increased life
expectancy of the general population, extended survival of patients with
immunodeficiency and chronic disorders, and advances in surgical techniques,
invasive diagnostic and therapeutic procedures, and bone marrow and solid organ
transplantation. These factors also increase the risk of VRE infection. Despite new
antibiotic drugs and a better understanding of drug resistance transmission patterns,
these organisms continue to cause significant morbidity and mortality, especially in
the health care setting.
There are as many as 9 recognized vancomycin-resistance phenotypes—VanA, VanB,
VanC, VanD, VanE, and VanG VanL, VanM, and VanN[3,4,5,6,7].The formation of
peptidoglycan receptors with reduced glycopeptide affinity is the mechanism of
vancomycin resistance in enterococci.This results in decreased binding of
vancomycin and decreased inhibition of cell wall synthesis[8].
Linezolid is an antibiotic with potent in vitro and in vivo activity against VRE strains.
It is the first approved oxazolidinone to be marketed and represents an antibiotic
mechanism of action that is characteristic of this class of compounds. Antibiotic
resistance surveillance data demonstrate that more than 99% of VRE isolates are
susceptible to linezolid [9]. The first isolates of Enterococcus resistant to Linezolid
were reported in 2002 [10]. The most common mechanism involves mutations in
domain V of 23S rRNA with the G2576T substitution being the most frequently
reported[11,12].Resistance to this drug is achieved by a G2576U mutation in domain
V of the 23S rRNA gene . Nearly all bacteria have multiple copies of the 23S rRNA
gene, which was thought to decrease the likelihood of resistance to linezolid;
Enterococcus faecium has six 23S rRNA operons, and E. faecalis has four[13,14].
Resistance levels correlate directly with the percentage of 23S rRNA genes possessing
the
G2576U
mutation[15].In
addition,plasmid-mediated
resistance
to
linezolid(mediated by Cfr methyltransferase) in E. faecalis of animal and human
origin has been reported ,which suggests that further spread of this resistance in VRE
is likely[16,17].
The mutant selection window (MSW) hypothesis postulates that drug-resistant mutant
subpopulations present before antimicrobial treatment are enriched and amplified
during therapy when antimicrobial concentrations fall within a specific range. The
upper boundary of the MSW is the minimum inhibitory concentration (MIC) of the
least drug-susceptible mutant subpopulation, a value called the mutant prevention
concentration (MPC). Closing the MSW would help limit drug resistance. One
strategy to closing the MSW is to combine two antibacterial agents with different
mechanisms of action. When treated with combination therapy, bacteria require two
simultaneous drug resistance mutations to grow[18,19].According to the mutant
selection window (MSW) hypothesis, we hope to select a appropriate drug combined
with linezolid for reducing linezolid resistance mutation.
Minocycline is a semi-synthetic second generation tetracycline . It act by binding to
the bacterial 30S ribosomal subunit and inhibiting protein synthesis. Tetracycline
resistance is due to the acquisition of new genes and is due to energy-dependent efflux
of tetracycline and protection of the ribosomes[20].
There is no cross-resistance between linezolid and minocycline. Based on the results
of preliminary experiments, we found out that linezolid and minocyline presented
synergistic effect when used in combination. So we chose minocycline with linezolid
for the test.
The aims of this study were to 1) determine whether linezolid combined with
minocycline can reduce the MSW against VRE, 2) determine the mechanism of drug
resistance in vitro, and 3) provide a theoretical basis for the rational use of drugs
against VRE.
2. MATERIALS AND METHODS
2.1 Materials
Thirty Enterococci isolates (including 10 vancomycin- susceptible Enterococci, and
20 VRE strains) were obtained from the Clinical Laboratory of Beijing Friendship
Hospital from August 2009 to September 2010. 22 of 30 strains are E.faecium, while
the rest ones are E. faecalis.Control strains included E. faecalis ATCC 29212 and E.
faecalis ATCC 51299. Resistance to vancomycin was defined by a zone of
inhibition >16 mm, as assessed by the Kirby Bauer method, and MIC >8 μg/ml, as
assessed by the microdilution method [21].
Linezolid and minocycline were obtained from the National Institutes for Food and
Drug Control. Brain-heart infusion and Mueller-Hinton agar were obtained from
Becton Dickinson and Co. (Cockeysville, MD). The restriction enzyme XspI was
obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Primer synthesis and
DNA sequencing was carried out by Invitrogen Corporation (Shanghai, China).
2.2 Minimum inhibitory concentrations of linezolid and minocycline
Determine the MICs of linezolid and minocycline against 30 Enterococci isolates
(including 20 VRE strains) by Broth microdilution according to Clinical and
Laboratory Standards Institute guidelines[21]. N27and N40 were isolated from two
different clinical blood samples which were resistance to macrolides, penicillins,
cephalosporins and glycopeptides.Both of them are E.faecium.
2.3 Checkerboard studies
Drug interactions were determined by using the broth microdilution checkerboard
method. Interactions were classified by fractional inhibitory concentration index(FICI)
The FICI was calculated for each combination using the following formula: FICA +
FICB = FICI, where FICA = MIC of drug A in combination/MIC of drug A alone, and
FICB = MIC of drug B in combination/MIC of drug B alone. The FICI was
interpreted as follows: synergy = FICI ≤0.5; indifference = 0.5<FICI ≤4; antagonism
= FICI > 4[22].All synergistic interactions were repeated to verify.
2.4 Time-kill studies
Time-kill studies were performed with selected antimicrobial combinations based on
the checkerboard test results .Synergy was defined as a ≥2-log10-unit decrease in
colony count after a 24-h incubation compared with the colony count after treatment
with the most active single antimicrobial.The drug combination was considered to be
antagonistic for = 2 log10 increase in CFU/ml and indifferent for < 2 log10 change in
CFU/ml[22].Only antibiotic combinations showing synergism or antagonism in both
chequerboard and time-kill assays were accepted as authentic synergistic or
antagonistic interactions,respectively.
2.5 Determination of the mutant prevention concentration
Single colonies of the isolates were inoculated on M-H agar plates and incubated for
24 hours at 35°C–37°C in O2. All bacterial growth was harvested from the plates,
added to 500 mL Mueller–Hinton broth, and incubated for an additional 24 h. The
resulting suspension was concentrated by centrifugation (5000×g for 30 min, 4°C).
Then the supernatant was discarded, and the cells were resuspended in
Mueller–Hinton broth to yield a concentration of 1010 CFU/mL. Finally, 100 μL of
this suspension was spread on 10 Mueller–Hinton agar plates containing antibiotics
and incubated for 96 h in O2. A series of two-fold dilutions of each antimicrobial
(alone or in combination) was tested. The lowest concentration that completely
inhibited colony formation was called the MPCpr. The drug concentration was then
lowered step-wise (20% each step) and retested. The lowest concentration that
completely inhibited colony formation was the MPC[23,24]. All MPC experiments
were performed in duplicate. MIC testing of colonies recovered from plates
containing growth was used to confirm the presence of resistant populations.
2.6 Polymerase chain reaction analysis of the 23S rRNA gene
Linezolid-susceptible and linezolid-resistant enterococci were then analyzed by
polymerase chain reaction (PCR). The cell lysate was extracted twice with
phenol-chloroform-isoamyl alcohol (24:25:1), and genomic DNA was precipitated
with ethanol. PCR amplification was carried out using Taq DNA polymerase and
primers
based
on
Enterococcus
23S
rRNA
gene
(5’-GCAGAAGGGAGCTTGACTGCGAG-3’and5’-ACCCAGCAATGCCCTTGGC
AG-3’). PCR was carried out under the following conditions: 95°C for 5 min,
followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min[13,25].
The PCR products were cloned and sequenced by Invitrogen Corporation (Shanghai,
China). Results were compared with GenBank sequences to further analyze the
mutation.
2.7 Determination of the number of mutation G2576U included in the 23rRNA
gene of resistant strains.
On the occurrence of the mutation G→U, a new restriction site XpnI was produced.
So the mutated copies produced two fragments, while wild-type strain could not be
cut. PCR product was purified using a PCR product purification kit (Qiagen, Valencia,
CA) and recovered in 40μL H2O. 5ul DNA was digested at 37°C for 2 h using
units of
10
XspI restriction enzyme(Fermentas, Hanover, MD) under conditions
recommended by the enzyme manufacturer. The purified PCR product (5μL) and
products of the restriction digest (5μL) were analyzed on a 1% (w/v) agarose gel. In
this study, we set the optical density of the 5μL PCR product (un-cut, 389-bp) as
100%. When the mutation caused restriction occurred, the 389-bp fragment could be
cut into two small fragments, 145-bp and 244-bp[13].And the remained optical
density of the 389-bp fragment was declined. What’s more, the degree of the decrease
was positive correlated with the amount of the mutation. Taken Enterococcus faceium
for example, which has 6 sites could mutate in domain V of the 23S rRNA. If all the 6
sites mutated simultaneously, the remained 389-bp was not exist any more after
restriction digestion, that’ to say, optical density decreased by 100%. So the ratio of
the 389-bp optical density was 100% compared with the optical density of original
PCR product. And if only 2 sites mutated simultaneously, 1/3 (2 sites / 6 sites) amount
of the original PCR product could be cut into two fragments, the rest 2/3 product were
present as 389-bp fragment. So the ratio of the optical density was about 33%, and so
on (as listed in Table 5). The optical density was scanned by the optical density
analysis software of BIO-RAD gel imaging system, and the ratio of the optical density
of 389-bp fragment was calculated automatically.
3 RESULTS
3.1 Susceptibility testing
The MIC values of the 30 enterococci isolates are shown in TABLE 1.The MIC of
ATCC29212,N27(VRE) and N40(VRE) of linazolid and
4μg/ml；2μg/ml、4μg/ml；2μg/ml、0.5μg/ml.
3.2 Checkerboard and time-kill assay results
minocycline is 2μg/ml、
In the checkerboard titration studies, the combination therapy showed synergy in 26.7%
strains of all the 30 Enterococci, while the rest ones presented indifference.
Antagonism was not observed. In this study,results of synergy or indifference are not
correlated to the susceptibility of the strains to minocycline.All 8 isolates in which the
drugs showed synergy and two isolates in which the drugs showed indifference were
randomly selected for time-kill studies, which confirmed the results of the
checkerboard tests. One of the effective combinations in Time-kill studies are shown
in FIGURE 1.
3.3 Mutant prevention concentration determination
MPC values for the individual drugs (linezolid and minocycline) are shown in
TABLE 2. In combination therapy with linezolid and minocycline, we found that
when the concentration of minocycline reached one-half of its MIC, the MPC of
linezolid was not affected. However, the MPC of linezolid was significantly reduced
for ATCC 29212 and N40 when the concentration of minocycline reached its MIC,
and was significantly reduced for N27 when the concentration of minocycline reached
2×MIC (Table 3). The MSW of linezolid was closed for ATCC 29212 when the
concentration of minocycline reached 4×MIC (i.e., the MPC for linezolid was equal to
its MIC), and the MSW for N27 and N40 was closed when the concentration of
minocycline reached 8×MIC.
3.4 MICs of linezolid-resistant mutants
We randomly selected 20 linezolid-resistant VRE strains from plates with 1MIC of
linezolid when MPC were detected alone and combination respectively to retest the
MIC. The average MIC of linezolid in strains treated with the drug combination was
4.2 μg/ml, MIC50 was 4 μg/ml, and MIC90 was 7 μg/ml, which was lower than of
strains treated with a single drug (MIC50, 4 μg/ml, MIC90, 8 μg/ml).
3.5 Mutation frequency:
Mutation frequency is the ratio of the resistant colonies relative to the total number of
bacteria. We found that the mutation frequency decreased considerably with
combination treatment compared with treatment with a single drug (TABLE 4).
3.6 Ratio of mutated G2576U in the 23 rRNA of resistant strains.
N27, N40 and ATCC 29212 and linezolid-resistant VRE strains selected from the last
test were analyzed by amplifying the 389-bp product in domain V of the 23S rRNA
gene. The amplification product was compared with the 23s rRNA gene of E. faecium
ATCC 27273 by using NCBI Blast. Mutations were not detected in N27, N40, and
ATCC 29212 (only one 389-bp fragment was observed after digestion with restriction
enzyme XspI). However, the G2576U mutation in domain V of the 23S rRNA gene
was detected in linezolid-resistant VRE strains isolated from antibiotic-containing
plates, but the mutation did not affect all copies of the genes (389-bp, 244-bp, and
145-bp fragments were observed in FIGURE 2). The purified PCR product was in line
1 while products after restricted digestion were in line2-9.Other mutations in this
region have not been detected. Mutation ratio after treatment with linezolid alone or
combined treatment with linezolid and minocyline was listed in TABLE5.
4 DISCUSSION
Linezolid represents the first member of a novel class of oxazolidinone derivatives,
which are effective against the most important Gram-positive organisms.
Oxazolidinones inhibit initiation of the synthesis of bacterial proteins by preventing
the formation of the ternary complex at the 70S ribosomal subunit[13].
Pharmacokinetic parameters for 600 mg oral doses of linezolid after multiple dosing
show Cmax values of approximately 21 μg/mL one hour after administration and a
half-life of 5.4 hours[26].At almost all doses, the in vivo concentration is higher than
the MIC for Enterococcus but below the MPC, which may explain why resistant
strains were produced and treatment ultimately failed after long-term therapy.
Minocycline is a broad-spectrum tetracycline antibiotic that inhibits protein synthesis
by targeting the 50S ribosome and is effective against Enterococcus. The
pharmacokinetics of minocycline are similar to that of linezolid, and there is no
cross-resistance. Pharmacokinetic parameters for 200 mg oral doses of minocycline
after multiple dosing show Cmax values of approximately 3-3.6 μg/mL 2-3 hour after
administration and a half-life of 12-18hours[27].
The results of this study show that rate of linezolid sensitivity for Enterococcus is
100%, which may be because it is not widely used in China. However, half of the
strains had resistance breakpoints near their MIC values. The combination of linezolid
and minocycline was synergistic for some strains, but showed additivity or
indifference in most strains.
Clinicians have a responsibility to prevent the selective growth of resistant mutants.
Pharmacokinetic/pharmacodynamic vitro model experiments show that when the
antibiotic concentration is maintained in the vicinity of the MIC, resistance occurs
more readily [19]. Thus antimicrobial drug selection and dosing regimens should be
based on the drug’s PK/PD parameters. In addition, understanding the mechanism of
resistance and the MPC is needed to combat the growing problem of bacterial
resistance.
Resistance to linezolid can be reduced by combining it with other antibiotics.
Resistant strains obtained in this test is much different from that of other
methods,such as resistant strains obtained from induction. Linezolid-resistant strains
obtained in this test is much different from that of other methods,such as resistant
strains obtained from induction.Many in vitro pharmacodynamic and animal
experiments have demonstrated that drug combination can decrease MPC values and
reduce the enrichment of drug-resistant mutant strains. Few studies have reported the
MPC and MSW of linezolid and the effect of combination treatment with minocycline
against VRE.
Our results show that the combination of linezolid and minocycline appears promising.
Minocycline increased the effects of linezolid against both susceptible and resistant
Enterococcus. The combination of linezolid plus minocycline significantly reduced
linezolid MPC for Enterococcus and reduced the generation of linezolid-resistant
strains. The data confirm that linezolid resistance was achieved by the G2576U
mutation in domain V of the 23S rRNA gene, and the level of resistance positively
correlated with the number of mutant alleles. Linezolid combined with minocycline
significantly reduced the number of mutations produced.
It is important to note that the PK/PD of minocycline in vivo is not entirely as same as
linezolid. In addition the Cmax values of minocycline are lower than the levels
required to be used in combination. The test results showed that, it was difficult to
close MSW drug concentration in vitro for Enterococci those with higher MIC values
of minocycline.So it will be necessary to combine in vitro findings with additional
pharmacokinetic and pharmacodynamic data in order to provide more meaningful
prediction of the in vivo efficacy of synergistic combinations in clinical
practice.Further studies are still needed to explore the molecular mechanisms
responsible for synergistic interactions of linezolid combined with minocycline to tap
their therapeutic potential.
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TABLE
TABLE 1. Minimum inhibitory concentrations of vancomycin-resistant enteroccocus study
isolates
Drug
S
I
R
Linezolid
100%
0
0
Minocycline
46.7%
6.7%
46.7%
Range (μg/ml)
MIC50 (μg/ml)
MIC90 (μg/ml)
0.25-2
2
2
0.25-32
8
16
NOTE:S=susceptible,I=intermediate,R=resistant.
Linezolid
breakpoints
:(S≤2μg/ml,I=4μg/ml,R≥8μg/ml);Minocycline
breakpoints
(S≤4μg/ml,I=8μg/ml,R≥16μg/ml); vancomycin breakpoints :(S≤4μg/ml,I=8-16μg/ml,R≥32μg/ml);
TABLE 2 MPC values of linezolid and minocycline.
ATCC 29212
N27
N40
MPC for linezolid (μg/ml)
7.2
8
10.2
MPC for minocycline (μg/ml)
51.2
57.6
14.4
TABLE 3 MPC linezolid for combination with minocycline.
ATCC 29212
N27
N40
MNO
LNZ-MPC
MNO
LNZ-MPC
MNO
LNZ-MPC
(μg/ml)
(μg/ml)
(μg/ml)
(μg/ml)
(μg/ml)
(μg/ml)
2
7.2
2
8
0.25
10.2
4
4
4
8
0.5
8
8
4
8
4
1
4
16
2
16
4
2
4
32
2
32
2
4
2
51.2
2
51.2
2
8
2
—
—
—
—
14.4
2
NOTE: MNO=minocycline, LNZ=linezolid
TABLE 4 Mutation frequencies
Mutation frequency with combination treatment
Mutation frequency
ATCC 29212
with a single drug
1/2MIC
1MIC
2MIC
1/2MIC
1MIC
2MIC
1/2MIC
1MIC
2MIC
MNO
MNO
MNO
MNO
MNO
MNO
MNO
MNO
MNO
8.7×10-8
2.5×10-8
6.8×10
9.2×10-8
3.1×10-8
7.3×10-9
4.4×10-8
1.8×10-8
3.5×10-9
3.5×10-8
6.9×10-9
5.0×10-8
6.6×10-9
2.6×10-9
4.3×10-9
1.6×10-9
1MIC
3.9×10-7
LNZ
~6.1×10-7
2MIC
1.5×10-7
LNZ
~8.5×10-8
N27
N40
-9
2.1×10-8
6.8×10-9
—
—
4MIC
LNZ
—
—
—
—
—
NOTE: MNO=minocycline，LNZ=linezolid
—
—
—
TABLE 5 Mutation ratios after treatment with linezolid alone or combination treatment.
Linezolid alone
Linezolid plus minocycline
Average
Ratio of
G2576U
Average
Ratio of
G2576U
MIC
optical
mutations /
MIC
optical
mutations /
(μg/ml)
density
total gene
(μg/ml)
density (%)
total gene
(%)*
copies
copies
ATCC 29212
4
48.0
2/4
3
25
1/4
N40
4
35.2
2/6
2.7
17.9
1/6
N27
4
31.6
2/6
2.1
16.7
1/6
NOTE:* the ratio of optical density = the optical density of 389-bp after restriction
digestion compared with the optical density of original PCR product.
FIGURE 1
Time-kill kinetics for confirmed synergistic interactions.
FIGURE 2
Line 1: cloned amplification products without digestion
Line2-9:XspI digestion of cloned amplification products