Carbapenems and Rifampin Exhibit Synergy against Mycobacterium

tuberculosis and Mycobacterium abscessus

Amit Kaushik,a Nayani Makkar,b Pooja Pandey,b Nicole Parrish,c Urvashi Singh,b Gyanu Lamichhanea
Taskforce to Study Resistance Emergence & Antimicrobial Development Technology, Department of Medicine, Johns Hopkins University, Baltimore, Maryland, USAa;
Department of Microbiology, All India Institute of Medical Sciences, New Delhi, Indiab; Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USAc

An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular
activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with syn-
ergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the
␤-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when
combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the
therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be ad-
ministered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of
resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of
rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-
resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also
exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. absces-
sus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they war-
rant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients.

E xisting treatment of tuberculosis (TB) requires multiple anti-

biotics to be taken daily for at least 6 months. Historically,
␤-lactam antibiotics have not been considered for treatment of
treatment, exhibit any synergy, indifference, or antagonism in ac-
tivity. We next compared frequencies of spontaneous resistant
mutants when M. tuberculosis is exposed to either rifampin or
TB, except for treatment of drug-resistant tuberculosis when faropenem or a combination of these two drugs. Finally, we stud-
available options are exhausted. Reasons that are often cited for ied antimicrobial activities of combinations containing rifampin
lack of activity of ␤-lactams against Mycobacterium tuberculosis and (carba)penems against drug-resistant clinical M. tuberculosis
include the chromosomally encoded extended-spectrum class A isolates. We also evaluated the abovementioned (carba)penems
␤-lactamase BlaC and poor permeability across the cell wall (1, 2). alone and in combination with rifampin for their antimicrobial
Clavulanic acid, by binding to and inhibiting the ␤-lactamase ac- activities against M. abscessus. Although the MIC of rifampin for
tivity of BlaC, has been shown to render M. tuberculosis susceptible M. abscessus is too high, we assessed if (carba)penems and rifam-
to ␤-lactams (3). Carbapenems, a subclass of ␤-lactams with a pin exhibited synergy and activity at levels that are therapeutically
five-membered ring next to the ␤-lactam core, are similar to pen- relevant.
icillins but with a carbon in the first position and a double bond
between C-2 and C-3 (4). They are poor substrates for BlaC and MATERIALS AND METHODS
are not affected by its ␤-lactamase activity (5). Recent reports have Bacterial strains and in vitro growth conditions. Drug-susceptible M.
demonstrated that carbapenems exhibit high antimicrobial po- tuberculosis H37Rv and drug-resistant M. tuberculosis clinical isolates
tency against M. tuberculosis; therefore, they may be considered (with known drug susceptibility profiles and multidrug-resistant [MDR]
for treatment of tuberculosis (5–9). strains defined as resistant to both rifampin and isoniazid), archived at the
Mycobacterium abscessus is a nontuberculous mycobacteria All India Institute of Medical Sciences, New Delhi, India, were used. The
that causes morbid infection in the lungs of a significant percent- clinical strains were obtained per institutional ethical guidelines and were
age of cystic fibrosis patients (10). In a recent study, imipenem,
meropenem, ertapenem, and doripenem (all belonging to an
older generation of carbapenems and none of which are orally Received 15 May 2015 Returned for modification 22 June 2015
Accepted 1 August 2015
bioavailable) were evaluated against M. abscessus, and it was de-
Accepted manuscript posted online 10 August 2015
termined that while imipenem, meropenem, and doripenem have
Citation Kaushik A, Makkar N, Pandey P, Parrish N, Singh U, Lamichhane G. 2015.
modest activities, other classes of ␤-lactams (penicillins and ceph- Carbapenems and rifampin exhibit synergy against Mycobacterium tuberculosis
alosporins except cefoxitin) are ineffective against this organism and Mycobacterium abscessus. Antimicrob Agents Chemother 59:6561–6567.
(11). doi:10.1128/AAC.01158-15.
In this study, we began by assessing potencies of the carbapen- Address correspondence to Gyanu Lamichhane, lamichhane@jhu.edu.
ems ertapenem, meropenem, imipenem, doripenem, biapenem, Supplemental material for this article may be found at http://dx.doi.org/10.1128
tebipenem, and panipenem and the penem faropenem against M. /AAC.01158-15.
tuberculosis by determining MICs and minimum bactericidal con- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
centrations (MBCs). We investigated if (carba)penems and isoni- doi:10.1128/AAC.01158-15
azid or rifampin, the two drugs that form the backbone of TB

October 2015 Volume 59 Number 10 Antimicrobial Agents and Chemotherapy aac.asm.org 6561
Kaushik et al.

deidentified. Strains were grown under standard conditions in Middle- index of ⱕ0.5 was interpreted as synergy, ⬎0.5 to 2.0 as indifference, and
brook 7H9 broth (Difco) supplemented with 0.5% glycerol, 10% oleic ⬎2 as antagonism.
acid-albumin-dextrose-catalase (OADC), and 0.05% Tween 80, with con- Time-kill assay. M. abscessus ATCC 19977 was grown in Middlebrook
stant shaking at 37°C or on Middlebrook 7H10 agar plates at 37°C. M. 7H9 broth to exponential phase, and a suspension at an A600 of 0.01 was
abscessus ATCC 19977 was grown in Middlebrook 7H9 broth with albu- prepared by serial dilution in the broth. Two hundred microliters of this
min-dextrose-catalase enrichment with constant shaking at 37°C or on suspension or approximately 105 CFU was inoculated into six tubes each
Middlebrook 7H10 agar plates at 37°C. All drugs were obtained from the with 5 ml of 7H9 broth. These samples contained the following drugs at
following commercial vendors: ertapenem (Toronto Research Chemi- subinhibitory concentrations (sub-MICs): rifampin (25 ␮g/ml), dorip-
cals); rifampin, isoniazid, meropenem, imipenem, doripenem, biapenem, enem (10 ␮g/ml), biapenem (10 ␮g/ml), rifampin and doripenem (25
faropenem, and tebipenem (Sigma-Aldrich); and panipenem (Ochem, ␮g/ml and 10 ␮g/ml, respectively), rifampin and biapenem (25 ␮g/ml and
Inc.). To assess the quality of these compounds, a few were randomly 10 ␮g/ml, respectively), and no drug as a positive control for M. abscessus
selected and assessed by liquid chromatography-mass spectrometry. The growth. The samples were cultured in a shaking incubator at 37°C. A
purity of compounds ranged from 95% to 99%. 100-␮l aliquot was removed from each sample at 0, 3, 6, 12, 24, 48, 72, 96,
MIC. MICs were determined using standard broth dilution methods 144, and 192 h. Following the initiation of this assay, appropriate serial
dilutions were plated onto Middlebrook 7H10 agar media, and CFU were
(12, 13). Briefly, 105 M. tuberculosis or M. abscessus bacilli grown to expo-
enumerated after 3 days of incubation at 37°C.
nential phase were inoculated into 15-ml sterile conical tubes containing
Determination of frequency of drug resistance emergence. M. tuber-
2.5 ml of 7H9 broth containing drug at 2-fold dilutions ranging from 80
culosis H37Rv was grown in Middlebrook 7H9 broth to exponential
␮g/ml to 0.16 ␮g/ml. Middlebrook 7H9 broth alone and without drug but
phase, a suspension with an A600 of 1.0 was prepared, and 1.0 ml of this
inoculated with 105 M. tuberculosis or M. abscessus bacilli were included as
suspension was inoculated onto each plate. The Middlebrook 7H10 agar
negative and positive controls, respectively. Rifampin and isoniazid were plates were supplemented with a rifampin/faropenem combination at
used as control drugs. Cultures were incubated at 37°C and evaluated for 3.0/0, 6.0/0, 3.0/50, 0.6/5.0, 0.6/0, 0/5.0, and 0/0 ␮g/ml, respectively, to
growth by visual inspection at 14 days for M. tuberculosis and at 3 days for select resistant mutants that were enumerated by counting CFU on plates
M. abscessus at 30°C per Clinical and Laboratory Standards Institute after 4 weeks of incubation at 37°C. The frequency of drug-resistant mu-
guidelines (14). Samples with no visible granulations were further as- tants was determined from the numbers of spontaneous mutants ob-
sessed by plating on Middlebrook 7H10 plates to verify the lack of bacte- served as a percentage of the input CFU inoculum.
rial growth. In this method, a single dose of drug is added at the beginning
of the assay. We also assessed the MIC by providing two doses of drug, one
RESULTS
at the beginning of the assay and one at the midpoint of the assay. Two sets
of tubes were used for each drug. In one set, drug was added from 80 MICs of (carba)penems against M. tuberculosis and M. absces-
␮g/ml to 0.16 ␮g/ml by 2-fold serial dilution. In the next set, half the sus. We evaluated the antimicrobial activity of (carba)penems
amount of drug was added in each tube. Therefore, this set started with 40 (i.e., ertapenem, meropenem, imipenem, doripenem, biapenem,
␮g/ml to 0.08 ␮g/ml drug. At the end of 1 week (midpoint of the assay), faropenem, tebipenem, and panipenem), alone and in combina-
the remaining half dose was added with a fresh preparation of the drug. tion with clavulanic acid, against M. tuberculosis and M. abscessus
We also assessed MIC90 using the broth microdilution method using 96- using broth dilution assay. For drug-susceptible wild-type M. tu-
well plates per Clinical and Laboratory Standards Institute recommenda- berculosis strain H37Rv, the potencies of these (carba)penems are
tions (14). Cation-adjusted Mueller-Hinton broth (Becton-Dickinson) as follows: tebipenem ⬎ faropenem ⫽ biapenem ⫽ doripenem ⬎
was used to assess antimicrobial activities of rifampin and (carba)penems meropenem ⬎ ertapenem ⬎ imipenem ⬎ panipenem, with tebi-
against M. abscessus strains. All MIC assessments were repeated to verify penem having MIC90 of 1.25 to 2.5, faropenem, biapenem, and
results.
doripenem having MIC90 of 2.5 to 5.0 ␮g/ml, and panipenem
MBC. Minimum bactericidal concentration (MBC) was determined
having a MIC90 of ⬎80 ␮g/ml (Table 1).
using a modification of the broth dilution method described above. For
M. tuberculosis, samples with no visible growth after 14 days of incubation
Clavulanic acid is a ␤-lactamase inhibitor. As M. tuberculosis
were plated on Middlebrook 7H10 agar plates, and CFU were enumerated and M. abscessus possess a chromosomally encoded ␤-lactamase
after incubation at 37°C for 21 days. Isoniazid was used as a positive (1, 2), we hypothesized that the activity of (carba)penems changes
control for bactericidal activity. The input inoculum is the CFU added to if supplemented with clavulanic acid; hence, we assessed MICs of
each well at the initiation of broth dilution assay and was determined by (carba)penems in combination with clavulanic acid. The addition
plating samples at the onset of the assay on Middlebrook 7H10 agar plates. of clavulanic acid did not alter the MIC90 of faropenem, while it
MBC99 refers to the concentration of the drug at which 99% of input reduced the MIC90 of biapenem and tebipenem by 4-fold. A 2-fold
bacilli were killed. reduction in MIC90 was observed for ertapenem, meropenem,
Checkerboard titration assay. The checkerboard titration assay is a imipenem, and doripenem (Table 1).
modification of the broth dilution assay and was undertaken as described (Carba)penems are known to undergo slow hydrolysis in aque-
previously (15). Two drugs were added to 2.5 ml of Middlebrook 7H9 ous solution as a result of the ␤-lactam ring opening mediated by
broth, each starting at its MIC and serially diluted 2-fold, so all possible nucleophilic attack on C-7 by oxygen from water (16). Due to this
2-fold dilution combinations below the respective MICs are represented. instability in an aqueous environment, the effective exposure of
After this step, 105 CFU of M. tuberculosis or M. abscessus was inoculated
(carba)penems to pathogen may change over time. Therefore, we
into each tube. The cultures were incubated at 37°C and evaluated for
assessed if dividing the drug into two dosages during the course of
growth by visual inspection of the broth at 14 days for M. tuberculosis and
at 3 days for M. abscessus. The fractional inhibitory concentration (FIC) of broth dilution assay affects its MIC. For this, we performed two
each drug in the combination was determined as described previously MIC assessments in parallel: in one we provided the drug in a
(15). Briefly, the FIC of a drug in a sample is computed as the concentra- single full dose at the onset of the assay, and in the second we
tion of the drug divided by the MIC of the drug when used alone. The FIC provided half the dose at the onset and the remaining half at the
index is the sum of the FIC of two drugs in a sample. The FIC index was midpoint of the assay. For instance, in the control set, 200 ␮g of
calculated for each mix of drugs that inhibited M. tuberculosis or M. ab- meropenem was added to a 2.5-ml final volume in the first tube
scessus growth, and an average FIC index was determined. An average FIC and assayed for 2 weeks. In the test set, 100 ␮g of meropenem was

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 Carbapenems and Rifampin Synergize

TABLE 1 Antimicrobial activity of (carba)penems against M. TABLE 3 Drug susceptibility of selected MDR M. abscessus clinical
tuberculosis H37Rva isolates
MIC90 (␮g/ml) Level of susceptibility toa:
MIC90 on double MBC99
Strain AMI CEF CIP CLR LZD MIN TOB TIG
Drug(s) (␮g/ml) dosingb (␮g/ml)
c ABS 1 I R R R R R R S
Ertapenem 10–20 10–20 ND
ABS 2 R R R R R R R R
Ertapenem ⫹ clavulanic acid 5–10 ND ND
ABS 3 R R R I R R R R
Meropenem 5–10 5–10 80
a
Meropenem ⫹ clavulanic acid 2.5–5.0 ND ND AMI, amikacin; CEF, cefoxitin; CIP, ciprofloxacin; CLR, clarithromycin; LZD,
linezolid; MIN, minocycline; TOB, tobramycin; and TIG, tigecycline. ABS1, M.
Imipenem 40–80 40–80 ND
abscessus isolate 1; ABS2, M. abscessus isolate 2; ABS3, M. abscessus isolate 3. R, resistant;
Imipenem ⫹ clavulanic acid 20–40 ND ND
I, intermediate; S, susceptible (per Clinical and Laboratory Standards Institute cutoffs).
Doripenem 2.5–5.0 1.25–2.5 20
Doripenem ⫹ clavulanic acid 1.25–2.5 ND ND
Biapenem 2.5–5.0 1.25–2.5 20
Biapenem ⫹ clavulanic acid 0.6–1.2 ND ND We next assessed the activity of the (carba)penems alone and in
Faropenem 2.5–5.0 2.5–5.0 20 combination with clavulanic acid against M. abscessus ATCC
Faropenem ⫹ clavulanic acid 2.5–5.0 ND ND 19977, a drug-susceptible strain (Table 2). Only doripenem and
Tebipenem 1.25–2.5 1.25–2.5 10 biapenem exhibited activity with a MIC90 in the range of 10 to 20
Tebipenem ⫹ clavulanic acid 0.31–0.62 ND ND ␮g/ml. The addition of clavulanic acid dramatically reduced the
Panipenem ⬎80 ND ND
MIC90 of meropenem: when used alone, this drug is not effective
Panipenem ⫹ clavulanic acid ND ND ND
up to 80 ␮g/ml, but in combination with clavulanic acid its MIC90
Clavulanic acid ⬎80 ND ND
a
diminished to 5 to 10 ␮g/ml, a decrease of ⬎16-fold. A 2-fold
(Carba)penems were tested alone and in combination with clavulanic acid, a
␤-lactamase inhibitor.
reduction in MIC90 was observed for doripenem, biapenem, faro-
b
Double dosing refers to providing half the dose at the onset and the remaining half at penem, and tebipenem, while the activities for ertapenem, imi-
the midpoint of the assay. penem, and panipenem remained unchanged in the presence of
c
ND, not determined. clavulanic acid. Encouraged by these results, we determined MICs
of carbapenems against three M. abscessus clinical isolates that are
resistant to a range of drugs used to treat M. abscessus infection
added at the onset of the assay, and an additional 100 ␮g was (Table 3). These strains were susceptible only to doripenem and
added at the beginning of the second week. If a drug is stable and biapenem, with a MIC90 in the range of 3.12 to 12.5 ␮g/ml (Table
does not undergo hydrolysis in the culture broth, the first set 4). The maximum concentration used in this assessment was 25
would provide a superior activity, as M. tuberculosis bacilli would ␮g/ml. Therefore, a MIC90 of ⬎25 ␮g/ml implies that the actual
be exposed to a greater dosage over 2 weeks. If the drug is unstable MIC90 is much higher, as full growth was observed in these wells.
over time, the addition of a fresh dose at the midpoint in the assay MBC. We evaluated if (carba)penems exhibited bactericidal
may result in superior activity in the second set. A twofold de- activity against M. tuberculosis. For this, we selected meropenem,
crease in MIC90 was observed for doripenem and biapenem upon doripenem, biapenem, faropenem, and tebipenem based on their
the addition of drug in two doses (Table 1). low MIC90 and determined MBC99 to be 80, 20, 20, 20, and 10
␮g/ml, respectively (Table 1).
Doripenem, biapenem, faropenem, and meropenem exhibit
TABLE 2 Antimicrobial activity of (carba)penems against M. abscessus synergy with rifampin against M. tuberculosis and M. abscessus.
ATCC 19977 We evaluated the potency of a combination of ertapenem, mero-
MIC90 for M. abscessus penem, imipenem, doripenem, biapenem, faropenem, and tebi-
Drug (␮g/ml) penem with rifampin or isoniazid against M. tuberculosis using
Ertapenem ⬎80 checkerboard titration assay (15). The fractional inhibitory con-
Ertapenem ⫹ clavulanic acid ⬎80 centration (FIC) index of isoniazid and any of the (carba)penems
Meropenem ⬎80 was in the 0.5 to 2.0 range, demonstrating that the activities of the
Meropenem ⫹ clavulanic acid 5–10 two drugs were indifferent/additive to each other. The assessment
Imipenem ⬎80 of activities of a combination containing two (carba)penems (all
Imipenem ⫹ clavulanic acid ⬎80 three possible pairs among biapenem, faropenem, and tebi-
Doripenem 10–20 penem) revealed FIC indexes in the range of 0.5 to 2.0, thereby
Doripenem ⫹ clavulanic acid 5–10
Biapenem 10–20
Biapenem ⫹ clavulanic acid 5–10
Faropenem 40–80 TABLE 4 Activity of carbapenems against MDR M. abscessus clinical
Faropenem ⫹ clavulanic acid 20–40 isolatesa
Tebipenem 40–80 MIC90 (␮g/ml)
Tebipenem ⫹ clavulanic acid 20–40
Strain Meropenem Imipenem Doripenem Biapenem Ertapenem
Panipenem ⬎80
Panipenem ⫹ clavulanic acid ⬎80 ABS 1 ⬎25 ⬎25 6.25 12.5 ⬎25
Clavulanic acid ⬎80 ABS 2 ⬎25 ⬎25 6.25 12.5 ⬎25
a
(Carba)penems were tested alone and in combination with 5 ␮g/ml clavulanic acid, a ABS 3 ⬎25 ⬎25 3.12 6.25 ⬎25
␤-lactamase inhibitor. a
ABS1, M. abscessus isolate 1; ABS2, M. abscessus isolate 2; ABS3, M. abscessus isolate 3.

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Kaushik et al.

FIG 1 Time-kill analysis of M. abscessus ATCC 19977 in Middlebrook 7H9 broth at 37°C when exposed to sub-MICs of drugs. The samples contained 25 ␮g/ml
rifampin (Rif), 10 ␮g/ml doripenem (Dori), 10 ␮g/ml biapenem (Bia), 25 ␮g/ml rifampin plus 10 ␮g/ml doripenem (Rif⫹Dori), 25 ␮g/ml rifampin plus 10
␮g/ml biapenem (Rif⫹Bia), and no drug as a control for growth of M. abscessus.

demonstrating indifference/additive activity. However, a combi- Mutants resistant to rifampin/faropenem combination were
nation of doripenem and rifampin exhibited a FIC of 0.33, biap- undetectable. We hypothesized that a synergistic combination
enem and rifampin exhibited a FIC of 0.24, meropenem and ri- would also decrease the frequency of mutants that are resistant to
fampin exhibited a FIC of 0.4, and faropenem and rifampin the combination. To test this hypothesis, we determined the fre-
exhibited a FIC of 0.19. In these combinations, wells containing quency of M. tuberculosis resistant mutants in the presence of ri-
either drug at less than 4- to 16-fold the respective MIC90 com- fampin and faropenem alone and in combinations (Table 5). At 10
pletely inhibited M. tuberculosis growth. For instance, rifampin/ times the MIC90 of rifampin, or 6 ␮g/ml, the frequency of resistant
doripenem combinations of 0.06/0.63 or 0.03/2.5 ␮g/ml, rifam- mutants was 1.8 ⫻ 10⫺8. Similarly, at 10 times the MIC90 of faro-
pin/biapenem concentrations of 0.06/0.4 or 0.03/1.56 ␮g/ml, and penem, or 50 ␮g/ml, mutants were selected at a frequency of 1 ⫻
rifampin/faropenem concentrations of 0.06/0.08, 0.03/0.16, or 10⫺6. However, we were unable to observe any CFU growth on
0.008/0.3 ␮g/ml completely inhibited M. tuberculosis growth. plates containing a combination of 0.6 ␮g/ml rifampin and 5.0
Similarly, we studied if rifampin and doripenem or biapenem, ␮g/ml faropenem, while plates containing either of the drugs at
the two carbapenems that we found to exhibit antimicrobial ac- these concentrations produced 1,000 to 5,000 CFU. Despite our
tivity against M. abscessus, exhibited synergistic activity against repeated efforts in which we increased the number of plates on
this pathogen. We assessed the MIC90 of rifampin to be 200 to 400 which to select spontaneous resistant mutants, we were unable to
␮g/ml, a concentration that is therapeutically irrelevant. How-
ever, a combination of rifampin and doripenem or biapenem ex-
hibited synergistic antimicrobial activity against M. abscessus with TABLE 5 Frequency of emergence of M. tuberculosis mutants resistant
a FIC of 0.48 and 0.50, respectively. Rifampin/doripenem combi- to rifampin and faropenem, alone or in combination
nations of 3.1/20, 6.2/20, and 50/10 ␮g/ml and rifampin/biap-
Concn (␮g/ml) of:
enem combinations of 6.2/20 or 25/10 ␮g/ml completely inhib- Total no. of Frequency of
ited M. abscessus growth. The concentrations of rifampin, Rifampin Faropenem resistant mutants resistant mutants
doripenem, and biapenem in these combinations are therapeuti- 3.0 0 22 2.2 ⫻ 10⫺8
cally relevant. We studied kinetics of growth inhibition or killing 6.0 0 18 1.8 ⫻ 10⫺8
of M. abscessus ATCC 19977 by rifampin and doripenem or biap- 0.6 0 1,000 1 ⫻ 10⫺6
enem at sub-MICs to assess the time course activity of synergy 0 50 1,000 1 ⫻ 10⫺6
between rifampin and doripenem or biapenem. The combina- 0 5.0 5,000 5 ⫻ 10⫺6
tions rifampin/doripenem and rifampin/biapenem yielded ⬃6 3.0 5.0 0 0
0.6 5.0 0 0
log10 fewer CFU than when drugs were used individually (Fig. 1).

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 Carbapenems and Rifampin Synergize

TABLE 6 Growth profile of rifampin monoresistant clinical isolate of M. tuberculosis in the presence of rifampin and (carba)penemsa
Growth profile with rifampin at (␮g/ml): Growth of control
Drug (concn [␮g/ml]) 1.0 0.5 0.25 0.12 0.063 0.031 0.016 Positive Negative
Meropenem (5) ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹⫹ ⫹⫹⫹
Imipenem (40) ⫺ ⫺ ⫺ ⫺ ⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
Doripenem (1.25) ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹
Biapenem (1.56) ⫺ ⫺ ⫺ ⫺ ⫹ ⫹⫹ ⫹⫹⫹ ⫺
Faropenem (0.63) ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺
No (carba)penem ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
a
For this strain, the rifampin MIC90 is ⬎1 ␮g/ml. ⫺ represents no visible growth, and ⫹⫹⫹ represents growth equivalent to that of the positive control (M. tuberculosis grown in
broth alone).

observe any growth in plates containing 0.6 ␮g/ml rifampin and results. In addition, for M. abscessus, we repeated the MIC90 as-
5.0 ␮g/ml faropenem. sessment using broth microdilution assay in 96-well format per
Rifampin/(carba)penem combinations are effective against Clinical and Laboratory Standards Institute guidelines (14). Data
drug-resistant clinical isolates of M. tuberculosis. We evaluated from these independent repeat assessments were highly consis-
the efficacy of rifampin in combination with meropenem, imi- tent. We observed that meropenem in solution is unstable and
penem, doripenem, biapenem, or faropenem for their activities loses potency even after a single freezing and thawing process.
against four clinical isolates of M. tuberculosis: one rifampin Therefore, only previously unthawed stock solutions of mero-
monoresistant, one isoniazid monoresistant, and two multidrug- penem were used in this study. It was beyond the scope of current
resistant strains (Tables 6 to 8; also see Table S1 in the supplemen- study or available resources to assess MBC, activity in combina-
tal material). Rifampin and (carba)penem combinations were tion with rifampin, and frequency of resistant mutants for all
growth inhibitory against all strains at sub-MIC levels of the commercially available (carba)penems. We also used only a single
drugs. The MIC90 of rifampin for rifampin monoresistant strains concentration of clavulanic acid, 5 ␮g/ml, an optimal concentra-
is 0.063 ␮g/ml when combined with 5 ␮g/ml meropenem. For tion derived from our titration of this agent with (carba)penems
multidrug-resistant strains, we assessed the MIC90 of rifampin at for activity against M. tuberculosis. This concentration is also
0.25 to 0.5 ␮g/ml when combined with 5 ␮g/ml meropenem. Of within one dilution of the concentration determined in a previous
the (carba)penems that we evaluated, faropenem afforded the
study (5).
most potent synergy with rifampin: when combined with 0.63
With the exception of panipenem, activities of all (carba)pen-
␮g/ml faropenem, only 0.063 to 0.031 ␮g/ml of rifampin was re-
ems evaluated in this study are therapeutically relevant, as their
quired to inhibit growth of both rifampin monoresistant and mul-
MIC90 range from 1.25 to 40 ␮g/ml. (Carba)penems are known to
tidrug-resistant strains (Tables 6 and 7; also see Table S1).
be either slow substrates or inhibitors of BlaC, a ␤-lactamase of M.
DISCUSSION tuberculosis (2, 5, 27–30). A 2- to 4-fold reduction in MIC90 was
␤-Lactams, one of the most widely used classes of antibiotics, are seen for all (carba)penems except faropenem when supplemented
known to inhibit bacterial D,D-transpeptidases (also known as with 5 ␮g/ml clavulanic acid. The MIC90 of doripenem and biap-
penicillin-binding proteins) (17, 18). It has been demonstrated enem against M. tuberculosis decreased 2-fold when the inoculat-
that ␤-lactams interfere with peptidoglycan biosynthesis and me- ing dosage was split into two halves. As (carba)penems are known
tabolism in multiple ways and eventually results in cell death (17, to exhibit time-dependent activity, our data suggest that a proper
19). Among ␤-lactams, carbapenems and penems are able to bind dosing interval for doripenem and biapenem has the potential to
and inhibit activities of D,D-transpeptidases, L,D-transpeptidases, further optimize activities of these drugs. Doripenem, biapenem,
and D,D-carboxypeptidases (6, 20–26). This versatile property of faropenem, and tebipenem exhibit bactericidal activity against M.
(carba)penems to target multiple enzymes prompted the current tuberculosis within therapeutically achievable concentrations. Due
study to test the clinical utility of commercially available (carba) to the poor oral bioavailability of ertapenem, meropenem, imi-
penems in M. tuberculosis and M. abscessus isolates from patients. penem, and doripenem, these drugs generally are administered
All MIC90 assessments were repeated at least twice to verify intravenously, a route that is not feasible for treatment of TB pa-

TABLE 7 Growth profile of multidrug-resistant clinical isolate 2 of M. tuberculosis in the presence of rifampin and (carba)penemsa
Growth profile with rifampin at (␮g/ml): Growth of control
Drug (concn [␮g/ml]) 1.0 0.5 0.25 0.12 0.063 0.031 0.016 Positive Negative
Meropenem (5) ⫺ ⫺ ⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹
Imipenem (40) ⫺ ⫺ ⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
Doripenem (1.25) ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
Biapenem (1.56) ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫺
Faropenem (0.63) ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺
No carba(penem) ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
a
For this strain, the rifampin MIC90 is ⬎1 ␮g/ml and the isoniazid MIC90 is ⬎0.1 ␮g/ml. ⫺ represents no visible growth, and ⫹⫹⫹ represents growth equivalent to that of the
positive control (M. tuberculosis grown in broth alone).

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Kaushik et al.

TABLE 8 Growth profile of an isoniazid monoresistant clinical isolate of M. tuberculosis in the presence of rifampin and (carba)penemsa
Growth profile with rifampin at (␮g/ml): Growth of control
Drug (concn [␮g/ml]) 1.0 0.5 0.25 0.012 0.063 0.031 0.016 Positive Negative
Meropenem (5) ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹⫹⫹
Imipenem (40) ⫺ ⫺ ⫺ ⫺ ⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
Doripenem (1.25) ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹⫹
Biapenem (1.56) ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺
Faropenem (0.63) ⫺ ⫺ ⫺ ⫺ ⫹ ⫹⫹ ⫹⫹⫹ ⫺
a
⫺ represents no visible growth, and ⫹⫹⫹ represents growth equivalent to that of the positive control (M. tuberculosis grown in broth alone).

tients en masse. However, newer (carba)penems, such as faro- (10). Recently, imipenem was reported to exhibit a MIC90 in the
penem and tebipenem, can be administered orally as prodrugs range of 16 to 32 ␮g/ml against a collection of M. abscessus isolates
and have the potential to be of high utility in treating tuberculosis from cystic fibrosis patients (33). We found doripenem and biap-
in a regimen containing rifampin, as it is likely that time above enem to be the most active among carbapenems against M. absces-
MIC would be prolonged due to synergy between these two drugs. sus ATCC 19977 and three clinical isolates that are resistant to a
The most significant observation of this study is the synergistic range of drugs used to treat M. abscessus infection. The addition of
activity of combinations containing a (carba)penem and rifam- clavulanic acid greatly enhanced the activity of meropenem, as its
pin. Since only sub-MIC levels are required when combined with MIC90 decreased from ⬎80 ␮g/ml to 5 to 10 ␮g/ml. The antimi-
rifampin, a further increase in the dosage of (carba)penems may crobial activity of doripenem, biapenem, faropenem, and tebi-
prolong the time above MIC and consequently make this combi- penem was modestly enhanced by clavulanic acid. More impor-
nation therapeutically valuable by enhancing the time-dependent tant is the synergy in antimicrobial activities of the combination of
activity of (carba)penems. The combination was effective against doripenem or biapenem and rifampin, as the concentrations of
drug-susceptible, rifampin monoresistant, isoniazid monoresis- these drugs in the combinations are therapeutically significant. As
tant, and two multidrug-resistant M. tuberculosis clinical isolates. the proportion of drug-resistant M. abscessus strains isolated from
As all evaluated strains were susceptible to rifampin and cystic fibrosis patients is gradually increasing (34), the potential
(carba)penems at concentrations that are readily achievable ther- impact of these synergistic combinations can be significant in the
apeutically, these data warrant studies to evaluate the clinical util- treatment of M. abscessus infection in this patient population.
ity of this combination for the treatment of drug-resistant TB. The
drug levels achievable with a 600-mg dose of rifampin is 1.5 ␮g/ml ACKNOWLEDGMENTS
of free drug, which is capable of inactivating strains with MIC90 We thank Jacques Grosset for critical discussions.
of up to 0.375 ␮g/ml. This study suggests that synergy with This study was supported by NIH awards 1R21AI111739-01 and
(carba)penems could lower the MIC of rifampin significantly. The 1DP2OD008459-01 to G.L.
data for rifampin-resistant strains are extremely promising and
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