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http://www.ncbi.nlm.nih.gov/pubmed/22560037
In vitro effects of citrus oils against Mycobacterium tuberculosis and non-tuberculous Mycobacteria of clinical importance
Philip G. Crandall a , Steven C. Ricke a , Corliss A. O’Bryan a & Nicole M. Parrish
Center for Food Safety and Department of Food Science, University of Arkansas, Fayetteville, Arkansas, USA
Division of Medical Microbiology, Johns Hopkins University, Baltimore, Maryland, USA
We evaluated the in vitro activity of citrus oils against Mycobacterium tuberculosis and other non-tuberculous Mycobacterium species. Citrus essential oils were tested against a variety of Mycobacterium species and strains using the BACTEC radiometric growth system. Cold pressed terpeneless Valencia oil (CPT) was further tested using the Wayne model of in vitro latency. Exposure of M. tuberculosis and M. bovis BCG to 0.025 % cold pressed terpeneless Valencia orange oil (CPT) resulted in a 3-log decrease in viable counts versus corresponding controls. Inhibition of various clinical isolates of the M. avium complex and M. abscessus ranged from 2.5 to 5.2-logs. Some species/strains were completely inhibited in the presence of CPT including one isolate each of the following: the M. avium complex, M. chelonae and M. avium subsp. paratuberculosis. CPT also inhibited the growth of BCG more than 99 % in an in vitro model of latency which mimics anaerobic dormancy thought to occur in vivo. The activity of CPT against drug-resistant strains of the M. avium complex and M. bscessus suggest that the mechanism of action for CPT is different than that of currently available drugs. Inhibition of latently adapted bacilli offers promise for treatment of latent infections of MTB. These results suggest that the antimycobacterial properties of CPT warrant further study to elucidate the specific mechanism of action and clarify the spectrum of activity.
Introduction
Tuberculosis (TB) is reported to cause more deaths than any other single infectious organism.[1]Worldwide in 2008 there were an estimated 8.9 to 9.9 million new cases of TB, 9.6 to13.3 million total cases of TB, 1.1 to 1.7 million deaths from TB among HIV-negative people and an additional 0.45 to 0.62 million TB deaths among HIV-positive people that may have been originally classified as death from AIDS.[2] Current TB treatment regimens comprise at least four drugs in combination given over a time period as long as 6 to 9 months.[3] This long course of treatment as well as expense often leads patients to discontinue treatment before completing the regime.[1] Multi-drug resistant TB (MDR-TB), defined as resistance to isoniazid (INH) and
rifampin (RIF),[4] is now common throughout the world, with an estimated 4.8 percent of all new and previously treated TB cases worldwide being MDR-TB.[2] Recently, newly adapted strains of TB have been identified throughout the world with an even more extensive resistance to
second-line drugs, known as eXtra drug resistant, XDRTB.[5] MDR- and XDR-TB strains are rapidly becoming the next global health emergency and are the harbingers of an impending pandemic.[6] The causative agent of TB is Mycobacterium tuberculosis (MTB). Mycobacteria other than MTB are known as non-tuberculous mycobacteria (NTM), and are common
environmental organisms found in water and soil that can cause disease in humans.[7] Some of the most commonly encountered NTM associated with human disease include: M. avium, M. intracellulare, M. kansasii, M. fortuitum, M. abscessus, and M. chelonae.[7] The NTM are of increasing importance as the number of clinically significant infections has increased, especially in immunocompromised patients.[8] However, compared to MTB, the NTM differ in susceptibility to antimycobacterial agents, and vary widely by species and drug. Unfortunately, resistance has emerged to all classes of traditional drugs used to combat these organisms, especially in M. abscessus, M. chelonae, and the M. avium complex.[9] M. avium subsp. paratuberculosis
(PTB) is the causative agent of Johne’s disease in domestic cattle, especially dairy animals, and wild ruminants, and may play a role in Crohn’s Disease in humans.[10] The need for developing new drugs for treatment of TB
aswell as theNTMiswell documented.[11–13] Plant essential oils have been widely used throughout human history for a variety of medicinal and cosmetic purposes. Aegicerin, a compound isolated from Clavija procera, has been found to have activity against theMTBstrainH37Rv as well as some
MDR-TB strains.[14] Earl et al.[1] examined the effects of extracts of native New Zealand plants including, Laurelia novae-zelandiae, which is reportedly used by indigenous Maori for the treatment of tubercular lesions. Many of the
extracts were inhibitory towards MTB including those of the Laurelia novae-zelandiae. Citrus oils have been used as a medicine since ancient times and many researchers have demonstrated bioactivity against bacteria, yeasts, and molds.[15] In this studywe evaluated the activity of citrus oils against M. tuberculosis and other NTM species of clinical importance.
Materials and methods
Essential oils
All of the citrus essential oilswere obtained as commercially
available products from FirmenichCitrus Center, Lakeland
FL and were stored per manufacturer’s recommendations
at 4◦C prior to use. For all assays, stock solutions of each
commercial extractwere diluted in ethanol resulting in final
test concentrations ranging from 0.025 % to 2.5 %. Fractions
tested were as follows: 1) five-fold concentrated orange
oil; 2) cold pressed orange oil; 3) highly concentrated
terpeneless Valencia orange oil (CPT); 4) high purity terpenes
from orange juice essence; 5) d-limonene; 6) terpenes
from cold pressed orange oil; 7) terpenes from orange juice
essence. All controls contained an equivalent volume of
ethanol diluent as used in test cultures.
Mycobacteria and susceptibility testing
A variety of Mycobacterium species and strains were
used, including MTB (ATCC H37Rv), the non-pathogenic
surrogate organism, M. bovis BCG (BCG, ATCC Pasteur
35734), and several other NTM species of clinical
importance: M. avium (ATCC 700898) and various clarithromycin
resistant clinical isolates, M. avium subspecies
paratuberculosis (ATCC 19698) and various drug resistant
clinical isolates of M. abscessus and M. chelonae. All
clinical isolates were identified to the species level with
subsequent susceptibility testing according to standard
methods.[16] Frozen stocks of all species/strains were
maintained at -70◦C. Working cultures were maintained
on Lowenstein Jensen slants and Middlebrook 7H11
agar plates (Difco, Detroit, Michigan). The BACTEC
radiometric growth system (Becton Dickinson, Sparks,
Maryland) was used to determine the minimal inhibitory
concentrations (MIC) of each extract against MTB. The
radiometric system is rapid and permits assessment of
susceptibility and determination ofMICs typically within 7
to 10 days versus 3 weeks required for agar-based methods.
MICs were defined using established protocols developed
and routinely used in our laboratory.[17,18] Briefly, a suspension
of each MTB strain to be tested was made using
commercially prepared diluting fluid (Becton Dickinson)
from growth on solid media. Suspensions were vortexed
with glass beads and allowed to settle for 30 minutes. The
supernatant was adjusted to a 1.0 McFarland turbidity
standard and 0.1 mL inoculated into BACTEC 12B vials
with and without test compound. Paired controls without
compound were set up in parallel including a growth
control and a 1:100 diluted control. All vials were read on
the BACTEC 460 instrument daily at 24-hour intervals.
When the growth index (GI) of the 1:100 control vial
reached 30, the GI change over the same 24-hour period
was calculated for each concentration of extract tested. The
MIC was defined as the lowest inhibitor concentration that
yielded a GI change less than that of the 1:100 control vial.
Wayne model of in vitro latenc
The activity of cold pressed terpeneless Valencia oil (CPT)
was assessed against BCG in the Wayne in-vitro model of
latency.[19] In this model, dormancy is triggered in the test
organisms by culturing them under conditions of gradual
oxygen depletion. Many of the first-line drugs such as
INHand RIF are not effective against oxygen-deprived “latent”
MTB organisms, whereas metronidazole (MET), an
anaerobe-specific drug is. Each test compound or combination
was added to “latently” adapted cultures ofMTB to
be tested. Since susceptibility can vary between aerobic and
anaerobically adapted bacilli, MICs were also determined
for all “latently” adapted cultures.
ATP assays
Either cold pressed terpeneless Valencia orange oil or diluent
was added to 120mLBCGcultures. Known respiratory
chain inhibitors tested included dicyclohexylcarbodiimide
(DCCD) (100 mg/L), an ATP synthase-specific inhibitor;
and dinitrophenol (DNP), an uncoupling agent. The ATP
molar concentration (ATP [M]) was determined using previously
established methods.[20,21] Briefly, single and multiple
time-point assays were conducted by removing culture
aliquots (30 mL) at 5 minutes, 30 minutes and 24 h, and
placing them immediately on ice. All subsequent manipulations
were conducted at 4◦C. Cells were harvested by
centrifugation and disrupted by bead-beating with 200 to
300 μm glass beads in an ATP extraction buffer (100 mM
Tris, 4 mM EDTA, pH 7.5) at maximum force for a total
of 2 min. Cellular debris was removed by centrifugation
(13000 x g for15min), andtheATP-containing supernatant
transferred to a clean tube. An ATP bioluminescence assay
(Roche Diagnostics, Mannheim, Germany) was used
to determine the ATP[M] in treated versus control samples.
Relative light units were measured on aWallac Victor
luminometer.
GC/MS analysis
An analysis of the principal components in the cold pressed
terpenelessValencia orange oilwas carried out using an Agilent
6890 gas chromatograph (GC)connected to an Agilent
5973N Mass Spectrometer (MS). The GC was equipped
with fused silica capillary column, DB-5 with a 30 m ×
0.25 mm x 0.25 um film thickness. The GC/MS was operated
at an initial temperature of 85◦ C, held for 6 min, ramp
of 4◦ C/min up to 210◦ C and held for 10 min. The carrier
gas was helium at a flow rate of 2 mL/min. One μLof sample
was injected into a split injector with a 200:1 split with
a temperature of 250◦ C. Identification of the compounds
was by comparison of their mass spectra with those of the
Wiley and two internal libraries, and chemical authentic
standards, when available, were run and spectra confirmed
compound identities. Samples were run in triplicate, with a
blank run between each sample. A known mixture was run
at the end of each day to calculate retention indices (RIs).
Results
Effects of oils on MTB
All seven citrus oil extracts demonstrated rapid bactericidal
activity against actively growing MTB in vitro (Fig. 1).
Data from a single run is shown because the inoculum and
hence instrument readings can vary between test runs. Extract
number 3, cold pressed terpeneless Valencia orange
oil (CPT), was the most potent against MTB and was selected
for additional testing using concentrations ranging
from 0.025 % to 2.5 %.
Effect of CPT
Exposure ofMTB and BCG to 0.025 % CPT resulted in a
3-log decrease in viable counts versus corresponding controls.
All species and strains were susceptible to CPT at the
concentration tested (0.2%) although differences in susceptibility
were observed between mycobacterial species and
strains. One clinical isolate of each of the M. avium complex
(MAI-4, Fig. 2) and M. chelonae (data not shown)
were completely inhibited following exposure to CPT. This
inhibition reflected a 6-log reduction in viable counts versus
untreated controls. For all other susceptible strains, viable
counts decreased an average of 3.3 logs (Figs. 2 and 3).
However, the range in inhibition varied between species.
For M. avium isolates, growth inhibition ranged from 2.5
to 5.2 logs (Fig. 2). For multi-drug resistant M. abscessus
isolates inhibition was more consistent with a narrower
range (2.5 logs to 4.3 logs) (Fig. 3).
Wayne model
In the Wayne model, CPT in concentrations ranging from
0.15 % to 1.5 % killed anaerobically growing BCG (Fig. 4).
Using a starting inoculum of approximately 106 CFU/mL,
the bactericidal activity was greater than 99 % with all concentrations
of CPT tested, whereas INH and RIF had little effect. OnlyMET (12.0 μg/mL) showed activity similar to
that of CPT.
ATP levels
Time-course studies over 24 hours were performed to examine
the effect of CPT (0.2 %) on ATP levels in BCG. As
shown in Figure 5, ATP[M] levels decreased rapidly (49.6
%) in CPT treated BCG compared to diluent controls at the decrease in ATP levels continued resulting in a 62 %
decrease at 30 minutes. At 24 hours, inhibition was 99.6 %
when compared to unexposed controls. We compared the
effect of CPT with two known ATP inhibitors,DCCD, and
DNP. Of the two, the activity of CPT more closely resembled
that of the ATP synthase-specific inhibitor, DCCD
versus the proton ionophore, DNP.
5 minutes post exposure. Following this initial rapid loss,
the decrease in ATP levels continued resulting in a 62 %
decrease at 30 minutes. At 24 hours, inhibition was 99.6 %
when compared to unexposed controls. We compared the
effect of CPT with two known ATP inhibitors,DCCD, and
DNP. Of the two, the activity of CPT more closely resembled
that of the ATP synthase-specific inhibitor, DCCD
versus the proton ionophore, DNP.
Quantitative and qualitative composition of CP terpeneless
Valencia orange oil
CPT is a commercial product obtained by a mechanical
extraction of the orange oil which is further concentrated
under vacuum. [22] The contents ( %) of the major components
of this orange fraction are presented in Table 1. The
most predominant compound is the alcohol linalool (20.2
%) followed by the aldehyde decanal (18 %). The amount
of the terpene limonene that is the principal component of
single-strength orange oil was very low at 0.3 %.
Discussion
Plant essential oils, including citrus oils, have been widely
used for a variety of medicinal and cosmetic purposes.
Many essential oils have been shown to have potent activity
against Gram-positive and Gram-negative organisms, yet
few studies have been conducted to date investigating the
effect of essential oils against the Mycobacteria. In this
study, we investigated the in vitro activity of seven essential
citrus oils against a variety of Mycobacteria including
ATCC strains of MTB, BCG, and several other clinical
species and strains of NTM. CPT, the most active fraction
tested, was bactericidal to all mycobacterial species tested
including drug-resistant strains of the M. avium complex
and M. abscessus. CPT demonstrated potent activity
against drug-resistant strains of the M. avium complex
and M. abscessus. More importantly, cross-resistance
was not observed for several strains resistant to multiple
antibiotics including: amikacin, clarithromycin, cefoxitin,
ciprofloxacin and higher fluoroquinolones, doxycycline,
minocycline, sulfamethoxazole, and linezolid. This suggests
the CPT-specific inhibition observed in this study
involves a mechanism of action different from that of these
currently available drugs.
Camacho-Corona et al.[23] evaluated the antimycobacterial
activity of extracts of nine plants used in Mexican
traditional medicine to treat tuberculosis and other
respiratory diseases. Of the plants tested Nasturtium
officinale showed the best activity against the sensitive
Mycobacterium tuberculosis, with Citrus sinensis and Citrus
aurantifolia active to a lesser extent. Activity against drugresistant
variants of M. tuberculosis was higher than for
the sensitive strains. These data point to the importance of
biological testing of extracts against drug-resistant M. tuberculosis
isolates, and the bioguided assay of these extracts
for the identification of lead compounds againstMDR-TB
isolates.
Although no drug-resistant isolates of MTB were tested
in this study, the unique activity of CPT was demonstrated
by inhibition of both actively growing and latently adapted
bacilli. MTB can cause an asymptomatic latent infection,
and in this dormant state, the bacilli may persist for years
before reactivation to active disease. Lawrence Wayne[19]
established an in vitro model which is able to trigger the
dormancy response of the bacillus through a process of
gradual oxygen depletion. This model has become a useful
tool for screening drugs for the ability to kill MTB and the
closely relatedBCGwhile in a state of anaerobic dormancy.
Only one antibiotic, MET, is known to be active against
MTB in theWayne model of in vitro latency; drugs such as
INH and RIF have little potency against latently adapted
MTBsince the bacilli are not in an actively growing state.[24]
The activity of CPT appears to be due to a mechanism
of action different from currently available antimycobacterial
drugs. This mechanismmay involve rapid disruption of
energy production and lipid synthesis. CPT disrupted ATP
metabolism via an unknown mechanism similar to that of a
known ATP synthase inhibitor, DCCD. Determination of
the specific mechanism of action was beyond the scope of
this investigation. Currently, this mechanism is thought to
involve direct interaction with the cell membrane and/or
inhibition of specific cellular processes or enzymes.[25]However,
the precise mechanism(s) of action remains unclear
andwill require further elucidation before definitive conclusions
can be made. Elucidation of the specific mechanism
of action may reveal a unique drug target(s) for which no
inhibitors currently exist. In addition, CPT and most of the
constituent compounds are generally regarded as safe by
the FDAand relatively inexpensive to produce compared to
current drugs, both extremely important considerations for
any new antimycobacterial treatment. The results collected
to date suggest that further study of CPT is warranted to
identify the most active compound(s) or combination of
compounds responsible for the rapid bactericidal activity
against the Mycobacteria.
In vitro effects of citrus oils against Mycobacterium tuberculosis and non-tuberculous Mycobacteria of clinical importance
Philip G. Crandall a , Steven C. Ricke a , Corliss A. O’Bryan a & Nicole M. Parrish
Center for Food Safety and Department of Food Science, University of Arkansas, Fayetteville, Arkansas, USA
Division of Medical Microbiology, Johns Hopkins University, Baltimore, Maryland, USA
We evaluated the in vitro activity of citrus oils against Mycobacterium tuberculosis and other non-tuberculous Mycobacterium species. Citrus essential oils were tested against a variety of Mycobacterium species and strains using the BACTEC radiometric growth system. Cold pressed terpeneless Valencia oil (CPT) was further tested using the Wayne model of in vitro latency. Exposure of M. tuberculosis and M. bovis BCG to 0.025 % cold pressed terpeneless Valencia orange oil (CPT) resulted in a 3-log decrease in viable counts versus corresponding controls. Inhibition of various clinical isolates of the M. avium complex and M. abscessus ranged from 2.5 to 5.2-logs. Some species/strains were completely inhibited in the presence of CPT including one isolate each of the following: the M. avium complex, M. chelonae and M. avium subsp. paratuberculosis. CPT also inhibited the growth of BCG more than 99 % in an in vitro model of latency which mimics anaerobic dormancy thought to occur in vivo. The activity of CPT against drug-resistant strains of the M. avium complex and M. bscessus suggest that the mechanism of action for CPT is different than that of currently available drugs. Inhibition of latently adapted bacilli offers promise for treatment of latent infections of MTB. These results suggest that the antimycobacterial properties of CPT warrant further study to elucidate the specific mechanism of action and clarify the spectrum of activity.
Introduction
Tuberculosis (TB) is reported to cause more deaths than any other single infectious organism.[1]Worldwide in 2008 there were an estimated 8.9 to 9.9 million new cases of TB, 9.6 to13.3 million total cases of TB, 1.1 to 1.7 million deaths from TB among HIV-negative people and an additional 0.45 to 0.62 million TB deaths among HIV-positive people that may have been originally classified as death from AIDS.[2] Current TB treatment regimens comprise at least four drugs in combination given over a time period as long as 6 to 9 months.[3] This long course of treatment as well as expense often leads patients to discontinue treatment before completing the regime.[1] Multi-drug resistant TB (MDR-TB), defined as resistance to isoniazid (INH) and
rifampin (RIF),[4] is now common throughout the world, with an estimated 4.8 percent of all new and previously treated TB cases worldwide being MDR-TB.[2] Recently, newly adapted strains of TB have been identified throughout the world with an even more extensive resistance to
second-line drugs, known as eXtra drug resistant, XDRTB.[5] MDR- and XDR-TB strains are rapidly becoming the next global health emergency and are the harbingers of an impending pandemic.[6] The causative agent of TB is Mycobacterium tuberculosis (MTB). Mycobacteria other than MTB are known as non-tuberculous mycobacteria (NTM), and are common
environmental organisms found in water and soil that can cause disease in humans.[7] Some of the most commonly encountered NTM associated with human disease include: M. avium, M. intracellulare, M. kansasii, M. fortuitum, M. abscessus, and M. chelonae.[7] The NTM are of increasing importance as the number of clinically significant infections has increased, especially in immunocompromised patients.[8] However, compared to MTB, the NTM differ in susceptibility to antimycobacterial agents, and vary widely by species and drug. Unfortunately, resistance has emerged to all classes of traditional drugs used to combat these organisms, especially in M. abscessus, M. chelonae, and the M. avium complex.[9] M. avium subsp. paratuberculosis
(PTB) is the causative agent of Johne’s disease in domestic cattle, especially dairy animals, and wild ruminants, and may play a role in Crohn’s Disease in humans.[10] The need for developing new drugs for treatment of TB
aswell as theNTMiswell documented.[11–13] Plant essential oils have been widely used throughout human history for a variety of medicinal and cosmetic purposes. Aegicerin, a compound isolated from Clavija procera, has been found to have activity against theMTBstrainH37Rv as well as some
MDR-TB strains.[14] Earl et al.[1] examined the effects of extracts of native New Zealand plants including, Laurelia novae-zelandiae, which is reportedly used by indigenous Maori for the treatment of tubercular lesions. Many of the
extracts were inhibitory towards MTB including those of the Laurelia novae-zelandiae. Citrus oils have been used as a medicine since ancient times and many researchers have demonstrated bioactivity against bacteria, yeasts, and molds.[15] In this studywe evaluated the activity of citrus oils against M. tuberculosis and other NTM species of clinical importance.
Materials and methods
Essential oils
All of the citrus essential oilswere obtained as commercially
available products from FirmenichCitrus Center, Lakeland
FL and were stored per manufacturer’s recommendations
at 4◦C prior to use. For all assays, stock solutions of each
commercial extractwere diluted in ethanol resulting in final
test concentrations ranging from 0.025 % to 2.5 %. Fractions
tested were as follows: 1) five-fold concentrated orange
oil; 2) cold pressed orange oil; 3) highly concentrated
terpeneless Valencia orange oil (CPT); 4) high purity terpenes
from orange juice essence; 5) d-limonene; 6) terpenes
from cold pressed orange oil; 7) terpenes from orange juice
essence. All controls contained an equivalent volume of
ethanol diluent as used in test cultures.
Mycobacteria and susceptibility testing
A variety of Mycobacterium species and strains were
used, including MTB (ATCC H37Rv), the non-pathogenic
surrogate organism, M. bovis BCG (BCG, ATCC Pasteur
35734), and several other NTM species of clinical
importance: M. avium (ATCC 700898) and various clarithromycin
resistant clinical isolates, M. avium subspecies
paratuberculosis (ATCC 19698) and various drug resistant
clinical isolates of M. abscessus and M. chelonae. All
clinical isolates were identified to the species level with
subsequent susceptibility testing according to standard
methods.[16] Frozen stocks of all species/strains were
maintained at -70◦C. Working cultures were maintained
on Lowenstein Jensen slants and Middlebrook 7H11
agar plates (Difco, Detroit, Michigan). The BACTEC
radiometric growth system (Becton Dickinson, Sparks,
Maryland) was used to determine the minimal inhibitory
concentrations (MIC) of each extract against MTB. The
radiometric system is rapid and permits assessment of
susceptibility and determination ofMICs typically within 7
to 10 days versus 3 weeks required for agar-based methods.
MICs were defined using established protocols developed
and routinely used in our laboratory.[17,18] Briefly, a suspension
of each MTB strain to be tested was made using
commercially prepared diluting fluid (Becton Dickinson)
from growth on solid media. Suspensions were vortexed
with glass beads and allowed to settle for 30 minutes. The
supernatant was adjusted to a 1.0 McFarland turbidity
standard and 0.1 mL inoculated into BACTEC 12B vials
with and without test compound. Paired controls without
compound were set up in parallel including a growth
control and a 1:100 diluted control. All vials were read on
the BACTEC 460 instrument daily at 24-hour intervals.
When the growth index (GI) of the 1:100 control vial
reached 30, the GI change over the same 24-hour period
was calculated for each concentration of extract tested. The
MIC was defined as the lowest inhibitor concentration that
yielded a GI change less than that of the 1:100 control vial.
Wayne model of in vitro latenc
The activity of cold pressed terpeneless Valencia oil (CPT)
was assessed against BCG in the Wayne in-vitro model of
latency.[19] In this model, dormancy is triggered in the test
organisms by culturing them under conditions of gradual
oxygen depletion. Many of the first-line drugs such as
INHand RIF are not effective against oxygen-deprived “latent”
MTB organisms, whereas metronidazole (MET), an
anaerobe-specific drug is. Each test compound or combination
was added to “latently” adapted cultures ofMTB to
be tested. Since susceptibility can vary between aerobic and
anaerobically adapted bacilli, MICs were also determined
for all “latently” adapted cultures.
ATP assays
Either cold pressed terpeneless Valencia orange oil or diluent
was added to 120mLBCGcultures. Known respiratory
chain inhibitors tested included dicyclohexylcarbodiimide
(DCCD) (100 mg/L), an ATP synthase-specific inhibitor;
and dinitrophenol (DNP), an uncoupling agent. The ATP
molar concentration (ATP [M]) was determined using previously
established methods.[20,21] Briefly, single and multiple
time-point assays were conducted by removing culture
aliquots (30 mL) at 5 minutes, 30 minutes and 24 h, and
placing them immediately on ice. All subsequent manipulations
were conducted at 4◦C. Cells were harvested by
centrifugation and disrupted by bead-beating with 200 to
300 μm glass beads in an ATP extraction buffer (100 mM
Tris, 4 mM EDTA, pH 7.5) at maximum force for a total
of 2 min. Cellular debris was removed by centrifugation
(13000 x g for15min), andtheATP-containing supernatant
transferred to a clean tube. An ATP bioluminescence assay
(Roche Diagnostics, Mannheim, Germany) was used
to determine the ATP[M] in treated versus control samples.
Relative light units were measured on aWallac Victor
luminometer.
GC/MS analysis
An analysis of the principal components in the cold pressed
terpenelessValencia orange oilwas carried out using an Agilent
6890 gas chromatograph (GC)connected to an Agilent
5973N Mass Spectrometer (MS). The GC was equipped
with fused silica capillary column, DB-5 with a 30 m ×
0.25 mm x 0.25 um film thickness. The GC/MS was operated
at an initial temperature of 85◦ C, held for 6 min, ramp
of 4◦ C/min up to 210◦ C and held for 10 min. The carrier
gas was helium at a flow rate of 2 mL/min. One μLof sample
was injected into a split injector with a 200:1 split with
a temperature of 250◦ C. Identification of the compounds
was by comparison of their mass spectra with those of the
Wiley and two internal libraries, and chemical authentic
standards, when available, were run and spectra confirmed
compound identities. Samples were run in triplicate, with a
blank run between each sample. A known mixture was run
at the end of each day to calculate retention indices (RIs).
Results
Effects of oils on MTB
All seven citrus oil extracts demonstrated rapid bactericidal
activity against actively growing MTB in vitro (Fig. 1).
Data from a single run is shown because the inoculum and
hence instrument readings can vary between test runs. Extract
number 3, cold pressed terpeneless Valencia orange
oil (CPT), was the most potent against MTB and was selected
for additional testing using concentrations ranging
from 0.025 % to 2.5 %.
Effect of CPT
Exposure ofMTB and BCG to 0.025 % CPT resulted in a
3-log decrease in viable counts versus corresponding controls.
All species and strains were susceptible to CPT at the
concentration tested (0.2%) although differences in susceptibility
were observed between mycobacterial species and
strains. One clinical isolate of each of the M. avium complex
(MAI-4, Fig. 2) and M. chelonae (data not shown)
were completely inhibited following exposure to CPT. This
inhibition reflected a 6-log reduction in viable counts versus
untreated controls. For all other susceptible strains, viable
counts decreased an average of 3.3 logs (Figs. 2 and 3).
However, the range in inhibition varied between species.
For M. avium isolates, growth inhibition ranged from 2.5
to 5.2 logs (Fig. 2). For multi-drug resistant M. abscessus
isolates inhibition was more consistent with a narrower
range (2.5 logs to 4.3 logs) (Fig. 3).
Wayne model
In the Wayne model, CPT in concentrations ranging from
0.15 % to 1.5 % killed anaerobically growing BCG (Fig. 4).
Using a starting inoculum of approximately 106 CFU/mL,
the bactericidal activity was greater than 99 % with all concentrations
of CPT tested, whereas INH and RIF had little effect. OnlyMET (12.0 μg/mL) showed activity similar to
that of CPT.
ATP levels
Time-course studies over 24 hours were performed to examine
the effect of CPT (0.2 %) on ATP levels in BCG. As
shown in Figure 5, ATP[M] levels decreased rapidly (49.6
%) in CPT treated BCG compared to diluent controls at the decrease in ATP levels continued resulting in a 62 %
decrease at 30 minutes. At 24 hours, inhibition was 99.6 %
when compared to unexposed controls. We compared the
effect of CPT with two known ATP inhibitors,DCCD, and
DNP. Of the two, the activity of CPT more closely resembled
that of the ATP synthase-specific inhibitor, DCCD
versus the proton ionophore, DNP.
5 minutes post exposure. Following this initial rapid loss,
the decrease in ATP levels continued resulting in a 62 %
decrease at 30 minutes. At 24 hours, inhibition was 99.6 %
when compared to unexposed controls. We compared the
effect of CPT with two known ATP inhibitors,DCCD, and
DNP. Of the two, the activity of CPT more closely resembled
that of the ATP synthase-specific inhibitor, DCCD
versus the proton ionophore, DNP.
Quantitative and qualitative composition of CP terpeneless
Valencia orange oil
CPT is a commercial product obtained by a mechanical
extraction of the orange oil which is further concentrated
under vacuum. [22] The contents ( %) of the major components
of this orange fraction are presented in Table 1. The
most predominant compound is the alcohol linalool (20.2
%) followed by the aldehyde decanal (18 %). The amount
of the terpene limonene that is the principal component of
single-strength orange oil was very low at 0.3 %.
Discussion
Plant essential oils, including citrus oils, have been widely
used for a variety of medicinal and cosmetic purposes.
Many essential oils have been shown to have potent activity
against Gram-positive and Gram-negative organisms, yet
few studies have been conducted to date investigating the
effect of essential oils against the Mycobacteria. In this
study, we investigated the in vitro activity of seven essential
citrus oils against a variety of Mycobacteria including
ATCC strains of MTB, BCG, and several other clinical
species and strains of NTM. CPT, the most active fraction
tested, was bactericidal to all mycobacterial species tested
including drug-resistant strains of the M. avium complex
and M. abscessus. CPT demonstrated potent activity
against drug-resistant strains of the M. avium complex
and M. abscessus. More importantly, cross-resistance
was not observed for several strains resistant to multiple
antibiotics including: amikacin, clarithromycin, cefoxitin,
ciprofloxacin and higher fluoroquinolones, doxycycline,
minocycline, sulfamethoxazole, and linezolid. This suggests
the CPT-specific inhibition observed in this study
involves a mechanism of action different from that of these
currently available drugs.
Camacho-Corona et al.[23] evaluated the antimycobacterial
activity of extracts of nine plants used in Mexican
traditional medicine to treat tuberculosis and other
respiratory diseases. Of the plants tested Nasturtium
officinale showed the best activity against the sensitive
Mycobacterium tuberculosis, with Citrus sinensis and Citrus
aurantifolia active to a lesser extent. Activity against drugresistant
variants of M. tuberculosis was higher than for
the sensitive strains. These data point to the importance of
biological testing of extracts against drug-resistant M. tuberculosis
isolates, and the bioguided assay of these extracts
for the identification of lead compounds againstMDR-TB
isolates.
Although no drug-resistant isolates of MTB were tested
in this study, the unique activity of CPT was demonstrated
by inhibition of both actively growing and latently adapted
bacilli. MTB can cause an asymptomatic latent infection,
and in this dormant state, the bacilli may persist for years
before reactivation to active disease. Lawrence Wayne[19]
established an in vitro model which is able to trigger the
dormancy response of the bacillus through a process of
gradual oxygen depletion. This model has become a useful
tool for screening drugs for the ability to kill MTB and the
closely relatedBCGwhile in a state of anaerobic dormancy.
Only one antibiotic, MET, is known to be active against
MTB in theWayne model of in vitro latency; drugs such as
INH and RIF have little potency against latently adapted
MTBsince the bacilli are not in an actively growing state.[24]
The activity of CPT appears to be due to a mechanism
of action different from currently available antimycobacterial
drugs. This mechanismmay involve rapid disruption of
energy production and lipid synthesis. CPT disrupted ATP
metabolism via an unknown mechanism similar to that of a
known ATP synthase inhibitor, DCCD. Determination of
the specific mechanism of action was beyond the scope of
this investigation. Currently, this mechanism is thought to
involve direct interaction with the cell membrane and/or
inhibition of specific cellular processes or enzymes.[25]However,
the precise mechanism(s) of action remains unclear
andwill require further elucidation before definitive conclusions
can be made. Elucidation of the specific mechanism
of action may reveal a unique drug target(s) for which no
inhibitors currently exist. In addition, CPT and most of the
constituent compounds are generally regarded as safe by
the FDAand relatively inexpensive to produce compared to
current drugs, both extremely important considerations for
any new antimycobacterial treatment. The results collected
to date suggest that further study of CPT is warranted to
identify the most active compound(s) or combination of
compounds responsible for the rapid bactericidal activity
against the Mycobacteria.
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