recently , the world health organization indicated that drug - resistant a. baumannii is defined as the first priority pathogen , in which researches and developments for new antibiotics are urgently needed .
the bacteria has been revealed to persist on dry surfaces for a month and presented several drug - resistant mechanisms including drug efflux pumps , drug - inactivating enzymes , and drug target mutations .
infected patients have many serious diseases including septicemia , pneumonia , and urinary tract infections .
the number of global drug - resistant a. baumannii was vary in estimation ; therefore , the high prevalence accounted to be approximately 54% and 77% of a. baumannii isolates have been revealed in italy and india , respectively . in thailand ,
baumannii collected from clinical specimens was approximately 59% . regarding the limit of antibiotic treatment ,
several studies revealed the effectiveness of extracted herbs on drug - resistant pathogens including methicillin - resistant staphylococcus aureus , vancomycin - resistant enterococci , and mdr - a .
herein , 10 volatile oils extracted from various medicinal plants were determined for their inhibitory effect on the growth of the most common human pathogens and mdr - a .
s. aureus atcc 25923 , escherichia coli atcc 25922 , pseudomonas aeruginosa atcc 27853 , and a. baumannii atcc 19606 were purchased from the department of medical sciences , ministry of public health , thailand .
baumannii were collected from the diagnostic laboratory , maharaj nakorn chiang mai hospital , chiang mai , thailand , during february - april , 2012 . both biochemical tests followed by constantiniu et al .   and
molecular biology test using amplified ribosomal dna - restriction enzyme analysis were performed for identification of a. baumannii .
primers used for 16s rdna gene amplification were designed as followed by the previous report .
the antimicrobial susceptibility testing was performed using disk diffusion method following the clinical and laboratory standards institute ( clsi ) guidelines .
the mdr was defined according to the unsusceptible of at least one in three agents of antimicrobial classes .
all 30 clinical isolates resisted to eight antibiotics in six antimicrobial classes consisting of amikacin , piperacillin / tazobactam , ciprofloxacin , cefoperazone / sulbactam , ceftazidime , trimethoprim / sulfamethoxazole , imipenem , and meropenem .
galangal , ginger , plai , lime , kaffir lime , sweet basil , tree basil , lemongrass , clove , and cinnamon were selected in this study [ table 1 ] .
the material was subjected to hydrodistillation using a clevenger - type glass apparatus for 3 - 5 h .
yields of the volatile oils obtained from the plants were calculated as the percent yield .
medicinal plants used in this study the antimicrobial activity testing was modified from prabuseenivasan et al . .
0.5 ( 1  10 cfu / ml ) and spread over the mueller - hinton agar ( mha ) plates using a sterile cotton swab .
each volatile oil was dissolved in 10% aqueous dimethyl sulfoxide ( dmso ) with 0.5% v / v tween 80 and sterilized by filtration .
sterilized disks ( whatman no . 5 , 6 mm diameter ) were impregnated with 20 l of volatile oils and placed on the surface of mha .
the volatile dissolving buffer ( 10% aqueous dmso , 0.5% v / v tween 80 ) and tea tree oil were used as negative and positive control , respectively .
after incubation at 37c for 16 - 18 h , the inhibition zone was measured .
all experiments were performed independently in triplicate and mean value was calculated . based on clsi guidelines ,
the preparation of water - insoluble volatile oils was slightly modified from the recommended clsi guidelines .
each volatile oil was dissolved with 50% dmso and serial 2-fold diluted in a 96-well microtiter plate ranging from 0.125 to 8 mg / ml .
the bacterial suspension was diluted into approximately 1  10 cfu / ml , and 100 l of bacterial suspension was applied to each well .
the inoculum with 2.5% dmso and media without inoculum were used as cell and media control , respectively .
the microplates were incubated at 35c for 20 h. due to the turbidity of volatile oil suspensions , iodonitrotetrazolium chloride ( int ) ( biochemica ) was used as color indicator to visualize the bacterial growth .
the mic was detected after added 50 l of 0.2 mg / ml int and further incubated at 35c for 30 min . to determine the mbc ,
10 l of bacterial inoculums were taken aseptically from the wells with no color change and plated onto mha plate and incubated at 35c for 20 - 24 h. all experiments were separately performed in triplicate and calculated as mode , median , and 90 percentile .
median mic value ( mic50 ) represented the mic value of one - half of the tested population .
the 90 percentile ( mic90 ) represented the mic value of 90% of the tested population .
likewise , mbc50 and mbc90 were the mbc values at which 50% or 90% of isolates in a tested population were killed , respectively . in this study , the inhibition zone of each volatile oil was compared with tea tree oil and statistically analyzed using independent student s t - test ( spss version 22 ) .
the mic and mbc values in each of the tested volatile oils and tea tree oil were statistically analyzed by mann 
s. aureus atcc 25923 , escherichia coli atcc 25922 , pseudomonas aeruginosa atcc 27853 , and a. baumannii atcc 19606 were purchased from the department of medical sciences , ministry of public health , thailand .
baumannii were collected from the diagnostic laboratory , maharaj nakorn chiang mai hospital , chiang mai , thailand , during february - april , 2012 . both biochemical tests followed by constantiniu et al .   and
molecular biology test using amplified ribosomal dna - restriction enzyme analysis were performed for identification of a. baumannii .
primers used for 16s rdna gene amplification were designed as followed by the previous report .
the antimicrobial susceptibility testing was performed using disk diffusion method following the clinical and laboratory standards institute ( clsi ) guidelines .
the mdr was defined according to the unsusceptible of at least one in three agents of antimicrobial classes .
all 30 clinical isolates resisted to eight antibiotics in six antimicrobial classes consisting of amikacin , piperacillin / tazobactam , ciprofloxacin , cefoperazone / sulbactam , ceftazidime , trimethoprim / sulfamethoxazole , imipenem , and meropenem .
galangal , ginger , plai , lime , kaffir lime , sweet basil , tree basil , lemongrass , clove , and cinnamon were selected in this study [ table 1 ] .
the material was subjected to hydrodistillation using a clevenger - type glass apparatus for 3 - 5 h .
yields of the volatile oils obtained from the plants were calculated as the percent yield .
0.5 ( 1  10 cfu / ml ) and spread over the mueller - hinton agar ( mha ) plates using a sterile cotton swab .
each volatile oil was dissolved in 10% aqueous dimethyl sulfoxide ( dmso ) with 0.5% v / v tween 80 and sterilized by filtration .
sterilized disks ( whatman no . 5 , 6 mm diameter ) were impregnated with 20 l of volatile oils and placed on the surface of mha .
the volatile dissolving buffer ( 10% aqueous dmso , 0.5% v / v tween 80 ) and tea tree oil were used as negative and positive control , respectively .
after incubation at 37c for 16 - 18 h , the inhibition zone was measured .
based on clsi guidelines , mics were determined by using broth microdilution method . the preparation of water - insoluble volatile oils was slightly modified from the recommended clsi guidelines .
each volatile oil was dissolved with 50% dmso and serial 2-fold diluted in a 96-well microtiter plate ranging from 0.125 to 8 mg / ml .
the bacterial suspension was diluted into approximately 1  10 cfu / ml , and 100 l of bacterial suspension was applied to each well .
the inoculum with 2.5% dmso and media without inoculum were used as cell and media control , respectively .
the microplates were incubated at 35c for 20 h. due to the turbidity of volatile oil suspensions , iodonitrotetrazolium chloride ( int ) ( biochemica ) was used as color indicator to visualize the bacterial growth .
the mic was detected after added 50 l of 0.2 mg / ml int and further incubated at 35c for 30 min . to determine the mbc ,
10 l of bacterial inoculums were taken aseptically from the wells with no color change and plated onto mha plate and incubated at 35c for 20 - 24 h. all experiments were separately performed in triplicate and calculated as mode , median , and 90 percentile .
median mic value ( mic50 ) represented the mic value of one - half of the tested population .
the 90 percentile ( mic90 ) represented the mic value of 90% of the tested population .
likewise , mbc50 and mbc90 were the mbc values at which 50% or 90% of isolates in a tested population were killed , respectively .
in this study , the inhibition zone of each volatile oil was compared with tea tree oil and statistically analyzed using independent student s t - test ( spss version 22 ) .
the mic and mbc values in each of the tested volatile oils and tea tree oil were statistically analyzed by mann 
the yields ranged from 0.1% to 4.3% w / w  ginger ( 0.1 ) , lemongrass ( 0.2 ) , tree basil ( 0.2 ) , galangal ( 0.3 ) , sweet basil ( 0.3 ) , cinnamon ( 0.9 ) , lime ( 1.0 ) , plai ( 1.1 ) , kaffir lime ( 2.1 ) , and clove ( 4.3 ) .
a disk diffusion method was performed to preliminarily evaluate the antibacterial activity of the volatile oils against four reference bacterial strains ( s. aureus , e. coli , p. aeruginosa , and a. baumannii ) . except p. aeruginosa ,
the difference in the inhibition zones between tea tree oil and each volatile oil was analyzed using independent student s t - test .
the results indicated that cinnamon oil exhibited a high potency of antibacterial activity against all bacterial strains tested ( p < 0.01 ) .
sweet basil and lemon grass were highly active against s. aureus and e. coli ; however , these volatile oils showed no significant activity when tested with both non - fermentative gram - negative bacilli , a. baumannii , and p. aeruginosa .
the volatile oils of clove , tree basil , lime , and ginger were moderately active against some bacterial strains ( p < 0.05 ) .
the antibacterial activity of plai and kaffir lime was rather inactive compared to tea tree oil .
the inhibition zones of various volatile oils against s. aureus , e. coli , p. aeruginosa , and a. baumannii standard strains were shown in figures 1 - 4 , respectively .
however , the mean of the inhibition zones of the cinnamon and clove oils was significantly higher than tea tree oil ( p < 0.01 ) . both standard strains of a. baumannii atcc 19606 and mdr - a .
the mic and mbc of the positive control tea tree oil against a. baumannii atcc 19606 were 2 and 4 mg / ml , respectively .
cinnamon oil was highly active , with mic and mbc values of 0.25 mg / ml .
the mics and mbcs of the volatile oils tested against a. baumannii atcc 19606 were shown in table 2 .
baumannii activity with mic90 and mbc90 of 2 and 4 mg / ml , respectively .
the mean mics of four volatile oils , cinnamon , clove , tree basil , and kaffir lime were significantly lower than the positive control tea tree oil with the mic90 of 0.25 , 0.5 , 1 , and 1 mg / ml , respectively ( p < 0.05 ) .
the mic and mbc of the volatile oils against 30 clinical strains of mdr - a .
inhibition zones of 11 volatile oils against staphylococcus aureus atcc 25923 . * indicated a significant difference at p < 0.05 , and * * indicated a highly significant difference at p < 0.01 inhibition zones of 11 volatile oils against escherichia coli atcc 25922 . * * indicated a highly significant difference at p < 0.01 inhibition zones of 11 volatile oils against pseudomonas aeruginosa atcc 27853 . *
* indicated a highly significant difference at p < 0.01 inhibition zones of 11 volatile oils against acinetobacter baumannii atcc 19606 .
* indicated a significant difference at p < 0.05 , and * * indicated a highly significant difference at p < 0.01 average inhibition zone in diameter obtained from various volatile oils against 30 multidrug - resistant - acinetobacter baumannii isolates . * * indicated a highly significant difference at p < 0.01 mics and mbcs of volatile oils against standard strain a. baumannii atcc 19606 the mics and mbcs of volatile oils against mdr - a .
the most problematic of a. baumannii infections nowadays are the mdr and it becomes a serious issue , in which most antibiotics drug therapy are unable to cure the diseases .
baumannii is urgent ; volatile oils are one such compound worth screening . in this study , 10 volatile oils were determined for antibacterial activity against s. aureus , e. coli , p. aeruginosa , a. baumannii , and 30 isolates of mdr - a .
the antimicrobial activity of tea tree oil against aerobic bacteria has previously been published ; the compounds involved in its antibacterial activity such as terpinen-4-ol , a - terpinene and g - terpinene have been characterized . similarly to carson and riley s study , an inactive effect of tea tree oil against p. aeruginosa was observed in this study .
baumannii isolates could be inhibited by tea tree oil with mic90 and mbc90 concentrations of 2 and 4 mg / ml , respectively .
the extracted volatile oils were preliminarily screened for antibacterial activity by disc diffusion method . among the medicinal plants tested , cinnamon oil exerted the highest activity to inhibit the growth of all bacteria while sweet basil and lemon grass strongly inhibited in some bacteria .
standard broth microdilution method was performed and revealed that the volatile oils of cinnamon , clove , tree basil , and kaffir lime showed strong antibacterial activity against mdr - a .
the antimicrobial activity of cinnamon oil against s. aureus , e. coli , acinetobacter 
 lwoffii , and p. aeruginosa has previously been demonstrated .
recently , rath and padhy indicated that the mic and mbc of methanolic extract of both clove and cinnamon against mdr - a .
the inhibition zones of tree basil and tea tree oil were indifferent ; the major constituents of tree basil volatile oil have previously been identified including thymol , g - terpinene , eugenol , and r - cymene . the mode of antibacterial action of thymol still unknown but it has been proposed to involve in outer and inner membrane disruption .
cinnamon oil possessed the highest inhibition effect against all bacterial strains and mdr - a .
mass spectrometry analysis was performed in this study to identify the active ingredients with antimicrobial activity .
thirteen peaks were observed and interpreted based on specific retention time compared to a reference database .
the major ingredients in cinnamon oil were cinnamaldehyde ( 75.89% ) , trans - cinnamyl acetate ( 7.07% ) , hydrocinnamaldehyde ( 2.39% ) , and 1,8-cineole ( 2.17% ) ( data not shown ) .
cinnamaldehyde has previously been reported to inhibit in both gram - positive and gram - negative bacteria .
noteworthy , aldehyde groups might be associated with the antimicrobial activity of cinnamon oil since these chemicals have an ability to covalently cross - link with the amine groups of dna and proteins and interfere their functions in the cells .
although the mode of action of cinnamaldehyde is inconclusive , gill and holley demonstrated that cinnamaldehyde at a concentration of 30 mm could kill l. monocytogenese through its effect on the energy generation and membrane permeability of the bacteria .
in addition , the interaction of cinnamaldehyde with essential enzymes and bacterial cell wall damage at high concentration has been investigated .
although cinnamaldehyde possesses potent antimicrobial activity against mdr pathogen , its cellular and in vivo cytotoxicity have been reported .
in addition , it has been reviewed to be a cause of allergic reaction in toothpaste .
consequently , a dosage level at which no adverse effects is indispensable determined before use in the future application .
our study indicated the antibacterial activity of volatile oils extracted from herbs against several bacteria , including mdr - a .
these plant extracts would be promising antimicrobial agents for further treating of human pathogens , including drug - resistant bacteria .
 aim : the aim of the study is to investigate the antibacterial activity of 10 volatile oils extracted from medicinal plants , including galangal ( alpinia galanga linn . ) , ginger ( zingiber officinale ) , plai ( zingiber cassumunar roxb . ) , lime ( citrus aurantifolia ) , kaffir lime ( citrus hystrix dc . ) , sweet basil ( ocimum basilicum linn . ) , tree basil ( ocimum gratissimum ) , lemongrass ( cymbopogon citratus dc . ) , clove ( syzygium aromaticum ) , and cinnamon ( cinnamomum verum ) against four standard strains of staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , acinetobacter baumannii , and 30 clinical isolates of multidrug - resistant a. baumannii ( mdr - a . 
 baumannii).materials and methods : agar diffusion , minimum inhibitory concentration , and minimum bactericidal concentration ( mbc ) were employed for the determination of bactericidal activity of water distilled medicinal plants . 
 tea tree oil ( melaleuca alternifolia ) was used as positive control in this study.results:the results indicated the volatile oil extracted from cinnamon exhibited potent antibacterial activity against the most common human pathogens , s. aureus , e. coli , p. aeruginosa , and a. baumannii . 
 most of volatile oil extracts were less effective against non - fermentative bacteria , p. aeruginosa . 
 in addition , volatile oil extracted from cinnamon , clove , and tree basil possessed potent bactericidal activity against mdr - a . 
 baumannii with mbc90 of 0.5 , 1 , and 2 mg / ml , respectively.conclusions:the volatile oil extracts would be useful as alternative natural product for the treatment of the most common human pathogens and mdr - a . 
 baumannii infections . 