Thai ethnomedicinal plants as resistant modifying agents for combating Acinetobacter baumannii infections

  • Pinanong Na Phatthalung1,

    Affiliated with

    • Sasitorn Chusri2 and

      Affiliated with

      • Supayang P Voravuthikunchai1Email author

        Affiliated with

        BMC Complementary and Alternative MedicineThe official journal of the International Society for Complementary Medicine Research (ISCMR)201212:56

        DOI: 10.1186/1472-6882-12-56

        Received: 19 March 2012

        Accepted: 26 April 2012

        Published: 26 April 2012

        Abstracts

        Background

        Acinetobacter baumannii is well-recognized as an important nosocomial pathogen, however, due to their intrinsic resistance to several antibiotics, treatment options are limited. Synergistic effects between antibiotics and medicinal plants, particularly their active components, have intensively been studied as alternative approaches.

        Methods

        Fifty-one ethanol extracts obtained from 44 different selected medicinal plant species were tested for resistance modifying agents (RMAs) of novobiocin against A. baumannii using growth inhibition assay.

        Results

        At 250 μg/ml, Holarrhena antidysenterica, Punica granatum, Quisqualis indica, Terminalia bellirica, Terminalia chebula, and Terminalia sp. that possessed low intrinsic antibacterial activity significantly enhanced the activity of novobiocin at 1 μg/ml (1/8xminimum inhibitory concentration) against this pathogen. Holarrhena antidysenterica at 7.8 μg/ml demonstrated remarkable resistant modifying ability against A. baumannii in combination with novobiocin. The phytochemical study revealed that constituents of this medicinal plant contain alkaloids, condensed tannins, and triterpenoids.

        Conclusion

        The use of Holarrhena antidysenterica in combination with novobiocin provides an effective alternative treatment for multidrug resistant A. baumannii infections.

        Background

        An underestimated nosocomial pathogen, Acinetobacter baumannii, is now widely acknowledged as a common bacterium in hospital irrigation and intravenous solutions. It possesses inherent multidrug-resistance (MDR) and the ability to rapidly colonize and infect patients. Moreover, the emergence of acquired MDR by A. baumannii to conventional antibiotics presents a serious therapeutic problem in the treatment of the infections [1, 2]. Several investigations suggested that synergy effects of plant secondary metabolites and conventional antibiotics could be an alternative way to increase the bacterial susceptibility [36].

        Plants, particularly ethnomedicinal plants are important sources of natural products. They are rich in a wide variety of secondary metabolites such as tannins, terpenoids, alkaloids, and flavonoids and have been well-established to possess antimicrobial properties [7]. Many plants have been evaluated not only for their inherent antimicrobial activity, but also for their action as a resistant modifying agent (RMA) [4].

        Novobiocin, a Gyr B inhibitor, is an effective aminocoumarin drug for the treatment of Gram-positive bacterial infections. However, its low level of activity against Gram-negative pathogens causes a major limitation [8]. Although, several investigations observed synergy and mechanisms of action between natural products and synthetic drugs in effectively combating Gram positive bacterial infections [5], there are a few RMA effective for use with A. baumannii[9, 10]. Therefore, the aim of this study was to further explore the resistant modifying activity of a wide range of medicinal plants according to their ethnobotanical basis in combination with novobiocin against A. baumannii.

        Methods

        Bacterial strain and culture condition

        Acinetobacter baumannii ATCC 19606 was employed in this study as a model reference strain. The strain was susceptible to ciprofloxacin, colistin, imipenem, and tobramycin and resistant to amikacin, ampicillin, azithromycin, erythromycin, and gentamicin which conducted by disc diffusion method [11]. Well-isolated colonies of A. baumannii ATCC 19606 were grown in Mueller Hinton Broth (MHB) (Difco Laboratories, Detroit, MI) at 37°C for 18–24 h. The culture density was adjusted to McFarland standards No. 0.5 and resuspended in MHB to obtain a final concentration of 1 × 106 cfu/ml.

        Medicinal plant materials

        Tested medicinal plants are shown in Table 1. Fifty-one ethanol extracts of 44 Thai medicinal plant species were kindly provided by the Natural Products Research Center, Prince of Songkla University, Hat Yai, Thailand [12]. Collected plant materials were washed with distilled water and dried at 60°C overnight. Ground plant material was macerated with 95% ethanol (1:2 w/v) for 7 days. The extract was filtered and evaporated using rotary evaporator at 45°C until it became completely dry. A stock solution (200 mg/ml) was prepared by dissolving 0.2 g of the dried extract in 1 ml of dimethylsulfoxide (DMSO) (Merck, Germany) and stored at −20°C.
        Table 1

        Intrinsic antibacterial activity and resistant modifying ability of crude extract (250 μg/ml) in combination with novobiocin (1/8xMIC) against Acinetobacter baumannii ATCC 19606

         

        Botanical names

        Family name

        Part used

        %Growth inhibitiona ± SDb

        Interpretationc

            

        PE

        PE + NOV

         

        1

        Aegle marmelos (L.) Corr. Serr.

        Rutaceae

        Fruit

        22.10 ± 0.68

        27.10 ± 1.38

        No synergy

        2

        Ardisia colorata Roxb.

        Primulaceae

        Fruit

        30.17 ± 2.56

        39.00 ± 6.09

        Synergy

        3

        Asclepias curassavica L.

        Asclepiadaceae

        Wood

        40.81 ± 0.28

        43.59 ± 1.78

        No synergy

        4

        Centella asiatica (L.) Urb.

        Apiaceae

        Whole

        19.09 ± 1.06

        23.93 ± 2.87

        No synergy

        5

        Cinnamomum bejolghota (Buch.-Ham.) Sweet

        Lauraceae

        Wood

        58.84 ± 1.37

        59.92 ± 1.78

        No synergy

           

        Bark

        55.62 ± 4.98

        62.44 ± 2.91

        No Synergy

        6

        Cinnamomum porrectum (Roxb.) Kosterm.

        Lauraceae

        Wood

        29.72 ± 6.54

        26.06 ± 5.21

        No synergy

           

        Bark

        56.88 ± 2.14

        63.31 ± 4.87

        No synergy

        7

        Curcuma longa L.

        Zingiberaceae

        Rhizome

        86.91 ± 2.64

        88.78 ± 2.08

        No synergy

        8

        Curcuma zedoaria (Christm.) Roscoe

        Zingiberaceae

        Rhizome

        77.73 ± 0.48

        79.59 ± 2.62

        No synergy

        9

        Derris scandens Benth.

        Leguminosea

        Stem

        49.01 ± 2.37

        47.31 ± 3.84

        No synergy

        10

        Dracaena loureiri Gagnep.

        Agavaceae

        Wood

        30.08 ± 0.99

        29.49 ± 3.19

        No synergy

        11

        Dryopteris syrmatica (Willd.) Kuntze

        Dryopteridaceae

        Stem

        17.59 ± 0.41

        26.66 ± 5.32

        Synergy

        12

        Eleutherine americana (Aubl.) Merr. ex K.

        Iridaceae

        Bulb

        17.87 ± 1.89

        22.26 ± 3.12

        No synergy

        13

        Euphorbia thymifolia L.

        Euphorbiaceae

        Whole plant

        53.64 ± 0.90

        73.99 ± 0.88

        Synergy

        14

        Garcinia mangostana L.

        Clusiaceae

        Pericarp

        93.25 ± 3.65

        90.48 ± 3.37

        No synergy

        15

        Gymnopetalum cochinchinensis (Lour.) Kurz

        Cucurbitaceae

        Fruit

        26.17 ± 0.59

        32.45 ± 4.39

        No synergy

        16

        Holarrhena antidysenterica (L.) Wall. ex A. DC.

        Apocynaceae

        Bark

        65.88 ± 0.11

        94.04 ± 0.59*

        Synergy

        17

        Impatiens balsamina L.

        Balsaminaceae

        Stem

        9.77 ± 0.30

        12.40 ± 1.56

        No synergy

        18

        Manilkara achras (Mill.) Fosb.

        Sapotaceae

        Fruit

        56.59 ± 1.02

        63.06 ± 2.97

        No synergy

        19

        Millingtonia hortensis L.f.

        Bignoniaceae

        Flower

        28.97 ± 4.30

        54.08 ± 0.83

        Synergy

        20

        Mitragyna speciosa Korth.

        Rubiaceae

        Leaf

        43.33 ± 2.40

        66.15 ± 0.26

        Synergy

        21

        Momordica charantia L.

        Cucurbitaceae

        Vine

        22.26 ± 0.85

        25.79 ± 3.10

        No synergy

        22

        Morinda citrifolia L.

        Rubiaceae

        Fruit

        16.96 ± 0.63

        25.86 ± 1.22

        Synergy

        23

        Murdannia loriformis (Hassk.) R. Rao & Kammathy

        Commilinaceae

        Whole plant

        16.42 ± 1.51

        22.04 ± 1.67

        No synergy

        24

        Oroxylum indicum (L.) Vent.

        Bignoniaceae

        Leaf

        67.18 ± 1.59

        71.30 ± 5.28

        No synergy

        25

        Peltophorum pterocarpum (DC.) Backer ex K. Heyne

        Fabaceae

        Flower

        42.80 ± 0.43

        47.83 ± 4.49

        No synergy

           

        Bark

        78.26 ± 0.60

        88.75 ± 6.10

        Synergy

        26

        Piper betle L.

        Piperaceae

        Leaf

        42.72 ± 0.13

        39.92 ± 3.43

        No synergy

        27

        Piper nigrum L.

        Piperaceae

        Fruit

        38.07 ± 1.96

        42.24 ± 2.60

        No synergy

           

        Seed

        29.07 ± 0.75

        31.47 ± 3.27

        No synergy

        28

        Piper retrofractum Vahl

        Piperaceae

        Fruit

        44.02 ± 1.08

        49.80 ± 4.19

        No synergy

        29

        Piper sarmentosum Roxb

        Piperaceae

        Leaf

        20.70 ± 0.88

        25.02 ± 0.62

        No synergy

        30

        Pluchea indica (L.) Less.

        Asteraceae

        Leaf

        26.64 ± 0.97

        53.59 ± 3.60*

        Synergy

        31

        Psidium guajava L.

        Myrtaceae

        Leaf

        71.24 ± 2.00

        81.19 ± 1.50*

        Synergy

        32

        Punica granatum L.

        Puniceaceae

        Pericarp

        72.58 ± 1.20

        99.29 ± 0.63*

        Synergy

        33

        Quercus infectoria G.Olivier

        Fagaceae

        Gall

        89.09 ± 0.15

        88.77 ± 1.00

        No synergy

        34

        Quisqualis indica L.

        Combretaceae

        Flower

        79.22 ± 0.28

        94.63 ± 2.62*

        Synergy

        35

        Rhizophora mucronata Lam.

        Rhizophoraceae

        Fruit

        44.64 ± 0.59

        53.35 ± 2.56

        Synergy

           

        Bark

        42.68 ± 8.20

        53.03 ± 4.95

        Synergy

        36

        Rhodomyrtus tomentosa (Aiton) Hassk.

        Myrtaceae

        Stem

        77.01 ± 1.28

        81.81 ± 4.01

        No synergy

        37

        Sandoricum indicum Cav.

        Meliaceae

        Root

        65.24 ± 1.32

        66.94 ± 2.13

        No synergy

        38

        Tamarindus indica L.

        Fabaceae

        Leaf

        19.76 ± 1.55

        25.03 ± 3.45

        No synergy

        39

        Terminalia bellirica (Gaertn.) Roxb.

        Combretaceae

        Fruit

        74.79 ± 0.53

        95.68 ± 1.14*

        Synergy

        40

        Terminalia chebula (Gaertn.) Retz.

        Combretaceae

        Fruit

        61.25 ± 0.42

        94.33 ± 1.95*

        Synergy

        41

        Terminalia sp.

        Combretaceae

        Fruit

        79.53 ± 0.24

        95.92 ± 1.10*

        Synergy

        42

        Theobroma cacao L.

        Sterculiaceae

        Pericarp

        17.35 ± 0.74

        22.81 ± 0.68

        No synergy

           

        Seed

        19.25 ± 1.08

        29.61 ± 4.13

        Synergy

        43

        Vitex trifolia L.

        Verbenaceae

        Leaf

        22.12 ± 0.68

        28.65 ± 3.57

        No synergy

        44

        Xylocarpus granatum J. Koenig.

        Meliaceae

        Pericarp

        52.39 ± 3.48

        53.27 ± 1.91

        No synergy

           

        Seed

        44.27 ± 5.13

        54.55 ± 3.66

        No synergy

        Percentage of growth inhibition of novobiocin against A. buamannii ATCC 19606 was 6.67%.

        aPercentage of growth inhibition in the present of plant extract (PE) and plant extract in combination with novobiocin (PE + NOV) against A. buamannii ATCC 19606.

        bSD Standard Deviation.

        cSynergy: (PE + NOV) > (PE) + (NOV); No synergy: (PE + NOV) < (PE) + (NOV) [6].

        *P < 0.01: Significantly different from the effect of plant extract.

        Determination of minimum inhibitory concentration (MIC) of novobiocin

        The MIC of novobiocin was determined by the broth microdilution method as described by the Clinical and Laboratory Standard Institute (CLSI) [13].

        Intrinsic antibacterial activity and resistant modifying ability of medicinal plant extracts

        Intrinsic antibacterial activities were determined by growth inhibition assays [9]. The bacterial culture (100 μl) was inoculated into a 96-well microtiter plate containing 50 μl of crude extracts (1,000 μg/ml) and 50 μl of MHB and then incubated at 37°C for 18 h. The intrinsic antibacterial activity was exhibited as the percentage of growth inhibition and calculated from the following equation:
        http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-12-56/MediaObjects/12906_2012_1052_Equ1_HTML.gif

        Where ODA is Optical density (OD) 595 nm of bacteria culture in MHB supplemented with 1%DMSO as positive control and ODB is OD 595 nm of the bacterial culture in MHB supplemented with plant extracts.

        Resistant modifying ability of the extracts was observed by adding of 50 μl novobiocin at a concentration of 1/8xMIC (1 μg/ml) into the tested plate instead of MHB. This biological activity was exhibited as the percentage of growth inhibition as well but calculated from the following equation, where ODC is OD 595 nm of the bacterial culture in MHB supplemented with the plant extract in combination with novobiocin:
        http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-12-56/MediaObjects/12906_2012_1052_Equ2_HTML.gif

        Effective medicinal plants that demonstrated a synergistic effect with novobiocin and exhibited bacterial growth inhibition more than 90% were selected for further experiments. The efficacy of combination therapy of the promising medicinal plants with novobiocin was additionally determined by measuring the resistant modifying capabilities of the extracts at varying concentrations ranging from 7.8 to 250 μg/ml.

        Phytochemical screening methods

        Phytochemical screening tests for alkaloids, condensed tannins, flavonoids, hydrolysable tannins, steroids, and triterpenes were qualitatively analyzed by standard colour tests as previously described [14].

        Results and discussion

        Intrinsic resistance of A. baumannii to novobiocin was observed with MIC value at 8 μg/ml. As shown in Table 1, 48 out of 51 tested ethanol extracts at concentration of 250 μg/ml had low inherent antibacterial activity (% of bacterial growth inhibition was less than 80%). In combination with the antibiotic, the extracts of 18 medicinal plants demonstrated synergistic interaction against A. baumannii. Interestingly, the bacterial growth inhibition in the presence of novobiocin in combination with the extracts of Holarrhena antidysenterica, Punica granatum, Quisqualis indica, Terminalia bellirica, Terminalia chebula, and Terminalia sp. extracts was significantly higher than the intrinsic antibacterial activity of the extracts (Table 1).

        To explore the potential of developing a more powerful combination therapy of these medicinal plants with novobiocin, we determined the resistant modifying ability of varying concentrations of the extracts from 7.8 to 250 μg/ml by growth inhibition assay as illustrated in Figure 1. Holarrhena antidysenterica extract which concentrations ranging from 7.8 to 62.5 μg/ml possessed no intrinsic anti-acinetobacter activity (Figure 1A) was demonstrated to be a powerful RMA in combination with novobiocin against this pathogen.
        http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-12-56/MediaObjects/12906_2012_1052_Fig1_HTML.jpg
        Figure 1

        Bacterial growth inhibition of Holarrhena antidysenterica (A), Punica granatum (B), Quisqualis indica (C), Terminalia bellirica (D), Terminalia chebula (E), and Terminalia sp. (F) ethanol extracts (○) and the extracts in combination with 1/8xMIC of novobiocin (●) against Acinetobacter baumannii ATCC 19606. Percentage of bacterial growth inhibition of 1/8xMIC of novobiocin on this pathogen was 6.67%.

        Our preliminary phytochemical test revealed that alkaloids were common principles among the effective extracts. In addition to alkaloids, other compounds including condensed tannins, triterpenoids, flavonoids, hydrolysable tannins, and steroids were detected (Table 2). Although the antibiotic resistant modifying ability of active principles of the effective medicinal plants has never been investigated, plant-derived alkaloids have been well-clarified as efflux pump inhibitors (EPIs) for Gram positive bacteria [15, 16]. Recent evaluation of 13 phyto-alkaloids for their EPI potential against staphylococcal isolates revealed that 60% and 30% of the tested compounds exhibited the activity against methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible S. aureus (MSSA), respectively [16]. Four plant-derived alkaloids consisting of reserpine, quinine, harmaline, and piperine possessed notable potential EPI activities on both MRSA and MSSA [16]. More importantly, their mechanisms of actions as a RMA have been proposed. Piperine was recorded as an inhibitor of MdeA [17] and NorA [18] efflux pumps of S. aureus and Rv1258c efflux pump of Mycobacterium tuberculosis[19]. Reserpine was found as an inhibitor of Bmr efflux pump in Bacillus subtilis, Tet(K) and NorA efflux pumps of S. aureus[20]. In addition to phyto-alkaloids, several plant-derived polyphenols such as epigallocatechin gallate of Camellia sinesis, tellimagrandin I and rugosin B isolated from Rosa canina have been established as useful RMAs with different mechanisms of actions including inhibitions of adapted drug target sites or enzymatic degradation of drugs [4]. Intensive investigations on plant-derived compounds as RMAs have been performed in Gram-positive, but relatively very few studies have been carried out to evaluated RMA activities of plant-derived compounds on Gram-negative bacteria [2123].
        Table 2

        Extraction yields and phytochemical constituents of tested medicinal plant extracts

         

        Botanical names

        Part used

        Yield (%; w/w)a

        Phytochemical constituentsb

            

        1

        2

        3

        4

        5

        6

        1

        Aegle marmelos (L.) Corr. Serr.

        Fruit

        5.3

        +

        +

        +

        -

        +

        -

        2

        Ardisia colorata Roxb.

        Fruit

        4.4

        +

        +

        -

        -

        +

        -

        3

        Asclepias curassavica L.

        Wood

        0.9

        +

        +

        -

        -

        -

        -

        4

        Centella asiatica (L.) Urb.

        Whole

        6.0

        +

        -

        -

        -

        +

        -

        5

        Cinnamomum bejolghota (Buch.-Ham.) Sweet

        Wood

        2.2

        +

        +

        -

        -

        +

        -

          

        Bark

        14.6

        +

        -

        -

        +

        +

        -

        6

        Cinnamomum porrectum (Roxb.) Kosterm.

        Wood

        11.2

        -

        -

        -

        -

        +

        -

          

        Bark

        7.0

        +

        +

        -

        -

        +

        -

        7

        Curcuma longa L.

        Rhizome

        13.9

        +

        +

        +

        -

        +

        -

        8

        Curcuma zedoaria (Christm.) Roscoe

        Rhizome

        13.9

        +

        +

        +

        -

        -

        +

        9

        Derris scandens Benth.

        Stem

        3.2

        -

        +

        -

        -

        +

        -

        10

        Dracaena loureiri Gagnep.

        Wood

        16.9

        -

        -

        -

        -

        -

        +

        11

        Dryopteris syrmatica (Willd.) Kuntze

        Stem

        4.5

        +

        +

        -

        -

        +

        -

        12

        Eleutherine americana (Aubl.) Merr. ex K.

        Bulb

        4.8

        +

        +

        -

        -

        -

        -

        13

        Euphorbia thymifolia L.

        Whole plant

        1.3

        -

        +

        -

        -

        +

        -

        14

        Garcinia mangostana L.

        Pericarp

        5.3

        -

        -

        -

        -

        -

        -

        15

        Gymnopetalum cochinchinensis (Lour.) Kurz

        Fruit

        7.6

        -

        -

        -

        -

        +

        -

        16

        Holarrhena antidysenterica (L.) Wall. ex A. DC.

        Bark

        2.1

        +

        +

        -

        -

        -

        +

        17

        Impatiens balsamina L.

        Stem

        5.2

        -

        +

        -

        -

        +

        -

        18

        Manilkara achras (Mill.) Fosb.

        Fruit

        26.7

        +

        -

        +

        -

        -

        +

        19

        Millingtonia hortensis L.f.

        Flower

        25.4

        +

        +

        +

        -

        -

        -

        20

        Mitragyna speciosa Korth.

        Leaf

        5.9

        +

        +

        -

        -

        +

        -

        21

        Momordica charantia L.

        Vine

        3.0

        +

        -

        -

        -

        +

        -

        22

        Morinda citrifolia L.

        Fruit

        7.3

        +

        -

        +

        -

        +

        -

        23

        Murdannia loriformis (Hassk.) R. Rao & Kammathy

        Whole plant

        7.6

        +

        -

        -

        -

        +

        -

        24

        Oroxylum indicum (L.) Vent.

        Leaf

        3.7

        +

        +

        -

        -

        +

        -

        25

        Peltophorum pterocarpum (DC.) Backer ex K. Heyne

        Flower

        7.1

        +

        -

        -

        -

        -

        -

          

        Bark

        7.1

        +

        +

        -

        -

        -

        +

        26

        Piper betle L.

        Leaf

        12.4

        -

        +

        -

        -

        +

        -

        27

        Piper nigrum L.

        Fruit

        4.2

        +

        -

        -

        -

        +

        -

          

        Seed

        4.2

        +

        -

        -

        -

        +

        -

        28

        Piper retrofractum Vahl

        Fruit

        7.0

        -

        -

        -

        -

        +

        -

        29

        Piper sarmentosum Roxb

        Leaf

        1.7

        +

        -

        -

        -

        +

        -

        30

        Pluchea indica (L.) Less.

        Leaf

        17.8

        +

        +

        -

        -

        +

        -

        31

        Psidium guajava L.

        Leaf

        8.0

        +

        +

        -

        -

        +

        -

        32

        Punica granatum L.

        Pericarp

        13.0

        +

        +

        +

        -

        -

        +

        33

        Quercus infectoria G.Olivier

        Gall

        37.8

        +

        -

        -

        +

        -

        -

        34

        Quisqualis indica L.

        Flower

        11.0

        +

        -

        +

        +

        +

        -

        35

        Rhizophora mucronata Lam.

        Fruit

        10.7

        +

        +

        -

        -

        -

        +

          

        Bark

        11.6

        -

        +

        -

        -

        -

        +

        36

        Rhodomyrtus tomentosa (Aiton) Hassk.

        Stem

        7.1

        +

        +

        -

        -

        -

        +

        37

        Sandoricum indicum Cav.

        Root

        4.0

        +

        -

        -

        -

        +

        -

        38

        Tamarindus indica L.

        Leaf

        4.8

        +

        +

        +

        -

        +

        -

        39

        Terminalia bellirica (Gaertn.) Roxb.

        Fruit

        14.8

        +

        -

        -

        -

        +

        -

        40

        Terminalia chebula (Gaertn.) Retz.

        Fruit

        5.9

        +

        +

        -

        -

        -

        +

        41

        Terminalia sp.

        Fruit

        23.9

        +

        -

        -

        +

        -

        -

        42

        Theobroma cacao L.

        Pericarp

        3.6

        +

        +

        -

        -

        +

        -

          

        Seed

        5.9

        -

        +

        +

        -

        -

        +

        43

        Vitex trifolia L.

        Leaf

        NDc

        +

        +

        -

        -

        +

        -

        44

        Xylocarpus granatum J. Koenig.

        Pericarp

        2.6

        +

        +

        -

        -

        +

        -

          

        Seed

        6.7

        +

        +

        +

        -

        -

        +

        aPercentage extract yields of medicinal plants were weight of crude extract per 100 g of dried plant materials.

        bPhytochemincal constituents: 1, alkaloids; 2, condensed tannins; 3, flavonoids; 4, hydrolysable tannins; 5, steroids and 6, triterpenoids; ‘-’ indicates absence of phytoconstituents ‘+’ indicates presence of phytoconstituents.

        c ND Not determined.

        In the last decade multidrug resistance in A. baumannii became a serious growing problem worldwide. Colistin, an old antibiotic with risk toxicity, has recently been brought back into use to treat MDR bacteria as a stopgap measure until new antibiotics can be developed [24]. A number of workers have proposed the synergistically action combination of conventional antibiotics with RMA act synergistically against MDR Gram-negative bacteria [4, 25, 26]. We have demonstrated that certain plant ethanol extracts significantly enhanced the activity of novobiocin against A. baumannii. Holarrhena antidysenterica is of interest since the extract at 7.8 to 62.5 μg/ml possessed no intrinsic antibacterial activity, but in combination with sub-MIC of novobiocin led to a marked decrease in the bacterial growth. Alkaloids were proposed as active principles of the plant that possessed antibacterial activity on S. aureus S. epidermidis Streptococcus faecalis B. subtilis Escherichia coli, and Pseudomonas aeruginosa[2729]. Some of the alkaloids such as pubadysone, pubescine, norholadiene, pubescimine, puboestrene, pubamide, and naringenin was isolated form bark, seeds, and leaves of this plant [3032].

        Our previous investigation demonstrated that ellagic acid which acts as an efflux pump inhibitor exhibited a synergistic effect with novobiocin and other aminocoumarins against both A. baumannii ATCC 19606 and MDR A. baumannii[9]. Ethylenediaminetetraacetic acid and polyethyleneimine that disturb outer membrane permeability have been reported as RMA for novobiocin against P. aeruginosa and Stenotrophomonas morelense[33, 34]. Similarly, berry-derived phenolic compounds that efficiently destabilized outer membrane permeability resulted in increase in novobiocin susceptibility of Salmonella enterica serotype Typhimurium [35].

        Since intrinsic novobiocin resistance in A. baumannii is related to the synergistic interaction between limited outer membrane permeability and energy-dependent multidrug efflux pumps [36, 37], the RMA for novobiocin possibly acts as a permeabilizer and/or an efflux pump inhibitor.

        Conclusion

        The RMA activity of Thai medicinal plants in combination with novobiocin against A. baumannii is reported for the first time. These findings led us to the development of a new generation of phytopharmaceuticals that using plant-derived compounds in combination with existing antibiotics to treat MDR A. baumannii that currently are almost untreatable. Its mechanism of action as well as the active constituents of a promising plant, Holarrhena antidysenterica should be further investigated.

        Declarations

        Acknowledgments

        This work was supported by the Thailand research Fund-the Commission on Higher Education (MRG 5480069, Fiscal year 2011–2013) and the Higher Education Research Promotion and National Research University of Thailand, Office of the Higher Education Commission.

        Authors’ Affiliations

        (1)
        Natural Products Research Center and Department of Microbiology, Faculty of Science, Prince of Songkla University
        (2)
        Faculty of Traditional Thai Medicine, Prince of Songkla University

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        38. Pre-publication history

          1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1472-6882/​12/​56/​prepub

        Copyright

        © Na Phatthalung et al.; licensee BioMed Central Ltd. 2012