Antimicrobial Survey of Local Herbal Drugs against Acinetobacter baumannii Isolated from Patients Admitted to a Level-I Trauma Center

Introduction

Trauma is a cellular disruption caused by an environmental energy that is beyond the body's resilience. Trauma remains the most common cause of death for all individuals between the ages of 1 and 44 years and is the third most common cause of death regardless of age. It is also the number one cause of years of productive life lost. Death after trauma has a trimodal distribution. Acute deaths (less than 24 hours) usually result from uncontrolled conditions, but infections and multiple organ dysfunction syndrome, which often arise from infection, are responsible for a significant proportion of late deaths. Indeed, infection is responsible for most deaths in patients who survive longer than 48 hours after trauma. Trauma infections can be divided into the injury and nosocomial infections. Most post traumatic infections are polymicrobial, involving a mixture of aerobic and anaerobic organisms. In one series, 37-45% of all trauma patients experienced infectious complications during their initial hospitalization. In that same study, 80% of all trauma patients who were in ICU at least 7 days met the criteria for SIRS [1]. Humans have evolved mechanisms to avoid infection despite presence of bacteria in our environment. Under normal circumstances there is a balance between our microbial, intact environmental barriers, and host defenses. Traumatic injury disrupts this balance and significantly increases the probability of developing an infection [1].
Acinetobacter baumannii is an aerobic Gram-negative bacillus which is non-fermentative, catalase-positive and oxidase-negative [2]. Considering the resistance to multiple antimicrobial agents along with the wide spreading nosocomial infections, A. baumannii has been evolved from being an underestimated microorganism to one of the most important factors causing the various infections at hospital environments, mainly in immunocompromised patients over the last 30 years [3-6]. Surprisingly, the intrinsic resistance of Acinetobacters could be related to not only the genetic materials including plasmids, transposons and integrons but also the ability to survive on anhydrous surfaces for the long interval periods [3,7-9]. Multidrug-resistant A. baumannii strains are frequently found to be responsible for epidemics of nosocomial infections, such as respiratory, bloodstream, urinary tract, skin, and soft tissue infections [10].
According to ethnopharmacological survey in Iranian folk medicine, we found some medical plants that used to prevent post-operative infection rate [11,12]. Therefore, we evaluated the antibacterial activity of some of these medical plants against Acinetobacters infection. The main purpose of this in vitro study was to evaluate the antibacterial activity of volatile oils obtained from some medicinal plants belonging to the Asteraceae/Compositae (Artemisia sieberi and Tanacetum dumosum) and Lamiaceae (Salvia mirzayanii, Mentha mozaffarianii and Satureja bachtiarica) on Acintobacters isolated from Trauma patients in Shahid Rajaee Hospital of Shiraz.

Materials and Methods

Plant material
The aerial parts of the plants were collected from South and Southwest of Iran in March until July 2017 in the flowering stage of the plants. The plants were identified by regional floras and authors with floristic and taxonomic references [13], and voucher specimens were deposited at the herbarium of Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. The shade-dried aerial parts of the plants were extracted by hydrodistillation for 3 h, giving relatively high yield oils in all collections (Table 1). The oils were dried over anhydrous sodium sulfate and kept at -4ºC.

Table 1. Chemical constituents (%) of the essential oil of S. bakhtiarica analyzed by GC-FID.

*The values are the means of three different FID area percentage ± SE; **The major compounds with more than 1.0% area concentration were formatted in bold font.

Gas Chromatography Analysis
Gas Chromatography was performed on a Varian CP-3800 chromatograph, with a FID and a HP-5 column (30 m × 0.25 mm i.d., 0.25 μm film thickness). The oven temperature was programmed from 60°C to 240°C at 5°C/min and finally kept 10 min at this temperature. The carrier gas was N2 with a flow rate of 0.9 mL/min. Injector and detector temperature were set at 250°C. The injection volume was 0.4 μl for the volatile oils and also n-alkanes for calculation of the retention indices was separately injected.

Gas Chromatography-Mass Spectroscopy Analyses
The GC-MS was carried out on an Agilent 7890N chromatograph, coupled to an Agilent 5975C mass spectrometer (Agilent Technologies, USA), operating at 70 eV ionization energy, 0.5 s/scan and the mass range: 35-400, equipped with a HP-5 MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm). Helium was used as the carrier gas at the constant flow of 0.9 ml/min and split ratio of 1:50. The oven temperature was 60°C rising to 250°C at a rate of 5°C /min, then held at 250°C for 10 min; transfer line temperature was set at 250°C.

Identification of compounds
Retention indices (RI) for all constituents were calculated according to Van den Dool approach [14], using n-alkanes (C6 – C22) as standards and the essential oils on a HP-5 column under the same chromatographic conditions. The identification of the components was made based on comparison of their mass spectra with those of the internal computer reference mass spectra libraries (Wiley 7.0 and Nist), as well as by comparison of their retention indices with data mentioned in the previous literatures [15].

Antibacterial Activity
The antimicrobial activity of the obtained essential oils was tested by using the disc-diffusion method on Muller Hinton Agar (MHA) [16] using determination of inhibition zones, and determining the minimal inhibitory concentration (MIC) using the macro dilution broth susceptibility assay as recommended by NCCLS [17].

Study Population and Sampling
All trauma patients admitted in Shahid Rajaee Hospital, the unique center of trauma in Shiraz, affiliated to Shiraz University of Medical Sciences (SUMS) hospital with single of sepsis and positive blood culture for Acintobacter baumannii. The hospital is a referral center of trauma for Fars Province situated in Southern part of Iran.

Microorganisms and Media
Disc diffusion assay:
Muller Hinton agar (MHA) was autoclaved at 121°C for 20 min. The plates (8-cm diameter) were prepared with 10 ml agar inoculated with 1 ml of each bacterial suspension. Microorganisms were cultured for 16 to 24 h at 37°C and prepared to turbidity equivalent to McFarland Standard No. 0.5. Sterile paper discs (6 mm in diameter) were impregnated with 20 μl of dilutions of known essential oil concentrations (5 μg/disc) and incubated at 37°C for 24 h. The essential oils were dissolved in dimethyl sulfoxid (DMSO, 15 μl) before the test for antimicrobial activity. Discs (6 mm diameter) of Colistin (10 μg) was used as positive controls. Bacterial growth inhibition was determined as the diameter of the inhibition zones around the discs (mm). The growth inhibition diameter was an average of three measurements, taken at three different directions. All tests were performed in triplicate.

Minimum Inhibitory Concentration (MIC) Using Muller Hinton Broth Micro-Dilution (MHB)
The minimal inhibitory concentration (MIC) values were evaluated using the broth serial dilution method according to standard methods [18]. The MIC was defined as the lowest concentration of the compound to inhibit the growth 50% of microorganisms. Bacterial strains were cultured overnight at 37 °C in Muller Hinton Broth (MHB, Oxoid). Stock solution of the essential oil was prepared in 33.3% (v/v) dimethyl sulfoxide (DMSO). Dilution series, using MHB, were prepared from 0.062-8 mg/ml. After incubation at 37 °C for 24 h, the microorganism growth inhibition was evaluated by measuring absorbance at 600 nm, and prepared to turbidity equivalent to McFarland Standard No. 0.5 using a spectrophotometer. An aliquot of the samples (5 μl) was added to 95 μl of fresh media followed by 100 μl of the bacterial suspension (OD=0.1 at 600 nm) in a 96-well plate. The plates were incubated at 37ºC for 24 h in a shaking incubator. Colistin was used as positive control in each assay. DMSO solution was used as a negative control. Control tubes were incubated under the same condition. Antibacterial activity was detected using a colorimetric method by adding 10 μl of 0.5% INT solution in water in each well at the end of the incubation period for further 30 min. Experiments were performed in triplicate but at three different times.

Results

In terms of measuring the antibacterial activity using disc diffusion method, the significant zones of inhibition around the discs were noted in Tables 2 and 3. Our examinations investigated the antimicrobial activities of medicinal plants belonging to the Asteraceae (Artemisia sieberi and Tanacetum dumosum) and Lamiaceae (Salvia mirzayanii, Mentha mozaffarianii and Satureja bachtiarica) families. Careful results on examined bacterial strains revealed that the essential oil of Satureja bachtiarica Bunge assayed by both Disk Diffusion and Minimum Inhibitory Concentration (MIC) methods was the most potent one against all of Acintobacters (Figures 1 and 2). Our findings showed that the aerial part of S. bachtiarica essential oil had high ability against Gram-negative Acintobacter strains. The essential oil seems to be active against all bacterial strains used (MIC values of 0.5 mg/ml). Hence, the most active concentration was 0.5 mg/ml inhibiting completely the growth of all the clinically isolated Acintobacters.  
The hydrodistillation of S. bachtiarica aerial parts led to a yellowish oil in 0.6% (w/w) yield. Then, the obtainedvolatile oil was analyzed by GC-FID and GC-MS. Therefore, the main constituents were identified as the oxygenated monoterpenes: carvacrol (48.57%) and linalool (5.25%); monoterpene hydrocarbons; p-cymene (16.55%) and γ-terpinene (6.86%) (Table 1) corresponding to 94.55% of the essential oil.

Table 2. Antimicrobial potential (MICa) of essential oil from S. bachtiarica, against 6 Acintobacters isolated from individual Trauma patients in Shahid rajaei Hospital of Shiraz, Iran as determined by nutrient-broth micro-dilution bioassay.

 *Minimum inhibitory concentration (MIC) the plant extracts in the bacterial suspension in the nutrient broth media (mg/ ml) determined in three replicates

Table 3. Disk diffusion results of five medical plants against 6 Acintobacters isolated from Trauma patients in Shahid rajaei Hospital of Shiraz, Iran.

 *Concentration of essential oil all medical plants is 2.5 mg/ml; ^aNA= not active

Fig. 1. Antimicrobial potential (MICa) of essential oil from S. bachtiarica, against Acintobacters. isolated from Trauma patients in Shahid rajaee Hospital of Shiraz, Iran as determined by nutrient-broth micro-dilution bioassay.

Fig. 2. Disk diffusion bioassay of five medical plants against Acintobacter mentioned in below.

1) Mentha mozaffarianii, 2) Artemisia sieberi, 3) Salvia mirzayanii, 4) Tanacetum dumosum, 5) Satureja bachtiarica, Positive control: Colestin

Discussion

Although the antibacterial activity and chemical constituents of the essential oil of S. bachtiarica have been previously reported [12,19,20], in agreement with the literature, the major constituents of the oil were characterized as carvacrol, p-cymene and thymol. Interestingly, our data revealed that the other major components after carvacrol and p-cymene were γ-terpinene and linalool. The Satureja species is commonly used as a spice and traditionally as a muscle pain reliever, tonic, and carminative in treating stomach and intestinal disorders such as cramps, nausea, indigestion, and diarrhea [21].
According to the phenyl backbone, the remarkable antimicrobial activity of S. bachtiarica may be because of the presence of mentioned compounds. It is suggested that these components could synergistically inhibited the various bacterial strains. Therefore, the good antimicrobial activity of carvacrol is certainly justified by the hypothesis proposed by A. Ben Arfa et al., [22] with related to its hydrophobic behavior in the membrane along with detoxifying the unwanted free radicals. The importance of the hydroxyl group in the phenolic structure was confirmed in terms of activity when carvacrol was compared to its methyl ether. Furthermore, the relative position of the hydroxyl group exerted an influence upon the components effectiveness as seen in the difference in activity between carvacrol and thymol against Gram-negative and Gram-positive bacteria. However, the significance of the phenolic ring was demonstrated by the lack of activity of the monoterpene cyclic hydrocarbon p-cymene. The high activity of the phenolic components may be further explained in terms of the alkyl substitution into the phenol nucleus, which is known to enhance the antimicrobial activity of phenols [23]. These compounds were strongly active despite their relatively low capacity to dissolve in water, which is in agreement with published data [24-35]. In another research, the antimicrobial activity of carvacrol was examined on various Gram-positive and Gram-negative bacterial strains. Results revealed that carvacrol was significantly inhibited all selected bacteria at different concentrations (400 and 800 μg/ml). The obtained data showed maximum inhibition against Serratia spp. (28 mm), followed by E. coli (26 mm) and Enterobacter spp. (25 mm), Klebsiella pneumonia (23 mm), Proteus mirabilis (22 mm), S. aureus (20 mm), S. epidermidis (22 mm) and St. pneumonia (16 mm), surprisingly, there are no effect on Pseudomonas aeroginosa [36]. It seems to be that the function of carvacrol as a phenolic essential oil could be due to the following manners including to disintegrate the outer membrane of bacterial cells, to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force (PMF), electron flow, active transport and coagulation of cell contents [37,38]. Likewise, the potency of carvacrol in disintegrating the cell membrane of gram negative bacteria were confirmed by means of enhancing lipopolysaccharides (LPS) accompanied by the increasing permeability of the cytoplasmic membrane to ATP [39]. On the basis of a comprehensive survey on essential oils isolated from various species of Satureja, some biological properties such as anti-bacterial, anti-fungal, anti-viral [40-42], anti-oxidant [43], antispasmodic, anti-diarrheal, anti-nociceptive, anti-inflammatory [44,45], anti-HIV-1 [40] and immunomodulatory effects [45] have been previously mentioned. S. bachtiarica known as Marzeh-e-Koohi is an Iranian endemic specie and aromatic plant that commonly observed among mountain in the southwestern part of Iran [46]. In the folk medicine, the plant is used as analgesic and antiseptic by Bakhtiari and Chaharmahali tribes of Iran [47]. The major phenyl backbone components of S. bachtiarica were carvacrol, thymol and p-cymene [48].
According to the considerable antibacterial activity of essential oils noted in the literatures and some problematic features existed by some bacterial strains, paying attention to explore the natural drugs from nature in order to repair various human defeats is worthwhile.
In conclusion, the S. bachtiarica (known as “Marzeh” in Persian)essential oil clearly demonstrates antibacterial properties, although the mechanistic processes are poorly understood. These activities suggest potential use as chemotherapeutic agents, food preserving agents and disinfectants.

Acknowledgment

The authors are grateful to Shahid Rajaee (Emtiaz) Trauma Hospital, Medicinal and Natural Products Chemistry Research Center (MNCRC) and Shiraz University of Medical Sciences for financial and kind support.

Conflict of interest: All authors declare there is no conflict of interest.