Antibacterial Activity of Pinus pinaster Bark Extract and its Components Against Multidrug-resistant Clinical Isolates of Acinetobacter baumannii

The aim of this research was to test the antibacterial activity of Pinus pinaster aqueous bark extract (PABE) and its basic components against multidrug-resistant isolates of Acinetobacter baumannii belonging to European clone I and II, isolated previously from the clinical outbreaks. The minimum bactericidal concentration of PABE against both clones of A. baumannii was 200 mg ml–1, while lower concentrations showed high antibacterial activity. After 24 h of treatment with 100, 50 or 10 mg ml–1 of extract, the reduction in the number of A. baumannii isolates belonging to European clone I and II was 85.8 ± 2.5 %, 78.5 ± 1.1 %, 66.3 ± 2.5 % and 90.2 ± 1.7 %, 78.6 ± 1.2 %, 69.8 ± 0.7 %, respectively. Several basic components: caffeic acid, catechin, epicatechin, gallic acid and vanillin, detected in the extract by high performance liquid chromatography, contributed to the antibacterial activity of the extract against both clones of A. baumannii. However, the antibacterial activity of extract was higher than that of each tested basic component suggesting that proanthocyanidins, which were present in quite a large amount in the extract, might have also contributed to the activity of the extract. Antibacterial activity of PABE against A. baumannii reveals that complex and inexpensive natural product might be useful in combat against naturally competent bacteria that easily acquire resistance against antibiotics.


INTRODUCTION
Bark extracts of P. pinaster (PBEs) have a long history of ethnomedicinal use. 1 As a mixture of a large variety of substances, PBEs are reported to exhibit a wide range of biological activities, including antioxidative, antiinflamatory, antitumor, antiatherogenic, antiviral, antimicrobial, etc. 2 In papers published thus far, different extraction procedures, using different solvents were applied, affecting the composition of extracts and biological activity.
Species of the genus Acinetobacter, like A. baumannii are naturally competent 3 and easily acquire DNA from the environment/other species.Therefore, it is not surprising that Acinetobacter spp.displays mechanisms of resistance to all existing antibiotic classes as well as a prodigious capacity to acquire new determinants of resistance. 4 A. baumannii is a nonfermentative, Gramnegative, nonmotile, oxidase-negative coccobacillus which is found in many health care environments 5 and is a very effective human colonizer.This species emerged over the last decade as a leading cause of hospital-acquired infec-tions; there are numerous reports of multidrug-resistant (MDR) A. baumannii from hospitals around the world 6 including Croatia. 7,8Problems caused by A. baumannii in the hospital setting are emphasized by high degree of resistance to drying and disinfectants, leading to longterm persistence and the occurrence of outbreaks in the hospital environment. 6Global outbreaks of infections caused by A. baumannii are also frequent. 4urrent concerns regarding inefficiency of antibiotics or deleterious effects of synthetic chemicals used for medical purposes have encouraged a worldwide research on natural products potentially useful in development of alternative treatments for common and emerging pathogens.Research on natural products represents convenient strategy to find extracts and bioactive compounds, based on which new, effective and less expensive treatments can be applied. 9The aim of this study was to determine the antibacterial and bactericidal activity of P. pinaster aqueous bark extract (PABE) and its components against MDR A. baumannii isolated from the clinical outbreaks.

EXPERIMENTAL Plant Material and Preparation of Extracts
In Croatia Pinus pinaster is mostly planted in coastal region.For this research bark was collected from 15 trees growing on the island of Rab.Bark samples from 15 trees were randomly pooled to form 3 samples (barks collected from 5 trees in each sample), air dried at 50-55 °C for eight days to constant weight and grinded to pieces (fraction < 1 mm) using Colortronic mill.Grinded bark (100 g) was extracted with 500 ml of water at 100 °C for 30 minutes.The aqueous extract was collected and grinded bark was once again extracted with 500 ml of fresh water at 100 °C for 30 minutes.The first and second extract were pooled and drypowdered extract was prepared from aqueous extract by spray-drying at 200 °C.From 100 g of grinded bark approximately 10 g of powdered extract was obtained.For treatment of bacteria dry-powdered extract was solved in sterile distilled water.

Determination of Proanthocyanidins (PA) Content
The proanthocyanidins were determined by hydrochloric acid-butanol method. 10Ten milligram of pine bark extract was dissolved in 10 ml of water.To 0.20 ml of this solution 3 ml of a 95 % solution of n-butanol / HCl (95/5; v/v) was added, followed by addition of 0.1 ml of a 1.4 % (w/v) Fe(SO4)  7 H2O in 2 M HCl.The tubes were incubated for 40 min at 95 °C.After incubation, the samples were cooled and analyzed by measuring absorbance at 550 nm.Cyanidin chloride (Sigma-Aldrich, Germany) was used as a standard.The final PA in pine bark extracts was expressed on a dry basis (mg/ gDW) and data were shown as a mean value of three biological and three technical replicates ± SE.

Antibacterial Activity Tests
The antibacterial activities of PABE against isolates of A. baumannii belonging to the EU clone I and II were tested according to the microdilution method, following the CLSI recommendations. 11The bacteria were pre-grown on nutrient agar (Biolife, Italy) for 16 h at 36.0 ± 0.1°C to obtain the cultures in log phase of growth.The bacterial biomass was then suspended in the nutrient broth (Biolife, Italy).For the experiments with powdered extract, 200, 100, 50, 10 or 1 mg of extract was added into 0.5 ml of sterile deionised water and vigorously shaken (40 Hz/10 min, Kartell TK3S).A 0.5 ml of bacterial suspension was added in the tubes, which resulted in following concentrations of powdered extract: 200, 100, 50, 10 or 1 mg ml -1 .The tubes which served as the positive control contained 0.5 ml of bacterial suspension and 0.5 ml of sterile deionised water.Both experiments were set in triplicate.The tubes were incubated in a dark during 24 h at 36.0 ± 0.1 °C with shaking at 120 rpm to assure the complete mixing.To confirm the antibacterial activity of basic components of extract, the experiments with pure caffeic acid, catechin, epicatechin, ferulic acid, gallic acid, taxifolin and vanillin solutions in 2 % ethanol were set up in triplicate.The tested concentrations of basic components were equivalent to those contained in 200 mg ml -1 of powdered extract.The positive controls were set up without and with 2 % of ethanol, which showed no difference in the number of bacteria (data not shown).
The number of viable cells was determined at the beginning of experiment and after 24 h of treatment.Tubes were vigorously shaken and 0.1 ml of sample was plated (spread plate method) directly onto the nutrient agar, while another sample was serially diluted (10 -1 to 10 -8 ) and inoculated onto the nutrient agar plates in triplicate.The inoculated plates were incubated at 36.0 ± 0.1 °C for 24 h.After the incubation period, the bacterial colonies were counted and the number of viable cells was reported as Colony forming units (CFU) ml -1 .The numbers of CFU were logarithmically transformed.The antibacterial activity of the extract, each compound and mixture of compounds (caffeic acid, catechin, epicatechin, ferulic acid, gallic acid, taxifolin and vanillin) was expressed as the reduction of log CFU and as the percent reduction of log CFU as compared to the corresponding control.The minimum bactericidal concentration (MBC) values were determined after 24 h of experiments according to the CLSI directions. 11The minimum inhibitory concentration (MIC) was not determined due to the turbidity and colour of extracts.The comparisons between the numbers of log CFU were done using the ANOVA and subsequently the post-hoc Duncan test was performed for the calculations concerning pair-wise comparisons.Statistical decisions were made at a significance level of p < 0.05.

RESULTS AND DISCUSSION
Concentrations of basic substances (determined by HPLC) as well as proanthocyanidins were expressed as mg g -1 of spray-dried powdered PABE (Table 1).Statistically significant differences in the concentrations of basic compounds and proanthocyanidins between extracts from 3 pooled samples (barks collected from 5 trees in each sample) were not determined, and therefore the extracts were pooled for further experiments/ treatments.
Antibacterial activities of PABE against isolates of A. baumannii belonging to EU clone I and II are shown in Table 2.The extract showed antibacterial activity with MBC of 200 mg ml -1 against both clones.
To elucidate which basic components contribute to the bactericidal activity of powdered extract at concentration of 200 mg ml -1 , experiments with pure solutions of components were performed (Table 3).For clone I caffeic acid, catechin, epicatechin, ferulic acid, gallic acid and vanillin resulted in statistically significant reduction of log CFU as compared to corresponding control, but the reduction of log CFU was generally low (0.4-0.9 log CFU reduction).The antibacterial activity of basic components against clone II (Table 3) was similar to those against clone I, except that the ferulic acid had no antibacterial activity against clone II.When basic substances were tested together against two clones of A. baumannii, the reductions of log CFU were 36.9 and 23.5 % for clone I and II, respectively.These results suggest that besides tested substances, some other substances such as proanthocyanidins, which were present in quite a large amount in the extract (Table 1), might have contributed to the antibacterial activity of extract, too.Several research groups published the results on antimicrobial activity of PBEs.As opposed to simple aqueous extract (PABE) which was used in this research, in those researches mostly Pycnogenol ® -commercial water/ethanol extract was tested and it was shown that 250 μg ml -1 of Pycnogenol ® counteracted the growth of several tested Gram-positive and Gramnegative bacteria, 13 inhibited growth of Helicobacter pylori in suspension (12.5 μg ml -1 ), while in ten times higher concentration reduced the adherence of the bacterium to gastric cells. 14However its efficiency against A. baumannii was not tested.Grimm et al. 15,16 showed that substances which are basic components of PBE are quickly absorbed after oral ingestion and their distribution in the body tissues is fast.Fourteen hours after volunteers were given 300 mg or 200 mg of PBE, fifteen compounds from PBE were detected in the their plasma. 15,16Maximum concentrations of four identified compounds in the plasma; catechin, caffeic acid, ferulic acid and taxifolin were 107 ng ml -1 , 17 ng ml -1 , 15 ng ml -1 and 33 ng ml -1 , respectively.Of those four substances, caffeic acid and catechin revealed antibacte-rial activity against A. baumannii clones in our research, but epicatechin, gallic acid and vanillin also contributed to the activity of PABE against both clones of A. baumanii, while ferulic acid slightly contributed to the activity of PABE against EU clone I.The above mentioned substances are known to possess antimicrobial activity against different pathogens.Vanillin is proved to express antifungal activity 17 and antibacterial activity against Cronobacter species; 18,19 catechin was effective against pathogenic bacteria, Escherichia coli O157:H7, Pseudomonas aeruginosa and the food spoilage fungus Penicillium chrysogenum 20,21 and, it also showed synergistic antibacterial effect with antibiotics. 22Furthermore, the combination of catechin and epicatechin gallate potentiated the activity of beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus. 23urthermore, epicatechin showed synergistic effect with the aflavin against clinical isolates of A. baumannii and Stenotrophomonas maltophilia. 24Caffeic acid is also known to exhibit antimicrobial activity, however its concentration in PABE was 0.05 % making it even less concentrated in solution which was applied to A. baumannii, Table 3. Reduction in the number of A. baumannii isolates belonging to European clone I and II after 24 h of treatment with basic components of Pinus pinaster bark extract (in concentration contained in 200 mg ml -1 of powdered extract) as compared to the corresponding control.and it was reported previously that concentrations higher than 0.4 % were needed to inhibit the growth of some microorganisms, including L. monocytogenes, E. coli, and S. aureus. 25Gallic acid which also contributed to antibacterial activity of PABE is a basic constituent of many other plant extracts.Tea extracts which contain gallic acid and catechins proved to be effective against H. pylori without affecting beneficial bacteria. 26These data suggest that PABE or its components like gallic acid and catechin could not only be efficient against MDR A. baumannii, but they could also be used in combination with probiotic bacteria.In our research the tested in vitro concentrations of basic PABE components that showed antibacterial activity against A. baumannii were at least three orders of the magnitude higher than the concentration of substances found in plasma of patients after oral ingestion of the same quantity of PBE. 15,16Therefore, the concern exists that the concentration of the bioactive substances in plasma of patients would not be high enough to treat bacteraemia, but since A. baumannii causes also skin and wound infections, 27,28,29 application of PABE in sufficient concentration in such cases is certainly possible.Except investigated basic substances, proanthocyanidins that were determined in PABE could have also contributed to the antibacterial activity.In grape seeds, proanthocyanidins were determined as active antibacterial agents toward 10 different pathogenic Gram-positive and Gram-negative bacterial strains. 30Plant-derived proanthocyanidins may inhibit the growth of pathogenic bacteria by binding strongly to proteins at bacterial cell surfaces. 31n this research we revealed that PABE at the concentration 200 mg ml -1 showed bactericidal activity against two MDR clinical isolates of A. baumannii belonging to EU clone I and II and antibacterial activity at lower concentrations, in vitro.This finding suggests that PABE and its components could potentially find an application in treatment of MDR A. baumannii-infected patients.

Table 1 .
Concentrations of basic substances and proanthocyanidins in Pinus pinaster aqueous bark extract (PABE) expressed as mg g -1 of spray-dried powdered PABE.Data shown are mean values of three biological and three technical replicates ± SE.

Table 2 .
Reduction in the number of A. baumannii isolates belonging to European clone I and II after 24 h of treatment with Pinus pinaster aqueous bark extract as compared to the corresponding control.c0 A. baumannii clone I (10 7 CFU ml -1 ) = 2.93 ± 0.57; c0 A. baumannii clone II (10 7 CFU ml -1 ) = 1.54 ± 0.26.Bactericidal activity was obtained at 200 mg ml -1 , and antibacterial activity at 10 mg ml -1 .Data shown are mean values of three biological and three technical replicates ± SE.