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ISSN 2304-3415, Russian Open Medical Journal
2018. Volume 7. Issue 1. Article CID e0104 DOI: 10.15275/rusomj.2018.0104_
Original article
Antibacterial activity of an aqueous extracts of Alkanna tinctoria roots against drug resistant aerobic pathogenic bacteria isolated from patients with burns infections
Ahmed Abduljabbar Jaloob Aljanaby
University of Kufa, Kufa, Iraq
Received 7 August 2017, Revised 24 December 2017, Accepted 28 December 2017
© 2017, Aljanaby A.A.J. © 2017, Russian Open Medical Journal
Abstract: Objective — Alkanna tinctoria (A. tinctoria) which is one of the most important medical plants worldwide, has been reported to have antimicrobial activities against some gram-positive and gram-negative bacteria.
Material and Methods — To investigate the ability of four different concentrations of cold-water and hot-water extracts of A. tinctoria roots to inhibit growth of 395 isolates of drug resistance bacteria isolated from patients with burns infections during the period of June 2015 to June 2016 in Iraq. Evaluation of A.tinctoria roots aqueous extract antibacterial activity (cold and boiling water) was done by use of agar well diffusion method.
Results — The results proved that out of 395 total bacterial isolates there were 321 (81.3%), 37 (9.4%) and 11 (2.8%) isolates with multidrug resistance (MDR), extensive drug resistance (XDR) and pan-drug resistance (PDR), respectively. Also, the results revealed that 300 mg/ml of the hot-water extract was the best effective inhibitor of the growth, it's inhibited growth of all MDR, XDR and PDR bacteria, the inhibition zone diameter for Pseudomonas aeruginosa (132 strains), Klebsiella pneumoniae (99 strains), methicillin sensitive Staphylococcus aureus (72 strains), methicillin resistant Staphylococcus aureus (41 strains), Escherichia coli (32 strains), Acinetobacter baumannii (11 strains) and Proteus ssp. (8 strains) were 28.44±0.11 mm, 28.46±0.25 mm, 28.53±0.24 mm, 28.37±0.12 mm, 27.88±0.09 mm, 28.001±0.001mm, and 27.59±0.23 mm, respectively. All inhibition zones diameter were not significantly different (P>0.05) from that for imipenem 10 mg, chosen as positive control.
Conclusion — From the overall results obtained it is evident that the hot-water extract (300 mg/ml) of A. tinctoria roots has excellent antibacterial activity against bacteria associated with burns infections. A. tinctoria roots may be considered as a raw material for the manufacture of ointment for treatment of burns infections.
Keywords: aqueous extracts, Alkanna tinctoria, multi-drug resistance, bacteria, burns infections
Cite as Aljanaby AAJ. Antibacterial activity of an aqueous extracts of Alkanna tinctoria roots against drug resistant aerobic pathogenic bacteria isolated from patients with burns infections. Russian Open Medical Journal 2018; 7: e0104.
Correspondence to Ahmed Abduljabbar Jaloob Aljanaby. Email: [email protected].
Introduction
Many microbial infections caused by multi-drug resistant (MDR) bacteria, extensive drug resistant (XDR) and pan-drug resistant (PDR) such as Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E.coli) and methicillin resistant Staphylococcus aureus (MRSA) are closely related with prolonged hospitalization and mortality rates due to the limited antimicrobial therapeutic options for infected patients [1, 2].
In recent years, control of infections acquired in communities and hospitals caused by MDR, XDR and PDR bacteria has become a major problem in both developing and developed countries [3, 4]. Drug resistant gram-negative and gram-positive bacteria produce enzymes that bestow resistance to most beta-lactam antibiotics such as Amoxicillin, Amoxclave, Cefotaxime, Ceftriaxone and Ceftazidime [5].
Plants are important source for antibacterial compounds. These compounds can be obtained from different parts of the
plant such as roots, leaves and flowers. Many herbal medicines derived from medical plant extracts are being used in the treatment of many bacterial infections such as burns infections. Many researchers have reported that plants contain very important constituents used as antibacterial compounds [6].
Many medical herbs are still being used by both rural and urban communities to treat many bacterial infections such as burns infections. Alkanna tinctoria (A. tinctoria) which is one of the most important medical plants worldwide, has been reported to have antimicrobial activities against some gram-positive and gram-negative bacteria [7] . However, in Iraq, there is no study on investigation of the antibacterial effects of A. tinctoria against drug resistance bacteria. Therefore, in this study, we investigated the antibacterial activity of the extract of A.tinctoria roots against multidrug resistance P. aeruginosa, K. pneumoniae, methicillin sensitive and methicillin resistant Staphylococcus aureus (S. aureus), E. coli, Acinetobacter baumannii (A. baumannii) and Proteus ssp. isolated from patients with burns infections.
ISSN 2304-3415, Russian Open Medical Journal
2018. Volume 7. Issue 1. Article CID e0104 DOI: 10.15275/rusomj.2018.0104_
Table1. Numbers and percentage of aerobic pathogenic bacteria isolated from patients with burns infections in central hospital of Al-Kufa City (Iraq)
P. aeruginosa MSSA MRSA K. pneumoniae E. coli A. baumannii Proteus ssp. Total
Single isolate 100 (25.3) 66 (16.7) 31 (7.8) 85 (21.5) 4 (1.0) 6 (1.5) 2 (0.5) 294 (74.4)
Mix isolates 32 (8.1) 6 (1.5) 10 (2.5) 14 (3.5) 28 (7.1) 5 (1.3) 6 (1.5) 101 (25.6)
Total isolates 132 (33.4) 72 (18.2) 41 (10.4) 99 (25.1) 32 (8.1) 11 (2.8) 8 (2.0) 395 (100)
N=395 isolates. Data presented as numbers and percentage (from N) - no. (%). MSSA, methicillin sensitive S. aureus; MRSA, methicillin resistant S. aureus.
Table 2. Resistance pattern of aerobic pathogenic bacteria isolated from patients with burns infections in central hospital of Al-Kufa City (Iraq)
Antimicrobials P. aeruginosa K. pneumoniae MSSA MRSA E. coli A. baumannii Proteus ssp. Total
(132 strains) (99 strains) (72 strains) (41 strains) (32 strains) (11 strains) 8 strains (395 strains)
AMC 30mg 132 (100) 95 (96.0) 63 (87.5) 38 (92.7) 27 (84.4) 8(72.7) 5 (62.5) 368 (93.2)
CTX 30mg 125 (94.7) 94 (94.9) 61 (84.7) 36 (87.8) 18 (56.3) 7 (63.6) 4 (50.0) 345 (87.3)
CRO 30mg 121 (91.7) 93 (93.9) 52 (72.2) 38 (83.7) 17 (53.1) 7 (63.6) 6 (75.0) 334 (84.6)
CAZ 30mg 119 (90.2) 95 (96.0) 58 (80.6) 37 (90.2) 19 (59.4) 8 (72.7) 6 (75.0) 342 (86.6)
IMP 10mg 25 (18.9) 10 (10.1) 2 (2.8) 7 (17.1) 0 (0) 0 (0) 0 (0) 44 (11.1)
GM 15mg 98 (74.2) 90 (90.9) 53 (72.6) 35 (85.5) 15 (46.9) 6 (54.5) 4 (50.0) 301 (76.2)
TM 10mg 118 (89.4) 89 (89.9) 58 (80.6) 32 (78.0) 15 (46.9) 6 (54.5) 4 (50.0) 322 (81.5)
AN 30mg 110 (83.3) 88 (88.9) 31 (43.1) 32 (78.0) 12 (37.5) 7 (63.6) 3 (37.5) 283 (71.6)
C 30mg 115 (87.1) 81 (81.8) 28 (38.9) 31 (75.6) 14 (43.8) 8 (72.7) 3 (37.5) 280 (70.9)
CIP 5mg 93 (70.5) 69 (69.7) 22 (30.6) 12 (29.3) 10 (31.3) 5 (45.5) 2 (25.0) 213 (53.9)
Data presented as numbers and percentage of aerobic pathogenic bacterial isolates that were resistant to antimicrobials - no. (%).
AMC, amoxicillin with clavulanic acid; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; IMP, imipenem; GM, gentamicin; TM, tobramycin; AN, amikacin; C, chloramphenicol; CIP, ciprofloxacin; MSSR, methicillin sensitive S. aureus; MRSA, methicillin resistant S. aureus.
Material and Methods
Bacterial isolates
A total of 395 swabs were collected from patients with burns infections in central hospital of Al-Kufa City (Iraq) during the period of June 2015 to June 2016. All swabs were streaked onto the surface of blood agar (Oxoid, UK), MacConkey agar (Oxoid, UK) and mannitol salt agar (Oxoid, UK) and incubated overnight at 37°C. All emergent colonies were identified by standard bacteriological methods (Growth on MacConkey Agar, Gram stain, Capsule stain, Triple Sugar Iron test, Catalase and oxidase test, Indole production test, Methyl Red test, Voges-Proskauer test, Simmons Citrate test and Coagulase test) [8] and then by cultivation on Chrome agar medium (Orientation Company, France). Finally all isolates were identified by using Vitek2 system (BioMerieux, France). Detection of methicillin resistant S. aureus strains were done by incubated all S. aureus strains overnight at 37°C in 5 ml of Mueller-Hinton broth (Oxoid, UK). All samples were streaked onto the surface of CHROMagar MRSA media (Orientation Company, France) by sterile swabs (Bioanalyse, Turkey) and incubated overnight at 37°C. The growth of more than two colonies will indicate methicillin-resistance [9].
Antimicrobials susceptibility test
Kirby-Bauer method was performed for antibiotic susceptibility testing according to the Clinical Laboratory Standards Institute (CLSI, 2014) [10]. Briefly, fresh bacterial strain with 0.5 McFarland turbidity was swabbed onto the Mueller-Hinton agar (Oxoid, UK) surface using sterile swab sticks. Antimicrobial discs (Bioanalyse, Turkey) were evenly embedded onto the inoculated agar incubated at 37°C overnight. Ten antimicrobials were used in this study: amoxicillin with clavulanic (AMC) acid 20/10 mg, cefotaxime (CTX) 30 mg, ceftriaxone (CRO) 30 mg, ceftazidime (CAZ) 30 mg, imipenem (IMP) 10 mg, gentamicin (GM) 15 mg, tobramycin (TM) 10 mg, amikacin (AN) 30 mg, Chloramphenicol (C) 30 mg, ciprofloxacin (CIP) 5 mg. Antimicrobial susceptibility and resistance was determined according to CLSI guidelines (2014) [10] according to strain growth zone diameter. E. coli ATCC 35218 and S. aureus
ATCC 25923 strains were used as control. According to the results of antimicrobials susceptibility test: any bacterial isolate resist to a minimum at least 3 different classes of antibiotics it is multi-drug resistance (MDR), any bacterial isolate remain susceptible to only one or two class of antibiotics it is extensive-drug resistance (XDR) and any bacterial isolate resistance to all sub classes in all classes of antibiotics it is pan-drug resistance (PDR) [10, 11].
Collection and identification of A. tinctoria and preparation of plant extract
A. tinctoria was obtained from medicinal plant herbarium. It was identified by the Department of Botany, Faculty of Science, University of Kufa (Iraq). A. tinctoria roots were freed from foreign particles, washed with water to remove dust, dried and ground to a powder. Fifty gram from roots powder was extracted with 500 ml cold sterile water and boiling water for overnight. The extracted solutions were then filtered through a 0.2 |m membrane filter (Whatman, USA) and evaporated to dryness at 45°C [6]. The extracts were kept sterile containers and stored at 4°C.
Antibacterial activity test
The antibacterial activity of aqueous extract (cold and boiling water) of A. tinctoria roots was done according to method by Rauha et al. (2000) [12] and Radji et al (2013) [3] by use of the agar well diffusion method as follow: Five or three fresh bacterial colony with 0.5 McFarland turbidity was swabbed onto the Mueller-Hinton agar surface (Oxoid, UK) using sterile swab (Bioanalyse, Turkey). Three wells (five mm in diameter) were made in each Mueller-Hinton agar (Oxoid, UK) plate by use of a sterile cork borer (Himedia-India). The crude roots of plant extracts were serially diluted to yield dilutions of 50, 100, 200, and 300 mg/ml, and 50 |l of each dilution was transferred to each well and left for three hours at 21-23°C to enable diffusion of the extract across the surface. The plates were then incubated at 37°C for 24 h. The inhibition zone around each well was measured in millimeters. All tests were carried out in triplicates.
ISSN 2304-3415, Russian Open Medical Journal
2018. Volume 7. Issue 1. Article CID e0104 DOI: 10.15275/rusomj.2018.0104_
Table 3. Resistance type of aerobic pathogenic bacteria isolated from patients with burns infections in central hospital of Al-Kufa City (Iraq)
Resistance type P. aeruginosa K. pneumoniae MSSA MRSA E. coli A. baumannii Proteus ssp. Total
(132 strains) (99 strains) (72 strains) (41 strains) (32 strains) (11 strains) (8 strains) (395 strains)
MDR 122 (92.4) 83 (83.8) 58 (80.6) 33 (80.5) 18 (56.3) 5 (45.5) 2 (25.0) 321 (81.3)
XDR 21 (15.9) 11 (11.1) 2 (2.8) 2 (4.9) 1 (3.1) 0 (0) 0 (0) 37 (9.4)
PDR_7 (5.3)_2 (2.0)_0J0_2 (4.9)_0J0_0J0_0J0_11 (2.8)
Data presented as numbers and percentage of aerobic pathogenic bacterial isolates that were resistant to antimicrobials - no. (%).
MDR, multi-drug resistance; XDR, extensive drug resistance; PDR, pan-drug resistance; MSSR, methicillin sensitive S. aureus; MRSA, methicillin resistant S. aureus.
Statistical analysis
Statistical analysis was performed with SPSS version 10 software by use of the t test. P <0.05 was regarded as indicative of statistical significance.
Results
Numbers and percentage of bacterial isolates
From the 395 swabs isolated from patients with burns infections, the most prevalent aerobic pathogenic bacteria was P. aeruginosa (132 isolates, 33.4%) while the least was Proteus ssp. (8 isolates, 2.0%) (Table 1).
Antibiotic susceptibility test
The results of this study proved that P. aeruginosa, K. pneumoniae, methicillin sensitive and methicillin resistant S. aureus were highly resistant to most antimicrobials especially to amoxicillin + clavulanic acid (20/10 mg) and third-generation cephalosporins, while E.coli, A. baumannii and Proteus ssp. were less resistant to these antimicrobials. The results indicated that out of the 395 total bacterial isolates there were 368 isolates (93.2%) were resistant to amoxicillin + clavulanic acid (20/10 mg), while there were only 44 isolates (11.1%) were resistant to imipenem (10 mg) (Table 2). The results of this study proved that imipenem (10 mg) was the best of the antibiotics tested. Imipenem (10 mg) was therefore used as positive control to compare with antibacterial activity of aqueous extract of A. tinctoria roots against bacterial isolates. On the other hand, the results proved out of the 395 total bacterial isolates there were 321 isolates (81.3%) were multi-drug resistance, 37 isolates (9.4%) were extensive drug resistance and 11 isolates (2.8%) were pan-drug resistance (Table 3).
Antibacterial activity of an aqueous extract of A. tinctoria roots
The results of the current study demonstrated that cold-water extracts 50 and 100 mg/ml of A. tinctoria roots had no any inhibitory activity against all bacterial isolates, while cold-water extracts 200 and 300 mg/ml, and hot-water extract 50 mg/ml of A. tinctoria roots had inhibitory activity against some bacterial isolates resulting in the inhibition zones ranging between 22.61±0.26 to 24.85±0.13 mm of cold-water extracts 200 mg/ml against P. aeruginosa and E.coli, respectively, 23.87±0.35 to 24.88±0.09 mm of cold-water extracts 300 mg/ml against methicillin resistant S. aureus and K. pneumoniae, respectively, 24.92±0.46 to 25.08±0.07 mm of hot-water extracts 50 mg/ml against E.coli and methicillin sensitive S. aureus, respectively.
Also, the results proved that hot-water extracts 100 and 200 mg/ml of A. tinctoria roots had inhibitory activity against large number of bacterial isolates resulting in the inhibition zones zones ranging between 24.83±0.30 to 26.09±0.47 mm of hot-water
extracts 100 mg/ml against methicillin sensitive S. aureus and E.coli, respectively, 26.08±0.08 to 26.77±0.23 mm of hot-water extracts 200 mg/ml against methicillin sensitive S. aureus and A. baumannii, respectively.
The study also demonstrated that hot-water extract 300 mg/ml of A. tinctoria roots has inhibitory activity against all bacterial resulting in the largest inhibition zones ranging between 27.59±0.23 to 28.53±0.24 mm against Proteus ssp. and methicillin sensitive S. aureus (Table 4).
The results were indicative of a significant difference P<0.05 between imipenem 10 mg/ml (Table 5) and hot-water extracts 50, 100, 200 mg/ml (Table 6). On the other hand, the result proved that there was non-significant difference P>0.05 between imipenem 10mg and the hot-water extract 300 mg/ml (Table 7).
Hot-water extract 300 mg/ml has the best antibacterial activity and produced large inhibition zones 28.18±0.09 mm, 28.21±0.11 mm and 28.32±0.11 mm against all multi-drug resistance, extensive drug resistance and pan-drug resistance of aerobic pathogenic bacterial isolates, respectively, and there was nonsignificant difference P>0.05 with imipenem 10mg (Table 7).
Discussion
Burns infections are one of the most dangerous problems in hospitals, and MDR gram-positive and gram-negative bacteria are the most dominant etiological agents of this infections include, P. aeruginosa, K. pneumoniae, S. aureus, E.coli, A. baumannii, Proteus ssp. and others [13-16].
Many classes of antimicrobials were used to prevent this type of infections such as beta-lactam, cephalosporins, aminoglycosides and others, but unfortunately, most pathogenic bacteria cause burns infections became more resistant to these antimicrobials by different means such as the vertical and horizontal transfer of antimicrobial resistance gene and became multi-drug resistance (one bacterial isolate resist to a minimum at least 3 different classes of antibiotics) or extensive-drug resistance (one bacterial isolate remain susceptible to only one or two class of antibiotics) or pan-drug resistance (one bacterial isolate resistance to all sub classes in all classes of antibiotics) [17-19].
Therefore, different medical plants were used by many researchers as alternative treatment against different bacterial infections because these medicinal plants are the rich source of traditional medicines [3, 6, 20-22].
A. tinctoria is one of the best medical plants used as plant herbal therapy for treating different infections worldwide [3, 23]. In Iraq there is no studies focused on the antibacterial activity of A. tinctoria, therefore, the present study was designed to evaluation the antibacterial activity of cold-water and hot-water extracts of A. tinctoria roots against drug resistance aerobic pathogenic bacteria isolated from patients with burns infections.
ISSN 2304-3415, Russian Open Medical Journal
2018. Volume 7. Issue 1. Article CID e0104 DOI: 10.15275/rusomj.2018.0104_
Table 4. Evaluation of the antibacterial activity of cold-water and hot-water extracts of A. tinctoria roots against aerobic pathogenic bacteria isolated
from patients with burns infections in central hospital of Al-Kufa City (Iraq)
Aerobic pathogenic bacteria Concentration, no. (%), M±SE mm
50 mg/ml 100 mg/ml 200 mg/ml 300 mg/ml
Cold-water extracts, R=3
P. aeruginosa (132 strains) K. pneumoniae (99 strains) MSSA (72 strains) MRSA (41 strains) E. coli (32 strains) A. baumannii (11 strains) Proteus ssp. (8 strains) 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 0 (0), Resistance 2 (1.5), 22.61±0.26 3 (3.0), 23.42±0.30 2 (2.8), 23.57±0.25 3 (7.3), 23.49±0.26 2 (6.3), 24.85±0.13 1 (9.1), 23.32±0.29 1 (12.5), 23.43±0.28 8 (6.1), 24.48±0.26 8 (8.1), 24.88±0.09 6 (8.3), 25.16±0.11 3 (7.3), 23.87±0.35 4 (12.5), 24.69±0.34 1 (9.1), 24.05±0.05 1 (12.5), 24.40±0.30
Hot-water extracts, R=3
P. aeruginosa (132 strains) K. pneumoniae (99 strains) MSSA (72 strains) MRSA (41 strains) E. coli (32 strains) A. baumannii (11 strains) Proteus ssp. (8 strains) 16(12.1), 24.67±0.28 18 (18.2), 24.54±0.24 17 (23.6), 24.51±.28 15 (36.6), 25.08±0.07 8 (25.0), 24.92±0.46 3 (27.3), 24.61±0.24 3 (37.5), 24.21±0.21 60 (45.5), 24.86±0.14 50 (50.5), 24.90±0.11 25 (34.7), 24.83±0.30 25 (61.0), 24.93±0.20 21 (65.6), 26.09±0.47 5 (45.5), 25.06±0.08 6 (75.0), 25.07±0.09 100(75.8), 26.14±0.07 90 (90.9), 26.43±0.16 66 (91.7), 26.08±0.08 35 (58.4), 26.45±0.29 32 (100), 26.85±0.15 9 (81.8), 26.77± 0.23 8 (100), 26.37±0.31 132 (100), 28.44±0.11 99 (100), 28.46±0.25 72 (100), 28.53±0.24 41 (100), 28.37±0.12 32 (100), 27.88±0.09 11 (100), 28.00±0.001 8 (100), 27.59±0.23
Data presented as numbers and percentage of aerobic pathogenic bacterial isolates that were sensitive to aqueous extract of A. tinctoria roots - no. (%). R, number of replicates; M, mean of diameter of inhibition zone (mm); SE, standard error of mean; MSSR, methicillin sensitive S. aureus; MRSA, methicillin resistant S. aureus.
Table 5. Comparison between imipenem 10mg (positive control) and cold-water extracts of A. tinctoria roots (200 and 300 mg/ml) against aerobic pathogenic bacteria isolated from patients with burns infections in central hospital of Al-Kufa City (Iraq)
Aerobic pathogenic bacteria Cold-water extracts, no. (%), M±SE mm 200 mg/ml, R=3 300 mg/ml, R=3 Imipenem 10mg, R=3, no. (%), M±SE mm P-valueA P-value B
P. aeruginosa (132 strains) 2 (1.5), 22.61±0.26 8 (6.1), 24.5±0.26 107 (81.1), 28.78±0.46 <0.001 <0.001
K. pneumoniae (99 strains) 3 (3.0), 23.42±0.30 8 (8.1), 24.88±0.09 88 (88.9), 28.47±0.27 <0.001 <0.001
MSSA (72 strains) 2 (2.8), 23.57±0.25 6 (8.3), 25.16±0.11 70 (97.2), 28.33±0.33 <0.001 <0.001
MRSA (41 strains) 3 (7.3), 23.49±0.26 3 (7.3), 23.87±0.35 34 (82.9), 28.47±0.26 <0.001 <0.001
E. coli (32 strains) 2 (6.3), 24.85±0.13 4 (12.5), 24.69±0.34 32 (100) 28.29±0.30 <0.001 0.001
A. baumannii (11 strains) 1 (9.1), 23.32±0.29 1 (9.1), 24.05±0.05 11 (100), 28.13±0.08 <0.001 <0.001
Proteus ssp. (8 strains) 1 (12.5), 23.43±0.28 1 (12.5), 24.40±0.30 8 (100), 28.16±0.43 <0.001 0.002
Data presented as numbers and percentage of aerobic pathogenic bacterial isolates that were sensitive to imipenem 10mg and aqueous extract of A. tinctoria roots - no. (%). A - P-value of imipenem 10mg and cold-water extract 200 mg/ml; B - P-value of imipenem 10mg and cold-water extract 300 mg/ml. R, number of replicates; M, mean of diameter of inhibition zone (mm); SE, standard error of mean. MSSR, methicillin sensitive S. aureus; MRSA, methicillin resistant S. aureus.
The results of the present study proved that P. aeruginosa, K. pneumoniae and S. areus were the most dominant pathogenic bacteria isolated from patients with burns infections (Table 1), also the result demonstrated that most bacterial isolates were highly resistant to most antimicrobials (Table 2) and out of total 395 isolates there were 321 isolates (81.3%) were MDR (Table 3).
In recent years, large numbers of gram-negative and grampositive bacteria become increasingly important pathogens in both community settings and hospitals, such as P. aeruginosa, K. pneumoniae, S. areus and others. These pathogenic bacteria play an important role in the colonization and infection of patients with burns infections [24, 25].
Treatment of those patients is often very difficult due to cross-resistance of drug-resistant bacteria with a large group of antimicrobials. Therefore, we need new sources of natural compounds with antibacterial activity against drug-resistance pathogenic bacteria.
This study aimed to evaluate the ability of different concentrations (50, 100, 200 and 300 mg/ml) of cold-water and hot-water extracts of A. tinctoria roots to inhibit growth of 395 drug resistance pathogenic bacteria isolated from patients with burns infections.
The results demonstrated that both cold-water 200 and 300 mg/ml and hot-water extracts 50, 100, 200 and 300 mg/ml of A. tinctoria roots had different inhibitory activity against bacterial isolates with the hot water extract at 300 mg/ml resulting in the largest inhibition zones (Table 4).
The results were indicative of a significant difference P<0.05 between imipenem 10mg (positive control) and the cold-water extracts 200 and 300 mg/ml (Table 5) and all hot-water extracts at different concentrations (Table 6). Also, the hot-water extract (300 mg/ml) inhibited all MDR, XDR and PDR bacterial isolates that were resistant to imipenem 10mg (Table 7).
This is an indication that hot water is a better than cold water as solvent for extracting the anti-bacterial compounds in A. tinctoria roots. These findings were indicative of the presence of active antibacterial compounds in this plant.
A. tinctoria is herbal medicines with antibacterial activity has been proved to possess health promotion properties and medicinal compounds, including the ability to inhibit the growth of gram-positive and gram-negative pathogenic bacteria and cause wound healing [7, 26, 27].
ISSN 2304-3415, Russian Open Medical Journal
2018. Volume 7. Issue 1. Article CID e0104 DOI: 10.15275/rusomj.2018.0104_
Table 6. Comparison between imipenem 10mg (positive control) and hot-water extracts of A. tinctoria roots (50, 100, 200, and 300 mg/ml) against aerobic pathogenic bacteria isolated from patients with burns infections in central hospital of Al-Kufa City (Iraq)
Aerobic Hot-water extracts, no. (%), M±SE mm Imipenem 10mg, R=3, P-value
pathogenic 50 mg/ml, 100 mg/ml, 200 mg/ml, 300 mg/ml, no. {%), M±SE mm A B C D
bacteria R=3 R=3 R=3 R=3
P. aeruginosa 16 (12.1), 60 (45.5), 100 (75.8), 132 (100), 107 (81.1), 0.002 0.001 0.005 0.509
(132 strains) 24.67±0.28 24.86±0.14 26.14±0.07 28.44±0.11 28.78±0.46
K. pneumoniae 18 (18.2), 50(50.5), 90 (90.9), 99 (100), 88 (88.9), <0.001 <0.001 0.003 0.971
(99 strains) 24.54±0.24 24.90±0.11 26.43±0.16 28.46±0.25 28.47±0.27
MSSA 17(23.6), 25 (34.7), 66 (91.7), 72 (100), 70 (97.2), <0.001 0.001 0.003 0.652
(72 strains) 24.51±0.28 24.83±0.30 26.08±0.08 28.53±0.24 28.33±0.33
MRSA 15 (36.6), 25 (61.0), 35 (58.4), 41 (100), 34 (82.9), <0.001 <0.001 0.007 0.751
(41 strains) 25.08±0.07 24.93±0.20 26.45±0.29 28.37±0.12 28.47±0.26
E. coli 8 (25.0), 21 (65.6), 32 (100), 32 (100), 32 (100), 0.004 0.013 0.013 0.263
(32 strains) 24.92±0.46 26.09±0.47 26.85±0.15 27.88±0.09 28.29±0.30
A. baumannii 3 (27.3), 5 (45.5), 9 (81.8), 11 (100), 11 (100), <0.001 <0.001 0.005 0.190
(11 strains) 24.61±0.24 25.06±0.08 26.77±0.23 28.00±0.01 28.13±0.08
Proteus ssp. 3 (37.5), 6(75.0), 8 (100), 8 (100), 8 (100) 0.001 0.002 0.028 0.299
(8 strains) 24.21±0.21 25.07±0.09 26.37±0.31 27.59±0.23 28.16±0.43
Data presented as numbers and percentage of aerobic pathogenic bacterial isolates that were sensitive to imipenem 10mg and aqueous extract of A. tinctoria roots - no. (%). A - P-value of imipenem 10mg and hot-water extract 50 mg/ml; B - P-value of imipenem 10mg and hot-water extract 100 mg/ml; C - P-value of imipenem 10mg and hot-water extract 200 mg/ml; D - P-value of imipenem 10mg and hot-water extract 300 mg/ml.
R, number of replicates; M, mean of diameter of inhibition zone (mm); SE, standard error of mean. MSSR, methicillin sensitive S. aureus; MRSA, methicillin resistant S. aureus.
Table 7. Comparison between imipenem 10mg (positive control) and hot-water extract of A. tinctoria roots 300 mg/ml against multi-drug resistance, extensive drug resistance and pan-drug resistance of aerobic pathogenic bacteria isolated from patients with burns infections in central hospital of AlKufa City (Iraq)
Resistance type Total, no. (%) Imipenem 10mg, R=3, no. (%), M±SE mm Hot-water extract 300 mg/ml, R=3, no. (%), M±SE mm P-value
MDR 321 (81.26) 290 (90.3), 28.18±0.09 321 (100), 28.18±0.09 0.953
XDR 37 (9.36) 31 (83.8), 28.19±0.10 37 (100), 28.21±0.11 0.917
PDR 11 (2.78) 0 (0), Resistant 11 (100), 28.32±0.11 ND
Data presented as numbers and percentage of aerobic pathogenic bacterial isolates that were sensitive to imipenem 10mg and hot-water extract of A. tinctoria roots - no. (%). R, number of replicates; M, mean of diameter of inhibition zone (mm); SE, standard error of mean; MDR, multi-drug resistance; XDR, extensive drug resistance; PDR, pan-drug resistance; ND, not done.
A. tinctoria root contain pharmaceutical compounds with a wide spectrum of biological properties such as hydroxynaphthoquinones (HNQ) and isohexenylnaphthazarins (IHN) are potent pharmaceutical substances with a well-established and wide spectrum of antibacterial, wound healing, anticancer and anti-inflammatory activities, in this study, the wide spectrum of anti-bacterial effect of A. tinctoria root extracts may be due to alkannin esters and shikonin semi-quinone radical formation (naphthoquinone), exhibiting cytotoxicity via the generation of endogenous superoxide anion radicals [28-32].
The results demonstrated that hot-water extract and a high concentration (300 mg/ml) of an active principle in the extracts of A. tinctoria; alkannin esters and shikonin might be responsible for this anti-bacterial activity.
Conclusion
The present study proved that hot-water extracted from A. tinctoria roots 300 mg/ml has very good antibacterial activity against all drug-resistance bacteria isolated from patients with burns infections. So, A. tinctoria roots may be considered as a raw material for the manufacture of ointment for treatment of burns infections.
Conflict of interest
We declare that we have no conflict of interest.
Acknowledgments
The authors are grateful to staff of microbiology laboratory in central hospital of Al-Kufa City (Iraq) for providing the burns swabs and all clinical samples.
Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For this type of study formal consent is not required.
References
1. Aljanaby AAJ and Medhat AR. Prevalence of some antimicrobials resistance associated-genes in Salmonella typhi Isolated from patients infected with typhoid fever. J Biol Sci 2017; 17(4): 171-184. https://dx.doi.org/10.3923/jbs.2017.171.184.
2. Aljanaby AAJ, Alhasnawi HMRJ. Phenotypic and molecular characterization of multidrug resistant klebsiella pneumoniae isolated from different clinical sources in Al-Najaf province - Iraq. Pak J Biol Sci 2017; 20(5): 217-232. https://dx.doi.org/10.3923/pjbs.2017.217.232.
3. Radji M, Agustama RA, Elya B, Tjampakasari CR. Antimicrobial activity of green tea extract against isolates of methicillinresistant Staphylococcus aureus and multi-drug resistant Pseudomonas Aeruginosa. Asian Pac J Trop Biomed 2013; 3(8): 663-667. https://dx.doi.org/10.1016/S2221-1691(13)60133-1.
ISSN 2304-3415, Russian Open Medical Journal
2018. Volume 7. Issue 1. Article CID e0104 DOI: 10.15275/rusomj.2018.0104_
4. Keen EF , Robinson BJ, Hospenthal DR, Aldous WK, Wolf SE, Chung KK, Murray CKP. Prevalence of multidrug-resistant organisms recovered at a military burn center. Burns. 2010 36(6): 819-825. https://dx.doi.org/10.1016/j.burns.2009.10.013.
5. Chishimba K, Hang'ombe BM, Muzandu K, Mshana SE, Matee MI, Nakajima C, Suzuki Y. Detection of extended-spectrum beta-lactamase-producing Escherichia coli in market-ready chickens in Zambia. Int J Microbiol 2016; 17: 5275724. https://dx.doi.org/10.1155/2016/5275724.
6. Aljanaby AAJJ. Antibacterial activity of an aqueous extract of Petroselinum crispum leaves against pathogenic bacteria isolated from patients with burns infections in Al-najaf Governorate, Iraq. Res Chem Intermed 2012; 38(9): 3709-3714. https://dx.doi.org/10.1007/s11164-012-0874-5.
7. Khan UA, Rahman H, Qasim M, Hussain A, Azizllah A, Murad W, Khan Z, et al. Alkanna tinctoria leaves extracts: a prospective remedy against multidrug resistant human pathogenic bacteria. BMC Complement Altern Med 2015; 15: 127. https://dx.doi.org/10.1186/s12906-015-0646-z.
8. MacFaddin JF. Biochemical tests for identification of medical bacteria. (3rd edition). USA: Lippincott Williams and Wilkins, 2000. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC493360.
9. Diederen B, van Duijn I, van Belkum A, Willemse P, van Keulen P, Kluytmans J. Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2005; 43(4): 1925-1927. https://dx.doi.org/10.1128/JCM.43A1925-1927.2005.
10. Performance standards for antimicrobial susceptibility testing; twenty-third informational supplement. USA: Clinical and Laboratory Standards Institute (CLSI), 2013. http://reflab.yums.ac.ir/uploads/clsi m100-s23-2013.pdf.
11. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012; 18(3): c268-c281. https://dx.doi.org/10.1111/j.1469-0691.2011.03570.x.
12. Rauha JP, Remes S, Heinonen M, Hopia A, Kähkönen M, Kujala T, et al. Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds. Int J Food Microbiol 2000; 56(1): 3-12. https://dx.doi.org/10.1016/S0168-1605(00)00218-X.
13. Agnihotri N, Gupta V, Joshi RM. Burns. Aerobic bacterial isolates from burn wound infections and their ntibiograms - a five-year study. Burn 2004; 30(3): 241-243. https://dx.doi.org/10.1016/j.burns.2003.11.010.
14. Brusselaers N, Monstrey S, Snoeij T, Vandijck D, Lizy C, Hoste E, et al. Morbidity and mortality of bloodstream infections in patients with severe burn injury. Am J Crit Care 2010; 19(6): 81-87. https://dx. doi.org/10.4037/a jcc2010341.
15. Sarhaddi N, Soleimanpour S, Farsiani H, Mosavat A, Dolatabadi S, Salimizand H, Amel Jamehdar S. Elevated prevalence of multidrug-resistant Acinetobacter baumannii with extensive genetic diversity in the largest burn centre of northeast Iran. J Glob Antimicrob Resist 2016; 21(8): 60-66. https://dx.doi.org/10.1016/Mgar.2016.10.009.
16. Devrim I, Kara A, Düzgöl M, Karkiner A, Bayram N, Temir G, et al. Burn-associated bloodstream infections in pediatric burn patients: Time distribution of etiologic agents. Burns 2017; 43(1): 144-148. https://dx.doi.org/10.1016/j.burns.2016.07.030.
17. Khosravi AD, Hoveizavi H, Mohammadian A, Farahani A, Jenabi A. Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR. Acta Cir Bras 2016; 31(3): 206-211. https://dx.doi.org/10.1590/S0102-865020160030000009.
18. Parhizgari N, Khoramrooz SS, Malek Hosseini SA, Marashifard M, Yazdanpanah M, Emaneini M, et al. High frequency of multidrug-resistant Staphylococcus aureus with SCCmec type III and Spa types t037 and t631 isolated from burn patients in southwest of Iran. APMIS 2016; 124(3): 221-228. https://dx.doi.org/10.1111/apm.12493.
19. Girerd-Genessay I, Benet T, Vanhems P. Multidrug-resistant bacterial outbreaks in burn units: a synthesis of the literature according to the ORION statement. J Burn Care Res 2016; 37(3): 172-180. https://dx. doi.org/10.1097/BCR.0000000000000256.
20. Bakht J, Khan S, Shafi M. Antimicrobial potentials of fresh Allium cepa against gram positive and gram negative bacteria and fungi. Pak J Botany 2013; 45: 1-6. https://www.pakbs.org/pjbot/PDFs/45(S1)/01.pdf.
21. Skata E, Rijo P, Garcia C, Sitarek P, Kalemba D, Toma M, et al. The essential oils of Rhaponticum carthamoides hairy roots and roots of soil-grown plants: chemical composition and antimicrobial, anti-inflammatory, and antioxidant activities. Oxid Med Cell Longev 2016; 2016: 8505384. https://dx.doi.org/10.1155/2016/8505384.
22. Sharifi-Rad J, Mnayer D, Roointan A, Shahri F, Ayatollahi SA, Sharifi-Rad M, et al. Antibacterial activities of essential oils from Iranian medicinal plants on extended-spectrum ß-lactamase-producing Escherichia coli. Cell Mol Biol (Noisy-le-grand) 2016; 62(9): 75-82. https://www.ncbi.nlm.nih.gov/pubmed/27650980.
23. Papageorgiou VP. Wound healing properties of naphthaquinone pigments from Alkanna tinctoria. Experientia 1978; 34(11): 1499-1501. https://www.ncbi.nlm.nih.gov/labs/articles/720485.
24. Cen H, Wu Z, Wang F, Han C. Pathogen distribution and drug resistance in a burn ward: a three-year retrospective analysis of a single center in China. Int J Clin Exp Med 2015; 8(10): 19188-19199. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694455.
25. Guo Q, Wu Q Bai D, Liu Y, Chen L, Jin S, et al. Potential use of dimethyl sulfoxide in treatment of infections caused by Pseudomonas aeruginosa. Antimicrob Agents Chemother 2016; 60(12): 7159-7169. https://dx.doi.org/10.1128/AAC.01357-16.
26. Ogurtan Z, Hatipoglu F, Ceylan C. The effect of Alkanna tinctoria Tausch on burn wound healing in rabbits. Dtsch Tierarztl Wochenschr 2002; 109(11): 481-485. https://www.ncbi.nlm.nih.gov/pubmed/12494554.
27. Ghosh PK, Gaba A. Phyto-extracts in wound healing. J Pharm Pharm Sci 2013; 16(5): 760-820. https://www.ncbi.nlm.nih.gov/pubmed/24393557.
28. Papageorgiou VP, Digenis GA. Isolation of two new alkannin esters from Alkanna tinctoria. Planta Med 1980; 39: 81-84. https://dx. doi.org/10.1055/s-2008-1074907.
29. Papageorgiou VP, Assimopoulou AN, Couladouros EA, Hepworth D, Nicolaou KC. The chemistry and biology of alkannin, shikonin, and related naphthazarin natural products. Angew Chem Int Ed 1999; 38: 270-300. https://dx.doi.org/10.1002/(SICI)1521-3773(19990201)38:3<270::AID-ANIE270>3.0.CQ;2-0.
30. Assimopouloua AN, Boskoub D, Papageorgiou VP. Antioxidant activities of alkannin, shikonin and Alkanna tinctoria root extracts in oil substrates. Food Chemistry 2004; 87(3): 433-438. https://doi.org/10.1016/j.foodchem.2003.12.017.
31. Spyros A, Assimopoulou AN, Papageorgiou VP. Structure determination of oligomeric alkannin and shikonin derivatives. Biomed Chromatogr 2005; 19(7): 498-505. https://dx.doi.org/10.1002/bmc.470.
32. Assimopoulou AN, Papageorgiou VP. Radical scavenging activity of Alkanna tinctoria root extracts and their main constituents, hydroxynaphthoquinones. Phytother Res 2005; 19(2): 141-147. https://dx. doi.org/10.1002/ptr.1645.
Authors:
Ahmed Abduljabbar Jaloob Aljanaby - Assistant Professor, Department of Microbiology, Faculty of Science, University of Kufa, Kufa, Iraq.
