Bacteriostatic and Bactericidal Activity of Deer Musk on Multidrug Resistance Bacteria

MATERIALS AND METHODS

Total time required to conduct this study about 4 months from September-December, 2017. Acquisition and analysis of data, drafting of article and revision January-April, 2018.

Musk: Black musk were provided as an oily solution commercially available at Abd-El Samad El Korashy Official stores treated in natural environmental conditions at temperature 25-28°C.

Microbial strain: Overnight cultures of the following 11 clinical MDR bacteria were used throughout the study: Staphylococcus capitis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Actinobacter baumanni, Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens, Streptococcus agalactiae, Streptococcus pyogenes and Enterococcus faecalis were obtained from Bio House Medical Lab. The inoculum prepared by making a direct NB (Oxoid, UK) suspension of isolated colonies selected from an 18-24 h NA (Oxoid, UK) agar plate. Adjusted the suspension turbidity spectrophotometrically 0.1 at 625 nm equivalent to a 0.5 McFarland standard, this results in suspension containing approximately 1.5×10⁸ CFU mL^–1.

Antibiotics: The antibiotics-standard Doxycycline (DO, 30 μg), Cefotaxime (CXM, 30 μg), Amoxicillin/clavulanic acid (AMC, 30 μg), sulfamethoxazole trimethoprim (SXT, 25 μg), Azithromycin (AZM, 15 μg), Penicillin G (PG, 10 units), Norfloxacin (NOR, 10 μg) and Clindamycin (DA, 10 μg) were used as a reference antibiotics in the disk diffusion method.

Antimicrobial activity of musk by agar disk diffusion method: Antimicrobial activity of deer musk at different concentrations on the growth MDR bacterial strains was carried out and determined by disk diffusion method protocol suggested in the CLSI¹⁸. Diffusion in solid media method was used for 11 MDR species with musk and it used with the antibiotics-standard as a reference antibiotics against a giving MDR strain. Commercially available antimicrobial disks and the filter paper discs (about 4 mm in diameter) impregnated with 50 μL of musk at the concentrations of (100% (v/v) and 10% (v/v)) stock solution of the musk to be used was freshly dissolved in 10% dimethyl sulfoxide (DMSO; BDH, UK) to obtain a stock concentration of 10% (v/v) applied on the surface of MHA (Oxoid, UK) agar plates were inoculated with a standardized inoculum of the test MDR bacteria using a sterile cotton swab by pressing slightly. The plates were incubated for overnight at 37°C and the diameters of inhibition zone were measured.

Minimum inhibitory concentrations: The MIC of musk was determined using a broth microdilution method on 11 MDR strains. In general, the protocol used followed that recommended by CLSI. Stock solution of the musk to be used was freshly dissolved in 10% dimethyl sulfoxide (DMSO; BDH, UK) in sterile MHB broth to obtain a stock concentration of 10% (v/v). Serial two-fold dilutions were prepared in 96-well microplates (Falcon, corning-life sciences, USA). This was serially diluted two fold to obtain concentration ranges of 10% (v/v) to 0.01% (v/v). An overnight culture of the 11 MDR microbial inoculum prepared in the same medium after dilution of standardized microbial suspension adjusted to 0.5 McFarland scale which each bacterial suspension was standardized at a cell density of 1.5×10⁸ (CFU mL^–1). After well-mixing, the 96-well microtitration plates have incubated for 18 h at 37°C. Two columns were left with the purpose to be used as a positive control (without antibiotic and inoculated with MDR) and a negative control (without antibiotic and not inoculated with the bacterium). Each MIC assay was performed thrice and the MIC value was considered as the lowest concentration of the musk causing an absence of bacterial growth and preventing visible turbidity and the MIC assay included ampicillin as a reference antibiotic.

Bactericidal activity of musk by kill-time test: Bactericidal component of musk effect determined by kill-time test. The geometric dilution method that has been standardized and described in CLSI¹⁸. In the presence of a selected concentration of the musk and determined by measuring the number of viable bacteria at various time intervals. The resulting graphic depiction is known as the time-kill curve. In this study, the killing curve method was prepared in duplicate, the 96-well microplates were used to determine the MBC. Two Gram-negative bacteria were selected one of them had the highest MIC value and two Gram-positive bacteria that had the lowest MIC values from all 11 MDR bacteria. About 10 μL of musk dilution added to 90 μL MHB in the first well containing an inoculum of around 10⁶ CFU mL^–1 of bacteria. In order carryover of musk dilution, 10-fold dilution series were prepared in sterile MHB and 10 μL of these dilutions from and the initial broth well were then removed and spread onto musk-free MH agar and incubated for 24 h at 35°C. The colony-forming units (CFU) were counted at fixed incubation time’s interval (30 min, 1, 2, 4 and 24 h). The minimal bactericidal concentration (MBC) was defined as that yielding growth of fewer than five colonies (>99% killing), time-killing curves was prepared at 37°C in duplicate at musk concentration 10 and 0.16% (v/v) and it used another row without musk and inoculated with the bacterium as a growth control.

RESULTS

Antimicrobial effect of musk by disk diffusion method: The antimicrobial effects of musk and reference antibiotics (RA) by using disc-diffusion method against the tested microorganism are presented in Table 1 and 2, respectively. According to the results a similar pattern of antimicrobial activity was observed in all of the bacteria and presented strong antimicrobial effect in wild types and MDR bacteria and the best result of antimicrobial activity was shown in all tested microorganism at 100% (v/v) and the bacteria reduced susceptibility to musk with the lowest concentration 10% (v/v). The maximum inhibition zone was observed in Streptococcus agalactiae. According to the antimicrobial susceptibility patterns shown in Table 1, Pseudomonas aeruginosa exhibited reduced susceptibility to musk. The inhibition zones of musk effect at 100 and 10% (v/v) against Streptococcus agalactiae and Staphylococcus aureus illustrated in Fig. 1.

Table 1:	In vitro antimicrobial activity of musk to several micro-organisms determined by diameter (mm) zone of inhibition

Fig. 1(a-c):	Effect of musk treatment by disk diffusion method at (a) Concentration 100% (v/v), (b) Concentration 10% against Streptococcus agalactiae and (c) References antibiotic treatment against Staphylococcus aureus

Table 2:	Antimicrobial effect of standard antibiotics comparing to deer musk against MDR bacteria by disk-diffusion method
DO: Doxycycline ( 30 µg), CXM: Cefotaxime (30 µg), AMC: Amoxicillin/clavulanic acid (30 µg), SXT: Sulfamethoxazole trimethoprim (25 µg), AZM: Azithromycin (15 µg), PG: Penicillin G (10 units), NOR: Norfloxacin (10 µg) and DA: Clindamycin (10 µg), Musk 100% v/v applied in the tested microorganisms, Susceptibility patterns: R: Resistant and S: Susceptible

Minimum inhibitory concentration (MIC): The mean MIC (v/v%) of the musk against the bacterial strains are shown in Table 3. The musk showed an inhibitory effect on the growth of the tested MDR which the MIC values of musk treatment getting between 10% v/v and 0.1 (v/v) for all MDR bacteria.

Fig. 2(a-f):	Alteration in the morphological form of the Streptococcus agalactiae colonies with time interval (a) 3 h, (b) 24, (c-e) Magnification of A (f) Magnification of B

Table 3:	Minimum inhibitory concentration values (%, v/v) of musk at 37°C against MDR microbial strains
MDR: Multidrug resistance bacteria

According to MIC values, the Gram-positive bacteria showed MIC values <2% (v/v), whereas the best activity was noted towards musk with Streptococcus agalactiae with the lowest MIC value <1% (v/v) and the MIC values of some strain of Gram-negative bacteria was <2% (v/v) and the Pseudomonas aeruginosa showed some resistance effect to musk at lowest concentration but interestingly it found to be active with musk by increased the incubation time and concentration of musk.

Cell morphology: Changes were observed in the morphological form of the bacterial colonies treated with musk in the most of MDR bacteria such as Staphylococcus aureus, Streptococcus agalactiae, Pseudomonas aeruginosa and Klebsiella baumanni indicating that the musk had an effect on bacterial cellular membranes Fig. 2. Figure 2a and b presented the alteration in the morphological form of the Streptococcus agalactiae colonies after musk treatment at different concentration with different time’s interval 3 and 24 h, Fig. 2c-f represented magnification of Fig. 2a and b for morphological changes resulting from musk treatment showed the alteration in the morphological form of the Streptococcus agalactiae colonies after musk treatment at different concentration with different time’s interval 3 and 24 h.

Fig. 3:	Time killing curve of musk against MDR bacteria musk concentration at 10% (v/v), a viable bacteria count CFU mL–1 was performed after 30 min, 1, 2, 4 and 24 h

Fig. 4:	Time killing curve of musk against MDR bacteria (MBC) at musk concentration 0.16% (v/v), a viable bacteria count CFU mL–1 was performed after 30 min, 1, 2, 4 and 24 h

Bactericidal activity: The bactericidal activity results were registered 99.9% killing (>3 log10 CFU mL^–1) of four selected MDR bacterial strains obtained in the time-killing curves within five-time intervals are summarized in Table 4 and highest MBC values were observed in Pseudomonas aeruginosa. The Killing curves were prepared in duplicate and the results were identical to one dilution. Colony-forming unit (CFU) were calculated at five different time points (30 min, 1, 2, 4 and 24 h) to determine the number of living cells (CFU mL^–1) of each well using the agar plate count method. Starts with the concentration 10% (v/v) of musk after 24 h incubation ending with concentration 0.16% (v/v), yielding a log CFU mL^–1 was a value of zero of selected MDR bacteria. According to time-killing curves that are shown in Fig. 3 and 4 remarked a perfectly bactericidal effect at both concentrations with Gram-positive Streptococcus agalactiae within 30 min at concentration <2% (v/v) and the Enterococcus faecalis showed exhibited good bactericidal activity within 30 min.

Table 4:	Minimum bactericidal concentration (MBC(%, v/v)) values of musk against a selected MDR bacteria at 37°C at different time periods (30 min, 1, 2 and 24 h), MBC values needed to achieve a reduction in numbers of 5 log CFU mL–1

However, the musk has demonstrated a perfectly bactericidal effect in Gram-negative bacteria Pseudomonas aeruginosa after 24 h incubation >2% (v/v) and present exhibited time-dependent bactericidal kinetics, whereas the bactericidal activity of musk increased by time against Pseudomonas aeruginosa and the Escherichia coli exhibited bactericidal activity <2% (v/v) after 24 h.

DISCUSSION

The main objective of the present study was to evaluate the ability of deer musk to inhibit the growth of pathogenic bacteria that are resistant to the different class of antibiotics and known as multidrug-resistant bacteria. Furthermore to provethe antibacterial effect of different concentrations of natural musk on the above-mentioned MDR bacteria. Additionally, herbal medicine based on oil products has shown the effect in literature with good safety profile¹⁹. Moreover, musk bacteriostatic and bactericidal effect has been previously discussed by Saddiq⁷ and many studies were carried out to investigate the use of musk to inhibit the growth of many pathogenic micro-organisms for human, animals and plants, so it is important to examine newer drugs with lesser resistance, wherefore the natural products may present a new source of antimicrobial agents with probably novel mechanisms of action, drugs originated from natural sources show a considerable role in the inhibition and treatment of human diseases. In many developing countries, old-style medicine is one of the main healthcare systems²⁰, around 61% of new drugs established between 1981 and 2002 were formed from natural products and they have been effective, particularly in the infectious disease and cancer²¹. Many studies cite the inhibitory activity of musk against Gram-negative and Gram-positive organisms as well as fungi. The deer musk showed a great activity in inhibiting the growth of MDR bacteria, probably due to the presence of active ingredients that inhibit bacterial growth. Among the tested micro-organisms, these results are similar to that obtained from a recent study^1,4-6,8. In the present study, it focused on the use of antimicrobial testing methods for the in vitro investigation of musk as a potential antimicrobial agent against MDR bacteria. The most known and basic method used in this study for antimicrobial susceptibility testing is the agar disk-diffusion method to see if there is any effect to musk on the bacteria and how effective it is and a microdilution method used to assess the MIC result for a musk. The MIC here is the lowest concentration of musk that inhibits more than 99% of the bacterial population. In addition, the time-killing test is used for determining the bactericidal effect of musk on the selected MDR microbial strain. The time-killing test reveals a time-dependent or a concentration-dependent antimicrobial effect, in this study, followed the time-dependent kill test to achieve the musk effect, also the musk showed effective against all tested micro-organisms especially against Gram-positive bacteria even in the low concentrations as Streptococcus agalactiae, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus capitis and Streptococcus pyogenes which also has a significant effective against all tested Gram-negative microorganisms Klebsiella pneumoniae, Escherichia coli, Actinobacter baumanni, Proteus mirabilis, Serratia marcescens, Pseudomonas aeruginosa. This differentiation in antimicrobial effects between tested bacteria may be due to bacterial strains differences used in this study. This result is in agreement with another study which showed that the musk had an antibacterial effect against, Lactobacillus acidophilus, Lactococcus lactis, Streptococcus thermophiles, Staphylococcus aureus and Penicillium puberulum fungus³. According to the MIC and MBC results showed that musk had a bacteriostatic effect against all MDR bacterial strains that used in this study at the lowest concentration especially against Streptococcus agalactiae registered the lowest MIC value was <0.5% (v/v) and the antimicrobial sensitivity by disk diffusion method values registered >40 mm. The Pseudomonas aeruginosa is notorious for its involvement in nosocomial infections and frequent resistance to antibiotics and there are several studies showed that the Pseudomonas aeruginosa the most highly resistant to EO (essential oil)^19,22,23, although the musk comes as an oily nature Pseudomonas aeruginosa showed some resistance against the lowest concentration of musk which the MIC value was >2% (v/v) and it has reduced susceptibility in 1:10 of musk dilution but the musk can effect and inhibit the growth the Pseudomonas aeruginosa with a low concentration and can kill it at the highest concentration depending on increasing the time. Another observation in this study musk and its oil form is a component extracted from genitalia of deer that confirm the role of essential oils in change the morphology of the bacterial colonies by increasing membrane permeability, after 24 h of treatment with musk, the breakdown of rod bacterial cells has been observed and it may perform as a lubricant, which caused branching patterns of the bacteria. Several bacterial strains show colonial branching patterns through growth on poor semisolid substrates, these patterns reflect the bacterial supportive self-organization²⁴. Other explanations, essential oils (EO) and their components have activity against a range of targets, mainly the membrane and cytoplasm and in certain cases, they totally change the morphology of the cells²². Moreover, the hydrophobicity of EO is reliable for the disruption of bacterial structures that leads to increased permeability of cell membrane because of a failure to discrete the EO from the bacterial cell membrane²⁵. The antimicrobial activity of EO comes from its toxic effects on membrane structure and function, damaging the cytoplasmic membrane, cytoplasm coagulation, destructive the membrane proteins, the degradation of the cell wall and raised permeability and causing leakage of the cell contents^26-30.

CONCLUSION

It could be concluded that musk presented antibacterial activity against different bacterial strains, but at different levels and all the tested multidrug-resistant bacteria were more or less sensitive to musk has a significant role in human medication, displaying antimicrobial activities. So the main aim to study of the synergistic effects of musk and their components could be applied both to produce the best use of its antibacterial activity and to decrease its concentrations compulsory to accomplish a specific antibacterial effect for food safety and for health purposes and to prove the potential future use of musk as alternatives to common antibiotics and to regulate their capability to improve the activity of antibiotics, according to some studies suggested that musk has a wide spectrum of antimicrobial activity including fungistatic, fungicidal, parasitostatic, bactericidal and bacteriostatic effects.

SIGNIFICANCE STATEMENT

Discovery of natural products such as musk with effective activity against pathogenic micro-organisms and some MDR strains in the current study as supported by earlier reports from Saudi Arabia, Iraq, Egypt and developed countries such as Germany, Switzerland and Korea further support the viability of observing for an alternative approach to manage drug resistant microbes.