Azalomycin F 5a, a 36-membered macrocyclic lactone isolated from several streptomyces strains, presented remarkable anti-methicillin-resistant Staphylococcus aureus (MRSA) activities. To improve its anti-MRSA potential and to evaluate the probability of MRSA resistant to it before development, the anti-MRSA activities of azalomycin F 5a in combination with vitamin K 3 were first evaluated using checkerboard assay. Then the minimal concentration inhibiting colony formation by 99% (MIC 99) and mutant prevention concentration (MPC) of azalomycin F 5a alone and in combination with vitamin K 3 against MRSA were determined using agar plates with linear antimicrobial concentration decrease. The fractional inhibitory concentration indexes (FICIs) of 0.25 - 0.50 showed the synergistic activity of azalomycin F 5a in combination with vitamin K 3. The mutant selection windows (MSWs, MIC 99-MPC) of azalomycin F 5a alone against MRSA tested were 2.07 - 6.40 μg/mL, and the MPCs of azalomycin F 5a in combination with vitamin K 3 against MRSA tested were 1.60 - 3.20 μg/mL. These indicated that the MPCs of azalomycin F 5a in combination could drop down to below its MIC 99 alone. According to the hypothesis of MSW, the narrower MSWs of azalomycin F 5a alone, even closed MSWs in combination with vitamin K 3, together with their synergistic anti-MRSA activities, indicated that azalomycin F 5a had a good potential to develop as a new antimicrobial agent.
Antimicrobials are a crucial defense against bacterial infections, while they also promote the evolution of bacteria, and even lead bacteria to be resistant to themselves. Now, infections from resistant bacteria are too common, and some pathogens have even become resistant to multiple types of antibiotics. As antimicrobial resistance seriously threatened human health, many strategies involving the development of new antibiotics, combination therapy with several antibiotics, the revival of old antimicrobials and the optimal use of clinic antibiotics were already put forward to fight or delay resistance [
Based on this, the hypotheses of mutant prevention concentration (MPC) and mutant selection window (MSW) were put forward [
Azalomycin F5a (
methicillin-resistant Staphylococcus aureus (MRSA) activities, together with those of vitamin K3, were evaluated by us [
MRSA ATCC 33,592 (Gentamycin and methicillin-resistant) were purchased from American Type Culture Collection, Manassas, VA, USA, and three clinic isolates MRSA HK01, HK02 and HK03 (Methicillin-resistant) were friendly presented by Hainan General Hospital, Haikou, China. Bacterial inocula were prepared in Mueller Hinton Broth (MHB) at 35˚C until the OD600 nm value was 0.60 before use. MHB used for MIC test and Mueller Hinton Agar (MHA) used for MIC99 and MPC test were purchased from Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China.
Azalomycin F5a was prepared in our laboratory according to our previous work [
The MICs of azalomycin F5a and vitamin K3 against MRSA ATCC 33,592 and three clinical isolates MRSA HK01, HK02, HK03 were respectively determined using broth microdilution method according to a standard procedure described by the Clinical and Laboratory Standards Institute (CLSI) in 2012 [
According to the MIC of azalomycin F5a and vitamin K3, the checkerboard method was designed to determine the fractional inhibitory concentration (FIC) indexes of azalomycin F5a in combination with vitamin K3 against MRSA ATCC 33,592 and three clinical isolates MRSA HK01, HK02 and HK03. The tests were performed on 96-well plate according to published methods [
The fractional inhibitory concentrations (FICs) were calculated as follows: FIC of azalomycin F5a = MIC of azalomycin F5a in combination/MIC of azalomycin F5a alone, and FIC of vitamin K3 = MIC of vitamin K3 in combination/MIC of vitamin K3 alone. The FIC index (FICI) was defined as the FIC of azalomycin F5a added to the FIC of vitamin K3. The effect of azalomycin F5a in combination with vitamin K3 against MRSA was interpreted as follows: Synergy, FICI ≤ 0.5; antagonism, FICI ≥ 4.0; and indifferent, 0.5 < FICI > 4.0.
According to the MIC of azalomycin F5a and vitamin K3 alone or in combination, their minimal concentrations that inhibit colony formation by 99% (MIC99s) against MRSA ATCC 33,592 were determined by utilizing linear antimicrobial concentration decrease (20% per sequential decrease) from MIC, plus a replicate. Referring the methods as described in previous papers [
Using MRSA ATCC 33,592, the MPCs were determined as described elsewhere [
The MICs of azalomycin F5a against all MRSA strains tested were 4.0 μg/mL, and those of vitamin K3 were 16.0 μg/mL except for 32.0 μg/mL against MRSA HK03. Further, checkerboard test showed that all the FICIs of azalomycin F5a in combination with vitamin K3 against MRSA strains tested were 0.25 - 0.50 (
Various ratios of azalomycin F5a to vitamin K3 could be selected to determine their MIC99s and MPCs in combination. Considering that
Checkerboard test showed synergistic anti-MRSA activity of azalomycin F5a in combination with vitamin K3, while the potential of MRSA be resistant to azalomycin F5a was
MRSA strains | Azalomycin F 5a | |||||
---|---|---|---|---|---|---|
MICs (μg/mL) | FICIs | |||||
Aa | Bb | C | b | c | ||
ATCC 33,592 | 4.0 | 1.0 | 1.0 | 0.375 | 0.50 | |
HK01 | 4.0 | 1.0 | 1.0 | 0.375 | 0.50 | |
HK02 | 4.0 | 1.0 | 1.0 | 0.375 | 0.50 | |
HK03 | 4.0 | 0.5 | 0.5 | 0.25 | 0.375 |
aA: MICs of azalomycin F5a alone. bB, C, b and c: The MICs (B and C) and FICIs (b and c) of azalomycin F5a in combination with vitamin K3 when the concentrations of vitamin K3 were respectively 0.125 and 0.25 MIC.
Antimicrobialsb | MIC (μg/mL) | Regression equation (rc) | MIC99 (μg/mL) | MPC (μg/mL) | MPC/MIC99d |
---|---|---|---|---|---|
AZF5a | 4.0 | y = 0.4251x + 0.0627 (0.996) | 2.18 | 5.12 | 2.35 |
Vit K3 | 16.0 | y = 0.0916x + 0.0660 (0.998) | 10.09 | 32.00 | 3.17 |
AZF5a/Vit K3 (1:2) | 1.0/2.0 | y = 2.0175x − 0.0947 (0.995) | 0.54 | 3.20 | 1.47 |
AZF5a/Vit K3 (1:4) | 1.0/4.0 | y = 2.2363x + 0.0400 (0.993) | 0.42 | 2.56 | 1.17 |
AZF5a/Vit K3 (1:8) | NDe | y =1.4602x + 0.0382 (0.994) | 0.65 | 2.05 | 0.94 |
AZF5a/Vit K3 (1:16) | ND | ND | ND | 1.60 | 0.73 |
aMRSA ATCC 33,592 was used as MRSA strain tested. bAZF5a and Vit K3 were abbreviations of azalomycin F5a and vitamin K3, respectively. cr, correlation coefficient of a binary regression equation. dMPC/MIC99 ratio is defined as the ratio of the MPC obtained to the original MIC99 alone. eND, not tested.
Antimicrobialsb | MIC (μg/mL) | Regression equation (rc) | MIC99 (μg/mL) | MPC (μg/mL) | MPC/MIC99d |
---|---|---|---|---|---|
AZF5a | 4.0 | y = 0.4929x − 0.0325 (0.999) | 2.07 | 6.40 | 3.09 |
Vit K3 | 32.0 | y = 0.0630x − 0.0440 (0.998) | 16.41 | 81.92 | 4.99 |
AZF5a/Vit K3 (1:2) | NDe | ND | ND | 3.20 | 1.54 |
AZF5a/Vit K3 (1:4) | ND | ND | ND | 3.20 | 1.54 |
AZF5a/Vit K3 (1:8) | 0.5/4.0 | ND | ND | 2.56 | 1.24 |
AZF5a/Vit K3 (1:16) | 0.5/8.0 | ND | ND | 1.60 | 0.77 |
aMRSA HK03 was used as MRSA strain tested. bAZF5a and Vit K3 were abbreviations of azalomycin F5a and vitamin K3, respectively. cr, correlation coefficient of a binary regression equation. dMPC/MIC99 ratio is defined as the ratio of the MPC obtained to the original MIC99 alone. eND, not tested.
unable to known in combination. Considering resistance would empirically emerge shortly after a new antibiotic is used, new antibiotics that MRSA be less resistant to was more value to develop. Thereby, the MIC99s and MPCs of azalomycin F5a and vitamin K3 alone and in combination were determined according the hypotheses of MPC and MSW [
To better understand the experimental results, researches on MPCs, MIC99s and MSWs of azalomycin F5a alone and in combination with vitamin K3 were further carried forward using the schematic representation of their mutant selection windows (
As the MPCs of one in combination will change as those of another, is there a certain correlation between the proportions of two antimicrobials and their MPCs in combinations? To understand it, experimental data shown in
the ratio of two antimicrobials and the sum of the MPC/MIC99Alone in combination to a certain pathogenic microorganism may be correlative although that observed between MIC and MPC was low (r2 = 0.39) [
Although the MPC of azalomycin F5a or vitamin K3 in combination can be dropped down to below its MIC99 alone by increasing the proportion of another, two new MSWs (MIC99Combination-MPCCombination) of azalomycin F5a and vitamin K3 against MRSA ATCC 33,592 emerged (
Antibiotic resistance, a part of natural evolution, can be significantly slowed but not be stopped. Developing new antibiotics is still an effective strategy in the battle against antibiotic resistance when we optimize the use of exist antimicrobial agents. According to the MPC and MSW hypotheses, new antimicrobials with narrow MSW will present more potential to prevent resistance. The synergistic anti-MRSA activities of azalomycin F5a in combination with vitamin K3, coupled with the narrow MSW of azalomycin F5a against MRSA tested, indicated that azalomycin F5a has a good potential to develop as a new antimicrobial agent [
Combination therapy with two or more drugs is the standard treatment for infections with Mycobacterium tuberculosis, human immunodeficiency virus (HIV) and Plasmodium falciparum [
1) Selecting remarkably synergistic and susceptible antimicrobial agents for combination: According to the MSW and MPC hypotheses, the less the MPC in combination, the larger the dosing range to prevent resistance is, and the easier maintaining antimicrobial concentration above their MPCs throughout combination therapy is. Perfectly, the MPC of each antimicrobial in combination drop down to below its MIC99 alone, and the MPCCombination/MIC99Combination of each antimicrobial in combination is equal to one. That is to say, no new MSW of each antimicrobial in combination emerge when synergistic combination closes the original MSW of two antimicrobials. Thereby, we can determine the FICIs of antimicrobial combination, and select those presenting less FICIs for combination. Further, two antimicrobials that possessed different action mechanisms to the same pathogenic bacteria should theoretically present synergistic activity, and display a significantly reduced MPC in combination. Thereby, we can select antimicrobials had different action mechanisms for the synergistic evaluation of antimicrobial combination.
2) As different proportion of each antimicrobial in synergistic combination would present their different MPCs, maintaining antimicrobial concentrations in vivo, especially in blood and in infection site, above their MPCs throughout combination therapy is a key for synergistic combination to prevent resistance, and this may be partly responsible for the conflicting results reported [
3) To prevent resistance, three interacted aspects include pathogenic microorganism, antimicrobial combination and human body should be systematically taken into account. As the absorption, distribution, metabolism and excretion of each antimicrobial in combination are different in human body, different proportion of each antimicrobial in synergistic combination would present their different MPC. If the antimicrobial concentration is less than its MPC in combination in the infection site or in blood of human body last for a long time, combination will accelerate the resistant mutant on the contrary. Moreover, antimicrobial concentration ranged from MIC99Combination to MPCCombination (especially to MICCombination) will stimulate the formation of biofilm and persisters, and this may enhance the resistance or tolerance to antimicrobials.
As we known, microorganisms in human body can promote and restrain each other to form balanced microorganism communities, and which can help us to defend pathogenic infection. Many antimicrobial agents can destruct the microorganism communities [
4) Although the general benefits of combination therapy compared with single or sequential administration of antibiotics for treating bacterial infections have been difficult to conclusively demonstrate, and some clinical data have been conflicting [
Azalomycin F5a, a 36-membered macrocyclic lactone, combined with vitamin K3 showed synergistic anti-MRSA activities. Simultaneously, the MPC of azalomycin F5a in combination could drop down to below its MIC99 alone when the proportion of vitamin K3 increased, and the narrow and even closed MSWs like this indicated that azalomycin F5a has a good potential to develop as a new antimicrobial agent. Further, the correlation between the ratio of azalomycin F5a/vitamin K3 and the sum of the MPC/MIC99 in combination was observed, and be expressed as a binary regression equations to a certain MRSA strain. Thus, the correlation between the ratio of two antimicrobials and the sum of the MPC/MIC99 was first found, which could be used to predict the MPCs and the perfect ratio of two antimicrobials in combination. Moreover, some opinions on antimicrobial combination and synergistic strategy were put forward to prevent drug resistance based on the analyses of experimental data and documents.
The authors declare no conflict of interest.
This study was supported by the National Natural Science Foundation of China [No. 81260476 and 81460529] and the University Science Research Project of Jiangxi, China [GJJ14277]. The authors would like to thank Hainan General Hospital, Haikou, China for providing three clinic isolates MRSA HK01, HK02 and HK03 (Methicillin-resistant).
Xu, X.J., Wu, X.J., Yuan, G.J., Xu, L. and Wang, Y.M. (2016) Mutant Selection Windows of Azalomycin F5a in Combination with Vitamin K3 against Methicillin-Resistant Staphylococcus aureus. Journal of Biosciences and Medicines, 4, 162-174. http://dx.doi.org/10.4236/jbm.2016.412020