Appl Biochem Biotechnol (2014) 173:116–128 DOI 10.1007/s12010-014-0808-3
In Vitro Antibacterial Screening of Six Proline-Based Cyclic Dipeptides in Combination with β-Lactam Antibiotics Against Medically Important Bacteria S. Nishanth Kumar & Ravi S. Lankalapalli & B. S. Dileep Kumar
Received: 18 November 2013 / Accepted: 12 February 2014 / Published online: 13 March 2014 # Springer Science+Business Media New York 2014
Abstract The in vitro synergistic antibacterial activity of six proline-based cyclic dipeptides [cyclo(D-Pro-L-Leu), cyclo(L-Pro-L-Met), cyclo(D-Pro-L-Phe), cyclo(L-Pro-L-Phe), cyclo(LPro-L-Tyr), and cyclo(L-Pro-D-Tyr)] in combination imipenem and ceftazidime was investigated in the present manuscript. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the cyclic dipeptides were compared with those of the standard antibiotics (imipenem and ceftazidime). The synergistic antibacterial activities of cyclic dipeptides with imipenem or ceftazidime were assessed using the checkerboard and time-kill methods. The results of the present study showed that the combined effect of six cyclic dipeptides with imipenem predominantly recorded synergistic interaction (FIC index 4 represented antagonism [14]. The fractional bactericidal concentration (FBC) is calculated in the same way described above by replacing the MIC values with the MBC values. Time-Kill Curves The time-kill assay against bacterial strains was exposed over time to six cyclic dipeptides and antibiotics alone as well as to their combinations according to the guideline provided by NCCLS [14, 15]. An overnight inoculum of 1×106 CFU/mL of each isolate was added along with the antimicrobials into tubes filled to a final volume of 3 mL with nutrient broth. The tubes were thereafter incubated at 37 °C, and viable counts were performed after 0, 2, 4, 6, 12, 24, and 48 h of incubation. At the above time intervals, one aliquot of 0.1-mL samples was removed, serially diluted with 0.85 % saline, and plated onto nutrient agar. The number of viable cells in each tube was estimated after counting plates and by multiplying by the appropriate dilution factor. Colony counts were performed in duplicate, and means were taken. Killing curves were constructed by plotting the log10 CFU/mL versus time over 48 h, and the change in bacterial concentration was determined. Bactericidal activity was defined as a reduction of 99.9 % (≥3 log10) of the total number of CFU/mL in the original inoculum. Bacteriostatic activity was defined as maintenance of the original inoculum concentration or a reduction of less than 99.9 % (≥3 log10) of the total number of CFU/mL in the original inoculums. Cytotoxicity Test The MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay was used to determine the cytotoxicity of cyclic dipeptides. A known cytotoxic drug cisplatin was used as positive control. VERO cell line was used for testing. MTT assay is based on the ability of mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the
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pale yellow MTT and to form dark blue formazan crystals which are largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of the crystals. The number of surviving cells is directly proportional to the level of the formazan product formed. The color can then be quantified by a simple colorimetric assay using a multi-well scanning spectrophotometer (ELISA reader). Briefly, cells (5×103 per well) were seeded in 0.2 mL of the medium (DMEM with 10 % PBS) on a 96-well plate, treated with cyclic dipeptides for 72-h incubation. Cytotoxicity was measured by removing the diketopiperazine containing media from the well, and 25 μL of MTT solution (5 mg/mL in PBS) and 75 μL of complete medium were added to wells (untreated and treated) and incubated. After 2 h, MTT lysis buffer was added to the wells (0.1 mL per well) and incubated for another 4 h at 37 °C. At the end of incubation, the optical densities at 570 nm were measured using a plate reader (Bio-Rad). The relative cell viability in percentage was calculated: (A570 of treated sample)/(A570 of untreated sample)×100 [16].
Result MIC and MBC MIC and MBC ranges of six proline-based cyclic dipeptides and antibiotics used alone for medically important bacteria are shown in Table 1. The ranges of MIC/MBC of cyclic dipeptides for the bacteria included in the study were 16 to 250 μg/mL for S. epidermis, 16 to 4,000 μg/mL for K. pneumoniae, 32 to 1,000 μg/mL for P. mirabilis, and 8 to 2,000 μg/mL for V. cholerae. DKP 5 recorded activity at higher concentration, i.e., between 125 and 4,000 μg/mL against the bacteria. Checkerboard Assay The combined activities of cyclic dipeptides with antibiotics from the in vitro checkerboard interactions against the medically important bacteria are summarized in Tables 2 and 3. FIC, FBC, FIC index, FBC index, and interpretations for the activities of cyclic dipeptides and antibiotics against the test bacteria predominantly showed a synergistic interaction. But some combination of cyclic dipeptides with ceftazidime recorded additive. Antagonism and indifference were not recorded for any of the combinations.
Table 1 Antibacterial activity of cyclic dipeptides and antibiotics against bacteria
Values represent mean of three replications
MIC/MBC (μg/mL) Test compounds
S. epidermis
K. pneumoniae
P. mirabilis
V. cholerae
DKP 1
32/64
250/500
250/500
500/1,000
DKP 2
16/32
8/16
32/64
64/125
DKP 3
125/125
125/250
32/64
64/125
DKP 4
250/250
32/64
64/125
125/250
DKP 5
125/125
2,000/4,000
500/1,000
1,000/2,000
DKP 6
16/16
16/32
32
8/16
Imipenem
4/4
2/4
1/1
2/4
Ceftazidime
8/8
4/4
8/8
8/8
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Table 2 Synergistic effects of the cyclic dipeptides with imipenem against bacteria Test bacteria
S. epidermis
K. pneumoniae
P. mirabilis
V. cholerae
Agent
MIC/MBC (μg/mL)
FIC/FBC
FICIb/FBCIc
Outcome
Alone
Combinationa
DKP 1 Imipenem
32/64 4/4
2/4 0.25/0.5
0.06/0.06 0.06/0.13
0.12/0.19
Synergistic/synergistic
DKP 2 Imipenem
16/32 4/4
4/4 1/1
0.25/0.16 0.25/0.25
0.5/0.41
Synergistic/synergistic
DKP 3 Imipenem
125/500 4/4
4/8 1/1
0.03/0.02 0.25/0.25
0.28/0.27
Synergistic/synergistic
DKP 4 Imipenem
250/500 4/4
8/8 0.5/0.5
0.03/0.02 0.16/0.16
0.19/0.18
Synergistic/synergistic
DKP 5 Imipenem
125/250 4/4
4/8 0.25/0.5
0.03/0.03 0.06/0.13
0.09/0.16
Synergistic/synergistic
DKP 6 Imipenem
16/32 4/4
2/4 0.12/0.25
0.13/0.13 0.03/0.06
0.16/0.16
Synergistic/synergistic
DKP 1 Imipenem
250/500 2/4
32/64 0.5/0.5
0.13/0.13 0.25/0.13
0.38/0.26
Synergistic/synergistic
DKP 2 Imipenem
8/8 2/4
1/1 0.25/0.25
0.13/0.13 0.13/0.06
0.26/0.19
Synergistic/synergistic
DKP 3 Imipenem
125/250 2/4
16/64 0.12/0.12
0.13/0.26 0.06/0.03
0.19/0.29
Synergistic/synergistic
DKP 4 Imipenem
32/64 2/4
2/4 0.06/0.12
0.06/0.06 0.03/0.03
0.09/0.09
Synergistic/synergistic
DKP 5 Imipenem
2,000/4,000 2/4
125/250 0.5/0.5
0.06/0.06 0.25/0.13
0.31/0.19
Synergistic/synergistic
DKP 6 Imipenem
16/32 2/4
2/4 0.06/0.12
0.13/0.13 0.03/0.03
0.16/0.19
Synergistic/synergistic
DKP 1 Imipenem
250/500 1/1
32/64 0.12/0.25
0.13/0.13 0.12/0.25
0.25/0.38
Synergistic/synergistic
DKP 2 Imipenem
32/64 1/1
8/16 0.03/0.06
0.25/0.25 0.03/0.06
0.28/0.31
Synergistic/synergistic
DKP 3 Imipenem
32/64 1/1
4/8 0.06/0.06
0.03/0.13 0.06/0.06
0.09/0.19
Synergistic/synergistic
DKP 4 Imipenem
64/125 1/1
8/8 0.12/0.12
0.13/0.06 0.12/0.12
0.25/0.18
Synergistic/synergistic
DKP 5 Imipenem
500/1,000 1/1
64/125 0.25/0.25
0.13/0.13 0.25/0.25
0.38/0.38
Synergistic/synergistic
DKP 6 Imipenem
32/64 1/1
8/8 0.25/0.25
0.25/0.13 0.25/0.25
0.5/0.38
Synergistic/synergistic
DKP 1 Imipenem
500/1,000 2/4
64/64 0.25/0.25
0.13/0.06 0.13/0.06
0.26/0.12
Synergistic/synergistic
DKP 2 Imipenem
64/125 2/4
16/16 0.12/0.25
0.25/0.13 0.06/0.06
0.31/0.18
Synergistic/synergistic
DKP 3 Imipenem
64/125 2/4
4/4 0.25/0.5
0.06/0.03 0.13/0.13
0.18/0.16
Synergistic/synergistic
DKP 4 Imipenem
125/250 2/4
32/64 0.5/1
0.26/0.26 0.25/0.25
0.51/0.51
Indifference/indifference
DKP 5 Imipenem
1,000/2,000 2/4
125/125 0.5/1
0.13/0.06 0.25/0.25
0.38/0.31
Synergistic/synergistic
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Table 2 (continued) Test bacteria
Agent
DKP 6 Imipenem a b c
MIC/MBC (μg/mL) Alone
Combinationa
8/16 2/4
1/2 0.25/0.25
FIC/FBC
FICIb/FBCIc
Outcome
0.13/0.13 0.13/0.06
0.26/0.19
Synergistic/synergistic
The MIC and MBC value of cyclic dipeptides with imipenem The fractional inhibitory concentration index (FIC index) The fractional bactericidal concentration index (FBC index)
Significant FICI/FBCI values are shown in bold
When DKP 4 was combined with imipenem for the inhibition of S. epidermis, an important synergistic effect (FIC=0.19) was observed, and the MIC values of DKP 4 and imipenem were reduced to 5 and 3 times below their individual MIC values, respectively (Table 2). Another notable synergism was observed (FIC=0.09) for DKP 1 and ceftazidime against P. mirabilis, and the concentration of the DKP1 and ceftazidime was reduced to 5 times below their individual MIC values (Table 3). Time-Kill Study The antibacterial effect of proline-based cyclic dipeptides with imipenem or ceftazidime against test bacteria was confirmed by time-kill curve experiments. Time-kill assays for the synergistic combinations (cyclic dipeptides and antibiotics) on test bacteria strains are shown in Fig. 2. The time-kill assay was conducted to determine the rates of killing of test bacteria when exposed to cyclic dipeptides with imipenem or ceftazidime (Fig. 2). When proline-based cyclic dipeptides with imipenem or ceftazidime were tested against S. epidermis (Fig. 2a), maximum reduction in the colony count was observed between 4 and 12 h when compared to the control. At 6 to 24 h, a complete bactericidal effect was observed with treatments of combination of cyclic dipeptides and antibiotics. In case of K. pneumoniae, maximum reduction was observed between 4 and 24 h (Fig. 2b). A complete bactericidal effect was observed within 24 h for cyclic dipeptides and imipenem and 48 h for cyclic dipeptides and ceftazidime. Similar results were observed for P. mirabilis and V. cholerae (Fig. 2c, d). Regrowth was observed for diketopiperazine and antibiotics alone after 24 h, whereas it was not observed for the combination even at 48 h (Fig. 2). Cytotoxicity Test The cytotoxicity of cyclic dipeptides was tested against VERO cell line by MTT assay. The results showed that there is no significant cytotoxicity up to 100 μg/mL except DKP 4 (Fig. 3). At 100 μg/mL concentration of cyclic dipeptides, approximately 80 to 85 % of cells were alive, whereas 44 % of cells were killed at 100 μg/mL concentration of cisplatin, which was used as a positive control in MTT assay (Supplementary Fig. 1). But DKP 4 recorded slight toxicity after 50 μg/mL. The results of the present study clearly indicated that cyclic dipeptides may be safe for the treatment of pathogenic bacteria. The cytotoxicity data clearly indicated that cyclic dipeptides may not affect the normal cell and its action may selectively to the test bacteria.
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Table 3 Synergistic effects of the cyclic dipeptides with ceftazidime against bacteria Test bacteria
S. epidermis
K. pneumoniae
P. mirabilis
V. cholerae
Agent
MIC/MBC (μg/mL)
FIC/FBC
FICIb/FBCIc
Outcome
Alone
Combinationa
DKP 1 Ceftazidime
32/64 4/4
4/8 1/1
0.13/0.13 0.25/0.25
0.38/0.38
Synergistic/synergistic
DKP 2 Ceftazidime
16/32 4/4
4/8 1/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
DKP 3 Ceftazidime
125/500 4/4
32/64 0.5/1
0.26/0.13 0.13/0.25
0.38/0.38
Synergistic/synergistic
DKP 4 Ceftazidime
250/500 4/4
32/32 0.25/0.5
0.13/0.06 0.06/0.13
0.19/0.19
Synergistic/synergistic
DKP 5 Ceftazidime
125/250 4/4
4/8 1/2
0.03/0.03 0.25/0.5
0.28/0.53
Synergistic/synergistic
DKP 6 Ceftazidime
16/32 4/4
2/4 0.5/0.5
0.13/0.13 0.13/0.13
0.26/0.26
Synergistic/synergistic
DKP 1 Ceftazidime
250/500 2/4
64/64 0.5/1
0.26/0.13 0.25/0.25
0.51/0.38
Synergistic/synergistic
DKP 2 Ceftazidime
8/8 2/4
1/2 0.25/0.5
0.13/0.25 0.13/0.13
0.26/0.38
Synergistic/synergistic
DKP 3 Ceftazidime
125/250 2/4
32/64 0.5/1
0.26/0.26 0.25/0.25
0.51/0.51
Indifference/indifference
DKP 4 Ceftazidime
32/64 2/4
16/16 1/2
0.5/0.25 0.5/0.5
1/0.75
Indifference/indifference
DKP 5 Ceftazidime
2,000/4,000 2/4
500/1,000 1/2
0.25/0.25 0.5/0.5
0.75/0.75
Indifference/indifference
DKP 6 Ceftazidime
16/32 2/4
4/8 0.25/0.25
0.25/0.25 0.13/0.06
0.38/0.31
Synergistic/synergistic
DKP 1 Ceftazidime
250/500 1/1
8/16 0.06/0.12
0.03/0.03 0.06/0.12
0.09/0.15
Synergistic/synergistic
DKP 2 Ceftazidime
32/64 1/1
2/4 0.25/0.5
0.06/0.06 0.25/0.5
0.31/0.56
Synergistic/indifference
DKP 3 Ceftazidime
32/64 1/1
8/16 0.25/0.5
0.25/0.25 0.25/0.5
0.5/0.75
Indifference/indifference
DKP 4 Ceftazidime
64/125 1/1
16/32 0.25/0.5
0.25/0.26 0.25/0.5
0.5/0.76
Indifference/indifference
DKP 5 Ceftazidime
500/1,000 1/1
250/250 0.25/0.5
0.5/0.25 0.25/0.5
0.75/0.75
Indifference/indifference
DKP 6 Ceftazidime
32/64 1/1
4/8 0.12/0.25
0.13/0.13 0.12/0.25
0.25.0.38
Synergistic/synergistic
DKP 1 Ceftazidime
500/1,000 2/4
250/500 0.5/1
0.5/0.5 0.25/0.25
0.75/0.75
Indifference/indifference
DKP 2 Ceftazidime
64/125 2/4
4/8 0.25/0.5
0.06/0.06 0.13/0.13
0.19/0.19
Synergistic/synergistic
DKP 3 Ceftazidime
64/125 2/4
2/4 0.25/0.5
0.03/0.03 0.13/0.13
0.16/0.16
Synergistic/synergistic
DKP 4 Ceftazidime
125/250 2/4
4/8 0.5/0.5
0.03/0.03 0.25/0.13
0.28/0.16
Synergistic/synergistic
DKP 5 Ceftazidime
1,000/2,000 2/4
250/500 1/1
0.25/0.25 0.5/0.25
0.75/0.75
Indifference/indifference
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Table 3 (continued) Test bacteria
Agent
DKP 6 Ceftazidime a b c
MIC/MBC (μg/mL) Alone
Combinationa
8/16 2/4
2/4 1/2
FIC/FBC
FICIb/FBCIc
Outcome
0.25/0.25 0.5/0.5
0.75/0.75
Indifference/indifference
The MIC and MBC value of cyclic dipeptides with ceftazidime The fractional inhibitory concentration index (FIC index) The fractional bactericidal concentration index (FBC index)
Significant FICI/FBCI values are shown in bold
Discussion The increasing occurrence, particularly in hospitals, of pathogenic-resistant bacteria especially Staphylococcus sp. to a wide range of antimicrobial agents, including all kinds of β-lactams, has made therapy more difficult. The increasing resistance to antibiotic represents the main factor justifying the need to find and/or develop new antimicrobial agents. Thus, many studies have been focused on antimicrobial agents and on the antimicrobial properties of naturalderived active principles [17–19]. Although many strategies have been proposed in an attempt to control the spread of drug resistance pathogenic bacteria, the search for new ways to treat infections stimulates the investigation for natural compounds as an alternative treatment to control these infections. Antimicrobial combination therapy may be used to extend spectrum coverage, prevent the emergence of resistant mutants, and gain synergy between antimicrobials [20]. Combination therapy is often recommended for empirical treatment of bacterial infections in intensive care units, where monotherapy is not likely to cover all potential pathogens, and the emergence of resistance is a potential threat [21]. In the last years, new antimicrobials specifically targeted against Gram-positive and Gramnegative strains have been launched, but strains with decreased susceptibility to these antibiotics have been increasingly isolated and, unfortunately, development of new antimicrobial agents in the next future seems to be declining [22, 23]. On the other hand, antimicrobial combination therapy should be useful in improving efficacy, providing broad-spectrum coverage, and preventing the emergence of resistant mutants [20]. Moreover, combination therapy is argued when empirical treatment of severe infections is needed, such as in severe pneumonia. As an etiological diagnosis with susceptibility results is almost never available to assist in the selection of the prompt therapy, the initial approach to treatment is largely empirical and is ordinarily guided by consensus guidelines. Moreover, early administration of appropriate empirical therapy for severe pneumonia has been demonstrated to significantly improve survival [24]. For this reason, antimicrobial therapy should cover both Gram-positive and Gram-negative bacteria until obtainment of laboratory results. But currently, no information regarding the antimicrobial activity of proline-based cyclic dipeptides in combination with antibiotics was available in literature. In the present study, proline-based cyclic dipeptides in combination with imipenem and ceftazidime exhibited significant synergistic effect against medically important bacteria. Synergism is a positive interaction created when two compounds combined and exert an inhibitory effect (on the targeted organisms) that is greater than the sum of their individual effects. Synergism has also
Appl Biochem Biotechnol (2014) 173:116–128
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Fig. 2 Time-kill curve of cyclic dipeptide and antibiotics alone and in combination. a Staphylococcus epidermis, b Klebsiella pneumoniae, c Proteus mirabilis, d Vibrio cholera. 1 DKP 1, 2 DKP 2, 3 DKP 3, 4 DKP 4, 5 DKP 5, and 6 DKP 6. Control, Corresponding DKPs, Imipenem, Ceftazidime, Corresponding DKPs + imipenem, Corresponding DKPs + ceftazidime
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Fig. 3 Cytotoxicity of cyclic dipeptides against VERO cell line by MTT assay
been defined as a phenomenon in which two different compounds are combined to enhance their individual activity. Antagonism results if the combination provides an effect less than the effect of either agent alone or less than the sum of the effects of the individual agents. Indifference results if the combination provides an effect equal to the effect of either agent alone [25]. Thus, evaluation of microbial-based antimicrobial agents for activity against medically important bacteria is imperious. We recently reported the efficacies of cyclo(D-Pro-L-Leu), cyclo(L-Pro-L-Met), cyclo(D-Pro-L-Phe), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(LPro-D-Tyr) against four test bacteria and five test fungi [11]. The structure of the compounds is shown in Table 1. Furthermore, we found that the combination of cyclo(L-Leu-L-Pro), cyclo(DLeu-L-Pro), and cyclo(L-Leu-L-Tyr) synergically inhibited four test bacteria [26]. To the best of our knowledge, the combination of cyclic dipeptides and antibiotics (cyclic dipeptides plus imipenem and ceftazidime) against medically important bacteria has never been shown before. We, therefore, examined the in vitro effect of cyclic dipeptides and antibiotics against four medically important bacteria (S. epidermis, K. pneumoniae, P. mirabilis, and V. cholerae). In the present study, data from the checkerboard assay indicates that cyclic dipeptides with ceftazidime showed the highest rate of synergy among the tested combinations. The ability of cyclic dipeptides and antibiotics combination to yield synergy was quite different for the various antibiotics. Time-killing curves also confirmed superior activity of cyclic dipeptides with ceftazidime. It is also evident that this ability was not a class effect since marked differences were found among β-lactams tested. Therefore, it seems rather difficult to compare our data with those of other studies in which different β-lactams have been used. Imipenem and ceftazidime affect bacteria through inhibiting cell wall synthesis of various Gram-positive and Gram-negative bacteria, and the mode of action of diketopiperazine is not known and it is supposed that its action may be through the novel route. Thus, the combination of cyclic dipeptides with imipenem and ceftazidime will also act the test bacteria through a novel route which may be different from the mechanism of action of cyclic dipeptides and antibiotics and that may be one of the reasons of the enhanced activity.
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Conclusion The results from the present study warrant further investigations on the possible synergistic effects of cyclic dipeptides with other class of antibacterial drugs. The observed synergism between cyclic dipeptides and antibiotics in vitro should also be investigated in in vivo animal model. In addition, further experiments could be performed in order to elucidate the molecular mechanisms underlying this synergistic effect. In addition, a broad screen of natural products is urgently needed, especially for those with no detectable activity as single agents but bearing synergistic effects with other compounds. Due to the general low toxicity of cyclic dipeptides, we speculate that cyclic dipeptides might be used to develop new synergistic and efficient associations with other antimicrobials for external use against antibiotic resistant strains. Acknowledgments The authors are grateful to KSCSTE and the Government of India for funding. Conflict of Interest The authors declare that they have no conflict of interest.
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In Vitro Antibacterial Screening of Six Proline-Based Cyclic Dipeptides in Combination with β-Lactam Antibiotics Against Medically Important Bacteria S. Nishanth Kumar & Ravi S. Lankalapalli & B. S. Dileep Kumar
Received: 18 November 2013 / Accepted: 12 February 2014 / Published online: 13 March 2014 # Springer Science+Business Media New York 2014
Abstract The in vitro synergistic antibacterial activity of six proline-based cyclic dipeptides [cyclo(D-Pro-L-Leu), cyclo(L-Pro-L-Met), cyclo(D-Pro-L-Phe), cyclo(L-Pro-L-Phe), cyclo(LPro-L-Tyr), and cyclo(L-Pro-D-Tyr)] in combination imipenem and ceftazidime was investigated in the present manuscript. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the cyclic dipeptides were compared with those of the standard antibiotics (imipenem and ceftazidime). The synergistic antibacterial activities of cyclic dipeptides with imipenem or ceftazidime were assessed using the checkerboard and time-kill methods. The results of the present study showed that the combined effect of six cyclic dipeptides with imipenem predominantly recorded synergistic interaction (FIC index 4 represented antagonism [14]. The fractional bactericidal concentration (FBC) is calculated in the same way described above by replacing the MIC values with the MBC values. Time-Kill Curves The time-kill assay against bacterial strains was exposed over time to six cyclic dipeptides and antibiotics alone as well as to their combinations according to the guideline provided by NCCLS [14, 15]. An overnight inoculum of 1×106 CFU/mL of each isolate was added along with the antimicrobials into tubes filled to a final volume of 3 mL with nutrient broth. The tubes were thereafter incubated at 37 °C, and viable counts were performed after 0, 2, 4, 6, 12, 24, and 48 h of incubation. At the above time intervals, one aliquot of 0.1-mL samples was removed, serially diluted with 0.85 % saline, and plated onto nutrient agar. The number of viable cells in each tube was estimated after counting plates and by multiplying by the appropriate dilution factor. Colony counts were performed in duplicate, and means were taken. Killing curves were constructed by plotting the log10 CFU/mL versus time over 48 h, and the change in bacterial concentration was determined. Bactericidal activity was defined as a reduction of 99.9 % (≥3 log10) of the total number of CFU/mL in the original inoculum. Bacteriostatic activity was defined as maintenance of the original inoculum concentration or a reduction of less than 99.9 % (≥3 log10) of the total number of CFU/mL in the original inoculums. Cytotoxicity Test The MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay was used to determine the cytotoxicity of cyclic dipeptides. A known cytotoxic drug cisplatin was used as positive control. VERO cell line was used for testing. MTT assay is based on the ability of mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the
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pale yellow MTT and to form dark blue formazan crystals which are largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of the crystals. The number of surviving cells is directly proportional to the level of the formazan product formed. The color can then be quantified by a simple colorimetric assay using a multi-well scanning spectrophotometer (ELISA reader). Briefly, cells (5×103 per well) were seeded in 0.2 mL of the medium (DMEM with 10 % PBS) on a 96-well plate, treated with cyclic dipeptides for 72-h incubation. Cytotoxicity was measured by removing the diketopiperazine containing media from the well, and 25 μL of MTT solution (5 mg/mL in PBS) and 75 μL of complete medium were added to wells (untreated and treated) and incubated. After 2 h, MTT lysis buffer was added to the wells (0.1 mL per well) and incubated for another 4 h at 37 °C. At the end of incubation, the optical densities at 570 nm were measured using a plate reader (Bio-Rad). The relative cell viability in percentage was calculated: (A570 of treated sample)/(A570 of untreated sample)×100 [16].
Result MIC and MBC MIC and MBC ranges of six proline-based cyclic dipeptides and antibiotics used alone for medically important bacteria are shown in Table 1. The ranges of MIC/MBC of cyclic dipeptides for the bacteria included in the study were 16 to 250 μg/mL for S. epidermis, 16 to 4,000 μg/mL for K. pneumoniae, 32 to 1,000 μg/mL for P. mirabilis, and 8 to 2,000 μg/mL for V. cholerae. DKP 5 recorded activity at higher concentration, i.e., between 125 and 4,000 μg/mL against the bacteria. Checkerboard Assay The combined activities of cyclic dipeptides with antibiotics from the in vitro checkerboard interactions against the medically important bacteria are summarized in Tables 2 and 3. FIC, FBC, FIC index, FBC index, and interpretations for the activities of cyclic dipeptides and antibiotics against the test bacteria predominantly showed a synergistic interaction. But some combination of cyclic dipeptides with ceftazidime recorded additive. Antagonism and indifference were not recorded for any of the combinations.
Table 1 Antibacterial activity of cyclic dipeptides and antibiotics against bacteria
Values represent mean of three replications
MIC/MBC (μg/mL) Test compounds
S. epidermis
K. pneumoniae
P. mirabilis
V. cholerae
DKP 1
32/64
250/500
250/500
500/1,000
DKP 2
16/32
8/16
32/64
64/125
DKP 3
125/125
125/250
32/64
64/125
DKP 4
250/250
32/64
64/125
125/250
DKP 5
125/125
2,000/4,000
500/1,000
1,000/2,000
DKP 6
16/16
16/32
32
8/16
Imipenem
4/4
2/4
1/1
2/4
Ceftazidime
8/8
4/4
8/8
8/8
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Table 2 Synergistic effects of the cyclic dipeptides with imipenem against bacteria Test bacteria
S. epidermis
K. pneumoniae
P. mirabilis
V. cholerae
Agent
MIC/MBC (μg/mL)
FIC/FBC
FICIb/FBCIc
Outcome
Alone
Combinationa
DKP 1 Imipenem
32/64 4/4
2/4 0.25/0.5
0.06/0.06 0.06/0.13
0.12/0.19
Synergistic/synergistic
DKP 2 Imipenem
16/32 4/4
4/4 1/1
0.25/0.16 0.25/0.25
0.5/0.41
Synergistic/synergistic
DKP 3 Imipenem
125/500 4/4
4/8 1/1
0.03/0.02 0.25/0.25
0.28/0.27
Synergistic/synergistic
DKP 4 Imipenem
250/500 4/4
8/8 0.5/0.5
0.03/0.02 0.16/0.16
0.19/0.18
Synergistic/synergistic
DKP 5 Imipenem
125/250 4/4
4/8 0.25/0.5
0.03/0.03 0.06/0.13
0.09/0.16
Synergistic/synergistic
DKP 6 Imipenem
16/32 4/4
2/4 0.12/0.25
0.13/0.13 0.03/0.06
0.16/0.16
Synergistic/synergistic
DKP 1 Imipenem
250/500 2/4
32/64 0.5/0.5
0.13/0.13 0.25/0.13
0.38/0.26
Synergistic/synergistic
DKP 2 Imipenem
8/8 2/4
1/1 0.25/0.25
0.13/0.13 0.13/0.06
0.26/0.19
Synergistic/synergistic
DKP 3 Imipenem
125/250 2/4
16/64 0.12/0.12
0.13/0.26 0.06/0.03
0.19/0.29
Synergistic/synergistic
DKP 4 Imipenem
32/64 2/4
2/4 0.06/0.12
0.06/0.06 0.03/0.03
0.09/0.09
Synergistic/synergistic
DKP 5 Imipenem
2,000/4,000 2/4
125/250 0.5/0.5
0.06/0.06 0.25/0.13
0.31/0.19
Synergistic/synergistic
DKP 6 Imipenem
16/32 2/4
2/4 0.06/0.12
0.13/0.13 0.03/0.03
0.16/0.19
Synergistic/synergistic
DKP 1 Imipenem
250/500 1/1
32/64 0.12/0.25
0.13/0.13 0.12/0.25
0.25/0.38
Synergistic/synergistic
DKP 2 Imipenem
32/64 1/1
8/16 0.03/0.06
0.25/0.25 0.03/0.06
0.28/0.31
Synergistic/synergistic
DKP 3 Imipenem
32/64 1/1
4/8 0.06/0.06
0.03/0.13 0.06/0.06
0.09/0.19
Synergistic/synergistic
DKP 4 Imipenem
64/125 1/1
8/8 0.12/0.12
0.13/0.06 0.12/0.12
0.25/0.18
Synergistic/synergistic
DKP 5 Imipenem
500/1,000 1/1
64/125 0.25/0.25
0.13/0.13 0.25/0.25
0.38/0.38
Synergistic/synergistic
DKP 6 Imipenem
32/64 1/1
8/8 0.25/0.25
0.25/0.13 0.25/0.25
0.5/0.38
Synergistic/synergistic
DKP 1 Imipenem
500/1,000 2/4
64/64 0.25/0.25
0.13/0.06 0.13/0.06
0.26/0.12
Synergistic/synergistic
DKP 2 Imipenem
64/125 2/4
16/16 0.12/0.25
0.25/0.13 0.06/0.06
0.31/0.18
Synergistic/synergistic
DKP 3 Imipenem
64/125 2/4
4/4 0.25/0.5
0.06/0.03 0.13/0.13
0.18/0.16
Synergistic/synergistic
DKP 4 Imipenem
125/250 2/4
32/64 0.5/1
0.26/0.26 0.25/0.25
0.51/0.51
Indifference/indifference
DKP 5 Imipenem
1,000/2,000 2/4
125/125 0.5/1
0.13/0.06 0.25/0.25
0.38/0.31
Synergistic/synergistic
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Table 2 (continued) Test bacteria
Agent
DKP 6 Imipenem a b c
MIC/MBC (μg/mL) Alone
Combinationa
8/16 2/4
1/2 0.25/0.25
FIC/FBC
FICIb/FBCIc
Outcome
0.13/0.13 0.13/0.06
0.26/0.19
Synergistic/synergistic
The MIC and MBC value of cyclic dipeptides with imipenem The fractional inhibitory concentration index (FIC index) The fractional bactericidal concentration index (FBC index)
Significant FICI/FBCI values are shown in bold
When DKP 4 was combined with imipenem for the inhibition of S. epidermis, an important synergistic effect (FIC=0.19) was observed, and the MIC values of DKP 4 and imipenem were reduced to 5 and 3 times below their individual MIC values, respectively (Table 2). Another notable synergism was observed (FIC=0.09) for DKP 1 and ceftazidime against P. mirabilis, and the concentration of the DKP1 and ceftazidime was reduced to 5 times below their individual MIC values (Table 3). Time-Kill Study The antibacterial effect of proline-based cyclic dipeptides with imipenem or ceftazidime against test bacteria was confirmed by time-kill curve experiments. Time-kill assays for the synergistic combinations (cyclic dipeptides and antibiotics) on test bacteria strains are shown in Fig. 2. The time-kill assay was conducted to determine the rates of killing of test bacteria when exposed to cyclic dipeptides with imipenem or ceftazidime (Fig. 2). When proline-based cyclic dipeptides with imipenem or ceftazidime were tested against S. epidermis (Fig. 2a), maximum reduction in the colony count was observed between 4 and 12 h when compared to the control. At 6 to 24 h, a complete bactericidal effect was observed with treatments of combination of cyclic dipeptides and antibiotics. In case of K. pneumoniae, maximum reduction was observed between 4 and 24 h (Fig. 2b). A complete bactericidal effect was observed within 24 h for cyclic dipeptides and imipenem and 48 h for cyclic dipeptides and ceftazidime. Similar results were observed for P. mirabilis and V. cholerae (Fig. 2c, d). Regrowth was observed for diketopiperazine and antibiotics alone after 24 h, whereas it was not observed for the combination even at 48 h (Fig. 2). Cytotoxicity Test The cytotoxicity of cyclic dipeptides was tested against VERO cell line by MTT assay. The results showed that there is no significant cytotoxicity up to 100 μg/mL except DKP 4 (Fig. 3). At 100 μg/mL concentration of cyclic dipeptides, approximately 80 to 85 % of cells were alive, whereas 44 % of cells were killed at 100 μg/mL concentration of cisplatin, which was used as a positive control in MTT assay (Supplementary Fig. 1). But DKP 4 recorded slight toxicity after 50 μg/mL. The results of the present study clearly indicated that cyclic dipeptides may be safe for the treatment of pathogenic bacteria. The cytotoxicity data clearly indicated that cyclic dipeptides may not affect the normal cell and its action may selectively to the test bacteria.
Appl Biochem Biotechnol (2014) 173:116–128
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Table 3 Synergistic effects of the cyclic dipeptides with ceftazidime against bacteria Test bacteria
S. epidermis
K. pneumoniae
P. mirabilis
V. cholerae
Agent
MIC/MBC (μg/mL)
FIC/FBC
FICIb/FBCIc
Outcome
Alone
Combinationa
DKP 1 Ceftazidime
32/64 4/4
4/8 1/1
0.13/0.13 0.25/0.25
0.38/0.38
Synergistic/synergistic
DKP 2 Ceftazidime
16/32 4/4
4/8 1/1
0.25/0.25 0.25/0.25
0.5/0.5
Synergistic/synergistic
DKP 3 Ceftazidime
125/500 4/4
32/64 0.5/1
0.26/0.13 0.13/0.25
0.38/0.38
Synergistic/synergistic
DKP 4 Ceftazidime
250/500 4/4
32/32 0.25/0.5
0.13/0.06 0.06/0.13
0.19/0.19
Synergistic/synergistic
DKP 5 Ceftazidime
125/250 4/4
4/8 1/2
0.03/0.03 0.25/0.5
0.28/0.53
Synergistic/synergistic
DKP 6 Ceftazidime
16/32 4/4
2/4 0.5/0.5
0.13/0.13 0.13/0.13
0.26/0.26
Synergistic/synergistic
DKP 1 Ceftazidime
250/500 2/4
64/64 0.5/1
0.26/0.13 0.25/0.25
0.51/0.38
Synergistic/synergistic
DKP 2 Ceftazidime
8/8 2/4
1/2 0.25/0.5
0.13/0.25 0.13/0.13
0.26/0.38
Synergistic/synergistic
DKP 3 Ceftazidime
125/250 2/4
32/64 0.5/1
0.26/0.26 0.25/0.25
0.51/0.51
Indifference/indifference
DKP 4 Ceftazidime
32/64 2/4
16/16 1/2
0.5/0.25 0.5/0.5
1/0.75
Indifference/indifference
DKP 5 Ceftazidime
2,000/4,000 2/4
500/1,000 1/2
0.25/0.25 0.5/0.5
0.75/0.75
Indifference/indifference
DKP 6 Ceftazidime
16/32 2/4
4/8 0.25/0.25
0.25/0.25 0.13/0.06
0.38/0.31
Synergistic/synergistic
DKP 1 Ceftazidime
250/500 1/1
8/16 0.06/0.12
0.03/0.03 0.06/0.12
0.09/0.15
Synergistic/synergistic
DKP 2 Ceftazidime
32/64 1/1
2/4 0.25/0.5
0.06/0.06 0.25/0.5
0.31/0.56
Synergistic/indifference
DKP 3 Ceftazidime
32/64 1/1
8/16 0.25/0.5
0.25/0.25 0.25/0.5
0.5/0.75
Indifference/indifference
DKP 4 Ceftazidime
64/125 1/1
16/32 0.25/0.5
0.25/0.26 0.25/0.5
0.5/0.76
Indifference/indifference
DKP 5 Ceftazidime
500/1,000 1/1
250/250 0.25/0.5
0.5/0.25 0.25/0.5
0.75/0.75
Indifference/indifference
DKP 6 Ceftazidime
32/64 1/1
4/8 0.12/0.25
0.13/0.13 0.12/0.25
0.25.0.38
Synergistic/synergistic
DKP 1 Ceftazidime
500/1,000 2/4
250/500 0.5/1
0.5/0.5 0.25/0.25
0.75/0.75
Indifference/indifference
DKP 2 Ceftazidime
64/125 2/4
4/8 0.25/0.5
0.06/0.06 0.13/0.13
0.19/0.19
Synergistic/synergistic
DKP 3 Ceftazidime
64/125 2/4
2/4 0.25/0.5
0.03/0.03 0.13/0.13
0.16/0.16
Synergistic/synergistic
DKP 4 Ceftazidime
125/250 2/4
4/8 0.5/0.5
0.03/0.03 0.25/0.13
0.28/0.16
Synergistic/synergistic
DKP 5 Ceftazidime
1,000/2,000 2/4
250/500 1/1
0.25/0.25 0.5/0.25
0.75/0.75
Indifference/indifference
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Table 3 (continued) Test bacteria
Agent
DKP 6 Ceftazidime a b c
MIC/MBC (μg/mL) Alone
Combinationa
8/16 2/4
2/4 1/2
FIC/FBC
FICIb/FBCIc
Outcome
0.25/0.25 0.5/0.5
0.75/0.75
Indifference/indifference
The MIC and MBC value of cyclic dipeptides with ceftazidime The fractional inhibitory concentration index (FIC index) The fractional bactericidal concentration index (FBC index)
Significant FICI/FBCI values are shown in bold
Discussion The increasing occurrence, particularly in hospitals, of pathogenic-resistant bacteria especially Staphylococcus sp. to a wide range of antimicrobial agents, including all kinds of β-lactams, has made therapy more difficult. The increasing resistance to antibiotic represents the main factor justifying the need to find and/or develop new antimicrobial agents. Thus, many studies have been focused on antimicrobial agents and on the antimicrobial properties of naturalderived active principles [17–19]. Although many strategies have been proposed in an attempt to control the spread of drug resistance pathogenic bacteria, the search for new ways to treat infections stimulates the investigation for natural compounds as an alternative treatment to control these infections. Antimicrobial combination therapy may be used to extend spectrum coverage, prevent the emergence of resistant mutants, and gain synergy between antimicrobials [20]. Combination therapy is often recommended for empirical treatment of bacterial infections in intensive care units, where monotherapy is not likely to cover all potential pathogens, and the emergence of resistance is a potential threat [21]. In the last years, new antimicrobials specifically targeted against Gram-positive and Gramnegative strains have been launched, but strains with decreased susceptibility to these antibiotics have been increasingly isolated and, unfortunately, development of new antimicrobial agents in the next future seems to be declining [22, 23]. On the other hand, antimicrobial combination therapy should be useful in improving efficacy, providing broad-spectrum coverage, and preventing the emergence of resistant mutants [20]. Moreover, combination therapy is argued when empirical treatment of severe infections is needed, such as in severe pneumonia. As an etiological diagnosis with susceptibility results is almost never available to assist in the selection of the prompt therapy, the initial approach to treatment is largely empirical and is ordinarily guided by consensus guidelines. Moreover, early administration of appropriate empirical therapy for severe pneumonia has been demonstrated to significantly improve survival [24]. For this reason, antimicrobial therapy should cover both Gram-positive and Gram-negative bacteria until obtainment of laboratory results. But currently, no information regarding the antimicrobial activity of proline-based cyclic dipeptides in combination with antibiotics was available in literature. In the present study, proline-based cyclic dipeptides in combination with imipenem and ceftazidime exhibited significant synergistic effect against medically important bacteria. Synergism is a positive interaction created when two compounds combined and exert an inhibitory effect (on the targeted organisms) that is greater than the sum of their individual effects. Synergism has also
Appl Biochem Biotechnol (2014) 173:116–128
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Fig. 2 Time-kill curve of cyclic dipeptide and antibiotics alone and in combination. a Staphylococcus epidermis, b Klebsiella pneumoniae, c Proteus mirabilis, d Vibrio cholera. 1 DKP 1, 2 DKP 2, 3 DKP 3, 4 DKP 4, 5 DKP 5, and 6 DKP 6. Control, Corresponding DKPs, Imipenem, Ceftazidime, Corresponding DKPs + imipenem, Corresponding DKPs + ceftazidime
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Fig. 3 Cytotoxicity of cyclic dipeptides against VERO cell line by MTT assay
been defined as a phenomenon in which two different compounds are combined to enhance their individual activity. Antagonism results if the combination provides an effect less than the effect of either agent alone or less than the sum of the effects of the individual agents. Indifference results if the combination provides an effect equal to the effect of either agent alone [25]. Thus, evaluation of microbial-based antimicrobial agents for activity against medically important bacteria is imperious. We recently reported the efficacies of cyclo(D-Pro-L-Leu), cyclo(L-Pro-L-Met), cyclo(D-Pro-L-Phe), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(LPro-D-Tyr) against four test bacteria and five test fungi [11]. The structure of the compounds is shown in Table 1. Furthermore, we found that the combination of cyclo(L-Leu-L-Pro), cyclo(DLeu-L-Pro), and cyclo(L-Leu-L-Tyr) synergically inhibited four test bacteria [26]. To the best of our knowledge, the combination of cyclic dipeptides and antibiotics (cyclic dipeptides plus imipenem and ceftazidime) against medically important bacteria has never been shown before. We, therefore, examined the in vitro effect of cyclic dipeptides and antibiotics against four medically important bacteria (S. epidermis, K. pneumoniae, P. mirabilis, and V. cholerae). In the present study, data from the checkerboard assay indicates that cyclic dipeptides with ceftazidime showed the highest rate of synergy among the tested combinations. The ability of cyclic dipeptides and antibiotics combination to yield synergy was quite different for the various antibiotics. Time-killing curves also confirmed superior activity of cyclic dipeptides with ceftazidime. It is also evident that this ability was not a class effect since marked differences were found among β-lactams tested. Therefore, it seems rather difficult to compare our data with those of other studies in which different β-lactams have been used. Imipenem and ceftazidime affect bacteria through inhibiting cell wall synthesis of various Gram-positive and Gram-negative bacteria, and the mode of action of diketopiperazine is not known and it is supposed that its action may be through the novel route. Thus, the combination of cyclic dipeptides with imipenem and ceftazidime will also act the test bacteria through a novel route which may be different from the mechanism of action of cyclic dipeptides and antibiotics and that may be one of the reasons of the enhanced activity.
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Conclusion The results from the present study warrant further investigations on the possible synergistic effects of cyclic dipeptides with other class of antibacterial drugs. The observed synergism between cyclic dipeptides and antibiotics in vitro should also be investigated in in vivo animal model. In addition, further experiments could be performed in order to elucidate the molecular mechanisms underlying this synergistic effect. In addition, a broad screen of natural products is urgently needed, especially for those with no detectable activity as single agents but bearing synergistic effects with other compounds. Due to the general low toxicity of cyclic dipeptides, we speculate that cyclic dipeptides might be used to develop new synergistic and efficient associations with other antimicrobials for external use against antibiotic resistant strains. Acknowledgments The authors are grateful to KSCSTE and the Government of India for funding. Conflict of Interest The authors declare that they have no conflict of interest.
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