The postantibiotic effect (PAE) and postantibiotic sub-minimum inhibitory concentration (MIC) effect (PA-SME) of valnemulin against Staphylococcus aureus were investigated in vitro using a spectrophotometric technique and classic viable count method. A standard curve was constructed by regression analysis of the number of colonies and the corresponding optical density (OD) at 630 nm of the inoculum. After exposure to valnemulin at different concentrations for an hour, the antibiotic was removed by centrifuging and washing. The PA-SMEs were measured after initial exposure to valnemulin at 4 × the MIC, and then, valnemulin was added to reach corresponding desired concentrations in the resuspended culture. Samples were collected hourly until the culture became turbid. The results were calculated by converting the OD values into the counts of bacteria in accordance with the curve. The MIC of valnemulin against eight strains was identically 0·125 μg ml−1. The mean PAEs were 2·12 h (1 × MIC) and 5·06 h (4 × MIC), and the mean PA-SMEs were 6·85 h (0·1 × MIC), 9·12 h (0·2 × MIC) and 10·8 h (0·3 × MIC). The results showed that the strains with identical MICs exhibited different PAEs and PA-SMEs. Valnemulin produced prolonged PAE and PA-SME periods for Staph. aureus, supporting a longer dosing interval while formulating a daily administration dosage.
Significance and Impact of the Study
In this study, valnemulin demonstrated prolonged postantibiotic effects and postantibiotic sub-MIC effects on strains of Staphylococcus aureus. The strains with identical MICs of valnemulin exhibited different PAEs and PA-SMEs. Staphylococcus aureus isolated from different species has little impact on the postantibiotic effect of valnemulin. The result suggests a longer dosing interval while formulating a daily administration dosage, and it may play a valuable role of valnemulin in treating Staph. aureus infections in animals.
Valnemulin is a pleuromutilin antimicrobial agent that inhibits protein synthesis by binding to the 50S ribosomal subunit of bacteria (Poulsen et al. 2001). It was exclusively developed for use in animal medicine and has been approved in some countries for treatment and prevention of swine dysentery, colonic spirochaetosis and enzootic pneumonia (Karlsson et al. 2001). Previous reports showed that Staphylococcus aureus, Streptococcus, Mycoplasma, Spirochaetes and Actinobacillus pleuropneumoniae are susceptible to valnemulin (Hogenauer 1975). Staphylococcus aureus is a common pathogen responsible for respiratory and skin infections in both human and animal medicine and often becomes resistant to multiple antibiotics (Wang et al. 2012). A pharmacodynamic study of valnemulin demonstrated superior antibacterial activity of valnemulin against Staph. aureus, which may give another treatment option to veterinarians. However, there are no adequate references regarding the optimal dosing intervals while developing a rational regimen. Postantibiotic effect (PAE) is defined as the persisting suppression of bacterial growth after short-term exposure and removal of an antimicrobial agent (Vogelman and Craig 1985). Moreover, during the PAE period, bacterial growth would be further suppressed if exposed to continuing subinhibitory concentrations of drug, which is defined postantibiotic sub-minimum inhibitory concentration (MIC) effect (PA-SME) (Cars and Odenholt-Tornqvist 1993). The PAE and PA-SME are inherent properties of most antimicrobial agents and are described by the length of time of their effect. The significance of these investigations is that a prolonged PAE and PA-SME are important pharmacodynamic parameters for determining longer dosing intervals, reducing adverse effects and lower costs while formulating a daily administration dosage (Craig and Vogelman 1987; Craig 1998). The aim of this study was to investigate in vitro the PAE as well as PA-SME of valnemulin against representative strains of Staph. aureus. This investigation may demonstrate a valuable role of valnemulin in the treatment of Staph. aureus infections in animals in the future.
Results and discussion
The standard curve was constructed by regression analysis of the number of colonies of the inoculum and its corresponding OD value (Fig. 1). The MIC of valnemulin against each of the study isolates was 0·125 μg ml−1. The PAEs and PA-SMEs for eight isolates are listed in Table 1. The PAE of valnemulin of 1 × MIC for all isolates was <3 h; however, the PAEs at 4 × MIC ranged from 3·2 to 5·9 h. After initial exposure to 4 × the MIC and then spiking the antibiotic with concentrations equal to 0·1 × MIC, 0·2 × MIC and 0·3 × MIC during the postantibiotic phase, the PA-SMEs ranged 5·3–8·0, 7·6–10·8 and 9·2–12·5 h, respectively. The mean PAE or PA-SME values and standard deviations (Mean ± SD, n = 8) at each drug concentration are shown in Fig. 2. The results indicate that strains with identical MICs of valnemulin exhibited different PAEs and PA-SMEs. Valnemulin caused longer regrowth periods in most clinical isolates from this study, as compared to the ATCC reference strain. Regrowth curves of the ATCC strain exposure to different levels of valnemulin are plotted in Fig. 3.
|Strain||PAE (h)||PA-SME (h)|
|1 × MIC||4 × MIC||0·1 × MIC||0·2 × MIC||0·3 × MIC|
|Mean ± SD||2·12 ± 0·53||5·06 ± 0·90||6·85 ± 0·91||9·12 ± 1·17||10·8 ± 1·22|
The mechanism for how antimicrobial agents produce the postantibiotic effect and postantibiotic sub-MIC effect is uncertain. Primarily, nonlethal lesions of bacteria resulting from antimicrobials persistently binding to the objective target site may cause a prolonged period of regrowth (Vogelman and Craig 1985; Karlowsky et al. 1994). Other potentially significant factors are the changes of bacterial morphology and physiological function while organisms are exposed to drugs (Lorian et al. 1989). Moreover, leucocytes would contribute more bactericidal activity to microorganism within the postantibiotic period. This might be the reason why PAEs of most antibiotics in vivo were generally prolonged in comparison with those in vitro (Minguez Minguez et al. 1992; Meng et al. 1995).
The PAEs and PA-SMEs have been studied extensively in different antibiotics not only in human medicine but in veterinary medicine (Bozkurt-Guzel and Gerceker 2012; Harada et al. 2012). The spectrophotometric technique and classic viable count method were extensively applied in the determination of PAE. Compared with viable count, the spectrophotometric assay has the advantages of convenience, simple operation and satisfactory reproducibility. Meanwhile, under the pressure of valnemulin, the regrowth of Staph. aureus was so slow that it would take a long time to reach 50% of the maximal OD value of the control culture (A50) (Lowdin et al. 1993). In the present study, the results were calculated by converting the OD values into the counts of bacteria with a standard curve. Our results showed that compared with 1 × MIC, the concentration at 4 × MIC contributed to an evidently prolonged duration of PAE, which was consistent with a previously published report (Bundtzen et al. 1981), where the PAE periods of a standard strain of Staph. aureus serially exposed to sixteen antimicrobials were investigated. Except for gentamicin, fifteen other antibacterial agents produced growth suppression periods lasting 1·4–3·1 h.
The persistent delayed regrowth of valnemulin against Staph. aureus might indicate less frequent administration in a therapeutic regimen. In contrast to the contribution of PAE, the PA-SME possibly reflects more clinical significance as a period of subinhibitory concentrations would always exist in vivo while the drug concentrations in plasma or tissue would be less than the MIC in the process of elimination (Cars and Odenholt-Tornqvist 1993; Odenholt 2001). Mirjani et al. (2010) studied the PA-SME of gentamicin and sophoraflavanone G against Staph. aureus and demonstrated that a sixfold increase in antimicrobial duration was achieved when Staph. aureus was exposed to 1/2× the synergistic MIC levels of gentamicin. In the present experiments, Staph. aureus during the postantibiotic phase was more sensitive to repeated exposure to valnemulin at various subinhibitory concentrations. Compared with the postantibiotic period of 4 × MIC, a much longer growth suppression effect could be obtained while, in the PA-phase, Staph. aureus was continuously exposed to valnemulin at a concentration of 0·3 × MIC. Following exposure to valnemulin at 4 × MIC for one hour, residual drug probably remains bound to the objective target site during a limited period (Vogelman and Craig 1985; Karlowsky et al. 1994). In the current study, seven isolates Staph. aureus were collected from swine and chickens. As showed in the Table 1, the PAEs and PA-SMEs of valnemulin exhibited in all study isolates independent of their sources (P < 0·05).
Further studies remain necessary for the impact of MIC on PAE and PA-SME. Previously, orbifloxacin and enrofloxacin were tested against E. coli or Pseudomonas aeruginosa and the strains with higher MICs contributed to shorter PAE and PA-SME versus those with lower MICs (Harada et al. 2012). However, for the eight isolates/strains used in this study, with the same MICs, they exhibited different persisting suppression periods of bacterial growth. The results obtained from this study were consistent with the conclusions of a previous report by Licata et al. (1997). BC-3205, a novel semi-synthetic pleuromutilin derivative, was studied in neutropenic murine infection model. Regrowth of Staph. aureus ATCC 29213 needed 4·5 h with free plasma concentrations below the MIC at a dosage of 40 mg kg−1, the corresponding duration extended to 8·5 h when the dose was adjusted to 160 mg kg−1 (Craig et al. 2009). Growth suppression periods may occur during the postantibiotic effect and sub-MIC effect in the PA-phase, because it was impossible to eliminate the drug all at once in vivo. The PAE and PA-SME of valnemulin in vivo were unknown. In view of the phagocytosis by leucocytes, we can anticipate a more prolonged period of delayed regrowth of Staph. aureus in vivo in the presence of valnemulin. In conclusion, this is the first study of PAE and PA-SME on valnemulin against Staph. aureus isolated from swine and chickens. The antibiotic induces a significant PAE and PA-SME in vitro, which may imply a longer dosing interval during treatment of Staph. aureus infection with valnemulin.
Material and methods
Seven clinical isolates of Staph. aureus were collected from wound muscle tissues of swine (SA18, SA19, SA22, SA46 and SA50) or chickens (4Y1W and 3YG2) in an affiliated veterinary hospital of South China Agricultural University (Table 1). Isolates were identified by Gram staining, catalase reaction, rabbit plasma coagulase test and the API Staph system (bioMérieux, Craponne, France). Staphylococcus aureus ATCC 25923 was used as a reference strain in this study.
Valnemulin was provided by Guangdong Dahuanong Animal Health Products CO., LTD (Guangdong, China). The stock solution at a concentration of 1280 mg l−1 was prepared by dissolving dry powders in sterile purified water and stored away from light at 4°C and used within 2 weeks of preparation.
Mueller-Hinton (MH) broth and MH agar were prepared for MIC determinations, PAE and PA-SME experiments. Pour plates of MH agar and quadrant hanging-drop technique were used to count colonies.
Primarily, a 0·1 ml-standard inoculum was added to 8 ml of MH broth, obtaining a concentration of c. 5 × 105 colony-forming units (CFU) ml−1 followed by incubation at 37°C in a constant temperature shaker. The optical density (OD) of the inoculum was determined hourly at 630 nm until the sixth hour by an automatic ELISA Reader (BioTek, Winooski, VT, USA). One hundred microlitres of the suspensions was withdrawn and subjected to serial 10-fold dilutions in MH broth simultaneously. Twenty-five microlitres of each tube contents was dropped onto quadrants of MH agar and incubated at 37°C for 24 h to be checked by viable colony count. The standard curve was constructed by regression analysis of the number of colonies and the corresponding OD value of the inoculum.
Determination of MIC
The MIC of valnemulin against each isolate was determined by the agar dilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI M31-A3 2008). All cultures were incubated in fresh MH broth to obtain a final concentration of c. 5 × 105 CFU ml−1. To make agar plates containing a given concentration, 2 ml aliquots of valnemulin solutions was prepared by 1 : 10-fold dilution from a working stock solution and mixed with 18 ml of MH agar before pouring onto Petri dishes. This process was repeated with various working stock solutions to make a series of plates containing the drug in a double-dilution scheme. The plates were inoculated with testing isolates of Staph. aureus and incubated for 24 h at 37°C. As a quality control strain, ATCC 25923 was included in the MIC determination. The assays were performed in triplicate.
Determination of the PAE
The PAE of valnemulin was performed by a combination of spectrophotometric method and the classic method of viable count on agar plate (Dominguez et al. 2001; Odenholt et al. 2003; Mirjani et al. 2010). All testing isolates were grown in MH medium at 37°C to the log-phase of growth, to produce a final concentration of c. 1 × 106 CFU ml−1. The organisms were exposed to valnemulin at concentrations of 1 × MIC and 4 × MIC, respectively. Growth controls were simultaneously inoculated without antibiotics. Tubes were placed in a 37°C shaker for 1 h. After exposure to valnemulin, the antibiotic was eliminated by centrifuging at 2 000 g for 10 min. Pellets were resuspended in prewarmed broth, centrifuged at 2000 g for 10 min and resuspended in fresh, drug-free MH broth. Samples were immediately obtained for determining the OD value and marked as 0 h, then checked hourly up to the time when broth became turbid. The OD values were converted into the number of bacteria (Athamna et al. 2004) in accordance with the standard curve that was determined previously. The PAE was calculated with the equation: PAE = T-C, where T is the time required for the number in the antibiotic-exposed culture to increase one log 10 above the number present immediately after centrifugation, and C is the corresponding time for the drug-free controls (Vogelman and Craig 1985). The experiments were performed in triplicate.
Determination of the PA-SME
Tubes for the determination of the PA-SME were carried out homogeneously. After eliminating 4 × MIC valnemulin and washing twice with MH broth, valnemulin was added to resuspended cultures to achieve concentrations of 0·1 × MIC, 0·2 × MIC and 0·3 × MIC, respectively. The procedure for the assay of the PA-SME was analogous to that used for PAE, except that after resuspension in antibiotic-free medium after 1 h, the resuspended culture was spiked with antibiotic at the stated sub-MIC levels. The bacterial growth was reflected by converting the OD values into the amount of bacteria based upon the identical standard curve. The PA-SME was defined as PA-SME = TPA-C, where TPA represents the time required for the counts of organisms in the sub-MIC-treated PA-phase to increase one log 10 and C is the corresponding time for unexposed organisms as described previously (Odenholt 2001).
This study was supported by National Science Fund for Distinguished Young Scholars Grants 31125026, Special Fund for Agro-scientific Research in the Public Interest Grants 201203040 and the 973 fund, the Ministry of Science and Technology of the People's Republic of China Grants 2009CB118805.
Conflict of interest
The authors declare no conflict of interest.