Abstract
The current study was conducted to evaluate the antibacterial combination efficacies, and whether the sub-inhibitory concentrations (sub-MIC) of antibiotics can influent on the biofilm formation of S. aureus. The minimum inhibitory concentration (MIC) of common antibacterial drugs was determined in vitro against clinical isolates of Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pasteurella multocida (P. multocida) alone and in combination with each other by using the broth microdilution method and the checkerboard micro-dilution method analyzed with the fractional inhibitory concentration index (FICI), respectively. Regarding these results, antibacterial drug combinations were categorized as synergistic, interacting, antagonistic and indifferent, and most of the results were consistent with the previous reports. Additionally, the effects of sub-MIC of seven antimicrobials (kanamycin, acetylisovaleryltylosin tartrate, enrofloxacin, lincomycin, colistin sulfate, berberine, and clarithromycin) on S. aureus biofilm formation were determined via crystal violet staining, scanning electron microscopy (SEM) and real-time PCR. Our results demonstrate that all antibiotics, except acetylisovaleryltylosin tartrate, effectively reduced the S. aureus biofilm formation. In addition, real-time reverse transcriptase PCR was used to analyze the relative expression levels of S. aureus biofilm-related genes such as sarA, fnbA, rbf, lrgA, cidA, and eno after the treatment at sub-MIC with all of the six antimicrobials. All antibiotics significantly inhibited the expression of these biofilm-related genes except for acetylisovaleryltylosin tartrate, which efficiently up-regulated these transcripts. These results provide the theoretical parameters for the selection of effective antimicrobial combinations in clinical therapy and demonstrate how to correctly use antibiotics at sub-MIC as preventive drugs.
Highlights
In recent years, there has been an increasing tendency regarding the misuse of antimicrobials in clinical veterinary treatments
Isolated strains of S. aureus (S. aureus Hb0206 isolated from pigs in Hubei, China), E. coli (E. coli Hd311 isolated from pigs in Hubei, China) and P. multocida (P. multocida 0625 isolated from pigs in Hubei, China) are provided by the microbiological lab of Huazhong Agriculture University
When enrofloxacin was combined with doxycycline, florfenicol or sulfadimidine, when rifampin was combined with kanamycin or florfenicol, or when lincomycin was combined with acetylisovaleryltylosin tartrate or colistin sulfate, we observed an antagonistic effect (FICI > 2)
Summary
There has been an increasing tendency regarding the misuse of antimicrobials in clinical veterinary treatments. There is increasing clinical evidence on the base of combination therapy of antimicrobials are a valuable treatment option against the mixed or multidrug-resistant bacterial infections (Bharat et al, 2016; Stein et al, 2016; Wei et al, 2016). Many previous studies of antibiotic combinations have demonstrated that the antimicrobial effects of beta-lactams in combination with macrolides are antagonistic, but the reports are conflicting (Yoshioka et al, 2016; Lee et al, 2017). A study by Lodise et al reported that beta-lactams were used in combination with macrolides in the treatment of community-acquired pneumonia (Lodise et al, 2007). The effects of common antibiotic combinations on S. aureus, E. coli, and P. multocida, representing both Gram-positive and Gram-negative bacteria, were investigated in vitro. Quinolones, rifamycins, lincosamides and an extract of Chinese medicine were included as experimental drugs in this report
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