C. difficile is the leading multidrug-resistant pathogen in hospital-acquired diarrhea [
1]. Currently, CDI is managed via the use of the two conventional antimicrobials, metronidazole and vancomycin, for the treatment of mild to moderate CDI and severe CDI, respectively. Fidaxomicin is a good alternative, especially in patients at risk of relapse [
8]. However, several issues are associated with the use of these agents, including a high recurrence rate of 20–25%.
C. difficile has developed resistance to several different classes of antimicrobials; however, the rate of resistance varies widely depending on the geographic regions and policies of antimicrobial use [
24]. Natural products derived from food and other plant extracts have great antimicrobial potential against multi-drug-resistant microorganisms [
25]. Berberine is a natural isoquinoline alkaloid that is increasingly drawing attention due to its multiple therapeutic effects, among others, on cancer, diabetes, hyperlipidemia, and cardiovascular diseases [
26‐
29]. In traditional Chinese medicine, berberine has been widely used to treat bacterial diarrhea and gastroenteritis [
30]. It has been previously indicated that, within the same species of bacteria, some strains may present distinct sensitivities to antimicrobial agents. Our research revealed that the MIC of berberine against
C. difficile strains ranged from 256 to 1.024 mg/L. In contrast, Tan et al. showed that the MIC values of berberine varied from 64 to 512 mg/L against
Staphylococcus aureus [
31]. Wang et al. found that the MIC value of berberine for
C. difficile spores was 640 mg/L [
17]. In this study, a higher MBC was observed, possibly due to the fact that some
C. difficile cells are found in the form of spores, which can wait later. It is likely that berberine will not enter the core of dormant spores due to the core’s extreme impermeability, consistent with the dormant spores’ resistance to antibiotics. The level of berberine accumulated at the berberine MICs in the individual germinated spores was heterogeneous for
C. difficile. These values were 25–50-fold higher than the MIC values. However, berberine did not affect the germination of
C. difficile spores, but did block the outgrowth of germinated spores. In our study, a higher MBC (in strain 1) was observed, at 16.384 mg/L. A certain amount of bacteria in special spores of
C. difficile was able to survive and persist at high berberine concentrations. Our study tested the sensitivity of 9 clinical strains (RT027) to berberine. As a result, we found that berberine presented significant antibacterial activity against all strains. Furthermore, synergism was observed in berberine at 1/2 MICs combined with vancomycin. The great diversity of MICs among the
C. difficile strains indicates the importance of determining the MIC value. Zuo et al. found that berberine significantly lowered the MIC values of a series of antibiotics against
S. aureus [
32].
In a previous study, the antimicrobial effect of berberine chloride in combination with various anti-staphylococcal drugs on reference CoNS strains was found to vary greatly depending on the bacterial strain and drug used. The most significant synergistic effects towards CoNS strains were noted when berberine was combined with linezolid, cefoxitin, and erythromycin [
33]. In another study, the combined use of fusidic acid (FA) and berberine chloride (BBR) was found to result in an in vitro synergistic action against 7 out of 30 clinical methicillin-resistant
Staphylococcus aureus (MRSA) strains [
34]. In the present study, we examined the sub-MICs of berberine and sub-MICs of vancomycin for biofilm formation in
C. difficile strains. The sub-MICs of BBR and VAN separately did not significantly increase biofilm formation in most strains except one. However, the sub-inhibitory concentration (1/2 MIC) of BBR with VAN was unexpectedly found to enhance biofilm formation in one clinical
C. difficile strain. This is a form of adaptation by
C. difficile to highly stressful environments produced by berberine with vancomycin, wherein the organism tended to live in its biofilm form instead of its planktonic form.
The flagella allow bacteria to move and contribute to bacterial colonization and pathogenesis by promoting adhesion to host cells, providing motor-driven nutrients and promoting biofilm formation [
35]. The clinical strains tested in this study showed significant motility. Here, three clinical strains were found to have an inhibition of motility after the addition of sub-MIC (1/2 MIC) doses of BBR. The mechanism of action of berberine on the motility of
C. difficile strains under the influence of sub-inhibitory doses of BBR is interesting: sub-inhibitory doses of BBR reduced the motility of clinical strains to varying degrees. To the best of our knowledge, this is the first study to focus on the biological effect of berberine both alone and in combination with vancomycin on
C. difficile and biofilm formation.
In conclusion, specific biological substances exhibit antimicrobial properties. Our work demonstrates the antimicrobial ability of berberine against C. difficile. Importantly, we observed that sub-MICs of BBR with VAN can enhanced biofilm formation in some C. difficile strains.