An in vitro study on the effects of nisin on the antibacterial activities of 18 antibiotics against Enterococcus faecalis

Abstract

Enterococcus faecalis rank among the leading causes of nosocomial infections worldwide and possesses both intrinsic and acquired resistance to a variety of antibiotics. Development of new antibiotics is limited, and pathogens continually generate new antibiotic resistance. Many researchers aim to identify strategies to effectively kill this drug-resistant pathogen. Here, we evaluated the effect of the antimicrobial peptide nisin on the antibacterial activities of 18 antibiotics against E. faecalis. The MIC and MBC results showed that the antibacterial activities of 18 antibiotics against E. faecalis OG1RF, ATCC 29212, and strain E were significantly improved in the presence of 200 U/ml nisin. Statistically significant differences were observed between the results with and without 200 U/ml nisin at the same concentrations of penicillin or chloramphenicol (p<0.05). The checkerboard assay showed that the combination of nisin and penicillin or chloramphenicol had a synergetic effect against the three tested E. faecalis strains. The transmission electron microscope images showed that E. faecalis was not obviously destroyed by penicillin or chloramphenicol alone but was severely disrupted by either antibiotic in combination with nisin. Furthermore, assessing biofilms by a confocal laser scanning microscope showed that penicillin, ciprofloxacin, and chloramphenicol all showed stronger antibiofilm actions in combination with nisin than when these antibiotics were administered alone. Therefore, nisin can significantly improve the antibacterial and antibiofilm activities of many antibiotics, and certain antibiotics in combination with nisin have considerable potential for use as inhibitors of this drug-resistant pathogen.

Figure 1. E. faecalis ATCC 29212 survival rates after treatment with different concentrations of penicillin (A), chloramphenicol (B), vancomycin (C), and linezolid (D) alone or in combination with 200 U/ml nisin.

Raw E. faecalis colony counts were transformed to log10 values to normalize the data. “*” denotes a statistically significant difference between the survival rates of E. faecalis after exposure to the same concentration of antibiotics alone and antibiotics in combination with 200 U/ml nisin (p<0.05). “#” also denotes a statistically significant difference between groups with and without 200 U/ml nisin (p<0.05), as well as the fact that E. faecalis was completely killed by the antimicrobial agents.

Figure 2. Morphological changes of E. faecalis ATCC 29212 observed by TEM after 12 hours of treatment with antibiotics in combination with nisin.

(A) Control; (B) 1024 mg/L penicillin; (C) 2000 U/ml nisin; (D) 1024 mg/L penicillin and 2000 U/ml nisin; (E) 1024 mg/L chloramphenicol; and (F) 1024 mg/L chloramphenicol and 2000 U/ml nisin.

Figure 3. CLSM images showed the effects of antibiotics in combination with nisin on E. faecalis ATCC 29212 biofilms.

(A) 512 mg/L penicillin; (B) 512 mg/L ciprofloxacin; (C) 512 mg/L chloramphenicol; (D) 400 U/ml nisin; (E) 512 mg/L penicillin and 400 U/ml nisin; (F) 512 mg/L ciprofloxacin and 400 U/ml nisin; (G) 512 mg/L chloramphenicol and 400 U/ml nisin. Bacteria with intact cell membranes are stained fluorescent green, whereas bacteria with damaged cell membranes are stained fluorescent red. All images were shown at 200× magnification and were collected using a Carl Zeiss CLSM.

Figure 4. Twenty-four hours old E. faecalis ATCC 29212 biofilms were treated with (A) control biofilm with no treatment, (B) 512 mg/L penicillin alone, (C) 400 U/ml nisin alone, and (D) the combination of 512 mg/L penicillin and 400 U/ml nisin for 12 h.

Their 3-dimensional images were scanned along the Z axis at different positions from bottom to top.