Limits...
In Vitro Activities of Nisin and Nisin Derivatives Alone and In Combination with Antibiotics against Staphylococcus Biofilms.

Field D, O' Connor R, Cotter PD, Ross RP, Hill C - Front Microbiol (2016)

Bottom Line: Growth curve-based comparisons established that combinations of derivatives nisin V + penicillin or nisin I4V + chloramphenicol had an enhanced inhibitory effect against S. aureus SA113 and S. pseudintermedius DSM21284, respectively, compared to the equivalent nisin A + antibiotic combinations or when each antimicrobial was administered alone.Furthermore, the metabolic activity of established biofilms treated with nisin V + chloramphenicol and nisin I4V + chloramphenicol combinations revealed a significant decrease in S. aureus SA113 and S. pseudintermedius DSM21284 biofilm viability, respectively, compared to the nisin A + antibiotic combinations as determined by the rapid colorimetric XTT assay.The results indicate that the activities of the nisin derivative and antibiotic combinations represent a significant improvement over that of the wild-type nisin and antibiotic combination and merit further investigation with a view to their use as anti-biofilm agents.

View Article: PubMed Central - PubMed

Affiliation: School of Microbiology, University College Cork Cork, Ireland.

ABSTRACT
The development and spread of pathogenic bacteria that are resistant to the existing catalog of antibiotics is a major public health threat. Biofilms are complex, sessile communities of bacteria embedded in an organic polymer matrix which serve to further enhance antimicrobial resistance. Consequently, novel compounds and innovative methods are urgently required to arrest the proliferation of drug-resistant infections in both nosocomial and community environments. Accordingly, it has been suggested that antimicrobial peptides could be used as novel natural inhibitors that can be used in formulations with synergistically acting antibiotics. Nisin is a member of the lantibiotic family of antimicrobial peptides that exhibit potent antibacterial activity against many Gram-positive bacteria. Recently we have used bioengineering strategies to enhance the activity of nisin against several high profile targets, including multi-drug resistant clinical pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, staphylococci, and streptococci associated with bovine mastitis. We have also identified nisin derivatives with an enhanced ability to impair biofilm formation and to reduce the density of established biofilms of methicillin resistant S. pseudintermedius. The present study was aimed at evaluating the potential of nisin and nisin derivatives to increase the efficacy of conventional antibiotics and to assess the possibility of killing and/or eradicating biofilm-associated cells of a variety of staphylococcal targets. Growth curve-based comparisons established that combinations of derivatives nisin V + penicillin or nisin I4V + chloramphenicol had an enhanced inhibitory effect against S. aureus SA113 and S. pseudintermedius DSM21284, respectively, compared to the equivalent nisin A + antibiotic combinations or when each antimicrobial was administered alone. Furthermore, the metabolic activity of established biofilms treated with nisin V + chloramphenicol and nisin I4V + chloramphenicol combinations revealed a significant decrease in S. aureus SA113 and S. pseudintermedius DSM21284 biofilm viability, respectively, compared to the nisin A + antibiotic combinations as determined by the rapid colorimetric XTT assay. The results indicate that the activities of the nisin derivative and antibiotic combinations represent a significant improvement over that of the wild-type nisin and antibiotic combination and merit further investigation with a view to their use as anti-biofilm agents.

No MeSH data available.


Related in: MedlinePlus

Live/dead staining confocal images of S. pseudintermedius DSM21284 biofilms (A) untreated, (B) chloramphenicol (Cm) 1X MIC (8 μg/ml), (C) nisin A 4X MIC (1.25 μM), (D) Nisin V (M21V) 4X MIC (1.25 μM), (E) nisin I4V 4X MIC (1.25 μM), (F) nisin A + Cm, (G) nisin V + Cm, and (H) nisin I4V + Cm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4834297&req=5

Figure 5: Live/dead staining confocal images of S. pseudintermedius DSM21284 biofilms (A) untreated, (B) chloramphenicol (Cm) 1X MIC (8 μg/ml), (C) nisin A 4X MIC (1.25 μM), (D) Nisin V (M21V) 4X MIC (1.25 μM), (E) nisin I4V 4X MIC (1.25 μM), (F) nisin A + Cm, (G) nisin V + Cm, and (H) nisin I4V + Cm.

Mentions: For nisin treatment alone of pre-formed S. pseudintermedius DSM21284 biofilms, a concentration of 4X MIC (1.25μM; 4.19μg/ml) was chosen since previous studies had established that no significant difference was observed in biofilm mass of S. pseudintermedius DSM21284 treated with 4X MIC of I4V peptide compared to the wild-type nisin A treated biofilms (Field et al., 2015b). Preliminary treatments of established S. pseudintermedius DSM21284 biofilms with 1/2X (4 μg/ml), 1X (8 μg/ml), and 2X MIC (16 μg/ml) of chloramphenicol concentrations revealed that only at 2X MIC was a minor reduction in the biofilm viability observed as determined by XTT assay (data not shown). Thus, for combinatorial experiments, nisin A and nisin derivatives were used alone at 4X MIC, chloramphenicol at 1X MIC and the relevant combinations thereof. Following addition of XTT and optical density readings at 492 nm (OD492), no substantial change in biofilm viability was observed following 24 h treatment with nisin A and nisin derivatives alone, chloramphenicol alone, or nisin/chloramphenicol combinations (Figure 4) with the exception of nisin I4V + chloramphenicol, where biofilm viability was virtually undetectable compared to the XTT (negative) control (Figure 4B). To assess the visual impact of nisin + antibiotic treatments, the experiment was repeated to enable visualization of the treated biofilms using confocal microscopy in conjunction with the BacLight LIVE/DEAD staining kit which facilitates differentiation between active and dead cells (Figure 5). The results revealed that the lower fluorescence signalsobserved for the nisin I4V + chloramphenicol treated biofilm compared to that for all other treatments was not due to less metabolic activity attributable to cell death alone, but also as a result of the reduction in biofilm density following treatment (Figure 5H).


In Vitro Activities of Nisin and Nisin Derivatives Alone and In Combination with Antibiotics against Staphylococcus Biofilms.

Field D, O' Connor R, Cotter PD, Ross RP, Hill C - Front Microbiol (2016)

Live/dead staining confocal images of S. pseudintermedius DSM21284 biofilms (A) untreated, (B) chloramphenicol (Cm) 1X MIC (8 μg/ml), (C) nisin A 4X MIC (1.25 μM), (D) Nisin V (M21V) 4X MIC (1.25 μM), (E) nisin I4V 4X MIC (1.25 μM), (F) nisin A + Cm, (G) nisin V + Cm, and (H) nisin I4V + Cm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834297&req=5

Figure 5: Live/dead staining confocal images of S. pseudintermedius DSM21284 biofilms (A) untreated, (B) chloramphenicol (Cm) 1X MIC (8 μg/ml), (C) nisin A 4X MIC (1.25 μM), (D) Nisin V (M21V) 4X MIC (1.25 μM), (E) nisin I4V 4X MIC (1.25 μM), (F) nisin A + Cm, (G) nisin V + Cm, and (H) nisin I4V + Cm.
Mentions: For nisin treatment alone of pre-formed S. pseudintermedius DSM21284 biofilms, a concentration of 4X MIC (1.25μM; 4.19μg/ml) was chosen since previous studies had established that no significant difference was observed in biofilm mass of S. pseudintermedius DSM21284 treated with 4X MIC of I4V peptide compared to the wild-type nisin A treated biofilms (Field et al., 2015b). Preliminary treatments of established S. pseudintermedius DSM21284 biofilms with 1/2X (4 μg/ml), 1X (8 μg/ml), and 2X MIC (16 μg/ml) of chloramphenicol concentrations revealed that only at 2X MIC was a minor reduction in the biofilm viability observed as determined by XTT assay (data not shown). Thus, for combinatorial experiments, nisin A and nisin derivatives were used alone at 4X MIC, chloramphenicol at 1X MIC and the relevant combinations thereof. Following addition of XTT and optical density readings at 492 nm (OD492), no substantial change in biofilm viability was observed following 24 h treatment with nisin A and nisin derivatives alone, chloramphenicol alone, or nisin/chloramphenicol combinations (Figure 4) with the exception of nisin I4V + chloramphenicol, where biofilm viability was virtually undetectable compared to the XTT (negative) control (Figure 4B). To assess the visual impact of nisin + antibiotic treatments, the experiment was repeated to enable visualization of the treated biofilms using confocal microscopy in conjunction with the BacLight LIVE/DEAD staining kit which facilitates differentiation between active and dead cells (Figure 5). The results revealed that the lower fluorescence signalsobserved for the nisin I4V + chloramphenicol treated biofilm compared to that for all other treatments was not due to less metabolic activity attributable to cell death alone, but also as a result of the reduction in biofilm density following treatment (Figure 5H).

Bottom Line: Growth curve-based comparisons established that combinations of derivatives nisin V + penicillin or nisin I4V + chloramphenicol had an enhanced inhibitory effect against S. aureus SA113 and S. pseudintermedius DSM21284, respectively, compared to the equivalent nisin A + antibiotic combinations or when each antimicrobial was administered alone.Furthermore, the metabolic activity of established biofilms treated with nisin V + chloramphenicol and nisin I4V + chloramphenicol combinations revealed a significant decrease in S. aureus SA113 and S. pseudintermedius DSM21284 biofilm viability, respectively, compared to the nisin A + antibiotic combinations as determined by the rapid colorimetric XTT assay.The results indicate that the activities of the nisin derivative and antibiotic combinations represent a significant improvement over that of the wild-type nisin and antibiotic combination and merit further investigation with a view to their use as anti-biofilm agents.

View Article: PubMed Central - PubMed

Affiliation: School of Microbiology, University College Cork Cork, Ireland.

ABSTRACT
The development and spread of pathogenic bacteria that are resistant to the existing catalog of antibiotics is a major public health threat. Biofilms are complex, sessile communities of bacteria embedded in an organic polymer matrix which serve to further enhance antimicrobial resistance. Consequently, novel compounds and innovative methods are urgently required to arrest the proliferation of drug-resistant infections in both nosocomial and community environments. Accordingly, it has been suggested that antimicrobial peptides could be used as novel natural inhibitors that can be used in formulations with synergistically acting antibiotics. Nisin is a member of the lantibiotic family of antimicrobial peptides that exhibit potent antibacterial activity against many Gram-positive bacteria. Recently we have used bioengineering strategies to enhance the activity of nisin against several high profile targets, including multi-drug resistant clinical pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, staphylococci, and streptococci associated with bovine mastitis. We have also identified nisin derivatives with an enhanced ability to impair biofilm formation and to reduce the density of established biofilms of methicillin resistant S. pseudintermedius. The present study was aimed at evaluating the potential of nisin and nisin derivatives to increase the efficacy of conventional antibiotics and to assess the possibility of killing and/or eradicating biofilm-associated cells of a variety of staphylococcal targets. Growth curve-based comparisons established that combinations of derivatives nisin V + penicillin or nisin I4V + chloramphenicol had an enhanced inhibitory effect against S. aureus SA113 and S. pseudintermedius DSM21284, respectively, compared to the equivalent nisin A + antibiotic combinations or when each antimicrobial was administered alone. Furthermore, the metabolic activity of established biofilms treated with nisin V + chloramphenicol and nisin I4V + chloramphenicol combinations revealed a significant decrease in S. aureus SA113 and S. pseudintermedius DSM21284 biofilm viability, respectively, compared to the nisin A + antibiotic combinations as determined by the rapid colorimetric XTT assay. The results indicate that the activities of the nisin derivative and antibiotic combinations represent a significant improvement over that of the wild-type nisin and antibiotic combination and merit further investigation with a view to their use as anti-biofilm agents.

No MeSH data available.


Related in: MedlinePlus