Chitosan can improve antimicrobial treatment independently of bacterial lifestyle, biofilm biomass intensity and antibiotic resistance pattern in non-aureus staphylococci (NAS) isolated from bovine clinical mastitis

Abstract

Bovine mastitis is the most frequent and costly disease that affects dairy cattle. Non-aureus staphylococci (NAS) are currently one of the main pathogens associated with difficult-to-treat intramammary infections. Biofilm is an important virulence factor that can protect bacteria against antimicrobial treatment and prevent their recognition by the host's immune system. Previously, we found that chronic mastitis isolates which were refractory to antibiotic therapy developed strong biofilm biomass. Now, we evaluated the influence of biofilm biomass intensity on the antibiotic resistance pattern in strong and weak biofilm-forming NAS isolates from clinical mastitis. We also assessed the effect of cloxacillin (Clx) and chitosan (Ch), either alone or in combination, on NAS isolates with different lifestyles and abilities to form biofilm. The antibiotic resistance pattern was not the same in strong and weak biofilm producers, and there was a significant association (p ≤ 0.01) between biofilm biomass intensity and antibiotic resistance. Bacterial viability assays showed that a similar antibiotic concentration was effective at killing both groups when they grew planktonically. In contrast, within biofilm the concentrations needed to eliminate strong producers were 16 to 128 times those needed for weak producers, and more than 1,000 times those required for planktonic cultures. Moreover, Ch alone or combined with Clx had significant antimicrobial activity, and represented an improvement over the activity of the antibiotic on its own, independently of the bacterial lifestyle, the biofilm biomass intensity or the antibiotic resistance pattern. In conclusion, the degree of protection conferred by biofilm against antibiotics appears to be associated with the intensity of its biomass, but treatment with Ch might be able to help counteract it. These findings suggest that bacterial biomass should be considered when designing new antimicrobial therapies aimed at reducing antibiotic concentrations while improving cure rates.

Keywords: antibiotic resistance; biofilm; biofilm and antibiotic resistance association; chitosan; cloxacillin; non-aureus staphylococcus.

Figure 1

Antibiotic resistance in SBP and WBP NAS isolates from clinical bovine mastitis. (A) Percentage of resistance to antibiotics in SBP and WBP NAS isolates. (B) Relative frequency of resistance to penicillin, ampicillin, cefoxitin, erythromycin and rifampicinin SBP and WBP NAS isolates.

Figure 2

Minimum bactericidal concentration of cloxacillin for SBP and WBP NAS isolates in planktonic cultures and preformed biofilms. The bar graph shows the cloxacillin MBC in planktonic cultures and biofilms measured by a plate count assay. Data are shown as the mean of each isolate ± SEM and box plot distribution. These experiments were performed three independent times with three biological replicates of each of the 10 SBP and WBP isolates. The p values *** < 0.001 were obtained with a Kruskal-Wallis test.

Figure 3

Bacterial viability of SBP and WBP NAS isolates in planktonic cultures and preformed biofilms after treatment with cloxacillin. (A) Bacterial viability of SBPs and WBPs in planktonic cultures grown in TSB or treated with different concentrations of cloxacillin, analyzed by flow cytometry using SYTO9 and PI dyes. (B) Bacterial viability percentages of SBPs and WBPs in planktonic cultures. (C) Bacterial viability of SBPs and WBPs in preformed biofilms grown in TSB or treated with different concentrations of cloxacillin, analyzed by flow cytometry using SYTO9 and PI dyes. (D) Bacterial viability percentages of SBPs and WBPs in preformed biofilms. These experiments were performed four independent times with three biological replicates of each of the 10 SBP and WBP isolates. Data were analyzed with one-way ANOVA followed by Bonferroni post-hoc, and are shown as mean ± SEM. The p values * < 0.05, ** < 0.01, and *** < 0.001 were considered significant.

Figure 4

Minimum bactericidal concentration of combined chitosan and cloxacillin in SBP and WBP NAS isolates in planktonic cultures and preformed biofilms. (A) Bacterial viability of SBPs and WBPs in planktonic cultures grown in TSB or treated with different concentrations of cloxacillin and chitosan, analyzed by flow cytometry using SYTO9 and PI dyes. (B) Bacterial viability percentages of SBPs and WBPs in planktonic cultures. (C) Bacterial viability of SBPs and WBPs in preformed biofilms grown in TSB or treated with different concentrations of cloxacillin and chitosan, analyzed by flow cytometry using SYTO9 and PI dyes. (D) Bacterial viability percentages of SBPs and WBPs in preformed biofilms. These experiments were performed four independent times with three biological replicates of each of the 10 SBP and WBP isolates. Data were analyzed with one-way ANOVA followed by Bonferroni post-hoc, and are shown as mean ± SEM. The p values * < 0.05, ** < 0.01, and *** < 0.001 were considered significant.