Effective biofilm eradication in MRSA isolates with aminoglycoside-modifying enzyme genes using high-concentration and prolonged gentamicin treatment

Abstract

Bone and soft tissue infections caused by biofilm-forming bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), remain a significant clinical challenge. While the control of local infection is necessary, systemic treatment is also required, and biofilm eradication is a critical target for successful management. Topical antibiotic treatments, such as antibiotic-loaded bone cement (ALBC), have been used for some time, and continuous local antibiotic perfusion therapy, a less invasive method, has been developed by our group. However, the optimal antibiotics and concentrations for biofilms of clinical isolates are still not well understood. We examined the efficacy of high concentrations of gentamicin against MRSA biofilms and the role of gentamicin resistance genes in biofilm eradication. We collected 101 MRSA samples from a hospital in Japan and analyzed their gene properties, including methicillin and gentamicin resistance, and their minimum biofilm eradication concentration (MBEC) values. Our results showed that high concentrations of gentamicin are effective against MRSA biofilms and that even concentrations lower than the MBEC value could eliminate biofilms after prolonged exposure. We also identified three aminoglycoside/gentamicin resistance genes [aac(6')-aph(2″), aph(3')-III, and ant(4')-IA] and found that the presence or absence of these genes may inform the selection of treatments. It was also found that possession of the aac(6')-aph(2″) gene correlated with the minimum inhibitory concentration/MBEC values of gentamicin. Although this study provides insight into the efficacy of gentamicin against MRSA biofilms and the role of gentamicin resistance genes, careful selection of the optimal treatment strategy is needed for clinical application.

Importance: Our analysis of 101 MRSA clinical isolates has provided valuable insights that could enhance treatment selection for biofilm infections in orthopedics. We found that high concentrations of gentamicin were effective against MRSA biofilms, and even prolonged exposure to concentrations lower than the minimum biofilm eradication concentration (MBEC) value could eliminate biofilms. The presence of the aac(6')-aph(2″) gene, an aminoglycoside resistance gene, was found to correlate with the minimum inhibitory concentration (MIC) and MBEC values of gentamicin, providing a potential predictive tool for treatment susceptibility. These results suggest that extended high concentrations of local gentamicin treatment could effectively eliminate MRSA biofilms in orthopedic infections. Furthermore, testing for gentamicin MIC or the possession of the aac(6')-aph(2″) gene could help select treatment, including topical gentamicin administration and surgical debridement.

Keywords: MRSA; aminoglycoside-modifying enzyme genes; biofilm; gentamicin; minimal biofilm eradication concentration (MBEC).

Fig 1

Distribution of MIC values of cefoxitin against clinical isolates of MRSA. Of the 101 isolates, 100 strains had MICs to cefoxitin of 8 µg/mL or higher, confirming that they were MRSA. The remaining one isolate was also confirmed as MRSA with MICs to oxacillin of 4 µg/mL, respectively.

Fig 2

Distribution of MIC and MBEC values of gentamicin against clinical isolates of MRSA. (A) Distribution of MIC of gentamicin against the 101 MRSA isolates. There were 66 (65.3%) strains that were resistant to gentamicin (≥ 16 µg/mL). Strains with the aac(6′)-aph(2″) gene (67 strains) are shown in blue, and those without the gene (34 strains) are shown in orange. All but one of the strains with aac(6′)-aph(2″) were resistant to gentamicin. (B) Distribution of MBEC of gentamicin against the 101 MRSA isolates. MBEC values for gentamicin were higher than MIC values and varied between strains: 52 (51.5%) strains had MBEC values above 2,048 µg/mL. Strains with the aac(6′)-aph(2″) gene (67 strains) are shown in blue, and those without the gene (34 strains) are shown in orange. (C) Relationship between MIC and MBEC values of gentamicin and the presence or absence of the aac(6′)-aph(2″) gene. Each dot represents the MIC and MBEC values against each MRSA strain. Strains with the aac(6′)-aph(2″) gene (67 strains) are indicated by blue dots and those without the gene (34 strains) by orange dots. The dashed lines indicate the borderline between MIC values of 8 and 16 µg/mL and MBEC values of 128 and 256 µg/mL, respectively.

Fig 3

Effect of prolonged gentamicin exposure on biofilms of MRSA clinical isolates. (A) Time–killing curve of MRSA biofilm after prolonged gentamicin exposure. Biofilms of one randomly selected clinical isolate of MRSA, carrying the aac(6′)-aph(2″) gene and an MBEC greater than 2,048 µg/mL, were exposed to gentamicin at concentrations of 0, 8, 128, and 2,048 µg/mL for 4, 8, 24, and 48 h to examine its bactericidal effect over time. The number of CFUs in the biofilm before exposure to gentamicin was set as 1, and the number of CFUs at each time point was expressed as a logarithm. The analysis was performed in triplicate. Two-way ANOVA with Bonferroni post hoc test was used to examine the statistical significance between groups. ***P < 0.001. (B) Percentage of MRSA biofilm eradicated by prolonged gentamicin exposure. A total of 51 strains with the aac(6′)-aph(2″) gene and MBEC values greater than 2,048 µg/mL were tested. All biofilms of all strains were exposed to gentamicin at a concentration of 1,024 µg/mL for 24, 48, and 72 h to determine the percentage of strains with viable bacteria in the biofilm. The percentage of strains surviving in the biofilm is shown in white, and the percentage of dead strains is shown in gray. Chi-squared test was used to examine the statistical significance between groups.