Biofilm is a Major Virulence Determinant in Bacterial Colonization of Chronic Skin Ulcers Independently from the Multidrug Resistant Phenotype

Abstract

Bacterial biofilm is a major factor in delayed wound healing and high levels of biofilm production have been repeatedly described in multidrug resistant organisms (MDROs). Nevertheless, a quantitative correlation between biofilm production and the profile of antimicrobial drug resistance in delayed wound healing remains to be determined. Microbial identification, antibiotic susceptibility and biofilm production were assessed in 135 clinical isolates from 87 patients. Gram-negative bacteria were the most represented microorganisms (60.8%) with MDROs accounting for 31.8% of the total isolates. Assessment of biofilm production revealed that 80% of the strains were able to form biofilm. A comparable level of biofilm production was found with both MDRO and not-MDRO with no significant differences between groups. All the methicillin-resistant Staphylococcus aureus (MRSA) and 80% of Pseudomonas aeruginosa MDR strains were found as moderate/high biofilm producers. Conversely, less than 17% of Klebsiella pneumoniae extended-spectrum beta-lactamase (ESBL), Escherichia coli-ESBL and Acinetobacter baumannii were moderate/high biofilm producers. Notably, those strains classified as non-biofilm producers, were always associated with biofilm producer bacteria in polymicrobial colonization. This study shows that biofilm producers were present in all chronic skin ulcers, suggesting that biofilm represents a key virulence determinant in promoting bacterial persistence and chronicity of ulcerative lesions independently from the MDRO phenotype.

Keywords: Acinetobacter baumannii; ESBL; Escherichia coli; Klebsiella pneumoniae; MDRO; MRSA; Pseudomonas aeruginosa; biofilm; skin ulcer; wound.

Figure 1

Bacterial isolates from patients with colonized skin ulcers: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumonia, Enterococcus faecalis, Enterobacter cloacae, Stenotrophomonas maltophilia, Citrobacter freundii, Morganella morganii, Streptococcus agalactiae, Klebsiella oxytoca, Enterobacter aerogenes, Proteus vulgaris and Serratia marcescens. Data in percentage. In brackets, the absolute occurrence of different bacterial species. Blue = Gram-positive; red = Gram-negative.

Figure 2

Quantity of not-MDRO (red) compared to its respective counterparts MDRO (blue): MRSA versus MSSA, MDRPA versus P. aeruginosa, ESBL-producing E. coli and K. pneumoniae versus E. coli and K. pneumoniae, respectively, and A. baumannii.

Figure 3

Biofilm production of bacterial isolates from patients with chronic skin ulcers. (A) biofilm production of the total bacterial isolates; (B) of MDRO and not-MDRO; (C) of the different bacterial species and according to multidrug resistance profile: (D) MRSA (n = 14) vs. MSSA (n = 23); (E) MDRPA (n = 5) vs. P. aeruginosa (n = 14); (F) K. pneumoniae ESBL (n = 6) vs. K. pneumoniae (n = 4), E. coli ESBL (n = 6) vs. E. coli (n = 9), and A. baumannii (n = 12) compared with the rest of isolates (n = 42). Biofilm formation was assessed by the cBRT and clinical isolates were classified as non-producers, weak, moderate and high biofilm producers. All results expressed as percentage of strains with the specific biofilm-forming ability.