Influences of biofilm structure and antibiotic resistance mechanisms on indirect pathogenicity in a model polymicrobial biofilm

Abstract

Indirect pathogenicity (IP), the commensal protection of antibiotic-sensitive pathogens by resistant microorganisms of low intrinsic virulence, can prevent the eradication of polymicrobial infections. The contributions of antibiotic resistance mechanisms and biofilm structure to IP within polymicrobial biofilms were investigated using a model two-member consortium. Escherichia coli ATCC 33456 was transformed with vectors conferring either ampicillin or spectinomycin resistance, creating two distinct populations with different resistance mechanisms. Each strain alone or the consortium was grown as biofilms in flow cells and planktonically in chemostats. Comparisons in survival and activity were made on the basis of MICs and minimum biofilm preventative concentrations, a newly introduced descriptor. In ampicillin-containing medium, commensal interactions were evident during both modes of cultivation, but the sensitive strain experienced a greater benefit in the chemostat, indicating that the biofilm environment limited the commensal interaction between the Amp(r) and Spt(r) strains. In spectinomycin-containing medium, growth of the sensitive strain in chemostats and biofilms was not aided by the resistant strain. However, green fluorescent protein expression by the sensitive strain was greater in mixed-population biofilms (9% +/- 1%) than when the strain was grown alone (2% +/- 0%). No comparable benefit was evident during growth in the chemostat, indicating that the biofilm structure contributed to enhanced activity of the sensitive strain.

FIG. 1.

Antibiotic concentrations required to prevent planktonic or biofilm growth. MICs for each strain were determined in batch culture. The antibiotic concentration at which attached cells were unable to form a biofilm within 48 h was termed the MBPC. A: Spt^r strain in ampicillin-containing medium. B: Amp^r strain in spectinomycin-containing medium. Filled symbols, MIC; open symbols, MBPC.

FIG. 2.

Prevention of morphological damage to antibiotic-sensitive bacteria during growth with a resistant strain. Biofilms of each strain alone or the two strains grown together were imaged by CLSM. Representative images of the substratum are shown. A: Spt^r strain in LB broth. B: Spt^r strain in LB broth plus 8 ppm ampicillin. C: Amp^r and Spt^r strains in LB broth plus 8 ppm ampicillin. D: Amp^r strain in LB broth. E: Amp^r strain in LB broth plus 20 ppm spectinomycin. F: Amp^r and Spt^r strains in LB broth plus 20 ppm spectinomycin. Magnification, ×400.

FIG. 3.

Flow cytometry scatter plots of Amp^r biofilms grown in spectinomycin-containing medium. Representative flow cytometry scatter plots showing the distribution of fluorescent events in Amp^r biofilms grown alone in medium containing subinhibitory concentrations of spectinomycin. x axis: SSC. y axis: GFP fluorescence (FL1-H).

FIG. 4.

Commensal protection in biofilms. Antibiotic-sensitive organisms grown in biofilms either alone (filled symbols) or with the resistant strain (open symbols) were enumerated by plate counts. A: Spt^r strain in ampicillin-containing medium. B: Amp^r strain in spectinomycin-containing medium.

FIG. 5.

Flow cytometry analyses of Amp^r populations grown alone or together with the Spt^r strain in spectinomycin-containing medium. Amp^r strain grown alone, filled circles. Amp^r and Spt^r strains grown together, open circles.

FIG. 6.

Representative scatter plots of biofilms grown in media containing 33.3 to 40 ppm spectinomycin. Top row: Spt^r strain alone. Middle row: Amp^r strain alone. Bottom row: mixed biofilms, with fluorescent populations indicated by the R1 region. x axis: SSC. y axis: GFP fluorescence (FL1-H).

FIG. 7.

Commensal protection in chemostats. Antibiotic-sensitive organisms grown in chemostats either alone (filled symbols) or with the resistant strain (open symbols) were enumerated by plate counts. A: Spt^r strain in ampicillin-containing medium. B: Amp^r strain in spectinomycin-containing medium.

FIG. 8.

Comparison of chemostat and biofilm commensalism in ampicillin-containing media. The log scale reduction of ampicillin-sensitive Spt^r cells grown in coculture with Amp^r cells was compared for chemostats (filled symbols) and biofilms (open symbols). Samples were taken from chemostats after three reactor volumes and from biofilms after 48 h. Cells were enumerated by dilution spread plating.