Effect of biofilms on recalcitrance of staphylococcal joint infection to antibiotic treatment

Abstract

The pathogenesis of joint infections is not well understood. In particular, we do not know why these infections respond poorly to antibiotic treatment. Here we show that methicillin-resistant Staphylococcus aureus, a major cause of joint infections, forms exceptionally strong biofilmlike aggregates in human synovial fluid (SF), to an extent significantly exceeding biofilm formation observed in growth medium or serum. Screening a transposon bank identified bacterial fibronectin- and fibrinogen-binding proteins as important for the formation of macroscopic clumps in SF, suggesting an important role of fibrin-containing clots in the formation of bacterial aggregates during joint infection. Pretreatment of SF with plasmin led to a strongly reduced formation of aggregates and increased susceptibility to antibiotics. These results give important insight into the pathogenesis of staphylococcal joint infection and the mechanisms underlying resistance to treatment. Furthermore, they point toward a potential novel approach for treating joint infections.

Keywords: MRSA; Staphylococcus aureus; antibiotic resistance; biofilm; joint infection; septic arthritis.

Figure 1.

Growth of Staphylococcus aureus in synovial fluid (SF), human serum, and growth medium (tryptic soy broth [TSB]). The methicillin-resistant S. aureus strain USA300 was grown in the indicated fluids and growth was measured according to colony-forming units (CFUs) (A) or quantitative polymerase chain reaction determination (B). The experiment was performed in triplicate, and error bars represent standard deviations.

Figure 2.

Staphylococcus aureus shows extreme aggregation in synovial fluid (SF). A–E, Washed overnight cultures of S. aureus (A, B, D, E: USA300) were incubated in the indicated fluids for 20 minutes, and aggregation was examined visually (A–C) or with light microscopy (D, E) in tryptic soy broth (TSB) (D) and SF (E). C, Red represents community associated methicillin-resistant S. aureus (MRSA) strains; purple, hospital-associated MRSA strains; black, methicillin-susceptible S. aureus strains. F, Scanning electron microscopy of an USA300 aggregate formed in a 24-hour culture grown in SF. G, Cell counting analysis of aggregates formed after 20-minute incubation in the indicated fluid, with SF obtained from 3 individuals (SF1, SF2, and SF3). Error bars represent standard deviations. Note that macroscopic clumps cannot be analyzed in the cell counter.

Figure 3.

Confocal laser scanning microscopy of biofilms grown in synovial fluid (SF), serum, or growth medium (tryptic soy broth [TSB]). Static 24-hour biofilms were grown in the indicated fluids, stained with propidium iodide, and visualized with confocal laser scanning microscopy. (In the TSB sample, small red clusters represent background staining, not cells.)

Figure 4.

Aggregation of USA300 methicillin-resistant Staphylococcus aureus in synovial fluid (SF) is predominantly protein dependent. A, Analysis for presence of polysaccharide intercellular adhesin (PIA) exopolysaccharide in USA300 biofilms using wheat germ agglutinin (WGA) staining. Propidium iodide (PI) staining shows the entire biofilm, and WGA staining shows PIA. In the merge images, PI- and WGA-stained areas appear yellow. Bottom, Biofilms of an ica mutant not expressing PIA do not stain with WGA. B, Macroscopic evaluation of the effect of enzymatic digestion on aggregates of USA300 formed in SF. Dispersin B is an enzyme specifically digesting PIA. C, Estimation of aggregate dispersion based on measurement of increased colony-forming unit (CFU) counts after enzymatic digestion (n = 7). ^†P < .01; error bars represent standard deviations; Abbreviation: NS, not significant.

Figure 5.

Factors affecting macroscopic aggregation of USA300 methicillin-resistant Staphylococcus aureus identified by screening of a transposon mutagenesis bank. The Nebraska USA300 transposon mutagenesis bank was screened for absence of aggregation after incubation in synovial fluid. Six clones showed no or strongly reduced aggregation (those with insertions in the fnbA, fnbB, clfA, clfB, rsbU, and trap genes). A, Macroscopic (left) and microscopic (right) evaluation of aggregation in USA300 wild-type and mutant strains. B, Analysis of remaining aggregates by cell counter.

Figure 6.

Plasmin treatment strongly increases susceptibility of Staphylococcus aureus aggregates in synovial fluid (SF) to antibiotics. A, Treatment of aggregates with plasmin before and after aggregate formation, macroscopic evaluation. Plasmin pretreatment was performed by incubating solutions before inoculation for 12 hours with 150 µg/mL plasmin; posttreatment was performed after 20-minute clump formation using the same concentration. B, Cellometry analysis. ^‡P < .001; error bars represent standard deviations. C, Antibiotic susceptibility was assessed by incubating the methicillin-resistant S. aureus strain USA300 for 6 hours in with cefazolin or vancomycin at 37°C, followed by brief proteinase K treatment and colony-forming unit (CFU) determination. CEF, cefazolin; TSB, tryptic soy broth. D, Alternatively, a luminescent S. aureus strain (Xen36) was used to monitor luminescence as readout under the same conditions. Plasmin pretreatment was performed by incubating solutions before inoculation for 12 hours with 150 µg/mL plasmin. C, D, Assays were performed in triplicate. *P < .05; ^†P < .01; ^‡P < .001; error bars represent standard deviations. Abbreviation: VAN, vancomycin.

Figure 7.

Model of Staphylococcus aureus biofilm formation in joint infection. S. aureus biofilms may form in a surface-attached form on the synovial membrane or on indwelling medical devices during prosthetic joint infection or exist as free-floating aggregates in synovial fluid (SF) within the joint cavity. Aggregate formation is dependent on host-derived fibrin, as indicated by preventing aggregate formation by plasmin treatment of SF, and bacterial fibrinogen- and fibronectin-binding proteins (ClfA, ClfB, FnbA, and FnbB). Plasmin treatment increases susceptibility of antibiotics and probably increases efficiency of host defenses by decreasing aggregate sizes for more efficient uptake by phagocytes. Abbreviations: eDNA, extracellular DNA; PIA, polysaccharide intercellular adhesin.