Association between methicillin susceptibility and biofilm regulation in Staphylococcus aureus isolates from device-related infections

Abstract

Production of icaADBC-encoded polysaccharide intercellular adhesin, or poly-N-acetylglucosamine (PIA/PNAG), represents an important biofilm mechanism in staphylococci. We previously described a glucose-induced, ica-independent biofilm mechanism in four methicillin-resistant Staphylococcus aureus (MRSA) isolates. Here, biofilm regulation by NaCl and glucose was characterized in 114 MRSA and 98 methicillin-sensitive S. aureus (MSSA) isolates from diagnosed device-related infections. NaCl-induced biofilm development was significantly more prevalent among MSSA than MRSA isolates, and this association was independent of the isolate's genetic background as assessed by spa sequence typing. Among MSSA isolates, PIA/PNAG production correlated with biofilm development in NaCl, whereas in MRSA isolates grown in NaCl or glucose, PIA/PNAG production was not detected even though icaADBC was transcribed and regulated. Glucose-induced biofilm in MRSA was ica independent and apparently mediated by a protein adhesin(s). Experiments performed with strains that were amenable to genetic manipulation revealed that deletion of icaADBC had no effect on biofilm in a further six MRSA isolates but abolished biofilm in four MSSA isolates. Mutation of sarA abolished biofilm in seven MRSA and eight MSSA isolates. In contrast, mutation of agr in 13 MRSA and 8 MSSA isolates substantially increased biofilm (more than twofold) in only 5 of 21 (23%) isolates and had no significant impact on biofilm in the remaining 16 isolates. We conclude that biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and Agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development.

FIG. 1.

Comparison of ica operon expression, PIA/PNAG production, and biofilm regulation in nine MSSA clinical isolates grown in BHI medium or in BHI supplemented with 4% NaCl or 1% glucose (Glu). Laboratory strain RN4220 and its isogenic Δica mutant were used as positive and negative controls, respectively. Transcription of gyrB (control) and icaA was measured by RT-PCR using RNA prepared from cultures grown at 37°C to an A₆₀₀ of 2.0. PIA/PNAG was measured in cell extracts from overnight cultures. Biofilm formation in tissue culture-treated 96-well plates was measured three times, and representative results are shown.

FIG. 2.

Comparison of ica operon expression, PIA/PNAG production, and biofilm regulation in 16 MRSA clinical isolates grown in BHI medium or in BHI supplemented with 4% NaCl or 1% glucose (Glu). Laboratory strain RN4220 and its isogenic Δica mutant were used as positive and negative controls, respectively. Transcription of gyrB (control) and icaA was measured by RT-PCR using RNA prepared from cultures grown at 37°C to an A₆₀₀ of 2.0. PIA/PNAG was measured in cell extracts from overnight cultures. Biofilm formation in tissue culture-treated 96-well plates was measured three times, and representative results are shown.

FIG. 3.

(A) Measurement of PIA/PNAG immunoreactivity in four representative wild-type (WT) MSSA clinical isolates and their corresponding Δica mutants grown in BHI medium supplemented with 4% NaCl. (B) Comparison of biofilm regulation in four representative WT MRSA clinical isolates and their corresponding Δica mutants. (C) Comparison of biofilm regulation in four WT MRSA isolates and their corresponding Δica mutants. Biofilm formation was analyzed by growing triplicate cultures in tissue culture-treated 96-well plates in BHI medium or in BHI medium supplemented with 4% NaCl or 1% glucose, followed by staining with crystal violet. Photographs of representative biofilm plates are shown.

FIG. 4.

Susceptibility of glucose-induced MRSA biofilms (left) and NaCl-induced MSSA biofilms (right) to treatment with sodium metaperiodate and proteinase K. MRSA biofilms were grown in BHI medium supplemented with 1% glucose, and MSSA biofilms were grown in BHI medium supplemented with 4% NaCl for 24 h. Each strain was grown in four individual wells. The laboratory strain RN4220 (wells 2A to 2D in each plate) was used a positive control for NaCl-induced biofilm formation in MSSA. The MRSA clinical isolate BH1CC (wells 3A to 3D in each plate) was used a positive control for glucose-induced biofilm formation in MRSA. The 15 clinical MSSA isolates used in the experiment were BH6(2005) (wells 5A to 5D), BH49(2004) (wells 6A to 6D), BH51(2004) (wells 6E to 6H), BH37(2004) (wells 7A to 7D), BH48(2004) (wells 7E to 7H), BH21(2004) (wells 8A to 8D), BH29(2004) (wells 8E to 8H), BH10(2004) (wells 9A to 9D), BH18(2002) (wells 9E to 9H), BH23(2003) (wells 10A to 10D), BH27(2003) (wells 10E to 10H), BH11(2003) (wells 11A to 11D), BH17(2003) (wells 11E to 11H), BH2(2003) (wells 12A to 12D), and BH8(2003) (wells 12E to 12D). The 14 clinical MRSA isolates used in this experiment were BH26(2004) (wells 5A to 5D), BH17(2005) (wells 5E to 5D), BH4(2004) (wells 6A to 6D), BH8(2004) (wells 6E to 6H), BH23(2003) (wells 7A to 7D), BH30(2004) (wells 7E to 7H), BH10(2003) (wells 8A to 8D), BH11b(2003) (wells 8E to 8H), BH14(2002) (wells 9A to 9D), BH16(2002) (wells 9E to 9H), BH10(2002) (wells 10A to 10D), BH12(2002) (wells 10E to 10H), BH2(2002) (wells 11A to 11D), and BH6(2002) (wells 11E to 11H).

FIG. 5.

Contribution of the sarA and agr loci to biofilm regulation in clinical isolates of MRSA (A) and MSSA (B). MRSA isolates were grown in BHI medium supplemented with 1% glucose for 24 h. MSSA isolates were grown in BHI medium supplemented with 4% NaCl for 24 h. WT, wild type.