Biofilm formation by Staphylococcus haemolyticus

Abstract

Infections due to coagulase-negative staphylococci (CoNS) most frequently occur after the implantation of medical devices and are attributed to the biofilm-forming potential of CoNS. Staphylococcus haemolyticus is the second most frequently isolated CoNS from patients with hospital-acquired infections. There is only limited knowledge of the nature of S. haemolyticus biofilms. The aim of this study was to characterize S. haemolyticus biofilm formation. We analyzed the biofilm-forming capacities of 72 clinical S. haemolyticus isolates. A detachment assay with NaIO(4), proteinase K, or DNase was used to determine the main biofilm components. Biofilm-associated genes, including the ica operon, were analyzed by PCR, and the gene products were sequenced. Confocal laser scanning microscopy (CLSM) was used to elucidate the biofilm structure. Fifty-three isolates (74%) produced biofilms after growth in Trypticase soy broth (TSB) with glucose, but only 22 (31%) produced biofilms after growth in TSB with NaCl. It was necessary to dissolve the biofilm in ethanol-acetone to measure the optical density of the full biofilm mass. DNase, proteinase K, and NaIO(4) caused biofilm detachment for 100%, 98%, and 38% of the isolates, respectively. icaRADBC and polysaccharide intercellular adhesin (PIA) production were found in only two isolates. CLSM indicated that the biofilm structure of S. haemolyticus clearly differs from that of S. epidermidis. We conclude that biofilm formation is a common phenotype in clinical S. haemolyticus isolates. In contrast to S. epidermidis, proteins and extracellular DNA are of functional relevance for biofilm accumulation, whereas PIA plays only a minor role. The induction of biofilm formation and determination of the biofilm mass also needed to be optimized for S. haemolyticus.

FIG. 1.

Dendrogram generated from the PFGE profiles of 72 Staphylococcus haemolyticus by the unweighted pair group method with arithmetic means. The Dice band-based similarity coefficient was calculated with a band position tolerance of 2%. Clusters were designated when the branching points exceeded 80% similarity (7) and contained three or more isolates. The scale bar at the top of the dendrogram represents similarity.

FIG. 2.

Organization of the ica operon, as described for S. epidermidis RP62A at the top. The percent identities between the individual ica genes or the complete ica operon from S. haemolyticus and the different published ica sequences from four staphylococcal species are shown below the organizational diagram. S. lugdunensis does not have the icaR gene. The GenBank accession numbers for the sequences are as follows: S. epidermidis, NC_002976.3; S. caprae, AF246926; S. lugdunensis, EF546621; S. haemolyticus, FJ472951. NA, not available.

FIG. 3.

Phylogenetic tree based on 372-bp icaA sequences from 10 staphylococcal species. The tree was generated by using the neighbor-joining algorithm. Bootstrap values for internal nodes are given. The GenBank accession numbers for the sequences are as follows: S. epidermidis, NC_002976.3; S. saprophyticus, AF500270; S. cohnii, AF500268; S. condimenti, AF500266; S. simulans, AF500263; S. caprae, AF246926; S. capitis, AF500269; S. lugdunensis, EF546621; and S. haemolyticus, FJ472951.

FIG. 4.

Percent biofilm detachment of 53 biofilm-producing S. haemolyticus isolates after treatment with NaIO₄ (light checks), proteinase K (dark checks), or DNase (stripes).

FIG. 5.

CLSM images of 24-h biofilms of S. epidermidis RP62A (A) and S. haemolyticus 51-10 (B) stained with DAPI (blue) and WGA (green). The S. epidermidis RP62A biofilm shows a complex three-dimensional structure, and polysaccharides dominate the extracellular matrix component. In comparison, the protein-rich biofilm of S. haemolyticus 51-10 is more condensed, and only small amounts of polysaccharides can be observed. Also, the RP62A biofilm is more than twice as thick as the S. haemolyticus biofilm.