Evaluation of the role of staphylococci in the pathomechanism of conjunctivitis

Abstract

Purpose: Determination of the association between ica genes and phenotypic biofilm formation in staphylococcal isolates involved in conjunctivitis, their antibiotic resistance as well as detection of selected virulence characteristics: adhesion to epithelial cells and in vitro cytotoxicity.

Methods: The study included 26 Staphylococcus aureus (SA) and 26 Staphylococcus epidermidis (SE) isolates. The presence of icaAD genes and ica operon was determined by the PCR assay. Phenotypic biofilm formation was verified using the microtitre plate assay. Antibiotic resistance was performed using the disc diffusion method. Staphylococcal ability to attach to host cells was assessed by flow cytometry. Cytotoxicity on epithelial cells was evaluated by LDH assay.

Results: The ica genes were detected in 26.9% of SE and in 42.3% of SA isolates. Only 15.3% of isolates (SE) were positive for both the icaAD and the ica operon. Phenotypically, 19.2% of SE isolates were strong biofilm producers, among which three were both icaAD- and ica operon-positive. About 26.9% of SA isolates were strong biofilm producers. Methicillin resistance (MR) was detected in 34.6% of SE and 26.9% of SA isolates. About 75% of MR isolates were multidrug resistant. SA isolates adhered to host cells more extensively than SE. SA isolates released higher level of LDH than SE.

Conclusions: Adherence abilities were commonly observed in staphylococci associated with conjunctivitis. However, low prevalence of isolates positive for a complete and functional ica locus and low prevalence of strong biofilm producers was detected. SA adhered to a greater extent to eukaryotic cells than SE and were more cytotoxic.

Keywords: Adhesion; Biofilm; Conjunctivitis; Cytotoxicity assay; Drug-resistance; Staphylococcus.

Fig. 1

PCR detection of the icaA and icaD genes in SA and SE isolates. Lane 1, absence of amplification product from a strong SE biofilm producer (31 III SE); Lane 2, icaA-positive, strong SE biofilm producer (21 SE); Lane 3, icaD-positive, strong SE biofilm producer (21 SE); Lane 4, icaA-positive, weak SA biofilm producer (40 SE); Lane 5, icaD-positive, weak SA biofilm producer (40 SE); Lanes 6–7, icaA- and icaD-negative (respectively) strong SA biofilm producer (54 SA); Lane 8, icaD-positive, weak SA biofilm producer (25 SA); Lanes 9–10, icaA- and icaD-negative (respectively) weak SE biofilm producer (42 SE); Lane 11, icaA-positive, weak SE biofilm producer (13 SE);

Fig. 2

PCR detection of the ica locus in SE isolates. Lanes 1–3, ica locus-positive, strong SE biofilm producers (21 SE, 31 III SE, 35 SE); Lane 4, ica locus-positive, weak SE biofilm producer (31 SE)

Fig. 3

Assessment of the attachment value in S. aureus and S. epidermidis strains (pooled data)

Fig. 4

Attachment to Detroit 562 cells by different staphylococcal clinical isolates. a represents attachment ability of S. epidermidis strains. b represents attachment ability of S. aureus strains Results expressed as mean % of attached bacterial cells/50 000 epithelial cells ± SD from at least three independent experiments

Fig. 5

Cytotoxic properties of Staphylococcus aureus strains and S. epidermidis strains (pooled data)

Fig. 6

Cytotoxicity of different S. aureus and S. epidermidis strains at MOI 30 on epithelial cell line. a represents cytotoxic ability of S. epidermidis strains. b represents cytotoxic ability of S. aureus strains. Results expressed as mean % cytotoxicity on epithelial cells ± SD from at least 3 independent experiments