Relationship of ST with genotypic and phenotypic data from the two sources.

<p>^a Represented by ST16, ST10, ST87, ST38, ST71, ST21, ST57 and ST46, each type with a single isolate.</p><p>^b Represented by lineage ST238 and ST118 (4 isolates for each one, 8.8%), ST2, ST4, ST9 (3 isolates for each one, 6.6%), ST23 (2 isolates for each one, 4.4%), ST26, ST135, ST494, ST43, ST48 and ST173 (one isolate for each one).</p><p>Relationship of ST with genotypic and phenotypic data from the two sources.</p

Molecular and Phenotypic Characterization of <i>Staphylococcus epidermidis</i> Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection

<div><p><i>Staphylococcus epidermidis</i> is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of <i>S</i>. <i>epidermidis</i> from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of <i>S</i>. <i>epidermidis</i> were used: one from ocular infection (n = 62), and another from healthy conjunctiva (n = 45). All isolates were genotyped by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome <i>mec</i> (SCC<i>mec</i>), detection of the genes <i>icaA</i>, <i>icaD</i>, IS256 and polymorphism type of <i>agr</i> locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%), while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%). By means of a principal coordinates analysis (PCoA) and a discriminant analysis (DA) it was found that ocular infection isolates had as discriminant markers <i>agr</i> III or <i>agr</i> II, SCC<i>mec</i> V or SCC<i>mec</i> I, <i>mecA</i> gene, resistance to tobramycin, positive biofilm, and IS256⁺. In contrast to the healthy conjunctiva isolates, the discriminating markers were <i>agr</i> I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66%) had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva isolates do not, in almost all cases, infect the eye due to their large genotypic and phenotypic difference with the ocular infection isolates.</p></div

Antibiotic resistance of healthy conjunctiva and ocular infection isolates.

<p>Oxa: oxacillin; Cip: ciprofloxacin; Ofl: ofloxacin; Lev: levofloxacin; Mox: moxifloxacin; Gat: gatifloxacin; Tob: tobramycin; Chl: chloramphenicol; Van: vancomycin.</p><p>Antibiotic resistance of healthy conjunctiva and ocular infection isolates.</p

Cluster analyses of the isolates from Ocular Infection and healthy conjunctiva.

<p>The colors represent the ST's. The black vertical line represents the isolates from healthy conjunctiva (HC) and green vertical line isolates from ocular infection (OI).</p

Statistical analysis of phenotypic and genotypic data.

<p>Principal Coordinates Analysis (PCoA) of <i>S</i>. <i>epidermidis</i> (A) from Ocular Infection (OI, green) and isolates from healthy conjunctiva (HC, black). The circles represent the confidence interval 95%. The first two coordinate axes represent 31% of the variance. Discriminant analysis (B) of the isolates of <i>S</i>. <i>epidermidis</i> from Ocular Infection (OI, green) and healthy conjunctiva (HC, black). Negative values belong to OI isolates and positive to HC isolates. The discriminant function properly allocated to 77% of isolates from HC and 84% from OI.</p

MrkH acts as repressor of <i>mr</i>k genes.

<p>Transcriptional expression (qRT-PCR) of <i>mrkI</i> (A), <i>mrkJ</i> (B) and <i>mrkA</i> (C) genes overexpressing the MrkH protein at different L(+)-arabinose concentrations in the WT strain. D. Transcriptional expression (qRT-PCR) of <i>mrkH</i> and <i>mrkI</i> in the WT, Δ<i>hns</i>, Δ<i>mrkJ</i> and Δ<i>hns</i> Δ<i>mrkJ</i> backgrounds. E. Quantification of biofilm formation by measuring violet crystal uptake under overexpression of MrkH in the WT and Δ<i>mrkJ</i> mutant. Results represent the mean and standard deviations of three independent experiments performed. Statistically significant with respect to the WT strain: *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

MrkH regulates the <i>mrkJ</i> promoter.

<p>A. Schematic representation of the <i>mrkJ</i> promoter. The panel shows the nucleotide sequence of the regulatory region, showing the previously reported transcription start site (+1) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173285#pone.0173285.ref024" target="_blank">24</a>]. The -35 and -10 promoter sequences and the transcription start site are underlined. Putative MrkH-binding site is boxed. B. Logo motif analysis using the MrkH-binding sites for <i>mrkA</i>, <i>mrkH</i> and <i>mrkJ</i> promoter regions. C. Transcriptional expression (qRT-PCR) of <i>mrkJ</i> gene in WT, Δ<i>mrkH</i> and complemented Δ<i>mrkH</i> backgrounds. D. <i>mrkJ</i> expression (qRT-PCR) in the wild-type (WT) and <i>mrkJ</i>ΔMrkHbox::FRT (<i>mrkJ</i>*). E. qRT-PCR assays determining the <i>cat</i> expression of <i>mrkJ</i> (pKK-<i>mrkJ</i>-wt) and a mutant variant within the MrkH-binding box (pKK-<i>mrkJ</i>-mut). Results represent mean and standard deviations of three independent experiments. ns, not significant; **, statistically significant with respect to the WT strain (<i>p</i><0.01).</p

The absence of MrkI does not affect the T3P.

<p>Transcriptional expression (qRT-PCR) of <i>mrkH-I-J</i> (A) and <i>mrkA</i> (B) genes in WT, Δ<i>mrkH</i> mutant, Δ<i>mrkI</i> mutant and Δ<i>mrkHI</i> double mutant. C. Quantification of biofilm formation by measuring violet crystal uptake in WT, Δ<i>mrkH</i> mutant, Δ<i>mrkI</i> mutant and Δ<i>mrkHI</i> double mutant. Results shown represent the mean and standard deviations of three independent experiments. ns, not significant; **, statistically significant with respect to the WT strain (<i>p</i><0.01).</p

Primers used in this study.

<p>Primers used in this study.</p