<i>Staphylococcus epidermidis</i> Isolated in 1965 Are More Susceptible to Triclosan than Current Isolates

<div><p>Since its introduction to the market in the 1970s, the synthetic biocide triclosan has had widespread use in household and medical products. Although decreased triclosan susceptibility has been observed for several bacterial species, when exposed under laboratory settings, no <i>in vivo</i> studies have associated triclosan use with decreased triclosan susceptibility or cross-resistance to antibiotics. One major challenge of such studies is the lack of strains that with certainty have not been exposed to triclosan. Here we have overcome this challenge by comparing current isolates of the human opportunistic pathogen <i>Staphylococcus epidermidis</i> with isolates collected in the 1960s prior to introduction of triclosan to the market. Of 64 current <i>S. epidermidis</i> isolates 12.5% were found to have tolerance towards triclosan defined as MIC≥0.25 mg/l compared to none of 34 isolates obtained in the 1960s. When passaged in the laboratory in the presence of triclosan, old and current susceptible isolates could be adapted to the same triclosan MIC level as found in current tolerant isolates. DNA sequence analysis revealed that laboratory-adapted strains carried mutations in <i>fabI</i> encoding the enoyl-acyl carrier protein reductase isoform, FabI, that is the target of triclosan, and the expression of <i>fabI</i> was also increased. However, the majority of the tolerant current isolates carried no mutations in <i>fabI</i> or the putative promoter region. Thus, this study indicates that the widespread use of triclosan has resulted in the occurrence of <i>S. epidermidis</i> with tolerance towards triclosan and that the adaptation involves FabI as well as other factors. We suggest increased caution in the general application of triclosan as triclosan has not shown efficacy in reducing infections and is toxic to aquatic organisms.</p></div

Susceptibility to triclosan among <i>S. epidermidis</i> isolates from 1965–66 (old) and from 2010–11 (current) as determined by their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).

<p>MIC/MBC50 = Minimum Inhibitory/Bactericidal Concentration required to inhibit the growth/kill of 50% of isolates. MIC/MBC90 = Minimum Inhibitory/Bactericidal Concentration required to inhibit the growth/kill of 90% of isolates</p

Triclosan MIC and MBC distributions for <i>S. epidermidis</i> isolates from 1965-66 and from 2010-11.

<p>A: Triclosan MIC (Mininmum inhibitory concentration) distributions. B: Triclosan MBC (Mininmum bactericidal concentration) distributions. Black bars = <i>S. epidermidis</i> blood isolates from 2010-11. White bars = <i>S. epidermidis</i> blood isolates from 1965-66. The lowest dilutions represent a MIC of≤0.0156 mg/l and a MBC of≤0.0652 mg/l. Eight isolates in the current group have a MIC ≥0.25 mg/l and the same eight isolates are the ones with a MBC = 8 mg/l.</p

Triclosan and antibiotic susceptibility of parental strains from 1965-66 (65–13 and 66–01) and from 2010–11 (BD-62, Van-1, BD-12 and BD-24) and their triclosan laboratory exposed descendents.

<p>Adapted strains are named with the parent name and the suffix a and b. The control strains passed along without triclosan have the suffix Ka and Kb. MIC_0/MBC_0, measured after ended adaptation. MIC_5/MBC_5, measured after 5 passages on triclosan free media. PEN = penicillin, FOX = cefoxitin, FA = fucidic acid, GEN = gentamicin, ERY = erythromycin, CLIN = clindamycin, RIF = rifampicin, LIN = linezolid, NOF = norfloxacin. ST = Sequence Type, determined by MLST.</p

Northern blot analysis of <i>fabI</i> transcripts in parental strains and their triclosan adapted descendant.

<p>Parental strains from 1965-66 (65-13 and 66-01) and from 2010-11 (BD-62, Van-1, BD-12 and BD-24) and the descendant strains isolated after triclosan adaptation. Adapted strains are named with the parent name and the suffix a.</p

Distribution of triclosan tolerance among <i>S. epidermidis</i> isolates from 1965-66 (old) and 2010-11 (current).

<p>MIC <0.25 mg/l is defined as susceptible and MIC ≥0.25 mg/l is defined as tolerant.</p>a<p>Old isolates versus current isolates.</p

Analysis of mutations in the <i>fabI</i> gene of <i>S. epidermidis</i> and the upstream promoter region.

<p>The suffix a* indicates adapted strains that have been passed for 5 days on triclosan free media. The <i>fabI</i> primers did not work on three isolates (65-13, BD-26 and BD-50). <i>S. epidermidis</i> ATCC 12228 is published on NCBI with its full sequence. It has a triclosan MIC <0.25 mg/l and its <i>fabI</i> gene is identical to all the current triclosan susceptible isolates we have sequenced. The putative promoter region of ATCC 12228 is different from the current triclosan susceptible isolates we have sequenced but those are all identical. ST, Sequence type determined by MLST. ND = not determined. Abbreviations for amino acids: H = histidine, Y = tyrosine, S = serine, A = alanine, G = glycine, F = phenylalanine, V = valine, Q = glutamine.</p

Comparison of morbidity and mortality after bloodstream infection with vancomycin-resistant versus -susceptible <i>Enterococcus faecium: a</i> nationwide cohort study in Denmark, 2010–2019

The emergence of bloodstream infections (BSI) caused by vancomycin-resistant Enterococci (VRE) has caused concern. Nonetheless, it remains unclear whether these types are associated with an excess risk of severe outcomes when compared with infections caused by vancomycin-susceptible Enterococci (VSE). This cohort study included hospitalized patients in Denmark with Enterococcus faecium-positive blood cultures collected between 2010 and 2019 identified in the Danish Microbiology Database. We estimated 30-day hazard ratio (HR) of death or discharge among VRE compared to VSE patients adjusted for age, sex, and comorbidity. The cohort included 6071 patients with E. faecium BSI (335 VRE, 5736 VSE) among whom VRE increased (2010–13, 2.6%; 2014–16, 6.3%; 2017–19; 9.4%). Mortality (HR 1.08, 95%CI 0.90–1.29; 126 VRE, 37.6%; 2223 VSE, 37.0%) or discharge (HR 0.89, 95%CI 0.75–1.06; 126 VRE, 37.6%; 2386 VSE, 41.6%) was not different between VRE and VSE except in 2014 (HR 1.87, 95% CI 1.18–2.96). There was no interaction between time from admission to BSI (1–2, 3–14, and >14 days) and HR of death (P = 0.14) or discharge (P = 0.45) after VRE compared to VSE, despite longer time for VRE patients (17 vs. 10 days for VSE, P E. faecium BSI warrants further study.</p