Staphylococcus aureus but not Listeria monocytogenes adapt to triclosan and adaptation correlates with increased fabI expression and agr deficiency.

BACKGROUND: The ability of pathogens to adapt to the widely used biocide, triclosan, varies substantially. The purpose of the study was to examine bacterial adaptation over an extended period of time to low increments of triclosan concentrations. Focus was two human pathogens, S. aureus and L. monocytogenes that previously have displayed inherent high and low adaptability, respectively. RESULTS: Three strains of L. monocytogenes and two strains of S. aureus including the community-acquired USA300 were exposed to increasing, sub-lethal concentrations of triclosan in triclosan-containing agar gradients. Following 25 days of exposure on agar plates to sub-lethal concentrations of triclosan with a twofold concentration increase every second day, minimum inhibitory concentration (MIC) for S. aureus increased from 0.125 (8325–4) and 0.0625 (USA 300) mg/L to 4 mg/L. The MIC of all three L. monocytogenes strains was initially 4 mg/L and remained unaltered by the exposure. The adapted S. aureus isolates retained normal colony size but displayed increased expression of fabI encoding an essential enzyme in bacterial fatty acid synthesis. Also, they displayed decreased or no expression of the virulence associated agrC of the agr quorum sensing system. While most adapted strains of USA300 carried mutations in fabI, none of the adapted strains of 8325–4 did. CONCLUSIONS: Adaptability to triclosan varies substantially between Gram positive human pathogens. S. aureus displayed an intrinsically lower MIC for triclosan compared to L. monocytogenes but was easily adapted leading to the same MIC as L. monocytogenes. Even though all adapted S. aureus strains over-expressed fabI and eliminated expression of the agr quorum sensing system, adaptation in USA300 involved fabI mutations whereas this was not the case for 8325–4. Thus, adaptation to triclosan by S. aureus appears to involve multiple genetic pathways

<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