Altered Competitive Fitness, Antimicrobial Susceptibility, and Cellular Morphology in a Triclosan-Induced Small-Colony Variant of Staphylococcus aureus

Abstract

Staphylococcus aureus can produce small-colony variants (SCVs) that express various phenotypes. While their significance is unclear, SCV propagation may be influenced by relative fitness, antimicrobial susceptibility, and the underlying mechanism. We have investigated triclosan-induced generation of SCVs in six S. aureus strains, including methicillin-resistant S. aureus (MRSA). Parent strains (P0) were repeatedly passaged on concentration gradients of triclosan using a solid-state exposure system to generate P10. P10 was subsequently passaged without triclosan to generate X10. Susceptibility to triclosan and 7 antibiotics was assessed at all stages. For S. aureus ATCC 6538, SCVs were further characterized by determining microbicide susceptibility and competitive fitness. Cellular morphology was examined using electron microscopy, and protein expression was evaluated through proteomics. Triclosan susceptibility in all SCVs (which could be generated from 4/6 strains) was markedly decreased, while antibiotic susceptibility was significantly increased in the majority of cases. An SCV of S. aureus ATCC 6538 exhibited significantly increased susceptibility to all tested microbicides. Cross-wall formation was impaired in this bacterium, while expression of FabI, a target of triclosan, and IsaA, a lytic transglycosylase involved in cell division, was increased. The P10 SCV was 49% less fit than P0. In summary, triclosan exposure of S. aureus produced SCVs in 4/6 test bacteria, with decreased triclosan susceptibility but with generally increased antibiotic susceptibility. An SCV derived from S. aureus ATCC 6538 showed reduced competitive fitness, potentially due to impaired cell division. In this SCV, increased FabI expression could account for reduced triclosan susceptibility, while IsaA may be upregulated in response to cell division defects. Staphylococcus aureus small-colony variants (SCVs) are characterized by low growth rate and the formation of small nonpigmented colonies (1, 2). They are commonly, but not exclusively, related to antibiotic exposure (3) and have been shown to display diverse phenotypic characteristics, including reduced beta-hemolysis, coagulase, and DNase activities (4), enhanced intracellular survival (5), impaired biofilm formation (6), reduced virulence (6), and low intrinsic susceptibility to certain antibiotics, cationic microbicides, and antimicrobial peptides (7,8). While all SCVs are not physiologically the same, certain SCVs have been reported to cause persistent skin, bone, and device-associated infections, and they have been isolated from patients undergoing prolonged antibiotic therapy (2, 9, 10). Due to their uncommon morphological features and pinpoint colony size, SCVs may be overlooked or misidentified in clinical microbiology laboratories, potentially confounding their identification.The phenotypic variation observed in S. aureus SCVs is often attributed to auxotrophy for menadione, hemin, or thiamine due to mutations in their respective genes. This results in impaired synthesis...

Loss of Function in Escherichia coli Exposed to Environmentally Relevant Concentrations of Benzalkonium Chloride

“…Minimum bactericidal concentrations were determined as described previously (14,33). Briefly, aliquots (10 l) from wells exhibiting no turbidity were transferred to sterile TSA prior to 4 days of incubation at 37°C to determine the MBC.…”

“…We have previously assessed the effect of formulation (29,30), growth in taxonomically diverse biofilm communities (31), and potential impairment of fitness in biocideadapted bacteria (32,33) in a series of studies intended to develop and apply methods for assessing the risk of biocide resistance based more closely on real-life conditions.…”

Small Colony Variants and Triclosan Resistance in Five International Clones of Methicillin Resistant Staphylococcus aureus

“…Susceptibility of SCVs to three widely used antimicrobials (penicillin, gentamicin and triclosan) has been shown to reduce notably (Seaman et al, 2007). Small colony variants of Staphylococcus aureus, first described 100 years ago are more resistant to antibiotics (Proctor et al, 1998) than normal colonies; however, Forbes et al (2015) found that triclosan susceptibility in all SCVs (which could be generated from 4/6 strains) was distinctly declined, while antibiotic susceptibility was considerably augmented in most of the cases. Clinical studies suggest a link between electron transport-defective strains and persistent infections, a defined hemB mutant with SCV phenotype provided strong additional evidence for these connections (Von Eiff C et al, 2000A).…”

“…Staphylococcus aureus can produce small-colony variants (SCVs) that express different phenotypes. While their importance is uncertain, SCV propagation may be influenced by relative fitness, antimicrobial susceptibility, and the underlying mechanism (Forbes et al, 2015). Some workers were unable to demonstrate development of resistance following exposure of S.aureus to sub-minimum inhibitory concentrations of triclosan (Suller & Russell, 2000) but low-level resistance had been reported previously in Escherichia coli and S. aureus including MRSA (Cookson et al, 1991); the resistance in these organisms is due to either fabI mutation leading to over-expression of reductase or over-expression of efflux pumps (Bayston et al, 2007).…”

“…Not only in hospital and community, Couto et al (2013) evaluated triclosan along with other biocides and antimicrobial susceptibility of methicillin-resistant staphylococcal isolates from horses. More recently, Forbes et al (2015) investigated triclosan-induced generation of SCVs in six S. aureus strains, including methicillin-resistant S. aureus (MRSA).…”