Abstract
Small Colony Variant (SCV) cells of bacteria are a slow-growing phenotype that result from specific defects in the electron transport chain. They form pinpoint colonies on agar plates and have a variety of phenotypic characteristics, such as altered carbon metabolism, decreased toxin and lytic enzyme production, aminoglycoside resistance, and increased intracellular persistence. They are clinically relevant in Staphylococcus aureus and Pseudomonas aeruginosa, serving as a reservoir for recurrent or prolonged infections. Here, we found that a SCV mutant in the foodborne pathogen Listeria monocytogenes (strain SCV E18), similar to the high persister mutant phenotype, survived significantly better than the wild type when exposed over a 48-h period to concentrations above Minimal Inhibitory Concentration for most tested antibiotics. SCV E18 survived more poorly than the wildtype in unactivated RAW264.7 macrophage cells, presumably because of its reduced listeriolysin O expression, however, it survived better in reactive oxygen species producing, phorbol 12-myristate 13-acetate-activated macrophages. Although SCV E18 was sensitive to oxygen as it entered the stationary phase, it was significantly more tolerant to H2O2 than the wild type, which may result from a shift in metabolism, however, further investigation is needed to resolve this. SCV E18 is a spontaneous mutant with a point mutation in the hemA gene. A wild type copy of hemA was complemented on plasmid pSOG30222, which restored the wild type phenotype. The results reported here suggest that the SCV of L. monocytogenes could be of clinical importance and highlight a need for adequate clinical screening for this phenotype, as it could affect antibiotic treatment outcomes.
Highlights
Listeria monocytogenes is a Gram-positive, foodborne pathogen that can cause the rare, but often lethal infection listeriosis
We have previously found that exposure to sub-lethal concentrations of triclosan with subsequent selection on gentamicin could generate stable L. monocytogenes Small Colony Variant (SCV) cells (Christensen et al, 2011), which all had mutations in one of the heme biosynthesis or metabolism genes and exhibited the same traits observed in SCVs of other species
Because L. monocytogenes SCV grow slower than the wild type (Kastbjerg et al, 2014), and because of the lowered oxidative phosphorylation observed in other SCV organisms, such as S. aureus (Proctor et al, 1998), we hypothesized that L. monocytogenes SCVs would have an increased tolerance to other classes of antibiotics, which was evaluated using time dependent killing experiments
Summary
Listeria monocytogenes is a Gram-positive, foodborne pathogen that can cause the rare, but often lethal infection listeriosis. By repeatedly exposing Escherichia coli to high concentrations of ampicillin, Fridman et al (2014) found that bacteria can tolerate antibiotics by extending the lag-phase by mutations in a variety of pathways, essentially rendering the cells dormant until the transient antibiotic pressure is removed Another potential reservoir of persistent and recurrent bacterial infections is the so-called Small Colony Variant (SCV) (Kahl et al, 2016). The phenotypic switching of the SCV and the persister cell are thought to be part of a bacterial bet-hedging strategy, allowing a small percentage of the population to survive and repopulate following stress exposure (Sousa et al, 2012) These two phenotypes share a number of other characteristics including slowed growth, intracellular persistence and a link to chronic infections. These results will help to determine the clinical significance of Small Colony Variants in L. monocytogenes
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