Subinhibitory concentrations of gentamicin reduce production of listeriolysin, the main virulence factor of Listeria monocytogenes

Abstract

The facultatively intracellular Gram-positive rod Listeria monocytogenes is the causative agent of severe infections in humans and animals, e.g. sepsis and meningoencephalitis. The treatment of choice is ampicillin in combination with gentamicin [1Jones EM MacGowan AP Antimicrobial chemotherapy of human infection due to Listeria monocytogenes.Eur J Clin Microbiol Infect Dis. 1995; 14: 165-175Crossref PubMed Scopus (99) Google Scholar]. Because of poor penetration through the blood-brain barrier and into the cytoplasm of cells where the listeriae multiply, these antibiotics are likely to reach concentrations below the levels obtained in the blood at the site of infection. Certain antibiotics are able to retard growth of bacteria at concentrations below the minimal inhibitory concentration assessed visually. In addition, some antibiotics are capable of selectively inhibiting the production of virulence factors by pathogenic bacteria [2Doss SA Tillotson GS Amyes SG Effect of sub-inhibitory concentrations of antibiotics on the virulence of Staphylococcus aureus.J Appl Bacteriol. 1993; 75: 123-128Crossref PubMed Scopus (31) Google Scholar]. Ampicillin has recently been shown to reduce the production of listeriolysin [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar]. Listeriolysin is an essential virulence factor of L. monocytogenes because it enables the bacterium to reach the cytoplasm of infected cells, and to start multiplication [4Portnoy DA Chakraborty T Goebel W Cossart P Molecular determinants of Listeria monocytogenes pathogenesis.Infect Immun. 1992; 60: 1263-1270Crossref PubMed Google Scholar]. Inhibition of the production of listeriolysin could also contribute to the therapeutic action of gentamicin on human listeriosis. Therefore, the effect of gentamicin in subinhibitory concentrations on the expression of this virulence factor was determined. This was done as previously described [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar], using an assay measuring the hemolytic activity in the culture supernatants of L. monocytogenes EGD with and without the plasmids containing the β-galactosidase fusions and measuring the activity of the listeriolysin promoter contained on a plasmid fused to the lacZ gene of Escherichia coli. This allowed the measurement of the activity of the listeriolysin promoter as β-galactosidase activity in the transformed strain. The expression of β-galactosidase from this promoter was compared with that of a plasmid contained in L. monocytogenes EGD harboring the promoter of a streptococcal protease fused at the same EcoRI site to the lacZ gene. Subinhibitory concentrations of gentamicin were able to suppress the expression of β-galactosidase from the listeriolysin promoter in concentrations that did not affect growth or the expression of β-galactosidase from the streptococcal promoter (Figure 1). Therefore, gentamicin is able to selectively suppress the production of this virulence factor. Because gentamicin inhibits bacterial protein synthesis by binding to the ribosome, this action might be due to a selective suppression of the translation of mRNAs deriving from the virulence gene itself or from the regulatory gene prfA, whose product regulates the production of listeriolysin and several other virulence factors of L. monocytogenes [4Portnoy DA Chakraborty T Goebel W Cossart P Molecular determinants of Listeria monocytogenes pathogenesis.Infect Immun. 1992; 60: 1263-1270Crossref PubMed Google Scholar]. In contrast to the β-galactosidase assay, the reduction of the hemolytic activity of culture supernatants of the wild-type strains and the transformed strains was not significant. This may be explained by the fact that in contrast to β-galactosidase, listeriolysin is rapidly inactivated at the incubation temperature of 37 °C ([5Geoffroy C Gaillard J-L Alouf JE Berche P Production of thiol-dependent haemolysins by Listeria monocytogenes and related species.J Gen Microbiol. 1989; 135: 481-487PubMed Google Scholar] and own unpublished data), which may have led to greater standard deviations in this assay that make it difficult to demonstrate minor effects on listeriolysin production. In conclusion, subinhibitory concentrations of gentamicin that do not affect growth are able to reduce the production of an essential virulence factor of L. monocytogenes. In combination with a similar effect of ampicillin [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar], this might contribute to the therapeutic action of the two antibiotics in human listeriosis.