Intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila

Abstract

The antimicrobial susceptibilities of Legionella pneumophila isolates grown either in U937 human monocytic cells or in Acanthamoeba polyphaga were studied after release from the host cells without further subculture. Time-survival studies showed that exposure of L. pneumophila cells, grown exclusively in vitro, to 5 g of rifampin per ml resulted in at least 99.9% killing after 6 h and no detectable survivors at 24 h. Similar rates of killing were observed for in vitro-grown cells tested by exposure to ciprofloxacin. Conversely, time-survival studies revealed that macrophage-grown and amoeba-grown cells were ca. 1,000-fold more resistant to the activities of both drugs. Macrophage-grown cells treated with 5 g of rifampin per ml showed 70 and 62% survival after 6 and 24 h, respectively. Intracellularly grown legionellae were also highly resistant to erythromycin (8 g/ml). After 24 h of exposure to the drug, there was 70 and 60% survival for amoeba-grown and macrophage-grown legionellae, respectively, whereas in vitro-grown cells showed a 2-log 10 reduction in viable count. When intracellularly grown L. pneumophila cells were subcultured in broth for 48 h, they reverted to the phenotype characteristic of in vitro growth. Morphologically, the cells were larger than their intracellularly grown counterparts and resistance characteristics were lost. The susceptibilities of the subcultured cells to all three drugs were similar to those of Legionella cells grown exclusively in vitro. In view of these findings, the successful treatment of Legionnaires' disease may be related as much to the resistance phenotype induced by intramacrophage growth as to the ability of the antibiotic to enter phagocytic cells.

Inorganic polyphosphate in the origin and survival of species

“…RpoS has been implicated in the virulence of bacterial pathogens for animals (11) and protozoa (12). Legionella pneumophilus emerges after replication within Acanthamoeba both more virulent (13) and more resistant to chemical attack (14,15). It would thus seem that poly P has a role in the merged pathways of both virulence and resistance.…”

Cellular Response of the Amoeba Acanthamoeba castellanii to Chlorine, Chlorine Dioxide, and Monochloramine Treatments

“…Some of them, such as Legionella pneumophila, are even able to multiply within amoeba trophozoites (23,29) and could be found within amoeba cysts (14,27). Intracellular L. pneumophila is more resistant to biocides (3,4,14). Consequently, free-living amoebae, such as Acanthamoeba, have been recently described as important targets in water treatment (19,30).…”

Effects of Oakmoss and its Components on <i>Acanthamoeba</i><i> castellanii</i> ATCC 30234 and the Uptake of <i>Legionella pneu</i><i>mophila</i> JCM 7571 (ATCC 33152) into <i><i>A. castellanii</i></i>

“…Therefore, it is very important to inhibit each stage of the L. pneumophila life cycle. In addition, Barker et al 1992 and1995 have reported that L. pneumophila grown in A. polyphage was less sensitive than L. pneumophila from cultures in broth to disinfectants and antibiotics because these bacteria undergo phenotype modifications upon intracellular growth.…”

“…in natural environments Greub and Raoult, 2004Molmeret et al, 2005Rowbotham, 1980Winiecka-Krusnell and Linder, 2001 . In addition, L. pneumophila co-cultured with amoeba is less sensitive to typical chlorine disinfectants Barker et al, 1992Dupuy et al, 2011García et al, 2007Kim et al, 2002 and L. pneumophila multiplied in amoebae possess greater resistance to disinfectants and antibacterial agents than L. pneumophila from cultures in broth Bandyopadhyay et al, 2004Barker et al, 1995 . L. pneumophila in biofilms is also resistant to stressful environmental conditions and the actions of antibacterial agents Cooper and Hanlon, 2010Kim et al, 2002Wright et al, 1991 .…”