Aminoglycosides Affect IntracellularSalmonella entericaSerovars Typhimurium and Virchow

Abstract

The high antibacterial activity and selectivity of aminoglycosides and their low activity against intracellular bacteria associated with eukaryotic cells make them the antibiotics of choice for the elimination of extracellular bacteria during intracellular studies. Given the evidence that aminoglycosides can penetrate the eukaryotic cell membrane, the goal of this study was to examine the influence of aminoglycosides on macrophage-associated Salmonella. Herein, we show that gentamicin, kanamycin, and tobramycin at concentrations between 15 to 150 g ml ؊1 do not kill intracellular Salmonella but have other effects on the bacterial physiology. By using Salmonella enterica serovars Typhimurium and Virchow harboring luciferase reporter plasmid, we observed that the light produced by intracellular Salmonella declined immediately upon exposure to aminoglycosides, indicating that the bacteria were under stress. The extent of this effect was dependent on the macrophage host, on the identity of the aminoglycoside and its concentration, on the exposure time, and on the Salmonella serovar. Salmonella associated with Nramp1-negative macrophages, in which the phagosomal pH is higher, were more susceptible to aminoglycosides than Salmonella associated with Nramp1-expressing macrophages. These results verify that aminoglycosides affect intracellular bacteria and that the extent of this effect is dependent on the acidity level within the phagosome, suggesting that for the study of intracellular bacteria, the aminoglycoside concentration should be limited to two to five times the MIC for the bacterial strain studied. This precaution should guarantee the complete execution of extracellular bacteria with minimal effects on the intracellular bacteria and the host cells.

Intracellular Listeria monocytogenes Comprises a Minimal but Vital Fraction of the Intestinal Burden following Foodborne Infection

“…Although this is a widely used technique in the field of bacterial pathogenesis, one must be cautious in interpreting data from gentamicin protection assays, since excessively high concentrations of gentamicin can kill or stress intracellular bacteria, possibly due to the pinocytosis of extracellular fluid containing antibiotics (30)(31)(32). As we showed here, even treatment with 25 g/ml gentamicin in vitro slightly underestimated the proportion of intracellular organisms in the MLN compared to using only 10 g/ml in tissue culture medium.…”

Construction of Aminoglycoside-Sensitive Burkholderia cenocepacia Strains for Use in Studies of Intracellular Bacteria with the Gentamicin Protection Assay

“…To overcome this limitation, several laboratories have modified this assay and utilized a combination of ceftazidime and gentamicin at very high concentrations ranging from 500 to 1,000 g ml Ϫ1 for ceftazidime and 250 to 500 g ml Ϫ1 for gentamicin to eliminate extracellular bacteria (6,47). These modifications are not ideal since aminoglycosides can penetrate eukaryotic cells by pinocytosis and may accumulate in phagolysosomes, causing bacterial cell death (19) or rapidly affecting the normal physiology of intracellular bacteria by causing bacterial stress (41). These effects depend on the vacuolar pH and the aminoglycoside concentration as the bacterial sensitivity to aminoglycosides decreases markedly at acidic pH (41).…”

“…These modifications are not ideal since aminoglycosides can penetrate eukaryotic cells by pinocytosis and may accumulate in phagolysosomes, causing bacterial cell death (19) or rapidly affecting the normal physiology of intracellular bacteria by causing bacterial stress (41). These effects depend on the vacuolar pH and the aminoglycoside concentration as the bacterial sensitivity to aminoglycosides decreases markedly at acidic pH (41). These observations are relevant to the intracellular survival of B. cenocepacia in macrophages, which occurs in a bacteria-containing vacuole that delays acidification and maintains a relatively high pH for several hours postinfection (29).…”

Interaction of Burkholderia pseudomallei and Burkholderia thailandensis with human monocyte-derived dendritic cells

“…Our use of the b-lactam, imipenem, is derived from reports in the literature that suggest that kanamycin is able to penetrate eukaryotic cells and may interfere with either host cell function or even intracellular bacterial replication, a situation that is potentially exacerbated by the high levels of kanamycin required to inhibit growth of B. pseudomallei (Menashe et al, 2008). Meanwhile, imipenem is lipid insoluble and penetrates cells poorly if at all (Menashe et al, 2008;Murdoch & Peterson, 1991;Sahly et al, 2008). Numerous other studies have used imipenem (Boddey et al, 2007;Brown et al, 2004;MacDonald & Speert, 2008) rather than kanamycin.…”