Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis

Abstract

The study of intracellular bacterial pathogens in cell culture hinges on inhibiting extracellular growth of the bacteria in cell culture media. Aminoglycosides, like gentamicin, were originally thought to poorly penetrate eukaryotic cells, and thus, while inhibiting extracellular bacteria, these antibiotics had limited effect on inhibiting the growth of intracellular bacteria. This property led to the development of the antibiotic protection assay to study intracellular pathogens in vitro. More recent studies have demonstrated that aminoglycosides slowly penetrate eukaryotic cells and can even reach intracellular concentrations that inhibit intracellular bacteria. Therefore, important considerations, such as antibiotic concentration, incubation time, and cell type need to be made when designing the antibiotic protection assay to avoid potential false positive/negative observations. Yersinia pestis, which causes the human disease known as the plague, is a facultative intracellular pathogen that can infect and replicate in macrophages. Y. pestis is sensitive to gentamicin and this antibiotic is often employed in the antibiotic protection assay to study the Y. pestis intracellular life cycle. However, a large variety of gentamicin concentrations and incubation periods have been reported in the Y. pestis literature without a clear characterization of the potential influences that variations in the gentamicin protection assay could have on intracellular growth of this pathogen. This raised concerns that variations in the gentamicin protection assay could influence phenotypes and reproducibility of data. To provide a better understanding of the potential consequences that variations in the gentamicin protection assay could have on Y. pestis, we systematically examined the impact of multiple variables of the gentamicin protection assay on Y. pestis intracellular survival in macrophages. We found that prolonged incubation periods with low concentrations of gentamicin, or short incubation periods with higher concentrations of the antibiotic, have a dramatic impact on intracellular growth. Furthermore, the degree of sensitivity of intracellular Y. pestis to gentamicin was also cell type dependent. These data highlight the importance to empirically establish cell type specific gentamicin protection assays to avoid potential artificial data in Y. pestis intracellular studies.

Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci

“…S3 and S5) suggests the possibility of intracellular S. aureus being killed by the internalized gentamicin. Consistent with our results, the possibility of gentamicin being internalized into host cells during the GPA was questioned previously (13,(17)(18)(19)(20). Therefore, we hypothesized that the effect of internalized gentamicin on intracellular bacteria might be the main cause for the discrepancy between the EPA and the GPA.…”

“…The GPA relies on the ability of gentamicin to kill all extracellular and membrane-bound bacteria and is also based on the assumption of the inability of gentamicin to penetrate eukaryotic cells (16). However, many reports suspected that higher concentrations of gentamicin for long incubation times possibly cause the nonspecific killing of intracellular bacteria (13,(17)(18)(19)(20), presumably by internalized gentamicin through pinocytosis (21). For this reason, the results of the GPA often exhibit significant variation (22).…”

Staphylococcus aureus Uses the GraXRS Regulatory System To Sense and Adapt to the Acidified Phagolysosome in Macrophages

“…Presumably, the discrepancy between previous studies and the data we present here, at least in terms of the role that Agr plays in intracellular survival of S. aureus , is due to strain variation, use of distinct cells and or cell lines, and methodological differences, namely, the prolonged use of antibiotics. The notion that aminoglycoside antibiotics such as gentamicin, which are commonly used for in vitro infection assays, do not enter host cells is not accurate and is beginning to recede ( 15 , 47 – 49 ). Nevertheless, several studies to date investigating macrophage infection by S. aureus have employed prolonged antibiotic treatment, which will have undoubtedly led, in some instances, to the characterization of intracellular antibiotic-treated bacteria.…”

Escalating Threat Levels of Bacterial Infection Can Be Discriminated by Distinct MAPK and NF-κB Signaling Dynamics in Single Host Cells

“…While major efforts are underway to understand and develop treatments to combat the growing threat of antimicrobial resistance, such efforts will be stymied by our inability to reprogram or mobilize the response of host cells in a controlled way during infection. Expanding our molecular knowledge of cellular responses during infection to include temporal patterns of signaling will be instrumental in developing a more comprehensive map of cellular decision making and will reveal new opportunities for VanCleave et al, 2017). To determine whether gentamicin could also influence signaling dynamics, for instance via the release of PAMPs from dead bacteria, we compared JNK and NF-kB signaling responses after exposure to S. typhimurium MOI 2.5 in the presence or absence of gentamicin over a period of 6 hours ( Figure S9).…”