Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi

Abstract

Background: Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families.

Methodology: A clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells.

Key results: Development of a standardised growth assay to quantify bacterial replication and viability in vitro. Quantitative comparison of different DNA extraction methods. Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement. Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds. Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing.

Conclusions: Here we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.

Fig 1. Panel A. A typical growth curve. An arrow indicates day 7, the time point used in subsequent experiments. Panel B. Inoculating dose ratio.

This figure shows the bacterial count after 7 days, from different relative amounts of bacteria added at day 0. A boiled sample was included, which was added at 1x relative amount.

Fig 2. Optimising quantification of O. tsutsugamushi grown in cultured mammalian cells.

Panel A. Levels of bacterial DNA as determined by qPCR, after releasing host cells from tissue culture flasks by different methods. Panel B. A comparison of DNA extraction methods. The graph shows the amount of O. tsutsugamushi-specific DNA detected by qPCR, and the table below shows the total concentration by qPCR or nanodrop analysis, the 260/280 absorption ratio and the 280/230 absorption ratio. Numbers in brackets indicate expected values for pure DNA. Panel C. Levels of bacterial DNA after boiling for different periods of time using the alkaline lysis DNA extraction method.

Fig 3. Optimising inoculation conditions for growth of O. tsutsugamushi in cultured L929 cells.

Panel A. The growth of O. tsutsugamushi after infecting host cells in an adherent or trypsinised state. Panel B. The growth of O. tsutsugamushi after infecting host cells at different levels of confluence.

Fig 4. Optimising media conditions for growth of O. tsutsugamushi in cultured L929 cells.

Panel A. The relationship between bacterial growth and FBS concentration. Panel B. The relationship between host cell (L929) growth and FBS concentration. Panel C. The growth of O. tsutsugamushi in RPMI or DMEM growth media. Panel D. The growth of O. tsutsugamushi in the presence of varying levels of daunorubicin. Panel E. The growth of L929 cells in the presence of varying levels of daunorubicin. Panel F. The growth of O. tsutsugamushi in the presence of different antibiotics. Antibiotic were used at the following concentrations: chloramphenicol (cam) 150 μg/ml, penicillin G (pen) 100 μg/ml (alone), penicillin G 125 μg/ml + streptomycin (strep) 200 μg/ml (combined).

Fig 5. Optimising lysis of L929 host cells infected with O. tsutsugamushi.

Panel A. Confocal fluorescence microscopy images showing the effect on host and bacterial cells of different lysis methods. Blue = nuclei (DAPI), red = host cells (Evans blue) and green = bacteria (Alexafluor 488-labelled antibody). Scale bar = 40 μm. Panel B. The effect of different host cell lysis methods on subsequent growth of bacteria. Panel C. The effect of purification temperature on subsequent growth of bacteria. Panel D. The yield of bacteria after different stages of purification. Panel E. the pelleting of bacteria after centrifugation at different g-forces. All samples were spun for 3 mins. Panel F. The effect of different centrifugation speeds on subsequent bacterial growth.

Fig 6. Quantifying the effect of short-term storage on bacterial viability.

Panel A. Growth of O. tsutsugamushi after storage at room temperature for 30 min or 120 min in a range of different buffers. Panel B. Growth of O. tsutsugamushi after storage for 1 day or 7 days in lysed or intact host cells. In this experiment bacteria were stored in the growth media in which they were previously grown.

Fig 7. Optimising freezing conditions for preserving bacterial viability.

Panel A. Comparing the growth of O. tsutsugamushi after being frozen in lysed or intact host cells, using SPG media. Panel B. Growth of bacteria after freezing in intact host cells in different buffers. Panel C. A comparison of different freezing buffers on the subsequent growth of purified bacteria and Panel D. Growth of O. tsutsugamushi after being frozen in SPG media at different freezing and thawing speeds. FF = fast freeze, SF = slow freeze, FT = fast thaw and ST = slow thaw. Panel E. Growth of O. tsutsugamushi after being frozen and stored for 40 mins or 1 week at-80°C. Bacteria were purified and stored in SPG buffer.