The data presented in this study showed that there are medium- and temperature-dependent growth differences in vitro between diverse isolates of the Bartonella genus. We compared three culturing techniques, the first using mammalian cell culture-based conditions with and without the presence of Vero E6 cells (M10-V and M10, respectively), the second using insect cell culture-based medium (S10), and the third using a combination of the mammalian and insect cell culture-based medium with and without Vero E6 cells (MS10-V and MS10, respectively).
Our data showed that the combination media, MS10 and MS10-V, were able to support the growth of all four Bartonella spp. well at both 35°C and 37°C. These growth curves were comparable to the curve with S10 medium for all cases except B. tamiae at 37°C, where by the end of the time course at day 9 the viability of the bacteria in S10 medium declined to a significantly lower level than that under the conditions with MS10 and MS10-V media.
Under the conditions with MS10 and MS10-V media, our data suggest that the presence of Vero E6 cells did not significantly affect the growth or sustainability of the bacterial cultures, suggesting that cell culture may not be necessary for culture of these bacterial species. However, due to the high bacterial loads that were reached for these growth curves, it is difficult to interpret this to be true, as monolayers had lifted off at between 5 and 7 days in most cases (data not shown). B. quintana
was the only strain for which the presence of mammalian cells in the culture (M10-V medium) was a significant benefit to growth compared to the growth achieved by culture under the same conditions but with cell-free (M10) medium. These results agree with those from previous work suggesting that under mammalian cell culture-based conditions, cell coculture is important for B. quintana
). Recent developments, however, have shown that cell culture is unnecessary when insect cell culture-based medium is used (28
). In the near future, our laboratory will be comparing the medium conditions described in this study with biological samples to more accurately test if cell coculture is necessary upon initial isolation of Bartonella
species that have not been adapted to laboratory conditions. For this reason, we see the ability of the MS10 medium to sustain mammalian cell culture as a benefit if coculture becomes necessary. Our laboratory also tested the combination MS10 medium with other cell lines and found that it was able to adequately support Huh-7 (human hepatocyte) and THP-1 (human monocyte) cell lines (microscopic observations; data not shown).
Vero E6 cells were chosen for this study as there is evidence in the literature that both B. henselae
and B. quintana
are able to adhere to different cell lines in vitro
, including monocytes, endothelial cells, and epithelial cells from various mammalian hosts (2
). Additionally, Vero E6 cells were used to successfully isolate B. tamiae
for the first time (24
). We acknowledge that endothelial cells are typically chosen for culturing Bartonella
species; however, they may not be necessary when the medium composition is changed, as shown in the present work with the use of combination medium or by replacement with insect cell culture-based medium. As vector-borne bacteria, Bartonella
species are required to survive in various environments. Clinically, Bartonella
species have been shown to cause a variety of clinical manifestations, such as bacteremia, endocarditis, bacillary angiomatosis, and peliosis, where the bacteria interact with multiple cell types within the same host (6
). Endothelial cells such as human umbilical vein endothelial cells provide an excellent in vitro
model with which to study the pathogenesis of Bartonella
, as there is evidence that endothelial cells are invaded in vivo
); however, for the purpose of a culturing protocol to isolate Bartonella
species from biological material, endothelial cells may not be optimal, as many of the cell lines require a higher passaging frequency and extra growth factors, which increase the cost substantially.
The bacterial strains for these experiments were used at the lowest passage possible, as B. henselae
has been shown to undergo phase variation after multiple laboratory passages and shows decreased autoagglutination in association with decreased cell adherence (2
). Immunofluorescence was used to assess the presence of the autoagglutination phenotype and also to examine if there were changes associated with temperature or medium differences under our test conditions (summarized in ). B. quintana
was the only strain that did not show agglutination under any condition. We still, however, observed medium- and cell-dependent differences in growth () which are similar to those found in the literature (22
). Specifically, we found that coculture with mammalian cells under mammalian cell culture-based conditions (M10-V medium) was required for growth; however, when insect cell culture-based medium is incorporated (S10 or MS10 medium), B. quintana
was able to grow in cell-free medium. Autoagglutination is associated with expression of the TAA proteins (26
). Although medium-dependent differences were seen with B. tamiae
and B. henselae
, it is unclear whether the agglutination phenotype correlates with growth or viability of the culture. It would be interesting to further investigate if there are changes in transcriptional regulation or if genes associated with host adaptability or pathogenicity are lost under these various culture conditions and also over a broader range of temperatures that includes lower temperatures more relevant to a vector host.
There is a discrepancy in the literature concerning the temperature for culturing Bartonella
species. The majority of studies that coculture Bartonella
species with mammalian cells use 37°C; in contrast, studies that use axenic insect cell culture-based medium and agar use a culture temperature of 35°C (15
). Although a 2°C difference may seem minor or trivial, it was interesting to see that there were statistically significant differences in peak growth between the two temperatures for three of the four strains tested (B. elizabethae
was the exception, showing no significant difference in peak growth between the two temperatures). Additionally, the drastic divergence in growth between these temperatures for B. tamiae
was surprising, as this strain was isolated from a febrile human patient. We are very interested to continue this investigation, as the temperature-dependent growth requirements may provide new insight into the life cycle and biological niche of this microorganism. This information is important to know because these two temperatures are very commonly used in the clinical laboratory setting and may sometimes be used interchangeably. Additionally, because of the minor difference in temperature, incubator settings should be calibrated properly, as the temperatures can fluctuate and may impact the growth of some Bartonella
The goals of this study were to compare key components of common Bartonella culturing methodologies, as well as combine them to investigate the best conditions under which to culture diverse Bartonella species of clinical importance. We have shown that by combining the mammalian and insect cell culture-based media (MS10 medium) we were able to achieve peak growth comparable to that achieved with the insect cell culture-based medium alone (S10 medium) at 35°C but that at 37°C the MS10 medium performed significantly better for both B. tamiae and B. quintana. Our data suggest that mammalian cell coculture is not necessary for growth of laboratory-adapted Bartonella strains; however, the combination medium is still able to support mammalian cell culture, which may remain an important variable during initial isolation of Bartonella from biological samples. Our data also show that B. tamiae has temperature-dependent growth requirements that are not adequately met with the mammalian or insect cell culture-based medium alone at 37°C, regardless of mammalian cell coculture.
This is the first study, to our knowledge, that has compared these methodologies using diverse Bartonella spp. These findings are important for clinical microbiologists, as Bartonella species are fastidious organisms and Bartonella is a diverse genus whose members are a growing concern as emerging or reemerging pathogens.