Low-dose irradiation affects the functional behavior of oral microbiota in the context of mucositis

Abstract

The role of host-microbe interactions in the pathobiology of oral mucositis is still unclear; therefore, this study aimed to unravel the effect of irradiation on behavioral characteristics of oral microbial species in the context of mucositis. Using various experimental in vitro setups, the effects of irradiation on growth and biofilm formation of two Candida spp., Streptococcus salivarius and Klebsiella oxytoca in different culture conditions were evaluated. Irradiation did not affect growth of planktonic cells, but reduced the number of K. oxytoca cells in newly formed biofilms cultured in static conditions. Biofilm formation of K. oxytoca and Candida glabrata was affected by irradiation and depended on the culturing conditions. In the presence of mucins, these effects were lost, indicating the protective nature of mucins. Furthermore, the Galleria melonella model was used to study effects on microbial virulence. Irradiated K. oxytoca microbes were more virulent in G. melonella larvae compared to the nonirradiated ones. Our data indicate that low-dose irradiation can have an impact on functional characteristics of microbial species. Screening for pathogens like K. oxytoca in the context of mucosits could be useful to allow early detection and immediate intervention.

Keywords: Galleria melonella; Irradiation; biofilm; mucositis; oral microbiota; virulence.

Figure 1

Growth curves of irradiated (10 Gy) and nonirradiated C. albicans, C. glabrata, K. oxytoca and S. salivarius planktonic cells cultured in different media (broth, DMEM, or DMEM + mucins). Vertical axis represents the OD of the cells measured at 600 nm using a spectrophotometer. Data of one experiment using two parallel cultures of each species are shown

Figure 2

Box and whiskers plot presentation of biofilms of irradiated (10 Gy) and nonirradiated C. albicans, C. glabrata, K. oxytoca, and S. salivarius cells formed during 24 h of culture in different media (broth, DMEM, or DMEM + mucins). Biofilms were generated both in static (24-well plates, first column) and dynamic conditions (24-well plates, second column; tubes, third column). Vertical axis represents the OD of dissolved crystal violet molecules measured at 540 nm using a spectrophotometer. Boxes and whiskers show median values and interquartile range of scores. Pooled data of at least four repetitions are shown. *P < 0.05

Figure 3

SYTO9-positive cells present in irradiated and nonirradiated biofilms after 24 h in different culture media. Biofilms were generated both in static and dynamic conditions (24-well plates with or without shaking). Vertical axis represents the SYTO9-positivity assessed by measurement of fluorescence intensity at Ex480/Em500 nm. Bars represent the means including the standard deviation. Pooled data of at least three repetitions are shown. *P < 0.05

Figure 4

Metabolic activity of irradiated and nonirradiated biofilm cells of K. oxytoca after 24 h of culturing in different media (without agitation) as determined by measuring the OD at 490 nm of the dissolved formazan crystals (vertical axis). Bars represent the means including the standard deviation. Data of one representative experiment are shown (three replicates)

Figure 5

Calcofluor staining of biofilms of irradiated (10 Gy) and nonirradiated C. albicans and S. salivarius formed within 24 h in static (white bars) or dynamic conditions (black bars) in 24-well plates and in presence of the three test media. Vertical axis represents the calcofluor fluorescence intensity at Ex355/Em430 nm. Bars represent the means including the standard deviation. Pooled data of two repetitions are shown. *P < 0.05

Figure 6

Survival curves of Galleria melonella after inoculation with K. oxytoca (a) or S. salivarius (b), plotted using the Kaplan–Meyer method. For experiments with K. oxytoca, two independent tests were performed and pooled results are presented. Test groups were PBS controls (n = 20), nonirradiated (0 Gy; n = 21) and irradiated (10 Gy; n = 21). Vertical axis represents the cumulative survival probability of the wax larvae during four days postinjection. For experiments with S. salivarius, three independent tests were performed and pooled results are presented. Test groups were PBS controls (n = 27), nonirradiated (0 Gy; n = 27) and irradiated (10 Gy; n = 27). Vertical axis represents the cumulative survival probability of the wax larvae during four days postinjection. Differences in survival were calculated using the log rank test. *P < 0.05