Effects of lipid emulsions on the formation of Escherichia coli–Candida albicans mixed-species biofilms on PVC

Patients receiving lipid emulsions are at increased risk of contracting catheter-related bloodstream infections (CRBSIs) in the clinic. More than 15% of CRBSIs are polymicrobial. The objective of this study was to explore the effects of lipid emulsions on the formation of Escherichia coli (E. coli)–Candida albicans (C. albicans) mixed-species biofilms (BFs) on polyvinyl chloride (PVC) surfaces and the underlying mechanism. Mixed-species BFs were produced by coculturing E. coli and C. albicans with PVC in various concentrations of lipid emulsions. Crystal violet staining and XTT assays were performed to test the mixed-species BF biomass and the viability of microbes in the BFs. The microstructures of the BFs were observed by an approach that combined confocal laser scanning microscopy, fluorescence in situ hybridization, and scanning electron microscopy. The study found that lipid emulsions could promote the formation of E. coli–C. albicans mixed-species BFs, especially with 10% lipid emulsions. The mechanism by which lipid emulsions promote mixed-species BF formation may involve significant upregulation of the expression of the flhDC, iha, HTA1, and HWP1 genes, which are associated with bacterial motility, adhesion, and BF formation. The results derived from this study necessitate strict aseptic precautions when handling lipid emulsions and avoiding the use of high concentrations of lipid emulsions for as long as possible.


Effects of lipid emulsions on microbial viability in mixed-species BFs. After 4 h, 12 h, 24 h, 48 h,
and 72 h of coculture, the viability of microbes in mixed-species BFs on the surfaces of PVC pieces was examined by using XTT in each group. There were significant differences between the lipid emulsion groups with various concentrations and the control group at 12-72 h (P < 0.05). The 10% lipid emulsion group showed the most significant effect of all groups, followed by the 15% and 20% lipid emulsion groups. The results showed that the microbial viability of each group increased with incubation time and was highest at 48 h (P < 0.001) (Fig. 2a,b).

Thickness and live/dead microbes of mixed-species BFs.
The formation of mixed-species BFs on the surfaces of PVC pieces was found to be a dynamic process. The thickness of the BFs in each group increased rapidly in 24 h of coculture and peaked at 48 h. There was a slight decline at 48-72 h in each group. The mixedspecies BFs of the lipid emulsion groups were more complex and denser than those of the control group. The thickness of mixed-species BFs in the 10%, 15%, and 20% lipid emulsion groups was thicker than that in the control group at 24 h, 48 h, and 72 h (P < 0.05) (Fig. 3a).
Both live and dead microbes in the mixed-species BFs were observed by confocal laser scanning microscopy (CLSM). The live microbes were stained green by SYTO9, whereas the dead microbes were stained red by propidium iodide (PI). At 24 h, there were only small proportions of dead microbes in the mixed-species BFs in each group. At 48-72 h, the proportions of live microbes decreased, and the proportions of dead microbes increased gradually from the outer layers to the inner layers of the mixed-species BFs (Fig. 3b,c).  Figure 1. Effect of lipid emulsions on microbial adhesion and mixed-species BF biomass. Compared with the control group at the same time, *P < 0.05, **P < 0.01, ***P < 0.001 (a). Compared with the 4 h at the same concentration, *P < 0.05, **P < 0.01, ***P < 0.001 (b). www.nature.com/scientificreports/

Composition of mixed-species BFs detected by fluorescence in situ hybridization (FISH). At
24 h, E. coli and C. albicans grew together in the BFs and formed clumps in the control group. The structure of mixed-species BFs in the lipid emulsion groups was more complex than that in the control group. At 72 h, the BFs were denser and more robust than those at 24 h in each group. After exposure to the 10%, 15%, and 20% lipid emulsions for 72 h, E. coli grew around the overlapping and interlaced C. albicans, and the mixed-species BFs looked like a "Christmas tree forest". The "Christmas tree forest"-like appearance indicates the formation of strong BFs. At 24 h and 72 h, in the control group and 5% lipid emulsion group, the proportions of C. albicans were greater than those of E. coli in the mixed-species BFs. The proportions of these two microbes in the mixedspecies BFs were similar in the 10% lipid emulsion group. When the concentration of lipid emulsions exceeded 15%, E. coli was obviously dominant in the mixed-species BFs (Fig. 4).

Microstructure of mixed-species BFs detected by scanning electron microscopy (SEM). It was
found that E. coli adhered to the chlamydospores, pseudohyphae, and mycelia of C. albicans. At 72 h, the BFs in each group were more complex and denser than those at 24 h, forming a three-dimensional network structure. The lipid emulsions remained a part of the mixed-species BFs and were attached to the surfaces of bacteria, mycelia and chlamydospores, forming more complex and mature BFs, especially in the 10%, 15%, and 20% lipid emulsion groups. With increasing lipid emulsion concentrations, an increasing amount of E. coli was found in the mixed-species BFs. C. albicans was closely surrounded by E. coli. The growth of C. albicans was inhibited when the concentration of lipid emulsions exceeded 15%. There was a significant decrease in the capacity of C. albicans to undergo the yeast-to-hypha switch after exposure to 10%, 15%, and 20% lipid emulsions for 72 h (Fig. 5).
Gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Specific differences were noted in the expression levels of various genes between the 10% lipid emulsion group and the control group at various time points. After 24 h of coculture, the expression levels of the flhDC, iha, HTA1, and HWP1 genes were upregulated in the 10% lipid emulsion group compared with those in the control group. The upregulation of the iha gene was the most obvious (P < 0.05) (Fig. 6a). The expression of the flhDC and HWP1 genes decreased after 48 h of coculture. The expression of the iha gene, which exhibited slight upregulation in the 10% lipid emulsion group after 48 h of coculture, had a similar pattern as that of the HTA1 gene (P < 0.05) (Fig. 6b). After 72 h of coculture, the expression levels of these four genes were downregulated in the 10% lipid emulsion group compared with the control group (P < 0.05) (Fig. 6c).

Discussion
In tumour patients with immune dysfunction and patients who consume glucocorticoids for long time periods, the BFs formed are usually a mixture of bacteria and fungi. E. coli and C. albicans are often coisolated in cases of BF-associated infections 18,19 . Microbes reside in mixed-species BFs, in which organism growth and metabolism are different from those in monospecies BFs 20,21 . Increased quantity and diversity of the species present increase the complexity of mixed-species BFs and enhance antimicrobial resistance 22,23 . The treatment of mixed-species BFs is more difficult and challenging than that of monospecies BFs. Therefore, research on factors influencing mixed-species BF formation has great clinical significance in reducing CRBSIs induced by lipid emulsions. Our experiments uniquely illustrated differences among various concentrations of lipid emulsions with regard to their effects on E. coli-C. albicans mixed-species BF formation. These data have the potential to impact the treatment of patients receiving PN. In our research, PVC was used as the CVC material, and an E. coli-C. albicans mixture was used as the source of opportunistic pathogen coinfections. TSB medium was used as a supplement for various concentrations of lipid emulsions because it could support both E. coli and C. albicans 24 . The results of CLSM, FISH, and SEM showed that E. coli and C. albicans could coexist in TSB medium or lipid emulsions of various concentrations www.nature.com/scientificreports/ and develop mixed-species BFs on the surface of PVC. Mixed-species BFs were the main cause of coinfection with E. coli-C. albicans via implanted biomaterials. The results of crystal violet (CV) and XTT assays, and the thickness of the mixed-species BFs showed that microbial adhesion, viability, and mixed-species BF formation were promoted by lipid emulsions, and 10% was the optimum lipid emulsion concentration among the four preset concentrations for the growth and formation of E. coli-C. albicans mixed-species BFs.
In addition, we used live/dead BacLight bacterial staining and CLSM to visualize the internal structure of mixed-species BFs. This was proven to be a reliable technique for the evaluation of microbial viability in BFs 25,26 .   www.nature.com/scientificreports/ The images revealed that the proportions of live pathogens declined gradually from the outer to inner layers due to the relatively hypoxic and nutrient-deficient environment. Dead pathogens were abundantly located at the bottom and inside of the mixed-species BFs. This was consistent with previous research 10,27 . Nucleic acids released into the extracellular environment by dead pathogens have been demonstrated to contribute to the formation and structural stability of BFs and provide abundant nutrients for live pathogens to support the sustainable growth of BFs 28 . This result suggested that over time, the number of dead cells at the bottom of the BFs increased, and the structure of the BFs became more stable and robust. Furthermore, by SEM and FISH, we observed a novel phenomenon that wherein E. coli was predominant in the mixed-species BFs after exposure to the 15% and 20% lipid emulsions for a long time, and the growth of C. albicans was inhibited and transformed from the hypha phase to the yeast phase. However, these results did not conclusively indicate that mixed-species BF formation was impaired when the concentration of the lipid emulsions exceeded 15% because we also observed the "Christmas tree" structure representing strong vitality in these groups. Swindell et al. pointed out that 10% Intralipid was conducive to the germination of C. albicans chlamydospores and the growth of mycelia, which was not entirely consistent with our experimental results 16 . The apparent differences between these two studies might be attributed to the BF species and different types of lipid emulsions. However, our findings are supported by other studies. These studies have proven that C. albicans has a unique sensitivity to medium-chain fatty acids with carbon chain lengths of C8, C10, and C12, represented by caprylic, capric, and lauric acid, respectively 29,30 . The abovementioned medium-chain fatty acids have been demonstrated to have antifungal effects 31,32 . The lipid emulsions used in our experiments contained mediumand long-chain fatty acids. The carbon chain lengths were C6-24. Moreover, the interactions between species are very complex, ranging from cooperation to competition and even predation 33,34 . E. coli can significantly influence candidal growth and induce hyphal death 35 . Interestingly, a study by Cabral et al. demonstrated that C. albicans could be killed by coculturing with E. coli in vitro and asserted that a soluble factor secreted by E. coli resulted in this effect 36 . However, other previous studies showed that E. coli could contribute to the colonization and invasiveness and the damaging effects of C. albicans 37,38 . These studies may help to explain the differences in the composition and architecture of mixed-species BFs grown in various concentrations of lipid emulsions. The interactions between species and the complex structure of mixed-species BFs can explain why infections caused by mixed-species BFs associated with biomaterials are extremely difficult to treat.
To better elucidate the mechanism underlying E. coli-C. albicans mixed-species BF formation enhanced by lipid emulsions, flhDC and iha gene expression in E. coli was measured. flhDC mainly regulates the biosynthesis of flagella and increases the motility and ability to colonize biomaterials 39,40 . The iha gene is the iron-regulated gene A homologue adhesin. The gene encodes a bacterial outer-membrane protein associated with the adhesion of bacteria 41 . In the E. coli strains causing primitive acute pyelonephritis, iha was associated with higher BF biomass formation 42 . Based on the qRT-PCR results and the functions of flhDC and iha, the current study confirmed a potential role of 10% lipid emulsions in promoting the movement and colonization of E. coli at the early stage of BF formation. After the initial adhesion was completed, the aggregation capacity of E. coli was enhanced by lipid emulsions during the middle stage of BF formation. In addition, HWP1 and HTA1 gene expression in C. albicans was measured. HWP1 encodes a fungal cell wall protein required for hyphal development and yeast adhesion to epithelial cells 43 . HWP1 is directly associated with BF formation in C. albicans. HTA1 encodes histone H2A of C. albicans and reflects the growth and reproductive ability of C. albicans cells to a certain extent 44 . The present study showed that HWP1 and HTA1 were expressed at a higher level in the 10% lipid emulsion group than in the control group at 24 h, indicating that lipid emulsions further the growth and hypha formation in C. albicans only at the early stage of BF formation. This result showed high concordance with SEM results. After 72 h of coculture, the four genes were downregulated in the 10% lipid emulsion group which might be related to nutrient consumption from the medium or most of the microbes in the mature mixed-species BFs entering the dormant stage.
In summary, the effects of lipid emulsions on E. coli-C. albicans mixed-species BFs were reported for the first time in our study. We demonstrated that lipid emulsions significantly stimulated E. coli-C. albicans mixedspecies BF formation by upregulating the expression of genes related to BF formation. Avoiding the use of high concentrations of lipid emulsions for as long as possible may be helpful in reducing CRBSIs during PN. Finally, further research on the possible mechanism by which lipid emulsions promote the formation of mixed-species BFs is needed to overcome microbial infections.

Microbes, reagents, and equipment. E. coli (MC1000) was a gift from the Yale Coli Genetic Stock
Center. C. albicans (ATCC10231) was purchased from the Institute of Microbiology, Chinese Academy of Sciences. PVC was purchased from Guangdong Kewei (China) Co., Ltd., processed into pieces with sizes of 5 mm × 5 mm or 8 mm × 8 mm, and sterilized by 24 h of formaldehyde fumigation for further experiments. Lipid emulsions (C6-24) were obtained from Fresenius Kabi (China). The TSB medium and XTT bacterial proliferation and cytotoxicity kit were supplied by Huankai (China) Microbial Technology Co., Ltd. The total RNA extraction kit was supplied by Tiangen Biotech (China) Co., Ltd. Primers were synthesized by Sangon Biotech (China) Co., Ltd. The cDNA synthesis kit was procured from Bio-Rad (USA). The FISH kit was purchased from Boxin Biology (China) Co., Ltd. The live/dead BacLight bacterial viability kit was obtained from Life (USA). The S-3000N scanning electron microscope was from Hitachi (Japan), and the FV1000 confocal laser scanning microscope was from Olympus (Japan). qRT-PCR assays were conducted using SuperReal PreMix Plus supplied by Tiangen Biotech (China) and were performed on an ABI 7500 PCR system (USA). www.nature.com/scientificreports/ Culture of microbes and experimental grouping. Standard strains of C. albicans and E. coli were inoculated on Sarpaul agar plates and MH agar plates, respectively, and incubated at 37 °C for 24 h. Subsequently, a single colony picked from the Sarpaul agar plate or MH agar plate with an inoculation ring was individually inoculated into a test tube containing 5 mL of TSB medium. These tubes were incubated in a constant-humidity oscillator at 37 °C and 200 r/min for 16-18 h. After the cells grew to the logarithmic growth phase, the concentration of the microbial solution in each tube was adjusted to 1.1 × 10 7 cells/mL using TSB medium in an ultraviolet spectrophotometer for later use. The mixed microbial solution in the study was prepared in a 1:1 ratio; that is, 2 mL of microbial solution was prepared by mixing 1 mL of C. albicans yeast solution and 1 mL of E. coli bacterial solution. The experiment included five groups: a control group and four groups with various concentrations of lipid emulsions (5%, 10%, 15%, or 20%). The TSB medium was mixed with the 20% lipid emulsions in various proportions to modulate the lipid emulsion concentration to 5%, 10%, 15%, or 20%. The mixed microbial solution with PVC pieces was treated with the above concentrations of lipid emulsions. As a control, samples were prepared similarly with TSB medium. All groups were incubated in a thermostatic incubator at 37 °C with 5% CO 2 .

Detection of microbial adhesion and BF biomass by crystal violet (CV) staining.
Then, 100 µL of the previously prepared lipid emulsions of various concentrations or TSB medium was added to a 96-well cell culture plate, and 10 µL of the mixed microbial solution and a 5 mm × 5 mm PVC piece were added into each well. Six wells were inoculated in each group in each plate. Plates were incubated for 4 h, 12 h, 24 h, 48 h, and 72 h in an incubator at 37 °C to allow E. coli and C. albicans to adhere to the surfaces of PVC pieces and induce mixed-species BF formation. After coculturing, the medium was removed from the 96-well plates, and 100 µL of PBS was added to wash and remove the floating microbes on the PVC piece three times. After gently washing and discarding the PBS, the BF was fixed with 95% methanol for 30 min. Then, 100 µL of 2% CV dye solution was added to each well after fixation, and the plate was incubated at 37 °C for 30 min. Then, the CV dye solution was discarded, and each well was rinsed with 100 µL of PBS three times. The bound CV was resolubilized in 100 µL of DMSO, and the absorbance was measured at 490 nm on a multifunctional marker to determine the microbial adhesion ability and quantify the biomass of BFs in the experimental wells. Wells containing TSB medium and a PVC piece were similarly processed and used as blank controls. The experiments were repeated three times independently.

Detection of microbial viability by XTT.
The mixed microbial solution was cocultured with various concentrations of lipid emulsions or TSB medium for 4 h, 12 h, 24 h, 48 h, and 72 h as described above and processed for the XTT assay as previously described 45 . Briefly, the PVC pieces in the wells were gently washed three times with cold PBS solution to remove the floating microbes on the PVC pieces. After gently washing and discarding the PBS from the 96-well plates, 100 µL of TSB medium and 20 µL of XTT solution were added to each well, and the plates were incubated at 37 °C for 2 h in the absence of light. Following incubation, the absorbance was measured at 450 nm to detect the viability of microbes in BFs by using a multifunctional marker. Wells containing TSB medium and a PVC piece were similarly processed and used as blank controls. The experiments were repeated three times independently.

Observation of the thickness and live/dead microbes of mixed-species BFs by CLSM. An
8 mm × 8 mm PVC piece and 100 µL of mixed microbial solution were added to each well of 24-well plates and cocultured with various concentrations of lipid emulsions (2 mL) in an incubator at 37 °C in the experimental groups. As a control, the PVC pieces and mixed microbial solution were treated with only TSB medium. The fluorescent stains for testing microbial viability in the BFs were identified using a live/dead BacLight bacterial viability kit. The protocol for sample preparation for the viability assay is described in detail in the supplementary data (see S1). CLSM observation was carried out using an argon laser. The green fluorescence excitation wavelength was 488 nm, and the emission wavelength was 519 nm. The red fluorescence excitation wavelength was 559 nm, and the emission wavelength was 567 nm. The live and dead microbes on the PVC pieces were evaluated according to the area occupied by the green fluorescence of live microbes and red fluorescence of dead microbes. Each PVC piece was scanned from the inside to the outside to measure the thickness of the BF.

Observation of mixed-species BF composition by FISH. FISH was used in combination with CLSM
to visualize the distribution of the two strains in the mixed-species BFs. The probe sequences of E. coli and C. albicans designed according to the sequences in GenBank are shown in Table 1. Probes for the 16S rRNA of E. coli and C. albicans were labelled with 5-carboxyfluorescein (FAM) and cyanine dye (Cy3), respectively. The protocol for sample preparation for FISH is described in detail in the supplementary data (see S2). After FISH and washing, the structure and composition of the mixed-species BFs on PVC pieces were observed by CLSM.

Quantitation of BF-related gene expression.
A 24-well cell culture plate was removed from the control group and 10% lipid emulsion group at 24 h, 48 h, and 72 h, and the BFs were scraped and transferred to 1.5 mL centrifuge tubes. Total RNA was extracted with a total RNA extraction kit for RNA quantitation. Reverse transcription was conducted with the extracted RNA samples by the Bio-Rad iScript cDNA Synthesis Kit. Primers were designed for flhDC, iha, HTA1, and HWP1, as well as for 16S rRNA and ACT1, which were used as the reference genes. The primer sequences are shown in Table 2. The primers and cDNA template synthesized from the reverse transcription reaction were used for qRT-PCR. The 2 −∆∆Ct method was used for comparison of the relative levels of mRNAs 47 .
Statistical analysis and image construction. SPSS 24.0 statistical software was used for statistical analyses. The experimental data conforming to the normal distribution or meeting the normal distribution after conversion were expressed as the mean ± standard deviation. Analysis of variance (ANOVA) was used for intragroup and intergroup comparisons, and a t-test was used for pairwise comparisons. A value of P < 0.05 was considered statistically significant. P < 0.01 and P < 0.001 indicated significant differences. All graphs were generated by GraphPad Prism 8.0.1 (https:// www. graph pad. com). Images were converted to appropriate format and resolution using Adobe Photoshop CS6 (https:// www. adobe. com).  GTT TGT TGA AAG TGG AT  GAT GGC GGT TGA CAT AAG C   iha  ATG ATA ACC GGG ATG GTC AA  CCC ATT TGT CGC TCT TCA GT   16S rRNA  GAG AGC AAG CGG ACC TCA TA  GCA GAC TCC ATT CCG GAC TAC   HTA1  ATG TCA GGT GGT AAA GGT AAAG CTA CAA TTC TTG AGA AGC CTT AAC   HWP1  GGT TGT GAG CCA TTA GGG TTA  GGT TGT GAG CCA TTA GGG TTA   ACT1  ACC ACC GGT ATT GTT TTG GA  TGG ACA AAT GGT TGG TCA  www.nature.com/scientificreports/