Effects of lipid emulsion on the formation of Escherichia coli –Candida albicans mixed species biofilm on PVC


 Patients receiving lipid emulsion have an increased risk of central venous catheter-related bloodstream infections (CRSBI) in the clinic. More than 15% of CRBSI are found to be polymicrobial. The objective of the study was to explore the mechanism and effects of lipid emulsion on the formation of Escherichia coli-Candida albicans mixed species biofilm (BF) on the surface of polyvinylchloride (PVC) materials. Mixed BFs were achieved by co-culturing Escherichia coli (E.coli) and Candida albicans (Ca) with PVC in various concentrations of lipid emulsion respectively. Crystal violet (CV) staining and XTT assay were performed to test the formation of BFs and the viability of bacteria in BFs. The microstructures of BFs were observed by a combined approach of fluorescence in situ hybridization (FISH), confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). The study found that lipid emulsion promoted the formation of Escherichia coli-Candida albicans mixed biofilm, especially 10% lipid emulsion. The mechnism of lipid emulsion prompting mixed biofilm formation may be significantly up-regulating the expression of flhDC, iha, HTA1, and HWP1 genes associated with bacterial motility, adhesion and biofilm formation. For prevention of central venous catheter-associated infections, medical professionals should maintain strict aseptic precautions when handling lipid emulsion and avoid using high concentrations of lipid emulsion for a long time as much as possible. The study provides interesting information for future research in biomaterial related infection.


Introduction
Polyvinylchloride (PVC) is one of the most used medical materials due to its unique properties, availability and low cost. Medical devices for infusion and arti cial nutrition are essentially made of plasticized PVC. Central venous catheters (CVCs) are no exception. CVCs are widely used in clinics and essential to patients that need central venous access for blood transfusion, chemotherapy, antibiotic infusion, uid management, stem cell infusion, long-term hemodialysis, and total parenteral nutrition 1-3 .
CVC-related bloodstream infections (CRBSI) are severe and joint complications in these patients requiring central venous catheterization. It is estimated that a total of 200,000~400,000 episodes of CRBSI occur annually in the USA 4,5 . A report published in 2013 estimated the cost associated with catheters related infections to be the highest among healthcare-associated infections 6 . Patients are at increased risk of CRBSI, resulting in a delay in primary disease treatment, increased morbidity and mortality, prolonged hospital stay, and higher hospital cost 7,8 . Prospective surveillance studies carried out during 2013~2020 reported the incidence of CRBSI to be 0.5~5.8% in adults 9,10 . The incidences of CRBSI were more than 10% in children, even with antimicrobial central venous catheters. According to recent reports, more patients were detected with CRBSI caused by Gram-negative bacteria than those caused by Gram-positive bacteria 11,12 . Gram-negative pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella spp. were the most common pathogenic bacteria isolated in CRBSI, of which E. coli was the number one 13,14 . Candida spp. is also a leading pathogen in infections caused by central venous catheters 15 . A large number of clinical reports have indicated that the incidence of CRBSI increases in patients undergoing gastrointestinal surgery or those with advanced cancer, intestinal failure, or severe pancreatitis receiving a long period of parenteral nutrition 16,17 . Lipid emulsion is an indispensable component of parenteral nutrition and can provide energy and essential fatty acids for patients receiving parenteral nutrient solution. The parenteral nutrient solution needs to be injected through the central venous catheter. It forms a high-glucose and high-lipid environment around the catheter, conducive to the adhesion, growth, and diffusion of pathogenic microorganism. After implantation of biomaterials in the human body, micropathogenic bacteria can more easily adhere and proliferate on the surface of biomaterials and form bio lms with complex structures. Once the bio lm forms, pathogenic microbes in mixed BFs can effectively resist immune destruction and antibiotic therapy, which can induce persistence of infection. Several studies have reported that patients receiving lipid emulsion have an increased risk of central venous catheter-related infections in the clinic. Still, these studies were limited to studying of infections caused by a single microorganism 18, 19 . In fact, pathogenic bacteria are often coinfected by two or more strains or species that have been shown to in uence disease virulence in nature. More than 15% of CRBSI caused during parenteral nutrition are found to be polymicrobial 20 . Studies have con rmed that 27~57% of Ca infections are associated with other pathogens or opportunistic pathogens. Ca and other pathogens can colonize on the surface of implants and accelerate the formation of mixed species BFs 21,22 . Clinical biomaterial infections that were polymicrobial had poorer clinical prognosis and double mortality rate than a single microbe infection.
Our research group has successfully constructed hybrid bio lm models of various biological implant materials, such as mixed bio lm of Staphylococcus epidermidis-Candida albicans on PVC 23 , mixed bio lm of Staphylococcus aureus-Escherichia coli on titanium plate 24 , and a tree shrew biomaterialcentered infection model 25 . In the present study, a model of the mixed bio lm of Escherichia coli-Candida albicans in vitro was constructed on the surface of PVC of a central venous catheter. In order to explore the pathogenic mechanism to reduce the infection associated with central venous catheters, we studied the effects of various concentrations of lipid emulsion on mixed bio lm formation and structure as well as the expression of hDC, iha, HTA1, and HWP1 genes that correlated with bio lm formation.

The structure of mixed BFs detected by FISH
The green channel is FAM, labeled by E.coli of 16s RNA. The red channel is Cy3, labeled by Ca of 18s RNA. The blue channel is the bacterial nucleus marked by DAPI. The 72 h bio lm is thicker and more complex than the 24 h bio lm. The structure of bio lm in the lipid emulsion groups was more complicated than that in the control group, especially in the 10% lipid emulsion group. In the lipid emulsion groups, E.coli were found to be growing around the mycelium of Ca overlapping and interlacing ( Figure 4).
The ultrastructure of mixed BFs detected by SEM It was found that the 72 h bio lm was more complex and denser than the 24 h bio lm-forming a threedimensional network structure. Lipid emulsion remained a part of the mixed bio lm and attached to the surface of bacteria, mycelia and spores forming more complicated and mature bio lms, especially in the 10%, 15%, 20% lipid emulsion groups. The microstructures of mixed BFs of lipid emulsion groups showed that E.coli was the superior strains and adhered to the spores, pseudohyphae and mycelium of Ca forming a mixed growth. With the increase lipid emulsion concentration, more and more E. coli was found in the mixed BFs, but the growth of Ca was inhibited when the concentration of lipid emulsion exceeded 15% ( Figure 5).

Bio lm-related gene expression
Certain 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 culture, the expression levels of hDC, iha, HTA1, and HWP1 genes were up-regulated in the 10% lipid emulsion group as compared to those in the control group (P<0.05) (Figure 6a). The expression of these four genes decreased gradually overtime after 48 h of culture (Figure 6b). At 72 h time point, the expression of these four genes was lower in the 10% lipid group than that in the control group (P<0.05) (Figure 6c).

Discussion
The opportunistic pathogens Staphylococcus epidermidis and Candida albicans that colonize the skin can easily enter the human body with the implantation of biological materials 26 . Conditioned pathogens in the intestinal tract such as Escherichia coli and Coprococcus can conduct bacterial translocation in the process of trauma, stress, shock, hypotension and parenteral nutrition, causing bacteria to adhere to the surface of biomaterials and even form bio lms 27,28 . Bio lms are lm-like structures of microbial communities attached to surfaces of implanted biomaterials, where aggregates of microbes adhere to each other, undergo reproduction and self-cloning, and are embedded in a self-secreted extracellular slime substance 23 . When a bacterial bio lm is formed, the bacterial family within it resists attack by the body's immune system, multiplying resistance to antibiotics and leading to persistent infections 29 . Especially found in tumor patients with immune dysfunction and patients consuming glucocorticoids for a long time, the bio lm is often a mixture of bacteria and fungus 30,31 . In nature and man-made environments, micropathogenic bacteria reside in mixed-species bio lms, in which the growth and metabolism of an organism are different from these behaviors in single-species bio lms 32 . Compared to single-species bacterial bio lm, the quantity and diversity of present species raise the complexity in mixed bio lm. The treatment of mixed bio lm is more di cult than that of a single. The management of long-term central venous catheter-associated infections by multidrug-resistant bacteria in these patients is a major challenge.
Clinically, central venous catheter-related infections are the most common. Removal of CVC is the main therapeutic intervention, especially encouraged and recommended in the case of sepsis or septic shock 33 . A recent retrospective study reported that patients with candidemia had lower survival if the CVC was not removed or removed after more than 72 h 34 . Antimicrobials should be continued for at least seven days, depending on the causative pathogen antibiotic treatment. However, there were also certain serious complications such as endocarditis, thrombophlebitis, and septic metastasis after adequate antimicrobial therapy. The risk of infection depends on host factors, catheter type, and routine care procedures.
In this paper, PVC was used as the carrier of the central venous catheter. Ca and E.coli mixture was used as the source of opportunistic pathogens coinfections. The results of XTT and crystal violet in this study showed that the bio lm formation ability, bacterial adhesion, and viability of the lipid emulsion groups were higher than that of the control group. The effects of different concentrations of lipid emulsions on the formation of mixed bio lm were observed and analyzed by SEM and CLSM. The activity of pathogenic bacteria and the thickness of bio lm formation were most superior when lipid emulsion concentration was 10%. With the increase lipid emulsion concentration, more and more E. coli was found in the mixed BFs, but the growth of Ca was inhibited and transformed from hyphae phase to yeast phase when the concentration of lipid emulsion exceeded 15%. The results indicated that there was interspecies competition between E.coli and Ca, and 10% of lipid emulsion was the optimum concentration for the growth and formation of E.coli-Ca mixed bio lm. Swindell K. pointed out that 10% of lipid emulsion was conducive to the germination of Ca spores and the growth of mycelia (18). The spores and mycelia of Ca could provide a lot of adhesion sites for E. coli, meanwhile, lipid emulsion provided rich nutrients, and promoted the growth of pathogenic bacteria and bio lm formation, which may partly explain why lipid emulsion has a catalytic effect on E. coli-Ca mixed bio lm formation 35 .
hDC mainly regulates the biosynthesis of E.coli agellum, bacterial division, and the expression of bacterial virulence genes 36 . Overexpressing hDC increased the motility and ability to colonize on biomaterials 37 . A number of K-12 strains exhibit limited motility due to low expression levels of hDC. Motility is bene cial for cells to access and explore new environments and to escape detrimental ones.
iha gene is iron-regulated gene homolog adhesion. The gene encodes a bacterial outer membrane protein associated with the adhesion of bacterial 38 . It mediates bacterial adhesion to host cells, and after iha gene knockout, the ability of bacterial cell bio lm formation is signi cantly reduced. HWP1 gene of Ca encodes for a fungal cell wall protein required for hyphal development and yeast adhesion to epithelial cells 39 . HWP1 is an important gene in Ca bio lm formation. When HWP1 gene was knocked out, its bacterial cells could not form a complete bio lm in vitro and in vivo. Doramectin S4 is a type of antimicrobial peptide, which can potentially be used in antifungal therapy. The mechanism of DS4 as an antifungal agent is via signi cantly decreasing the expression of HWP1 gene as evidenced by the gene expression analyses 40 .
HTA1 gene encodes the Ca histone H2A. Histones play an essential role in DNA replication. Therefore, the expression of HTA1 gene re ects the growth and reproduction ability of Ca cells to a certain extent 41 .
qRT-PCR results showed that hDC, iha, HWP1, and HTA1 genes were up-regulated in the 10% lipid emulsion group compared with that of the control group at 24 h. This indicated that lipid emulsion could promote the early formation of E. coli-Ca mixed bio lm, especially the signi cant promotion of movement and adhesion of E. coli at early stage. In the middle stage, lipid emulsion promotes the adhesion and aggregation of E.coli and Ca. This may be the molecular mechanism by which lipid emulsion promotes the formation of bio lm. Some studies demonstrated that pathogenic Ca could enhance virulence determinants of a bacterium in vivo with devastating consequences to the host 42,43 . After 72 h culture, hDC, iha, HWP1, and HTA1 genes were downregulated in the 10% lipid emulsion group compared with that of the control group, which may be related to nutrient consumption the medium.
Thus, lipid emulsion can potentially prompt the formation of Escherichia coli-Candida albicans mixed bio lm. To prevent central venous catheter-associated infections, medical professionals should maintain strict aseptic precautions when handling lipid emulsion and avoid using high concentrations of lipid emulsion for a long time as much as possible.

Methods
Bacterial strains, reagents, and equipment Escherichia coli (MC1000) was gifted by The Yale Coli Genetic Stock Center. Candida albicans (ATCC10231) was purchased from the Institute of Microbiology, Chinese Academy of Sciences. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. In situ hybridization kit was purchased from Boxin Biology (Guangzhou) Co., Ltd. The cDNA Synthesis kit was procured from Bio-Rad Inc. Live&Dead bacterial staining kit was obtained from Life, USA. Polypropylene 24-well and 96-well cell culture plates were purchased from Corning, USA. MH agar plates and Shapaul agar plates were from Tu'an Biological Engineering (Zhengzhou) Co., Ltd. Tryptic Soy Broth (TSB) medium was from Huankai Microbiological Reagent (Guangdong) Co., Ltd. S-3000N scanning electron microscope was from HITACHI, Japan and FV1000 confocal laser scanning microscope from Olympus, Japan. Total RNA extraction kit was supplied by Tiangen Biotech (Beijing) Co., Ltd. The qRT-PCR assay was conducted using the SuperReal PreMix Plus (Tiangen, China),and was performed on the ABI 7500 PCR system (ABI, USA).

Bacterial culture and Experimental grouping
The standard strains of Ca and E.coli were inoculated on Sarpaul agar plates and MH agar plates respectively and incubated at 37°C for 24 h. Subsequently, 5 mL TSB culture medium was added into test tube, and the liquid level junction in the test tube was gently vibrated. An inoculation ring was used to select a single colony on the bacterial plate of each group and inoculate into the test tube containing 5 mL TSB medium. These tubes were incubated in a constant humidity oscillator at 37°C and 200 r/min for 16~18 h. After the growth of bacterial cells to the logarithmic growth phase, the concentration of the bacterial solution in each group was adjusted to 1.1 × 10 7 CFU/mL using TSB medium in an ultraviolet spectrophotometer for later use. The mixed bacterial solution in the study was prepared in 1:1 ratio, that is, 2 mL of bacterial solution was prepared by mixing 1 mL Ca bacterial solution and 1 mL E. coli bacterial solution. Bacterial suspensions treated with various concentrations of lipid emulsion were added to a 96-well cell culture plate and incubated in a 37 °C thermostatic incubator. The experiment included ve groups: a control group with TSB medium alone and four lipid emulsion groups. The concentrations of lipid emulsion in the other four groups were 5%, 10%, 15%, and 20%. The TSB medium was mixed with lipid emulsion in various proportions to modulate the lipid emulsion concentration to 0%, 5%, 10%, 15%, and 20%.

Detection of bacterial adhesion and bio lm formation by CV
A total of 100 µL of the previously prepared lipid emulsion of different concentrations was inoculated into 96-well cell culture plates, and then 10 µL of the prepared bacterial solution and a PVC piece were added into each well. Six wells were inoculated in each group at each time point and incubated in a 37 °C incubator. The medium was removed from the 96-well plates at 4 h, 12 h, 24 h, 48 h, and 72 h after coculturing. 100 µL PBS was added to wash and remove the oating bacteria on the PVC piece for three times. After gently washing and discarding PBS, 100 μL 2% CV dye solution was added to each well, and incubated at 37°C for 30 minutes. Then, CV dye solution was sucked and discarded, and 100 µL PBS was added to rinse for three times. The ability of bacterial adhesion and mixed bio lm formation was determined by crystal violet staining, and measured at an absorbance of 490 nm wavelength by using a multifunctional marker in various concentrations of lipid emulsion. Experiments were repeated three times.

Detection of bacterial viability by XTT
After 4 h, 12 h, 24 h, 48 h, and 72 h co-culture with different concentrations of lipid emulsion, the PVC pieces were gently washed three times with cold PBS solution to remove the oating bacteria on the PVC pieces. After gently washing and discarding PBS, 100 µL TSB culture medium and 20 µL XTT solution were added to each well and incubated at 37°C for 2 h in the absence of light. Following incubation, 120 µL medium was removed from each well to measure the viability of bacteria at an absorbance of 450 nm wavelength by using a multifunctional marker in various concentrations of lipid emulsion. Experiments were repeated three times.

Observation of mixed bio lm thickness and live/dead bacteria by CLSM
The uorescent stains for bacterial viability in the BFs were identi ed using Live&Dead Bacterial viability kit. The PVC pieces were taken out for 24 h, 48 h, and 72 h after culture and gently washed with normal saline three times to remove the oating bacteria on the PVC pieces. They were then immersed in the uorescent stains and stained for 20 min at room temperature in the absence of light. After absorbing excess uorescent dye, the PVC pieces were put on a uorescent image slide under the CLSM. CLSM observation was carried out using an argon laser. The green uorescence excitation wavelength is 488 nm, and the emission wavelength is 519 nm. The red uorescence excitation wavelength is 559 nm, and the emission wavelength is 567 nm. The live and dead bacteria on the PVC pieces at each time point were evaluated according to the area occupied by the green uorescence of live bacteria and red uorescence of dead bacteria. Each PVC piece was scanned from internal to external to measure the thickness of the bio lm.

Observation of mixed bio lm composition by FISH
The probe sequences of E. coli and Ca were designed according to GenBank (Table 1). The PVC pieces were taken out for FISH after 24 h and 72 h incubation according to the manufacturer's instructions. All the samples of PVC pieces were observed under the CLSM. The green uorescence excitation wavelength is 488 nm, and the emission wavelength is 519 nm. The red uorescence excitation wavelength is 559 nm, and the emission wavelength is 567 nm. The blue uorescence excitation wavelength is 340 nm, and the emission wavelength is 488 nm. Each PVC piece was scanned from internal to external to observe the structure and composition of mixed BFs.

Observation of mixed bio lm ultrastructure by SEM
The PVC pieces were taken out after 24 h and 72 h co-culture, and gently washed with PBS solution three times and xed with 2% glutaraldehyde phosphate buffer on slides for SEM analysis. Then, the PVC pieces were washed three times with PBS and dried in critical CO 2 . The surface of PVC pieces turned to golden brown because of ions sputtering on the surface. The ultrastructure of the mixed bio lm was observed by SEM.

Quantitation of bio lm-related gene expression
A 24-well cell culture plate was removed each 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 by the 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 hDC, iha, HTA1, HWP1, and 16 sRNA, act1 which were used as the reference gene. Primer sequences are shown in (Table 2). The primers and cDNA template synthesized from the reverse transcription reaction were used for the quantitative RT-PCR reaction. The 2 −∆∆Ct method was used for the comparison of the relative levels of mRNAs.
Statistical Analysis 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 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 signi cant difference. P<0.01 and P<0.001 suggested signi cant difference.