Development and application of a polymicrobial, in vitro, wound biofilm model

Aims: The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements. Methods and Results: The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip‐flow reactor (DFR), and a three‐species biofilm model was established using methicillin‐resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Cl. perfringens in the colony‐drip‐flow reactor model. Plate counts revealed that MRSA, Ps. aeruginosa and Cl. perfringens grew to 7·39 ± 0·45, 10·22 ± 0·22 and 7·13 ± 0·77 log CFU per membrane, respectively. The three‐species model was employed to evaluate the efficacy of two antimicrobial dressings, Curity? AMD and Acticoat?, compared to sterile gauze controls. Microbial growth on Curity? AMD and gauze was not significantly different, for any species, whereas Acticoat? was found to significantly reduce growth for all three species. Conclusions: Using the colony‐DFR, a three‐species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species. Significance and Impact of the Study: The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment.     

Unravelling interspecies interactions across heterogeneities in complex biofilm communities

The importance of microbial biofilms has been well-recognized for several decades, and focus is now shifting towards investigating multispecies biofilm communities rather than mono- or dual-species biofilms. Therefore, the demand for techniques that provide a sufficient amount of information at adequate resolution is increasing. One major challenge for multispecies studies is that diversity and spatial organization often lead to a high degree of spatial and chemical heterogeneity. Many current approaches do not account for such heterogeneity and therefore only provide average information (−omics techniques in particular), which could obscure important information about the community. Here, we bring attention to the issues of heterogeneity when analysing synthetic multi-species biofilms, in vitro, and the importance of multi-scale approaches. We provide an overview of current and newer approaches that can be applied to biofilm communities, in order to elucidate interactions at the appropriate scale.     

Interactions of microorganisms within a urinary catheter polymicrobial biofilm model

Biofilms are often polymicrobial in nature, which can impact their behavior and overall structure, often resulting in an increase in biomass and enhanced antimicrobial resistance. Using plate counts and locked nucleic acid/2′-O-methyl-RNA fluorescence in situ hybridization (LNA/2′OMe-FISH), we studied the interactions of four species commonly associated with catheter-associated urinary tract infections (CAUTI): Enterococcus faecalis, Escherichia coli, Candida albicans, and Proteus mirabilis. Eleven combinations of biofilms were grown on silicone coupons placed in 24-well plates for 24 h, 37°C, in artificial urine medium (AUM). Results showed that P. mirabilis was the dominant species and was able to inhibit both E. coli and C. albicans growth. In the absence of P. mirabilis, an antagonistic relationship between E. coli and C. albicans was observed, with the former being dominant. E. faecalis growth was not affected in any combination, showing a more mutualistic relationship with the other species. Imaging results correlated with the plate count data and provided visual verification of species undetected using the viable plate count. Moreover, the three bacterial species showed overall good repeatability SD (S_r) values (0.1–0.54) in all combinations tested, whereas C. albicans had higher repeatability S_r values (0.36–1.18). The study showed the complexity of early-stage interactions in polymicrobial biofilms. These interactions could serve as a starting point when considering targets for preventing or treating CAUTI biofilms containing these species.     

Role of interspecies interactions in dual-species biofilms developed in vitro by uropathogens isolated from polymicrobial urinary catheter-associated bacteriuria

Most catheter-associated urinary tract infections are polymicrobial. Here, uropathogen interactions in dual-species biofilms were studied. The dual-species associations selected based on their prevalence in clinical settings were Klebsiella pneumoniae–Escherichia coli, E. coli–Enterococcus faecalis, K. pneumoniae–E. faecalis, and K. pneumoniae–Proteus mirabilis. All species developed single-species biofilms in artificial urine. The ability of K. pneumoniae to form biofilms was not affected by E. coli or E. faecalis co-inoculation, but was impaired by P. mirabilis. Conversely, P. mirabilis established a biofilm when co-inoculated with K. pneumoniae. Additionally, E. coli persistence in biofilms was hampered by K. pneumoniae but not by E. faecalis. Interestingly, E. coli, but not K. pneumoniae, partially inhibited E. faecalis attachment to the surface and retarded biofilm development. The findings reveal bacterial interactions between uropathogens in dual-species biofilms ranged from affecting initial adhesion to outcompeting one bacterial species, depending on the identity of the partners involved.     

Metabolite cross-feeding enhances virulence in a model polymicrobial infection

Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.     

Individual growth detection of bacterial species in an in vitro oral polymicrobial biofilm model

Most in vitro studies on the antibacterial effects of antiseptics have used planktonic bacteria in monocultures. However, this study design does not reflect the in vivo situation in oral cavities harboring different bacterial species that live in symbiotic relationships in biofilms. The aim of this study was to establish a simple in vitro polymicrobial model consisting of only three bacterial strains of different phases of oral biofilm formation to simulate in vivo oral conditions. Therefore, we studied the biofilm formation of Actinomyces naeslundii (An), Fusobacterium nucleatum (Fn), and Enterococcus faecalis (Ef) on 96-well tissue culture plates under static anaerobic conditions using artificial saliva according to the method established by Pratten et al. that was supplemented with 1 g l^?1 sucrose. Growth was separately determined for each bacterial strain after incubation periods of up to 72 h by means of quantitative real-time polymerase chain reaction and live/dead staining. Presence of an extracellular polymeric substance (EPS) was visualized by Concanavalin A staining. Increasing incubation times of up to 72 h showed adhesion and propagation of the bacterial strains with artificial saliva formulation. An and Ef had significantly higher growth rates than Fn. Live/dead staining showed a median of 49.9 % (range 46.0–53.0 %) of living bacteria after 72 h of incubation, and 3D fluorescence microscopy showed a three-dimensional structure containing EPS. An in vitro oral polymicrobial biofilm model was established to better simulate oral conditions and had the advantage of providing the well-controlled experimental conditions of in vitro testing.     

Development of an oral mucosa model to study host-microbiome interactions during wound healing

Crosstalk between the human host and its microbiota is reported to influence various diseases such as mucositis. Fundamental research in this area is however complicated by the time frame restrictions during which host-microbe interactions can be studied in vitro. The model proposed in this paper, consisting of an oral epithelium and biofilm, can be used to study microbe-host crosstalk in vitro in non-infectious conditions up to 72 h. Microbiota derived from oral swabs were cultured on an agar/mucin layer and challenged with monolayers of keratinocytes grown on plastic or collagen type I layers embedded with fibroblasts. The overall microbial biofilm composition in terms of diversity remained representative for the oral microbiome, whilst the epithelial cell morphology and viability were unaffected. Applying the model to investigate wound healing revealed a reduced healing of 30 % in the presence of microbiota, which was not caused by a reduction of the proliferation index (52.1–61.5) or a significantly increased number of apoptotic (1–1.13) or necrotic (32–30.5 %) cells. Since the model allows the separate study of the microbial and cellular exometabolome, the biofilm and epithelial characteristics after co-culturing, it is applicable for investigations within fundamental research and for the discovery and development of agents that promote wound healing.     

Antimicrobial effects of a pulsed electromagnetic field: an in vitro polymicrobial periodontal subgingival biofilm model

Abstract

The objective was to test the influence of a pulsed electromagnetic field (PEMF) on bacterial biofilm colonization around implants incorporated with healing abutments. Healing abutments with (test group) and without (control group) active PEMF devices were placed in a multispecies biofilm consisting of 31 different bacterial species. The biofilm composition and total bacterial counts (x10⁵) were analyzed by checkerboard DNA-DNA hybridization. After 96?h, the mean level of 7 out of the 31 bacterial species differed significantly between groups, namely Eubacterium nodatum, Fusobacterium nucleatum ssp. nucleatum, Streptococcus intermedius, Streptococcus anginosus, Streptococcus mutans, Fusobacterium nucleatum ssp. Vicentii and Capnocytophaga ochracea were elevated in the control group (p?<?0.05). The mean total bacterial counts were lower in the Test group vs the control group (p?<?0.05). An electromagnetic healing cap had antimicrobial effects on the bacterial species and can be used to control bacterial colonization around dental implants. Further clinical studies should be conducted to confirm these findings.     

An HLA-A2.1-transgenic rabbit model to study immunity to papillomavirus infection

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition, vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy.     

Development and characterisation of a novel three-dimensional inter-kingdom wound biofilm model

Chronic diabetic foot ulcers are frequently colonised and infected by polymicrobial biofilms that ultimately prevent healing. This study aimed to create a novel in vitro inter-kingdom wound biofilm model on complex hydrogel-based cellulose substrata to test commonly used topical wound treatments. Inter-kingdom triadic biofilms composed of Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus were shown to be quantitatively greater in this model compared to a simple substratum when assessed by conventional culture, metabolic dye and live dead qPCR. These biofilms were both structurally complex and compositionally dynamic in response to topical therapy, so when treated with either chlorhexidine or povidone iodine, principal component analysis revealed that the 3-D cellulose model was minimally impacted compared to the simple substratum model. This study highlights the importance of biofilm substratum and inclusion of relevant polymicrobial and inter-kingdom components, as these impact penetration and efficacy of topical antiseptics.     

A porcine model of skin wound infected with a polybacterial biofilm

A clinically relevant porcine model of a biofilm-infected wound was established in 10 minipigs. The wounds of six experimental animals were infected with a modified polymicrobial Lubbock chronic wound biofilm consisting of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Bacillus subtilis. Four animals served as uninfected controls. The wounds were monitored until they had healed for 24 days. The biofilm persisted in the wounds up to day 14 and significantly affected healing. The control to infected healed wound area ratios were: 45%/21%, 66%/37%, and 90%/57% on days 7, 10 and 14, respectively. The implanted biofilm prolonged inflammation, increased necrosis, delayed granulation and impaired development of the extracellular matrix as seen in histological and gene expression analyses. This model provides a therapeutic one-week window for testing of anti-biofilm treatments and for research on the pathogenesis of wound infections in pig that is clinically the most relevant animal wound healing model.