Polymicrobial Biofilms Research Articles

Patients with obstructive sleep apnea can favor the predisposing factors of periodontitis by the presence of P. melaninogenica and C. albicans, increasing the severity of the periodontal disease.

ObjectiveThe aim of this study was to analyze the cultivable oral microbiota of patients with obstructive sleep apnea (OSA) and its association with the periodontal condition.MethodsThe epidemiology profile of patients and their clinical oral characteristics were determined. The microbiota was collected from saliva, subgingival plaque, and gingival sulcus of 93 patients classified into four groups according to the periodontal and clinical diagnosis: Group 1 (n = 25), healthy patients; Group 2 (n = 17), patients with periodontitis and without OSA; Group 3 (n = 19), patients with OSA and without periodontitis; and Group 4 (n = 32), patients with periodontitis and OSA. Microbiological samples were cultured, classified, characterized macroscopically and microscopically, and identified by MALDI-TOF-MS. The distribution of complexes and categories of microorganisms and correlations were established for inter- and intra-group of patients and statistically evaluated using the Spearman r test (p-value <0.5) and a multidimensional grouping analysis.ResultThere was no evidence between the severity of OSA and periodontitis (p = 0.2813). However, there is a relationship between the stage of periodontitis and OSA (p = 0.0157), with stage III periodontitis being the one with the highest presence in patients with severe OSA (prevalence of 75%; p = 0.0157), with more cases in men. The greatest distribution of the complexes and categories was found in oral samples of patients with periodontitis and OSA (Group 4 P-OSA); even Candida spp. were more prevalent in these patients. Periodontitis and OSA are associated with comorbidities and oral conditions, and the microorganisms of the orange and red complexes participate in this association. The formation of the dysbiotic biofilm was mainly related to the presence of these complexes in association with Candida spp.ConclusionPeriodontopathogenic bacteria of the orange complex, such as Prevotella melaninogenica, and the yeast Candida albicans, altered the cultivable oral microbiota of patients with periodontitis and OSA in terms of diversity, possibly increasing the severity of periodontal disease. The link between yeasts and periodontopathogenic bacteria could help explain why people with severe OSA have such a high risk of stage III periodontitis. Antimicrobial approaches for treating periodontitis in individuals with OSA could be investigated in vitro using polymicrobial biofilms, according to our findings.

Quorum Sensing and Quorum Quenching with a Focus on Cariogenic and Periodontopathic Oral Biofilms.

Numerous in vitro studies highlight the role of quorum sensing in the pathogenicity and virulence of biofilms. This narrative review discusses general principles in quorum sensing, including Gram-positive and Gram-negative models and the influence of flow, before focusing on quorum sensing and quorum quenching in cariogenic and periodontopathic biofilms. In cariology, quorum sensing centres on the role of Streptococcus mutans, and to a lesser extent Candida albicans, while Fusobacterium nucleatum and the red complex pathogens form the basis of the majority of the quorum sensing research on periodontopathic biofilms. Recent research highlights developments in quorum quenching, also known as quorum sensing inhibition, as a potential antimicrobial tool to attenuate the pathogenicity of oral biofilms by the inhibition of bacterial signalling networks. Quorum quenchers may be synthetic or derived from plant or bacterial products, or human saliva. Furthermore, biofilm inhibition by coating quorum sensing inhibitors on dental implant surfaces provides another potential application of quorum quenching technologies in dentistry. While the body of predominantly in vitro research presented here is steadily growing, the clinical value of quorum sensing inhibitors against in vivo oral polymicrobial biofilms needs to be ascertained.

Effect of Myrtenol and Its Synergistic Interactions with Antimicrobial Drugs in the Inhibition of Single and Mixed Biofilms of Candida auris and Klebsiella pneumoniae.

The increased incidence of mixed infections requires that the scientific community develop novel antimicrobial molecules. Essential oils and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities and are attracting more and more attention. Therefore, the aim of this study was to evaluate myrtenol (MYR), one of the constituents commonly found in some essential oils, for its potential to inhibit biofilms alone and in combination with antimicrobial drugs against Candida auris/Klebsiella pneumoniae single and mixed biofilms. The antimicrobial activity of MYR was evaluated by determining bactericidal/fungicidal concentrations (MIC), and biofilm formation at sub-MICs was analyzed in a 96-well microtiter plate by crystal violet, XTT reduction assay, and CFU counts. The synergistic interaction between MYR and antimicrobial drugs was evaluated by the checkerboard method. The study found that MYR exhibited antimicrobial activity at high concentrations while showing efficient antibiofilm activity against single and dual biofilms. To understand the underlying mechanism by which MYR promotes single/mixed-species biofilm inhibition, we observed a significant downregulation in the expression of mrkA, FKS1, ERG11, and ALS5 genes, which are associated with bacterial motility, adhesion, and biofilm formation as well as increased ROS production, which can play an important role in the inhibition of biofilm formation. In addition, the checkerboard microdilution assay showed that MYR was strongly synergistic with both caspofungin (CAS) and meropenem (MEM) in inhibiting the growth of Candida auris/Klebsiella pneumoniae-mixed biofilms. Furthermore, the tested concentrations showed an absence of toxicity for both mammalian cells in the in vitro and in vivo Galleria mellonella models. Thus, MYR could be considered as a potential agent for the management of polymicrobial biofilms.

Visualization and elimination of polymicrobial biofilms by a combination of ALA-carvacrol-blue light.

Chronic wound infections caused by multidrug-resistant (MDR) bacteria are one of the serious threats to public health due to limited therapeutic options and lengthy care. This investigation combines 5-aminolevulinic acid (ALA), blue light (BL), and phytochemical carvacrol, named ABC cocktail or trio-therapy, to efficiently eliminate wound-related MDR pathogens. Both planktonic cells and biofilms of blue light-refractory Escherichia (E.) coli and Klebsiella (K.) pneumoniae succumbed to the trio-therapy partly due to porphyrin accumulations following ALA incubation. ALA either alone or alongside carvacrol could vigorously trigger bursts of reactive oxygen species (ROS) upon blue light irradiation in K. pneumoniae, but not in mammalian cells. The robust antimicrobial activity was extended to polymicrobial biofilms composed of five MDR pathogens (Staphylococcus aureus, E. coli, K. pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) in vitro and in vivo. Strikingly, polymicrobial biofilm in mouse wounds became readily visible in the presence of ALA owing to the increasing generation of porphyrins that exhibited bright red fluorescence in response to blue light. Thus, ALA not only enhances killing efficacy, but also helps to pinpoint the infections for guiding debridement, precise phototherapy, and timely assessment of treatment effectiveness. Featuring a broadened antimicrobial spectrum and advantages of bacterial/biofilm imaging, the trio-therapy can be used either alone or adjunctive to other wound management modalities to effectively combat MDR bacteria in wounds.

Dual species biofilms are enhanced by metabolite cross-feeding.

Enterococcus faecalis and Staphylococcus aureus are frequently co-isolated from biofilm-associated infections. A new study by Ch'ng et al. revealed that S. aureus-released heme feeds E. faecalis respiration, augmenting E. faecalis growth and overall biofilm biomass. Their finding further supports the theory that metabolite cross-feeding is a critical aspect shaping polymicrobial biofilm interactions.

Treponema denticola Induces Interleukin-36γ Expression in Human Oral Gingival Keratinocytes via the Parallel Activation of NF-κB and Mitogen-Activated Protein Kinase Pathways.

The oral epithelial barrier acts as both a physical barrier to the abundant oral microbiome and a sentry for the immune system that, in health, constrains the accumulation of the polymicrobial plaque biofilm. The immune homeostasis during gingivitis that is largely protective becomes dysregulated, unproductive, and destructive to gingival tissue as periodontal disease progresses to periodontitis. The progression to periodontitis is associated with the dysbiosis of the oral microbiome, with increasing prevalences and abundances of periodontal pathogens such as Treponema denticola. Despite the association of T. denticola with a chronic inflammatory disease, relatively little is known about gingival epithelial cell responses to T. denticola infection. Here, we characterized the transcriptome of gingival keratinocytes following T. denticola challenge and identified interleukin-36γ (IL-36γ) as the most differentially expressed cytokine. IL-36γ expression is regulated by p65 NF-κB and the activation of both the Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways downstream of Toll-like receptor 2 (TLR2). Finally, we demonstrate for the first time that mitogen- and stress-activated kinase 1 (MSK1) contributes to IL-36γ expression and may link the activation of MAPK and NF-κB signaling. These findings suggest that the interactions of T. denticola with the gingival epithelium lead to elevated IL-36γ expression, which may be a critical inducer and amplifier of gingival inflammation and subsequent alveolar bone loss.

Co-culture with Acinetobacter johnsonii enhances benzalkonium chloride resistance in Salmonella enterica via triggering lipid A modifications

Salmonella enterica is one of the leading causes of foodborne gastroenteritis worldwide. In the food production environment, many bacterial species co-exist on surfaces in biofilm structures, which can act as reservoirs of microbial contamination of food products. Polymicrobial biofilms have been shown to have greater tolerance to antimicrobials, such as disinfectants, however the mechanistic basis of this is poorly understood. In this study, S. enterica subsp. enterica serovar Liverpool was co-cultured in mixed-species biofilms with bacteria isolated from the food production environment and challenged with the cationic biocide disinfectant, benzalkonium chloride (BC). Co-culture with the common environmental bacterium Acinetobacter johnsonii resulted in >200-fold higher resistance of S. Liverpool to BC, compared to mono-culture biofilms. The transcriptional response of S. enterica to biofilm co-culture was determined using a dual RNA-seq strategy. Genes controlled by the PhoPQ and PmrAB two-component systems, involved in lipid A modification and associated with cationic antimicrobial peptide resistance (CAMP) of S. Liverpool, were significantly upregulated. Deletion of either the phoP or pmrA genes resulted in an increase in susceptibility to BC, suggesting that activation of their regulons during co-culture enhances BC resistance. S. Liverpool lipid A profiles changed significantly upon co-culturing, with greater incorporation of both phosphoethanolamine and palmitate, which was dependent upon activation of PhoPQ and PmrAB. We conclude that when grown in the presence of A. johnsonii, S. Liverpool increases its tolerance to cationic BC disinfection by remodelling its cell envelope including reducing the net negative charge of lipid A and increasing lipid A acyl density.

Tachyplesin I Analogue Peptide as an Effective Antimicrobial Agent against Candida albicans-Staphylococcus aureus Poly-Biofilm Formation and Mixed Infection.

Microbial biofilms are difficult to tackle in many infectious diseases. Candida albicans and Staphylococcus aureus are prevalent symbiotic strains in polymicrobial biofilms, which showed enhanced antimicrobial resistance and made identifying effective treatment techniques more difficult. The antibiofilm abilities of tachplesin I analogue peptide (TP11A) and tachplesin I were investigated quantitatively in this study. Both inhibited C. albicans monomicrobial, S. aureus monomicrobial, and C. albicans-S. aureus polymicrobial biofilms quite well. TP11A suppressed the biofilm- and virulence-related genes of C. albicans (hwp 1) and S. aureus (ica A, fnb B, agr A, hla, nor A, and sig B) in the mixed biofilm, according to quantitative reverse transcription polymerase chain reaction analysis. We created an injectable thermosensitive in situ PLEL@TP11A gel system by simply adding TP11A into poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PLEL). Using C. albicans-S. aureus mixed infected wound models of mice, the in vivo therapeutic effect of TP11A and PLEL@TP11A in polymicrobial infections was investigated. The findings revealed that TP11A and PLEL@TP11A could efficiently reduce bacterial and fungal burden in wound infections, as well as accelerated wound healing. Based on above findings, TP11A might be an effective antimicrobial against C. albicans-S. aureus poly-biofilm formation and mixed infection.

Synthesis and evaluation of novel furanones as biofilm inhibitors in opportunistic human pathogens

Diseases caused by biofilm-forming pathogens are becoming increasingly prevalent and represent a major threat to human health. This trend has prompted a search for novel inhibitors of microbial biofilms which could, for example, be used to potentiate existing antibiotics. Naturally-occurring, halogenated furanones isolated from marine algae have proven to be effective biofilm inhibitors in several bacterial species. In this work, we report the synthesis of a library of novel furanones and their subsequent evaluation as biofilm inhibitors in several opportunistic human pathogens including S. enterica, S. aureus, E. coli, S. maltophilia, P. aeruginosa and C. albicans. A number of the most potent compounds were subjected to further analysis by confocal laser-scanning microscopy for their effects on P. aeruginosa and C. albicans biofilms individually, in addition to mixed polymicrobial biofilms. Lastly, we investigated the impact of a promising candidate on survival rates in vivo using a Galleria mellonella model.

Effects of Surface Prereacted Glass on Saliva-Derived Polymicrobial Biofilms in an Active Attachment Biofilm Model

Bioactive restorative materials are being developed to either influence the de/remineralization balance of the dental hard tissues locally or to release components that interact with the oral microbiota. Surface prereacted glass (S-PRG, Shofu, Japan) is a material that may influence both processes. S-PRG releases fluoride, which can interact with the de/remineralization process, and a range of other compounds that may influence the oral microbiota. In the current study, several experiments were performed to investigate the potential of S-PRG to influence both the growth and lactic acid production of saliva-derived polymicrobial biofilms. Biofilm formation was studied using the Amsterdam Active Attachment model. An eluate of the S-PRG particles was tested by adding it to the growth medium or by exposing the biofilms to it for 1 h. The effect of S-PRG particles was tested by adding the particles to the growth medium. The current experiments showed that the presence of S-PRG eluate in the medium influenced biofilm growth and lactic acid production even at low concentrations. The composition of the biofilms changed in the presence of S-PRG eluate, even at concentrations of S-PRG eluate at which biofilm viability was not affected. Treatment of developing biofilms with S-PRG eluate did neither show an effect on biofilm viability nor on lactic acid production. The addition of S-PRG particles to the growth medium resulted in both a lower biofilm viability and lower lactic acid production, indicating that the release of ions from the particles was fast enough to influence biofilm formation. From the current experiments, it can be concluded that S-PRG has the potential to influence biofilm growth, but the presence of the released ions during biofilm formation is required to show an effect.

A GIANT TONSILLOLITH IN A 13-YEAR-OLD PATIENT

Tonsillar stones or tonsilloliths are polymicrobial biofilms formed in the crypts of palatine tonsils, which become calcified by mineral deposition, especially of calcium salts. Large calcifications can cause odynophagia, dysphagia, and otalgia and are rare in young patients. A 13-year-old male patient was referred by the orthodontist for investigation of an extensive radiopaque area in the region of the left ascending ramus of the mandible observed on panoramic radiograph. The patient was asymptomatic and did not report any other disease. On clinical examination, no alterations in the parotideomasseteric region or oral lesions were observed. Blood tests and ultrasonography of the parotideomasseteric and submandibular regions and fan beam tomography were requested. Ultrasonography and blood tests were unremarkable, whereas tomography revealed a large, calcified mass in the soft tissues of the parapharyngeal space, measuring 1.9 × 2.5 cm and compatible with a tonsillolith. The patient was then referred for tonsillectomy.

ANTIMICROBIAL PHOTODYNAMIC THERAPY AS AN ALTERNATIVE TREATMENT FOR DENTURE STOMATITIS WITH CANDIDA SPP.: CASE REPORT

Antimicrobial photodynamic therapy (aPDT) is a treatment option to reduce pathogenic microorganisms such as bacteria, viruses, and fungi and can be effective in dental stomatitis (DS), because a complex polymicrobial biofilm can proliferate on the denture surface, and <i>Candida</i> spp. is one of the most relevant agents. The aim was to report the case of an 83-year-old edentulous man, denture user, with DS. A microbiological exam identified cfu/mL of <i>Candida</i> spp. Then, 5 sessions of aPDT with 0.01% methylene blue solution and a red laser source (aluminium indium gallium phosphide, 660 nm, 9 J output energy, fluence of 300 J/cm², 100 mW output power, irradiance of 3.33 W/cm², 90 seconds) were performed. After 5 weeks of aPDT, there was remission of DS and reduction of in colony-forming units per milliliter of fungi and <i>Candida</i> spp. aPDT can be an effective therapy in DS associated with <i>Candida</i> spp.

Detection of Streptococcus Mutans and Streptococcus sobrinus by Polymerase Chain Reaction (PCR) in Saliva Samples

Background: Dental caries has been a major problem in Bangladesh for decades. Oral diseases are the most common non-communicable diseases related to severe local and systemic disorders. Oral microorganisms can grow and spread in the oral mucosae and most commonly in biomaterials under polymicrobial biofilms, leading to several diseases such as dental caries and periodontal disorders. Aims: This study aimed to detect S. mutants and S. sobrinus by polymerase chain reaction (PCR) amplification and to relate the link between their presence and dental caries.&#x0D; Materials and Methods: Streptococcusmutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) strains were isolated from individuals with no caries. However, they were isolated with high counts of those organisms in saliva samples. The saliva samples were collected from the patients who attended the outdoor Dhaka Dental College Hospital from March 2018 to December 2018. PCR was performed to detect the presence of different organisms.&#x0D; Results: Prevalence of S. mutans and S. sobrinus was, respectively, 19% and 04%. About 68% of the saliva sample was not either positive for both bacterial species, whereas 09% was positive for both bacterial species.&#x0D; Conclusion: This study demonstrated that PCR is an easy, quick, and reliable method for detecting S. mutans and S. sobrinus in epidemiological studies. It was also observed that S. mutans was the most common organism than S. sobrinus to develop caries.&#x0D; TAJ 2022; 35: No-1: 07-10

In vitro evaluation of cytotoxicity in human osteoblastic cells and antimicrobial activity in the biofilm of different root canal sealers

Objective: Evaluate the cytotoxicity in human osteoblastic cells and antimicrobial activity in different root canal sealers in vitro. Methods: BioRoot RCS, TotalFill BC Sealer, and Bio-C Sealer were used in experimental groups, and AH Plus was used as a control. Human osteoblast-like cells and MTT quantitative colorimetric assay were used to evaluate cytotoxicity. Saos-2 cells were exposed to undiluted sealer extracts for 24 h. The supernatant was then collected and the formazan crystals resulting from MTT reduction were dissolved in pure dimethyl sulfoxide. Absorbance was measured in an automated spectrophotometer at a wavelength of 540 nm. Antimicrobial activity was analyzed by the direct contact test using a polymicrobial biofilm composed of Enterococcus faecalis, Candida albicans, and Streptococcus mutans. At 24, 48, and 72 h, colony-forming units were counted on agar plates. A nonparametric Kruskal-Wallis test was used for the statistical analysis. The level of significance was set at 5%. Results: AH Plus showed the lowest cytotoxicity after 24 h, with a significant difference in relation to BioRoot RCS and Bio-C Sealer (p ≤ 0.01). There was no significant difference in cytotoxicity between TotalFill BC Sealer and Bio-C Sealer (p &gt; 0.05). At 24 h, TotalFill BC Sealer and AH Plus showed the least microbial growth compared to Bio-C Sealer (p &lt; 0.05). At 48 and 72 h, there were no significant differences between sealers (p &gt; 0.05). Conclusions: AH Plus had the lowest cytotoxicity. TotalFill BC Sealer and AH Plus yielded greater reductions in microbial counts in the first 24 h compared to Bio-C Sealer. Clinical Relevance: 2c.

Engineering Antimicrobial Polymer Nanocomposites: In Situ Synthesis, Disruption of Polymicrobial Biofilms, and In Vivo Activity.

The increasing incidence of microbial infections and a limited arsenal of effective antibacterial and antifungal agents have entailed the need for new broad-spectrum therapeutics. Polymer-inorganic nanocomposites have emerged as an integral choice of antimicrobials but are limited by complicated synthesis, narrow-spectrum activity, and poor in vivo efficacy. Herein, chloride counterions of a nontoxic, moderately antibacterial polymer have been explored for in situ nanoprecipitation-based synthesis of water-soluble polymer-silver chloride nanocomposites. With the controlled release of silver ions, the nanocomposites were highly active against multidrug-resistant bacteria as well as fluconazole-resistant fungi. Alongside the elimination of metabolically inactive bacterial cells, the nanocomposites disrupted polymicrobial biofilms, unlike antibiotics and only silver-based ointments. This underlined the role of the engineered composite design, where the polymer interacted with the biofilm matrix, facilitating the penetration of nanoparticles to kill microbes. Further, the nanocomposite diminished Pseudomonas aeruginosa burden in mice skin infection (>99.9%) with no dermal toxicity proving its potential for clinical translation.

Disinfection and Biocompatibility of Titanium Surfaces Treated with Glycine Powder Airflow and Triple Antibiotic Mixture: An In Vitro Study.

The aim of this in vitro study was to compare three disinfection protocols of biofilm-coated machined (MAC) and acid etched (SLA) commercial pure Grade 4 Titanium disks. Samples were infected with a vial of polymicrobial biofilm to simulate peri-implantitis in vitro. Seventeen MAC and twenty SLA titanium disks were randomly assigned to: (1) glycine powder air-flow (GYPAP) for 1 min; (2) a local delivered triple paste antibiotic composed by a gel mixture with ciprofloxacin, metronidazole, and clarithromycin (3MIX) for 1 h; and (3) a combination of both (GYPAP + 3MIX). Biocompatibility of the titanium disks after each treatment protocol was assessed by measurement of adhesion and growth of adipose-derived mesenchymal stem cells (ASCs) after 24 and 72 h. A confocal laser scanning microscope (CLSM) assessed the antibacterial effect of each treatment. Data of the antibacterial efficacy and cell viability were presented as mean with standard deviation and calculated by one-way ANOVA with multiple comparisons via Bonferroni tests. Results were considered significant with p < 0.05. The higher cell viability was achieved by the 3MIX and GYPAP combination on the SLA surfaces after 72 h. CLSM analysis showed a mean ratio of dead bacteria statistically higher in the 3MIX + GYPAP group compared with the GYPAP and 3MIX subgroups (p < 0.05). In conclusion, data showed that the combination of GYPAP and 3MIX could be preferred to the other protocols, especially in presence of SLA titanium surface.

Polymicrobial biofilms of ocular bacteria and fungi on ex vivo human corneas

Microbes residing in biofilms confer several fold higher antimicrobial resistances than their planktonic counterparts. Compared to monomicrobial biofilms, polymicrobial biofilms involving multiple bacteria, multiple fungi or both are more dominant in nature. Paradoxically, polymicrobial biofilms are less studied. In this study, ocular isolates of Staphylococcus aureus, S. epidermidis and Candida albicans, the etiological agents of several ocular infections, were used to demonstrate their potential to form mono- and polymicrobial biofilms both in vitro and on human cadaveric corneas. Quantitative (crystal violet and XTT methods) and qualitative (confocal and scanning electron microscopy) methods demonstrated that they form polymicrobial biofilms. The extent of biofilm formation was dependent on whether bacteria and fungi were incubated simultaneously or added to a preformed biofilm. Additionally, the polymicrobial biofilms exhibited increased resistance to different antimicrobials compared to planktonic cells. When the MBECs of different antibacterial and antifungal agents were monitored it was observed that the MBECs in the polymicrobial biofilms was either identical or decreased compared to the monomicrobial biofilms. The results are relevant in planning treatment strategies for the eye. This study demonstrates that ocular bacteria and fungi form polymicrobial biofilms and exhibit increase in antimicrobial resistance compared to the planktonic cells.

A microplate‐based Response Surface Methodology model for growth optimization and biofilm formation on polystyrene polymeric material in a Candida albicans and Escherichia coli co‐culture

AbstractBiofilm formation is one of the crucial factors for virulence in Candida albicans, Escherichia coli and their polymicrobial biofilm formed on medical prosthetics. In this study, high biofilm forming C. albicans M207 from a case of invasive candidiasis and a standard strain of bacterial culture E. coli ATCC 39936 were used for initial screening and media optimization using One Variable at a time (OVAT) and Response Surface Methodology (RSM) models, respectively, in polystyrene microtiter plates for in vitro biofilm studies. Sucrose and peptone significantly affected biofilm formation when screened by OVAT. Media optimization by Central Composite Design (CCD) showed that pH significantly affected biofilm formation for all the cultures which was further validated at critical parameters. Biofilm was quantified by growth OD measurements, MTT and CV assays in the presence and absence of FBS precoating and compared with standard Trypticase Soy Broth (TSB) media which did not show any significant difference. This high throughput polystyrene microtiter plate assay for preliminary screening of nutritional components using OVAT and media optimization using RSM model is being reported for the first time for biofilm formation by C. albicans and E. coli in a single and dual species environment. This study is essential for functional analysis of in‐vitro growth and biofilm formation by these two pathogenic strains on polymeric hospital equipments and to develop suitable control measures to inhibit biofilm formation by C. albicans and E. coli single species and their co‐culture.

In vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure) against polymicrobial communities and individual bacterial strains derived from diabetic foot infections.

Introduction. Diabetic foot infection (DFI) is the main reason for diabetes-related hospitalisation and is a major cause of diabetes-related amputation. DFIs are often complicated by ischaemia in the affected limb, the presence of polymicrobial biofilms and increasingly the occurrence of antibiotic resistant bacteria.Hypothesis/Gap statement. Antibiotic loaded beads could inhibit the growth of polymicrobial DFI communities with differing compositions in vitro.Aim. This study investigates the in vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure, Biocomposites Ltd., UK) against polymicrobial DFI communities and individual bacterial strains derived from DFIs.Methodology. Debrided tissue obtained from the base of infected diabetic foot ulcers was homogenised and spread over the surface of Columbia blood agar (CBA) and fastidious anaerobe agar (FAA) plates. Calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar and following incubation, zones of inhibition (ZOI) were measured. For individual bacterial strains isolated from the infected tissue, calcium sulfate beads containing vancomycin, gentamicin, flucloxacillin or rifampicin and beads containing a combination of vancomycin and gentamicin or flucloxacillin and rifampicin were tested for their ability to inhibit growth.Results. Calcium sulfate beads loaded with a combination of vancomycin and gentamicin were able to inhibit bacterial growth from all polymicrobial tissue homogenates tested, with ZOI diameters ranging from 15 to 40 mm. In the case of individual bacterial strains, beads containing combinations of vancomycin and gentamicin or flucloxacillin and rifampicin were able to produce ZOI with Gram-positive facultatitive anaerobic strains such as Staphylococcus aureus and Enterococcus faecalis, Gram-negative facultative anaerobic strains such as Pseudomonas aeruginosa and obligate anaerobic strains such as Finegoldia magna even where acquired resistance to one of the antibiotics in the combination was evidenced.Conclusion. The local use of calcium sulfate beads containing a combination of two antibiotics demonstrated high efficacy against polymicrobial DFI communities and individual DFI bacterial strains in in vitro zone of inhibition tests. These results show promise for clinical application, but further research and clinical studies are required.

The Antibiofilm Role of Biotics Family in Vaginal Fungal Infections.

“Unity in strength” is a notion that can be exploited to characterize biofilms as they bestow microbes with protection to live freely, escalate their virulence, confer high resistance to therapeutic agents, and provide active grounds for the production of biofilms after dispersal. Naturally, fungal biofilms are inherently resistant to many conventional antifungals, possibly owing to virulence factors as their ammunitions that persistently express amid planktonic transition to matured biofilm state. These ammunitions include the ability to form polymicrobial biofilms, emergence of persister cells post-antifungal treatment and acquisition of resistance genes. One of the major disorders affecting vaginal health is vulvovaginal candidiasis (VVC) and its reoccurrence is termed recurrent VVC (RVVC). It is caused by the Candida species which include Candida albicans and Candida glabrata. The aforementioned Candida species, notably C. albicans is a biofilm producing pathogen and habitually forms part of the vaginal microbiota of healthy women. Latest research has implicated the role of fungal biofilms in VVC, particularly in the setting of treatment failure and RVVC. Consequently, a plethora of studies have advocated the utilization of probiotics in addressing these infections. Specifically, the excreted or released compounds of probiotics which are also known as postbiotics are being actively researched with vast potential to be used as therapeutic options for the treatment and prevention of VVC and RVVC. These potential sources of postbiotics are harnessed due to their proven antifungal and antibiofilm. Hence, this review discusses the role of Candida biofilm formation in VVC and RVVC. In addition, we discuss the application of pro-, pre-, post-, and synbiotics either individually or in combined regimen to counteract the abovementioned problems. A clear understanding of the role of biofilms in VVC and RVVC will provide proper footing for further research in devising novel remedies for prevention and treatment of vaginal fungal infections.