Real-time monitoring of Pseudomonas aeruginosa biofilm formation on endotracheal tubes in vitro.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for both acute and chronic infections in humans. In particular, its ability to form biofilm, on biotic and abiotic surfaces, makes it particularly resistant to host's immune defenses and current antibiotic therapies as well. Innovative antimicrobial materials, like hydrogel, silver salts or nanoparticles have been used to cover new generation catheters with promising results. Nevertheless, biofilm remains a major health problem. For instance, biofilm produced onto endotracheal tubes (ETT) of ventilated patients plays a relevant role in the onset of ventilation-associated pneumonia. Most of our knowledge on Pseudomonas aeruginosa biofilm derives from in vitro studies carried out on abiotic surfaces, such as polystyrene microplates or plastic materials used for ETT manufacturing. However, these approaches often provide underestimated results since other parameters, in addition to bacterial features (i.e. shape and material composition of ETT) might strongly influence biofilm formation. RESULTS: We used an already established biofilm development assay on medically-relevant foreign devices (CVC catheters) by a stably transformed bioluminescent (BLI)-Pseudomonas aeruginosa strain, in order to follow up biofilm formation on ETT by bioluminescence detection. Our results demonstrated that it is possible: i) to monitor BLI-Pseudomonas aeruginosa biofilm development on ETT pieces in real-time, ii) to evaluate the three-dimensional structure of biofilm directly on ETT, iii) to assess metabolic behavior and the production of microbial virulence traits of bacteria embedded on ETT-biofilm. CONCLUSIONS: Overall, we were able to standardize a rapid and easy-to-perform in vitro model for real-time monitoring Pseudomonas aeruginosa biofilm formation directly onto ETT pieces, taking into account not only microbial factors, but also ETT shape and material. Our study provides a rapid method for future screening and validation of novel antimicrobial drugs as well as for the evaluation of novel biomaterials employed in the production of new classes of ETT.

Pneumocystis jirovecii genotyping: experience in a tertiary-care hospital in Northern Italy.

Respiratory samples from Pneumocystis jirovecii pneumonia (PJP) cases collected at a tertiary-care university hospital in Modena were analyzed for the presence of specific polymorphisms in the mitochondrial large subunit ribosomal RNA (mtLSU-rRNA). Retrospectively, 57 cases were selected in a six-year period and 34 out of the 57 processed BAL samples returned PCR positive results, thus allowing further molecular analysis. The following P.jirovecii genotype distribution was observed: genotype 3 (50%), genotype 2 (23%), genotype 1 (18%), genotypes 1 or 4 (9%). These data add novel insights on P.jirovecii epidemiology, investigating a previously unstudied area of Northern Italy. A peculiar local distribution is highlighted with respect to other areas within the national panorama, thus encouraging further in-depth studies in an attempt to better understand the overall situation concerning P.jirovecii genotype circulation.

Candida albicans survival, growth and biofilm formation are differently affected by mouthwashes: an in vitro study.

Candida albicans is the most common cause of oral mycoses. The aim of the present study was to investigate in vitro the susceptibility of C. albicans to mouthwashes, in terms of growth, survival and biofilm formation. Candida albicans, laboratory strain SC5314, and 7 commercial mouthwashes were employed: 3 with 0.2% chlorhexidine digluconate; 1 with 0.06% chlorhexidine digluconate and 250 ppm F- sodium fluoride; 3 with fluorine-containing molecules. None of the mouthwashes contained ethanol in their formulations. The anti-Candida effects of the mouthwashes were assessed by disk diffusion, crystal violet and XTT assays. By using five protocols combining different dilutions and contact times the mouthwashes were tested against: 1) C. albicans growth; 2) biofilm formation; 3) survival of fungal cells in early, developing and mature Candida biofilm. Chlorhexidine digluconate-containing mouthwashes consistently exhibited the highest anti-Candida activity, irrespective of the protocols employed. Fungal growth, biofilm formation and survival of Candida cells within biofilm were impaired, the effects strictly depending on both the dilution employed and the time of contact. These in vitro studies provide evidence that mouthwashes exert anti-Candida activity against both planktonic and biofilm fungal structures, but to a different extent depending on their composition. This suggests special caution in the choice of mouthwashes for oral hygiene, whether aimed at prevention or treatment of oral candidiasis.

Leveraging Dental Stem Cells for Oral Health during Pregnancy: A Concise Review.

Pregnancy induces significant changes in oral health because of hormonal fluctuations, making it a crucial period for preventive measures. Dental stem cells (DSCs), particularly those derived from the dental pulp and periodontal ligaments, offer promising avenues for regenerative therapies and, possibly, preventive interventions. While the use of DSCs already includes various applications in regenerative dentistry in the general population, their use during pregnancy requires careful consideration. This review explores recent advancements, challenges, and prospects in using DSCs to address oral health issues, possibly during pregnancy. Critical aspects of the responsible use of DSCs in pregnant women are discussed, including safety, ethical issues, regulatory frameworks, and the need for interdisciplinary collaborations. We aimed to provide a comprehensive understanding of leveraging DSCs to improve maternal oral health.

The Spr1875 protein confers resistance to the microglia-mediated killing of Streptococcus pneumoniae.

By screening a whole-genome λ-display library of Streptococcus pneumoniae, we have previously identified a novel surface protein, named Spr1875, that exhibited immunogenic properties and was closely related to pneumococcal virulence. In the present study, we investigated the role of the Spr1875 antigen in the interaction of S. pneumoniae with microglia, the resident brain macrophages. By using an in vitro infection model, the BV2 microglial cell line was challenged with the S. pneumoniae strain DP1004 and its isogenic spr1875-deleted mutant (Δspr1875). Both strains were phagocytosed by microglia efficiently and to a similar extent; however, the DP1004 strain was more resistant than the Δspr1875 mutant to the intracellular killing, as assessed by antibiotic protection and phagosome maturation assays. Moreover, significant differences between the two strains were also observed in terms of susceptibility to microglia-mediated killing. Taken together, these results indicate that S. pneumoniae-microglial cell interplay is influenced by the presence of Spr1875, suggesting that this protein may play a role in the pathogenesis of pneumococcal meningitis.

Contribution of different pneumococcal virulence factors to experimental meningitis in mice.

BACKGROUND: Pneumococcal meningitis (PM) is a life-threatening disease with a high case-fatality rate and elevated risk for serious neurological sequelae. In this study, we investigated the contribution of three major virulence factors of Streptococcus pneumoniae, the capsule, pneumococcal surface protein A (PspA) and C (PspC), to the pathogenesis of experimental PM. METHODS: Mice were challenged by the intracranial route with the serotype 4 TIGR4 strain (wt) and three isogenic mutants devoid of PspA, PspC, and the capsule. Survival, bacterial counts, and brain histology were carried out. To study the interaction between S. pneumoniae mutants and microglia, phagocytosis and survival experiments were performed using the BV2 mouse microglial cell line. RESULTS: Virulence of the PspC mutant was comparable to that of TIGR4. In contrast, survival of animals challenged with the PspA mutant was significantly increased compared with the wt, and the mutant was also impaired at replicating in the brain and blood of infected mice. Brain histology indicated that all strains, except for the unencapsulated mutant, caused PM. Analysis of inflammation and damage in the brain of mice infected with TIGR4 or its unencapsulated mutant demonstrated that the rough strain was unable to induce inflammation and neuronal injury, even at high challenge doses. Results with BV2 cells showed no differences in phagocytic uptake between wt and mutants. In survival assays, however, the PspA mutant showed significantly reduced survival in microglia compared with the wt. CONCLUSIONS: PspA contributed to PM pathogenesis possibly by interacting with microglia at early infection stages, while PspC had limited importance in the disease. The rough mutant did not cause brain inflammation, neuronal damage or mouse death, strengthening the key role of the capsule in PM.

Attenuation of Pseudomonas aeruginosa Virulence by Pomegranate Peel Extract.

Pseudomonas aeruginosa is an opportunistic pathogen often responsible for biofilm-associated infections. The high adhesion of bacterial cells onto biotic/abiotic surfaces is followed by production of an extracellular polysaccharidic matrix and formation of a sessile community (the biofilm) by the release of specific quorum-sensing molecules, named autoinducers (AI). When the concentrations of AI reach a threshold level, they induce the expression of many virulence genes, including those involved in biofilm formation, motility, pyoverdine and pyocyanin release. P. aeruginosa embedded into biofilm becomes resistant to both conventional drugs and the host's immune response. Accordingly, biofilm-associated infections are a major clinical problem underlining the need for new antimicrobial therapies. In this study, we evaluated the effects of pomegranate peel extract (PomeGr) in vitro on P. aeruginosa growth and biofilm formation; moreover, the release of four AI was assessed. The phenolic profile of PomeGr, exposed or not to bacteria, was determined by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis. We found that bacterial growth, biofilm production and AI release were impaired upon PomeGr treatment. In addition, the PomeGr phenolic content was also markedly hampered following incubation with bacterial cells. In particular, punicalagin, punicalin, pedunculagin, granatin, di-(HHDP-galloyl-hexoside) pentoside and their isomers were highly consumed. Overall, these results provide novel insights on the ability of PomeGr to attenuate P. aeruginosa virulence; moreover, the AI impairment and the observed consumption of specific phenolic compounds may offer new tools in designing innovative therapeutic approaches against bacterial infections.

Pomegranate Extract Affects Fungal Biofilm Production: Consumption of Phenolic Compounds and Alteration of Fungal Autoinducers Release.

Candida albicans expresses numerous virulence factors that contribute to pathogenesis, including its dimorphic transition and even biofilm formation, through the release of specific quorum sensing molecules, such as the autoinducers (AI) tyrosol and farnesol. In particular, once organized as biofilm, Candida cells can elude conventional antifungal therapies and the host's immune defenses as well. Accordingly, biofilm-associated infections become a major clinical challenge underlining the need of innovative antimicrobial approaches. The aim of this in vitro study was to assess the effects of pomegranate peel extract (PomeGr) on C. albicans growth and biofilm formation; in addition, the release of tyrosol and farnesol was investigated. The phenolic profile of PomeGr was assessed by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis before and after exposure to C. albicans. Here, we showed that fungal growth, biofilm formation and AI release were altered by PomeGr treatment. Moreover, the phenolic content of PomeGr was substantially hampered upon exposure to fungal cells; particularly pedunculagin, punicalin, punicalagin, granatin, di-(HHDP-galloyl-hexoside)-pentoside and their isomers as well as ellagic acid-hexoside appeared highly consumed, suggesting their role as bioactive molecules against Candida. Overall, these new insights on the anti-Candida properties of PomeGr and its potential mechanisms of action may represent a relevant step in the design of novel therapeutic approaches against fungal infections.

Plasminogen- and fibronectin-binding protein B is involved in the adherence of Streptococcus pneumoniae to human epithelial cells.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading frame spr0075 of the strain R6 genome. This locus encodes for an approximately 120-kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and six repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, because fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C-terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, whereas sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggest that, by directly interacting with fibronectin, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.

Role of the (Mn)superoxide dismutase of Enterococcus faecalis in the in vitro interaction with microglia.

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severe nosocomial and community-acquired infections. Although enterococcal meningitis is rare, mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of this infection remain poorly understood, even though the ability of E. faecalis to avoid or survive phagocytic attack in vivo may be very important during the infection process. We previously showed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis was implicated in oxidative stress responses and, interestingly, in the survival within mouse peritoneal macrophages using an in vivo-in vitro infection model. In the present study, we investigated the role of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. By using an in vitro infection model, murine microglial cells were challenged in parallel with the wild-type strain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosed by microglia efficiently and to a similar extent, the ΔsodA mutant was found to be significantly more susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protection assay. In addition, a significantly higher percentage of acidic ΔsodA-containing phagosomes was found and these also underwent enhanced maturation as determined by the expression of endolysosomal markers. In conclusion, these results show that the MnSOD of E. faecalis contributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity, and this could even be important for intracellular killing in neutrophils and thus for E. faecalis pathogenesis.

The protein "mycoarray": a novel serological assay for the laboratory diagnosis of primitive endemic mycoses.

A protein microarray containing fungal antigens (the "mycoarray") has been set up to provide rapid and appropriate serodiagnosis of primitive endemic mycoses, an important cause of morbidity and mortality in an increasingly high number of patients. The mycoarray consists of three antigen extracts (histoplasmin, coccidioidin and Coccidioides "TP") and antibody dilution curves were spotted on microarray slides. The arrays were processed with coccidioidomycosis and histoplasmosis patients� sera or with control sera and the occurring immunocomplexes were detected by indirect immunofluorescence. In agreement with clinical and microbiological diagnosis, the results distinguished between histoplasmosis and coccidioidomycosis patients. In addition, the assay could clearly discriminate between IgM and IgG antibody reactivity. No reactivity was ever observed in the arrays processed with negative control sera. Therefore, this pilot study demonstrates that the "mycoarray" is sensitive and specific enough to discriminate between healthy individuals and patients with histoplasmosis or coccidioidomycosis. Because of miniaturization and multiparametricity, the new assay cuts costs and processing time. Thus, once clinically validated and implemented as a large-scale array, the "mycoarray" will be ready to be applied to the daily clinical practice.

The ß-Lactamase Inhibitor Boronic Acid Derivative SM23 as a New Anti-Pseudomonas aeruginosa Biofilm.

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great capacity to form biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), a cell-to-cell communication mechanism used by many bacteria. Selective inhibition of QS-controlled pathogenicity without affecting bacterial growth may represent a novel promising strategy to overcome the well-known and widespread drug resistance of P. aeruginosa. In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as ß-lactamase inhibitor, on biofilm formation and virulence factors production by P. aeruginosa. Our results indicated that SM23: (1) inhibited biofilm development and production of several virulence factors, such as pyoverdine, elastase, and pyocyanin, without affecting bacterial growth; (2) decreased the levels of 3-oxo-C12-HSL and C4-HSL, two QS-related autoinducer molecules, in line with a dampened lasR/lasI system; (3) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; and (4) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model closely mimicking clinical settings. Taken together, our results indicate that, besides inhibiting ß-lactamase, SM23 can also act as powerful inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.

Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA) Impair Neutrophil Candidacidal Activity and Are Increased in the Cellular Fraction of Vaginal Samples from Women with Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) is primarily caused by Candida albicans and affects 75% of childbearing age women. Although C. albicans can colonize asymptomatically, disease is associated with an increased Candida burden, a loss of epithelial tolerance and a breakdown in vaginal microbiota homeostasis. VVC symptoms have been ascribed to a powerful inflammatory response associated with the infiltration of non-protective neutrophils (PMN). Here, we compared the immunological characteristics of vaginal fluids and cellular protein extracts obtained from 28 VVC women and from 23 healthy women colonized by Candida spp. We measured the levels of antibodies against fungal antigens and human autoantigens (anti-Saccharomyces cerevisiae antibodies (ASCA), C. albicans germ tube antibodies (CAGTAs) and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA)), in addition to other immunological markers. Our results show that the pANCA levels detected in the cellular protein extracts from the vaginal fluids of symptomatic women were significantly higher than those obtained from healthy colonized women. Consistent with a potential physiologically relevant role for this pANCA, we found that specific anti-myeloperoxidase antibodies could completely neutralize the ex vivo killing capacity of polymorphonuclear cells. Collectively, this preliminary study suggests for the first time that pANCA are found in the pathogenic vaginal environment and can promptly impair neutrophil function against Candida, potentially preventing a protective response.

Safety and immunogenicity of an intramuscular Helicobacter pylori vaccine in noninfected volunteers: a phase I study.

INTRODUCTION: Helicobacter pylori infection is among the most common human infections and the major risk factor for peptic disease and gastric cancer. Immunization with vaccines containing the H pylori vacuolating cytotoxin A (VacA), cytotoxin-associated antigen (CagA), and neutrophil-activating protein (NAP), alone or in combination, have been shown to prevent experimental infection in animals. AIM: We sought to study the safety and immunogenicity of a vaccine consisting of recombinant VacA, CagA, and NAP given intramuscularly with aluminium hydroxide as an adjuvant to noninfected healthy subjects. METHODS: This controlled, single-blind Phase I study randomized 57 H pylori-negative volunteers into 7 study arms exploring 2 dosages (10 and 25 microg) of each antigen and 3 schedules (0, 1, 2 weeks; 0, 1, 2 months; and 0, 1, 4 months) versus alum controls. All participants were followed for 5 months. Thirty-six subjects received a booster vaccination 18-24 months after the completion of the primary vaccination. RESULTS: Local and systemic adverse reactions were mild and similar in placebo and vaccine recipients on the monthly schedules. All subjects responded to 1 or 2 of the antigens and 86% of all vaccines mounted immunoglobulin G antibody responses to all 3 antigens. Vaccinees exhibited an antigen-specific cellular response. Vaccination 18-24 months later elicited anamnestic antibody and cellular responses. CONCLUSIONS: This intramuscular H pylori vaccine demonstrated satisfactory safety and immunogenicity, produced antigen-specific T-cell memory, and, therefore, warrants further clinical study.

The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation.

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 (AFR1) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1-overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1Delta mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1-overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans-microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.

Antimicrobial and antibiofilm efficacy of a copper/calcium hydroxide-based endodontic paste against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans.

Endodontic biofilm is a microbial community, enclosed in a polymeric matrix of polysaccharide origin where are found pathogens, like bacteria and opportunistic fungi responsible for various endodontic pathologies. As clinical importance is the fact, that biofilm is extremely resistant to common intracanal irrigants, antimicrobial drugs and host immune responses. The aim of this study was to evaluate the in vitro efficacy of a Cu/CaOH2-based endodontic paste, against bacteria and fungi, such as Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. We found that such compound significantly reduced microbial replication time and cell growth. Moreover, biofilm formation and persistence were also affected; treated biofilms showed both a reduced number of cells and levels of released pyoverdine. This study provides the first evidence on effectiveness of this endodontic compound against microbial biofilms. Given its wide range of action, its use in prevention and treatment of the main oral biofilm-associated infections will be discussed.

Longitudinal Survey of Fungi in the Human Gut: ITS Profiling, Phenotyping, and Colonization.

The fungal component of the intestinal microbiota of eight healthy subjects was studied over 12 months using metagenome survey and culture-based approaches. Aspergillus, Candida, Debaryomyces, Malassezia, Penicillium, Pichia, and Saccharomyces were the most recurrent and/or dominant fungal genera, according to metagenomic analysis. The biodiversity of fungal communities was lower and characterized by greater unevenness, when compared to bacterial microbiome. The dissimilarities both among subjects and over the time within the same subject suggested that most of the fungi passed through the gastro-intestinal tract (GIT) without becoming stable colonizers. Certain genera, such as Aspergillus and Penicillium, were isolated in a minority of cases, although they recurred abundantly and frequently in the metagenomics survey, likely being environmental or food-borne fungi that do not inhabit the GIT. Candida genus was recurrently detected. Candida albicans isolates dominated among the cultivable mycobiota and longitudinally persisted, likely as commensals inhabiting the intestine or regularly reaching it from Candida-colonized districts, such as the oral cavity. Other putative colonizers belonged to Candida zeylanoides, Geotrichum candidum, and Rhodotorula mucilaginosa, with persisting biotypes being identified. Phenotyping of fungal isolates indicated that C. albicans adhered to human epithelial cells more efficiently and produced greater amounts of biofilm in vitro than non-albicans Candida (NAC) and non-Candida fungi (NCF). The C. albicans isolates also induced the highest release of HBD-2 by human epithelial cells, further differing from NAC and NCF. Nine representative isolates were administered to mice to evaluate the ability to colonize the intestine. Only two out of three C. albicans strains persisted in stools of animals 2 weeks after the end of the oral administration, whereas NAC and NCF did not. These results confirm the allochthonous nature of most the intestinal fungi, while C. albicans appears to be commonly involved in stable colonization. A combination of specific genetic features in the microbe and in the host likely allow colonization from fungi normally present solely as passengers. It remains to be established if other species identified as potential colonizers, in addition to Candida, are true inhabitants of the GIT or rather reach the intestine spreading from other body districts.

Identification and characterization of an aspartyl protease from Cryptococcus neoformans.

Cryptococcosis, caused by Cryptococcus neoformans, is an invasive infection often occurring in AIDS patients. Potent therapy against HIV, which includes protease inhibitors (PIs), has beneficial effects also on opportunistic infections by pathogens such as C. neoformans and C. albicans. PIs inhibit growth of C. albicans by affecting the activity of its aspartyl proteases. We identified, cloned and sequenced a cDNA from C. neoformans encoding for a putative aspartyl protease (CnAP1), and the corresponding genomic region. The gene cnap1 codifies for a protein of 505 aa, with a canonical aspartyl protease structure. We purified the recombinant protein and analyzed its activity in the presence of PIs (Indinavir, Lopinavir, Ritonavir), but did not evidence any inhibition of protease activity. The transcriptional level of cnap1 in C. neoformans is constant in different media. The absence of any inhibition activity by PIs suggests that other targets for PIs might exist in C. neoformans.

Genomic and Phenotypic Variation in Morphogenetic Networks of Two Candida albicans Isolates Subtends Their Different Pathogenic Potential.

The transition from commensalism to pathogenicity of Candida albicans reflects both the host inability to mount specific immune responses and the microorganism's dimorphic switch efficiency. In this study, we used whole genome sequencing and microarray analysis to investigate the genomic determinants of the phenotypic changes observed in two C. albicans clinical isolates (YL1 and YQ2). In vitro experiments employing epithelial, microglial, and peripheral blood mononuclear cells were thus used to evaluate C. albicans isolates interaction with first line host defenses, measuring adhesion, susceptibility to phagocytosis, and induction of secretory responses. Moreover, a murine model of peritoneal infection was used to compare the in vivo pathogenic potential of the two isolates. Genome sequence and gene expression analysis of C. albicans YL1 and YQ2 showed significant changes in cellular pathways involved in environmental stress response, adhesion, filamentous growth, invasiveness, and dimorphic transition. This was in accordance with the observed marked phenotypic differences in biofilm production, dimorphic switch efficiency, cell adhesion, invasion, and survival to phagocyte-mediated host defenses. The mutations in key regulators of the hyphal growth pathway in the more virulent strain corresponded to an overall greater number of budding yeast cells released. Compared to YQ2, YL1 consistently showed enhanced pathogenic potential, since in vitro, it was less susceptible to ingestion by phagocytic cells and more efficient in invading epithelial cells, while in vivo YL1 was more effective than YQ2 in recruiting inflammatory cells, eliciting IL-1ß response and eluding phagocytic cells. Overall, these results indicate an unexpected isolate-specific variation in pathways important for host invasion and colonization, showing how the genetic background of C. albicans may greatly affect its behavior both in vitro and in vivo. Based on this approach, we propose that the co-occurrence of changes in sequence and expression in genes and pathways driving dimorphic transition and pathogenicity reflects a selective balance between traits favoring dissemination of the pathogen and traits involved in host defense evasion. This study highlights the importance of investigating strain-level, rather than species level, differences, when determining fungal-host interactions and defining commensal or pathogen behavior.

The synthetic killer peptide KP impairs Candida albicans biofilm in vitro.

Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections.