Transcription Factors of CAT1, EFG1, and BCR1 Are Effective in Persister Cells of Candida albicans-Associated HIV-Positive and Chemotherapy Patients

BackgroundBiofilm is an accumulation of cells, which are formed on mucosal surfaces of the host as well as on medical devices. The inherent resistance of Candida strains producing biofilms to antimicrobial agents is an important and key feature for biofilm growth, which can lead to treatment failure. This resistance is due to the regulatory increase of the output pumps, the presence of extracellular matrix, and the existence of persister cells. Persister cells are phenotypic variants that have MICs similar to antibiotic-sensitive populations and are able to tolerate high doses of antibiotics. The current study investigated the possible role of EFG1, BCR1, and CAT1 in the establishment or maintenance of persister cells in Candida albicans strains that produce biofilms.MethodsAfter identifying Candida isolates by molecular methods, C. albicans isolates were confirmed by sequencing. Isolation of persister cells and determination of their MIC were performed by microdilution method. Then, RNA extraction and cDNA synthesis were performed from 60 C. albicans isolates under promoting and inducing conditions. Afterward, the mean expression of BCR1, EFG1, and CAT1 genes in both persister and non-persister groups was calculated using real-time qPCR. Phylogeny tree of persister and non-persister group isolates was drawn using ITS fragment.ResultsA total of 77 persister isolates were taken from the oral cavity of HIV patients as well as from patients undergoing chemotherapy. Biofilm intensity in persister isolates separated from HIV-infected patients was different from the non-persister group. The mean fold change of BCR1 (10.73), CAT1 (15.34), and EFG1 (2.41) genes in persister isolates was significantly higher than these genes in isolates without persister.ConclusionIt can be concluded that the most important factor in the production of persister cells is biofilm binding and production, not biofilm development or mature biofilm production, which was found in the expression of BCR1 gene without change in the expression of EFG1 gene in the persister group. Also, catalase plays an essential role in the production of persister in C. albicans biofilm producers with ROS detoxification.

Download Full-text

Improvement of XTT assay performance for studies involving Candida albicans biofilms

Brazilian Dental Journal ◽

10.1590/s0103-64402008000400014 ◽

2008 ◽

Vol 19 (4) ◽

pp. 364-369 ◽

Cited By ~ 68

Author(s):

Wander José da Silva ◽

Jayampath Seneviratne ◽

Nipuna Parahitiyawa ◽

Edvaldo Antonio Ribeiro Rosa ◽

Lakshman Perera Samaranayake ◽

...

Keyword(s):

Candida Albicans ◽

Biofilm Formation ◽

Cell Activity ◽

Growth Yield ◽

Xtt Assay ◽

Biofilm Growth ◽

Assay Performance ◽

Reduction Assay ◽

Cell Layers ◽

Biofilm Development

2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used to study Candida biofilm formation. However, considering that the XTT reduction assay is dependent on cell activity, its use for evaluating mature biofilms may lead to inaccuracies since biofilm bottom cell layers tend to be relatively quiescent at later stages of biofilm formation. The aim of this study was to improve XTT reduction assay by adding glucose supplements to the standard XTT formulation. Candida albicans ATCC 90028 was used to form 24-, 48- and 72-h biofilms. The oxidative activity at 90, 180 and 270 min of incubation was evaluated. The control consisted of standard XTT formulation without glucose supplements, and was modified by the addition of 50, 100 and 200 mM of glucose. The XTT assay with 200 mM glucose showed more accurate and consistent readings correlating with biofilm development at 24, 48 and 72 h. Biofilm growth yield after 180 min incubation, when evaluated with the 200 mM glucose supplemented XTT, produced the most consistent readings on repetitive testing. It may be concluded that glucose supplementation of XTT could minimize variation and produce more accurate data for the XTT assay.

Download Full-text

Candida albicansbiofilms are generally devoid of persister cells

10.1101/420596 ◽

2018 ◽

Author(s):

Iryna Denega ◽

Christophe d’Enfert ◽

Sophie Bachellier-Bassi

Keyword(s):

Extracellular Matrix ◽

Candida Albicans ◽

Antimicrobial Agents ◽

Laboratory Strain ◽

Clinical Isolates ◽

Wild Type ◽

Persister Cells ◽

High Concentrations ◽

Ambiguous Data ◽

Very High

AbstractCandida albicansis known for its ability to form biofilms – communities of microorganisms embedded in an extracellular matrix developing on different surfaces. Biofilms are highly tolerant to antifungal therapy. This phenomenon has been partially explained by the appearance of so-called persister cells, phenotypic variants of wild-type cells, capable of surviving very high concentrations of antimicrobial agents. Persister cells inC. albicanswere found exceptionally in biofilms while none were detected in planktonic cultures of this fungus. Yet, this topic remains controversial as others could not observe persister cells in biofilms formed by theC. albicansSC5314 laboratory strain. Due to ambiguous data in the literature, this work aimed to reevaluate the presence of persister cells inC. albicansbiofilms. We demonstrated that isolation ofC. albicans“persister cells” as described previously was likely to be the result of survival of biofilm cells that were not reached by the antifungal. We tested biofilms of SC5314 and its derivatives, as well as 95 clinical isolates, using an improved protocol, demonstrating that persister cells are not a characteristic trait ofC. albicansbiofilms. Although some clinical isolates are able to yield survivors upon the antifungal treatment of biofilms, this phenomenon is rather stochastic and inconsistent.

Download Full-text

Patients with Long-Term Oral Carriage Harbor High-Persister Mutants of Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00860-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 39-44 ◽

Cited By ~ 139

Author(s):

Michael D. LaFleur ◽

Qingguo Qi ◽

Kim Lewis

Keyword(s):

Candida Albicans ◽

Amphotericin B ◽

Antimicrobial Agents ◽

Oral Candidiasis ◽

Wild Type ◽

Persister Cells ◽

Microtiter Plates ◽

High Concentrations

ABSTRACT Fungal biofilms produce a small number of persister cells which can tolerate high concentrations of fungicidal agents. Persisters form upon attachment to a surface, an important step in the pathogenesis of Candida strains. The periodic application of antimicrobial agents may select for strains with increased levels of persister cells. In order to test this possibility, 150 isolates of Candida albicans and C. glabrata were obtained from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day. Persister levels were measured by exposing biofilms growing in the wells of microtiter plates to high concentrations of amphotericin B and plating for survivors. The persister levels of the isolates varied from 0.2 to 9%, and strains isolated from patients with long-term carriage had high levels of persisters. High-persister strains were isolated from every patient with Candida carriage of more than 8 consecutive weeks but from no patients with transient carriage. All of the high-persister isolates had an amphotericin B MIC that was the same as that for the wild type, indicating that these strains were drug-tolerant rather than drug-resistant mutants. Biofilms of the majority of high-persister strains also showed an increased tolerance to chlorhexidine and had the same MIC for this antimicrobial as the wild type. This study suggests that persister cells are clinically relevant, and antimicrobial therapy selects for high-persister strains in vivo. The drug tolerance of persisters may be a critical but overlooked component responsible for antimicrobial drug failure and relapsing infections.

Download Full-text

Hypoxic Adaptation by Efg1 Regulates Biofilm Formation by Candida albicans

Applied and Environmental Microbiology ◽

10.1128/aem.00098-09 ◽

2009 ◽

Vol 75 (11) ◽

pp. 3663-3672 ◽

Cited By ~ 55

Author(s):

Catrin Stichternoth ◽

Joachim F. Ernst

Keyword(s):

Candida Albicans ◽

Biofilm Formation ◽

Transcriptional Control ◽

Sulfur Metabolism ◽

Systemic Infection ◽

Inducible Promoter ◽

Biofilm Growth ◽

Hypoxic Adaptation ◽

Biofilm Development

ABSTRACT Hypoxia is encountered frequently by Candida albicans during systemic infection of the human host. We tested if hypoxia allows biofilm formation by C. albicans, which is a major cause of perseverance and antifungal resistance in C. albicans infections. Using an in vitro biofilm system, we unexpectedly discovered that several positive regulators of biofilm formation during normoxia, including Tec1, Ace2, Czf1, Och1, and Als3, had little or no influence on biofilm development during hypoxia, irrespective of the carbon dioxide level, indicating that C. albicans biofilm pathways differ depending on the oxygen level. In contrast, the Efg1 and Flo8 regulators were required for both normoxic and hypoxic biofilm formation. To explore the role of Efg1 during hypoxic and/or biofilm growth, we determined transcriptome kinetics following release of EFG1 expression by a system under transcriptional control of a doxycycline-inducible promoter. During hypoxia, Efg1 rapidly induced expression of all major classes of genes known to be associated with normoxic biofilm formation, including genes involved in glycolysis, sulfur metabolism, and antioxidative and peroxisome activities, as well as genes for iron uptake. The results suggest that hypoxic adaptation mediated by the Efg1 and Flo8 regulators is required even during normoxic biofilm development, while hypoxic biofilm formation in deep tissues or in organs may generate foci of C. albicans infections.

Download Full-text

Trichosporon asahii and Trichosporon inkin Biofilms Produce Antifungal-Tolerant Persister Cells

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.645812 ◽

2021 ◽

Vol 11 ◽

Author(s):

Rossana de Aguiar Cordeiro ◽

Ana Luiza Ribeiro Aguiar ◽

Bruno Nascimento da Silva ◽

Lívia Maria Galdino Pereira ◽

Fernando Victor Monteiro Portela ◽

...

Keyword(s):

Inhibitory Concentration ◽

Growth Control ◽

Persister Cells ◽

Biofilm Growth ◽

Trichosporon Asahii ◽

Microbial Biofilms ◽

Microtiter Plates ◽

Trichosporon Inkin ◽

Dormant Cells ◽

High Doses

Persister cells are metabolically inactive dormant cells that lie within microbial biofilms. They are phenotypic variants highly tolerant to antimicrobials and, therefore, associated with recalcitrant infections. In the present study, we investigated if Trichosporon asahii and T. inkin are able to produce persister cells. Trichosporon spp. are ubiquitous fungi, commonly found as commensals of the human skin and gut microbiota, and have been increasingly reported as agents of fungemia in immunocompromised patients. Biofilms derived from clinical strains of T asahii (n=5) and T. inkin (n=7) were formed in flat-bottomed microtiter plates and incubated at 35°C for 48 h, treated with 100 μg/ml amphotericin B (AMB) and incubated at 35°C for additional 24 h. Biofilms were scraped from the wells and persister cells were assayed for susceptibility to AMB. Additionally, we investigated if these persister cells were able to generate new biofilms and studied their ultrastructure and AMB susceptibility. Persister cells were detected in both T asahii and T. inkin biofilms and showed tolerance to high doses of AMB (up to 256 times higher than the minimum inhibitory concentration). Persister cells were able to generate biofilms, however they presented reduced biomass and metabolic activity, and reduced tolerance to AMB, in comparison to biofilm growth control. The present study describes the occurrence of persister cells in Trichosporon spp. and suggests their role in the reduced AMB susceptibility of T. asahii and T. inkin biofilms.

Download Full-text

Fibrin Biopolymer Incorporated with Antimicrobial Agents: A Proposal for Coating Denture Bases

Materials ◽

10.3390/ma14071618 ◽

2021 ◽

Vol 14 (7) ◽

pp. 1618

Author(s):

Helena Sandrini Venante ◽

Ana Paula Chappuis-Chocano ◽

Oscar Oswaldo Marcillo-Toala ◽

Rafaela Alves da Silva ◽

Rodrigo Moreira Bringel da Costa ◽

...

Keyword(s):

Laser Scanning ◽

Antimicrobial Agents ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Alcoholic Extract ◽

Base Surface ◽

Biofilm Growth ◽

Denture Stomatitis ◽

Colony Forming Unit ◽

Biofilm Development

The characteristics of the denture base surface, in combination with the oral environment, promote the colonization and development of Candida albicans biofilm, which is the main cause of denture stomatitis. This study evaluated the effectiveness of fibrin biopolymer with digluconate chlorhexidine or Punica granatum alcoholic extract to prevent C. albicans biofilm. Conventional heat polymerized and pre-polymerized poly(methyl methacrylate) (PMMA) circular specimens (10 × 2 mm) were fabricated (n = 504) and randomly divided into groups: no treatment (control—CT), fibrin biopolymer coating (FB), fibrin biopolymer with P. granatum (FBPg), or digluconate of chlorhexidine (FBCh) coating. The specimens were inoculated with C. albicans SC5314 (1 × 107 cells/mL) and incubated for 24, 48, and 72 h. Crystal violet and colony-forming unit assays were used to quantify the total biofilm biomass and biofilm-living cells. A qualitative analysis was performed using confocal laser scanning microscopy. Data obtained are expressed as means and standard deviations and were statistically analyzed using a three-way analysis of variance (α = 0.05). The FBPg and FBCh groups inhibited the growth of C. albicans biofilm in both PMMA materials analyzed, with FBCh performing better in all periods evaluated (p < 0.0001). The colony forming unit (CFU) assay showed that the FB group favored the C. albicans biofilm growth at 24 h and 48 h (p < 0.0001), with no differences with CT group at 72 h (p = 0.790). All groups showed an enhancement in biofilm development up to 72 h (p < 0.0001), except the FBCh group (p = 0.100). No statistical differences were found between the PMMA base materials (p > 0.050), except in the FB group (p < 0.0001). Fibrin biopolymer, albeit a scaffold for the growth of C. albicans, when combined with chlorhexidine digluconate or P. granatum, demonstrated excellent performance as a drug delivery system, preventing and controlling the formation of denture biofilm.

Download Full-text

Bifunctional Composites for Biofilms Modulation on Cervical Restorations

Journal of Dental Research ◽

10.1177/00220345211018189 ◽

2021 ◽

pp. 002203452110181

Author(s):

A.A. Balhaddad ◽

I.M. Garcia ◽

L. Mokeem ◽

M.S. Ibrahim ◽

F.M. Collares ◽

...

Keyword(s):

Free Energy ◽

Surface Free Energy ◽

Contact Angles ◽

Bacterial Biofilm ◽

Dental Composite ◽

Log P ◽

Rrna Gene ◽

Cervical Lesions ◽

Biofilm Growth ◽

Biofilm Development

Cervical composites treating root carious and noncarious cervical lesions usually extend subgingivally. The subgingival margins of composites present poor plaque control, enhanced biofilm accumulation, and cause gingival irritation. A potential material to restore such lesions should combine agents that interfere with bacterial biofilm development and respond to acidic conditions. Here, we explore the use of new bioresponsive bifunctional dental composites against mature microcosm biofilms derived from subgingival plaque samples. The designed formulations contain 2 bioactive agents: dimethylaminohexadecyl methacrylate (DMAHDM) at 3 to 5 wt.% and 20 wt.% nanosized amorphous calcium phosphate (NACP) in a base resin. Composites with no DMAHDM and NACP were used as controls. The newly formulated 5% DMAHDM–20% NACP composite was analyzed by micro-Raman spectroscopy and transmission electron microscopy. The wettability and surface-free energy were also assessed. The inhibitory effect on the in vitro biofilm growth and the 16S rRNA gene sequencing of survival bacterial colonies derived from the composites were analyzed. Whole-biofilm metabolic activity, polysaccharide production, and live/dead images of the biofilm grown over the composites complement the microbiological assays. Overall, the designed formulations had higher contact angles with water and lower surface-free energy compared to the commercial control. The DMAHDM-NACP composites significantly inhibited the growth of total microorganisms, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum by 3 to 5-log ( P < 0.001). For the colony isolates from control composites, the composition was typically dominated by the genera Veillonella, Fusobacterium, Streptococcus, Eikenella, and Leptotrichia, while Fusobacterium and Veillonella dominated the 5% DMAHDM–20% NACP composites. The DMAHDM-NACP composites contributed to over 80% of reduction in metabolic and polysaccharide activity. The suppression effect on plaque biofilms suggested that DMAHDM-NACP composites might be used as a bioactive material for cervical restorations. These results may propose an exciting path to prevent biofilm growth and improve dental composite restorations’ life span.

Download Full-text

Evaluation of Antifungal Activity by Mixed Oxide Metallic Nanocomposite against Candida spp.

Processes ◽

10.3390/pr9050773 ◽

2021 ◽

Vol 9 (5) ◽

pp. 773

Author(s):

Ayodeji Precious Ayanwale ◽

Brenda Lizbeth Estrada-Capetillo ◽

Simón Yobanny Reyes-López

Keyword(s):

Antifungal Activity ◽

Antimicrobial Agents ◽

Mononuclear Cells ◽

Promising Result ◽

Sol Gel ◽

Group Analysis ◽

Candida Spp ◽

Human Mononuclear Cells ◽

Viability Study ◽

High Doses

High doses of antimicrobial agents are a huge threat due to the increasing number of pathogenic organisms that are becoming resistant to antimicrobial agents. This resistance has led to a search for alternatives. Therefore, this study presents the synthesis and characterization of ZrO2-Ag2O nanoparticles (NPs) by sol-gel. The NPs were analyzed by dynamic light scattering (DLS), UV-visible (UV-vis), Raman and scanning electron microscopy (SEM). The NPs were later evaluated for their antifungal effects against Candidaalbicans, Candida dubliniensis, Candida glabrata, and Candida tropicalis, using disc diffusion and microdilution methods, followed by the viability study. The DLS showed sizes for ZrO2 76 nm, Ag2O 50 nm, and ZrO2-Ag2O samples between 14 and 42 nm. UV-vis shows an absorption peak at 300 nm for ZrO2 and a broadband for Ag2O NPs. Raman spectra were consistent with factor group analysis predictions. SEM showed spherically shaped NPs. The antifungal activity result suggested that ZrO2-Ag2O NPs were effective against Candida spp. From the viability study, there was no significance difference in viability as a function of time and concentration on human mononuclear cells. This promising result can contribute toward the development of alternative therapies to treat fungal diseases in humans.

Download Full-text

Intravenous lidocaine reduces perioperative opioids without negatively affecting the electrical stapedial reflex threshold in pediatric cochlear implants

Ain-Shams Journal of Anesthesiology ◽

10.1186/s42077-021-00153-7 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

W. Z. Bakhet ◽

L. M. El Fiky ◽

H. A. Debis

Keyword(s):

Placebo Group ◽

Cochlear Implants ◽

Opioid Consumption ◽

Intravenous Lidocaine ◽

Lidocaine Group ◽

Registration Number ◽

Stapedial Reflex ◽

The Mean ◽

Reflex Threshold ◽

High Doses

Abstract Background Total intravenous anesthesia (TIVA) with propofol and remifentanil is frequently used for pediatric cochlear implants (CIs) surgery as it does not suppress the electrical stapedial reflex threshold (ESRT). However, high doses of remifentanil exacerbate postoperative pain and increase opioid consumption. Intravenous lidocaine reduces pain and opioid requirement. This study investigated the effect of intravenous lidocaine on perioperative opioid consumption and ESRT in pediatric CIs. Results The mean (95% CI) remifentanil consumption was significantly lower in lidocaine group than in placebo group [0.57 (0.497–0.643) vs 0.69 (0.63–0.75)] μg/kg/min, P = 0.016. The mean (95% CI) propofol consumption was significantly lower in lidocaine group than in placebo group [155.5 (146–165) vs 171 (161–181) μg/kg/min, P = 0.02. MBP and HR were significantly lower after surgical incision, laryngeal mask airway (LMA) removal, and at PACU admission in the lidocaine group compared with the placebo group. The PACU pain score was significantly lower in the lidocaine group compared to the placebo group. The mean (95% CI) pethidine consumption in PACU was significantly lower in the lidocaine group than in the placebo group 7.0 (6.17–7.83) vs. 8.9 (7.84–9.96) mg, P = 0.012. There were no differences between groups regarding ESRT response. Conclusions Intravenous lidocaine infusion reduced perioperative opioid requirements without altering the ESRT in pediatric CIs. Trial registration Clinical registration number: NCT04194294.

Download Full-text

Arginine Is a Critical Substrate for the Pathogenesis of Pseudomonas aeruginosa in Burn Wound Infections

mBio ◽

10.1128/mbio.02160-16 ◽

2017 ◽

Vol 8 (2) ◽

Cited By ~ 16

Author(s):

Jake Everett ◽

Keith Turner ◽

Qiuxian Cai ◽

Vernita Gordon ◽

Marvin Whiteley ◽

...

Keyword(s):

Antimicrobial Agents ◽

Hospital Setting ◽

Multidrug Resistant ◽

Burn Patients ◽

Burn Wound ◽

Burn Wounds ◽

Resistant Strains ◽

High Doses ◽

Ubiquitous Presence

ABSTRACT Environmental conditions affect bacterial behavior and can greatly influence the course of an infection. However, the environmental cues that elicit bacterial responses in specific infection sites are relatively unknown. Pseudomonas aeruginosa is ubiquitous in nature and typically innocuous. However, it is also one of the most prevalent causes of fatal sepsis in burn wound patients. The aim of this study was to determine the impact of environmental factors, specifically the availability of arginine, on the pathogenesis of P. aeruginosa in burn wound infections. Comparison of burned versus noninjured tissue revealed that l-arginine (l-Arg) was significantly depleted in burn wounds as a consequence of elevated arginase produced by myeloid-derived suppressor cells. We also observed that l-Arg was a potent chemoattractant for P. aeruginosa, and while low concentrations of l-Arg increased P. aeruginosa’s swimming motility, high concentrations resulted in diminished swimming. Based on these observations, we tested whether the administration of exogenous l-Arg into the burn wound could attenuate the virulence of P. aeruginosa in thermally injured mice. Administration of l-Arg resulted in decreased P. aeruginosa spread and sepsis and increased animal survival. Taken together, these data demonstrate that the availability of environmental arginine greatly influences the virulence of P. aeruginosa in vivo and may represent a promising phenotype-modulating tool for future therapeutic avenues. IMPORTANCE Despite our growing understanding of the pathophysiology of burn wounds and the evolution of techniques and practices to manage infections, sepsis remains a significant medical concern for burn patients. P. aeruginosa continues to be a leader among all causes of bacteremic infections due to its tendency to cause complications in immunocompromised patients and its ubiquitous presence in the hospital setting. With the unforgiving emergence of multidrug-resistant strains, it is critical that alternative strategies to control or prevent septic infections in burn patients be developed in parallel with novel antimicrobial agents. In this study, we observed that administration of l-Arg significantly reduced bacterial spread and sepsis in burned mice infected with P. aeruginosa. Given the safety of l-Arg in high doses and its potential wound-healing benefits, this conditionally essential amino acid may represent a useful tool to modulate bacterial behavior in vivo and prevent sepsis in burn patients. IMPORTANCE Despite our growing understanding of the pathophysiology of burn wounds and the evolution of techniques and practices to manage infections, sepsis remains a significant medical concern for burn patients. P. aeruginosa continues to be a leader among all causes of bacteremic infections due to its tendency to cause complications in immunocompromised patients and its ubiquitous presence in the hospital setting. With the unforgiving emergence of multidrug-resistant strains, it is critical that alternative strategies to control or prevent septic infections in burn patients be developed in parallel with novel antimicrobial agents. In this study, we observed that administration of l-Arg significantly reduced bacterial spread and sepsis in burned mice infected with P. aeruginosa. Given the safety of l-Arg in high doses and its potential wound-healing benefits, this conditionally essential amino acid may represent a useful tool to modulate bacterial behavior in vivo and prevent sepsis in burn patients.

Download Full-text