Biofilm Formation Pattern Research Articles

Patterns of Biofilm Formation by Members of Listeria, Salmonella, and Pseudomonas at Various Temperatures and the Role of Their Synergistic Interactions in the Formation of Biofilm Communities

Patterns of Biofilm Formation by Members of Listeria, Salmonella, and Pseudomonas at Various Temperatures and the Role of Their Synergistic Interactions in the Formation of Biofilm Communities

Dynamics of Nakaseomyces glabratus biofilm formation

Formation of biofilms of microorganisms, including those of Nakaseomyces glabratus, is responsible for the development of local and systemic pathologies in humans and animals. The system of gene expression coordination (quorum sensing) in the representation of signaling molecules allows regulation of the amount and composition of biofilm populations thus expanding the adaptive capacity of microorganisms. In the presence of gingivitis and odontolithiasis clinical signs in dogs, excessive growth of gram-positive yeast microorganisms is a differential sign of the decreased resistance of the digestive system mucous membranes to colonization. Examination of the densitometric and morphometric parameters revealed general patterns of biofilm formation, regardless of the source of Nakaseomyces glabratus isolates. Depending on the time of cultivation of the microorganisms, a gradual increase in the optic density absolute values was established. Intercellular communications were achieved by coaggregation of the heteromorphic structures, which formed clusters with rounded liquid-containing formations detected among them. The population immobilization of the architectonics of the mature three-dimensional biofilm, as consistent with cultivation conditions, was accompanied by the differentiation of numerous cells of different sizes and shapes depending on the stage of the cell cycle. Results of the examination of the general patterns of the heterogeneous micromycete population development are promising for expanding the boundaries of knowledge of the adaptation mechanisms of ubiquitous microorganisms to long-term in vivo and in vitro persistence. Methods for studying morphometric and densitometric indicators avoiding interfering into the natural biofilm architectonics are recommended to optimize the long-term and retrospective mycological studies, as well as to develop effective mycosis treatment and prevention regimens.

Characterization and tolerance of foodborne pathogenic bacteria in microplastic biofilm

Microplastics have the ability to selectively accumulate microorganisms from their surrounding environments, leading to the formation of biofilms. The potential for microplastic biofilm in food processing environments to contaminate the processing environment and food matrices poses a threat to food safety. This study validated the biofilm formation of three commonly encountered foodborne pathogens, Salmonella enterica serovar Typhimurium (ST), Staphylococcus aureus (SA), and Listeria monocytogenes (LM) on microplastics, and investigated their tolerance to disinfectant and gastrointestinal tract. Three strains showed distinct biofilm formation patterns, with biomass reaching a steady state at 2 d. Meanwhile, the particle size of microplastics exhibited a specific impact on biofilm formation, with smaller microplastics sizes leading to increased biofilm formation. Sodium hypochlorite (NaClO) can effectively eliminate most biofilm-forming bacteria, except for ST. After treatment with 50 ppm NaClO, 34% cells in ST biofilm survived. Only SA survived in simulated gastric fluid, with around 3 log reduction after 1 h. ST and LM were not detected. All three strains showed a slight increase after treatment with simulated intestinal fluid for 6 h. These results highlighted the capacity of microplastics to transport pathogens through biofilms, emphasizing the potential synergistic effects between microplastics and pathogens in food-associated environments.

Quantitative and Qualitative Determination of Biofilm Formation Pattern in Multidrug Resistance Acinetobacter baumannii in Correlation with COVID-19 and Respiratory Infected Patients

Respiratory tract infection (RTI) is one of the triggering factors that cause severe and life-threatening health problems. Acinetobacter baumannii frequently causes nosocomial infections, especially in intensive care units (ICUs). Multidrug-resistant (MDR) A. baumannii encodes multiple virulence factors that contribute to chronic diseases, as well as antibiotic resistance; among them is the capacity to colonize and produce Biofilm on biotic and abiotic surfaces that is the primary source of worry in hospital environments. The study assessed the role of antibiotic resistance and biofilm formation patterns caused by MDR A. baumannii between isolates from COVID-19 and respiratory-infected patients in Baghdad Medical City hospitals and Teaching laboratories. This study included isolating and identifying A. baumannii from the sputum samples of respiratory tract-infected patients. (some of them infected with COVID-19) . Diagnosis of isolates was done by conventional cultural, microscopical and biochemical methods and confirmed by API 20E and VITEK2 system. Investigation of the susceptibility profile of the isolates against 18 types of antibiotics was tested, and the determination of A. baumannii biofilm formation was recorded by qualitative and quantitative methods. Results showed that 36 bacterial isolates were identified as A. baumannii. Most isolates were resistant to antibiotics, including all penicillins and cephalosporins (including inhibitor combinations), fluoroquinolones and aminoglycosides, followed by carbapenems antibiotics. In contrast, all isolates were susceptible to colistin (94.4%), followed by minocycline (36.1%), and the proportions of MDR, XDR, and PDR were 19.4%, 77.8 %, and 2.8 %, respectively. All isolates of A. baumannii showed biofilm formation (100%) by Quantitative method(MTP) and 91.6%. By the qualitative approach, we can conclude that there were no significant changes in biofilm values after 48 hours between groups with and without COVID-19 infection. However, there was a significant difference in adherence levels of bacteria between COVID-19 groups, with a higher proportion of solid adherence in the COVID-19 group compared to moderate adherence in the COVID-19-free group patients. Keywords: Acinetobacter baumannii, Antibiotic Susceptibility, Biofilm formation patterns, COVID-19 and respiratory infected patients.

The effects of temperature, salt, and phosphate on biofilm and exopolysaccharide production by Azotobacter spp.

Inoculation of agriculturally important biofilms to plants under stress conditions has been of great interest in recent years. Therefore, in this study, biofilm- and exopolysaccharide (EPS)-forming ability of Azotobacter spp. was examined under different temperatures, NaCl concentrations, and phosphate levels. Azotobacter strains formed varying levels of biofilm and EPS depending on the tested factors. The pattern of biofilm formation was similar to that of EPS production under the conditions tested. Biofilm and EPS production at 28°C was consistently higher than at either 18 or 37°C. Biofilm production significantly increased in A. chroococcum strains (SBS2, SBS4, and SBS12) and A. vinelandii SBS6 with increasing salinity. Furthermore, a strong negative correlation was observed between biofilm or EPS production and increasing phosphate concentrations. Higher phosphate concentrations decreased biofilm and EPS production. In conclusion, contrary to temperature and phosphate effect, salinity differently affected biofilm and EPS production by Azotobacter strains.

In situ detection of Cu2+, Fe3+ and Fe2+ ions at the microbe-mineral interface during bioleaching of chalcopyrite by moderate thermophiles

Bioleaching of metal sulfides represents an interfacial process in which the spatial relationship of metal ions with microbial cells and extracellular polymeric substances (EPS) is complex. The dissolution of chalcopyrite, cell attachment, and biofilm formation patterns were assessed during bioleaching with mixed cultures at 45 °C. The analysis of confocal laser scanning microscopy (CLSM) indicated that EPS were laid flat on the surface of chalcopyrite coupons, forming monolayer biofilms, with significant amounts of Fe3+, Fe2+ and Cu2+ enriched at the cell-chalcopyrite interface. In addition, the metal ions from mineral surfaces were extracted by ultrasonication. The results showed that the mineral surface is enriched with large amounts of Fe3+ (12.07 mg/g) and Cu2+ (10.82 mg/g) during the final stage of bioleaching, and more Cu2+ ions and iron maters from bio-oxidizing maybe were enclosed in the EPS space. It was inferred that the EPS with jarosite on the surface of chalcopyrite gradually acted as a weak diffusion barrier for Cu2+, and Fe3+ ions transference during bioleaching. These results can provide important evidence to reveal the hypothesis of ''metal ions enrichment on the mineral surface'' and benefit a further understanding of the bioleaching mechanism.

Biofilm formation by ESBL-producing uropathogenic E. coli

Introduction: E. coli is considered to be the leading cause of urinary tract infections. (UTIs) and is responsible for both community acquired and hospital acquired UTIs. The ability of this bacterium to form biofilm on various devices like catheters plays an important role in progression of the disease. Biofilm production in extended spectrum beta lactamases (ESBLs) producing organisms provides a favourable environment for the exchange of resistance genes and may also facilitate widespread dissemination.
 Methods: The urine samples were collected from the suspected cases of urinary tract infections and were cultured on cystine lactose electrolyte deficient (CLED) agar plates to isolate the desired UPEC strains. All the isolated E. coli strains were subjected to ESBL detection by double disk approximation method and to biofilm formation by tissue culture plate method of biofilm detection. All the results were statistically analysed using k2 test with p value at <0.05 level of significance.
 Results: Out of all the gram negative bacilli isolated from urine samples, E. coli alone accounted for 90% of the isolates. Of all the UPEC isolates subjected to ESBL detection 50% of the strains turned out to be the ESBL producers and 35% of the strains showed biofilm formation.
 Conclusion:
 
 The majority of the work in literature sites the data samples collected from indoor patients, where these isolates coli strains which produced ESBLs were mainly a strong biofilm former as compared to non-ESBL producer strains.
 In this study in spite of all the coli strains being outpatient isolates still the correlation between ESBL production and biofilm formation holds true.
 We found a significant correlation between biofilm formation and ESBL production. The detection of ESBL production and biofilm formation pattern will help in development of newer and more effective treatment.

Variable pH and subsequent change in pCO2 modulates the biofilm formation, synthesis of extracellular polymeric substances, and survivability of a marine bacterium Bacillus stercoris GST-03

Biofilm-forming bacteria adhere to the substrates and engage in the nutrient cycling process. However, environmental conditions may interrupt the biofilm formation ability, which ultimately may affect various biogeochemical cycles. The present study reports the effect of varying pH and subsequent change in pCO2 on the survivability, biofilm formation, and synthesis of extracellular polymeric substances (EPS) of a biofilm-forming marine bacterium Bacillus stercoris GST-03 isolated from the Bhitarkanika mangrove ecosystem, Odisha, India. Understanding the pH-dependent alteration in EPS constituents, and associated functional groups of a marine bacterium will provide better insight into the adaptability of the bacteria in future ocean acidification scenarios. The strain was found to tolerate and form biofilm up to pH 4, with the maximum biofilm formation at pH 6. EPS yield and the synthesis of the key components of the EPS, including carbohydrate, protein, and lipid, were found maximum at pH 6. Changes in biofilm formation patterns and various topological parameters at varying pH/pCO2 conditions were observed. A cellular chaining pattern was observed at pH 4, and maximum biofilm formation was obtained at pH 6 with biomass of 5.28582 ± 0.5372 μm3/μm2 and thickness of 9.982 ± 1.5288 μm. Structural characterization of EPS showed changes in various functional groups of constituent macromolecules with varying pH. The amorphous nature of the EPS and the changes in linkages and associated functional groups (-R2CHOR, –CH3, and –CH2) with pH variation was confirmed. EPS showed a two-step degradation with a maximum weight loss of 59.147% and thermal stability up to 480 °C at pH 6. The present work efficiently demonstrates the role of EPS in providing structural and functional stability to the biofilm in varying pH conditions. The findings will provide a better understanding of the adaptability of marine bacteria in the future effect of ocean acidification.

Ethanol at Subinhibitory Concentrations Enhances Biofilm Formation in Salmonella Enteritidis.

The survival of Salmonella Enteritidis in the food chain is relevant to its biofilm formation capacity, which is influenced by suboptimal environmental conditions. Here, biofilm formation pattern of this bacterium was assessed in the presence of ethanol at sub-minimal inhibitory concentrations (sub-MICs) by microtiter plate assays, cell characteristic analyses, and gene expression tests. It was observed that ethanol at subinhibitory concentrations (1/4 MIC, 2.5%; 1/2 MIC, 5.0%) was able to stimulate biofilm formation in S. Enteritidis. The OD595 value (optical density at 595 nm) used to quantify biofilm production was increased from 0.14 in control groups to 0.36 and 0.63 under 2.5% and 5.0% ethanol stresses, respectively. Ethanol was also shown to reduce bacterial swimming motility and enhance cell auto-aggregation ability. However, other cell characteristics such as swarming activity, initial attachment and cell surface hydrophobicity were not remarkedly impacted by ethanol. Reverse transcription quantitative real-time PCR (RT-qPCR) analysis further revealed that the luxS gene belonging to a quorum-sensing system was upregulated by 2.49- and 10.08-fold in the presence of 2.5% and 5.0% ethanol, respectively. The relative expression level of other biofilm-related genes (adrA, csgB, csgD, and sdiA) and sRNAs (ArcZ, CsrB, OxyS, and SroC) did not obviously change. Taken together, these findings suggest that decrease in swimming motility and increase in cell auto-aggregation and quorum sensing may result in the enhancement of biofilm formation by S. Enteritidis under sublethal ethanol stress.

Insight into biofilm-forming patterns: biofilm-forming conditions and dynamic changes in extracellular polymer substances.

The microbial biofilm adheres to the surface of the carrier, which protects the pollutant-degrading bacteria and resists harsh environments; thus, research on biofilm-forming patterns will help promote the application of biofilms in wastewater treatment. Herein, univariate analysis and response surface methodology (RSM) confirmed that glucose and mannose at 3-5g/L promoted biofilm formation. Notably, the microplate method demonstrated that compared to trivalent cations, divalent cations could more greatly enhance the activity (especially magnesium) of the biofilm matrix, and the period of biofilm formation in the three strains was divided into the following stages: initial attachment (0-10h), microcolony (10-24h), maturation (24-48h), and dispersion (36-72h). During maturation, large amounts of extracellular polysaccharides (EPs) and extracellular DNA (eDNA) were distributed in the extracellular and intracellular spaces, respectively, as observed by super-resolution structured illumination microscopy (SR-SIM). This study enhances the understanding of the characteristics and patterns of biofilm formation and can facilitate the application of biofilms in wastewater treatment.

The impact of Nitrogen and Carbon Sources on the Biofilm Formation of Micrococcus luteus

This study is conducted to show the influence of different media on the extent and pattern of biofilm formation. Trends of newly emerging pathogens continue steadily. Micrococcus luteus is one of those emerging pathogens. Incidental isolations of this bacteria have been recorded from patients with urinary tract infection and/or immunocompromised conditions. Biofilm formation on the surfaces of wound drainage and urinary catheters has been reported to be the source of recurrence and colonization of the pathogen in those patients. The current study's approach assesses the role of nutrient availability on the patterns of attachment till detachment and dispersion of the biofilms. Different species of bacteria are used to correlate their biofilm formation trend. Micrococcus luteus was chosen in the study due to its emerging pathogenic potential. Validation of biofilm formation is provided by involving Proteus mirabilis; which is an ideal biofilm producer, in parallel with Micrococcus luteus throughout the entire experimental settings. The findings of this study confirm statistically significant differences in biofilm formation patterns when nutritionally different culture media have been utilized to resemble possible environments for the pathogen. Micrococcus luteus has been found to possess the highest potential to produce biofilm in peptone water media where it over paced Proteus mirabilis. Results of the study reveal that both availability and scarcity of carbon and nitrogen sources can influence both positively and negatively on the patterns of biofilm formation by different strains of bacteria and incubation time. Biofilm assessment is an inevitable technique for nosocomial infections due to the complications of antibiotic susceptibility trends that prolong the hospitalization process, which limits treatment capacity.

Comparative study of biofilm formation in tubotympanic and atticoantral ear disease

<p class="abstract"><strong>Background:</strong> Biofilm is an organized bacterial community that may be homogeneous or heterogeneous. It plays a significant role in the pathogenesis of chronic rhinosinusitis, chronic tonsillitis and chronic suppurative otitis media. Biofilms may explain the recurrence and recalcitrant episodes of otorrhea in chronic otitis media (COM). The objective of this study was to detect biofilm formation in chronic suppurative otitis media and compare the pattern of biofilm formation in tubotympanic and atticoantral ear diseases.</p><p class="abstract"><strong>Methods:</strong> This was a prospective cross sectional study at a tertiary care hospital in Varanasi, Uttar Pradesh, India. Patients included in the study ranged from 10 to 65 years of age from both the sexes and varying socioeconomic status. Samples were collected using sterile precautions in cases of tubotympanic as well as atticoantral COM. The collected samples were directly inoculated onto MacConkey and blood agar media. LisaScan® EM microtitter plate reader was used to identify the presence of biofilm.</p><p class="abstract"><strong>Results:</strong> The most common organism was <em>P. aeruginosa</em> and <em>S. aureus</em> followed by <em>Moraxella catarrhalis</em> whereas in cases with tubotympanic COM, most common organism was <em>S. aureus</em>. We found that, in atticoantral type of COM biofilm formation was significantly more common as compared to that in tubotympanic type of COM.</p><p class="abstract"><strong>Conclusions:</strong> Biofilm formation in COM is even higher when atticoantral disease is present.</p>

СОВРЕМЕННЫЕ СПОСОБЫ ИССЛЕДОВАНИЯ МИКРОБНЫХ БИОПЛЕНОК КИШЕЧНИКА

The article outlines the main methods of studying microbial biofilms, which make it possible to reveal: the patterns of biofilm formation by microorganisms of the Enterobacteriaceae family, genetic programs regulating the processes of film formation, qualitative and quantitative characteristics of the components of microbial communities, the influence of external factors on the stages of biofilm formation and dispersion. The study of the phenomenon of film formation in combination with monitoring the resistance of intestinal microorganisms in the biofilm to antibacterial drugs will make it possible to get closer to understanding the role of biofilms in the course of infectious processes of a microbial origin.

Comparison of biofilm formation between non-pathogenic Listeria strains under different stress conditions

AbstractMicro-organisms can attach to food surfaces and develop biofilms which present a concern in food and environmental safety. The main goal of the current study was to investigate the biofilm formation of six non-pathogenic Listeria strains under different stress conditions using a microplate assay. The effect of the weak biofilm-forming non-pathogenic Listeria strains on the biofilm formation of a strong biofilm-forming pathogenic Listeria strain (Listeria monocytogenes #8) was also examined. Listeria innocua CCM4030, Listeria innocua 2885 and Listeria seeligeri/welshimeri 292 showed the same patterns of biofilm formation with increasing NaCl concentrations from 0.05 to 15%, but all the other strains showed a continuously decreasing trend of OD595 in the same conditions. This study showed that in the case of non-pathogenic Listeria strains, higher concentrations of NaCl do not present a stress condition that enhances biofilm formation. Decrease in pH inhibited biofilm formation for all the non-pathogenic Listeria strains. The weak biofilm forming non-pathogenic Listeria strains (Listeria innocua 2885 and Listeria innocua CCM4030) overgrew the strong biofilm-forming Listeria strain (Listeria monocytogenes #8) during biofilm formation. This phenomenon could be beneficial and potentially be used as a novel control strategy to prevent the colonization of the pathogenic Listeria at food processing facilities such as in meat industry.

Investigation of the association of virulence genes and biofilm production with infection and bacterial colonization processes in multidrug-resistant Acinetobacter spp.

The aim of this study was to evaluate the phenotypic and molecular patterns of biofilm formation in infection and colonization isolates of Acinetobacter spp. from patients who were admitted in a public hospital of Recife-PE-Brazil in 2018-2019. For the biofilm phenotypic analysis, Acinetobacter spp. isolates were evaluated by the crystal violet staining method; the search of virulence genes (bap, ompA, epsA, csuE and bfmS) was performed by PCR; and the ERIC-PCR was performed for molecular typing. Amongst the 38 Acinetobacter spp. isolates, 20 were isolated from infections and 18 from colonization. The resistance profile pointed that 86.85% (33/38) of the isolates were multidrug-resistant, being three infection isolates, and two colonization isolates resistant to polymyxin B. All the isolates were able to produce biofilm and they had at least one of the investigated virulence genes on their molecular profile, but the bap gene was found in 100% of them. No clones were detected by ERIC-PCR. There was no correlation between biofilm formation and the resistance profile of the bacteria, neither to the molecular profile of the virulence genes. Thus, the ability of Acinetobacter spp. to form biofilm is probably related to the high frequency of virulence genes.

In vitro Antibiotic Susceptibility, Virulence Genes Distribution and Biofilm Production of Staphylococcus aureus Isolates from Bovine Mastitis in the Liaoning Province of China.

PurposeThe aim of this study was to identify the subtype, characterize the antimicrobial resistance, determine the virulence gene distribution, and analyze the biofilm production of Staphylococcus aureus isolates from bovine mastitis milk samples in the Liaoning Province of China.Materials and MethodsIn total, 56 Staph. aureus isolates were collected and identified in this study; the isolates were divided into different spa types based on the sequence of the polymorphic X region of the spa gene. Additionally, antimicrobial susceptibility was investigated using the broth microdilution method, and 18 virulence genes were detected using PCR. Biofilm formation was measured by spectrophotometry with crystal violet staining and observed using confocal laser scanning microscopy.ResultsThere were 12.12% (56/462) milk samples that were positive for Staph. aureus. These isolates were nonsusceptible to sulfamethoxazole (100%), penicillin (76.9%), daptomycin (76.79%), clindamycin (69.64%), and oxacillin (60.71%); however, the majority of the isolates (80.4%) were susceptible to amoxicillin/clavulanate. The predominant virulence genes encoded the cytotoxins, hla (94.64%) and hlb (89.29%), and the adhesion factors clfA (89.29%), clfB (89.29%), and fnbB (80.36%). Comparatively, virulence genes related to other adhesion factors such as cna (8.93%) and enterotoxins, such as seg (26.79%), sea (16.07%), seb (7.14%), and sec (7.14%) were detected at relatively lower rates. The following eight spa types were identified: t267 (35.84%), t730 (22.64%), t518 (15.09%), t1190 (11.32%), t1456 (9.43%), t224 (1.88%), t9129 (1.88%), and t177 (1.88%). The highest biofilm production was observed for t267. Staph. aureus exhibited various patterns of biofilm formation, with the biofilm often being associated with a tower-shaped structure or a thicker biofilm.ConclusionOur results indicated that Staph. aureus isolates from dairy cows with mastitis in the Liaoning Province of China were non-susceptible to sulfamethoxazole, penicillin, daptomycin, oxacillin, and clindamycin. Additionally, the most prevalent subtype was t267, which displayed resistance to multiple antimicrobial agents and harbored several virulence genes, including clfA, clfB, fnbB, hla, and hlb.

Ecological insights into the underlying evolutionary patterns of biofilm formation from biological wastewater treatment systems: Red or Black Queen Hypothesis?

Interspecies interactions and phylogenetic distances were studied to reveal the underlying evolutionary adaptations of biofilms sourced from wastewater treatment processes. Based on 380 pairwise cocultures of 40 strains from two microbial aggregates (surface-attached and mobile aggregates [flocs]) at two substrate concentrations (LB broth and 0.1× LB broth), interspecies interactions were explored using biofilm classification schemes. There was a strong source-dependence of biofilm development formed by the monocultures, that is, a higher biofilm formation potential for strains from attached aggregates than for those from sludge flocs at both substrate concentrations. Interestingly, the results showed that total biofilm reduction was dominant in the dual-species biofilm sourced from flocs in both LB broth (67.37%) and 0.1× LB broth (64.21%), indicating high interspecific competition in mobile aggregates and the independence of substrate concentrations. However, biofilm reduction was higher (33.68%) than induction (19.37%) for the biofilms formed by surface-attached aggregates in LB broth, while the opposite trend was apparent in 0.1× LB broth, suggesting the occurrence of indeterministic processes for biofilm formation and important roles of substrate concentrations. In addition, the more closely related phylogenetic relationships of cocultures from mobile aggregates were consistent with higher competition compared with those from surface-attached aggregates. Overall, the underlying evolutionary patterns of biofilms formed from mobile aggregates consistently followed the essence of the "Red Queen Hypothesis," while biofilms developed from surface-attached aggregates were not deterministic. This study advanced our understanding of biofilm-related treatment processes using the principles of microbial ecology.

Mechanism of biofilm formation on a hydrophobic polytetrafluoroethylene membrane during the purification of surface water using direct contact membrane distillation (DCMD), with especial interest in the feed properties

The impact of feed water quality on biofilm formation during membrane distillation (MD) was investigated in this study, particularly emphasizing the interrelationship between organics, salts, and microbes. Two types of typical natural surface waters in Nanjing, China, were chosen as feed solutions for long-term MD operation, including the Qinhuai River and Xuanwu Lake. The biofilms that developed under different feed water qualities exhibited distinct Foulant compositions and structures, causing different flux decline trends for the MD system. Accordingly, two typical patterns of biofilm formation were suggested for the MD operation of the two different kinds of surface waters in this study. Organics from a primal feed solution and dead bacteria were the key to the establishment of a biofilm on the membrane, and this needs to be effectively removed from the MD system through pre-treatment and process control strategies. Finally, a feasible strategy for MD biofouling control was suggested.

Screening of Candida albicans GRACE library revealed a unique pattern of biofilm formation under repression of the essential gene ILS1

Candida albicans biofilm formation is governed by a regulatory circuit comprising nine transcription factors which control a network of target genes. However, there are still unknown genes contributing to biofilm features. Thus, the GRACE library was screened to identify genes involved in mature biofilm development. Twenty-nine conditional mutants were selected for a second screening revealing three groups of genes: twenty- two conditional mutants were defective for normal growth and unable to form biofilms; six strains, conditionally defective in genes ARC40, ARC35, ORF19.2438, SKP1, ERG6, and ADE5,7 that are likely essential or involved in general cell processes, grew normally as free-floating cells but produced less biofilm; finally, the conditional strain for a putative essential isoleucyl- tRNA synthetase gene, ILS1, was unable to grow as yeast-phase cells but was capable of producing a tridimensional biofilm structure in spite of reduced metabolic activity. This unique biofilm still relied on the classical biofilm genes, while it differentially induced groups of genes involved in adhesion, protein synthesis, cell wall organization, and protein folding. Although the conditional mutant repressed genes annotated for morphology and homeostasis processes affecting morphology and metabolism, the dynamic cell growth enabled the formation of a complex biofilm community independent of ILS1.

An Azole-Resistant Candida parapsilosis Outbreak: Clonal Persistence in the Intensive Care Unit of a Brazilian Teaching Hospital.

The incidence of candidemia by the Candida parapsilosis complex has increased considerably in recent decades, frequently related to use of indwelling intravascular catheters. The ability of this pathogen to colonize healthcare workers (HCW)' hands, and to form biofilm on medical devices has been associated with the occurrence of nosocomial outbreaks and high mortality rates. Fluconazole has been the leading antifungal drug for the treatment of invasive candidiasis in developing countries. However, azole-resistant C. parapsilosis isolates are emerging worldwide, including in Brazil. Few studies have correlated outbreak infections due to C. parapsilosis with virulence factors, such as biofilm production. We thus conducted a microbiological investigation of C. parapsilosis complex isolates from a Brazilian teaching hospital. Additionally, we identified a previously unrecognized outbreak caused by a persistent azole-resistant C. parapsilosis (sensu stricto) clone in the intensive care unit (ICU), correlating it with the main clinical data from the patients with invasive candidiasis. The molecular identification of the isolates was carried out by PCR-RFLP assay; antifungal susceptibility and biofilm formation were also evaluated. The genotyping of all C. parapsilosis (sensu stricto) was performed by microsatellite analysis and the presence of ERG11 mutations was assessed in the azole non-susceptible isolates. Fourteen C. parapsilosis (sensu stricto) isolates were recovered from patients with invasive candidiasis, eight being fluconazole and voriconazole-resistant, and two intermediate only to fluconazole (FLC). All non-susceptible isolates showed a similar pattern of biofilm formation with low biomass and metabolic activity. The A395T mutation in ERG11 was detected exclusively among the azole-resistant isolates. According to the microsatellite analysis, all azole non-susceptible isolates from the adult ICU were clustered together indicating the occurrence of an outbreak. Regarding clinical data, all patients infected by the clonal non-susceptible isolates and none of the patients infected by the susceptible isolates had been previously exposed to corticosteroids (p = 0.001), while the remaining characteristics showed no statistical significance. The current study revealed the persistence of an azole non-susceptible C. parapsilosis clone with low capacity to form biofilm over two years in the adult ICU. These results reinforce the need of epidemiological surveillance and monitoring antifungal susceptibility of C. parapsilosis isolates in hospital wards.