Micro ecosystems from feed industry surfaces: a survival and biofilm study of <Emphasis Type="Italic">Salmonella</Emphasis>versus host resident flora strains

Background  

The presence of Salmonella enterica serovars in feed ingredients, products and processing facilities is a well recognized problem worldwide. In Norwegian feed factories, strict control measures are implemented to avoid establishment and spreading of Salmonella throughout the processing chain. There is limited knowledge on the presence and survival of the resident microflora in feed production plants. Information on interactions between Salmonella and other bacteria in feed production plants and how they affect survival and biofilm formation of Salmonella is also limited. The aim of this study was to identify resident microbiota found in feed production environments, and to compare the survival of resident flora strains and Salmonella to stress factors typically found in feed processing environments. Moreover, the role of dominant resident flora strains in the biofilm development of Salmonella was determined.     

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H₂O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4—used as an indicator strain, B. licheniformis T6-5, and B. firmus H₂O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H₂O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H₂O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H₂O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.     

Biofilm Production Potential of Salmonella Serovars Isolated from Chickens in North West Province,South Africa

Bacterial biofilms have recently gained considerable interest in the food production and medical industries due to their ability to resist destruction by disinfectants and other antimicrobials. Biofilms are extracellular polymer matrices that may enhance the survival of pathogens even when exposed to environmental stress. The effect of incubation temperatures (25°C, 37°C, and 40°C) and Salmonella serotype on biofilm-forming potentials was evaluated. Previously typed Salmonella serotypes (55) isolated from the gut of chickens were accessed for biofilms formation using a standard assay. Salmonella Typhimurium ATCC 14028^TM and Salmonella Enteritidis ATCC 13076^TM (positive controls), Escherichia coli (internal control) and un-inoculated Luria Bertani (LB) broth (negative control) were used. The isolates formed no biofilm (11.86–13.56%), weak (11.86–45.76%), moderate (18.64–20.34%), strong biofilms (23.73–54.24%) across the various temperatures investigated. Serotypes, Salmonella Heidelberg and Salmonella Weltevreden were the strongest biofilm formers at temperatures (25°C, 37°C, and 40°C, respectively). The potential of a large proportion (80%) of Salmonella serotypes to form biofilms increased with increasing incubation temperatures but decreased at 40°C. Findings indicate that average temperature favours biofilm formation by Salmonella serotypes. However, the influence of incubation temperature on biofilm formation was greater when compared to serotype. A positive correlation exists between Salmonella biofilm formed at 25°C, 37°C and 40°C (p ≥ 0.01). The ability of Salmonella species to form biofilms at 25°C and 37°C suggests that these serotypes may present severe challenges to food-processing and hospital facilities.Key words: Salmonella, biofilm, biofilm production potential, crystal violet microtitre     

Zeta Potential of Selected Bacteria in Drinking Water When Dead, Starved, or Exposed to Minimal and Rich Culture Media

The zeta potentials of E. coli, GFP (green fluorescence protein)-labeled E. coli, Salmonella Newport, and Pseudomonas sp. in different states (nutrient-starved and dead) and grown in rich and minimal media were measured. Capillary electrophoresis experiments were conducted to measure the zeta potential of the different cells suspended in a drinking water sample. Salmonella Newport strain showed a lower zeta potential compared to E. coli, GFP-labeled E. coli, and Pseudomonas sp. Starved E. coli cells had a lower zeta potential compared to E. coli cells grown under rich media conditions. Salmonella Newport cells grown in minimal media also had a lower zeta potential compared to rich, starved, and dead cells. The different bacterial cell types exhibited differences in size as well. These results suggest that when bacterial cells are present in drinking water they can exhibit significant heterogeneity in the size and zeta potential, depending on their physiological state.     

Production of H<Subscript>2</Subscript> from sucrose by <Emphasis Type="Italic">Escherichia coli</Emphasis> strains carrying the pUR400 plasmid,which encodes invertase activity

Escherichia coli HD701, a hydrogenase-upregulated strain, has the potential for industrial-scale H₂ production but is unable to metabolise sucrose, which is a major constituent of many waste materials that could be used as feedstocks for H₂ production processes. A 70 kb plasmid (pUR400), which carries the genes necessary for sucrose transport into the cell and its metabolism, was conjugated into E. coli strains HD701 and FTD701 [a derivative of HD701 which has a deletion of the tatC gene of the twin arginine transport (Tat) protein system] from an E. coli K12 strain. Comparative studies on H₂ evolution by FTD701 and HD701, with and without the pUR400 plasmid, were made using sucrose as substrate. The parental strains did not evolve H₂, although HD701/pUR400 and FTD701/pUR400 evolved 1.27 ± 0.09 and 1.38 ± 0.05 ml H₂ mg dry wt^–1 l culture^–1, respectively over 10 h. This work provides the choice for using a recombinant E. coli strain, which produces H₂ from sucrose, as an alternative to coupling-in an upstream invertase, and hence this provides a simpler method for the bioproduction of H₂ from sucrose.Revisions requested 24 August 204; Revisions received 21 October 2004     

Formation of nesquehonite and other minerals as a consequence of biofilm dehydration

A microbial biofilm community was established over 971 days within gravel in an aquarium so as to model biofouling of an aquifer. When the water was allowed to evaporate slowly, white crystalline deposits, containing several carbonate and sulphate minerals including nesquehonite (MgCO₃.3H2O), were seen at the highest points on the surface of the biofouled gravel. No such deposits occurred in regions lacking biofilms. These crystals appeared to originate from evaporation of dissolved salts which had migrated through the biofilm. Surfaceadherent microbial biofilms may conceivably provide a conduit for solute transport in porous media such as soils and aquifers.     

Functional properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from dairy products

Twenty-four acid- and bile-tolerant lactobacilli isolates from dairy products were identified and further in vitro characterized for the presence of functional traits potentially useful for probiotic applications, which included desirable and undesirable traits, such as biofilm formation, ability to inhibit intestinal pathogens, antibiotic susceptibility, and enzyme activity. The majority of examined strains were susceptible to certain antimicrobial agents (streptomycin, gentamicin, clindamycin, erythromycin, tetracycline, quinupristin–dalfopristin), except for three strains of Lactobacillus rhamnosus with minimal inhibitory concentration levels for streptomycin higher than the microbiological breakpoints (≥32 μg/mL), which are considered as resistant. Undesirable traits such as α-chymotrypsin or N-acetyl-β-glucosaminidase activities were not detected, but low β-glucuronidase, and moderate and high β-glucosidase activities were recorded in nine strains, which were eliminated from further examination together with three isolates showing unsuitable antibiotic resistance. Of the remaining 12 isolates, 4 (Lactobacillus fermentum 202, Lactobacillus gallinarum 7001, L. rhamnosus 183, and Lactobacillus plantarum L2-1) manifested an outstanding potential to inhibit selected intestinal pathogens in an agar spot test, including Escherichia coli and Salmonella spp., and simultaneously demonstrated strong biofilm-forming capacity. In conclusion, the results of our in vitro experiments showed that the above four strains had a potential probiotic value and met the criteria to be identified as a possible probiotic microorganism, with the necessity of verification through well-designed in vivo experimental, clinical, and technological studies before the strains can be used as probiotics or as starter probiotic cultures.     

Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

VIM‐1 carbapenemase‐producing Escherichia coli in gulls from southern France

Acquired carbapenemases currently pose one of the most worrying public health threats related to antimicrobial resistance. A NDM‐1‐producing Salmonella Corvallis was reported in 2013 in a wild raptor. Further research was needed to understand the role of wild birds in the transmission of bacteria resistant to carbapenems. Our aim was to investigate the presence of carbapenem‐resistant Escherichia coli in gulls from southern France. In 2012, we collected 158 cloacal swabs samples from two gull species: yellow‐legged gulls (Larus michahellis) that live in close contact with humans and slender‐billed gulls (Chroicocephalus genei) that feed at sea. We molecularly compared the carbapenem‐resistant bacteria we isolated through culture on selective media with the carbapenem‐susceptible strains sampled from both gull species and from stool samples of humans hospitalized in the study area. The genes coding for carbapenemases were tested by multiplex PCR. We isolated 22 carbapenem‐resistant E. coli strains from yellow‐legged gulls while none were isolated from slender‐billed gulls. All carbapenem‐resistant isolates were positive for bla_VIM‐1 gene. VIM‐1‐producing E. coli were closely related to carbapenem‐susceptible strains isolated from the two gull species but also to human strains. Our results are alarming enough to make it urgently necessary to determine the contamination source of the bacteria we identified. More generally, our work highlights the need to develop more bridges between studies focusing on wildlife and humans in order to improve our knowledge of resistant bacteria transmission routes.     

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new Y ehD f imbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.     

Biofilms as a microbial hazard in the food industry: A scoping review

Biofilms pose a serious public health hazard with a significant economic impact on the food industry. The present scoping review is designed to analyse the literature published during 2001–2020 on biofilm formation of microbes, their detection methods, and association with antimicrobial resistance (if any). The peer-reviewed articles retrieved from 04 electronic databases were assessed using PRISMA-ScR guidelines. From the 978 preliminary search results, a total of 88 publications were included in the study. On analysis, the commonly isolated pathogens were Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli, Bacillus spp., Vibrio spp., Campylobacter jejuni and Clostridium perfringens. The biofilm-forming ability of microbes was found to be influenced by various factors such as attachment surfaces, temperature, presence of other species, nutrient availability etc. A total of 18 studies characterized the biofilm-forming genes, particularly for S. aureus, Salmonella spp., and E. coli. In most studies, polystyrene plate and/or stainless-steel coupons were used for biofilm formation, and the detection was carried out by crystal violet assays and/or by plate counting method. The strain-specific significant differences in biofilm formation were observed in many studies, and few studies carried out analysis of multi-species biofilms. The association between biofilm formation and antimicrobial resistance was not clearly defined. Further, viable but non-culturable form of the foodborne pathogens is posing an unseen (by conventional cultivation techniques) but potent threat to the food safety. The present review recommends the need for carrying out systematic surveys and risk analysis of biofilms in food chain to highlight the evidence-based public health concerns, especially in regions where microbiological food hazards are quite prevalent.     

Characterization of Enterobacteria Producing the Low-Molecular-Weight Antibiotics Microcins

A comparative study of the morphological, cultural, physiological, and biochemical properties of the microcinogenic strains EcS 5/98, EcS 6/98, and EcB 214/99 and the known microcin C51 producer Escherichia coli M17(p74) showed that these strains belong to the species E. coli. The strains produced microcins with molecular masses lower than 10 kDa. Microcin biosynthesis was stimulated by a deficiency of nutrients in the cultivation media. The microcins were found to be resistant to thermolysin but were degraded by pronase, protolichetrem, and the Bacillus mesentericus metalloproteinase. This indicated that the microcins are peptides or contain peptides in their molecules. The study of cross immunity to the microcins and the sequencing of their genetic determinants showed that the microcins of strains EcS 5/98 and EcS 6/98 are of B type, whereas the microcin of strain EcB 214/99 presumably belongs to another type, since it suppresses the growth of the producers of C and B-type microcins. The new microcin producers possess antibacterial activity against natural isolates belonging to the genera Escherichia and Salmonella, against a wide range of colicinogenic Escherichia strains, and against collection Salmonella cultures.     

Role of nutrient limitation in the competition between uropathogenic strains of Klebsiella pneumoniae and Escherichia coli in mixed biofilms

Klebsiella pneumoniae and Escherichia coli form mixed species biofilms in catheter-associated urinary tract infections. Recently, a detrimental effect of K. pneumoniae over E. coli was observed in mixed species biofilms grown in an artificial urine medium. The mechanism behind this competitive interaction was studied. K. pneumoniae partially outcompeted E. coli in early-stage batch-fed biofilms, whereas both microorganisms co-exist at longer times (K. pneumoniae:E. coli ratio, 55:1), as shown by cell counts and confocal microscopy. E. coli cells were scattered along the K. pneumoniae biofilm. Biofilm supernatants did not appear to contain either antimicrobial or anti-biofilm activities against E. coli. Biofilms grown under continuous flow prevented interspecies competition. K. pneumoniae showed both increased siderophore production and better growth in iron-limited media compared to E. coli. In summary, these results indicate the importance of nutrient (particularly iron) competition in the modulation of the bacterial composition of mixed species biofilms formed by uropathogenic K. pneumoniae and E. coli.     

Comparative susceptibility of planktonic and 3‐day‐old Salmonella Typhimurium biofilms to disinfectants

Aims: To compare the susceptibility of a 3‐day‐old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella. Methods and Results: The susceptibility of planktonic cells to disinfectants was tested by a modified version of the Council of Europe suspension test EN 1276. Salmonella biofilms were formed using the Calgary Biofilm Device. Results show that 3‐day‐old Salmonella biofilms are less susceptible to the disinfectants benzalkonium chloride, chlorhexidine gluconate, citric acid, quaternary ammonium compounds, sodium hypochlorite (SH) and ethanol, compared to planktonic Salmonella. Surprisingly, the results also demonstrate that low concentrations of SH were more effective against a 3‐day‐old biofilm compared to high concentrations of SH. Conclusions: While all the disinfectants evaluated were able to reduce biofilm‐associated cells at concentrations and contact times sufficient to eliminate planktonic cells, there were still sufficient viable cells remaining in the biofilm to cause further contamination and potential infection. Significance and Impact of the Study: Protocols for the use of chemical disinfectants need to include biofilm susceptibility testing. There is a requirement for an effective and standardized tool for determining the susceptibility of biofilms to disinfectants.     

Long tail fibres of the novel broad‐host‐range T‐even bacteriophage S16 specifically recognize Salmonella OmpC

We report isolation and characterization of the novel T4‐like Salmonella bacteriophage vB_SenM‐S16. S16 features a T‐even morphology and a highly modified 160 kbp dsDNA genome with 36.9 mol % G+C, containing 269 putative coding sequences and three tRNA genes. S16 is a virulent phage, and exhibits a maximally broad host range within the genus Salmonella, but does not infect other bacteria. Synthesis of functional S16 full‐length long tail fibre (LTF) in Escherichia coli was possible by coexpression of gp37 and gp38. Surface plasmon resonance analysis revealed nanomolar equilibrium affinity of the LTF to its receptor on Salmonella cells. We show that OmpC serves as primary binding ligand, and that S16 adsorption can be transferred to E. coli by substitution of ompC with the Salmonella homologue. S16 also infects ‘rough’ Salmonella strains which are defective in lipopolysaccharide synthesis and/or its carbohydrate substitution, indicating that this interaction does not require an intact LPS structure. Altogether, its virulent nature, broad host range and apparent lack of host DNA transduction render S16 highly suitable for biocontrol of Salmonella in foods and animal production. The S16 LTF represents a highly specific affinity reagent useful for cell decoration and labelling, as well as bacterial immobilization and separation.     

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC)

Background  

Crohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes.     

Role of planktonic and sessile extracellular metabolic byproducts on <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> intra and interspecies relationships

Bacterial species are found primarily as residents of complex surface-associated communities, known as biofilms. Although these structures prevail in nature, bacteria still exist in planktonic lifestyle and differ from those in morphology, physiology, and metabolism. This study aimed to investigate the influence of physiological states of Pseudomonas aeruginosa and Escherichia coli in cell-to-cell interactions. Filtered supernatants obtained under planktonic and biofilm cultures of each single species were supplemented with tryptic soy broth (TSB) and used as the growth media (conditioned media) to planktonic and sessile growth of both single- and two-species cultures. Planktonic bacterial growth was examined through OD₆₄₀ measurement. One-day-old biofilms were evaluated in terms of biofilm biomass (CV), respiratory activity (XTT), and CFU number. Conditioned media obtained either in biofilm or in planktonic mode of life triggered a synergistic effect on planktonic growth, mainly for E. coli single cultures growing in P. aeruginosa supernatants. Biofilms grown in the presence of P. aeruginosa biofilms-derived metabolites presented less mass and activity. These events highlight that, when developed in biofilm, P. aeruginosa release signals or metabolites able to prejudice single and binary biofilm growth of others species and of their own species. However, products released by their planktonic counterparts did not impair biofilm growth or activity. E. coli, living as planktonic or sessile cultures, released signals and metabolites or removed un-beneficial compounds which promoted the growth and activity of all the species. Our findings revealed that inter and intraspecies behaviors depend on the involved bacteria and their adopted mode of life.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Constructing multispecies biofilms with defined compositions by sequential deposition of bacteria

Rationally-assembled multispecies biofilms could benefit applied processes including mixed waste biodegradation and drug biosynthesis by combining complementary metabolic pathways into single functional communities. We hypothesized that the cellular composition of mature multispecies biofilms could be manipulated by controlling the number of each cell type present on newly colonized surfaces. To test this idea, we developed a method for attaching specific numbers of bacteria to a flow cell by recirculating cell suspensions. Initial work revealed a nonlinear relationship between suspension cell density and areal density when two strains of Escherichia coli were simultaneously recirculated; in contrast, sequential recirculation resulted in a predictable deposition of cell numbers. Quantitative analysis of cell distributions in 48-h biofilms comprised of the E. coli strains demonstrated a strong relationship between their distribution at the substratum and their presence in mature biofilms. Sequentially depositing E. coli with either Pseudomonas aeruginosa or Bacillus subtilis determined small but reproducible differences in the areal density of the second microorganism recirculated relative to its areal density when recirculated alone. Overall, the presented method offers a simple and reproducible way to construct multispecies biofilms with defined compositions for biocatalytic processes.     

Inhibition on <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm formation using divalent cation chelators (EDTA)

Candida albicans can readily form biofilms on both inanimate and biological surfaces. In this study we investigated a means of inhibiting biofilm formation using EDTA (Ethylenediaminetetra-acetic acid), a divalent cation chelating agent, which has been shown to affect C. albicans filamentation. Candida albicans biofilms were formed in 96-well microtitre plates. Cells were allowed to adhere for 1, 2, and 4 h at 37°C, washed in PBS, and then treated with different concentrations of EDTA (0, 2.5, 25, and 250 mM). EDTA was also added to the standardized suspension prior to adding to the microtiter plate and to a preformed 24 h biofilm. All plates were then incubated at 37°C for an additional 24 h to allow for biofilm formation. The extent and characteristics of biofilm formation were then microscopically assessed and with a semi-quantitative colorimetric technique based on the use of an XTT-reduction assay. Northern blot analysis of the hyphal wall protein (HWP1) expression was also monitored in planktonic and biofilm cells treated with EDTA. Microscopic analysis and colorimetric readings revealed that filamentation and biofilm formation were inhibited by EDTA in a concentration dependant manner. However, preformed biofilms were minimally affected by EDTA (maximum of 31% reduction at 250 mM). The HWP1 gene expression was reduced in EDTA-treated planktonic and biofilm samples. These results indicate that EDTA inhibits C. albicans biofilm formation are most likely through its inhibitory effect on filamentation and indicates the potential therapeutic effects of EDTA. This compound may serve a non-toxic means of preventing biofilm formation on infections with a C. albicans biofilm etiology.