New Methods for Analysis of Spatial Distribution and Coaggregation of Microbial Populations in Complex Biofilms

In biofilms, microbial activities form gradients of substrates and electron acceptors, creating a complex landscape of microhabitats, often resulting in structured localization of the microbial populations present. To understand the dynamic interplay between and within these populations, quantitative measurements and statistical analysis of their localization patterns within the biofilms are necessary, and adequate automated tools for such analyses are needed. We have designed and applied new methods for fluorescence in situ hybridization (FISH) and digital image analysis of directionally dependent (anisotropic) multispecies biofilms. A sequential-FISH approach allowed multiple populations to be detected in a biofilm sample. This was combined with an automated tool for vertical-distribution analysis by generating in silico biofilm slices and the recently developed Inflate algorithm for coaggregation analysis of microbial populations in anisotropic biofilms. As a proof of principle, we show distinct stratification patterns of the ammonia oxidizers Nitrosomonas oligotropha subclusters I and II and the nitrite oxidizer Nitrospira sublineage I in three different types of wastewater biofilms, suggesting niche differentiation between the N. oligotropha subclusters, which could explain their coexistence in the same biofilms. Coaggregation analysis showed that N. oligotropha subcluster II aggregated closer to Nitrospira than did N. oligotropha subcluster I in a pilot plant nitrifying trickling filter (NTF) and a moving-bed biofilm reactor (MBBR), but not in a full-scale NTF, indicating important ecophysiological differences between these phylogenetically closely related subclusters. By using high-resolution quantitative methods applicable to any multispecies biofilm in general, the ecological interactions of these complex ecosystems can be understood in more detail.     

Revisiting the DNA C-values of the genome size-standards used in plant flow cytometry to choose the "best primary standards"

Flow cytometry (FCM) techniques have enabled characterization of the genome size for various plant species. In order to measure the nuclear genome size of a species, reference standards with well-established DNA content are necessary. However, different 2C-values have been described for the same species used as reference standard. This fact has brought about inaccurate genome measurements, making relevant the establishment of optimal DNA reference standards for plant cytometric analyses. Our work revisited the genome size of Arabidopsis thaliana and other seven plant standards, which were denominated ??Dole?el??s standard set?? and have been widely used in plant DNA measurements. These eight plant standards were reassessed for a comparative measurement of their DNA content values, using each plant species as primary standard in a cascade-like manner, from A. thaliana to Allium cepa. The genome size values obtained here were compared to those reported in the literature by statistical analyses. As a result, Raphanus sativus and Drosophila melanogaster were considered the most inadequate primary standards, whereas A. thaliana, Solanum lycopersicum and Pisum sativum were found to be the most suitable.     

Comparing DNA Extraction Methods for Analysis of Botanical Materials Found in Anti-Diabetic Supplements

A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A ₂₆₀/A ₂₈₀ between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A ₂₆₀/A ₂₈₀ ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A ₂₆₀/A ₂₈₀ measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin^® plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.     

The Effects from DNA Extraction Methods on the Evaluation of Microbial Diversity Associated with Human Colonic Tissue

Potentially valuable sources of DNA have been extracted from human colonic tissues and are retained in biobanks throughout the world, and might be re-examined to better understand host–microbe interactions in health and disease. However, the published protocols for DNA extraction typically used by gastroenterologists have not been systematically compared in terms of their recovery of the microbial fraction associated with colonic tissue. For this reason, we examined how three different tissue DNA extraction methods (the QIAGEN AllPrep DNA/RNA kit, salting out and high molecular weight (HMW) methods of DNA extraction) employed in past clinical trials, and the repeated bead beating and column (RBB+C) method might impact the recovery of microbial DNA from colonic tissue, using a custom designed phylogenetic microarray for gut bacteria and archaea. All four methods produced very similar profiles of the microbial diversity, but there were some differences in probe signal intensities, with the HMW method producing stronger probe intensities for a subset of the Firmicutes probes including Clostridium and Streptococcus spp. Real-time PCR analysis revealed that the HMW and RBB+C extracted DNA contained significantly more DNA of Firmicutes origin and that the different DNA extraction methods also gave variable results in terms of host DNA recovery. All of the methods tested recovered DNA from the archaeal community although there were some differences in probe signal intensity. Based on these findings, we conclude that while all four methods are efficacious at releasing microbial DNA from biopsy tissue samples, the HMW and RBB+C methods of DNA extraction may release more DNA from some of the Firmicutes bacteria associated with colonic tissue. Thus, DNA archived in biobanks could be suitable for retrospective profiling analyses, provided the caveats with respect to the DNA extraction method(s) used are taken into account.     

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.     

Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into “dead” cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.     

Microbial diversity in the anaerobic tank of a full-scale produced water treatment plant

Microbial characteristics in the anaerobic tank of a full-scale produced water treatment plant capable of anaerobic hydrocarbon removal were analyzed and compared to those in the influent produced water using cultivation-independent molecular methods. Clones related to methanogens including the methylotrophic Methanomethylovorans thermophila and hydrogen- and the formate-utilizing Methanolinea tarda were in abundance in both samples, but greater numbers of M. tarda-like clones were detected in the biofilm library. Both DGGE and cloning analysis results indicated that the archaea in the biofilm were derived from the influent produced water. Bacterial communities in the influent and biofilm samples were significantly different. Epsilonproteobacteria was the dominant bacterial group in the influent while Nitrospira and Deltaproteobacteria were the predominant groups in the biofilm. Many clones related to syntrophic bacteria were found among the Deltaproteobacteria. One Deltaproteobacteria clone was related to Syntrophus, which is commonly found in methanogenic hydrocarbon-degrading consortia. A number of Deltaproteobacteria clones were assigned to the clone cluster group TA, members of which predominate in various methanogenic consortia that degrade aromatic compounds. These results suggest that a microbial community associated with methanogenic hydrocarbon degradation may have been established in the biofilm.     

Metagenomic and metaproteomic analyses of Accumulibacter phosphatis‐enriched floccular and granular biofilm

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory‐scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high‐stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter‐enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.     

种子微生物生态学研究进展

植物种子微生物生态学是研究与种子相联合的微生物的组成﹑功能﹑演替、它们之间关系及其与宿主之间相互关系的科学。种子中蕴含着丰富的微生物资源,它们对种子以及植物的健康具有重要的影响。不同种类植物种子联合的微生物群落由于受到种子本身及外界环境因素的影响而有所差异。论述了种子微生物生态学的概念、主要研究方法、种子微生物生态系统中的微生物种类、相关影响因素,以及种子微生物生态学研究的发展方向。种子微生物生态学的研究对生产实践有重要意义,同时也将丰富种子生物学的内容,对种子科学的发展起到促进作用。     

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.     

Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria,bacteri-polymer mixtures and biofilms

Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis for freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at ～ 1740 cm^?1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggests that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled to composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum (amide I/carbohydrate C-O-～ 1150 cm^?1) and bacteria-poly-β-hydroxybutyrate (amide I/carbonyl ～ 1740 cm^?1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR sepectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at ～ 1740 cm^?1 was seen in FT-IR spectra of V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactioni within adherent microbial consortia.     

Influence of Commonly Used Primer Systems on Automated Ribosomal Intergenic Spacer Analysis of Bacterial Communities in Environmental Samples

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.     

High-Throughput Screening of Multispecies Biofilm Formation and Quantitative PCR-Based Assessment of Individual Species Proportions,Useful for Exploring Interspecific Bacterial Interactions

Multispecies biofilms are predominant in almost all natural environments, where myriads of resident microorganisms interact with each other in both synergistic and antagonistic manners. The interspecies interactions among different bacteria are, despite the ubiquity of these communities, still poorly understood. Here, we report a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms, based on the Nunc-TSP lid system and crystal violet staining. The relative proportion of the individual species in a four-species biofilm was assessed using quantitative PCR based on SYBR Green I fluorescence with specific primers. The results indicated strong synergistic interactions in a four-species biofilm model community with a more than 3-fold increase in biofilm formation and demonstrated the strong dominance of two strains, Xanthomonas retroflexus and Paenibacillus amylolyticus. The developed approach can be used as a standard procedure for evaluating interspecies interactions in defined microbial communities. This will be of significant value in the quantitative study of the microbial composition of multispecies biofilms both in natural environments and infectious diseases to increase our understanding of the mechanisms that underlie cooperation, competition and fitness of individual species in mixed-species biofilms.     

Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration

Sewage sludge is the solid, organic material remaining after wastewater is treated and discharged from a wastewater treatment plant. Sludge is treated to stabilize the organic matter and reduce the amount of human pathogens. Once government regulations are met, including material quality standards (e.g., E. coli levels and heavy metal content) sludge is termed “biosolids”, which may be disposed of by land application according to regulations. Live-culture techniques have traditionally been used to enumerate select pathogens and/or indicator organisms to demonstrate compliance with regulatory requirements. However, these methods may result in underestimates of viable microorganisms due to several problems, including their inability to detect viable but non-culturable (VBNC) cells. Real-time quantitative polymerase chain reaction (qPCR) is currently under investigation as a fast, sensitive, and specific molecular tool for enumeration of pathogens in biosolids. Its main limitation is that it amplifies all target DNAs, including that from non-viable cells. This can be overcome by coupling qPCR with propidium monoazide (PMA), a microbial membrane-impermeant dye that binds to extracellular DNA and DNA in dead or membrane-compromised cells, inhibiting its amplification. PMA has successfully been used to monitor the presence of viable pathogens in several different matrices. In this review the use of PMA-qPCR is discussed as a suitable approach for viable microbial enumeration in biosolids. Recommendations for optimization of the method are made, with a focus on DNA extraction, dilution of sample turbidity, reagent concentration, and light exposure time.     

Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration

Sewage sludge is the solid, organic material remaining after wastewater is treated and discharged from a wastewater treatment plant. Sludge is treated to stabilize the organic matter and reduce the amount of human pathogens. Once government regulations are met, including material quality standards (e.g., E. coli levels and heavy metal content) sludge is termed “biosolids”, which may be disposed of by land application according to regulations. Live-culture techniques have traditionally been used to enumerate select pathogens and/or indicator organisms to demonstrate compliance with regulatory requirements. However, these methods may result in underestimates of viable microorganisms due to several problems, including their inability to detect viable but non-culturable (VBNC) cells. Real-time quantitative polymerase chain reaction (qPCR) is currently under investigation as a fast, sensitive, and specific molecular tool for enumeration of pathogens in biosolids. Its main limitation is that it amplifies all target DNAs, including that from non-viable cells. This can be overcome by coupling qPCR with propidium monoazide (PMA), a microbial membrane-impermeant dye that binds to extracellular DNA and DNA in dead or membrane-compromised cells, inhibiting its amplification. PMA has successfully been used to monitor the presence of viable pathogens in several different matrices. In this review the use of PMA–qPCR is discussed as a suitable approach for viable microbial enumeration in biosolids. Recommendations for optimization of the method are made, with a focus on DNA extraction, dilution of sample turbidity, reagent concentration, and light exposure time.     

Modulation of eDNA Release and Degradation Affects Staphylococcus aureus Biofilm Maturation

Recent studies have demonstrated a role for Staphylococcus aureus cidA-mediated cell lysis and genomic DNA release in biofilm adherence. The current study extends these findings by examining both temporal and additional genetic factors involved in the control of genomic DNA release and degradation during biofilm maturation. Cell lysis and DNA release were found to be critical for biofilm attachment during the initial stages of development and the released DNA (eDNA) remained an important matrix component during biofilm maturation. This study also revealed that an lrgAB mutant exhibits increased biofilm adherence and matrix-associated eDNA consistent with its proposed role as an inhibitor of cidA-mediated lysis. In flow-cell assays, both cid and lrg mutations had dramatic effects on biofilm maturation and tower formation. Finally, staphylococcal thermonuclease was shown to be involved in biofilm development as a nuc mutant formed a thicker biofilm containing increased levels of matrix-associated eDNA. Together, these findings suggest a model in which the opposing activities of the cid and lrg gene products control cell lysis and genomic DNA release during biofilm development, while staphylococcal thermonuclease functions to degrade the eDNA, possibly as a means to promote biofilm dispersal.     

Differential effect of sample preservation methods on plant and arbuscular mycorrhizal fungal DNA

A wide range of methods are commonly used for preserving environmental samples prior to molecular analyses. However, the effect of these preservation methods on fungal DNA is not understood. The objective of this study was to test the effect of eight different preservation methods on the quality and yield of DNA extracted from Bromus inermis and Daucus carota roots colonized by the arbuscular mycorrhizal (AM) fungus, Glomus intraradices. The total DNA concentration in sample extracts was quantified using spectrophotometry. Samples that were frozen (− 80 ºC and − 20 ºC), stored in 95% ethanol, or silica gel dried yielded total (plant and fungal) DNA concentrations that were not significantly different from fresh samples. In contrast, samples stored in CTAB solution or freeze-dried resulted in significantly reduced DNA concentrations compared with fresh samples. The preservation methods had no effect on the purity of the sample extracts for both plant species. However, the DNA of the dried samples (silica gel dried, freeze-dried, heat dried) appeared to be slightly more degraded compared with samples that remained hydrated (frozen, stored in ethanol or CTAB solutions) during storage when visualized on a gel. The concentration of AM fungal DNA in sample extracts was quantified using TaqMan real time PCR. Methods that preserved samples in hydrated form had similar AM fungal DNA concentrations as fresh samples, except D. carota samples stored in ethanol. In contrast, preservation methods that involved drying the samples had very low concentrations of AM fungal DNA for B. inermis, and nearly undetectable for D. carota samples. The drying process appears to be a major factor in the degradation of AM fungal DNA while having less of an impact on plant DNA. Based on these results, samples that need to be preserved prior to molecular analysis of AM fungi should be kept frozen to minimize the degradation of plant and AM fungal DNA.     

Influence of an Oyster Reef on Development of the Microbial Heterotrophic Community of an Estuarine Biofilm

We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the γ- and δ-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium -Bacteroides cluster, and the phyla Planctomyces and Holophaga-Acidobacterium. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the Planctomyces phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.     

Peppermint (Mentha piperita) inhibits microbial biofilms in vitro

Microbial biofilms have become increasingly problematic in the food processing and medical industries where they cause food and surface contamination. Biofilms have also been implicated as the cause of serious infections in humans as their occurrence makes it difficult to treat common infections and the likelihood of recurrent infections is high. Due to emerging resistance, conventional control methods are fast becoming ineffective. In this study, the use of a selection of commercial plant extracts is investigated. The inhibitory effects of eight herbal extracts on the development of microbial biofilms was investigated against clinical and reference strains of Pseudomonas aeruginosa and Candida albicans. The antimicrobial activity was investigated on the planktonic forms using the minimum inhibitory concentration assay. The extracts that showed the highest antimicrobial activity against the two test organisms were Echinacea angustifolia (cone flower), Mentha piperita (peppermint) and Rosmarinus officinalis (rosemary) with minimum inhibitory concentration values between 0.38 and 2.5 mg/ml. The crystal violet assay was used to assess the effect of pre-treating a surface with plant extracts on cell attachment and the extent of biofilm development following exposure to extracts (biofilm biomass). Most of the extracts reduced microbial colonization by at least 50%. In contrast, preformed biofilms were less responsive to the majority of extracts, thus growth inhibition was more difficult to achieve. Mentha piperita was the only extract that showed some antibiofilm activity against both pathogens.