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PubMed2006

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.

Bacteria↗
PubMed2004

Evaluation of chlorines' impact on biofilms on scratched stainless steel surfaces.

Biofilms of a wild type Escherichia coli were grown on 316 stainless steel slides in a nutrient starved medium. The stainless steel surfaces were either polished to a smooth finish or scribed. The scribes consisted of lines and crosses. Biofilm samples were taken after 3, 6, 12, 24 and 48 h of growth. After sampling, the slides were soaked in deionized water or 50 or 200 ppm free chlorine prior to vital staining. Images were captured and the areas of viable and total biofilm were estimated. The individual biofilm patches, circularities, total percentage coverage, and viability percentage coverage were analyzed. The biofilms tended to increase in size between 6 and 24 h. A 3-6 h old biofilm on a polished stainless steel surface detached when 200 ppm sodium hypochlorite was applied. When grown in scribes, the circularity decreased up to 24 h, but thereafter increased. As the film grew older, it detached with or without a sodium hypochlorite treatment from the part of the surface that was polished, but remained in the neighborhood of the scribe. Based on the results, we recommend sanitizing at intervals of less than 12 h for this and similar strains of bacteria and protection of stainless steel surfaces to minimize scratching.

Analysis of Variance↗
PubMed2004

ComX activity of Streptococcus mutans growing in biofilms.

In many streptococci, including Streptococcus mutans, genetic competence is regulated by a quorum sensing system mediated by a competence stimulating peptide (CSP) pheromone, encoded by the comC gene. In Streptococcus pneumoniae, a central component of this system is ComX, which acts as an alternative sigma factor to activate competence genes involved in DNA uptake and processing. The quorum sensing system responsible for genetic competence induction in S. mutans has been linked to biofilm formation and the acid tolerance response. To examine the response of comX to CSP in S. mutans, a transcriptional fusion of the comX promoter (pcomX) with lacZ was constructed to generate reporter vector pcomx::pALH122 (replicative vector) and transformed into S. mutans UA159 comC-, which is unable to produce endogenous CSP. CSP was added and pcomX::lacZ relative expression index (REI) examined, revealing a 2-fold increase in maximal beta-gal activity 5 and 10 min after CSP addition. The effect of endogenous CSP on pcomX::lacZ expression was also examined by measuring REI in cells grown as a biofilm; peak pcomX activity was observed at 3 h. To determine the temporal pattern of transformation frequency, pMA2, a Spr shuttle vector, was transformed into biofilm-grown cells, with maximal transformation frequency observed at 3 h. Confocal microscopy was performed to examine pcomX activity using a similarly constructed green fluorescent protein reporter vector, pcomX::gfp, in a 4-h biofilm, revealing active pcomX activity in high cell density areas within the biofilm population. These results demonstrated a positive correlation between pcomX activity, natural transformation and competence development in biofilms.

Adaptation, Physiological↗
PubMed2006

Identification of Helicobacter pylori biofilms in human gastric mucosa.

The purpose of this study was to use endoscopically directed biopsies and scanning electron microscopy (SEM) to document the existence of Helicobacter pylori biofilms in human gastric mucosa. Patients underwent flexible esophagogastroduodenoscopies with three gastric mucosal biopsies. Rapid urease testing was performed to determine the presence or absence of H pylori. Urease-positive and urease-negative control specimens were imaged with SEM to obtain detailed images of gastric mucosa for the identification of biofilm colonies. Samples were obtained from patients who underwent esophagogastroduodenoscopies. Eleven were found to be H pylori positive and nine were H pylori negative. These were imaged at 500x and 1000x with electron microscopy. Dense, mature biofilms were present and attached to the cell surface of H pylori-positive specimens and were absent in urease-negative controls. Photomicrographs were obtained. Biofilms are complex microbiological ecosystems where sessile bacteria surround themselves in a protective matrix. This lifestyle affords protection, allows for growth in hostile environments, and alters host physiology. Many have hypothesized that H pylori infections resulting in gastric ulcers may be a manifestation of biofilms. Our investigation is the first to photographically document the existence of H pylori biofilms on human gastric mucosa. This elucidation of the ecology and pathophysiology of the mucosa of the organism is important to our understanding of a potential mechanism of this organism's resistance to current therapy and how to better eradicate it in the future.

Biofilms↗
PubMed2006

Presence and density of Helicobacter pylori biofilms in human gastric mucosa in patients with peptic ulcer disease.

Our purpose was to use endoscopically directed biopsies and scanning electron microscopy to quantify Helicobacter pylori biofilm density on the surface of human gastric mucosa in urease-positive and -negative patients. Participating patients underwent flexible esophago-gastro-duodenoscopies coupled with gastric mucosal biopsies. Rapid urease testing was performed on all specimens to determine the presence of H. pylori, followed by scanning electron microscopy to identify the existence of biofilms. Samples were then analyzed using Carnoy Image Analysis Software to determine percent biofilm coverage of the total surface area. These data were compared to control specimens that were urease negative. Of the patients who tested urease positive for H. pylori, the average percent of total surface area covered by biofilms was 97.3%. Those testing negative had an average surface area coverage of only 1.64%. These differences were determined to be statistically significant at the 0.0001 level. This study demonstrates that compared with controls, urease-positive specimens have significant biofilm formation, whereas urease-negative specimens have little to none. This was reflected in the significantly increased biofilm surface density in urease positive specimens compared with urease-negative controls.

Biofilms↗
PubMed2004

Prevalence of microbial biofilms on selected fresh produce and household surfaces.

Investigations of biofilms in domestic environments are sparsely represented in the literature. In this study, samples of various household surfaces, including food, laundry and kitchen items, were analyzed for evidence of biofilm presence. Visualization of the surfaces was carried out using cryostage scanning electron microscopy (CSEM) and light microscopy. Qualitative evidence of the presence of biofilm formation was obtained from all of the sample groups analyzed, suggesting the widespread existence of microorganisms in biofilms on domestic surfaces. This suggests that biofilms may be important in household hygiene, and highlights the need for standardized, approved biofilm methods suitable for consumer products testing.

Bacteria↗
PubMed2005

A novel method to control the balance of skin microflora. Part 1. Attack on biofilm of Staphylococcus aureus without antibiotics.

BACKGROUND: Staphylococcus aureus (SA) is usually present in atopic dry skin, and not only in regions seriously affected by atopic dermatitis. SA discharges various toxins and enzymes that injure the skin, and forms a biofilm from fibrin fiber and glycocalyx; the biofilm is important for adhesion of SA to the skin and for resistance to anti-microbial agents. Even highly effective moisturizers do not work perfectly on atopic dry skin. Staphylococcus epidermidis (SE) is a major constituent of skin microflora on healthy human skin, and provides protection against the growth of pathogenic bacteria. OBJECTIVES: Since treatment with anti-microbials may lead to re-growth of SA, which grows faster than other Staphylococci and often shows antibiotic resistance, we searched for novel approaches to control the skin-microfloral balance without using conventional anti-microbials. METHOD: Biofilm formation by SA in vitro was observed in detail using scanning electron microscopy. Approximately 500 substances were screened for a selective effect on SA growth and SA biofilm. RESULTS: We found that xylitol inhibited the formation of glycocalyx, and farnesol dissolved fibrin fibers. Farnesol suppressed the growth of only SA, and did not affect that of SE. Xylitol and farnesol synergistically inhibited biofilm formation by SA. CONCLUSION: Xylitol and farnesol have potential for controlling the skin-microfloral balance because of their selective effects and inhibition of biofilm formation. They might provide a useful and safe method to care for skin colonized by SA, without using antibiotics.

Administration, Cutaneous↗
PubMed2006

Low-biodegradable composite chemical wastewater treatment by biofilm configured sequencing batch reactor (SBBR).

Biofilm configured system with sequencing/periodic discontinuous batch mode operation was evaluated for the treatment of low-biodegradable composite chemical wastewater (low BOD/COD ratio approximately 0.3, high sulfate content: 1.75 g/l) in aerobic metabolic function. Reactor was operated under anoxic-aerobic-anoxic microenvironment conditions with a total cycle period of 24 h [fill: 15 min; reaction: 23 h (aeration along with recirculation); settle: 30 min; decant: 15 min] and the performance of the system was studied at organic loading rates (OLR) of 0.92, 1.50, 3.07 and 4.76 kg COD/cum-day. Substrate utilization showed a steady increase with increase in OLR and system performance sustained at higher loading rates. Maximum non-cumulative substrate utilization was observed after 4h of the cycle operation. Sulfate removal efficiency of 20% was observed due to the induced anoxic conditions prevailing during the sequence phase operation of the reactor and the existing internal anoxic zones in the biofilm matrix. Biofilm configured sequencing batch reactor (SBR) showed comparatively higher efficiency to the corresponding suspended growth and granular activated carbon (GAC) configured systems studied with same wastewater. Periodic discontinuous batch mode operation of the biofilm reactors results in a more even distribution of the biomass throughout the reactor and was able to treat large shock loads than the continuous flow process. Biofilm configured system coupled with periodic discontinuous batch mode operation imposes regular variations in the substrate concentration on biofilm organisms. As a result, organisms throughout the film achieve maximum growth rates resulting in improved reaction potential leading to stable and robust system which is well suited for treating highly variable wastes.

Biodegradation, Environmental↗
PubMed2005

Detection of icaA and icaD loci by polymerase chain reaction and biofilm formation by Staphylococcus epidermidis isolated from dialysate and needles in a dialysis unit.

Staphylococcus epidermidis, a coagulase-negative staphylococcus, is a major cause of infections associated with indwelling medical devices. Certain strains produce slime and form biofilm on polymer surfaces, where their pathogenicity is associated with biofilm formation. In this report, we investigated the presence or absence of the intercellular adhesion icaA and icaD genes by polymerase chain reaction, and phenotypic biofilm production was examined by qualitative Congo red agar (CRA) assay. A total of 32 strains of S. epidermidis were identified from dialysates and needles 4h after the initiation of dialysis. Qualitative biofilm production revealed that 16 (50%) strains produced slime on CRA plates. Among the 23 strains positive for the ica operon, 15 were biofilm positive on CRA, eight were biofilm negative, and one was icaA and icaD negative but produced slime. These results show that the ability of S. epidermidis to produce slime is not associated with the presence of icaA and icaD genes.

Bacterial Adhesion↗
PubMed2006

Effect of different stabilized preparations of peracetic acid on biofilm.

The effect of three stabilized peracetic acid (PAA) preparations (Bioxal M), with or without surfactants, on an Escherichia coli biofilm model was studied. The biofilm was prepared in glass tubes, and was evaluated indirectly using spectrophotometry. The ability of the products to fix or remove the biofilm was determined by their detergent activity (DA). None of the preparations tested fixed the biofilm. The effect of Bioxal M-1 on the biofilm was equivalent to the control (sterile water). Bioxal M-2 and Bioxal M-3 displayed slightly positive DAs. Non-ionic surfactant improved the DA of the products. Regardless of disinfectant activity, PAA agents display different DAs depending on their formulation. This criterion could be used to select the weakest biofilm-fixing agents. Users should therefore be concerned about the efficiency of the cleaning stage of medical devices. When choosing PAA products, non-fixing ability should be considered in addition to antimicrobial activity.

Biofilms↗
PubMed2006

The effect of exposure to irrigant solutions on apical dentin biofilms in vitro.

This study assessed the effectiveness of different concentrations of sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) (Vista Dental Products, Racine, WI), and BioPure MTAD (Dentsply Endodontics-Tulsa Dental, Tulsa, OK). Intracanal contents were collected from 10 patients diagnosed with chronic apical periodontitis. The samples were cultured on hemisections of root apices to generate a polymicrobial biofilm. Each biofilm was separately immersed in 6% NaOCl, 3% NaOCl, 1% NaOCl, 2% CHX, 1% NaOCl followed by BioPure MTAD, and sterile phosphate buffered solution (PBS). SEM analysis showed 6% NaOCl and 3% NaOCl were capable of disrupting and removing the biofilm; 1% NaOCl and 1% NaOCl followed by MTAD were capable of disrupting the biofilm, but not eliminating bacteria; 2% CHX was not capable of disrupting the biofilm. Viable bacteria could not be cultured from specimens exposed to 6% NaOCl, 2 % CHX, or 1% NaOCl followed by BioPure MTAD. These results indicate that 6% NaOCl was the only irrigant capable of both rendering bacteria nonviable and physically removing the biofilm.

Biofilms↗
PubMed2007

Biofilm formation in uropathogenic Escherichia coli strains: relationship with prostatitis, urovirulence factors and antimicrobial resistance.

PURPOSE: Escherichia coli strains are the most frequent cause of urinary tract infections. Biofilm formation allows the strains to persist a long time in the genitourinary tract and interfere with bacterial eradication. We determined the possible relationships between the different urinary tract infections, and in vitro biofilm formation, the presence of urovirulence factors and nalidixic acid resistance. MATERIALS AND METHODS: A total of 151 E. coli strains collected from patients with cystitis (44 strains), pyelonephritis (75) and prostatitis (32) were analyzed for in vitro biofilm formation, the phylogenetic group, the presence of several urovirulence factors and resistance to nalidixic acid. RESULTS: E. coli strains causing prostatitis produced biofilm in vitro more frequently than those causing other urinary tract infections and had a higher frequency of hemolysin (p = 0.03 and 0.0002, respectively). However, only hemolysin was independently associated with prostatitis. On the other hand, strains forming biofilm presented a significantly higher frequency of hemolysin and type 1 fimbriae expression. CONCLUSIONS: Although hemolysin is the main virulence factor by which E. coli causes acute prostatitis, the association between hemolysin and biofilm formation may result in increased ability of E. coli strains to persist in the prostate.

Biofilms↗
PubMed2006

A multi-phase mathematical model of quorum sensing in a maturing Pseudomonas aeruginosa biofilm.

It is well known that sessile bacteria have a strong tendency to exist in a biofilm phenotype, whereby bacterial cells aggregate and produce a gel-like extracellular matrix, which, in an infection scenario, offers a significant barrier to attack by conventional antibiotics and the immune system. In this paper we develop a multi-phase model of a maturing Pseudomonas aeruginosa biofilm, allowing for the production and secretion of exopolysaccharide (EPS). The primary quorum-sensing system of P. aeruginosa (namely the lasR system) is believed to be required for full biofilm development, and we thus take the synthesis of EPS to be regulated by the cognate signal molecule, 3-oxo-C12-HSL. We also take EPS and signal production, along with bacterial growth, to be limited by oxygen availability, thus factoring in the nutrient poor conditions deep inside the biofilm. We use simulations to examine the role played by quorum sensing in the biofilm maturation process, and to investigate the effect of anti-quorum sensing and antibiotic treatments on EPS concentration, signal level, bacterial numbers and biofilm growth rate. In addition, we undertake analysis of the associated travelling-wave behaviour.

Biofilms↗
PubMed2006

Quorum sensing and DNA release in bacterial biofilms.

The multicellular behavior of bacteria has been the subject of much recent interest. This behavior includes coordinated control of virulence, luminescence, competence and biofilm formation; each of these appears to be regulated or influenced by quorum sensing. An understanding of what biofilms constitute, and how they develop, is emerging. It is clear that biofilm formation is a carefully orchestrated process that is dependent on quorum sensing. Somewhat surprisingly, several independent observations have noted an important role for DNA in the structure of biofilms. Recent studies describe a mechanism for linking DNA release to quorum sensing, providing a possible mechanism for the coordinated release of DNA, and its integration into a biofilm. A review of the literature reveals that similar observations have been made for biofilms of both Gram-positive and Gram-negative organisms. Further study will determine whether this is a general trend, however.

4-Butyrolactone↗
PubMed2006

Influence of magnesium ions on biofilm formation by Pseudomonas fluorescens.

Mg(2+) can potentially influence bacterial adhesion directly through effects on electrostatic interactions and indirectly by affecting physiology-dependent attachment processes. However, the effects of Mg(2+) on biofilm structure are largely unknown. In this study, Pseudomonas fluorescens was used to investigate the influence of Mg(2+) concentration (0, 0.1 and 1.0mM MgCl(2)) on biofilm growth. Planktonic and attached cells were enumerated (based on DAPI staining) while biofilm structures were examined via confocal laser scanning microscopy and three-dimensional structures were reconstructed. Mg(2+) concentration had no influence on growth of planktonic cells but, during biofilm formation, Mg(2+) increased the abundance of attached cells. For attached cells, the influence of Mg(2+) concentration changed over time, suggesting that the role of Mg(2+) in bacterial attachment is complex and dynamic. Biofilm structures were heterogeneous and surface colonization and depth increased with increasing Mg(2+) concentrations. Overall, for P. fluorescens, Mg(2+) increased initial attachment and altered subsequent biofilm formation and structure.

Bacterial Adhesion↗
PubMed2005

Biofilm formation by Escherichia coli is stimulated by synergistic interactions and co-adhesion mechanisms with adherence-proficient bacteria.

Laboratory strains of Escherichia coli do not show significant ability to attach to solid surfaces and to form biofilms. We compared the adhesion properties of the E. coli PHL565 laboratory strain to eight environmental E. coli isolates: only four isolates displayed adhesion properties to glass significantly higher than PHL565. The ability of the adhesion-proficient isolates to attach to glass tubes strongly correlated with their ability to express curli (thin aggregative fimbriae), thus suggesting that curli are a common adhesion determinant in environmental strains. Despite its inability to attach to solid surfaces, growth of E. coli PHL565 in mixed cultures with Pseudomonas putida MT2 resulted in co-adhesion and in formation of a mixed E. coli/P. putida biofilm, which was able to colonize glass surfaces with dramatic efficiency compared to P. putida alone. E. coli/P. putida interactions stimulate initial adhesion to glass, and the presence of both bacterial species in the mature biofilm was confirmed by quantitative PCR. In contrast, no synergistic biofilm formation was observed in mixed cultures of E. coli with the Gram-positive bacterium Staphylococcus epidermidis. Interestingly, E. coli PHL565 also stimulated biofilm formation by bacterial communities isolated from drinking water distribution systems. Our results strongly suggest that co-adhesion and synergistic interaction with biofilm-forming species might represent an important mechanism, and a possible alternative strategy to production of adhesion determinants, for persistence and propagation of E. coli in the environment.

Bacterial Adhesion↗
PubMed2005

Biofilm formation and dispersal and the transmission of human pathogens.

Several pathogenic bacterial species that are found in the environment can form complex multicellular structures on surfaces known as biofilms. Pseudomonas aeruginosa, Vibrio cholerae and certain species of nontuberculous mycobacteria are examples of human pathogens that form biofilms in natural aquatic environments. We suggest that the dynamics of biofilm formation facilitates the transmission of pathogens by providing a stable protective environment and acting as a nidus for the dissemination of large numbers of microorganisms; both as detached biofilm clumps and by the fluid-driven dispersal of biofilm clusters along surfaces. We also suggest that emerging evidence indicates that biofilm formation conveys a selective advantage to certain pathogens by increasing their ability to persist under diverse environmental conditions.

Bacteria↗
PubMed2005

Finding gene-expression patterns in bacterial biofilms.

The production of biofilms by bacteria is a lifestyle that is thought to require or involve a differential gene expression compared with that of planktonic bacteria. Recently, we have witnessed a change of focus from the simple hunt for hypothetical essential biofilm genes to the identification of late and more complex biofilm functions. However, finding common bacterial biofilm gene-expression patterns through global expression analysis remains difficult. Owing to the apparently minimal overlap between functions involved in biofilm formation by different bacteria, exploring the biofilm lifestyle could prove to be a case-by-case task for which global approaches show their limits.

Adaptation, Physiological↗