Conjugative Plasmid Transfer and Adhesion Dynamics in an Escherichia coli Biofilm

A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-related infections in nosocomial settings.Bacterial biofilms are complex communities of bacterial cells living in close association with a surface (17). Bacterial cells in these protected environments are often resistant to multiple factors, including antimicrobials, changes in the pH, oxygen radicals, and host immune defenses (19, 38). Biofilm formation is a property of many bacterial species, and a range of molecular mechanisms that facilitate this process have been described (2, 3, 11, 14, 16, 29, 33, 34). Often, the ability to form a biofilm is dependent on the production of adhesins on the bacterial cell surface. In Escherichia coli, biofilm formation is enhanced by the production of certain types of fimbriae (e.g., type 1 fimbriae, type 3 fimbriae, F1C, F9, curli, and conjugative pili) (14, 23, 25, 29, 33, 39, 46), cell surface adhesins (e.g., autotransporter proteins such as antigen 43, AidA, TibA, EhaA, and UpaG) (21, 34, 35, 40, 43), and flagella (22, 45).The close proximity of bacterial cells in biofilms creates an environment conducive for the exchange of genetic material. Indeed, plasmid-mediated conjugation in monospecific and mixed E. coli biofilms has been demonstrated (6, 18, 24, 31). The F plasmid represents the best-characterized conjugative system for biofilm formation by E. coli. The F pilus mediates adhesion to abiotic surfaces and stabilizes the biofilm structure through cell-cell interactions (16, 30). Many other conjugative plasmids also contribute directly to biofilm formation upon derepression of the conjugative function (16).One example of a conjugative system employed by gram-negative Enterobacteriaceae is the type 4 secretion (T4S) system. The T4S system is a multisubunit structure that spans the cell envelope and contains a secretion channel often linked to a pilus or other surface filament or protein (8). The Agrobacterium tumefaciens VirB-VirD4 system is the archetypical T4S system and is encoded by 11 genes in the virB operon and one gene (virD4) in the virD operon (7, 8). Genes with strong homology to genes in the virB operon have also been identified on other conjugative plasmids. For example, the pilX1 to pilX11 genes on the E. coli R6K IncX plasmid and the virB1 to virB11 genes are highly conserved at the nucleotide level (28).We recently described identification and characterization of the mrk genes encoding type 3 fimbriae in a uropathogenic strain of E. coli isolated from a patient with a nosocomial catheter-associated urinary tract infection (CAUTI) (29). The mrk genes were located on a conjugative plasmid (pMAS2027) and were strongly associated with biofilm formation. In this study we determined the entire sequence of plasmid pMAS2027 and revealed the presence of conjugative transfer genes homologous to the pilX1 to pilX11 genes of E. coli R6K (in addition to the mrk genes). We show here that biofilm formation is driven primarily by type 3 fimbriae and that the T4S apparatus is unable to mediate biofilm growth in the absence of the mrk genes. Finally, we demonstrate that conjugative transfer of pMAS2027 within a mixed biofilm confers biofilm formation properties on recipient cells due to acquisition of the type 3 fimbria-encoding mrk genes.     

Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing

Subtilosin, the cyclic lantibiotic protein produced by Bacillus subtilis KATMIRA1933, targets the surface receptor and electrostatically binds to the bacterial cell membrane. In this study, subtilosin was purified using ammonium sulfate ((NH₄)₂SO₄) precipitation and purified via column chromatography. Subtilosin’s antibacterial minimum and sub-minimum inhibitory concentrations (MIC and sub-MIC) and anti-biofilm activity (biofilm prevention) were established. Subtilosin was evaluated as a quorum sensing (QS) inhibitor in Gram-positive bacteria using Fe(III) reduction assay. In Gram-negative bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter. The results showed that Gardnerella vaginalis was more sensitive to subtilosin with MIC of 6.25 μg/mL when compared to Listeria monocytogenes (125 μg/mL). The lowest concentration of subtilosin, at which more than 90% of G. vaginalis biofilm was inhibited without effecting the growth of planktonic cells, was 0.78 μg/mL. About 80% of L. monocytogenes and more than 60% of Escherichia coli biofilm was inhibited when 15.1 μg/mL of subtilosin was applied. Subtilosin with 7.8–125 μg/mL showed a significant reduction in violacein production without any inhibitory effect on the growth of C. violaceum. Subtilosin at 3 and 4 μg/mL reduced the level of Autoinducer-2 (AI-2) production in G. vaginalis. However, subtilosin did not influence AI-2 production by L. monocytogenes at sub-MICs of 0.95–15.1 μg/mL. To our knowledge, this is the first report exploring the relationship between biofilm prevention and quorum sensing inhibition in G. vaginalis using subtilosin as a quorum sensing inhibitor.     

Inactivation of Efflux Pumps Abolishes Bacterial Biofilm Formation

Bacterial biofilms cause numerous problems in health care and industry; notably, biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Bacteria rely on efflux pumps to get rid of toxic substances. We discovered that efflux pumps are highly active in bacterial biofilms, thus making efflux pumps attractive targets for antibiofilm measures. A number of efflux pump inhibitors (EPIs) are known. EPIs were shown to reduce biofilm formation, and in combination they could abolish biofilm formation completely. Also, EPIs were able to block the antibiotic tolerance of biofilms. The results of this feasibility study might pave the way for new treatments for biofilm-related infections and may be exploited for prevention of biofilms in general.     

激光扫描共聚焦显微镜观察细菌生物膜形成的方法学研究

目的:建立激光扫描共聚焦显微镜观察生物膜形成过程的方法,为进一步研究生物膜的形成机制奠定基础。方法:以临床分离金葡菌X428为研究对象,在盖玻片上形成生物膜,分别于接种后的4、8、12、16、24和48h取出玻片,采用免疫荧光技术标记多糖和细菌,激光扫描共聚焦显微镜(CLSM)观察生物膜形成情况。结果:取得了生物膜形成过程的不同时间点的CLSM图像,4h时细菌在盖玻片上粘附形成小菌落,8h和12h细菌聚集成簇,多糖基质产生并逐渐增多,至16h形成成熟生物膜结构;24h和48h生物膜已经播散,其结构变小。结论:应用免疫荧光技术和激光扫描共聚焦显微镜技术研究生物膜形成过程是一种简便可行的方法。     

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

Bacterial Adhesion and Biofilm Formation in the Presence of Chitosan and Its Derivatives

Microbiology - The effect of chitosan and its derivatives on adhesion and biofilm formation by bacteria with diametrically opposite properties of cell surfaces was studied. Treatment of polystyrene...     

High Frequency Conjugative Mobilization Accomplished by a Natural Bacillus subtilisStrain Carrying a Large Plasmid

Conjugative properties of the strain Bacillus subtiliscarrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis168 strain with a frequency of approximately 10^–2. The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugative mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37°C, but conjugation also proceeded at lower temperatures, up to 21°C.     

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.     

Comparative Effect of Chlorhexidine and Some Mouthrinses on Bacterial Biofilm Formation on Titanium Surface

The aim of the present study was to evaluate the effectiveness of chlorhexidine digluconate (CHX) and commonly used mouthrinses to single- and poly-species biofilms by S. mutans, S. aureus and P. aeruginosa, on titanium discs of grade IV. The formation of single- and poly-species biofilms at 16.5, 40.5 and 64.5-h incubation on titanium surface was evaluated by plate count (CFU ml⁻¹) before and after exposure to CHX and four mouthrinses (Curasept, Listerine, Meridol and Buccagel) and expressed as percentage of Inhibitory Activity (IA%). The application of the different anti-plaque formulations on biofilm can reduce the adhesion of bacteria to titanium surface with different degrees. The higher efficacy was observed for Listerine that shows IA% = 100 on the biofilm formed by S. mutans at 16.5 h. Log count of CFU was dependent to culture time and four mouthrinses for S. mutans and S. aureus, whilst was not dependent to culture time but to mouthrinses for P. aeruginosa. In general, the efficacy was particularly lesser to poly-species biofilms; no statistical differences were evidenced between all the mouthrinses and CHX as control group. The tested mouthrinses, compared to reference CHX 0.2%, have demonstrated a significant lower antibacterial activity than Listerine towards the experimental biofilms. This “in vitro” biofilm model should prove extremely useful for pre-clinical testing of anti-plaque agents, which inhibit biofilm formation, can prevent subsequent implant failure.     

The Mechanism of Fractal-Like Structure Formation by Bacterial Populations

Three types of population growth and development of chemotaxic motile bacteria Escherichia coli on semi-solid nutrient media are investigated: a) stable development – circular symmetrical waves; b) bursts; c) fractal-like self-organization. Experimental investigation of the burst formation is presented. The microscopic analysis of growing, fractal-like structures is carried out, and a mechanism for such structure formation is suggested. It is supposed that fractal-like bacterial structures growth is based on the principle of successively forming multiple micro-bursts. A mathematical model has been suggested to reproduce the experimental results. The structures obtained by numerical modeling of population growth in the parameter space substrate concentration - bacterial movement rate reproduce the corresponding experimental structures in the space nutrient concentration in the media – the density of the media.     

Analysis of Bacterial Spatial Patterns at the Initial Stage of Biofilm Formation

Using sophisticated microscopy techniques, we observed the spatial pattern of bacteria colonizing a sterile 316L stainless steel coupon as bulk water containing bacteria flowed across the coupon. The experiments used stainless steel of differing roughness and surface chemistry. The ultimate goal was to identify surface features which influence bacterial adsorption. The immediate statistical goal was to distinguish patterns consistent with complete spatial randomness from patterns showing regularity or aggregation. This goal was accomplished by using modified analyses of distance functions commonly applied in field ecology. The method protected against a potential multiple comparisons problem. For the null value of the distance function, we calculated tolerance envelopes such that the tolerance level was simultaneous for all distances of concern. Computer simulation experiments showed that the nominal level was accurate. The methodology was effective for detecting and describing patterns of colonization known not to be completely spatially random.     

Effects of Norspermidine and Spermidine on Biofilm Formation by Potentially Pathogenic Escherichia coli and Salmonella enterica Wild-Type Strains

Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.     

Study of the Response of a Biofilm Bacterial Community to UV Radiation

We have developed a bioluminescent whole-cell biosensor that can be incorporated into biofilm ecosystems. RM4440 is a Pseudomonas aeruginosa FRD1 derivative that carries a plasmid-based recA-luxCDABE fusion. We immobilized RM4440 in an alginate matrix to simulate a biofilm, and we studied its response to UV radiation damage. The biofilm showed a protective property by physical shielding against UV C, UV B, and UV A. Absorption of UV light by the alginate matrix translated into a higher survival rate than observed with planktonic cells at similar input fluences. UV A was shown to be effectively blocked by the biofilm matrix and to have no detectable effects on cells contained in the biofilm. However, in the presence of photosensitizers (i.e., psoralen), UV A was effective in inducing light production and cell death. RM4440 has proved to be a useful tool to study microbial communities in a noninvasive manner.     

Biosorption of Copper by a Bacterial Biofilm on a Flexible Polyvinyl Chloride Conduit

Inexpensive technologies with less-than-optimal efficiencies as a strategy for countering economic restraints to pollution control have been evaluated by using a laboratory-scale biotreatment process for copper-containing effluent. Economizing measures include the use of polyvinyl chloride (PVC) cylinders fashioned from commercially available flexible PVC conduit to support a biofilm that was cultured in an inexpensive medium prepared in wastewater. The biofilm was challenged by aqueous copper solution in a bioreactor and subsequently analyzed under a scanning electron microscope with energy-dispersive X-ray microanalysis.     

Effect of Homocysteine on Biofilm Formation by Mycobacteria

Mycobacteria show peculiar aggregated outgrowth like biofilm on the surface of solid or liquid media. Biofilms harbor antibiotic resistant bacteria in a self-produced extracellular matrix that signifies the bacterial fate to sedentary existence. Despite years of research, very little is known about the mechanisms that contribute to biofilm formation. LuxS has been previously known to play a role in biofilm formation in Autoinducer-2 dependent manner. We here show the effect of LuxS product-homocysteine, on the biofilm forming ability of non-tuberculous mycobacteria, Mycobacterium smegmatis and Mycobacterium bovis BCG showing AI-2 independent phenotypic effect of LuxS. Exogenous supplementation of homocysteine in the culture media leads to aberrant cording, pellicle outgrowth, and biofilm formation. Thus, our study contributes to the better understanding of the mechanism of mycobacterial biofilm formation and sheds light on the role of LuxS product homocysteine. In addition, we highlight the contribution of activated methyl cycle in bacterial quorum sensing.     

Influence of Exudates of the Kelp Laminaria Digitata on Biofilm Formation of Associated and Exogenous Bacterial Epiphytes

Wild populations of brown marine algae (Phaeophyta) provide extensive surfaces to bacteria and epiphytic eukaryotes for colonization. On one hand, various strategies allow kelps prevent frond surface fouling which would retard growth by reducing photosynthesis and increasing pathogenesis. On the other hand, production and release of organic exudates of high energy value, sometimes in association with more or less selective control of settlement of epiphytic strains, allow bacteria to establish surface consortia not leading to macrofouling. Here, we present the analysis of adhesion and biofilm formation of bacterial isolates from the kelp Laminaria digitata and of characterized and referenced marine isolates. When they were grown in flow cell under standard nutrient regimes, all used bacteria, except one, were able to adhere on glass and then develop as biofilms, with different architecture. Then, we evaluated the effect of extracts from undisturbed young Laminaria thalli and from young thalli subjected to oxidative stress elicitation; this latter condition induced the production of defense molecules. We observed increasing or decreasing adhesion depending on the referenced strains, but no effects were observed against strains isolated from L. digitata. Such effects were less observed on biofilms. Our results suggested that L. digitata is able to modulate its bacterial colonization. Finally, mannitol, a regular surface active component of Laminaria exudates was tested individually, and it showed a pronounced increased on one biofilm strain. Results of these experiments are original and can be usefully linked to what we already know on the oxidative halogen metabolism peculiar to Laminaria. Hopefully, we will be able to understand more about the unique relationship that bacteria have been sharing with Laminaria for an estimated one billion years.     

Influence of Plasmid pO157 on Escherichia coli O157:H7 Sakai Biofilm Formation

The role of plasmid pO157 in biofilm formation was investigated using wild-type and pO157-cured Escherichia coli O157:H7 Sakai. Compared to the wild type, the biofilm formed by the pO157-cured mutant produced fewer extracellular carbohydrates, had lower viscosity, and did not give rise to colony morphology variants that hyperadhered to solid surfaces.Enterohemorrhagic Escherichia coli serotype O157:H7 is a major food-borne pathogen causing hemorrhagic colitis and the hemolytic-uremic syndrome (17). Many E. coli O157:H7 outbreaks have been associated with contaminated undercooked ground beef, vegetables, fruits, and sprouts (20, 31). One of the largest disease outbreaks occurred in Sakai City, Japan, in 1996 with nearly 8,000 confirmed cases. The E. coli isolate responsible for this outbreak, referred to as “Sakai,” is one of the best-characterized isolates and one of only three O157 strains for which the genome has been fully sequenced (8, 16). Because of its importance as a human pathogen and its characterization, Sakai was the focus of this investigation.There is significant phenotypic diversity among E. coli O157:H7 strains, including the ability to form biofilm. Previous studies show that certain E. coli O157:H7 strains form biofilm on various surfaces, and biofilm on food or food-processing surfaces can serve as a source or vehicle of contamination that may result in human infection (6, 18, 25). Biofilm is an organized and structured community of microorganisms that attaches to solid surfaces and contains cells embedded in an extracellular polymer matrix (4, 26). Exopolysaccharide (EPS) is a major component of the biofilm matrix and is required for the development of characteristic biofilm architecture (5, 29). Bacteria gain a variety of advantages from biofilm formation that include attachment, colonization, and protection from adverse environments (4, 11).E. coli O157:H7 carries a 92-kb virulence plasmid (pO157) encoding a number of putative virulence determinants, including ehxA, etpC to etpO, espP, katP, toxB, ecf, and stcE (31). However, the biological role of pO157 is not fully understood, and only 19 genes among the 100 open reading frames (ORFs) in pO157 have been characterized (2, 15). Our previous work indicates that pO157 is a colonization factor in cattle and may regulate several chromosomal genes (14, 24, 31).To investigate the role of pO157 in biofilm formation, we characterized the biofilm of wild-type E. coli O157:H7 strain Sakai and an isogenic pO157-cured Sakai (Sakai-Cu). Both strains were kindly provided by C. Sasakawa (University of Tokyo). Sakai-Cu was generated using a plasmid incompatibility method (27). This method is not prone to secondary mutations and requires minimal passage in laboratory medium. The mini-R plasmid pK2368, harboring a chloramphenicol (CM) resistance gene and being in the same plasmid incompatibility group as pO157, was introduced into wild-type Sakai by transformation. Transformants were isolated on LB agar containing CM and selected for loss of pO157 by agarose gel electrophoresis analysis. CM-resistant transformants were cured of pKP2368 by subculturing in LB broth without CM. The absence of pO157 was confirmed by Southern blot hybridization with a pO157-specific gene probe (derived from ecf1), and chromosomal DNA integrity was confirmed by pulsed-field gel electrophoresis (data not shown).Because E. coli O157:H7 strains are generally not strong biofilm producers, the condition most conducive to biofilm production, a fluorometric flow cell method, was used to compare separately grown Sakai and Sakai-CU (3). The biofilm cultivation systems consisted of seven parts: (i) medium reservoir, (ii) multichannel pump (205S; Watson Marlow, United Kingdom), (iii) bubble trap (BioSurface Technologies Co., Bozeman, MT), (iv) flow cell, (v) outflow reservoir, (vi) air pump (DrsFosterSmith, Rhinelander, WI), and (vii) flow meter (Gilmont, BC Group, St. Louis, MO). The flow cell was constructed from two rectangular acrylic plates that were 104 by 48 mm. Sidewalls (62 by 26 by 5 mm) were glued to the top plate to form an elongated hexagonal growth chamber. There were 56- by 20-mm square openings in the top and bottom rectangular plates that were sealed with 60- by 24-mm glass slides (Fisher, Pittsburgh, PA). The upper and lower plates were assembled with screws and sealed using a microseal B film (MJ Research, Waltham, MA). The flow cell volume was about 10.4 ml, the medium flow rate was 10.5 ml/h, and the hydraulic retention time was 1 h. Under these conditions, the linear surface velocity was about 80 mm/h at the center of the flow cell. The biofilm was grown with BGM2 medium (21). To prepare the inoculums, Sakai and Sakai-Cu were grown at 37°C in BGM2 medium to mid-exponential phase, and cells were harvested by centrifugation and resuspended in 0.85% NaCl. One hundred μl of the resuspended cell solution was inoculated from the effluent side of flow cells through a long stainless steel needle (Fisher, Pittsburgh, PA). The cells were incubated for at least 3 h without supplying fresh medium, and then fresh medium was supplied to the biofilm cultivation system at 30°C.At various times, the resulting biofilms were stained with a green fluorescent dye, wheat germ agglutinin (WGA)- Alexa Fluor 488 (Invitrogen, Carlsbad, CA), and analyzed using the Olympus FluoView confocal laser scanning microscopy system (Olympus, Tokyo, Japan). Using the Olympus FluoView software program, version 1.7b, for analysis, the fluorescence intensities of Sakai and Sakai-Cu biofilm matrices were each analyzed from >20 three-dimensional-complexity images. Fluorescence was greater for Sakai than for Sakai-Cu, with average values of 2,448 ± 668 and 2,022 ± 619, respectively (Student''s t test; P < 0.05). Overhead images from the Sakai-Cu strain biofilm revealed more-compact cell clusters than images from wild-type Sakai (Fig. 1A and B). Comparisons of images taken sideways indicated that the Sakai-Cu biofilms were not as thick as those of wild-type Sakai (Fig. 1C and D), and typical ratios were consistently 9:11, respectively (P < 0.05). A previous study demonstrated that the biofilm of a wcaF::can mutant of E. coli K-12, which is deficient in EPS production, lacked depth and complex architecture (5). Sakai-Cu showed a similar but less dramatic phenomenon. These observations indicated that pO157 influenced biofilm formation and architecture.Open in a separate windowFIG. 1.Wild-type Sakai (A and C) or Sakai-Cu (B and D) biofilms after 3 days of incubation. Both strains were grown at 30°C in an individual flow cell apparatus. The biofilm was stained with WGA-Alexa Fluor 488 and examined by confocal microscopy. Representative overhead (A and B) or sagittal (C and D) images are shown and were generated using the deconvolution software. Bar, 50 μm.To quantitatively compare Sakai and Sakai-Cu biofilms, the contents of each flow cell apparatus were collected at various times and analyzed for bacterial cell number, viscosity, and EPS production. Biofilms were harvested by a standard technique that preserves cell numbers and minimizes viscosity changes (9). Briefly, floating cells in the biofilm were carefully collected with a pipet, and the remaining cells were scraped from the flow cell apparatus with sterilized applicator sticks. Biofilm samples were collected on days 1, 3, 5, 8, and 12, and measurements were means ± standard deviations (SD) of at least triplicate measurements from separately grown biofilms. There was no significant difference in bacterial number (CFU/ml) from Sakai and Sakai-Cu biofilms at any of the times measured (data not shown). A Cannon-Fenske routine viscometer (Size 100; Cannon Instrument Co., Pennsylvania) was used to determine biofilm viscosity. The conversion constant was 0.015 cSt/s (mm²/s²), and viscosities were measured according to the manufacturer''s instructions. Briefly, the viscometer was aligned vertically in the holder, and the sample was charged into the viscometer tube until the sample reached the “F” mark in the tube. A suction bulb was used to draw the sample slightly above mark “E.” The sample was allowed to flow freely, and the efflux time was measured as the time for the meniscus to pass from mark “E” to mark “F.” Measurements were repeated at least six times, and the kinematic viscosity in mm²/s (cSt) of the samples was calculated by multiplying the efflux time in seconds by the viscometer constant. The viscosity of Sakai biofilm was dramatically increased after 8 days (P < 0.001), while there was no significant change in the viscosities of Sakai-Cu biofilms through day 12 (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Comparison of Sakai and Sakai-Cu biofilm viscosity. Three or four separately grown biofilms were each harvested on the days indicated, and viscosity was measured using a Cannon-Fenske Routine viscometer.Bacterial EPS are associated with attachment to both inanimate surfaces and host cells (29). EPS can be categorized as extracellular carbohydrate complexes (ECC) that are loosely associated with cells and easily removed, referred to as slime (fraction I), or ECC that are closely associated with cells and removed only after heat treatment, referred to as capsule (fraction II) (22). No significant difference in ECC was observed until days eight and 12, when the level of total ECC produced from Sakai biofilms was significantly higher than that from the Sakai-Cu biofilms (P < 0.05) (Fig. ​(Fig.3).3). Also, by days eight and 12, levels of Sakai ECC fraction I, representing primarily secreted slime carbohydrates, were 5 and 10 times higher than Sakai-Cu ECC fraction I, respectively. These results correlated with the results of increased viscosity in Sakai biofilm samples that had aged for 8 or 12 days.Open in a separate windowFIG. 3.Comparison of Sakai and Sakai-Cu biofilm extracellular carbohydrate (ECC) production. ECC I was collected from cells by centrifugation, and ECC II was collected by centrifugation after heat treatment on each indicated day. Bar height represents total ECC production from each biofilm sample. The proportion of total ECC that was either ECC I (dark gray) or ECC II (light gray) is shown. Asterisks indicate significant differences between wild-type Sakai (Wt) and Sakai-Cu (Cu); day 8, P < 0.05; day 12, P < 0.001.Interestingly, during biofilm sampling, two colony morphology variants were isolated that are referred to here as sticky and mucoid. These variants were found only in wild-type Sakai biofilms that had aged for ≥8 days and were not found in Sakai-Cu biofilms even after screening of 10⁴ colonies and even among biofilms aged for 18 days. The percentages of sticky and mucoid variants in Sakai biofilms ranged from 5 to 30% and 0 to 5%, respectively. The differences in colony morphology were readily distinguished, as shown in Fig. ​Fig.4.4. The sticky variant was raised in elevation and shinier than the Sakai parent strain but was not difference in size. When single bacterial colonies grown on agar plates were touched with a sterilized toothpick and that toothpick was gently lifted up, the colonies had a hyperadherence phenotype and elongated to approximately 1 cm between the plate and the toothpick. This phenomenon was unique to the sticky colony variants and was not observed among colonies of the parent Sakai strain (Fig. ​(Fig.4D).4D). The mucoid colony variants were convex in elevation and shiny in texture, had irregular colony shapes, and were larger than the Sakai parent strain but were not hyperadherent. The motility of variants was determined using 0.3% soft agar, and both sticky and mucoid variants exhibited 30- to 90%-reduced motility compared to the parent Sakai strain (data not shown). The characteristics of both sticky and mucoid variants were inherited, and the variant characteristics were maintained in laboratory subculture through 15 generations.Open in a separate windowFIG. 4.Colony morphologies of wild-type, mucoid, and sticky variants. The wild-type E. coli O157:H7 Sakai strain formed small, flat, and nonsticky colonies on LB agar (A). The mucoid variant formed irregular, large, shiny, mucoid, convex, and nonsticky colonies (B). The sticky variant formed small, slightly raised, and sticky colonies (C). The sticky variant adheres to a toothpick touched to the colony surface (D). Bar, 1 cm.It is known that mutation is a powerful mechanism of adaptation when bacteria are faced with environmental change (1). Like other bacterial variants, the sticky and mucoid phenotypic biofilm variants may provide a survival advantage in specific niches (10, 19). Pseudomonas aeruginosa is a well-known biofilm model, and colony morphology variants are a common biofilm-related phenomenon. Both reduced-motility and hyperadherence variants have been described (10) and have characteristics similar to those of the E. coli O157:H7 biofilm variants described here. However, unlike the P. aeruginosa biofilm variants, the sticky and mucoid Sakai variants were not smaller, rougher, or more wrinkled than the parent colony.Although it is possible that the changes measured in biofilm formation and the generation of hyperadherent variants were not due to the plasmid, it is highly unlikely. The method of plasmid curing by incompatibility is gentle and is not prone to secondary mutation. A powerful and common approach to address possible secondary mutations is complementation; however, it was not used here because reintroduction of the plasmid requires the manipulation of a very large piece of DNA (92 kb) and the procedure itself is likely to introduce mutation. Also, reintroduction of the large 92-kb pO157 plasmid would require antibiotic resistance for efficient selection, and this may influence biofilm formation.Many regulatory mechanisms are involved in biofilm formation (7, 12, 13, 28, 30, 32). Among those mechanisms, the relationship between biofilm formation and acid resistance is well known. Biofilm formation is upregulated after the deletion of the gad or hde gene, which allows bacteria to survive under acidic conditions (12). Previously we showed that an isogenic pO157-cured strain of E. coli O157:H7, ATCC 43894, enhanced acid resistance through increased expression of Gad (14). Similarly, Sakai-Cu has enhanced acid resistance compared to wild-type Sakai (data not shown and J. Y. Lim, B. Hong, H. Sheng, S. Shringi, R. Kaul, and C. J. Hovde, submitted for publication). The link between increased acid resistance and reduced biofilm formation, reduced ESP production, reduced viscosity, and lack of colony morphology variants was not explored here. Comparisons of biofilm formation were not made between these two strains because neither wild-type E. coli O157:H7 ATCC 43894 nor its plasmid-cured strain form significant biofilm under the laboratory conditions tested (data not shown).Two pO157-cured E. coli O157 strains (ATCC 43894 and Sakai) do not colonize cattle as well as their wild-type counterpart (14, 24). The mechanism for this difference may be related to pO157 encoding a set of putative type II secretion genes, etpC to etpM, etpO, and etpS, and these etp genes may be associated with protein secretion required for efficient adherence (23). Tatsuno et al. reported that the toxB gene encoded on pO157 is required for the full epithelial cell adherence phenotype (27). These results may relate to the defect of Sakai-Cu in biofilm formation.In conclusion, this is the first report that pO157 affects biofilm formation of E. coli O157:H7 Sakai through increased EPS production and generation of hyperadherent variants. Further study of biofilm formation under a variety of conditions and comparisons of Sakai with other E. coli O157:H7 strains will be important for understanding the relationship between biofilm formation and E. coli O157:H7 virulence and survival on foods and in the farm environment.     

Effect of Bacterial Interference on Biofilm Development by Legionella pneumophila

In the ecology of Legionella pneumophila a crucial role may be played by its relationship with the natural flora; thus we investigated the interactions between Legionella and other aquatic bacteria, particularly within biofilms. Among 80 aquatic bacteria screened for the production of bacteriocin-like substances (BLSs), 66.2% of them were active against L. pneumophila. The possible effect of some of these aquatic bacteria on the development and stability of L. pneumophila biofilms was studied. Pseudomonas fluorescens, the best BLS producer, showed the greatest negative effect on biofilm formation and strongly enhanced the detachment of Legionella. Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Aeromonas hydrophila, and Stenotrophomonas maltophilia, although producing BLSs at different levels, were less active in the biofilm experiments. Acinetobacter lwoffii did not produce any antagonistic compound and was the only one able to strongly enhance L. pneumophila biofilm. Our results highlight that BLS production may contribute to determining the fate of L. pneumophila within ecological niches. The interactions observed in this study are important features of L. pneumophila ecology, which knowledge may lead to more effective measures to control the persistance of the germ in the environment.