Inactivation of biofilm bacteria.

The current project was developed to examine inactivation of biofilm bacteria and to characterize the interaction of biocides with pipe surfaces. Unattached bacteria were quite susceptible to the variety of disinfectants tested. Viable bacterial counts were reduced 99% by exposure to 0.08 mg of hypochlorous acid (pH 7.0) per liter (1 to 2 degrees C) for 1 min. For monochloramine, 94 mg/liter was required to kill 99% of the bacteria within 1 min. These results were consistent with those found by other investigators. Biofilm bacteria grown on the surfaces of granular activated carbon particles, metal coupons, or glass microscope slides were 150 to more than 3,000 times more resistant to hypochlorous acid (free chlorine, pH 7.0) than were unattached cells. In contrast, resistance of biofilm bacteria to monochloramine disinfection ranged from 2- to 100-fold more than that of unattached cells. The results suggested that, relative to inactivation of unattached bacteria, monochloramine was better able to penetrate and kill biofilm bacteria than free chlorine. For free chlorine, the data indicated that transport of the disinfectant into the biofilm was a major rate-limiting factor. Because of this phenomenon, increasing the level of free chlorine did not increase disinfection efficiency. Experiments where equal weights of disinfectants were used suggested that the greater penetrating power of monochloramine compensated for its limited disinfection activity. These studies showed that monochloramine was as effective as free chlorine for inactivation of biofilm bacteria. The research provides important insights into strategies for control of biofilm bacteria.     

Inactivation of bacteria by Purogene

The bacteriocidal efficacy of Purogene, a stabilized aqueous solution of chlorine dioxide (ClO2) was examined using bacteria of concern to public health. The organisms tested were: Escherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella pneumoniae, Streptococcus pyogenes Group A, Salmonella typhimurium and Bacillus subtilis. The test organisms responded differently to inactivation by Purogene. At least a 4 log reduction in bacterial counts was noted when Purogene was applied at a concentration of 0.75 mg/l. Since Purogene is a stabilized complex, it was necessary to provide a chemical environment suitable for the release of ClO2 in this solution. This was done by varying the pH of Purogene from 3.5 to 8.6 (pH of Purogene is 8.6) while keeping the pH of the experimental medium constant (pH 7.0). The results showed that Purogene was most efficacious at the lowest pH tested (pH 3.5). This indicates that as chlorine dioxide solutions were reduced to chlorite (which predominates at pH 8.6), their bacteriocidal efficacy was reduced, suggesting free chlorine dioxide as the active disinfecting species.     

Inactivation of bacteria by Purogene

The bacteriocidal efficacy of Purogene, a stabilized aqueous solution of chlorine dioxide (ClO₂) was examined using bacteria of concern to public health. The organisms tested were: Escherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella pneumoniae, Streptococcus pyogenes Group A, Salmonella typhimurium and Bacillus subtilis . The test organisms responded differently to inactivation by Purogene. At least a 4 log reduction in bacterial counts was noted when Purogene was applied at a concentration of 0.75 mg/l. Since Purogene is a stabilized complex, it was necessary to provide a chemical environment suitable for the release of ClO₂ in this solution. This was done by varying the pH of Purogene from 3.5 to 8.6 (pH of Purogene is 8.6) while keeping the pH of the experimental medium constant (pH 7.0). The results showed that Purogene was most efficacious at the lowest pH tested (pH 3.5). This indicates that as chlorine dioxide solutions were reduced to chlorite (which predominates at pH 8.6), their bacteriocidal efficacy was reduced, suggesting free chlorine dioxide as the active disinfecting species.     

Coaggregation amongst aquatic biofilm bacteria

In a comparative study, the PCR-based RAPD and ERIC fingerprint methods were evaluated for their resolving power to discriminate among 21 isolates of a natural Rhizobium meliloti population. PCR fingerprint patterns were analysed by using an automated laser fluorescent (ALF) DNA sequencer, thus allowing the automated on-line storage of data. Results obtained were compared to a classification system using insertion sequence (IS) fingerprinting. Both PCR fingerprint methods were comparable in their ability to resolve differences amongst Rh. meliloti isolates. Grouping of strains on the basis of their RAPD as well as their ERIC fingerprints correlated with grouping of strains according to their IS fingerprints. Moreover, strains displaying identical PCR patterns could be further differentiated according to their IS fingerprints, thus allowing a detailed insight into phylogenetic relationship among strains. The automated evaluation of strain-specific fingerprint patterns has the potential to become a valuable tool for studies of bacterial population genetics. Moreover, the rapid identification of single strains, e.g. pathogens in epidemiological studies seems feasible.     

Inactivation of Pseudomonas aeruginosa biofilm by dense phase carbon dioxide

Dense phase carbon dioxide (DPCD) is one of the most promising techniques available to control microorganisms as a non-thermal disinfection method. However, no study on the efficiency of biofilm disinfection using DPCD has been reported. The efficiency of DPCD in inactivating Pseudomonas aeruginosa biofilm, which is known to have high antimicrobial resistance, was thus investigated. P. aeruginosa biofilm, which was not immersed in water but was completely wet, was found to be more effectively inactivated by DPCD treatment, achieving a 6-log reduction within 7 min. The inactivation efficiency increased modestly with increasing pressure and temperature. This study also reports that the water-unimmersed condition is one of the most important operating parameters in achieving efficient biofilm control by DPCD treatment. In addition, observations by confocal laser scanning microscopy revealed that DPCD treatment not only inactivated biofilm cells on the glass coupons but also caused detachment of the biofilm following weakening of its structure as a result of the DPCD treatment; this is an added benefit of DPCD treatment.     

Inactivation of bacteria by decay of incorporated radioactive phosphorus

Cultures of Escherichia coli will not grow in media containing very high specific activities of radiophosphorus P³², the inhibition of growth being due to the decay of assimilated P³² atoms. Experiments with a differentially labeled thymineless strain of E. coli show that the P³² disintegrations which occur in the bacterial deoxyribonucleic acid, i.e. in the nucleus, are mainly responsible for the inactivation of the cell. The kinetics with which radioactive bacterial populations are inactivated indicate that the function of several nuclei per bacterial cell must be eliminated by P³² decay before the ability to generate a colony is lost. The efficiency with which each P³² disintegration inactivates the nucleus in which it has occurred is calculated to be 0.02 (at –196°), i.e., similar in magnitude to the killing efficiency of P³² decay in bacteriophages. P³² decay and thymine starvation cooperate in bringing about the death of individuals of the thymineless strain, from which observation it is inferred that "thymineless death" is likewise a nuclear inactivation. The descendants of a non-radioactive bacterial culture grown for several generations in the presence of P³² and the descendants of a radioactive culture grown in the absence of P³² are inactivated by P³² decay in a manner which indicates that the phosphorus atoms of bacterial nuclei are dispersed among the progeny nuclei in their line of descendance.     

Inactivation of bacteria in seawater by low-amperage electric current

Seawater used in mariculture has been suspected of being a potential source of infection. In this study, the lethal effects of low-amperage electric treatment on microorganisms were examined in natural seawater and in seawater inoculated with Vibrio parahaemolyticus. In both cases, bacteria including V. parahaemolyticus in seawater were completely eliminated in 100 ms by a 0.5-A, 12-V direct current. Electron microscopic investigation of the electrically treated bacteria revealed substantial structural damage at the cellular level. In conclusion, our results indicate that low-amperage electric treatment is effective for rapid inactivation of microorganisms in seawater.     

Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria

The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria.     

Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria

Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain, Blastomonas natatoria 2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development.     

Quorum-quenching potential of recombinant PvdQ-engineered bacteria for biofilm formation

International Microbiology - Quorum sensing (QS) is a core mechanism for bacteria to regulate biofilm formation, and therefore, QS inhibition or quorum quenching (QQ) is used as an effective and...     

Inactivation of alkene oxidation by epoxides in alkene-and alkane-grown bacteria

Summary The oxidation of propene by resting-cells of ethene-grown Mycobacterium E3 was inactivated by 1,2-epoxypropane. Inactivation increased with increasing epoxide concentrations with 50% inactivation at approximately 30 mM epoxide. Other lower epoxides as epoxyethane and 1,2-epoxybutane also inactivated oxidation of propene as well as of other alkenes. Propene oxidation by resting-cells of ethane-grown Mycobacterium E20 and resting-cells of methane-grown Methylosinus trichosporium OB3b was inactivated for 50% at much lower 1,2-epoxypropane concentrations of approximately 1 and 3 mM respectively. It was demonstrated that in vivo the predominant effect of 1,2-epoxypropane was on the epoxidizing enzyme, i.e. alkene mono-oxygenase (strain E3), alkane mono-oxygenase (strain E20) and methane mono-oxygenase (methylotroph) and that the effect of the epoxide on the alkene mono-oxygenase was irreversible.     

Coaggregation between freshwater bacteria within biofilm and planktonic communities

The coaggregation ability of bacteria isolated from a freshwater biofilm was compared to those derived from the coexisting planktonic population. Twenty-nine morphologically distinct bacterial strains were isolated from a 6-month-old biofilm, established in a glass tank under high-shear conditions, and 15 distinct strains were isolated from the associated re-circulating water. All 44 strains were identified to genus or species level by 16S rDNA sequencing. The 29 biofilm strains belonged to 14 genera and 23.4% of all the possible pair-wise combinations coaggregated. The 15 planktonic strains belonged to seven genera and only 5.8% of all the possible pair-wise combinations coaggregated. Therefore, compared to the planktonic population, a greater proportion of the biofilm strains coaggregated. It is proposed that coaggregation influences biofilm formation and species diversity in freshwater under high shear.     

Detachment and emission of airborne bacteria in gas-phase biofilm reactors

Three laboratory scale biofilters filled with different packing materials (peat and sieved sugarcane bagasse) and operating with different microbial cultures (allochthonous and autochthonous bacteria) were run and monitored in parallel to assess the emission rate of airborne bacteria in the biofiltration of benzene-contaminated air streams. The effect of the fluid dynamic and loading conditions on the rate of microbial emission in the air environment was investigated by performing continuous experiments at different inlet benzene concentrations and superficial gas velocities. The experiments prove that the concentration of airborne bacteria in the effluent air from lab-scale biofilters is only slightly higher than in the ambient air. The emission rate is not dependent on superficial gas velocity because of low shear stress exerted by the gas flow. On the other hand, the loading conditions have a strong effect on the emission rate, which increases with increasing growth and degradation rate, and different packing media show remarkably different behaviors.     

Influence of growth environment on coaggregation between freshwater biofilm bacteria

AIM: To characterize the expression of coaggregation between Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 following growth in liquid culture, on agar and in an artificial biofilm matrix composed of poloxamer hydrogel. METHODS AND RESULTS: The ability of B. natatoria 2.1 and M. luteus 2.13 to coaggregate with one another was assessed following growth in liquid culture as colonies on agar or within a poloxamer hydrogel matrix. In all these environments a cycle of gain and loss of coaggregation occurred when the two cell types were aged simultaneously, with optimum expression occurring in early stationary phase. Blastomonas natatoria 2.1 cells only coaggregated maximally after entry into stationary phase. Conversely, M. luteus 2.13 cells only coaggregated in exponential phase and early stationary phase and coaggregation ability was lost in late stationary phase. Maximal coaggregation therefore only occurred between the two strains if both were in early stationary phase, when the surface properties of the two cell types were optimal for coaggregation. CONCLUSION: In addition to occurring between cells grown in liquid culture, coaggregation between aquatic bacteria occurs after growth as a biofilm on agar and in an artificial biofilm matrix in poloxamer. Under all conditions, the B. natatoria 2.1 coaggregation adhesin and complementary receptor on M. luteus 2.13 were only expressed simultaneously during early stationary phase.     

Characterization of extracellular polymers synthesized by tropical intertidal biofilm bacteria

AIM: This study was performed to determine the potential of tropical intertidal biofilm bacteria as a source of novel exopolymers (EPS). METHODS AND RESULTS: A screening procedure was implemented to detect EPS-producing biofilm bacteria. Isolates MC3B-10 and MC6B-22, identified respectively as a Microbacterium species and Bacillus species by 16S rDNA and cellular fatty acids analyses, produced different EPS, as evidenced by colorimetric and gas chromatographic analyses. The polymer produced by isolate MC3B-10 displays significant surfactant activity, and may chelate calcium as evidenced by spectroscopic analysis. CONCLUSIONS: Polymer MC3B-10 appears to be a glycoprotein, while EPS MC6B-22 seems to be a true polysaccharide dominated by neutral sugars but with significant concentrations of uronic acids and hexosamines. EPS MC3B-10 possesses a higher surfactant activity than that of commercial surfactants, and given its anionic nature, may chelate cations thus proving useful in bioremediation. The chemical composition of polymer MC6B-22 suggests its potential biomedical application in tissue regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a Microbacterium species producing EPS with surfactant properties, which expands our knowledge of the micro-organisms capable of producing these biomolecules. Furthermore, this work shows that tropical intertidal environments are a nonpreviously recognized habitat for bioprospecting EPS-producing bacteria, and that these molecules might be involved in ecological roles protecting the cells against dessication.     

Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm

The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium. Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas europaea -like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1–2 orders of magnitude lower than those of N. europaea . Nitrospira sp. were virtually absent in this part of the biofilm, whereas they were most abundant at the oxic–anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.     

硫酸盐还原细菌生物被膜的形成与调控研究进展

硫酸盐还原细菌(sulfate-reducing bacteria,SRB)形成的生物被膜是微生物导致金属锈蚀行为的主要原因,同时也是重金属污水微生物修复技术的关键因子。生物被膜形成及调控机制研究对SRB的防治和利用均十分重要。本文综述了近年来SRB生物被膜的研究进展,包括SRB生物被膜的胞外多聚物组成和控制因子,并着重阐述了目前已知的调控因子对SRB生物被膜形成的影响。     

Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria.

The speed of recovery of cell suspensions and biofilm populations of the ammonia oxidizer Nitrosomonas europaea, following starvation was determined. Stationary-phase cells, washed and resuspended in ammoniumfree inorganic medium, were starved for periods of up to 42 days, after which the medium was supplemented with ammonium and subsequent growth was monitored by measuring nitrite concentration changes. Cultures exhibited a lag phase prior to exponential nitrite production, which increased from 8.72 h (no starvation) to 153 h after starvation for 42 days. Biofilm populations of N. europaea colonizing sand or soil particles in continuous-flow, fixed column reactors were starved by continuous supply of ammonium-free medium. Following resupply of ammonium, starved biofilms exhibited no lag phase prior to nitrite production, even after starvation for 43.2 days, although there was evidence of cell loss during starvation. Biofilm formation will therefore provide a significant ecological advantage for ammonia oxidizers in natural environments in which the substrate supply is intermittent. Cell density-dependent phenomena in a number of gram-negative bacteria are mediated by N-acyl homoserine lactones (AHL), including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Addition of both ammonium and OHHL to cell suspensions starved for 28 days decreased the lag phase in a concentration-dependent manner from 53.4 h to a minimum of 10.8 h. AHL production by N. europaea was detected by using a luxR-luxAB AHL reporter system. The results suggest that rapid recovery of high-density biofilm populations may be due to production and accumulation of OHHL to levels not possible in relatively low-density cell suspensions.     

Inactivation of N-TIMP-1 by N-terminal acetylation when expressed in bacteria

The high-affinity binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is essential for regulation of the turnover of the extracellular matrix during development, wound healing, and progression of inflammatory diseases, such as cancer, atherosclerosis, and arthritis. Bacterially expressed N-terminal inhibitory domains of TIMPs (N-TIMPs) have been used extensively for biochemical and biophysical study of interactions with MMPs. Titration of N-TIMP-1 expressed in E. coli indicates, however, that only about 42% of the protein is active as an MMP inhibitor. The separation of inactive from fully active N-TIMP-1 has been achieved both by MMP affinity and by high-resolution cation exchange chromatography at an appropriate pH, based on a slight difference of charge. Purification by cation exchange chromatography with a Mono S column enriches the active portion of N-TIMP-1 to >95%, with K(i) of 1.5 nM for MMP-12. Mass spectra reveal that the inactive form differs from active N-TIMP-1 in being N-terminally acetylated, underscoring the importance of the free alpha-NH(2) of Cys1 for MMP inhibition. N(alpha)-acetylation of the CTCVPP sequence broadens the N-terminal sequence motifs reported to be susceptible to alpha-amino acetylation by E. coli N-acetyl transferases. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 960-968, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.