Biofilm formation by Salmonella Typhimurium and Staphylococcus aureus on stainless steel under either mono- or dual-species multi-strain conditions and resistance of sessile communities to sub-lethal chemical disinfection

Intercellular interactions encountered within and between different bacterial species are believed to play key roles in both biofilm formation and antimicrobial resistance. In this study, Salmonella Typhimurium and Staphylococcus aureus (3 strains per species) were left to form biofilms on stainless steel coupons incubated at 20 °C for 144 h (i.e. 6 days), in periodically renewable growth medium, under either mono- or dual-species conditions. Subsequently, the developed sessile communities were exposed for 6 min to sub-lethal concentrations of: (i) benzalkonium chloride (BC, 50 ppm), (ii) sodium hypochlorite (NaClO, 10 ppm), or (iii) peroxyacetic acid (PAA, 10 ppm). The dominance of each strain in the mono- and dual-species biofilm communities, both before and after disinfection, was monitored by pulsed field gel electrophoresis (PFGE). Results revealed that dual-species conditions led to a significant (ca. 10-fold) reduction in the number of sessile cells for both species, compared to mono-species ones, with interspecies interactions however found to not exert any significant effect on the disinfection resistance of each species as a whole. However, PFGE analysis revealed that the different strains here employed behaved differently with regard to biofilm formation and disinfection resistance, with this effect to be also strongly dependent on the culture conditions (mono-/dual-species) and the disinfectant applied. Such results expand our knowledge on multi-species biofilms formed by foodborne pathogenic bacteria and could hopefully be helpful in our efforts to develop effective elimination strategies and thus improve food safety.     

Distribution of bacteria in a domestic hot water system in a Danish apartment building

Bacterial growth in hot water systems seems to cause problems such as bad odor of the water, skin allergies and increased heat transfer resistance in heating coils. In order to establish a basis for long-term suppression of bacterial growth, we studied the distribution of bacteria in a Danish domestic hot water system. Heterotrophic plate counts (HPC) were measured in both water and biofilm samples from various sampling sites in the system. In hot water samples, where the temperature was 55-60 degrees C, the HPC were 10(3)-10(4)CFU/mL at incubation temperatures of 25 degrees C or 37 degrees C and 10(5)CFU/mL at 55 degrees C or 65 degrees C. In the cold water (10 degrees C) supplying the hot water system, the HPC at 25 degrees C or 37 degrees C was lower than in the hot water, and no bacteria were found after incubation at 55 degrees C or 65 degrees C. HPC constituted from 38% to 84% of the AODC results in hot water but only 2% in cold water, which showed a high ratio of culturable bacteria in hot water. Biofilm samples from the hot water tank and the inner surface of the pipes in the cold and hot water distribution system were collected by specially designed sampling devices, which were exposed in the system for 42 days. The quasi-steady-state number of bacteria in the biofilm, measured as the geometric mean of the HPC obtained between 21 and 42 days, was five-fold higher in the hot water pipe (13x10(5)CFU/cm(2) at 55 degrees C) than in the cold water pipe (2.8x10(5)CFU/cm(2) at 25 degrees C). There was no significant difference between the number of bacteria in the biofilm samples from the top, middle and bottom of the hot water tank, and the number of bacteria in the biofilm counted at 55 degrees C ranged from 0.6x10(4) to 1.7x10(4)CFU/cm(2). The surfaces of the sacrificial aluminum anodes and the heating coils in the hot water tank also contained high bacterial numbers. The measured number of bacteria in water and biofilm samples was related to the dimensions of the hot water system, and calculations showed that the majority of bacteria (72%) were located in the biofilm especially in the distribution system, which accounts for the greatest surface area. Free-living bacteria accounted for 26% and only a minor part of the bacteria were in the sludge in the hot water tank (2%).     

Optimisation and significance of ATP analysis for measuring active biomass in granular activated carbon filters used in water treatment

A method for determining the concentration of active microbial biomass in granular activated carbon (GAC) filters used in water treatment was developed to facilitate studies on the interactions between adsorption processes and biological activity in such filters. High-energy sonication at a power input of 40 W was applied to GAC samples for the detachment of biomass which was measured as adenosine triphosphate (ATP). Modelling of biomass removal indicated that a series of six to eight sonication treatments of 2 min each yielded more than 90% of the attached active biomass. The ATP concentrations in 30 different GAC filters at nine treatment plants in The Netherlands ranged from 25 to 5000 ng ATP cm(-3) GAC, with the highest concentrations at long filter run times and pretreatment with ozone. A similar concentration range was observed in nine rapid sand (RS) filters. ATP concentrations correlated significantly (p<0.05) with total direct bacterial cell counts in each of these filter types, but the median value of the ATP content per cell in GAC filters (2.1 x 10(-8) ng ATP/cell) was much lower than in the RS filters (3.6 x 10(-7) ng ATP/cell). Average biofilm concentrations ranging from 500 to 10(5) pg ATP cm(-2) were calculated assuming spherical shapes for the GAC particles but values were about 20 times lower when the surface of pores >1 microm diameter is included in these calculations. The quantitative biomass analysis with ATP enables direct comparisons with biofilm concentrations reported for spiral wound membranes used in water treatment, for distribution system pipes and other aquatic environments.     

Influence of biofilms on the movement of colloids in porous media. Implications for colloid facilitated transport in subsurface environments

Colloid transport through porous media can be influenced by the presence of biofilms. Sterile and non-sterile sand columns were investigated using Laponite RD as model colloid and a highly mucoid strain of Pseudomonas aeruginosa as model biofilm former. Laponite RD was marked specifically by fluorescent complexes with rhodamine 6G. Breakthrough curves (BTCs) were used as parameters for determination of colloid transport characteristics. In the sterile columns, the colloid was mobile (collision efficiencies from 0.05 to 0.08) both after the presence of Na(+) and Ca(2+) ions followed by deionised water influent. In the biofilm-grown column, the same treatment did not result in colloid retention in the case of Na(+) exposure, but in altered or enhanced colloid transport. In the case of Ca(2+) ions exposure, colloid retention increased with biofilm age. After 3 weeks, almost complete retention was observed. Similar observations were made in columns packed with material from slow sand filtration units. These data reveal the complex interactions between biofilms, cations and colloid transport. Changes in the electrolyte composition of water percolating the subsurface can frequently occur and will result in different colloid transport characteristics with regard to the dominating species of ions and the relative abundance of microbial biofilms. This has to be considered when modelling colloid transport through the subsurface.     

Resistance and recovery of river biofilms receiving short pulses of Triclosan and Diuron

The effects of the herbicide Diuron (DIU) and the bactericide Triclosan (TCS) were assessed on laboratory-grown stream biofilms. Four week-old biofilms were exposed in mesocosms to 48-hours of short pulses of either DIU or TCS. The direct and indirect effects of each toxicant on the biofilms, and the subsequent recovery of the biofilms, were evaluated according to structural and functional biomarkers. These parameters were analyzed immediately before exposure, immediately after exposure, and 9 and 16 days post-exposure. DIU caused an increase in diatom mortality (+ 79%), which persisted until the end of the experiment. TCS also affected diatom mortality (+ 41%), although the effect did not appear until 1 week post-exposure. TCS caused an increase in bacterial mortality (+ 45%); however, this parameter returned to normal values 1 week post-exposure. TCS compromised the cellular integrity of the green alga Spirogyra sp., whereas DIU did not. TCS also strongly inhibited phosphate uptake (− 71%), which did not return to normal values until 2 weeks post-exposure. DIU directly affected algae, but barely affected the heterotrophs, whereas TCS seriously impaired bacteria (direct effect) as well as autotrophs (indirect effect). However, the biofilms recovered their normal structure and function within only a few days to a few weeks. These findings demonstrate the capacity of biofilms to cope with periodic inputs of toxicants, but also the risks associated to repeated exposure or multi-contamination in aquatic ecosystems.     

Synthesis,characterization, and antimicrobial activity of chitosan–zinc oxide/polyaniline composites

Organic–inorganic materials of chitosan–zinc oxide/polyaniline (CS–ZnO/PANI) composite were prepared via precipitation with a polymerization method and characterized by FT-IR, XRD, EDXS and TEM analysis, thereby providing evidence of composite formation. The size of the prepared CS–ZnO/PANI composite was found to be 100–200 nm, thereby rendering the morphology suitable for biomedical applications. Antibacterial activities of chitosan–ZnO (CS–ZnO), polyaniline (PANI) and CS–ZnO/PANI composites were determined against Gram-positive bacterium, Staphylococcus aureus (S. aureus), and Gram-negative bacterium, Pseudomonas aeruginosa (P. aeruginosa) and were tested in-vitro at 5–50 μg/mL. Results showed that CS–ZnO/PANI composite had broad-spectrum antibacterial activity that was greatly enhanced in comparison with CS–ZnO. In addition, CS–ZnO/PANI composite has tested fungal strains of Candida albicans (C. albicans) and relatively higher activities were observed than the known antibiotics. Finally, the antimicrobial activity of CS–ZnO/PANI composite against established biofilms was also examined and resulted in more than 95% inhibition in biofilm formation.     

Dynamics of lead immobilization in sulfate reducing biofilms

We have evaluated the effects of selected minerals present in subsoil environment on the efficiency of lead removal from contaminated groundwaters using biofilms composed of sulfate-reducing microorganisms, and examined the stability of metal deposits after the biofilms had been temporarily exposed to the air. To quantify the studied effects, lead was immobilized in biofilms of Desulfovibrio desulfuricans grown anaerobically in two flat-plate flow reactors, one filled with hematite and the other with quartz. While the biofilms in both reactors were heterogeneous and consisted of voids and channels, the biofilms grown on hematite were denser, thicker, and more porous than those grown on quartz. The average H₂S concentrations, measured using microelectrodes, were higher in the biofilms grown on quartz than those measured in the biofilms grown on hematite. During 18 weeks of operation, iron was continuously released from the hematite. Lead was immobilized more efficiently in the biofilms grown on quartz than it was in the biofilms grown on hematite. Lead deposits were partially reoxidized, especially in biofilms grown on hematite, and the biofilms in both reactors responded to the presence of oxygen by lowering their density and increasing the H₂S production rate.     

Factors affecting bulk to total bacteria ratio in drinking water distribution systems

Bacteria in drinking water systems can grow in bulk water and as biofilms attached to pipe walls, both causing regrowth problems in the distribution system. While studies have focused on evaluating the factors influencing the bacteria in bulk water and in biofilms separately, there is a need for understanding biofilm characteristics relative to the bulk water phase. The current study evaluated the effects of chlorine and residence time on the presence of culturable bacteria in biofilms relative to that in bulk water. The results showed that when no chlorine residual was present in the system, the median ratio of bulk to total bacteria was 0.81, indicating that 81% of the bacteria were present in bulk water, whereas only 19% were present in the biofilm. As chlorine concentration increased to 0.2, 0.5, and 0.7 mg/L, the median percentage of bacteria present in bulk water decreased to 37, 28, and 31, respectively. On the other hand, as the residence times increased to 8.2, 12, 24, and 48h, the median percentage of bacteria present in bulk water increased to 7, 37, 58, and 88, respectively, in the presence of a 0.2mg/L chlorine residual. The common notion that biofilms dominate the distribution system is not true under all conditions. These findings suggest that bulk water bacteria may dominate in portions of a distribution system that have a low chlorine residual.     

Attachment and biofilm formation by Escherichia coli O157:H7 at different temperatures, on various food-contact surfaces encountered in beef processing

Escherichia coli O157:H7 attached to beef-contact surfaces found in beef fabrication facilities may serve as a source of cross-contamination. This study evaluated E. coli O157:H7 attachment, survival and growth on food-contact surfaces under simulated beef processing conditions. Stainless steel and high-density polyethylene surfaces (2 × 5 cm) were individually suspended into each of three substrates inoculated (6 log CFU/ml or g) with E. coli O157:H7 (rifampicin-resistant, six-strain composite) and then incubated (168 h) statically at 4 or 15 °C. The three tested soiling substrates included sterile tryptic soy broth (TSB), unsterilized beef fat-lean tissue (1:1 [wt/wt]) homogenate (10% [wt/wt] with sterile distilled water) and unsterilized ground beef. Initial adherence/attachment of E. coli O157:H7 (0.9 to 2.9 log CFU/cm²) on stainless steel and high-density polyethylene was not affected by the type of food-contact surface but was greater (p < 0.05) through ground beef. Adherent and suspended E. coli O157:H7 counts increased during storage at 15 °C (168 h) by 2.2 to 5.4 log CFU/cm² and 1.0 to 2.8 log CFU/ml or g, respectively. At 4 °C (168 h), although pathogen levels decreased slightly in the substrates, numbers of adherent cells remained constant on coupons in ground beef (2.4 to 2.5 log CFU/cm²) and increased on coupons in TSB and fat-lean tissue homogenate by 0.9 to 1.0 and 1.7 to 2.0 log CFU/cm², respectively, suggesting further cell attachment. The results of this study indicate that E. coli O157:H7 attachment to beef-contact surfaces was influenced by the type of soiling substrate and temperature. Notably, attachment occurred not only at a temperature representative of beef fabrication areas during non-production hours (15 °C), but also during cold storage (4 °C) temperatures, thus, rendering the design of more effective sanitation programs necessary.     

Reinigung von lösemittelhaltiger Prozessabluft in Biorieselbettreaktoren

In numerous industrial processes, solvent-containing exhaust gas is generated and has to be purified to comply with the emission limits prescribed by law. However, conventional exhaust gas purification technologies, such as thermal post-combustion, require high temperatures of 1200 °C and often fossil fuels. As a significantly more sustainable alternative, biotrickling filters enable biodegradation at 20 °C. The pollutants are converted into harmless substances by bacteria in a biofilm on lava stones.