Effect of enterocin AS-48 singly or in combination with biocides on planktonic and sessile B. cereus

Enterocin AS-48 was tested singly or in combination with biocides on a cocktail of six Bacillus cereus strains in planktonic state and in biofilms formed on polystyrene microtiter plates. The biocides tested were benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, triclosan, chlorhexidine, polyhexamethylen guanidinium chloride and commercial sanitizers P3 oxonia and P3 topax 66. The numbers of survivors were determined after 60 min incubation with biocides or the biocide-bacteriocin combinations. Addition of enterocin AS-48 (25 mg/l) increased the inactivation of planktonic cells by the quaternary ammonium compounds, bisphenols and biguanines tested in a range of biocide concentrations from 0.25 to 2.5 g/l, and by 4 g/l polyguanine. Increased inactivation of the bacilli was also observed for the combination of enterocin AS-48 with 2.5% P3 oxonia, but not by P3 topax 66. In the sessile state, the bacilli were more resistant to biocides and also to the bacteriocin-biocide combinations. Hexadecylpyridium chloride was the most active biocide on biofilms in the single treatments. In the combined treatments with 50 mg/l bacteriocin, hexadecylpyridinium (2.5 g/l), polyhexamethylen guanidinium chloride (4 g/l) and P3 oxonia (2.5%) achieved complete inactivation of bacilli populations. P3 topax 66 showed the lowest performance among all treatments tested, either singly or in combination with bacteriocin. A cocktail of endospores was challenged with biocides and enterocin AS-48 for 60 min at temperatures of 22 °C, 40 °C, and 60 °C. Enterocin AS-48 did not significantly (p > 0.05) reduce viable counts or increase the lethal effect of biocides. However, treatments with 5 g/l benzalkonium chloride at 60 °C, 2.5 g/l hexadecypyridinium at 60 °C or P3 oxonia at 0.025% and 60 °C or at 0.25% at 22–60 °C achieved complete inactivation of bacterial endospores, both singly and in combination with bacteriocin. Significant reductions of viable counts (1–2 log cycles) were also obtained for some treatments with cetrimide, triclosan or polyhexamethylen guanidinium chloride, but not for chlorhexidine (up to 5 g/l) or P3 topax 66 (up to 1%). Polystyrene surfaces dosed with enterocin AS-48 (25 or 50 mg/l) remained free of detectable bacilli from 2 to 24 h after being inoculated with a cocktail of endospores, but stainless steel surfaces dosed with 50 mg/l bacteriocin did not prevent bacterial growth from endospores. Results from this study suggest that enterocin AS-48 could be applied as enhancer of biocide activity against planktonic and sessile B. cereus cells.     

Corrosion of brass in natural and artificial seawater under anaerobic conditions

The corrosion of brass in deoxygenated non-buffered and buffered artificial and natural seawater was studied. The weight gains and the average corrosion rates of brass samples, immersed in both media during periods of 1 week, 1 and 3 months, were determined. The morphology of the corroded surfaces, with and without the corrosion products, was analyzed by visual observation and scanning electron microscopy (SEM). Energy dispersive spectroscopy (EDS) was used for the identification of the corrosion products and X-ray diffraction (XRD) for the identification of the crystalline corrosion products. In general it was concluded that deoxygenating and buffering, at pH 9, both contribute to enhance the corrosion of brass in seawater.     

Interactions between laponite and microbial biofilms in porous media: implications for colloid transport and biofilm stability

Quartz sand columns and sand-filled microscope flow cells were used to investigate the transport characteristics of the clay colloid laponite, and a biofilm-forming bacterium, Pseudomonas aeruginosa SG81. Separate experiments were performed with each particle to determine their individual transport characteristics in clean sand columns. In a second set of experiments, bacterial biofilms were formed prior to introduction of the clay colloids. In the independent transport experiments, bacteria and laponite each conformed to known physicochemical principles. A sodium chloride concentration of 7 x 10(-2) M caused complete retention of the laponite within the sand columns. P. aeruginosa SG81 was generally less influenced by ionic strength effects; it showed relatively low mobility at all ionic strengths tested and some (albeit reduced) mobility when introduced to the columns in 1M NaCl, the highest concentration tested, but nevertheless showed reproducible trends. Under conditions favourable to laponite retention and biofilm stability (7 x 10(-2) MNaCl), laponite suspensions were able to remobilise a portion of the attached bacterial biomass. At low ionic strength, the profile of laponite elution was also altered in the presence of a P. aeruginosa biofilm. These observations suggest that while a reduction in ionic strength has a dominant influence on the mobilisation of biological and inorganic colloids, the presence of laponite and biomass can have a distinct influence on the mobility of both types of colloids. Since these events are likely to occur in subsurface environments, our results suggest that colloid-biofilm interactions will have implications for colloid-bound contaminant transport and the remobilisation of pathogens.     

Potential use of carvacrol and citral to inactivate biofilm cells and eliminate biofouling

Biofouling (i.e., accumulation of microorganisms on wetted surfaces) represents a major problem in the food industries, since bacterial biofilms are common sources of persistent infections due to their resilience to cleaning and disinfection treatments. Therefore, alternative treatments based on the use of essential oils or their individual compounds against this bacterial adaptation phenomenon are currently being studied. This work presents a quantitative comparison of the disinfectant potential of 500–2000 μL/L of carvacrol or citral against mature biofilms of Staphylococcus aureus SC-01, Listeria monocytogenes EGD-e or Escherichia coli MG1655. Treatments with 1000 ppm of carvacrol or citral at 45 °C for 60 min were capable of reducing more than 5 logarithmic cycles of the sessile cells forming part of mature biofilms of all the three species. Furthermore, the synergism observed between carvacrol and heat allowed for the physical removal of biofilms by treatments simulating in situ wash conditions (80 °C/60 s). These results demonstrate the great potential of the essential oils’ constituents citral and carvacrol in the eradication of biofilms formed by foodborne pathogenic microorganisms.     

The effects of biofilms formed on whey reverse osmosis membranes on the microbial quality of the concentrated product

The study investigated the development of bacterial biofilms on spiral wound reverse osmosis (RO) whey concentration membranes and their influence on the microbial quality and safety of concentrated whey (retentate). Used RO membranes, obtained from a commercial whey processing plant, were evaluated at intervals of 2 months for a total duration of 14 months using standard techniques. Results confirmed the presence of multi‐species bacterial biofilms on whey RO membranes. Considerable variations were noticed in the distribution pattern of biofilms constitutive microflora as the membranes aged. A greater increase in retentate counts as compared to feed suggested the possibilities of cross‐contamination from the membrane biofilms.     

Self-bioremediation of cork-processing wastewaters by (chloro)phenol-degrading bacteria immobilised onto residual cork particles

Cork manufacturing is a traditional industry in Southern Europe, being the main application of this natural product in wine stoppers and insulation. Cork processing begins at boiling the raw material. As a consequence, great volumes of dark wastewaters, with elevated concentrations of chlorophenols, are generated, which must be depurated through costly physicochemical procedures before discarding them into public water courses. This work explores the potential of bacteria, isolated from cork-boiling waters storage ponds, in bioremediation of the same effluent. The bacterial population present in cork-processing wastewaters was analysed by DGGE; low bacterial biodiversity was found. Aerobic bacteria were isolated and investigated for their tolerance against phenol and two chlorophenols. The most tolerant strains were identified by sequencing 16S rDNA. The phenol-degrading capacity was investigated by determining enzyme activities of the phenol-degrading pathway. Moreover, the capacity to form biofilms was analysed in a microtitre plate assay. Finally, the capacity to form biofilms onto the surface of residual small cork particles was evaluated by acridine staining followed by epifluorescence microscopy and by SEM. A low-cost bioremediation system, using phenol-degrading bacteria immobilised onto residual cork particles (a by-product of the industry) is proposed for the remediation of this industrial effluent (self-bioremediation).     

Bacterial activity in plant (Schoenoplectus validus) biofilms of constructed wetlands

Biofilm-bacterial communities have been exploited in the treatment of wastewater in ‘fixed-film’ processes. Our understanding of biofilm dynamics requires a quantitative knowledge of bacterial growth-kinetics in these microenvironments. The aim of this paper was to apply the thymidine assay to quantify bacterial growth without disturbing the biofilm on the surfaces of emergent macrophytes (Schoenoplectus validus) of a constructed wetland. The isotope was rapidly and efficiently taken-up and incorporated into dividing biofilm-bacteria. Isotope diffusion into the biofilm did not limit the growth rate measurement. Isotope dilution was inhibited at >12 μM thymidine. Biofilm-bacterial biomass and growth rates were not correlated to the plant surface area (r² < 0.02). The measurements of in situ biofilm-bacterial growth rates both displayed, and accommodated, the inherent heterogeneity of the complex wetland ecosystem. Biofilm-bacterial respiratory activities, measured using the redox dye CTC, and growth rates were measured simultaneously. The dye did not interfere with bacterial growth. Biofilm-bacterial specific growth rates ranged from 1.4 ± 0.6 d⁻¹ to 3.3 ± 1.3 d⁻¹. In the constructed wetlands of this study biofilm-bacterial specific growth rates, compared to those of natural ecosystems, could be markedly improved through changes in wetland design that increased bacterial respiration while minimising biofilm growth.     

Escherichia coli O157:H7 survival,biofilm formation and acid tolerance under simulated slaughter plant moist and dry conditions

Microorganisms persisting in slaughter plant environments may develop acid resistance and be translocated to other environmental surfaces or products. The objective of this study was to evaluate the potential of Escherichia coli O157:H7 to form biofilms and maintain acid resistance, under different culture habituation scenarios, on stainless steel coupons (2 × 5 × 0.08 cm), in the presence of beef carcass decontamination runoff fluids (washings). Coupons were stored in test tubes with unsterilized water washings (WW; pH 6.94) or lactic acid washings (LAW; pH 4.98), which were inoculated with E. coli O157:H7 (10³–10⁴ CFU/ml) and incubated at 15 (24 or 48 h) or 35 °C (7 or 24 h), simulating different habituation scenarios on sites of a slaughter plant, including sanitation and overnight drying, during consecutive operational shifts. Acid resistance (AR) of planktonic and detached E. coli O157:H7 cells was assessed in tryptic soy broth adjusted to pH 3.5 with lactic acid. The highest pre-drying attachment and AR of E. coli O157:H7 were observed after 24 h at 35 °C and 48 h at 15 °C. Drying reduced (P < 0.05) recovery of attached E. coli O157:H7 cells; however, exposure of dried coupons to uninoculated washings allowed recovery of attached E. coli O157:H7, which restored AR, especially under conditions that favored post-drying growth. Exposure of attached cells to 50 ppm PAA for 45 s before drying, as well as habituation in LAW, reduced the recovery and AR of E. coli O157:H7. Therefore, incomplete removal of biofilms may result in cells of increased AR, especially in sites within a slaughter plant, in which liquid meat wastes may remain for long periods of time.     

Comparison of uranium(VI) removal by Shewanella oneidensis MR-1 in flow and batch reactors

To better understand the interactions among metal contaminants, nutrients, and microorganisms in subsurface fracture-flow systems, biofilms of pure culture of Shewanella oneidensis MR-1 were grown in six fracture-flow reactors (FFRs) of different geometries. The spatial and temporal distribution of uranium and bacteria were examined using a tracer dye (brilliant blue FCF) and microscopy. The results showed that plugging by bacterial cells was dependent on the geometry of the reactor and that biofilms grown in FFRs had a limited U(VI)-reduction capacity. To quantify the U(VI)-reduction capacity of biofilms, batch experiments for U(VI) reduction were performed with repetitive U(VI) additions. U(VI)-reduction rates of stationary phase cultures decreased after each U(VI) addition. After the fourth U(VI) addition, stationary phase cultures treated with U(VI) with and without spent medium yielded gray and black precipitates, respectively. These gray and black U precipitates were analyzed using high-resolution transmission electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. Data for randomly selected areas of black precipitates showed that reduced U particles (3-6 nm) were crystalline, whereas gray precipitates were a mixture of crystalline and amorphous solids. Results obtained in this study, including a dramatic limitation of S. oneidensis MR-1 and its biofilms to reduce U(VI) and plugging of FFRs, suggest that alternative organisms should be targeted for stimulation for metal immobilization in subsurface fracture-flow systems.     

Role of discontinuous chlorination on microbial production by drinking water biofilms

Microbial quality in water distribution systems is strongly affected by the development of microbial biofilms. Production and release of microbial cells by the biofilm affect microbial levels in the water column and in some cases this fact constitutes a public health concern. In this study, we attempt to analyze in which way the existence of different episodes of chlorine depletion affects both biofilm formation and microbial load of an artificial laboratory system. The work was carried out using two parallel packed bed reactors both supplied with running tap water. One of the reactors was used as a control and was permanently exposed to the action of chlorine. In the other reactor, chlorine was neutralized at selected times during the experiment and for periods of variable length. During the experiment the concentration of total and viable cells from the effluent was monitored at the exit of each of the reactors. The data obtained were used to estimate microbial production from the biofilms. As an average, release of microbial cells to the water phase increased tenfold in the absence of chlorine. The results also indicate that disinfectant efficiency against the biofilm was not recovered when chlorine returned to normal levels after each event of chlorine neutralization. Cell viability in the water phase in the presence of chlorine was low at the beginning of the experiment but increased 4 orders of magnitude after five neutralization periods. Therefore, subsequent episodes of chlorine depletion may accelerate the development of microbial communities with reduced susceptibility to disinfection in real drinking water systems.