Comparative susceptibility of Salmonella Typhimurium biofilms of different ages to disinfectants

There is a general consensus that with increasing age a biofilm shows increased resistance to antimicrobials. In this study the susceptibility of 3-, 5- and 7-day-old Salmonella enterica serovar Typhimurium biofilms to disinfectants was evaluated. It was hypothesized that 7-day-old biofilms would be more resistant to disinfectants compared to 3- and 5-day-old biofilms. Biofilms were formed using the MBEC? system and treated with six chemical disinfectants for 1 and 5 min. Four disinfectants at the highest concentration available showed 100% reduction in viable cells from all ages of biofilms after exposure for 5 min, and ethanol at 70% v/v was the least effective against biofilms, followed by chlorhexidine gluconate (CG). At the recommended user concentrations, only sodium hypochlorite showed 100% reduction in viable cells from all ages of biofilms. Benzalkonium chloride and CG were the least effective against biofilms, followed by quaternary ammonium compound which only showed 100% reduction in viable cells from 5-day-old biofilms. Overall, the results from this study do not display enhanced resistance in 7-day-old biofilms compared to 3- and 5-day-old biofilms. It is concluded that under the conditions of this study, the age of biofilm did not contribute to resistance towards disinfectants. Rather, the concentration of disinfectant and an increased contact time were both shown to play a role in successful sanitization.     

Effectiveness of disinfectants in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Effectiveness of Disinfectants in Killing Enterobacter sakazakii in Suspension, Dried on the Surface of Stainless Steel, and in a Biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

Ginseng aqueous extract attenuates the production of virulence factors, stimulates twitching and adhesion, and eradicates biofilms of Pseudomonas aeruginosa

This study was carried out to examine the antimicrobial activity of the aqueous extract of Panax quinquefolius from North American ginseng (NAGE) root against Pseudomonas aeruginosa . The minimum inhibitory concentrations of reference and clinical isolates of Pseudomonas aeruginosa were measured by a standard agar-dilution method. At subinhibitory NAGE concentrations, the secretion of virulence factors, motility on agar, and adhesion to 96-well microplates were studied on the nonmucoid Pseudomonas aeruginosa O1 strain. At suprainhibitory concentrations, the activity of NAGE against mature biofilm complexes formed in the Calgary Biofilm Device and the Stovall flow cell were assessed. NAGE possessed an antibacterial activity against all the Pseudomonas aeruginosa strains at 1.25%-2.5% w/v. NAGE also significantly attenuated pyocyanin, pyoverdine, and lipase concentrations, stimulated twitching, and attenuated swarming and swimming motility. At 1.25% w/v, NAGE augmented adhesion, and at 5% w/v detached 1-day-old biofilms in microplates. The extract also eradicated 6-day-old mature biofilms (5% w/v), and fluorescence microscopy displayed a reduction of live cells and biofilm complexes compared with nontreated biofilms. These data suggest that the aqueous extract from North American ginseng possesses antimicrobial activities in vitro.     

Microbiological evaluation of a range of disinfectant products to control mixed-species biofilm contamination in a laboratory model of a dental unit water system

Dental unit water system (DUWS) tubing harbors complex multispecies biofilms that are responsible for high microbial levels at the distal outlet. The aim of this study was to use an established biofilm laboratory model to simulate biofouling of DUWS to evaluate practical, cost-effective, and evidence-based methods of microbial decontamination. Reproducible biofilms were developed in the model over 14 days; decontamination was assessed using total viable counts (TVC) and microscopic-image analysis techniques to view the inner surface of tubing. Flushing did not reduce the biofilm coverage or TVC. Combizyme and ozone did not completely eliminate the viable bacteria (70 and 65% reduction in biofilm TVC, respectively), nor did they remove the biofilm (45 and 57% reduction in biofilm coverage, respectively). Chlorhexidine and Bio2000 (active agent: ethanol and chlorhexidine), Tegodor and Gigasept Rapid (aldehyde based), and Grotanol (hydroxide based) completely eliminated the TVC but did not completely remove biofilm (31, 53 33, 34, and 64.9% reduction of biofilm coverage, respectively). Other products including Grotanol Flussig (phenol based), Betadine (povidone-iodine based), Alpron (chlorite based), and the hydroxide-containing products Sporklenz, Sterilex Ultra, Dialox, Sterilox, Sanosil, Oxigenal, and Grotanat Bohrerbad resulted in a 100% reduction in the biofilm TVC and a >95% reduction in biofilm coverage. The study demonstrated that while many disinfectants achieve a sufficient reduction in TVC they may not necessarily remove unwanted biofilm from the tubing surfaces as tested in this laboratory-controlled biofilm model.     

Microbiological Evaluation of a Range of Disinfectant Products To Control Mixed-Species Biofilm Contamination in a Laboratory Model of a Dental Unit Water System

Dental unit water system (DUWS) tubing harbors complex multispecies biofilms that are responsible for high microbial levels at the distal outlet. The aim of this study was to use an established biofilm laboratory model to simulate biofouling of DUWS to evaluate practical, cost-effective, and evidence-based methods of microbial decontamination. Reproducible biofilms were developed in the model over 14 days; decontamination was assessed using total viable counts (TVC) and microscopic-image analysis techniques to view the inner surface of tubing. Flushing did not reduce the biofilm coverage or TVC. Combizyme and ozone did not completely eliminate the viable bacteria (70 and 65% reduction in biofilm TVC, respectively), nor did they remove the biofilm (45 and 57% reduction in biofilm coverage, respectively). Chlorhexidine and Bio2000 (active agent: ethanol and chlorhexidine), Tegodor and Gigasept Rapid (aldehyde based), and Grotanol (hydroxide based) completely eliminated the TVC but did not completely remove biofilm (31, 53 33, 34, and 64.9% reduction of biofilm coverage, respectively). Other products including Grotanol Flussig (phenol based), Betadine (povidone-iodine based), Alpron (chlorite based), and the hydroxide-containing products Sporklenz, Sterilex Ultra, Dialox, Sterilox, Sanosil, Oxigenal, and Grotanat Bohrerbad resulted in a 100% reduction in the biofilm TVC and a >95% reduction in biofilm coverage. The study demonstrated that while many disinfectants achieve a sufficient reduction in TVC they may not necessarily remove unwanted biofilm from the tubing surfaces as tested in this laboratory-controlled biofilm model.     

Comparative susceptibility of planktonic and 3‐day‐old Salmonella Typhimurium biofilms to disinfectants

Aims: To compare the susceptibility of a 3‐day‐old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella. Methods and Results: The susceptibility of planktonic cells to disinfectants was tested by a modified version of the Council of Europe suspension test EN 1276. Salmonella biofilms were formed using the Calgary Biofilm Device. Results show that 3‐day‐old Salmonella biofilms are less susceptible to the disinfectants benzalkonium chloride, chlorhexidine gluconate, citric acid, quaternary ammonium compounds, sodium hypochlorite (SH) and ethanol, compared to planktonic Salmonella. Surprisingly, the results also demonstrate that low concentrations of SH were more effective against a 3‐day‐old biofilm compared to high concentrations of SH. Conclusions: While all the disinfectants evaluated were able to reduce biofilm‐associated cells at concentrations and contact times sufficient to eliminate planktonic cells, there were still sufficient viable cells remaining in the biofilm to cause further contamination and potential infection. Significance and Impact of the Study: Protocols for the use of chemical disinfectants need to include biofilm susceptibility testing. There is a requirement for an effective and standardized tool for determining the susceptibility of biofilms to disinfectants.     

Effect of growth temperature,surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.     

Pseudomonas aeruginosa biofilms react with and precipitate toxic soluble gold

The interaction between biofilms of Pseudomonas aeruginosa PAO1 and 0.01-5 mM gold chloride was investigated using flow-cells. Scanning confocal laser microscopy (SCLM) of these biofilms revealed the formation of two distinct structural features: (i) confluent areas of uniform thickness and (ii) cell clusters which often emerged as 30-40 micro m, tall narrow pillars (or pedestals) of cells and exopolymeric substance (EPS). When 5-day-old, quasi-steady state biofilms (as indicated by the stability of film thickness and overall structure) were exposed to relatively high AuCl3 (i.e. 0.5-5 mM) for 30 min at 20 degrees C, reduction of the auric ion resulted in the formation of both extracellular and intracellular metallic gold colloids, as revealed by transmission electron microscopy (TEM). Most mineralization occurred on cell surfaces with lesser amounts within cells and little throughout the EPS. Little to no mineralization of gold was seen at 0.01-0.1 mM concentrations. As initial AuCl3 concentrations approached 0.5 mM or greater, more gold particles were seen and cell viability, as determined by a BacLight live/dead viability probe, approached zero. At an intermediate concentration of 0.1 mM, the live:dead ratio increased to 4:1. However, when planktonic cells were exposed to this same 0.1 mM concentration, it resulted in a 4-log reduction in viable counts as determined by plating. The higher resistance of biofilm cells to 0.1 mM gold can be attributed to its binding to the EPS and cell surfaces of the biofilm which ensured a (presumably) low effective cytoplasmic concentration of gold (i.e. no gold crystals were seen in cells by TEM). In addition, SCLM revealed the formation of larger extracellular gold crystals at the substratum (coverslip) level of the biofilms, with a higher proportion of crystals detected beneath pillars (cell cluster structures), suggesting the possibility of unique cell types, more reduced microenvironments at the base of each cluster, or a combination of both. These results suggest that the biomineralization of gold is impacted by biofilm structure.     

Disinfectant test against monoculture and mixed-culture biofilms composed of technological, spoilage and pathogenic bacteria: bactericidal effect of essential oil and hydrosol of Satureja thymbra and comparison with standard acid-base sanitizers

Aims: To assess the antimicrobial action of three natural‐derived products (essential oil, decoction and hydrosol of Satureja thymbra) against biofilms, composed of useful, spoilage and pathogenic bacteria (formed as monoculture or/and mixed‐culture), and to compare their efficiency with three standard acid and alkaline chemical disinfectants. Methods and Results: Two acids (hydrochloric and lactic, pH 3), one alkali (sodium hydroxide, pH 11), the essential oil of S. thymbra (1% v/v) and the two by‐products of the essential oil purification procedure (the decoction and the hydrosol fraction of essential oil, 100%), were tested against biofilms formed by five bacterial species, either as monospecies, or as mixed‐culture of all species. The tested bacterial species were Staphylococcus simulans and Lactobacillus fermentum (useful technological bacteria), Pseudomonas putida (spoilage bacterium), Salmonella enterica and Listeria monocytogenes (pathogenic bacteria). Biofilms were left to be formed on stainless steel coupons for 5 days at 16°C, before the application of disinfection treatments, for 60 and 180 min. The disinfection efficiency was evaluated by detaching the remaining viable biofilm cells and enumerating them by agar plating, as well as by automated conductance measurements (using Rapid Automated Bacterial Impedance Technique). Both these methods revealed that the essential oil and the hydrosol of S. thymbra exhibited a strong antimicrobial action against both monospecies and mixed‐culture biofilms. Surprisingly, the efficiency of the other three acid–base disinfectants was not adequate, although a long antimicrobial treatment was applied (180 min). Conclusions: The essential oil of S. thymbra (1%), as well as its hydrosol fraction (100%), presents sufficient bactericidal effect on bacterial biofilms formed on stainless steel. Significance and Impact of the Study: Use of natural antimicrobial agents could provide alternative or supplemented ways for the disinfection of microbial‐contaminated industrial surfaces.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p>0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p<0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p<0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1-0.5%) and exposure period were noted (p<0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p>0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.     

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.     

Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.     

Heterogeneity in the efficacy of dental chemical disinfectants on water-derived biofilms in vitro

Abstract

Conditions in dental unit waterlines are favourable for biofilm growth and contamination of dental unit water. The aim of this study was to assess the effect of several chemical disinfectants on bacteria in a biofilm model. Water-derived biofilms were grown in a static biofilm model (Amsterdam Active Attachment model), using two growth media. Biofilms were challenged with Alpron/Bilpron, Anoxyl, Citrisil, Dentosept, Green & Clean, ICX and Oxygenal in shock dose and maintenance doses. The concentration and the composition of the chemical disinfectants influenced the number of culturable bacteria in the biofilms. The application of a single shock dose followed by a low dose of the same chemical disinfectants resulted in the greatest suppression of viable bacteria in the biofilms. Exposure to Citrisil and ICX consistently resulted in failure to control the biofilms, while Alpron/Bilpron had a substantial and relevant effect on the number of bacteria in the biofilms.     

Development of a Standard Test To Assess the Resistance of Staphylococcus aureus Biofilm Cells to Disinfectants

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.     

The elimination effects of lavender essential oil on Listeria monocytogenes biofilms developed at different temperatures and the induction of VBNC state

Listeria monocytogenes is a typical foodborne pathogen that causes hard-to-treat bacterial infections, mainly due to its ability to form biofilm and enter into a viable but non-culturable state (VBNC). In this study, we investigated the removal effects of four antimicrobial agents on L. monocytogenes biofilms formed at 32°C and 10°C, analysed the resistances of the mature biofilms to antimicrobial agents, and explored the VBNC state of cells in mature biofilms induced by lavender essential oil (LEO). The results showed that the growth of L. monocytogenes was completely inhibited when 1·6% (v/v) of the LEO was added. Meanwhile, the results of the crystal violet staining and XTT reduction method indicated that different concentrations of LEO significantly reduced L. monocytogenes biofilms biomass and metabolic activities, followed by sodium hypochlorite, lactic acid, and hydrogen peroxide. Moreover, the confocal laser scanning microscopy (CLSM) images confirmed that the treated biofilms became thinner, the structure was sparse, and the appearance was blurry. More interestingly, L. monocytogenes biofilms developed at 10°C were less susceptible to the sanitizers than those formed at 32°C. In addition, LEO presented a more significant dispersing effect on the biofilm cells, and 1/2 MIC to 4 MIC of LEO could induce fewer VBNC state cells in biofilm and plankton compared with sodium hypochlorite. This study indicated that the LEO could be considered as an ideal antibiofilm agent for controlling L. monocytogenes. But we should pay attention to the resistance of the biofilms developed at low temperatures.