Inactivation of Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus on stainless steel and glass surfaces by neutral electrolysed water

AIM: To ascertain the efficacy of neutral electrolysed water (NEW) in reducing Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes on glass and stainless steel surfaces. Its effectiveness for that purpose is compared with that of a sodium hypochlorite (NaClO) solution with similar pH, oxidation-reduction potential (ORP) and active chlorine content. METHODS AND RESULTS: First, the bactericidal activity of NEW was evaluated over pure cultures (8.5 log CFU ml-1) of the abovementioned strains: all of them were reduced by more than 7 log CFU ml-1 within 5 min of exposure either to NEW (63 mg l-1 active chlorine) or to NaClO solution (62 mg l-1 active chlorine). Then, stainless steel and glass surfaces were inoculated with the same strains and rinsed for 1 min in either NEW, NaClO solution or deionized water (control). In the first two cases, the populations of all the strains decreased by more than 6 log CFU 50 cm-2. No significant difference (P相似文献   

Effect of chlorination on the development of marine biofilms dominated by diatoms

This study addressed the antifouling efficiency of commercially available chlorine at different concentrations (0.5%, 1%, and 2%) and exposure times (0.5?min, 1?min, 5?min, and 15 min). The rapid and non-destructive FIRe (fluorescence induction and relaxation) technique was used to evaluate the effects of the biocide on diatom dominated biofilms. The efficiency of chlorine in removing diatoms from the developed biofilms increased with an increase in concentration and exposure time. The fluorescence measurements revealed low F(v)/F(m) and high σ(PSII) values for chlorine-treated Navicula and Amphora biofilms indicating that chlorination was efficient in damaging the photosystem-II reaction centers. Chlorination also caused mortality of diatom cells by damaging the cell body. In natural biofilms, the biocidal effect of chlorine was species specific; species of Amphiphrora, Navicula, Cylindrotheca, and Coscinodiscus showed an increase in the density of the population, but species of Pleurosigma, Amphora, and Thalassionema did not increase in density after chlorine treatment. It was also demonstrated that diatoms can colonize, grow and photosynthesize on chlorine-treated surfaces. Under pulse chlorination (treatment every 6 h), irrespective of chlorine concentration, the development of biofouling decreased with an increase in exposure time. Differences between exposure times of 1 to 15?min were not significant. Additionally, transmission levels of the control (non-chlorine-treated) fouled coupons reduced significantly (～20%) compared to the chlorine-treated fouled coupons (<2%). These results suggest that chlorine can be used as a biocide to control the development of diatom biofilms.     

Effectiveness of chlorine washing disinfection and effects on the appearance of artichoke and borage

AIM: Optimal conditions for chlorine application to obtain a reasonable decrease in the microbial counts without damaging the appearance of artichoke and borage have been established. METHODS AND RESULTS: The influence of chlorine concentration (0-200 mg l(-1)), pH, addition of organic acids, contact time and presence of protective structures on the microflora and vegetal appearance were studied. When pH was not controlled the effect of chlorine depended on its concentration until the pH increase caused by addition of chlorine reached 8.8. Any further increase in chlorine concentration was nullified by the pH increase. When pH was adjusted to 4.5 with acetic acid, the effectiveness increased with concentration. However, the use of citric acid to control pH caused a sharp decrease in effectiveness at concentration about 250 mg l(-1). The higher effectiveness of chlorine on homogenized plant extracts compared with the whole plant showed the impact of the vegetal structures on the resistance of the microorganisms. For artichoke, a relationship between the effectiveness of chlorine disinfection and its structures was also found. Extended washing times did not affect the total counts. However, in both vegetables, the appearance was affected by the extended contact times. CONCLUSIONS: The solutions rendering the highest microbial reduction with minimum damages were: 50 mg l(-1) free chlorine without pH control for artichoke and 100 mg l(-1) free chlorine at pH 7.0 for borage. SIGNIFICANCE AND IMPACT OF THE STUDY: Specific conditions for chlorine disinfection of artichoke and borage were determined to reduce the microorganisms in minimally processed artichoke and borage without damaging their appearance.     

Survival of Listeria monocytogenes in commercial food-processing equipment cleaning solutions and subsequent sensitivity to sanitizers and heat

AIMS: To determine the ability of Listeria monocytogenes to survive exposure to commercial food-processing equipment cleaning solutions and subsequent treatment with sanitizers or heat. METHODS AND RESULTS: Cells of five strains of L. monocytogenes were suspended in 1% solutions of eight commercial cleaners (pH 7.1-12.5) or in water (control) and incubated at 4 degrees C for 30 min or 48 h before populations were determined by plating on tryptose phosphate agar. After exposure of cells to cleaning solutions for 30 min, populations of the most resistant strain of L. monocytogenes were reduced by < or = 1.63 log10 cfu ml(-1). In only three highly alkaline cleaning solutions (pH 11.6-12.4) were populations reduced significantly (P < or = 0.05) compared with reductions in water. After 48 h, populations were significantly higher in one cleaning solution (pH 10.4) than in water, while populations in six of the other seven cleaning solutions were reduced by > or = 4.72 log10 cfu ml(-1). Cells exposed to cleaning solutions for 30 min became sensitive to 4.0 or 6.0 mg l(-1) free chlorine and to 50 or 100 mg l(-1) benzalkonium chloride and cetylpyridinium chloride, common components of quaternary ammonium sanitizers. Cells exposed to four of the five test cleaners had D56 degrees C values less than or equal to those of the control cells. CONCLUSIONS: Listeria monocytogenes tolerates exposure to a high concentration of alkaline cleaning solutions but consequently becomes sensitized to sanitizers. SIGNIFICANCE AND IMPACT OF THE STUDY: The elimination of L. monocytogenes surviving exposure to alkaline cleaning solutions widely used for food-processing equipment is essential and the appropriate use of sanitizers for subsequent application to equipment is important in achieving this goal.     

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.     

Enhanced inactivation of salmonella and pseudomonas biofilms on stainless steel by use of T-128, a fresh-produce washing aid, in chlorinated wash solutions

The effect of the washing aid T-128 (generally recognized as safe [GRAS] formulation, composed mainly of phosphoric acid and propylene glycol) on inactivation of Salmonella and Pseudomonas populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads in chlorinated wash solutions dominated by hypochlorous acid. Biofilms were formed statically on stainless steel coupons suspended in 2% lettuce extract after inoculation with Salmonella enterica serovar Thompson or Newport or with Pseudomonas fluorescens. Coupons with biofilms were washed in chlorine solutions (0, 0.5, 1, 2, 5, 10, or 20 mg/liter at pH 6.5, 5.0 and 2.9), with or without T-128, and with increasing loads of organic matter (0, 0.25, 0.5, 0.75, or 1.0% lettuce extract). Cell populations on coupons were dispersed using intermittent, pulsed ultrasonication and vortexing and enumerated by colony counts on XLT-4 or Pseudomonas agars. Cell responses to fluorescent viability staining of biofilm treatment washing solutions were examined using confocal laser scanning microscopy. Results showed that 0.1% T-128 (without chlorine) reduced P. fluorescens biofilm populations by 2.5 log(10) units but did not reduce Salmonella populations. For both Salmonella and Pseudomonas, the sanitizing effect of free chlorine (1.0 to 5.0 mg/liter) was enhanced (P < 0.05) when it was combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters and significantly (P < 0.05) augmented inactivation of bacteria in biofilms compared to treatments without T-128. Image analysis of surfaces stained with SYTO and propidium iodide corroborate the cultural assay results showing that T-128 can aid in reducing pathogen viability in biofilms and thus can aid in sanitizing stainless steel contact surfaces during processing of fresh-cut produce.     

Effect of Chlorine on Giardia lamblia Cyst Viability

The effect of chlorine concentration on Giardia lamblia cyst viability was tested under a variety of conditions. The ability of Giardia cysts to undergo excystation was used as the criterion of viability. The experimental variables employed included temperature (25, 15, and 5°C), pH (6, 7, and 8), chlorine-cyst contact time (10, 30, and 60 min), and chlorine concentration (1 to 8 mg/liter). In the pH range studied, cyst survival generally was observed to increase as buffer pH increased. Water temperature coupled with chlorination proved to be important in cyst survival. Results of these experiments at the three temperatures studied can be summarized as follows: at 25°C, exposure to 1.5 mg/liter for 10 min killed all cysts at pH 6, 7, and 8. At 15°C, 2.5 mg of chlorine per liter for 10 min killed all cysts at pH 6, but at pH 7 and 8 small numbers of cysts remained viable after 30 min but not after 60 min. At 5°C, 1 mg of chlorine per liter for 60 min failed to kill all the cysts at any pH tested. At this temperature, 2 mg of chlorine per liter killed all cysts after 60 min at pH 6 and 7, but not at pH 8. A chlorine concentration of 4 mg/liter killed all the cysts at all three pH values after 60 min, but not after 30 min. A chlorine concentration of 8 mg/liter killed all Giardia cysts at pH 6 and 7 after contact for 10 min, and at pH 8 after 30 min. This study points up the role of temperature, pH, and chlorine demand in the halogen treatment of drinking water to destroy cysts. It also raises an epidemiological problem, namely: low water temperatures, where killing of Giardia requires relatively high chlorine concentrations and long contact times, are (i) to be expected in many areas where epidemic waterborne giardiasis has been reported and (ii) particularly conducive to the long-term survival of Giardia cysts.     

The effect of a commercial UV disinfection system on the bacterial load of shell eggs

AIMS: To study the effect of UV irradiation on the bacterial load of shell eggs and of a roller conveyor belt. METHODS AND RESULTS: The natural bacterial load on the eggshell of clean eggs was significantly reduced by a standard UV treatment of 4.7 s; from 4.47 to 3.57 log CFU per eggshell. For very dirty eggs no significant reduction was observed. Eggs inoculated with Escherichia coli and Staphylococcus aureus (4.74 and 4.64 log CFU per eggshell respectively) passed the conveyor belt and were exposed to UV for 4.7 and 18.8 s. The reduction of both inoculated bacteria on the eggshell was comparable and significant for both exposure times (3 and 4 log CFU per eggshell). Escherichia coli was reduced but still detectable on the conveyor rollers. The internal bacterial contamination of eggs filled up with diluent containing E. coli or S. aureus was not influenced by UV irradiation. Conclusions: There is a significant lethal effect of UV irradiation on the bacterial contamination of clean eggshells and recent shell contamination, contamination of rollers can be controlled and the internal contamination of eggs is not reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: The penetration of UV into organic material appears to be poor and UV disinfection can be used as an alternative for egg washing.     

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.     

Use of Integrated Cell Culture-PCR To Evaluate the Effectiveness of Poliovirus Inactivation by Chlorine

Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.     

Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine

Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.     

Relationship between inactivation kinetics of a Listeria monocytogenes suspension by chlorine and its chlorine demand

AIMS: Chlorine demand by Listeria monocytogenes cells and inactivation of L. monocytogenes by chlorine (0.6-1.0 mg l(-1)) at different temperatures (4, 20 and 30 degrees C) have been investigated in a batch reactor. METHODS AND RESULTS: Chlorine demand depended on the microbial concentration and was independent on the initial chlorine concentration and temperature. Chlorine decay was modelled by the addition of two first-order decay equations. Inactivation of L. monocytogenes by chlorine depended on the initial microbial concentration, initial chlorine concentration and temperature. A mathematical model based on a biphasic inactivation properly described survival curves of L. monocytogenes and a tertiary model was developed that satisfactorily predicted the inactivation of L. monocytogenes by different concentrations of initial chlorine at different temperatures. CONCLUSIONS: Both available chlorine decay and inactivation of L. monocytogenes by chlorine were biphasic and can be modelled by a two-term exponential model. SIGNIFICANCE AND IMPACT OF THE STUDY: The biphasic nature of survival curves of L. monocytogenes did not reflect the effect of a change of available chlorine concentration during the treatment. The microbial inactivation was caused by successive reactions that occur after the consumption of the chlorine by the bacterial cell components.     

Efficacy of electrolysed oxidizing water in inactivating Vibrio parahaemolyticus on kitchen cutting boards and food contact surfaces

AIM: To determine the efficacy of electrolysed oxidizing (EO) water in inactivating Vibrio parahaemolyticus on kitchen cutting boards and food contact surfaces. METHODS AND RESULTS: Cutting boards (bamboo, wood and plastic) and food contact surfaces (stainless steel and glazed ceramic tile) were inoculated with V. parahaemolyticus. Viable cells of V. parahaemolyticus were detected on all cutting boards and food contact surfaces after 10 and 30 min, respectively, at room temperatures. Soaking inoculated food contact surfaces and cutting boards in distilled water for 1 and 3 min, respectively, resulted in various reductions of V. parahaemolyticus, but failed to remove the organism completely from surfaces. However, the treatment of EO water [pH 2.7, chlorine 40 ppm, oxidation-reduction potential 1151 mV] for 30, 45, and 60 s, completely inactivated V. parahaemolyticus on stainless steel, ceramic tile, and plastic cutting boards, respectively. CONCLUSIONS: EO water could be used as a disinfecting agent for inactivating V. parahaemolyticus on plastic and wood cutting boards and food contact surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: Rinsing the food contact surfaces with EO water or soaking cutting boards in EO water for up to 5 min could be a simple strategy to reduce cross-contamination of V. parahaemolyticus during food preparation.     

Residence time and food contact time effects on transfer of Salmonella Typhimurium from tile, wood and carpet: testing the five-second rule

AIMS: Three experiments were conducted to determine the survival and transfer of Salmonella Typhimurium from wood, tile or carpet to bologna (sausage) and bread. METHODS AND RESULTS: Experiment 1. After 28 days, 1.5 to 2.5 log(10) CFU cm(-2) remained on tile from and the more concentrated media facilitated the survival of S. Typhimurium compared with the more dilute solutions. Experiments 2 and 3. The bacterial transfer rate to food decreased as the bacterial residence time on the surface increased from 2, 4, 8 to 24 h with transfers of 6.5, 4.8, 4.6 and 3.9 log CFU ml(-1) in the rinse solutions, respectively. Over 99% of bacterial cells were transferred from the tile to the bologna after 5 s of bologna exposure to tile. Transfer from carpet to bologna was very low (<0.5%) when compared with the transfer from wood and tile (5-68%). CONCLUSIONS: (i) Salmonella Typhimurium can survive for up to 4 weeks on dry surfaces in high-enough populations to be transferred to foods and (ii) S. Typhimurium can be transferred to the foods tested almost immediately on contact. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the ability of bacteria to survive and cross-contaminate other foods even after long periods of time on dry surfaces, thus reinforcing the importance of sanitation on food contact to minimize the risk of foodborne illness.     

Interaction of Desulfovibrio desulfuricans biofilms with stainless steel surface and its impact on bacterial metabolism

AIMS: To study the influence of some metallic elements of stainless steel 304 (SS 304) on the development and activity of a sulfate-reducing bacterial biofilm, using as comparison a reference nonmetallic material polymethylmethacrylate (PMMA). METHODS AND RESULTS: Desulfovibrio desulfuricans biofilms were developed on SS 304 and on a reference nonmetallic material, PMMA, in a flow cell system. Steady-state biofilms were metabolically more active on SS 304 than on PMMA. Activity tests with bacteria from both biofilms at steady state also showed that the doubling time was lower for bacteria from SS 304 biofilms. The influence of chromium and nickel, elements of SS 304 composition, was also tested on a cellular suspension of Des. desulfuricans. Nickel decreased the bacterial doubling time, while chromium had no significant effect. CONCLUSIONS: The following mechanism is hypothesized: a Des. desulfuricans biofilm grown on a SS 304 surface in anaerobic conditions leads to the weakening of the metal passive layer and to the dissolution in the bulk phase of nickel ions that have a positive influence on the sulfate-reducing bacteria metabolism. This phenomenon may enhance the biocorrosion process. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the interactions between metallic surfaces such as stainless steel and bacteria commonly implied in the corrosion phenomena which is primordial to fight biocorrosion.     

pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis

AIMS: To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. METHODS AND RESULTS: The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6.5 to 9.25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6.5, growth at pH 9.25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6.5. CONCLUSIONS: A change in the operating pH from 6.5 to 9.25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release.     

Bactericidal effect of chlorine on Mycobacterium paratuberculosis in drinking water

AIMS: One possible route of transmission of Mycobacterium paratuberculosis from cattle to humans is via contaminated water supplies. The aim of this work was to determine whether this organism can survive standard water treatment processes. METHODS AND RESULTS: Two strains of M. paratuberculosis (bovine strain, NCTC 8578 and human strain Linda, ATCC 43015) were subjected to various chlorine concentrations (0.5, 1.0 and 2.0 microg ml(-1)) for 15 and 30 min. Chlorine test solutions were made up in two types of water, sterile water that had been deionized and subjected to reverse osmosis (DRO) and DRO water containing MgCl(2), CaCl(2), NaHCO(3) and bovine serum albumin (0.3% w/v), the latter to mimic conditions the organism would experience in commercial water treatment operations. CONCLUSION: The data showed that when initial inoculum levels were high (10(6) cfu ml(-1)) neither M. paratuberculosis strain was completely killed at the free chlorine concentrations and contact times applied. Log10 reductions in the range 1.32-2.82 were observed. The greatest log(10) reduction in cell numbers (2.82 and 2.35 for the bovine and human strains, respectively) was observed at the highest chlorine concentration (2 microg ml(-1)) and longest contact time (30 min). SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the need for further research into the survival of M. paratuberculosis during water treatment.     

Augmenting effect of acetic acid for acidification on bactericidal activity of hypochlorite solution

AIMS: Bactericidal activity of chlorine solution is enhanced by weak acidification. We compared the effects of various acids on the bactericidal activity of hypochlorite solution to establish a method for safe and effective use of an acidic hypochlorite solution. METHODS AND RESULTS: The bactericidal activities of acidic hypochlorite solutions that had been adjusted to pH 5.0 with hydrochloric acid, acetic acid, citric acid, lactic acid, formic acid, phosphoric acid or sulphuric acid against Bacillus subtilis spores were compared. The acidic solutions prepared with hydrochloric acid and acetic acid showed the highest bactericidal activity, and all of the spores (5 x 106 cfu ml(-1)) were killed within 10 min. On the other hand, the solutions prepared with citric acid and lactic acid showed no bactericidal activity against any bacterial strains tested in this study despite the low pH. The amount of chlorine gas produced by the preparation using acetic acid was sixfold less than that produced from the preparation using hydrochloric acid. CONCLUSIONS: Acetic acid is the most suitable and safe acid for the preparation of an acidic hypochlorite solution. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for establishing a method for safe and effective use of an acidic hypochlorite solution.     

A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.     

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.