季铵盐类消毒剂及大肠杆菌对其耐药性研究进展

研究表明,食源性细菌消毒剂耐药严重,大肠杆菌是食品污染状况及耐药性监测的指示菌,对季铵盐类消毒剂表现出比革兰氏阳性菌更强的抗性,且大肠杆菌对消毒剂与抗生素耐药性可共传播。鉴于此,本文综述了季铵盐类消毒剂的结构与种类、作用机制、大肠杆菌消毒剂耐药产生、耐药基因、基因型与表型的关系以及与抗生素耐药共传播机制等的研究进展。食源性大肠杆菌对季铵盐类消毒剂抗性的耐药机制研究很少,研究大肠杆菌对季铵盐类消毒剂耐药性,可为消毒剂的规范使用以及食源性大肠杆菌的防控提供科学依据及理论基础。     

Resuscitation of the viable but non-culturable state of Salmonella enterica serovar Oranienburg by recombinant resuscitation-promoting factor derived from Salmonella Typhimurium strain LT2

A gene encoding the resuscitation-promoting factor (Rpf) from Salmonella Typhimurium LT2 was cloned and characterized. The amino acid sequence encoded by S. Typhimurium LT2 rpf gene shares 24.2% homology with Micrococcus luteus Rpf, which is secreted by growing cells, and required to resuscitate from viable but non-culturable (VNC) state. The S. Typhimurium LT2 rpf gene is 696 bp long, and shared a conserved segment with Salmonella enterica serovar Oranienburg (99.4%). Recombinant Rpf (rRpf) proteins of S. Typhimurium LT2 after expression in E. coli BL21 harboring the pET15-b plasmid was approximately 25 kDa. Since S. Oranienburg cells are relatively quick to enter the VNC state just after incubating in the presence of 7% NaCl at 37 degrees C for 3 days, we evaluated the biological effect of rRpf by using S. Oranienburg VNC cells. The rRpf not only promoted proliferation but also induced resuscitation of VNC cells to the culturable state in a dose-dependent manner. Therefore, rRpf may be useful for detection of bacterial contaminants present in the VNC form in food samples and the environment.     

INFLUENCE OF STARVATION, TEMPERATURE, AND pH ON CULTURABILITY OF ESCHERICHIA COLI O157:H7

In farm and food processing environments Escherichia coli O157:H7 can survive for days to months. In this study, we investigated the influence of starvation, pH (pH 4 or 7), temperature (4, 10 and 22C), and exposure to chlorine (50, 100, 150, 200 ppm) on culturability of E. coli O157:H7. The culturable cell population in PBS decreased to an undetectable level at 4C and 10C, but not at 22C during the experimental period. Cells suspended in PBS adjusted to pH 4 lost culturability more rapidly than at pH 7. Culturable cells were not detectable after exposure to chlorine, however, viable cell populations in the range of 1 – 2 log remained stable for 5 days. During loss of culturability two distinct morphological cell populations emerged; typical rod shaped and coccoid shaped cells. In this study, independent of exposure to chlorine, conditions of low temperature and low pH had the greatest influence on entry of E. coli O157:H7 into a VNC state.     

Inactivation strategy for Clostridium perfringens spores adhered to food contact surfaces

The contamination of enterotoxigenic Clostridium perfringens spores on food contact surfaces posses a serious concern to food industry due to their high resistance to various preservation methods typically applied to control foodborne pathogens. In this study, we aimed to develop an strategy to inactivate C. perfringens spores on stainless steel (SS) surfaces by inducing spore germination and killing of germinated spores with commonly used disinfectants. The mixture of l-Asparagine and KCl (AK) induced maximum spore germination for all tested C. perfringens food poisoning (FP) and non-foodborne (NFB) isolates. Incubation temperature had a major impact on C. perfringens spore germination, with 40 °C induced higher germination than room temperature (RT) (20 ± 2 °C). In spore suspension, the implementation of AK-induced germination step prior to treatment with disinfectants significantly (p < 0.05) enhanced the inactivation of spores of FP strain SM101. However, under similar conditions, no significant spore inactivation was observed with NFB strain NB16. Interestingly, while the spores of FP isolates were able to germinate with AK upon their adhesion to SS chips, no significant germination was observed with spores of NFB isolates. Consequently, the incorporation of AK-induced germination step prior to decontamination of SS chips with disinfectants significantly (p < 0.05) inactivated the spores of FP isolates. Collectively, our current results showed that triggering spore germination considerably increased sporicidal activity of the commonly used disinfectants against C. perfringens FP spores attached to SS chips. These findings should help in developing an effective strategy to inactivate C. perfringens spores adhered to food contact surfaces.     

Susceptibility of Listeria monocytogenes strains to disinfectants and chlorinated alkaline cleaners at cold temperatures

Minimal inhibitory concentration (MIC), suspension and biofilm tests were used in evaluating the disinfecting efficacy of eight commercially available disinfectants and four chlorinated alkaline cleaners against 10 strains of Listeria monocytogenes at refrigerated temperatures. The adaptive response and cross-adaptation of L. monocytogenes to the disinfectants and chlorinated alkaline cleaners were investigated. The bactericidal components in the agents used were chlorine, quaternary ammonium compound (QAC), peracetic acid, ethanol and isopropanol. With some exceptions the disinfectants were efficient against the L. monocytogenes strains. One alkaline hypochlorite containing disinfectant was not efficient in the suspension and MIC tests at the lowest concentration recommended by the manufacturer. The chlorinated alkaline cleaners were effective against L. monocytogenes. A QAC-based disinfectant was found to be the least-effective agent on both glass bead-blasted polyethylene and stainless-steel surfaces. Adaptive and cross-adaptive responses of L. monocytogenes strains were observed towards the QAC-based agent, but over 2-fold increases to other agents were not observed. These results suggest that the adaptive responses of L. monocytogenes to disinfectants or chlorinated alkaline cleaners are of a minor concern.     

Effect of surface properties of different food contact materials on the efficiency of quaternary ammonium compounds residue recovery and persistence

Residues of quaternary ammonium compounds (QACs) remaining after sanitising were evaluated for a number of materials used in food plants. The residues were collected by swabs and measured using a spectrophotometric method. Surface topography and energy affected the QACs recovery. Highest percentage of QACs recovery was achieved for the tile material (102.2%) which had the most hydrophilic properties and least irregularities in surface topology, followed by stainless steel (82.1%). Meanwhile, the lowest recovery occurred in PVC (42.1%) and resin (44.3%) that exhibited hydrophobic characteristics and abrupt changes in height profile in a given surface area. Monitoring of QACs residues deposited on the surfaces after 7 days showed that the recovery of QACs in PVC and resin reduced significantly (P < 0.05), supposing that QACs might be degraded or interacted with the materials. However, no significant changes in residue recovery were observed for tiles and stainless steel surfaces after 7 days.     

Control of Salmonella in food related environments by chemical disinfection

Salmonella may be transferred to food through cross-contamination during processing and preparation. To minimise the risk of cross-contamination, proper cleaning and disinfection is essential for the food industry. Recently, disinfection of areas for preparation and storage of food has also gained increased popularity in households. There is a range of disinfectants available with different properties and usage areas, and care must be taken to choose the proper disinfectant for the specific application.There are many methods for testing the antimicrobial effect of disinfectants. To evaluate whether a disinfectant will be effective in practical settings, the test method should model real-life situations. Most disinfectants are effective against Salmonella at recommended user concentration in suspension tests. However, a number of factors may reduce the biocidal effect of disinfectants under practical conditions. This include properties of the surface to be disinfected, presence of soiling on the surface, the physiological state of the bacteria exposed to disinfection, including bacteria embedded in biofilms, and the effects of other stresses (e.g. desiccation, starvation and temperature).Here we review the effects of disinfectants used in food related areas in industries and in households against Salmonella. A general overview is given for disinfectants in use and methods used to evaluate effects. Effects of disinfectants against Salmonella in suspension and on surfaces, including biofilms, are presented and compared. Novel control strategies such as use of electrolysed water, antimicrobial surfaces, and anti-biofilm compounds are also covered. Finally, we review the ability of Salmonella to gain reduced susceptibility to disinfectants through adaptation and other physiological responses like biofilm formation.     

Electrochemistry of free chlorine and monochloramine and its relevance to the presence of Pb in drinking water

The commonly used disinfectants in drinking water are free chlorine (in the form of HOCl/OCl-) and monochloramine (NH2Cl). While free chlorine reacts with natural organic matter in water to produce chlorinated hydrocarbon byproducts, there is also concern that NH2Cl may react with Pbto produce soluble Pb(II) products--leading to elevated Pb levels in drinking water. In this study, electrochemical methods are used to compare the thermodynamics and kinetics of the reduction of these two disinfectants. The standard reduction potential for NH2Cl/Cl- was estimated to be +1.45 V in acidic media and +0.74 V in alkaline media versus NHE using thermodynamic cycles. The kinetics of electroreduction of the two disinfectants was studied using an Au rotating disk electrode. The exchange current densities estimated from Koutecky-Levich plots were 8.2 x 10(-5) and 4.1 x 10(-5) A/cm2, and by low overpotential experiments were 7.5 +/- 0.3 x 10(-5) and 3.7 +/- 0.4 x 10(-5) A/cm2 for free chlorine and NH2Cl, respectively. The rate constantforthe electrochemical reduction of free chlorine at equilibrium is approximately twice as large as that for the reduction of NH2Cl. Equilibrium potential measurements show that free chlorine will oxidize Pb to PbO2 above pH 1.7, whereas NH2Cl will oxidize Pb to PbO2 only above about pH 9.5, if the total dissolved inorganic carbon (DIC) is 18 ppm. Hence, NH2Cl is not capable of producing a passivating PbO2 layer on Pb, and could lead to elevated levels of dissolved Pb in drinking water.     

Aeromonas biofilm on stainless steel: efficiency of commonly used disinfectants

Aeromonas spp. are ubiquitous bacteria widely distributed among aquatic environments that have the ability to form biofilms. This aptitude allows them to persist in water distribution systems, contaminating drinking water, food processing surfaces and ultimately food. For this study, the biofilm‐forming ability of aeromonads was evaluated after 48‐h incubation on stainless steel discs at both 4 and 20 °C. Subsequently, disinfectants based on amphoteric surfactants and chlorine compounds were evaluated regarding the capacity to eradicate preformed biofilm and inhibit biofilm formation. Results obtained demonstrated that all strains under analysis were able to form biofilm at both room and refrigeration temperatures. The chlorine‐based disinfectant demonstrated to be efficient in removing preformed biofilm, but both were unsuccessful in preventing biofilm formation, highlighting the importance of adequate cleaning and disinfection procedures, with emphasis on food processing surfaces.     

Roles of hydroxyl radicals in electrolyzed oxidizing water (EOW) for the inactivation of Escherichia coli

The food industry has recognized electrolyzed oxidizing water (EOW) as a promising alternative decontamination technique. However, there is not a consensus about the sanitizing mechanism of EOW. In this study, we evaluated the disinfection efficacy of different types of EOW on Escherichia coli. Based on the hypothesis of hydroxyl radicals existing in EOW, in the present study, the hydroxyl radicals existed in slightly acidic electrolyzed water (SAEW) and acidic electrolyzed water (AEW) diluted to different levels were detected quantitatively. An ultraviolet (UV) spectrophotometer was used to scan EOW with different pH values. Accounting for the results of UV scanning to EOW with different pH value and the disinfection efficacy of different types of EOW, it can be concluded that considering the lower chlorine concentration of EOW compared with traditional chlorine disinfectants, the existing form of chlorine compounds rather than the hydroxyl radicals played important role in the disinfection efficacy of EOW.     

Susceptibility of Vibrio parahaemolyticus to disinfectants after prior exposure to sublethal stress

In the present study, Vibrio parahaemolyticus 690 in phosphate buffered-saline containing 3% NaCl was subjected to sublethal stresses: heat shock at 42 °C for 15 min, acid adaptation at pH 5.0 for 30 min, or cold shock at 20 °C for 4 h. The effect of sublethal stress on the susceptibility of V. parahaemolyticus to a chlorine-containing disinfectant (Clidox-S) and a quaternary ammonium compound (Quatricide) at 25 and 40 °C was investigated. It was found that the sublethal stresses examined enhanced the resistance of V. parahaemolyticus 690 to both disinfectants. Depending on the kinds of sublethal stress, V. parahaemolyticus 690 showed various degrees of enhanced resistance to disinfectants. Furthermore, the phenomenon of enhanced resistance to the disinfectants was more marked at 40 than at 25 °C.     

The effects of various disinfectants againstListeria monocytogeneson fresh-cut vegetables

Demand for fresh, healthier convenience-type foods has stimulated sales of fresh-cut vegetables in North America. As part of an overall program to better define the microbiological safety of these products, studies were done to examine the effects of various disinfectants againstListeria monocytogeneson fresh-cut vegetables. Some of the more established disinfectants such as chlorine and chlorine dioxide, as well as some of the newer ones including Salmide^Rand trisodium phosphate were tested againstL. monocytogeneson lettuce and cabbage. Different exposure times, as well as concentrations of the disinfectants were evaluated. In addition, organic acids such as lactic and acetic were examined for their ability to inactivateL. monocytogenespresent on the surface of vegetables. Chlorine was also tested in combination with the organic acids and various surfactants. For chlorine (200ppm, 10min), the maximum observed log₁₀reduction ofL. monocytogenesat 4 and 22°C, respectively, was 1.3 and 1.7 for lettuce and 0.9 and 1.2 for cabbage. Chlorine dioxide treatment (5ppm, 10min) of lettuce and cabbage at 4 and 22°C, respectively, resulted in a maximum observed log₁₀reduction ofL. monocytogenesof 1.1 and 0.8 for lettuce and 0.4 and 0.8 for cabbage. In comparison, the highest levels of Salmide^Rused on cabbage and lettuce (200ppm) at 22°C led to maximum reductions of 1.8 and 0.6 logs, respectively. None of the surfactants tested improved the disinfectant efficiency of chlorine, and actually proved to be antagonistic. Trisodium phosphate had almost no effect on reducing numbers ofL. monocytogenes, at levels that did not affect the organoleptic quality of lettuce. Lactic acid proved to be more effective than acetic acid in reducing numbers ofL. monocytogenes, although maximum reductions of only 0.5 and 0.2 logs were observed after a 10min exposure to 1% solutions of each organic acid. In general, therefore, one can expect approximately a 1log reduction in numbers ofL. monocytogenes, regardless of the disinfectant used.     

Quaternary ammonium compound stresses induce specific variations in fatty acid composition of Pseudomonas aeruginosa

The involvement of cell membrane fatty acids in resistance of Pseudomonas aeruginosa to Quaternary Ammonium Compounds (QACs) stresses was investigated. The strain was grown in a medium with increasing concentrations of different biocides: two QACs, and two non-QACs. In the presence of two QACs only, the strain was able to grow with increasing concentrations. During cellular adaptation to QACs, the resistance to the same biocide increased. A principal component analysis was performed with whole of fatty acid compositions which highlighted a specific variation for the cultures in presence of QACs. These modifications gave evidence of the outer membrane involvement in cellular response to the presence of QACs.     

食品防腐剂、消毒剂和臭氧对香蕉货架寿命的影响

炭疽病是影响香蕉采后货架寿命限制因子。为了寻找国家标准允许使用的控制香蕉采后炭疽病的方法,从腐烂香蕉上分离炭疽病菌,并进行鉴定。对纯水中炭疽病菌添加消毒剂和臭氧进行处理,在培养基上分别添加多种食品防腐剂、消毒剂和臭氧处理,抑制该菌孢子的萌发和菌丝的生长,筛选出抑制效果好的处理。对香蕉用所选防腐剂、消毒剂和臭氧处理进行保鲜试验,结果发现3 mg/kg的桂醛、50 mg/kg的戊二醛、10 mg/kg的二氧化氯、10 g/h臭氧处理对纯水中炭疽病菌的消毒效果很好,但对培养基上炭疽病菌消毒效果不好。0.24%亚硫酸氢钠可以完全抑制炭疽病孢子萌发,且对炭疽病菌丝生长抑制作用最强。12 mg/kg对羟基苯甲酸乙酯有较强的抑制炭疽病孢子萌发和菌丝生长作用。山梨酸钾、双乙酸钠、苯甲酸钠、脱氢醋酸钠、丙酸钙对香蕉炭疽病菌抑制效果较差。10 g/h臭氧处理香蕉10 min,12 mg/kg对羟基苯甲酸乙酯洗果3 min,与500 mg/kg多菌灵抑菌效果类似,消毒处理均显著好于对照。先臭氧处理或戊二醛、桂醛、二氧化氯处理,后对羟基苯甲酸乙酯处理,可以控制香蕉炭疽病,延长香蕉货架寿命。     

Microbial testing methods for detection of residual cleaning agents and disinfectants-prevention of ATP bioluminescence measurement errors in the food industry

The ATP luminescence measurement is based on the presence of an enzymatic reaction and may significantly be affected by cleaning agents and disinfectants. In addition, disinfectants can also reduce the activity of the luciferase enzyme and also act as ATP-releasing agents. The agents disrupt the cell walls but preserve ATP in measurable form, and therefore correlation with culture methods can be poor. Therefore, if a rapid method is used to detect ATP, a control must be used for reliable results. The possible effect of disinfectants can be eliminated with a rapid test to minimize sources of error. In the present study a microbiological residue testing method that is nonspecific for residues was developed. The effects of a total of 38 commercial cleaning agents and disinfectants of various types were assessed using two microbiological methods, the Vibrio fischeri photobacteria test and Micrococcus luteus inhibition zone technique. The results show that the V. fischeri photobacteria test is very sensitive. This test can therefore be used for testing cleaning agent residues on surfaces in very small amounts. A small study was also carried out in a food factory to show applicability in processing facilities. The study showed, that a need for this type of method exists in food processing.     

Heat shock and cold shock treatments affect the survival of Listeria monocytogenes and Salmonella Typhimurium exposed to disinfectants

The foodborne pathogens Listeria monocytogenes and Salmonella Typhimurium were subjected to heat shock at 48°C for 10 and 30 min, respectively, and then cold shocked at 15°C for 3 h. The effect of these shocks on the viability of test organisms exposed to chlorine dioxide and quaternary ammonium compounds was then determined. After exposure to the disinfectants, the viable population of each test organism, regardless of heat shock or cold shock treatment, decreased as the exposure period was extended. Both heat shock and cold shock treatments reduced the susceptibility of L. monocytogenes to both disinfectants at 25°C. However, for Salmonella Typhimurium, exposure to the chlorine dioxide disinfectant or quaternary ammonium compounds at 25°C significantly reduced (P < 0.05) survival of heat-shocked cells but significantly increased (P < 0.05) survival of cold-shocked cells compared with control cells. Survival of both L. monocytogenes and Salmonella Typhimurium generally was reduced after exposure to disinfectants at 40°C compared with 25°C.     

SYNERGISTIC EFFECT OF VITAMIN B1 ON SANITIZER AND DISINFECTANT TREATMENTS FOR REDUCTION OF BACILLUS CEREUS IN RICE

The synergistic bactericidal effects of vitamin B₁ (thiamine dilauryl sulfate) and the efficacy of commercial sanitizers for minimization of Bacillus cereus contamination in cooked rice were investigated. Sanitizer-treated rice exhibited a greater reduction than water-treated rice, while sanitizer-treated rice with Vitamin B₁ produced an even greater reduction. The treatments for B. cereus in rice included (1) 100 ppm hydrogen peroxide with 500 ppm vitamin B₁; (2) 200 ppm hydrogen peroxide with 100 ppm vitamin B₁; (3) 400 ppm hydrogen peroxide; (4) 50 ppm chlorine with 500 ppm vitamin B₁; (5) 60 ppm chlorine with 300 ppm vitamin B₁; (6) 70 ppm chlorine with 100 ppm vitamin B₁; (7) 80 ppm chlorine; and (8) 100,000 ppm ethanol with 500 vitamin B_1. All treatments completely eliminated B. cereus in rice. The sensory properties of all sanitizer-treated cooked rice did not differ significantly from the same properties for water-treated cooked rice.

PRACTICAL APPLICATIONS

The prevalence of Bacillus cereus in rice and rice products has been documented and control of the growth of B. cereus in rice is an important consideration. The results obtained from this study can be of use to rice producers in the manufacture of safe products. Although chemical disinfectants can be used to reduce the amount of B. cereus in rice, vitamin B₁ can also be used as an effective additive that reduces the amount of disinfectant use via a synergistic antimicrobial effect. The increasing use of chemical disinfectants for safety in the food industry can be reversed using our method.     

单核细胞增生李斯特菌对季铵盐类消毒剂耐药机制研究进展

单核细胞增生李斯特菌（Listeria monocytogenes,Lm）是一种重要的食源性致病菌,能够在食品加工环境中存活数月乃至数年之久。季铵盐类消毒剂在食品工业中的广泛应用导致Lm对此类消毒剂产生耐药性,从而影响消毒效果,增加食品被环境中Lm污染的机率。本文综述了Lm对季铵盐类消毒剂的耐药现状、耐药机制以及Lm对季铵盐类的适应性耐受机制的研究进展,阐明Lm对季铵盐类消毒剂耐药和适应性耐受机制,为指导食品从业人员合理规范化使用此类消毒剂、预防和控制食源性致病菌季铵盐类耐药提供理论依据,对保障食品质量安全也具有重要的现实意义。     

细菌活的非可培养状态的研究现状及发展趋势

微生物是危害食品质量安全的重要因素。细菌活的非可培养状态的概念提出,对于微生物学、食品质量安全、水质管理、流行病学等方面,都有重大意义。对细菌活的非可培养的研究现状及发展趋势进行了综述。     

Induction of Viable but Nonculturable State in Beer Spoilage Lactic Acid Bacteria

Strong beer spoilage strains Lactobacillus lindneri DSM 20692 and Lactobacillus paracollinoides JCM 11969^T were repeatedly subcultured in degassed beer and their culturability on MRS agar was examined. As a result, the two strains were found to show decreased culturability, suggesting that the prolonged contact with beer reduces the culturability of beer spoilage lactic acid bacteria (LAB). After 30 subcultures in degassed beer, both strains were subjected to sublethal heat treatment. As a consequence, L. lindneri DSM 20692 and L. paracollinoides JCM 11969^T were no longer detectable on MRS agar despite the presence of 460 viable cells, indicating that the viable but nonculturable (VNC) states were induced for both strains. Problematically, the heat treated VNC strains were shown to exhibit beer spoilage ability, suggesting that spoilage incidents can occur without detection by culture media. It was also shown that, once acquired, the VNC states are stably maintained in beer without further heat treatment. These results suggest the possibility that beer spoilage LAB strains remain hidden in pitching yeast and work‐in‐process products without detection. Furthermore L. lindneri DSM 20692 and L. paracollinoides JCM 11969^T in VNC states were successfully stored at ?80°C with 10% dimethyl‐sulfoxide as a cryoprotectant and reconstituted in degassed beer without losing VNC characteristics. Taken together, these findings show that valuable bioresources can be acquired from culturable beer spoilage LAB strains and maintained for long‐term storage as frozen culture stocks.