Characterization of micro-organisms isolated from dairy industry after cleaning and fogging disinfection with alkyl amine and peracetic acid

AIMS: To characterize micro-organisms isolated from Norwegian dairy production plants after cleaning and fogging disinfection with alkyl amine/peracetic acid and to indicate reasons for survival. METHODS AND RESULTS: Microbial samples were collected from five dairy plants after cleaning and fogging disinfection. Isolates from two of these production plants, which used fogging with alkylamino acetate (plant A), and peracetic acid (plant B), were chosen for further characterization. The sequence of the 16S ribosomal DNA, fatty acid analysis and biochemical characteristics were used to identify isolates. Three isolates identified as Rhodococcus erythropolis, Methylobacterium rhodesianum and Rhodotorula mucilaginosa were isolated from plant A and one Sphingomonas sp. and two M. extorquens from plant B. Different patterns of resistance to seven disinfectants in a bactericidal suspension test and variable degree of attachment to stainless steel were found. The strains with higher disinfectant resistance showed lower degree of attachment than susceptible strains. CONCLUSIONS: The study identifies and characterizes micro-organisms present after cleaning and fogging disinfection. Both surface attachment and resistance were shown as possible reasons for the presence of the isolates after cleaning and disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the awareness of disinfectant resistance as well as attachment as mechanisms of survival in dairy industry. It also strengthens the argument of frequent alternation of disinfectants in the food processing industry to avoid the establishment of resistant house strains.     

Efficacy of some commerical disinfectants against the bacterial isolates from a poultry slaughterhouse in Turkey

In this study, efficacy of seven different commercial disinfectant preparations was investigated against characteristic bacteria of a poultry slaughterhouse in Ankara (Turkey) by using paper disc-agar diffusion method and surface effectiveness test. According to paper disc-agar diffusion method, some disinfectants had wide efficacy against the test bacteria. The disinfectant effectiveness generally increased by the increasing of disinfectant concentration. However, some disinfectants were ineffective even though at their highest concentrations. The most effective disinfectant was B which contains QAC as active agent.Staphylococcus spp.,Staphyloccus aureus andEscherichia coli were the most sensitive bacteria to the disinfectants. Some pathogenic isolates, especiallySalmonella spp. andCampylobacter spp., were the most resistant ones to many of the disinfectants tested. The results of paper disc-agar diffusion method indicated the importance of characteristic bacterial strains of food plants as the test bacteria for disinfectant efficacy tests. Conversely of paper disc-agar diffusion method, all disinfectants were effective against the isolates by surface effectiveness test depending on exposure time. The disinfectants, except A and F, produced at least 3 log unit reduction during 5 min of exposure. However, all disinfectants at their lowest concentrations were effective against all tested bacteria during 15 and 30 min by this test.     

Adaptation and growth of Serratia marcescens in contact lens disinfectant solutions containing chlorhexidine gluconate.

Serratia marcescens (11 of 12 strains) demonstrated an ability to grow in certain chlorhexidine-based disinfecting solutions recommended for rigid gas-permeable contact lenses. For a representative strain, cells that were grown in nutrient-rich medium, washed, and inoculated into disinfecting solution went into a nonrecoverable phase within 24 h. However, after 4 days, cells that had the ability to grow in the disinfectant (doubling time, g = 5.7 h) emerged. Solutions supporting growth of S. marcescens were filter sterilized. These solutions, even after removal of the cells, showed bactericidal activity against Pseudomonas aeruginosa and a biphasic survival curve when rechallenged with S. marcescens. Adaptation to chlorhexidine by S. marcescens was not observed in solutions formulated with borate ions. For chlorhexidine-adapted cells, the MIC of chlorhexidine in saline was eightfold higher than that for unadapted cells. Cells adapted to chlorhexidine showed alterations in the proteins of the outer membrane and increased adherence to polyethylene. Cells adapted to chlorhexidine persisted or grew in several other contact lens solutions with different antimicrobial agents, including benzalkonium chloride.     

A rapid method for assessing the suitability of quenching agents for individual biocides as well as combinations

AIMS: To develop a novel, rapid method for testing the ability of quenching agents to neutralize disinfectants. METHODS AND RESULTS: Tests were performed to determine the suitability of different neutralizers for a range of disinfectants, using a new method based on the Bioscreen optical density analyser. Results showed that during disinfection tests, efficacy could be over-estimated due to poor, or no, neutralization of the disinfectant after a specified time of exposure to the bacteria. The failure to distinguish adequately between bacteriostatic and bactericidal effects can lead to false results during disinfectant testing. Experiments also showed that dilution of the disinfectant, following exposure to the bacteria, was not always sufficient to stop the activity of the disinfectant for chemicals with low dilution coefficients. CONCLUSIONS: The quench test proved to be very quick and easy to perform, with results being available within 18 h. Using the Bioscreen, the test is automated and determines whether dilution into a particular neutralizer is able to inactivate a disinfectant within 30 s. SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach allows the efficacy of quenching agents to be determined, prior to undertaking each disinfection study, and can help in the development of more suitable quenching solutions. The test has also been used to find suitable neutralizers for mixtures of disinfectants which might be used during studies on synergistic biocide combinations.     

Model systems allowing quantification of sensitivity to disinfectants and comparison of disinfectant susceptibility of persistent and presumed nonpersistent Listeria monocytogenes

Aims:  To determine if Listeria monocytogenes persistent strains differ from presumed nonpersistent strains in disinfection susceptibility and to examine the influence of attachment and NaCl on susceptibility.
Methods and Results:  Two model-systems that allowed quantitative inter-strain comparison of disinfectant sensitivity were developed. Persistent L. monocytogenes were not more tolerant to the disinfectants Incimaxx DES and Triquart SUPER than presumed nonpersistent isolates. When calibrating the systems with respect to presence of biological material and cell density, attached bacteria were as sensitive to disinfectants as were planktonic bacteria. Growth with 5% NaCl increased the tolerance of planktonic cells to Incimaxx DES. All strains of spot inoculated L. monocytogenes survived well 20 h of drying when protected by growth media and 5% NaCl, but were not protected by NaCl against disinfection.
Conclusions:  Persistent strains of L. monocytogenes are as susceptible to disinfectants as are presumed nonpersistent strains and attachment does not render the strains more tolerant to disinfectants. Growth with NaCl affected the susceptibility of the planktonic L. monocytogenes to Incimaxx DES and protected spot inoculated cells during drying.
Significance and Impact of the Study:  Attachment to surfaces does not per se offer protection to L. monocytogenes against disinfectants and disinfection tolerances do not appear to influence the ability of a strain to persist.     

A comparison of the bactericidal efficacy of 18 disinfectants used in the food industry against Escherichia coli O157:H7 and Pseudomonas aeruginosa at 10 and 20 degrees C

A number of proprietary disinfectant products (18) used in the food industry were tested for their bactericidal efficacy against Pseudomonas aeruginosa and Escherichia coli O157:H7 at 20 and 10 degrees C according to the BS EN 1276 (1997) quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas. At 20 degrees C, 13 products passed at their in-use concentration (under clean and dirty conditions) against Ps. aeruginosa and 15 passed against E. coli O157:H7. The number of products passing the test at 10 degrees C was 11 and 14 for Ps. aeruginosa and E. coli O157:H7, respectively. The products exhibiting reduced efficacy at the lower temperature were amphoterics and quaternary ammonium compounds although some of these types of products were effective at both temperatures. Products that passed against Ps. aeruginosa generally also passed against E. coli O157:H7. Taking all the results together, only 11 of the total of 18 products achieved a pass result under all the parameters tested. This work demonstrates the need for final verification of disinfectant efficacy by undertaking field trials in the food-processing environment in which the product is intended for use.     

多药耐药大肠杆菌对不同消毒剂的抗性探究

目的：研究多药耐药大肠杆菌对不同消毒剂的抗力水平,为临床上合理选择消毒剂提供依据。方法：采用营养肉汤稀释法及悬液定量杀菌实验分别测定临床分离出的具有多重耐药性的大肠杆菌对临床常用消毒剂的最低抑茵浓度（minimum inhibitory concentration,MIC）及不同作用时间消毒剂对菌株的杀灭率,并与大肠杆菌标准株的测定结果进行比较。结果：不同消毒剂对多药耐药大肠杆菌的MIC值不同,洁芙柔消毒凝胶的MIC值为2．8±1．9,绿莎新爱尔施牌消毒片的MIC值为2583．4±1107．2,爱护佳免洗手消毒液的MIC值为144．63：86．9,安尔碘II型皮肤消毒剂的MIC值为16．1±7．7。4种常用消毒剂对多要耐药大肠杆菌的MIC值均高于标准菌,绿莎新爱尔施牌消毒片及爱护佳免洗手消毒液的MIC值与标准大肠杆菌较为接近。另外,4种常用消毒剂在常用浓度下对标准大肠杆菌和多药耐药大肠杆菌作用5min的杀灭率均能打到100％,标准大肠杆菌的被杀灭率略高于多药耐药大肠杆菌。结论：多药耐药菌株对消毒剂的抗性日益受到关注,使用时调整浓度、加强消毒剂科学管理对控制医院感染具有重要意义。     

Impact of prescribed cleaning and disinfectant use on microbial contamination in the home

Aims: To identify and quantify the presence of Escherichia coli, Staphylococcus aureus, Salmonella, hepatitis A and norovirus in households and to assess the effect of chlorine and quaternary ammonium–based disinfectants following a prescribed use. Methods and Results: Eleven sites distributed in kitchen, bathroom, pet and children′s areas of two groups of 30 homes each: (i) a nonprescribed disinfectant user group and (ii) a disinfectant protocol user group. During the 6‐week study, samples were collected once a week except for week one when sample collection occurred immediately before and after disinfectant application to evaluate the disinfectant protocol. The concentration and occurrence of bacteria were less in the households with prescribed use of disinfectants. The greatest reductions were for E. coli (99%) and Staph. aureus (99·9999%), respectively. Only two samples were positive for HAV, while norovirus was absent. Disinfection protocols resulted in a significant (P < 0·05) microbial reduction in all areas of the homes tested compared to homes not using a prescribed protocol. Conclusions: The study suggests that disinfectant product application under specific protocol is necessary to achieve greater microbial reductions. Significance and Impact of the Study: Prescribed protocols constitute an important tool to reduce the occurrence of potential disease‐causing micro‐organisms in households.     

Wide variation in effectiveness of laboratory disinfectants against bacteriophages

Aims: The purpose of this study was to identify an effective disinfectant for the inactivation of the bacteriophages (phages) being used in our laboratory, as published studies on phage inactivation are far from unanimous in their conclusions. Methods and Results: The phages studied were three closely related strains of Myoviridae and three strains of Siphoviridae. Three disinfectants which are used commonly in microbiology laboratories were evaluated: Virkon (1%), ethanol (75%) and sodium hypochlorite (2500 ppm available chlorine). The most effective of these was Virkon, which inactivated all six phages rapidly. Ethanol was effective against the Myoviridae but had little effect on the Siphoviridae. Sodium hypochlorite was the least effective of the disinfectants evaluated. Conclusions: The findings of this study demonstrate a wide diversity in the effectiveness of disinfectants tested for inactivation of phages. Significance and Impact of the Study: Of the disinfectants tested Virkon is the most suitable choice for those unable to carry out disinfection validation studies, or where a broad spectrum disinfectant against phages is required. All of the phages in this study showed resilience to inactivation by sodium hypochlorite, and therefore this disinfectant is an unwise choice for use against phage without first assessing its effectiveness.     

细菌对消毒剂抗性机理研究进展

消毒剂通过抑制膜的主动运输、抑制微生物的代谢、扰乱微生物的DNA复制、使微生物细胞裂解而导致胞内成分渗漏以及使胞内物质凝结等方面发挥灭菌作用.消毒剂在杀灭细菌和控制细菌污染方面具有重要作用,但细菌对消毒剂也会产生抗性.细菌对消毒剂的抗性机制主要通过形成生物被膜,阻挡或外排机制减少消毒剂分子进入细胞,使消毒剂分子失活,以及其他表型性耐药等4个途径来实现,其中通过形成生物被膜阻止消毒剂分子进入细菌细胞或在进入细胞前使消毒剂失去效用,在细菌对消毒剂的抗性机制中具有重要作用.以实际污染的主要菌株为对象,研究它们对常用消毒剂的抗性机制,对控制细菌污染具有极大的应用价值和现实意义.     

细菌抵抗消毒剂及其对抗生素共耐药

消毒剂是一种可杀灭物体表面、器材设备、皮肤、空气和水源等传播媒介上携带的病原微生物的有机分子。它在体外能杀灭病原微生物,切断其传播途径,进而达到控制污染的目的,在生命安全防控中起着重要的作用。但是不合理地使用消毒剂导致细菌对消毒剂产生耐药。消毒剂耐药基因在不同种属间的水平转移加剧其传播风险,使消毒剂耐药情况进一步恶化。更令人担忧的是,细菌对消毒剂的耐药可能会导致对抗生素产生共耐药,给公共安全带来巨大的威胁。但目前为止,对消毒剂耐药以及共耐药的认识还不够全面。本文总结了关于细菌对消毒剂耐药的研究报道,对消毒剂的作用机制、细菌对消毒剂的耐药机制进行了论述,另外针对消毒剂耐药基因的传播以及细菌对消毒剂和抗生素的共耐药进行了综述,为减少消毒剂耐药性的产生和制定合理的消毒剂使用规范奠定基础。     

The testing of disinfectants

Although all disinfectant tests have the same final purpose, namely measuring the antimicrobial activity of a chemical substance or preparation, a large number of testing methods has been described. They are subdivided into suspension tests, carrier and surface disinfection tests and other practice-mimicking tests. The suspension tests comprise qualitative and quantitative suspension tests, and, as derived tests, the determination of the phenol coefficient and capacity tests. There is an essential difference between a carrier test and a surface disinfectant test: in the former case the carrier is submerged in the disinfectant solution during the whole exposure time, whereas in the latter case the disinfectant is applied on the carrier for the application time and thereafter the carrier is drying during the exposure.The basic principle now widely accepted is that the antimicrobial efficiency of a disinfectant is examined at three stages of testing.The first stage concerns laboratory tests in which it is verified whether a chemical compound or a preparation possesses antimicrobial activity. For these preliminary screening tests, suspension tests are considered.In the second stage of tests, disinfection procedures and not disinfectants are examined. It is determined under which conditions and at which use-dilution for a given application the preparation is active: the tests simulate real-life situations; such tests are carrier tests for the disinfection of materials by submersion and surface disinfection tests.The last stage takes place in the field, and comprises the in-loco or in-situ tests with as variants the in-use tests, which examine whether, after a normal period of use, germs are still killed by the disinfectant solution.It is the task of the European and international standardization organisations to develop new standards and to elaborate tests, which predict the effectiveness of a preparation in practice under variable circumstances.     

Effects of disinfectants on Campylobacter jejuni

Because of the increasing recognition that Campylobacter jejuni is an important enteric pathogen of humans, we studied the effects of widely used disinfectants on the viability of this organism. At an inoculum size of 10(3) to 10(4) CFU/ml, 1.25 mg of hypochlorite per liter killed three strains within 1 min. At an inoculum size of 10(6) to 10(7) CFU/ml, 5 mg of hypochlorite per liter killed three strains within 15 min. Killing of similar concentrations of C. jejuni and Escherichia coli by hypochlorite was approximately the same. At the high inoculum, 0.15% phenolic compound, 10 mg of iodophor per liter, 1:50,000 quaternary ammonium compound, 70% ethyl alcohol, and 0.125% glutaraldehyde killed all three strains within 1 min. These studies demonstrate that, under the conditions we tested (pH 7.0; 24 to 26 degrees C), the recommended standard concentrations of disinfecting agents are adequate to destroy C. jejuni.     

Effects of disinfectants on Campylobacter jejuni.

Because of the increasing recognition that Campylobacter jejuni is an important enteric pathogen of humans, we studied the effects of widely used disinfectants on the viability of this organism. At an inoculum size of 10(3) to 10(4) CFU/ml, 1.25 mg of hypochlorite per liter killed three strains within 1 min. At an inoculum size of 10(6) to 10(7) CFU/ml, 5 mg of hypochlorite per liter killed three strains within 15 min. Killing of similar concentrations of C. jejuni and Escherichia coli by hypochlorite was approximately the same. At the high inoculum, 0.15% phenolic compound, 10 mg of iodophor per liter, 1:50,000 quaternary ammonium compound, 70% ethyl alcohol, and 0.125% glutaraldehyde killed all three strains within 1 min. These studies demonstrate that, under the conditions we tested (pH 7.0; 24 to 26 degrees C), the recommended standard concentrations of disinfecting agents are adequate to destroy C. jejuni.     

Isolation of Serratia marcescens in the midgut of Rhodnius prolixus: impact on the establishment of the parasite Trypanosoma cruzi in the vector

The effects of resident bacteria in the stomach of 5th-instar larvae of Rhodnius prolixus on the erythrocyte lysis and Trypanosoma cruzi infection were studied. The bacteria population increased approximately 10,000-fold after feeding. Hemolysis rose to approximately 28% within 24h postfeeding and then linearly grew until day 4 attaining almost 100%. The number of surviving Y strain of T. cruzi in the stomach declined drastically, while the infection with Dm28c clone was maintained stable. Five days after feeding, few different types of bacterial colonies were obtained when stomach content suspensions were spread onto BHI agar plates. The hemolytic bacteria were isolated and identified as Serratia marcescens biotype A1a (referenced as RPH), which produces the pigment prodigiosin. In vitro experiments, comparing incubations of RPH with S. marcescens SM365, a prodigiosin pigment producer, and S. marcescens DB11, a nonpigment variant, as a control, with erythrocytes and T. cruzi demonstrated that: (i) at 30 degrees C, SM365 and RPH diminished the populations of Y strain, but not DM28c clone, and DB11 was unable to lyse both T. cruzi strains; (ii) at 0 degrees C, SM263 and RPH killed the flagellates, but DB11 did not; and (iii) all three strains of S. marcescens were able to lyse erythrocytes. These results suggest that S. marcescens trypanolytic activity from the SM365 and RPH strains is distinct from the hemolytic activity and that prodigiosin is an important factor for the trypanolytic action of the bacteria.     

Prolonged survival of Serratia marcescens in chlorhexidine.

During an outbreak of Serratia marcescens infections at our hospital, we discovered widespread contamination of the 2% chlorhexidine hand-washing solution by S. marcescens. Examination by electron microscopy of the sides of bottles in which this solution was stored revealed that microorganisms were embedded in a fibrous matrix. Bacteria, free in the liquid, were morphologically abnormal, showing cell wall disruption or cytoplasmic changes. Furthermore, bacteria adherent to the walls of the storage jugs and embedded in this fibrous matrix also had morphologically abnormal cytoplasm. Despite these changes, viable S. marcescens organisms were recovered from the fluid during a storage period of 27 months. The concentration of chlorhexidine required to inhibit these strains of Serratia was 1,024 microgram/ml; however, the organism could survive in concentrations of up to 20,000 micrograms/ml. Additional studies are needed to define the mechanism(s) that allows such bacteria to contaminate and survive in disinfectants.     

Evaluation of the efficacy of chemical disinfectants for disinfection of heat‐polymerised acrylic resin

doi: 10.1111/j.1741‐2358.2010.00400.x
Evaluation of the efficacy of chemical disinfectants for disinfection of heat‐polymerised acrylic resin Objective: This study evaluated the efficacy of disinfectants on the internal aspect of heat‐polymerised acrylic resin contaminated with microbial strains. Background: Dentures absorb oral fluids and become contaminated by different microorganisms. Methods: Two hundred and fifty rectangular specimens were made of heat‐polymerised acrylic resin, and then divided into five groups corresponding to the microbial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, S. mutans and Enterococcus faecalis). After contamination, the specimens were immersed in 1 and 2% sodium hypochlorite and 2% glutaraldehyde for periods of 5, 10 and 15 min. The specimens were placed into tubes containing different broths and incubated at 35°C and then visually analysed. Turbidity in the medium indicated microbial growth. The Fisher’s exact test was used in the analysis of the results. Results: The strain E. faecalis was the most resistant to the disinfectant solutions, and among them, glutaraldehyde was more effective than 2 and 1% hypochlorite for disinfection for 5 min; in the 10‐min period there were no differences between the disinfectants. In 15 min of immersion, 1% hypochlorite and glutaraldehyde were more effective than 2% hypochlorite. Conclusions: Disinfection for 10 min with 1% hypochlorite and glutaraldehyde is effective in disinfecting the internal aspect of heat‐polymerised acrylic resin.     

Bacterial growth in ampholytic disinfectant solutions.

Large numbers of live bacteria were isolated from solutions of 2 ampholytic disinfectants supplied from a built-in predosing system. They were resistant to and proliferated in these disinfectants. Bacteria were eradicated by employing a 3rd type of ampholytic disinfectant in the system.     

Disinfection in Food Processing – Efficacy Testing of Disinfectants

The key to effective cleaning and disinfection of food plants is the understanding of the type of the soil to be removed from the surfaces. An efficient cleaning and disinfection procedure consists of a sequence of rinses using good quality water with application of detergents and disinfectants. Disinfection is required in food plant operations, where wet surfaces provide favourable conditions for the growth of microbes. The efficacy of disinfectants is usually determined in suspensions, which do not mimic the growth conditions on surfaces where the agents are required to inactivate the microbes. Therefore, the suspension tests do not give adequate information and reliable carrier tests, which mimic surface growth, are needed. In developing a proposal for the testing of disinfectants on surfaces to an analytical standard, it is important to identify the major sources of variation in the procedure. In response to the need for a relatively realistic, simple and reliable test for disinfectant efficacy a method for culturing laboratory model biofilms has developed. The use of artificial biofilms i.e. biofilm-constructs inoculated with process contaminants in disinfectant testing can also be used for screening the activity of various disinfectants on biofilm cells. Both biofilm carrier tests showed clearly that the biofilm protects the microbes against the disinfectants. The chemical cleanliness is also essential in food plants. The total cleanliness of the process lines is mainly based on measuring the microbial load using culturing techniques. These results can give an incorrect picture of the total cleanliness, because the viable microbes do not grow when disinfectants are left on the surface. The luminescent bacteria light inhibition method offers a useful alternative for testing chemical residue left on surfaces after cleaning and disinfection operations.     

Farm disinfectants select for cyclohexane resistance, a marker of multiple antibiotic resistance, in Escherichia coli

AIMS: The aim of this study was to determine if three classes of farm disinfectants were able to select for ciprofloxacin or cyclohexane tolerant [indicative of a multiple antibiotic resistance (MAR) phenotype] Escherichia coli and if cyclohexane-tolerant E. coli could be isolated from farms. METHODS AND RESULTS: Chicken slurry containing ca 1 : 99 ratio ciprofloxacin resistant : susceptible E. coli (10 different resistant strains examined) was treated for 24 h with each of the disinfectants and examined for survival of resistant : susceptible strains. Ciprofloxacin-sensitive (n=5) and -resistant (n=5) E. coli were grown with sublethal concentrations of the disinfectants and then plated to agar containing ciprofloxacin or overlaid with cyclohexane. Escherichia coli (n=389) isolated from farms were tested for cyclohexane tolerance. Minimum inhibitory concentrations (MIC) were determined against representative isolates and mutants. The disinfectants did not select for the ciprofloxacin-resistant E. coli in poultry slurry but following growth with each of the three disinfectants, higher numbers (P < or = 0.023) of cyclohexane-tolerant E. coli were isolated and these had a MAR phenotype. Of the 389 farm E. coli tested, only one was cyclohexane tolerant. CONCLUSIONS: It is possible that in a farm environment, E. coli could be exposed to similar concentrations of the disinfectants that are selected for MAR type organisms under these laboratory conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study suggest that cyclohexane-resistant E. coli are not common on farms, but in view of the ease of isolating them in the laboratory with farm disinfectants, further investigations on farms are warranted.