Development and application of a tool to assess laboratory hygiene in contained-use facilities

To gain information on laboratory hygiene in contained-use laboratories, a method was developed to study the presence of microorganisms on laboratory equipment. Focusing detection on genetically modified organisms (GMOs) containing the universal M13 primer binding sites enabled the detection of a broad range of GMOs using a single PCR. Swabbing surfaces in three different contained-use laboratories led to detection of M13-containing PCR products in 26 out of 34 swabs. Most sequences (up to five per sample) were detected in swabs from the centrifuge and sink, followed by swabs taken from the bin and incubator (up to four sequences per sample). The obtained sequences varied in length from 171 nucleotides (nt) to 878 nt. In most cases, sequences were only partially similar to sequences published in GenBank. The lengths of the regions with high similarity varied from 94 nt to 795 nt, and these similarities ranged from 81% to 100%. Similarities with more than one sequence were commonly found, complicating the identification of detected sequences. Nonetheless, 84% of the detected sequences were actually handled in the laboratory at the time of sampling. This demonstrates that the method may be used as a quality control tool to assess the efficacy of decontamination and cleaning of commonly used surfaces, such as laboratory benches, freezer doors, and centrifuge rotors, without prior knowledge of the identity or characteristics of the GMOs.     

BACTERIOLOGICAL STUDIES OF IMMERSION CLEANING OF MILKING EQUIPMENT ON COMMERCIAL FARMS

SUMMARY: A study has been made of the immersion cleaning technique as applied on commercial farms. In an initial survey bacteriological results for rinses and swabs of the milking equipment were satisfactory on most farms, but on one farm the results were very disappointing. Further investigations at this farm showed that the poor results were caused partly by sporing bacteria which survived the caustic soda treatment, presumably in the spore state, and partly by non-sporing organisms, chiefly chromobacteria and micrococci.
These non-sporing organisms are found in abundance on the surface of dirty wash troughs, and under certain conditions re-contamination of the milking equipment can occur during the rinsing operation. Suggested modifications to the original immersion cleaning technique include the use of hot water for the before-milking hypochlorite rinse, brushing the equipment in the after-milking rinse to remove extraneous dirt, regular cleansing of the wash trough and the monthly cleansing of the basket and bin.     

Use of ultrasonic energy in assessing microbial contamination on surfaces

Ultrasonic tanks were evaluated for their ability to remove viable microorganisms from various surfaces for subsequent enumeration. Test surfaces were polished stainless steel, smooth glass, frosted glass, and electronic components. The position of contaminated surfaces in relation to the ultrasonic energy source, distance of the ultrasonic source from the test surfaces, and temperature of the rinse fluid were some of the factors which influenced recovery. Experimental systems included both naturally occurring microbial contamination and artificial contamination with spores of Bacillus subtilis var. niger. The results showed that ultrasonic energy was more reliable and efficient than mechanical agitation for recovering surface contaminants. Conditions which increased the number and percentage of microorganisms recovered by ultrasonic energy were: using a cold rinse fluid, placing the sample bottle on the bottom of the ultrasonic tank, and facing the contaminated surfaces toward the energy source. It was also demonstrated that ultrasonic energy could be effectively used for eluting microorganisms from cotton swabs.     

Thermoduric and Psychrotrophic Organisms on Poorly Cleansed Milking Plants and Farm Bulk Milk Tanks

S ummary . Poorly cleansed pipeline milking plants gave much higher total and thermoduric colony counts than the internal surfaces of refrigerated farm bulk milk tanks, higher counts being recorded for milking plants after circulation cleaning than after cleansing by the acidified boiling water (ABW) method. Psychrotrophic counts were highest for farm milk tanks and lowest for milking plants cleansed by the ABW method. There was much variation in the proportion of thermoduric and psychrotrophic organisms in the total bacterial content of milking plants determined at 30°, thermodurics being much more prevalent in 21% of the rinses and psychrotrophs in 16%. Both groups formed about equal proportions in 9% of the pipeline rinses. The bacterial content of the farm bulk milk tanks was characterized by many psychrotrophs and few thermodurics.     

Sporicidal effect of disinfectants on Bacillus cereus isolated from the milk processing environment

The sporicidal efficacy of sodium hypochlorite and a combination of peracetic acid and hydrogen peroxide on Bacillus cereus spores isolated from the milk processing environment was examined using the European Suspension Test and by a surface disinfection test on stainless steel and rubber. The results of the laboratory tests were compared with field trials in a milking installation. In general, it was difficult to obtain consistent results, as the repeatability and reproducibility of the tests were found to vary according to the test strain, spore suspension preparation, disinfectant test solution, organic load, contact time and temperature. The sporulation medium used to obtain spores influenced the sporicidal effect considerably. To overcome this problem a standard method for preparation of spore suspensions should be prescribed. The various disinfectants were more effective in suspensions than on surfaces and in field trials. For the suspension tests SE values ranging from 1.0 to 3.0 were reached within 10 min at 50°C, depending on the disinfectant used. Sodium hypochlorite-based products were most effective. The activity on spores on surfaces and in field trials was limited. In surface tests reductions of 0.4–0.8 were observed within 10 min at 50°C depending on the type of surface. The SE values obtained for rubber were lower compared with stainless steel. The decrease in spore levels found in the milking installation was comparable with the surface experiments, i.e. 0.4–1.0. It is important to develop standard test procedures to assess the sporicidal efficacy of disinfectants used in food hygiene. Surface tests should be included to reflect the in-use conditions more closely and minimum standards should be determined for both suspension tests and surface tests.     

Effect of calf suckling on oxytocin,prolactin, growth hormone and milk yield in crossbred Gir x Holstein cows during milking

This study evaluated the effect of different milking managements on oxytocin, prolactin and growth hormone release in Gir x Holstein cows. Six cows were suckled by their calves, before and after milking (SM group); another six cows were submitted to exclusive milking (M group). High OT levels were observed during suckling of the SM group, however both groups had similar levels of OT during milking. The SM group presented PRL levels significantly higher than the M group, however only during suckling. For GH levels, the SM group showed higher levels than the M group, however this difference was significant only during the first suckling, cleaning of the teats and at the beginning of machine milking. Despite these results, SM cows produced more milk (milking plus suckling) than M cows, however milk obtained by milking was similar for both groups.     

Bacteriological Examination of Pipeline Milking Machines by Rinsing the Entire System

The results of bacteriological examination of rinses of the entire systems of 12 pipeline milking machines were compared with a similar examination of the milk collected while they were used for milking. The machines were cleaned in three different ways and showed varying degrees of complexity. There was a close correlation between the results when the milk was collected with the maximum of cleanliness during milking. This is attributed to the localization of most of the micro-organisms present in the milking machine in the junctions and complex parts of the machine and not on the surface of the equipment, so they are removed with the same efficiency by cold water or warm milk. The number of micro-organisms retrieved by water during successive rinses (or by the milk during milking) decreased at a rate which depended either on the level of initial contamination of the equipment, its design or on the types of micro-organisms present. Recommendations are made for the interpretation of the results of rinsing.     

Disinfection and hygiene in the veterinary field and disinfection of animal houses and transport vehicles

Disinfection in animal houses means always a combination between cleaning and disinfection because the high amount of organic material present in such an environment will neutralize rapidly each disinfectant brought to the surface if no cleaning step had been done before. Depending on the kind of material, cleaning gives a 3 log reduction of total bacterial count on the surface and disinfection another 3 log reduction. This means that under practical conditions generally 10³ cfu of bacteria remain per cm² of surface, mostly sporeformers. The choice of disinfectant depends on the purpose of disinfection. In the case of notifiable diseases, it must be active against a defined pathogen. In the case of prophylactic disinfection, it must be active against a broad spectrum of microorganisms.Only disinfectants which are tested for their activity on surfaces which are representative for animal houses e.g. wood should be used, otherwise the failure under practical conditions can be predicted and work, as well as money, will be wasted. The optimal temperature for the liquids used in cleaning and disinfection is 40°C and the optimal temperature of surfaces is 20°C. Colder surfaces require higher concentrations of active substances in the solution, below 10°C the effect of practical disinfection is incomplete. Low air humidity and high air velocity have a negative influence on the action of most disinfectants on surfaces. The amount of disinfectant necessary to disinfect a surface in an animal house is at least 0.4 l/m². Transport vehicles are very difficult to disinfect, especially in winter-time. They should be cleaned with hot water, the remaining liquid should be removed by a suitable vacuum cleaner and the concentration of the disinfectant should be elevated at least for the factor 3. Mechanic action will improve the disinfecting effect in vehicles as well as preliminary disinfection prior cleaning.     

Achieving hygiene in the domestic kitchen: the effectiveness of commonly used cleaning procedures

AIMS: To quantify the transmission of Salmonella and Campylobacter to hands, cloths, and hand- and food-contact surfaces during the preparation of raw poultry in domestic kitchens, and to examine the impact on numbers of these bacteria of detergent-based cleaning alone, or in conjunction with thorough rising. METHODS AND RESULTS: Groups of volunteers prepared chickens for cooking. Surfaces were sampled either before cleaning or after cleaning using water and detergent with or without thorough rinsing. Although cleaning followed by rinsing consistently achieved decontamination of surfaces contaminated with Campylobacter, significant numbers of surfaces were still contaminated with low numbers of Salmonella. Where cloths contaminated with Salmonella were stored overnight, a reduction in the efficacy of detergent-based cleaning regimes was observed. CONCLUSIONS: Rinsing is the critical step in ensuring that bacteria are removed from surfaces during cleaning, but this may still leave residual contamination. Growth of Salmonella occurs in some contaminated cloths during overnight storage; Salmonella on cloths stored overnight are also more difficult to remove by washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Rinsing, as part of the cleaning process, is a critical step in achieving hygiene in the kitchen. However, to achieve completely hygienic surfaces, the use of an antimicrobial agent may be necessary.     

Relationship of goat milk flow emission variables with milking routine,milking parameters,milking machine characteristics and goat physiology

The aim of this paper was to study the relationship between milk flow emission variables recorded during milking of dairy goats with variables related to milking routine, goat physiology, milking parameters and milking machine characteristics, to determine the variables affecting milking performance and help the goat industry pinpoint farm and milking practices that improve milking performance. In total, 19 farms were visited once during the evening milking. Milking parameters (vacuum level (VL), pulsation ratio and pulsation rate, vacuum drop), milk emission flow variables (milking time, milk yield, maximum milk flow (MMF), average milk flow (AVMF), time until 500 g/min milk flow is established (TS500)), doe characteristics of 8 to 10 goats/farm (breed, days in milk and parity), milking practices (overmilking, overstripping, pre-lag time) and milking machine characteristics (line height, presence of claw) were recorded on every farm. The relationships between recorded variables and farm were analysed by a one-way ANOVA analysis. The relationships of milk yield, MMF, milking time and TS500 with goat physiology, milking routine, milking parameters and milking machine design were analysed using a linear mixed model, considering the farm as the random effect. Farm was significant (P<0.05) in all the studied variables. Milk emission flow variables were similar to those recommended in scientific studies. Milking parameters were adequate in most of the farms, being similar to those recommended in scientific studies. Few milking parameters and milking machine characteristics affected the tested variables: average vacuum level only showed tendency on MMF, and milk pipeline height on TS500. Milk yield (MY) was mainly affected by parity, as the interaction of days in milk with parity was also significant. Milking time was mainly affected by milk yield and breed. Also significant were parity, the interaction of days in milk with parity and overstripping, whereas overmilking showed a slight tendency. We concluded that most of the studied variables were mainly related to goat physiology characteristics, as the effects of milking parameters and milking machine characteristics were scarce.     

Kinetics of conditioning layer formation on stainless steel immersed in seawater

Adhesion of microorganisms to surfaces in marine environments leads to biofouling. The deleterious effects of biofilm growth in the marine environment are numerous and include energy losses due to increased fluid frictional resistance or to increased heat transfer resistance, the risk of corrosion induced by microorganisms, loss of optical properties, and quality control and safety problems. Antifouling agents are generally used to protect surfaces from such a biofilm. These agents are toxic and can be persistent, causing harmful environmental and ecological effects. Moreover, the use of biocides and regular cleaning considerably increase the maintenance costs of marine industries. An improved knowledge of bio‐film adhesion mechanisms is needed for the development of an alternative approach to the currently used antifouling agents. The aim of this study is to characterise the chemical composition of the molecules first interacting with stainless steel during the period immediately following immersion in natural seawater and to elucidate the kinetics of the adsorbtion process. Proteins are shown to adhere very rapidly, closely followed by carbohydrates. The distribution on the surface of organic molecules is also examined. The ad‐sorbate on the surface is not a continuous film but a heterogeneous deposit, whose average thickness varies widely. The cleaning procedures used affect the adsorption kinetics. In particular, cleaning with hexane results in slower adsorption of nitrogen‐containing species than does cleaning in acetone.     

STUDIES ON THE USE OF BACTERICIDAL RUBBER IN MILKING MACHINE CLUSTERS

SUMMARY: Bactericidal rubber containing tetramethylthiuram disulphide was used in milking machine clusters cleaned by immersion in caustic soda on four farms, and by ordinary chemical sterilization on eight farms. Some reduction in the bacterial content of clusters was brought about by the bactericidal rubber, but high bacterial counts were not prevented, particularly where cleaning was inadequate. 'Fatigue splitting' of bactericidal rubber liners occurred after two to six weeks use.     

Evaluation of procedures for the collection, processing, and analysis of biomolecules from low-biomass surfaces

To comprehensively assess microbial diversity and abundance via molecular-analysis-based methods, procedures for sample collection, processing, and analysis were evaluated in depth. A model microbial community (MMC) of known composition, representative of a typical low-biomass surface sample, was used to examine the effects of variables in sampling matrices, target cell density/molecule concentration, and cryogenic storage on the overall efficacy of the sampling regimen. The MMC used in this study comprised 11 distinct species of bacterial, archaeal, and fungal lineages associated with either spacecraft or clean-room surfaces. A known cellular density of MMC was deposited onto stainless steel coupons, and after drying, a variety of sampling devices were used to recover cells and biomolecules. The biomolecules and cells/spores recovered from each collection device were assessed by cultivable and microscopic enumeration, and quantitative and species-specific PCR assays. rRNA gene-based quantitative PCR analysis showed that cotton swabs were superior to nylon-flocked swabs for sampling of small surface areas, and for larger surfaces, biological sampling kits significantly outperformed polyester wipes. Species-specific PCR revealed differential recovery of certain species dependent upon the sampling device employed. The results of this study empower current and future molecular-analysis-based microbial sampling and processing methodologies.     

Hard Surface Biocontrol in Hospitals Using Microbial-Based Cleaning Products

Background

Healthcare-Associated Infections (HAIs) are one of the most frequent complications occurring in healthcare facilities. Contaminated environmental surfaces provide an important potential source for transmission of many healthcare-associated pathogens, thus indicating the need for new and sustainable strategies.

Aim

This study aims to evaluate the effect of a novel cleaning procedure based on the mechanism of biocontrol, on the presence and survival of several microorganisms responsible for HAIs (i.e. coliforms, Staphyloccus aureus, Clostridium difficile, and Candida albicans) on hard surfaces in a hospital setting.

Methods

The effect of microbial cleaning, containing spores of food grade Bacillus subtilis, Bacillus pumilus and Bacillus megaterium, in comparison with conventional cleaning protocols, was evaluated for 24 weeks in three independent hospitals (one in Belgium and two in Italy) and approximately 20000 microbial surface samples were collected.

Results

Microbial cleaning, as part of the daily cleaning protocol, resulted in a reduction of HAI-related pathogens by 50 to 89%. This effect was achieved after 3–4 weeks and the reduction in the pathogen load was stable over time. Moreover, by using microbial or conventional cleaning alternatively, we found that this effect was directly related to the new procedure, as indicated by the raise in CFU/m² when microbial cleaning was replaced by the conventional procedure. Although many questions remain regarding the actual mechanisms involved, this study demonstrates that microbial cleaning is a more effective and sustainable alternative to chemical cleaning and non-specific disinfection in healthcare facilities.

Conclusions

This study indicates microbial cleaning as an effective strategy in continuously lowering the number of HAI-related microorganisms on surfaces. The first indications on the actual level of HAIs in the trial hospitals monitored on a continuous basis are very promising, and may pave the way for a novel and cost-effective strategy to counteract or (bio)control healthcare-associated pathogens.     

Processes of bioadhesion on stainless steel surfaces and cleanability: A review with special reference to the food industry

Biofouling of equipment surfaces in the food industry is due initially to physico-chemical adhesion processes, and subsequently to the proliferation of microbes within an extracellular polymer matrix. Two physico-chemical theories can be applied to predict simple cases of bacterial adhesion. However, these models are limited in their applicability owing to the complexity of bacterial surfaces and the surrounding medium. Various factors that can affect the bacterial adhesion process have been listed, all directly linked to the solid substratum, the suspension liquid or the microorganism. For stainless steel surfaces, it is important to take into account the grade of steel, the type of finish, surface roughness, the cleaning procedures used and the age of the steel. Regarding the suspension fluid within which adhesion takes place, pH, ionic composition and the presence of macromolecules are important variables. In addition, the adhering microorganisms have extremely complex surfaces and many factors must be taken into account when conducting adhesion tests, such as the presence of cell appendages, the method of culture, the contact time between the microorganism and the surface, and exopolymer synthesis. Research on biofilms growing on stainless steel has confirmed results obtained with other materials, regarding resistance to disinfectants, the role of the extracellular matrix and the process by which the biofilm forms. However, it appears that the bactericidal activity of disinfectants on biofilms differs according to the type of surface on which they are growing. The main cleaners and disinfectants used in the food industry are alkaline and acid detergents, peracetic acid, quaternary ammonium chlorides and iodophors. The cleanability and disinfectability of stainless steel surfaces have been compared with those of other materials. According to the published research findings, stainless steel is comparable in its biological cleanability to glass, and significantly better than polymers, aluminium or copper. Moreover, microorganisms in a biofilm developing on a stainless steel surface can be killed with lower concentrations of disinfectant than those on polymer surfaces.     

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.     

THE BACTERIAL CONTENT OF FARM DAIRY EQUIPMENT

SUMMARY: The results obtained during the examination of 2,310 advisory rinses of farm dairy equipment by means of colony count on Yeastrel milk agar at 30°, coli-aerogenes test at 30° and milk spoilage organisms (MSO) test at 22°, are discussed in relation to attainment of proposed satisfactory colony count levels. A much higher proportion of rinses of milking machine clusters gave unsatisfactory results than those of metal equipment. The MSO test (3 days, 22°) was a more sensitive indicator of the presence of milk spoilage organisms than the coli-aerogenes test, and is recommended for routine use. Results for milking machine clusters sterilized with steam were much better than those for clusters claimed to have been cleansed by chemical methods.     

Microbiological Aspects of Cold Cleaning with an lodophor of Milk Pipeline Installations

Over a period of three years bacteriological comparisons were made of a method of cleaning pipeline milking machines with (1) cold iodophor solution (cold method), (2) a chlorinated alkaline solution at 55-60°C circulated for 10 min (hot method), and (3) acidified boiling water (ABW method). Rinse samples from the entire installations gave the best results with the ABW method. The cold method favoured a psychrotrophic flora, coliform bacteria or lactic streptococci, depending on the part of the milking machine rinsed; the hot method tended to cause a build-up of the heat-resistant flora. In spite of this, the two methods can give acceptable results provided that the installations are correctly designed and constructed.     

Natural roles of biosurfactants

Microorganisms produce a variety of surface-active agents (or surfactants). These can be divided into low-molecular-weight molecules that lower surface and interfacial tensions efficiently and high-molecular-weight polymers that bind tightly to surfaces. These surfactants, produced by a wide variety of microorganisms, have very different chemical structures and surface properties. It is therefore reasonable to assume that different groups of biosurfactants have different natural roles in the growth of the producing microorganisms. Moreover, as their chemical structures and surface properties are so different, each emulsifier probably provides advantages in a particular ecological niche. Several bioemulsifiers have antibacterial or antifungal activities. Other bioemulsifiers enhance the growth of bacteria on hydrophobic water-insoluble substrates by increasing their bioavailability, presumably by increasing their surface area, desorbing them from surfaces and increasing their apparent solubility. Bioemulsifiers also play an important role in regulating the attachment–detachment of microorganisms to and from surfaces. In addition, emulsifiers are involved in bacterial pathogenesis, quorum sensing and biofilm formation. Recent experiments indicate that a high-molecular-weight bioemulsifier that coats the bacterial surface can be transferred horizontally to other bacteria, thereby changing their surface properties and interactions with the environment.     

Stomaching: a new concept in bacteriological sample preparation

An entirely new mixing device, particularly suitable for preparing bacterial suspensions from foods, fabrics, swabs, and other fairly soft materials, has been developed. With this technique the sample and diluent are put into an inexpensive, sterile plastic bag which is vigorously pounded on its outer surfaces by paddles when placed inside the machine. The resulting compression and shearing forces effectively remove even deep-seated bacteria. After samples are taken for analysis the bag and its remaining contents are thrown away. Labor involved in cleaning and sterilizing reusable homogenizer cups or probes is eliminated, and the device is immediately ready for reuse. Running costs are thus drastically reduced, compared with conventional homogenizers. Additional advantages of this device, which is simple and inexpensive to manufacture, are low noise level, negligible temperature rise, and the small storage space required for bags.