Dean O. Cliver

Microorganisms in honey.

Knowledge of the moisture and temperature conditions influencing growth of microorganisms in honey has long been used to control the spoilage of honey. However, the need for additional microbiological data on honey will increase as new technologies for, and uses of honey develop. Microorganisms in honey may influence quality or safety. Due to the natural properties of honey and control measures in the honey industry, honey is a product with minimal types and levels of microbes. Microbes of concern in post-harvest handling are those that are commonly found in honey (i.e., yeasts and spore-forming bacteria), those that indicate the sanitary or commercial quality of honey (i.e., coliforms and yeasts), and those that under certain conditions could cause human illness. Primary sources of microbial contamination are likely to include pollen, the digestive tracts of honey bees, dust, air, earth and nectar, sources which are very difficult to control. The same secondary (after-harvest) sources that influence any food product are also sources of contamination for honey. These include air, food handlers, cross-contamination, equipment and buildings. Secondary sources of contamination are controlled by good manufacturing practices. The microbes of concern in honey are primarily yeasts and spore-forming bacteria. Total plate counts from honey samples can vary from zero to tens of thousands per gram for no apparent reason. Most samples of honey contain detectable levels of yeasts. Although yeast counts in many honey samples are below 100 colony forming units per gram (cfu/g), yeasts can grow in honey to very high numbers. Standard industry practices control yeast growth. Bacterial spores, particularly those in the Bacillus genus, are regularly found in honey. The spores of C. botulinum are found in a fraction of the honey samples tested-normally at low levels. No vegetative forms of disease-causing bacterial species have been found in honey. Bacteria do not replicate in honey and as such high numbers of vegetative bacteria could indicate recent contamination from a secondary source. Certain vegetative microbes can survive in honey, at cool temperatures, for several years. However, honey has anti-microbial properties that discourage the growth or persistence of many microorganisms. Typically, honey can be expected to contain low numbers and a limited variety of microbes. A routine microbiological examination of honey might include several different assays. A standard plate count provides general information. Specialized tests, such as a count of yeasts and an assay for bacterial spore-formers, may also be useful. An indicator of sanitary quality as provided by coliform counts might be included. Additional tests, to explain unusually high counts or address a certain problem, may be needed. The use of honey in products that receive no or limited heat treatment may require additional tests. More information on the source and control of microbes in honey is needed to answer the concerns currently facing the industry.

Pretreatment to avoid positive RT-PCR results with inactivated viruses

Enteric viruses that are important causes of human disease must often be detected by reverse transcription-polymerase chain reaction (RT-PCR), a method that commonly yields positive results with samples that contain only inactivated virus. This study was intended to develop a pretreatment for samples, so that inactivated viruses would not be detected by the RT-PCR procedure. Model viruses were human hepatitis A virus, vaccine poliovirus 1 and feline calicivirus as a surrogate for the Norwalk-like viruses. Each virus was inactivated (from an initial titer of approximately 10(3) PFU/ml) by ultraviolet light, hypochlorite or heating at 72 degrees C. Inactivated viruses, that were treated with proteinase K and ribonuclease for 30 min at 37 degrees C before RT-PCR, gave a negative result, which is to say that no amplicon was detected after the reaction was completed. This antecedent to the RT-PCR method may be applicable to other types of viruses, to viruses inactivated in other ways and to other molecular methods of virus detection.

Capsid Functions of Inactivated Human Picornaviruses and Feline Calicivirus

ABSTRACT The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72°C), and physiological temperature (37°C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37°C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72°C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37°C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72°C inactivation is the capsid and that the target of thermal inactivation (37°C versus 72°C) is temperature dependent.

Ultraviolet Inactivation of Feline Calicivirus, Human EntericViruses and Coliphages¶

Norwalk and Norwalk‐like viruses (NLV) are major causes of food‐ and water‐related disease in the United States. There is no host cell line in which the NLV can be tested for infectivity. Feline calicivirus (FCV) and NLV both belong to the family Caliciviridae. FCV can be assayed for infectivity in the Crandell Reese feline kidney cell line, so FCV serves as a surrogate for NLV. This study is the first report of UV inactivation of FCV and also of using the plaque technique, in contrast to the 50% tissue culture infectious dose end point technique, to determine the FCV infectivity titer. The infectivity titers (log10 plaque‐forming units/mL) of UV‐inactivated FCV, hepatitis A virus (HAV), poliovirus type 1 (PV1) and two small, round coliphages were plotted as a function of UV dose and analyzed by regression analysis and analysis of variance. These fitted straight‐line curves represent exponential inactivation, so UV inactivation can be said to show “one‐hit kinetics.” The decimal inactivation doses of UV for FCV, HAV, PV1, MS2 and ϕX174 were 47.85, 36.50, 24.10, 23.04 and 15.48 mW s/cm2, respectively. FCV appears to be the most UV resistant among the tested viruses.

Decontamination of Plastic and Wooden Cutting Boards for Kitchen Use

Decontamination of Plastic and wooden cutting boards was studied, with a view to preventing cross-contamination of foods in home kitchens. New and used Plastic (four polymers plus hard rubber) and wood (nine hardwoods) boards were cut into 5-cm square blocks (25 cm2 area) for these experiments. Bacterial contaminants-- Escherichia coli (two nonpathogenic strains plus serotype O157:H7), Listeria innocua , L. monocytogenes , or Salmonella typhimurium --applied to the block surface in nutrient broth or chicken juice, were recovered by soaking the surface in nutrient broth or pressing the block onto nutrient agar, within minutes or ≥12 h later. Persistence and overnight multiplication of bacteria on plastic surfaces depended on maintenance of humidity so as to prevent drying of the contaminant. New plastic cutting surfaces were relatively easy to clean and were microbiologically neutral, but plastic boards with extensive knife scars were difficult to clean manually, especially if they had deposits of chicken fat on them. Fewer bacteria were generally recovered from wooden blocks than from plastic blocks. Clean wood blocks rapidly absorbed all of the inoculum, after which the bacteria could not be recovered within 3 to 10 min. If the board surface was coated with chicken fat, some bacteria might be recovered even after 12 h at room temperature and high humidity. Cleaning with hot water and detergent generally removed these bacteria, regardless of bacterial species, wood species, and whether the wood was new or used.

Infectivity of RNA from inactivated poliovirus.

ABSTRACT During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72°C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbeccos modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22°C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.

Survival of Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure and manure slurry at sublethal temperatures.

Exponential inactivation was observed for Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure with decimal reduction times ranging from half a day at 37 C to 1-2 wk at 4 C. There was no material difference in inactivation rates between S. typhimurium and E. coli O157:H7. Inactivation was slower in slurries made by mixing two parts of water with one part of manure; decimal reduction times (time required for 90% destruction) ranged from 1-2 days at 37 C to 6-22 wk at 4 C. Escherichia coli O157:H7 consistently exhibited slightly slower inactivation than S. typhimurium. Log decimal reduction time for both strains was a linear function of storage temperature for manure and slurries. Chemical analysis indicated that accumulation of free ammonia in poultry manure was an important factor in inactivation of the pathogens. This finding was experimentally confirmed for S. typhimurium by adding ammonia directly to peptone water or to bovine manure, which was naturally low in ammonia, and adjusting pH to achieve predetermined levels of free ammonia.

Cryptosporidium parvum studies with dairy products.

Cryptosporidium parvum is a protozoan parasite capable of causing massive waterborne outbreaks. This study was conducted to model the transfer of C. parvum oocysts from contaminated water via food contact surfaces into yogurt and ice-cream, as well as to examine oocyst survival. Propidium iodide staining, combined with a direct immunofluorescence assay, was used for oocyst viability determination. Oocysts were recovered from milk products by a sucrose flotation-based procedure, with average recoveries of 82.3, 60.7, and 62.5% from low (1%) fat milk, 9% fat ice-cream, and 98% fat-free yogurt, respectively. Oocysts were also recovered, by rinsing with tap water, from stainless steel surfaces inoculated with oocyst suspension, with average recoveries of 93.1% when the surface was still wet and 69.0% after the surface had air-dried at room temperature. Viability of oocysts on the surface was significantly affected by desiccation; 5% of the oocysts remained viable after 4 h of air-drying at room temperature, while the proportion of viable oocysts was 81, 69, and 45% after air-drying for 10 min, 1 h, and 2 h, respectively. In contrast, oocyst viability only dropped from 82 to 75% after 30 min contact at room temperature with 5% bleach solution (equivalent to 0.26% NaOCl). Transfer of oocysts from milk and stainless steel surfaces into yogurt, and oocyst survival during the process were analyzed. Yogurt was made from pasteurized low fat milk and live yogurt starter by incubating at 37 degrees C for 48 h and then stored at 4 degrees C. Oocyst viability decreased from 83% (80%) to approximately 60% after 48 h at 37 degrees C and to approximately 58% following 8 days of storage, similar to oocyst survival in the controls using pasteurized milk without the addition of live yogurt. Oocyst survival in ice-cream was investigated by inoculating oocysts into ice-cream mix, and mixing and freezing in an ice-cream freezer, and hardening at -20 degrees C. Although approximately 20% (25 and 18%) of oocysts were viable before hardening, none were viable after 24 h at -20 degrees C. Control samples of oocysts suspended in distilled water and stored at -20 degrees C were taken at the same time intervals and 8% of the oocysts were still viable after 24 h.

Coliphages as indicators of human enteric viruses in groundwater

Due to a lack of dependable routine methods for direct analysis of pathogenic microorganisms, tests for bacteria that are supposed to be of intestinal origin are used to indicate the presence and extent of fecal pollution in water. Current indicators are not accurate monitors of fecal pollution and do not adequately reflect the presence of human enteric viruses. Coliphages (viruses that infect the bacterium Escherichia coli which occurs in the feces of all warm‐blooded animals) have been proposed as indices of water quality. Coliphages are readily recovered from sewage from all parts of the world. In most cases, the persistence of coliphages in surface waters, groundwaters, and sewage is greater than that of human enteric viruses and enteric bacteria. Coliphages have a number of unique characteristics which permit selective analytical techniques. On the basis of these techniques, a system for predicting the presence of human enteric viruses in groundwater can be developed.

Reduction of Escherichia coli O157:H7 and Salmonella Typhimurium in Artificially Contaminated Alfalfa Seeds and Mung Beans by Fumigation with Ammonia

Sprouts eaten raw are increasingly perceived as hazardous foods because they have been vehicles in outbreaks of foodborne disease, often involving Escherichia coli O157:H7 and Salmonella Typhimurium. Although the source of these pathogens has not been established, it is known that the seeds usually are already contaminated at the time sprouting begins. Earlier studies had shown that ammonia was lethal to these same pathogens in manure, so it seemed reasonable to determine whether ammonia was effective against them when associated with seeds to be used for sprouting. Experimentally contaminated (10(8) to 10(9) CFU/g) and dried seeds, intended for sprouting, were sealed in glass jars in which 180 or 300 mg of ammonia/liter of air space was generated by action of ammonium sulfate and sodium hydroxide. Samples were taken after intervals up to 22 h at 20 degrees C. Destruction of approximately 2 to 3 logs was observed with both bacteria associated with alfalfa seeds, versus 5 to 6 logs with mung beans. Greater kills are apparently associated with lower initial bacterial loads. Germination of these seeds was unaffected by the treatment. It appears that this simple treatment could contribute significantly to the safety of sprout production from alfalfa seeds and mung beans.