Bacterial tracking in a dairy production system using phenotypic and ribotyping methods

A systematic sampling plan was designed to collect raw and pasteurized milk samples throughout a single-raw milk source, dairy-processing operation experiencing reduced product shelf lives due to bacterial contamination. The objectives were to track bacterial contamination sources throughout a complete dairy production system and use this information to reduce bacterial spoilage losses in processed fluid products. Over a 5-week period, 233 bacterial isolates were collected, representative of different colony morphologies on psychrotrophic bacteria count (PBC) plates. Forty-five isolates (19%) were obtained from pasteurized milk and 188 (81%) were isolated from raw product. Thirty isolates were identified as Pseudomonas spp. by Gram stain and biochemical methods. Of these, 27 (90%) were postpasteurization isolates and 3 (10%) were raw milk isolates. Automated ribotyping revealed that raw and pasteurized Pseudomonas fluorescens isolates were indistinguishable (similarity index > 0.93), suggesting the possibility of postpasteurization contamination with bacteria from raw product. In the plant, filler nozzles were identified as the primary reservoirs of postpasteurization contamination. Nozzle replacement produced significantly lower finished-product PBCs at 7 days postprocessing (> 4-log reduction) and extended fluid product shelf life.     

Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes

Detection of psychrotrophic strains (those able to grow at or below 7 degreesC) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7 degreesC) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Differential induction of interleukin-12, interleukin-18, and interleukin-1beta converting enzyme mRNA in experimental autoimmune encephalomyelitis of the Lewis rat

Four out of ten Bacillus cereus strains produced spores able to adhere to monolayers of Caco-2 cells (human epithelial cells). One of these strains has been involved in an outbreak of food poisoning where the symptoms were more severe and persisted for longer than a normal B. cereus food poisoning. The hydrophobicity of the spores is a contributing factor for the adhesion to occur. The spores are able to germinate in an environment similar to that of the small intestine and then the vegetative cells can produce the enterotoxin directly at the target place. A concentrated and active form of the enterotoxin will be taken up by the epithelial cells in the small intestine. Spore adhesion could be an important virulence factor for some B. cereus strains.     

HIV testing for HIV prevention: a comparative analysis of policies in Britain, Hungary and Sweden

Growth of vegetative cells and outgrowth of spores of enterotoxigenic psychrotrophic Bacillus cereus in refrigerated minimally processed food products is a public health concern. A study was undertaken to determine the combined effects of pH, nisin, and temperature on growth and survival of 20 strains of B. cereus. The minimum growth temperatures in tryptic soy broth (pH 7.3) and brain heart infusion broth (BHI broth, pH 7.4) were 5 degrees C for two strains and 8 degrees C for five other strains. Vegetative cells of four of eight strains grew at 8 degrees C in BHI broth (pH 6.01 and 6.57) containing 10 micrograms of nisin per ml. At 15 degrees C, all strains grew at pH 5.53 to 6.57; three strains tolerated nisin at 50 micrograms/ml (pH 6.57), whereas two other strains had a maximum tolerance of 10 micrograms of nisin per ml. Tolerance of vegetative cells of B. cereus to nisin increased as the pH of the broth was increased from 5.53 to 6.01 and again to pH 6.57. Outgrowth of spores (six of six strains tested) was inhibited by 5 and 50 micrograms of nisin per ml at 8 and 15 degrees C, respectively. At 15 degrees C, outgrowth of spores of two strains occurred at pH 6.52 in BHI broth containing 10 micrograms of nisin per ml. The effectiveness of nisin in controlling the growth of psychrotrophic strains of B. cereus capable of causing human illness was more pronounced at 8 degrees C than at 15 degrees C and as the pH was decreased from 6.57 to 5.53. Studies to determine the effectiveness of nisin in controlling growth of psychrotrophic B. cereus in nonpasteurized foods held at refrigeration temperatures are warranted.     

Identification and detection of Bacillus sporothermodurans spores in 1, 10, and 100 milliliters of raw milk by PCR

A PCR method was developed to detect spores of Bacillus sporothermodurans in 1, 10, and 100 ml of raw milk. Two primers were derived from a unique sequence after subtractive hybridization of B. sporothermodurans DNA with DNA of MB 397, a not yet identified spore-forming bacterium isolated from raw milk, closely related to B. sporothermodurans. Specific identification was proven on a large collection of Bacillus strains and on strains from relevant taxa. The detection of B. sporothermodurans in raw milk is based on activation, germination, and outgrowth of the spores, followed by PCR identification. Spores from 10 and 100 ml were concentrated by centrifugation after chemical extraction of the milk components. The total test takes 28 h. The detection limits are 9, 0.4, and 0.22 CFU/ml for 1, 10, and 100 ml, respectively.     

Testing of raw milk for tetracycline residues

A newly improved Bacillus calidolactis tube diffusion test and two postscreening test systems--a receptor assay (Charm HVS; Charm Sciences, Inc., Malden, MA) and a newly developed Bacillus cereus ATCC 11778 mycoides test system--were evaluated for the detection and identification of tetracycline residues using 973 samples of bulk milk taken at random in The Netherlands. All milk samples were assayed with the B. calidolactis tube and the receptor test. The milk samples testing as suspect or positive with one or both of the test systems were analyzed with HPLC (limit of detection, 10 ng/ml) and the recently developed B. cereus test system. The B. calidolactis tube diffusion test detected tetracycline residues > 45 ng/ml in milk. With the B. cereus test plate, residues of oxytetracycline and tetracycline of > 30 ng/ml milk were detected; for chlortetracycline and doxycycline, the detection limit was 10 ng/ml. Raw milk exhibiting inhibition diameters of < 20 mm on the B. cereus test plate fulfilled the European Union criterion for maximum residue level for tetracyclines of < 100 ng/ml (including their 4-epimer derivatives). The detection limits of the receptor assay depended on the type of milk used. The scintillation counts that were obtained for control samples of bulk milk were considerably lower than for the milks obtained from Charm Sciences, Inc. or processed using UHT pasteurization. One of 973 milk samples was suspect for tetracycline residues by means of the B. calidolactis tube test as well as by the receptor assay; 8 other samples were also considered to be positive using the receptor assay alone. The presence of tetracycline residues could not be proved for these 9 samples (residue concentration, < 10 ng/ml) with HPLC. We concluded that the receptor assay was not reliable to detect tetracycline residues in raw milk at < 150 ng/ml. The B. cereus test plate was determined to be an inexpensive, reliable alternative for the receptor assay for detection of tetracycline residues.     

Environmental reservoirs for Serratia marcescens intramammary infections in dairy cows

Via special media, Serratia marcescens isolates were found in 3 bedding pack samples and in 2 milking parlor floor samples, and in milk samples from 19 cows during an episode of mastitis in a dairy cow herd. Chromosomal digest patterns of isolated S marcescens were indistinguishable for 18 of the milk samples and all bedding pack samples. Our findings provide strong evidence that the bedding pack was the reservoir of S marcescens associated with the outbreak of intramammary infections. Additionally, our ability to match digest patterns of isolates in the bedding pack and milk confirms the theory that S marcescens is an environmental pathogen capable of causing mastitis.     

Prevention of Listeria monocytogenes contamination on dairy farms and in the cheese industry

Listeria monocytogenes remains a pathogen bacteria the prevention of which, in food products, stays delicate. A complete organization, from the farmer's production to the industry and the consumer is necessary to eliminate Listeria in a type of food product. Listeria monocytogenes is susceptible of contaminate raw milk. The silage may be a major source of the occurrence of Listeria monocytogenes in raw milk. In this case, the contamination sources in a dairy farm and the good hygienic practices must be well-defined. In dairy industry, the contamination must be managed by the application of a systematic and methodically system like H.A.C.C.P. (Hazard Analysis Critical Control Points). It is useful for the study of Listeria monocytogenes contamination and involve the hazard analysis of each industry plant. In fact, the raw products, food products, manufacturing process, environmental conditions, process equipment, materials and staff organizational are specific and characteristic.     

Inactivation of Bacillus Spores by Ultraviolet or Gamma Radiation

Bacillus anthracis spores represent an important bioterrorism agent that can be dispersed in air or water. Existing decontamination practices based on these spores have focused on chemical disinfectants; however, the basic characteristics of radiation-based disinfectants suggest potential advantages in their application for control of Bacillus spores. Experiments were conducted to examine the effectiveness of ultraviolet (UV254) radiation and γ radiation for inactivation of Bacillus spores. Spores of Bacillus cereus were used for most experiments because of their similarity to B. anthracis. A limited number of experiments were also conducted using B. anthracis Sterne spores. In aqueous suspension, B. anthracis Sterne spores were observed to be slightly more resistant to UV254 than the spores of B. cereus. For the conditions of culture and assay used in these experiments, both spore types were more sensitive to UV254 radiation in aqueous suspension than the spores of B. subtilis, which are commonly used to characterize the performance of UV disinfection systems for water. Dried spores on surfaces were observed to be more resistant to UV254 than the same spores in aqueous suspension; it is likely that the increased resistance to UV of the dried spores was attributable to surface characteristics (porosity and texture) of the solid materials. γ radiation was shown to accomplish similar rates of inactivation for spores in aqueous suspension and for dried spores on surfaces. Collectively, these results suggest that the application of UV or ionizing radiation may hold promise for decontamination following bioterrorism events.     

Identification of Bacillus cereus by Fourier transform infrared spectroscopy (FTIR)

The objective of this study was to evaluate the potential of Fourier transform infrared spectroscopy (FTIR) for rapid identification of Bacillus cereus isolates. Ten B. cereus group isolates (comprising B. cereus, Bacillus mycoides, and Bacillus thuringiensis strains), five other Bacillus spp., and five non-Bacillus spp. were used. Two types of media, brain heart infusion (BHI) and Trypticase soy agar (TSA), were tested. The results indicated that all B. cereus group isolates produced characteristic absorbance peaks at wave numbers between 1738 and 1740 cm-1. These peaks were not affected by the growth medium. None of the other bacteria tested showed a similar peak after growth on BHI or TSA. Absorbance peaks between 1800 and 1500 cm-1 of members of the B. cereus group had different shapes and sizes, suggesting that FTIR may be useful for rapid identification of species within the B. cereus group.     

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.     

The SUVIMAX (France) study: the role of antioxidants in the prevention of cancer and cardiovascular disorders

Endospores of Clostridium spp. capable of producing gas in a lactate-containing medium were enumerated from 14 pasteurized milk samples from Wisconsin cheese plants. Concentrations of endospores of lactate-fermenting, gas-producing Clostridium spp. were between 5.0 x 10(-2) and 1.7 x 10(0) MPN ml(-1). Concentrations of presumptive C. tyrobutyricum endospores (defined by subterminal endospore position and lactate dehydrogenase activity) were lower, not exceeding 2.0 x 10(-2) MPN ml(-1). Based on subterminal endospore position, lactate dehydrogenase activity, and a carbohydrate fermentation profile identical to C. tyrobutyricum strain ATCC 25755, five isolates (Ct) were initially characterized as C. tyrobutyricum, a known cause of late-blowing in high-pH cheeses. Twenty-eight other isolates (Cx) produced gas from lactate, but differed from ATCC 25755 in either endospore position, lactate dehydrogenase activity or carbohydrate fermentation profile. When inoculated at high concentrations in Gouda cheese, strain ATCC 25755, two Ct isolates and 18 Cx isolates tested produced gas during ripening. Among the five Ct isolates obtained and two reference strains confirmed as C. tyrobutyricum, there were four qualitatively different volatile organic acid byproduct profiles. Each of the two confirmed C. tyrobutyricum reference strains and five Ct isolates had distinct quantitative cell membrane fatty acid (CMFA) profiles. The Cx isolates represented 14 different volatile organic acid byproduct profiles and each isolate had a unique CMFA profile. Pulsed field gel electrophoresis (PFGE) of DNA from the two confirmed reference C. tyrobutyricum strains, four Ct and three Cx isolates, showed a low degree of relatedness. The results of this study suggest that a heterogeneous group of lactate-fermenting, gas-producing Clostridium spp. may be found in milk. Gas chromatographic analysis of volatile organic acid byproducts or CMFA, and PFGE of DNA are highly discriminating methods for differentiating Clostridium spp. that may cause late blowing in high-pH cheeses.     

Infections caused by Flavobacterium meningosepticum in patients in a neonatal intensive care unit

During a period of three and a half months 7 neonates from a neonatal intensive care unit became infected by F. meningosepticum, serotype B. The pathogens could be isolated from the tracheal secretions and--less frequently--from throat swabs, gastric juice and nose swabs. Environmental sampling led to the isolation of F. meningosepticum from the humidification fluid of the respirator, from vaporizers as well as from the artificial ventilation tubing. F. mengingosepticum was found in the water of humidifiers from 3 children, who developed neither a colonization nor an infection. In a number of cases the patients' environment was contaminated with F. meningosepticum prior to colinization or infection. Nearly identical resistance patterns against the antibiotics tested, could be demonstrated for the isolated strains. The primary source of infection could not be identified, it is supposed however that the index patient had been admitted to the hospital nine months before. A strict hygienic policy, like consequently performed hand hygiene, adequate processing of ventilation equipment and application of sterile tubings led to extinction of the infections. During subsequent environmental controls, F. meningosepticum could not be isolated.     

Technical design and assessment of tube equipment using two-phase flow for cleaning and disinfection

Most pipeline systems in dairy and food processing plants are cleaned by circulating cleaning solutions under pressure with a liquid pump. The flow of the circulated solutions is single-phase or flooded flow. Milking system pipelines are subject to special requirements which distinguish them from those in dairy and other food processing plants. Milking system pipelines are considerably larger in diameter than product lines in dairy plants because they must carry both milk and air in a stratified flow condition during the milking process. Milking machine Clean-In-Place (CIP) systems have historically used flooded flow to circulate cleaning solutions. The force to move liquid, however, is typically the vacuum provided by the same vacuum pump used during milking, rather than a positive pressure liquid pump. As the size and complexity of milking machines has increased in recent years, flooded flow CIP systems have become inadequate. The amount of water required to fully flood a milking system becomes impractical with very long and/or large diameter pipelines. The power available to achieve adequate flow velocity is also limited. Air admission has been used to produce two-phase (air/water) slug flow and overcome some of the limitations of fully flooded CIP. Cycled air admission can reduce the amount of water required for circulation and increase flow velocities and thus enhance mechanical cleaning action. Cycled air admission has been implemented in the field largely through trial and error methods. There has been a lack of fundamental design information and testing protocols for air-injected milking machine CIP systems. This has resulted in mixed success in the application of air injected systems. This paper summarizes both laboratory and field research conducted at the University of Wisconsin Milking Research and Instruction lab to provide basic information for the design of air injected CIP systems and methods for field assessment of these systems. Just as properly implemented air-injection can improve cleaning and sanitation in milking machines with less water and energy, so to it may have applications in the cleaning of other types of tube equipment.     

Non operative treatment of liver and spleen trauma. Clinical experience

A commercially available ELISA kit was used for the detection of Bacillus diarrhoeal enterotoxin (BDE) in a variety of foods and faeces. The ability of isolates of Bacillus spp., including Bacillus cereus, to produce BDE in Brain Heart Infusion broth containing 0.1% glucose was also checked by use of the kit. Results show that 29 out of 31 B. cereus isolates were enterotoxigenic. Foods positive for performed BDE were always contaminated with > 10(5) B. cereus cfu g-1, but not all foods contaminated with large numbers of B. cereus were positive for BDE. Bacillus sp., other than one isolate which closely resembled B. subtilis, were negative for BDE production. Criteria for the confirmation of Bacillus-mediated diarrhoea should now include reports of symptoms and incubation periods consistent with the diarrhoeal form of food-poisoning by Bacillus spp., together with the results of tests for enterotoxigenicity of the Bacillus isolate, and detection of BDE in either the food and/or faeces.     

Isolation of multiple Salmonella serovars from a dairy two years after a clinical salmonellosis outbreak

Samples were obtained for bacteriologic culturing of salmonellae from cows and calves on, and the environment of, a large California dairy that used free-stall housing and a flush system for manure handling. Two years previously, the dairy had an outbreak of clinical salmonellosis in the lactating herd; however, since that time, it had not had problems with clinical salmonellosis. On the basis of mean annual milk production per cow, this dairy was consistently ranked in the top 25% of dairies in the area enrolled in the Dairy Herd Improvement Association. Results of bacteriologic culture of 76% (108/142) of environmental samples and 48% (639/1,339) of fecal samples were positive for salmonellae. Eighty-two percent of the isolates were serovar C1, subclassified as Salmonella montevideo, and 17% were serovar E. Results of bacteriologic culture of 85% of samples of recycled flush water being pumped into the free-stall alleys were positive, as were results of bacteriologic culture of 78% of samples of herd bulk milk filters, 97% of fecal samples collected from calves being fed nonsalable milk, and 25% of fecal samples collected from cows at the time of breeding. These findings suggest that freedom from clinical salmonellosis and comparatively high measures of herd performance do not indicate the absence of salmonellae from a premises, and that hardy infectious agents transmitted by ingestion of feces can become established in the environment of modern free-stall dairies that use recycled water in their manure flush systems.     

Sensitivity of aerobic spore-forming species of the genus Bacillus to the pteridine compound O129

Discs containing the pteridine compound O129 at various concentrations may be useful in differentiating the following closely related species belonging to the genus Bacillus, viz. B. subtilis, B. licheniformis and B. pumilus; B. polymyxa and B. macerans; together with B. cereus and its subspecies B. cereus var. mycoides. At concentrations of 10 micrograms O129, the ATCC type strain of B. subtilis was resistant whereas B. licheniformis and B. pumilus were sensitive. However, B. subtilis was sensitive to O129 at 150 micrograms. Similar reactions differentiated the ATCC type strains of B. polymyxa and B. macerans. The ATCC type strain of B. cereus was resistant to O129 at both 150 micrograms and 10 micrograms, but its subspecies B. cereus var. mycoides (ATCC 28) was partially sensitive at 150 micrograms concentration. When clinical isolates of B. cereus var. mycoides were subsequently tested, this partial sensitivity was found to be a variable characteristic.     

Interferon-induced protein 10 and interleukin 8. C-X-C chemokines present in proliferative diabetic retinopathy

From May through November 1997, 1,011 samples of raw milk from bulk storage tanks were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. The samples originated from 1,011 different dairy herds located throughout the Netherlands. O157 VTEC was not isolated from any of the milk samples examined. Additionally, survival of O157 VTEC in raw and UHT-sterilized cow's milk at 7 and 15 degrees C was studied, both in the absence and presence of an activated lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS). Results indicated that the O157 VTEC strain tested was able to grow in raw milk at 7 degrees C as well as at 15 degrees C. Naturally occurring amounts of thiocyanate and hydrogen peroxide in the raw milk tested were not sufficient to activate the LPS. Although the LPS exhibited an antimicrobial activity against O157 VTEC in LPS-activated sterilized milk, O157 VTEC populations were not (or not as obviously) reduced in LPS-activated raw milk. Possibly background microflora were more sensitive to the LPS than the O157 VTEC test strain. It was concluded that raw milk contaminated with O157 VTEC will remain a hazard if kept at 7 or 15 degrees C. Effective pasteurization and avoiding postpasteurization contamination are necessary to ensure the safety of milk.     

Intracoronary stent implantation using a single high-pressure perfusion balloon catheter

INTRODUCTION: A factory producing lead ingots, located in Ca?apava, caused lead and cadmium contamination of the environment, in the Paraiba Valley region of Southeastern, Brazil, through the discharge of industrial waste and the recycling of batteries. The factory, set in a rural, dairy cattle breeding area, worried sanitary authorities who envisaged the possibility of these metals' having entered the food chain. For the purpose of assessing the levels of contamination of the milk produced in the region, due to the cattle's possible consumption of contaminated grass and water, the amounts of cadmium and lead present in the milk were verified. MATERIAL AND METHOD: Major producers, covering an area of up to 20 km from the contaminated source, authorized collection of 218 samples of both pasteurized and non-pasteurized milk, which were analysed. Lead and cadmium levels were determined by flame atomic absorption spectrophotometry, the lead being pre-concentrated by complexation with APDC (ammonium 1-pyrrolidinecarbodithioate) and further extraction with isobutyl methylketone. RESULTS AND CONCLUSIONS: Of the total number of samples, 43 presented lead levels over the maximum limit of 0.05 mg/kg established by Brazilian legislation. The median value found for lead was 0.04 mg/L. The variance analysis, with 95% confidence level, found no significant difference among the types of milk studied with regard to lead levels. As for cadmium, all samples showed levels below the 0.02 mg/L quantification limit of the method. In spite of the environmental contamination, the levels of cadmium found in the milk were below the 1.0 mg/kg limit established by Brazilian legislation.