Control of microbial contamination of Franz diffusion cell receptor phase in the development of transcutaneous breast cancer therapeutics

Aims:  To determine the micro-organism contamination of excised porcine (pig) ear, and evaluate the use of Cyclopore track-etched membranes (CTEM) for preventing ingress into Franz-type diffusion cells.
Methods:  Swabs were taken from four locations and used to inoculate Tryptone Soya Agar (TSA) and Sabouraud Dextrose Agar (SDA) plates. Diffusion cells were assembled to include porcine skin with and without CTEM, and the receptor phase sampled periodically and spread onto plates.
Results:  Five distinct colony types were isolated after incubation of all swabs on TSA plates at 37°C; on SDA plates, one fungal colony was found at 30°C and one at 37°C. The SDA agar plate incubated at 30°C resulted in the growth of a large diffused white fungal colony. No regional differences were observed. Without the CTEM, the receptor phase became contaminated within 6 h. With the CTEM present, microbial ingress was substantially retarded with visible presumptive fungal growth occurring at 24 h and detectable contamination on both microbiological media at 48 h.
Conclusions:  As expected, the native porcine ears were considerably contaminated. The ingress of contamination into the diffusion cell receptor phases can be largely, but not entirely, eliminated using CTEM. The addition of antimicrobial agents was necessary to eliminate micro-organisms that were observed at later time points.
Significance and Impact of the Study:  This article, while highlighting the presence of a high number of micro-organisms on native porcine skin, presents a practical means to reduce the risk of microbial contamination in transdermal/transcutaneous permeation studies, particularly in the study of cell cultures grown within Franz diffusion cell receptor compartments.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

Thermal ecology of western tent caterpillars Malacosoma californicum pluviale and infection by nucleopolyhedrovirus

Abstract 1. Western tent caterpillars hatch in the early spring when temperatures are cool and variable. They compensate for sub-optimal air temperatures by basking in the sun.
2. Tent caterpillars have cyclic population dynamics and infection by nucleopolyhedrovirus (NPV) often occurs in populations at high density.
3. To determine whether climatic variation might influence viral infection, the environmental determinants of larval body temperature and the effects of temperature on growth and development rates and larval susceptibility to NPV were examined.
4. In the field, larval body temperature was determined by ambient temperature, irradiance, and larval stage. The relationship between larval body temperature and ambient temperature was curvilinear, a property consistent with, but not necessarily limited to, behaviourally thermoregulating organisms.
5. Larvae were reared at seven temperatures between 18 and 36 °C. Larval growth and development increased linearly with temperature to 30 °C, increased at a lower rate to 33 °C, then decreased to 36 °C. Pupal weights were highest for larvae reared between 27 and 30 °C.
6. The pathogenicity (LD₅₀) of NPV was not influenced by temperature, but the time to death of infected larvae declined asymptotically as temperature increased.
7. Taking into account larval growth, the theoretical yield of the virus increased significantly between 18 and 21 °C then decreased slightly as temperatures increased to 36 °C.
8. Control and infected larvae showed no difference in temperature preference on a thermal gradient. The modes of temperature preference were similar to those for optimal growth and asymptotic body temperatures measured in the field on sunny days.
9. Warmer temperatures attained by basking may increase the number of infection cycles in sunny springs but do not protect larvae from viral infection.     

Factors which affect the abundance of an invasive cladoceran, Daphnia lumholtzi, in U.S. reservoirs

1.  Daphnia lumholtzi is a subtropical, Old World species which is rapidly spreading throughout reservoirs in the southern U.S.A. It was first recorded in Lake Texoma (Oklahoma–Texas) in September 1991.
2. Southern U.S. reservoirs typically have strong spatial and temporal gradients in temperature, conductivity, turbidity and in the distribution of organisms. Therefore, the present experiments examined the reproductive and moulting rates, and survival of D . lumholtzi in relation to extremes of food concentration, temperature, conductivity and turbidity.
3. Increases in temperature (range = 15–29 °C) and decreases in turbidity (range = 0.6–470 NTU) increased reproductive and moulting rates, whereas food concentration only affected the rate of reproduction, while conductivity had no effects. Survival was affected only by temperature.
4.  Daphnia lumholtzi tolerates high temperatures (27–30 °C) at which other cladocerans in Lake Texoma disappear. Therefore, D . lumholtzi may exploit resources in midsummer, when there are few potential competitors.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Optimization of fertilization success in Sander vitreus is influenced by the sperm : egg ratio and ova storage

Walleye Sander vitreum eggs could be stored at 7° C for up to 30 h with 60% fertilization rates. Fertilization rates at 1 and 12° C fell to 17 and 5%, respectively. At spermatozoa : egg ratios <25 000 : 1, survival to the eyed stage declined precipitously. Beyond a ratio of 25 000 : 1, survival to the eyed stage did not increase > c . ∼ 70%. Therefore, a standard spermatozoa : egg ratio of 25 000 : 1 was established and is recommended for studies concerning fertilizing success.     

The preservation of micro-organisms in biological specimens stored at – 70°C

The preservation of micro-organisms that may be found on the skin was studied by storage in liquid media at -70°C. In the first part of the study the performance of 12 varieties of suspending media was evaluated with pure cultures of 17 species of micro-organisms maintained in the laboratory. After storage for 1 year the best medium (Oxoid Nutrient Broth with 15% glycerol) showed a mean survival for all organisms studied of 83.8%, with no significant differences between organisms. Even the worst medium (distilled water) permitted greater than 40% survival at 1 year. No changes in the characteristics of these micro-organisms were detected after 6 months storage in glycerol broth. In the second part of the study nose swabs were suspended in one representative medium (Bacto Nutrient Broth containing 7% glycerol). The mean percentage survival of staphylococci in these suspensions after 1 year's storage at -70°C was 75.4%. These results indicate that coagulase-negative staphylococci in samples of skin flora may be stored under these conditions for long periods, greatly reducing the work-load in epidemiological studies of infection.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

H umphrey , T.J. 1990. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating. Journal of Applied Bacteriology 69 493–497.
Storage of cultures of Salmonella enteritidis PT4 at either 4° or 8°C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4–7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60° rather than 55°C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37°C prior to heating enabled the organisms to recover heat resistance.     

Survival at low temperature of larvae of the pine processionary moth Thaumetopoea pityocampa from an area of range expansion

1 Larvae of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) develop throughout the winter, although their feeding activity and survival can be impaired by adverse climatic factors. The present study investigated the survival at low temperature of larvae originating from a population with range expansion in an alpine valley in Northern Italy.
2 The supercooling point of individually analysed larvae averaged at −7 °C. This value insufficiently described the cold hardiness of the larvae; 39% of the tested larvae were alive when returned to room temperature immediately after freezing. When larval colonies inside their nest were exposed to −17 °C for 1 h after gradual temperature decrease, survival was 70.4%.
3 Rearing of larvae in the laboratory at different day/night temperatures indicated an effect of cumulative chill injury on larvae. A logistic regression explained the relationship between negative thermal sum (h°C below 0 °C) received in the laboratory experiment and larval survival. A similar relationship was demonstrated between negative thermal sum and survival of larval colonies in the field.
4 In the laboratory experiment, some tested larvae were able to survive for up to 8 weeks without feeding depending on rearing temperature. As expected, feeding occurred only when larvae were reared at temperatures of 9 °C day/0 °C night.
5 We classify the larvae of T. pityocampa as being moderate freezing tolerant. The winter behaviour allows this species to track climate warming by a rapid expansion into those areas that become compatible with the insect's development.     

Survival of Salmonella on refrigerated chicken carcasses and subsequent transfer to cutting board

Objective:  To determine the effect of refrigeration time and temperature on Salmonella cell numbers on inoculated chicken carcasses and their transfer to a plastic cutting board.
Methods and Results:  The survival of Salmonella on chicken skin and the transfer to a plastic cutting board when exposed to different refrigeration temperatures (2, 6 or 8°C) for 9 days were the two main issues on which this work focused. Two scenarios were carried out to ascertain these effects: carcasses treated with a decontaminating acetic acid solution and untreated carcasses. All of the contaminated carcasses remained contaminated after 9 days of refrigeration. However, on untreated samples, while Salmonella numbers increased almost 1·5 log at 8°C, the pathogen numbers decreased about 1 log at 2 and 6°C. On acid-treated samples, cell numbers slightly decreased at all of the temperatures studied. Temperature did not affect salmonellae transfer to the cutting board, but time did. Acid decontamination increased cell numbers transferred to the cutting board compared with untreated samples.
Conclusion:  Proper refrigeration at low temperatures did not allow Salmonella numbers to rise, regardless of which carcasses had been, or had not been, acid treated. Despite the fact that the rate of transfer was not affected by temperature, the acid treatment detached Salmonella cells from the chicken skin and, therefore, the probability of greater cross-contamination should be studied further.
Significance and Impact of the Study:  The results of this study may provide better information about the refrigeration conditions for fresh chicken storage and also determine if these, along with acetic acid decontamination of broiler chicken, would affect the pathogen transfer to a cutting board.     

Changes in Escherichia coli O157:H7 numbers during holding on excised lean, fascia and fat beef surfaces at different temperatures

Aim:  To investigate changes in Escherichia coli O157:H7 numbers on excised beef carcass surfaces over 72 h at different temperatures.
Methods and Results:  Excised lean meat, fascia and fat were inoculated with E. coli O157:H7 and held in an environmental chamber for 72 h, at air speed 0·5 m s⁻¹, relative humidity (RH) 90%, and temperatures 4, 8 and 12°C. On lean, pathogen counts increased significantly at 12°C. On fascia, significant reductions in counts occurred at 4 and 8°C. Pathogen numbers were significantly reduced on fat at 4, 8 and 12°C (64 h). Counts on fat were significantly less at all temperatures, compared to lean or fascia and surface water activity, a_w, decreased significantly over time on fat at 4°C. Significant decreases in surface pH values were recorded on all meat substrates.
Conclusions:  The survival of E. coli O157:H7 varied in relation to the meat substrate and the holding temperature. Reductions in counts on fat surfaces appeared to be related to low surface a_w values. No relationship between pathogen survival and surface pH was established.
Significance and Impact of the Study:  The use of excised meat pieces in an environmental cabinet offers a more flexible approach to determining the use of different chilling regimes in the production of safe meat.     

Resistance of Escherichia coli grown at different temperatures to various environmental stresses

Aims:  To study the influence of growth temperature on the resistance of Escherichia coli to three agents of different nature: heat, pulsed electric field (PEF) and hydrogen peroxide.
Methods and Results:  Escherichia coli cells were grown to stationary phase at 10°C, 20°C, 30°C, 37°C and 42°C. Survival curves to a heat treatment at 57·5°C, to a PEF treatment at 22 kV cm⁻¹ and to 40 mmol l⁻¹ hydrogen peroxide were obtained and fitted to a model based on the Weibull distribution to describe and compare the inactivation. Time to inactivate the first log cycle of the population at 57·5°C of cells grown at 42°C was sixfold higher than that corresponding to cells grown at 10°C. On the contrary, cells grown at 10°C and 20°C were more resistant to PEF and hydrogen peroxide treatments.
Conclusions:  The influence of growth temperature on bacterial resistance depends on the stress applied. Cells grown at higher temperatures were more heat resistant, but more sensitive to PEF and hydrogen peroxide.
Significance and Impact of the Study:  Results obtained in this investigation help in understanding the physiology of bacterial resistance and the inactivation mechanisms of different technologies.     

Nonisothermal heat resistance determinations with the thermoresistometer Mastia

Aims:  To design and build a thermoresistometer, named Mastia, which could perform isothermal and nonisothermal experiments.
Methods and Results:  In order to evaluate the thermoresistometer, the heat resistance of Escherichia coli vegetative cells and Alicyclobacillus acidoterrestris spores was explored. Isothermal heat resistance of E. coli was characterized by D _60°C = 0·38 min and z =  4·7°C in pH 7 buffer. When the vegetative cells were exposed to nonisothermal conditions, their heat resistance was largely increased at slow heating and fast cooling rates. Isothermal heat resistance of A. acidoterrestris was characterized by D _95°C = 7·4 min and z =  9·5°C in orange juice. Under nonisothermal conditions, inactivation was reasonably well predicted from isothermal data.
Conclusions:  The thermoresistometer Mastia is a very suitable instrument to get heat resistance data of micro-organisms under isothermal and nonisothermal treatments.
Significance and Impact of the Study:  The thermoresistometer Mastia can be a helpful tool for food processors in order to estimate the level of safety of the treatments they apply.     

Anhydrobiosis quotient: a novel approach to evaluate stability in desiccated bacterial cells

Aim:  The major objective of this study was the development of a methodology to quantify the anhydrobiotic ability of bacteria and its application to evaluate the stability of desiccated bacterial cells using the biocontrol agent Tsukamurella paurometabola C-924 as a model of anhydrobiote.
Methods and Results:  Tsukamurella paurometabola C-924 was desiccated by spray-drying. Samples of desiccated cells were stored at several temperatures and viability and residual moisture were measured at different intervals of time. The term anhydrobiosis quotient (ε) was defined, and a scale of anhydrobiotic ability for classifying micro-organisms in terms of tolerance to desiccation was established (1 ≤  ε  ≤ 15). The anhydrobiosis quotient was used to evaluate the stability of the anhydrobiotic cells. As a main result, changes in the anhydrobiosis quotient at several temperatures were fitted using a reparameterized Weibull model, which was found to be robust for the prediction of the stability at 4°C.
Conclusions:  A novel methodology was developed to evaluate the desiccated state in bacteria. The anhydrobiosis quotient allows the quantitative estimation of the anhydrobiotic ability, and the mathematical model developed allows the prediction of the desiccated state of bacterial populations.
Significance and Impact of the Study:  The new methodology could be applied in studying the anhydrobiosis state of bacterial populations as a predictive tool for industrial and environmental microbiology.     

Lactic acid bacteria associated with a heat-processed pork product and sources of variation affecting chemical indices of spoilage and sensory characteristics

Aims:  To evaluate the potential for developing a quality index for a Danish modified atmosphere packaged (MAP) heat-processed and naturally contaminated pork meat product stored at 5°C.
Methods and Results:  The composition of the predominating microflora and changes in contents of tyramine, arginine, organic acids and sensory characteristics were analysed. The microflora was predominated by Lactobacillus sakei , Leuconostoc carnosum and Carnobacterium divergens . The presence of each species varied between products and batches resulting in limited usefulness of the concentrations of these bacteria or their metabolites as indices of quality. Furthermore, the three species differed in their metabolic activities as shown by use of a model meat extract. However, when MAP storage of the processed pork product was followed by aerobic storage then acetic acid showed some potential as a chemical indicator of sensory quality.
Conclusion:  Variation in processing parameters and spoilage microbiota limited the usefulness of concentrations of micro-organisms and their metabolites as indices of spoilage for the studied processed MAP pork product.
Significance and Impact of the Study:  The present study contributes to an understanding of the difficulties experienced in developing quality indices to be used in the control of microbial spoilage of processed MAP meat products.     

Extracellular protectants produced by Clostridium perfringens cells at elevated temperatures

Aim:  The mechanisms of adaptation of Clostridium perfringens to high temperatures are not well understood. In this work, the involvement of extracellular compounds in protection to heat was determined.
Methods and Results:  Cells were grown in fluid thioglycollate medium or chicken broth. When mid-log phase was reached, they were heat-shocked at 50°C for 30 min. Then cultures were centrifuged and supernatants were transferred to nonshocked cells. Heat tolerance of these cells was performed at 55°C. Viable cells were determined. In some cases, supernatants were heated at 65°C or 100°C or treated with trypsin. Supernatants were fractionated and PAGE was made of fractions showing heat-protective activity. When C. perfringens was exposed to a heat shock at 50°C, extracellular factors were found in the culture supernatant that provided protection to cells not exposed to a heat shock. The extracellular factors were sensitive to heat and trypsin treatment suggesting a protein component. SDS-PAGE analysis of supernatant fractions from heat-treated cells revealed two induced proteins (56 and 125 kDa) that could be involved in heat tolerance.
Conclusion:  In this work, the presence and thermoprotective activity of extracellular factors produced by C. perfringens under a heat shock was demonstrated.
Significance and Impact of the Study:  The detection of thermoprotective extracellular factors of C . perfringens will aid in our understanding of the physiology of survival of C. perfringens in foods.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

Sanitation of faeces from source-separating dry toilets using urea

Aim:  To develop a reliable and simple method to produce safe fertilizers from human excreta using urea for sanitation of faeces.
Methods and Results:  Urea was added to faecal matter (17% dry matter) at concentrations of 0·5–2% (w/w) and inactivation of Salmonella enterica subspecies 1 serovar Typhimurium (Salm. Typhimurium), Enterococcus spp . and the Salm. Typhimurium bacteriophage 28B was monitored at 14, 24 and 34°C. Urea additions enhanced inactivation and inactivation rates were positively related to increasing NH₃ (aq) concentration and temperature. Salm. Typhimurium was the most sensitive of the organisms studied, while Enterococcus spp. showed more persistence, especially at lower temperatures. The bacteriophage was the most resistant organism studied.
Conclusions:  Salmonella reduction levels that meet requirements for safe reuse of faeces as fertilizer (i.e. 6 log₁₀ reduction) can be achieved for 1% urea within 2 months at 14°C or within 1 week at 24°C and 34°C.
Significance and Impact of the Study:  The relationships between organism inactivation rates and temperature, ammonia and pH were identified. Urea treatment proved to be a robust and efficient option for safe recycling of plant nutrients.     

Survival capacity in water of Arcobacter species under different temperature conditions

Aims:  To assess the survival capacity in vitro of arcobacters in water at temperatures applied in the food industry.
Methods and Results:  Four strains of each Arcobacter species were inoculated in potable water and water with 1% organic material and stored at 4, 7, 20, 52, 56 and 60°C. Samples were taken at known time points and the numbers of bacteria were determined on Arcobacter -selective medium. All Arcobacter species remained viable for a temperature-dependent period of time, although Arcobacter butzleri displayed a significant longer survival and heat resistance . No significant intraspecies differences were detected, resulting in no definite identification of origin or strain dependency. The survival period for all species was prolonged in the presence of the organic material only for the low temperatures.
Conclusions:  The present study demonstrates that water can act as a reservoir and as a potential source of Arcobacter contamination to humans and animals.
Significance and Impact of the Study:  This study assessed for the first time the survival of all human-related Arcobacter species in water. Particularly A. butzleri showed to be the most robust species with regard to temperature which is interesting as that species is often found in human clinical specimens.     

Intergenerational acclimation in aphid overwintering

Abstract.  1. When first instar nymphs and adults of the grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphidiae) were maintained in long-term cultures (>6 months) at 20 °C and 10 °C, the LT₅₀ decreased from −8 and −8.8 °C to −16.0 and −13.5 °C, respectively.
2. When aphids from the 20 °C culture were transferred to 10 °C, there was a progressive increase in cold tolerance through three successive generations. Transfer of newly moulted pre-reproductive adults reared at 10 °C for three generations back to 20 °C resulted in a rapid loss of cold hardiness in their nymphal offspring.
3. In all generations reared at 10 °C, first born nymphs were more cold hardy than those born later in the birth sequence. The LT₅₀ of nymphs produced on the first day of reproduction in the first, second and third generations maintained at 10 °C were −14.8, −17.0 and −16.6 °C, respectively. Thereafter, nymphal cold hardiness decreased over the subsequent 14 days of reproduction in each generation at 10 °C with mean LT₅₀ values of −10.3, −12.6 and −14.8 °C, respectively. By contrast, the cold tolerance of first born nymphs of aphids reared continuously at 20 °C did not differ in comparison with later born siblings. The LT₅₀ of adult aphids was also unaffected by ageing.
4. The ecological relevance of these findings is discussed in relation to the overwintering survival of aphids such as S. avenae .