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.     

Salmonella DNA persistence in natural seawaters using PCR analysis

The risks of false-positive responses were examined when using the polymerase chain reaction (PCR) method for the detection of Salmonella in the marine environment (water and shellfish). The degradation rates of DNA, both free and from dead Salmonella , were evaluated in natural seawaters maintained at 10° and 20°C, using PCR with Vir and invA primers. The DNA of dead Salmonella was detected up to 55 d in seawater collected in winter and stored at 10°C. But in summer, the persistence was shorter: 10 d or even 2 d for a smaller inoculum (3 × 10³ Salmonella ml⁻¹). The role of the planktonic organisms present in spring and summer was pinpointed. For free DNA, the persistence times were shorter: from 2 to 4 d at 20°C, and from 3 to 8 d at 10°C showing that the nuclease activity of marine organisms is higher at warm temperatures. These data led us to recommend careful interpretations of direct PCR results, especially during cold periods and for samples collected close to terrestrial discharges of high concentrations of live, dead or lysed Salmonella . PCR is a rapid, specific and sensitive method, but should be applied with care to marine samples, in order to avoid false-positive responses.     

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.     

Interactions of Salmonella enterica with lettuce leaves

Aims:  To investigate the interactions of Salmonella enterica with abiotic and plant surfaces and their effect on the tolerance of the pathogen to various stressors.
Methods and Results:  Salmonella strains were tested for their ability to form biofilm in various growth media using a polystyrene plate model. Strong biofilm producers were found to attach better to intact Romaine lettuce leaf tissue compared to weak producers. Confocal microscopy and viable count studies revealed preferential attachment of Salmonella to cut-regions of the leaf after 2 h at 25°C, but not for 18 h at 4°C. Storage of intact lettuce pieces contaminated with Salmonella for 9 days at 4°C resulted only in small changes in population size. Exposure of lettuce-associated Salmonella cells to acidic conditions (pH 3·0) revealed increased tolerance of the attached vs planktonic bacteria.
Conclusions:  Biofilm formation on polystyrene may provide a suitable model to predict the initial interaction of Salmonella with cut Romaine lettuce leaves. Association of the pathogen with lettuce leaves facilitates its persistence during storage and enhances its acid tolerance.
Significance and Impact of the Study:  Understanding the interactions between foodborne pathogens and lettuce might be useful in developing new approaches to prevent fresh produce-associated outbreaks.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

The destruction of Salmonella typhimurium in chicken exudate by different freeze-thaw treatments

Antibacterial treatments for frozen poultry, including holding at -5°C and slow thawing at 4°C to which exponential phase cells of Salmonella typhimurium were susceptible, were found to be relatively ineffective against stationary phase cells. Exposure of the latter, however, to a pre-freezing triple stress treatment of cold-shock exposure at 5°C to a solution containing 5 mg/l of free available chlorine in 1% succinic acid (pH 2.5) for 20 min substantially lowered the resistance of the cells to subsequent freezing, storage and thawing in poultry flesh exudate. Cell survival was further decreased by storage of exudate at - 18°C for 28 d and this reduced the proportion of stationary phase cells to less than 1% of initial numbers, with a concomitant increase in sensitivity to deoxycholate. Such a combined pre-treatment may have practical potential for salmonella decontamination in the production of frozen poultry.     

Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods

DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non- Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non- Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37°C and/or 43°C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella .     

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.     

Cloning of the him genes encoding the integration host factor from Salmonella typhirmurium in E. coli

Abstract By hybridization of him A and him D gene probes from E. coli to chromosomal DNA of Salmonella typhimurium cross-hybridization was obtained in both cases. A gene bank of Salmonella DNA was isolated using the mini-Mu cloning system. This gene bank was transformed into either a prototrophic E. coli him A or him D mutant. Transformants complementing either the him A or him D defect were isolated on minimal medium plates supplemented with 40 μg/ml leucin at 42°C. The Salmonella him genes on these plasmids were further verified by their ability to plate phage Mu and to yield turbid plaques with phage lambda and by the ability of the recombinant plasmids to hybridize to E. coli him gene probes.     

Salmonella in animal slurry can be destroyed by aeration at low temperatures

Cattle and other animals infected by Salmonella can emit high numbers of these bacteria. To determine an effective means for reducing this bacterial group in animal slurry, samples were subjected to aeration in laboratory experiments and in farm-scale slurry tanks. A clear reduction in Salmonella levels was found in laboratory experiments at temperatures from 4 to 40 °C. Aeration in farm-scale slurry tanks increased the temperature above the ambient temperatures (often less than 0 °C) to maxima ranging between 19 and 40 °C. Farm-scale aeration resulted in similar reductions in Salmonella as those achieved in laboratory experiments. Thus, reductions, ranging from greater than 99% of the initial number to no detectable Salmonella , could be reached after 2–5 weeks using aeration processes with cattle slurries contaminated by Salm. infantis or pig slurry contaminated by Salm. typhimurium . These results suggest that farmers can control the spread of Salmonella from slurry to agricultural fields. The reduction mechanisms remain unknown, though the increase in pH (to 7·6–9·0) found in slurries after aeration might exert a decreasing effect on these bacteria.     

Opposite effects of temperature on breaking of dormancy and on induction of frost resistance in apple embryos

Apples ( Pyrus mains L., ev. Golden Delicious) were placed at 0, 12 and 35°C. At different intervals, seeds were taken from fruits, and the ability of isolated embryos to germinate was determined. In parallel, the survival of similar embryos frozen for 30 min at -7°C was estimated. Storage of apples at 0°C led to complete removal of embryo dormancy within ca 10 weeks. At 12°C, dormancy was only partly eliminated, and at 35°C breaking of dormancy did not occur. The onset of secondary embryo dormancy was thus evident as the result of 35 and 12°C treatments. On the other hand, the storage of apples at 35°C was the most effective treatment for induction of frost resistance in the embryos. At 12°C the same level of frost resistance was reached much slower. Even a prolonged storage of apples at 0°C did not allow frost survival of all embryos. The above relations were confirmed in experiments with transfer of fruits from one temperature of storage to another. The temperature of fruit storage affected the dormancy removal in embryos and induction of resistance to rapid freezing in opposite directions.     

Growth of Bacteria on Meat at Room Temperatures

The development of spoilage flora and the growth of individual psychrotrophs and pathogens on meat held at 20 or 30°C were studied. Under aerobic conditions psychrotrophic pseudomonads accounted for 60% of the spoilage flora at 20°C, but <20% at 30°C where they were displaced by species of Acinetobacter and Enterobacteriaceae which included both mesophilic and psychrotrophic strains. Mesophiles dominated the anaerobic spoilage flora at 30°C when clostridia were the major species, but at 20° the flora contained mesophiles and psychrotrophs in similar proportions and was dominated by Enterobacteriaceae. These results were largely predictable from the growth rate data for individual organisms.
Interactions between species occurred more frequently at 20°C than at 30°C. When pathogenic species were grown at 20 or 30°Cin competition with equal numbers of psychrotrophic spoilage organisms, no interactions were observed. When pathogens were grown in competition with high numbers of psychrotrophs, only Lactobacillus growing anaerobically inhibited Salmonella typhimurium and Escherichia coli , but other pathogens were inhibited to varying degrees depending on the competing species and the incubation conditions. In general, the degree of inhibition was greater at 20 than at 30°C and facultative organisms were more susceptible under anaerobic than aerobic conditions. It appears that the cumulative stresses of low pH, suboptimal temperatures and competition with large numbers of saprophytic organisms can inhibit many of the pathogens likely to be present on meat. The organisms least affected by the conditions on meat surfaces, Salmonella and Esch. coli , are likely to be the main hazards on meat of normal pH held at room temperatures.     

Observations on the Microbiology of Cooked Chicken Carcasses

S ummary . The residual microbial flora and the flora developing during storage at 1–3° and at 16°, of chicken carcasses cooked in a circulating moist air oven operated at 85°, have been studied. All parts of the carcasses reached and maintained 85° for at least 50 min, and the residual flora consisted largely of spore forming bacteria. The predominant residual species were Bacillus subtilis and Clostridium bifermentans. Non-sporing bacteria were not detected after cooking nor after storage at 1–3° for up to 7 days. Storage at 16° for 3 days markedly increased the number of non-sporing organisms although off-odours typical of spoilage were not apparent until at least 10 days. Staphylococcus aureus and Salmonella spp. were not detected after cooking and storage and Cl. welchii was rarely isolated. It is concluded that poultry cooked by this method present a minimal risk of food-borne infection or intoxication by these organisms if contamination after cooking is avoided, the carcasses are cooled rapidly to c , 3° and stored at this temperature or frozen.     

Effect of the DNA replication velocity on the response of Salmonella typhimurium to mild heating

Changes in the DNA replication velocity of Salmonella typhimurium following mild heat stress (52°C) were studied independently of the major physiological parameter of growth rate, using thymine-requiring mutant strains derived from Salm. typhimurium LT2. The isolated mutant strains BM1 or BM2, grown either as batch or chemostat cultures, showed a greater sensitivity to 52°C heat stress when grown on a minimal medium containing near-limiting concentrations of thymine, compared with growth in the presence of excess thymine. Radiolabelling experiments provided evidence for alterations in the velocity of DNA replication upon growth on different thymine concentrations, independent of the growth rate. Thus, replicating DNA was implicated as the major site of injury after mild moist heat stress. and accepted 3 June 1989     

Elevation of the heat resistance of Salmonella typhimurium by sublethal heat shock

The survival of Salmonella typhimurium after a standard heat challenge at 55°C for 25 min increased by several orders of magnitude when cells grown at 37°C were pre-incubated at 42°, 45° or 48°C before heating at the higher temperature. Heat resistance increased rapidly after the temperature shift, reaching near maximum levels within 30 min. Elevated heat resistance persisted for at least 10 h. Preincubation of cells at 48°C for 30 min increased their resistance to subsequent heating at 50°, 52°, 55°, 57° or 59°C. Survival curves of resistant cells were curvilinear. Estimated times for a '7D' inactivation increased by 2.6- to 20-fold compared with cells not pre-incubated before heat challenge.     

Efficiency of different enrichment and isolation procedures for the detection of Salmonella serotypes in edible offal

Rapid detection systems for Salmonella in foodstuffs are currently being developed. However, existing standards still call for application of traditional methods employing pre-enrichment followed by selective enrichment and isolation. The efficacy of various methods was tested using 264 chicken and lamb organ meats. Pre-enrichment was carried out in Tryptone Soy Broth (TSB) and enrichment in Tetrathionate Brilliant Green Broth (TTB) at 37°C, Selenite Broth with Brilliant Green and Sulphapyridine at 37°C and 43°C, and Rappaport-Vassiliadis Broth (RV 10) at 42°C. The isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen Enteric Agar (HEA) and Salmonella-Shigella Agar.
Enrichment in RV/42°C followed by isolation on BGA as recommended by ISO standard no. 6579 and enrichment in TTB/37°C followed by isolation in HEA, no longer recommended by that standard, produced the best results. Low percentages of positive samples and difficulties in detecting Salmonella are the result of interference by competing organisms (Enterobacteriaceae) and the number of salmonellas present after enrichment.
A total of 528 samples (TSB, eggs, lamb liver and chicken liver) were inoculated with Salm. enteritidis, Salm. kapemba and Salm. virchow , and the preceding experiment was repeated. All the TSB and egg samples tested positive, but the percentage of positive samples from the lamb and chicken liver was only 81–92%. Recovery of the salmonellas did not depend upon the method employed or the serotype inoculated but instead on interference by competing flora and the numbers of Salmonella present in the samples.     

Chilling Injury,Physicochemical Properties,and Antioxidant Enzyme Activities of Red Pitahaya (<i>Hylocereus polyrhizus</i>) Fruits under Cold Storage Stress

Low-temperature storage is extensively used to optimize the postharvest life of various fresh fruits. However, red pitahaya (Hylocereus polyrhizus) fruits are sensitive to chilling injury (CI), which leads to the limitation of low-temperature storage. In this study, red pitahaya fruits were stored at 2, 4, 6, 8, and 10°C, respectively, for 27 days to determine the appropriate storage temperature. During the storage of red pitahaya fruits, storage at 8°C was more effective in suppressing decay and maintaining quality than other low temperatures. Low-temperature (2, 4, and 6°C) storage decreased weight loss (WL) and maintained higher content of titratable acidity (TA), soluble sugars (SS), and total phenolics (TP) but different degrees of CI were detected. No CI was observed at 8°C and 10°C. Red pitahay as stored at 8 and 10°C were associated with better color evaluation, lower electrolyte leakage (EL), respiration rate, and lipoxygenase (LOX) activity, and higher fruit firmness, superoxide dismutase (SOD) activity, and catalase (CAT) activity. However, higher storage temperature (10°C) resulted in higher metabolic activity leading to lower quality and antioxidant capacities compared with 8°C. Therefore, our results demonstrated that red pitahaya stored at 8°C exhibited a protective effect on fruit quality and resisted CI development during storage.     

Survival of enteric bacteria and coliphage MS2 in pure human urine

Aims:  The survival of Escherichia coli , Salmonella enterica serovar Typhimurium, Enterococcus faecalis and coliphage MS2 was studied in stored, fresh and diluted (1 : 1) human urine at 15 and 30°C.
Methods and Results:  Survival rate was studied by the plate count method. All the organisms showed rapid inactivation in stored urine, but they survived better in diluted and fresh urine. The high pH level and temperature were the major factors found to influence the survival of the micro-organisms with the survival rate being higher at 15°C than at 30°C.
Conclusions:  The destruction of all micro-organisms in stored urine required <1 week at 30°C. Thus, the storage of urine is a useful way to reduce the risk of contamination while using urine as a fertilizer.
Significance and Impact of the Study:  The urine fertilization is aimed for the developing countries and the high temperatures in these countries may hasten the destruction of micro-organisms in urine. On the contrary, a higher survival rate of these organisms in fresh and diluted urine is a public health concern because the dilution of urine with water is likely to happen during flushing.     

Physiological response of cut rose flowers to cold storage

The effects of low temperature storage on the physiology of cut rose flowers ( Rosa hybridaL. cv. Mercedes) were studied. Extension of cold storage or increase in temperature (from 3 to 8°C) was accompanied by shortening of vase life and advancement of petal senescence, as reflected in an advance in the timing of the rise in ethylene production and an increase in membrane permeability (ion leakage). Although storage at a relative humidity (RH) of 65% reduced petal water content by 20% in comparison with flowers stored at 95% RH, it did not shorten vase life. The progression of petal senescence was measured during storage at 3°C and during aging at 22°C. Both ethylene production rates and membrane microviscosity measured by fluorescence depolarization increased with time at 3°C and at 22°C, but more slowly at 3°C. At 3°C membrane permeability measured by ion leakage did not increase. Following cold storage the rate of ethylene production in the petals was increased by up to eight times the rate in unstored flowers. Silver thiosulphate extended the vase life of both stored and fresh flowers equally by 2 days, but did not increase the life of stored flowers to that of treated fresh flowers. It is concluded that the primary effect of cold storage on roses is to slow down senescence and that the continued slow senescence leads to shorter vase life. The possible occurrence of sequential processes during senescence and the effects of temperature on these processes is discussed.