Survival of pathogenic bacteria during mesophilic anaerobic digestion of animal waste

The survival of pathogenic bacteria was investigated during the operation of a full-scale anaerobic digester which was fed daily and operated at 28°C. The digester had a mean hydraulic retention time of 24 d. The viable numbers of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes and Campylobacter jejuni were reduced during mesophilic anaerobic digestion. Echerichia coli had the smallest mean viable numbers at each stage of the digestion process. Its mean T₉₀ value was 76.9 d. Yersinia enterocolitica was the least resistant to the anaerobic digester environment; its mean T₉₀ value was 18.2 d. Campylobacter jejuni was the most resistant bacterium; its mean T₉₀ value was 438.6 d. Regression analysis showed that there were no direct relationships between the slurry input and performance of the digester and the decline of pathogen numbers during the 140 d experimental period.     

Metabolic activity of pathogenic bacteria during semicontinuous anaerobic digestion.

In natural environments such as anaerobic digesters, bacteria are frequently subjected to the stress of nutrient fluxes because of the continual changes in the flow of nutrients, and to survive, they must be capable of adapting readily to nutrient changes. In this study, the metabolic activities of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes, and Campylobacter jejuni were studied within culture bags (Versapor-200 filters, 0.22-microns pore size) in laboratory anaerobic digesters. The metabolic activity of these bacteria was indicated by their adenylate energy charge (EC) ratios and their ability to incorporate [3H]thymidine, which was related to the respective changes in viable numbers within the culture bags during anaerobic digestion. Fluctuations in the adenylate EC ratios, the uptake of [3H]thymidine, and the viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes cells were probably due to constant changes in the amount of available nutrients within the anaerobic digesters. The viability of S. typhimurium increased quickly after a fresh supply of nutrients was added to the system as indicated by the uptake of [3H]thymidine and an increase in the adenylate EC ratios. The viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes organisms declined rapidly from 10(7) to 10(8) CFU/ml to 10(3) to 10(4) CFU/ml and remained at this level for an indefinite period. The decimal reduction time calculated during the period of exponential decline ranged from 0.8 to 1.2 days for these bacteria. C. jejuni had the greatest mean decimal reduction time value (3.6 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

Radiation inactivation of some food-borne pathogens in fish as influenced by fat levels

The influence of low (0·39–1·1%), medium (4·25%) and high (7·1–32·5%) fat levels in fish on radiation inactivation of four food-borne pathogens was investigated. Cells of Listeria monocytogenes 036, Yersinia enterocolitica F5692, Bacillus cereus and Salmonella typhimurium at logarithmic phase were inoculated in 10% fish homogenates and subjected to gamma irradiation at ice temperature (0–1 °C) with doses ranging from 0·05 to 0·8 kGy. The radiation survival curves of L. monocytogenes and B. cereus were characterized by shoulders, while a tailing effect was depicted by cells of Y. enterocolitica and B. cereus . The D₁₀ values in kGy calculated on the exponential part of the curve ranged from 0·2 to 0·3, 0·15 to 0·25, 0·1 to 0·15 and 0·09 to 0·1 for L. monocytogenes 036, B. cereus, Salm. typhimurium and Y. enterocolitica F5692, respectively. This order (D₁₀) of radiation resistance of each organism was not affected by the fat content of the fish. Inoculated pack studies carried out separately with each pathogen in fatty (Indian sardine, 7·1%) and lean (Golden anchovy, 0·39%) fish showed no difference in their survival after exposure to 1 kGy and 3 kGy doses, which corroborated the above observation. The practical significance of these results in the application of the technology is discussed.     

Thermal inactivation of Listeria monocytogenes during a process simulating temperatures achieved during microwave heating

Conventional heating was used to expose cells of Listeria monocytogenes , either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10⁶ upon reaching 70°C. Simulated microwave cooking of L. monocytogenes in situ , on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85°C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10⁶. To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70°C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10⁶ and 10⁸. These results show that when a temperature of 70°C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes . The survival of this organism during microwave heating when temperatures of over 70°C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5·8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15°C in (1) aerobic conditions; (2) 30% CO₂+ air; (3) 30% CO₂+ N₂; and (4) 100% CO₂. When samples were held at 1°C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6°C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1°C rather than 37°C. In CO₂ atmospheres growth of L. monocytogenes was inhibited on meat held at 6°C, especially under 100% CO₂. By contrast, storage at 15°C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.     

Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive

The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10³ cfu g^-1 than on leaves inoculated with about 10⁵ cfu g^-1, but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested.     

Characterization of the Thyroid Hormone Transport System of Cerebrocortical Rat Neurons in Primary Culture

Abstract: The uptake of 3',3,5-triiodo- l -thyronine (T₃) and l -thyroxine (T₄) by primary cultures derived from rat brain hemispheres was studied under initial velocity conditions, at 25°C. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The K _m of T₃ uptake was very similar to that of T₄, and did not vary significantly from day 1 to 4 in culture (310–400 n M ). The maximal velocity ( V _max) of T₃ uptake nearly doubled between day 1 and 4 of culture (41 ± 3 vs. 70 ± 5 pmol/min/mg of DNA, respectively). The V _max of T₄ uptake did not change (28 ± 8 and 31 ± 4 pmol/min/mg of DNA on days 1 and 4, respectively). The rank order of unlabeled thyroid hormone analogues to compete with labeled T₃ or T₄ uptakes were the same (T₃ > T₄ > 3',5',3-triiodo- l -thyronine > 3',3,5-triiodo- d -thyronine > triiodothyroacetic acid), indicating that the transport system is stereospecific. Unlabeled T₄ was a stronger competitor of labeled T₄ uptake than of labeled T₃ uptake, whereas unlabeled T₃ had the same potency for both processes. These results suggest that T₃ and T₄ are transported either by two distinct carriers or by the same carrier bearing separate binding sites for each hormone. They also indicate that the efficiency of T₃ uptake increases during neuronal maturation.     

Listeria monocytogenes isolation from human faecal specimens: experiments with the selective media, PALCAM and L-PALCAMY

The selective media PALCAM and L-PALCAMY were evaluated for their potential ability to detect Listeria monocytogenes in faeces. Recovery on PALCAM was almost total, and similar at 30°C and 35°C with or without CO₂ incubation. Warm enrichment in L-PALCAMY was necessary in order to detect low numbers (<10²/ml faeces). Faeces in excess of 0.25 ml/10 ml L-PALCAMY was inhibitory. The results point to L-PALCAMY and PALCAM as an epidemiological tool.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Sensitivity of bacteria to irradiation on poultry meat under various atmospheres

The sensitivity of seven bacterial species inoculated on to sterile poultry meat and irradiated under various atmospheres (air, CO₂, vacuum and nitrogen) was investigated. The irradiation sensitivities of Streptococcus faecalis and Staphylococcus aureus were unaffected by atmosphere. The other micro-organisms ( Pseudomonas putida, Salmonella typhimurium, Escherichia coli, Moraxella phenylpyruvica and a Lactobacillus sp.) were more sensitive (their D₁₀ values decreased) when irradiated in atmospheres other than air. In general, a vacuum or CO₂ atmosphere during irradiation was found to have the most lethal effect. Plating media were found to have a significant effect on the recovery of irradiated E. coli and Salm. typhirium.     

Effects on temperature on spontaneous and thyroxine-stimulated locomotor activity of Atlantic cod

Spontaneous locomotor activity of cod Gadus morhua maintained at 6° C tripled from February to May. In contrast, locomotor activity of cod held at 2° C was significantly lower than at 6° C (between 25 and 65% lower) and the seasonal increase was smaller. Plasma levels of both thyroxine (T₄) and triiodothyronine (T₃) did not differ between 2 and 6° C. T₄ injection increased locomotor activity by 10% for both temperature regimes. These data indicate that low water temperature reduces locomotor activity associated with migration in cod and that thyroid hormones are not involved in this decrease. This study provides a possible mechanism through which cold waters may affects migration and distribution of cod via its Effects on locomotor activity and swimming speed.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

Survival, physiological response and recovery of enteric bacteria exposed to a polar marine environment.

Survival, sublethal injury, and recoverability of Escherichia coli, Enterococcus faecalis, Salmonella typhimurium, and Yersinia enterocolitica were investigated by using diffusion chambers over 54 to 56 days of in situ exposure to a polar marine environment (-1.8 degrees C; salinity, 34.5 ppt) at McMurdo Station, Antarctica. Plate counts were used to assess recoverability and injury, whereas direct viable counts (DVCs) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction were utilized to determine substrate responsiveness and respiratory activity, respectively. T90 values (times for 10-fold decreases in numbers of recoverable cells) on nonselective medium were ca. 216 to 259 h for E. coli, S. typhimurium, and Y. enterocolitica and 432 h for E. faecalis. Sublethal injury was greater in populations of indicator bacteria than in pathogens. DVCs, CTC reduction, and plate counts indicated progressive increases in viable but nonculturable cells in E. coli, S. typhimurium, and Y. enterocolitica cultures throughout the 54-day exposure. Forty-eight-day exposure of E. coli, S. typhimurium, and Y. enterocolitica resulted in decreased optimal incubation temperatures for colony formation and inability to form colonies at 37 degrees C. The detection of responsive E. coli, S. typhimurium, and Y. enterocolitica by the DVC and CTC methods remained within 1% of inoculum values during 54 days of exposure, indicating some long-term persistence in the viable-but-nonculturable state. Percentages of respiring E. coli and S. typhimurium increased significantly upon addition of nutrients at all temperatures tested, indicating that nutrient availability rather than temperature limited enteric bacterial activity in this very cold environment. Large nutrient inputs to low-temperature marine environments may thus allow for the long-term persistence of enteric bacteria in a nonrecoverable state.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.     

Effect of rising temperature on the heat resistance of Listeria monocytogenes in meat emulsion

The heat resistance of a strain of L. monocytogenes was determined both in broth and in meat emulsion. The D -values for meat emulsion were approximately two to three times higher than those for broth and also the z -value increased significantly. The micro-organism proved to be more resistant when the cells were heated up slowly (0·5°C/min) to constant temperatures of 60, 63 and 66°C in meat emulsion. The D ₆₀, D ₆₃ and D ₆₆ were, respectively 12·95, 5·4 and 2·3 min. Results may have implications in the survival of Listeria monocytogenes in particular food preparations.