Effects of sewage treatment on the removal of Listeria monocytogenes

Two sewage treatment plants in Baghdad, Iraq, were investigated to assess the effects of the different treatment stages on the removal of Listeria monocytogenes . The bacteria were severely affected after the activation and digestion stages at both plants. A dramatic decrease in numbers of listerias after each of these two stages was noticed during the cold months (September-January). The organisms were able to survive these treatments and were present in the final effluent and even in low numbers in the sewage sludge cake. Sufficient dewatering of sewage sludge is recommended to obtain sewage free of listerias. Improvements in the isolation procedure of L. monocytogenes from such heavily contaminated material is also discussed.     

Listeria monocytogenes contamination of crops grown on soil treated with sewage sludge cake

Listeria monocytogenes was found in the sewage sludge cake which is commonly used as an agricultural fertilizer in Iraq. Soils treated with this material were contaminated with the organism. Pot and field experiment showed that crops grown on treated soil became contaminated with L. monocytogenes and when alfalfa plant was grown on farmland soil treated with sewage sludge cake, listerias were found on 10% of 50 plants sampled at harvest, but the organism was detected only in low numbers on these crops (less than or equal to 5 cells/g). This could add to the risk to animals and man.     

Listeria monocytogenes contamination of crops grown on soil treated with sewage sludge cake

A l -G hazali , M.R. & A l -A zawi , S.K. 1990. Listeria monocytogenes contamination of crops grown on soil treated with sewage sludge cake. Journal of Applied Bacteriology 69 , 642–647.
Listeria monocytogenes was found in the sewage sludge cake which is commonly used as an agricultural fertilizer in Iraq. Soils treated with this material were contaminated with the organism. Pot and field experiment showed that crops grown on treated soil became contaminated with L. monocytogenes and when alfalfa plant was grown on farmland soil treated with sewage sludge cake, listerias were found on 10% of 50 plants sampled at harvest, but the organism was detected only in low numbers on these crops (≤ 5 cells/g). This could add to the risk to animals and man.     

Detection and enumeration of Listeria monocytogenes in a sewage treatment plant in Iraq

Listeria monocytogenes was isolated from a sewage treatment plant in Baghdad, Iraq, at all stages of treatment. The treatment processes did not yield a sewage sludge cake or a final discharge free of listerias. The agricultural practice of using such sewage products as fertilizers could become a route of spreading the organism in Iraq, particularly by infecting animals that consume vegetation in fields spread with such sewage. Dewatering of sewage reduced the number of L. monocytogenes but long periods of exposure to sun would be needed to obtain a 'safe' sewage sludge cake.     

Detection and enumeration of Listeria monocytogenes in a sewage treatment plant in Iraq

Listeria monocytogenes was isolated from a sewage treatment plant in Baghdad, Iraq, at all stages of treatment. The treatment processes did not yield a sewage sludge cake or a final discharge free of listerias. The agricultural practice of using such sewage products as fertilizers could become a route of spreading the organism in Iraq, particularly by infecting animals that consume vegetation in fields spread with such sewage. Dewatering of sewage reduced the number of L. monocytogenes but long periods of exposure to sun would be needed to obtain a 'safe' sewage sludge cake.     

Occurrence of Listeria sp and L monocytogenes in sewage sludge used for land application: effect of dewatering,liming and storage in tank on survival of Listeria species

The application of sewage sludge to agricultural land is widely used in France. To determine the impact of sludge treatments, concentrations of Listeria sp., Listeria monocytogenes and faecal indicators were monitored in five types of sludge from three sewage treatment plants in Angers (France) and its suburbs over a 1-year period. On the whole, bacteria were reduced in numbers through sludge treatments. Apart from liming, which leads to reduced levels of bacteria below detection limits, other sludge treatments did not eliminate Listeria sp. and faecal indicators. Listeria sp. and L. monocytogenes were found respectively in 87% and 73% of dewatered sludges and in 96% and 80% of sludges stored in tanks. Concentrations of L. monocytogenes, ranging from 0.15 to 20 MPN g(-1) dry matter in dewatered sludge and from 1 to 240 MPN g(-1) dry matter in sludge stored in tanks, did not show seasonal variations. Spreading of sanitised sludge onto agricultural land results in the addition of 10(6)-10(8) L. monocytogenes per hectare per year, which may contribute to the increase in the dissemination of this pathogenic species in the environment.     

Storage effects of sewage sludge cake on the survival of Listeria monocytogenes

Sewage sludge cake is widely used as an agricultural fertilizer in Iraq. Listeria monocytogenes was shown to be present in small numbers in this material despite sewage treatments. In an attempt to reduce the numbers of this pathogen in this sewage end product, the survival of L. monocytogenes was monitored in a heap of sewage sludge cake stored for over 23 weeks on farm land. The organisms were reduced in numbers and eliminated to undetectable limits during 8 weeks of storage under subtropical weathering and did not recover even 2 months after disappearance. Dewatering processes seem to have some affect on the survival of the bacteria. Therefore, solar dewatering by heaping the sewage sludge cake and exposing it to sun for no less than 8 weeks is recommended to obtain a listeria-free product.     

Storage effects of sewage sludge cake on the survival of Listeria monocytogenes

Sewage sludge cake is widely used as an agricultural fertilizer in Iraq. Listeria monocytogenes was shown to be present in small numbers in this material despite sewage treatments. In an attempt to reduce the numbers of this pathogen in this sewage end product, the survival of L. monocytogenes was monitored in a heap of sewage sludge cake stored for over 23 weeks on farm land. The organisms were reduced in numbers and eliminated to undetectable limits during 8 weeks of storage under subtropical weathering and did not recover even 2 months after disappearence. Dewatering processes seem to have some affect on the survival of the bacteria. Therefore, solar dewatering by heaping the sewage sludge cake and exposing it to sun for no less than 8 weeks is recommended to obtain a listeria-free product.     

Isolation and Enumeration of Listeria monocytogenes from Sewage, Sewage Sludge and River Water

Listeria monocytogenes in sewage, sewage sludge and river water was isolated by enrichment at 4°C with subculture and enrichment in thiocyanate, naladixic acid broth and plating on to Tryptose Agar. The results indicated that L. monocytogenes is present in sewage and sewage sludge in considerable numbers and that this organism survives longer than Salmonella spp. on land sprayed with sewage sludge.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69° to 73°C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73°C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69 degrees to 73 degrees C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73 degrees C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.     

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.     

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 10(6) upon reaching 70 degrees 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 degrees C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10(6). To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70 degrees C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10(6) and 10(8). These results show that when a temperature of 70 degrees 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 degrees 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.     

Behaviour of Listeria monocytogenes in meat and its control by a bacteriocin-producing strain of Lactobacillus sake

Lactobacillus sake Lb 706 can release a bacteriocin inhibitory to Listeria monocytogenes . In MRS broth, viable counts decreased rapidly when Lact. sake Lb 706 was added, whereas growth of the listerias was not affected by a bacteriocin-negative variant of the same Lactobacillus strain. Inhibition of L. monocytogenes was also observed in pasteurized minced meat inoculated with Lact. sake Lb 706. The bacteriocin produced is apparently effective in meat. However, the effect of the bacteriocin producer was less evident in minced meat than in broth. In comminuted cured raw pork filled into casings (German-type 'fresh Mettwurst'), L. monocytogenes was able to grow at a pH of 6.3, but addition of Lact. sake Lb 706 prevented the growth of listerias during the first few days after manufacture. At normal pH (5.7) L. monocytogenes did not multiply and addition of Lact. sake Lb 706 reduced viable counts of listerias by about one log cycle. Lactobacillus sake Lb 706 therefore may have some potential as a protective culture in meat products.     

Behaviour of Listeria monocytogenes in meat and its control by a bacteriocin-producing strain of Lactobacillus sake

Lactobacillus sake Lb 706 can release a bacteriocin inhibitory to Listeria monocytogenes. In MRS broth, viable counts decreased rapidly when Lact. sake Lb 706 was added, whereas growth of the listerias was not affected by a bacteriocin-negative variant of the same Lactobacillus strain. Inhibition of L. monocytogenes was also observed in pasteurized minced meat inoculated with Lact. sake Lb 706. The bacteriocin produced is apparently effective in meat. However, the effect of the bacteriocin producer was less evident in minced meat than in broth. In comminuted cured raw pork filled into casings (German-type 'fresh Mettwurst'), L. monocytogenes was able to grow at a pH of 6.3, but addition of Lact. sake Lb 706 prevented the growth of listerias during the first few days after manufacture. At normal pH (5.7) L. monocytogenes did not multiply and addition of Lact. sake Lb 706 reduced viable counts of listerias by about one log cycle. Lactobacillus sake Lb 706 therefore may have some potential as a protective culture in meat products.     

Isolation of Listeria monocytogenes from raw milk and its environment at dairy farms in Japan

The incidence of Listeria monocytogenes contamination in raw milk from farm bulk tanks and silage was investigated monthly at 20 dairy farms in Aomori prefecture, Japan, from January to July in 1989. Listeria sp. was isolated from one sample (5%) of raw milk collected in May, but L. monocytogenes was not isolated from raw milk. L. monocytogenes was isolated from two samples of silage (10%) collected in June. Silage, cow feces, sawdust bedding, dust and soil in these farms were tested for listerias, and we confirmed listerias contamination of the environment in the dairy farms.     

Effect of sewage sludge treatment and additional aerobic post-stabilization revealed by infrared spectroscopy and multivariate data analysis

Sewage sludge samples representing different stages during waste water and sewage sludge treatment were collected at four Austrian municipal waste water treatment plants. Changes of sludge composition are reflected by a specific infrared spectroscopic pattern. Anaerobically digested sludge was subjected to aeration in lab-scale reactors in order to find out if post-aeration after anaerobic digestion provides enhanced organic matter degradation and stabilization. Spectral data were evaluated by means of multivariate statistics. Similar spectral characteristics of sludge degradation stages were visualized by principal component analysis. The effect of additional aerobic treatment of anaerobically stabilized sludge was revealed by discriminant analysis that distinguishes additionally aerated sludge from all the other degradation stages of sludge because of changes in the spectral pattern by increasing stabilization. Based on partial least squares regression (PLSR) a correlation coefficient of R² = 0.91 was found between spectral characteristics and the chemical oxygen demand (COD).     

Comparison of a cultural method with ListerScreen plus Rapid'L.mono or PCR-ELISA methods for the enumeration of L. monocytogenes in naturally contaminated sewage sludge

Cultural methods used to count Listeria monocytogenes in sewage sludge are laborious and time consuming, and alternative methods are needed to reduce analysis time and improve detection limits. In this study, a survey of L. monocytogenes in sewage sludge is presented with a comparative study between a cultural method and immunomagnetic separation using a ListerScreen test followed by identification of L. monocytogenes with Rapid'L.mono agar or PCR-ELISA. These two alternative methods improved the detection of L. monocytogenes in different types of sludge, irrespective of their physical and chemical characteristics. The ListerScreen method coupled with detection of L. monocytogenes on Rapid'L.mono offers the advantage of being less sophisticated than the molecular method and allows isolation of the organism, which may be useful in epidemiological studies. However, ListerScreen coupled with PCR-ELISA proved best for high-sensitivity detection of L. monocytogenes in sewage samples.     

Examination of Bacterial Characteristics of Anaerobic Membrane Bioreactors in Three Pilot-Scale Plants for Treating Low-Strength Wastewater by Application of the Colony-Forming-Curve Analysis Method

Characteristic sludge ecosystems arising in anaerobic membrane bioreactors of three pilot-scale plants treating low-strength (less than 1 g of biological oxygen demand per liter) sewage or soybean-processing wastewater were examined by analysis of the colony-forming-curves (CFC) obtained by counting colonies at suitable intervals. The wastewaters, containing high amounts of suspended solids (SS) (SS/chemical oxygen demand ratio, 0.51 to 0.80), were treated by using two types of bioreactors: (i) a hydrolyzation reactor for solubilization and acidification of SS in wastewater and (ii) a methane fermentation reactor for producing methane. The colony counts for the two sewage treatment plants continued to increase even after 3 weeks of incubation, whereas those for soybean-processing wastewater reached an approximately constant level within 3 weeks of incubation. The CFCs were analyzed by correlating the rate of colony appearance on roll tubes with the physiological types of bacteria present in the bioreactors. It was found that there were large numbers of slow-colony-forming anaerobic bacteria within the bioreactors and that the viable populations consisted of a few groups with different growth rates. It is considered that the slow-growing colonies appearing after 10 days of incubation were the dominant microflora in the sewage treated by hydrolyzation reactors. In particular, highly concentrated sludge (30.0 g of mixed-liquor volatile SS per liter) retained by the membrane separation module contained a large number of such bacteria. Slow-growing colonies of these bacteria could be counted by using a sludge extract medium prepared from only the supernatant of autoclaved sludge. In addition, the highest colony counts were almost always obtained with the sludge extract medium, meaning that most of the anaerobic bacteria in these sludges have complex nutrient requirements for growth. This report also indicates the usefulness of application of the CFC analysis method to the study of bacterial populations of anaerobic treatment systems.