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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Long-term survival of pathogenic and sanitation indicator bacteria in experimental biowaste composts

For economic, agricultural, and environmental reasons, composting is frequently used for organic waste recycling. One approach to limiting the potential risk from bacterial food-borne illnesses is to ensure that soil amendments and organic fertilizers are disinfected. However, more knowledge concerning the microbiological safety of composted substrates other than sludge and manure is necessary. Experimental in-vessel biowaste composts were used to study the survival of seeded Listeria monocytogenes, Salmonella enterica subsp. enterica serotype Enteritidis, and Escherichia coli. Four organic waste mixtures, containing various proportions of paper and cardboard, fruits and vegetables, and green waste, were composted in laboratory reactors with forced aeration. The physicochemical and microbiological parameters were monitored for 12 weeks during composting. The survival of bacteria over a 3-month period at 25 degrees C was assessed with samples collected after different experimental composting times. Strain survival was also monitored in mature sterilized composts. Nonsterile composts did not support pathogen growth, but survival of seeded pathogens was observed. Salmonella serovar Enteritidis survived in all composts, and longer survival (3 months) was observed in mature composts (8 and 12 weeks of composting). Mature biowaste composts may support long-term survival of Salmonella serovar Enteritidis during storage at room temperature. E. coli and L. monocytogenes survival was observed only in 4-week-old composts and never in older composts. Proper composting may prevent long-term survival of E. coli and L. monocytogenes. These results suggest that like composted sewage sludge or manure, domestic waste composts may support pathogen survival. Survival was not related to the physicochemical characteristics of the composts.     

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.     

Decrease of enteric micro-organisms from rural sewage sludge during their composting in straw mixture

AIMS: To study the decrease of enteric micro-organisms including viable nematode eggs, enteroviruses, faecal indicators (Escherichia coli and enterococci) and pathogenic bacteria (Listeria monocytogenes, Salmonella sp. and Clostridium perfringens) of a rural sewage sludge when it is composted for 7 months in mixture with straw. METHODS AND RESULTS: Numbers of the test organisms and the physico-chemical parameters were measured on a monthly basis on the mixture, on the compost after being turned, and on the pile in three positions representing the part by which air is incoming, the bottom of the pile and the part through which air is outgoing. The lowest temperature in the pile was observed at the bottom, where it did not exceed 50 degrees C against 66 degrees C in the two other areas. There were no significant differences between the three areas in terms of micro-organism survival. Infectious enteroviruses were inactivated rapidly and were not found after the first turning whereas some genomes were detected until after the third turning. Escherichia coli and enterococci presented a similar survival rate and their number decreased by 4 log(10) whereas Salmonella decayed at a greater rate than L. monocytogenes. The numbers of C. perfringens decreased gradually to reach a final concentration in the mature compost of about 10(2) CFU g(-1) dry matter (d.m.), which was similar to that of the faecal indicators. CONCLUSIONS: The hygienic effect of sludge composting in mixture with straw results in a significant reduction of enteric micro-organisms, the concentration of the faecal indicators in the final product being < 64 most probable number g(-1) d.m. The concentrations of Salmonella, enteroviruses and viable nematode eggs in the final product were not detectable which is in accordance with the French legislation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results which pointed out the different behaviour of the test micro-organisms reflect the difficulty to propose a relevant indicator of hygienization. Otherwise, they show that composting is an efficient means for hygienization of sludge of rural wastewater treatment, where the straw is available close to their place of production.     

Survival of Escherichia coli and Salmonella spp. after application of sewage sludge to a Pinus radiata forest

AIMS: This study investigated the survival of Escherichia coli and Salmonella spp. in sewage sludge applied to young and old Pinus radiata forest in Spring and Autumn/Winter. METHODS AND RESULTS: Large numbers of E. coli were present in sludge applied to the forest blocks but Salmonella spp. numbers were low or nondetectable. In the mature stand in Spring, numbers of E. coli returned to back-ground after 3 weeks and die-off was significantly correlated with per cent solids of sludge. E. coli survived longer in mature and young stands in Autumn/Winter where numbers did not significantly decrease until weeks 5 and 13, respectively. Salmonella spp. was detectable in the mature stand until week 4 and in the young stand until week 11 in Autumn/Winter. CONCLUSIONS: Microbial die-off was related to desiccation of the sewage sludge, and was faster in warmer, drier conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: In many countries, environmental and health risks associated with the application of sewage sludge to land are minimized by 'best management practice' guidelines, where risks are managed by restriction of public access to these sites. This study provides supporting evidence that withholding periods of greater than 6 months are sufficient to reduce microbial contaminants to background levels.     

Oxygen diffusion and microbial activity in the composting of dehydrated sewage sludge cakes

The effective diffusivity for oxygen within sewage sludge cakes was measured, and in turn used for the study of microbial activity in the sludge cake composting. The diffusivity was found to be proportional to the one and a half power of the sludge cake porosity. Composting experiments were done using two types of sludge cakes, 5 mesh sieved and pelletized. It was found that the microbial activity in the inner core region of the pelletized sludge cake was significantly less than that in the outer region, even through the rate of oxygen diffusion was fast enough to support the microorganisms' activities. It appears, therefore, that microorganisms acting to degrade organic matter grow primarily on the macroscopic interface between solids and air.     

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.     

A comparison of methods used to enumerate Escherichia coli in conventionally treated sewage sludge

AIMS: This study examined the suitability of three analytical methods for isolating and enumerating Escherichia coli from conventionally treated sewage sludge. METHODS AND RESULTS: Crude sewage, mesophilic anaerobic digested (MAD) sludge, and final product sludge samples were taken from six sewage treatment works for analysis. Two of the three methods tested were membrane filtration techniques, utilizing chromogenic E. coli/coliform (CEC) media and membrane-lactose glucuronide agar (MLGA); the third method was a most probable number (MPN) technique utilizing Colilert in Quantitray 2000 (Idexx). The methods were evaluated for variation, consistency, false-positive and false-negative results, as well as method correlation. The methods gave good and consistent recovery of E. coli for a range of conventionally treated sewage matrices. All of the methods had a false-positive rate of <3%, although MLGA had a high false-negative rate (35.5%) compared with Colilert (3.81%) and the CEC method (6.75%). This resulted in slightly lower presumptive counts but comparable numbers of confirmed counts. CONCLUSIONS: The three detection methods tested, chromogenic, MLGA and Colilert gave comparable recoveries, and did not vary by greater than one order of magnitude (1 log). SIGNIFICANCE AND IMPACT OF THE STUDY: Forthcoming revisions to the Use of Sludge in Agriculture Regulations (1989) will categorize sewage sludge as untreated, conventionally treated or enhanced treated in accordance to microbiological standards. The standard will be based upon numbers of E. coli removed through the sludge treatment process and the numbers remaining in the final product. It is recommended that the Colilert 2000 (Idexx, Westbrook, Maine) and CEC methods would be equally suitable to assess the reduction of indigenous E. coli in conventionally treated sludges, and that MLGA be used with follow-up confirmatory testing.     

Comparison of sludge and clinical isolates of Listeria monocytogenes

AIMS: Listeria monocytogenes strains isolated in the same geographical area from sewage sludge and from patients presenting with listeriosis were compared. METHODS AND RESULTS: All isolates were typed by serotyping, phage typing and SmaI/ApaI pulsed-field gel electrophoresis (PFGE). Among the sludge isolates (n=32), 22 subtypes could be distinguished by the combination of all typing methods. The human isolates (n=11) were distributed into 10 subtypes which clearly differed from those observed among sludge isolates, except for one cluster formed by two related human isolates which showed high similarity in PFGE patterns (SmaI: 92%; ApaI: 89.5%) with one sludge isolate. CONCLUSION: These results suggest the existence of an epidemiological link between sludge and human isolates, but they may also be reflecting the distribution of L. monocytogenes types within the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Sludge and human L. monocytogenes may be related but further epidemiological studies are necessary to elucidate this point.     

Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion.

The extent of reduction in selected microorganisms was tested during both aerobic wastewater treatment and anaerobic digestion of sludge at the wastewater treatment plant in Ottawa to compare the removal of two encysted pathogenic protozoa with that of microbial indicators. Samples collected included the raw wastewater, the primary effluent, the treated wastewater, the mixed sludge, the decanted liquor, and the cake. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested. During aerobic wastewater treatment (excluding the anaerobic sludge digestion), Cryptosporidium and Giardia were reduced by 2.96 log10 and 1.40 log10, respectively. Clostridium perfringens spores, Clostridium perfringens total counts, somatic coliphages, and heterotrophic bacteria were reduced by approximately 0.89 log10, 0.96 log10, 1.58 log10, and 2.02 log10, respectively. All of the other microorganisms were reduced by at least 3.53 log10. Sludge samples from the plant were found to contain variable densities of microorganisms. Variability in microbial concentrations was sometimes great between samples, stressing the importance of collecting a large number of samples over a long period of time. In all cases, the bacterial concentrations in the cake (dewatered biosolids) samples were high even if reductions in numbers were observed with some bacteria. During anaerobic sludge digestion, no statistically significant reduction was observed for Clostridium perfringens, Enterococcus sp., Cryptosporidium oocysts, and Giardia cysts. A 1-2 log10 reduction was observed with fecal coliforms and heterotrophic bacteria. However, the method utilized to detect the protozoan parasites does not differentiate between viable and nonviable organisms. On the other hand, total coliforms and somatic coliphages were reduced by 0.35 log10 and 0.09 log10, respectively. These results demonstrate the relative persistence of the protozoa in sewage sludge during wastewater treatment.