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.     

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 and Enterococcus faecium in sludge evaluated by real-time PCR and culture methods

AIMS: This study evaluates the behaviour in spiked sludge of a pathogenic bacteria, Listeria monocytogenes, by cultural and molecular techniques, and compares its survival with the one of a faecal indicator, Enterococcus faecium. METHODS AND RESULTS: Listeria monocytogenes strain Scott A and E. faecium(T) were followed for 17 days after inoculation in sludge. Kinetics of survival depended on the bacteria and on the technique used [most probable number method, direct plate count or real-time quantitative PCR (qPCR)]. The concentration of L. monocytogenes decreased rapidly regardless of the technique, but the decrease was much more dramatic with culture techniques than with qPCR. On the contrary, the concentrations of culturable E. faecium(T) were stable. CONCLUSIONS: The results suggest that the cells of L. monocytogenes strain Scott A might have entered a viable, but nonculturable (VBNC) status, whereas cells of the indicator bacteria, E. faecium(T), maintained themselves better and stayed culturable. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference of survival kinetics in the sludge of a faecal indicator (E. faecium) and a pathogenic bacterium (L. monocytogenes) may be linked to the fact that they either enter or do not enter into a VBNC status.     

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.     

The risk of Listeria monocytogenes infection in beef cattle operations

Aim:  To determine the prevalence of Listeria monocytogenes and associated risk factors among beef operations (cow-calf and feedlot) in central and southern California.
Methods and Results:  A repeated cross-sectional study where faecal and environmental samples were collected from 50 operations three times a year at different seasons was carried out. Samples were tested for presence of L. monocytogenes using a combination of enrichment and polymerase chain reaction tests. Data on putative risk factors were also collected. Listeria monocytogenes was detected in faecal samples from cows, calves and other animals on calf-cow operations at proportions of 3·1%, 3·75% and 2·5%, respectively. The organism was detected in 5·3% of cut-grass, 5·3% of soil, 14·3% of irrigation ditches, 3·1% of the ponds and 6·5% of water troughs samples. Listeria monocytogenes was less common in faecal (0·3%) and soil (0·75%) samples collected from feedlots.
Conclusions:  Listeria monocytogenes was present at a higher proportion among cow-calf operations than feedlots. There was no significant seasonal variation in the occurrence of this pathogen within the two types of operations.
Significance and Impact of the Study:  If risk mitigation strategies were implemented to reduce the public health risk these should focus in cow-calf operations.     

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.     

Behavior of Listeria monocytogenes during fabrication and storage of experimentally contaminated smoked salmon

Experiments were carried out to examine the behavior of Listeria monocytogenes in the course of fabrication and storage of smoked salmon. In three trials, raw salmon fillets were surface inoculated with L. monocytogenes, marinated, smoked at 26 to 30 degrees C, and stored at 4 or 10 degrees C for up to 30 days. At different times during the fabrication and storage, samples were taken and, by means of the three-tube most probable number (MPN) method, quantitatively analyzed for the concentration of L. monocytogenes. The initial Listeria levels in the raw fillets were 10(4) MPN/g in trial 1, 10(1) MPN/g in trial 2, and 10(2) MPN/g in trial 3. During the fabrication, neither an increase nor a decrease of the inoculated quantities was observed. During the storage, however, a significant growth was measured in two of three trials; in trial 1, a 2.5 log10 MPN/g increase and in trial 3, an increase of even 4.5 log10 MPN/g. In the second trial, the Listeria level remained about the same. The results indicate the importance of preventing pre- and postprocessing contamination of L. monocytogenes in raw and smoked salmon. Because a significant increase of L. monocytogenes was measured during storage, there might be an increasing risk of infection for the consumer by storing such fish for a long time.     

Quantification of Listeria monocytogenes in fermented sausages by MPN-PCR method

AIMS: To combine the principles of most-probable-number (MPN) statistics and the conventional PCR technique to enumerate Listeria monocytogenes in fermented sausages. METHODS AND RESULTS: A simple method to enumerate L. monocytogenes in fermented sausages was developed and compared with direct plating in Palcam agar. Species-specific MPN-PCR, but not direct plating, made the enumeration of L. monocytogenes possible in all assayed samples. CONCLUSIONS: MPN-PCR proved to be a rapid and reliable method for enumerating L. monocytogenes in fermented sausages, including low contaminated samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This MPN-PCR technique may facilitate the enumeration of L. monocytogenes for routine analyses in fermented sausages without excessive work.     

Behavior of Listeria monocytogenes during fabrication and storage of experimentally contaminated smoked salmon.

Experiments were carried out to examine the behavior of Listeria monocytogenes in the course of fabrication and storage of smoked salmon. In three trials, raw salmon fillets were surface inoculated with L. monocytogenes, marinated, smoked at 26 to 30 degrees C, and stored at 4 or 10 degrees C for up to 30 days. At different times during the fabrication and storage, samples were taken and, by means of the three-tube most probable number (MPN) method, quantitatively analyzed for the concentration of L. monocytogenes. The initial Listeria levels in the raw fillets were 10(4) MPN/g in trial 1, 10(1) MPN/g in trial 2, and 10(2) MPN/g in trial 3. During the fabrication, neither an increase nor a decrease of the inoculated quantities was observed. During the storage, however, a significant growth was measured in two of three trials; in trial 1, a 2.5 log10 MPN/g increase and in trial 3, an increase of even 4.5 log10 MPN/g. In the second trial, the Listeria level remained about the same. The results indicate the importance of preventing pre- and postprocessing contamination of L. monocytogenes in raw and smoked salmon. Because a significant increase of L. monocytogenes was measured during storage, there might be an increasing risk of infection for the consumer by storing such fish for a long time.     

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.     

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.     

Behaviour of the pathogen surrogates Listeria innocua and Clostridium sporogenes during production of parsley in fields fertilized with contaminated amendments

The survival and transfer of Listeria innocua and Clostridium sporogenes, used as surrogates of the food borne pathogens Listeria monocytogenes and Clostridium botulinum, were quantitatively assessed under field conditions. In the soil, spores of C. sporogenes declined by less than 0.7 log cycles within 16 months and were detected on parsley leaves throughout the experiment. In contrast, L. innocua in the soil declined by 7 log cycles in 90 days and was detected on leaves in low numbers (>0.04 MPN g(-1)) during the first 30 days. Rates of decline in soil were similar in the laboratory at 20 degrees C for two strains of L. innocua and L. monocytogenes ; and in the field for L. innocua over two different years. L. innocua survived better in winter, indicating an important influence of temperature. The major cause of transfer of L. innocua from soil to parsley leaves was splashing due to rain and irrigation. As few as 1 CFU g(-1) Listeria in soil led to contamination of parsley leaves. Internalisation of Listeria through parsley roots was not observed. Under the conditions of soil and climate studied, a delay of 90 days between application of potentially contaminated fertilizer and harvest should be sufficient to eliminate L. monocytogenes.     

Listeria monocytogenes in spontaneous abortions in humans and its detection by multiplex PCR

AIM: To assess the extent of Listeria monocytogenes in causation of human spontaneous abortions by isolation methods and PCR analysis for the presence of virulence-associated genes. METHODS AND RESULTS: A total of 305 samples comprising blood, urine, placental bits, faecal and vaginal swabs were collected from 61 patients with spontaneous abortions. Listeria spp. were isolated from 10 samples collected from nine (14.8%) patients. Confirmation of these isolates was based on biochemical tests, haemolysis on blood agar, CAMP test, phosphatidylinositol-specific phospholipase C (PI-PLC) assay followed by in vivo pathogenicity tests and multiplex PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap). Three isolates were confirmed as L. monocytogenes. Of these, two isolates turned out to be pathogenic and found to posses all five genes. However, the remaining two haemolytic L. monocytogenes isolates lacking the plcA gene and activity in the PI-PLC assay were found to be nonpathogenic by in vivo tests. CONCLUSIONS: The occurrence of pathogenic L. monocytogenes in cases of spontaneous abortions was 3.3%. It seems that the plcA gene and its expression have an important role as essential virulence determinants in pathogenic Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The recovery of pathogenic L. monocytogenes isolates from cases of spontaneous abortion indicates the significance of listeric infection in pregnant women.     

Listeria monocytogenes occurrence and characterization in meat-producing plants

AIMS: The prevalence, level of contamination and epidemiological profile of Listeria monocytogenes were investigated in two meat-producing plants during a 20-month period. METHODS AND RESULTS: Sampling for L. monocytogenes was carried out in a cattle slaughterhouse (n = 72) and a swine meat-processing plant (n = 68) during a 20-month period. Swabs and food samples were analysed with the most probable number (MPN) technique for L. monocytogenes and the isolated strains were characterized by AscI-restriction analysis pulsed-field gel electrophoresis (REA-PFGE). Contamination of meat and meat products was always at low level (below 50 MPN per gram). The seven L. monocytogenes positive samples isolated in the bovine slaughterhouse yielded strains with the same REA-PFGE profile. However, the seven strains isolated in the swine meat processing plant showed six different profiles. Two of them showed indistinguishable profiles with L. monocytogenes strains collected from other meat processing facilities located in the same area. SIGNIFICANCE AND IMPACT OF THE STUDY: The genotyping method is a valuable tool to investigate contamination sources. The study of REA-PFGE profiles indicated that environmental contamination was probably responsible for the persistence of over 16 months of one strain of L. monocytogenes in the cattle slaughterhouse. Several meat suppliers could be responsible for the contamination in the pig meat processing facility, and this is confirmed by the finding of some identical strain in other meat processing facilities located in the same area.     

Behaviour of Listeria monocytogenes in packaged fresh mushrooms (Agaricus bisporus)

AIMS: The aim of this study was to evaluate the potential of Listeria monocytogenes to grow in mushrooms packaged in two different types of PVC films when stored at 4 degrees C and 10 degrees C. METHODS AND RESULTS: Mushrooms were packed in two polymeric films (perforated and nonperforated PVC) and stored at 4 degrees C and 10 degrees C. The carbon dioxide and oxygen content inside the packages, aerobic mesophiles, psychrotrophs, Pseudomonas spp., Listeria monocytogenes, faecal coliforms, Escherichia coli, anaerobic spores and major sensory factors were determined. The mushrooms packaged in nonperforated film and stored at 4 degrees C had the most desirable quality parameters (texture, development stage and absence of moulds). Listeria monocytogenes was able to grow at 4 degrees C and 10 degrees C in inoculated mushrooms packaged in perforated and nonperforated films between 1 and 2 log units during the first 48 h. After 10 d of storage, the populations of L. monocytogenes were higher in mushrooms packaged in nonperforated film and stored at 10 degrees C. CONCLUSIONS: MAP followed by storage at 4 degrees C or 10 degrees C extends the shelf life by maintaining an acceptable appearance, but allows the growth and survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: According to this study additional hurdles must be studied in order to prevent the growth of L. monocytogenes.     

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.