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.

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.

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.

Study of Listeria monocytogenes survival during the preparation and the conservation of two kinds of dairy product.

We tested yoghurts and soft cheeses for survival of Listeria monocytogenes during their manufacture and their storage at 4 degrees C. In yoghurt, even when the concentration of germs is high, the bacterial population decreased rapidly and the life of time of L. monocytogenes in this product depends on the sample acidity. The microorganisms disappear when the pH falls to 3.5. In soft white cheeses, only a fabrication with chemical and bacterial ferments allows an acidity which is compatible with destruction of majority of L. monocytogenes. Under these conditions, the pH decreases to 4-4.5 according to the series of production.

Persistence of Listeria monocytogenes in three sorts of soil.

Life time of Listeria monocytogenes (strain PS 10401, serovar 4b) was studied in three sorts of soil: a chalky soil, poor in organic matters (pH 8.3) a peaty soil rich in organic matters (pH 5.5) a mixture of a chalky and peaty soil (pH 7.9). About 10(5) colony forming units (c.f.u.) per g of dry material were inoculated to each sort of soil which was incubated at 4 degrees C or at 20 degrees C. Periodically samples were taken and a numeration of germs was made on Tryptose Soy Agar. When direct numeration was negative, an enrichment was made at low temperature. In peaty soil, L. monocytogenes disappeared after incubation of 162 days at 4 degrees C and 156 days at 20 degrees C. In chalky soil, L. monocytogenes disappeared after 394 days of incubation at 20 degrees C, whereas at 4 degrees C, 10(4) c.f.u./g of dry matter were found 1500 days later. In mixture of chalky and peaty soil, 10(4) c.f.u. were found 1500 days after inoculation at 4 degrees C whereas 10(3) c.f.u. were found at 20 degrees C. Acid soils do not allow the persistence of L. monocytogenes for more than 160 days. Low temperature are significantly favourable to the persistence of L. monocytogenes.

Preservation of the virulence of Listeria monocytogenes in different sorts of soil.

Listeria monocytogenes (strain PS 10401, serovar 4b) whose virulence was well known, has been placed for 600 days in a sterile chalky soil (pH 8.3) and in a mixture of chalky and peaty soil (pH 7.9) and incubated at 4 degrees C and 20 degrees C. Groups of six OF1 mice were challenged by subcutaneous injection in the rear left hind foodpad with each sort of germs. Three days after inoculation, mice were killed by cervical disruption and numeration of L. monocytogenes was made in the spleen. Chemical composition of soil does not change virulence of germs. But low temperatures significantly increase virulence.