Abstract
Listeria spp. were found in most treated waters (84.4%) and raw sludge (89.2%) of six French urban wastewater treatment plants and one composting facility, examined monthly over a 1-year period. Most strains belonged to Listeria monocytogenes, serotypes 4b/4e being predominant. Sludge composting and liming reduced or prevented Listeria contamination.
In most communities of developed countries, liquid wastes are transformed by wastewater treatment plants in treated waters which are discharged in rivers and in sludge which is disposed of in landfills, incinerated, or, increasingly, recycled as soil amendment (3, 12, 24). Municipal wastewater contains substantial numbers of various microorganisms, including pathogens (22). The numbers and types of pathogens in wastewater treatment plant effluents depend on the initial level of contamination of the influent and on the efficiency of subsequent treatment processes (22). Most microorganisms are eliminated from water through primary (physical) and secondary (biological) treatments, while they are concentrated in primary sludge by settling, and decreased in number by secondary wastewater and sludge treatments (3, 12, 24). At present in France, no microbial controls are mandatory for wastewater treatment plant effluents before their discharge into the environment.
Over the past few years, France has experienced large outbreaks of food-borne listeriosis (7), which have received much attention from the media and put pressure on regulatory agencies to prevent future cases. Discharge of wastewater treatment plant effluents into the environment is likely to enrich the soil-plant primary habitat of Listeria spp. Previous surveys on the occurrence of Listeria spp. in wastewater treatment plant effluents (1, 2, 4-6, 8, 11, 13, 14, 16, 20, 25), most dating before 1990 (1, 2, 8, 13, 14, 20, 25), have generally focused on either treated waters (4, 5, 8, 13, 14) or sludge (6, 11, 20), in one (1, 4-6, 8, 13, 14, 16, 20) to three (2, 11) wastewater treatment plants. The aim of the present study was to investigate, using novel methods, the current occurrence of Listeria spp. in both types of effluents produced by a series of representative French wastewater treatment plants. In addition, the effect of composting on removal of Listeria spp. from sludge was compared to liming.
Sampling campaigns were conducted monthly over a 1-year period (February 2001 to February 2002), in six wastewater treatment plants (A to F) of the Bordeaux area (southwestern France), selected for their various environments and the different treatments they used (Table 1), and a composting facility, which used the aerated pile method, with a rotting phase of 3 weeks and a curing phase in windrows of 4 weeks. Samples, taken at three different points, were collected in sterile boxes of 1 liter and processed within the following 6 hours.
TABLE 1.
Characteristics of the six treatment plants and the composting facility
| Treatment plant or facility | Environment
|
Treatment
|
Sludge disposal | ||
|---|---|---|---|---|---|
| Capacity (no. of inhabitants) | Type of activities | Waters | Sludgea | ||
| Treatment plants | |||||
| A | 15,000 | Urban area, 2 slaughterhouses upstream, 1 bathing place downstream | Activated sludge process with nitrogen removal | Dewatering (belt filter presses) (17% DM) aerobic stabilization | 50% land application after 1 year storage; 50% composting |
| B | 21,000 | Urban area | Activated sludge process | Dewatering (belt filter presses) (13% DM) | 100% composting |
| C | 50,000 | Urban area, truck farming downstream | Activated sludge process | Dewatering (belt filter presses) (23% DM) lime conditioning | 100% incineration |
| D | 14,000 | Urban area | Filter bed | Dewatering (belt filter presses) (13% DM) | 100% composting |
| E | 5,000 | Rural area | Activated sludge process | Grate draining (4.5% DM) | 100% dumping |
| F | 30,000 | Urban area, 1 hospital upstream | Activated sludge process | Dewatering (centrifugation) liming (1.5% DM before treatment, 24% DM after treatment) | 100% land application |
| Composting facility | Composting (45% DM before treatment, 55% DM at half treatment, 60% DM after treatment) | 100% land application, sale as fertilizer | |||
DM, dry matter (mean value of monthly readings within the year).
Listeria spp. were quantified by a three-tube most probable number (MPN) assay (www.jlindquist.net/genera/micro/102dil3.htm) using the enrichment Fraser broth (Bio-Rad) and the selective PALCAM agar (Bio-Rad), found to be more sensitive and reliable than direct enumeration on selective medium (data not shown). Bacterial cells from characteristic colonies grown on PALCAM agar were identified at the genus level by Gram staining and catalase reaction, and further tested for hemolysis and phosphatidylinositol-specific phospholipase C production (ALOA medium, AES). Then, phenotypically identical colonies were pooled, and cells were identified at the species level by restriction fragment length polymorphism (RFLP) of PCR-amplified (PCR-RFLP) 23S rRNA gene fragments (17). Briefly, after amplification of two 23S rDNA fragments, a restriction scheme using two or three enzymes leads to the identification of each Listeria species, while mixtures of species yield complex or not interpretable patterns (17).
After separation and reidentification, isolates were serotyped using all commercially available O and H antisera (Eurobio), according to the Seeliger classification scheme (21). Statistical analyses, performed on SAS Statistical Software version 8.2 (SAS Institute), were based on Fisher exact chi-squared tests (independent samples) and exact McNemar tests (paired samples) for association between qualitative variables, and a Wilcoxon rank sum test for association between quantitative variables.
Listeria spp. were found in most treated water samples (84.4%), at low levels (<0.3 to 2.1 × 101 MPN/ml) (Table 2). In the single survey (4) where Listeria spp. have been researched by a method similar to ours, and where these data are clearly indicated, prevalence (83.3%) and counts (0 to 1.6 × 101 MPN/ml) were within our ranges. In another recent study (5), the prevalence was unknown, but much lower counts were observed (5.5 × 10−1 ± 3.6 × 10−1 MPN/ml). In two earlier studies (1, 2), using the cold-enrichment procedure, similar counts (<3 to 2.8 × 101 or 3.9 × 101 MPN/ml) but higher prevalence rates (100%) were reported. The prevalence of Listeria spp. significantly varied from 61.5% (plant A) to 100% (C and E) in the five sites using the same activated sludge method, and reached 76.9% in the single wastewater treatment plant using the filter bed method (D). Similarly, the quantities of Listeria spp. were statistically lower in the A plant (P = 0.005) and higher in the C plant (P = 0.004) compared to the other sites. Nevertheless, the small numbers of Listeria spp. detected in treated waters, as much as they are going to be further diluted, should not represent a health hazard.
TABLE 2.
Prevalence and amount of Listeria spp. in treated waters according to site and sampling campaign
| Parameter | Treatment plant
|
No. of positive sewage waters/no. tested (% positive) | |||||
|---|---|---|---|---|---|---|---|
| A | B | C | D | E | F | ||
| No. positive samples/no. testeda (% positive) | 8/13 (61.5) | 10/13 (76.9) | 13/13 (100.0) | 10/13 (76.9) | 12/12 (100.0) | 12/13 (92.3) | 65/77 (84.4) |
| No. of MPN/mlb | |||||||
| February 2001 | <0.3 | 0.9 | 0.9 | 2.1 | 0.9 | 0.9 | 5/6 (83.3) |
| March | <0.3 | <0.3 | <0.3 | <0.3 | <0.3 | <0.3 | 6/6 (100.0) |
| April | <0.3 | <0.3 | 2.0 × 101 | 0.4 | ND | 0.4 | 5/5 (100.0) |
| May | <0.3 | <0.3 | <0.3 | 9.0 | 0.4 | <0.3 | 5/6 (83.3) |
| June | <0.3 | <0.3 | 2.3 | <0.3 | <0.3 | 0.9 | 4/6 (66.7) |
| July | <0.3 | <0.3 | <0.3 | <0.3 | <0.3 | <0.3 | 5/6 (83.3) |
| August | <0.3 | <0.3 | 2.3 | <0.3 | <0.3 | 0.9 | 4/6 (66.7) |
| September | <0.3 | 2.3 | <0.3 | <0.3 | <0.3 | <0.3 | 6/6 (100.0) |
| October | <0.3 | 0.9 | <0.3 | <0.3 | <0.3 | <0.3 | 6/6 (100.0) |
| November | <0.3 | <0.3 | 4.0 | <0.3 | <0.3 | <0.3 | 4/6 (66.7) |
| December | <0.3 | <0.3 | 2.3 | <0.3 | <0.3 | <0.3 | 5/6 (83.3) |
| January 2002 | <0.3 | 0.4 | 2.1 × 101 | 0.9 | 0.9 | <0.3 | 5/6 (83.3) |
| February 2002 | <0.3 | <0.3 | <0.3 | 0.9 | <0.3 | <0.3 | 5/6 (83.3) |
Proportions of samples positive for Listeria spp. were significantly different according to the treatment plant (Fisher's chi-square test, P = 0.03) but did not vary significantly according to the calendar period (June, July, and August versus others, exact McNemar test for paired samples, P = 0.26).
Concentrations of Listeria spp. were significantly different according to the treatment plant (P = 0.02), but did not vary significantly according to the calendar period (June, July, and August versus others, Wilcoxon rank sum test, P = 0.16). ND, not determined.
Listeria spp. were found in most raw sludge samples (89.2%), generally at high levels: <7 to ca. 5 × 104 MPN/g of dry matter (Table 3). Comparison to the literature is difficult because most of the studies have examined the effect of the different steps of sludge treatment, and because the denomination of each type of sludge is often ambiguous. In the latest study (14), dewatered sludge, which appears to be similar to our raw sludge, showed an analogous prevalence of Listeria spp. (87%), but considerably lower counts of these organisms (10−1 to 2 × 101 MPN/g of dry matter), maybe related to the use of a modified and highly selective Fraser broth. The levels of Listeria spp. were statistically lower in sludge of the A plant compared to the other sites (P < 0.0001), and the highest mean concentration was found in plant C. These variations could not be ascribed to specific environments as previously suggested (1, 11) since the lowest numbers of Listeria spp. were found in the wastewater treatment plants downstream from two slaughterhouses (A) or a hospital (F), although wastewater from these facilities are known to be enriched in pathogenic microorganisms (19, 23). The role of sludge treatment was no more evident since the lowest concentrations of Listeria spp. were observed in the wastewater treatment plant using the mesophilic aerobic stabilization (A), while the highest ones were detected in a plant (C) performing the theoretically more efficient lime stabilization. Our data highlight the need to standardize treatments in French wastewater treatment plants. Significantly lower prevalence and densities of Listeria spp. were observed in raw sludge in Summer in contradiction with previous studies, possibly due to insufficient monthly samplings and/or lack of adequate statistical tests (2, 6, 11, 16).
TABLE 3.
Prevalence and amount of Listeria spp. in raw and hygienized sludge according to site and sampling campaign
| Parameter | Raw sludge
|
Limited sludge (F) | Composted sludge (composting facility)
|
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | C | D | E | F | Composting facility | No. positive/no. tested (% positive) | Half composted | Final product | ||
| No. of positive samples/no. testeda (% positive) | 9/12 (75.0) | 13/13 (100.0) | 10/11 (90.9) | 12/13 (92.3) | 12/12 (100.0) | 9/10 (90.0) | 9/12 (75.0) | 74/83 (89.2) | 0/12 (0) | 2/6 (33.3) | 1/9 (11.1) |
| No. of MPN/g DMb | |||||||||||
| February 2001 | 1.4 × 102 | 1.5 × 103 | 4.8 × 103 | 1.5 × 103 | 1.1 × 104 | ND | 4.4 × 102 | 6/6 (100.0) | <1.3 × 101 | ND | <5.0 |
| March | 2.4 × 102 | 3.1 × 102 | 4.8 × 104 | 6.9 × 102 | 1.6 × 104 | ND | 4.4 × 101 | 6/6 (100.0) | <1.3 × 101 | ND | <5.0 |
| April | 2.4 × 102 | 6.9 × 102 | 3.0 × 104 | 1.2 × 103 | ND | ND | 2.0 × 103 | 5/5 (100.0) | <1.3 × 101 | ND | <5.0 |
| May | 1.4 × 102 | 6.9 × 102 | 1.7 × 103 | 1.5 × 103 | 5.1 × 103 | 1.5 × 103 | 4.4 × 103 (α) | 7/7 (100.0) | <1.3 × 101 | ND | <5.0 |
| June | <1.8 × 101 | 3.1 × 102 | 3.9 × 103 | 1.5 × 103 | 5.1 × 102 | <2.0 × 101 | 6.4 × 102 | 5/7 (71.4) | <1.3 × 101 | <6.0 (α) | ND |
| July | <1.8 × 101 | 1.8 × 102 | ND | <2.3 × 101 | 8.9 × 102 | 1.5 × 102 | <7.0 (β) | 3/6 (50.0) | ND | ND | <5.0 |
| August | 5.2 × 101 | 6.9 × 103 | <1.3 × 101 | 1.5 × 103 | 8.8 × 101 | 2.7 × 102 | <7.0 | 5/7 (71.4) | <1.3 × 101 | <6.0 (β) | <5.0 |
| September | 1.4 × 102 | 1.2 × 102 | 5.2 × 102 | 1.8 × 102 | 5.1 × 102 | 7.3 × 101 | 2.0 × 102 (γ) | 7/7 (100.0) | <1.3 × 101 | ND | <5.0 (β) |
| October | 1.4 × 102 | 6.9 × 102 | 1.7 × 102 | 3.1 × 103 | 5.1 × 102 | 6.0 × 102 | 1.3 × 102 | 7/7 (100.0) | <1.3 × 101 | <6.0 (γ) | ND |
| November | <1.8 × 101 | 1.8 × 102 | 6.5 × 102 | 1.5 × 104 | 4.4 × 103 | 6.0 × 102 | <7.0 | 5/7 (71.4) | <1.3 × 101 | ND | <5.0 (γ) |
| December | 1.4 × 102 | 1.2 × 103 | ND | 1.5 × 103 | 8.8 × 101 | 6.0 × 103 | 1.1 × 103 | 6/6 (100.0) | <1.3 × 101 | 2.0 × 101 | ND |
| January | ND | 1.5 × 104 | 8.7 × 102 | 6.9 × 102 | 5.1 × 102 | 1.0 × 103 | >2.4 × 104 (δ) | 6/6 (100.0) | <1.3 × 101 | <6.0 | ND |
| February 2002 | 1.7 × 102 | 2.2 × 103 | 6.5 × 101 | 3.1 × 102 | 3.3 × 103 | 1.3 × 103 | ND | 6/6 (100.0) | <1.3 × 101 | <6.0 | <5.0 (δ) |
Proportions of raw sludge samples positive for Listeria spp. were not significantly different according to the treatment plant (Fisher's chi-square test, P = 0.11), but significantly varied according to the calendar period (June, July, and August versus others, exact McNemar test for paired samples, P < 0.0001); proportions of samples positive for Listeria spp were significantly different between raw sludge and composted sludge as well as between raw sludge and limed sludge (Fisher's chi-square test, P < 0.0001).
Concentrations of Listeria spp. in raw sludge were significantly different according to the treatment plant (Wilcoxon rank sums test, P = 0.003) and to the calendar period (June, July, and August versus others, Wilcoxon rank sum test, P = 0.01). Concentrations of Listeria spp. were significantly different between raw and composted sludge as well as between raw and limed sludge (Wilcoxon rank sum test, P = 0.049 and P = 0.045, respectively).
Of the 187 samples analyzed, 142 (75.9%) contained Listeria spp., including 105 (73.9%) that provided a single strain and 37 that were mixtures of two (33 samples, 23.2%) or more strains (Table 4). Among the 181 nonredundant strains, the most pathogenic species, L. monocytogenes, predominated (59.1% of the strains, including seven non hemolytic and phosphatidylinositol-specific phospholipase C negative, 71.8% of the samples), followed by L. innocua (46.5 and 37.6%, respectively), both species often coexisting (20.4% of the samples). In earlier studies (1, 2, 8, 25), only L. monocytogenes, identified by unknown methods (8, 25) or conventional tests including hemolysis (1, 2), was looked for. When Listeria species were differentiated by similar tests, the API Listeria kit or a heteroduplex mobility assay which does not detect mixtures of species, L. monocytogenes was generally found to be predominant, followed by L. innocua (4, 5, 10, 11, 13, 14, 20). However, the reverse was observed in one survey (6), and L. seeligeri (5), L. ivanovii (4), or the rare L. grayi (6) was occasionally reported in high proportions. Most L. monocytogenes strains belonged to the 4b/4e (49.5%) and 1/2a and 1/2b (33.6%) serovars (Table 5), in accordance with the only two studies where sludge strains were fully serotyped, which were performed in France (11, 20). Serotypes 4b, 1/2a, and 1/2b are those essentially involved in human and animal listeriosis (9, 15, 18). In particular, the two consecutive nationwide outbreaks of listeriosis in France in 1999 to 2000 were caused by the 4b serotype (7).
TABLE 4.
Distribution of Listeria species according to sample
| Samples (no. positive) | No. of samples with Listeria sp.
|
No. of strains | ||||
|---|---|---|---|---|---|---|
| L. monocytogenes | L. innocua | L. seeligeri | L. welshimeri | Listeria sp. | ||
| Treated waters (65)a | 49 | 40 | 1 | 1 | 91 | |
| Raw sludge (74)b | 56 | 27 | 2 | 1 | 1 | 87 |
| Half-composted sludge (2) | 1 | 1 | 2 | |||
| Composted sludge (1) | 1 | 1 | ||||
| Total (142) | 107 | 68 | 3 | 1 | 2 | 181 |
41 samples with one strain and 23 samples with two strains (20 with L. monocytogenes plus L. innocua, 1 with L. innocua plus L. seeligeri, 1 with two L. innocua strains, and 1 with two L. monocytogenes). One sample had four strains (two L. monocytogenes plus two L. innocua).
62 samples with one strain and 11 samples with two strains (1 with two L. monocytogenes strains, 8 with L. monocytogenes plus L. innocua, 1 with L. monocytogenes plus L. welshimeri, and 1 with L. monocytogenes plus L. seeligeri). One sample had three strains (L. monocytogenes).
TABLE 5.
Distribution of serotypes according to Listeria species
| Listeria species (no. of strains) | No. of strains with indicated serotype(s)
|
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1/2a | 1/2b | 1/2c | 3a | 3b | 4a | 4e | 4b/4e | 4b/4e/6a | 6a | 6b | 6a/6b | NTa | |
| L. monocytogenes (107) | 18 | 18 | 1 | 2 | 3 | 2 | 4 | 53 | 6 | ||||
| L. innocua (68) | 41 | 8 | 17 | 2 | |||||||||
| L. seeligeri (3) | 2 | 1 | |||||||||||
| L. welshimeri (1) | 1 | ||||||||||||
| Listeria sp. (2) | 2 | ||||||||||||
| Total (181) | 18 | 20 | 1 | 2 | 3 | 2 | 4 | 54 | 2 | 41 | 9 | 17 | 8 |
NT, not typeable.
Composting significantly reduced both prevalence and numbers of Listeria spp. present in 75% of the mixtures of sludge and vegetable wastes prepared in the composting facility, at <7 to >2 × 104 MPN/g of dry matter (Table 3). This was particularly evident for the four batches followed throughout the different steps of the composting process (Table 3). However, two of the six half-composted samples (end of the intensive rotting phase) and one of the nine final products (end of the curing phase) contained low levels (<6 to 2 × 101 and <5 MPN/g of dry matter, respectively) of Listeria spp., after procedure incidents (Table 3). The efficiency of sludge composting on Listeria removal, based on a limited sample size, needs to be further confirmed and optimized. Composting mainly inactivates pathogenic microorganisms by self-heating during the intensive rotting phase, and the control of many conditions is critical for achieving optimal decontamination (3, 12). However, this process changes organic wastes into a valuable peat-like product that can serve as soil conditioner for many horticultural, landscape and nursery uses (24). Liming completely eliminated Listeria spp. present in 90% of the raw sludge obtained in the F site, at <2 × 101 to 6 × 103 MPN/g of dry matter (Table 3). Liming, which leads to a considerable increase in pH and temperature, regularly provides total Listeria destruction (11), but limed sludge can only be used as agricultural limestone for soil pH control and artificial soil for daily landfill cover (12, 22, 24). The number of Listeria spp. in sludge did not correlate with those of bacterial indicators of fecal contamination (data not shown), in agreement with a previous survey (11), but in disagreement with two others (4, 6). Thus, specific detection and enumeration of Listeria spp. are required for controls.
In conclusion, Listeria spp. were generally present in wastewater treatment plant effluents and at high levels in raw sludge. Since the most clinically important L. monocytogenes species and 4b/4e, 1/2a, and 1/2b serotypes predominated, sludge hygienization, by composting under strictly supervised conditions or liming, should be useful before land application.
Acknowledgments
We are grateful to Isabelle Déportes from the Agence de l'Environnement et de la Maîtrise de l'Energie (ADEME) and Gérard Deviers from the Direction Départementale de l'Action Sanitaire et Sociale (DDASS) for helpful discussions and comments.
This work was funded by research grants from the ADEME from the Ministère de l'Education Nationale et de la Recherche (EA 525), the DDASS, the Conseil Regional d’Aquitaine, and the Fond Européen de Développement Régional. D.P. was a recipient of a scholarship from the ADEME.
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