Abstract

The aim of the present study was to assess performance of waste stabilization ponds (WSPs) on the removal of Listeria spp. in Isfahan, Iran. A total of 104 samples were taken from eight sampling locations from influent and effluent of a wastewater treatment plant (WWTP). Sewage samples were analyzed for the presence of Listeria spp. using selective enrichment protocol. Listeria isolates were also identified by biochemical and polymerase chain reaction (PCR) amplification. Listeria spp. was enumerated by a three tube most probable number (MPN) for total coliform counts (TC), fecal coliform counts (FC), total suspended solids (TSS), and total dissolved solids (TDS). In total, 54/104 (51.92%), 49/104 (47.11%), 36/104 (34.61%), and 27/104 (25.95%) samples were positive for Listeria spp., L. monocytogenes, L. innocua, and L. seeligeri, respectively. The mean MPN/100 mL enumeration of L. monocytogenes for influent, anaerobic, facultative ponds 1, 2, 3, 4 and maturation ponds 1 and 2 were 21.54, 10.61, 8, 5.77, 4, 2.54, 1.38, and 0.46, respectively. The removal percentage of Listeria spp. in the anaerobic, facultative, and maturation ponds were 44.71, 76.5, and 81.4%, respectively. Results showed that the WSPs were able to decrease the Listeria spp. levels significantly, although unable to remove them completely.

INTRODUCTION

Waste stabilization ponds (WSPs) are widely used for domestic and municipal wastewater treatment, especially in developing countries, where the climate is most favorable for the operation of these facilities (Abdel-Aatty & Karnel 2008). WSPs are low-cost, highly efficient, and simple in construction, operation, and maintenance (Peña Varón & Mara 2004; Yi et al. 2009). Additionally, they are usually designed as a sequence of anaerobic, facultative, and maturation pond systems (Babu et al. 2010). The ponds consist of complex communities of bacteria, algae, viruses, protozoa, rotifers, insects, crustaceans, and fungi (Kehl et al. 2009). Furthermore, the use of WSPs is widespread because of the high biochemical oxygen demand (BOD) and high pathogenic micro-organism removal capacity. Moreover, the effluent and sludge of WSPs are valuable and can be safely reused in agricultural areas. Therefore, the removal efficiency of pathogens in WSPs is an important line of inquiry in order to avoid health risk problems (Kehl et al. 2009; Kerstens et al. 2009).

Listeria monocytogenes is a Gram-positive, intracellular (Dongyou 2008; Taherkhani et al. 2013), psychotropic (Vaid et al. 2010), facultative anaerobic, and non-spore forming bacterium (Jadhav et al. 2012). It is an important food-borne pathogen which may exist in various types of raw or cooked food stuff such as milk and dairy products, fish and seafood, meat and meat products, and vegetables and agricultural products (Jadhav et al. 2012; Szlavik et al. 2012). This bacterium can cause listeriosis (Ramaswamy et al. 2007), with a case-fatality rate of about 30% (Moreno et al. 2011). It has severe clinical consequences (e.g., meningitis, encephalitis, septicemia) (Dongyou 2008), especially in high-risk persons such as the elderly, pregnant women, newborns, and immunocompromised individuals (McCarthy & Burkhardt 2012; Prencipe et al. 2012). L. monocytogenes is commonly distributed in a diversity of environments and can be found in wastewater at high levels (Moreno et al. 2011). This bacterium has also been isolated from soil, surface water, sewage, vegetation, fecal matter, animal feed, agricultural ecosystems, and domestic environments (Vaid et al. 2010).

Prevention of L. monocytogenes contamination is particularly difficult, since the pathogen is ubiquitous in the environment, and capable of growth in diverse conditions including temperatures between 1 and 45 °C, pH 4–9, with high concentration of sodium chloride and reduced water activity (aw 0.90–0.92; 11.5% NaCl) (Vaid et al. 2010; Karina et al. 2011; Prencipe et al. 2012).

L. monocytogenes tends to survive in wastewater and municipal sewage sludge; therefore, it poses a significant health risk to humans via sludge applications or by irrigating crops with treated wastewater on agricultural land (Garrec et al. 2003a, 2003b; Moreno et al. 2011). The prevalence and contamination of Listeria spp. in various foods including meat and meat products, vegetables, ready-to-eat foods (Jalali & Abedi 2008), raw milk and dairy products (Mahmoodi 2010; Rahimi et al. 2010), fresh and frozen fish and shrimp (Rahimi et al. 2012) have been reported in Iran. However, few, if any, studies have been conducted on the prevalence of Listeria spp. in wastewater treatment plants (WWTPs) or their effluents in the region. Therefore, the objective of this study was to assess the performance of the WSPs in removal of Listeria spp.

MATERIALS AND METHODS

Description of the sampling site

The WSPs used for municipal wastewater treatment are located in Isfahan, Iran. The domestic wastewater flow and the population equivalent of the WSPs were about 570 m3/h and 72,000 persons, respectively. Table 1 shows the operating conditions of the WSPs.

Table 1

Operating conditions of the WSP

Pond Surface area (m2Length (m) Width (m) Volume (m3Depth (m) HRT (d) 
Anaerobic pond (1–8) 1,500 50 30 4,500 2.7 
Facultative pond (1–4) 10,000 100 100 28,000 2.8 5.5 
Facultative pond (5–8) 15,000 150 100 42,000 2.8 4.9 
Maturation pond (1–2) 11,250 150 75 21,375 1.9 1.6 
Pond Surface area (m2Length (m) Width (m) Volume (m3Depth (m) HRT (d) 
Anaerobic pond (1–8) 1,500 50 30 4,500 2.7 
Facultative pond (1–4) 10,000 100 100 28,000 2.8 5.5 
Facultative pond (5–8) 15,000 150 100 42,000 2.8 4.9 
Maturation pond (1–2) 11,250 150 75 21,375 1.9 1.6 

The WSPs consist of 18 ponds which include eight anaerobic, eight facultative, and two maturation ponds. The facultative and maturation ponds are in series. Figure 1 shows the WSP and sampling sites. In this study, samples were taken from eight points during different WSP stages. These points include the influent of the WSPs (1 point), the effluent of the anaerobic ponds (1 point), the effluent of the facultative ponds (4 points), and the effluent of the maturation ponds (2 points). A total of 13 samples were collected from each point. Therefore, the total number of samples analyzed in this study was 104. Samples were tested without any pre-treatment, and all the conditions were repeated at the same condition as before.

Figure 1

Diagram of WSP and sampling sites. A, anaerobic pond; F, facultative pond; M, maturation pond; SP, sampling point.

Figure 1

Diagram of WSP and sampling sites. A, anaerobic pond; F, facultative pond; M, maturation pond; SP, sampling point.

Wastewater sampling

A total of 104 samples were taken biweekly over four months from October to February, from eight discrete sampling locations from the influent and effluent of the WSPs (Figure 1). The reason for selecting these months for sampling was due to the fact that temperature can significantly affect the efficiency of the stabilization ponds. Therefore, sampling was carried out during the cold months to evaluate the efficiency of the stabilization ponds in the worst weather conditions. The samples were transferred in portable insulated cool boxes to the laboratory an hour approximately after sampling and were analyzed immediately after transporting to the laboratory.

Microbial analysis

Wastewater samples were analyzed for the presence of Listeria spp. using selective enrichment and isolation protocol recommended by the United States Department of Agriculture (USDA) (McClain & Lee 1988). Primary enrichment was done with Listeria enrichment broth medium (UVMΙ, Merck, Germany). Ten mL of wastewater was aseptically transferred into 100 mL of UVMΙ then incubated for 24 h at 30 °C. Then, 100 μL of primary enrichment broth was transferred into 9.9 mL of Fraser broth (Merck) and incubated for 24–48 h at 35 °C. Fifty μL of secondary enrichment was streaked on to polymyxin-acriflavin-lithium-chloride-ceftazidime-aesculin-mannitol (PALCAM) agar (Merck) supplemented with selective supplement (HC784958 Merck) and incubated for 24–48 h at 37 °C.

The plates were examined for typical Listeria colonies (black colonies with black sunken) and at least 3–5 typical colonies were sub-cultured on Tryptone Soy Agar supplemented with 0.6% of yeast extract (TSAYE) and incubated at 37 °C for 24 h. All of the isolates were subjected to Gram staining catalase and motility test at 25 and 37 °C, acid production from glucose, manitol, rhamnose, xylose, α-methyl-d-mamoside, nitrate reduction, hydrolysis of esculin, and MR/VP test. For further confirmations of Listeria spp., all isolates were subjected to other biochemical reactions, β-hemolytic activity, and CAMP test (Moreno et al. 2011). L. monocytogenes isolates were further confirmed by polymerase chain reaction (PCR) amplification.

Listeria spp. were also enumerated by a three tube most probable number (MPN) in Fraser broth. Samples of influent and final effluent were also analyzed for total coliform counts (TC), fecal coliform counts (FC), total suspended solids (TSS), and total dissolved solids (TDS). Analyses of these parameters were conducted according to the Standard Methods for the Examination of Water and Wastewater (APHA) (Eaton & Franson 2005).

PCR confirmation

Isolates of L. monocytogenes were analyzed for the presence of the hlyA gene that encodes listeriolysin O. Bacterial strains were cultured in BHI broth at 37 °C for 18 h, and genomic DNA of bacterial strains were extracted as described previously (Fitter et al. 1992). Primer 234 (5′-CATCGACGG CAACCTCGGAGA-3′) and primer 319 (5′-ATCAATTACCGTTCTCCACCATT-3′) were selected as published by Fitter et al. (1992). These primers allowed amplification of a 417 bp internal fragment of the hlyA gene (Mengaud et al. 1988). PCR was achieved as described by Fitter et al. (1992), and 15 μL of the PCR amplified reaction mixtures were subjected to horizontal gel electrophoresis in 1.8% agarose gels run in 1 × Tris-borate (TBE). PCR products were stained with a 1% (1 μg/mL) solution of ethidium bromide, and tested under UV illumination. DNA extracted from IRTCC1293, L. monocytogenes 4a, and deionized water were used as the positive and negative controls, respectively.

Statistical analysis

Paired t-test and test of significance (one-way ANOVA) were performed using SPSS 17.0. All of the significances and correlations were considered statistically significant at P values of <0.05 or <0.01. Also paired t-test was performed to calculate the removal efficiency of the WSPs.

RESULTS

One hundred and four samples from eight sampling sites (13 samples from each site) were collected over a four-month period from October to February. Table 2 shows the number and percentage of Listeria-positive samples. Totally, 54 out of 104 (51.92%), 49 out of 104 (47.11%), 36 out of 104 (34.61%), and 27 out of 104 (25.95%) samples were positive for Listeria spp. L. monocytogenes, L. innocua, and L. seeligeri, respectively. The most common Listeria spp. isolated from WSP influent was L. monocytogenes (84.61%) followed by L. seeligeri (53.84%) and L. innocua (46.15%).

Table 2

The prevalence of positive samples (percentages) of Listeria spp. in wastewater samples collected at various sites

Sampling sites Total Listeria spp. (%) L. monocytogenes (%) L. innocua (%) L. seeligeri (%) 
Influent 13 12 (92.3) 11 (84.61) 6 (46.15) 7 (53.84) 
Anaerobic pond 13 8 (61.54) 8 (61.53) 6 (46.15) 4 (30.76) 
Facultative pond 1 13 8 (61.54) 8 (61.53) 6 (46.15) 4 (30.76) 
Facultative pond 2 13 7 (53.84) 6 (46.15) 6 (46.15) 4 (30.76) 
Facultative pond 3 13 7 (53.84) 6 (46.15) 5 (38.46) 3 (23.07) 
Facultative pond 4 13 7 (53.84) 5 (38.46) 4 (30.77) 3 (23.07) 
Maturation pond 1 13 3 (23.077) 3 (23.077) 2 (15.38) 1 (7.69) 
Maturation pond 2 13 2 (15.38) 2 (15.38) 1 (7.69) 1 (7.69) 
Total 104 54 (51.92) 49 (47.11) 36 (34.61) 27 (25.95) 
Sampling sites Total Listeria spp. (%) L. monocytogenes (%) L. innocua (%) L. seeligeri (%) 
Influent 13 12 (92.3) 11 (84.61) 6 (46.15) 7 (53.84) 
Anaerobic pond 13 8 (61.54) 8 (61.53) 6 (46.15) 4 (30.76) 
Facultative pond 1 13 8 (61.54) 8 (61.53) 6 (46.15) 4 (30.76) 
Facultative pond 2 13 7 (53.84) 6 (46.15) 6 (46.15) 4 (30.76) 
Facultative pond 3 13 7 (53.84) 6 (46.15) 5 (38.46) 3 (23.07) 
Facultative pond 4 13 7 (53.84) 5 (38.46) 4 (30.77) 3 (23.07) 
Maturation pond 1 13 3 (23.077) 3 (23.077) 2 (15.38) 1 (7.69) 
Maturation pond 2 13 2 (15.38) 2 (15.38) 1 (7.69) 1 (7.69) 
Total 104 54 (51.92) 49 (47.11) 36 (34.61) 27 (25.95) 

Figure 2 shows the mean bacteria quantity (MPN/100 mL) of different Listeria spp. for each sampling site. Thus, mean bacteria quantity (MPN/100 mL) of Listeria spp. was 25.46 for influent wastewater and 0.46 for maturation effluent at pond 2. The mean MPN/100 mL enumeration of L. monocytogenes in influent, anaerobic, facultative effluent ponds 1, 2, 3, 4, and maturation effluent ponds 1 and 2 were 21.54, 10.61, 8, 5.77, 4, 2.54, 1.38, and 0.46, respectively (Figure 2). The mean bacteria quantity (MPN/100 mL) of L. seeligeri and L. innocua of influent wastewater were 7 and 8.69, respectively. These figures for maturation effluent at pond 2 for both species were 0.23 (Figure 2). The TS, TSS, and TDS in influent and effluent of WSP are shown in Table 3. Table 4 shows the removal performance of Listeria spp. in each step of the WSPs. Meanwhile, removal efficiency (percentage) of Listeria spp., TC, FC, and TSS are indicated in Table 4. All of the isolated L. monocytogenes were also confirmed by PCR.

Figure 2

Mean bacteria quantity (MPN/100 mL) of Listeria spp. from eight sampling sites. 1: influent, 2: anaerobic pond, 3: facultative pond 1, 4: facultative pond 2, 5: facultative pond 3, 6: facultative pond 4, 7: maturation pond 1, 8: maturation pond 2.

Figure 2

Mean bacteria quantity (MPN/100 mL) of Listeria spp. from eight sampling sites. 1: influent, 2: anaerobic pond, 3: facultative pond 1, 4: facultative pond 2, 5: facultative pond 3, 6: facultative pond 4, 7: maturation pond 1, 8: maturation pond 2.

Table 3

Mean MPN/100 mL of TC and FC and average of TS, TSS, and TDS (mg/L) in influent and effluent of the WSP

Sampling sites Influent
 
Effluent
 
Mean SD Mean SD 
TC (MPN/100 mL) 1.6 × 108 284 × 106 6.54 × 103 3,478.87 
FC (MPN/100 mL) 4.39 × 107 32,633,275 4.61 × 103 1,980 
TS (mg/L) 537 132 521 42 
TSS (mg/L) 431 102 235 89 
TDS (mg/L) 764 208 762 98 
Sampling sites Influent
 
Effluent
 
Mean SD Mean SD 
TC (MPN/100 mL) 1.6 × 108 284 × 106 6.54 × 103 3,478.87 
FC (MPN/100 mL) 4.39 × 107 32,633,275 4.61 × 103 1,980 
TS (mg/L) 537 132 521 42 
TSS (mg/L) 431 102 235 89 
TDS (mg/L) 764 208 762 98 
Table 4

Removal efficiency (percentage) of Listeria spp., TC, FC, and TSS in the WSP

Listeria species Anaerobic pond Facultative pond 1 Facultative pond 2 Facultative pond 3 Facultative pond 4 Maturation pond 1 Maturation pond 2 Overall removal efficiency 
Listeria spp. 44.7 25.1 29.9 32.2 33.8 55.8 68.4 98.1 
L. monocytogenes 50.7 24.6 27.8 30.6 36.5 45.4 66.6 97.8 
L. innocua 36.2 26.3 24.5 30 39.2 58.8 57.1 97.3 
L. seeligeri 57.1 28.2 14.2 25 22.2 57.1 50 96.7 
Total coliform – – – – – – – 99.9 
Fecal coliform – – – – – – – 99.9 
TSS – – – – – – – 45.4 
Listeria species Anaerobic pond Facultative pond 1 Facultative pond 2 Facultative pond 3 Facultative pond 4 Maturation pond 1 Maturation pond 2 Overall removal efficiency 
Listeria spp. 44.7 25.1 29.9 32.2 33.8 55.8 68.4 98.1 
L. monocytogenes 50.7 24.6 27.8 30.6 36.5 45.4 66.6 97.8 
L. innocua 36.2 26.3 24.5 30 39.2 58.8 57.1 97.3 
L. seeligeri 57.1 28.2 14.2 25 22.2 57.1 50 96.7 
Total coliform – – – – – – – 99.9 
Fecal coliform – – – – – – – 99.9 
TSS – – – – – – – 45.4 

DISCUSSION

The performance of WSPs was investigated for Listeria spp. removal during a four-month period. A total 104 samples were taken from different units of the WSPs. The prevalence of Listeria spp. was up to 51.92% (Table 2). In a study in Spain, L. monocytogenes, L. innocua, and L. seeligeri were isolated from 53, 12, and 13% of river samples, respectively (Combarro et al. 1997). Paillard et al. (2005) showed that in six French urban activated sludge WWTPs and one composting facility in the treated water and raw sludge, the isolation rates of Listeria spp. were 84.4 and 89.2%, respectively. They also showed that most of the species were L. monocytogenes and concluded that the application of sludge in composting under certain conditions and liming could reduce or prevent Listeria contamination (Paillard et al. 2005). In another study in the Netherlands (Czeszejko et al. 2003), conducted on untreated wastewater, the L. monocytogenes isolation rate was 90% and both L. seeligeri and L. grayi were 5%. In the Czeszejko et al. (2003) study, similar to our findings, L. monocytogenes was the most common species in the influent and effluent wastewater. In a similar study in Italy, the prevalence of Listeria species in various types of water including river, brackish water, and urban wastewater, 72.4% of L. monocytogenes was reported (Bernagozzi et al. 1994). Therefore, we can conclude that L. monocytogenes is the most common species isolated from wastewater (Bernagozzi et al. 1994; Czeszejko et al. 2003; Paillard et al. 2005). In another study in Egypt, isolation of Listeria in influent wastewater, anaerobic effluent, facultative and maturation ponds was reported to be 1.3 × 104, 4.9 × 103, 7.6 × 102, and 1.1 × 102 CFU/100 mL, respectively (Abdel-Aatty & Karnel 2008). Bernagozzi et al. (1994) showed that 74.4% of their samples contained Listeria spp. at concentrations of 2 and 1,320 MPN/100 mL in fresh surface water and untreated sewage, respectively. Our results show the reduction trend for L. innocua and L. seeligeri during the WSP process (Figure 2).

These results show that WSPs are more efficient than the activated sludge for removal of L. monocytogenes. Al-ghazali & Al-Azawi (1986) reported removal efficiency of Listeria spp. of about 85–99.7% in an activated sludge WWTP during cold seasons (Al-ghazali & Al-azawi 1986). Combarro et al. (1997) showed 92% removal efficiency for L. monocytogenes in extended aeration activated sludge. Additionally, Garrec et al. (2003a) isolated 95% of L. monocytogenes from activated sludge, and 73% and 80% from dewatered and stored sludge, respectively. In another study in Iran, L. monocytogenes was isolated from 69.6% of an activated sludge (Navidjouy et al. 2013). In this study, Listeria spp. was found in 96–100% (mean 98.18%). L. monocytogenes was isolated from 93.2–100% (mean 97.85%) during the WSP process. Another study shows that combined mechanical and biological treatment of domestic and industrial sewage, including that of poultry processing industry does not eliminate L. monocytogenes (Czeszejko et al. 2003).

Abdel-Aatty & Karnel (2008) reported 99.9998 and 99.9976% removal for TC and FC, respectively. No significant relation was observed on removal of Listeria and TC/FC (P value >0.05). Therefore, it seems that both TC and FC are not appropriate indicators for Listeria spp. Other studies confirm that there is no significant relation between Listeria spp. removal and fecal indicators, other water quality parameters such as pH, seasonal variation, BOD5, organic loading rate (Al-ghazali & Al-azawi 1986; Combarro et al. 1997; Czeszejko et al. 2003). Based on the results of the present study, WSPs are not capable of removing Listeria spp. completely. Non-pathogenic species of Listeria spp. were found in the WSP effluent. L. monocytogenes is a pathogenic bacterium and due to its pathogenicity, USEPA covered this bacterium in the new standard of effluent quality for reuse in agricultural lands and other uses (Council 2002). Pathogenic species of Listeria spp. have high resistance in water resources and can be an important health risk for humans and animals. WSPs become overloaded over time; therefore, it is necessary to upgrade and maintain processing controls to enhance the efficacy of this system in terms of bacteria removal, especially pathogenic micro-organisms like L. monocytogenes.

CONCLUSION

The present WSP effluent did not have an acceptable quality for unrestricted agricultural use in the case of Listeria spp., especially L. monocytogenes. Through effluent of the WSP, it is used on agricultural land; therefore, it could increase the environmental load of pathogenic Listeria and, consequently, enhance the risk of widely spreading diseases to human and animals.

ACKNOWLEDGEMENTS

This study is registered at the Isfahan University of Medical Sciences (IUMS) (number 387280). The authors would like to thank the vice-chancellor for research of IUMS for financial support. The authors declare that they have no conflict of interests.

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