DETECTION OF SALMONELLA IN SLUDGE SEDIMENTS

E. Otuszak-Walczak, P. Walczak, B. Wiera Institute of Fermentation Technology and Microbiology, Technical University of Lodz, Lodz, Poland Correspondence to: Elbieta Otuszak-Walczak E-mail: oltuszak@p.lodz.pl

ABSTRACT
A total of 145 sewage sediments samples from municipal and industrial wastewater treatment plants were tested for the presence of Salmonella using horizontal and PCR-based methods. Samples were pre-enriched overnight in buffered peptone water (BPW) at 37C followed by 3 h enrichment in BHI broth at 37oC. Obtained samples from BPW were selectively enriched in RVS broth (24 h, 42oC) and MKTTn (24 h at 37oC). Resulting cultures were plated onto XLD and BGA agar plates, incubated at 37oC for 24h. Presumptive Salmonella isolates were biochemically and serologically confirmed. As a comparison method, samples from BHI were lysed to break down the cell walls and release DNA and tested as described by the BAX Q7 Salmonella PCR Assay (Dupont). Among 145 samples derived from the 134 sludge sediments analyzed with horizontal and PCR-based methods, identical results were obtained for 136 samples (93,8% identity) of which 110 pairs of results were Salmonella negative, 26 samples were Salmonella positive for both analytical methods. Results obtained with traditional method were opposite to that obtained with PCR method only in nine samples. In conclusion, PCR method using Bax Q7 system should be further optimized for such unusual matrix as sludge sediments in order to eliminate false negative results. Keywords: sediments Real Time PCR, Salmonella, sanitation, wasteland bioremediation. Another 381.3 thousand tons were deposited in landfill depots, sediment fields, lagoons and sediment ponds. Total amount of deposited sludge sediments at the end of 2006 was 8.7 million tons including production from the previous years (7). Low utilization of sludge sediments in agriculture is due the presence of pathogenic microflora, especially Salmonella spp. that can contaminate harvesting plants and being source of infection for the livestock. Epidemiological data analyzed by Reilly et al (1987) (8) showed that among 26 outbreaks of salmonellosis, 13 was caused by the wastes and waste sediments. Sanitation of sediments from the wastewater treatment plants is the prerequisite of their safe disposal in the environment and use as fertilizer in agriculture. According to the existing regulations, sediments should be free of Salmonella and its presence ought to be tested by traditional or alternative indirect methods prior to their disposal. Recommended horizontal procedure for the detection of Salmonella and its identification requires 3 to 6 days for completion and therefore alternative reliable methods should be applied in order to reduce time of analysis. BaxQ7 Salmonella PCR Assay is recommended as an alternative method for detection of Salmonella in food

Introduction
Commonly used method for the purification of industrial and municipal wastewater is based on the aerobic activated sludge process combined with anaerobic methane fermentation. This generates large quantities of sediments, which should be safely stored in landfill depots, sediment fields and lagoons located at the site of sewage treatment plants. However, in modern installations, waste sediments are further processed by burning or composting. Burning method is usually applied when sediments contain high amount of heavy metals and other toxic substances, thus not allowing for direct use of sludge sediments in agriculture. Composting of sediments is the most appropriate method since obtained compost can be further utilized in agriculture as natural fertilizer or used for bioremediation of industrially degraded grounds. Agricultural use of sediments is the common practice in the Western European Countries where 54 to 66% of total sediment production is utilized for that purpose (5). Polands production of sludge sediments in 2006 was 1,064.7 thousand tons of which only 106.8 thousand tons were used as fertilizers in agriculture and 287.3 thousand tones used for
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samples and environmential (2). The aim of work was application of BaxQ7 (DuPont Qualicon) real time PCR system as an alternative method for the reliable detection of Salmonella in the lime treated sediments from wastewater treatment plants.

Materials and methods

Lime treated samples of sludge sediments were taken from the municipal and industrial wastewater treatment plants from the territory of the whole Poland. They were collected by trained personnel and sent to the analytical laboratory. Microbiological tests for the presence of Salmonella were started at the day of receiving sample. Samples were tested with traditional microbiological method according to the ISO-6579: 2003, or analyzed with BaxQ7 real time PCR system (1,3). The first stage of analysis was common for both methods. Preenrichment of Salmonella cells were carried out in BPW (buffered peptone water) 225 ml inoculated with 25g of sediment samples and incubated at 37oC for 18-24h. Resulting cultures were analyzed for the presence of Salmonella by the horizontal method or with PCR method, after the second enrichment step (3h incubation at 37oC in BHI broth). Traditional method 0.1 ml of enriched culture in BPW was transferred into 10 ml of RVS broth and incubated for 24 h at 42oC. Simultaneously, 1 ml of culture in BPW was used for the inoculation of 10 ml of MKTTn broth and incubated for 24 h at 37oC. Resulting cultures were sub cultured onto XLD and BGA plates for the isolation of single colonies and incubated at 37oC for 24 h. Bacterial colonies with typical Salmonella morphology were tested with slide agglutination method for Salmonella antigen group O, Vi and H using commercially available antiserum. Simultaneously, biochemical tests using API20E strips, Biomrieux, were carried out for confirmation of their taxonomic relatedness (1).

PCR based method 5 l of enriched culture from BHI broth was transferred to the lysis tube with 0.2 ml of protease containing lysis buffer (BL) and incubated at 37oC for 20 min. At this stage cells were lysed to break open the cell walls and release DNA. In the next stage, denaturation of protease activity was carried out by heating of lysis tubes for 10 min. at 95 oC and then cooling them down to 5oC for 5 min. 50 l of obtained lysates were used to hydrate the PCR tablets containing all necessary PCR reagents and DNA template for the positive internal control. Samples were processed in the automated thermocycler BaxQ7 according to the manufacturers instruction in about 3.5 h (3).

Results and Discussion

Comparison of two analytical methods based either on cultivation or PCR technique for the detection of Salmonella in sludge sediment samples were carried out. Table 1 summarizes results of analysis of 145 samples derived from the 134 sludge sediments originated from municipal and industrial wastewater treatment plants in Poland. Before estimation there was no data for the presence of Salmonella in the tested samples as well as for the possible contamination level. Of the 145 samples tested in this study, 136 (93,8%) were identical for Salmonella by either cultivation or PCR-based method. Among them, 110 pairs of results were Salmonella negative and 26 samples were Salmonella positive for both analytical methods.

TABLE 1. Comparison between horizontal and PCR based methods of analysis of lime treated sludge sediments. BAX System Q7 Method ISO 6579:2003 Total No. of analysis 145 (100%) No. of identical results 136 ((93,8%) No. of positive results 28 (19,3%) 33 (22,7%) No. of negative results 117 (80,7%) 112 (77,3%) In nine cases results obtained with cultivation method were different to that obtained with PCR based method (6.2% samples). Results obtained with BaxQ7 in two samples were positive whereas horizontal method showed absence of
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Salmonella. On the other hand, 7 results obtained with horizontal method were positive whereas obtained with PCR method were negative. Two samples estimated as Salmonella positive with
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cultivation method and negative with BaxQ7 method were subjected to the repeated analysis. In the first sample, enrichment procedure was carried out for the newly weighted 25 g sludge sediment and results of estimation confirmed absence of Salmonella evaluated by BaxQ7 method. Since presence of Salmonella in this sample was unquestionably confirmed by serological and biochemical methods, therefore negative result obtained with PCR-based method may be due

to the unequal distribution of bacterial cells in the sample or very low level of contamination. In the second sample repeated evaluation of Salmonella presence gave two positive results with both methods of analysis. It shows that sludge sediment analysis of Salmonella with BaxQ7 system is sensitive to the applied matrix and therefore should be further optimized in order to eliminate false negative results.

107

106

Salmonella +

Salmonella +

105

104

Salmonella +

Salmonella +

103

102

Salmonella

Salmonella -

Temperature, T (C) Fig.1. Profiles of melting curves for Salmonella specific amplification product (Salmonella) and internal positive control product (IPC) obtained for cell suspensions from 107 to 102 c.f.u./ml.

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Separated PCR experiments with defined Salmonella cell density showed that detection limit for the BAX Q7 Salmonella PCR Assay has been set by the manufacturer at the level of 104 c.f.u./ml and the presence of pathogen in 103 to 102 c.f.u/ml is interpreted by the system as negative results Fig.1. At the same time, presence of 103 to 102 c.f.u/ml of Salmonella cells in the enrichment medium allows for their detection with traditional cultivation method. Comparative studies of Feder et al 2001 concerning detection of Salmonella in porcine fecal samples and water with horizontal and PCR based methods showed also false negative results obtained with PCR (4, 6). One of the possible reason which reflects on false negative results in PCR method is low homogeneity of sludge sediments, especially those treated with solid lime causing formation of local alkaline zones whereas other parts of sediments are not alkalised. In such cases pre-enrichment stage may be inhibited by high alkalinity of BPW caused by addition of lime lumps with weighted sample Presence of bacterial growth inhibitors in sludge sediments will also reduce cell enrichment level and may prevent multiplication of Salmonella to the point exceeding 104 c.f.u./ml, lower detection limit for BaxQ7 system. High concentration of native accompanying microflora in sludge sediments compared to the number of Salmonella cells makes enrichment method more favourable for competitors and therefore growth of Salmonella can be retarded. Modification of enrichment procedure for PCR method by introducing selective enrichment step with RVS or MKTTn media followed by short cultivation step in BHI broth can eliminate problems of false negative results. This however requires further studies. Presence of inhibitors of PCR reaction in the sludge sediments can be another reason for false negative results obtained by this method. Therefore metabolites produced in BPW by native sludge sediment microflora can strongly influence of PCR reaction. During adaptation of

PCR-based procedures developed for food matrices to very unusual sludge sediment matrix all that factors should be taken into account.

REFERENCES
1. Anonymous (2003) Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Salmonella (ISO 6579:2003). International Organization for Standardization, Geneva, Switzerland. Anonymous 2003. Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Salmonella (ISO 6579:2003). International Organization for Standardization, Geneva, Switzerland. Anonymous (2006) Product information on BAX Q7 System PCR Assay, DuPont Qualicon. Available at http://www.dupont.com Bennett A,R. et al (1998) Letters in Applied Microbiology 26: 437-441 Butarewicz A (2003) Higieniczne aspekty procesu kompostowania osadw ciekowych. Nowe spojrzenie na osady ciekowe Odnawialne rda energii. Wydawnictwo Politechniki Czstochowskiej. Czstochowa 3-5 luty 2003:243-252. Feder I. et al (2001) J Clin Microbiol 39:2477-2484 Ochrona rodowiska (2007) Gwny Urzd Statystyczny, Informacje i opracowania statystyczne, Warszawa Reilly W.J. et al (1987)Veterinary record 108:553-555

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