e:> Pergamon

PIT: S0273-1 223(98)008 I2-9

WaL Sci. Tech. Vol. 38. No. 12. pp. 123-126. 1998.(01998IAWQ

Published byElsevierScienceLtdPrinted in Greal Britain. All rights reserved

0273-1223/98 S19'00 + 0'00

DETECTION AND SURVIVAL OFPATHOGENS DURING TWO-STAGETHERMOPHILICIMESOPHILICANAEROBIC TREATMENT OFSUSPENDED ORGANIC WASTE

C. Burtscher, P. A. Fall, O. Christ, P. A. Wilderer andS. Wuertz

Instituteo/Water QualityControland WasteManagement, Technical UniversityofMunich,Am Coulombwall, D-85748Garching, Germany

ABSTRACT

The food-borne pathogens Salmonella typhimurium and listeria monocytogenes were effectively eliminatedfrom suspended organic waste during the Ihermophilic (55°C) phase of a two-stage Ihermophilic/mesophilicanaerobic reactor. The feasibility of PeR technology was evaluated in order to improve the speed ofdetection and reduce the effort involved in detecting the presence of a range of pathogenic bacteria. Ourresults show direct extraction of nucleic acids followed by purification and PeR to be a promising meIhodIhat will enable the rapid verification of hygienic performance during waste treatment , © 1998 IAWQ.Published by Elsevier Science Ltd. All rights reserved

KEYWORDS

Anaerobic digestion; hygienic performance; nucleic acid extraction; organic waste; pathogens; polymerasechain reaction.

INTRODUCflON

Currentlegislation in Germany mandates the reuseof wastematerials whenever possibleeven if this requirespretreatment (Rummier, 1996). This has led to increased emphasis on composting of biological householdwasteand other organic materials of biological origin. An alternative to composting is anaerobic digestionofsuspendedorganic waste. In bothcases the question of survival of food-borne pathogens has been addressedby specifying that compost must be free of Salmonella spp. This requires time-eonsuming isolationtechniques which preclude the use of a battery of tests for the routine testingof other bacteria. However, thegenus Salmonella does not have the characteristics necessary to justify its use as a general indicatororganism due to the fact that other human pathogens differ both in their physiology and in their effectivedose. Potential contaminants are Salmonella typhimurtum; Listeria monocytogenes, Yersinia enterocolitica,Clostridium perfringens and Campylobaeter jejuni among others. We present data on the efficiency ofhygienic performance in a two-stage thermophilic/mesophilic anaerobic digestion systembased on detectionof bacteria by classical enrichment procedures. In order to assure quality control of the produced compost

123

124 C. BURTSCHER et al.

and broaden its marketing potential, for example as garden compost to relieve precious peat resources, it isdesirable to have a method that will allow rapid detection of a range of microorganisms. We report resultsrelating to the application of a nucleic acid extraction method and the polymerase chain reaction (PeR) forthe rapid detection of pathogens in anaerobically treated suspended organic waste.

MATERIALS AND METHODS

Operation of the pilot-scale plant - biological waste, that had been suspended to a total solids content of10%, was passed through a two-stage thermophilic/mesophilic anaerobic digester. The two reactors had acapacity of 500L each. The thermophilic reactor was operated at 55°C with a hydraulic retention time of 5dand a mean cell residence time of 48h. The addition of untreated suspended organic waste occurred in 6hintervals. Samples were taken through an outlet installed on the reactor.

Bacterial species, culture medium, and growth conditions - Salmonella typhimurium strain 96 BR 385 andListeria monocytogenes strain I HE/9211104/666-2 serovar 1/2a were maintained on Standard I and LSMmedia, respectively. Isolation procedures for Salmonella spp were essentially as described by Kauffmann(1935). Enrichment broths were streaked on XLD agar. For detection of Listeria monocytogenes, ImL ofsuspension was enriched in UVM I broth and subsequently in Fraser broth. Cells were plated on Oxfordselective agar. Presumptive cells were confirmed by PCR. Clostridium spp were isolated in DRCM mediumand identified microscopically based on the production of endospores. Clostridium perfringens wasconfirmed on TSY agar supplemented with Fluorocult. For detection of Campylobacter spp, cells wereenriched in Preston selective enrichment broth and spread on Preston agar plates. Yersinia enterocoliticawas isolated by cold enrichment and cells were subsequently streaked on CIN agar. Presumptive cells we~eidentified by API 20E (Biomerieux). All nutrient media were supplied by Merck except for Preston media(Oxoid) , Enrichment procedures and biochemical confirmation were generally performed as described bythe manufacturers.

Extraction of nucleic acids and PCR - direct extraction of nucleic acids was performed as previouslydescribed by Zhou et al. (1996). The method was downsized to one fifth of the volume. When an enrichmentstep was included, ImL of suspended organic waste was put into 9mL of peptone water and incubated at37°C overnight (about 17h). The suspension was concentrated by centrifugation at 9,OOOxg and extracted asdescribed previously. Crude DNA extracts were passed through ImL Wizard Miniprep PCR purificationresin (Promega, Madison. Wis.). The resulting DNA concentration was estimated by agarose gelelectrophoresis. PCR was used directly on colonies isolated on agar plates and on nucleic acid extracts . Inthe case of Salmonella typhimurium the primer pair LHNS-53I (5'·TACCAAAGCfAAACGCGCAGCT-3')and RHNS-682 (5'·TGATCAGGAAATcrTCCAGTTGC-3') were utilised for the amplification of a 0.152­kbp region of the hns gene using the conditions given by the authors (Jones et al., 1993). The primer pairLL5 (S'·AACCTATCCAGGTGCTC-3') and LL6 (S'·CTGTAAGCCATTTCGTC-3'). which are specific forthe hlyA gene for Jisteriolysin O. were used for Listeria monocytogenes as previously reported (Thomas etal. , 1991). The resulting fragment was 0.267 kbp in size. All PeR products were identified by sizeestimation after agarose gel electrophoresis.

RESULTS AND DISCUSSION

Monitoring of pathogens in a pilot-scale reactor - the incoming suspended organic waste and thethermophilic stage of a two-stage reactor were sampled during a period of eight months using classicalenrichment and isolation methods. Salmonella typhlmurium was detected occasionally and 3/4 feed samplestested positive for Listeria monocytogenes. All treated samples were negative for Salmonella typhimuriumand Listeria monocytogenes indicating that the anaerobic digestion process at 55°C effectively removed allviable cells. The genus Clostridium is widespread in nature. It is anaerobic and capable of formingendospores. Using anaerobic enrichment in DRCM medium, Clostridium spp were detectable in all samplestaken. Occasionally, the pathogen Clostridium perfringens was found in treated samples. Since Clostridiumis highly heat resistant, elimination during thermophilic treatment was not expected. Yersinia enterocoliticawas not detected in any sample. Campy/obaeter spp were found both in feed samples and post-treatment

Detectionand survivalof pathogens 125

samples. Further identification of the colonies was not undertaken. It is possible that Campylobacter jejuniwas present since this organism is known to be thermophilic.

Survival ofpathogens under simulated conditions - in order to estimate whether the thermophilic treatmenttime in the pilot scale reactor was adequate for the elimination of pathogens a laboratory-scale experimentwas performed. High cell numbers of pathogens mixed with suspended organic waste (the input into the pilotscale reactor) were incubated under anaerobic conditions at 55°C. After 6h no live cells of Listeriamonocytogenes or Salmonella typhimurium could be demonstrated using classical isolation methods. Thisexposure period corresponds to the time lapsed between additions of fresh suspended organic waste to thepilot scale reactor. Even a 2h incubation was sufficient to kill all viable cells (Table I). The state ofanaerobiosis was not a critical factor since aerobic treatment had the same effect. At lower temperatures(37°C) the celIs remained viable as would be expected based on their optimum growth temperatures.

Table I. CelI viability under anaerobic and thermophilic conditions

Species

L monocytogenesIStyphimurium

Length ofincubation (h)

2222

PresenceofQz

++

Temperature(0C)

55375537

Viable cells*detected

+

+*The cell density was lOi/mL waste suspension.

Detection ofpathogens by PCR after nucleic acid extraction - direct extraction of nucleic acids from organicwaste suspensions was successfully used to amplify specific genes. Crucial to the procedure was a simplepurification step involving a commercially available resin (Wizard PCR Preps DNA Purification system,Promega, Wis.). In samples amended with physiologically active cells of Salmonella typhimurium afterdirect extraction of DNA 104 cells were detected in ImL of suspended organic waste. Figure I shows thatafter overnight enrichment in peptone water DNA from less than 10 cells of Salmonella typhimuriumlmLorganic waste could be amplified by PCR and visualized on an agarose gel.

2 .' -1 5 (, 7 S Q I0

Figure I. Agarosegel electrophoresisof PCR-amplified DNA followingnucleicacid extractionafter an enrichmentstep (17h).

[5L DNA extract was used for each PCR reaction.Bandscorrespondto a O.152-kbp regionof the hns gene ofS typhimurium. Lanes I & 10=lOO-bp ladder size marker;Lane 2 =pure cultureof S typhimurium positivecontrol;

Lanes 3-7 =extract from organic wasteamendedwith 105, \04, \03, \02, 10cellslmL;Lane 8 =extract ofunamendedorganic waste;Lane9 =wateras negativecontrolI

126

CONCLUSIONS

C.BURTSCHER et al.

The elimination of pathogens during anaerobic digestion of suspended organic waste is critical to asuccessful implementation of this increasingly important technology. Based on our results it can beconcluded that Salmonella typhimurium and Listeria monocytogenes are efficiently removed in the two­stage thermophilic/mesophilic anaerobic digestion scheme tested. The time and effort required to follow thefate of other food-borne pathogens is too great when only classical enrichment and isolation methods areconsidered. Furthermore, the results obtained on selective or differential media are often ambiguous andmisleading necessitating further testing by biochemical or serological markers. For this reason we attemptedto apply PCR methodology to suspended organic waste samples. peR after nucleic acid extraction andpurification (Zhou et al., 1996) was successful. This method is promising since it does not rely on severalenrichment steps and enables sample analysis within two working days. Further work should investigate thedetection limit for different pathogens.

ACKNOWLEDGEMENT

Thanks are due to l-M. Collard of the Institute of Hygiene & Epidemiology, Brussels, Belgium, for hisgenerous gift of Salmonella typhimurium strain 96 BR 385 and Listeria monocytogenes strain IHF192JIl04/666-2 serovar 1/2a. This work was supported in part by BayFORREST grant number F78.

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Kauffmann . F. (1935). Weitere Erfahrungen mit dem kombinierten Anreicherungsverfahren rur Salmonellenbazillen. Z. Hyg.

Infekt·Krkh.. 117. 26-32 .Rummier. W. (1996) . Paper on " TIle environment policy concept of the closed substance cycle and waste management act".

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in milk and ground beef with the polymerase chain reaction. Appl. Environ. Mlcrobiol., 57. 2576-2580.Zhou, J.• Bruns. M. A. and Tiedje , J. M. (1996) . DNA recovery from soils of diverse compos ition. Appl. Environ. Microbiol., 62.

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