<\1o/eel//ur <\4icrohiu/ Ec%gr ,\4alll/u/1.1.3: I-10, 1995 c 19<)5 Kho\'cr Acad,'lHic Puhlislu:rs. Printed in the NeTher/ands.

Extraction of microbial DNA from sewage and manure slurries

KORNELIA SMALLA Federal Institute o( Biology for Agriculture and Forestry, Biochemistry and Planl Virology Division, Messelt<ej[ I II12, 38104 Braunschll<eif{, Germany

Introduction

Sewage and manure slurries are environments where rather high numbers of micro-organisms are found, The microbial composition and metabolic activity is strongly dependent on several biotic and abiotic factors (e,g" nutrient availability, duration of storage, pretreatment), Sewage and manure slurries are supposed to play a central role in the natural circulation of pathogens, plasmid-encoded antibiotic or heavy metal resistance genes, Therefore, these habitats are of great interest in view of microbial ecology and hygiene, However, different bacteria of hygienic importance like Shigella dysenteria [7], Salmonella typhimurium [24], Salmonella enteritidis [16], Campylohacter jejuni [8,13,14], Escherichia coli [2,3], Vibrio cholerae [5] or Vihrio vulniflcus [26] were described to enter the viable but nonculturable state, The potential health hazard presented by pathogens existing in the nonculturable state may be significant because it has been shown that nonculturable bacteria can be resuscitated [II] and remain potentially pathogenic [4], Furthermore, genetically modified micro-organisms used in industrial settings might be undeliberately released via sewage into the environment. In view of the fact that genetically modified strains like E coli K 12 producing biologically active substances might enter the viable but nonculturable state under environmental stress one should not rely on cultivation methods only when tracking the fate of recombinant micro-organisms in the environment. Therefore, microbial ecologists have recently developed methods obviating the need for cultivation by applying molecular techniques to directly extracted nucleic acids from different environmental habitats [6,12,21],

Experimental approach

The methodology of well-established protocols for nucleic acid extraction [18,28] from isolates is applied to the environmental sample directly or

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after an appropriate concentration step. Directly extracted DNA does not contain only microbial nucleic acids but also DNA of different origin (algae, protozoa). Depending on the environmental matrix, coextraction of substances like humic acids or other organic and inorganic compounds might occur which can disturb subsequent DNA analysis [17,22,23]. Therefore, not only efficient recovery of nucleic acids from an environmental sample but also sufficient purity of the obtained DNA is of importance. PCR-assisted amplification of DNA sequences specific for pathogenic bacteria [1,10], for genetically modified micro-organisms, or antibiotic resistance genes [20] and· for genetically modified organisms [25] has considerably improved our ability to detect noncultured organisms (e.g., certain pathogenic bacteria or released genetically modified micro­organisms) in sewage or manure slurries. Furthermore, direct nucleic acid extraction methods are extremely helpful for detection of viruses, e.g., enteroviruses [9] where standard methods are very time-consuming and not applicable for viruses which are not amenable to culture.

F or nucleic acid extraction from sewage and manure, different protocols can be adapted. The method of choice will clearly be determined by the sample characteristics, e.g., content of particles and flocks, microorganism density, contaminations with low-molecular substances which have to be removed as well as by the general objective of the investigation. For DNA extraction from manure samples with a very high particle content, protocols originally developed for sediments and soil might be useful. On the other hand, DNA from sewage with low content of particulate matter can be extracted after an appropriate filtration step as applied for nucleic acid extraction from aquatic samples [21]. However, prefiltration steps usually applied to reduce clogging of the filter will diminish the DNA yield when microorganisms are attached to the flocks.

The following protocols were successfully applied to the extraction of genomic DNA from sewage and manure slurries which served as a target for subsequentPCR amplification of antibiotic resistance genes and replication or transfer related sequences of broad host range plasmids. The limit of detection has to be determined separately for each DNA extraction method and the respective target/primer system. In addition, the detection sensitivity of a certain sequence might be influenced by the Taq-polymerase used and by the addition of stabilizing proteins (bovine serum albumin, T4-protein: [15,23]). The following protocols should be regarded just as examples implying that other protocols described in the manual might be useful as well.

Nucleic acid extraction from sewage using the Sterivex protocol [21,27 J

The Sterivex protocol was originally developed for nucleic acid extraction from aquatic habitats. The first step of the procedure is filtration of the liquid sample through a bacterial filter (Sterivex, Millipore, 0.22 /lm). The

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volume of sewage which can be pumped through the Sterivex cartridge with a peristaltic pump depends on the content of cellular aggregates. For sewage, volumes of 10 ml (influent of a sewage treatment plant) to 100 ml (sewage treatment plant effluent) should be used. The advantage of the Sterivex procedure is that several steps can be performed in the filter housing thus reducing the probability of contaminations from the laboratory. A further advantage is that the filter can be stored at -20°C after filtration of sewage.

Procedure

Steps in the procedure

- Sewage with a low content of particulates is pumped through a

Sterivex filter (Millipore, 0.22 ~m) without prior prefiltration using

a peristaltic pump (flow rate will decrease during the filtration due

to clogging of the filter).

- Wash the filter with 10 ml sterile SET buffer; close the inlet and

the outlet of the filter unit with parafilm and store at -20°C.

- Thaw filter unit before adding 1.8 ml SET buffer and 67 ~I freshly

dissolved lysozyme with a syringe; after inverting, incubate on ice

for 30 min.

- Add 16 1-11 SDS solution and incubate the filter on a roller (Spira­

mix) to keep the entire filter in contact with the reagents for 1 h

(approx. 32 r.p.m.).

- Add 50 1-11 proteinase K solution and continue incubation on the

roller for 3-4 h.

- Remove the lysate from the filter with a syringe; add 1 ml SET

buffer to the filter and move on a roller for approx. 5 min.

- Pool the crude lysate with the SET wash buffer in a centrifuge

tube.

- Add 0.5 vol 7.5 M ammonium acetate and incubate for 15 min at

room temperature after careful mixing to precipitate proteins.

- Centrifuge at 16,000 x g for 15 min at room temperature.

- Place the supernatant in a new centrifuge tube and precipitate

DNA by adding 2.5 vol ethanol (at least for 2 h at -20°C).

- Centrifuge at 26,000 x g for 30 min at 4 cC.

- Resuspend the pellet without drying in 600 1-11 TE, place the

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solution in a microtube and precipitate with 0.6 vol isopropanol

for 30 min at room temperature.

Centrifuge at 26,000 x g for 20 min at 4°C.

Wash the pellet with 70% ethanol and air dry it.

Dissolve the pellet in an appropriate volume of TE.

In case the DNA extracted is not amplifiable by PCR (testing by

addition of a positive control to the target DNA) purification with

glassmilk is recommended (Geneclean II, 810101, Vista, CAl.

Notes

Controls. To control for contamination of the resulting DNA during the nucleic

extraction procedure, as a negative control one Sterivex filter is processed exactly the

same way as described above but with filtration of sterile distilled water instead ofthe

environmental sample; the resulting solution is used as a control for peR in addition

to the water control, which ensures the absence of the respective amplified target

sequence in the peR reagents.

Determination of the detection limit. The detection limit of a naturally occurring

sequence can be determined by adding defined cell numbers of the microorganism

containing the sequence to be monitored without sewage to the Sterivex procedure.

Usually bacterial cells of an overnight culture are harvested by centrifugation. The

resulting cell pellet is washed twice with sterile saline before diluting the cells in

saline. For an estimate of the cell number a counting chamber is used. The dilutions

containing approx. 108 , 105 , 103 , 102 and 0 cells are filtered through Sterivex filters

(two parallels for each cell density) and are plated in parallel to determine the c.f.u.

counts of each dilution. The nucleic acids are recovered as described in the protocol.

Usually the limit of detection is determined by Southern blot hybridization of the peR

product with a specific probe. The actual detection limit will depend on the volume

used for harvesting bacteria, the copy number of the target sequence per cell and the

proportion of recovered DNA used as target in the peR. We determined the limit of

detection for a chromosomally Tn5 labelled P. syringae strain applying the Sterivex

procedure. When using 1/10 vol of the extracted DNA we obtained strong PCR signals

after Southern blot hybridization of amplification products specific for Tn5 for cell

numbers corresponding to 4 x 106and 4 x 103 c.f.u. Faint signals were obtained for the

other cell densities used, except for the control without added P. syringae (chr::Tn5)

(see Fig. 1).

The determination of the detection limit for genetically modified micro-organisms by

amplification of a sequence not naturally occurring is done as described above except

for resuspending the harvested cell pellet in sewage.

DNA yield. Somerville et al. [21] determined for the protocol described above (with

the only modification being 5% sucrose instead of 20%) a yield of about 1 ng high­

molecular weight DNA per 106 cells. According to Byrd and Colwell [2], free DNA does

not contribute to the DNA extracted with the Sterivex protocol.

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1 2 3 4 5 6 7 R 9 10 11 12 13 14 15

Figure I. Determination of the extraction efficiency for chromosomally Tn5 labelled P. srringae after peR amplification and Southern blot hybridization: lanes: I: Dig-ladder (Boehringer 1218603): 2, 3: 4.2 x lOr, c.f.u.; 4, 5: 3.6 x 103 c.f.u.; 6, 7: 3.2 x 10 1 c.f.u.: 8, 9: 4.5 x IOu c.f.u.: 10, II: <10 c.f.u.; 12, 13: 0 c.f.u.: 14: water control: 15: Dig-ladder.

Solutions

- SET buffer

- 5% sucrose.

- 50 mM EDTA.

- 50 mM Tris-HCI pH 7.6.

- lysozyme solution

5 mg lysozyme freshly dissolved in:

10 mM Tris-HCI pH 8.0.

1 mM EDTA.

10 mM NaCI.

- SDS solution

- 25% in double-distilled water.

- proteinase K solution

- 20 mg/ml in double-distilled water.

All reagent buffers and water used are to be prepared RNase free

as described by Sambrook et al. [18] for the extraction of RNAs.

Genomic DNA extraction from manure slurries or sewage using the

Qiagen bacterial DNA isolation kit

The protocol was originally designed for the rapid, easy preparation

and purification of genomic DNA from pure cultures using nontoxic

reagents. Therefore the following instruction is an adaptation of the

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Oiagen protocol to directly extract genomic DNA from manure slur­

ries or sewage. With the protocol described only DNA from micro­

organisms recovered by the harvesting method is obtained.

At first, the volume of sewage or manure which can be used for

genomic DNA extraction without overloading the Oiagen tips has to

be determined. According to Oiagen, approximate cell numbers

should not exceed 8 x 1010 using the maxi preparation protocol.

The approximate cell number can be determined using a counting

chamber or by plating. If the sewage or manure slurry contains

flocks, a pretreatment with a blending step to dislodge adsorbed bac­

teria followed by prefiltration through a Miracloth filter is recom­

mended. Complete resuspension of the bacterial pellet obtained by

centrifugation and the absence of particulate matter is important for

efficient lysis and to avoid clogging of the Oiagen tips used for

pu rification.

Steps in the procedure

Centrifuge sewage or manure slurry samples containing a

maximum of approx. 8 x 1010 cells. Spin the bacteria down at

5,000 x g for 20 min; higher x g-forces are recommended to

recover also dwarf cells from the environmental sample.

- Resuspend the bacterial pellet thoroughly with 11 ml 81 buffer

with dissolved RNase by vortexing at highest speed (no particles

should remain).

- Add 300 ~I lysozyme solution and 500 ~I proteinase K stock

solution.

Incubate at 37°C for at least 30 min (longer incubation is

recommended when particles are still visible).

- Add 4 ml 82 buffer and mix the solutions thoroughly by inverting

or vortexing briefly.

- Incubate at 50°C for at least 15 min; it is necessary that the lysate

becomes clear at this stage (if not, extend the incubation time or

remove remaining partiCles by a short centrifugation step).

- Equilibrate a Oiagen tip with 10 ml 08T buffer and drain the tip

completely by gravity flow.

- Vortex the lysate for 5-10 sec and load it, if clear and particle-free,

onto the equilibrated Oiagen tip (flow can be assisted by a gentle

positive pressure with flow rates not exceeding 20-40 drops/min).

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- Wash the Oiagen tip 2-3 times with 15 ml OC buffer by gravity flow.

- Elute the genomic DNA with 15 ml OF buffer.

- Add 0.7 volumes of isopropanol equilibrated to room temperature

and precipitate by inverting 10-20 times.

- Centrifuge at >5,000 x g at 4 °C for 20 min.

- Resuspend the DNA pellet in TE buffer and transfer to microfuge

tube.

- Wash centrifuged DNA in 70% ethanol, air-dry the pellet for 10

min and resuspend in a suitable volume, overnight or for 1-2 h at

55°C by gentle shaking.

Solutions - B1 buffer (bacterial lysis buffer) pH 8.0 containing:

- 50 mM EDTA.

- 50 mM Tris-HCI.

- 0.5% Tween-20.

- 0.5% Triton X-100.

- B2 buffer (bacterial lysis buffer) pH 5.5 containing:

- 3 M Guanidine Hydrochloride.

- 20% Tween-20.

- OBT buffer (equilibration buffer) pH 7.0 containing:

- 750 mM NaCI.

- 50 mM MOPS (3-(N-Morpholino)propanesulfonic acid).

- 0.15% Triton X-100.

- OC buffer (wash buffer) pH 7.0

- 1.0 M NaCI.

- 50 mM MOPS.

- 15 % ethanol.

- OF buffer (elution buffer) pH 8.5

- 1.25 M NaCI. - 50 mM Tris-HCI.

- 15% ethanol. - RNase A solution

- 200 IJg/ml B1 buffer (stable for 6 months stored at 4°C). - lysozyme solution

- 100 mg/ml distilled water (aliquots should be stored at -20°C).

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proteinase K solution

Note

20 mg/ml in distilled water (stable for 3-6 months at 4 ec or

store aliquots at -20 ec).

Lysozyme and detergents in S1 buffer added to resuspended cells ensure complete

lysis of bacterial cells. RNA is degraded by RNase A. Proteinase K and S2 buffer strip

all proteins from the DNA. The lysate loaded onto the Qiagen tips should be clear. The

salt and pH conditions in the lysate and the selectivity of the Qiagen resin ensure that

only DNA is bound while RNA, proteins and other low-molecular compounds are not

retained. Wash buffer QC containing 1 M NaCI elutes any residual RNA and proteins

bound to nucleic acid. The genomic DNA is efficiently eluted with buffer QF containing

1.25 M NaCI at pH 8.5. The binding, washing and elution conditions for the Qiagen

procedure are strongly influenced by pH and salt concentration. Concentrated DNA

might diminish the gravity flow rate. Dilution of the DNA with QST buffer is an

alternative. Isopropanol precipitation should be performed at room temperature to

prevent salt precipitation. The DNA pellet should be dissolved at slightly alkaline pH

(8.0-8.5).

Genomic DNA prepared on Qiagen tips is sufficiently pure to allow restriction

enzyme digestion and PCR.

All buffers should be equilibrated at room temperature before

usage.

Freezing and thawing of enzymes should be avoided.

DNA extraction from manure slurries with a protocol developed for

soil DNA extraction

For manure samples with a rather high content of particulate mat­

ter the protocols described above might not be suitable due to

clogging of the Sterivex filter or the Qiagen tips. The protocol for

DNA extraction from soil described in detail in Chapter 1.3.3. is with

some modifications applicable to manure slurry. Micro-organisms

and particulate matter from 20-100 ml manure slurry are pelleted by

centrifugation (16,000 x g, 20 min, 4 ec) . The resulting pellet is

washed twice with sterile saline. The pellet is resuspended and

processed further as described by Smalla et al. [19]. However, for

DNA recovered from manure the purification is much easier than for

soil DNA. In most cases, only a one step purification with glass milk

(Geneclean II, 810101, Vista, CAl was needed to obtain PCR amplifiable DNA.

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The advantage of this protocol is that the rather harsh cell

disruption by applying a bead beating step will result in a more

efficient lysis of bacterial cells or spores. Furthermore, a prefiltration

step which will definitely reduce the number of cells subjected to

DNA extraction is omitted. The resulting DNA is somewhat sheared

due to the bead beating treatment which might be an advantage for

the amplification of peR products smaller than 1 kb. However,

phenol and phenol/chloroform extraction are applied which is critical

with regard to hazardous waste disposal.

Acknowledgement

The work is funded by the EC BIOTECH (grant no. BI02-CT92-0491).

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