Ž .Journal of Microbiological Methods 47 2001 355–368www.elsevier.comrlocaterjmicmeth

ž /Application of polymerase chain reaction PCR and TaqManePCR techniques to the detection and identification of

Rhodococcus coprophilus in faecal samples

Marion G. Savill a,), Sonya R. Murray a, Paula Scholes a, Els W. Maas a,Rachel E. McCormick a, Edward B. Moore b, Brent J. Gilpin a

a ( )Christchurch Science Centre, Institute of EnÕironmental Science and Research ESR Ltd., PO Box 29-181, Christchurch, New Zealandb DiÕision of Microbiology, GBF-National Research Center for Biotechnology, D-38124 Braunschweig, Germany

Accepted 27 August 2001

Abstract

ŽRhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal as.opposed to human faecal contamination of aquatic environments. However, conventional detection methods limit its use for

this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNAX Ž .from faecal samples is described. PCR and 5 -nuclease TaqMane PCR methods were developed to allow the detection and

enumeration of R. coprophilus in faecal samples within 2–3 days. Both PCR methods targeted the 16S rRNA gene,producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce anamplification product by conventional PCR, while as little as one cell was required for the TaqMane PCR method. Thelatter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successfulamplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative forsamples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus todistinguish faecal pollution of farmed herbivores from human pollution. q 2001 Published by Elsevier Science B.V.

Keywords: Rhodococcus coprophilus; Faecal source identification; TaqMane

1. Introduction

Farmed animals contribute large quantities of fae-cal material as pollution of natural waterways. How-ever, it is often difficult to quantify and apportionthese inputs, particularly in the lower reaches ofwaterways where human inputs also occur. In New

) Corresponding author. Tel.: q64-33516019; fax: q64-33510010.

Ž .E-mail address: marion.savill@esr.cri.nz M.G. Savill .

Zealand, this is regarded as a significant water man-Ž .agement issue Sinton et al., 1998 because over half

the land area is devoted to agriculture accommodat-ing a population of approximately 45 million sheep

Ž .and 9 million cattle Anonymous, 2000 . Althoughepidemiological evidence is lacking, there is awidespread perception that water contaminated byhuman faeces constitutes a greater health risk than

Žwater contaminated by animal faeces Sinton et al.,.1998 . The management and mitigation of faecal

pollution in water would be assisted by assays that

0167-7012r01r$ - see front matter q 2001 Published by Elsevier Science B.V.Ž .PII: S0167-7012 01 00343-8

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368356

could determine whether the contamination was ofhuman or animal origin.

The potential microbial human faecal source indi-cators that have been investigated include Pseudo-

Ž .monas aeruginosa Wheater et al., 1980 , someŽmembers of the Bacteroides fragilis group Allsop

.and Stickler, 1985; Fiksdal et al., 1985 , sorbitol-fer-Žmenting bifidobacteria Mara and Oragui, 1985; Ja-

.gals et al., 1995; Rhodes and Kator, 1999 , andŽbacteriophages of B. fragilis strain HSP40 Jofre et

.al., 1986; Tartera and Jofre, 1987 .Suggested indicators of grazing animals include

ŽStreptococcus boÕis Oragui and Mara, 1981; Oragui. Žand Mara, 1983 , thermophilic bifidobacteria Gavini

. Žet al., 1991 , and Rhodococcus coprophilus Rowbo-.tham and Cross, 1977; Mara and Oragui, 1981 . R.

coprophilus is a natural inhabitant of the faeces ofherbivores. It is a nocardioform actinomycete, form-ing a fungus-like mycelium, which breaks up into

ŽGram-positive, aerobic, bacterial cells Sneath et al.,.1986 . These bacteria contaminate nearby grass and

hay, are eaten by herbivores, survive passage throughtheir digestive systems, and thus again infect the

Ž .voided dung Al-Diwany and Cross, 1978 .The potential use of R. coprophilus as an indica-

tor of herbivorous faecal pollution in water was firstŽ .recognised by Rowbotham and Cross 1977 , who

noted its presence in the faeces of domesticatedherbivores, pasture run-off and associated receivingwaters and sediments, but its absence from humanfaecal wastes. It has since been frequently isolatedfrom the dung of herbivores such as cows, donkeys,goats, horses and sheep, from poultry reared in prox-imity to farm animals, and from fresh waters andwastewaters polluted with animal faecal materialŽ .Mara and Oragui, 1981; Oragui and Mara, 1983 .

Ž .Al-Diwany and Cross 1978 examined the incidenceŽ .of various actinomycetes in mostly rural aquatic

habitats, and found that R. coprophilus counts werereasonably well correlated with faecal streptococci,suggesting a faecal origin. In Africa, Mara and Or-

Ž . Ž .agui 1985 and Jagals et al. 1995 found R. co-prophilus to be more strongly associated with ani-mal, rather than human, faecal pollution of water.

A major impediment to the use of R. coprophilusas an indicator of animal faecal pollution is thecumbersome and time-consuming culturing method

Žassociated with this organism Rowbotham and

Cross, 1977; Mara and Oragui, 1981; Sneath et al.,.1986 . Conventional culturing takes up to 21 days to

establish a result, which severely limits its applica-tion. Species identification by biochemical means is

Ž .also difficult Bell et al., 1998 .Ž .Ribosomal ribonucleic acid rRNA genes, partic-

ularly the 16S rRNA gene, appear to be most usefulfor identifying and distinguishing specific microbes,since they provide sufficient information to allow themeasurement of both close and distant phylogenetic

Ž .relationships Woese, 1987 . Fast, sensitive and ac-curate identification methods using PCR targeted tospecies-specific regions in the 16S rRNA gene have

Žbeen described for a wide range of microbes Spring.et al., 2000; Whitehead and Cotta, 2000 . Recently,

Ž .Bell et al. 1999 showed the potential to use PCR-based tests for the identification of various species ofrhodococci using species-specific regions of the 16SrRNA gene, but R. coprophilus was not included intheir study.

For a number of purposes the mere detection ofan organism does not provide sufficient information,and the enumeration of R. coprophilus is importantto assist in determining the degree of animal faecalpollution. Quantitative, Areal timeB, 5X-nuclease orTaqMane PCR exploits the 5X nuclease activity ofTaq polymerase to cleave a dual-labelled fluorogenicprobe that is annealed to the target sequence during

Ž .amplification Lee et al., 1993; Livak et al., 1995 .The 5X end is labeled with the reporter dye FAMŽ . X6-carboxyfluoresceine and the 3 end is labeled

Ž .with TAMARA 6-carboxytetramethylrhodaminewhich serves as a quencher dye. During PCR, thefluorogenic probe is cleaved, separating the reporterdye from the quencher, and thereby generating afluorescent emission. Measurements of this fluores-cence increase throughout the PCR cycles, enablingquantitative estimates of the initial quantity of DNAto be made. TaqMan quantitative assays have beendeveloped for a number of micro-organisms includ-

Ž .ing Campylobacter jejuni Nogva et al., 2000 , Lis-Ž .teria monocytogenes Norton and Batt, 1999 ,Ž .Yersinia enterocolitica Sen, 2000 and hepatitis B

Ž .Pas et al., 2000 .This paper describes the development of a PCR

assay for the detection of R. coprophilus in animalfaecal samples and the conversion of this assay intoa quantitative TaqMane PCR assay.

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368 357

2. Materials and methods

2.1. Bacterial strains

The culture collection sources of the 13Rhodococcus species, 8 related actinomycete genera,and 8 other bacterial genera used in this study, arelisted in Tables 1–3, respectively. All Rhodococcusspp. were grown in Tryptic Soy broth at 30 8C,except R. coprophilus which was grown in Brain

Ž .Heart Infusion broth BHI at 30 8C, and R. equiwhich was grown in BHI at 35 8C. Related actino-mycetes were grown in BHI at 30 8C except A.naeslundii, C. xerosis, G. bronchialis, N. brasilien-sis and S. griseus which were grown at 37 8C.Non-actinomycetes were grown in BHI at 35 8C.Incubation periods for each species are listed inTables 1–3.

2.2. Plate counts

Pure cultures of R. coprophilus were culturedwith gentle agitation at 30 8C in BHI broth for 8days. Ten-fold serial dilutions in sterile quarter-

Žstrength Ringer solution pH 7.0, Oxoid, Bas-.ingstoke, Hampshire, UK were prepared from the

broth down to 10y5. Each dilution was spread platedŽ . Žout 100 ml onto MM3 agar plates Mara and

.Oragui, 1981 using 1% Bovine serum albuminŽ .BSA, Sigma coated plastic pipette tips to minimisethe binding of R. coprophilus to the plastic. Theplates were incubated for 18–25 days at 30 8Cfollowed by 4–5 days in sunlight. Colonies of R.coprophilus were presumptively identified by therough, mycelial appearance of redrorange colonies.

2.3. Determination of naturally occurring R. co-prophilus leÕels in faeces

Faecal samples were collected from six cows,three sheep, five humans, and from one each of deer,horse, pig, rabbit, duck and possum. Fresh animal

Ž .and human faeces 1 g were added to 9 ml ofŽ .quarter-strength Ringer solution pH 7.0 and heated

at 55 8C for 6 min after which they were seriallydiluted in quarter-strength Ringer solution to a 1 in10,000 dilution. Each dilution was plated in triplicateon MM3 agar. The plates were incubated as de-

scribed above. Colonies were taken from the platesŽand their identity confirmed by PCR see DNA

extraction from pure cultures and PCR methods sec-.tions . Isolated colonies were placed in 20% glycerol

and stored at y80 8C.

2.4. Sequencing of enÕironmental isolates of R. co-prophilus

Individual isolates of R. coprophilus recoveredfrom MM3 agar plates inoculated with cow faeceswere resuspended in 100 ml of ddH O and incubated2

at 100 8C for 10 min. The tubes were centrifuged at12,000=g for 10 min and the clear supernatantused as DNA template to amplify almost the com-plete 16S rRNA gene using primers and PCR condi-

Ž .tions as previously described Karlson et al., 1993 .The resulting product was purified and sequenced as

Ž .previously reported Karlson et al., 1993 . All se-quences obtained from the 16S rRNA genes werealigned with 16S rRNA and rDNA sequences usingthe e-mail servers at the Ribosomal Database ProjectŽ . Ž .Maidak et al., 1997 and using FASTA version 3

Žto search the EMBL database Pearson and Lipman,.1988 .

2.5. DNA extraction methods

2.5.1. Pure cultureCells were cultured using standard culture condi-

Ž .tions specific for each genus Tables 1–3 . Thecultures were then centrifuged at 1600=g for 40min in a MSE Mistral 2000 centrifuge. The cellpellet was resuspended in 600 ml of extraction bufferŽ25 mM Tris, 10 mM EDTA, 50 mM glucose at pH

.8.0 and transferred to a sterile tube. To this wasŽ y1 .added 40 ml of lysozyme 50 mg ml , and the

tube incubated for 5 min at room temperature.Ž .Sodium dodecyl sulphate 20% SDS; 24 ml and

Ž y1 .proteinase K 10 mg ml ; 8 ml were then added,and incubated at 37 8C for 30 min. The top aqueousphase was washed twice with Tris buffered phenolr

Ž . w xchloroformriso-amyl alcohol IAA 25:24:1 byŽmixing for 10 min and centrifuging Heraeus sepat-

.ech Biofuge A for 15 min at 15,000=g. Theaqueous phase was then extracted with chloroformr

Ž .IAA 24:1 . Finally, the upper aqueous phase wascollected and DNA precipitated using the sodium

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368358

Table 1Rhodococcus species and growth conditions used in this study

Species Source EMBL No. Days growtha,bR. coprophilus ATCC 29080 X80826 8

c.R. coprophilus DSM 43347 X80626a,b,cR. chubuensis DSM 44019 X80627 8

a,bR. equi NCTC 1621 X80614 5aR. equi ATCC33707 X80594aR. equi ATCC6939 X80604cR. equi DSM 20307 X80614cR. equi Unpublished

cR. erythreus DSM43066 X79289 8b,cR. erythropolis DSM 43066 X79289aR. erythropolis ATCC4277 X81929aR. erythropolis NCIMB11148 X76691 5

a,b,cR. fascians DSM 20669 X79186 7a,bŽ .R. fascians R. luteus DSM 43673 X81932

aR. fascians ATCC12974 X81930cR. fascians DSM 20131 X53204 10

a,b,cR. globerulus DSM 43954 X80619a,c a a,cR. globerulus NCIMB12315 X81931 , X77779aR. globerulus DSM43953 X80620cR. globerulus Unpublished

cR. luteus DSM 43673 X79187a,b,cR. marinonascens DSM 43752 X80617 5a,cR. marinonascens ATCC35653 X81933

a,b,cR. opacus DSM 43205 X80630 8a,cR. opacus DSM 43206 X80631cR. opacus X89710a,bR. rhodnii DSM 43959 X80623 6aR. rhodnii ATCC35071 X81935aR. rhodnii DSM 43337 X80622a,cR. rhodnii DSM 43336 X80621

a,bR. rhodochrous DSM 43241 X79288 6cR. rhodochrous X70295aR. rhodochrous CIP1759.88 X81936 6

a,bR. roseus DSM 43274 X81921 6a,b,cR. ruber DSM 43338 X80625 6

cRhodococcus sp. NZRM4028cRhodococcus sp. NZRM4029cRhodococcus sp. X89240cRhodococcus sp. DSM 43943 X80616

All Rhodococcus sp. grown in Tryptic Soy at 30 8C except R. coprophilus, which was grown with Brain Heart infusion at 30 8C, and R.equi, which was grown with Brain Heart infusion at 35 8C.Key: ATCC—American Type Culture Collection, NCTC—National Collection of Type Cultures, DSM—Deutsche Sammlung vonMikroorganismen, NCDO—National Collection of Dairy Organisms, CIP—Collection of Institute Pasteur, NCIMB—National Collectionof Industrial and Marine Bacteria.

a DNA sequences used to design primers.bSpecies tested by PCR.c DNA sequences used to design TaqMan probe.

Ž .acetate ethanol precipitation Sambrook et al., 1989 .The DNA was dissolved in 20 ml of double deionisedŽ .dd H O and stored at y20 8C.2

The amount of DNA in all samples was quanti-fied, and the purity determined using a ShimadzuUV240 spectrophotometer at two absorbance wave-

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368 359

Table 2The actinomycete genera related to Rhodococcus, and growth conditions, as used in this study

Genus and species Source EMBL No. Days growthbActinomyces naeslundii NCTC 10301 6

cCorynebacterium glutamicum ATCC 13032 Z46753a,bCorynebacterium xerosis ATCC 373 X81914 6a,cCorynebacterium xerosis DSM20743 X84446

c( )Dietzia maris R. maris DSM43672 X79290c( )Gordonia aichiensis R. aichiensis DSM43978 X80633

b( )Gordonia bronchialis R. bronchialis NZRM3171 6a a a( )Gordonia bronchialis R. bronchialis DSM43247 X79287 , X75903a( )Gordonia bronchialis R. bronchialis CIP1780.88 X81919

b( )Gordonia rubropertincta R. rubropertincta DSM 43197 X80632c( )Gordonia sputi R. chubuensis DSM 44019 X80627 8

b,c c b,c( )Gordonia terrae R. terrae DSM 43249 X53202 , X79286cNocardia asteroides DSM43757 X80606 7

bNocardia brasiliensis NCTC 10300aNocardia brasiliensis DSM43758 X80680 6

a,bNocardia calcarea DSM 43188 X80618b,cNocardia corynebacteroides DSM20151 X80615 7

cNocardia otidiscaÕiarum Unpublisheda,bNocardia transÕalensis DSM 43405 X80609

cNorcardioides simplex Unpublished 10bStreptomyces griseus NCTC 7807

bTsukamurella paurometabola DSM 20162 X80628 6cTsukamurella paurometabolum Unpublished 7

cTsukamurella wratislaÕiensis NCIMB13082 Z37138

All species were grown in Brain Heart infusion at 30 8C except A. naeslundii, C. xerosis, G. bronchialis, N. brasiliensis and S. griseus,which were grown at 37 8C. Key as in Table 1.

lengths, 260 and 280 nm according to the method ofŽ .Sambrook et al. 1989 . Samples of approximately

200 mg mly1 were prepared for PCR, whereverpossible.

2.5.2. From faecal samplesThree different methods of extracting seeded R.

coprophilus DNA from human and animal faecalsamples were tested. They were those of Boom et al.Ž . Ž . Ž .1990 , Cohen et al. 1994 , and Kreader 1995 .

Ž .From the results of these tests see Results , a methodwas developed which was largely a composite of the

Ž . Ž .methods of Cohen et al. 1994 and Kreader 1995 .In addition, the cell lysis steps were further modifiedby the addition of 10% hexadecyl trimethyl ammo-

Ž .nium bromide CTAB r0.7 M NaCl and 10% b-mercaptoethanol during incubation.

One gram of a faecal sample was resuspended inŽ .2.5–5 ml of TE containing 0.5% vrv Triton-X

100. The sample was left overnight at 2–8 8C, and 2ml of supernatant was centrifuged at 12,000=g.The pellet was resuspended in 34 ml 5 M NaCl, andthe following solutions added: 150 ml lysing solutionŽ50 mM EDTA, 50 mM Tris HCl, 20% sucrose in

.ddH O , 27 ml 10% CTABr0.7 M NaCl, 5 ml 10%2Žb-mercaptoethanol, and 20 ml lysozyme 50 mg

y1 .ml . Incubation was at 37 8C for 30 min.Ž .A solution of 5 M NaCl 166 ml was added,Žfollowed by 1 ml lysing buffer 100 mM NaCl, 25

.mM EDTA, 10 mM Tris HCl and 0.5% SDS , 20 mlŽ y1 .proteinase K 50 mg ml , 134 ml 10% CTABr0.7

M NaCl, and 40 ml 10% b-mercaptoethanol, andincubated at 60 8C for 60 min. Thereafter, the method

Ž .of Kreader 1995 was followed—incubation at 658C for 10 min, extraction with chloroform, incuba-tion with CTABrNaCl, chloroform extraction, phe-nolrchloroform extraction and ethanol precipitation.The DNA was subjected to a final clean up proce-

Ždure using the DNA-bind method 1% wrv diatoma-

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368360

Table 3The bacterial genera not closely related to Rhodococcus, andgrowth conditions, as used in this study

Genus and species Source EMBL No. Daysgrowth

a,bAeromonas hydrophila ATCC 7966 X74677 2a,bAeromonas hydrophila DSM30187 X87271a,bAeromonas hydrophila ATCC7966 X60404a,bAeromonas hydrophila ATCC7966 X74676

bBacillus cereus ATCC 10702 2bBacillus subtilis ATCC 6051 2aBacillus subtilis NCDO1769 X60646

bEnterobacter aerogenes ATCC 13048 1bEnterococcus faecalis ATCC 19433 2

bEscherichia coli ATCC 25922 X80724 1bMorganella morganii ATCC 25830 2

bPseudomonas aeruginosa ATCC 25668 2bStaphylococcus aureus ATCC 25923 2

bStaphylococcus epidermidis ATCC 12228 2

All species were grown in Brain Heart infusion at 35 8C. Key asin Table 1.

.ceous earth-Sigma D5384 of Carter and MiltonŽ .1993 . The supernatant was transferred to a cleantube and centrifuged to pellet any remaining diatomsthat would inhibit PCR. All controls were performedusing the same method.

This extraction method was applied to 1-g sam-ples from cows, sheep, deer, horse, pig, rabbit, duck,possum and human faeces to determine the presenceor absence of naturally occurring R. coprophilus. A

Ž .duplicate sample of each faecal sample 1 g wasŽ 6seeded with R. coprophilus cells 1.6=10 CFU as

.determined by the plate count method as positiveextraction controls. A sample of a pure culture of R.

Ž 6 .coprophilus 1.6=10 CFU was extracted as apositive control, and a reagent blank, which con-tained all components of the extraction procedureexcept R. coprophilus cells or faecal material, wasused as a negative control.

2.6. ConÕentional PCR system design and optimiza-tion

DNA sequences from 36 bacterial isolates repre-senting 16 different species were aligned using two

Žsequence alignment packages—DNAMAN Lynnon.Biosoft, Version 2.5 , and the Wisconsin Package

Ž .GCG, Madison, WI, USA . Details of the organisms

Ž .including EMBL numbers are presented in Tables1–3. The related genera were chosen from both a16S rRNA gene dendrogram generated in this study,

Žand a published 16S rRNA gene dendrogram Rai-.ney et al., 1995 . Species that had similar 16S rRNA

gene sequences, particularly in the two priming re-gions, were selected for laboratory testing by PCR.

Two primers potentially specific for R. coprophi-lus were selected: a forward primer 5X-GGG TCT

X Ž .AAT ACC GGA TAT GAC CAT-3 Tm 58 8C anda reverse primer 5X-GCA GTT GAG CTG CGG

X Ž .GAT TTC ACA C-3 Tm 71 8C . The primer se-quences were checked using Basic Local Alignment

ŽTool BLAST: http:rr www.ncbi.nlm.nih.govr.BLASTr to ensure no fortuitous sequence comple-

mentarities, and the primers were synthesized byŽ .Life Technologies USA .

The amplification conditions were obtained byindividually optimising the annealing temperatureŽ . Ž .55–65 8C , MgCl concentration 1–3.5 mM ,2

Ž .primer concentration 2–10 pmol , template DNAŽ . Žconcentration 0.2–40 ng , dNTP concentration 5–

. Ž .300 mM , and AmpliTaq Applied Biosystems, USAŽ .concentration 0.5–3.0 units . The final format cho-

sen for amplification was: 2 ng template DNA, 10w xmM Tris HCl pH 8.4 , 50 mM KCl, 2.5 mM

MgCl , 150 pmol of each dNTP, 2.5 units AmpliTaq2

and 5 pmol of each primer. The 100 ml reactionmixture was overlaid with a drop of sterile mineraloil and thermal cycled in a Hybaid Omnigene ther-mal cycler. The thermocycle program consisted ofthe 40 cycles of 1 min at 94 8C, 1 min at 65 8C, 1min at 72 8C with a final 8 min extension at 72 8C.

A reagent blank that contained all the componentsof the reaction mixture, with the exception of tem-

Ž .plate DNA which was substituted by sterile ddH O ,2

was included in every set of samples as a negativePCR control. Similarly, a sample containing a DNAextract from a pure culture of R. coprophilus wasincluded as a positive PCR control. The PCR was

Žtested against other Rhodococcus species 13 species,. ŽTable 1 , related actinomycetes eight genera, Table

. Ž2 and distantly related micro-organisms seven gen-.era, Table 3 .

Reaction products were detected by the appear-ance of the expected 443 bp fragment upon elec-

Ž .trophoresis in 2% wrv agarose gels stained withethidium bromide and visualised under UV light.

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368 361

2.7. Determination of conÕentional PCR detectionlimit

R. coprophilus was cultured for 8 days and seri-ally diluted in quarter-strength Ringer solution to10y7, and the inoculum count determined. A volumeŽ .500 ml of each dilution was then inoculated into 1

Ž .g of either gamma-irradiated dosage: 26.7 kilograyscow faeces, non-irradiated rabbit faeces, or Ringer

Ž .solution as a control and extracted as described asabove. All samples were then tested using PCR todetermine the presencerabsence of R. coprophilus.

2.8. TaqMane PCR system design

The conventional PCR system was modified bythe addition of a new TaqMane probe. The probewas designed based on the alignment of 16S rDNAsequences of two R. coprophilus wild type strains

Ž .with 39 related species Tables 1 and 2 . The se-quences were aligned using evolutionarily conserved

Ž .primary and secondary structures Woese et al., 1983Žusing the Olsen Sequence Editor Gary J. Olsen,

.University of Wisconsin . The TaqMane probe se-Žlected 6FAM-ATG CAT GTC CTG TGG TGG

.AAA GGT TTA CTG-TAMARA, Tm of 69 8C wassynthesised by Applied Biosystems.

2.9. TaqMane PCR optimisation, amplification andanalysis of data

TaqMane PCR amplification was performed in atotal reaction volume of 25 ml containing 1= Mas-

Ž .termix Applied Biosystems cat a 4304437 , 200nM probe, 300 nM of forward primer, 300 nM of

Ž .reverse primer and 2 ml template 100 ng . Theoptimum concentrations of primers and probe weredetermined by titration experiments with varyingamounts of primers and probe as recommended byApplied Biosystems. Thermal cycling was performedin an ABI Prism 7700 and consisted of two initialholds of 50 8C for 2 min and 95 8C for 10 min. Thefirst hold is required for optimal AmpErase uracil-

Ž .N-glycosylase UNG activity which prevents theamplification of previously amplified DNA. The sec-ond hold is required to activate AmpliTaq GoldDNA Polymerase. This was followed by 40–60 cy-cles of denaturation at 95 8C for 15 s and an

annealrextend step of 62 8C for 1 min. Each testwas performed in duplicate in the same run. Datawere analysed using the SDS 1.6.3 application soft-

Ž .ware Applied Biosystems . The threshold was set ata level which intersected the amplification curve atthe steepest exponential point of the plot, and whichresulted in equivalent Y intercepts for standardcurves.

Ž .All Rhodococcus 12 species and 11 strains fromŽeight related genera eight genera: marked in Tables

.1 and 2 except R. roseus but including R.Ž .rhodocrous DSM 432740 , were tested on the Taq-

Mane system to ensure no false positives wereobtained.

DNA standard curves were generated by amplifi-cation on 12 separate occasions of serial 10-folddilution of R. coprophilus DNA. Dilutions of theDNA were stored at y20 8C between use. Standardcurves of R. coprophilus cells were prepared byserial 10-fold dilution of freshly grown cells. DNAwas extracted using the pure culture extractionmethod as described above, and was added to thereactions. The C ’s were plotted against log input ofT

DNA or cells as appropriate.

3. Results

3.1. Sequences of 16S genes in isolates of R. co-prophilus from cow faeces

The 16S rRNA gene sequence from the two NewŽZealand cow isolates EMBL accession numbers

.X89784 and X89785 had a 99.6% homology withthe 16S rRNA gene from R. coprophilus DSM

Ž .43347 strain EMBL X80626 . The sequence aroundthe primers and TaqMane probe designed to iden-tify R. coprophilus were the same in the two cow

Ž .isolates as the type strain DSM 43347 used todesign the primers.

3.2. PCR specificity

The two primers potentially specific for R. co-prophilus were selected from the 16S rRNA genesequences and amplified a 443-bp fragment from R.

Ž .coprophilus Fig. 2 . No amplification product wasŽobtained with any other Rhodococcus species 12

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368362

Table 4Plate count results showing natural level of R. coprophilus pergram of faeces

y1Faecal sample cfu g5Cow 1 3.3=106Cow 2 2.3=106Cow 3 3.6=105Cow 4 5.8=105Cow 5 3.9=105Cow 6 4.2=105Sheep 1 3.2=105Sheep 2 3.1=105Sheep 3 1.7=105Horse 2.7=103Deer 5.5=10

. Žspecies, Table 1 , related actinomycetes eight gen-.era, Table 2 or more distantly related micro-

Ž .organisms seven genera, Table 3 .

3.3. Naturally occurring leÕels of R. coprophilus inanimal faecal samples

Serial dilutions of animal faecal samples wereŽ .plated out on MM3 media Table 4 . The six cow

faecal samples tested gave an average of 1.3=106

R. coprophilus CFU gy1 of faecal material. Themean of three sheep faecal samples was 2.7=105

CFU gy1. The single horse sample was similar to thecow and sheep samples and contained 2.7=105

CFU gy1, while the count in deer was lower, at3 y1 Ž5.5=10 CFU g . All other animals tested pos-

.sum, duck, pig, rabbit and the five human samples,were negative. All colonies tested from the positivespread plates were confirmed as R. coprophilus byconventional PCR.

3.4. DNA extraction from faecal samples

With R. coprophilus inoculated human faecalŽ .samples the method of Boom et al. 1990 produced

no amplification product whilst the method ofŽ .Kreader 1995 produced a very weak amplification

Ž .product. The method of Cohen et al. 1994 gave astrong positive result with inoculated human faeceseven before the final purification using GlassMaxŽ .Life Technologies . However, when this methodwas applied to seeded and unseeded herbivore faecalsamples, no successful PCR amplification was ob-served. To obtain DNA suitable for PCR from herbi-vore faeces, it was necessary to add CTABrNaCland b-mercaptoethanol during incubation with eachof the proteolytic enzymes. Early addition of NaClalong with the CTAB kept the salt concentrationabove 0.5 M, thus preventing the formation andprecipitation of CTABrnucleic acid complexes, as

Ž .reported by Wilson 1994 . The addition of a thirdincubation of CTABrNaCl at 65 8C for 10 minŽ .Wilson, 1994 also further improved the purity of

Ž .the DNA results not shown . These additional treat-ments allowed DNA to be obtained that could beamplified from herbivore faecal material, whereasother methods tested, did not. We also substituted

Ždiatomaceous earth DNA bind, Carter and Milton,

Fig. 1. PCR results for DNA extracted from animal faecal samples which showed the presence of natural R. coprophilus. Lanes: 1 and 17,123 bp ladder; 2, positive PCR control; 3, negative PCR control; 4, positive R. coprophilus control; 5, negative control; 6, deer; 7, horse;8–10, sheep; 11–16, cow.

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368 363

Fig. 2. PCR results for DNA extracted from animal and human faecal samples which showed no natural R. coprophilus. Lanes: 1 and 20,123 bp ladder; 2, positive PCR control; 3, negative PCR control; 4, positive R. coprophilus control; 5, negative control; 6, seeded duckfaeces; 7, duck; 8, seeded possum; 9, possum; 10, seeded rabbit; 11, rabbit; 12, seeded pig; 13, Pig; 14, seeded human; 15–19, human.

. Ž1993 for GlassMax, observing superior results re-.sults not shown .

Using the modified extraction method, R. co-prophilus was identified in all six cow, three sheep,single horse, and single deer faecal samples tested,

Ž .as well as inoculated human faeces Fig. 1 , but notin possum, duck, pig, rabbit and the five uninocu-

Ž .lated human samples Fig. 2 .

3.5. Detection limit of conÕentional PCR in herbi-Õore faecal samples and pure cultures

Irradiation of the cow faecal sample did not com-pletely denature R. coprophilus DNA naturally pre-sent in the sample, since an uninoculated sampleyielded a R. coprophilus PCR product. We weretherefore unable to use the irradiated cow faecalsample as a means of determining the PCR detectionlevel. To overcome this problem non-irradiated rab-bit faeces were inoculated with a dilution series ofR. coprophilus. The detection limit was 60 CFU perPCR, when the dilution series was performed inrabbit faeces or Ringers solution.

3.6. R. coprophilus fluorogenic TaqMane speci-ficity

The specificity of the R. coprophilus primerswith the fluorogenic TaqMane probe was confirmedagainst the same species as the conventional PCRexcept R. roseus, which was not tested, and an

additional R. rhodocrous strain which was. Again,only R. cophrophilus produced a positive result.

3.7. QuantitatiÕe aspects and detection limits

The TaqMane system could reproducibly detectlevels of DNA as low as 10 pg from serially dilutedDNA and approximately one CFU from serially di-luted cells. Linear responses in TaqMane PCR were

Ž .observed over at least five log cycles Fig. 3a and b .Freshly diluted DNA, frozen and thawed up to

three times over a month’s period could be repro-ducibly amplified using the TaqMane system withcorrelation coefficients 0.997 and above, and slopes

Ž .ranging from y3.4 to y3.8 and Fig. 3a, runs 1–3 .The efficiencies of PCR amplification as determined

Žby the slope perfect slope indicating 100% effi-ciency y3.32192, John Pfeifer, personal communi-

.cation, Applied Biosystems for these three runswere all above 86%. The use of DNA frozen andthawed more than this resulted in progressively moredegraded DNA with C at each dilution increasing,T

Ž .and slopes above y4 for example run 8, Fig. 3a .Serial 10-fold dilutions of R. coprophilus cells

were extracted and amplified using the TaqMan sys-tem on four separate occasions. The plotted slopesclustered together with an average Y intercept of

Ž . Ž .42.19 s.d. 0.323 Fig. 3b . Correlation coefficientsabove 0.976 were observed on all occasions. Theefficiencies of PCR amplification, as determined bythe slope, were all above 90%.

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368364

Ž .Fig. 3. a Standard curves of TaqMane amplification of R. coprophilus DNA. A dilution series was prepared and frozen betweenŽ . Ž . Ž . Ž .amplifications. Runs 1 open triangles , 2 circles , and 3 squares took place within a month while run 8 closed triangles was amplified

Ž .three months later. b Standard curves of TaqMane amplification of R. coprophilus cells. Four separate dilution series of R. coprophilusŽ . Ž . Ž . Ž .cells were extracted and amplified. Run A open triangles , B circles , C squares and D closed triangles .

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368 365

4. Discussion

Assays to distinguish between animal and humansources would be useful, both as general faecalpollution management tools and because of a percep-tion of a greater risk associated with human inputs.Although R. coprophilus was first proposed as ananimal source indicator around 20 years agoŽ .Rowbotham and Cross, 1977 it has not been adoptedfor this purpose, primarily because of a lengthy

Ž .culture procedure Jagals et al., 1995 . PCR has thepotential to overcome this time-consuming cultureproblem.

This study addressed two issues associated withPCR-based methods for detecting R. coprophilus:firstly, the selection of primer and probe sequenceswhich allowed the amplification of a portion of DNAspecific to the organism, and secondly the develop-ment of techniques to overcome the PCR inhibitorspresent in the faeces of herbivores.

The DNA primer sequences selected from the 16SrRNA gene region appear to be highly specific for R.coprophilus. Both conventional and TaqMane PCRreactions, with the specific primers and probe, wereable to distinguish R. coprophilus from all the otherorganisms tested. The range of organisms testedprovided a representation of genetically closely re-lated genera and other micro-organisms common inthe environment.

Faecal material from herbivores causes greaterinhibition of PCR than human faeces. This may bebecause of the large amounts of plant material pre-sent containing phenolic compounds which areknown to bind to DNA, so preventing its use as a

Ž .PCR substrate Kreader, 1995 . It is possible that theearly addition of b-mercaptoethanol in the protocolthat we developed protected the released DNA fromphenolic compounds since it is used as an OHØ

Ž .radical scavenger Stanton et al., 1993 . Polysaccha-rides and tannins present in plants have also been

Žreported to co-purify with DNA Murray and.Thompson, 1980 and are potentially strong PCR

Ž .inhibitors Kreader, 1995; Muhammad et al., 1994 .CTAB is normally added after lysis during the

Ž .extraction step Kreader, 1995 to complex and pre-cipitate any such proteinrpolysaccharides presentŽ .Wilson, 1994 . We decided that the same level ofprotection should be applied during the actual lysis

of the organism, when the DNA was first released.This would reduce the time the DNA was exposed tothe phenolic compounds, polysaccharide and tannins,hence the addition of NaClrCTAB and b-mercapto-ethanol during both proteolytic steps.

All animal faecal samples determined positive byplate count were also positive by conventional PCR,indicating that no false negative results were ob-tained by this molecular method. The levels of R.coprophilus, as indicated by the plate count in sheepand cows were within, or slightly higher than the

Ž . 4range stated by Mara and Oragui 1981 of 6.9=10to 6.9=105 for cows and 1.7=104 to 2.5=106

CFU gy1 for sheep. Since multiple sampling wasonly conducted for cattle and sheep, it is probablyinappropriate to draw conclusions from results ofsingle samples of the remaining animal species tested.However, it was noted that all faecal samples thatcontained R. coprophilus were from ground grazingherbivores that generally congregate in high num-bers. The one horse sample examined was slightlyhigher with 2.7=105 CFU gy1 than the published

4 5 y1 Žcount of 1.8=10 to 2.2=10 CFU g Mara and.Oragui, 1981 . We present here what appears to be

the first publication of a farmed deer sample that hasbeen tested for R. coprophilus. Deer, pigs, horses,rabbits, possums and ducks were included in thesurvey because they are likely to contribute signifi-cantly to water pollution, particularly in rural NewZealand.

The five humans and single pig, possum, rabbitand duck samples were all negative for R. co-prophilus by the plate count method and conven-tional PCR. The animals in this study, whose faeceswere found not to contain R. coprophilus, do notfeed on pasture impacted by a large amount of faecalmaterial. Possums are largely arboreal feeders andare unlikely to ingest significant quantities of R.coprophilus. Similarly the rabbit sample was takenfrom a park devoid of livestock, and potentially withlittle exposure to R. coprophilus. Since ducks gener-ally feed in waterways and vegetation at the water’sedge the presence of R. coprophilus may depend onthe faecal load of the water. This is most likely to bethe cause of the discrepancy between our negativeresult for duck and the positive obtained by Mara

Ž .and Oragui 1981 . The pig in our study was housedin a piggery and pellet fed. Although Mara and

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368366

Ž .Oragui 1981 reported a positive result for pigs, itwas not clear whether the pigs were free range orhoused in piggeries. The former situation may ex-plain the discrepancy between these results.

The negative results for the human faecal samplesillustrate the potential use of R. coprophilus as anindicator of domesticated herbivore faecal pollution.

ŽThe detection limit of the PCR assays 60 CFU per.conventional and 1 CFU for TaqMane PCR is

sufficiently sensitive to detect R. coprophilus infaecal samples, since 1 g of faecal material containsbetween 5.5=103 and 3.6=106 CFU depending onthe animal.

By converting a conventional PCR system to aTaqMane PCR system, the sensitivity has beenincreased by an order of magnitude, the use of aprobe added another level of discrimination, and theidentification turned into an enumerative identifica-tion system. The correlation coefficients indicate aconsistent relationship existed between the amount

Ž .of template log input DNA or cells and the amountŽ .of product represented by C in the standard curves.T

The linearity apparent in Fig. 3a and b, of thestandard curves, and the fact that the PCR operates ata constant efficiency confirm that the assay is suitedfor quantitative measurements. Although R. co-prophilus has multiple copies of the 16S rRNA gene,this has not interfered with the linear relationshipbetween the amount of template and amount ofproduct. The detection limit of approximately 1 CFUper PCR is similar to those in other reports using afluorogenic 5X-nuclease PCR assay for endpoint de-

Ž .tection. Nogva et al. 2000 obtained a detection ofapproximately 1 CFU of C. jejuni and Chen et al.Ž .1997 reported a detection limit of 2 CFU forSalmonella enterica.

TaqMan amplification of diluted DNA standardshighlighted the danger of repeated freeze–thawing ofDNA. The DNA as measured by threshold cycle,became progressively more degraded with eachfreeze–thaw cycle, particularly in the lowest dilu-tions of DNA. Conventional PCR often makes use ofDNA standards of 100 ngrml, whose repeatedfreeze–thawing, while ill-advised, has little practical

Žconsequence as degradation is relatively slow Fig..3a, 100 ng point and in endpoint non-quantitative

PCR, undetectable. TaqMan PCR, however, uses adilution series of DNA down to 10 pg or less of

DNA, which is used to generate a standard curve andquantify unknowns. Severe degradation of these lowconcentration standards makes quantitation impossi-ble. The clearest indicator of an unusable standardcurve is the slope, which reflects the PCR amplifica-tion efficiency. From our observations slopes varyingby more than 15% from the optimum slope ofy3.32192 should not be used. Freshly diluted DNAstandards should ideally be frozen in aliquots andthawed only once. Experiments are currently under-way to assess the stability of dilute R. coprophilusDNA solutions in extended storage at y20 and y808C.

Extraction and amplification of serial 10-fold dilu-tions of R. coprophilus cells produced more usefulstandard curves. Equivalent numbers of cells whenextracted and amplified on different occasions pro-duced C s which could be plotted within a narrowT

Ž .band Fig. 3b . Quantitation is accurate to at least thelog level. This should be sufficient for water moni-toring purposes, where the key information requiredis whether there are small or large numbers of R.coprophilus present, and whether the number is in-creasing or decreasing.

It should be noted that there are two significantproblems associated with the use of R. coprophilusas an animal faecal indicator. First, the organismappears to be more persistent in the environmentthan traditional faecal indicators such as Escherichia

Ž .coli and faecal streptococci Mara and Oragui, 1983 .Thus, it may need to be used in conjunction withshort-lived animal indicators, such as S. boÕis to

Žindicate the proximity of the faecal source Oragui.and Mara, 1983 . Second, the species has been found

Ž .in activated sludge scums Sezgin et al., 1988 ,which can be composed of both human and animalwaste and its reported presence in river foams proba-

Žbly still originated from animal sources Al-Diwany.and Cross, 1978 . The development of this enumera-

tive TaqMane PCR method should help clarify thesetwo latter points.

PCR methods for the detection and enumerationof R. coprophilus provide a means to overcome amajor impediment to the use of this organism as ananimal faecal indicator, namely that of a lengthyculture period. Further research is now being under-taken in our laboratory to extend the study intoenvironmental effluents and waters.

( )M.G. SaÕill et al.rJournal of Microbiological Methods 47 2001 355–368 367

Acknowledgements

The authors wish to thank Miss Emily Gerard forher work in establishing the Rhodococcus spp. cul-tures; Dr. Andrew Hudson and Mr. Lester Sinton fortheir comments and suggestions on this work; andMr. Nick Garret and Mr. Jeff Foote for their statisti-cal analysis and guidance. This work was fundedfrom the New Zealand Public Good Science Fund,administered by the Foundation for Research Scienceand Technology.

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