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Detection of faecal pollution in water by an Escherichia coli uidA gene probe
Journal of. Microbiological Methods 13 ~,|. oql . . . . .~. 7f~7 . . . - 9 mA © 1991 Elsevier Science Publishers BN. 0167-7012/91/$ 3 50
~.v'~C~'7
MIMET 00429
Detection of faecal pol|ution in water by Escherichia coii uidA gene probe
an
David H. Green t, Gillian D. LeWiS2, Sureelak Rodtong ~ and Margaret W. l_ot~tit ~ 1Department o f MlcroOiology, University o f 9tago, Dunedin, N e w Zealand; 2Enviro~mental Science Programme, University oJ Auckland, Auckland, New Zealand
(Received 22 November 1990; revision received 28 February 1991; accepted 7 March 1991)
Summary This paper describes a method for the detection of faecal pollution in water, using a gene probe based an the Escherichia c~li K-12 gene, uidA, which encodes/3-glucuronidase (GUD). GUD has been reported to be produced by 97-99.5°70 ofE. coli and we report that all E. coil and Shigella strains tested, were reactive with the uidA gene probe, whether or not they were actively producing GU D. Other faecal and environmental bacteria tested were not reactive with the uidA gene probe. The water testing procedure described is based on the membrane filter technique and uses a non-selective enrichment period of 10 h at 37 °C prior to the membranes being hybridised with the uidA gene probe, after which up to 500 faecal coliforms could be counted per membrane.
Key words: Escherichia coli; Fecal pollution; Gene probe
Introduction
Development of a specific test to detect faecal pollution in water as an indicator of the possibie presence of disease-causing organisms is important in the interest of public health. Most methods currently available, detect not only bacteria from faeces but also from soil and veget,:,ion. Even those tests designed to detect the indicator E. coli, a bacterium which grows only in the gut of man and other warm-blooded animals, suffer from the deficiency that they do not detect cells that are viable but non-culturable [1]. Some methods incorporate a resuscitation step supposedly to break nop-growth or to revive damaged cells, as in the mTEC method [2]. However it has been our experience that mTEC medium gave a 10-fold lower recovery of E. coil stressed by exposure to seawater than did standard plate count agar (G. Gray-Young, pers. comm.). Correspondence to: M. W. Loutit, Department of Microbiology, University of Otago, Box 56, Dunedin, New Zealand.
208 Methods based on detection of specific nucleic acid sequences would seem to offer the best option to overcome these deficiences and the polymerase chain reaction (PCR), a method which enzymatically amplifies specific DNA sequences [3], has been suggested for application to water testing. Bej et al. [4] reported a method using PCR to ~est for coliform bacteria and E. coli based on amplification of iacZ ana lamb genes. Although highly sensitive, this method may not be suitable for waters in which environmental factors, such as humic acids, could inhibit enzymatic reactions and the method is not specific for faecal bacteria. Methods based on histochemical detection of the enzyme ~-glucuronidase (GUD) have been described for detection of E. coli [5-7], but detection of a translational product may not be entirely satisfactory. For example, Rice et al. [7] reported that 97-99.5°/o of E. coli produce GUD, and Chang et al. [8] reported the occurrence of a significant proportion (34°7o) of E. coli as GUD-negative. This paper describes an alternative approach in which the E. coli GUD coding gene, uidA, is the target for E. coli detection. Use of non-selective enrichment and a specific gene probe to detect non-culturable as well as growing E. coli in water samples, is the basis for the test to detect faecal pollution. Materials and Methods
Bacterial isolates Salmonella typhimurium strains were obtained from the Dunedin Public Hospital culture collection and the remaining strains (Table 1) from the University of Otago Microbiology Depactment cult..:;c c~l!ection. Strains of E. coli, Citrobacter, Enterobacter and Klebsiella (Table 2) were isolated from flesh and marine waters on mTEC (Difco~ a~ar and Simmon's Citrate agar (Difco) and identified on the basis of biochemical tests and API 20E analysis (La Balme Les Grottes, France). All isolates were stored at 4°C and subcultured on tryptic soy agar (TSA) (Difco). uidA gene probe preparation and labelling The uidA gene probe was prepared from the plasmid pGL1 by simultaneous restriction endonuclease digestion with XhoI and EcoRI. The resultant 4.3-kb uidA containing fragment was separated by agaro~e gel electrophoresis in Tris acetate buffer [9], purified by glass powder elution (GeneCiean kit, Bio 101, La Jolla, USA) and labelled with digoxigenin-ll-dUTP (DIG) by random priming (DIG DNA Labelling kit Boehringer Mannheim, FRG). Plasmid pGL1 was generated from pUC9 [10] and a 6.9-kb EcoRI-HindIII fragment from E. coil JM83 [10]. Presence of the uidA gene in pGLI was confirmed by complementation of the manA-uidA deletion of E. coli PK803 [11], restoring GUD expression, and by hybridisation (Southern blot) with the uidA fragment derived from pBKman (PK803 and pBKman kindly donated by P. Keumpel). uidA gene probe hybridisation Bacterial cells were grown on the positively charged nylon membrane (HybondN +, Amersham, UK) or dotted onto Hybond-N + membrane u~:inga vacuum dot blot
209 apparatus (BioRad, USA) The DNA of nlembrane-bound cells was released by alkaline treatment (0.5 M NaOH for 8 min) and the DNA was fixed to the hybridisation support (0.4 M NaOH for 20 rain). Membranes were prehybridised for 2 h at 68 °C and hybridised for 18 h at 68°C using solutions prepared as indicated fo~ "-- " " ~ DNA Detection kit (Bgehringer Mannheim)~ and the uidA gene probe was added to the t..,t..~n~.,~,.~,, ~nh,~iOn at a f i n a l concentration ot ~ 2 5 - 5 0 ng.ml -~ Posthybridisation washes and immunologic colour development were performed as recommended (Boehrin~er Mam, bei~). Specificity of uidA gene probe Specificity of the uidA gene probe was established by probing a range of organisms likely to be recovered from water by non-selective, aerobic enrichment at 37 °C. E. coli 611 and Vibrio anguillarum 775(A), were used as the positi~,e and negative controls, respectively. Washed cells of the bacterial strains to be tested (Table !) were diluted in 10xSSC (0.15 M NaCI, 0.015 M sodium citrate, pI-; 7) and dotted onto HybondN+ membrane and washed through with an equal volume of 10×SSC. The membranes were air-dried, treated with alkali and hybridised with the uidA gene probe, as described above. ~glucuronidase in situ agar plate assay Specificity of the uidA gene probe was paired with the absence or presence of GUD production. GUD production was determined by an in situ agar plate assay in which tempered TSA was supplemented with 5-bromo-4-chloro-3-indoyl-~-D-glucuronide (BCIG) at 100/zg.m! -~ (kindly donated by Biosynth Ag, Switzerland). The BCIG stock solution was prepared as described by Watkins et al. [6]. Bacterial species (Tables 1, 2) were inoculated onto the surface of the n£~ r by sterile tooth-pick and incubated at 37 °C for 24 h. Elaboration of GUD was scored as positive if the colony colouration was blue-green. Non-selective enrichment The optimum incubation time required for enrichment of cells was determined by using marine and freshwater san'iples and washed E. coli 611 cells diluted in sterile tap water to a concentration of = 1 cell .ml -j. Volumes (10O ml) were passed through a 47-mm diameter Hybond-N+ membrane (0.45-/zm porosity) held in a Swinn~.x-47 filter holder (Millipore, USA). The membranes were enriched on sterile absorbent paas saturated with tryptic soy broth (TSB) (Difco) and inct, bated at 37 °C for between 0 and 12 h. The membranes were treated with alkali and then hybridised with the uidA gene probe, as described. Analysis o f environmental water samples Water samples were collected from two fresh and three marine sites and testecl ~vithin 4 h of collection by the mTEC membrane filter [2] and the uidA gene probe methods. Freshwater (20 ml) and marine water (50 ml) Samples were filtered in duplicate, through 0.2-#m porosity membrane filters (Advantec, MFS, USA) and 47-mm diameter Hybor, d-N+ membranes held in a Swinnex-47 filter holder. Filtered samples
210 were then washed with 50 ml of sterile tap water. Advantec membrane filters were placed aseptically on mTEC agar and incubated for 2 h at 37 °C, then at 44.5 °C for 2 0 - 22 h. Each filter was further incubated on urease substrate for 10-15 min and yellow colonies counted as presumpti',e E. col; Hybond-N+ membranes were placed aseptically on a sterile pad of Whatman (Qualitative 1) filter paper saturated with TSB and incubated in a sealed container for 10 h at 37 °C. Following incubation, membranes were treated with alkali, fixed and hybridised with the uidA gene probe at SO ng.m! -I. Mu!t,.'~!e membranes p~-ocessed concurrently were prehybridised and hybridised by stacking each membrane between Whatman 504 filter paper (5.5 x 5.5 cm) soaked in the appropriate solution. Colonies reactive with the uidA gene probe were counted directly from the filter following colour development.
,'ia.dysis o f non-growing cells Non-growing E. coil cells were induced by inoculation of washed, log phase E. coil cells at 1 × 10~ cells .ml -l (E. coil AB 259 (Table 1) and E. coli V~ an environmental isolate) into artificial seawater [12] and incubated with the exclusion of light at 15 °C for a total of 15 wk (G. Gray-Young, pers. comm.). Duplicate samples (10 ml) were removed from these cultures and filtered onto Hybond-N+ of Advantec membrane filters and incubated for 18 h at 37 °C on TSB saturated pads or mTEC agar, respectively. Enumeration of E. coii was by either uidA gene probe or incubation on urease substrate. Results
The DNA sequence chosen for the detection of faecal pollution was the E. coli K-12 gene, uidA. A 4.3-kb XhoI-EcoRI fragment containing uidA was isolated from the plasmid pGLI and used as the gene probe. The uidA gene resides in the first 2 kb of DNA sequence and the remaining DNA sequence (2.3 kb) is E. coli K-12 chromosomal D N A d o w n s t r e a m nf . . . . . . . . . . . . . . . . . . . . .
.
uidA )~-~)
r@t~in~rl *~***'~u
tr~ l,v
n~t-~m~ . . . . . . ~,e;..~;,,. ÷~ Vl..,i.zaaaao)... a l j ~ l t l ~ . l L y tO
17'. ~ l : ^--..i .... :--:--~td,. LI./Lg (;I.IIU l l l g [ ~ . ~ l l l l ~
sensitivity. The 4.3-kb uidA gene probe was reactive with all laboratory strains (Table 1) of E. coli and Shigella (S. boydii and S. dysenteriae) tested, all of which produced GUD. Salmonella isolates, S. typhimurium, S. arizonae, S. enteritidis and remaining bacteria in Table 1 were not reactive with the uidA gene probe and did not produce GUD. Of the presumptive E. coli isolates (Table 2), 83 of the 89 were confirmed as E. coli by biochemical tests and API 20E analysis. The remaining six were shown to be species of Klebsieila and Enterobacter, equating to a 6.7o/0 false-positive result. All 83 E. coli ~olates were reactive with the uidA gene ~.robe even though eight (9.60/0) did not produce GUD. No reaction with the uidA gene probe or GUD production was observed in any Citrobacter, Enterobacter or Klebsiella isolates tested (Table 2). The incubation time required to grow microcolonies, which gave a visible hybridisation signal was 8 h for E. coli 611 and l0 h for bacteria from water samples (data not shown). Incubation of membrane filters for longer periods did not improve the hybridisation signal when high numbers of faecal coliforms were present in the water sample, as interference between microcolonies resulted in a poorly defined hybridisation signal.
211 TABLE 1 B A C T E R I A L S T R A I N S T E S T E D F O R / 5 - G L U C U R O N I D A S E (GUD) P R O D U C T I O N A N D R E A C TIVITY WlTH uidA GENE PROBE ~act:ria! s~ec!es
:,*
E s c h e r i c h i a coil 611 ( N C T C 8622) E. coli AB259 Salmonella typhimurium S. a r i z o n a e S. enterittdis Shigella b o y d i i type 3 S h . b o y d i i type 7 Sh. d y s e n t e r i a e E n t e r o b a c t e r aerogenes ( N C T C 8197) C i t r o b a c t e r f r e u n d i i ( N C T C 9750) K l e b s i e i l a p n e u m o n i a e ( N C T C 9633) Serratia m a r c e s c e n s Vibrio a n g u i l l a r u m 775(A) P r o t e u s vulgaris Enterococcus faecalis
1 1 16
*Total number o f isolates;
,_,,_,D
,.,U~
1 1 -
uidA +
uidA -
|
1
! 1
16 !
-
16
-
!
1
-
1
--
I
--
1 1
1 1
--
l
--
1
!
--
1
- -
1 1 !
-
1
-
1
-
!
I
-
1
1
-
1
-
!
1
-
1
-
1
1 1
-
1
-
1
1
-
1
1
- , negative result.
TABLE 2 ENVIRONMENTAL ENTEROBACTERIACEAE ISOLATES TESTED FOR/~-GLUCURONIDASE (GUD) P R O D U C T I O N A N D R E A C T I V I T Y W I T H u i d A G E N E P R G B E Bacterial species*
n
GUD ÷
GUD-
uidA +
uidA -
E s c h e r i c h ! a coil C i t r o b a c t e r sp. Klebsieila pneumoniae Klebsiella oxytocae E n t e r o b a c t e r cloacae E n t e r o b a c t e r sp.
83 ii
75 -
8 II
83
!I
3 1 1
-
3 1 1
-
3 1 1
42
-
42
-
42
*Bacteria were isolated f r o m fresh and marine water using m T E C and S i m m o n ' s c-'.trate agar ~nd identity by biochemical tests and A P I 20E analyses. -, negative result.
confirmed
An initial study compared mTEC agar and TSB for resuscitation of non-culturable E. coli. Detection of E. coli AB 259 (mTEC, 27 cfu.10 ml-~; uidA 93 cfu.10 m1-1) and E. coli V ! (mTEC 90 cfu.10 ml-~; uidA 1056 cfo .10 ml -I) resulted in the uidA gene probe method enumerating 3.4- and ll.7-fold mo~e E. coli than did mTEC agar. A further study directly compared the mTEC [2] and uidA gene probe methods by examination of marine and freshwater samples; the results are given in Table 3. Figure I shows an mTEC membrane filter and a uidA probed membrane filter after a water sample has been processed. Discolouration evident on the mTEC membrane filter did not cause any problem in hybridisation and reading, of the uidA probed membrane.
212 TABI E 3 DE ~E(2l ION OF E. C O L I BY MTEC METHOD AND uidA GENE PROBE ANALYSIS OF FRESH AND MARINE WATER SAMPLES Sample type"
Freshwater A B C D E Marine water F G H
Presumptive E. coli on mTEC medium cfu- I00 m l - I
Confirmed E. coli bv uidA gone probe colonies. I00 ml I
595 398 318 170 110
680 238 208 143 133
24 102 142
30 81 332
*Samples B - E , G and H were collected after heavy rain.
~
J
Fig. !. Comparison of E. coli detection fro"n marine samples by mTEC and uidA gene probe. Note heavy precipitate of solids on mTEC membrane filter. This solid did not affect detection of colonies by uidA gene probe.
Discussion Membrane filtration and DNA hybridisation techniques, have been combined in a
213 sensitive and specific method lbr detection of faecal pollution in fresh and marine waters. The DNA sequence on which the gone probe is based contains the E. coil uidA gene and downstream E. coli chromosomal DNA. The uidA gone probe is specific for all E. coli examined and the enteric pathogen Shige(la. In this study none of the Salmonella isolates were reactive with the uidA gene probe, though, between 17 and 30°70 of Sabnonella have been reported to produce GUD [5], additionall); Le Minor lt~ reported that production of GUD by Saimonefia was restricted to specific serotypes. The Salmonella tested in this study were of species reported not to produce • ,,,po, t,~n,y, ,::heability ,,,'-~'*~"~,,~uidA gone probe to detect Shigella and perhaps some serotypes of Salmonella does not detract from the use of this probe as an indicator of faecal pollution. Environmentally derived members of the coliform group (Citrobacter, Enterobacter and Klebsiella) were not reactive with the uidA gene probe, although some species of Citrobacter and Enterc, bacter have been reported to produce GUD [6]. Analysis of water samples (Table 3) resulted in the mTEC method recording higher numbers of presumptive E. colt than the uidA probe for half of the water samples analysed. This occurred most notably in, freshwater samples collected after heavy rain. The mTEC method is recognised to produce i0 - 40% false-positive colonies (predominantly Klebsiella and Enterobacter species) in temperate and tropical waters [2, 14] and the absence of uidA gene probe reactivity with these soil and vegetation derived organisms suggests that following heavy rain or at times of high organic loading the mTEC method may overestimate the number of E. coil in water. The resuscitation of artificial seawater "stressed" E. coil on non ~elective medium (TSB) and uidA gene probe detection, was 3 and 11 times more effective than on mTEC medium, suggesting that a higher proportion of injured or non-culturable cells in water samples would be detected by the uidA gone probe method; this wa,; apparent in both marine and freshwater samples in this study (Table 3). The sensitivity of the uiclA gene probe requires that the membrane and adhering ~_eLsbc _~,_... . . . . ~_._,j_, iij n to produce a visible hybridisation signal. H_owever,the small area of the hybridisation signal was not adversely affected by background signal due c ¢'x¢'X to organic material in the water sample. In our work an upper limit of as many as .,vv uidA reactive colonies per 47 mm diameter membrane allowed reliable enumeration and were in general, more easily counted than those on mTEC medium on which the often large mucoid colonies tended to overgrow one another and only up to 50 cfu could be reliably counted. Use of the uidA gone probe method to analyse water samples for faecal pollution has the sensitivity to detect if present, a single faecal isolate after a period of 10 h enrichment. The uidA gene probe is specific for E. coil, Shigella and possibly some Salmonella, making this procedur,, a suitable alternative to the existing procedures tbr the detection of faecal pollution in water samples.
Acknowledgements We wish to thank P. Keumpel for generously donating pBKman and PK803 (University of Colorado, Boulder, Colorado) and Gillian Gray-Young (M.Sc. thesis), Jan Hesketh and Karen Robert for assistance in completion of this study.
214
References I Roszak, D.B. and Colwell, R.R. (1987) Survival strategies of bacteria in the natural environment. Microbial. Rev. 51, 365-379. 2 Dufour, A. P., Strickland, E. R. and Cabelli, V.J. (1981) Membrane filter method for enumerating Escherichia coiL A ~ ! . Environ. Microbial. 41, 1152-1158. 3 Saiki, R. K., Gelfand, D. H., Stoffe!, S., Scharf, S. J., Higuchi, R., Horn, G.T., Mullis, K. B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. 4 Bei, " ~ L. and Atlas, R.M. (1990) Detection . . . . " ,,l . . . cuhiorm .... . A.K.,Steffan, R.J.,~eCe~aJe,J., r~ - - " Hiatt, bacteria in water by polymerase chain reaction and gene probes. Appi. Environ. Micr,3biol. 56, 307-314. 5 Feng, P. C. S. and Hartman, P.A. (1982) Fluorogenic assays for immediate confirmation of Escherichia coiL Appl. Environ. Microbial. 43, 1320-1329. 6 Watkins, W. D., Rippey, S. R., Claw.i, C. R., Kel!ey-Reitz, D. J. and Burkhardt III, W. (1988) Novel compound for identifying Escherichia coiL Appl. Environ. Microbial. 54, 1874-1875. 7 Rice, E.W., Allen, M.J. and Edberg, S.C. (1990) Efficacy of B-glucuronidase assay for identification of Escherichia coil by the defined-substrate technology. Appl. Environ. Microbial. 56, 1203-1205. 8 Chang, G. W., Brill, J. and Lum, R. (1989) Proportion of/3-D-glucuronidase-negative Escherichia coli in human ;'ecai samples. Appi. Environ. Microbioi. 55, 335-339. 9 Dillion, J.A.R., Nasim. A. and Nestmann, E. R. (195) Recombinant DNA Methodology, 20 pp., John Wiley and Sons, USA. 10 Vieira, E and Messing, J. (1982) The pUC plasmids, an Ml3mp7-derived system for insertion mutagenesis and sequencing with synthetic primers. Gene 19, 259-268. 11 Jefferson, R.A., Burgess, S. M. and Hirsh, D. (1986) B-glucuronidase from Escherichia coil as a genefusion marker. Proc. Natl. Acad. Sci. U.S.A. 83, 8447-8451. 12 Austin, B. (1988) Marine Microbiology, p. 24, Cambridge University Press. 13 LeMin~r~ L. (~979) Tetrathi~nate-reductase~ ~g~ucur~nidase~ and ~NPG-test in the genus Sa~m~ne~a. Zbl. Bakt. Hyg. Abt. Orig. A 243, 321-325. 14 Santiago-Mercado, J. and Hazen, T. C. (1987) Comparison of four membrane filter methods for fecal coliform enumeration in tropical waters. Appl. Environ. Microbial. 53, 2922-2928.
~.v'~C~'7
MIMET 00429
Detection of faecal pol|ution in water by Escherichia coii uidA gene probe
an
David H. Green t, Gillian D. LeWiS2, Sureelak Rodtong ~ and Margaret W. l_ot~tit ~ 1Department o f MlcroOiology, University o f 9tago, Dunedin, N e w Zealand; 2Enviro~mental Science Programme, University oJ Auckland, Auckland, New Zealand
(Received 22 November 1990; revision received 28 February 1991; accepted 7 March 1991)
Summary This paper describes a method for the detection of faecal pollution in water, using a gene probe based an the Escherichia c~li K-12 gene, uidA, which encodes/3-glucuronidase (GUD). GUD has been reported to be produced by 97-99.5°70 ofE. coli and we report that all E. coil and Shigella strains tested, were reactive with the uidA gene probe, whether or not they were actively producing GU D. Other faecal and environmental bacteria tested were not reactive with the uidA gene probe. The water testing procedure described is based on the membrane filter technique and uses a non-selective enrichment period of 10 h at 37 °C prior to the membranes being hybridised with the uidA gene probe, after which up to 500 faecal coliforms could be counted per membrane.
Key words: Escherichia coli; Fecal pollution; Gene probe
Introduction
Development of a specific test to detect faecal pollution in water as an indicator of the possibie presence of disease-causing organisms is important in the interest of public health. Most methods currently available, detect not only bacteria from faeces but also from soil and veget,:,ion. Even those tests designed to detect the indicator E. coli, a bacterium which grows only in the gut of man and other warm-blooded animals, suffer from the deficiency that they do not detect cells that are viable but non-culturable [1]. Some methods incorporate a resuscitation step supposedly to break nop-growth or to revive damaged cells, as in the mTEC method [2]. However it has been our experience that mTEC medium gave a 10-fold lower recovery of E. coil stressed by exposure to seawater than did standard plate count agar (G. Gray-Young, pers. comm.). Correspondence to: M. W. Loutit, Department of Microbiology, University of Otago, Box 56, Dunedin, New Zealand.
208 Methods based on detection of specific nucleic acid sequences would seem to offer the best option to overcome these deficiences and the polymerase chain reaction (PCR), a method which enzymatically amplifies specific DNA sequences [3], has been suggested for application to water testing. Bej et al. [4] reported a method using PCR to ~est for coliform bacteria and E. coli based on amplification of iacZ ana lamb genes. Although highly sensitive, this method may not be suitable for waters in which environmental factors, such as humic acids, could inhibit enzymatic reactions and the method is not specific for faecal bacteria. Methods based on histochemical detection of the enzyme ~-glucuronidase (GUD) have been described for detection of E. coli [5-7], but detection of a translational product may not be entirely satisfactory. For example, Rice et al. [7] reported that 97-99.5°/o of E. coli produce GUD, and Chang et al. [8] reported the occurrence of a significant proportion (34°7o) of E. coli as GUD-negative. This paper describes an alternative approach in which the E. coli GUD coding gene, uidA, is the target for E. coli detection. Use of non-selective enrichment and a specific gene probe to detect non-culturable as well as growing E. coli in water samples, is the basis for the test to detect faecal pollution. Materials and Methods
Bacterial isolates Salmonella typhimurium strains were obtained from the Dunedin Public Hospital culture collection and the remaining strains (Table 1) from the University of Otago Microbiology Depactment cult..:;c c~l!ection. Strains of E. coli, Citrobacter, Enterobacter and Klebsiella (Table 2) were isolated from flesh and marine waters on mTEC (Difco~ a~ar and Simmon's Citrate agar (Difco) and identified on the basis of biochemical tests and API 20E analysis (La Balme Les Grottes, France). All isolates were stored at 4°C and subcultured on tryptic soy agar (TSA) (Difco). uidA gene probe preparation and labelling The uidA gene probe was prepared from the plasmid pGL1 by simultaneous restriction endonuclease digestion with XhoI and EcoRI. The resultant 4.3-kb uidA containing fragment was separated by agaro~e gel electrophoresis in Tris acetate buffer [9], purified by glass powder elution (GeneCiean kit, Bio 101, La Jolla, USA) and labelled with digoxigenin-ll-dUTP (DIG) by random priming (DIG DNA Labelling kit Boehringer Mannheim, FRG). Plasmid pGL1 was generated from pUC9 [10] and a 6.9-kb EcoRI-HindIII fragment from E. coil JM83 [10]. Presence of the uidA gene in pGLI was confirmed by complementation of the manA-uidA deletion of E. coli PK803 [11], restoring GUD expression, and by hybridisation (Southern blot) with the uidA fragment derived from pBKman (PK803 and pBKman kindly donated by P. Keumpel). uidA gene probe hybridisation Bacterial cells were grown on the positively charged nylon membrane (HybondN +, Amersham, UK) or dotted onto Hybond-N + membrane u~:inga vacuum dot blot
209 apparatus (BioRad, USA) The DNA of nlembrane-bound cells was released by alkaline treatment (0.5 M NaOH for 8 min) and the DNA was fixed to the hybridisation support (0.4 M NaOH for 20 rain). Membranes were prehybridised for 2 h at 68 °C and hybridised for 18 h at 68°C using solutions prepared as indicated fo~ "-- " " ~ DNA Detection kit (Bgehringer Mannheim)~ and the uidA gene probe was added to the t..,t..~n~.,~,.~,, ~nh,~iOn at a f i n a l concentration ot ~ 2 5 - 5 0 ng.ml -~ Posthybridisation washes and immunologic colour development were performed as recommended (Boehrin~er Mam, bei~). Specificity of uidA gene probe Specificity of the uidA gene probe was established by probing a range of organisms likely to be recovered from water by non-selective, aerobic enrichment at 37 °C. E. coli 611 and Vibrio anguillarum 775(A), were used as the positi~,e and negative controls, respectively. Washed cells of the bacterial strains to be tested (Table !) were diluted in 10xSSC (0.15 M NaCI, 0.015 M sodium citrate, pI-; 7) and dotted onto HybondN+ membrane and washed through with an equal volume of 10×SSC. The membranes were air-dried, treated with alkali and hybridised with the uidA gene probe, as described above. ~glucuronidase in situ agar plate assay Specificity of the uidA gene probe was paired with the absence or presence of GUD production. GUD production was determined by an in situ agar plate assay in which tempered TSA was supplemented with 5-bromo-4-chloro-3-indoyl-~-D-glucuronide (BCIG) at 100/zg.m! -~ (kindly donated by Biosynth Ag, Switzerland). The BCIG stock solution was prepared as described by Watkins et al. [6]. Bacterial species (Tables 1, 2) were inoculated onto the surface of the n£~ r by sterile tooth-pick and incubated at 37 °C for 24 h. Elaboration of GUD was scored as positive if the colony colouration was blue-green. Non-selective enrichment The optimum incubation time required for enrichment of cells was determined by using marine and freshwater san'iples and washed E. coli 611 cells diluted in sterile tap water to a concentration of = 1 cell .ml -j. Volumes (10O ml) were passed through a 47-mm diameter Hybond-N+ membrane (0.45-/zm porosity) held in a Swinn~.x-47 filter holder (Millipore, USA). The membranes were enriched on sterile absorbent paas saturated with tryptic soy broth (TSB) (Difco) and inct, bated at 37 °C for between 0 and 12 h. The membranes were treated with alkali and then hybridised with the uidA gene probe, as described. Analysis o f environmental water samples Water samples were collected from two fresh and three marine sites and testecl ~vithin 4 h of collection by the mTEC membrane filter [2] and the uidA gene probe methods. Freshwater (20 ml) and marine water (50 ml) Samples were filtered in duplicate, through 0.2-#m porosity membrane filters (Advantec, MFS, USA) and 47-mm diameter Hybor, d-N+ membranes held in a Swinnex-47 filter holder. Filtered samples
210 were then washed with 50 ml of sterile tap water. Advantec membrane filters were placed aseptically on mTEC agar and incubated for 2 h at 37 °C, then at 44.5 °C for 2 0 - 22 h. Each filter was further incubated on urease substrate for 10-15 min and yellow colonies counted as presumpti',e E. col; Hybond-N+ membranes were placed aseptically on a sterile pad of Whatman (Qualitative 1) filter paper saturated with TSB and incubated in a sealed container for 10 h at 37 °C. Following incubation, membranes were treated with alkali, fixed and hybridised with the uidA gene probe at SO ng.m! -I. Mu!t,.'~!e membranes p~-ocessed concurrently were prehybridised and hybridised by stacking each membrane between Whatman 504 filter paper (5.5 x 5.5 cm) soaked in the appropriate solution. Colonies reactive with the uidA gene probe were counted directly from the filter following colour development.
,'ia.dysis o f non-growing cells Non-growing E. coil cells were induced by inoculation of washed, log phase E. coil cells at 1 × 10~ cells .ml -l (E. coil AB 259 (Table 1) and E. coli V~ an environmental isolate) into artificial seawater [12] and incubated with the exclusion of light at 15 °C for a total of 15 wk (G. Gray-Young, pers. comm.). Duplicate samples (10 ml) were removed from these cultures and filtered onto Hybond-N+ of Advantec membrane filters and incubated for 18 h at 37 °C on TSB saturated pads or mTEC agar, respectively. Enumeration of E. coii was by either uidA gene probe or incubation on urease substrate. Results
The DNA sequence chosen for the detection of faecal pollution was the E. coli K-12 gene, uidA. A 4.3-kb XhoI-EcoRI fragment containing uidA was isolated from the plasmid pGLI and used as the gene probe. The uidA gene resides in the first 2 kb of DNA sequence and the remaining DNA sequence (2.3 kb) is E. coli K-12 chromosomal D N A d o w n s t r e a m nf . . . . . . . . . . . . . . . . . . . . .
.
uidA )~-~)
r@t~in~rl *~***'~u
tr~ l,v
n~t-~m~ . . . . . . ~,e;..~;,,. ÷~ Vl..,i.zaaaao)... a l j ~ l t l ~ . l L y tO
17'. ~ l : ^--..i .... :--:--~td,. LI./Lg (;I.IIU l l l g [ ~ . ~ l l l l ~
sensitivity. The 4.3-kb uidA gene probe was reactive with all laboratory strains (Table 1) of E. coli and Shigella (S. boydii and S. dysenteriae) tested, all of which produced GUD. Salmonella isolates, S. typhimurium, S. arizonae, S. enteritidis and remaining bacteria in Table 1 were not reactive with the uidA gene probe and did not produce GUD. Of the presumptive E. coli isolates (Table 2), 83 of the 89 were confirmed as E. coli by biochemical tests and API 20E analysis. The remaining six were shown to be species of Klebsieila and Enterobacter, equating to a 6.7o/0 false-positive result. All 83 E. coli ~olates were reactive with the uidA gene ~.robe even though eight (9.60/0) did not produce GUD. No reaction with the uidA gene probe or GUD production was observed in any Citrobacter, Enterobacter or Klebsiella isolates tested (Table 2). The incubation time required to grow microcolonies, which gave a visible hybridisation signal was 8 h for E. coli 611 and l0 h for bacteria from water samples (data not shown). Incubation of membrane filters for longer periods did not improve the hybridisation signal when high numbers of faecal coliforms were present in the water sample, as interference between microcolonies resulted in a poorly defined hybridisation signal.
211 TABLE 1 B A C T E R I A L S T R A I N S T E S T E D F O R / 5 - G L U C U R O N I D A S E (GUD) P R O D U C T I O N A N D R E A C TIVITY WlTH uidA GENE PROBE ~act:ria! s~ec!es
:,*
E s c h e r i c h i a coil 611 ( N C T C 8622) E. coli AB259 Salmonella typhimurium S. a r i z o n a e S. enterittdis Shigella b o y d i i type 3 S h . b o y d i i type 7 Sh. d y s e n t e r i a e E n t e r o b a c t e r aerogenes ( N C T C 8197) C i t r o b a c t e r f r e u n d i i ( N C T C 9750) K l e b s i e i l a p n e u m o n i a e ( N C T C 9633) Serratia m a r c e s c e n s Vibrio a n g u i l l a r u m 775(A) P r o t e u s vulgaris Enterococcus faecalis
1 1 16
*Total number o f isolates;
,_,,_,D
,.,U~
1 1 -
uidA +
uidA -
|
1
! 1
16 !
-
16
-
!
1
-
1
--
I
--
1 1
1 1
--
l
--
1
!
--
1
- -
1 1 !
-
1
-
1
-
!
I
-
1
1
-
1
-
!
1
-
1
-
1
1 1
-
1
-
1
1
-
1
1
- , negative result.
TABLE 2 ENVIRONMENTAL ENTEROBACTERIACEAE ISOLATES TESTED FOR/~-GLUCURONIDASE (GUD) P R O D U C T I O N A N D R E A C T I V I T Y W I T H u i d A G E N E P R G B E Bacterial species*
n
GUD ÷
GUD-
uidA +
uidA -
E s c h e r i c h ! a coil C i t r o b a c t e r sp. Klebsieila pneumoniae Klebsiella oxytocae E n t e r o b a c t e r cloacae E n t e r o b a c t e r sp.
83 ii
75 -
8 II
83
!I
3 1 1
-
3 1 1
-
3 1 1
42
-
42
-
42
*Bacteria were isolated f r o m fresh and marine water using m T E C and S i m m o n ' s c-'.trate agar ~nd identity by biochemical tests and A P I 20E analyses. -, negative result.
confirmed
An initial study compared mTEC agar and TSB for resuscitation of non-culturable E. coli. Detection of E. coli AB 259 (mTEC, 27 cfu.10 ml-~; uidA 93 cfu.10 m1-1) and E. coli V ! (mTEC 90 cfu.10 ml-~; uidA 1056 cfo .10 ml -I) resulted in the uidA gene probe method enumerating 3.4- and ll.7-fold mo~e E. coli than did mTEC agar. A further study directly compared the mTEC [2] and uidA gene probe methods by examination of marine and freshwater samples; the results are given in Table 3. Figure I shows an mTEC membrane filter and a uidA probed membrane filter after a water sample has been processed. Discolouration evident on the mTEC membrane filter did not cause any problem in hybridisation and reading, of the uidA probed membrane.
212 TABI E 3 DE ~E(2l ION OF E. C O L I BY MTEC METHOD AND uidA GENE PROBE ANALYSIS OF FRESH AND MARINE WATER SAMPLES Sample type"
Freshwater A B C D E Marine water F G H
Presumptive E. coli on mTEC medium cfu- I00 m l - I
Confirmed E. coli bv uidA gone probe colonies. I00 ml I
595 398 318 170 110
680 238 208 143 133
24 102 142
30 81 332
*Samples B - E , G and H were collected after heavy rain.
~
J
Fig. !. Comparison of E. coli detection fro"n marine samples by mTEC and uidA gene probe. Note heavy precipitate of solids on mTEC membrane filter. This solid did not affect detection of colonies by uidA gene probe.
Discussion Membrane filtration and DNA hybridisation techniques, have been combined in a
213 sensitive and specific method lbr detection of faecal pollution in fresh and marine waters. The DNA sequence on which the gone probe is based contains the E. coil uidA gene and downstream E. coli chromosomal DNA. The uidA gone probe is specific for all E. coli examined and the enteric pathogen Shige(la. In this study none of the Salmonella isolates were reactive with the uidA gene probe, though, between 17 and 30°70 of Sabnonella have been reported to produce GUD [5], additionall); Le Minor lt~ reported that production of GUD by Saimonefia was restricted to specific serotypes. The Salmonella tested in this study were of species reported not to produce • ,,,po, t,~n,y, ,::heability ,,,'-~'*~"~,,~uidA gone probe to detect Shigella and perhaps some serotypes of Salmonella does not detract from the use of this probe as an indicator of faecal pollution. Environmentally derived members of the coliform group (Citrobacter, Enterobacter and Klebsiella) were not reactive with the uidA gene probe, although some species of Citrobacter and Enterc, bacter have been reported to produce GUD [6]. Analysis of water samples (Table 3) resulted in the mTEC method recording higher numbers of presumptive E. colt than the uidA probe for half of the water samples analysed. This occurred most notably in, freshwater samples collected after heavy rain. The mTEC method is recognised to produce i0 - 40% false-positive colonies (predominantly Klebsiella and Enterobacter species) in temperate and tropical waters [2, 14] and the absence of uidA gene probe reactivity with these soil and vegetation derived organisms suggests that following heavy rain or at times of high organic loading the mTEC method may overestimate the number of E. coil in water. The resuscitation of artificial seawater "stressed" E. coil on non ~elective medium (TSB) and uidA gene probe detection, was 3 and 11 times more effective than on mTEC medium, suggesting that a higher proportion of injured or non-culturable cells in water samples would be detected by the uidA gone probe method; this wa,; apparent in both marine and freshwater samples in this study (Table 3). The sensitivity of the uiclA gene probe requires that the membrane and adhering ~_eLsbc _~,_... . . . . ~_._,j_, iij n to produce a visible hybridisation signal. H_owever,the small area of the hybridisation signal was not adversely affected by background signal due c ¢'x¢'X to organic material in the water sample. In our work an upper limit of as many as .,vv uidA reactive colonies per 47 mm diameter membrane allowed reliable enumeration and were in general, more easily counted than those on mTEC medium on which the often large mucoid colonies tended to overgrow one another and only up to 50 cfu could be reliably counted. Use of the uidA gone probe method to analyse water samples for faecal pollution has the sensitivity to detect if present, a single faecal isolate after a period of 10 h enrichment. The uidA gene probe is specific for E. coil, Shigella and possibly some Salmonella, making this procedur,, a suitable alternative to the existing procedures tbr the detection of faecal pollution in water samples.
Acknowledgements We wish to thank P. Keumpel for generously donating pBKman and PK803 (University of Colorado, Boulder, Colorado) and Gillian Gray-Young (M.Sc. thesis), Jan Hesketh and Karen Robert for assistance in completion of this study.
214
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