School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK1
Author for correspondence: Alan J. McCarthy. Tel: +44 151 794 4413. Fax: +44 151 794 4401. e-mail: AJ55M{at}liverpool.ac.uk
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ABSTRACT |
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Keywords: rRNA, fluorometry, RNADNA ratio
a Present address: Clean Environment Management Centre, University of Teesside, Middlesborough TS1 3BA, UK.
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INTRODUCTION |
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Conventional methods for detection of L. monocytogenes use enrichment and selective isolation followed by biochemical tests (Farber & Perkin, 1991 ; McLauchlin & Pini, 1989
). Such procedures are valuable, but speed and sensitivity can be improved by the use of PCR-based methods (Bessesen et al., 1990
; Cooray et al., 1994
; Scheu et al., 1999
). However, detection of DNA does not discriminate between viable and non-viable cells and DNA can be associated with detrital material (DellAnno et al., 1998
). In theory, RNA detection is indicative of metabolically active organisms, but often requires an enrichment step prior to the molecular biological analysis (Blais et al., 1997
; Klein & Juneja, 1997
). This precludes simple, rapid quantification and suffers from poor sensitivity when attempted directly on food samples (Powell et al., 1994
; Wang et al., 1992
; Cano et al., 1995
; Makino et al., 1995
).
It has been established that bacterial growth rate and cellular RNA concentration are positively correlated in a number of bacterial species (Kjellgaard & Kurland 1963 ; Rosset et al., 1966
; Gausing, 1977
; Kerkhof & Ward, 1993
; Amann et al., 1990a
, b
; Muttray & Mohn, 1998
). Increased levels of rRNA associated with increased growth rates (Bremer & Dennis, 1987
) would effectively increase L. monocytogenes detection sensitivity if rRNA targeted probes were used. However, the correlation is not absolute for the growth of all bacterial species and the relationship is poor at low growth rates (Kramer & Singleton, 1992
; Kerkhof & Ward, 1993
; Kerkhof & Kemp, 1999
). Under such conditions the cellular rRNA concentration approached the detection limits of the techniques used.
Definition of the relationship between L. monocytogenes growth and cellular RNA content is required prior to the development of meaningful RNA-based detection methods. In this paper, we use fluorometry and quantitative nucleic acid probing to determine RNADNA ratios for L. monocytogenes in relation to the batch culture growth cycle, and culture viability in the presence and absence of pH control.
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METHODS |
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Agarose gels (0·81·2%, w/v) were prepared in 1x TAE buffer (2 ml 50x TAE; 0·5 M Tris/HCl, pH 7·6; 0·05 M EDTA; 57·1 ml glacial acetic acid) and 2 µl ethidium bromide (10 mg ml-1) was added to 100 ml molten agarose prior to pouring the gel. Gel equipment was suitably treated to inactivate RNases and used exclusively for RNA work. Nucleic acid samples (115 µl) were mixed with 4 µl loading buffer (50%, v/v, glycerol, 50%, v/v, TE buffer, 0·05%, w/v, bromophenol blue) prior to loading onto the gel. Nucleic acids were separated by electrophoresis at 90 V cm-1 for 1 h and visualized on a UV transilluminator.
Fluorometric quantification of nucleic acid extracts.
Standard curves for fluorometric determination of DNA or RNA concentration were prepared using serial dilutions of chromosomal DNA from L. monocytogenes ATCC 19111 (undiluted concentration 5·68 µg ml-1 determined by A260) and E. coli 16S and 23S rRNA mixture (Roche Diagnostics 206938, 4 µg ml-1) in molecular grade water. Fluorescence of nucleic acid samples was recorded on a Perkin Elmer 3000 Spectrofluorometer. Samples (10 µl) were added to fluorometry cuvettes and to each was added 3 µl staining solution (10 mg ethidium bromide ml-1 in 5 mM Tris/HCl, pH 8·0, 10 mM EDTA) prior to addition of PBS to a final volume of 3 ml. Samples were mixed and equilibrated at 37 °C for 15 min and fluorescence measured (excitation 260, emission
590; LePecq & Paoletti, 1966
) using slit widths of 10 and 20 nm for excitation and emission, respectively. No signal expansion was required throughout and all solutions were checked prior to use for quenching or enhancement of fluorescent signal. Unstained nucleic acids and PBS mixed with stain solution were used as negative controls. The data were subjected to linear regression analysis by a least squares method. Correlation coefficients were calculated by a Pearsons product moment coefficient (Sokal & Rohlf, 1995
).
L. monocytogenes nucleic acid extracts were digested with either DNase or RNase, as appropriate, before fluorescent quantification. To digest RNA, DNase-free RNase (Roche Diagnostics 1119915) was added (315 U µl-1). Nucleic acids were also incubated in the presence of 3 µl RNase-free DNase (Roche Diagnostics 776785) after addition of DNase buffer (20 mM Tris/HCl and 10 mM MgCl2, pH 7·4) to each sample. All digestions were performed for 4 h at 37 °C. The efficacy of DNA or RNA selective digestion had been assessed previously (data not shown). Fluorescence was determined as described above.
Quantitative oligonucleotide probe hybridization.
Nucleic acids were applied to positively charged nylon membranes (Positive; Appligene) with a Minifold II vacuum manifold (Schleicher and Schuell) which had a sample footprint size of 6 mm2. For RNA immobilization, the manifold was soaked overnight in 0·5% (v/v) diethylpyrocarbonate (DEPC)-treated water to inhibit RNase activity. Samples of RNA (maximum volume 50 µl) were mixed with 3 vols of a solution that contained 70% (v/v) deionized formamide, 24% (v/v) of a 37% (v/v) filtered formaldehyde solution and 6% (v/v) 20x SSC (3 M NaCl, 0·3 M sodium citrate, dissolved in 800 ml DEPC-treated water and adjusted to pH 7·0 before adjusting the volume up to 1 l). Samples were heated for 15 min at 68 °C, chilled on ice and 2 vols of ice cold 20x SSC added before storage on ice until required. RNA was applied to the manifold according to manufacturers instructions and slots were rinsed twice with 200 µl 20x SSC under vacuum.
DNA samples (maximum volume 50 µl) were heated to 95 °C, chilled on ice and 1 vol. ice cold 20x SSC added before storage on ice until required. Subsequent application of DNA to the membrane followed the procedure described above for RNA. Both DNA and RNA were fixed onto the nylon membrane by air-drying for 1 h followed by cross-linkage at 80 °C for 1 h. Membranes were wrapped in aluminium foil and stored at 4 °C prior to further treatment.
A probe, MV9RP2, was designed to be specific for L. monocytogenes rRNA (5'-ATAGTTTTATGGGATTAGCTC-3', position 13011281). This probe was designed by comparison of rDNA sequences deposited in the EMBL database (accession nos X56148X56154; Collins et al., 1991 ). The CHECKPROBE package (Ribosomal Database Project, http://www.cme.msu.edu/RDP) was used to screen candidate sequences for diagnostic value and possible artefact formation. Experimental evaluation of this probe against rDNA obtained from a large collection of Listeria reference strains and clinical isolates showed cross-reactivity with a type strain of Listeria innocua ATCC 33090 (data not shown). Probes were end-labelled with [
-32P]dATP (ICN Supplies) and efficiency of labelling was determined using the procedure described by Hiorns et al. (1995)
. It was intended to use probe MV9RP2 to specifically detect L. monocytogenes DNA also, but preliminary experiments showed this to have poor sensitivity (data not shown). Consequently, oligonucleotide probe pA, designed to be specific for all Eubacteria (5'-AGAGTTTGATCCTGGCTCAG-3'; position 828; Edwards et al., 1989
), was used to detect L. monocytogenes rDNA. To monitor the relationship between L. monocytogenes rRNA and rDNA, subsequent experiments were performed using this organism in pure culture.
Membranes were prehybridized for at least 1 h in a buffer that comprised 5x SSPE (0·78 M NaCl, 0·155 M Na2HPO4 . H2O, 74 mM EDTA, pH 7·4, made up to 800 ml and adjusted to pH 7·4 prior to dilution to 1 l), 20% (v/v) deionized formamide, 0·02% SDS, 0·1% (w/v) N-lauryl sarcosine and blocking reagent [2%, w/v, of a solution that comprised 0·1 M maleic acid and 0·15 M NaCl adjusted to pH 8·0 prior to addition of blocking reagent (10%, w/v, Roche Diagnostics 1096176)]. Prehybridization solution was removed and membranes rinsed briefly in hybridization solution prior to addition of hybridization solution that contained 5x SSPE, 20% (v/v) deionized formamide, 0·02% SDS, 0·1% N-lauryl sarcosine and 10 pM radiolabelled oligonucleotide probe. Membranes were hybridized overnight at 40 °C prior to washing twice for 15 min at room temperature in the above solution. Filters were wrapped in cling film and X-ray film exposed by autoradiography for 2 and 7 d at -70 °C. The autoradiograph signal was quantified with ImageQuant version 3.2 software running on a Molecular Dynamics Personal Densitometer. Signal (pixel) intensity above background was transferred to Microsoft Excel version 3.0 software where data manipulation was performed.
A preliminary experiment determined the response of oligonucleotide probe signal in relation to nucleic acid concentration. Briefly, titration series of either L. monocytogenes RNA or DNA were applied to nylon membranes and hybridized overnight in the presence of 10 pM radioactively labelled oligonucleotide probe pA (DNA) or MV9RP2 (RNA) as outlined above. Autoradiographs were quantified and data analysed as described above.
Growth of L. monocytogenes in shake flask and pH-controlled batch culture.
Conical flasks (5 l volume) containing 2 l Tryptone Soya broth (30 g l-1) supplemented with 0·3% (w/v) yeast extract and 0·5% (w/v) D-glucose (TSYGB) were inoculated with 2·5x1010 c.f.u. of an overnight culture of L. monocytogenes ATCC 19111. Cultures were incubated at 37 °C with shaking at 100 r.p.m. and samples (10 ml) removed aseptically at intervals. Growth was determined by measurement of OD660 and viable counts determined on NAB plates (30 g Nutrient broth l-1, pH 7·2; 12 g Agar No.2 Lab M l-1) supplemented with 0·5% (v/v) defibrinated horse blood (Sigma Aldrich). In addition, pH was recorded by insertion of a pH probe into an aliquot of growth medium. Biomass was harvested by centrifugation of 1 ml aliquots at 740 g for 5 min. Nucleic acid extraction was performed prior to fluorescent and oligonucleotide assessment of RNA or DNA concentrations as described in detail above.
Fermenter vessels (LH Inceltech 501 series) that contained 1·5 l TSYGB medium buffered with KH2PO4 (8·5 g l-1) were prepared and maintained at 37 °C, pH 6·8, for 24 h prior to inoculation with 1010 c.f.u. of an overnight culture of L. monocytogenes ATCC 19111. Constant rates of agitation, air flow and pH were maintained throughout. Samples were removed to determine OD660, viable counts and nucleic acid concentrations at intervals up to 456 h, as described above.
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RESULTS AND DISCUSSION |
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Titration series of RNA showed a sigmoidal relationship between the logarithm of the nucleic acid concentration and pixel intensity (Fig. 2a). At the lowest amounts of nucleic acids applied (<740 ng rRNA), the hybridization signal was below the detection threshold. This rRNA detection limit was equivalent to about 100 c.f.u. of a late-exponential-phase culture. Saturation of the nylon membrane occurred at >5 µg per slot. A linear relationship was observed between L. monocytogenes DNA concentration and pixel intensity for the range studied (Fig. 2b
). Pixel intensities obtained for DNA were significantly lower than those obtained from RNA analysis (Fig. 2a
) and the detection limit was about 1 µg DNA. This is significantly higher than that determined by Kerkhof & Ward (1993)
and may be due to the many variables involved when preparing filters for oligonucleotide probing. However, the difference in amount of target sequence as a proportion of total nucleic acid applied is significant. This is estimated to be 0·4% of total RNA and 0·004% of total DNA (assuming lengths of 5S+16S+23S rRNA to be 4566 bp and an L. monocytogenes genome size of 3·15x106 bp) (Michel & Cossart, 1992
). In subsequent experiments, titration series of nucleic acids of known concentration and experimental samples were immobilized onto nylon membranes simultaneously. Calibration curves for DNA and RNA were prepared for each experiment and used to determine the nucleic acid concentration from experimental samples. These values were used to determine the RNADNA ratio.
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Growth of L. monocytogenes in pH-controlled batch culture
To determine the effects of prolonged culture of L. monocytogenes on survival and nucleic acid content, a pH-controlled batch culture was inoculated and growth was monitored. Growth of the culture showed a predictable exponential phase of about 10 h duration (Fig. 4a) followed by a prolonged death phase. Viable counts were 1010 c.f.u. ml-1 after 10 h and 109 c.f.u. ml-1 at the end of the experiment (456 h). The promotion of L. monocytogenes survival by maintaining the pH close to neutrality would appear to be correlated with the role of buffered dairy products as a vector for this human pathogen. During exponential growth, intact RNA and DNA were recovered (Fig. 4b
) in accordance with the data from batch culture in the absence of pH control (Fig. 3b
). The rapid degradation of DNA in prolonged shake flask culture (Fig. 3b
) was not apparent under pH control where intact DNA was maintained for the 96 h duration of the experiment (Fig. 4b
), while 16S rRNA and 23S rRNA were lost. Estimation of RNADNA ratios by fluorometry and oligonucleotide probing gave similar patterns (Fig. 5
). Nucleic acid ratios increased rapidly to a maximum value observed towards the end of the exponential growth phase and decreased rapidly thereafter. Nucleic acid ratios obtained from samples taken between 96 and 456 h were generally <3, although viable counts were >109 c.f.u. ml-1. Nucleic acid ratios estimated from the starvation phase by fluorometry were consistently greater than those determined by oligonucleotide probing. This could be due to partial nucleic acid degradation and loss of oligonucleotide probe targets in samples taken from the starvation phase.
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The rate and degree of ribosomal loss at the onset of bacterial starvation appears to vary, depending upon the species studied and the nature of the starvation imposed. For example, E. coli cells have been reported to lose functional ribosomes rapidly after the onset of starvation (Davis et al., 1986 ). However in Vibrio alginolyticus, RNA concentration decreased by up to 99% of exponential levels, but gradually over a 15 d starvation period (Kramer & Singleton, 1992
). Starvation responses can be remarkably specific, as Kramer & Singleton (1992)
also reported that Vibrio furnissii lost most of its rRNA during only 3 d of starvation. Maintenance of an active ribosomal pool would appear to be essential for cell survival and recovery. Starvation of E. coli cells resulted in rapid loss and degradation of functional ribosomes that led to rapid loss of viability (Kaplan & Apirion, 1975a
, b
; Davis et al., 1986
), but a pool in excess of translational requirements was maintained in marine Vibrio spp. (Flardh et al., 1992
). Ribosomes may represent a valuable source of metabolites to starved cells, but a critical number must be retained to maintain cell viability (Davis et al., 1986
). Differential retention of ribosomes shown by different bacterial species may also affect recovery rates from a stressed environment (Kramer & Singleton, 1992
). It is possible that ribosome dimers (100S ribosomes) reported to be of increased resistance to protease and nuclease degradation in E. coli (Wada et al., 1990
) formed during the prolonged stationary phase, although these were not found in starved Vibrio spp. cells (Flardh et al., 1992
).
In this paper, we have demonstrated that L. monocytogenes behaves in a similar manner to E. coli, i.e. viability and rRNA are lost rapidly once exponential growth has ceased. Viability can be maintained under buffered or pH-controlled conditions, enabling persistence of about 10% of the maximum exponential population, supported by the recovery of intact DNA, but not RNA.
In conclusion, the data presented here for L. monocytogenes show that it is possible to assess RNADNA ratios by fluorometry and oligonucleotide probing. In particular, we have shown that L. monocytogenes populations exhibit maximal RNA content late in the exponential growth phase and maintain viability over an extended period in pH-controlled batch culture, with greatly reduced cellular ribosome content when exponential growth has ceased. Having defined the L. monocytogenes RNADNA characteristics in relation to the growth cycle and maintenance of culture viability, modification of molecular detection methods for this pathogen to also provide information on their physiological status can be addressed.
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ACKNOWLEDGEMENTS |
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Received 4 December 2000;
revised 14 May 2001;
accepted 31 May 2001.
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