Journal of Microbiological Methods 30 (1997) 153–160
Journal
of
Microbiological
Methods
Rapid detection of Enterobacteriaceae in urine by fluorescent 16S
rRNA in situ hybridization on membrane filters
M.W. Mittelman
a
a,b,
*, M. Habash 1 , a , J.-M. Lacroix 2 , a , A.E. Khoury c , M. Krajden b , d
Centre for Infection and Biomaterials Research, Bell G-631, The Toronto Hospital, University of Toronto, 200 Elizabeth St.,
Toronto, Ontario M5 G 2 C4, Canada
b
Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada
c
Hospital for Sick Children, Toronto, Ontario, Canada
d
Department of Microbiology, The Toronto Hospital, Toronto, Ontario, Canada
Received 6 May 1997; received in revised form 30 June 1997; accepted 30 June 1997
Abstract
The detection and enumeration of putatively pathogenic bacteria in urine is the single-most important diagnostic criterion
for the diagnosis of urinary tract infections (UTI). We have developed a fluorescent in situ hybridization (FISH) assay which
utilizes a fluorescently-tagged 16S rDNA oligonucleotide probe specific for 16S rRNA of the Enterobacteriaceae family.
The technique involves fixation of a urine specimen, filtration through a 0.2 m m polycarbonate membrane, staining with the
nucleic acid dye, 49,6-diamidino-2-phenylindole (DAPI), hybridization with a fluorescently tagged nucleic acid probe, and
examination under epifluorescence microscopy. The technique was found to be sensitive and specific, recovering #10 3
Escherichia coli / ml within 4 h, both in spiked PBS and in filter-sterilized urine. Two non-Enterobacteriaceae, Pseudomonas
aeruginosa and Staphylococcus aureus, did not react with the probe but were visualized via DAPI staining. Eighty-three
urine specimens were randomly selected from the clinical laboratory and assayed using this new method. A total of 10
specimens were identified by the hospital laboratory as containing members of the Enterobacteriaceae family, including E.
coli and Proteus mirabilis. All 10 of these specimens were also positive by the membrane-based FISH assay. Of those
specimens characterized as either ‘no growth’ or ‘mixed organisms’ by the hospital laboratory, 24 were positive using the
membrane-based FISH assay. This FISH assay for bacteriuria shows promise as a rapid technique for use in clinical
diagnosis of urinary tract infections. 1997 Elsevier Science B.V.
Keywords: Bacteriuria; FISH assay; Fluorescence microscopy; Membrane filter; UTI
1. Introduction
Urinary tract infections (UTIs) are a significant
*Corresponding author. Tel.: 11 416 3404564; fax: 11 416
3404607; e-mail: marc.mittelman@utoronto.ca
1
Present address: Department of Microbiology and Immunology,
University of Western Ontario, London, Ontario.
2
Present address: Visible Genetics, Inc., Toronto, Ontario.
cause of morbidity and mortality world-wide. Although over 90% of UTIs are relatively simple
infections in anatomically normal patients, they
nevertheless result in considerable morbidity, lost
work time, and a financial burden to the health-care
system [1]. In complicated UTIs, renal damage
leading to kidney failure and death can occur. UTIs
account for a significant proportion of all antibiotic
prescriptions written in North America; indeed, in
0167-7012 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved.
PII S0167-7012( 97 )00061-4
154
M.W. Mittelman et al. / Journal of Microbiological Methods 30 (1997) 153 – 160
1988, two million prescriptions were written for
UTIs in Canada alone [2].
The majority of patients with UTIs complain of
lower urinary tract symptoms such as painful urination, frequency, and suprapubic pain. However, these
symptoms are also seen with other conditions such as
vaginitis and sexually transmitted diseases. Confirmation of a UTI is made upon microbiological
culture of urine from patients presenting with urinary
tract symptoms. Approximately 90% of all cases are
caused by Gram-negative bacteria of fecal origin [1].
The current standard for the microbiological diagnosis of UTI involves quantitative culture of the
urine using a calibrated loop on selective and / or
non-selective culture media. Following an 18–24 h
incubation, colonies are counted and ordinarily reported in gradations of 0, or 10 000 to .10 5 cfu / ml
[3]. Among symptomatic patients, particularly
women, a count of $10 5 cfu / ml has been adopted as
a criterion to distinguish infection from adventitious
‘contamination’. However, as many as 45% of
women presenting with clinical symptoms and subsequently treated with antibiotics are culture negative
with routine isolation procedures [2].
Although the culture technique remains an important diagnostic tool, there are two major problems
associated with the viable plate count method. Firstly, the long incubation time means that therapy is
often initiated prior to diagnosis. Secondly, the
viable-count techniques may not detect fastidious
pathogens or those bacteria which are viable, but not
culturable [4,5]. Patients on antibiotic therapy may
have antibiotic resistant microorganisms that are
capable of causing disease, but incapable of growth
on standard laboratory media. While the risks associated with acute infections may be sufficient to
warrant initiation of antibiotic therapy prior to the
availability of culture results, the clinician would
benefit from a definitive diagnosis — or exclusion —
of bacteriuria. Definitive diagnosis of bacteriuria can
also be critical for patients suffering from end-stage
renal failure or drug allergies, where prescribing of
antibiotics must be carefully weighed against the risk
of side effects.
A number of rapid screening techniques for bacteriuria have been evaluated. These include endotoxin [6], leukocyte esterase / nitrate reduction [7], catal-
ase [8], DNA probe [9] and substrate mineralization
[10] assays. Each of these methods suffers from one
or more disadvantages, however, including poor
sensitivity and / or specificity. Direct, viable-count
techniques have been employed for detecting viable,
but non-culturable microorganisms in the environment. These types of techniques have been used for a
number of years to detect both bacterial and
eucaryotic cellular activity. The need to recover
slow-growing, chlorine-damaged, and fastidious bacteria has been the driving force behind development
of direct, viable-count assays [11]. Stains employed
have included 2-( p-iodophenyl)-3-( p-nitrophenyl)5-phenyltetrazolium chloride [12], fluorescein diacetate [13], and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). None of these techniques specifically
identify putative pathogen groups; such a technique
would be important for making an accurate diagnosis
of UTI.
Fluorescent in situ hybridization (FISH) of bacterial 16S rRNA with oligonucleotide probes has
been used for the detection of bacteria in environmental and clinical specimens. Identification of
single bacterial cells within natural microbial communities has been accomplished using a series of
fluorescently tagged oligonucleotide probes [14,15].
Gersdorf et al. [16] used a FISH assay to detect
putative periodontal pathogens in subgingival plaque
in humans. The uropathogenic protozoan, Trichomonas vaginalis, was detected in clinical samples
using an in situ DNA hybridization assay. Probes
specific to the species, genus, and family levels have
been developed for several human pathogens [17]. It
is the combination of highly conserved and variable
regions within the 16S ribosome which enable the
synthesis of specific probes targeted to various
phylogenetic levels [18].
We have developed a membrane-based FISH assay
which utilizes fluorescently-tagged 16S rDNA oligonucleotide probes specific for 16S rRNA of the
Enterobacteriaceae family. This rapid, membranebased assay demonstrates good specificity and selectivity for a significant group of uro-pathogens. The
assay can be designed to target various putative
uropathogens and specifically quantify the total
number of microorganisms, allowing for laboratoryclinical correlations.
M.W. Mittelman et al. / Journal of Microbiological Methods 30 (1997) 153 – 160
2. Materials and methods
2.1. Reagents and materials
2.1.1. Bacteria
Overnight cultures of clinical strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were prepared from nutrient broth
tubes incubated at 378C. Spiking experiments were
carried out over a range of bacterial concentrations,
from 10 2 –10 7 cfu / ml.
2.1.2. Hybridization buffer
The buffer consisted of 63 SSC (13 SSC: 0.15 M
NaCl, 0.015 M sodium citrate), 20 mM Tris–HCl
(pH 7.0, 258C), 0.1% SDS, 0.01% poly (A), and
0.2% diethylpyrocarbonate in 100 ml of deionized
water. Double distilled water was treated with diethylpyrocarbonate (0.2%, w / v), and autoclaved for
RNAse inactivation.
2.1.3. Wash buffer
The buffer consisted of 0.9 M NaCl, 20 mM
Tris–HCl (pH 7.2, 258C), 0.1% SDS, and 0.2%
diethylpyrocarbonate in 500 ml of deionized water.
2.1.4. Fluorescent-probe
The probe had the following sequence:
59 2
CAT GAA TCA CAA AGT GGT AAG CGC C 2 39
(synthesized by BioMolecular Assays, Inc., Woburn,
MA) and labeled on its 59 end with tetramethylrhodamine isothiocyanate. This sequence is
homologous to the bases 1458 to 1482 of the positive
strand 16S rDNA sequence of Escherichia coli [19].
A search of 329 017 sequences in the BLASTn
database [20] revealed that this sequence is highly
conserved among members of the Enterobacteriaceae family (including the common UTI pathogen genera of Escherichia, Proteus, Yersinia, Klebsiella). A few non-Enterobacteriaceae (Plesiomonas
shigelloides and an insect endosymbiont) also
showed 100% homology to this oligonucleotide
sequence. However, these organisms have not been
previously associated with UTIs.
155
2.1.5. Gelatin-coated slides
Plastic coated slides (Cell Tek Inc., Glenview, IL)
were precleaned in a solution of 10% KOH (w / v in
95% ethanol) for 1 h at room temperature. The slides
were rinsed with distilled water and air dried. The
slides were then dipped twice in a gelatin solution
(0.1% gelatin 150 bloom, BDH Chemicals Toronto,
and 0.01% chromium potassium sulfate, w / v in
distilled water) maintained at 708C. Following
gelatin coating, the slides were dried in a vertical
position overnight at 258C.
2.2. Specimen collection
A total of 83 patient urine specimens were randomly selected over a 2-week period from the
microbiology laboratory at The Toronto Hospital,
General Division. Specimens were assigned a study
number; both identity of the patients and the preliminary diagnoses were blinded to the investigators.
The majority of specimens were obtained from inpatients, both with and without indwelling urinary
catheters. Ten-ml aliquots were removed from specimens within 4 h of patient collection. Specimens
were maintained at 48C during the interval between
patient collection and aliquoting.
2.3. Methods
2.3.1. Specimen fixation and filtration
One ml of the urine specimen was added to 3 ml
of a 4% (w / v in PBS) paraformaldehyde solution.
Paraformaldehyde was best suited for fixing the
bacteria as it produced no background fluorescence,
unlike glutaraldehyde which fluoresces in the same
region of the spectrum as the rhodamine label found
on the probe. Cloudy specimens or those containing
particulate material were diluted 1:10 in PBS to
facilitate filtration. After a 90 min fixation, the
suspensions were transferred to a Nuclepore filtration unit containing a 0.2 m m black membrane
(Corning Separations Division, Acton, MA), a 10
m m filter paper and a metal grid (Fig. 1). The fixed
specimen was filtered in vacuo, then rinsed with 2 ml
of RNAse-free water to remove the paraformaldehyde.
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M.W. Mittelman et al. / Journal of Microbiological Methods 30 (1997) 153 – 160
Fig. 1. Nuclepore disposable filtration unit. The filter funnel
contains up to 15 ml.
2.3.2. Staining with DAPI
One ml of a 5 m g / ml 49,6-diamidino-2-phenylindole (DAPI, Polysciences, Warrington, PA) solution
was placed in the filtration unit, covered with
aluminium foil, and allowed to stand, for 5 min at
room temperature. The DAPI solution was filtered in
vacuo and the membrane was rinsed with 2 ml of
RNAse-free water. The filtration unit was disassembled and the membrane removed for hybridization.
Unstained samples were also prepared to determine
if the fixed cells autofluoresced.
2.3.3. Transfer of the bacteria to a gelatin-coated
slide
Three m l of RNAse-free water were added to the
well of a gelatin-coated slide. The membrane was
deposited on top of the slide, bacteria-side down.
The slide and membrane were placed in a vacuum
desiccator for |15 min until the membrane had
dried. Once dry, the membrane was carefully peeled
from the surface and discarded. In conjunction with
the slides from the hybridization procedure, parallel
samples were stained with DAPI alone to determine
the efficiency of transfer from the membrane to the
gelatin-coated slides.
2.3.4. Hybridization of the fluorescent probe
Eight m l of the hybridization buffer and 2 m l of
the fluorescent probe (25 ng /m l) were added to the
well of the gelatin-coated slides containing the
transferred bacteria. The slide was placed in a 50-ml
disposable centrifuge tube which was equilibrated
with 5 ml of the wash buffer in an oven maintained
at 458C. The hybridization reaction was allowed to
proceed for 2 h at 458C. After hybridization, the slide
was gently rinsed with 5 ml of the wash buffer,
immersed in wash buffer, and returned to the hybridization oven for 10 min at 55.58C. (Following a
series of validation experiments conducted at temperatures between 37 and 658C, the optimal temperature for post-hybridization rinsing was found to be
55.560.58C.) The rinsing step was repeated a second
time. After the final rinsing, the slide was washed
with 5 ml of RNAse-free water and allowed to air
dry at 258C.
2.3.5. Microscopic observation
A drop of low-fluorescing immersion oil was
placed in the well of the slide and a coverslip was
added. The slides were stored in the dark until
observed under the microscope. Samples were observed under epifluorescence microscopy (LeitzLeico DMRB, Frankfurt). This microscope contained
a 100 W mercury lamp, Leico PL Fluorstar 1003 /
1.3 oil immersion objective, and a fluor cluster with
filters specific for DAPI and the rhodamine probe.
2.3.6. Bacterial enumeration
Culturable cells were enumerated on nutrient agar
by a calibrated inoculating loop technique [3]. The
standard hospital laboratory technique involved direct plating of urine specimens to blood and MacConkey agars via a calibrated platinum loop (loop
count). All plates were incubated 24 h at 358C.
3. Results
The optimal specimen processing conditions for
maximal sensitivity and specificity are summarized
in Table 1.
Cell fixation prior to the hybridization procedure
was essential, as non-fixed cells did not hybridize
with the probe. The use of gelatin-coated slides was
found to be necessary for successful hybridization of
the cultures. When the technique was first applied,
the hybridization and washing steps were performed
inside the filtration unit and the bacteria were not
fixed using paraformaldehyde; no labeled or DAPIstained bacteria were found on the membrane. In the
absence of gelatin, no bacteria were observed. The
transfer efficiency of bacteria from membrane to
M.W. Mittelman et al. / Journal of Microbiological Methods 30 (1997) 153 – 160
157
Table 1
Optimal FISH assay processing conditions for urine specimens
Assay step
Reagent
Processing time
Processing temperature, 8C
Specimen fixation
DAPI staining
Membrane transfer
16S rRNA hybridization
Washing
Formaldehyde, 3% final concentration
DAPI, 5 m g / ml
Gelatin-coated slide
16S rRNA probe, 8 m l of 25 ng /m l solution
Wash buffer solution
90
5
15
2
10
25
25
25
45
55.560.5
gelatin-coated slide was 5565% (n55). It may be
possible to further optimize transfer efficiency
through direct application of gelatin to the membrane
immediately following specimen filtration.
Hybridization stringency was significantly influenced by the washing temperature. At lower temperatures (#508C), all bacteria tested were labeled,
including S. aureus and P. aeruginosa. If higher
temperatures ($608C) were employed, no labeling
(greater than background) was observed on S. aureus
and P. aeruginosa, and only a faint labeling was
observed for E. coli. At higher temperatures (658C),
no labeling was observed for E. coli. The optimal
temperature was found to be 55–568C; at this
temperature, E. coli was strongly labeled and the
other bacteria were not labeled above the level of
background fluorescence. Similar results were observed when bacteria were added to filter-sterilized
urine and the procedure was performed using patient
urine samples spiked with |10 5 E. coli / ml. With
patient urines, a slightly higher level of background
fluorescence was observed. The assay required |4 h
min
min
min
h
min
from sample collection to imaging under the epifluorescent microscope and recording of data.
Assay specificity findings are shown in Table 2.
The probe hybridized with E. coli in both PBS,
filter-sterilized urine, and in unfiltered patient urine
specimens. Although P. aeruginosa and S. aureus
demonstrated weak staining with the probe, the cells
were clearly non-reactive relative to E. coli. All three
test organisms were visualized with DAPI staining.
Validation assay results for the three test organisms
were similar over the range of bacterial titers evaluated, from 10 2 –10 7 cfu / ml. Autofluorescence of
unstained control specimens (patient urine containing
either Enterobacteriaceae or non-Enterobacteriaceae) was not observed.
The clinical findings for both the hospital-based
(culture) method and the membrane-based FISH
assay are summarized in Table 3. A total of 83
specimens were tested. Enterobacteriaceae identified
by the hospital were E. coli, Enterobacter aerogenes,
Citrobacter koseri, Serratia marcescens, Proteus
mirabilis, and Hafnia alvei. All 10 of those speci-
Table 2
Results of assay validation
Organism
Experimental conditions
16S rRNA probe
reaction a
DAPI reaction b
E. coli
E. coli
E. coli
P. aeruginosa
P. aeruginosa
P. aeruginosa
S. aureus
E. coli
(negative assay control)
Spiked PBS
Spiked filter-sterilized urine
Spiked patient specimens
Spiked PBS
Spiked filter-sterilized urine
Spiked patient specimen
Spiked PBS
Spiked filter-sterilized urine
(unstained preparation)
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Negative
(no probe added)
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Negative
(DAPI staining
not performed)
a
Observation of orange-red fluorescing cells under epifluorescence microscopy.
Observation of blue fluorescing cells under epifluorescence microscopy.
b
M.W. Mittelman et al. / Journal of Microbiological Methods 30 (1997) 153 – 160
158
Table 3
Summary of FISH assay findings for clinical specimens
Technique
Sample
size
Number positive for
Enterobacteriaceae
(%)
Number positive for
non-Enterobacteriaceae
putative pathogens
Hospital-based,
selective culturing
83
10
12.0
9
10.8
Membrane-based
probe assay
83
34
41.0
0
,1.2
mens containing Enterobacteriaceae identified by
the hospital laboratory also hybridized with the
probe; an additional 24 specimens hybridized with
the probe, but were culture-negative (Table 3).
Coagulase-negative staphylococci or Enterococcus
spp., which are putative urinary tract pathogens,
were reported by the hospital laboratory in 9 / 83
specimens. None of these specimens hybridized with
the probe. Three of the 24 culture negative patients
(%)
Comments
Non-Enterobacteriaceae
putative pathogens were
either Enterococcus spp.
or coagulase-negative
staphylococci
100% specificity for
culture-positive
Enterobacteriaceae
(all with urine specimens that hybridized with the
probe) had been previously reported as positive for
E. coli by the hospital laboratory.
Cell counts obtained by the membrane-based
probe assay were in the same range as or greater than
those obtained by the hospital-based culture method
(Fig. 2). Total counts (DAPI) were significantly
higher than the culture values for specimens that
hybridized with the probe (Fig. 2) and those speci-
Fig. 2. Bacteria counts associated with patient urine specimens containing one or more Enterobacteriaceae spp. The hospital culture counts
are in cfu / ml; the FISH and total counts are direct microscopic counts calculated as cells / ml. The reported hospital culture values are
presented as the midranges of reported values; e.g. a reported range of .10 000,50 000 cfu / ml is presented as 30 000 cfu / ml. CNS,
coagulase-negative staphylococci.
M.W. Mittelman et al. / Journal of Microbiological Methods 30 (1997) 153 – 160
mens with reported non-Enterobacteriaceae culture
counts. White blood cells, when present in the urine,
were readily stained by the DAPI; however, hybridization with the oligonucleotide probe was not observed. Stained bacterial cells were easily differentiated from white blood cells.
4. Discussion
Rapid detection of urinary tract pathogens is
important for the rationale and effective use of
antibiotics in treating UTIs. Since the majority of
urine specimens taken in general practitioners’ offices are reported as ‘culture negative’ [1,21], it is
clear that significant numbers of patients may be
improperly treated with antibiotics. In the absence of
more rapid and quantitative assays, the risk of failing
to treat symptomatic patients has outweighed the risk
of unnecessary antibiotic dosing. The purpose of this
study was to develop a quantitative, direct, microscopic assay for a major group of urinary tract
pathogens, the Enterobacteriaceae. We have demonstrated that this new FISH assay is both specific and
sensitive for members of this pathogen family;
significantly, results are available in less than 4 h
from sample acquisition.
A significant proportion of hospital and community urine specimens submitted for bacterial culture are
negative following 1–2 days incubation [21]. In our
study of 83 patient urine specimens, routine culture
assays were positive for putative UTI pathogens in
only 19 cases (22.9%). The results of the FISH
assays, while recovering significantly greater numbers of Enterobacteriaceae, also demonstrated that a
significant number of clinical specimens were negative. The high specificity of this FISH assay, based
on DAPI enumeration and FISH identification, suggests that this assay is reliable for prescreening the
large number of specimens that would otherwise be
subject to culturing. This could reduce the frequency
of inappropriate antibiotic usage for uninfected patients or suggest another etiology for the clinical
symptomatology. The assay should be amenable to
automation via application of automated aliquoting /
filtration combined with image analysis. Preliminary
observations (data not shown) also suggest that white
159
blood cells can be differentiated from bacteria and
enumerated by an image analysis system.
Probe specificity is the single-most important
criterion in assessing the utility of hybridizationbased assays. In this study, we used three clinical
isolates, E. coli, P. aeruginosa, and S. aureus, to
optimize the experimental conditions for specimen
processing, hybridization, and washing stringency
associated with this new, membrane-based assay.
Although the majority of urinary tract infections are
caused by members of the Enterobacteriaceae family, Enterococcus spp. (e.g. E. faecalis) and
coagulase-negative staphylococci (e.g. S. saprophyticus) are responsible for a significant number
of infections [3]. Therefore, consideration should be
given to the development of other probes, perhaps
tagged with different fluorescent markers, for use in
improved membrane-based FISH assays for bacteriuria. Additional screening studies with much
larger numbers of clinical UTI isolates are clearly
needed to develop this new method for routine
clinical diagnostic use.
The use of the nucleic acid-specific dye, DAPI,
enabled detection of total bacterial numbers in
patient urine specimens. Total bacterial counts may
be helpful in correlating the relationship between
bacterial counts, the types of microorganisms, and
the clinical manifestations, particularly in compromised patient populations. The significantly higher
total counts seen relative to viable counts are not
surprising, given the inadequacy of routine culturing
techniques for recovering fastidious and / or damaged
bacteria from a variety of environments [22]. In the
absence of significant viable bacteria, the finding of
large numbers of bacteria in urine using a directcounting technique may be of significance to the
clinician. For example, this situation could indicate
the presence of atypical, fastidious organisms requiring specialized culturing conditions.
Fluorescence-based microbial direct-count techniques offer a number of advantages over cultural
techniques in the enumeration of bacteria from
clinical specimens. These include: (1) rapid availability of quantitative results; (2) broad range of
detection; (3) the ability to detect non-culturable
cells (e.g. antibiotic damaged cells); (4) relatively
low costs per assay. This membrane-based FISH
assay for bacteriuria shows promise as a rapid
160
M.W. Mittelman et al. / Journal of Microbiological Methods 30 (1997) 153 – 160
technique (,4 h) for use in clinical diagnosis of
urinary tract infections. The assay is specific, and has
a broad dynamic range which will allow it to assess
the relationship between bacterial counts, the types
of microorganisms present, and the clinical manifestations associated with UTIs.
Acknowledgements
This research was supported by grants from
Corning Inc., Separations Division and the Natural
Sciences and Engineering Research Council of
Canada. The technical assistance of Mr. S. Sinnadurai and Dr. F. Soboh is acknowledged.
References
[1] J.C. Nickel, A practical approach to urinary tract infections,
Can. J. Diag. 10 (1993) 64–80.
[2] W.E. Stamm, Protocol for the diagnosis of urinary tract
infection: reconsidering the criterion for significant bacteriuria, Urology 32 (1988) 6–10.
[3] B.A. Forbes, P.A. Granato, Processing specimens for bacteria, in: P. Murray, E.S. Baron, M.A. Pfaller, F.C. Tenover,
R.H. Yolken (Eds.), Manual of Clinical Microbiology, 6th ed.
ASM, Washington, DC, 1995, pp. 265–279.
[4] R.R. Colwell, Nonculturable but still viable and potentially
pathogenic, Zentralbl. Bacteriol. 279 (1993) 154–156.
[5] M. Pommepuy, M. Butin, A. Derrien, M. Gourmelon, R.R.
Colwell, M. Cormier, Retention of enteropathogenicity by
viable but nonculturable Escherichia coli exposed to seawater and sunlight, Appl. Environ. Microbiol. 62 (1996)
4621–4626.
[6] M. Pezzlo, Detection of urinary tract infections by rapid
methods, Clin. Microbiol. Rev. 1 (1988) 268–280.
[7] M.T. Pezzlo, D. Amsterdam, J.P. Anhalt, T. Lawrence, N.J.
Stratton, E.A. Vetter, E.M. Peterson, L.M.D.L. Maza, Detection of bacteriuria and pyuria by uriscreen, a rapid
enzymatic screening test, J. Clin. Microbiol. 30 (1992) 680–
684.
[8] M.T. Dalton, S. Comeau, B. Rainnie, K. Lambert, K.R.
Forward, A comparison of the API Uriscreen with the Vitek
urine identification-3 and the leukocyte esterase or nitrite
strip as a screening test for bacteriuria, Diag. Microbiol.
Infect. Dis. 16 (1993) 93–97.
[9] C. Koenig, L.J. Tick, B.A. Hanna, Analyses of the Flashtrack
DNA probe and UTIscreen bioluminescence tests for bacteria, J. Clin. Microbiol. 30 (1992) 342–345.
[10] D. Colombrita, G. Ravizzola, F. Pirali, M. Manni, N. Manca,
E. Savoldi, A. Turano, Evaluation of a Bactec system for
urine culture screening, J. Clin. Microbiol. 27 (1989) 118–
119.
[11] F.P. Yu, G.A. McFeters, Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent
probes, J. Microbiol. Methods 20 (1994) 1–10.
[12] P.S. Tabor, R.A. Neihof, Improved method for determination
of respiring individual microorganisms in natural waters,
Appl. Environ. Microbiol. 43 (1982) 1249–1255.
[13] P.E. Jorgensen, T. Ericksen, B.K. Jensen, Estimation of
viable biomass in wastewater and activated sludge by
determination of ATP, oxygen utilization rate, and FDA
hydrolysis, Water Res. 11 (1992) 1495–1501.
[14] R.I. Amann, L. Krumholz, D.A. Stahl, Fluorescent oligonucleotide probing of whole cells for determinative,
phylogenic, and environmental studies in microbiology, J.
Bacteriol. 172 (1990) 762–770.
[15] R.E. Hicks, R.I. Amann, D.A. Stahl, Dual staining of natural
bacterial plankton with 49,6-diamidino-2-phenylindole and
fluorescent oligonucleotide probes targeting kingdom-level
16S rRNA sequences, Appl. Environ. Microbiol. 58 (1992)
2158–2163.
[16] H. Gersdorf, K. Pelz, U.B. Gobel, Fluorescence in situ
hybridization for direct visualization of gram-negative anaerobes in subgingival plaque samples, FEMS Immunol.
Med. Microbiol. 6 (1993) 109–114.
[17] H.J. Monstein, E. Kihlstrom, A. Tiveljung, Detection and
identification of bacteria using in-house broad range 16S
rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA
genes, APMIS 104 (1996) 451–458.
[18] D.J. Lane, B. Pace, G.J. Olsen, D.A. Stahl, M.L. Sogin, N.R.
Pace, Rapid determination of 16S ribosomal RNA sequences
for phylogenetic analysis, Proc. Natl. Acad. Sci. USA 82
(1985) 6955–6959.
[19] K. Greisen, M. Loeffelholz, A. Purohit, D. Leong, PCR
primers and probes for the 16s rRNA gene of most species of
pathogenic bacteria, including bacteria found in cerebrospinal fluid, J. Clin. Microbiol. 32 (1994) 335–351.
[20] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman,
Basic local alignment search tool, J. Mol. Biol. 215 (1990)
403–410.
[21] P. Murray, P. Traynor, D. Hopson, Evaluation of microbiological processing of urine specimens: comparison of
overnight versus two-day incubation, J. Clin. Microbiol. 30
(1992) 1600–1601.
[22] G.A. McFeters, F.P. Yu, B.H. Pyle, P. Stewart, Physiological
assessment of bacteria using fluorochromes, J. Microbiol.
Methods 21 (1995) 1–13.