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From the Departments of 1 Clinical Ophthalmology and 2 Pathology, The Institute of Ophthalmology and University College London, London, United Kingdom; and 3 Department of Ophthalmology, Whipps Cross Hospital, London, United Kingdom.
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Abstract |
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METHODS. PCR-RFLP was performed on 53 strains of 14 bacterial species (eight Gram positive and five Gram negative) collected from both keratitis and endophthalmitis patients. Two pairs of oligonucleotide primers based on the 16S rDNA gene were used to PCR-amplify 1.2- and 1.0-kb fragments of bacterial genomic DNA. RFLPs within the PCR product were used to speciate the organisms.
RESULTS. The sensitivity of the nested PCR amplification reaction was one organism. All bacteria tested could be identified and speciated using RFLP analysis except for Escherichia coli and Serratia marcescens, which could not be interdifferentiated using RFLP. Molecular analysis of two vitreous samples from two eyes with typical signs of bacterial endophthalmitis confirmed the presence of E. coli in the vitreous from a culture-positive case with E. coli endophthalmitis and revealed the presence of Staphylococcus epidermidis in the vitreous of a culture-negative case.
CONCLUSIONS. It is expected that this technique will provide a useful laboratory tool for future microbiologic diagnosis of patients presenting with endophthalmitis, especially for those eyes that prove culture negative.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Samples
All bacteria isolated from clinical ocular samples collected at
Moorfields Eye Hospital, whether from cases of keratitis or
endophthalmitis, were included for purposes of completion and
comparison. A total of 54 strains of 14 bacterial species were tested,
of which 43 were clinical isolates and 11 were National Collection of
Type Culture (NCTC) strains. Species and strain details appear in Table 1
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Procedure for Endophthalmitis Cases.
Intraocular (aqueous and vitreous) sampling was performed on every
patient under aseptic conditions. Aqueous sampling was undertaken using
a 27-gauge (0.33 mm) needle using topical anesthesia, and 100 to 200
µl was aspirated. Vitreous sampling was undertaken after
subconjunctival injection of anesthetic. Vitreous (200–400 µl) was
aspirated using a 23-gauge needle that was inserted through the pars
plana 3 mm behind the limbus in aphakic eyes and 4 mm behind the limbus
in phakic eyes.
Intraocular samples were examined by Gram’s stain and immediately cultured on solid (blood agar) and liquid media (Robertson’s cooked meat broth and Brain Heart infusion (Difco Laboratories UK, West Molesey, Surrey, United Kingdom) under both aerobic and anaerobic conditions. Aerobic organisms isolated from an intraocular sample as heavy growth from one solid medium or as growth of the same organism in more than one medium (solid and/or liquid) were considered responsible for causing endophthalmitis. All cultures were maintained for up to 14 days.
Procedure for Keratitis Cases.
Corneal scrapes were performed at the slit lamp under topical
anesthesia. Sterile 27-gauge needles were used to scrape off corneal
epithelium and anterior stroma in the region of the leading edge of the
corneal ulcer. Samples were placed on a slide for Gram and Giemsa
staining and also were plated immediately on Blood and Sabauraud’s
dextrose agar (Difco Laboratories UK) before transport to the
microbiology laboratory. Plates were incubated under aerobic conditions
at 30°C and 37°C.
Identification of Isolates.
Bacterial isolates were identified using standard microbiologic
methods. After isolation by culture the API biochemical identification
system (API Analytab Products, Division of Sherwood Medical,
NY) was used for identification. Organisms were subsequently
stored on beads (Mast Diagnostics, Bootle, Merseyside, United Kingdom)
at -70°C.
Collection of Normal and Inflamed Vitreous.
Vitreous was collected by sterile technique at the time of vitrectomy
during planned surgical procedures in patients with no evidence of
intraocular infection or inflammation or medical history of uveitis
and/or diabetes mellitus ("normal" vitreous). Vitreous also was
collected from patients with other causes of posterior segment
inflammation not associated with bacterial infection ("inflamed"
vitreous). Samples of vitreous were aliquoted in a sterile manner and
stored at -20°C.
DNA Extraction
Bacteria.
Method 1 was the full extraction procedure. Previous work in this
laboratory has successfully used glass beads and a bead beater
apparatus (Stratech Scientific, Bedfordshire, United Kingdom)
to effectively release DNA from bacterial cells in
suspension.11
Briefly, a 2-mm colony of bacteria was
diluted into 300 µl sterile phosphate-buffered saline solution, with
50 µg/ml proteinase-K and 0.5 g glass beads (0.1-mm size). This
mixture was beaten on the bead beater for 10 seconds after which it was
incubated at 50°C for 30 minutes. Phenol (500 µl, pH 8.0) was then
added, and the samples were vortexed for 30 seconds and centrifuged for
2 minutes at 14,000g. The aqueous phase was removed and
extracted twice, the first time using an equal volume of
phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0) and subsequently
with an equal volume of chloroform. The DNA was subsequently
precipitated with 2.5 volumes of ethanol in the presence of 300 mM
sodium acetate (pH 5.2). The DNA was pelleted at 14,000g for
20 minutes, washed with ice cold 80% ethanol, air-dried at 65°C for
10 minutes, and resuspended in 25 µl of sterile TE buffer (10 mM
Tris-HCl, pH 8.0, 1 mM EDTA). The DNA was diluted to a working
concentration of 10 ng/µl for use in PCR reactions and stored at
-20°C.
Method 2 was direct PCR. Bacteria were suspended in the distilled water component of the PCR cocktail and used directly in PCR reactions.
Fungi.
DNA was extracted by a previously described method.12
Briefly, DNA extraction tubes were prepared containing a single 1-mm
colony of C. albicans or scrapings of A.
fumigatus, or F. solani in 100 µl of 0.05 M Tris, pH
7.5, 0.01 M EDTA, 0.028 M ß-mercaptoethanol, and 0.3 mg/ml zymolase
(ICN Biomedicals, Aurora OH). Samples were incubated for 30 minutes at
37°C, followed by addition of 0.1% SDS and 15 µg/ml proteinase K,
and incubation continued for a further 5 minutes. The mixture was
subsequently heated to 95°C for 5 minutes and cooled on ice for 15
minutes. Samples were extracted with an equal volume of
phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0) and subsequently
with an equal volume of chloroform and precipitated with 2.5 volumes of
ethanol in the presence of 300 mM sodium acetate (pH 5.2). The DNA was
pelleted at 14,000g for 20 minutes, washed with ice-cold
80% ethanol, air-dried at 65°C for 10 minutes, and resuspended in 25
µl sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The DNA was
diluted to a working concentration of 10 ng/µl for use in PCR
reactions.
Human Leukocytes.
DNA was extracted from 10 ml human whole blood using the Machery–Nagel
Nucleospin-Blood DNA extraction kit (Biogene, Cambridge, United
Kingdom) according to the manufacturer’s instructions. The DNA was
diluted to a working concentration of 10 ng/µl and stored at
-20°C.
PCR Amplification of Bacterial 16S rDNA
Design and Optimization of Primers.
Multiple copies of the 16S rDNA genes are present in all bacterial
genomes. Details of the four primer pairs used in this study appear in
Table 2 .
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Pretreatment of Taq DNA Polymerase to Remove
Contaminating Bacterial DNA.
Taq DNA polymerase is known to be contaminated with low
levels of bacterial DNA not originating from either Thermus
aquaticus or E. coli and is easily amplified using
universal bacterial primers based on ribosomal gene
sequences.13
14
15
The Replitherm Taq DNA
polymerase (Replitherm Thermostable DNA polymerase; Cambio Ltd.) used
in this study is one of a number of commercially available
Taq polymerases with known low levels of DNA contamination.
Although this level of contamination is insufficient to give a
detectable amplification product after just one round of PCR, it is
easily detected after nested amplification. Therefore, in this study,
before first-round PCR amplification, the Replitherm Taq was
treated with AluI restriction endonuclease (ratio of 3:1
units of Taq:AluI; Promega UK Ltd., Southampton,
United Kingdom), which recognizes a specific nucleotide sequence AGCT.
The criteria for choosing this restriction enzyme were (1) its known
high cutting frequency within the 16S rDNA genomic template, which was
evident after sequence analysis of the amplified fragments from the 14
bacterial species and (2) the ability to heat-inactivate the enzyme
before addition of template DNA. Before PCR amplification, the water,
PCR buffer, magnesium, and Taq components were mixed and
incubated at 37°C with AluI for 30 minutes. The
restriction enzyme was subsequently inactivated by incubation at 95°C
for 2 minutes, after which the dNTPs, primers, and template DNA were
added, and the PCR cycle commenced.
DNA Sequencing and Restriction Analysis
Amplified DNAs from PCR reactions were agarose gel
purified, excised, and recovered into sterile water (Geneclean II Kit;
BIO 101, La Jolla, CA). PCR fragments were directly cycle-sequenced in
both directions using an ABI prism automated DNA sequencer (model 377,
version 2.1.1; PE Biosystems, Warrington, UK). Sequences were
aligned and scanned both manually and using database and software
programs available through the HGMP computer center. A search was made
for a total of 268 restriction enzyme recognition sites.
Cloning of PCR Products
Amplified DNA from PCR reactions was purified on agarose/TBE gels,
excised, and recovered into solution (Geneclean II Kit; BIO 101). In
some cases PCR products were directly cloned into pCR II (Cat. No.
K2000–01; Invitrogen BV, Leek, The Netherlands) to aid sequencing.
Restriction Enzyme Analysis
After PCR amplification, the concentration of PCR product was
estimated using ethidium bromide staining of agarose/TBE gels.
Restriction enzyme combinations were used that yielded fragments that
allowed easy identification of species after separation of
digested DNA on polyacrylamide/TBE gels. One restriction
enzyme cocktail was developed to directly speciate organisms. All
enzymes were purchased from Promega UK Ltd. (Southampton, United
Kingdom) except for DraIII and AflIII, which were
purchased from New England Biolaboratories Ltd. (Hitchin,
Hertfordshire, United Kingdom).
RFLP Protocol
The restriction enzyme cocktail (5 units each enzyme) was added to
approximately 1 µg of DNA/PCR product in PCR buffer that had been
adjusted to contain 100 mM NaCl, 1 mM dithiothreitol, and a final
concentration of 7 mM MgCl2. A restriction enzyme
cocktail containing the following nine restriction endonucleases was
used to achieve speciation: AflIII, BssHII,
ClaI, DraI, DraIII, HpaI,
NdeI, NsiI, and SalI. Restriction
enzyme digests were performed at 37°C for 18 hours. The reaction was
halted by freezing, and restriction fragments were analyzed on 10%
TBE/polyacrylamide gels.
Electrophoresis and Imaging
After PCR, amplification products were resolved on a 1%
agarose/TBE gel and visualized using ethidium bromide under UV
illumination. A molecular weight ladder was included in each run
(1.0-kb ladder, Cat. No. 15615-016; Gibco BRL, Paisley, Scotland).
Restriction enzyme digests were resolved on 10% polyacrylamide/TBE gels and visualized under UV illumination after staining with ethidium bromide. A molecular weight ladder was included in each run (100 bp, Cat no. SLL-100; Advanced Biotechnologies, Leatherhead, Surrey, United Kingdom).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nested PCR Protocol and Controls
In each case, 1 µl of first-round PCR product was added to the
nested amplification reaction as template. Control-negative (no DNA)
samples included water and vitreous (normal or inflamed as
appropriate). Control negatives were included in each protocol, and the
first-round negatives were included as test samples in the nested PCR
reaction. Control-negative samples from both rounds of PCR were
consistently negative after two rounds of amplification.
Control-positive samples included extracted genomic DNA (10 ng and 10 fg) or live organisms (1–5 organisms by dilution from fresh overnight culture) in both water and vitreous. Conrtol-positive samples were consistently positive after one round (10 ng extracted DNA only) and two rounds of amplification (10 fg extracted genomic DNA or 1–5 live organisms).
Sensitivity of PCR Reactions
The sensitivity of the first-round PCR was routinely found to be
10 pg from dilutions of DNA from E. coli, coagulase-negative
staphylococci, Staphylococcus aureus and
Klebsiella pneumoniae, starting from a
concentration of 10 ng/µl (Fig. 2a
). The sensitivity of the reaction was improved to 1 fg after nested
PCR (Fig. 2b) . Assuming a total DNA content of 5 fg per
organism,16
this is approximately equivalent to a
sensitivity of one organism.
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DNA Sequencing
The sequencing reaction was performed three times for each PCR
product using the same template but different PCR reactions. Comparison
of the DNA sequences obtained made with the full and partial sequences
of the genes already available in GenBank demonstrated that they were
derived from the 16S rDNA gene (sequences 97–100% identical). The
sequences obtained were submitted to GenBank and have been assigned the
following accession numbers: Streptococcus pyogenes:
AFO76028, Streptococcus viridans: AFO76036,
Streptococcus faecalis: AFO76027, P. acnes: AFO76032,
S. aureus: AFO76030, Streptococcus pneumoniae:
AFO76029, Bacillus cereus: AFO76031, E. coli:
AFO76037, Serratia marcescens: AFO76038, Haemophilus
influenzae: AFO76035, K. pneumoniae: AFO76033,
Proteus mirabilis: AFO76034, and Pseudomonas
aeruginosa: AFO76039.
Strain Testing
A set of control patterns was obtained from all bacterial species.
Subsequently, PCR-RFLP and sequence analysis was performed on all
clinical isolates to ensure the reproducibility of the procedure. All
clinical isolates of streptococci, S. aureus, and each of
the Enterobacteriacae, yielded results from PCR, RFLP, and
sequencing that were in agreement with culture results. RFLP analysis
of coagulase-negative staphylococci yielded three patterns. Two of
these were still identified as coagulase-negative staphylococci by a
masked observer, but a third pattern was found to be unidentifiable.
Sequence analysis of clinical isolates of coagulase-negative
staphylococci yielded 98% base identity in each case, with several of
the following staphylococcal species that belong to this group:
S. epidermidis,S. caprae, S. capitis, S. warneri, S.
lugdunensis and S. pateurii. These sequences were also
found to be 97% identical with that of S. aureus. These new
RFLP patterns have been added to the "control patterns" to aid
future identification of these species.
Cloning of PCR Products
Sequencing direct from PCR product did not yield adequate
sequencing data for the following bacteria: P. mirabilis, H.
influenzae, S. viridans, and P. acnes. PCR fragments
were gene-cleaned and cloned into pCRII. Subsequent PCR and sequencing
was then performed successfully using primers complementary to Sp6 and
T7 sites present in the pCRII vector.
RFLP Analysis of Amplified PCR Products Differentiates Bacterial
Species
The combination of AflIII, BssHII,
ClaI, DraI, DraIII, HpaI,
NdeI, NsiI, and SalI successfully
differentiated between the PCR-amplified products from 13 of 14
bacterial species (Fig. 4)
. E. coli and S. marcescens have identical RFLP
patterns, reflecting the similarity of the 16S rDNA gene sequences for
these two species (96.7% identical). The only stretch of nucleotides
that would reliably identify one from the other, however, has a
sequence that is not recognized by any of the restriction enzymes
commercially available.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The choice of primers was affected by the knowledge that 16S rDNA genes are highly conserved in bacterial genomes and may be present in multiple copies.17 18 Bacteria were differentiated by exploiting the variable stretches of DNA sequence in the 16S rRNA gene. The study of sequence variability also can be carried out using other methods, for example, by the synthesis of species-specific oligonucleotide probes, which would identify DNA sequences through hybridization, or by design of PCR primers and amplification protocols, which discriminate for the organism of interest.19 20 However, probes and primers would need to be designed for individual species, and as such these methods were unsuitable for our purposes.
Standard DNA extraction methods are time-consuming and involve multiple tube transfers wherein organisms or DNA may be lost. The method used simply involves heating to 95°C as part of the PCR-cycling protocol and shows great potential because it reduces the potential loss of organisms or DNA in transfer and is found to be reliable, reproducible, simple, and rapid. A highly sensitive approach was required to confirm the presence of any bacteria present. The ability to detect 10 pg of genomic DNA after one round of PCR may be slightly less than the sensitivity reported by other authors21 and would be improved by reducing the annealing temperature. We have opted for the highest annealing temperature possible, to ensure specificity, which may have reduced the sensitivity of the reaction, but the use of a second-round nested approach improved sensitivity, allowing detection of as little as 1 fg, while maintaining the specificity of PCR detection.
Thirteen RFLP patterns were obtained from 14 bacterial species and only 12 of the 14 bacterial species had unique RFLP patterns that could identify them from all other species tested. The patterns obtained by gel electrophoresis were found to be reproducible. However, species identification could be made difficult if the minor and fainter bands were not clearly visible on all gels. The identical patterns obtained from E. coli and S. marcescens reflect the similarity in the gene sequence of the 16S rDNA from these organisms. Although the minor sequence difference was not useful in differentiating these two organisms by RFLP analysis, a stretch of nine nucleotides is present, which is more than sufficient to allow the design of oligonucleotide primers that would allow differentiation of these organisms using additional PCR protocols. Because the treatment of these two organisms would be identical (whether endophthalmitis is caused by E. coli or S. marcescens), further identification was not pursued.
Because this PCR/RFLP protocol is designed to detect the presence of all bacteria, if the organism present is not one of the series studied here, it is conceivable that this specific restriction enzyme digestion may be unable to identify the pathogen. As a precaution, therefore, all amplified PCR products were sequenced to confirm the identity of the bacterium. This technique has been used successfully to confirm the identity of the organism detected by culture in one case and to confirm bacterial involvement in a culture-negative case. No inhibitory effects of vitreous were observed in the analysis of these two samples. To investigate inhibitory effects further, larger numbers of clinical samples (aqueous and vitreous) have been analyzed. Results are reported elsewhere and indicate that the level of PCR inhibition varied from sample to sample and that dilution of the sample was required in the analysis of 8/18 (44%) intraocular samples (5 aqueous and 3 vitreous) before a positive PCR result was obtained (Okhravi N, Adamson P, Carroll N, Dunlop A, Matheson M, Towler HMA, Lightman S, unpublished results).
The detection of bacterial DNA by PCR-based methodologies, in body sites that are considered sterile, has been used to improve the rate of microbiologic diagnosis for cerebral-spinal fluid,19 22 23 synovial fluid,24 and vitreous (Okhravi N, Adamson P, Carroll N, Dunlop A, Matheson M, Towler HMA, Lightman S, unpublished results).11 Results demonstrate that samples from cases clinically suspected as harboring infection contain bacterial DNA, whereas samples from clinically noninfective cases do not (Okhravi N, Adamson P, Carroll N, Dunlop A, Matheson M, Towler HMA, Lightman S, unpublished results).22 23 25
At this center, 63% of cultures from cases of postsurgical, presumed bacterial endophthalmitis proved to be negative.1 This is a reflection of the large number of cases referred to this tertiary referral center having had prior antibiotic treatment (topical, systemic, and intraocular) for endophthalmitis. The use of antibiotics may affect the culture-positive rate but should not affect the ability to PCR-amplify DNA in the short term. However, because of the lack of quantification, it is not possible to judge the number of organisms present and therefore to differentiate contamination from clear evidence of infection. Although PCR-based methods are able to provide the reliability and sensitivity required, data regarding antibiotic drug sensitivities currently can only be obtained after positive culture. In culture-negative cases, however, information regarding etiology can only be obtained using PCR technology. The presence of multiple organisms in the sample (either as true causative agents of infection or after contamination) is always a possibility, especially in samples collected from the cornea or from cases with endophthalmitis after penetrating injury. The inability of PCR-RFLP techniques to deal effectively with the presence of multiple organisms is a potential drawback of this methodology. A PCR-based study of postoperative endophthalmitis, however, has revealed the presence of multiple organisms in the anterior chamber much more frequently than in the vitreous cavity, allowing unambiguous species identification from the vitreous sample in these cases (Okhravi N, Adamson P, Carroll N, Dunlop A, Matheson M, Towler HMA, Lightman S, unpublished results). It is expected that PCR-based technology will prove to be a useful adjunct to microbiologic culture techniques, especially in culture-negative cases, and provide a useful addition to the diagnostic tools available to the clinician.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 14, 1999; revised September 15 and November 2, 1999; accepted November 16, 1999.
Commercial relationships policy: N.
Corresponding author: Narciss Okhravi, Department of Clinical Ophthalmology, The Institute of Ophthalmology and University College London, Bath Street, London EC1V 9EL, UK. nokhravi{at}hgmp.mrc.ac.uk
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