Abstract:
The invention provides a method for preparation of probes for use in detecting enteroinvasive Escherichia coli and Shigella species. The small probes provide reliable and inexpensive means for detecting the pathogens in food samples and in environmental and clinical samples.

Description:
The invention described herein provides a method for preparation of probes for use in detecting enteroinvasive Escherichia coli and Shigellae species. The small probes of the invention provide reliable and inexpensive means for detecting such pathogens in food samples and in environmental and clinical samples. 
     BACKGROUND OF THE INVENTION 
     Invasive strains of Shigella and enteroinvasive E. coli have been shown to carry a plasmid (the invasiveness plasmid) that encodes genes necessary for virulence. (Hale, et al., Infect. Immun. 40: 340-350 (1983)) Based on identification of critical regions on this plasmid, DNA probes have been developed that identify potentially invasive isolates (Boileau, et al., J. Clin. Microbiol. 20:(1984) Small and Falkow (In L. Leive (ed.), Microbiology-1986. American Society for Microbiology, Washington, D.C., pp. 121-124) had described a 2.5 kilobase HindIII fragment of the invasiveness plasmid from an enteroinvasive E. coli strain for use in testing invasivness of the enteric organisms. Previously, it had been demonstrated that virulence of these organisms could be predicted by inoculating the conjunctival sac of a guinea pig with a strain of Shigella. Invasive strains produced a purulent keratoconjunctivitis associated with invasion of corneal epitheial cells and infiltration with polymorphonuclear leukocytes similar to that seen in intestinal lesions caused by Shigellae. 
     The use of guinea pigs in the identification of pathogenic organisms in the food supply and in clinical speciments is clearly expensive and time-consuming. The use of large fragments such as the 17-kilobase EcoRI fragment of Boileau and the 2.5-kilobase HindIII fragment of Small and Falkow are expensive to make and use. 
    
    
     DESCRIPTION OF THE FIGURES 
     FIG. 1: Sequencing Strategy 
     A partial restriction map of the 2.5 kilobase pair HindIII fragment is shown. Restriction sites HindIII (H), PstI (P), and DraI (D) are marked. The box below the map is the 627 base pair open reading frame (ORF) and the box above is the sequence used to synthesize an oligonucleotide for probe analysis. Arrows indicate the direction and length of sequence generated by the Sanger dideoxynucleotide chain termination method. For double-stranded sequencing, plasmid templates are generated by the Erase-a-base system of Promega. 
     FIG. 2. Northern Hybridization Blots 
     Total RNA from an invasive Shigella flexneri strain (270) and an avirulent strain (354) were hybridized against a 2.0 kilobase pair HindIII/PstI fragment. Two transcripts of 700 and 500 bases were detected as indicated by the arrows. 
     FIG. 3. Southern Hybridization Blots 
     The 230 kilobase pair plasmid was isolated from Shigella flexneri 270, digested with HindIlI, BamHI, and EcoRI, run on an agarose gel, transferred to nitrocellulose filter paper, and hybridized with the 2.5 kilobase pair HindIII fragment from E. coli. As seen in the Southern blots, the 2.5 kilobase pair from the large plasmid in enteroinvasive E. coli hybridized with a 2.5 kilobase pair HindIII fragment and &gt;20 kilobase pair EcoRI and BamHI fragments from the plasmid isolated from Shigella flexneri. Sequence data from the 2.5 kilobase pair HindIII fragment from Shigella flexneri is identical with its counter part from E. coli. 
     FIG. 4. Southern Blots of Shigella and E. coli Hybridized with Shigella probes. 
     To confirm that the Shigella probe was specific for the 230-kilobase pair plasmid that is associated with Sereny-positive Shigellae and E. coli organisms, plasmid DNA was isolated from several of the strains tested as shown in Table 1 and hybridized with the Shigella probe. Only Sereny-positive strains hybridized with the probe. Lanes marked 1-5 are E. coli. Lanes 6, 10, and 24 are Sh. flexneri, lanes 7, 11, 12,14, and 15 are Sh. sonnei. lane 9 is Sh. dysenteriae, and lane 8 is a Sh. species. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The 2.5-kilobase fragment of Small and Falkow has now been sequenced and has been found to have the described sequence of bases. An open reading frame (ORF) is seen beginning at position 108 to 738. The peptide sequence encoded by the open reading frame is indicated below the base sequence of this peptide. The presence of this reading frame has been supported by Northern blotting. It has now been found that probes consisting of oligoneoxyribonucleotides complementary to a specific sequence in this open reading frame can be used to detect enteroinvasive E. coli and Shigella species in foods and from environmental and clinical isolates. Such probes must be at least 18 bases long and should, ideally, encompass one of the more diverse portions of complementary strand. In no case should a sequence employed as a probe have more than four consecutive identical bases. It is preferred that no more than two consecutive bases be identical. A preferred sequence tested in this laboratory is the sequence 5&#39;-TAATACTCCTGAACGGCG-3&#39;. Probes of such length can easily be made by known means in a nucleotide sythesizer. 
     The synthetic DNA probe may be used in nucleic acid hybridizations either using lysed bacteria on a solid support (e.g. filter papers or nitrocellulose or nylon membranes) or using total fractionated nucleic acid isolated from bacteria which is transferred to a solid support. The probe so designed specifically detects enteroinvasive E. coli, and Shigella species, including Sh. flexneri, Sh. sonnei, Sh. dysenteria and Sh. boydii. Nucleic acid hybridizations entail use of a radioactively or non-radioactively labelled DNA (probe) which binds to a complementary strand of DNA or RNA in the target. (e.g. nucleic acid from lysed bacterial cells or isolated nucleic acid.) 
     The use of the probes described herein have two advantages over the current technology: 1) The probes discriminate between virulent and non-virulent enteroinvasive E. coli and Shigella species which classical microbiological techniques are not able to distinguish. 2) Since the sequences of the oligodeoxyribonucleotides are known, they are not only more stable, but are also easier and less expensive to make and distribute than the 17 and 2.5 kilobase pair fragments previously known. 
     The 2.5-kilobase fragment received from Small and Falkow was found to have the following sequence: ##STR1## 
     The following examples are provided to exemplify the means of practicing the claimed invention. These examples are not to be viewed as limiting the scope of the invention, since one or ordinary skill in the art would the art understand that equivalent methods known in the art can be used to practice the invention taught herein. 
     EXAMPLE 1 
     Filter Preparation 
     1. Prepare a culture of organisms from the food, environmental, or clinical samples. 
     2. After the desired time for colonial growth (approximately 20-24  hours growth at 37° C. on agar medium) a Whatman No. 541 filter was applied to the surface of the plate. The filter was gently pressed onto the surface of the growth to insure that all air bubbles were removed from beneath the surface of the filter and to assure that the filter was thoroughly wetted. The orientation of the filter was designated with a soft lead pencil. After the filter was allowed to remain in contact with the colonies on the agar surface for about 5-10 minutes it was removed with a smooth motion. 
     3. The filter was placed growth side up in a 15×100 mm petri dish over a Whatman No. 1 filter paper soaked with 3.0 ml of 0.85% saline. The filter was smoothed to assure that no air bubbles were trapped between the filters. 
     4. The Petri dish was covered and exposed to 650 to 700 watts of microwave irradiation for 30 seconds. 
     5. The Whatman No. 541 filter was transferred to another Whatman No. 1 filter soaked with 3.0 ml of lysing solution #1 (preparation described below) with forceps by gently sliding the Whatman #541 filter onto the second filter and was allowed to remain in place for 5 minutes. 
     6. The top filter described in section 5 above was then transferred to another Whatman No. 1 filter which had been soaked in 3.0 ml of lysing solution #2 (see below) and allowed to sit for 5 minutes. 
     7. Filter containing colonial material from step 6 was then transferred to a sheet of chromatography paper (an equivalent method may be used) and air dried. The filter may be stored in a vacuum desiccator at room temperature until use. 
     Lysing Solutions 
     Solution #1: 0.5M NaOH; 1.5M NaCl. 
     Solution #2: 1.0M Tris, pH7; 2.0M NaCl. 
     (See Plasmid 10:296-298) 
     EXAMPLE 2 
     Oligonucleotide Labelling 
     1. A reaction mixture is made by combining: 
     
         ______________________________________Distilled H.sub.2 O       18.8   ulDNA (300 ng/reaction)     1.7    ul1OX Kinase Buffer         2.5    ulgamma-AT.sup.32 P (150 uCi; 3000 Ci/mmol)                     1.0    ulT.sub.4 Kinase (10 units/ul)                     1.0    ul______________________________________ 
    
     2. After incubating at 37° C. for 60 minutes 4.0 ul 0.25M EDTA, pH 8 was added, followed by addition of 1.6 ul 4.0M NH 4  OAc. 
     3. A NACS PREPAC column was prepared by hydrating for 1 hour using 0.25M NH 4  OAc. The prepared mixture was then loaded on the column. Then the column was washed with 3-5 ml 0.25M NH 4  OAc. 
     4. The DNA was eluted from the column using 200 ul portions of 4.0M NH 4  OAc and the amount of radioactivity in each fraction was determined. 
     EXAMPLE 3 
     Hybridization 
     1. Hybridization reaction mixture was prepared by mixing the following: 
     
         ______________________________________Distilled H.sub.2 O       28.9   ml20X SSC (3.0M NaCl., 0.3M Na Citrate)                     15.0   ml50X Denhardt&#39;s Solution. (1 g. each of                     5.0    mlbovine serum albumin, focol, and poly-vinylpyrrolidone in 100 ml distilledwater)0.5M EDTA, pH 8.0         0.1    ml______________________________________ 
    
     2. 1.0 ml calf thymus DNA (Mol. Wt. 300,000 to 500,000; 10 mg/ml) was boiled for 5-10 minutes, added to the hybridization reaction mixture, and stirred. 
     3. 8-10 ml of the hybridization mix prepared in step 2 was added to a prepared Whatman #541 filter prepared in Example 1. 
     4. Immediately thereafter one million cpm of DNA probe of the invention was added to the treated filter or membrane prepared in step 4 and aggitated to assure full exposure of the filter to the probe. The receptical containing the filter was then placed in an incubator at 37° C. or 18-24 hours. 
     EXAMPLE 4 
     Filter washing 
     1. The filter prepared above, after required incubation, was removed from the mixture, rinsed for 5-10 seconds in 6X SSC and placed in 5-10 ml of fresh 6X SSC for 60 minutes. The filter was then removed from the liquid. After addition of fresh 6X SSC, the filter in a receptical was incubated for 60 minutes. 
     2. Liquid was removed and the filter was washed briefly in 2X SSC, removed from solution, and allowed to air dry on chromatography paper or some equivalent surface. 
     EXAMPLE 5 
     Film Exposure 
     1. The dried filters were placed in an X-ray cassette with intensifying screens. (Screens were protected by covering the filters with plastic film wrap.) In a dark environment, a sheet of X-ray film was placed over the filters in the cassette, which was then exposed for 18-24 hours at -70° C. 
     2. After the film had warmed to room temperature, it was developed in accord with instructions provided by the film manufacturer. 
     The invention may also be practiced using nucleic acid isolates following known nucleic acid blotting and hybridization techniques. Examples of such techniques are discussed in Davis, et al. Basic Methods in Molecular Biology, New York, 1986, pp. 62-65 and 75-78. RNA blotting techniques such as Northern blotting and hybridization techniques known to those of ordinary skill in the art may also be used. See Davis, et al, (cited above) at pages 143-146 and 150-151. The above references are incorporated herein by reference. DNA may be isolated by the modified alkaline denaturation method of Birnboim and Doly, Nucleic Acids Res., 7: 1513-1523 (1979), which is incorporated herein by reference. 
     EXAMPLE 6 
     RNA Isolation 
     1. Cells (100 ml) were grown to the end of the logarithmic phase, then centrifuged at 6000 rpm for 5 minutes. The resulting pellet was suspended in 5 ml Tris-EDTA buffer. 
     2. The suspension prepared in step 1 was centrifuged at 6000 rpm for 5 minutes. Cells were then resuspended in 2.5 ml Tris-EDTA. 
     3. To the suspension prepared in step 2, 1 ml lysozyme (0.4 mg/ml) was added. The resulting mixture was incubated 5-20 minutes at 37° C. 
     4. After incubation, 0.25 ml 10% sodium dodecyl sulfate was added followed by mixing. Phenol/chloroform (1:1, v/v) was added. The mixture was vortexed on ice for about 20 minutes, then centrifuged 20 minutes at 15,000 rpm. 
     5. The lower layer of the mixture resulting from step 4 was transferred to a sterile 30 ml beaker on ice. An equal volume of isopropanol was slowly added while spooling nucleic acid onto a glass rod. 
     6. Nucleic acid was dissolved in 1 ml Tris-EDTA and the phenol/chloroform extraction and isopropanol spooling was repeated. The spooled nucleic acid was then redissolved in 1 ml Tris-EDTA. 
     7. To the dissolved nucleic acid prepared in step 6 was added 0.1 volume 3 molar NaOAc, pH 4.5 and 2.0 volumes ethanol. The resulting mixture was placed on ice for 30 Minutes, then centrifuged at 10,000 rpm for 10 minutes. The supernatant was removed and 1.0 ml 70% ethanol was added to the residue. The resulting ethanol mixture was centrifuged as before and the supernatant removed. The residue was dried. The resulting product can be stored at -20° C. until needed. 
     Oligonucleotides of bases which are complementary to a specific sequence in an open reading frame of the 2.5 kilobase HindII fragment disclosed above may be used as probes by the method of the invention. However, probes of 18 to 24 bases are preferred. Probes from a variable region are preferred. For purposes of the invention, probes should have no more than 4 identical bases consecutively. Preferably, no more than two consecutive bases should be identical. 
     The bases complementary to the first reading frame identified have the following sequence: ##STR2## 
     Table 1 indicates that the enteroinvasive organisms were readily identified by the probe having the sequence 5&#39;-TAATACTCCTGAACGGCG-3&#39;. 
     
                       TABLE 1______________________________________BACTERIAL STRAINS HYBRIDIZED WITH SHIGELLAPROBESeveral Shigella and enteroinvasive E. coli stains werehybridized against the synthetic oligonucleotide derived from theopen reading frame of the 2.5 kilobase HindIII fragment. Asexpected, only those strains that carry the 230 kilobase pairplasmid hybridized to the probe. Other gram-negative and gram-positive bacteria tested failed to hybridize with the probe. Thenumber in parenthesis indicates the number of strains tested.BACTERIA TESTED BY HYBRIDIZATION WITHSHIGELLA PROBE            Results of Presence ofBacteria tested  Hybridization                       Plasmid______________________________________Shigella flexneri (4)            +          +Sh. sonnei (1)   +          +Sh. dysentariae (2)            +          +Sh. boydii (1)   +          +Sh. flexneri 354 (1)            -          -Sh. sonnei (2)   -          -Sh. boydii (1)   -          -Escherichia coli (6)            +          +E. coli (3)      -          -Other gram negativesEnterobacter cloacae            -Klebsiella pneumoniae            -Proteus vulgaris -Salmonella typhimurium            -Serratia marcescens            -Yersinia enterocolitica (3)            -Gram positivesStaphlycoccus aureus            -Staphlycoccus epidermidis            -Streptococcus pyogenes            -Streptococcus faecalis            -Listeria monocytogenes            -Listeria ivanovii            -Listeria seeligeri            -______________________________________