Sequences of E. coli O157

Details Sequences of E. coli O157 Sequences of E. coli O157

1999-12-03

2002-04-02

Fredman, Jeffrey (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06365723

ABSTRACT:

BACKGROUND OF THE INVENTION
Escherichia coli
is a common enteric bacterial strain that has both laboratory and human health importance. One particular strain of
E. coli,
designated O157:H7 is a human enteric pathogen that causes acute hemorrhagic colitis. Young children and the elderly are particularly susceptible to disease caused by this bacteria, which is usually contracted by eating contaminated food such as undercooked meat. In the most vulnerable patients, the colitis frequently develops into hemolytic uremic syndrome (HUS), a condition that is often fatal. The disease has a very rapid progression, and is consequently very difficult to treat. Often, patients are severely ill by the time the disease is diagnosed. Once a diagnosis has been made, appropriate antibiotics may be administered to kill the infective bacteria. Sometimes, however, by the time a diagnosis has been rendered, toxic proteins secreted by the bacteria have damaged mucosal cells and entered the blood stream. Recently, clinical isolates of O157:H7 have been found to exhibit resistance to an increasing spectrum of antibiotics, which will further complicate treatment.
The source of the bacteria in the several recent cases of disease caused by this organism were traced to hamburgers purchased in fast food restaurants. The bacteria are extremely proficient at establishing an infection; ingestion of as few as 10 live bacteria is sufficient to establish an infection. The highly infective nature of O157:H7 and the devastating sequelae associated with infection by this bacteria, together with the extensive public attention given to outbreaks of hemorrhagic colitis, has generated a great deal of interest among medical professionals and the general public in developing the means for early diagnosis and treatment of the disease. Farmers are desirous of an effective treatment of infections in cattle and pigs, which are the main reservoirs for
E. coli
enteric pathogens. The ability to diagnose and treat livestock infected by this organism will prevent the loss of livestock and the transmission of the organism from animals to humans. Meat suppliers and those in the food industry are very much interested in a means for detecting the organism in tainted meat. Because the infective dose of O157:H7 is extremely low, a highly sensitive test is needed to identify contaminating organisms in food.
Modern geneticists have been working to resolve the genetic code of many organisms. Efforts to sequence the human genome are ongoing. The effort to sequence the genomes of whole organisms began with an effort to sequence the genome of
E. coli.
For the original effort to sequence the
E. coli
genome, a useful and common laboratory strain, designated K12, was chosen. The entire genome of that strain was sequenced and published.
Science,
277:1453-1462 (1997). Since the genes which are responsible for the pathogenicity of
E. coli
0157:H7 are missing from strain K12, the sequence of the K12 genome is of limited help in developing tools to detect, hinder or destroy
E. coli
0157:H7.
Some efforts have been directed toward the sequencing of specific genes from 0157:H7. U.S. Pat. No. 5,798,260 describes the sequence of one specific gene, named adhesion, from that genome. The development of additional sequence information from
E. coli
0157:H7 would be needed for comprehensive efforts at detection, diagnosis, prophylaxis and therapeutic approaches to infections caused by the organism.
BRIEF SUMMARY OF THE INVENTION
It is an object of the invention to provide essentially the entire sequence of
E. coli
0157:H7 to enable detection, diagnosis, prophylaxis and therapeutic tools to combat bacterial infections.
It is another object of the present invention to provide a means to detect low numbers of
Escherichia coli
O157:H7 in a contaminated food source.
It is yet another object of this invention to provide a means for the early diagnosis of humans and livestock infected with O157:H7.
Another object of the present invention is to provide a means of treating humans and livestock infected with 0157:H7.
It is a further object of the present invention to provide a means for the prevention of infection by O157:H7.
The present invention includes many DNA sequences that are unique to
E. coli
O157:H7.
One aspect of the present invention is a DNA sequence comprising an open reading frame (ORF), designated 03169, that encodes a putative cytotoxin 3169 amino acids in length that resembles the clostridial cytotoxins ToxA and ToxB of
C. difficile
and cytotoxin L of
C. sordelli.
Another aspect of the present invention is a DNA sequence that constitutes an urease gene cluster.
A third aspect of the present invention is a chromosomal gene that encodes a toxin related to the RTX family of cytotoxins and associated transport proteins.
Another aspect of the present invention are genes that are found in the Locus of Enterocyte Effacement (LEE), a 45-kb cluster of genes that are involved in the attachment of pathogens to intestinal epithelial cells and other related functions necessary to establish infection.
Another aspect of the present invention is a hypothetical serine/threonine kinase (stk) encoded by phage 933W, a lysogenic bacteriophage found in O157:H7.
The present invention is also a putative tail fiber gene, which is found on phage 933W.
Another aspect of the present invention is a method for detecting
E. coli
O157:H7 and distinguishing the strain from other strains of
E. coli
by genetic analysis and testing.
It is a feature of the invention disclosed here that virtually the entire genome of
E. coli
0157:H7 is set forth in the data contained here, combined with the information already published in the field.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Not applicable.
DETAILED DESCRIPTION OF THE INVENTION
The investigators here have sequenced virtually the entire genome of
E. coli
0157:H7. Presented in this specification is essentially all the DNA sequence which is contained in strain 0157:H7 and not found in the prviously sequenced
E. coli
strain K12. The genome sequence is essentially complete, lacking only an occasional presumably small sequence linkage between established long sequences known. The availability of the sequence data presented here will enable intelligent design of diagnostic detection, prophylaxis and therapeutic tools for disease and infections caused by this organisms.
The sequence of
E. coli
0157:H7 was, in brief, performed by shotgun cloning in the M13 Janus vector (Burland et al.
Nucl. Acids Res.
21:3385-3390 (1993)). Genomic DNA was prepared for library construction by nebulization, end-repair and size fractionation as described in Mahillon et al.
Gene
223:47-54 (1998). Recovered DNA fragments were ligated into the M13 Janus vectors. Library subclones were picked as plaques, from which template DNAs were prepared and then sequenced by Prism-terminator Cycle Sequencing chemistry and analyzed on ABI377 automated sequencers. Sequences were assembled by the Seqman II program (DNASTAR), and finishing employed a combination of PCR and primer walking techniques. Open reading frames were identified and analyzed as described in Blattner et al.
Science
277:1453-1474 (1997). All the sequences presented in this specification are unique to strain 0157:H7 as compared to strain K12. This sequence data, when combined with the sequence of K12, resolves all of the genetic sequence of 0157:H7. The information on the K12 sequence, contained in
Science,
277:1453-62 (1997) is hereby incorporated by reference as if set forth in full herein.
An important analysis which has been begun on this sequence data is the identification of genetic sequences associated with the pathogenesis of infection, which sequences provide information essential to the diagnosis, treatment, and prevention of infection by that organism. In order to facilitate the identification of genes involved in the pathogenesis of infection by enterohemorrhagic
E. coli
(EHEC) for use in detection of the pathogen, and in the diagnosis, treatm

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