Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-13
2003-09-23
Benzion, Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100
Reexamination Certificate
active
06623931
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of diagnostics based on DNA sequence information.
BACKGROUND
Antimicrobial resistance to multiple antibiotics is a significant and well described clinical problem; however, a less well-characterized phenomenon, antimicrobial tolerance, has emerged in pathogenic isolates of
Streptococcus pneumoniae
with potentially serious effects on patient outcome. Tolerance describes the ability of bacteria to stop growing in the presence of an antibiotic, while surviving to resume growth once the antibiotic is remove; Incidence of tolerance to vancomycin, the antibiotic of last resort for Gram-positive infections, has increased to 8% in the past few years. Tolerance has also been implicated in-poor patient outcome with pneumococcal meningitis, mortality 30% versus non-tolerant 5% (unpublished data).
In 1997, a locus was identified that is believed to control the activation of the major pneumococcal autolytic enzyme LytA, which is the enzyme whose loss of function is associated with tolerance. Novak R. B. et al, “Emergence of vancomycin tolerance in
Streptococcus pneumoniae’, Nature
399:590-593 (1999). The operon, vex/pep27/vncr/s, encodes for a signal peptide, Pep27, that is transported out of the cell via the Vex dedicated transporter. Novak, R. et al., “Signal transduction by a death signal peptide: uncovering the mechanism of bacterial killing by penicillin”,
Molec Cell.
5:49-57 (2000). Pep27 is believed to be a quorum sensing peptide. Novak et al., id. (2000). Once it reaches a critical density in the supernatant, it signals through the two-component regulatory system, VncS and VncR, which subsequently induces activation of LytA. Novak et al., id. (2000).
It has been demonstrated that mutating any one of the genes of the vex/pep27/vncr/s operon prevents proper signaling, resulting in lack of LytA activation and tolerance to penicillin and vancomycin. Novak et al., id. (2000). However, the genetic basis for naturally occurring vancomycin tolerance in the community has not been determined.
SUMMARY OF THE INVENTION
The present invention provides a rapid diagnostic assay to identify strains of bacteria, particularly strains of
Streptococcus pneumoniae
, which are likely to have acquired tolerance to antibiotics. The assay is based on the identification of allelic variations with the vncS, vex2 and pep27 genes that are closely associated with tolerance to penicillin and vancomycin when present.
In one aspect, combinations of vex2 and pep27 alleles which are associated with antibiotic tolerance are taught.
In another aspect, an allele of the vncS gene which is associated with antibiotic tolerance is taught.
Single nucleotide polymorphisms (SNPs) which identify the various vncS, vex2 and pep27 alleles which are relevant to the present invention are taught. According to the present invention, the likely presence of antibiotic tolerant bacterial strains can be identified by determining the presence or absence of these distinctive SNPs in the vncS, vex2 and pep27 genes that are associated with antibiotic tolerance.
Various methods for identifying the SNPs taught herein or and kits providing the components needed to perform such methods are included herein as part of the invention.
DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID No. 1 is a portion of the Type 4 allele of the vex2 gene. The presence of a “G” nucleotide at position 41 and an “A” nucleotide at position 67 identify this as a Type 4 allele.
SEQ ID No. 2 is a portion of the R6 allele of the vex2 gene. The presence of an “A”nucleotide at position 41 and a “G” nucleotide at position 67 identify this as an R6 allele.
SEQ ID No. 3 is a portion of the Type 4 allele of the pep27 gene. The presence of a “G” nucleotide at position 35 and a “G” nucleotide at position 46 identify this as a Type 4 allele.
SEQ ID No. 4 is a portion of the R6 allele of the pep27 gene. The presence of an “A” nucleotide at position 35 and an “A” nucleotide at position 46 identify this as an R6 allele.
SEQ ID No. 5 is a portion of the wildtype allele for the vncS gene. The presence of a “T” nucleotide at position 79 identifies this as a wildtype allele.
SEQ ID No. 6 is a portion of the vancomycin tolerant allele for the vncS gene. The presence of a “C” nucleotide at position 79 identifies this as a vancomycin tolerant allele.
SEQ ID No. 7 is a forward primer for the vex2 gene which hybridizes to a region approx. 255 nucleotides upstream of the SNPs which distinguish the Type 4 allele from the R6 allele.
SEQ ID No. 8 is a reverse primer for the vex2 gene which hybridizes to a region approx. 160 nucleotides downstream of the SNPs which distinguish the Type 4 allele from the R6 allele.
SEQ ID No.9 is a forward primer for the pep27 gene which hybridizes to a region about 90 nucleotides upstream of the SNPs which distinguish the Type 4 allele from the R6 allele.
SEQ ID No. 10 is a reverse primer for the pep27 gene which hybridizes to a region about 90 nucleotides downstream of the SNPs which distinguish the Type 4 allele from the R6 allele.
SEQ ID No. 11 is a forward primer for the vncS gene which hybridizes to a region about 380 nucleotides upstream of the SNP which distinguishes the wildtype allele from the antibiotic tolerant allele.
SEQ ID No. 12 is a reverse primer for the vncS gene which hybridizes to a region about 30 nucleotides downstream of the SNP which distinguishes the wildtype allele from the antibiotic tolerant allele.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A Laboratory Handbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocols in Immunology” Volumes I-III [Coligan, J. E., ed. (1994)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984).
Definitions: The terms and phrases used herein to describe and claim the present invention shall have the meanings set forth below.
By “antibiotic tolerance” or “antibiotic tolerant” is meant the ability of bacteria to stop growing in the presence of an antibiotic, while surviving to resume growth once the antibiotic is removed. In contrast to antibiotic resistance, bacterial strains which are antibiotic tolerant cannot be killed by increasing the amount of antibiotic used.
By “oligonucleotide,” is meant a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. The oligonucleotides of the invention are preferably from 10 to 50 nucleotides in length, even more preferably from 20-30 nucleotides in length or from 15-25 nucleotides in length, and may be DNA, RNA or synthetic nucleic acid, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be appreciated by those skilled in the art. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence to form a stable hybrid. Such molecules are known in the art and include, for example, peptide nucleic acids (PNAs) in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
By “primer” is meant an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation o
Atkinson Robyn M.
Tuomanen Elaine I.
Benzion Gary
Chakrabarti Arun K.
Elmer J. Scott
St. Jude Children's Research Hospital
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