Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-03
2004-03-30
Horlick, Kenneth R. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091500, C536S023200, C536S023700, C536S024320, C536S024320
Reexamination Certificate
active
06713254
ABSTRACT:
The present invention relates to DNA probes, a method and a kit for identifying antibiotic-resistant strains of bacteria.
The occurrence of antibiotic-resistant strains of bacteria, particularly of streptococcus strains, represents an increasing problem. So far, antibiotic susceptibility tests have been carried out by isolating bacteria and establishing a culture to define the minimum antibiotic inhibitory concentration in a biological test. This method takes at least 1 to 2 days. Well-calculated and thus optimum treatment is not possible within this period. Therefore, there is a need for a faster identification of existing resistances.
The object of the present invention consists in providing products and methods by means of which bacterial strains, particularly streptococcus strains, can be tested fast and reliably for existing antibiotic resistances.
This object is achieved by the subject matters defined in the claims.
The invention is described below by way of penicillin resistance of
Streptococcus pneumoniae.
However, this principle also applies in correspondingly general fashion to bacteria and resistances to other antibiotics. Neisserias and MRSA strains (methicillin-resistant
Staphylococcus aureus
), which do not produce &bgr;-lactamase, are mentioned by way of example.
All of the penicillin-resistant
S. pneumoniae
strains have modified penicillin target proteins (penicillin-binding proteins, PBP). The DNA sequences of genes which play a decisive part in the development of penicillin resistance in
Streptococcus pneumoniae
have meanwhile been determined in a number of penicillin-resistant streptococcus strains. Three genes were identified where differences between sensitive and resistant strains occur in connection with the development of penicillin resistance: PBP2x, PBP1a and PBP2b.
A comparison between the DNA sequences shows within the genes regions which are present in all of the sensitive
S. penumoniae
strains but are modified in resistant strains. In this connection, reference is made to
FIG. 1
which shows that the resistant strains differ more or less markedly from the sensitive strain R6 in the PBP2x gene but also differ among themselves.
Because of the above finding that differences between penicillin-sensitive and penicillin-resistant strains occur within certain genes, the applicant developed DNA probes by means of which resistant and sensitive strains can be differentiated. In this connection, reference is made to FIG.
4
. The probes which are specific to sensitive sequences discriminate genes which code for low-affinity PBP variants responsible for penicillin resistance. The probes which are specific to resistant sequences react with a very frequently occurring class of PBP variants and can also be used for epidemiological purposes.
The applicant identified the following DNA probes [SEQ ID NOS.:1-8 ]:
a) Sensitivity-specific probes for PBP2x. The numerals in the column “nucleotide” refer to the nucleotides of the published sequence (Laible et al., Mol. Microbiol. 5, pp. 1993-2002 (1991)). The numerals in parentheses refer to the codon and the position (1, 2 or 3) within the codon of the structural gene. The number of bases in the nucleotide is given by “meric”.
Nucleotide (codon)
oligonucleotide
−meric
314-330
(105.2-110.3)
AGT CAG CAA CGG GTA AG
(1)
17
758-774
(253.2-258.3)
AAC GAA CGA TGG ACG GT
(2)
17
792-809
(264.3-270.2)
CAT TTC CAG NCC CCT CCA
(3)
18 (N: preferably C)
1098-1114
(366.3-372.1)
TGC AGA TGC CAC GAT TC
(4)
17
1302-1317
(434.2-439.3)
CTG GTC AGC TTC CTG CG
(5)
17
1677-1696
(559.3-566.1)
TGG TTA TCT AGT CGG GTT AA
(6)
20
1715-1731
(572.2-577.3)
CTG TAT CGA TGA GTC CG
(7)
17
2011-2029
(671.1-677.1)
AAC AGT TCT GCT GAA GAA G
(8)
19
b) Resistance-specific probes [SEQ ID NOS.: 14-17] for PB2x (as above; sequences in parentheses [SEQ ID NOS.: 20-23] are in accordance with the corresponding sections of sensitive strains).
1065-1084
(AGG AGA AGT CTT TAA TAG T)
(355.3-361.3)
TGG AGA ATA NTT CAA TAG N
(I)
19 (N: preferably C)
1202-1221
(CCC TCC TTG AGC AAA AGA TG)
(401.2-407.3)
GTC TAC TTG AAC AAA AAA TG
(II)
20
1549-1566
(TTG GTA GGG ACG GAT CCG)
(517.1-522.3)
TTA GTT GGG ACG GAC CCT
(III)
18
1759-1776
(GTG ACG GTC CAA CAA CCT)
(587.1-592.3)
GTA ACN NTT CAA CAG CCT
(IV)
18 (N: preferably G)
c) Sensitivity-specific probes [SEQ ID NOS.: 9-12] for PBP1a (values refer to the nucleotides of the published sequence of the structural gene; Martin et al., EMBO J. 11, pp. 3831-3836 (1992))
(1034-1051)
TAG GAG CAC GCC ATC AGT
18
(specific in most known sequences)
1631-1648
GAC GAA ATG CCT ATC TTG
18
1722-1740
CTC TCA ATT TGT AGC ACC T
19
1794-1812
CTA TTC TAA CCG TCT GAC A
19
d) Resistant specific probes [SEQ ID NOS.: 18-19] for PBP1a ([SEQ ID NOS.: 24-25] in parentheses).
(TAC AGA CGA ATA CGT TGC C)
945-963
CTC CGA NCA ATA CGT CTC T
19 (N: preferably T)
(GCA CCT GAT GAA CTA TTT GC)
1735-1754
GCT CCA GAT NAA ATG TTT GT
20 (N: preferably G)
e) Sensitivity-specific probes [SEQ ID NO.: 13] for PBP2b (values refer to the nucleotides of the published sequence of the structural gene; Hakenbeck, R., Matrin, C., Dowsen, C., Grebe, T., J. Bacteriol. 176, pp. 5574-5577 (1996))
1329-1348
ATC AAA TAC CTA TAT GGT CC
20
N=any nucleotide
The above probes and those differing therefrom by one or several nucleotides, preferably up to 4 nucleotides, respectively, are perfectly suited to test unknown
Streptococcus pneumoniae
strains for resistance to penicillin.
For this purpose, bacteria according to the invention are centrifuged off a sample and in the case of
S. pneumoniae
the PBP genes (the resistance determinants) are amplified directly via PCR (polymerase chain reaction) as described in the literature (Grebe and Hakenbeck (1996), Antimicrob. Agents Chemother. 40, pp. 829-834). The advantage in connection with
S. pneumoniae
consists in that a detergence-induced lysis occurs rapidly and thus PCR can be carried out without long-winding DNA preparations. Since this step fails with other streptococci, only pneumococcus DNA is amplified specifically by means of this step. As an alternative, bacterial DNA (chromosomal and/or extrachromosomal) is isolated according to standard methods. This DNA is hybridized with at least one sensitivity-specific probe and with at least one resistance-specific probe under standard conditions with which a person skilled in the art is sufficiently familiar (see e.g. Maniatis et al., Molecular Cloning, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory). The hybridization is preferably carried out under stringent conditions such as 20° C. below the melting point of the hybridizing DNA. The oligonucleotides are preferably chosen such that they have similar melting temperatures and thus several of them can be tested in the same hybridization batch under the same conditions (see FIG.
2
). The oligonucleotides are preferably labeled when offered (P
32
, S
35
, biotin/avidin system; dioxygenine (DIG)-labeled; fluorescein-labeled) and hybridized against immobilized DNA. As an alternative, the oligonucleotides are offered on an oligonucleotide microarray in non-labeled fashion and the DNA to be hybridized is obtained via PCR and labeled while amplified.
It can be concluded from the hybridization result whether or not the unknown strain is sensitive to antibiotics. Depending on the resistance gene, at least one sensitivity-specific probe and one resistance-specific probe should be used for the hybridization. However, the DNA of the unknown strain is hybridized advantageously with several sensitivity-specific and resistance-specific probes in succession, since evaluation o
Burns Doane Swecker & Mathis L.L.P.
Horlick Kenneth R.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften E.V.
Wilder Cynthia
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