Patent Application: US-53115903-A

Abstract:
the preferred therapeutic for human anthrax infections are therapeutics from the fluoroquinolone class , in particular , ciprofloxacin . this invention discloses the molecular basis for fluoroquinolone action and provides the molecular signatures which form the basis of diagnostic assays . this invention further discloses nucleotide signatures associated with cip - resistance are useful in diagnostic tests to rapidly identify cip resistant b . anthracis and to infer the level of resistance of these mutant strains . according to this invention , the diagnostic potential of the molecular signatures is illustrated using a primer extension assay . further , pcr and extension primers which allow the detection of these signatures are disclosed .

Description:
the present invention discloses a molecular assay for screening b . anthracis for single nucleotide polymorphism ( snps ) associated with ciprofloxacin ( cip ) resistance . this diagnostic approach provides a rapid screening of b . anthracis samples for cip resistance in situations where culturing the sample is not possible . it is anticipated that in the event of any bioterrorist activity , this rapid assay will make possible the early detection of malicious spread of anthrax . a study of multiple mutant b . anthracis strains showed that the primary target of cip in wild - type b . anthracis is gyra , the secondary target is parc and the tertiary targets are yet to be fully determined . the target order of cip appears to be determined by the amino acid residues of the gyrase and toposiomerase iv subunit qrdrs . assays are presented for determining fluoroquinolones resistant b . anthracis based on the mutational status of the six gyra and three parc nucleotides . primers for multiplexing to amplify nine loci are disclosed in table 3 and fig2 . these nine loci represent the most common mutants and may be quickly assayed using the present methods . because these nine mutations play a critically important role in determining the level of cip resistance , snp information may be used in developing an appropriate antibiotic treatment strategy at an early stage of an outbreak . a newly acquired strain could be genotyped in just a few hours . strains of cip resistant b . anthracis that arise either by misuse of antibiotics or malevolence , can be rapidly genotyped using the disclosed snp assay this assay was developed using the snapshot ™ technology ( abi prismt ™ applied biosystems inc .) but other snp assays known in the art are available and may be used . in the present assay , the mutational status of the six gyra and three parc nucleotides is easily observed in a single lane on an abi377 or abi3100 . “ polymerase chain reaction ” or “ pcr ” a technique in which cycles of denaturation , annealing with primer , and extension with dna polymerase are used to amplify the number of copies of a target dna sequence by approximately 106 times or more . the polymerase chain reaction process for amplifying nucleic acid is disclosed in u . s . pat . nos . 4 , 683 , 195 and 4 , 683 , 202 , which are incorporated herein by reference . “ primer ” a single - stranded oligonucleotide or dna fragment which hybridizes with a dna strand of a locus in such a manner that the 3 ′ terminus of the primer may act as a site of polymerization using a dna polymerase enzyme . “ primer pair ” two primers including , primer 1 that hybridizes to a single strand at one end of the dna sequence to be amplified and primer 2 that hybridizes with the other end on the complementary strand of the dna sequence to be amplified . “ primer site ”: the area of the target dna to which a primer hybridizes . “ multiplexing ” is an assay system for simultaneous , multiple determinations in a single assay process in a thermocycling instrument ( pcr ). a process to implement such a capability in a process is a “ multiplexed assay .” systems containing several loci are called multiplex systems described , for example , in u . s . pat . no . 6 , 479 , 235 to schumm , et al ., u . s . pat . no . 6 , 270 , 973 to lewis , et al . and u . s . pat . no . 6 , 449 , 562 to chandler , et al . “ isolated nucleic acid ” is a nucleic acid which may or may not be identical to that of a naturally occurring nucleic acid . when “ isolated nucleic acid ” is used to describe a primer , the nucleic acid is not identical to the structure of a naturally occurring nucleic acid spanning at least the length of a gene . the primers herein have been designed to bind to both sequences flanking . certain primers also may bind internally to the dna sequence of interest . it is to be understood that primer sequences containing insertions or deletions in these disclosed sequences that do not impair the binding of the primers to these flanking sequences are also intended to be incorporated into the present invention . method for rapid assay of b . anthracis for detection of fluoroquinolones - resistant strains . dna extraction . dna was extracted as described in keim et . al ., 2000 . briefly , dna was extracted from each resistant mutant by suspending ˜ 1 mg of cellular material from blood agar plates in 150 μl of heat - soak buffer ( 10 mm tris - hcl , 1 mm edta , ph 8 . 0 ) and heating to 85 ° c . for 30 min . cellular debris was pelleted by centrifugation and the supernatant was used as template in pcr reactions . pcr amplification . all primers ( table 1 ) were designed from the incomplete b . anthracis genome sequence generally provided by the institute for genomic research , rockville , md ., usa . pcr products were amplified in 50 μl pcr reactions and prepared as follows : 1 × pcr buffer ( 20 mm tris ph 8 . 4 , 50 mm kcl ) ( gibco / brl , bethesda , md ., usa ), 0 . 10 mm dntps , 2 mm mgcl 2 , 2 μl heat - soak supernatant as template , 0 . 04 u / μl tag dna polymerase ( gibco / brl , bethesda , md ., usa ), 0 . 2 μm forward and reverse primers , adjusted to 50 μl with filtered ( 0 . 2 μm ) 17 . 8 mohm e - pure water . reactions were heated to 94 ° c . for 5 min , then subjected to 35 cycles of 20 s at 94 ° c ., 20 s at 60 ° c . and 20 s at 72 ° c . this was followed by heating to 72 ° c . for 5 min to complete primer extension . pcr products were quantified on . etbr stained 1 . 5 % synergel ™ ( diversified biotech , boston , mass ., usa )/ 0 . 7 % agarose ( gibco / brl , bethesda , md ., usa ). quantified pcr products were sequenced as follows . dna sequencing . pcr products were diluted 1 : 5 in water and sequenced on an abi377 fluorescent sequencer using the abi prism ® ready reaction bigdye ™ terminator cycle sequencing kit ( both from perkin - elmer / applied biosystems inc ., foster city , calif ., usa ). when necessary , contiguous gene sequences were prepared from the individual sequences using seqman ™ software ( dnastar , inc ., madison , wis ., usa ). contiguous sequences were aligned with the wild - type sequences using megalign ™ software ( dnastar , inc ., madison , wis ., usa ). snp multiplex assay . a single nucleotide polymorphism ( snp ) assay was developed to rapidly identify the nine observed resistance mutations ( table 2 ). flanking primers ( table 3 ) were designed to amplify bases 203 - 323 of gyra ( 122 by product ) and bases 175 - 320 of parc ( 146 by product ). pcr products were amplified in 10 μl singleplex or duplex pcrs with final concentrations of 1 × pcr buffer ( above ), 3 mm mgcl 2 , 0 . 1 mm dntps , forward and reverse primer pairs ( 0 . 1 μm gyra primers / 0 . 4 μm parc primers ), 1 u platinum ® taq dna polymerase ( gibco / brl , bethesda , md ., usa ), and 1 μl heat - soak supernatant as template . reactions were heated to 94 ° c . for 5 min , then subjected to 30 cycles of 20 s at 94 ° c ., 30 s at 60 ° c . and 30 s at 72 ° c . the remainder of the procedure was carried out according to methods known in the art . preferred instructions are given in the abi prism ® snapshot ™ multiplex kit and run on an ab3100 genetic analyzer ( applied biosystems , foster city , calif ., usa ). single base extension ( sbe ) primers ( table 3 ) were designed with polynucleotide tails ( poly - cs and single as ) to customize amplicon lengths to 4 - bp intervals such that when separated electrophoretically , the six gyra snps were detected in the 5 ′ to 3 ′ order in which mutations are found , followed by the three parc snps ( also in 5 ′ to 3 ′ order of occurrence ). since primers gyra265 and gyra254 overlapped 1 and 2 snp loci respectively , they were designed with degenerate base pairs at sites 266 and 265 to allow annealing on templates with step 3 mutations . despite primer degeneracy , amplification of gyra265 in the 13 - primer multiplex was weak on s3 - 2 mutants . this locus was therefore targeted individually by performing a second sbe containing only the two gyra265 primers when a template was suspected to be an s3 - 2 mutant . the order of sbe products enabled multiplexing of sbe pcrs and facilitated scoring , eliminating the need for a size standard . therefore , this assay can be performed on a 4 - dye abi377 if a 5 - dye capillary machine is not available . table 3 external and sbe primers used in the snp assay product seq id # name sequence ( 5 ′→ 3 ′) size snp external primers seq id # 37 bagyra01f_flanking tcagcacgtattgttggtgaag 122 — seq id # 38 bagyra01r_flanking tgcccatcaacaagcatataac 122 — seq id # 39 baparc02f_flanking aaagcgttccgtaagtcgg 145 — seq id # 40 baparc02r_flanking ttattaccatgcatctcaact 145 — aaaac sbe primers seq id # 41 bagyrasnp247f_ atcggtaagtatcaccctcat 22 g / t internal seq id # 42 bagyrasnp248f_ ccccccggtaagtatcaccctc 26 g / a internal atg seq id # 43 bagyrasnp250f_ cccccccggtaagtatcaccctc 30 g / a internal atggt seq id # 44 bagyrasnp254r_ cccccccccccccccatcgtttcat 34 c / t † internal aaacagct seq id # 45 bagyrasnp254r ( g ) _ cccccccccccccccatcgttgcat 34 c / t † internal aaacagct seq id # 46 bagyrasnp254r ( t ) _ cccccccccccccccatcgttttat 34 c / t † internal aaacagct seq id # 47 bagyrasnp254r ( gt ) _ cccccccccccccccatcgttgtat 34 c / t † internal aaacagct seq id # 48 bagyrasnp265r_ cccccccccccccccccccgccatacg 38 g / a † internal taccatcgttt seq id # 49 bagyrasnp265r ( g ) _ cccccccccccccccccccgccatacg 38 g / a † internal taccatcgttg seq id # 50 bagyrasnp266r_ cccccccccccccccccccccccccgcca 42 a / c † internal tacgtaccatcgtt seq id # 51 baparcsnp242f_ ccccccccccccccccccccccccccccccc 47 c / t / a internal cacccgcacggtgatt seq id # 52 baparcsnp253r_ ccccccccccccccccccccccccccccga 51 g / a † internal cttaaacgtaccatcgctt seq id # 53 baparcsnp254r_ cccccccccccccccccccccccccccccccc 54 a / g † internal cagcgatggtacgtttaagtc † snps will be detected as reverse complements in the snapshot ™ assay when reverse sbe primers are used . selection of mutant b . anthracis strains and identification of fluoroquinolones - resistant sites bacterial strains . selections were performed on the non - virulent , px01 -/ px02 -, ames strain of b . anthracis ( ivins et al ., 1986 ). all dna samples used for the diversity study came from our b . anthracis dna collection ( keim , et al ., 2000 ). b . anthrocis strains were selected sequentially at increasing ctp concentrations to produce a resulting stepwise accumulation of mutations . mutant strains were isolated with mics as high as 64 μg cip / ml ( 1000 - fold higher than wild - type ) these results are given in table 2 . the accumulation of mutations occurred in a distinctive and ordered manner . first level mutants , selected on 0 . 25 μg cip / ml , developed at a rate of 6 . 6 × 10 − 10 and had one of five mutations within the gyra qrdr ( table 2 ). a disproportionate number ( 71 %) of these mutants possessed the c254 → t missense mutation in gyra ( table 2 ). the level of resistance conferred by this mutation was similar to that of other s1 mutations . since this mutation provided no selective advantage over the other s1 mutations , it is reasonable to call the c254 nucleotide of b . anthracis gyra a mutational hotspot . second level mutants , selected on 1 . 5 μg cip / ml , developed at a rate of 1 . 0 × 10 − 8 and possessed one of four mutations within the parc qrdr ( table 2 ). as with the s1 mutants one s2 genotype , c242 → t , was overrepresented ( 71 %) ( table 2 ). while it is likely that the c242 nuelcotide of parc represents another mutational hotspot , the overrepresentation could also be a result of the disproportionate level of resistance conferred to the strain by the mutation ( table 2 ). third level mutants , selected on 24 μg cip / ml , developed at a rate of 4 . 8 × 10 − 10 . two third - level mutants were identified with novel mutations within the gyra qrdr ( table 2 ). however , the other 21 third - level mutants had no additional alterations in either the gyra or parc qrdrs . potential qrdrs in gyrb and pare were also sequenced from these strains and revealed no additional mutations in these regions . the targeted stepwise accumulation of mutations ( s1 gyra → s2 parc → s3 gyra /?) give further evidence to support the hypothesis that particular fluoroquinolones have different primary topoisomerase targets within various bacterial species ( ferrero et al ., 1995 ; ng et al ., 1996 ; pan and fisher et al ., 1998 ). b . anthracis has the ability to develop a number of different missense mutations that enable it to grow in the presence of cip . the stepwise phenotypic rates at which b . anthracis develops resistance to cip ( 4 . 8 × 10 − 10 to 1 . 0 × 10 − 8 ) are similar to those reported for fluoroquinolone resistance in other species . the rarity of human anthrax cases and the carcass - dependent transmission cycle of this pathogen make the development and spread of cip resistant b . anthracis through patient non - compliance unlikely . however , the agricultural practice of antimicrobial growth promotion does have this potential outcome . cip regimens targeted at serum and tissue concentrations of ≧ 0 . 38 μg cip / ml would reduce the chances for developing cip resistant b . anthracis by requiring the statistically unlikely event ( 6 . 6 × 10 − 18 ) of a bacterium to develop advantageous mutations in the gyra and parc qrdrs simultaneously . the serum and tissue concentrations resulting from the low - level feeding of antibiotics as antimicrobial growth promoters in livestock would not reach the mpc and likely fall short of the mic for wild - type b . anthracis . therefore , this practice could present a potential risk for the development of resistant strains , particularly in those livestock regions to which b . anthracis is endemic . diversity study . the qrdrs of gyra and parc were sequenced from eight major diversity groups and analyzed for point mutations as described below . the eight strains , 3 ( 74 - 42c - 8 ), 25 ( 14185 ), 39 ( 46 ), 45 ( 2b80 ), 62 ( oct - 321 ), 77 ( vollum ), 80 stepwise mutant selection . b . anthracis ames −/− strain was taken from a frozen stock , streaked onto blood agar plates and grown overnight at 35 ° c . cells from isolated colonies were used to inoculate culture tubes containing 5 ml of mueller - hinton broth . cultures were incubated overnight at 37 ° c . in a g24 environmental incubator shaker ( new brunswick scientific , edison , n . j ., usa ) shaking at 225 rpm . each of these cultures ( mean od 625 ˜ 1 . 4 or 1 . 43 × 10 8 cfu / ml ) was transferred to a 0 . 45 μm nitrocellulose membrane filter ( millipore , bedford , mass . usa ). membranes were placed cell - side - up onto mueller - hinton agar containing 0 . 25 μg cip / ml and incubated for ˜ 40 h . cells from a single colony from each positive plate were streaked onto blood agar and grown overnight at 35 ° c . cells from these plates were used to prepare frozen stocks and to isolate dna for sequencing ( see below ). the most common unique genotype , s1 - 1 , was subjected to a subsequent round of selection on mueller - hinton agar containing 1 . 5 μg cip / ml . likewise , the most common genotype from this selection , s2 - 1 , was subjected to a third and final selection on agar containing 24 μg cip / ml . mutation rates . mutation rates for steps 1 , 2 and 3 ciprofloxacin resistant mutants were determined using 96 independent cultures of the wild type ames −/−, s1 - 1 , and s2 - 1 , respectively . a single colony of the starting isolate was suspended in lb broth and used to inoculate each of the independent cultures with approximately 1 , 000 cells . for steps 1 and 3 mutants , 96 1 ml cultures were grown in lb broth in four 24 - well plates ( costar ). for step 2 , 96 100 μi were grown in lb broth cultures in a single 96 - well plate ( costar ). all plates were incubated overnight at 37 ° c . in a g24 environmental incubator shaker ( new brunswick scientific , edison , n . j ., usa ) shaking at 225 rpm . six cultures were chosen at random for each step and used to determine the average total number of cells present in each culture . the remaining 90 cultures were plated onto mueller - hinton ciprofloxacin plates with concentrations of 0 . 25 μg cip / ml , 1 . 5 μg cip / ml , and 24 μg cip / ml for steps 1 , 2 , and 3 , respectively . for step 2 , the 100 μl cultures were directly plated . for steps 1 and 3 , the 1 ml cultures were transferred to sterile 1 . 5 ml microcentrifuge tubes and centrifuged at 3 , 000 × g for 5 min . approximately 850 μl of the supernatant was removed , the pellet was resuspended in the remaining broth and plated . all of the plates were incubated at 37 ° c . for ˜ 48 h . up to four putative resistant colonies from each positive plate were transferred to fresh selective medium , and incubated at 37 ° c . for ˜ 48 h to confirm resistance . the number of plates devoid of resistant mutants represents zero mutational events . this value was used with the cell count in the poisson distribution to estimate the mutation rate for each step . susceptibility testing . mics were determined by the mueller - hinton agar dilution method according to the guidelines of the national committee for clinical laboratory standards ( national committee , 1997 ). the e - 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