TmRNA

Example 2

In the following examples, the method to perform antimicrobial sensitivity testing of bacteria in whole blood sample after partial lysis of blood cells and enrichment of the blood culture was described. Klebsiella pneumoniae species was used as an example. The blood cells in the K. pneumoniae spiked whole blood were partially lysed by a lysing reagent, and most of the supernatant was removed after centrifugation. The remaining minimal volume of the liquid was enriched with TSB growth medium and pre-incubated at 37° C. for to allow the bacteria in the mixture multiply. At the end the pre-incubation period, the antimicrobial sensitivity test was performed by aliquoting the culture into multiple tubes, one without antibiotic and the remaining with different antibiotics.

K. pneumoniae cells were spiked into 2 mL of EDTA-treated whole blood at 100 CFU/mL concentrations. Blood cell lysing reagent containing 40 mg/mL Saponin, 10 mg/mL SPS and 1% PPG in water of 200 uL was added to the blood. The content was thoroughly mixed by inverting followed by vortexing at low speed for 10 sec, centrifugation at 12,000 rpm for 2 min and removal of 1.8 mL of the supernatant, leaving 200 μL of the liquid and the sedimented remaining blood cells and the microbial cells. One wash cycle was performed by adding 1.8 mL of TSB medium to the remaining liquid supernatant of 200 uL, mixing by inverting, centrifugation at 12,000 rpm for 2 min and removal of 1.8 mL of the supernatant, 200 μL of the liquid and the sedimented remaining blood cells and the microbial cells. The remaining minimal volume of the liquid was supplemented with 3.8 mL TSB growth medium, mixed thoroughly by inverting and pre-incubated at 37° C. for 2 hours to allow the bacteria in the mixture multiply. At the end the pre-incubation period, 1 mL each of the culture was distributed into multiple tubes. One tube was designated as non-treated growth control tube without any antibiotic, and 8 ug/mL final concentrations of norfloxacin and tetracycline were added to the remaining respective tubes. The culture tubes were incubated at 37° C. for 2 hours and at the end of the incubation time the respective TSB-enriched blood culture of 1 mL was subjected to blood sample pretreatment procedure which includes blood cell lysis step followed by 4 wash cycles was performed as described above. K. pneumoniae cells in the resulting pretreated sample was lysed by electrical lysis and the cell lysate of 1 uL containing nominal 1 cell was subjected to RT-PCR.

The rRNA primers used in this example are rRNA Primer #1 (enterob2) forward (5′-GTGCCCTTGAGGCGTGGCTTC-3′) (SEQ. ID. 5), rRNA Primer #1 (enterob2) reverse (5′-GCGGGACTTAACCGAACATTCAC-3′) (SEQ. ID. 6), rRNA Primer #2 (enterob4) forward (5′-ACAAGCGGTGGAGCATGTGG-3′) (SEQ. ID. 7), rRNA Primer #2 (enterob4) reverse (5′-GCGGGACTTAACCCAACATTTCAC-3′) (SEQ. ID. 8), rRNA Primer #3 (ebGN3) forward (5′-ACTTTCAGCGGGGAGGAAGG-3′) (SEQ. ID. 9) and rRNA Primer #3 (ebGN3) reverse (5′-GCGGGACTTAACCCAACATTTCAC-3′) (SEQ. ID. 10). The 16S rRNA fragments of 203 base pairs (nucleotides 504 to 707 using K. pneumoniae str. Kp52.145 as a reference) were amplified by primer pair #1, 166 base pairs by primer pair #2 and 666 base pairs by primer pair #3.

The tmRNA primers used in this example are tmRNA Primer #A forward (5′-GCAAACGACGAAAACTACGCTTTAGC-3′) (SEQ. ID 11), tmRNA Primer #A reverse (5′-GCTTAGTCAGTCTTTACATTCGC-3′) (SEQ. ID 12), tmRNA Primer #B forward (5′-GCAAACGACGAAAACTACGCTTTAGC-3′) (SEQ. ID 13), tmRNA Primer #B reverse (5′-CGGACGGACACGCCACTAAC-3′) (SEQ. ID 14), tmRNA Primer #C forward (5′-GCAAACGACGAAAACTACGCTTTAGC-3′) (SEQ. ID 15), tmRNA Primer #C reverse (5′-CCTACATCCTCGGTACTACATGC-3′) (SEQ. ID 16), tmRNA Primer #D forward (5′-GGGATTTGCGAAACCCAAGGTGC-3′) (SEQ. ID 17), tmRNA Primer #D reverse (5′-GTTTTAACGCTTCAACCCCAGGC-3′) (SEQ. ID 18), tmRNA Primer #E forward (5′-GGGATTTGCGAAACCCAAGGTGC-3′) (SEQ. ID 19), tmRNA Primer #E reverse (5′-GCTTAGTCAGTCTTTACATTCGC-3′) (SEQ. ID 20), tmRNA Primer #F forward (5′-GGGATTTGCGAAACCCAAGGTGC-3′) (SEQ. ID 21), tmRNA Primer #F reverse (5′-CGGACGGACACGCCACTAAC-3′) (SEQ. ID 22), tmRNA Primer #G forward (5′-GGGATTTGCGAAACCCAAGGTGC-3′) (SEQ. ID 23), tmRNA Primer #G reverse (5′-CCTACATCCTCGGTACTACATGC-3′) (SEQ. ID 24). The tmRNA fragments of 218 base pairs (nucleotides 97 to 315 using K. pneumoniae str. Kp52.145 as a reference) were amplified by primer pair #A, 183 base pairs by primer pair #B, 240 base pairs by primer pair #C, 221 base pairs by primer pair #D, 293 base pairs by primer pair #E, 258 base pairs by primer pair #F and 315 base pairs by primer pair #G.

The real time fluorescence signal versus cycle number is presented in FIGS. 8 and 9 to demonstrate the sensitivity tests using norfloxacin and tetracycline. In this example, the reduction of rRNA and tmRNA in response to the antibiotic was demonstrated compared to without antibiotic (Non-treated) culture.