Figure 1PCR-RFLP, parC gene. (A) M, represents 100 bp DNA marker; 1, uncut PCR product (360 bp); and 2, negative control. (B) M, represents 100 bp DNA marker; 1, products of wild-type strain digestion by Sau3A enzyme giving 200 bp and 160 bp fragments; and 2, products of mutant strain digestion by Sau3A enzyme with 200 bp, 80 bp, and 80 bp fragments. PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism.
Figure 2PCR-RFLP, parE gene. (A) M, represents 100 bp DNA marker; 1. negative control; and 2, uncut PCR product (290 bp). (B) M, represents 100 bp DNA marker; 1, MspI restriction enzyme digestion products of wild-type strain giving 160 bp and 130 bp fragments; and 2, products of mutant strain digestion by MspI enzyme representing 130 bp, 130 bp, and 30 bp fragments. PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism.
Figure 3PCR-RFLP, gyrA gene. (A) M, represents 100 bp DNA marker; 1, products of mutant strains digestion by AluI enzyme representing 180 bp, 140 bp, and 60 bp fragments; and 2, AluI restriction enzyme digestion products of wild-type strain giving 200 bp and 180 bp fragments. (B) M, represents 100 bp DNA marker; 1, negative control; and 2, uncut PCR product (380 bp). PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism.
Table 1PCR primers for QRDRs of the fluoroquinolone resistance genes.
PCR conditions |
Amplicon size (bp) |
Primer (5′–3′) |
Gene |
One cycle of 5 min at 94°C; 32 cycles of 1 min at 94°C, 1 min at 61°C, 1 min at 72°C; one cycle of 8 min at 72°C |
360 |
F: 5′-TGG GTT GAA GCC GGT TCA-3′ R: 5′-TGC TGG CAA GAC CGT TGG-3′ |
parC
|
One cycle of 5 min at 94°C; 32 cycles of 1 min at 94°C, 1 min at 61°C, 1 min at 72°C; one cycle of 8 min at 72°C |
290 |
F: 5′-AAG GCG CGT GAT GAG AGC-3′ R: 5′-TCT GCT CCA ACA CCC GCA-3′ |
parE
|
One cycle of 5 min at 94°C; 32 cycles of 1 min at 94°C, 1 min at 57°C, 1 min at 72°C; one cycle of 8 min at 72°C |
380 |
F: 5′-CCG TCG CAT TCT TTA CG-3′ R: 5′-AGT TGC TCC ATT AAC CA-3′ |
gyrA
|
Table 2Antibiotic susceptibility of S. pneumoniae.
Susceptible (%) |
Intermediate (%) |
Resistance (%) |
Antibiotic |
4 (8.89) |
4 (8.89) |
37 (82.22) |
Nalidixic acid |
5 (11.11) |
7 (15.56) |
33 (73.33) |
Ciprofloxacin |
6 (13.33) |
15 (33.33) |
24 (53.33) |
Ofloxacin |
8 (7.78) |
15 (33.33) |
22 (48.89) |
Norfloxacin |
14 (31.11) |
12 (26.67) |
19 (42.22) |
Levofloxacin |
Table 3Mutation pattern in clinical samples based on quinolone resistance.
LEV
|
OFL
|
NOR
|
CIP
|
NA
|
Mutation |
SR |
R |
SR |
R |
SR |
R |
SR |
R |
SR |
R |
1 (2.22) |
0 (0) |
2 (4.44) |
1 (2.22) |
2 (4.44) |
0 (0) |
2 (4.44) |
2 (4.44) |
1 (2.22) |
3 (6.66) |
parC+/parE–/gyrA–
|
1 (2.22) |
0 (0) |
4 (8.88) |
1 (2.22) |
3 (6.66) |
1 (2.22) |
1 (2.22) |
4 (8.88) |
2 (4.44) |
3 (6.66) |
parC–/parE–/gyrA+
|
2 (4.44) |
16a (35.55) |
1 (2.22) |
17a (37.77) |
1 (2.22) |
17a (37.77) |
0 (0) |
18a (40) |
0 (0) |
18a (40) |
parC+/parE–/gyrA+
|
4a (8.88) |
0 (0) |
6a (13.33) |
0 (0) |
6a (13.33) |
0 (0) |
2 (4.44) |
4 (8.88) |
1 (2.22) |
6 (13.33) |
parC+/parE+/gyrA–
|
3a (6.66) |
0 (0) |
1 (2.22) |
2 (4.44) |
2 (4.44) |
1 (2.22) |
2 (4.44) |
1 (2.22) |
0 (0) |
3 (6.66) |
parC–/parE+/gyrA+
|
0 (0) |
3 (6.66) |
1 (2.22) |
3 (6.66) |
1 (2.22) |
3 (6.66) |
0 (0) |
4 (8.88) |
4 (8.88) |
4 (8.88) |
parC+/parE+/gyrA+
|
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
parC–/parE–/gyrA–
|