CHAPTER 1 INTRODUCTION
5.7 Limitations and future studies
41 reservoir of tet efflux gene and may further emerge to become antimicrobial resistant in the future (Boerlin et al., 2005).
42 CHAPTER 6
CONCLUSION
Overall, UPEC isolates are predominantly recovered from female patients. The highest resistance rate of UPEC isolates was found to be against ampicillin, followed by tetracycline and nalidixic acid, while the isolates showed the highest susceptibility to imipenem and minocycline. The current study revealed a high prevalence of tetA genes as compared to tetB genes, which may propose the higher occurrence of widespread and dissemination of tetA efflux genes among the UPEC strains in hospital settings. Both tet genes showed negative association to age and gender of patients, however, there is a significant positive association between genotypic profile and phenotypic profile of tetracycline antibiotic class. All tetracycline-resistant isolates harboured at least one tet efflux gene, while one minocycline-resistant isolate harboured tetB gene.
Although minocycline phenotypically showed a better efficacy in comparison to tetracycline, the high prevalence of tet genes carriage in minocycline- susceptible isolates revealed that the reservoir for tet genes has expanded in UPEC strains despite the low level or no expression of tet genes, which promotes gene dissemination among species. Meanwhile, tetA genes also correlate with the resistance profiles of other drugs, which is in line with the fact where tet genes are capable in encoding multidrug MFS transporters.
43 In a nutshell, the continued emergence of efflux mediated multidrug resistance among UPEC strains will be detrimental to the current therapeutic paradigm for UTIs. To impede the emergence of antimicrobial resistance, therapeutic strategy shift from broad-spectrum antimicrobials into antibiotics that target selectively to core resistance determinants has to be highlighted. Since the findings obtained in this study only focus on the basic molecular standpoints of efflux gene carriage in conferring to corresponding resistance traits, further in-depth studies are preferable to expand the understanding of specific important determinants in order to improvise and idealise the therapeutic strategy for UTI treatment.
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49 APPENDICES
Appendix A
Table 1: Interpretive categories and zone diameter breakpoints (in millimetres) of each antibiotic based on CLSI guidelines.
Tetracycline (30µg)
Minocycline (30 µg)
Trimethoprim- sulfamethoxazole
(25 µg)
Ciprofloxacin (5 µg)
Nalidixic acid (30 µg)
Levofloxacin (5 µg)
Imipenem (10 µg)
Ampicillin (10 µg)
Chloramphenicol (30 µg)
Susceptible ≥15 ≥16 ≥16 ≥26 ≥19 ≥21 ≥23 ≥17 ≥18
Intermediate 12-14 13-15 11-15 22-25 14-18 17-20 20-22 14-16 13-17
Resistant ≤11 ≤12 ≤10 ≤21 ≤13 ≤16 ≤19 ≤13 ≤12
(Adapted from Clinical and Laboratory Standard Institute, 2021)
50 Appendix B
Table 2: Demographic data of each UPEC clinical isolates.
Sample Gender Age
UTIPS 1 M 2
UTIPS 2 F 78
UTIPS 3 F 48
UTIPS 4 F 26
UTIPS 5 F 67
UTIPS 6 F 36
UTIPS 7 F 77
UTIPS 8 F 76
UTIPS 9 M 69
UTIPS 10 M 52
UTIPS 12 F 66
UTIPS 13 F 24
UTIPS 14 F 74
UTIPS 15 M 40
UTIPS 16 F 41
UTIPS 17 F 30
UTIPS 18 F 51
UTIPS 19 M 63
UTIPS 20 F 88
UTIPS 21 F 53
UTIPS 22 F 63
UTIPS 23 F 63
UTIPS 24 M 77
UTIPS 25 F 79
UTIPS 26 F 61
UTIPS 27 F 76
UTIPS 28 F 66
UTIPS 29 F 38
51 Table 2 (continued)
UTIPS 30 F 73
UTIPS 31 M 63
UTIPS 32 M 68
UTIPS 33 F 68
UTIPS 34 F 64
UTIPS 35 F 56
UTIPS 36 F 64
UTIPS 37 F 59
UTIPS 38 F 79
UTIPS 39 F 86
UTIPS 40 F 35
UTIPS 41 F 70
UTIPS 42 F 17
UTIPS 43 F 17
UTIPS 44 M 57
UTIPS 45 F 30
UTIPS 46 F 48
UTIPS 47 F 26
UTIPS 48 M 82
UTIPS 49 M 87
UTIPS 50 F 53
UTIPS 51 F 75
UTIPS 52 M 13
UTIPS 53 F 5
UTIPS 54 F 39
UTIPS 55 M 53
UTIPS 56 M 49
UTIPS 57 F 67
UTIPS 58 F 44
UTIPS 59 M 82
UTIPS 60 F 3
UTIPS 61 M 68
‘M’ stands for male and ‘F’ stands for female.
52 Appendix C
Table 3: Antimicrobial susceptibility profile of UPEC isolates.
Sample NAL CIP LEX TET MIN SXT AMP CHL IPM
UTIPS 1 S S S S S S S R S
UTIPS 2 R R S R S S R S S
UTIPS 3 S S S S S S S S S
UTIPS 4 R R R R S R R S S
UTIPS 5 R R R R S S R S S
UTIPS 6 S S S S S S R S S
UTIPS 7 R R R R S R R R S
UTIPS 8 S S S R S S R S S
UTIPS 9 R R R R S R R S S
UTIPS 10 R S S R S R R R S
UTIPS 12 S S S S S S R R S
UTIPS 13 S S S R S S R S S
UTIPS 14 R R R R S R R S S
UTIPS 15 R R R R S R R S S
UTIPS 16 R R R R S R R S S
UTIPS 17 S S S R S R R S S
UTIPS 18 R R R R S R R S S
UTIPS 19 R S S R S R R R S
53 Table 3 (continued)
UTIPS 20 S S S R S S R R S
UTIPS 21 R R R S S S R S S
UTIPS 22 S S S R S R S S S
UTIPS 23 S S S S S S S S S
UTIPS 24 R R R S S S R S S
UTIPS 25 S S S S S S R S S
UTIPS 26 S S S S S S R S S
UTIPS 27 R S S S S S R S S
UTIPS 28 S S S S S S S S S
UTIPS 29 S S S R S S R R S
UTIPS 30 R S S S S S R S S
UTIPS 31 R R R S S S R S S
UTIPS 32 R R R R S S R R S
UTIPS 33 R S S S S S R S S
UTIPS 34 R R R R S R R S S
UTIPS 35 R S S S S S S S S
UTIPS 36 S S S S S S S S S
UTIPS 37 S S S S S S S R S
UTIPS 38 S S S R S S R S S
UTIPS 39 R S S R S S R S S
UTIPS 40 S S S S S R R S S
UTIPS 41 R R S R S R R S S
54 Table 3 (continued)
UTIPS 42 R S S R S R R S S
UTIPS 43 R S S R S R R S S
UTIPS 44 S S S S S S R S S
UTIPS 45 R S S S S R S S S
UTIPS 46 S S S S S S S S S
UTIPS 47 R R S R S S R S S
UTIPS 48 S S S S S S S S S
UTIPS 49 R R R S S S S S S
UTIPS 50 S S S R S R R S S
UTIPS 51 S S S S S S S S S
UTIPS 52 S S S R S S R S S
UTIPS 53 S S S S S S R R S
UTIPS 54 R S S R S R R S S
UTIPS 55 R R R R S R R S S
UTIPS 56 S S S R R S S S S
UTIPS 57 S S S S S S S S S
UTIPS 58 S S S S S S S S S
UTIPS 59 S S S S S S S S S
UTIPS 60 S S S R S S R S S
UTIPS 61 R R R S S S R S S
‘R’ stands for resistant, and ‘S’ stands for susceptible.
‘NA’ denotes nalidixic acid, ‘CIP’ denotes ciprofloxacin, ‘LEX’ denotes levofloxacin, ‘TET’ denotes tetracycline, ‘MIN’ denotes minocycline,
‘SXT’ denotes trimethoprim-sulfamethoxazole, ‘AMP’ denotes ampicillin, ‘CHL’ denotes chloramphenicol, ‘IMP’ denotes imipenem.
55 Appendix D
Table 4: The concentration and purity of DNA extracts from UPEC clinical isolates.
Sample DNA concentration
(ng/µl) A260/A280 ratio
UTIPS 1 182.52 1.84
UTIPS 2 195.10 1.90
UTIPS 3 211.00 1.88
UTIPS 4 223.60 1.76
UTIPS 5 186.97 1.84
UTIPS 6 196.85 1.90
UTIPS 7 244.06 1.82
UTIPS 8 189.48 1.86
UTIPS 9 222.32 1.93
UTIPS 10 149.31 1.84
UTIPS 12 222.47 1.77
UTIPS 13 247.61 1.88
UTIPS 14 265.59 1.75
UTIPS 15 228.27 1.81
UTIPS 16 209.98 1.89
UTIPS 17 268.06 1.84
UTIPS 18 228.72 1.85
UTIPS 19 151.45 1.93
UTIPS 20 169.07 1.83
UTIPS 21 199.92 1.97
UTIPS 22 175.11 1.75
UTIPS 23 167.75 1.92
UTIPS 24 237.89 1.90
UTIPS 25 187.85 1.78
UTIPS 26 134.36 1.92
UTIPS 27 195.65 1.81
UTIPS 28 147.94 1.93
UTIPS 29 173.10 1.75
UTIPS 30 150.39 1.76
UTIPS 31 186.12 1.92
UTIPS 32 210.41 1.97
UTIPS 33 186.08 1.93
UTIPS 34 179.50 1.81
UTIPS 35 167.08 1.81
56 Table 4 (continued)
UTIPS 36 188.58 1.98
UTIPS 37 206.35 1.78
UTIPS 38 131.62 1.91
UTIPS 39 192.39 1.91
UTIPS 40 224.02 1.97
UTIPS 41 158.59 1.88
UTIPS 42 160.28 1.82
UTIPS 43 156.76 1.82
UTIPS 44 159.63 1.83
UTIPS 45 184.43 1.96
UTIPS 46 122.98 1.99
UTIPS 47 198.54 1.91
UTIPS 48 223.63 1.85
UTIPS 49 164.18 1.92
UTIPS 50 156.13 1.94
UTIPS 51 207.73 1.92
UTIPS 52 150.46 1.83
UTIPS 53 134.55 1.80
UTIPS 54 197.75 1.87
UTIPS 55 170.40 2.00
UTIPS 56 211.77 1.95
UTIPS 57 153.31 1.81
UTIPS 58 208.89 1.90
UTIPS 59 190.67 1.95
UTIPS 60 182.55 1.92
UTIPS 61 225.89 1.88
57 Appendix E
Table 5: PCR screening of clinical isolates and the antimicrobial susceptibility profiles towards tetracycline and minocycline.
Sample TET MIN tetA tetB
UTIPS 1 S S - -
UTIPS 2 R S ＋ -
UTIPS 3 S S - -
UTIPS 4 R S ＋ -
UTIPS 5 R S ＋ -
UTIPS 6 S S - -
UTIPS 7 R S - ＋
UTIPS 8 R S ＋ -
UTIPS 9 R S ＋ -
UTIPS 10 R S ＋ -
UTIPS 12 S S ＋ -
UTIPS 13 R S ＋ -
UTIPS 14 R S ＋ ＋
UTIPS 15 R S ＋ -
UTIPS 16 R S ＋ -
UTIPS 17 R S ＋ -
UTIPS 18 R S ＋ -
UTIPS 19 R S ＋ -
UTIPS 20 R S - ＋
UTIPS 21 S S - -
UTIPS 22 R S ＋ -
UTIPS 23 S S - -
UTIPS 24 S S - -
UTIPS 25 S S - -
UTIPS 26 S S - -
UTIPS 27 S S - -
UTIPS 28 S S - -
UTIPS 29 R S ＋ -
UTIPS 30 S S ＋ -
UTIPS 31 S S - -
UTIPS 32 R S ＋ -
UTIPS 33 S S - ＋
58 Table 5 (continued)
UTIPS 34 R S ＋ -
UTIPS 35 S S - -
UTIPS 36 S S - -
UTIPS 37 S S - -
UTIPS 38 R S ＋ -
UTIPS 39 R S - ＋
UTIPS 40 S S - -
UTIPS 41 R S - ＋
UTIPS 42 R S ＋ -
UTIPS 43 R S ＋ -
UTIPS 44 S S - -
UTIPS 45 S S - -
UTIPS 46 S S - -
UTIPS 47 R S ＋ -
UTIPS 48 S S - -
UTIPS 49 S S - -
UTIPS 50 R S - ＋
UTIPS 51 S S - -
UTIPS 52 R S ＋ -
UTIPS 53 S S - -
UTIPS 54 R S ＋ -
UTIPS 55 R S ＋ -
UTIPS 56 R R - ＋
UTIPS 57 S S - -
UTIPS 58 S S - -
UTIPS 59 S S - -
UTIPS 60 R S ＋ -
UTIPS 61 S S - -
‘R’ stands for resistant, and ‘S’ stands for susceptible.
‘+’ signifies positive for the gene screened while ‘-’ signifies negative for the gene screened.
59 Appendix F
Table 6: Representative data of statistical analysis on the negative association between tetA gene and gender of patients.
tetA
Total absent present
sex Female Count 24 20 44
% within sex 54.5% 45.5% 100.0%
% within tetA 72.7% 74.1% 73.3%
% of Total 40.0% 33.3% 73.3%
Male Count 9 7 16
% within sex 56.3% 43.8% 100.0%
% within tetA 27.3% 25.9% 26.7%
% of Total 15.0% 11.7% 26.7%
Total Count 33 27 60
% within sex 55.0% 45.0% 100.0%
% within tetA 100.0% 100.0% 100.0%
% of Total 55.0% 45.0% 100.0%
Chi-Square Tests
Value df
Asymptotic Significance (2-sided)
Exact Sig. (2- sided)
Exact Sig. (1- sided)
Pearson Chi-Square .014a 1 .907
Continuity Correctionb .000 1 1.000
Likelihood Ratio .014 1 .907
Fisher's Exact Test 1.000 .571
N of Valid Cases 60
a. 0 cells (.0%) have expected count less than 5. The minimum expected count is 7.20.
b. Computed only for a 2x2 table
Symmetric Measures
Value
Approximate Significance
Nominal by Nominal Phi -.015 .907
Cramer's V .015 .907
N of Valid Cases 60
60 Appendix G
Table 7: Representative data of statistical analysis on the positive association between tetA gene and tetracycline resistance profile.
tetA
Total absent present
tetracycline resistant Count 6 25 31
% within tetracycline 19.4% 80.6% 100.0%
% within tetA 18.2% 92.6% 51.7%
% of Total 10.0% 41.7% 51.7%
susceptible Count 27 2 29
% within tetracycline 93.1% 6.9% 100.0%
% within tetA 81.8% 7.4% 48.3%
% of Total 45.0% 3.3% 48.3%
Total Count 33 27 60
% within tetracycline 55.0% 45.0% 100.0%
% within tetA 100.0% 100.0% 100.0%
% of Total 55.0% 45.0% 100.0%
Chi-Square Tests
Value df
Asymptotic Significance (2-
sided)
Exact Sig. (2- sided)
Exact Sig. (1- sided)
Pearson Chi-Square 32.926a 1 .000
Continuity Correctionb 30.014 1 .000
Likelihood Ratio 37.559 1 .000
Fisher's Exact Test .000 .000
N of Valid Cases 60
a. 0 cells (.0%) have expected count less than 5. The minimum expected count is 13.05.
b. Computed only for a 2x2 table
Symmetric Measures
Value
Approximate Significance
Nominal by Nominal Phi -.741 .000
Cramer's V .741 .000
N of Valid Cases 60