Thus, the current study was undertaken to detect the prevalence of tetA and tetB genes in correlation with efflux pump resistance mechanisms among UPEC isolates by molecular screening. A total of 60 UPEC isolates obtained from Raja Permaisuri Bainun Hospital, Ipoh were subjected to antimicrobial susceptibility testing followed by molecular detection of tetA and tetB genes by duplex PCR assay. We hereby confirm that LEE SHUET YI (ID no.: 18ADB01539) has completed the final year project entitled "MOLECULAR DISCOVERY OF TETRACYCLINE RESISTANCE GENES ASSOCIATED WITH EFFLUENT PUMP MECHANISM IN ISOLATES OF UROPATHOGENIC Escherichie coli (UPEC)" under the supervision of Dr.
INTRODUCTION
Urinary tract infections (UTIs) .1 Overview
- Mechanism of action of tetracycline
- Tetracycline resistance (Tcr) determinants
- Tetracycline and minocycline
The transport can be categorized into uniport (transport of a single substrate), symport (co-transport of two substrates in one direction) and antiport (transport of two substrates in opposite directions), of which the process is driven by the existing electrochemical or ionic (H+) gradient (Choudhuri, 2014; Kumar et al., 2020). 9 binding specificity and enhance hydrophobicity to drive the antimicrobial efflux and expel lipophilic drugs (Kumar et al., 2020). 11 membrane diffusivity and ribosomal binding affinity compared to tetracycline (Asadi et al., 2020; Roy et al., 2021).
Chemicals and Reagents
Magnesium Chloride (MgCl₂) Promega Corporation, United States 5x GreenGoTaq® Flexi Buffer Promega Corporation, United States GoTaq® G2 Flexi DNA Polymerase Promega Corporation, United States tetA Forward and Reverse Primers Eurogentec Ait Pte Ltd., Singapore tetB Forward and Reverse Primers Integrated DNA technologies.
Methodology .1 Sample collection
- Antimicrobial susceptibility test
- Optimisation of duplex PCR condition
- Agarose gel electrophoresis
- Statistical analysis
Samples were plated on TSA, incubated overnight at 37°C, and then stored at 4°C for further assays. The diameter of the zone of inhibition was measured in millimeters and the results were interpreted as resistant (R), intermediate (I) and sensitive (S) according to the efficacy standard provided by the Clinical and Laboratory Standards Institute (CLSI) guidelines (2021 ). Prior to duplex PCR screening of tet genes among UPEC isolates, PCR conditions were optimized to ensure optimal amplification of the two target genes tetA and tetB.
17 3.2.5 Duplex PCR detection of tetracycline resistance genes in samples Optimized duplex PCR conditions were applied to subsequent duplex PCR screening of DNA extracts to detect the carriage of tetA and tetB genes in UPEC isolates. The PCR reaction mixture with a total volume of 25 µl was prepared as shown in Table 3.2 below. The primer sequences and expected product size of the respective tet resistance genes are shown in Table 3.3, while the cycle condition used is shown in the following Table 3.4.
After PCR amplification, gel electrophoresis was performed to detect the presence of amplified tet genes. The 100 bp DNA scale of SMOBIO was used as a molecular weight size marker to estimate the product size of the amplicons. The distribution of UPEC isolates and the prevalence of tetA and tetB genes were evaluated according to the demographic profiles of the patients.
Furthermore, the association of tet genes with demographic profiles and the association between genotypic traits and antibiotic susceptibility profiles were also analyzed.
Overview
Antimicrobial susceptibility test
It was found that 21 had the least resistance to imipenem (0.00%) followed by minocycline (1.67%), of which only one out of 60 samples was minocycline resistant and none of the strains showed resistance to imipenem.
Concentration and purity of DNA extracts
Optimisation of duplex PCR condition
The optimized cycling condition of duplex PCR was as follows: 5 minutes at 94°C for initial denaturation, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 30 seconds and extension at 72°C for 60 seconds, while the final extension step was for 8 minutes at 72°C. All UPEC clinical isolates were subjected to duplex PCR detection of tetA and tetB genes. In general, the UPEC isolates illustrated a higher prevalence of tetA gene compared to tetB gene.
Distribution of UPEC clinical isolates in relation to gender and age groups of patients
The representative data of the association between tet genes and the demographic profiles are listed in Appendix F. Both tetA and tetB genes show no association with either gender or age groups as the p-values are greater than 0.05. The gene prevalence in both sexes shows similarities whereby the frequency of tetA gene is higher than that of tetB gene.
It was found that the percentage of isolates carrying the tetA gene is the same in the young and working (50.00%), which is higher than in the elderly (37.50%). Meanwhile, tetB genes were found to be most prevalent in the age group (25.00%), followed by the working age group (6.25%), while none of the younger patients had the tetB gene. Compared to Figure 4.7, the prevalence of the tetA gene is greater than that of the tetB gene in all age groups.
Association between phenotypic antimicrobial resistance traits and genotypic profile of clinical isolates
31 In contrast to the tetracycline resistance profile, UPEC clinical isolates exhibited a high susceptibility to minocycline as only one out of 60 samples was minocycline resistant and was found to contain the tetB gene (100.00%). Despite the majority (59 out of 60) of UPEC isolates being susceptible to minocycline, duplex PCR screening revealed resistance gene carriage in 34 strains (57.63%). Regarding the association between phenotypic and genotypic variables, the minocycline susceptibility profile shows positive correlation with tetB (p = 0.010) but negative association with tetA (p = 0.362).
Apart from the tetracycline antibiotic class, the tetA gene was also found to be positively associated with other antimicrobials, including nalidixic acid (p = 0.020), trimethoprim-sulfamethoxazole (p = 0.001) and ampicillin (p = 0.000). Meanwhile, considering that all UPEC isolates were susceptible to imipenem, the association between tet genes and imipenem susceptibility profile was not able to be calculated since the variable of imipenem susceptibility profile is constant. In any case, the negative association of tet genes with antimicrobial susceptibility profiles to the other antibiotics including ciprofloxacin, levofloxacin, chloramphenicol was also observed as p-values > 0.050.
Overview
Antimicrobial susceptibility test
This result agrees with the findings obtained by Dehkordi et al. 2020), where UPEC samples showed the lowest resistance rate of 9.09% to imipenem. In addition, imipenem/cilastatin/relebactam combination therapy was also approved to treat cUTIs and cIAI (Chen et al., 2020). Despite its ideal antimicrobial action, it may suggest that imipenem may be a potential long-term drug treatment for UTIs, imipenem, which is a carbapenem, often serves as a last resort for MDR strains due to increasing rates of resistance. to β-lactam antibiotics (Sekyere, 2016; Chen et al., 2020).
Thus, minocycline is able to demonstrate better efficacy in eradicating a Gram-negative pathogen (Asadi et al., 2020). The present study showed a predominant distribution of the tetA gene (41.67%) compared to the tetB gene (11.67%), which is in agreement with the findings of Olowe et al. In addition, the findings reported by Skočková et al. 2012), also in line with the above-mentioned statement, since the shift in distribution from the tetB gene to the tetA gene in E.
Consistent with this study, Olowe's study also revealed a low coexistence incidence of resistance genes, with only 4.40% of isolates carrying both tetA and tetB genes (Olowe et al., 2013). This observation is in line with the findings of Dormanesh et al. 2014), who stated that the main rationale behind the predominance of the pathogenic E. Moreover, an imbalance in the vaginal flora can also endanger the women (Dormanesh et al., 2014).
The UPEC isolate occurrence shows agreement with the findings reported by Vargová et al. 2017) which revealed the increased incidence of urinary tract infection in patients over 60 years of age.
Association between phenotypic antimicrobial susceptible profiles and the tetracycline resistance genes prevalence
This is because the tetB gene is the only efflux gene capable of encoding large facilitator superfamily transporters that are effective against minocycline (Chorpra and Roberts, 2001; Asadi et al., 2020). This statement is consistent with the research reported by Huys et al. 2005 ), whereby an increase in minimal inhibitory concentrations (MICs) of minocycline was detected in tetracycline-resistant multidrug-resistant (MDR) Gram-negative strains screened positive for tetB genes. However, this finding contradicts the current study, as seven out of 59 susceptible isolates were found to have the tetB gene.
In any case, a study reported by Tuckman et al. 2007) showed agreement with the present results, where 16 minocycline-susceptible isolates (MICs ≤ 64 µg/ml) screened positive for the tetB gene. The possible explanation behind the transport of the tetB gene in susceptible isolates is that the tetB genes were not expressed or poorly expressed (Tuckman et al., 2007). This also suggests that the transport of resistance genes in UPEC strains does not necessarily reflect the phenotypic expression of resistance, and one of the reasons may be due to the gene regulation mechanism in the bacteria (Møller et al., 2016).
This suggests that the MFS efflux pump encoded by tetA is not only selective for the tetracycline class, but is also able to expel other drugs, further leading to multi-drug resistance (Warburton et al., 2012). In addition, the UPEC strains may also carry other genes responsible for various resistance mechanisms, including ribosomal protection, enzymatic inactivation, and drug target site modification, resulting in the appearance of resistance features shown in the current study (Chhetri et al. , 2015; Grossman, 2016). This suggests the acquisition of resistance genes among the susceptible isolates, which also implies that the UPEC strains may have become significant.
41 tet efflux genes and may further emerge in the future to become antimicrobial resistant (Boerlin et al., 2005).
Limitations and future studies
Virulence factors and antimicrobial resistance of uropathogenic Escherichia coli (UPEC) isolated from urinary tract infections: a systematic review and meta-analysis. Uropathogenic Escherichia coli in the high vaginal swabs of fertile and infertile women: virulence factors, O serogroups, and phenotyping and genotypic characterization of antibiotic resistance. Patterns of efflux pump genes among tetracycline resistance uropathogenic Escherichia coli isolates obtained from human urinary tract infections.
Distribution of tetracycline resistance genes in genotypically related and unrelated multidrug-resistant strains of Acinetobacter baumannii from different European hospitals. Antimicrobial resistance and prevalence of tetracycline resistance genes in Escherichia coli isolated from colibacillosis lesions in broilers in Sistan, Iran. Identification and sequence of a tet(M) tetracycline resistance-determining homologue in clinical isolates of Escherichia coli.
Distribution and transfer of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products. Occurrence of TET genes mediating tetracycline resistance in Escherichia coli clinical isolates in Osun State, Nigeria. Uropathogenic Escherichia coli (UPEC) infections: virulence factors, bladder responses, antibiotic and non-antibiotic antimicrobial strategies.
Occurrence of tetracycline resistance genes among Escherichia coli isolates from the phase 3 clinical trials for tigecycline. TetAB (46), a predicted heterodimeric ABC transporter conferring tetracycline resistance in Streptococcus australis isolated from the oral cavity. NA' stands for nalidixic acid, 'CIP' stands for ciprofloxacin, 'LEX' stands for levofloxacin, 'TET' stands for tetracycline, 'MIN' stands for minocycline.
