Plasmid Replicon Diversity of Clinical Uropathogenic Escherichia coli isolates from Riyadh, saudi Arabia

The aim of this study was to identify and compare the plasmid replicons of clinical uropathogenic Escherichia coli (UPEC) isolates, involving extended spectrum β-lactamase (ESBL)-positive and ESBL-negative, E. coli ST131 and non-ST131 and various ST131 subclones. Plasmid replicon typing on 24 clinical UPEC isolates was carried out using polymerase chain reaction-based replicon typing. A statistical analysis was performed to assess the associations between plasmid replicon types and ESBL carriage, and to evaluate the link between st131 isolates and high replicon carriage. eight replicons, I1α, N2, Iγ, X1, FIIS, K, FIA, and FII were detected. The FII was the most common replicon identified here. ESBL-positive E. coli isolates were highly associated with I1α, N2, I γ , X1, and FIIS replicons, while FIA was present only in ESBL-negative group. ST131 isolates were highly associated with I1α and N2 replicons compared to non-ST131. No link was found between replicon carriage and the number or type of ESBLs in E. coli isolates. The diversity observed in replicon patterns of our clinical E. coli isolates indicates that they might be originated from different sources. The presence of replicons reported previously in animal sources suggests a possible transfer of antimicrobial resistance between animal and human bacterial isolates.


iNtRODUCtiON
Urinary tract infections (UTIs) are commonly caused by Escherichia coli (E.coli), 1 and it is shown that uropathogenic E. coli (UPEC) subset are responsible for approximately 80% of UTIs. 2 Over the past two decades, the levels of antibiotic resistance and extended spectrum β-lactamases (ESBLs) carriage of UPEC have increased markedly. 3,4SBLs are enzymes capable of degrading β-lactams antibiotics, rendering these agents inactive. 5ESBL genes have evolved into several hundreds of variants, complicating the fight against ESBL-producing bacterial isolates causing UTIs. 6For instance, bla CTX-M gene has nearly 170 gene variants so far. 7Among CTX-M gene variants, bla CTX-M-15 is the most common and it belongs to the CTX-M-1 phylogroup. 8Plasmids carry the vast majority of ESBL genes, and they are major players in the transfer and dissemination of ESBL-mediated resistance among clinical bacterial strains. 9There has been a link between particular plasmid types and ESBL-encoding genes.For instance, IncFII plasmids frequently carry bla CTX-M-15 , while IncK plasmids commonly harbor bla CTX-M-14 . 10he worldwide dissemination of the multidrug resistant (MDR) E. coli sequence type 131 (E. coli ST131) clone represents a major challenge to public health. 11E. coli ST131 is often fluoroquinolone (FQ) insusceptible and commonly carry CTX-M-15 ESBL on IncFII plasmids. 12,13ST131 isolates are subdivided into different subclones, and H30 is among the most common ST131 subclone.H30 comprises two subsets, H30R and H30Rx. 115][16] The identification of plasmid replicon types is important in understanding the epidemiological perspective of prevalence and transmission of ESBL encoding genes in E. coli, and this can help in tracking their origin by comparing them to environmental or animal isolates and in the diagnosis of clinically important ESBL-producing E. coli. 17Given that information on plasmid replicon diversity of clinical E. coli isolates is very scarce in Saudi Arabia, this study sought to describe and compare the replicon diversity of clinical E. coli urine isolates, including ST131 and non-ST131, ESBL-positive and ESBL-negative, and different ST131 subclones.The link between plasmid replicon and ESBL carriage was also evaluated.

Bacterial isolates
This study involved a total of 24 E. coli isolates that obtained from urine specimens of hospitalized in-patients at a tertiary hospital in Riyadh, Saudi Arabia.Details on these E. coli isolates are shown in Table 1.

Plasmid replicon typing assays
Plasmid replicon typing was performed by polymerase chain reaction (PCR)-based assay using 2.0 kit (Diatheva, Italy) following the manufacturer's instructions.Replicon typing was done through eight multiplex PCR reactions that can detect up to 30 replicon types (Table 2).Each reaction mix has 25µl containing 1U DNA polymerase.The setup of the PCR reactions included: 1 denaturation cycle for 10 minutes at 95°C; 25 denaturation cycles for 60 sec at 95°C, annealing for 30 sec at 60°C, and extension for 60 sec at 72°C; and 1 cycle of final extension for 5 minutes at 72°C.PCR products were visualized after being run on 2.5% agarose gel containing ethidium bromide.The assays were performed in triplicate showing fully concordant results.

Statistical analysis
Prism GraphPad (version 9.3.0)was used for statistical analysis.Fisher's exact test was used for comparisons of various isolate groups, while Mann-Whitney U test was used to determine the mean replicon scores.The threshold for statistical significance was a P-value ≤ 0.05.

Comparison of plasmid replicon carriage between ESBL-positive and ESBL-negative isolates
Our plasmid replicon typing results a MDR phenotype refers to displaying resistance to at least 1 antibiotic in ≥3 antibiotic groups.demonstrated a similarity between these isolate groups in their capability of harboring two replicon types: K and FII (Fig. 2).However, some differences in their replicon carriage were identified.Four replicons: N2, 1γ, X1 and FIIS were only carried by ESBL-positive isolates, while ESBL-negative isolates were exclusively carried the FIA replicon (Fig. 2).The ability of ESBL-positive isolates to harbor I1α replicon was more than that of ESBL-negative E. coli isolates.The difference between ESBL-positive and ESBL-negative isolates in carrying the N2 replicon was significant (P = 0.01) (Fig. 2).

Comparison of plasmid replicon carriage of st131 and non-ST131 isolates
Our data demonstrated a similarity between both isolate groups to carry three replicon types: FIA, FIIS and FII (Fig. 3).Nonetheless, few insignificant differences in their replicon carriage were observed.Non-ST131 isolates exclusively harbored 1γ, X1 and K replicons, with at least one isolate showing capability of carrying these replicons.However, ST131 isolates were more able to carry I1α and N2 compared to non-ST131.No statistical difference in the median replicon carriage score between these two isolate groups was detected (P = 0.60) (Fig. 3).

Comparison of plasmid replicon carriage of E. coli st131 subclones
All ST131 isolates were similar in their incapability of carrying three replicons: 1γ, X1 and K, however there was a slight variability between ST131 subclones in carrying five replicons: I1a, FIA, N2, FIIS and FII.In general, the of replicon carriage of H30 isolates were nearly comparable to non-H30 isolates, and there was no specific replicon profile of a particular ST131 subclone (Fig. 4).The replicon carriage of H30 non-Rx subclone was higher than H30Rx, and a significant difference one ESBL type, such as U46 and U82, did not carry more replicons in comparison to those carrying single ESBL type (Table 4).

DisCUssiON
Characterizing bacterial plasmids in MDR bacteria, such as ExPEC, is essential in elucidating the role these plasmids play in the global spread of antimicrobial resistance.Several techniques have successfully been used to characterize plasmids with variable degrees of applicability.Among these techniques, PCR-based replicon typing is an easy useful tool with proven specificity and sensitivity. 18,19In Enterobacteriaceae, IncF, IncI, IncA/C, IncL, IncN and IncH are among the most frequently reported plasmids associated with carrying antibacterial resistance genes. 20n our analysis, eight replicons were detected in the 24 isolates, and there was a high degree of plasmid replicon diversity among these isolates.This is common for E. coli isolates and between these subclades in their carriage of I1α and N2 replicons was found (P = 0.05) (Fig. 4).Non-H30 isolates was not significantly associated with a specific replicon type compared to H30 isolates (Fig. 4).

Relating the plasmid replicon carriage to ESBL carriage
The results of relating the plasmid replicon carriage of the twenty-four E. coli isolates to their ESBL carriage are shown in Table 4.In general, there was no specific relationship between replicon carriage and the number or type of ESBLs carried by E. coli isolates.Our data showed that FII was found in 10 (83.3%)CTX-M-15producing isolates, however this replicon type was not exclusively harbored by ESBL-positive isolates, as it was also detected in 10 (83.3%)ESBL-negative isolates.Additionally, isolates carrying more than concurs with a previous report demonstrating high diversity in replicon types among UPEC isolates. 21he diversity observed in replicon patterns of our clinical E. coli isolates indicates that they might be originated from different sources.With respect to the distribution of replicons, our data found that IncF plasmids were the most encountered group.FII replicons were found alone in 11 isolates and combined with FIA in two isolates.4][25] However, it is in contrast to a previous study, showing that FIB and B/O were the most common replicon types among a collection of UPEC isolates. 21e also found four isolates showing the replicon combination I1a, N2 & FII.The presence of such a combination is not uncommon and the cointegration of IncF plasmids with IncI1 and IncN in E. coli was previously reported. 26,27ur study compared the replicon types of ESBL-positive and negative isolates, and demonstrated that K and FII replicons were detected in both isolate groups in similar frequency, and that ESBL-positive isolates were more probable to carry FIIS, I1a, N2, 1γ and X1 isolates in comparison to ESBL-negative isolates.It has been shown that ESBL genes are mostly found on IncF plasmids, that also encode for  carbapenemases, aminoglycoside-modifying enzymes, and quinolone resistance, 10 which agrees with our findings.However, we showed that FII carriage was comparable between ESBL-positive and negative isolates, suggesting that this replicon is not specific to ESBL-producing E. coli.Given the little focus towards characterizing plasmid replicon carriage of non-ESBL producing E. coli strains in the literature, it is crucial to perform comparative large-scale studies on the epidemiology of IncF plasmid carriage of ESBL-positive and ESBLnegative E. coli isolates.
Our analysis also showed the ability of some isolates to carry replicons that are harbored by E. coli isolates from animal sources.For example, IncIγ, the replicon detected in one isolate in this study, was also reported to carry bla CMY-2 gene in E. coli isolate from animal source. 280][31][32] In this study, N2 replicon was found in six isolates, all of them also have FII replicons.This is in accordance with a previous study reported the colocalization of IncN with IncF plasmids in E. coli. 33Several ESBL genes were carried on IncN plasmids in E. coli isolates including bla CTX-M-1 , 34 bla CTX-M-14 and bla CTX-M-27 . 35IncN2 plasmids were reported in E. coli from human hosts and carried bla NDM-1 gene in Thailand, 36 Australia, 37 and China. 38ncX1 plasmid was present in two isolates, both are ESBL-producing non-ST131 isolates.IncX1 encoding qnrS1 gene was identified in a previous study in a quinolone-resistant E. coli isolate from animal source. 39urrently, E. coli ST131 is considered as the main contributor for the spread of multidrug resistance and certain genes coding for CTX-M ESBLs on IncFII plasmids.Surprisingly, our data has not found higher plasmid replicon carriage of ST131 compared to non-ST131, although almost all our ST131 isolates were MDR and CTX-M-15 producing.Moreover, FII replicon was detected in similar frequencies in both ST131 and non-ST131 isolates.Despite our sample size is low, this finding is important and highlights the importance of performing comparative studies on different successful ExPEC clones to evaluate the difference in their plasmid replicon carriage.Two fluoroquinolone-resistance clades are believed to lead the global expansion of ST131.The two clades are H30R (Clade 1), and H30Rx (Clade 2), both have fimH30 allele. 40,41A recent plasmidome meta-analysis-based evidence has indicated that CTX-M-encoding ST131 plasmids are clade-specific, meaning that clonal expansion is the cause of the global expansion of ST131 rather than horizontal gene transfer.The analysis found that IncF plasmids coding for CTX-M-27 are found only in clade 1, whereas IncF plasmids coding for CTX-M-15 are found only in clade 2. 42 Our analysis showed that, among the 12 ST131 isolates included in the study, five are H30R, five are H30Rx and two are non-H30.All H30R isolates have identical replicons (I1α, N2 and FII) except one isolate that possessed only FIIS replicon.All H30Rx isolates posses FII replicon except one of them that had both FII and FIA.However, in contrast to the recent evidence, most of these subclones were having CTX-M-15 regardless of the subclone.
With respect to the limitations of this study; it examined a small number of UPEC isolates, therefore the associations observed in the current analysis might not be fully definitive.It also provided a description and a comparison of the replicon diversity of E. coli urine isolates collected from Riyadh city, which does not necessarily reflect the plasmid replicon diversity of E. coli isolates collected from other geographical parts in Saudi Arabia.

CONClUsiON
This study is the first to identify and compare the plasmid replicon diversity of different UPEC isolate groups from Saudi Arabia.This diversity observed in replicon patterns of our isolates indicates that they might be originated from different sources.The presence of replicons reported previously in animal sources suggests a possible transfer of antibiotic resistance between animal and human bacterial isolates.The lack of specific replicon carriage of the globally disseminated MDR ExPEC clone, ST131, or its subclones was also demonstrated here.In future, studying the plasmid replicon diversity of major MDR E. coli clones is essential to define the role of these plasmids in making MDR ExPEC clones such successful pathogens.

Fig. 1 .
Fig. 1.The overall distribution of plasmid replicons detected in this study.

Fig. 2 .
Fig.2.The plasmid replicon types detected for ESBL-producing and non-ESBL-producing isolates.The asterisk refers to presence of significant difference between the two isolate groups for N2 replicon carriage (P = 0.01).

Fig. 4 .
Fig. 4. The plasmid replicon types detected for isolates belonging to ST131 subclones.The asterisks refer to the presence of significant difference between the H30Rx and H30 non-Rx isolate groups for I1a and N2 replicon carriage (P = 0.05).

table 1 .
Information on the E. coli isolates used in this study

table 2 .
Information on replicon types provided by PBRT kit

table 3 .
Plasmid replicon types detected for all tested E. coli isolates

table 4 .
Relating the plasmid replicon type(s) to ESBL carriage