Phylogeny, Sequence-typing and virulence profile of uropathogenic E. coli (UPEC) strains from Pakistan

Escherichia coli lineage ST131 predominates across various spectra of extraintestinal infections, including urinary tract infection (UTI). The distinctive resistance profile, diverse armamentarium of virulence factors and rapid global dissemination of ST131 E. coli makes it an intriguing pathogen. However, not much is known about the prevalence and genetic attributes of ST131 lineage in Pakistan. We estimated the prevalence and genetic attributes of E. coli ST131 isolates causing UTI among 155 randomly selected samples. Samples were analyzed by phylogenetic grouping, O-typing, fumC/fimH typing. Isolates were further tested for ESBL and virulence factors using PCR. Conclusively, this study provides important insights into the genetic and virulence attributes of pandemic MDR ST131 strains involved in UTIs. It also highlights high prevalence of ST131-O25b-H30 UPEC isolates in local population, which was previously unreported from this part of globe.


Methods
We estimated the prevalence and genetic attributes of E. coli ST131 isolates causing UTI among 155 randomly selected samples. Samples were analyzed by phylogenetic grouping, O-typing, fumC/fimH typing. Isolates were further tested for ESBL and virulence factors using PCR.

Conclusion
Conclusively, this study provides important insights into the genetic and virulence attributes of pandemic MDR ST131 strains involved in UTIs. It also highlights high prevalence of ST131-O25b-H30 UPEC isolates in local population, which was previously unreported from this part of globe. Keywords: ST131, VF genes, ESBL, UPEC, MDR Background Extra-intestinal E. coli is the major cause of urinary tract infections and resistance among UTI strains has been mounting against different antibiotics, including trimethoprim-sulfamethoxazole, fluoroquinolones extended spectrum cephalosporins and amoxicillin clavulanic acid [1][2][3]. Due to the emergence of specific clonal groups such as ST131, global dissemination of fluoroquinolone-resistance was highlighted across different geographical regions [4][5][6]. Clonal group ST131 predominates across various spectra of infections including cystitis, pyelonephritis, bacteremia, meningitis, septic shock, epididymo-orchitis and osteoarticular infection. [7,8]. In addition, ST131 strains harbor diverse armamentarium of virulence factors and their genetic homogeneity regarding virulence potential and resistance profile has been endorsed [8]. Notably, a subgroup of ST131 strains, known as H30-Rx has remarkable tendency to encode bla- 9,10]. In the current scenario of global urgency related to the antibiotic resistance, underlying epidemiological factors related to the fitness and fast emergence of ST131 across different regions are under intensive scrutiny. However, in Pakistan phylogenetic grouping, sequence types, virulence attributes and antibiotic susceptibility profile of UPEC strains remains unexplored [11,12]. Therefore, data related to the clonal types and Phylogeny, serotyping, and fumC/ fimH Typing We used the procedure reported by Clermont et al, 2000 to perform phylogenetic analysis of 155 isolates [ 14]. FumC/fimHtyping (CH typing) was performed as previously described [15]. Briefly, PCR amplifications were carried out in 25µl (12.5µl GoTaq DNA polymerase (Promega) 7.5µl water, 1 µl bacterial DNA, 2 µl of each forward and reverse primers). The amplified products were analyzed on 2% agarose gel. The confirmed PCR products were purified using PCR purification kit (QIAquick, PCR Purification Kit, QIAGEN) and all the fragments were sequenced (ABI 3130, Perkin-Elmer Applied Biosystems, Foster City, California). The forward and reverse sequences were aligned, trimmed off using Codon Code Aligner and results were compiled according to the standard procedures [15,16]. Additionally, by targeting 347bp of pabB gene fragment, clonal group ST131 was scrutinized for serogroup O25b [17]. Previously typed O25b-ST131 and K-12 E. coli strains were included as experimental controls in this study.

Detection of β-lactamases and Virulence factor genes
In order to detect extra-chromosomally encoded ESBL factors, plasmid DNA was isolated by commercially available kit (Thermo-Scientific Gene Jet plasmid Miniprep Kit). ESBL factors including bla -TEM , bla -SHV and genes bla -OXA , bla -PSE were also amplified by PCR as described elsewhere [18]. Amplified products were purified (Gel Band Purification Kit, Amersham, USA) and sequencing was done by automated DNA sequencer (ABI 3130, Perkin-Elmer Applied Biosystems, Foster City, California).
Sequences were reported to the Gene Bank database (accession number; KX171170-171195). PCR amplifications and sequencing of bla -CTX-M allele was carried out, bla -CTX-M types were determined by comparing DNA sequences available in the database [19]. A total of 18 different Virulence factor (VF) genes corresponding to the main classes of extra-intestinal virulence associated genes (VAGs) including adhesins, toxins, siderophores, capsular proteins and uropathogenic-specific protein (usp) were scrutinized in 155 isolates. VF genes were amplified by previously reported sets of primers and amplification conditions [20].

Statistical analysis
The statistical analysis was performed using Graph Pad Prism, version 7. Both Chi square and Fisher exact tests were used to assess differences by assuming cut-off value of P < 0.05 as significant.

Phylogeny and sequence typing
Overall, phylogenetic group B2 showed highest representation, 92(59%) followed by D 43 (28%), B1 12 (8%) and A 8(5%) ( MDR among ST131 strains Significant number of the isolates assigned to phylogenetic group B2 and D were multi-drug resistant (Table 3). Similarly, among different STs including ST131, ST405, ST168, ST29, ST69 and ST89, significant number of the isolates were multidrug resistant ( Table 3). The tendency of ESBL production and the fluoroquinolone resistance was relatively higher among ST131 isolates and majority of the isolates were multi-drug resistant (Table 3). Likewise, resistance against nitrofurantoin was significantly higher among ST131 isolates in comparison to other sequence types ( Table 4). One of the frequently prevalent sequence types, ST168 strains showed significant resistance to levofloxacin (Table 4). Resistance against carbapenemes has not been evaluated for the scrutinized strains; hence it is beyond the scope of this discussion.
Some virulence factors such as sfa/foc, fyuA and feoBwere detected frequently among ST131 isolates whereas VF-genes papEF, sfa/focand hlyAwere frequently associated with H30 sub-clone (Table 5). Overall, virulence genes such as sfa/foc, fyuA and feoB were associated significantly ( p < 0.05) with strains ST131, while papEF had significant presence among clonal group ST131-H30.  [4,5]. In this study, clonal group ST131 was the most prevalent lineage, comprising 46% of the isolates and majority of these isolates (59%) belonged to the phylogenetic group B2. Prevalence of two other lineages ST405 and ST168 were 18% and 10% respectively. Involvement of these lineages in UTIs has been described earlier and recently ST405 was confirmed as an emerging uropathogenic E. coli clone in Saudi Arabia [21,22]. Urinary tract infections caused by E. coli pose considerable challenge and are associated with high morbidity and mortality [5]. Due to their resistance against variety of antibiotics, including βlactams, aminoglycosides and fluoroquinolones, infections caused by pandemic clonal group ST131 are challenging to treat [23,24]. In this context, epidemiological significance of a sub group ST131 H30-Rx has been well described [5 , 25]. In this study, 50% of the ST131 strains carried H30 variant of fimH gene and 61% belonged to the serogroup O25b. Moreover, all the isolates belonging to sub-group ST131-H30-O25b carried ESBL bla-CTX-M-15 however, overall prevalence of these strains constituted 10% of the total isolates. In this study overall resistance against fluoroquinolones remained 60%, which was remarkably higher among ST131 isolates (60% vs 82%). Generally, for the treatment of UTIs commonly prescribed antibiotics include sulphamethoxazole-trimethoprim and fluoroquinolones. Due to the emerging resistance to these antibiotics alternative therapeutic choices such as nitrofurantoin, fosfomycin and β-lactam inhibitors can be used.
In this study, prevalence of ESBL genes was higher among ST131 isolates and 90% of the ST-131 isolates were resistant to ceftazidime and cefotaxime. Likewise, resistance to ceftriaxone was confirmed in 77% of these isolates. Because of their favorable safety, cephalosporins are considered important therapeutic choice for the treatment of uncomplicated UTIs among pregnant women [ 26].
Nitrofurantoin is a fluoroquinolone-sparing alternative antibiotic used for uncomplicated cystitis [27]. In recent years use of nitrofurantoin has increased steadily, due to resistance against trimethoprim/sulfamethoxazole and aminopencillins. Contraindication of ciprofloxacin in pregnancy and adverse impact on gut flora favored the use of nitrofurantoin as a treatment option for UTIs. In this study 13% of the ST-131 isolates were resistant to nitrofurantoin.
We found that majority of the isolates belonging to the lineages ST405, ST168,

Availability of data and material
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests
We declare that there is no conflict of interest.      ST, VF and resistance-updated.xlsx