Phylogeny, sequence-typing and virulence profile of uropathogenic Escherichia coli (UPEC) strains from Pakistan

Background Escherichia coli lineage ST131 predominates across various spectra of extra-intestinal 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. Methods We estimated prevalence and genetic attributes of E. coli ST131 isolates causing UTI among 155 randomly selected samples. Samples were analyzed for phylogenetic grouping, O-typing and fumC/fimH typing. Isolates were further tested for the ESBL and virulence factors using PCR. Results Overall, 59% of the UPEC isolates belonged to the phylogenetic group B2, followed by D = 28%, B1 = 8% and A = 5%. Among 18 different Sequence-types, ST131 was the dominant lineage (n = 71; 46%) out of which 72% of the isolates were assigned to the phylogenetic group B2, while 61% adhered to the serogroup O25b. FumC/fimH typing confirmed 49% of the ST131 as H30 sub-types. In this study, significant numbers of the identified ST131 isolates were MDR and 42% showed ESBL phenotypes, out of which 37% carried bla-CTX-M-15. Moreover, different virulence factors were detected in following percentages: fimH,155(100%), iutA 86 (55%), feoB 76 (49%), papC 75 (48%), papGII 70 (45%), kpsMTII 40 (26%), papEF 37 (24%), fyuA 37 (24%), usp 22 (14%), papA 20 (13%), sfa/foc20 (13%), hlyA 18 (12%), afa 15 (10%), cdtB 11 (7%), papGI 6 (4%), papGIII 6 (4%), kpsMTIII 4 (3%) and bmaE2 (1%). Conclusion Conclusively, this study provides important insight into the genetic and virulence attributes of pandemic MDR ST131 strains involved in UTIs. It also highlights higher prevalence of ST131-O25b-H30 UPEC isolates in patients, which was previously unreported from this part of globe.

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 resistance profile of the strains involved in urinary tract infections in Pakistan is extremely scarce. This study fills the gap and provides important insights into the genetic and virulence attributes of pandemic MDR ST131 strains involved in UTIs in Pakistan.

Sample collection and antibiotic susceptibility testing
Altogether n = 155 identified uropathogenic E. coli (UPEC) were collected during the period of August 2012 to August 2014, from Pakistan Institute of Medical Sciences. Ethical Review Board (ERB) of Pakistan Institute of Medical Sciences approved this study. Ethical Review Board approved verbal consent taken from all the patients. Important patient data such as name, age, gender, location was recorded and unique identification number were assigned to each patient. Samples were from community-acquired urinary tract infections. Antibiotic testing and phenotypic detections of ESBL were performed by disc diffusion methods according to the guidelines CLSI, 2014 [13]. Isolates were tested for the susceptibility to 12 different classes of antibiotics including β-lactamase inhibitors (piperacillin tazobactam, amoxicillin-clavulanic acid), cephalosporins (ceftazidime, cefotaxime, ceftriaxone), fluoroquinolone (ciprofloxacin, levofloxacin), aminoglycosides (amikacin), trimethoprim sulfonamides, nitrofurantoin, and fosfomycin (BIOANA-LYSE, Turkey). Control strain E. coli ATCC 25922 was used in this assay.
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 25 μl (12.5 μl GoTaq DNA polymerase (Promega), 7.5 μl 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, QIAGEN) and all the amplified DNA 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 347 bp of pabB gene fragment, clonal group ST131 was scrutinized for serogroup O25b [17]. Previously typed O25b-ST131 strains and K-12 E. coli 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 PCR amplified as described elsewhere [18]. Amplified products were then 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 factors (VF) 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 all 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.

MDR among ST131 strains
pt?>Significant number of the isolates assigned to the phylogenetic group B2 and D were multi-drug resistant (Table 2). Similarly, among different STs including ST131, ST405, ST168, ST29, ST69 and ST89, significant number of the isolates were multi-drug resistant ( Table 2). The tendency of ESBL production and the fluoroquinolone resistance was relatively higher among ST131 isolates and majority of these isolates were multi-drug resistant ( Table 2). Resistance against nitrofurantoin was significantly higher among ST131 isolates in comparison to the other sequence types, whereas one of the frequently prevalent sequence types, ST168 strains were significantly resistant to levofloxacin (Table 3). Resistance against carbapenemes has not been evaluated for the scrutinized strains in this study; hence it is beyond the scope of this discussion.
In this study, prevalence of ESBL genes was higher among ST131 and 90% of these strains were resistant to ceftazidime and cefotaxime. Likewise, resistance to ceftriaxone was confirmed in 77% of these strains. 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 the treatment of uncomplicated cystitis [27]. In recent years use of nitrofurantoin has increased steadily, particularly due to the resistance against trimethoprim/sulfamethoxazole and aminopencillins. Contraindication of ciprofloxacin in pregnancy and adverse impact on the gut flora favored the use of nitrofurantoin as an alternative treatment option for UTIs. In this study 13% of the ST-131 strains were resistant to nitrofurantoin.
We found that majority of the isolates belonging to the lineages ST405, ST168, ST29, ST69 and ST89 were multi-drug resistant. Percentage of MDR isolates was particularly higher among fluoroquinolonesresistant ST131 strains. Overall 59% of the isolates   belonged to the phylogenetic group B2. A previous study from Pakistan confirmed that 50% of the UPEC isolates belonged to the phylogenetic group B2 [28]. Likewise, another study conducted in Pakistan reported that only 12% of the E. coli strains belonged to this phylogenetic group. These findings suggest that prevalence of phylogenetic group B2 may vary across different regions [29]. Few studies conducted previously in this region included phylogentic analysis of UPEC strains. Phylogenetic group B2 strains are equipped with various VF genes relating to the extra-intestinal infections. These genes include P-fimbriae, S-fimbriae, haemolysin, aerobactin, K1 and K5 antigens and capsular antigen genes [30,31]. A previous report focusing on the UPEC, in Pakistan described prevalence of various VF genes, including hlyA, sfaDE, papC,cnf1, eaeA and afaBC [29] While another study conducted on the rectal floral isolates of Pakistani children confirmed that virulence factors such as S-fimbriae, haemolysin, K-1 antigens and class III PapG adhesins were either very rare or completely absent [29]. In current study, UPEC strains of phylogenetic group B2, carried range of virulence factors, including genes for adhesins (fimH 100%, papA 13%, papC 47%, papEF 21% papGI 3%, papGII 40%, papGIII 4%, sfa/foc 14%, afa 11%, bmaE 1%), toxins (hlyA 7%, cdtB 7%) iron acquisition system (iutA 57%, feoB43%, fyuA 23%) capsular proteins (kpsMTII 26%, kpsMTIII 3%) and uropathogenic specific protein (usp 14%). We observed that the gene papGII was significantly associated with phylogenetic group B2 strains and association of papGII with pyelonephritis and bacteraemia in human has been confirmed earlier [32][33][34]. In the current study, fimbriae associated gene fimH was detected among 100% of the UPEC isolates Role of fimH in adhesion, invasion and formation of the intracellular bacterial communities (IBCs) has been described previously and its importance in the host pathogen interaction was confirmed by higher vulnerabilities of premenopausal women to UPEC infections [35]. In this study genes related to the adhesins (papEF, sfa/foc) and toxins (hlyA) were found to be strongly associated with ST131 H30 sub-clone. Recently hlyA in interaction with natural killer (NK) cells of urinary bladder was described [36]. Likewise, we witnessed significant association of the iron acquisition genes ((fyuA and feoB) with ST131 lineage. The importance of gentic factors related to the iron acquisition system was shown by strong upregulation of these genes during UTIs [37]. Generally, E. coli strains causing UTI share similar properties in terms of phylogeny, sero-grouping and VF genes. However, other than genetic attributes of the virulence strains, host factors may play important role in the outcome of infection [38].

Conclusion
In conclusion it is the first report that highlights MDR ST131 as a predominant linage associated with UTI in Pakistan. ST131 and other scrutinized sequence types having MDR status among UTI isolates in Pakistan indicate considerable constraints on the empirical choice for the treatment of UTI. Alternative therapies and identification of effective prevention strategies-including antibiotic stewardshipare needed. As antibiotic resistance can be transferred from UPEC to other pathogens, more judicious use of antibiotics is required.