Advertisement

BMC Infectious Diseases

, 19:620 | Cite as

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

  • Ihsan Ali
  • Zara Rafaque
  • Ibrar Ahmed
  • Faiza Tariq
  • Sarah E. Graham
  • Elizabeth Salzman
  • Betsy Foxman
  • Javid Iqbal DastiEmail author
Open Access
Research article
Part of the following topical collections:
  1. Healthcare-associated infection control

Abstract

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.

Keywords

ST131 VF genes ESBL UPEC MDR 

Abbreviations

ESBL

Extended spectrum beta lactamases

IBCs

Intracellular bacterial communities

MDR

Multidrug resistance

NK

Natural killer

ST

Sequence type

UPEC

Uropathogenic E. coli

UTI

Urinary tract infections

VF

Virulence factors

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 widely endorsed [8]. Notably, a subgroup of ST131 strains, known as H30-Rx has remarkable tendency to encode bla-CTX-M-15 gene [7, 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 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.

Methods

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 (BIOANALYSE, 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.

Results

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%) (Table 1). Eighteen different sequence types (STs) of 152 isolates were confirmed, constituting 98% of all the isolates; the remaining 2% of the isolates were un-typeable. Clonal group ST131 comparised 71(46%) of all the isolates, followed by two other lineages, ST405 28(18%) and ST168 16(10%) (Table 2). Majority of the ST131 strains 51(72%) belonged to the phylogenetic group B2, while 43(61%) were assigned to serogroup O25b. CH typing confirmed 35(49%) as ST131-H30 sub-group of strains, out of which 22(31%) belonged to the serogroup O25b.
Table 1

Distribution of ESBL factors, antibiotic resistance and VF genes in MDR UPEC

Numbers and percentagesof isolates and their respective traits (n = 155)

Resistance traits

Total Isolates (n = 155)n (%)

Group A

(n = 8)n(%)

Group B1

(n = 12) n(%)

Group B2

(n = 92) n(%)

Group D

(n = 43)n(%)

p value

ESBL phenotypes

65(42)

1(13)

3(25)

36(39)

25(58)

0.0268

blaCTX-M-15

57(37)

1(13)

3(25)

32(35)

21(49)

0.1333

blaTEM

23(15)

1(13)

3(25)

13(14)

6(14)

0.7823

blaSHV

6(4)

00

00

3(3)

3(7)

0.0907

blaOXA

10(6)

00

1(8)

4(4)

5(12)

0.3610

blaPSE

1(0.6)

00

00

1(1)

00

0.8783

Piperacillin tazobactam

7(5)

00

1(8)

4(4)

2(5)

0.8514

Ceftazidime

96(62)

3(38)

7(58)

54(59)

32(74)

0.1483

Cefotaxime

101(65)

3(38)

7(58)

59(64)

32(74)

0.2028

Ceftriaxone

99(64)

3(38)

8(67)

59(64)

29(67)

0.4001

Ciprofloxacin

95(61)

6(75)

7(58)

56(90)

26(60)

0.4001

Levofloxacin

97(63)

7(88)

8(67)

56(90)

26(60)

0.4929

Amikacin

7(5)

1(13)

00

5(5)

1(2)

0.4920

Gentamicin

47(30)

4(50)

6(50)

24(26)

13(30)

0.2171

Amoxicillin-clavulanic acid

111(72)

5(63)

7(58)

66(72)

26(60)

0.5224

Trimethoprim sulfonamides

130(84)

7(88)

11(92)

77(84)

35(81)

0.8461

Nitrofurantoin

9(6)

1(13)

1(8)

6(7)

1(2)

0.6075

Fosfomycin

15(10)

1(13)

2(17)

8(9)

4(9)

0.8370

fimH

155 (100)

8 (100)

12 (100)

92 (100)

43 (100)

> 0.9999

papA

20 (13)

1 (13)

1 (8)

12 (13)

6 (14)

0.9659

papC

75 (48)

2 (25)

8 (67)

43 (47)

22 (51)

0.3092

papEF

37 (24)

0

6 (50)

19 (21)

12 (28)

0.0476

papGI

6 (4)

0

0

3 (3)

3 (7)

0.5699

papGII

70 (45)

2 (25)

7 (58)

37 (40)

24 (53)

0.1695

papGIII

6 (4)

0

0

4 (4)

2 (5)

0.8177

sfa/foc

20 (13)

2 (25)

1 (8)

13 (14)

4 (9)

0.5968

Afa

15 (10)

0

1 (8)

10 (11)

4 (9)

0.7919

bmaE

2 (1)

0

1 (8)

1 (1)

0

0.1466

fyuA

37 (24)

1 (13)

2 (17)

21 (23)

13 (30)

0.5882

iutA

86 (55)

5 (63)

5 (42)

52 (57)

24 (56)

0.7701

feoB

76 (49)

3 (38)

6 (50)

40 (43)

27 (63)

0.1852

kpsmtII

40 (26)

0

4 (33)

21 (23)

15 (35)

0.1438

kpsmtIII

4 (3)

0

1 (8)

3 (3)

0

0.3765

Usp

22 (14)

2 (25)

2 (17)

14 (15)

4 (9)

0.6256

hlyA

18 (12)

1 (13)

3 (25)

6 (7)

8 (19)

0.0908

cdtB

11 (7)

0

3 (25)

6 (7)

2 (5)

0.0758

Distribution of resistance and virulence traits among different phylogroups of uropathogenic E. coli (N = 155). The p values were calculated by comparing different traits among phylogroups

Table 2

Distribution of MDR and fluoroquinolone resistant MDR strains in different phylogroups and ST-types

Numbers and percentages of the sequence typed isolates (n = 155)

No of isolates in group n (%)

No of MDR isolates n (%)

ESBL+FQR-MDR isolates n (%)

p value

Group A 8(5)

7(88)

2(25)

0.0117

Group B112(8)

10(83)

5(42)

0.0350

Group B2 92(59)

71(77)

33(36)

< 0.0001

Group D 43(28)

36(84)

11(26)

< 0.0001

ST131 71(46)

57(82)

22(31)

< 0.0001

H30 35(49)

28(80)

10(29)

< 0.0001

Non H30 36(51)

29(81)

12(33)

< 0.0001

ST405 28(18)

24(86)

10(36)

0.0001

ST168 16(10)

14(88)

7(44)

0.0092

ST2913(8)

9(69)

5(38)

0.1156

ST69 5(3)

5(100)

2(40)

0.0384

ST95 2(1)

2(100)

00

 

ST31 2(1)

00

00

 

ST10 2(1)

1(50)

00

 

ST4482(1)

1(50)

00

 

ST892(1)

2(100)

1(50)

0.2482

ST7032(1)

2(100)

00

 

ST910 1(1)

1(100)

00

 

ST5451(1)

00

00

 

ST9711(1)

00

00

 

ST1531(1)

00

00

 

ST1521(1)

1(100)

00

 

ST121(1)

1(100)

00

 

ST8381(1)

0(100)

00

 

NSC3(2)

2(67)

2(67)

> 0.9999

Phylogenetic and sequence type distribution of co-resistance among uropathogenic E. coli (N = 155). The p values were calculated by comparing total number of MDR producers and ESBL producers FQR MDR

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.
Table 3

Chi-squareddistribution of ESBL factors and antibiotic resistance in different ST-types

No (%) of the isolates

ST131; n(%)

Other STs; n (%)

Resistance traits

All isolates (n = 155)

All ST131 (n = 71)

Non H30 (n = 35)

H30 (n = 36)

ST405 (n = 28)

ST168 (n = 16)

ST29 (n = 13)

ST69 (n = 5)

ST95 (n = 2)

ST31 (n = 2)

ST10 (n = 2)

ST448 (n = 2)

ST89 (n = 2)

ST703 (n = 2)

ST910 (n = 1)

ST545 (n = 1)

ST971 (n = 1)

ST153 (n = 1)

ST152 (n = 1)

ST12 (n = 1)

ST838 (n = 1)

NSC (n = 3)

ESBL phenotypes

65(42)

30(42)

15(43)

15(42)

13(46)

8(50)

7(54)

2(40)

00

00

1(50)

00

1(50)

00

00

00

00

00

1(100)

00

00

2(67)

blaCTX-M-15

57(39)

27(38)

12(34)

15(42)

11(39)

8(50)

6(46)

2(40)

00

00

1(50)

00

00

00

00

00

00

00

00

00

00

2(67)

blaTEM

23(15)

8(11)

5(14)

3(8)

5(18)

5(31)

3(23)

2(40)

00

00

1(50)

00

1(50)

00

00

00

00

00

1(100)

00

00

00

blaSHV

6(4)

3(4)

1(3)

2(6)

1(4)

2(13)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

blaOXA

10(6)

6(8)

3(9)

3(8)

3(11)

1(6)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

blaPSE

1(0.6)

00

00

00

00

00

00

00

00

00

00

00

00

00

1(100)

00

00

00

00

00

00

00

Piperacillin tazobactam

7(5)

3(4)

2(6)

0(0)

3(11)

2(13)

00

00

1(50)

00

00

00

00

00

00

00

00

00

00

00

00

00

Ceftazidime

96(62)

43(61)

20(57)7

23(64)

20(71)

10(63)

9(69)

5(100)

1(50)

00

00

00

1(50)

2(100)

1(100)

00

00

00

00

1(100)

00

00

Cefotaxime

101(65)

46(65)

22(63)

24(67)

20(71)

10(63)

9(69)

5(100)

2(100)

00

1(50)

1(50)

1(50)

2(100)

1(100)

00

00

00

1(100)

00

00

00

Ceftriaxone

99(64)

46(65)

25(71)

21(58)

21(75)

10(63)

9(69)

2(40)

1(50)

00

00

1(50)

1(50)

1(50)

1(100)

1(100)

00

00

1(100)

1(100)

1(100)

2(67)

Ciprofloxacin

95(61)

45(63)

23(66)

22(61)

18(64)

13(81)

6(46)

4(80)

2(100)

1(50)

1(50)

1(50)

00

2(100)

00

00

00

00

00

00

00

2(67)

Levofloxacin

97(63)

45(63)

24(69)

21(58)

19(68)

14*(88)

6(46)

4(80)

2(100)

1(50)

2(100)

00

2(100)

00

00

00

00

00

00

00

00

2(67)

Amikacin

7(5)

4(6)

2(6)

2(6)

00

1(6)

1(8)

1(20)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Gentamicin

47(30)

21(30)

11(31)

10(28)

10(36)

5(31)

3(23)

1(20)

2(100)

00

1(50)

1(50)

00

2(100)

00

00

00

00

00

00

00

1(33)

Amoxicillin-clavulanic acid

111(72)

48(68)

25(71)

23(64)

22(79)

10(63)

9(69)

2(40)

00

00

00

1(50)

1(50)

1(50)

1(100)

1(100)

00

00

1(100)

1(100)

1(100)

2(67)

Trimethoprim sulfonamides

130(84)

61(86)

30(86)

31(86)

24(86)

13(81)

10(77)

4(80)

2(100)

1(50)

1(50)

1(50)

2(100)

2(100)

1(100)

1(100)

1(100)

1(100)

1(100)

1(100)

00

3(100)

Nitrofurantoin

9(6)

9(13)***

6(17)***

3(8)

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

00

Fosfomycin

15(10)

9(13)

3(9)

6(17)

00

2(13)

00

00

00

1(50)

00

1(50)

1(50)

00

00

00

00

00

1(100)

00

00

00

Distribution of antibiotic resistance of uropathogenic E. coli (n = 155) among different sequence types. The p values were calculated by comparing individual STswith each other. The table correlates different traits in vertical columns among different sequence types. The percentages were calculated with reference to total number of sequence types

*P < 0.05, *** P ≤ 0.001

Occurrence of β-lactamases among ST131

Overall, occurrence of ESBL was higher among clonal group ST131, constituting 42% of the total ESBL phenotypes (Table 3). Moreover, 78% of the ESBL phenotypes showed resistance to at least one fluoroquinolone and 95% were resistant to at least one cephalosporin. The occurrence of ESBL genes remained as follows, bla-CTX-M-15, 57(39%), bla-TEM 23(15%) and bla-SHV 6(4%). Prevalence of other β-lactamases genes such as bla-OXA and bla-PSE 1 remained 6 and 0.6% respectively. In comparison to other sequence types, overall prevalence of β-lactamases genes was higher among ST131 strains, 27(38%) of the bla-CTX-M-15, followed by bla-TEM 8(11%), bla-SHV 3(4%) and bla-OXA 6(8%). Presence of bla-CTX-M-15 was highest (100%) among ST131 H30-O25b and 91% of the bla-CTX-M-15 positive ST131 H30-O25b isolates were resistant to fluoroquinolones (Data not shown). ESBL producing isolates were frequently found resistant to ceftazidime, cefotaxime, ceftriaxone, ciprofloxacin, levofloxacin, amoxicillin-clavulanic acid and trimethoprim sulfonamides (Table 4).
Table 4

Distribution of drug resistance and VF genes among ESBL producers and non ESBLUPEC

Numbers and percentages of the isolates andtheir respective traits

Resistance traits

All isolates (n = 155); n(%)

Non ESBL producers (n = 90) n(%)

ESBL producers (n = 65) n(%)

p value

Piperacillin tazobactam

7(5)

4(4)

3(5)

> 0.9999

Ceftazidime

96(62)

36(40)

60(92)

< 0.0001

Cefotaxime

101(65)

40(44)

61(94)

< 0.0001

Ceftriaxone

99(64)

49(54)

50(77)

0.0040

Ciprofloxacin

95(61)

45(50)

50(77)

0.000684

Levofloxacin

97(63)

48(53)

49(75)

0.0051

Amikacin

7(5)

4(4)

3(5)

0.9597

Gentamicin

47(30)

30(33)

17(26)

0.3373

Amoxicillin-clavulanic acid

111(72)

50(56)

61(94)

< 0.0001

Trimethoprim sulfonamides

130(84)

67(74)

63(97)

0.0002

Nitrofurantoin

9(6)

6(7)

3(5)

0.5900

Fosfomycin

15(10)

8(9)

7(11)

0.6960

fimH

155 (100)

90 (100)

65 (100)

> 0.9999

papA

20 (13)

12 (13)

8 (12)

0.8509

papC

75 (48)

42 (47)

33 (51)

0.6140

papEF

37 (24)

18 (20)

19 (29)

0.1834

papGI

6 (4)

3 (3)

3 (5)

0.6831

papGII

70 (45)

33 (37)

37 (57)

0.0124

papGIII

6 (4)

4 (4)

2 (3)

0.6632

sfa/foc

20 (13)

11 (12)

9 (14)

0.7660

Afa

15 (10)

7 (8)

8 (12)

0.3466

bmaE

2 (1)

1 (1)

1 (2)

0.8246

fyuA

37 (24)

20 (22)

17 (26)

0.5710

iutA

86 (55)

45 (50)

41 (63)

0.1060

feoB

76 (49)

40 (44)

36 (55)

0.1788

kpsmtII

40 (26)

19 (21)

21 (32)

0.1160

kpsmtIII

4 (3)

2 (2)

2 (3)

0.4729

Usp

22 (14)

10 (9)

12 (18)

0.1957

hlyA

18 (12)

7 (8)

11 (17)

0.0795

cdtB

11 (7)

6 (7)

5 (8)

0.8062

Distribution of resistance and virulence traits among ESBL and non-ESBL producing uropathogenic E. coli (N = 155). The p values were calculated by comparing different traits among ESBL producer’s and non-ESBL producers

Distribution of VF genes among different sequence types

A total of 18 different virulence factors were scrutinized among 155 isolates. Percntages of VF genes were as follows: fimH,155(100%), iutA86 (55%), feoB 76 (49%), papC 75 (48%), papGII70 (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%). Some virulence factors such as sfa/foc, fyuA and feoB were detected frequently among ST131 isolates whereas VF-genes papEF, sfa/focand hlyA were 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 ST131 strains, while papEF had significant presence among clonal group ST131-H30.
Table 5

Chi-squared distribution of virulence factor genes in different ST-types

Number of the isolates with traits n(%)

Traits

Total n = 155

ST-131 n = 71

NonH30 n = 35

H30 n = 36

ST-05 n = 28

ST-168 n = 16

ST-29 n = 13

ST-69 n = 5

ST-95

n = 2

ST-31 n = 2

ST-10 n = 2

ST-448 n = 2

ST-89 n = 2

ST-703 n = 2

ST-910 n = 1

ST-545 n = 1

ST-971 n = 1

ST-153 n = 1

ST-152 n = 1

ST-12 n = 1

ST-838 n = 1

NSC n = 3

fimH

155 (100)

71 (100)

35 (100)

36 (100)

28 (100)

16 (100)

13 (100)

5 (100)

2 (100)

2 (100)

2 (100)

2 (100)

2 (100)

2 (100)

1 (100)

1 (100)

1 (100)

1 (100)

1 (100)

1 (100)

1 (100)

3 (100)

papA

20 (13)

12 (17)

5 (14)

7 (19)

4 (14)

1 (6)

0

0

0

0

0

1 (50)

0

0

0

0

0

1(100)

0

0

0

1 (33)

papC

75 (48)

33 (46)

17 (49)

16 (44)

15 (54)

9 (56)

5 (38)

3 (60)

1 (50)

1 (50)

0

0

1 (50)

1 (50)

1 (100)

0

1 (100)

1 (100)

0

0

1 (100)

2 (67)

papEF

37 (24)

20 (28)

7 (20)

13* (36)

7 (25)

3 (19)

3 (23)

1 (20)

0

0

0

1 (50)

0

0

0

1 (100)

1 (100)

0

0

0

0

1 (33)

papGI

6 (4)

2 (3)

0

2 (6)

1 (4)

1 (6)

0

1 (20)

0

0

0

0

0

0

0

0

0

0

0

0

0

1 (33)

papGII

70 (45)

32 (45)

17 (49)

15 (42)

12 (43)

6 (38)

4 (31)

4 (80)

2 (100)

0

2 (100)

1 (50)

2 (100)

1 (50)

0

1 (100)

0

0

0

1 (100)

0

2 (67)

papGIII

6 (4)

4 (6)

1 (3)

3 (8)

1 (4)

1 (6)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

sfa/foc

20 (13)

13* (18)

4 (11)

9* (25)

2 (7)

1 (6)

1 (8)

0

1 (50)

0

1 (50)

0

0

0

0

0

0

0

0

0

0

1 (33)

afa

15 (10)

7 (10)

4 (11)

3 (8)

6*(21)

1 (6)

1 (8)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

bmaE

2 (1)

1 (1)

1 (3)

0

0

1 (6)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

fyuA

37 (24)

12*(17)

7 (20)

5 (14)

10 (36)

6 (38)

2 (15)

2 (40)

0

0

2(100)

0

0

0

0

0

0

0

1 (100)

1 (100)

0

1 (33)

iutA

86 (55)

41 (58)

22*** (63)

19 (53)

14 (50)

10 (63)

5 (38)

3 (60)

2 (100)

1 (50)

2 (100)

1 (50)

1 (50)

1 (50)

1 (100)

0

1 (100)

0

1 (100)

0

0

2 (67)

feoB

76 (49)

28* (39)

13 (37)

15 (42)

12 (43)

10 (63)

6 (46)

4 (80)

2 (100)

1 (50)

2 (100)

1 (50)

2 (100)

2 (100)

1 (100)

0

1 (100)

0

1 (100)

1 (100)

0

2 (67)

kpsmtII

40 (26)

20 (28)

9 (26)

11 (31)

8 (29)

2 (13)

2 (15)

2 (40)

0

0

0

1 (50)

0

0

0

0

0

0

1 (100)

1 (100)

1 (100)

2 (67)

kpsmtIII

4 (3)

2 (3)

0

2 (6)

0

0

0

1 (20)

0

0

0

0

0

0

0

1 (100)

0

0

0

0

0

0

usp

22 (14)

13 (18)

5 (14)

8 (22)

2 (7)

3 (19)

1 (8)

0

0

0

1 (50)

0

0

0

0

1(100)

0

0

0

0

0

1 (33)

hlyA

18 (12)

8 (11)

1 (3)

7* (32)

5 (18)

1 (6)

1 (8)

0

1 (50)

0

0

0

0

0

0

0

0

0

0

1 (100)

0

1 (33)

cdtB

11 (7)

4 (6)

1 (3)

3 (8)

4 (14)

1 (6)

0

0

1(50)

0

0

0

0

0

0

0

0

0

0

0

0

1 (33)

Distribution of virulence traits of uropathogenic E. coli (n = 155) among different sequence types. The p values were calculated by comparing individual STs with each other. The table correlates different traits in vertical columns among different sequence types. The percentages were calculated with reference to total number of sequence types

*P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Discussion

E. coli ST131 was reported from three different continents [8]. However, recently it has become the most predominant lineage associated with variety of infections around the globe. ST131 strains have a tendency to harbor ESBL enzymes bla-CTX-M-15, which play a significant role in mounting resistance against  β-lactam class of antibiotics [8]. Moreover, ST131 strains show remarkable resistance to the fluoroquinolones and demonstrate greater abilities to adhere bladder, kidneys and epithelial cells [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 higher morbidity and mortality [5]. Due to their resistance against variety of antibiotics, including β-lactams, aminoglycosides and fluoroquinolones, infections caused by pandemic clonal group ST131 are particularly 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. All the isolates belonging to the sub-group ST131-H30-O25b carried ESBL bla-CTX-M-15 however, overall prevalence of these particular strains constituted only 10% of the total isolates. Resistance against fluoroquinolones in ST131 strains remained 60%, that was remarkably higher. For the treatment of UTIs commonly prescribed antibiotics include sulphamethoxazole-trimethoprim and fluoroquinolones. However, due to the emerging resistance to these antibiotics alternative therapeutic choices such as nitrofurantoin, fosfomycin and β-lactam inhibitors can be prescribed.

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 fluoroquinolones-resistant 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 stewardship – are needed. As antibiotic resistance can be transferred from UPEC to other pathogens, more judicious use of antibiotics is required.

Notes

Acknowledgements

We thank Prof. Betsy Foxman, Hunein F. and Hilda Maassab Endowed Professor of Epidemiology Director, Center for Molecular and Clinical Epidemiology of Infectious Diseases at University of Michigan, USA for supporting this work.

Authors’ contributions

DIJ and FB designed and supervised the study. AI and RZ conducted the bench work and assembled the data. TF helped in MIC determination. AI, GES, and ES helped in statistical and bioinformatics analysis. DIJ did analysis, interpretation and drafted the manuscript. All authors have read, contributed and approved the final manuscript.

Funding

Higher Education Commission Pakistan provided 6 month stipend and bench fee for the doctoral work of Ihsan Ali at Center for Molecular and Clinical Epidemiology of Infectious Diseases at University of Michigan, USA which enabled us to perform ST typing of strains (International Research Support Initiative Program IRSIP/2016). Work of Zara Rafaque was funded by HEC indigenous PhD scholarships covering monthly stipend University fee and lab reagents that helped us to screen VF and ESBL genes (HEC-213-53961-2BM2–093). Higher Education Commission Pakistan played no direct role in collection, analysis, interpretation and publication of this work.

Ethics approval and consent to participate

Ethical Review Board (ERB) of Pakistan Institute of Medical Sciences approved this study. Ethical Review Board approved verbal consent taken from all the patients.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

  1. 1.
    Dehbanipour R, Rastaghi S, Sedighi M, Maleki N, Faghri J. High prevalence of multidrug-resistance uropathogenic Escherichia coli strains, Isfahan, Iran. J Nat Sci Biol Med. 2016;7(1):22.CrossRefGoogle Scholar
  2. 2.
    Mittal S, Sharma M, Chaudhary U. Biofilm and multidrug resistance in uropathogenic Escherichia coli. Pathog Glob Health. 2015;109(1):26–9.CrossRefGoogle Scholar
  3. 3.
    Tanvir R, Hafeez R, Hasnain S. Prevalence of multiple drug resistant Escherichia coli in patients of urinary tract infection registering at a diagnostic laboratory in Lahore Pakistan. Pak J Zool. 2012;44(3):707–12.Google Scholar
  4. 4.
    Johnson JR, Tchesnokova V, Johnston B, Clabots C, Roberts PL, Billig M, Riddell K, Rogers P, Qin X, Butler-Wu S. Abrupt emergence of a single dominant multidrug-resistant strain of Escherichia coli. J Infect Dis. 2013;207(6):919–28.CrossRefGoogle Scholar
  5. 5.
    Petty NK, Zakour NLB, Stanton-Cook M, Skippington E, Totsika M, Forde BM, Phan M-D, Moriel DG, Peters KM, Davies M. Global dissemination of a multidrug resistant Escherichia coli clone. Proc Natl Acad Sci U S A. 2014;111(15):5694–9.CrossRefGoogle Scholar
  6. 6.
    Shahzad N, Aslam B, Hussain I, Ijaz M, Rasool MH, Tasneem F, Hamid T, Tayyeb A, Hussain T. Distribution and Phylogenetic Analysis of Bacterial Isolates from Urinary Tract Infection Patients of Pakistan. Pak J Zool. 2016;48(6):1925–30.Google Scholar
  7. 7.
    Johnson JR, Johnston B, Clabots C, Kuskowski MA, Castanheira M. Escherichia coli sequence type ST131 as the major cause of serious multidrug-resistant E. coli infections in the United States. Clin Infect Dis. 2010;51(3):286–94.CrossRefGoogle Scholar
  8. 8.
    Nicolas-Chanoine M-H, Bertrand X, Madec J-Y. Escherichia coli ST131, an intriguing clonal group. Clin Microbiol Rev. 2014;27(3):543–74.CrossRefGoogle Scholar
  9. 9.
    Johnson JR, Johnston B, Thuras P, Launer B, Sokurenko EV, Miller LG. Escherichia coli Sequence Type 131 H30 Is the Main Driver of Emerging Extended-Spectrum-β-Lactamase-Producing E. coli at a Tertiary Care Center. mSphere. 2016;1(6):e00314–6.PubMedPubMedCentralGoogle Scholar
  10. 10.
    Johnson JR, Menard M, Johnston B, Kuskowski MA, Nichol K, Zhanel GG. Epidemic clonal groups of Escherichia coli as a cause of antimicrobial-resistant urinary tract infections in Canada, 2002 to 2004. Antimicrob Agents Chemother. 2009;53(7):2733–9.CrossRefGoogle Scholar
  11. 11.
    Cagnacci S, Gualco L, Debbia E, Schito GC, Marchese A. European emergence of ciprofloxacin-resistant Escherichia coli clonal groups O25: H4-ST 131 and O15: K52: H1 causing community-acquired uncomplicated cystitis. J Clin Microbiol. 2008;46(8):2605–12.CrossRefGoogle Scholar
  12. 12.
    Hefzy EM, Hassuna NA. Fluoroquinolone-resistant sequence type 131 subgroups O25b and O16 among Extraintestinal Escherichia coli isolates from community-acquired urinary tract infections. Microb Drug Resist. 2017;23(2):224–9.CrossRefGoogle Scholar
  13. 13.
    Clinical and Laboratory Standards Institute, 2014. Performance standards for antimicrobial susceptibility testing: 24th informational supplement. . Document M100–S24.Google Scholar
  14. 14.
    Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of theEscherichia coli phylogenetic group. Appl Environ Microbiol. 2000;66(10):4555–8.CrossRefGoogle Scholar
  15. 15.
    Weissman SJ, Johnson JR, Tchesnokova V, Billig M, Dykhuizen D, Riddell K, Rogers P, Qin X, Butler-Wu S, Cookson BT. High-resolution two-locus clonal typing of extraintestinal pathogenic Escherichia coli. Appl Environ Microbiol. 2012;78(5):1353–60.CrossRefGoogle Scholar
  16. 16.
    Tchesnokova V, Billig M, Chattopadhyay S, Linardopoulou E, Aprikian P, Roberts PL, Skrivankova V, Johnston B, Gileva A, Igusheva I. Predictive diagnostics for Escherichia coli infections based on the clonal association of antimicrobial resistance and clinical outcome. J Clin Microbiol. 2013;51(9):2991–9.CrossRefGoogle Scholar
  17. 17.
    Clermont O, Dhanji H, Upton M, Gibreel T, Fox A, Boyd D, Mulvey MR, Nordmann P, Ruppé E, Sarthou JL. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J Antimicrob Chemother. 2009;64(2):274–7.CrossRefGoogle Scholar
  18. 18.
    del Castillo BR, Vinué L, Román EJ, Guerra B, Carattoli A, Torres C, Martínez-Martínez L. Molecular characterization of multiresistant Escherichia coli producing or not extended-spectrum β-lactamases. BMC Microbiol. 2013;13(1):84.CrossRefGoogle Scholar
  19. 19.
    Bush KTP JG: CTX-M-type Beta-lactamases. Lahey Clinic. 2015.Google Scholar
  20. 20.
    Yun KW, Kim HY, Park HK, Kim W, Lim IS. Virulence factors of uropathogenic Escherichia coli of urinary tract infections and asymptomatic bacteriuria in children. J Microbiol Immunol Infect. 2014;47(6):455–61.CrossRefGoogle Scholar
  21. 21.
    Alghoribi MF, Gibreel TM, Farnham G, Al Johani SM, Balkhy HH, Upton M. Antibiotic-resistant ST38, ST131 and ST405 strains are the leading uropathogenic Escherichia coli clones in Riyadh, Saudi Arabia. J Antimicrob Chemother. 2015;70(10):2757–62.CrossRefGoogle Scholar
  22. 22.
    Peirano G, Pitout JD. Molecular epidemiology of Escherichia coli producing CTX-M β-lactamases: the worldwide emergence of clone ST131 O25: H4. Int J Antimicrob Agents. 2010;35(4):316–21.CrossRefGoogle Scholar
  23. 23.
    Banerjee R, Robicsek A, Kuskowski MA, Porter S, Johnston BD, Sokurenko E, Tchesnokova V, Price LB, Johnson JR. Molecular epidemiology of Escherichia coli sequence type 131 and its H30 and H30-Rx subclones among extended-spectrum-β-lactamase-positive and-negative E. coli clinical isolates from the Chicago region, 2007 to 2010. Antimicrob Agents Chemother. 2013;57(12):6385–8.CrossRefGoogle Scholar
  24. 24.
    Lau SH, Reddy S, Cheesbrough J, Bolton FJ, Willshaw G, Cheasty T, Fox AJ, Upton M. Major uropathogenic Escherichia coli strain isolated in the northwest of England identified by multilocus sequence typing. J Clin Microbiol. 2008;46(3):1076–80.CrossRefGoogle Scholar
  25. 25.
    Johnson JR, Clermont O, Johnston B, Clabots C, Tchesnokova V, Sokurenko E, Junka AF, Maczynska B, Denamur E. Rapid and specific detection, molecular epidemiology, and experimental virulence of the O16 subgroup within Escherichia coli sequence type 131. J Clin Microbiol. 2014;52(5):1358–65.CrossRefGoogle Scholar
  26. 26.
    Nicolle L. Management of asymptomatic UTIs in women. Medscape Womens Health. 1996;1(3):4–4.PubMedGoogle Scholar
  27. 27.
    Hooton TM. Fluoroquinolones and resistance in the treatment of uncomplicated urinary tract infection. Int J Antimicrob Agents. 2003;22:65–72.CrossRefGoogle Scholar
  28. 28.
    Bashir S, Haque A, Sarwar Y, Ali A, Anwar MI. Virulence profile of different phylogenetic groups of locally isolated community acquired uropathogenic E. coli from Faisalabad region of Pakistan. Ann Clin Microbiol Antimicrob. 2012;11(1):23.CrossRefGoogle Scholar
  29. 29.
    Nowrouzian F, Östblom A, Wold A, Adlerberth I. Phylogenetic group B2 Escherichia coli strains from the bowel microbiota of Pakistani infants carry few virulence genes and lack the capacity for long-term persistence. Clin Microbiol Infect. 2009;15(5):466–72.CrossRefGoogle Scholar
  30. 30.
    Abdi HA, Rashki A. Comparison of virulence factors distribution in uropathogenic E. coli isolates from phylogenetic groups B2 and D. Int J Enteric Pathog. 2014;2(4):1–5.CrossRefGoogle Scholar
  31. 31.
    Er DK, Dundar D, Uzuner H, Osmani A. Relationship between phylogenetic groups, antibiotic resistance and patient characteristics in terms of adhesin genes in cystitis and pyelonephritis isolates of Escherichia coli. Microb Pathog. 2015;89:188–94.CrossRefGoogle Scholar
  32. 32.
    Johnson JR. papG alleles among Escherichia coli strains causing urosepsis: associations with other bacterial characteristics and host compromise. Infect Immun. 1998;66(9):4568–71.PubMedPubMedCentralGoogle Scholar
  33. 33.
    Johnson JR, Kuskowski MA, Gajewski A, Soto S, Horcajada JP, de Anta MTJ, Vila J. Extended virulence genotypes and phylogenetic background of Escherichia coli isolates from patients with cystitis, pyelonephritis, or prostatitis. The J Infect Dis. 2005;191(1):46–50.CrossRefGoogle Scholar
  34. 34.
    Otto G, Sandberg T, Marklund B-I, Ulleryd P, Svanborg C. Virulence factors and pap genotype in Escherichia coli isolates from women with acute pyelonephritis, with or without bacteremia. Clin Infect Dis. 1993;17(3):448–56.CrossRefGoogle Scholar
  35. 35.
    Foxman B, Brown P. Epidemiology of urinary tract infections: transmission and risk factors, incidence, and costs. Infect Dis Clin N Am. 2003;17(2):227–41.CrossRefGoogle Scholar
  36. 36.
    Gur C, Coppenhagen-Glazer S, Rosenberg S, Yamin R, Enk J, Glasner A, Bar-On Y, Fleissig O, Naor R, Abed J. Natural killer cell-mediated host defense against uropathogenic E. coli is counteracted by bacterial hemolysinA-dependent killing of NK cells. Cell Host Microbe. 2013;14(6):664–74.CrossRefGoogle Scholar
  37. 37.
    Hagan EC, Lloyd AL, Rasko DA, Faerber GJ, Mobley HL. Escherichia coli global gene expression in urine from women with urinary tract infection. PLoS Pathog. 2010;6(11):e1001187.CrossRefGoogle Scholar
  38. 38.
    Takahashi A, Kanamaru S, Kurazono H, Kunishima Y, Tsukamoto T, Ogawa O, Yamamoto S. Escherichia coli isolates associated with uncomplicated and complicated cystitis and asymptomatic bacteriuria possess similar phylogenies, virulence genes, and O-serogroup profiles. J Clin Microbiol. 2006;44(12):4589–92.CrossRefGoogle Scholar

Copyright information

© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Ihsan Ali
    • 5
  • Zara Rafaque
    • 1
  • Ibrar Ahmed
    • 2
  • Faiza Tariq
    • 1
  • Sarah E. Graham
    • 3
  • Elizabeth Salzman
    • 4
  • Betsy Foxman
    • 4
  • Javid Iqbal Dasti
    • 1
    Email author
  1. 1.Department of Microbiology, Faculty of Biological SciencesQuaid-i-Azam UniversityIslamabadPakistan
  2. 2. Alpha Genomics (Pvt) LtdIslamabadPakistan
  3. 3.Department of BiophysicsUniversity of MichiganAnn ArborUSA
  4. 4.Department of Epidemiology, School of Public HealthUniversity of MichiganAnn ArborUSA
  5. 5.Department of Medical Laboratory Technology (MLT)the University of HaripurAbbottabadPakistan

Personalised recommendations