Abstract

Objectives

To evaluate the current prevalence of the three clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69 (where ST stands for sequence type) among Escherichia coli isolates causing extraintestinal infections in Spain and to characterize their virulence background, 500 consecutive non-duplicate E. coli isolates causing extraintestinal infections were analysed.

Methods

The 500 isolates were collected during February 2009 from five hospitals in different Spanish regions. Phylogenetic groups, STs, serotypes, virulence genes, PFGE profiles, antimicrobial resistance and extended-spectrum β-lactamase (ESBL) enzymes were determined.

Results

The three clonal groups accounted for 19% of the 500 isolates. Furthermore, they accounted for 37% of the isolates exhibiting trimethoprim/sulfamethoxazole plus ciprofloxacin resistance, 34% of aminoglycoside-resistant isolates and 30% of multidrug-resistant isolates. Clonal group ST131 was the most prevalent, and accounted for 12% of isolates overall and for 23% of multidrug-resistant isolates. The ST131 isolates exhibited a significantly higher virulence score (mean of virulence genes 8.1) compared with the ST393 (6.0) and ST69 (5.4) isolates. The prevalence of ESBL-producing isolates was 7%. Six (10%) of the 59 ST131 isolates were positive for CTX-M-15 and one (6%) of the 16 ST393 isolates was positive for CTX-M-14, whereas none of the 22 ST69 isolates produced ESBL enzymes.

Conclusions

The three clonal groups investigated accounted for 30% of the multidrug-resistant isolates, which gives evidence of an important clonal component in the emergence of resistances among extraintestinal pathogenic E. coli. Notably, a single high virulence clonal group (O25b:H4-B2-ST131) causes approximately 1 in every 10 extraintestinal infections in Spain, representing an important public health threat. A new variant of the ST131 clonal group, which is non-ESBL-producing but trimethoprim/sulfamethoxazole resistant and with high virulence content, is reported.

Introduction

The intercontinental Escherichia coli clonal group producing CTX-M-15 with a high virulence potential, O25b:H4-B2-ST131 (where ST stands for sequence type), has been reported recently all over the world, representing a major public health problem.1,2 This clonal group belongs to the B2 phylogenetic group, to the serotype O25b:H4 and to the multilocus ST131. The O25b:H4-B2-ST131 clonal group is characterized by co-resistance to several classes of antibiotics and is able to acquire different mechanisms of resistance. Although commonly associated with the dissemination of CTX-M-15 extended-spectrum cephalosporin resistance, E. coli O25b:H4-B2-ST131 also occurs as a fluoroquinolone-resistant but cephalosporin-susceptible pathogen.3–12

Since the outbreak of 1986 in London, numerous studies have reported evidence of the continued clinical importance and global distribution of another fluoroquinolone-resistant clonal group that belongs to the D phylogenetic group, to the serotype O15:H1 and to the multilocus ST393.4,6,13,14 Olesen et al.15 recently published an epidemiological analysis with the conclusion that the O15:H1-D-ST393 clonal group has maintained a fairly stable virulence profile since its first known appearance, but, in contrast, its antimicrobial resistance profile has become progressively more extensive. Similarly, Mora et al.16 recently reported the emergence of CTX-M-14-producing isolates of this clonal group in Spain.

Clonal group A (CGA) belongs to the D phylogenetic group and to the multilocus ST69, and the isolates exhibit distinctive O groups (O11, O17, O73 and O77). CGA-D-ST69 accounted for up to 50% of trimethoprim/sulfamethoxazole-resistant urinary tract infections (UTIs) due to E. coli in the USA in the 1990s and also has a worldwide distribution.6,17–20

Despite the clinical importance and worldwide distribution of these three drug-resistant clonal groups, as far as we know, they have been screened in only one study carried out in Canada from 2002 to 2004.6 For this reason, the present study was carried out to evaluate the current prevalence of these three clonal groups among E. coli isolates causing extraintestinal infections in Spain and to understand their contribution to the emergence of antimicrobial resistance. This type of study has a special interest due to the dramatic increase in ciprofloxacin and trimethoprim/sulfamethoxazole resistances observed in Spain during recent years.21,22

Materials and methods

Bacterial isolates

Five hospitals in different Spanish regions (Lugo, LU; Barcelona, BA; Santander, SA; Madrid, MA; and Seville, SE) participated in the present study. In the study period (February 2009), 100 consecutive non-duplicate clinically relevant E. coli isolates were obtained in each hospital from ambulatory (71%) and hospital-admitted (29%) patients. Clinical sources included urine (421 isolates), blood (25 isolates), surgical wounds (14 isolates), respiratory tract samples (5 isolates), bile (5 isolates) and other sources (30 isolates).

Antimicrobial susceptibility and extended-spectrum β-lactamase (ESBL) typing

Susceptibility to antibiotics was analysed by broth microdilution and/or disc diffusion. Minimal inhibitory concentrations were determined using a MicroScan WalkAway automated system (Siemens, Madrid, Spain) according to the manufacturer's instructions. Intermediate susceptibility was not considered as resistant. Resistance was interpreted based on the recommended breakpoints of the CLSI.23 Multidrug-resistant isolates were those resistant to at least one representative of three or more antimicrobial classes, including fluoroquinolones (ciprofloxacin), trimethoprim/sulfamethoxazole, aminoglycosides (gentamicin and tobramycin), β-lactam/β-lactamase inhibitors (amoxicillin/clavulanic acid and piperacillin/tazobactam) and extended-spectrum cephalosporins (cefotaxime, ceftazidime and cefepime).

Suggestive evidence of ESBL production was defined as synergy between amoxicillin/clavulanate and at least one of cefotaxime, ceftazidime, aztreonam or cefepime. To determine the genotype of the ESBLs, PCR was performed using the TEM, SHV, CTX-M-1 and CTX-M-9 group-specific primers, as reported previously.7,24 Sequencing of both strands of amplicons was performed using specific primers.

Detection of clonal groups

For detection of the three drug-resistant clonal groups, all 500 isolates were screened by PCR for the O25b rfb variant (O25b:H4-B2-ST131 associated),25 for the O15 rfb allele and a single nucleotide polymorphism (SNP) in fumC specific for CC31 (where CC stands for clonal complex) (O15:H1-D-ST393 associated),6,26 and for an SNP in fumC and gyrB specific for CC69 (CGA-D-ST69 associated).6,27

Phylogenetic grouping and multilocus sequence typing (MLST)

To confirm the presumptive clonal group assignments, all isolates of the three clonal groups detected by the PCR screening were analysed for their phylogenetic group (A, B1, B2 and D) by the multiplex PCR-based method of Clermont et al.28 and MLST. MLST was achieved as previously described by gene amplification and sequencing of the seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) according to the protocol and primers specified at the E. coli MLST web site (http://mlst.ucc.ie/mlst/dbs/Ecoli). The allelic profile of the seven gene sequences and the STs were obtained via the electronic database at the E. coli MLST web site.29

O and H typing

Determination of O and H antigens was carried out using the method previously described by Guinée et al.30 with all available O (O1–O181) and H (H1–H56) antisera. Isolates that did not react with O and H antisera were classified as non-typeable (ONT and HNT, respectively), and those non-motile were denoted as HNM. Additionally, the specific O25a and O25b molecular subtypes were determined by PCR.25

Virulence factors

The presence of 30 virulence genes was analysed as documented previously,31–33 using primers specific for genes and operons that encode extraintestinal virulence factors characteristic of extraintestinal pathogenic E. coli (ExPEC), i.e. fimH, fimAvMT78, papEF (positive results were tested for papG I, papG II, papG III and papG IV alleles), sfa/focDE, afa/draBC (positive results were tested for afa operon FM955459), bmaE, gafD, cnf1, cdtB, sat, hlyA, iucD, iroN, kpsM II (establishing neuC-K1, -K2 and -K5 variants), kpsM III, cvaC, iss, traT, ibeA, malX, usp and tsh.

PFGE

XbaI-PFGE analysis was performed as previously described.31 Profiles were analysed with the BioNumerics fingerprinting software (Applied Maths, St-Martens-Latem, Belgium). Dendrograms were generated by the unweighted pair-group method using arithmetic averages, based on the Dice similarity coefficient with a 1.0% band position tolerance.

Statistical analysis

Comparisons of proportions and scores (continuous variables) were tested using Fisher's exact test and the Mann–Whitney U-test, respectively. For each comparison, a P value of <0.05 was considered to denote significant differences.

Results and discussion

Prevalence of clonal groups

According to molecular typing, the clonal group O25b:H4-B2-ST131 accounted for 59 (12%) of the 500 study isolates, the clonal group O15:H1-D-ST393 accounted for 16 (3%) and CGA-D-ST69 accounted for 22 (4%). Thus, the three clonal groups collectively accounted for 19% of the total isolates analysed. Hospital Vall d'Hebron in Barcelona city exhibited the highest prevalence (30%) of the three clonal groups. ST131 and ST69 occurred in all five hospitals, and ST393 in four. ST131 was significantly more prevalent than either of the other two groups in the five hospitals (P < 0.001 for each comparison and collectively) (Table 1). The three clonal groups were detected with a similar prevalence among isolates obtained from ambulatory and hospital-admitted patients: ST131 (12% versus 12%, respectively); ST393 (3% versus 4%, respectively); and ST69 (4% versus 4%, respectively).

Table 1.

Prevalence of the three clonal groups among 500 E. coli isolates from five hospitals

Hospital City Total isolates Prevalence of clonal groups, number of isolates
 
O25b:H4-B2-ST131 O15:H1-D-ST393 CGA-D-ST69 
Hospital Lucus Augustia Lugo 100 
Hospital Vall d'Hebron Barcelona 100 16 
Marqués de Valdecilla Santander 100 
12 de Octubre Madrid 100 15 
Virgen Macarena Seville 100 11 
Total  500 59 (12%) 16 (3%) 22 (4%) 
Hospital City Total isolates Prevalence of clonal groups, number of isolates
 
O25b:H4-B2-ST131 O15:H1-D-ST393 CGA-D-ST69 
Hospital Lucus Augustia Lugo 100 
Hospital Vall d'Hebron Barcelona 100 16 
Marqués de Valdecilla Santander 100 
12 de Octubre Madrid 100 15 
Virgen Macarena Seville 100 11 
Total  500 59 (12%) 16 (3%) 22 (4%) 

aPreviously named Complexo Hospitalario Xeral-Calde.7

In Canada during 2002–04, Johnson et al.6 found that the three clonal groups contributed substantially to the study population (37%); however, in the Canadian study four subsets of ∼50 isolates (all from UTIs) were selected according to combined trimethoprim/sulfamethoxazole and fluoroquinolone phenotypes, and so it is difficult to make a detailed comparison with our results. Nevertheless, E. coli ST131, like in Spain, was the most prevalent clonal group in Canada (23%). In another study including mostly invasive isolates, Johnson et al.8 estimated that ST131 caused 17% of E. coli infections in patients hospitalized across the USA in 2007.

Distribution of the three clonal groups by resistance subgroup

In the present study, the three clonal groups accounted for 37% of the isolates exhibiting trimethoprim/sulfamethoxazole plus ciprofloxacin resistance, 34% of the aminoglycoside-resistant isolates and 30% of the multidrug-resistant isolates (Table 2). CGA-D-ST69 was concentrated within the trimethoprim/sulfamethoxazole-resistant and ciprofloxacin-susceptible group (10 of 81 isolates, 12%), whereas the O25b:H4-B2-ST131 (23%) and O15:H1-D-ST393 (10%) clonal groups were concentrated within the trimethoprim/sulfamethoxazole-resistant and ciprofloxacin-resistant group. Notably, clonal group ST131 was the most prevalent in all resistance subgroups and accounted for 23% of multidrug-resistant isolates. Similar results were found by Johnson et al.6 in the Canadian study (2002–04). In another study, Johnson et al.8 estimated that clonal group ST131 accounted for >40% of all isolates exhibiting any resistance, >50% of trimethoprim/sulfamethoxazole- plus ciprofloxacin-resistant, aminoglycoside-resistant and multidrug-resistant isolates, and 67%–69% of extended-spectrum cephalosporin-resistant and fluoroquinolone-resistant isolates from patients hospitalized across the USA in 2007. Therefore, although the ST131 clonal group is highly prevalent among Spanish resistant isolates, its frequency in 2009 was significantly lower than that estimated in the USA during 2007. In the US study, however, 73% of isolates were obtained from blood cultures, while in our study blood isolates represented only 5% of the isolates studied. In view of our results, we cannot discount the existence of other successful clonal groups in Spain.

Table 2.

Distribution of the three clonal groups by resistance subgroup

Resistance phenotypesa Total isolates Prevalence of clonal groups, number of isolates (%)
 
O25b:H4-B2-ST131 O15:H1-D-ST393 CGA-D-ST69 Any 
Trimethoprim/sulfamethoxazole 174 31 (18%) 10 (6%) 14 (8%) 55 (32%) 
Fluoroquinolones 155 41 (26%) 14 (9) 4 (3%) 59 (38%) 
Trimethoprim/sulfamethoxazole plus fluoroquinolones 93 21 (23%) 9 (10%) 4 (4%) 34 (37%) 
Aminoglycosides 61 17 (28%) 4 (7%) 21 (34%) 
Amoxicillin/clavulanic acid 39 6 (15%) 6 (15%) 
Extended-spectrum cephalosporins 38 6 (16%) 1 (3%) 7 (19%) 
Any of the above 256 51 (20%) 14 (5%) 14 (5%) 79 (31%) 
Multidrug resistance (three or more of the above drug classes) 61 14 (23%) 1 (2%) 3 (5%) 18 (30%) 
Resistance phenotypesa Total isolates Prevalence of clonal groups, number of isolates (%)
 
O25b:H4-B2-ST131 O15:H1-D-ST393 CGA-D-ST69 Any 
Trimethoprim/sulfamethoxazole 174 31 (18%) 10 (6%) 14 (8%) 55 (32%) 
Fluoroquinolones 155 41 (26%) 14 (9) 4 (3%) 59 (38%) 
Trimethoprim/sulfamethoxazole plus fluoroquinolones 93 21 (23%) 9 (10%) 4 (4%) 34 (37%) 
Aminoglycosides 61 17 (28%) 4 (7%) 21 (34%) 
Amoxicillin/clavulanic acid 39 6 (15%) 6 (15%) 
Extended-spectrum cephalosporins 38 6 (16%) 1 (3%) 7 (19%) 
Any of the above 256 51 (20%) 14 (5%) 14 (5%) 79 (31%) 
Multidrug resistance (three or more of the above drug classes) 61 14 (23%) 1 (2%) 3 (5%) 18 (30%) 

aFluoroquinolones, resistant to ciprofloxacin; aminoglycosides, resistant to gentamicin, tobramycin and/or amikacin; extended-spectrum cephalosporins, resistant to cefepime, ceftazidime and/or cefotaxime.

In the present study, the prevalence of ESBL-producing isolates was 7% (35 of 500 isolates). This prevalence is higher than that observed in a multicentre study performed in 2006 in Spain (4%).21 Six (10%) of the 59 ST131 isolates were positive for CTX-M-15 (55SA, 167LU, 6MA, 99SE, 85MA and 29BA isolates) and one (6%) of the 16 ST393 isolates was positive for CTX-M-14 (114LU isolate), whereas none of the 22 ST69 isolates produced ESBL enzymes. Thus, in Spain in 2009, the ST131 clone occurred frequently as a fluoroquinolone-resistant but cephalosporin-susceptible pathogen. Cagnacci et al.4 characterized 148 E. coli isolates displaying reduced susceptibility to ciprofloxacin and causing uncomplicated UTIs in eight European countries during 2003 to 2006. About one-third (51 isolates) belonged to two clonal groups (ST131 and ST393), but only 12 (6%) were ESBL positive.

Virulence factors

ExPEC strains have specialized virulence factors that enable them to colonize host surfaces, injure host tissues and avoid host defence systems. The 97 isolates belonging to the clonal groups (O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69) of the present study were analysed by PCR for the presence of 30 genes encoding virulence factors typical of ExPEC that cause UTIs, sepsis and meningitis. Comparisons of the virulence gene prevalence values among the three clonal groups identified significant differences (Table 3).

Table 3.

Virulence genes of the three clonal groups

Virulence genes Clonal groups, number of isolates (%)
 
P valuea
 
O25b:H4-B2-ST131 (n = 59) O15:H1-D-ST393 (n = 16) CGA-D-ST69 (n= 22) ST131 versus ST393 ST131 versus ST69 ST393 versus ST69 
Adhesins 
fimH 58 (98%) 16 (100%) 22 (100%)    
fimAvMT78 16 (100%) <0.001  <0.001 
papEF 15 (25%) 14 (88%) 12 (55%) <0.001 0.011 0.029 
papG I    
papG II 3 (5%) 14 (88%) 8 (36%) <0.001 0.005 <0.001 
papG III 12 (20%) 0.043 0.016  
papG IV 3 (14%)  0.018  
sfa/focDE    
afa/draBC 7 (12%) 1 (5%)    
afa FM955459 4 (7%)    
bmaE 1 (5%)    
gafD 1 (5%)    
Toxins 
cnf1 10 (17%)  0.033  
cdtB 6 (10%)    
sat 39 (66%) 15 (94%) 11 (50%) <0.001  0.004 
hlyA 10 (17%)  0.033  
Siderophores 
iucD 52 (88%) 15 (94%) 19 (86%)    
iroN 22 (37%) 5 (23%) 0.002   
Capsula 
kpsM II 43 (73%) 16 (100%) 11 (50%) 0.013 0.034 0.001 
kpsM II-K2 6 (10%) 1 (5%)    
kpsM II-K5 36 (61%) 16 (100%) 10 (45%) 0.001  <0.001 
neuC-K1 1 (2%)    
kpsM III 6 (27%)  <0.001 0.027 
Miscellaneous 
cvaC 14 (24%) 5 (23%) 0.024   
iss 21 (36%) 5 (23%) 0.002   
traT 51 (86%) 3 (19%) 19 (86%) <0.001  <0.001 
ibeA 16 (27%) 0.013 0.003  
malX (PAI) 58 (98%) <0.001 <0.001  
usp 59 (100%) <0.001 <0.001  
tsh 1 (2%) 2 (9%)    
ExPEC status 38 (64%) 16 (100%) 13 (59%) 0.002  0.003 
Strains with ≥8 virulence genes 25 (42%) 1 (5%) <0.001 <0.001  
Virulence genes Clonal groups, number of isolates (%)
 
P valuea
 
O25b:H4-B2-ST131 (n = 59) O15:H1-D-ST393 (n = 16) CGA-D-ST69 (n= 22) ST131 versus ST393 ST131 versus ST69 ST393 versus ST69 
Adhesins 
fimH 58 (98%) 16 (100%) 22 (100%)    
fimAvMT78 16 (100%) <0.001  <0.001 
papEF 15 (25%) 14 (88%) 12 (55%) <0.001 0.011 0.029 
papG I    
papG II 3 (5%) 14 (88%) 8 (36%) <0.001 0.005 <0.001 
papG III 12 (20%) 0.043 0.016  
papG IV 3 (14%)  0.018  
sfa/focDE    
afa/draBC 7 (12%) 1 (5%)    
afa FM955459 4 (7%)    
bmaE 1 (5%)    
gafD 1 (5%)    
Toxins 
cnf1 10 (17%)  0.033  
cdtB 6 (10%)    
sat 39 (66%) 15 (94%) 11 (50%) <0.001  0.004 
hlyA 10 (17%)  0.033  
Siderophores 
iucD 52 (88%) 15 (94%) 19 (86%)    
iroN 22 (37%) 5 (23%) 0.002   
Capsula 
kpsM II 43 (73%) 16 (100%) 11 (50%) 0.013 0.034 0.001 
kpsM II-K2 6 (10%) 1 (5%)    
kpsM II-K5 36 (61%) 16 (100%) 10 (45%) 0.001  <0.001 
neuC-K1 1 (2%)    
kpsM III 6 (27%)  <0.001 0.027 
Miscellaneous 
cvaC 14 (24%) 5 (23%) 0.024   
iss 21 (36%) 5 (23%) 0.002   
traT 51 (86%) 3 (19%) 19 (86%) <0.001  <0.001 
ibeA 16 (27%) 0.013 0.003  
malX (PAI) 58 (98%) <0.001 <0.001  
usp 59 (100%) <0.001 <0.001  
tsh 1 (2%) 2 (9%)    
ExPEC status 38 (64%) 16 (100%) 13 (59%) 0.002  0.003 
Strains with ≥8 virulence genes 25 (42%) 1 (5%) <0.001 <0.001  

PAI, pathogenicity island.

aP values by Fisher's exact test are shown where P < 0.05.

The virulence gene profiles identified in each clonal group were different, with 24 profiles (P1–P24) among the 59 ST131 isolates (Figure 1), 4 profiles (P25–P28) among the 16 ST393 isolates (Figure 2) and 16 profiles (P29–P44) among the 22 ST69 isolates (Figure 3). However, isolates belonging to the same clonal groups showed similar virulence gene profiles. Among the 28 ESBL-producing isolates not belonging to the three clonal groups, 18 different virulence profiles (P45–P62) were identified (Figure 4) and only 4 isolates showed virulence profiles (P25, P35 and P36) previously observed.

Figure 1.

PFGE of XbaI-digested DNA from the 59 O25b:H4-B2-ST131 isolates included in this study. Clusters with ≥85% similarity are indicated in bold. Isolate designation, virulence profile designation-number of virulence genes, virulence genes and associated resistances are shown on the right. Isolates 55SA, 167LU, 6MA, 99SE, 85MA and 29BA of Group II were CTX-M-15 producing. CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; TOB, tobramycin.

Figure 1.

PFGE of XbaI-digested DNA from the 59 O25b:H4-B2-ST131 isolates included in this study. Clusters with ≥85% similarity are indicated in bold. Isolate designation, virulence profile designation-number of virulence genes, virulence genes and associated resistances are shown on the right. Isolates 55SA, 167LU, 6MA, 99SE, 85MA and 29BA of Group II were CTX-M-15 producing. CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; TOB, tobramycin.

Figure 2.

PFGE of XbaI-digested DNA from the 16 O15:H1-D-ST393 isolates included in this study. Clusters with ≥85% similarity are indicated in bold. Isolate designation, serotype, virulence profile designation-number of virulence genes, virulence genes and associated resistances are shown on the right. Isolate 114LU was positive for CTX-M-14. CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole.

Figure 2.

PFGE of XbaI-digested DNA from the 16 O15:H1-D-ST393 isolates included in this study. Clusters with ≥85% similarity are indicated in bold. Isolate designation, serotype, virulence profile designation-number of virulence genes, virulence genes and associated resistances are shown on the right. Isolate 114LU was positive for CTX-M-14. CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole.

Figure 3.

PFGE of XbaI-digested DNA from the 22 CGA-D-ST69 isolates included in this study. Clusters with ≥85% similarity are indicated in bold. Isolate designation, serotype, virulence profile designation-number of virulence genes, virulence genes and associated resistances are shown on the right. CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; TOB, tobramycin.

Figure 3.

PFGE of XbaI-digested DNA from the 22 CGA-D-ST69 isolates included in this study. Clusters with ≥85% similarity are indicated in bold. Isolate designation, serotype, virulence profile designation-number of virulence genes, virulence genes and associated resistances are shown on the right. CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; TOB, tobramycin.

Figure 4.

PFGE of XbaI-digested DNA from the 28 ESBL-producing isolates not belonging to clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69. Clusters with ≥85% similarity are indicated in bold. Isolate designation, serotype, type of ESBL enzyme produced, phylogenetic groups, virulence profile designation-number of virulence genes, virulence genes and associated resistances are shown on the right. CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; TOB, tobramycin.

Figure 4.

PFGE of XbaI-digested DNA from the 28 ESBL-producing isolates not belonging to clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69. Clusters with ≥85% similarity are indicated in bold. Isolate designation, serotype, type of ESBL enzyme produced, phylogenetic groups, virulence profile designation-number of virulence genes, virulence genes and associated resistances are shown on the right. CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; TOB, tobramycin.

Notably, the ST131 isolates exhibited a significantly higher virulence score (mean/median/range 8.1/7/4–13) compared with the ST393 (6.0/6/3–7; P = 0.01), ST69 (5.4/6/2–9; P < 0.001) and ESBL-producing isolates belonging to other clonal groups (4.0/4/1–7; P < 0.001). In particular, the ST131 isolates carrying the ibeA gene showed the highest virulence score (11.4/13/8–13). In contrast, Johnson et al.6 studying Canadian E. coli isolates from UTIs (2002–04) observed that the virulence scores differed only slightly among the three clonal groups. In the present study, 16 (27%) of 59 ST131 isolates carried the ibeA gene associated with a high virulence score, whereas in the Canadian study only 1 (2%) of 46 ST131 was positive for the ibeA gene. This could explain the differences observed in both studies with respect to the virulence scores.

Sixty-seven (69%) of the 97 isolates belonging to the three clonal groups analysed in this study satisfied the criteria for ExPEC status according to the definition of Johnson et al.34 Thus, the prevalence of ExPEC status was higher within the three clonal groups (69%) than within the 28 ESBL-producing isolates not belonging to them (14%) (P < 0.001).

Macrorestriction profiles by PFGE

Figure 1 shows a dendrogram with the XbaI macrorestriction profiles obtained by PFGE of the 59 ST131 isolates with a similarity of 58.5%. The strains remained distributed in four groups (I–IV) having identities of 67.6%, 71.5%, 72.6% and 75.0%, respectively, characteristically defined by the virulence and resistance profiles of their isolates. Among the 59 ST131 isolates, a total of 11 clusters with ≥85% similarity were observed. Interestingly, two of these clusters included CTX-M-15-positive and -negative isolates. Previously, Nicolas-Chanoine et al.1 studied the virulence genotypes of 36 CTX-M-15-producing ST131 isolates from eight countries and three continents, and found similar pathotypes to those observed in the present study among the ST131 isolates of Group II.

The novelty of our findings is shown by the eight ST131 ibeA isolates of Group I, all of them with identical high virulence gene content and co-trimoxazole resistance. These eight isolates were obtained from each of the five hospitals included in the study. The virulence profile of this group had been previously described only in seven ESBL-producing strains isolated in the hospital of Barcelona in 200812 and one human ST131 strain non-ESBL producer isolated in the hospital of Lugo in 2009.11 Comparing the PFGE profiles of those strains with the eight strains of the present study (Group I), the seven ESBL-producing strains from Barcelona formed a cluster having 88.5% similarity with three ST131 strain non-ESBL producers of Group I (28MA, 8MA and 143LU) and clustered with 82.1% similarity with three other isolates of Group I (18SE, 54BA and 84MA) (dendrogram not shown). Thus, we report for first time the spread of a new variant of the ST131 clonal group with high virulence content and that is non-ESBL-producing but co-trimoxazole resistant. The similarity of the PFGE profiles and the same virulence profile would be indicative of recent emergence. In fact, among a collection of 28 non-ESBL-producing ST131 strains with the ibeA gene obtained from 2464 E. coli blood cultures of patients admitted to the Hospital Lucus Augusti (previously named Complexo Hospitalario Xeral-Calde) from 1998 to 2009 (prevalence of 1.1%),11 only one strain isolated in 2009 showed the same virulence profile as the eight ST131 isolates of Group I from the present study.

Figure 2 shows a dendrogram obtained by PFGE of the 16 O15:H1-D-ST393 isolates identified in the present study that showed a similarity of 72.2%, with a cluster of ≥85% similarity that includes 13 isolates. Comparing the PFGE profiles of these 16 isolates with seven CTX-M-14-producing strains of this clonal group isolated in Spanish hospitals between 2005 and 2008,12,16 five of the seven CTX-M-14-producing strains formed a cluster having 85.8% similarity with the 13 isolates of the present study (dendrogram not shown). The virulence profiles of this clonal group were very similar, including the fimH, fimAvMT78, pagG II, sat, iucD and kpsM II-K5 genes.

Figure 3 shows a dendrogram with the profiles obtained by PFGE of the 22 isolates belonging to CGA-D-ST69, which showed a similarity of 68.2%, with four clusters having ≥85% similarity.

In contrast, the 28 ESBL-producing isolates not belonging to any of the three clonal groups showed 48.2% similarity, with only two clusters of ≥85% similarity (Figure 4).

Conclusions

The three clonal groups investigated in this study accounted for 37% of isolates exhibiting trimethoprim/sulfamethoxazole plus ciprofloxacin resistance, 34% of aminoglycoside-resistant isolates and 30% of multidrug-resistant isolates, which gives evidence of an important clonal component in the emergence of resistance among ExPEC. Notably, a single high virulent clonal group (O25b:H4-B2-ST131) causes approximately 1 in every 10 extraintestinal infections in Spain. The ST131 clonal group was significantly more prevalent than the other two clonal groups characterized in this study, and exhibited a significantly higher virulence score compared with the ST393 and ST69 isolates. This is consistent with its high virulence potential in a mouse model of septicaemia35 and perhaps partly explains the higher epidemiological success of the ST131 clonal group. Interestingly, a new variant of the ST131 clonal group, which is non-ESBL-producing but trimethoprim/sulfamethoxazole resistant and with high virulence content, is reported.

The high prevalence of the ST131 clonal group, particularly among multidrug-resistant isolates, has important clinical and public health implications, due to the real risk of treatment failure. Especially problematic is the dual fluoroquinolone and trimethoprim/sulfamethoxazole resistance (36% of the ST131 isolates), and these drugs constitute the empirical oral UTI therapy. Hence, alternative prophylactic strategies, such as the development of vaccines against successful clonal groups, are urgently needed.

Funding

This work was partially supported by Red Española de Investigación en Patología Infecciosa (REIPI RD06/0008/1018-1016-0011-0017-0026-0031) and grants PS09/01273, PS07/0971, PI070190 and PI10/01955 (Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, Gobierno de España), AGL-2008-02129 (Ministerio de Ciencia e Innovación, Gobierno de España), 09TAL007261PR, 10MRU261023PR and 2007/000044-0 (Xunta de Galicia and The European Regional Development Fund, ERDF) and CTS-5259 (Consejería de Innovación, Junta de Andalucía).

A. M. acknowledges the Ramón y Cajal programme from the Spanish Ministerio de Ciencia e Innovación. R. M. acknowledges the grant of Agencia Española de Cooperación Internacional (AECI) (Ministerio de Asuntos Exteriores y de Cooperación).

Transparency declarations

None to declare.

Acknowledgements

We thank Monserrat Lamela for skilful technical assistance.

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Author notes

These authors made equal contributions to this study.