JAC Advance Access published online on November 2, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm401
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Virulence factors in Escherichia coli with CTX-M-15 and other extended-spectrum ß-lactamases in the UK
Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK
* Corresponding author. E-mail: edi.karisik{at}wanadoo.fr
Received 8 June 2007; returned 31 July 2007; revised 26 September 2007; accepted 28 September 2007
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Abstract |
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Objectives: Multiresistant Escherichia coli with CTX-M-15 extended-spectrum ß-lactamase (ESBL) are widespread in the UK. We examined their phylogenetic groups and virulence factors.
Methods: Clinical E. coli isolates (n = 114), collected between 2003 and 2006, were phylogenetically grouped and screened by PCR for 33 virulence factor genes. They included representatives of the five major UK epidemic E. coli strains with CTX-M-15 enzyme, as well as non-clonal isolates with CTX-M or other types of ESBLs.
Results: All representatives of the epidemic E. coli strains belonged to the virulent extra-intestinal phylogenetic group B2, as did 60% (34/56) of the non-clonal isolates with CTX-M-15-like enzymes and 75% (15/20) of those with non-CTX-M ESBLs. Half of those with CTX-M-9-like enzymes belonged to virulence group D. Within phylogenetic group B2, the prevalence of most virulence factors was comparable among clonal and non-clonal isolates with CTX-M enzymes, and among those with non-CTX-M ESBLs. The most frequent virulence genes were PAI, fimH, fyuA, iutA, kpsMTII, K5, traT, uidA and usp. Among the five epidemic clones, afa/draBC was specific to strain A, whereas P fimbriae were only detected in strain D, and only representatives of the B–C–E group specifically harboured sfaS, kpsMTII and K5. However, afa/draBC was also found in 30% of non-clonal isolates with CTX-M ESBLs, and no virulence gene was unique to the epidemic strains.
Conclusions: Most E. coli with CTX-M ESBLs belonged to virulent phylogenetic groups, mainly B2. The successful epidemic strains did not appear more virulent, but iutA and fyuA were significantly more prevalent among these than in non-clonal isolates also belonging to phylogenetic group B2. The most successful clone with CTX-M-15 enzyme (A) differed from other epidemic clones in harbouring afa/draBC, but this was also found in non-clonal isolates with CTX-M-15 enzyme.
Key Words: virulence , phylogenetic group , CTX-M , E. coli
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Introduction |
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Escherichia coli is a major cause of extra-intestinal infections in humans, being the major agent of urinary tract infections (UTIs) and one of the two commonest agents of bacteraemia. Isolates of the species divide into four phylogenetic groups: A, B1, B2 and D,1 and most isolates causing extra-intestinal infections belong to phylogenetic group B2 and, less often, to group D. Commensal strains largely belong to groups A and B1.
Numerous virulence factors have been identified in extra-intestinal E. coli. These include adhesins and fimbriae, toxins, siderophores, polysaccharide coatings and invasins.2 The presence of two or more of a specific subset of virulence genes, including papA, papC, sfa/foc, afa/draBC, iutA and kpsMTII, defines an isolate as an ‘extra-intestinal pathogenic E. coli' (ExPEC). Such strains typically belong to phylogenetic groups B2 or D and are often responsible for acute UTIs.3
E. coli isolates from bacteraemias and complicated UTIs are rapidly becoming more resistant to modern antibiotics, with dramatic recent rises in resistance to cephalosporins and fluoroquinolones across Europe (http://www.rivm.nl/earss/). More generally, multiresistant E. coli with CTX-M extended-spectrum ß-lactamases (ESBLs) are now endemic worldwide,4 except maybe in the US. The particular CTX-M ESBLs that are prevalent vary with the country,5 as does the clonality of the producer strains. In the UK, five major related E. coli strains (A–E), all with CTX-M-15 enzyme, have been distinguished by PFGE, along with many unrelated producers.6 Strain A is the most disseminated lineage, having been recorded from over 50 different centres and being dominant in parts of e.g. Shropshire, Lancashire, Hampshire and Ulster. Although distinct by PFGE, the five epidemic strains A–E share between 70% and 85% banding pattern similarity based on UPGMA and Dice coefficient parameters and are more closely related to one another than are the diverse producers, which predominate, e.g. around London.
We compared the presence of 33 virulence-associated genes among clonal and non-clonal UK E. coli isolates with CTX-M-15-like ß-lactamases and other ESBLs, seeking to evaluate their potential role in epidemiological success.
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Materials and methods |
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Bacterial isolates
One hundred and fourteen E. coli isolates were investigated and divided into four groups comprising: (i) representatives of the five national epidemic strains with CTX-M-15 ß-lactamase (n = 14);6 (ii) non-clonal isolates with group-1 CTX-M enzymes (mostly CTX-M-15) from across the UK (n = 56); (iii) non-clonal E. coli isolates with CTX-M-9-like enzymes (mostly CTX-M-9 and CTX-M-14, n = 24); and (iv) isolates with non-CTX-M-type ESBLs (n = 20, exhibiting the resistance phenotype typically associated with TEM or SHV ESBL production). The isolates were collected from various centres across the UK between January 2003 and January 2006 and were selected to represent a larger collection of E. coli isolates with ESBLs (>2000) referred to the Antibiotic Resistance Monitoring and Reference Laboratory over the same period. Also, most of the 114 isolates were isolated from UTIs, with a few from bacteraemias. Control E. coli strains for virulence factors (2H16, 2H25, J96, L31, PM9 and V27)2 were gifts from Dr James R. Johnson (Minneapolis, MN, USA). blaCTX-M genes were sought and phylogenetically grouped by PCR, as described previously.7
Phylogenetic typing of clinical isolates and virulence genotyping
Phylogenetic typing of E. coli clinical isolates was performed by a published method.1 Thirty-three known or suspected virulence factor genes of extra-intestinal E. coli, including a pathogenicity-associated island (PAI), were sought using five multiplex PCR assays.2
We sought to compare the prevalence of the 33 individual virulence genes between: (i) isolates with and without CTX-M ESBLs; (ii) producers of CTX-M15-like and of CTX-M-9-like enzymes; and finally (iii) virulence group B2 isolates with and without CTX-M ESBLs. For each of the 99 comparisons, the prevalence of virulence factor genes between the groups was either compared using Fisher’s exact test (http://www.graphpad.com/quickcalcs/index), or was dismissed as obviously not significant. To allow for the multiple comparisons made, which increases the random chance that some non-significant associations will appear significant, the Bonferroni correction was applied; the criterion for significance was therefore taken as 5% probability divided by 99.8,9
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Results and discussion |
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Irrespective of ESBL type or epidemic status, most (88/114; 77%) of the isolates investigated were found to belong to phylogenetic groups B2 or D, which are well known to be associated with extra-intestinal infections. All 14 representatives of the five epidemic E. coli strains (A–E) belonged to phylogenetic group B2, as did 60% of non-clonal isolates with CTX-M-15-like ESBLs and 75% of those with non-CTX-M ESBLs. In contrast, half the producers of CTX-M-9-like enzymes belonged to phylogenetic group D, also virulent, whereas the remaining isolates in this group were about equally distributed between groups A and B2. The proportion of isolates belonging to the commensal phylogenetic groups A and B1 was similar among the non-clonal isolates with CTX-M-15-like enzymes (28% and 4%, respectively) and among those with CTX-M-9-like enzymes (25% and 2%, respectively).
Twenty-nine of the 33 virulence factor genes sought were identified in at least one clinical isolate. The exceptions were hlyA (haemolysin), cvaC (colicin V), cdtB (cytolethal distending toxin) and papG allele I (J96-associated papG variant), which were not detected in any of the 114 isolates by PCR, though they were confirmed in their respective control strains. Organisms belonging to the virulent phylogenetic groups B2 and D had averages of 6.5 and 5.6 virulence factor genes each, respectively, compared with 2.9 and 1.5, respectively, for isolates belonging to phylogenetic groups A and B1 (Table 1). Among 88 isolates of phylogenetic groups B2 and D, 76 (86%) counted as ExPEC, compared with only 3/26 (12%) of those belonging to groups A and B1. All the representative isolates of epidemic strains A–E were ExPEC of phylogenetic group B2, as were 32/56 (57%) of non-clonal isolates with group-1 CTX-M enzymes; 5/24 (21%) of those with group-9 CTX-M enzymes; and 15/20 (75%) of those with non-CTX-M ESBLs. The key marker iutA was detected in all ExPEC isolates.
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The epidemic lineages with CTX-M-15 enzyme did not produce more virulence factors than the non-epidemic isolates of the same phylogenetic group (B2), regardless of ESBL type. All four sets of ESBL-positive isolates belonging to this phylogenetic group had between 5 and 10 virulence genes per isolate.
Different representatives of the same epidemic strain exhibited consistent virulence profiles (Table 2) but the patterns varied among these five strains, with specific profiles for strains A, D and the B–C–E group. The afa/draBC gene, which encodes a urinary tract adhesin,10 was specific to strain A, P fimbriae were only detected in strain D and only representatives of the B–C–E group carried sfaS, encoding S fimbriae, also kpsMTII and K5. None of the virulence genes sought was found exclusively in the epidemic strains. Most of the non-clonal isolates with group-1 CTX-M enzymes had virulence repertoires that more closely resembled that of the B–C–E group of epidemic strains than those of strains A and D. Nevertheless, a sizeable minority (30%) also had the afa/draBC gene, which was unique to strain A among the five epidemic lineages. The similarity between epidemic and non-epidemic strains with CTX-M-15 enzymes was even more apparent if only non-clonal isolates belonging to phylogenetic group B2 were considered (Table 1). More broadly, similarities were also apparent between the virulence factor profiles of the phylogenetic group B2 isolates with CTX-M-15-like ß-lactamases (whether epidemic strains or not) and those with non-CTX-M ESBLs. Among phylogenetic group B2 isolates with group-1 CTX-M and those with non-CTX-M ESBLs: 12/33 (36%) and 8/15 (53%), respectively, had all of PAI, fimH (type 1 fimbriae), fyuA (yersiniabactin), iutA (aerobactin receptor), kpsMTII (group-2 capsule LPS), traT (serum survival gene), uidA (ß-D-glucuronidase) and usp (uropathogenic-specific bacteriocin). The genes iutA and fyuA, which encode siderophores, were universal among the clonal isolates with CTX-M-15 enzymes (Table 2), and were produced by 94% and 88% of non-clonal B2 isolates with CTX-M-15-like enzymes and by 93% and 73% of those with non-CTX-M ESBLs, respectively. However, the combination kpsMTII/K5—genes involved in the synthesis of group II capsules—was significantly more prevalent in phylogenetic group B2 isolates with non-CTX-M enzymes than in those with CTX-M enzymes (93% versus 50%, P < 0.001), with a similar pattern also observed for PAI (80% in those with non-CTX-M enzymes versus 32% in those with CTX-M ESBLs, P < 0.001). The afa/draBC gene pair was significantly associated with the production of CTX-M ß-lactamases, regardless of their phylogenetic type (29% prevalence among CTX-M-positive isolates versus 0% among CTX-M-negative isolates, P < 0.001) and it was only detected among isolates that belonged to a virulent phylogenetic group (B2 or D). Although fyuA was similarly prevalent in non-clonal isolates with CTX-M-15-like enzymes and those with CTX-M-9-like enzymes, iutA was significantly more common among those with CTX-M-15 ß-lactamases (86% versus 54%, P < 0.001).
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Overall, virulence was not restricted to the major UK epidemic clones with CTX-M-15 enzymes. Their similar profiles suggested them to be more closely related than suggested previously by PFGE. All five clones were serotyped as O25 previously6 and typed as ST131 by MLST (Andrew Fox, personal communication), further confirming their relatedness and therefore clonal expansion as a major contributor in the dissemination of CTX-M-15 enzyme in the UK.
In summary, the vast majority of E. coli isolates with CTX-M-type ß-lactamases, including all members of the five epidemic strains A–E, belonged to virulent extra-intestinal phylogenetic groups, mostly B2. These findings agree with previous investigations of the phylogenetic background of CTX-M producers in Canada and in France.3,11 As expected from UTI isolates, most also met the definition of ExPEC. Isolates belonging to phylogenetic groups B2 and D had more virulence factors than those belonging to A and B1; the vast majority had seven to nine virulence traits, regardless of the ESBL-type they produced and regardless of whether they belonged to one of the five successful epidemic lineages with CTX-M-15 ß-lactamase. The virulence profiles did however vary slightly between strains A (the most successful lineage), D and the B–C–E cluster. Strain A consistently had afa/draBC, which encodes a urinary tract adhesin. This virulence gene was absent in the other four major epidemic E. coli lineages, but was found in 39% of non-clonal phylogenetic group B2 isolates with group-1 CTX-M enzymes.
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This study was funded by AstraZeneca.
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None to declare.
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We wish to thank AstraZeneca for financially supporting this work. We are also grateful to Dr James R. Johnson, MD (Minneapolis, MN, USA) for supplying E. coli-positive control strains for virulence screening and to Andrew Grant (Statistics Unit, Centre for Infections, Health Protection Agency, London) for helpful advice and comments. Part of this work was presented at the Forty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, 2006 (Poster B1314/6).
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