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JAC Advance Access published online on July 27, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm284
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Most Escherichia coli strains overproducing chromosomal AmpC ß-lactamase belong to phylogenetic group A

Stéphane Corvec1,2, Adèle Prodhomme1, Cécile Giraudeau1, Sandie Dauvergne2, Alain Reynaud1,2 and Nathalie Caroff2,*

1 Laboratoire de Bactériologie-Hygiène, CHU, Nantes, France 2 JE 2437, Génétique des Interactions hôte-microorganisme, Faculté de Pharmacie, Université de Nantes, France

* Correspondence address. Laboratoire de Bactériologie, JE 2437, Faculté de Pharmacie, Université de Nantes, 1 rue Gaston Veil, 44035 Nantes Cédex 01, France. Tel: +33-2-40-41-29-48; Fax: +33-2-40-41-29-52; E-mail: nathalie.caroff{at}univ-nantes.fr

Received 11 May 2007; returned 4 July 2007; revised 11 June 2007; accepted 6 July 2007


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Objectives: To determine the phylogenetic group and the production of different virulence factors (VFs) of a collection of Escherichia coli strains overproducing their chromosomal AmpC cephalosporinase.

Methods: Fifty-five E. coli strains, isolated over a 12 year period, and previously identified as AmpC overproducers by increased MICs of third-generation cephalosporins without extended-spectrum ß-lactamase production (negative double-disc synergy test), were phylogrouped by multiplex PCR. As a comparison, 100 E. coli clinical isolates, susceptible to all ß-lactams, were also tested by the same method. The ampC promoter sequence was determined for all these isolates. ERIC-2 PCR (where ERIC stands for enterobacterial repetitive intergenic consensus) was used to compare the isolates. Search for virulence-associated genes (papG alleles, sfa/foc, hly and iucC) was performed by multiplex PCR for the 55 AmpC overproducers.

Results: Most of the AmpC overproducers (47/55) belonged to phylogenetic group A, correlated with a low prevalence of the main VFs in these strains. The –32, –42 and –11 mutations, responsible for AmpC overproduction, were usually associated with DNA polymorphisms at positions –88, –82, –18, +1 and +58 in the ampC promoter. In the control susceptible isolates, these polymorphisms were detected in 13 ampC promoters (9 group B1 and 4 group A). These polymorphisms were never associated with the main phylogenetic group B2, representing 66% of the susceptible isolates.

Conclusions: AmpC overproduction was clearly correlated with non-virulent commensal phylogenetic groups A and B1, and absence of the main E. coli VFs. Susceptible isolates harbouring the same sequence polymorphisms as AmpC overproducers also belonged to commensal phylogenetic groups.

Key Words: E. coli , virulence , cephalosporinase , promoter


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Escherichia coli usually produces only very small amounts of a chromosomal, non-inducible class C cephalosporinase. However, laboratory mutants and clinical strains overproducing this enzyme have been described in detail. Overproduction is linked to various mutations in strategic parts of the ampC promoter leading to increased transcription rates and, as a consequence, increased production of the enzyme and high-level resistance to numerous ß-lactams, including third-generation cephalosporins.1

The most frequent mutation is located at position –42, creating a new –35 box, with perfect homology with the consensus hexamer. Other mutations have also been described at positions –32 and –11, and at various positions of the transcriptional attenuator. The latter are supposed to destabilize the hairpin structure of the attenuator, resulting in increased ampC expression. An 8–280-fold increase in ampC expression has been detected by RT–PCR according to the different mutations present in the promoters.2

E. coli is by far one of the most important bacterial pathogens, according to the number and diversity of community and nosocomial infections it can cause. Most E. coli strains causing human infections belong to the virulent, extraintestinal phylogenetic group B2 (or D) and possess a panel of virulence factors (VFs) such as adhesins or haemolysins.3

Recently, fluoroquinolone-resistant E. coli isolates have been shown to belong essentially to non-virulent, commensal A or B1 phylogenetic groups.4,5 This study was conducted to determine the phylogenetic group of non-related E. coli strains overproducing chromosomal AmpC.


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Bacterial strains and antimicrobial susceptibility

Fifty-five E. coli strains presenting a ß-lactam resistance pattern compatible with AmpC overproduction were collected at Nantes University Hospital during a 12 year period. AmpC overproduction was suspected on reduced susceptibility to cefoxitin and third-generation cephalosporins, particularly ceftazidime, and absence of synergy with clavulanate (negative double-disc synergy test). The MICs of ceftazidime were determined using the Etest method. For most strains, spectrophotometric data were determined previously using cefalotin as a substrate.1 The source of isolation of these strains was mostly urine (35), but also blood (9), wounds of different localizations (5), bile (1), amniotic fluid (2), placenta (1), peritoneal fluid (1) and vaginal swab (1).

These strains were compared with 100 non-repeat E. coli susceptible to all antibiotics including ß-lactams and isolated from urine (25), blood cultures (25), peritoneal fluid (30), respiratory samples (10) and gastric fluid (10).

Amplification and sequencing of the ampC promoter

Primers AB1 (5'-GATCGTTCTGCCGCTGTG-3') and ampC2 (5'-GGGCAGCAAATGTGGAGCAA-3') corresponding to nucleotides –151/–134 and +120/+101, respectively (GenBank accession number J01611) were used to amplify a 271 bp fragment of the ampC promoter. Boiled lysates were used as template DNA. PCRs were performed in a final volume of 50 µL containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 µM of each nucleotide, 0.5 µM of each primer and 2.5 U of Taq DNA polymerase. PCR conditions were as follows: 90 s of denaturation at 94°C, 30 s of denaturation at 57°C and 30 s of extension at 72°C, with a final extension step of 7 min at 72°C.

The PCR fragment was purified and sequenced with the ABI PRISM Big dye terminator v1.1 cycle sequencing ready reaction kit (Applied Biosystems, Courtaboeuf, France). Sequence analysis was performed on an 3130XL genetic analyser DNA sequencer (Applied Biosystems). The ampC promoter sequences were compared with the ampC promoter sequence of E. coli K-12.6

Determination of phylogenetic group by multiplex PCR

Phylogenetic group was determined using a triplex-PCR targeting chuA, yja and TSPE4.7 The pattern of PCR products obtained allowed the classification into one of the four phylogenetic groups: A, B1, B2 and D.

Virulence-associated genes

Five virulence-associated genes, papG alleles II and III, hly, sfa/foc and the siderophore gene iucC, were searched for by multiplex PCR as previously described3 for the 55 AmpC-overproducing strains.

Molecular typing by ERIC-2 PCR (where ERIC stands for enterobacterial repetitive intergenic consensus)

DNA was extracted with the QIAGEN DNA kit and measured by Nanodrop® spectrophotometer. Ten nanograms was used for PCR with the ERIC-2 primer (5'-AAGTAAGTGACTGGGGTGAGCG-3'). Conditions of amplification were: initial denaturation of 90 s at 94°C; followed by 44 cycles of 15 s at 94°C, 15 s at 40°C and 60 s at 72°C; with a final extension step of 7 min at 72°C.


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Most AmpC overproducers belong to phylogroup A

In this study, the phylogenetic group of a collection of E. coli strains overproducing their chromosomal cephalosporinase has been determined. Strains were collected during a 12 year period from different patients hospitalized in various clinical units and compared by an ERIC-2 method, excluding an epidemic situation (data not shown). As a comparison, 100 non-repeat ß-lactam-susceptible clinical isolates were grouped by the same method.

AmpC overproducers belonged much more frequently to phylogenetic group A than the susceptible isolates [47/55 (85%) versus 13/100 (13%), P < 0.0001]. Out of the 55 AmpC overproducers, 47 belonged to phylogenetic group A (85.5%), 5 to group B1 (9.1%), 2 to group D (3.6%) and 1 to group B2 (1.8%). Out of the 100 susceptible isolates, 66 belonged to phylogenetic group B2, 11 belonged to phylogenetic group D and 13 and 10 to phylogenetic groups A and B1, respectively.

Various mutations known to increase ampC transcription rate were present in the ampC promoter of the 55 AmpC overproducers: at positions –42 (45 isolates, 81.8%), –32 (6 isolates, 10.9%) and –11 (3 isolates, 5.5%); a single isolate had an insertion between the –35 and –10 boxes. In 19 isolates, these mutations/insertion were associated with a genetic modification in the transcriptional attenuator (positions +17 to +37).

Among the 45 isolates presenting the most frequent –42 (C->T) mutation in the ampC promoter, 39 belonged to phylogenetic group A, 5 to group B1 and 1 to group D (Table 1). Five out of six isolates presenting a –32 mutation in the ampC promoter were also from phylogenetic group A, the last one being from phylogenetic group D. The three isolates presenting the mutation at position –11 in the Pribnow box belonged to group A. The strain presenting an insertion of 1 bp between the –35 and –10 boxes, inducing a 17 bp interbox distance instead of 16 in the native promoter, belonged to phylogenetic group B2.


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  		Table 1. Phylogenetic groups and ampC promoter sequences for 55 E. coli strains overproducing AmpC

 
Genetic framework

Mutations leading to AmpC overproduction usually occurred on a specific genetic framework, with polymorphisms at positions –88, –82, –18, –1 and +58. These polymorphisms were found in each isolate harbouring the –42 mutation in the ampC promoter. Five out of six isolates with a –32 mutation also had the same polymorphisms. Interestingly, the AmpC overproducer presenting a single change at position –32, without the –88, –82, –18, –1 and +58 mutations, belonged to phylogenetic group D. A single strain classified in phylogenetic group D had a genetic framework similar to other AmpC overproducers (–88, –82, –18, –1 and +58).

These polymorphisms were also detected in 13% of the ampC promoters from susceptible isolates (Table 2). The susceptible isolates with these –88, –82, –18, –1 and +58 mutations also belonged to non-virulent phylogenetic groups, particularly B1.


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  		Table 2. Comparison between phylogenetic groups and ampC promoter polymorphism in 100 susceptible and 55 resistant E. coli isolates

 
Among susceptible isolates, all B2 isolates presented polymorphisms at positions –73 (C->T) and +81 (G->A) in the ampC promoter, which were never detected in phylogenetic groups A or B1. In addition, the polymorphism at position –28 (G->A) occurred in 50% of susceptible isolates belonging to phylogenetic group B2. On the other hand, the single polymorphism at position +70 (C->T) was associated with phylogenetic group D.

VFs

The following step was the search for five selected VFs by multiplex PCR to characterize the virulence pattern of the 55 AmpC overproducers. Among the aerobactin system, the hydroxamate siderophore (iucC) was detected in 70% of the isolates (74% of the isolates from group A), but none of them, except the group B2 isolate, produced sfa/foc adhesin. Haemolysin was only detected in three strains (5%), one from phylogenetic group B2, one from group A and the another one from phylogenetic group B1; two of them were isolated from blood cultures. Finally, papGII and papGIII alleles were detected in three and two isolates, respectively, all of them being from phylogenetic group A, isolated from urine and presenting a –42 mutation in the ampC promoter.


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It has been demonstrated recently that fluoroquinolone-resistant E. coli isolates belonged to non-pathogenic phylogenetic group A or B1.4,5 Conversely, Pitout et al. have recently demonstrated that extended-spectrum ß-lactamase producers mostly belong to group D and B2. In particular, CTX-M14-producing strains were more likely to belong to phylogenetic group D, although CTX-M15 strains mostly belonged to group B2.8

The study presented here showed that E. coli strains overproducing chromosomal AmpC were significantly more likely to belong to phylogenetic group A than susceptible isolates. Prevalence of the different phylogenetic groups in the 100 susceptible isolates was in perfect concordance with what is usually described for extraintestinal clinical isolates, i.e. a predominance of B2 isolates, and to a lesser extent group D isolates.3

The most frequent mutation leading to AmpC overproduction was located at position –42, creating a new displaced –35 box, modifying the ampC transcription start. This mutation was particularly associated with phylogenetic group A. Other genetic events responsible for AmpC overproduction occurred either in the –35 and –10 boxes, increasing the homology with the consensus TTGACA and TATAAT hexamers, or in the interbox sequence. In 35% of the strains, an additional genetic event modifying the ampC attenuator (positions +17 to +37 in the ampC gene) was detected. This hairpin structure is a transcriptional terminator for the fumarate-reductase operon located upstream of the ampC gene. The mutations in this structure are supposed to increase the transcription rate by decreasing the stability of the hairpin. However, Tracz et al.2 have recently demonstrated by quantifying ampC transcripts that these mutations in the attenuator have a very mild impact on ampC transcription rate. To support this hypothesis, here we detected two susceptible isolates with mutations at positions +22 (C->T), +26 (T->G), +27 (A->T) and +32 (G->A) in the attenuator. Both belonged to phylogroup D. Among the resistant strains mutated in the attenuator, 17 out of 19 isolates belonged to phylogenetic group A (one D and one B2). It is interesting to notice that, in our study, the most resistant isolates (MIC of ceftazidime >16 mg/L) were always associated with a –42 or –32 mutation with a substitution in the attenuator. We cannot exclude that the destabilization can be different and the impact on transcription may vary, depending on the localization of the genetic event in the hairpin.

None of the isolates had an insertion sequence in the ampC promoter, as recently described by Tracz et al.2 in two clinical strains and one laboratory-selected strain.

An interesting point was that mutations responsible for AmpC overproduction were mostly associated with polymorphisms in the ampC gene at positions –88, –82, –18, –1 and +58.

The same polymorphisms were detected in 13% of the susceptible isolates, belonging only to phylogenetic group A or B1.

It has been demonstrated that fluoroquinolone-resistant phylogenetic group A isolates have a marked decrease of VFs. This is probably not due to a ‘loss’ of VFs during conversion from fluoroquinolone susceptibility to resistance, but more likely to the co-existence of two distinct populations, the less virulent being selected from the gut flora during fluoroquinolone treatment.9 It has been suggested that veterinary use of fluoroquinolones could play a role in the selection.

For AmpC overproducers, we confirmed the very low prevalence of the main VFs, in perfect concordance with what was expected for strains belonging to phylogenetic group A. Our results for VFs were in correlation with the data of Bingen-Bidois et al.3 iucC was the only VF ubiquitous in this group, detected in 70% of the isolates, compared with 55% of the group A isolates tested by Bingen-Bidois et al. sfa/foc adhesin, frequent in B2 isolates, was absent in all the 47 isolates from phylogenetic group A tested here. Haemolysin was detected only in three isolates, two of them being from blood. It has been shown that haemolysin is most frequently produced by bacteraemia isolates.10

We can suspect that AmpC overproducers also represent a quite distinct population of E. coli, according to the polymorphisms detected by sequencing in the ampC promoter. The data on polymorphisms confirm that loss of virulence does not occur when the strains become resistant by cephalosporinase overproduction, but more probably it represents a distinct population from the virulent B2 isolates, characterized by a different genetic framework. As suggested by Pitout et al.8 for CTX-M producers, these various patterns reveal a complex evolutionary history for E. coli isolates.

It has been established that increased resistance linked with chromosomal mutations can have a biological cost for bacteria, resulting in reduced fitness without antibiotic selection. This phenomenon has been particularly studied for mutations occurring in gyrA, gyrB, parC and parE for fluoroquinolone-resistant strains.11 However, this loss of fitness could be cancelled by compensatory mutations in clinical isolates. It would be of particular interest to study the fitness of these isolates presenting various mutations or combinations of mutations in the ampC promoter.


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This work was funded by the Ministère de l'Education Nationale et de la Recherche (UPRES-JE2437).


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None to declare.


   Acknowledgements
 
This work was presented in part at the Seventeenth European Congress of Clinical Microbiology and Infectious Diseases, Munich, Germany, 2007 (Abstract P1016).


   References
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1 . Caroff N, Espaze E, Bérard I, et al. Mutations in the ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without extended spectrum ß-lactamase production. FEMS Microb Lett (1999) 173:459–65.[Web of Science][Medline]

2 . Tracz DM, Boyd DA, Bryce E, et al. ampCgene expression in promoter mutants of cefoxitin-resistant Escherichia coli clinical isolates. FEMS Microbiol Lett (2007) 270:265–71.[CrossRef][Web of Science][Medline]

3 . Bingen-Bidois M, Clermont O, Bonacorsi S, et al. Phylogenetic analysis and prevalence of urosepsis strains of Escherichia coli bearing pathogenicity island-like domains. Infect Immun (2002) 70:3216–26.[Abstract/Free Full Text]

4 . Moreno E, Prats G, Sabaté M, et al. Quinolone, fluoroquinolone and trimethoprim/sulfamethoxazole resistance in relation to virulence determinants and phylogenetic background among uropathogenic Escherichia coli. J Antimicrob Chemother (2006) 57:204–11.[Abstract/Free Full Text]

5 . Johnson JR, Kuskowski MA, O'Bryan TT, et al. Virulence genotype and phylogenetic origin in relation to antibiotic resistance profile among Escherichia coli urine sample isolates from Israeli women with acute uncomplicated cystitis. Antimicrob Agents Chemother (2005) 49:26–31.[Abstract/Free Full Text]

6 . Jaurin B, Grundström T. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of ß-lactamases of the penicillinase type. Proc Natl Acad Sci USA (1981) 78:4897–901.[Abstract/Free Full Text]

7 . Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of Escherichia coli phylogenetic group. Appl Environ Microbiol (2000) 66:4555–8.[Abstract/Free Full Text]

8 . Pitout JD, Laupland KB, Church DL, et al. Virulence factors of Escherichia coli isolates that produce CTX-M-type extended-spectrum ß-lactamases. Antimicrob Agents Chemother (2005) 49:4667–70.[Abstract/Free Full Text]

9 . Johnson JR, Johnston B, Kuskowski MA, et al. Spontaneous conversion to quinolone and fluoroquinolone resistance among wild-type Escherichia coli isolates in relation to phylogenetic background and virulence genotype. Antimicrob Agents Chemother (2005) 49:4739–44.[Abstract/Free Full Text]

10 . Bonacorsi S, Houdouin V, Mariani-Kurkdjian P, et al. Comparative prevalence of virulence factors in Escherichia coli causing urinary tract infection in male infants with and without bacteremia. J Clin Microbiol (2006) 44:1156–8.[Abstract/Free Full Text]

11 . Komp Lindgren P, Marcusson LL, Sandvang D, et al. Biological cost of single and multiple norfloxacin resistance mutations in Escherichia coli implicated in urinary tract infections. Antimicrob Agents Chemother (2005) 49:2343–51.[Abstract/Free Full Text]


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