Detection of clonally related Escherichia coli isolates producing different CMY ß-lactamases from a cystic fibrosis patient.

OBJECTIVES: This study reports details on Escherichia coli isolates recovered from a cystic fibrosis (CF) patient in order to understand how this pathogen adapts to and resists broad-spectrum antipseudomonal therapy in this context. METHODS: Five E. coli isolates were obtained from various clinical samples (airways, urine or dialysis catheter) over a 7 month period covering a double-lung transplantation. All isolates were analysed in terms of clonality [enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing], virulence profiles (phylogroup and search for 15 virulence genes), growth patterns (morphotype, biofilm-forming ability and growth rate), hypermutability and antimicrobial susceptibility, with molecular characterization of ß-lactamases and porins. RESULTS: The five isolates shared similar ERIC-PCR profiles and sequence types (ST1193) and exhibited the same virulence profile. The respiratory isolates were strong mutators, exhibited mucoid or small-colony morphotypes, exhibited strong biofilm-forming ability and grew slowly compared with the others. All isolates were highly resistant to ceftazidime. The respiratory isolates showed reduced susceptibility to cefepime and high resistance to aztreonam. The patient had received a 31 day course of ceftazidime/aztreonam until transplantation. All isolates harboured the same wild-type chromosomal AmpC. A CMY-2 enzyme was detected in the non-respiratory isolates. The respiratory isolates harboured L293S and V211A/L293S new CMY-2 variants, which were designated CMY-94 and CMY-95, respectively. OmpF porin loss was observed in the non-respiratory isolates. CONCLUSIONS: Our study shows that, similarly to Pseudomonas aeruginosa, E. coli can undergo phenotypic and genomic changes in the CF context. For the first time, we identified an in vivo expanded-spectrum evolution of the CMY-2 ß-lactamase, during bacterial persistence in the CF lung.

Occurrence of ST23 complex phylogroup A Escherichia coli isolates producing extended-spectrum AmpC beta-lactamase in a French hospital.

Extended-spectrum AmpC beta-lactamase (ESAC) Escherichia coli producers were investigated over a 5-year period. Eleven isolates presenting a strong ampC promoter and different strategic AmpC mutations, including two newly described modifications (A292V and an L-A-A insertion at 295), were characterized. All the isolates belonged to phylogenetic group A and to the ST23 complex.

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

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 beta-lactamase production (negative double-disc synergy test), were phylogrouped by multiplex PCR. As a comparison, 100 E. coli clinical isolates, susceptible to all beta-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.

Detection of an IS21 insertion sequence in the mexR gene of Pseudomonas aeruginosa increasing beta-lactam resistance.

To understand the regulation of the MexAB OprM efflux system in a clinical strain of Pseudomonas aeruginosa presenting a decreased susceptibility to ticarcillin and aztreonam, the mexR repressor gene was amplified by polymerase chain reaction (PCR) and was shown to be disrupted by an insertion sequence of more than 2 kb, with characteristic direct and inverted repeat sequences. Sequencing revealed a 2131-bp IS21 insertion sequence. A reverse transcription PCR method was used to quantify mexA transcripts and showed an increased transcription rate of mexA in this strain, compared with a PAO1 control strain. The nalB phenotype in P. aeruginosa may be due to point mutations, but also to the presence of an insertion sequence in the mexR regulator gene.

AmpC cephalosporinase hyperproduction in Acinetobacter baumannii clinical strains.

OBJECTIVE: To compare the genetic environments of ampC genes in different Acinetobacter baumannii isolates showing different levels of beta-lactam resistance. METHODS: The patterns of beta-lactam resistance and beta-lactamase production were investigated for 42 A. baumannii clinical strains. The MICs of various beta-lactams were determined in the presence or absence of the class C cephalosporinase inhibitor, cloxacillin (500 mg/L). The ampC gene and its 5' adjacent sequence were analysed by PCR and DNA sequencing. An RT-PCR method was developed to evaluate ampC transcript levels. RESULTS: Strains fell into three resistance groups: first, strains with a ceftazidime MIC < or =8 mg/L (20 strains, 47.6%); secondly, strains with a ceftazidime MIC 32 mg/L, which was reduced four-fold in the presence of cloxacillin (eight strains, 19%); and thirdly, strains with a ceftazidime MIC > or =256 mg/L, which did not decrease in the presence of cloxacillin (14 strains, 33.4%). In all of the resistant isolates (groups II and III), but not in any of the ceftazidime-susceptible isolates (group I), a 1180 bp insert showing all the characteristics of an insertion sequence was detected upstream from the ampC gene. Isolates having this insert overexpress ampC, according to RT-PCR experiments. CONCLUSION: Presence of an insertion sequence upstream of ampC in A. baumannii clinical isolates, possibly including a strong promoter, has the potential to cause over-expression of AmpC, resulting in high-level ceftazidime resistance.