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Keywords:

  • AmpC;
  • porins;
  • carbapenems;
  • β-lactamase;
  • Escherichia coli

Abstract

  1. Top of page
  2. Abstract
  3. Conclusion
  4. References

ESAC β-lactamases have increased catalytic efficiencies toward extended-spectrum cephalosporins and to a lesser extent toward imipenem as compared with the wild-type cephalosporinases. We show here that ESAC expression associated with the loss of both OmpC and OmpF porins conferred in Escherichia coli a high level of resistance to ertapenem and reduced the susceptibility to imipenem. On the contrary, ESAC expressed in the OmpC- or OmpF-deficient E. coli strains or narrow-spectrum cephalosporinase expressed in the OmpC-and OmpF-deficient strain do not confer reduced susceptibility to any of the carbapenems. The production of ESAC β-lactamase in favorable E. coli background may represent an additional mechanism of resistance to ertapenem.

Overexpression of chromosomal ampC genes in enterobacterial strains (Hanson & Sanders, 1999) confers resistance to aminopenicillins and early generation cephalosporins and reduces the susceptibility to several extended-spectrum cephalosporins (ESCs) such as ceftazidime, cefotaxime and ceftriaxone (Livermore & Brown, 2001). Zwitterionic cephalosporins (cefepime and cefpirome) and carbapenems (imipenem, ertapenem, meropenem), which penetrate very efficiently through the outer membrane of Gram-negative bacteria (Hancock & Bellido, 1996) and are poor substrates for AmpC β-lactamases, remain active in vitro against enterobacterial isolates producing high levels of cephalosporinases.

Recently, a novel mechanism of resistance due to the production of variant cephalosporinases has been reported (Nordmann & Mammeri, 2007). These extended-spectrum AmpC (ESAC) β-lactamases, which are structurally related to wild-type cephalosporinases by insertions, deletions or substitutions (Nordmann & Mammeri, 2007), display a broadened hydrolysis spectrum (Kim et al., 2006; Mammeri et al., 2007). They constitute an emerging mechanism of resistance in Escherichia coli as suggested by a recent epidemiological study (H. Mammeri, F. Eb, A. Berkani & P. Normann), which showed that the prevalence of clinical isolates producing ESAC β-lactamases was almost identical to those harboring plasmid-mediated cephalosporinases such as CMY-2 or ACC-1 (Philippon et al., 2002).

At the biochemical level, ESAC β-lactamases exhibited increased catalytic efficiency against ESCs and slightly against imipenem, which was correlated to increased minimal inhibitory concentration (MIC) values of ESCs, whereas those of imipenem remained unaffected (Lee et al., 2003, 2004; Kim et al., 2006; Mammeri et al., 2007). Therefore, we have investigated the combined effect of ESAC and additional outer membrane protein defects in carbapenem resistance.

Four recombinant plasmids, pEC14, pEC18, pBER and pEC2, which coded for three ESAC β-lactamases, AmpC EC14, AmpC EC18 and AmpC BER, and a narrow-spectrum cephalosporinase, AmpC EC2, respectively, were used in this study. These plasmids, characterized in previous works (Mammeri et al., 2006, 2007), were extracted using the Qiagen midi kit (Qiagen, Courtaboeuf, France) and subsequently introduced by transformation into three porin-deficient E. coli strains, E. coli JF701, E. coli JF703 and E. coli HB4 (Sambrook & Russell, 2000).

Escherichia coli JF701 and E. coli JF703 (Nikaido et al., 1983) lack porins OmpC and OmpF, respectively, whereas E. coli HB4, which lack both porins OmpC and OmpF, was an in vitro obtained derivative of E. coli 4.

Escherichia coli 4 was resistant to all the extended-spectrum β-lactams due to the production of the plasmid-mediated CMY-2 β-lactamase combined with the loss of membrane permeability. Sequence analysis of the ompC and ompF genes together with sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the outer membrane proteins revealed that the loss of permeability was related to the complete absence of OmpC and OmpF expression (Poirel et al., 2004).

Esherichia coli 4 was cultured for 10 days in trypticase soy (TS) broth at room temperature in an attempt to cure its resident plasmid. Then, the broth was subcultured on TS agar plates and the colonies were tested for their susceptibility to ESCs. The resulting strain, E. coli HB4, remained resistant to narrow-spectrum cephalosporins but was susceptible to ESCs, although the MICs of these compounds were slightly increased as compared with those for wild-type E. coli strain (Table 1). The PCR experiment failed to detect the blaCMY−2 gene in the derivative strain, thus confirming the loss of the plasmid-borne resistance marker. Moreover, the chromosome-borne ampC gene of E. coli HB4 was amplified and sequenced, as previously described, to determine its contribution in the resistance pattern (Mammeri et al., 2006). The analysis of its promoter region failed to detect mutation as compared with the wild-type promoter of the ampC gene of E. coli K12 (Caroff et al., 2000), thus indicating that E. coli HB4 had an ampC gene with a weak promoter (Tracz et al., 2007). Furthermore, the contribution of overexpressed efflux systems in the resistance pattern of this strain was ruled out because Phe–Arg β-naphthylamide, which inhibits this mechanism of resistance, failed to decrease MIC values (data not shown).

Table 1.   MIC values of β-lactams for transformants of Escherichia coli JF703, E. coli JF701 and E. coli HB4
β-LactamsMICs (μg mL−1)
E. coli JF703 (pEC2)*E. coli JF703 (pEC14)*E. coli JF703 *E. coli JF701 (pEC2) *E. coli JF701 (pEC14) *E. coli JF701 *E. coli HB4 (pEC2) *E. coli HB4 (pEC14) *E. coli HB4 *
  • *

    Escherichia coli JF703, E. coli JF701 and E. coli HB4 are recipient strains that lacked porin OmpF, OmpC and OmpF+OmpC, respectively. Recombinant plasmids pEC2 and pEC14 encoded narrow-spectrum cephalosporinase and ESAC β-lactamases, respectively.

Ticarcillin1283243281256644
Piperacillin3264432161641284
Cefoxitin2561284128321>256>256>256
Ceftazidime82560.51640.06416>2564
Cefotaxime240.1250.12520.0324>1284
Cefepime0.12540.060.06410.00811281
Aztreonam480.1250.0310.0164324
Imipenem0.1250.250.1250.1250.250.1250.520.25
Ertapenem0.0640.1250.0060.0040.0060.0042321
Meropenem0.0320.0320.0320.0320.0320.0320.0320.0320.032

Transformation experiments gave rise to 12 recombinant clones, introducing each of the four plasmids (pEC14, pEC18, pEC2 and pBER) in the three bacterial strains (JF701, JF703 and HB4).

MICs of β-lactams for the transformants and the recipient strains were determined by an agar dilution technique (Bellais et al., 2000). Because the MICs for the ESAC-producing transformants were almost identical, only those for the transformants producing the ESAC β-lactamase AmpC EC14 and the transformants producing the narrow-spectrum AmpC EC2 were presented in Table 1.

MICs for the OmpF-deficient transformants (E. coli JF703) mirrored those for the OmpC-deficient transformants (E. coli JF701), although they were slightly increased. These phenotypic results were in agreement with the spectrophotometric study conducted by Nikaido et al. showing that OmpF has a ten-time higher permeability coefficient than OmpC (Nikaido et al., 1983).

Escherichia coli JF701 (pEC2) and E. coli JF703 (pEC2), which produced the narrow-spectrum cephalosporinase AmpC EC2, were susceptible to ESCs, whereas E. coli HB4 (pEC2), which produced the same narrow-spectrum β-lactamase but lacked both porins OmpC and OmpF, had a reduced susceptibility for these compounds (Table 1) (Clinical and Laboratory Standards Institute, 2005). These results indicated that the lack of both porins is required to decrease significantly the outer membrane permeability in E. coli. Therefore, the reduced penetration of ESCs into the periplasmic space compensated for the weak hydrolytic activity of narrow-spectrum cephalosporinases toward these compounds, thus leading to a significant level of resistance.

The ESAC-producing E. coli HB4 transformants were resistant to ertapenem (MICs≥32 mg L−1) and had a reduced susceptibility to imipenem (Table 1), whereas E. coli HB4 (pEC2), which produced a narrow-spectrum cephalosporinase, remained susceptible to carbapenems. Our study demonstrates that ESAC β-lactamases could significantly increase the levels of resistance to carbapenems. The loss of membrane permeability constitutes a favorable background that magnifies the phenotypic expression of the carbapenemase activity exhibited by these variant enzymes.

Conclusion

  1. Top of page
  2. Abstract
  3. Conclusion
  4. References

This report indicates that ESAC β-lactamases may provide a favorable background for selection of carbapenem resistance among E. coli isolates. It showed that the susceptibility to imipenem and meropenem is less affected than susceptibility to ertapenem, thus indicating that this recently introduced carbapenem may constitute a risk to select novel mechanisms of resistance such as ESAC β-lactamases.

References

  1. Top of page
  2. Abstract
  3. Conclusion
  4. References