This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by González-López, J. J.
Right arrow Articles by Prats, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by González-López, J. J.
Right arrow Articles by Prats, G.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, September 2006, p. 3175-3178, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.00273-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo Reversion to the Wild-Type ß-Lactam Resistance Phenotype Mediated by a Plasmid Carrying ampR and qnrA1 in Enterobacter cloacae

J. J. González-López, M. Sabaté, S. Lavilla, M. N. Larrosa, R. M. Bartolomé, and G. Prats*

Servicio de Microbiología, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain

Received 3 March 2006/ Returned for modification 1 May 2006/ Accepted 25 June 2006


arrow
ABSTRACT
 
Resistance to ß-lactams and quinolones in two isogenic Enterobacter cloacae isolates was studied. One was susceptible to cefoxitin and amoxicillin-clavulanate. The other one showed its natural ß-lactam resistance pattern. Both isolates had a nonfunctional AmpR regulator. However, within the second one, the presence of a plasmid carrying ampR and qnrA1 allowed reversion to the wild-type ß-lactam resistance phenotype and decreased susceptibility to fluoroquinolones.


arrow
TEXT
 
The production of the chromosomally encoded AmpC ß-lactamase in many members of the family Enterobacteriaceae, including Enterobacter cloacae, Citrobacter freundii, Providencia stuartii, Serratia marcescens, Morganella morganii, and Yersinia enterocolitica, is induced by the presence of some ß-lactams, resulting in a broad range of resistance to this family of antibiotics (1, 2). This induction is due mainly to the presence of AmpR, a regulator belonging to the LysR family.

The production of AmpC is closely related to the recycling of peptidoglycan. This process implies the activity of the transmembrane permease AmpG and the cytoplasmic amidase AmpD, a protein involved in the turnover and recycling of muropeptides (6, 8, 13). During normal bacterial growth, in the absence of ß-lactams, anhydromuropeptides released from the bacterial peptidoglycan in the periplasm are imported into the cytoplasm by AmpG and hydrolyzed by the amidase AmpD, and the resulting components are used for the synthesis of murein precursors. One of these precursors, the UDP-MurNac pentapeptide (uridine-pyrophosphoryl-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid-D-alanyl-D-alanine), binds to AmpR, causing AmpR to assume a configuration which does not activate the ampC promoter, thereby resulting in a low level of ampC expression. In contrast, in the presence of ß-lactam, anhydromuropeptides accumulate in the cytoplasm, displacing UDP-MurNac pentapeptide from its AmpR-binding site. AmpR thereby assumes an active configuration and enhances ampC expression (7).

Two E. cloacae isolates, Ecl-834 and Ecl-835, were obtained from the same urine sample from a patient with urinary tract infection. Antibiotic susceptibility was tested by disk diffusion according to the Clinical and Laboratory Standards Institute guidelines for Enterobacteriaceae (3). For selected antibiotics, MICs were determined by Etest (Table 1). Both isolates were resistant to ampicillin, cephalothin, and cefuroxime and showed synergy of cefotaxime, ceftazidime, cefepime, and aztreonam with amoxicillin-clavulanate, suggesting the production of an extended-spectrum ß-lactamase (ESBL). Ecl-834 was also resistant to cefoxitin, amoxicillin-clavulanate, and trimethoprim-sulfamethoxazole and showed induction of resistance to aztreonam by imipenem. However, Ecl-835 was susceptible to cefoxitin, amoxicillin-clavulanate, and trimethoprim-sulfamethoxazole and did not show induction of resistance to aztreonam by imipenem (Fig. 1). By Etest, the ciprofloxacin MIC was 1.5 and 0.25 µg/ml for Ecl-834 and Ecl-835, respectively (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. MICs of different antibiotics for E. cloacae isolates


Figure 1
View larger version (96K):
[in this window]
[in a new window]
  		FIG. 1. Susceptibility test for E. cloacae isolates. AMC, amoxicillin-clavulanic acid; AMK, amikacin; AMP, ampicillin; ATM, aztreonam; CAZ, ceftazidime; CEF, cephalothin; CIP, ciprofloxacin; COL, colistin; CTX, cefotaxime; CXM, cefuroxime; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; SXT, trimethoprim-sulfamethoxazole; TZP, piperacillin-tazobactam. Ecl-834 and Ecl-835, clinical isolates; Ecl-835 (pAMPRDHA1), Ecl-835 with a recombinant plasmid carrying ampRDHA-1; Ecl-835 (pQNRA1), Ecl-835 with a recombinant plasmid carrying qnrA1; Ecl-834 CQR, Ecl-834 derivative without qnrA1-ampRDHA-1; Ecl-834 CQRE, Ecl-834 derivative without qnrA1-ampRDHA-1 and the blaCTX-M-9 genes.

In order to determine whether the two isolates were isogenic, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) was performed as previously described (17). We included in the ERIC-PCR analysis an unrelated E. cloacae strain (harboring qnrA1) as control. Amplified PCR products were analyzed by agarose gel electrophoresis. The banding patterns revealed genomic identity between the two isolates (Fig. 2).


Figure 2
View larger version (109K):
[in this window]
[in a new window]
  		FIG. 2. Agarose gel electrophoresis of the ERIC-PCR. Lanes 1 and 6, molecular size markers; lane 2, Ecl-834; lane 3, Ecl-835; lane 4, Enterobacter cloacae nonrelated strain; lane 5, water blank.

Analytical isoelectric focusing of the crude cell extract (15) showed in both isolates the same ß-lactam-hydrolyzing bands, one of pI 8.0, compatible with the presence of one ESBL (12, 14, 16), and another of pI >8.5, compatible with the chromosomal AmpC ß-lactamase (4). blaCTX-M-9 was detected by PCR amplification (16), and direct sequencing of amplicons showed 100% sequence identity with blaCTXM-9 in both isolates. The pI and susceptibility test results showed that production of the chromosomal ampC ß-lactamase in Ecl-835 could be detected by isoelectric focusing but not by disk diffusion, suggesting that AmpC was produced at a low level in this isolate.

To elucidate why Ecl-834 and Ecl-835 exhibited different pattern of chromosomal ß-lactamase production, ampC and its regulatory gene ampR were amplified and sequenced using primers designed based on homologous regions of ampC and ampR sequences from E. cloacae. There was no difference in the ampC or ampR sequences between the two isolates. However, in comparisons of these ampC and ampR sequences with those from E. cloacae isolates with a normal resistance phenotype, including (i) one from public databases and (ii) one obtained from our collection, an insertion of a C between bases 92 and 93 of the ampR gene was found. This insertion is predicted to generate a new stop codon 18 bp downstream, resulting in a putative peptide comprising 37 amino acids instead of the 291 that form the native AmpR protein. Therefore, Ecl-834 and Ecl-835 presumably presented a truncated ampR despite Ecl-834 showing an inducible AmpC phenotype. We did not find any deleterious mutation in ampC (nonsense, insertion, or deletion) which could lead to a nonfunctional AmpC protein, as happens with AmpR.

During a separate ongoing prospective study, the presence of plasmid-mediated quinolone-resistance determinant qnr was evaluated among ESBL-producing enterobacterial isolates, including Ecl-834 and Ecl-835. The qnrA1 gene was found in Ecl-834 but not in Ecl-835. Wang et al. reported in 2003 two different complex class1 integrons, In36 and In37, containing the qnr gene and also an ampR gene of ß-lactamase DHA-1 (ampRDHA-1) between orf513 and the second copy of the 3' conserved sequence, which include the sulI gene (18). To determine whether this was true also for the integron in strain Ecl-834, PCR amplification and sequencing of the genes located downstream of qnrA1 revealed an ampRDHA-1 gene in this strain, as reported for integrons In36 and In37. We speculated that this gene conceivably could complement the defective chromosomally encoded ampR gene in strain Ecl-834.

In order to prove that in strain Ecl-834 the plasmid-encoded ampRDHA-1 substituted for the defective chromosomal ampR gene, the ampRDHA-1 gene was cloned using a pGEM-T Easy vector system (Promega Corp., Madison, WI), and the resulting recombinant plasmid was electroporated into Ecl-835 electrocompetent cells. The resulting Ecl-835 (pAMPRDHA1) strain showed the same antibiogram disk diffusion pattern as Ecl-834 except for trimethoprim-sulfamethoxazole, to which the derivative remained susceptible (Fig. 1). In addition to this assay, plasmid curing was performed with Ecl-834 and acridine orange in order to demonstrate that the plasmid containing the qnrA1-ampR genes was responsible for conferring on the wild-type strain the phenotype of resistance to ß-lactams. Ecl-834 was growth in brain heart infusion medium with 80 µg/ml acridine orange (Sigma-Aldrich Inc., Steinheim, Germany) for 24 h with shaking at 42°C. Cells which had lost the plasmid containing the qnrA1-ampR DHA-1 genes and the plasmid with the ESBL gene were selected by negative selection-replica plating on LB with cefoxitin (100 µg/ml) and ampicillin (100 µg/ml). The Ecl-834 derivatives that had lost the plasmid or the integron containing the qnrA1-ampR DHA-1 genes but not the blaCTX-M-9 gene (Ecl-834 CQR) exhibited the same antibiogram as did Ecl-835. The Ecl-834 derivatives that had lost both qnrA1-ampRDHA-1 and the blaCTX-M-9 gene (Ecl-834 CQRE) were susceptible to all antibiotics tested (Fig. 1). Overall, these experiments proved that the wild-type resistance phenotype of Ecl-834 is due to the presence of the plasmid-mediated ampRDHA-1.

We next sought to assess how the presence of the plasmid bearing the qnrA1-ampR genes could affect quinolone and fluoroquinolone resistance in these isolates. By Etest, the MIC of nalidixic acid was ≥256 µg/ml for both isolates, whereas the MIC of ciprofloxacin was 1.5 and 0.25 µg/ml for Ecl-834 and Ecl-835, respectively, representing a sixfold difference (Table 1). PCR amplification and sequencing of the quinolone resistance-determining region of the gyrA, gyrB, parC, and parE genes, as previously described (11), showed in both isolates a single amino acid change in gyrA, Ser83->Phe, which previously has been described for E. cloacae as responsible for resistance to nalidixic acid (5).

qnrA1, including its promoter, was cloned into a pGEM-T Easy vector system (Promega). Electroporation of the resulting recombinant plasmid into Ecl-835 electrocompetent cells yielded strain Ecl-835 (pQNRA1). This strain showed the same ciprofloxacin MIC, 1.5 µg/ml, as strain Ecl-834, demonstrating that QnrA1 is able to increase the ciprofloxacin MIC sixfold (Table 1).

In our laboratory, we have found that around 1.5% of isolates of E. cloacae do not express phenotypically the intrinsic resistance of this species to ß-lactams (unpublished data). Previous works have described the constitutive expression of AmpC in E. cloacae strains due to chromosomal ampR mutations (9, 10). To our knowledge, this work reports the first isolation of a clinical strain defective for AmpR production due to chromosomal ampR mutation which in vivo reverts to its presumed ancestral phenotype as a result of the acquisition of an exogenous ampR gene embedded in a plasmid which also carries a qnrA1 gene.


arrow
ACKNOWLEDGMENTS
 
We thank J. R. Johnson (Minneapolis VA Medical Center, Minneapolis, MN) and F. Navarro (Hospital de la Santa Creu i Sant Pau, Barcelona, Spain) for critically reading the manuscript and providing helpful comments. We are grateful to L. Martínez-Martínez (Hospital Universitario Marqués de Valdecilla, Santander. Spain) for providing Klebsiella pneumoniae UAB1 harboring the qnrA1 gene, which was used as a control strain.

This work was supported by a grant from the "Fondo de Investigación Sanitaria" (PI 050289) and from the "Red Española para la Investigación en Patología Infecciosa." J.J.G.-L. received a grant from "Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica."


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Vall d'Hebron, Pg. Vall d'Hebron 119-129, 08035 Barcelona, Spain. Phone: 34-93-2746817. Fax: 34-93-2746801. E-mail:gprats{at}vhebron.net. Back


arrow
REFERENCES
 
    1
  1. Bennett, P. M., and I. Chopra. 1993. Molecular basis of ß-lactamase induction in bacteria. Antimicrob. Agents Chemother. 37:153-8.[Free Full Text]
    2. 2
  2. Bush, K., G. A. Jacoby, and A. A. Medeiros. 1995. A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-33.[Medline]
    3. 3
  3. Clinical and Laboratory Standards Institute. 2004. Performance standards for antimicrobial susceptibility testing: M100-S15, 15th informational supplement, vol. 25. Clinical Laboratory Standards Institute, Wayne, Pa.
    4. 4
  4. Conceiçao, T., N. Faria, L. Lito, J. Melo Cristino, M. J. Salgado, and A. Duarte. 2004. Diversity of chromosomal AmpC beta-lactamases from Enterobacter cloacae isolates in a Portuguese hospital. FEMS Microbiol. Lett. 230:197-202.[CrossRef][Medline]
    5. 5
  5. Deguchi, T., M. Yasuda, M. Nakano, S. Ozeki, E. Kanematsu, Y. Nishino, S. Ishihara, and Y. Kawada. 1997. Detection of mutations in the gyrA and parC genes in quinolone-resistant clinical isolates of Enterobacter cloacae. J. Antimicrob. Chemother. 40:543-9.[Abstract/Free Full Text]
    6. 6
  6. Hanson, N. D., and C. C. Sanders. 1999. Regulation of inducible AmpC beta-lactamase expression among Enterobacteriaceae. Curr. Pharm. Des. 5:881-94.[Medline]
    7. 7
  7. Jacobs, C., J. M. Frere, and S. Normark. 1997. Cytosolic intermediates for cell wall biosynthesis and degradation control inducible beta-lactam resistance in gram-negative bacteria. Cell 88:823-32.[CrossRef][Medline]
    8. 8
  8. Jacobs, C., L. J. Huang, E. Bartowsky, S. Normark, and J. T. Park. 1994. Bacterial cell wall recycling provides cytosolic muropeptides as effectors for beta-lactamase induction. EMBO J. 13:4684-94.[Medline]
    9. 9
  9. Kaneko, K., R. Okamoto, R. Nakano, S. Kawakami, and M. Inoue. 2005. Gene mutations responsible for overexpression of AmpC ß-lactamase in some clinical isolates of Enterobacter cloacae. J. Clin. Microbiol. 43:2955-8.[Abstract/Free Full Text]
    10. 10
  10. Kuga, A., R. Okamoto, and M. Inoue. 2000. ampR gene mutations that greatly increase class C ß-lactamase activity in Enterobacter cloacae. Antimicrob. Agents Chemother. 44:561-7.[Abstract/Free Full Text]
    11. 11
  11. Linde, H. J., F. Notka, C. Irtenkauf, J. Decker, J. Wild, H. H. Niller, P. Heisig, and N. Lehn. 2002. Increase in MICs of ciprofloxacin in vivo in two closely related clinical isolates of Enterobacter cloacae. J. Antimicrob. Chemother. 49:625-30.[Abstract/Free Full Text]
    12. 12
  12. Mugnier, P., I. Podglajen, F. W. Goldstein, and E. Collatz. 1998. Carbapenems as inhibitors of OXA-13, a novel, integron-encoded beta-lactamase in Pseudomonas aeruginosa. Microbiology 144:1021-31.[Abstract/Free Full Text]
    13. 13
  13. Park, J. T. 1995. Why does Escherichia coli recycle its cell wall peptides? Mol. Microbiol. 17:421-6.[Medline]
    14. 14
  14. Poirel, L., T. Naas, I. Le Thomas, A. Karim, E. Bingen, and P. Nordmann. 2001. CTX-M-type extended-spectrum ß-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrob. Agents Chemother. 45:3355-61.[Abstract/Free Full Text]
    15. 15
  15. Sabate, M., E. Miro, F. Navarro, C. Verges, R. Aliaga, B. Mirelis, and G. Prats. 2002. Beta-lactamases involved in resistance to broad-spectrum cephalosporins in Escherichia coli and Klebsiella spp. clinical isolates collected between 1994 and 1996, in Barcelona (Spain). J. Antimicrob. Chemother. 49:989-97.[Abstract/Free Full Text]
    16. 16
  16. Sabate, M., R. Tarrago, F. Navarro, E. Miro, C. Verges, J. Barbe, and G. Prats. 2000. Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing ß-lactamase (CTX-M-9) from Escherichia coli in Spain. Antimicrob. Agents Chemother. 44:1970-3.[Abstract/Free Full Text]
    17. 17
  17. Versalovic, J., T. Koeuth, and J. R. Lupski. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:6823-31.[Abstract/Free Full Text]
    18. 18
  18. Wang, M., J. H. Tran, G. A. Jacoby, Y. Zhang, F. Wang, and D. C. Hooper. 2003. Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrob. Agents Chemother. 47:2242-8.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, September 2006, p. 3175-3178, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.00273-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by González-López, J. J.
Right arrow Articles by Prats, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by González-López, J. J.
Right arrow Articles by Prats, G.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»