False susceptibility of Klebsiella oxytoca to some extended-spectrum cephalosporins

S. A. Granier1,2, M. H. Nicolas-Chanoine2, J. C. Nguyen Van1, V. Leflon-Guibout2, M. D. Kitzis1 and F. W. Goldstein1,*

1 Laboratoire de Microbiologie Médicale, Fondation Hôpital Saint-Joseph, 185, rue Raymond Losserand, 75674 Paris cedex 14; 2 Service de Microbiologie-Hygiène, Hôpital Ambroise Paré AP-HP, Université Versailles-Saint-Quentin-en-Yvelines-UFR Médicale Paris-Ile-de-France-Ouest, Boulogne-Billancourt, France

Sir,

Klebsiella oxytoca is an opportunistic pathogen that has been involved in various nosocomial infections.1 K. oxytoca carries a chromosomal class A ß-lactamase called K1, or KOXY. When the corresponding bla gene is under the control of a strong promoter, KOXY is overproduced and responsible for the loss of susceptibility to ceftriaxone and aztreonam.2 However, because of the presence of synergy between ceftriaxone, cefotaxime, cefpirome, cefepime or aztreonam and clavulanic acid, loss of susceptibility can be mistaken for plasmid-mediated, extended-spectrum ß-lactamase (ESBL). This mistake leads to interpreting such isolates as non-susceptible to any third generation cephalosporins as well as aztreonam. This is because several Antibiogram Committees, notably the French Antibiogram Committee, have recommended interpreting in this manner the ß-lactam susceptibility of ESBLA-producing isolates. Another difficulty encountered for assessing the ß-lactam susceptibility of KOXY-overproducing isolates is the poor reproducibility of their antibiograms, which seems to be related to a significant inoculum effect.

The present study was therefore performed to evaluate the MICs of different ß-lactam molecules for KOXY-overproducing K. oxytoca isolates tested with increasing inocula.

The sixteen K. oxytoca clinical isolates studied displayed a decreased susceptibility to aztreonam and no synergy between ceftazidime and clavulanic acid. The overproduction of the KOXY ß-lactamases in these isolates was assessed by promoter sequencing. We found in the KOXY promoter of each isolate one of the two following mutations: G-8A or G-12T in the –10 region. As previously described by Fournier et al.,3 these mutations are responsible for overproduction of KOXY. The absence of other ß-lactamases was checked by using the electro-focusing method. Escherichia coli ATCC 25922 was used as a control strain.

MICs of ceftriaxone, cefotaxime, cefpirome, cefepime, aztreonam and ceftazidime were determined for three different inocula (104, 105 and 106 cfu/mL) by using the Mueller–Hinton broth dilution method as recommended by the NCCLS. Antibiotic powders were provided by their manufacturers.

As indicated in Table 1, an increase in inoculum from 104 to 106 cfu/mL resulted in a strong increase in MIC50 of all the ß-lactams tested, except for ceftazidime. However, the individual isolate analysis showed that MICs varied widely independently of the ß-lactam molecule and the inoculum tested. For the lowest (104 cfu/mL) as well as the standard (105 cfu/mL) inocula, certain isolates were susceptible (MIC < 4 mg/L) and others resistant to the different ß-lactams tested. The exception to this was ceftazidime, to which all of the isolates remained susceptible. Using an inoculum >=106 cfu/mL should clearly discriminate antibiotics that are stable to KOXY ß-lactamase (ceftazidime) from those that are inactivated by K. oxytoca overproducing this ß-lactamase (ceftriaxone, cefotaxime, cefepime, cefpirome and aztreonam).


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Table 1.  MICs of six ß-lactams for 16 K. oxytoca clinical isolates overproducing their chromosomal ß-lactamase
 
Our study demonstrates that the bacterial inoculum plays a significant role in the ß-lactam susceptibility test and, hence, in the interpretation of MICs against KOXY-overproducing isolates.4,5 In most studies, these isolates are considered to be resistant to aztreonam and ceftriaxone but susceptible to cefotaxime, cefepime, cefpirome and ceftazidime.2 How-ever, assuming that the in vivo inoculum is often very high (107–109 cfu/mL), this study strongly suggests that KOXY-overproducing K. oxytoca isolates should not be reported as susceptible to cefotaxime, cefepime and cefpirome, and that the use of ceftazidime might be safer in the treatment of infections due to these isolates. Nevertheless, to increase the strength of these suggestions, in vivo experiments are mandatory.

Acknowledgements

This paper was presented in part at the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA, 2001 (poster D-179).

Footnotes

* Corresponding author. Tel: +33-1-44-12-36-50; Fax: +33-1-44-12-32-34; E-mail: fgoldstein{at}hopital-saint-joseph.org Back

References

1 . Jeong, S. H., Kim, W. M., Chang, C. L., Kim, J. M., Lee, K., Chong, Y. et al. (2001). Neonatal intensive care unit outbreak caused by a strain of Klebsiella oxytoca resistant to aztreonam due to overproduction of chromosomal ß-lactamase. Journal of Hospital Infection 48, 281–8.[ISI][Medline]

2 . Fournier, B., Lagrange, P. H. & Philippon, A. (1996). In-vitro susceptibility of Klebsiella oxytoca strains to 13 ß-lactams in the presence and absence of beta-lactamase inhibitors. Journal of Antimicrobial Chemotherapy 37, 931–42.[Abstract]

3 . Fournier, B., Lu, C. Y., Lagrange, P. H., Krishnamoorthy, R. & Philippon, A. (1995). Point mutation in the Pribnow box, the molecular basis of ß-lactamase overproduction in Klebsiella oxytoca. Antimicrobial Agents and Chemotherapy 36, 1365–8.

4 . Courvalin, P. (1992). Interpretative reading of antimicrobial susceptibility tests. ASM News 58, 368–75.

5 . Livermore, D. M., Winstanley, T. G. & Shannon, K. P. (2001). Interpretative reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. Journal of Antimicrobial Chemotherapy 48, 87–102.[Abstract/Free Full Text]