Distribution of ß-lactamases in Acinetobacter baumannii clinical isolates and the effect of Syn 2190 (AmpC inhibitor) on the MICs of different ß-lactam antibiotics

Cristina Danes, Margarita M. Navia, Joaquim Ruiz, Francesc Marco, Angels Jurado, M. Teresa Jimenez de Anta and Jordi Vila*

Servei de Microbiologia, Institut Clínic Infeccions i Immunologia (ICII), Hospital Clínic-IDIBAPS, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain

Received 19 September 2001; returned 28 December 2001; revised 28 February 2002; accepted 21 April 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The distribution of ß-lactamases in a group of 20 epidemiologically well defined Acinetobacter baumannii clinical isolates and the in vitro activity of Syn 2190, a novel ß-lactamase AmpC inhibitor, were determined. Twenty-five per cent of the strains carried and expressed a TEM-type ß-lactamase, whereas 35% had an OXA-type ß-lactamase. In nine out of 11 (82%) ceftazidime-resistant and four out of 13 (30.7%) cefepime-resistant strains, the MIC of these ß-lactam antibiotics decreased when determined in the presence of Syn 2190. Thus, our results suggest that in a high percentage of A. baumannii clinical isolates the increased production of AmpC, in combination or not with other resistance mechanisms, contributes to the resistance pattern in A. baumannii to ß-lactams.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Acinetobacter baumannii is an emergent opportunistic nosocomial pathogen, which very often causes nosocomial outbreaks that are difficult to control due to the ease with which this microorganism disseminates and its persistence in the hospital environment. Successive surveys have reported increased resistance in A. baumannii clinical isolates.1

A. baumannii is currently resistant to most ß-lactam antibiotics, particularly penicillins and cephalosporins, and lately to carbapenems.1,2 Ceftazidime, piperacillin and imipenem are among the most active ß-lactam antibiotics against A. baumannii. Several authors have studied the presence of ß-lactamases as a mechanism of resistance to these antibiotics.13 Some of these reports were performed using Acinetobacter calcoaceticus, since they were carried out before the studies that defined 13 genospecies, including one species for A. calcoaceticus and another for A. baumannii.1 This study aimed to investigate the distribution of ß-lactamases in a group of epidemiologically well defined A. baumannii clinical isolates. Moreover, the in vitro activity of Syn 2190, a novel AmpC inhibitor,4,5 in combination with various penicillins and cephalosporins was also determined.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

The 20 A. baumannii clinical isolates analysed during this study are part of a collection from the Hospital Clinic, and were isolated in Spanish hospitals during a 10 year period.6,7

Susceptibility testing

All susceptibility testing was by Etest (AB Biobisk, Sölna, Sweden) following the manufacturer’s recommendations. The MICs of ampicillin, piperacillin, ceftazidime, imipenem and cefepime were determined in both the presence and absence of 4 mg/L Syn 21904,5 (Naeja Pharmaceutical Inc., Edmonton, Canada). In addition, the MICs of ticarcillin, cefotaxime and cefoxitin were determined. The MIC of imipenem was also determined in the presence of 25 mg/L reserpine. Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used as quality controls.

Characterization of ß-lactamase

Determination of isoelectric points was performed as described previously.8 The presence of tem, oxa 2-like, oxa 3-like and shv-type genes was determined by PCR with primers and conditions described previously.3,8 Determination of the presence of oxa 20 and oxa 37 genes was carried out under the same conditions as the other oxa genes, but using the following primers: 5'-CACATCGGTTTATAATGAAT-3' and 5'-TTGGGTGGCAAAGCATTGACG-3'.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Twenty A. baumannii clinical isolates were studied. These isolates were identified as A. baumannii based on biochemical criteria and by amplified ribosomal DNA restriction analysis. Previous studies have shown that, with the exception of strains 6R and 67384, the isolates were epidemiologically unrelated.6,7 Susceptibility to different ß-lactam antibiotics is shown in Table 1. All strains were resistant to ampicillin. Eleven out of 20 A. baumannii strains had an MIC of ampicillin >256 mg/L, and in nine, this high level of resistance was correlated with the presence of an OXA and/or TEM ß-lactamase (Table 2). Twenty-five per cent of the strains carried and expressed a TEM-type ß-lactamase, whereas 35% of the strains had an OXA-type ß-lactamase (Table 2). Plasmid-mediated TEM-1- and TEM-2-type ß-lactamases have been described previously.1,9 In a previous study, Joly-Guillou et al.9 established a prevalence of TEM-1-type ß-lactamase in Acinetobacter spp. of c. 34%. Several OXA-type ß-lactamases have been described in A. baumannii, with most being located in an integron.2,3 The remaining 11 strains not presenting an OXA- or TEM-type ß-lactamase had a MIC range of ampicillin between 32 and >256 mg/L, and a decrease in the MIC of ampicillin was observed for all when it was determined in the presence of 4 mg/L Syn 2190, an AmpC inhibitor. These results suggest that at least 50% of the strains present an overexpressed chromosomal AmpC ß-lactamase, with different levels of expression depending on the strain. Syn 2190 is an inhibitor of AmpC ß-lactamases but does not affect other types of these enzymes.4,5 Although a CARB-type ß-lactamase has been found in an A. calcoaceticus subsp. anitratus strain,1 in our study this type of ß-lactamase was not detected. Susceptibility to piperacillin was also low, with 35% (seven out of 20) of the strains being resistant and 45% (nine out of 20) being intermediately resistant. Strains carrying the same set of ß-lactamases, for instance strains 6R, 661 and 704R producing TEM and OXA ß-lactamases, or 67384, 875 and 74I producing an OXA ß-lactamase, showed different susceptibility to the piperacillin plus Syn 2190 combination. In strains 6R, 67384 and 875, the MIC of piperacillin was >256 mg/L, in both the presence and absence of Syn 2190, whereas strains 661, 704R and 74I showed a decrease in the MIC of piperacillin from >256 to 32–64 mg/L when it was determined in the presence of the AmpC inhibitor. Three possible explanations could be posed for this phenomenon: (i) there may be a decrease in Syn 2190 penetration in strains 6R, 67384 or 875 so that it would not inhibit AmpC; (ii) a complementary mechanism of resistance to piperacillin besides the expression of ß-lactamases in these strains, such as a decrease in permeability, increased efflux or modifications in the penicillin-binding proteins; or (iii) various chromosomal cephalosporinases with different levels of inhibition by Syn 2190 could be synthesized by each strain. In fact, several cephalosporinases, which are very heterogeneous from a functional point of view, have been described.1,10


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Table 1.  MICs of different ß-lactam antibiotics, some plus Syn 2190, for 20 A. baumannii clinical isolates
 

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Table 2.  Distribution of ß-lactamases in the 20 A. baumannii clinical isolates
 
The proportion of cefotaxime-resistant strains (90%) was higher than that of ceftazidime-resistant strains (55%), whereas in general cefepime showed low activity, with 60% of the strains being resistant. For nine out of 11 (82%) ceftazidime-resistant strains, the MIC of this ß-lactam antibiotic decreased at least four-fold when determined in the presence of Syn 2190, whereas this was the case for only three of the 12 (25%) cefepime-resistant or intermediate strains. Recently, Bou & Martinez-Beltrán10 cloned an AmpC ß-lactamase from A. baumannii with a typical cephalosporinase profile that slightly affected cefepime. However, in Pseudomonas aeruginosa, a total de-repression of the AmpC enzyme compromises cefepime.11 This could be the case in the above-mentioned strains, whereas in those strains showing no change in the MIC of cefepime (>32 mg/L) in the presence of Syn 2190, the resistance could be related to decreased permeability or increased efflux. This is further supported by the fact that strains such as 77 or 31 are highly resistant to cefepime but susceptible to ceftazidime, whereas neither MIC is affected by Syn 2190.

Three out of 20 (15%) A. baumannii strains showed a MIC of imipenem >32 mg/L, indicating the presence of a carbapenemase. Several ß-lactamases affecting imipenem have been described,1,2 some of which have been located in a plasmid that can be transferred, such as ARI-1, which is now OXA-23.1 The combination of imipenem plus Syn 2190 did not modify the activity of this carbapenem, suggesting that AmpC has no effect on this antimicrobial agent. A set of oxacillinases with activity against carbapenems has recently been described.2 The imipenem-susceptible strains presented a range of imipenem MICs between 0.06 and 4 mg/L. All four strains with a MIC of imipenem of 1 mg/L had an MIC of 0.19 (three strains) and 0.38 mg/L (one strain) in the presence of reserpine (25 mg/L). Reserpine is an efflux pump inhibitor, in both Gram-positive and -negative bacteria. These results suggest that an efflux pump, inhibited by reserpine, could be involved in the moderate increase in resistance to imipenem.

In summary, despite the fact that other mechanisms of resistance to ß-lactam antibiotics, such as decreased permeability or increased efflux, may contribute to the final MIC, our results indicate that a high percentage of A. baumannii clinical isolates show increased production of AmpC, and that other mechanisms, such as OXA- and TEM-type ß-lactamases, play an important role in resistance to ß-lactam antibiotics.


    Acknowledgements
 
We would like to thank Naeja Pharmaceutical Inc. (Edmonton, Canada) for kindly providing Syn 2190. This work has been supported by grant FIS00/0997 from Fondo de Investigaciones Sanitarias.


    Footnotes
 
* Corresponding author. Tel: +34-93-2275522; Fax: +34-93-2275454; E-mail: vila{at}medicina.ub.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Vila, J. (1998). Mechanisms of antimicrobial resistance in Acinetobacter baumannii. Review of Medical Microbiology 9, 87–97.

2 . Bou, G., Oliver, A. & Martinez-Beltrán, J. (2000). OXA-24, a novel class D ß-lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain. Antimicrobial Agents and Chemotherapy 44, 1556–61.[Abstract/Free Full Text]

3 . Vila, J., Navia, M., Ruiz, J. & Casals, C. (1997). Cloning and nucleotide sequence analysis of a gene encoding an OXA-derived ß-lactamase in Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 41, 2757–9.[Abstract]

4 . Babini, G. S. & Livermore, D. M. (2000). Effect of conalbumin on the activity of Syn 2190, a 1.5 dihydroxy-4-pyridon monobactam inhibitor of AmpC ß-lactamases. Journal of Antimicrobial Chemotherapy 45, 105–9.[Abstract/Free Full Text]

5 . Nishida, K., Kunugita, C., Uji, T., Higashitani, F., Hyodo, A., Unemi, N. et al. (1999). In vitro and in vivo activities of Syn2190, a novel ß-lactamase inhibitor. Antimicrobial Agents and Chemotherapy 43, 1895–900.[Abstract/Free Full Text]

6 . Marcos, M. A., Jimenez de Anta, M. T. & Vila, J. (1995). Correlation of six methods for typing nosocomial isolates of Acinetobacter baumannii. Journal of Medical Microbiology 42, 328–35.[Abstract]

7 . Vila, J., Ruiz, J., Navia, M., Becerril, B., Garcia, I., Perea, S. et al. (1999). Spread of amikacin resistance in Acinetobacter baumannii strains isolated in Spain due to an epidemic strain. Journal of Clinical Microbiology 37, 758–61.[Abstract/Free Full Text]

8 . Navia, M. M., Capitano, L., Ruiz, J., Vargas, M., Urassa, H., Schellemberg, D. et al. (1999). Typing and characterization of mechanisms of resistance of Shigella spp. isolated from feces of children under 5 years of age from Ifakara, Tanzania. Journal of Clinical Microbiology 37, 3113–7.[Abstract/Free Full Text]

9 . Joly-Guillou, M. L., Vallée, E., Bergogne-Bérézin, E. & Philippon, A. (1988). Distribution of ß-lactamases and phenotype analysis in clinical strains of Acinetobacter calcoaceticus. Journal of Antimicrobial Chemotherapy 22, 597–604.[Abstract]

10 . Bou, G. & Martinez-Beltrán, J. (2000). Cloning, nucleotide sequencing and analysis of the gene encoding an AmpC ß-lactamase in Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 44, 428–32.[Abstract/Free Full Text]

11 . Livermore, D. M. (1995). ß-Lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 557–84.[Abstract]