J Antimicrob Chemother 1999; 44: 135

Sir,

Although the carbapenem antibiotics have the broadest antibacterial activity of all β -lactams, strains of Pseudomonas aeruginosa resistant to these agents have been reported. The mechanisms of the acquired imipenem resistance in this species may result from a decrease in the uptake of carbapenems and from an increased expression of the pump efflux. In P. aeruginosa imipenem proved to be capable of penetrating cells via an outer membrane protein, the OprD porin, and the absence of this channel resulted in the acquisition of imipenem resistance, reducing meropenem susceptibility, but not affecting the activity of other β -lactams. 1 Recently, plasmid mediated carbapenemase, which confers high-level resistance to several β-lactams including carbapenems, was found. 2,3 The report of these enzymes was restricted to Japan, although recently, a carbapenemase producer in a colonizer isolate in the UK was detected. 4

In the present work we describe the carbapenemase activity observed in three imipenem-resistant P. aeruginosa isolates clinically responsible for urinary and lower respiratory tract infections in patients in a Portuguese hospital. MICs for these isolates were determined by Etest. Isoelectric focusing and β-lactamase activity were evaluated as detailed previously. 5

High-level MICs of imipenem (.32 mg/L), piperacillin–tazobactam (48–256 mg/L) and cefpirome (64–256 mg/L), and susceptibility to aztreonam (6–8 mg/L) were found. In contrast to the previous reports of carbapenemases, 3,4 meropenem and ceftazidime were more active, 0.5–0.75 mg/L and 24–48 mg/L, respectively. A multiresistant phenotype, which includes amikacin, tobramycin, gentamicin, ciprofloxacin and chloramphenicol, was shared by all the isolates.

Carbapenemase activity was demonstrated in the crude sonicates of cell suspensions by UV spectrophotometry with 0.1 mM of imipenem in 50 mM phosphate buffer, pH 7.0. Imipenem was hydrolysed (36.5–59.6 U/mg protein) and when the suspension was incubated with EDTA 2 mM, the activity of the enzyme was completely lost (0.7–1 U/mg protein). Activity was restored by addition of divalent cations, such as Zn2+ (9.4–19.6 U/mg protein). One unit of enzyme activity was defined as the amount of enzyme needed to hydrolyse 1 µ mol of imipenem per min at 30°C. These results were further confirmed by a biological method. The extracts and imipenem 10 mg/L were incubated together for 15 min before being transferred to wells cut into a bioassay plate containing Mueller–Hinton agar, the surface of which was inoculated with Escherichia coli ATCC 25922. The extracts of all isolates hydrolysed the imipenem. Inhibition of the activity over imipenem was obtained when the extracts were incubated with EDTA 2 mM. This metallo-β-lactamase with a pI >.9.0, as predicted by isoelectric focusing, was observed in the three nosocomial P. aeruginosa isolates. As OprD expression has been implicated as a resistance mechanism in imipenem-resistant isolates, a normal expression of this protein in these clinical isolates allowed us to exclude the contribution of this porin in the behaviour observed.

Apart from Japan, this is the first report of evidence of carbapenemase production by P. aeruginosa isolates responsible for nosocomial infections. As the previously reported carbapenemases showed different MICs from this one, a molecular and enzymatic characterization of this new enzyme is being undertaken.

The emergence of this new carbapenemase is of great concern, as carbapenems are often used as treatment of last resort against infections caused by Gram-negative bacteria that are resistant to other antimicrobial agents.

*
Corresponding author. Tel: +35-1-39-852567; Fax: +35-1-39-852569; E-mail: ocardoso@ci.uc.pt

References

1.
Hancock, R. E. W. (
1998
). Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative Gram-negative bacteria.
Clinical Infectious Disease
 
27, Suppl. 1
,
S93
–9.
2.
Watanabe, M., Iyobe, S., Inoue, M. & Mitsuhashi, S. (
1991
). Transferable imipenem resistance in Pseudomonas aeruginosa.
Antimicrobial Agents and Chemotherapy
 
35
,
147
–51.
3.
Woodford, N., Palepou, M.-F. I., Babini, G. S., Bates, J. & Livermore, D. M. (
1998
). Carbapenemase-producing Pseudomonas aeruginosa in UK.
Lancet
 
15
,
546
–7.
4.
Senda, K., Arakawa, Y., Nakashima, K. et al. (
1996
). Multifocal outbreaks of metallo-β-lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum β-lactams, including carbapenems.
Antimicrobial Agents and Chemotherapy
 
40
,
349
–53.
5.
Livermore, D. M. & Williams, J. D. (
1996
). β-lactams: mode of action and mechanisms of resistance. In Antibiotics in Laboratory Medicine, 4th edn (Victor Lorian, Ed.), pp. 502–78. Baltimore MD.