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Letters to the Editor / International Jour

Specific mechanisms of resistance that affect meropenemo a greater degree, such as overexpression of efflux pumps,ave not been detected in our A. baumannii collection.n the other hand, carbapenemases such as OXA-58 andIM-1, which hydrolyse imipenem more efficiently thaneropenem, have been disseminated among our A. bau-annii clones [9,10]. However, the mechanisms underly-

ng resistance to carbapenems in A. baumannii are usuallyultiple and those associated with �-lactamase-independent

esistance, such as low permeability of the outer membrane,educed affinity of penicillin-binding proteins and/or upreg-lated efflux, are not easily defined in acinetobacters [3].herefore, much of the resistance in our isolates may dependn impermeability or other combined mechanisms that affectainly imipenem.The present results support the observation that pat-

erns of resistance to carbapenems in A. baumannii mayary markedly between distinct geographic regions and thatusceptibility status provided by systems which use onlymipenem should not be used to predict susceptibility for

eropenem, or vice versa. Moreover, they underline the needor extension of valuable tools such as MYSTIC and SEN-RY in order to monitor carbapenem susceptibility trendsnd to guide local interventions.

eferences

[1] Wroblewska MM, Rudnicka J, Marchel H, Luczak M. Multidrug-resistant bacteria isolated from patients hospitalised in Intensive CareUnits. Int J Antimicrob Agents 2006;27:285–9.

[2] Pournaras S, Markogiannakis A, Ikonomidis A, et al. Outbreak of mul-tiple clones of imipenem-resistant Acinetobacter baumannii isolatesexpressing OXA-58 carbapenemase in an intensive care unit. J Antimi-crob Chemother 2006;57:557–61.

[3] Coelho J, Woodford N, Turton J, Livermore DM. Multiresistantacinetobacter in the UK: how big a threat? J Hosp Infect 2004;58:167–9.

[4] Jones RN, Sader HS, Fritsche TR, Rhomberg PR. Carbapenem sus-ceptibility discords among Acinetobacter isolates. Clin Infect Dis2006;42:158.

[5] Rhomberg PR, Jones RN. Antimicrobial spectrum of activity formeropenem and nine broad spectrum antimicrobials: report from theMYSTIC Program (2002) in North America. Diagn Microbiol InfectDis 2003;47:365–72.

[6] Garcia-Rodriguez JA, Jones RN. Antimicrobial resistance in gram-negative isolates from European intensive care units: data from theMeropenem Yearly Susceptibility Test Information Collection (MYS-TIC) programme. J Chemother 2002;14:25–32.

[7] Jones RN, Sader HS, Beach ML. Contemporary in vitro spectrumof activity summary for antimicrobial agents tested against 18569strains non-fermentative Gram-negative bacilli isolated in the SENTRYAntimicrobial Surveillance Program (1997–2001). Int J AntimicrobAgents 2003;22:551–6.

[8] Clinical and Laboratory Standards Institute. Performance standardsfor antimicrobial susceptibility testing; 15th informational supplement.

M100-S15. Wayne, PA: CLSI; 2005.

[9] Poirel L, Marque S, Heritier C, Segonds C, Chabanon G, NordmannP. OXA-58, a novel class D �-lactamase involved in resistance to car-bapenems in Acinetobacter baumannii. Antimicrob Agents Chemother2005;49:202–8.

ctbo

ntimicrobial Agents 28 (2006) 374–378 377

10] Franceschini N, Caravelli B, Docquier J-D, et al. Purification and bio-chemical characterization of the VIM-1 metallo-�-lactamase. Antimi-crob Agents Chemother 2000;44:3003–7.

Alexandros IkonomidisSpyros Pournaras

Antonios N. ManiatisDepartment of Clinical Microbiology,

University of Thessalia, Mezourlo,Larissa, Greece

Nicholas J. LegakisAthanassios Tsakris∗

Department of Microbiology, Medical School,University of Athens, 115 27 Athens, Greece

∗ Corresponding author. Tel.: +30 210 746 2140fax: +30 210 746 1489

E-mail address: atsakris@med.uoa.gr(A. Tsakris)

oi:10.1016/j.ijantimicag.2006.07.007

esistance to imipenem and zinc in clinical isolates ofseudomonas aeruginosa

Sir,Pseudomonas aeruginosa is an opportunistic pathogen

apable of causing severe infections in immunocompro-ised patients [1]. It is characterised by a naturally high

evel of resistance to antimicrobial agents, heavy metalsnd solvents [2] owing to the low permeability of its outerembrane together with the presence of multiple efflux

umps [3] belonging to the resistance, nodulation, cell divi-ion (RND) transporter family. The RND type efflux pumpzcCBA, recently described by Perron et al. [4], contributes

o the intrinsic resistance of P. aeruginosa to zinc and cad-ium as well as to the carbapenem antibiotic imipenem.o-regulation between carbapenem influx and heavy metalfflux was found to be responsible for the cross-resistancebserved in P. aeruginosa. CzcCBA is regulated by thewo-component system czcR–czcS. In that study, the authorselected mutants resistant both to heavy metals (zinc, cad-ium and cobalt) and to imipenem by exposing a wild-

ype strain to high concentrations of zinc (20, 25 or 30 mMnCl2). Sequencing the czcR–czcS DNA region in zinc-nd imipenem-resistant mutants revealed the presence of a194L mutation in the CzcS sensor protein. A second muta-

ion (G197D), also located in the CzcS protein, was foundn an environmental isolate obtained from a polluted riverhat was further exposed to zinc in the laboratory. Resistant

utants show increased transcriptional levels of czcR and

zcC, whilst oprD expression is reduced. Overexpression ofhe mutated CzsS protein in a susceptible wild-type strain,ut not of the wild-type form, resulted in a decreased level ofprD expression.

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78 Letters to the Editor / International Jour

On the other hand, zinc has been found to be releasedrom urinary catheters, resulting in antibiotic resistance [5–7]ediated by repression of OprD. Perron et al. [4] reported that

o stable mutant constitutively resistant to zinc and imipenemould be obtained after 6 days of growth in a latex urinaryatheter, but they suggest that such mutants might appear inivo at a low frequency.

In this study, susceptibility to zinc was tested in 57mipenem-resistant clinical isolates of P. aeruginosa col-ected from 1998 to 2005 in our hospital. The maximumolerable concentration to zinc was determined by com-arison with the wild-type strain PA01. The clinical iso-ates, as well as the control strain PA01, were grown in

ueller–Hinton supplemented with 1, 5, 10, 15, 20, 25 and0 mM ZnCl2 in double spots. The highest concentrationolerated by the control strain and by 54 of the 57 clin-cal isolates was 15 mM ZnCl2. Only three isolates wereble to grow in media containing 20 mM ZnCl2, the low-st concentration of zinc used by Perron et al. [4] to obtainutants resistant to both imipenem and zinc. To determinehether the resistance phenotype to zinc and imipenem wasue to mutations in the czcS gene, as described by Perront al. [4], we sequenced the region of the gene that con-ained both of the mutations. None of the three isolates con-ained mutations despite being resistant to both imipenem andinc.

Zinc concentrations released into urine from siliconizedatex urinary catheters reached 1 mM, which is significantlyower than the concentration used by Perron et al. [4] tobtain stable, constitutively resistant mutants. The presencef micromolar amounts of zinc in urine or in Mueller–Hintons sufficient to induce repression of oprD and to causeesistance to imipenem. In P. aeruginosa, imipenem resis-ance is mainly due to mutations affecting expression of theprD porin [8]. In our opinion, concentrations of ZnCl2 of0 mM or higher are able to exist in metal-polluted envi-onmental ecological niches, but such levels of zinc are nothysiologically tolerable or possible in humans because ofheir toxicity. Our results do not call into question a co-egulation mechanism between imipenem and zinc resis-

ance but they do suggest that P. aeruginosa is able toownregulate the expression of oprD by another mechanismhat does not necessarily imply the occurrence of mutationsn czcS. d

ntimicrobial Agents 28 (2006) 374–378

cknowledgment

This study was supported by grant number SAF2003-1241 from the Spanish Ministry of Education and Science.

eferences

1] Rosenfeld M, Ramsey BW, Gibson RL. Pseudomonas acquisition inyoung patients with cystic fibrosis: pathophysiology, diagnosis and man-agement. Curr Opin Pulm Med 2003;9:492–7.

2] Wang CL, Michels PC, Dawson SC, et al. Cadmium removal by a newstrain of Pseudomonas aeruginosa in aerobic culture. Appl EnvironMicrobiol 1997;63:4075–8.

3] Nikaido H. Prevention of drug access to bacterial targets: permeabilitybarriers and active efflux. Science 1994;264:382–8.

4] Perron K, Caille O, Rossier C, van Delden C, Dumas JL, KohlerT. CzcR–CzcS, a two-component system involved in heavy metaland carbapenem resistance in Pseudomonas aeruginosa. J Biol Chem2004;279:8761–8.

5] de Haan KE, Woroniecka UD, Boxma H, de Groot CJ, van den HamerCJ. Urinary zinc excretion in a patient with burns: a caveat when usingbladder catheters in urine zinc studies. Burns 1990;16:393–5.

6] Conejo MC, Garcia I, Martinez-Martinez L, Picabea L, Pascual A.Zinc eluted from siliconized latex urinary catheters decreases OprDexpression, causing carbapenem resistance in Pseudomonas aeruginosa.Antimicrob Agents Chemother 2003;47:2313–5.

7] Martınez-Martınez L, Pascual A, Conejo MC, Picabea L, Perea EJ.Resistance of Pseudomonas aeruginosa to imipenem induced by elu-ates from siliconized latex urinary catheters is related to outer mem-brane protein alterations. Antimicrob Agents Chemother 1999;43:397–9.

8] Yoneyama H, Nakae T. Mechanism of efficient elimination of proteinD2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa.Antimicrob Agents Chemother 1993;37:2385–90.

Carmen Velasco a,∗Jose Manuel Rodrıguez a

M. Carmen Conejo a

Alvaro Pascual a,b

a Department of Microbiology, School of Medicine,Av. Doctor Fedriani s/n, 41071 Seville, Spain

b University Hospital Virgen Macarena, Seville, Spain

∗ Corresponding author. Tel.: +34 954 552 863fax: +34 954 377 413

E-mail address: cvelasco@us.es(C. Velasco)

oi:10.1016/j.ijantimicag.2006.08.001