5. Fontana R, Lo Cascio G, Ligozzi M, Friscia O, Oldoni T, Italian Epidemiological Observatory Collaborative Group. Antimicrobial susceptibility of respiratory isolates of Enterobacteriaceae and Staphylococcus aureus in Italy: incidence and trends over the period 1997–1999.Eur J Clin Microbiol Infect Dis2001;20: 854–863.
6. Karlowsky JA, Jones ME, Thornsberry C, Friedland IR, Sahm DF. Trends in antimicrobial susceptibilities among Enterobacteriaceae isolated from hospitalized patients in the United States from 1998 to 2001. Antimicrob Agents Chemother1998;47: 1672–1680.
7. Sader HS, Jones RN, Andrade-Baiocchi S, Biedenbach DJ, SENTRY Participants Group (Latin America). Four-year evaluation of frequency of occurrence and antimicrobial susceptibility patterns of bacteria from bloodstream infections in Latin American medical centers. Diagn Microbiol Infect Dis2002;44: 273–280.
8. Turnidge J, Bell J, Biedenbach DJ, Jones RN. Pathogen occurrence and antimicrobial resistance trends among urinary tract infection isolates in the Asia–Western Pacific Region: report from the SENTRY Antimicrobial Surveil-lance Program, 1998–1999.Int J Antimicrob Agents2002;20: 10–17.
9. Vila J, Navia M, Ruiz J, Casals C. Cloning and nucleotide sequence analysis of a gene encoding an OXA-derived
b-lactamase inAcinetobacter baumannii. Antimicrob Agents Chemother1997;41: 2757–2759.
10. Ploy MC, Giamarellou H, Bourlioux P, Courvalin P, Lambert T. Detection ofaac(6¢)-I genes in amikacin-resist-antAcinetobacterspp. by PCR.Antimicrob Agents Chemother
1994;38: 2925–2928.
11. Navia MM, Ruiz J, Sanchez-Cespedes J, Vila J. Detection of
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12. Gallardo F, Ruiz J, Marco F, Towner KJ, Vila J. Increase in incidence of resistance to ampicillin, chloramphenicol and trimethoprim in clinical isolates of Salmonella serotype Typhimurium with investigation of molecular epidemiol-ogy and mechanisms of resistance.J Med Microbiol1999;
48: 367–374.
13. Miro´ E, Navarro F, Mirelis Bet al.Prevalence of clinical isolates of Escherichia coli producing inhibitor-resistant
b-lactamases at a university hospital in Barcelona, Spain, over a 3-year period.Antimicrob Agents Chemother2002;46: 3991–3994.
14. Hannecart-Pokorni E, Depuydt F, de Wit L, van Bossuyt E, Content J, Vanhoof R. Characterization of the 6¢ -N-ami-noglycoside acetyltransferase geneaac (6¢)-Im associated with a sulI-type integron. Antimicrob Agents Chemother
1997;41: 314–318.
15. Vourli S, Tzouvelekis LS, Tzelepi E, Lebessi E, Legakis NJ, Miriagou V. Characterization of In111, a class 1 integron that carries the extended-spectrum beta-lacta-mase gene bla(IBC-1). FEMS Microbiol Lett 2003; 225: 149–153.
R E S E A R C H N O T E
IMPs, VIMs and SPMs: the diversity of
metallo-
b
-lactamases produced by
carbapenem-resistant
Pseudomonas
aeruginosa
in a Brazilian hospital
H. S. Sader1,2, A. O. Reis1, S. Silbert1and A. C. Gales11Laboratorio Alerta e Laboratorio Especial de
Microbiologia Clı´nica, Disciplina de Doenc¸as Infecciosas e Parasita´rias, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil and2The Jones Group⁄JMI Laboratories, North Liberty, IA, USA
A B S T R A C T
Pseudomonas aeruginosa isolates (n= 183), collec-ted from bacteraemic patients hospitalised in Sa˜o Paulo Hospital (Brazil) during 2000–2001, were screened for susceptibility to antimicrobial agents. The polymyxins were the most active compounds
(100% susceptibility), followed by amikacin and
cefepime (59.0%), meropenem (57.4%), and
imi-penem and gentamicin (55.2%).
Imipenem-resist-ant isolates were ribotyped and screened for
production of metallo-b-lactamases (MBLs) by
PCR with primers for blaIMP, blaVIM and blaSPM. MBL production was detected in 36 isolates
(19.7% of the entire collection; 43.9% of the
imipenem-resistant isolates) and the MBLs inclu-ded SPM-1-like (55.6%), VIM-2-like (30.6%) and
IMP-1-like (8.3%) enzymes.
Keywords Carbapenem resistance, imipenem resist-ance, metallo-b-lactamases, polymyxin, Pseudomonas aeruginosa
Original Submission:28 May 2004; Revised Submis-sion:6 July 2004; Accepted:9 August 2004
Clin Microbiol Infect2005; 11: 73–76 10.1111/j.1469-0691.2004.01031.x
Pseudomonas aeruginosa is a leading cause of hospital-acquired infections that are often difficult
Corresponding author and reprint requests: H. S. Sader, The Jones Group⁄JMI Laboratories, 345 Beaver Kreek Center, Suite A, North Liberty, IA 52317, USA
E-mail: helio-sader@jmilabs.com
Research Note
73
to treat because of the limited choice of effective antimicrobial agents. Carbapenems, mainly imi-penem and meroimi-penem, are potent agents for the treatment of infections caused by multiresistant Gram-negative bacilli, especially P. aeruginosa.
Resistance to carbapenems in P. aeruginosa has
mostly been low level (MIC 8–32 mg⁄L), and
associated with reduced uptake as a result of OprD porin loss [1]. Resistance by this mechanism depends on continued expression of the
chromo-somal AmpCb-lactamase. Low-level carbapenem
resistance can also arise via over-expression of efflux pumps. However, production of
metallo-b-lactamases (MBLs) is now being reported
increasingly as a cause of high-level carbapenem resistance (MIC > 32 mg⁄L) inP. aeruginosa[1–3].
The MBL types are IMP, VIM and the recently described SPM [2–4]. The objective of the present study was to detect and characteriseP. aeruginosa
isolates producing MBLs in a teaching hospital located in Sao Paulo, Brazil.
The present study investigated allP. aeruginosa
isolates (n= 183) from bloodstream infections
(one isolate⁄patient) during 2000 and 2001 at the
Sa˜o Paulo Hospital complex, which is a 600-bed tertiary care university hospital. The isolates were tested initially by disk diffusion [5] against imipenem, meropenem, aztreonam, cefepime, ceftazidime, amikacin, gentamicin, ciprofloxacin and polymyxin B. All isolates that were non-susceptible to imipenem or meropenem were then retested by broth microdilution (Trek Diagnostics Systems, East Grinstead, UK) according to NCCLS protocols [6] against the same group of antimicrobial agents (except that polymyxin B was replaced by colistin) plus piperacillin–tazo-bactam. Quality control strains wereP. aeruginosa
ATCC 27853, Escherichia coli ATCC 25922 and
Staphylococcus aureusATCC 29213. Isolates resist-ant to both imipenem and meropenem were screened initially for MBL production by the disk approximation test, with ceftazidime and imipe-nem as substrates, and EDTA and 2-mercapto-propionic acid as b-lactamase inhibitors [7]. The results were confirmed with Etest MBL strips (AB Biodisk, Solna, Sweden) [8].
All isolates positive for MBL production were
analysed by PCR for blaIMP-1 (sense 5¢
-CTA-CCGCAGCAGAGTCTTTGC-3¢ and antisense
5¢-GAACAACCAGTTTTGCCTTACC-3¢ primers),
blaVIM-1 (sense 5¢
-TCTACATGACCGCGTCTGTC-3¢and antisense 5¢-TGTGCTTTGACAACGTTCGC-3¢
primers), blaVIM-2 (sense 5¢
-ATGTTCAAACTTTT-GAGTAGTAAG-3¢ and antisense 5¢
-CTACTCAAC-GACT-GAGCG-3¢ primers) and bla
SPM-1 (sense
5¢-CCTACAATCTAACGGCGACC-3¢ and antisense
5¢-TC-GCCGTGTCCAGGTATAAC-3¢primers) under
standard conditions [4].
The genetic similarity of the 47
imipenem-resistant isolates (MIC‡16 mg⁄L) isolated in
2001 was investigated by automated ribotyping with the RiboPrinter Microbial Characterization System (DuPont de Nemours, Wilmington, DE, USA) [9]. In brief, this automated process includes lysis of overnight cell cultures, cleavage of DNA with the restriction enzymePvuII, size separation by means of gel electrophoresis, and modified Southern blotting. Results were analysed with the Riboprinter system, and isolates were considered to have an identical ribotype if the similarity coefficient was ‡0.93.
Overall, 82 (44.8%) of 183P. aeruginosaisolates
from bloodstream infections in 2000 and 2001 were resistant to imipenem. The highest suscep-tibility (by disk diffusion) was to polymyxin B
(100%), followed by amikacin and cefepime
(59.0%), meropenem (57.4%), imipenem and
gen-tamicin (55.2%), piperacillin–tazobactam (54.6%),
ciprofloxacin (53.5%), ceftazidime (51.4%) and
aztreonam (47.5%). Against imipenem-resistant
isolates (n= 82), the most active compound was
colistin or polymyxin E (MIC90 £0.5 mg⁄L; 100%
susceptible), followed by aztreonam (MIC50, 32 mg⁄L; 34.1% susceptible), cefepime (MIC50,
16 mg⁄L; 29.2% susceptible) and amikacin
(MIC50, > 64 mg⁄L; 29.2%susceptible).
MBL production was detected in 36 isolates,
representing 19.7% of the 183 isolates
investi-gated, or 43.9% of the 82 imipenem-resistant
isolates. Among the MBL-producing isolates, 20
(55.6%) were positive by PCR for blaSPM-1, 11
(30.6%) were positive for blaVIM-2, and three
(8.3%) were positive for blaIMP-1. Two (5.6%)
isolates did not produce PCR amplification prod-ucts with any of the primers used (Table 1). Apart from the polymyxins (100%susceptibility),
aztre-onam (55.5% susceptibility) was the most active
antimicrobial agent against the MBL-producing isolates. All isolates producing an SPM-1-like amplification product were susceptible to aztreo-nam. Susceptibilities to piperacillin–tazobactam
and amikacin were 27.7% and 25.0%,
respect-ively. All other compounds tested were active against < 15%of the 36 MBL-producing isolates.
74
Clinical Microbiology and Infection, Volume 11 Number 1, January 2005Analysis of genetic similarity by ribotyping
showed great clonal diversity among the
47 imipenem-resistant isolates collected in 2001. Twenty-five ribogroups were identified, and five of those were represented by more than one isolate (Table 1). Seven ribogroups were identi-fied among blaSPM-1-positive isolates, three
ribo-groups were found among blaVIM-2-positive
isolates, and all three blaIMP-1-positive isolates had distinct ribogroups.
Although the production of MBL is uncommon among clinical isolates of P. aeruginosa in other parts of the world, this mechanism is becoming increasingly more common in Brazil and other Latin American countries [10,11]. After the first
report of SPM-1 (Sa˜o Paulo
metallo-b-lactamase) in a P. aeruginosa isolate from this hospital (isolate 1088 in Table 1) [4], this enzyme has disseminated throughout Brazil [10].
The present study showed that P. aeruginosa
isolates causing bloodstream infections in the Sa˜o Paulo Hospital complex had high rates of resist-ance to most antimicrobial agents evaluated, and only the polymyxins showed consistent in-vitro activity. However, the role of aztreonam in the treatment of infections caused by isolates produ-cing SPM-1-like enzymes should be evaluated further.
The correlation between carbapenem usage and resistance to these compounds has been difficult to establish [12]. Although the use of carbapenems in the Sa˜o Paulo Hospital complex is restricted to treatment of infections caused by Gram-negative bacilli resistant to all other compounds, or for very special conditions, both imipenem and merope-nem are now used routinely to treatP. aeruginosa
infections because of the high rates of resistance to other compounds. The yearly usage of these two carbapenems combined varied from 1.7 to 3.8 g⁄1000 patient-days in the entire hospital,
and from 16.1 to 20.7 g⁄1000 patient-days in the
intensive care unit, during 1996–2001. In the same period, several clusters of infections caused by imipenem-resistant isolates with an identical or similar molecular fingerprinting pattern were identified. Thus, both factors (high usage of carbapenems and clonal dissemination of resistant strains) may be contributing to the increasing frequency of carbapenem-resistantP. aeruginosain this hospital.
Production of MBLs represents an important mechanism of resistance to carbapenems and
other b-lactams among P. aeruginosa isolates in
this hospital. One in five P. aeruginosa isolates
from bloodstream infections produced an MBL, and three types of MBL (IMP, VIM and SPM) were detected. The observed genetic diversity
among isolates producing each type of MBL
indicates that both horizontal and clonal dis-semination of these genes has occurred in the hospital. Strict infection control measures should be implemented to prevent further spread of these potent MBLs.
A C K N O W L E D G E M E N T S
We are very grateful to T. Walsh and M. Tolleman for providing the blaSPM-1 primers and to M. C. Tognin, L. C.
Menezes and A. P. Penteado for excellent technical support. The study received financial support from the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP, no. 2001⁄033497) and the Conselho Nacional de Desenvolvimento
Table 1. Demographics and molecular results of the 36 metallo-b-lactamase-producing isolates of Pseudomonas aeruginosa
Isolate Date
(day/month/year) Hospital unit Ribogroup PCR
1191 26⁄3⁄2001 Internal Medicine 87-3 IMP-1-like 1260 24⁄4⁄2001 Internal Medicine 90-5 IMP-1-like 955 16⁄1⁄2001 Internal Medicine 92-3 IMP-1-like 1111 20⁄2⁄2001 Internal Medicine 87-2 SPM-1-like 1991 18⁄12⁄2001 Dialysis 97-7 SPM-1-like 1971 12⁄12⁄2001 Pneumology 97-7 SPM-1-like 1088a 25⁄2⁄2001 Oncohaematology 2 87-2 SPM-1-like 1104 21⁄2⁄2001 Oncohaematology 2 87-2 SPM-1-like
1110 17⁄2⁄2001 Oncohaematology 2 87-2 SPM-1-like 1129 8⁄3⁄2001 Oncohaematology 2 87-2 SPM-1-like 1212 11⁄4⁄2001 Oncohaematology 2 87-2 SPM-1-like 1228 17⁄4⁄2001 Oncohaematology 2 87-2 SPM-1-like 1621 8⁄9⁄2001 Oncohaematology 2 89-5 SPM-1-like
1638 9⁄9⁄2001 Oncohaematology 2 89-6 SPM-1-like 929 3⁄1⁄2001 Oncohaematology 2 92-1 SPM-1-like 956 15⁄1⁄2001 Oncohaematology 2 92-4 SPM-1-like 971 19⁄1⁄2001 Oncohaematology 2 92-5 SPM-1-like 700 5⁄11⁄2000 Oncohaematology 2 NT SPM-1-like 813 1⁄12⁄2000 Oncohaematology 2 NT SPM-1-like 867 14⁄12⁄2000 Oncohaematology 2 NT SPM-1-like 318 10⁄5⁄2000 Paediatric ICU NT SPM-1-like 376 8⁄6⁄2000 Paediatric ICU NT SPM-1-like 172 21⁄3⁄2000 Unknown NT SPM-1-like 1395 24⁄6⁄2001 Adult ICU 71-2 VIM-2-like 1280 1⁄5⁄2001 Gastroenterology 71-2 VIM-2-like 1231 17⁄14⁄2001 Internal Medicine 71-2 VIM-2-like 1364 12⁄6⁄2001 Internal Medicine 71-2 VIM-2-like 1483 22⁄7⁄2001 Oncohaematology 2 71-2 VIM-2-like 1211 11⁄4⁄2001 Adult ICU 87-4 VIM-2-like 1384 20⁄6⁄2001 Adult ICU 89-2 VIM-2-like 1916 24⁄11⁄2001 Internal Medicine 97-6 VIM-2-like 1665 14⁄9⁄2001 Paediatric ICU 89-2 VIM-2-like
290 4⁄5⁄2000 Paediatric ICU NT VIM-2-like 483 10⁄8⁄2000 Infectious Diseases NT VIM-2-like 1747b 12⁄10⁄2001 Oncohaematology 1 71-2 Negative
1918b 26⁄11⁄2001 Paediatric ICU 94-7 Negative
ICU, intensive care unit; NT, not typed.
aThe prototype SPM-1 producer [4].
bIsolates with positive phenotypic test results and negative PCR results for all three
primers.
Research Note
75
Cientı´fico e Tecnolo´gico (CNPq, no. 303316⁄2002-0). The results were presented in preliminary form at the 43rd Interscience Conference on Antimicrobial Agents and Chemo-therapy (ICAAC), Chicago, IL, 14–17 September 2003.
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1. Livermore DM. Multiple mechanisms of antimicrobial resistance inPseudomonas aeruginosa: our worst nightmare?
Clin Infect Dis2002;34: 634–640.
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3. Bush K. Metallo-beta-lactamases: a class apart.Clin Infect Dis1998;27(suppl 1): S48–S53.
4. Toleman MA, Simm AM, Murphy TA et al. Molecular characterization of SPM-1, a novel metallo-beta-lactamase isolated in Latin America: report from the SENTRY anti-microbial surveillance programme.J Antimicrob Chemother
2002;50: 673–679.
5. National Committee for Clinical Laboratory Standards.
Performance standards for antimicrobial disk susceptibility tests, 8th edn. Approved standard M2-A8. Wayne, PA: NCCLS, 2003.
6. National Committee for Clinical Laboratory Standards.
Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically, 6th edn. Approved standard M7-A6. Wayne, PA: NCCLS, 2003.
7. Arakawa Y, Shibata N, Shibayama Ket al.Convenient test for screening metallo-beta-lactamase-producing gram-negative bacteria by using thiol compounds.J Clin Micro-biol, 2000;38: 40–43.
8. Walsh TR, Bolmstrom A, Qwarnstrom A, Gales A. Eval-uation of a new Etest for detecting metallo-beta-lactamases in routine clinical testing.J Clin Microbiol2002;40: 2755– 2759.
9. Pfaller MA, Wendt C, Hollis RJet al.Comparative evalu-ation of an automated ribotyping system versus pulsed-field gel electrophoresis for epidemiological typing of clinical isolates ofEscherichia coliandPseudomonas aerugi-nosafrom patients with recurrent gram-negative bactere-mia.Diagn Microbiol Infect Dis1996;25: 1–8.
10. Gales AC, Menezes LC, Silbert S, Sader HS. Dissem-ination in distinct Brazilian regions of an epidemic carbapenem-resistant Pseudomonas aeruginosa producing SPM metallo-beta-lactamase.J Antimicrob Chemother2003;
52: 699–702.
11. Sader HS, Mendes RE, Castanheira Met al.The emergence of metallo-b-lactamases and increasing carbapenem resistance amongP. aeruginosaandAcinetobacterspp. from bloodstream infections (BSI): report from the SENTRY Antimicrobial Surveillance Program [abstract C2-2028]. In:
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J Antimicrob Chemother2004;53: 290–296.
R E S E A R C H N O T E
Use of high-performance liquid
chromatography (HPLC) to monitor
b
-lactam plasma concentrations during the
treatment of endocarditis
P. Tattevin1, O. Tribut2, C. Arvieux1, M. Dupont1, R. Flicoteaux1, L. Desbordes3, Y. Le Tulzo1and C. Michelet1
1Infectious Diseases and Intensive Care Unit,
2Pharmacology and3Bacteriology, Pontchaillou
University Hospital, Rennes, France
A B S T R A C T
Guidelines recommend high doses of b-lactams
for the therapy of endocarditis. This report des-cribes a retrospective study of 15 endocarditis patients (median age 64 years), treated according
to guidelines, whose b-lactam trough plasma
concentrations were measured with high-per-formance liquid chromatography because of tol-erance or efficacy concerns. For amoxycillin, the mean level was 86.8 mg⁄L (range: 30–212 mg⁄L);
five (45%) patients had concentrations > 1000·
MIC. For cloxacillin, the mean level was
47.9 mg⁄L (range: 16.7–104 mg⁄L). The
conse-quences of high and unpredictedb-lactam trough
plasma concentrations for a prolonged period have not yet been thoroughly evaluated.
Keywords b-Lactams, endocarditis, HPLC, pharma-cokinetics, therapeutic drug monitoring, tolerance
Original Submission:15 June 2004; Revised Submis-sion:18 July 2004; Accepted:10 August 2004
Clin Microbiol Infect2005; 11: 76–79 10.1111/j.1469-0691.2004.01030.x
High doses of b-lactams are used for the
treat-ment of endocarditis [1–3], but treattreat-ment guide-lines do not take into account many factors related to the host, such as age, sex and liver function, that may affect b-lactam pharmacokinetics [3,4].
Corresponding author and reprint requests: P. Tattevin, Clinique des Maladies Infectieuses, CHU Pontchaillou, 35033 Rennes Cedex, France
E-mail: pierre.tattevin@chu-rennes.fr
