1 Infectious Diseases Service, Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil; 2 Microbiolgy Unit, Clinical Pathology Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil
Received 20 March 2005; returned 15 September 2005; revised 21 September 2005; accepted 3 October 2005
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: From January to October 2004, carbapenem-resistant P. aeruginosa (CRPA) were recovered from hospitalized patients. Mortality, site of infection/colonization, patient location and susceptibility profiles were analysed. A sample of CRPA was screened for MBL production, evaluated for the presence of blaSPM-1, blaIMP-1 and blaVIM-2 genes by PCR and submitted for molecular typing by DNA macrorestriction.
Results: A total of 135 CRPA (one isolate per patient) were recovered. Two major antibiotic susceptibility profiles comprised 63.7% of the isolates (susceptibility to polymyxin B and aztreonam, and susceptibility only to polymyxin B). Thirty-five CRPA were screened for MBL production (10 isolates from April, June and July, and 25 from September and October) and 27 (77.1%) proved to be positive for MBL production. Twenty-one of the 24 CRPA tested carried the blaSPM-1 gene. The mortality of patients with CRPA was 48.1% and no variable was associated with death. Molecular typing revealed the presence of a clone with four related subtypes among the blaSPM-1-positive CRPA.
Conclusions: The prevalence of MBL production by CRPA is high and horizontal transmission is a major determinant for the spread of SPM-1 CRPA among patients in this institution. As infection control measures failed to control the spread of CRPA, continuous surveillance for MBL production is warranted.
Keywords: P. aeruginosa , ß-lactamases , antibiotic resistance , carbapenems , nosocomial infections
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The dissemination of an epidemic P. aeruginosa producing SPM-1, an MBL described in Brazil, has been demonstrated in distinct regions of this country.4 At the Hospital São Lucas (HSL), a 600-bed tertiary-care Brazilian teaching hospital, high carbapenem resistance rates have been reported among P. aeruginosa.5 The aim of this study was to describe the first nosocomial outbreak of P. aeruginosa producing SPM-1 MBL in southern Brazil.
![]() |
Patients and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The study was conducted at a 600-bed tertiary-care teaching hospital in Porto Alegre, southern Brazil. HSL has a 13-bed adult general ICU, two (15- and 7-bed) adult cardiac ICUs, a 12-bed paediatric ICU and a 38-bed neonatal ICU. From January to October 2004, P. aeruginosa isolates resistant to imipenem and/or meropenem were recovered from hospitalized patients. Information about the patients was obtained from medical records and from the local infection control committee's database. The following variables were analysed: mortality, site of infection/colonization, patient location and susceptibility profile.
Characterization of isolates
Biochemical tests were used to identify P. aeruginosa which included: oxidase, oxidation of glucose on OF-medium, arginine and nitrate, growth in cetrimide agar and the production of characteristic pigments (blue and green). Susceptibility was determined by the disc-diffusion method according to NCCLS guidelines.6 Selected isolates were tested with ceftazidime in the presence of 2-mercaptopropionic acid to screen for MBLs.7
Carbapenemase identification
PCR for detection of the blaSPM-1 gene was performed on total DNA using SPM primers (forward: 5'-CCTACAATCTAACGGCGACC-3', reverse: 5'-TCGCCGTGTCCAGGTATAAC-3'). The cycling parameters were 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 1 min and extension at 68°C for 1 min, and a final extension at 68°C for 5 min. PCR products were visualized by electrophoresis on 2.0% agarose gels stained with 1% ethidium bromide. Isolates producing MBL in the screening test but without PCR amplification product for blaSPM-1 were further tested using blaIMP-1 and blaVIM-2 primers as previously described.8
Molecular typing
DNA macrorestriction using SpeI followed by PFGE was performed as previously described.9 Restriction fragment profiles were compared visually and interpreted according to the criteria of Tenover et al.10
Statistical analysis
All statistical analyses were carried out using SPSS for Windows, Version 10.0. Bivariate analysis was performed for each variable. P values were calculated by 2 test. A P value <0.05 was considered to be significant. The 95% confidence interval (CI) of the prevalence of MBL production was calculated.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Susceptibility rates of the 135 CRPA were as follows: aztreonam, 57.7%; ceftazidime, 23.7%; piperacillin/tazobactam, 17.0%; amikacin, 14.0%; cefepime, 11.8%; and ciprofloxacin, 5.9%. Five (3.7%) of the 135 isolates were resistant to imipenem and susceptible to meropenem; the remaining isolates were resistant to both carbapenems. All CRPA were susceptible to polymyxin B. Nineteen antibiotic susceptibility profiles were detected among the CRPA: five major profiles comprised 79.2% of the isolates and two comprised 63.7% (Table 1). The remaining 14 profiles accounted for 20.8% of the CRPA (2.2% or less each one).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since 2000 it has been noted at HSL that there is an increasing incidence of CRPA, with resistance rates to imipenem and meropenem of 58.9% and 50.0%, respectively, in 200203.5 This fact has lead the local infection control committee's members to adopt contact precautions for patients colonized or infected by CRPA. Regardless of these measures, resistance rates did not decrease. According to the present study, 57% of P. aeruginosa presented resistance to imipenem and/or meropenem.
The present report showed an extremely high prevalence of MBL production by CRPA isolates in HSL, as 77.1% of tested CRPA proved to be MBL producers (80% of CRPA from April, June and July, and 76.0% from September and October). Excluding polymyxin B, aztreonam was the agent which displayed the highest in vitro activity against CRPA (57.7%), and antibiotic profile 1 (susceptibility only to polymyxin B and aztreonam) was the most common (37.0%). This profile is compatible with MBL production, since aztreonam is stable against these carbapenemases.3 However, a considerable number of CRPA isolates (26.7%) had antibiotic profile 2 (similar to profile 1, but also fully or intermediately resistant to aztreonam), suggesting the co-existence of another mechanism of resistance among these isolates.
The molecular typing indicated the presence of a single clone with four related subtypes among blaSPM-1-positive CRPA. This finding suggests that horizontal transmission is a major determinant of the spreading of CRPA producing SPM-1 among adult patients in HSL. No environmental reservoirs were examined in any hospital ward and this warrants attention in further studies.
All CRPA producing MBL from September and October proved to carry the blaSPM-1 gene, except one from October (Table 2). This isolate was further tested for the presence of the blaIMP-1 and blaVIM-2 genes, but it did not produce PCR amplification products with any of the primers used (data not shown), suggesting the presence of another MBL gene among CRPA from HSL.
The high mortality (48.1%) among the patients with CRPA may reflect difficulties in antimicrobial therapy choices. However, as neither potential confounding variables were analysed nor data from carbapenem-susceptible P. aeruginosa patients were compared, such hypothesis must be confirmed with appropriate methodology.
A limitation of the present study was that MBL screening was not applied to all the CRPA, and we could not precisely determine the prevalence of MBL production among all isolates. However, the calculated 95% CI (59.989.6%) indicates that this prevalence remains high even considering the lower limit of the interval as the true prevalence of MBL production.
MBL production is a major clinical and public health problem since it has been increasingly reported and poses a challenge to antimicrobial therapy. This study described the dissemination of CRPA producing SPM-1 MBL in a tertiary-care teaching hospital. The failure of infection control measures in reducing the rates of carbapenem-resistant isolates is worrying, as this may lead to an endemic presence of MBL producers in this institution. Continuous surveillance for MBL production is needed in institutions with endemic or epidemic carbapenem-resistant non-fermentative Gram-negative bacilli or Enterobacteriaceae.
![]() |
Transparency declarations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Livermore DM. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare? Clin Infect Dis 2002; 34: 63440.[CrossRef][ISI][Medline]
3.
Walsh TR, Toleman MA, Poirel L et al. Metallo-ß-lactamases: the quiet before the storm? Clin Microbiol Rev 2005; 18: 30625.
4.
Gales AC, Menezes LC, Silbert S et al. Dissemination in distinct Brazilian regions of an epidemic carbapenem-resistant Pseudomonas aeruginosa producing SPM metallo-ß-lactamase. J Antimicrob Chemother 2003; 52: 699702.
5. Zavascki AP, Cruz RP, Goldani LZ. High rate of antimicrobial resistance in Pseudomonas aeruginosa at a tertiary-care teaching hospital in southern Brazil. Infect Control Hosp Epidemiol 2004; 25: 8057.[ISI][Medline]
6. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility TestingTwelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA, 2002.
7.
Arakawa Y, Shibata N, Shibayama K et al. Convenient test for screening metallo-ß-lactamase-producing gram-negative bacteria by using thiol compounds. J Clin Microbiol 2000; 38: 403.
8. Sader HS, Reis AO, Silbert S et al. IMPs, VIMs and SPMs: the diversity of metallo-ß-lactamases produced by carbapenem-resistant Pseudomonas aeruginosa in a Brazilian hospital. Clin Microbiol Infect 2005; 11: 736.[CrossRef][ISI][Medline]
9. Kaufmann ME. Pulsed-field gel electrophoresis. In: Woodford N, Johnson AP, eds. Molecular Bacteriology. Protocols and Clinical Applications. New Jersey: Humana Press Inc., 1998; 3350.
10.
Tenover F, Arbeit R, Goering R et al. Interpreting chromossomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 22339.
|