The influence of metallo-b-lactamase production on mortality in nosocomial Pseudomonas aeruginosa infections

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1 Journal of Antimicrobial Chemotherapy Advance Access published June 3, 2006 Journal of Antimicrobial Chemotherapy doi: /jac/dkl239 The influence of metallo-b-lactamase production on mortality in nosocomial Pseudomonas aeruginosa infections Alexandre Prehn Zavascki 1,2 *, Afonso Luís Barth 2,3, Ana Lúcia Saraiva Gonçalves 3, Ana Lúcia Didonet Moro 4, Juliana Fernandez Fernandes 5, Andreza Francisco Martins 3, Fabiano Ramos 1 and Luciano Zubaran Goldani 2,6 1 Infectious Diseases Service, Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil; 2 Medicine: Medical Sciences Postgraduate Program, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; 3 Microbiology Unit, Clinical Pathology Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 4 Medical School, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil; 5 Medical School, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; 6 Division of Infectious Diseases, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil Received 8 February 2006; returned 21 April 2006; revised 25 April 2006; accepted 12 May 2006 Objectives: To assess the effect of metallo-b-lactamase (MBL) production on Pseudomonas aeruginosa nosocomial infection mortality and to identify the determinants of such effect. Methods: A cohort study of patients with P. aeruginosa nosocomial infections was conducted at two teaching hospitals. MBL was detected by ceftazidime/2-mercaptopropionic disc approximation test and selected isolates were submitted to PCR using bla SPM-1 primer. Molecular typing was performed by DNA macrorestriction. To evaluate the influence of MBL on mortality a Cox proportional hazards model was performed using a hierarchized framework of the variables. Results: A total of 298 patients with P. aeruginosa infections were included. Infections by MBL-carrying Pseudomonas aeruginosa (MBL-PA) resulted in higher in-hospital mortality than those by non-mbl-pa (51.2% versus 32.1%, respectively; relative risk 1.60, 95% CI ) and higher mortality rates [17.3 per 1000 versus 11.8 per 1000 patient-days, respectively; hazard ratio (HR) 1.55, 95% CI ]. In the final multivariat model, severe sepsis or septic shock [adjusted HR (AHR) 3.62, 95% CI ], age (AHR 1.02, 95% CI ) and use of appropriate therapy 72 h (AHR 0.49, 95% CI ) were significantly associated with mortality. Fourteen MBL-PA tested carried the bla SPM-1 gene. Clonal dissemination was documented in both hospitals. Conclusions: MBL-PA infections resulted in higher mortality rates most likely related to the severity of these infections and less frequent early institution of appropriate antimicrobial therapy. Empirical treatments should be reviewed at institutions with high prevalence of MBL. Keywords: P. aeruginosa, resistance, b-lactamases, nosocomial infections Introduction Pseudomonas aeruginosa is a leading cause of nosocomial infections. 1 Infections by this organism are usually associated with high mortality regardless of appropriate antimicrobial therapy. 1 Treatment options for P. aeruginosa infections are usually scarce owing to a combination of inherent resistance to many drug classes and its ability to acquire resistance to many drug classes. 1 In recent years, the metallo-b-lactamases (MBLs) have emerged as one of the most feared resistance mechanisms because of their ability to hydrolyse virtually all b-lactam agents, including the carbapenems, and because their genes are carried on highly mobile elements. 2 So far, five major clinically important groups of MBLs have been identified: IMP, VIM, SPM, GIM and the recently described SIM. 2,3 Initially limited to south-east Asia, MBLs have rapidly spread through Europe, followed by Latin... *Correspondence address. Serviço de Infectologia, Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul, 6690 Ipiranga Avenue, , Porto Alegre, Brazil. Tel/Fax: ; apzavascki@terra.com.br... Page 1 of 6 Ó The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 Zavascki et al. America, especially after Recently, MBLs have been described in North America and Oceania, while their prevalence has continued to increase in many countries from the other continents, giving the problem a worldwide dimension. 2,4 The spread of these enzymes in nosocomial Gram-negative rods, particularly among P. aeruginosa isolates, severely limits therapeutic options for infections by these pathogens. 2 Although the potential threat of acquired MBLs is no longer questioned, the clinical impact of and the optimal therapy for infections caused by organisms producing these enzymes remain unknown. 2 Two studies have compared mortality between P. aeruginosa producing MBLs (MBL-PA) and non-mbl- PA. 5,6 However, these studies have neither controlled for potential confounding factors associated with mortality nor evaluated antimicrobial therapeutic options for these infections. The aim of the present study was to assess the influence of MBL production on the mortality of patients with P. aeruginosa nosocomial infections and to investigate which factors determine this effect through the construction of a multivariate model using a hierarchized framework of the potential confounding variables. Patients and methods Study design A prospective, longitudinal study of consecutive patients with P. aeruginosa nosocomial infections was performed at two tertiarycare teaching hospitals in Porto Alegre, southern Brazil. The study period was from September 2004 to June 2005 at São Lucas Hospital (SLH), a 600 bed hospital, and from January to June 2005 at Hospital de Clínicas de Porto Alegre (HCPA), a 1200 bed hospital. Patients with positive cultures for P. aeruginosa were eligible for the study. They were excluded if they were 18 years of age, if they had cystic fibrosis, if P. aeruginosa was recovered <48 h after hospital admission (unless the patient had been hospitalized in the past 60 days) or if they did not fulfil the CDC criteria for infections. 7 Patients were followed from the first isolation of P. aeruginosa to discharge from hospital or death. Data were collected from medical charts and/or hospital computer system databases, during and after the patients hospitalization. The researchers were blinded for the MBL status of P. aeruginosa isolates. The study was approved by ethics review boards of both the hospitals. Written informed consent was obtained from each participant. Microbiology, MBL identification and molecular typing Biochemical tests which included oxidase, oxidation of glucose on OF-medium, arginine and nitrate, growth in cetrimide agar and production of characteristic pigments (blue and green) were used to identify P. aeruginosa. Susceptibility to amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem and piperacillin tazobactam was determined by the disc diffusion method according to CLSI (formerly NCCLS) guidelines. 8 Polymyxin B susceptibility was determined using the interpretative criteria ( 14 mm) proposed elsewhere. 9 All isolates resistant to ceftazidime were screened for MBL production with ceftazidime in the presence of 2- mercaptopropionic acid as described previously. 10 A sample of randomly selected MBL-PA isolates was submitted to PCR for detection of the bla SPM-1 gene. 11 DNA macrorestriction using SpeI followed by PFGE was performed for molecular typing of randomly selected isolates. 12 Restriction fragment profiles were compared visually and were analysed using the Tenover criteria. 13 Variables and definitions The primary outcome was in-hospital mortality for any cause. The variable studied was MBL production. Secondary outcomes were occurrence of bacteraemia, microbiological response (at least one negative culture from the infection site after successful treatment of infection), length of hospital stay after infection, length of need for vasoactive drugs and length of mechanical ventilation (the latter three outcomes were assessed in survivors). Potentially confounding variables were categorized into three groups. (i) Variables related to the patients baseline status: age; sex; Charlson comorbidity score; 14 baseline diseases; iatrogenic immunosuppression, such as chemotherapy-induced neutropenia (neutrophil count < 1000 cells/mm 3 ) and/or receipt of corticoid drugs (prednisolone 10 mg/daily or equivalent doses) or other immunosuppressive agents for >14 days; presence of other concomitant infections (infections by other organisms at a site other than that infected by P. aeruginosa, excluding coagulase-negative staphylococci in a single blood culture); and previous surgical procedure during hospital stay. (ii) Variables related to the infection: presentation with severe sepsis or septic shock; 15 site of infection; infection by P. aeruginosa at more than one site (not including patients with an identified primary site and bacteraemia); polymicrobial infection (isolation of another organism from the same site of infection); and associated bacteraemia (isolation of P. aeruginosa from one or more blood samples). (iii) Variables related to the treatment of infections: receiving appropriate empirical therapy (defined as the administration of an antimicrobial agent to which the isolate was susceptible in vitro in 24 h of sample collection); time to adequacy of therapy (only for those who have not received appropriate empirical therapy: time in days from the sample collection to the first dose of appropriate therapy); surgical procedure for treatment of the infection, when it was needed; development of treatment toxicities: nephrotoxicity, hepatotoxicity and haematotoxicity (only those which were not explained by other causes and led to interruption of treatment were considered); and treatment with antibiotic combinations (treatment with more that one agent with in vitro susceptibility). Aminoglycosides in monotherapy were not considered appropriate treatment despite in vitro susceptibility. 16 Statistical analysis All statistical analyses were carried out using SPSS for Windows, version Bivariate analysis was performed separately for each of the variables. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated for binomial variables. P values were calculated using the c 2 or Fisher s exact test for categorical variables and the Student s t-test or the Wilcoxon rank-sum test for continuous variables. Survival curves were prepared with a Kaplan Meier estimation and compared using the log-rank test. A Cox proportional hazards model was used to assess the effect of MBL production on mortality rate adjusting for potential confounders. Adjusted hazard ratios (AHRs) and 95% CIs were calculated. The model was constructed using a hierarchized framework of the variables as proposed elsewhere. 17 Variables for which the P value was 0.20 in bivariate analysis were included in the model according to the following sequence: Step 1: variables related to the patients baseline status; Step 2: variables accepted in step 1 plus those related to the infection; Step 3: variables accepted after step 2 plus those related to the treatment. A P value of 0.05 was set as the limit for acceptance or removal of the new terms in each step. MBL production was included and remained in the model independent of the P value. Proportional hazards assumption was graphically Page 2 of 6

3 Influence of metallo-b-lactamase on P. aeruginosa infection mortality checked. Tests for interactions were not performed. All tests were two-tailed and a P 0.05 was considered significant. The rationale for the hierarchized approach was to assess which factors mediate the effect of MBL production on mortality. Each step shows the effect of MBL production on mortality which is not mediated by the specific group of variables included in the model as well as those accepted from previous steps. Our previous hypothesis was that MBL-PA infections were significantly associated with increased mortality and, using the hierarchized approach, it would be expected that MBL production would lose its statistical significance or substantially decrease its magnitude of the effect when variables which mediate such effect were included in the model. Cox proportional hazards models were performed for both the subgroup of patients with MBL-PA infections using the above criteria and for those who received appropriate therapy, including variables that were a priori judged clinically relevant, in order to evaluate different antibiotic agents for the treatment of MBL-PA. These subgroup analyses were a priori planned, and no other subgroup analysis was performed. Results A total of 532 patients presented the isolation of P. aeruginosa (287 from HCPA and 245 from SLH). Of them 234 patients were excluded due to the following criteria: age 18 years (n = 86), isolate recovered <48 h of admission (n = 59), cystic fibrosis (n = 52) and did not fulfil the CDC criteria for infection (n = 37). A total of 298 patients were analysed. Eighty-six (28.9%) patients presented MBL-PA infections. The overall in-hospital mortality of patients with P. aeruginosa infections was 37.6% (112 of 298): 51.2% (44 of 86) for patients with MBL-PA infections and 32.1% (68 of 212) for patients with non-mbl-pa infections (RR 1.60, 95% CI , P = 0.003). The mortality rate was 17.3 per 1000 patient-days among MBL-PA-infected patients and 11.8 per 1000 patientdays among non-mbl-pa-infected ones (HR 1.55, 95% CI , P = 0.02) (Figure 1). The characteristics of patients as well as variables related to presentation of infections and treatments according to in-hospital mortality are shown in Table 1. Table 2 presents the results of multivariable analysis. Step 1 showed that MBL production (AHR 1.49, 95% CI , P = 0.05), higher age (AHR 1.02, 95% CI , P < 0.001) and higher Charlson score (AHR 1.09, 95% CI , P = 0.02) were significantly associated with death. Malignancy, neurological disease and cardiac disease were not included in the model because they were highly associated with Charlson score (data not shown). With the inclusion of variables related to infection in the model (step 2), the effect of MBL production on mortality lost its statistical significance (AHR 1.37, 95% CI , P = 0.12). Severe sepsis or septic shock (AHR 3.45, 95% CI , P < 0.001) and age (AHR 1.02, 95% CI , P = 0.003) were significantly associated with death. In the last step, variables related to the treatment were included in the model. MBL production remained statistically non-significant and the magnitude of its effect on mortality decreased substantially (AHR 1.07, 95% CI , P = 0.73). Variables associated with in-hospital mortality in the final model were severe sepsis or septic shock (AHR 3.62, 95% CI , P < 0.001), age (AHR 1.02, 95% CI , P = 0.006) and initiation of Survival probability (%) MBL negative MBL positive P = Days Number at risk MBL positive MBL negative Figure 1. Survival curves of 298 patients with Pseudomonas aeruginosa infection according to metallo-b-lactamase (MBL) production. appropriate antimicrobial therapy 72 h of the onset of infection (AHR 0.49, 95% CI , P = 0.001). Occurrence of bacteraemia was more frequent among MBL- PA than non-mbl-pa (24.4% versus 10.8%, respectively; RR 2.25, 95% CI , P = 0.004). The number of cultures performed per days of hospitalization after infection ratio did not differ between the groups (median 0.48 for MBL-PA versus 0.44 for non-mbl-pa; P = 0.59). Five (26.3%) of 19 MBL-PA patients presented microbiological response versus 15 (39.5%) of 38 non-mbl-pa (RR 0.67, 95% CI , P = 0.49). Among survivors, MBL-PA patients presented a significantly longer length of hospital stay [median 28 days, interquartile range (IQR) versus 20 days, IQR 12 35, P = 0.009] and need for vasoactive drug therapy (median 15 days, IQR 3 20 versus 2 days, IQR 2 6; P = 0.006) than non-mbl-pa ones. The length of mechanical ventilation was also longer among MBL-PAinfected patients, although without statistical significance (median 15 days, IQR 7 20 versus 8 days, IQR 3 12, P = 0.25). A total of 63 antibiotic resistance profiles were identified among the isolates, but only 6 among the 86 MBL-PA, including susceptibility only to polymyxin B and aztreonam (34 isolates, 39.5%); susceptibility only to polymyxin B (30, 34.9%); susceptibility to polymyxin B, aztreonam and amikacin (11, 12.7%); susceptibility to polymyxin B and piperacillin tazobactam (7, 8.1%); susceptibility to polymyxin B, aztreonam and piperacillin tazobactam (3, 3.8%); and susceptibility to polymyxin B and ciprofloxacin (1, 1.2%). Fourteen MBL-PA were tested for the presence of MBL genes and all were positive for bla SPM-1. Of 18 MBL-PA isolates from SLH submitted to molecular typing, 17 (94.9%) were clonally related and only 1 showed a distinct DNA macrorestriction profile. Ten (100%) of ten MBL-PA from HCPA submitted to molecular typing comprised a single clone. A subgroup analysis was performed for the 86 patients with MBL-PA infections. Variables included in the Cox model were Page 3 of 6

4 Zavascki et al. Table 1. Characteristics of 298 patients, and variables related to presentation of infections and treatments, according to in-hospital mortality Variables non-survivors (n = 112) Hospital mortality survivors (n = 186) P Related to the patient age, years sex, male 75 (67.0) 107 (57.5) Charlson score, median (25 75% 4 (2 6) 3 (1 5) percentile) Comorbidities neurological 45 (40.2) 52 (28.0) 0.04 cardiac 64 (57.1) 88 (47.3) 0.13 pulmonary 40 (35.7) 53 (28.5) 0.37 malignancy 33 (29.5) 32 (17.2) 0.02 diabetes 29 (25.9) 46 (24.7) 0.93 renal 25 (22.3) 43 (23.1) 0.99 cirrhosis 13 (11.6) 14 (7.5) 0.33 AIDS 9 (8.2) 10 (5.4) 0.51 Iatrogenic immunosuppression 42 (37.5) 60 (32.3) 0.42 Other infections 44 (39.3) 38 (20.4) Previous surgery 53 (47.3) 94 (50.5) 0.68 Related to the infection severe sepsis or septic shock 76 (67.9) 34 (18.3) site of infection lower respiratory tract 73 (65.2) 77 (41.4) urinary tract 16 (14.3) 45 (24.2) 0.21 skin and soft tissue 13 (11.6) 34 (18.3) 0.27 central venous catheter 6 (5.4) 17 (9.1) 0.37 intra-abdominal 7 (6.3) 18 (9.7) 0.43 primary bloodstream 6 (5.4) 6 (3.2) 0.55 others 3 (2.7) 8 (4.3) 0.38 >1 site 11 (9.9) 18 (9.7) 0.87 polymicrobial 52 (46.4) 65 (34.9) 0.07 bacteraemia 24 (21.4) 20 (10.8) 0.02 Related to the treatment appropriate empirical therapy 18 (16.1) 62 (33.3) time to adequacy of therapy, days antibiotic combinations 11 (9.8) 27 (14.5) 0.29 surgical procedure 15 (13.2) 44 (23.7) 0.05 toxicity 3 (3.2) 8 (4.9) 0.47 Values are number (%) or mean SD unless otherwise indicated. age (P = 0.09), Charlson score (P = 0.03), severe sepsis or septic shock (RR 3.14, 95% CI , P < 0.001), lower respiratory tract infection (RR 1.83, 95% CI , P = 0.009) and appropriate therapy 72 h (RR 0.44, 95% CI , P = 0.04). The variables independently associated with in-hospital mortality among MBL-PA were appropriate therapy 72 h (AHR 0.33, 95% CI , P = 0.001) and severe sepsis or septic shock (AHR 4.38, 95% CI , P < 0.001) (Figure 2). Table 2. Multivariate analysis of factors associated with Pseudomonas aeruginosa nosocomial infections mortality Variables AHR (95% CI) P Step 1 MBL production 1.49 ( ) 0.05 age 1.02 ( ) <0.001 Charlson score 1.09 ( ) 0.02 sex, male 1.23 ( ) 0.31 other infection 1.03 ( ) 0.88 Step 2 MBL production 1.37 ( ) 0.12 age 1.02 ( ) severe sepsis or septic shock 3.45 ( ) <0.001 Charlson score 1.08 ( ) 0.06 polymicrobial infection 1.31 ( ) 0.18 lower respiratory tract infection 1.30 ( ) 0.21 bacteraemia 0.92 ( ) 0.73 Step 3 MBL production 1.07 ( ) 0.73 age 1.02 ( ) severe sepsis or septic shock 3.62 ( ) <0.001 appropriate therapy 72 h 0.49 ( ) surgical procedure 0.69 ( ) 0.19 AHR, adjusted hazard ratio; MBL, metallo-b-lactamase. Of these 86 patients 39 received appropriate antibiotic therapy (Table 3). No specific antimicrobial scheme was significantly associated with lower in-hospital mortality (P = 0.37). Patients treated only with b-lactams had lower in-hospital mortality than patients treated with polymyxin B, although without statistical significance (30.8% versus 45.5%, respectively; RR 0.68, 95% CI , P = 0.31). Mortality rates were 7.1 per 1000 patientdays and 10.3 per 1000 patient-days for those treated with b- lactams and polymyxin B, respectively (P = 0.38). Two patients who received appropriate combination therapy with polymyxin B plus aztreonam were excluded from this latter analysis. There was also no statistically significant difference in mortality rates between those treated with b-lactams (AHR 0.69, 95% CI , P = 0.51) and those treated with polymyxin B after adjusting for severe sepsis or septic shock and appropriate therapy 72 h. Discussion The present study showed that MBL-PA infections resulted in higher mortality rates than non-mbl-pa infections. The severity of MBL-PA infections partially mediated the effect of MBL production on mortality, since the inclusion of variables related to the infection in the Cox model, such as severe sepsis or septic shock, has made MBL production statistically non-significant and has decreased the AHR from 1.49 to Nevertheless, it seems that the inappropriateness of early therapy was the major determinant of higher mortality among MBL-PA patients, because the magnitude of the effect of MBL production on mortality decreased substantially with the inclusion of appropriate therapy 72 h in the multivariate model (AHR 1.49 in step 1, 1.37 in step 2, and 1.07 in step 3). Moreover, despite the loss of statistical Page 4 of 6

5 Influence of metallo-b-lactamase on P. aeruginosa infection mortality Survival probability (%) Appropriate therapy 72 h No appropriate therapy 72 h P < Days Number at risk Therapy 72 h No therapy 72 h Figure 2. Estimated survival curves of 86 patients with metallo-b-lactamaseproducing Pseudomonas aeruginosa infections according to appropriate therapy 72 h, adjusted for severe sepsis or septic shock. Table 3. Antibiotic therapy and mortality of 39 patients with metallo-b-lactamase-producing Pseudomonas aeruginosa infections who received appropriate therapy Treatment Total n = 39 (%) Non-survivors n = 15 (%) DDD, mean SD a ATM 15 (38.5) 5 (33.3) POB 6 (15.4) 4 (66.7) TZP 7 (17.9) 3 (42.9) POB + b-lactam without 5 (12.8) 1 (20.0) susceptibility POB + ATM b 2 (5.1) 2 (100.0) ATM + AMK 1 (2.6) 0 (0.0) ATM + b-lactam without 1 (2.6) 0 (0.0) susceptibility ATM + (AMK + 1 (2.6) 0 (0.0) b-lactam, both without susceptibility) TZP + b-lactam without susceptibility 1 (2.6) 0 (0.0) DDD, defined daily dose; ATM, aztreonam; POB, polymyxin B; TZP, piperacillin/tazobactam; AMK, amikacin. a DDDs were polymyxin, B 2.0 mg/kg; aztreonam, 6.0 g; and piperacillin/ tazobactam, 13.5 g. b DDD for polymyxin B. significance in step 2 of multivariate analysis, MBL production clearly tended to maintain its effect on mortality regardless of the inclusion of infection-related variables (AHR 1.37; 95% CI , P = 0.12). Hirakata et al. 5 have shown that infection-related death was significantly higher among MBL-PA patients than non-mbl-pa patients (5.8% versus 1.2%, respectively; Odds Ratio 5.00, 95% CI ). Laupland et al. 6 have reported a higher case-fatality among MBL-PA patients than non-mbl-pa patients (25% versus 13%; RR 1.98, 95% CI ). In both studies, it was suggested that MBL-PA isolates presented higher virulence than non-mbl-pa ones, based on higher mortality rates among MBL- PA patients and higher frequency of infections in such group. 5,6 Unfortunately, both were limited by the lack of a multivariable analysis to control for important confounders. Assuming that severe sepsis or septic shock would be in some degree caused by the virulence of the organisms, a potential higher virulence of MBL-PA isolates might be inferred from the fact that such manifestations partially mediated the effect of MBL on mortality. In addition, MBL-PA-infected patients from our study presented more bacteraemia and survivors presented a significantly longer need for vasoactive drug therapy than those infected by non-mbl-pa. Most of isolates were susceptible only to aztreonam and polymyxin B, or only to polymyxin B, and only a few remained susceptible to ciprofloxacin and amikacin. Susceptibility to piperacillin tazobactam was detected in some MBL-PA isolates. This interesting finding has previously been reported in some IMP- and VIM-producing P. aeruginosa. 18,19 We also showed that the only MBL type among our isolates was SPM-1, which is the most prevalent enzyme in Brazil. 11 The finding that almost all MBL-PA genotyped isolates comprised a single strain indicates that horizontal transmission continues to be a major determinant of the spread of MBL as reported previously. 11 This is a worrisome finding because patient-to-patient transmission may render MBL-PA strains endemic in our institutions. Patients treated with b-lactam-based therapies had mortality rates comparable to those treated with polymyxin B-based therapies, suggesting that aztreonam, and maybe piperacillin tazobactam, might be useful agents in the armamentarium against these infections, if there was in vitro susceptibility. Besides the fact that only a few isolates were tested for the presence of MBL genes and submitted to molecular typing, a limitation of our study was that only 39 patients with MBL-PA infections received appropriate antimicrobial therapy, thereby limiting the study power to detect differences between treatments. This subgroup multivariable model had fewer than 10 outcome events per independent variable, thus, potentially compromising its accuracy due to model overfitting, and its results should be parsimoniously analysed. 20 Although small differences in susceptibility profiles can occur, MBL-PA isolates usually have similar profiles regardless of the type of enzyme. Thus, we think that many of our findings could be generalized for organisms producing other MBL types, although virulence factors can differ among distinct organisms and MBL types. In conclusion, patients with MBL-PA infections presented higher mortality than those with non-mbl-pa infections. Early appropriate antimicrobial therapy may be the only modifiable factor able to decrease mortality. Hospitals with high prevalence of MBL-PA should review empirical therapeutic approaches in order to include organisms producing these enzymes. Aztreonam and, perhaps, piperacillin tazobactam seem to be effective options for the treatment of MBL-PA infections if there is in vitro susceptibility. Since novel therapeutics against Gram-negative rods are not a near prospect and the emergence of this resistance mechanism is a real threat for almost all Page 5 of 6

6 Zavascki et al. available options, measures to control the spread of MBLmediated resistances are urgently required. Acknowledgements We are grateful to Patrick Barcelos Gaspareto, Cláudia Meirelles Leite, Larissa Lutz, Denise Pires Machado and Rodrigo Pires dos Santos for their support with the microbiological tests. This study has received financial support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Ministry of Education, Brazil, and Fundação de Incentivo a Pesquisa e Eventos (FIPE), Hospital de Clínicas de Porto Alegre. Transparency declarations None to declare. References 1. Livermore DM. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare? Clin Infect Dis 2002; 34: Walsh TR, Toleman MA, Poirel L et al. Metallo-b-lactamases: the quiet before the storm? Clin Microbiol Rev 2005; 18: Lee K, Yum JH, Yong D et al. Novel acquired metallo-b-lactamase gene, bla(sim-1), in a class 1 integron from Acinetobacter baumannii clinical isolates from Korea. Antimicrob Agents Chemother 2005; 49: Peleg AY, Franklin C, Bell JM et al. Dissemination of the metallob-lactamase gene blaimp-4 among Gram-negative pathogens in a clinical setting in Australia. Clin Infect Dis 2005; 41: Hirakata Y, Yamaguchi T, Nakano M et al. Clinical and bacteriological characteristics of IMP-type metallo-b-lactamase-producing Pseudomonas aeruginosa. Clin Infect Dis 2003; 37: Laupland KB, Parkins MD, Church DL et al. Population-based epidemiological study of infections caused by carbapenem-resistant Pseudomonas aeruginosa in the Calgary health region: importance of metallo-b-lactamase (MBL)-producing strains. J Infect Dis 2005; 192: Garner JS, Jarvis WR, Emori TG et al. CDC definitions for nosocomial infections. Am J Infect Control 1988; 16: National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA, Gales AC, Reis AO, Jones RN. Contemporary assessment of antimicrobial susceptibility testing methods for polymyxin B and colistin: review of available interpretative criteria and quality control guidelines. J Clin Microbiol 2001; 39: Arakawa Y, Shibata N, Shibayama K et al. Convenient test for screening metallo-b-lactamase-producing Gram-negative bacteria by using thiol compounds. J Clin Microbiol 2000; 38: Zavascki AP, Gaspareto PB, Martins AF et al. Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing SPM-1 metallo-b-lactamase in a teaching hospital in southern Brazil. J Antimicrob Chemother 2005; 56: Kaufmann ME. Pulsed-field gel electrophoresis. Methods Mol Med 1998; 15: Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: Charlson ME, Pompei P, Ales KL et al. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis 1987; 40: Bone RC, Balk RA, Cerra FB et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992; 101: Giamarellou H. Prescribing guidelines for severe Pseudomonas infections. J Antimicrob Chemother 2002; 49: Victora CG, Huttly SR, Fuchs SC et al. The role of conceptual frameworks in epidemiological analysis: a hierarchical approach. Int J Epidemiol 1997; 26: Gibb AP, Tribuddharat C, Moore RA et al. Nosocomial outbreak of carbapenem-resistant Pseudomonas aeruginosa with a new bla(imp) allele, bla(imp-7). Antimicrob Agents Chemother 2002; 46: Crespo MP, Woodford N, Sinclair A et al. Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-8, a novel metallo-b-lactamase, in a tertiary care center in Cali, Colombia. J Clin Microbiol 2004; 42: Concato J, Feinstein AR, Holford TR. The risk of determining risk with multivariable models. Ann Intern Med 1993; 118: Page 6 of 6