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JAC Advance Access originally published online on August 8, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):882-885; doi:10.1093/jac/dkl327
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Risk factors for nosocomial infections due to Pseudomonas aeruginosa producing metallo-ß-lactamase in two tertiary-care teaching hospitals

Alexandre Prehn Zavascki1,2,*, Afonso Luís Barth2,3, Patrick Barcelos Gaspareto3, Ana Lúcia Saraiva Gonçalves3, Ana Lúcia Didonet Moro4, Juliana Fernandez Fernandes5 and Luciano Zubaran Goldani2,6

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

*Correspondence address. Serviço de Infectologia, Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul, 6690 Ipiranga Avenue, 90610-000, Porto Alegre, Brazil. Tel/Fax: +55-51-33621850; E-mail: apzavascki{at}terra.com.br

Received 13 May 2006; returned 12 June 2006; revised 6 July 2006; accepted 15 July 2006


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Objectives: To assess risk factors for nosocomial infections due to Pseudomonas aeruginosa producing metallo-ß-lactamase (MBL-PA) in two teaching hospitals where horizontal dissemination has been demonstrated.

Methods: A case–control study was performed in both hospitals (assigned as hospital 1 and 2). Cases were patients with MBL-PA infections and controls were those with non-MBL-PA infections. Multivariate analysis was performed to identify independent risk factors.

Results: A total of 86 cases and 212 controls were included in the study. A logistic regression model showed that exposure to ß-lactams [odds ratio (OR) 3.21; 95% confidence interval (CI) 1.74–5.93] or fluoroquinolones (OR 3.50; 95% CI 1.46–8.37) was associated with MBL-PA infections. Other independent risk factors were neurological disease (OR 3.00; 95% CI 1.61–5.58), urinary tract infection (OR 2.48; 95% CI 1.21–5.09) and renal failure (OR 2.29; 95% CI 1.13–4.65). Admission to hospital 1 (OR 5.97; 95% CI 3.45–14.09) and intensive care unit stay (OR 2.07; 95% CI 1.46–3.96) were also associated with increased risk for MBL-PA infections.

Conclusions: ß-Lactam exposure is an important risk factor for MBL-PA infections even in a setting where patient-to-patient transmission plays a major role in the spread of the isolates. Other risk factors deserve further investigation, particularly exposure to fluoroquinolones.

Keywords: bacterial drug resistance , multiple bacterial drug resistance , fluoroquinolones , SPM-1 ß-lactamase , P. aeruginosa , ß-lactamases , case–control studies


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The metallo-ß-lactamases (MBLs) have recently emerged as one of the most feared resistance mechanisms because of their ability to hydrolyse virtually all ß-lactam agents, including the carbapenems, and because their genes are carried on highly mobile elements.1 The prevalence of MBL production in Pseudomonas aeruginosa, a leading cause of nosocomial infections, has been increasing in many countries from Southeast Asia, Europe, Latin America and, more recently, North America and Oceania.1

We have recently shown that P. aeruginosa producing MBL (MBL-PA) infections were associated with higher mortality rates than non-MBL-PA infections.2 We have also demonstrated that patient-to-patient transmission played a major role in the dissemination of MBL-PA isolates in institutions at which such studies were carried out.2,3

The identification of exposures associated with infection by resistant pathogens has been attempted in order to potentially correct modifiable variables and to improve knowledge of both the epidemiology and pathogenesis of resistant organisms. Only two case–control studies have assessed risk factors for acquiring MBL-PA.4,5 However, they were limited either by the lack of a multivariable analysis4 or by the small number of case patients, thereby importantly decreasing its power to detect differences between exposures.5 Moreover, risk factors specifically for infections by such organisms were not evaluated. To the best of our knowledge, this is the first study aiming to assess risk factors for nosocomial infections by MBL-PA.


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Study design

Two nested case–control studies with incident cases were performed in two tertiary-care teaching hospitals in Porto Alegre, Southern Brazil: Hospital São Lucas (hospital 1), a 600 bed hospital (from September 2004 to June 2005), and Hospital de Clínicas de Porto Alegre (hospital 2), a 1200 bed hospital (from January to June 2005). All patients with positive cultures for P. aeruginosa were eligible for the study. They were detected by daily surveillance of laboratory records. Cultures were solicited by the attendant physician when infection was suspected to occur. Those with nosocomial infections, according to the CDC criteria,6 who were aged >18 years, and without cystic fibrosis were included in the study.2

Data were collected from medical charts and/or hospital computer system databases during patients' hospitalization, in order to avoid possible missing data. The researchers were blinded for the MBL status of P. aeruginosa isolates. The ethics review boards of both hospitals have approved the study. Written informed consent was obtained from each participant.

Bacterial identification, antimicrobial susceptibility and screening for MBL production

Conventional biochemical tests were used to identify P. aeruginosa. Susceptibility was determined by the disc diffusion method according to CLSI guidelines. All isolates resistant to ceftazidime were screened for MBL production with ceftazidime in the presence of 2-mercaptopropionic acid.2

Definitions

Cases were those patients with nosocomial infections by MBL-PA and controls were those infected by non-MBL-PA. The variables potentially associated with the MBL-PA infections included: age; sex; Charlson comorbidity score;7 baseline diseases; iatrogenic immunosuppression, such as chemotherapy-induced neutropenia (neutrophil count <1000 cells/mm3), and/or receipt of corticoid drugs or other immunosuppressive agents for >14 days; time at risk (length of hospital stay before P. aeruginosa recovery); previous surgical procedure during hospital stay; hospital of admission; intensive care unit stay; site of infection; infection due to P. aeruginosa at more than one site; source from which the isolate was recovered; presence of devices (central venous catheter, endotracheal tube and urinary catheter); and antimicrobial exposure [only those used for at least 48 h during the last 14 days were analysed: ß-lactams (aztreonam, ceftriaxone, cefepime, ceftazidime, ertapenem, imipenem, meropenem or piperacillin/tazobactam), fluoroquinolones (ciprofloxacin or levofloxacin), aminoglycosides (gentamicin or amikacin), anaerobicidal agents (clindamycin or metronidazole) and vancomycin].

Statistical analysis

All statistical analyses were carried out using SPSS for Windows, Version 13.0. Univariate analysis was performed separately for each of the variables. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for binomial variables. P values were calculated using the {chi}2 test or Fisher's exact test for categorical variables and Student's t-test or Wilcoxon rank-sum test for continuous variables. Variables for which the P value was ≤0.2 in univariate analysis were included one by one in a logistic regression model according to their P value and the magnitude of their effect. A P value of 0.05 was set as the limit for acceptance or removal of the new terms in the model. All tests were two-tailed, and a P value ≤0.05 was considered significant.


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A total of 532 patients had positive cultures for P. aeruginosa. Of these, 234 patients were excluded due to the following criteria: age ≤18 years (n = 86); isolate recovered within 48 h of the patient’s admission (n = 59); cystic fibrosis (n = 52); and did not fulfil the CDC criteria for infection (n = 37). A total of 298 patients were included in the study: 159 from hospital 1 and 139 from hospital 2. Eighty-six (28.9%) patients were cases, 66 (41.5%) of 159 patients from hospital 1 and 20 (14.4%) of 139 from hospital 2, and 212 (71.1%) were controls, 93 (58.5%) from hospital 1 and 119 (85.6%) from hospital 2. Complete data were obtained from all included patients.

The incidence of infections due to MBL-PA and non-MBL-PA isolates was not significantly different in any of the months of the study at both hospitals (P = 0.11 for hospital 1 and P = 0.69 for hospital 2). The lung was the most frequent site of nosocomial infection (150 patients, 50.3%), followed by urinary tract (61, 20.5%), skin and soft tissue (47, 15.8%), intra-abdominal (25, 8.4%), central venous catheter (23, 7.7%), primary bloodstream (12, 4.0%) and others (7, 2.4%). Twenty-seven (9.1%) patients presented infections at more than one site. Resistance to antimicrobial agents of MBL-PA and non-MBL-PA isolates are summarized in Table 1.


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  		Table 1. Resistance to antimicrobial agents of case and control Pseudomonas aeruginosa isolates

 
The results of univariate analysis of risk factors for MBL-PA infections are shown in Table 2. The results of the multivariate analysis showed that recent use of a ß-lactam (OR 3.21; 95% CI 1.74–5.93) or a fluoroquinolone (OR 3.50; 95% CI 1.46–8.37) was a significant risk factor for MBL-PA infections. Other variables non-related to antibiotic exposure that were statistically significant in the multivariate model were neurological disease (OR 3.00; 95% CI 1.61–5.58), urinary tract infection (OR 2.48; 95% CI 1.21–5.09), renal failure (OR 2.29; 95% CI 1.13–4.65) and intensive care unit stay (OR 2.07; 95% CI 1.46–3.96). Admission to hospital 1 was also a significant factor associated with MBL-PA infection (OR 5.97, 95% CI 3.45–14.09). The results of multivariate analysis are presented in Table 3.


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  		Table 2. Univariate analysis of risk factors for metallo-ß-lactamase-producing Pseudomonas aeruginosa infections

 


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  		Table 3. Multivariate analysis of risk factors for metallo-ß-lactamase-producing Pseudomonas aeruginosa infections

 

   Discussion
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As it was expected, since MBLs determine resistance to virtually all ß-lactams, exposure to this class of drugs was a significant risk factor for infections by MBL-PA at both hospitals. Notably, our results indicated that the selective pressure imposed by antibiotic use, especially to ß-lactams, remains an important risk factor for infections by antibiotic-resistant P. aeruginosa when patient-to-patient transmission has been documented.2,3 It is possible that, in the setting of patient-to-patient transmission, antimicrobial exposure increases the risk for resistant organisms by creating a microbiologically favourable environment for bacterial strains, which are brought from external sources to the host, more than selecting resistant strains within a bacterial population. Dose and duration of antimicrobial therapy have also been associated with increased risk for resistant organisms, including MBL-PA.4 Unfortunately, we did not evaluate such variables, and their potential association with MBL-PA infections could not be determined in our study. Other ß-lactam agents with narrower spectrum of activity, such as first- and second-generation therapy, were not evaluated. However, this fact probably had no impact on our results, since such agents are not usually prescribed for hospitalized patients, unless for surgical prophylaxis.

Exposure to fluoroquinolones was also a risk factor for MBL-PA infections. Interestingly, fluoroquinolones have been previously reported as a risk factor for SPM-1 MBL-PA,5 which was the MBL type produced by the isolates of the present study (data regarding molecular analysis of the isolates are described in detail elsewhere).2 Such an association might be accounted for by the fact that the blaSPM-1 gene is surrounded by mobile genetic regions closely related to Salmonella enterica serovar Typhimurium, which are in turn associated with other mobile elements called STX regions.1 These regions can be mobilized under bacterial stress, and it has been shown that resistance elements linked to STX regions increased 300-fold when bacteria were exposed to fluoroquinolones.8

Admission to hospital 1 was an independent risk factor for infections by MBL-PA, probably owing to the higher prevalence of MBL production by P. aeruginosa isolates from this hospital. Intensive care unit stay also increased the risk for MBL-PA infections even adjusting for potential confounders such as antimicrobial exposure. We believe that a higher rate of cross-transmission could have occurred at these units, despite the adoption of contact isolation. No other specific medical or surgical ward was associated with higher incidence of MBL-PA infections at both hospitals (data not shown).

Renal failure and urinary tract infections were interesting findings of our study. Renal failure has been found to be a risk factor for imipenem-resistant P. aeruginosa in a previous study conducted at hospital 1,9 and the urinary catheter has also been reported as a risk factor for multidrug-resistant P. aeruginosa10 and for IMP-1 MBL-PA.4 In addition, resistance among isolates recovered from urine has usually been reported as higher than from other sites,4 and chronic renal failure and dialysis were among the few variables significantly associated with SPM-1 MBL in univariate analysis of the study by Nouér et al.5 It has been demonstrated that substances eluted from siliconized latex urinary catheters were related to the loss of the OprD outer membrane protein, leading to imipenem resistance,11 but there is no reported mechanism associating MBL-mediated resistance with urinary catheter or another factor related to renal diseases.

Neurological disease was a significant risk factor for MBL-PA infections. There is no obvious explanation for such an association but it might be possible that an unknown factor could mediate it. No geographical or temporal clustering was found among these patients (data not shown). However, being bedridden, a frequent condition among neurological patients, has been found as a risk factor for multidrug-resistant P. aeruginosa.10,12 Bedridden status might favour a higher rate of cross-transmission as a result of a more frequent contact with healthcare professionals, because of their limited capacity to perform usual activities.

We used patients with non-MBL-PA infections as controls rather than a sample of hospitalized patients selected by a criterion other than infection by P. aeruginosa, as proposed elsewhere,13 because an important limitation of the latter design is that it is not possible to distinguish whether the variables are risk factors for the resistant pathogen or for the pathogen regardless of the resistance profile.14 However, it must be noted that the design adopted in our study tends to overestimate the magnitude of the effect of antibiotic exposure.13,14

In conclusion, exposure to ß-lactams is a significant risk factor for MBL-PA infections even when patient-to-patient transmission plays a major role in the dissemination of isolates. Fluoroquinolones may be a risk factor for SPM-1 MBL through a mobilization of SPM-1 genetic apparatus. The findings that variables related to urinary tract diseases were risk factors for resistant P. aeruginosa in this and other studies deserve further investigation. Strategies improving infection control measures, including antibiotic use policies and isolation of patients with MBL-PA, are urgently required to prevent an increase in MBL-PA nosocomial infections.


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None to declare.


   Acknowledgements
 
We are grateful to Andreza Francisco Martins, Cláudia Meirelles Leite and Fabiano Ramos for their contributions to this work. This work was supported by 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.


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1 Walsh TR, Toleman MA, Poirel L, et al. (2005) Metallo-ß-lactamases: the quiet before the storm? Clin Microbiol Rev 18:306–25.[Abstract/Free Full Text]

2 Zavascki AP, Barth AL, Gonçalves ALS, et al. (2006) The influence of metallo-ß-lactamase production on mortality in nosocomial Pseudomonas aeruginosa infections. J Antimicrob Chemother 58:387–92.[Abstract/Free Full Text]

3 Zavascki AP, Gaspareto PB, Martins AF, et al. (2005) Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing SPM-1 metallo-ß-lactamase in a teaching hospital in southern Brazil. J Antimicrob Chemother 56:1148–51.[Abstract/Free Full Text]

4 Hirakata Y, Yamaguchi T, Nakano M, et al. (2003) Clinical and bacteriological characteristics of IMP-type metallo-ß-lactamase-producing Pseudomonas aeruginosa. Clin Infect Dis 37:26–32.[CrossRef][Web of Science][Medline]

5 Nouér SA, Nucci M, de-Oliveira MP, et al. (2005) Risk factors for acquisition of multidrug-resistant Pseudomonas aeruginosa producing SPM metallo-ß-lactamase. Antimicrob Agents Chemother 49:3663–7.[Abstract/Free Full Text]

6 Garner JS, Jarvis WR, Emori TG, et al. (1988) CDC definitions for nosocomial infections. Am J Infect Control 16:128–40.[CrossRef][Web of Science][Medline]

7 Charlson ME, Pompei P, Ales KL, et al. (1987) A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis 40:373–83.[CrossRef][Web of Science][Medline]

8 Beaber JW, Hochhut B, Waldor MK. (2004) SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427:72–4.[CrossRef][Medline]

9 Zavascki AP, Cruz RP, Goldani LZ. (2005) Risk factors for imipenem-resistant Pseudomonas aeruginosa: a comparative analysis of two case-control studies in hospitalized patients. J Hosp Infect 59:96–101.[CrossRef][Web of Science][Medline]

10 Defez C, Fabbro-Peray P, Bouziges N, et al. (2004) Risk factors for multidrug-resistant Pseudomonas aeruginosa nosocomial infection. J Hosp Infect 57:209–16.[CrossRef][Web of Science][Medline]

11 Conejo MC, Garcia I, Martinez-Martinez L, et al. (2003) Zinc eluted from siliconized latex urinary catheters decreases OprD expression, causing carbapenem resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 47:2313–5.[Abstract/Free Full Text]

12 Aloush V, Navon-Venezia S, Seigman-Igra Y, et al. (2006) Multidrug-resistant Pseudomonas aeruginosa: risk factors and clinical impact. Antimicrob Agents Chemother 50:43–8.[Abstract/Free Full Text]

13 Harris AD, Karchmer TB, Carmeli Y, et al. (2001) Methodological principles of case-control studies that analysed risk factors for antibiotic resistance: a systematic review. Clin Infect Dis 32:1055–61.[CrossRef][Web of Science][Medline]

14 Zavascki AP. (2004) Assessing risk factors for acquiring antimicrobial-resistant pathogens: a time for a comparative approach. Clin Infect Dis 39:871–2.[CrossRef][Web of Science][Medline]


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