INTRODUCTION: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has been isolated with increasing frequency in Brazilian hospitals. Since June 2003, its detection in a teaching hospital in the city of Florianópolis, Brazil, has increased. This study aimed to investigate the minimal inhibitory concentration (MIC), presence of Metallo-β-lactamase (MβL) and a possible clonal relationship among the isolates.
METHODS: The study included 29 CRPA and seven isolates with reduced susceptibility. The MIC was determined by agar-dilution. Detection of MβL was performed by Double Disk Sinergism (DDS) and Combined Disk (CD). The MβL gene was verified by PCR and nucleotide sequence analysis. Epidemiological typing was performed by pulsed-field gel electrophoresis.
RESULTS: Among the 29 carbapenem-resistant isolates, polymyxin B presented 100% susceptibility and piperacillin/tazobactam 96.7%. Seventeen (62%) strains were verified as clonal (A clone) and among these, six isolates indicated phenotypically positive tests for MβL and harbored the blaSPM-1 gene. The first CRPA isolates were unrelated to clone A, harbored blaIMP-16and were phenotypically positive only by CD.
CONCLUSIONS: The spread of a high-level of resistance clone suggests cross transmission as an important dissemination mechanism and has contributed to the increased rate of resistance to carbapenems. This study emphasizes the need for continuous surveillance and improved strategies
Key-words: Pseudomonas aeruginosa. Carbapenem resistance. Nosocomial infections.
INTRODUÇÃO: O isolamento de Pseudomonas aeruginosa resistente aos carbapenêmicos (PARC) tem sido cada vez mais frequente nos hospitais brasileiros. O presente estudo investigou a concentração inibitória mínina (CIM), a presença de metalo-β-lactamases (MβL), e uma possível relação clonal entre PARC isoladas entre junho de 2003 a junho de 2005, em um hospital escola na cidade de Florianópolis, Brasil.
MÉTODOS: O estudo incluiu 29 PARC e sete isolados com suscetibilidade reduzida. A CIM foi determinada por diluição em ágar. A detecção de MβL foi realizada por sinergismo de duplo disco (SDD) e disco combinado (DC). Genes para MβL foram pesquisados por PCR e confirmados pela análise da sequência de nucleotídeos. A tipagem epidemiológica foi realizada por gel de eletroforese em campo pulsátil.
RESULTADOS: Entre os 29 isolados resistentes aos carbapenêmicos, 100% apresentaram suscetibilidade a polimixina B, e 96,7% a piperacilina/tazobactam. Dezessete (62%) destes isolados pertenciam a um mesmo clone (clone A); entre estes, seis isolados apresentaram testes fenotípicos positivos para MβL e carreavam o gene blaSPM-1. O primeiro isolado PARC não foi relacionado ao clone A, carreava o gene blaIMP-16 e foi fenotipicamente positivo somente por DC.
CONCLUSÕES: A propagação de um clone com alto nível de resistência sugere a transmissão cruzada como um importante mecanismo de disseminação e tem contribuído para o aumento nos níveis de resistência aos carbapenêmicos. Este estudo enfatiza a necessidade de vigilância contínua e melhoramento nas estratégias de controle de infecção nesta instituição.
Palavras-chaves: Pseudomonas aeruginosa. Resistência aos carbapenêmicos. Infecção nosocomial.
Pseudomonas aeruginosa, one of the main microorganisms that cause nosocomial infections1,2, is known for its intrinsic resistance to a range of antimicrobial agents3. It can also become resistant to all commercially available antimicrobial agents by developing numerous resistance mechanisms2,3. For two decades, carbapenems were considered an excellent therapeutic choice for such infections. However, Pseudomonas aeruginosa resistant to this class of antimicrobial agent has been isolated with increasing frequency in Brazilian hospitals, mainly in Intensive Care Units4,5. Studies suggest that infection caused by carbapenem-resistant Pseudomonas aeruginosa(CRPA) significantly increases mortality in critically ill patients6,7. The most common form of resistance is through either lack of drug penetration (i.e. porin mutations and efflux pumps) and/or carbapenem-hydrolyzing β-lactamases, such as metallo-β-lactamases (MβL)3. It has been suggested that in the absence of MβL, high imipenem resistance rates in isolates can show great genomic variability, which can be associated with continuous selection of resistant mutants8. However, in many geographic regions of Brazil, this has been associated with the dissemination of an epidemic clone that produces SPM MβL9. Clinical isolates of CRPA have been increasingly detected since June 2003 in a teaching hospital in the city of Florianópolis, Santa Catarina, Brazil. The aim of this study was to investigate the minimum inhibitory concentration (MIC) for antipseudomonas antimicrobials, the presence of MβL and a possible clonal relationship among isolates.
This study included clinical isolates of CRPA from patients at the University Hospital, Federal University of Santa Catarina (HU/UFSC), between June 2003 and June 2005. Twenty-nine isolates showed high-level resistance to carbapenems and seven isolates presented reduced susceptibility. The samples came mainly from patients in the Intensive Care Unit (32.8%) and Internal Medicine Unit III (19.8%), but isolates from other inpatient units were also collected. The bacteria were identified by conventional biochemical tests in accordance with the published recommendations10.
Antimicrobial susceptibility test
The MIC of bacterial isolates was determined for each of nine antimicrobial agents (amikacin, ceftazidime, aztreonam, cefepime, ciprofloxacin, imipenem, meropenem, piperacillin/tazobactam and polymyxin B), performed by the agar dilution method and interpreted in accordance with CLSI11. Pseudomonas aeruginosa ATCC 27853 was used for quality control.
Phenotypic detection of metallo-β-lactamase
Two methods were used to screen the isolates for MβL detection: the double-disk synergy test12(DDS), using 2-mercaptopropionic acid (MPA) (Sigma, Steinheim, Germany), and the ceftazidime disk (CAZ – 30µg), placed 20 mm away; and the combined disk13 (CD) test, using disk of imipenem (IMI – 10µg) with and without ethylenediaminetetraacetic acid (EDTA) (930µg) (Invitrogen, SanDiego, USA). DDS test results were considered positive if the growth inhibition zone increased or if a ghost zone appeared, while the CD test was considered positive if the increase in zone diameter was > 7mm. SPM-1-producing Pseudomonas aeruginosa and IMP-producing Acinetobacter baumannii were used as positive controls.
Molecular detection of metallo-β-lactamase genes
All isolates were tested for the presence of blaSPM-1, blaIMP, blaVIM genes by polymerase chain reaction (PCR) using primers, as previously described14. Total DNA was obtained by boiling bacterial cells. PCR conditions used for blaSPM and blaVIM were performed according to Toleman et al15, while the conditions used to detect the blaIMP gene were previously described by Gales et al16. Positive controls for blaSPM-1, blaVIM-1 and blaIMP genes were run simultaneously.
Amplicons obtained from an SPM-producing isolate and from the IMP-producing isolate were sequenced, using the set of primers previously described. The amplification products for the blaIMP-1 and blaSPM-1 genes were purified using a GFX-TM PCR purification kit (Amersham Bioscience, Piscataway, USA). The sequences were identified with MegaBACETM (Amersham Bioscience, Piscataway, USA), analyzed with ChromasPro version 1.33 (Technelysium Pty LTDA), and compared with GenBank database sequences using BLAST tool (http://www.ncbi.nih.gov/BLAST).
Pulsed-field gel electrophoresis (PFGE)
DNA of all isolates was prepared as described previously17 and cleaved with SpeI (10U) (Fermentas, Glen Burnie, USA) at 37°C. Electrophoresis was performed on a CHEF – DRIII (Bio-rad Laboratories, Hercules, USA) for 23h at 6V/cm at 12ºC and pulse times from 5 to 60 s. The gels were analyzed with Gel-Pro Analyzer 4.0 and NTSYS 2.02 software. Clusters of possibly related isolates were identified using the Dice similarity coefficient and unweighted pair-group method with arithmetic averages (UPGMA). Identical isolates were assigned the same capital letter. Isolates with more than 90% similarity were assigned as a subtype of the major type, which was designated with the same capital letter followed by an Arabic number (e.g. A1, A2, A3, A4).
Pseudomonas aeruginosa resistant to carbapenems (imipenem and meropenem), were isolated from the urinary tract (37.9%), bloodstream (31%), respiratory tract (13.8%) and from other anatomical sites (17.3%). All of them were susceptible to polymyxin B, and 96.7% to piperacillin/tazobactam. Susceptibility to the other antimicrobial agents tested was infrequent, 6% to aztreonam and cefepime; 10% to ciprofloxacin; 16.7% to amikacin and 43% to ceftazidime. Six out of the seven isolates that showed reduced susceptibility to carbapenems presented reduced susceptibility only to meropenem (MIC 8µg/mL), while the remaining isolate showed reduced susceptibility to both carbapenems tested (meropenem and imipenem) (MIC 8µg/mL).
Isolates that showed intermediate susceptibility to carbapenems presented negative results in the phenotypic test for MβL. When the 29 carbapenem-resistant isolates were tested, seven MβL-producing isolates were detected by the CD test and six isolates by DDS. When PCR was used to detect MβL genes, six of the seven isolates phenotypically positive for MβL yielded a 650 bp product compatible with a fragment amplified from blaSPM-1, while one isolate yielded a 590 bp product compatible with a fragment amplified from blaIMP. The remaining isolates did not generate PCR products. The results of PCR for blaSPM-1 confirmed the findings of both phenotypic methods used (DDS and CD). The positive isolate for blaIMP by PCR was also positive in the CD test, but produced a false negative result in DDS. Sequencing of the respective PCR products confirmed that the genes implicated were blaSPM-1 and blaIMP-16 (Table 1).
PFGE genotyping of the samples, isolated at the HU/UFSC during the period studied (Figure 1), revealed the presence of a clone (clone A) that included 17 (62%) of the 29 samples of CRPA and comprised all SPM-1 positive strains. The remaining eight were unrelated, except for two strains that were clonal (clone J).
IMP-16 producing Pseudomonas aeruginosa was the first strain resistant to carbapenems at the HU/UFSC and was not related to any other isolates.
The first Pseudomonas aeruginosa representatives of A clone (subtype A3), isolated in March 2004, harbored blaSPM-1 gene. In the following months, five additional positive blaSPM-1 isolates belonging to A clone subtypes were isolated in different units of the hospital.
Pseudomonas aeruginosa was one of the main pathogens involved in nosocomial infection during the study period. Carbapenems were considered an excellent therapeutic choice for treatment of these infections; however, the increasing resistance to these agents verified in the institution (13% in 2003, 32% in 2004 and 44% in 2005) was concerning.
All isolates were susceptible to polymyxin B, currently used for empirical treatment of Pseudomonas aeruginosa infections in severely ill patients, particularly those in Intensive Care Units in Brazilian hospitals. Piperacillin-tazobactam was also a viable alternative treatment (96.7% susceptibility) and all isolates producing MβL were susceptible to this drug. According to previous studies, piperacillin-tazobactam could be a reliable treatment option for MβL-producing Pseudomonas aeruginosa when used appropriately7,18,19. Expression of high-level resistance to various antimicrobial agents may involve different mechanisms, such as the production of enzymes, a reduction in the permeability of the external membrane and overexpression of efflux systems2,20,21.
MβL-producing isolates presented high MICs for both carbapenems (>128µg/mL) and other β-lactams (>256µg/mL), except aztreonam (16µg/mL). Aztreonam is not a good substrate for MβL, including SPM-115; however, its reduced susceptibility could be explained by the possible additional mechanisms of resistance to β-lactams carried by this strain9. The isolates belonging to clone A, which do not carry MβLs, showed lower MICs, especially to Imipenem (32 to 64µg/mL), and somewhat higher for meropenem (64 to 128µg/mL).The overexpression of the MexAB-OprM efflux system could explain this finding, since the hydrophobic chains of meropenem seem to be a better substrate than imipenem in this system22. Four isolates sensitive to ceftazidime (MIC 8µg/mL), were resistant to cefepime and carbapenems. According to Hocquet et al21, this phenotype could be due to overexpression of ampC and the MexXY-OprM21 efflux system. This clone probably has multiple resistance mechanisms, which, according to Maniati et al23, would explain the high MIC for carbapenems23.
Unrelated isolates presented variable susceptibility profiles, some of which showed resistance only to carbapenems. A possible resistance mechanism of these isolates could be due to the loss of porin (OprD)24. The expression of different resistance mechanisms to Pseudomonas aeruginosareveals the diverse sensitivity of phenotypic profiles in the susceptibility test of this microorganism.
Attempts to standardize phenotyping techniques to detect MβL have encountered various obstacles, such as the differences observed among this class of enzymes and the variation in test results according to the species of bacteria studied. Picão et al25 suggested that DDS is the best method for testing different species of bacteria with diverse MβLs25. The phenotypic methods used in this study proved to be satisfactory in identifying SPM-1-producing isolates, the main MβL detected in Brazilian hospitals. Nevertheless, DDS failed to identify the isolate carrying blaIMP-16. The IMP-16 enzyme was characterized in a Pseudomonas aeruginosa isolate in a hospital in the city of Brasília in 200126. Pseudomonas aeruginosa which produces this enzyme was also isolated in Santa Catarina in 2003, suggesting that the blaIMP-16 variant is circulating in Brazil. These findings justify the use of both methods, DDS and CD, to improve the sensitivity of MβL phenotyping.
One clonal type (clone A) was verified as predominant among the carbapenem-resistant isolates and was identified in all units at the HU/UFSC. This finding suggests that this clone is better adapted to the hospital environment. Alternatively, a continuous source of new acquisition of this microorganism may have occurred, since the other CRPA isolates did not disseminate clonally. The presence of a predominant clone among the resistant strains also indicates that cross-transmission between patients is an important mechanism for the dissemination of CRPA in this hospital. The main isolation site for this microorganism was the urinary tract, followed by the catheter (Table 1). These two sites are frequently identified in infection associated with inappropriate manipulation of invasive devices. Other Brazilian authors also reported the urinary tract as the main isolation of clonal strains CRPA6,9. Another interesting finding was the isolation of clone A predominantly in general medicine clinics (52.9%), mainly in the Internal Medicine Unit III (35%) (Table 1), in contrast to that described by other reports4,5. These results highlight the need for improved measures to control nosocomial infection and show that the manipulation of invasive devices is one of the main procedures that require intervention measures.
Clonal dissemination of SPM-producing Pseudomonas aeruginosa has been described in several Brazilian states: São Paulo, Ceará, Bahia, Paraná and the Federal District9. The same clone was also described in Rio de Janeiro in a subsequent study27. Reports of isolates from Pernambuco, Amazonas and Minas Gerais states described clonal dissemination of SPM-1-producing Pseudomonas aeruginosa4,28,29. The isolates from Santa Catarina were compared with the clone described by Gales et al9 and it proved to be the same clone, known as the Brazilian epidemic clone. Identification of a single clone in a country of continental dimensions like Brazil, could be explained by environmental dissemination9, community dissemination30 or even dissemination by some common source distributed nationally.
The production of MβL did not represent a frequent mechanism of carbapenem resistance in HU/UFSC. However, 62% clonality in CRPA suggests cross transmission as an important mechanism of dissemination, especially outside the Intensive Care Unit. The spread of this clone, with a high-level of resistance and the challenge to eliminate it from the hospital environment, has contributed to the increased rate of resistance to carbapenems. The results of this study emphasize the need for continuous surveillance and improved strategies for infection control in this institution.
The authors thank Laboratório Alerta, Division of Infectious Diseases, São Paulo School of Medicine (EPM/Unifesp) Federal University of São Paulo, for providing the control strains.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
1. Sader HS, Gales AC, Pfaller MA, Mendes RE, Zoccoli C, Barth A, et al. Pathogen frequency and resistance patterns in Brazilian hospitals: summary of results from three years of the SENTRY Antimicrobial Surveillance Program. Braz J Infect Dis 2001; 5:200-214. [ Links ]
2. Rossolini GM, Mantengoli E. Treatment and control of severe infections caused by multiresistant Pseudomonas aeruginosa. Clin Microbiol Infect 2005; 4(suppl 11):17-32. [ Links ]
3. Livermore DM. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare? Clin Infect Dis 2002; 34:634-640. [ Links ]
4. Cezario RC, Duarte De Morais L, Ferreira JC, Costa-Pinto RM, da Costa Darini AL, Gontijo-Filho PP. Nosocomial outbreak by imipenem-resistant metallo-β-lactamase-producing Pseudomonas aeruginosa in an adult intensive care unit in a Brazilian teaching hospital. Enferm Infecc Microbiol Clin 2009; 27:269-274. [ Links ]
5. Zavascki AP, Gaspareto PB, Martins AF, Goncalves AL, Barth AL. Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing SPM-1 metallo-β-lactamase in a teaching hospital in southern Brazil. J Antimicrob Chemother 2005; 56:1148-1151. [ Links ]
6. Furtado GH, Bergamasco MD, Menezes FG, Marques D, Silva A, Perdiz LB, et al. Imipenem-resistant Pseudomonas aeruginosa infection at a medical-surgical intensive care unit: Risk factors and mortality. J Crit Care 2009; 24:625.e9-14. [ Links ]
7. Zavascki AP, Barth AL, Gonçalves AL, Moro AL, Fernandes JF, Martins AF, et al. The influence of metallo-beta-lactamase production on mortality in nosocomial Pseudomonas aeruginosa infections. J Antimicrob Chemother 2006; 58:387-392. [ Links ]
8. Ribeiro J, Mendes RE, Domingos R, Franca E, Silbert S, Jones RN, et al. Microbiological and epidemiological characterization of imipenem-resistant Pseudomonas aeruginosa strains from a Brazilian tertiary hospital: report from the SENTRY Antimicrobial Surveillance Program. J Chemother 2006; 18:461-467. [ Links ]
9. Gales AC, Menezes LC, Silbert S, Sader HS. Dissemination in distinct Brazilian regions of an epidemic carbapenem-resistant Pseudomonas aeruginosa producing SPM metallo-β-lactamase. J Antimicrob Chemother 2003; 52:699-702. [ Links ]
10. York MK, Schreckenberger PC, Miller JM. Identification of Gram-negative bacteria. In: Isenberg HD, editors. Clinical Microbiology Procedures Handbook. 2th ed. Washington, (DC): ASM press; 2004. p.18.104.22.168-21. [ Links ]
11. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. 7th ed. M7-A7, Wayne, PA: CLSI; 2006. [ Links ]
12. Arakawa Y, Shibata N, Shibayama K, Kurokawa H, Yagi T, Fujiwara H, et al. Convenient test for screening metallo-β-lactamase-producing gram-negative bacteria by using thiol compounds. J Clin Microbiol 2000; 38:40-43. [ Links ]
13. Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-β-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002; 40:3798-3801. [ Links ]
14. Sader HS, Reis AO, Silbert S, Gales AC. IMPs, VIMs and SPMs: the diversity of metallo-β-lactamases produced by carbapenem-resistant Pseudomonas aeruginosa in a Brazilian hospital. Clin Microbiol Infect 2005; 11:73-76. [ Links ]
15. Toleman MA, Simm AM, Murphy TA, Gales AC, Biedenbach DJ, Jones RN, et al. Molecular characterization of SPM-1 a novel metallo-β-lactamase isolated in Latin America: report from the SENTRY Antimicrobial Surveillance Program. J Antimicrob Chemother 2002; 50:673-679. [ Links ]
16. Gales AC, Tognim MCB, Reis AO, Jones RN, Sader HS. Emergence of an IMP-like metallo-enzyme in an Acinetobacter baumannii clinical strain from a Brazilian teaching hospital. Diagn Microbiol Infect Dis 2003; 45:77-79. [ Links ]
17. Kaufmann M. Pulsed-field-gel-electrophoresis. In: Woodford N, Johnson AP, editors. Molecular Bacteriology: protocols and clinical applications. New Jersey: Human Press; 1988. p. 33-51. [ Links ]
18. Parkins MD, Pitout JD, Church DL, Conly JM, Laupland KB. Treatment of infections caused by metallo-β-lactamase-producing Pseudomonas aeruginosa in the Calgary Health Region. Clin Microbiol Infect 2007;13:199-202. [ Links ]
19. Corvec S, Poirel L, Espaze E, Giraudeau C, Drugeon H, Nordmann P. Long-term evolution of a nosocomial outbreak of Pseudomonas aeruginosa producing VIM-2 metallo-enzyme. J Hosp Infect 2008; 68:73-82. [ Links ]
20. Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-β-lactamases: the quiet before the storm? Clin Microbiol Rev 2005; 18:306-325. [ Links ]
21. Hocquet D, Berthelot P, Roussel-Delvallez M, Favre R, Jeannot K, Bajolet O, et al. Pseudomonas aeruginosa may accumulate drug resistance mechanisms without losing its ability to cause bloodstream infections. Antimicrob Agents Chemother 2007; 51:3531-3536. [ Links ]
22. Quale J, Bratu S, Gupta J, Landman D. Interplay of efflux system, ampC and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemoter 2006; 50:1633-1641. [ Links ]
23. Maniati M, Ikonomidis A, Mantzana P, Daponte A, Maniatis AN, Pournaras S. A highly carbapenem-resistant Pseudomonas aeruginosa isolate with a novel blaVIM-4/blaP1b integron overexpresses two efflux pumps and lacks OprD. J Antimicrob Chemother 2007; 60:132-135. [ Links ]
24. Hancock RE. Resistence mechanisms in Pseudomonas aeruginosa and other non fermentative gram-negative bacteria. Clin Infect Dis 1998; 27:93-99. [ Links ]
25. Picao RC, Andrade SS, Nicoletti AG, Campana EH, Moraes GC, Mendes RE, et al. Metallo-β-Lactamase Detection: Comparative Evaluation of Double-Disk Synergy versus Combined Disk Tests for IMP, GIM, SIM, SPM or VIM-producing isolates. J Clin Microbiol 2008; 46:2028-2037. [ Links ]
26. Mendes RE, Toleman MA, Ribeiro J, Sader HS, Jones RN, Walsh TR. Integron carrying a novel metallo-β-lactamase gene, blaIMP-16, and a fused form of aminoglycoside-resistant gene aac(6′)-30/aac(6′)-Ib’: report from the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother 2004; 48:4693-4702. [ Links ]
27. Pellegrino FL, Casali N, Dos Santos KR, Nouer SA, Scheidegger EM, Riley LW, et al. Pseudomonas aeruginosa epidemic strain carrying bla(SPM) metallo-β-lactamase detected in Rio de Janeiro. Brazil. J Chemother 2006; 18:151-156. [ Links ]
28. Poirel L, Magalhaes M, Lopes M, Nordmann P. Molecular analysis of metallo-β-lactamase gene bla(SPM-1)-surrounding sequences from disseminated Pseudomonas aeruginosa isolates in Recife, Brazil. Antimicrob Agents Chemother 2004; 48:1406-1409. [ Links ]
29. Cipriano R, Vieira VV, Fonseca EL, Rangel K, Freitas FS, Vicente AC. Coexistence of epidemic colistin-only-sensitive clones of Pseudomonas aeruginosa, including the blaSPM clone, spread in hospitals in a Brazilian Amazon City. Microb Drug Resist 2007; 13:142-146. [ Links ]
30. Carvalho AP, Albano RM, de Oliveira DN, Cidade DA, Teixeira LM, Marques-Ede A. Characterization of an epidemic carbapenem-resistant Pseudomonas aeruginosa producing SPM-1 metallo-β-lactamase in a hospital located in Rio de Janeiro, Brazil. Microb Drug Resist 2006; 12:103-108. [ Links ]
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Received in 13/04/2010
Accepted in 12/05/2010