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Revista da Sociedade Brasileira de Medicina Tropical

Print version ISSN 0037-8682

Rev. Soc. Bras. Med. Trop. vol.45 no.6 Uberaba Nov./Dec. 2012

http://dx.doi.org/10.1590/S0037-86822012000600010 

MAJOR ARTICLE

 

Phenotypic and molecular characterization of antimicrobial resistance and virulence factors in Pseudomonas aeruginosa clinical isolates from Recife, State of Pernambuco, Brazil

 

Caracterização fenotípica e molecular de fatores de resistência a antimicrobianos e virulência de isolados clínicos de Pseudomonas aeruginosa de Recife, Estado de Pernambuco, Brasil

 

 

Paula Regina Luna de Araújo JácomeI; Lílian Rodrigues AlvesI; Adriane Borges CabralI; Ana Catarina Souza LopesI; Maria Amélia Vieira MacielI

IDepartamento de Medicina Tropical, Programa de Pós-Graduação em Medicina Tropical, Universidade Federal de Pernambuco, Recife, PE

Correspondence

 

 


ABSTRACT

INTRODUCTION: The emergence of carbapenem resistance mechanisms in Pseudomonas aeruginosa has been outstanding due to the wide spectrum of antimicrobial degradation of these bacteria, reducing of therapeutic options.
METHODS: Sixty-one clinical strains of P. aeruginosa isolated from five public hospitals in Recife, Pernambuco, Brazil, were examined between 2006 and 2010, aiming of evaluating the profiles of virulence, resistance to antimicrobials, presence of metallo-β-lactamase (MBL) genes, and clonal relationship among isolates.
RESULTS: A high percentage of virulence factors (34.4% mucoid colonies; 70.5% pyocyanin; 93.4% gelatinase positives; and 72.1% hemolysin positive) and a high percentage of antimicrobial resistance rates (4.9% pan-resistant and 54.1% multi-drug resistant isolates) were observed. Among the 29 isolates resistant to imipenem and/or ceftazidime, 44.8% (13/29) were MBL producers by phenotypic evaluation, and of these, 46.2% (6/13) were positive for the blaSPM-1 gene. The blaIMP and blaVIM genes were not detected. The molecular typing revealed 21 molecular profiles of which seven were detected in distinct hospitals and periods. Among the six positive blaSPM-1 isolates, three presented the same clonal profile and were from the same hospital, whereas the other three presented different clonal profiles.
CONCLUSIONS: These results revealed that P. aeruginosa is able to accumulate different resistance and virulence factors, making the treatment of infections difficult. The identification of blaSPM-1 genes and the dissemination of clones in different hospitals, indicate the need for stricter application of infection control measures in hospitals in Recife, Brazil, aiming at reducing costs and damages caused by P. aeruginosa infections.

Keywords: Pseudomonas aeruginosa. Carbapenemase. Multidrug resistance. Virulence factors.


RESUMO

INTRODUÇÃO: A emergência de mecanismos de resistência aos carbapenêmicos em Pseudomonas aeruginosa tem se destacado devido ao amplo espectro de degradação de antimicrobianos, reduzindo as opções terapêuticas.
MÉTODOS: Sessenta e um isolados de P. aeruginosa procedentes de cinco hospitais públicos de Recife, Pernambuco, Brasil, entre 2006 e 2010, foram analisadas, com o objetivo de avaliar o perfil de virulência, resistência aos antimicrobianos, a presença de genes metalo-β-lactamase (MBL) e a relação clonal entre os isolados.
RESULTADOS: Foi observada uma elevada produção de fatores de virulência na amostra (34,4% colônias mucoides; 70,5% piocianina; 93,4% gelatinase e 72,1% hemolisina), bem como um elevado percentual de resistência (4,9% isolados panresistentes e 54,1% multirresistentes). Dentre os 29 isolados resistentes ao imipenem e/ou ceftazidima, 44,8% (13/29) apresentaram MBL por meio da pesquisa fenotípica, e destes, 46,2% (6/13) foram positivos para o gene blaSPM-1, não havendo detecção dos genes blaIMP e blaVIM. A tipagem molecular revelou 21 perfis genéticos dos quais sete foram detectados em hospitais e períodos distintos, e dos isolados blaSPM-1 positivos, três apresentaram o mesmo perfil clonal e foram procedentes do mesmo hospital, enquanto que os outros três isolados blaSPM-1 positivos apresentaram perfis clonais distintos.
CONCLUSÕES: Estes resultados revelam que a P. aeruginosa é capaz de acumular diferentes fatores de virulência e resistência, dificultando o tratamento das infecções. A identificação de genes blaSPM-1 e disseminação de clones sugere a necessidade de aplicação mais rigorosa de medidas de controle de infecção nos hospitais de Recife, visando reduzir custos e danos provocados por este tipo de infecção.

Palavras-chaves: Pseudomonas aeruginosa. Carbapenemase. Multirresistência. Fatores de virulência.


 

 

INTRODUCTION

Pseudomonas aeruginosa is a bacterium of environmental origin considered an essentially opportunistic pathogen infecting hospitalized and immune-compromised patients1,2. In Brazil, P. aeruginosa is an important cause of nosocomial infections and is considered the first cause of nosocomial pneumonia and the third cause of bloodstream primary infection in intensive care units (ICU)3,4.

Some virulence factors favor this pathogen's infection, such as the formation of pyocyanin, hemolysin, gelatinase, and biofilm, which act increasing tissue damage and protecting P. aeruginosa against the recognition of the immune system and the action of antibiotics5-7. The pathogenesis of P. aeruginosa infections is multifactorial, as suggested by the number and wide array of virulence determinants possessed by the bacterium. Pili, lipopolysaccharide (LPS), flagella, elastase, alkaline protease, siderophores, siderophore uptake systems and extracellular protein toxins (exoenzyme S and exotoxin A) are examples of other virulence factors5.

Some regulatory mechanisms of virulence factors has excelled as the loss-of-function mutations in mucA that blocks the production of many invasive virulence factors (including type III secretion system [T3SS], exotoxin A, protease IV, and type IV pili [TFP]) by inhibiting cAMP-Vfr-dependent signaling (CVS) at the level of vfr expression involving AlgU and the response regulator AlgR8,9. Furthermore, together, AlgU and AlgR activate the transcription of genes encoding the biosynthetic enzymes for alginate production, resulting in the mucoid phenotype8.

Pseudomonas aeruginosa can also harbor several mechanisms of resistance, which generates multi-drug resistant isolates (resistance to three or more classes of anti-Pseudomonas agents), or pan-resistant isolates (resistance to all antimicrobials clinically used)4,10.

An alternative treatment to infections caused by multi-drug resistant organisms is the use of carbapenems, such as the imipenem, meropenem, and more recently the doripenem; however, they are not indicated as the first choice for empirical treatment of hospital or community infections. Moreover, the indiscriminate use of carbapenems in the hospital environment leads to the selection of resistant strains to this class of antimicrobials11,12.

The production of carbapenemase enzymes became the mechanism of greater relevance towards carbapenem resistance due to the growing enzyme diversity, especially featuring the metallo-β-lactamases (MBL). These enzymes have high versatility and wide hydrolytic activity over almost all β-lactam antibiotics, with the exception of monobactams13,14.

Nine subclasses of MBL have been described yet, IMP-1 (Imipemenase), VIM-1 (Verona imipenemase), SPM-1 (São Paulo metallo-β-lactamase), GIM-1 (German imipenemase), SIM-1 (Seoul imipenemase), AIM-1 (Australian imipenemase) KHM-1 (Kyorin Hospital metallo-β-lactamase), NDM-1 (New Delhi metallo-β lactamase), DIM-1 (Dutch imipenemase)13, of which only the first three were identified in different Brazilian states45.

In addition to the research of resistance genes, another indispensable tool in controlling infections is the application of genotyping methods for the determination of genetic relationships among the microorganisms, thus allowing mapping the dynamics of infection transmission16,17. The utilization of the enterobacterial repetitive intergenic consensus sequence by polymerase chain reaction (ERIC-PCR) has demonstrated its efficacy, efficiency, and usefulness for epidemiological surveillance17-19.

This study characterized, phenotypically and genotypically,
P. aeruginosa isolates   obtained from public hospitals in Recife, Brazil, with the objective of identifying resistance and virulence markers and establishing the clonal dissemination of the isolates.

 

METHODS

Bacterial isolates

Were analyzed 61 P. aeruginosa isolates from various sites of infection (blood, urine, tracheal secretions, wound, eye discharge and ulcer) of hospitalized patients from five teaching hospitals in Recife, between 2006 and 2010. Among the microbiological characteristics were investigated colony morphology, pigment production, hemolysin production2 and gelatinase production20.

Pigments production and colony morphology

The strains (pure culture) were streaked on Cetrimide agar and incubated for 24h at 37°C to visual analysis of pigment production (pyocyanin, pioverdin, piorubin and piomelanin) and colony morphology (mucoid and not mucoid)2.

Antimicrobial susceptibility

The antibiotic susceptibility testing was evaluated by agar diffusion using amikacin, gentamicin, ciprofloxacin, ticarcillin/clavulanic acid, aztreonam, cefepime, ceftazidime, imipenem, meropenem and polymyxin, as recommended by the Clinical and Laboratory Standards Institute (CLSI)21. Imipenem and / or ceftazidime resistant isolates were investigated  for   metallo-β-lactamases (MBLs) producing, using disk approximation test described by Arakawa hard et al.22, performed in duplicate for each isolate.

Metallo-β-lactamase PCR

To search the related carbapenemase-encoding genes, the total DNA of  isolates was obtained using Brazol kit (LGC Biotecnologia), according to manufacturer's instructions, and quantified by spectrophotometry (Ultraspec 3000, Pharmacia Biotech) at wavelengths 260 to 280nm. The MBL genes were surveyed using the following primers: blaSPM-1 F (5'-CCTACAATCTAACGGCGA CC-3'), blaSPM-1 R (5'-TCGCCGTGTCCAGGTATAAC-3')9, blaIMP F (5'-GGAATAGAGTG GCTTAATTCTC-3') and blaIMP R (5'-CGTGTGATGCYCCAAYTTCACT-3'), blaVIM F (5'-CAGATTGCCGATGGTGTTTGG-3') and blaVIM R (5'-AGGTGGGCCATTCAGCCAGA-3')23. Amplification reactions were prepared in a total volume of 25µl  per tube, comprising: 25ng of genomic DNA, 10pmol of primers, 1x buffer, 100μM of each deoxyribonucleotide triphosphate (Ludwig Biotec), 1.5mM MgCl2 and 1.0U Taq DNA polymerase (Promega). In each round of amplification positive and negative controls for genes blaSPM-1 (P. aeruginosa PSA319), blaIMP (P. aeruginosa 48-1997A) and blaVIM (P. aeruginosa VIM-1) were included. The cycles parameters were 95°C for 5minutes, followed by 30 cycles of denaturation at 95°C for 1minute, annealing for 1min at 50.6ºC, 55.3°C and 62ºC, to blaIMP, blaSPM-1 and blaVIM, respectively; extension at 68°C for 1minute, and finally 10minutes at 68°C. PCR products were stained with blue-green (LGC Biotecnologia - São Paulo) and subjected to electrophoresis on agarose gel 1% in TBE buffer
(Tris-borate 0.089 M EDTA and 0.002M) under constant voltage of 100V11.

ERIC-PCR

To assess the genetic relationship of the isolates, ERIC-PCR was carried out. Amplification reactions were prepared in a total volume of 25µl per tube, comprising: 100ng of genomic DNA, 10pmol of primers (ERIC-1 [5'-ATGTAAGCT CCTGGGGATTCAC-3']; ERIC-2 [5'-AAGTAAGTGACTGGGGTGAGCG-3'])12, 1x buffer, 200μM of each deoxyribonucleotide triphosphate (Promega), 1.5mM MgCl2 and 1.0U Taq DNA polymerase (Promega). The amplification parameters used were 95°C for 3minutes, followed by 30 cycles of denaturation at 92°C for 1minute, annealing at 36°C for 1minute and extension at 72°C for 8minutes, followed by a final extension 16minutes at 72°C. PCR products were stained with blue-green (LGC biotec) and subjected to electrophoresis on agarose gel to 1.5% in TBE buffer under constant voltage of 100V24. Data analysis and dendrograms were made ​​using the Darwin 5.0 software.

 

RESULTS

The analysis of virulence factors revealed that out of the 61 P. aeruginosa isolates studied, 34.4% presented mucoid colonies, 70.5% were pyocyanin producers, 93.4% were hemolysin and gelatinase (72.1%) producers (Table 1).

 

 

Antimicrobial susceptibility testing data are shown in Figure 1. The highest level of resistance was recorded against gentamicin and ciprofloxacin, 41% (25/61), whereas polymyxin B and aztreonam presented the lowest percentages of resistance, 13.1% (8/61) and 29.5% (18/61), respectively.

The multidrug resistance profile was observed in 54.1% (33/61) of the isolates, of which, 27.3% (9/33) were susceptible only to polymyxin B and 9.1% (3/33) revealed the pan-resistant profile, as described in Table 1.

Among the 29 isolates resistant to imipenem and/or ceftazidime, 44.8% (13/29) were MBL producers, of which, 46.2% (6/13) revealed the blaSPM-1 gene; two of them (P7A, P15A) were isolated from the same hospital, and were collected in 2006. The other ones (P6BL, P3R, P4R, P7R) were collected in 2010 and originated from two other hospitals. The blaIMP and blaVIM genes were not detected.

The molecular typing of the isolates revealed 21 genetic profiles with predominance of clones A, B, and C, as shown in the dendrogram (Figure 2). Among the SPM-1 positive isolates, three shared the same clonal profile (clone D), while the other three presented distinct clonal profiles (clones C, G, and P). Seven clones (A, B, C, D, E, J, and K) were detected in more than one hospital and were detected in distinct periods.

 

 

DISCUSSION

The phenotypic characterization performed in this study revealed a high percentage of virulence factors among the studied isolates, with the exception of the mucoid phenotype, associated with the formation of biofilm present in only 34.4% of the colonies. Deptuła and Gospodarek23 observed even smaller frequencies, when comparing the frequency of virulence factors among multi-drug susceptible and multidrug-resistant (MDR) isolates with biofilm formation in 9.3% and 8%, respectively.

The biofilm is composed of alginate and confers a mucoid consistency to P. aeruginosa isolates, acting as a protecting niche for the bacterium against the recognition of the immune system and the action of antibiotics, thus increasing the possibility of infection chronicity6. The biofilm production has been well studied in patients with cystic fibrosis because this is a chronic pulmonary infection, allowing verifying the transformation from non mucoid to mucoid isolates during the process of disease evolution6,26.

The production of pyocyanin was observed in 70.5% of the studied isolates. This virulence factor consists of a blue phenazinic pigment produced exclusively by P. aeruginosa and related to tissue damage through the formation of reactive hydroxyl radicals and superoxides, mainly in the respiratory epithelium6,27. Some studies report the production of pyocyanin in P. aeruginosa isolates ranging between 41.3% and 81.5%28,29. The association of this production with the production of biofilm26 and the presence of multidrug resistance were also investigated in these studies25, and no significant difference in either of the two cases was reported.

The production of hemolysin and gelatinase, which accounted for 93.4% and 72.1%, respectively, correspond to other virulence factors associated with tissue injury. Stehling et al.31, in an analytical study to assess the production of virulence factors among non-mucoid and mucoid isolates, did not observe significant difference in the production of hemolysin (p = 0.5911) and gelatinase
(p = 0.5542), reporting average frequencies of 53.6% and 37.5%, respectively.

The antimicrobial susceptibility profiles obtained in this study showed that the most active drug against P. aeruginosa was polymyxin B (82%), followed by ceftazidime (67.2%), imipenem (63.9%), and meropenem (62.3%). Pires et al.32-35,38 reported, in a study conducted between January and June of 2009 in the Hospital das Clínicas de Pernambuco, Brazil, the observation that the most active antimicrobials against P. aeruginosa were amikacin (84.6%), imipenem (81.8%), meropenem (79.3%), and aztreonam (74.4%). Nevertheless, this comparative evaluation between antimicrobials was impaired because there was variation in the choice of antibiotic tested in the different studies. Figueiredo et al.33 investigated 304 isolates of P. aeruginosa, from two public hospitals in Recife (Hospital das Clínicas de Pernambuco and Hospital Agamenon Magalhães), in the period between September 2004 and January 2006, and observed that aztreonam (56.5%), amikacin (55.9%), meropenem (55.7%), and imipenem (51.7%) showed increased activity. In these studies, the carbapenems  were among the four most active antimicrobials, with susceptibility percentages ranging from 51.5% to 79.3%. On the other hand, both Pires et al.32 and Figueiredo et al.33 observed higher antimicrobial susceptibility compared to our results, where aztreonam showed the lower antimicrobial activity, with more expressive resistance rates over the past two years.

A high percentage of multi-drug resistant isolates observed in this study, is in agreement with previous findings of multidrug resistance in P. aeruginosa in the same municipality33-35. However, several studies have reported lower rates of multi-drug resistant organisms3,10,38, which highlights the importance of the results revealed in our study.

The detection of P. aeruginosa isolates, producers of MBL, has been described worldwide with a predominance of the IMP and VIM types14. In Brazil, the most prevalent enzyme is the SPM-138, which description remained restricted to the Brazilian territory until 2010 when the first report of an SPM-1 isolate, identified in 2007, in Europe, from a Swiss patient who had been in a university hospital in Recife-PE, Brazil, was published37.

The blaSMP-1 gene has been reported in P. aeruginosa isolates from the northeast, midwest, southeast and southern states in Brazil, with prevalences varying from 3.1% to 77.1% in isolates resistant to imipenem and/or ceftazidime11,13,30,40-51. Despite not having been identified in this study, the blaIMP-type genes have already been detected in others brazilian states: Rio de Janeiro, Brasília, João Pessoa, São Paulo, Porto Alegre, and Santa Catarina40,43,45,48,51-53, whilst, the blaVIM-2 gene has been reported only in São Paulo 48,49.

Reconciling research including the identification of genes for the production of MBL and molecular typing for the identification of clonal relationships between isolates of P. aeruginosa has been quite frequent in the recent years. This approach has been considered an important methodology because it allows verifying cross transmission in cases of outbreaks54. Lee et al.55, observed the production of MBL in 55% of imipenem resistant isolates from Taiwan where the isolates involved in the outbreak were not related. Mansour et al.54, in Tunisia, detected the production of MBL in only 7% of isolates resistant to imipenem, with prevalence of a unique genetic profile, indicating that the outbreak was caused from the same source of infection. In this study, a clonal dissemination of one isolate of P. aeruginosa carrying the blaSMP-1 gene was identified involving three inpatients in the HR in the second half of 2010. These isolates showed the same virulence factors investigated as well as the same resistance profile and being susceptible to polymyxin B. On the other hand, the other blaSMP-1 positive isolates were not genetically related and presented distinct virulence and resistance profiles.

Out of the total sample set, 21 genetic profiles were identified, with dissemination of seven clones in more than one hospital and during different periods. Similar results are frequent, particularly in studies involving patients with serious and chronic infection, which represent favorable conditions for the occurrence of random non-lethal genetic mutations56.  

This study concluded that P. aeruginosa is a pathogen able to accumulate several virulence factors, which are often associated with multidrug resistance and pan-resistance, making the treatment of infections caused by this bacterium difficult. The identification of blaSPM-1 genes detected in more than one hospital (interhospital dissemination), suggests the implementation of the infection control measures in hospitals in Recife, Brazil, aiming at reducing costs and damages caused by this invasive microorganism especially in patients presenting serious health conditions.

 

ACKNOWLEDGMENTS

We gratefully acknowledge Prof. Dr. Marcia M.C. Morais from the Microbial Resistance Laboratory, Institute of Biological Sciences, University of Pernambuco for kindly providing the positive controls for detection of metallo-β-lactamases genes.

 

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

 

FINANCIAL SUPPORT

This work was supported by FACEPE (Fundação de Amparo Ciência do Estado de Pernambuco) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) granted in APQ 0579-2.12/07, and PROPESQ (Pró-Reitoria para Assuntos de Pesquisa e Pós-Graduação) granted in Edital de Multiusuários/PROPESQ-2007.

 

REFERENCES

1. Pitt TL, Simpson AJ. Pseudomonas and Burkholderia spp. In: Gillespie SH, Hawkey PM, editors. Principles and Practice of Clinical Bacteriology. 2nd ed. London: John Wiley & Sons;  2006. p. 427-443.         [ Links ]

2. Winn Jr WC, Allen SD, Janda WM, Koneman E, Procop G, Schreckenberger PC, et al. Bacilos gram-negativos não fermentadores. In: Toros EF, editor. Koneman Diagnostico Microbiológico, Texto e Atlas Colorido, 6th ed. Rio de Janeiro: Guanabara Koogan; 2008. p. 302-386.         [ Links ]

3. Zavascki AP, Barth AL, Fernandes JF, Moro ALD, Gonçalves ALS, Goldani LZ. Reappraisal of Pseudomonas aeruginosa hospital-acquired pneumonia mortality in the era of metallo-β-lactamase-mediated multidrug resistance: a prospective observational study. Crit Care 2006; 10:100-110.         [ Links ]

4. Layeux B, Taccone FS, Fagnoul D, Vincent JL, Jacobs F. Amikacin monotherapy for sepsis caused by panresistant Pseudomonas aeruginosa. Antimicrob Agents Chemother  2010; 54:4939-4941.         [ Links ]

5. Todar K. The mechanisms of bacterial pathogenicity. Todar's Online Textbook of Bacteriology.2009 [Internet]. [Cited 2010 August 28]. Available from: http://textbookbacteriology.net/themicrobialword/pathogenesis.html.         [ Links ]

6. Ciofu O, Lee B, Johannesson M, Hermansen NO, Meyer P, Høiby N. Scandinavian cystic fibrosis study consortium. Investigation of the algT operon sequence in mucoid and non-mucoid Pseudomonas aeruginosa isolates from 115 scandinavian patients with cystic fibrosis and in 88 in vitro non-mucoid revertants. Microbiology 2008; 154:103-113.         [ Links ]

7. Cevahir N, Demir M, Kaleli I, Gurbuz M, Tikvesli S. Evalution of biofilm production, gelatinase activity, and mannose-resistent hemagglutination Acinetobacter baumannii strains. J Microbiol Immunol Infect 2008; 41:513-518.         [ Links ]

8. Jones AK, Fulcher NB, Balzer GJ, Urbanowski ML, Pritchett CL, Schurr MJ, et al. Activation of the Pseudomonas aeruginosa AlgU regulon through mucA mutation inhibits cyclic AMP/Vfr signaling. J Bacteriol. 2010; 192:5709-5717.         [ Links ]

9. Balasubramanian D, Schneper L, Merighi M, Smith R, Narasimhan G, Lory S, et al. The regulatory repertoire of Pseudomonas aeruginosa AmpC ß- lactamase regulator AmpR includes virulence genes. PLoS One 2012; 7:e34067.         [ Links ]

10.  Tam VH, Chang KT, Abdelraouf K, Brioso CG, Ameka M, McCaskey LA, et al. Prevalence, resistance mechanisms, and susceptibility of multidrug-resistant bloodstream isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2010; 54:1160-1164.         [ Links ]

11.  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 ]

12.  Mendes RE, Castanheira M, Pignatari ACC, Gales AC. Metallo-β-lactamases. J Bras Patol Med Lab 2006; 42:103-113.         [ Links ]

13.  Gräf T, Fuentefria DB, Corção G. Ocorrência de isolados de Pseudomonas aeruginosa multirresistentes produtoras de metallo-β-lactamase blaSPM-1 em amostras clínicas. Rev Soc Bras Med Trop 2008; 41:306-308.         [ Links ]

14.  Walsh TR. Emerging carbapenemases: a global perspective. Int J Antimicrob Agents 2010; 36:8-14.         [ Links ]

15.  Samuelsen O, Buaro L, Toleman MA, Giske CG, Hermansen NO, Walsh TR, et al. The first metallo-beta-lactamase identified in Norway is associated with a TniC-like transposon in a Pseudomonas aeruginosa isolate of sequence type 233 imported from Ghana. Antimicrob Agents Chemother 2010; 53:331-332.         [ Links ]

16.  Spacov ICG, Silva SAM, Morais Júnior MA, Morais MMC. Polymorphism of the rDNA and tDNA loci in clinical isolates of Pseudomonas aeruginosa: A perspective for molecular epidemiology surveillance. Genet Mol Biol 2006; 29:722-729.         [ Links ]

17.  Hafiane A, Ravaoarinoro M. Différentes méthodes de typage des souches de Pseudomonas aeruginosa isolées des patients atteints de mucoviscidose.
Med Mal Infect 2008; 38:238-247.         [ Links ]

18.  Pinna A, Usai D, Sechi LA, Molicotti P, Zanetti S, Carta A. Detection of virulence factors in Pseudomonas aeruginosa strains isolated from contact lens-associated corneal ulcers. Cornea 2008; 27:320-326.         [ Links ]

19.  Wolska K, Szweda P. A comparative evaluation of PCR Ribotiping and ERIC PCR for determining the diversity of clinical Pseudomonas aerugnosa isolates.
Pol J Microbiol 2008;  57:157-163.         [ Links ]

20.  Garrity G. Bergey's Manual® of Systematic Bacteriology. Vol 2: The Proteobacteria, Part A Introductory Essays.  2nd ed. Softcover; 2005.         [ Links ]

21.  Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing. 20th informational supplement. M100-S20. Wayne, Pennsylvania: CLSI;  2010.         [ Links ]

22.  Arakawa Y, Shibata S, 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 ]

23.  Dong F, Xu XW, Song WQ, Lü P, Yu SJ, Yang YH, et al. Characterization of multidrug-resistant and metallo-betalactamase-producing Pseudomonas aeruginosa isolates from a paediatric clinic in China. Chin Med J 2008; 121:1611-1616.         [ Links ]

24.  Duan H, Chai T, Liu J, Zhang X, Qi C, Gao J, et al. Source identification of airborne Escherichia coli of swine house surroundings using ERIC-PCR and REP-PCR. 
Environ Res 2009; 109:511-517.         [ Links ]

25.  Deptuła A, Gospodarek E. Reduced expression of virulence factors in multidrug-resistant Pseudomonas aeruginosa strains. Arch Microbiol 2010; 192:79-84.         [ Links ]

26.  Lee B, Haagensen JAJ, Ciofu O, Andersen JB, Høiby N, Molin N. Heterogeneity of biofilms formed by nonmucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J Clin Microbiol 2005; 43: 5247-5255.         [ Links ]

27.  Kong F, Young L, Chen Y, Ran H, Meyers M, Joseph P. Pseudomonas aeruginosa pyocyanin inactivates lung epithelial vacuolar ATPase-dependent cystic fibrosis transmembrane conductance regulator expression and localization. Cell Microbiol  2006; 8:1121-1133.         [ Links ]

28.  Fothergill JL, Panagea S, Hart CA, Walshaw MJ, Pitt TL, Winstanley C. Widespread pyocyanin over-production among isolates of a cystic fibrosis epidemic strain.
BMC Microbiol 2007; 7:1-10.         [ Links ]

29.  Iwalokun BA, Akinsinde KA, Lanlenhin O, Onubogu CC. Bacteriocinogenicity and production of pyocins from Pseudomonas species isolated in Lagos, Nigeria.
Afr J Biotechnol 2006; 5:1072-1077.         [ Links ]

30.  Magalhães V, Lins AK, Magalhães M. Metallo-β-lactamase producing Pseudomonas aeruginosa strains isolated in hospitals in Recife, PE, Brazil. Braz J Microbiol
2005; 36:123-125.         [ Links ]

31.  Stehling EG, Silveira WD, Leite DS. Study of Biological Characteristics of Pseudomonas aeruginosa Strains Isolated from Patients with Cystic Fibrosis
and from Patients with Extra-Pulmonary Infections. Braz J Infect Dis 2008;
12:86-88.         [ Links ]

32.  Pires EJVC, Silva Júnior VV, Lopes ACS, Veras DL, Leite LE, Maciel MAV. Epidemiologic analysis of clinical isolates of Pseudomonas aeruginosa from an university hospital. Rev Bras Ter Intensiva 2009; 21:384-390.         [ Links ]

33.  Figueiredo EAP, Ramos H, Maciel MAV, Vilar MCM, Loureiro NG, Pereira RG. Pseudomonas aeruginosa: Freqüência de resistência a múltiplos fármacos e resistência cruzada entre antimicrobianos no Recife/PE. Rev Bras Ter Intensiva 2007; 19:421-427.         [ Links ]

34.  McGowan Jr JE. Resistance in nonfermenting gram-negative bac­teria: multidrug resistance to the maximum. Am J Med 2006; 119:29-36.         [ Links ]

35.  Raja NS, Singh NN. Antimicrobial susceptibility pattern of clinical iso­lates of Pseudomonas aeruginosa in a tertiary care hospital. J Microbiol Immunol Infect 2007; 40:45-49.         [ Links ]

36.  Neves MT, Lorenzo MEP, Almeida RAMB, Fortaleza CMCB Antimicrobial use and incidence of multidrug-resistant Pseudomonas aeruginosa in a teaching hospital: an ecological approach.  Rev Soc Bras Med Trop 2010; 43:629-632.         [ Links ]

37.  Amutha R, Padmakrishnan, Murugan T, Renuga devi MP. Studies on multidrug resistant Pseudomonas aeruginosa from pediatric population with special reference to extended spectrum beta lactamase. Indian J Sci Technol 2009; 2:11-13.         [ Links ]

38.  Figueiredo DQ, Castro LFS, Santos KRN, Teixeira LM, Mondino SSB. Detecção de metalo-beta-lactamases em amostras hospitalares de Pseudomonas aeruginosa e Acinetobacter baumannii. J Bras Patol Med Lab 2009; 45:177-184.         [ Links ]

39.  Salabi AE, Toleman MA, Weeks J, Bruderer T, Frei R, Walsh TR. First report of the metallo-β-lactamase SPM-1 in Europe. Antimicrob Agents Chemother 2010; 54:582.         [ Links ]

40.  Santos-Filho L, Santos I, Xavier DE, Menezes LC. Tipagem molecular de amostras de Pseudomonas aeruginosa produtoras de metalo-β-lactamases isoladas em
João Pessoa/PB, Brasil. Rev Bras Anal Clin 2003; 35:127-131.

41.  Poirel L, Magalhães M, Lopes M, Nordmann P. Molecular analysis of metallo-β-lactamase gene blaSPM-1-surrounding sequences from disseminated Pseudomonas aeruginosa isolates in Recife, Brazil. Antimicrob Agents Chemother 2004; 48:1406-1409.         [ Links ]

42.  Cipriano R, Vieira VV, Fonseca EL, Rangel K, Freitas FS, Vicente ACP. 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 ]

43.  Mendes RE, Toleman MA, Ribeiro J, Sader HS, Ronald N, Jones RN, et al. 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 ]

44.  Zavascki AP, Gaspareto PB, Martins AF, Gonçalves 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 ]

45.  Martins AF, Zavascki AP, Gaspareto PB, Barth AL. Dissemination of Pseudomonas aeruginosa producing SPM-1-like and IMP-1-like metallo-β-lactamases in hospitals from southern Brazil. Infection 2007; 35:457-460.         [ Links ]

46.  Wirth FW, Picoli SU, Cantarelli VV, Gonçalves ALS, Brust FR, Santos LMO, et al. Metallo-β-lactamase-producing Pseudomonas aeruginosa in two hospitals from Southern Brazil. Braz J Infect Dis 2009; 13:170-172.         [ Links ]

47.  Cezário RC, De-Morais LD, Jferreira JC, Costa-Pinto RM, Darini ALC, 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 ]

48.  Sader HS, Reis AO, Silber S, Gales AC. IMPs, VIMs, 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 ]

49.  Franco MRG, Caiaffa-Filho HH, Burattini MN, Rossi F. Metallo-beta-lactamases among imipenem-resistant Pseudomonas aeruginosa in a brazilian university hospital. Clin Sci  2010; 65:825-829.         [ Links ]

50.  Carvalho APDA, Albano RM, Oliveira DN, Cidade DAP, Teixeira LM, Marques EA. 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 ]

51.  Scheffer MA, Gales AC, Barth AL, Filho JRC. Dalla-Costa LM. Carbapenem-resistant Pseudomonas aeruginosa - clonal spread in southern Brazil and in the state of Goiás. Braz J Infect Dis 2010; 14:508-509.         [ Links ]

52.  Pellegrino FLP, Moreira MB, Nouer AS. Antimicrobial resistance and genotype characterization of Pseudomonas aeruginosa isolates from a university affiliated hospital in Rio de Janeiro, Abstract L-14. 101th ASM General Metting; 2001.         [ Links ]

53.  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 ]

54.  Mansour W, Poirel L, Bettaieb D, Bouallegue O, Boujaafar N, Nordmann P. Metallo-β-lactamase-producing Pseudomonas aeruginosa isolates in Tunisia.  Diagn Microbiol Infect Dis 2009; 64:458-461.         [ Links ]

55.  Lee MF, Peng CF, Hsu HJ, Chen YH. Molecular characterisation of the metallo-β-lactamase genes in imipenem-resistant gram-negative bacteria from a university hospital in southern Taiwan. Int J Antimicrob Agents 2008; 32:475-480.         [ Links ]

56.   Freitas AL, Barth AL. Typing of Pseudomonas aeruginosa from hospitalized patients:
a comparison of susceptibility and biochemical profiles with genotype. Braz J Med Biol Res 2004; 37:77-82.         [ Links ]

 

 

Address to:
Dra. Paula Regina Luna de Araújo Jácome. Depto MedTrop/PPG MedTrop/UFPE. Av. Moraes Rego 1235, Cidade Universitária, 50670-901 Recife, PE, Brasil.

Phone: 55 81 2126-8526; Fax: 55 81 2126-8528
e-mail: paulajacome@ibest.com.br

Received in 12/03/2012
Accepted in 23/10/2012

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