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

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.

Pseudomonas aeruginosa is a bacterium of environmental origin considered an essentially opportunistic pathogen infecting hospitalized and immune-compromised patients 1,2 .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 antibiotics [5][6][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 factors 5 .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 AlgR 8,9 .Furthermore, together, AlgU and AlgR activate the transcription of genes encoding the biosynthetic enzymes for alginate production, resulting in the mucoid phenotype 8 .
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 antimicrobials 11,12 .
The production of carbapenemase enzymes became the mechanism of greater relevance towards carbapenem resistance due to the growing enzyme diversity, especially featuring the metalloβ-lactamases (MBL).These enzymes have high versatility and wide hydrolytic activity over almost all β-lactam antibiotics, with the exception of monobactams 13,14 .
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 transmission 16,17 .The utilization of the enterobacterial repetitive intergenic consensus sequence by polymerase chain reaction (ERIC-PCR) has demonstrated its efficacy, efficiency, and usefulness for epidemiological surveillance [17][18][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.

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 production 2 and gelatinase production 20 .

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.
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 bla SPM-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 bla IMP and bla VIM 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.
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 Gospodarek 23 observed even smaller frequencies, when comparing the frequency of virulence factors among multidrug 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 chronicity 6 .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 evolution 6,26 .
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 epithelium 6,27 .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 biofilm 26 and the presence of multidrug resistance were also investigated in these studies 25 , 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 nonmucoid 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][33][34][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%). Neertheless, 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 municipality [33][34][35] .However, several studies have reported lower rates of multi-drug resistant organisms 3,10,38 , 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 types 14 .In Brazil, the most prevalent enzyme is the SPM-1 38 , 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 published 37 .
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 outbreaks 54 .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 bla SMP-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 bla SMP-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 nonlethal genetic mutations 56 .
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 bla SPM-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.The authors declare that there is no conflict of interest.

ACKNOWLEDGMENTS FINANCIAL SUPPORT
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.

FIGURE 2 -
FIGURE 2 -Dendrogram estimated by the enterobacterial repetitive intergenic consensus sequence by polymerase chain reaction for 61 Pseudomona aeruginosa isolates from five public hospitals in Recife, State of Pernambuco, Brazil, isolated between years 2006 and 2010.

TABLE 1 -Frequency of the microbiological characteristics of Pseudomonas aeruginosa isolates from hospitals in Recife, Brazil, obtained from years 2006 to 2010. Characteristic Number /Sample Percentage (%)
Number: number of isolates presenting a particular characteristic; Sample: sample size used for the study of a particular characteristic; MDR: multidrug-resistant isolates.