Production of metallo-β-lactamase among Pseudomonas aeruginosa strains isolated in the State of Sergipe , Brazil

Introduction: Acquired production of metallo-β-lactamases is an important mechanism of resistance in Pseudomonas aeruginosa. The objective of this study was to investigate the production of metallo-β-lactamase and the genetic diversity among ceftazidimeresistant P. aeruginosa isolates from State of Sergipe, Brazil. Methods: Metallo-β-lactamase was investigated using the disk approximation test and polymerase chain reaction (PCR). Genetic diversity was evaluated by pulsed-fi eld gel electrophoresis (PFGE). Results: A total of 48 (51.6%) isolates were resistant to ceftazidime. Six (12.2%) of these were positive for metallo-βlactamase production. Only two (4.1%) of the ceftazidime-resistant isolates carried the blaSPM-1 gene. Conclusions: Production of metallo-β-lactamases was not the main mechanism of resistance to ceftazidime and carbapenems among P. aeruginosa strains in Sergipe, Brazil.

Carbapenem resistance has increased among Pseudomonas aeruginosa strains worldwide.Production of metalloβ-lactamases (MβL) has been identified as an important mechanism of carbapenem resistance among P. aeruginosa (1) .
Thus, the objective of the present study was to investigate the occurrence of MβL-producing strains and the genetic diversity of ceftazidime-resistant P. aeruginosa isolates from three institutions in the State of Sergipe, Brazil.
Pseudomonas aeruginosa isolates (n = 95) were recovered from March 2008 to December 2009 from patients attending two tertiary healthcare institutions [Hospital Primavera (HP), a private hospital with 45 (47.4%) isolates and Hospital de Urgência do Estado de Sergipe (HUSE), a public hospital with 10 (10.5%) isolates], and from the Laboratório Central de Saúde Pública do Estado de Sergipe (LACEN), a public clinical laboratory, which receives clinical specimens from patients attending hospitals throughout Sergipe with 40 (42.1%)isolates.Most (62.1%) isolates were from patients admitted to the intensive care units of the two hospitals included in this study.Isolates were obtained from the following clinical specimens: 24 (25.3%) from lower respiratory tract secretions, 21 (22.1%) from urine, 11 (11.6%) from cerebrospinal fl uid, eight (8.4%) from surgical wound secretions, three (3.2%)from blood, and 28 (29.4%) from various other clinical sources.Only one isolate was selected from each patient.
Isolates with resistance to ceftazidime (CAZ-R) were phenotypically screened for MβL production using the disk approximation (DA) test, as described by Arakawa et al. (7) .Undiluted 2-mercaptopropionic acid (2-MPA) was used as an inhibitor of MβL, and a 30-μg ceftazidime disk was used as the substrate.For the DA test, 3μL of 2-MPA was added to a blank fi lter disk, placed 2cm away from a ceftazidime disk.One ceftazidime disk was also placed 5 cm away.After incubation, the presence of an enlarged zone of inhibition was interpreted as a positive test.A metallo-β-lactamase-producing strain (P1088) and P. aeruginosa ATCC 27853 were used as positive and negative controls, respectively, for the MβL phenotypic screening tests.
Pseudomonas aeruginosa CAZ-R isolates were typed by pulsed-fi eld gel electrophoresis (PFGE) using SpeI (Invitrogen, São Paulo, Brazil), as described elsewhere (11) .Band profi les were analyzed using the Gel-Compar II Program (Applied Maths, Kortrijk, Belgium).Isolates with a coeffi cient of similarity of 85% of more (with PFGE patterns differing in one to six bands) were included in the same genotype designated with a capital letter.A bla SPM-1 -positive P. aeruginosa strain representative of the SP clone (P1088) was included for comparison.A clinical bla SPM-1 -positive strain isolated in Niterói, State of Rio de Janeiro (365h2) that was part of our culture collection was also used for comparison.
Phenotypic tests were positive in six (12.2%) of the 49 CAZ-R isolates using the CAZ-2MPA combination.However, based on the genes screened by PCR, only two (4.1%) isolates carried the bla SPM-1 gene.Interestingly, one of these bla SPM-1positive isolates [isolate 65, carbapenem-susceptible, with a minimum inhibitory concentration (MIC) for imipenem of 0.75μg/mL, as determined by E-test] yielded a positive DA test result, while the other (isolate 69, carbapenem-resistant) was negative.The phenotypic tests of both SPM-1-positive isolates were repeated.No other carbapenemase-encoding genes were found.The two SPM-1-positive isolates showed resistance to six or seven antimicrobial agents.The characteristics of the six Mβl-positive isolates according to the phenotypic tests are shown in Table 1.
A total of 41 of the 49 CAZ-R isolates were analyzed by PFGE.Thirty-seven unique PFGE band profi les were identifi ed (Figure 1); among these, genotypes A, D, and H included three isolates each, while genotypes B, C, E, F, G, and I included two isolates each.The three isolates belonging to genotype A were isolated from the three institutions involved in the study between August and September 2009.The two bla SPM-1 -positive isolates (65 and 69) included in genotype F were obtained from two different sectors of HP and were genotypically related to the bla SPM-1 -positive controls strains (> 95% similarity).One band differed among isolates 65 and 69.The bla SPM-1 -positive control P1088 and isolate 65 exhibited indistinguishable PFGE patterns, whereas isolate 69 and the other bla SPM-1 -positive control strain (365h2) were also indistinguishable by PFGE (Figure 2).
Detection of MβL-producing P. aeruginosa is important for controlling the dissemination of MβL isolates and for the correct choice of antimicrobial regimens; these enzymes are able to hydrolyze most β-lactams but do not always exhibit carbapenem resistance (12) , as observed in one of the two SPM-1-positive isolates.This result emphasizes the utilization of ceftazidime resistance as a criterion for selecting isolates for phenotypic MβL production tests.Among the CAZ-R isolates, six (12.2%) were positive for MβL production according the phenotypic tests results.However, only two isolates carried an MβL-encoding gene, one of which was considered negative in the DA tests, suggesting that these tests were not useful for screening MβL in the isolates studied, yielding false-positive and false-negative results.This discrepancy among the results  obtained in phenotypic and genotypic tests has also been reported in other studies (13) (14) and may be related to diffi culties in reading and interpreting tests, variations in the sensitivity and specifi city of the method according to the combination of substrate and chelating agent, the concentration of the chelating agent, and the presence of other resistance mechanisms that may affect the results of the phenotypic tests.We found a low (4.1%)rate of isolates harboring the bla SPM-1 gene.Different rates of SPM-producing P. aeruginosa isolates have been reported in Brazil, varying according to geographic region as follows: 7.5% in São Luis (5) (in Northeastern Brazil), 20.7% in Recife (4) (in Northeastern Brazil), and 42% in Goiás (13) (in Mid-west Brazil).

Rev
The two SPM-1-positive isolates showed electrophoretic profi les indistinguishable from or closely related to the bla SPM-1 -positive control strains (P1088 and 365h2).These results confi rmed the data from other studies indicating that the SP clone was widespread in Brazil (3) .However, the MβL-negative isolates showed high genetic diversity and were unrelated to the SP clone.Interestingly, one of the bla SPM-1 -positive isolates was susceptible to carbapenem.One possible reason for this could be the low expression of bla SPM-1 .A carbapenem-susceptible P. aeruginosa isolate harboring the bla SPM-1 gene has previously been described in Brazil (15) .
To the best of our knowledge, this is the fi rst report of SPM-1 P. aeruginosa isolates identifi ed in Sergipe.However, the results obtained in the present study suggested that resistance to ceftazidime and carbapenem among the P. aeruginosa isolates analyzed in this study was a result of resistance mechanisms other than MβL production.