Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo- β -lactamase-producing isolates

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL) genes bla SPM-1 , bla IMP and bla VIM . The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

was used per patient for a total of 73 isolates.The bacterial species were identified by standard biochemical tests.Susceptibility testing was performed by means of the disk-diffusion method with the following antimicrobial agents: imipenem, ceftazidime, ciprofloxacin, amikacin, gentamicin, piperacillin/tazobactam, aztreonam and polymyxin B, in compliance with the Clinical and Laboratory Standards Institute guidelines (CLSI 2010).The minimal inhibitory concentration (MIC) for carbapenems of MBL-negative isolates was determined by the automated BD Phoenix system and interpreted in accordance with CLSI.Selected isolates were screened for MBL production by the disk approximation test as previously described (Arakawa et al. 2000).
Presumptive MBL producers were further tested for the bla SPM-1 , bla IMP and bla VIM genes.Bacterial DNA was extracted by using the Brazol kit (LGC Biotecnologia, Brazil) following the recommendations of the manufacturer and analysed by polymerase chain reaction (PCR) using the primer pairs bla SPM-1 (forward: 5'-CCTACAATCTAACGGCGACC-3', reverse: 5'-TCGCCGTGTCCAGGTATAAC-3'), bla IMP (forward: 5'-GGAATAGAGTGGCTTAATTCTC-3', reverse: 5'-GTGATGCGTCYCCAAYTTCACT-3') and bla VIM (forward: 5'-TGCGCATTCGACCGACAATC-3', reverse: 5'-GTCGAATGCGCAGCACCAGG-3') (Migliavacca et al. 2002, Gales et al. 2003, Toleman et al. 2005).Positive controls for the P. aeruginosa bla SPM-1 , bla IMP and bla VIM genes were kindly provided by Special Clinical Microbiology Laboratory and ALERTA Laboratory (São Paulo, Brazil).The amplicons were purified with the aid of a PCR purification kit (Promega Co, USA) and submitted to DNA sequencing by the platform of the Aggeu Magalhães Research Centre, Oswaldo Cruz Foundation, Recife.The nucleotide sequences were evaluated with the BioEdit TM program and analysed by on-line BLASTn at GenBank dataset (National Centre for Biotechnology and Information).In addition, all of the isolates were genotyped by DNA macrorestriction followed by pulsedfield gel electrophoresis (PFGE) using the endonuclease SpeI.A representative isolate of the SPM Brazilian clone (from São Paulo Hospital) and the sequenced strain PA01 (kindly provided by the Pseudomonas Genome Project, Boston, MA, USA) were included as a reference point.
Clonal relationships among the isolates were established using the criteria of Tenover et al. (1995).
The isolates from the present work revealed an important change in the epidemiology of carbapenem-resistant P. aeruginosa isolates between the periods 2002-2003 and 2008-2009, showing a decreasing prevalence of the epidemic SPM-1-producing clone in the final period of bacterial recovery (Table ).Moreover, the antimicrobial susceptibility test revealed that these bacteria were co-resistant to many other anti-Pseudomonas drugs, particularly the most recent strains (2008/2009).On the other hand, all of the bacterial samples were susceptible to polymyxin B (Table ).
A high prevalence of the MBL phenotype was observed among the 2002/2003 isolates (98.4%) (Table ).This coincided with a higher incidence of bla SPM-1 than those found in previous studies conducted on a national scale (Sader et al. 2004, Gräf et al. 2008).Nevertheless, this high incidence of the bla SPM-1 gene decreased in the 2008/2009 isolates (Table ), suggesting that carbapenemresistance mechanisms other than MBL must be present and are being spread in the hospital under study.Moreover, these other mechanisms, such as efflux pump over expression (associated or not with porin down-regulation), may also be involved in the overall increased resistance to anti-Pseudomonas drugs observed in 2008/2009.None of the isolates showed amplification of the bla IMP or bla VIM gene (data not shown).Thus, two presumptive MBL producers, from 2002/2003, did not carry any of the MBL genes tested.As expected, none of the 10 MBLnegative isolates indicated the presence of MBL genes.
Molecular typing indicated the prevalence of bacterial isolates (herein designated as genotype A) closely re- gene belonged to the clonal pattern A. Interestingly, the increase in bacterial variation was also accompanied by an increase in bacterial resistance.It is noteworthy that the MIC values for imipenem and meropenem were not high among the more recent isolates (MIC > 8 μg/mL), which corroborates the hypothesis that there are alternative resistance mechanisms.This is supported by the fact that MBL enzymes increase antimicrobial MICs more effectively than does either efflux pump over-expression or porin down-regulation alone (Xavier et al. 2010).
In conclusion, the population of carbapenem-resistant P. aeruginosa in the hospital under study was replaced by MBL-negative, genetically unrelated bacterial isolates.This finding emphasises the need for continuous surveillance strategies and an improvement of the infection control measures in this institution.

TABLE Microbiological
(Gales et al. 2003)05, Fonseca et al. 2010997A, which was widely disseminated in the 2002/2003 group of isolates (Table).The existence of common PFGE types among carbapenem-resistant P. aeruginosa isolates from distinct geographical locations has been reported and indicates clonal dispersion(Zavascki et al. 2005, Fonseca et al. 2010).In Brazil, there have been previous reports of the spread of a unique SPM-type MBL-positive clone(Gales et al. 2003).As expected, MBL-negative isolates from 2008/2009 were unrelated to the epidemic Brazilian clone and showed six distinct PFGE types (Table).Thus, new clones could be responsible for the dissemination of other resistance mechanisms to carbapenems.The three 2008/2009 MBL-positive isolates that carried the bla SPM-1 lated