Diversity of β-lactamases produced by ceftazidime-resistant ...

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    MS-AAC00453-09 Version 2 1

    Diversity of -lactamases produced by ceftazidime-resistant 2

    Pseudomonas aeruginosa causing bloodstream infections in Brazil 3


    Renata C. Pico,1,2

    Laurent Poirel,1 Ana C. Gales,

    2 and Patrice Nordmann

    1* 5

    Service de Bactriologie-Virologie, INSERM U914 Emerging Resistance to Antibiotics, 6

    Hpital de Bictre, Assistance Publique/Hpitaux de Paris, Facult de Mdecine Paris 7

    Sud, K.-Bictre, France1 and Laboratrio ALERTA, Universidade Federal de So Paulo, 8

    So Paulo, Brazil2 9



    Keywords: Pseudomonas aeruginosa, Brazil, ESBL, MBL.



    Running title: MBL and ESBL-producing P. aeruginosa in Brazil 14


    *Corresponding author. Mailing address: Service de Bactriologie-Virologie, Hpital de 16

    Bictre, 78 rue du Gnral Leclerc, 94275 Le Kremlin-Bictre cedex, France. Phone: 33-1-17

    45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice@bct.aphp.fr 18

    Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00453-09 AAC Accepts, published online ahead of print on 13 July 2009

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    A retrospective survey was conducted to characterize -lactamases in a 20

    collection of 43 ceftazidime-resistant Pseudomonas aeruginosa isolates recovered from 21

    patients with bloodstream infections hospitalized at a Brazilian teaching hospital 22

    between January and December 2005. Resistance rates for carbapenems, 23

    aminoglycosides and quinolones were over 80%, only colistin remaining active 24

    against all isolates. Pulsed-field gel electrophoresis analysis identified seven different 25

    genotypes. AmpC overproduction was found to be the sole -lactamase-mediated 26

    mechanism responsible for ceftazidime resistance in four isolates (9.3%). Nine isolates 27

    (20.9%) produced an extended-spectrum -lactamase (ESBL), being GES-1 (n=7, 28

    16.3%) and CTX-M-2 (n=2, 4.6%). A carbapenemase activity was detected in 30 29

    (70%) additional isolates. Among those isolates, two isolates (4.6%) produced the 30

    ESBL GES-5 possessing the ability to hydrolyze imipenem, a single isolate (2.3%) 31

    produced the metallo--lactamase (MBL) IMP-1, and 27 isolates produced the MBL 32

    SPM-1 (62.8%). None of the isolates co-produced both ESBL and MBL. Insertion 33

    sequence elements ISCR4 and ISCR1 were associated with blaSPM-1 and blaCTX-M-2 34

    genes, respectively, whereas the blaGES-1 and blaGES-5 genes were part of class 1 35

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    integron structures. This study underlines the spread of MBL- and ESBL-producing 36

    P. aeruginosa as an important source of ceftazidime resistance in Brazil. 37

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    Pseudomonas aeruginosa is a leading cause of hospital-acquired infections. 38

    Acquisition of -lactamases by P. aeruginosa nosocomial isolates, such as class A 39

    extended-spectrum--lactamases (ESBL), and class B metallo--lactamases (MBL) is 40

    critical for antimicrobial therapy in hospitalized patients (18). 41

    The ESBLs reported in P. aeruginosa are SHV, TEM, PER, VEB, BEL, GES, and 42

    more recently CTX-M types (1, 7, 8, 15, 19, 22, 28). The GES-type enzymes are peculiar 43

    since point amino acid changes in their active sites may extend their hydrolytic activity to 44

    carbapenems (30, 39, 40). ESBL production in P. aeruginosa has been documented in 45

    Brazil (2, 5, 20), but its prevalence remains unknown. 46

    Five types of acquired MBLs have been identified in P. aeruginosa: IMP, VIM, 47

    SPM, GIM, and AIM (41, 42). In Brazil, IMP-, VIM- and SPM-producing P. aeruginosa 48

    clinical isolates have been identified (34). In addition, SPM producers have been reported 49

    as endemic in Brazilian territory due to dissemination of a single clone (10). 50

    The aim of this study was to investigate diversity and frequency of both ESBL and 51

    MBL production and to characterize the genetic support of those acquired -lactamase 52

    genes in a collection of ceftazidime-resistant P. aeruginosa clinical isolates from Brazil, 53

    taken as a model of developing country. 54

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    Bacterial strains. A total of 154 consecutive P. aeruginosa isolates were recovered 56

    from patients with bloodstream infections hospitalized at Hospital So Paulo between 57

    January and December 2005. A single isolate per patient was retained for this study. 58

    Among those isolates, 43 (28%) were ceftazidime-resistant by the CLSI disk diffusion 59

    method (inhibition zone 14 mm, MIC 32 g/ml) and thus were further characterized. 60

    Escherichia coli TOP10 was used as recipient strain in cloning experiments (22). 61

    Transformation experiments were performed using both E. coli TOP10 and P. aeruginosa 62

    PAO1 as the recipients. 63

    Clinical data. Clinical data including age, co-morbidities, ward of hospitalization, 64

    site of infection, therapeutic regimen and final disposition (death or discharge) have been 65

    collected for each patient. 66

    Susceptibility testing, screening for AmpC overproducers and/or ESBL 67

    production. Antibiotic susceptibility profiles of the 43 P. aeruginosa isolates were 68

    determined by the agar-dilution method, according to the CLSI guidelines (3). AmpC 69

    overproducers were identified by testing susceptibility to ceftazidime on Mueller-Hinton 70

    (MH) plates supplemented with 250 g/ml cloxacillin (17, 32). Detection of ESBL 71

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    production was carried out by a double disk synergy method testing ceftazidime, 72

    aztreonam, cefepime distant 15 mm from ticarcillin/clavulanic acid disks, on MH plates 73

    supplemented or not with cloxacillin-containing plates (27). 74

    Screening for carbapenemase activity. Hydrolysis of imipenem was assessed by 75

    UV spectrophtotometry assays, as described (10, 11, 24). Briefly, 10 ml of an overnight 76

    broth culture was harvested and then disrupted by sonication. Whole-protein extracts were 77

    obtained after centrifugation. Hydrolytic activity of 20 l of the crude extract was 78

    determined against 100 M imipenem in 100 mM phosphate buffer (pH 7.0), and 79

    measurements were carried out at a wavelength of 297 nm. 80

    PCR amplification for detection of ESBL and MBL genes; analysis of the genetic 81

    environment and sequencing. Specific primers were used in standard PCR conditions to 82

    detect ESBL and MBL encoding genes, namely blaTEM, blaSHV, blaCTX-M, blaGES, blaPER, 83

    blaVEB, blaBEL, blaKPC, blaIMP, blaVIM, blaSPM, blaGIM, blaAIM (7, 12, 15, 16, 19, 22, 25, 28, 84

    29, 35, 38, 42). The genetic environment of blaIMP was determined by PCR using the 85

    previously published specific primers to anneal at the 5and 3conserved sequences (CS) of 86

    class 1 integrons, followed by sequencing (23). The genetic environment of blaCTX-M-2 was 87

    determined by PCR and further sequencing using specific primers for the insertion 88

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    sequence ISCR1 located upstream and for qacE1/sul1 tandem (16). The genetic context 89

    of blaSPM-1 was determined by using primers hybridizing the ISCR4, as described (26). For 90

    direct DNA sequencing, PCR products were purified using PCR purification columns 91

    (Qiagen, Courtaboeuf, France). Sequencing reactions were performed using specific 92

    primers and an automated ABI 337 sequencer (Applied Biosystems, Foster city CA). The 93

    nucleotide and deduced protein sequences were analyzed with software available over the 94

    internet at the National Center for Biotechnology Information website 95

    (http://www.ncbi.nlm.nih.gov). 96

    PFGE analysis. Genetic relatedness among the ceftazidime-resistant P. aeruginosa 97

    was evaluated by pulsed-field gel electrophoresis (PFGE) using restriction enzyme SpeI 98

    (GE Healthcare, Orsay, France) as described (26). Analysis of PFGE patterns was


    performed by visual inspection of photographs of ethidium bromide-stained gels. The 100

    isolates were classified according to the criteria described by Tenover et al. (36). The P. 101

    aeruginosa isolate 48-1997 (38), corresponding to the SPM-producing national clone, was 102

    included in PFGE experiments for direct comparison of genotypes. 103

    Genetic support of -lactamase-encoding genes. Plasmid extraction was performed 104

    by the Kieser technique (13). E. coli NCTC50192 harboring four plasmids of 154-, 66-, 38- 105

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    and 7-kb was used as size marker for plasmids. Transformation assays were performed by 106

    electroporation with plasmid extracts from