Novel Carbapenem-Hydrolyzing Oxacillinase OXA-62 from Pandoraea pnomenusa

Abstract

Pandoraea spp. are gram-negative, glucose nonfermenting rods detectable in blood cultures and sputa of cystic fibrosis patients. They are resistant to various antibiotic groups, with imipenem being the only active ␤-lactam. We isolated an imipenem-resistant (MIC, 64 g/ml) Pandoraea pnomenusa strain from a cystic fibrosis patient. Cloning and sequencing identified two ␤-lactamases of Bush group 2d, namely, the known OXA-33, located on an integron, and the novel carbapenem-hydrolyzing oxacillinase OXA-62. OXA-62 is only distantly related to other oxacillinases (OXA-50 being closest with 43% amino acid identity). It hydrolyzes penicillins, oxacillin, imipenem, and meropenem but not expanded-spectrum cephalosporins. The bla OXA-62 gene is chromosome located. No transposable elements were found in its genetic neighborhood. With OXA-62-specific primers, bla OXA-62 could be identified in all P. pnomenusa strains and appears to be species specific. This additional mechanism of carbapenem resistance further complicates the treatment of infections caused by P. pnomenusa.The genus Pandoraea was established by Coenye et al. in 2000 (6) as a result of reanalyzing strains not definitely identified to the species level which were phenotypically closest to Burkholderia cepacia, Ralstonia pickettii, or Ralstonia paucula. The genus Pandoraea includes five named species (Pandoraea apista, P. norimbergensis, P. pnomenusa, P. pulmonicola, and P. sputorum) and four unnamed genomospecies (9). Pandoraea sp. strains have been isolated from patients with septicemia and respiratory tract infections (mostly cystic fibrosis), as well as from food, water, and soil (6,9,14,17,21).Antibiotic therapy of infections caused by Pandoraea spp. is impaired by their resistance to multiple antibiotics, including penicillins, cephalosporins, cefoxitin, meropenem, aminoglycosides, and chloramphenicol. Their resistance to fluoroquinolones is variable. Only tetracycline, trimethoprim-sulfamethoxazole, and imipenem were found to be active against the majority of isolates (9,14,17,21). We recently cultured a P. pnomenusa strain from a cystic fibrosis patient. For this multiresistant strain, the MIC of imipenem was unusually high. A novel carbapenemhydrolyzing oxacillinase, OXA-62, turned out to be involved in the mechanism of resistance to imipenem.(Part of this work was presented at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy [abstr. C1-666 and C2-2180].) MATERIALS AND METHODSStrains and plasmids. The strains used in this study are characterized in Table 1. The Pandoraea spp. were isolated from sputa of cystic fibrosis patients between 1997 and 2004 in different centers in Germany, partly as Burkholderia spp. not identifiable to the species level. They were identified by phenotypic (API 20 NE [BioMérieux, Marcy l'Etoile, France] and additional biochemical tests) and genotypic methods (PCR with species-specific oligonucleotides) with Pandoraea sp. type strains obtained from the Laboratorium voor Microbiologie (Universiteit Ghe...

Carbapenemases: the Versatile β-Lactamases

“…This demonstrates the presence of other mechanisms contributing to the carbapenem resistance in P. aeruginosa, such as intrinsic impermeability, possibly coupled with an efflux mechanism. This effect of lowlevel transferable resistance has also been observed in K. pneumoniae and A. baumannii with metallo-␤-lactamases (39,184), as well as with OXA carbapenemases (12,170,192). Many OXA carbapenemases have been found in A. baumannii, where MICs for imipenem are usually higher than 8 g/ml, but E. coli cells expressing these enzymes have imipenem MICs of Յ2 g/ml (225).…”

“…These PoxB oxacillinases have been reported to be commonly present on the chromosomes of many strains of P. aeruginosa (40 of 70 strains tested) (96) and are thought to be part of the natural component of ␤-lactamases in that species, but they may not be expressed in all strains and do not cause carbapenem resistance (imipenem MIC, Յ1 g/ml) (56,96). Species-specific OXA enzymes include the subgroup 8 OXA-60 family, considered to be a natural component of the genome of Ralstonia pickettii (57), and the subgroup 9 OXA-62, identified as a species-specific oxacillinase in Pandoraea pnomenusa (192).…”

“…This carbamate activates the catalytic serine side chain so that the acyl intermediate can form (131,153). To ensure that this modification occurs in studies with isolated enzymes, some researchers add 10 mM NaHCO 3 to their reactions (57,69,192).…”

Inhibition of co-colonizing cystic fibrosis-associated pathogens by Pseudomonas aeruginosa and Burkholderia multivorans

“…More recently, genus Pandoraea has been implicated as the cause of declining lung function and even death in some chronically colonized patients and has recently been reported as a virulent pathogen with multiple mechanisms of pathogenicity (Costello et al, 2011;Jørgensen et al, 2003;Stryjewski et al, 2003). Pandoraea colonization has been associated with CF and non-CF patients and often occurs when the patient has already succumbed to chronic colonization with another pathogen (Schneider et al, 2006). Nine species have been identified within the genus to date Daneshvar et al, 2001;Sahin et al, 2011).…”

Género Pandoraea

“…Es variable para fluoroquinolonas y presenta un patrón singular de resistencia a carbapenems: son resistentes a meropenem pero susceptibles a imipenem 1,4 . En un estudio reciente efectuado en Alemania, se ha encontrado una cepa de P. pnomenusa resistente a imipenem 5 , lo que hace importante su identificación hasta el nivel de especie. …”