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Naturally occurring Class A ss-lactamases from the Burkholderia cepacia complex.

ABSTRACT: Chromosomally encoded ss-lactamases from the Burkholderia cepacia complex species (formerly Pseudomonas cepacia) were characterized. Cloning and sequencing identified an Ambler class A ss-lactamase (PenB) from B. cenocepacia. It shares 82% amino acid identity with the PenA ss-lactamases previously identified from B. multivorans 249. Its expression was dependent upon a LysR-type regulatory protein. Its narrow-spectrum hydrolysis activity mostly included penicillins but also included expanded-spectrum cephalosporins and aztreonam at lower levels. In that study, Pen-like ss-lactamases (PenC, PenD, PenE, PenF) that shared 63 to 92% identity with PenB from B. cenocepacia were identified from other Burkholderia species. The corresponding ss-lactamase genes might be used as genetic tools for accurate Burkholderia species identification.

SUBMITTER: Poirel L

PROVIDER: S-EPMC2650541 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Naturally occurring Class A ss-lactamases from the Burkholderia cepacia complex.

Poirel Laurent L Rodriguez-Martinez José-Manuel JM Plésiat Patrick P Nordmann Patrice P

Antimicrobial agents and chemotherapy 20081215 3

Chromosomally encoded ss-lactamases from the Burkholderia cepacia complex species (formerly Pseudomonas cepacia) were characterized. Cloning and sequencing identified an Ambler class A ss-lactamase (PenB) from B. cenocepacia. It shares 82% amino acid identity with the PenA ss-lactamases previously identified from B. multivorans 249. Its expression was dependent upon a LysR-type regulatory protein. Its narrow-spectrum hydrolysis activity mostly included penicillins but also included expanded-spec ...[more]

PMID: 19075063

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Project description:Burkholderia pseudomallei, the founding member of the B. pseudomallei complex (Bpc), is a biothreat agent and causes melioidosis, a disease whose treatment mainly relies on ceftazidime and meropenem. The concern is that B. pseudomallei could enhance its drug resistance repertoire by the acquisition of DNA from resistant near-neighbor species. Burkholderia ubonensis, a member of the B. cepacia complex (Bcc), is commonly coisolated from environments where B. pseudomallei is present. Unlike B. pseudomallei, in which significant primary carbapenem resistance is rare, it is not uncommon in B. ubonensis, but the underlying mechanisms are unknown. We established that carbapenem resistance in B. ubonensis is due to an inducible class A PenB ?-lactamase, as has been shown for other Bcc bacteria. Inducibility is not sufficient for high-level resistance but also requires other determinants, such as a PenB that is more robust than that present in susceptible isolates, as well as other resistance factors. Curiously and diagnostic for the two complexes, both Bpc and Bcc bacteria contain distinct annotated PenA class A ?-lactamases. However, the protein from Bcc bacteria is missing its essential active-site serine and, therefore, is not a ?-lactamase. Regulated expression of a transcriptional penB'-lacZ (?-galactosidase) fusion in the B. pseudomallei surrogate B. thailandensis confirms that although Bpc bacteria lack an inducible ?-lactamase, they contain the components required for responding to aberrant peptidoglycan synthesis resulting from ?-lactam challenge. Understanding the diversity of antimicrobial resistance in Burkholderia species is informative about how the challenges arising from potential resistance transfer between them can be met.IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is highly fatal if not properly treated. Our data show that, in contrast to B. pseudomallei, B. ubonensis ?-lactam resistance is fundamentally different because intrinsic resistance is mediated by an inducible class A ?-lactamase. This includes resistance to carbapenems. Our work demonstrates that studies with near-neighbor species are informative about the diversity of antimicrobial resistance in Burkholderia and can also provide clues about the potential of resistance transfer between bacteria inhabiting the same environment. Knowledge about potential adverse challenges resulting from the horizontal transfer of resistance genes between members of the two complexes enables the design of effective countermeasures.

Project description:Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread.ImportanceBenzyldimethylalkylammonium chloride is commonly used as an antiseptic in the United States. Several recent microbial outbreaks were linked to antiseptics that were found to contain strains of the Burkholderia cepacia complex. Burkholderia species survived in antiseptics, possibly because of the degradation of antiseptic molecules or regulation of relevant gene expression. In this study, we assessed the efflux pump and the potential of B. cepacia complex bacteria to degrade benzyldimethylalkylammonium chloride and improved our understanding of the resistance mechanisms, by using proteomic and metabolic information. To our knowledge, this is the first systematic report of the intrinsic mechanisms of B. cepacia complex strain resistance to benzyldimethylalkylammonium chloride, based on the metabolic and proteomic evidence for efflux pumps and the complete biodegradation of benzyldimethylalkylammonium chloride.

Project description:The objective of the present study was to provide an updated classification for Burkholderia cepacia complex (Bcc) taxon K isolates. A representative set of 39 taxon K isolates were analyzed through multilocus sequence typing (MLST) and phylogenomic analyses. MLST analysis revealed the presence of at least six clusters of sequence types (STs) within taxon K, two of which contain the type strains of Burkholderia contaminans (ST-102) and Burkholderia lata (ST-101), and four corresponding to the previously defined taxa Other Bcc groups C, G, H and M. This clustering was largely supported by a phylogenomic tree which revealed three main clades. Isolates of B. contaminans and of Other Bcc groups C, G, and H represented a first clade which generally shared average nucleotide identity (ANI) and average digital DNA-DNA hybridization (dDDH) values at or above the 95-96% ANI and 70% dDDH thresholds for species delineation. A second clade consisted of Other Bcc group M bacteria and of four B. lata isolates and was supported by average ANI and dDDH values of 97.2 and 76.1% within this clade and average ANI and dDDH values of 94.5 and 57.2% toward the remaining B. lata isolates (including the type strain), which represented a third clade. We therefore concluded that isolates known as Other Bcc groups C, G, and H should be classified as B. contaminans, and propose a novel species, Burkholderia aenigmatica sp. nov., to accommodate Other Bcc M and B. lata ST-98, ST-103, and ST-119 isolates. Optimized MALDI-TOF MS databases for the identification of clinical Burkholderia isolates may provide correct species-level identification for some of these bacteria but would identify most of them as B. cepacia complex. MLST facilitates species-level identification of many taxon K strains but some may require comparative genomics for accurate species-level assignment. Finally, the inclusion of Other Bcc groups C, G, and H into B. contaminans affects the phenotype of this species minimally and the proposal to classify Other Bcc group M and B. lata ST-98, ST-103, and ST-119 strains as a novel Burkholderia species is supported by a distinctive phenotype, i.e., growth at 42°C and lysine decarboxylase activity.

Dataset Information

Naturally occurring Class A ss-lactamases from the Burkholderia cepacia complex.

Publications

Naturally occurring Class A ss-lactamases from the Burkholderia cepacia complex.

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