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Evolution of TEM-type enzymes: biochemical and genetic characterization of two new complex mutant TEM enzymes, TEM-151 and TEM-152, from a single patient.

ABSTRACT: Two clinical isolates of Escherichia coli, CF1179 and CF1295, were isolated from a patient hospitalized in the hematology unit of the University Hospital of Clermont-Ferrand, Clermont-Ferrand, France. They were resistant to penicillin-clavulanate combinations and to ceftazidime. The double-disk synergy test was positive only for isolate CF1179. Molecular comparison of the isolates showed that they were clonally related. E. coli recombinant strains exhibiting the resistance phenotype of the clinical strains were obtained by cloning. The clones corresponding to strains CF1179 and CF1295 produced TEM-type beta-lactamases with pI values of 5.7 and 5.3, respectively. Sequencing analysis revealed two novel blaTEM genes encoding closely related complex mutant TEM enzymes, designated TEM-151 (pI 5.3) and TEM-152 (pI 5.7). These two genes also harbored a new promoter region which presented a 9-bp deletion. The two novel beta-lactamases differed from the parental enzyme, TEM-1, by the substitution Arg164His, previously observed in extended-spectrum beta-lactamases (ESBLs), and by the substitutions Met69Val and Asn276Asp, previously observed in the inhibitor-resistant penicillinase TEM-36/IRT-7. They differed by two amino acid substitutions: TEM-152 harbored a Glu240Lys ESBL-type substitution and TEM-151 had an Ala284Gly substitution. Functional analysis of TEM-151 and TEM-152 showed that both enzymes had hydrolytic activity against ceftazidime (kcat, 5 and 16 s-1, respectively). TEM-152 was more resistant than TEM-151 to the inhibitor clavulanic acid (50% inhibitory concentrations, 1 versus 0.17 microM). These results confirm the evolution of TEM-type enzymes toward complex enzymes harboring the two kinds of substitutions which confer an extended spectrum of action against beta-lactam antibiotics and resistance to inhibitors.

SUBMITTER: Robin F

PROVIDER: S-EPMC1855492 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Evolution of TEM-type enzymes: biochemical and genetic characterization of two new complex mutant TEM enzymes, TEM-151 and TEM-152, from a single patient.

Robin Frédéric F Delmas Julien J Schweitzer Cédric C Tournilhac Olivier O Lesens Olivier O Chanal Catherine C Bonnet Richard R

Antimicrobial agents and chemotherapy 20070112 4

Two clinical isolates of Escherichia coli, CF1179 and CF1295, were isolated from a patient hospitalized in the hematology unit of the University Hospital of Clermont-Ferrand, Clermont-Ferrand, France. They were resistant to penicillin-clavulanate combinations and to ceftazidime. The double-disk synergy test was positive only for isolate CF1179. Molecular comparison of the isolates showed that they were clonally related. E. coli recombinant strains exhibiting the resistance phenotype of the clini ...[more]

PMID: 17220412

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Project description:BACKGROUND: Calcium carbonate biominerals form often complex and beautiful skeletal elements, including coral exoskeletons and mollusc shells. Although the ability to generate these carbonate structures was apparently gained independently during animal evolution, it sometimes involves the same gene families. One of the best-studied of these gene families comprises the ?- carbonic anhydrases (CAs), which catalyse the reversible transformation of CO2 to HCO3 - and fulfill many physiological functions. Among Porifera -the oldest animal phylum with the ability to produce skeletal elements- only the class of calcareous sponges can build calcitic spicules, which are the extracellular products of specialized cells, the sclerocytes. Little is known about the molecular mechanisms of their synthesis, but inhibition studies suggest an essential role of CAs. In order to gain insight into the evolution and function of CAs in biomineralization of a basal metazoan species, we determined the diversity and expression of CAs in the calcareous sponges Sycon ciliatum and Leucosolenia complicata by means of genomic screening, RNA-Seq and RNA in situ hybridization expression analysis. Active biomineralization was located with calcein-staining. RESULTS: We found that the CA repertoires of two calcareous sponge species are strikingly more complex than those of other sponges. By characterizing their expression patterns, we could link two CAs (one intracellular and one extracellular) to the process of calcite spicule formation in both studied species. The extracellular biomineralizing CAs seem to be of paralogous origin, a finding that advises caution against assuming functional conservation of biomineralizing genes based upon orthology assessment alone. Additionally, calcareous sponges possess acatalytic CAs related to human CAs X and XI, suggesting an ancient origin of these proteins. Phylogenetic analyses including CAs from genomes of all non-bilaterian phyla suggest multiple gene losses and duplications and presence of several CAs in the last common ancestor of metazoans. CONCLUSIONS: We identified two key biomineralization enzymes from the CA-family in calcareous sponges and propose their possible interaction in spicule formation. The complex evolutionary history of the CA family is driven by frequent gene diversification and losses. These evolutionary patterns likely facilitated the numerous events of independent recruitment of CAs into biomineralization within Metazoa.

Project description:Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe.IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today.

Dataset Information

Evolution of TEM-type enzymes: biochemical and genetic characterization of two new complex mutant TEM enzymes, TEM-151 and TEM-152, from a single patient.

Publications

Evolution of TEM-type enzymes: biochemical and genetic characterization of two new complex mutant TEM enzymes, TEM-151 and TEM-152, from a single patient.

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