Characterization of a Novel AmpC β-Lactamase Produced by a Carbapenem-Resistant Cedecea davisae Clinical Isolate

ABSTRACTACedecea davisaeisolate, which was intermediate or resistant to third-generation cephalosporins and carbapenems, was recovered from a urine sample. Susceptibility testing, isoelectric focusing, and analysis of outer membrane proteins showed that AmpC β-lactamase expression combined with porin deficiency accounted for the carbapenem resistance. A cloning experiment followed by phenotypic and enzymatic characterization identified a novel class C enzyme that was phylogenetically and biochemically close to the chromosome-borne β-lactamases of the generaEnterobacterandCitrobacter.

Developmental potential of pig embryos reconstructed by use of sow versus pre-pubertal gilt oocytes after somatic cell nuclear transfer

SummaryIn this study, the developmental ability of cloned embryos using gilt versus sow oocytes was evaluated under the hypothesis that the efficiency of nuclear transfer using gilt oocytes was lower than that of sow oocytes, but that it could be optimized. Five experiments were performed with routine production of cloned embryos with sow oocytes serving as the control. Results showed that: Experiment 1: Blastocyst rates of cloned embryos with gilt oocytes was about half compared with control. Experiment 2: An extended maturation time of 48 h used for gilt oocytes resulted in lower blastocyst rates after cloning. Experiment 3: Development of cloned embryos with gilt oocytes was improved by co-culture with sow oocytes. Experiment 4: After maturation of gilt oocytes using follicular fluid from gilt instead of sow, the oocytes were sorted into large and small oocytes, and after cloning, blastocyst rates were higher using large gilt oocytes compared with small oocytes; however, the rate remained lower compared with control. Experiment 5: Six sow recipients received a total of 503 morulae and blastocysts cloned from gilt oocytes (four recipients) and 190 cloned from sow oocytes (two recipients). All recipients became pregnant and went to term, resulting in 26 (gilt oocytes) and six (sow oocytes) piglets. In conclusion, results confirmed that nuclear transfer efficiency was higher using sow versus gilt oocytes, but the use of gilt oocytes can be optimized by sorting after ooplasm size following maturation and by maturing gilt and sow oocytes together.

Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop

ABSTRACTTheCitrobacter freundiiisolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) β-lactamase. The chromosome-borneblaAmpC-CHAgene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded theEscherichia coliTOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resultingE. coliTOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase ofC. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficientE. colistrain. Moreover, it exhibited increased catalytic efficiency (kcat/Km) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (kcat), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A β-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.

Coproduction of Novel 16S rRNA Methylase RmtD and Metallo-β-Lactamase SPM-1 in a Panresistant Pseudomonas aeruginosa Isolate from Brazil

ABSTRACT Serious infections with Pseudomonas aeruginosa are frequently treated with the combination of a β-lactam antimicrobial and an aminoglycoside. P. aeruginosa strain PA0905 was isolated in 2005 from an inpatient in Brazil. It showed a panresistant phenotype that included resistance to β-lactams, aminoglycosides, and fluoroquinolones. The β-lactam resistance was conferred by the production of the metallo-β-lactamase SPM-1. No inhibitory zone was observed when a disk diffusion test was performed with the semisynthetic aminoglycoside arbekacin, raising suspicion of 16S rRNA methylase production. A cloning experiment subsequently revealed the presence of a novel 16S rRNA methylase, RmtD, which accounted for the high-level resistance to all 4,6-disubstituted deoxystreptamine aminoglycosides, such as amikacin, tobramycin, and gentamicin. RmtD shared a moderate degree of identity with RmtA, another 16S rRNA methylase that was initially reported to occur in P. aeruginosa in Japan in 2003. This is the first identification of aminoglycoside resistance mediated by a 16S rRNA methylase in South America. This is also the first report to document coproduction of a metallo-β-lactamase and a 16S rRNA methylase, a combination that would severely compromise therapeutic options for the infected patients.

Molecular Characterization of a Cephamycin-Hydrolyzing and Inhibitor-Resistant Class A β-Lactamase, GES-4, Possessing a Single G170S Substitution in the Ω-Loop

ABSTRACT The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type β-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new β-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 μg/ml) and β-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 ± 1.7 μM). The GES-4 enzyme had a single G170S substitution in the Ω-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of β-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla GES-4 genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla GES-4 genes found in strain KG502 were almost identical to that of bla GES-3 in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla GES-4 gene found in strain KG502 might well emerge from a point mutation in the bla GES-3 gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.

Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

ABSTRACT A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.