Contribution of asparagine 346 residue to the carbapenemase activity of CMY-2 ß-lactamase.

Only a few plasmid-borne AmpC (pAmpC) ß-lactamases, such as CMY-2, can account for carbapenem resistance in Enterobacteriaceae in combination with outer membrane impermeability. The aim of this study was to elucidate the contribution of Asn-346, which is well conserved among carbapenem-hydrolyzing pAmpCs, to the hydrolysis spectrum of CMY-2. Site-directed mutagenesis experiments were carried out to replace Asn-346 with glycine, alanine, valine, glutamate, aspartate, serine, threonine, glutamine, tyrosine, isoleucine, lysine, and histidine. The recombinant plasmids were transferred into wild-type and porin-deficient Escherichia coli strains. Asn-346 replacement reduced significantly the MICs of all ß-lactams, except the Asn-346-Ile substitution that increased the MICs of cephalosporins, whereas it decreased those of carbapenems. The biochemical characterization, along with a molecular modeling study, showed that the size and the polarity of the side chain at position 346 assisted substrate binding and turnover. This study shows for the first time that the amino acid at position 346 contributes to the ß-lactamase activity of cephalosporinases. Asparagine and isoleucine residues, which are well conserved at position 346 among AmpC-type enzymes, modulate their hydrolysis spectrum in an opposing sense. Ile-346 confers higher level of cephalosporins resistance, whereas Asn-346 confers carbapenem resistance in combination with outer membrane impermeability.

Value of Xpert MRSA/SA blood culture assay on the Gene Xpert® Dx System for rapid detection of Staphylococcus aureus and coagulase-negative staphylococci in patients with staphylococcal bacteremia.

The Xpert MRSA/SA BC assay was examined prospectively in patients with staphylococcal bacteremia including 6 patients with blood culture bottles inoculated with biological fluid (synovial fluid in 4 cases and peritoneal fluid in 2 cases). Among the 56 Staphylococci species isolated, 80.3% were coagulase-negative staphylococci (CoNS) and 19.7% were S. aureus. Methicillin susceptibility test results showed that 77.8% of isolates were methicillin-resistant CoNS (MRCoNS) and 22.2% of isolates were methicillin-susceptible CoNS (MSCoNS). Of 11 S. aureus isolates, 63.7% were methicillin-susceptible S. aureus (MSSA) and 36.3% were methicillin-resistant S. aureus (MRSA). Xpert MRSA/SA BC results showed that genotypic results were in concordance with phenotypic results in 94.6% of cases versus 5.4% of discordant cases. Of these 3 discordant cases, 1 S. aureus isolate had an MRSA phenotype and an SPA(+)mec(+)SCCmec(-) genotype and another S. aureus isolate was phenotypically MSSA and genotypically SPA(+)mec(+)SCCmec(-), and 1 S. epidermidis isolate was phenotypically MSCoNS and genotypically SPA(-)mec(+)SCCmec(-).

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord.

BACKGROUND: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin+ Sox2+ neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). RESULTS: Here we report the isolation and long term propagation of another population of Nestin+ cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. CONCLUSION: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.

Emergence of ertapenem resistance in an Escherichia coli clinical isolate producing extended-spectrum beta-lactamase AmpC.

Escherichia coli isolate MEV, responsible for a bloodstream infection, was resistant to penicillins, cephalosporins, and ertapenem. Molecular and biochemical characterization revealed the production of a novel, chromosome-borne, extended-spectrum AmpC (ESAC) ß-lactamase with a Ser-282 duplication and increased carbapenemase activity. This study demonstrates for the first time that chromosome-borne ESAC ß-lactamases can contribute to the emergence of ertapenem resistance in E. coli clinical isolates.

Phenotypic and biochemical comparison of the carbapenem-hydrolyzing activities of five plasmid-borne AmpC ß-lactamases.

The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) ß-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of ß-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 ß-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 ß-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low K(m) values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type ß-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.

Characterization of CSP-1, a novel extended-spectrum beta-lactamase produced by a clinical isolate of Capnocytophaga sputigena.

The Capnocytophaga sputigena isolate NOR, responsible for septicemia, was resistant to amoxicillin and narrow-spectrum cephalosporins. In a cloning experiment, a new gene, bla(CSP-1), was identified; this gene encodes a novel extended-spectrum beta-lactamase (ESBL) that shares only 52% and 49% identities with the CME-1 and VEB-1 beta-lactamases, respectively. The G+C content of this gene, its genetic environment, the absence of conjugation transfer, and its detection in two reference strains suggested that it was an intrinsic resistance gene located on the chromosome.

Lectin affinity electrophoresis of serum alkaline phosphatase in metastasized breast cancer.

The use of serum alkaline phosphatase (ALP) isoenzymes as markers of breast cancer metastases and treatment efficacy has received little attention. Twenty-six breast cancer women (56+/-13 years, all post-menopausal) were prospectively evaluated during their first and third course of chemotherapy (4-week interval). Serum samples were analyzed for ALP isoenzymes (bone, liver, and intestine) using a lectin affinity electrophoresis kit (Hydragel 15 ISO-PAL, Sebia) adapted on a semi-automated Hydrasys system (Sebia). Results were compared with imaging techniques for the presence of metastases; bone ALP isoenzyme (B-ALP) results were compared with C-Terminal degradation products of type I collagen (S-CTX) (CrossLaps, IDS Nordic). Serum B-ALP, but not S-CTX, confirmed the presence of bone metastases (BM) (n=15) with 67/100% sensitivity/specificity (using a 69 UI/L ROC cut-off); ROC AUC was 0.806 (P=0.0004) (NS for S-CTX). Chemotherapy reduced serum B-ALP by 24% over 4 weeks (P=0.0012); there was no change for S-CTX. There was no specific clinical pattern for other ALP isoenzymes (liver and intestine). In conclusion, serum B-ALP, but not S-CTX, could help confirm the presence of BM in breast cancer patients.

Crossover and noncrossover pathways in mouse meiosis.

During meiosis, recombination between homologous chromosomes generates crossover (CR) and noncrossover (NCR) products. CRs establish connections between homologs, whereas intermediates leading to NCRs have been proposed to participate in homologous pairing. How these events are differentiated and regulated remains to be determined. We have developed a strategy to detect, quantify, and map NCRs in parallel to CRs, at the Psmb9 meiotic recombination hot spot, in male and female mouse germ lines. Our results report direct molecular evidence for distinct CR and NCR pathways of DNA double-strand break (DSB) repair in mouse meiosis based on three observations: both CRs and NCRs require Spo11, NCR products have shorter conversion tracts than CRs, and only CRs require the MutL homolog Mlh1. We show that both products are formed from middle to late pachytene of meiotic prophase and provide evidence for an Mlh1-independent CR pathway, where mismatch repair does not require Mlh1.

An initiation site for meiotic crossing-over and gene conversion in the mouse.

During meiosis, the reductional segregation of homologous chromosomes at the first meiotic division requires reciprocal exchange (crossing over) between homologs. The number of crossovers is tightly regulated (one to two per homolog in mice), and their distribution in the genome is not random-recombination 'hot' and 'cold' regions can be identified. We developed a molecular assay to study these events directly in mouse germ cells. This analysis was developed with reference to the proteosome subunit beta type 9 (Psmb9, previously called Lmp2) hot-spot region identified through genetic analysis. Here we show that this hot spot is an initiation site of meiotic recombination on the basis of two observations: (i) crossover density is maximal in an interval of 210 bp and decreases on both sides of this region; (ii) a high frequency of gene conversion is found in the region of highest crossover density. We then used this strategy to carry out the first temporal analysis of meiotic recombination in mouse spermatogenesis and demonstrate that crossover events occur during the pachytene stage of meiotic prophase.