Ian Phillips

Prudent Use of Antibiotics: Are Our Expectations Justified?

Prudent use of antibiotics has 3 components, rational use, adherence to local guidelines and policies, and avoidance or reversal of upward demographic trends in antibiotic resistance. Although rational use is mandatory, it must be recognized that adherence to policies will reduce clinical freedom, for good or ill. Expectations that prudent use will deliver reversals in resistance trends should be accepted with caution. Sound, pertinent data are lacking, and our ability to detect change, if it does occur, and to attribute its cause correctly, is questionable.

Reevaluation of Antibiotic Breakpoints

Differences in antibiotic breakpoints have resulted from differences in clinical practice and in interpretation of the parameters that are considered when breakpoints are set. Differences in the first of these are less common for newer agents, whereas differences in the latter could be resolved by consensus discussions. Greater difficulty arises when common pathogens acquire resistance, especially when this occurs by degrees, as has happened with penicillin for gonococci and the pneumococci, and for the fluoroquinolones in relation to a wide variety of bacterial species. Changes to breakpoints under such circumstances should be introduced only after the most careful consideration and the education of diagnostic microbiologists and clinicians.

Deletion of Genes From the Mouse Genome Using Cre/loxP Technology.

The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmo1, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technology. Genomic clones corresponding to the genes to be deleted are first isolated, and then appropriate genomic fragments are cloned into vectors containing a loxP site. This produces targeting vectors, which are electroporated into mouse embryonic stem (ES) cells to allow a homologous recombination event to take place between the mouse genomic fragment, present within the vector, and the homologous sequences in the ES cell genome. Screening of ES cells for recombinants in which loxP sites have been inserted on either side of the gene cluster to be deleted is described. Recombination by Cre recombinase to produce ES cell lines carrying the deletion on chromosome 1 is also described.

Microinjection of Targeted Embryonic Stem Cells and Establishment of Knockout Mouse Lines for Fmo Genes

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mixed coat color (the chimera). Also described are methods to establish the success of blastocyst injection and implantation of germ-line transmission of the knockout (KO) mutation. Breeding strategies to produce congenic and isogenic KO mouse lines are outlined. Simple methods for the isolation of tail DNA, the tagging of mice, and record keeping of the line are also given.

Expression of recombinant flavin-containing monooxygenases in a baculovirus/insect cell system.

The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodopterafrugiperda (Sf) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins.

Determination of cellular localization of expression of flavin-containing monooxygenase genes in mouse tissues by in situ hybridization.

Methods are described for the cellular localization of expression of flavin-containing monooxygenase (FMO) genes in various mouse tissues by in situ hybridization. These include the production of digoxigenin (DIG)-labeled antisense and sense RNA probes by transcription from FMO cDNA templates, the preparation of paraffin wax-embedded and cryostat tissue sections, the hybridization of RNA probes to tissue sections, and the specific detection of hybridized probes using an antibody to DIG.