Sheila I. Hull

Pre-inoculation of urinary catheters with Escherichia coli 83972 inhibits catheter colonization by Enterococcus faecalis.

PURPOSE The capacity of a preexisting coating of Escherichia coli 83972 to reduce catheter colonization by Enterococcus faecalis 210 was investigated. Enterococcus was chosen for these trials since it is a common urinary pathogen in patients with an indwelling urinary catheter. MATERIALS AND METHODS Each experiment tested 3 growth conditions. Group 1 or E. coli plus Enterococcus catheters were exposed to E. coli 83972 for 24 hours and then to Enterococcus for 30 minutes. Group 2 or E. coli alone catheters were incubated in E. coli for 24 hours and then in sterile broth for 30 minutes. Group 3 or Enterococcus alone catheters did not undergo the initial incubation with E. coli before the 30-minute incubation with Enterococcus: All catheters were then incubated in sterile human urine for 24 hours. Catheters were washed with saline and cut into 5, 1 cm. segments. Each segment was sonicated and the sonication fluid was diluted and plated. The results of each of the 5 segments were averaged and the set of experiments was repeated 7 times. RESULTS A preexisting coating of E. coli 83972 reduced catheter colonization by E. faecalis 210 more than 10-fold. Enterococcus alone catheters had a median of 9.7 x 10(5) enterococci per cm., whereas E. coli plus Enterococcus catheters had a median of 0.38 x 10(5) enterococci per cm. (p = 0.016). CONCLUSIONS Pre-inoculating urinary catheters with E. coli 83972 significantly impedes catheter colonization by Enterococcus: These promising in vitro results prompt the clinical investigation of this particular application of bacterial interference.

Increased Expression of Type-1 Fimbriae by Nonpathogenic Escherichia coli 83972 Results in an Increased Capacity for Catheter Adherence and Bacterial Interference

BACKGROUND In vitro, urinary catheter colonization by avirulent Escherichia coli 83972 impedes subsequent catheter colonization by a variety of uropathogenic organisms. However, E. coli 83972 shows a low efficacy of adherence to silicone urinary catheter material, possibly because the fim operon encoding adhesive type 1 fimbriae is incomplete. We hypothesized that improving the catheter adherence of E. coli 83972 would improve its bacterial interference properties. METHODS We created adhesive mutants by transforming wild-type E. coli 83972 with fim(+) plasmids. Adherence to urinary catheters and ability to prevent uropathogenic E. coli from colonizing urinary catheters were studied by use of a sonication assay. RESULTS The addition of a single-copy fim(+) plasmid increased adherence to urinary catheters 10-fold, and addition of an 18-copy fim(+) plasmid increased adherence 100-fold. The more adherent 18-copy fim(+) plasmid strain was more effective at blocking catheter colonization by pathogenic E. coli than was the wild-type parental strain. Neither Deltafim nor fim(+) E. coli 83972 adhered to shed urinary epithelial cells. CONCLUSIONS Our results indicate that improving urinary catheter adherence augments the bacterial interference capabilities of benign E. coli 83972. Increased expression of type-1 fimbriae may enhance bacterial interference without conferring virulence on E. coli 83972.

Molecular cloning and expression of the O4 polysaccharide gene cluster from Escherichia coli.

The Escherichia coli O4 serotype is common among isolates from urinary tract infections. The genes responsible for the biosynthesis of the O4 polysaccharide in a human uropathogenic E. coli were cloned and expressed in E. coli K-12. The recombinant plasmid pGH60, which conferred the O4 phenotype, encoded eight proteins with apparent molecular weights of 39, 36.5, 35, 32.8, 26, 24, 20.7 and 13 kDa.

Genetic characterization of the O4 polysaccharide gene cluster from Escherichia coli.

The Escherichia coli O4 serotype is among those commonly isolated from urinary tract infections. In order to study the genetics of the O-antigen, the O4 biosynthesis genes from a uropathogenic E. coli have previously been cloned into E. coli K-12. A subclone, GH58, has been identified which reacts with antisera against the O4 serotype. In contrast to the wild-type parental strain, lipopolysaccharide (LPS) from this clone is devoid of rhamnose and does not cross-react with O18 antisera. The recombinant plasmid from GH58, pGH58, was used to transform the rfb deletion strain HU1190. The resultant strain agglutinates in O4 antisera, but produces unpolymerized LPS. Escherichia coli K-12 strains HB101 and RC712 containing pGH58 produce polymerized LPS, indicating that the genetic background of the host can influence the LPS encoded by recombinant molecules. A cosmid, pGH84, has been identified which encompasses the entire pGH58 gene sequences and includes an additional 34 kilobases of DNA. HU1190 containing this cosmid agglutinates in O4 antisera and produces a polymerized LPS. By constructing several deletion subclones of pGH84, we have localized the genes necessary for polymerized LPS to a 5.5 kb ClaI-BamHI fragment. P1 transductants that make polymerized and unpolymerized O4 LPS have also been identified.

Contribution of the traT Gene to Serum Resistance Among Clinical Isolates of Enterobacteriaceae

Antimicrobial resistance plasmids containing the traT gene confer resistance to serum bactericidal activity upon some laboratory strains of Escherichia coli. We have examined the DNA of Enterobacteriaceae from human extraintestinal infections to determine the frequency of traT-like genes. DNA sequences homologous with traT were found among 58% of Escherichieae but among none of the Klebsielleae or Proteeae tested and were found as frequently among serum-sensitive E. coli as among serum-resistant strains. Sequences related to traT were always associated with large plasmids. The potential contribution of traT-containing plasmids to serum resistance of clinical isolates is discussed.

Sequences of the genes encoding the minor tip components of Pap-3 pili of Escherichia coli

We report the sequence of the papE, papF and papG genes from the O75:K5 uropathogenic P pili variant, Pap-3. Comparison of the deduced amino acid sequences with those of other P pili variants reveals regions of complete homology, as well as regions of variation. Analysis of the variations in the hydrophilic domains of these proteins will help elucidate the residues which determine binding specificity.

Molecular cloning and expression of the 01 rfb region from a pyelonephritic Escherichia coli 01:H1:K7

The genes responsible for the biosynthesis of the O1 polysaccharide from a human pyelonephritic Escherichia coli were cloned and expressed in a rfb-deleted E. coli K-12 strain. Deletion analysis of the clone demonstrated that a DNA fragment size larger than 6.7 kb and smaller than 10 kb is responsible for O1-antigen biosynthesis.

[22] Molecular cloning of adhesion genes

Publisher Summary The molecular cloning of genes associated with bacterial adhesion requires methods different from the cloning of single genes. Adhesion of gram-negative bacteria to mammalian tissue is often promoted by complex structures called pili, or fimbriae, on the surface of the bacterium. Expression of fully functional adhesion may require expression of up to 10 genes spaced over a 10- to 15-kb region of DNA. Successful cloning methods must result in chimeric molecules with high-molecular-weight inserts that represent contiguous regions of the donor chromosome. Inserts must be sufficiently random such that the entire chromosome is represented in 1,000–2,000 recombinant molecules. The chapter presents methods for preparing recombinant DNA libraries that have been used successfully for cloning large bacterial operons and functional gene clusters in the 10- to 25-kb range such as adhesion organelles and carbohydrate antigens. Methods useful in screening for adhesin gene clones are also presented.