Orally administered beta-lactamase enzymes represent a novel strategy to prevent colonization by Clostridium difficile.

OBJECTIVES: Antibiotics that are excreted into the intestinal tract and that disrupt the indigenous microbiota may promote infection by Clostridium difficile. We previously demonstrated that oral administration of a proteolysis-resistant, recombinant class A beta-lactamase inactivates ampicillin or piperacillin excreted into the small intestine during parenteral treatment. We hypothesized that oral administration of this beta-lactamase in conjunction with parenteral ampicillin or piperacillin would preserve the colonic microbiota, thus preventing the overgrowth of and toxin production by C. difficile in mice. METHODS: Subcutaneous ampicillin, subcutaneous piperacillin or either of these plus oral beta-lactamase or either of these plus tazobactam-inactivated oral beta-lactamase were administered to mice 24 and 12 h prior to harvest of caecal contents. Contents were inoculated with one of four strains of C. difficile, and growth and toxin production were assessed after 24 h of incubation under anaerobic conditions. To assess changes in stool microbiota, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal RNA genes was performed. RESULTS: Mice treated with ampicillin, piperacillin or either of these plus tazobactam-inactivated oral beta-lactamase developed high-density colonization with C. difficile, whereas those treated with ampicillin or piperacillin plus the beta-lactamase did not. DGGE demonstrated that antibiotic treatment resulted in significant alteration of the indigenous stool microbiota, whereas antibiotic plus beta-lactamase treatment did not. CONCLUSIONS: Administration of oral recombinant beta-lactamase preserved the colonic microbiota of mice during parenteral beta-lactam antibiotic treatment and prevented the overgrowth of and toxin production by C. difficile in caecal contents. Oral beta-lactamase therapy may represent a novel approach towards preventing C. difficile infections in healthcare settings.

Inhibition of ampicillin-induced emergence of resistance in intestinal coliforms by targeted recombinant beta-lactamase.

The aim of the present study was to determine whether oral targeted recombinant beta-lactamase (TRBL) administration could overcome the development of ampicillin-induced resistance in the gut microbiota. Eighteen laboratory beagles with permanent jejunal fistula were randomised to receive ampicillin + placebo, ampicillin + TRBL or placebo. A total of 982 coliform isolates, collected from jejunal and faecal samples before, during and after the treatment were tested against nine antimicrobials. The proportion of ampicillin resistance (multi-resistance) among coliform isolates increased from 20 to 36% in the ampicillin + placebo group but far less, 20-36%, in the ampicillin + TRBL group. These results indicate that TRBL may prevent the emergence of beta-lactam-associated resistance in coliforms in the gut.

Enzymic degradation of a beta-lactam antibiotic, ampicillin, in the gut: a novel treatment modality.

Antibiotics can cause severe alterations in the gut microflora and promote diarrhoea and overgrowth of pathogenic bacteria. The present study investigated the potency of targeted recombinant beta-lactamase (TRBL) to degrade a beta-lactam antibiotic in the jejunum of fistula-operated beagles. We used different peroral doses of purified beta-lactamase (PenP) of Bacillus licheniformis in enteric-coated pellets together with intravenous ampicillin. Serum and jejunal samples were collected for ampicillin and beta-lactamase analysis. A dose-response effect of TRBL on ampicillin concentrations in the jejunal samples could be observed. The highest doses applied decreased the jejunal ampicillin concentrations to undetectable levels. In the serum samples, the ampicillin concentrations were not affected by the beta-lactamase dose used. Our results indicate that it may be possible to evolve a targeted treatment to degrade beta-lactam antibiotics intestinally and, thus, decrease antibiotic-induced adverse effects on the gut microflora.

Polyethyleneimine is an effective permeabilizer of gram-negative bacteria.

The effect of the polycation polyethyleneimine (PEI) on the permeability properties of the Gram-negative bacterial outer membrane was investigated using Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium as target organisms. At concentrations of less than 20 micrograms ml-1, PEI increased the bacterial uptake of 1-N-phenylnaphthylamine, which is a hydrophobic probe whose quantum yield is greatly increased in a lipid environment, indicating increased hydrophobic permeation of the outer membrane by PEI. The effect of PEI was comparable to that brought about by the well-known permeabilizer EDTA. Permeabilization by PEI was retarded but not completely inhibited by millimolar concentrations of MgCl2. PEI also increased the susceptibility of the test species to the hydrophobic antibiotics clindamycin, erythromycin, fucidin, novobiocin and rifampicin, without being directly bactericidal. PEI sensitized the bacteria to the lytic action of the detergent SDS in assays where the bacteria were pretreated with PEI. In assays where PEI and SDS were simultaneously present, no sensitization was observed, indicating that PEI and SDS were inactivating each other. In addition, a sensitizing effect to the nonionic detergent Triton X-100 was observed for P. aeruginosa. In conclusion, PEI was shown to be a potent permeabilizer of the outer membrane of Gram-negative bacteria.

Oral administration of beta-lactamase preserves colonization resistance of piperacillin-treated mice.

We hypothesized that orally administered, recombinant class A beta-lactamase would inactivate the portion of parenteral piperacillin excreted into the intestinal tract, preserving colonization resistance of mice against nosocomial pathogens. Subcutaneous piperacillin or piperacillin plus oral beta-lactamase were administered 24 and 12 h before orogastric inoculation of piperacillin-resistant pathogens. Oral administration of beta-lactamase reduced piperacillin-associated alteration of the indigenous microflora and prevented overgrowth of pathogens.

Orally administered targeted recombinant Beta-lactamase prevents ampicillin-induced selective pressure on the gut microbiota: a novel approach to reducing antimicrobial resistance.

Antibiotics that are excreted into the intestinal tract promote antibiotic resistance by exerting selective pressure on the gut microbiota. Using a beagle dog model, we show that an orally administered targeted recombinant beta-lactamase enzyme eliminates the portion of parenteral ampicillin that is excreted into the small intestine, preventing ampicillin-induced changes to the fecal microbiota without affecting ampicillin levels in serum. In dogs receiving ampicillin, significant disruption of the fecal microbiota and the emergence of ampicillin-resistant Escherichia coli and TEM genes were observed, whereas in dogs treated with ampicillin in combination with an oral beta-lactamase, these did not occur. These results suggest a new strategy for reducing antimicrobial resistance in humans.