Pertti Koski

Orally Administered Targeted Recombinant Beta-Lactamase Prevents Ampicillin-Induced Selective Pressure on the Gut Microbiota: a Novel Approach to Reducing Antimicrobial Resistance

ABSTRACT 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 β-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 β-lactamase, these did not occur. These results suggest a new strategy for reducing antimicrobial resistance in humans.

Oral Administration of β-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 Recombinant Metallo-β-Lactamase Preserves Colonization Resistance of Piperacillin-Tazobactam-Treated Mice

Antibiotics that are excreted into the intestinal tract may disrupt colonization resistance (i.e., the ability of the indigenous microflora to provide resistance against colonization by potentially pathogenic microorganisms) (1, 5). Because antibiotic activity within the intestinal lumenis is not needed for the treatment of most infections, we hypothesized that the enzymatic inactivation of the portion of antibiotic that is excreted into the intestinal tract would result in the preservation of colonization resistance during parenteral antibiotic therapy. We previously demonstrated that an orally administered recombinant class A β-lactamase preserved the colonization resistance of piperacillin-treated mice against nosocomial pathogens (4). Here, we show that a recombinant class B metallo-β-lactamase that is resistant to tazobactam inactivation preserves colonization resistance during treatment with the more widely used broad-spectrum antibiotic piperacillin-tazobactam. Targeted recombinant β-lactamase 2 (TRBL-2) containing amino acid residues 31 to 257 of the metalloenzyme of a clinical Bacillus cereus isolate (98ME 1552 from Helsinki University Hospital) was overproduced in Bacillus subtilis using a bacillar secretion vector (4). The hydrolysis rate of TRBL-2 for piperacillin with or without tazobactam was 275 μg/minute/μg of enzyme. Individually housed female CF-1 mice weighing 25 to 30 g (Harlan Sprague-Dawley, Indianapolis, Indiana) were used to examine the efficacy of TRBL-2 in the preservation of colonization resistance against vancomycin-resistant enterococci (VRE). At 24 h and again at 12 h prior to the orogastric inoculation of 104 CFU of VRE strain C68 (4), mice received either subcutaneous (0.2 ml) and orogastric (0.5 ml) phosphate-buffered saline (PBS), subcutaneous piperacillin-tazobactam (4 mg) and orogastric PBS, subcutaneous piperacillin-tazobactam (4 mg) and orogastric TRBL-2 (60 mg/kg of body weight), or subcutaneous piperacillin-tazobactam (4 mg) and orogastric TRBL-2 (60 mg/kg) that had been inactivated by boiling for 10 min. Stool VRE density was monitored as previously described (2). Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial rRNA genes was performed on stool samples collected 3 days after the inoculation of VRE in order to monitor changes in the indigenous microflora (3, 4). One-way analysis of variance was performed to compare VRE densities among treatment groups, with P values adjusted for multiple comparisons using the Scheffe correction. DGGE similarity indices were compared using Students t test. Computations were performed using Stata software (version 5.0; Stata, College Station, Texas). Mice treated with piperacillin-tazobactam developed high-density VRE stool colonization; saline controls and mice treated with piperacillin-tazobactam in conjunction with TRBL-2 did not (P < 0.001) (Fig. ​(Fig.1).1). The protective effect of TRBL-2 was eliminated by heat inactivation. DGGE analysis showed that piperacillin-tazobactam caused a significant disruption of the indigenous microflora but that piperacillin-tazobactam in conjunction with TRBL-2 caused a relatively minor alteration of the microflora (mean similarity indices of mice treated with piperacillin-tazobactam and mice treated with piperacillin-tazobactam and TRBL-2 in comparison to those of saline controls were 33% and 86%, respectively; P < 0.001) (Fig. ​(Fig.22). FIG. 1. Efficacy of oral β-lactamase in preventing piperacillin-tazobactam-induced overgrowth of VRE. The densities (log10 CFU/g) of stool VRE are shown after the orogastric inoculation of 104 CFU of VRE on day 0. Prior to inoculation, none of the mice ... FIG. 2. DGGE analysis of stool microflora of individual mice. Lane 1, controls containing rRNA genes amplified from strains of Bacteroides thetaiotaomicron, Bacteroides uniformis, and Escherichia coli (top to bottom); lanes 2 to 4, saline control mice; lanes ... These findings provide further evidence that oral β-lactamase treatment may be an effective means to preserve colonization resistance during therapy with broad-spectrum, parenteral β-lactam antibiotics. Additional studies are needed to determine the efficacy of this strategy as a means to limit the dissemination of nosocomial pathogens in humans.

Orally administered β-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.