Vancomycin-resistant Enterococci may obtain nutritional support by scavenging carbohydrate fragments generated during mucin degradation by the anaerobic microbiota of the colon.

Vancomycin-resistant Enterococcus (VRE) is an important nosocomial pathogen that colonizes the intestinal tract. The substrates that provide nutritional support for VRE in the colon are not known. We tested the hypothesis that enzymatic breakdown of complex polysaccharides and glycoconjugates by other members of the indigenous microbiota could provide a source of nutrients for VRE. Nine vancomycin-resistant E. faecium strains were unable to ferment complex plant polysaccharides or hog gastric or bovine submaxillary mucin; however, each of the strains was able to ferment monosaccharides that are components of mucins and plant polysaccharides. Preincubation of hog gastric mucin with partially purified enzyme mixtures obtained from supernatants of Ruminococcus torques or a human stool specimen resulted in release of monosaccharides that supported growth of VRE. These results suggest that enzymatic breakdown of complex polysaccharides such as mucin by members of the indigenous microbiota may provide a source of nutritional support for VRE.

Enterococcal surface protein Esp does not facilitate intestinal colonization or translocation of Enterococcus faecalis in clindamycin-treated mice.

Enterococcal surface protein (Esp) is a cell wall-associated protein of Enterococcus faecalis that has been identified as a potential virulence factor. We used a mouse model to examine whether Esp facilitates intestinal colonization or translocation of E. faecalis to mesenteric lymph nodes. After clindamycin treatment, similar levels of high-density colonization were established after orogastric inoculation of an E. faecalis isolate containing the esp gene within a large pathogenicity island and an isogenic mutant created by allelic replacement of the esp gene with a chloramphenicol resistance cassette (P=0.7); translocation to mesenteric lymph nodes was detected in 3 of 12 (25%) mice in both groups. Isogenic mutants of FA2-2 (a plasmid-free derivative of E. faecalis strain JH2) with or without the esp gene failed to establish colonization of clindamycin-treated mice. These results suggest that Esp does not facilitate intestinal colonization or translocation of E. faecalis.

Coexistence of vancomycin-resistant enterococci and Staphylococcus aureus in the intestinal tracts of hospitalized patients.

The potential for transfer of vancomycin-resistance genes from enterococci to Staphylococcus aureus exists when these organisms share an ecologic niche. We performed an 8-month prospective study to determine the frequency at which S. aureus and vancomycin-resistant enterococci (VRE) coexist in the intestinal tracts of VRE-colonized patients and evaluated whether antianaerobic antibiotic therapy promoted increased density of S. aureus colonization. Of 37 patients colonized with vancomycin-resistant Enterococcus faecium, 23 (62%) had S. aureus recovered from stool specimens and 20 (87%) had methicillin-resistant strains. There was no significant difference in the mean density (+/- standard deviation) of S. aureus during versus > or =1 month after discontinuation of antianaerobic antibiotic therapy (5.1+/-1.5 vs. 4.7+/-1.6 log10 colony-forming units per gram of stool; P=.34). No S. aureus isolates were resistant to vancomycin. S. aureus and VRE often coexist in the intestinal tract, providing a potential reservoir for the emergence of vancomycin-resistant S. aureus isolates.

Increased susceptibility to vancomycin-resistant Enterococcus intestinal colonization persists after completion of anti-anaerobic antibiotic treatment in mice.

BACKGROUND: Antibiotic-associated disruption of the indigenous intestinal microflora may persist beyond the treatment period. Although piperacillin/tazobactam inhibits the establishment of vancomycin-resistant Enterococcus (VRE) stool colonization in mice during treatment, we hypothesized that this agent and other anti-anaerobic antibiotics would increase susceptibility to colonization during the period of recovery of the intestinal microflora. DESIGN: Mice received 10(4) colony-forming units of vancomycin-resistant E. faecium by orogastric inoculation 2, 5, or 10 days after completing 5 days of subcutaneous antibiotic treatment, or both during and 2 days after the completion of treatment. Denaturing gradient gel electrophoresis (DGGE) was performed to assess changes in the intestinal microflora. RESULTS: Anti-anaerobic antibiotics (ie, piperacillin/ tazobactam, cefoxitin, and clindamycin) caused significant disruption of the indigenous microflora (mean DGGE similarity indices < or = 27% in comparison with saline controls) and promoted the establishment of high-density colonization when VRE was inoculated 2 or 5, but not 10, days following treatment (P < .001). Piperacillin/tazobactam exhibited a biphasic effect on the establishment of colonization (ie, inhibition when exposed to VRE during treatment and promotion when exposed to VRE after discontinuation of treatment), resulting in greater overall promotion of colonization than did agents with minimal anti-anaerobic activity (ie, levofloxacin, cefepime, and aztreonam) when VRE was inoculated both during and 2 days after treatment (P < .001). CONCLUSION: Patients receiving anti-anaerobic antibiotics, including piperacillin/tazobactam, may be susceptible to the establishment of high-density VRE colonization during the period of recovery of the anaerobic microflora.

Acquisition of nosocomial pathogens on hands after contact with environmental surfaces near hospitalized patients.

We examined the frequency of acquisition of bacterial pathogens on investigators' hands after contacting environmental surfaces near hospitalized patients. Hand imprint cultures were positive for one or more pathogens after contacting surfaces near 34 (53%) of 64 study patients, with Staphylococcus aureus and vancomycin-resistant Enterococcus being the most common isolates.

Effect of the increasing use of piperacillin/tazobactam on the incidence of vancomycin-resistant enterococci in four academic medical centers.

BACKGROUND: The substitution of piperacillin/tazobactam, ampicillin/sulbactam, or both for third-generation cephalosporins has been associated with reduced vancomycin-resistant enterococci (VRE). However, piperacillin/tazobactam came into widespread use during a period in which the prevalence of VRE increased. We hypothesized that the increasing use of piperacillin/tazobactam and other agents with relatively enhanced anti-enterococcal activity (ie, piperacillin, ampicillin/sulbactam, and ampicillin) has been associated with increased or unchanged rates of VRE in some hospitals. DESIGN: We retrospectively evaluated the correlation between hospital antibiotic use (defined daily doses per 10,000 patient-days of care) and incidence of stool or non-stool VRE isolation. We assessed whether a high or increasing proportion of use of beta-lactam agents with relatively enhanced versus minimal (ie, third-generation cephalosporins and ticarcillin/clavulanate) anti-enterococcal activity would prevent increased VRE. SETTING: Four academic medical centers. RESULTS: With the increasing use of piperacillin/tazobactam, the use of beta-lactam agents with enhanced activity against enterococci surpassed the combined use of third-generation cephalosporins and ticarcillin/clavulanate in each hospital. In one hospital, the incidence of VRE was positively correlated with the use of piperacillin/tazobactam or beta-lactam agents with enhanced anti-enterococcal activity (P < .0001). The incidence of VRE rose steadily in another hospital despite relatively high use of beta-lactam agents with enhanced versus minimal anti-enterococcal activity. A negative correlation between VRE and piperacillin/tazobactam or beta-lactam agents with enhanced anti-enterococcal activity was observed in one hospital, but this correlation was not statistically significant. CONCLUSION: Increasing the hospital use of piperacillin/tazobactam and other beta-lactams with relatively enhanced anti-enterococcal activity may not be an effective control measure for VRE.

Antianaerobic antibiotic therapy promotes overgrowth of antibiotic-resistant, gram-negative bacilli and vancomycin-resistant enterococci in the stool of colonized patients.

BACKGROUND AND OBJECTIVE: Antianaerobic antibiotic therapy promotes persistent high-density growth of vancomycin-resistant enterococci (VRE) in the stool of colonized patients. We tested the hypothesis that antibiotic regimens with potent antianaerobic activity promote overgrowth of coexisting antibiotic-resistant, gram-negative bacilli in the stool of VRE-colonized patients. DESIGN: Eight-month prospective study examining the effect of antibiotic therapy on the stool density of gram-negative bacilli resistant to ceftazidime, ciprofloxacin, or piperacillin/tazobactam. SETTING: A Department of Veterans Affairs medical center including an acute care hospital and nursing home. PATIENTS: All VRE-colonized patients with at least 3 stool samples available for analysis. RESULTS: One-hundred forty stool samples were obtained from 37 study patients. Forty-nine (61%) of 80 stool samples obtained during therapy with an antianaerobic regimen were positive for an antibiotic-resistant, gram-negative bacillus, where-as only 14 (23%) of 60 samples obtained 4 or more weeks after completion of such therapy were positive (P < .001). Twenty-four (65%) of the 37 patients had one or more stool cultures positive for a gram-negative bacillus resistant to ciprofloxacin, ceftazidime, or piperacillin/tazobactam. The density of these organisms was higher during therapy with antianaerobic regimens than in the absence of such therapy for at least 2 weeks (mean +/- standard deviation, 5.6 +/- 1.4 and 3.9 +/- 0.71 log10 organisms/g; P < .001). CONCLUSION: Limiting the use of antianaerobic antibiotics in VRE-colonized patients may reduce the density of colonization with coexisting antibiotic-resistant, gram-negative bacilli.

Inhibition of methicillin-resistant Staphylococcus aureus by an in vitro continuous-flow culture containing human stool microflora.

We used an in vitro continuous-flow culture model of human stool microflora to examine the ability of human stool microflora to inhibit growth of two methicillin-resistant S. aureus (MRSA) strains. Continuous-flow cultures consistently eliminated MRSA inocula of 10(6) cfu/mL within 4 days, and addition of continuous-flow culture resulted in elimination of a pre-established MRSA culture ( approximately 10(8) cfu/mL) within 6-8 days. Anaerobic or "aerobic" (i.e., continuous bubbling of room air to eliminate obligate anaerobes) cultures eliminated MRSA at similar rates. The MRSA strains were unable to replicate under anaerobic conditions in sterile filtrates produced from the continuous-flow culture, but rapid growth occurred when glucose was added. These data demonstrate that indigenous stool microflora efficiently eliminate MRSA colonization and obligate anaerobes are not essential for inhibition. Our findings also suggest that inhibition of MRSA in continuous-flow cultures is due to depletion of nutrients rather than production of inhibitory conditions.

Use of denaturing gradient gel electrophoresis for analysis of the stool microbiota of hospitalized patients.

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal RNA gene amplicons was used to study the stool microbiota of hospitalized patients and to examine the effect of antibiotic therapy. For one patient, 16 anaerobic species identified by random cloning and sequencing of PCR-amplified rRNA genes from stool were represented by bands on the DGGE gel. DGGE analysis and similarity index comparisons demonstrated that the anaerobic microbiota of this individual remained stable in the absence of antibiotic therapy, was minimally affected by ciprofloxacin but markedly reduced by clindamycin therapy, and recovery of some organisms was evident within days after discontinuation of clindamycin. DGGE analysis of additional patients demonstrated similar disruptions of the intestinal microbiota associated with antibiotic therapy. The DGGE banding patterns of nine patients showed considerable variability, but several bands were shared among patients. Thus, our findings are consistent with previous studies that utilized culture techniques, and suggest that DGGE is a useful technique for analysis of the stool microbiota of hospitalized patients.

Effect of carbapenem administration on establishment of intestinal colonization by vancomycin-resistant enterococci and Klebsiella pneumoniae in mice.

In a mouse model, ertapenem inhibited the anaerobic intestinal microflora and promoted overgrowth of enterococci, whereas imipenem-cilastatin had no effect on the indigenous microflora. Ertapenem, but not imipenem-cilastatin, promoted modest overgrowth of vancomycin-resistant enterococci when exposure occurred during treatment. Neither agent promoted colonization with extended-spectrum beta-lactamase-producing Klebsiella pneumoniae.

In vitro killing of nosocomial pathogens by acid and acidified nitrite.

Exposure to pH 1 or 2 buffers or acidic gastric contents resulted in the killing of vancomycin-resistant Enterococcus sp., Klebsiella pneumoniae, Staphylococcus aureus, and Candida glabrata but not Clostridium difficile spores. Nitrite enhanced killing under acidic conditions, but significant killing of C. difficile spores required nitrite concentrations above usual physiological levels.

Adhesion of vancomycin-resistant enterococcus to human intestinal mucus.

The intestinal mucus layer provides a potential niche for colonization by vancomycin-resistant Enterococcus faecium (VREF). We therefore examined the ability of six VREF strains to adhere to human intestinal mucus and determined binding kinetics. Four of six (67%) VREF strains demonstrated significant adhesion to immobilized intestinal mucus compared with a Salmonella typhimurium-negative control strain, but the level of adherence was low compared with Lactobacillus rhamnosus GG. Binding kinetics studies demonstrated that the maximum number of these four VREF strains that could adhere to a unit surface area of immobilized mucus was similar to or higher than the maximum number of L. rhamnosus GG that could adhere; however, L. rhamnosus GG demonstrated 20- to 130-times higher affinity than the VREF strains. These results demonstrate that VREF strains may adhere to human intestinal mucus and suggest that L. rhamnosus GG might be able to displace VREF strains.

Effect of antibiotic treatment on growth of and toxin production by Clostridium difficile in the cecal contents of mice.

In mice, subcutaneous administration of antibiotics that disrupt the anaerobic microflora (i.e., clindamycin, piperacillin-tazobactam, and ceftriaxone) facilitated in vitro growth of and toxin production by Clostridium difficile in cecal contents, whereas antibiotics that cause minimal disruption of the anaerobic microflora (i.e., levofloxacin, cefepime, and aztreonam) did not.

Effects of daptomycin, linezolid, and vancomycin on establishment of intestinal colonization with vancomycin-resistant enterococci and extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae in mice.

In mice, vancomycin and linezolid treatment disrupted the anaerobic intestinal microflora, based on denaturing gradient gel electrophoresis analysis, and promoted colonization by Klebsiella pneumoniae and vancomycin-resistant enterococci. However, the effects varied depending on dose and duration of treatment. Daptomycin treatment did not disrupt the anaerobic microflora or promote either pathogen.

Growth in cecal mucus facilitates colonization of the mouse intestinal tract by methicillin-resistant Staphylococcus aureus.

Intestinal colonization by methicillin-resistant Staphylococcus aureus (MRSA) is common in some groups of hospitalized patients and has been associated with an increased risk of staphylococcal infection. We tested the hypothesis that growth of MRSA in the colonic mucus layer is required for establishment of intestinal colonization. Mice treated with oral streptomycin before oral administration of MRSA developed persistent intestinal colonization, and the cecal mucus layer contained high concentrations of MRSA. MRSA strains grew rapidly when inoculated into cecal mucus in vitro but were unable to replicate under anaerobic conditions in cecal contents of saline- or streptomycin-treated mice. Oral vancomycin treatment reduced the density of 1 MRSA strain in stool but had no effect on a second strain. These results suggest that the cecal mucus layer provides an important niche that facilitates intestinal colonization by MRSA. Oral nonabsorbed antibiotics may be ineffective in eradicating some MRSA strains from the intestinal tract.

Effect of parenteral antibiotic administration on establishment of intestinal colonization by Candida glabrata in adult mice.

We examined the effect of antibiotic treatment on establishment of intestinal colonization by Candida glabrata in adult mice. Subcutaneous ceftriaxone, piperacillin-tazobactam, clindamycin, and metronidazole promoted increased density of stool colonization, whereas cefepime, levofloxacin, and aztreonam did not. These findings suggest that antibiotics that inhibit intestinal anaerobes promote C. glabrata colonization.

Mechanisms by which anaerobic microbiota inhibit the establishment in mice of intestinal colonization by vancomycin-resistant Enterococcus.

We used a mouse model to test the hypothesis that anaerobic microbiota in the colon inhibit the establishment of vancomycin-resistant enterococci (VRE) colonization by depleting nutrients within cecal contents and limiting the association of VRE with the mucus layer. Anaerobic growth of VRE was assessed in cecal contents and cecal mucus of mice that had received treatment with subcutaneous clindamycin or saline. VRE grew to high concentrations in cecal contents of clindamycin-treated mice and in cecal mucus of both groups but not in cecal contents of saline-treated mice, unless the cecal contents were autoclaved or converted into sterile filtrates. After orogastric inoculation of VRE, clindamycin-treated mice acquired high concentrations of VRE within the mucus layer, whereas saline-treated mice did not. These results suggest that colonic microbiota inhibit VRE by producing inhibitory substances or conditions rather than by depleting nutrients. The colonic mucus layer provides a potential niche for growth of VRE.

Effect of parenteral fluoroquinolone administration on persistence of vancomycin-resistant Enterococcus faecium in the mouse gastrointestinal tract.

We examined the effect of subcutaneous fluoroquinolone antibiotic administration on persistence and density of vancomycin-resistant Enterococcus faecium stool colonization in mice. Levofloxacin and ciprofloxacin did not promote colonization in comparison to saline controls, whereas moxifloxacin and gatifloxacin promoted persistent overgrowth in a dose-dependent fashion.

Efficacy of oral ramoplanin for inhibition of intestinal colonization by vancomycin-resistant enterococci in mice.

Ramoplanin is a glycolipodepsipeptide antibiotic with activity against gram-positive bacteria that is in clinical trials for prevention of vancomycin-resistant Enterococcus (VRE) bloodstream infections and treatment of Clostridium difficile diarrhea. Orally administered ramoplanin suppresses VRE intestinal colonization, but recurrences after discontinuation of treatment have frequently been observed. We used a mouse model to examine the efficacy of ramoplanin for inhibition of VRE colonization and evaluated the etiology of recurrences of colonization. Eight days of treatment with ramoplanin (100 microg/ml) in drinking water suppressed VRE to undetectable levels, but 100% of mice developed recurrent colonization; a higher dose of 500 microg/ml in water was associated with recurrent colonization in 50% of mice. Two of eight (25%) mice treated with the 100-microg/ml dose of ramoplanin had low levels of VRE in their cecal tissues on day 8 despite undetectable levels in stool and cecal contents. Mice that received prior ramoplanin treatment did not develop VRE overgrowth when challenged with 10(7) CFU of oral VRE 1, 2, or 4 days later. In communal cages, rapid cross-transmission and overgrowth of VRE was observed among clindamycin-treated mice; ramoplanin treatment effectively suppressed VRE overgrowth in such communal cages. Ramoplanin treatment promoted increased density of indigenous Enterobacteriaceae and overgrowth of an exogenously administered Klebsiella pneumoniae isolate. These results demonstrate the efficacy of ramoplanin for inhibition of VRE colonization and suggest that some recurrences occur due to reexpansion of organisms that persist within the lining of the colon. Ramoplanin treatment may be associated with overgrowth of gram-negative bacilli.

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.