IMPACT OF TIGECYCLINE VERSUS OTHER ANTIBIOTICS ON THE FECAL METABOLOME AND ON COLONIZATION RESISTANCE TO CLOSTRIDIUM DIFFICILE IN MICE.

BACKGROUND: The glycylcycline antibiotic tigecycline may have a relatively low propensity to promote Clostridium difficile infection in part because it causes less disruption of the indigenous intestinal microbiota than other broad-spectrum antibiotics. We used a mouse model to compare the effects of tigecycline versus other commonly used antibiotics on colonization resistance to C. difficile and on the metabolic functions of the intestinal microbiota. METHODS: To assess in vivo colonization resistance to C. difficile, mice were challenged with oral C. difficile spores 1, 7, or 12 days after completion of 3 days of treatment with subcutaneous saline, tigecycline, ceftriaxone, piperacillin-tazobactam, or linezolid. Levels of bacterial metabolites in fecal specimens of mice treated with the same antibiotics were analyzed using non-targeted metabolic profiling by gas chromatograph (GC)/mass spectrometry (MS) and ultra-high performance liquid chromatography-tandem MS (UPLC-MS/MS). RESULTS: All of the antibiotics disrupted colonization resistance to C. difficile when challenge occurred 2 days after treatment. Only piperacillin/tazobactam mice had disturbed colonization resistance at 7 days after treatment. All of the antibiotics altered fecal metabolites in comparison to controls, but tigecycline caused significantly less alteration than the other antibiotics, including less suppression of multiple amino acids, bile acids, and lipid metabolites. CONCLUSIONS: Tigecycline, linezolid, and ceftriaxone caused transient disruption of colonization resistance to C. difficile, whereas piperacillin/tazobactam caused disruption that persisted for 7 days post-treatment. Tigecycline caused less profound alteration of fecal bacterial metabolites than the other antibiotics, suggesting that the relatively short period of disruption of colonization resistance might be related in part to reduced alteration of the metabolic functions of the microbiota.

Effect of Fidaxomicin versus Vancomycin on Susceptibility to Intestinal Colonization with Vancomycin-Resistant Enterococci and Klebsiella pneumoniae in Mice.

The use of oral vancomycin or metronidazole for treatment of Clostridium difficile infection (CDI) may promote colonization by health care-associated pathogens due to disruption of the intestinal microbiota. Because the macrocyclic antibiotic fidaxomicin causes less alteration of the intestinal microbiota than vancomycin, we hypothesized that it would not lead to a loss of colonization resistance to vancomycin-resistant enterococci (VRE) and extended-spectrum-ß-lactamase-producing Klebsiella pneumoniae (ESBL-Kp). Mice (8 per group) received orogastric saline, vancomycin, or fidaxomicin daily for 5 days at doses resulting in stool concentrations in mice similar to those measured in humans. The mice were challenged with 10(5) CFU of orogastric VRE or ESBL-Kp on day 2 of treatment and concentrations of the pathogens in stool were monitored. The impact of drug exposure on the microbiome was measured by cultures, real-time PCR for selected anaerobic bacteria, and deep sequencing. In comparison to saline controls, oral vancomycin promoted establishment of high-density colonization by VRE and ESBL-Kp in stool (8 to 10 log10 CFU/g; P < 0.001), whereas fidaxomicin did not (<4 log10 CFU; P > 0.5). Vancomycin treatment resulted in significant reductions in enterococci, Bacteroides spp., and Clostridium leptum, whereas the population of aerobic and facultative Gram-negative bacilli increased; deep-sequencing analysis demonstrated suppression of Firmicutes and expansion of Proteobacteria during vancomycin treatment. Fidaxomicin did not cause significant alteration of the microbiota. In summary, in contrast to vancomycin, fidaxomicin treatment caused minimal disruption of the intestinal microbiota and did not render the microbiota susceptible to VRE and ESBL-Kp colonization.

Effect of Surotomycin, a Novel Cyclic Lipopeptide Antibiotic, on Intestinal Colonization with Vancomycin-Resistant Enterococci and Klebsiella pneumoniae in Mice.

Surotomycin (formerly called CB-183,315) is a novel, orally administered cyclic lipopeptide antibacterial in development for the treatment of Clostridium difficile infection (CDI) that has potent activity against vancomycin-resistant enterococci (VRE) but limited activity against Gram-negative bacilli, including Bacteroides spp. We used a mouse model to investigate the impact of surotomycin exposure on the microbiome, and to test the consequences of the disruption on colonization by vancomycin-resistant enterococci (VRE) and extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (ESBL-KP), in comparison with the effects of oral vancomycin and metronidazole. Mice (8 per group) received saline, vancomycin, metronidazole, or surotomycin through an orogastric tube daily for 5 days and were challenged with 10(5) CFU of VRE or ESBL-KP administered through an orogastric tube on day 2 of treatment. The concentrations of the pathogens in stool were determined during and after treatment by plating on selective media. A second experiment was conducted to determine if the antibiotics would inhibit established VRE colonization. In comparison to controls, oral vancomycin promoted VRE and ESBL-KP overgrowth in stool (8 log10 to 10 log10 CFU/g; P < 0.001), whereas metronidazole did not (<4 log10 CFU/g; P > 0.5). Surotomycin promoted ESBL-KP overgrowth (>8 log10 CFU/g; P, <0.001 for comparison with saline controls) but not VRE overgrowth. Surotomycin suppressed preexisting VRE colonization, whereas metronidazole and vancomycin did not. These results suggest that treatment of CDI with surotomycin could reduce levels of VRE acquisition and overgrowth from those with agents such as vancomycin and metronidazole. However, surotomycin and vancomycin may promote colonization by antibiotic-resistant Gram-negative bacilli.

Community-Acquired Pyelonephritis in Pregnancy Caused by KPC-Producing Klebsiella pneumoniae.

Carbapenem-resistant Enterobacteriaceae (CRE) usually infect patients with significant comorbidities and health care exposures. We present a case of a pregnant woman who developed community-acquired pyelonephritis caused by KPC-producing Klebsiella pneumoniae. Despite antibiotic treatment, she experienced spontaneous prolonged rupture of membranes, with eventual delivery of a healthy infant. This report demonstrates the challenge that CRE may pose to the effective treatment of common infections in obstetric patients, with potentially harmful consequences to maternal and neonatal health.

Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients.

No new acquisition of Clostridium difficile occurred among 12 hospitalised patients receiving tigecycline, and pre-existing colonisation was reduced to undetectable levels in 2 patients. Moreover, 91% of stool suspensions obtained during tigecycline therapy exhibited inhibitory activity against C. difficile. These results suggest that tigecycline achieves sufficient concentrations to inhibit intestinal colonisation by C. difficile.

Unexpected challenges in treating multidrug-resistant Gram-negative bacteria: resistance to ceftazidime-avibactam in archived isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of ß-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel ß-lactam/ß-lactamase inhibitor combination. The isolates were systematically analyzed through a variety of genetic, biochemical, genomic, and microbiological methods to understand how resistance manifests to a unique drug combination that is not yet clinically released. We discovered that avibactam was able to inactivate different AmpC ß-lactamase enzymes and that blaPDC regulatory elements and penicillin-binding protein differences did not contribute in a major way to resistance. By using carefully selected combinations of antimicrobial agents, we deduced that the greatest barrier to ceftazidime-avibactam is membrane permeability and drug efflux. To overcome the constellation of resistance determinants, we show that a combination of antimicrobial agents (ceftazidime/avibactam/fosfomycin) targeting multiple cell wall synthetic pathways can restore susceptibility. In P. aeruginosa, efflux, as a general mechanism of resistance, may pose the greatest challenge to future antibiotic development. Our unexpected findings create concern that even the development of antimicrobial agents targeted for the treatment of multidrug-resistant bacteria may encounter clinically important resistance. Antibiotic therapy in the future must consider these factors.

Evaluation of a sporicidal peracetic acid/hydrogen peroxide-based daily disinfectant cleaner.

OxyCide Daily Disinfectant Cleaner, a novel peracetic acid/hydrogen peroxide-based sporicidal disinfectant, was as effective as sodium hypochlorite for in vitro killing of Clostridium difficile spores, methicillin-resistant Staphylococcus aureus, and vancomcyin-resistant enterococci. OxyCide was minimally affected by organic load and was effective in reducing pathogen contamination in isolation rooms.

Metabolomics analysis identifies intestinal microbiota-derived biomarkers of colonization resistance in clindamycin-treated mice.

BACKGROUND: The intestinal microbiota protect the host against enteric pathogens through a defense mechanism termed colonization resistance. Antibiotics excreted into the intestinal tract may disrupt colonization resistance and alter normal metabolic functions of the microbiota. We used a mouse model to test the hypothesis that alterations in levels of bacterial metabolites in fecal specimens could provide useful biomarkers indicating disrupted or intact colonization resistance after antibiotic treatment. METHODS: To assess in vivo colonization resistance, mice were challenged with oral vancomycin-resistant Enterococcus or Clostridium difficile spores at varying time points after treatment with the lincosamide antibiotic clindamycin. For concurrent groups of antibiotic-treated mice, stool samples were analyzed using quantitative real-time polymerase chain reaction to assess changes in the microbiota and using non-targeted metabolic profiling. To assess whether the findings were applicable to another antibiotic class that suppresses intestinal anaerobes, similar experiments were conducted with piperacillin/tazobactam. RESULTS: Colonization resistance began to recover within 5 days and was intact by 12 days after clindamycin treatment, coinciding with the recovery bacteria from the families Lachnospiraceae and Ruminococcaceae, both part of the phylum Firmicutes. Clindamycin treatment caused marked changes in metabolites present in fecal specimens. Of 484 compounds analyzed, 146 (30%) exhibited a significant increase or decrease in concentration during clindamycin treatment followed by recovery to baseline that coincided with restoration of in vivo colonization resistance. Identified as potential biomarkers of colonization resistance, these compounds included intermediates in carbohydrate or protein metabolism that increased (pentitols, gamma-glutamyl amino acids and inositol metabolites) or decreased (pentoses, dipeptides) with clindamycin treatment. Piperacillin/tazobactam treatment caused similar alterations in the intestinal microbiota and fecal metabolites. CONCLUSIONS: Recovery of colonization resistance after antibiotic treatment coincided with restoration of several fecal bacterial metabolites. These metabolites could provide useful biomarkers indicating intact or disrupted colonization resistance during and after antibiotic treatment.

Sensitive and selective culture medium for detection of environmental Clostridium difficile isolates without requirement for anaerobic culture conditions.

Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile, but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l-cystine, termed C. difficile brucella broth with thioglycolic acid and l-cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non-C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions.

The role of a second-shell residue in modifying substrate and inhibitor interactions in the SHV beta-lactamase: a study of ambler position Asn276.

Inhibitor-resistant class A beta-lactamases of the TEM and SHV families that arise by single amino acid substitutions are a significant threat to the efficacy of beta-lactam/beta-lactamase inhibitor combinations. To better understand the basis of the inhibitor-resistant phenotype in SHV, we performed mutagenesis to examine the role of a second-shell residue, Asn276. Of the 19 variants expressed in Escherichia coli, only the Asn276Asp enzyme demonstrated reduced susceptibility to ampicillin/clavulanate (MIC increased from 50/2 --> 50/8 microg/mL) while maintaining high-level resistance to ampicillin (MIC = 8192 microg/mL). Steady-state kinetic analyses of Asn276Asp revealed slightly diminished k(cat)/K(m) for all substrates tested. In contrast, we observed a 5-fold increase in K(i) for clavulanate (7.4 +/- 0.9 microM for Asn276Asp vs 1.4 +/- 0.2 microM for SHV-1) and a 40% reduction in k(inact)/K(I) (0.013 +/- 0.002 microM(-1 )s(-1) for Asn276Asp vs 0.021 +/- 0.004 microM(-1) s(-1) for SHV-1). Timed electrospray ionization mass spectrometry of clavulanate-inhibited SHV-1 and SHV Asn276Asp showed nearly identical mass adducts, arguing for a similar pathway of inactivation. Molecular modeling shows that novel electrostatic interactions are formed between Arg244Neta2 and both 276AspOdelta1 and Odelta2; these new forces restrict the spatial position of Arg244, a residue important in the recognition of the C(3)/C(4) carboxylate of beta-lactam substrates and inhibitors. Testing the functional consequences of this interaction, we noted considerable free energy costs (+DeltaDeltaG) for substrates and inhibitors. A rigid carbapenem (meropenem) was most affected by the Asn276Asp substitution (46-fold increase in K(i) vs SHV-1). We conclude that residue 276 is an important second-shell residue in class A beta-lactamase-mediated resistance to substrates and inhibitors, and only Asn is able to precisely modulate the conformational flexibility of Arg244 required for successful evolution in nature.