Extracellular SalB Contributes to Intrinsic Cephalosporin Resistance and Cell Envelope Integrity in Enterococcus faecalis

ABSTRACT Enterococci are major causes of hospital-acquired infections. Intrinsic resistance to cephalosporins is a universal trait among clinically relevant enterococci. Cephalosporin resistance enables enterococci to proliferate to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. However, the genetic and biochemical mechanisms of intrinsic cephalosporin resistance in enterococci are not well understood. A two-component signal transduction system, CroR/S, is required for cephalosporin resistance in enterococci. Although the CroR/S regulon is not well defined, one gene reported to be CroR dependent in Enterococcus faecalis JH2-2 encodes an extracellular putative peptidoglycan hydrolase, SalB. To test the hypothesis that SalB is responsible for CroR-dependent cephalosporin resistance, we examined ΔsalB mutants in multiple genetic lineages of E. faecalis, revealing that SalB is required not only for intrinsic cephalosporin resistance but also for maintenance of cell envelope integrity in the absence of antibiotic stress. The N-terminal signal sequence is necessary for SalB secretion, and secretion is required for SalB to promote cephalosporin resistance. Functional dissection revealed that the C-terminal SCP domain of SalB is essential for biological activity and identified three residues within the SCP domain that are required for the stability and function of SalB. Additionally, we found that in contrast to what is seen in E. faecalis JH2-2, SalB is not regulated by the CroR/S two-component system in E. faecalis OG1, suggesting diversity in the CroR/S regulon among distinct lineages of E. faecalis. IMPORTANCE Resistance to cephalosporins is universal among clinically relevant enterococci, enabling enterococcal proliferation to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. Disabling cephalosporin resistance could therefore reduce the incidence of enterococcal infections. However, the genetic and biochemical mechanisms of cephalosporin resistance are not well understood. The significance of this work is the identification of a novel extracellular factor (SalB) that promotes cephalosporin resistance in E. faecalis, which could potentially serve as a target for therapeutics that impair enterococcal cephalosporin resistance. Additionally, our work highlights the importance of the C-terminal SCP domain of SalB, including several conserved residues within the SCP domain, for the ability of SalB to promote cephalosporin resistance.

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Oxidative Stress Enhances Cephalosporin Resistance of Enterococcus faecalis through Activation of a Two-Component Signaling System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03984-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 159-169 ◽

Cited By ~ 28

Author(s):

Dušanka Djorić ◽

Christopher J. Kristich

Keyword(s):

Oxidative Stress ◽

Enterococcus Faecalis ◽

Cell Envelope ◽

Signaling System ◽

Intrinsic Resistance ◽

Content Type ◽

Prior Therapy ◽

Two Component ◽

Cephalosporin Resistance ◽

Hospital Acquired

ABSTRACTEnterococcus faecalisis a low-GC Gram-positive bacterium, a normal resident of the gastrointestinal (GI) tract, and an important hospital-acquired pathogen. An important risk factor for hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporins, antibiotics that impair cell wall biosynthesis by inhibiting peptidoglycan cross-linking. Enterococci are intrinsically resistant to cephalosporins; however, environmental factors that modulate cephalosporin resistance have not been described. While searching for the genetic determinants of cephalosporin resistance inE. faecalis, we unexpectedly discovered that oxidative stress, whether from external sources or derived from endogenous metabolism, drives enhanced intrinsic resistance to cephalosporins. A particular source of oxidative stress, H2O2, activates signaling through the CroR-CroS two-component signaling system, a known determinant of cephalosporin resistance inE. faecalis. We find that CroR-CroS is required for adaptation to H2O2stress and that H2O2potentiates the activities of cephalosporins againstE. faecaliswhen the CroR-CroS signaling system is nonfunctional. Rather than directly detecting H2O2, our data suggest that the CroR-CroS system responds to cell envelope damage caused by H2O2exposure in order to promote cell envelope repair and enhanced cephalosporin resistance.

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IreB, a Ser/Thr Kinase Substrate, Influences Antimicrobial Resistance in Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01472-13 ◽

2013 ◽

Vol 57 (12) ◽

pp. 6179-6186 ◽

Cited By ~ 31

Author(s):

Cherisse L. Hall ◽

Michael Tschannen ◽

Elizabeth A. Worthey ◽

Christopher J. Kristich

Keyword(s):

Signaling Pathway ◽

Enterococcus Faecalis ◽

Intrinsic Resistance ◽

Uncharacterized Protein ◽

Gram Positive ◽

Content Type ◽

Kinase Substrate ◽

Cephalosporin Resistance ◽

Endogenous Substrate ◽

Conserved Gene

ABSTRACTEnterococcus faecalisis a Gram-positive bacterium that is a major cause of hospital-acquired infections, in part due to its intrinsic resistance to cephalosporins. The mechanism that confers intrinsic cephalosporin resistance in enterococci remains incompletely defined. Previously, we have shown that the Ser/Thr protein kinase and phosphatase pair IreK and IreP act antagonistically to regulate cephalosporin resistance inE. faecalis. We hypothesize that IreK senses antibiotic-induced cell wall damage and activates a signaling pathway leading to antibiotic resistance. However, the factors downstream of IreK have not yet been identified. To discover such factors, suppressor mutations that restored resistance to a ΔireKkinase mutant were identified. Mutations were found in IreB, a highly conserved gene of unknown function that is widespread among low-GC Gram-positive bacteria. We show that IreB plays a negative regulatory role in cephalosporin resistance and is an endogenous substrate of both IreK and IreP. IreB is phosphorylated on conserved threonine residues, and mutations at these sites impair cephalosporin resistance. Our results are consistent with a model in which the activity of IreB is modulated by IreK-dependent phosphorylation in a signaling pathway required for cephalosporin resistance and begin to shed light on the function of this previously uncharacterized protein.

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Convergence of PASTA Kinase and Two-Component Signaling in Response to Cell Wall Stress inEnterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.00086-18 ◽

2018 ◽

Vol 200 (12) ◽

Cited By ~ 12

Author(s):

Stephanie L. Kellogg ◽

Christopher J. Kristich

Keyword(s):

Gene Expression ◽

Cell Wall ◽

Antimicrobial Resistance ◽

Enterococcus Faecalis ◽

Opportunistic Pathogen ◽

Intrinsic Resistance ◽

Content Type ◽

Signaling Systems ◽

Changing Environments ◽

Two Component

ABSTRACTTwo common signal transduction mechanisms used by bacteria to sense and respond to changing environments are two-component systems (TCSs) and eukaryote-like Ser/Thr kinases and phosphatases (eSTK/Ps).Enterococcus faecalisis a Gram-positive bacterium and a serious opportunistic pathogen that relies on both a TCS and an eSTK/P pathway for intrinsic resistance to cell wall-targeting antibiotics. The TCS consists of a histidine kinase (CroS) and a response regulator (CroR) that become activated upon exposure of cells to cell wall-targeting antibiotics, leading to a modulation of gene expression. The eSTK/P pathway consists of a transmembrane kinase (IreK) and its cognate phosphatase (IreP), which act antagonistically to mediate antibiotic resistance through an unknown mechanism. Because both CroS/R and IreK/P contribute to enterococcal resistance toward cell wall-targeting antibiotics, we hypothesized that these signaling systems are intertwined. To test this hypothesis, we analyzed CroR phosphorylation and CroS/R-dependent gene expression to probe the influence of IreK and IreP on CroS/R signaling. In addition, we analyzed the phosphorylation state of CroS, which revealed the IreK-dependent phosphorylation of a Thr residue important for CroS function. Our results are consistent with a model in which IreK positively influences CroR-dependent gene expression through the phosphorylation of CroS to promote antimicrobial resistance inE. faecalis.IMPORTANCETwo-component signaling systems (TCSs) and eukaryote-like Ser/Thr kinases (eSTKs) are used by bacteria to sense and adapt to changing environments. Understanding how these pathways are regulated to promote bacterial survival is critical for a more complete understanding of bacterial stress responses and physiology. The opportunistic pathogenEnterococcus faecalisrelies on both a TCS (CroS/R) and an eSTK (IreK) for intrinsic resistance to cell wall-targeting antibiotics. We probed the relationship between CroS/R and IreK, revealing the convergence of IreK and the sensor kinase CroS to enhance signaling through CroS/R and increase antimicrobial resistance inE. faecalis. This newly described example of eSTK/TCS convergence adds to our understanding of the signaling networks mediating antimicrobial resistance inE. faecalis.

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The NtrYX Two-Component System Regulates the Bacterial Cell Envelope

mBio ◽

10.1128/mbio.00957-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 3

Author(s):

Kimberly C. Lemmer ◽

François Alberge ◽

Kevin S. Myers ◽

Alice C. Dohnalkova ◽

Ryan E. Schaub ◽

...

Keyword(s):

Bacterial Cell ◽

Cell Envelope ◽

Structure And Function ◽

Two Component System ◽

Component System ◽

Content Type ◽

Bacterial Cell Envelope ◽

Two Component ◽

Assembly Structure ◽

And Function

ABSTRACT Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides, loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria. IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

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MurAA Is Required for Intrinsic Cephalosporin Resistance of Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05984-11 ◽

2012 ◽

Vol 56 (5) ◽

pp. 2443-2451 ◽

Cited By ~ 20

Author(s):

Dušanka Vesić ◽

Christopher J. Kristich

Keyword(s):

Enterococcus Faecalis ◽

Unique Property ◽

Intrinsic Resistance ◽

Positive Bacterium ◽

Content Type ◽

Peptidoglycan Synthesis ◽

Cephalosporin Resistance ◽

Transposon Mutants ◽

Mechanistic Basis ◽

Increased Susceptibility

ABSTRACTEnterococcus faecalisis a low-GC Gram-positive bacterium that is intrinsically resistant to cephalosporins, antibiotics that target cell wall biosynthesis. To probe the mechanistic basis for intrinsic resistance, a library of transposon mutants was screened to identifyE. faecalisstrains that are highly susceptible to ceftriaxone, revealing a transposon mutant with a disruption inmurAA. murAAis predicted to encode a UDP-N-acetylglucosamine 1-carboxyvinyl transferase that catalyzes the first committed step in peptidoglycan synthesis: phosphoenolpyruvate (PEP)-dependent conversion of UDP-N-acetylglucosamine to UDP-N-acetylglucosamine-enolpyruvate. In-frame deletion ofmurAA, but not its homolog in theE. faecalisgenome (murAB), led to increased susceptibility ofE. faecalisto cephalosporins. Furthermore, expression ofmurAAenhanced cephalosporin resistance in anE. faecalismutant lacking IreK (formerly PrkC), a key kinase required for cephalosporin resistance. Further genetic analysis revealed that MurAA catalytic activity is necessary but not sufficient for this role. Collectively, our data indicate that MurAA and MurAB have distinct roles inE. faecalisphysiology and suggest that MurAA possesses a unique property or activity that enables it to enhance intrinsic resistance ofE. faecalisto cephalosporins.

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Reciprocal Regulation of Resistance-Nodulation-Division Efflux Systems and the Cpx Two-Component System in Vibrio cholerae

Infection and Immunity ◽

10.1128/iai.00025-14 ◽

2014 ◽

Vol 82 (7) ◽

pp. 2980-2991 ◽

Cited By ~ 24

Author(s):

Dawn L. Taylor ◽

X. Renee Bina ◽

Leyla Slamti ◽

Matthew K. Waldor ◽

James E. Bina

Keyword(s):

Antimicrobial Resistance ◽

Vibrio Cholerae ◽

Cell Envelope ◽

Two Component System ◽

Reciprocal Regulation ◽

Component System ◽

Content Type ◽

Expression Of Genes ◽

Two Component ◽

Resistance Nodulation Division

ABSTRACTThe Cpx two-component regulatory system has been shown inEscherichia colito alleviate stress caused by misfolded cell envelope proteins. TheVibrio choleraeCpx system was previously found to respond to cues distinct from those in theE. colisystem, suggesting that this system fulfills a different physiological role in the cholera pathogen. Here, we used microarrays to identify genes that were regulated by theV. choleraeCpx system. Our observations suggest that the activation of theV. choleraeCpx system does not induce expression of genes involved in the mitigation of stress generated by misfolded cell envelope proteins but promotes expression of genes involved in antimicrobial resistance. In particular, activation of the Cpx system induced expression of the genes encoding the VexAB and VexGH resistance-nodulation-division (RND) efflux systems and their cognate outer membrane pore protein TolC. The promoters for these loci contained putative CpxR consensus binding sites, and ectopiccpxRexpression activated transcription from the promoters for the RND efflux systems. CpxR was not required for intrinsic antimicrobial resistance, but CpxR activation enhanced resistance to antimicrobial substrates of VexAB and VexGH. Mutations that inactivated VexAB or VexGH efflux activity resulted in the activation of the Cpx response, suggesting thatvexABandvexGHand thecpxP-cpxRAsystem are reciprocally regulated. We speculate that the reciprocal regulation of theV. choleraeRND efflux systems and the Cpx two-component system is mediated by the intracellular accumulation of an endogenously produced metabolic by-product that is normally extruded from the cell by the RND efflux systems.

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Identification and Characterization of a Bacitracin Resistance Network in Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02111-13 ◽

2013 ◽

Vol 58 (3) ◽

pp. 1425-1433 ◽

Cited By ~ 28

Author(s):

Susanne Gebhard ◽

Chong Fang ◽

Aishath Shaaly ◽

David J. Leslie ◽

Marion R. Weimar ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Enterococcus Faecalis ◽

Abc Transporter ◽

Regulatory System ◽

Proteome Analysis ◽

Peptide Antibiotic ◽

Content Type ◽

Resistance Network ◽

Two Component ◽

Host Origin

ABSTRACTResistance ofEnterococcus faecalisagainst antimicrobial peptides, both of host origin and produced by other bacteria of the gut microflora, is likely to be an important factor in the bacterium's success as an intestinal commensal. The aim of this study was to identify proteins with a role in resistance against the model antimicrobial peptide bacitracin. Proteome analysis of bacitracin-treated and untreated cells showed that bacitracin stress induced the expression of cell wall-biosynthetic proteins and caused metabolic rearrangements. Among the proteins with increased production, an ATP-binding cassette (ABC) transporter with similarity to known peptide antibiotic resistance systems was identified and shown to mediate resistance against bacitracin. Expression of the transporter was dependent on a two-component regulatory system and a second ABC transporter, which were identified by genome analysis. Both resistance and the regulatory pathway could be functionally transferred toBacillus subtilis, proving the function and sufficiency of these components for bacitracin resistance. Our data therefore show that the two ABC transporters and the two-component system form a resistance network against antimicrobial peptides inE. faecalis, where one transporter acts as the sensor that activates the TCS to induce production of the second transporter, which mediates the actual resistance.

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Induction of Daptomycin Tolerance in Enterococcus faecalis by Fatty Acid Combinations

Applied and Environmental Microbiology ◽

10.1128/aem.01178-20 ◽

2020 ◽

Vol 86 (20) ◽

Author(s):

William Brewer ◽

Johnathan Harrison ◽

Holly E. Saito ◽

Elizabeth M. Fozo

Keyword(s):

Fatty Acids ◽

Antibiotic Resistance ◽

Fatty Acid ◽

Stearic Acid ◽

Enterococcus Faecalis ◽

Cell Envelope ◽

Negative Effects ◽

Gram Positive ◽

Bacterial Populations ◽

Content Type

ABSTRACT Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid. IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

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Chemical Genetic Interaction Profiling Reveals Determinants of Intrinsic Antibiotic Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01334-17 ◽

2017 ◽

Vol 61 (12) ◽

Cited By ~ 24

Author(s):

Weizhen Xu ◽

Michael A. DeJesus ◽

Nadine Rücker ◽

Curtis A. Engelhart ◽

Meredith G. Wright ◽

...

Keyword(s):

Antibiotic Resistance ◽

Mycobacterium Tuberculosis ◽

Antibiotic Susceptibility ◽

Genetic Interaction ◽

Cell Envelope ◽

Mycolic Acid ◽

Protein Characterization ◽

Intrinsic Resistance ◽

Content Type ◽

Chemical Genetic

ABSTRACT Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.

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Next-Generation Probiotics Targeting Clostridium difficile through Precursor-Directed Antimicrobial Biosynthesis

Infection and Immunity ◽

10.1128/iai.00303-17 ◽

2017 ◽

Vol 85 (10) ◽

Cited By ~ 22

Author(s):

Jennifer K. Spinler ◽

Jennifer Auchtung ◽

Aaron Brown ◽

Prapaporn Boonma ◽

Numan Oezguen ◽

...

Keyword(s):

Clostridium Difficile ◽

Rational Design ◽

Lactobacillus Reuteri ◽

Human Microbiome ◽

Disease Recurrence ◽

Next Generation ◽

Intrinsic Resistance ◽

Content Type ◽

Public Health Burden ◽

And Function

ABSTRACT Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.

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