Dimeric Stilbene Antibiotics Target the Bacterial Cell Wall in Drug-Resistant Gram-Positive Pathogens.

The prevalence of antibiotic resistance has been increasing globally, and new antimicrobial agents are needed to address this growing problem. We previously reported that a stilbene dimer from Photorhabdus gammaproteobacteria exhibits strong activity relative to its monomer against the multidrug-resistant Gram-positive pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis. Here, we show that related dietary plant stilbene-derived dimers also have activity against these pathogens, and MRSA is unable to develop substantial resistance even after daily nonlethal exposure to the lead compound for a duration of three months. Through a systematic deduction process, we established the mode of action of the lead dimer, which targets the bacterial cell wall. Genome sequencing of modest resistance mutants, mass spectrometry analysis of cell wall precursors, and exogenous lipid II chemical complementation studies support the target as being lipid II itself or lipid II trafficking processes. Given the broad distribution of stilbenes in plants, including dietary plants, we anticipate that our mode of action studies here could be more broadly applicable to multipartite host-bacterium-plant interactions.

Vancomycin-Dependent Response in Live Drug-Resistant Bacteria by Metabolic Labeling.

The surge in drug-resistant bacterial infections threatens to overburden healthcare systems worldwide. Bacterial cell walls are essential to bacteria, thus making them unique targets for the development of antibiotics. We describe a cellular reporter to directly monitor the phenotypic switch in drug-resistant bacteria with temporal resolution. Vancomycin-resistant enterococci (VRE) escape the bactericidal action of vancomycin by chemically modifying their cell-wall precursors. A synthetic cell-wall analogue was developed to hijack the biosynthetic rewiring of drug-resistant cells in response to antibiotics. Our study provides the first in vivo VanX reporter agent that responds to cell-wall alteration in drug-resistant bacteria. Cellular reporters that reveal mechanisms related to antibiotic resistance can potentially have a significant impact on the fundamental understanding of cellular adaption to antibiotics.

Incorporation of a Valine-Leucine-Lysine-Containing Substrate in the Bacterial Cell Wall.

The emergence of antibiotic-resistant bacteria is a major public health threat, and therefore novel antimicrobial targets and strategies are urgently needed. In this regard, cell-wall-associated proteases are envisaged as interesting antimicrobial targets due to their role in cell wall remodeling. Here, we describe the discovery and characteristics of a protease substrate that is processed by a bacterial cell-wall-associated protease. Stationary-phase grown Gram-positive bacteria were incubated with fluorogenic protease substrates, and their cleavage and covalent incorporation into the cell wall was analyzed. Of all of the substrates used, only one substrate, containing a valine-leucine-lysine (VLK) motif, was covalently incorporated into the bacterial cell wall. Linkage of the VLK-peptide substrate appeared unrelated to sortase A and B activity, as both wild-type and sortase A and B knock out Staphylococcus aureus strains incorporated this substrate into their cell wall with comparable efficiency. Additionally, the VLK-peptide substrate showed significantly higher incorporation in the cell wall of VanA-positive Enterococcus faecium strains than in VanB- and vancomycin-susceptible isolates. In conclusion, the VLK-peptide substrate identified in this study shows promise as a vehicle for targeting antimicrobial compounds and diagnostic contrast agents to the bacterial cell wall.

Mass transfer characterization of gamma-aminobutyric acid production by Enterococcus faecium CFR 3003: encapsulation improves its survival under simulated gastro-intestinal conditions.

Gamma-aminobutyric acid (GABA) production by free and Ca-alginate encapsulated cells of Enterococcus faecium CFR 3003 was investigated. Mass transfer rates characterizing the GABA production process using encapsulated cells were investigated. Experiments were performed to investigate external film and internal pore diffusion mass transfer rates. The Damkohler and Thiele analysis provides a good description of external film and internal pore diffusion resistances, respectively. The experiments revealed that the external film effects could be neglected but the process is affected to the greater extent by internal mass transfer effects and was found to be the principal rate-controlling step. Protective effect of encapsulation on cell survivability was tested under digestive environment, when challenged to salivary α-amylase, simulated gastric fluid and intestinal fluid. Viability of encapsulated cells was significantly higher under simulated gastro-intestinal conditions and could produce higher GABA than those observed with free cells. The results indicate that the Ca-alginate encapsulated probiotics could effectively be delivered to the colonic site for effective inhibitory action.

Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content.

BACKGROUND: The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains. METHODOLOGY: Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs. PRINCIPAL FINDINGS: Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM. CONCLUSION: Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.

Inhibitors of aminoglycoside resistance activated in cells.

The most common mechanism of resistance to aminoglycoside antibiotics entails bacterial expression of drug-metabolizing enzymes, such as the clinically widespread aminoglycoside N-6'-acetyltransferase (AAC(6')). Aminoglycoside-CoA bisubstrates are highly potent AAC(6') inhibitors; however, their inability to penetrate cells precludes in vivo studies. Some truncated bisubstrates are known to cross cell membranes, yet their activities against AAC(6') are in the micromolar range at best. We report here the synthesis and biological activity of aminoglycoside-pantetheine derivatives that, although devoid of AAC(6') inhibitory activity, can potentiate the antibacterial activity of kanamycin A against an aminoglycoside-resistant strain of Enterococcus faecium. Biological studies demonstrate that these molecules are potentially extended to their corresponding full-length bisubstrates by enzymes of the coenzyme A biosynthetic pathway. This work provides a proof-of-concept for the utility of prodrug compounds activated by enzymes of the coenzyme A biosynthetic pathway, to resensitize resistant strains of bacteria to aminoglycoside antibiotics.

Method for rapid and sensitive detection of Enterococcus sp. and Enterococcus faecalis/faecium cells in potable water samples.

We have developed a rapid and robust technological solution including a membrane filtration and dissolution method followed by a molecular enrichment and a real-time PCR assay, for detecting the presence of Enterococcus sp. or Enterococcus faecalis/faecium per 100 mL of water in less than 5 h and we compared it to Method 1600 on mEI agar in terms of specificity, sensitivity, and limit of detection. The mEI and the Enterococcus sp.-specific assay detected respectively 73 (64.0%) and 114 (100%) of the 114 enterococcal strains tested. None of the 150 non-enterococcal strains tested was detected by both methods with the exception of Tetragenococcus solitarius for the Enterococcus sp. assay. The multiplexed E. faecalis/faecium assay efficiently amplified DNA from 47 of 47 (100%) E. faecalis and 27 of 27 (100%) E. faecium strains tested respectively, whereas none of the 191 non-E. faecalis/faecium strains tested was detected. By simultaneously detecting the predominant fecal enterococcal species, the E. faecalis/faecium-specific assay allows a better distinction between enterococcal strains of fecal origin and those provided by the environment than Method 1600. Our procedure allows the detection of 4.5 enterococcal colony forming units (CFU) per 100 mL in less than 5 h, whereas the mEI method detected 2.3 CFU/100 mL in 24 h (95% confidence). Thus, our innovative and highly effective method provides a rapid and easy approach to concentrate very low numbers of enterococcal cells present in a 100 mL water sample and allows a better distinction between fecal and environmental enterococcal cells than Method 1600.

Method revealing bacterial cell-wall architecture by time-dependent isotope labeling and quantitative liquid chromatography/mass spectrometry.

The molecular details of the biosynthesis and resulting architecture of the bacterial cell wall remain unclear but are essential to understanding the activity of glycopeptide antibiotics, the recognition of pathogens by hosts, and the processes of bacterial growth and division. Here we report a new strategy to elucidate bacterial cell-wall architecture based on time-dependent isotope labeling of bacterial cells quantified by liquid chromatography/accurate mass measurement mass spectrometry. The results allow us to track the fate of cell-wall precursors (which contain the vancomycin-binding site) in Enterococcus faecium, a leading antibiotic-resistant pathogen. By comparing isotopic enrichments of postinsertionally modified cell-wall precursors, we find that tripeptides and species without aspartic acid/asparagine (Asp/Asn, Asx) bridges are specific to mature cell wall. Additionally, we find that the sequence of cell-wall maturation varies throughout a cell cycle. We suggest that actively dividing E. faecium cells have three zones of unique peptidoglycan processing. Our results reveal new organizational characteristics of the bacterial cell wall that are important to understanding tertiary structure and designing novel drugs for antibiotic-resistant pathogens.

Lactoferrin-induced reduction of vanB vancomycin resistance in enterococci.

The mucosal protein lactoferrin (LF) reduces the MIC of vancomycin for staphylococcal isolates sensitive to this glycopeptide. The purpose of this research was to investigate the effect of LF on the MIC of vancomycin for vanB resistant isolates of Enterococcus faecalis (Efs1) and E. faecium (Efm1). At a concentration of 2048 microg/ml, LF reduced the MIC of vancomycin 16-fold for Efs1 and eight-fold for Efm1. The cell wall precursors of Efs1 were examined following growth in media supplemented with LF, D-alanine or D-lactate. The precursors were extracted from harvested cells by ether and ion exchange chromatography and the amino acid and lactate composition was determined. Compared with that of unsupplemented media, D-alanine or LF supplementation caused an increase in the D-alanine content of the precursors. Concomitantly, the D-lactate content was reduced. Exogenous D-lactate did not affect the composition of the precursors. This suggests that LF caused an increase in the pool of pentapeptide cell wall precursors. The LF-induced reduction in the vancomycin resistance of enterococci at in vivo concentrations suggests a potential use for this protein as an adjunctive agent to vancomycin.

Clonal dissemination and colony morphotype variation of vancomycin-resistant Enterococcus faecium isolates in metropolitan Detroit, Michigan.

Thirty-two isolates of vancomycin-resistant Enterococcus faecium (VRE) recovered from 25 patients hospitalized at six hospitals in the metropolitan Detroit, Mich., area over a 32-month period were examined for relatedness by repetitive-sequence PCR (rep-PCR). All isolates were shown to carry the vanA gene by PCR. The rep-PCR patterns generated from each isolate showed that the first three VRE isolates obtained from hospital A between June 1992 and February 1994 were distinct strains. Thereafter, all VRE isolates originating from hospital A and those collected from five other area hospitals had identical rep-PCR patterns. On detailed examination, subcultures of 25 of the 32 VRE isolates produced two distinct colony types characterized phenotypically by a rough and a smooth appearance, respectively. Both colony types retained the vanA locus and the rep-PCR pattern of the primary isolate. These data suggest that a single strain of VRE with the capacity to produce two colonial variants has been disseminated to several Detroit-area hospitals. The clinical significance of the colonial morphotypes is unclear.