Synergy Mechanisms of Daptomycin-Fosfomycin Combinations in Daptomycin-Susceptible and -Resistant Methicillin-Resistant S. aureus : In vitro , Ex vivo and In vivo Metrics

Increased usage of daptomycin (DAP) for methicillin-resistant Staphylococcus aureus (MRSA) infections has led to emergence of DAP-resistant (DAP-R) strains, resulting in treatment failures. DAP-fosfomycin (Fosfo) combinations are synergistically active against MRSA, although the mechanism(s) of this interaction are not fully understood. The current study explores four unique, but likely interrelated activities of DAP-Fosfo combinations: i ) synergistic killing; ii ) prevention of evolution of DAP-R; iii ) resensitization of already DAP-R subpopulations to a DAP-susceptible (DAP-S) phenotype; and iv ) perturbations of specific cell envelope phenotypes known to correlate with DAP-R in MRSA. Using an isogenic DAP-S (CB1483) / DAP-R (CB185) clinical MRSA strain-pair, we demonstrated that DAP + Fosfo combinations: i ) enhanced killing of both strains in vitro and ex vivo ; ii ) increased target tissue clearances of the DAP-R strain in an in vivo model of experimental infective endocarditis (IE); iii ) prevented emergence of DAP-R in the DAP-S parental strain both in vitro and ex vivo ; and iv ) resensitized the DAP-R strain to a DAP-S phenotype ex vivo . Phenotypically, following exposure to sub-MIC Fosfo, the DAP-S/ DAP-R strain-pair exhibited distinct modifications in: i ) net positive surface charge (p<0.0001); ii ) quantity (p<0.0001) and localization of cell membrane cardiolipin (CL); iii ) DAP surface binding; and iv ) membrane fluidity (p <0.0001). Furthermore, pre-conditioning to this strain-pair to DAP +/- Fosfo sensitized these organisms to killing by the human host defense peptide, LL37. These data underscore the notion that DAP-Fosfo combinations can impact MRSA clearances within multiple microenvironments, likely based on specific phenotypic adaptations.

Within-Host Adaptation of Staphylococcus aureus in a Bovine Mastitis Infection Is Associated with Increased Cytotoxicity

Within-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen’s strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype–phenotype associations under different infection-relevant growth conditions.

Varied Contribution of Phospholipid Shedding From Membrane to Daptomycin Tolerance in Staphylococcus aureus

It has been suggested that daptomycin can be inactivated by lipids released by Staphylococcus aureus and that this effect is antagonized by phenol soluble modulins (PSMs), which bind to the shed lipids. PSM production is regulated by the Agr system, and others have shown that loss of the Agr function enhances S. aureus survival in the presence of daptomycin. Here we assessed the impact of Agr function on daptomycin activity and lipid metabolism under various conditions. Daptomycin activity was evaluated against three sets of isogenic strain series with wild-type or dysfunctional Agr using static daptomycin time-kills over 24 h and against one strain pair using in vitro pharmacokinetic/pharmacodynamic (PK/PD) models simulating clinical daptomycin exposure for 48 h. We performed comprehensive lipidomics on bacterial membranes and the spent media to correlate lipid shedding with survival. In static time-kill experiments, two agr-deficient strains (SH1000- and USA300 LAC ΔagrA) showed improved survival for 8 h compared with their corresponding wild-type strains as seen in previous studies, but this difference did not persist for 24 h. However, four other agr-deficient strains (SH1001 and JE2 agr KOs) did not demonstrate improved survival compared to isogenic wild-type strains at any time in the time-kills. Lipidomics analysis of SH1000, SH1001, and SH1000- strains showed daptomycin exposure increased lipid shedding compared to growth controls in all strains with phosphatidylglycerols (PGs), lysylPGs and cardiolipins predominating. In the cell pellets, PGs and lysylPGs decreased but cardiolipins were unchanged with daptomycin exposure. The shed lipid profiles in SH1001 and SH1000- were similar, suggesting that the inability to resist daptomycin by SH1001 was not because of differences in lipid shedding. In the PK/PD model, the agr mutant SH1000- strain did not show improved survival relative to SH1000 either. In conclusion, inactivation of daptomycin by shed lipids may be dependent on genetic background, the specific agr mutations, or the techniques used to generate these KOs rather than the overall function of the Agr system, and its contribution to daptomycin tolerance seems to be varied, transient, and growth-condition dependent.

Quantitative trait locus mapping identifies the Gpnmb gene as a modifier of mouse macrophage lysosome function

We have previously shown that the DBA/2J versus AKR/J mouse strain is associated with decreased autophagy-mediated lysosomal hydrolysis of cholesterol esters. Our objective was to determine differences in lysosome function in AKR/J and DBA/2J macrophages, and identify the responsible genes. Using a novel dual-labeled indicator of lysosome function, DBA/2J versus AKR/J bone marrow derived macrophages had significantly decreased lysosome function. We performed quantitative trait loci mapping of lysosome function in bone marrow macrophages from an AKR/J × DBA/2J strain intercross. Four distinct lysosome function loci were identified, which we named macrophage lysosome function modifier (Mlfm) Mlfm1 through Mlfm4. The strongest locus Mlfm1 harbors the Gpnmb gene, which has been shown to recruit autophagy protein light chain 3 to autophagosomes for lysosome fusion. The parental DBA/2J strain has a nonsense variant in Gpnmb. siRNA knockdown of Gpnmb in AKR/J macrophages decreased lysosome function, and Gpnmb deletion through CRISP/Cas9 editing in RAW 264.7 mouse macrophages also demonstrated a similar result. Furthermore, a DBA/2 substrain, called DBA/2J-Gpnmb+/SjJ, contains the wildtype Gpnmb gene, and macrophages from this Gpnmb-preserved DBA/2 substrain exhibited recovered lysosome function. In conclusion, we identified Gpnmb as a causal modifier gene of lysosome function in this strain pair.

Mechanistic Fingerprinting Reveals Kinetic Signatures of Resistance to Daptomycin and Host Defense Peptides in Streptococcus mitis-oralis

Streptococcus mitis-oralis (S. mitis-oralis) infections are increasingly prevalent in specific populations, including neutropenic cancer and endocarditis patients. S. mitis-oralis strains have a propensity to evolve rapid, high-level and durable resistance to daptomycin (DAP-R) in vitro and in vivo, although the mechanism(s) involved remain incompletely defined. We examined mechanisms of DAP-R versus cross-resistance to cationic host defense peptides (HDPs), using an isogenic S. mitis-oralis strain-pair: (i) DAP-susceptible (DAP-S) parental 351-WT (DAP MIC = 0.5 µg/mL), and its (ii) DAP-R variant 351-D10 (DAP MIC > 256 µg/mL). DAP binding was quantified by flow cytometry, in-parallel with temporal (1–4 h) killing by either DAP or comparative prototypic cationic HDPs (hNP-1; LL-37). Multicolor flow cytometry was used to determine kinetic cell responses associated with resistance or susceptibility to these molecules. While overall DAP binding was similar between strains, a significant subpopulation of 351-D10 cells hyper-accumulated DAP (>2–4-fold vs. 351-WT). Further, both DAP and hNP-1 induced cell membrane (CM) hyper-polarization in 351-WT, corresponding to significantly greater temporal DAP-killing (vs. 351-D10). No strain-specific differences in CM permeabilization, lipid turnover or regulated cell death were observed post-exposure to DAP, hNP-1 or LL-37. Thus, the adaptive energetics of the CM appear coupled to the outcomes of interactions of S. mitis-oralis with DAP and selected HDPs. In contrast, altered CM permeabilization, proposed as a major mechanism of action of both DAP and HDPs, did not differentiate DAP-S vs. DAP-R phenotypes in this S. mitis-oralis strain-pair.

Acinetobacter lanii sp. nov., Acinetobacter shaoyimingii sp. nov. and Acinetobacter wanghuae sp. nov., isolated from faeces of Equus kiang

Six aerobic, non-motile, non-haemolytic, Gram-stain-negative, oxidase-negative strains (185T, 187, 323-1T, 194, dk386T and dk771) were recovered from different faecal samples of Equus kiang on the Qinghai–Tibet Plateau. In the 16S rRNA gene sequences, one strain pair, 185T/187, shared highest similarity to Acinetobacter equi 114T (97.9 %), and the other two (323-1T/194 and dk771T/dk386) to Acinetobacter harbinensis CGMCC 1.12528T (98.6 and 97.0 %, respectively). Phylogenomic tree analysis showed that these six strains formed three separate clades in the genus Acinetobacter . Digital DNA–DNA hybridization values of each pair of the isolates with all members of the genus Acinetobacter were far below 70 %. The main cellular fatty acids of all six strains were C18 : 1 ω9c, C16 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Q-9 was the predominant respiratory quinone for strains 185T, 323-1T and dk386T. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Based on the genotypic, phenotypic and biochemical analyses, these six strains represent three novel species of the genus Acinetobacter , for which the names Acinetobacter lanii sp. nov., Acinetobacter shaoyimingii sp. nov. and Acinetobacter wanghuae sp. nov. are proposed. The type strains are 185T (=CGMCC 1.13636T=JCM 33607T), 323-1T (=CGMCC 1.13940T=JCM 33608T) and dk386T (=CGMCC 1.16589T=JCM 33592T), respectively.

903. Resensitization to β-Lactams in Enterococci Depends on Penicillin-Binding Protein (PBP) Mislocalization and Is Mediated by a Single Protein That Modulates Cell Membrane (CM) Adaptation to Daptomycin (DAP)

Background DAP disrupts bacterial CM by binding to septal anionic phospholipids (APLs). LiaX, an effector of the LiaFSR stress system, modulates DAP-R by diverting APLs away from the septum. Enterococci are intrinsically resistant to β-lactams due to the presence of PBPs (e.g., PBP5) with low affinity to these drugs. However, emergence of DAP-R leads to increased susceptibility to β-lactams, a phenomenon designated as the see-saw effect. Here, we dissect the molecular mechanism of this phenomenon. Methods We studied a clinical strain pair of DAP-S (S613) and DAP-R (R712) E. faecalis strains recovered from a patient before and after DAP therapy. We generated deletions of liaX and PBPs (ponA and pbp5) in DAP-susceptible (DAP-S) E. faecalis OG1RF and JH2-2. APLs and membrane structures were visualized with NAO and/or FM4-64. PBPs and LiaX localization were evaluated with bocillin-FL or immunofluorescence. PBP transcripts and PBP5 protein levels were measured by qRT-PCR or immunoblotting, respectively. β-Lactam binding affinity of PBPs was assessed by SDS-PAGE of bocillin-FL stained membranes and a LiaX–PBP5 interaction was evaluated by the bacterial two-hybrid (BACTH) system. MICs were determined via E-test. Results Deletion of liaX led to DAP-R and redistribution of APL microdomains (nonseptal foci with CM aberrations; Figure 1A) in all strains, with a marked decrease in ceftriaxone (CRO) MICs. Only PBP5 was essential for β-lactam resistance but not for DAP-R. DAP-R was associated with mislocalization of PBPs to the sites of CM aberrations (Figure 2). Notably, LiaX and PBP5 were localized to the septum in DAP-S strains but redistributed away from septal areas upon development of DAP-R (Figure 3). An interaction of LiaX and PBP5 was confirmed by the BACTH system. Mislocalized PBPs, most notably PonA and PBP5, had increased affinity for β-lactams in all DAP-R strains. The increased affinity of PBPs to β-lactams was not associated with increased transcripts or PBP5 levels. Conclusion LiaX regulates CM adaptation and cell wall synthesis via membrane remodeling and direct interactions with key PBPs. Changes in LiaX that cause DAP-R results in mislocalization of PBPs to nonseptal areas and likely increases access of β-lactam to the active site, explaining the see-saw effect. Disclosures All Authors: No reported Disclosures.

Influence of Extracellular Cellulose and Colanic Acid Production on the Survival of Shiga Toxin–Producing Escherichia coli on Spinach and Lettuce after Chlorine Treatment

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) strains produce extracellular cellulose and colanic acid, which may influence stress tolerance. This study investigates the role of these extracellular polymers on the tolerance of STEC to chlorine treatment after attachment to lettuce and spinach. Four STEC strains, two wild-type cellulose-producing and their cellulose-deficient derivatives, were used. One strain pair produced colanic acid in addition to cellulose. Spinach and lettuce with attached cells were treated with chlorinated water (50 and 150 ppm of free chlorine). The production of the extracellular polymers by the planktonic cells had small, but significant, effects on the survival of the attached pathogen when subjected to chlorine treatment. On the lettuce surface, the colanic acid–producing, cellulose-negative mutant (49d) was most susceptible to the treatment, declining significantly (P &lt; 0.05) in population by 0.9 and 1.4 log units after treatment with 50 and 150 ppm of chlorine, respectively. Chlorine treatment reduced populations of cellulose-deficient cells on the intact spinach surface 1.2 log units more than the wild type when treated with 150 ppm of chlorine (P &lt; 0.05). However, populations of cellulose-producing cells were reduced by 1.5 log units more than their mutant counterparts when the cells also produced colanic acid (P &lt; 0.05). A greater proportion of cells attached to the spinach leaf edge were injured by chlorine treatment compared with attached to the leaf surface. These results indicate that extracellular polymers do not generally increase the ability of STEC to survive chlorine treatment and that any effects on survival are influenced by location of attachment, type of leafy green, and concentration of chlorine.

Dynamics of Lewis b Binding and Sequence Variation of the babA Adhesin Gene during Chronic Helicobacter pylori Infection in Humans

ABSTRACTHelicobacter pyloriundergoes rapid microevolution during chronic infection, but very little is known about how this affects host interaction factors. The best-studied adhesin ofH. pyloriis BabA, which mediates binding to the blood group antigen Lewis b [Le(b)]. To study the dynamics of Le(b) adherence during human infection, we analyzed pairedH. pyloriisolates obtained sequentially from chronically infected individuals. A complete loss or significant reduction of Le(b) binding was observed in strains from 5 out of 23 individuals, indicating that the Le(b) binding phenotype is quite stable during chronic human infection. Sequence comparisons ofbabAidentified differences due to mutation and/or recombination in 12 out of 16 strain pairs analyzed. Most amino acid changes were found in the putative N-terminal extracellular adhesion domain. One strain pair that had changed from a Le(b) binding to a nonbinding phenotype was used to study the role of distinct sequence changes in Le(b) binding. By transformations of the nonbinding strain with ababAgene amplified from the binding strain,H. pyloristrains with mosaicbabAgenes were generated. Recombinants were enriched for a gain of Le(b) binding by biopanning or for BabA expression on the bacterial surface by pulldown assay. With this approach, we identified several amino acid residues affecting the strength of Le(b) binding. Additionally, the data showed that the C terminus of BabA, which is predicted to encode an outer membrane β-barrel domain, plays an essential role in the biogenesis of this protein.IMPORTANCEHelicobacter pyloricauses a chronic infection of the human stomach that can lead to ulcers and cancer. The bacterium can bind to gastric epithelial cells with specialized outer membrane proteins. The best-studied protein is the BabA adhesin which binds to the Lewis b blood group antigen. SinceH. pyloriis a bacterium with very high genetic variability, we asked whetherbabAevolves during chronic infection and how mutations or recombination inbabAaffect binding. We found that BabA-mediated adherence was stable in most individuals but observed a complete loss of binding or reduced binding in 22% of individuals. One strain pair in which binding was lost was used to generatebabAsequences that were mosaics of a functional allele and a nonfunctional allele, and the mosaic sequences were used to identify amino acids critically involved in binding of BabA to Lewis b.