Viridans group streptococci (VGS), especially the Streptococcus mitis-oralis subgroup, are pivotal pathogens in a variety of invasive endovascular infections, including "toxic shock" in neutropenic cancer patients and infective endocarditis (IE). Previously, we showed that the serial in vitro passage of S. mitis-oralis strains in sublethal daptomycin (DAP) resulted in rapid, high-level and stable DAP-resistance (DAP-R), which is accompanied by distinct changes in several genotypic and phenotypic signatures: (1) the disappearance of two key membrane phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL); (2) increased membrane fluidity; (3) increased positive surface charge; (4) single nucleotide polymorphisms (SNPs) in two loci involved in CL biosynthesis (pgsA; cdsA); and (5) DAP hyperaccumulation. The current study examined these same metrics following in vitro serial DAP passages of a separate well-characterized S. mitis-oralis bloodstream isolate (SF100). Although some metrics seen in prior DAP post-passage strains were recapitulated with SF100 (e.g., pgsA SNPs, enhanced membrane fluidity), we observed the following major differences (comparing the parental versus post-passage variant): (1) no change in PG content; (2) reduced, but not absent, CL, with enhancement in phosphatidic acid (PA) content; (3) an unusual pattern of CL localization; (4) significantly decreased positive surface charge; (5) no difference in DAP accumulation; and (6) no cdsA SNPs. Thus, S. mitis-oralis strains are not "pre-programmed" phenotypically and/or genotypically to adapt in an identical manner during the evolution of the DAP-R.

Weekly oritavancin plus ampicillin continuous infusion combination therapy was used to successfully treat a deep spine vancomycin-resistant Enterococcus faecium infection associated with hardware. Checkerboard and time-kill assays confirmed synergy between these two antibiotics. Further synergies of oritavancin and ampicillin with rifampin or the endogenous human antimicrobial peptide cathelicidin LL-37 were demonstrated.

Daptomycin is a lipopeptide antibiotic that is used clinically against many gram-positive bacterial pathogens and is considered a key frontline bactericidal antibiotic to treat multidrug-resistant enterococci. Emergence of daptomycin resistance during therapy of serious enterococcal infections is a major clinical issue. In this work, we show that deletion of the gene encoding the response regulator, LiaR (a member of the LiaFSR system that controls cell envelope homeostasis), from daptomycin-resistant Enterococcus faecalis not only reversed resistance to 2 clinically available cell membrane-targeting antimicrobials (daptomycin and telavancin), but also resulted in hypersusceptibility to these antibiotics and to a variety of antimicrobial peptides of diverse origin and with different mechanisms of action. The changes in susceptibility to these antibiotics and antimicrobial peptides correlated with in vivo attenuation in a Caenorhabditis elegans model. Mechanistically, deletion of liaR altered the localization of cardiolipin microdomains in the cell membrane. Our findings suggest that LiaR is a master regulator of the enterococcal cell membrane response to diverse antimicrobial agents and peptides; as such, LiaR represents a novel target to restore the activity of clinically useful antimicrobials against these organisms and, potentially, increase susceptibility to endogenous antimicrobial peptides.

Streptococcus mitis/oralis is an important pathogen, causing life-threatening infections such as endocarditis and severe sepsis in immunocompromised patients. The β-lactam antibiotics are the usual therapy of choice for this organism, but their effectiveness is threatened by the frequent emergence of resistance. The lipopeptide daptomycin (DAP) has been suggested for therapy against such resistant S. mitis/oralis strains due to its in vitro bactericidal activity and demonstrated efficacy against other Gram-positive pathogens. Unlike other bacteria, however, S. mitis/oralis has the unique ability to rapidly develop stable, high-level resistance to DAP upon exposure to the drug both in vivo and in vitro Using isogenic DAP-susceptible and DAP-resistant S. mitis/oralis strain pairs, we describe a mechanism of resistance to both DAP and cationic antimicrobial peptides that involves loss-of-function mutations in cdsA (encoding a phosphatidate cytidylyltransferase). CdsA catalyzes the synthesis of cytidine diphosphate-diacylglycerol, an essential phospholipid intermediate for the production of membrane phosphatidylglycerol and cardiolipin. DAP-resistant S. mitis/oralis strains demonstrated a total disappearance of phosphatidylglycerol, cardiolipin, and anionic phospholipid microdomains from membranes. In addition, these strains exhibited cross-resistance to cationic antimicrobial peptides from human neutrophils (i.e., hNP-1). Interestingly, CdsA-mediated changes in phospholipid metabolism were associated with DAP hyperaccumulation in a small subset of the bacterial population, without any binding by the remaining larger population. Our results indicate that CdsA is the major mediator of high-level DAP resistance in S. mitis/oralis and suggest a novel mechanism of bacterial survival against attack by antimicrobial peptides of both innate and exogenous origins.

Objectives

Among viridans group streptococcal infective endocarditis (IE), the Streptococcus mitis group is the most common aetiological organism. Treatment of IE caused by the S. mitis group is challenging due to the high frequency of β-lactam resistance, drug allergy and intolerability of mainstay antimicrobial agents such as vancomycin or gentamicin. Daptomycin has been suggested as an alternative therapeutic option in these scenarios based on its excellent susceptibility profile against S. mitis group strains . However, the propensity of many S. mitis group strains to rapidly evolve stable, high-level daptomycin resistance potentially limits this approach.

Methods

We evaluated the activity of 6 mg/kg/day daptomycin alone or in combination with gentamicin, ceftriaxone or ceftaroline against two daptomycin-susceptible S. mitis group strains over 96 h in a pharmacokinetic/pharmacodynamic model of simulated endocardial vegetations.

Results

Daptomycin alone was not bactericidal and high-level daptomycin resistance evolved at 96 h in both organisms. Combinations of daptomycin + ceftriaxone and daptomycin + ceftaroline demonstrated enhanced killing activity compared with each antibiotic alone and prevented emergence of daptomycin resistance at 96 h. Use of gentamicin as an adjunctive agent neither improved the efficacy of daptomycin nor prevented the development of daptomycin resistance.

Conclusions

Addition of ceftriaxone or ceftaroline to daptomycin improves the bactericidal activity against S. mitis group strains and prevents daptomycin resistance emergence. Further investigation with combinations of daptomycin and β-lactams in a large number of strains is warranted to fully elucidate the clinical implications of such combinations for treatment of S. mitis group IE.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

We report a case of infective endocarditis (IE) caused by ceftaroline-resistant, daptomycin-tolerant, and heterogeneous vancomycin-intermediate methicillin-resistant S. aureus (MRSA). Resistance to ceftaroline emerged in the absence of drug exposure, and the E447K substitution in the active site of PBP2a previously associated with ceftaroline resistance was identified. Additionally, we present evidence of patient-to-patient transmission of the strain within the same unit. This case illustrates the difficulties in treating MRSA IE in the setting of a multidrug-resistant phenotype.

The viridans group streptococci (VGS) are a heterogeneous group of organisms which are important components of the normal human oral flora. Among the VGS, the Streptococcus mitis/oralis subgroup is one of the most common causes of infective endocarditis (IE). Daptomycin (DAP) is a potential alternative therapeutic option for invasive S. mitis infections, given high rates of β-lactam resistance and vancomycin tolerance in such strains. However, the ability of these strains to rapidly evolve high-level and durable DAP resistance (DAP-R) is problematic. Recent data suggest that combination DAP-β-lactam therapy circumvents this issue. Human-simulated dose-escalating DAP-alone dose regimens (6, 8, 10, or 12 mg/kg/day times 4 days) versus DAP (6 mg/kg/day) plus ceftriaxone (CRO) (2 g once daily times 4 days or 0.5 g, single dose) were assessed against two prototypical DAP-susceptible (DAP-S) S. mitis/oralis strains (SF100 and 351), as measured by a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). No DAP-alone regimen was effective, with regrowth of high-level DAP-R isolates observed for both strains over 96-h exposures. Combinations of DAP-CRO with either single- or multidose regimens yielded significant reductions in log10 CFU/g amounts within SEVs for both strains (∼6 log10 CFU/g) within 24 h. In addition, no DAP-R strains were detected in either DAP-CRO combination regimens over the 96-h exposure. In contrast to prior in vitro studies, no perturbations in two key cardiolipin biosynthetic genes (cdsA and pgsA) were identified in DAP-R SEV isolates emerging from strain 351, despite defective phospholipid production. The combination of DAP-CRO warrants further investigation for treatment of IE due to S. mitis/oralis.

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistant Enterococcus faecalis, an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in the Caenorhabditis elegans model, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host-pathogen interactions.