Klebsiella pneumoniae exhibiting a phenotypic hyper-splitting phenomenon including the formation of small colony variants.

In this study, we characterized a Klebsiella pneumoniae strain in a patient with shrapnel hip injury, which resulted in multiple phenotypic changes, including the formation of a small colony variant (SCV) phenotype. Although already described since the 1960s, there is little knowledge about SCV phenotypes in Enterobacteriaceae. The formation of SCVs has been recognized as a bacterial strategy to evade host immune responses and compromise the efficacy of antimicrobial therapies, leading to persistent and recurrent courses of infections. In this case, 14 isolates with different resisto- and morpho-types were distinguished from the patient's urine and tissue samples. Whole genome sequencing revealed that all isolates were clonally identical belonging to the K. pneumoniae high-risk sequence type 147. Subculturing the SCV colonies consistently resulted in the reappearance of the initial SCV phenotype and three stable normal-sized phenotypes with distinct morphological characteristics. Additionally, an increase in resistance was observed over time in isolates that shared the same colony appearance. Our findings highlight the complexity of bacterial behavior by revealing a case of phenotypic "hyper-splitting" in a K. pneumoniae SCV and its potential clinical significance.

Sensitizing methicillin-resistant Staphylococcus aureus (MRSA) to cefuroxime: the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most ß-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO3-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care ß-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such "NaHCO3-responsive" isolates can be effectively cleared by ß-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3-responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting ß-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific ß-lactam agents.

Membrane Phenotypic, Metabolic and Genotypic Adaptations of Streptococcus oralis Strains Destined to Rapidly Develop Stable, High-Level Daptomycin Resistance during Daptomycin Exposures.

The Streptococcus mitis-oralis subgroup of viridans group streptococci are important human pathogens. We previously showed that a substantial portion of S. mitis-oralis strains (>25%) are 'destined' to develop rapid, high-level, and stable daptomycin (DAP) resistance (DAP-R) during DAP exposures in vitro. Such DAP-R is often accompanied by perturbations in distinct membrane phenotypes and metabolic pathways. The current study evaluated two S. oralis bloodstream isolates, 73 and 205. Strain 73 developed stable, high-level DAP-R (minimum inhibitory concentration [MIC] > 256 µg/mL) within 2 days of in vitro DAP passage ("high level" DAP-R [HLDR]). In contrast, strain 205 evolved low-level and unstable DAP-R (MIC = 8 µg/mL) under the same exposure conditions in vitro ("non-HLDR"). Comparing the parental 73 vs. 73-D2 (HLDR) strain-pair, we observed the 73-D2 had the following major differences: (i) altered cell membrane (CM) phospholipid profiles, featuring the disappearance of phosphatidylglycerol (PG) and cardiolipin (CL), with accumulation of the PG-CL pathway precursor, phosphatidic acid (PA); (ii) enhanced CM fluidity; (iii) increased DAP surface binding; (iv) reduced growth rates; (v) decreased glucose utilization and lactate accumulation; and (vi) increased enzymatic activity within the glycolytic (i.e., lactate dehydrogenase [LDH]) and lipid biosynthetic (glycerol-3-phosphate dehydrogenase [GPDH]) pathways. In contrast, the 205 (non-HLDR) strain-pair did not show these same phenotypic or metabolic changes over the 2-day DAP exposure. WGS analyses confirmed the presence of mutations in genes involved in the above glycolytic and phospholipid biosynthetic pathways in the 73-D2 passage variant. These data suggest that S. oralis strains which are 'destined' to rapidly develop HLDR do so via a conserved cadre of genotypic, membrane phenotypic, and metabolic adaptations.

Diflunisal and Analogue Pharmacophores Mediating Suppression of Virulence Phenotypes in Staphylococcus aureus.

Invasive methicillin-resistant Staphylococcus aureus (MRSA) infections are leading causes of morbidity and mortality that are complicated by increasing resistance to conventional antibiotics. Thus, minimizing virulence and enhancing antibiotic efficacy against MRSA is a public health imperative. We originally demonstrated that diflunisal (DIF; [2-hydroxy-5-(2,4-difluorophenyl) benzoic acid]) inhibits S. aureus virulence factor expression. To investigate pharmacophores that are active in this function, we evaluated a library of structural analogues for their efficacy to modulate virulence phenotypes in a panel of clinically relevant S. aureus isolates in vitro. Overall, the positions of the phenyl, hydroxyl, and carboxylic moieties and the presence or type of halogen (F vs. Cl) influenced the efficacy of compounds in suppressing hemolysis, proteolysis, and biofilm virulence phenotypes. Analogues lacking halogens inhibited proteolysis to an extent similar to DIF but were ineffective at reducing hemolysis or biofilm production. In contrast, most analogues lacking the hydroxyl or carboxylic acid groups did not suppress proteolysis but did mitigate hemolysis and biofilm production to an extent similar to DIF. Interestingly, chirality and the substitution of fluorine with chlorine resulted in a differential reduction in virulence phenotypes. Together, this pattern of data suggests virulence-suppressing pharmacophores of DIF and structural analogues integrate halogen, hydroxyl, and carboxylic acid moiety stereochemistry. The anti-virulence effects of DIF were achieved using concentrations that are safe in humans, do not impair platelet antimicrobial functions, do not affect S. aureus growth, and do not alter the efficacy of conventional antibiotics. These results offer proof of concept for using novel anti-virulence strategies as adjuvants to antibiotic therapy to address the challenge of MRSA infection.

Gp05, a Prophage-Encoded Virulence Factor, Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a serious public health threat. We recently demonstrated that the presence of a novel prophage ϕSA169 was associated with vancomycin (VAN) treatment failure in experimental MRSA endocarditis. In this study, we assessed the role of a ϕSA169 gene, ϕ80α_gp05 (gp05), in VAN-persistent outcome using gp05 isogenic MRSA strain sets. Of note, Gp05 significantly influences the intersection of MRSA virulence factors, host immune responses, and antibiotic treatment efficacy, including the following: (i) activity of the significant energy-yielding metabolic pathway (e.g., tricarboxylic acid cycle); (ii) carotenoid pigment production; (iii) (p)ppGpp (guanosine tetra- and pentaphosphate) production, which activates the stringent response and subsequent downstream functional factors (e.g., phenol-soluble modulins and polymorphonuclear neutrophil bactericidal activity); and (iv) persistence to VAN treatment in an experimental infective endocarditis model. These data suggest that Gp05 is a significant virulence factor which contributes to the persistent outcomes in MRSA endovascular infection by multiple pathways. IMPORTANCE Persistent endovascular infections are often caused by MRSA strains that are susceptible to anti-MRSA antibiotics in vitro by CLSI breakpoints. Thus, the persistent outcome represents a unique variant of traditional antibiotic resistance mechanisms and a significant therapeutic challenge. Prophage, a critical mobile genetic element carried by most MRSA isolates, provides their bacterial host with metabolic advantages and resistance mechanisms. However, how prophage-encoded virulence factors interact with the host defense system and antibiotics, driving the persistent outcome, is not well known. In the current study, we demonstrated that a novel prophage gene, gp05, significantly impacts tricarboxylic acid cycle activity, stringent response, and pigmentation, as well as vancomycin treatment outcome in an experimental endocarditis model using isogenic gp05 overexpression and chromosomal deletion mutant MRSA strain sets. The findings significantly advance our understanding of the role of Gp05 in persistent MRSA endovascular infection and provide a potential target for development of novel drugs against these life-threatening infections.

Diflunisal Attenuates Virulence Factor Gene Regulation and Phenotypes in Staphylococcus aureus.

Virulence factor expression is integral to pathogenicity of Staphylococcus aureus. We previously demonstrated that aspirin, through its major metabolite, salicylic acid (SAL), modulates S. aureus virulence phenotypes in vitro and in vivo. We compared salicylate metabolites and a structural analogue for their ability to modulate S. aureus virulence factor expression and phenotypes: (i) acetylsalicylic acid (ASA, aspirin); (ii) ASA metabolites, salicylic acid (SAL), gentisic acid (GTA) and salicyluric acid (SUA); or (iii) diflunisal (DIF), a SAL structural analogue. None of these compounds altered the growth rate of any strain tested. ASA and its metabolites SAL, GTA and SUA moderately impaired hemolysis and proteolysis phenotypes in multiple S. aureus strain backgrounds and their respective deletion mutants. Only DIF significantly inhibited these virulence phenotypes in all strains. The kinetic profiles of ASA, SAL or DIF on expression of hla (alpha hemolysin), sspA (V8 protease) and their regulators (sigB, sarA, agr (RNAIII)) were assessed in two prototypic strain backgrounds: SH1000 (methicillin-sensitive S. aureus; MSSA) and LAC-USA300 (methicillin-resistant S. aureus; MRSA). DIF induced sigB expression which is coincident with the significant inhibition of RNAIII expression in both strains and precedes significant reductions in hla and sspA expression. The inhibited expression of these genes within 2 h resulted in the durable suppression of hemolysis and proteolysis phenotypes. These results indicate that DIF modulates the expression of key virulence factors in S. aureus via a coordinated impact on their relevant regulons and target effector genes. This strategy may hold opportunities to develop novel antivirulence strategies to address the ongoing challenge of antibiotic-resistant S. aureus.

Role of the NaHCO3 Transporter MpsABC in the NaHCO3-ß-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care ß-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO3 responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to ß-lactams such as cefazolin and oxacillin in the presence of NaHCO3. A bicarbonate transporter, MpsAB (membrane potential-generating system), was recently found in S. aureus, where it plays a role in concentrating NaHCO3 for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype. Radiolabeled NaH14CO3 uptake profiling revealed significantly higher accumulation in NaHCO3-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO2 conditions, NaHCO3-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO3 supplementation under 5% CO2 conditions. NaHCO3-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO3-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO3 transporter MpsABC is a key contributor to the NaHCO3-ß-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most ß-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO3 responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to ß-lactams in the presence of NaHCO3. A recently described S. aureus NaHCO3 transporter, MpsAB, is involved in intracellular NaHCO3 concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO3-ß-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using ß-lactams.

Combinations of Daptomycin plus Ceftriaxone, but Not Ascending Daptomycin Dose-Regimens, Are Effective in Experimental Endocarditis Caused by Streptococcus mitis-oralis Strains: Target Tissue Clearances and Prevention of Emergence of Daptomycin-Resistance.

The Streptococcus mitis-oralis subgroup of the viridans group streptococci (VGS) are the most common cause of infective endocarditis (IE) in many parts of the world. These organisms are frequently resistant in vitro to standard ß-lactams (e.g., penicillin; ceftriaxone [CRO]), and have the notable capacity for rapidly developing high-level and durable daptomycin resistance (DAP-R) during exposures in vitro, ex vivo, and in vivo. In this study, we used 2 prototypic DAP-susceptible (DAP-S) S. mitis-oralis strains (351; and SF100), which both evolved stable, high-level DAP-R in vitro within 1 to 3 days of DAP passage (5 to 20 µg/mL DAP). Of note, the combination of DAP + CRO prevented this rapid emergence of DAP-R in both strains during in vitro passage. The experimental rabbit IE model was then employed to quantify both the clearance of these strains from multiple target tissues, as well as the emergence of DAP-R in vivo under the following treatment conditions: (i) ascending DAP-alone dose-strategies encompassing human standard-dose and high-dose-regimens; and (ii) combinations of DAP + CRO on these same metrics. Ascending DAP-alone dose-regimens (4 to 18 mg/kg/d) were relatively ineffective at either reducing target organ bioburdens or preventing emergence of DAP-R in vivo. In contrast, the combination of DAP (4 or 8 mg/kg/d) + CRO was effective at clearing both strains from multiple target tissues (often with sterilization of bio-burdens in such organs), as well as preventing the emergence of DAP-R. In patients with serious S. mitis-oralis infections such as IE, especially caused by strains exhibiting intrinsic ß-lactam resistance, initial therapy with combinations of DAP + CRO may be warranted.

Impact of Bicarbonate on PBP2a Production, Maturation, and Functionality in Methicillin-Resistant Staphylococcus aureus (MRSA).

Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit ß-lactam-susceptibility in vitro, ex vivo and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Herein, we investigate the impact of NaHCO3 on factors required for PBP2a functionality. Prototype NaHCO3-responsive and -nonresponsive MRSA strains (as defined in vitro) were assessed for the impact of NaHCO3 on: expression of genes involved in PBP2a production-maturation pathways (mecA, blaZ, pbp4, vraSR, prsA, sigB, and floA); membrane PBP2a and PrsA protein content; and membrane carotenoid content. Following NaHCO3 exposure in NaHCO3-responsive (vs - nonresponsive) MRSA, there was significantly reduced expression of: i) mecA and blaZ; ii) the vraSR-prsA gene axis; and iii) pbp4 Carotenoid production was reduced, while floA expression was increased by NaHCO3 exposure in all MRSA strains. This work underscores the distinct regulatory impact of NaHCO3 on a cadre of genes encoding factors required for maintenance of the MRSA phenotype through PBP2a functionality and maturation.

Influence of Sodium Bicarbonate on Wall Teichoic Acid Synthesis and ß-Lactam Sensitization in NaHCO3-Responsive and Nonresponsive Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) strains pose major treatment challenges due to their innate resistance to most ß-lactams under standard in vitro antimicrobial susceptibility testing conditions. A novel phenotype among MRSA, termed "NaHCO3 responsiveness," where certain strains display increased susceptibility to ß-lactams in the presence of NaHCO3, has been identified among a relatively large proportion of MRSA isolates. One underlying mechanism of NaHCO3 responsiveness appears to be related to decreased expression and altered functionality of several genes and proteins involved in cell wall synthesis and maturation. Here, we studied the impact of NaHCO3 on wall teichoic acid (WTA) synthesis, a process intimately linked to peptidoglycan (PG) synthesis and functionality, in NaHCO3-responsive versus -nonresponsive MRSA isolates. NaHCO3 sensitized responsive MRSA strains to cefuroxime, a specific penicillin-binding protein 2 (PBP2)-inhibitory ß-lactam known to synergize with early WTA synthesis inhibitors (e.g., ticlopidine). Combining cefuroxime with ticlopidine with or without NaHCO3 suggested that these latter two agents target the same step in WTA synthesis. Further, NaHCO3 decreased the abundance and molecular weight of WTA only in responsive strains. Additionally, NaHCO3 stimulated increased autolysis and aberrant cell division in responsive strains, two phenotypes associated with disruption of WTA synthesis. Of note, studies of key genes involved in the WTA biosynthetic pathway (e.g., tarO, tarG, dltA, and fmtA) indicated that the inhibitory impact of NaHCO3 on WTA biosynthesis in responsive strains likely occurred posttranslationally. IMPORTANCE MRSA is generally viewed as resistant to standard ß-lactam antibiotics. However, a NaHCO3-responsive phenotype is observed in a substantial proportion of clinical MRSA strains in vitro, i.e., isolates which demonstrate enhanced susceptibility to standard ß-lactam antibiotics (e.g., oxacillin) in the presence of NaHCO3. This phenotype correlates with increased MRSA clearance in vivo by standard ß-lactam antibiotics, suggesting that patients with infections caused by such MRSA strains might be amenable to treatment with ß-lactams. The mechanism(s) behind this phenotype is not fully understood but appears to involve mecA-PBP2a production and maturation axes. Our study adds significantly to this body of knowledge in terms of additional mechanistic targets of NaHCO3 in selected MRSA strains. This investigation demonstrates that NaHCO3 has direct impacts on S. aureus wall teichoic acid biosynthesis in NaHCO3-responsive MRSA. These findings provide an additional target for new agents being designed to synergistically kill MRSA using ß-lactam antibiotics.

Evidence of Neutralizing and Non-Neutralizing Anti-Glucosaminidase Antibodies in Patients With S. Aureus Osteomyelitis and Their Association With Clinical Outcome Following Surgery in a Clinical Pilot.

Staphylococcus aureus osteomyelitis remains a very challenging condition; recent clinical studies have shown infection control rates following surgery/antibiotics to be ~60%. Additionally, prior efforts to produce an effective S. aureus vaccine have failed, in part due to lack of knowledge of protective immunity. Previously, we demonstrated that anti-glucosaminidase (Gmd) antibodies are protective in animal models but found that only 6.7% of culture-confirmed S. aureus osteomyelitis patients in the AO Clinical Priority Program (AO-CPP) Registry had basal serum levels (>10 ng/ml) of anti-Gmd at the time of surgery (baseline). We identified a small subset of patients with high levels of anti-Gmd antibodies and adverse outcomes following surgery, not explained by Ig class switching to non-functional isotypes. Here, we aimed to test the hypothesis that clinical cure following surgery is associated with anti-Gmd neutralizing antibodies in serum. Therefore, we first optimized an in vitro assay that quantifies recombinant Gmd lysis of the M. luteus cell wall and used it to demonstrate the 50% neutralizing concentration (NC50) of a humanized anti-Gmd mAb (TPH-101) to be ~15.6 µg/ml. We also demonstrated that human serum deficient in anti-Gmd antibodies can be complemented by TPH-101 to achieve the same dose-dependent Gmd neutralizing activity as purified TPH-101. Finally, we assessed the anti-Gmd physical titer and neutralizing activity in sera from 11 patients in the AO-CPP Registry, who were characterized into four groups post-hoc. Group 1 patients (n=3) had high anti-Gmd physical and neutralizing titers at baseline that decreased with clinical cure of the infection over time. Group 2 patients (n=3) had undetectable anti-Gmd antibodies throughout the study and adverse outcomes. Group 3 (n=3) had high titers +/- neutralizing anti-Gmd at baseline with adverse outcomes. Group 4 (n=2) had low titers of non-neutralizing anti-Gmd at baseline with delayed high titers and adverse outcomes. Collectively, these findings demonstrate that both neutralizing and non-neutralizing anti-Gmd antibodies exist in S. aureus osteomyelitis patients and that screening for these antibodies could have a value for identifying patients in need of passive immunization prior to surgery. Future prospective studies to test the prognostic value of anti-Gmd antibodies to assess the potential of passive immunization with TPH-101 are warranted.

The NaHCO3-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus (MRSA) Is Influenced by mecA Genotype.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their in vitro resistance to most ß-lactams on standard laboratory testing. A novel phenotype frequently identified in MRSA strains, termed 'NaHCO3-responsiveness', is a property whereby strains are susceptible in vitro to many ß-lactams in the presence of NaHCO3. Specific mecA genotypes, repression of mecA/PBP2a expression and perturbed maturation of PBP2a by NaHCO3 have all been associated with this phenotype. The aim of this study was to define the relationship between specific mecA genotypes and PBP2a substitutions, on the one hand, with NaHCO3-responsiveness in vitro. Mutations were made in the mecA ribosomal binding site (RBS -7) and at amino acid position 246 of its coding region in parental strains MW2 (NaHCO3-responsive) and C36 (NaHCO3- nonresponsive) to generate 'swap' variants, each harboring the other's mecA-RBS/coding region genotypes. Successful swaps were confirmed by both sequencing, as well as predicted swap of in vitro penicillin-clavulanate susceptibility phenotypes. MW2 swap variants harboring the nonresponsive mecA genotypes became NaHCO3-nonresponsive (resistant to the ß-lactam, oxacillin [OXA]), in the presence of NaHCO3. Moreover, these swap variants had lost NaHCO3-mediated repression of mecA/PBP2a expression. In contrast, C36 swap variants harboring the NaHCO3-responsive mecA genotypes remained NaHCO3-nonresponsive phenotypically, and still exhibited nonrepressible mecA/PBP2a expression. These data demonstrate that in addition to the mecA genotype, NaHCO3-responsiveness may also depend on strain-specific genetic backgrounds.

Impacts of NaHCO3 on ß-Lactam Binding to PBP2a Protein Variants Associated with the NaHCO3-Responsive versus NaHCO3-Non-Responsive Phenotypes.

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to ß-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many ß-lactam antibiotics with less affinity than PBPs which are predominant in methicillin-susceptible (MSSA) strains. A novel, rather frequent in vitro phenotype was recently identified among clinical MRSA bloodstream isolates, termed "NaHCO3-responsiveness". This phenotype features ß-lactam susceptibility of certain MRSA strains only in the presence of NaHCO3. Two distinct PBP2a variants, 246G and 246E, have been linked to the NaHCO3-responsive and NaHCO3-non-responsive MRSA phenotypes, respectively. To determine the mechanistic impact of PBP2a variants on ß-lactam susceptibility, binding profiles of a fluorescent penicillin probe (Bocillin-FL) to each purified PBP2a variant were assessed and compared to whole-cell binding profiles characterized by flow cytometry in the presence vs. absence of NaHCO3. These investigations revealed that NaHCO3 differentially influenced the binding of the fluorescent penicillin, Bocillin-FL, to the PBP2a variants, with binding intensity and rate of binding significantly enhanced in the 246G compared to the 246E variant. Of note, the NaHCO3-ß-lactam (oxacillin)-responsive JE2 strain, which natively harbors the 246G variant, had enhanced Bocillin-FL whole-cell binding following exposure to NaHCO3. This NaHCO3-mediated increase in whole-cell Bocillin-FL binding was not observed in the NaHCO3-non-responsive parental strain, COL, which contains the 246E PBP2a variant. Surprisingly, genetic swaps of the mecA coding sites between JE2 and COL did not alter the NaHCO3-enhanced binding seen in JE2 vs. COL. These data suggest that the non-coding regions of mecA may be involved in NaHCO3-responsiveness. This investigation also provides strong evidence that the NaHCO3-responsive phenotype in MRSA may involve NaHCO3-mediated increases in both initial cell surface ß-lactam binding, as well as ultimate PBP2a binding of ß-lactams.

Proteomic Correlates of Enhanced Daptomycin Activity following ß-Lactam Preconditioning in Daptomycin-Resistant, Methicillin-Resistant Staphylococcus aureus.

Clinical treatment options for daptomycin (DAP)-resistant (DAP-R), methicillin-resistant Staphylococcus aureus (MRSA) infections are relatively limited. Current therapeutic strategies often take advantage of potential synergistic activity of DAP plus ß-lactams; however, the mechanisms underlying their combinatorial efficacy are likely complex and remain incompletely understood. We recently showed that in vitro ß-lactam passaging can resensitize DAP-R strains to a DAP-susceptible (DAP-S) phenotype. To further investigate the implications of selected ß-lactam pretreatments on DAP plus ß-lactam combination efficacy, we utilized DAP-R strain D712. We studied six such combinations, featuring ß-lactams with a broad range of penicillin-binding protein-targeting profiles (PBP-1 to -4), using DAP-R strain D712. Of note, preconditioning with each ß-lactam antibiotic (sequential exposures), followed by DAP exposure, yielded significantly enhanced in vitro activity compared to either DAP treatment alone or simultaneous exposures to both antibiotics. To explore the underpinnings of these outcomes, proteomic analyses were performed, with or without ß-lactam preconditioning. Relative proteomic quantitation comparing ß-lactam pretreatments (versus untreated controls) identified differential modulation of several well-known metabolic, cellular, and biosynthetic processes, i.e., the autolytic and riboflavin biosynthetic pathways. Moreover, these differential proteomic readouts with ß-lactam preconditioning were not PBP target specific. Taken together, these studies suggest that the cellular response to ß-lactam preconditioning in DAP-R MRSA leads to distinct and complex changes in the proteome that appear to resensitize such strains to DAP-mediated killing.

Synergy Mechanisms of Daptomycin-Fosfomycin Combinations in Daptomycin-Susceptible and -Resistant Methicillin-Resistant Staphylococcus 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 is not fully understood. The current study explored 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 combinations of DAP plus Fosfo (DAP+Fosfo) (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 < 0.05), (ii) quantity (P < 0.0001) and localization of cell membrane cardiolipin (CL), (iii) DAP surface binding, and (iv) membrane fluidity (P < 0.05). Furthermore, preconditioning this strain pair to DAP with or without Fosfo (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.

Staphylococcus aureus adaptive evolution: Recent insights on how immune evasion, immunometabolic subversion and host genetics impact vaccine development.

Despite meritorious attempts, a S. aureus vaccine that prevents infection or mitigates severity has not yet achieved efficacy endpoints in prospective, randomized clinical trials. This experience underscores the complexity of host-S. aureus interactions, which appear to be greater than many other bacterial pathogens against which successful vaccines have been developed. It is increasingly evident that S. aureus employs strategic countermeasures to evade or exploit human immune responses. From entering host cells to persist in stealthy intracellular reservoirs, to sensing the environmental milieu and leveraging bacterial or host metabolic products to reprogram host immune responses, S. aureus poses considerable challenges for the development of effective vaccines. The fact that this pathogen causes distinct types of infections and can undergo transient genetic, transcriptional or metabolic adaptations in vivo that do not occur in vitro compounds challenges in vaccine development. Notably, the metabolic versatility of both bacterial and host immune cells as they compete for available substrates within specific tissues inevitably impacts the variable repertoire of gene products that may or may not be vaccine antigens. In this respect, S. aureus has chameleon phenotypes that have alluded vaccine strategies thus far. Nonetheless, a number of recent studies have also revealed important new insights into pathogenesis vulnerabilities of S. aureus. A more detailed understanding of host protective immune defenses versus S. aureus adaptive immune evasion mechanisms may offer breakthroughs in the development of effective vaccines, but at present this goal remains a very high bar. Coupled with the recent advances in human genetics and epigenetics, newer vaccine technologies may enable such a goal. If so, future vaccines that protect against or mitigate the severity of S. aureus infections are likely to emerge at the intersection of precision and personalized medicine. For now, the development of S. aureus vaccines or alternative therapies that reduce mortality and morbidity must continue to be pursued.

New Mechanistic Insights into Purine Biosynthesis with Second Messenger c-di-AMP in Relation to Biofilm-Related Persistent Methicillin-Resistant Staphylococcus aureus Infections.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinically challenging subset of invasive, life-threatening S. aureus infections. We have recently demonstrated that purine biosynthesis plays an important role in such persistent infections. Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger that regulates many cellular pathways in bacteria. However, whether there is a regulatory connection between the purine biosynthesis pathway and c-di-AMP impacting persistent outcomes was not known. Here, we demonstrated that the purine biosynthesis mutant MRSA strain, the ΔpurF strain (compared to its isogenic parental strain), exhibited the following significant differences in vitro: (i) lower ADP, ATP, and c-di-AMP levels; (ii) less biofilm formation with decreased extracellular DNA (eDNA) levels and Triton X-100-induced autolysis paralleling enhanced expressions of the biofilm formation-related two-component regulatory system lytSR and its downstream gene lrgB; (iii) increased vancomycin (VAN)-binding and VAN-induced lysis; and (iv) decreased wall teichoic acid (WTA) levels and expression of the WTA biosynthesis-related gene, tarH. Substantiating these data, the dacA (encoding diadenylate cyclase enzyme required for c-di-AMP synthesis) mutant strain (dacAG206S strain versus its isogenic wild-type MRSA and dacA-complemented strains) showed significantly decreased c-di-AMP levels, similar in vitro effects as seen above for the purF mutant and hypersusceptible to VAN treatment in an experimental biofilm-related MRSA endovascular infection model. These results reveal an important intersection between purine biosynthesis and c-di-AMP that contributes to biofilm-associated persistence in MRSA endovascular infections. This signaling pathway represents a logical therapeutic target against persistent MRSA infections. IMPORTANCE Persistent endovascular infections caused by MRSA, including vascular graft infection syndromes and infective endocarditis, are significant and growing public health threats. A particularly worrisome trend is that most MRSA isolates from these patients are "susceptible" in vitro to conventional anti-MRSA antibiotics, such as VAN and daptomycin (DAP), based on Clinical and Laboratory Standards Institute breakpoints. Yet, these antibiotics frequently fail to eliminate these infections in vivo. Therefore, the persistent outcomes in MRSA infections represent a unique and important variant of classic "antibiotic resistance" that is only disclosed during in vivo antibiotic treatment. Given the high morbidity and mortality associated with the persistent infection, there is an urgent need to understand the specific mechanism(s) of this syndrome. In the current study, we demonstrate that a functional intersection between purine biosynthesis and the second messenger c-di-AMP plays an important role in VAN persistence in experimental MRSA endocarditis. Targeting this pathway may represent a potentially novel and effective strategy for treating these life-threatening infections.

Impact of Bicarbonate-ß-Lactam Exposures on Methicillin-Resistant Staphylococcus aureus (MRSA) Gene Expression in Bicarbonate-ß-Lactam-Responsive vs. Non-Responsive Strains.

Methicillin-resistant Staphylococcus aureus (MRSA) infections represent a difficult clinical treatment issue. Recently, a novel phenotype was discovered amongst selected MRSA which exhibited enhanced ß-lactam susceptibility in vitro in the presence of NaHCO3 (termed 'NaHCO3-responsiveness'). This increased ß-lactam susceptibility phenotype has been verified in both ex vivo and in vivo models. Mechanistic studies to-date have implicated NaHCO3-mediated repression of genes involved in the production, as well as maturation, of the alternative penicillin-binding protein (PBP) 2a, a necessary component of MRSA ß-lactam resistance. Herein, we utilized RNA-sequencing (RNA-seq) to identify genes that were differentially expressed in NaHCO3-responsive (MRSA 11/11) vs. non-responsive (COL) strains, in the presence vs. absence of NaHCO3-ß-lactam co-exposures. These investigations revealed that NaHCO3 selectively repressed the expression of a cadre of genes in strain 11/11 known to be a part of the sigB-sarA-agr regulon, as well as a number of genes involved in the anchoring of cell wall proteins in MRSA. Moreover, several genes related to autolysis, cell division, and cell wall biosynthesis/remodeling, were also selectively impacted by NaHCO3-OXA exposure in the NaHCO3-responsive strain MRSA 11/11. These outcomes provide an important framework for further studies to mechanistically verify the functional relevance of these genetic perturbations to the NaHCO3-responsiveness phenotype in MRSA.

A Combined Phenotypic-Genotypic Predictive Algorithm for In Vitro Detection of Bicarbonate: ß-Lactam Sensitization among Methicillin-Resistant Staphylococcus aureus (MRSA).

Antimicrobial susceptibility testing (AST) is routinely used to establish predictive antibiotic resistance metrics to guide the treatment of bacterial pathogens. Recently, a novel phenotype termed "bicarbonate (NaHCO3)-responsiveness" was identified in a relatively high frequency of clinical MRSA strains, wherein isolates demonstrate in vitro "susceptibility" to standard ß-lactams (oxacillin [OXA]; cefazolin [CFZ]) in the presence of NaHCO3, and in vivo susceptibility to these ß-lactams in experimental endocarditis models. We investigated whether a targeted phenotypic-genotypic screening of MRSA could rule in or rule out NaHCO3 susceptibility upfront. We studied 30 well-characterized clinical MRSA bloodstream isolates, including 15 MIC-susceptible to CFZ and OXA in NaHCO3-supplemented Mueller-Hinton Broth (MHB); and 15 MIC-resistant to both ß-lactams in this media. Using a two-tiered strategy, isolates were first screened by standard disk diffusion for susceptibility to a combination of amoxicillin-clavulanate [AMC]. Isolates then underwent genomic sequence typing: MLST (clonal complex [CC]); agr; SCCmec; and mecA promoter and coding region. The combination of AMC disk susceptibility testing plus mecA and spa genotyping was able to predict MRSA strains that were more or less likely to be NaHCO3-responsive in vitro, with a high degree of sensitivity and specificity. Validation of this screening algorithm was performed in six strains from the overall cohort using an ex vivo model of endocarditis. This ex vivo model recapitulated the in vitro predictions of NaHCO3-responsiveness vs. nonresponsiveness above in five of the six strains.