Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally attached fatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolarization. It has long been assumed that the mode of action of daptomycin involves the formation of oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not been experimentally demonstrated. In the work described in this thesis, I have used fluorescence energy transfer (FRET) between native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the antibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly and is stable for a length of time longer than required for the bactericidal effect. Through variation of the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have determined that the oligomer consists of approximately 6–7 molecules, or, depending on the structure of the oligomer, possibly up to twice that number. Oligomer formation on liposomes and on bacterial membranes was confirmed using excimer fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was also used to demonstrate a stoichiometric interaction between daptomycin and PG. It has previously been shown that the bactericidal activity of daptomycin requires calcium and correlates with the concentration of PG in the bacterial cell membrane; these requirements mirror those observed here for oligomer formation. Furthermore, membrane permeabilization is selective, and electron microscopy of bacterial membranes exposed to daptomycin has revealed no discontinuities or accretions of electron density. Both of these findings suggest formation of a small membrane lesion, which is compatible with the small size of the oligomer that was determined here. In conjunction with these previous findings, the experiments contained in my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin.

Daptomycin (Dap) is a cyclic lipodepsipeptide antibiotic that is currently used in the treatment of bacterial infections caused by Staphylococcus aureus including the strains that are methicillin-resistant (MRSA), as well as vancomycin-resistant (VRSA). Although it is known that daptomycin inserts into the membrane of the bacteria causing cell death very few details of its specific mechanism of action have been elucidated. Studies on Dap-resistant bacteria suggest that Dap may interact with specific proteins as well as specific lipids. It is anticipated that a photoaffinity label (PAL) of daptomycin (Dap-PAL) will be useful in determining what bacterial membrane components interact with Dap. The objective of this proposal is to prepare a photoaffinity label of daptomycin. Starting from 4,4’-dimethylbenzophenone, a benzophenone derivative was prepared that contained a 4-aldehyde moiety and a 4’-alkyl tail which contained a terminal alkyne group. This derivative was attached to the Orn residue in Dap via reductive amination to give the desired Dap-PAL. The minimal inhibitory concentration (MIC) of the Dap-PAL with Bacillus subtillus was determined to be 1.5 μg/mL which is only two times greater than the MIC of Dap.

Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally attached fatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolarization. It has long been assumed that the mode of action of daptomycin involves the formation of oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not been experimentally demonstrated. In the work described in this thesis, I have used fluorescence energy transfer (FRET) between native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the antibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly and is stable for a length of time longer than required for the bactericidal effect. Through variation of the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have determined that the oligomer consists of approximately 6–7 molecules, or, depending on the structure of the oligomer, possibly up to twice that number. Oligomer formation on liposomes and on bacterial membranes was confirmed using excimer fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was also used to demonstrate a stoichiometric interaction between daptomycin and PG. It has previously been shown that the bactericidal activity of daptomycin requires calcium and correlates with the concentration of PG in the bacterial cell membrane; these requirements mirror those observed here for oligomer formation. Furthermore, membrane permeabilization is selective, and electron microscopy of bacterial membranes exposed to daptomycin has revealed no discontinuities or accretions of electron density. Both of these findings suggest formation of a small membrane lesion, which is compatible with the small size of the oligomer that was determined here. In conjunction with these previous findings, the experiments contained in my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin.

Context: Daptomycin is the only antibiotic available with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). Its increased use has resulted in cases of decreased daptomycin efficacy. Recent in vitro studies have shown effective use of beta (beta)-lactam and daptomycin antibiotics, as a combination therapy, in the treatment of VRE. We describe a case of effective treatment in a patient with VRE infection using dual ampicillin and daptomycin therapy that shows bench-to-bedside application of the abovementioned finding. Case Report: A 76-year-old gentleman with a history of bilateral arthroplasty was admitted with a swollen left knee. Blood cultures were positive for Enterococcus faecium. Left knee joint aspiration showed leukocytosis and alpha defensins. Extensive imaging did not show any other source of infection. Culture sensitivity results showed multidrug-resistant enterococci sensitive to daptomycin. The patient was started on intravenous (IV) daptomycin. His left knee prosthesis was explanted and a spacer was placed. The patient continued to be bacteremic for 10 days after removing the knee prosthesis. The patient was trialed on combination IV ampicillin and daptomycin. His blood culture turned negative 2 days later. The patient was discharged home to continue 6 weeks of IV ampicillin and daptomycin. Conclusion: The exact mechanism of the daptomycin/ampicillin synergy effect is unclear. Current hypothesis suggests that ampicillin causes a reduction in the net positive charge of the bacterial surface, possibly by releasing lipoteichoic acid (LTA) from the cell wall. This process increases the ability of the cationic daptomycin/calcium complex to bind to the cell wall more effectively. Our case shows the clinical application of the same. A prospective randomized control trial to explore the effectiveness of dual antibiotic therapy in vivo is needed. If proven, daptomycin/-lactam can become a standard of care to treat VRE and decrease daptomycin nonsusceptibility.

Methicillin-resistant S.aureus (MRSA) is a highly virulent pathogen associated with serious healthcare-associated (HCA-MRSA) and community-associated (CA-MRSA) infections. MRSA is an increasingly important cause of skin and skin-structure, bloodstream and other invasive infections including pneumonia and endocarditis. The pharmacodynamics of existing treatments and anovel cephalosporin with activity against MRSA were studied in an in vitro infection model comparing the antibacterial effects of monotherapy and combination therapy under standard and altered environmental conditions. The activity of monotherapy with vancomycin, daptomycin, linezolid and ceftobiprole against clinical MRSA isolates were tested along with combinations of vancomycin-ceftobiprole, daptomycin-ceftobiprole and linezolid-ceftobiprole. Antibacterial response under standard conditions supporting optimal bacterial growth were compared to altered conditions with acidic pH 5.5, diluted nutrient broth (1:2) and increased temperature 40°C. Two clinical isolates including one HCA-MRSA (#81655) and one CA-MRSA (#79002) were studied in an in vitro pharmacodynamic model (IPDM) over 24 hours. Clinical dosing regimens equivalent to vancomycin 1500 mg intravenously every 12 hours (peak =24.4 mg/L, trough =7.4 mg/L), daptomycin 6 mg/kg (420 mg) intravenously every 24 hours (peak =8.2 mg/L, trough =0.8 mg/L) and linezolid 600 mg intravenously every 12 hours (peak =9.2 mg/L, trough =2.8 mg/L) were tested. Ceftobiprole was administrated as a bolus dose followed by constant infusion of 10 mg/L. Antibacterial effects were quantified as initial bacterial kill rate over 4 hours (KR4) and absolute bacterial kill at 24 hours (BK24). Minimum inhibitory concentrations (MIC) were measured via E-test® methods using initial isolates and those recovered after 24 hours of therapy. The KR4 with daptomycin and vancomycin were equivalent (P=0.14), yet daptomycin was more rapid than ceftobiprole (P=0.03) and linezolid (P<0.0001). The BK24 was greatest with ceftobiprole and vancomycin which were superior to linezolid (P<0.0001, P<0.0001, respectively) and daptomycin (P=0.0001, P=0.0001, respectively). Daptomycin was associated with bacterial re-growth and increasing MICs from 0.25 mg/L to 2-4 mg/L during therapy for isolate #79002 under standard conditions. Furthermore, daptomycin activity against both isolates was significantly reduced under altered conditions (KR4, P=0.0001; BK24, P=0.04). Combination therapy with vancomycin-ceftobiprole was indifferent compared with either agent alone. Although daptomycin-ceftobiprole prevented daptomycin non-susceptibility during therapy and resulted in significantly greater BK24 compared with daptomycin alone (BK24 difference of 4.07 log10 cfu/mL, P=0.0001), the combination was indifferent from ceftobiprole alone. Finally, linezolid-ceftobiprole was similar to linezolid but significantly less active than ceftobiprole alone (BK24 difference of 1.39 log10 cfu/mL, P=0.005) raising concerns of potential antagonism with this combination. In conclusion, this study provides important data regarding antimicrobial pharmacodynamics against MRSA. Overall, monotherapy with either ceftobiprole or vancomycin was most active. Combination therapy with ceftobiprole prevented the emergence of daptomycin non-susceptibility during therapy, but demonstrated potential antagonism with linezolid.

Methicillin-resistant S.aureus (MRSA) is a highly virulent pathogen associated with serious healthcare-associated (HCA-MRSA) and community-associated (CA-MRSA) infections. MRSA is an increasingly important cause of skin and skin-structure, bloodstream and other invasive infections including pneumonia and endocarditis. The pharmacodynamics of existing treatments and anovel cephalosporin with activity against MRSA were studied in an in vitro infection model comparing the antibacterial effects of monotherapy and combination therapy under standard and altered environmental conditions. The activity of monotherapy with vancomycin, daptomycin, linezolid and ceftobiprole against clinical MRSA isolates were tested along with combinations of vancomycin-ceftobiprole, daptomycin-ceftobiprole and linezolid-ceftobiprole. Antibacterial response under standard conditions supporting optimal bacterial growth were compared to altered conditions with acidic pH 5.5, diluted nutrient broth (1:2) and increased temperature 40°C. Two clinical isolates including one HCA-MRSA (#81655) and one CA-MRSA (#79002) were studied in an in vitro pharmacodynamic model (IPDM) over 24 hours. Clinical dosing regimens equivalent to vancomycin 1500 mg intravenously every 12 hours (peak =24.4 mg/L, trough =7.4 mg/L), daptomycin 6 mg/kg (420 mg) intravenously every 24 hours (peak =8.2 mg/L, trough =0.8 mg/L) and linezolid 600 mg intravenously every 12 hours (peak =9.2 mg/L, trough =2.8 mg/L) were tested. Ceftobiprole was administrated as a bolus dose followed by constant infusion of 10 mg/L. Antibacterial effects were quantified as initial bacterial kill rate over 4 hours (KR4) and absolute bacterial kill at 24 hours (BK24). Minimum inhibitory concentrations (MIC) were measured via E-test® methods using initial isolates and those recovered after 24 hours of therapy. The KR4 with daptomycin and vancomycin were equivalent (P=0.14), yet daptomycin was more rapid than ceftobiprole (P=0.03) and linezolid (P<0.0001). The BK24 was greatest with ceftobiprole and vancomycin which were superior to linezolid (P<0.0001, P<0.0001, respectively) and daptomycin (P=0.0001, P=0.0001, respectively). Daptomycin was associated with bacterial re-growth and increasing MICs from 0.25 mg/L to 2-4 mg/L during therapy for isolate #79002 under standard conditions. Furthermore, daptomycin activity against both isolates was significantly reduced under altered conditions (KR4, P=0.0001; BK24, P=0.04). Combination therapy with vancomycin-ceftobiprole was indifferent compared with either agent alone. Although daptomycin-ceftobiprole prevented daptomycin non-susceptibility during therapy and resulted in significantly greater BK24 compared with daptomycin alone (BK24 difference of 4.07 log10 cfu/mL, P=0.0001), the combination was indifferent from ceftobiprole alone. Finally, linezolid-ceftobiprole was similar to linezolid but significantly less active than ceftobiprole alone (BK24 difference of 1.39 log10 cfu/mL, P=0.005) raising concerns of potential antagonism with this combination. In conclusion, this study provides important data regarding antimicrobial pharmacodynamics against MRSA. Overall, monotherapy with either ceftobiprole or vancomycin was most active. Combination therapy with ceftobiprole prevented the emergence of daptomycin non-susceptibility during therapy, but demonstrated potential antagonism with linezolid.

Background: Staphylococcus aureus is an aerobic, Gram positive commensal organism that is capable of causing a wide spectrum of disease. This study contributes to previously published literature regarding daptomycin versus vancomycin use in S. aureus bacteremia (SAB). Methods: Adult patients admitted between 2010 and 2014, billed for ICD-9 code V09.0, 038.11, 038.12, 041.11, or 041.12, and received vancomycin and daptomycin were included in this retrospective analysis. Patients were stratified by time to change in antibiotics from vancomycin to daptomycin to the early switch (1-3 days), intermediate switch (4-7 days), or late switch (8 days or later) group. The primary outcome was treatment failure defined as 30-day recurrence, 60-day all-cause mortality, and 90-day all-cause readmission. Results: 193 patients were enrolled in the final cohort. The overall treatment failure rate was 18% with no differences between early switch, intermediate switch, and late switch (P=0.72) groups. Independent predictors of treatment success were length of stay (OR=1.035) and time to positive culture (OR=0.961). Conclusions: Results of this study did not demonstrate a difference in treatment failure based on time to switch from vancomycin to daptomycin. Future research should focus on optimizing use of vancomycin and daptomycin and medical management of SAB.

MRSA podem ser resistentes à maioria dos antimicrobianos por aquisição de elementos genéticos móveis ou mutações e algumas linhagens representam um grande desafio para o tratamento de suas infecções. Além disso, resistência heterogênea pode existir em taxas subestimadas e causar falha em tratamento. A investigação de disseminação de linhagens específicas de MRSA, adoção de medidas que diminuam a possibilidade de surtos e políticas que evitem uso excessivo de antibióticos são ações importantes que devem ser tomadas em hospitais. Este estudo objetivou caracterizar MRSA, de um hospital de São Carlos-SP, genotipica e fenotipicamente. No período de julho de 2011 a janeiro de 2012, foram isolados 34 S. aureus de diferentes pacientes, sendo que 27 (79,4%) foram identificados como MRSA. Tipagem por PFGE resultou em três pulsotipos e prevalência das linhagens ST105/ST5-SCCmecII. Beta hemólise e presença simultânea dos genes seh/sei/sem/seo/sem/lukDE/hla/hlb/hlg foram encontrados em 96,3% e 85% dos isolados, respectivamente, com nenhum isolado alta produção de biofilme, pelo método utilizado. Nos ensaios de sensibilidade, o isolado SCMSC29 foi caracterizado como S. aureus com resistência intermediária heterogênea à vancomicina (hVISA) e SCMSC29 e SCMSC35 como S. aureus não-sensíveis heterogenêos à daptomicina (hDNSSA), todos confirmados por análise de população. Na tentiva de elucidar os mecanismos de heterorresistência, foram realizados ensaios comparando os isolados heterogêneos com o sensível SCMSC31, relacionado por similaridade. O fenótipo hVISA e hDNSSA apresentado pelo isolado SCMSC29, aparenta estar relacionado com um aumento da expressão dos genes graR, vraR, rpoB, mprF e dltA, sutil aumento da espessura de parede celular, redução de autólise e uma mutação no gene mprF (T551A). O fenótipo hDNSSA de SCMSC35, pode estar associado a mutação encontrada nos genes rpoB (T622A), mprF (M347L, L720F) e aumento da espessura da parede celular. Apesar destes preocupantes fenótipos encontrados, alternativas de tratamento testadas (teicoplanina, linezolida, tetraciclina, tigeciclina, quinupristinadalfopristina e tedizolida) foram ativas contra todos os isolados. Assim, hVISA e hDNSSA foram encontrados entre isolados ST105/ST5-SCCmecII, que demonstraram mutações pontuais e tendência de espessamento de parede celular. O novo antimicrobiano tedizolida, ainda não utilizado no Brasil, possuiu alta eficiência para estes isolados, mostrada in vitro por ensaios de concentração inibitória mínima. A aparição dos perfis de heteroresistência é um achado preocupante e devem ser tomadas medidas para melhorar o diagnóstico deste fenótipo nos laboratórios clínicos além de se evitar disseminação. A adoção de programas de vigilância e a cautela no uso destes antibióticos são importantes para monitorar possível disseminação e para não haver seleção de clones resistentes e co-resistentes a vários outros antibióticos. / MRSA may be resistant to most antimicrobials by the acquisition of mobile genetic elements or mutations and some strains represent a huge challenge for infection treatment. In addition, heterogeneous resistance may exist at underestimated rates and cause treatment failure. Actions such as research of specific MRSA strains spread, adoption of measures that reduce the possibility of outbreaks and policies that avoid excessive use of antibiotics are important and must be taken in hospitals. This study aimed to characterize genotypically and phenotypically, MRSA from a São Carlos-SP hospital. From July 2011 to January 2012, 34 S. aureus from different patients were isolated, among those 27 (79.4%) were identified as MRSA. Typing by PFGE resulted in three pulsotypes and prevalence of ST105/ST5-SCCmecII strains. Beta hemolysis and simultaneous presence of the seh / sei / sem / seo / sem / / lukDE / hla / hlb / hlg genes were found in 96.3% and 85% of the isolates, respectively, with no good biofilm forming sample. In the sensitivity assays, SCMSC29 isolate was characterized as S. aureus heterogeneous vancomycin intermediate resistant S. aureus (hVISA) and SCMSC29 and SCMSC35 as heterogeneous daptomycin non-susceptible S. aureus (hDNSSA), all confirmed by population analysis profile. In order to elucidate mechanisms of heteroresistance, we performed several comparative analyses between heterogeneous samples and a related sensitive isolate (SCMSC31). The hVISA and hDNSSA phenotype presented by the SCMSC29 isolate appeared to be related to increased expression of graR, vraR, rpoB, mprF and dltA genes, slight increase in cell wall thickness, reduction of autolysis and a mutation in the mprF (T551A), when compared to SCMSC31. The hDNSSA phenotype of SCMSC35 may be associated with a mutation found in rpoB (T622A), mprF (M347L, L720F) and increased cell wall thickness. Despite these worrying phenotypes found, treatment alternatives tested (teicoplanin, linezolid, tetracycline, tigecycline, quinupristindalfopristine and tedizolide) were active against all isolates. In conclusion, hVISA and hDNSSA were found to be among ST105/ST5-SCCmecII lineage and demonstrated cell wall thickening. The new antimicrobial tedizolide, not yet used in Brazil, had greater efficiency, shown in vitro by tests of minimum inhibitory concentration. The appearance of heteroresistance profiles is a troubling finding and measures should be taken to improve the diagnosis of this phenotype in clinical laboratories in addition to avoiding dissemination. The adoption of surveillance programs and the caution in the use of these antibiotics are important to monitor possible dissemination and to avoid the selection of resistant clones and coresistant to several other antibiotics.

MRSA podem ser resistentes à maioria dos antimicrobianos por aquisição de elementos genéticos móveis ou mutações e algumas linhagens representam um grande desafio para o tratamento de suas infecções. Além disso, resistência heterogênea pode existir em taxas subestimadas e causar falha em tratamento. A investigação de disseminação de linhagens específicas de MRSA, adoção de medidas que diminuam a possibilidade de surtos e políticas que evitem uso excessivo de antibióticos são ações importantes que devem ser tomadas em hospitais. Este estudo objetivou caracterizar MRSA, de um hospital de São Carlos-SP, genotipica e fenotipicamente. No período de julho de 2011 a janeiro de 2012, foram isolados 34 S. aureus de diferentes pacientes, sendo que 27 (79,4%) foram identificados como MRSA. Tipagem por PFGE resultou em três pulsotipos e prevalência das linhagens ST105/ST5-SCCmecII. Beta hemólise e presença simultânea dos genes seh/sei/sem/seo/sem/lukDE/hla/hlb/hlg foram encontrados em 96,3% e 85% dos isolados, respectivamente, com nenhum isolado alta produção de biofilme, pelo método utilizado. Nos ensaios de sensibilidade, o isolado SCMSC29 foi caracterizado como S. aureus com resistência intermediária heterogênea à vancomicina (hVISA) e SCMSC29 e SCMSC35 como S. aureus não-sensíveis heterogenêos à daptomicina (hDNSSA), todos confirmados por análise de população. Na tentiva de elucidar os mecanismos de heterorresistência, foram realizados ensaios comparando os isolados heterogêneos com o sensível SCMSC31, relacionado por similaridade. O fenótipo hVISA e hDNSSA apresentado pelo isolado SCMSC29, aparenta estar relacionado com um aumento da expressão dos genes graR, vraR, rpoB, mprF e dltA, sutil aumento da espessura de parede celular, redução de autólise e uma mutação no gene mprF (T551A). O fenótipo hDNSSA de SCMSC35, pode estar associado a mutação encontrada nos genes rpoB (T622A), mprF (M347L, L720F) e aumento da espessura da parede celular. Apesar destes preocupantes fenótipos encontrados, alternativas de tratamento testadas (teicoplanina, linezolida, tetraciclina, tigeciclina, quinupristinadalfopristina e tedizolida) foram ativas contra todos os isolados. Assim, hVISA e hDNSSA foram encontrados entre isolados ST105/ST5-SCCmecII, que demonstraram mutações pontuais e tendência de espessamento de parede celular. O novo antimicrobiano tedizolida, ainda não utilizado no Brasil, possuiu alta eficiência para estes isolados, mostrada in vitro por ensaios de concentração inibitória mínima. A aparição dos perfis de heteroresistência é um achado preocupante e devem ser tomadas medidas para melhorar o diagnóstico deste fenótipo nos laboratórios clínicos além de se evitar disseminação. A adoção de programas de vigilância e a cautela no uso destes antibióticos são importantes para monitorar possível disseminação e para não haver seleção de clones resistentes e co-resistentes a vários outros antibióticos. / MRSA may be resistant to most antimicrobials by the acquisition of mobile genetic elements or mutations and some strains represent a huge challenge for infection treatment. In addition, heterogeneous resistance may exist at underestimated rates and cause treatment failure. Actions such as research of specific MRSA strains spread, adoption of measures that reduce the possibility of outbreaks and policies that avoid excessive use of antibiotics are important and must be taken in hospitals. This study aimed to characterize genotypically and phenotypically, MRSA from a São Carlos-SP hospital. From July 2011 to January 2012, 34 S. aureus from different patients were isolated, among those 27 (79.4%) were identified as MRSA. Typing by PFGE resulted in three pulsotypes and prevalence of ST105/ST5-SCCmecII strains. Beta hemolysis and simultaneous presence of the seh / sei / sem / seo / sem / / lukDE / hla / hlb / hlg genes were found in 96.3% and 85% of the isolates, respectively, with no good biofilm forming sample. In the sensitivity assays, SCMSC29 isolate was characterized as S. aureus heterogeneous vancomycin intermediate resistant S. aureus (hVISA) and SCMSC29 and SCMSC35 as heterogeneous daptomycin non-susceptible S. aureus (hDNSSA), all confirmed by population analysis profile. In order to elucidate mechanisms of heteroresistance, we performed several comparative analyses between heterogeneous samples and a related sensitive isolate (SCMSC31). The hVISA and hDNSSA phenotype presented by the SCMSC29 isolate appeared to be related to increased expression of graR, vraR, rpoB, mprF and dltA genes, slight increase in cell wall thickness, reduction of autolysis and a mutation in the mprF (T551A), when compared to SCMSC31. The hDNSSA phenotype of SCMSC35 may be associated with a mutation found in rpoB (T622A), mprF (M347L, L720F) and increased cell wall thickness. Despite these worrying phenotypes found, treatment alternatives tested (teicoplanin, linezolid, tetracycline, tigecycline, quinupristindalfopristine and tedizolide) were active against all isolates. In conclusion, hVISA and hDNSSA were found to be among ST105/ST5-SCCmecII lineage and demonstrated cell wall thickening. The new antimicrobial tedizolide, not yet used in Brazil, had greater efficiency, shown in vitro by tests of minimum inhibitory concentration. The appearance of heteroresistance profiles is a troubling finding and measures should be taken to improve the diagnosis of this phenotype in clinical laboratories in addition to avoiding dissemination. The adoption of surveillance programs and the caution in the use of these antibiotics are important to monitor possible dissemination and to avoid the selection of resistant clones and coresistant to several other antibiotics.

The objectives of this research were to identify the individual ionization constants (pKa values) of lipopeptide (daptomycin), evaluate the factors of pH, concentration, temperature, and calcium ions on daptomycin aggregation in aqueous solutions, and elucidate the effects of conformation and aggregation on ionization and the interaction mechanism between polyamidoamine (PAMAM) dendrimers and daptomycin. Daptomycin is a cyclic anionic lipopeptide antibiotic. It is composed of 13 amino acids with six ionizable groups, four side-chain carboxylic acids and two side-chain amine residues. The pKa values for individual daptomycin residues have not been elucidated. The sequence-specific pKa values for the four acidic residues and one aromatic amine (Kyn-13) in daptomycin were determined in the monomeric state by TOCSY 2D 1H NMR. From the NMR pH titration, the estimated pKa values for Asp-3, Asp-9, and mGlu-12 were determined to be 4.15, 3.85, and 4.55 in the absence of salt, and 4.07, 3.83, and 4.39 in the presence of 150 mM NaCl, respectively. The pKa value for Asp-7 is estimated to be ~1.01 in the absence of salt and 1.31 in the presence of salt. The estimated Hill coefficients for Asp-7 were 0.72 and 1.31 in the absence and presence of salt, respectively. The increase in Hill coefficients from 0.72 to 1.31 with increasing salt concentration is consistent with the estimated lower pKa in the absence of salt and suggests that a salt bridge is formed in solution possibly between Asp-7 acidic group and the neighboring Orn-6 basic group. The pKa value of the aromatic amine (Kyn-13) was confirmed using UV and fluorescence spectroscopic titrations. Aggregation behavior and critical aggregation concentration (CAC) values of daptomycin were evaluated in the different pH aqueous solutions by using the complementary analytical techniques, fluorescence, dynamic and static light scattering, and NMR spectroscopy. Based on fluorescence resonance energy transfer (FRET) from donor Trp-1 to acceptor Kyn-13, the CAC values were determined by an upward inflection of the intrinsic fluorescence emission from Kyn-13 at 460 nm as a function of increasing daptomycin concentration. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy. The effects of temperature and calcium ion on daptomycin aggregation were also discussed. The interaction mechanism between daptomycin and PAMAM dendrimers generation 5 and 6 was studied using fluorescence spectroscopy. The shapes of binding isotherms daptomycin were quantitatively described by one- and two-site binding models to estimate binding capacity and dissociation constants. Both solvent pH values and PAMAM generation size were shown to affect the binding model and parameters. The interaction between daptomycin and PAMAM dendrimer was proposed wherein the ionized Asp-3 and Asp-9 residues of daptomycin interact with PAMAM cationic surface amine.