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.

Ubiquitous bacterial species with the potential to serve as reservoir for antibiotic resistance genes are fanning the flames of antimicrobial resistance. Shortly after discovering the high-level daptomycin (DAP) resistant Mammaliicocci sciuri (M. sciuri) strain TS92 this threat was apparent, as first genetic studies of the Ziebuhr group pointed towards a novel resistance mechanism. The elucidation of this resistance mechanism was therefore urgently required and was addressed by investigating the biological and the chemical background. The aim of this work was to explore the chemical background by applying LC-(HR)MS techniques. Non-specific adsorption was identified as the reason for the initial observation of highly deviating results when working with DAP. The extent was therefore evaluated in a study including several solvents and matrices as well as several syringe filter, ultrafiltration, and reaction container materials. It was shown, that the adsorption behavior of DAP is strongly dependent on the solvent and the contact surface but does not follow any general rule. It became apparent that a preliminary empirical study is always necessary to select an individually suitable combination. Moreover, it could be demonstrated that polypropylene containers are entirely unsuitable for DAP in Mueller-Hinton (MH) medium, as the adsorption loss is already > 25% after one transfer. Within the frame of this study, the use of Protein LoBind® or, with reservation, laboratory glass surfaces was highly recommended instead, whenever possible. The most suitable storage solvent for DAP stock solutions was found in 50% methanol. Following these recommendations resulted in precisely reproducible results in both the biological and chromatographic studies. In previous experiments, a degradation of DAP incubated together with M. sciuri TS92 was suspected. To confirm this, a suitable LC-MS/MS method for the quantification of DAP in a bacterial growth medium and in presence of M. sciuri TS92 was therefore developed. The sample preparation consisted of sterile filtration followed by solvent-induced protein precipitation and centrifugation. The reversed phase chromatography assay made use of a gradient elution and covered the concentrations from 1 to 20 μg/mL by a 1/x² weighted linear regression model, that was calculated from eight calibration levels within the range. All requirements of the internationally approved European Medicines Agency (EMA) guideline on bioanalytical method validation were met. It was subsequently applied to study the time-dependent amount of DAP in presence or absence of M. sciuri TS92 in MH medium. The initial suspicion was confirmed, as the amount of DAP decreased significantly after 24 h of incubation only in presence of M. sciuri TS92. Thus, antibiotic inactivation was hypothesized to be the defense mechanism against DAP. The novel two-gene operon drcAB was found by the Ziebuhr group to mediate the resistance of M. sciuri TS92 against DAP. Moreover, the group provided a heterologous expressible clone of this operon on a plasmid vector, which was transformed into DAP-sensitive Staphylococcus aureus (S. aureus), generating S. aureus RN4220_Pxyl/tet-drcAB. The previously developed DAP quantification assay was applied here to monitor the time dependent amount of DAP in a liquid MH medium culture of this strain upon heterologous drcAB expression. The assay remained applicable and valid for the cultures of this artificial S. aureus strain and it could be demonstrated that the presence of DrcAB leads to a significant loss of DAP after 24 h of incubation. Along with the results of the Ziebuhr group, this was evidence that drcAB expression indeed confers DAP resistance to S. aureus, namely by structural modification(s) and thus antibiotic inactivation. For the qualification of these structural modification(s), drcAB expressing and non-expressing S. aureus RN4220_Pxyl/tet-drcAB cultures in MH medium were again spiked with DAP. After 24 h of incubation, their supernatants were subjected to untargeted LC-HRMS analysis. Apart from the detection principle, this method was a slightly modified version of the previously quantification method. HRMS and MS/HRMS data were simultaneously obtained from information-dependent acquisition runs providing comprehensive characterization of the sample compositions. The subsequently performed GUCS approach revealed two substances that emerge in the presence of DrcAB. Under consideration of the corresponding (MS/)HRMS information, the isotope pattern and the in silico plausibility, the data allowed structural elucidation of both substances. The structures found suggested a two-step modification mechanism of DAP, comprising the N-substitution of the arylamine moiety of kynurenine with C3H4NO and subsequently the hydrolysis of the lactone moiety. This resulted in the postulation that DAP is deprived of its antibiotic effect by the enzymatic transfer of dehydroalanine. / Ubiquitär vorkommende bakterielle Spezies, die als Genreservoir für Antibiotikaresistenzen dienen können, sind Öl für das Feuer der antimikrobiellen Resistenz. Kurz nach der Entdeckung des hochgradig Daptomycin (DAP)-resistenten Mammaliicocci sciuri (M. sciuri) TS92, als die ersten genetischen Studien der Arbeitsgruppe Ziebuhr auf einen neuartigen Resistenzmechanismus hindeuteten, war diese Bedrohung erkennbar. Die Aufklärung dieses Resistenzmechanismus war daher dringend erforderlich und wurde durch Untersuchungen des biologischen als auch des chemischen Hintergrunds adressiert. Das Ziel der vorliegenden Arbeit war die Untersuchung des chemischen Hintergrundes unter Anwendung verschiedener flüssigchromatographischer sowie massenspektrometrischer Techniken. Eine unspezifische Adsorption wurde als Grund für die, zu Beginn der Untersuchungen, häufige Beobachtung voneinander abweichender Ergebnisse bei der Arbeit mit DAP identifiziert. Das Ausmaß wurde daher in einer Studie bewertet, in der unterschiedliche Lösungsmittel und Matrices, sowie unterschiedliche Materialien für Spritzenfilter, Ultrafiltrationsmembranen und Reaktionsgefäßen einbezogen wurden. Es wurde gezeigt, dass das Adsorptionsverhalten von DAP stark vom Lösungsmittel und der Kontaktoberfläche abhängig ist, dabei jedoch keiner allgemeinen Regel folgt. Daraus wurde ersichtlich, dass zur Auswahl einer individuell geeigneten Kombination immer eine empirische Voruntersuchung notwendig ist. Darüber hinaus konnte festgestellt werden, dass Polypropylen-Reaktionsgefäße völlig ungeeignet für DAP in Müller-Hinton (MH) Medium sind und dass bei dessen Verwendung bereits nach einem Transferschritt ein Adsorptionsverlust von mehr als 25% auftritt. Im Rahmen dieser Studie wurde stattdessen die Verwendung von Protein LoBind® oder, unter Vorbehalt, Laborglas Oberflächen wann immer möglich empfohlen. Das geeignetste Lösungsmittel zur Lagerung von DAP-Stammlösungen war 50% Methanol. Die Einhaltung dieser Empfehlungen führte sowohl bei den biologischen als auch bei den chromatographischen Studien zu gut reproduzierbaren Ergebnissen. In früheren Experimenten wurde der Abbau von DAP bei Inkubation mit M. sciuri TS92 vermutet. Um dies zu bestätigen, wurde deshalb eine geeignete LC-MS/MS-Methode zur Quantifizierung von DAP in bakteriellen Wachstumsmedien und in Anwesenheit von M. sciuri TS92 entwickelt. Die Probenvorbereitung bestand zunächst aus einer Sterilfiltration mit anschließender lösungsmittelinduzierten Proteinfällung und Zentrifugation. Die Umkehrphasen-chromatographische Prüfung verwendete ein Gradient und umfasste den Konzentrationsbereich von 1 bis 20 μg/mL. Dieser wurde durch ein lineares Regressionsmodell aus acht Kalibrierpunkten mit einer Gewichtung von 1/x² abgedeckt. Alle Anforderungen der international anerkannten European Medicines Agency (EMA)-Leitlinie „Guideline on bioanalytical method validation“ wurden eingehalten. Anschließend wurde die Methode zur zeitabhängigen Untersuchung der Menge an DAP in MH-Medium bei An- bzw. Abwesenheit von M. sciuri TS92 verwendet. Der initiale Verdacht wurde bestätigt, nachdem die Menge an DAP nach einer Inkubationszeit von 24 h nur bei Anwesenheit von M. sciuri TS92 signifikant abfiel. Daher konnte die Inaktivierung des Antibiotikums als Abwehrmechanismus gegen DAP angenommen werden. Die Arbeitsgruppe Ziebuhr zeigte, dass das neuartige und aus zwei Genen bestehende drcAB Operon die DAP-Resistenz an M. sciuri TS92 vermittelt. Außerdem stellte die Gruppe einen heterolog exprimierbaren Klon des Operons auf einem Plasmidvektor bereit, der in einen DAP-sensitiven Staphylococcus aureus (S. aureus) transformiert wurde, wodurch S. aureus RN4220_Pxyl/tet-drcAB entstand. Die zuvor entwickelte DAP-Quantifizierungsmethode wurde verwendet, um die zeitabhängige DAP-Menge in einer flüssigen MH-Kultur dieses Stamms nach heterologer drcAB-Expression zu untersuchen. Die Methode blieb anwendbar und valide für die MH-Kultur dieses künstlichen S. aureus Stamms und es konnte gezeigt werden, dass die Anwesenheit von DrcAB zu einem signifikanten DAP-Verlust nach einer Inkubationszeit von 24 h führte. Zusammen mit den Ergebnissen der Arbeitsgruppe Ziebuhr weist dies daraufhin, dass die Expression von drcAB tatsächlich eine DAP-Resistenz in S. aureus erzeugt, und zwar durch strukturelle Modifikation(en) und somit durch Inaktivierung des Antibiotikums. Zur Qualifizierung dieser strukturellen Modifikation(en) wurden drcAB exprimierende und nicht exprimierende S. aureus RN4220_Pxyl/tet-drcAB Kulturen in MH-Medium erneut mit DAP dotiert. Nach einer Inkubationszeit von 24 h wurden die Überstände einer ungerichteten LC-HRMS Analyse unterzogen. Abgesehen vom Detektionsprinzip wurde eine geringfügig veränderte Methode der Quantifizierungsmethode verwendet. HRMS- und MS/HRMS-Daten wurden simultan durch IDA-Messläufe generiert, wodurch eine umfassende Charakterisierung der Probenbestandteile erhalten wurde. Der im Anschluss durchgeführte GUCS Ansatz offenbarte zwei Substanzen, die bei Anwesenheit von DrcAB auftreten. Unter Berücksichtigung der dazugehörigen (MS/)HRMS-Informationen, der Isotopenmuster und der in silico-Plausibilität, erlaubten diese Daten die Strukturaufklärung beider Substanzen. Diese lassen einen zweistufigen Modifizierungsmechanismus vermuten, in dem zunächst die N-Substitution des Arylaminrestes von Kynurenin mit C3H4NO und anschließend die Hydrolyse des Lactons stattfindet. Dies führte zur Postulierung, dass DAP durch die enzymatische Übertragung von Dehydroalanin seiner antibiotischen Wirkung beraubt wird.

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.