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.

Virulence Potential of Biofilm-Producing Staphylococcus pseudintermedius, Staphylococcus aureus and Staphylococcus coagulans Causing Skin Infections in Companion Animals.

Coagulase-positive staphylococci (CoPS) account for most bacteria-related pyoderma in companion animals. Emergence of methicillin-resistant strains of Staphylococcus pseudintermedius (MRSP), Staphylococcus aureus (MRSA) or Staphylococcus coagulans (MRSC), often with multidrug-resistant (MDR) phenotypes, is a public health concern. The study collection comprised 237 staphylococci (S. pseudintermedius (n = 155), S. aureus (n = 55) and S. coagulans (n = 27)) collected from companion animals, previously characterized regarding resistance patterns and clonal lineages. Biofilm production was detected for 51.0% (79/155), 94.6% (52/55) and 88.9% (24/27) of the S. pseudintermedius, S. aureus and S. coagulans, respectively, and was a frequent trait of the predominant S. pseudintermedius and S. aureus clonal lineages. The production of biofilm varied with NaCl supplementation of the growth media. All S. pseudintermedius and S. aureus strains carried icaADB. Kaplan-Meier survival analysis of Galleria mellonella infected with different CoPS revealed a higher virulence potential of S. aureus when compared with other CoPS. Our study highlights a high frequency of biofilm production by prevalent antimicrobial-resistant clonal lineages of CoPS associated with animal pyoderma, potentially related with a higher virulence potential and persistent or recurrent infections.

Carbapenemase-Producing Extraintestinal Pathogenic Escherichia coli From Argentina: Clonal Diversity and Predominance of Hyperepidemic Clones CC10 and CC131.

Extraintestinal pathogenic Escherichia coli (ExPEC) causes infections outside the intestine. Particular ExPEC clones, such as clonal complex (CC)/sequence type (ST)131, have been known to sequentially accumulate antimicrobial resistance that starts with chromosomal mutations against fluoroquinolones, followed with the acquisition of bla CTX-M-15 and, more recently, carbapenemases. Here we aimed to investigate the distribution of global epidemic clones of carbapenemase-producing ExPEC from Argentina in representative clinical isolates recovered between July 2008 and March 2017. Carbapenemase-producing ExPEC (n = 160) were referred to the Argentinean reference laboratory. Of these, 71 were selected for genome sequencing. Phenotypic and microbiological studies confirmed the presence of carbapenemases confirmed as KPC-2 (n = 52), NDM-1 (n = 16), IMP-8 (n = 2), and VIM-1 (n = 1) producers. The isolates had been recovered mainly from urine, blood, and abdominal fluids among others, and some were from screening samples. After analyzing the virulence gene content, 76% of the isolates were considered ExPEC, although non-ExPEC isolates were also obtained from extraintestinal sites. Pan-genome phylogeny and clonal analysis showed great clonal diversity, although the first phylogroup in abundance was phylogroup A, harboring CC10 isolates, followed by phylogroup B2 with CC/ST131, mostly H30Rx, the subclone co-producing CTX-M-15. Phylogroups D, B1, C, F, and E were also detected with fewer strains. CC10 and CC/ST131 were found throughout the country. In addition, CC10 nucleated most metalloenzymes, such as NDM-1. Other relevant international clones were identified, such as CC/ST38, CC155, CC14/ST1193, and CC23. Two isolates co-produced KPC-2 and OXA-163 or OXA-439, a point mutation variant of OXA-163, and three isolates co-produced MCR-1 among other resistance genes. To conclude, in this work, we described the molecular epidemiology of carbapenemase-producing ExPEC in Argentina. Further studies are necessary to determine the plasmid families disseminating carbapenemases in ExPEC in this region.

Rapid detection of the widely circulating B.1.617.2 (Delta) SARS-CoV-2 variant.

The emergence of the B.1.617.2 (Delta) variant of the severe acute syndrome coronavirus (SARS-CoV-2) that emerged in 2019 (COVID-19), resulted in a surge of cases in India and has expanded and been detected across the world, including in the United States. The B.1.617.2 (Delta) variant has been seen to be twice more transmissible coupled with potential increases in disease severity and immune escape. As a result, case numbers and hospitalisations are once again on the rise in the USA. On 16 July 2021, the Centers for Disease Control and Prevention (CDC) reported a 7-day average 69.3% increase in new cases and a 35% increase in hospitalisations. Although the gold standard for SARS-CoV-2 variants identification remains genomic sequencing, this approach is not accessible to many clinical laboratories. The main goal of this study was to validate and implement the detection of the B.1.617.2 (Delta) variant utilising an open reverse transcription polymerase chain reaction (RT-PCR) platform by explicitly detecting the S-gene target failure (SGTF) corresponding to the deletion of two amino acids (ΔE156/ΔF157) characteristic of B.1.617.2 (Delta) variant. This approach was conceived as a rapid screening of B.1.617.2 (Delta) variant in conjunction with CDC's recommended N1 (nucleocapsid gene), N2, and RP (human RNase P) genes, as a pre-screening tool prior to viral genomic sequencing. We assessed 4,937 samples from 5 July to 5 September 2021. We identified the B.1.617.2 (Delta) variant in 435 of 495 positive samples (87.8%); the additional positive samples (7 samples, 1.4%) were found to belong to the B.1.1.7 (Alpha, UK) lineage and the remaining 53 samples (10.7%) were reported as 'other' lineages. Whole genome sequencing of 46 randomly selected samples validated the strains identified as positive and negative for the B.1.617.2 (Delta) variant and confirmed the S gene deletion in addition to B.1.617.2 characteristic mutations including L452R, T478K, P681R and D950N located in the spike protein. This modality has been used as routine testing at the Riverside University System Health (RUHS) Medical Center as a method for detection of B.1.617.2 (Delta) to pre-screen samples before genome sequencing. The assay can be easily implemented in clinical laboratories, most notably those with limited economic resources and access to genomic platforms.

Staphylococcus pseudintermedius's PBP4 Is Directly Associated with the Dissociated Oxacillin and Cefoxitin Phenotype.

Staphylococcus pseudintermedius is an important pathogen responsible for infections in dogs and in humans. The emergence and dissemination of methicillin-resistant S. pseudintermedius (MRSP) and the multidrug resistance frequently seen in this species make difficult the treatment of these pathogens. The cefoxitin disk is widely used as a marker of methicillin resistance mediated by the mecA gene in Staphylococcus aureus and other staphylococcal species; however, it is not useful to detect ß-lactam resistance of MRSP in clinical microbiology laboratories. The purpose of this study was to elucidate the molecular bases of the dissociated phenotype between oxacillin and cefoxitin antibiotics. By using a combinatorial approach that included the Penicillin-Binding Proteins' (PBP) profile, their affinity for different ß-lactam antibiotics and the analyses of PBPs' sequence, we provide evidence that PBP4 showed still affinity for its target cefoxitin, impairing its phenotypic resistant detection in MRSP. Together, these findings provide evidence that S. pseudintermedius PBP4 is directly associated with the dissociated oxacillin and cefoxitin phenotype.

Impact of PrsA on membrane lipid composition during daptomycin-resistance-mediated ß-lactam sensitization in clinical MRSA strains.

BACKGROUND: The cyclic anionic lipopeptide daptomycin is used in the treatment of severe infections caused by Gram-positive pathogens, including MRSA. Daptomycin resistance, although rare, often results in treatment failure. Paradoxically, in MRSA, daptomycin resistance is usually accompanied by a concomitant decrease in ß-lactam resistance in what is known as the 'see-saw effect'. This resensitization is extensively used for the treatment of MRSA infections, by combining daptomycin and a ß-lactam antibiotic, such as oxacillin. OBJECTIVES: We aimed: (i) to investigate the combined effects of daptomycin and oxacillin on the lipid composition of the cellular membrane of both daptomycin-resistant and -susceptible MRSA strains; and (ii) to assess the involvement of the post-translocational protein PrsA, which plays an important role in oxacillin resistance in MRSA, in membrane lipid composition and remodelling during daptomycin resistance/ß-lactam sensitization. RESULTS: The combination of microbiological and biochemical studies, with fluorescence microscopy using lipid probes, showed that the lipid composition and surface charge of the daptomycin-resistant cells exposed to daptomycin/oxacillin were dependent on antibiotic concentration and directly associated with PrsA, which influenced cardiolipin remodelling/relocation. CONCLUSIONS: Our findings show that PrsA, in addition to its post-transcriptional role in the maturation of PBP 2a, is a key mediator of cell membrane remodelling connected to the see-saw effect and may have a key role in the resensitization of daptomycin-resistant strains to ß-lactams, such as oxacillin.

Carotenogenesis of Staphylococcus aureus: New insights and impact on membrane biophysical properties.

Staphyloxanthin (STX) is a saccharolipid derived from a carotenoid in Staphylococcus aureus involved in oxidative-stress tolerance and antimicrobial peptide resistance. STX influences the biophysical properties of the bacterial membrane and has been associated to the formation of lipid domains in the regulation of methicillin-resistance. In this work, a targeted metabolomics and biophysical characterization study was carried out to investigate the biosynthetic pathways of carotenoids, and their impact on the membrane biophysical properties. Five different S. aureus strains were investigated, including three wild-type strains containing the crtM gene related to STX biosynthesis, a crtM-deletion mutant, and a crtMN plasmid-complemented variant. LC-DAD-MS/MS analysis of extracts allowed the identification of 34 metabolites related to carotenogenesis in S. aureus at different growth phases (8, 24 and 48 h), showing the progression of these metabolites as the bacteria advances into the stationary phase. For the first time, 22 members of a large family of carotenoids were identified, including STX and STX-homologues, as well as Dehydro-STX and Dehydro-STX-homologues. Moreover, thermotropic behavior of the CH2 stretch of lipid acyl chains in live cells by FTIR, show that the presence of STX increases acyl chain order at the bacterial growth temperature. Indeed, the cooperative melting event of the bacterial membrane, which occurs around 15 °C in the native strains, shifts with increased carotenoid content. These results show the diversity biosynthetic of carotenoids in S. aureus, and their influence on membrane biophysical properties.

Carbapenems drive the collateral resistance to ceftaroline in cystic fibrosis patients with MRSA.

Chronic airways infection with methicillin-resistant Staphylococcus aureus (MRSA) is associated with worse respiratory disease cystic fibrosis (CF) patients. Ceftaroline is a cephalosporin that inhibits the penicillin-binding protein (PBP2a) uniquely produced by MRSA. We analyzed 335 S. aureus isolates from CF sputum samples collected at three US centers between 2015-2018. Molecular relationships demonstrated that high-level resistance of preceding isolates to carbapenems were associated with subsequent isolation of ceftaroline resistant CF MRSA. In vitro evolution experiments showed that pre-exposure of CF MRSA to meropenem with further selection with ceftaroline implied mutations in mecA and additional mutations in pbp1 and pbp2, targets of carbapenems; no effects were achieved by other ß-lactams. An in vivo pneumonia mouse model showed the potential therapeutic efficacy of ceftaroline/meropenem combination against ceftaroline-resistant CF MRSA infections. Thus, the present findings highlight risk factors and potential therapeutic strategies offering an opportunity to both prevent and address antibiotic resistance in this patient population.

Characterization of the First mecA-Positive Multidrug-Resistant Staphylococcus pseudintermedius Isolated from an Argentinian Patient.

Staphylococcus pseudintermedius is commonly associated with colonization or infection in dogs, and was identified as a novel species within the genus Staphylococcus in 2006. Methicillin resistance emerged in S. pseudintermedius during the last decade. We describe here a genomic characterization of the first methicillin-resistant S. pseudintermedius (MRSP) recovered from a human patient in Argentina. The strain was phenotypically identified as MRSP 8510 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and antimicrobial susceptibility testing. We assessed genetic characterization by mecA PCR, SCCmec (staphylococcal chromosomal cassette) typing, and whole-genome sequencing. MRSP 8510 was phenotypically resistant to six classes of antimicrobial agents, consistent with the genes found in its genome. We concluded that MRSP 8510 was a multidrug-resistant ST1412 isolate. This study highlights the importance of the detection and characterization of pathogens with potential risks of zoonotic transmission to humans, as they may constitute a reservoir of genes associated with antimicrobial resistance.

Tedizolid is a promising antimicrobial option for the treatment of Staphylococcus aureus infections in cystic fibrosis patients.

BACKGROUND: Tedizolid is a protein synthesis inhibitor in clinical use for the treatment of Gram-positive infections. Pulmonary MRSA infections are a growing problem in patients with cystic fibrosis (CF) and the efficacy of tedizolid-based therapy in CF pulmonary infections is unknown. OBJECTIVES: To evaluate the in vitro and in vivo activity of tedizolid and predict the likelihood of tedizolid resistance selection in CF-background Staphylococcus aureus strains. METHODS: A collection of 330 S. aureus strains (from adult and paediatric patients), either of normal or small colony variant (SCV) phenotypes, gathered at three CF centres in the USA was used. Tedizolid activity was assessed by broth microdilution, Etest and time-kill analysis. In vivo tedizolid efficacy was tested in a murine pneumonia model. Tedizolid in vitro mutants were obtained by 40 days of exposure and progressive passages. Whole genome sequencing of clinical S. aureus strains with reduced susceptibility to tedizolid was performed. RESULTS: MRSA strain MIC90s were tedizolid 0.12-0.25 mg/L and linezolid 1-2 mg/L; for MSSA strains, MIC90s were tedizolid 0.12 mg/L and linezolid 1-2 mg/L. Two strains, WIS 441 and Seattle 106, with tedizolid MICs of 2 mg/L and 1 mg/L, respectively, had MICs above the FDA tedizolid breakpoint (0.5 mg/L). Tedizolid at free serum concentrations exhibited a bacteriostatic effect. Mean bacterial burdens in lungs (log10 cfu/g) for WIS 423-infected mice were: control, 11.2±0.5; tedizolid-treated (10 mg/kg), 3.40±1.87; linezolid-treated (40 mg/kg), 4.51±2.1; and vancomycin-treated (30 mg/kg), 5.21±1.93. For WIS 441-infected mice the (log10 cfu/g) values were: control, 9.66±0.8; tedizolid-treated, 3.18±1.35; linezolid-treated 5.94±2.19; and vancomycin-treated, 4.35±1.7. CONCLUSIONS: These results suggest that tedizolid represents a promising therapeutic option for the treatment of CF-associated MRSA/MSSA infections, having potent in vivo activity and low resistance potential.

Efficacy of newly generated short antimicrobial cationic lipopeptides against methicillin-resistant Staphylococcus aureus (MRSA).

INTRODUCTION: Infection caused by methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) is a serious clinical challenge and research to develop new antimicrobials is imperative. METHODS: This study investigated the in vitro and in vivo efficacy of the short cationic dialkyl lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2. The antibacterial efficacy of (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 was evaluated in representative clinical methicillin-susceptible S. aureus and MRSA strains by both in vitro (MIC, time-kill curve) and in vivo (wax worms model) approaches. RESULTS: These studies revealed that both (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 have rapid bactericidal activity, with a decrease of > 3 log10 colony forming units (CFU)/mL achieved in the first 6 hours of treatment. Furthermore, (C10)2-KKKK-NH2 performed similarly to daptomycin, with a sustained bacterial killing after 24 hours. Wax worms infected and treated with these lipopeptides showed a decreased survival rate of 90% to 50% within the first day of treatment. Scanning electron microscopy determined that the effect of the short lipopeptides in S. aureus was associated with important morphological structural changes that may suggest cell membrane perturbation. CONCLUSION: These findings suggest that the short lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 may be potential new options for treating MRSA infections.

Identification and molecular epidemiology of methicillin resistant Staphylococcus pseudintermedius strains isolated from canine clinical samples in Argentina.

BACKGROUND: Staphylococcus pseudintermedius is the leading cause of pyoderma in dogs and the frequent use of antimicrobial treatment is associated to the development of resistance to nearly all classes of antibiotics. Despite S. pseudintermedius significance, our understanding of the molecular mechanism of ß-lactam resistance and its genetic diversity remains limited. We aimed to: i) determine the phenotypic resistance profile of methicillin resistant Staphylococcus pseudintermedius (MRSP) isolated from infected dogs in three different veterinary hospitals in Buenos Aires, Argentina; ii) identify the SCCmec elements and resistance genes; and iii) analyze the clonal relationship between isolates and in regard of dominant lineages found in the world. RESULTS: In addition to the differential levels of ß-lactam resistance, MRSP isolates (n = 10) showed resistance to 5-6 families of antibiotics, and were therefore categorized as multidrug-resistant. All the isolates were variant of SCCmec V homologous to S. aureus; additional SCCmecFinder analysis classified five of the genomes as SCCmec type V (5C2&5) with mecA (encodes for PBP2a), mecRI and mecI and all the genes closely related to the reference SCCmec type V S. aureus TSGH17 strain. In the remaining five strains, mecA was present, although other genes associated with SCCmec V including mecR1 and mecI were missing. PBP2a was inducible in low level resistance strains (MRSP 8151), and constitutively expressed in MRSP 8150, suggesting different mecA regulatory mechanisms. MRSP isolates showed significant genetic diversity: eight PFGE clonal types and six multilocus-sequence typing (MLST) sequence types (STs) (339, 649, 919, 920, 921 and 922), including four new STs genetically distinct from STs reported in other geographic areas. Comparative genomics and phylogenetic analyses of the MRSP showed a correlation between the genetic content and the phenotypes, and established the genetic relationship between the isolates. CONCLUSIONS: MRSP could be a threat to animal health due to it concerning level of antimicrobial resistance. Our study highlights genetic and epidemiological aspects of multidrug-resistant MRSP strains from Argentina showing high degree of correlation between the resistance genes and the phenotype of the isolates and, furthermore, they appeared evolutionary closer to major worldwide reported ST68 and ST71.

VraSR and Virulence Trait Modulation during Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus Infection.

Methicillin-resistant Staphylococcus aureus (MRSA) threatens human health in hospital and community settings. The lipopeptide antibiotic daptomycin (DAP) is a frequently used treatment option for MRSA infection. DAP exposure can cause bacterial resistance because mutations are induced in genes implicated in cell membrane and cell wall metabolism. Adaptations aimed at surviving antimicrobial pressure can affect bacterial physiology and modify in vivo aptitude and pathogenesis. In this study, clinical DAP-susceptible (DAPs) and DAP-resistant (DAPr) MRSA isolates were used to investigate associations between DAP resistance and staphylococcal virulence. We previously found that VraSR is a critical sensor of cell membrane/wall homeostasis associated with DAP acquisition during MRSA infection. The present study found that DAPr CB1634 and CB5014 MRSA strains with vraSR upregulation were less virulent than their susceptible counterparts, CB1631 and CB5013. Differential gene-transcription profile analysis revealed that DAPr CB1634 had decreased agr two-component system expression, virulence factors, and highly suppressed hemolysis activity. Functional genetic analysis performed in DAPr CB1634 strains using vraSR inactivation followed by gene complementation found that vraSR acted as a transcriptional agrA regulator. These results indicated that VraSR has a broad range of regulatory functions. VraSR also appeared to affect DAPr adherence to epithelial cells, which would affect DAPr strain colonization and survival in the host. The correlation between DAP resistance and decreased virulence was also found in the CB5013 (DAPs) and CB5014 (DAPr) pair. Taken together, these findings are the first evidence that DAP resistance and MRSA virulence are tightly connected and involve compromised expression of regulatory and virulence determinants.IMPORTANCE Methicillin-resistant S. aureus continues to develop resistance to antimicrobials, including those in current clinical use as daptomycin (DAP). Resistance to DAP arises by mutations in cell membrane and cell wall genes and/or upregulation of the two-component VraSR system. However, less is known about the connection between the pathogen and virulence traits during DAP resistance development. We provide new insights into VraSR and its regulatory role for virulence factors during DAP resistance, highlighting coordinated interactions that favor the higher persistence of MRSA DAP-resistant strains in the infected host.

Activity of Telavancin against Staphylococcus aureus Isolates, Including Those with Decreased Susceptibility to Ceftaroline, from Cystic Fibrosis Patients.

Methicillin-resistant Staphylococcus aureus (MRSA) acquisition in cystic fibrosis (CF) patients confers a clinical outcome worse than that in non-CF patients with an increased rate of declined lung function. Telavancin, an approved lipoglycopeptide used to treat infections due to S. aureus, has a dual mode of action causing inhibition of peptidoglycan synthesis and membrane depolarization. MRSA infections in CF patients remain an important problem with no foreseeable decline in prevalence rates. Although telavancin is currently in clinical use for the treatment of complicated skin infections and hospital-acquired pneumonia, the activity against S. aureus infections in CF patients has not been investigated. In this work, we studied the activity of telavancin against CF patient-derived S. aureus strains collected from geographically diverse CF centers in the United States. We found that the telavancin MIC90 was 0.06 µg/ml, 8-fold lower than the ceftaroline or daptomycin MIC90 and 25-fold lower than the linezolid and vancomycin MIC90 We demonstrate that telavancin at serum free concentrations has rapid bactericidal activity, with a decrease of more than 3 log10 CFU/ml being achieved during the first 4 to 6 h of treatment, performing better in this assay than vancomycin and ceftaroline, including against S. aureus strains resistant to ceftaroline. Telavancin resistance was infrequent (0.3%), although we found that it can occur in vitro in both CF- and non-CF patient-derived S. aureus strains by progressive passages with subinhibitory concentrations. Genetic analysis of telavancin-resistant in vitro mutants showed gene polymorphisms in cell wall and virulence genes and increased survival in a Galleria mellonella infection model. Thus, we conclude that telavancin represents a promising therapeutic option for infections in CF patients with potent in vitro activity and a low resistance development potential.

Combination Antibiotic Exposure Selectively Alters the Development of Vancomycin Intermediate Resistance in Staphylococcus aureus.

Invasive methicillin-resistant Staphylococcus aureus (MRSA) treated with vancomycin (VAN) is associated with reduced VAN susceptibility and treatment failure. VAN combination therapy is one strategy to improve response, but comprehensive assessments of combinations to prevent resistance are limited. This study identifies optimal combinations to prevent the emergence of VAN-intermediate Staphylococcus aureus (VISA). Two standard MRSA and two heterogeneous VISA (hVISA) strains were exposed for 28 days in vitro to VAN alone, VAN with cefazolin (CFZ), fosfomycin, gentamicin, meropenem, rifampin, piperacillin-tazobactam (TZP), or trimethoprim-sulfamethoxazole. In addition to VAN susceptibility testing, cell wall thickness (CWT), carotenoid content, and membrane fluidity were determined for Mu3. VAN plus any ß-lactam limited the VAN MIC increase to 1 to 4 mg/liter throughout the 28-day exposure, with CFZ and TZP being the most effective agents (VAN MIC = 1 to 2 mg/liter). Similar MIC trends occurred with the lipo-/glycopeptide agents daptomycin and telavancin, where ß-lactam combinations with VAN prevented MIC increases to these agents as well. Combinations with non-ß-lactams were ineffective in preventing VAN MIC increases with VAN MICs of 4 to 16 mg/liter emerging during weeks 2 to 4 of treatment. VAN plus ß-lactam decreased CWT significantly, whereas VAN plus other antibiotics significantly increased the CWT. No correlation was observed between carotenoid content or membrane fluidity and antibiotic exposure. Only the combination exposures of VAN plus ß-lactam suppress the development of VISA. Rational selection of VAN plus ß-lactam should be further explored as a long-term combination treatment of MRSA infections due to their ability to suppress VAN resistance.

Daptomycin Resistance in Clinical MRSA Strains Is Associated with a High Biological Fitness Cost.

Daptomycin remains as one of the main treatment options for Methicillin-Resistant Staphylococcus aureus (MRSA). Sporadic resistance cases reported in patients treated with either daptomycin or glycopeptides are a growing concern. In a previous study, we described a clinical case of a patient with a community-acquired MRSA infection resistant to daptomycin and with intermediate resistance to vancomycin who developed a recurrent infection with a susceptible isogenic strain. In the present work, we further investigated the sequential events to determine whether the switch from a daptomycin resistance to a susceptible phenotype was due to a phenomenon of resistance reversion or recurrent infection with a susceptible strain. Pairwise competition experiments showed that the susceptible clinical recurrent SA6850 strain had increased fitness when compared to the resistant counterpart SA6820 strain. In fact, although we have demonstrated that reversion of daptomycin resistance to daptomycin susceptible can occur in vitro after serial passages in drug-free media, phylogenetic analysis suggested that the in vivo process was the result of a recurrent infection with a previous susceptible isolate carried by the patient rather than a resistance reversion of the strain. Whole genome sequence of evolved strains showed that daptomycin resistance in MRSA is associated with a high fitness cost mediated by mutations in mprF gene, revealed as a key element of the biological cost. Moreover, we determined that daptomycin resistance-associated fitness cost was independent of vancomycin intermediate resistance phenotype, as demonstrated in additional clinical MRSA vancomycin susceptible strains. This study highlights important observations as, despite daptomycin offers a useful treatment option for the patients with persistent infections, it has to be carefully monitored. The high fitness cost associated to daptomycin resistance may explain the reduced dissemination of daptomycin resistance and the absence of daptomycin reported outbreaks.

Molecular Bases Determining Daptomycin Resistance-Mediated Resensitization to ß-Lactams (Seesaw Effect) in Methicillin-Resistant Staphylococcus aureus.

Antimicrobial resistance is recognized as one of the principal threats to public health worldwide, yet the problem is increasing. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains are among the most difficult to treat in clinical settings due to the resistance of MRSA to nearly all available antibiotics. The cyclic anionic lipopeptide antibiotic daptomycin (DAP) is the clinical mainstay of anti-MRSA therapy. The decreased susceptibility to DAP (DAP resistance [DAPr]) reported in MRSA is frequently accompanied by a paradoxical decrease in ß-lactam resistance, a process known as the "seesaw effect." Despite the observed discordance in resistance phenotypes, the combination of DAP and ß-lactams has been proven to be clinically effective for the prevention and treatment of infections due to DAPr MRSA strains. However, the mechanisms underlying the interactions between DAP and ß-lactams are largely unknown. In the study described here, we studied the role of mprF with DAP-induced mutations in ß-lactam sensitization and its involvement in the effective killing by the DAP-oxacillin (OXA) combination. DAP-OXA-mediated effects resulted in cell wall perturbations, including changes in peptidoglycan insertion, penicillin-binding protein 2 (PBP 2) delocalization, and reduced membrane amounts of PBP 2a, despite the increased transcription of mecA through mec regulatory elements. We have found that the VraSR sensor-regulator is a key component of DAP resistance, triggering mutated mprF-mediated cell membrane (CM) modifications that result in impairment of PrsA location and chaperone functions, both of which are essential for PBP 2a maturation, the key determinant of ß-lactam resistance. These observations provide for the first time evidence that synergistic effects between DAP and ß-lactams involve PrsA posttranscriptional regulation of CM-associated PBP 2a.

The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A.

Expression of the methicillin-resistant S. aureus (MRSA) phenotype results from the expression of the extra penicillin-binding protein 2A (PBP2A), which is encoded by mecA and acquired horizontally on part of the SCCmec cassette. PBP2A can catalyze dd-transpeptidation of peptidoglycan (PG) because of its low affinity for ß-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase protein as a regulator of ß-lactam resistance expression. Deletion of prsA altered oxacillin resistance in three different SCCmec backgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affecting mecA mRNA levels. The N- and C-terminal domains of PrsA were found to be critical features for PBP2A protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of the mecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents.

Impact of efflux in the development of multidrug resistance phenotypes in Staphylococcus aureus.

BACKGROUND: Efflux has been recognized as a resistance mechanism to antimicrobials in Staphylococcus aureus; however its role on the development of clinically relevant resistance is still poorly characterized. This study aimed to examine the impact of efflux on development of resistance to fluoroquinolones and other antimicrobials in S. aureus strains representing relevant phenotypes in terms of antibiotic susceptibility and efflux activity. METHODS: Two closely related methicillin- and ciprofloxacin-resistant Staphylococcus aureus clinical strains, with different efflux capacity and the pan-susceptible strain ATCC25923 were exposed to constant concentrations of the efflux pump (EP) substrates ciprofloxacin, ethidium bromide and cetrimide. Parental and exposed strains were tested regarding their susceptibility towards antibiotics, biocides and ethidium bromide, efflux capacity and levels of EP gene expression. Occurrence of resistance-associated mutations was screened by sequencing. RESULTS: Multidrug resistance phenotypes emerged upon exposure, independently of the substrate or its concentration, which were correlated with increased efflux capacity of the exposed strains. The temporal pattern of EP gene expression disclosed an early-response with high expression of several genes, followed by a late-response, characterized by overexpression of specific genes. The overall cell response was more pronounced for strains with an initial basal efflux activity. Remarkably, detection of the IS256 element in the promoter regions of mgrA and norA, in some cases associated with increased gene expression, suggests that these genes may be hot spots for IS256 insertion events. The results obtained with exposure of ATCC25923 to ciprofloxacin were particularly striking, revealing a step-wise development of fluoroquinolone resistance, with a first efflux-mediated response, followed by the occurrence of a mutation in grlA that resulted in phenotypic resistance. Additionally, challenge by non-fluoroquinolone agents, particularly cetrimide, promoted cross resistance to fluoroquinolones, revealing the potential role of biocides as selective pressure for the emergence of resistance to these antibiotics. CONCLUSIONS: This study reveals efflux as a significant component of S. aureus resistance to fluoroquinolones and biocides and as a primary mechanism to withstand stress imposed by antimicrobials. This efflux-mediated response can result in the emergence of multidrug resistance in healthcare environments and should be taken into account in the management of this major pathogen.

PBP2a mutations causing high-level Ceftaroline resistance in clinical methicillin-resistant Staphylococcus aureus isolates.

Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.