Dataset Information

Host gene expression profiling and in vivo cytokine studies to characterize the role of linezolid and vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) murine sepsis model.

ABSTRACT: Linezolid (L), a potent antibiotic for Methicillin Resistant Staphylococcus aureus (MRSA), inhibits bacterial protein synthesis. By contrast, vancomycin (V) is a cell wall active agent. Here, we used a murine sepsis model to test the hypothesis that L treatment is associated with differences in bacterial and host characteristics as compared to V. Mice were injected with S. aureus USA300, and then intravenously treated with 25 mg/kg of either L or V at 2 hours post infection (hpi). In vivo alpha-hemolysin production was reduced in both L and V-treated mice compared to untreated mice but the reduction did not reach the statistical significance [P?=?0.12 for L; P?=?0.70 for V). PVL was significantly reduced in L-treated mice compared to untreated mice (P?=?0.02). However the reduction of in vivo PVL did not reach the statistical significance in V- treated mice compared to untreated mice (P?=?0.27). Both antibiotics significantly reduced IL-1? production [P?=?0.001 for L; P?=?0.006 for V]. IL-6 was significantly reduced with L but not V antibiotic treatment [P<0.001 for L; P?=?0.11 for V]. Neither treatment significantly reduced production of TNF-?. Whole-blood gene expression profiling showed no significant effect of L and V on uninfected mice. In S. aureus-infected mice, L altered the expression of a greater number of genes than V (95 vs. 42; P?=?0.001). Pathway analysis for the differentially expressed genes identified toll-like receptor signaling pathway to be common to each S. aureus-infected comparison. Expression of immunomodulatory genes like Cxcl9, Cxcl10, Il1r2, Cd14 and Nfkbia was different among the treatment groups. Glycerolipid metabolism pathway was uniquely associated with L treatment in S. aureus infection. This study demonstrates that, as compared to V, treatment with L is associated with reduced levels of toxin production, differences in host inflammatory response, and distinct host gene expression characteristics in MRSA sepsis.

SUBMITTER: Sharma-Kuinkel BK

PROVIDER: S-EPMC3614971 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Host gene expression profiling and in vivo cytokine studies to characterize the role of linezolid and vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) murine sepsis model.

Sharma-Kuinkel Batu K BK Zhang Yurong Y Yan Qin Q Ahn Sun Hee SH Fowler Vance G VG

PloS one 20130402 4

Linezolid (L), a potent antibiotic for Methicillin Resistant Staphylococcus aureus (MRSA), inhibits bacterial protein synthesis. By contrast, vancomycin (V) is a cell wall active agent. Here, we used a murine sepsis model to test the hypothesis that L treatment is associated with differences in bacterial and host characteristics as compared to V. Mice were injected with S. aureus USA300, and then intravenously treated with 25 mg/kg of either L or V at 2 hours post infection (hpi). In vivo alpha- ...[more]

PMID: 23565251

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Project description:Although methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (RVS-MRSA; including vancomycin-intermediate S. aureus [VISA] and heterogeneous VISA [hVISA]) have been linked with vancomycin treatment failure, it is unclear whether they are more pathogenic than vancomycin-susceptible MRSA (VS-MRSA). We prospectively assessed patients with clinical MRSA isolates during a 10-month period to determine clinical status (infection versus colonization) and therapeutic outcome before correlating these findings with the results of detailed in vitro assessment of vancomycin susceptibility, including population analysis profile (PAP) testing. hVISA and VISA were defined by standard PAP criteria (area-under-the-curve ratio compared to that of the reference hVISA strain Mu3 [>or=0.9]) and routine CLSI criteria (vancomycin MIC, 4 to 8 microg/ml), respectively. Among the 117 patients assessed, 58 had RVS-MRSA isolates (56 hVISA and 2 VISA) and 59 had VS-MRSA isolates; the patient demographics and comorbidities were similar. RVS-MRSA was associated with a lower rate of infection than VS-MRSA (29/58 versus 46/59; P = 0.003), including a lower rate of bacteremia (3/58 versus 20/59, respectively; P < 0.001). The cure rates in RVS-MRSA and VS-MRSA groups were not statistically different (16/26 versus 31/42; P = 0.43), but the post hoc assessment of treatment regimes and study size made detailed conclusions difficult. The results of the macro method Etest correlated well with the PAP results (sensitivity, 98.3%, and specificity, 91.5%), but broth microdilution and our preliminary RVS-MRSA detection method correlated poorly. All isolates were susceptible to linezolid and daptomycin. These data suggest that detailed prospective laboratory identification of RVS-MRSA isolates may be of limited value and that, instead, such in vitro investigation should be reserved for isolates from patients who are failing appropriate anti-MRSA therapy.

Project description:The genomic evolution in vivo in persistent infection was critical information for understanding how methicillin-resistant Staphylococcus aureus (MRSA) was adapted to host environments with high antibiotic selective pressure. Thirty-two successive MRSA blood isolates with incremental non-susceptibility to vancomycin (VISA), daptomycin (DRSA), and/or linezolid (LRSA) were isolated from a patient failing multiple courses of antimicrobial therapy during 1,356 days of bacteremia. Whole genome sequencing (WGS) for all consecutive isolates were conducted to characterize the evolutionary pathways, resistance-associated mutations and their temporal relationship with antimicrobial treatment. The WGS-based phylogeny categorized the isogenic strains into three major clades, I (22 isolates), II (7 isolates), and III (3 isolates), respectively, harboring a median (range) of 7 (1-30), 62 (53-65), and 118 (100-130) non-synonymous mutations when compared to the very first isolate. Clade I strains were further grouped into early and late subclades, which, respectively, shared the most recent common ancestor with Clade III strains at day 393.7 and Clade II strain at day 662.5. Clade I and Clade III strains were characterized, respectively, with high rates of VISA (9/22, 40.9%) and VISA-and-DRSA phenotype (2/3, 66.7%). Linezolid-resistance including VISA-DRSA-and-LRSA phenotype was exclusively identified in Clade II strains after eight courses of linezolid treatment. The LRSA displayed a small colony variant phenotype and were associated with G2576T mutations in domain V region of 23S rRNA. Substantial loss of mobile elements or alleles mediating resistance or virulence were identified during the evolution of multi-resistance. However, the gene loss might not be correlated to the development of VISA, DRSA, or LRSA phenotype. In conclusion, MRSA in persistent bacteremia was adapted to harsh host environment through multiple pathways involving both resistance-associated mutations and extensive gene loss.

Project description:The precise mechanism and effects of antibiotics in host gene expression and immunomodulation in MRSA infection is unknown. Using a well characterized Methicillin Resistant Staphylococcus aureus (MRSA) isolate USA300 in a murine model of infection, we determined that linezolid and vancomycin induced differential production of bacterial toxins and host cytokines, differences in host gene expression, and differences in immunomodulators during MRSA bloodstream infection. A total of 35 A/J mice, categorized into seven groups (no infection; no infection with linezolid; no infection with vancomycin; 2 hour post-infection (hpi) S. aureus; 24 hpi S. aureus; 24 hpi S. aureus with linezolid; and 24 hpi S. aureus with vancomycin), were used in this study. Mice were injected with USA300 (6 x 106 CFU/g via i.p. route), then intravenously treated with linezolid (25 mg/kg) or vancomycin (25 mg/kg) at 2 hpi. Control and S. aureus infected mice were euthanized at each time point (2 h or 24h) following injection. Whole blood RNA was used for microarray; three cytokines and two S. aureus toxins [PantonValentine Leukocidin (PVL) and alpha hemolysin] were quantified in mouse serum by ELISA. S. aureus CFUs were significantly reduced in blood and kidney after linezolid or vancomycin treatment in S. aureus-infected mice. In vivo IL-1β in mouse serum was significantly reduced in both linezolid (p=0.001) and vancomycin (p=0.006) treated mice compared to untreated ones. IL-6 was significantly reduced only in linezolid treated (p<0.001) but not in vancomycin treated mice. However, another proinflammatory cytokine, TNF-α, did not exhibit altered levels in either linezolid or vancomycin treated mice (p=0.3 and p=0.51 respectively). In vivo level of bacterial toxin, Panton-Valentine leukocidin, in mouse serum was significantly reduced only in linezolid treated mice (p=0.02) but not in vancomycin treated mice. There was no significant effect of either treatment in in vivo level of alpha hemolysin production. Unsupervised hierarchical clustering using the gene expression data from 35 microarrays revealed distinct clustering based on infection status and treatment group. Study of the antibiotic-specific difference in gene expression identified the number of genes uniquely expressed in response to S. aureus infection, infection with linezolid treatment, and infection with vancomycin treatment. Pathway associations study for the differentially expressed genes in each comparison group (Control vs. 24 h S. aureus infection, 24 h S. aureus infection vs. 24 h S. aureus linezolid, and 24 h S. aureus infection vs. 24 h S. aureus vancomycin) in mice using Kyoto Encyclopedia of Genes and Genomes (KEGG) identified toll-like receptor signaling pathway to be common to every comparison groups studied. Glycerolipid metabolism pathway was uniquely associated only with linezolid treatment comparison group. The findings of this study provide the evidence that protein synthesis inhibitor like linezolid does a better job in treating MRSA sepsis compared to cell wall acting antibiotics like vancomycin.

Project description:The herbal products proved to be more promising antimicrobials even though their antimicrobial activity is milder than commercially available antibiotics. Moreover, herbal drugs may act synergistically with antibiotics to kill microbes. In this study, we aimed to enhance the activity of penicillin against MRSA through combination with the active saponin fraction isolated from the Zygophyllum album plant. Three different types of metabolites (saponins, sterols, and phenolics) have been extracted from Zygophyllum album with ethanol and purified using different chromatographic techniques. The antibacterial activity of crude extract and the separated metabolites were checked against MRSA isolates, Saponin fraction (ZA-S) was only the active one followed by the crude extract. Therefore, the compounds in this fraction were identified using ultra-high-performance liquid chromatography connected to quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) operated in positive and negative ionization modes. UHPLC/QTOF-MS revealed the presence of major six ursane-type tritepenoidal saponins (Quinovic acid, Quinovic acid 3β-O-β-D-quinovopyranoside, Zygophylloside C, Zygophylloside G, Zygophylloside K and Ursolic acid), in addition to Oleanolic acid. Interaction studies between saponin fraction and penicillin against MRSA were performed through the checkerboard method and time-kill assay. According to checkerboard results, only three combinations showed a fractional inhibitory concentration index less than 0.5 at concentrations of (62.5 + 312.5, 62.5 + 156.25, and 62.5 + 78.125 of penicillin and ZA-S, respectively). Time kill assay results showed that the highest reduction in log10 colony-forming unit (CFU)/ml of initial inoculum of MRSA after 24 h occurred by 3.7 at concentrations of 62.5 + 312.5 (µg/µg)/ml of penicillin and ZA-S, respectively. Thus, the combination between saponin fraction of Zygophyllum album and penicillin with these concentrations could be a potential agent against MRSA that can serve as possible model for new antibacterial drug.

Dataset Information

Host gene expression profiling and in vivo cytokine studies to characterize the role of linezolid and vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) murine sepsis model.

Publications

Host gene expression profiling and in vivo cytokine studies to characterize the role of linezolid and vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) murine sepsis model.

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