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Staphylococcal Small Colony Variants Have Novel Mechanisms for Antibiotic Resistance

Over the past 4 years, a variant subpopulation of Staphylococcus aureus has been characterized that is defective in electron transport. These organisms grow slowly and are typical of the previously described small colony variants (SCVs). Indeed, many earlier papers included data that are consistent with defective respiratory activity in SCVs. We present a hypothesis that serves as biochemical basis for the development of SCVs. These variants are particularly interesting because they have been associated with very persistent infections, and they are more resistant to many antibiotics than normal S. aureus. Because of their slow growth, atypical colonial morphology, and unusual biochemical profile, they are easily missed or misidentified in the clinical laboratory. This is of some significance, as this subpopulation is more resistant to antibiotics than the parent population from which they arose. When an infection is particularly resistant to therapy, persists for a long period, or fails to respond to apparently adequate antimicrobial therapy, clinicians and clinical laboratory personnel should consider special efforts to search for SCV

Transcriptomic and metabolic responses of Staphylococcus aureus exposed to supra-physiological temperatures

<p>Abstract</p> <p>Background</p> <p>Previous evaluation by different molecular and physiological assays of <it>Staphylococcus aureus </it>(<it>S. aureus</it>) responses to heat shock exposure yielded a still fragmentary view of the mechanisms determining bacterial survival or death at supra-physiological temperatures. This study analyzed diverse facets of <it>S. aureus </it>heat-shock adjustment by recording global transcriptomic and metabolic responses of bacterial cultures shifted for 10 min from 37°C to a sub-lethal (43°C) or eventually lethal (48°C) temperature. A relevant metabolic model of the combined action of specific stress response mechanisms with more general, energy-regulating metabolic pathways in heat-shocked <it>S. aureus </it>is presented.</p> <p>Results</p> <p>While <it>S. aureus </it>cultures shifted to 43°C or left at 37°C showed marginal differences in growth and survival rates, bacterial cultures exposed to 48°C showed a rapid growth arrest followed by a subsequent decline in viable counts. The most substantial heat shock-induced changes at both 43°C and 48°C occurred in transcript levels of HrcA- and CtsR-regulated genes, encoding classical chaperones DnaK and GroESL, and some Hsp100/Clp ATPases components, respectively. Other metabolic pathways up-regulated by <it>S. aureus </it>exposure at 48°C included genes encoding several enzymes coping with oxidative stress, and DNA damage, or/and impaired osmotic balance. Some major components of the pentose phosphate cycle and gluconeogenesis were also up-regulated, which reflected depletion of free glucose by bacterial cultures grown in Mueller-Hinton broth prior to heat shock. In contrast, most purine- and pyrimidine-synthesis pathway components and amino acyl-tRNA synthetases were down-regulated at 48°C, as well as arginine deiminase and major fermentative pathway components, such as alcohol, lactate and formate dehydrogenases. Despite the heat-induced, increased requirements for ATP-dependent macromolecular repair mechanisms combined with declining energy sources, intracellular ATP levels remained remarkably constant during heat shock.</p> <p>Conclusion</p> <p>The sequential loss of replication and viability at 48°C cannot be explained by significant reductions in intracellular ATP levels, but may reflect ATP rerouting for macromolecular repair mechanisms and cell survival. Our metabolic model also suggests that heat-stressed <it>S. aureus </it>should down-regulate the production of potential, DNA-damaging reactive oxygen species that might result from electron transport-generated ATP, involving excessive levels of free heavy metals, in particular iron.</p

Scope and Predictive Genetic/Phenotypic Signatures of Bicarbonate (NaHCO3) Responsiveness and β-Lactam Sensitization in Methicillin-Resistant Staphylococcus aureus.

Addition of sodium bicarbonate (NaHCO3) to standard antimicrobial susceptibility testing medium reveals certain methicillin-resistant Staphylococcus aureus (MRSA) strains to be highly susceptible to β-lactams. We investigated the prevalence of this phenotype (NaHCO3 responsiveness) to two β-lactams among 58 clinical MRSA bloodstream isolates. Of note, ∼75% and ∼36% of isolates displayed the NaHCO3 responsiveness phenotype to cefazolin (CFZ) and oxacillin (OXA), respectively. Neither intrinsic β-lactam MICs in standard Mueller-Hinton broth (MHB) nor population analysis profiles were predictive of this phenotype. Several genotypic markers (clonal complex 8 [CC8]; agr I and spa t008) were associated with NaHCO3 responsiveness for OXA

Fibronectin Is More Active than Fibrin or Fibrinogen in Promoting Staphylococcus aureus Adherence to Inserted Intravascular Catheters

To further define the role of fibrin(ogen) and fibronectin in Staphylococcus aureus adherence to central venous catheters, the amount, chemical integrity, and biologic activity of these proteins adsorbed on lines inserted in hospitalized patients were prospectively studied. Polyurethane cannulas promoted a significantly lower adherence of S. aureus than polyvinyl chloride (P < .01) or Hickman (P < .001) cannulas and contained the lowest amount of immunologically assayed fibronectin but not of fibrin(ogen). Fibrinogen showed an extensive loss of adherence-promoting activity on inserted cannulas, which was related to its proteolytic breakdown, as detected by SDS-PAGE and immunoblots with antifibrinogen antibodies and confirmed by in vitro studies with purified protein fragments. In contrast, either intact or fragmented fibronectin, although present in much lower amounts than fibrin(ogen), could actively promote S. aureus adherence onto intravenous catheter

Proteomic and Membrane Lipid Correlates of Reduced Host Defense Peptide

We previously described a transposon mutant in Staphylococcus aureus strain SH1000 that exhibited reduced susceptibility to cationic thrombin-induced platelet microbicidal proteins (tPMPs). The transposon insertion site was mapped to the gene snoD, the staphylococcal nuo orthologue. Hence, further studies have been performed to understand how this mutation impacts susceptibility to tPMP, by comparing proteomics profiling and membrane lipid analyses of the parent vs. mutant strains. Surprisingly, the mutant showed differential regulation of only a single protein when cultivated aerobically (FadB), and only a small number of proteins under anaerobic growth conditions (AdhE, DapE, Ddh, Ald1, IlvA1, AgrA, Rot, SA2366, and SA2367). Corresponding to FadB impact on lipid remodeling, membrane fatty acid analyses showed that the snoD mutant contained more short chain anteiso-, but fewer short chain iso-branched chain fatty acids under both aerobic and anaerobic conditions vs. the parental strain. Based upon these proteomic and membrane compositional data, a hypothetical "network" model was developed to explain the impact of the snoD mutation upon tPMP susceptibility

Bloodstream Infections Caused by Small-Colony Variants of Coagulase-Negative Staphylococci Following Pacemaker Implantation

Small-colony variants (SCVs) of Staphylococcus aureus cause persistent and relapsing infections. Relatively little is known regarding infections caused by SCVs of coagulase-negative staphylococci. We report two cases of pacemaker electrode infections due to SCVs of Staphylococcus epidermidis and Staphylococcus capitis. Sequence analysis of a portion of the 16S rRNA gene (16S rDNA) confirmed the identity of the staphylococcal species as S. capitis and S. epidermidis. Isolates from cultures of blood obtained over at least a 2-week interval were compared by pulsed-field gel electrophoresis and found to be clonal even though the colony morphology was very different. Analysis for auxotrophism revealed hemin dependencies for all isolated SCVs. The two cases have several clinical and laboratory characteristics (which are also seen with S. aureus SCV infections) and strongly suggest that SCVs of coagulase-negative staphylococci must be actively sought, because they grow very slowly and can be easily misse

Derived neutrophil lymphocyte ratio is predictive of survival from intermittent therapy in advanced colorectal cancer: a post hoc analysis of the MRC COIN study

BACKGROUND: The phase III COntinuous or INtermittent (COIN) trial failed to show non-inferiority of intermittent compared with continuous chemotherapy for advanced colorectal cancer in overall survival (OS). The present analysis evaluated whether the derived neutrophil to lymphocyte ratio (dNLR) could predict the effect of intermittent vs continuous chemotherapy on OS in patients with advanced colorectal cancer. METHODS: A post hoc exploratory analysis of COIN arms A and C was performed. Landmark analysis was conducted on all patients with available WBC and neutrophils data. The dNLR was calculated using a formula which has previously demonstrated predictive power in cancer patients: dNLR=ANC/(WBC−ANC). A high dNLR was defined using a cut-off value of ⩾2.22. Derived neutrophil to lymphocyte ratio was then correlated with clinical outcomes. Survival curves were generated based on dNLR using the Kaplan–Meier method. Comparison between groups was performed using Cox regression. RESULTS: A total of 1630 patients were assigned to the continuous (N=815) or intermittent (N=815) arms. There was a strong association between dNLR level and OS. The median survival times in the ITT population were 18.6 months and 12.5 months for patients with low and high dNLR, respectively (HR=1.70; 95% CI=1.52–1.90; P<0.001). The estimate of the hazard ratio did not alter substantially (HR=1.54) after adjusting for treatment, tumour status, number of metastatic sites, alkaline phosphate and platelet count. CONCLUSIONS: Derived neutrophil to lymphocyte ratio is strongly prognostic for survival in the COIN intermittent vs continuous treatment arms. Derived neutrophil to lymphocyte ratio does not predict for detrimental survival in patients treated with intermittent therapy

Advancing the application, quality and harmonization of implementation science measures

BACKGROUND: The field of implementation science (IS) encompasses a broad range of constructs and uses measures from a variety of disciplines. However, there has been little standardization of measures or agreement on definitions of constructs across different studies, fields, authors, or research groups. METHODS: We describe a collaborative, web-based activity using the United States National Cancer Institute’s (NCI) Grid-Enabled Measures (GEM) portal that uses a wiki platform to focus discussion and engage the research community to enhance the quality and harmonization of measures for IS health-related research and practice. We present the history, process, and preliminary data from the GEM Dissemination & Implementation (D&I) Campaign on IS measurement. RESULTS: The GEM D&I Campaign has been ongoing for eight weeks as of this writing, and has used a combination of expert opinion and crowd-sourcing approaches. To date it has listed definitions for 45 constructs and summarized information on 120 measures. Usage of the website peaked at a rate of 124 views from 89 visitors on week seven. Users from seven countries have contributed measures and/or constructs, shared experience in using different measures, contributed comments, and identified research gaps and needs. CONCLUSION: Thus far, this campaign has provided information about different IS measures, their associated characteristics, and comments. The next step is to rate these measures for quality and practicality. This resource and ongoing activity have potential to advance the quality and harmonization of IS measures and constructs, and we invite readers to contribute to the process