Publications by Year: 2016

Asplenic patients are at increased risk for sepsis and fulminant infection. Sepsis in these patients is typically secondary to encapsulated bacteria, with Streptococcus pneumoniae being the most frequent pathogen. Rare complications of severe sepsis include purpura fulminans and bilateral adrenal hemorrhage (Waterhouse-Friderichsen syndrome). We present the case of a 36-year-old woman, healthy except for splenectomy years prior for idiopathic thrombocytopenic purpura treatment, who presented with fever. Upon presentation to our hospital, three hours after symptoms onset, she had purpura fulminans and shock. Despite timely antimicrobials and maximal resuscitative efforts, her disease progressed and she expired 12 hours after symptoms onset. Autopsy revealed bilateral adrenal hemorrhage; acute adrenal crisis likely contributed to her refractory shock. Prior to her presentation, she had not received guideline-based post-splenectomy care. Sepsis in asplenic patients can be fulminant and rapidly fatal. Streptococcus pneumoniae remains the most frequent cause, despite decreasing rates in recent years related to widespread pneumococcal vaccination. Guideline-based vaccinations and "pill-in-pocket" therapy can be life-saving for asplenic patients. Purpura fulminans represents an extreme manifestation of disseminated intravascular coagulation, is more common in asplenic patients, and portends a poor prognosis. Waterhouse-Friderichsen syndrome can be seen concurrently with purpura fulminans and further portends a poor prognosis; pre-mortem diagnosis requires a high index of suspicion.

With rapid emergence of multidrug-resistant bacteria, there is often a need to perform susceptibility testing for less commonly used or newer antimicrobial agents. Such testing can often be performed only by using labor-intensive, manual dilution methods and lies outside the capacity of most clinical labs, necessitating reference laboratory testing and thereby delaying the availability of susceptibility data. To address the compelling clinical need for microbiology laboratories to perform such testing in-house, we explored a novel, automated, at-will broth microdilution-based susceptibility testing platform. Specifically, we used the modified inkjet printer technology in the HP D300 digital dispensing system to dispense, directly from stock solutions into a 384-well plate, the 2-fold serial dilution series required for broth microdilution testing. This technology was combined with automated absorbance readings and data analysis to determine MICs. Performance was verified by testing members of the Enterobacteriaceae for susceptibility to ampicillin, cefazolin, ciprofloxacin, colistin, gentamicin, meropenem, and tetracycline in comparison to the results obtained with a broth microdilution reference standard. In precision studies, essential and categorical agreement levels were 96.8% and 98.3%, respectively. Furthermore, significantly fewer D300-based measurements were outside ±1 dilution from the modal MIC, suggesting enhanced reproducibility. In accuracy studies performed using a panel of 80 curated clinical isolates, rates of essential and categorical agreement and very major, major, and minor errors were 94%, 96.6%, 0%, 0%, and 3.4%, respectively. Based on these promising initial results, it is anticipated that the D300-based methodology will enable hospital-based clinical microbiology laboratories to perform at-will broth microdilution testing of antimicrobials and to address a critical testing gap.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

We evaluated activity of apramycin, a non-ototoxic/non-nephrotoxic aminocyclitol against 141 clinical Enterobacteriaceae isolates, 51% of which were non-susceptible to carbapenems (CRE). Among CRE, 70.8% were apramycin susceptible, which compared favorably to aminoglycosides in current clinical use. Our data suggest that apramycin deserves further investigation as a repurposed, anti-CRE therapeutic.