Behavioral screening of conserved RNA-binding proteins reveals CEY-1/YBX RNA-binding protein dysfunction leads to impairments in memory and cognition.

RNA-binding proteins (RBPs) regulate translation and plasticity which are required for memory. RBP dysfunction has been linked to a range of neurological disorders where cognitive impairments are a key symptom. However, of the 2,000 RBPs in the human genome, many are uncharacterized with regards to neurological phenotypes. To address this, we used the model organism C. elegans to assess the role of 20 conserved RBPs in memory. We identified eight previously uncharacterized memory regulators, three of which are in the C. elegans Y-Box (CEY) RBP family. Of these, we determined that cey-1 is the closest ortholog to the mammalian Y-Box (YBX) RBPs. We found that CEY-1 is both necessary in the nervous system for memory ability and sufficient to increase memory. Leveraging human datasets, we found both copy number variation losses and single nucleotide variants in YBX1 and YBX3 in individuals with neurological symptoms. We identified one predicted deleterious YBX3 variant of unknown significance, p.Asn127Tyr, in two individuals with neurological symptoms. Introducing this variant into endogenous cey-1 locus caused memory deficits in the worm. We further generated two humanized worm lines expressing human YBX3 or YBX1 at the cey-1 locus to test evolutionary conservation of YBXs in memory and the potential functional significance of the p.Asn127Tyr variant. Both YBX1/3 can functionally replace cey-1, and introduction of p.Asn127Tyr into the humanized YBX3 locus caused memory deficits. Our study highlights the worm as a model to reveal memory regulators and identifies YBX dysfunction as a potential new source of rare neurological disease.

Growth of Acinetobacter baumannii impacted by iron chelation.

Acinetobacter baumannii (AB) has become multidrug-resistant (MDR) in recent years, and, currently, there are limited effective treatment options. Nutrient metals (e.g. iron) are essential to the metabolic functions of AB. This study examined the impact of iron chelation on the growth of AB in vitro and in vivo. Susceptible and MDR-AB bloodstream isolates (n = 9) were recovered from different patients between 2011 and 2018. Clonal diversity was ascertained by Fourier-transform infrared spectroscopy. In vitro bacterial densities were measured over 20 h to determine growth profiles. Variable amounts of a chelating agent [deferiprone (DFP)] were added to create a concentration gradient. Galleria mellonella larvae were inoculated with an isolate, with and without DFP. Quantitative culture was used to ascertain the bacterial burden of aggregate larvae immediately and 4 h post-infection. Increasing concentrations of DFP caused a transient and concentration-dependent hindrance to in vitro growth, compared to the no-treatment group. In vivo bacterial burden immediately post-infection in both groups was comparable. After 4 h, the burden was much higher in the control group comparatively (8.7 and 6.7 log CFU g-1). These results support that micro-nutrient limitation has the potential of being a novel approach for treating high-risk infections due to MDR-AB.

Deciphering longitudinal optical-density measurements to guide clinical dosing regimen design: A model-based approach.

BACKGROUND: Model-based analysis of longitudinal optical density measurements from a bacterial suspension exposed to antibiotics has been proposed as a potentially efficient and effective method for extracting useful information to improve the individualized design of treatments for bacterial infections. To that end, the authors developed in previous work a mathematical modeling framework that can use such measurements for design of effective dosing regimens. OBJECTIVES: Here we further explore ways to extract information from longitudinal optical density measurements to predict bactericidal efficacy of clinically relevant antibiotic exposures. METHODS: Longitudinal optical density measurements were collected in an automated instrument where Acinetobacter baumannii, ATCC BAA747, was exposed to ceftazidime concentrations of 1, 4, 16, 64, and 256 mg/L and to ceftazidime/amikacin concentrations of 1/0.5, 4/2, 16/8, 64/32, and 256/128 (mg/L)/(mg/L) over 20 h. Calibrated conversion of measurements produced total (both live and dead) bacterial cell concentration data (CFU/mL equivalent) over time. Model-based data analysis predicted the bactericidal efficacy of ceftazidime and of ceftazidime/amikacin (at ratio 2:1) for periodic injection every 8 h and subsequent exponential decline with half-life of 2.5 h. Predictions were experimentally tested in an in vitro hollow-fiber infection model, using peak concentrations of 60 and 150 mg/L for injected ceftazidime and of 40/20 (mg/L)/(mg/L) for injected ceftazidime/amikacin. RESULTS: Model-based analysis predicted low (<62%) confidence in clinically relevant suppression of the bacterial population by periodic injections of ceftazidime alone, even at high peak concentrations. Conversely, analysis predicted high (>95%) confidence in bacterial suppression by periodic injections of ceftazidime/amikacin combinations at a wide range of peak concentrations ratioed at 2:1. Both predictions were experimentally confirmed in an in vitro hollow fiber infection model, where ceftazidime was periodically injected at peak concentrations 60 and 150 mg/L (with predicted suppression confidence 38% and 59%, respectively) and a combination of ceftazidime/amikacin was periodically injected at peak concentrations 40/20 (mg/L)/(mg/L) (with predicted suppression confidence 98%). CONCLUSIONS: The paper highlights the potential of clinicians using the proposed mathematical framework to determine the utility of different antibiotics to suppress a patient-specific isolate. Additional studies will be needed to consolidate and expand the utility of the proposed method.

Clinical and genomic analysis of virulence-related genes in bloodstream infections caused by Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a common cause of bloodstream infections, which is associated with high mortality and long periods of hospitalization. To advance the medical care of our patients, the study was designed to identify microbial characteristics associated with poor clinical outcomes. A collection of 32 A. baumannii bloodstream isolates with diverse genetic backgrounds (as determined by multilocus sequence typing) was studied. These isolates were recovered by unique patients (18 males, 14 females; age range: 17 days to 87 years) between 2011 and 2018. A sequential screening approach (cross-referencing analyses using different endpoints) was used to identify isolates with the best correlation between bacterial virulence and clinical prognosis. Isolates associated with more rapid in vitro growth rate, shorter median survival time in pre-clinical infection models, and hospital mortality were selected as candidates for high virulence, while those with opposite characteristics were selected as controls with low virulence. Whole genome sequencing was undertaken in the most promising clinical isolates. We found five virulence genes (beta-hemolysin/cytolysin, Cpi-1a + Cpi-1 (SPI-1 like), enhanced entry proteins, FbpABC, Paa) and 1 secretory system (T6SS) only present in a highly virulent isolate (AB23), compared to a low virulence control isolate (AB6). These genetic elements could be associated with the poor prognosis of A. baumannii bacteraemia and further investigations are warranted.

Optimal ceftazidime/avibactam dosing exposure against KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Infections due to carbapenem-resistant Enterobacterales are considered urgent public health threats and often treated with a ß-lactam/ß-lactamase inhibitor combination. However, clinical treatment failure and resistance emergence have been attributed to inadequate dosing. We used a novel framework to provide insights of optimal dosing exposure of ceftazidime/avibactam. METHODS: Seven clinical isolates of Klebsiella pneumoniae producing different KPC variants were examined. Ceftazidime susceptibility (MIC) was determined by broth dilution using escalating concentrations of avibactam. The observed MICs were characterized as response to avibactam concentrations using an inhibitory sigmoid Emax model. Using the best-fit parameter values, %fT>MICi was estimated for various dosing regimens of ceftazidime/avibactam. A hollow-fibre infection model (HFIM) was subsequently used to ascertain the effectiveness of selected regimens over 120 h. The drug exposure threshold associated with bacterial suppression was identified by recursive partitioning. RESULTS: In all scenarios, ceftazidime MIC reductions were well characterized with increasing avibactam concentrations. In HFIM, bacterial regrowth over time correlated with emergence of resistance. Overall, suppression of bacterial regrowth was associated with %fT>MICi ≥ 76.1% (100% versus 18.2%; P < 0.001). Using our framework, the optimal drug exposure could be achieved with ceftazidime/avibactam 2.5 g every 12 h in 5 out of 7 isolates. Furthermore, ceftazidime/avibactam 2.5 g every 8 h can suppress an isolate deemed resistant based on conventional susceptibility testing method. CONCLUSIONS: An optimal drug exposure to suppress KPC-producing bacteria was identified. The novel framework is informative and may be used to guide optimal dosing of other ß-lactam/ß-lactamase inhibitor combinations. Further in vivo investigations are warranted.

SIMULTANEOUS IN VITRO SIMULATION OF MULTIPLE ANTIMICROBIAL AGENTS WITH DIFFERENT ELIMINATION HALF-LIVES IN A PRE-CLINICAL INFECTION MODEL.

Combination therapy for treatment of multi-drug resistant bacterial infections is becoming common. In vitro testing of drug combinations under realistic pharmacokinetic conditions is needed before a corresponding combination is eventually put into clinical use. The current standard for design of such in vitro simulations for drugs with different half-lives is heuristic and limited to two drugs. To address that void, we develop a rigorous design method suitable for an arbitrary number of N drugs with different half-lives. The method developed offers substantial flexibility and produces novel designs even for two drugs. Explicit design equations are rigorously developed and are suitable for immediate use by experimenters. These equations were used in experimental verification using a combination of three antibiotics with distinctly different half-lives. In addition to antibiotics, the method is applicable to any anti-infective or anti-cancer drugs with distinct elimination pharmacokinetics.

Optimizing pharmacokinetics/pharmacodynamics of ß-lactam/ß-lactamase inhibitor combinations against high inocula of ESBL-producing bacteria.

OBJECTIVES: Reduced in vitro ß-lactam activity against a dense bacterial population is well recognized. It is commonly attributed to the presence of ß-lactamase(s) and it is unknown whether the inoculum effect could be diminished by a ß-lactamase inhibitor. We evaluated different ß-lactam/ß-lactamase inhibitor combinations in suppressing a high inoculum of ESBL-producing bacteria. METHODS: Three clinical isolates expressing representative ESBLs (CTX-M-15 and SHV-12) were examined. The impact of escalating ß-lactamase inhibitor (tazobactam or avibactam) concentrations on ß-lactam (piperacillin or ceftazidime) MIC reduction was characterized by an inhibitory sigmoid Emax model. The effect of various dosing regimens of ß-lactam/ß-lactamase inhibitor combinations was predicted using %T>MICi and selected exposures were experimentally validated in a hollow-fibre infection model over 120 h. The threshold exposure to suppress bacterial regrowth was identified using recursive partitioning. RESULTS: A concentration-dependent reduction in ß-lactam MIC was observed (r2 ≥0.93). Regrowth could be suppressed in all six experiments using %T>MICi ≥73.6%, but only one out of six experiments below the threshold (P = 0.015). The exposures to suppress regrowth might be attained using the clinical dose of avibactam, but a much higher dose than the standard dose would be needed for tazobactam. CONCLUSIONS: A dense population of ESBL-producing bacteria could be suppressed by an optimized dosing regimen of selected ß-lactam/ß-lactamase inhibitor combinations. The reversibility of enzyme inhibition could play an important role in diminishing the inoculum effect. In vivo investigations to validate these findings are warranted.

MIC profiling of ceftazidime/avibactam against two carbapenemase-producing Klebsiella pneumoniae isolates.

OBJECTIVES: The aim of this study was to correlate the results of a modified susceptibility testing method with outcomes of ceftazidime/avibactam (CAZ/AVI) therapy. METHODS: Two bloodstream K. pneumoniae isolates (CAZ/AVI-susceptible) from an abdominal source were recovered from two unrelated patients. Both patients were treated with CAZ/AVI but had discordant outcomes: KP118 (eradication within 24 h) and KP286 (persistent bacteraemia for over 30 days). Carbapenemase production in the two isolates was confirmed by Carba NP test. The CAZ minimum inhibitory concentration (MIC) was determined with escalating AVI concentrations (0-16 mg/L). The concentration-response was characterised by the sigmoid inhibitory maximum effect model. The best-fit parameter values were used to predict %T > MICi associated with CAZ/AVI exposures expected in peritoneal fluid after standard dosing (2.5 g every 8 h). These CAZ/AVI exposures were simulated in a hollow-fibre infection model (HFIM), and the bacterial responses were correlated with observed clinical outcomes. RESULTS: The AVI-dependent reduction in CAZ MIC was well characterised in both bacterial isolates (r2 ≥ 0.98). In the HFIM, sustained suppression of KP118 (T > MICi = 100%) was observed over 5 days, but not with KP286 (T > MICi < 100%). These observations are consistent with the clinical course of the patients. CONCLUSIONS: The discordant patient outcomes could be potentially explained by MIC profiling of CAZ/AVI. This method appears to be more robust than conventional susceptibility testing in predicting positive clinical outcome of CAZ/AVI therapy, and the clinical utility of this approach should be further investigated.

The impact of serum protein binding on bacterial killing of minocycline.

OBJECTIVES: Minocycline is increasingly used clinically for treating infections due to multidrug resistant bacteria. We previously reported that the serum protein binding of minocycline atypically correlated with total concentration using microdialysis, but the therapeutic implications of this finding remained unclear. The objective of this study was to ascertain the functional impact of serum protein binding on bacterial killing. METHODS: Time-kill experiments using 4 strains of Acinetobacter baumannii were conducted comparing the activity of minocycline in mouse serum (50 mg/L) and 50% cation-adjusted Mueller-Hinton broth (CA-MHB) (4 mg/L). As a control, similar experiments were also conducted for a clinically achievable levofloxacin concentration (4 mg/L) in serum and 50% CA-MHB (2 mg/L). Serial samples were collected in duplicate over 6 hours, and bacterial burden was determined by quantitative culture. RESULTS: Minocycline exhibited concentration-dependent bactericidal activity against the reference strain in mouse and human serum. Despite using approximately 10× the peak concentration associated with clinical dosing, only moderate bacterial killing was observed. All the minocycline killing profiles in serum were inferior to those observed in CA-MHB. In contrast, the reduction in bactericidal activity seen with levofloxacin was less dramatic. CONCLUSION: Antimicrobial activity of minocycline was dramatically reduced in the presence of serum, which corroborated with our atypical serum protein binding findings. If validated, these results implied dose escalation might not the best approach to improve the clinical efficacy of minocycline for bacteremia. Future investigations will focus on the specificity and mechanism(s) of minocycline protein binding.