Ironing out Persisters? Revisiting the Iron Chelation Strategy to Target Planktonic Bacterial Persisters Harboured in Carbapenem-Resistant Escherichia coli.

Antibiotic resistance is a global health crisis. Notably, carbapenem-resistant Enterobacterales (CRE) pose a significant clinical challenge due to the limited effective treatment options. This problem is exacerbated by persisters that develop upon antibiotic exposure. Bacteria persisters can tolerate high antibiotic doses and can cause recalcitrant infections, potentially developing further antibiotic resistance. Iron is a critical micronutrient for survival. We aimed to evaluate the utility of iron chelators, alone and in combination with antibiotics, in managing persisters. We hypothesized that iron chelators eradicate CRE persisters in vitro, when administered in combination with antibiotics. Our screening revealed three clinical isolates with bacteria persisters that resuscitated upon antibiotic removal. These isolates were treated with both meropenem and an iron chelator (deferoxamine mesylate, deferiprone or dexrazoxane) over 24 h. Against our hypothesis, bacteria persisters survived and resuscitated upon withdrawing both the antibiotic and iron chelator. Pursuing our aim, we next hypothesized that iron chelation is feasible as a post-antibiotic treatment in managing and suppressing persisters' resuscitation. We exposed bacteria persisters to an iron chelator without antibiotics. Flow cytometric assessments revealed that iron chelators are inconsistent in suppressing persister resuscitation. Collectively, these results suggest that the iron chelation strategy may not be useful as an antibiotic adjunct to target planktonic bacteria persisters.

The cost-effectiveness of a real-time seizure detection application for people with epilepsy.

OBJECTIVES: Automated seizure detection modalities can increase safety among people with epilepsy (PWE) and reduce seizure-related anxiety. We evaluated the potential cost-effectiveness of a seizure detection mobile application for PWE in Singapore. METHODS: We used a Markov cohort model to estimate the expected changes to total costs and health outcomes from a decision to adopt the seizure detection application versus the current standard of care from the health provider perspective. The time horizon is ten years and cycle duration is one month. Parameter values were updated from national databases and published literature. As we do not know the application efficacy in reducing seizure-related injuries, a conservative estimate of 1% reduction was used. Probabilistic sensitivity analysis, scenario analyses, and value of information analysis were performed. RESULTS: At a willingness-to-pay of $45,000/ quality-adjusted life-years (QALY), the incremental cost-effectiveness ratio was $1,096/QALY, and the incremental net monetary benefit was $13,656. Probabilistic sensitivity analyses reported that the application had a 99.5% chance of being cost-effective. In a scenario analysis in which the reduction in risk of seizure-related injury was 20%, there was a 99.8% chance that the application was cost-effective. Value of information analysis revealed that health utilities was the most important parameter group contributing to model uncertainty. CONCLUSIONS: This early-stage modeling study reveals that the seizure detection application is likely to be cost-effective compared to current standard of care. Future prospective trials will be needed to demonstrate the real-world impact of the application. Changes in health-related quality of life should also be measured in future trials.

A decision-support tool for funding health innovations at a tertiary academic medical center.

OBJECTIVES: To report the processes used to design and implement an assessment tool to inform funding decisions for competing health innovations in a tertiary hospital. METHODS: We designed an assessment tool for health innovation proposals with three components: "value to the institution," "novelty," and "potential for adoption and scaling." The "value to the institution" component consisted of twelve weighted value attributes identified from the host institution's annual report; weights were allocated based on a survey of the hospital's leaders. The second and third components consisted of open-ended questions on "novelty" and "barriers to implementation" to support further dialogue. Purposive literature review was performed independently by two researchers for each assessment. The assessment tool was piloted during an institutional health innovation funding cycle. RESULTS: We used 17 days to evaluate ten proposals. The completed assessments were shared with an independent group of panellists, who selected five projects for funding. Proposals with the lowest scores for "value to the institution" had less perceived impact on the patient-related value attributes of "access," "patient centeredness," "health outcomes," "prevention," and "safety." Similar innovations were reported in literature in seven proposals; potential barriers to implementation were identified in six proposals. We included a worked example to illustrate the assessment process. CONCLUSIONS: We developed an assessment tool that is aligned with local institutional priorities. Our tool can augment the decision-making process when funding health innovation projects. The tool can be adapted by others facing similar challenges of trying to choose the best health innovations to fund.

Neuropilin-2 Signaling Modulates Mossy Fiber Sprouting by Regulating Axon Collateral Formation Through CRMP2 in a Rat Model of Epilepsy.

Programmed neural circuit formation constitutes the foundation for normal brain functions. Axon guidance cues play crucial roles in neural circuit establishment during development. Whether or how they contribute to maintaining the stability of networks in mature brains is seldom studied. Upon injury, neural rewiring could happen in adulthood, of which mossy fiber sprouting (MFS) is a canonical example. Here, we uncovered a novel role of axon guidance molecule family Sema3F/Npn-2 signaling in MFS and epileptogenesis in a rat model of epilepsy. Dentate gyrus-specific Npn-2 knockdown increased seizure activity in epileptic animals along with increased MFS. Hippocampal culture results suggested that Npn-2 signaling modulates MFS via regulating axon outgrowth and collateral formation. In addition, we discovered that Sema3F/Npn-2 signal through CRMP2 by regulating its phosphorylation in the process of MFS. Our work illustrated that Npn-2 signaling in adult epilepsy animals could potentially modulate seizure activity by controlling MFS. MFS constitutes the structural basis for abnormal electric discharge of neurons and recurrent seizures. Therapies targeting Npn-2 signaling could potentially have disease-modifying anti-epileptogenesis effects in epilepsy treatment.

Conditional knockout of ASK1 in microglia/macrophages attenuates epileptic seizures and long-term neurobehavioural comorbidities by modulating the inflammatory responses of microglia/macrophages.

BACKGROUND: Apoptosis signal-regulating kinase 1 (ASK1) not only causes neuronal programmed cell death via the mitochondrial pathway but also is an essential component of the signalling cascade during microglial activation. We hypothesize that ASK1 selective deletion modulates inflammatory responses in microglia/macrophages(Mi/Mϕ) and attenuates seizure severity and long-term cognitive impairments in an epileptic mouse model. METHODS: Mi/Mϕ-specific ASK1 conditional knockout (ASK1 cKO) mice were obtained for experiments by mating ASK1flox/flox mice with CX3CR1creER mice with tamoxifen induction. Epileptic seizures were induced by intrahippocampal injection of kainic acid (KA). ASK1 expression and distribution were detected by western blotting and immunofluorescence staining. Seizures were monitored for 24 h per day with video recordings. Cognition, social and stress related activities were assessed with the Y maze test and the three-chamber social novelty preference test. The heterogeneous Mi/Mϕ status and inflammatory profiles were assessed with immunofluorescence staining and real-time polymerase chain reaction (q-PCR). Immunofluorescence staining was used to detect the proportion of Mi/Mϕ in contact with apoptotic neurons, as well as neuronal damage. RESULTS: ASK1 was highly expressed in Mi/Mϕ during the acute phase of epilepsy. Conditional knockout of ASK1 in Mi/Mϕ markedly reduced the frequency of seizures in the acute phase and the frequency of spontaneous recurrent seizures (SRSs) in the chronic phase. In addition, ASK1 conditional knockout mice displayed long-term neurobehavioral improvements during the Y maze test and the three-chamber social novelty preference test. ASK1 selective knockout mitigated neuroinflammation, as evidenced by lower levels of Iba1+/CD16+ proinflammatory Mi/Mϕ. Conditional knockout of ASK1 increased Mi/Mϕ proportion in contact with apoptotic neurons. Neuronal loss was partially restored by ASK1 selective knockout. CONCLUSION: Conditional knockout of ASK1 in Mi/Mϕ reduced seizure severity, neurobehavioral impairments, and histological damage, at least via inhibiting proinflammatory microglia/macrophages responses. ASK1 in microglia/macrophages is a potential therapeutic target for inflammatory responses in epilepsy.

Cost effectiveness of temporary isolation rooms in acute care settings in Singapore.

OBJECTIVES: To estimate the change to health service costs and health benefits from a decision to adopt temporary isolation rooms that are effective at isolating the patient within a general ward environment. We assess the cost-effectiveness of a decision to adopt an existing temporary isolation room in a Singapore setting. METHOD: We performed a model-based cost-effectiveness analysis to evaluate the impact of a decision to adopt temporary isolation rooms for infection prevention. We estimated changes to the costs from implementation, the number of cases of healthcare associated infection, acute care bed days used, they money value of bed days, the number of deaths, and the expected change to life years. We report the probability that adoption was cost-effective by the cost by life year gained, against a relevant threshold. Uncertainty is addressed with probabilistic sensitivity analysis and the findings are tested with plausible scenarios for the effectiveness of the intervention. RESULTS: We predict 478 fewer cases of HAI per 100,000 occupied bed days from a decision to adopt temporary isolation rooms. This will result in cost savings of $SGD329,432 and there are 1,754 life years gained. When the effectiveness of the intervention is set at 1% of cases of HAI prevented the incremental cost per life year saved is $16,519; below the threshold chosen for cost-effectiveness in Singapore. CONCLUSIONS: We provide some evidence that adoption of a temporary isolation room is cost-effective for Singapore acute care hospitals. It is plausible that adoption is a positive decision for other countries in the region who may demonstrate fewer resources for infection prevention and control.

Whole genome sequencing reveals hidden transmission of carbapenemase-producing Enterobacterales.

Carbapenemase-producing Enterobacterales (CPE) infection control practices are based on the paradigm that detected carriers in the hospital transmit to other patients who stay in the same ward. The role of plasmid-mediated transmission at population level remains largely unknown. In this retrospective cohort study over 4.7 years involving all multi-disciplinary public hospitals in Singapore, we analysed 779 patients who acquired CPE (1215 CPE isolates) detected by clinical or surveillance cultures. 42.0% met putative clonal transmission criteria, 44.8% met putative plasmid-mediated transmission criteria and 13.2% were unlinked. Only putative clonal transmissions associated with direct ward contact decreased in the second half of the study. Both putative clonal and plasmid-mediated transmission associated with indirect (no temporal overlap in patients' admission period) ward and hospital contact did not decrease during the study period. Indirect ward and hospital contact were identified as independent risk factors associated with clonal transmission. In conclusion, undetected CPE reservoirs continue to evade hospital infection prevention measures. New measures are needed to address plasmid-mediated transmission, which accounted for 50% of CPE dissemination.

Predictors and Outcomes of Healthcare-Associated Infections Caused by Carbapenem-Nonsusceptible Enterobacterales: A Parallel Matched Case-Control Study.

Objectives: The increasing incidence of carbapenem-nonsusceptible Enterobacterales as major pathogens in healthcare associated infections (HAIs) is of paramount concern. To implement effective prevention strategies against carbapenem-nonsusceptible Enterobacterales (CnSE) HAIs, it is crucial to identify modifiable factors associated with these infections. We identified risk factors for CnSE-HAIs, and compared clinical outcomes of CnSE-HAI and carbapenem-sensitive Enterobacterales (CSE)-HAI patients. Methods: We conducted a multi-centre parallel matched case-control study in two 1700-bedded Singapore acute-care hospitals from 2014-2016. Patients with CnSE-HAIs and CSE-HAIs were compared to a common control group without HAIs (1:1:3 ratio), matched by time-at-risk and patient ward. Carbapenem nonsusceptible was defined as non-susceptibility to either meropenem or imipenem. Presence of healthcare associated infections were defined by the criteria provided by the European Centre for Disease Prevention and Control. Outcomes of CnSE-HAI and CSE-HAI patients were compared using multivariable logistic and cox regression; the models were adjusted for infection and treatment characteristics. Results: Eighty CnSE-HAI and 80 CSE-HAI patients were matched to 240 patients without HAIs. All CRE-HAIs patients had prior antibiotic exposure, with 44 (55.0%) with prior carbapenem exposure. The most common CnSE-HAIs were intra-abdominal infections (28.8%) and pneumonia (23.8%). The most common CnSE species was Klebsiella spp. (63.8%). In the risk factor analysis, presence of drainage devices [adjusted odds ratio (aOR), 2.19; 95% CI, 1.29 - 3.70] and prior carbapenem exposure (aOR,17.09; 95% CI, 3.06 - 95.43) independently predicted CnSE-HAIs. In the crude outcomes analysis, CnSE-HAI patients had higher all-cause in-hospital mortality and longer time to discharge compared to CSE-HAI patients. After adjusting for differences in receipt of antibiotics with reported susceptibility to the Enterobacterales, there was no significant difference in all-cause in-hospital mortality between the two groups (aOR, 1.76; 95% CI, 0.86-3.58). Time to discharge remained significantly longer in patients with CnSE-HAI (adjusted hazard ratio, 0.71; 95% CI, 0.51 - 0.98) after adjusting for disease severity, receipt of antibiotics with reported susceptibility and receipt of appropriate source control. Conclusion: Appropriate management of deep-seated Enterobacterales infections and reducing exposure to carbapenems may reduce risk of CnSE-HAIs in Singapore. Efforts to improve antimicrobial therapy in CnSE-HAI patients may improve patient outcomes.

Estimating the excess bed days and economic burden of healthcare-associated infections in Singapore public acute-care hospitals.

We estimated the annual bed days lost and economic burden of healthcare-associated infections to Singapore hospitals using Monte Carlo simulation. The mean (standard deviation) cost of a single healthcare-associated infection was S$1,809 (S$440) [or US$1,362 (US$331)]. This translated to annual lost bed days and economic burden of 55,978 (20,506) days and S$152.0 million (S$37.1 million) [or US$114.4 million (US$27.9 million)], respectively.

Stepwise membrane binding of extended synaptotagmins revealed by optical tweezers.

Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca2+- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER-PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca2+- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides.

Estimating the economic cost of carbapenem resistant Enterobacterales healthcare associated infections in Singapore acute-care hospitals.

Quantifying the costs of hospital associated infections (HAIs) caused by carbapenem-resistant Enterobacterales (CRE) can aid hospital decision makers in infection prevention and control decisions. We estimate the costs of a CRE HAI by infection type and the annual costs of CRE HAIs to acute-care hospitals in Singapore. We used tree diagrams to estimate the costs (in Singapore dollar) of different CRE HAI types from the health service perspective and compared them to the costs of carbapenem-susceptible HAIs. We used two approaches to estimate costs-direct costs of consumables for infection prevention and treatment; and costs associated with lost bed days. Cost of a HAI were extrapolated to annual CRE HAI incidence in Singapore acute-care hospitals to estimate the annual cost to the hospitals. We found that the cost of a CRE HAI based on direct cost and lost bed days are SGD$9,913 (95% CI, SGD$9,431-10,395) and SGD$10,044 (95% CI, SGD$9,789-10,300) respectively. CRE HAIs are markedly higher than the carbapenem-susceptible HAIs for all infection types. In both approaches, CRE pneumonia was the costliest infection. Based on a CRE HAI incidence of 233 per 100,000 inpatient admissions, CRE HAIs costed SGD$12.16M (95% CI, SGD$11.84-12.48M) annually based on direct costs, and SGD$12.33M (95% CI, SGD$12.01-12.64M) annually based on lost bed days. In conclusion, we described the cost of CRE HAIs in Singapore hospitals and identified infections with the highest costs. The findings may be useful in informing future economic evaluations of competing CRE HAI prevention and treatment programmes.

In vitro Bactericidal Activities of Combination Antibiotic Therapies Against Carbapenem-Resistant Klebsiella pneumoniae With Different Carbapenemases and Sequence Types.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is becoming increasingly problematic due to the limited effectiveness of new antimicrobials or other factors such as treatment cost. Thus, combination therapy remains a suitable treatment option. We aimed to evaluate the in vitro bactericidal activity of various antibiotic combinations against CRKP with different carbapenemase genotypes and sequence types (STs). Thirty-seven CRKP with various STs and carbapenemases were exposed to 11 antibiotic combinations (polymyxin B or tigecycline in combination with ß-lactams including aztreonam, cefepime, piperacillin/tazobactam, doripenem, meropenem, and polymyxin B with tigecycline) in static time-kill studies (TKS) using clinically achievable concentrations. Out of the 407 isolate-combination pairs, only 146 (35.8%) were bactericidal (≥3 log10CFU/mL decrease from initial inoculum). Polymyxin B in combination with doripenem, meropenem, or cefepime was the most active, each demonstrating bactericidal activity in 27, 24, and 24 out of 37 isolates, respectively. Tigecycline in combination with ß-lactams was rarely bactericidal. Aside from the lower frequency of bactericidal activity in the dual-carbapenemase producers, there was no apparent difference in combination activity among the strains with other carbapenemase types. In addition, bactericidal combinations were varied even in strains with similar STs, carbapenemases, and other genomic characteristics. Our findings demonstrate that the bactericidal activity of antibiotic combinations is highly strain-specific likely owing to the complex interplay of carbapenem-resistance mechanisms, i.e., carbapenemase genotype alone cannot predict in vitro bactericidal activity. The availability of WGS information can help rationalize the activity of certain combinations. Further studies should explore the use of genomic markers with phenotypic information to predict combination activity.

Biallelic PI4KA variants cause neurological, intestinal and immunological disease.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα/PI4KA/OMIM:600286) is a lipid kinase generating phosphatidylinositol 4-phosphate (PI4P), a membrane phospholipid with critical roles in the physiology of multiple cell types. PI4KIIIα's role in PI4P generation requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. Here we show that biallelic PI4KA sequence alterations in humans are associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, affected individuals may present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Our cellular, biochemical and structural modelling studies indicate that PI4KA-associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

Inhibition of Gasdermin D-Mediated Pyroptosis Attenuates the Severity of Seizures and Astroglial Damage in Kainic Acid-Induced Epileptic Mice.

Objective: Our study aimed to explore whether gasdermin D (GSDMD)-mediated pyroptosis is involved in the mechanism of kainic acid-induced seizures. Methods: C57BL/6 mice were randomly divided into sham and epilepsy groups. The epilepsy group was intrahippocampally injected with kainic acid to induce status epilepticus (SE), and the sham group was injected with an equal volume of saline. Dimethyl fumarate (DMF) was used as the GSDMD N-terminal fragments (GSDMD-N) inhibitor and suspended in 0.5% sodium carboxymethyl cellulose (CMC) for orally administration. The epilepsy group was divided into SE + CMC and SE + DMF groups. In the SE + DMF group, DMF was orally administered for 1 week before SE induction and was continued until the end of the experiment. An equal volume of CMC was administered to the sham and SE + CMC groups. Recurrent spontaneous seizures (SRSs) were monitored for 21 days after SE. Western blot analysis and immunofluorescent staining was performed. Results: The expression of GSDMD increased at 7-21 days post-SE, and GSDMD-N expression was significantly elevated 7 days after SE in both ipsilateral and contralateral hippocampus. GSDMD-positive cells were co-labeled with astrocytes, but not neurons or microglia. Astroglial damage occurs following status epilepticus (SE). Damaged astrocytes showed typical clasmatodendrosis in the CA1 region containing strong GSDMD expression at 7-21 days post-SE, accompanied by activated microglia. In the SE + DMF group, the expression of GSDMD-N was significantly inhibited compared to that in the SE + CMC group. After administration of DMF, SRSs at 7-21 days after SE were significantly decreased, and the number of clasmatodendritic astrocytes, microglia, and the expression of inflammatory factors such as IL-1ß and IL-18 were significantly attenuated. Conclusion: GSDMD-mediated pyroptosis is involved in the mechanism of kainic acid-induced seizures. Our study provides a new potential therapeutic target for seizure control.

GABAergic Interneuron and Neurotransmission Are mTOR-Dependently Disturbed in Experimental Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) is a major cause for drug-resistant epilepsies. The molecular and cellular mechanisms of epileptogenesis in FCD are still poorly understood. Some studies have suggested that deficiencies of γ-aminobutyric acid (GABA) system may play an important role in type II FCD, but it remains controversial. In order to examine whether and how GABAergic interneurons and synaptic function are affected, we generated a somatic mTOR hyperactivation-based mouse model of type II FCD by in utero electroporation, quantified densities of interneurons in the malformed cortices, and recorded miniature inhibitory postsynaptic currents in dysmorphic neurons. We detected 20-25% reduction of GABAergic interneurons within malformed cortices, independent of cortical regions and cell subtypes but proportionate to the decrease of global neuron counts. GABAergic synaptic transmission from interneurons to mTOR hyperactivated dysmorphic neurons was dramatically disrupted, outweighing the decrease of interneuron counts. Postnatal mTOR inhibition partially rescued these alterations of GABAergic system. We also quantified the expression of GABAA receptor, GABA transporter, and chloridion transporter encoding genes and found that their expression was relatively intact within the malformed cortices. Taken together, these results confirmed that GABAergic interneuron and synapse transmission are disturbed profoundly in an mTOR-dependent manner in type II FCD. Our study suggests that postsynaptic mechanisms independent of interneuron reduction or altered expression of GABA synapse genes might be accountable for the impaired GABAergic neurotransmission in type II FCD as well as other mTOR-related epilepsies.

Treatment and Outcomes of Infections Caused by Diverse Carbapenemase-Producing Carbapenem-Resistant Enterobacterales.

Background: Diverse sequence types (ST) and various carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) infections, which complicate treatment strategies, have emerged in Singapore. We aim to describe these CP-CRE infections and clinical outcomes according to their carbapenemase types and determine the hierarchy of predictors for mortality that are translatable to clinical practice. Methods: Clinically significant CP-CRE infections were identified in Singapore General Hospital between 2013 and 2016. Retrospectively, all clinically relevant data were retrieved from electronic medical records from the hospital. Univariate analysis was performed. To further explore the relationship between the variables and mortality in different subsets of patients with CP-CRE, we conducted recursive partitioning analysis on all study variables using the "rpart" package in R. Results: One hundred and fifty five patients were included in the study. Among them, 169 unique CP-CRE were isolated. Thirty-day all-cause in-hospital mortality was 35.5% (n = 55). There was no difference in the severity of illness, or any clinical outcomes exhibited by patients between the various carbapenemases. Root node began with patients with Acute Physical and Chronic Health Evaluation (APACHEII) score ≥ 15 (n = 98; mortality risk = 52.0%) and <15 (n = 57; mortality risk = 9.0%). Patients with APACHEII score ≥ 15 are further classified based on presence (n = 27; mortality risk = 23.0%) and absence (n = 71, mortality risk = 62.0%) of bacterial eradication. Without bacterial eradication, absence (n = 54) and presence (n = 17) of active source control yielded 70.0 and 35.0% mortality risk, respectively. Without active source control, the mortality risk was higher for the patients with non-receipt of definite combination therapy (n = 36, mortality risk = 83.0%) when compared to those who received (n = 18, mortality risk = 47.0%). Overall, the classification tree has an area under receiver operating characteristic curve of 0.92, with a sensitivity of 0.87 and specificity of 0.91. Conclusion: Different mortality risks were observed with different treatment strategies. Effective source control and microbial eradication were associated with a lower mortality rate but not active empiric therapy for CP-CRE infection. When source control was impossible, definitive antibiotic combination appeared to be associated with a reduction in mortality.

Elimination of Extracellular Adenosine Triphosphate for the Rapid Prediction of Quantitative Plate Counts in 24 h Time-Kill Studies against Carbapenem-Resistant Gram-Negative Bacteria.

Traditional in vitro time-kill studies (TKSs) require viable plating, which is tedious and time-consuming. We used ATP bioluminescence, with the removal of extracellular ATP (EC-ATP), as a surrogate for viable plating in TKSs against carbapenem-resistant Gram-negative bacteria (CR-GNB). Twenty-four-hour TKSs were conducted using eight clinical CR-GNB (two Escherichia coli, two Klebsiella spp., two Acinetobacter baumannii, two Pseudomonas aeruginosa) with multiple single and two-antibiotic combinations. ATP bioluminescence and viable counts were determined at each timepoint (0, 2, 4, 8, 24 h), with and without apyrase treatment. Correlation between ATP bioluminescence and viable counts was determined for apyrase-treated and non-apyrase-treated samples. Receiver operator characteristic curves were plotted to determine the optimal luminescence threshold to discriminate between inhibitory/non-inhibitory and bactericidal/non-bactericidal combinations, compared to viable counts. After treatment of bacteria with 2 U/mL apyrase for 15 min at 37 °C, correlation to viable counts was significantly higher compared to untreated samples (p < 0.01). Predictive accuracies of ATP bioluminescence were also significantly higher for apyrase-treated samples in distinguishing inhibitory (p < 0.01) and bactericidal (p = 0.03) combinations against CR-GNB compared to untreated samples, when all species were collectively analyzed. We found that ATP bioluminescence can potentially replace viable plating in TKS. Our assay also has applications in in vitro and in vivo infection models.

Clinical Experience with High-Dose Polymyxin B against Carbapenem-Resistant Gram-Negative Bacterial Infections-A Cohort Study.

Population pharmacokinetic studies have suggested that high polymyxin B (PMB) doses (≥30,000 IU/kg/day) can improve bacterial kill in carbapenem-resistant Gram-negative bacteria (CR-GNB). We aim to describe the efficacy and nephrotoxicity of patients with CR-GNB infections prescribed high-dose PMB. A single-centre cohort study was conducted from 2013 to 2016 on septic patients with CR-GNB infection and prescribed high-dose PMB (~30,000 IU/kg/day) for ≥72 h. Study outcomes included 30-day mortality and acute kidney injury (AKI) development. Factors associated with AKI were identified using multivariable regression. Forty-three patients with 58 CR-GNB received high-dose PMB; 57/58 (98.3%) CR-GNB were susceptible to PMB. The median daily dose and duration of high-dose PMB were 32,051 IU/kg/day (IQR, 29,340-34,884 IU/kg/day) and 14 days (IQR, 7-28 days), respectively. Thirty-day mortality was observed in 7 (16.3%) patients. AKI was observed in 25 (58.1%) patients with a median onset of 8 days (IQR, 6-13 days). Higher daily PMB dose (aOR,1.01; 95% CI, 1.00-1.02) and higher number of concurrent nephrotoxins (aOR, 2.14; 95% CI; 1.03-4.45) were independently associated with AKI. We observed that a sizable proportion developed AKI in CR-GNB patients described high-dose PMB; hence, the potential benefits must be weighed against increased AKI risk. Concurrent nephrotoxins should be avoided to reduce nephrotoxicity.

The impact of healthcare associated infections on mortality and length of stay in Singapore-A time-varying analysis.

OBJECTIVE: Methods that include the time-varying nature of healthcare-associated infections (HAIs) avoid biases when estimating increased risk of death and excess length of stay. We determined the excess mortality risk and length of stay associated with HAIs among inpatients in Singapore using a multistate model that accommodates the timing of key events. DESIGN: Analysis of existing prospective cohort study data. SETTING: Seven public acute-care hospitals in Singapore. PATIENTS: Inpatients reviewed in a HAI point-prevalence survey (PPS) conducted between June 2015 and February 2016. METHODS: We modeled each patient's admission over time using 4 states: susceptible with no HAI, infected, died, and discharged alive. We estimated the excess mortality risk and length of stay associated with HAIs, with adjustment for the baseline characteristics between the groups for mortality risk. RESULTS: We included 4,428 patients, of whom 469 had ≥1 HAI. Using a multistate model, the expected excess length of stay due to any HAI was 1.68 days (95% confidence interval [CI], 1.15-2.21 days). Surgical site infections were associated with the longest excess length of stay of 4.68 days (95% CI, 2.60-6.76 days). After adjusting for baseline differences, HAIs were associated with increased hazards of in-hospital mortality (adjusted hazard ratio [aHR], 1.32; 95% CI, 1.09-1.65) and decreased hazards in being discharged (aHR, 0.75; 95% CI, 0.67-0.84). CONCLUSIONS: HAIs are associated with increased length of hospital stay and mortality in hospitalized patients. Avoiding nosocomial infections can improve patient outcomes and free valuable bed days.

In Vitro Pharmacodynamics of Fosfomycin against Carbapenem-Resistant Enterobacter cloacae and Klebsiella aerogenes.

The increase of carbapenem-resistant Enterobacterales (CRE) and lack of therapeutic options due to the scarcity of new antibiotics has sparked interest toward the use of intravenous fosfomycin against systemic CRE infections. We aimed to investigate the in vitro pharmacodynamics of fosfomycin against carbapenem-resistant Enterobacter cloacae and Klebsiella aerogenes Time-kill studies and population analysis profiles were performed with eight clinical CRE isolates, which were exposed to fosfomycin concentrations ranging from 0.25 to 2,048 mg/liter. The 24-h mean killing effect was characterized by an inhibitory sigmoid maximum effect (Emax) model. Whole-genome sequencing was performed to elucidate known fosfomycin resistance mechanisms. Fosfomycin MICs ranged from 0.5 to 64 mg/liter. The isolates harbored a variety of carbapenemase genes including blaIMP, blaKPC, and blaNDM Five out of eight isolates harbored the fosA gene, while none harbored the recently discovered fosL-like gene. Heteroresistant subpopulations were detected in all isolates, with two out of eight isolates harboring heteroresistant subpopulations at up to 2,048 mg/liter. In time-kill studies, fosfomycin exhibited bactericidal activity at 2 to 4 h at several fosfomycin concentrations (one isolate at ≥16 mg/liter, two at ≥32 mg/liter, two at ≥64 mg/liter, two at ≥128 mg/liter, and one at ≥512 mg/liter). At 24 h, bactericidal activity was only observed in two isolates (MICs, 0.5 and 4 mg/liter) at 2,048 mg/liter. From the Emax model, no significant bacterial killing was observed beyond 500 mg/liter. Our findings suggest that the use of fosfomycin monotherapy may be limited against CRE due to heteroresistance and rapid bacterial regrowth. Further optimization of intravenous fosfomycin dosing regimens is required to increase efficacy against such infections.