Acinetobacter baumannii: Envelope Determinants That Control Drug Resistance, Virulence, and Surface Variability.

Acinetobacter baumannii has emerged as an important nosocomial pathogen, particularly for patients in intensive care units and with invasive indwelling devices. The most recent clinical isolates are resistant to several classes of clinically important antibiotics, greatly restricting the ability to effectively treat critically ill patients. The bacterial envelope is an important driver of A. baumannii disease, both at the level of battling against antibiotic therapy and at the level of protecting from host innate immune function. This review provides a comprehensive overview of key features of the envelope that interface with both the host and antimicrobial therapies. Carbohydrate structures that contribute to protecting from the host are detailed, and mutations that alter these structures, resulting in increased antimicrobial resistance, are explored. In addition, protein complexes involved in both intermicrobial and host-microbe interactions are described. Finally we discuss regulatory mechanisms that control the nature of the cell envelope and its impact on host innate immune function.

Preclinical safety and biodistribution of CRISPR targeting SIV in non-human primates.

In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 1012, 1.4 × 1013 and 1.4 × 1014 genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.

Enterococcus faecalis Strains with Compromised CRISPR-Cas Defense Emerge under Antibiotic Selection for a CRISPR-Targeted Plasmid.

Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Non-MDR E. faecalis strains frequently possess CRISPR-Cas systems, which reduce the frequency of MGE acquisition. We demonstrated in previous studies that E. faecalis populations can transiently maintain both a functional CRISPR-Cas system and a CRISPR-Cas target. In this study, we used serial passage and deep sequencing to analyze these populations. In the presence of antibiotic selection for the plasmid, mutants with compromised CRISPR-Cas defense and enhanced ability to acquire a second antibiotic resistance plasmid emerged. Conversely, in the absence of selection, the plasmid was lost from wild-type E. faecalis populations but not E. faecalis populations that lacked the cas9 gene. Our results indicate that E. faecalis CRISPR-Cas can become compromised under antibiotic selection, generating populations with enhanced abilities to undergo horizontal gene transfer. IMPORTANCE Enterococcus faecalis is a leading cause of hospital-acquired infections and disseminator of antibiotic resistance plasmids among Gram-positive bacteria. We have previously shown that E. faecalis strains with an active CRISPR-Cas system can prevent plasmid acquisition and thus limit the transmission of antibiotic resistance determinants. However, CRISPR-Cas is not a perfect barrier. In this study, we observed populations of E. faecalis with transient coexistence of CRISPR-Cas and one of its plasmid targets. Our experimental data demonstrate that antibiotic selection results in compromised E. faecalis CRISPR-Cas function, thereby facilitating the acquisition of additional resistance plasmids by E. faecalis.

5' Untranslated mRNA Regions Allow Bypass of Host Cell Translation Inhibition by Legionella pneumophila.

Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a 2-h time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low-abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling data sets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5' untranslated regions (5' UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5' UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.

A Quantitative Metal-Encoded Conjugate Platform for Targeting Ligand Discovery.

The indiscriminate biodistribution of therapeutics can be a key barrier to their safety and efficacy. Localization of compounds into non-diseased tissues often leads to both toxic and dose-limiting effects. To overcome this barrier, nanomedicine implements targeting agents to localize or selectively uptake drugs at disease sites. However, to date there are only a small number of targeting agents with limited scope for targeting tissues. Small-molecule ligands are particularly attractive as targeting agents due to their relatively low cost, tunability, and ease of conjugation. Currently, there are no systematic approaches to the discovery of new small-molecule targeting ligands. Here, we developed a quantitative metal-encoded conjugate platform to determine the biodistribution of multiple small molecules in vivo. By utilizing lanthanide metal complexes, this platform successfully distinguished known ligands with differential tissue targeting in vivo. This system will facilitate the discovery of small molecules as targeting ligands and can accelerate the identification of novel biological targets for tissue-targeted drug delivery.

Chromosomal Resistance to Metronidazole in Clostridioides difficile Can Be Mediated by Epistasis between Iron Homeostasis and Oxidoreductases.

Chromosomal resistance to metronidazole has emerged in clinical Clostridioides difficile isolates, but the genetic mechanisms remain unclear. This is further hindered by the inability to generate spontaneous metronidazole-resistant mutants in the lab to interpret genetic variations in clinical isolates. We therefore constructed a mismatch repair mutator in nontoxigenic ATCC 700057 to survey the mutational landscape for de novo resistance mechanisms. In separate experimental evolutions, the mutator adopted a deterministic path to resistance, with truncation of the ferrous iron transporter FeoB1 as a first-step mechanism of low-level resistance. Deletion of feoB1 in ATCC 700057 reduced the intracellular iron content, appearing to shift cells toward flavodoxin-mediated oxidoreductase reactions, which are less favorable for metronidazole's cellular action. Higher-level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin/flavodoxin oxidoreductase (PFOR; encoded by nifJ), a synonymous codon change to putative xdh (xanthine dehydrogenase; encoded by CD630_31770), likely affecting mRNA stability, and last, frameshift and point mutations that inactivated the iron-sulfur cluster regulator (IscR). Gene silencing of nifJ, xdh, or iscR with catalytically dead Cas9 revealed that resistance involving these genes occurred only when feoB1 was inactivated; i.e., resistance was seen only in the feoB1 deletion mutant and not in the isogenic wild-type (WT) parent. Interestingly, metronidazole resistance in C. difficile infection (CDI)-associated strains carrying mutations in nifJ was reduced upon gene complementation. This observation supports the idea that mutation in PFOR is one mechanism of metronidazole resistance in clinical strains. Our findings indicate that metronidazole resistance in C. difficile is complex, involving multigenetic mechanisms that could intersect with iron-dependent and oxidoreductive metabolic pathways.

MTGIpick allows robust identification of genomic islands from a single genome.

Genomic islands (GIs) that are associated with microbial adaptations and carry sequence patterns different from that of the host are sporadically distributed among closely related species. This bias can dominate the signal of interest in GI detection. However, variations still exist among the segments of the host, although no uniform standard exists regarding the best methods of discriminating GIs from the rest of the genome in terms of compositional bias. In the present work, we proposed a robust software, MTGIpick, which used regions with pattern bias showing multiscale difference levels to identify GIs from the host. MTGIpick can identify GIs from a single genome without annotated information of genomes or prior knowledge from other data sets. When real biological data were used, MTGIpick demonstrated better performance than existing methods, as well as revealed potential GIs with accurate sizes missed by existing methods because of a uniform standard. Software and supplementary are freely available at http://bioinfo.zstu.edu.cn/MTGI or https://github.com/bioinfo0706/MTGIpick.

A Type I Restriction-Modification System Associated with Enterococcus faecium Subspecies Separation.

The gastrointestinal colonizer Enterococcus faecium is a leading cause of hospital-acquired infections. Multidrug-resistant (MDR) E. faecium isolates are particularly concerning for infection treatment. Previous comparative genomic studies revealed that subspecies referred to as clade A and clade B exist within E. faecium MDR E. faecium isolates belong to clade A, while clade B consists of drug-susceptible fecal commensal E. faecium isolates. Isolates from clade A are further grouped into two subclades, clades A1 and A2. In general, clade A1 isolates are hospital-epidemic isolates, whereas clade A2 isolates are isolates from animals and sporadic human infections. Such phylogenetic separation indicates that reduced gene exchange occurs between the clades. We hypothesize that endogenous barriers to gene exchange exist between E. faecium clades. Restriction-modification (R-M) systems are such barriers in other microbes. We utilized a bioinformatics analysis coupled with second-generation and third-generation deep-sequencing platforms to characterize the methylomes of two representative E. faecium strains, one from clade A1 and one from clade B. We identified a type I R-M system that is clade A1 specific, is active for DNA methylation, and significantly reduces the transformability of clade A1 E. faecium Based on our results, we conclude that R-M systems act as barriers to horizontal gene exchange in E. faecium and propose that R-M systems contribute to E. faecium subspecies separation.IMPORTANCEEnterococcus faecium is a leading cause of hospital-acquired infections around the world. Rising antibiotic resistance in certain E. faecium lineages leaves fewer treatment options. The overarching aim of this work was to determine whether restriction-modification (R-M) systems contribute to the structure of the E. faecium species, wherein hospital-epidemic and non-hospital-epidemic isolates have distinct evolutionary histories and highly resolved clade structures. R-M provides bacteria with a type of innate immunity to horizontal gene transfer (HGT). We identified a type I R-M system that is enriched in the hospital-epidemic clade and determined that it is active for DNA modification activity and significantly impacts HGT. Overall, this work is important because it provides a mechanism for the observed clade structure of E. faecium as well as a mechanism for facilitated gene exchange among hospital-epidemic E. faecium isolates.

Perioperative activation of spinal α7 nAChR promotes recovery from preoperative stress-induced prolongation of postsurgical pain.

Preoperative stress could delay the recovery of postoperative pain and has been reported to be a risk factor for chronic postsurgical pain. As stress could facilitate the proinflammatory activation of microglia, we hypothesized that these cells may play a vital role in the development of preoperative stress-induced pain chronification after surgery. Our experiments were conducted in a rat model that consists of a single prolonged stress (SPS) procedure and plantar incision. A previous SPS exposure induced anxiety-like behaviors, prolonged incision-induced mechanical allodynia, and potentiated the activation of spinal microglia. Based on the results from ex vivo experiments, spinal microglia isolated from SPS-exposed rats secreted more proinflammatory cytokines upon challenge with LPS. Our results also demonstrated that microglia played a more important role than astrocytes in the initiation of SPS-induced prolongation of postsurgical pain. We further explored the therapeutic potential of agonism of α7 nAChR, an emerging anti-inflammatory target, for SPS-induced prolongation of postsurgical pain. Multiple intrathecal (i.t.) injections of PHA-543613 (an α7 nAChR agonist) or PNU-120596 (a type II positive allosteric modulator) during the perioperative period shortened the duration of postsurgical pain after SPS and suppressed SPS-potentiated microglia activation, but their effects were abolished by pretreatment with methyllycaconitine (an α7 nAChR antagonist; i.t.). Based on the results from ex vivo experiments, the anti-inflammatory effects of PHA-543613 and PNU-120596 may have been achieved by the direct modulation of microglia. In conclusion, stress-induced priming of spinal microglia played a key role in the initiation of preoperative stress-induced prolongation of postsurgical pain, and PHA-543613 and PNU-120596 may be potential candidates for preventing pain chronification after surgery.

Imbalanced spinal infiltration of Th17/Treg cells contributes to bone cancer pain via promoting microglial activation.

Increasing evidence suggests that T cells participate in the pathology of neuropathic pain, as well as the activation of microglia. However, whether T cells infiltrate into the spinal cord and contribute to the development of bone cancer pain (BCP) remains unknown. Here, we used a mouse model of BCP to show that numbers of T cells infiltrated into the spinal cord after sarcoma cell implantation with increased BCP, and most infiltrating T cells in the spinal cord were CD3+CD4+ T cells. Both Th17 and Treg subpopulations were analyzed by immunofluorescence. Treg cells in the spinal cord were transiently up-regulated, followed by an imbalance towards Th17 afterwards, and elevated IL-17/IL-17A levels were observed in both blood and spinal cord. Meanwhile, TGF-ß, IL-6, and IL-23, the factors which regulate Th17/Treg differentiation, increased their expressions during the development of BCP. Additionally, IL-17A receptor (IL-17AR) was found to be expressed on microglia, and the level of IL-17AR increased with activated microglia during BCP development. Furthermore, BCP was ameliorated when IL-17/IL-17A neutralizing antibodies were intrathecally injected, accompanied with inhibited Th17/Treg infiltration and suppressed microglial activation. In conclusion, T cells infiltrated into the spinal cord with the imbalance of Th17/Treg towards Th17 during the development of BCP, which could promote the microglial activation and further increased BCP, while neutralizing IL-17/IL-17A in the spinal cord could ameliorate BCP. Our results suggest that targeting the imbalanced Th17/Treg infiltration in the spinal cord could be a novel strategy for BCP therapy.

The Ubiquitination of Spinal MrgC Alleviates Bone Cancer Pain and Reduces Intracellular Calcium Concentration in Spinal Neurons in Mice.

Mas-related G-protein-coupled receptor subtype C (MrgC) has been shown to play an important role in the development of bone cancer pain. Ubiquitination is reported to participate in pain. However, whether MrgC ubiquitination plays a role in bone cancer pain remains unclear. To answer this question, we designed and performed this study. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer pain. MrgC agonist bovine adrenal medulla 8-22 (BAM 8-22) or MrgC antagonist anti-MrgC antibody were injected intrathecally on day 14 after bone cancer pain was successfully induced. The pain behaviors, the MrgC ubiquitination levels and intracellular calcium concentration in spinal neurons were measured before and after injection, respectively. With comparison to normal and sham group, mice in tumor group exhibited serious bone cancer pain on day 14, and the level of MrgC ubiquitination and intracellular calcium concentration in spinal neurons was significantly higher. Intrathecal injection of BAM 8-22 significantly alleviated bone cancer pain, increased the MrgC ubiquitination level and decreased intracellular calcium concentration in spinal neurons; however, these effects were reversed by administration of anti-MrgC antibody. Our study reveals that MrgC ubiquitination participates in the production and maintenance of bone cancer pain in mice, possibly through the regulation of intracellular calcium concentration in mice spinal neurons.

Dehydrocorydaline attenuates bone cancer pain by shifting microglial M1/M2 polarization toward the M2 phenotype.

Bone cancer pain remains a major challenge in patients with primary or metastatic bone cancer due to a lack of understanding the mechanisms. Previous studies have revealed the two distinct functional polarization states of microglia (classically activated M1 and alternatively activated M2) in the spinal cord in nerve injury-induced neuropathic pain. However, whether microglia in the spinal cord polarize to M1 and M2 phenotypes and contribute to the development of bone cancer pain remains unclear. In this study, we used a mouse model with bone cancer to characterize the M1/M2 polarization of microglia in the spinal cord during the development of bone cancer pain, and investigated the antinociceptive effects of dehydrocorydaline, an alkaloidal component isolated from Rhizoma corydalis on bone cancer pain. Our results show that microglia in the spinal cord presented increased M1 polarization and decreased M2 polarization, while overproduction of IL-1ß and inhibited expression of IL-10 was detected during bone cancer pain development. Intraperitoneal administration of dehydrocorydaline (10 mg/kg) had significant antinociceptive effects on day 14 after osteosarcoma cell implantation, accompanied by suppressed M1 phenotype and upregulated M2 phenotype of microglia in the spinal cord, while alleviated inflammatory response was observed then. These results suggest that the imbalanced polarization of microglia toward the M1 phenotype in the spinal cord may contribute to the development of bone cancer pain, while dehydrocorydaline helps to attenuate bone cancer pain, with microglial polarization shifting toward the M2 phenotype in the spinal cord.

Loss-of-Function Mutations in epaR Confer Resistance to ϕNPV1 Infection in Enterococcus faecalis OG1RF.

Enterococcus faecalis is a Gram-positive opportunistic pathogen that inhabits the human gastrointestinal tract. Because of the high frequency of antibiotic resistance among Enterococcus clinical isolates, interest in using phage to treat enterococcal infections and to decolonize high-risk patients for antibiotic-resistant Enterococcus is rising. Bacteria can evolve phage resistance, but there is little published information on these mechanisms in E. faecalis In this report, we identified genetic determinants of E. faecalis resistance to phage NPV1 (ϕNPV1). We found that loss-of-function mutations in epaR confer ϕNPV1 resistance by blocking phage adsorption. We attribute the inability of the phage to adsorb to the modification or loss of an extracellular polymer in strains with inactivated epaR Phage-resistant epaR mutants exhibited increased daptomycin and osmotic stress susceptibilities. Our results demonstrate that in vitro spontaneous resistance to ϕNPV1 comes at a cost in E. faecalis OG1RF.

Genome Modification in Enterococcus faecalis OG1RF Assessed by Bisulfite Sequencing and Single-Molecule Real-Time Sequencing.

UNLABELLED: Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged, reducing treatment options for these infections. MDR E. faecalis strains have large genomes containing mobile genetic elements (MGEs) that harbor genes for antibiotic resistance and virulence determinants. Bacteria commonly possess genome defense mechanisms to block MGE acquisition, and we hypothesize that these mechanisms have been compromised in MDR E. faecalis. In restriction-modification (R-M) defense, the bacterial genome is methylated at cytosine (C) or adenine (A) residues by a methyltransferase (MTase), such that nonself DNA can be distinguished from self DNA. A cognate restriction endonuclease digests improperly modified nonself DNA. Little is known about R-M in E. faecalis. Here, we use genome resequencing to identify DNA modifications occurring in the oral isolate OG1RF. OG1RF has one of the smallest E. faecalis genomes sequenced to date and possesses few MGEs. Single-molecule real-time (SMRT) and bisulfite sequencing revealed that OG1RF has global 5-methylcytosine (m5C) methylation at 5'-GCWGC-3' motifs. A type II R-M system confers the m5C modification, and disruption of this system impacts OG1RF electrotransformability and conjugative transfer of an antibiotic resistance plasmid. A second DNA MTase was poorly expressed under laboratory conditions but conferred global N(4)-methylcytosine (m4C) methylation at 5'-CCGG-3' motifs when expressed in Escherichia coli. Based on our results, we conclude that R-M can act as a barrier to MGE acquisition and likely influences antibiotic resistance gene dissemination in the E. faecalis species. IMPORTANCE: The horizontal transfer of antibiotic resistance genes among bacteria is a critical public health concern. Enterococcus faecalis is an opportunistic pathogen that causes life-threatening infections in humans. Multidrug resistance acquired by horizontal gene transfer limits treatment options for these infections. In this study, we used innovative DNA sequencing methodologies to investigate how a model strain of E. faecalis discriminates its own DNA from foreign DNA, i.e., self versus nonself discrimination. We also assess the role of an E. faecalis genome modification system in modulating conjugative transfer of an antibiotic resistance plasmid. These results are significant because they demonstrate that differential genome modification impacts horizontal gene transfer frequencies in E. faecalis.

Quantifying unrecognised replication present in reports of HIV diagnoses.

New diagnoses of HIV infection were reported confidentially to the Public Health Laboratory Service AIDS Centre under a national voluntary surveillance scheme. Two sets of data drawn from the national data sets were made available to us for analysis, the first in 1991 and the second in 1994, by which time the replication of reports had been reduced. The data used in the analyses consisted of the numbers of replications of the reported full date of birth in the individual records (one, two, three and so on), for each year of birth. This paper uses a nonparametric maximum likelihood estimation method for quantifying the amount of replication in the data. The estimated amount of replication was 3.37% (95% confidence interval (0.98%, 11.83%)) in the 1991 data set and 0.58% (95% confidence interval (0%, 2.64%)) in the 1994 data set.

Identification of essential genes that support fitness of Acinetobacter baumannii efflux pump overproducers in the presence of fluoroquinolone.

Acinetobacter baumannii is a nosocomial pathogen often associated with multidrug resistance (MDR) infections. Fluoroquinolone resistance (FQR) due to drug target site mutations and elevated expression of RND drug transporters is common among clinical isolates. We describe here a CRISPRi platform that identifies hypomorphic mutations that preferentially altered drug sensitivity in RND pump overproducers. An sgRNA library against essential genes of A. baumannii was constructed with single and double nucleotide mutations that produced titratable knockdown efficiencies and introduced into multiple strain backgrounds. Other than nusG depletions, there were few candidates in the absence of drug treatment that showed lowered fitness specifically in strains overexpressing clinically relevant RND efflux pumps AdeAB, AdeIJK, or AdeFGH. In the presence of ciprofloxacin, the hypomorphs causing hypersensitivity were predicted to result in outer membrane dysfunction, to which the AdeFGH overproducer appeared particularly sensitive. Depletions of either the outer membrane assembly BAM complex, LOS biogenesis proteins, or Lpt proteins involved in LOS transport to the outer membrane caused drug hypersensitivity in at least two of the three pump overproducers. On the other hand, depletions of translation-associated proteins, as well as components of the proton-pumping ATP synthase pump resulted in fitness benefits for at least two pump-overproducing strains in the presence of the drug. Therefore, pump overproduction exacerbated stress caused by defective outer membrane integrity, while the efficacy of drug resistance in efflux overproducers was enhanced by slowed translation or defects in ATP synthesis linked to the control of proton movement across the bacterial membrane.

Identification of Determinants that Allow Maintenance of High-Level Fluoroquinolone Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is associated with multidrug resistant (MDR) infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates. We here identify proteins that contribute to the fitness of FQR strains overexpressing three known RND systems using high-density insertion mutagenesis. Overproduction of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced LOS biosynthesis and blocked production of the major A. baumannii porin. In contrast, AdeAB pump overproduction, which does not affect the outer membrane pump component, was relatively tolerant to loss of these functions, consistent with outer membrane protein overproduction being the primary disruptive component. Surprisingly, overproduction of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overproduction, resulting in activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from peroxide stress. These results indicate that the RND outer membrane protein overproduction is compensated by cytoplasmic buffering and maintenance of outer membrane integrity in A. baumannii to facilitate fitness of FQR isolates.

Effect of remimazolam on electroencephalogram burst suppression in elderly patients undergoing cardiac surgery: Protocol for a randomized controlled noninferiority trial.

Background: Postoperative delirium (POD) is a common complication following cardiac surgery and increases postoperative morbidity and mortality. Intraoperative electroencephalogram (EEG) burst suppression suggests excessively deep anesthesia and predicts POD. Use of remimazolam provides a stable hemodynamic status and an appropriate depth of anesthesia. We aim to assess remimazolam administered for anesthesia and sedation in elderly patients having cardiac surgery. Methods: This is a randomized controlled clinical trial with noninferiority design. A total of 260 elderly patients aged equal to or greater than 60 years undergoing cardiac surgery will be randomly allocated to receive remimazolam or propofol (1:1) for general anesthesia and postoperative sedation until extubation. The primary outcome is the cumulative time with EEG burst suppression which is obtained from the SedLine system. The noninferiority margin is 2.0 min. The secondary outcomes include the POD occurrence within the first 5 days postoperatively and the duration of perioperative hypotension. Discussion: This noninferiority trial is the first to evaluate the effect of perioperative remimazolam administration on EEG burst suppression, POD occurrence, and duration of hypotension in elderly patients who undergo cardiac surgery. Trial registration: Chinese Clinical Trial Registry (ChiCTR2200056353).

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.