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.

Hyphema and vitreous hemorrhage after micropulse cyclophotocoagulation a case report.

INTRODUCTION: We present two cases of vitreous hemorrhage after micropulse cyclophotocoagulation one of which had concurrent hyphema. To the best of our knowledge, these are the first cases of vitreous hemorrhage due to micropulse CPC in the United States. CASE DESCRIPTION: The first case is an 82-year-old woman with bilateral severe primary open angle glaucoma. BCVA in the right eye was 20/25, and 10-2 Humphrey visual field showed severe peripheral defects. The patient underwent MPCPC of the right eye and at one week, a settled 2 mm hyphema and vitreous hemorrhage confirmed by B-scan were noted. At three months, the patient had a BCVA of 20/80 with an IOP of 12 and retina consultation deferred a PPV. The second case is of a patient with bilateral moderate stage POAG who underwent MPCPC in both eyes. His original VA was 20/200 bilaterally. At 2 weeks, RE VA was count fingers at one foot and LE was 20/150-1. At two months, a RE B scan revealed dense vitreous opacities. Retina consultation revealed vitreous hemorrhage but a PPV was deferred. CONCLUSION: Clinicians should be aware of the risks of bleeding and the potential need for additional surgical interventions after MPCPC.

Gender Disparities Among Academic Vitreoretinal Specialists in the United States With Regard to Scholarly Impact and Academic Rank.

Background and objective While men outnumber women in the specialty of ophthalmology in general, the subspecialty of vitreoretinal surgery in particular has the highest percentage of men across all ophthalmic subspecialties. This study aimed to analyze the gender disparities regarding the publication productivity and academic rank of academic vitreoretinal specialists in the United States (US). Methods This cross-sectional study evaluated 116 ophthalmology residency programs in the US participating in the 2022 San Francisco Match. The academic vitreoretinal faculty from each ophthalmology residency program was included. The information on gender, academic rank, and publication activity in terms of the h-index were collected from institutional websites, the Scopus database, and the National Library of Medicine PubMed website. Results A total of 467 academic vitreoretinal specialists were identified. Among them, 345 (73.9%) were men, and 122 (26.1%) were women (p<0.001). When the academic ranks were analyzed, a higher number of men (43.8%) were found to hold the rank of full professor as compared to women. Furthermore, a higher number of women (47.5%) were found to hold the rank of assistant professor as compared to their male colleagues. Regarding the number of publications, in all academic rank categories, women had a significantly lower number of publications compared to men (p<0.001). Men also had a higher publication productivity or scholarly impact [h-index=15.2 ± 0.82 standard error of the mean (SEM)] compared to women (h-index=12.8 ± 0.99 SEM) (p=0.0004). Higher h-index correlated with higher academic rank, from assistant professor through full professor (p<0.001). Conclusion The field of vitreoretinal surgery has significantly fewer women compared to men, with women producing fewer publications and having less scholarly impact. H-index and total number of publications are also associated with a higher academic rank. Furthermore, full professors are more likely to be men, while assistant professors are more likely to be women. Future efforts should be aimed at reducing the gender disparity in vitreoretinal surgery.

The impact of primary repair timing on longitudinal visual outcomes after open globe injury.

BACKGROUND: The urgency with which to repair open globe injuries is a debated topic that lacks grounding in longitudinal visual outcomes data. We aim to test the association between primary repair timing and visual recovery potential following OGI. SUBJECTS/METHODS: We performed a retrospective cohort study of medical records from a US academic medical center (7/2017 to 11/2021). We included all patients with a principal diagnosis of OGI, a documented date and time of injury, presentation, repair, and ≥ 3 months of complete follow-up data on visual outcomes. We excluded those with prior OGI in the same eye. We also tested the correlation of injury to repair time (ITR) in hours with best corrected visual acuity (BCVA, in logMAR units) at last follow-up in the general cohort and select subpopulations and the impact of repair delay on visual improvement over the follow-up period. RESULTS: One hundred twenty-nine patients with OGI were analyzed (91 patients with ≥ 3 months of follow-up). The majority were male (105/129, 81%) with a median age of 45 years. Most OGI involved zone 1 (57%), followed by zone 3 (24%), and zone 2 injuries (19%). Median ocular trauma score (OTS) was 60 (IQR 37-70); mean presenting BCVA was logMAR 1.9 (median 2.3, IQR 1.0-2.7). Median ITR was 22 h (IQR 15-30 h, range 5-199 h). ITR time did not significantly correlate with final BCVA (n = 91, ß = - 0.003, 95% CI - 0.009-0.002, P = 0.233), nor did it significantly increase the odds of developing ocular complications or requiring secondary ocular surgeries (OR 0.985, 95% CI 0.967-1.002, P = 0.085). Additionally, the rate of BCVA improvement over subsequent months of follow-up did not significantly differ based on ITR time. Presenting BCVA (R2 = 0.701, P < 0.001) and OTS (R2 = 0.477, P < 0.001) significantly correlated with final BCVA, independent of repair delays. CONCLUSIONS: In this cohort of OGI patients, repair timing does not significantly correlate with final BCVA, and delays beyond 24 h do not significantly correlate with worse visual recovery potential. Repair time alone should be emphasized to a lesser extent as a prognosticator of visual potential, in favor of significant predictors such as the ocular trauma score and presenting visual acuity.

Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo.

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.

Initial Post-Operative Visit Absenteeism is Associated With Worse Amputation-Free Survival after Tibial Angioplasty.

OBJECTIVES: Tibial revascularization is often performed in the setting of critical limb ischemia and tissue loss requiring close patient monitoring in the early post-operative period for worsening gangrene and/or ischemia. Multiple studies have shown loss to follow-up is an independent risk factor for poor outcomes in several vascular procedures. Therefore, we evaluated the risk factors relating to loss to follow up against outcomes in patients undergoing tibial endovascular procedures with the hypothesis that poor post-operative visit compliance is associated with decreased amputation-free survival rates. METHODS: We performed a single-institution retrospective chart review of patients who underwent therapeutic endovascular tibial revascularization between 2014-2018. Patient follow-up and outcomes of death or major amputation (trans-tibial/trans-femoral) were followed up to 36-months post-operatively. Patients who had undergone previous infra-geniculate interventions or reached mortality/major amputation within 30-days post-operatively were excluded from analysis. RESULTS: We identified 89 patients who met inclusion criteria. The overall rate of attendance at less than <1 month, 1-6 months, 6-15 months and 15-36 months post-operatively were 60%, 64%, 60 and 40% respectively. 16% of patients had complete loss to follow-up. Patients without tissue loss (≤ Rutherford 4) were less likely to attend early <1 month and 1-6 month follow-up intervals. Notably, absenteeism from the first immediate post-operative visit was a significant risk factor for further absenteeism at 1-6 months (51% vs. 26%; P = 0.01) and at greater than 6-month follow-up (48% vs. 31%; P = 0.05). Compared to the cohort of all patients, failure to follow-up within 1 month was associated with a decrease in attendance from 64% to 26% at 1-6 months and 63-31% at more than 6 months. Missing the first post-operative visit was also associated with decreased amputation-free survival (P = 0.04). CONCLUSIONS: Absenteeism from the first post-operative visit is associated with worse amputation-free survival and a significant risk factor for further absenteeism from post-operative care. Given these results, ensuring close immediate post-operative follow up is essential to improving outcomes in patients undergoing tibial revascularization.

Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference.

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Development of rationally designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii, and we developed a CRISPR interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. IMPORTANCE New approaches are urgently needed to control A. baumannii, one of the most drug-resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

Author Correction: Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

Reactive oxygen species signaling in primordial germ cell development in Drosophila embryos.

REDOX mechanisms that induce biosynthesis of the reactive oxygen species (ROS) have attracted considerable attention due to both the deleterious and beneficial responses elicited by the reactive radical. In several organisms including Drosophila melanogaster, modulation of ROS activity is thought to be crucial for the maintenance of cell fates in developmental contexts. Interestingly, REDOX mechanisms have been shown to be involved in maintaining progenitor fate of stem cells as well as their proliferation and differentiation. Here, we have explored the possible functions of ROS during proper specification and developmental progression of embryonic primordial germ cells (PGCs). Indicating its potential involvement in these processes, ROS can be detected in the embryonic PGCs and the surrounding somatic cells from very early stages of embryogenesis. Using both "loss" and "gain" of function mutations in two different components of the REDOX pathway, we show that ROS levels are likely to be critical in maintaining germ cell behavior, including their directed migration. Altering the activity of a putative regulator of ROS also adversely influences the ability of PGCs to adhere to one another in cellular blastoderm embryos, suggesting potential involvement of this pathway in orchestrating different phases of germ cell migration.

The Landscape of Phenotypic and Transcriptional Responses to Ciprofloxacin in Acinetobacter baumannii: Acquired Resistance Alleles Modulate Drug-Induced SOS Response and Prophage Replication.

The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis.IMPORTANCE Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.