The diagnostic value of [18F]FAPI-42 PET/CT for pulmonary artery masses: comparison with [18F]FDG PET/CT.

OBJECTIVES: To investigate the potential utility of [18F]fibroblast activation protein inhibitor (FAPI) positron emission tomography/computed tomography (PET/CT) for evaluating pulmonary artery (PA) masses, and compare it with [18F]fluorodeoxyglucose (FDG) PET/CT. METHODS: Participants with clinically suspected PA malignancy were prospectively enrolled and underwent dual-tracer PET/CT ([18F]FAPI-42 and [18F]FDG) imaging. Visual analysis and semi-quantitative parameters were compared between the two types of radiotracers. The tissue specimen underwent immunohistochemical staining to verify FAP expression in the tissue. RESULTS: Thirty-three patients (18 males/15 females; mean age 53.1 ± 15.4 years) were enrolled. All 21 patients with malignant PA masses were FDG-positive (100%), whereas 20 out of 21 patients were FAPI-positive (95.2%). All 12 patients with benign PA masses were both negative in FDG and FAPI PET. The mean maximum standardized uptake value (SUVmax) and target-to-background ratio (TBR) of FAPI and FDG in malignant PA masses were significantly higher than those of benign masses. Although there was no significant difference in SUVmax between FDG and FAPI in malignant PA masses (11.36 vs. 9.18, p = 0.175), the TBR (liver) and TBR (left ventricle) were more favorable for FAPI than for FDG (13.04 vs. 5.17, p < 0.001); (median: 7.75 vs. 2.75, p = 0.007). Immunohistochemical analysis (n = 16) validated that the level of FAP expression corresponded strongly to the uptake of FAPI in PET/CT scans (rs = 0.712, p = 0.002). For clinical management, FAPI PET found more metastatic lesions than FDG PET in 4 patients, with 2 patients upgrading and 1 patient changing treatment decisions. CONCLUSIONS: FAPI PET/CT is feasible in the diagnosis of PA masses. Although not superior to FDG PET/CT, FAPI PET/CT showed better target-to-background contrast. CLINICAL RELEVANCE STATEMENT: This study found that FAPI PET/CT is not superior to FDG PET/CT in diagnosing PA masses, but FAPI PET/CT displays better target-to-background contrast and more positive lesions, which may help improve disease management. KEY POINTS: Pulmonary malignancies lack specificity in clinical manifestations, laboratory tests, and routine imaging examinations. FAPI PET/CT is not diagnostically better than FDG PET/CT but displays better target-to-background contrast and more positive lesions. Dual-tracer PET/CT ([18F]FAPI-42 and [18F]FDG) imaging improves clinical management of pulmonary artery masses.

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.

In Vitro Activities of Oxazolidinone Antibiotics Alone and in Combination with C-TEMPO against Methicillin-Resistant Staphylococcus aureus Biofilms.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a global health concern. The propensity of MRSA to form biofilms is a significant contributor to its pathogenicity. Strategies to treat biofilms often involve small molecules that disperse the biofilm into planktonic cells. Linezolid and, by extension, theoxazolidinones have been developed to treat infections caused by Gram-positive bacteria such as MRSA. However, the clinical development of these antibiotics has mainly assessed the susceptibility of planktonic cells to the drug. Previous studies evaluating the anti-biofilm activity of theoxazolidinones have mainly focused on the biofilm inhibition of Enterococcus faecalis and methicillin-sensitive Staphylococcus aureus, with only a few studies investigating the activity of oxazolidinones for eradicating established biofilms for these species. Very little is known about the ability of oxazolidinones to eradicate MRSA biofilms. In this work, five oxazolidinones were assessed against MRSA biofilms using a minimum biofilm eradication concentration (MBEC) assay. All oxazolidinones had inherent antibiofilm activity. However, only ranbezolid could completely eradicate MRSA biofilms at clinically relevant concentrations. The susceptibility of the MRSA biofilms to ranbezolid was synergistically enhanced by coadministration with the nitroxide biofilm dispersal agent C-TEMPO. We presume that ranbezolid acts as a dual warhead drug, which combines the mechanism of action of the oxazolidinones with a nitric oxide donor or cytotoxic drug.

[Analysis of Clinicopathological Features on Spread Through Air Spaces
of Lung Adenocarcinoma].

BACKGROUND: The biological and molecular characteristics of spread through air spaces (STAS), a newly recognized invasive mode of lung cancer, remain controversial. The aim of this study was to investigate the clinicopathological features and molecular characteristics of STAS in patients with pulmonary adenocarcinoma. METHODS: A total of 694 resected invasive non-mucinous lung adenocarcinomas diagnosed by clinicopathology from July 2019 to March 2021 in the First Affiliated Hospital of Guangzhou Medical University were collected, and the relationship between STAS and clinicopathological factors was analyzed. The state of protein expression of anaplastic lymphoma kinase (ALK) was detected by immunohistochemical method. Epidermal growth factor receptor (EGFR) was detected by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). ROS proto-oncogene 1-receptor (ROS1) was detected by reverse transcription-PCR (RT-PCR). RESULTS: A total of 344 STAS positive cases and 350 STAS negative cases were collected. By univariate analysis, STAS positivity was statistically associated with tumor maximum diameter (P<0.001), pleural invasion (P<0.001), lymphovascular invasion (P<0.001), nerve invasion (P=0.013), lymph node metastasis (P<0.001), clinical stage (P<0.001) and histological type (P<0.001). There was a statistical correlation between STAS and ALK protein expression (P=0.001). Multivariate analysis showed that STAS positive was correlated with pleural invasion (P=0.001), vascular invasion (P<0.001), lymph node metastasis (P=0.005)and ALK protein expression (P=0.032). CONCLUSIONS: STAS is associated with highly aggressive biological behavior of lung adenocarcinoma, suggesting a poor prognosis.

Preclinical Evaluation of Nitroxide-Functionalised Ciprofloxacin as a Novel Antibiofilm Drug Hybrid for Urinary Tract Infections.

Urinary tract infections (UTIs) are the second most common bacterial infection with high recurrence rates and can involve biofilm formation on patient catheters. Biofilms are inherently tolerant to antimicrobials, making them difficult to eradicate. Many antibiofilm agents alone do not have bactericidal activity; therefore, linking them to antibiotics is a promising antibiofilm strategy. However, many of these hybrid agents have not been tested in relevant preclinical settings, limiting their potential for clinical translation. Here, we evaluate a ciprofloxacin di-nitroxide hybrid (CDN11), previously reported to have antibiofilm activity against uropathogenic Escherichia coli (UPEC) strain UTI89 in vitro, as a potential UTI therapeutic using multiple preclinical models that reflect various aspects of UTI pathogenesis. We report improved in vitro activity over the parent drug ciprofloxacin against mature UTI89 biofilms formed inside polyethylene catheters. In bladder cell monolayers infected with UTI89, treatment with CDN11 afforded significant reduction in bacterial titers, including intracellular UPEC. Infected mouse bladders containing biofilm-like intracellular reservoirs of UPEC UTI89 showed decreased bacterial loads after ex vivo bladder treatment with CDN11. Activity for CDN11 was reported across different models of UTI, showcasing nitroxide-antibiotic hybridization as a promising antibiofilm approach. The pipeline we described here could be readily used in testing other new therapeutic compounds, fast-tracking the development of novel antibiofilm therapeutics.

O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, providing a plausible explanation for its evolutionary loss.

Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for inactivation of OAg biogenesis during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS primarily contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-linked OAg intermediates in the periplasmic leaflet of the inner membrane interfere with peptidoglycan sacculus biosynthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for as an evolutionary fitness benefit. Our work thus provides a plausible explanation for the long-held mystery of the selective pressure that may have led to the loss of OAg biogenesis in E. coli K-12; this opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.

Simple and Efficient Synthesis of 3-Aryl-2-oxazolidinone Scaffolds Enabling Increased Potency toward Biofilms.

Infectious diseases caused by bacterial pathogens are a leading cause of mortality worldwide. In particular, recalcitrant bacterial communities known as biofilms are implicated in persistent and difficult to treat infections. With a diminishing antibiotic pipeline, new treatments are urgently required to combat biofilm infections. An emerging strategy to develop new treatments is the hybridization of antibiotics. The benefit of this approach is the extension of the useful lifetime of existing antibiotics. The oxazolidinones, which include the last resort antibiotic linezolid, are an attractive target for improving antibiofilm efficacy as they present one of the most recently discovered classes of antibiotics. A key step in the synthesis of new 3-aryl-2-oxazolidinone derivatives is the challenging formation of the oxazolidinone ring. Herein we report a direct synthetic route to the piperazinyl functionalized 3-aryl-2-oxazolidinone 17. We also demonstrate an application of these piperazine molecules by functionalizing them with a nitroxide moiety as a strategy to extend the useful lifetime of oxazolidinones and improve their potency against Methicillin-resistant Staphylococcus aureus (MRSA) biofilms. The antimicrobial susceptibility of the linezolid-nitroxide conjugate 11 and its corresponding methoxyamine derivative 12 (a control for biofilm dispersal) was assessed against planktonic cells and biofilms of MRSA. In comparison to linezolid and our lead compound 10 (a piperazinyl oxazolidinone derivative), the linezolid-nitroxide conjugate 11 displayed a minimum inhibitory concentration that was 4-16-fold higher. The opposite effect was seen in biofilms where the linezolid-nitroxide hybrid 11 was >2-fold more effective (160 µg/mL versus >320 µg/mL) in eradicating MRSA biofilms. The methoxyamine derivative 12 performed on par with linezolid. The drug-likeness of the compounds was also assessed, and all compounds were predicted to have good oral bioavailability. Our piperazinyl oxazolidinone derivative 10 was confirmed to be lead-like and would be a good lead candidate for future functionalized oxazolidinones. The modification of antibiotics with a dispersal agent appears to be a promising approach for eradicating MRSA biofilms and overcoming the antibiotic resistance associated with the biofilm mode of growth.

Extensive Diversity in Escherichia coli Group 3 Capsules Is Driven by Recombination and Plasmid Transfer from Multiple Species.

Bacterial capsules provide protection against environmental challenges and host immunity. Historically, Escherichia coli K serotyping scheme, which relies on the hypervariable capsules, has identified around 80 K forms that fall into four distinct groups. Based on recent work by us and others, we predicted that E. coli capsular diversity is grossly underestimated. We exploited group 3 capsule gene clusters, the best genetically defined capsule group in E. coli, to analyze publicly available E. coli sequences for overlooked capsular diversity within the species. We report the discovery of seven novel group 3 clusters that fall into two distinct subgroups (3A and 3B). The majority of the 3B capsule clusters were found on plasmids, contrary to the defining feature of group 3 capsule genes localizing at the serA locus on the E. coli chromosome. Other new group 3 capsule clusters were derived from ancestral sequences through recombination events between shared genes found within the serotype variable central region 2. Intriguingly, flanking regions 1 and 3, known to be conserved areas among capsule clusters, showed considerable intra-subgroup variation in clusters from the 3B subgroup, containing genes of shared ancestry with other Enterobacteriaceae species. Variation of group 3 kps clusters within dominant E. coli lineages, including multidrug-resistant pathogenic lineages, further supports that E. coli capsules are undergoing rigorous change. Given the pivotal role of capsular polysaccharides in phage predation, our findings raise attention to the need of monitoring kps evolutionary dynamics in pathogenic E. coli in supporting phage therapy. IMPORTANCE Capsular polysaccharides protect pathogenic bacteria against environmental challenges, host immunity, and phage predations. The historical Escherichia coli K typing scheme, which relies on the hypervariable capsular polysaccharide, has identified around 80 different K forms that fall into four distinct groups. Taking advantage of the supposedly compact and genetically well-defined group 3 gene clusters, we analyzed published E. coli sequences to identify seven new gene clusters and revealed an unexpected capsular diversity. Genetic analysis revealed that group 3 gene clusters shared closely related serotype-specific region 2 and were diversified through recombination events and plasmid transfer between multiple Enterobacteriaceae species. Overall, capsular polysaccharides in E. coli are undergoing rigorous change. Given the pivotal role capsules play in phage interactions, this work highlighted the need to monitor the evolutionary dynamics of capsules in pathogenic E. coli for effective phage therapy.

A Buried Water Network Modulates the Activity of the Escherichia coli Disulphide Catalyst DsbA.

The formation of disulphide bonds is an essential step in the folding of many proteins that enter the secretory pathway; therefore, it is not surprising that eukaryotic and prokaryotic organisms have dedicated enzymatic systems to catalyse this process. In bacteria, one such enzyme is disulphide bond-forming protein A (DsbA), a thioredoxin-like thiol oxidase that catalyses the oxidative folding of proteins required for virulence and fitness. A large body of work on DsbA proteins, particularly Escherichia coli DsbA (EcDsbA), has demonstrated the key role that the Cys30-XX-Cys33 catalytic motif and its unique redox properties play in the thiol oxidase activity of this enzyme. Using mutational and functional analyses, here we identify that a set of charged residues, which form an acidic groove on the non-catalytic face of the enzyme, further modulate the activity of EcDsbA. Our high-resolution structures indicate that these residues form a water-mediated proton wire that can transfer protons from the bulk solvent to the active site. Our results support the view that proton shuffling may facilitate the stabilisation of the buried Cys33 thiolate formed during the redox reaction and promote the correct direction of the EcDsbA-substrate thiol-disulphide exchange. Comparison with other proteins of the same class and proteins of the thioredoxin-superfamily in general suggest that a proton relay system appears to be a conserved catalytic feature among this widespread superfamily of proteins. Furthermore, this study also indicates that the acidic groove of DsbA could be a promising allosteric site to develop novel DsbA inhibitors as antibacterial therapeutics.

Repeat-Unit Elongations To Produce Bacterial Complex Long Polysaccharide Chains, an O-Antigen Perspective.

The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the protective outer membrane of Gram-negative bacteria. Most species produce one of the structurally diverse O-antigens, with nearly all the polysaccharide components having complex structures made by the Wzx/Wzy pathway. This pathway produces repeat-units of mostly 3-8 sugars on the cytosolic face of the cytoplasmic membrane that is translocated by Wzx flippase to the periplasmic face and polymerized by Wzy polymerase to give long-chain polysaccharides. The Wzy polymerase is a highly diverse integral membrane protein typically containing 10-14 transmembrane segments. Biochemical evidence confirmed that Wzy polymerase is the sole driver of polymerization, and recent progress also began to demystify its interacting partner, Wzz, shedding some light to speculate how the proteins may operate together during polysaccharide biogenesis. However, our knowledge of how the highly variable Wzy proteins work as part of the O-antigen processing machinery remains poor. Here, we discuss the progress to the current understanding of repeat-unit polymerization and propose an updated model to explain the formation of additional short chain O-antigen polymers found in the lipopolysaccharide of diverse Gram-negative species and their importance in the biosynthetic process.

Cysteine-Dependent Conformational Heterogeneity of Shigella flexneri Autotransporter IcsA and Implications of Its Function.

Shigella IcsA is a versatile surface virulence factor required for early and late pathogenesis stages extracellularly and intracellularly. Despite IcsA serving as a model Type V secretion system (T5SS) autotransporter to study host-pathogen interactions, its detailed molecular architecture is poorly understood. Recently, IcsA was found to switch to a different conformation for its adhesin activity upon sensing the host stimuli by Shigella Type III secretion system (T3SS). Here, we reported that the single cysteine residue (C130) near the N terminus of the IcsA passenger had a role in IcsA adhesin activity. We also showed that the IcsA passenger (IcsAp) existed in multiple conformations, and the conformation populations were influenced by a central pair of cysteine residues (C375 and C379), which was not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues altered the exposure of IcsA epitopes to polyclonal anti-IcsA antibodies previously shown to block Shigella adherence, yet without loss of IcsA intracellular functions in actin-based motility (ABM). Anti-IcsA antibody reactivity was restored when the IcsA-paired cysteine substitution mutants were expressed in an ΔipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovered a novel molecular switch empowered by a centrally localized, short-spaced cysteine pair in the Type V autotransporter IcsA that ensured conformational heterogeneity to aid IcsA evasion of host immunity. IMPORTANCE Shigella species are the leading cause of diarrheal-related death globally by causing bacillary dysentery. The surface virulence factor IcsA, which is essential for Shigella pathogenesis, is a unique multifunctional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we showed that the three cysteine residues in IcsA contributed to the protein's distinct functions. The N-terminal cysteine residue within the IcsA passenger domain played a role in adhesin function, while a centrally localized cysteine pair provided conformational heterogeneity that resulted in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserved biological function in distinct IcsA conformations for cell adhesion, actin-based motility, and autophagy escape, providing a potential strategy by which Shigella evades host immunity and targets this essential virulence factor.

Conserved FimH mutations in the global Escherichia coli ST131 multi-drug resistant lineage weaken interdomain interactions and alter adhesin function.

The binding of the type 1 fimbrial adhesin FimH to mannosylated receptors is allosterically regulated to enhance the fitness of uropathogenic Escherichia coli (UPEC) during urinary tract infection (UTI). Mutations in the two FimH domains (pilin and lectin) located outside the mannose binding pocket have been shown to influence mannose binding affinity, yet the details of the allostery mechanism are not fully elucidated. Here we characterised different FimH conformational states (termed low-affinity tense and high-affinity relaxed conformations) of natural FimH variants using molecular dynamics (MD) simulation techniques and report key structural dynamics differences between them. The clinically dominant FimH30 variant from the pandemic multidrug resistant E. coli ST131 lineage contains an R166H mutation that weakens FimH interdomain interactions and allows enhanced mannose interactions with pre-existing high-affinity relaxed conformations. When expressed in an isogenic ST131 strain background, FimH30 mediated high human cell adhesion and invasion, and enhanced biofilm formation over other variants. Collectively, our computational and experimental findings support a model of FimH protein allostery that is mediated by shifts in the pre-existing conformational equilibrium of FimH, additional to the sequential step-wise process of structural perturbations transmitted from one site to another within the protein. Importantly, it is the first study to shed light into how natural mutations in a clinically dominant FimH variant influence the protein's conformational landscape optimising its function for ST131 fitness at intestinal and extraintestinal niches.

A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide.

The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a fivefold increase in the yield of engineered mutant colonies obtained in S. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by eightfold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA-protein complexes into clinically important bacteria.

Loss of ß-Ketoacyl Acyl Carrier Protein Synthase III Activity Restores Multidrug-Resistant Escherichia coli Sensitivity to Previously Ineffective Antibiotics.

Antibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organization list of bacterial pathogens that urgently require new antibiotics, 9 out of 12 are Gram-negative, with four being of "critical priority." One crucial barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. While fatty acids are a shared constituent of all structural membrane lipids, their biosynthesis pathway in bacteria is distinct from eukaryotes, making it an attractive target for new antibiotic development that remains less explored. Here, we interrogated the redundant components of the bacterial type II fatty acid synthesis (FAS II) pathway, showing that disrupting FAS II homeostasis in Escherichia coli through deletion of the fabH gene damages the cell envelope of antibiotic-susceptible and antibiotic-resistant clinical isolates. The fabH gene encodes the ß-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show that fabH null mutation potentiated the killing of multidrug-resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemase kpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infection in vitro. Our findings showcase the potential of FabH as a promising target that could be further explored in the development of therapies that may repurpose currently ineffective antibiotics or rescue failing last-resort antibiotics against Gram-negative pathogens. IMPORTANCE Gram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor toward antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy. Fatty acids are the fundamental building blocks of structural membrane lipids, and their synthesis constitutes an attractive antimicrobial target, as it follows distinct pathways in prokaryotes and eukaryotes. Here, we identified a component of fatty acid synthesis, FabH, as a gate-keeper of outer membrane barrier function. Without FabH, Gram-negative bacteria become susceptible to otherwise impermeable antibiotics and are resensitized to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.

Co-Occurrence of Multidrug Resistant Klebsiella pneumoniae Pathogenic Clones of Human Relevance in an Equine Pneumonia Case.

The global epidemiology of multidrug resistant Klebsiella pneumoniae, a serious threat to both animal and human health, is dominated by the spread of pathogenic clones, each separately evolving via acquisition of transferable antibiotic resistance or niche-specific virulence determinants. In horses, K. pneumoniae infection can lead to severe respiratory illness. Here, we characterized multiple isolates recovered from bronchial aspirates of a mare with pneumonia refractory to antibiotics. First, we used a combination of standard microbiology, bacteriophage cross-susceptibility and antibiotic resistance testing to profile the infecting K. pneumoniae population. The genomes of isolates with distinct fingerprints (pulsed-field gel electrophoresis) and unique combined bacteriophage/antibiotic profiles were then further analyzed using whole-genome sequencing. Adhesion to human epithelial cells and biofilm production were also measured as virulence indicators. Although it is commonly expected for one clone to dominate an infection episode, we identified five coexisting multidrug resistant K. pneumoniae sharing the same niche. One was a novel sequence type (ST4656), while the other four were all members of emerging human pathogenic clonal groups (ST307, ST628, ST893 and ST392). These isolates did not display significant differences from one another in terms of virulence or resistance and differed only in plasmid content from isolates implicated in severe human infections, with equal potential to prolong duration and severity of infection when sharing the same niche. This study highlights the importance of more precise surveillance and detection measures to uncover bacterial heterogeneity, reminding us that the "single clone" concept is not an absolute in invasive bacterial infections. IMPORTANCE Multidrug resistant Klebsiella pneumoniae are agents of life-threatening infections in animals and humans, with several multidrug resistant clones causing outbreaks of disease worldwide. It is generally accepted that only one clone will be dominant in an infection episode. In this study, we investigated K. pneumoniae isolates from a horse with severe pneumonia and demonstrated co-occurrence of multiple sequence types previously identified as emerging human pathogens. The equine isolates are not significantly different from one another in terms of virulence or resistance, with equal potential to prolong duration and severity of infection, and are indistinguishable from isolates recovered from humans, except for plasmid content. Our study highlights how the "one dominant clone" concept is not an absolute in severe infection, illustrating the need for improved diagnostics to track heterogeneity of infection, and reinforces the importance of cross-monitoring of environmental and human reservoirs of multidrug resistant pathogens.

Interdependence of Shigella flexneri O Antigen and Enterobacterial Common Antigen Biosynthetic Pathways.

Outer membrane (OM) polysaccharides allow bacteria to resist harsh environmental conditions and antimicrobial agents, traffic to and persist in pathogenic niches, and evade immune responses. Shigella flexneri has two OM polysaccharide populations, being enterobacterial common antigen (ECA) and lipopolysaccharide (LPS) O antigen (Oag); both are polymerized into chains by separate homologs of the Wzy-dependent pathway. The two polysaccharide pathways, along with peptidoglycan (PG) biosynthesis, compete for the universal biosynthetic membrane anchor, undecaprenyl phosphate (Und-P), as the finite pool of available Und-P is critical in all three cell wall biosynthetic pathways. Interactions between the two OM polysaccharide pathways have been proposed in the past where, through the use of mutants in both pathways, various perturbations have been observed. Here, we show for the first time that mutations in one of the two OM polysaccharide pathways can affect each other, dependent on where the mutation lies along the pathway, while the second pathway remains genetically intact. We then expand on this and show that the mutations also affect PG biosynthesis pathways and provide data which supports that the classical mutant phenotypes of cell wall mutants are due to a lack of available Und-P. Our work here provides another layer in understanding the complex intricacies of the cell wall biosynthetic pathways and demonstrates their interdependence on Und-P, the universal biosynthetic membrane anchor. IMPORTANCE Bacterial outer membrane polysaccharides play key roles in a range of bacterial activities from homeostasis to virulence. Two such OM polysaccharide populations are ECA and LPS Oag, which are synthesized by separate homologs of the Wzy-dependent pathway. Both ECA and LPS Oag biosynthesis join with PG biosynthesis to form the cell wall biosynthetic pathways, which all are interdependent on the availability of Und-P for proper function. Our data show the direct effects of cell wall pathway mutations affecting all related pathways when they themselves remain genetically unchanged. This work furthers our understanding of the complexities and interdependence of the three cell wall pathways.

Development of 3D Printed Biodegradable Mesh with Antimicrobial Properties for Pelvic Organ Prolapse.

To address the increasing demand for safe and effective treatment options for pelvic organ prolapse (POP) due to the worldwide ban of the traditional polypropylene meshes, this study introduced degradable polycaprolactone (PCL)/polyethylene glycol (PEG) composite meshes fabricated with melt-electrowriting (MEW). Two PCL/PEG mesh groups: 90:10 and 75:25 (PCL:PEG, wt%) were fabricated and characterized for their degradation rate and mechanical properties, with PCL meshes used as a control. The PCL/PEG composites showed controllable degradation rates by adjusting the PEG content and produced mechanical properties, such as maximal forces, that were higher than PCL alone. The antibacterial properties of the meshes were elicited by coating them with a commonly used antibiotic: azithromycin. Two dosage levels were used for the coating: 0.5 mg and 1 mg per mesh, and both dosage levels were found to be effective in suppressing the growth of S. aureus bacteria. The biocompatibility of the meshes was assessed using human immortalized adipose derived mesenchymal stem cells (hMSC). In vitro assays were used to assess the cell viability (LIVE/DEAD assay), cell metabolic activity (alamarBlue assay) and cell morphology on the meshes (fluorescent and electron microscopy). The cell attachment was found to decrease with increased PEG content. The freshly drug-coated meshes showed signs of cytotoxicity during the cell study process. However, when pre-released for 14 days in phosphate buffered saline, the initial delay in cell attachment on the drug-coated mesh groups showed full recovery at the 14-day cell culture time point. These results indicated that the PCL/PEG meshes with antibiotics coating will be an effective anti-infectious device when first implanted into the patients, and, after about 2 weeks of drug release, the mesh will be supporting cell attachment and proliferation. These meshes demonstrated a potential effective treatment option for POP that may circumvent the issues related to the traditional polypropylene meshes.

Pulmonary artery intimal sarcoma - A primeval or rediscovered tumor? A report of 14 new cases with literature review.

Pulmonary artery sarcomas (PAS) are rare with many being undifferentiated pleomorphic or spindle cell (UPSC) sarcomas with variable atypia. The term pulmonary artery intimal sarcoma (PAIS) was rarely coined in the early literature and sometimes used for luminal sarcomas. With the advent of immunohistochemistry and molecular genetics, many of these UPSC sarcomas were found to frequently overpress MDM2 and/or CDK4 and PDGFRA with genetic alterations in 12q12-15 and 4q12, where the MDM2, CDK4 and PDGFRA genes are located. These recent developments enabled refinement in diagnosis of PAIS. We diagnosed 14 cases of PAIS (6 males and 8 females, mean age 44 years) in 2015 - 2020 in our institution. Six were initially misdiagnosed as thromboembolism and the remaining pulmonary artery tumors. The tumors were pulmonary artery intraluminal polypoid masses with histology of spindle cell sarcomas exhibiting immunohistochemical positivity for MDM2 (100%) and CDK4 (79%) with MDM2 gene amplification (100%). Ten surgically treated patients fared better than four other biopsy only and not surgically treated patients, who died of disease within 5-11 months. PAIS needs to be differentiated from other spindle cell tumors and those exhibiting MDM2 gene amplification, especially dedifferentiated liposarcoma. The use of biopsy to provide diagnostic material poses a sampling error problem and correlation with clinical, radiologic, histologic, immunophenotypic and genotypic features are essential for accurate diagnosis and early surgical intervention of PAIS.