Clinical Setting Comparative Analysis of Uropathogens and Antibiotic Resistance: A Retrospective Study Spanning the Coronavirus Disease 2019 Pandemic.

Background: Antimicrobial resistance (AMR) in uropathogens has been increasing in Australia. Many nations observed heightened AMR during the coronavirus disease 2019 (COVID-19) pandemic, but it is not known how this may vary across clinical settings and in nations with lower infection rates. Methods: We investigated the uropathogen composition and corresponding antibiotic resistance of 775 559 Australian isolates from the community, hospitals, and aged care facilities before (2016-2019) and during (2020-2022) the COVID-19 pandemic. A mathematical model was developed to predict the likelihood of resistance to currently recommended antibiotics for treating urinary tract infections (UTIs). Results: Among uropathogens originating from the community, hospitals, and aged care facilities, Escherichia coli accounted for 71.4%, 57.6%, and 65.2%, respectively. During the COVID-19 pandemic period, there was an increase in UTIs caused by E coli across all settings. Uropathogens from aged care and hospitals frequently showed higher resistance to antibiotics compared to those isolated from the community. Interestingly, AMR among uropathogens showed a declining trend during the COVID-19 pandemic. Based on the resistance patterns of the past 3 years, our modeling predicted that 30%, 42.6%, and 38.8% of UTIs in the community, hospitals, and aged care facilities, respectively, would exhibit resistance to trimethoprim treatment as empirical therapy. In contrast, resistance to nitrofurantoin was predicted to be 14.6%, 26%, and 24.1% from these 3 respective settings. Conclusions: Empirical therapy of UTIs in Australia with trimethoprim requires evaluation due to high rates of resistance observed across clinical settings.

BAF facilitates interphase nuclear membrane repair through recruitment of nuclear transmembrane proteins.

Nuclear membrane rupture during interphase occurs in a variety of cell contexts, both healthy and pathological. Membrane ruptures can be rapidly repaired, but these mechanisms are still unclear. Here we show barrier-to-autointegration factor (BAF), a nuclear envelope protein that shapes chromatin and recruits membrane proteins in mitosis, also facilitates nuclear membrane repair in interphase, in part through recruitment of the nuclear membrane proteins emerin and Lem-domain-containing protein 2 (LEMD2) to rupture sites. Characterization of GFP-BAF accumulation at nuclear membrane rupture sites confirmed BAF is a fast, accurate, and persistent mark of nucleus rupture whose kinetics are partially dictated by membrane resealing. BAF depletion significantly delayed nuclear membrane repair, with a larger effect on longer ruptures. This phenotype could be rescued by GFP-BAF, but not by a BAF mutant lacking the Lap2, emerin, Man1 (LEM)-protein binding domain. Depletion of the BAF interactors LEMD2 or emerin, and to a lesser extent lamin A/C, increased the duration of nucleus ruptures, consistent with LEM-protein binding being a key function of BAF during membrane repair. Overall our results suggest a model where BAF is critical for timely repair of large ruptures in the nuclear membrane, potentially by facilitating membrane attachment to the rupture site.

Methods to Illuminate the Role of Salmonella Effector Proteins during Infection: A Review.

Intracellular bacterial pathogens like Salmonella enterica use secretion systems, such as the Type III Secretion System, to deliver virulence factors into host cells in order to invade and colonize these cells. Salmonella virulence factors include a suite of effector proteins that remodel the host cell to facilitate bacterial internalization, replication, and evasion of host immune surveillance. A number of diverse and innovative approaches have been used to identify and characterize the role of effector proteins during infection. Recent techniques for studying infection using single cell and animal models have illuminated the contribution of individual effector proteins in infection. This review will highlight the techniques applied to study Salmonella effector proteins during infection. It will describe how different approaches have revealed mechanistic details for effectors in manipulating host cellular processes including: the dynamics of effector translocation into host cells, cytoskeleton reorganization, membrane trafficking, gene regulation, and autophagy.

Optimized Fluorescence Complementation Platform for Visualizing Salmonella Effector Proteins Reveals Distinctly Different Intracellular Niches in Different Cell Types.

The bacterial pathogen Salmonella uses sophisticated type III secretion systems (T3SS) to translocate and deliver bacterial effector proteins into host cells to establish infection. Monitoring these important virulence determinants in the context of live infections is a key step in defining the dynamic interface between the host and pathogen. Here, we provide a modular labeling platform based on fluorescence complementation with split-GFP that permits facile tagging of new Salmonella effector proteins. We demonstrate enhancement of split-GFP complementation signals by manipulating the promoter or by multimerizing the fluorescent tag and visualize three effector proteins, SseF, SseG, and SlrP, that have never before been visualized over time during infection of live cells. Using this platform, we developed a methodology for visualizing effector proteins in primary macrophage cells for the first time and reveal distinct differences in the effector-defined intracellular niche between primary macrophage and commonly used HeLa and RAW cell lines.

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single-cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here, we establish a pipeline for long-term (17 h) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyper-replication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models.

6-Oxopyriphlorins.

Reaction of 2,6-pyridinedicarbaldehyde with a series of tripyrranes in the presence of trifluoroacetic acid, followed by oxidation of a pyriphlorin intermediate with silver(I) acetate, gave 6-oxopyriphlorins in moderate to good yields. The oxophlorin analogues were nonaromatic porphyrinoids that gave bright green colored solutions. Protonation with TFA afforded species that were attributed to mono- and dicationic structures. The proton NMR spectra in TFA­CDCl3 showed downfield shifts to the meso-protons and upfield shifts to the interior NH resonances that indicated the presence of a weakly diatropic dicationic species. Reaction of a 6-oxopyriphlorin with nickel(II) acetate in DMF or palladium(II) acetate in acetonitrile afforded the corresponding metallo-derivatives.

Pyrazole analogues of porphyrins and oxophlorins.

A series of porphyrin analogues with pyrazole rings replacing one of the usual pyrrole subunits have been synthesized. This was accomplished by reacting 1-phenyl, 1-methyl and 1-ethyl pyrazole-1,3-dicarbaldehydes with a tripyrrane in the presence of TFA, followed by an oxidation step. The initially formed phlorin product was sufficiently stable for the N-phenyl system to be isolated and characterized, although the related N-alkyl phlorin analogues were less stable. Attempts to dehydrogenate the intermediary phlorins with DDQ resulted in decomposition, but the N-alkylphlorins could be oxidized with 0.2% aqueous ferric chloride solutions. Although the phenyl-substituted phlorin could not be oxidized under these conditions, it did afford the pyrazoloporphyrin upon treatment with silver acetate under acidic conditions. Oxidations with silver acetate also afforded oxophlorin analogues where the oxo-linkage was selectively formed at the 5-position. The pyrazole-containing porphyrin analogues are cross-conjugated and exhibit only a small degree of diatropic character. The internal CH resonances were observed between 5.27 and 5.87 ppm, while the external meso-protons fell into a range of 6.84-7.88 ppm. The borderline overall aromatic character was attributed to dipolar resonance contributors. Protonation considerably increased the diatropicity and the diprotonated dications formed from these porphyrin analogues gave the internal CH resonance at upfield values of 2.65-3.20 ppm. The aromatic character was enhanced by the presence of an electron-donating alkyl substituent on the nitrogen compared to the phenyl-substituted species. The pyrazoloporphyrins reacted with nickel(II) acetate in DMF, or palladium(II) acetate in acetonitrile, to give the corresponding organometallic derivatives. The metal complexes showed increased diatropic character but protonation afforded nonaromatic cations. The oxophlorin analogues were also nonaromatic in the free base and protonated forms. This work extends our understanding of carbaporphyrinoid systems and provides the first detailed studies on pyrazole-containing porphyrin analogues.

In vitro and in vivo studies of the utility of dimethyl and diethyl carbaporphyrin ketals in treatment of cutaneous leishmaniasis.

Carbaporphyrin ketals are porphyrinoid compounds in which a pyrrole ring of a typical porphyrin macrocycle has been replaced by a ketal-substituted indene ring. It was recently demonstrated that these compounds are effective in vitro against Leishmania tarentolae. Their in vitro effectiveness is increased when they are exposed to visible light; they act as photosensitizers capable of mediating the production of reactive oxygen species (ROS). Following on this evidence, the effectiveness and cytotoxicity of the dimethyl and diethyl carbaporphyrin ketals (CKOMe and CKOEt, respectively) were determined in vitro using pathogenic Leishmania species with and without exposure to visible light (2 and 4 h). The effectiveness against various pathogenic Leishmania species was determined to be in a micromolar range. Additionally, the effect of encapsulating the carbaporphyrin ketals in liposome formulations was tested. Liposomal delivery diminished their toxicity, while the effectiveness was enhanced upon exposure to visible light (photodynamic effect). The cytotoxicity levels for human U937 cells and hamster peritoneal macrophages were in the ranges of 0.3 to 9 µM and 7 to 330 µM, respectively. When tested in vivo, using a hamster (Mesocricetus auratus) model of cutaneous leishmaniasis, CKOMe was active even in the dark, suggesting that the compound, once metabolized in the animal tissue, produces an active ingredient that does not seem to be photosensitive. Reduction in lesion size, histopathologic analyses, and smears confirmed the in vivo effectiveness of the compound, since the parasitic load was diminished without noticeable toxic effects.

Naphthiporphyrins.

Benziporphyrins, cross-conjugated porphyrin analogues with a benzene ring in place of one of the usual pyrrole units, have varying degrees of macrocyclic aromaticity because the 6π electron arene needs to give up its aromatic characteristics to facilitate conjugation over the entire system. As naphthalene would lose less resonance stabilization energy in giving up one of its benzene units, it was proposed that naphthiporphyrins would exhibit enhanced diatropicity compared to the related benziporphyrins. A naphthiporphyrin was prepared using the "3 + 1" variant of the MacDonald condensation by reacting 1,3-naphthalenedicarbaldehyde with a tripyrrane in the presence of TFA, followed by oxidation with DDQ. Although the free base form of naphthiporphyrin showed no overall diatropicity, the corresponding dication in TFA-CDCl(3) demonstrated a significant diatropic ring current where the internal CH shifted upfield to between 4.0 and 4.6 ppm. Naphthiporphyrin was converted to the corresponding palladium(II) complexes by reaction with Pd(OAc)(2) in acetonitrile, and the complex was further characterized by X-ray crystallography. Oxynaphthiporphyrins were similarly prepared by the "3 + 1" methodology from 4-methoxy-1,3-naphthalene-dicarbaldehyde, and these showed slightly enhanced diatropic character compared to oxybenziporphyrins. Reaction of oxybenziporphyrins or oxynaphthiporphyrins with silver(I) acetate afforded the corresponding silver(III) organometallic derivatives. A meso-tetraphenyl naphthiporphyrin was also synthesized in 4% yield by reacting a 1,4-naphthalene dicarbinol with 2 equiv of benzaldehyde and 3 equiv of pyrrole in the presence of BF(3).Et(2)O, followed by oxidation with DDQ. However, this 1,4-naphthiporphyrin showed reduced diatropic character compared to the corresponding p-benziporphyrin system. The NMR spectra for the 1,4-naphthiporphyrin show that the naphthalene unit pivots over the macrocycle and this presumably leads to further steric interactions that reduce the planarity of the macrocycle. These results demonstrate that while naphthiporphyrins can show enhanced aromatic properties as predicted, other factors may overwhelm this effect.

Adding to the confusion! Synthesis and metalation of pyrazole analogues of the porphyrins.

N-substituted pyrazole dialdehydes are shown to react with a tripyrrane under '3 + 1' conditions to give aza-analogues of the N-confused porphyrins; these novel porphyrinoids show borderline aromatic properties and readily afford organometallic derivatives with Ni(OAc)2 and Pd(OAc)2.

Carbaporphyrin ketals as potential agents for a new photodynamic therapy treatment of leishmaniasis.

Dimethyl and diethyl carbaporphyrin ketals inhibit the growth of Leishmania tarentolae promastigotes in vitro. The concentration dependency of the inhibitory effect was tested using the MTT assay. The presence of reactive oxygen species, such as singlet oxygen and superoxide, was detected using electron paramagnetic resonance spectroscopy with selected spin traps and confocal microscopy in cultures exposed to these carbaporphyrin ketals. These unique porphyrinoids show promise as potent inhibitors of Leishmania.