ACS Infectious Diseases最新文献

Antibiotic resistance is a serious health concern worldwide, predominantly driven by β-lactamase enzymes that can inactivate most prescribed β-lactam drugs. Carbapenemases are specialized β-lactamases that hydrolyze carbapenems, considered last-resort antibiotics. Insertion variants of Klebsiella pneumoniae carbapenemase (KPC) play a major role in this threat, showing alarming resistance to ceftazidime-avibactam (CZA), a key combination therapy to treat this menace. KPC-107 is a variant with an exceptional 28-residue insertion raised by duplication mutation (Ser-Ser-Pro-Arg-Ala-Val-Thr-Glu-Ser-Leu-Gln-Lys-Leu-Thr-Leu-Gly-Ser-Ala-Leu-Ala-Ala-Pro-Gln-Arg-Gln-Gln-Phe-Val) between Ambler positions 180 and 181, conferring resistance to CZA. Our study employs comprehensive biophysical, biochemical, and biological analyses accompanied by molecular docking and MD simulations, reporting that KPC-107 exhibits 10 times enhanced catalytic efficiency and 4-fold increased MIC value for ceftazidime compared to KPC-2, while its activity against other tested β-lactams is diminished. The reduced K_i value (0.23 ± 0.05 μM) of avibactam against KPC-107, in contrast with KPC-2 (K_i = 0.83 ± 0.1 μM), is further explained by carbamylation and decarbamylation rates. Insertion-induced active site modification might lead to a better binding with ceftazidime, which is reduced in the case of avibactam, which could possibly explain the contrasting behavior of KPC-107 against these ligands, as confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Molecular docking and MD simulations, including free energy landscape (FEL), principal component analysis (PCA), and radial distribution function (RDF), may further support the binding affinity and the mechanisms underlying ceftazidime resistance. Our findings suggest that KPC-107 adapts distinct mechanistic strategies to evade CZA armor by enhanced ceftazidime hydrolysis while compromising increased inhibition by avibactam, strengthening its role in antibiotic resistance evolution.

In 2024, an estimated 10 million people developed Tuberculosis (TB), nearly half a million of whom were infected with drug-resistant tuberculosis (DR-TB). Early detection of infection and drug resistance enables rapid engagement in effective care. Bacterial culture and nucleic acid testing remain the primary diagnostic methods, with smear microscopy being phased out. However, these methods present significant limitations for diagnosing drug resistance, such as lengthy time-to-result for phenotypic tests, as well as the need for prior knowledge of resistance mutations and prohibitive cost for molecular tests. To address this, we developed a rapid phenotypic TB drug susceptibility test, termed Tre-DST, based on novel metabolically incorporated trehalose probes, which specifically detect live mycobacteria. We used the nonpathogenic Mycobacterium smegmatis and the virulence-attenuated Mycobacterium tuberculosis (Mtb) H37Ra or auxotrophic Mtb to demonstrate a strong correlation between cost-effective plate reader results and flow cytometry data, suggesting that the plate reader is a suitable fluorescence detector for Tre-DST. We determined that adding a 1-week incubation step allowed Mtb samples originally seeded at 10⁴ CFU/mL to become detectable, over 2 weeks earlier than colony-forming unit analysis. We found that Tre-DST reports on drug susceptibility in a drug-agnostic manner, demonstrating loss of fluorescence with frontline TB drugs as well as the newer drug bedaquiline. Tre-DST distinguished RIF- and INH-resistant auxotrophs from susceptible controls and accurately reported the resistance activity. Ultimately, because Tre-DST is agnostic to mechanisms of drug resistance, this assay is likely compatible with all WHO-recommended and future DR-TB drugs as a diagnostic in reference laboratories.

Multidrug-resistant (MDR) Acinetobacter baumannii is a major clinical threat with limited treatment options, as current therapies rely on polymyxins such as colistin. Targeting the lipopolysaccharide (LPS) biosynthetic pathway offers a new target for new antibiotic discovery, yet most efforts have focused on enzyme-based assays that do not reflect cell level physiology. Here, we developed a cell-based screening strategy that links colistin resistance to inhibition of lipooligosaccharide (LOS) biogenesis in A. baumannii. Using colistin, we established a phenotypic platform in which compounds that inhibit LOS synthesis rescue bacterial growth from colistin’s mode of action. This approach allows direct identification of inhibitors acting on essential LPS enzymes, including LpxC. Screening a library of about 7000 small molecules discovered non-hydroxamate compounds that restored growth under colistin stress. Hit compounds were validated through LpxC enzyme assays and protein-compound binding assays. Furthermore, our molecular docking study suggests that the hit compounds bind to the LpxC catalytic pocket similarly to CHIR-090. Together, our work introduces a novel phenotypic screening strategy for discovering LPS targeted inhibitors and provides new chemical scaffolds for developing antibiotics against A. baumannii and other Gram-negative pathogens.

Antimicrobial resistance poses a significant threat to medicine, exemplified by methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all antibiotics. We designed chimeric antisense oligonucleotides (ASOs) targeting the conserved flavin mononucleotide (FMN) riboswitch, a regulator of essential bacterial metabolic pathways. Three chemically modified ASOs were optimized for stability, binding affinity, and RNase H recruitment, and conjugated to the pVEC cell-penetrating peptide for uptake. Antibacterial activity was tested against MRSA ATCC 6538, with growth monitored by optical density. pVEC-ASO-3 showed the most potent effect, with MIC₅₀ and MIC₉₀ values of 1.25 and 7.5 nM, respectively. No inhibition was observed in Escherichia coli, which lacks the FMN riboswitch, confirming specificity. Cytotoxicity testing in human A549 cells revealed 95% viability at the highest concentration. These results demonstrate that riboswitch-targeted ASOs can selectively inhibit MRSA growth with minimal toxicity, supporting their potential as a new class of antibacterial agents against drug-resistant pathogens.

The L1 capsid protein of Human papilloma virus 16 (HPV16) forms pentamers that assemble into virus-like particles (VLPs). With many viruses, interaction of the virus with polysaccharides modulates association of the virus with a host cell. For HPV16, soluble polyanionic saccharides heparin and carrageenan bind to L1 and inhibit cell entry. Based on the observed multivalent binding of heparin, we hypothesized that anionic polysaccharides could act as scaffolds to promote VLP assembly. We observed that heparin, a highly sulfated, flexible polymer, increased the initial rate of assembly and stabilized assembled L1 VLPs. Heparin also notably increased the susceptibility of L1 pentamers to proteolysis. Conversely, κ-carrageenan, a less sulfated, rigid polymer, blocked assembly and destabilized VLPs. We examined the effects of a small panel of anions on assembly to identify their important features. We observed that smaller anions required much higher concentration for an effect, and that their chemical nature also had an impact. These data demonstrate that artificial binding partners can control VLP assembly through their interaction with the exterior of capsid protein and that structural details beyond size and electrostatics are critical to accelerating or inhibiting self-assembly.

Pseudomonas aeruginosa is a notorious bacterial pathogen causing chronic pulmonary infections in people with cystic fibrosis (CF) due to its high tolerance to antibiotics and ability to form recalcitrant biofilms. A newer approach to attenuate the virulence of P. aeruginosa in CF could be the local reinforcement of a resilient community of competing bacteria in the lung. Lactobacilli can mediate antagonistic effects against P. aeruginosa by production of organic acids, but it is not entirely clear if they can exert this beneficial effect locally at the site of infection. While the nutritional environment of the airways in CF promotes P. aeruginosa, it does not support robust growth of lactic acid bacteria, thus attenuating their probiotic potential. To overcome this obstacle, we hypothesized that prebiotic fructooligosaccharides (FOS) could selectively stimulate Lactiplantibacillus plantarum during culture in synthetic cystic fibrosis sputum medium (SCFM2). Indeed, FOS supported the growth of L. plantarum and led to increased acid production. Co-cultivation of L. plantarum and P. aeruginosa reduced biofilm formation and FOS enabled L. plantarum to grow to higher densities in dual-species biofilms. However, this came at the cost of an increased production of the cytotoxic metabolite pyocyanin by P. aeruginosa. To examine whether L. plantarum would influence the pathogenicity of P. aeruginosa, we developed a dual-bacterial species infection model using a CF – relevant airway cell line exposed to the nutritional environment of SCFM2. L. plantarum, grown in SCFM2 or SCFM-FOS, did not inhibit the adhesion of P. aeruginosa. In contrast, the presence of live as well as heat-inactivated L. plantarum, or sterile L. plantarum supernatants drastically enhanced the cell damage during coinfection with P. aeruginosa. This effect was not exclusively dependent on differences in the proliferation of P. aeruginosa or addition of SCFM2 to the cell culture medium. Our data indicate that a potential benefit of bacteriotherapy is determined by the nutritional environment of the diseased body site and that the use of L. plantarum in the context of chronic pulmonary infections must be carefully evaluated.

The cysteine 3C-like proteases (3CL^pro) of caliciviruses, coronaviruses, and picornaviruses are essential for viral replication. In this study, we report the development of potent broad-spectrum peptidomimetic antiviral agents that target the 3CL^pro of caliciviruses (NS6), coronaviruses (M^pro), and a picornavirus (3C). Based upon previously reported inhibitors, a small library of compounds was designed, synthesized and tested to identify a core structure, which was then derivatized with a focus upon P3 and P4 positions to afford new inhibitors with improved potency against the respective viral enzymes and enhanced binding as determined by X-ray crystallography. These compounds were tested against a range of viruses in culture, revealing minimal toxicity while exhibiting broad-spectrum potent nanomolar activities against noroviruses and several coronavirus species, including alpha and omicron variants of SARS-CoV-2 and Middle East Respiratory Syndrome virus (MERS).

The rise of multidrug-resistant (MDR) Neisseria gonorrhoeae is a growing health concern. New treatments are urgently needed as resistance to last-line antibiotics increases. The zinc ionophore, PBT2, has previously been shown to facilitate zinc uptake in bacteria and restore susceptibility to existing antibiotics in combination therapies. In contrast, we demonstrate that PBT2 alone is sufficient to inhibit the growth of N. gonorrhoeae. Guided by this finding, we synthesized a series of 8-hydroxyquinoline-based new chemical entities, herein termed a “ionophoroantibiotics (IP-antibiotics).” These compounds displayed potent activity against MDR N. gonorrhoeae, with several exhibiting greater efficacy than PBT2. Proteomic profiling studies suggested IP-antibiotics disrupt metal and phosphate metabolism in N. gonorrhoeae, upregulating iron transport and downregulating phosphate pathways. PBT2 broadly affects metal and metabolic proteins, whereas the new compounds act more selectively. This distinct mode of action circumvents established resistance mechanisms and targets key bacterial vulnerabilities. Collectively, these findings identify IP-antibiotics as a promising new class of antibiotics for the treatment of MDR N. gonorrhoeae.

The limitations of the current standard therapy for pulmonary diseases caused by Mycobacterium avium complex (MAC) underscore the urgent need for novel therapeutic agents. Among potential candidates, thiopeptide antibiotics have attracted attention due to their antibacterial activity but their poor solubility has limited clinical application. In this study, we developed AJ-099, a thiopeptide derivative with improved solubility and enhanced antibacterial potency. The in vitro antibacterial activity of AJ-099 was confirmed across macrolide-susceptible and- resistant clinical isolates, with minimum inhibitory concentration values consistently falling within the range of ≤0.125–0.5 μg/mL. In addition, AJ-099 exhibited a marked growth inhibition against both macrolide-susceptible and -resistant MAC clinical isolates in macrophages. Importantly, AJ-099 displayed synergistic effects in combination with clarithromycin (CLR), a macrolide drug, which resulted in significantly greater reductions in intracellular MAC burden compared to either agent alone. The synergistic effect was consistently observed in vivo, where the AJ-099 and CLR combination achieved superior bacterial clearance and reduced lung inflammation to the current standard therapy consisting of CLR, ethambutol, and rifampicin. Collectively, these results highlight that AJ-099 in combination with macrolides is a promising candidate for treating MAC pulmonary infections. Moreover, its potent activity against macrolide-resistant MAC strains suggests it may offer an effective therapeutic option for refractory MAC pulmonary infections.