Anthranilate Acts as a Signal to Modulate Biofilm Formation, Virulence, and Antibiotic Tolerance of Pseudomonas aeruginosa and Surrounding Bacteria

Pseudomonas aeruginosa is a notorious pathogen with high antibiotic resistance, strong virulence, and ability to cause biofilm-mediated chronic infection. We found that these characteristics change profoundly before and after the time when anthranilate is produced as an “anthranilate peak”.

Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli

With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics’ efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

Photothermal Therapy may be a Double-edge Sword by Inducing the Formation of Bacterial Antibiotic Tolerance

Photothermal nanoparticles are thought to be the most potential candidates against infectious disease, by disrupting cell membrane and inhibiting metabolism. However, subpopulation survived with this low-activity state may be endowed...

Autoinducer Analogs Can Provide Bactericidal Activity to Macrolides in Pseudomonas aeruginosa through Antibiotic Tolerance Reduction

Macrolide antibiotics are used in treating Pseudomonas aeruginosa chronic biofilm infections despite their unsatisfactory antibacterial activity, because they display several special activities, such as modulation of the bacterial quorum sensing and immunomodulatory effects on the host. In this study, we investigated the effects of the newly synthesized P. aeruginosa quorum-sensing autoinducer analogs (AIA-1, -2) on the activity of azithromycin and clarithromycin against P. aeruginosa. In the killing assay of planktonic cells, AIA-1 and -2 enhanced the bactericidal ability of macrolides against P. aeruginosa PAO1; however, they did not affect the minimum inhibitory concentrations of macrolides. In addition, AIA-1 and -2 considerably improved the killing activity of azithromycin and clarithromycin in biofilm cells. The results indicated that AIA-1 and -2 could affect antibiotic tolerance. Moreover, the results of hydrocarbon adherence and cell membrane permeability assays suggested that AIA-1 and -2 changed bacterial cell surface hydrophobicity and accelerated the outer membrane permeability of the hydrophobic antibiotics such as azithromycin and clarithromycin. Our study demonstrated that the new combination therapy of macrolides and AIA-1 and -2 may improve the therapeutic efficacy of macrolides in the treatment of chronic P. aeruginosa biofilm infections.

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.

Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa, a multidrug-resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may potentially overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth, pyocyanin production, and biofilm formation of P. aeruginosa. Methods: Pseudomonas aeruginosa ATCC 10145 and clinical isolates were cultured in a royal jelly-containing medium to test the antibacterial activity. Pyocyanin production was observed by measuring the absorbance at 690 nm after 36 h culture and determined using extinction coefficient 4310 M-1 cm-1. Static microtiter plate biofilm assay performed to detect the biofilm formation, followed by scanning electron microscopy. Results: Royal jelly effectively inhibited the viability of both strains from a concentration of 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC 10145, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates. Concentrations of 12.5% and 6.125% significantly induced biofilm formation of P. aeruginosa ATCC 10145, in line with the results of the SEM analysis. Conclusions: The royal jelly concentration of 25% or higher inhibits bacterial growth; however, the subinhibitory concentration increases pyocyanin production and biofilm formation in P. aeruginosa. It is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

Modelling of filamentous phage-induced antibiotic tolerance of P.aeruginosa

Filamentous molecules tend to spontaneously assemble into liquid crystalline droplets with a tactoid morphology in the environments with the high concentration on non-adsorbing molecules. Tactoids of filamentous Pf bacteriophage, such as those produced by Pseudomonas aeruginosa, have been linked with increased antibiotic tolerance. We modelled this system and show that tactoids, composed of filamentous Pf virions, can lead to antibiotic tolerance by acting as an adsorptive diffusion barrier. The continuum model, reminiscent of descriptions of reactive diffusion in porous media, has been solved numerically and good agreement was found with the analytical results, obtained using a homogenisation approach. We find that the formation of tactoids significantly increases antibiotic diffusion times leading to stronger antibiotic resistance.

Peptidoglycan recycling contributes to outer membrane integrity and carbapenem tolerance in Acinetobacter baumannii

The Gram-negative cell envelope is an essential structure that not only protects the cell against lysis from the internal turgor, but also forms a barrier to limit entry of antibiotics. Some of our most potent bactericidal antibiotics, the β-lactams, exploit the essentiality of the cell envelope by inhibiting its biosynthesis, typically inducing lysis and rapid death. However, many Gram-negative bacteria exhibit antibiotic tolerance, the ability to sustain viability in the presence of β-lactams for extended time periods. Despite several studies showing that antibiotic tolerance contributes directly to treatment failure, and is a steppingstone in acquisition of true resistance, the molecular factors that promote intrinsic tolerance are not well-understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. While typically reserved to combat multidrug resistant infections, carbapenem β-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate high intrinsic tolerance to meropenem, form spheroplasts with the antibiotic and revert to normal growth after antibiotic removal. Using transcriptomics and genetics screens, we characterized novel tolerance factors and found that outer membrane integrity maintenance, drug efflux and peptidoglycan homeostasis collectively contribute to meropenem tolerance in A. baumannii. Furthermore, outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to meropenem tolerance in A. baumannii.

128. Mutations that Inactivate the Tricarboxylic Acid Cycle in Staphylococcus aureus Arise During Persistent MRSA Bacteremia

Background Persistent MRSA bacteremia is common with high morbidity and mortality despite appropriate antibiotics. Persistent infections are associated with antibiotic tolerance and can arise from perturbations in cellular pathways. We performed whole genome sequencing of clinical isolates to identify the genetic bases of antibiotic tolerance. Methods Whole genomes of MRSA from patients with persistent bacteremia were sequenced, which identified 8 isolates harboring different citZ mutations. To assess the effect of the mutations directly on citrate synthase activity, purified recombinant enzymes were assayed using a commercially available kit. The same kit was used to measure activity in whole cell lysates of MRSA isolates harboring wild-type and mutant citZ alleles. Enzyme kinetic parameters were determined by fitting initial rate data to the Michaelis-Menten equation using nonlinear regression. Figure 1. Whole Genome Sequencing of Persistent MRSA Bacteremia Whole genome sequencing of 206 blood cultures from 20 patients with persistent MRSA bacteremia (defined as >7 days) under the hypothesis that continuous antibiotic exposure will give rise to - and enrich for - mutations that convey antibiotic tolerance through in-host evolution. Results Analysis of whole genomes from 206 blood cultures from 20 patients with persistent MRSA bacteremia identified citZ, which encodes for citrate synthase, the first step of the tricarboxylic acid cycle, as the most repeatedly mutated gene. The data revealed parallelism in its evolution, as citZ was mutated 8 independent times, a rate far greater than from chance alone. To characterize the impact of the mutations on enzyme activity, recombinant proteins were expressed in E. coli and purified for enzymatic assays. As compared to wildtype enzyme, two mutants had reduced activity, and four mutants had near-absent activity; two mutants were unable to be expressed due to destabilizing mutations. Michaelis-Menten analysis of the two mutants with residual activity show that, as compared to wildtype, both had lower affinity for its substrates, and lower maximum activity. Furthermore, we found the cell was unable to compensate for the loss of citrate synthase activity, as lysates harboring these mutations also displayed analogous reductions in activity. Figure 2. Purified citrate synthase mutant proteins have reduced enzyme activity Citrate synthase catalyzes the reaction between acetyl-CoA and oxaloacetic acid to form citric acid with CoA-SH as a byproduct. CoA-SH can interact with DTNB to form TNB, which can be measured spectrophotometrically at A412nm to indirectly measure the activity of citrate synthase. Wildtype citrate synthase and the D141N mutant have comparable activity whereas the remaining study mutants have either reduced (mutants A313P and A313V) or near-absent (mutants G7D, G7D + D141N, S201P, P354S) activity. The three active site mutants (H248G, D309G, H219G) also have near-absent activity and serve as negative controls. Positive control is citrate synthase provided in the Sigma Citrate Synthase Assay Kit. No enzyme control replaces enzyme with assay buffer. All error bars are ± 1 SD, calculated from at least three replicate experiments. Figure 3. A313P and A313V mutants have decreased substrate affinity and decreased maximal activity Panels A and B show plots of the initial rate of citrate synthase activity (Y-axis, in units of µmole/mL/sec) versus OAA/AcCoA substrate concentration (X-axis, in millimolar units), for wildtype citrate synthase and the A313P and A313V mutants. Panel (A) shows enzymatic parameters for wildtype, A313P mutant, and A313V mutant with variable OAA (0-0.625 mM) and fixed AcCoA (0.3 mM). Panel (B) shows enzymatic parameters for wildtype, A313P mutant, and A313V mutant with fixed OAA (0.5 mM) and variable AcCoA (0-2.5 mM). Three replicates were performed for each condition, with error bars showing ± 1 SD. Figure 4. Citrate synthase mutants disrupt functional dimerization and destabilize alpha-helix packing Identified citrate synthase mutants mapped onto the crystal structure of T. thermophilus citrate synthase (PDB 1IOM) show that the mutations are located either at dimerization interfaces or within the interior of hydrophobic packing interfaces. Conclusion Cellular metabolism and virulence regulation are interconnected in S. aureus, as alterations in TCA cycle activity lead to increased persister formation and host macrophage inactivation. Our findings that inactivating citZ mutations are enriched can provide a potential explanation for the mechanism of persistent bacteremia. Disclosures All Authors: No reported disclosures