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Background: Trans-translation is a ribosome rescue system that plays an important role in bacterial tolerance to environmental stresses. It is absent in animals, making it a potential treatment target. However, its role in antibiotic tolerance in Pseudomonas aeruginosa remains unknown.Methods: The role and activity of trans-translation during antibiotic treatment were examined with a trans-translation-deficient strain and a genetically modified trans-translation component gene, respectively. In vitro assays and murine infection models were used to examine the effects of suppression of trans-translation.Results: We found that the trans-translation system plays an essential role in P. aeruginosa tolerance to azithromycin and multiple aminoglycoside antibiotics. We further demonstrated that gentamicin could suppress the azithromycin-induced activation of trans-translation. Compared with each antibiotic individually, gentamicin and azithromycin combined increased the killing efficacy against planktonic and biofilm-associated P. aeruginosa cells, including a reference strain PA14 and its isogenic carbapenem-resistance oprD mutant, the mucoid strain FRD1, and multiple clinical isolates. Furthermore, the gentamicin-azithromycin resulted in improved bacterial clearance in murine acute pneumonia, biofilm implant, and cutaneous abscess infection models.Conclusions: Combination treatment with gentamicin and azithromycin is a promising strategy in combating P. aeruginosa infections. [ABSTRACT FROM AUTHOR]

Objectives: Bacterial persisters are a small subpopulation of cells that are highly tolerant of antibiotics and contribute to chronic and recalcitrant infections. Global gene expression in Pseudomonas aeruginosa persister cells and genes contributing to persister formation remain largely unknown. The objective of this study was to examine the gene expression profiles of the persister cells and those that regained growth in fresh medium, as well as to identify novel genes related to persister formation.Methods: P. aeruginosa persister cells and those that regrew in fresh medium were collected and subjected to RNA sequencing analysis. Genes up-regulated in the persister cells were overexpressed to evaluate their roles in persister formation. The functions of the persister-contributing genes were assessed with pulse-chase assay, affinity chromatography, fluorescence and electron microscopy, as well as a light-scattering assay.Results: An operon containing PA2282-PA2287 was up-regulated in the persister cells and down-regulated in the regrowing cells. PA2285 and PA2287 play key roles in persister formation. PA2285 and PA2287 were found to bind to RpoC and FtsZ, which are involved in transcription and cell division, respectively. Pulse-chase assays demonstrated inhibitory effects of PA2285 and PA2287 on the overall transcription. Meanwhile, light-scattering and microscopy assays demonstrated that PA2285 and PA2287 interfere with cell division by inhibiting FtsZ aggregation. PA2285 and PA2287 are conserved in pseudomonads and their homologous genes in Pseudomonas putida contribute to persister formation.Conclusions: PA2285 and PA2287 are novel bifunctional proteins that contribute to persister formation in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes various acute and chronic infections. It is intrinsically resistant to a variety of antibiotics. However, production of pyocins during SOS response sensitizes P. aeruginosa to quinolone antibiotics by inducing cell lysis. The polynucleotide phosphorylase (PNPase) is a conserved phosphate-dependent 3′–5′ exonuclease that plays an important role in bacterial response to environmental stresses and pathogenesis by influencing mRNA and small RNA stabilities. Previously, we demonstrated that PNPase controls the type III and type VI secretion systems in P. aeruginosa. In this study, we found that mutation of the PNPase coding gene (pnp) increases the bacterial resistance to ciprofloxacin. Gene expression analyses revealed that the expression of pyocin biosynthesis genes is decreased in the pnp mutant. PrtR, a negative regulator of pyocin biosynthesis genes, is upregulated in the pnp mutant. We further demonstrated that PNPase represses the expression of PrtR on the post-transcriptional level. A fragment containing 43 nucleotides of the 5′ untranslated region was found to be involved in the PNPase mediated regulation of PrtR. Overall, our results reveled a novel layer of regulation on the pyocin biosynthesis by the PNPase in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Bacterial biofilms are surface‐attached communities of non‐replicating bacteria innately tolerant to antibiotics. Biofilms display differential gene expression profiles and physiologies as compared to their planktonic counterparts; however, their biology remains largely unknown. In this study, we used a halogenated phenazine (HP) biofilm eradicator in transcript profiling experiments (RNA‐seq) to define cellular targets and pathways critical to biofilm viability. WoPPER analysis with time–course validation (RT‐qPCR) revealed that HP‐14 induces rapid iron starvation in MRSA biofilms, as evident by the activation of iron‐acquisition gene clusters in 1 hour. Serine proteases and oligopeptide transporters were also found to be up‐regulated, whereas glycolysis, arginine deiminase, and urease gene clusters were down‐regulated. KEGG analysis revealed that HP‐14 impacts metabolic and ABC transporter functional pathways. These findings suggest that MRSA biofilm viability relies on iron homeostasis. Exploring ways to end a film: A halogenated phenazine probe molecule was used in transcript‐profiling investigations with RNA‐seq technology to define cellular targets and pathways critical to biofilm survival (see picture; MRSA, methicillin‐resistant Staphylococcus aureus). The results suggest that MRSA biofilm viability relies on iron homeostasis and establish a framework for the development of next‐generation therapeutics to eradicate biofilms. [ABSTRACT FROM AUTHOR]

Agents capable of eradicating bacterial biofilms are of great importance to human health as biofilm-associated infections are tolerant to our current antibiotic therapies. We have recently discovered that halogenated quinoline (HQ) small molecules are: 1) capable of eradicating methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, and 2) synthetic tuning of the 2-position of the HQ scaffold has a significant impact on antibacterial and antibiofilm activities. Here, we report the chemical synthesis and biological evaluation of 39 HQ analogues that have a high degree of structural diversity at the 2-position. We identified diverse analogues that are alkylated and aminated at the 2-position of the HQ scaffold and demonstrate potent antibacterial (MIC≤0.39 μ m) and biofilm eradication (MBEC 1.0-93.8 μ m) activities against drug-resistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium strains while demonstrating <5 % haemolysis activity against human red blood cells (RBCs) at 200 μ m. In addition, these HQs demonstrated low cytotoxicity against HeLa cells. Halogenated quinolines are a promising class of antibiofilm agents against Gram-positive pathogens that could lead to useful treatments against persistent bacterial infections. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii infection is a serious threat to burn patients. Bacteremia due to A. baumannii is becoming the most common cause of mortality following burn. However, the epidemiology of A. baumannii causing burn-related bloodstream infections has rarely been reported. We retrospectively collected 81 A. baumannii isolates from the bloodstream of burn patients over a three-year period. Antibiotic susceptibility tests, the prevalence of antibiotic-resistant genes and sequence typing (ST) were conducted to characterize these strains. Most of the isolates showed an extensive drug-resistant phenotype. The resistance frequencies to imipenem and meropenem were 94% and 91%, respectively. The blaOXA-23-like gene, AmpC, IS-AmpC, PER and SIM are the five most prevalent resistant genes, and their prevalence rates are 93% (75/81), 86% (70/81), 73% (59/81), 73% (59/81) and 52% (42/81), respectively. The 81 isolates were grouped into 10 known and 18 unknown ST types, with ST368 (38%) being the most prevalent. Except for ST457 and four new types (STn2, STn6, STn11 and STn14), the remaining 23 ST types belonged to one clonal complex 92, which is most common among clinical isolate in China. The above results indicated that ST368 isolates possessing both the blaOXA-23-like gene and ampC gene were the main culprits of the increasing nosocomial A. baumannii infection in this study. More attention should be paid to monitoring the molecular epidemiology of A. baumannii isolates from burn patients to prevent further distribution. Such information may help clinicians with therapeutic decisions and infection control in the Burns Institute. [ABSTRACT FROM AUTHOR]