Tissue remodeling by an opportunistic pathogen triggers allergic inflammation.

Different effector arms of the immune system are optimized to protect from different classes of pathogens. In some cases, pathogens manipulate the host immune system to promote the wrong type of effector response-a phenomenon known as immune deviation. Typically, immune deviation helps pathogens to avoid destructive immune responses. Here, we report on a type of immune deviation whereby an opportunistic pathogen, Pseudomonas aeruginosa (P. aeruginosa), induces the type 2 immune response resulting in mucin production that is used as an energy source by the pathogen. Specifically, P. aeruginosa-secreted toxin, LasB, processed and activated epithelial amphiregulin to induce type 2 inflammation and mucin production. This "niche remodeling" by P. aeruginosa promoted colonization and, as a by-product, allergic sensitization. Our study thus reveals a type of bacterial immune deviation by increasing nutrient supply. It also uncovers a mechanism of allergic sensitization by a bacterial virulence factor.

The modification landscape of Pseudomonas aeruginosa tRNAs.

RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here, we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in Escherichia coli, which enabled us to identify similar modifications in P. aeruginosa Our analysis supports a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA-modifying enzymes, some of which play roles in determining virulence and pathogenicity.

Development of innovative multi-epitope mRNA vaccine against Pseudomonas aeruginosa using in silico approaches.

The rising issue of antibiotic resistance has made treating Pseudomonas aeruginosa infections increasingly challenging. Therefore, vaccines have emerged as a viable alternative to antibiotics for preventing P. aeruginosa infections in susceptible individuals. With its superior accuracy, high efficiency in stimulating cellular and humoral immune responses, and low cost, mRNA vaccine technology is quickly replacing traditional methods. This study aimed to design a novel mRNA vaccine by using in silico approaches against P. aeruginosa. The research team identified five surface and antigenic proteins and selected their appropriate epitopes with immunoinformatic tools. These epitopes were then examined for toxicity, allergenicity and homology. The researchers also checked their presentation and identification by major histocompatibility complex cells and other immune cells through valuable tools like molecular docking. They subsequently modeled a multi-epitope protein and optimized it. The mRNA was analyzed in terms of structure and stability, after which the immune system's response against the new vaccine was simulated. The results indicated that the designed mRNA construct could be an effective and promising vaccine that requires laboratory and clinical trials.

ADARs regulate cuticle collagen expression and promote survival to pathogen infection.

BACKGROUND: In all organisms, the innate immune system defends against pathogens through basal expression of molecules that provide critical barriers to invasion and inducible expression of effectors that combat infection. The adenosine deaminase that act on RNA (ADAR) family of RNA-binding proteins has been reported to influence innate immunity in metazoans. However, studies on the susceptibility of ADAR mutant animals to infection are largely lacking. RESULTS: Here, by analyzing adr-1 and adr-2 null mutants in well-established slow-killing assays, we find that both Caenorhabditis elegans ADARs are important for organismal survival to gram-negative and gram-positive bacteria, all of which are pathogenic to humans. Furthermore, our high-throughput sequencing and genetic analysis reveal that ADR-1 and ADR-2 function in the same pathway to regulate collagen expression. Consistent with this finding, our scanning electron microscopy studies indicate adr-1;adr-2 mutant animals also have altered cuticle morphology prior to pathogen exposure. CONCLUSIONS: Our data uncover a critical role of the C. elegans ADAR family of RNA-binding proteins in promoting cuticular collagen expression, which represents a new post-transcriptional regulatory node that influences the extracellular matrix. In addition, we provide the first evidence that ADAR mutant animals have altered susceptibility to infection with several opportunistic human pathogens, suggesting a broader role of ADARs in altering physical barriers to infection to influence innate immunity.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

In vitro development of resistance against antipseudomonal agents: comparison of novel ß-lactam/ß-lactamase inhibitor combinations and other ß-lactam agents.

We subjected seven P. aeruginosa isolates to a 10-day serial passaging against five antipseudomonal agents to evaluate resistance levels post-exposure and putative resistance mechanisms in terminal mutants were analyzed by whole-genome sequencing analysis. Meropenem (mean, 38-fold increase), cefepime (14.4-fold), and piperacillin-tazobactam (52.9-fold) terminal mutants displayed high minimum inhibitory concentration (MIC) values compared to those obtained after exposure to ceftolozane-tazobactam (11.4-fold) and ceftazidime-avibactam (5.7-fold). Fewer isolates developed elevated MIC values for other ß-lactams and agents belonging to other classes when exposed to meropenem in comparison to other agents. Alterations in nalC and nalD, involved in the upregulation of the efflux pump system MexAB-OprM, were common and observed more frequently in isolates exposed to ceftazidime-avibactam and meropenem. These alterations, along with ones in mexR and amrR, provided resistance to most ß-lactams and levofloxacin but not imipenem. The second most common gene altered was mpl, which is involved in the recycling of the cell wall peptidoglycan. These alterations were mainly noted in isolates exposed to ceftolozane-tazobactam and piperacillin-tazobactam but also in one cefepime-exposed isolate. Alterations in other genes known to be involved in ß-lactam resistance (ftsI, oprD, phoP, pepA, and cplA) and multiple genes involved in lipopolysaccharide biosynthesis were also present. The data generated here suggest that there is a difference in the mechanisms selected for high-level resistance between newer ß-lactam/ß-lactamase inhibitor combinations and older agents. Nevertheless, the isolates exposed to all agents displayed elevated MIC values for other ß-lactams (except imipenem) and quinolones tested mainly due to alterations in the MexAB-OprM regulators that extrude these agents.

NorA efflux pump mediates Staphylococcus aureus response to Pseudomonas aeruginosa pyocyanin toxicity.

Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.

Pseudomonas aeruginosa producing FIM-1-metallo-ß-lactamase: emergence and cross-transmission in a long-term acute care rehabilitation hospital.

FIM-1 metallo-ß-lactamase was previously detected in sporadic Pseudomonas aeruginosa clinical isolates. Here, we report on FIM-1-positive P. aeruginosa from two patients who had shared the same ward in a long-term acute care rehabilitation hospital. Whole-genome sequencing analysis revealed close relatedness of these isolates, which belonged to an ST235 sublineage (clade 8/14) different from those previously reported. Results highlighted the occurrence of clonal diversity among FIM-positive strains and the possibility of their cross-transmission in some healthcare settings.

Phenazine-1 carboxylic acid of Pseudomonas aeruginosa induces the expression of Staphylococcus aureus Tet38 MDR efflux pump and mediates resistance to phenazines and antibiotics.

In this study, we showed that phenazine-1 carboxylic acid (PCA) of Pseudomonas aeruginosa induced the expression of Tet38 efflux pump triggering Staphylococcus aureus resistance to tetracycline and phenazines. Exposure of S. aureus RN6390 to supernatants of P. aeruginosa PA14 and its pyocyanin (PYO)-deficient mutants showed that P. aeruginosa non-PYO phenazines could induce the expression of Tet38 efflux pump. Direct exposure of RN6390 to PCA compound at 0.25× MIC led to a five-fold increase in tet38 transcripts. Expression of Tet38 protein was identified through confocal microscopy using RN6390(pRN-tet38p-yfp) that expressed YFP under control of the tet38 promoter by PCA at 0.25× MIC. The MICs of PCA of a Tet38-overexpressor and a Δtet38 mutant showed a three-fold increase and a two-fold decrease, respectively, compared with that of wild-type. Pre-exposure of RN6390 to PCA (0.25× MIC) for 1 hour prior to addition of tetracycline (1× or 10× MIC) improved bacteria viability of 1.5-fold and 2.6-fold, respectively, but addition of NaCl 7% together with tetracycline at 10× MIC reduced the number of viable PCA-exposed RN6390 of a 2.0-log10 CFU/mL. The transcript levels of tetR21, a repressor of tet38, decreased and increased two-fold in the presence of PCA and NaCl, respectively, suggesting that the effects of PCA and NaCl on tet38 production occurred through TetR21 expression. These data suggest that PCA-induced Tet38 protects S. aureus against tetracycline during coinfection with P. aeruginosa; however, induced tet38-mediated S. aureus resistance to tetracycline is reversed by NaCl 7%, a nebulized treatment used to enhance sputum mobilization in CF patients.

Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan.

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ2 test). Only 4.1% of CRPA isolates carried the carbapenemase genes: blaGES (2) and blaIMP (13). One ST235 isolate carried the novel blaIMP variant blaIMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

Effects of quorum sensing-interfering agents, including macrolides and furanone C-30, and an efflux pump inhibitor on nitrosative stress sensitivity in Pseudomonas aeruginosa.

Long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infection, despite how limited the clinically achievable concentrations are, being far below their MICs. An increase in the sub-MIC of macrolide exposure-dependent sensitivity to nitrosative stress is a typical characteristic of P. aeruginosa. However, a few P. aeruginosa clinical isolates do not respond to sub-MIC of macrolide treatment. Therefore, we examined the effects of sub-MIC of erythromycin (EM) on the sensitivity to nitrosative stress together with an efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN). The sensitivity to nitrosative stress increased, suggesting that the efflux pump was involved in inhibiting the sub-MIC of macrolide effect. Analysis using efflux pump-mutant P. aeruginosa revealed that MexAB-OprM, MexXY-OprM, and MexCD-OprJ are factors in reducing the sub-MIC of macrolide effect. Since macrolides interfere with quorum sensing (QS), we demonstrated that the QS-interfering agent furanone C-30 (C-30) producing greater sensitivity to nitric oxide (NO) stress than EM. The effect of C-30 was decreased by overproduction of MexAB-OprM. To investigate whether the increase in the QS-interfering agent exposure-dependent sensitivity to nitrosative stress is characteristic of P. aeruginosa clinical isolates, we examined the viability of P. aeruginosa treated with NO. Although treatment with EM could reduce cell viability, a high variability in EM effects was observed. Conversely, C-30 was highly effective at reducing cell viability. Treatment with both C-30 and PAßN was sufficiently effective against the remaining isolates. Therefore, the combination of a QS-interfering agent and an EPI could be effective in treating P. aeruginosa infections.

Evaluation of fortimicin antibiotic combinations against MDR Pseudomonas aeruginosa and resistome analysis of a whole genome sequenced pan-drug resistant isolate.

BACKGROUND: Multidrug-resistant (MDR) P. aeruginosa is a rising public health concern, challenging the treatment of such a ubiquitous pathogen with monotherapeutic anti-pseudomonal agents. Worryingly, its genome plasticity contributes to the emergence of P. aeruginosa expressing different resistant phenotypes and is now responsible for notable epidemics within hospital settings. Considering this, we aimed to evaluate the synergistic combination of fortimicin with other traditional anti-pseudomonal agents and to analyze the resistome of pan-drug resistant (PDR) isolate. METHODS: Standard methods were used for analyzing the antimicrobial susceptibility tests. The checkerboard technique was used for the in vitro assessment of fortimicin antibiotic combinations against 51 MDR P. aeruginosa and whole genome sequencing was used to determine the resistome of PDR isolate. RESULTS: Out of 51 MDR P. aeruginosa, the highest synergistic effect was recorded for a combination of fortimicin with ß-lactam group as meropenem, ceftazidime, and aztreonam at 71%, 59% and 43%, respectively. Of note, 56.8%, 39.2%, and 37.2% of the tested MDR isolates that had synergistic effects were also resistant to meropenem, ceftazidime, and aztreonam, respectively. The highest additive effects were recorded for combining fortimicin with amikacin (69%) and cefepime (44%) against MDR P. aeruginosa. Resistome analysis of the PDR isolate reflected its association with the antibiotic resistance phenotype. It ensured the presence of a wide variety of antibiotic-resistant genes (ß-lactamases, aminoglycosides modifying enzymes, and efflux pump), rendering the isolate resistant to all clinically relevant anti-pseudomonal agents. CONCLUSION: Fortimicin in combination with classical anti-pseudomonal agents had shown promising synergistic activity against MDR P. aeruginosa. Resistome profiling of PDR P. aeruginosa enhanced the rapid identification of antibiotic resistance genes that are likely linked to the appearance of this resistant phenotype and may pave the way to tackle antimicrobial resistance issues shortly.

Oligoribonuclease mediates high adaptability of P. aeruginosa through metabolic conversion.

BACKGROUND: Oligoribonuclease (orn) of P. aeruginosa is a highly conserved exonuclease, which can regulate the global gene expression levels of bacteria through regulation of both the nanoRNA and c-di-GMP. NanoRNA can regulate the expression of the bacterial global genome as a transcription initiator, and c-di-GMP is the most widely second messenger in bacterial cells. OBJECTIVE: This study seeks to elucidate on the regulation by orn on pathogenicity of P. aeruginosa. METHODS: P. aeruginosa with orn deletion was constructed by suicide plasmid homologous recombination method. The possible regulatory process of orn was analyzed by TMT quantitative labeling proteomics. Then experiments were conducted to verify the changes of Δorn on bacterial motility, virulence and biofilm formation. Bacterial pathogenicity was further detected in cell and animal skin trauma models. ELISA detection c-di-GMP concentration and colony aggregation and biofilm formation were observed by scanning electron microscope. RESULTS: orn deletion changed the global metabolism of P. aeruginosa and reduced intracellular energy metabolism. It leads to the disorder of the quorum sensing system, the reduction of bacterial motility and virulence factors pyocyanin and rhamnolipids. But, orn deletion enhanced pathogenicity in vitro and in vivo, a high level of c-di-GMP and biofilm development of P. aeruginosa. CONCLUSION: orn regulates the ability of P. aeruginosa to adapt to the external environment.

Vitamin D and vitamin K1 as novel inhibitors of biofilm in Gram-negative bacteria.

BACKGROUND: The persistent surge in antimicrobial resistance represents a global disaster. The initial attachment and maturation of microbial biofilms are intimately related to antimicrobial resistance, which in turn exacerbates the challenge of eradicating bacterial infections. Consequently, there is a pressing need for novel therapies to be employed either independently or as adjuvants to diminish bacterial virulence and pathogenicity. In this context, we propose a novel approach focusing on vitamin D and vitamin K1 as potential antibiofilm agents that target Gram-negative bacteria which are hazardous to human health. RESULTS: Out of 130 Gram-negative bacterial isolates, 117 were confirmed to be A. baumannii (21 isolates, 17.9%), K. pneumoniae (40 isolates, 34.2%) and P. aeruginosa (56 isolates, 47.9%). The majority of the isolates were obtained from blood and wound specimens (27.4% each). Most of the isolates exhibited high resistance rates to ß-lactams (60.7-100%), ciprofloxacin (62.5-100%), amikacin (53.6-76.2%) and gentamicin (65-71.4%). Approximately 93.2% of the isolates were biofilm producers, with 6.8% categorized as weak, 42.7% as moderate, and 50.4% as strong biofilm producers. The minimum inhibitory concentrations (MICs) of vitamin D and vitamin K1 were 625-1250 µg mL-1 and 2500-5000 µg mL-1, respectively, against A. baumannii (A5, A20 and A21), K. pneumoniae (K25, K27 and K28), and P. aeruginosa (P8, P16, P24 and P27) clinical isolates and standard strains A. baumannii (ATCC 19606 and ATCC 17978), K. pneumoniae (ATCC 51503) and P. aeruginosa PAO1 and PAO14. Both vitamins significantly decreased bacterial attachment and significantly eradicated mature biofilms developed by the selected standard and clinical Gram-negative isolates. The anti-biofilm effects of both supplements were confirmed by a notable decrease in the relative expression of the biofilm-encoding genes cusD, bssS and pelA in A. baumannii A5, K. pneumoniae K28 and P. aeruginosa P16, respectively. CONCLUSION: This study highlights the anti-biofilm activity of vitamins D and K1 against the tested Gram-negative strains, which emphasizes the potential of these vitamins for use as adjuvant therapies to increase the efficacy of treatment for infections caused by multidrug-resistant (MDR) strains and biofilm-forming phenotypes. However, further validation through in vivo studies is needed to confirm these promising results.

Dual action of benzaldehydes: Inhibiting quorum sensing and enhancing antibiotic efficacy for controlling Pseudomonas aeruginosa biofilms.

Quorum sensing (QS) has a central role in biofilm lifestyle and antimicrobial resistance, and disrupting these signaling pathways is a promising strategy to control bacterial pathogenicity and virulence. In this study, the efficacy of three structurally related benzaldehydes (4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde)) in disrupting the las and pqs systems of Pseudomonas aeruginosa was investigated using bioreporter strains and computational simulations. Additionally, these benzaldehydes were combined with tobramycin and ciprofloxacin antibiotics to evaluate their ability to increase antibiotic efficacy in preventing and eradicating P. aeruginosa biofilms. To this end, the total biomass, metabolic activity and culturability of the biofilm cells were determined. In vitro assays results indicated that the aromatic aldehydes have potential to inhibit the las and pqs systems by > 80 %. Molecular docking studies supported these findings, revealing the aldehydes binding in the same pocket as the natural ligands or receptor proteins (LasR, PQSA, PQSE, PQSR). Benzaldehydes were shown to act as virulence factor attenuators, with vanillin achieving a 48 % reduction in pyocyanin production. The benzaldehyde-tobramycin combination led not only to a 60 % reduction in biomass production but also to a 90 % reduction in the metabolic activity of established biofilms. A similar result was observed when benzaldehydes were combined with ciprofloxacin. 4-Hydroxybenzaldehyde demonstrated relevant action in increasing biofilm susceptibility to ciprofloxacin, resulting in a 65 % reduction in biomass. This study discloses, for the first time, that the benzaldehydes studied are potent QS inhibitors and also enhancers of antibiotics antibiofilm activity against P. aeruginosa.

Repression of resistance mechanisms of Pseudomonas aeruginosa: implications of the combination of antibiotics and phytoconstituents.

Antimicrobial resistance is a prevalent problem witnessed globally and creating an alarming situation for the treatment of infections caused by resistant pathogens. Available armaments such as antibiotics often fail to exhibit the intended action against resistant pathogens, leading to failure in the treatments that are causing mortality. New antibiotics or a new treatment approach is necessary to combat this situation. P. aeruginosa is an opportunistic drug resistant pathogen and is the sixth most common cause of nosocomial infections. P. aeruginosa due to its genome organization and other factors are exhibiting resistance against drugs. Bacterial biofilm formation, low permeability of outer membrane, the production of the beta-lactamase, and the production of several efflux systems limits the antibacterial potential of several classes of antibiotics. Combination of phytoconstituents with antibiotics is a promising strategy to combat multidrug resistant P. aeruginosa. Phytoconstituents such as flavonoids, terpenoids, alkaloids, polypeptides, phenolics, and essential oils are well known antibacterial agents. In this review, the activity of combination of the phytoconstituents and antibiotics, and their corresponding mechanism of action was discussed elaborately. The combination of antibiotics and plant-derived compounds exhibited better efficacy compared to antibiotics alone against the antibiotic resistance P. aeruginosa infections.

Performance evaluation of rhamnolipid biosurfactant produced by Pseudomonas aeruginosa and its effect on marine oil-spill remediation.

This study aimed to reveal that the effect of biosurfactant on the dispersion and degradation of crude oil. Whole genome analysis showed that Pseudomonas aeruginosa GB-3 contained abundant genes involved in biosurfactant synthesis and metabolic processes and had the potential to degrade oil. The biosurfactant produced by strain GB-3 was screened by various methods. The results showed that the surface tension reduction activity was 28.6 mN·m-1 and emulsification stability was exhibited at different pH, salinity and temperature. The biosurfactant was identified as rhamnolipid by LC-MS and FTIR. The fermentation conditions of strain GB-3 were optimized by response surface methodology, finally the optimal system (carbon source: glucose, nitrogen source: ammonium sulfate, C/N ratio:16:1, pH: 7, temperature: 30-35 °C) was determined. Compared with the initial fermentation, the yield of biosurfactant increased by 4.4 times after optimization. In addition, rhamnolipid biosurfactant as a dispersant could make the dispersion of crude oil reach 38% within seven days, which enhanced the bioavailability of crude oil. As a biostimulant, it could also improve the activity of indigenous microorganism and increase the degradation rate of crude oil by 10-15%. This study suggested that rhamnolipid biosurfactant had application prospect in bioremediation of marine oil-spill.

Bacterial biofilm growth and perturbation by serine protease from Bacillus sp.

In nature, bacteria are ubiquitous and can be categorized as beneficial or harmless to humans, but most bacteria have one thing in common which is their ability to produce biofilm. Biofilm is encased within an extracellular polymeric substance (EPS) which provides resistance against antimicrobial agents. Protease enzymes have the potential to degrade or promote the growth of bacterial biofilms. In this study, the effects of a recombinant intracellular serine protease from Bacillus sp. (SPB) on biofilms from Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were analyzed. SPB was purified using HisTrap HP column and concentrated using Amicon 30 ultra-centrifugal filter. SPB was added with varying enzyme activity and assay incubation period after biofilms were formed in 96-well plates. SPB was observed to have contrasting effects on different bacterial biofilms, where biofilm degradations were observed for both 7-day-old A. baumannii (37.26%) and S. aureus (71.51%) biofilms. Meanwhile, SPB promoted growth of P. aeruginosa biofilm up to 176.32%. Compatibility between protein components in S. aureus biofilm with SPB as well as a simpler membrane structure morphology led to higher biofilm degradation for S. aureus compared to A. baumannii. However, SPB promoted growth of P. aeruginosa biofilm due likely to its degrading protein factors that are responsible for biofilm detachment and dispersion, thus resulting in more multi-layered biofilm formation. Commercial protease Savinase which was used as a comparison showed degradation for all three bacterial biofilms. The results obtained are unique and will expand our understanding on the effects that bacterial proteases have toward biofilms.

Saccharomyces cerevisiae ß-glucan improves the response of trained macrophages to severe P. aeruginosa infections.

OBJECTIVE P. AERUGINOSA: (PA), the major pathogen of lung cystic fibrosis (CF), polarizes macrophages into hyperinflammatory tissue damaging phenotype. The main aim of this study was to verify whether training of macrophages with ß-glucan might improve their response to P. aeruginosa infections. METHODS: To perform this task C57BL/6 mice sensitive to infections with P. aeruginosa were used. Peritoneal macrophages were trained with Saccharomyces cerevisiae ß-glucan and exposed to PA57, the strong biofilm-forming bacterial strain isolated from the patient with severe lung CF. The release of cytokines and the expression of macrophage phenotypic markers were measured. A quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. The effect of in vivo ß-glucan-trained macrophages in the air pouch model of PA57 infection was investigated. In all experiments the effect of trained and naïve macrophages was compared. RESULTS: Trained macrophages acquired a specific phenotype with mixed pro-inflammatory and pro-resolution characteristics, however they retained anti-bacterial properties. Most importantly, transfer of trained macrophages into infected air pouches markedly ameliorated the course of infection. PA57 bacterial growth and formation of biofilm were significantly suppressed. The level of serum amyloid A (SAA), a systemic inflammation biomarker, was reduced. CONCLUSIONS: Training of murine macrophages with S. cerevisiae ß-glucan improved macrophage defense properties along with inhibition of secretion of some detrimental inflammatory agents. We suggest that training of macrophages with such ß-glucans might be a new therapeutic strategy in P. aeruginosa biofilm infections, including CF, to promote eradication of pathogens and resolution of inflammation.

Activity of imipenem/relebactam and comparators against KPC-producing Klebsiella pneumoniae and imipenem-resistant Pseudomonas aeruginosa.

PURPOSE: Relebactam is a novel ß-lactamase inhibitor, which, when combined with imipenem/cilastatin, is active against both class A and class C ß-lactamases. To evaluate in vitro antimicrobial activity of imipenem/relebactam against a collection of recent clinical isolates of carbapenem-non-susceptible P. aeruginosa and K. pneumoniae ST258 and ST512 KPC producers belonging to different lineages from hospitals in Southern Spain. METHODS: Six hundred and seventy-eight isolates were tested: 265 K. pneumoniae (230 ST512/KPC-3 and 35 ST258/KPC-3) and 413 carbapenem-non-susceptible P. aeruginosa. Imipenem, piperacillin/tazobactam, ceftazidime, cefepime, aztreonam, ceftolozane/tazobactam, meropenem, amikacin, ciprofloxacin, colistin, and ceftazidime/avibactam were used as comparators against P. aeruginosa. Against K. pneumoniae ceftazidime, cefepime, aztreonam, and ceftolozane/tazobactam were not tested, and tigecycline was studied instead. MICs were determined in duplicate by broth microdilution according to EUCAST guidelines. RESULTS: Imipenem/relebactam displayed potent in vitro activity against both sequence types of KPC-3-producing K. pneumoniae. MIC50 and MIC90 values were 0.25 mg/L and 1 mg/L, respectively, with percent of susceptible isolates >97%. Only three K. pneumoniae ST512/KPC-3 isolates and one ST258/KPC-3 were resistant to imipenem/relebactam. Relebactam sensitized 98.5% of K. pneumoniae isolates resistant to imipenem. The activity of imipenem/relebactam against P. aeruginosa was moderate (susceptibility rate: 62.7%). Analysis of the acquired and mutational resistome of isolates with high levels of resistance to imipenem/relebactam has not shown a clear association between them. CONCLUSION: Imipenem/relebactam showed excellent activity against K. pneumoniae KPC-3. The activity of imipenem/relebactam against imipenem-resistant P. aeruginosa was moderate.