Extending the Potency and Lifespan of Antibiotics: Inhibitors of Gram-Negative Bacterial Efflux Pumps.

Efflux is a natural process found in all prokaryotic and eukaryotic cells that removes a diverse range of substrates from inside to outside. Many antibiotics are substrates of bacterial efflux pumps, and modifications to the structure or overexpression of efflux pumps are an important resistance mechanism utilized by many multidrug-resistant bacteria. Therefore, chemical inhibition of bacterial efflux to revitalize existing antibiotics has been considered a promising approach for antimicrobial chemotherapy over two decades, and various strategies have been employed. In this review, we provide an overview of bacterial multidrug resistance (MDR) efflux pumps, of which the resistance nodulation division (RND) efflux pumps are considered the most clinically relevant in Gram-negative bacteria, and describe over 50 efflux inhibitors that target such systems. Although numerous efflux inhibitors have been identified to date, none have progressed into clinical use because of formulation, toxicity, and pharmacokinetic issues or a narrow spectrum of inhibition. For these reasons, the development of efflux inhibitors has been considered a difficult and complex area of research, and few active preclinical studies on efflux inhibitors are in progress. However, recently developed tools, including but not limited to computational tools including molecular docking models, offer hope that further research on efflux inhibitors can be a platform for research and development of new bacterial efflux inhibitors.

Epidemiology, Clinical, and Microbiological Characteristics of Multidrug-Resistant Gram-Negative Bacteremia in Qatar.

Antimicrobial resistance is a global healthcare threat with significant clinical and economic consequences peaking at secondary and tertiary care hospitals where multidrug-resistant Gram-negative bacteria (MDR GNB) lead to poor outcomes. A prospective study was conducted between January and December 2019 for all invasive bloodstream infections (BSIs) secondary to MDR GNB in Qatar identified during routine microbiological service to examine their clinical, microbiological, and genomic characteristics. Out of 3238 episodes of GNB BSIs, the prevalence of MDR GNB was 13% (429/3238). The predominant MDR pathogens were Escherichia coli (62.7%), Klebsiella pneumoniae (20.4%), Salmonella species (6.6%), and Pseudomonas aeruginosa (5.3%), while out of 245 clinically evaluated patients, the majority were adult males, with the elderly constituting almost one-third of the cohort and with highest observed risk for prolonged hospital stays. The risk factors identified included multiple comorbidities, recent healthcare contact, previous antimicrobial therapy, and admission to critical care. The in-hospital mortality rate was recorded at 25.7%, associated with multiple comorbidities, admission to critical care, and the acquisition of MDR Pseudomonas aeruginosa. Resistant pathogens demonstrated high levels of antimicrobial resistance but noticeable susceptibility to amikacin and carbapenems. Genomic analysis revealed that Escherichia coli ST131 and Salmonella enterica ST1 were the predominant clones not observed with other pathogens.

Current Epidemiological Status and Antibiotic Resistance Profile of Serratia marcescens.

The spread of antibiotic resistance represents a serious worldwide public health issue, underscoring the importance of epidemiology research in determining antimicrobial strategies. The purpose of this research was to investigate antibiotic resistance in Serratia marcescens isolates from clinical samples over seven years at the University Hospital "San Giovanni di Dio e Ruggi d'Aragona" in Salerno, Italy. S. marcescens is an important opportunistic pathogen associated with a wide spectrum of clinical diseases, including pneumonia, keratitis, meningitis, and urinary tract and wound infections. Outbreaks of nosocomial infections by S. marcescens strains have been documented in high-risk settings, mainly affecting immunocompromised patients and newborns. The primary objective of this study is to assess the rates of antibiotic resistance over the years to deal with a future emergency which includes the failure of various therapies due to antibiotic resistance. During the investigation, a total of 396 species of S. marcescens were isolated from various clinical samples, mainly from broncho-aspirates and sputum (31.6%) and blood cultures (21.5%). Antibiotics that showed the greatest susceptibility included ceftazidime/avibactam, amikacin, trimethoprim/sulfamethoxazole, and selected members of the cephalosporin class. However, a disconcerting trend of increasing rates of carbapenem resistance was outlined over the observation period. The absence of effective countermeasures, combined with growing antibiotic resistance that negates the effectiveness of multiple antibiotics, highlights the potential for S. marcescens infections to trigger serious clinical complications and increased mortality rates. The surveillance of Serratia marcescens infections constitutes a pivotal element in refining empiric therapy to mitigate the dissemination of antimicrobial resistance.

Hospital Wastes as Potential Sources for Multi-Drug-Resistant ESBL-Producing Bacteria at a Tertiary Hospital in Ethiopia.

The hospital environment is increasingly becoming an important reservoir for multi-drug-resistant (MDR) Gram-negative bacteria, posing serious challenges to efforts to combat antimicrobial resistance (AMR). This study aimed to investigate the role of hospital waste as a potential source of MDR ESBL-producing bacteria. Samples were collected from multiple sources within a hospital and its vicinity, including surface swabs, houseflies, and sewage samples. The samples were subsequently processed in a microbiology laboratory to identify potential pathogenic bacteria and confirmed using MALDI-TOF MS. Bacteria were isolated from 87% of samples, with the predominant isolates being E. coli (30.5%), Klebsiella spp. (12.4%), Providencia spp. (12.4%), and Proteus spp. (11.9%). According to the double disc synergy test (DDST) analysis, nearly half (49.2%) of the bacteria were identified as ESBL producers. However, despite exhibiting complete resistance to beta-lactam antibiotics, 11.8% of them did not test positive for ESBL production. The characterization of E. coli revealed that 30.6% and 5.6% of them carried blaCTX-M group 1 type-15 and blaNDM genes, respectively. This finding emphasizes the importance of proper hospital sanitation and waste management practices to mitigate the spread of AMR within the healthcare setting and safeguard the health of both patients and the wider community.

BacteSign: Building a Findable, Accessible, Interoperable, and Reusable (FAIR) Database for Universal Bacterial Identification.

With the increasing incidence of diverse global bacterial outbreaks, it is important to build an immutable decentralized database that can capture regional changes in bacterial resistance with time. Herein, we investigate the use of a rapid 3D printed µbiochamber with a laser-ablated interdigitated electrode developed for biofilm analysis of Pseudomonas aeruginosa, Acinetobacter baumannii and Bacillus subtilis using electrochemical biological impedance spectroscopy (EBIS) across a 48 h spectrum, along with novel ladder-based minimum inhibitory concentration (MIC) stencil tests against oxytetracycline, kanamycin, penicillin G and streptomycin. Furthermore, in this investigation, a search query database has been built demonstrating the deterministic nature of the bacterial strains with real and imaginary impedance, phase, and capacitance, showing increased bacterial specification selectivity in the 9772.37 Hz range.

Equinins as Novel Broad-Spectrum Antimicrobial Peptides Isolated from the Cnidarian Actinia equina (Linnaeus, 1758).

Sea anemones are valuable for therapeutic research as a diversified source of bioactive molecules, due to their diverse bioactive molecules linked to predation and defence mechanisms involving toxins and antimicrobial peptides. Acid extracts from Actinia equina tentacles and body were examined for antibacterial activity against Gram-positive, Gram-negative bacteria, and fungi. The peptide fractions showed interesting minimum inhibitory concentration (MIC) values (up to 0.125 µg/mL) against the tested pathogens. Further investigation and characterization of tentacle acid extracts with significant antimicrobial activity led to the purification of peptides through reverse phase chromatography on solid phase and HPLC. Broad-spectrum antimicrobial peptide activity was found in 40% acetonitrile fractions. The resulting peptides had a molecular mass of 2612.91 and 3934.827 Da and MIC ranging from 0.06 to 0.20 mg/mL. Sequencing revealed similarities to AMPs found in amphibians, fish, and Cnidaria, with anti-Gram+, Gram-, antifungal, candidacidal, anti-methicillin-resistant Staphylococcus aureus, carbapenemase-producing, vancomycin-resistant bacteria, and multi-drug resistant activity. Peptides 6.2 and 7.3, named Equinin A and B, respectively, were synthesized and evaluated in vitro towards the above-mentioned bacterial pathogens. Equinin B exerted interesting antibacterial activity (MIC and bactericidal concentrations of 1 mg/mL and 0.25 mg/mL, respectively) and gene organization supporting its potential in applied research.

A comprehensive review of recent developments in the gram-negative bacterial UDP-2,3-diacylglucosamine hydrolase (LpxH) enzyme.

The emergence of multidrug resistance has provided a great challenge to treat nosocomial infections, which have become a major health threat around the globe. Lipid A (an active endotoxin component), the final product of the Raetz lipid A metabolism pathway, is a membrane anchor of lipopolysaccharide (LPS) of the gram-negative bacterial outer membrane. It shields bacterial cells and serves as a protective barrier from antibiotics, thereby eliciting host response and making it difficult to destroy. UDP-2,3-diacylglucosamine pyrophosphate hydrolase (LpxH), a crucial peripheral membrane enzyme of the Raetz pathway, turned out to be the potential target to inhibit the production of Lipid A. This review provides a comprehensive compilation of information regarding the structural and functional aspects of LpxH, as well as its analogous LpxI and LpxG. In addition, apart from by providing a broader understanding of the enzyme-inhibitor mechanism, this review facilitates the development of novel drug candidates that can inhibit the pathogenicity of the lethal bacterium.

Ceftazidime-Avibactam Combination Therapy versus Monotherapy for the Treatment Carbapenem-Resistant Gram-Negative Bacterial Infections: A Retrospective Observational Study.

Purpose: Since the introduction of ceftazidime-avibactam (CZA) in the Chinese market, accumulating clinical evidence has substantiated its efficacy in the treatment of infections caused by carbapenem-resistant gram-negative bacteria (CR-GNB). Nevertheless, an ongoing debate persists concerning the choice between monotherapy and combination therapy when devising clinical anti-infection protocols. Patients and Methods: This retrospective, single-center observational study enrolled patients with CR-GNB infections who received CZA treatment between December 2019 and August 2023. The primary outcome assessed was 30-day mortality, and the secondary outcome measured was 14-day bacterial clearance. A multivariate Cox regression model was used to identify variables that were independently associated with 30-day mortality rate. Results: Eighty-three patients were enrolled in the study; of which, 45 received CZA monotherapy, whereas 38 received combination therapy. The overall 30-day mortality rate was 31.3%, and no significant difference was observed in the 30-day mortality rates between the CZA combination therapy and monotherapy groups (31.6% vs 31.1%, p=0.963). After adjustment by propensity score matching, the 30-day mortality rate was not significantly different between the two groups (28.6% vs 31.4%, p=0.794). Multivariate COX analysis revealed that age and SOFA score were independent predictors of 30-day mortality. Conclusion: Combination therapy with CZA and other antimicrobials was not found to have an advantage over monotherapy in reducing the 30-day mortality rate.

Differential development of antibiotic resistance and virulence between Acinetobacter species.

The two species that account for most cases of Acinetobacter-associated bacteremia in the United Kingdom are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and Acinetobacter baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii, and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30, respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defense systems and harbored a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm, and infect both in vivo and in in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesize that the fact that it only harbors a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram-negative bacteria. IMPORTANCE: Acinetobacter lwoffii is often a harmless commensal but is also an emerging pathogen and is the most common cause of Acinetobacter-derived bloodstream infections in England and Wales. In contrast to the well-studied and often highly drug-resistant A. baumannii, A. lwoffii has remained susceptible to antibiotics. This study explains why this organism has not evolved resistance to antibiotics. These new insights are important to understand why and how some species develop antibiotic resistance, while others do not, and could inform future novel treatment strategies.

Clinical characteristics and antimicrobial therapy of healthcare-associated carbapenem-non-susceptible gram-negative bacterial meningitis: a 16-year retrospective cohort study.

OBJECTIVE: Healthcare-associated Gram-negative bacterial meningitis is a substantial clinical issue with poor outcomes, especially for neurosurgical patients. Here, we aimed to study the characteristics and treatment options of patients with healthcare-associated carbapenem-non-susceptible (Carba-NS) Gram-negative bacterial meningitis. METHODS: This observational cohort study was conducted at a teaching hospital from 2004 to 2019. The clinical characteristics of patients with meningitis with Carba-NS and carbapenem-susceptible (Carba-S) bacilli were compared, and the antimicrobial chemotherapy regimens and outcomes for Carba-NS Gram-negative bacterial meningitis were analyzed. RESULTS: A total of 505 patients were included, of whom 83.8% were post-neurosurgical patients. The most common isolates were Acinetobacter spp. and Klebsiella spp., which had meropenem-resistance rates of 50.6% and 42.5%, respectively, and showed a markedly growing carbapenem-resistance trend. Kaplan-Meier curve analysis revealed that Carba-NS Gram-negative bacilli were associated with a significantly higher in-hospital mortality rate (18.8%, 35/186) compared to the Carba-S group (7.4%, 9/122; P = 0.001). For Carba-NS Enterobacterales meningitis, aminoglycoside-based and trimethoprim-sulfamethoxazole-based regimens yielded significantly higher clinical efficacy rates than non-aminoglycoside-based and non-trimethoprim-sulfamethoxazole-based regimens (69.0% vs. 38.7%, P = 0.019 and 81.8% vs. 46.9%, P = 0.036, respectively). For Carba-NS A. baumannii complex meningitis, tetracycline-based (including doxycycline, minocycline, or tigecycline) therapy achieved a significantly higher clinical efficacy rate (62.9%, 22/35) than the non-tetracycline-based therapy group (40.4%, 19/47; P = 0.044). CONCLUSIONS: Our findings revealed that Carba-NS Gram-negative bacilli are associated with higher in-hospital mortality in patients with healthcare-associated meningitis. The combination therapies involving particular old antibiotics may improve patients' outcome. TRIAL REGISTRATION: This study was registered on the Chinese Clinical Trial Register under ChiCTR2000036572 (08/2020).

The synergistic antibacterial activity and mechanism of colistin-oxethazaine combination against gram-negative pathogens.

Background: The rapid spread of bacteria with plasmid-mediated resistance to antibiotics poses a serious threat to public health. The search for potential compounds that can increase the antibacterial activity of existing antibiotics is a promising strategy for addressing this issue. Methods: Synergistic activity of the FDA-approved agent oxethazine combined with colistin was investigated in vitro using checkerboard assays and time-kill curves. The synergistic mechanisms of their combination of oxethazine and colistin was explored by fluorescent dye, scanning electron microscopy (SEM) and LC-MS/MS. The synergistic efficacy was evaluated in vivo by the Galleria mellonella and mouse sepsis models. Results: In this study, we found that oxethazine could effectively enhance the antibacterial activity of colistin against both mcr-positive and -negative pathogens, and mechanistic assays revealed that oxethazine could improve the ability of colistin to destruct bacterial outer membrane and cytoplasmic membrane permeability. In addition, their combination triggered the accumulation of reactive oxygen species causing additional damage to the membrane structure resulting in cell death. Furthermore, oxethazine significantly enhanced the therapeutic efficacy of colistin in two animal models. Conclusion: These results suggested that oxethazine, as a promising antibiotic adjuvant, can effectively enhance colistin activity, providing a potential strategy for treating multidrug-resistant bacteria.

Evaluation to Determine Antibiotic Resistance Reduction in Gram-Negative Bacteria from 2019 to 2021 by the Saudi Arabia National Plan.

Introduction: The main objective of this study was to investigate the three-year evaluation of antibiotic resistance (AR) of multi-drug-resistant organisms and extended-spectrum beta-lactamase (ESBL)-resistant rate of gram-negative bacteria in one of the largest hospitals by the Saudi Arabia Nation Plan. Methods: This study was conducted in the Department of Laboratory Medicine, in a private hospital in Riyadh City, Saudi Arabia, from January 2019 to December 2021 in 120-bed private hospitals. A total of 4700 gram-negative isolated organisms were obtained from the various specimens of the patients, and antibiotic sensitivity tests were performed. According to the manufacturer's instructions, the inoculum prepared was applied to two test cards, one for the identification system VITEK 2 ID-GNB and another for susceptibility testing antimicrobial susceptibility testing (AST) No. 12. Result: The most common gram-negative bacteria isolated was Escherichia coli (2706/4700; 57.57%), followed by Klebsiella pneumoniae (905/4700; 19.25%) and Pseudomonas aeruginosa (395/4700; 8.40%). Escherichia coli's highest AR reduction was reported for cefotaxime (CTX) of 29% (295/1018; 29%, 172/818; 21%, 0/870; 0%) for 2019, 2020, and 2021, respectively. Except for Salmonella species, which displayed enhanced AR, the ESBL and multidrug-resistant (MDR) rates decreased significantly (p 0.05) for most bacteria. Conclusion: This study helps to understand the maximum number of gram-negative bacteria susceptible to the Saudi National Action Plan (SNAP) to decrease the prevalence of AR, ESBL, and MDR. To comprehensively understand SNAP's effectiveness, other trials involving gram-positive bacteria should be considered.

Efficacy of Random Forest Models in Predicting Multidrug-Resistant Gram-Negative Bacterial Nosocomial Infections Compared to Traditional Logistic Regression Models.

This study evaluates whether random forest (RF) models are as effective as traditional Logistic Regression (LR) models in predicting multidrug-resistant Gram-negative bacterial nosocomial infections. Data were collected from 541 patients with hospital-acquired Gram-negative bacterial infections at two tertiary-level hospitals in Urumqi, Xinjiang, China, from August 2022 to November 2023. Relevant literature informed the selection of significant predictors based on patients' pre-infection clinical information and medication history. The data were split into a training set of 379 cases and a validation set of 162 cases, adhering to a 7:3 ratio. Both RF and LR models were developed using the training set and subsequently evaluated on the validation set. The LR model achieved an accuracy of 84.57%, sensitivity of 82.89%, specificity of 80.10%, positive predictive value of 84%, negative predictive value of 85.06%, and a Yoden index of 0.69. In contrast, the RF model demonstrated superior performance with an accuracy of 89.51%, sensitivity of 90.79%, specificity of 88.37%, positive predictive value of 87.34%, negative predictive value of 91.57%, and a Yoden index of 0.79. Receiver operating characteristic curve analysis revealed an area under the curve of 0.91 for the LR model and 0.94 for the RF model. These findings indicate that the RF model surpasses the LR model in specificity, sensitivity, and accuracy in predicting hospital-acquired multidrug-resistant Gram-negative infections, showcasing its greater potential for clinical application.

Rhizoma Paridis saponins attenuate Gram-negative bacteria-induced inflammatory acne by binding to KEAP1 and modulating Nrf2 and MAPK pathways.

Acne vulgaris represents a chronic inflammatory condition, the pathogenesis of which is closely associated with the altered skin microbiome. Recent studies have implicated a profound role of Gram-negative bacteria in acne development, but there is a lack of antiacne agents targeting these bacteria. Polyphyllins are major components of Rhizoma Paridis with great anti-inflammatory potential. In this study, we aimed to evaluate the antiacne effects and the underlying mechanisms of PPH and a PPH-enriched Rhizoma Paridis extract (RPE) in treating the Gram-negative bacteria-induced acne. PPH and RPE treatments significantly suppressed the mRNA and protein expressions of interleukin (IL)-1ß and IL-6 in lipopolysaccharide (LPS)-induced RAW 264.7 and HaCaT cells, along with the intracellular reactive oxygen species (ROS) generation. Furthermore, PPH and RPE inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in LPS-induced RAW 264.7 cells. Based on molecular docking, PPH could bind to kelch-like ECH-associated protein 1 (KEAP1) protein. PPH and RPE treatments could activate nuclear factor erythroid 2-related factor 2 (NRF2) and upregulate haem oxygenase-1 (HO-1). Moreover, RPE suppressed the mitogen-activated protein kinase (MAPK) pathway. Therefore, PPH-enriched RPE showed anti-inflammatory and antioxidative effects in vitro, which is promising for alternative antiacne therapeutic.

In vitro activity of the newly approved antimicrobial agent Cefiderocol against Carbapenem resistant Gram negative clinical isolates.

INTRODUCTION: Carbapenem resistant Gram negative bacteria have emerged as priority pathogens in recent years. Cefiderocol is a siderophore cephalosporin licensed in 2019 with claimed activity against ESBL producing and carbapenem resistant bacteria with much better safety margin compared to colistin. The present study was undertaken to assess the in vitro activity of cefiderocol against carbapenem resistant clinical isolates, compared to some select antimicrobial agents including colistin. MATERIALS AND METHODS: Seventy-seven isolates of Gram negative bacteria belonging to the three commonly encountered groups of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter spp were included. Susceptibility testing for Cefiderocol was determined by Kirby-Bauer's disk diffusion technique as per CLSI guidelines using Cefiderocol disc (30 µg). Sensitivity for the other agents were determined using automated system. RESULTS: Of the 77 isolates, 58.4% belonged to Enterobacterales, followed by P.aeruginosa (27.3%) and Acinetobacter spp (14.3%). Three out of 45 Enterobacterales isolates, one out of 21 P.aeruginosa and none in the Acinetobacter group were found resistant to cefiderocol. All the isolates were intermediate sensitive (I) for colistin since the "susceptible" interpretive category has been eliminated. Tigecycline showed good activity (80.0% sensitive) against Enterobacterales followed by aztreonam (71.1% sensitive). CONCLUSION: Cefiderocol is not yet available in India and our study is possibly the second one from this country demonstrating in vitro resistance to this important antimicrobial agent. However, with a relatively better safety profile compared to colistin, cefiderocol can be an important agent to combat these highly resistant pathogens.

A rapid, inexpensive and effective method for the efficient isolation of genomic DNA from Gram-negative bacteria.

Currently, there are several protocols to extract bacterial DNA based on different principles. However, the quantity and the quality of the DNA obtained by each method are highly variable and microorganism dependent. In most of these classical crude methods, highly toxic and hazardous organic solvents such as phenol and chloroform are used for deproteinization, whereas in certain protocols, expensive enzymes including RNases and Proteinases are used. This study was designed to introduce a simple, rapid, inexpensive and effective genomic DNA isolation procedure for Gram-negative bacteria, without the usage of toxic chemicals and costly enzymes. This novel method was compared with another classical method known as the salting-out method, which uses proteinase-K. Concentration and yield of the extracted DNA were determined by gel electrophoresis by comparing the gel band intensity of the sample DNA to that of a DNA quantitation standard and by the Quantus™ fluorometer. According to the results, the yield of extracted DNA was higher in the novel method compared to the salting-out method. Moreover, the entire process was accomplished in less than 2 h with the novel method. Purity and integrity of extracted genomic DNA by both methods were similar. In addition, the quality of DNA was determined using Multicopy Associated Filamentation (MAF) gene amplification by polymerase chain reaction (PCR). Thus, the described technique is non-toxic, less time and fund consuming, efficient and a well-suited method for routine DNA isolation from Gram negative bacteria.

Potential of using medicinal plant extracts as photosensitizers for antimicrobial photodynamic therapy.

Antimicrobial photodynamic therapy (APDT) is a promising approach to overcome antimicrobial resistance. However, for widespread implementation of this approach, approved photosensitizers are needed. In this study, we used commercially available preparations (Calendulae officinalis floridis extract, Chamomillae recutitae floridis extract, Achillea millefolii herbae extract; Hypericum perforatum extract; Eucalyptus viminalis folia extract) as photosensitizers for inactivation of gram-negative (Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacteria. Spectral-luminescent analysis has shown that the major chromophores are of chlorophyll (mainly chlorophyll a and b) and hypericin nature. The extracts are efficient generators of singlet oxygen with quantum yield (Î³Δ ) from 0.40 to 0.64 (reference compound, methylene blue with Î³Δ = 0.52). In APDT assays, bacteria before irradiation were incubated with extracts for 30 min. After irradiation and 24 h of incubation, colony-forming units (CFU) were counted. Upon exposure of P. aeruginosa to radiation of 405 nm, 590 nm, and 660 nm at equal energy dose of 30 J/cm2 (irradiance - 100 mW/cm2 , exposure time - 5 min), the most pronounced effect is observed with blue light (>3 log10 reduction); in case of S. aureus, the effect is approximately equivalent for light of indicated wavelengths and dose (>4 log10 reduction).

Examining Protein Translocation by ß-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.

ß-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded ß-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species.

Dissecting the Organization of a ß-Barrel Assembly Machinery (BAM) Complex by Neutron Reflectometry.

The ß-barrel assembly machinery (BAM) complex in Gram-negative bacteria facilitates the assembly of ß-barrel proteins into the outer membrane. Understanding the protein-protein interactions within this complex is essential for unravelling its functional mechanisms. Here, we present the use of neutron reflectometry for investigating the organization of ß-barrel membrane protein complexes in the membrane environment. The spatial organization, protein positioning, protein-lipid interactions, and conformational changes within the complex can be elucidated by this method.

Silencing gram-negative bacteria binding protein 1 decreases the immunity of Tribolium castaneum against bacteria.

Gram-negative bacteria binding proteins (GNBPs) have the ability to recognize molecular patterns associated with microbial pathogens (PAMPs), leading to the activation of immune responses downstream. In the genome of Tribolium castaneum, three GNBP genes have been identified; however, their immunological roles remain unexplored. In our study, a GNBP1, designated as TcGNBP1, were identified from the cDNA library of T. castaneum. The coding sequence of TcGNBP1 consisted of 1137 bps and resulted in the synthesis of a protein comprising 378 amino acids. This protein encompasses a signal peptide, a low-complexity region, and a glycoside hydrolase 16 domain. TcGNBP1 was strongly expressed in early adult stages, and mainly distributed in hemolymph and gut. Upon being challenged with Escherichia coli or Staphylococcus aureus, the transcript levels of TcGNBP1 were significantly changed at different time points. Through molecular docking and ELISA analysis, it was observed that TcGNBP1 has the ability to interact with lipopolysaccharides, peptidoglycan, and ß-1, 3-glucan. Based on these findings, it was further discovered that recombinant TcGNBP1 can directly bind to five different bacteria in a Ca2+-dependent manner. After knockdown of TcGNBP1 with RNA interference, expression of antimicrobial peptide genes and prophenoloxidase (proPO) activity were suppressed, the susceptibility of T. castaneum to E. coli or S. aureus infection was enhanced, leading to low survival rate. These results suggest a regulatory mechanism of TcGNBP1 in innate immunity of T. castaneum and provide a potential molecular target for dsRNA-based insect pest management.