Synthesis and structure-activity relationship of novel thiazole aminoguanidines against MRSA and Escherichia coli.

Novel lead thiazole aminoguanidines exhibited strong activity against Gram-positive bacteria. The potential targets of these substances are undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP). Here, we report the synthesis and antibacterial evaluation of a library of thiazole aminoguanidines analogues, wherein the rotatable bond is inserted between the C2 position of thiazole and hydrophobic group. The molecular flexibility is increased, and new analogues with strong activity against MRSA and E. coli are produced. The best compound 4i showed rapid sterilization and low tendency to induce bacterial resistance. The IC50 of compound 4i to EcUPPS enzyme is 145 µmol L-1 (58 µg mL-1). Compound 4i can also inhibit and destroy bacterial biofilms. These thiazole aminoguanidines can be developed as potential therapeutic candidates in the future.

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification.

The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa, which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa. The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.

Antimicrobial Peptide-Inspired Design of Amino-Modified Lignin with Improved Antimicrobial Activities.

A major challenge to make use of lignin as an antimicrobial material is the weak antimicrobial activity of industrial lignin. Inspired by the antimicrobial mechanism of actions of antimicrobial peptides, alkyldiamines were employed as lysine mimics for lignin modifications. Accordingly, aminoalkyl-modified lignins with different degrees of substitution of amino groups and different hydrophobicity were synthesized. The chemical structure, properties, and antimicrobial activities of the as-prepared aminoalkyl lignins were thoroughly characterized with state-of-the-art technologies. The results indicated that aminobutyl lignin showed enhanced antimicrobial activity against S. aureus and E. coli and performed even better than copper ions. The antimicrobial mechanism of action of the as-prepared aminobutyl lignin was similar to that of polylysine, which damaged the cell membrane, leading to the leakage of intracellular molecules and death of the cell. This study provides a feasible approach to afford modified lignin with enhanced antimicrobial performance, which would facilitate the high-value valorization of lignin as biological materials.

Fundicoccus culcitae sp. nov., a novel potential bacteriocin producing bacterium isolated from a spoiled eye mask.

A Gram-positive, facultatively anaerobic, oval beaded-shape, oxidase-negative, and non-motile bacterium designated DM20194951T was isolated from a spoiled eye mask obtained from Guangdong, China. Based on the 16S rRNA gene sequence, phylogenetic analysis indicated that strain DM20194951T showed the highest sequence similarity (95.8%) to Fundicoccus ignavus WS4937T. Meanwhile, strain DM20194951T could be distinguished from the type strains in the genus Fundicoccus by distinct phenotypic and genotypic traits. Strain DM20194951T grew variably with 1-2% (w/v) NaCl and tolerated pH 6.0-10.0. Growth was observed from 28 to 37 °C. The diagnostic diamino acids in the cell-wall peptidoglycan consisted of aspartic and glutamic acids as well as alanine. The predominant fatty acids were C18:1 ω9c, C16:0, and C16:1 ω9c. In the polar lipid profile, two glycolipids, three phospholipids, one phosphatidylglycerol, and one diphosphatidylglycerol were found. No respiratory quinones were detected. The DM20194951T genome is 3.2 Mb in size and contains a G + C content of 38.1%. A gene cluster for lactococcin 972 family bacteriocin production was found in the DM20194951T genome. Based on morphological, genotypic, and phylogenetic data, strain DM20194951T should be considered to represent a novel species in the genus Fundicoccus, for which the name Fundicoccus culcitae sp. nov. is proposed with the type strain DM20194951T (= KCTC 43472T = GDMCC 1.3614T).

[Clinical Characteristics and Prognostic Factors of Patients with Primary Parotid Gland Lymphoma].

OBJECTIVE: To explore the clinical characteristics and prognostic factors of patients with primary parotid gland lymphoma, and construct a prognostic model nomogram for patients with primary diffuse large B-cell lymphoma (DLBCL) of parotid gland. METHODS: Primary parotid gland lymphoma and primary DLBCL of parotid gland patients from 1984 to 2016 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. Univariate and multivariate Cox regression analysis were conducted to determine the independent prognostic factors of primary parotid gland lymphoma and primary DLBCL of parotid gland, respectively. According to the established independent prognostic factors of primary DLBCL of parotid gland, nomogram was built to predict 3- and 5-year survival, and the discrimination and calibration of the model were evaluated by concordance index (C-index) and calibration plots. RESULTS: A total of 2 610 patients with primary parotid gland lymphoma were identified. Their median age was 66(15-99) years old, the male to female ratio was 1∶1.8, and 20.5% of them was primary DLBCL of parotid gland, which was the most common histological subtype in aggressive lymphomas. Multivariate Cox regression analysis showed that sex, age, Ann Arbor stage, years of diagnosis, marital status, histological subtype, surgery, and radiation were the independent prognostic factors of primary parotid gland lymphoma, while age, marital status, surgery, and chemotherapy were the independent prognostic factors of primary DLBCL of parotid gland. The C-index of the prediction model was 0.702(95%CI: 0.696-0.768), reflecting a good discrimination ability. The predicted value probability of the calibration plots was close to the actual value probability, reflecting a good accuracy ability. CONCLUSIONS: Sex, age, Ann Arbor stage, years of diagnosis, marital status, histological subtype, surgery, and radiation were the independent prognostic factors of primary parotid gland lymphoma. The nomogram survival prediction model for primary DLBCL of parotid gland patients can assist clinical decision effectively.

Geraniol attenuates virulence factors by inhibiting quorum sensing of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can cause severe respiratory tract infections. Geraniol, a chemical component of essential oils, has antimicrobial and anti-inflammatory activities, along with low toxicity. However, the effect and mechanism of geraniol against P. aeruginosa virulence factors are rarely studied. In this study, we investigated the quorum sensing (QS) inhibitory effects and mechanisms of geraniol against P. aeruginosa PAO1, using physiological and biochemical techniques, quantitative reverse transcription polymerase chain reaction, and transcriptomics. Geraniol slightly affected P. aeruginosa PAO1 growth, prolonged the lag phase, and delayed growth periods in a concentration-dependent manner. Geraniol inhibited three QS systems of P. aeruginosa, las, rhl, and pqs by suppressing the expression level of their key genes, including the three signal synthetase encoding genes of lasI, rhlI, and pqsABCDEH, and the corresponding signal receptor encoding genes of lasR, rhlR, and pqsR. Geraniol also suppressed certain virulence genes regulated by these three QS systems, including rhlABC, lasAB, lecAB, phzABMS, and pelABG, resulting in the attenuation of the related virulence factors, rhamnolipids, exoprotease LasA, elastase, lectin, pyocyanin, and biofilm. In conclusion, geraniol can suppress the virulence factors of P. aeruginosa PAO1 by inhibiting the three QS systems of las, rhl, and pqs. This study is significant for improving the treatment of bacterial infections caused by P. aeruginosa.

High value valorization of lignin as environmental benign antimicrobial.

Lignin is a natural aromatic polymer of p-hydroxyphenylpropanoids with various biological activities. Noticeably, plants have made use of lignin as biocides to defend themselves from pathogen microbial invasions. Thus, the use of isolated lignin as environmentally benign antimicrobial is believed to be a promising high value approach for lignin valorization. On the other hand, as green and sustainable product of plant photosynthesis, lignin should be beneficial to reduce the carbon footprint of antimicrobial industry. There have been many reports that make use of lignin to prepare antimicrobials for different applications. However, lignin is highly heterogeneous polymers different in their monomers, linkages, molecular weight, and functional groups. The structure and property relationship, and the mechanism of action of lignin as antimicrobial remains ambiguous. To show light on these issues, we reviewed the publications on lignin chemistry, antimicrobial activity of lignin models and isolated lignin and associated mechanism of actions, approaches in synthesis of lignin with improved antimicrobial activity, and the applications of lignin as antimicrobial in different fields. Hopefully, this review will help and inspire researchers in the preparation of lignin antimicrobial for their applications.

ompX contribute to biofilm formation, osmotic response and swimming motility in Citrobacter werkmanii.

Citrobacter werkmanii, an aerobe and mesophilic Proteobacterium, is universal in industrial putrefaction, coastal water, and human blood. Our previous studies have discovered that outer membrane protein X (OmpX) of C. werkmanii is involved in calcium response, but the underlying mechanisms and its molecular characteristics remain elusive. To that end, the ompX gene was deleted from the genome of C. werkmanii and its phenotypic variations were thoroughly investigated in conjunction with the wild type (WT) and complementary strains using biochemical and molecular techniques such as RNA-Seq, respectively. The results demonstrated that deleting ompX reduces biofilm formation on polystyrene and glass surfaces. Meanwhile, ΔompX's swimming ability but not for its twitching or swarming abilities, was also reduced on semi-solid plates compared with WT, which was caused by inhibition of flagellar assembly genes, such as flgC, flhB, and fliE, etc. Furthermore, ompX inactivation altered susceptibility to various bactericide classes, as well as responses to Ca2+ and Mg2+ stress. In addition, when compared to WT, ΔompX captures a total of 1,357 deferentially expressed genes (DEGs), of which 465 were up-regulated and 892 were down-regulated, which can be enriched into various GO ontology and KEGG pathway terms. Furthermore, ompX, as well as ompD and ompW, can be modulated at the transcriptional levels by rbsR and tdcA. Overall, the ompX gene contributed to a variety of biological functions in C. werkmanii and could be served as a targeted site for controlling biofilm formation and developing new bactericides.

Proteomic signatures of synergistic interactions in antimicrobials.

Mounting evidence has shown that antimicrobial agents can interfere synergistically with bacterial viability and proliferation when acting together at both the planktonic and biofilm levels without clear underlying molecular mechanisms. Here, multiplexed proteomics by iTRAQ was used to study the interplay between two biocides, the isothiazolone 1,2-benzisothiazolin-3-one (BIT) and the chelating agent disodium ethylenediaminetetraacetic acid (EDTA-2Na), employing the Citrobacter werkmanii as a model system. We first confirmed that these two biocides act synergistically on this bacterial species and then extracted the proteomic profiles of C. werkmanii cells in the presence of BIT, EDTA-2Na, and their combinations. In particular, we identified 43 core proteins that are differentially expressed in all three conditions simultaneously. Meanwhile, we found that these core proteins are consistently up-regulated when these two biocides are present, but not for single biocides, where we found a balanced mix of up- and down-regulation. Meanwhile, most of the deletion mutants of the core DEPs exhibited biofilm growth inhibition under joint biocide action, while their response was very heterogenous, with respect to the wild-type strain. Together, our results show that while BIT and EDTA-2Na act on multiple protein targets, they interact synergistically at the protein level in a very consistent manner. SIGNIFICANCE: Our preliminary experiments have demonstrated that a combination of 1,2-benzisothiazolin-3-one (BIT) and EDTA-2Na shows higher inhibitory effects on planktonic growth and biofilm formation in both C. werkmanii and Staphylococcus aureus than when these two biocides act alone. However, the mechanistic basis of such synergistic interaction is still unknown. Therefore, the key proteins involved in the above-mentioned enhanced antimicrobial synergy were elucidated using multiplexed proteomics analysis by isobaric tags for relative and absolute quantification (iTRAQ). Our results reveal that the joint action of BIT and EDTA-2Na induces consistent protein expression alteration in a set of core proteins of C. werkmanii, which underlies a strong synergistic antimicrobial effect, which increase our understanding of the action modes of BIT and EDTA-2Na as well as their combinations.

The interplays between epigallocatechin-3-gallate (EGCG) and Aspergillus niger RAF106 based on metabolism.

Epigallocatechin-3-gallate (EGCG) is a vital kind of catechin with high bioactive activities, however, limited research has been conducted to elucidate the molecular basis of EGCG biotransformation by Aspergillus niger and the underlying regulatory mechanisms. In this study, A. niger RAF106, isolated from Pu-erh tea, was applied to conduct the EGCG fermentation process, and the samples were collected at different fermentation times to determine the intermediary metabolites of EGCG and the metabolome as well as physiological activity changes of A. niger RAF106. The results demonstrated that EGCG enhances the growth of A. niger RAF106 by promoting conidial germination and hyphae branching. Meanwhile, metabolomic analyses indicated that EGCG significantly regulates the amino acid metabolism of A. niger RAF106. Furthermore, metabolomic analyses also revealed that the levels of original secondary metabolites in the supernatant of the cultures changed significantly from the fermentation stage M2 to M3, in which the main differentially changed metabolites (DCMs) were flavonoids. Most of these flavonoids exhibited antioxidant properties and thus increased the radical scavenging activity of the supernatant of the cultures. In addition, we also found several intermediary metabolites of EGCG, GA, and EGC, including oolonghomobisflavan A, (-)-Epigallocatechin 3, 5-di-gallate, (-)-Epigallocatechin 3-(3-methyl-gallate) (-)-Catechin 3-O-gallate, 4'-Methyl-(-)-epigallocatechin 3-(4-methyl-gallate), myricetin, prodelphinidin B, 7-galloylcatechin, and 3-hydroxyphenylacetic acid. These findings contribute to improving the bioavailability of EGCG and help mine highly active metabolites, which can be used as raw materials for the development of pharmaceutical intermediates or functional foods. In addition, the results also provide a theoretical basis for better control of the risk of A. niger origin and the regulatory mechanisms of the biotransformation process mediated by A. niger.

[Efficacy Analysis of Bendamustine-Based Combination Regimen in Treatment of Patients with Relapsed/Refractory Non-Hodgkin Lymphoma].

OBJECTIVE: To investigate the efficacy and safety of bendamustine combined with gemcitabine, vinorelbine，glucocorticoids (BeGEV)±X regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma. METHODS: A total of 18 relapsed/ refractory non-Hodgkin lymphoma patients at the age of 18 years or older hospitalized in the First People's Hospital of Changzhou from March 2020 to March 2021 were selected. They received two or more cycles of BeGEV±X regimen. X could be anti-CD20 monoclonal antibody, PD-1-blocking antibodies, lenalidomide, BTK inhibitor, Bcl-2 inhibitor and so on according to patients' disease feature. The clinical efficacy and adverse effects were observed. RESULTS: In total, 18 patients completed two or more cycles of BeGEV±X regimen, including 14 with diffuse large B-cell lymphoma, one with low-grade follicular lymphoma, one with follicular lymphoma grade 3b, one with angioimmunoblastic T-cell lymphoma and one with peripheral T-cell lymphoma, not otherwise specified. 11 patients were male. The median age of the patients was 64 years old. 17 patients had modified Ann Arbor stage Ⅲ/Ⅳ disease. 13 patients had high- intermediate risk or high risk IPI score, while 15 patients had high-intermediate high risk or high risk NCCN-IPI score. 14 cases had extranodal sites of disease. And 6 cases had bulky disease. 12 patients experienced refractory disease, while 8 patients had received 3 line or more prior treatment. After two or three cycles of chemotherapy, the complete response rate was 6/18, the partial response rate was 3/18, and the objective response rate was 9/18. From the beginning of salvage chemotherapy to the end of follow-up, the median progression-free survival time was 130 days, and the median overall survival was 152 days. The most common grade 3 to 4 adverse events were hematologic toxicities, infection and febrile neutropenia. CONCLUSION: BeGEV±X is an effective salvage regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma, while adverse events such as hematologic toxicities and infection should be closely monitored.

Outer membrane protein of OmpF contributes to swimming motility, biofilm formation, osmotic response as well as the transcription of maltose metabolic genes in Citrobacter werkmanii.

Bacterial outer membrane proteins (Omps) are essential for environmental sensing, stress responses, and substance transport. Our previous study discovered that OmpA contributes to planktonic growth, biocide resistance, biofilm formation, and swimming motility in Citrobacter werkmanii, whereas the molecular functions of OmpF in this strain are largely unknown. Thus, in this study, the ompF gene was firstly knocked out from the genome of C. werkmanii using a homologous recombination method, and its phenotypical alternations of ∆ompF were then thoroughly characterized using biochemical and molecular approaches with the parental wild type (WT) and complementary (∆ompF-com) strains. The results demonstrated that the swimming ability of ∆ompF on semi-solid plates was reduced compared to WT due to the down-regulation of flgC, flgH, fliK, and fliF. Meanwhile, ompF deletion reduces biofilm formation on both glass and polystyrene surfaces due to decreased cell aggregation. Furthermore, ompF inactivation induced different osmotic stress (carbon sources and metal ions) responses in its biofilms when compared to WT and ∆ompF-com. Finally, a total of 6 maltose metabolic genes of lamB, malE, malK, malG, malM, and malF were all up-regulated in ∆ompF. The gene knockout and HPLC results revealed that the MalEFGK2 cluster was primarily responsible for maltose transport in C. werkmanii. Furthermore, we discovered for the first time that the upstream promoter of OmpF and its transcription can be combined with and negatively regulated by MalT. Overall, OmpF plays a role in a variety of biochemical processes and molecular functions in C. werkmanii, and it may even act as a targeted site to inhibit biofilm formation.

The role of structural evolution in the complexation and flocculation of heavy metals by the microbial product poly-γ-glutamic acid.

The process and mechanism of heavy metal flocculation with extracellular polymeric substances (EPS) secreted by microorganisms, are crucial to their fate in natural environment, wastewater treatment and soil bioremediation applications. However, the structural features of EPS and the relationship between these features and the flocculation process and mechanism remain unclear. In the present study, structural features of the microbial product poly-γ-glutamic acid (γ-PGA) complexed with the heavy metal ions Pb2+ and Cu2+ were characterized and the evolution of these features was identified as having a key role in the flocculation process and mechanism. The secondary structure of the γ-PGA-Pb complex changed significantly, while that of the γ-PGA-Cu complex was only slightly altered. The significant structural change in γ-PGA-Pb was found to be responsible for the combination of residual COOH and Pb2+, promoting the bridging of inter-colloids and faster growth of hydrodynamic diameter. If the conformation changed sufficiently, such as with the γ-PGA-Pb complex in the pH range 4.6-6.2, pH had no impact on the conversion ratio. The unchanged structure of γ-PGA-Cu prevented the flocculation process, although the coordination mode of γ-PGA-Cu resulted in a higher biosorption capacity. This in-depth molecular-level study provides insight into the γ-PGA flocculation mechanism, promoting the use of γ-PGA and γ-PGA producing microorganisms for application in various remediation strategies.

Pseudomonas aeruginosa heteroresistance to levofloxacin caused by upregulated expression of essential genes for DNA replication and repair.

Pseudomonas aeruginosa (P. aeruginosa), a common cause of severe chronic infections, has developed heteroresistance to several antibiotics, thus hindering successful treatment. In this study, we aimed to investigate the characteristics and mechanisms underlying levofloxacin (LVX) heteroresistance in P. aeruginosa PAS71 and PAS81 clinical isolates using a combination of physiological and biochemical methods, bacterial genomics, transcriptomics, and qRT-PCR. The six P. aeruginosa strains, namely PAS71, PAS72, PAS81, PAS82, ATCC27853, and PAO1, were studied. The Kirby-Bauer (K-B), minimum inhibitory concentration (MIC) test, and population analysis profile (PAP) experimental results showed that PAS71, PAS81, ATCC27853, and PAO1 were heteroresistant to LVX, with MIC of 0.25, 1, 0.5, and 2 µg/ml, respectively; PAS72 and PAS82 were susceptible to LVX with a MIC of 0.25 and 0.5 µg/ml, respectively. The resistance of PAS71 and PAS81 heteroresistant subpopulations was unstable and had a growth fitness cost. Genomic and transcriptomic results proved that the unstable heteroresistance of PAS71 and PAS81 was caused by elevated expression of essential genes involved in DNA replication and repair, and homologous recombination, rather than their genomic single-nucleotide polymorphism (SNP) and insertion-deletion (InDel) mutations. Additionally, PAS71 and PAS81 enhanced virulence and physiological metabolism, including bacterial secretion systems and biosynthesis of siderophore group nonribosomal peptides, in response to LVX stress. Our results suggest that the upregulation of key genes involved in DNA replication and repair, and homologous recombination causes unstable heteroresistance in P. aeruginosa against LVX. This finding provides novel insights into the occurrence and molecular regulation pathway of P. aeruginosa heteroresistant strains.

Metagenomic analysis of microbial communities and antibiotic resistance genes in spoiled household chemicals.

Numerous attempts have been utilized to unveil the occurrences of antibiotic resistance genes (ARGs) in human-associated and non-human-associated samples. However, spoiled household chemicals, which are usually neglected by the public, may be also a reservoir of ARGs because of the excessive and inappropriate uses of industrial drugs. Based upon the Comprehensive Antibiotic Research Database, a metagenomic sequencing method was utilized to detect and quantify Antibiotic Resistance Ontology (AROs) in six spoiled household chemicals, including hair conditioner, dishwashing detergent, bath shampoo, hand sanitizer, and laundry detergent. Proteobacteria was found to be the dominant phylum in all the samples. Functional annotation of the unigenes obtained against the KEGG pathway, eggNOG and CAZy databases demonstrated a diversity of their functions. Moreover, 186 types of AROs that were members of 72 drug classes were identified. Multidrug resistance genes were the most dominant types, and there were 17 AROs whose resistance mechanisms were categorized into the resistance-nodulation-cell division antibiotic efflux pump among the top 20 AROs. Moreover, Proteobacteria was the dominant carrier of AROs with the primary resistance mechanism of antibiotic efflux. The maximum temperature of the months of collection significantly affected the distributions of AROs. Additionally, the isolated individual bacterium from spoiled household chemicals and artificial mixed communities of isolated bacteria demonstrated diverse resistant abilities to different biocides. This study demonstrated that there are abundant microorganisms and a broad spectrum profile of AROs in spoiled household chemicals that might induce a severe threat to public healthy securities and merit particular attention.

Synthesis, molecular docking, and evaluation of antibacterial activity of 1,2,4-triazole-norfloxacin hybrids.

A series of 1,2,4-triazole-norfloxacin hybrids was designed, synthesized, and evaluated for in vitro antibacterial activity against common pathogens. All the newly synthesized compounds were characterized by Fourier-transform infrared spectrophotometry, proton and carbon nuclear magnetic resonance, and electrospray ionization-mass spectrometry. Representative compounds from each step of the synthesis were further characterized by X-ray crystallography. Many of the compounds synthesized exhibited antibacterial activity superior to that of norfloxacin toward both, gram-positive and gram-negative bacteria. The toxicity of the 1,2,4-triazole-norfloxacin hybrids toward bacterial cells was 32-512 times higher than that toward mouse fibroblast cells. Moreover, hemolysis was not observed at concentrations of 64 µg/mL, suggesting good biocompatibility. Molecular docking showed a least binding energy of -9.4 to -9.7 kcal/mol, and all compounds were predicted to show remarkable affinity for the bacterial topoisomerase IV.

Roles of ompA of Citrobacter werkmanii in bacterial growth, biocide resistance, biofilm formation and swimming motility.

The genus Citrobacter is commonly found in environmental and industrial settings, some members of which have been used for bioremediation of heavy metals owing to the absorption ability of their biofilms. Although our previous studies have found that the outer membrane protein A (OmpA) contributes to the process of Citrobacter werkmanii biofilm formation, the underlying mechanisms remain elusive. Therefore, we deleted ompA from the genome of C. werkmanii and investigated its phenotypes in comparison to the wild type strain (WT) and the complementary strain using biochemical and molecular techniques including RNA-Seq. Our results demonstrated that the deletion of ompA led to an increase in biofilm formation on both polystyrene and glass surfaces due to upregulation of some biofilm formation related genes. Meanwhile, swimming ability, which is mediated by activation of flagellar assembly genes, was increased on semi-solid plates in the ∆ompA strain when compared with WT. Additionally, inactivation of ompA also caused increased 1,2-benzisothiazolin-3-one (BIT) resistance, differential responses to Ca2+ stress, curli protein expression and cellulose production. Finally, ∆ompA caused differential expression of a total of 1470 genes when compared with WT, of which 146 were upregulated and 1324 were downregulated. These genes were classified into different Gene Ontology (GO) and KEGG pathways. In summary, ompA in C. werkmanii contributes to a variety of biological functions and may act as a target site to modulate biofilm formation. KEY POINTS: • ompA is a negative regulator for biofilm formation by C. werkmanii. • ompA inhibits swimming motility of C. werkmanii. • ompA deletion causes different expression profiles in C. werkmanii.

Detoxification of ionic liquids using glutathione, cysteine, and NADH: Toxicity evaluation by Tetrahymena pyriformis.

Ionic liquids (ILs), also known as green solvents, are widely acknowledged in several fields, such as chemical separation, synthesis, and electrochemistry, owing to their excellent physiochemical properties. However, their poor biodegradability may lead to environmental and health risks, posing a severe threat to humans, thus requiring further research. In this study, the biotoxicities of the imidazolium-based ILs were evaluated in Tetrahymena pyriformis. Moreover, IL detoxification was investigated by addition of glutathione (GSH), cysteine, and nicotinamide adenine dinucleotide (NADH). Reactive oxygen species (ROS) initiated by different IL types caused damage to Tetrahymena, while glutathione, cysteine, and NADH eliminated ROS, achieving the detoxification purposes. Detoxification results showed that NADH exhibited the best detoxification ability, followed by glutathione and cysteine. Finally, RT-PCR results suggested that metallothionein might have participated in IL detoxification.

Crystal structure of bis-{2-hy-droxy-N'-[1-(pyrazin-2-yl)ethyl-idene]benzohydrazidato}cadmium(II).

In the title complex mol-ecule, [Cd(C13H11N4O2)2], the Cd atom is coordinated in a distorted octa-hedral geometry by two tridentate ligands synthesized from 2-hy-droxy-benzohydrazide and 1-(pyrazin-2-yl)ethan-1-one. The mol-ecule has twofold crystallographic symmetry and is isomorphous to its Mn, Co, Ni, Cu and Zn counterparts.

Diallyl sulfide from garlic suppresses quorum-sensing systems of Pseudomonas aeruginosa and enhances biosynthesis of three B vitamins through its thioether group.

Diallyl sulfide (DAS) and diallyl disulfide (DADS), two constituents of garlic, can inhibit quorum sensing (QS) systems of Pseudomonas aeruginosa. However, the differences in the mechanism of QS inhibition between DAS and DADS, and the functional chemical groups of these sulfides that contribute in QS inhibition have not been elucidated yet. We assumed that the sulfide group might play a key role in QS inhibition. To prove this hypothesis and to clarify these unsolved problems, in this study, we synthesized diallyl ether (DAE), and compared and investigated the effects of DAS and DAE on the growth and production of virulence factors, including Pseudomonas quinolone signal (PQS), elastase and pyocyanin, of P. aeruginosa PAO1. Transcriptome analysis and qRT-PCR were used to compare and analyse the differentially expressed genes between the different treatment groups (DAS, DAE and control). The results indicated that DAS did not affect the growth dynamics of P. aeruginosa PAO1; however, DAS inhibited transcription of most of the QS system genes, including lasR, rhlI/rhlR and pqsABCDE/pqsR; thus, biosynthesis of the signal molecules C4 -HSL (encoded by rhlI) and PQS (encoded by pqsABCDE) was inhibited. Furthermore, DAS inhibited the transcription of virulence genes regulated by the QS systems, including rhlABC, lasA, lasB, lecA and phzAB, phzDEFG, phzM and phzS that encode for rhamnolipid, exoprotease, elastase, lectin and pyocyanin biosynthesis respectively. DAS also enhanced the expression of the key genes involved in the biosynthesis of three B vitamins: folate, thiamine and riboflavin. In conclusion, DAS suppressed the production of some virulence factors toxic to the host and enhanced the production of some nutrition factors beneficial to the host. These actions of DAS may be due to its thioether group. These findings would be significant for development of an effective drug to control the virulence and pathogenesis of the opportunistic pathogen P. aeruginosa.