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).

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.

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.

Biological functions of nirS in Pseudomonas aeruginosa ATCC 9027 under aerobic conditions.

Through our previous study, we found an up-regulation in the expression of nitrite reductase (nirS) in the isothiazolone-resistant strain of Pseudomonas aeruginosa. However, the definitive molecular role of nirS in ascribing the resistance remained elusive. In the present study, the nirS gene was deleted from the chromosome of P. aeruginosa ATCC 9027 and the resulting phenotypic changes of ΔnirS were studied alongside the wild-type (WT) strain under aerobic conditions. The results demonstrated a decline in the formations of biofilms but not planktonic growth by ΔnirS as compared to WT, especially in the presence of benzisothiazolinone (BIT). Meanwhile, the deletion of nirS impaired swimming motility of P. aeruginosa under the stress of BIT. To assess the influence of nirS on the transcriptome of P. aeruginosa, RNA-seq experiments comparing the ΔnirS with WT were also performed. A total of 694 genes were found to be differentially expressed in ΔnirS, of which 192 were up-regulated, while 502 were down-regulated. In addition, these differently expressed genes were noted to significantly enrich the carbon metabolism along with glyoxylate and dicarboxylate metabolisms. Meanwhile, results from RT-PCR suggested the contribution of mexEF-oprN to the development of BIT resistance by ΔnirS. Further, c-di-GMP was less in ΔnirS than in WT, as revealed by HPLC. Taken together, our results confirm that nirS of P. aeruginosa ATCC 9027 plays a role in BIT resistance along with biofilm formation and further affects several metabolic patterns under aerobic conditions.

Functional roles of norCB in Pseudomonas aeruginosa ATCC 9027 under aerobic conditions.

Nitric oxide (NO) reductase (NorCB) of Pseudomonas aeruginosa is an essential enzyme that metabolizes NO and alleviates anaerobic NO toxicity during denitrification processes under anaerobic conditions. However, the molecular functions of norCB in the presence of oxygen are poorly understood. This study utilized norCB knockout from P. aeruginosa ATCC 9027 to analyze the resulting phenotypic changes of ΔnorCB in comparison to the wild-type parental strain (WT) and the complementary strain (ΔnorCB-com). The results demonstrated an increase in planktonic growth and biofilm formation by ΔnorCB compared to WT and ΔnorCB-com in the presence of isothiazolones under aerobic conditions. Deletion of norCB led to increased swimming ability and decreased pyocyanin production. Inactivation of norCB also led to an increase of cellular H2 O2 concentration due to decreased activity of its catalases. In addition, the deletion of norCB also influenced the relative expressions of several other genes, including norD, nirS, hmgA, and hpd. These findings provide preliminary evidence that norCB in P. aeruginosa plays an essential role in bacterial life process under aerobic conditions and improves the application of denitrification in the next step.

Diallyl disulfide from garlic oil inhibits Pseudomonas aeruginosa virulence factors by inactivating key quorum sensing genes.

Garlic oil can disrupt the quorum sensing (QS) pathways of the opportunistic pathogen Pseudomonas aeruginosa; however, the underlying mechanisms for this effect are unclear. Diallyl disulfide (DADS) is one of the most abundant sulfur-containing compounds in garlic oil. This study investigated the effects of DADS on the growth, virulence factor production (elastase, pyocyanin, biofilm, and swarming motility), and essential gene expression of P. aeruginosa PAO1, particularly as they apply to QS and virulence. DADS at 1.28 mg/mL did not affect P. aeruginosa PAO1 growth, although it decreased elastase and pyocyanin production, biofilm formation, and swarming motility. Each of these phenomena is regulated by the three QS systems of P. aeruginosa PAO1 (las, rhl, and pqs). Real-time q-PCR revealed that DADS down-regulated the transcription levels of several important QS genes (lasI, lasR, rhlI, rhlR, pqsA, and pqsR) in the three systems. Furthermore, the transcription levels of QS-regulated virulence genes were also down-regulated. The lasB gene, encoding LasB elastase, is co-regulated by the las, rhl, and pqs systems, and thus the down-regulation of genes across the three systems further down-regulated lasB. Additionally, phzM (encoding pyocyanin), pslB (responsible for the production of a biofilm matrix polysaccharide), and chiC (encoding chitinase) were positively activated by LasR, and a decrease in lasR transcription further down-regulated the transcription of phzM, pslB, and chiC. Hence, DADS inhibits P. aeruginosa PAO1 virulence factors by inactivating the transcription of key genes across three different QS systems.

Role of Ttca of Citrobacter Werkmanii in Bacterial Growth, Biocides Resistance, Biofilm Formation and Swimming Motility.

To screen, identify and study the genes involved in isothiazolone resistance and biofilm formation in Citrobacter werkmanii strain BF-6. A Tn5 transposon library of approximately 900 mutants of C. werkmanii strain BF-6 was generated and screened to isolate 1,2-benzisothiazolin-3-one (BIT) resistant strains. In addition, the tRNA 2-thiocytidine (32) synthetase gene (ttcA) was deleted through homologous recombination and the resulting phenotypic changes of the ΔttcA mutant were studied. A total of 3 genes were successfully identified, among which ΔttcA mutant exhibited a reduction in growth rate and swimming motility. On the other hand, an increase in biofilms formation in ΔttcA were observed but not with a significant resistance enhancement to BIT. This work, for the first time, highlights the role of ttcA gene of C. werkmanii strain BF-6 in BIT resistance and biofilm formation.

Complete genome sequence of Citrobacter werkmanii strain BF-6 isolated from industrial putrefaction.

BACKGROUND: In our previous study, Citrobacter werkmanii BF-6 was isolated from an industrial spoilage sample and demonstrated an excellent ability to form biofilms, which could be affected by various environmental factors. However, the genome sequence of this organism has not been reported so far. RESULTS: We report the complete genome sequence of C. werkmanii BF-6 together with the description of the genome features and its annotation. The size of the complete chromosome is 4,929,789 bp with an average coverage of 137×. The chromosome exhibits an average G + C content of 52.0%, and encodes 4570 protein coding genes, 84 tRNA genes, 25 rRNA operons, 3 microsatellite sequences and 34 minisatellite sequences. A previously unknown circular plasmid designated as pCW001 was also found with a length of 212,549 bp and a G + C content of 48.2%. 73.5%, 75.6% and 92.6% of the protein coding genes could be assigned to GO Ontology, KEGG Pathway, and COG (Clusters of Orthologous Groups) categories respectively. C. werkmanii BF-6 and C. werkmanii NRBC 105721 exhibited the closest evolutionary relationships based on 16S ribosomal RNA and core-pan genome assay. Furthermore, C. werkmanii BF-6 exhibits typical bacterial biofilm formation and development. In the RT-PCR experiments, we found that a great number of biofilm related genes, such as bsmA, bssR, bssS, hmsP, tabA, csgA, csgB, csgC, csgD, csgE, and csgG, were involved in C. werkmanii BF-6 biofilm formation. CONCLUSIONS: This is the first complete genome of C. werkmanii. Our work highlights the potential genetic mechanisms involved in biofilm formation and paves a way for further application of C. werkmanii in biofilms research.

Comparison of transcriptomes of wild-type and isothiazolone-resistant Pseudomonas aeruginosa by using RNA-seq.

Isothiazolone biocides (such as Kathon) are widely used in a variety of industrial and domestic applications. However, the mechanisms through which bacteria develop resistance to these biocides are not completely clear. A better understanding of these mechanisms can contribute to optimal use of these biocides. In this study, transcription profiles of a Kathon-resistant strain of Pseudomonas aeruginosa (Pa-R) and the wild-type strain were determined using RNA sequencing (RNA-Seq) with the Illumina HiSeq 2000 platform. RNA-Seq generated 18,657,896 sequence reads aligned to 7093 genes. In all, 1550 differently expressed genes (DEGs, log2 ratio ≥1, false discovery rate (FDR) ≤0.001) were identified, of which 482 were up-regulated and 1068 were down-regulated. Most Kathon-induced genes were involved in metabolic and cellular processes. DEGs significantly enriched nitrogen metabolism and oxidative phosphorylation pathways. In addition, Pa-R showed cross-resistance to triclosan and ciprofloxacin and showed repressed pyocyanin production. These results may improve our understanding of the resistance mechanisms of P. aeruginosa against isothiazolones, and provide insight into the development of more efficient isothiazolones.

The dynamics and mechanism of the antimicrobial activity of tea tree oil against bacteria and fungi.

Tea tree oil (TTO) is a yellow liquid extracted from Melaleuca alternifolia. Although the antimicrobial activity of TTO has been known for a long time, its specific antimicrobial effects and mechanism underlying these remain poorly characterized. The present study investigated the chemical composition of TTO and the dynamics and mechanism of its antimicrobial activities in two bacterial and two fungal strains. Gas chromatography-mass spectrometry analysis identified alkenes and alcohols as the main constituents of TTO. Terpinen-4-ol was the most abundant individual component, accounting for approximately 23 % of the TTO. Poisoned food technique assessment showed that the minimum inhibitory concentrations of TTO for bacterial strains (Escherichia coli and Staphylococcus aureus) and fungal strains (Candida albicans and Aspergillus niger) were 1.08 and 2.17 mg/mL, respectively. Antimicrobial dynamic curves showed that with increasing concentrations of TTO, the rate of cell killing and the duration of growth lag phase increased correspondingly. These data indicated that TTO produced concentration and time-dependent antimicrobial effects. The minimum bactericidal and fungicidal concentrations of TTO were 2.17, 4.34, and 4.34 against E. coli, S. aureus, and C. albicans, respectively. However, A. niger conidia were not completely eradicated, even after 3 days in the presence of 17.34 mg/mL TTO. Transmission electron microscopy images indicated that TTO penetrated the cell wall and cytoplasmic membrane of all the tested bacterial and fungal strains. TTO may also penetrate fungal organelle membrane. These findings indicated that TTO maybe exerts its antimicrobial effects by compromising the cell membrane, resulting in loss of the cytoplasm and organelle damage, which ultimate leads to cell death.

The Three Bacterial Lines of Defense against Antimicrobial Agents.

Antimicrobial agents target a range of extra- and/or intracellular loci from cytoplasmic wall to membrane, intracellular enzymes and genetic materials. Meanwhile, many resistance mechanisms employed by bacteria to counter antimicrobial agents have been found and reported in the past decades. Based on their spatially distinct sites of action and distribution of location, antimicrobial resistance mechanisms of bacteria were categorized into three groups, coined the three lines of bacterial defense in this review. The first line of defense is biofilms, which can be formed by most bacteria to overcome the action of antimicrobial agents. In addition, some other bacteria employ the second line of defense, the cell wall, cell membrane, and encased efflux pumps. When antimicrobial agents permeate the first two lines of defense and finally reach the cytoplasm, many bacteria will make use of the third line of defense, including alterations of intracellular materials and gene regulation to protect themselves from harm by bactericides. The presented three lines of defense theory will help us to understand the bacterial resistance mechanisms against antimicrobial agents and design efficient strategies to overcome these resistances.

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.

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.

[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.

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.

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.

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.