Host Range,Morphology and Sequence Analysis of Ten Temperate Phages Isolated from Pathogenic Yersinia enterocolitica Strains

Yersinia enterocolitica is a heterogeneous species comprising highly pathogenic, weakly pathogenic and non-pathogenic strains. Previous data suggest that gene exchange may occur in Yersinia. Only scarce information exists about temperate phages of Y. enterocolitica, even though many prophage sequences are present in this species. We have examined 102 pathogenic Y. enterocolitica strains for the presence of inducible prophages by mitomycin C treatment. Ten phages were isolated from nine strains belonging to the bio (B)/serotypes (O) B2/O:5,27, B2/O:9 and 1B/O:8. All phages are myoviruses showing lytic activity only at room temperature. Whole-genome sequencing of the phage genomes revealed that they belong to three groups, which, however, are not closely related to known phages. Group 1 is composed of five phages (type phage: vB_YenM_06.16.1) with genome sizes of 43.8 to 44.9 kb, whereas the four group 2 phages (type phage: vB_YenM_06.16.2) possess smaller genomes of 29.5 to 33.2 kb. Group 3 contains only one phage (vB_YenM_42.18) whose genome has a size of 36.5 kb, which is moderately similar to group 2. The host range of the phages differed significantly. While group 1 phages almost exclusively lysed strains of B2/O:5,27, phages of group 2 and 3 were additionally able to lyse B4/O:3, and some of them even B2/O:9 and 1B/O:8 strains.     

Metabolomics Comparison of Drug-Resistant and Drug-Susceptible Pseudomonas aeruginosa Strain (Intra- and Extracellular Analysis)

Pseudomonas aeruginosa is a common human pathogen belonging to the ESKAPE group. The multidrug resistance of bacteria is a considerable problem in treating patients and may lead to increased morbidity and mortality rate. The natural resistance in these organisms is caused by the production of specific enzymes and biofilm formation, while acquired resistance is multifactorial. Precise recognition of potential antibiotic resistance on different molecular levels is essential. Metabolomics tools may aid in the observation of the flux of low molecular weight compounds in biochemical pathways yielding additional information about drug-resistant bacteria. In this study, the metabolisms of two P. aeruginosa strains were compared—antibiotic susceptible vs. resistant. Analysis was performed on both intra- and extracellular metabolites. The ¹H NMR method was used together with multivariate and univariate data analysis, additionally analysis of the metabolic pathways with the FELLA package was performed. The results revealed the differences in P. aeruginosa metabolism of drug-resistant and drug-susceptible strains and provided direct molecular information about P. aeruginosa response for different types of antibiotics. The most significant differences were found in the turnover of amino acids. This study can be a valuable source of information to complement research on drug resistance in P. aeruginosa.     

Salmonella Typhimurium and Pseudomonas aeruginosa Respond Differently to the Fe Chelator Deferiprone and to Some Novel Deferiprone Derivatives

The ability to obtain Fe is critical for pathogens to multiply in their host. For this reason, there is significant interest in the identification of compounds that might interfere with Fe management in bacteria. Here we have tested the response of two Gram-negative pathogens, Salmonella enterica serovar Typhimurium (STM) and Pseudomonas aeruginosa (PAO1), to deferiprone (DFP), a chelating agent already in use for the treatment of thalassemia, and to some DFP derivatives designed to increase its lipophilicity. Our results indicate that DFP effectively inhibits the growth of PAO1, but not STM. Similarly, Fe-dependent genes of the two microorganisms respond differently to this agent. DFP is, however, capable of inhibiting an STM strain unable to synthesize enterochelin, while its effect on PAO1 is not related to the capability to produce siderophores. Using a fluorescent derivative of DFP we have shown that this chelator can penetrate very quickly into PAO1, but not into STM, suggesting that a selective receptor exists in Pseudomonas. Some of the tested derivatives have shown a greater ability to interfere with Fe homeostasis in STM compared to DFP, whereas most, although not all, were less active than DFP against PAO1, possibly due to interference of the added chemical tails with the receptor-mediated recognition process. The results reported in this work indicate that DFP can have different effects on distinct microorganisms, but that it is possible to obtain derivatives with a broader antimicrobial action.     

Giving Drugs a Second Chance: Antibacterial and Antibiofilm Effects of Ciclopirox and Ribavirin against Cystic Fibrosis Pseudomonas aeruginosa Strains

Drug repurposing is an attractive strategy for developing new antibacterial molecules. Herein, we evaluated the in vitro antibacterial, antibiofilm, and antivirulence activities of eight FDA-approved “non-antibiotic” drugs, comparatively to tobramycin, against selected Pseudomonas aeruginosa strains from cystic fibrosis patients. MIC and MBC values were measured by broth microdilution method. Time–kill kinetics was studied by the macro dilution method, and synergy studies were performed by checkerboard microdilution assay. The activity against preformed biofilms was measured by crystal violet and viable cell count assays. The effects on gene expression were studied by real-time quantitative PCR, while the cytotoxic potential was evaluated against IB3-1 bronchial CF cells. Ciclopirox, 5-fluorouracil, and actinomycin D showed the best activity against P. aeruginosa planktonic cells and therefore underwent further evaluation. Time–kill assays indicated actinomycin D and ciclopirox, contrarily to 5-fluorouracil and tobramycin, have the potential for bacterial eradication, although with strain-dependent efficacy. Ciclopirox was the most effective against the viability of the preformed biofilm. A similar activity was observed for other drugs, although they stimulate extracellular polymeric substance production. Ribavirin showed a specific antibiofilm effect, not dependent on bacterial killing. Exposure to drugs and tobramycin generally caused hyperexpression of the virulence traits tested, except for actinomycin D, which downregulated the expression of alkaline protease and alginate polymerization. Ciclopirox and actinomycin D revealed high cytotoxic potential. Ciclopirox and ribavirin might provide chemical scaffolds for anti-P. aeruginosa drugs. Further studies are warranted to decrease ciclopirox cytotoxicity and evaluate the in vivo protective effects.     

Specific Maltose Derivatives Modulate the Swarming Motility of Nonswarming Mutant and Inhibit Bacterial Adhesion and Biofilm Formation by Pseudomonas aeruginosa

We have demonstrated that specific synthetic maltose derivatives activate the swarming motility of a Pseudomonas aeruginosa nonswarming mutant (rhlA) at low concentration, but inhibit it at high concentration. Although these molecules are not microbicidal, active maltose derivatives with bulky hydrocarbon groups inhibited bacterial adhesion, and exhibited biofilm inhibition and dispersion (IC₅₀ ～20 μM and DC₅₀ ～30 μM , respectively). Because the swarming motility of the rhlA mutant is abolished by the lack natural rhamnolipids, the swarming activation suggests that maltose derivatives are analogues of rhamnolipids. Together, these results suggest a new approach of controlling multiple bacterial activities (bacterial adhesion, biofilm formation, and swarming motility) by a set of disaccharide‐based molecules.     

Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa

In recent years, the effectiveness of antimicrobials in the treatment of Pseudomonas aeruginosa infections has gradually decreased. This pathogen can be observed in several clinical cases, such as pneumonia, urinary tract infections, sepsis, in immunocompromised hosts, such as neutropenic cancer, burns, and AIDS patients. Furthermore, Pseudomonas aeruginosa causes diseases in both livestock and pets. The highly flexible and versatile genome of P. aeruginosa allows it to have a high rate of pathogenicity. The numerous secreted virulence factors, resulting from its numerous secretion systems, the multi-resistance to different classes of antibiotics, and the ability to produce biofilms are pathogenicity factors that cause numerous problems in the fight against P. aeruginosa infections and that must be better understood for an effective treatment. Infections by P. aeruginosa represent, therefore, a major health problem and, as resistance genes can be disseminated between the microbiotas associated with humans, animals, and the environment, this issue needs be addressed on the basis of an One Health approach. This review intends to bring together and describe in detail the molecular and metabolic pathways in P. aeruginosa’s pathogenesis, to contribute for the development of a more targeted therapy against this pathogen.     

EGCG-Mediated Potential Inhibition of Biofilm Development and Quorum Sensing in Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa), one of the dangerous multidrug resistance pathogens, orchestrates virulence factors production through quorum sensing (QS). Since the exploration of QS inhibitors, targeting virulence to circumvent bacterial pathogenesis without causing significant growth inhibition is a promising approach to treat P. aeruginosa infections. The present study has evaluated the anti-QS and anti-infective activity of epigallocatechin-3-gallate (EGCG), a bioactive ingredient of the traditional green tea, against P. aeruginosa. EGCG showed significant inhibitory effects on the development of biofilm, protease, elastase activity, swimming, and swarming motility, which was positively related to the production of C4-AHL. The expression of QS-related and QS-regulated virulence factors genes was also evaluated. Quantitative PCR analysis showed that EGCG significantly reduced the expression of las, rhl, and PQS genes and was highly correlated with the alterations of C4-AHL production. In-vivo experiments demonstrated that EGCG treatment reduced P. aeruginosa pathogenicity in Caenorhabditis elegans (C. elegans). EGCG increased the survival of C. elegans by 23.25%, 30.04%, and 36.35% in a dose-dependent manner. The findings of this study strongly suggest that EGCG could be a potential candidate for QS inhibition as an anti-virulence compound against bacterial infection.     

Expanding the Spectrum of Antibiotics Capable of Killing Multidrug-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

Infections from antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa are a serious threat because reduced antibiotic efficacy complicates treatment decisions and prolongs the disease state in many patients. To expand the arsenal of treatments against antimicrobial-resistant (AMR) pathogens, 600-Da branched polyethylenimine (BPEI) can overcome antibiotic resistance mechanisms and potentiate β-lactam antibiotics against Gram-positive bacteria. BPEI binds cell-wall teichoic acids and disables resistance factors from penicillin binding proteins PBP2a and PBP4. This study describes a new mechanism of action for BPEI potentiation of antibiotics generally regarded as agents effective against Gram-positive pathogens but not Gram-negative bacteria. 600-Da BPEI is able to reduce the barriers to drug influx and facilitate the uptake of a non-β-lactam co-drug, erythromycin, which targets the intracellular machinery. Also, BPEI can suppress production of the cytokine interleukin IL-8 by human epithelial keratinocytes. This enables BPEI to function as a broad-spectrum antibiotic potentiator, and expands the opportunities to improve drug design, antibiotic development, and therapeutic approaches against pathogenic bacteria, especially for wound care.     

Serratiopeptidase Affects the Physiology of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

Pseudomonas aeruginosa is frequently involved in cystic fibrosis (CF) airway infections. Biofilm, motility, production of toxins and the invasion of host cells are different factors that increase P. aeruginosa’s virulence. The sessile phenotype offers protection to bacterial cells and resistance to antimicrobials and host immune attacks. Motility also contributes to bacterial colonization of surfaces and, consequently, to biofilm formation. Furthermore, the ability to adhere is the prelude for the internalization into lung cells, a common immune evasion mechanism used by most intracellular bacteria, such as P. aeruginosa. In previous studies we evaluated the activity of metalloprotease serratiopeptidase (SPEP) in impairing virulence-related properties in Gram-positive bacteria. This work aimed to investigate SPEP’s effects on different physiological aspects related to the virulence of P. aeruginosa isolated from CF patients, such as biofilm production, pyoverdine and pyocyanin production and invasion in alveolar epithelial cells. Obtained results showed that SPEP was able to impair the attachment to inert surfaces as well as adhesion/invasion of eukaryotic cells. Conversely, SPEP’s effect on pyocyanin and pyoverdine production was strongly strain-dependent, with an increase and/or a decrease of their production. Moreover, SPEP seemed to increase swarming motility and staphylolytic protease production. Our results suggest that a large number of clinical strains should be studied in-depth before drawing definitive conclusions. Why different strains sometimes react in opposing ways to a specific treatment is of great interest and will be the object of future studies. Therefore, SPEP affects P. aeruginosa’s physiology by differently acting on several bacterial factors related to its virulence.     

Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DPPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.     

Anti-Virulence Potential of a Chionodracine-Derived Peptide against Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Cystic Fibrosis Patients

Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa. A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.     

BSA Hydrogel Beads Functionalized with a Specific Aptamer Library for Capturing Pseudomonas aeruginosa in Serum and Blood

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG₄-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.     

重组铜绿假单胞菌外毒素A工程菌发酵及表达产物的纯化

目的建立稳定、高效表达重组铜绿假单胞菌外毒素A(rEPA)的工程菌发酵及表达产物纯化工艺。方法规模化发酵表达rEPA的重组大肠杆菌rPE553D,离心收集菌体,渗透压调解使菌体周质间隙蛋白释放后,高速离心收集蛋白溶液。经DEAESepharoseFF、PhenylSepharose6FF疏水层析和SOURCE30Q强离子交换层析,超滤浓缩纯化rEPA。用HPLC、SDS-PAGE和Westernblot等方法检定生化和免疫学特性,并用小鼠和Vero细胞检定毒性。结果每55L培养基中rEPA产量超过4g,纯度在95%以上,细胞毒性降低了至少32000倍。其余各项指标均符合《中国生物制品规程》要求。结论已建立了收率高、纯度好、稳定、适合规模化生产rEPA的工艺。     

Enhanced removal of selected hydrocarbons from soil by Pseudomonas aeruginosa UG2 biosurfactants and some chemical surfactants

The ability of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa UG2 to wash a model hydrocarbon mixture from unsaturated soil columns was studied. Both aliphatic and aromatic hydrocarbons were effectively removed without soil clogging with non-recirculating biosurfactant solutions. Recirculation of wash solutions did not substantially affect washing efficiency. Of the several chemical surfactants tested, only Triton X-100 provided comparable hydrocarbon washing efficiency without soil clogging. The results suggest that UG2 biosurfactants have the potential for remediation of hydrophobic pollutants in unsaturated soil.     

Optimization and Characterization of Biosurfactant Rhamnolipid Production by Pseudomonas aeruginosa Isolated from an Artificially Contaminated Soil

Biosurfactants are surfactants biologically produced by microorganisms, presenting several advantages when compared to synthetic surfactants. Pseudomonas aeruginosa is known for producing rhamnolipids, considered one of the most interesting types of biosurfactants due to their high yields, when compared to other types. In this work, the production of rhamnolipid from P. aeruginosa was optimized. At first, the Plackett–Burman design was used to select most significant variables affecting the biosurfactant production yield among nine variables—carbon–nitrogen ratio, carbon concentration, nitrogen source, pH, cultivation time, potassium and magnesium concentrations, agitation, and temperature. Then, using main variables, a central point experimental design aiming to optimize rhamnolipid production was performed. The maximum biosurfactant concentration obtained was 0.877 mg L⁻¹. The rhamnolipid also displayed a great emulsification rate, reaching approximately 67%, and the ability to reduce water surface tension from 72.02 to 35.26 mN m⁻¹ at a critical micelle concentration (CMC) of 127 mg L⁻¹, in addition to presenting a good stability when exposed to wide pH and salinity ranges. The results suggest that rhamnolipids are promising substitutes for synthetic surfactants, especially due to lower impacts on the environment.     

原油降解菌株Pseudomonas.aeruginosa AS1生理生化性质及分类研究

原油降解菌株AS1筛选自延长油田油水样,对延长轻质原油具有良好降解能力,通过生理生化指标和16SrDNA基因序列分析进行了菌种鉴定,原油降解菌株AS1的16SrDNA基因序列与Pseu-domonas aeruginosa的相似度为99.1%,因而将其命名为P.aeruginosa AS1。该菌株的最适生长温度为37℃,能以延长轻质原油、液体石蜡为唯一碳源生长,并能合成鼠李糖脂类生物表面活性剂,该表面活性剂对柴油、煤油和原油等均有很好的乳化效果,在常温下形成EI24值为100%的乳状液。鉴于P.aerugi-nosa AS1的良好生物属性,该菌株有进一步进行微生物矿场试验的潜力。     

The MexJK Multidrug Efflux Pump Is Not Involved in Acquired or Intrinsic Antibiotic Resistance in Pseudomonas aeruginosa,but Modulates the Bacterial Quorum Sensing Response

Multidrug efflux pumps are critical elements in both intrinsic and acquired antibiotic resistance of bacterial populations. Consequently, most studies regarding these protein machineries focus on this specific phenotype. Nevertheless, different works show that efflux pumps participate in other aspects of bacterial physiology too. Herein, we study the Pseudomonas aeruginosa multidrug efflux pump MexJK. Previous studies, using model strains lacking MexAB-OprM and MexCD-OprJ efflux pumps, support that MexJK can extrude erythromycin, tetracycline, and triclosan. However, the results here reported indicate that this potential increased extrusion, in a mutant overexpressing mexJK, does not alter the antibiotics susceptibility in a wild-type genetic background where all intrinsic multidrug efflux pumps remain functional. Nevertheless, a clear impact on the quorum sensing (QS) response, mainly in the Pqs-dependent QS regulation network and in the expression of Pqs-regulated virulence factors, was observed linked to mexJK overexpression. The production of the siderophore pyoverdine strongly depended on the level of mexJK expression, suggesting that MexJK might participate in P. aeruginosa pyoverdine-dependent iron homeostasis. All in all, the results presented in the current article support that the functions of multidrug efflux pumps, as MexJK, go beyond antibiotic resistance and can modulate other relevant aspects of bacterial physiology.