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IntroductionAccurate neurological impairment assessment is crucial for the clinical treatment and prognosis of patients with acute ischemic stroke (AIS). However, the original perfusion parameters lack the deep information for characterizing neurological impairment, leading to difficulty in accurate assessment. Given the advantages of radiomics technology in feature representation, this technology should provide more information for characterizing neurological impairment. Therefore, with its rigorous methodology, this study offers practical implications for clinical diagnosis by exploring the role of ischemic perfusion radiomics features in assessing the degree of neurological impairment.MethodsThis study employs a meticulous methodology, starting with generating perfusion parameter maps through Dynamic Susceptibility Contrast-Perfusion Weighted Imaging (DSC-PWI) and determining ischemic regions based on these maps and a set threshold. Radiomics features are then extracted from the ischemic regions, and the t-test and least absolute shrinkage and selection operator (Lasso) algorithms are used to select the relevant features. Finally, the selected radiomics features and machine learning techniques are used to assess the degree of neurological impairment in AIS patients.ResultsThe results show that the proposed method outperforms the original perfusion parameters, radiomics features of the infarct and hypoxic regions, and their combinations, achieving an accuracy of 0.926, sensitivity of 0.923, specificity of 0.929, PPV of 0.923, NPV of 0.929, and AUC of 0.923, respectively.ConclusionThe proposed method effectively assesses the degree of neurological impairment in AIS patients, providing an objective auxiliary assessment tool for clinical diagnosis.

Abstract CRISPR based technologies have been used for fast and sensitive detection of pathogens. To test the possibility of CRISPR based detection strategy in Pseudomonas aeruginosa infections, a combined method of recombinase polymerase amplification followed by Cas12a-mediated detection via fluorescence reader or lateral flow biosensor (named Cas12a-RCFL) has been established in this study. The Cas12a-RCFL can detect as low as 50 CFU/mL Pseudomonas aeruginosa. The whole detection process can be finished within one hour with satisfied detection specificity. Cas12a-RCFL also shows good sensitivity of detecting Pseudomonas aeruginosa inStaphylococcus aureus and Acinetobacter baumannii contaminated samples. For the detection of 22 clinical samples, Cas12a-RCFL matches with PCR sequencing result exactly without DNA purification. This Cas12a-RCFL is rapid and sensitive with low cost, which shows good quality to be adopted as a point-of-care testing method.

Abstract Acinetobacter baumannii has become one of the most challenging conditional pathogens in health facilities. It causes various infectious diseases in humans, such as wound or urinary tract infections and pneumonia. Phage therapy has been used as an alternative strategy for antibiotic-resistant A. baumannii infections and has been approved by several governments. Previously, we have reported two potential phage therapy candidates, Abp1 and Abp9, both of which are narrow-host-range phages. In the present study, we screened and isolated 22 A. baumannii bacteriophages from hospital sewage water and determined that Abp95 has a wide host range (29%; 58/200). The biological and genomic characteristics and anti-infection potential of Abp95 were also investigated. Abp95 belongs to the Myoviridae family, with a G+C content of 37.85% and a genome size of 43,176 bp. Its genome encodes 77 putative genes, none of which are virulence, lysogeny, or antibiotic resistance genes. Abp95 was found to accelerate wound healing in a diabetic mouse wound infection model by clearing local infections of multidrug-resistant A. baumannii. In conclusion, the lytic phage Abp95, which has a wide host range, demonstrates potential as a candidate for phage therapy against multiple sequence types of carbapenem-resistant A. baumannii.

Antimicrobial-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens represent a global threat to human health. ESKAPE pathogens are the most common opportunistic pathogens in nosocomial infections, and a considerable number of their clinical isolates are not susceptible to conventional antimicrobial therapy. Therefore, innovative therapeutic strategies that can effectively deal with ESKAPE pathogens will bring huge social and economic benefits and ease the suffering of tens of thousands of patients. Among these strategies, CRISPR (clustered regularly interspaced short palindromic repeats) system has received extra attention due to its high specificity. Regrettably, there is currently no direct CRISPR-system-based anti-infective treatment. This paper reviews the applications of CRISPR-Cas system in the study of ESKAPE pathogens, aiming to provide directions for the research of ideal new drugs and provide a reference for solving a series of problems caused by multidrug-resistant bacteria (MDR) in the post-antibiotic era. However, most research is still far from clinical application.

The rapid increase in antibiotic resistance presents a dire situation necessitating the need for alternative therapeutic agents. Among the current alternative therapies, phage therapy (PT) is promising. This review extensively summarizes preclinical PT approaches in various in-vivo models. PT has been evaluated in several recent clinical trials. However, there are still several unanswered concerns due to a lack of appropriate regulation and pharmacokinetic data regarding the application of phages in human therapeutic procedures. In this review, we also presented the current state of PT and considered how animal models can be used to adapt these therapies for humans. The development of realistic solutions to circumvent these constraints is critical for advancing this technology.

A 36-year-old healthy male patient was presented to the emergency room 3 h after experiencing a laceration to the left foot caused by a porcelain shard. The defect measured 7.5 × 6.0 × 0.8 cm, and the composite amputated tissue consisted of skin and subcutaneous layers. The terminal branch of the lateral calcaneal artery was first anastomosed end-to-end to the corresponding artery in the wound defect. The lateral calcaneal nerve was anastomosed after blood flow was restored. The two lateral veins were anastomosed end-to-end to the corresponding veins in the wound defect. Postoperatively, 1.0 × 1.5 cm area of skin necrosis was present at the distal end of the tissue, which healed smoothly after two weeks of dressing changes. The patient had retained excellent aesthetic and functionality by the 37 month follow up. Although such isolated amputation is relatively rare, microsurgical replantation is, thus, a feasible option in the management of heel amputation.

Acinetobacter baumannii (A. baumannii) has emerged as one of the most troublesome pathogens in health care institutions. A. baumannii can cause a wide range of diseases in humans, including pneumonia and septicemia. Phage therapy has drawn great interest from medical researchers as a potential way to control infections by antibiotic-resistant A. baumannii. Using a pandrug-resistant clinical A. baumannii isolate ABZY9 as an indicator, we isolated a lytic phage Abp9 from hospital sewage. Abp9 belongs to myoviridae family and shows a wider host range of 12%. Abp9 contains a linear double-stranded DNA genome of 44,820 bp with a G + C content of 37.69%. The Abp9 genome contains 80 open reading frames, but lacks any known virulence genes or lysogen-formation genes. In a systemic A. baumannii infection mouse models, Abp9 treatment showed good therapeutic effects. We have also observed an excellent lytic activity against A. baumannii in biofilm form of growth in vitro. All of these suggest that Abp9 is a good candidate for the phage therapy against drug-resistant A. baumannii infections.

Background/Aims: N-acetylcysteine (NAC) is a novel and promising agent with activity against bacterial biofilms. Human serum also inhibits biofilm formation by some bacteria. We tested whether the combination of NAC and human serum offers greater anti-biofilm activity than either agent alone. Methods: Microtiter plate assays and confocal laser scanning microscopy were used to evaluate bacterial biofilm formation in the presence of NAC and human serum. qPCR was used to examine expression of selected biofilm-associated genes. Extracellular matrix (ECM) was observed by transmission electron microscopy. The antioxidants GSH or ascorbic acid were used to replace NAC, and human transferrin, lactoferrin, or bovine serum albumin were used to replace serum proteins in biofilm formation assays. A rat central venous catheter model was developed to evaluate the effect of NAC on biofilm formation in vivo. Results: NAC and serum together increased biofilm formation by seven different bacterial strains. In Staphylococcus aureus, expression of genes for some global regulators and for genes in the ica-dependent pathway increased markedly. In Pseudomonas aeruginosa, transcription of las, the PQS quorum sensing (QS) systems, and the two-component system GacS/GacA increased significantly. ECM production by S. aureus and P. aeruginosa was also enhanced. The potentiation of biofilm formation is due mainly to interaction between NAC and transferrin. Intravenous administration of NAC increased colonization by S. aureus and P. aeruginosa on implanted catheters. Conclusions: NAC used intravenously or in the presence of blood increases bacterial biofilm formation rather than inhibits it.

Background/Aims: As an “ESKAPE” pathogen, Acinetobacter baumannii is one of the leading causes of drug-resistant infections in humans. Phage therapy may be a useful strategy in treating infections caused by drug-resistant A. baumannii. Among 21 phage strains that were isolated and described earlier, we investigated the therapeutic efficacy of Abp1 because of its relatively wide host range. Methods: Phage stability assays were used to evaluate thermal and pH stability of Abp1. Abp1 was co-cultured with A. baumannii (AB1) over a range of multiplicities of infection to determine its bactericidal efficacy. HeLa or THP-1 cells were used in the cytotoxicity and protection assays. Finally, the therapeutic effects of Abp1 on local and systemic A. baumannii infection in mice were determined. Results: We found that Abp1 exhibits high thermal and pH stability and has a low frequency of lysogeny. Bacteriophage resistance also occurs at a very low frequency (3.51±0.46×10-8), and Abp1 can lyse almost all host cells at a MOI as low as 0.1. Abp1 has no detectable cytotoxicity to HeLa or THP-1 cells as determined by LDH release assay. Abp1 can rescue HeLa cells from A. baumannii infection, even if introduced 2 hours post infection. In both local and systemic A. baumannii infection mouse models, Abp1 treatment exhibits good therapeutic effects. Conclusion: Abp1 is an excellent candidate for phage therapy against drug-resistant A. baumannii infections.

Background: Thermal injury is the main cause of pulmonary disease in stroke after burn and can be life threatening. Heat-induced inflammation is an important factor that triggers a series of induces pathological changes. However, this mechanism underlying heat-induced inflammation in thermal inhalation injury remains unclear. Studies have revealed that astragaloside-IV (AS-IV), a natural compound extracted from Astragalus membranaceus, has protective effects in inflammatory diseases. Here, we investigated whether the protective effects of AS-IV occur because of the suppression of heat-induced endoplasmic reticulum (ER) stress and excessive autophagy Methods: AS-IV was administered to Wistar rats after thermal inhalation injury and 16HBE140-cells were treated with AS-IV. TNF-α, IL-6, and IL-8 levels were determined by ELISA and real-time PCR. ER stress and autophagy were determined by western blot. Autophagic flux was measured by recording the fluorescence emission of the fusion protein mRFP-GFP-LC3 by dynamic live-cell imaging. Results: AS-IV had protective effects against heat-induced reactive oxygen species production and attenuated ER stress. AS IV alleviated heat-induced excessive autophagy in vitro and in vivo. Excessive autophagy was attenuated by the PERK inhibitor GSK2656157 and eIF2α siRNA, suggesting that heat stress-induced autophagy can activate the PERK-eIF2α pathway. Beclin 1 and Atg5 siRNAs inhibited the upregulation of the inflammatory cytokines TNF-α, IL-6, and IL-8 after heat exposure. Conclusions: Thus, AS-IV may attenuate inflammatory responses by disrupting the crosstalk between autophagy and the PERK-eIF2α pathway and may be an ideal agent for treating inflammatory pulmonary diseases.

Background/Aims: Skin transplantation aims to cover skin defects but often fails due to immune rejection of the transplantated tissue. Immature dendritic cells (imDCs) induce immune tolerance but have a low migration rate. After stimulation, imDCs transform into mature DCs, which activate immune rejection. Thus, inducing imDC to obtain a high migration counteracts development of immune tolerance. Methods & Results: We transfected imDCs with a recombinant adenovirus carrying the CCR7 gene (Ad-CCR7) and a small interfering RNA targeting RelB (RelB-siRNA) to concurrently overexpress CCR7 and downregulate RelB expression. Functionally, such cells showed a significantly enhanced migration rate in the chemotactic assay and decreased T-cell proliferation after lipopolysaccharide stimulation in mixed lymphocyte reactions. Cotransfected cells showed an increased ability to induce immune tolerance by upregulating T regulatory (Treg) cells and shifting the Th1/Th2 ratio. Cotransfection of Ad-CCR7 and RelB-siRNA endowed imDCs with resistance to apoptosis and cell death. CCR7 overexpression and RelB knockdown (KD) in imDCs improve skin-graft survival in a murine skin-transplantation model. Conclusion: Transfection with Ad-CCR7 and RelB KD in imDCs may be an effective approach inducing immune tolerance, thus being potentially valuable for inhibiting allograft rejection.

Abstract Unlike individual, free-floating planktonic bacteria, biofilms are surface-attached communities of slow- or non-replicating bacteria encased within a protective extracellular polymeric matrix enabling persistent bacterial populations to tolerate high concentrations of antimicrobials. Our current antibacterial arsenal is composed of growth-inhibiting agents that target rapidly-dividing planktonic bacteria but not metabolically dormant biofilm cells. We report the first modular synthesis of a library of 20 halogenated phenazines (HP), utilizing the Wohl-Aue reaction, that targets both planktonic and biofilm cells. New HPs, including 6-substituted analogues, demonstrate potent antibacterial activities against MRSA, MRSE and VRE (MIC = 0.003–0.78 µM). HPs bind metal(II) cations and demonstrate interesting activity profiles when co-treated in a panel of metal(II) cations in MIC assays. HP 1 inhibited RNA and protein biosynthesis while not inhibiting DNA biosynthesis using 3H-radiolabeled precursors in macromolecular synthesis inhibition assays against MRSA. New HPs reported here demonstrate potent eradication activities (MBEC = 0.59–9.38 µM) against MRSA, MRSE and VRE biofilms while showing minimal red blood cell lysis or cytotoxicity against HeLa cells. PEG-carbonate HPs 24 and 25 were found to have potent antibacterial activities with significantly improved water solubility. HP small molecules could have a dramatic impact on persistent, biofilm-associated bacterial infection treatments.

ABSTRACT Acinetobacter baumannii is a growing threat, although lytic bacteriophages have been shown to effectively kill A. baumannii. However, the interaction between the host and the phage has not been fully studied. We demonstrate the global profile of transcriptional changes in extensively drug-resistant A. baumannii AB1 and the interaction with phage φAbp1 through RNA sequencing (RNA-seq) and bioinformatic analysis. Only 15.6% (600/3,838) of the genes of the infected host were determined to be differentially expressed genes (DEGs), indicating that only a small part of the bacterial resources was needed for φAbp1 propagation. Contrary to previous similar studies, more upregulated rather than downregulated DEGs were detected. Specifically, φAbp1 infection caused the most extensive impact on host gene expression at 10 min, which was related to the intracellular accumulation phase of virus multiplication. Based on the gene coexpression network, a middle gene (gp34, encoding phage-associated RNA polymerase) showed a negative interaction with numerous host ribosome protein genes. In addition, the gene expression of bacterial virulence/resistance factors was proven to change significantly. This work provides new insights into the interactions of φAbp1 and its host, which contributes to the further understanding of phage therapy, and provides another reference for antibacterial agents. IMPORTANCE Previous research has reported the transcriptomic phage-host interactions in Escherichia coli and Pseudomonas aeruginosa, leading to the detailed discovery of transcriptomic regulations and predictions of specific gene functions. However, a direct relationship between A. baumannii and its phage has not been previously reported, although A. baumannii is becoming a rigorous drug-resistant threat. We analyzed transcriptomic changes after φAbp1 infected its host, extensively drug-resistant (XDR) A. baumannii AB1, and found defense-like responses of the host, step-by-step control by the invader, elaborate interactions between host and phage, and elevated drug resistance gene expressions of AB1 after phage infection. These findings suggest the detailed interactions of A. baumannii and its phage, which may provide both encouraging suggestions for drug design and advice for the clinical use of vital phage particles.

Mesenchymal stem cells (MSCs) possess a capacity to enhance cutaneous wound healing that is well characterized. However, the therapeutic effect of MSCs appears to be limited. Modifying MSC target genes to increase necessary biological effects is a promising strategy for wound therapy. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that has a therapeutic effect on wound healing. In this study, we modified human amniotic mesenchymal stem cells (hAMSCs) using recombinant lentiviral vectors for expressing IL-10 and evaluated the therapeutic effects of hAMSCs-IL-10 in wound healing. We elucidated the mechanisms underlying the effects. We found that promoting wound healing was maintained by synergistic effects of hAMSCs and IL-10. hAMSCs-IL-10 showed stronger biological effects in accelerating wound closure, enhancing angiogenesis, modulating inflammation, and regulating extracellular matrix remodeling than hAMSCs. hAMSCs-IL-10 would be better at promoting wound healing and improving healing quality. These data may provide a theoretical foundation for clinical administration of hAMSCs-IL-10 in cutaneous wound healing and skin regeneration.

Pseudomonas aeruginosa is the leading cause of infection in burn patients. The increasing carbapenem resistance of P. aeruginosa has become a serious challenge to clinicians. The present study investigated the molecular typing and carbapenem resistance mechanisms of 196 P. aeruginosa isolates from the bloodstream and wound surface of patients in our burn center over a period of 6 years. By multilocus sequence typing (MLST), a total of 58 sequence types (STs) were identified. An outbreak of ST111, a type that poses a high international risk, occurred in 2014. The isolates from wound samples of patients without bacteremia were more diverse and more susceptible to antibiotics than strains collected from the bloodstream or the wound surface of patients with bacteremia. Importantly, a large proportion of the patients with multisite infection (46.51%) were simultaneously infected by different STs in the bloodstream and wound surface. Antimicrobial susceptibility testing of these isolates revealed high levels of resistance to carbapenems, with 35.71% susceptibility to imipenem and 32.14% to meropenem. To evaluate mechanisms associated with carbapenem resistance, experiments were conducted to determine the prevalence of carbapenemase genes, detect alterations of the oprD porin gene, and measure expression of the ampC β-lactamase gene and the mexB multidrug efflux gene. The main mechanism associated with carbapenem resistance was mutational inactivation of oprD (88.65%), accompanied by overexpression of ampC (68.09%). In some cases, oprD was inactivated by insertion sequence element IS1411, which has not been found previously in P. aeruginosa. These findings may help control nosocomial P. aeruginosa infections and improve clinical practice.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are prevalent in burn wards, and are especially serious in S. aureus bacteremia (SAB) patients. Glycopeptides and daptomycin are effective against MRSA infections, but MIC creeps can reduce their efficacy. Our object was to perform a molecular epidemiological investigation of S. aureus isolates in our burn center and to evaluate MICs for antimicrobials against SAB-associated MRSA isolates. A total of 259 S. aureus isolates, obtained from August 2011 to July 2016, were used in this study. Multiple molecular typing was used for molecular epidemiological analysis. E-tests were used to determine MICs of vancomycin, teicoplanin, and daptomycin for SAB-associated MRSA isolates. MIC values were stratified by collection date or source and compared. Spearman's test was used to analyze MICs correlations amongst tested antimicrobials. ST239-MRSA-III-t030-agrI clone was found to be dominant in both SAB and non-SAB patients, and significantly more in SAB patients (P < 0.0001). SAB-MRSA isolates exhibited decreased MICs for vancomycin, teicoplanin, and daptomycin during the 5-year period. Compared to those isolated from catheters or wounds, SAB-MRSA isolates from the bloodstream were less susceptible to vancomycin and daptomycin, but more susceptible to teicoplanin. MICs Correlation was found only between vancomycin and daptomycin in MRSA isolates from the bloodstream (rho = 0.250, P = 0.024). In conclusion, our results suggest that MRSA infections are still serious problems in burn centers. In contrast to most other studies, we observed increased susceptibility to glycopeptides and daptomycin against SAB-associated MRSA in our center from 2011 to 2016, suggesting the use of glycopeptides does not lead to MIC creeps. Isolates from different sites of the body may exhibit different levels of susceptibility and change trend over time for different antimicrobials, antimicrobials selection for MRSA infections should be considered comprehensively.

The gastrointestinal microbiome is crucial in human health. With greater than 10 times the cell count of an individual, the gastrointestinal microbiome provides many benefits to the host. It plays an important role in chronic illnesses and immune diseases and also following burns and trauma. This study aimed to determine whether severe burns affect the gastrointestinal microbiome during the early stages of after burn injury and the extent to which the microbiome is disturbed by such burns. We used a rat burn model to investigate any changes occurring in the microbiome after the burn trauma using 16S rRNA sequencing and downstream α-diversity, β-diversity, and taxonomy analysis. With 128631 and 143694 clean sequence reads, an average of 2287 and 2416 operational taxonomic units (OTUs) were recognized before and after the burn injury, respectively. Bacterial diversity within the pre- and post-burn groups was similar according to OTU richness, Chao 1 index, Shannon index and ACE index. However, the constituents of the gastrointestinal microbiota changed after the burn injury. Compared with the pre-burn samples, the post-burn samples showed a tendency to cluster together. The ratio of Firmicutes to Bacteroidetes decreased after the burn injury. Also, the abundance of some probiotic organisms (i.e., butyrate-producing bacteria and Lactobacillus) decreased after the burn injury. In contrast, opportunistic pathogenic bacteria, such as those of the genera Escherichia and Shigella and the phylum of Proteobacteria are more abundant post-burn. In conclusion, dysbiosis in the gastrointestinal microbiome was observed after the burn injury. Although the total number of species in the gastrointestinal microbiome did not differ significantly between the pre- and post-burn injury groups, the abundance of some bacterial components was affected to various extents.

Staphylococcus aureus is a common pathogen isolated from burn patients that can form biofilms on burn wounds and implanted deep vein catheters, which often leads to refractory infections or even biofilm-related sepsis. As biofilm formation is usually regulated by environmental conditions, we hypothesized that serum composition may be altered after burn injury, potentially affecting the ability of infecting bacteria to form biofilms. As predicted, we observed that serum from burn-injured rats increases biofilm formation by S. aureus and also induces bacterial aggregation and adherence to human fibronectin and fibrinogen. Analysis of potential regulatory factors revealed that exposure to burn serum decreases expression of the quorum-sensing agr system and increases mRNA levels of some biofilm inducers such as sarA and icaA. In addition, we also observed that burn serum imposes oxidative stress and increases expression of key oxidoreductase genes (sodA, sodM, katA, and ahpC) in S. aureus. Importantly, the ability of burn serum to enhance biofilm formation and bacterial cell aggregation can be abrogated by treatment with an antioxidant. Taken together, these findings indicate that burn serum increases S. aureus biofilm formation via elevated oxidative stress, and may lead to novel strategies to control biofilm formation and infection in burn patients.

Acinetobacter baumannii is a leading pathogen responsible for nosocomial infections. The emergence of carbapenem-resistant A. baumannii (CRAB) has left few effective antibiotics for clinicians to use. To investigate the temporal evolutionary relationships among CRAB strains, we collected 248 CRAB isolates from a Chinese burns institute over 3 years. The prevalence of the OXA-23 gene was detected by polymerase chain reaction. Multilocus sequence typing was used to type the CRAB strains and eBURST was used to analyze their evolutionary relationships. Wood surfaces (41%), sputa (24%), catheters (15%) and bloods (14%) were the four dominant isolation sources. Except for minocycline (33.5%) and sulbactam/cefoperazone (74.6%), these CRAB strains showed high resistance rates (> 90%) to 16 tested antibiotics. The 248 isolates fall into 26 sequence types (ST), including 9 known STs and 17 unknown STs. The majority (230/248) of these isolates belong to clonal complex 92 (CC92), including eight isolates belonging to seven unreported STs. A new clonal complex containing 11 isolates grouped into four new STs was identified. The OXA-23 gene was detected at high prevalence among the CRAB isolates and the prevalence rate among the various STs differed. The majority of the isolates displayed a close evolutionary relationship, suggesting that serious nosocomial spreading and nosocomial infections of CRAB have occurred in the burn unit. In conclusion, the main clonal complex for CRAB in this Chinese burn unit remain unchanged during the 3-year study period, and a new clonal complex was identified. CC92 is the dominant complex, and more attention should be directed towards monitoring the new clonal complex we have identified herein.

The impact of phage infection on the host cell is severe. In order to take over the cellular machinery, some phage proteins were produced to shut off the host biosynthesis early in the phage infection. The discovery and identification of these phage-derived inhibitors have a significant prospect of application in antibacterial treatment. This work presented a phage protein, gp70.1, with nonspecific inhibitory effects on Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). Gp70.1 was encoded by early gene – orf 70.1 from P. aeruginosa phage PaP3. The P. aeruginosa with a plasmid encoding gp70.1 showed with delayed growth and had the appearance of a small colony. The combination of multifaceted analysis including microarray-based transcriptomic analysis, RT-qPCR, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics and phenotype experiments were performed to investigate the effects of gp70.1 on P. aeruginosa. A total of 178 genes of P. aeruginosa mainly involved in extracellular function and metabolism were differentially expressed in the presence of gp70.1 at 3 examined time points. Furthermore, our results indicated that gp70.1 had an extensive impact on the extracellular phenotype of P. aeruginosa, such as motility, pyocyanin, extracellular protease, polysaccharide, and cellulase. For the metabolism of P. aeruginosa, the main effect of gp70.1 was the reduction of amino acid consumption. Finally, the RNA polymerase sigma factor RpoS was identified as a potential cellular target of gp70.1. Gp70.1 was the first bacterial inhibitor identified from Pseudomonas aeruginosa phage PaP3. It was also the first phage protein that interacted with the global regulator RpoS of bacteria. Our results indicated the potential value of gp70.1 in antibacterial applications. This study preliminarily revealed the biological function of gp70.1 and provided a reference for the study of other phage genes sharing similarities with orf70.1.

The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.

We isolated and characterized a new Pseudomonas aeruginosa myovirus named PaP1. The morphology of this phage was visualized by electron microscopy and its genome sequence and ends were determined. Finally, genomic and proteomic analyses were performed. PaP1 has an icosahedral head with an apex diameter of 68-70 nm and a contractile tail with a length of 138-140 nm. The PaP1 genome is a linear dsDNA molecule containing 91,715 base pairs (bp) with a G+C content of 49.36% and 12 tRNA genes. A strategy to identify the genome ends of PaP1 was designed. The genome has a 1190 bp terminal redundancy. PaP1 has 157 open reading frames (ORFs). Of these, 143 proteins are homologs of known proteins, but only 38 could be functionally identified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-mass spectrometry allowed identification of 12 ORFs as structural protein coding genes within the PaP1 genome. Comparative genomic analysis indicated that the Pseudomonas aeruginosa phage PaP1, JG004, PAK_P1 and vB_PaeM_C2-10_Ab1 share great similarity. Besides their similar biological characteristics, the phages contain 123 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore propose that these four phages be classified as PaP1-like phages, a new phage genus of Myoviridae that infects Pseudomonas aeruginosa.