The rising prevalence of multidrug-resistant pathogens, such as Enterobacter cloacae, poses significant challenges to public health and food safety, particularly in the poultry industry, where contamination risks are high, leading to foodborne diseases. This study investigated the isolation and characterization of a bacteriophage, vB_Ecl_id4496, targeting E. cloacae. Electron micrographs revealed that the phage had an icosahedral head of 92 ± 10 nm in diameter and a long contractile tail with an approximate length of 125 ± 8 nm. Bacterial Lysis kinetics demonstrated phage-mediated lysis of E. cloacae, whereas thermal stability analysis showed reduced activity at elevated temperatures. Whole-genome sequencing revealed a linear double-stranded DNA genome of 172,413 bp with a GC content of 43.25

Multidrug-resistant (MDR) Enterobacter cloacae is a growing public health issue worldwide, highlighting the urgent need for alternative antimicrobial strategies. This study reports on a lytic phage, designated B1, isolated from sewage, which exhibits specificity and lytic efficiency against MDR E. cloacae. Morphological observation revealed that B1 possesses an icosahedral head (~54 nm) and a short tail (~13 nm). Phage B1 showed a narrow host range, demonstrated stability within a temperature range of 4-37 °C, tolerance to pH values between 5 and 11, and showed an excellent bacteriolytic capacity with a short latent period of less than 10 min and a burst size of approximately 150 PFU/initially infected cell, indicating a rapid lytic cycle and efficient replication capability. Whole-genome sequencing revealed that the phage genome consists of 40,163 base pairs of double-stranded DNA containing 52 open reading frames (ORFs) with a GC content of 52%. Comparative genome-wide analysis using VIRIDIC revealed that B1 shares 75% to 92% similarity with Escherichia phage IMM-002 (accession: NC_048071), Citrobacter phage SH4, and Cronobacter phage Dev2 (accession: NC_023558), but shares less than 70% similarity with other Enterobacter phages. According to ICTV criteria, B1 represents a new species within the same genus as T7-like phages belonging to Autographiviridae, subfamily Studiervirinae, genus Kayfunavirus. In addition, B1 lacks lysogeny-associated or virulence genes and exhibits potent lytic activity against multidrug-resistant E. cloacae, making it a promising candidate for phage therapy. These findings opened up our understanding of the diversity of T7-like phages and provided insights into their evolutionary adaptability and therapeutic potential.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that poses a significant threat due to its increasing multidrug resistance, particularly in clinical settings. This study aimed to isolate and characterize a novel bacteriophage, phiLCL12, from hospital wastewater and evaluate its potential in combination with antibiotics to combat P. aeruginosa infections and biofilm formation. Transmission electron microscopy revealed that phiLCL12 possesses a long contractile tail. The isolated phage exhibited a broad host range of 82.22% and could adsorb up to 98% of its target within 4 min. It was effective against multidrug-resistant strains at both high and low multiplicities of infection (MOIs) levels in lysis tests. Taxonomic classification was determined using PhaGCN2 and Whole genomic analysis, and the results identified phiLCL12 as a member of the Pbunavirus. In vitro experiments demonstrated that phiLCL12 significantly enhanced biofilm clearance and inhibited biofilm formation when combined with sub-inhibitory concentrations of imipenem. Furthermore, in vivo experiments using a zebrafish model showed that phage-antibiotic synergy (PAS) improved survival rate compared to antibiotic treatment alone. This study demonstrates that phiLCL12 is effective in both eradicating and preventing P. aeruginosa biofilm formation. The combination of phiLCL12 and imipenem provides a synergistic effect, significantly enhancing survival outcomes in a zebrafish model. These findings highlight the potential of phage-antibiotic synergy as a promising therapeutic strategy against biofilm-associated infections.

The global rise of multidrug-resistant Enterobacter cloacae strains, especially those that are resistant to carbapenems and produce metallo-β-lactamases, poses a critical challenge in clinical settings owing to limited treatment options. While bacteriophages show promise in treating these infections, their use is hindered by scarce resources and insufficient genomic data. In this study, we isolated ECLFM1, a novel E. cloacae phage, from sewage water using a carbapenem-resistant clinical strain as the host. ECLFM1 exhibited rapid adsorption and a 15-min latent period, with a burst size of approximately 75 PFU/infected cell. Its genome, spanning 172,036 bp, was characterized and identified as a member of Karamvirus. In therapeutic applications, owing to a high multiplicity of infection, ECLFM1 showed increased survival in zebrafish infected with E. cloacae. This study highlights ECLFM1's potential as a candidate for controlling clinical E. cloacae infections, which would help address challenges in treating multidrug-resistant strains and contribute to the development of alternative treatments.

The rise of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a significant global challenge in clinical and healthcare settings, severely limiting treatment options. This study aimed to utilize a bacteriophage as an alternative therapy against carbapenem-resistant K. pneumoniae. A novel lytic N4-like Klebsiella phage, vB_kpnP_KPYAP-1 (KPYAP-1), was isolated from sewage. It demonstrated efficacy against the K62 serotype polysaccharide capsule of blaOXA-48-producing K. pneumoniae. KPYAP-1 forms small, clear plaques, has a latent period of 20 min, and reaches a growth plateau at 35 min, with a burst size of 473 plaque-forming units (PFUs) per infected cell. Phylogenetic analysis places KPYAP-1 in the Schitoviridae family, Enquatrovirinae subfamily, and Kaypoctavirus genus. KPYAP-1 employs an N4-like direct terminal repeat mechanism for genome packaging and encodes a large virion-encapsulated RNA polymerase. It lacks integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and toxins, ensuring its safety for therapeutic use. Comparative genome analysis revealed that the KPYAP-1 genome is most similar to the KP8 genome, yet differs in tail fiber protein, indicating variations in host recognition. In a zebrafish infection model, KPYAP-1 significantly improved the survival rate of infected fish by 92% at a multiplicity of infection (MOI) of 10, demonstrating its potential for in vivo treatment. These results highlight KPYAP-1 as a promising candidate for developing phage-based therapies targeting carbapenemase-producing K. pneumoniae.

Multiple drug-resistant (MDR) Pseudomonas aeruginosa commonly causes severe hospital-acquired infections. The gradual emergence of carbapenem-resistant P. aeruginosa has recently gained attention. A wide array of P. aeruginosa-mediated pathogenic mechanisms, including its biofilm-forming ability, limits the use of effective antimicrobial treatments against it. In the present study, we isolated and characterized the phenotypic, bio-logical, and genomic characteristics of a bacteriophage, vB_PaP_phiPA1-3 (phiPA1-3). Biofilm eradication and phage rescue from bacterial infections were assessed to demonstrate the efficacy of the application potential. Host range spectrum analysis revealed that phiPA1-3 is a moderate host range phage that infects 20% of the clinically isolated strains of P. aeruginosa tested, including carbapenem-resistant P. aeruginosa (CRPA). The phage exhibited stability at pH 7.0 and 9.0, with significantly reduced viability below pH 5.0 and beyond pH 9.0. phiPA1-3 is a lytic phage with a burst size of 619 plaque-forming units/infected cell at 37 degrees C and can effectively lyse bacteria in a multiplicity of infection-dependent manner. The genome size of phiPA1-3 was found to be 73,402 bp, with a G+C content of 54.7%, containing 93 open reading frames, of which 62 were annotated as hypothetical proteins and the remaining 31 had known functions. The phage possesses several proteins similar to those found in N4-like phages, including three types of RNA polymerases. This study concluded that phiPA1-3 belongs to the N4-like Schitoviridae family, can potentially eradicate P. aeruginosa biofilms, and thus, serve as a valuable tool for controlling CRPA infections.

Acinetobacter baumannii is an opportunistic pathogen that significantly causes hospital-acquired infections. Due to its multidrug resistance, treating infections caused by this pathogen is challenging. Recently, phages have gained attention as a potential alternative to antibiotics in treating bacterial infections. While lytic phages are preferred in therapy, the use of temperate phages for this purpose has received less attention. This study characterized a novel temperate phage vB_AbaM_ABMM1 (ABMM1) with antibacterial activity toward A. baumannii. ABMM1 adsorbs quickly, has short latent periods, and is relatively stable at various temperatures and neutral pH. ABMM1 has an icosahedral head and a contractile tail. It has a 75,731 kb circular permuted dsDNA genome containing 86 gene products with 37.3% G + C content and a mosaic arrangement typical of temperate phages. Genomic analysis confirmed that ABMM1 does not have antibiotic-resistance genes or virulence-related factors. The packaging strategy was predicted in silico, suggesting that ABMM1 represents a headful phage. Only truncated ABMM1 prophage was detected and has similarity in the genome of several A. baumannii strains. Despite its ability to integrate into the host chromosome, the high MOI of ABMM1 (MOI 10) effectively killed the host bacterial cells and reduced the fatality rate of bacterial infection in the zebrafish model. These findings indicate that ABMM1 can be an alternative treatment for A. baumannii infection.

Multidrug-resistant Acinetobacter baumannii (MDRAB) is a pathogen recognized as antimicrobial-resistant bacteria involved in healthcare-associated infections. Resistance to antibiotics has made alternative therapies necessary. Bacteriophage therapy is considered a potential solution to treat MDRAB. In this study, we isolated and characterized the phage vB_AbaS_TCUP2199 (TCUP2199), which can infect MDRAB. Morphological analysis revealed that TCUP2199 belongs to the Siphoviridae family. TCUP2199 has a wide host range, can adsorb rapidly (68.28% in 2 min), and has a burst size of 196 PFU/cell. At least 16 distinct structural proteins were visualized by SDS polyacrylamide gel electrophoresis. A stability test showed that TCUP2199 was stable at 37 °C and pH 7. Genome analysis of TCUP2199 showed that it consists of a double-stranded DNA genome of 79,572 bp with a G+C content of 40.39%, which contains 98 putative open reading frames, none of which is closely related to the bacteriophage genome sequence that was found in the public database. TCUP2199 shows similarity in genomic organization and putative packaging mechanism with Achromobacter phage JWF and Pseudoalteromonas phage KB12-38 based on protein BLAST and phylogenetic analysis. Because of those unique characteristics, we consider TCUP2199 to be a novel phage that is suitable for inclusion in a phage cocktail to treat A. baumannii infection.

Carboxy-terminal processing protease (Ctp) is a serine protease that controls multiple cellular processes through posttranslational modification of proteins. Acinetobacter baumannii ATCC 17978 ctp mutant, namely MR14, is known to cause cell wall defects and autolysis. The objective of this study was to investigate the role of ctp mutation–driven autolysis in regulating biofilms in A. baumannii and to evaluate the vesiculation caused by cell wall defects. We found that in A. baumannii, Ctp is localized in the cytoplasmic membrane, and loss of Ctp function enhances the biofilm-forming ability of A. baumannii. Quantification of the matrix components revealed that extracellular DNA (eDNA) and proteins were the chief constituents of MR14 biofilm, and the transmission electron microscopy further indicated the presence of numerous dead cells compared with ATCC 17978. The large number of MR14 dead cells is potentially the result of compromised outer membrane integrity, as demonstrated by its high sensitivity to sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). MR14 also exhibited the hypervesiculation phenotype, producing outer-membrane vesicles (OMVs) of large mean size. The MR14 OMVs were more cytotoxic toward A549 cells than ATCC 17978 OMVs. Our overall results indicate that A. baumanniictp negatively controls pathogenic traits through autolysis and OMV biogenesis.

Biofilm formation is one of the main causes of increased antibiotic resistance in Acinetobacter baumannii infections. Bacteriophages and their derivatives, such as tail proteins with depolymerase activity, have shown considerable potential as antibacterial or antivirulence agents against bacterial infections. Here, we gained insights into the activity of a capsular polysaccharide (CPS) depolymerase, derived from the tailspike protein (TSP) of φAB6 phage, to degrade A. baumannii biofilm in vitro. Recombinant TSP showed enzymatic activity and was able to significantly inhibit biofilm formation and degrade formed biofilms; as low as 0.78 ng, the inhibition zone can still be formed on the bacterial lawn. Additionally, TSP inhibited the colonization of A. baumannii on the surface of Foley catheter sections, indicating that it can be used to prevent the adhesion of A. baumannii to medical device surfaces. Transmission and scanning electron microscopy demonstrated membrane leakage of bacterial cells treated with TSP, resulting in cell death. The therapeutic effect of TSP in zebrafish was also evaluated and the results showed that the survival rate was significantly improved (80%) compared with that of the untreated control group (10%). Altogether, we show that TSP derived from φAB6 is expected to become a new antibiotic against multi-drug resistant A. baumannii and a biocontrol agent that prevents the formation of biofilms on medical devices.

Motility plays an essential role in the host–parasite relationship of pathogenic bacteria, and is often associated with virulence. While many pathogenic bacteria use flagella for locomotion, Acinetobacter baumannii strains do not have flagella, but have other features that aid in their motility. To study the genes involved in motility, transposon mutagenesis was performed to construct A. baumannii mutant strains. Mutant strain MR14 was found to have reduced motility, compared to wild-type ATCC 17978. NCBI BLAST analysis revealed that the Tn10 transposon in the MR14 genome is integrated into the gene that encodes for carboxy-terminal processing protease (Ctp). Additionally, MR14 exhibits a mucoidy, sticky phenotype as the result of increased extracellular DNA (eDNA) caused by bacterial autolysis. Transmission and scanning electron microscopy revealed cytoplasmic content leaving the cell and multiple cell membrane depressions, respectively. MR14 showed higher sensitivity to environmental stressors. Mutation of the ctp gene reduced invasion and adhesion of A. baumannii to airway epithelial cells, potentially due to increased hydrophobicity. In the zebrafish model of infection, MR14 increased the survival rate by 40% compared to the wild-type. Taken together, the ctp gene in A. baumannii has a pivotal role in maintaining membrane integrity, adaptation to environmental stress, and controlling virulence.

Acinetobacter baumannii (A. baumannii) strains are common nosocomial pathogens that can cause infections and can easily become resistant to antibiotics. Thus, analytical methods that can be used to rapidly identify A. baumannii from complex samples should be developed. Tail fiber proteins derived from the tail fibers of bacteriophages can recognize specific bacterial surface polysaccharides. For example, recombinant tail proteins, such as TF2 and TF6 derived from the tail fibers of bacteriophages phi AB2 and phi AB6, can recognize A. baumannii clinical isolates M3237 and 54149, respectively. Thus, TF2 and TF6 can be used as probes to target specific A. baumannii strains. Generally, TF2 and TF6 are tagged with a hexahistidine (His(6)) for ease of purification. Given that His(6) possesses specific affinity toward alumina through His(6)-Al chelation, TF2- and TF6-immobilized alumina-coated magnetic nanoparticles (Fe3O4@Al2O3 MNPs) were generated through chelation under microwave heating (power, 900 W) for 60 s in this study. The as-prepared TF2-Fe3O4@Al2O3 and TF6-Fe3O4@Al(2)O(3 )MNPs were used as affinity probes to trap trace A. baumannii M3237 and 54149, respectively, from sample solutions. Matrix-assisted laser desorption/ionization mass spectrometry capable of identifying bacteria on the basis of the obtained fingerprint mass spectra of intact bacteria was used as the detection tool. Results demonstrated that the current approach can be used to distinguish A. baumannii M3237 from A. baumannii 54149 by using TF2-Fe3O4@Al2O3 and TF6-Fe3O4@Al(2)O(3 )MNPs as affinity probes. Furthermore, the limits of detection of the current method for A. baumannii M3237 and 54149 are similar to 10(5) and similar to 10(4) cells mL(-1), respectively. The feasibility of using the developed method to selectively detect A. baumannii M3237 and 54149 from complex serum samples was demonstrated.

Pseudaminic acid (Pse) has been known for participating in crucial bacterial virulence and thus is an attractive target in the development of glycoconjugate vaccine. Particularly, this therapeutic alternative was suggested to be a potential solution against antibiotic resistant Acinetobacter baumannii that poses a serious global health threat. Also, Pse was found to be involved in the exopolysaccharide (EPS) of mild antibiotic resistant A. baumannii strain 54149 (Ab-54149) of which specific glycosyl linkage can be depolymerized by phage ΦAB6 tailspike protein (ΦAB6TSP). In this study, we found that the antibodies induced by Ab-54149 EPS was capable of recognizing a range of EPS of other clinical A. baumannii strains, and deemed as a great potential material for vaccination. To efficiently acquire homogeneous EPS-derived oligosaccharide with significant immunogenic activity for the production of glycoconjugate, we used the ΦAB6TSP for the fragmentation of Ab-54149 EPS instead of chemical methods. Moreover, insight into the ligand binding characterization of ΦAB6TSP suggested the branched Pse on the Ab-54149 EPS served as a recognition site of ΦAB6TSP. The serum boosted by ΦAB6TSP-digested product and carrier protein CRM197 conjugate complex displayed specific sensitivity toward Ab-54149 EPS with bacterial killing activity. Strikingly, Pse is an ideal epitope with strong antigenicity, profiting the application of the probe for pathogen detection and glyco-based vaccine.

With an increase in antibiotic-resistant strains, the nosocomial pathogen Acinetobacter baumannii has become a serious threat to global health. Glycoconjugate vaccines containing fragments of bacterial exopolysaccharide (EPS) are an emerging therapeutic to combat bacterial infection. Herein, we characterize the bacteriophage ΦAB6 tailspike protein (TSP), which specifically hydrolyzed the EPS of A. baumannii strain 54149 (Ab-54149). Ab-54149 EPS exhibited the same chemical structure as two antibiotic-resistant A. baumannii strains. The ΦAB6 TSP-digested products comprised oligosaccharides of two repeat units, typically with stoichiometric pseudaminic acid (Pse). The 1.48-1.89-Å resolution crystal structures of an N-terminally-truncated ΦAB6 TSP and its complexes with the semi-hydrolyzed products revealed a trimeric β-helix architecture that bears intersubunit carbohydrate-binding grooves, with some features unusual to the TSP family. The structures suggest that Pse in the substrate is an important recognition site for ΦAB6 TSP. A region in the carbohydrate-binding groove is identified as the determinant of product specificity. The structures also elucidated a retaining mechanism, for which the catalytic residues were verified by site-directed mutagenesis. Our findings provide a structural basis for engineering the enzyme to produce desired oligosaccharides, which is useful for the development of glycoconjugate vaccines against A. baumannii infection.

Concerns of Acinetobacter baumannii infection have increased due to the emergence of multi-drug resistance. In the present study, we determined the capsular polysaccharide (CPS) structure of A. baumannii SK44, a clinical isolate from Taiwan, to consist of pentasaccharide repeats. We found that CPS-induced antibody provided 55% protection against challenge in an animal model. The CPS-specific antibody reacted with the surface components of about 62% clinical isolates (342/554 strains) from cross-sectional and longitudinal studies by dot-immunoassay. Pulsed-field gel electrophoresis of positive strains showed the antibody covered different clonalites of A. baumannii clinical isolates. Meanwhile, using the CPS antibody as a probe, we found a number of outer membrane proteins bound to the antibody, including OmpA/motB, TonB-dependent receptor, and Omp38, indicating their association with CPS. These results might lead to the use of the capsular polysaccharide as a vaccine to prevent A. baumannii infection.

The increase in the prevalence of multidrug-resistant Acinetobacter baumannii (MDRAB) strains is a serious public health concern. Antimicrobial peptides (AMPs) are a possible solution to this problem. In this study, we examined whether AMPs could be derived from phage endolysins. We synthesized four AMPs based on an amphipathic helical region in the C-terminus of endolysin LysAB2 encoded by the A . baumannii phage ΦAB2. These peptides showed potent antibacterial activity against A . baumannii (minimum inhibitory concentration, 4–64 μM), including some MDR and colistin-resistant A . baumannii . Of the four peptides, LysAB2 P3, with modifications that increased its net positive charge and decreased its hydrophobicity, showed high antibacterial activity against A . baumannii but little haemolytic and no cytotoxic activity against normal eukaryotic cells. The results of electron microscopy experiments and a fluorescein isothiocyanate staining assay indicated that this peptide killed A . baumannii through membrane permeabilization. Moreover, in a mouse intraperitoneal infection model, at 4 h after the bacterial injection, LysAB2 P3 decreased the bacterial load by 13-fold in ascites and 27-fold in blood. Additionally, LysAB2 P3 rescued sixty percent of mice heavily infected with A . baumannii from lethal bacteremia. Our results confirmed that bacteriophage endolysins are a promising resource for developing effective AMPs.

Aeromonas hydrophila is an opportunistic pathogenic bacterium causing diseases in human and fish. The emergence of multidrug-resistant A. hydrophila isolates has been increasing in recent years. In this study, we have isolated a novel virulent podophage of A. hydrophila, designated as Ahp1, from waste water. Ahp1 has a rapid adsorption (96% adsorbed in 2 min), a latent period of 15 min, and a burst size of 112 PFU per infected cell. At least eighteen Ahp1 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 36-kDa protein being the predicted major capsid protein. Genome analysis of Ahp1 revealed a linear doubled-stranded DNA genome of 42,167 bp with a G + C content of 58.8%. The genome encodes 46 putative open reading frames, 5 putative phage promoters, and 3 transcriptional terminators. Based on high degrees of similarity in overall genome organization and among most of the corresponding ORFs, as well as phylogenetic relatedness among their DNAP, RNAP and major capsid proteins, we propose a new subgroup, designated Ahp1-like subgroup. This subgroup contains Ahp1 and members previously belonging to phiKMV-like subgroup, phiAS7, phi80-18, GAP227, phiR8-01, and ISAO8. Since Ahp1 has a narrow host range, for effective phage therapy, different phages are needed for preparation of cocktails that are capable of killing the heterogeneous A. hydrophila strains.

Acinetobacter baumannii is a non-fermenting, gram-negative bacterium. In recent years, the frequency of A. baumannii infections has continued to increase, and multidrug-resistant strains are emerging in hospitalized patients. Therefore, as therapeutic options become limited, the potential of phages as natural antimicrobial agents to control infections is worth reconsidering. In our previous study, we isolated ten virulent double-stranded DNA A. baumannii phages, ϕAB1-9 and ϕAB11, and found that each has a narrow host range. Many reports indicate that receptor-binding protein of phage mediates host recognition; however, understanding of the specific interactions between A. baumannii and phages remains very limited. In this study, host determinants of A. baumannii phages were investigated. Sequence comparison of ϕAB6 and ϕAB1 revealed high degrees of conservation among their genes except the tail fiber protein (ORF41 in ϕAB1 and ORF40 in ϕAB6). Furthermore, we found that ORF40ϕAB6 has polysaccharide depolymerase activity capable of hydrolyzing the A. baumannii exopolysaccharide and is a component of the phage tail apparatus determining host specificity. Thus, the lytic phages and their associated depolymerase not only have potential as alternative therapeutic agents for treating A. baumannii infections but also provide useful and highly specific tools for studying host strain exopolysaccharides and producing glycoconjugate vaccines.

BackgroundThe lon gene of Helicobacter pylori strains is constitutively expressed during growth. However, virtually nothing is understood concerning the role of Lon in H.pylori. This study examined the function and physiological role of Lon in H.pylori (HpLon) using a trapping approach to identify putative Lon binding partners in the bacterium.Materials and MethodsProtease-deficient Lon was expressed and served as the bait in trapping approach to capture the interacting partners in H.pylori. The antibiotic susceptibility of wild-type and lon derivative mutants was determined by the E test trips and the disc diffusion assay. The effect of HpLon on RdxA activity was detected the change in NADPH oxidation and metronidazole reduction by spectrophotometer.ResultsLon in Helicobacter pylori (HpLon) interacting partners are mostly associated with metronidazole activation. lon mutant presents more susceptible to metronidazole than that of the wild type, and this phenotype is recovered by complementation of the wild-type Lon. We found that the ATPases associated with a variety of cellular activities (AAA(+)) module of HpLon causes a decrease in both NADPH oxidase and Mtz reductase activity in RdxA, a major Mtz-activating enzyme in H.pylori.ConclusionMetronidazole resistance of H.pylori causes the serious medical problem worldwide. In this study, HpLon is involved in metronidazole susceptibility among H.pylori strains. We provide the evidence that HpLon alters RdxA activity in vitro. The decrease in metronidazole activation caused by HpLon is possibly prior to accumulate mutation in rdxA gene before the metronidazole-resistant strains to be occurred.