Tail Fiber Protein Research Articles

Ackermannviridae bacteriophage against carbapenem-resistant Klebsiella pneumoniae of capsular type 64.

Lytic bacteriophages (phages) are promising clinically viable therapeutic options against carbapenem-resistant Klebsiella pneumoniae (CRKP). In China, the predominant strains are those assigned to sequence type 11 and capsular type 64 (ST11-KL64). The emergence of phage resistance is a major bottleneck hindering effective phage therapy, requiring more new phages to provide the flexibility for creating different phage cocktails. However, the majority of phages against ST11-KL64 CRKP belong to the genus Przondovirus of the family Autographiviridae, which limits the options for constructing cocktails. We recovered a novel lytic phage of the genus Taipeivirus within the family Ackermannviridae against ST11-KL64 CRKP from a river in China. We phenotypically characterized this phage and obtained its genome sequence for analysis. This phage can inhibit the growth of ST11-KL64 CRKP for 6.5 h at a 0.1 multiplicity of infection and exhibits a narrow host range, being unable to attack CRKP strains of the other 30 capsular types. This phage carries no genes encoding antimicrobial resistance, virulence, or lysogeny. It is stable across a wide range of temperatures and pH values, making it suitable for phage therapy. Unlike other Taipeivirus phages, P01 has two tail spike proteins and a unique tail fiber protein. The distinct tail composition of this phage contributes to its activity against ST11-KL64 CRKP and its narrow host range. Taken together, we recovered a phage of a novel viral species with the potential for therapy, which expands the phage biobank against CRKP.

In Vitro and In Vivo Assessments of Newly Isolated N4-like Bacteriophage against ST45 K62 Capsular-Type Carbapenem-Resistant Klebsiella pneumoniae: vB_kpnP_KPYAP-1.

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.

Proposition of a phagosensor with a unique Teseptimavirus SAL_R1S on a carbon nanotube platform for efficient detection of typhoid pathogen

The detection of the typhoid pathogen, Salmonella enterica serotype Typhi (S. Typhi), holds massive clinical, public health, and epidemiological significance around the globe. Conventional diagnosis relies on bacterial isolation having a set of challenges when it comes to accurate detection, therapeutic intervention and disease management. Substantial reviews and reports exist on the advantages of bacteriophage-based biosensors (phagosensors) concerning Salmonella. However, phagosensor for Salmonella Typhi point of care detection at a lower limit of detection (LOD) has yet to be reported. This study is the earliest endeavor to develop a multi-wall carbon nanotubes (MWCNTs) based electrochemical phagosensor utilizing a unique bacteriophage SAL_R1S as a biomolecular recognition element, selectively binding S. Typhi DMS_A1 at LOD of 1 CFU/ml. S. Typhi DMS_A1, retrieved from patient's blood, consists of 10 pathogenicity islands and a wide range of efflux pump genes in its whole genome, which has not yet been documented for Salmonella. Subsequent screening for its specific bacteriophage from a sewage sample pinpointed the phage SAL_R1S of class Caudoviricetes, family Autographiviridae and genus Teseptimavirus. The whole genome- and tail-fiber protein- based alignment was close to Salmonella phage Vi06 covering 88.8 % and 90 % similarity, respectively. SAL_R1S exclusively binds S. Typhi in a specific manner and also possess excellent genetic feature as a candidate for developing a highly sensitive electrochemical phagosensor. Therefore, it was covalently immobilized onto a modified SPE/MWCNT/PANI-based electrode surface, allowing charge-directed, oriented immobilization which then confirmed through scanning electron microscopy. The electrode surface was featured via field emission scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. The pathogen detection process of the phagosensor is quick (∼ 20 min). It has exceptional selectivity for typhoid pathogens from blood, wastewater or within mixed populations, indicating the application of this proposed phagosensor in clinical settings as a rapid, alternative to available conventional detection techniques, and low-cost surveillance tool.

A Novel Dhillonvirus Phage against Escherichia coli Bearing a Unique Gene of Intergeneric Origin.

Antibiotics resistance is expanding amongst pathogenic bacteria. Phage therapy is a revived concept for targeting bacteria with multiple antibiotics resistances. In the present study, we isolated and characterized a novel phage from hospital treatment plant input, using Escherichia coli (E. coli) as host bacterium. Phage lytic activity was detected by using soft agar assay. Whole-genome sequencing of the phage was performed by using Next-Generation Sequencing (NGS). Host range was determined using other species of bacteria and representative genogroups of E. coli. Whole-genome sequencing of the phage revealed that Escherichia phage Ioannina is a novel phage within the Dhillonvirus genus, but significantly diverged from other Dhillonviruses. Its genome is a 45,270 bp linear double-stranded DNA molecule that encodes 61 coding sequences (CDSs). The coding sequence of CDS28, a putative tail fiber protein, presented higher similarity to representatives of other phage families, signifying a possible recombination event. Escherichia phage Ioannina lytic activity was broad amongst the E. coli genogroups of clinical and environmental origin with multiple resistances. This phage may present in the future an important therapeutic tool against bacterial strains with multiple antibiotic resistances.

Antibacterial activity evaluation of a novel K3-specific phage against Acinetobacter baumannii and evidence for receptor-binding domain transfer across morphologies

Acinetobacter baumannii (A. baumannii) poses a serious public health challenge due to its notorious antimicrobial resistance, particularly carbapenem-resistant A. baumannii (CRAB). In this study, we isolated a virulent phage, named P1068, from medical wastewater capable of lysing CRAB, primarily targeting the K3 capsule type. Basic characterization showed that P1068 infected the A. baumannii ZWAb014 with an optimal MOI of 1, experienced a latent period of 10 ​min and maintained stability over a temperature range of 4–37 ​°C and pH range of 3–10. Phylogenetic and average nucleotide identity analyses indicate that P1068 can be classified as a novel species within the genus Obolenskvirus of the Caudoviricetes class as per the most recent virus classification released by the International Committee on Taxonomy of Viruses (ICTV). Additionally, according to classical morphological classification, P1068 is identified as a T4-like phage (Myoviridae). Interestingly, we found that the tail fiber protein (TFP) of P1068 shares 74% coverage and 88.99% identity with the TFP of a T7-like phage (Podoviridae), AbKT21phiIII (NC_048142.1). This finding suggests that the TFP gene of phages may undergo horizontal transfer across different genera and morphologies. In vitro antimicrobial assays showed that P1068 exhibited antimicrobial activity against A. baumannii in both biofilm and planktonic states. In mouse models of intraperitoneal infection, P1068 phage protected mice from A. baumannii infection and significantly reduced bacterial loads in various tissues such as the brain, blood, lung, spleen, and liver compared to controls. In conclusion, this study demonstrates that phage P1068 might be a potential candidate for the treatment of carbapenem-resistant and biofilm-forming A. baumannii infections, and expands the understanding of horizontal transfer of phage TFP genes.

Regulation of anti-phage defense mechanisms by using cinnamaldehyde as a quorum sensing inhibitor.

Multidrug-resistant bacteria and the shortage of new antibiotics constitute a serious health problem. This problem has led to increased interest in the use of bacteriophages, which have great potential as antimicrobial agents but also carry the risk of inducing resistance. The objective of the present study was to minimize the development of phage resistance in Klebsiella pneumoniae strains by inhibiting quorum sensing (QS) and thus demonstrate the role of QS in regulating defense mechanisms. Cinnamaldehyde (CAD) was added to K. pneumoniae cultures to inhibit QS and thus demonstrate the role of the signaling system in regulating the anti-phage defense mechanism. The QS inhibitory activity of CAD in K. pneumoniae was confirmed by a reduction in the quantitative expression of the lsrB gene (AI-2 pathway) and by proteomic analysis. The infection assays showed that the phage was able to infect a previously resistant K. pneumoniae strain in the cultures to which CAD was added. The results were confirmed using proteomic analysis. Thus, anti-phage defense-related proteins from different systems, such as cyclic oligonucleotide-based bacterial anti-phage signaling systems (CBASS), restriction-modification (R-M) systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, and bacteriophage control infection (BCI), were present in the cultures with phage but not in the cultures with phage and CAD. When the QS and anti-phage defense systems were inhibited by the combined treatment, proteins related to phage infection and proliferation, such as the tail fiber protein, the cell division protein DamX, and the outer membrane channel protein TolC, were detected. Inhibition of QS reduces phage resistance in K. pneumoniae, resulting in the infection of a previously resistant strain by phage, with a significant increase in phage proliferation and a significant reduction in bacterial growth. QS inhibitors could be considered for therapeutic application by including them in phage cocktails or in phage-antibiotic combinations to enhance synergistic effects and reduce the emergence of antimicrobial resistance.

Binding activity and specificity of tail fiber protein 35Q for Salmonella pullorum.

Salmonella, a prevalent pathogen with significant implications for the poultry industry and food safety, presents a global public health concern. The rise in antibiotic resistance has exacerbated the challenge of prevention. Accurate and sensitive detection methods are essential in combating Salmonella infections. Bacteriophages, viruses capable of targeting and destroying bacteria, leverage their host specificity for accurate microbial detection. Notably, the tail fiber protein of bacteriophages plays a crucial role in recognizing specific hosts, making it a valuable tool for targeted microbial detection. This study focused on the tail fiber protein 35Q of Salmonella pullorum (SP) bacteriophage YSP2, identified through protein sequencing and genome analysis. Bioinformatics analysis revealed similarities between 35Q and other Salmonella bacteriophage tail fiber proteins. The protein was successfully expressed and purified using an Escherichia coli expression system, and its binding activity and specificity were confirmed. ELISA assays and adsorption experiments demonstrated that 35Q interacts with the outer membrane protein (OMP) receptor on bacterial surfaces. This investigation provides valuable insights for targeted Salmonella detection, informs the development of specific therapeutics, and enhances our understanding of the interaction between Salmonella bacteriophages and their hosts.

Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

Utility of protein–protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display

Over the past three decades a diverse collection of small protein domains have been used as scaffolds to generate general-purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK), and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and Next Generation Sequencing (NGS), we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low uM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were insertion into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general-purpose protein-binding reagents.

A novel phage putative depolymerase, Depo16, has specific activity against K1 capsular-type Klebsiella pneumoniae.

Klebsiella pneumoniae, especially hypervirulent K. pneumoniae (hvKP), is a common opportunistic pathogen that often causes hospital- and community-acquired infections. Capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. Some phages encode depolymerases that can recognize and degrade bacterial polysaccharides. In this study, the lytic bacteriophage vB_KpnP_ZK1 (abbreviated as ZK1) was isolated using serotype K1 hvKP as the host. Although amino acid sequence BLAST analysis indicated that the tail fiber protein Depo16 of phage ZK1 showed no significant similarity to any reported phage depolymerases, it displayed enzymatic activities that are characteristic of phage depolymerases. After expression and purification, Depo16 could efficiently remove the capsular polysaccharide layer that surrounds the surface of serotype K1 K. pneumoniae. Although no bactericidal activity was detected, Depo16 makes serotype K1 K. pneumoniae sensitive to peritoneal macrophages (PMs). In addition, in a mouse bacteremia model of serotype K1 K. pneumoniae, 25 µg of Depo16 was effective in significantly prolonging survival. Depo16 treatment can reduce the bacterial load in blood and major tissues and alleviate tissue damage in mice. This indicates that the putative depolymerase Depo16 is a potential antibacterial agent against serotype K1 K. pneumoniae infections.IMPORTANCEKlebsiella pneumoniae often causes hospital-acquired infections and community-acquired infections. Capsular polysaccharide (CPS) is one of the crucial virulence factors of K. pneumoniae. K1 and K2 capsular-type K. pneumoniae strains are the most prevalent serotypes of hypervirulent K. pneumoniae (hvKP). In this study, a novel K. pneumoniae phage named vB_KpnP_ZK1 was isolated, and its putative depolymerase Depo16 showed low homology with other reported phage depolymerases. Depo16 can specifically degrade the K. pneumoniae K1 capsule making this serotype sensitive to peritoneal macrophages. More importantly, Depo16 showed a significant therapeutic effect in a mouse bacteremia model caused by serotype K1 K. pneumoniae. Thus, Depo16 is a potential antibacterial agent to combat serotype K1 K. pneumoniae infections.

Host range expansion of Acinetobacter phage vB_Ab4_Hep4 driven by a spontaneous tail tubular mutation.

Bacteriophages (phages) represent promising alternative treatments against multidrug-resistant Acinetobacter baumannii (MDRAB) infections. The application of phages as antibacterial agents is limited by their generally narrow host ranges, so changing or expanding the host ranges of phages is beneficial for phage therapy. Multiple studies have identified that phage tail fiber protein mediates the recognition and binding to the host as receptor binding protein in phage infection. However, the tail tubular-dependent host specificity of phages has not been studied well. In this study, we isolated and characterized a novel lytic phage, vB_Ab4_Hep4, specifically infecting MDRAB strains. Meanwhile, we identified a spontaneous mutant of the phage, vB_Ab4_Hep4-M, which revealed an expanded host range compared to the wild-type phage. A single mutation of G to C was detected in the gene encoding the phage tail tubular protein B and thus resulted in an aspartate to histidine change. We further demonstrated that the host range expansion of the phage mutant is driven by the spontaneous mutation of guanine to cytosine using expressed tail tubular protein B. Moreover, we established that the bacterial capsule is the receptor for phage Abp4 and Abp4-M by identifying mutant genes in phage-resistant strains. In conclusion, our study provided a detailed description of phage vB_Ab4_Hep4 and revealed the tail tubular-dependent host specificity in A. baumannii phages, which may provide new insights into extending the host ranges of phages by gene-modifying tail tubular proteins.

A hybrid receptor binding protein enables phage F341 infection of Campylobacter by binding to flagella and lipooligosaccharides.

Flagellotropic bacteriophages are interesting candidates as therapeutics against pathogenic bacteria dependent on flagellar motility for colonization and causing disease. Yet, phage resistance other than loss of motility has been scarcely studied. Here we developed a soft agar assay to study flagellotropic phage F341 resistance in motile Campylobacter jejuni. We found that phage adsorption was prevented by diverse genetic mutations in the lipooligosaccharides forming the secondary receptor of phage F341. Genome sequencing showed phage F341 belongs to the Fletchervirus genus otherwise comprising capsular-dependent C. jejuni phages. Interestingly, phage F341 encodes a hybrid receptor binding protein (RBP) predicted as a short tail fiber showing partial similarity to RBP1 encoded by capsular-dependent Fletchervirus, but with a receptor binding domain similar to tail fiber protein H of C. jejuni CJIE1 prophages. Thus, C. jejuni prophages may represent a genetic pool from where lytic Fletchervirus phages can acquire new traits like recognition of new receptors.

Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa.

Bacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance. In this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains. Phage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia. We show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy's evolutionary impact that may be exploited to generate robust therapy schemes.

Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

Contracting the Host Range of Bacteriophage T7 Using a Continuous Evolution System.

Bacteriophage T7 is an intracellular virus that recognizes its host via tail and tail fiber proteins known as receptor-binding proteins (RBPs). The RBPs attach to a specific lipopolysaccharide (LPS) displayed on the host. While there are various reports of phage host range expansion resulting from mutations in the RBP encoding genes, there is little evidence for contraction of host range. Notably, most experimental systems have not monitored changes in host range in the presence of several hosts simultaneously. Here, we use a continuous evolution system to show that T7 phages grown in the presence of five restrictive strains and one permissive host, each with a different LPS, gradually cease to recognize the restrictive strains. Remarkably, this result was obtained in experiments with six different permissive hosts. The altered specificity is due to mutations in the RBPs as determined by gene sequencing. The results of using this system demonstrate a major role for RBPs in restricting the range of futile infections, and this process can be harnessed to reduce the host range in applications such as recognition and elimination of a specific bacterial serotype by bacteriophages.

Bacteriophage long tail fibre proteins as a biorecognition element in electrochemical biosensors for the detection of Salmonella in lake water

We developed an electrochemical impedance spectroscopy (EIS) biosensor based on bacteriophage long tail fibre proteins (LTFPs) to capture and detect Salmonella. First, based on the genetic information of a Salmonella enterica bacteriophage (SEP37), we found that the genes ORF99, ORF100, and ORF256 encode proteins associated with host recognition. We used the pETdue1 plasmid to construct ORF99–ORF100 and ORF256 co-expression vectors to express (LTFPs) in Escherichia coli and used purified LTFPs as specific biorecognition elements. Then, we fixed gold nanoparticles (AuNPs), cysteamine (Cys), and LTFPs on a gold disk electrode surface via layer-by-layer assembly to construct an EIS biosensor. Salmonella in the sample can be captured by LTFPs on the electrode surface and reduce the electron transfer sites. We applied the EIS biosensor to detect Salmonella spiked in lake water. It showed a good linear relationship within the concentration range of 10 ∼ 1 × 106 colony-forming units (CFU)/mL. The detection limit was 9 CFU/mL. This method based on bacteriophage LTFPs does not require complex pretreatment steps and extends the application of tail fibre proteins as a biorecognition element in the detection field.

Specific separation and sensitive detection of foodborne pathogens by phage-derived bacterial-binding protein-nano magnetic beads coupled with smartphone-assisted paper sensor

Foodborne pathogen infection poses a significant threat to public health and is considered as one of the most serious hazards in global food safety. Herein, a sensitive and efficient method for on-site monitoring of foodborne pathogens was developed by using a smartphone-assisted paper-sensor combined with phage-derived bacterial-binding proteins-nano magnetic beads (PBPs-MBs). PBPs including tail fiber protein (TFP:gp13), cell-wall binding domain (CBD) of endolysin and tailspike protein (TSP) coated on the surface of MBs were applied for rapid separation and enrichment of targeted bacteria (Escherichia coli O157:H7, Staphylococcus aureus and Salmonella typhimurium, respectively) from food samples in 20 min before detection on paper-based sensors. The paper-based sensor was loaded with the lytic agent (polymyxin B) to induce bacterial lysis and release specific endogenous enzymes. Subsequently, three distinct chromogenic substrates were hydrolyzed by their corresponding enzymes, resulting in characteristic color changes on the paper, respectively. In addition, a smartphone APP for red-green-blue (RGB) color analysis of paper was able to directly detect three foodborne pathogens. As a result, the limit of detection (LOD) values for three foodborne pathogens were found to be 2.44 × 102, 2.68 × 104 and 4.62 × 103 CFU/mL, respectively, which were much lower than other studies (106–108 CFU/mL) based on enzymes. Moreover, the feasibility of this approach was further assessed through the successful detection of targeted bacteria in real samples with satisfactory recovery rates. In conclusion, this smartphone-assisted biosensor offers promising application potential for point-of-care testing (POCT) of foodborne pathogens in resource-scarce areas.

Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Effect of a Depolymerase Encoded by Phage168 on a Carbapenem-Resistant Klebsiella pneumoniae and Its Biofilm.

Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are becoming increasingly common within clinical settings, requiring the development of alternative therapies. In this study, we isolated, characterized, and sequenced the genome of a CRKP phage, Phage168. The total genomic DNA of Phage168 was 40,222 bp in length, encoding 49 predicted proteins. Among these proteins, Dep40, the gene product of ORF40, is a putative tail fiber protein that exhibits depolymerase activity based on the result of bioinformatics analyses. In vitro, we confirmed that the molecular weight of the Phage168 depolymerase protein was about 110 kDa, the concentration of the produced phage 168 depolymerase protein was quantified as being 1.2 mg/mL, and the depolymerase activity was still detectable after the dilution of 1.2 µg/mL. This recombinant depolymerase exhibited enzyme activity during the depolymerization of the formed CRKP biofilms. We also found that depolymerase, when combined with polymyxin B, was able to enhance the bactericidal effect of polymyxin B on CRKP strains by disrupting their biofilm. When recombinant depolymerase was used in combination with human serum, it enhanced the sensitivity of the CRKP strain UA168 to human serum, and the synergistic bactericidal effect reached the strongest level when the ratio of depolymerase to human serum was 3:1. Our results indicated that depolymerase encoded by Phage168 may be a promising strategy for combating infections caused by drug-resistant CRKP formed within the biofilm.

Coevolutionary phage training andJoint application delays the emergence ofphage resistance inPseudomonas aeruginosa.

Antibiotic-resistant bacteria are current threats to available antibiotic therapies, and this has renewed interest in the therapeutic use of phage as an alternative. However, development of phage resistance has led to unsuccessful therapeutic outcomes. In the current study, we applied phage training to minimize bacterial phage resistance and to improve treatment outcome by adapting the phage to their target hosts during co-evolution. We isolated and characterized a novel Pseudomonas aeruginosa N4-like lytic phage (PWJ) from wastewater in Yangzhou, China. PWJ is a double-stranded DNA podovirus that can efficiently lyse the model strain ATCC 27,853 and opportunistic pathogen PAO1. Genome sequencing of PWJ revealed features similar to those of the N4-like P. aeruginosa phage YH6. We used PWJ to screen for an evolved trained phage (WJ_Ev14) that restored infectivity to PWJ phage bacterial resisters. BLASTN analysis revealed that WJ_Ev14 is identical to its ancestor PWJ except for the amino acid substitution R1051S in its tail fiber protein. Moreover, phage adsorption tests and transmission electron microscopy of resistant bacteria demonstrated that the R1051S substitution was most likely the reason WJ_Ev14 could re-adsorb and regain infectivity. Furthermore, phage therapy assays in vitro and in a mouse P. aeruginosa lung infection model demonstrated that PWJ treatment resulted in improved clinical results and a reduction in lung bacterial load whereas the joint phage cocktail (PWJ+ WJ_Ev14) was better able to delay the emergence of resister bacteria. The phage cocktail (PWJ +WJ_Ev14) represents a promising candidate for inclusion in phage cocktails developed for clinical applications.