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BACKGROUND: This study addresses the urgent need for infection control agents driven by the rise of drug-resistant pathogens such as Acinetobacter baumannii. Our primary aim was to develop and assess a novel endolysin, Tha-PA90, designed to combat these challenges. METHODS: Tha-PA90 incorporates an antimicrobial peptide (AMP) called thanatin at its N-terminus, enhancing bacterial outer membrane permeability and reducing host immune responses. PA90 was selected as the endolysin component. The antibacterial activity of the purified Tha-PA90 was evaluated using an in vitro colony-forming unit (CFU) reduction assay and a membrane permeability test. A549 cells were utilized to measure the penetration into the cytosol and the cytotoxicity of Tha-PA90. Finally, infection control was monitored in A. baumannii infected mice following the intraperitoneal administration of Tha-PA90. RESULTS: Tha-PA90 demonstrated remarkable in vitro efficacy, completely eradicating A. baumannii strains, even drug-resistant variants, at a low concentration of 0.5 µM. Notably, it outperformed thanatin, achieving only a < 3-log reduction at 4 µM. Tha-PA90 exhibited 2-3 times higher membrane permeability than a PA90 and thanatin mixture or PA90 alone. Tha-PA90 was found within A549 cells' cytosol with no discernible cytotoxic effects. Furthermore, Tha-PA90 administration extended the lifespan of A. baumannii-infected mice, reducing bacterial loads in major organs by up to 3 logs. Additionally, it decreased proinflammatory cytokine levels (TNF-α and IL-6), reducing the risk of sepsis from rapid bacterial lysis. Our findings indicate that Tha-PA90 is a promising solution for combating drug-resistant A. baumannii. Its enhanced efficacy, low cytotoxicity, and reduction of proinflammatory responses render it a potential candidate for infection control. CONCLUSIONS: This study underscores the significance of engineered endolysins in addressing the pressing challenge of drug-resistant pathogens and offers insights into improved infection management strategies.
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Infecções por Acinetobacter , Acinetobacter baumannii , Endopeptidases , Animais , Camundongos , Peptídeos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos , Antibacterianos/farmacologia , Infecções por Acinetobacter/tratamento farmacológico , Testes de Sensibilidade MicrobianaRESUMO
Alizarin (1,2-dihydroxyanthraquinone) is an anthraquinone reddish dye widely used for painting and textile dyeing. As the biological activity of alizarin has recently attracted increasing attention from researchers, its therapeutic potential as complementary and alternative medicine is of interest. However, no systematic research has been conducted on the biopharmaceutical and pharmacokinetic aspects of alizarin. Therefore, this study aimed to comprehensively investigate the oral absorption and intestinal/hepatic metabolism of alizarin using a simple and sensitive tandem mass spectrometry method developed and validated in-house. The present method for the bioanalysis of alizarin has merits, including a simple pretreatment procedure, small sample volume, and adequate sensitivity. Alizarin exhibited pH-dependent moderate lipophilicity and low solubility with limited intestinal luminal stability. Based on the in vivo pharmacokinetic data, the hepatic extraction ratio of alizarin was estimated to be 0.165-0.264, classified as a low level of hepatic extraction. In an in situ loop study, considerable fractions (28.2%-56.4%) of the alizarin dose were significantly absorbed in gut segments from the duodenum to ileum, suggesting that alizarin may be classified as the Biopharmaceutical Classification System class II. An in vitro metabolism study using rat and human hepatic S9 fractions revealed that glucuronidation and sulfation, but not NADPH-mediated phase I reactions and methylation, are significantly involved in the hepatic metabolism of alizarin. Taken together, it can be estimated that the fractions of oral alizarin dose unabsorbed from the gut lumen and eliminated by the gut and liver before reaching the systemic circulation are 43.6%-76.7%, 0.474%-36.3%, and 3.77%-5.31% of the dose, respectively, resulting in a low oral bioavailability of 16.8%. Therefore, the oral bioavailability of alizarin depends primarily on its chemical degradation in the gut lumen and secondarily on first-pass metabolism.
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Produtos Biológicos , Espectrometria de Massas em Tandem , Ratos , Humanos , Animais , Disponibilidade Biológica , Cromatografia Líquida , Ratos Sprague-Dawley , Antraquinonas , Administração OralRESUMO
Enterobacter aerogenes is a member of the ESKAPE group of bacteria, and multi-drug-resistant strains are increasingly being found. In this study, a novel bacteriophage, ATCEA85, which infects E. aerogenes, has been isolated and characterized. ATCEA85 is seen to have a circularly permuted linear double-stranded DNA genome of 47,484 base pairs in length. The closest related phage found in the databases is the Klebsiella phage Kp3, which exhibits 77% identity over a 34% query coverage. The G+C content of ATCEA85 is 56.2%, and 15 putative open reading frames are functionally annotated.
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DNA Viral/genética , Enterobacter aerogenes/virologia , Genoma Viral , Fases de Leitura Aberta , Filogenia , Siphoviridae/genética , Composição de Bases , DNA/genética , Ontologia Genética , Anotação de Sequência Molecular , Siphoviridae/classificação , Siphoviridae/isolamento & purificação , Sequenciamento Completo do GenomaRESUMO
An increasing number of Klebsiella pneumoniae isolates have been found to be multi-drug resistant. A novel bacteriophage, PBKP05, which infects K. pneumoniae, was isolated and characterized. It has a linear double-stranded DNA genome of 30,240 base pairs in length. Its G+C content is 53%, and 47 putative open reading frames are functionally annotated. This phage can be a candidate material for phage therapy.
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Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Genoma Viral , Klebsiella pneumoniae/virologia , Bacteriófagos/classificação , Composição de Bases , Sequência de Bases , DNA Viral/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Podoviridae , Análise de Sequência de DNARESUMO
Unfortunately, the original article was published with incorrect accession number and the correct version is updated here.
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When phages infect bacteria cultured in the presence of sublethal doses of antibiotics, the sizes of the phage plaques are significantly increased. This phenomenon is known as phage-antibiotic synergy (PAS). In this study, the observation of PAS was extended to a wide variety of bacterium-phage pairs using different classes of antibiotics. PAS was shown in both Gram-positive and Gram-negative bacteria. Cells stressed with ß-lactam antibiotics filamented or swelled extensively, resulting in an increase in phage production. PAS was also sometimes observed in the presence of other classes of antibiotics with or without bacterial filamentation. The addition of antibiotics induced recA expression in various bacteria, but a recA deletion mutant strain of Escherichia coli also showed filamentation and PAS in the presence of quinolone antibiotics. The phage adsorption efficiency did not change in the presence of the antibiotics when the cell surfaces were enlarged as they filamented. Increases in the production of phage DNA and mRNAs encoding phage proteins were observed in these cells, with only a limited increase in protein production. The data suggest that PAS is the product of a prolonged period of particle assembly due to delayed lysis. The increase in the cell surface area far exceeded the increase in phage holin production in the filamented host cells, leading to a relatively limited availability of intracellular holins for aggregating and forming holes in the host membrane. Reactive oxygen species (ROS) stress also led to an increased production of phages, while heat stress resulted in only a limited increase in phage production.IMPORTANCE Phage-antibiotic synergy (PAS) has been reported for a decade, but the underlying mechanism has never been vigorously investigated. This study shows the presence of PAS from a variety of phage-bacterium-antibiotic pairings. We show that increased phage production resulted directly from a lysis delay caused by the relative shortage of holin in filamented bacterial hosts in the presence of sublethal concentrations of stress-inducing substances, such as antibiotics and reactive oxygen species (ROS).
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Antibacterianos/farmacologia , Bacteriófagos/efeitos dos fármacos , Bacteriófagos/fisiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bacteriófagos/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Negativas/virologia , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/virologia , Quinolonas/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis.
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Carcinoma Hepatocelular/terapia , Hepatite C/metabolismo , Histona Desacetilases/metabolismo , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Proteínas Repressoras/metabolismo , Vesiculovirus/fisiologia , Proteínas do Core Viral/metabolismo , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Resultado do Tratamento , Estomatite Vesicular/virologiaRESUMO
In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria.
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Cápsulas Bacterianas/metabolismo , Colífagos/enzimologia , Colífagos/crescimento & desenvolvimento , Escherichia coli/virologia , Polissacarídeos/metabolismo , Escherichia coli/genética , Perfilação da Expressão Gênica , Hidrólise , Análise em MicrossériesRESUMO
OBJECTIVES: Clostridioides difficile has emerged as a major cause of life-threatening diarrheal disease. Conventional antibiotics used in current standards of care exacerbate the emergence of antibiotic-resistant strains and pose a risk of recurrent C. difficile infection (CDI). Thus, there is an urgent need for alternative therapeutics that selectively eliminate C. difficile without disturbing the commensal microbiota. This study aimed to explore the potential of endolysins as an alternative therapeutic agent to antibiotics. Endolysin is a bacteriophage-derived peptidoglycan hydrolase that aids in the release of phage progeny during the final stage of infection. METHODS: In order to exploit endolysin as a therapeutic agent against CDI, the bactericidal activity of 23 putative endolysins was compared and ΦCD27 endolysin CD27L was selected and modified to CD27L_EAD by cleaving the cell-wall binding domain of CD27L. RESULTS: CD27L_EAD exhibited greater bacteriolytic activity than CD27L and its activity was stable over a wide range of salt concentrations and pH conditions. CD27L_EAD was added to a co-culture of human gut microbiota with C. difficile and the bacterial community structure was analyzed. CD27L_EAD did not impair the richness and diversity of the bacterial population but remarkably attenuated the abundance of C. difficile. Furthermore, the co-administration of vancomycin exerted synergistic bactericidal activity against C. difficile. ß-diversity analysis revealed that CD27L_EAD did not significantly disturb the composition of the microbial community, whereas the abundance of some species belonging to the family Lachnospiraceae decreased after CD27L_EAD treatment. CONCLUSIONS: This study provides insights into endolysin as a prospective therapeutic agent for the treatment of CDI without damaging the normal gut microbiota.
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Antibacterianos , Clostridioides difficile , Infecções por Clostridium , Endopeptidases , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Endopeptidases/farmacologia , Endopeptidases/genética , Endopeptidases/uso terapêutico , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Microbioma Gastrointestinal/efeitos dos fármacos , Bacteriófagos/genética , Bacteriólise/efeitos dos fármacosRESUMO
Endolysins are lytic enzymes produced by bacteriophages at the end of their lytic cycle and degrade the peptidoglycan layer of the bacterial cell wall. Thus, they have been extensively explored as a promising antibacterial agent to replace or supplement current antibiotics. Gram-negative bacteria, however, are prone to resist exogenous endolysins owing to their protective outer membrane. We previously engineered endolysin EC340, encoded by the Escherichia coli phage PBEC131, by substituting its seven amino acids and fusing an antimicrobial peptide cecropin A at its N-terminus. The engineered endolysin LNT113 exerted superior activity to its intrinsic form. This study investigated how cecropin A fusion facilitated the bactericidal activity of LNT113 toward Gram-negative bacteria. Cecropin A of LNT113 markedly increased the interaction with lipopolysaccharides, while the E. coli defective in the core oligosaccharide was less susceptible to endolysins, implicating the interaction between the core oligosaccharide and endolysins. In fact, E. coli with compromised lipid A construction was more vulnerable to LNT113 treatment, suggesting that the integrity of the lipid A layer was important to resist the internalization of LNT113 across the outer membrane. Cecropin A fusion further accelerated the inner membrane destabilization, thereby enabling LNT113 to deconstruct it promptly. Owing to the increased membrane permeability, LNT113 could inactivate some Gram-positive bacteria as well. This study demonstrates that cecropin A fusion is a feasible method to improve the membrane permeability of endolysins in both Gram-negative and Gram-positive bacteria.
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Peptídeos Catiônicos Antimicrobianos , Escherichia coli , Lipídeo A , Escherichia coli/metabolismo , Endopeptidases/química , Bactérias Gram-Negativas/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Bactérias Gram-Positivas/metabolismo , OligossacarídeosRESUMO
BACKGROUND: Concerns about the rise in antimicrobial resistance have led to renewed interest in phage therapy worldwide, but perceptions among relevant medical professionals in Korea remain largely unknown. MATERIALS AND METHODS: We conducted a semi-quantitative online survey to evaluate the Korean infectious disease specialists' perception of phage therapy. RESULTS: We sent out the link to the questionnaire to 380 subjects and received 91 replies, with 90/91 respondents identifying as Korean infectious diseases specialists or trainees. Ten out of 91 (11.0%) respondents scored themselves as well-informed about phage therapy. The majority (93.4%) of respondents would consider using phage therapy if the safety of the phage formulation is guaranteed, and 80% of respondents would consider participating in clinical trials with phage therapy given adequate support. The biggest concern was uncertainty about safety (73.6%) and efficacy (65.9%). Acinetobacter baumannii was ranked as a high priority for phage therapy research, as were bone and joint infections. CONCLUSION: Korean infectious diseases specialists are receptive to phage therapy, but a better understanding of safety, efficacy and clinical trials are warranted to progress phage therapy within the Korean healthcare system.
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A novel lytic bacteriophage, SA11, infecting Staphylococcus aureus was isolated, and the whole genome was sequenced. It belongs to the siphoviridae based on electron microscopic observation. It has a linear double-stranded DNA genome of 136,326 bp. Genomic analysis showed that it is distantly related to Staphylococcus phages A5W, K, ISP, Sb-1, and G1.
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Siphoviridae/genética , Fagos de Staphylococcus/genética , Staphylococcus aureus/virologia , DNA Viral/genética , Genoma Viral , Dados de Sequência Molecular , Siphoviridae/classificação , Siphoviridae/isolamento & purificação , Fagos de Staphylococcus/classificação , Fagos de Staphylococcus/isolamento & purificaçãoRESUMO
PA26, a novel lytic bacteriophage infecting Pseudomonas aeruginosa, was isolated, and the whole genome was sequenced. It was found to belong to the myoviridae by an electron microscopic observation. It had a linear double-stranded DNA genome of 72,321 bp. Genomic analysis showed that it resembled another Pseudomonas phage, LIT1.
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Myoviridae/genética , Fagos de Pseudomonas/genética , Pseudomonas aeruginosa/virologia , DNA Viral/genética , Genoma Viral , Dados de Sequência Molecular , Myoviridae/classificação , Myoviridae/isolamento & purificação , Fagos de Pseudomonas/classificação , Fagos de Pseudomonas/isolamento & purificação , República da Coreia , Microbiologia da ÁguaRESUMO
Escherichia coli O157:H7 is a human pathogen. We isolated a novel bacteriophage infecting this bacterium from a sewage water treatment facility. Phage PBECO4 belongs to the family Myoviridae, having an isometric head and a contractile tail. It has a linear double-stranded DNA genome of 348,113 base pairs in length with a GC content of 34.09 %. Whole-genome sequencing revealed that PBECO4 is distantly related to enterobacteria phage vB_KleM_RaK2, with 10 % similarity, and Cronobacter phage vB_CsaM_GAP32 with 6 % similarity. Five hundred fifty-one putative open reading frames (ORFs) and six tRNA genes were found. Eight ORFs are related to genes encoding structural proteins, nine to DNA packaging, two to DNA lysis activity, and 42 to replication and regulation. Four hundred ninety ORFs have not been functionally annotated.
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Colífagos/genética , Escherichia coli O157/virologia , Genoma Viral/genética , Genômica , Myoviridae/genética , Colífagos/isolamento & purificação , Colífagos/fisiologia , Humanos , Dados de Sequência Molecular , Myoviridae/isolamento & purificação , Myoviridae/fisiologia , Fases de Leitura Aberta/genética , Filogenia , RNA de Transferência/genética , Análise de Sequência de DNA , Proteínas Virais/genéticaRESUMO
[This corrects the article DOI: 10.3389/fmicb.2022.821936.].
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[This corrects the article DOI: 10.3389/fmicb.2022.1093670.].
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Genes encoding endolysins were identified and cloned from three different Escherichia coli bacteriophages, 10-24(13), PBEC30, and PBEC56. Putative antimicrobial peptide (AMP)-like C-terminal alpha helix structures with amphipathic natures were predicted from the three endolysins. Each gene was cloned and expressed as hexahistidine-tagged forms, and the products were purified and characterized. The purified endolysins exhibited antibacterial activities against a variety of Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumonia. Their antibacterial activities were improved by N-terminal fusion with an antimicrobial peptide, cecropin A. Minimum inhibitory concentrations (MIC) were as low as 4 µg/mL, depending on the targeted strain. The endolysins' enzymatic activities were not affected by changes in pH at ranges from 5 to 10 and were stable at temperatures between 4 and 65 °C. The in vivo efficacies of the three endolysins were also demonstrated using Galleria melonella for infection models.
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Bacteriófagos , Endopeptidases , Endopeptidases/genética , Endopeptidases/farmacologia , Endopeptidases/química , Antibacterianos/farmacologia , Antibacterianos/química , Escherichia coli/genética , Bactérias Gram-NegativasRESUMO
Antimicrobial agents targeting peptidoglycan have shown successful results in eliminating bacteria with high selective toxicity. Bacteriophage encoded endolysin as an alternative antibiotics is a peptidoglycan degrading enzyme with a low rate of resistance. Here, the engineered endolysin was developed to defeat multiple drug-resistant (MDR) Acinetobacter baumannii. First, putative endolysin PA90 was predicted by genome analysis of isolated Pseudomonas phage PBPA. The His-tagged PA90 was purified from BL21(DE3) pLysS and tested for the enzymatic activity using Gram-negative pathogens known for having a high antibiotic resistance rate including A. baumannii. Since the measured activity of PA90 was low, probably due to the outer membrane, cell-penetrating peptide (CPP) DS4.3 was introduced at the N-terminus of PA90 to aid access to its substrate. This engineered endolysin, DS-PA90, completely killed A. baumannii at 0.25 µM, at which concentration PA90 could only eliminate less than one log in CFU/ml. Additionally, DS-PA90 has tolerance to NaCl, where the â¼50% of activity could be maintained in the presence of 150 mM NaCl, and stable activity was also observed with changes in pH or temperature. Even MDR A. baumannii strains were highly susceptible to DS-PA90 treatment: five out of nine strains were entirely killed and four strains were reduced by 3-4 log in CFU/ml. Consequently, DS-PA90 could protect waxworm from A. baumannii-induced death by â¼70% for ATCC 17978 or â¼44% for MDR strain 1656-2 infection. Collectively, our data suggest that CPP-fused endolysin can be an effective antibacterial agent against Gram-negative pathogens regardless of antibiotics resistance mechanisms.
Assuntos
Acinetobacter baumannii , Anti-Infecciosos , Peptídeos Penetradores de Células , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Farmacorresistência Bacteriana Múltipla , Endopeptidases , Testes de Sensibilidade Microbiana , Peptidoglicano , Cloreto de SódioRESUMO
Endolysins are bacteriophage enzymes required for the eruption of phages from inside host bacteria via the degradation of the peptidoglycan cell wall. Recombinant endolysins are increasingly being seen as potential antibacterial candidates, with a number currently undergoing clinical trials. Bacteriophage PBPA90 infecting Pseudomonas aeruginosa harbors a gene encoding an endolysin, lysPA90. Herein, recombinant LysPA90 demonstrated an intrinsic antibacterial activity against Escherichia coli in vitro. It was observed that a sub-inhibitory concentration of the recombinant protein induced the upregulation of genes related to flagella biosynthesis in a commensal E. coli strain. Increases in the number of bacterial flagella, and in motility, were experimentally substantiated. The treatment caused membrane stress, leading to the upregulation of genes rpoE, rpoH, dnaK, dnaJ, and flhC, which are upstream regulators of flagella biosynthesis. When adherent invasive Escherichia coli (AIEC) strains were treated with subinhibitory concentrations of the endolysin, bacterial adhesion and invasion into intestinal epithelial Caco-2 cells was seen to visibly increase under microscopic examination. Bacterial counting further corroborated this adhesion and invasion of AIEC strains into Caco-2 cells, with a resultant slight decrease in the viability of Caco-2 cells then being observed. Additionally, genes related to flagella expression were also upregulated in the AIEC strains. Finally, the enhanced expression of the proinflammatory cytokine genes TNF-α, IL-6, IL-8, and MCP1 in Caco-2 cells was noted after the increased invasion of the AIEC strains. While novel treatments involving endolysins offer great promise, these results highlight the need for the further exploration of possible unanticipated and unintended effects.
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The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 µM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 µM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.