Search Results (211)

Listeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food-processing facilities for years. Although phages can control L. monocytogenes during food production, phage-resistant bacterial subpopulations can regrow in phage-treated environments. In this study, an L. monocytogenes hly defective strain, NJ05-Δhly, was produced, which considerably regulated the interactions between L. monocytogenes and phages. Specifically, we observed a 76.92-fold decrease in the efficiency of plating of the defective strain following infection with the Listeria phage vB-LmoM-NJ05. The lytic effect was notably diminished at multiplicities of infection of 1 and 10. Furthermore, the inactivation of LLO impaired biofilm formation, which was completely suppressed and eliminated following treatment with 10⁸ PFU/mL of phage. Additionally, phages protected cells from mitochondrial membrane damage and the accumulation of mitochondrial reactive oxygen species induced by L. monocytogenes invasion. Transcriptomic analysis confirmed these findings, revealing the significant downregulation of genes associated with phage sensitivity, pathogenicity, biofilm formation, and motility in L. monocytogenes. These results underscore the vital role of LLO in regulating the pathogenicity, phage susceptibility, and biofilm formation of L. monocytogenes. These observations highlight the important role of virulence factors in phage applications and provide insights into the potential use of phages for developing biosanitizers. Full article

Pseudomonas aeruginosa (P. aeruginosa) is a common antibiotic-resistant pathogen, posing significant public health threats worldwide. It is a major cause of ocular infections, mostly linked to contact lens wear. P. aeruginosa often produces biofilm during infections, and these are also associated with antibiotic resistance. Bacteriophage (phage) therapy is emerging as a promising approach for treating multidrug-resistant P. aeruginosa. Objective: This study aimed to assess the antibiofilm effects of six phages against P. aeruginosa biofilms isolated from patients with corneal infections. Method: This study examined P. aeruginosa strains for their ability to form biofilms using crystal violet assay. Six P. aeruginosa bacteriophages (DiSu1 to DiSu6) were used, which were isolated from sewage water in Melbourne, Australia. Spot tests were used to assess phage sensitivity. The effect of phages against P. aeruginosa strains was determined using time–kill assay and efficiency of plating. The ability of phage to inhibit biofilm formation over 24 h or reduce preformed biofilms was also studied and confirmed using confocal laser scanning microscopy with Live/Dead staining. Result: After 24 h of incubation, all tested P. aeruginosa strains formed moderate to strong biofilms. All P. aeruginosa strains were sensitive to at least four of the six phages. The highest level of bacterial growth inhibition in the liquid infection model was observed when phages were applied at a multiplicity of infection (MOI) of 100. Certain bacteria/phage combinations were able to inhibit biofilm formation over 24 h, with the combination of strain PA235 and phage DiSu3 producing the highest inhibition (83%) at a MOI of 100. This was followed by the combinations of PA223/DiSu3 (56%), and PA225/DiSu5 (52%). For the reduction in preformed biofilms, the best combinations were PA235 (90%), PA221 (61%), and PA213 and PA225 (57% each), all with DiSu3 after 3 h. However, exposing the biofilm with phages for over 24 h appeared to promote phage resistance as there was evidence of biofilm growth, with the only combination still showing a significant reduction being PA221/DiSu3 (58%) at MOI of 100. Conclusions: This study showed that the effect of phages against P. aeruginosa is concentration (MOI) dependent. Phages at higher MOI have the ability to disrupt, inhibit, and reduce P. aeruginosa biofilms. However, prolonged exposure of the biofilm with phages appeared to promote phage resistance. To enhance phage efficacy and address this form of resistance, further studies utilizing phage cocktails or a combination of phages and antibiotics is warranted. Full article

Bacteriophages can be used as biocontrol agents in agriculture to improve food safety, provided they can remain viable in food environments. The viability of ten Shigella phages (AShi, Shi3, Shi22, Shi30, Shi33, Shi34, Shi40, Shi88, Shi93, and Shi113) was evaluated against different additives and biocides used daily in food applications. In addition, the influence of additives on phage viability in a food matrix was investigated. Treatments with lactic and citric acid were the most effective to inactivate phages. In addition, the acetic acid was the most phage-friendly treatment evaluated. Preservatives such as acetate, lactate, benzoate, sorbate, and propionate proved to be highly compatible with all the phages tested. Regarding the influence of the food matrix on phage viability, an equal or higher viability was found for most phages tested when compared with the corresponding organic acid. Finally, when phages were exposed to sodium hypochlorite, ethanol, quaternary ammonium chloride (QAC), and H₂O₂, most of them were sensitive to long incubations and high concentrations. However, when biocide concentrations employed are low, 10³–10⁴ PFU mL⁻¹ phage particles remains viable. Thus, the phages evaluated could be used in combination with additives and biocides as a biocontrol tool against the foodborne pathogen S. flexneri in agricultural products. Full article

Bacteriophages (or phages) remain the leading cause of failure in dairy fermentations. Thereby, phage-resistant Lactococcus lactis and Lactococcus cremoris dairy starters are in continuous demand. In this work, our goal was to identify phage defense mechanisms against ceduoviruses encoded by two wild isolates of dairy origin named L. lactis IPLA517 and IPLA1064. These strains were previously subjected to experimental evolution to select derivatives that are resistant to the bacteriocin Lcn972. It was observed that the Lcn972^R derivatives became sensitive to phage infection; however, the underlying mechanism was not defined. The long-read sequencing technologies applied in this work reveal that all of the Lcn972^R derivatives shared the loss of a 41 kb endogenous plasmid (p41) that harbors a putative exopolysaccharide (EPS) gene cluster with significant homology to one described in Lactococcus garvieae. Using a CRISPR-Cas9-based approach, p41 was selectively cured from L. lactis IPLA1064. Phage infection assays with three ceduoviruses demonstrated that curing p41 restored phage sensitivity at levels comparable to the Lcn972^R-IPLA1064 derivatives. Phage adsorption to Δp41 cells was also increased, consistent with the hypothesis of EPS production hindering access to the phage receptor protein Pip. Our results reinforce the role of EPSs in protecting Lactococcus against phage infection, a phenomenon that is rarely reported for ceduoviruses. Moreover, the results also exemplify the likely horizontal gene transfer that can occur between L. lactis and L. garvieae in a dairy environment. Full article

Background/Objectives: The viral pandemic caused by the SARS-CoV-2 virus has affected millions of people. However, it was noticed that high mortality was often a result of bacterial co-infections. One of the main pathogens responsible for secondary infections in patients with viral respiratory tract infections, including COVID-19, is Staphylococcus aureus. In recent years, the number of infections caused by drug-resistant strains of S. aureus has been growing rapidly, often exceeding the number of infections caused by antibiotic-sensitive strains. In addition, biofilm-related infections are more difficult to treat due to the lower sensitivity of biofilm structure to antibiotics. Bacteriophages are seen as alternative treatment of bacterial infections. Therefore, in our work, we have analyzed the efficacy of three Kayviruses against S. aureus strains isolated from COVID-19 patients. Methods: We analyzed the ability of tested phages to remove S. aureus biofilm both from polystyrene plates as well as from the surface of pulmonary epithelial cells. Results: We have observed that tested Kayviruses had a broad host range. Furthermore, phages were able to effectively reduce biofilm biomass and number of viable cells in pure culture. During our research, none of the tested phages was shown to have a negative effect on cell viability and were able to inhibit the negative effect S. aureus had on cell condition. Conclusions: Our results show tested phages were effective in reducing the biofilm of S. aureus strains isolated from COVID-19 patients, had no adverse effect on lung epithelial cell viability. Therefore, it should be recognized that the properties of three studied Kayviruses give them an advantage in the selection of phages for treatment of staphylococcal infections. Full article

Zearalenone (ZEN), a toxic estrogenic mycotoxin in cereals, threatens human and animal health through reproductive, immune, and cytotoxic effects, necessitating sensitive detection methods. While nanobodies offer advantages over conventional antibodies for on-site ZEN detection, their application remains unexplored. This study aimed to develop an anti-ZEN nanobody derived from an anti-ZEN phage display nanobody library. An alpaca was immunized with a ZEN-bovine serum albumin (ZEN-BSA) antigen, achieving peak serum antibody titers (1:25,600) following four immunizations. A high-capacity phage display nanobody library (1.0 × 10¹¹ plaque-forming units/mL) was constructed. Following four rounds of biopanning, an enrichment factor of 479 was achieved. Phage ELISA screening identified six phage display nanobodies with specific ZEN-binding activity, and multiple sequence alignment revealed four unique nanobody sequences. The selected phage display nanobody, designated phage-V44, was expressed and purified, and its presence was validated by SDS-PAGE and western blotting, which detected a single approximately 17 kDa band consistent with the expected nanobody size. We established a working curve for an indirect competitive enzyme-linked immunoassay (ELISA) for ZEN, which showed an IC50 value of 7.55 ng/mL. The specificity and affinity of the V44 were also verified. Collectively, the study successfully constructed an anti-ZEN phage display nanobody library, screened four specific ZEN-binding phage display nanobodies, and prepared the anti-ZEN nanobody V44. Thereby establishing a foundation for the nanobody’s future integration into rapid on-site detection methods for ZEN in both animal feed and human food products. Full article

Zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, contaminates animal feed and grain crops, thereby entering the food chain and posing a significant threat to human health. Consequently, there is an urgent need for a sensitive and rapid method for detecting trace levels of ZEN. In this study, we developed a phage-displayed antigen-binding fragment (Fab-phage) and established a Fab-phage-based enzyme-linked immunosorbent assay (Fab-pELISA) for ZEN detection. Under optimal conditions, this method exhibits a half-maximal inhibitory concentration of 0.36 ng/mL, with a linear range from 0.07 to 3.89 ng/mL and a detection limit of 0.03 ng/mL. The method demonstrates high selectivity towards ZEN and good recovery rates of 97.35–122.66% with relative standard deviations not exceeding 3.5%. Furthermore, the detection results obtained using Fab-pELISA on real cereal samples are consistent with those from high-performance liquid chromatography, meeting practical application requirements. Therefore, the Fab-phage serves as a valuable biochemical reagent, and the established Fab-pELISA represents a promising analytical strategy for detecting ZEN and other trace toxic contaminants in cereals. Full article

Staphylococcus aureus is a common pathogen, often recovered from children’s infections. Βiofilm formation, antimicrobial resistance and production of adhesins and toxins contribute to its virulence. As resistance to antimicrobials rises worldwide, alternative therapies like bacteriophages (among them the well-studied Bacteriophage K) can be helpful. The aim of this study was to determine the bacteriophage and antimicrobial susceptibility and the presence of virulence genes among S. aureus from infections in children and adolescents. Eighty S. aureus isolates were tested for biofilm formation and antimicrobial susceptibility. The presence of two genes of the ica operon (icaA, icaD), adhesin’s (fnbA, fnbB, sasG) and toxin’s genes (PVL, tst, eta, etb) was tested by PCRs. Susceptibility to Bacteriophage K was determined using a spot assay. Thirteen isolates were methicillin-resistant (MRSA) and 41 were multi-resistant. Twenty-five S. aureus (31.3%) were resistant to Bacteriophage K, mostly from ocular and ear infections. Twelve S. aureus (15%) were PVL-positive, seven (8.8%) positive for tst, 18 (22.5%) were eta-positive and 46 were (57.5%) etb-positive. A total of 66 (82.5%) isolates carried fnbA, 16 (20%) fnbB and 26 (32.5%) sasG. PVL, tst and sasG carriage were more frequent in MRSA. Bacteriophage-susceptible isolates carried more frequently eta (32.7%) and etb (69.1%) compared to phage-resistant S. aureus (0% and 32%, respectively). Although mainly methicillin-sensitive, S. aureus from pediatric infections exhibited high antimicrobial resistance and carriage of virulence genes (especially for exfoliative toxins and fnbA). MRSA was associated with PVL, tst and sasG carriage, whereas Bacteriophage susceptibility was associated with eta and etb. The high level of Bacteriophage K susceptibility highlights its potential use against staphylococcal infections. Full article

Porcine edema disease (ED), which causes enormous economic losses in pig farms, is caused by Shiga toxin type 2e (Stx2e) Escherichia coli (STEC), which frequently occurs in young piglets. In this study, we aimed to express a fused Stx2e peptide on a phage surface to generate an innovative sandwich ELISA for the detection of STEC antigen in field pig farming samples. The amino acid sequences at positions 241–319 were selected for capture antibody (T1D2) production. T1D2 was selected after the third round of biopanning, and it showed a high yield with no major impurities. T1D2-ELISA can detect recombinant modified Stx2e antigen, and the detection limit of the antigen was approximately below 20 pg/mL. The sensitivity of T1D2-ELISA was determined using five different stool samples, with a total of 25 stool samples. Positive Stx2e antigen samples were detected only in one of the 25 samples using T1D2-ELISA. The ELISA values of positive stool samples were >300 pg and <600 pg. In conclusion, we developed an innovative ELISA for the detection of STEC antigens in field pig farming samples. It can also be used to easily detect STEC antigens in porcine stool samples. We anticipate that our novel T1D2-ELISA method will enable the effective monitoring of STEC antigen content during industrial vaccine production. By leveraging this approach, we aimed to enhance production efficiency and ensure high-quality vaccines. Full article

Foodborne viruses are significant contributors to global food safety incidents, posing a serious burden on human health and food safety. In this study, a multiplex reverse transcription–droplet digital PCR (RT-ddPCR) assay based on the MS2 phage as a process control virus (PCV) was developed to achieve the simultaneous detection of hepatitis A virus (HAV) and hepatitis E virus (HEV) in bivalve shellfish. By optimizing the reaction system and procedures, the best reaction conditions were selected, and the specificity, sensitivity, and reproducibility of the method were assessed. Additionally, the MS2 phage’s recovery rate was utilized as an indicator to evaluate the optimal sample nucleic acid enrichment method. The results indicated that the RT-ddPCR assay exhibited optimal amplification efficiency with primer concentrations of 900 nmol/L, probe concentrations of 350 nmol/L for HAV and HEV, and 500 nmol/L for MS2, an annealing temperature of 53.1 °C, an extension time of 90 s, and 45 cycles. Additionally, the developed multiplex RT-ddPCR assay demonstrated high specificity, with quantitation limits of 12.6, 8.9, and 7.8 copies/reaction being observed for HAV, HEV, and the MS2 phage, respectively. A total of 240 bivalve samples were analyzed, of which 4 were positive for HAV and 12 for HEV. The viral loads for HAV ranged from 3048 to 6528 copies/2 g, while those for HEV ranged from 3312 to 20,350 copies/2 g. This assay provides a vital tool for enhancing food safety monitoring. Full article

Non-small cell lung cancer (NSCLC) is the predominant form of lung cancer and poses a significant public health challenge. Early detection is crucial for improving patient outcomes, with serum biomarkers such as carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCAg), and cytokeratin fragment 19 (CYFRA 21-1) playing a critical role in early screening and pathological classification of NSCLC. However, due to being mainly based on corresponding antibody binding reactions, existing detection technologies for these serum biomarkers have shortcomings such as complex operations, high false positive rates, and high costs. This study aimed to develop new methods for detecting CEA, SCCAg, and CYFRA 21-1 to assist in the diagnosis of NSCLC. Affinity peptides of CEA, SCCAg, and CYFRA 21-1, respectively, were screened by phage display technology, and the peptides’ binding affinities were determined by enzyme-linked immunosorbent assay and biolayer interferometry. Peptides with high affinity were then integrated as binding domains into biosensors by fusing them with circularly permuted fluorescent proteins (cpFPs) through genetic coding. The resulting biosensors, C4 biosensor for CEA, S1 biosensor for SCCAg, and Y3 biosensor for CYFRA 21-1, demonstrated robust sensitivity and specificity even at concentrations as low as 1 ng/mL for their respective tumor markers. When applied to clinical samples and recalibrated for the upper limit of normal concentrations, the biosensors exhibited enhanced sensitivity and specificity for NSCLC diagnosis. This study introduced innovative biosensors for the detection of CEA, SCCAg, and CYFRA 21-1, providing a highly sensitive, specific, rapid, and cost-effective diagnostic alternative that could significantly improve NSCLC screening rates. Full article

The treatment of infections caused by Staphylococcus aureus is currently complicated by the increasing number of strains resistant to antimicrobial agents. One promising way to solve this problem is phage therapy. Due to the lack of data on the effectiveness and safety of phage preparations, STAFAL^® is the only registered phage preparation for the treatment of infectious diseases in the Slovak Republic and the entire European Union. The aim of this work was to determine the effectiveness of the STAFAL^® phage preparation against S. aureus strains of different origins with variable sensitivity to antimicrobial substances and with different genetic backgrounds. For this purpose, 111 carrier strains, 35 clinical isolates from bloodstream infections, and 46 strains from skin and soft tissue infections were analysed. The effectiveness of STAFAL^® was determined by the plaque forming method. STAFAL^® was effective against 74.0% of the strains tested. Susceptibility to this phage preparation was significantly higher in strains resistant to methicillin (MRSA), erythromycin and clindamycin (p < 0.05). The high efficiency of the STAFAL^® preparation was confirmed against spa types t003, t024 and t032, typical of the hospital environment. The in vitro results indicate high therapeutic potential of the STAFAL^® antistaphylococcal phage preparation, especially against MRSA strains. Full article

Background: Introducing unnatural base pairs into a natural, double-stranded DNA construct is a powerful tool within synthetic biology. Accordingly, the ability to detect these unnatural base pairs has many applications, including the study and detection of semisynthetic organisms. Objective and Methods: The work described here aimed to select human antibodies for the specific recognition of Hirao’s base pair dDs–dPn in various natural DNA contexts by using a combination of phage and yeast display technologies. Results: We selected a total of six antibodies in yeast-displayed scFv format, and further tested three of these antibodies in soluble form as minibodies and IgGs. We also describe an assay that can be used to detect plasmids containing dDs–dPn pair. Conclusions: Our antibodies did not afford the desired specificity or sensitivity for detection of a single unnatural base pair among thousands of natural. However, our data indicate not only that such detection is possible but also that these antibodies may be candidates for further affinity and specificity maturation. Full article

Escherichia coli and its bacteriophages are among the most studied model microorganisms. Bacteriophages for various E. coli strains can typically be easily isolated from environmental sources, and many of these viruses can be harnessed to combat E. coli infections in humans and animals. However, some relatively rare E. coli strains pose significant challenges in finding suitable phages. The uropathogenic strain E. coli UPEC124, isolated from a patient suffering from neurogenic bladder dysfunction, was found to be resistant to all coliphages in our collections, and initial attempts to isolate new phages failed. Using an improved procedure for phage enrichment, we isolated the N4-related phage Mimir124, belonging to the Gamaleyavirus genus, which was able to lyse this “difficult” E. coli strain. Although Mimir124 is a narrow-spectrum phage, it was effective in the individualized treatment of the patient, leading to pathogen eradication. The primary receptor of Mimir124 was the O antigen of the O101 type; consequently, Mimir124-resistant clones were rough (having lost the O antigen). These clones, however, gained sensitivity to some phages that recognize outer membrane proteins as receptors. Despite the presence of nine potential antiviral systems in the genome of the UPEC124 strain, the difficulty in finding effective phages was largely due to the efficient, non-specific cell surface protection provided by the O antigen. These results highlight the importance of an individualized approach to phage therapy, where narrow host-range phages—typically avoided in pre-fabricated phage cocktails—may be instrumental. Furthermore, this study illustrates how integrating genomic, structural, and functional insights can guide the development of innovative therapeutic strategies, paving the way for broader applications of phage therapy in combating multidrug-resistant bacterial pathogens. Full article

Food contamination with non-typhoidal Salmonella (NTS) presents a significant public health risk, underscoring the critical need for rigorous food safety measures throughout the production, distribution, preparation, and consumption stages. Conventional diagnostic strategies are time-consuming and labor-intensive and are thus sub-optimal for throughput NTS detection. Bacteriophages (phages) are highly specialized bacterial viruses and exhibit extreme specificity for their hosts. This organic phage/bacterial interaction provides an invaluable tool that can potentially replace or complement existing S. enterica detection methods. Here, we explored work in this area and reviewed data from PubMed/MEDLINE, Embase, and ScienceDirect up to 4 November 2024. Thirty-five studies were selected from 607 retrieved articles using the JBI Critical Appraisal Checklist to ensure quality. Salmonella enrichment, rapid detection, and effective recovery in diverse food sources for various NTS serovars were targeted. Utilizing phages as bio-probes alongside lateral flow immunoassays, surface-enhanced Raman spectroscopy, fluorescence, and electrochemistry assays enabled rapid and highly sensitive detection of NTS, achieving limits as low as 7 to 8 CFU/mL within 30 min. Balancing detection sensitivity with rapid analysis time is essential. Further research and development will be pivotal to overcoming challenges and maximizing the efficiency of NTS phage-based detection to ensure optimal food safety. Full article