Individual Phage Research Articles

Isolation, characterization, and application of lytic bacteriophages for controlling Enterobacter cloacae complex (ECC) in pasteurized milk and yogurt

Reducing bacterial pathogen contamination not only improves overall global public health but also diminishes food waste and loss. The use of lytic bacteriophages (phages) that infect and kill bacteria could be a beneficial tool for suppressing bacterial growth during dairy products storage time. Four Enterobacter cloacae (E. cloacae) complex isolates which were previously isolated from contaminated dairy products were used to identify lytic phages in wastewater. Phages specific to multi-drug resistant (MDR) E. cloacae complex 6AS1 were isolated from local sewage. Two novel phages vB_EclM-EP1 and vB_EclM-EP2 were identified as myoviral particles and have double-stranded DNA genome. Their host range and lytic capabilities were detected using spot test and efficiency of plating (EOP) against several bacterial isolates. The phages had a latent period of 30 min, and a large burst size of about 100 and 142 PFU/cell for vB_EclM-EP1 and vB_EclM-EP2, respectively. Both phages were viable at pH ranging 5–9 and stable at 70 °C for 60 min. The individual phages and their cocktail preparations (vB_EclM-EP1 and vB_EclM-EP2) reduced and inhibited the growth of E. cloacae complex 6AS1 during challenge test in milk and yogurt samples. These results indicate that the E. cloacae complex-specific phages (vB_EclM-EP1 and vB_EclM-EP2) have a potential application as microbicidal agents in packaged milk and milk derivatives during storage time. In addition, our environment is a rich sources of lytic phages which have potential use in eliminating multidrug-resistant isolates in food industry as well as in biocontrol.

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL−1 (1.5 μg kg−1) and an IC50 value of 0.36 ng mL−1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

Isolation, Characterization, Genome Analysis and Host Resistance Development of Two Novel Lastavirus Phages Active against Pandrug-Resistant Klebsiella pneumoniae.

Klebsiella pneumoniae is a global health threat and bacteriophages are a potential solution in combating pandrug-resistant K. pneumoniae infections. Two lytic phages, LASTA and SJM3, active against several pandrug-resistant, nosocomial strains of K. pneumoniae were isolated and characterized. Their host range is narrow and latent period is particularly long; however, their lysogenic nature was refuted using both bioinformatic and experimental approaches. Genome sequence analysis clustered them with only two other phages into the new genus Lastavirus. Genomes of LASTA and SJM3 differ in only 13 base pairs, mainly located in tail fiber genes. Individual phages, as well as their cocktail, demonstrated significant bacterial reduction capacity in a time-dependent manner, yielding up to 4 log reduction against planktonic, and up to 2.59 log on biofilm-embedded, cells. Bacteria emerging from the contact with the phages developed resistance and achieved numbers comparable to the growth control after 24 h. The resistance to the phage seems to be of a transient nature and varies significantly between the two phages, as resistance to LASTA remained constant while resensitization to SJM3 was more prominent. Albeit with very few differences, SJM3 performed better than LASTA overall; however, more investigation is needed in order to consider them for therapeutic application.

Phage and phage cocktails formulations.

Antibiotic-resistant bacterial infection is a major global problem and can be life-threatening. Bacteriophages or phages can be substituted choice over traditional antibiotics treatments. Phages are natural obligate parasites viruses of bacteria, and they can infect and kill antibiotic-sensitive and -resistant bacteria. Further, phages can be utilised as antibacterial agents against various kinds of bacterial infectious diseases. As compared to antibiotics, phages can show a more variety of modes of action and can also be safe in several cases. Phages as a mixture (cocktail) of viral strains are usually used in clinical practices. Generally, to propagate phage cocktails, the individual phage is grown and then mixed to prepare phage cocktails. Antibiotic resistance and biofilm formation can be controlled through formulating phage cocktails that comprise phages infecting single species or by combining phages with non-phages (antibiotics), which may result in a broad spectrum of activity. This chapter briefly highlights the formulations and application of phage cocktails, which are being used to treat various bacterial infections.

Selection and characterization of bacteriophages specific to Salmonella Choleraesuis in swine.

Salmonella Choleraesuis is the most common serotype that causes salmonellosis in swine. Recently, the use of bacteriophages as a potential biocontrol strategy has increased. Therefore, this study aimed to isolate and characterize bacteriophages specific to S. Choleraesuis associated with swine infection and to evaluate the efficacy of individual phages and a phage cocktail against S. Choleraesuis strains in simulated intestinal fluid (SIF). Three strains of S. Choleraesuis isolated from pig intestines served as host strains for phage isolation. The other 10 Salmonella serovars were also used for the phage host range test. The antibiotic susceptibility of the bacterial strains was investigated. Water samples from natural sources and drain liquid from slaughterhouses were collected for phage isolation. The isolated phages were characterized by determining the efficiency of plating against all Salmonella strains and the stability at a temperature range (4°C-65°C) and at low pH (2.5-4.0) in simulated gastric fluids (SGFs). Furthermore, morphology and genomic restriction analyses were performed for phage classification phages. Finally, S. Choleraesuis reduction in the SIF by the selected individual phages and a phage cocktail was investigated. The antibiotic susceptibility results revealed that most Salmonella strains were sensitive to all tested drugs. Salmonella Choleraesuis KPS615 was multidrug-resistant, showing resistance to three antibiotics. Nine phages were isolated. Most of them could infect four Salmonella strains. Phages vB_SCh-RP5i3B and vB_SCh-RP61i4 showed high efficiency in infecting S. Choleraesuis and Salmonella Rissen. The phages were stable for 1 h at 4°C-45°C. However, their viability decreased when the temperature increased to 65°C. In addition, most phages remained viable at a low pH (pH 2.5-4.0) for 2 h in SGF. The efficiency of phage treatment against S. Choleraesuis in SIF showed that individual phages and a phage cocktail with three phages effectively reduced S. Choleraesuis in SIF. However, the phage cocktails were more effective than the individual phages. These results suggest that the newly isolated phages could be promising biocontrol agents against S. Choleraesuis infection in pigs and could be orally administered. However, further in vivo studies should be conducted.

Modeling the Directed Evolution of Broad Host Range Phages.

Background: The host ranges of individual phages tend to be narrow, yet many applications of phages would benefit from expanded host ranges. Empirical methods have been developed to direct the evolution of phages to attack new strains, but the methods have not been evaluated or compared for their consequences. In particular, how do different methods favor generalist (broad host range) phages over specialist phages? All methods involve exposing phages to two or more novel bacterial strains, but the methods differ in the order in which those hosts are presented through time: Parallel presentation, Sequential presentation, and Mixed presentation. Methods: We use a combination of simple analytical methods and numerical analyses to study the effect of these different protocols on the selection of generalist versus specialist phages. Results: The three presentation protocols have profoundly different consequences for the evolution of generalist versus specialist phages. Sequential presentation favors generalists almost to the exclusion of specialists, whereas Parallel presentation does the least so. However, other protocol attributes (the nature of dilution between transfers of phages to new cultures) also have effects on selection and phage maintenance. It is also noted that protocols can be designed to enhance recombination to augment evolution and to reduce stochastic loss of newly arisen mutants.

Bacteriophage cocktail as a substitute for antimicrobials in companion animal dermatology

The present study focuses on the use of phage cocktails as a substitute for antibiotics in companion animal dermatology. For this purpose, a systematic search was carried out in the Scopus database, with the search criteria: "veterinary" and "bacteriophage" and "dermatology" in article title, abstract and keywords during the period 2010-2021. Seven in vitro studies and one in vivo study in companion animals, for which those carried out in laboratory animals were added. In this review, the use of non-transducing lytic phage cocktails as therapeutics for pyodermas is discussed and projected, as well as the resistance to phages and the strategies to overcome it, the comparison with antibiotics, the use of cocktails in other animal species, as well as the use of individual phages and cocktails in veterinary dermatology, and autochthonous phages as a strategy when phage collections from previous studies do not have the desired effects. It is concluded that non-transducing lytic autophage cocktails are an alter-native against antimicrobial resistance in companion animal dermatology. Finally, it is recommended to compare the use of these cocktails with other antibiotic substitutes and evaluate their possible syner-gism to reduce pathogenic bacteria on the skin.

Imaging the Infection Cycle of T7 at the Single Virion Level.

T7 phages are E. coli-infecting viruses that find and invade their target with high specificity and efficiency. The exact molecular mechanisms of the T7 infection cycle are yet unclear. As the infection involves mechanical events, single-particle methods are to be employed to alleviate the problems of ensemble averaging. Here we used TIRF microscopy to uncover the spatial dynamics of the target recognition and binding by individual T7 phage particles. In the initial phase, T7 virions bound reversibly to the bacterial membrane via two-dimensional diffusive exploration. Stable bacteriophage anchoring was achieved by tail-fiber complex to receptor binding which could be observed in detail by atomic force microscopy (AFM) under aqueous buffer conditions. The six anchored fibers of a given T7 phage-displayed isotropic spatial orientation. The viral infection led to the onset of an irreversible structural program in the host which occurred in three distinct steps. First, bacterial cell surface roughness, as monitored by AFM, increased progressively. Second, membrane blebs formed on the minute time scale (average ~5 min) as observed by phase-contrast microscopy. Finally, the host cell was lysed in a violent and explosive process that was followed by the quick release and dispersion of the phage progeny. DNA ejection from T7 could be evoked in vitro by photothermal excitation, which revealed that genome release is mechanically controlled to prevent premature delivery of host-lysis genes. The single-particle approach employed here thus provided an unprecedented insight into the details of the complete viral cycle.

Formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing Klebsiella pneumoniae in vitro in Kenya.

BackgroundThe development of alternative control measures, such as phage therapy or adjunctive therapy, is urgently needed to manage the dissemination of carbapenemase-producing Klebsiella pneumoniae.ObjectiveThis study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of carbapenemase-producing K. pneumoniae clinical isolate in vitro in Kenya.MethodsThe study was conducted from February 2021 to October 2021 at the Institute of Primate Research, Nairobi, Kenya. Phage cocktails were formulated based on the morphology and biological properties of precipitated Klebsiella phages. The efficacy of individual bacteriophages and phage cocktails as well as their combination with antibiotics were determined for their inhibitory activity on carbapenemase-producing K. pneumoniae (KP20).ResultsThe precipitated bacteriophages were members of Myoviridae, Siphoviridae and Podoviridae. Regarding the evaluation of the phage cocktails, the absorbances at 600 nm of the bacterial culture treated with the two-phage cocktail (2φ MA) ranged from 0.173 to 0.246 at 16 h and 20 h whereas it peaked from 2.116 to 2.190 for the positive control. Moreover, the results of the adjunctive therapy showed that the optical density at 600 nm of the bacterial culture treated with 2φ MA was 0.186 at 24 h post-incubation time while it was 0.099 with the bacterial culture treated with imipenem in combination with 2φ MA.ConclusionThis study demonstrated that the two-phage cocktail in combination with imipenem was able to synergistically delay the increase in carbapenemase-producing K. pneumoniae growth in vitro.

Comparison between the Biofilm Desorption Abilities of T4 and MS2 Coliphages

Biofilms are a collection of microorganisms that adhere to a surface where they continue to grow. Firmly established biofilms can be hazardous to human health. Chemical and biological as well as combination methods are being tested to control biofilms. The elucidation of the biofilm disruption capabilities of individual bacteriophages has received limited attention. Although the treatment of biofilms with a combination of bacteriophages is effective, the extent to which DNA and RNA coliphages individually desorb biofilms is not well understood. Here, we show that both T4 and MS2 coliphages desorbed natural biofilms. Individual incubations of the equivalent viral load of T4 and MS2 coliphages with natural biofilms resulted in similar desorption of these biofilms. We also note that the nutrient deprivation significantly reduced biofilm growth. However, the biofilm desorption upon nutrient deprivation was similar to that observed with both T4 and MS2 phages.

Application of phage therapy against red-fleshed kiwifruit canker

The global production and trade of kiwifruit have been severely impacted by bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa), and the rare cultivar red-fleshed kiwifruit is particularly susceptible to this disease. Available treatments, such as spraying of copper-based bactericides or antibiotics, may contaminate the environment or promote bacterial resistance. Phage therapy may be an alternative approach to inactivating Psa. This study investigated the potential application of a phage cocktail to the control of Psa infection. The pathogenicity of Psa was evaluated by artificially contaminating red-fleshed kiwifruit seedlings with a biovar III strain (Psa BJ530). Four lytic bacteriophages were isolated, and the data showed that they had strong biological control characteristics. The phage cocktail prepared from the four phages had better biological treatment efficacy both in vitro and in vivo compared with each individual phage. Our results showed that the isolated phages effectively controlled Psa infection and provided a basis for the biological control of bacterial canker in red-fleshed kiwifruit orchards.

Surface conditioning with bacteriophages reduces biofilm formation of Salmonella Heidelberg.

Salmonella remains one of the most common foodborne pathogens worldwide, and its resistance to antimicrobials has increased considerably over the years. In this context, was evaluated the action of three bacteriophages isolated or combined in inhibiting the adhesion and removal of Salmonella Heidelberg biofilm on a polystyrene surface. The bacteriophages UPF_BP1, UPF_BP2, UPF_BP3 and a pool of them were used for biocontrol of Salmonella Heidelberg biofilms on polystyrene surface in the action times of 3, 6 and 9 h. Individual and combined phages exhibited reductions in Salmonella Heidelberg adhesion of up to 83.4% and up to 64.0% in removal of preformed biofilm. The use of synergistic combinations between the phages is the most indicated option due to its potential to reduce biofilms. The use of the bacteriophage pool in surface conditioning is an alternative in the control of Salmonella Heidelberg biofilms.

1276. Assessment of Anti-biofilm Activity of Staphylococcus aureus Bacteriophages Against Clinical Isolates from Patients with Left Ventricular Assist Device Infections

Abstract Background Staphylococcus aureus is a common cause of biofilm-mediated left ventricular device (LVAD) infections which are difficult to resolve with antibiotics alone and are associated with substantial morbidity and mortality. Recently, bacteriophages (phage) therapy has been used to resolve LVAD infections in a few cases. Our goal was to assess in vitro susceptibility and anti-biofilm activity of two S. aureus bacteriophages against clinical isolates from patients with S. aureus LVAD infections in order to develop a S. aureus phage cocktail for clinical use. Methods Two bacteriophages, OMS1 and OMS2, from the Israeli Phage Bank, were assessed for lytic activity against 15 S.aureus isolates (9 methicillin resistant, MRSA and 6 methicillin susceptible, MSSA) via agar overlay method and plaque forming units (PFU)/mL were enumerated. We then formed bacterial biofilms after overnight incubation at 120 rpm in a 96-well plates in duplicate; experiments were repeated thrice. Wells were then treated with tryptic soy broth (TSB, control) or TSB containing phage at 109 PFU/mL for 24 hours. After washing, biofilms were stained with crystal violet and biomass quantified via optical density at 570mm. Results All bacterial isolates were susceptible to both phages via agar overlay to varying degrees as determined by phage titers obtained via serial dilutions (Figure 1a, 1b). OMS1 led to significant reduction in biofilm of 7/9 MRSA and 3/6 MSSA isolates and OMS2 reduced biofilms in 9/9 MRSA and 4/6 MSSA isolates (Figure 1c). Phage titers and Biofilm Biomass Figure 1a) Phage titers in PFU/ml obtained from each bacterial isolate via agar overlay method. 1b) Representative agar plate demonstrating lytic plaques from OMS1 and OMS2 on a Staphylococcus aureus LVAD isolate. 1c) Biofilm biomass of Staphylococcus aureus isolates alone (SA) or with individual phage (SA+OMSA1 and SA=OMS2) assessed by optical density readings at 570 nm; error bars represent standard error of the mean. Conclusion We demonstrated in vitro lytic and anti-biofilm activity of 2 S. aureus phages against clinical S. aureus isolates from patients with LVAD infection. Our data suggests that phage susceptibility measured with agar overlay does not always correlate with phage susceptibility of S. aureus biofilms, suggesting that more than one method should be used to assess in vitro activity. We plan to assess for synergistic activity with the phage combination. Disclosures Ryan K. Shields, PharmD, MS, Shionogi (Consultant, Research Grant or Support) Ran Nir-Paz, MD, BiomX (Consultant)Technophage (Scientific Research Study Investigator, Advisor or Review Panel member) Saima Aslam, MD, MS, BioMx (Consultant)Cystic Fibrosis Foundation (Grant/Research Support)Gilead (Consultant)Johnson and Johnson (Consultant)Merck (Consultant)

Ontogeny of the Alloimmune Anti-Canine Factor VIII Inhibitor Response in Severe Hemophilia Α Dogs

The development of inhibitors to Factor VIII (FVIII) is a major complication of hemophilia A (HA) treatment. Most preclinical studies in HA animals have been limited to a xenoprotein response, e.g., evaluating the murine immune response to human FVIII (hFVIII). Severe HA dogs are a naturally occurring outbred model that recapitulates the spontaneous bleeding phenotype of the disease; their severe HA is due to intron 22 inversion (INV22) of the canine F8 gene, analogous to the INV22 found in 45% of severe HA patients. Importantly, similar to HA INV22 patients, about 20% of these animals develop an anti-canine FVIII (cFVIII) inhibitor response. This ability of the HA dogs to develop inhibitors in a species-specific manner provides an opportunity to probe the molecular ontogeny of an alloimmune anti-FVIII response.Here, we use antibody phage display to capture the humoral anti-cFVIII IgG repertoire in a dog with severe HA. This dog developed and maintained high titers of cFVIII inhibitor (89 Bethesda Units, BU) after exposure to cFVIII protein. The dog then received AAV liver-directed gene therapy encoding a cFVIII transgene, and after an initial anamnestic response characterized by rapid increase of cFVIII inhibitor titer (peak titer of 182 BU), the dog exhibited successful inhibitor eradication and immune tolerance induction, similar to our previous report (Finn et al., Blood 2010).Canine peripheral blood mononuclear cells were used to construct a single-chain variable fragment (scFv) phage display library which was sequentially selected four times against immobilized recombinant B-domain deleted (BDD) cFVIII protein. The amount of enrichment per round of selection plateaued during the third round from which 55 scFv/phage clones were isolated for characterization. Individual phage clones were sequenced by Sanger sequencing and screened for binding to both canine and human BDD-FVIII by ELISA. ScFv/phage clones were tested for inhibitory activity in a modified Bethesda assay.We identified 26 distinct scFv clones binding cFVIII based on heavy chain/light chain composition comprising 16 distinct CDRH3 sequences and 23 distinct CDRL3 sequences. The heavy chains of the 26 clones were derived from six canine germline IGHV genes, namely IGHV3-2, IGHV3-5, IGHV3-9, IGHV3-19, IGHV3-38, and IGHV3-41. The 16 distinct CDRH3 sequences had a mean length of 10.1 ± 3.6 amino acids, shorter than the 13.5 ± 3.6 amino acids previously reported for total canine CDRH3 repertoires (Steiniger et al., Mol. Immunol. 2014). Only 10 of the 26 FVIII-specific clones contained a lambda light chain, despite canine IgG repertoires dominated by the use of lambda light chains (Steiniger et al.). Eight of the 26 clones exhibited inhibitory activity. Although this dog was never exposed to hFVIII, 12/26 (46%) clones bound both cFVIII and hFVIII, consistent with the high degree of homology between the two orthologues with 85% identity. For three clones, framework region mutations and different light chain pairings resulted in altered inhibitory activity and hFVIII binding. Interestingly, one clone in particular comprised the majority of randomly screened scFv from the third and fourth rounds of phage library selection.These results suggest that cFVIII-specific B cells arise from multiple germline VH genes and exhibit high CDRH3 diversity. While the IgG-FVIII interaction has previously been suggested to be mostly influenced by the IgG heavy chain, our data suggest that for some antibodies, the IgG light chain may influence both inhibitory activity and epitope reactivity. Future studies will assess B cell clonal evolution via deep sequencing and longitudinal epitope specificity during multi-year immune tolerance induction by continuous exposure to cBDD-FVIII gene therapy. This is the first in-depth assessment of FVIII immunogenicity in a large HA model that avoids the use of a non-species specific antigen. Direct comparison with ongoing studies in HA inhibitor patients will inform the immunodominant epitopes of FVIII across species and thus provide insights on FVIII immunogenicity. DisclosuresDoshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau. Samelson-Jones: Spark: Research Funding; Pfizer: Consultancy, Research Funding. Siegel: Verismo Therapeutics, Inc: Other: Co-Founder and Equity Holder; Vetigenics, LLC: Other: Co-Founder and Equity Holder.

Search for Peptides Specifically Binding with the B7-2 Costimulatory Molecule

—The interaction of B7-1/B7-2 ligands with CD28/CTLA-4 receptors plays a key role in the regulation of the immune response. The aim of this study was to find and study peptides that interact with the human B7-2 molecule. In the course of the work, three rounds of affinity selection were carried out and individual phage clones were selected, which include peptides with varying degrees of interaction with the coregulatory target B7-2. As a result of DNA sequencing of selected phages, nucleotide sequences encoding peptides that specifically bind to B7-2 were obtained. The identified peptides can be used as a basis for the development of immunotherapeutic drugs for regulating the immune response in the treatment of oncological diseases.

ACTIVITY RANGE OF COMMERCIAL PHAGE PREPARATIONS AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS ISOLATES

Methicillin-resistant Staphylococcus aureus (MRSA) was first discovered in 1962, since then it has been recognized as a major causative agent of healthcare-associated infections. MRSA infections are usually divided into three groups: hospital- (HA), community- (CA) and livestock-associated (LA) MRSA. The incidence of LA-MRSA infections among livestock and companion animals is a great concern because these sources could potentially be reservoirs for zoonotic infections. In Ukraine, recent statistics indicated approximately 82 % of tested livestock were S. aureus positive with 53.8 % of resistance. In addition, the diversity of MRSA in Ukraine has not been investigated.
 Antibiotic resistance during treatment in both clinical practice and veterinary medicine remains a challenge and requires alternative solutions. Bacteriophage-based treatment represents a promising biocontrol strategy with proven effectiveness against MRSA. The purpose of our research was to evaluate the activity of two commercial phage preparations against a local collection of LA-MRSA host strains and to characterize and adapt individual phages isolated from these preparations with the desired goal of enhancing their efficacy.
 In our study, we tested two commercial phage preparations (Eliava BioProduction, Georgia) Staphylococcus Bacteriophage and Intesti Bacteriophage on local LA-MRSA isolates. In addition, we utilized two single phages we isolated from these preparations. Both commercial phage preparations showed variable activity against the tested isolates. Intesti Bacteriophage cocktail lysed 60 % of isolates, whereas the single phage isolated from it was found to be ineffective. Staphylococcus Bacteriophage preparation showed activity against 30 % of MRSA tested, while the isolated phage was active against 70 % of the tested isolates. For the first time in Ukraine, in this study we assessed host range of commercial phages on local circulating livestock-associated MRSA. Further host range analysis on an expanded collection of Ukrainian MRSA isolates as well as taxonomic and genomic characterization of the phages is planned.

Efficacy of Novel Bacteriophages against Escherichia coli Biofilms on Stainless Steel

Biofilm formation by E. coli is a serious threat to meat processing plants. Chemical disinfectants often fail to eliminate biofilms; thus, bacteriophages are a promising alternative to solve this problem, since they are widely distributed, environmentally friendly, and nontoxic to humans. In this study, the biofilm formation of 10 E. coli strains isolated from the meat industry and E. coli ATCC BAA-1430 and ATCC 11303 were evaluated. Three strains, isolated from the meat contact surfaces, showed adhesion ability and produced extracellular polymeric substances. Biofilms of these three strains were developed onto stainless steel (SS) surfaces and enumerated at 2, 12, 24, 48, and 120 h, and were visualized by scanning electron microscopy. Subsequently, three bacteriophages showing podovirus morphology were isolated from ground beef and poultry liver samples, which showed lytic activity against the abovementioned biofilm-forming strains. SS surfaces with biofilms of 2, 14, and 48 h maturity were treated with mixed and individual bacteriophages at 8 and 9 log10 PFU/mL for 1 h. The results showed reductions greater than 6 log10 CFU/cm2 as a result of exposing SS surfaces with biofilms of 24 h maturity to 9 log10 PFU/mL of bacteriophages; however, the E. coli and bacteriophage strains, phage concentration, and biofilm development stage had significant effects on biofilm reduction (p < 0.05). In conclusion, the isolated bacteriophages showed effectiveness at reducing biofilms of isolated E. coli; however, it is necessary to increase the libraries of phages with lytic activity against the strains isolated from production environments.

Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant bacteria, novel antibacterial agents, are urgently needed. A bacteriophage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens in agricultural settings. Phage biocontrol is increasingly accepted as a sustainable technology, effective at decontaminating foodborne pathogens. To ensure effective biocontrol outcomes, systematic screening of phage combinations against targeted bacteria under required environmental conditions is crucial. Antibacterial efficacy of phage cocktails may be affected by phage genera and combination, targeted bacterial strains, the multiplicity of infection, temperature, and time. To formulate a phage cocktail with superior efficacy, the proposed method was to systematically evaluate the effectiveness of individual phages and phage cocktails in killing foodborne bacterial pathogens under targeted conditions. Bacterial killing efficacy was monitored by measuring optical density at desired temperatures and durations. Superior phage efficacy was determined by complete inhibition of bacterial growth. The proposed method is a robust, evidence-based approach to facilitate formulating phage cocktails with superior antibacterial efficacy.

Bacteriophage cocktail and phage antibiotic synergism as promising alternatives to conventional antibiotics for the control of multi-drug-resistant uropathogenic Escherichia coli

Infections related to antibiotic resistant bacteria are accelerating on a global scale, and hence to encounter this problem in case of urinary tract infections; bacteriophages were isolated for biocontrol of multi-drug resistant (MDR) uropathogenic Escherichia coli (UPECs) isolates. Four lytic phages were purified, characterized, and evaluated for their effectiveness in the form of cocktail and in synergy with antibiotics. Morphological features and other life cycle specifications of phages revealed that two phages Escherichia phage FS11 and Escherichia phage FS17 belonged to Myoviridae and the other two phages Escherichia phage PS8 and Escherichia phage PS6 belonged to Siphoviridae family of order Caudovirales. One step growth curve analysis demonstrated that phage FS11 and phage FS17 had latent time of 24 min and 26 min, and a burst size of ~121 and 98 phage particles/ cell respectively; while for phage PS8 and phage PS6, the latent time was 42 min and 35 min, and the burst size was 87 and 78 particles/ cell, respectively; depicting the lytic nature of phages. The use of all four phages together in the form of a cocktail resulted into a considerable enhancement in the lytic ability; the phage cocktail lysed 86.7% of the clinical isolates, compared to lysis in the range of 50%-66% by individual phages. Studies on in vitro evaluation of phage-antibiotic combinations revealed synergism between antibiotics and the phage cocktail (phage PS6 and phage FS17), wherein the phage cocktail was observed to efficiently inhibit the strains in the presence of sub-lethal doses of antibiotics. The study thus concludes that the use of multiple phages and phage-antibiotic combinations could prove beneficial in the era of rapidly increasing drug-resistant strains.

Phage Biocontrol of Pseudomonas aeruginosa in Water.

Bacteriophage control of harmful or pathogenic bacteria has aroused growing interest, largely due to the rise of antibiotic resistance. The objective of this study was to test phages as potential agents for the biocontrol of an opportunistic pathogen Pseudomonas aeruginosa in water. Two P. aeruginosa bacteriophages (vB_PaeM_V523 and vB_PaeM_V524) were isolated from wastewater and characterized physically and functionally. Genomic and morphological characterization showed that both were myoviruses within the Pbunavirus genus. Both had a similar latent period (50–55 min) and burst size (124–134 PFU/infected cell), whereas there was variation in the host range. In addition to these environmental phages, a commercial Pseudomonas phage, JG003 (DSM 19870), was also used in the biocontrol experiments. The biocontrol potential of the three phages in water was tested separately and together as a cocktail against two P. aeruginosa strains; PAO1 and the environmental strain 17V1507. With PAO1, all phages initially reduced the numbers of the bacterial host, with phage V523 being the most efficient (>2.4 log10 reduction). For the environmental P. aeruginosa strain (17V1507), only the phage JG003 caused a reduction (1.2 log10) compared to the control. The cocktail of three phages showed a slightly higher decrease in the level of the hosts compared to the use of individual phages. Although no synergistic effect was observed in the host reduction with the use of the phage cocktail, the cocktail-treated hosts did not appear to acquire resistance as rapidly as hosts treated with a single phage. The results of this study provide a significant step in the development of bacteriophage preparations for the control of pathogens and harmful microbes in water environments.