Scientific Publications
Bacteriophage Research
Proteon Pharmaceuticals is committed to driving innovation in bacteriophage research. Our scientific publications contribute to the global understanding of phage applications in microbiology, animal health, and antimicrobial resistance.
Advancing Science
Our Research Publications and Impact
2024
Escherichia coli is a diverse and ubiquitous strain of both commensal and pathogenic bacteria. In this study, we propose the use of multiplex polymerase chain reaction (PCR), using amplification of three genes (cydA, lacY, and ydiV), as a method for determining the affiliation of the tested strains to the E. coli species. The novelty of the method lies in the small number of steps needed to perform the diagnosis and, consequently, in the small amount of time needed to obtain it. This method, like any other, has some limitations, but its advantage is fast, cheap, and reliable identification of the presence of E. coli. Sequences of the indicated genes from 1,171 complete E. coli genomes in the NCBI database were used to prepare the primers. The developed multiplex PCR was tested on 47,370 different Enterobacteriaceae genomes using in silico PCR. The sensitivity and specificity of the developed test were 95.76% and 99.49%, respectively. Wet laboratory analyses confirmed the high specificity, repeatability, reproducibility, and reliability of the proposed test. Because of the detection of three genes, this method is very cost and labor-effective, yet still highly accurate, specific, and sensitive in comparison to similar methods.
Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.
The studies on phage therapy have shown an overall protective effect of phages in bacterial infections, thus providing an optimistic outlook on the future benefits of phage-based technologies for treating bacterial diseases. However, the therapeutic effect is highly affected by the proper composition of phage cocktails. The rational approach to the design of bacteriophage cocktails, which is the subject of this study, allowed for development of an effective anti-mastitis solution, composed of virulent bacteriophages acting on Escherichia coli and Staphylococcus aureus. Based on the in-depth bioinformatic characterization of bacteriophages and their in vitro evaluation, the cocktail of five phages against E. coli and three against S. aureus strains was composed. Its testing in the milk model experiment revealed a reduction in the number of S. aureus of 45% and 30% for E. coli strains, and in the study of biofilm prevention, it demonstrated 99% inhibition of biofilm formation for all tested S. aureus strains and a minimum of 50% for 50% of E. coli strains. Such insights justify the need for rational design of cocktails for phage therapy and indicate the potential of the developed cocktail in the treatment of diseased animals, but this requires further investigations to evaluate its in vivo efficacy.
Pseudomonas aeruginosa is a pathogen that causes infections in animals and humans, with veterinary implications including ear infections in dogs, respiratory diseases in cats, and mastitis in ruminants. In humans, it causes severe hospital-acquired infections, particularly in immunosuppressed patients. This study aimed to identify and assess the prevalence of specific virulence factors in Pseudomonas aeruginosa isolates. Methods: We analyzed 98 Pseudomonas aeruginosa isolates from various animal samples (dogs, cats, ruminants, fowl) from northeastern Poland in 2019–2022 for virulence-related genes (toxA, exoU, exoT, exoS, lasB, plcN, plcH, pldA, aprA, gacA, algD, pelA, endA, and oprF) by PCR and assessed biofilm formation at 48 and 72 h. Genomic diversity was assessed by ERIC-PCR. Results: The obtained results showed that all strains harbored the pelA gene (100%), while the lowest prevalence was found for pldA (24%) and exoU (36%). Regardless of the animal species, strong biofilm forming ability was prevalent among the strains after both 48 h (75%) and 72 h (74%). We obtained as many as 87 different genotyping profiles, where the dominant one was profile ERIC-48, observed in four strains. Conclusions: No correlation was found between presence or absence of determined genes and the nature of infection. Similarly, no correlation was found between biofilm-forming genes and biofilm strength. The high genetic diversity indicates challenges for effective prevention, emphasizing the need for ongoing monitoring and research.
2022
Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.
Bovine mastitis is a common disease worldwide, and staphylococci are one of the most important etiological factors of this disease. Staphylococcus aureus show adaptability to new conditions, by which monitoring their virulence and antibiotic resistance mechanisms is extremely important, as it can lead to the development of new therapies and prevention programs. In this study, we analyzed Staphylococcus aureus (n = 28) obtained from dairy cattle with subclinical mastitis in Poland. The sensitivity of the isolated strains to antibiotics were confirmed by the disc diffusion method. Additionally, minimum inhibitory concentration values were determined for vancomycin, cefoxitin and oxacillin. Genotyping was performed by two methods: PCR melting profile and MLVF-PCR (multiple-locus variable-number tandem-repeat fingerprinting). Furthermore, the presence of antibiotic resistance and virulence genes were checked using PCR reactions. The analyzed strains showed the greatest resistance to penicillin (57%), oxytetracycline (25%) and tetracycline (18%). Among the analyzed staphylococci, the presence of 9 of 15 selected virulence-related genes was confirmed, of which the icaD, clfB and sea genes were confirmed in all staphylococci. Biofilm was observed in the great majority of the analyzed bacteria (at least 70%). In the case of genotyping among the analyzed staphylococci (combined analysis of results from two methods), 14 patterns were distinguished, of which type 2 was the dominant one (n = 10). This study provides new data that highlights the importance of the dominance of biofilm over antibiotic resistance among the analyzed strains.
2021
Salmonella infections (salmonellosis) pose serious health risks to humans, usually via food-chain contamination. This foodborne pathogen causes major food losses and human illnesses, with significant economic impacts. Overuse of antibiotics in the food industry has led to multidrug-resistant strains of bacteria, and governments are now restricting their use, leading the food industry to search for alternatives to secure food chains. Bacteriophages, viruses that infect and kill bacteria, are currently being investigated and used as replacement treatments and prophylactics due to their specificity and efficacy. They are generally regarded as safe alternatives to antibiotics, as they are natural components of the ecosystem. However, when specifically used in the industry, they can also make their way into humans through our food chain or exposure, as is the case for antibiotics. In particular, agricultural workers could be repeatedly exposed to bacteriophages supplemented to animal feeds. To our knowledge, no studies have investigated the effects of such exposure to bacteriophages on the human gut microbiome. In this study, we used a novel in-vitro assay called RapidAIM to investigate the effect of a bacteriophage mixture, BAFASAL®, used in poultry farming on five individual human gut microbiomes. Multi-omics analyses, including 16S rRNA gene sequencing and metaproteomic, revealed that ex-vivo human gut microbiota composition and function were unaffected by BAFASAL® treatment, providing an additional measure for its safety. Due to the critical role of the gut microbiome in human health and the known role of bacteriophages in regulation of microbiome composition and function, we suggest assaying the impact of bacteriophage-cocktails on the human gut microbiome as a part of their safety assessment.
2020
Probiotics are considered an alternative to antibiotics in the prevention and treatment of Salmonella diseases in poultry. However, to use probiotics as proposed above, it is necessary to evaluate their properties in detail and to select the most effective bacterial strains in the application targeted. In this study, probiotic properties of new Lactobacillus sp. strains were investigated and their antimicrobial activity against 125 environmental strains of Salmonella sp. was determined using the agar slab method. Furthermore, their survival in the presence of bile salts and at low pH, antibiotics susceptibility, aggregation and coaggregation ability, adherence to polystyrene and Caco-2 cells, and cytotoxicity were investigated. Each strain tested showed antagonistic activity against at least 96% of the environmental Salmonella sp. strains and thus representing a highly epidemiologically differentiated collection of poultry isolates. In addition, the probiotic properties of new Lactobacillus strains are promising. Therefore, all strains examined showed a high potential for use in poultry against salmonellosis.
Phage therapy, a promising alternative to antimicrobial treatment of bacterial diseases, is getting more and more popular, especially due to the rising awareness of antibiotic resistance and restrictions in antibiotics’ use. During recent years, we observed a growing trend of bacteriophages’ application in aquaculture, which in each year reports high losses due to bacterial diseases. This review provides an update of the status of bacteriophage therapy for the treatment and prevention of infections in the aquatic environment. As it is still mostly in the scientific stage, there are a few constraints that may prevent effective therapy. Therefore, specific characteristics of bacteriophages, that can act in favor or against their successful use in treatment, were described. We underlined aspects that need to be considered: specificity of phages, bacterial resistance, safety, immune response of the host organism, formulation, administration and stability of phage preparations as well as bacteriophages’ influence on the environment. The biggest challenge to overcome is finding the right balance between the desired and problematic characteristics of bacteriophages. Finally, regulatory approval challenges may be encountered by bacteriophage manufacturers. Even though there are still some technical constraints connected with the global use of bacteriophage therapy, it was concluded that it can be successfully applied in aquaculture.
Supplementing animal food with bacteriophages active against animal bacterial pathogens can be an excellent way of mildly influencing the quality of food and the health condition of animal breeding. Polysaccharides were used to immobilize biologically active cultures, and the technique of low-temperature drying under atmospheric pressure was chosen to maintain microbiological activity. Periodically dried material had the form of spheres with initial diameter 3 mm and moisture content 97%. The average initial concentration of bacteriophages of Siphoviridae family was 1×108 PFU/g (plaque-forming units per gram). Drying tests were carried out in temperatures range from -10°C up to 30°C. The activity of bacteriophages was checked prior and after drying, by enumeration by double agar overlay plaque assay.
IDS’2020 – 22nd International Drying Symposium Worcester, Massachusetts, USA
June 28 – July 1, 2020
Bacteriophages are bacterial predators, which are garnering much interest nowadays vis-à-vis the global phenomenon of antimicrobial resistance. Bacteriophage preparations seem to be an alternative to antibiotics, which can be used at all levels of the food production chain. Their safety and efficacy, however, are of public concern. In this study, a detailed evaluation of BAFASAL® preparation was performed. BAFASAL® is a bacteriophage cocktail that reduces Salmonella in poultry farming. In vivo acute and sub-chronic toxicity studies on rats and tolerance study on targeted animals (chicken broiler) conducted according to GLP and OECD guidelines did not reveal any signs of toxicity, which could be associated with BAFASAL® administration. In addition, no evidences of genotoxicity were observed. The tolerance study with 100-times concentrated dose also did not show any statistically significant differences in the assessed parameters. The in vitro crop assay, mimicking normal feed storage and feed application conditions showed that BAFASAL® reduced the number of Salmonella bacteria in experimentally contaminated feed. Moreover, reductions were observed for all examined forms (liquid, powder, spray). Furthermore, the in vivo efficacy study showed that treatment with BAFASAL® significantly decreased Salmonella content in caeca of birds infected with Salmonella Enteritidis. Detailed examination of BAFASAL® in terms of safety and efficacy, adds to the body of evidence that bacteriophages are harmless to animals and effective in the struggle against bacteria.
Salmonellosis is one of the most important bacterial diseases in pigeons. This study aimed to estimate the prevalence of Salmonella spp. in domestic pigeons (Columba livia f. domestica) in Poland, its antimicrobial susceptibility (both phenotypic and genotypic), and its capability for biofilm formation. The presence of selected virulence genes, nucleotide homology of selected genes, and susceptibility to bacteriophages were investigated as well. From the 585 pigeons tested, 5.47% turned out positive. All isolated strains were recognized as Salmonella enterica ser. Typhimurium. The asymptomatic pigeons were carriers of 37.5% of the isolates. The dominant variants were as follows: 1,4,[5],12,:i:1,2 (53.13%) and 1,4,[5],12,:-:- (31.25%). Most of the strains analysed showed the ability to produce biofilm after 24 and 48 hr of incubation (59.38% and 53.13%, respectively). Over 90% of the strains were confirmed for lpfA, agafA, invA, sivH, and avrA virulence genes. Also, of the thirteen antimicrobial susceptibility genes, the following were confirmed: sul1, tet(A), blaTEM-1, floR, strA, and strB. The most common were the strB (18%) and tet(A) (12%) genes that are responsible for coding resistance to aminoglycosides and tetracyclines, respectively. Most of the strains were phenotypically resistant to oxytetracycline (46.88%), neomycin (53.13%) and tylosin (100%). The susceptibility of the investigated Salmonella strains to the bacteriophages was between 33% and 100%. MLST, PCR MP and ERIC PCR analyses indicated a very high genetic similarity of the investigated strains (over 99%). Results of our study indicate that Salmonella enterica ser. Typhimurium is still an important agent in domestic pigeons and that its antimicrobial resistance increases. Alarming is also the confirmation of a single-phase variant 1,4,[5],12:i,-, which could have increased virulence and multi-drug resistance encoded on the plasmid. Most importantly, however, such strains have been isolated from humans with clinical symptoms of Salmonella infection.
2019
Aquaculture is the fastest growing sector of food production worldwide. However, one of the major reasons limiting its effectiveness are infectious diseases among aquatic organisms resulting in vast economic losses. Fighting such infections with chemotherapy is normally used as a rapid and effective treatment. The rise of antibiotic resistance, however, is limiting the efficacy of antibiotics and creates environmental and human safety concerns due to their massive application in the aquatic environment. Bacteriophages are an alternative solution that could be considered in order to protect fish against pathogens while minimizing the side-effects for the environment and humans. Bacteriophages kill bacteria via different mechanisms than antibiotics, and so fit nicely into the ‘novel mode of action’ concept desired for all new antibacterial agents.
Inland fishery belongs to those branches of animal production that use very large amounts of chemotherapeutics, in particular antibiotics. The accumulation of chemotherapeutic agents in bottom sediments is a direct threat to the aquatic environment and directly affects the condition and health of the fish. Finding a preparation that could be used both prophylactically to increase the resistance of fish and therapeutically in case of infection with pathogenic bacteria, without side effects for fish and aquatic environment could be a great solution to this problem. Our aim was to determine influence of BAFADOR® the new bacteriophage-based preparation on European eel immunity and survival after experimental challenge. Application of BAFADOR® increased total protein level, immunoglobulin level, lysozyme activity and ceruloplasmin level in European eel serum. Potential killing activity and metabolic activity of spleen phagocytes as well as pronephros lymphocyte proliferation of was higher compared to control. The preparation also reduced mortality after experimental infections with the pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. Our results showed that preparation BAFADOR® is well tolerated by the fish organism causing stimulation of cellular and humoral immunity parameters and reduces the mortality of the European eel after experimental challenge.
Recently, a rapid increase in the resistance of pathogenic bacteria to antibiotics and chemotherapeutics admitted for use in aquaculture has been observed. This happens especially often in intensive breeding. The use of drugs in closed circuits is problematic because it can damage biological filters. Therefore, in recent years, there has been a growing interest in natural methods of combating pathogens. These include bacteriophages. The aim of the study was to determine the safety of the new BAFADOR® bacteriophage-based preparation, its effect on selected immunological parameters and the effectiveness of prophylactic and therapeutic use after experimental infections with pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. The use of BAFADOR® increased the activity of lysozyme, total protein level and immunoglobulin level. The level of ceruloplasmin in the rainbow trout serum remained unchanged regardless of the route of administration of the preparation. Potential killing activity and metabolic activity of spleen phagocytes and proliferation of pronephros lymphocytes were higher compared to the control group. Both therapeutic and prophylactic application of the preparation after mixed experimental infection of A. hydrophila and P. fluorescens limited the mortality of rainbow trout.
2017
Introduction: Colibacillosis – the most common disease of poultry, is caused mainly by avian pathogenic Escherichia coli (APEC). However, thus far, no pattern to the molecular basis of the pathogenicity of these bacteria has been established beyond dispute. In this study, genomes of APEC were investigated to ascribe importance and explore the distribution of 16 genes recognised as their virulence factors.
Material and methods: A total of 14 pathogenic for poultry E. coli strains were isolated, and their DNA was sequenced, assembled de novo, and annotated. Amino acid sequences from these bacteria and an additional 16 freely available APEC amino acid sequences were analysed with the DIFFIND tool to define their virulence factors.
Results: The DIFFIND tool enabled quick, reliable, and convenient assessment of the differences between compared amino acid sequences from bacterial genomes. The presence of 16 protein sequences indicated as pathogenicity factors in poultry resulted in the generation of a heatmap which categorises genomes in terms of the existence and similarity of the analysed protein sequences.
Conclusion: The proposed method of detection of virulence factors using the capabilities of the DIFFIND tool may be useful in the analysis of similarities of E. coli and other sequences deriving from bacteria. Phylogenetic analysis resulted in reliable segregation of 30 APEC strains into five main clusters containing various virulence associated genes (VAGs).
2016
Urinary tract infections (UTIs) caused by P. mirabilis are difficult to cure because of the increasing antimicrobial resistance of these bacteria. Phage therapy is proposed as an alternative infection treatment. The aim of this study was to isolate and differentiate uropathogenic P. mirabilis strain specific polyvalent bacteriophages producing polysaccharide depolymerases (PDs). 51 specific phages were obtained. The plaques of 29 bacteriophages were surrounded by halos, which indicated that they produced PDs. The host range analysis showed that, except phages 58B and 58C, the phage host range profiles differed from each other. Phages 35 and 45 infected all P. mirabilis strains tested. Another 10 phages lysed more than 90% of isolates. Among these phages, 65A, 70, 66 and 66A caused a complete lysis of the bacterial lawn formed by 62% to 78% of strains. Additionally, phages 39A and 70 probably produced PDs. The phages’ DNA restriction fragment length polymorphism (RFLP) analysis demonstrated that genomes of 51 isolated phages represented 34 different restriction profiles. DNA of phage 58A seemed to be resistant to selected EcoRV endonuclease. The 33 RFLP-EcoRV profiles showed a Dice similarity index of 38.8%. 22 RFLP patterns were obtained from single phage isolates. The remaining 12 restriction profiles consisted of 2 to 4 viruses. The results obtained from phage characterization based on the pattern of phage host range in combination with the RFLP method enabled effective differentiation of the studied phages and selection of PD producing polyvalent phages for further study.
2015
Background: Salmonellosis is of great economic concern in all phases of the poultry industry, from production to marketing, leading to severe economic losses. Monitoring the source of the bacterial contamination has fundamental importance in the spreading of salmonellosis.
Results: We applied a ligation-mediated PCR method, PCR MP (PCR melting profile), to type S. enterica ssp. enterica ser. Enteritidis (56 strains) and 43 control strains classified to other serovars isolated from poultry. We demonstrated the PCR MP potential for salmonellosis spreading monitoring. Our rapid test presents higher discriminatory power (0.939 vs. 0.608) compared to current molecular subtyping tool such as pulsed-field gel electrophoresis (PFGE), which ineffectiveness underlies the high degree of clonality of S. Enteritidis.
Conclusions: PCR MP was found to be a highly discriminating, sensitive and specific method that could be a valuable molecular tool, particularly for analyzing epidemiological links of limited number of S. enterica ser. Enteritidis strains.