Host-specific Bacteriophages Research Articles

FENko-Kae01 is a flagellum-specific jumbo phage infecting Klebsiella aerogenes

BackgroundKlebsiella aerogenes is an opportunistic pathogen that causes a wide variety of infections. Due to the rising problem of antibiotic resistance, novel antibiotics and strategies to combat bacterial infections are needed. Host-specific bacteriophages are natural enemies of bacteria and can be used in phage therapy as an alternative form of treatment against bacterial infections. Jumbo phages are defined as phages with genomes larger than 200 kb. Relatively few studies have been done on jumbo phages compared to smaller phages.ResultsA novel phage, fENko-Kae01, was isolated from a commercial phage cocktail. Genomic analysis revealed that fENko-Kae01 is a lytic jumbo phage with a 360 kb genome encoding 578 predicted genes. No highly similar phage genomes were identified and fENko-Kae01 may be a completely new genus representative. No known genes associated with lysogenic life cycle, bacterial virulence, or antibiotic resistance were identified. The phage had myovirus morphology and a narrow host range. Phage resistant bacterial mutants emerged under phage selection. Whole genome sequencing revealed that the biogenesis of the flagellum was affected in four mutants and the lack of functional flagellum was confirmed in motility assays. Furthermore, phage fENKo-Kae01 failed to adsorb on the non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor.ConclusionsfENko-Kae01 is a novel jumbo bacteriophage that is considered safe for phage therapy. fENko-Kae01 uses the flagellum as the phage-binding receptor and may represent a completely novel genus.

Impact of effluent parameters and vancomycin concentration on vancomycin resistant Escherichia coli and its host specific bacteriophage lytic activity in hospital effluent

Vancomycin resistance in bacteria has been classified under high priority category by World Health Organization (WHO) and its presence in hospital effluent is reported to be increasing owing to excess antibiotics use. Among various strategies, bacteriophage has been recently considered as a promising biological agent for combating such antimicrobial resistant bacteria (ARB). However, the influence of effluent's properties on phage-ARB interaction in actual hospital effluent is not completely understood. The present works intends to study this influence of hospital effluent and its parameters on the interaction between vancomycin resistant E. coli (VRE) and its host specific bacteriophage. The isolated VRE was identified by 16S rRNA sequencing, matrix-assisted laser desorption/ionization–time of flight (MALDI – TOF) and whole genome sequencing. The infectivity of phage onto host bacteria was investigated using electron microscopic techniques, dynamic light scattering (DLS), spectrofluorophotometer and confirmed using double agar overlay method. The monovalency and polyvalency of isolated phage against various bacterial species were determined. The phage morphology was identical to T7 phage belonging to Podoviridae. The phage lysis was maximum at pH 7 (90.2%), 37 °C (91.6%) and vancomycin concentration of 50 μg/mL in both synthetic media (89.13%) and effluent (100%). At a maximum vancomycin concentration of 100 μg/mL, decrease in Ca, K, Mg and P (up to 19.70, 14.18, 28, and 15.82% respectively) concentration in effluent was observed due to phage infectivity when compared to control. The whole genome sequencing was performed and the bioinformatics analysis presented the role of mdfA gene encoding the efflux pump in causing vancomycin resistance in E. coli. It also depicted the presence of multiple genes responsible for mercury, cobalt, zinc and cadmium resistance in VRE. These results clearly indicate that bacteriophage mediated combating of VRE is possible in actual hospital effluent and can be used as one of the treatment methods.

History of Discovery and Environmental Role of Ice Nucleating Bacteria.

The phenomenon of biological ice nucleation that is exhibited by a variety of bacteria is a fascinating phenotype, which has been shown to incite frost damage to frost-sensitive plants and has been proposed to contribute to atmospheric processes that affect the water cycle and earth's radiation balance. This review explores the several possible drivers for the evolutionary origin of the ice nucleation phenotype. These bacteria and the gene required for this phenotype have also been exploited in processes as diverse as reporter gene assays to assess environmentally responsive gene expression in various plant pathogenic and environmental bacteria and in the detection of foodborne human pathogens when coupled with host-specific bacteriophage, whereas ice nucleating bacteria themselves have been exploited in the production of artificial snow for recreation and oil exploration and in the process of freezing of various food products. This review also examines the historical development of our understanding of ice nucleating bacteria, details of the genetic determinants of ice nucleation, and features of the aggregates of membrane-bound ice nucleation protein necessary for catalyzing ice. Lastly, this review also explores the role of these bacteria in limiting the supercooling ability of plants and the strategies and limitations of avoiding plant frost damage by managing these bacterial populations by bactericides, antagonistic bacteria, or cultural control strategies.

Simple Two‐step, High Yield Protocol for Isolation and Amplification of Bacteriophages Against Methicillin‐resistant Staphylococcus Aureus (MRSA)

Bacteriophages are bacteria-targeting viruses that may prove useful as therapeutic agents against multidrug-resistant bacterial strains. Though phage therapy is a century-old concept, there is very limited progress on its therapeutic application due to the rapid expansion of antibiotics portfolios in the last few decades. However, the emergence of multidrug-resistant organisms has brought our attention back to bacteriophages. The first step towards developing effective phage therapy against multidrug-resistant bacteria is isolation, amplification, and purification of specific bacteriophages. There are many reported protocols for isolating host-specific bacteriophages from the environment. However, most of them are complex, multistep, low-yielding, resource-intensive protocols, requiring elaborate laboratory setup. We have demonstrated a simple two-step, high-yielding protocol for isolating and amplifying bacteriophages against methicillin-resistant Staphylococcus aureus (MRSA). We have shown that mixing various environmental samples (i.e., sample pooling) and phage amplification at two different temperatures significantly enhance the yield of MRSA phages. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of water sample filtrate for isolation of bacteriophages Basic Protocol 2: Bacterial strain and culture conditions Basic Protocol 3: Native bacteriophage count in water sample filtrate Basic Protocol 4: Isolation and enrichment of MRSA-specific bacteriophages Basic Protocol 5: Quantification of bacteriophages by drop cast method Basic Protocol 6: Effect of incubation temperature and heat shock on bacteriophage yield.

Modeling human pollution in water bodies using somatic coliphages and bacteriophages that infect Bacteroides thetaiotaomicron strain GA17

The ability to detect human fecal pollution in water is of great importance when assessing the associated health risks. Many microbial source tracking (MST) markers have been proposed to determine the origin of fecal pollution, but their application remains challenging. A range of factors, not yet sufficiently analyzed, may affect MST markers in the environment, such as dilution and inactivation processes. In this work, a statistical framework based on Monte Carlo simulations and non-linear regression was used to develop a classification procedure for use in MST studies. The predictive model tested uses only two parameters: somatic coliphages (SOMCPH), as an index of general fecal pollution, and human host-specific bacteriophages that infect Bacteroides thetaiotaomicron strain GA17 (GA17PH). Taking into account bacteriophage dilution and differential inactivation, the threshold concentration of SOMCPH was calculated to be around 500 PFU/100 mL for a limit of detection of 10 PFU/100 mL. However, this threshold can be lowered by increasing the analyzed volume sample, which in turn lowers the limit of detection. The resulting model is sufficiently accurate for application in practical cases involving MST and could be easily used with markers other than those tested here.

Comparing the Efficacy of Bacteriophages and Antibiotics in Treating Salmonella enterica Serovar Typhimurium on Streptomycin-pretreated Mice

The misuse of antibiotics had contributed significantly to the development of antibiotic resistance, hence decreasing its efficacy. Non-typhoidal Salmonella is a global problem causing gastroenteritis in humans. Since there are various antibiotics in treating Salmonella infections, its potential to become multidrug-resistant remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. Isolated and purified from sewage water, a dose of 4.30 x 1010 PFU (plaque-forming units) Salmonella phages were used to treat gastroenteritis on streptomycin-mouse models. Lack of treatment yielded 2.06 x 106 CFU (colony-forming units)/g of Salmonella 24 h post-infection, which exponentially dropped to 3.80 x 103 CFU/g 24 h post-infection in bacteriophage treatment before gradually decreasing. However, the antibiotic treatment inhibited growth with 3.58 x 102 CFU/g 24 h post-infection, but it did not retain the decrease of the pathogen. Results also showed that the microflora of the gut was preserved with the appearance of various colonies in phage treatment, which aided recovery in a phenomenon known as “microbial interference.” In contrast, antibiotic treatment depleted the normal microflora, allowing colonization of Salmonella and resurgence of pathogenic growth in the gut. Bacteriophage therapy demonstrated a significant decrease in bacterial load when compared to the antibiotic ciprofloxacin. The high specificity of the treatment also allowed the phages to synergize with the gut microbiota, thereby inhibiting colonization and further increase in bacterial growth.

Cell Wall Lytic Enzymes And Their Role In Bacteriophages Infection

Use of chemical pesticides has been shown to have many negative side effects, such as insecticide resistance and resurgence, an outbreak of secondary pests and diseases, the disappearance of parasitoid and predator species as well as residual effects on food crops and on the environment. Over the past 60 years, both the number of agricultural toxins in the environment and incidence rates of toxin-related diseases has increased dramatically. The most effective way to combat this problem is through the use of natural predators. One of the best examples of this is the use of host-specific bacteriophages to control bacterial diseases. The mechanism of infection is a very interesting one. To break through the bacterial cell wall the bacteriophages must produce a range of lytic enzymes. This review will examine and discuss studies of these site-specific cell wall lytic enzymes and their roles in the infection of bacteriophages.

Characteristics of and variation in airborne ARGs among urban hospitals and adjacent urban and suburban communities: A metagenomic approach

Environmental antibiotic resistance genes (ARGs) have received much attention, while the characteristics of ARGs carried by particulate matter (PM) as a function of urban functional region are almost unknown. In this study, ARGs carried by PM2.5 and PM10 in an urban hospital, a nearby urban community and the nearest suburban community were detected using metagenomics. In total, 643 ARG subtypes belonging to 22 different ARG types were identified. The chloramphenicol exporter gene, sul1, bacA, and lnuA were the most abundant ARG subtypes in all air samples. The hospital exhibited higher ARG abundance and richness than the nearby communities. ARG profiles depended on functional region: hospital and suburban samples clustered separately, and samples from the nearby urban community interspersed among them. The representation of multidrug and quinolone resistance genes decayed with distance from the hospital to the urban community to the suburban community, indicating that hospital PM may be a hotspot for ARGs encoding proteins conferring multidrug and quinolone resistance. Airborne ARGs carried by PM in the hospital environment were more closely associated with clinically important pathogens than were those in nearby communities. In particular, carbapenemase genes, including blaNDM,blaKPC,blaIMP,blaVIM,and blaOXA-48, were discovered in hospital PM. In the suburban community, crAssphage, a human host-specific bacteriophage, was applied to predict ARG abundance and found to be enriched due to anthropogenic pollution but showed no clear evidence for ARG selection. In the hospital and the nearby urban community, the drivers of ARGs were complex. Our results highlighted that PM ARGs were closely related to human activities and revealed a potential hotspot, which could provide new evidence for further research and consequently mitigate the formation of airborne ARGs and transfer risks.

Improving the identification of the source of faecal pollution in water using a modelling approach: From multi-source to aged and diluted samples

The last decades have seen the development of several source tracking (ST) markers to determine the source of pollution in water, but none of them show 100% specificity and sensitivity. Thus, a combination of several markers might provide a more accurate classification. In this study Ichnaea® software was improved to generate predictive models, taking into account ST marker decay rates and dilution factors to reflect the complexity of ecosystems. A total of 106 samples from 4 sources were collected in 5 European regions and 30 faecal indicators and ST markers were evaluated, including E. coli, enterococci, clostridia, bifidobacteria, somatic coliphages, host-specific bacteria, human viruses, host mitochondrial DNA, host-specific bacteriophages and artificial sweeteners. Models based on linear discriminant analysis (LDA) able to distinguish between human and non-human faecal pollution and identify faecal pollution of several origins were developed and tested with 36 additional laboratory-made samples. Almost all the ST markers showed the potential to correctly target their host in the 5 areas, although some were equivalent and redundant. The LDA-based models developed with fresh faecal samples were able to differentiate between human and non-human pollution with 98.1% accuracy in leave-one-out cross-validation (LOOCV) when using 2 molecular human ST markers (HF183 and HMBif), whereas 3 variables resulted in 100% correct classification. With 5 variables the model correctly classified all the fresh faecal samples from 4 different sources. Ichnaea® is a machine-learning software developed to improve the classification of the faecal pollution source in water, including in complex samples. In this project the models were developed using samples from a broad geographical area, but they can be tailored to determine the source of faecal pollution for any user.

Isolation and Enrichment of Bacteriophages by Membrane Filtration Immobilization Technique

The method described here enables rapid bacteriophage isolation and enrichment of host-specific bacteriophages from an environmental sample. This is achieved by using a simple 0.45-µm Millipore membrane where a specific host is immobilized on the membrane and a sample suspected of containing bacteriophages is exposed to the immobilized cells with the help of a membrane filtration unit. This filtration step facilitates host-specific interaction of bacteriophages with the host and maximization of this interaction using a classic membrane filtration method. Under the effect of vacuum from a vacuum pump, a filter assembly provides a chance for every bacteriophage in the sample to interact with the specific host on the membrane filter. Our technique allows retaining specific bacteriophages on the membrane along with its host cells via adsorption; these adsorbed bacteriophages (along with their hosts) on a filter disc are then enriched in regular nutritive broth, tryptone soya broth (TSB), by incubation. With help of a plaque assay method, host-specific phages of various bacterial species can be isolated, segregated, and enriched. © 2018 by John Wiley & Sons, Inc.

Membrane filtration immobilization technique-a simple and novel method for primary isolation and enrichment of bacteriophages.

To develop a method for the isolation and enrichment of bacteriophages selectively against specific bacteria coupled with a membrane filtration technique. Rapid isolation and concentration of host-specific bacteriophages was achieved by exposure of the sample suspected to contain bacteriophages to a specific host immobilized on a 0·45μm membrane in a membrane filtration unit. The principle behind this method is the exploitation of host-specific interaction of bacteriophages with their host and maximizing this interaction using a classic membrane filtration method. This provides a chance for each bacteriophage in the sample to interact with the specific host on the membrane filter fitted with a vacuum pump. Specific bacteriophages of the host are retained on the membrane along with its host cells due to the effect of adsorption and these adsorbed bacteriophages (along with their hosts) on the filter disc are then amplified and enriched in regular nutritive broth tryptose soya broth by incubation. With the help of the plaque assay method, host-specific phages of various bacterial species were isolated, segregated and enriched. The phage concentration method coupled with membrane filtration immobilization of host bacteria was able to isolate and enrich the host-specific bacteriophages by several fold using a lower quantity of an environmental water sample, or other phage suspensions. Enrichment of phages from single plaques was also achieved. The isolation and detection of host-specific bacteriophages from a low density bacteriophage water sample in a single step by the use of a simple and basic microbiological technique can be achieved. Enrichment of phages from low phage titre suspensions is also achieved very effectively.

Biocontrol of Salmonella Enteritidis in spiked chicken cuts by lytic bacteriophages ΦSP-1 and ΦSP-3.

The ability of host specific bacteriophages ΦSP-1 and ΦSP-3 to lyse Salmonella in artificially contaminated cuts of pressure cooked chicken meat was evaluated at different temperatures -4 °C, room temperature (28 ± 0.5 °C) and 37 °C applying low and high multiplicity of infection (MOI). Bacteriophages were able to significantly reduce the bacterial counts at all the temperatures studied. At 4 °C, individual application of Φ SP-1 and Φ SP-3 resulted in significant drop in bacterial counts (log10 2.46 and 2.1 CFU/ml, respectively) at high MOI and (log10 0.98 and 0.52 CFU/ml, respectively) at low MOI, when compared to the untreated control on day 3. Similarly at room temperature the drop was log10 3.99 and 3.46 CFU/ml at high MOI and log10 2.51 and 2.3 CFU/ml at low MOI. At 37 °C the drop was log10 1.98 and 2.38 at high MOI and at low MOI it was log10 1.52 and 1.98 CFU/ml. Increased efficiency was observed when phages where applied as cocktail at high MOI as the bacterial counts at the end of day 3 dropped by log10 3.52 CFU/ml at 37 °C and to beyond detectable level at 4 °C and room temperature. The average reduction of bacterial load in the same group was -4 °C (79%), room temperature (92%) and 37 °C (78%).

Isolation of Host-Specific Bacteriophages from Sewage Against Human Pathogens

Isolation of Host-Specific Bacteriophages from Sewage Against Human Pathogens

Efficient point mutagenesis in mycobacteria using single‐stranded DNA recombineering: characterization of antimycobacterial drug targets

Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis. We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide-derived single-stranded DNA recombineering and mycobacteriophage-encoded proteins. Phage Che9c gp61-mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10,000-fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage lambda Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host-specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin.

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by > or = 4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by > or = 2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.

High-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes

With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an approximately 100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.

Management of Tomato Bacterial Spot in the Field by Foliar Applications of Bacteriophages and SAR Inducers.

Various combinations of the harpin protein, acibenzolar-S-methyl, and bacteriophages were compared for controlling tomato bacterial spot in field experiments. Harpin protein and aciben-zolar-S-methyl were applied every 14 days beginning twice before transplanting and then an additional four applications throughout the season. Formulated bacteriophages were applied prior to inoculation followed by twice a week at dusk. A standard bactericide treatment, consisting of copper hydroxide plus mancozeb, was applied once prior to inoculation and then every 7 days, while untreated plants served as an untreated control. Experiments were conducted in north and central Florida fields during fall 2001, spring 2002, and fall 2002. In three consecutive seasons, acibenzolar-S-methyl applied in combination with bacteriophage or bacteriophage and harpin significantly reduced bacterial spot compared with the other treatments. However, it did not significantly affect the total yield compared with the standard or untreated control. Application of host-specific bacteriophages was effective against the bacterial spot pathogen in all three experiments, providing better disease control than copper-mancozeb or untreated control. When results of the disease severity assessments or harvested yield from the bacteriophage-treated plots were grouped and compared with the results of the corresponding nonbacteriophage group, the former provided significantly better disease control and yield of total marketable fruit.

Application of host-specific bacteriophages to the surface of chicken skin leads to a reduction in recovery of Campylobacter jejuni.

Retail poultry products are widely purported as the major infection vehicle for human campylobacteriosis. Numerous intervention strategies have sought to reduce Campylobacter contamination on broiler carcasses in the abattoir. This study reports the efficacy of bacteriophage in reducing the number of recoverable Campylobacter jejuni cells on artificially contaminated chicken skin.

A simple method for comparison of bacterial populations in a large number of samples by phage technique

A simple method for the comparison of bacterial populations from a large number of samples using the host-specific bacteriophage was described. Xanthomouas oryzae No. 60 and its specific phage were used for this purpose. Samples of bacteria are suspended in Ca Vf Ch medium or potato semi-synthetic medium, and added with phage suspensions to make the final concentration of 103-5 per ml. The samples were then incubated under optimum temperatures and under shaking at 80 rpm to propagate the added phage. Three hours later, aliquots were pipetted out from the centrifuged (6000 rpm, 5min.) supernatant and plated with the indicator strain of bacteria, to make plaque countings. It was confirmed that the number of plaques are usually proportional to the number of bacteia initially contained in the sample. This method will be favorably applied for studies on plant resistance against bacterial diseases, effects of bacteriocides, and other subjects, where problems may be dissolved on the basis of bacterial number contained in the plant tissues.