Phage-resistant Bacterial Mutants Research Articles

Application of a novel lytic Jerseyvirus phage LPSent1 for the biological control of the multidrug-resistant Salmonella Enteritidis in foods.

Non-typhoidal Salmonella is the tremendously predominant source of acquired foodborne infection in humans, causing salmonellosis which is a global threat to the healthcare system. This threat is even worse when it is combined with the incidence of multidrug-resistant Salmonella strains. Bacteriophage therapy has been proposed as a promising potential candidate to control a diversity of foodborne infective bacteria. The objective of this study designed to isolate and characterize lytic phages infecting zoonotic multi-drug resistant and strong biofilm producer Salmonella enterica serovar Enteritidis EG.SmE1 and then apply the isolated phage/s as a biocontrol agent against infections in ready-to-eat food articles including milk, water, apple juice, and chicken breasts. One lytic phage (LPSent1) was selected based on its robust and stable lytic activity. Phage LPSent1 belonged to the genus Jerseyvirus within the Jerseyvirinae subfamily. The lysis time of phage LPSent1 was 60 min with a latent period of 30 min and each infected cell burst about 112 plaque-forming units. Phage LPSent1 showed a narrow host range. Furthermore, the LPSent1 genome did not encode any virulence or lysogenic genes. In addition, phage LPSent1 had wide pH tolerance, prolonged thermal stability, and was stable in food articles lacking its susceptible host for 48 h. In vitro applications of phage LPSent1 inhibited free planktonic cells and biofilms of Salmonella Enteritidis EG.SmE1 with a lower occurrence to form phage-resistant bacterial mutants which suggests promising applications on food articles. Application of phage LPSent1 at multiplicities of infections of 100 or 1000 showed significant inhibition in the bacterial count of Salmonella Enteritidis EG.SmE1 by 5 log10/sample in milk, water, apple juice, and chicken breasts at either 4°C or 25°C. Accordingly, taken together these findings establish phage LPSent1 as an effective, promising candidate for the biocontrol of MDR Salmonella Enteritidis in ready-to-eat food.

Developing and Characterizing Bacteriophage for Therapeutic Use Against Carbapenem‐Resistant Enterobacteriaceae in a Multiple Drug Resistant Klebsiella pneumoniae Model

Carbapenem‐resistant Enterobacteriaceae (CRE) are a group of Gram‐negative bacterial pathogens which carry resistance to a large proportion of available antibiotics and are a significant cause of nosocomial infection in the United States. Within this group, Klebsiella pneumoniae carrying the blaKPC carbapenemase gene (KPC+) are of particular concern in the hospital setting among immunocompromised patients, and strains of sequence type (ST) 258 are prevalent in the US, South America and Europe. Bacteriophages, bacterial viruses, have been proposed as a potential alternative form of treatment for infections caused by multidrug‐resistant pathogens, including KPC+ K. pneumoniae. Bacteriophage therapy has many appealing qualities including ease of isolation, specificity, and a low production cost. One limitation, however, is the quick appearance of bacteriophage resistant bacterial mutants. In this study, we examine the genetic modifications responsible for bacteriophage resistance in several ST 258 K. pneumoniae strains and investigate how bacteriophage resistance might be overcome in a model of tri‐bacteriophage combination therapy for K. pneumoniae. Identification, evaluation and characterization of candidate phages was based on in vitro virulence assays, whole genome sequencing, complementation, transmission electron microscopy and traditional plaque assays. Isolation of 27 bacteriophages from environmental sources resulted in 9 bacteriophages (primary phages) which infect the ST258 KPC+ K. pneumoniae model strain 39827. Further isolation of phages specific for “primary” phage resistant bacterial mutants yielded “secondary” phages and subsequent double phage resistant mutants. Whole genome sequencing, SNP and INDEL identification, and complementation of these in vitro generated bacteriophage resistant K. pneumoniae strains characterizes the bacterial capsule as a probable common primary phage receptor in ST258 KPC+ K. pneumoniae and mutational loss of this primary receptor exposes outer membrane proteins and lipopolysaccharide (LPS) as secondary phage receptors. A tri‐phage cocktail representing multiple receptor types was more effective at suppressing growth of the wild‐type KPC+ K. pneumoniae host strain than a single phage in vitro. Additionally, single and double phage resistant bacterial mutants exhibited growth defects, suggesting a loss of fitness associated with phage resistance mutations. In an in vivo murine model of GI decolonization the tri‐phage cocktail was able to lower bacterial K. pneumoniae concentrations in both fecal and cecal samples by 1–2 log units. These data indicate a promising treatment for K. pneumoniae using genomics to overcome bacteriophage resistance and increase the effectiveness of bacteriophage therapy.Support or Funding InformationFunding for this project is generously provided by NIAID Project R33‐AI121689

Designing phagebodies.

A study reports the generation of synthetic ‘phagebodies’ with a broadened host range that were able to target naturally occurring phage-resistant bacterial mutants.

Phage Reduce Stability for Regaining Infectivity during Antagonistic Coevolution with Host Bacterium.

The coevolution between phage and host bacterium is an important force that drives the evolution of the microbial community, yet the coevolution mechanisms have still not been well analyzed. Here, by analyzing the interaction between a Bacillus phage vB_BthS_BMBphi and its host bacterium, the coevolution mechanisms of the first-generation phage-resistant bacterial mutants and regained-infectivity phage mutants were studied. The phage-resistant bacterial mutants showed several conserved mutations as a potential reason for acquiring phage resistance, including the mutation in flagellum synthesis protein FlhA and cell wall polysaccharide synthesis protein DltC. All the phage-resistant bacterial mutants showed a deleted first transmembrane domain of the flagellum synthesis protein FlhA. Meanwhile, the regain-infectivity phage mutants all contained mutations in three baseplate-associated phage tail proteins by one nucleotide, respectively. A polymorphism analysis of the three mutant nucleotides in the wild-type phage revealed that the mutations existed before the interaction of the phage and the bacterium, while the wild-type phage could not infect the phage-resistant bacterial mutants, which might be because the synchronized mutations of the three nucleotides were essential for regaining infectivity. This study for the first time revealed that the synergism mutation of three phage baseplate-associated proteins were essential for the phages’ regained infectivity. Although the phage mutants regained infectivity, their storage stability was decreased and the infectivity against the phage-resistant bacterial mutants was reduced, suggesting the phage realized the continuation of the species by way of “dying to survive”.

Reviving Phage Therapy for the Treatment of Cholera.

Cholera remains a major risk in developing countries, particularly after natural or man-made disasters. Vibrio cholerae El Tor is the most important cause of these outbreaks, and is becoming increasingly resistant to antibiotics, so alternative therapies are urgently needed. In this study, a single bacteriophage, Phi_1, was used to control cholera prophylactically and therapeutically in an infant rabbit model. In both cases, phage-treated animals showed no clinical signs of disease, compared with 69% of untreated control animals. Bacterial counts in the intestines of phage-treated animals were reduced by up to 4 log10 colony-forming units/g. There was evidence of phage multiplication only in animals that received a V. cholerae challenge. No phage-resistant bacterial mutants were isolated from the animals, despite extensive searching. This is the first evidence that a single phage could be effective in the treatment of cholera, without detectable levels of resistance. Clinical trials in human patients should be considered.

Psychrotrophic Bacteriophages for Beef Spoilage Pseudomonads

A total of 40 beef spoilage pseudomonads was used as bacterial hosts for the isolation of psychrotrophic bacteriophages (phages) from spoiled rib steaks. Thirty-eight homologous phages, lytic for 25 of these hosts, were isolated and purified. An additional 12 bacterial isolates were susceptible to heterologous phage lysis and only three of the bacteria examined were resistant to lysis by any of the phage tested. On the basis of heterologous cross-sensitivity to phages, the meatborne Pseudomonas strains and 4 identified ATCC Pseudomonas hosts were differentiated into 37 distinct phage lysotypes. In addition, Pseudomonas phages were found to inhibit bacterial growth in tryptic soy broth by significantly extending the lag phase under psychrotrophic conditions (7°C). However, the incubation of bacteria with phages resulted in the selection of phage resistant bacterial mutants.

Resistance relationships in Mycobacterium smegmatis ATCC 607 to phages sensitive or resistant to both chloroform and streptomycin sulphate.

Four of eight mycobacteriophages did not form plaques after they were exposed to chloroform. Phages sensitive to chloroform did not produce plaques when plated on media containing 1000 microgram/ml of streptomycin sulphate. The same concentration of dihydrostreptomycin sulphate did not interfere with plaque formation. Mutants of Mycobacterium smegmatis resistant to each of the eight phages were isolated. Sensitivity or resistance to chloroform and streptomycin sulphate and phage resistant bacterial mutants may provide a basis for classifying the mycobacteriophages.

A STUDY OF THE DISTRIBUTION AND THE EFFECTS OF BACTERIOPHAGE OF ROOT NODULE BACTERIA IN THE SOIL

Bacteriophage for clover nodule bacteria can be found on roots and nodules of all naturally grown clover plants and also in the soil surrounding the roots, but not in soil without clover plants. Alternative hosts for the phage of clover bacteria are pea bacteria, and vice versa. The bacteria and the phage are heterogeneous in the sense that only a proportion of strains of clover bacteria and of pea bacteria are susceptible to lysis by a given race of phage, and only a proportion of races of phage can lyse a given bacterial strain. There does not seem to be any association between the susceptibility of bacterial strains to lysis by phage and any other features such as antigenic structure and effectiveness in nitrogen fixation. There may be an association with avirulence, i.e. inability to infect the host plant. The behavior of phage–bacterial mixtures depends on the surrounding medium. The longevity of phage in soil or in a soil-like medium such as a vermiculite mixture is relatively short, and the effect of phage can be localized so that phage-susceptible bacteria and the phage can exist close to each other without any apparent interaction. However, as long as the phage is present, phage-resistant bacterial mutants are usually present also. The phage-resistant mutants may also be mutants in other respects such as effectiveness in nitrogen fixation. In the presence of weakened phage, bacterial mutants were found to occur that differ from the parent form in effectiveness but resemble it in susceptibility to the phage.