Phage & predatory bacteria Flashcards
causes and challenges of AB resistance
Overuse in humans
Inappropriate prescribing/unregulated purchase
Inadequate diagnostics
Rapid diagnostics could make therapy more targeted
Extensive agricultural use
In the US, ~80% of antibiotic use is in animals
Insufficient new antibiotics being developed
Anti-infective development is not profitable
types of new antimicrobials
Quorum sensing inhibition
Cell division inhibitors (e.g FtsZ inhibitor)
Sigma-factor inhibitors
Bacteriophage therapy
Predatory bacteria
Bacteriophages (or ‘phages’)
Can be classified according to morphology or host specificity, but also by their biological cycle:
Lytic (replication and lysing - virulent)
Lysogenic (latent - integration into the genome)
Viruses can either be “naked” or “enveloped”
Lipid bilayer of enveloped viruses is derived from the host cell membrane
Transduction is the
transfer of host genes from one cell to another by a virus
Bacteriophage play a key role in transfer of genetic material between bacterial cells
Mobilisation of antibiotic resistance genes
Cholera toxin produced by Vibrio cholerae is actually encoded by a lysogenic phage (CTX phage)
The Lytic Life Cycle of Bacteriophage T4
Following attachment of the bacteriophage to the bacterium (mediated by tail fibres), the baseplate settles down on the bacterial surface. Contraction of the sheath is accompanied by injection of the phage DNA through the bacterial cell envelope.
Within two minutes, the E. coli RNA polymerase starts synthesizing T4 mRNA – called early mRNA. This early mRNA encodes gene products involved in:
phage DNA replication, nucleases (to degrade host cell DNA) and a phage-encoded sigma factor.
Late mRNA encodes the structural proteins of the virion, and other proteins required for phage release.
Phage therapeutics
Therapeutic potential of bacteriophages was recognized soon after their discovery in the early 20th century
Decline of phage therapy in the Western world due to:
mixed therapeutic results
poor understanding of phage biology
advent of broad-spectrum antibiotics
language barriers
frequent lack of appropriate control groups within studies
Phage therapy continued to be studied in Eastern Europe & Soviet Union
Bacteriophage therapy – case study (1)
Slopek S et al., (1987). Results of bacteriophage treatment of suppurative bacterial infections in the years 1981-1986.
550 patients:
1 wk – 86 yrs of age, from 10 clinical departments/hospitals in 3 cities
Antibiotic treatment deemed ineffective in 518/550 patients
Causative agents were staphylococci, Pseudomonas, Escherichia, Klebsiella, Salmonella
92% success rate overall
No control groups in study
Bacteriophage therapy – case study (2)
Babalova EG et al., (1968). Preventative value of dried dysentery bacteriophage.
Prophylactic use of bacteriophage to protect against bacterial dysentery
30,769 patients: 6 months – 7 yrs of age
Control (placebo) groups within each street studied:
Children on one side of streets received phage (n = 17,044)
Children on other side of streets did not (n = 13,725)
Shigella phages administered orally, once-a-week; 109 day study period
The fact that there was a reduction in the number of cases of diarrhea of undetermined cause suggests that either (a) some dysentery cases were not correctly identified as such, or (b) that the phage preparation, although developed against Shigella species, was also active against other gastrointestinal pathogens.
Challenges of bacteriophage therapy
Bacteriophage have a very limited spectrum of activity
Necessitates the need for bacteriophage cocktails?
Challenge of producing & testing well-defined mixtures of phage for regulatory approval
Bacteria can become resistant to bacteriophage, as they can to antibiotics
Bacteriophage cocktails may help combat this
As live biological entities, phage can evolve as well – potentially circumventing resistance
Economical considerations
As natural entities, phages are difficult to patent
Using predatory bacteria to fight infections
Bdellovibrio bacteriovorus is a Gram-negative bacterial species that is a predator of other Gram-negative bacteria
while in attack phase, bacteria collide with prey, and form a temporary attachment to the prey cell. They then enter the prey cell through the outer membrane, gaining access to the periplasm. Within the periplasm, the Bdellovibrio modifies the linkage of the peptidoglycan to make it more flexible to accommodate its growth. The resulting structure is referred to as a bdelloplast – essentially, a rounded-up prey cell containing a periplasmic Bdellovibrio.
Bdellovibrio-produced hydrolytic enzymes bring about the hydrolysis of macromolecules of the prey cell, supporting Bdellovibrio growth. This growth is filamentous, and when bdelloplast resources are exhausted, this filamentous cell septates to produce multiple new motile progeny that lyse the bdelloplast and re-commence the predation cycle on other cells. This septation is a rare example of ‘multiple fission’ rather than ‘binary fission’
Zebrafish embryo infection model
Injected ZFEs into the hindbrain ventricle to establish localized infection with shigella
Bdellovibrio controlled Shigella numbers in vivo and conferred partial protection from lethal Shigella infection (increased survival by ~35%)
Combination of bacterial predation and host immunity was required for maximal beneficial effect
Bdellovibrio itself is non-pathogenic to ZFEs and is self-limiting - because it has no prey bacteria on which to feed, and so there is no replication cycle. Additionally, the authors showed that Bdellovibrio is ingested by macrophages and neutrophils of the ZFE.
Phage extra reading
In contrast to their ‘forgotten’ application as therapeutic
tools, phages were used – mainly throughout the second half of last century – as key reagents in order to understand the basic principles of molecular biology. Several biotechnological applications are also phage-derived. These include phage display, production of recombinant antibodies, gene therapy and drug delivery.
Predatory bacteria such as Bdellovibrio bacteriovorus or Micavibrio aeruginosavorus, antimicrobial peptides isolated from frogs, alligators and cobras that destroy bacteria or metals like copper and silver are some of the most interesting new lines of investigation
In mammals, many phages are found on skin surfaces, mucous membranes and the digestive tract. The human gut is inhabited by a high number of bacterial
cells representing thousands of strains and a large number of harmless bacteriophages. This suggests that phage therapy using natural viruses could be, in theory, safe. Moreover, bacteriophages infect and destroy only a narrow range of bacteria, if not single species, in opposite to large spectrum antibiotics.
This represents a positive argument with regards to the specificity of the agent to be targeted and the overall maintenance of the host’s natural microbiota
The recent development of phage expressing an RNA-guided nuclease Cas9 enables sequence-specific targeting of a bacterial strain. This design of such phages, which have been dubbed ‘intelligent antibiotics’, now offers the possibility to precisely manipulate the microbiota
Interestingly, broad-host range phages have been reported as for instance in the case of Staphylococcus
aureus or diseases that are linked to a specific pathogen
such as dysentery which is associated to a specific sub-group of Shigella. Because phages need a specific host to replicate, most of them are cleared quickly from the body if they do not find their cognate bacterial host, meaning that
(i) the administration route should be of special interest and
(ii) once the infection is treated, phages will be naturally cleared without secondary effects for the host.
The occurrence of antibiotic-resistant mutations favors the idea that bacteria could also develop resistance against phages. But unlike antibiotics, phages can evolve as well (even with higher substitution rates than bacteria), meaning that bacterial resistance should not be a major problem impairing phage therapy
Phages are easy to isolate and cheap and quick to produce. Furthermore, phages replicate only in the
presence of their specific host, meaning that they proliferate according to the bacterial (infectious) load after administration. Together with the fact that phages can be stable in various environmental conditions (equivalent to those tolerated by bacterial spores), makes them interesting treatments against bacterial infections, especially in developing countries.
However, phages are natural entities making them difficult to patent, which can represent a hurdle for their marketing as treatment against bacterial infections. For these reasons, pharmaceutical companies, with few exceptions, have shown limited interest for phage therapy, which, combined to the lack of clarity about regulatory issues concerning their use, makes funding difficult to gather for this type of research
clinical trials, such as the Phagoburn project, which is the first large, multi-center clinical trial of phage therapy for human infections funded by the European Commission have been recently set-up. This project is dedicated to the elaboration of a treatment with phage cocktails obtained from sewage or river water to treat burned patients infected by E coli and P aeruginosa. Up to now, phages cocktails are approved for the US Food and Drug Administration in the food processing industry. These preparations are accepted for ready-to-eat meat and poultry products as antibacterial food additives. For example, a commercial product of phages targeting the foodborne pathogen Listeria sp. is used in dairy industry and in aquaculture to reduce the intensive use of antibiotics
Finally, phages can be relevant in the treatment of biofilms. Indeed some bacteria are able to create biofilms in which antibiotics are inefficacious even against genetically sensitive targets. Phages have been reported to disrupt biofilms and to kill bacteria present inside the structure, as a result of active penetration into the bacterial biofilm. Anti-biofilm activity of phages is of special interest in hospitals, where catheters or other medical devices can be infected. This biocontrol can be used prior to biofilm formation, to already formed biofilms or synergistically with additional disruptive mechanisms.
