Lecture 10 - Novel Antimicrobial Strategies Flashcards
The challenge of antibiotic resistance
Currently 700,000 people die of resistant infections every year, and it has been estimated that by 2050, 10 million lives will be at risk each year due to drug-resistant infections
Causes and challenges include:
Overuse in humans
Inappropriate prescribing; unregulated purchase (incl. online)
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
The quest for new antimicrobial strategies
Novel antimicrobial targets have been identified through the study of bacterial physiology and virulence:
Quorum sensing inhibition
Cell division inhibitors (e.g FtsZ inhibitor)
Sigma-factor inhibitors
Bacteriophage therapy
Predatory bacteria
Bacteriophages (or ‘phages’)
Viruses that infect bacteria
Believed to be the most abundant biological entity on the planet
Found in all ecosystems
Estimated 107 phages per mL seawater and 109 in sediments
Human gut estimated to contain approx. 100 phage species, totalling 1015 phage particles
Can be classified according to morphology or host specificity, but also by their biological cycle:
Lytic
Lysogenic
Viral structure
Viral particles are structurally diverse but do share common features:
Nucleic acid component is always located within the particle, surrounded by the capsid (a protein coat)
Capsid is composed of a number of individual protein molecules, arranged in a repetitive pattern around the DNA/RNA
Complex of nucleic acid and protein coat is referred to as the nucleocapsid
Viruses can either be “naked” or “enveloped”
Lipid bilayer of enveloped viruses is derived from the host cell membrane
Bacteriophages – lytic vs. lysogenic
Bacteriophage (also called phage) can be lytic or lysogenic.
In the lytic mode, the viruses multiply inside the bacterial host cell, with the release of the assembled phage particles causing lysis of the bacterial cell.
In contrast, in the lysogenic mode, phages integrate their genome into the bacterial chromosome, where it remains latent. The phage genome is therefore replicated as part of the normal process of replication of the bacterial chromosome. Viral particles are not produced, and the host cell comes to no harm. Bacterial cells that harbour temperate phage are called lysogens. Under certain stressful conditions, temperate viruses may revert to the lytic pathway and produce virions, which will then result in lysis of the host cell.
The roles of lysogenic bacteriophage
Bacteriophage play a key role in transfer of genetic material between bacterial cells
Transduction – the transfer of host genes from one cell to another by a virus
Clearly, if mediated by a lysogenic bacteriophage, such genetic material will be stably inserted into the genome of the new host
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.
Bacteriophage therapy
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
Non-clinical applications of bacteriophage
In the Western world, many companies & researchers pursued non-clinical uses of bacteriophage, including food safety, agricultural, industrial and clinical diagnostic applications
ListShieldTM and LISTEXTM P100
Phage cocktails targeted at Listeria monocytogenes; designed as sterilising agents when processing foods, particularly meat, poultry & dairy products
Omnilytics’ AgriphageTM
Used to treat crop pathogens including Xanthomonas campestris and Pseudomonas syringae
Bacteriophage therapy – case study (1)
Slopek S et al., (1987). Results of bacteriophage treatment of suppurative bacterial infections in the years 1981-1986.
Arch Immunol Ther Exp 35(5): 569-83.
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
Causative agents were isolated, and a panel of 250 lytic phages were screened for activity against them
Dosing:
Administered orally (using baking soda to neutralise gastric acid), locally via phage-containing dressings, or via drops (eye, ear, nasal)
Treatment lasted 1-16 weeks
Results:
Success deemed as complete patient recovery with negative cultures:
92% success rate overall; 94% success when considering those 518 patients for whom antibiotic therapy was ineffective
No control groups in study
Bacteriophage therapy – case study (2)
Babalova EG et al., (1968). Preventative value of dried dysentery bacteriophage.
Zh Mikrobiol. Epidemiol. Immunobiol. 2: 143-145.
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
Along the x-axis of the graph:
“Dysentery” refers to the number of cases of clinical dysentery
“Culture-confirmed” refers to the number of cases of dysentery which were confirmed by culture of Shigella spp.
“Diarrhea” refers to the number of cases of diarrhea of undetermined cause.
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
The Phagoburn project
“The first international clinical study on phages in the world that meets international standards in clinical evaluation”
EU-funded project (France, Belgium & Switzerland)
Phase I/II trial aimed to evaluate tolerance & effectiveness of two phage treatments in 220 burn patients (P. aeruginosa and E. coli)
Aimed to compare the effect of the bacteriophages against a reference treatment
Phagoburn – results of Phase I/II clinical trial
Would phage treatment have been any different from a placebo?
Question marks over phage resistance, and whether dose was too low
Using predatory bacteria to fight infections?
Bdellovibrio bacteriovorus is a Gram-negative bacterial species that is a predator of other Gram-negative bacteria
There is a rare host-independent (HI) growth phase that can be observed in laboratory conditions, but the primary growth phase is host-dependent.
The small, motile Bdellovibrio are said to be in “attack phase”. They do not divide, except when replicating within the periplasm of prey bacteria. These motile bacteria collide with prey, and form a temporary attachment during 5-10 minutes of a recognition phase. 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 prey 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’.
Predation by Bdellovibrio
Attachment to prey cell
Replication within the prey
