MicrobiologyBytes

Traditional approaches to phage therapy rely on the ability of viruses to kill their bacterial prey. However, the narrow host range or most bacteriophages and the ability of bacteria to become resistant to infection mean that in practice, using phage to simply replace antibiotics is not feasible. We need smarter approaches, which is where a recent paper comes in. Using phages to engineer sensitivity to antibiotics is a promising approach, but whether this proof-of-principle experiment ever makes it to the clinic is another matter.

Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes. (2011)Appl. Environ. Microbiol. 23 Nov 2011 doi: 10.1128/AEM.05741-11
Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that will resensitize pathogens to approved antibiotics therefore holds key advantages. We present a proof-of-principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages encoding the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene’s sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage’s ability to kill pathogens in the infected host, but instead, to deliver genetic constructs into the bacteria, and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phages will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

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This review gives an overview of the current state-of-the-art of bacteriophage therapy and discusses the challenges to be overcome before phage therapy can become a part of Western medicine.

The next generation of bacteriophage therapy Curr Opin Microbiol. (2011)14(5): 524-531
Bacteriophage therapy for bacterial infections is a concept with an extensive but controversial history. There has been a recent resurgence of interest into bacteriophages owing to the increasing incidence of antibiotic resistance and virulent bacterial pathogens. Despite these efforts, bacteriophage therapy remains an underutilized option in Western medicine due to challenges such as regulation, limited host range, bacterial resistance to phages, manufacturing, side effects of bacterial lysis, and delivery. Recent advances in biotechnology, bacterial diagnostics, macromolecule delivery, and synthetic biology may help to overcome these technical hurdles. These research efforts must be coupled with practical and rigorous approaches at academic, commercial, and regulatory levels in order to successfully advance bacteriophage therapy into clinical settings.

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Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. A new paper has examined the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. The authors set out to find out how many protein-protein interactions remain to be found in this phage.

In order to map lambda’s interactions, they cloned 68 out of 73 lambda open reading frames (the “ORFeome”) into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions, including 16 out of 30 previously published interactions (53%). They also also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda.

Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins.

The protein interaction map of bacteriophage lambda. BMC Microbiology 2011, 11:213 doi:10.1186/1471-2180-11-213

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The mechanism by which dsDNA is packaged into tailed bacteriophage virions has fascinated molecular biologists since it was realized, over four decades ago, that these structures contain long dsDNA molecules that are hundreds of times more compact than dsDNA in solution. Experiments in the 1970s showed that, rather than condensing the DNA first and then assembling a shell around this DNA core, tailed-phage DNA is inserted into a preformed protein container (called a prohead or procapsid). Mmolecular genetics studies indicate that the basic machinery of dsDNA packaging is similar in all tailed phages.

The recent burst of structural progress concerning phage DNA-packaging proteins, as well as the introduction of optical tweezer technology into this field, has allowed the formulation of much more detailed ideas for the mechanism by which the DNA-packaging motor pumps DNA into phage procapsids. Potential applications of packaging nanomotors in nanotechnology and biology are beginning to be described, for example these nanomotors could be used for efficient delivery of nucleic acids or related molecules across barriers such as cell membranes or for applications in single-molecule DNA sequencing. There is little doubt that this will be a fertile research area for some time into the future.

The DNA-packaging nanomotor of tailed bacteriophages. 2011 Nat Rev Microbiol. 9(9):647-57 doi: 10.1038/nrmicro2632
Tailed bacteriophages use nanomotors, or molecular machines that convert chemical energy into physical movement of molecules, to insert their double-stranded DNA genomes into virus particles. These viral nanomotors are powered by ATP hydrolysis and pump the DNA into a preformed protein container called a procapsid. As a result, the virions contain very highly compacted chromosomes. Here, I review recent progress in obtaining structural information for virions, procapsids and the individual motor protein components, and discuss single-molecule in vitro packaging reactions, which have yielded important new information about the mechanism by which these powerful molecular machines translocate DNA.

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E. coli has been in the media a lot recently (Latest News), so MicrobiologyBytes thinks it’s time for:

10 things you should know about E. coli:

1. Escherichia coli (E. coli) is a normal inhabitant of the human gut. It’s been with us for millions of years and overall does us a lot of good, e.g. helping with digestion and providing vitamins we can’t make for ourselves.

2. There are many different strains of E. coli, which all look much alike. They are identified by the antigens on the surface of the cell. These include somatic (O antigens) on the surface of the cell, flagellar (H antigen) and capsular (K antigens) associated with polysaccharide capsules on some strains.

3. A few strains of E. coli are pathogenic and cause disease. Enterotoxigenic (ETEC) strains cause diarrhea but are non-invasive and do not leave the intestine. Enteropathogenic (EPEC) strains also cause diarrhea and enter epithelial cells around the intestine. Enteroinvasive (EIEC) strains cause severe diarrhea and high fever. Enterohemorrhagic (EHEC) strains such as E. coli O157:H7 cause bloody diarrhea, hemolytic-uremic syndrome and kidney failure.

4. E. coli O157:H7 infections often case to bloody diarrhea and occasionally acute kidney failure, especially in young children and elderly people.

5. Most infections are associated with eating undercooked, contaminated ground beef (e.g. burgers), drinking unpasteurized milk, swimming in or drinking contaminated water, and eating contaminated salad vegetables. Infection can also be aquired via direct contact with animal faeces, for example on farms.

6. A bit of dirt never did me any harm… E. coli O157:H7 is new. It was first recognized around 25 years ago and is now widespread, possibly due to agricultural practices.

7. Where did it come from? This strain of E. coli contains lysogenic bacteriophages which encode Shiga toxins (these strains are known as STECs: Shiga Toxin Producing Escherichia coli). E. coli O157:H7 has two stx toxins, stx1 and stx2.

8. How does it cause disease? E. coli O157:H7 is an EHEC strain which kills epithelial cells in the gut, resulting in bloody diarrhea. It also invades the urinary tract causing an ascending infection which damages the kidneys. But it gets worse. Broad spectrum fluoroquinolone antibiotics such as ciprofloxacin which are often used to treat infections cause an SOS response in E. coli cells which in turn induces the lytic cycle of the lysogenic toxin-carrying phages. This results in a thousand-fold increase in toxin expression. Treatment with some some beta-lactam antibiotics also increase stx toxin production.

9. Many people recover without antibiotics or other specific treatment in 5–10 days. There is no clinical evidence that antibiotics improve the course of disease, and some may make it much worse (see above). Haemolytic-uremic syndrome is a life-threatening condition usually treated in an intensive care unit. Blood transfusions and kidney dialysis are often required. Even with intensive care, the death rate for haemolytic uremic syndrome is 3%–5%.

10. Wash your hands thoroughly with soap and warm water after contact with animals. Wash raw vegetables such as salads well before eating. Cook meat thoroughly all the way through, especially burgers and sasuages where external contamination of meat is transferred to the inside by mincing.

11. E. coli is a Gram-negative bacterium. IT’S NOT A VIRUS! So next time a journalist talks or writes about “the E. coli virus” – do us all a favour and yell at them!

Related:

• The continuing evolution of E. coli O157:H7
• E. coli O157:H7 – getting to the bottom of the burger bug
• Bacterial toxins

A number of ecological studies have revealed that microbial viruses predominate in the biosphere and outnumber their hosts by at least one order of magnitude. Due to their abundance and consequent influence on the composition and diversity of microbial communities, viruses can be rightfully considered to be the “major players in the global ecosystem”. Until recently, the majority of viruses in the environment were believed to possess double-stranded DNA genomes. However, technological advances in single-stranded (ss) DNA amplification and sequencing from environmental samples revealed that viruses with ssDNA genomes are more prevalent in both soil and marine environments than previously recognized. This realization precipitated an interest amongst environmental virologists in the diversity and distribution of ssDNA bacterial viruses in nature. Among ssDNA viruses that are most often identified in the environment using metagenomic approach are those belonging to the family Microviridae. However, the host organisms have yet to be determined.

Unexplored diversity and abundance of the Microviridae viruses in the environment fuels interest in this virus group. In order to obtain more information about these viruses, researchers analyzed the genomic sequences available in public databases for the presence of proviruses related to Microviridae. The rationale behind this approach is that a provirus, defective or not, represents a molecular record that a cell has been in contact with a particular virus. This study identified seven proviruses that are related to members of the Microviridae. The proviruses are integrated in the genomes of different species of the order Bacteroidales (phylum Bacteroidetes). The identified proviruses are only distantly related to the previously characterized microviruses and gokushoviruses and may represent a new group or subfamily within the Microviridae. Searches against metagenomic databases suggest that these new viruses might be associated with the human gut microbiota. This extends our knowledge of the evolution, diversity and host range of microviruses.

Microviridae Goes Temperate: Microvirus-Related Proviruses Reside in the Genomes of Bacteroidetes. 2011 PLoS ONE 6(5): e19893. doi:10.1371/journal.pone.0019893
The Microviridae comprises icosahedral lytic viruses with circular single-stranded DNA genomes. The family is divided into two distinct groups based on genome characteristics and virion structure. Viruses infecting enterobacteria belong to the genus Microvirus, whereas those infecting obligate parasitic bacteria, such as Chlamydia, Spiroplasma and Bdellovibrio, are classified into a subfamily, the Gokushovirinae. Recent metagenomic studies suggest that members of the Microviridae might also play an important role in marine environments. In this study we present the identification and characterization of Microviridae-related prophages integrated in the genomes of species of the Bacteroidetes, a phylum not previously known to be associated with microviruses. Searches against metagenomic databases revealed the presence of highly similar sequences in the human gut. This is the first report indicating that viruses of the Microviridae lysogenize their hosts. Absence of associated integrase-coding genes and apparent recombination with dif-like sequences suggests that Bacteroidetes-associated microviruses are likely to rely on the cellular chromosome dimer resolution machinery. Phylogenetic analysis of the putative major capsid proteins places the identified proviruses into a group separate from the previously characterized microviruses and gokushoviruses, suggesting that the genetic diversity and host range of bacteriophages in the family Microviridae is wider than currently appreciated.

Although Archaea inhabit the human body and possess some characteristics of pathogens, there is a notable lack of pathogenic archaeal species identified to date. This paper proposes that the scarcity of disease-causing Archaea is due, in part, to mutually-exclusive phage and virus populations infecting Bacteria and Archaea, coupled with an association of bacterial virulence factors with phages or mobile elements. The ability of bacterial phages to infect Bacteria and then use them as a vehicle to infect eukaryotes may be difficult for archaeal viruses to evolve independently. Differences in extracellular structures between Bacteria and Archaea would make adsorption of bacterial phage particles onto Archaea (i.e. horizontal transfer of virulence) exceedingly hard. If phage and virus populations are indeed exclusive to their respective host Domains, this has important implications for both the evolution of pathogens and approaches to infectious disease control.

The proportional lack of archaeal pathogens: Do viruses/phages hold the key? (2011) BioEssays 1521-1878 doi: 10.1002/bies.201000091

Related:

• Archaea – timeline of the third domain
• Archaea – a microbial cockatrice?

Pseudomonas aeruginosa is the second most common pathogen responsible for hospital-acquired bacterial pneumonia as well as ventilator-associated pneumonia, and the first causative agent of morbidity and mortality in cystic fibrosis (CF) patients. Although antibiotics are still an effective means of treating bacterial lung infections, the alarming rise of multidrug-resistant bacteria in hospitals has highlighted the need for new therapies. Bacteriophages – viruses infecting bacteria – have been proposed to treat human bacterial infections since their discovery in the early 20th century. However, after a short period of development, the advent of antibiotics led to this therapeutic approach being abandoned, except in Eastern Europe where bacteriophages are still used today to treat patients. During the past 20 years, studies in animal models have demonstrated the potential of bacteriophages. Recently the first phase II clinical trial on bacteriophage treatments of chronic otitis was published, and demonstrated the interest of using bacteriophages on multidrug resistant infections.

The effects of bacteriophage therapy on lung infections has only very recently been addressed in animal models. On the one hand, a proof of concept with a bioluminescent strain of P. aeruginosa showed that bacteriophages administrated intranasally had a rapid efficacy with respect to preventing and curing deadly lung infections. On the other hand, a clinical strain of Burkholderia cenocepacia isolated from a CF patient was used to show that the intraperitoneal administration of bacteriophages was more effective than intranasal applications in a non-deadly infectious model. This paper reports an evaluation in an animal model of the efficacy of curative and preventive bacteriophage treatments of lung infections using a multidrug resistant mucoid P. aeruginosa strain isolated from a CF patient.

Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention. (2011) PLoS ONE 6(2): e16963. doi:10.1371/journal.pone.0016963
Multidrug-resistant bacteria are the cause of an increasing number of deadly pulmonary infections. Because there is currently a paucity of novel antibiotics, phage therapy – the use of specific viruses that infect bacteria – is now more frequently being considered as a potential treatment for bacterial infections. Using a mouse lung-infection model caused by a multidrug resistant Pseudomonas aeruginosa mucoid strain isolated from a cystic fibrosis patient, we evaluated bacteriophage treatments. New bacteriophages were isolated from environmental samples and characterized. Bacteria and bacteriophages were applied intranasally to the immunocompetent mice. Survival was monitored and bronchoalveolar fluids were analysed. Quantification of bacteria, bacteriophages, pro-inflammatory and cytotoxicity markers, as well as histology and immunohistochemistry analyses were performed. A curative treatment (one single dose) administrated 2 h after the onset of the infection allowed over 95% survival. A four-day preventive treatment (one single dose) resulted in a 100% survival. All of the parameters measured correlated with the efficacy of both curative and preventive bacteriophage treatments. We also showed that in vitro optimization of a bacteriophage towards a clinical strain improved both its efficacy on in vivo treatments and its host range on a panel of 20 P. aeruginosa cystic fibrosis strains. This work provides an incentive to develop clinical studies on pulmonary bacteriophage therapy to combat multidrug-resistant lung infections.

Related:

• Saturday Cimema: Bacteriophage Therapy
• Can the Common Cold Cure Cystic Fibrosis?
• AAV cystic fibrosis gene therapy
• Molecular basis for Pseudomonas aeruginosa persistent infections in CF patients

Recognized as a global problem, antibiotic resistance increases the morbidity and mortality caused by bacterial infections, as well as the cost of treating infectious diseases. The threat from resistance (particularly multiple resistance in bacterial strains that are widely disseminated) is serious. The key factors contributing to this threat are the pressure of increased antibiotic usage (in both human and animal medicine), greater mobility of the population and industrialization. Many potentially life-threatening infections, generally regarded as diseases from the past due to the success of antibiotics and vaccines, have returned as resistance increasingly hampers successful therapy and prophylaxis.

Several studies have focused on antibiotic resistance codification in plasmids or transposons, and there is also interesting information about the extent of antibiotic resistance genes in a given environment (the so-called “resistome”). However, there is less information on the potential contribution of phages to antibiotic resistance-gene transfer, despite calls for research in this field. Recent reports conclude that the horizontal transfer of genetic information by phages is much more prevalent than previously thought, and that the environment plays a crucial role in the phage-mediated transfer of antibiotic-resistance genes. This paper highlights the potential role of phages in the spread of these genes in the aquatic environment.

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples. (2011) PLoS ONE 6(3): e17549. doi:10.1371/journal.pone.0017549
Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.