Abstract Bachelor Project 1 FBT 2020-2021: Finding mycobacteriophages through phage hunting for the treatment of diseases related to antibiotic resistant Mycobacterium strains

The Mycobacterium genus contains several pathogenic species that affect human health. Some of these mycobacterial infections are treated successfully with different antibiotics, but the emergence of multidrug resistant strains has become a major global threat. The use of bacteriophages, viruses that infect bacteria, is considered an alternative to the current antibiotics. These phages are present everywhere in the environment, because phages are the most common entities in the biosphere. This research project focusses on finding phages that infect the model mycobacteria M. smegmatis, to eventually treat diseases that are related to antibiotic resistant strains of the Mycobacterium genus.

The experiments in this project were implemented by using the M. smegmatis mc2155 strain. Firstly, optimisation of the protocols for M. smegmatis growth and subsequent infection was sought by trying three possible methods to eliminate the clumps and create a smooth bacterial lawn. Then, phage hunting was performed by taking environmental samples. With these samples, the plaque assay was performed to determine whether there were potentially effective phages present in the environmental samples, and if these phages could infect the host of interest. The found phages were purified and amplified to obtain a monoclonal phage population. Lastly, a micrograph of the phages was taken by transmission electron microscopy to observe the phages and their morphology.

After testing the three optimisation protocols, the best and easiest protocol to optimise a liquid culture of M. smegmatis, and thus to eliminate clumps, was to vortex the liquid culture with glass beads. In total 21 samples were taken by phage hunting in several locations in Valencia. After phage hunting, eight samples with plaques were obtained by implementing the plaque assay. These samples all originated from the WWTP’s of Valencia, which are artificial environments. Plaques were then purified by using the phage purification protocol, so that purified phage samples were obtained. In total 25 purified phages were obtained. These purified phages originated from five different environmental samples and were amplified to obtain concentrated phage samples. Six of these purified phages were used for the application of TEM. The micrographs of the phages, taken by TEM, showed that all the isolated phages belonged to the family of the Siphoviridae, which have a long, flexible and non-contractile tail. Five phages had isometric icosahedral capsid structures, whereas one phage had a prolate capsid. This difference in morphology, however, is an interesting factor for further research, such as sequencing.

Because M. smegmatis functions as a model for other mycobacterial species, the phages found in this research project might also be effective against pathogenic Mycobacterium species, such as M. tuberculosis or M. abscessus. However, additional host range experiments should be done to further explore this possibility. Finally, further characterization of the isolated phages, such as sequencing, will provide interesting information for creating phage cocktails as a promising therapeutic tool against mycobacterial infections.

Abstract Bachelor Project 2 FBT 2020-2021: Isolation of phages infecting different bacterial species obtained from human axilla

The aim of this research is to find phages that can infect odour causing bacteria or commensals that become pathogens, starting from the human axilla. Antibiotic resistance is more and more emerging in the daily life and smelling good is a must. Phages could be beneficial against those two problems. It could infect the pathogens and deodorant can be manufactured with phages against the bacteria that cause an odorous smell. The microbiome of the human axilla is isolated and 16S rRNA PCR is executed, followed by Sanger sequencing. After identification of the bacteria, phages could be searched. This happens with a spot test or plaque assay.

The seventy-one isolated bacteria resulted in thirty-seven amplified ones. After sequencing and data analysis, seventeen different species were identified. The difference between the sexes is that a higher differentiation was found in the male samples. The species that were most common with the female were S. epidermidis strain Fussel, while the males had approximately the same percentage S. epidermidis strain Fussel and S. lugdunensis strain ATCC 43809. S. hominis and Corynebacterium are known to cause an odorous smell. In the males, S. hominis is more represented than in the females and Corynebacterium is not present with the females. The microbiome of healthy individuals can be divided into 4 phyla: Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. The moist areas of the skin are dominated by Staphylococcus and Corynebacterium. The only phylum, not present in the identified bacteria was Bacteroidetes. Staphylococcus could be identified as the most dominant genus. Corynebacterium was noted as dominant in moist areas, but there was only one species present. In the lab, there was already another intern who had found phages against S. epidermidis. After purifying the phages, eleven remained. Those phages were against S. epidermidis strain Fussel, S. capitis, S. epidermidis strain NBRC, D. profundi, B. soli, M. yunnanensis, P. marinus and B. frigoritolerans.

It can be concluded that with those found phage, different infections could potentially be treated. The phages that were found are not phages that can be used to eliminate bad odour. This because the bacteria that cause those odour are Corynebacterium and S. hominis and no phages were found against those. If further research wants to be conducted, the phages first need to have an optimal titre.

Abstract Bachelor Project FBT 2018-2019: Finding a bacteriophage as potential killer for antibiotic-resistant bacteria

There are many diseases that lead to the death of which chronic obstructive pulmonary disease (COPD) is the third most common one. It is determined that bacteria are possible causative agents for this disease. Bacterial airway infections play a role in many cases of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and can be successfully treated with antibiotics. In some cases, antibiotics are not always the right treatment because emerging bacterial resistant variants arise. Antibiotic-resistant bacteria lead to higher medical costs, prolonged hospital stays and increased mortality. Phage therapy, which uses phages (natural predators of bacteria) to treat bacterial infections can be a potential solution to treat antibiotic-resistant bacteria.

The aim of this bachelor project is to find phages who would be a potential killer for antibiotic- resistant bacteria. Phages can be alternative tools against antibiotic-resistant bacteria. The project focuses on Klebsiella pneumoniae as a pathogenic bacteria relevant in COPD. Escherichia coli and Mycobacterium smegmatis are going to be used here as controls for detection of environmental phages.

New phages are searched around Valencia in Spain. Phages are sought especially near hospitals, sometimes in sewers and close to water recycling systems. At these places, there is the best chance to find phages for K. pneumoniae. After collecting the sample (including the potential phage) different tests are carried out. Phage hunting was done sixth times, but didn’t always give positive results. Potential phages were found from the first and the third time phage hunting. In total, six lytic phages have been found who can kill a clinical strain of K. pneumoniae.

In the future, phages found in this project will be further characterized by transmission electron microscopy (TEM) and next-generation sequencing to provide more insight about the novelty of these phages. In addition, experimental evolution will be done in order to obtained improved phages, more lytic, or with increased host range, that will be potential tools against COPD involved bacteria.