Study Plan
The use of bacteriophage and its Endolysin as therapeutic agents
against multidrug-resistant Streptococcus Pneumoniae
BY
FAZAL MEHMOOD Khan
Wuhan Institute of Virology
University of Chinese Academy of Sciences
2017
Title: The use of bacteriophage and its Endolysin as therapeutic agents against multidrug-
resistant Streptococcus Pneumoniae
Background:
Streptococcus pneumoniae is a Gram-positive bacterium, which is the leading cause of life-
threatening infection which is called pneumonia. The organism was first isolated in 1881 from
two independent works of the U.S. Army physician George Sternberg (Sternberg 1881) and the
French chemist Louis Pasteur (Pasteur 1881). Streptococcus pneumoniae causes an estimated
one million childhood deaths each year, mostly in developing countries (O Brien et al., 2009).
Currently, the most common treatment for Streptococcus pneumoniae infection is antibiotics.
The S. pneumoniae continues to cause significant illness worldwide, and with challenging
antibiotic resistance issues (Collignon, 1992). The persistence of high antibiotic selective
pressure in the community and international spread of epidemic or countrywide circulation of
endemic Multi-resistant clones have substantially contributed to the crisis of resistance. This
resistance has complicated treatment options and increased the likelihood of treatment failure
(Kaplan et al., 1998).
Emergence of Streptococcus species that are resistant to multiple antibiotics is a serious global
problem and suggests that alternative treatment and prevention strategies are needed to fight
against Streptococcus species infections.
Bacteriophages are not only important members of microbial ecosystems, but their potential
usefulness is being recognized. For example, the idea of phage therapy has been attracting broad
interest, as reviewed by (Merril et al., 2005). One of the key advantages for phage therapy is that
phages are active against antibiotic-resistant bacteria and they usually do not disturb beneficial
microbiota (Baker-Austin et al., 2008, Jiang et al., 2014). Recently, there has been renewed
interest in the application of bacteriophage as a non-antibiotic approach to control bacterial
infections in various fields including human infections, food safety, agriculture, and veterinary
applications (Wittebole et al., 2014).
Phage Endolysins, which are enzymes encoded by bacteriophages to degrade the bacterial
peptidoglycan cell wall, usually exhibit a wider antimicrobial spectrum compared to their host
bacteriophages (Schmelcher et al., 2012). Many studies have shown that Endolysins possess
strong antimicrobial activity against Gram-positive bacteria. However, the efficacy of externally
applied Endolysins against Gram-negative bacteria is limited because the outer membrane blocks
the access of Endolysins to the peptidoglycan cell wall (Fischetti, 2010). Only a few studies on
purified Endolysins from phage targeting Gram-negative bacteria have been reported
(Borysowski et al., 2006). Endolysins are also successfully applied as therapeutic agents in
animal model studies against many human pathogens (Courchesne et al., 2009). Although,
Endolysins have been used as antibacterial compounds to control antibiotic resistant bacteria in
diverse applications.
Bacteriophages and their Endolysins have been expected to be promising alternative
antimicrobials. However, compared with Endolysins, bacteriophages have some disadvantages,
including phage-resistant bacteria and presence of bacterial toxins in phage lysates (Gupta and
Prasad, 2011). To avoid these limitations, the use of purified Endolysins will be studied. There
are currently about 60 Vibrio phage genome sequences available in the GenBank database
(http://www.ncbi.nlm.nih.gov/genbank/), with no more than 22 of these phages infecting Vibrio
parahaemolyticus. A fair number of Streptococcus pneumoniae phages have been isolated and
characterized in greater detail. In this study, the genome of the Streptococcus pneumoniae
-infecting bacteriophage will be sequenced and preliminary bioinformatic analysis will be
performed, mostly focusing on genes related to lytic activity. The Endolysin from the
Streptococcus pneumoniae-infecting bacteriophage will be cloned and purified and its lytic
activity will be analyzed. These data will be fundamental for the prospective use of
Streptococcus pneumoniae-infecting bacteriophage and its endolysin as therapeutic agents
against Streptococcus pneumoniae in the future.
Aims and Objective
1. To screen out new antimicrobial agents for multidrug-resistant Streptococcus
pneumoniae.
2. To isolate and characterize the lytic bacteriophage specific to Streptococcus pneumoniae.
This will provide essential information for the development of novel phage-based
biocontrol agents against Streptococcus pneumoniae.
3. To obtain endolysin from specific lytic bacteriophage will be purified and analyze their
antibacterial activity to use them as alternative therapeutic agents against Streptococcus
pneumoniae.
Study Methodology
Streptococcus pneumoniae strains will be collected and cultured on agar plates as
described by (Weiyu W et al., 2016).
The Streptococcus pneumoniae -infecting bacteriophage will be isolated from aquatic
sewage water or marine water.
For the morphology of bacteriophage electron microscope will be used as described by
(Weiyu W et al., 2016).
The specific vector will be used for cloning and expression of the putative endolysin
protein in Escherichia coli BL21 (DE3). (Weiyu W et al., 2016)
The DNA extraction, sequencing and bioinformatics analysis will be performed by using
the protocol of (Weiyu W et al., 2016).
The Endolysin gene will be amplified from genomic DNA and will be cloned into the
plasmid for expression as described by (Weiyu W et al., 2016).
The recombinant Endolysin will be purified using the protocol of (Weiyu W et al., 2016).
The Coomassie brilliant blue G-250 assay developed by (Bradford MM, 1976) will be
used to determine the purified Endolysin concentration.
Purified endolysin activity will be tested by the turbidity reduction assay described by
(Nelson et al., 2001).
Antimicrobial spectrum of purified Lysqdvp001 The ten reference strains used for the
antimicrobial spectrum test are listed above and preparation of cell suspensions and
activity tests are described
REFERENCES
Baker-Austin C, McArthur JV, Tuckfield RC, et al., 2008. Antibiotic resistance in the shellfish
pathogen Vibrio parahaemolyticus isolated from the coastal wat er and sediment of
Georgia and South Carolina, USA. J Food Prot 71: 25522558.
Borysowski J, Weber-Dabrowska B and Gorski A, 2006. Bacteriophage endolysins as a novel
class of antibacterial agents. Exp Biol Med (Maywood) 231:366377.
Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254
Collignon P, Bell J (1996). Drug resistant S. pneumonia; the beginning of the end for many
antibiotics? MJA, 164:64.
Courchesne NM, Parisien A and Lan CQ, 2009.Production and application of bacteriophage and
bacteriophage-encoded lysins. Recent Pat Biotechnol 3:3745.
Fischetti VA, 2010. Bacteriophage endolysins: a novel anti-infective to control Gram-positive
pathogens. Int J Med Microbiol 6:357362.
Fuenzalida L, Armijo L and Zabala A et al., 2007. Vibrio parahaemolyticus strains isolated
during investigation of the summer 2006 seafood related diarrhea outbreaks in two
regions of Chile. Int J Food Microbiol 3:270275.
Gupta R and Prasad Y, 2011. P-27/HP endolysin as antibacterial agent for antibiotic resistant
Staphylococcus aureus of human infections. Curr Microbiol 1:3945.
Kaplan SL, Mason EO Jr. Management of infections due to antibiotic-resistant Streptococcus
pneumoniae. Clin Microbiol Rev 1998;11:628 44.
Merril CR, Scholl D and Adhya SL, 2005 Phage therapy. In The Bacteriophages ed. Calendar, R.
and Calendar, R.L. Oxford: Oxford University Press.
Nelson D, Loomis L and Fischetti VA, 2001. Prevention and elimination of upper respiratory
colonization of mice by group A streptococci by using a bacteriophage lytic enzyme.
Proc Natl Acad Sci USA 7:41074112.
O'Brien KL, Wolfson LJ, Watt JP, Henkle E, Deloria-Knoll M, Mccall N, et al. Burden of
disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates.
Lancet. 2009; 374 (9693):893902. doi: 10.1016/S0140-6736(09)61204-6 PMID: 19748398 .
Schmelcher M, Donovan DM and Loessner MJ, 2012. Bacteriophage endolysins as novel
antimicrobials. Future Microbiol 10:11471171.
Weiyu W, Mengzhe L, Hong L, et al., 2016. The Vibrio parahaemolyticus-infecting
bacteriophage qdvp001:genome sequence and endolysin with a modular structure. Arch
Virol 161:26452652.
Wittebole X, Rock SD and Opal SM, 2014. A historical overview of bacteriophage therapy as an
alternative to antibiotics for the treatment of bacterial pathogens. Virulence 5: 226235.
