Genomic Investigation of Methicillin-Resistant Staphylococcus aureus Isolates from Pakistan and Development of Multi epitope Vaccine

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant and opportunistic pathogen. The emergence of new clones of MRSA in both healthcare settings and community warrants serious attention and epidemiological surveillance. However, epidemiological data of MRSA isolates from Pakistan is limited. We performed a whole genome-based comparative analysis of MRSA strains isolated from different regions of Pakistan to understand the genetic diversity, Sequence type (ST), and distribution of virulence and antibiotic-resistance genes. Of the four sequenced strains, two strains (P10 and R46) belong to ST113 and harbour SCCmec type IV encoding mecA gene. Both strains contain two plasmids, and three and two complete prophage sequences are present in P10 and R46, respectively. The specific antibiotic resistance determinants in P10 include two aminoglycoside-resistance genes aph(3')-IIIa and aad(6), a streptothrin-resistance gene sat-4, a tetracycline-resistance gene tet(K), a mupirocin-resistance gene mupA, and a point mutation in fusA conferring resistance to fusidic acid and strain R46 has a specific plasmid associated gene ant(4')-Ib. The strains harbour many virulence factors common to MRSA; however, no Panton-Valentine leucocidin (lukF-PV/lukS-PV) and Toxic shock syndrome toxin (tsst) genes were found in any genomes. The phylogenetic relationship of P10 and R46 with other prevailing MRSA strains suggests that ST113 strains are closely related to ST8 strains and ST113 strains are a single-locus variant of ST8. We also performed genome-based analysis of MRSA strain Lr2 belongs to ST22 and determined comparative analysis with other ST22 strains. The genomic data showed that Lr2 belongs to spa-type t2986, and harbors SCCmec type IVa(2B), one complete plasmid, and seven prophages or prophage-like elements. The strain harbours several prophage-associated virulence factors, Abstract xxiii including Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin (TSST). The SNP phylogenetic analysis revealed that strain Lr2 exhibits the nearest identities to a South African CA-MRSA ST22 strain and makes a separate clade with an Indian CA MRSA ST22 strain. Although most ST22 strains carry blaZ, mecA, and mutations in gyrA, Lr2 strain does not have the blaZ gene. But unlike other ST22 strains, Lr2 carries the antibiotic resistance genes erm(C) and aac(6′)-Ie-aph(2′′)-Ia. Among ST22 strains analysed, only the local strain Lr2 possesses both PVL and TSST, which possibly makes this ST22 clone highly virulent and can serves as a reservoir of virulence factors for other CA-MRSA. One strain (Lr12) was identified to have novel MLST profile, spa type t442, and harbours SCCmec type V. The MLST phylogenetic analysis with all publicly available complete genomes of global MRSA also positioned the Lr12 strain in a separate branch and hence a new ST number 5352 was assigned. These findings provide important information of emerging MRSA clone in Pakistan and the sequenced strains can be used as reference strains for comparative genomic analysis of other MRSA strains in Pakistan. However, further studies are needed on a large collection of MRSA isolates from Pakistan to better understanding of the genomic epidemiology and evolution of these clones in Pakistan. The second part of the thesis is to design a multi-epitope vaccine against S. aureus, because despite ongoing efforts, no clinically approved vaccines against this pathogen exist due to the complexities of its infection process. Therefore, we designed multi-epitope vaccines (CTB-B and B) against S. aureus by employing immunoinformatics approaches. The designed vaccines composed of a linear B-cell epitope (20-mer) each from eight well characterized S. aureus virulence factors, including ClfB, FnbpA, Hla, IsdA, IsdB, LukE, Abstract xxiv SdrD, and SdrE. The designed vaccines were expressed, purified, and administered to C57BL/6 mice with Freund adjuvant to evaluate the immunogenicity and protective efficacy. The results revealed that the immunized mice provoked high IgG against the designed immunogens and the antibody titer increased significantly following the second immunization. However, the generated antibodies did not protect mice from infection. The interaction of anti-B antibodies with source virulence factors showed that generated antibodies have no binding affinity with any of the corresponding virulence factors. Our results demonstrate the limitation of in silico designed B-cell multi-epitope vaccine and suggest that a protein domain carrying both linear and conformational B-cell epitopes might be a better strategy to develop an effective multi-epitope vaccine against S. aureus