Genotyping of Methicillin Resistant Staphylococcus aureus (MRSA) from Local Hospital of Rawalpindi/Islamabad, Pakistan

Abstract

Methicillin resistant S. aureus (MRSA) is a versatile and dangerous human’s pathogen and is a common cause of nosocomial and community acquired infections. S. aureus causes several types of infections such as bacteremia, folliculitis, sepsis, mastitis, meningitis and toxic shock syndrome and staphylococcal pneumonia. S. aureus has developed resistance to the antibiotic ‘methicillin’ and continued spread of methicillin-resistant S. aureus (MRSA) strains poses a significant risk to patients and contributes to a substantial financial burden on healthcare resources. HA-MRSA isolates usually belong to six lineages (CC1, CC5, CC8, CC22, CC30 and CC45) out of ten dominant clonal complexes (CCs) or lineages. Various methods have been employed to identify and characterize S. aureus strains. Phenotypic techniques have been replaced by more robust and accurate molecular techniques. The commonly used molecular techniques are Pulse Field Gel Electrophoresis (PFGE), Multilocus variable number of Tandem repeat analysis (MLVA), Restriction modification tests (RM), multilocus sequence typing (MLST) and spa typing. Present work focuses on genotyping of clinical MRSA isolates from three tertiary care hospital located in “Rawalpindi/Islamabad”, in order to examine the types and phylogenetic relationship of the isolates. In order to gain the understanding of the distribution of MRSA clones in Pakistan, where unregulated antibiotic use is widespread and distributions of MRSA is supposed to be high, an epidemiological relationships between 123 methicillin resistant Staphylococcus aureus strains, isolated between 2006 and 2008 from three tertiary care hospitals of Rawalpinidi and Islamabad, were examined using MLVA scheme, combined with RM typing, PVL sceening, STAR element analysis, spa typing and MLST to investigate the phylogenetic relationships of the Pakistani MRSA isolates. Six loci (clfA, clfB, sdrC, sdrD, spa and sspa) were used in a multilocus variable number tandem repeat analysis (MLVA). A total of 63 MTs/haplotypes were obtained by MLVA. Analysis of restriction modification (RM) genes detected, an RM3 type, associated with CC8, in most strains and an RM1 type, associated with CC30, in only two strains. On further typing of selected strains by Spa typing and MLST, it was found that the RM3/CC8 isolates were ST113-t064, ST113-t451 or ST239, with one of four spa types, whilst the RM1/CC30 isolates were ST30-t021. Analysis of STAR element of these strains for three loci showed their close resemblance; only the strains belonging to CC30 showed no STAR motif in gapR upstream region, confirming their genetic homology to other CC30 strains. Furthermore, the ST30 strains were also found positive for PVL gene. The present genotypic study showed that in Pakistan, the isolates belonging to clonal complex eight (CC8) are dominant in clinical settings. They belong to ST239, ST113 or ST8. The other clonal complex found was CC30 with presence of PVL gene and isolates belong to ST30. The use of MLVA in resource poor laboratories as a rapid and robust method for grouping noscomial MRSA isolates into clusters for identification of localized outbreaks is quite fruitful and MLVA may also provide an understanding of the evolutionary processes as changes in the number of repeats at different loci, may be indicative of which loci are prone to natural selection resulting in higher levels of variation, thus VNTRs serve as evolutionary clock for investigating an outbreaks and transmission events. In this study, we observed more variation in clfA and clfB than in sdrC, sdrD, spa and sspa. We also found that a change in repeat number was not necessarily gradual but may have occurred as a result of large jumps. Some isolates with significant differences in repeat numbers at single locus but being identical numbers in all other loci. Further evidence is provided by the spa typing results, i.e. with a loss of four repeats resulting in a shift from t987 to t030 and a two repeat difference changed t021 to t275. These large jumps might be due to deletions or insertions mediated by recombination or as result of deletions due to slip-strand impairing during DNA replication. Thus MRSA infections have become a challenge across the globe. The MRSA isolates which were once endemic to Europe and America, Africa has now been reported from Asia and thus suggesting that the MRSA isolates which once was endemic to a certain geographical area are no more confined to those boundaries. More over the pandemic spread of one type of MRSA clone across the globe is the result of antibiotic resistance. Therefore a joint global effort will be effective for the xvii control of MRSA infection. Although this study was carried out on limited number of isolated but it is quite useful to strengthen the MRSA data in Pakistan and to develop the genetic profile of MRSA in Pakistan and then to link it globally. This study also helped to under stand that, although there are only two lineages in these hospitals as in most of other Asian countries but there is a diversity at subspecies level as some of the isolates assumed a specific genetic profile as they evolved locally after they were imported to this region. The work presented in the thesis has been published in the following articles: 1- 2- Arfat Y1*, Johnson M2, Malik SA1, Morrissey J2 and Bayliss CD2 (2013). Epidemiology of Methicillin Resistant Staphylococcus aureus (MRSA) Isolates from Pakistan. African Journal of Microbiology Research; Vol.7 (7): 568-576. H5 Index: 15. 2- Joanne Purvees1*, Mathew Blades2, Yasrab Arfat3, Salman A Malik1, Christopher D Bayliss1 and Julie Morrissey1 (2012). Variation in the genomic locations and sequence conservation of STAR elements among Staphylococcus aureus species provides insight into DNA repeat evolution. Genomics; 13: 515. Impact factor 4.07.