4.0 RESULTS. 4.1 Isolation and identification

Transcription

1 4.0 RESULTS 4.1 Isolation and identification The isolation of s. aureus from the sample of human patients was done at by department of microbiology, Indira Gandhi Medical College, Shimla. They were confirmed in the microbiology laboratory, Shoolini University. A total of 150 clinical isolates recovered for human patients during the period July, 2011 to December, 2012 at Indira Gandhi Medical College and Hospital (IGMC), Shimla. The details of the isolates are given in the Table 4.1: pus 92 (61%), urine 25(17%), blood 21(14%), and catheter associated 12 (8%). Of these, 135 were confirmed as S. aureus, whereas rest of the samples had G+ve cocci other than S. aureus (5) and G-ve rods (10). The identification was based on colony morphology, microscopic examination of stained preparations and biochemical tests. On mannitol salt agar, the colonies of S. aureus were one mm in diameter with entire margin, opaque, smooth and in gram stained preparations they appeared as gram positive cocci arranged in small grape like clusters under microscope (Fig.4.1). Biochemically, the isolates were indole negative, methyl red and voges-proskauer positive and positive for catalase, coagulase and oxidase. The strains fermented mannitol with the formation of yellow pigment (Fig 4.2). On blood agar, the colonies of this organism produced β- haemolysis. 4.2 Screening for methicillin resistance by MRSA detection kit Employing MRSA detection kit, MRSA strains produced colour change from pink to yellow with agglutination of bacterial cells (Fig.4.3). Colour change was however, not observed in case of the methicillin sensitive strains. Out of a total of 135 isolates examined, 62 (45.92%) were detected as MRSA. Of these, 32% MRSA were detected positive both by the MRSA kit as well as antibiotic sensitivity assay (Sharma, 2014). The detection kit thus, appeared more sensitive than the antibiotic culture sensitivity assay as 13.9% additional MRSA strains could be detected by the kit. 4.3 Extraction of genomic DNAs of MRSA The genomic DNAs were extracted from 44 MRSA isolates showing resistance to several antibiotics belonging to different groups. The extracted DNAs in a volume of 8µl was 1

2 electrophoresed in 0.8 % agarose in 1x TAE, and stained with ethidium bromide and band of DNA visualized under ultraviolet tramsilluminator (Fig.4.4). All the extracted DNAs after testing for the quality suitable for PCR amplification. The DNAs were stored at -20 C until their use in experiment. 4.4 PCR amplification of mec-a hypervariable gene segment The genomic DNAs of 44 isolates were used as templates for PCR amplification of mec-a hypervariable region. A band of expected size of 550 ± 50 base pairs (bps) of the amplicons was observed in agarose gel (Fig.4.5). Amplification product variation found may due to presences of repeated unit. The amplification was achieved in case of 40 isolates. MRSA isolates, no.34 and 82 which originated from urine and isolate no. 43 and 61 from pus were not amplified in the PCR assays. 4.5 PCR amplification of van A gene region The genomic DNAs of 44 isolates were used as templates for PCR amplification of van A gene segment. The expected size of the amplicon 400 to 500 base pairs (bps) was observed in agarose gel (Fig.4.6). PCR amplification of van-a gene segment was achieved in 38/44 isolates. MRSA isolate no. 34 and 82 from urine while 61, 67 and 102 from pus and isolate 40 originating from blood were not amplified in the PCR assays. 4.6 PCR Amplification of pvl gene region The genomic DNAs of 44 isolates were used as templates for PCR amplification of pvl gene. The expected size of 400±50 base pairs (bps) of the amplicon was observed in agarose gel (Fig.4.7). The amplification was achieved in 38 samples. However, six could not be amplified. MRSA isolate no.34 from urine and 30, 59, 61, 73, and 78 originating from pus was not amplified in the PCR assays. 2

3 4.7 Restriction Enzyme digestion of selective amplicon Restriction Enzyme analysis of amplicon of van-a amplified from MRSA The amplified DNA of each MRSA isolate was digested with SmaI. The digested products were separated by agarose gel electrophoresis. Single distinct pattern were observed: ~300bp to ~200pb (Fig.4.8) Also single band of variable size ~275bp and ~350bp were also seen which may represent undigested DNA of the amplicon or such isolates might have no SmaI restriction site Restriction Enzyme analysis of amplicon of pvl amplified from MRSA The PCR product of pvl amplicon was digested with the AluI. Two distinct patterns were observed in agarose gel on electrophoresis: Pattern I: ~400bp to ~50bp, pattern II: ~375bp to ~100bp (Fig.4.9). 4.8 Nucleotide sequence homology of mec-a-h gene and pvl gene amplicons of MRSA The amplicon of three selected strains of S. aureus recovered one each from blood, urine and pus were sequenced for their nucleotides and sequence homology to the published NCBI sequences was determined by BLAST n analysis. The results of BLAST analysis are presented below Nucleotide sequence homology of mec-a-h gene amplicon of MRSA isolate-64 (blood origin) with the standard NCBI sequences The BLAST analysis of nucleotide sequence of mec-a-h gene amplicon of MRSA isolate-64 recovered from blood showed 98% homology with standard clinical isolates of S. aureus strains CMF 3119 (accession no. HF ), M299 (accession no. HM ) and S. aureus strain M1 (accession no. HM ), mec type IV S. aureus cassette (SCC) sequences (Fig & 4.11). The nucleotide sequences of the amplicon were submitted to NCBI and have been assigned accession number KJ

4 Nucleotide sequence homology of mec-a-h gene amplicon of MRSA isolate-135 (urine origin) with the standard NCBI sequences The BLAST analysis of nucleotide sequence of mec-a-h gene amplicon of MRSA isolate-135 recovered from urine origin showed 97% homology with standard clinical isolates S. aureus strain CMF 3119 (accession no. HF ), M299 (accession no. HM ) and S. aureus strain M1 (accession no. HM ) mec type IV S. aureus cassette (SCC) sequences respectively (Fig-4.12 & 4.13). The nucleotide sequences of the amplicon were submitted to NCBI and have been assigned accession number KJ Nucleotide sequence homology of mec-a-h gene amplicon of MRSA isolate-97(pus origin) with the standard NCBI sequences The BLAST analysis of nucleotide sequence of mec-a-h gene amplicon of MRSA isolate-97 recovered from pus showed 99% homology with standard clinical isolates S. aureus strain CMF 3119 (accession no. HF ), M299 (accession no. HM ) and S. aureus strain M1 (accession no. HM ) mec type IV S. aureus cassette (SCC) nucleotide sequences (Fig & 4.15). The nucleotide sequences of the amplicon were submitted to NCBI and have been assigned accession number KJ Nucleotide sequence homology of pvl gene amplicon of MRSA isolate-64 (blood origin) with the standard NCBI sequences The BLAST analysis of nucleotide sequence of pvl gene amplicon of MRSA isolate-64 recovered from blood showed 99% homology with all S. aureus strains 7401 (accession no. AP ), FD SB11 (accession no. AB ) and S. aureus strain BJ-12 (accession no. AB ) panton valentine leukocidin (LulS-Pv, LuckF-PV) nucleotide sequences (Fig-4.16& 4.17). The nucleotide sequences of the amplicon were submitted to NCBI and have been assigned accession number KJ

5 Nucleotide sequence homology of pvl gene amplicon of MRSA isolate-135 (urine origin) with the standard NCBI sequences The BLAST analysis of nucleotide sequence of pvl gene amplicon of MRSA isolate-135 recovered from urine showed 94% homology with all S. aureus strain of 7401 (accession no. AP ), FD SB11 (accession no. AB ) and BJ-216 (accession no. AB ) panton valentine leukocidin (LulS-Pv, LuckF-PV) gene nucleotide sequences respectively (Fig & 4.19). The nucleotide sequences of the amplicon were submitted to NCBI and have been assigned accession number KJ Nucleotide sequence homology of pvl gene amplicon of MRSA isolate-97 (pus origin) with the standard NCBI sequences The BLAST analysis of nucleotide sequence of pvl gene of MRSA isolate-97 recovered pus origin showed 92% homology with all S. aureus strain 7401 (accession no. AP ), FD SB11 (accession no. AB ) and BJ-216 (accession no. AB ) panton valentine leukocidin (LulS-Pv, LuckF-PV) gene nucleotide sequences respectively (Fig-4.20 & 4.21). The nucleotide sequences of the amplicon were submitted to NCBI and have been assigned accession number KJ Alignment of nucleotide sequences of amplicons of mec-a-h genes of MRSA isolates studied. The nucleotide sequences of amplicons of mec-a-h genes of MRSA isolates: blood (isolate no.64), urine (isolate no.135) and pus (isolates no.97) were aligned to determine sequence homology or relatedness. Distinct conserved sequences were observed among the three isolates. The results of alignment are presented through Fig 4.22 & Alignment of nucleotide sequences of amplicons of pvl genes of MRSA isolates studied The amplicons of pvl genes of different MRSA isolates as mentioned under 4.9 were aligned to determine sequence homology or relatedness. The blood isolate-64 differed from the other two 5

6 isolates in that the former had larger amplicon size. The results of alignment are presented through Fig 4.24 & The three isolates were related Predicted amino acid sequences of mec-a-h gene of selective MRSA isolates The nucleotide sequences of the amplicons of mec-a-h gene of MRSA isolates were converted into predicted amino acid sequences using EXPASY Translate Tool. The predicted amino acid sequences of all three MRSA isolates studied were deduced from the nucleotide sequences of the amplicons as presented below under to Amino acid sequences of MRSA (isolate_64) of blood origin ASALTKSVVIFIFIRLLTEIILIRGIVKAILSKIADKRFVKTSFK-NCR-EVC-KQFSVKLQIRGTLKAV LSKIADKRFVKSSSK-NCR-EVR-KQFHAKLLIRGKLKAVLSKIADKRYVKSSSRQNCR-EVRKQF SVKLLIRGKLKAILG-NCR-GVQKN-T-L Amino acid sequences of MRSA (Isolate_135) of urine origin VKAILSKIADKRFVKSSSK-NCR-EVC-KQFSVKLQIRGTLKAVLSKIAVKRFVKSSSK-NCR-EVR- KQFHAKLLIRGKLKAVLSKIADKRYVKSSSRQNCR-EVR-KQFSVKLLIRGKLKAIRESKIADKG YR KTRLD Amino acid sequences of MRSA (isolate_97) of pus origin IVKAILSKIADKRFVKSSSK-NCR-EVC-KQFSVKLQIRGTLKAVLSKIADKRFVKSSSK-NCR-EVR- KQFHAKLLIRGKLKAVLSKIADKRYVKSSSRQNCR-EVR-KQFSVKLLIRGKLKAILSKI ADKGY RKTRLDYKNW Amino acid sequences of standard stain of S. aureus M299 KQF-VKLQIRGLLKAVLSKIADKRFVKSSSQ-NYR-EVR-KQF-VKLQIRGLLKAVLSKIADKRY VKSNSMQNC--GVS-KQFSVKLQIRGTLKAVLGKIADKRCVKSSSQ-NC GVS 4.12 Predicted amino acid sequences of pvl gene of MRSA isolates The nucleotide sequences of the amplicons of pvl gene of MRSA isolates were converted into predicted amino acid sequence using EXPASY Translate Tool. The predicted amino acid sequences of all three MRSA isolates are as presented below Amino acid sequences of MRSA (isolate_64) of blood origin 6

7 PYSQNPRDYFVPDNELPPLVHSGFNPSFIATVSHEKGSGDTSEFEITYGRNMDVTHATRRTTHYG NSYLEGSRIHNAFVNRNYTVKYEVNWKTHEIKVKGHN-YEK NSQIISCYINCIAFAIQYI Amino acid sequences of MRSA (isolate_135) of urine origin GIPPHYYTVGFNPSFIATVSHEKGSGNTSEFEIT-KNMDVTHATKKTTHYGNIYLKGSKIH NAFVNKIYTVKYEVNWKTHEIKVKGHN-YEKNSQIIICYIICFVFAIQN Amino acid sequences of MRSA (isolate_97) of pus origin FWWPNNELPFYEHGGFNPSFIATVSQEKGSGDTSEFEIT-GKNMDVTQATRKTTHYGNS YLEGSRKHNAFVNRIYTFNYEVNGKTQEIKVKGHN-YEKNSQIIICYIIGFAFAI Amino acid sequences of standard strain FD SB11 Staphylococcus aureus luks-pv, lukf-pv genes for panton-palentine leukocidin S, Panton valentine leukocidin F, complete cds, strain: FD SB11 LFVGYKPYSQNPRDYFVPDNELPPLVHSGFNPSFIATVSHEKGSGDTSEFEITYGRNMDVTHATR RTTHYGNSYLEGSRIHNAFVNRNYTVKYEVNWKTHEIKVKGHN-YEKNSQIISCYINCIAFAIQYS 4.13 Comparative analysis of amino acid sequences of mec-a-h gene of MRSA isolates The amino acid sequences of the three MRSA isolates of different origins were compared with that of mec-a positive standard strains M299. The major amino acid substitutions were seen at position 66, 85, 98, 122, 145 and161 which are as follows: T66L, Q 85K, V98A, N122S, T145K and S161K in these strains (Fig.4.26 &4.27) Comparative analysis of amino acid sequences of pvl gene of MRSA isolates The amino acid sequences of three MRSA isolates of different origins were compared with that of pvl positive standard strains FD SB11. The major amino acid substitutions were seen at positions: 20, 21, 22, 40, 65, 76, 87 and 98 which are as follows: N20G (urine-135), E21I (urine- 135), L22P (urine-135), H40Q (pus-97), K65R (urine-135), K76E (urine-135), K87R (urine-135) and H98Q (pus-97) (Fig & 4.29). 7

8 4.15 Plasmid profiling As shown in Figure 4.30, a single Plasmid of 2kb was observed in ethidium bromide stained agarose gel. The plasmid was present in 36/40 MRSA isolates tested Bacteriophage Typing The bacteriophage typing is used for precise identification method of S. aureus and provides valuable information in epidemiological studies of Staphylococcal infection in the community. The results of bacteriophage typing are presented in Table 4.2. Out of 37 MRSA isolates tested, 32 (86.48%) were typeable and (13.5%) non-typeable. The most prevalent phage group was mixed phage group (46.87%) followed by phage group III (37.5%), phage group II (12.5%) and phage group I (3.13), None of the isolates were lysed by phages of group NA and V. Table4.1. Originwise details of isolates Sr. Name of Isolates Pus Urine Blood C.A. * No. 1. Staphylococcus aureus 2. Other Bacteria Total *Catheter Associated 8

9 Table 4.2 Results of bacteriophage typing of MRSA isolates of different origins Sr. No. Phage Group Number Isolates (%) Phage type (number isolates) at R.T.D. X I 1 (3.13) 29/52/52A (1) 2 II 4 (12.50) 3C/55/71 (4) 3 III 12 (37.50) 47/77/84/85 (1), 77 (2), 77/84 (3), 47/77 (3), 47/77/84 (3) 4 NA 0 (0.0) - 5 V 0 (0.0) - 6 Mixed Phage 15 (46.87) 3C/55/77 (1), 52A/79/42E/47/77/48 (1), 53A/42E/77/84 (1), 52A/42E/47/77/84 (1), 52A/79/6/42E/47/77/84 (1), 29/52/77/84/85/81 (1), 29/52/77/84 (1), 29/52/79/80/47/54/77/84 (1), 52A/47/77/84 (2), 29/52/52A/79/80/47/77/84 (2), 29/52/52A (3) 9

10 Fig.4.1 Gram positive cocci in grape like clusters (S. aureus) under light microscope (100X) A B Fig.4.2 Production yellow pigment by S. aureus isolate on Mannitol salt agar (A). The isolate in B is not S. aureus since there is not any production of yellow pigment Fig.4.3 MRSA detection by the MRSA kit. 10

11 Samples 2-5 are MRSA positive which is indicated by change of colour from pink to yellow. The sample in tube 1 is not MRSA L Genomic DNA >10kb 8kb Fig4.4. Gel electrophoresis of DNAs of MRSA isolates. The extracted genomic DNA band is visible in lane 1 to 15 L contains markers of 10 kb ladder). 11

12 Amplicons Fig.4.5 Gel electrophoresis of PCR product of mec-a H gene amplicon of MRSA isolates. DNA band of 550±50bp is visible. Lane L- molecular markers of 1kb, lane 1 amplicon of standard MRSA strain, ATCC4300. Amplification of the gene is seen in lanes 2 to 6. Lane 2 (isolate-113), Lane 3 (isolate-115), Lane 4 (isolate-127), Lane 5 (isolate-128) indicates expected amplicon of clinical isolates and No amplification is seen in Lane 6 (isolate-43) and 7(isolate -61). 12

13 Amplicons Fig.4.6 Gel electrophoresis of PCR product of van-a gene amplicon. DNA bands of 400bp to 500bps are visible in case of MRSA isolates. Lane L- molecular markers of 1kb. Amplification is seen in lanes 1 to 12. Lane 1 (isolate-64), Lane 2 (isolate-135), Lane 3 (isolate-73), Lane 4 (isolate-78), Lane 5 (isolate-97), Lane 6 (isolate-101), Lane 8 (isolate-105), Lane 9 (isolate-113), Lane 10 (isolate-115), Lane 11 (isolate- 127), Lane 12 (isolate-128) and Lane 13 MRSA positive standard strain ATCC

14 Amplicons Fig.4.7 Gel electrophoresis of PCR product pvl amplicon. A band of 420 bps is visible. Lane- L molecular markers of 1kb, Lane 1 Standard MRSA strain ATCC Amplification is seen in Lanes 3 to 7. Lane 2 (isolate-79) Lane 3 (isolate-82) Lane 4 (isolate-97) Lane 5 (isolate-113) Lane 6 (isolate-115) Lane 7 (isolate-127) 14

15 Fig 4.8 RE analysis of van-a amplicon digested with smai. A single restriction pattern is visible which consists of two bands ~300bps and ~200bps in lanes 2, 4, 5 and 6. Lane 1 and 3 a single band is visible which might represent undigested DNA or might lack restriction site for SmaI. In lanes 1 (~275bps) and lane 3 (~350bps) sized band are visible lane 7-blank. Lane L- Molecular marker 1kb ladder. Fig 4.9 RE analysis of pvl gene amplicon of MRSA isolates. Lane L -Molecular markers of 1kb. Lanes 1 to 7 contain digested amplicons of MRSA isolates. Two restriction patterns are visible. Pattern I- ~400 bps and ~50bps (lane 1), Pattern II- ~375bps to ~100bps and ~50bps (lanes 2, 4 and 5) and lane 3 contains partially digested products. Lane 6 and Lane 7- blank. Nucleotide sequences of mec-a-h gene amplicon of MRSA isolate-64 (blood origin) GCCTTCCCAATTTTGTAATCAAGTCTAGTTTTTCTGTACCCCTTATCTGCAATTTTACCCTAGGATTGCTTTTAA CTTACCCCTTATCAGCAATTTTACTGAGAACTGCTTTTAACGCACCTCTTATCTGCAATTTTGCCTAGAACTGCT TTTAACGTACCTCTTATCTGCAATTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTGCATGGAA TTGCTTTTAACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTATCTGCAATTTTACT TAGAACTGCTTTTAACGTACCTCTTATCTGTAATTTTACTGAGAACTGCTTTTAACAAACCTCTTATCTGCAATT TTACTTAAAACTGGTTTTAACAAACCTCTTATCTGCAATTTTACTTAGAATTGCTTTTACGATTCCTCTTATCAG TATAATCTCAGTAAGAAGGCGTATAAAAATGAAAATTACTACCGATTTTGTAAGTGCGGACGCCTGAAGGAA ACCTAAAAATAAGTT 64_MEC_CUS-220_B07_061.ab GCCTTCCCAATTTTGTAATCAAGTCTAGTTTTTCTGTACCCCTTATCTGCA gi emb HF : TTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTT-----CATTTTTATACGCA gi emb HF : gi gb HM : TTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTT-----CATTTTTATACGCA gi gb HM : gi gb HM : TTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTT-----CATTTTTATACGCA gi gb HM : _MEC_CUS-220_B07_061.ab1 gi emb HF : gi emb HF : gi gb HM : gi gb HM : gi gb HM : gi gb HM : ATTTTACCCTAGGATTGCTTTTAACTTACCCCTTATCAGCAATTTTACTGAGAACTGCTT TTCTTACTGAGATTATACTAATAAGAGGAATAGTAAAAGCAATTCTAAGTAAAATTGCAG G TTCTTACTGAGATTATACTAATAAGAGGAATAGTAAAAGCAATTCTAAGTAAAATTGCAG G TTCTTACTGAGATTATACTAATAAGAGGAATAGTAAAAGCAATTCTAAGTAAAATTGCAG G 64_MEC_CUS-220_B07_061.ab1 TTAACGCACCTCTTATCTGCAATTTTGCCTAGAACTGCTTTTAACGTACCTCTTATCTGC 15

16 gi emb HF : gi emb HF : gi gb HM : gi gb HM : gi gb HM : gi gb HM : _MEC_CUS-220_B07_061.ab1 gi emb HF : gi emb HF : gi gb HM : gi gb HM : gi gb HM : gi gb HM : _MEC_CUS-220_B07_061.ab1 gi emb HF : gi emb HF : gi gb HM : gi gb HM : gi gb HM : gi gb HM : _MEC_CUS-220_B07_061.ab1 gi emb HF : gi emb HF : gi gb HM : gi gb HM : gi gb HM : gi gb HM : _MEC_CUS-220_B07_061.ab1 gi emb HF : gi emb HF : gi gb HM : gi gb HM : gi gb HM : gi gb HM : ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGTTAAAAGC :***.. * ***:.:***.** *.. **.** ***: :***.. * ***:.:** AATTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTGCATGGAATTGCTT AGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAAATTGCAG AGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAAATTGCAG AGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAAATTGCAG AGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAAATTGCAG AGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAAATTGCAG AGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAAATTGCAG *.** *.. *.** *.*: :*** :..* ***:.****.** *...*..******: TTAACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTATCTGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGTTAAAAGC ATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGTTAAAAGC :***.. * ***:.:***.** **. **.** ***: :*** *... ***:.:** AATTTTACTTAGAACTGCTTTTAACGTACCTCTTATCTGTAATTTTACTGAGAACTGCTT AATTCCATGCAAAATTGCTGATAAGG GGTA AATTCCATGCAAAATTGCTGATAAGG GGTA AATTCCATGCAAAATTGCTGATAAGG GGTA AATTCCATGCAAAATTGCTGATAAGG GGTA AATTCCATGCAAAATTGCTGATAAGG GGTA AATTCCATGCAAAATTGCTGATAAGG GGTA **** * *.** **** :*** * * *: TTAACAAACCTCTTATCTGCAATTTTACTTAAAACTGGTTTTAACAAACCTCTTATCTGC AGTTAAAAGCAGTTCTCAGTAAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGC AGTTAAAAGCAGTTCTCAGTAAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGC AGTTAAAAGCAGTTCTCAGTAAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGC AGTTAAAAGCAGTTCTCAGTAAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGC AGTTAAAAGCAGTTCTCAGTAAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGC AGTTAAAAGCAGTTCTCAGTAAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGC : ::.*** *: **.**:* **::**.*: *:** :***: ::.*** *: **.*. ** Cont... 64_MEC_CUS-220_B07_061.ab1 gi emb HF : gi emb HF : gi gb HM : gi gb HM : gi gb HM : gi gb HM : AATTTTACTTAGAATTGCTTTTACGATTCCTCTTATCAGTATAATCTCAGTAAGAAGGCG AAAATTGCAGATAAGAGGTGCGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTA AAAATTGCAGATAAGAGGTGCGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTA AAAATTGCAGATAAGAGGTGCGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTA AAAATTGCAGATAAGAGGTGCGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTA AAAATTGCAGATAAGAGGTGCGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTA AAAATTGCAGATAAGAGGTGCGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTA **::**.*: * ** :* * :.*:: *: **.******:*** *:*::*...**. 64_MEC_CUS-220_B07_061.ab1 gi emb HF : gi emb HF : gi gb HM : gi gb HM : gi gb HM : gi gb HM : TATAAAAATGAAAATTACTACCGATTTTGTAAGTGCGGACGCCTGAAGGAAACCTAAAAA AGTTAAAAGCAATCCTA--AGTAAAATTGCAGATAAG---GGGTACAGAAAAACTAGACT AGTTAAAAGCAATCCTA--AGTAAAATTGCAGATAAG---GGGTA AGTTAAAAGCAATCCTA--AGTAAAATTGCAGATAAG---GGGTACAGAAAAACTAGACT AGTTAAAAGCAATCCTA--AGTAAAATTGCAGATAAG---GGGTA AGTTAAAAGCAATCCTA--AGTAAAATTGCAGATAAG---GGGTACAGAAAAACTAGACT AGTTAAAAGCAATCCTA--AGTAAAATTGCAGATAAG---GGGTA :.*:**** **:. ** *.*::*** *..*..* * *. 64_MEC_CUS-220_B07_061.ab1 TAAGTT gi emb HF : TGATTACAAAAT gi emb HF : gi gb HM : TGATTACAAAAT gi gb HM : gi gb HM : TGATTACAAAAT gi gb HM :

17 Fig.4.10 Multiple sequences alignment of sequences of mec-a-h gene amplicon of MRSA isolate-64 (blood origin), S. aureus standard strain CMF 3119, M299 and M1 (CLUSTAL OMEGA analysis) 17

18 Cont... Fig.4.11 Jal view Multiple sequences alignment of sequences of mec-a-h gene amplicon of MRSA isolate-64 (blood origin), S. aureus standard strain CMF 3119, M299 and M1 (CLUSTAL OMEGA analysis) 18

19 Nucleotide sequences of mec-a-h gene amplicon of MRSA isolate -135 (urine origin) AAGGCCCCTTCCCATTTTTGTAATCAAGTCTAGTTTTTCTGTACCCCTTATCTGCAATTTTACTCTCACGGATTG CTTTTAACTTACCCCTTATCAGCAATTTTACTGAGAACTGCTTTTAACGCACCTCTTATCTGCAATTTTGCCTAG AACTGCTTTTAACGTACCTCTTATCTGCAATTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTG CATGGAATTGCTTTTAACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTAACTGCAA TTTTACTTAGAACTGCTTTTAACGTACCTCTTATCTGTAATTTTACTGAAAACTGCTTTTAACAAACCTCTTATC TGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTATCTGCAATTTTACTTAGAATTGCTTTTACTATTCCTC TTATTAGTATAATCTCAATAAGAAGGCTTATAAAAACGAAAATTACAACCGATTTTGTAAGTGCGGAGCCAGA AAGAACAGTAAATCAGTT 135_MEC_CUS-220_C07_059.ab1 135_MEC_CUS-220_C07_059.ab1 135_MEC_CUS-220_C07_059.ab1 135_MEC_CUS-220_C07_059.ab1 135_MEC_CUS-220_C07_059.ab AAGGCCCCTTCCCATTTTTGTAATCAAGTCTAGTTTTTCTGTACCCCTTAT TACTATTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTTC-----ATTTTTAT TACTATTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTTC-----ATTTTTAT TACTATTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTTC-----ATTTTTAT.:. * *. ** **:.:****.. * **.****. **** CTGCAATTTTACTCTCACGGATTGCTTTTAACTTACCCCTTATCAGCAATTTTACTGAGA ACGCATTCT--TACTGAGATTATACTAATAAGAGGAATAGTAAAAGCAATTCTAAGTAAA ACGCATTCT--TACTGAGATTATACTAATAAGAGGAATAGTAAAAGCAATTCTAAGTAAA ACGCATTCT--TACTGAGATTATACTAATAAGAGGAATAGTAAAAGCAATTCTAAGTAAA. ***:* * :** *. ::*.**::*** :.... **:.******* **. *.* ACTGCTTTTAACGCACCTCTTATCTGCAATTTTGCCTAGAACTGCTTTTAACGTACCTCT ATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGT ATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGT ATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTTTGT * ***: :***.. * ***:.:***.** *.. **.** ***: :***.. * * TATCTGCAATTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTGCATGGA TAAAAGCAGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAA TAAAAGCAGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAA TAAAAGCAGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGTAAA **:.:***.** *.. *.** *.*: :*** :..* ***:.****.** *...*..* ATTGCTTTTAACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCT ATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGT ATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGT ATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTACGT *****: :***.. * ***:.:***.** **. **.** ***: :*** *... * 19

20 135_MEC_CUS-220_C07_059.ab1 135_MEC_CUS-220_C07_059.ab1 135_MEC_CUS-220_C07_059.ab1 135_MEC_CUS-220_C07_059.ab1 135_MEC_CUS-220_C07_059.ab1 TAACTGCAATTTTACTTAGAACTGCTTTTAACGTACCTCTTATCTGTAATTTTACTGAAA TAAAAGCAATTCCATGCAAAATTGCTGATAAGGGGTAAGTTAAAAGCAGTTCTCAGTAAA TAAAAGCAATTCCATGCAAAATTGCTGATAAGGGGTAAGTTAAAAGCAGTTCTCAGTAAA TAAAAGCAATTCCATGCAAAATTGCTGATAAGGGGTAAGTTAAAAGCAGTTCTCAGTAAA ***.:****** * *.** **** :*** *..: ***:.:* *.** *.. *** ACTGCTTTTAACAAACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCT ATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGCAAAATTGCAGATAAGAGGTGCGT ATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGCAAAATTGCAGATAAGAGGTGCGT ATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGCAAAATTGCAGATAAGAGGTGCGT * ***: :*** *... ***:.:***.** ** *.** ***: :*** *.. * TATCTGCAATTTTACTTAGAATTGCTTTTACTATTCCTCTTATTAGTATAATCTCAATAA TAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGG TAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGG TAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGG **:.:***.** *.. **.******* :**.. GAAGGCTTATAAAAACGAAAATTACAACCGATTTTGTAAGTGCGGACGCCAGAAAGAA TAAGTTAAAAG--CAATCCTAAGTAAAATTGCAGATAAGGGGTACAGAAAAACTA -----TAAGTTAAAAG--CAATCCTAAGTAAAATTGCAGATAAGGGGTACAGAAAAACTA -----TAAGTTAAAAG--CAATCCTAAGTAAAATTGCAGATAAGGGGTACAGAAAAACTA ::.*:****.***. **.*::*** *..*..**..******.*. CAGTAAATCAGTT----- GACTTGATTACAAAATGG GACTTGATTACAAAATGG GACTTGATTACAAAATGG * *:.** * :: Fig.4.12 Multiple nucleotide sequences alignment of mec-a-h gene amplicon of MRSA isolate-135 (urine origin), S. aureus standard strain CMF 3119, M299 and M1 (CLUSTAL OMEGA analysis). 20

21 Cont... 21

22 Fig.4.13 Jal view Multiple nucleotide sequences alignment of mec-a-h gene amplicon of MRSA isolate-135 (urine origin), S. aureus standard strain CMF 3119, M299 and M1 (CLUSTAL OMEGA analysis). Nucleotide sequences of mec-a-h gene amplicon of MRSA isolate -97(pus origin) TCCCAATTTTTGTAATCAAGTCTAGTTTTTCTGTACCCCTTATCTGCAATTTTACTTAGGATTGCTTTTAACTTAC CCCTTATCAGCAATTTTACTGAGAACTGCTTTTAACGCACCTCTTATCTGCAATTTTGCCTAGAACTGCTTTTAA CGTACCTCTTATCTGCAATTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTGCATGGAATTGCT TTTAACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTATCTGCAATTTTACTTAGAA CTGCTTTTAACGTACCTCTTATCTGTAATTTTACTGAGAACTGCTTTTAACAAACCTCTTATCTGCAATTTTACT TAGAACTGCTTTTAACAAACCTCTTATCTGCAATTTTACTTAGAATTGCTTTTACGATTCCTCTTATTAGTATAA TCTCAGTAAGAAGGCGTATAAAAATGAAAATTACAACCGATTTTGTAAGTGCGGACGCCTGAGGGAAAACGT AATTAAGTG 97_MEC_CUS-220_A07_063.ab1 97_MEC_CUS-220_A07_063.ab1 97_MEC_CUS-220_A07_063.ab1 97_MEC_CUS-220_A07_063.ab1 97_MEC_CUS-220_A07_063.ab1 97_MEC_CUS-220_A07_063.ab1 97_MEC_CUS-220_A07_063.ab1 97_MEC_CUS-220_A07_063.ab TCCCAATTTTTGTAATCAAGTCTAG------TTTTTCTGTACC TACTATTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTTCATTTTTATACGCA TACTATTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTTCATTTTTATACGCA TACTATTCCCTCAGGCGTCAGCACTTACAAAATCGGTTGTAATTTTCATTTTTATACGCA **..* **:.:****.. * **. *****.*..*. CCTTATCTGCAATTTTACTTAGGATTGCTTTTAACTTACCCCTTATCAGCAATTTTACTG TTCTTACTGAGATTATACTAATAAGAGGAATA GTAAAAGCAATTCTAAGT TTCTTACTGAGATTATACTAATAAGAGGAATA GTAAAAGCAATTCTAAGT TTCTTACTGAGATTATACTAATAAGAGGAATA GTAAAAGCAATTCTAAGT *::***..***:****:*.* :* ::*: **:.******* **. AGAACTGCTTTTAACGCACCTCTTATCTGCAATTTTGCCTAGAACTGCTTTTAACGTACC AAAATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTT AAAATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTT AAAATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTT *.** ***: :***.. * ***:.:***.** *.. **.** ***: :***.. TCTTATCTGCAATTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTGCAT TGTTAAAAGCAGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGT TGTTAAAAGCAGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGT TGTTAAAAGCAGTTCTCAGTAAAATTACAGATAAGAGGTACGTTAAAAGCAGTTCTAAGT * ***:.:***.** *.. *.** *.*: :*** :..* ***:.****.** *...* GGAATTGCTTTTAACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACC AAAATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTA AAAATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTA AAAATTGCAGATAAGAGGTTTGTTAAAAGCAGTTCTAAGTAAAATTGCAGATAAGAGGTA..******: :***.. * ***:.:***.** **. **.** ***: :*** *... TCTTATCTGCAATTTTACTTAGAACTGCTTTTAACGTACCTCTTATCTGTAATTTTACTG CGTTAAAAGCAATTCCATGCAAAATTGCTGATAAGGGGTAAGTTAAAAGCAGTTCTCAGT CGTTAAAAGCAATTCCATGCAAAATTGCTGATAAGGGGTAAGTTAAAAGCAGTTCTCAGT CGTTAAAAGCAATTCCATGCAAAATTGCTGATAAGGGGTAAGTTAAAAGCAGTTCTCAGT ***:.:****** * *.** **** :*** *..: ***:.:* *.** *.. AGAACTGCTTTTAACAAACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACC AAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGCAAAATTGCAGATAAGAGGTG AAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGCAAAATTGCAGATAAGAGGTG AAAATTGCAGATAAGAGGTACGTTAAAAGCAGTTCTAGGCAAAATTGCAGATAAGAGGTG *.** ***: :*** *... ***:.:***.** ** *.** ***: :*** *.. TCTTATCTGCAATTTTACTTAGAATTGCTTTTACGATTCCTCTTATTAGTATAATCTCAG CGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTAAG CGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTAAG

23 97_MEC_CUS-220_A07_063.ab1 97_MEC_CUS-220_A07_063.ab1 CGTTAAAAGCAGTTCTCAGTAAAATTGCTGATAAGGGGTAAG ***:.:***.** *.. **.******* :**.*..: TAAGAAGGCGTATAAAAATGAAAATTACAACCGATTTTGTAAGTGCGGACGCCT--GAGG TTAAAAGCA--ATCCTAAGTAAAATTGCAGATAAGGGGTACAGAAAAA TTAAAAGCA--ATCCTAAGTAAAATTGCAGATAAGGGGTACAGAAAAA TTAAAAGCA--ATCCTAAGTAAAATTGCAGATAAGGGGTACAGAAAAA *:**** * **. **.*::*** *..*..**..*:.*.. GAAAACGTAATTAAGTG---- CTAGACTTGATTACAAAATGG CTAGACTTGATTACAAAATGG CTAGACTTGATTACAAAATGG :*.** *.****..:. Fig.4.14 Multiple nucleotide sequences alignment of sequences of mec-a-h gene amplicon of MRSA isolate- 97 (pus origin), S. aureus standard strain CMF 3119, M299 and M1 (CLUSTAL OMEGA analysis) 23

24 Cont... Fig.4.15 Jal view of Multiple nucleotide sequences alignment of sequences of mec-a-h gene amplicon of MRSA isolate- 97 (pus origin), S. aureus standard strain CMF 3119, M299 and M1 (CLUSTAL OMEGA analysis).. 24

25 Nucleotide sequences of pvl gene amplicon of MRSA isolate-64 (blood origin) GGCCCCATTATTTTGTTGGAATAACCATATAGTCAAAATCCGAGAGACTATTTTGTGCCAGACAATGAATTAC CCCCATTAGTACACAGTGGTTTCAATCCTTCATTTATTGCAACTGTTTCTCATGAAAAAGGCTCAGGAGATACA AGTGAATTTGAAATAACGTATGGCAGAAATATGGATGTTACTCATGCTACTAGAAGAACAACACACTATGGCA ATAGTTATTTAGAAGGATCTAGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGAAGTGAA CTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAGTCAAATCATCAGTTGTTA CATCAATTGCATTGCTTTTGCTATCCAATACATTTGATGAAAAAGAAAC gi dbj AP : gi dbj AB : gi dbj AB : _pvl gi dbj AP : gi dbj AB : gi dbj AB : _pvl gi dbj AP : gi dbj AB : gi dbj AB : _pvl gi dbj AP : gi dbj AB : gi dbj AB : _pvl gi dbj AP : gi dbj AB : gi dbj AB : _pvl gi dbj AP : gi dbj AB : gi dbj AB : _pvl gi dbj AP : gi dbj AB : gi dbj AB : _pvl TTATTTGTTGGATATAAACCATATAGTCAAAATCCGAGAGACTATTTTGTGCCA TTATTTGTTGGATATAAACCATATAGTCAAAATCCGAGAGACTATTTTGTGCCA TTATTTGTTGGATATAAACCATATAGTCAAAATCCGAGAGACTATTTTGTGCCA GGCCCCATTATTTTGTTGGAATAACCATATAGTCAAAATCCGAGAGACTATTTTGTGCCA :*::** *. *::************************************** GACAATGAATTACCCCCATTAGTACACAGTGGTTTCAATCCTTCATTTATTGCAACTGTT GACAATGAATTACCCCCATTAGTACACAGTGGTTTCAATCCTTCATTTATTGCAACTGTT GACAATGAATTACCCCCATTAGTACACAGTGGTTTCAATCCTTCATTTATTGCAACTGTT GACAATGAATTACCCCCATTAGTACACAGTGGTTTCAATCCTTCATTTATTGCAACTGTT ************************************************************ TCTCATGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTATGGCAGAAATATG TCTCATGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTATGGCAGAAATATG TCTCATGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTATGGCAGAAATATG TCTCATGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTATGGCAGAAATATG ************************************************************ GATGTTACTCATGCTACTAGAAGAACAACACACTATGGCAATAGTTATTTAGAAGGATCT GATGTTACTCATGCTACTAGAAGAACAACACACTATGGCAATAGTTATTTAGAAGGATCT GATGTTACTCATGCTACTAGAAGAACAACACACTATGGCAATAGTTATTTAGAAGGATCT GATGTTACTCATGCTACTAGAAGAACAACACACTATGGCAATAGTTATTTAGAAGGATCT ************************************************************ AGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGAAGTGAACTGGAAA AGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGAAGTGAACTGGAAA AGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGAAGTGAACTGGAAA AGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGAAGTGAACTGGAAA ************************************************************ ACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAGTCAAATCATCAGT ACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAGTCAAATCATCAGT ACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAGTCAAATCATCAGT ACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAGTCAAATCATCAGT ************************************************************ TGTTACATCAATTGCATTGCTTTTGCTATCCAATACAGTTGATG TGTTACATCAATTGCATTGCTTTTGCTATCCAATACAGTTGATG TGTTACATCAATTGCATTGCTTTTGCTATCCAATACAGTTGATG TGTTACATCAATTGCATTGCTTTTGCTATCCAATACATTTGATGAAAAAGAAAC ************************************* ****** Fig.4.16 Multiple sequences alignment of nucleotide sequences of pvl gene amplicon of MRSA isolate- 64 (blood origin), S. aureus standard strain 7401, FD SB11 and BJ-12. (CLUSTAL OMEGA analysis) 25

26 Fig.4.17 Jal view of Multiple sequences alignment of nucleotide sequences of pvl gene amplicon of MRSA isolate- 64 (blood origin), S. aureus standard strain 7401, FD SB11 and BJ-12. (CLUSTAL OMEGA analysis) 26

27 Nucleotide sequences of pvl gene amplicon of MRSA isolate-135 (urine origin) CCCCAAAAAAACAAATGGAATTCCCCCCCATTATTACACGGTGGGTTTCAATCCTTCATTTATTGCAACTGTTT CTCATGAAAAAGGCTCAGGAAATACAAGTGAATTTGAAATAACGTAGGGCAAAAATATGGATGTTACTCATG CTACTAAAAAAACAACACACTATGGCAATATTTATTTAAAAGGATCTAAAATACACAACGCATTTGTAAACAA AATTTACACAGTTAAATATGAAGTGAACTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATTGATATGA AAAAAATAGTCAAATCATCATTTGTTACATCATTTGCTTTGTTTTTGCTATCCAAAACATTTAAAACAAAAAAA A gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_H06_034.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_H06_034.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_H06_034.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_H06_034.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_H06_034.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_H06_034.ab AATGAATTACCCCC-ATTAGTACACAGTGGTTTCAATCCTTCATT AATGAATTACCCCC-ATTAGTACACAGTGGTTTCAATCCTTCATT AATGAATTACCCCC-ATTAGTACACAGTGGTTTCAATCCTTCATT CCCCAAAAAAACAAATGGAATTCCCCCCCATTATTACACGGTGGGTTTCAATCCTTCATT :..*:*.***** :*:: :.... ***************** TATTGCAACTGTTTCTCATGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTA TATTGCAACTGTTTCTCATGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTA TATTGCAACTGTTTCTCATGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTA TATTGCAACTGTTTCTCATGAAAAAGGCTCAGGAAATACAAGTGAATTTGAAATAACGTA **********************************.************************* TGGCAGAAATATGGATGTTACTCATGCTACTAGAAGAACAACACACTATGGCAATAGTTA TGGCAGAAATATGGATGTTACTCATGCTACTAGAAGAACAACACACTATGGCAATAGTTA TGGCAGAAATATGGATGTTACTCATGCTACTAGAAGAACAACACACTATGGCAATAGTTA GGGCAAAAATATGGATGTTACTCATGCTACTAAAAAAACAACACACTATGGCAATATTTA ****.**************************.**.******************** *** TTTAGAAGGATCTAGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGA TTTAGAAGGATCTAGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGA TTTAGAAGGATCTAGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGA TTTAAAAGGATCTAAAATACACAACGCATTTGTAAACAAAATTTACACAGTTAAATATGA ****.*********.***********************.**:****************** AGTGAACTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAG AGTGAACTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAG AGTGAACTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAG AGTGAACTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAG ************************************************************ TCAAATCATCAGTTGTTACATCAATTGCATTGCTTTTGCTATCCAATACA TCAAATCATCAGTTGTTACATCAATTGCATTGCTTTTGCTATCCAATACA TCAAATCATCAGTTGTTACATCAATTGCATTGCTTTTGCTATCCAATACA TCAAATCATCATTTGTTACATCATTTGCTTTGTTTTTGCTATCCAAAACATTTAAAACAA *********** ***********:****:*** *************:*** gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_H06_034.ab1 AAAAAA Fig.4.18 Multiple sequences alignment of nucleotide sequences of pvl gene amplicon of MRSA isolates (urine origin), S. aureus standard strains 7401, FD SB11 and BJ-216. (CLUSTAL OMEGA analysis) 27

28 Fig.4.19 Jal view multiple sequences alignment of nucleotide sequences of pvl gene amplicon of MRSA isolate 135 (urine origin), S. aureus standard strains 7401, FD SB11 and BJ (CLUSTAL OMEGA analysis) Nucleotide sequences of pvl gene amplicon of MRSA isolate-97 (pus origin) AGAAAGAAAAAAACTTATTTTGGTGGCCAAACAATGAATTACCCTTTTATGAACACGGTGGTTTCAATCCTTC ATTTATTGCAACTGTTTCTCAGGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTAGGGCAAAAAT ATGGATGTTACTCAGGCTACTAGAAAAACAACACACTATGGCAATAGTTATTTAGAAGGATCTAGAAAACAC AACGCATTTGTAAACAGAATTTACACATTTAATTATGAGGTGAACGGGAAAACTCAGGAAATTAAGGTGAAG 28

29 GGACATAATTGATATGAAAAAAATAGTCAAATCATCATTTGTTACATCATTGGCTTTGCTTTTGCTATCCAAAA CATTAAAAAAAAAAAAAA 97_PVL_CUS-220_G06_036.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_G06_036.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_G06_036.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_G06_036.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_G06_036.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_G06_036.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_G06_036.ab1 gi dbj AP : gi dbj AB : gi dbj AB : _PVL_CUS-220_G06_036.ab1 AGAAAGAAAAAAACTTATTTTGGTGGCCAAACAATGAATTACCCTTTTATGAACACGGTG TATTTTGTGCCAGACAATGAATTACCCCCATTAGTACACAGTG TATTTTGTGCCAGACAATGAATTACCCCCATTAGTACACAGTG TATTTTGTGCCAGACAATGAATTACCCCCATTAGTACACAGTG *:** ****.************** :*::*:****.*** GTTTCAATCCTTCATTTATTGCAACTGTTTCTCAGGAAAAAGGCTCAGGAGATACAAGTG GTTTCAATCCTTCATTTATTGCAACTGTTTCTCATGAAAAAGGCTCAGGAGATACAAGTG GTTTCAATCCTTCATTTATTGCAACTGTTTCTCATGAAAAAGGCTCAGGAGATACAAGTG GTTTCAATCCTTCATTTATTGCAACTGTTTCTCATGAAAAAGGCTCAGGAGATACAAGTG ********************************** ************************* AATTTGAAATAACGTAGGGCAAAAATATGGATGTTACTCAGGCTACTAGAAAAACAACAC AATTTGAAATAACGTATGGCAGAAATATGGATGTTACTCATGCTACTAGAAGAACAACAC AATTTGAAATAACGTATGGCAGAAATATGGATGTTACTCATGCTACTAGAAGAACAACAC AATTTGAAATAACGTATGGCAGAAATATGGATGTTACTCATGCTACTAGAAGAACAACAC **************** ****.****************** **********.******** ACTATGGCAATAGTTATTTAGAAGGATCTAGAAAACACAACGCATTTGTAAACAGAATTT ACTATGGCAATAGTTATTTAGAAGGATCTAGAATACACAACGCATTTGTAAACAGAAATT ACTATGGCAATAGTTATTTAGAAGGATCTAGAATACACAACGCATTTGTAAACAGAAATT ACTATGGCAATAGTTATTTAGAAGGATCTAGAATACACAACGCATTTGTAAACAGAAATT *********************************:***********************:** ACACATTTAATTATGAGGTGAACGGGAAAACTCAGGAAATTAAGGTGAAGGGACATAATT ACACAGTTAAATATGAAGTGAACTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATT ACACAGTTAAATATGAAGTGAACTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATT ACACAGTTAAATATGAAGTGAACTGGAAAACTCATGAAATTAAAGTGAAAGGACATAATT ***** ****:*****.****** ********** ********.*****.********** GATATGAAAAAAATAGTCAAATCATCATTTGTTACATCATTGGCTTTGCTTTTGCTATCC GATATGAAAAAAATAGTCAAATCATCAGTTGTTACATCAATTGCATTGCTTTTGCTATCC GATATGAAAAAAATAGTCAAATCATCAGTTGTTACATCAATTGCATTGCTTTTGCTATCC GATATGAAAAAAATAGTCAAATCATCAGTTGTTACATCAATTGCATTGCTTTTGCTATCC *************************** ***********:* **:*************** AAAACATTAAAAAAAAAAAAAA AATACA AATACA AATACA AAAACATTAAAAAAAAAAAAAA **:*** Fig.4.20 Multiple sequences alignment of nucleotide sequences of pvl gene amplicon of MRSA isolate-97 (pus origin), S. aureus standard strains 7401, FD SB11 and BJ-216. (CLUSTAL OMEGA analysis) 29

30 Fig.4.21 Jal view of multiple sequences alignment of nucleotide sequences of pvl gene amplicon of MRSA isolate-97 (pus origin), S. aureus standard strains 7401, FD SB11 and BJ-216. (CLUSTAL OMEGA analysis) 135_MEC_CUS-220_C07_059.ab1 AAGGCCCCTTCCCATTTTTGTAATCAAGTCTAGTTTTTCTGTACCCCTTATCTGCAATTT 60 64_MEC_CUS-220_B07_061.ab GCCTTCCCAATTTTGTAATCAAGTCTAGTTTTTCTGTACCCCTTATCTGCAATTT 55 97_MEC_CUS-220_A07_063.ab TCCCAATTTTTGTAATCAAGTCTAGTTTTTCTGTACCCCTTATCTGCAATTT 52 * **.*:********************************************* 135_MEC_CUS-220_C07_059.ab1 TACTCTCACGGATTGCTTTTAACTTACCCCTTATCAGCAATTTTACTGAGAACTGCTTTT _MEC_CUS-220_B07_061.ab1 TACC--CTAGGATTGCTTTTAACTTACCCCTTATCAGCAATTTTACTGAGAACTGCTTTT _MEC_CUS-220_A07_063.ab1 T-AC--TTAGGATTGCTTTTAACTTACCCCTTATCAGCAATTTTACTGAGAACTGCTTTT 109 *. :.*************************************************** 135_MEC_CUS-220_C07_059.ab1 AACGCACCTCTTATCTGCAATTTTGCCTAGAACTGCTTTTAACGTACCTCTTATCTGCAA _MEC_CUS-220_B07_061.ab1 AACGCACCTCTTATCTGCAATTTTGCCTAGAACTGCTTTTAACGTACCTCTTATCTGCAA _MEC_CUS-220_A07_063.ab1 AACGCACCTCTTATCTGCAATTTTGCCTAGAACTGCTTTTAACGTACCTCTTATCTGCAA 169 ************************************************************ 135_MEC_CUS-220_C07_059.ab1 TTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTGCATGGAATTGCTTTT _MEC_CUS-220_B07_061.ab1 TTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTGCATGGAATTGCTTTT _MEC_CUS-220_A07_063.ab1 TTTTACTGAGAACTGCTTTTAACTTACCCCTTATCAGCAATTTTGCATGGAATTGCTTTT 229 ************************************************************ 135_MEC_CUS-220_C07_059.ab1 AACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTAACTGCAA _MEC_CUS-220_B07_061.ab1 AACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTATCTGCAA

31 97_MEC_CUS-220_A07_063.ab1 AACGTACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTATCTGCAA 289 *****************************************************:****** 135_MEC_CUS-220_C07_059.ab1 TTTTACTTAGAACTGCTTTTAACGTACCTCTTATCTGTAATTTTACTGAAAACTGCTTTT _MEC_CUS-220_B07_061.ab1 TTTTACTTAGAACTGCTTTTAACGTACCTCTTATCTGTAATTTTACTGAGAACTGCTTTT _MEC_CUS-220_A07_063.ab1 TTTTACTTAGAACTGCTTTTAACGTACCTCTTATCTGTAATTTTACTGAGAACTGCTTTT 349 *************************************************.********** 135_MEC_CUS-220_C07_059.ab1 AACAAACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTATCTGCAA _MEC_CUS-220_B07_061.ab1 AACAAACCTCTTATCTGCAATTTTACTTAAAACTGGTTTTAACAAACCTCTTATCTGCAA _MEC_CUS-220_A07_063.ab1 AACAAACCTCTTATCTGCAATTTTACTTAGAACTGCTTTTAACAAACCTCTTATCTGCAA 409 *****************************.***** ************************ 135_MEC_CUS-220_C07_059.ab1 TTTTACTTAGAATTGCTTTTACTATTCCTCTTATTAGTATAATCTCAATAAGAAGGCTTA _MEC_CUS-220_B07_061.ab1 TTTTACTTAGAATTGCTTTTACGATTCCTCTTATCAGTATAATCTCAGTAAGAAGGCGTA _MEC_CUS-220_A07_063.ab1 TTTTACTTAGAATTGCTTTTACGATTCCTCTTATTAGTATAATCTCAGTAAGAAGGCGTA 469 ********************** *********** ************.********* ** 135_MEC_CUS-220_C07_059.ab1 TAAAAACGAAAATTACAACCGATTTTGTAAGTGCGGACGCCAGAAAGAACAGTAAATCAG _MEC_CUS-220_B07_061.ab1 TAAAAATGAAAATTACTACCGATTTTGTAAGTGCGGACGCCTGAAGGAAACCTAAAAATA _MEC_CUS-220_A07_063.ab1 TAAAAATGAAAATTACAACCGATTTTGTAAGTGCGGACGCCTGAGGGAAAACGTAATTAA 529 ****** *********:************************:**..***.. :**: :. 135_MEC_CUS-220_C07_059.ab1 TT _MEC_CUS-220_B07_061.ab1 AGTT _MEC_CUS-220_A07_063.ab1 GTG- 532 Fig Nucleotide sequences homology between mec-a-h genes of MRSA isolates 64 (blood origin), 135 (urine origin), and 97 (pus origin). Blue line indicates deletion where as the variability is shown by nucleotide marked in red. 31

32 Fig Jal view of nucleotide sequences homology between mec-a-h genes of MRSA isolates 64 (blood origin), 135 (urine origin), and 97 (pus origin). 64PVL_CUS-220_G06_036.ab1 GGCCCCATTATTTTGTTGGAATAACCATATAGTCAAAATCCGAGAGACTATTTTGTGCCA _PVL_CUS-220_H06_034.ab CCCCAAAAAAACA 13 97_PVL_CUS-220_G06_036.ab AGAAAGAAAAAAACTTATTTTGGTGGCCA 29. :..** 64PVL_CUS-220_G06_036.ab1 GACAATGAATTACCCCCATTAGTACACAGTGGTTTCAATCCTTCATTTATTGCAACTGTT _PVL_CUS-220_H06_034.ab1 AATGGAATTCCCCCCCATTATTACACGGTGGGTTTCAATCCTTCATTTATTGCAACTGTT 73 97_PVL_CUS-220_G06_036.ab1 AACAATGAATTACCCTTTTATGAACACGGTGGTTTCAATCCTTCATTTATTGCAACTGTT 89.*..:.::.*** :*:: :.... ****************************** 64PVL_CUS-220_G06_036.ab1 TCTCATGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTATGGCAGAAATATG _PVL_CUS-220_H06_034.ab1 TCTCATGAAAAAGGCTCAGGAAATACAAGTGAATTTGAAATAACGTAGGGCAAAAATATG _PVL_CUS-220_G06_036.ab1 TCTCAGGAAAAAGGCTCAGGAGATACAAGTGAATTTGAAATAACGTAGGGCAAAAATATG 149 ***** ***************.************************* ****.******* 64PVL_CUS-220_G06_036.ab1 GATGTTACTCATGCTACTAGAAGAACAACACACTATGGCAATAGTTATTTAGAAGGATCT _PVL_CUS-220_H06_034.ab1 GATGTTACTCATGCTACTAAAAAAACAACACACTATGGCAATATTTATTTAAAAGGATCT _PVL_CUS-220_G06_036.ab1 GATGTTACTCAGGCTACTAGAAAAACAACACACTATGGCAATAGTTATTTAGAAGGATCT 209 *********** *******.**.******************** *******.******** 64PVL_CUS-220_G06_036.ab1 AGAATACACAACGCATTTGTAAACAGAAATTACACAGTTAAATATGAAGTGAACTGGAAA _PVL_CUS-220_H06_034.ab1 AAAATACACAACGCATTTGTAAACAAAATTTACACAGTTAAATATGAAGTGAACTGGAAA _PVL_CUS-220_G06_036.ab1 AGAAAACACAACGCATTTGTAAACAGAATTTACACATTTAATTATGAGGTGAACGGGAAA 269 *.**:********************.**:******* ****:*****.****** ***** 64PVL_CUS-220_G06_036.ab1 ACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAGTCAAATCATCAGT _PVL_CUS-220_H06_034.ab1 ACTCATGAAATTAAAGTGAAAGGACATAATTGATATGAAAAAAATAGTCAAATCATCATT _PVL_CUS-220_G06_036.ab1 ACTCAGGAAATTAAGGTGAAGGGACATAATTGATATGAAAAAAATAGTCAAATCATCATT 329 ***** ********.*****.************************************* * 64PVL_CUS-220_G06_036.ab1 TGTTACATCAATTGCATTGCTTTTGCTATCCAATACATTTGATGAAAAAGAAAC

33 135_PVL_CUS-220_H06_034.ab1 TGTTACATCATTTGCTTTGTTTTTGCTATCCAAAACATTTAAAACAAAAAAAA _PVL_CUS-220_G06_036.ab1 TGTTACATCATTGGCTTTGCTTTTGCTATCCAAAACATTAAAAAAAAAAAAAA- 382 **********:* **:*** *************:*****:.*:..****.*** Fig Nucleotide sequences homology between pvl genes of MRSA isolates 64 (blood origin), 135 (urine origin), and 97 (pus origin). Blue line indicates deletion where as the variability is shown by nucleotide marked in red. 33

34 Fig Jal view of nucleotide sequences homology between pvl genes of MRSA isolates 64 (blood origin), 135 (urine origin), and 97 (pus origin). Stand KQF-VK 5 64_mec_MRSA ASALTKSVVIFIFIRLLTEIILIRGIVKAILSKIADKRFVKTSFK-NCR-EVC-KQFSVK _mec_MRSA VKAILSKIADKRFVKSSSK-NCR-EVC-KQFSVK 31 97_mec_MRSA IVKAILSKIADKRFVKSSSK-NCR-EVC-KQFSVK 32 * * **** ** Stand LQIRGLLKAVLSKIADKRFVKSSSQ-NYR-EVR-KQF-VKLQIRGLLKAVLSKIADKRYV 61 64_mec_MRSA LQIRGTLKAVLSKIADKRFVKSSSK-NCR-EVR-KQFHAKLLIRGKLKAVLSKIADKRYV _mec_MRSA LQIRGTLKAVLSKIAVKRFVKSSSK-NCR-EVR-KQFHAKLLIRGKLKAVLSKIADKRYV 88 97_mec_MRSA LQIRGTLKAVLSKIADKRFVKSSSK-NCR-EVR-KQFHAKLLIRGKLKAVLSKIADKRYV 89 ***** ********* ********:** *********.** *** ************** Stand KSNSMQNC--GVS-KQFSVKLQIRGTLKAVLGKIADKRCVKSS-SQ-NC--GVS _mec_MRSA KSSSRQNCR-EVR-KQFSVKLLIRGKLKAILG-NCR--GVQKN-T-L _mec_MRSA KSSSRQNCR-EVR-KQFSVKLLIRGKLKAIRESKIADKGYRKTRLD _mec_MRSA KSSSRQNCR-EVR-KQFSVKLLIRGKLKAIL-SKIADKGYRKTRLDYKNW **.* *** * * ******** ***.***: :.. * Fig Amino acid sequences alignment of mec-a-h gene amplicons of MRSA isolates-64 (blood origin), 135 (urine origin), and 97 (pus origin).the highlighted portion reveal substitutions of amino acid at these positions. 34

35 Fig Jal view of amino acid sequences alignment of mec-a-h gene amplicons of MRSA isolates 64 (blood origin), 135 (urine origin), and 97 (pus origin). 135_pvl_MRSA GIPPHYYTVGFNPSFIATVSHEKGSGNTSEFEIT-GKNMDV 40 64_pvl_MRSA PYSQNPRDYFVPDNELPPLVHSGFNPSFIATVSHEKGSGDTSEFEITYGRNMDV 54 Stand_FD-SB11 LFVGYKPYSQNPRDYFVPDNELPPLVHSGFNPSFIATVSHEKGSGDTSEFEITYGRNMDV 60 97_pvl_MRSA FWWPNNELPFYEHGGFNPSFIATVSQEKGSGDTSEFEIT-GKNMDV 45 * ***********:*****:******* *:**** 135_pvl_MRSA THATKKTTHYGNIYLKGSKIHNAFVNKIYTVKYEVNWKTHEIKVKGHN-YEKNSQIIICY 99 64_pvl_MRSA THATRRTTHYGNSYLEGSRIHNAFVNRNYTVKYEVNWKTHEIKVKGHN-YEKNSQIISCY 113 Stand_FD-SB11 THATRRTTHYGNSYLEGSRIHNAFVNRNYTVKYEVNWKTHEIKVKGHN-YEKNSQIISCY _pvl_MRSA TQATRKTTHYGNSYLEGSRKHNAFVNRIYTFNYEVNGKTQEIKVKGHN-YEKNSQIIICY 104 *:**::****** **:**: ******: **.:**** **:***************** ** 135_pvl_MRSA IICFVFAIQN _pvl_MRSA INCIAFAIQYI 124 Stand_FD-SB11 INCIAFAIQYS _pvl_MRSA IIGFAFAI * :.*** Fig Amino acid sequences alignment of pvl gene amplicons of MRSA isolates 64 (blood origin), 135 (urine origin), and 97 (pus origin) with standard MRSA strain FD SB11. The highlighted portion reveals substitutions of amino acid at these positions. 35

36 Fig Jal view of amino acid sequences alignment of pvl gene amplicons of MRSA isolates 64 (blood origin), 135 (urine origin), and 97 (pus origin) with standard MRSA strain FD SB11 2kb Extracted plasmid Fig.4.30 Gel electrophoresis of plasmid DNAs from MRSA isolates. A single band of plasmid DNA of <2 kb is demonstrable in the extracted plasmid DNAs of isolates mentioned at the top. Isolate 34 in Lane 1 does not contain any plasmid DNA. Lane M- Molecular marker of 2kb-8kb. 36