The discovery of penicillin in 1928 was followed by the discovery and commercial production of many other antibiotics. We now take for granted that any infectious disease is curable by antibiotic therapy. Antibiotics are manufactured at an estimated scale of about 100,000 tons annually worldwide, and their use had a profound impact on the life of bacteria on earth. More strains of pathogens have become antibiotic resistant, and some have become resistant to many antibiotics and chemotherapeutic agents, the phenomenon of multidrug resistance. Multidrug-resistant organisms develop when antibiotics are taken longer than necessary or when they are not needed.  Multidrug resistance in bacteria occurs by the accumulation, on resistance (R) plasmids or transposons, of genes, this will lead to Mutational Alteration of the Target Protein Development of resistance in many human pathogens has emerged into a large scale and resistance has evolved into multidrug resistance in the present regime.

1. Aims & Objectives
• To collect samples from hospital sewage and Municipal drains
• To isolate and identification of multidrug resistant bacteria
• To study of urbanisation is influence to turn resistant
• To Characterisation of gene to responsible for multi drug resistant
• To study of control measures

2. Introduction
• The rapid global urbanization and other extensive anthropogenic activities indiscriminate use of
antibiotics in human and veterinary medicine is often associated with increased bacterial resistant
and the ARB have created serious problems in the treatment of infectious diseases
• These bacteria and their antibiotic resistant gene enter the environment through varies sources like
Hospital waste water and municipal waste water are potential source for entry of ARB and ARG in to
the natural environment
• Antibiotic resistance in the environment can be transferred form pathogenic bacteria to non
pathogenic bacteria , which impair water ecology throughout change in, population dynamics and
physiology.
• A study has show that a large number of bacteria and resistant genes particularly in hospital strains
have been found in hospital waste water discharge
• ARGs can be transferred among the bacteria in the environment through Plasmids, Transposons and
Integrans and mechanism involved is, Permeability changes , Active efflux of microbial cell,
Degradation of the antimicrobial agent, Enzymatic modification of antibiotic, Modification of
antibiotic target and over production of target

3. Introduction
• The discovery of penicillin in 1928 was followed by the commercial production of many other antibiotics at
an esteemed scale of 1,00,000 tons annually worldwide, and their use had a profound impact on the life of
bacteria on earth.
• More strains of pathogens have become antibiotic resistant, and some have become resistant to many
antibiotics and chemotherapeutic agents, the phenomenon of multidrug resistance.
• Indeed, some strains have become resistant to practically all of the commonly available agents. A notorious
case is the methicillin-resistant Staphylococcus aureus (MRSA), which is resistant not only to methicillin
(which was developed to fight against penicillinase-producing S. aureus)

4. Some bacteria showing resistance to Antibiotics
S.No Name of the pathogen Resistance to antibiotic
1 Klebsiella pneumoniae Amikacin (30µg), Cifroflaxacin (5µg), Gentamicin (10µg)
Cefotoxime (30µg), Norflaxacin (10µg), Vankomycin
(30µg), Co-Trimaxazole (25µg)
2 Pseudomonas aeruginosa Amphicillin(10µg), Cefroflaxacin (5µg), Salbactam(10µg),
Ceftazadime(30µg), Vankomycin (30µg)
3 Escherichia coli Amikacin (30µg), Cefroflaxacin (5µg), Levoflaxacin (5µg),
Ceftazadime(30µg), Piperacillin(100µg), Vankomycin
(30µg), Co-Trimaxazole (25µg)
4 Staphylococcus aureus Amikacin (30µg), Cefazoline (10µg), Gentamicine (10µg),
Ceftazadime(30µg), Methicilin (30ug), Co-Trimaxazole
(25µg)

5. Materials &Methods
 Sample collection:
• A total of 100 samples were collected from different areas of hospitals sewage and
municipal drainages located in Warangal in 30th march 2018 to 29th April 2019.
• All samples were collected in a sterile container .
• After collection, samples were stored in ice cold sterile containers during
transportation from the collection point to the laboratory.
• The samples collected from hospitals sewage and municipal drainages were
transferred in LB medium (Himedia, India) to the department of Microbiology
Kakatiya university, Warangal. For the isolation and identification all the samples
collected from different sources

6. Geographical distribution of Samples collection points
1.MGM Hospital sewage (25 nos)
2.Prashanthi Hospital Sewage (25 nos)
3.Municipal wastes in streams (25 nos)
4.Municipal Drains at varies places of Warangal (25 nos)

7. Bacterial isolation
• Samples collected from source were inoculated (5 μL inoculums) in LB medium
and stored at 4°C.
• The isolates were further characterized by streaking on Nutrient, MacConkey agar
and incubated overnight at 37°C.
• The phylogenetic classification of the bacteria isolates was conducted by PCR
and assigned to the 5 major bacteria groups SRKU20, SRKU42, SRKU55, SRKU78
and group SRKU99 according to the 16S rRNA analysis.
• Based on the initial biochemical tests, a total of 288 isolates from 100 samples
were selected and stored in sterile NA slants for further tests.

8. Antibiotic sensitivity
• All the bacterial isolates were tested for antibiotics resistance including
• Amikacin (30µg) Amoxyclav (30µg) Amphicillin (10µg), Cifroflaxacin (5µg)
• Ofloxacin (5µg) Levofloxacin (5µg) Gentamicin (10µg), Cefixime(30µg),
• Cefotoxime (30µg) Ceftazadime (30µg) Oxacillin (1µg)
• Piperacillin (100µg) Tobramicin (10µg) ,Gentamicin (10µg) , Penicillin G (10 U)
• Colistin (10µg) Erythromycin (15µg) Amoxicillin(10µg) Tetracycline(30µg)
• Vankomycine (30µg) Co-Trimaxazole (25µg)
• All antibiotic discs were purchased from Hi media Laboratories, India

9. Morphological appearance bacterial isolates having Multi drug resistant
A.
B.
A. SRKU 20
B. SRKU 42
C. SRKU 55
D. SRKU 99
C.
D.

10. Colony morphology for antibiotic resistant strains
S No. Sample ID Gram Stain Shape Nutrient agar MecConkey Agar
1 SRKU 20 Negative Rod Circular , Greyish white
mucoid colonies (2-3mm)
Large shinny and dark pink in
color with mucoid colonies
(2-3mm)
2 SRKU 42 Negative Rod Irregular, Smooth, Greenish
blue colonies (2-4mm))
Circular, Smooth, Mucoid,
colourles colonies (2-3mm)
3 SRKU 55 Negative Rod Large, Thick, Greyish,
White, Moist and Smooth
(1-3mm)
Bright pink with mucoid
colonies (2-3mm)
4 SRKU 78 Negative Rod Circular , Greyish white
mucoid colonies (2-3mm)
Large shinny and dark pink in
color with mucoid colonies
(2-3mm)
5 SRKU 99 Negative Rod Irregular,Smooth, Greenish
blue colonies (2-4mm))
Circular, Smooth, Mucoid,
colourles colonies (2-3mm)

11. Biochemical tests for antibiotic resistant strains
Sno Sample ID Indole Methyl
Red
Voges–
Proskauer
Citrate Catalase Oxidase H 2S
production
1 SRKU 20 Negative Negative Positive Positive Positive Negative Negative
2 SRKU 42 Negative Negative Negative Positive Positive Positive Positive
3 SRKU 55 Positive Positive Negative Negative Positive Negative Negative
4 SRKU 78 Negative Negative Positive Positive Positive Negative Negative
5 SRKU 99 Negative Negative Negative Positive Positive Positive Positive

12. Plate: II Antibiotic sensitivity containing selected
multidrug resistance strains
SRKU20. Klebsiella pneumonia
SRKU42 Pseudomonas aeruginosa
SRKU55 Escherichia coli
SRKU78 Klebsiella pneumonia
SRKU99 Pseudomonas aeruginosa

13. Plate: II Antibiotic sensitivity containing selected
multidrug resistance strains
Sno Sample ID Source of Sample
Amikacin
(30µg)
Amphicillin(10µg)
Cefazoline(10µg)
Cefroflaxacin
(5µg)
Ofloxacin
(5µg)
LeVoflaxacin
(5µg)
Gentamycine
(10µg)
Salbactam(10µg)
Cefixime(30µg)
Cefotoxime
(30µg)
Ceftazadime(30µg)
Piperacillin(100µg)
Nitrofurointain(30ug)
Norflaxacin
(10µg)
Cefifime(10µg)
Vankomycin
(30µg)
Co-Trimaxazole
(25µg)
1 SRKU20 Hospital Sewage (MGM Hos)
R S S R S S R S S R S S S R S
R R
2 SRKU42
Municipal Drainage (Alankar
Junction)
S R S R S S S R S S R S S S S
R S
3 SRKU55 Municipal Drainage (Gandhi agar)
R S S R S R S S S S R R S S S
R R
4 SRKU78 Hospital Sewage (Prashanthi) R S R S S S R S S S R S S S S R R
5 SRKU99 Hospital Sewage (Prashanthi)
S R S R S R S S S R S S S S S
R R
R = Resistant S = Sensitive

14. Molecular identification
• 1. Klebsiella pneumoniae (SRKU20)
gDNA and 16S Amplicon QC data: (SRKU20)
Source: Hospital Sewage (MGM Hospital)
Characteristic features:
1. Non motile, non spore forming, Gram
negative bacteria with a prominent
polysaccharide capsule,
2. The size of Klebsiella pneumoniae is
about 1–2 µm × 0.5–0.8 µm
3. In MecConkey agar - Large shinny and
dark pink in color with mucoid colonies
4. Colony s are circular 2-3mm diametre
5. IMViC (- - + +)
6. Opportunistic pathogen

15. • AGKSSGGCAGGCTACACATGCAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGAT
GGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAAYGTCGCAAGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGA
TTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACG
GGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGG
AGGAAGGCGKTRAGGTTAATAACCTYRYCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGC
GTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTYAACCTGGGAACTGCATTCGAAACTGGCAGGCT
AGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGA
CGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTT
CCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT
AATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTWCCAGAGATGCWTTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGC
TGTCGTCAGCTCGTGTTGTGAATGTTGGGTAGTCCCGCACGAGCGCAACCCTTATCTTGTGCAGCGGATCAGGCCGGCAACTCAAAGCAGACTGGCAGTGATA
ACTGGCAGAAGGTGGGGGATKAACGTCA
• Reverse Seq Data
• CTAYMAAAGTGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATT
CACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCT
CGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACT
GGCAGTCTCCTTTGAGTTCCCGGCCKRACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACA
GCCATGCAGCACCTGTCTCACAGTTCCCGAAGGCACCAAWSCATCTCTGSWAAGTTCTGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTA
AACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGC
CACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCT
TTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTT
TCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCG
TATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGMYGAGGTTATTAACCTCACGCCTTCCTCCCCGCTGAAAGTGCTT
TACACCCGACGCTTCTTCACACACGCCGCATGCCTGCATCAGCTGCGCCAATGTGCATATCCCACTGCTGCATCCCGCTAGATCTGATCGGTATCTCAGCTATCA
Sanger Sequence Chromatogram
Forward Seq data

16. 2. Pseudomonas aeruginosa (SRKU42)
Source: Municipal Drainage
Characteristic features:
1. Aerobic, Non sporeing , motile Gram
negative bacilli
2. which produce water soluble pigments
which diffuse through the media,
the best-known pigments are pyocyanin
(blue green)
3. Produce large , opaque ,non lactose
fermenting organisms on MecConkey agar
4. On NAM produce greenish blue colonies
5. IMVP (- - -), H2S (+) Catalase (+) and
Oxydase (+)
6. Normal flora in the gastro intestinal tract

17. Sanger Sequence Chromatogram
• CGGCGCGGCTACACATGCAAGTCGARCGGATGASGGAGCTWGYTCCTGGATTCRSCGSCGGACGGGTGARTAATGCCTARGAATCTGMCTGGTARTGGGGG
ATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTGAGGGAGAAAGTGGGGGATCTTCGGACCTCRCGCTATCRGATGAGCCTARGTCGGATTAKCT
AKTTGGTGGGGTAAAGGCCTACCMAGGCGACKATCCGTAWCTGGWCTGAGAGGATGATCARTCACACTGGAACTGASACACRGKCCAGACTCCTACGGSA
GGCAGCAGYGGGGAATATTGSACAATGGGCGAAAGCCTGATCCAGCCRTGCCSCGTGTGTGAAGAAGGTCTTCGGATTGWAMAGCACTTTAAGTTGGGAG
GAAGGGCASKRAKTTAATACCTTGCTGTTTTGACRTTAYCRACASAATAAGCACCGGMTAACTTCGTGCCAGCASCCGCGGTAATACGAAGGGTGCAAGCG
TTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCAGCAWGTTGGATGTGAAAATCCCCGGGCTCAACCTGGSAACTGCATCCAAAACTACTGAG
CTASAGTACGGWAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCRTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGKACTGATACT
GACACTGAGGTGCRAAAGCGTGGGGAGCARACASGATTAGATACCCTGGTAGGCCACGCCRTAACGATGWCTACTARCCCTTGGGRATCCTTGASATCWTT
AGWGGMGCAGCTAACGCGATAAGGKCGACCGCCYGTSSGAGKACGGSMCGCAASGTWAAACTCAAACTGAATTGGASRGGGGSSMCGCCCAAGCGGKR
GAGCAWGTGGRTTKAATTSGAGCCACGCGAAGAACCTWACCTGKCTTTGAMTTGCTGRCAACTTACAGAGCATGGATTGGGTGCYGTCKSACTCAAMMM
CAGTGACWGSATGGCTTGTCGTCAGSTCMWGACYGARAKGTGGGGTAGGTYCCGTACARCSACCTRKYCTTRCTTMCAKCCTCGGGAACCTAGTGACTGC
CGKCAWCCGAGGAGTGG CATGASTCAGKCATYAATAGC
Reverse Seq Data
• CTAAATCAWCGTTGGAAASCGKCCTCCTTGCGGTTAGACTASCTACTTCTGGAGMAACCCAMTCCCATGGKGTGACGGGSGGKGTGTACAAGGCCCGGGA
ACGTATTCWCCGYGACATTCTGATTCMCGATTACTAGCGATTCCSACTTCACGYAGTCGAGTTGCAGACTGCRATCCGGACTACGATCGGKTTTWTGGGATT
AGCTCCACCTCGCGGSTTGGCAACCCTTTGTACCSACCATTGTAKCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCC
TCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCCGARGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACR
ACACRAGCTGACGACAGCCATGCAGCACCTGTGTCTGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCAGCATGTCAAGGCCAGGKAAGGTTC
TTCGCGTTGCTTCKAAWTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGARTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGAC
TTATCGCGTTAGCTGCGCCACTAARATCTCAAAGATCCCAMCRGCTAGTCGACATCGTTTACGGCGTGGACTACCASGGTATCTAATCCTGTTTGCTCCCCAC
GCTTTYKCACCTCASYGTCAGTATYASWCCASGWGGTCGCCTTCKCCACTGGTGTTCCTTCCTATATCTACRCATWTTCACCGCTACACAGGARRATTCYMC
CAYCCTCTAYYGTACTCTMGCTYWSYMGTTTTGGATRYRSYTCYCWKSGTTGAGMSCCGGGRGATTTYWCATCYCASATTGMWGAACCASCTAYGCGCKC
GTTAYRCCCAGTAAATACYCGAKTAAMMGSTTTGCACCCYTMGTATWTCCGCGGCTGCTGGGCACGARGTTAGGCCSGTGCTYATCTGTGRTAACGTCAAA
MYCAGCRAAGYATTACTWACTGAYCGTTCCTCCTACWTAAAASTGMCTTACAGWCCGAGACGTYCTTCACACWCGCGCATGGCTGCATCAGGACTCGCAT
GCGAAGTCCATTGCTGCTCCGTAGGAGT
Forward Seq data

18. 3. Escherichia coli (SRKU55)
Source: Municipal Drainage (Gandhi agar)
Characteristic features:
1. Non sporing, capsulated, motile gram
negative bacteria.
2. The size of Escherichia coli is about 1–3
µm × 0.4–0.7 µm
3. Circular, Smooth, mucoid bright pink
colonies due to lactose fermentation
are produced in MecConkey Agar
4. IMViC (+ + - - )

19. • TSCGGGCAGCTACACATGCAAGTCGAACGGTAACAGRAAGCAGCTTGCTGYTTYGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATG
GAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGA
TTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTAC
GGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGG
GAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAG
CGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGC
TTGAGTCTCGTAGAGGGGGGTAGAATTCCAGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGCGGCCCCCTGGACGAGACTGACG
CTCAGTGCGAAAGCGTGAGAGCAACAGATAGAWACCTGGTAGTCCMCGCCGTAACGATKTTCGACTKGGAGGTKGKKGCCCTKGRRGGCSGTKGCC
Reverse Seq Data
• TATMAAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTAT
TCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTC
TCGCGAGRTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCAC
TGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGAC
AGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACATTCTCATCTCTGAAAACTTCCGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAA
ACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCC
ACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGCGTGACTACCAGGTATCTAATCCTGTTTGCTCCCACGCTTTCSCMCTGACGTCAGTCTCGTCAGG
GCGCTCGCACGTATCTCAGATCTYTACGCATTCACCGCCWMMCTGGAATTCTACCCCCCCCTCYWMSARRACYTMAGCCT
Sanger Sequence Chromatogram
Forward Seq data

20. 4. Klebsiella pneumoniae (SRKU78)
Source: Hospital Sewage (MGM Hospital)
Characteristic features:
1. Non motile, non spore forming, Gram
negative bacteria with a prominent
polysaccharide capsule,
2. The size of Klebsiella pneumoniae is
about 1–2 µm × 0.5–0.8 µm
3. In MecConkey agar - Large shinny and
dark pink in color with mucoid colonies
4. Colony s are circular 2-3mm diametre
5. IMViC (- - + +)
6. opportunistic pathogen

21. Sanger Sequence Chromatogram
TSCGGGCAGCTACACATGCAAGTCGAACGGTAACAGRAAGCAGCTTGCTGYTTYGCTGACGAGTGGCGGACGGGTGAGTA
ATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAG
GGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACG
ATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGG
GAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGC
GGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAG
CCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGT
GAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGTGTA
GCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGCGGCCCCCTGGACGAGACTGACG
Reverse Seq Data
TATMAAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGT
GTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAG
ACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGRTCGCTTCTCTTTGTATGCGCCATTGTAGCAC
GTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGA
GTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCT
GACGACAGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACATTCTCATCTCTGAAAACTTCCGTGGATGTCAAGACCAG
GTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCT
TGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACA
TCGTTTACGCGTGACTACCAGGTATCTAATCCTGTTTGCTCCCACGCTTTCSCMCTGACGTCAGTCTCGTCAGGGCGCTCGCA
CGTATCTCAGATCTYTACGCATTCACCGCCWMMCTGGAATTCTACCCCCCCCTCYWMSARRACYTMAGCCT
Forward Seq data

22. 5. Pseudomonas aeruginosa (SRKU99)
Source: Hospital Sewage (Prashanthi hospital)
Characteristic features:
Characteristic features:
1. Aerobic, Non sporeing , motile Gram
negative bacilli
2. which produce water soluble pigments
which diffuse through the media,
the best-known pigments are pyocyanin
(blue green)
3. Produce large , opaque ,non lactose
fermenting organisms on MecConkey agar
4. On NAM produce greenish blue colonies
5. IMVP (- - -), H2S (+) Catalase (+) and
Oxidase (+)
6. Normal flora in the gastro intestinal tract

23. Sanger Sequence Chromatogram
• TGSGGCAGGCCTACACATGCAAGTCGARCGGATGAASGRAGCTTGCTCCTGGATTCRGCGGCGGACGGGTGAGTAATGCCTAG
GAATCTGCCTGGTAGTGGGGGATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTGAGGGAGAAAGTGGGGGATCTTC
GGACCTCRCGCTATCAGATGAGCCTAGGTCGGATTAKCTAGTTGGTGGGGTAAAGGCCTACCMAGGCSACGATCCGTAWCTGGT
CTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGSAGGCAGCAGTGGGGAATATTGGACAATGG
GCGAAAGCCTGATCCAGCCRTGCCSCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTR
AGTTAATACCTTGCTGTTTTGACGTTAYCRACASAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGC
AAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGG
AACTGCATCCRAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCMTGTGTAGCGGTGAAATGCRTAGATATAGGAA
TGAACACCAGTGGCGAGCGACMCTGGACTGATACTGACACTGAGTGCGAAGCGTGGGAGCGAAMGATAGATACCCTGCTAGT
CAGTCGTAACGATKTCAACTAGCCKKKTGGGAWCCTKMMRAATCTTTWATTGAGC
• Reverse Seq Data
• TAYCCCWCCSYTGGTACCKKCCYCCTTGCGGTTAGACTASCTACTTCTGGAGCAACCCACTCCCWTGGKGTGACGGGSGGKGTGT
ACAAGGCCCGGGAACGTATTCACCGYGACATTCTGATTCMCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGC
RATCCGGACTACGATCGGKTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAG
CCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTARAGTGCCCACCCG
AGGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGC
AGCACCTGTGTCTGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCAGSATGTCAAGRCCAGGTAAGGTTCTTCGCGTT
GCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGG
CGGWCGACTTATCGCGTTAGCTGCGCCACTAAGATSTCAARGATCCCAACRGSCTAGWCGACATCGTTTACGGCGTGGACTACC
AGGTATCTAATCCTGTTTGCTCCCCMMGCTTCGCACTCRCTGTCAGTATCAGTCAAGRWGTCGCTTCGCCACTGGTGTTCTTCCC
WTATYCTACSCATTTMMCGSCCTMMMCMRGGGGGGA
Forward Seq data

24. Thank you for giving the space
• Prof .G.Gangadhar reddy sir, Dean,
• faculty of Sciences
• Head of Dept Dr Sujatha maam
• Board of Studies Chairman Dr Raja Komuraiah sir
• Dr Venkataiah Sir
• All the mentors
• Co scholars and all PG students
thank you