“MOLECULAR CHARACTERIZATION OF MULTI DRUG RESISTENT STREPTOCOCCUS SPECIES USING AFLP (AMPLIFIED FRAGMENT LENGTH POLYMORPHISM )”

SYNOPSIS FOR

M. PHARM DISSERTATION

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

BENGALOORU, KARNATAKA.

SUBMITTED BY,

PIDATALA NAGARJUNA REDDY

I M. PHARM

DEPARTMENT OF PHARMACOLOGY

P.E.SCOLLEGE OF PHARMACY

BENGALOORU – 560050

(2010-2012)

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

BENGALOORU, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION

1.0 / NAME AND ADDRESS OF THE CANDIDATE. / PIDATALA NAGARJUNA REDDY
PERMANENT ADDRESS
#454, 1ST FLOOR,2NDD-MAIN ROAD 4THCROSS MUTYALANAGAR BANGALORE-560054
LOCAL ADDRESS
SAME AS ABOVE
2.0 / NAME OF THE INSTITUTION. / P.E.S.COLLEGE OF PHARMACY
Hanumanthanagar, 50 feet road, B.S.K 1st stage, Bengalooru – 50,
3.0 / COURSE OF STUDY AND SUBJECT. / M.PHARM.
PHARMACOLOGY.
4.0 / DATE OF ADMISSION TO COURSE. / 28th May 2010
5.0 / TITLE OF THE TOPIC:
“MOLECULAR CHARACTERIZATION OF MULTI DRUG RESISTENT SPECTOCOCCUS SPECIES BY USING AFLP (AMPLIFIED FRAGMENT LENGTH POLYMORPHISM )”
6.0
7.
8. / 6.1. NEED FOR THE STUDY:
Multidrug resistance is a condition enabling a disease-causing organism to resist distinct drugs or chemicals of a wide variety of structure and function targeted at eradicating the organism. Organisms that display multidrug resistance can be pathologic cells, including bacterial and neoplastic (tumor) cells.
Various microorganisms have survived for thousands of years by their being able to adapt to antimicrobial agents. They do so via spontaneous mutation or by DNA transfer. It is this very process that enables some bacteria to oppose the assault of certain antibiotics, rendering the antibiotics ineffective. These microorganisms employ several mechanisms in attaining multidrug resistance.
Large amount of antibiotics used for human therapy in farm animal and aquaculture, resulted in the solution of pathogenic bacteria resistant to multiple drugs. Multidrug resistance in bacteria may be generated by bacteria may accumulate multiple genes on resistant (R) plasmids or by increased expression of genes that code for multidrug efflux pumps extruding wide range of drugs .
Streptococcus is a genous of spherical gram +ve bacteria belongs to phylumfirmicutes. There are about 26 species present in this genus. These species are responsible for many cases of life threatening infections, bacteramia, meningitis, pneumonia, endocarditis, erysipelas and necrotizing fasciitis. Since the late 1970’s and 1980’s antibiotic resistance among the pneumococci has become emerging problem. Several multi drug resistant clones have rapidly spread throughout the world. They also resistant to other drug vizpenicillin, beta-lactum and non beta-lactum has been rapidly increasing and resistance expanding to multiple antimicrobial agents in many parts of the world. Similarly there is increase in erythromycin resistance among strepto coccus pyrogen species cause more concerned. In India the prevalence of multi drug resistant streptococcus in 1997 was about 3.8%.
The development of molecular techniques for genetic analysis has led to great increasing our knowledge of microbial genetics and understanding the structure and behavior of bacterial genomes. These molecular techniques, in particularly use of molecular markers, have been used to monitor DNA sequence variation in and among the species and create new sources of genetic variation by introducing to control the diseases.By using pulsed field gel electrophoresis(PFGE), ribotyping and finger printing are used for molecular characterization [ref]. On the literature survey
it was found that various species of streptococcus bacteria producing / showing multi-drug resistance. Since we are doing this project in collaboration with M/s Azyme bioscience pvt ltd. Bangaloreat Biotechnology department. At this point of time it is not possible to disclose the name of the streptococcus species and detailed procedures for which the molecular characterization is to be performed.
6.2REVIEW OF THE LITERATURE:
Ashraf M. et al. reported the quinolone-resistance-determining region of parC of topoisomerase IV and may be responsible for the reduced susceptibility to ciprofloxacin in this strain.[1]
Mohammed et al.The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in streptococcus species.[2]
Rosmari Horner et al Describethe fi rst case of Spontaneous bacterial peritonitis (SBP) at the University Hospital of Santa Maria (HUSM) caused by S. bovis, resistant to the antibiotics erythromycin and clindamycin (inducible clindamycin resistance detected by disk diffusion test using the D-zone test).[3]
Kelly G. et alThe relationship of S bovis to endocarditis, meningitis, brain abscesses, and peritonitis has also been well described. However, S bovis is a rare pathogen infecting joint prostheses and should raise the possibility of a gastrointestinal lesion.[4]
Han-Tan Chai et alStreptococcus bovis endocarditis complicated by a superiormesenteric artery mycotic aneurysm and systemic septic emboli in a patient with colon diverticulitis and they develop multidrug resistance in patient. [5]
Ste´phan Ellmerich et alThere are numerous reports documenting the correlation between Streptococcus bovis bacteraemia and endocarditis in conjunction with colonic diseases. [6]
Amplification method:[7]
Identication of envelope proteins with lytic activity Most bacterial species described so far
contain several lytic enzymes. However, in spite of this fact, only a limited number of these enzymes have been puri®ed because of their presence in small amounts and/or because of their high-af®nity binding to the cell wall. Using a procedure previously published to characterize the pneumococcalglucosaminidase (GarcõÂa et al., 1989), we identi®ed, by SDS±PAGE, four protein bands with apparently strong choline-binding af®nity . Residual murein hydrolase activity was detected when the major protein band (Mr of about 55 000) was excised and eluted from the gel, renatured and assayed on choline-labelled cell walls (data not shown). The N-terminal amino acid sequence of this protein was determined yielding Asn±Glu±Thr±Glu±Val± Ala±Lys±Thr±Ser±Gln. This sequence was compared with the translated version of the partial nucleotide sequence of the genome of a pneumococcal strain that has been recently released (see below), and a perfect match with an internal peptide of a gene product was found. This gene (hereafter designated lytC ) was located in the 3832 bp contig no. 4270. Analysis of this contig (Fig. 2A) revealed that the lytC gene is preceded by an open reading frame, whose product showed strong similarity (66% identity, 78% similarity) to the Tpi triosephosphate isomerase of Lactobacillus delbrueckii (accession no. AJ000339). Other genes further upstream of tpi±lytC were also preliminarily identi®ed on the basis of sequence similarities: the ®rst putative gene product of the contig, albeit incomplete, is 48% identical (66% similar) to the homoserine O-succinyltransferase (MetA) of Escherichia coli (accession no. U00006), whereas the gene immediately preceding tpi appears to code for DnaD (58% identity, 76% similarity),a protein involved in the initiation of DNA replication in Streptococcus mutans.
[8]To monitor epidemiological spread of resistant pneumococci, dependable and efficient identification techniques are a prerequisite. However, accumulating data indicate that identification of Streptococcus pneumoniae by molecular biology andconventional biochemical methods leads to controversial results [10, 14, 18, 19].Various fingerprinting techniques for S. pneumoniae have been described previously, including pulsed-field gel electrophoresis (PFGE) [9]and several PCR-
based genomic profiling assays [13,14]. PFGE is still considered to be the “gold standard” for determination of epidemiological relationships between pneumococcal isolates. However, it is a
laborious and time-inefficient method. For obvious reasons, PCR-based protocols are much more favored than the classical PFGE protocol. In this study, we explored the use of amplified fragment length polymorphism (AFLP) for epidemiological fingerprinting of macrolide- resistant S. pneumoniae isolates with emphasis on the comparison with PFGE.
We analyzed 85 erythromycin-resistant S. pneumoniae clinical isolates collected from several laboratories throughout The Netherlands between December 2001 and April 2002. Strains were stored in polypropylene vials at _70°C until testing. Strains were identified by the participating laboratories by their own standard identification techniques. All isolates were analyzed by 16S rRNA sequencing (described below). Strains were grown overnight on blood agar plates at 37°C under 5% CO2 conditions. AFLP, PFGE, and data analysis were performed according to previously described procedures with minor modifications [16].The identification of all S. pneumonia strains was confirmed by sequence analysis of part of the 16S rRNA gene. Amplicons were generated under standard PCR conditions with the primers 5_-CGGCGTGCCTAATACATGC-3_ and 5_-CGTATTACCGCGGCTGCT-3_. After purification of the amplicons by High Pure chemistry (Roche Diagnostics), they were subjected to sequence analysis on an ABI3700 platform under the conditions recommended by the manufacturer (Applied Biosystems). The sequences obtained were compared to sequences in the public DNA libraries by using the web-based BLAST interface [9].
Fingerprints of the 85 strains analyzed in this study, obtained by using PFGE and AFLP. As can be observed, nearly identical clusters of closely related strains can be identified by both typing methods. AFLP analysis showed, in addition to the clusters of epidemiological related strains, the formation of two clearly discernible megaclusters of strains in the dendrogram. We speculated that this collection of strains contained different species. Therefore, the identity of all strains was analyzed by 16S rRNA sequencing. All but one of the strains in AFLP cluster I (n _ 58; 68% of all strains) were confirmed to be S. pneumoniae. All isolates that were not grouped in cluster I (n _ 26; 31% of all strains) proved to be nonpneumococcal strains. Of these 26 isolates, 17 (65%) were Streptococcus mitis strains, which were all grouped in cluster II, and the remaining 9 (35%) were unidentifiable streptococcal species. Remarkably, one strain in the pneumococcal cluster (cluster I) in the AFLP dendrogram could not unequivocally be identified as S. pneumoniae by 16Ssequencing but could only be designated as streptococcal species.
The emergence of penicillin- and multiresistant pneumococcal isolates worldwide [12]necessitates continuous monitoring of the epidemiological spread of such strains. For this purpose, time-efficient and dependable fingerprinting techniques are essential. In the present study, we used AFLP versus PFGE for molecular typing of macrolide-resistant S. pneumoniae strains.Establishment of epidemiological relationships between pneu-mococcal strains was more easily established by AFLP than by PFGE. Moreover, AFLP analysis showed, in contrast to PFGE, the formation of clusters on a species level, allowing simultaneous discrimination between pneumococci and closely related species like S. mitis. Highly specific molecular biological methods like 16S sequencing provide new possibilities for more definite identification of S. pneumoniae. However, naturally occurring sequence variations within the 16S rRNA gene may complicate identification procedures, and it is not always clear where to draw the line between S. pneumoniae and genotypically similar species like S. mitis [14, 19]. Clearly, this is not restricted to 16S analysis but holds true for any other single-gene method. In contrast, AFLP is a genome-wide analysis technique, much less influenced by naturally occurring minor sequence variations. In our study, one strain in the pneumococcal cluster (cluster I) in the AFLP dendrogram could not unequivocally be identified as S. pneumoniae by 16S sequencing but was only designated to be a streptococcus species. However, AFLP analysis showed that this isolate was closely related to the other pneumococci in cluster I. Therefore, this particular isolate should be designated as S. pneumoniae. AFLP analysis, showing a clear separation between pneumococcal and nonpneumococcal clusters in the dendrogram, can be used as an alternative method to 16S rRNA sequencing for a more definite identification of streptococcal isolates. Phylogenetically related species like S. mitis have reduced antimicrobial susceptibility patterns compared to S.pneumoniae, and failure to differentiate between these two species will significantly influence pneumococcal resistance rates [11, 18]. This underscores the need for specific and dependable techniques for identification of S. pneumoniae in epidemiological studies. In contrast to PFGE, AFLP offers fully computerized data acquisition, which allows large numbers of isolates to be processed in a relatively short period of time. This makes this technique an efficient and dependable method for epidemiological fingerprinting of pneumococci. In summary, AFLP is an efficient alternative to PFGE for assessment of epidemiological relationships between pneumococcal isolates and is, in contrast to PFGE, effective in distinguishing S. pneumoniae from phylogenetically related species
like S.mitis. In our opinion, AFLP analysis should be the preferred method for epidemiological fingerprinting of pneumococci.

MATERIAL AND METHODS:

7.1 SOURCE OF DATA :
Experiment will be performed as described in the standard bibliography, may be obtained from standard journals and text books available within the college or from other pharmacy colleges or from libraries of National Institutes or through internets from industry.



7.2 OBJECTIVES OF THE STUDY:
  1. To isolate and identification of streptococcus species from patients.
  2. Estimation of antimicrobial activity.
  3. Molecular characterization of isolated organism by using AFLP
7.3 METHODOLOGY:
  1. To isolate and identify pathogenic organism from patients
  • collection of samples from hospital
  • Isolate organism by agar plating method
  • Identification of organism Bergyal’s manual
  • Estimation of antimicrobial properties by difco agar method
  1. Estimation of antimicrobial activity
  1. Molecular characterization of isolated organism by using AFLP
  • Isolation of DNA
  • Quantification of DNA by using UV spectrophotometer
  • Restriction digestion
  • Amplify the DNA by using PCR
  • Poly acryl amide gel electrophoresis
  • Analysis the result by using static software
REFERENCES :
1.Ashraf M, Ahmed, Shin-ichi Miyoshi, Sumio S, . Molecular characterization of a multidrug-resistant strain of enteroinvasive Escherichia coli O164isolated in Japan. Journal of Medical Microbiology. 2005;54:273-8.
2.Mohammed SA, Ana BF, Angela HAM, Van H, G. C. Molecular characterization of Intrinsic and Acquired Antibiotic Resistance in Lactic Acid Bacteria and Bifidobacteria. journal of molecular biology and biotechnology 2008;14:6-15..
3.Rosmari H, Adenilde S, Loiva OD, Oliveira, Nara LFD, Forno, et al. Spontaneous bacterial peritonitis caused by streptococcus bovis Braz J Infect Dis. 2010;14(3):294-6.
4.Kelly G, Vince, Stephen R, Kantor, Javier D, 2003. VN. Late Infection of a total knee arthroplasty with streptococcus bovis in association with carcinoma of the large Intestine. The Journal of Arthroplasty 2003;18(6).
5.Han TC, Boon LT, Hsu TY, CC. M. Infective endocarditis caused by Streptococcus bovis complicated by a superior mesenteric artery mycotic aneurysm and systemic septic emboli in a patient with colon diverticulitis. International Journal of Infectious Diseases. 2010;14:317-8.
6.Stephan E, Nabil D, Marie S, PK. J. Production of cytokines by monocytes, epithelial and endothelial cells activated by streptococcus bovis Academic press. 1999;26:26-31.
7.Pedro G, Ernesto G, Jose LG, Rubens L. The molecular characterization of the ®rst autolytic lysozyme of Streptococcus pneumoniae reveals evolutionary mobile domains Molecular Microbiology. Journal of Medical Microbiology. 1999;33:128-38.
8.Chris Neeleman, Corne´ H. W. Klaassen, Hanneke A. de Valk, Maaike T. de Ruiter, Mouton JW. Amplified Fragment Length Polymorphism Fingerprinting Is an Effective Technique To Distinguish Streptococcus pneumonia from Other Streptococci and an Efficient Alternative to Pulsed-Field Gel Electrophoresis for Molecular Typing of Pneumococci journal of clinical microbiology. Jan. 2004:369–71.
9.Altschul SF, T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, et al. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 1997;25:3389–402.
10.Chandler LJ, B. S. Reisner, G. L. Woods, Jafri. AK. Comparison of four methods for identifying Streptococcus pneumoniae. Diagn MicrobiolInfect Dis. 2000;37:285–7.
11.Dobay O, F. Rozgonyi, E. Hajdu, E. Nagy, M. Knausz, Amyes. SG. Antibiotic susceptibility and serotypes of Streptococcus pneumonia isolates from Hungary. J Antimicrob Chemother. 2003
51:887–93.
12.Farrell DJ, I. Morrissey, S. Bakker, Felmingham. D. Molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae and Streptococcus pyogenes isolated from the PROTEKT 1999– 2000 study. J Antimicrob Chemother. 2002;50(Suppl. 1):39–47.
13.Hermans PW, M. Sluijter, T. Hoogenboezem, H. Heersma, A. van Belkum, Groot. Rd. Comparative study of five different DNA fingerprint techniques for molecular typing of Streptococcus pneumoniae strains. J Clin Microbiol. 1995; 33:1606–12.
14.Kaijalainen T, S. Rintamaki, E. Herva, Leinonen. M. Evaluation of gene-technological and conventional methods in the identification of Streptococcus pneumoniae. J Microbiol Methods. 2002; 51:111–8.
15.Kell CM, J. Z. Jordens, M. Daniels, T. J. Coffey, J. Bates, J. Paul, et al. Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya. Infect Immun 1993;61:4382–91.
16.Klaassen, C. H. W., H. A. van Haren, Horrevorts. AM. Molecular fingerprinting of Clostridium difficile isolates: pulsed-field gel electrophoresis versus amplified fragment length polymorphism. J Clin Microbiol. 2002; 40:101– 4.
17.Lefevre JC, G. Faucon, A. M. Sicard, Gasc. AM. DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis. J Clin Microbiol. 1993; 31:2724–8.
18.Wester CW, D. Ariga, C. Nathan, T. W. Rice, J. Pulvirenti, R. Patel, et al. Possible overestimation of penicillin resistant Streptococcus pneumoniae colonization rates due to misidentification of oropharyngeal streptococci Diagn Microbiol Infect Dis 2002;42:263–68.
19.Whatmore AM, A. Efstratiou, A. P. Pickerill, K. Broughton, G. Woodard, D. Sturgeon, et al. Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of “atypical” pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes. Infect Immun. 2000; 68:1374–82.
9.

10.

11.

12. /
Signature of the candidate:
(PIDATALA NAGARJUNA REDDY.)
Remarks of the guide:
Name And Designation of:
11.1 Guide Mr. Mukund Handral.
Asst. Professor
Dept., Pharmacology
PES college of pharmacy
Bangalore-560050
11.2 Signature

11.3 Co-Guide Mr.mahesh .M
CEO of azyme Biosciences Pvt.Ltd.
11.4 Signature

11.5 Head of the department Mr. R. Srinath
Asst. Professor & HOD
Dept., Pharmacology
PES college of pharmacy
Bangalore-560050
11.6 signature
12.1 Remarks of the Principal :
12.2 Signature of the principle:
Prof. Dr. S. Mohan
Principal
PES college of pharmacy, Bangalore
560050