Metagenomic Approach to Identifying Novel Antibiotics

(and other gene products).

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, otitis media, and sinusitis. 40% of strains causing disease in adults are resistant to penicillin. 25% are resistant to multiple drugs. Vancomycin is the only agent against which resistance has not emerged.

Jacobs MR.Am J Med. 2004 Aug 2;117 Suppl 3A:3S-15S.

1996 – levofloxacin introduced.1999- 0.4% strains resistant.2000- 0.9% resistant.2001- 1.2% of the S. pneumoniae bacteria in Canada were resistant to levofloxacin.

Are we entering a post-antibiotic era?

Methicillin-resistant Staphylococcus aureus (MRSA) with sensitivity only to vancomycin increased from 22.8 percent in 1987 to 56.2 percent in 1997. J Am Board Fam Pract. 2004 May-Jun;17(3):220-6.

1997- 1st case of S.aureus with reduced sensitivity to vancomycin.

2003- Identified isolate which was completely resistant to vancomycin.

microvet.arizona.edu/

Enterococcus faecium and Enterococcus faecalis normally found in the bowel and the female genito-urinary tract. In hospitalised patients, infections may be associated with intravascular devices, and life threatening bloodstream infections may result.

1987 Vancomycin-resistant enterococci (VRE)were first reported in England and France.

1989 reported in one New York City hospital.

1991, 38 hospitals in the United States reported VRE strains.

1993, 14 percent of patients with enterococcus in intensive-care units in some hospitals had vancomycin-resistant strains, a 20-fold increase from 1987.

Cases of infection with VRE have resulted in mortality rates as high as 73%.

http://www.molbio.princeton.edu/

Vancomycin-resistant enterococci

‘8 9 ’9 0 ’9 1 ’9 2 ’9 3 ’ 9 4 ’9 5 9 6 ’9 7 ‘9 8

CDC's National Nosocomial Infections Surveillance (NNIS) system

Necrotizing Fasciitis Caused by Community-Necrotizing Fasciitis Caused by Community-Associated Methicillin-Resistant Associated Methicillin-Resistant

Staphylococcus aureus in Los AngelesStaphylococcus aureus in Los Angeles

Volume 352:1445-1453Volume 352:1445-1453

Necrotizing Fasciitis

• 1928: Alexander Fleming discovered the first antibiotic.

• He observed that Penicillium fungus made an antibiotic, penicillin, that killed S. aureus.

• 1940s: Penicillin was tested clinically and mass produced.

The Birth of Modern Chemotherapy

Figure 1.5

Selman WaksmanSelman Waksman

actinomycin (1940), clavacin, streptothricin (1942), streptomycin (1943),streptomycin (1943), grisein (1946), neomycin (1948),neomycin (1948), fradicin, candicidin, candidin, and others.

Nov. 7, 1949Nov. 7, 1949

““Soil microorganisms have been Soil microorganisms have been the source of many useful the source of many useful biomolecules. However, various biomolecules. However, various molecular analyses … have molecular analyses … have confirmed that less than 1% of the confirmed that less than 1% of the microorganisms in soil can be microorganisms in soil can be cultured by conventional cultured by conventional methods.”methods.”

Yun, Jiae. (2004) Applied and Environmental Microbiology p.7229-7235

Prokaryotic Gene Prokaryotic Gene ExpressionExpression

Construction of a Genomic Library

1. Extract DNA from organism.

2. Digest with restriction enzyme.

3. Ligate into plasmids digested with same restriction enzyme.

4. Transform recombinant plasmids into bacteria (E. coli)

MetagenomicsMetagenomics

GENE DISCOVERY AND GENE DISCOVERY AND BIOTECHNOLOGYBIOTECHNOLOGY

One definition of biotechnology is "the One definition of biotechnology is "the deliberate manipulation of DNA molecules to deliberate manipulation of DNA molecules to

produce commercial products from living produce commercial products from living organisms."organisms."

SCREEN FOR NOVEL LIPASESSCREEN FOR NOVEL LIPASES

Ravi Ranjan , Amit Grover , Raj Kishor Kapardar , Rakesh Sharma, Biochemical and Biophysical Research Communications, 335 (2005) 57–65

1 ggatcccggc gacccggctg cactggtacc gcggcgagta ccgtaccgcc tcgcggctga 61 aggacgtcgc gggcaagggc ctgggcaagc tcgtggacgc cttccgcatc gcggcctggg 121 tgcgccggca cgacgtggtg gtcgtgccgg gcacgggcat cctggagagc acgctgccgc 181 tgaggccctg gggcctgccg tactcgctgt tcctgctgtg cgggtggggc cgggtgctgc 241 gcgcccgggt cgcgctcgtc gccgtcggcg cctccgaaac cggcagccgg ccggtgcggg 301 tgctgacccg ctgggcggcg cggctggccg cgtaccggtc gtaccgggac accctgtccc 361 gtgacgcgat gcgggccatg ggcgtggaca ccacgggaga ccaggttcac acggacctgg 421 tgttcgccct gccgaccccg ccgcggggcg acctgtcggg cccgcccggg ccggtctgtg 481 tcggcatcat ggacttccac ggcagcaacg acgagcggga ccgggcgcgg gagctgaaca 541 gccgctacct ggacggcatc acgcggttcg tcggcgcgct ggccgcggag ggcaggccgg 601 tgcggctgct gacgggtgac gacgtggacc ggccggtggc cgaggcgatc atcgaggcgg 661 tggactcgcc gatggtcacc

Copyright ©2002 by the National Academy of Sciences

Zengler, Karsten et al. (2002) Proc. Natl. Acad. Sci. USA 99, 15681-15686

Phylogenic Tree