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2009 OMIG, Abstract 17

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American and Indian clinical isolates of P. aeruginosa harbor CRISPR genes from multiple subtypes.
J.H. Hammond1, K.C. Cady1, P. Lalitha3, R..S. Karthikeyan3 , R.T. Allar2, G.A. O’Toole1,  M. E. Zegans1,2
1Dept of Microbiology and Immunology,
2Dept of Surgery (Ophthalmology) Dartmouth Medical School, Hanover, NH,
3Dept of Ocular Microbiology, Aravind Eye Hospital, Madurai, India  

Purpose: Bacteriophages exert profound effects on bacterial ecology and pathogenesis, but there has been little investigation into their role during Pseudomonas aeruginosa (PA) infections of the ocular surface. CRISPRs have been reported to modulate some of the interactions between bacteria and these viruses. In PA there appear to be two CRISPR system subtypes; an Escherichia coli subtype is presumed to mediate resistance to infection, and a Yersinia pestis subtype has been shown to interact with bacteriophage in a way that impacts swarming motility and biofilm formation – two group behaviors with roles in virulence and antibiotic resistance. We studied strains of PA from both the USA and India to determine how common these CRISPR subtypes are in clinical isolates of PA.
Methods: All genetic assays were performed following previously published protocols. Clinical isolates were obtained from Dartmouth Medical School, the Campbell Lab at UPMC, and the Aravind Eye Hospital. 73 ocular isolates, 10 CF isolates, 23 sputum isolates and 16 urine isolates of PA were studied. Using PCR to amplify cas genes specific to each CRISPR, we were able to measure the frequency of each subtype in these isolates. Additionally, the CRISPRs in a subset of isolates were sequenced to obtain data regarding their spacer content.






Y. pestis subtype

29/73 (40%)

7/23 (30%)

2/16 (13%)

2/10 (20%)

 E. coli subtype

4/73 (5%)


1/16 (6%)

2/10 (20%)

Clinical isolate CRISPR sequence reveals spacers with homology to a variety of bacteriophage.
Conclusions: Clinical isolates of PA commonly contain CRISPR, with the Yersinia subtype being found more frequently. Subtypes were found in similar proportions in isolates from either the USA or India. Spacer sequence indicates exposure of isolates to a diversity of different bacteriophage. Given the ubiquity of bacteriophage in the environment, this suggests that CRISPRs may play a role in the pathology of strains infecting the eye.
Funding provided by NIH.    Disclosure: N

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