Molecular Identification of Bacterial Pathogen Infecting Coconut Leaf Beetle

ABSTRACT Many species of microorganisms can cause diseases and mortality of insect pests. Accurate detection and identification of the entomophatogens are essential for development of biological control agent to the pest. Brontispa longissima, a serious and invasive pest of coconut, was infected by bacterium causing mortality of the larvae and pupae in coconut field. Objective of the research was to identify bacterium as a causal agent of the field-infected B. longissima using molecular technique. Research was conducted between April and August 2011. Molecular identification using polymerase chain reaction (PCR) amplification of 16s ribosomal RNA of the infected larvae and sequencing of the gene showed that Serratia marcescens is the causal agent of the disease.Keywords: Brontispa longissima, coconut, 16s rRNA, Serratia marcescens. Identifikasi Molekular Bakteri Pathogen yang Menginfeksi Hama Daun Kelapa Brontispa longissima(Coleoptera:Chrysomelidae)ABSTRAK Banyak mikroorganisme dapat menimbulkan penyakit pada serangga hama. Deteksi dan identifikasi yang akurat dari pathogen penyebab penyakit pada serangga (entomopathogen) hama merupakan tahap yang penting dalam pengembangan pengendalian biologi untuk hama tersebut. Brontispa longissima sebagai hama penting dan bersifat invasif pada tanaman kelapa diinfeksi oleh sejenis bakteri yang menyebabkan kematian larva dan pupa dari serangga tersebut di lapangan. Penelitian ini bertujuan untuk mengidentifikasi organisme penyebab penyakit pada hama B. longissima dengan menggunakan teknik molekuler. Penelitian dilaksanakan pada bulan April sampai dengan Agustus 2011. Identifikasi bakteri dilakukan dengan mengamplifikasi 16s ribosomal RNA dari larva yang terinfeksi dengan menggunakan PCR (polymerase chain reaction). Hasil analisis sekuens nukleotida 16s ribosomal RNA dari larva yang terinfeksi menunjukkan bahwa Serratia marcescens adalah bakteri penyebab dari penyakit tersebut.Kata kunci: Brontispa longissima, kelapa, 16s rRNA, Serratia marcescens.

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1834. Incremental Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz359.096 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S44-S44

Author(s):

Sarwat Khalil ◽

Madiha Fida ◽

Douglas W Challener ◽

Omar Abu Saleh ◽

Muhammad R Sohail ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Clinical Practice ◽

16S Rrna ◽

Ribosomal Rna ◽

Mayo Clinic ◽

Sanger Sequencing ◽

Diagnostic Value ◽

Chain Reaction ◽

Material Support ◽

Polymerase Chain

Abstract Background Polymerase chain reaction (PCR)/sequencing targeting the 16S ribosomal RNA (rRNA) gene to detect bacteria in normally sterile tissues and fluids has become increasingly popular in clinical medicine. This culture-independent technique can detect bacteria that are nonviable or difficult to cultivate using conventional methods. The clinical value of this type of testing is not well defined. We aimed to assess the diagnostic value of 16S rRNA PCR/Sanger sequencing as a clinical diagnostic assay at Mayo Clinic. Methods This is an interim analysis of the first 173 of 478 patients who had 16S rRNA PCR/Sanger sequencing done on sterile tissues or fluids at our institution from April, 2017 to November, 2018 as part of routine clinical practice. Medical records are being retrospectively reviewed, with results compared with those of culture. Results We reviewed 207 specimens from 173 patients (musculoskeletal 79%, cardiovascular 7%, central nervous system 4%, other 9%) that underwent 16S rRNA PCR/Sanger sequencing by clinical request (Table 1). In 90% of these specimens, the test was pre-planned rather than added-on. Nine specimens were excluded from analysis, as cultures were not performed. Overall concordance of culture with PCR/sequencing was 81% (160/197; P < 0.0001). Of 44 culture-positive specimens, PCR detected the same bacterium in 21 (48%) (Table 2). 45% (20/44) of those with positive cultures and 46% of those with positive PCR/sequencing results had received prior antimicrobial therapy (Table 3). PCR was negative in 139/144 specimens that were culture-negative (97%). PCR/sequencing was helpful in detecting a putative bacterial pathogen in 4 patients with negative cultures (Table 4). Conclusion Overall, 16S rRNA PCR/Sanger sequencing improved diagnostic yield compared with culture in a minority of cases. The described assay is limited by its inability to detect polymicrobial infections, a technical limitation that could possibly be addressed using massive parallel sequencing. Careful selection of cases and a save and add-on approach may be more cost-effective than upfront testing, although this was requested in a minority of cases. Disclosures Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

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