Infectious Disease Reports 2019; volume 11:8132 Janibacter species with most reliable diagnostic tool. However, evidence of genomic sometimes a diagnosis cannot be made Correspondence: Ying Bai, Division of using blood culture because of poor labora- Vector-Borne Diseases, Centers for Disease polymorphism isolated tory infrastructure, presence of uncommon Control and Prevention, 3156 Rampart Road, from resected heart valve pathogens, or pretreatment of the patient Fort Collins, Colorado 80521 USA. in a patient with aortic stenosis with antibiotics prior to sample collection. Tel. 1-970-266-3555. Up to 35% of all infective endocarditis E-mail: [email protected] cases remain blood culture negative either Lile Malania,1 Ying Bai,2 Key words: aortic stenosis; bacteria; genome 3 4 due to slow growth of the bacteria or inap- polymorphism; Janibacter. Kamil Khanipov, Marika Tsereteli, 2 5 1 propriate media. Mikheil Metreveli, David Tsereteli, Calcific aortic stenosis is also a major 1 1 Acknowledgements: The authors would like Ketevan Sidamonidze, Paata Imnadze, problem in many countries. In the United to thank Natalia Abazashvili, Tamriko 3 2 Yuriy Fofanov, Michael Kosoy States, the incidence rate of aortic stenosis Giorgadze, and Nazibrola Chitadze for their 1National Center for Disease Control has been reported as 27.1 per 10,000.3 The assistance with laboratory tests; Guram and Public Health, Tbilisi, Georgia; mechanism of calcification remains unclear. Katstadze, and Neli Chakvetadze for their par- ticipation in investigation of the human case; 2Division of Vector-Borne Diseases, The hypothesis is that low grade chronic or recurrent bacterial endocarditis with specif- Levent Albayrak and George Golovko for the Centers for Disease Control and assistance with genome analysis; and ic calcifiable bacteria is a cause of calcifica- Prevention, Fort Collins, CO, USA; 4 Christina Nelson for careful revision of the 3Department of Pharmacology and tion of the aortic valves. Along those lines, manuscript, thoughtful comments, and con- an association between Chlamydia pneumo- Toxicology, University of Texas Medical structive suggestions. nia and Mycoplasma pneumonia and calci- Branch, Galveston, TX, USA; 4Tbilisi fication in aortic stenosis has been Contributions: LM, YB, and KK contributed State Medical University, Tbilisi, reported.5,6 More recently, a case of equally. LM, MK, conception and design; LM, Georgia; 5Department of Cardiology, Bartonella endocarditis on a bioprosthetic MT, MM, DT, data collecting; LM, KS, bacte- High Technology Medical Center, aortic valve that caused rapidly progressive riologicalonly analysis; YB, KS, sequence analy- University Clinic, Tbilisi, Georgia aortic stenosis was reported.7 ses; KH, YF, genome analysis; LM, YB, MK, Due to recent descriptions of Bartonella manuscript writing; MK, PI, manuscript reviewing and final approval. infections in the country of Georgia8 and the importance of identifying sources of bacte- Abstract useConflict of interests: the authors declare no The authors report isolation and identi- rial endocarditis, a collaboration was estab- potential conflict of interests. fication of two strains of bacteria belonging lished between hospitals in Tbilisi (the cap- to the genus Janibacter from a human ital of Georgia) and the National Center for Funding: the work was supported by the patient with aortic stenosis from a rural area Disease Control and Public Health International Science and Technology Center Project (ISTC G-1683-p). of the country of Georgia. The microorgan- (NCDC&PH), where culturing of fastidious isms were isolated from aortic heart valve. microorganisms from endocarditis cases Received for publication: 1 April 2019. Two isolates with slightly distinct colony has been performed. In this paper, we report isolation and identification of two bacterial Revision received: 10 July 2019. morphologies were harvested after sub-cul- Accepted for publication: 11 July 2019. turing from an original agar plate. strains, both belonging to the genus Preliminary identification of the isolates is Janibacter, from heart valve tissues collect- This work is licensed under a Creative based on amplification and sequencing of a ed during an investigation of a human case, Commons Attribution-NonCommercial 4.0 fragment of 16SrRNA. Whole genome which originally was suspected as endo- International License (CC BY-NC 4.0). sequencing was performed using the carditis, but later diagnosed as a case of aor- ©Copyright: the Author(s), 2019 Illumina MiSeq instrument. Both isolates tic stenosis. Detailed characterization of the Licensee PAGEPress, Italy were identified as undistinguished strains of full genomes of the both isolates was per- formed with identification of a significant Infectious Disease Reports 2019; 11:8132 the genus Janibacter. Characterization of doi:10.4081/idr.2019.8132 whole genome sequences of eachNon-commercial culture difference in gene composition between has revealed a 15% difference in gene pro- cultures of these two Janibacter. file between the cultures and confirmed that both strains belong to the genus Janibacter blood were used to inoculate chocolate agar with the closest match to J. terrae. Genomic plates. The inoculated plates were incubated comparison of cultures of Janibacter Materials and Methods at 35oC with 5% carbon dioxide for 30 days. obtained from human cases and from envi- Bacterial culture Each individual colony from agar was sep- ronmental sources presents a promising Surgically removed cardiac valve mate- arately streaked onto secondary agar plates. direction for evaluating a role of these bac- rial and whole blood were collected from a The subcultures were harvested separately teria as human pathogens. patient with suspected endocarditis. The by scraping bacterial growth after sufficient clinical part of the work was approved by growth was observed. DNA was heat the Ethical Committee of the High extracted at 95oC for 10 min from whole Technology Medical Center, Tbilisi, bacterial cells. The prepared DNA was Introduction Georgia. The material was frozen, trans- stored at -80oC. Bacteria are a common source of infec- ferred to the National Center for Diseases tive endocarditis with inflammation of the Control and Public Health, Tbilisi, Georgia, PCR amplification and sequencing endothelia cells and valves of the heart, usu- and stored at -80oC until it was plated. The Amplification of the 16S rRNA frag- ally caused by bacteria.1 Blood culture is the homogenized cardiac tissue and whole ments from isolated DNA of both cultures [page 22] [Infectious Disease Reports 2019; 11:8132] Article was performed by conventional broad- sion, the patient’s fevers became more per- slightly different morphology were range PCR assay using 63F and 1187R sistent and she was evaluated in an outpa- observed on the agar plate inoculated with primers in a C1000 Touch Thermal Cycler tient clinic. She was prescribed empiric heart tissues. Both colonies had circular (Bio-Rad, Hercules, CA). The PCR prod- antibiotics of unknown type, but did not shape and light cream color with a slight ucts were analyzed for the presence of improve. She was admitted to the intensive difference in size. No hemolysis on choco- amplicons of the appropriate size by elec- care unit in May 2014 due to periodic late agar was observed. These two colonies trophoresis in 1.5% agar gels containing squeezing pain in the chest that radiated were separately sub-cultured onto new agar GelGreen stain (Biotium, Hayward, CA). under the left shoulder blade with the origi- plates. Microscopy indicated Gram-positive Positive and negative controls were includ- nal diagnosis of endocarditis. The patient coccobacilli. When bacterial mass on both ed in each PCR assay. PCR products with lived in a rural area near the town of Kareli plates was sufficient, colonies were harvest- appropriate sizes of amplicons were puri- in the region of Shida Kartli, Georgia ed on brain heart infusion medium and fied using a QIAquick PCR Purification Kit (South Caucasus). She was engaged in agri- stored at –70oC until the strains were (Qiagen, Valencia, CA) according to manu- cultural work and reported frequent con- shipped to CDC-Fort Collins for additional facturer’s instructions, then sequenced in tacts with farm animals (pigs and cattle) and characterization. both directions using ABI 3130 Genetic pets at home. By Mega BLAST search, the ribosomal Analyzer (Applied Biosystems, Foster City, Clinical signs upon hospital admission amplicons from both isolates were identi- o CA). included fever of 38 C, ischemic disease, fied as a Janibacter sp. based on the frag- angina, and arterial hypertension. No rash ment amplification and sequence analysis Genome analysis or lymphadenopathy was reported. with the highest match to Janibacter hoylei. Whole genome sequencing (WGS) was Coronary angiographyshowed unaltered The 16S rRNA sequences from both iso- performed using the Illumina MiSeq instru- coronary vessels. Echocardiography detect- lates were undistinguished. ment. Sequencing of the two samples result- ed critical stenosis of the aortic valve with Sequencing of each isolate genome ed in 8 million 51 nucleotide long sequenc- high gradient and critical opening. On aus- resulted in 2 and 6 million sequencing reads ing reads. The sequencing reads were fil- cultation there was rough systolic murmur 51 nucleotides long in size trimmed to 32 tered to exclude: (a) nucleotides below the at every auditory point, with epicenter on basesonly long non-overlapping