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Characterization and Whole Genome Sequencing of a Novel Strain of Bergeyella Cardium Related to Infective Endocarditis Hongwei Pan, Wei Li, Enhua Sun and Yi Zhang*

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Characterization and Whole Genome Sequencing of a Novel Strain of Bergeyella Cardium Related to Infective Endocarditis Hongwei Pan, Wei Li, Enhua Sun and Yi Zhang* Pan et al. BMC Microbiology (2020) 20:32 https://doi.org/10.1186/s12866-020-1715-0 RESEARCH ARTICLE Open Access Characterization and whole genome sequencing of a novel strain of Bergeyella cardium related to infective endocarditis Hongwei Pan, Wei Li, Enhua Sun and Yi Zhang* Abstract Background: Bergeyella cardium infection is becoming increasingly prevalent in patients with infective endocarditis, suggesting its significance in disease pathogenesis. However, few studies have fully characterized this species. Results: Herein, we report the morphological and physiological characteristics, as well as whole genome sequencing of a newly identified B. cardium HPQL strain isolated from a patient with infective endocarditis. Results from the cellular morphology and biochemical analysis provide basic knowledge on the new pathogen. The whole genome sequencing of B. cardium HPQL consists of a circular chromosome with a total length of 2,036,890 bp. No plasmid was detected. Comparative genomics were carried out then. Antibiotics resistance related genes, pathogenesis related genes, predicted insertion sequences, genome islands and predicted CRISPRs sequences were demonstrated. To our knowledge, this is the first study to provide a complete genome sequence for Bergeyella spp. Conclusions: This study provides fundamental phenotypic and genomic information for the newly identified fastidious infective endocarditis causative bacteria, B. cardium. Our results provide insights into effective clinical diagnosis and treatment of this pathogen. Keywords: Bergeyella cardium, Infective endocarditis, Genome sequencing Background humans [6, 7]. B. zoohelcum is usually isolated from the Infective endocarditis is a serious infectious disease with normal oral microflora of animals such as cats and dogs high associated morbidity and mortality. Identification of [8]. Hence, animal bites and prolonged exposure to pets the causative agents is, therefore, crucial for improving are the primary causes of human infection with B. zoo- the clinical outcome [1]. Clinically, infective endocarditis helcum [4]. In addition, a patient suffered B. zoohelcum is generally diagnosed based on positive blood cultures, bacteremia after eating food prepared with coagulated removed leads, and/or infected pocket material [1]. goat blood [9, 10]. A case of cellulitis due to B. zoohel- Species belonging to the genera Streptococcus, Staphylo- cum infection was also reported in a tsunami victim [9]. coccus, and Enterococcus are the primary causative or- Alternatively, infections caused by other Bergeyella spp. ganisms of infective endocarditis [2]. However, recently are rarely reported. A previously uncultivated Bergeyella new pathogens are emerging as additional etiological sp. (clone AF14) with strong homology to a previously agents, such as Bergeyella spp. [3–5]. reported uncultivated oral Bergeyella strain was sus- Bergeyella spp. are non-fermenting gram-negative pected to be an opportunistic pathogen during preterm bacilli, belonging to the family Flavobacteriaceae [3]. B. birth [11]. Further, the isolation of two Bergeyella strains zoohelcum, known to cause cellulitis, leg abscess, teno- was reported from patients with infective endocarditis. synovitis, septicemia, pneumonia, and meningitis, is one Both strains shared 94.9% homology with B. zoohelcum, of the best described zoonotic pathogens afflicting suggesting that they are a new species belonging to of the genus Bergeyella. The two strains were designated as Bergeyella cardium13-07T and Bergeyella cardium13–16 * Correspondence: [email protected] Department of Clinical Laboratory, Qilu Hospital of Shandong University, [3]. Meanwhile, another case study reported the isolation Jinan 250012, Shandong Province, China of a Bergeyella strain from an infective endocarditis © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pan et al. BMC Microbiology (2020) 20:32 Page 2 of 9 patient that had 98.2% shared identity with B. zoohel- the presence of gram-negative bacilli. However, no cum, which was slightly lower than the ≥99.0% hom- reliable identification was made by matrix-assisted laser ology required for two organisms to be considered the desorption/ionization-time of flight mass spectrometry same species [4]. Recently, a novel Bergeyella sp. was (MALDI-TOF MS). Thus, to accurately identify the isolated from a patient with infective endocarditis. The pathogenic species, a 1425 bp sequence from the 16S organism was determined to be genetically most closely rRNA gene of the isolated strain was amplified and related to B. cardium [12]. Moreover, the first case of B. sequenced via polymerase chain reaction (PCR). The cardium prosthetic valve endocarditis was also reported sequence was then submitted to NCBI BLASTN to iden- quite recently [5]. tify matched bacterial sequences. Results revealed that The increasing number of cases of B. cardium infec- the sequence with the highest homology (approximately tion in patients with infective endocarditis suggests its 99%) was isolated from B. cardium. To further verify the importance in disease pathogenesis. However, studies nucleotide BLAST results, a detailed phylogenetic tree examining the microbial characteristics and genetic was constructed as shown in Fig. 1. Results showed the features of this species are very rare. In this study, we isolated strain clustering with two B. cardium strains therefore, sought to describe the isolation, identification that had been isolated from two infective endocarditis and characterization of a new B. cardium sp. from blood patients in Korea [3]. Hence, the phylogenetic analysis cultures of a patient with infective endocarditis. We also supported the finding that the isolated strain was a novel performed whole genome sequencing and, through strain of B. cardium. We, therefore designated the iso- phylogenetic analysis, we were able to predict the pos- lated strain in our study as B. cardium HPQL (identified sible origin of this newly identified species. by Hongwei Pan from QiLu hospital). Results General microbial characteristics of B. cardium HPQL Phylogenetic analysis identified the isolate as a novel Morphological, physiological and biochemical characterization species of B. cardium of the newly isolated strain was performed. We ob- Four days after the initial blood culture was obtained served that the bacterial cells aggregate together in from a 63-year-old man with infective endocarditis, the blood cultures (Fig. 2a). Moreover, small colonies of growth of microorganisms was reported through an B. cardium HPQL were observed on blood agar after automated blood culture system. Gram staining revealed 48 h of incubation at 35 °C (Fig. 2c). However, the Fig. 1 Phylogenetic tree based on 16S rRNA gene sequences. Phylogenetic analysis was performed on B. cardium HPQL and closely related species based on 16S rRNA gene sequences. The HPQL strain was determined to cluster with B. cardium spp. The phylogenetic tree was created using the Neighbor-Joining algorithm. The branches are scaled in terms of the expected number of substitutions per site Pan et al. BMC Microbiology (2020) 20:32 Page 3 of 9 Fig. 2 Morphological characterization of B. cardium HPQL. a Bacterial cells cultured from positive blood cultures. The bar is equal to 5 μm. b Gram staining properties of the HPQL strain. The bar is equal to 20 μm. c Bacterial colonies after culturing for 48 h on Columbia blood agar. d Scanning electron microscope observation of the bacterial cells strain did not grow on MacConkey agar or Mueller- Genomic features of the B. cardium HPQL strain Hinton agar plates, indicating that this organism exhibits The whole genome of the newly isolated B. cardium fastidious growth patterns. Further, individual colonies HPQL strain was sequenced due to its potential clinical grown on blood agar appeared as non-pigmented, circular, importance in patients with infective endocarditis. The shiny, and smooth with entire edges (Fig. 2c). Microscopic genomic details are provided in Table 2 and Fig. 3a. and scanning electron microscope observation revealed ir- Table 1 Minimum inhibitory concentration of antimicrobial regularly rod-shaped bacterial cells (Fig. 2b and d). agents of B. cardium HPQL Biochemical characteristic of the strain were further Antimicrobial agent (μ g/mL) MICs after 96 h incubation analyzed. According to the results procured from API Penicillin 0.032 NH, this bacterial strain did not produce penicillinase, Ceftriaxone 0.048 omithine decarboxylase, urease, β-galactosidase, proline arylamidase or gamma glutamyl transferase. However, it Cefepime 0.016 was positive for lipase, alkaline phosphatase and oxidase Cefotaxime 0.024 activity, and negative for catalase activity and indole pro- Meropenem 0.012 duction. Additionally, acid was found to be produced Imipenem 0.032 from D-glucose, D-fructose, D-maltose, D-sucrose. Tigecycline 0.032 Amoxicillin/Clavulafiate 0.016 Antimicrobial susceptibility analysis Sulfamethoxazole
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