Genome sequence and analysis of resistance and virulence determinants in a strain of Neisseria mucosa causing native-valve endocarditis

Correspondence Brian L. Hollenbeck bhollenb@bidmc.harvard.edu ygrad@hsph.harvard.edu Division of Infectious Diseases, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, SL435, Boston, MA 02215, USA Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA Department of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA Department of Immunology and Infectious Diseases, Harvard TH Chan School of Public Health, 665 Huntington Avenue, Boston, MA 02115 Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, 75 Francis Street, PBB A-4, Boston, MA 02115, USA


Introduction
Neisseria mucosa and other commensal Neisseria colonize mucosal surfaces in humans but only rarely cause disease, mostly in patients with predisposing factors.In spite of their typically benign phenotype, these species possess a number of resistance and virulence determinants and may transfer these genes to Neisseria meningitidis and Neisseria gonorrhoeae with relative ease via plasmids or uptake of naked DNA (Bowler et al., 1994;Maiden, 2008).This latter mechanism of genetic exchange has been successful in Neisseria as a mechanism for horizontal gene exchange because the genome contains an abundance of DNA uptake sequences (DUSs) (Maiden, 2008;Snyder et al., 2007), which facilitate transfer of minimal mobile elements by homologous recombination (Davidsen et  2004; Qvarnstrom & Swedberg, 2006).The host and bacterial virulence factors involved in the transformation of N. mucosa from a commensal organism into a pathogen have not been previously evaluated.We present a case of native-valve endocarditis in a previously healthy 27-yearold female and report resistance and virulence factors harboured within the genome.

Case report
A 27-year-old Caucasian female presented with fever, wrist and knee pain, abdominal pain, and headache.Three weeks prior to admission the patient experienced a sore throat which resolved over 5 days.She recovered completely, but 1 week later developed fever (40 uC), headache, myalgias, polyarthritis, nausea and vomiting.Her medical history included attention deficit disorder and bilateral augmentation mammoplasty.Her medications included methylphenidate and an oral contraceptive.She reported consuming six alcoholic beverages per week, no tobacco use, and no intravenous drug use.She had been monogamous with a male sexual partner over the prior several years.On physical examination, she was afebrile.The oropharynx did not reveal erythema, ulceration or piercings.Her wrists were swollen bilaterally, with tenderness to palpation, warmth and decreased range of motion.She had a new systolic murmur at the lower left sternal border.Skin examination revealed a few scattered macular lesions on the hands and feet (Fig. 1a,b).She did not have meningismus.Laboratory values were significant for white blood cell count of 18 400 ml 21 (normal range 4000-10 000) with 85.9 % polymorphonuclear leukocytes (50-70 %), 8.2 % lymphocytes (18-42 %) and 5.6 % monocytes (2-11 %), haemoglobin 11.6 g dl 21 (12-16), platelet count 274 000 ml 21 (150-440), erythrocyte sedimentation rate 78 mm h 21 (0-20), C-reactive protein 194.4 mg l 21 (v5.0), alkaline phosphatase 258 IU l 21 (35-105), lactate dehydrogenase 427 IU l 21 (135-214).Computerized tomography scan of the abdomen and pelvis demonstrated hypodensities in the spleen and kidneys consistent with multiple infarctions.Thirty-eight hours after admission, two sets (three out of four bottles) of blood cultures were positive for Gramnegative diplococci.The patient was empirically treated with intravenous ceftriaxone 2 g daily and oral doxycycline 100 mg twice daily for a presumptive diagnosis of disseminated N. gonorrhoeae infection.Transoesophageal echocardiogram revealed a mitral valve vegetation and mitral valve prolapse.On hospital day 3, the patient developed an embolic lesion on the tip of her nose (Fig. 1c).By modified Duke's criteria (Durack et al., 1994), the patient was diagnosed with endocarditis.
The Gram-negative diplococcus was identified as N. mucosa by API NH kit (bioMe ´rieux) and nitrate reaction (Remel) at Mayo Medical Laboratory (Rochester, MN).Antimicrobial susceptibility testing by agar dilution at Mayo Medical Laboratory revealed ceftriaxone MIC 0.125 mg ml 21 , penicillin MIC 2 mg ml 21 , and levofloxacin MIC j1 mg ml 21 .Although there are no Clinical and Laboratory Standards Institute (CLSI) standard method and interpretation for susceptibility testing for this organism, we performed additional antimicrobial susceptibility testing by CLSI standard microdilution method (with 5 % sheep blood) for N. meningitidis, which demonstrated erythromycin MIC w4 mg ml 21 , ampicillin MIC 0.5 mg ml 21 , tetracycline MIC 2 mg ml 21 , and trimethoprim/sulfamethoxazole MIC 0.5/0.9mg ml 21 .Blood cultures were negative after starting ceftriaxone, and the patient's symptoms improved.During week 3 of treatment with ceftriaxone, she developed severe neutropenia (absolute neutrophil count 125 ml 21 ).Ceftriaxone was discontinued and her neutrophil count recovered within 2 weeks.She completed therapy for endocarditis with three additional weeks of oral levofloxacin and recovered uneventfully.Human immunodeficiency virus antibody was negative and antinuclear antibody titre was v1 : 20; CH50 and serum immunoglobulin levels measured after resolution of her infection were normal.
For genome sequencing, the strain (termed LVG-13) was cultured on chocolate agar plates and the DNA was isolated using the Qiagen EZ1 system (Qiagen).The genome was sequenced using the Illumina MiSeq platform (Illumina) to produce paired-end reads 100 nt in length.The genome was assembled using Velvet with k-mer optimization and expected coverage values as described previously (Croucher et al., 2011;Zerbino & Birney, 2008), and yielded a genome 2.2 Mb in length with approximately 30-fold coverage.The genome sequence was annotated via the RAST server (Aziz et al., 2008).DUSs, including both canonical (GCCGTCTGAA) and non-canonical (GtCGTCTGAA), were identified using the 'fuzznuc' application of EMBOSS (Rice et al., 2000).The DUS1 sequence GtCGTCTGAA appears 1855 times, and the canonical uptake sequence GCCGTCTGAA appears 145 times.Using 53 ribosomal protein subunit (rps) genes (Bennett et al., 2012) defined in the isolate by top BLAST match (Altschul et al., 1990) and downloaded from BIGSdb for 55 Neisseria species (Jolley & Maiden, 2010), a neighbour-joining phylogeny was constructed in SeaView (Gouy et al., 2010).The phylogeny shows that the isolate clusters with other N. mucosa (Fig. 2), providing sequence-based confirmation of biochemical identification.Presence of known or putative virulence factors was assessed using a cut-off of 50 % identity to the query protein over at least 75 % of the query length from a list compiled by Marri et al. (2010) (Table S1, available in the online Supplementary Material).There were a total of 86 known or putative virulence factors identified in this strain.

Discussion
As N. mucosa is almost always commensal, the invasive infection observed in this case raised the possibility that this strain had acquired a gene or genes that contributed to a more aggressive phenotype.The genetic basis of pathogenic phenotypes for Neisseria remains incompletely defined, and it has been postulated that pathogenicity is polygenic and that multiple different combinations of genes or allelic variants could confer increased probability of invasion (Bennett et al., 2010;Joseph et al., 2011;Marri et al., 2010).We identified the presence of 86 known or putative Neisseria virulence factors in this isolate, findings that are consistent with analysis of previously published sequences of N. mucosa (Marri et al., 2010).This isolate contains more copies of DUS1 than of the canonical DUS sequence, consistent with previous studies.The phylogeny illustrates that N. mucosa is closely related to Neisseria perflava and Neisseria sicca, which also preferentially contain DUS1 over the canonical Neisseria DUS (Marri et al., 2010;Qvarnstrom & Swedberg, 2006).The apparent polyphyly of the three species may result from genetic exchange using DUS1 regions, an exchange mechanism that may preferentially result in homologous recombination of housekeeping genes (Bennett et al., 2013;Davidsen et al., 2004;Maiden, 2008).Additional similar cases of invasive disease accompanied by genetic sequencing would be necessary to determine if the identified DUS regions and putative virulence factors are necessary or sufficient to cause disease.
The N. mucosa isolate in this study exhibited relatively high MIC values to b-lactams, and lacks a TEM-1 b-lactamase.This likely reflects the previously reported intrinsic relative resistance to penicillin through production of penicillinbinding protein 2 (encoded by penA) with low affinity for b-lactams (Spratt et al., 1992).In N. gonorrhoeae, reduced susceptibility to cephalosporins is associated with mosaic penA genes, reflecting recombination of N. gonorrhoeae with commensal Neisseria species in the transpeptidase region of the gene (Spratt et al., 1992).LVG-13 possesses a penA gene most similar to that found in a number of other commensal isolates, but has less than 30 % amino acid identity over the length of the protein to those found in N. gonorrhoeae and in N. meningitidis.
Host factors may also predispose to infection with commensal Neisseria species.Several reviews of N. mucosa endocarditis cases have highlighted that a majority of patients had underlying structural heart disease, and lesions involving the mitral valve were particularly common (Ingram et al., 1992;Pilmis et al., 2014).The patient in this case had no known underlying structural heart disease, but was found to have mitral valve prolapse at the time of diagnosis.These clinical case-series highlight that embolic disease and mycotic aneurysms are common in Neisseria endocarditis and approach rates seen with Staphylococcus aureus endocarditis, findings that are in contrast to the typically benign nature of this species.
Although generally regarded as a benign part of the human microbiota, any of the commensal Neisseria species can infrequently cause invasive infections, including meningitis, pericarditis, peritonitis and endocarditis (Ingram et al., 1992;Johnson, 1983;Pilmis et al., 2014), and can also serve as a reservoir of resistance determinants and virulence factors for N. meningitidis and N. gonorrhoeae.The transformation of these generally benign organisms into aggressive pathogens may be a matter of host factors, bacterial virulence factors that predispose to invasion, or a combination of the two.The rarity of invasive disease caused by N. mucosa highlights the importance of genome sequencing and cataloguing of commensal and pathological strains, together with description of the host and clinical course, to elucidate the factors that contribute to pathogenic potential of commensal Neisseria species.

Fig. 1 .
Fig. 1. (a,b) Initial presentation with Janeway lesions located on the hallux and plantar aspect of the right foot, respectively.(c) On hospital day 3, the patient developed an Osler's node on the nose.