Abiotrophia spp. and Granulicatella spp. Infective Endocarditis: A Contemporary Perspective.

BACKGROUND: Abiotrophia spp. and Granulicatella spp. are Gram-positive cocci, formerly known as nutritionally variant or deficient Streptococcus. Their role as causative agents of infective endocarditis (IE) is numerically uncertain, as well as diagnostic and clinical management of this infection. The aim of our study is to describe the clinical, microbiological, therapeutic, and prognosis of patients with IE caused by these microorganisms in a large microbiology department. METHODS: Retrospective analysis of all the patients with Abiotrophia spp. and Granulicatella spp. IE registered in our centre in the period 2004-2021. RESULTS: Of the 822 IE in the study period, 10 (1.2%) were caused by Abiotrophia spp. (7) or Granulicatella spp. (3). The species involved were A.defectiva (7), G.adiacens (2) and G.elegans (1). Eight patients were male, their mean age was 46 years and four were younger than 21 years. The most frequent comorbidities were congenital heart disease (4; 40%) and the presence of intracardiac prosthetic material (5; 50%). IE occurred on 5 native valves and 5 prosthetic valve or material. Blood cultures were positive in 8/10 patients, within a mean incubation period of 18.07 hours. In the other two patients, a positive 16SPCR from valve or prosthetic material provided the diagnosis. Surgery for IE was performed in seven patients (70%) and in all cases positive 16S rRNA PCR and sequencing from valve or prosthetic material was demonstrated. Valves and/or prosthetic removed material cultures were positive in four patients. Nine patients received ceftriaxone (4 in monotherapy and 5 in combination with other antibiotics). The mean length of treatment was 6 weeks and IE-associated mortality was 20% at one year follow-up. CONCLUSIONS: Abiotrophia spp. or Granulicatella spp. IE were infrequent but not exceptional in our environment and particularly affected patients with congenital heart disease or prosthetic material. Blood cultures and molecular methods allowed the diagnosis. Most of them required surgery and the associated mortality, in spite of a mean age of 46 years, was high.

Abiotrophia defectiva DnaK Promotes Fibronectin-Mediated Adherence to HUVECs and Induces a Proinflammatory Response.

Abiotrophia defectiva is a nutritionally variant streptococci that is found in the oral cavity, and it is an etiologic agent of infective endocarditis. We have previously reported the binding activity of A. defectiva to fibronectin and to human umbilical vein endothelial cells (HUVECs). However, the contribution of some adhesion factors on the binding properties has not been well delineated. In this study, we identified DnaK, a chaperon protein, as being one of the binding molecules of A. defectiva to fibronectin. Recombinant DnaK (rDnaK) bound immobilized fibronectin in a concentration-dependent manner, and anti-DnaK antiserum reduced the binding activity of A. defectiva with both fibronectin and HUVECs. Furthermore, DnaK were observed on the cell surfaces via immune-electroscopic analysis with anti-DnaK antiserum. Expression of IL-8, CCL2, ICAM-1, and VCAM-1 was upregulated with the A. defectiva rDnaK treatment in HUVECs. Furthermore, TNF-α secretion of THP-1 macrophages was also upregulated with the rDnaK. We observed these upregulations in rDnaK treated with polymyxin B, but not in the heat-treated rDnaK. The findings show that A. defectiva DnaK functions not only as an adhesin to HUVECs via the binding to fibronectin but also as a proinflammatory agent in the pathogenicity to cause infective endocarditis.

Abiotrophia defectiva adhere to saliva-coated hydroxyapatite beads via interactions between salivary proline-rich-proteins and bacterial glyceraldehyde-3-phosphate dehydrogenase.

Abiotrophia defectiva is a species of nutritionally variant streptococci that is found in human saliva and dental plaques and that has been associated with infective endocarditis. In our previous study, it was found that A. defectiva could bind specifically to saliva-coated hydroxyapatite beads (SHA). This study identified a cell surface component of A. defectiva that promotes adherence to SHA beads. The binding of A. defectiva to SHA was reduced in the presence of antibodies against human proline-rich protein (PRP); these results suggested that PRP may be a critical component mediating interactions between A. defectiva and the salivary pellicle. Two-dimensional gel electrophoresis of whole A. defectiva cells followed by Far-Western blotting was conducted by probing with synthetic peptides analogous to the binding region of PRP known as PRP-C. The results indicate that an A. defectiva protein of 37 kDa interacts with PRP-C. The results of amino-terminal sequencing of the adhesive A. defectiva protein revealed significant similarity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Recombinant GAPDH bound to immobilized PRP-C in a dose-dependent manner and binding of A. defectiva to SHA or to PRP was reduced in the presence of anti-GAPDH antiserum. Western blotting or electron immunomicroscopic observations with anti-GAPDH antiserum revealed that this protein was expressed in both cytosolic and cell wall fractions. These results suggest that A. defectiva could specifically bind to PRP via interactions with cell surface GAPDH; the findings suggest a mechanism underlying A. defectiva-mediated adherence to saliva-coated tooth surfaces.

Abiotrophia defectiva endarteritis with infective spondylodiscitis in an adult patient with patent ductus arteriosus.

Endarteritis is a major complication in patients with patent ductus arteriosus, causing significant morbidity and mortality. We report an adult patient with asymptomatic patent ductus arteriosus and endarteritis involving the main pulmonary artery and secondary infective spondylodiscitis at the L5-S1 intervertebral disc caused by Abiotrophia defectivaA. defectiva, commonly referred to as nutritionally variant streptococci, cannot be identified easily by conventional blood culture techniques from clinical specimens. Its isolation was confirmed by 16S ribosomal RNA sequencing. The patient was successfully managed with a combination of penicillin G and gentamicin, pending surgical repair of the patent ductus arteriosus.

Antimicrobial Susceptibilities of Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans.

Nutritionally variant streptococci (NVS) are fastidious Gram-positive cocci comprised of the species Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans. NVS are an important cause of bacteremia and infective endocarditis (IE) associated with significant morbidity and mortality. Antimicrobial susceptibility testing (AST) was performed for 14 antimicrobials using the broth microdilution MIC method described in the Clinical and Laboratory Standards Institute (CLSI) M45 guideline. A total of 132 clinical NVS blood isolates collected from 2008 to 2014 were tested. Species level identification of NVS isolates was achieved by 16S rRNA gene sequencing and/or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Ninety isolates were identified as G. adiacens, 37 as A. defectiva, and 5 as G. elegans. All isolates were susceptible to vancomycin (MIC90 = 1 µg/ml), and none displayed high-level resistance to aminoglycosides. G. adiacens was considerably more susceptible to penicillin than A. defectiva (38.9% versus 10.8% of isolates susceptible) but was less susceptible to cephalosporins than was A. defectiva (43.3% versus 100% of isolates susceptible to ceftriaxone). Several isolates were resistant to levofloxacin (6%), erythromycin (51%), and clindamycin (10%). The MIC90 for daptomycin was ≥ 4 µg/ml for G. adiacens and A. defectiva. G. elegans isolates were 100% susceptible to all antimicrobials tested, with the exception of erythromycin, to which only 20% were susceptible. This study provides antimicrobial susceptibility data for a recent collection of NVS and demonstrates important NVS species-related differences with respect to susceptibility to penicillin, cephalosporins, carbapenems, and daptomycin. Species-level identification of NVS organisms when susceptibility testing is not readily available may aid in treatment decisions.

Aortic Valve Endocarditis Caused by Abiotrophia defectiva: Case Report and Literature Overview.

UNLABELLED: Abiotrophia defectiva or nutritionally variant Streptococcus (NVS) are a rare but important cause of infectious endocarditis, with high rates of bacteriological failure and mortality. We report the case of a 74-year-old man admitted for fever, fatigue and general malaise in the absence of any underlying cardiac, immunosuppressive illness and previous dental manipulations. Transthoracic and transesophageal echocardiogram revealed bacterial vegetation and significant aortic stenosis and regurgitation. Initial blood culture reported gram-positive cocci in chains, subsequently identified as A. defectiva. The patient completed 6 weeks of antibiotic therapy with ampicillin, with a significant decrease of serum inflammatory markers. He refused cardiac surgery and had relapsing endocarditis with positive blood culture for the same pathogen. The patient was then submitted to double-valve cardiac surgery, obtaining a prompt resolution of clinical signs and symptoms, without other relapse or any complications. CONCLUSION: Infectious diseases caused by A. defectiva are extremely rare illnesses. Due to the difficult isolation of the pathogen and the slow clinical progression, clinicians should be aware of this bacterium when dealing with blood culture-negative infective endocarditis.

Metagenomic analysis for detecting pathogens in culture-negative infective endocarditis.

Pathogen identification is important for proper diagnosis and optimal treatment of infective endocarditis (IE). Blood and valve cultures are the gold standard for detecting pathogens responsible for IE. However, these tests only detect culturable pathogens, and have low sensitivity, especially in patients previously treated with antibiotics. Culture-negative IE is still a major clinical problem and a diagnostic challenge. Recently, metagenomic analysis using next generation sequencing has been used to detect pathogens directly from clinical samples. However, there are very few reports of the use of metagenomic analysis for pathogen identification in culture-negative IE cases and the usefulness of this new method is unknown. Here, we report a case of successful pathogen detection with metagenomic analysis in a patient of culture-negative IE. The patient underwent valve replacement surgery and received antibiotics for 5 weeks and survived. Using metagenomic analysis of resected vegetation, we detected Abiotrophia defectiva, which is often associated with culture-negative IE due to its fastidious growth. This method may be useful for pathogen identification in future cases of culture-negative IE.

[A case with Abiotrophia defectiva isolated from postoperative blood culture of a patient with diffuse peritonitis].

We have recently detected Abiotrophia defectiva (A. defectiva) from postoperative blood culture of a patient with diffuse peritonitis. Pleomorphic Gram positive rod appearance and no growth on a standard sheep blood agar led us to suspect aerotolerant anaerobe. From the test results of simplified identification kit for anaerobes, we tentatively reported Clostridium tertium (C. tertium) to the clinician. However, the analysis of 16SrRNA sequence proved it to be A. defectiva. When Gram positive coccus or pleomorphic Gram positive rod are observed on blood culture, it is recommended to incubate Brucella HK (RS) blood agar plates for 24 hours at 35 degrees C both aerobically and anaerobically. The growth both aerobically and anaerobically suggests A. defectiva and Satellitism test and Rapid ID 32 STREP(SYSMEX) testing should be performed to identify the responsible bacteria. A. defectiva is Gram positive streptococcus and requires pyridoxal hydrochloride or L-cysteine to grow. It could be unidentified or misidentified and its particularity often leads incomplete report to the clinician. Our study shows that more careful examination will increase the detection of A. defectiva.

Identification and characterization of a fibronectin-binding protein from Granulicatella adiacens.

The interaction of microorganisms with fibronectin plays an important role in infective endocarditis. Granulicatella adiacens is a member of the oral microbiota, formerly known as nutritionally variant streptococci, and is often isolated from endocarditis patients. In the present study we identified a surface protein, designated Cha, which binds to fibronectin, by a plaque hybridization procedure using the cshA sequence as probe, which encodes a fibronectin-binding molecule of Streptococcus gordonii DL1. The cha sequence was highly homologous to cshA and encoded a product of 2351 amino acid residues. The protein comprised a unique sequence in the N-terminal half region. The C-terminal region contained nine complete, and one incomplete, 115-amino acid residue repeat blocks. Among eight strains of nutritionally variant streptococci, three G. adiacens strains and one Abiotrophia defectiva strain carried the cha gene. Heterologous expression studies suggested that Cha adhered to immobilized fibronectin, and that this function was located in the unique region. Recombinant Cha protein also adhered to immobilize fibronectin and partially inhibited adherence of G. adiacens to fibronectin in a dose-dependent manner. These results suggest that Cha is a cell surface protein that mediates adherence of G. adiacens to fibronectin.

Abiotrophia defectiva infection of a total hip arthroplasty diagnosed by 16S rRNA gene sequencing.

We describe a case of a total hip arthroplasty infection caused by Abiotrophia defectiva, identified by 16S rRNA gene sequencing. Removal of the prosthesis followed by antibiotic treatment resulted in a good clinical outcome. 16S rRNA gene sequencing can be a useful tool in diagnosing infection with this fastidious microorganism that can easily be misidentified using phenotypic identification methods.

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms.

BACKGROUND: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. RESULTS: As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. CONCLUSIONS: Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.

Use of groESL as a target for identification of Abiotrophia, Granulicatella, and Gemella species.

We determined the groESL sequences of three species of nutritionally variant streptococci (Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans) and three Gemella species (Gemella morbillorum, Gemella haemolysans, and Gemella sanguinis). The nucleotide sequence similarities between the groES and groEL genes of the above genera were 41.7 to 85.9% and 63.7 to 84.3%, respectively. The intraspecies similarities of groESL sequences for the isolates of Abiotrophia and Granulicatella species were 94.4 to 97.8% for groES and 94.0 to 98.2% for groEL. For Ge. morbillorum and Ge. sanguinis, all strains showed the same groESL spacer length (8 bp), and sequence identities within species were >97.8% for groES and >96.1% for groEL. However, higher intraspecies heterogeneity was observed in Ge. haemolysans. Phylogenetic analysis of groEL sequences separated the 6 isolates of Ge. haemolysans into two subgroups. Among these isolates, three isolates with the same groESL spacer region length (45 bp) clustered together but were distant from the ATCC reference strain (with a spacer length of 8 bp). The remaining three isolates, with a spacer length of 50 or 8 bp, clustered together. Although 16S rRNA gene sequence analysis did not provide enough discrimination for the 6 Ge. haemolysans isolates, rpoB gene sequence analysis supported the subgrouping. Based on the obtained groESL sequences, we developed a multiplex PCR that enables simple, rapid, and accurate identification of Abiotrophia, Granulicatella, and Gemella at the genus level. This assay would be helpful for identifying these fastidious and slow-growing organisms in clinical laboratories.