Endocarditis caused by Abiotrophia and Granulicatella species, formerly known as nutritionally variant streptococci (NVS) is rare. It is associated with increased complications such as heart failure, systemic emboli, valve replacement surgery, treatment failures and mortality. The diagnosis of these infections is challenging due to specific nutritional growth requirements although modern techniques such as 16S rRNA sequence analysis and Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are particularly useful. Penicillin resistance among these organisms is a growing problem. Penicillin and gentamicin combination or alternatively Vancomycin alone are the recommended treatment options, however there is increasing data regarding susceptibilities to other antibiotics. Varying susceptibilities to antibiotics among different species of NVS needs to be studied further.
- Abiotrophia and Granulicatella endocarditis
- aorto-right ventricular fistula
- Abiotrophia endocarditis
Nutritionally variant (deficient) streptococci (NVS) were first described by Frankel and Hirsch in 1961 . These gram positive bacteria resembled streptococci but had specific nutritional growth requirements. Since their first identification, over the years nomenclature of NVS changed several times based on DNA-DNA hybridization studies and 16S rRNA sequence analysis. They were first included in the genus
NVS are members of the normal flora of human pharynx, human urogenital and intestinal tracts .
Infective endocarditis caused by NVS is rare, causing approximately 2% of all cases of infective endocarditis . Over 125 cases of infective endocarditis caused by
Although endocarditis and bacteremia are the most common infections associated by
Ophthalmological infections have been encountered, ranging from keratitis to endophthalmitis . NVS are known to cause corneal ulcers , vitreous infections  and infectious crystalline keratopathy . Orthopedic infections, including prosthesis infection, septic arthritis, discitis and sacroiliitis have been reported [10, 11, 12]. Synovial biopsy sample from a patient with culture negative endocarditis also yielded NVS . NVS are also associated with central nervous system infections; more commonly brain abscesses but rarely meningitis , subarachnoid hemorrhage  and intracranial aneurysms  have been reported. CNS infections have been commonly linked to embolic phenomena, neurosurgical instrumentation and immunosuppression [10, 16]. NVS have been isolated from patients with otitis media , otitis externa , sinusitis , parapneumonic effusion , cirrhosis , peritonitis , pancreatic abscess , bacteremia associated with postpartum or postabortal sepsis , tubo-ovarian abscess , breast implant associated infection , wound infections, and vaginal discharge . Endarteritis caused by
Nutritionally variant streptococci were first described by Frenkel and Hirsch in 1961 from blood cultures of cases of subacute bacterial endocarditis and from otitis media. These cell wall deficient, L form ‘streptococci’ were noted to grow in satellite colonies around other bacteria requiring substances secreted by other bacteria for growth . ‘Abiotrophia’ means life nutrition deficiency, referring to the need of specific nutrients in media for growth of these bacteria . They are catalase-negative, oxidase-negative, facultative anaerobic gram positive bacteria . They often form white-gray, non-hemolytic colonies. These organisms hardly grow in culture media that streptococci ordinarily grow, such as sheep blood agar. They require supplementation of L-cysteine or pyridoxal HCl. In the absence of these supplements, NVS can also grow forming satellite colonies adjacent to streaks of helper bacteria such as
Bouvet et al. in 1989 showed that NVS could be divided into two groups,
In 1995, Kawamura et al. proposed that these distinct species be transferred to a new genus,
Finally, in 2000, Collins et al. proposed the taxonomy of NVS that we use today. They pointed out that genus
Bacterial attachment to damaged heart valves is the key factor in infective endocarditis. Intact vascular endothelium can resist the development of endocarditis . Experimental animal models showed that when catheter induced endocardial damage is produced; these endocardial lesions can be infected by direct inoculation of bacteria or by intravenous inoculation . Pathophysiology of infective endocarditis typically would start with endothelial cell denudation, followed by exposure of underlying extracellular matrix (ECM) and finally binding of fibrin and platelets . Extracellular matrix proteins are exposed during damage to the cardiac endothelium providing potential sites of attachment for virulent organisms 
Some groups of NVS are more pathogenic and other groups are less pathogenic. Highly pathogenic
By binding to the extracellular matrix proteins, bacteria are able to adhere to the damaged endocardium and subsequently producing colonization and infection. ECM binding ability however is not the sole indicator of pathogenicity. Some strains of NVS have high infectivity without significant binding to the ECM proteins suggesting other mechanisms involved in pathogenesis. Other mechanisms of endocardial infectivity of NVS remains to be discovered .
As a group, NVS have heterogenous properties of pathogenicity.
Okada et al. noted that  NVS isolates from endocarditis patients and from normal oral flora both had the ability to cause infective endocarditis.
NVS show morphologic variations depending on the pyridoxal concentrations in the growth medium . Due to the difficulties in identification of these bacteria, it is crucial for microbiology staff to be vigilant and be aware of the pleomorphic nature of the NVS to prevent misidentification.
NVS should be suspected when gram stain shows microbial cells but cultures are negative . Once their nutritional growth requirements are supplemented in media, NVS convert to streptococci-like cells  and gram positivity making them easier to identify, although it was also shown that correcting nutritional deficiency may not convert all abnormalities . For
Contemporary blood culture methods enable
Cargill et al. noted that anaerobic blood culture bottles became positive sooner than aerobic blood cultures bottles; (3.56 h, standard deviation 8.49 h) although they noted that this was not significant or reliable .
Specific phenotypic characteristics of NVS can be identified by examining their patterns of production of α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase and β-glucuronidase, and fermentation of trehalose, pullulan, tagatose and sucrose [32, 38].
Molecular diagnostic techniques can be used for rapid and accurate diagnosis of NVS in blood or tissue samples. PCR amplification of 16S rRNA and restriction fragment length polymorphism (RFLP) for routine detection of NVS was developed by Ohara-Nemoto et al. in 1997 . For culture negative infective endocarditis, molecular techniques appear to be more sensitive in resected valvular tissue compared to blood samples .
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable and cost-effective technique used to identify microorganisms by utilizing MALDI-TOF MS devices in the clinical microbiology labs . These devices carry the potential to complement or replace the phenotypic identification of various microorganisms including bacteria . MALDI-TOF MS is a rapid and accurate diagnostic tool that has been used to identify and timely diagnose NVS .
In culture negative endocarditis,
5. Clinical presentation and complications
Endocarditis caused by NVS typically follows a slow and indolent course. Endocarditis develops as a result of bacteremia.
Mortality rate associated with endocarditis caused by NVS is 17% which is higher than that of viridans streptococci (0–12%) and enterococci (9%) .
Underlying valvular disease is commonly seen as a predisposing factor for development of endocarditis. Over 90% of the cases have preexisting heart disease and 10% of patients have prosthetic heart valves . Newer data however, suggest that there is increased involvement of normal heart valves in the past decade .
Embolization is a common complication of Abiotrophia endocarditis affecting one-third of patients. Typical peripheral manifestations of endocarditis such as petechia, digital clubbing, Osler nodes are not frequently found .
It has been known that infective endocarditis caused by NVS carries a higher risk of embolization, treatment failure and increased mortality as compared to infective endocarditis caused by viridans streptococci .
Stein et al. reviewed 30 published case reports of endocarditis caused by NVS and found that 17% of patients had relapses after antibiotic therapy. Bacteriologic failure rate was 41% (defined as positive blood cultures after 7 days of appropriate antibiotic therapy, relapse following a course of therapy with appropriate antibiotics, or a positive valve culture). It is notable that bacteriologic failure was seen despite the sensitivity of the organisms to the antibiotics used in two thirds of the cases. About 31% of the patients required surgery. Mortality rate was 17% which was higher than that of endocarditis caused by enterococci or viridans streptococci .
Similarly a more recent review of 29 cases of solely
Large vegetation sizes are associated with increased risk of systemic embolism in infective endocarditis. Case studies reveal large vegetation sizes with infective endocarditis caused by NVS (greater than 10 mm in 7 out of 8 cases reviewed by Lin et al., and average vegetation size of 16 mm in a case series of 29 patients by Adam et al.) . These findings correlate with the high rates of systemic embolism seen in endocarditis caused by NVS.
Endocarditis caused by NVS is associated with high rates of infectious intracranial cerebral aneurysms although the exact incidence is unknown. Having a low threshold for obtaining imaging of the CNS is reasonable even for patients with vague complaints such as severe localized headaches or mild confusion . Many infectious intracranial cerebral aneurysms resolve by antibiotic treatment with reductions in size in the first 1–2 weeks. The risk of rupture decreases with time on antibiotic therapy .
Endocarditis caused by NVS is associated with 13% of aortic valve damage and 11% of mitral valve damage. If not recognized on a timely basis, these patients may present with congestive heart failure as the first presenting manifestation of the infection . Congestive heart failure is a potential complication of valvular destruction which can necessitate heart valve replacement surgery.
Aorto-RV fistula is a rare complication of
Endocarditis caused by NVS has rarely been reported in children. According to a review of 13 pediatric cases in children, 69% had underlying heart disease . Similar to adult patients, endocarditis caused by NVS in pediatric populations also appears to be associated with high complication rates including severe valvular damage, surgical valve replacement and systemic embolization [54, 55].
Antimicrobial susceptibility testing is very difficult for
Recommended treatment regimen is Ampicillin (12 g/d in divided doses) or penicillin (18–30 million U/D in divided doses or by continuous infusion) plus gentamicin 3 mg/kg/d in 2–3 divided doses).
For those patients who are intolerant to penicillin, Vancomycin alone without the use of gentamicin can be given for therapy. This is in contrast to enterococcal endocarditis treatment where Vancomycin is combined with gentamicin .
The duration of treatment for
The treatment duration is 4 weeks for native valve endocarditis with symptoms or illness ≤3 months. 6 week therapy is recommended for patients with symptoms >3 months. For prosthetic valve or other prosthetic cardiac material infections, minimum 6 weeks of antibiotic therapy is recommended .
Historically, in animal models it was shown that Penicillin alone was inferior to Penicillin plus aminoglycoside or Vancomycin alone for the treatment of infective endocarditis caused by NVS [57, 58]. it was shown that penicillin plus low dose (0.32 mg/kg) vs. high dose (1.05 mg/kg) gentamicin treatment results were virtually identical .
There is encouraging data to suggest that shortened courses of aminoglycosides in the treatment regimens (median 15 days) may result is similar clinical outcomes in treatment of enterococcal endocarditis. However this particular issue requires further study and it is not yet known how this would apply to treatment of infective endocarditis caused by
Given the growing concerns over antibiotic resistance among NVS, poor treatment outcomes and high rates of treatment failures it is important to look into data for susceptibilities of a broad range of antibiotics. There is however limited data available regarding the antibiotic susceptibilities of
NVS have the highest in vitro penicillin resistance compared to any other streptococci. The rate of penicillin resistance among NVS appears to be rising over the years. While an earlier study by Cooksey and Swenson in 1979  and Gephart and Washington in 1982  showed no isolates had a penicillin MIC >1 μg/ml, subsequent studies showed significantly increasing penicillin resistance; Bosley and Facklam in 1990  noted 9% rate of resistance to penicillin and Alberti in 2016  reported 14% rate of penicillin resistance among NVS. It is also notable that the method of penicillin susceptibility testing has changed over the years. Douglas et al. (1994)  found that while historical method of penicillin susceptibility testing by reference dilution method did not find penicillin resistance, when same NVS isolates were tested with E test, 7% penicillin resistance was detected. The high rate of penicillin resistance among VNS appears also to be consistent among NVS isolates from pediatric infections .
According to antibiotic susceptibility testing of 132 isolates by Albierti et al. in 2016, only 33% of the 132 isolates were susceptible to penicillin and 14% were resistant with an MIC ≥4 μg/ml. The remaining 53% of the isolates had penicillin MICs in the intermediate category (0.25–2 μg/ml) . Liao et al. reported 50% of their isolates (14 out of 28 isolates) had intermediate susceptibility to penicillin .
There appears to be differences in penicillin susceptibilities among different species of NVS. Albierti et al. showed that penicillin susceptibility is much less among
In an earlier study by Touhy et al. in a review of 39 isolates from 1995 to 1999, similar to Albierti et al.’s findings,
7.1. Penicillin tolerance
It is notable that clinical failures of treatment have frequently been described even for penicillin susceptible strains when appropriate antibiotics are given. Holloway et al. described a phenomenon of penicillin tolerance among NVS which minimum bactericidal concentration (MBC) significantly exceeded (greater than 32) the minimum inhibitory concentration (MIC) that would lead to a slower antibiotic effect and potentially a worse clinical response. In addition to the usual nutritional supplements of vitamin B6 and cysteine to the plates, by adding penicillinase to the subculture medium and a staphylococcal streak across the plates they showed that even though all tested isolates were susceptible to penicillin (MICs of the strains ranged from 0.05 to 0.4 U of penicillin per ml), 100% of the isolates were penicillin tolerant. The isolates did not show any penicillin tolerance if the subculture was supplemented only with pyridoxal and cysteine . Therefore, in order to identify penicillin tolerance and not misidentify the strains as penicillin sensitive, it is necessary to add penicillinase to the medium in addition to the usual growth supplements, pyridoxal HCl, cysteine and staphylococcal streak.
The slow growth rate of NVS is also thought to be responsible from poor response to antibiotic treatment. NVS generation time is 2–3 h while viridans streptococci generation time is 40–50 min [2, 41, 68].
7.2. Susceptibility testing
According to the latest consensus guidelines from the Clinical and Laboratory Standards Institute (CLSI) for antimicrobial susceptibility testing for infrequently isolated or fastidious bacteria, disk diffusion test for
E test is proven to be a rapid and simple method for MIC estimation for NVS, comparable to broth microdilution MIC testing .
CLSI consensus guidelines also emphasize that cases of
Penicillin resistance is often associated with resistance or decreased susceptibility to other beta-lactam antibiotics including ceftriaxone .
However, overall cephalosporin susceptibility among NVS appears to be higher compared to penicillin. In addition, A
Albierti et al. noted that some of the isolates that were resistant to ceftriaxone still remained susceptible to Ceftaroline. (51.6% of
Species related differences of penicillin or cephalosporin sensitivities in determining antibiotic choices remains to be investigated. The high rates of ceftriaxone resistance among
European Society of Cardiology (ESC) Clinical Practice Guidelines include Ceftriaxone in their recommendations for treatment of endocarditis caused by
Iv Vancomycin is recommended as an alternative regimen to iv penicillin for those patients who are not able to tolerate penicillin or ampicillin .
Bouvet et al. by using an experimental animal model found that Vancomycin alone was as good as combination of Vancomycin and Gentamicin for treatment of endocarditis caused by NVS .
Vancomycin susceptibility breakpoint is typically MIC <1 μg/ml and no resistance to Vancomycin among NVS has been reported thus far [62, 64, 66]. It is notable however that MIC90 for Vancomycin is 2 times higher for
NVS remains susceptible to aminoglycosides (MICs for Gentamicin and streptomycin≤4 μg/ml), high level aminoglycoside resistance has not been reported. As per AHA and BSAC guidelines for treatment of infective endocarditis caused by
Macrolide resistance is common among
Resistance to Meropenem or Imipenem among
Resistance to quinolones among NVS is rare. 8
13. Daptomycin and linezolid
There are no CLSI defined sensitivity breakpoints for Daptomycin and Linezolid for NVS. Albierti et al. found that Daptomycin MICs appear to be relatively high for NVS. Daptomycin MIC90 was >4 μg/ml for
When the breakpoint of Linezolid susceptibility for viridans group streptococci (MIC ≤2 μg/ml) is applied to NVS, all NVS would be considered susceptible to Linezolid according to one study of 132 isolates [62, 69]. It was noted that
Rifampin appears to be one of the most effective antibiotics against NVS although the data is limited. It was shown that Rifampin had a minimal bactericidal concentration of 2 μg/ml while that of penicillin was 1 μg/ml . Combination of Vancomycin and Rifampin showed synergy in in vitro studies . According to one review of 15 isolates of
15. Role of surgery
Endocarditis caused by NVS is associated with high rates of complications including heart failure, embolization and valvular damage. The need for surgery and time of surgery remains to be determined. However based on outcomes of several cases published in the literature, the rate of surgical treatment is very high especially due to development of heart failure.
he rate of valve surgery is high; 51% (in a review of 29 cases of
A vegetation size of 10 mm or more is associated with increased mortality and increased risk of embolic events . EASE Trial showed that early surgery in infective endocarditis in patients with large vegetations significantly reduced the mortality, risk of systemic embolism or recurrence of infective endocarditis (3% in the early surgery group vs. 28% in the conventional treatment group) . Lin et al.  reported 7 out of 8 cases of endocarditis caused by NVS had large vegetation sizes (10 mm). In the same review, 7 out of 8 cases required surgery (4 out of 8 cases required early valve replacement due to severe heart failure, while 3 cases underwent mitral valve repair 2,4, and 7 months after the diagnosis of endocarditis).
Combined approach with antibiotic treatment and surgery provides the best outcomes in endocarditis caused by NVS. Specifically, early surgical intervention should be considered for those patients with heart failure due to valvular destruction , hemodynamic compromise  or large vegetation sizes [68, 77].
Infective endocarditis caused by NVS has posed tremendous diagnostic and therapeutic challenges and continue to do so even in the era of modern medicine. Delays in diagnosis due to difficulties in identification frequently cause delays in treatment and poor treatment outcomes. Treatment failure and high complication rates associated with
Interpretation of the medical literature of
Differences in pathogenicity and susceptibility to antimicrobials have been demonstrated among these heterogeneous group of bacteria. More studies are needed to determine if there are further species specific differences of these fascinating microorganisms which would help us improve our understanding, diagnosis and the treatment outcomes of infections caused by NVS.