Abstract
Granulicatella species, along with the genus Abiotrophia, were originally known as ‘nutritionally variant streptococci’. They are a normal component of the oral flora, but have been associated with a variety of invasive infections in man and are most noted as a cause of bacterial endocarditis. It is often advised that Granulicatella endocarditis should be treated in the same way as enterococcal endocarditis. We review here the published data concerning diagnosis and treatment of Granulicatella infection, and include some observations from local cases, including four cases of endocarditis.
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Introduction
Nutritionally variant streptococci (NVS), first described in the early 1960s from cases of endocarditis (Frenkel & Hirsch, 1961), were transferred to a separate genus, Abiotrophia, in the mid-1990s (Kawamura et al., 1995). Since then, this genus has been divided into the genera Abiotrophia and Granulicatella on the basis of 16S rRNA gene sequencing (Collins & Lawson, 2000). We review here the published data concerning diagnosis and treatment of Granulicatella infection. The PubMed database () was searched for ‘Granulicatella’ and individual references were reviewed for relevant clinical data. The reference lists of these publications were also reviewed. For specific infections, e.g. infective endocarditis, searches for ‘Abiotrophia’ were also undertaken: the species identification was reviewed and relevant cases were included. We also include some observations from local cases, including four cases of endocarditis, two cases of infection of prosthetic materials, and two cases considered to be either transient bacteraemias or contamination of blood cultures at the point of collection. Both of these final cases were also associated with the presence of prosthetic materials. Of the six active infections, successful therapy was possible in four.
Granulicatella species are catalase-negative and oxidase-negative, facultatively anaerobic, Gram-positive cocci (Collins & Lawson, 2000), and belong to the Carnobacteriaceae (Ludwig et al., 2008). The Streptococcaceae (including the genus Streptococcus), Enterococcaceae (including Enterococcus) and Aerococcaceae (including Abiotrophia) are related families within the order Lactobacilliales (Ludwig et al., 2008). Granulicatella and Abiotrophia were known as NVS because of their requirement for pyridoxal or other additional agents to be incorporated into standard media for successful laboratory isolation (Frenkel & Hirsch, 1961; Ruoff, 1991). Recovery of NVS from blood samples can be achieved using thiol broths (Ruoff, 1991).
Three species of Granulicatella have been described: G. adiacens, G. elegans and G. balaenopterae (Collins & Lawson, 2000). A further species, Abiotrophia para-adiacens, related to Abiotrophia adiacens, was proposed prior to separation of the Granulicatella and Abiotrophia genera (Kanamoto et al., 2000), and is occasionally reported (Dowd et al., 2008; Senn et al., 2006a, b), but the name is not currently validly published (Garrity et al., 2004). G. balaenopterae has not been described from human samples (Collins & Lawson, 2000).
Granulicatella species are a normal component of the oral flora (Aas et al., 2005; Sato et al., 1999), are found in dental plaque (Mikkelsen et al., 2000), endodontic infection (Siqueira & Rôças, 2006) and dental abscesses (Robertson & Smith, 2009), but can also cause a variety of serious infections.
Frequency
Granulicatella species are uncommon clinical isolates, with the majority of reported isolates recovered from blood culture. In the largest series to include the clinical source of isolates, 39 of 43 G. adiacens isolates with a known source were isolated from blood, with the remainder isolated from sinus, bone marrow, abscesses and eye samples (Christensen & Facklam, 2001). The NVS as a group have been estimated to cause between 5 and 6 % of all cases of streptococcal endocarditis (Brouqui & Raoult, 2001; Roberts et al., 1979), although this is often over-reported as 5 % of all cases of endocarditis (Facklam, 2002; Ruoff, 1991; Wijetunga et al., 2002). A review of local cases revealed eight episodes of possible bloodstream infection caused by G. adiacens between 2005 and 2009. For comparison, a total of six episodes caused by Abiotrophia species were identified during the same time period.
Clinical significance
Endocarditis
Seventeen cases of Granulicatella endocarditis (rather than nutritionally variant streptococcal endocarditis) have been described in the literature since 1997 (see Table 1). Cases have been reported for both G. adiacens (Chang et al., 2008; Christensen et al., 1999; Jeng et al., 2005; Lin & Hsu, 2007; Perkins et al., 2003; Hernando Real et al., 2007; Rosenthal et al., 2002; Schwede et al., 2007; Vandana et al., 2010; Woo et al., 2003) and G. elegans (Al-Tawfiq et al., 2007; Casalta et al., 2002; Ohara-Nemoto et al., 2005; Roggenkamp et al., 1998). These include infections of prosthetic valves (Christensen et al., 1999; Jeng et al., 2005; Schwede et al., 2007) and of pacemaker leads (Rosenthal et al., 2002). Locally, four cases of endocarditis were identified, in two of which the patients had known cardiac abnormalities. In each case, between two and six sets of blood cultures were positive for G. adiacens after less than 24 h incubation (Bactec 9240 automated blood culture system; Becton Dickinson). It has been suggested that Granulicatella may be under-reported and diagnosed as ‘culture-negative’ endocarditis (Brouqui & Raoult, 2001); however, the largest case series of culture-negative endocarditis cases identified only one case (0.3 %; G. elegans; Houpikian & Raoult, 2005).
Endocarditis caused by G. adiacens is more common than that caused by Abiotrophia species, with G. elegans being comparatively rare (Christensen & Facklam, 2001). Strains of G. adiacens from endocarditis have been reported to display fibrinonectin binding, unlike those not associated with endocarditis or G. elegans (Dowd et al., 2008; Okada et al., 2000). This difference between G. adiacens and G. elegans may account for the different prevalence in endocarditis. Mixed valvular disease is not uncommon (5 of 21 cases; Lin & Hsu, 2007; Schwede et al., 2007), and many cases have required surgical intervention (Al-Tawfiq et al., 2007; Casalta et al., 2002; Chang et al., 2008; Christensen et al., 1999; Lin & Hsu, 2007; Roggenkamp et al., 1998). Combinations of either benzylpenicillin (penicillin G) (Christensen et al., 1999; Lin & Hsu, 2007; Ohara-Nemoto et al., 2005; Schwede et al., 2007) or amoxicillin (Casalta et al., 2002; Perkins et al., 2003) with gentamicin are the most frequent antibiotic interventions. The British Society for Antimicrobial Chemotherapy (BSAC), American Heart Association (AHA) and the European Society for Cardiology (ESC) all make recommendations for the treatment of NVS or Granulicatella in their endocarditis guidelines (Baddour et al., 2005; Gould et al., 2012; Habib et al., 2009). The AHA guidelines explicitly recommend that NVS endocarditis should be treated as for enterococci (Baddour et al., 2005); the BSAC guidelines previously contained the same explicit recommendation (Elliott et al., 2004), although the latest guidelines no longer make this link between treatments (Gould et al., 2012). Locally, therapeutic success has been achieved with both vancomycin (1 g 12-hourly) and amoxicillin (2 g 4-hourly), with the addition of gentamicin (1 mg kg−1 to a maximum of 80 mg 12-hourly) when the isolates were provisionally identified. Three patients were treated with 6 weeks of antibiotic therapy, although one required aortic and mitral valve replacement after 4 weeks.
NVS endocarditis has been considered to have a high relapse rate (Stein & Nelson, 1987), and relapses following treatment have been reported for Granulicatella endocarditis (Christensen et al., 1999; Schwede et al., 2007). To date, no relapses are known for our local patients who survived the initial episode. Complications, which may necessitate surgery, are not uncommon, and include valvular damage (Al-Tawfiq et al., 2007; Roggenkamp et al., 1998), heart failure (Lin & Hsu, 2007), distal embolization (Lin & Hsu, 2007; Vandana et al., 2010), cerebral mycotic aneurysm (Chang et al., 2008; Lin & Hsu, 2007) and extension of vegetations to the atrial wall (Al-Tawfiq et al., 2007; Jeng et al., 2005). One of our local cases required surgery following the development of cardiac failure. The overall mortality for reported cases of Granulicatella endocarditis (rather than NVS endocarditis) is 9.5 %, with two deaths (see Table 1).
Other bloodstream infection
Bacteraemia has been reported in the absence of endocarditis (Abdul-Redha et al., 2007; Christensen & Facklam, 2001; Senn et al., 2006a; Woo et al., 2003). G. adiacens bacteraemia has also been reported in association with early onset neonatal sepsis, with maternal vaginal carriage also identified (Bizzarro et al., 2011). Infected aortic atheroma, with an associated dissection, has also been reported (Woo et al., 2003). This patient did not survive their infection. Two episodes of probable transient bacteraemia were identified locally. In both episodes, a single blood culture taken from an arterial line grew G. adiacens, and both cases were considered to be either colonizers of the lines or coincidental transient bacteraemia.
Other infection
Two patients were identified locally with a probable focus of infection involving prosthetic material. The first had radiological evidence of infection of an abdominal aortic graft. One of six sets of blood cultures grew G. adiacens. The patient did not survive attempted surgical repair of the infected graft and targeted antimicrobial therapy was never started. The second patient had four sets of blood cultures taken on the same day, both peripherally and from the lumens of a Tesio line, which grew G. adiacens, and morphologically identical colonies grew from the Tesio line tip. Transthoracic echocardiography showed no evidence of valvular lesions, and the patient was treated with a short course of vancomycin.
G. adiacens has also been reported to cause infection of the central nervous system. As well as mycotic aneurysms arising from endocarditis (Chang et al., 2008; Lin & Hsu, 2007), Granulicatella infections following neurosurgery have been reported to cause both brain abscesses (Biermann et al., 1999) and meningitis with epidural abscess (Cerceo et al., 2004). One case of brain abscess without surgery has been reported; the child had congenital heart defects but no vegetations were seen on imaging (Michelow et al., 2000).
G. adiacens has been reported to cause septic arthritis (Hepburn et al., 2003; Riede et al., 2004), vertebral osteomyelitis (Rosenthal et al., 2002; Fukuda et al., 2010; Heath et al., 1998) and discitis (Heath et al., 1998). There are also reports of infections of breast implants (del Pozo et al., 2008) and of peritoneal dialysis related peritonitis (Altay et al., 2008). Locally, one further G. adiacens was isolated from a pericardial fluid sample; this patient was transferred to another centre and no further information was available.
Identification
Locally, Granulicatella isolates are identified by either biochemical testing (API Strep; bioMérieux) and/or molecular confirmation. In several cases, identification was delayed as the isolates were thought to be poorly growing streptococci before pyridoxal dependence was considered. In one case, direct antimicrobial susceptibility testing from positive blood cultures was noted to show growth only in the vicinity of the Staphylococcus aureus strain used as a control for Stokes’ susceptibility testing, and the possibility of Abiotrophia species or Granulicatella species was then considered. Molecular identification locally is made by an in-house 16S rRNA gene sequencing method using the primers fD1 (Weisburg et al., 1991) and UR (Hendolin et al., 2000).
In all but one case locally, blood cultures became positive within 24 h of incubation; the exception was considered to represent a transient bacteraemia. In all but three cases, both the aerobic and anaerobic blood culture bottles became positive; of the three remaining cases, two were considered transient events. On average, anaerobic blood cultures became positive slightly earlier than the aerobic bottle, but this was not significant or reliable (3.56 h, standard deviation 8.49 h). Where an acceptable API 20 Strep result was not available, isolates seemed to identify as Gemella species.
Biochemical testing has been reported to potentially lead to incorrect identification, with G. elegans being misidentified as Streptococcus acidominimus, Gemella morbillorum and G. adiacens (Abdul-Redha et al., 2007; Al-Tawfiq et al., 2007) and G. adicens as Gemella as happened locally. Identification of isolates by 16S rRNA gene sequencing of either bacterial isolates (Biermann et al., 1999; Ohara-Nemoto et al., 2005; Hernando Real et al., 2007; Woo et al., 2003) or of valvular tissue (Casalta et al., 2002), or identification by biochemical methods including API (Perkins et al., 2003; del Pozo et al., 2008; Rosenthal et al., 2002) and VITEK (bioMérieux; Altay et al., 2008), or combinations of approaches (Abdul-Redha et al., 2007; Al-Tawfiq et al., 2007; Lin & Hsu, 2007), have been previously reported.
A variety of molecular techniques have been developed to help accelerate the identification of Gram-positive cocci, including Granulicatella. As well as 16S rRNA gene sequencing, sequencing of the rpoB gene (Drancourt et al., 2004) and the ribosomal 16S–23S intergenic spacer region (Tung et al., 2007) have been used to identify Granulicatella, Abiotrophia, Enterococcus and Streptococcus species. Fluorescence in situ hybridization has been used for rapid identification of Gram-positive cocci, including Granulicatella, from positive blood cultures (Gescher et al., 2008). Oligonucleotide arrays have also been used, using the ribosomal intergenic spacer region (Tung et al., 2006). The increasing use of molecular methods in the diagnosis of endocarditis, including 16S (Breitkopf et al., 2005) and 23S (Vollmer et al., 2010) rRNA gene sequencing, may mean an increasing recognition of formerly rare organisms in endocarditis. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been reported to identify G. adiacens to genus level in one-third of tests only (Neville et al., 2011).
Susceptibilities
Antibiotic susceptibility testing by disc diffusion is not recommended for Granulicatella isolates (Jorgensen & Hindler, 2007). Broth microdilution (using a supplemented Mueller–Hinton broth) is suggested (Jorgensen & Hindler, 2007), although a disc diffusion method utilizing S. aureus culture supernatants incorporated into the test media has also been reported (Tantimavanich et al., 2002). Etest results have been shown to be similar to broth dilution for penicillin, although there is a higher rate of intermediate susceptibility reported (Douglas et al., 1994). In our isolates, penicillin MICs ranged from 0.016 to 1.0 mg l−1 by Etest (AB Biodisk/bioMérieux). Four of the five local isolates with MIC data were susceptible to benzylpenicillin by EUCAST streptococcal breakpoint values (European Committee on Antimicrobial Susceptibility Testing, 2011). Resistance to β-lactam antibiotics has been reported in both G. adiacens (susceptibility rates of 55–67 % to penicillin, 0–63 % to ceftriaxone, 81 % to amoxicillin and 96 % to meropenem) and G. elegans (susceptibility rates of 100 % to penicillin, 0 % to cefuroxime and 33 % to ceftriaxone) (Tuohy et al., 2000; Zheng et al., 2004). Resistance has also been reported to clindamycin, tetracycline, erythromycin and ciprofloxacin, but not to vancomycin or rifampicin (Tuohy et al., 2000; Zheng et al., 2004). The need for routine susceptibility testing is unclear as many infections may be managed without such results (Jorgensen & Hindler, 2007).
Summary
Granulicatella species are an uncommon cause of infection. We suggest that Granulicatella (and Abiotrophia) species should be considered in patients where slow-growing α-haemolytic streptococci are isolated from blood cultures or other sterile sites. For isolates which are showing poor growth after 48 h, we suggest that a presumptive identification should be made by examining for satellitism around an S. aureus strain if pyridoxal-supplemented blood agar is not available (Reimer & Reller, 1981). Where antimicrobial susceptibilities will affect clinical management, centres should consider using reference facilities to confirm the results of local laboratory testing. It has been advised that NVS endocarditis should be treated in the same way as enterococcal endocarditis, using combinations of benzylpenicillin (Baddour et al., 2005; Gould et al., 2012; Habib et al., 2009), ampicillin (Baddour et al., 2005)/amoxicillin (Gould et al., 2012; Habib et al., 2009) or vancomycin (Baddour et al., 2005; Habib et al., 2009) alongside gentamicin (Baddour et al., 2005; Gould et al., 2012; Habib et al., 2009). Reviewing the cases of specific Granulicatella endocarditis shows that these antimicrobial combinations are clinically effective in practice.
Acknowledgements
We thank Mr Mark Liddington, Consultant in Plastic Surgery, Mr Andrew Mavor, Consultant in Vascular Surgery, and Dr Wazir Baig, Consultant in Cardiology, all of the Leeds Teaching Hospitals NHS Trust.