Bloodstream and endovascular infections due to Abiotrophia defectiva and Granulicatella species

Background Abiotrophia and Granulicatella species, previously referred to as nutritionally variant streptococci (NVS), are significant causative agents of endocarditis and bacteraemia. In this study, we reviewed the clinical manifestations of infections due to A. defectiva and Granulicatella species that occurred at our institution between 1998 and 2004. Methods The analysis included all strains of NVS that were isolated from blood cultures or vascular graft specimens. All strains were identified by 16S rRNA sequence analysis. Patients' medical charts were reviewed for each case of infection. Results Eleven strains of NVS were isolated during the 6-year period. Identification of the strains by 16S rRNA showed 2 genogroups: Abiotrophia defectiva (3) and Granulicatella adiacens (6) or "para-adiacens" (2). The three A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas 7 of 8 Granulicatella spp. strains were isolated from immunosuppressed patients, mainly febrile neutropenic patients. We report the first case of "G. para-adiacens" bacteraemia in the setting of febrile neutropenia. Conclusion We propose that Granulicatella spp. be considered as a possible agent of bacteraemia in neutropenic patients.


Background
Nutritionally variant streptococci (NVS), first described in 1961 by Frenkel and Hirsch [1], were classified on the basis of growth characteristics such as nutrient requirements (pyridoxal) and presence of satellitism. In 1989, based on DNA-DNA hybridisation, Bouvet et al. showed that NVS could be divided in two groups, Streptococcus defectivus and Streptococcus adiacens [2]. In 1995, based on the genetic and phylogenetic analysis of the 16S rRNA sequences, the genus Abiotrophia and the two species A. defectiva and A. adiacens were proposed by Kawamura [3]. In 1998, Roggenkamp et al. proposed the new species A. elegans [4], and in 1999, Lawson et al. proposed the species A. balaenopterae [5]. Based on 16S rRNA heterogeneity and phenotypic differences, Kanamoto et al. proposed an additional species "Abiotrophia para-adiacens" [6]. In 2000, A. adiacens, A. balaenopterae and A. elegans were reclassified in the new genus Granulicatella by Collins and Lawson [7].
In this study, we reviewed the clinical manifestations of infections due to A. defectiva and Granulicatella species that occurred at our institution over a 6-year period.

Bacterial strains
The analysis included all strains of NVS that were isolated from blood cultures or vascular graft specimens from patients admitted to our 800-bed University Hospital from January 1998 to December 2004. The automated blood culture system used in the microbiology laboratory during the study period was the Bactec 9240 (Becton Dickinson, Sparks, Md.) with the Plus aerobic/F and Lytic Phylogenetic analysis of the strains Figure 1 Phylogenetic analysis of the strains. Phylogenetic tree showing the affiliation of 3 isolates to A. defectiva, 6 isolates to G. adiacens and 2 isolates to "G. para-adiacens". The tree was inferred from 1315 base pairs 16S rRNA sequence data by the neighbour-joining method using the Kimura-corrected p-distance. Streptococcus oralis was used as outgroup. Genbank accession numbers are shown in parentheses. anaerobic/F vials (Becton Dickinson). The strains were identified to the species level using the Rapid ID32 STREPT system (Bio Mérieux SA, Marcy-l'Etoile, France).

16S rRNA gene sequencing
All strains were also identified by 16S rRNA sequence analysis. DNA was extracted with the MagNA Pure LC DNA isolation Kit I (Roche Diagnostics, Mannheim, Germany) according to the instructions of the manufacturer. Polymerase chain reaction (PCR) amplification of the 16S RNA gene was performed with primers fD1 and rP2 [29] and Taq DNA polymerase (Gibco BRL, Life Technologies) followed by electrophoresis of the PCR products on ethidium bromide-stained 1% agarose gel. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France). Sequencing was performed by using the dRhodamine Terminator Cycle Sequencing Ready Reaction kit with one of six different primers and AmpliTaq DNA (Perkin-Elmer Biosystems, Warrington, England) with a 3100 ABI Prism automated sequencer (Applied Biosystems, Courtaboeuf, France). Sequences derived from each primer were aligned and combined into a single 16S rRNA sequence by using Contig Express, a component of the Vector NTI suite 9.0 (Informax, Frederick, MD). Each sequence was compared with all eubacterial 16S rRNA sequences available in the GenBank database by using the BLASTN 2.2.2 program available on the National Center for Biotechnology Information website [30,31]. The 16S rRNA sequences of Abiotrophia and Granulicatella isolates were aligned with those of other members of the genus Abiotrophia and Granulicatella by using the CLUSTWAL W program supported by the DDBJ website [32]. Sequences were edited by removal of the longer 5' and 3' ends so that their lengths matched that of the shortest sequence and then analysed by neighbourjoining, parsimony and minimum evolution methods (Kimura's correction, pairwise deletion option) using the Mega 2.1 software [33]. GenBank accession numbers are shown in Figure 1.

Clinical data
The patients' medical charts were reviewed and clinical characteristics (age, sex, clinical diagnosis, underlying conditions, predisposing factors, antibiotic treatment and outcome) were recorded for each case of infection due to NVS. Neutropenia was defined as a neutrophil count of <500 cells/mm 3 .

Ethics
The design of this study was in accordance with the ethical standards of our hospital ethics committee. Because the study was retrospective, informed consent was not required.

Results
During the six-year study period, ten patients had positive blood cultures for NVS and one had a positive culture of a vascular graft fragment. All but one positive blood culture were detected within the first twenty-four hours of incubation. The strains grew equally from both vials. All strains showed satellitism around streaks of Staphylococcus aureus. Seven of 11 strains were identified successfully to the species level using the Rapid ID32 STREPT system.
The epidemiological and clinical characteristics of the patients are shown in Additional file 1. Two patients with A. defectiva infections had endocarditis, one of which also had sacroileitis. The third patient had polymicrobial (A. defectiva plus Escherichia coli) vascular graft infection related to an aorto-enteric fistula.
Seven of 8 patients with bacteraemia due to Granulicatella spp. were immunosuppressed. Underlying conditions included haematological malignancies (5), lung cancer (1) and advanced metastatic oesophageal cancer (1). Three patients had polymicrobial infections (G. adiacens plus Clostridium sordellii, Staphylococcus epidermidis or Lactobacillus rhamnosus). Six patients presented with febrile chemotherapy-induced neutropenia and mucositis, including one patient with possible infection of a catheter and one with possible endocarditis. One of the six patients with neutropenia died 10 days after bacteraemia from gastrointestinal bleeding in the setting of refractory thrombopenia. The other 5 neutropenic patients clinically improved clinically with intravenous antibiotic therapy. The 3 patients with primary bacteraemia due to Granulicatella spp. with intermediate susceptibility or resistance to penicillin were successfully treated (cases 7, 10 and 11).

Discussion
A. defectiva and Granulicatella spp. are now considered as two distinct genera based on 16S rRNA tree topology and sequence divergence considerations [7]. The review of these clinical cases suggests that each species is associated with a distinct clinical presentation: A. defectiva infections were seen in immunocompetent patients with endovascular infections, whereas 7 of 8 Granulicatella spp. bacteraemia occurred in immunosuppressed, mainly febrile neutropenic patients. To date, no cases of A. defectiva and only five cases of Granulicatella spp. bacteraemia in neutropenic patients have been reported. Pierard et al. described one case of G. adiacens bacteraemia among 62 cases of streptococcal bacteraemia in neutropenic patients [35]. In a small series published by Woo et al., three cases of G. adiacens bacteraemia were associated with febrile neutropenia in cancer patients [36]. Finally, one case of G. elegans bacteraemia was reported by Murray et al. in a febrile neutropenic cancer patient [37]. We report here the first case of "G. para-adiacens" infection in the setting of febrile neutropenia.
One and two of 8 Granulicatella spp. strains, respectively, showed reduced susceptibility and resistance to penicillin. This rate of resistance is similar to the prevalence of penicillin resistance recently described [26,28]. This suggests that antimicrobial susceptibility testing should be systematically done in order to select appropriate antimicrobial therapy. In severely ill patients or those with a suboptimal response to initial therapy with beta-lactam antibiotics, treatment with vancomycin should be considered. We did not observed therapeutic failures; the fatal outcome in two cases was not attributed to the infection.
All Abiotrophia and Granulicatella strains were susceptible to vancomycin. However, depending on the culture medium used for E-test method, we observed discrepant results. Overestimation of vancomycin E-test values have previously been reported for Strepococci spp., when compared to values obtained with broth or agar dilution methods [38][39][40]. Vancomycin E-test values should thus be interpreted with caution.
As observed in this series, Granulicatella spp. bacteraemia may occur in the setting of chemotherapy-associated mucositis and neutropenia. Oro-intestinal colonisation by Granulicatella spp. and subsequent mucositis may represent predisposing factors for bacteraemia in neutropenic patients, as it is well documented for viridans streptococci [41,42]. The absence of cases of bacteraemia due to A. defectiva in neutropenic patients could reflect a lower frequency of oral colonisation by this species in comparison to Granulicatella spp. In one study, the rates of oral colonisation in healthy students were 11.8% and 87.1% for A. defectiva and G. adiacens, respectively [9].

Conclusion
We report six cases of bacteraemia due to Granulicatella spp. in febrile neutropenic patients. Chemotherapyinduced neutropenia and oral mucositis may represent predisposing factors. Granulicatella spp. should be considered as a possible agent of bacteraemia in neutropenic cancer patients.

Competing interests
The author(s) declare that they have no competing interests.

Authors' contributions
LS collected the clinical data, carried out the 16S rRNA sequencing and wrote the draft of the manuscript. AW, JME and GP did the microbiological studies. KJ and GG participated in the 16S rRNA sequencing. JME, GG, JB, TC and GP provided input into subsequent drafts of this manuscript. All authors read and approved the final version of manuscript.