Introduction

During the past years, invasive infections due to Streptococcus dysgalactiae (SD) have been increasingly reported worldwide [1]. Human pathogenic SD most commonly belongs to the subspecies equisimilis and expresses Lancefield group C, G, or infrequently group A antigen [2]. Species determination of SD has been considerably improved by the introduction of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) [3]. Further typing of SD is based on the sequence of the 5′-part of the emm gene encoding the hypervariable NH2-terminal part of the cell wall-attached M protein [4, 5]. Several molecular epidemiological studies have investigated the distribution of emm types among SD isolates in attempt to link different emm types to invasive or non-invasive infections [1, 6, 7].

SD can cause both superficial and invasive infections often with a focus in soft tissues [8]. A severe type of infection caused by SD is infective endocarditis (IE). Oppegaard et al. recently described nine cases of SD-caused IE and reported the onset of symptoms to be rapid and the disease course to be severe [9]. In addition, Lother et al. identified 12 cases with group C- or G-caused IE, four cases of definitive, and eight cases of possible IE, out of 209 events of groups C and G bacteremia. Of these 12 cases, a majority had embolic events with rapid and progressive disease and considerable mortality rate [10]. Other studies have focused on all β-hemolytic streptococci (BHS) and typically the majority of cases have been attributed to group B streptococci, with no species determination performed to determine if some isolates were SD. In one such study, El Rafei et al. showed large vegetations and high rate of systemic embolization in BHS-caused IE [11]. Another study by Lefort et al. presented high rate of extra cardiac complications in IE caused by BHS [12].

Intravenous antibiotic treatment for several weeks is the backbone of IE treatment, but occasionally valve surgery is needed. Previous studies have implied that penicillin and aminoglycosides (AG) have a synergistic effect on SD in vitro [13]. Swedish guidelines [14] recommend β-lactam monotherapy in IE caused by BHS based on a lack of benefit from combination therapy in group B streptococcal IE [15] whereas international guidelines advocate combination therapy [16].

Aminoglycosides are well known for their nephrotoxicity and ototoxicity [17, 18] but are still recommended in BHS-caused IE [16, 19]. In a retrospective study based on 40 patients with IE caused by group G streptococci, Smyth et al. showed that patients treated with combination therapy had better outcome than single therapy [20]. Another more recent study could not demonstrate any difference in outcome for patients treated with β-lactam monotherapy compared to combination therapy in BHS-caused IE [11].

This study aimed to delineate clinical features of SD in IE, to describe which emm types are prevalent in the condition and to investigate the antibiotic susceptibility and possible synergistic effect between penicillin and gentamicin on SD isolates from IE.

Material and methods

Collection of bacterial isolates

In 1995, the Swedish Registry of Infective Endocarditis (SRIE) organized by the Swedish Society of Infectious Disease was established. All 30 departments of infectious diseases (ID) in Sweden have participated since its inception. The ID departments have regional responsibility for care of patients with severe infections and report cases treated at the respective clinic. In order to identify cases of IE with SD, cases caused by BHS of groups A, C, and G were identified from the SRIE between the years 2008 and 2016. Stored bacterial isolates were collected from relevant laboratories and were cultivated on blood agar plates in 5% CO2 at 37 °C. To determine the species identity, they were reanalyzed with Microflex MALDI-TOF MS (Bruker, Bremen, Germany) using the direct transfer or the extraction protocol described elsewhere [21] and the software FlexControl and MBT Compass 4.1, with reference database MBT-BDAL-7321 (MALDI Biotyper; Bruker). A score value above 2.0 was required for species determination.

emm typing

Typing based on the sequence of the emm gene was performed as described at (http://www.cdc.gov/streplab) and the emm gene sequences were compared with those in the CDC emm sequence database. An emm sequence that presented > 98% identity with the CDC reference strain was identified as that certain emm type.

Antibiotic synergy

Minimal inhibitory concentration (MIC) was determined by broth microdilution and Etest (BioMerieux, Marcy l’Etoile, France, Sweden). Mueller-Hinton agar (MHA), with defibrinated horse blood and β-NAD, plates were used for Etest MIC determination and the Etest synergy method. Mueller-Hinton broth enriched with β-NAD and defibrinated horse blood was prepared [22] to define MIC and minimal bactericidal concentration (MBC) through the broth microdilution method [23, 24]. Antibiotics used were benzyl-penicillin (Astra Zeneca) and gentamicin (Schering-Plough). Etest synergy was performed according to the MIC:MIC ratio method and cross or 90° angle method [25]. Etest synergy methods were performed on all isolates and the summation fractional inhibitory concentration (ΣFIC) was calculated for each set of MICs as described [25]. Synergy was defined as ΣFIC ≤ 0.5. In order to test bactericidal synergy, the time-kill technique was applied as described by Weinstein [26] with certain modifications [27]. Synergy was defined if a > 2 log10 cfu/ml difference between the drugs in combination, as compared to the most effective active single antibiotic alone, at one given time point (6 or 24 h).

Data collection

Clinical information on patients with SD-caused IE and on patients with IE caused by Staphylococcus aureus, alpha-hemolytic streptococci, and enterococci was extracted from the SRIE. Data from the SRIE on IE caused by SD and the other pathogens were compared. In addition, detailed information on SD-caused IE was obtained from the medical records for patients that gave informed consent. Epidemiological, clinical parameters and microbiological results were recorded.

Statistics

Fisher’s exact test was applied for categorical data and comparison of continuous variables was analyzed utilizing Mann-Whitney U test where p values were adjusted according to the Bonferroni correction. Significance was defined as a p value less than 0.05. Microsoft Excel 2016 (Microsoft Corporation) was used for data collection and Graph Pad Prism, version 7.0b (GraphPad Software) and the SPSS software system, version 24 (SPSS) were utilized for statistical calculation.

Results

Inclusion of patients with IE caused by SD

From 2008 to 2016, 104 cases of IE with BHS of groups A, C, and G, that potentially could be SD, had been reported to SRIE. Twenty-seven of these cases were excluded since the corresponding blood isolates were not available for reanalysis and 11 cases were excluded since they were found to be alpha-hemolytic. Sixty-six BHS isolates of groups A, C, and G streptococci were species-determined with MALDI-TOF MS of which three strains were classified as Streptococcus agalactiae, Streptococcus canis (S. canis), and Streptococcus equi (S. equi). Thirteen isolates were Streptococcus pyogenes (all of which had group A carbohydrate). The remaining 50 cases of SD comprised the study group.

In 17, cases the SD isolate was grouped as Lancefield group C and in 33 cases as group G.

Distribution of the emm gene

The emm types of all isolates are given in Fig. 1. Isolates which expressed group G antigen were predominantly of stC74a (n = 6) or stG643 (n = 5) whereas isolates that expressed group C most often were stG62647 (n = 6).

Fig. 1
figure 1

The distribution of emm types among patients with SD-caused IE expressed as proportion of total isolates (n = 17 for GCS and n = 33 for GGS). Black bars represent GCS and white bars represent GGS

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Antibiotic susceptibility

The MICs, MBCs, ΣFIC, and the results of the time-kill assays for nine representative isolates of SD are given in Table 1. All isolates were susceptible to inhibition and killing action of both penicillin and gentamicin. Applying the Etest-based synergy methods gave similar results and did not indicate synergy. ΣFIC was between 0.8 and 2. Synergy was detected using the time-kill method in four out of nine isolates. Of these four isolates, synergy was detected at 6 h and in one case synergy was also distinguished at 24 h. In seven cases, the killing action of penicillin alone was so pronounced at 24 h that additional killing using the combination was technically difficult to detect.

Table 1 In vitro antibiotic susceptibility of SD IE isolates (n = 9)
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Clinical characteristics of cases of IE due to SD

Out of 50 patients with SD-caused IE identified in the registry, the episodes of 46 patients were studied in detail through the medical records at the respective hospitals. Four patients were excluded since informed consent was not obtained and two patients were excluded as IE could be rejected after reviewing the medical records. Table 2 describes the clinical presentation of the 44 patients with SD-caused IE and a further detailed account for all patients is given in the Appendix Table 4. Twenty-nine percent of the patients (n = 13) were treated at the intensive care unit due to circulatory failure. Three patients with indications for surgery were not operated due to age and severe underlying diseases such as liver encephalopathy and cardiovascular disease. The in-hospital mortality among the studied patients was 11% (n = 5).

Table 2 Clinical and microbiological features of patients with SD-caused IE (n = 44) obtained from medical records
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Comparison of IE due to SD and IE caused by other major IE-pathogens

Cases of IE due to SD were compared with cases of IE caused by S. aureus (n = 1378), alpha-hemolytic streptococci (n = 934), and enterococci (n = 414) reported to the same registry (Table 3). The median age was significantly higher among patients with SD-caused IE compared to S. aureus (74 vs 65 years) (p = 0.003). The onset of SD-caused IE was acute as median time from onset of symptoms to initiation of treatment was only 1 day which was significantly less compared to enterococci and alpha-hemolytic streptococci (1 day vs 7–15 days) (p < 0.0001) but not to S. aureus (1 day vs 2 days). The in-hospital mortality was 8% and embolization was seen in 46% in patients with SD-caused IE.

Table 3 Clinical characteristics of cases of SD-caused IE compared to cases of IE due to other pathogens obtained from the SRIE
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Discussion

This study is the largest report of SD-caused IE and describes the clinical presentation and outcome of 50 cases of IE due to SD. The condition has an acute onset, high rate of embolization, and aggressive course often including organ dysfunction. Previous studies have demonstrated SD-caused IE as having an acute onset of illness and high mortality similar to S. aureus-caused IE [9, 11]. Furthermore, Lother et al. showed that 6% of patients with GCS or GGS bacteremia developed IE with extensive need of intensive care unit treatment but of these cases only two were verified at the species level as SD [10]. As with other types of invasive SD infections [29], old males are the typical patients.

In the present study, we demonstrate synergy between penicillin and gentamicin in vitro against some SD isolates with the time-kill method. However, in the 24-h samples, there was an efficient killing action by penicillin alone resulting in difficulties in evaluating the effect of combination with aminoglycosides. Etest-based methods were not able to demonstrate synergy. Interestingly, according to the medical records, 27 of the 44 patients received treatment with aminoglycosides of which 11 patients had one dose whereas 16 patients had several dosages of aminoglycosides during time of care. There was no statistical significant difference in clinical outcome between patients treated with long-term addition of aminoglycosides and those receiving monotherapy but the study was likely under-powered to detect such difference. There is to date no firm results to guide the use of addition of aminoglycosides and potential additional killing of bacteria must be weighed against the risk of side effects.

We found SD-caused IE to be much more common than IE caused by S. pyogenes which is in line with previous studies [11, 12]. According to the SRIE, and also in line with previous reports, group B streptococci was the most common BHS-causing endocarditis comprising 76 cases, though these isolates were not confirmed to the species level by us. In the present study, the emm types stC74a and stG62647 were the most commonly encountered in SD isolates with groups G and C carbohydrate respectively. The distribution of emm types was similar to that described previously in Sweden [6]. A previous study has indicated that rare emm types are associated to poor prognosis [7] but four of the five fatal cases described here were caused by common types (stG480, stG652, stG2078, and stC74a).

This study is limited by its retrospective design and a possible bias in the inclusion of patients in the SRIE. It has previously been estimated that around 75–80% of cases of IE in Sweden are included in the SRIE [30]. When comparing the medical records to the data reported to the registry, some inaccuracies were noted. For example, the SRIE was found to contain several episodes reported as caused by BHS of group A that were instead due to alpha-hemolytic streptococci. However, we chose not to correct the SRIE data after analysis of the medical records since no corresponding corrections were possible for IE caused by the other pathogens. Thus, there are discrepancies between the information on episodes extracted from the SRIE (Table 3), the medical records (Table 2), and the Appendix Table 4.

MALDI-TOF MS can identify SD to the species level but subspecies determination can formally not be made with this method. The vast majority of human isolates of SD are Streptococcus dysgalactiae subspecies equisimilis (SDSE) but formally, we cannot exclude that we also had isolates of Streptococcus dysgalactiae subspecies dysgalactiae though such isolates generally are alpha-hemolytic [5]. There are also isolates of SDSE exhibiting alpha-hemolysis; however, this is a rare occurrence [5]. Interestingly in our study population, one isolate was S. canis and another isolate was S. equi, both of which are of animal origin and are rare causes to human infections. These species, however, have been reported as IE-pathogens [31, 32].

Conclusion

SD-endocarditis has an acute onset of symptoms and causes severe infection in elderly people. Synergy of penicillin and gentamicin could be shown in vitro but it was not distinguished utilizing Etest methodology.