
==== Front
New Microbes New Infect
New Microbes New Infect
New Microbes and New Infections
2052-2975
Elsevier

S2052-2975(24)00257-9
10.1016/j.nmni.2024.101473
101473
Original Article
Identification and characterisation of carbapenem-resistant Streptococcus nidrosiense sp. nov. isolated from blood culture
Rønning Torunn Gresdal Torunn.Gresdal.Ronning@stolav.no
ab⁎1
Olaisen Camilla a1
Ås Christina Gabrielsen ab
Afset Jan Egil ab
Haugan Maria Schei ab
a Department of Medical Microbiology, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway
b Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway
⁎ Corresponding author. Department of Medical Microbiology, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway. Torunn.Gresdal.Ronning@stolav.no
1 These are co-first authors and contributed equally to this work.

27 8 2024
12 2024
27 8 2024
62 10147310 6 2024
2 7 2024
26 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Background

This study aimed to investigate a highly resistant strain of Streptococcus sp. isolated from a patient with bloodstream infection and determine its taxonomic classification.

Methods

The strain was isolated from blood culture from a 65-year-old male patient admitted to St. Olavs University hospital, Trondheim, Norway, in 2023. Antimicrobial susceptibility testing as well as phenotypic and biochemical characterization were performed. Whole genome sequencing was conducted and genomic comparison to Streptococcus type strains was carried out.

Results

The strain was initially identified as Streptococcus mitis/oralis but showed significant genetic differences, suggesting that it belonged to an undescribed species within the Streptococcus genus. Phenotypic and biochemical characterization identified the strain as a non-motile, facultative anaerobic bacterium with α-hemolysis. Antimicrobial susceptibility testing showed resistance to all beta-lactams tested. Genomic analyses confirmed the classification of the strain as a novel species, which was designated Streptococcus nidrosiense.

Conclusion

This study combines conventional phenotypic tests with whole genome sequencing for accurate taxonomic classification of a bacterial strain isolated from blood culture. The identification of a novel species within the Streptococcus genus contributes to the understanding of microbial diversity and antibiotic resistance of the Streptococcus genus in clinical settings.

Highlights

• We isolated a novel strain of Streptococcus sp. from a patient with BSI.

• Unusual pattern of antibiotic resistance, including carbapenem-resistance.

• Carbapenemase antibiotic resistance in VGS is a concerning healthcare issue.

• Accurate taxonomic classification by phenotypic and genotypic analysis.

• We designate the novel species Streptococcus nidrosiense.

Keywords

Streptococcus nidrosiensis
Viridans streptococci
Carbapenem resistance
Handling Editor: Patricia Schlagenhauf
==== Body
pmc1 Introduction

The viridans group streptococci (VGS) encompass a diverse array of catalase-negative, Gram-positive cocci that are capable of serving as commensals within the human body. These organisms have the ability to colonize various anatomical sites such as the gastrointestinal and genitourinary tracts, in addition to the oral mucosa [1]. However, the VGS can also be human pathogens, especially in immunocompromised individuals and neonates, causing invasive infections such as endocarditis, meningitis, sepsis and pneumonia [2]. Taxonomy of the VGS group has been inconsistent and even controversial, due to large variation in hemolytic activity, differences in substrate metabolism as well as high level of sequence homology between 16S rRNA genes within the group. There are however currently more than 30 recognized species of VGS [1,3], which are categorized into six significant groups: the S. mutans group, the S. salivarius group, the S. anginosus group, the S. mitis group, the S. sanguinis group, and the S. bovis group [4]. Recently, whole genome sequencing-based methods, including Average Nucleotide Identity (ANI) [5,6] and digital DNA–DNA Hybridization (dDDH), have emerged as effective tools for the classification of a large number of species, particularly when conventional biochemical tests are insufficient to accurately assign a strain to its appropriate species designation [7]. These methods have thus been able to distinguish more clearly between the VGS, as well as aid in the designation of novel species [8,9].

In 2023, a highly resistant strain (hereafter termed SO-23-1) of Streptococcus sp. was isolated from blood culture from a patient who suffered from a severe polymicrobial bloodstream infection with fatal outcome. Initially, routine analyses identified the strain as Streptococcus oralis. Due to its high level of beta-lactam resistance, which is unusual for streptococci, whole-genome sequencing was conducted for further investigation. Data analysis revealed that the isolated bacterial strain was genetically closely related to S. oralis, but with significant differences, suggesting that the strain belongs to a novel bacterial species within the Streptococcus genus. The aim of this study was to characterize and determine the taxonomic classification of this strain.

2 Materials and methods

2.1 Case presentation and strain identification

Strain SO-23-1 was isolated from blood culture from a 65-year-old male patient admitted to St. Olavs University hospital, Trondheim, Norway, in 2023. The patient had lung cancer, adenocarcinoma stage T4N0M0, with extensive lesions including tumor growth into the mediastinum and a bronchopleural fistula. He was initially admitted to the hospital due to community-acquired pneumonia with pleural effusion. Initial culture of sputum and pleural fluid both showed polymicrobial growth of a mixture of aerobic and anaerobic bacteria, likely belonging to the oral microbiota. The patient's condition was later, during hospital admission, complicated by pneumothorax, pleural empyema and bacteraemia. Strain SO-23-1 was isolated after two months hospitalisation, together with Rothia mucilaginosa and Lacticaseibacillus rhamnosus, from blood culture (BACTEC Plus/F Aerobic) after overnight incubation at 35 °C in a Becton Dickinson BACTEC FX blood culture system. Repeated bacterial culture of the pleural fluid showed similar polymicrobial growth of what was considered a mixture of upper airway bacteria, and Pneumocystis jirovecii DNA was detected in a sputum sample. Prior to the isolation of bacteria from blood culture, the patient had received empirical treatment with meropenem for several weeks without significant clinical improvement. Due to the lack of treatment effect and the isolation of streptococci in blood culture, antimicrobial treatment was changed to piperacillin-tazobactam. Two days later, treatment was further changed to clindamycin, as well as metronidazole, due to detection of unusual beta-lactam resistance of the Streptococcus species. In addition, trimethoprim-sulfamethoxazole was given to cover infection with Pneumocystis jirovecii. Due to lack of treatment response and advanced stage of cancer disease, active treatment was discontinued after 24 h and the patient died the following day.

Identification of the bacterial strain was performed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) [10], with a MALDI Biotyper Sirius instrument and the Bruker Daltonics database V12.0.0.0_10833–11897 on colonies obtained from sheep blood agar COLS+ (Oxoid). Log (score) values above 2.0 were accepted as probable species-level identification, while scores between 1.7 and 1.9 were accepted as probable genus-level identification [11]. Subsequent investigations involved ten replicates of the SO-23-1 strain using the same MALDI-ToF MS technology.

2.2 Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was initially performed using the EUCAST standardised disc diffusion method, followed by MIC confirmation by broth microdilution (Sensititre™ Streptococcus STP6F and Sensititre™ Gram Positive GPALL1, Thermo Scientific) according to EUCAST guidelines [12]. In silico detection of antibiotic resistance genes was performed using AMRFinder Plus [13], and possible mutations in genes encoding penicillin-binding proteins (PBPs) were analysed using Geneious Prime v2023.0.4 software (Biomatters).

2.3 Phenotypical and biochemical characterisation

The culture was grown on sheep blood agar COLS+ in aerobic and anaerobic atmosphere with 24- and 48-h incubation at 35 °C. The strain was also cultured in tryptic soy broth (TSB) (SIGMA-ALDRICH) for 24 h at 35 °C, in normal atmosphere and in 5 % CO2. Light microscopy of wet mount and fixed Gram stained [14] liquid culture was performed to investigate motility and morphology of the bacterium.

Catalase- (bioMérieux) and coagulase-tests (in-house plasma) [15] were performed as previously described.

A NaCl tolerance test [16] was performed by preparing a dilution series of NaCl in TSB, with the following NaCl concentrations: 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 6.5 and 7.0 %. Briefly, 1–3 bacterial colonies were inoculated in triplicate in TSB with added NaCl, and observation of visible turbidity after 24 h of incubation at 35 °C in 5 % CO2 atmosphere was considered a positive result.

Biochemical characteristics of the strain SO-23-1 were determined by API 20 STREP according to the manufacturer's instructions (bioMérieux).

2.4 DNA extraction and whole genome sequencing

Bacterial colonies were dissolved in 200 μL TE buffer containing 1.5 mg/mL lysozyme, 0.5 mg/mL proteinase K (Qiagen) and 250 U/mL mutanolysin (Merck), before 15 min incubation at 37 °C, 15 min at 65 °C, and addition of 100 mg/mL RNaseA. Genomic DNA was subsequently extracted using the EZ1 DNA Tissue kit (Qiagen) with an EZ1 Advanced XL extractor (Qiagen). A short-read sequencing library was prepared using Illumina DNA prep, and sequenced using the MiSeq reagent kit v3 (2x300 bp) on a MiSeq Instrument (Illumina). A long-read sequencing library was prepared using the rapid sequencing kit SQK-RAD004 and sequenced on a flongle flow cell (FLO-FLG001, R.9.4.1) using a MinION Mk1b instrument (Oxford Nanopore Technologies) [17]. Long-read data were basecalled using Guppy v5.1.15 (Oxford Nanopore Technologies) and assembled using the Flye assembler v2.7 [18]. The assembly was furthermore circulated with circlator v1.5.5 [19] and polished with long read data using using Racon v1.4.20 [20], before polishing with short-read data using Pilon v1.23 [21].

2.5 Genomic annotation and comparison

Reference genomes for Streptococcus mitis group type strains were downloaded from RefSeq Database (NCBI) and annotated using Prokka v1.14.6 [22]. Complete nucleotide sequences of housekeeping genes 16S rDNA, 23S rDNA, rpoB, sodA and gyrA were extracted, aligned with MUSCLE v5.1 and Neighbour-joining phylogenies inferred with the Tamura-Nei distance model using the Geneious treebuilder in Geneious Prime v2023.0.4 (Biomatters). Pangenome analysis was performed using Roary v3.13.0 [23] with identity treshold 70 %, and a core genome phylogeny inferred using FastTree [24] with the GTR substitution model. Calculation of the percentage of conserved proteins (POCP) was performed using the POCP Nextflow pipeline [25,26]. The genome of strain SO-23-1 was taxonomically classified and closely related strains identified using the Type Strain Genome Server (TYGS) pipeline as well as the Genome Taxonomy Database Toolkit (GTDB-tk). Finally, BLAST-based Average Nuclotide Identity (ANIb) was calculated using Jspecies WS [5] and digital DNA-DNA hybridisation was performed using the Genome-to-Genome Distance Calculator (GGCD) webserver [27], with the recommended formula 2 (sum of all identities/HSP length) used for score calculation.

The genome sequence data were registered under GenBank BioProject PRJNA1101760 (accession numbers: CP152419 (whole genome sequence), PP761413, PP761414, PP761415, PP761416 (16S rRNA sequences)).

3 Results

3.1 Phenotypic and biochemical characterisation

SO-23-1 exibited facultative anaerobic growth with α-haemolysis on sheep blood agar (Fig. 1a). Here, the colonies were dry and adherent. Both coagulase and catalase tests were negative. The isolate was identified by MALDI-ToF MS as S. mitis/oralis from culture grown on sheep blood agar. Further testing by MALDI-ToF MS identified strain SO-23-1 as S. mitis/oralis in 9 out of 10 replicates with score values ranging from 1.84 to 2.14 (median 1.98) and in one replicate as S. infantis with score value 1.95, all members of the S. mitis group.Fig. 1 a) Growth of Streptococcus sp. strain SO-23-1 on sheep blood agar. Image was captured after 48h growth in CO2 atmosphere, using backlight. b) Gram-stain microscopy of the strain from culture in TSB after 24h incubation in CO2 atmosphere, and c) after 24h in normal atmosphere. Images b) and c) were captured with Olympus cellSens Entry software version 2.3 with 1000 X amplification on an Olympus BX43 microscope with an Olympus DP26 digital camera.

Fig. 1

The bacterium was found to be Gram-positive and non-motile. Morphologically, the bacteria appeared as rod-shaped cocci in chains with a diphteroides-like appearance (Fig. 1b) after incubation with CO2. However, after incubation in normal atmosphere, the bacterial cells showed a more characteristic viridans streptococci apperance, with cocci in long chains (Fig. 1c). This is similar to how S. mutans can display cellular dimorphism as cocci under neutral or basic growth conditions, and as short rods or diphteroid appearance in acidic culture media [28,29].

The SO-23-1-strain grew well in TSB supplemented with NaCl concentrations ranging from 0.5 % to 3 %. Scarce growth was observed when the NaCl concentration in TSB was increased to 4.0 %. No growth was observed in TSB containing 5.0 % NaCl or higher.

By interpretation of the API 20 STREP test, SO-23-1 was identified as Streptococcus intermedius (91.20 % similarity). The strain had positive biochemical reactions towards the substrates ESC, βGAL, PAL, LAP, ADH (negative after 4 h, positive after 24 h), as well as LAC and GAL in the API 20 STREP test. Tests were negative towards substrates HIP, PYRA, αGAL, βGUR, RIB, ARA, SOR, TRE, RAF, AMD and GLYG. The results for VP and MAN were inconclusive (Table 1). Furthermore, type strains for Streptococcus oralis, subsp. oralis (CCUG 13229T), Streptococcus oralis, subsp. dentisani (CCUG 66492T), Streptococcus oralis, subsp. tigurinus (CCUG 66514T), Streptococcus mitis (CCUG 31611T), Streptococcus pneumoniae (CCUG 33638), Streptococcus pseudopneumoniae (CCUG 49455T), Streptococcus infantis (CCUG 39817T) and Streptococcus peroris (CCUG 39814T) were tested in parallel. However, results were inconclusive, and varied between replicates. Most strains were misidentified in the API database (results not shown). Alpha haemolytic streptococci are known to be difficult to identify using biochemical tests such as the API system [30].Table 1 API 20 Strep results for the SO-23-1 strain.

Table 1Test	Active ingredient	Reaction/Enzyme	Result	
VP	sodium pyruvate	acetoin production (Voges Proskauer)	Inconclusive	
HIP	hippuric acid	hydrolysis (HIPpuric acid	Negative	
ESC	esculin ferric citrate	β-glucosidase hydrolysis (ESCulin)	Positive	
PYRA	pyroglutamic acid-β-naphthylamide	PYRrolidonyl Arylamidase	Negative	
αGAL	6-bromo-2-naphtyl-αD-galactopyranoside	α-GALactosidase	Negative	
βGUR	naphthol ASBI-glucuronic acid	β-GlUcuRonisidase	Negative	
βGAL	2-naphtyl-βD-galactopyranoside	β-GALactosidase	Positive	
PAL	2-naphthyl phosphate	ALKaline Phosphatase	Positive	
LAP	L-laucine-β-naphthylamide	Leucine AminoPeptidase	Positive	
ADH	L-arginine	Arginine DiHydrogenase	Negative after 4h, positive after 24h	
RIB	D-ribose	acidification (RIBose)	Negative	
ARA	L-arabinose	acidification (ARAbinose)	Negative	
MAN	D-mannitol	acidification (MANnitol)	Inconclusive	
SOR	D-sorbitol	acidification (SORbitol)	Negative	
LAC	D-lactose	acidification (LACtose)	Positive	
TRE	D-trehalose	acidification (TREhalose)	Negative	
INU	inulin	acidification (INUlin)	Negative	
RAF	D-raffinose	acidification (RAFfinose)	Negative	
AMD starch	starch	acidification (AmiDon)	Negative	
GLYG	glycogen	acidification (GLYcoGen)	Negative	

3.2 Antimicrobial susceptibility and detection of resistance genes

Strain SO-23-1 showed phenotypic resistance to all beta-lactams tested, including penicillins, cephalosporins and carbapenems (Table 2).Table 2 Results from antimicrobial susceptibility testing of strain SO-23-1. SIR categories were determined using the EUCAST breakpoints for the viridans group streptococci (VGS). ND: breakpoints not defined by EUCAST. HLAR: high-level aminoglycoside resistance. MLSB: macrolide-lincosamide-streptogramin B. *Inferred from ampicillin.

Table 2Antibiotic	MIC (μg/mL)	Interpretation (EUCAST)	
Benzylpenicillin	>4	Resistant	
Ampicillin	>8	Resistant	
Amoxicillin-clavulanate	16	Resistant	
Piperacillin-tazobactam	–	Resistant*	
Ceftriaxone	>2	Resistant	
Cefuroxime - parenteral	>4	Resistant	
Cefepime	4	Resistant	
Cefotaxime	>4	Resistant	
Meropenem	>2	Resistant	
Ertapenem	>4	Resistant	
Moxifloxacin	≤1	ND	
Levofloxacin	1	ND	
Gentamicin	8	Resistant	
Gentamicin HLAR	≤500	Negative	
Vancomycin	1	Susceptible	
Clindamycin	≤0.125	Susceptible	
Erythromycin	1	ND	
Azithromycin	≤0.25	ND	
Tetracycline	≤1	ND	
Tigecycline	≤0.015	ND	
Linezolid	0.5	ND	
Trimethoprim-sulfamethoxazole	≤0.5	ND	
Daptomycin	0.5	ND	
Chloramphenicol	2	ND	
Rifampicin	2	ND	
Inducible MLSB resistance	–	Negative	

No known beta-lactamase genes were detected in the strain, but the intrinsic pbp2b-gene (Table 3) was reported by AMRFinder Plus as a probable cause of beta-lactam resistance. The pbp2b-gene in strain SO-23-1 contains the previously described T446A- and E476G-amino acid substitutions that are commonly found in clinical penicillin-resistant streptococci [31]. Furthermore, the SO-23-1 strain had multiple mutations in pbp2b shown to confer meropenem resistance in S. pneumoniae: V225I, S412P, N422Y, T426K, Q427L, S473T, S480A, G497P, N606D, L609T, A619G, N659K, G660N, S664A [32]. Mutations associated with meropenem resistance in S. pneumoniae were also found in pbp1a (P4Q, L9I, I10A, V21F, N58S, Q61E) and pbp2x (A347S) [32]. Two additional resistance genes were detected in strain SO-23-1, mrs(D) and mef(A) (Table 3), encoding a macrolide efflux transport system described in different Streptococci including S. oralis [33]. Phenotypic macrolide resistance was however not observed. Strain SO-23-1 did, as reported for other streptococci [34], display intrinsic low-level resistance to gentamicin (Table 2).Table 3 Antibiotic resistance genes detected in strain SO-23-1. Results obtained from AMRFinder plus.

Table 3Gene symbol	Sequence name	Antibiotic resistance class	Antibiotic resistance subclass	% Coverage of reference sequence	% Identity to reference sequence	Accession number of closest sequence	
pbp2b	Streptococcus pneumoniae beta-lactam resistant PBP2B	Beta-lactam	Beta-lactam	100.00	90.29	WP_001224884.1	
mef(A)	macrolide efflux MFS transporter Mef(A)	Macrolide	Erytromycin	100.00	100.00	WP_000417519.1	
msr(D)	ABC-F type ribosomal protection protein Msr(D)	Macrolide	Erytromycin	100.00	100.00	WP_000420313.1	

3.3 Bioinformatic analyses

While strain SO-23-1 was initially identified as Streptococcus mitis/oralis by MALDI-ToF MS, it only shared 98.1–99.2 % sequence similarity of the 16S rRNA gene with S. oralis type strains, the highest similarity being to S. oralis subsp. dentisani 7747T (Fig. 2). The closest matching Streptococcus type strain based on 16S rRNA gene homology was the newly described species S. bouchesdurhonensis Marseille-Q6994, with 99.2–99.4 % similarity. However, it has been established that the 16S rRNA gene is insufficient for discrimination between closely related species within the Streptococcus genus [35]. Phylogenies of other genes including 23S rDNA, rpoB, sodA and gyrA, similarly showed that the strain clustered separately from other Streptococcus reference genomes (results not shown).Fig. 2 Sequence similarity matrix of 16S rRNA genes of different Streptococcus species compared to the four 16S rRNA genes of strain SO-23-1. Scale represents substitutions per site.

Fig. 2

The complete genome of strain SO-23-1 consisted of a circular chromosome of 2,018,446 bp with a GC content of 39.7 %. It encoded 1838 protein-coding genes, as well as four rRNA operons and 59 tRNA genes. The SO-23-1 strain had two known virulence genes, pavA and psaA, which encode a fibronectin-binding protein and a lipoprotein component of an Mn2+ transporter, respectively, both previously described in S. pneumoniae [36,37].

Comparisons between the genome of strain SO-23-1 to those of type strains of the Streptococcus mitis group showed >50 % percentage of protein homology, confirming that the strain indeed belonged to the genus Streptococcus. Pangenome analysis and core genome phylogeny (Fig. 3) initially identified S. infantis ATCC 700779 and S. peroris ATCC 700780 as the most closely related type strains, however with protein homology of only 78.9 and 81.1 % accordingly.Fig. 3 Core genome phylogeny, annotated with results from ANIb, dDDH and POCP analyses of strain SO-23-1 compared to the genetically closest type strains of the Streptococcus mitis group. Coloured legend showing lowest (green) to highest (red) percent similarity. Figure made using iTOL [38]. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Average Nucleotide Indentity (ANI) analysis within GTDB identified the strain Streptococcus infantis UC6950A as the best match, notably with taxonomy status marked as « inconclusive». Another close match identified by ANI was Streptococcus sp. HMSC074B11, which was taxonomically classified only to genus level. Furthermore, as shown in Fig. 3, the ANIb distances were below the commonly used treshold for within-species designation of 95 %.

There were identified no good matches by digital DNA-DNA hybridisation (dDDH) in the TYGS database, the highest match being S. infantis ATCC 700779 with a dDDH score of 34.3 %. Including the closest strains by ANI distance in the analysis, the highest dDDH score was 59.4 % against S. infantis UC6950A, which is also below the established 70 % threshold for within-species classification.

4 Discussion

Here, we present an as-yet undescribed Streptococcus species with unusual carbapenem resistance. The isolate was obtained from blood culture of a patient with severe underlying conditions who suffered from a polymicrobial bloodstream infection that resulted in a fatal outcome. Due to unusual phenotypic resistance to all beta-lactams tested, further phenotypic and genotypic characterization of the strain was performed.

The use of conventional phenotypic methods to identify alpha-haemolytic streptococci in general [30] and viridans streptococci such as Mitis streptococci in particular, has previously been shown to have limited utility [39,40]. MALDI-ToF MS also exhibits suboptimal performance in distinguishing between species within the S. mitis-group [39,40]. This limitation is evident in the case of SO-23-1, as this strain was misidentified as Streptococcus mitis/oralis. SO-23-1 exhibited anticipated growth patterns when cultivated in Tryptic Soy Broth supplemented with various concentrations of NaCl, up to a maximum of 4 %. The NaCl-test serves as a means of distinguishing between Streptococci and Enterococci. Streptococci, in contrast to Enterococci, fail to thrive in NaCl concentrations of 6.5 % or above.

SO-23-1 was classified as Streptococcus intermedius with a 91.20 % similarity (<80 % for API 20 Strep is considered unacceptable for identification), as determined through the analysis of the API 20 STREP test. However, due to the test's limited efficacy in accurately distinguishing between strains within the S. mitis group [30], we did not rely on this result.

Genotypic characterization of the housekeeping gene 16S rRNA revealed 98.1–99.2 % sequence similarity with S. oralis type strains, with the highest similarity to S. oralis subsp. dentisani 7747T. However, it has been established that the 16S rRNA gene is insufficient for discrimination between closely related species within the Streptococcus genus. New bioinformatics techniques, such as dDDH and ANI, offer better accuracy in discriminating between closely related species. Using these tools and relevant databases, we were unable to identity other Streptococci as above threshold for within-species classification, indicating that strain SO-23-1 was a novel species.

Genes encoding beta-lactam antibiotic resistance in Streptococci is uncommon, but mutations that alter the penicillin-binding proteins (PBPs), the targets for all β-lactam drugs, have been previously described [32]. Although decreased susceptibility against penicillins and cephalosporins is not uncommon in VGS, carbapenem antibiotic resistance is still rare [41,42]. Carbapenemase antibiotic resistance in VGS represents a concerning healthcare issue due to its impact on patient treatment. The standard treatment of severe infections, such as infective endocarditis and septicemia, caused by Streptococci in Norway is benzylpenicillin, possibly in combination with gentamicin. Here, several weeks of treatment with meropenem might have contributed to the mutations conferring carbapenem-resistance in the penicillin-binding proteins in strain SO-23-1. Alternatively, the strain could have possessed the variants of the penicillin-binding genes from the outset. Mosaic variants of such genes have been reported to be shared between various Streptococcal species [43]. Nevertheless, we believe that the highly unusual carbapenem resistance in strain SO-23–1 may have been a contributing factor to the patient's death due to delayed adequate antibiotic treatment.

In conclusion, combined results, including core genome phylogeny, protein homology, 16S similarity, ANI and dDDH suggest that strain SO-23-1 is indeed a novel species, which we designate Streptococcus nidrosiense (Latin adj.; pertaining to Nidaros, the historical name of the city of Trondheim, from where the strain was isolated).

Ethical considerations

Ethical approval was obtained from the Norwegian Regional Committees for Medical and Health Research Ethics (REC), REC-number: 623773. This ethical approval includes a waiver of informed consent.

CRediT authorship contribution statement

Torunn Gresdal Rønning: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Camilla Olaisen: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Christina Gabrielsen Ås: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Jan Egil Afset: Writing – review & editing, Validation, Project administration, Methodology, Investigation, Conceptualization. Maria Schei Haugan: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

There are no conflict of interest for any of the authors.

This project was supported by internal funds from the Clinic of Laboratory Medicine, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway.
==== Refs
References

1 Doern C.D. Burnham C.A. It's not easy being green: the viridans group streptococci, with a focus on pediatric clinical manifestations J Clin Microbiol 48 11 2010 3829 3835 10.1128/jcm.01563-10 20810781
2 Shenep J.L. Viridans-group streptococcal infections in immunocompromised hosts Int J Antimicrob Agents 14 2 2000 129 135 10.1016/s0924-8579(99)00172-7 10720803
3 Jlssl K.V. A review on updated species list of viridans streptococci causing infective endocarditis J Pure Appl Microbiol 16 3 2022;2022 1590 1594
4 Facklam R. What happened to the streptococci: overview of taxonomic and nomenclature changes Clin Microbiol Rev 15 4 2002 613 630 10.1128/cmr.15.4.613-630.2002 12364372
5 Richter M. Rossello-Mora R. Oliver Glockner F. Peplies J. JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison Bioinformatics 32 6 2016 929 931 10.1093/bioinformatics/btv681 26576653
6 Lee I. Ouk Kim Y. Park S.C. Chun J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity Int J Syst Evol Microbiol 66 2 2016 1100 1103 10.1099/ijsem.0.000760 26585518
7 Goris J. Konstantinidis K.T. Klappenbach J.A. Coenye T. Vandamme P. Tiedje J.M. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities Int J Syst Evol Microbiol 57 Pt 1 2007 81 91 10.1099/ijs.0.64483-0 17220447
8 Zoaiter M. Magdy Wasfy R. Caputo A. Fenollar F. Zeaiter Z. Fournier P.E. Houhamdi L. Streptococcus bouchesdurhonensis sp. nov. isolated from a bronchoalveolar lavage of a patient with pneumonia Arch Microbiol 205 1 2022 3 10.1007/s00203-022-03348-0 36436132
9 Wajima T. Hagimoto A. Tanaka E. Kawamura Y. Nakaminami H. Identification and characterisation of a novel multidrug-resistant streptococcus, Streptococcus toyakuensis sp. nov., from a blood sample J Glob Antimicrob Resist. 29 2022 316 322 10.1016/j.jgar.2022.04.018 35500839
10 Clark A.E. Kaleta E.J. Arora A. Wolk D.M. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology Clin Microbiol Rev 26 3 2013 547 603 10.1128/CMR.00072-12 23824373
11 Sauer S. Freiwald A. Maier T. Kube M. Reinhardt R. Kostrzewa M. Geider K. Classification and identification of bacteria by mass spectrometry and computational analysis PLoS One 3 7 2008 e2843 10.1371/journal.pone.0002843
12 Breakpoint tables for interpretation of MICs and zone diameters, version 14.0 The European committee on antimicrobial susceptibility testing 2024
13 Feldgarden M. Brover V. Gonzalez-Escalona N. Frye J.G. Haendiges J. Haft D.H. AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence Sci Rep 11 1 2021 12728 10.1038/s41598-021-91456-0
14 Coico R. Gram staining Curr Protoc Microbiol 2005 10.1002/9780471729259.mca03cs00 Appendix 3:Appendix 3C
15 Sperber W.H. Tatini S.R. Interpretation of the tube coagulase test for identification of Staphylococcus aureus Appl Microbiol 29 4 1975 502 505 10.1128/am.29.4.502-505.1975 164821
16 Facklam R.R. Comparison of several laboratory media for presumptive identification of enterococci and group D streptococci Appl Microbiol 26 2 1973 138 145 10.1128/am.26.2.138-145.1973 4490481
17 Wang Y. Zhao Y. Bollas A. Wang Y. Au K.F. Nanopore sequencing technology, bioinformatics and applications Nat Biotechnol 39 11 2021 1348 1365 10.1038/s41587-021-01108-x 34750572
18 Kolmogorov M. Yuan J. Lin Y. Pevzner P.A. Assembly of long, error-prone reads using repeat graphs Nat Biotechnol 37 5 2019 540 546 10.1038/s41587-019-0072-8 30936562
19 Hunt M. Silva N.D. Otto T.D. Parkhill J. Keane J.A. Harris S.R. Circlator: automated circularization of genome assemblies using long sequencing reads Genome Biol 16 2015 294 10.1186/s13059-015-0849-0 26714481
20 Vaser R. Sovic I. Nagarajan N. Sikic M. Fast and accurate de novo genome assembly from long uncorrected reads Genome Res 27 5 2017 737 746 10.1101/gr.214270.116 28100585
21 Walker B.J. Abeel T. Shea T. Priest M. Abouelliel A. Sakthikumar S. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement PLoS One 9 11 2014 e112963 10.1371/journal.pone.0112963
22 Seemann T. Prokka: rapid prokaryotic genome annotation Bioinformatics 30 14 2014 2068 2069 10.1093/bioinformatics/btu153 24642063
23 Page A.J. Cummins C.A. Hunt M. Wong V.K. Reuter S. Holden M.T. Roary: rapid large-scale prokaryote pan genome analysis Bioinformatics 31 22 2015 3691 3693 10.1093/bioinformatics/btv421 26198102
24 Price M.N. Dehal P.S. Arkin A.P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix Mol Biol Evol 26 7 2009 1641 1650 10.1093/molbev/msp077 19377059
25 Qin Q.L. Xie B.B. Zhang X.Y. Chen X.L. Zhou B.C. Zhou J. A proposed genus boundary for the prokaryotes based on genomic insights J Bacteriol 196 12 2014 2210 2215 10.1128/JB.01688-14 24706738
26 Hölzer M. POCP-nf: an automatic nextflow pipeline for calculating the percentage of conserved proteins in bacterial taxonomy Bioinformatics 2024 10.1093/bioinformatics/btae175
27 Meier-Kolthoff J.P. Carbasse J.S. Peinado-Olarte R.L. Goker M. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes Nucleic Acids Res 50 D1 2022 D801 D807 10.1093/nar/gkab902 34634793
28 Ruoff K.L. Miscellaneous catalase-negative, gram-positive cocci: emerging opportunists J Clin Microbiol 40 4 2002 1129 1133 10.1128/jcm.40.4.1129-1133.2002 11923320
29 Schelenz S. Page A.J. Emmerson A.M. Streptococcus mutans endocarditis: beware of the 'diphtheroid' J R Soc Med 98 9 2005 420 421 10.1177/014107680509800911 16140857
30 Hoshino T. Fujiwara T. Kilian M. Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients J Clin Microbiol 43 12 2005 6073 6085 10.1128/jcm.43.12.6073-6085.2005 16333101
31 Zapun A. Contreras-Martel C. Vernet T. Penicillin-binding proteins and β-lactam resistance FEMS (Fed Eur Microbiol Soc) Microbiol Rev 32 2 2008 361 385 10.1111/j.1574-6976.2007.00095.x
32 Varghese R. Basu S. Neeravi A. Pragasam A. Aravind V. Gupta R. Emergence of meropenem resistance among cefotaxime non-susceptible Streptococcus pneumoniae: evidence and challenges Front Microbiol 12 2021 810414 10.3389/fmicb.2021.810414
33 Iannelli F. Santoro F. Santagati M. Docquier J.-D. Lazzeri E. Pastore G. Type M resistance to macrolides is due to a two-gene efflux transport system of the ATP-binding cassette (ABC) superfamily Front Microbiol 9 2018 10.3389/fmicb.2018.01670
34 Krause K.M. Serio A.W. Kane T.R. Connolly L.E. Aminoglycosides: an overview Cold Spring Harb Perspect Med. 6 6 2016 10.1101/cshperspect.a027029
35 Church D.L. Cerutti L. Gürtler A. Griener T. Zelazny A. Emler S. Performance and application of 16S rRNA gene cycle sequencing for routine identification of bacteria in the clinical microbiology laboratory Clin Microbiol Rev 33 4 2020 10.1128/cmr.00053-19
36 Huang L.-D. Yang M.-J. Huang Y.-Y. Jiang K.-Y. Yan J. Sun A.-H. Molecular characterization of predominant serotypes, drug resistance, and virulence genes of Streptococcus pneumoniae isolates from east China Front Microbiol 13 2022 10.3389/fmicb.2022.892364
37 Johnston J.W. Myers L.E. Ochs M.M. Benjamin W.H. Jr. Briles D.E. Hollingshead S.K. Lipoprotein PsaA in virulence of Streptococcus pneumoniae: surface accessibility and role in protection from superoxide Infect Immun 72 10 2004 5858 5867 10.1128/iai.72.10.5858-5867.2004 15385487
38 Letunic I. Bork P. Interactive Tree of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation Nucleic Acids Res 49 W1 2021 W293 W296 10.1093/nar/gkab301 33885785
39 Teles C. Smith A. Ramage G. Lang S. Identification of clinically relevant viridans group streptococci by phenotypic and genotypic analysis Eur J Clin Microbiol Infect Dis 30 2 2011 243 250 10.1007/s10096-010-1076-y 20981464
40 Davies A.P. Reid M. Hadfield S.J. Johnston S. Mikhail J. Harris L.G. Identification of clinical isolates of α-hemolytic streptococci by 16S rRNA gene sequencing, matrix-assisted laser desorption ionization–time of flight mass spectrometry using MALDI biotyper, and conventional phenotypic methods: a comparison J Clin Microbiol 50 12 2012 4087 4090 10.1128/jcm.02387-12 22993176
41 Singh N. Poggensee L. Huang Y. Evans C.T. Suda K.J. Bulman Z.P. Antibiotic susceptibility patterns of viridans group streptococci isolates in the United States from 2010 to 2020 JAC Antimicrob Resist 4 3 2022 dlac049 10.1093/jacamr/dlac049
42 van Prehn J. van Triest M.I. Altorf-van der Kuil W. van Dijk K. Third-generation cephalosporin and carbapenem resistance in Streptococcus mitis/oralis. Results from a nationwide registry in The Netherlands Clin Microbiol Infect 25 4 2019 518 520 10.1016/j.cmi.2018.11.021 30528371
43 Dowson C.G. Hutchison A. Woodford N. Johnson A.P. George R.C. Spratt B.G. Penicillin-resistant viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains of Streptococcus pneumoniae Proc Natl Acad Sci USA 87 15 1990 5858 5862 10.1073/pnas.87.15.5858 2377622
