The Aerococcus genus was first described in 1953, with the first studied representative thereof being Aerococcus viridans isolated from the air and street dust [1]. Currently, we know of eight species comprising the Aerococcus genus: A. viridians, A. urinae, A. sanguinicola, A. christensenii, A. urinaehominis, A. urinaeequi, A. suis, A. vaginalis [2].

Aerococcus bacteria are commonly associated with the urinary tract infection and urosepsis [3]. At the same time, within the last 10 years there were many reports of complications caused by the representatives of this genus, such complications manifesting as bloodstream infection and infective endocarditis, the etiology of which is most often linked to A. urinae and A. sanguinicola [2, 3]. Moreover, it was established that aerococci can cause invasive infections, such as osteomyelitis, meningitis, septic arthritis, peritonitis, and soft tissue infections, with isolated Aerococcus-like microorganisms and A. urinae often assumed to be behind the etiology thereof [2]. Since 1987, 17 cases of bacteremia/septicemia caused by Aerococcus-like microorganisms (pure culture isolated from the blood)  have been reported in Denmark, with 6 of them being endocarditis cases and 11 — septicemia cases; despite adequate antimicrobial therapy, 7 patients died [4]. Other authors, considering diseases of the urinary system, took isolation of Aerococcus in blood culture as an etiological factor of bacteremia [5].

The virulence of Aerococcus species is associated with their ability to build biofilms (in particular, on heart valves in vivo), form aggregation of platelets, and adhere to surfaces using capsular polysaccharide the presence of which was confirmed by comparative genomic analysis that revealed a wide intraspecific diversity of loci synthesizing it [2, 6, 7].

This study aimed to genetically characterize the Aerococcus sp. 1KP-2016 strain isolated from the blood of a patient with a bloodstream infection.

METHODS

We cultivated the Aerococcus sp. 1KP-2016 strain under microaerophilic conditions for 24–48 h at 37±1°C on Columbia agar base (Conda; Spain) with 10% of sheep blood. The cultural and morphological properties of the resulting colonies were uncovered using a SteREO Discovery V12 stereoscopic microscope (Carl Zeiss; Germany) with a PlanApo S 1.0 × FWD 60 mm lens objective and a PI 10 × 23 Br foc eyepiece. Gram staining (ZAO ECOlab; Russia) allowed establishing stain-related properties. The stained smears were examined through an Axio Scope A1 light microscope with EC Plan-NEOFLUAR 100 × 1.3 lens and PI 10 × 23 Br foc eyepiece (Carl Zeiss; Germany). The biochemical properties of the bacteria were studied with the help of Micro-LA-Test STREPTOtest 16 (Lakhema, Czech Republic), a commercially available biochemical test system, and a catalase test prepared in the laboratory.

As per the guidelines by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), we studied the colonies' susceptibility to antibiotics with the disk diffusion test using the commercial standardized paper disks (HiMedia Laboratories Pvt Limited; India).

The cells were boiled to release the chromosomal DNA. The 16S rRNA gene fragment was amplified as per the generally accepted protocol [8]. The PCR reaction mixture contained 1.5 mM of MgCl₂, 10 mM of Tris-HCl (pH 8.3), 50 mM of KCl, 0.1 µM of primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGYTACCTTGTTACGACTT -3'), 200 mM of each nucleoside triphosphate and 1 unit of Taq DNA polymerase (Thermo Fisher Scientific; Lithuania). The amplification was done with 1 µl of the DNA preparation in the total volume of the reaction mixture (25 µl) using the Tertsik amplifier (DNK-Technologiya; Russia). The PCR products were purified and sequenced at ZAO Evrogen (Moscow, Russia) (http://evrogen.ru/). To process the results of sequencing, we used the ChromasLite software (Technelysium Pty Ltd, Australia) (for the chromatogram format); the sequences were collated with the EMBL/NCBI (http://www.ncbi.nlm.nih.gov/nuccore) international online database using the megablast algorithm.

The genome of the Aerococcus sp. 1KP-2016 strain was sequenced using the Ion Proton system (Thermo Fisher Scientific; USA) at Gabrichevsky Research Institute for Epidemiology and Microbiology. The genome was assembled de novo with the help of the SPAdes software [9]. Contest16S [10] and CheckM [11] enabled checking of the assembly for contamination. In the search for CRISPR-Cas systems we relied on CRISPRCasFinder [12], for integrated phage genomes — on PHASTER [13], for antibiotic resistance genes — ResFinder [14].

We used all publicly available genomes of the Aerococcus genus from the Refseq database (as of October 4, 2021) for comparative analysis, and Abiotrophia defectiva ATCC 49176T as an outgroup. As per the annotations given in the databases, protein-coding regions of the genomes were clustered into groups of orthologs with the help of the ProteinOrtho software (standard settings) [15]. Ultimately, we obtained a conservative part of the proteome consisting of 543 such groups, each containing a single-copy protein-coding gene from each genome. The amino acid sequences of proteins in these groups of orthologs were aligned using MUSCLE [16] and concatenated. RapidNJ algorithm [17] guided the phylogenetic reconstruction based on the resulting concatenate. To calculate the average nucleotide identity (ANI) between genomes, we applied the ANIb approach and used the JSpeciesWS online service [18]. The ANI data are represented by two values divided by a slash in order to show the differences between mapping of fragments of the first genome onto the second one and second onto the first, respectively. As of the time of writing of this article, the release of the GTDB database, which contains an alternative taxonomy based on a purely phylogenetic approach, was 202 [19].

RESULTS

The Aerococcus sp. 1KP-2016 strain was isolated from the buffy coat of blood collected from a patient with a bloodstream infection (36 years old, city of Stavropol) in August 2016; the patient had subfebrile temperature for a long period of time (over a year).

Individual small smooth colonies less than 1 mm in size with uneven edges, a raised center, translucent grayish-white in color, with a small hemolysis zone formed on Columbia agar in 24–48 hours (fig. 1). Gram-stained smear contained Grampositive cocci forming tetrads (fig. 2). Biochemical tests of the isolated culture returned positive for galactosidase, esculin, lactose, trehalose, and negative for catalase, hippurate, phosphatase, leucine, alpha-arginine, urease, mannitol, sorbitol, raffinose, inulin, melibiose, ribose. The culture proved to be resistant to ciprofloxacin, ofloxacin, penicillin, erythromycin, doxycycline, showed intermediate resistance to clindamycin and susceptibility only to imipenem.

The Aerococcus sp. 1KP-2016 strain genome assembled to the level of contigs was deposited in the DBJ/EMBL/ GenBank under identifier NEEY00000000; subsequently, it was included in the NCBI Refseq as NZ_NEEY00000000. The assembly yielded 119 contigs with the mean coverage of 78x; the resulting genomic sequence consisted of 2.042 million bps, with the GC content at 38.5%. The Contest16S algorithm did not reveal any differing fragments of the 16S rRNA gene, which would have signaled of contamination. Verification of the set of conserved genes with CheckM has shown that the genome was 98.9% complete with contamination at 1.1%, which confirms high quality of the assembly. There were no CRISPR loci found in the genome. According to the PHASTER data, the genome contains two putative prophages, one of which includes intact genes of the major capsid protein (B9P78_00230), tail protein (B9P78_00200), phage terminase (B9P78_00240), and primase (B9P78_00325), which indicates its intactness. Despite the low susceptibility of the strain to antibacterial drugs, the search for antibiotic resistance genes yielded only the gene encoding chloramphenicol-Oacetyltransferase (B9P78_09255). A region at the beginning of the NEEY01000023 contig encodes a number of enzymes participating in biosynthesis of polysaccharides (B9P78_05530B9P78_05565) that form part of the cell wall or capsule.

The sequence of the Aerococcus sp. 1KP-2016 strain's 16S rRNA was 98.7% and 98.6% identical to the sequences of the type A. viridans and A. urinaeequi strains, respectively. Currently, the bacterial species differentiation threshold is accepted at 98.7% [20], which disallows relying on the 16S rRNA's similarity to make unambiguous conclusions about the taxonomic position. At the same time, the ANI between the sequenced genomic sequence and the genomes of the type strains A. viridans ATCC 11563 and A. urinaeequi DSM 20341 strains is 77.38/77.39% and 76.49/76.26%, respectively. This is significantly below the generally accepted threshold of 95–96% that allows assigning bacteria to a species [20, 21], which indicates the need to introduce a new species. The result is additionally confirmed by the GTDB, an alternative taxonomy database based on the comparison of genomes, in which the 1KP-2016 strain belongs to the separate Aerococcus sp002252085 species.

Reconstruction of the phylogeny (90 are marked with circles" data-note="" >fig. 3) of Aerococcus sp. 1KP-2016 strain shows that its closest relative is the Aerococcus sp. SJQ22 (RKMG01000000) strain, which was isolated from the soil and is currently not assigned to any validated species of the Aerococcus genus. However, the ANI between Aerococcus sp. 1KP-2016 and this strain was only 87.87/88.05%, which is below the generally accepted level of similarity within the species.

DISCUSSION

Aerococcus cause sporadic urinary tract disease, endocarditis, CSF and bloodstream infections. The most commonly isolated strains are A. urinae (55–60%) and A. sanguinicola (26–46%); A. viridians is isolated less frequently. The prevalent strain in Europe and the US is A. sanguinicola, A. viridians is found less often. Aerococcus bacteria are considered as part of the normal microbiota of the urogenital tract. They are isolated from the urine in the absence of clinical symptoms of the disease. In 2010-2015, a retrospective cohort study showed the etiological role played by the Aerococcus bacteria in urinary tract infections and asymptomatic bacteriuria (mainly in elderly women), with a noteworthy presence of other microorganisms in 35% of the cases [5, 22]. According to a retrospective cohort study of 2005–2020, 22.4% of the involved patients had a proven clinical picture of the aerococci blood infection, and their mortality depended on the duration of the disease: at 30 days, it was 17%, and at three months — 24% [2]. For microbiologists, it is difficult to properly identify the Aerococcus bacteria when they are isolated from urine or blood samples: the morphology often leads to their confusion with staphylococci, and hemolysis — with α-hemolytic streptococci. Given their low biochemical activity, biochemical tests do not yield reliable results. In this regard, it is possible to identify the species using mass spectrometry, but this method allows identification of only five species of the aerococci: A. christensenii, A. sanguinicola, A. urinae, A. urinaehominis, A. viridians. Therefore, sequencing of the 16S rRNA gene is used to identify strains of aerococci isolated from blood [5, 22].

This study is the first successful attempt at identification of the Aerococcus sp. 1KP-2016 strain isolated from the buffy coat that has a 16S rRNA sequence that matches the previously described representatives of this genus 98.7% or less. The whole genome sequencing and the subsequent phylogenetic reconstruction we undertook have shown that this strain is a representative of a new species of the Aerococcus genus, one closest to A. viridans and A. urinaeequi.

Consequently, aerococci are still the microorganisms the epidemiological and clinical characteristics of which have not been studied in full. There are many open fundamental questions about the pathogenetic role played by them in the invasive infectious pathologies. The complexity of identification of species within the Aerococcus genus requires use of the modern molecular genetic technologies that enable identification of the new types of microorganisms and expansion of the etiological spectrum of pathogens of severe invasive diseases.

CONCLUSIONS

The genotypic features of the studied strain allow attributing the 1KP-2016 strain isolated from the blood as a new species of the Aerococcus genus that is evolutionarily close to A.viridans and A. urinaeequi yet different from them. It is necessary to further study its phenotypic and chemotaxonomic properties while applying the polyphasic approach to bacterial taxonomy.