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31 Evolution I Research Article
Staphylococcus brunensis sp. nov. isolated from human clinical
specimens with a staphylococcal cassette chromosome-related
genomic island outside of the rlmH gene bearing the ccrDE
recombinase gene complex
Vojtěch Kovařovic,1
Adéla Finstrlová,1
Ivo Sedláček,2
Petr Petráš,3
Pavel Švec,2
Ivana Mašlanová,1
Meina Neumann-Schaal,4
Ondřej
Šedo,5
Tibor Botka,1
Eva Staňková,2
Jiří Doškář,1
Roman Pantůček1
AUTHOR AFFILIATIONS See affiliation list on p. 17.
ABSTRACT Novel species of coagulase-negative staphylococci, which could serve as
reservoirs of virulence and antimicrobial resistance factors for opportunistic pathogens
from the genus Staphylococcus, are recognized in human and animal specimens due to
advances in diagnostic techniques. Here, we used whole-genome sequencing, extensive
biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize five
coagulase-negative strains from the Staphylococcus haemolyticus phylogenetic clade
obtained from human ear swabs, wounds, and bile. Based on the results of polyphasic
taxonomy, we propose the species Staphylococcus brunensis sp. nov. (type strain NRL/St
16/872T
= CCM 9024T
= LMG 31872T
= DSM 111349T
). The genomic analysis revealed
numerous variable genomic elements, including staphylococcal cassette chromosome
(SCC), prophages, plasmids, and a unique 18.8 kb-long genomic island SbClc c r D£
integrated into the ribosomal protein L7 serine acetyltransferase gene rimL SbClccroF has
a cassette chromosome recombinase (ccr) gene complex with a typical structure found
in SCCs. Based o n nucleotide and amino acid identity to other kno wn ccr genes and
the distinct integration site that differs from the canonical methyltransferase gene rlmH
exploited by SCCs, we classified the ccr genes as novel variants, ccrDE. The comparative
genomic analysis o f SbClccrDF with related islands sho ws that they can accumulate
virulence and antimicrobial resistance factors creating no vel resistance elements, which
reflects the evolution of SCC. The spread o f these resistance islands into established
pathogens such as Staphylococcus aureus wo uld po se a great threat to the healthcare
system.
IMPORTANCE The co agulase-negative staphylo co cci are impo rtant o ppo rtunistic
human patho gens, which cause blo o dstream and foreign bo dy infectio ns, mainly
in immuno co mpro mised patients. The mobile elements, primarily the staphylococcal
cassette chro mo so me mec, which co nfers resistance to methicillin, are the key to the
successful dissemination of staphylococci into healthcare and community settings. Here,
we present a novel species o f the Staphylococcus genus iso lated from human clinical
material. The detailed analysis of its genome revealed a previously undescribed genomic
island, which is closely related to the staphylococcal cassette chromosome and has the
potential to accumulate and spread virulence and resistance determinants. The island
harbors a set of conserved genes required for its mobilization, which we recognized
as no vel cassette chromosome recombinase genes ccrDE. Similar islands were revealed
not o nly in the genomes o f coagulase-negative staphylo co cci but also in S. aureus.
The co mparative genomic study co ntributes substantially to the understanding o f the
evolution and pathogenesis of staphylococci.
Editor Shannon D. Manning, Michigan State
University, East Lansing, Michigan, USA
Address correspondence to Roman Pantůček,
pantucek@sci.muni.cz
Vojtěch Kovařovic and Adéla Finstrlová contributed
egually to this article. To determine the order of the
authors we flipped the coin.
The authors declare no conflict of interest.
See the funding table on p. 18.
Received 29 March 2023
Accepted 3 July 2023
Published 15 September 2023
Copyright © 2023 Kovařovic et al. This is an openaccess
article distributed under the terms of the
Creative Commons Attribution 4.0 International
license.
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KEYWORDS coagulase-negative staphylococci, phylogenetic analysis, comparative
genomics, mobile genetic elements, genomic islands, cassette chromosome recombinase,
polyphasic taxonomy, gram-positive pathogens
Staphylococci, mainly coagulase-positive Staphylococcus aureus, are the leading cause
of a broad spectrum of diseases in humans and animals. Over the last three decades,
coagulase-negative staphylococcal species (CoNS), with the most significant being
Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis,
have been recognized as opportunistic pathogens, especially in immunocompromised
patients. CoNS are a frequent cause of nosocomial infections related to catheters or
medical devices aided by their ability to form a biofilm (1). Additionally, the gene pool
of substrate utilization pathways and resistance determinants enables CoNS to occupy
various niches, providing favorable conditions for the emergence of multidrug-resistant
CoNS and their subsequent spread in healthcare environments (2).
The adaptation of S. haemolyticus strains to diverse environments is facilitated by
frequent recombination among numerous insertion sequences (ISSha 7) (3). Therefore,
the standard microbial and molecular diagnostic tools have limited discriminatory
power to reliably distinguish species closely related to S. haemolyticus (4). Only recently,
molecular diagnostics approaches, mainly in-depth whole-genome characterization,
have assigned atypical S. haemolyticus strains isolated from clinical specimens into the
new species Staphylococcus borealis (5) and Staphylococcus taiwanensis (6). The core
genome phylogeny also led to the reclassification of the S. petrasii phylogenetic complex
(7, 8), which now consists of three species—S. petrasii, S. croceilyticus, and S. pragensis
(9). All these species were isolated from various human biological samples, mainly from
wounds, eye and ear infections, urinary infections, and blood samples (10).
The versatility of CoNS is associated with a significant reservoir of mobile genetic
elements (MGEs). Notably, the methicillin resistance encoded by the staphylococcal
chromosomal cassette (SCC) mec (SCCmec) significantly complicates healthcare and
increases the need to use second-line antimicrobial drugs to treat staphylococcal
infections (11). Apart from mec genes responsible for methicillin resistance, SCCmec
carries numerous genes for virulence, such as phenol-soluble modulins (PSM-mec),
plasmin, or heavy-metal and other resistance genes contributing to the survival of these
strains in an environment (12-14). It is suspected that the SCCmec originates from CoNS
species (15), but the natural mechanism of SCC transmission is still unknown. It can be
transferred intra- and even interspecies by transduction (16), conjugation (17), or natural
competence (18). The transfer of SCCmec is mediated by ccr chromosome cassette genes
of the serine recombinase family. Three phylogenetically distinct ccr genes, namely
ccrA, ccrB, and ccrC, have been delineated with nucleotide identities below 50%. These
recombinases recognize a specific off site in the bacterial rlmH gene for ribosomal
RNA large-subunit methyltransferase H (19). The recombinases CcrA and CcrB function
together as a heterotetramer in the specific excision of the SCC (20), whereas the CcrC
recombinase enables mobilization of the element without another recombinase (21).
The recent increase in the association of CoNS with nosocomial infections and
improvements in diagnostic approaches make it possible to recognize other often
overlooked species of this group related to human diseases. Investigation of these CoNS
as a pool of genes for antimicrobial resistance and virulence can reveal the molecular
mechanisms that lead to these opportunistic pathogens' evolution, adaptation, and
success. This article reports the polyphasic characterization of five Staphylococcus sp.
isolates from human clinical material to delineate a novel species. Whole-genome
sequence analyses of the strains revealed a remarkable non-SCC genomic island
harboring ccrDE cassette chromosome recombinase types.
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RESULTS
Phylogenetic relationship of the strains
Five unidentified Staphylococcus sp. strains were collected from various human clinical
specimens from both mixed culture and monoculture between 2016 and 2022 (Table
1) and transferred to the National Reference Laboratory for Staphylococci (National
Institute of Public Health, Prague) for long-term storage and further study. The phylogenetic
analysis of complete 16S rRNA gene sequences consistently placed the five isolates
in S. haemolyticus cluster group defined previously (22). The closest relatives were
Staphylococcus petrasii, Staphylococcus croceilyticus, and Staphylococcus pragensis, with
16S rRNA gene sequence similarities ranging from 99.80 to 99.59%, while other species
were below 99.4% similarity. The topology of the neighbor-joining (NJ) phylogenetic
tree constructed with 16S rRNA gene sequences was similar to that of the maximum
likelihood (ML) tree (Fig. 1A). Because the 16S rRNA analysis has limited discriminatory
power in the genus Staphylococcus (5, 6, 23), the phylogenetic position of the new
isolates was also assessed using the concatenated multilocus sequence data of six
routinely used conserved housekeeping genes: rpoB, hsp60, dnaJ, tufA, gap, and sodA
for discrimination of staphylococcal species (Fig. 1B). The ML phylogenetic tree for the
housekeeping genes had a very similar topology to that of the 16S rRNA gene tree and to
the additional phylogenetic trees constructed using the up-to-date bacterial core gene
(UBCG) at the nucleotide and protein level (Fig. 1C and D).
The whole-genome phylogenetic distance from the related staphylococcal taxa with
an ANI value of <92.3% (Table S3) and digital DNA-DNA hybridization (dDDH) of <45.6%
determined the closest species S. petrasii; however, the values were below the species
delineation thresholds, which are 95-96% and 70%, respectively (24). The whole-genome
phylogeny thus confirmed that the five isolates represent a distinct Staphylococcus
species designated Staphylococcus brunensis sp. nov.
Growth, morphological, biochemical, and chemotaxonomical characterization
of analyzed isolates
All five isolates exhibited Gram-positive stain, irregular cells ranging in diameter from 433
nm to 1,210 nm (Fig. S1) arranged in pairs, tetrads, and clusters. They grew very well on
TABLE 1 Origin of the strains of Staphylococcus brunensis sp. nov. characterized in this study
Strain Date of isolation Locality Specimen Sex Age Diagnosis Other microflora
NRL/St" 16/872T
Oct 2016 Karlovy Vary Swab of ear M 3y Acute otitis externa Monoculture
= CCM" 9024T
= LMGC
31,872T
= DSMd
111349T
NRL/St0
19/737 = Jul 2019 Prague Wound pus M 32 y Surgical wound S. aureus
CCM" 9023 infection
NRL/St" 18/288 = Mar 2018 Lyon Wound pus M 51 y Leg wound infection NAe
P12563
NRL/St0
21/187 = Jul 2021 Karlovy Vary Bile M 83 y Cholelithiasis and E. coli, Klebsiella
P13332 cholecystitis pneumoniae,
Citrobacter
freundii, and
Enterococcus
faecalis
NRL/St0
22/194 = Apr 2022 Prague Swab of ear M 6 m Acute otitis media Monoculture
P13326
"NRL/St, National Reference Laboratory for Staphylococci, National Institute for Public Health, Prague.
k
CCM, Czech Collection of Microorganisms.
C
LMG, Bacteria Collection at the Laboratorium voor Microbiologie Universiteit Gent.
''DSM, German Collection of Microorganisms and Cell Cultures.
e
NA, not analyzed.
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Research Article Microbiology Spectrum
S. petrasii NCTC 13835T
S. brunensis sp. nov. NRL 18/288
S. brunensis sp. nov. NRL 16/872'
S. brunensis sp. nov. NRL 21/287
S. brunensis sp. nov. NRL 19/737
S. brunensis sp. nov. NRL 22/149
r S. croceilytious CCM 8421T
77/71^— s. pragensis CCM 8529"
59/«^- S. haemolytlcus ATCC 29970T
S. íaíwař7ens/sNTUH-S172T
S. iorea//s51-48'
B
97/85
S. aureus DSM 20231T
S. devriesei NCTC 13828T
S. lugdunensis NCTC 12217T
I S. hominis subsp. hominis NCTC 11320T
100/100"— s. hominis subsp. novobiosepticus CCUG 42399'
S. pasteuri DSM 10656'
S. warnen NCTC 11044'
S. brunensis sp. nov. NRL 21/187
S. brunensis sp. nov. NRL 22/194
100/98 1
f S. brunensissp. nov. NRL 18/288
S. brunensis sp. nov. NRL 19/737
S. brunensis sp. nov. NRL 167872*
L
S. pe/ras« NCTC 13835'
S. croceilyticus CCM 8421'
S. pragensis CCM 8529T
S. caledonicus H8/1'
— S. devriesei NCTC 13828'
S. taiwanensis NTUH-S172'
S. borealis 51-48'
S. haemolyticus P i l C C 29970'
r- S. hominis subsp. hominis NCTC 11320'
*— S. hominis subsp. novobiosepticus CCUG 42399'
S. lugdunensis NCTC 12217'
S. pasteuri DSM 10656'
— S. warnen NCTC 11044'
S. aureus DSM 202311
S. brunensis sp. nov. NRL 16/872' [
8 7 S. brunensis sp. nov. NRL 18/288
S. brunensis sp. nov. NRL 21/187
S. brunensis sp. nov. NRL 22/194
S. brunensis sp. nov. NRL 19/737
— S. petrasii NCTC 13835'
S. croceilyticus CCM 8421T
— S. pragensis CCM 8529'
j S. haemolyticus ATCC 29970'
' S. borealis 51-48*
S. taiwanensis NTUH-S172*
921 S. atew'ese/NCTC 13828*
1
S. caledonicus H8/1'
92 r—S. hominis subsp. hom/n/s NCTC 11320'
I— S. hominis subsp. novobiosepticus CCUG 42399'
S. lugdunensis NCTC 12217*
j S. pasteuri DSM 10656*
1
S. warneri NCTC 11044*
g 2 S. brunensis sp. nov. NRL 16/872*
— S. brunensis sp. nov. NRL 18/288
92 i S. brunensis sp. nov. NRL 21/187
r- S. brunensis sp. nov. NRL 22/194
S. brunensis sp. nov. NRL 19/737
L
S. pefras//' NCTC 13835*
— S. croceilyticus CCM 8421*
— S. pragensis CCM 8529*
92 S. haemolyticus ATCC 29970*
S. borealis 51-48*
S. ía/Vvanens/s NTUH-S172'
92 S. dew/ese/NCTC 13828'
' — S. caledonicus H8/11
2
-cS. hominis subsp. novobiosepticus CCUG 42399*
S. lugdunensis NCTC 12217'
92j S. pasteuri DSM 10656'
~^ S. warnen NCTC 11044'
FIG 1 Evolutionary analyses of Staphylococcus brunensis sp. nov. and its closest relatives. GenBank accession numbers of used sequences are listed in Table SI.
(A) Evolutionary history inferred using neighbor-joining (NJ) method based on complete 16S rRNA gene sequences extracted from whole-genome assemblies.
Filled circles indicate that the corresponding nodes were also identified from analysis by the maximum likelihood (ML) method. The percentage of replicate trees
above 50% in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches for the NJ and ML methods. S.
aureus DSM 20231T
was used as an outgroup. The evolutionary distances are in the units of the number of base substitutions per site. All ambiguous positions
were removed for each sequence pair. There were a total of 1,552 positions in the final data set. (B) Unrooted ML tree based on multilocus sequence analysis
of concatenated nucleotide sequences from six loci—rpoB, hsp60, dnaJ, tufA, gap, and sodA—were extracted from whole-genome assemblies. Filled circles
indicate that the corresponding nodes were also identified from analysis by the NJ method. There were a total of 3,952 positions in the final data set. Bootstrap
probability values (percentages of 500 tree replications) greater than 50% are shown at branch points. The evolutionary distances are given as the number
of substitutions per site. (C) Nucleotide sequence-based and (D) protein sequence-based phylogenetic tree of the concatenated alignment of 92 core genes
constructed using up-to-date bacterial core gene (UBCG) set. The ML tree was inferred using RAxML software and 100 replicates. The threshold for the gene
support index was set to 94% for the nucleotide and 80% for the protein-based tree. Gene support indices are given at branching points (the maximal possible
value is 92). The bar indicates the number of substitutions per position.
tryptone soy agar (TSA), Columbia agar with blood, plate count agar, P agar, and nutrient
agar, and did not grow in a thioglycollate medium. The Congo red agar method showed
negativity in the production of polysaccharide intercellular adhesin (PIA) associated
with biofilm formation. Phenotypic identification based on bacitracin resistance and
sensitivity to furazolidone, positive catalase test, growth in the presence of NaCI above
10%, and microscopic morphology assigned five isolates as Staphylococcus sp. In contrast
to the main characteristics of staphylococci, isolate NRL/St 19/737 exhibited atypical
negative catalase activity. Test-dependent results were also observed for the Voges-Proskauer
(VP) test (acetoin) and for (3-glucuronidase. With detection of acetoin production, S
we only obtained positive results in all five strains when pyruvic acid served as substrate
using the commercial VP test (Erba Lachema) instead of glucose as substrate for the •§
_o
c
%
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TABLE 2 Differentiation of Staphylococcus brunensis sp. nov. from closely related staphylococci occurring in human clinical material
Test'' Result obtained for indicated type strain7result from species description
S. brunensis sp. S. petrasii S. croceilyticus S. pragensis S. haemolyticus S. borealis S. taiwanensis
nov.6
CCM 8418T
CCM 8421T
CCM 8529T
CCM 2737T
CCM9145T
CCM
Arginine dihydrolase + +/+ +/+ - / - +/+ +/+ +/+
Voges-Proskauer test + +/+ +/+ +/+ w/+ - / - +/+
Urease - +/d +/+ - / - - / - +/+ +/+
(5-Glucuronidasec
+ - / - +/+ - / - +/d +/d - / DNA
hydrolysis - w/d - / - +/+ +/d w/- - / Acid
from: lactose + -16 - / - - / - -/d - / - - / Galactose
+ -16 - / - - / - +/d -Art - / Mannose
- +/+ - / - - / - - / - -/d - / Ribose
- -/w w/w - / - -/d +/+ +/+
D-arabinose - - / - +/+ - / - - / - - / - - / N-Acetylglucosamine
- - / - - / - - / - +/+ +/d - / Pale
yellow pigment - - / - +/+ - / - -/- +/+ - / "All
data were taken from this study (in two replications).
'Presented data are uniform for all isolates of S. brunensis sp. nov.
c
STAPHYtest 24 kit.
*+, positive; -, negative; w, weak reaction; d, 11-89% strains positive; nt, not tested.
conventional VP test. Similar results were obtained with the (3-glucuronidase test, where
all isolates were positive in the STAPHYtest 24 kit, but negative in the API ZYM kit due
to a different substrate for enzyme detection. The differentiation of novel staphylococcal
isolates from similar and/or closely related staphylococci occurring in human clinical
material is shown in Table 2. The species S. petrasii, 5. pragensis, and S. haemolyticus were
phenotypically the most similar taxa to the aforementioned isolates. Strain-dependent
utilization results are specified in Table S4. Complete characteristics of S. brunensis sp.
nov. are stated in the protologue given subsequently.
Antibiotic susceptibility testing showed that all five strains are susceptible to cefoxitin,
clindamycin, gentamicin, chloramphenicol, linezolid, oxacillin, rifampicin, tobramycin,
trimethoprim, sulphamethox/trimethoprim (cotrimoxazole), tetracycline, and fusidic
acid. Susceptibility to ciprofloxacin and levofloxacin was intermediate. Susceptibility to
ampicillin, penicillin G, tigecycline, and erythromycin was strain-dependent (Table S4).
By using cluster analysis of matrix-assisted laser-desorption/ionization-time-of-flight
mass spectrometry (MALDI-TOF MS) protein profiles, all five S. brunensis sp. nov. strains
were separated into a coherent cluster distinguished from phylogenetically related
j — S.caledonicus CCM 9192T
'— S.devriesei CCM 7896T
S.taiwanensis CCM 9267T
S.6orea//sCCM9145T
- S.haemolyticus CCM 2737T
- S.pasteuri CCM 4389T
- S.lugdunensis CCM 4064T
r S.hominis subsp. novobiosepticus CCM 47861
L
S.hominis subsp. hominis CCM 3474T
- S.wameriCCM 2730T
- S.pragensis CCM 8529T
- S.petrasii CCM 8418T
- S. croceilyticus CCM 8421T
NRL/St 22/194 ^
NRL/St 19/737
NRL/St 16/872T
NRL/St 21/187
NRL/St 18/288 S.
brunensis sp. nov.
1000 900 800 700 600 500 400 300 200 100
Distance Level
FIG 2 Dendrogram based on MALDI-TOF MS profiles of Staphylococcus brunensis sp. nov. and other
phylogenetically related species. The dendrogram was generated using the correlation distance measure
with the average linkage algorithm (UPGMA) settings of the software BioTyper version 3.1 (Bruker
Daltonics).
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Staphylococcus spp., as shown in Fig. 2. All five strains share 35 signals within the m/z
range 2-11 kDa, while five of these signals (m/z = 4130, 6660, 7614, 8258, and 10625)
were found to be specific for S. brunensis sp. nov., being absent in the MALDI-TOF MS
protein profiles of a comprehensive set of 65 S. petrasii, 5. croceilyticus, and S. pragensis
strains analyzed previously (10).
The chemotaxonomic analyses of type strain S. brunensis NRL/St 16/872T
showed
predominantly menaquinone-7 (MK-7, 95%) and a small amount of MK-6 (4%) and MK-8
(1%). The major fatty acids were C is:o anteiso (38.6%) and C i7:rj anteiso (19.6%), followed
by C 1 9 : 0 anteiso (8.3%), C 1 5 : 0 iso (6.3%), C ,7 : 0 iso (6.9%), C 1 8 : 0 (6.8%), C 1 9 : 0 i s o (4.4%), and
C 20:0 (3.0%). A small amount of C 1 6 : 0 i S 0 (1 -8%), C 1 6 : 0 (1.2%), and C 1 8 : 0 i S 0 (1 -6%) fatty
acids were also present. The detected peptidoglycan type is A3a (L-Lys-Gly3.4, A11.2). All
chemotaxonomic data are in-line with previous reported patterns (7).
DNA fingerprinting of S. brunensis sp. nov.
Screening of the investigated bacterial group by rep-PCR fingerprinting with primer
(GTG)5 showed their genotypic coherence. All five isolates had visually similar fingerprints,
which were grouped into a single cluster and separated from the other entries
in the in-house fingerprint database, which includes members of all recognized species
of the genus Staphylococcus, including type strains of phylogenetically closely related
4000
3000
2000
1500
1000
B
CM tCO
CM
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FIG 3 Cluster analysis of rep-PCR fingerprints and ribotype patterns obtained from Staphylococcus brunensis sp. nov. strains and the type strains of phylogenetically
closely related Staphylococcus species. (A) Dendrogram based on (GTG)5-PCR fingerprints. (B) Dendrogram based on cluster analysis of EcoRI ribotype
patterns obtained using a RiboPrinter system. The dendrograms were calculated with Pearson's correlation coefficients with the UPGMA clustering method (r,
expressed as percentage similarity values).
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species (Fig. 3A). The obtained results also suggest that this rapid and easy-to-perform
method can be used for the identification of S. brunensis sp. nov., similarly to how we
demonstrated for the identification of Staphylococcus spp. in our previous studies (8, 23,
25).
In contrast to the rep-PCR method, automated ribotyping with the restriction enzyme
FcoRI revealed heterogeneity among the five examined strains (Fig. 3B). Three isolates,
NRL/St 16/872T
, NRL/St 22/194, and NRL/St 18/288, exhibited unique fingerprint patterns
that allowed differentiation at the strain level and indicated that they were not clonally
related. The remaining two strains, NRL/St 19/737 and NRL/St 21/187, had visually
identical ribotypes, despite being isolated in 2019 and 2021, respectively, at different
locations and were therefore not considered clonally related. These results suggest that
automated ribotyping with FcoRI can separate isolates of S. brunensis sp. nov. at the strain
level, although some strains may have similar ribotype patterns. However, a reliable
assessment of the discriminatory power of this technique for typing S. brunensis sp. nov.
requires the analysis of a more significant number of strains from different localities and
sources.
Genome characterization of S. brunensis sp. nov.
The comparison of sequenced genomes (Table S5) revealed a high degree of similarity
between the isolates (Fig. 4). The S. brunensis sp. nov. genomes were 2.5-2.6 Mb long
with GC content 33.3-33.4% encoding 2,500-2,700 CDS, 61-62 tRNAs,and 19 rRNAs. The
pangenome consists of 2,230 core and 416 accessory, and 569 unique genes in total.
The sequenced genomes differ in variable genomic elements, which constitute 5-8%
of the genome and are associated with virulence and antimicrobial resistance genes,
predominantly located at plasmids. All S. brunensis sp. nov. genomes possess a type
IIU CRISPR-Cas complex with 15-20 variable spacers, some of which target siphoviral
prophages as determined by blastn search. The core gene kat encoding catalase in the
strain NRL/St 19/737 (locus tag MT339_07575) has a 1 bp deletion in homopolymer
polyA, which introduces a premature stop codon at the 3' end of the gene. It is possible
that the catalase-negative phenotype of the strain is associated with the loss of function
of this gene, although a truncated protein without the first 22 amino acids could be
produced.
N R L / S t 1 9 / 7 3 7 N
R L / S t 1 6 / 8 7 2 ' N
R L / S t 2 1 / 1 8 7
N R L / S t 2 2 / 1
N R L / S t 1 8 / 2 8 8
A ISShaf | SCC | transponson • CRISPR-Cas locus
A IS [| prophage • S b C l c c r D £
500 kbp
64%
1
I 100%
FIG 4 Genome comparison of Staphylococcus brunensis sp. nov. isolates. Mobile genetic elements are shown and color coded as in the legend. The ISSha? loci
that are divergent for the respective strains are marked with black circles. Only nucleotide blast hits above 64% identity and longer than 2 kb are shown.
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TABLE 3 Plasmid contigs of Staphylococcus brunensis sp. nov. grouped by replication gene and gene content
Replication genes Strain Size (kb) Antimicrobial resistance Virulence Saccharide Mobilization/toxin-antifactors
utilization genes toxin systems
rep39 NRL/St 16/872T
31.2 copZ, csoR, czrB, qacR, qacA dp - mobA
NRL/St 18/288 21.9 copZ, csoR, arsM, qacR, qacA, - - fstP
NRL/St 21/187 22.9 copZ, copA, csoR, arsR, arsM dp - mobA
rep20 NRL/St 16/872T
27.2 arsR, arsM isaB, essG - mobC, relaxaseP, mobP
NRL/St 19/737 and NRL/St 33.9 arsR, arsM isaB, essG mqo, hxIR, hxIA, hxll mobC, relaxaseP, mobP
18/288
NRL/St 21/187 22.9 qacR, qacA isaB, essG rep39,
rep5b, rep21, NRL/St 19/737 32.0 blaZ, blaR, blal, ermC, cadX, - - mobC, relaxaseP, mobP
rep10,rep20 cadA, cadD
rep21 NRL/St 16/872T
, NRL/St 2.8 cadD, cadX - - -
18/288, NRL/St 21/187, and
NRL/St 22/194
rep 13 NRL/St 16/872T
and NRL/St
21/187
2.6 - - - rep5b
NRL/St 18/288 7.9 - - - mobC, relaxase P
repW NRL/St 22/194 44.7 ermC
The S. brunensis sp. nov. genomes harbor several large plasmids, which can be
grouped by the presence of either rep39 or rep20 gene. Due to high sequence variability
and mosaicism caused by interspersed \S431, it was not possible to determine
a complete consensus sequence for all plasmids. The plasmid contigs comprise betalactam,
heavy metal, and disinfectant resistance as well as virulence and saccharide
utilization genes as shown in Table 3. All analyzed strains harbor a small rep21 plasmid
conferring cadmium resistance. The strain NRL/St 22/194 contains an additional replO
plasmid harboring the ermC gene similar to the pUSA05-1 plasmid from S. aureus (26).
These two small plasmids are integrated into the rep39-type plasmid contig of strain
NRL/St 19/737. Furthermore, two cryptic plasmids, one containing the rep13 gene and
the other containing rep5b gene were identified (Table 3).
The resistance to penicillin in S. brunensis sp. nov. NRL/St 18/288 is encoded by the
WoZgene located at a 9 kb Tn554-like transposon (Fig. 4) integrated into the chromosomal
isaB gene. The chromosomes of S. brunensis sp. nov. harbor several copies of various
full and partial IS elements from the IS3, IS6, IS30, IS7 782, IS200/IS605, and ISha7 families
(Fig. 4). The IS437 flanked composite transposon localized downstream of the cspC gene
contains the gene for a short-chain dehydrogenase associated with survival in stress
conditions (27). Each strain carries six to eight ISSha 7 copies integrated in six conserved
and six variable loci, usually adjacent to rRNA genes. One copy of ISSha 7 is inserted in the
SCC element of S. brunensis sp. nov. NRL/St 21/287 strain (Fig. 4).
The S. brunensis sp. nov. strains have one or two complete prophages in their
genomes integrated into two different 18 bp off sites. The OSBR-1 prophage of NRL/St
16/872T
is integrated in the off site AATCCCTTACTTCCCGTT, located in the tRNA-Ser(gga)
gene. The other strains encompass one or two CRISPR spacers homologous to the
(DSBR-1, and thus are immune to infection by this phage. The same off site is used by
(DSBR-4 in the genome of NRL/St 19/737. Both prophages are 45.4 kb long and share
79.3% nt identity. The next off site, AATCCCTCCGTTTCCGTT in tRNA-Ser(gct), is occupied
by either OSBR-2 or OSBR-3. Prophage OSBR-2, which is 47 kb long, is integrated into
the genomes of strains NRL/St 16/872T
, NRL/St 19/288, and NRL/St 21/287. The strain
NRL/St 19/737 harbors a 43.5 kb OSBR-3 prophage, which shares 79% nt identity with
(DSBR-2 genome along 57% of its length; the difference is mainly in the morphogenesis
module. Both OSBR-2 and OSBR-3 carry a putative accessory virulence factor sialic acid
transporter (28) in the lysis gene module.
Two S. brunensis sp. nov. strains NRL/St 16/872T
and NRL/St 21/287 comprise an
approximately 18.2 kb-long genomic island in the rRNA methyltransferase H gene rlmH
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(orfX). The genomic island was almost identical in both strains; the only difference is
the presence of an insertion sequence ISSha7 in strain NRL/St 21/287. The island is
bordered by imperfect 18 bp-long direct repeats GAAGC(A/G)TATCATAA(G/A)TGA and
harbor ccrA1B2 genes; thus, according to the rules of the IWG-SCC (29) the element was
designated SCCNRivst 16/872- The most similar element to SCC NRL/St 16/872 i s a
composite
island in genome of S. hominis C34847 (30), sharing 95.2% nt identity along 75% of their
length. The SCCccM9024 carries genes for the type I restriction-modification (RM) system,
sharing high homology with genes encoding a restriction (hsdR) and methylation (hsdM)
subunit with the RM system of S. hominis C34847 and S. aureus NTUH-4729 (31).
Interestingly, the putP gene, which encodes a sodium proline symporter, was wedged
between the ccrB2 gene and a set of three short hypothetical genes conserved in the
ccr gene complex, thus disrupting the canonical organization of the complete ccr gene
complex. Similarly, the putP gene and the flanking sequence from the ccr gene complex
were found in SCC elements of various CoNS, S. epidermidis 11PPP121 (GenBank accession
no. MH188479), 5. haemolyticus BC05211 (KX181861), 5. hominis J6 (LT963442), and S.
hominis J11 (LT963438), and in plasmids of S. warneri 16A (CP031267) and S. pasteuri 3C
(CP031281), pointing to a high degree of recombination occurring in staphylococci.
The strain NRL/St 22/194 also harbors an SCC element inserted into the rlmH gene
bordered by the 21 bp imperfect repeat GG(C/A)GAAGC(A/G)TATCATAA(G/A)GTGA. The
26.6 kb pseudo-SCC element named ipSCCNRivst 22/194-p/s has n o
recombinase genes,
but carries several virulence genes, including the gene encoding plasmin sensitive
protein (p/s), poly(glycerol-phosphate) a-glycosyltransferase (tagE), and UDP-N-acetylmuramate-L-alanine
ligase (murF), which are also found in S. aureus composite island
SCCmecWAMRSA40 (32).
Novel genomic island harboring cassette chromosome recombinase genes
ccrDE
The strains NRL/St 16/872T
, NRL/St 21/187 and NRL/St 22/194 harbor a mobile element of
size 18.8 kb with cassette chromosome recombinase genes, integrated in the rimJ/rimL
gene orthologous to the the ribosomal-protein-serine acetyltransferase gene riml in
Escherichia coli and ydaF in Bacillus subtilis (UniProt accession no. P13857 and P96579,
respectively). The island designated S. brunensis chromosomal island ccrDE (SbClc c r Df)
harbors another homologue of riml with 70% nt identity to the original riml; thus, it
may complement the function of the truncated riml gene. SbClc c r of is bordered by the
tetranucleotide motif GAAA. Additionally, the 19 bp direct repeat ATTCCACAATGAAATCCAT
was found on the integration site in riml and inside the riml homologue on the
island, which suggests a complex recombination event.
SbClc c r DF possesses a cluster of genes homologous to the ccr gene complex from
SCC elements. The core of the ccr complex consists of two ccr genes, similar to SCC
elements type IV or II. Additional genes in the ccr complex, that is the putative primase
polA, cassette chromosome helicase ccA>2, three short proteins with the domains SAUGI,
DUF960, and DUF1643, and a hypothetical protein (Fig. 5), were homologous to those
in SCCmec type V, which has only one ccrC gene in the ccr complex. SbClc c r Df ccr
genes share more than 98% DNA sequence identity to ccrA8B9 recombinases (Table S6)
discovered recently in the S. haemolyticus genome (33). The values of nucleotide identity
to currently known ccrAl-7, ccrBl-8, and ccrCl-2 genes from SCCs range from 38.0 to
53.3% (Table S6). Although it is slightly above the threshold of 50.0% for the definition
ccr gene complex putative transposon
MmL polA cch2 ccrD ccrE SAUGl| DUF1643 mcrC mcr& yeiH cysL mqo
DUF960 IR1I R 2
IR3 IR4 A"mL
FIG 5 Annotated map of genes and possible functions in genomic island SbClc c r rj£ harbored by Staphylococcus brunensis sp. nov. Genes are labeled according
to known or putative function. Sequence of direct repeats DR1, DR2: 5'-ATTCCACAATGAAATCCAT-3'; sequence of inverted repeats IR1HR4: 5'-TGGTTCTGTTGC-
AAAGT-3'.
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Research Article Microbiology Spectrum
of a gene variant according to the rules of IWG-SCC (29), the amino acid (aa) identity
level of SbClc c r DF recombinases to known CcrA, CcrB, and CcrC reaches a maximum of
39.9%, which is substantially lower than aa identity among Ccr allotypes, which range
from 50.9 to 92.3%. Therefore, based on the borderline nucleotide identities and low
protein identities to CcrA, CcrB, and CcrC allotypes, we propose designating the SbClc c r
recombinases as new allelic types ccrD and ccrE and reclassifying the described ccrA8B9
recombinases accordingly (33). The CcrDE recombinases are able to excise the SbClc c r D£
element from its insertion sequence site, which has been detected by sequence read
alignment and PCR analysis (Fig. S2).
Apart from the ccr gene complex, the island SbClc c r of carries the 5-methylcytosinespecific
restriction enzyme mcrB and specificity subunit mcrC genes for the putative RM
system (Fig. 5), commonly found on SCC elements, chromosomal islands, and prophages
that help to maintain the mobile element in the genome (34). Approximately 5.8 kbp of
the SbClc c r of is occupied by a putative transposon bordered by a perfect 17 bp inverted
repeat of TGGTTCTGTTGCAAAGT. The transposon has one copy of the insertion sequence
from the IS6 family (sharing 95% nt identity to \S431mec) and accessory genes encoding
putative membrane protein iyeiH), putative transcriptional regulator (cysL), and malate
chinonine oxidoreductase (mqo) (Fig. 5), which are frequently found on plasmids.
We surveyed the GenBank database for sequences resembling SbClccrof. In addition
to S. haemolyticus BC5211 (33), we found related genomic islands with ccrDE in
the genomes of S. haemolyticus (GenBank accession nos. CP033814 and CP102568), S.
hominis subsp. hominis K1 (CP020618), and 5. borealis GDY8P80P (35), and in S. aureus
ER04332 and ER11327 (36), and related clones. The island is consistently inserted in
the rimL gene, in a locus downstream of the conserved metE gene (Fig. 6A), which
is 17-56 kb counterclockwise from the replication origin in CoNS species. However,
in S. aureus strains, the rimL gene is located at the end of the oriC environ due to
large-scale chromosomal inversion. The variable region of the islands comprises genes
for an RM system (Fig. 6A). In S. borealis GDY8P80P, the island harbors many transposons
with resistance genes to beta-lactam antibiotics, tetracycline, aminoglycosides, and
other antimicrobials. The phylogenetic analysis of ccrD and ccrE genes from the related
genomic islands (Fig. 6B) and the pairwise nucleotide identity comparison (Table S6)
clearly distinguished two allotypes designated ccrDIEI, present in the genomes of S.
brunensis sp. nov., S. haemolyticus, and S. aureus, and ccrD2E2, found in the genomes of S.
borealis and S. hominis (Fig. 6B; Table S6).
Taxonomic description of S. brunensis sp. nov.
Staphylococcus brunensis (bru.nensis L. adj. brunensis from Bruna, the Roman name of
the city of Brno, Czech Republic, where this and other staphylococcal species were first
described).
Cells are Gram stain positive cocci, occurring predominantly in pairs and clusters,
non-spore-forming, and nonmotile. Colonies on TSA agar are circular, whole margin,
flat, smooth, shiny, 2 mm in diameter, aerobic, and white. Hemolytic activity on sheep
blood agar, production of delta-hemolysin revealed in synergistic test with a beta-hemolytic
producing strain (S. pseudintermedius CCM 4710). Growth in the presence of 12%
NaCI, at 20°C and 45°C, but not at 15°C and 48°C. They are positive for pyrrolidonyl
arylamidase, arginine dihydrolase, and nitrate reduction and negative for coagulase,
clumping factor, oxidase, urease, VP test (acetoin, conventional tube test), hyaluronidase,
thermostable nuclease, and ornithine decarboxylase; susceptible to furazolidon
(100 ug) and novobiocin (5 ug), and resistant to bacitracin (10 IU); partially resistant to
lysostaphin (200 mg L"1
) and resistant to lysozyme; negative for hydrolysis of esculin,
gelatine, DNA, and Tween 80; positive on API ZYM for acid phosphatase, alkaline
phosphatase (weak), esterase (C 4), and esterase lipase (C 8); negative on API ZYM for
lipase (C14), valine arylamidase, cystine arylamidase, trypsin, ct-chymotrypsin, naphtholAS-BI-phosphohydrolase,
a-galactosidase, (3-galactosidase, (3-glucuronidase, (3-glucosidase,
N-acetyl-p-glucosaminidase, a-mannosidase, and a-fucosidase. S. brunensis sp. nov.
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I Iff II « 7 &
CP119327 (2444051..2509421)
S. haemolyticus BC5211
CP090475 ( 2 5 0 6 3 2 1 . 2 5 6 6 2 0 2 )
S. haemolyticus FDAARGOS 517
CP033814 (1200995..1246296)
S. haemolyticus strain 6
CP102568 ( 2 5 5 7 5 9 . 3 1 8 1 2 4 )
I IfII
S. borealis GDY8P80P
CP063443 (1061825..1158400)
S. hominis K1
CP020618 (1275794..1308911)
S. aureus ER04332
CP030518 (357269..398037)
S. aureus ER11327
CP051909 ( 4 1 9 1 1 9 . 4 7 0 6 9 7 )
I I chromosomal
f B hypothetical
~J chromosomal island
I I restriction modification system
I I ccr complex
| recombinase
| transponsase
I I antimicrobials resistance
20 Kbp
1 0 o j ccrD1 S. brunensis NRL/St 16/8721
(MT340J 2205)
n ccrD1 S. haemolyticus FDAARGOS517 (AYX83926)
100 W ccrA8 (ccrD1) S. haemolyticus BC5211 (LUR16_RS12245)
ggiOT07S. haemolyticus strain 6 (UUY81088)
J ccrD1 S. aureus ER11327 (QOA42770)
9 8 [
ccrD1 S. aureus ER04332 (QFL25609)
lD0QCcrD2 S. hominis K1 (AUJ52226)
ccrD2 S. borealis GDY8P80P (CP063443)
- ccrA1 S. aureus NCTC 10442 (BAA8664B)
35 r ccrE1 S. brunensis NRL/St 16/872'(MT340J2215)
54J- ccrE1 S. aureus ER04332 (QFL25607)
KccrSS (ccrE1) S. haemolyticus BC5211 (LUR16_RS12255)
99I ccrEt S.haemolyticus strain 6 (UUY81090)
ccrEi S. haemolyticus FDAARGOS517 (AYX83928)
ccrE1 S. aureus ER11327 (QOA42768)
991- ccrE2 S.hominis K1 (AUJ52228)
100
100
ccrE2 S.borealis GDY8P80P (QOV86914)
rcrßfS. 8ureusNCTC10442(BAA94328)
0.10
FIG 6 Comparative analysis of chromosomal islands harboring ccrDE cassette chromosome recombinases (CIc c ,oe). (A) Comparison of CIc c ,oe and flanking
regions from different staphylococcal species. The genomic island CIc c ,oe is highlighted with a gray background. Genes are labeled according to known or
putative functions, as shown in the legend. Only nucleotide blast hits above 65% identity and longer than 2 kb are shown. The position of the island is provided
in parentheses next to the GenBank accession number. (B) Maximum likelihood trees of nucleotide sequences of ccrD and ccrA8 recombinases with ccrA1 as an
outgroup, and ccrE and ccrB9 recombinases with ccrB1 as an outgroup, respectively. The trees were constructed using Tamura-Nei model with 500 bootstrap
replicates. The evolutionary distances are in the number of base substitutions per site. The protein GenBank accession numbers or locus tags are shown in
parentheses.
produce acid from glycerol (weak), galactose, D-glucose, D-fructose, maltose, lactose,
sucrose, and trehalose. They do not produce acid from erythritol, D-arabinose, L-arabinose,
ribose, D-xylose, L-xylose, adonitol, (3-methyl-D-xyloside, mannose, sorbose,
rhamnose, dulcitol, inositol, mannitol, sorbitol, ct-methyl-D-mannoside, ct-methyl-D-glucoside,
A/-acetyl glucosamine, amygdaline, arbutine, salicin, cellobiose, melibiose, inulin,
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Research Article Microbiology Spectrum
melezitose, D-raffinose, starch, glycogen, xylitol, (3-gentiobiose, D-turanose, D-lyxose,
D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-keto-gluconate, and
5-keto-gluconate.
Variable biochemical reactions were obtained for catalase (4 of 5 positive), leucine
arylamidase (2 of 5 positive), ct-glucosidase (1 of 5 positive), and growth in 15% NaCI (4 of
5 positive) (Table S4).
Utilization (Biolog MicroPlate GEN III, protocol A) is positive for D-maltose, D-trehalose,
D-turanose, ct-D-glucose, D-fructose, D-galactose, glycerol, L-alanine, L-arginine,
L-glutamic acid, L-serine, D-gluconic acid, acetic acid, and formic acid. Negative
utilization for D-cellobiose, gentiobiose, stachyose, D-raffinose, D-melibiose, (3-methylD-glucoside,
D-salicin, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl
neuraminic acid, 3-methyl glucose, D-fucose, L-fucose, L-rhamnose, inosine, D-sorbitol,
D-mannitol, myo-inositol, D-glucose-6-P04, D-aspartic acid, D-serine, gelatin, glycyl-Lproline,
L-histidine, D-galacturonic acid, D-galactonic acid lactone, D-glucuronic acid,
glucuronamide, mucic acid, quinic acid, D-saccharic acid, p-hydroxy phenylacetic acid,
D-lactic acid methyl ester, citric acid, ct-keto glutaric acid, D-malic acid, bromo-succinic
acid, y-amino-butyric acid, ct-hydroxy-butyric acid, (3-hydroxy-D,L-butyric acid, and
propionic acid.
The type strain is NRL/St 16/872T
(= OCM 9024T
= DSM 111349T
= LMG 31872T
). The
major respiratory quinone is menaquinone-7. The major fatty acids are C i 5 : u anteiso
a n
d
Ci7:0 anteiso- The peptidoglycan type is A3ct (A11.2). The DNA G+C content of strain
NRL/St 16/872T
is 33.40 mol%, calculated from the whole-genomic sequence. The species
description is based on the characterization of five strains isolated from various human
clinical materials. Most of the characteristics of the type strain NRL/St 16/872T
agree with
the species description. The GenBank/ENA/DDBJ accession number for the 16S rRNA
gene is OQ401401. The complete chromosome sequence of the type strain is available
under GenBank accession number CP119327.
DISCUSSION
The recent molecular diagnostic methods and polyphasic taxonomic approach, including
whole-genome sequencing, allow for more effective species differentiation of CoNS
from various sources. Here, we described 5. brunensis sp. nov., which occupies similar
niches to the closely related species from the S. petrasii phylogenetic complex (10).
The available isolates of S. brunensis sp. nov. were associated with the human ear and
wound infections, whereas S. petrasii and S. pragensis predominated in wounds, blood, or
urinary tract infections (Table S7). However, it is challenging for clinicians to determine
whether these CoNS are causative agents of human diseases or suspected contaminants
associated with the occurrence of these commensal bacteria on human skin.
The pathogenic potential of CoNS is related to immune evasion, invasion of host
tissues, and biofilm formation, allowing them to persist on the surfaces of indwelling
medical devices and thus cause chronic infections (1). The intercellular adhesion operon
(ica), biofilm-associated protein (bap/bhp), and fibronectin-binding protein genes (fnbA/
fbe) directly associated with biofilm production in S. epidermidis (37) have not been
identified in either S. brunensis sp. nov. genomes or the other species from 5. petrasii
complex. Likewise, the ica operon homologue has not been detected in S. haemolyticus
(3) and may be missing in this phylogenetic clade. Biofilm formation in the initial phase
is influenced by molecules involved in surface adhesion. In staphylococci, these are
microbial surface components recognizing adhesion matrix molecules (MSCRAMMs) (38).
Homologs of cell wall-anchored serine-aspartate repeat-containing protein genes sdrC,
sdrG, and sdrH have been identified in 5. petrasii genomes (10) but not in S. brunensis sp.
nov. However, gene homologues for elastin-binding protein (ebp), thermonuclease (nuc),
autolysin E {atlE), and gene clusters (cap5 or cap8) involved in the synthesis of capsular
polysaccharides have been found in genomes of both S. petrasii (10) and S. brunensis sp.
nov.
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A comparison of representative genome sequences revealed that the species S.
brunensis sp. nov., S. croceilyticus, 5. petrasii, and 5. pragensis differ only marginally in
genomic G+C content, genome size, and the median protein count encoded by the core
genome. The major cause of interspecies differences in the S. petrasii complex is the
accessory genome and variable genetic elements (Fig. S3). The role of these elements
is best described in 5. aureus, where HGT contributes to adaptation and evolution into
successful lineages (39-41).
More than 90% of clinical staphylococcal isolates harbor plasmids of various size
that can be classified to small multicopy plasmids or larger plasmids carrying several
resistance determinants. However, only 5% (42) of staphylococcal plasmids are large
multiresistance elements with the ability to mobilize or undergo conjugative transfer.
Strains of S. brunensis sp. nov. harbor several large plasmids that encode genes for
mobilization (mobA, mobC, mobP) and their spread by HGT is possible. The resistance
genes are usually cointegrated between two copies of ISs that promote their spread (43).
An example is IS437 previously described in plasmids pSK41 and pG01 (44), harboring
linezolid and high-level resistance to vancomycin (45). It is probable that IS437, identified
in 5. brunensis plasmid sequences, is responsible for the mosaic structure of the elements
and also promotes the spread of resistance genes across the genus Staphylococcus.
The plasmid-borne resistance to antibiotics and disinfectants suggests enhanced
survival in healthcare environments. The resistance to beta-lactam (blaZ) and macrolide
(ermQ antibiotics in 5. brunensis sp. nov. strains correlates with resistance genes on
plasmids and the Tn554-like transposon. Resistance to quaternary ammonium compounds
(qacA), copper and heavy metals, and the hexulose utilization operon (hxl) in
plasmids of S. brunensis sp. nov. suggests adaptation to various ecological niches similar
to nosocomial and community-associated isolates of S. haemolyticus (46,47).
The genomes of S. brunensis sp. nov. contain more insertion sequence elements than
the closest relatives from S. petrasii complex (7, 8). The copy number variability of IS
implies their recent capture and propagation and increases the genome plasticity among
the strains. The most abundant ISSha7 elements with 98% similarity to ISSha 7 from
S. haemolyticus (3) are often located near rRNA genes and may contribute to variable
ribotype patterns.
A significant MGE in the genome of S. brunensis sp. nov. is the genomic island
SbClc c r of with unique recombinase genes ccrDIEI. Xiao et al. (33) reported the existence
of these recombinases as ccrA8B9 allotypes. Nevertheless, their amino acid sequences
that were highly different from other known ccrAB variants place them among novel
ccrDE alleles. This reclassification also made it possible to distinguish another apparent
variant ccrD2E2, present in S. hominis and S. borealis. The ccrDE genes in all inspected
staphylococcal genomes were present on a genomic island related to SbClc c r Df.
Chromosomal islands with ccrDE (Clc c r DfS) share gene structure similar to SCCs, such
as conserved ccr complex, presence of variable regions, and flanking by direct repeats.
However, unlike SCCs, which are canonicaly inserted in the rlmH (orfX) gene in the oriC
environ (48), Clc c r DfS are inserted in the rimL gene. The SCC-like islands with Ccr or
closely related large serine recombinase were inserted in the rlmH in other genera of
Gram-positive bacteria, that is the genera Mammaliicoccus and Macrococcus, Enterococcus
faecium, Bacillus cereus, Geobacillus vulcani, and Clostridioides difficile (49-52). Hence,
Clc c r DfS might represent a new class of SCC elements integrated into the rimL gene due
to the altered specificity of the ccrDE recombinases.
The molecular mechanism of the action of ccrAB recombinases requires specific
sequences of around 60-70 bp and the presence of the central dinucleotide GA in the
integration site, which are essential for the integration of the cassettes into the highly
conserved rlmH gene (20). However, the ccrAB recombinases also act on non-canonical
less conserved recombination sites (20). Xiao et al. (33) showed that the ccrDE recombinases
are functional in excision and transfer of an SCC from rlmH, but they do not move
together with the cassette. Here, we proved that the OCcr0E bearing the ccrDE genes is
excised from the chromosome. Since OCcr0E has a
similar size as pathogenicity islands
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and possesses a set of genes required for its replication (53), the element would perfectly
fit into the phage capsid to be spread via transduction, which is a common transmission
route of MGE in staphylococci (54, 55). The high sequence conservation of the ccrDE
gene complex in various species corresponds with their spread through horizontal gene
transfer.
The proportion and diversity of 0CaDE w a s
l ° w i n t n e
sequenced genomes deposited
in the public databases, making it extremely difficult to identify the donor of ccrDE
complex responsible for the formation of this element. Since we found ccrDlEI and
ccrD2E2 predominantly in CoNS genomes, it is possible that CoNS species are predisposed
to forming this element and it can subsequently be transferred to S. aureus.
The variable regions of Clc c r of are highly diverse in gene content, suggesting that the
Clc c r of element evolved only recently in independent acquisition events, which are also
common in the evolution of SCCmec (56) or staphylococcal pathogenicity islands (57).
The benefit of Q\CaDE f ° r t n e
bacterial host might be only marginal, so there is no
selective pressure to maintain established element and disseminate it to more strains.
The only widespread genes identified in all C\ccrDES that help to preserve the element are
the RM system genes that differ in the Clc c r DfS, suggesting that they come from various
sources similar to canonical SCC elements (30, 31).
With the high selective pressure exerted on staphylococci by the use of antimicrobials,
this element is a perfect candidate for the acquisition and spread of resistance
and virulence genes. The SCCmec element underlying the successful spread of MRSA
clones originated through joining of ccr gene complex with mec gene complex coming
from multiple sources. The first signs of the accumulation of resistance genes in C\CCrDE
were observed in S. borealis, previously misidentified as S. haemolyticus (35), where the
island comprised several transposons. A similar cluster of drug-resistance genes to that
in C\caDE of S. borealis was also reported in S. aureus on an SCC element (58) and a
plasmid (59). We conclude that the gene structure of Clc c r of indicates its ability to act as
a primordial element to accumulate virulence and antimicrobial resistance factors. The
spread of the island to established pathogens such as S. aureus would thus represent a
new threat to the healthcare system.
Conclusion
The identification of the new species S. brunensis within the Staphylococcus genus
expands our understanding of the diversity of coagulase-negative staphylococci. The
number of the strains available is still limited, but similar to its closest relative S. petrasii,
it can be expected that more strains will be captured as better diagnostic methods
are developed. Genome analysis of the new isolates has important implications for
studying the role of coagulase-negative staphylococci as a reservoir of transmissible
genes that can facilitate improved survival in the environment, resistance to antibiotic
treatment, or increased virulence following horizontal transfer. Characterization of a
previously unexplored genomic island closely related to the SCC indicates the potential
for its interspecies transfer enabled by unique ccrDE recombinase genes in both
coagulase-negative staphylococci and the more clinically significant S. aureus. Identifying
the new type of MGE thus opens up new possibilities for future research of gene
transfer mechanisms in the emergence of multidrug-resistant staphylococcal strains
with implications for clinical practice. These findings deepen our understanding of the
evolution and pathogenesis of staphylococci, shedding light on how these bacteria
acquire and disseminate virulence traits and antibiotic resistance.
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MATERIALS AND METHODS
Bacterial strains, cultivation and phenotypic identification, and antimicrobial
susceptibility testing
Simultaneously analyzed reference strains of Staphylococcus spp. were obtained from the
Czech Collection of Microorganisms (CCM, Masaryk University, Brno). The strains grew
well in a basic set of staphylococcal media at a temperature of 30-37°C. The morphological,
biochemical, and physiological characterization was performed as previously
mentioned (7, 60-62). Antimicrobial susceptibility testing by disc diffusion method
on Mueller-Hinton agar with adherence to EUCAST guidelines (63) was performed as
described previously (60).
Genome sequencing and bioinformatics analyses
The short-read sequencing was conducted for the type strain NRL/St 16/872T
(LGC
Genomics, Berlin, Germany). The genomic DNA was isolated with a GenElute Bacterial
Genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA) from pure culture colonies
cultivated on Colombia sheep blood agar (Oxoid). The library was prepared using a
Nextera XT DNA Library Preparation Kit (lllumina, San Diego, CA, USA) and sequenced
externally by LGC Genomics (Berlin, Germany) on the NextSeq platform with 2 x
150 bp reads (lllumina). Genomes of all novel isolates were sequenced by Oxford
Nanopore Technology (ONT). The genomic DNA was extracted as described previously
(64). Sequencing libraries were prepared using an SQK-RAD004 rapid barcoding kit and
sequenced with a FLO-FLG001 cell in a MinlON device and MinKnow V21.10.4 software
(Oxford Nanopore Technologies, Oxford, UK).
The software Guppy version 6.0.0 (Oxford Nanopore Technologies) with config
dna_r9.4.1_450bps_sup.cfg and default settings was used for basecalling, demultiplexing,
and barcode trimming. The ONT reads were filtered by quality mapping to lllumina
reads using Filtlong version 0.2.1 (https://github.com/rrwick/Filtlong) with a minimum
length of 1,500 bp and quality threshold set to 95% and mapping on lllumina reads
where applicable. The quality of both long and short reads was assessed with FastQC
version 0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and NanoStat
(65). Complete chromosome and partial plasmid sequences were obtained using either
a hybrid assembly with Unicycler version 0.4.9 (66) or a long-read-only assembly with
Trycycler version 0.5.3 (67). The resulting contigs were further polished with Medaka
version 1.6.1 (https://github.com/nanoporetech/medaka) and Polypolish version 0.5.0
(68). Sequences were manipulated and inspected in the cross-platform bioinformatics
software Ugene version 38.1 (69). The genomes were annotated using the NCBI
Prokaryotic Genome Annotation Pipeline (70). Nucleotide and protein multiple sequence
aligment was performed with Clustal Omega (71). The multiple sequence alignment
was visualized using EasyFig version 2.2.5 (72) and IslandCompare version 1.0 (73).
Variable genetic content was identified with PhiSpy version 3.4 (74), MobilomeFinder
(75), PlasmidFinder (76), IslandViewer 4 (77), ISFinder (78), and SCCmecFinder (79). The
CRISPR/Cas system was characterized by CRISPRCasTyper (80). Virulence and resistance
genes were predicted using Abricate (https://github.com/tseemann/abricate) with the
CARD (81), Resfinder (82), and VFDB (83) databases.
Phylogenetic and pangenomic analyses
The partial 16S rRNA gene was sequenced by Sanger sequencing in the Eurofins MWG
Operon sequencing facility (Ebersberg, Germany) with previously described primers (84).
Whole-genome sequences of related staphylococcal species were obtained from the
NCBI database (Table S1).The multilocus sequence data of six housekeeping genes (rpoB,
groEL, dnaJ, tufA, sodA, and gap) that are commonly used in phylogenetic studies of the
Staphylococcaceae were extracted from whole-genome sequence assemblies as follows
(gene coordinates of S. aureus): 1420..1974 for rpoB, 270..826 for hsp60, 23..911 for dnaJ,
49..929 for gap, 383..1032 for tufA, and 50..480 for the sodA gene. The phylogenetic
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analyses were performed with the software MEGA version 11 (85). The UBCG collection
of 92 conservative genes (86), the average nucleotide identity (ANI) (87), and digital
DNA-DNA hybridization (dDDH) values by the d4 formula using the web-based genometo-genome
distance calculator (GGDC) version 3.0 (88) were used for calculations of
overall genome relatedness indices. The pangenome was calculated with the OrthoVenn2
pipeline with proteins clustered at the default threshold (89).
DNA fingerprinting
For genotypic characterization of the investigated bacterial group, fingerprinting by
repetitive sequence-based PCR (rep-PCR) with the primer (GTG)s (90) and automated
ribotyping with the restriction enzyme FcoRI were performed. The isolation of DNA
for rep-PCR fingerprinting, PCR conditions, and fingerprint analysis were performed as
previously described (91). Automated ribotyping was performed using the RiboPrinter
microbial characterization system (DuPont Qualicon) according to the manufacturer's
instructions. Numerical analysis of rep-PCR fingerprints and FcoRI ribotype patterns
was performed using BioNumerics version 7.6 (Applied Maths, Belgium). The ribotype
patterns were imported into the BioNumerics software using the load samples import
script provided by the manufacturer.
PCR analysis of mobilizable genomic island encoding Ccr recombinases
To determine whether a genomic island is mobilizable, we designed primers spanning
the excision site and primers targeting the key genes present in the genomic island
(Table S2). The PCR reaction was conducted using Quick-Load 2x master mix with
standard buffer (New England Biolabs, Ipswich, MA, USA) and a 200 nM concentration
of each primer. The genomic island product was further analyzed by Sanger sequencing
(Eurofins Genomics, Germany).
Transmission electron microscopy
A 200-mesh carbon/formvar-coated grid was placed on a drop of suspension of bacteria
in water for 20 min. Bacterial cells on the grid were negatively stained with 2% ammonium
molybdate and treated with UV light. A Morgagni 268D Philips (ThermoFisher
Scientific, The Netherlands) transmission electron microscope was used to visualize
bacterial cells.
MALDI-TOF MS
Protein fingerprinting by means of MALDI-TOF MS using an Ultraflextreme instrument
(Bruker Daltonics, Germany) was conducted after a standard extraction protocol (92).
MALDI-TOF mass spectra were obtained using an UltrafleXtreme instrument (Bruker
Daltonics) operated in linear positive mode using the software FlexControl version 3.4.
Signals present in at least seven out of nine independent mass spectra acquired per
sample were taken into account. Mass spectra were processed using FlexAnalysis version
3.4 (Bruker Daltonics) and BioTyper version 3.1 software (Bruker Daltonics) supplemented
with database version 10.0.0.0 (9,607 entries).
Chemotaxonomic characterization
Respiratory quinones were extracted and analyzed as previously described (93). Identity
was confirmed by mass spectrometry, as described by Schumann et al. (94). Analysis
of the cellular fatty acid profile was performed using a Microbial Identification System
(MIDI, Newark, DE) according to the Standard Protocol of the Sherlock Microbial
Identification System software, version 6.1 (95). The fatty acids were identified using gas
chromatography-mass spectrometry (GC-MS) according to the study of Vieira et al. (93).
Isolation and structural analysis of the peptidoglycan was performed according to
published protocols with some modifications. Briefly, the amino acid composition of the
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total hydrolysate (4 N HCl, 100°C for 16 h) of the peptidoglycan was analyzed by GC/MS
(protocol 10 by Schumann [96]). The partial hydrolysate (4 N HCl, 100°C, 0.75 h) of the
peptidoglycan was analyzed by high-resolution liquid chromatography-mass spectrometry
(LC-MS) as described previously (94, 96). Enantiomeric analysis was performed by
liquid chromatography as described by reference 97.
ACKNOWLEDGMENTS
Daniel Krsek (National Reference Laboratory for Diagnostic Electron Microscopy of
Infectious Agents, National Institute of Public Health, Prague, Czech Republic) is
gratefully acknowledged for transmission electron microscopy, Anika Wasner, Anja
Frühling, and Gesa Martens for technical assistance; Peter Schumann for helpful
discussions (all Leibniz Institute DSMZ); and Tereza Gelbicovä (Department of Public
Health, Faculty of Medicine, Masaryk University) for valuable help.
This work was supported by the project National Institute of Virology and Bacteriology
(Programme EXCELES, ID Project No. LX22NP05103)—Funded by the European
Union-Next Generation EU to R. P. CIISB, Instruct-CZ Centre of Instruct-ERIC EU
consortium, funded by MEYS CR infrastructure project LM2023042 and European
Regional Development Fund-Project "UP CIISB" (No. CZ.02.1.01/0.0/0.0/18 046/0015974)
is gratefully acknowledged for the financial support of the measurements at the CEITEC
Proteomics Core Facility. Computational resources were supplied by the project e-INFRA
CZ project (ID:90140), supported by the Ministry of Education, Youth and Sports of
the Czech Republic. T. B. gratefully acknowledges funding from the Ministry of Health
of the Czech Republic (NU21J-05-00035). P. P. was supported by a conceptual development
of research organization funded by the Ministry of Health of the Czech Republic
(NIPH, 75010330). The Grant Agency of Masaryk University (MUNI/A/1287/2022) to J. D.
supported the research activities of students. This study was partly funded by the Czech
Collection of Microorganisms.
AUTHOR AFFILIATIONS
'Department of Experimental Biology, Division of Genetics and Molecular Biology,
Faculty of Science, Masaryk University, Brno, Czech Republic
department of Experimental Biology, Czech Collection of Microorganisms, Faculty of
Science, Masaryk University, Brno, Czech Republic
reference Laboratory for Staphylococci, National Institute of Public Health, Praha, Czech
Republic
4
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany
5
Central European Institute of Technology, Masaryk University, Brno, Czech Republic
AUTHOR ORCIDs
Adéla Finstrlová " http://orcid.org/0000-0003-2343-4357
Ivo Sedláček " http://orcid.org/0000-0003-1717-187X
Pavel Švec ©http://orcid.org/0000-0002-1051-5177
Ivana Mašlaňová " http://orcid.org/0000-0002-2597-2848
Meina Neumann-Schaal " http://orcid.org/0000-0002-1641-019X
Tibor Botka " http://orcid.org/0000-0003-2708-3718
Jiří Doškář " http://orcid.org/0000-0001-5216-9586
Roman Pantůček " http://orcid.org/0000-0002-3950-675X
September/October 2023 Volume 11 Issue 5 10.1128/spectrum.01342-23 17 o
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FUNDING
Funder Grant(s) Author(s)
Ministerstvo Školství,
Mládeže a Tělovýchovy
(MŠMT)
LX22NPO5103, LM2023042,
CZ.02.1.01 /0.0/0.0/18_046/0015974, e-l N FRA
CZID:90140
Roman Pantůček
Jiří Doškář
Tibor Botka
Ondrej Šedo
Ivana Mašlaňová
Adéla Finstrlová
Vojtěch Kovařovic
Ministerstvo
Zdravotnictví České
Republiky (MZCR)
NU21J-05-00035, NI 75010330 Petr Petráš
Tibor Botka
Masarykova Univerzita
(MU)
MUNI/A/1287/2022 Roman Pantůček
Jiří Doškář
Adéla Finstrlová
Vojtěch Kovařovic
AUTHOR CONTRIBUTIONS
Vojtěch Kovařovic, Conceptualization, Data curation, Formal analysis, Investigation,
Methodology, Software, Validation, Visualization, Writing - original draft, Writing review
and editing | Adéla Finstrlová, Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing original
draft, Writing - review and editing | Ivo Sedláček, Conceptualization, Data
curation, Formal analysis, Investigation, Methodology, Resources, Supervision, Validation,
Visualization, Writing - original draft, Writing - review and editing | Petr Petráš,
Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation,
Resources, Validation, Writing - original draft, Writing - review and editing | Pavel
Švec, Conceptualization, Data curation, Formal analysis, Investigation, Methodology,
Validation, Visualization, Writing - review and editing | Ivana Mašlaňová, Data curation,
Formal analysis, Investigation, Software, Supervision, Validation, Writing - review
and editing | Meina Neumann-Schaal, Conceptualization, Data curation, Investigation,
Methodology, Resources, Validation, Visualization, Writing - review and editing | Ondřej
Šedo, Conceptualization, Data curation, Funding acquisition, Investigation, Methodology,
Validation, Visualization, Writing - review and editing | Tibor Botka, Funding acquisition,
Investigation, Methodology, Software, Validation, Writing - review and editing | Eva
Staňková, Data curation, Investigation, Validation, Writing - review and editing | Jiří
Doškář, Conceptualization, Funding acquisition, Supervision, Writing - original draft,
Writing - review and editing | Roman Pantůček, Conceptualization, Data curation, Formal
analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision,
Visualization, Writing - original draft, Writing - review and editing
DATA AVAILABILITY
The complete genome sequences of new isolates NRL/St 16/872T
, NRL/St 19/737,
NRL/St 18/288, NRL/St 21/187, and NRL/St 22/194 have been deposited in GenBank/ENA/
DDBJ database under accession numbers CP119327-CP119331, JALGRI000000000,
JALGRH000000000, JALGRG000000000, and CP116597-CP116599, respectively. The
associated BioProject number is PRJNA779217. The accession number for the 16S rRNA
gene of the type strain NRL/St 16/872T
is OQ401401.
ADDITIONAL FILES
The following material is available online.
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Supplemental Material
Supplemental tables and figures (Spectrum01342-23-S0001.pdf). Tables S1 to S5,
Table S7, Figures S1 to S3.
Table S6 (Spectrum01342-23-S0002.xlsx). Pairwise identity matrices of nucleotide and
amino acid sequences of cassette recombinases.
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