foods

Article
Whole-Genome Analysis of Staphylococcus aureus Isolates from
Ready-to-Eat Food in Russia
Yulia Mikhaylova † , Andrey Shelenkov *,† , Aleksey Chernyshkov, Marina Tyumentseva, Stepan Saenko,
Anna Egorova , Igor Manzeniuk and Vasiliy Akimkin

Central Research Institute of Epidemiology, Novogireevskaya str., 3a, 111123 Moscow, Russia
* Correspondence: fallandar@gmail.com
† These authors contributed equally to this work.

Abstract: This study provides a thorough investigation of a diverse set of antimicrobial resistant
(AMR) Staphylococcus aureus isolates collected from a broad range of ready-to-eat (RTE) food in
various geographic regions of Russia ranging from Pskov to Kamchatka. Thirty-five isolates were
characterized using the whole genome sequencing (WGS) analysis in terms of clonal structure,
the presence of resistance and virulence determinants, as well as plasmid replicon sequences and
CRISPR/Cas systems. To the best of our knowledge, this is the first WGS-based surveillance of
Russian RTE food-associated S. aureus isolates. The isolates belonged to fifteen different multilocus
sequence typing (MLST)-based types with a predominant being the ones of clonal complex (CC)
22. The isolates studied can pose a threat to public health since about 40% of the isolates carried
at least one enterotoxin gene, and 70% of methicillin-resistant (MRSA) isolates carried a tsst1 gene
encoding a toxin that may cause severe acute disease. In addition, plasmid analysis revealed some
Citation: Mikhaylova, Y.; Shelenkov,
important characteristics, e.g., Rep5 and Rep20 plasmid replicons were a “signature” of MRSA CC22.
A.; Chernyshkov, A.; Tyumentseva,
By analyzing the isolates belonging to the same/single strain based on cgMLST analysis, we were
M.; Saenko, S.; Egorova, A.;
able to identify the differences in their accessory genomes marking their dynamics and plasticity.
Manzeniuk, I.; Akimkin, V.
Whole-Genome Analysis of
This data is very important since S. aureus isolates studied and RTE food, in general, represent an
Staphylococcus aureus Isolates from important route of transmission and dissemination of multiple pathogenic determinants. We believe
Ready-to-Eat Food in Russia. Foods that the results obtained will facilitate performing epidemiological surveillance and developing
2022, 11, 2574. https://doi.org/ protection measures against this important pathogen in community settings.
10.3390/foods11172574
Keywords: foodborne pathogen; antibiotic resistance; virulence determinants; multidrug resistance;
Academic Editors: Sarah Azinheiro,
pathogenic potential; CRISPR/Cas system; genomic epidemiology
Alejandro Garrido-Maetsu and
Eugenio Parente

Received: 30 June 2022

Accepted: 22 August 2022 1. Introduction
Published: 25 August 2022
The urgency of the food safety issue as one of the main risk factors for public health
Publisher’s Note: MDPI stays neutral and gene pool maintenance increases every year [1]. The vast majority of foodborne
with regard to jurisdictional claims in outbreaks caused by antimicrobial-resistant pathogens are the result of the consumption
published maps and institutional affil- of contaminated foods of either animal origin or multi-ingredient foods [2]. Foodborne
iations. illnesses are considered a major source of morbidity and mortality, mainly in such suscep-
tible groups as infants, the elderly, and immunocompromised people. In healthy adults,
foodborne infections and intoxications are usually mild and self-limiting [3].
Staphylococcus aureus is a versatile opportunistic pathogen, which can survive in
Copyright: © 2022 by the authors.
diverse environments, grow in many types of foods, and cause food poisoning [2]. The
Licensee MDPI, Basel, Switzerland.
wide application of molecular genotyping techniques revealed that S. aureus is a clonal
This article is an open access article
distributed under the terms and
bacterium with a limited number of genetic lineages, or clonal complexes (CCs), being
conditions of the Creative Commons
predominant in the staphylococcal population worldwide [4]. This species represents
Attribution (CC BY) license (https://
a serious threat to human health and constitute one of the main challenges to the food
creativecommons.org/licenses/by/ industry [5]. In particular, staphylococcal intoxications are ranked the third in the world
4.0/). among all foodborne infections in terms of occurrence frequency [6,7]. This bacterium

Foods 2022, 11, 2574. https://doi.org/10.3390/foods11172574 https://www.mdpi.com/journal/foods

Foods 2022, 11, 2574 2 of 15

has an extraordinary capacity for acquiring new antimicrobial resistance characteristics.

Thus, the problem of food intoxication became more complicated due to the presence
of S. aureus isolates harboring a broad spectrum of antibiotic resistance and virulence
determinants. Moreover, in such cases, ready-to-eat (RTE) food, which does not require
thermal processing before consumption, represents a vehicle for spreading antibiotic-
resistant microorganisms [8].
The presence of antibiotic-resistant staphylococci in RTE food usually is not investi-
gated, especially by whole genome analysis, and only the data from a small number of
studies are currently available. In most studies, the detection of molecular characteristics
of antimicrobial resistance, virulence, and genotypes, as well as typing of the food strains,
were performed by PFGE and PCR analyses [8–12]. Detection of methicillin-resistant S.
aureus (MRSA) strains in Chinese food products has been reported for a variety of foods
such as raw meat, rice flour, vegetable salads, sandwiches, meat products, and eggs [13].
This study showed the geographical variation of S. aureus isolates from sushi sold in Beijing
and Copenhagen. In addition, a large-scale microarray analysis describing the genome
composition of 267 S. aureus isolates sampled from 244 RTE-foods in Switzerland was
performed, which showed the pathogenic potential of the isolates studied [14]. The results
revealed that one-third of the tested isolates had at least one major enterotoxin gene (sea-see),
and the toxic shock syndrome gene (tsst), while various genes associated with antimicrobial
resistance, including genes involved in resistance to beta-lactams (blaZ), methicillin (mecA),
and vancomycin (vanB), were also detected. These S. aureus strains mostly belonged to
clonal complexes commonly detected in humans colonized or infected with S. aureus (CC8,
15, 30 and 45).
Whole genome analysis was applied in a previous study in China examining the
prevalence and characterization of antimicrobial-resistant S. aureus in sushi and pork from
a large number of outlets in Beijing [15]. Studying a large heterogeneous data set of
S. aureus isolates collected from food and from individuals from many provinces of China
over a nine-year period showed that Chinese and European strains of MRSA have evolved
differently [16].
There is a lack of information available on the epidemiology and whole genome char-
acterization of S. aureus sampled from RTE food in Russia. In this paper, we described for
the first time, to the best of our knowledge, the variation in lineages, antimicrobial resis-
tance (AMR) and virulence genes, plasmid sequences, and CRISPR/Cas system carriage
for S. aureus isolates from RTE food collected in various geographic regions of Russia using
whole genome sequencing (WGS) analysis. The main goals of investigations were to obtain
epidemiologically related data, such as to perform isolate typing using various molecular
classification schemes, as well as to reveal the presence of antibiotic resistance genes, viru-
lence factors, plasmids, and CRISPR/Cas systems in the genomes of the isolates studied.

2. Materials and Methods

2.1. Determination of Antibiotic Susceptibility
All isolates were identified down to a species level by time-of-flight mass spectrome-
try (MALDI-TOF MS) using the VITEK MS system (bioMerieux, Marcy-l’Étoile, France).
The susceptibility was determined by the disc diffusion method with the Mueller–Hinton
medium (bioMerieux, Marcy-l’Étoile, France) and disks with antibiotics (BioRad, Mar-
neslaCoquette, France), as well as by the boundary concentration method on VITEK2
Compact 30 analyzer (bioMerieux, Marcy-l’Étoile, France). The isolates were tested for
susceptibility/resistance to the following groups of antimicrobial drugs: aminoglycosides,
beta-lactams, fluoroquinolones, glycopeptides, lincosamides, penicillins, and tetracyclines.
Specific antibiotics are presented in Figure 1, including additional antimicrobial compounds,
such as fosfomycin, fusidic acid, trimethoprim-sulfamethoxazole, and linezolid. The panel
of antimicrobial compounds included for testing in this study was chosen according to
the EUCAST and CLSI recommendations. To interpret the results obtained, the EUCAST
Foods 2022, 11, 2574 3 of 15

clinical breakpoints, version 11.0 was used https://www.eucast.org/clinical_breakpoints/

(accessed on 20 April 2022) where available.

Figure 1. Typing, geography, and phylogenetic analysis of S. aureus isolates from ready-to-eat (RTE)
foodFigure
studied.
1. Typing, geography, and phylogenetic analysis of S. aureus isolates from ready-to-eat (RTE)
food studied.
2.2. Sample Collection, DNA Isolation, Sequencing, and Genome Assembly
Thirty-five
2.2. isolates DNA
Sample Collection, were Isolation,
obtainedSequencing,
from different types ofAssembly
and Genome ready-to-eat food during the
period of 2019–2020isolates
Thirty-five (Table were
S1). Sample
obtainedcollection was carried
from different outready-to-eat
types of in cafes and restaurants
food during
by eight Russian Federal Centers of Hygiene and Epidemiology located in Moscow,
the period of 2019–2020 (Table S1). Sample collection was carried out in cafes and restau- Saint-
Petersburg, Nizhny Novgorod, Ekaterinburg, Rostov-on-Don, Stavropol, Novosibirsk
rants by eight Russian Federal Centers of Hygiene and Epidemiology located in Moscow,
andSaint-Petersburg,
Khabarovsk forNizhny
the purposes of food
Novgorod, poisoningRostov-on-Don,
Ekaterinburg, monitoring and epidemiological
Stavropol, Novosi-
surveillance. The majority of the samples were isolated from the meat and chicken
birsk and Khabarovsk for the purposes of food poisoning monitoring and epidemiological products
(11 isolates), ten isolates were collected from salads, and six samples were isolated from fish
products including rolls, while the remaining eight samples were obtained from potatoes,
porridge, soup, yogurt, omelets, and bread (Table S1). All the isolates mentioned above
were selected for WGS based on their resistance to two or more antimicrobial compounds
from different groups.
Genomic DNA was isolated with DNeasy Blood and Tissue kit (Qiagen, Hilden,
Germany) and used for paired-end library preparation with Nextera™ DNA Sample
Prep Kit(Illumina® , San Diego, CA, USA) and WGS of all isolates on Illumina® Hiseq
platform (Illumina® , San Diego, CA, USA). Assemblies were obtained using SPAdes version
3.11 and 3.12 [17]. Genome sequences were uploaded to Genbank under the project
number PRJNA823522.

2.3. Data Processing

Assembled genomes were processed using a custom software pipeline including a set
of scripts for seamless integration of various available software tools [18,19]. Resfinder 4.0
database was used for antimicrobial gene identification (https://cge.cbs.dtu.dk/services/
ResFinder/, accessed on 20 April 2022) and VFDB (http://www.mgc.ac.cn/VFs/main.htm,
accessed on 20 April 2022) for virulence factor determination. CRISPRCasFinder [20] was
used to identify the presence of CRISPR/Cas systems and spacers in the genomes studied.
The methods of multilocus sequencing typing (MLST) and Spa-typing, which are
commonly used to study the global epidemiology and population structure of S. aureus,
were applied to isolate typing. Agr (Accessory gene regulator) typing was also performed
for our S. aureus isolates. This method involves the determination of variable regions from
the cluster consisting of five genes (agrABCD and d-hemolysin). Four groups of agr (I–IV)
are defined based on the types of variable regions [21].
Foods 2022, 11, 2574 4 of 15

The analysis of cgMLST schemes was performed using MentaList software (https:
//github.com/WGS-TB/MentaLiST, version 0.2.4), and the tree was built using PHYLOViz
online (http://online.phyloviz.net, accessed on 20 April 2022).

3. Results
3.1. Isolate Typing
The results of the isolate typing using MLST and Spa-types are presented in Table
S1 and in Figure 1. The isolates belonged to fifteen different MLST-based sequence types,
while a major part of them belonged to seven clonal complexes (CCs). The dominant
lineage in our collection was CC22 comprising ten isolates from five geographic regions
of the European part of Russia. The CC22 isolates from the Pskov region differed by ST
(6110), and the Dagestan isolates of the same lineage had a distinct SpaII type (t2571). The
majority of CC22 isolates were characterized by the same SpaII (t223), and all members of
this clonal group in our collection belonged to AgrI type.
The isolates of CC5 were collected from distant regions. The samples from the Tumen
region and Yamal-Nenets autonomous district were characterized by the same typing
pattern, while the isolate from the Sverdlovsk region differed from the Karelian sample by
ST, and the Bashkir isolate had a distinct SpaII type.
Four isolates collected in Moscow, Penza, Vladimir, and Kemerovo regions belonged
to ST7. The latter region is located quite far from the first three but was characterized by the
same typing patterns as the isolates from Penza and Vladimir. The sample from Moscow
differed by SpaII type.
The lineage CC45 was presented by three isolates collected from the Far Eastern part
of Russia (Altai, Khabarovsk, and Kamchatka) which differed from each other by SpaII
types. The clonal groups CC30 and CC398 included three samples, which also had different
SpaII types. It is worth noting that two isolates of these clonal complexes were collected
from the Vologda region. Two isolates of CC97 obtained from the Moscow and Sverdlovsk
regions had the same typing patterns.
The results of Spa-typing of four isolates (Crie-F280, 372, 374, and 380) collected from
different regions and belonging to different STs (5814, 580, 398, and 88, respectively) were
particularly interesting. They were characterized by unique four respective sequences of
short Spa-gene repeats, which were not presented in spaserver.ridom.de, indicating novel
Spa-types (Table S1).
Typing analysis of the samples studied did not show a correlation between the typing
patterns and the geographical origin of the isolates (Figure 1). Antimicrobial-resistant
S. aureus isolates of RTE food origin of some clonal lineages (CC5, CC97, and ST7) were
collected from very distant geographic regions of Russia, while CC30 and CC22 were
observed in the European part only (Table S1 and Figure 1).
More detailed typing that allows better isolate discrimination can be achieved by
cgMLST profiling, which involves a comparison of 1861 genes common to the most existing
isolates (https://www.cgmlst.org/ncs/schema/schema/141106/, accessed on 20 April
2022). The minimum spanning tree for the isolates is presented in Figure S1, and the
complete profiles are listed in Table S2. The threshold for the number of allele differences
sufficient for assigning a particular isolate to a single strain can be determined ad hoc,
but usually, the value of 15 is used for S. aureus [22]. Four ST6110 isolates from the Pskov
region obtained from rolls differed from each other by the only allele of the cgMLST profile
and only in Crie-F524/Crie-F527 pair. Three ST22 and two ST398 isolates from Dagestan
and Khabarovsk regions, respectively, did not have any differences in cgMLST profiles,
while two Karelian ST30 isolates (Crie-F267 and Crie-F343) differed by three cgMLST
alleles. The latter two isolates were considered to belong to a single strain according to the
threshold defined for S. aureus [22]. It was interesting that the samples from Dagestan and
the Republic of Karelia were collected from different types of RTE food (bread, soup, salad,
and trout caviar). Such a variety of sources may indicate the contamination with S. aureus
of kitchen facilities located in the cafés/restaurants producing this RTE food.
Foods 2022, 11, 2574 5 of 15

3.2. Antimicrobial Resistance Phenotypes and Genotypes

The results of antibiotic susceptibility testing of the isolates included in the study
and their antimicrobial resistance gene profiles are presented in Figure 2. All the isolates
except Crie-F279 were susceptible to benzylpenicillin. According to phenotypic analysis,
eleven S. aureus isolates belonged to MRSA. Since some of them belonged to the same
clones (highlighted by color in the table), the actual number of MRSA strains was six.
One of them (Crie-F374, ST398) was multidrug-resistant (MDR) as it was also resistant to
gentamicin, trimethoprim/sulfamethoxazole, clindamycin, and tetracycline. This isolate
was collected in the Vologda region from minced pork cutlet produced by one of the largest
manufacturers of semi-finished meat products in Russia. ST398 isolates were also collected
earlier from pork meat in Denmark and China [15,23]. The other five MRSA strains in our
collection belonged to ST22.
Benzylpenicillin

aac(6')-aph(2'')
Ciprofloxacin

Clindamycin

Tetracycline
Gentamicin

cat(pC194)

cat(pC221)
Additional
Trime-sulf

Sample ID
Linezolid
Cefoxitin
Oxacillin

ant(6)-Ia

ant(9)-Ia
aadA24

erm(A)

erm(C)
erm(B)

erm(T)

lnu(A)

lnu(B)

tet(M)
tet(K)
tet(A)

tet(C)
mecA

tet(L)
aadD

dfrG

dfrK
blaZ

fexA
str

R S - S S S R S S S Crie-F267 - - - - + - + - - - - - - + - - - - - - - - - -
R S - S S S R S S S Crie-F343 - - - - + - + - - - - - - + - + - - - - + - -
R S - S S S S R S S Crie-F271 - - - - - - + - - - + - - - - - - - - - - - - +
R S - S S S R S S S Crie-F272 - - - - - - + - - + - - - - - + - - - - - - - -
R S - S R S S S S S Crie-F273 + - - - - - + - + - - - - - - - - - - - - + - -
R S - S S S S R S S Crie-F274 - - - - - - - - - - - - - - - - - - - - - - - -
R S - S S S S S S S Crie-F278 - - - - - - + - - - - - - - - - - - - - - - - -
S S - S S S R S R S Crie-F279 - - - - - - + - - - - - - - - + - - - - - - - -
R S - R S S S S S S Crie-F280 - - - - - - + - + - - - - - - - - - - - - + - -
R S - S S S S R S S Crie-F346 - - - - - - + - - - + - - - - + - - - - - + - +
R S - S S S S R S S Crie-F347 - - - - - - + - + - - - - - - - - - - - - + - -
R S - S S S R S S S Crie-F371 - - - - - - + - - - - - - - - + - - - - - - - -
R S - S S S S S S S Crie-F372 - - - - - - + - - - - - - - - - - - - - - - - -
R S - S S S S R S S Crie-F373 - - - - - - + - - - - - - - - - - - - - - + - -
R S - S S S R S S S Crie-F375 - - - - - - + - - - - - - - - + - - - - - - - -
R S - R S S S S S S Crie-F378 - + - - - - + - + - - - - - - - - - - - - - - -
R S - S S S S R S S Crie-F380 - - - - - - + - - - - - - - - + - - - - - + - -
R S - S S S S R S S Crie-F381 - - - - - - + - + - - - - - - + - - - - - - - -
R S - S S S R S S S Crie-F384 - - - - - - + - - - - - - - - - + - - - - - - -
R S - S S S R S S S Crie-F385 - - - - - - + - - - - - - - - - + - - - - - - -
R S S S S R S S S S Crie-F528 - - - - - - + - - - - + - - - - - - - - - - - -
R S R S S S S S S S Crie-F595 - - - - - - + - - - - - - - - - - - - - - - - -
R S R S S S S S S S Crie-F596 - - - - - - + - - - - - - - - - - - - - - - - -
R S R S S S S S S S Crie-F597 - - - - - - + - - - - - - - - - - - - - - - - -
R R - S S S S S S S Crie-F275 - - - - - - + + - - - - - - - - - + - - - - - -
R R - S S S S S S S Crie-F276 - - - - - - + + - - - - - - - - - + - - - - - -
R R - S S S S S S S Crie-F277 - - - - - - + + - - - - - - - - - + - - - - - -
R R - S R R R R S S Crie-F374 - - + + - + + + - - - - + - + - - - + - - + + +
R R - S S S S S S S Crie-F377 - - - - - - + + + - - - - - - + - + - - - - - -
R R - S S S S S S S Crie-F379 - - - - - - + + + - - - - - - - - + - - - - - -
R R - S S S S S S S Crie-F382 - - - - - - + + - - - - - - - - - + - - - - - -
R R S S S S S S S S Crie-F524 - - - - - - + + - - - - - - - - - - - - - - - -
R R S S S S S S S S Crie-F525 - - - - - - + + - - - - - - - - - - - - - - - -
R R S S S S S S S S Crie-F526 - - - - - - + + - - - - - - - - - - - - - - - -
R R S S S S S S S S Crie-F527 - - - - - - + + - - - - - - - - - - - - - - - -

Figure 2. Phenotypic and genomic antimicrobial resistance profiles of S. aureus isolates from ready-to-
eat food. ‘Trime-sulf’ represents trimethoprim-sulfamethoxazole. ‘Additional’ antibiotics, to which
all isolates were susceptible, include fosfomycin, fusidic acid, and tigecycline. Antibiotics belonging
to the same group are colored with the same color. ‘+’ means that the gene is present in the isolate,
‘-‘ – that the gene was not found. ‘R’ means that a given isolate was resistant to particular antibiotic,
‘S’ – that an isolate was susceptible to this drug. The isolates found to belong to the same strain are
highlighted with color.

All remaining methicillin-susceptible S. aureus (MSSA) isolates differed from each

other by having additional resistance to at least one antibiotic—clindamycin or tetracycline.
Separate isolates were characterized by additional resistance to linezolid, ciprofloxacin, or
cefoxitin (Figure 2). It is worth noting that the latter antimicrobial drug was added to the
testing antibiotic panel in 2020.
Foods 2022, 11, 2574 6 of 15

In silico searching for antimicrobial resistance determinants indicated that MRSA

isolates from our collection were characterized by a very limited set of AMR genes (blaZ
and mecA), except for the only MDR isolate Crie-F374 possessing 11 AMR genes providing
resistance to aminoglycosides, beta-lactams, trimethoprim, macrolides, lincosamide, and
tetracycline. AMR gene and phenotypic profiles for this isolate were in good correspon-
dence. MRSA isolates belonging to the Dagestan strain (Crie-F275-277) and a sample
collected from the Vologda region (Crie-F382) carried the same AMR genes including an
additional inuA gene determining resistance to lincosamide. Notably, only MRSA isolates
possessed inu genes in our S. aureus collection. The isolates Crie-F377 and Crie-F379 were
also positive for cat gene (resistance to phenicols) and the former isolate additionally had
an ermC gene (resistance to macrolides). All MRSA samples under investigation harbored
staphylococcal cassette chromosome mec (SCCmec) IVc.
As for the MSSA RTE food isolates, five of them (CrieF-278, 372, 595, 596, and 597)
had the only AMR gene—blaZ. The maximum number of AMR genes (5) representing
the whole spectrum of antimicrobial compounds mentioned above were carried by two
isolates Crie-F343 and Crie-F346, respectively, which were collected from distant geographic
regions. Their genomic AMR profiles differed from each other by the set of erm genes
(erm(A) and erm(C) for CrieF-343 vs. erm(C) only for CrieF-346), as well as by tet cluster
genes and ant(9)-Ia revealed in CrieF-343 only. Moreover, the isolate Crie-F343 differed
significantly from its relative sample (Crie-F267) by carrying additional erm(C) and tet(K)
genes. These findings may indirectly indicate the plasmid localization of additional AMR
genes in the Crie-F343 isolates, which hypothesis was additionally confirmed by BLASTing
the contigs containing these genes to the ‘nt’ database (https://www.ncbi.nlm.nih.gov/
nucleotide/, accessed on 20 April 2022). Another close isolate Crie-F347 was characterized
by 3 AMR genes (blaZ, cat(pC194) and tet(K)). Separate MSSA isolates differed by AMR
genes determining resistance to aminoglycosides (aaC, aaD, and ant(9)-Ia), phenicols (cat
and fexA), trimethoprim (dfrG), macrolides (genes of erm cluster), and tetracycline (genes
of tet cluster). Interestingly, the ST45 isolate CrieF-274 did not carry any AMR genes. At
the same time, only CrieF-528 (MSSA) and CrieF-374 (MRSA) carried the trimethoprim
resistance genes (dfrG and dfrK, respectively), which corresponded with their phenotypes.

3.3. Virulence Gene Profiles

The set of virulence factors in S. aureus strains is extensive and includes both structural
components of the cell and secreted products that play a role in the pathogenesis of
infectious diseases [24]. The peculiarity of staphylococci is that a single virulence factor
can perform several functions in pathogenesis, as well as multiple virulence factors can
perform the same function [25].
We identified the virulence factors in the RTE food S. aureus isolates investigated
by bioinformatics analysis of WGS data. The complete list of detected virulence genes is
shown in Table S3. An extensive spectrum of identified 116 virulence genes included 31
clusters determining genes encoding surface cell-bound proteins of the MSCRAMM family
recognizing adhesive matrix molecules (clfA, clfB, fnbA, emp, efb, eap/map, cna, sdrC-E), the
genes whose products are the part of immune evasion mechanisms (ads, coa, spa, sbi, VWbp),
the genes encoding different toxins and extracellular enzymes. The genes involved in the
formation of the polysaccharide matrix of biofilms (icaA-D, icaR), capsule biosynthesis
(cap8), as well as regulatory proteins, were also identified.
The most common virulence genes found in all isolates under study were adsA, aur,
cap8A-G, cap8L-P, esaAB, essAB, esxA, icaADR, isd, lip and sspBC. In order to elucidate the
presence of distinctive virulence gene patterns, the virulence factors will be discussed
for three groups of the isolates: MRSA isolates, isolates with the maximum number of
virulence factors, and the remaining MSSA isolates.
Virulence gene patterns of MRSA isolates were very similar (except for several isolates)
and quite poor, namely, the isolates carried from 13 to 29 determinants in addition to
common virulence genes. No virulence determinants specific to MRSA isolates were found
Foods 2022, 11, 2574 7 of 15

in our S. aureus collection. Expectedly, four isolates from the Pskov region belonging
to the same ST22 S. aureus strain were characterized by identical profiles of virulence
determinants. However, one of the three Dagestan isolates of another ST22 MRSA strain
(Crie-F277) differed from its two relatives (Crie-F275 and 276) by the presence of clfB and
prgl genes. It was the only sample from our RTE food MRSA collection possessing the
latter virulence determinant. These three Dagestan isolates differed from other ST22 MRSA
samples due to the absence of spa and tsst1 genes. The isolate Crie-F379 of the same
ST draws attention to the presence of the esxB gene and seven additional genes of the
esaG cluster. Both genes encode extracellular proteins important for the establishment of
infection in the host. This isolate was characterized by the maximal number of virulence
determinants in our MRSA isolates’ subset. MDR MRSA ST398 isolate Crie-F374 collected
from the Vologda region differed from other MRSA RTE food samples in our collection
by the absence of chp, sak, and tsst1 genes and by the presence of esaG1, esaG5, and esaG6
(together with Crie-F379) virulence determinants, which represent antitoxin proteins of
type VII secretion system.
In silico search for virulence determinants allowed revealing four isolates (Crie-F267,
343, 346, and 347) in our S. aureus sample set possessing a slightly broader spectrum of the
genes analyzed. The first two ST30 isolates belonged to the same strain and possessed 79
and 80 virulence determinants, respectively. The isolate Crie-F343 differed from its relative
by the presence of the cna gene.
The isolate Crie-F346 (ST5) was characterized by the greatest number of virulence
genes among all samples (86), and it simultaneously carried the genes sea, sec, and selk
encoding staphylococcal enterotoxins.
The remaining group of RTE food MSSA isolates included 20 samples and two of
them (Crie-F384, 385) belonging to the same strain carried a comparatively low number of
virulence determinants and differed from each other by the presence of the fnbB gene in the
Crie-F384 genome. Three isolates of ST5 (Crie-F271, 375 and 378) possessed very similar vir-
ulence gene profiles and were characterized by minor differences in the absence/presence
of single genes. For instance, Crie-F271 did not have chp and sea genes; Crie-F375 was lack
of hemolysine-encoding gene hly and sed gene encoding enterotoxin. Crie-F378 was the
only isolate from this group carrying the tsst1 gene.
Four isolates of ST7 (Crie-F279, 371, 381, and 595) had more differences in the absence
or presence of separate genes than those of ST5, namely, esaG5 and esaG7, fnbA, map, spa,
and ssa. The isolate Crie-F371 was the only carrying pefB gene, while all of the ST7 MSSA
isolates possessed lukD and sea genes encoding leuko- and enterotoxins, respectively.
From a food safety concept, S. aureus virulence factors encoding toxin production are
of special interest since they may be associated with staphylococcal food poisoning [26]. In
our S. aureus isolates of RTE food origin, all samples except Crie-F375 carried hly/hla gene
encoding exotoxin alpha-hemolysin, while all isolates had an hlb gene of beta-hemolysin.
Leukotoxin gene lukD was observed in 14 isolates of our collection regardless of
geographic origin and types of RTE food. The enterotoxin gene sea that is associated with
the severity of infections (sepsis and shock) [27] and promotes bacterial survival in vivo [28]
was detected in ten RTE food samples. Different pairs of isolates (Crie-F272, 346; Crie-F271,
378 and Crie-F272, 346) also carried other enterotoxin genes (sec, sed, and sel, respectively).
The gene tsst-1 encoding toxic shock syndrome toxin was observed in 10 isolates, seven
of which were ST22 MRSA, including the four isolates from the Pskov region belonging
to a single strain. The pathogenic potential of the isolates possessing the genes encoding
different types of toxins is notable. For example, apart from the genes of exotoxins, the
isolate Crie-F378 carried three enterotoxin genes sea, sed, and tsst-1. The ST5 isolate Crie-
F346 from Tumen regions possessed three of the enterotoxin genes mentioned above (sea,
sec, and sel).
Foods 2022, 11, 2574 8 of 15

3.4. Plasmid Sequences

The genes providing antibiotic resistance are usually located on mobile genetic ele-
ments (MGE) [29]. S. aureus contains many types of MGEs, including plasmids, transposons,
insertion sequences, bacteriophages, pathogenicity islands, and staphylococcal cassette
chromosomes. Plasmids are the major source of dissemination of resistance determinants
and virulence factors.
The classification of plasmids has been determined by incompatibility groups based
on the finding that two plasmids with the same replication (Rep) proteins cannot be stably
maintained in the same cell [30]. This method has been developed based on the sequence
of the Rep genes [31]. In 35 samples analyzed in the present study, the PlasmidFinder has
indicated 69 occurrences of 10 different plasmid replicon sequences (Figure 3).

repUS43
repUS5
Clonal

rep10

rep13
rep16

rep19
rep20
rep23
rep5

rep7
complex ID/Replicon
CC30 Crie-F267 + - - - + - - - - -
CC30 Crie-F343 + + - - + - - - - -
CC30 Crie-F373 + - - - + - - - - -
CC5 Crie-F271 - - - - - - + - - +
CC5 Crie-F278 - - - + - - + - - -
CC5 Crie-F375 + - - - + - - - - -
CC5 Crie-F346 - - + - + + + + - +
CC5 Crie-F378 - - - + - - + - - -
CC45 Crie-F272 + + + - + - - - - -
CC45 Crie-F274 - - - - - - - - - -
CC45 Crie-F596 - - - - + - - - + -
CC22 Crie-F275 - - - - - - - - - -
CC22 Crie-F276 - - - - - - - - - -
CC22 Crie-F277 - - - + - - - - - -
CC22 Crie-F524 + - - - - - + - - -
CC22 Crie-F525 + - - - - - + - - -
CC22 Crie-F526 + - - - - - + - - -
CC22 Crie-F527 + - - - - - + - - -
CC22 Crie-F377 + - + - - - + - - -
CC22 Crie-F379 + - + - + - + - - -
CC22 Crie-F382 + - - - - - + - - -
CC398 Crie-F374 - + - - - - - - - +
CC97 Crie-F347 - - - + - - + - - -
CC97 Crie-F597 - - - - - - + - - -
CC15 Crie-F528 + - - - + - - - - -
ST398 Crie-F384 - - - - - - - - - -
ST398 Crie-F385 - - + - - - - - - -
ST7 Crie-F279 + - - - + - - - - -
ST7 Crie-F371 + - + - - - - - - -
ST7 Crie-F381 + - + - + - - - - -
ST7 Crie-F595 + - - - + - - - - -
ST20 Crie-F273 - - - + + - - - - -
ST88 Crie-F380 + - - - - - + - - -
ST580 Crie-F372 - - - - - - - - + -
ST5814 Crie-F280 - + - - - - + - - -

Figure 3. Plasmid replicon sequences profiles of RTE food S. aureus isolates studied. ‘+’ means that
the given replicon is present in the isolate, ‘-‘ – that the replicon was not found. The isolates found to
belong to the same strain are highlighted with color.

Four isolates (Crie-F274, 275, 276, and Crie-F384) did not have any Rep-sequences. The
isolate Crie-F346 was characterized by the maximal number of Rep-sequences comprising
6 out of 10 types observed. The predominant plasmid replicon sequence found was Rep5
Foods 2022, 11, 2574 9 of 15

detected in 18 from 35 isolates under investigation. Four MRSA isolates from the Pskov
region (Crie-F524-527) belonging to the same strain had the same set of Rep-sequences
(Rep5 and Rep20) while two Karelian isolates (Crie-F267 and Crie-F343) presented another
epidemiological strain differed by the Rep composition. Apart from the common Rep5 and
Rep16, the isolate Crie-F343 carried an additional Rep7 plasmid replicon sequence. Two
Dagestan isolates, Crie-F275 and Crie-F276, did not have Rep sequences, while their relative
Crie-277 belonging to the same strain possessed Rep13. A similar situation was observed
in the case of two Khabarovsk isolates (Crie-F384 and 385) belonging to the same strain.
Crie-F385 carried Rep10 only, while Crie-F343 was lack of any plasmid replicon sequences.
Two isolates (Crie-F528 and 595) having different STs and collected from different regions
and different types of RTE food possessed the same set of Rep sequences (Rep5 and Rep16)
representing a major part of the replicons revealed in S. aureus isolates studied (18 for Rep5
and 13 for Rep16, respectively).

3.5. CRISPR/Cas Systems

Bioinformatics analysis of CRISPR in foodborne pathogens is crucial for assessing
their potential evolution in order to predict the outbreaks, which is very important for
food safety. At present, the investigations of the CRISPR/Cas systems of pathogenic
bacteria of foodborne origin are scarce. In fact, the CRISPR/Cas system in addition to the
immune defense function has also been found to be involved in the virulence regulation
and formation of biofilms in foodborne pathogens [32].
In our RTE food S. aureus collection, three isolates carrying CRISPR/Cas systems of IE
type were revealed. Interestingly, the CRISPR/Cas loci of two Karelian samples (Crie-F267
and Crie-F343) belonging to the same strain were significantly different. The Cas cassette of
the isolate Crie-F343 consisted of seven genes encoding Cas1 endonuclease, Cas2 integrase,
Cas5, Cas6, Cas7, Cse1, and Cse2 proteins. However, the CRISPR/Cas locus of Crie-F267
was deficient and comprised only Cas1 endonuclease and Cas2 integrase. The CRISPR/Cas
cassette composition of the third isolate Crie-F346 from the Tumen region was similar to
the one of Crie-F343.
Additionally, the genes encoding potential Cas proteins (Cas3_0_I) similar to the
CRISPR/Cas type I system were found in the genomes of five isolates (Crie-F271, 278,
375, 378, and 384). The similarity was determined using the CRISPRCasFinder tool [20].
The analysis of these proteins by BLASTp algorithm revealed that Cas3-like proteins of
the isolates Crie-F271, 278, 375, and 384 were identical and shared 100% identity with
DEAD/DEAH box helicase of S. aureus, while Cas3-like protein of the isolate Crie-F378
shared 99% identity with another S. aureus DEAD/DEAH box helicase.

4. Discussion
The identification of the origin of RTE food-related S. aureus strains and evaluation
of their pathogenic potential represent very important tasks in the field of food safety
investigations. During a one-year period (2019–2020), RTE food samples were collected
from cafes and restaurants in different geographic regions of Russia (from Moscow to
Kamchatka), corresponding to eight Russian Federal Centers of Hygiene and Epidemiology.
In this study, a WGS was used to determine virulence and antimicrobial resistance gene
profiles, plasmid sequences, and CRISPR/Cas systems of antibiotic-resistant S. aureus
isolates collected from various types of RTE foods. To the best of our knowledge, this is the
first WGS analysis of this pathogen of RTE food origin in Russia.
The 35 samples analyzed belonged to 15 different MLST-based sequence types and
to 20 SpaII types, including four new SSR1 sequences of the SpaII gene, and three groups
of AGR system (I-III). The MLST- and Spa-based typing methods provide a high level of
standardization for the research and the possibility of effective data exchange between
laboratories throughout the world. In addition, Spa-typing allows investigating separate
local outbreaks and molecular evolution in general. Notably, 11 out of 35 S. aureus isolates
under investigation were MRSA isolated from different types of products (bread, salad,
Foods 2022, 11, 2574 10 of 15

meat products, and rolls). The majority of MRSA isolates belonged to CC22 clonal group,
while a single MRSA sample (Crie-F374) was characterized by ST398. These results conform
well with other studies highlighting that the MSSA population is more heterogeneous than
the MRSA population since the former isolates usually constitute a larger number of
different clones and lineages [33–35].
The MSSA isolates of our sample collection were assigned to CC5, CC30, CC45, CC97,
CC398, and other singleton genetic lines. It is well known that strains of CC30 and CC45 are
commonly detected in human subjects colonized or infected with S. aureus [14]. Moreover,
the S. aureus CC30 clonal complex has caused a major impact on global human health,
triggering three pandemic waves and an epidemic of toxic shock syndrome (TSS). This
clonal group is the most common human-colonizing MSSA lineage from which several
MRSA clones have emerged [36].
The characterization of phenotypic and genotypic antimicrobial resistance profiles in
our RTE-food S. aureus isolates revealed a single MDR/MRSA sample (Crie-F374) which
was resistant to antimicrobial compounds of five different groups (penicillins, aminogly-
cosides, trimethoprim/sulfamethoxazole, lincosamide, and tetracycline) and carried a
wide repertoire of resistance determinants corresponding to its phenotypic profile. This
isolate was collected from a pork cutlet and belonged to CC398 which is associated with
food production animals (i.e., LA-MRSA (livestock-associated MRSA)). Our data are in
agreement with previous studies highlighting that CC398 MRSA strains usually possess
a large variety of resistance genes and show higher levels of multiple drug resistance in
comparison to non-CC398 MRSA isolates [37,38]. For instance, CC22 MRSA isolates from
our collection were resistant to penicillins only and carried just 2–5 antibiotic resistance
determinants (Figure 2). S. aureus strains of CC398 were found in livestock around the
globe [39]. LA-MRSA strains were also documented in humans proving a zoonotic trans-
mission from animals to humans [5]. Moreover, LA-MRSA prevalence in livestock remains
high in many geographical regions, and the acquisition of new virulence and resistance
determinants constitutes a growing threat to human health [40]. CC398 MRSA isolates
found in humans are usually characterized by two spa-types (t011 and t034) [41], while
the isolate Crie-F374 was characterized by a new SSR1 sequence of SpaII gene (Table S1)
that has not been found in the respective database (BURP; [42]). Moreover, sak and scn
genes, which are considered to be associated with human infections [14,43,44], were not
found in its genome. Thus, one may suppose the livestock origin of this MDR MRSA isolate
collected from RTE food proved to be very stable to various “farm-to-fork” conditions.
In our study, all but one (CrieF-274) S. aureus isolates from RTE foods were positive
for the blaZ gene. This rate is very close to the prevalence of blaZ among human carriers in
Switzerland [14,45] and Germany [46].
The majority of our MRSA isolates carried only a limited number of virulence factors,
including the ones facilitating specific adhesion, colonization, invasion, and toxin formation.
Such virulence markers as sak and scn genes were also detected in most isolates studied (29
and 32 samples, respectively), which is likely to indicate their origin from food handlers.
Four samples (Crie-F267, 343, 346, and 347) among our MSSA isolates (n = 24) were
characterized by a medium variety of AMR determinants (3–5 genes) and a slightly broader
spectrum of virulence factors (up to 86 genes). At the same time, these isolates were resistant
to two antibiotics only. Interestingly, two Karelian isolates (Crie-F267, 343) representing
a single strain differed by AMR gene content. The differences included erm(c) and tet(K)
genes in the genome of the Crie-F343 isolate indicating non-chromosomal localization of
these determinants and, therefore, the possibility of their transmission and spreading. The
spectra of the virulence determinants of these two isolates were almost the same, except for
gene composition in several clusters.
Additionally, three isolates carried putative CRISPR/Cas system of IE-type, which
is quite rare for S. aureus species [47]. Some pathogens have only a small fraction of the
sequenced isolates with CRISPR/Cas, and S. aureus is one of them. Only 0.6% (45 of 7865)
samples, mainly clinical isolates, had CRISPR/Cas systems [32,47]. For several species it
Foods 2022, 11, 2574 11 of 15

was shown that CRISPR/Cas restricted horizontal gene transfer: Enterococcus faecalis [48],
Pseudomonas aeruginosa [49], and Acinetobacter baumannii [50].
In spite of the same system type, the Cas-cassette composition of the isolates represent-
ing the same strain differed by a deletion in the case of the Crie-F267 isolate. These isolates
also differed by plasmid replicon composition. It is well known that the CRISPR/Cas
system has also been found, in addition to the immune defense function, to play the role
in hindering the uptake of antibiotic resistance genes, regulating the virulence of bacteria,
and influencing the formation of biofilms by foodborne pathogens [32,47,51]. However,
three isolates harboring putative CRISPR/Cas systems in our S. aureus RTE-food collection
carried a broad variety of antibiotic-resistant genes and a rather extensive spectrum of
virulence factors. According to the UniProtKB database, Cas3-like proteins of the isolates
Crie-F271, 278, 375, and 384 shared homology with DEAD-box ATP-dependent RNA he-
licase CshA, which played an important role in quorum sensing of S. aureus [52]. At the
same time, the Cas3-like protein of Crie-F378 was similar to putative ComF operon 1 of
Staphylococcus epidermidis encoding the purine salvage pathway enzyme. Thus, it could be
assumed that the first four isolates possessed additional pathogenic determinants.
We would like to emphasize that the low number of CRISPR/Cas systems in S. aureus
isolates of different geographic origins may indicate that the presence of such immune
systems in this species is a spontaneous biological phenomenon, namely, that such systems
could be acquired from some other bacterial species sharing the environment with S.
aureus [53].
The analysis of plasmid sequences showed that six isolates from our set harbored
multiple types (more than 2) of plasmid replicons simultaneously, while only four isolates
did not have any Rep-sequences at all. An association between resistance genotype and
Rep families, as well as a relationship between separate CC and some Rep sequences, were
described for S. aureus strains in the previous studies [31,54]. In spite of a small sample set
for each CC in our S. aureus RTE food collection, the plasmid sequence data obtained are in
accordance with the results of the works mentioned above. For instance, the combination
of Rep5 and Rep16 families was common for the isolates belonging to CC30 and ST7. At
the same time, the presence of Rep5 and Rep20 plasmid replicons was a “signature” of
MRSA CC22 isolates.
It is well known that Staphylococci typically carry one or more plasmids per cell and
these plasmids include varied gene content [55]. S. aureus plasmids can confer resistance
to antimicrobials, biocides, and heavy metals [56], and may also encode host survival
elements, virulence factors, and toxins [55]. The acquisition of these different genetic
elements in a single large plasmid can enhance the adaptation and dissemination of S.
aureus in different environments due to co-selective advantage [57]. In order to obtain
reliable whole-plasmid assemblies, long-read sequencing would be preferable since it
provides the necessary resolution to separate genomic and plasmid sequences on the stage
of contig assembly [58]. We are planning to sequence the isolates using a MinIon device
(Oxford Nanopore Technologies, Oxford, UK) to get deeper insights into plasmid sequences
and structure that could facilitate elucidating resistance transfer mechanisms.
At a first glance, the isolates from our collection are mostly non-MDR and do not
seem to represent a significant danger to public health. On the other hand, however, given
the fact that they were isolated from RTE food, the samples and their sources represent a
“means of transfer and dissemination” of important pathogenicity determinants. Moreover,
the pathogenic potential of separate isolates studied cannot be ignored, especially for those
carrying enterotoxin genes. Our WGS results revealed that 14 samples (40%) carried at
least one enterotoxin gene (sea, sec, sed, or sel). Additionally, major portion of the isolates
harboring the tsst1 gene were MRSA, and seven out of 10 of them were assigned to CC22
being one of the most important disease-causing clones transmitting rapidly within and
between hospitals globally [59]. Therefore, the presence of these pathogenic factors in S.
aureus isolates from RTE food can pose a significant threat to public health. We can also
suggest that such pathogenic potential, including an arsenal of mechanisms providing
Foods 2022, 11, 2574 12 of 15

resistance to antimicrobial drugs, leads to the long-time circulation, transmission, and

dissemination of S. aureus in the community and, in particular, within the RTE food supply
chains. Therefore, the development of monitoring criteria for MRSA and MSSA, as well as
for other selected foodborne bacteria, may provide a valuable option for controlling the
dissemination of antimicrobial resistance and other pathogenic factors. Such criteria could
be based on microbiological properties or antimicrobial resistance potential of the isolates
studied.

5. Conclusions
This study provides the WGS-based investigation of 35 S. aureus from RTE food in var-
ious geographic regions of Russia. The isolates were characterized by their phenotypic and
genomic AMR profiles, their population structure, the presence of virulence determinants,
as well as by plasmid replicon sequences and CRISPR/Cas systems.
To the best of our knowledge, this is the first WGS-based study of such a diverse S.
aureus population obtained from RTE food in Russia.
We believe that the WGS data obtained will greatly facilitate further studies of food-
borne S. aureus epidemiology, as well as its genome plasticity, in terms of the acquisition of
various genetic elements related to host adaption, antimicrobial resistance, and virulence.
In turn, the results of such studies will help to develop preventive measures against human
infections caused by this important pathogen in community settings.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/foods11172574/s1, Table S1: Origin and typing of RTE
food S. aureus isolates studied; Table S2. cgMLST profiles for S. aureus isolates from ready-to-eat food;
Table S3: Virulence factors revealed in S. aureus isolates from ready-to-eat food; Figure S1: Minimum-
spanning tree (MST) of cgMLST allelic profiles for S. aureus isolates; if a group of the isolates possessed
completely the same profiles, only one isolate from this group was shown for clarity.
Author Contributions: Conceptualization, Y.M., A.S. and A.C.; Data curation, Y.M. and A.S.; Formal
analysis, Y.M. and A.S.; Funding acquisition, V.A.; Investigation, Y.M., A.S., A.C., M.T. and A.E.;
Methodology, Y.M., A.S., A.C. and M.T.; Project administration, I.M.; Resources, A.C. and A.E.;
Software, A.S.; Supervision, I.M. and V.A.; Validation, A.E.; Visualization, Y.M., A.S. and S.S.; Writing—
original draft, Y.M.; Writing—review & editing, A.S. and M.T. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by a grant from the Ministry of Science and Higher Education of
the Russian Federation (agreement No. 075-15-2019-1666).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The assembled genome sequences for all isolates were uploaded to the
NCBI Genbank under the project number PRJNA823522.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

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