RESULTS AND DISCUSSION
Ten of Salmonella Enteritidis were isolated from 293 samples.
Among them, seven of Salmonella Enteritidis classified as MDR
showed resistance to five antibiotic classes including Quinolones (NAL),
Polymyxin (COL), Aminoglycosides (GEN), third generation cephalosporins
(FOT, AXO, TAZ), Penicillins (AMP) and Tetracyclines (TET) and those
with intermediate resistance to Quinolones (Table 1). All MDR isolates
were susceptible to Carbapenems (IMI), Phenicols (CHL), Macrolides
(AZI), Aminoglycoside (STR, AMI) and third generation cephalosporins
(FOX) (Table 1).
Genome assembly of all the sequenced Salmonella Enteritidis
strains generated 2 circular contigs consisted of a chromosome and a
plasmid. The size of chromosome ranged from 4,678,918 to 4,68,786bp with
a GC content of 52.2%. Six of the seven isolates had a plasmid size of
110,273 with a GC content of 51.7% and Z0719SL0013 had a plasmid size
of 109,445 with a GC content of 51.7%. All sequence files are available
from NCBI under BioProject PRJNA658425.
All of the sequenced Salmonella Enteritidis strains harboured
antibiotic resistance genes and chromosomal mutations that coincided
with antibiotic resistance phenotypes (Table 1). The antibiotic
resistance gene aac(6’)-laa_1 (aminoglycoside) was detected on
the chromosome, whereas the aac(3’)-lld_1 (gentamicin),blaCTX-M-15 (cephalosporin), and tet(A) (tetracycline)
were detected on the plasmid found in all of the MDR isolates.
Chromosomal mutations in the gyrA (quinolone) and phoQ(colistin) were also found (Table1).
The whole genome SNP tree generated by the RAxML based on results of the
PhaME analysis showed that all of the isolated SalmonellaEnteritidis strains were genetically close with a median pair-wise
distance of 8 (range 1–16) SNPs
(Figure 1). Further, all of the sequenced isolates were clustered
together with the FORC_019 strain, isolated from human blood with
salmonellosis, showing monophyletic relationship with bootstrap support
of 100. The median pair-wise SNP distance of the FORC_019 strain from
the other isolates was 11.2 (range 10–16). A pairwise SNP distance of
≤20 and monophyletic relationship with a bootstrap support value
>0.9 indicated that these strains originated from the same
genetic ancestor . Results of whole genome SNP analysis betweenSalmonella Enteritidis strains isolated from human and poultry
sources were consistent with those of the previous PFGE and MLVA studies
.
All of the MDR strains isolated in this study harboured an
~110 kb plasmid (pSE-VMDR) with the IncFIB and IncFII
replicons and 51.7% GC content, including 146 ORFs. Six of the seven
isolates had a plasmid with the same genomic structure. Compared with
other plasmids, an inverted genomic region of ~52 kb in
size was detected in the pZ0719SL0013 (Figure 2a). An inverted genomic
region ~59.5 kb in size like that in the pZ0719SL0013
was found in the pFORC_019. The inverted region of the pFORC_019
contained a 7.5 kb insertion sequence comprising a Tn6029 remnant and
Tn4352, which encoded multiple antibiotic resistance genes, i.e.,sul2-strA-strB and APH(3) , respectively.
BLAST comparison between the pSE-VMDR and the pFORC_019 revealed 99%
of sequence identity and 93.5% of coverage (Figure 2b). This comparison
also revealed that the pSE-VMDR was a novel fusion plasmid consisting of
the pSEJ and the FORC_038 chromosome, which contains genes encoding
virulence factors and genes encoding antibiotic resistance proteins,
respectively. Salmonella Enteritidis contains a 59 kb serotype
specific virulence plasmid, pSENV . Studies of clinicalSalmonella Enteritidis isolates have shown that the antibiotic
resistance gene has been introduced into the virulence plasmid .
Nucleotide sequence identity of the antibiotic resistance region in the
pSE-VMDR was 100% with that of the chromosome of the FORC_038 strain
of Salmonella Virchow isolated from raw chicken meat in Korea
except for a 7.5 kb insertion sequence in pFORC_019. SalmonellaEnteritidis, Salmonella Montevideo, and Salmonella Virchow
are the most prevalent serotypes of Salmonella entericaisolated from chicken meats and chicken slaughterhouses in Korea .
Clonal dissemination of Salmonella Virchow carrying the CTX-M-15
gene may have occurred in contaminated food in Korea . SalmonellaEnteritidis strains isolated from chicken and human stools in Korea
during 2009 carried a 95 kb of conjugative plasmid harbouring the
CTX-M-15 gene . The antibiotic resistance phenotype, plasmid size, and
plasmid replicon type in the seven MDR isolates studied herein were
consistent with those of the 2009 isolates. The existence of the
ISEcp1 upstream from the CTX-M-15 was also consistent.
In conclusion, Salmonella Enteritidis strains that can infect
human and poultry have been circulating through clonal or horizontal
transmission for a long time in Korea. Since we showed that the
antibiotic resistance genes can be transmitted via plasmids betweenSalmonella enterica strains in an intraspecies manner,
antibiotics against which this plasmid confers resistance have been
specifically avoided at poultry farms to prevent the further spread of
such plasmids among Salmonella Enteritidis strains. This is
critically important to prevent the prevalence of MDR Salmonellafrom increasing