Non-typhoidal Salmonella enterica is responsible for 88,000 cases of gastroenteritis in Canada each year. The symptoms of gastroenteritis can be mild to severe depending on the health conditions of individuals. Generally, the patient may recover without antibiotic treatment. However, antibiotic intervention may be necessary for children, the elderly, and immunosuppressed patients.
genus belongs to the Enterobacteriaceae
family and includes two species, bongori
. According to the Kauffman–White scheme, more than 2500 serotypes have been characterized [1
]. As reported by the US Centers for Disease Control and Prevention (CDC), although all S. enterica
serotypes can cause disease in humans, less than 100 serotypes account for much of the infections.
In 2014, a global report by the World Health Organization (WHO) on the surveillance of antimicrobial resistance (AMR) revealed that increasing resistance across many different infections has become a serious concern for public health worldwide [2
]. AMR can be acquired by either spontaneous mutations or by horizontal gene transfer (HGT), in which plasmids are known to play a key role [3
]. Plasmids are mobile genetic elements (MGE) encoding for their self-replication and transfer. The genes responsible for plasmid maintenance and transmission form a “backbone” that is a core set of genes encoding for essential plasmid functions [4
]. Plasmids also provide non-essential cellular functions, such as virulence factors, AMRs, metabolic pathways, and unknown functions that are defined by genes encoding hypothetical and unknown proteins, which all confer competitive advantages to the bacterial host in specific situations. Once an AMR gene becomes stable on a plasmid through environmental pressures, it can quickly spread across species and ecosystems that can lead to its transfer from the surrounding environment to human pathogens [5
Plasmids are amenable to detailed analysis using data from whole genome sequencing (WGS), using complementary software including mlplasmid, PlasmidFinder, cBar, plasmidSPAdes, Recycler, and PLACNET [7
]. A recent comparison between five bioinformatics software showed that plasmidSPAdes was capable of fully or partially predicting 84% of plasmids used as references [12
]. However, as plasmidSPAdes separates plasmids from chromosomes based on read coverage, plasmid contigs with a similar coverage to the chromosomal contigs are often mislabelled [8
Plasmids are widespread in S. enterica,
where they are known to carry nonessential genes involved in AMR and virulence [13
]. Given that the spreading of AMR genes through microorganisms is a major issue for public health worldwide, the prediction of plasmid-carrying AMR genes will give insight into their dissemination across bacterial strains. In the current study, we expanded our knowledge of AMR genes carried by the plasmidome of 1750 S. enterica
genomes. These genomes sequenced by Illumina MiSeq as part of a Salmonella
Syst-OMICS project were analyzed by Plasmid-Gather, a pipeline designed to predict plasmid scaffolds based on the presence of essential genes for plasmid replication, mobility, and sequence similarity to a reference plasmid.
By combining a collection of public and SalFoS data, we identified a high proportion of plasmid contigs in Illumina MiSeq WGS assemblies using two databases containing essential conserved plasmid elements (PlasmidFinder-DB and MOBs-DB) combined with known reference plasmids. One of the added values will be to increase the plasmid sequences identified. The databases can be regularly updated to include new ORIs and mobility genes for future analyses.
By using Plasmid-Gather and the combined strategies described here, IncFII and IncFIB were the most frequent ORIs predicted in S. enterica
; this was presumably caused by the over-representation of the S.
Enteritidis and S.
Typhimurium isolates in the dataset (Table 1
). These two serotypes accounted for more than 23% of all the isolates and carried together 61% and 87%, respectively, of all th eIncFII and IncFIB (Table 1
). The distribution of ORIs among the S. enterica
serotypes showed that IncFII, IncI1, and ColpVC were found in a broad range of serotypes, whereas IncA/C2 and IncX1 are restricted to a dozen serotypes (Table 1
). Interestingly, Lindsey et al. (2009) demonstrated by a cluster-based analysis using the pulsed field gel electrophoresis (PFGE) of 216 multidrug resistance S. enterica
that IncI1 is not clonally distributed, whereas IncA/C is commonly observed in the same serotypes. Hence, IncI1 is presumably much more mobile than IncA/C [42
]. IncI1 incompatibility was often associated with multi-drug resistance and with the widespread distribution of Beta-lactam resistance genes [28
]. Likewise, we observed that plasmids with IncI1 are among the most important carriers of AMR genes (Supplementary Table S5 and Supplementary Table S9
). Hence, one may assume that the mobility of IncI1 also leads to the spread of AMR genes in many S. enterica
serotypes, whereas IncA/C2 seems more serotype restricted, but were associated with several AMR genes.
Large plasmids, representing different Inc ORIs, are known to integrate and carry transposons or integrons conferring AMR [3
]. Several multi-resistance plasmids have been identified in Salmonella
. Among them is the Inc group A/C (IncA/C and A/C2), consisting of 150 kb plasmids [31
]. In our study, 49 of the 53 IncA/C2 plasmid scaffolds had less than 54 kb. This may be due to the limitations of plasmid assembly, as demonstrated in Figure 3
. Three of the four IncA/C2 assembled with expected sizes encoding seven AMR genes each (aph(3
) (Supplementary Table S5
). In addition, the IncA/C2 plasmid reconstructed by hybrid assembly from the S624 isolate possessed 12 AMR genes, a greater number than the other plasmids carrying AMR genes obtained using hybrid assemblies (Supplementary Table S9
). Furthermore, in the Illumina MiSeq data we noted that nearly all the S. enterica
isolates with IncA/C2 (52/53) had one or more AMR encoded by scaffolds in their plasmidome. Isolates with IncA/C2 plasmids carried on average five AMR genes (up to 11 for the S628 isolate); IncA/C2 is the only ORI predicted in 23/53 genomes. Multidrug resistance isolates have been linked previously to IncA/C [42
Regarding the 52 AMR genes significantly enriched amongst S. enterica
chromosomes, 34 were part of what we call "the core resistome"—i.e., AMR genes found in more than 95% of S. enterica
genomes (described in Supplementary Table S8
). The predominant AMR mechanism in the so-called core resistome is antibiotic efflux (26/34). Efflux transporters exist as either single- (e.g., Tet) or multi-component pumps (e.g., MdsABC complex) [45
]. Multidrug efflux pumps are common resistance mechanisms among Gram-negative bacteria [45
]. However, due to various efflux pumps that can compensate with wide substrate specificity, it remains a challenge to identify which drug efflux pump confers AMR. For other less frequent AMR genes found in S. enterica
sharing a 99% amino acid identity were found in different serotypes (e.g., Typhimurium, Braenderup, and I 4 [5
];12;i;- for aac(6’)-Iaa
; Enteritidis, Newport, and Heidelberg for aac(6’)-Iy
). Together, these 2 N
-acetyltranferases (AAC) were encoded within 1738 chromosomes (99.3%), as shown in Supplementary Table S8
. The gene fosA7
, conferring resistance to fosfomycin, was predicted in 100% of the S.
Heidelberg (51/51) isolates. In alignment with these results, fosfomycin resistance has previously been found in S.
Heidelberg isolated from broiler chickens [46
]. Similarly, fosA7
was observed predominantly in almost all the isolates from the same serotype: in 96% of S.
Agona (25/26), in 100% of the S.
Telelkebir (8/8), in 67% of the S.
Derby (8/12), and in 70% of the S.
Alachua (7/10). The remaining fosA7
= 23) were distributed among 14 under-represented serotypes.
Although considered as chromosome encoded, some efflux pumps have been identified on plasmids, such as the tetA
gene encoding tetracycline resistance [47
]. As shown in Table 2
, 3 of the 13 AMR genes enriched in the Salmonella
plasmidome encoded efflux pumps, 2 conferred resistance to tetracycline (tet(C)
) and the last one was the resistance to chloramphenicol/florfenicol (floR
). Tetracycline has been overused in human and veterinary medicines as growth promoters in animals [48
]. The tet(C)
AMR genes were often reported on MGEs, such as genomic islands (GEIs), as part of conjugative elements and in plasmids [50
]. We observed a low abundance of tet(C)
in isolates carrying plasmid scaffolds (7.9% and 2.2%, respectively) (Table 2
). Previous studies have shown the rare occurrence of these AMR genes in Salmonella enterica
]. The serotypes of S. enterica
from SalFoS harboring tet
genes were mostly S.
Newport (29.9%, n
= 26) and S.
Typhimurium (23%, n
= 20) for tet(C)
Kentucky (41.7%, n
= 10) for tet(D)
. The floR
gene is the only significant plasmid gene conferring resistance to chloramphenicol (Table 2
). We also noticed that 92.3% of the plasmidomes coding for floR
also carried an IncA/C2, thereby leading to the conclusion that this ORI is likely to be strongly associated with its dissemination. The connection between floR
and IncA/C2 can also be seen in hybrid assemblies, because floR
was only predicted once in an IncA/C2 plasmid (Supplementary Table S9
). The floR
gene was already highlighted as the most common in Salmonella
chloramphenicol-resistant strains [56
In examining the AMR genes detected in plasmids (Table 2
), the most common resistance encoded was resistance to streptomycin (strA
resistance genes). In addition to being used for human medicine, streptomycin is used as a feed supplements for pigs and as a pesticide for agriculture [48
]. Likely because it is extensively used in agriculture, resistance to streptomycin was frequently found in environmental and pathogenic isolates [59
]. Moreover, aminoglycoside antibiotic was the most prevalent drug class identified.
In this study, we were also interested in AMR genes that may complicate the treatment of salmonellosis and cause possible public health issues by the HGT of AMR genes. In 2014, of all antimicrobials prescribed in human medicine used for treating bacterial infections, the beta-lactam amoxicillin represented the largest proportion used (26%), followed by azithromycin (9%) and ciprofloxacin (8%) [61
]. In the same year, 5% of the non-typhoidal Salmonella
isolates were resistant to amoxicillin, while no resistance to azithromycin or ciprofloxacin was observed; these last two antimicrobials were largely prescribed to treat severe and invasive salmonellosis [61
]. Three AMR genes identified in the S. enterica
plasmidome confer resistance to either amoxicillin (blaTEM-1
= 47)), azithromycin (mphA
= 4)) or ciprofloxacin (aac(6’)-Ib-cr
= 1)) (Supplementary Table S7
); the last two antibiotics are used to treat severe and invasive Salmonella
]. Fortunately, these three AMR genes are infrequent in the S. enterica
plasmidome, except for blaTEM-1
, and are not co-carried by the same isolate. However, once an AMR gene is plasmid-stable, AMR resistance can quickly spread through bacterial communities, and so this is something that may need future monitoring. In contrast, there is no clear pattern among S. enterica
isolates harboring blaTEM-1
; these strains were isolated from 1981 to 2011 in five countries from eight species representing 15 Salmonella
serotypes. Furthermore, nine different ORIs were found to be associated with scaffolds carrying blaTEM-1