Class 1 integron-borne cassettes harboring blaCARB-2 gene in multidrug-resistant and virulent Salmonella Typhimurium ST19 strains recovered from clinical human stool samples, United States

International lineages, such as Salmonella Typhimurium sequence type (ST) 19, are most often associated with foodborne diseases and deaths in humans. In this study, we compared the whole-genome sequences of five S. Typhimurium strains belonging to ST19 recovered from clinical human stool samples in North Carolina, United States. Overall, S. Typhimurium strains displayed multidrug-resistant profile, being resistance to critically and highly important antimicrobials including ampicillin, ticarcillin/clavulanic acid, streptomycin and sulfisoxazole, chloramphenicol, tetracycline, respectively. Interestingly, all S. Typhimurium strains carried class 1 integron (intl1) and we were able to describe two genomic regions surrounding blaCARB-2 gene, size 4,062 bp and 4,422 bp for S. Typhimurium strains (HS5344, HS5437, and HS5478) and (HS5302 and HS5368), respectively. Genomic analysis for antimicrobial resistome confirmed the presence of clinically important genes, including blaCARB-2, aac(6’)-Iaa, aadA2b, sul1, tetG, floR, and biocide resistance genes (qacEΔ1). S. Typhimurium strains harbored IncFIB plasmid containing spvRABCD operon, as well as rck and pef virulence genes, which constitute an important apparatus for spreading the virulence plasmid. In addition, we identified several virulence genes, chromosomally located, while the phylogenetic analysis revealed clonal relatedness among these strains with S. enterica isolated from human and non-human sources obtained in European and Asian countries. Our results provide new insights into this unusual class 1 integron in virulent S. Typhimurium strains that harbors a pool of genes acting as potential hotspots for horizontal gene transfer providing readily adaptation to new surrounds, as well as being crucially required for virulence in vivo. Therefore, continuous genomic surveillance is an important tool for safeguarding human health.

Introduction Non-typhoidal Salmonella (NTS) is one of the most important foodborne pathogens with unprecedented impact on global health [1]. Among NTS, Salmonella enterica subsp. enterica serovar Typhimurium represents a major threat, since its worldwide spread has been associated with a broad host range, which includes mostly humans and food-related sources [1,2]. Besides that, the emergence of multidrug-resistant (MDR) S. enterica is another crucial aspect for food-related outbreaks globally, limiting our therapeutic options [3].
In addition to the high global burden of salmonellosis, extended-spectrum β-lactamase (ESBL)-producing S. enterica strains have been recognized as high-priority bacteria causing serious public health issue (https://www.who.int/news-room/detail/27-02-2017-whopublishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed). Aside from this, the emergence of mobile genetic elements (MGEs), for instance, class 1 integrons play an essential role in the global spread of antimicrobial resistance [4,5]. Another aspect to be considered is the wide range of virulence package that is typically associated with Salmonella Pathogenicity Islands (SPI), contributing to the infection process among diverse hosts [6][7][8]. In this context, while the surveys with genomic approach have helped in the development of mitigation strategies and clinical management, continuous active surveillance is urgently required.
Here, we describe the genomic characteristics of five MDR and virulent S. Typhimurium strains carrying the bla CARB-2 gene recovered from clinical human stool samples in North Carolina, United States.

Ethics approval and consent to participate
The human patients from whom Salmonella were recovered were completely anonymous and even after all the analysis and tests, the human sample remained anonymous. As such, the NC State IRB (FWA: 00003429) indicated the study research did not need IRB approval because it does not meet the definition for human subjects research.

Bacterial strains and antimicrobial susceptibility testing
We conducted a genomic investigation on five clinical S. Typhimurium strains collected in 2014 in North Carolina, United States. The strains were subjected to phenotypic characterization using the microdilution panel susceptibility approach on Gram-negative Sensititre plates (CMV3AGNF and GNX2F, Trek Diagnostic Systems, OH, USA) following the interpretative criteria of Clinical and Laboratory Standards Institute [9, 10]. The MDR profile was defined as resistant to three or more classes of antimicrobials [11]. All S. Typhimurium strains underwent molecular screening for class 1 integron by PCR [12, 13] and were subsequently characterized by whole-genome sequencing (WGS) according to Pornsukarom et al. [14]. CA, USA) at a paired-end read (300 bp). Resulted raw sequence reads underwent a strict quality control, as well as we obtained the draft genomes by using default settings in CLC workbench 10.1.1 (Qiagen) as per Monte et al. [15]. The sequencing data were deposited in NCBI (PRJNA613764). For each strain, we uploaded the sequences into Center for Genomic Epidemiology (http://genomicepidemiology.org/) to detect multilocus sequence typing (MLST), resistome, plasmid incompatibility groups and Salmonella Pathogenicity Islands.
Virulome analyzes were performed by using default settings available in VFanalyzer [16]. Additionally, the genetic context of bla CARB-2 and presence of virulence genes were investigated using BLASTn analysis against the non-redundant (NR) database and manually curated using Geneious v. 11.1.5 (Biomatters Ltd., Auckland, New Zealand).
Interestingly, these strains possess a highly conserved spv operon composed by spvR, spvA, spvB, spvC, and spvD genes which are located upstream of the genes pefA (plasmid-encoded fimbriae) and rck (resistance to complement killing) in a virulence plasmid as shown in Fig 2. In addition, in silico analyses confirmed that these virulence genes were located on IncFIB plasmid.
To achieve a better understanding of the clonal spread of these MDR strains, we reconstructed a phylogenetic tree based on SNPs. Indeed, these strains were found to be genetically related. The phylogenetic tree framed a major cluster composed by five S. Typhimurium strains from this study (HS5478, HS5344, HS5437, HS5302, and HS5368), which nested together with S. enterica strains from different sources (Human, poultry, ovine) and countries, including Denmark (SAMEA4349586), Scotland (SAMEA773504 and SAMEA773551), France (SAMN07734943), and China (SAMN09759463) as shown in Fig 3. Interestingly, S. Typhimurium strains within same cluster shared the same resistance phenotype and genotype profile.

Discussion
The continuous dispersal of MDR S. enterica strains frequently deserves attention of the public health authorities, particularly the international lineages as S. Typhimurium ST19 that most often causes diseases and deaths [18,19]. Owing to their importance, the ST19 members have been globally identified in a variety of sources, such as human clinical samples, animals, food products, and environmental samples [20][21][22]. Moreover, S. Typhimurium ST19 has shown broad resistance to a variety of critically important antimicrobials [23], including colistin (an antibiotic of last resort for some MDR infections) [24,25]. Besides that, the occurrence of intermediate resistance reported here implies in possible treatment failure that should be noted by public health authorities.
It is important to note that these strains can easily acquire such genes through mobile genetic elements such as plasmids, integrons, and genomic islands from other MDR clones, resulting in their rapid dissemination. The presence of class 1 integron in all S. Typhimurium ST19 strains constitutes a risk factor to the rapid spread of antimicrobial resistance (AMR) genes. Indeed, class 1 integron coding various resistance profiles has been widely reported in S. Typhimurium as well as in multiple serovars [5,21,[26][27][28][29][30]. This genetic frame is crucial for the spread resistance markers, since they are able to capture AMR genes through chromosomal cassettes incorporating them by site-specific recombination [4, 12, 31]. Additionally, resistance genes located in class 1 integrons are often within Salmonella genomic islands (SGI), such as the conjugative Salmonella genomic island 1 (SGI1) (~43-kb) and its variants [32,33].
The detection of quaternary ammonium compounds (QACs) raises a particular concern, since this qac-containing integrons typically harbors a pool of genes that are hotspots for   horizontal gene transfer providing readily adaptation to new surrounds [34,35]. The co-resistance of critically important antimicrobials and disinfectants QACs reinforces the evidence of the overuse of biocides in clinical settings [34], and their spread have been also described in Salmonella serotypes isolated from livestock [36].
The bla CARB-2 gene, earlier identified as bla PSE-1 , is most often a part of the chromosomal cassette [37,38]. To date, the occurrence of this carbenicillinase gene has been limited to a few reports in different bacteria species and countries, including Acinetobacter pittii and Salmonella serovars in Australia [38,39], Salmonella Typhimurium from England and Wales [40], Salmonella Senftenberg in Mexico [41], S. Typhimurium in Canada [42], Pseudomonas aeruginosa in Netherlands [43], and Escherichia coli in Pakistan [44]. It is noteworthy that such genetic element has the ability to move among different lineages of S. enterica serovars on a global scale, contributing to AMR spread [28]. Indeed, the genetic contexts surrounding bla-CARB-2 gene in this study are typically found in SGI1 and its variant SGI1-B.
Drug-resistant variants of SGI1 have been identified in numerous S. enterica serovars, and strains harboring them may be more virulent and have a tendency to rapidly disseminate [33,39]. In fact, S. Typhimurium strains within this survey demonstrate to possess several virulence factors, which have been reported earlier [45][46][47][48]. Furthermore, we confirmed the presence of several plasmid-borne virulence genes (spvR, spvA, spvB, spvC, spvD, rck, and pefA) that denotes an important genomic apparatus for the spreading of this plasmid, and may provide fitness benefit as previously reported [28,49,50]. Increasing evidences have demonstrated that the spv operon affects the formation of autophagosomes, as well as highlight its association in killing of macrophages and neutrophils [6], being crucially required for virulence in vivo [8], including aggravated damage in zebrafish infection model [7]. Furthermore, the PhoP-regulated gene mig-14 that is required for virulence and resistance to antimicrobial peptides was detected in these strains. Yet, mig-14 contributes to Salmonella persistence in hosts, being also associated with resistance against polymyxin B and cathelin-related antimicrobial peptide (CRAMP) [51][52][53][54]. Thus, the clonal dissemination of MDR S. Typhimurium (mostly the invasive clones) constitutes an important issue to public health [55], especially S. Typhimurium ST19, which have been circulating worldwide (http://enterobase.warwick.ac.uk/) as demonstrated in this study, since our S. Typhimurium strains nested with international lineages from at least four countries (Fig 2).
In summary, we report the genomic features of virulent and MDR S. Typhimurium ST19 strains carrying the bla CARB-2 gene recovered from clinical human samples in United States. Our results provide new insights into this genetic environment that besides bla CARB-2 , contains genes, coding resistance to quaternary ammonium compounds (qacEΔ1) and sulfonamides (sul1). Furthermore, our findings could aid in understanding the epidemiology of S. Typhimurium ST19, which are of great value to initiate preventive measures to safeguard human health. Given the high spread of this international lineage, especially among the young and the elderly or immunocompromised people, public health authorities and regulatory food agencies need to be aware of the potential impact in public health and in economy caused by such pandemic MDR S. Typhimurium ST19 lineage, with particular attention in high-burden areas.