New Multidrug-Resistant Salmonella enterica Serovar Anatum Clone, Taiwan, 2015–2017

In 2011, a Salmonella enterica serovar Anatum clone emerged in Taiwan. During 2016–2017, infections increased dramatically, strongly associated with emergence and spread of multidrug-resistant strains with a plasmid carrying 11 resistance genes, including blaDHA-1. Because these resistant strains infect humans and food animals, control measures are urgently needed.


The Study
To investigate the epidemiologic trend, we analyzed the data in the Salmonella fingerprint database constructed by the Taiwan Centers for Disease Control. The database comprises demographic and experimental data, including pulsed-field gel electrophoresis (PFGE) fingerprints obtained by using the PulseNet standardized PFGE protocol (3), serotypes obtained using PFGE pattern comparison and conventional methods (4), and antimicrobial drug susceptibility testing results for isolates collected from hospitals nationwide. We conducted whole-genome sequencing for 68 Salmonella Anatum isolates from humans and animals and 9 isolates from chicken carcasses and abbatoir environments by using the Illumina MiSeq platform (https://www.illumina.com) and identified resistance genes, incompatibility groups of plasmids, and sequence types by using the whole-genome sequencing data. To investigate clonal relationships and locations of resistance genes, we constructed a dendrogram for Salmonella Anatum strains with whole-genome singlenucleotide polymorphism profiles to assess genetic relatedness among strains and determined the complete genomic sequence of Salmonella Anatum strain R16.0676 with whole-genome sequencing data generated by using a MinION nanopore sequencer (https://nanoporetech. com/products/minion) and an Illumina MiSeq sequencer. To investigate mobility of resistance plasmids, we conducted conjugation experiments to transfer the resistance genes-carrying (R) plasmid from Salmonella Anatum strain R16.0676 into recipient Escherichia coli C600 and transferred an R plasmid from an E. coli transconjugant back to a rifampin-resistant mutant of Salmonella Anatum strain R13.0957 (Appendix, https://wwwnc.cdc.gov/EID/ article/25/1/18-1103-App1.pdf).
The Salmonella fingerprint database of the Taiwan Centers for Disease Control contained PFGE fingerprints for 34,160 Salmonella isolates recovered during 2004-2017, of which antimicrobial drug sensitivity test results were available for 23,018. Salmonella Anatum was not a prevalent serovar among those collected during 2004-2014 ( Figure 1). However, the number of Salmonella Anatum infections increased in 2015 and subsequently underwent another sharp increase in 2016 and 2017. In 2017, Salmonella Anatum accounted for 14.2% of Salmonella infections in Taiwan and ranked as the third most frequently identified serovar.
All MDR SL3_2 isolates, including the 4 isolates recovered from the abattoirs, harbored an IncC plasmid and the same 11 resistance genes identified in strain R16.0676. Strain R17.0132 acquired an additional mcr-1 gene and was resistant to colistin ( Figure 2). We did not obtain any transconjugants with pR16.0676_90k, but we did obtain a transconjugant with a composite plasmid, which had the same sequences as pR16.0676_90k and pR16.0676_34k (Appendix Figure, panel C). This 125kb composite plasmid probably resulted from insertion of pR16.0676_90k into pR16.0676_34k through an insertion sequence 26-mediated transposition process. The resulting plasmid acquired an additional copy of insertion sequence 26 and an 8-bp tandem repeat in the insertion site. More than a dozen genes are typically required for conjugation (6). pR16.0676_90k harbored only 3 genes, and pR16.0676_34k contained at least 12 genes related to conjugation. Fusion of the 2 plasmids caused the composite plasmid to become self-transmissible. When the composite plasmid was transferred back into a rifampinresistant mutant of Salmonella Anatum strain R13.0957, we obtained transconjugants harboring only a 58-kb or 83-kb R plasmid, which were derived from the 125-kb plasmid through deletions (Appendix Figure, panel C). Accordingly, the composite plasmid was unstable in Salmonella Anatum.

Conclusions
We identified a new Salmonella Anatum clone that emerged in Taiwan in 2011. During 2011-2014, strains of the new clone were not resistant and caused few infections. The dramatic increase in Salmonella Anatum infections that occurred during 2016-2017 was strongly associated with the emergence of MDR strains in 2015. The most crucial concern regarding emergence of the MDR Salmonella Anatum clone was that all MDR strains carry bla DHA-1 , which encodes AmpC β-lactamase and confers resistance to β-lactam drugs, including third-generation cephalosporins. This resistance cannot be overcome by using β-lactam inhibitors. Because these MDR strains can cause numerous infections in humans and are prevalent in animals used for food, urgent control measures are needed.

Sequencing of complete genome of S. Anatum strain R16.0676 and plasmids
We used a MinION nanopore sequencer (Oxford Nanopore Technologies, UK) to obtain long reads for S. Anatum strain R16.0676 and plasmids from transconjugants, an Albacore basecaller (Oxford Nanopore Technologies) to execute base calling of nanopore reads, Canu (2) to assemble reads, Pilon (3) to polish the Canu-assembled contigs with the Illumina reads, and Nanopolish (https://github.com/jts/nanopolish) to polish the Canu-assembled contigs with raw nanopore reads. Subsequently, we used PCR and Sanger sequencing techniques to correct the uncertain sequences and RAST (http://rast.nmpdr.org/) to annotate the complete chromosome and plasmid sequences of the strain R16.0676 (4).

Construction of a dendrogram for S. Anatum strains using wgSNP profiles
We used the tools provided in BioNumerics version 7.6.3 for construction of a dendrogram with wgSNP profiles of S. Anatum strains. The sequences of raw reads were mapped to the reference genomic sequence of S. Anatum strain GT-38 (GenBank accession no. CP013226) and the mapped sequences of strains and the reference were aligned for SNP calling by using the option of strict SNP filtering (closed SNP set). By using this SNP calling criteria, SNPs are called by removing positions with at least one ambiguous base (non-ATGC base), one unreliable base (N), one gap and non-informative SNPs. Each retained SNP position has minimum 5x coverage, at least covered once in both forward and reverse direction. The minimum distance between retained SNP position is 12 bp.
A dendrogram was constructed with the whole genome SNP profiles using the categorical (SNPs) option for similarity coefficient and single linkage algorithm for cluster analysis.

Conjugation
We conducted conjugation experiments to transfer the resistance genes-carrying (R) plasmid from strain R16.0676 into Escherichia coli C600 recipients by using LB medium with 50 mg/L ampicillin and 2,000 mg/L streptomycin for transconjugant selection. Subsequently, we transferred an R plasmid from an E. coli transconjugant back to a rifampicin-resistant mutant of S.
Anatum strain R13.0957 by using LB medium with 50 mg/L ampicillin and 150 mg/L rifampicin for transconjugant selection. The Page 3 of 6 plasmids and their sizes were estimated using a S1-PFGE method (5). The sequences of R plasmids from transconjugants were determined using MinION nanopore sequencer or/and Illumina MiSeq sequencer.