Does Salmonella diarizonae 58:r:z53 Isolated from a Mallard Duck Pose a Threat to Human Health?

Salmonella diarizonae (IIIb) is frequently isolated from reptiles and less frequently from birds and mammals. However, its isolation from invasive human infections has not been widely reported. Migratory mallard ducks are excellent bioindicators of pathogen presence and pathogen antibiotic resistance (AMR). We present the first isolation from a mallard duck in central Europe of the antibiotic-resistant Salmonella enterica subsp. diarizonae with the unique antigenic pattern 58:r:z53 and report its whole-genome sequencing, serosequencing, and genotyping, which enabled the prediction of its pathogenicity and comparison with phenotypic AMR. The isolated strain was highly similar to S. diarizonae isolated from humans and food. Twenty-four AMR genes were detected, including those encoding aminoglycoside, fluoroquinolone, macrolide, carbapenem, tetracycline, cephalosporin, nitroimidazole, peptide antibiotic, and disinfecting agent/antiseptic resistance. Six Salmonella pathogenicity islands were found (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, and SPI-13). An iron transport system was detected in SPI-1 centisome C63PI. Plasmid profile analyses showed three to be present. Sequence mutations in the invA and invF genes were noted, which truncated and elongated the proteins, respectively. The strain also harbored genes encoding type-III secretion-system effector proteins and many virulence factors found in S. diarizonae associated with human infections. This study aims to elucidate the AMR and virulence genes in S. enterica subsp. diarizonae that may most seriously threaten human health.


Introduction
The Salmonella genus, containing 2659 serovars, is divided into two species: Salmonella enterica with 2637 and Salmonella bongori with the remaining 22.There are six subspecies of Salmonella enterica: enterica (I) containing 1586 serovars, salamae (II) with 522, arizonae (IIIa) with 102, diarizonae (IIIb) with 338, houtenae (IV) with 76, and indica (VI) with the final 13 [1].The Salmonella enterica species is a widely known pathogen causing serious illness in humans and animals.Some serotypes of Salmonella enterica subsp.enterica are particularly well described and are known to cause a wide range of food-and water-borne illnesses [2][3][4][5][6][7].Salmonella enterica subsp.diarizonae is the third most abundant subspecies of the genus.The diarizonae (IIIb) subspecies is commonly isolated from a variety of cold-blooded animals, including snakes, turtles, and lizards, as well as from birds, sheep, and even humans [8][9][10][11].Although human infection caused by S. diarizonae seems to be rare, more and more cases of gastroenteritis and bacteremia are being described in the global literature [12][13][14].
Salmonella diarizonae 58:r:z 53 was first isolated in Germany in 1985 from snake feces and in 2021 it was isolated in Poland as a multidrug-resistant bacterium from a mallard duck (Anas platyrhynchos).In this study, we present a genome analysis of this rare multidrugresistant strain to demonstrate its potential human pathogenicity and virulence features.

Source Animal and Antimicrobial Resistance of the Isolate
The mallard duck from the intestine of which the S. enterica subsp.diarizonae 58:r:z 53 (S.IIIb 58:r:z 53 ) strain was isolated had no pathological changes when given an anatomopathological examination and was in good physical condition.The isolated strain was found to be resistant to 14 of the 33 antibiotics tested [15].It was sensitive to ampicillin, amoxicillin, trimethoprim-sulfamethoxazole, third-generation cephalosporin, and imipenem.

Genotypic Serotyping
Multidirectional whole-genome sequencing (WGS) analysis predicted the O antigen to be the serotype 58 variant, the H1 antigen (fliC) to be r, and the H2 antigen (fljB) to be z53.The strain was tentatively classified as Salmonella enterica subsp.diarizonae (subspecies IIIb) serotype 58:r:z53 and named Salmonella IIIb 58:r:z53.

Genotypic Serotyping
Multidirectional whole-genome sequencing (WGS) analysis predicted the O antigen to be the serotype 58 variant, the H1 antigen (fliC) to be r, and the H2 antigen (fljB) to be z 53 .The strain was tentatively classified as Salmonella enterica subsp.diarizonae (subspecies IIIb) serotype 58:r:z 53 and named Salmonella IIIb 58:r:z 53 .

Antimicrobial Resistance of Salmonella IIIb 58:r:z 53
A total of 20 different antimicrobial resistance genes were identified, all having been detected in the chromosome.Additionally, the tolC, marA, sdiA, and emrD genes were detected, which are associated with resistance to disinfecting agents and antiseptics including triclosan (Table 2, Supplementary Table S1).The concordance between phenotypic resistance and the presence of known AMR genes was not consistent with the genotype for all antimicrobials.Most of the detected AMR genes were associated with aminoglycoside resistance and were in a Salmonella isolate, which was phenotypically gentamycin-, streptomycin-, and neomycin-resistant.Similarly, the present macrolide-resistance genes were associated with phenotypic resistance to erythromycin and tylosin in the IIIb 58:r:z 53 strain.Fluoroquinolone-resistance genes were present in the S. IIIb 58:r:z 53 strain, which was nevertheless sensitive to enrofloxacin and marbofloxacin but not to flumequine.Salmonella IIIb 58:r:z 53 showed intermediate resistance to florphenicol and four genes (emrD, sdiA, acrA, and acrB) from the multidrug efflux pump family were identified.The carriage of tolC and marA, which confer carbapenem resistance, was detected in Salmonella IIIb 58:r:z 53 but did not prevent the strain from being sensitive to imipenem.

Phylogenetic Analysis of Salmonella IIIb 58:r:z 53
To infer the phylogenetic affinities of the Salmonella IIIb 58:r:z 53 isolate, the maximumlikelihood phylogeny was estimated using kSNP4 ( https://sourceforge.net/projects/ksnp/) and based on the core single-nucleotide polymorphisms computed from 31 genomes downloaded from the GenBank database (Supplementary Table S2).Additionally, whole-genomeand whole-proteome-based trees were inferred using the TYGS strain-typing server (Supplementary Figure S4).Phylogenetic analysis using both tools showed that the strains named in the National Center for Biotechnology Information BioSample database as Salmonella enterica diarizonae serovar Salmonella arizonae 341279 and Salmonella enterica diarizonae serovar Salmonella diarizonae 605789 isolated from humans in the UK formed a single distinct clade with the tested strain.This single clade also included strain FMA0161 (Salmonella enterica subsp.diarizonae IIIb 58:r:-) isolated from cream-filled wafers in Taiwan in 2011, which showed the highest average nucleotide identity (99.65%) and conserved DNA threshold (84.15%) (Figure 9) (Supplementary Table S2).
serovar Salmonella arizonae 341279 and Salmonella enterica diarizonae serovar Salmonella diarizonae 605789 isolated from humans in the UK formed a single distinct clade with the tested strain.This single clade also included strain FMA0161 (Salmonella enterica subsp.diarizonae IIIb 58:r:-) isolated from cream-filled wafers in Taiwan in 2011, which showed the highest average nucleotide identity (99.65%) and conserved DNA threshold (84.15%) (Figure 9) (Supplementary Table S2).
Figure 9. Phylogenetic analysis of the Salmonella enterica subsp.diarizonae IIIb 58:r:z53 strain as a maximum-likelihood tree of S. diarizonae isolates built using core genome single-nucleotide polymorphisms.The inner ring represents the country from which each isolate originated and the outer ring indicates the isolation source.Closely related strains are marked by the blue oval.Salmonella bongori N268-08 was used as the outgroup.All strains are presented in Table S2.Singlenucleotide polymorphisms were identified with kSNP4 [22].

Discussion
Salmonella enterica subsp.enterica is responsible for over 99% of cases of human salmonellosis and is therefore the subject of extensive research.However, there is still only very limited published research and genomic information about the non-enterica subspecies [12,13,23,24].Our whole-genome analysis revealed that there were six known Salmonella pathogenicity islands in Salmonella IIIb 58:r:z53 (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, and SPI-13) and there was also centisome C63PI, an iron transport system.Genes from SPI-2, SPI-3, and SPI-13 are reported to be required for S. Typhimurium survival and replication in macrophages [24].All of them were found in Salmonella IIIb 58:r:z53.In addition, SPI-1 and SPI-5, which are crucial for the intracellular lifestyle of the pathogen, were present in Salmonella IIIb 58:r:z53.The iron transport system C63PI in SPI-1, crucial for the entry of Salmonella into host cells, was also found in Salmonella IIIb 58:r:z53 [25].
Plasmids P1 and P2 contained many of the SPI-7 genes predicted to be related to virulence (the type IVb pilus pil and tra regions).Therefore, the presence of the pil locus in diarrheagenic bacteria may have contributed to their ability to infect humans.
The comparison of Salmonella IIIb 58:r:z53 with S. diarizonae isolated from newborn child infections revealed that 11 out of 12 type-III effector genes were present in all strains, Figure 9. Phylogenetic analysis of the Salmonella enterica subsp.diarizonae IIIb 58:r:z 53 strain as a maximum-likelihood tree of S. diarizonae isolates built using core genome single-nucleotide polymorphisms.The inner ring represents the country from which each isolate originated and the outer ring indicates the isolation source.Closely related strains are marked by the blue oval.Salmonella bongori N268-08 was used as the outgroup.All strains are presented in Table S2.Single-nucleotide polymorphisms were identified with kSNP4 [22].

Discussion
Salmonella enterica subsp.enterica is responsible for over 99% of cases of human salmonellosis and is therefore the subject of extensive research.However, there is still only very limited published research and genomic information about the non-enterica subspecies [12,13,23,24].Our whole-genome analysis revealed that there were six known Salmonella pathogenicity islands in Salmonella IIIb 58:r:z 53 (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, and SPI-13) and there was also centisome C63PI, an iron transport system.Genes from SPI-2, SPI-3, and SPI-13 are reported to be required for S. Typhimurium survival and replication in macrophages [24].All of them were found in Salmonella IIIb 58:r:z 53 .In addition, SPI-1 and SPI-5, which are crucial for the intracellular lifestyle of the pathogen, were present in Salmonella IIIb 58:r:z 53 .The iron transport system C63PI in SPI-1, crucial for the entry of Salmonella into host cells, was also found in Salmonella IIIb 58:r:z 53 [25].
Plasmids P1 and P2 contained many of the SPI-7 genes predicted to be related to virulence (the type IVb pilus pil and tra regions).Therefore, the presence of the pil locus in diarrheagenic bacteria may have contributed to their ability to infect humans.
The comparison of Salmonella IIIb 58:r:z 53 with S. diarizonae isolated from newborn child infections revealed that 11 out of 12 type-III effector genes were present in all strains, namely steC, sseJ, sseG, sseF, sptP, sopE2, sopB, sipA, sipB, sipD, and avrA [26].Moreover, Salmonella IIIb 58:r:z 53 harbored the gene encoding the glycosyltransferase SseK2, which interferes with the proper immune response to infection through tumor necrosis factoralpha-stimulated nuclear factor kappa B cell signaling.The host immune response is also hampered by the E3 ubiquitin ligase SlrP, known to have been produced by S. diarizonae and isolated from human and animal infections and by Salmonella IIIb 58:r:z 53 because it inhibits the release of interleukin-1β.Fimbriae on the Salmonella cell surface mediate adhesion to host cells [27].Salmonella IIIb 58:r:z 53 bore the mig-14 gene coding antimicrobial resistance protein Mig-14, important in bacterial resistance to antimicrobial peptides and necessary for replication of the S. Typhimurium serovar in the liver and spleen [28].
The Salmonella IIIb 58:r:z 53 P1 plasmid bore the samA gene, part of the samAB operon.The operon of which samA is part efficiently promotes UV mutagenesis when carried on a high-copy-number 60-MDa cryptic plasmid, as observed in research concerning the samAB operon in the Typhimurium serovar chromosome [29].
Aminoglycosides interfere with bacterial protein synthesis by binding to the bacterial 30S or ribosomal subunit.Aminoglycoside 6 ′ -N-acetyltransferase (AAC(6 ′ )) inactivates aminoglycoside antibiotics through acetylation of the 6-amino-acid group of the compound.One aminoglycoside found in S. enterica, AAC(6 ′ )-Iy, is a cryptic chromosomally encoded aminoglycoside acetyltransferase that has been shown to confer extensive aminoglycoside resistance in strains expressing the structural gene.
Most of the Salmonella IIIb 58:r:z 53 AMR mechanism was associated with its antibiotic efflux pump.Genes involved in the pump mechanism-marR encoding the MarR regulator of the AcrAB multidrug efflux pump and msbA encoding the multidrug resistance transporter-were found in Salmonella IIIb 58:r:z 53 and S. diarizonae isolated from invasive newborn child infections [26].Additionally, the Salmonella IIIb 58:r:z 53 genome also contained tolC, marA, sdiA, and emrD associated with resistance to disinfecting agents and antiseptics including triclosan.A human isolate of S. enterica subsp.diarizonae serovar IIIb 48:i:z was found to contain the marA gene [26].
Three approaches to phylogenetic relationship reconstruction were taken in this study: core SNP identification and whole-genome-and whole-proteome-based strategies.They revealed that the S. IIIb 58:r:z 53 strain was clustered with a Taiwanese strain (isolated from food) and S. enterica subsp.diarizonae from the UK (isolated from humans).This shows that the isolate presented in this study could unquestionably infect humans and the fact that it could be present in meat for human consumption indicates that it could be widespread and a real threat to public health.

Sampled Animal
The analyzed S. enterica spp.diarizonae (S.IIIb 58:r:z 53 ) strain was isolated from the intestine of a mallard duck (Anas platyrhynchos) shot as prey in accordance with the hunting law in force in Poland [30,31].The mallard duck was in good physical condition, with no pathological changes observed when given an anatomopathological examination.

Salmonella spp. Isolation and Identification
Salmonella spp.were isolated in accordance with PN-EN ISO 6579-1:2017-04 Microbiology of the food chain-horizontal method for the detection, enumeration, and serotyping of Salmonella-Part 1: Detection of Salmonella spp.[32].The microbiological media used were described in a publication by Pławi ńska-Czarnak et al. [15].Biochemical strain identification was performed using a VITEK ® 2 GN (Gram-Negative) card and API20E test (BioMérieux, Craponne, France) according to the manufacturer's instructions.For serological typing, the strains were originally grown on 2% nutrient agar slants and reisolated on Salmonella-Shigella agar, Hektoen agar, Bismuth sulfite agar, and Xylose Lysine Deoxycholate agar (Merck, Darmstadt, Germany) before serotyping.The presumptive colonies of Salmonella strains were chosen and were cultured on Enrichment LAB-AGAR TM (BioMaxima, Lublin, Poland) at 37 • C overnight.These cultures were used for serological identification.The antigenic formula of the Salmonella strains was determined with the use of standard agglutination methods and the serotype name was assigned according to the White-Kaufmann-LeMinor (WKL) scheme [1,33].A small amount of bacterial mass from one colony was first checked by slide agglutination for a positive reaction with polyvalent HM serum and next, somatic O antigen was identified by slide agglutination in a drop of serum.Then, each strain was grown on swarm agar plates (BioMaxima, Lublin, Poland) at 37 • C overnight to test phases 1 and 2 of H antigens by slide agglutination.Polyvalent and monovalent anti-O and anti-H diagnostic sera for Salmonella antigens were purchased from SSI Diagnostica A/S (Hillerød, Denmark), Sifin Diagnostics GmbH (Berlin, Germany), and BIOMED (Kraków, Poland).Salmonella antigens were classified into serotypes by the WKL scheme.When a serovar had not been previously recorded in Poland, the strain representing this newly recognized serovar was sent to the WHO Collaborating Centre for Reference and Research on Salmonella (Institut Pasteur, Paris, France) for the identification to be verified and confirmed [15].

Antimicrobial Sensitivity Testing
The Salmonella strain was subcultured as described previously.From an 18-24 h culture, a suspension was prepared to 0.5 McFarland turbidity with a DensiCHEK Plus instrument (BioMérieux, Marcy-l'Étoile, France) and the inoculum was transferred to another VITEK ® tube containing 3 mL of 0.45% saline.The card was automatically filled by a vacuum device and automatically sealed.It was manually inserted in the VITEK2 Compact reader-incubator module (BioMérieux, Craponne, France) and the card was automatically subjected to a kinetic fluorescence measurement every 15 min.This is a test methodology based on the minimum inhibitory concentration (MIC) technique reported by MacLowry and Marsh [34] and Gerlach [35].To analyze MIC patterns of S. enterica subsp.diarizonae, a MERLIN MICRONAUT system (MERLIN Diagnostika GmbH, Bremen, Germany) was used.The MICs were interpreted according to the Clinical and Laboratory Standards Institute and Food and Drug Administration breakpoints [36].The antimicrobial susceptibility was assessed by determining the MIC values using 96-well MICRONAUT Special Plates in a protocol described by Pławi ńska-Czarnak et al. in 2022 [37].

Phylogenetic Analysis
The phylogenetic analysis of S. IIIb strain 58:r:z 53 was performed including the genomic sequences of other S. enterica subsp.diarizonae members that are available in GenBank.
In total, 31 sequences were selected using the Referenceseeker tool [45] and based on an ANI value of 99% and conserved DNA threshold of 0.69.Those 31 comprised 10 incomplete genome sequences (drafts) and 21 complete ones.Salmonella bongori N268-08 was used as the outgroup.The sequences were uploaded to the TYGS typing server [46] and a whole-genome sequence-based tree was constructed.
Phylogenetic analysis was also performed taking a reference-free SNP-based approach using kSNP4 software [22].A phylogenetic tree was constructed based on the maximumlikelihood method and visualized using the ggtree R package 3.12.0( https://bioconductor .org/packages/release/bioc/html/ggtree.html, accessed on 29 February 2024).
for the purposes of the study.The hunt took place in accordance with Polish hunting law (Act of the Polish Parliament dated 13 October 1995, item 713, the Hunting law, Chapter 3, Art.8 Hunt) during the 2021 hunting season.
Informed Consent Statement: Not applicable.

18 Figure 1 .
Figure 1.Illustration of the distribution of the genome annotations of Salmonella enterica subsp.diarizonae 58:r:z53 isolated from a mallard duck in Poland.The protein-coding sequence (CDS) elements of the figure are shown in gray for the position label (Mbp: megabase pairs); GC: guanine and cytosine; tRNA: transfer RNA; rRNA: ribosomal RNA.Visualized with GenoVi [16], accessed on 6 March 2024.

Figure 1 .
Figure 1.Illustration of the distribution of the genome annotations of Salmonella enterica subsp.diarizonae 58:r:z 53 isolated from a mallard duck in Poland.The protein-coding sequence (CDS) elements of the figure are shown in gray for the position label (Mbp: megabase pairs); GC: guanine and cytosine; tRNA: transfer RNA; rRNA: ribosomal RNA.Visualized with GenoVi [16], accessed on 6 March 2024.

Figure 2 .
Figure 2. Genome map of S. enterica subsp.diarizonae 58:r:z53.The innermost rings show genome positions (Mbp: megabase pairs) and GC content, shown in black.The outer rings represent the coding orientation, with the forward strand on the outside and the reverse strand on the inside.Created with Proksee [17].

Figure 2 .
Figure 2. Genome map of S. enterica subsp.diarizonae 58:r:z 53 .The innermost rings show genome positions (Mbp: megabase pairs) and GC content, shown in black.The outer rings represent the coding orientation, with the forward strand on the outside and the reverse strand on the inside.Created with Proksee [17].

Figure 3 .
Figure 3. Illustration of the S. diarizonae 58:r:z53 plasmid 1 (p28P1, length 170,536 base pairs (bp)).The outermost and innermost rings represent the coding orientation, with the forward and reverse strands, respectively.The two central rings present mobile genetic element (MGE) annotation with the mobile orthologous groups database (Mobile OG DB).Regions involved in stability/transfer/defense are shown in blue, plasmid transfer in green, integration/excision in orange, and replication/recombination/repair in pink.Created with Proksee [17].

Figure 3 .
Figure 3. Illustration of the S. diarizonae 58:r:z 53 plasmid 1 (p28P1, length 170,536 base pairs (bp)).The outermost and innermost rings represent the coding orientation, with the forward and reverse strands, respectively.The two central rings present mobile genetic element (MGE) annotation with the mobile orthologous groups database (Mobile OG DB).Regions involved in stability/transfer/defense are shown in blue, plasmid transfer in green, integration/excision in orange, and replication/recombination/repair in pink.Created with Proksee [17].

Figure 4 .
Figure 4. Illustration of S. diarizonae 58:r:z53 plasmid 2 (p28P2, length 102,826 bp).The outermost and innermost rings represent the coding orientation, with the forward and reverse strands, respectively.The two central rings present MGE annotation with Mobile OG DB.Regions involved in stability/transfer/defense are shown in blue, plasmid transfer in green, integration/excision in orange, and replication/recombination/repair in pink.Created with Proksee [17].

Figure 4 .
Figure 4. Illustration of S. diarizonae 58:r:z 53 plasmid 2 (p28P2, length 102,826 bp).The outermost and innermost rings represent the coding orientation, with the forward and reverse strands, respectively.The two central rings present MGE annotation with Mobile OG DB.Regions involved in stability/transfer/defense are shown in blue, plasmid transfer in green, integration/excision in orange, and replication/recombination/repair in pink.Created with Proksee [17].

Figure 5 .
Figure 5. Illustration of S. diarizonae 58:r:z53 plasmid 3 (p28P3, 6462 bp).The outermost and innermost rings represent the coding orientation, with the forward and reverse strands, respectively.Regions involved in stability/transfer/defense are shown in blue, plasmid transfer in green, and replication/recombination/repair in pink.Created with Proksee [17].

Figure 5 .
Figure 5. Illustration of S. diarizonae 58:r:z 53 plasmid 3 (p28P3, 6462 bp).The outermost and innermost rings represent the coding orientation, with the forward and reverse strands, respectively.Regions involved in stability/transfer/defense are shown in blue, plasmid transfer in green, and replication/recombination/repair in pink.Created with Proksee [17].

Figure 6 .
Figure 6.Three-dimensional view of (a) the full-length InvA protein (685 amino acids (aa)), (b) the truncated S. diarizonae 58:r:z 53 InvA protein (665 aa), and (c) the truncated S. diarizonae 58:r:z 53 InvA protein with no loss of transmembrane structure.Created with Phyre2, Protein Homology/analogY Recognition Engine V 2.0, Structural Bioinformatics Group, Imperial College, London, UK. (a,b) are rainbow-colored from the N to C terminus.

Table 1 .
Salmonella pathogenicity islands (SPIs) detected in the genome of Salmonella enterica subsp.diarizonae 58:r:z 53 isolated from a mallard duck in Poland and the GenBank references of previous detections of those islands.

Table 2 .
Genes related to antibiotic resistance found in S. diarizonae 58:r:z 53 .