Salmonella enterica serovar Infantis from Food and Human Infections, Switzerland, 2010–2015: Poultry-Related Multidrug Resistant Clones and an Emerging ESBL Producing Clonal Lineage

Objectives: The aim of this study was to characterize a collection of 520 Salmonella enterica serovar Infantis strains isolated from food (poultry meat), human infections and environmental sources from the years 2010, 2013 and 2015 in Switzerland. Methods: We performed antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) analysis on all 520 S. Infantis isolates, and whole genome sequencing (WGS) on 32 selected isolates. Results: The majority (74.8%) of the isolates was multidrug resistant (MDR). PFGE analysis revealed that 270 (51.9%) isolates shared an identity of 90%. All isolates subjected to WGS belonged to sequence type (ST) 32 or a double-locus variant thereof (one isolate). Seven (21.9%) of the sequenced isolates were phylogenetically related to the broiler-associated clone B that emerged in Hungary and subsequently spread within and outside of Europe. In addition, three isolates harboring blaCTX-M-65 on a predicted large (∼320 kb) plasmid grouped in a distinct cluster. Conclusion: This study documents the presence of the Hungarian clone B and related clones in food and human isolates between 2010 and 2015, and the emergence of a blaCTX-M-65 harboring MDR S. serovar Infantis lineage.


INTRODUCTION
Non-typhoidal Salmonella enterica (NTS) are one of the most important etiological agents of foodborne diarrheal diseases in humans worldwide and cause an estimated 80.3 million foodborne illnesses a year (Majowicz et al., 2010). Although most cases of salmonellosis are self-limiting episodes of gastro-enteritis, severe cases of infection, including bacteremia and meningitis require antimicrobial treatment. Ciprofloxacin is a common first-line antimicrobial for treating salmonellosis but because fluoroquinolones are not used for treating children, β-lactams (ampicillin or third-generation cephalosporins) are of equal importance (Medalla et al., 2016). Multidrugresistant (MDR) NTS is associated with higher morbidity and mortality outcomes compared to drug-susceptible strains and is a major public health concern (Mølbak, 2005). Salmonella enterica subsp. enterica serovar Infantis (S. serovar Infantis) has emerged as the fourth most common serovar causing human salmonellosis in Europe, with 1,846 cases reported by the EU/EEA countries in 2014 (European Food Safety Authority [EFSA] and European Centre for Disease Prevention [ECDC], 2016). Poultry, especially from layer and broiler farms, as well as pigs are the main animal reservoirs for S. serovar Infantis (Nógrády et al., 2012). This serovar is also dominant in broiler meat, accounting for 35.9% of all Salmonella isolates reported from EU countries in 2014 (European Food Safety Authority [EFSA] and European Centre for Disease Prevention [ECDC], 2016). Over the last few years, antimicrobial resistance has emerged in S. serovar Infantis isolates from human and animal sources in various European countries and consequently, this serovar, together with S. Kentucky, contributes significantly to the numbers of MDR Salmonella in Europe (Nógrády et al., 2008;Dionisi et al., 2011). Closely related MDR clones of S. serovar Infantis have disseminated among broiler populations and associated animal growing environments, ultimately being disseminated into the food chain and then into humans in European countries such as Hungary, Poland and Austria (Nógrády et al., 2012). Isolates belonging to these clones are characterized by their resistance to nalidixic acid, sulfamethoxazole, streptomycin and tetracycline (NaSSuT). Recently, resistance to third generation cephalosporins has emerged in S. serovar Infantis isolates in Italy, due to the circulation of an extended-spectrum β-lactamase (ESBL) producing, MDR clone with additional reduced susceptibility to ciprofloxacin (Franco et al., 2015). The spread of MDR S. serovar Infantis clones throughout the food production system (mainly poultry and poultry meat) and in humans is highly worrisome and warrants improved understanding of its epidemiology. In Switzerland, S. serovar Infantis ranks among the top five of Salmonella serovars registered by the National Centre for Enteropathogenic Bacteria and Listeria (NENT). However, currently no data on antimicrobial resistance patterns or clonal relationships of the isolates exist, despite its clinical importance.
The aim of this study was to characterize a collection of 520 S. serovar Infantis strains isolated from food (poultry meat), human infections and environmental sources from the years 2010, 2013 and 2015 in Switzerland (i) by determining their phenotypic antibiotic resistance profiles using the disk diffusion method and (ii) by assessing genotypic characteristics and clonal relatedness using molecular methods including pulsed-field gel electrophoresis (PFGE), PCR, and whole genome sequencing (WGS).

Bacterial Strains
A total of 520 non-duplicate S. serovar Infantis isolates from human infections (n = 84), poultry meat (n = 418) and other sources (n = 18) were collected during 2010, 2013 and 2015 at the National Centre for Enteropathogenic Bacteria and Listeria (NENT), Switzerland. The isolates had been forwarded by hospitals, diagnostic laboratories or surveillance programs of retail markets and food or feed producing facilities for final species-level identification according to the White-Kauffmann-Le Minor scheme (Grimont and Weill, 2008).

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed using the disk-diffusion method and the antibiotics ampicillin (AM), amoxicillin-clavulanic acid (AMC), cefotaxime (CTX), nalidixic acid (Na), ciprofloxacin (CIP), gentamicin (GM), kanamycin (K), streptomycin (S), sulfamethoxazole (Su), trimethoprim (TMP), tetracycline (T), and chloramphenicol (C) (Becton Dickinson, Heidelberg, Germany). Results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) performance standards (CLSI, 2016). For sulfamethoxazole, for which breakpoints are not listed separately from trimethoprim, an inhibition zone of ≤10 mm was interpreted as resistant. Isolates displaying resistance to three or more classes of antimicrobials (counting β-lactams as one class) were defined as multidrug-resistant (MDR). Synergistic effects between AMC and CTX were regarded as an indication of the presence of an ESBL producer (Kaur et al., 2013).

Detection and Characterization of Extended-Spectrum β-Lactamase (bla) Genes
Putative ESBL producers were grown on Brilliance TM ESBL agar (Oxoid, Hampshire, United Kingdom). The presence of bla ESBLs was confirmed by PCR by screening for bla TEM , bla SHV , and bla CTX−M alleles belonging to CTX-M groups 1, 2, 8, 9, and 25 as described previously (Woodford et al., 2006;Geser et al., 2012;Zurfluh et al., 2015). Synthesis of primers and DNA custom sequencing was carried out by Microsynth (Balgach, Switzerland) and nucleotide sequences were analyzed with CLC Main Workbench 6.6.1. For database searches the BLASTN program of NCBI 1 was used.

Pulsed-Field Gel Electrophoresis
Pulsed-field gel electrophoresis was performed according to the PulseNet protocol of the Centers for Disease Control and Prevention (CDC) 2 , using the restriction enzyme XbaI (Roche, Mannheim, Germany) and Salmonella serovar Braenderup strain H9812 (ATCC BAA 664) as the reference strain. Restricted DNA was separated in a 1% agarose gel (BioRad, Cressier, Switzerland) at 12 • C for 20 h at 6 V/cm under linear ramp with switch times from 2 to 64 s and 120 • included angle using a CHEF-DR III system (BIO-RAD, Munich, Germany). Gels were stained with ethidium bromide and visualized under UV light with Gel Doc (BIO-RAD, Munich, Germany). GelCompar II software (Applied Maths NV, Sint-Martens-Latem, Belgium) was used for analysis. Pairwise similarities between the XbaI PFGE patterns were calculated by the DICE similarity coefficient.
Clustering was based on the unweighted pair-group method with averages, setting tolerance at 1% and optimization at 0.5%.
recommended workflow on CLC Genomics Work bench version 8.0 (CLC bio, Aarhus, Denmark). Genomic contigs were annotated using the RAST annotation server (Aziz et al., 2008). The assembled sequences were uploaded to the http://www.genomicepidemiology.org/ server. Sequences of the seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA and thrA) were analyzed to identify multilocus sequence types (MLST) 3 , antibiotic resistance genes 4 , and plasmid replicon types 5 , using each website's algorithms and databases. Routine processing of genome datasets was carried out by in-house perl scripts (available upon request). A local customized database of Salmonella chromosomal and plasmid genomes from NCBI was created and used for annotation, plasmid analysis, and homology detection with BLAST suite (Altschul et al., 1990). Phylogenetic analysis was conducted using a multiloci analysis based on a published core gene dataset (Leekitcharoenphon et al., 2012). Alleles in 2780 core gene loci across all the genomes were identified with Salmonella enterica subsp. enterica serovar Typhimurium LT2 genome as the reference. Of these loci, 1,500 core gene loci were randomly chosen and alleles were concatenated. The data matrix of alleles was subject to multiple alignment and phylogenetic analysis using tools available in MEGA7 suite (Kumar et al., 2016). The phylogenetic tree was built using the Neighbor-Joining algorithm in the MEGA 7 suite with 9,562 positions across 50 genomes for the final analysis. For genome comparison, whole genome draft sequences of Salmonella enterica serovar Infantis strains from public databases were retrieved, including four recent strains of the prevalent Hungarian clone B of S. serovar Infantis from Hungary (Wilk et al., 2016(Wilk et al., , 2017, five bla ESBL harboring, and eight non-bla ESBL harboring strains from Italy, Israel and the United States. (Aviv et al., 2014;Franco et al., 2015), and one fully susceptible strain from the United Kingdom (Olasz et al., 2015). An overview of the strains and their GenBank accession numbers is given in Table 1. For plasmid comparison, annotations were obtained from RAST (Aziz et al., 2008), and compared with pCSAM042077 (Tate et al., 2017) on the SEED server for additional verification (Overbeek et al., 2013).

Antimicrobial Susceptibility Testing
The distribution of resistance phenotypes among the S. serovar Infantis isolates is summarized in the Table 2 and shown in detail in technical Supplementary Tables S1-S3.
Multidrug resistant was detected in 389 (74.8%) of the isolates. Thereof, the majority (379/520, 72.9% of all isolates) showed a combined resistance pattern to nalidixic acid, sulfamethoxazole and tetracycline, with (NaSSuT) or without streptomycin (NaSuT). The NaSSut pattern was detected in 189 (36.3% of all isolates) and the NaSuT pattern in 190 (36.5% of all isolates). Resistance to ampicillin and ciprofloxacin was verified in 20 (3.8%) and 24 (4.6%) of the isolates, respectively, and showed a rising prevalence between 2010 and 2015 for both antimicrobials, as illustrated in Figure 1. A total of 496 (95.4% of all isolates) tested within the intermediate range for ciprofloxacin, according to CLSI breakpoints. An ESBL phenotype and growth on Brilliance TM ESBL agar was recorded for three strains (0.6% of all isolates), whereof two originated from humans (isolates 21-13 and 125-15) and one (isolate 144-13) from food ( Table 3 and  Supplementary Tables S2, S3).
Frontiers in Microbiology | www.frontiersin.org Whole genome sequencing did not detect any resistance genes in six non-MDR isolates ( Table 3 and Supplementary  Tables S1-S3), except for human isolate 153-13 which showed intermediate resistance to streptomycin and harbored aadA1 and sul1 (Table 3 and Supplementary Table S2).

Detection of Plasmids and Replicon Types among the Isolates
By mapping WGS assemblies to the 320 kb plasmid pCFSAN42077 from bla CTX−M−65 carrying food isolate CFSAN42077 (Tate et al., 2017), contigs or regions similar to FIGURE 2 | Multiple sequence alignment with pCSAM042077 from S. Infantis from food from the United States (FSIS150291), of a bla CTX−M−65 cassette on putative ∼320 kb plasmids from three S. Infantis strains from food (144-13) and diseased humans (21-13 and 125-15) from Switzerland and one S. Infantis strain from Italy (ERR1014119). The ∼5 kb cassette consists of bla CTX−M−65 followed by an IS5/IS1182 family transposase and a gene encoding a TonB-dependent siderophore receptor, and one or two hypothetical genes. The cassette is flanked on either side by the IS6 transposase. The fipA gene is of varied lengths, or absent. this plasmid in isolates 21-13, 144-13 and 125-15 were detected (data not shown). Specific contigs bearing the bla CTX−M−65 gene and surrounding loci were identified (Figure 2). The ∼5 kb cassette consisted of bla CTX−M−65 followed by an IS5/IS1182 family transposase and a gene encoding a TonB-dependent siderophore receptor. The 5 -end is flanked by an IS6 family transposase upstream of the fipA gene, which is truncated in pCFSAN42077. By comparison, fipA is absent in p14026835, a bla CTX−M−65 harboring plasmid from human S. Infantis isolate 14026835 from Italy (ERR1014119), as shown in Figure 2. As predicted, no replicons were detected in the genomes of these three bla CTX−M−65 harboring isolates (Franco et al., 2015).

MLST and Phylogenetic Analysis
All 32 isolates subjected to WGS belonged to sequence type (ST) ST32, except isolate 21-13, which was a double-locus variant (purE and sucA) of ST32 (Table 3).
Genetic relatedness was investigated by mapping the 32 genomes to the genome sequences of isolates belonging to the Hungarian clone B from broilers, the bla ESBL , and non-bla ESBL harboring strains from Italy, Israel and United States. The resulting unrooted phylogenetic tree is shown in Figure 3. The isolates segregated into eight major clusters. Clusters determined by PFGE and by WGS did not correlate.
Close phylogenetic relatedness with strains 3337 12 H, 757 13 H, 786 13H, 1070 16 belonging to the Hungarian clone B was detected in seven (21.9%) of the sequenced strains and included human and food isolates from 2010, 2013 and 2015 (Figure 3). The bla CTX−M−65 harboring isolates 21-13, 144-13 and 125-15 were more distantly related to the isolates that clustered with the Hungarian clone B. All three isolates were closely related to, and formed a distinct cluster with the bla CTX−M−65 harboring human isolate (italy 2014) from Italy and the food isolate CFSAN 042077 from the United States.

Accession Numbers
The accession numbers of the 32 sequenced isolates are listed in Table 3 and Supplementary Tables S1-S3. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession NAOX00000000. The version described in this paper is version NAOX00000000.1.

DISCUSSION
S. serovar Infantis has emerged as an important disseminator of MDR in the food chain, representing a threat to human health (Nógrády et al., 2012). The majority of strains in this study exhibited either the NaSuT pattern or the characteristic NaSSuT pattern previously identified in the MDR S. Infantis clone B that emerged in broilers in farms in Hungary and subsequently spread within and outside of Europe, including to the countries of Poland, Austria, Germany, Israel and Japan (Hauser et al., 2012;Nógrády et al., 2012). WGS performed in this study detected clonality in strains deemed different by PFGE and provides evidence that the Hungarian clone B has persisted since at least 2010 in Switzerland within the food chain and is associated with human disease. Strains belonging to three other closely related clusters were less prevalent but showed similar resistance patterns and persistence within food and human isolates.
In addition, we observed the occurrence of S. serovar Infantis harboring bla CTX−M−65 . This bla gene has been detected previously in one human S. serovar Infantis isolate in Great Britain and in one from Italy (Burke et al., 2014), and furthermore, in an outbreak of S. Infantis in Ecuador (Cartelle Gestal et al., 2016), as well as in food in the United States. The results presented in this study show that the bla CTX−M−65 harboring S. serovar Infantis from Switzerland belong to a unique lineage but are similar to the strains from Italy and the United States, suggesting the emergence of a bla CTX−M−65 harboring, MDR S. serovar Infantis lineage in Europe as well as in North and South America. Moreover, this study shows that this lineage has been present in food and humans at least as early as 2013 in Europe.
Whole genome sequencing of these strains indicate the presence of a ∼320 kb plasmid similar to the pESI plasmid, a megaplasmid carrying multiple resistance and virulence genes originally detected in S. serovar Infantis in Israel in 2008 (Aviv et al., 2014). In addition, resistance genes drfA14 and fosA (trimethoprim and phenicol resistance genes, respectively) which are also found on pESI-like plasmids (Franco et al., 2015), were detected in all bla CTX−M−65 harboring strains. Several other non-bla ESBL harboring strains from this study had a close phylogenetic-relatedness to strains from Israel and Italy which harbor non-bla ESBL pESI or pESIlike plasmids (Aviv et al., 2014), suggesting that certain S. serovar Infantis clones or lineages are acquiring these plasmids independently.
Our results correlate with the recent detection of a clone harboring a pESI-like plasmid and the bla CTX−M−1 gene in the broiler industry and humans in Italy (Franco et al., 2015), and suggest that some S. serovar Infantis clones harboring pESIlike plasmids may be undergoing a microevolution by acquiring bla ESBL genes. While in Europe bla CTX−M−1 is prevalent within the poultry industry and has been well documented (Zurfluh et al., 2014), bla CTX−M−65 has rarely been described. By contrast, it is a prevalent bla ESBL gene in animal and human E. coli strains and S. serovar Indiana isolates in China (Bai et al., 2016), from where it may have disseminated via horizontal transfer. However, further studies are needed to clarify the origins of this bla ESBL gene and to characterize and compare the plasmids carrying bla ESBL genes in S. serovar Infantis.
The emergence of MDR S. Infantis with resistance to third generation cephalosporins in food and in humans is of great concern, particularly since these strains show intermediate resistance to ciprofloxacin. The use of fluoroquinolones for the treatment of infections caused by such strains may be associated with unfavorable treatment outcomes and the selection of high-level ciprofloxacin resistance (Humphries et al., 2012).
This study extends our knowledge on clones of S. serovar Infantis circulating in food and causing disease in humans and provides evidence for the emergence of an MDR, ESBL-producing clone harboring bla CTX−M−65 in Switzerland. Our results highlight the necessity of strategies to reduce the prevalence of S. serovar Infantis within the food producing industry.

AUTHOR CONTRIBUTIONS
DH, BT, and RS designed the study. KZ, DH, and DA carried out the microbiological and molecular biological tests. GG, HC, FN, and BT carried out whole genome sequencing. MN-I, RS, DH, GG, and BT analyzed and interpreted the data. MN-I drafted the manuscript. All authors read and approved the final manuscript.