National Prevalence of Salmonella enterica Serotype Kentucky ST198 with High-Level Resistance to Ciprofloxacin and Extended-Spectrum Cephalosporins in China, 2013 to 2017

Ciprofloxacin and extended-spectrum cephalosporins are the choice for treatment of severe nontyphoidal S. enterica infections in adults. S. enterica serotype Kentucky ST198 has gained epidemiological importance globally, because the clone is frequently resistant to both of these high-level-resistance drug groups. The genetic and epidemiological characterization of S. Kentucky has been well studied in Western countries; however, the information is unclear for China.

necessity of surveillance for the high-risk clone to prevent its further dissemination in China. KEYWORDS Salmonella enterica serotype Kentucky, ST198, fluoroquinolone resistance, extended-spectrum cephalosporin resistance, Salmonella genomic island N ontyphoidal Salmonella enterica (NTS) is among the most prevalent zoonotic pathogens, causing an estimated 93.8 million infections per year and resulting in 155,000 deaths globally (1). Numerous serotypes of NTS have been identified, and most of them inhabit animal intestines. However, a few commonly infect humans, including Salmonella enterica serotypes Enteritidis, Typhimurium, Virchow, Hadar, Heidelberg, Agona, and Indiana. Currently, the most common serotypes associated with human disease are S. Enteritidis and S. Typhimurium, while others appear to be becoming more visible on the global NTS landscape (2). Ciprofloxacin and extended-spectrum cephalosporins (ESCs) are the most frequent choices for the treatment of severe NTS infections in adults; thus, the emergence of resistance to ciprofloxacin and ESCs in S. enterica has become a serious public health concern. In 2017, fluoroquinolone-resistant Salmonella spp. and extended-spectrum-b-lactamase (ESBL)-producing Enterobacteriaceae were listed by the World Health Organization among the highest-priority pathogens posing a risk to human health.
Recently, the convergence of these high-level resistances has been found in a single serotype of S. enterica, Kentucky, which largely limits clinical treatment strategies (3). Epidemiological studies by using multilocus sequence typing (MLST) reveal that ciprofloxacin-resistant (CIP r ) S. Kentucky is a single clone belonging to sequence type 198 (ST198). S. Kentucky ST198 has gained epidemiological importance globally (4)(5)(6), and the clone is frequently resistant to a variety of antibiotics, especially to ciprofloxacin at a high level (i.e., MIC . 4 mg/liter), a rare trait in NTS. CIP r ST198 was first isolated in a French person who traveled back from Egypt in 2002 (5). Since then, this clone has been detected with high prevalence in Africa, the Middle East, and southern Asia, and it is widely disseminated to Europe and North America via travel-related infections (4)(5)(6)(7)(8). In addition, S. Kentucky is a foodborne pathogen, and domestic poultry has largely contributed to its global spread (most recently in South Asia and Europe) (3). S. Kentucky ST198 has been established in poultry in France, Poland, the United States, and Vietnam (3,(7)(8)(9)(10)(11). In some cases, the clone is also detected in bovines (10,11). Therefore, CIP r ST198 represents a significant risk to food safety and public health.
To understand the origin and evolutionary trajectory, a recent study analyzed a global collection of multidrug-resistant (MDR) S. Kentucky isolates by using whole-genome sequencing (WGS) for the first time (3). The results show that all of MDR S. Kentucky isolates belonged to ST198, which is estimated to have emerged circa 1989 in Egypt following the acquisition of the antimicrobial resistance (AMR)-associated Salmonella genomic island 1 (SGI1), conferring resistance to ampicillin, streptomycin, gentamicin, sulfamethoxazole, and tetracycline (3). In the early 2000s, amino acid substitutions in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC conferring high-level resistance to fluoroquinolones were accumulated in ST198, further facilitating its wide dissemination (5). However, we noted that the WGS study lacked Chinese isolates, and only one historical genome retrieved from GenBank was included. More recently, a Chinese group performed an epidemiological investigation of S. Kentucky strains collected from clinical cases and the poultry supply chain between 2010 and 2016 in China (12). Using XbaI pulsed-field gel electrophoresis and MLST, the study determined that 35.0% (63/180) of S. Kentucky strains were ST198 and that 60.3% of ST198 isolates (38/63) were resistant to ciprofloxacin. However, due to the lack of WGS data, the study was unable to fully provide a genetic and epidemiological characterization of the isolates.
To fill in the knowledge gap, we analyzed in this study a national collection of S.
Kentucky strains isolated from humans and food in China by using WGS. We aimed to provide a genetic and epidemiological characterization of S. Kentucky ST198 in China.

RESULTS AND DISCUSSION
S. Kentucky ST198 was detected in food and clinical samples in multiple provinces. A total of 12 S. Kentucky strains (0.097%) were detected by retrospectively screening 12,379 S. enterica strains isolated from patients with acute infectious diarrhea in 5 of 10 provinces (Fujian, Guangxi, Hunan, Sichuan, and Zhejiang) (Fig. 1). Eight of them were assigned to ST198, and the other four were ST314. Epidemiological investigations showed that none of the ST198-positive patients had records of traveling abroad within 3 months prior to their admissions, suggesting that acquisitions of S. Kentucky ST198 by these patients were not related to traveling abroad. This is different from the situation in Europe and North America, where more than half of ST198 infections are related to travel outside Europe and North America (5,6).
As food has been identified as a potential major vehicle for infections by CIP r ST198 in Europe, Africa, and North America, we further screened 3,026 serotyped S. enterica strains isolated from poultry, meat products, aquatic products, and eggs. Twenty-one S. Kentucky strains (0.69%) were isolated from poultry (n = 17; chicken and duck) and meat products (n = 4; pork, mutton, and pig liver), and they were collected from three provinces (Guangxi, Hunan, and Zhejiang), where positive human isolates were detected as well (Fig. 1). Nineteen of them were assigned to ST198, and the others were ST314. Together, our findings show that the positivity ratio for S. Kentucky ST198 among S. enterica isolates was 0.18% (27/15,405) in China, which is ca. 2-fold lower than that reported in a previous study (0.39% [40/16,247]) (12). This difference could be caused by different regions included in the sample collection. The data suggest that the positivity ratio for S. Kentucky ST198 was lower in China than in Europe (0.5% to 1%) and comparable to that in the United States (0.2%) (4,5). The higher positivity ratio detected in food isolates than in clinical isolates is consistent with the previous findings that domestic poultry and poultry sources largely contribute to the global spread of ST198 (4,12,13). Additionally, our data showed that ST314 was the second most prevalent subpopulation in S. Kentucky.
Occurrences of inter-/intraregion and cross-host transmission. We performed a phylogenomic analysis on the 27 isolates to further understand their genetic relationship. The results based on 4.3-Mbp core genome showed the division of isolates into two clades: one clade consists of 25 isolates, and the other one includes the remaining 2 isolates (Fig. 2). A previous study showed that ST198 was diversified into two major clades, ST198.1 and ST198.2 (11). Clade 198.1 consists of isolates recovered from agricultural sources in the United States, and clade 198.2 is composed of human clinical isolates collected in Kuwait and the United States (11). In this study, the 25 isolates were classified into clade 198.2, and the other 2 isolates were assigned to clade 198.1 (Fig. 2), suggesting that clade 198.2 was much more prevalent than clade 198.1 in China. The two clades differed by 195 core genome single-nucleotide polymorphisms (SNPs), of which 147 were in coding regions. Clade 198.2 was further diversified into two subclades (198.2-1 and 198.2-2) differing by 44 SNPs, of which 40 were in coding regions.
A few intraregional clonal transmission events were detected, including (i) 10 strains (zj-f72, zj-f75, zj-f77, zj-f79, zj-f78, zj-f82, zj-f87, zj-f94, zj-f95, and zj-f100) isolated from chicken in Zhejiang province differing by 0 to 6 SNPs, (ii) 2 strains (zj-f54 and zj-f60) isolated from chicken in Zhejiang province differing by 4 SNPs, (iii) 2 strains (gx-f7 and gx-f9) isolated from pork and duck in Guangxi province with identical genetic backgrounds (i.e., 0 SNPs), and (iv) 2 strains (sc-h42 and sc-h43) isolated from human in Sichuan province with identical genetic backgrounds. Very few SNPs (#10 SNPs) detected among strains in each event suggests a recent occurrence. Further epidemiological investigation showed that the chicken isolates were from the same slaughterhouse in Zhejiang province, the two strains (gx-f7 and gx-f9) isolated from pork and duck were obtained from two cities in Guangxi province, and the two human isolates (sc-h42 and sc-h43) were obtained from the same family. The results indicate that most intraregional clonal transmission events are caused by epidemiological contacts.
Origins of Chinese ST198 isolates might be diverse. To further understand the genetic relationship of isolates collected from China and other countries, we performed phylogenetic analysis with additional 116 genomes retrieved from GenBank, representative of a global collection (3,13). The 19 strains of subclade 198.2-1 clustered with historical strains isolated from Egypt (26 to 86 SNPs) (Fig. 3). The amino acid mutations in QRDRs detected in these 19 strains (gyrA, TCC to TTC, Ser83Phe; gyrA, GAC to GGC, Asp87Gly; and parC, AGC to ATC, Ser80Ile) were identical to those in the Egypt historical strains (Fig. 3). The six strains of subclade 198.2-2 clustered with strains isolated from Southeast Asia (Cambodia, Indonesia, Myanmar, and Vietnam) (30 to 52 SNPs) (Fig. 3). They shared identical amino acid mutations in the QRDRs (gyrA, TCC to TTC, Ser83Phe; gyrA, GAC to AAC, Asp87Asn; and parC, AGC to ATC, Ser80Ile), of which the mutation of gyrA codon 87 was different from that in subclade 198.2-1. The two isolates of clade 198.1 clustered with strains isolated from North America (Canada and the United States) (65 to 72 SNPs). Mutation of the QRDR (gyrA, TCC to TTC, Ser83Phe) was exclusively identified in two Chinese isolates from clade 198.1 (Fig. 3).
It is suggested that high-level resistance of ST198 strains to fluoroquinolones likely first emerged in Egypt in the early 2000s; this resistance is mainly caused by a combination of three amino acid substitutions in the QRDRs of gyrA and parC (5). GyrA-Ser83Phe (TCC to TTC) is the first mutation obtained by ST198, circa 1992, followed by ParC-Ser80Ile (AGC to ATC) circa 1996. Ciprofloxacin resistance appeared after the occurrence of mutations GyrA-Asp87Gly (GAC to GGC), Asp87Asn (GAC to AAC or), and Asp87Tyr (GAC to TAC) (3,5). Previous epidemiological studies demonstrated that strains carrying the GyrA-Asp87Tyr (TAC) mutation mainly spread from Egypt and Northern Africa into East Africa, Middle Africa, South Asia, Europe, and Western Asia, and those with the GyrA-Asp87Asn (AAC) mutation spread to Southeast Asia, Europe, and West Africa (3)(4)(5). Taken together, the data suggest that the Chinese isolates collected in this study might have various geographic origins and evolved locally.
Various resistomes were detected in ST198 isolates. All ST198 isolates (n = 27) collected in this study were MDR (Table 1) demonstrates that a recent clonal dissemination occurred among three provinces (Zhejiang, Guangxi, and Hunan). The ESBL gene bla CTX-M-9 carried by the 19 isolates is located on the chromosome. More resistance genes were detected in isolates of 198.2-2, except for gx-h1 and gx-f8, than in isolates of 198.2-1, and no acquired resistance genes were found in the two isolates of 198.1 (Fig. 2). Of concern, plasmid-mediated quinolone resistance (PMQR) genes aac(69)-Ib-cr and qnrS1 were detected in zj-f50, zj-f60, and zj-f54. The qnrS1 gene was flanked by a Tn3-like transposon, a relic of an IS2like insertion sequence upstream, and a truncated tnpR gene downstream. The genetic context of qnrS1 is identical to that carried by plasmid pINF5 (GenBank accession number AM234722) detected in an S. Infantis strain (see Fig. S1 in the supplemental material). However, a conjugation assay failed in getting transconjugants with ciprofloxacin resistance. It is supposed that recombination via Tn3 might mediate the capture of qnrS1 by the plasmid (14). Previous studies show that PMQR genes are rarely carried by ST198 isolates, while they seem more common in Chinese isolates. A recent report FIG 3 Core genome phylogeny of global S. Kentucky CIP r ST198. The phylogenetic tree is mid-point rooted. A total of 116 genomes of ST198 representing a global data set were retrieved from GenBank and were included in the analysis in this study. Leaf nodes are colored by region of origin (see map). Colored branches indicate inferred geographical distribution of internal branches. Data columns indicate country of origin, source of isolate (H, human; N, nonhuman; ?, unknown), SGI type, obtained resistance genes of beta-lactams and quinolones, and quinolone resistance-related codons, with resistanceassociated alleles highlighted. The first two letters of the name of our isolates represent their origins; the year of sampling is shown by the number behind the isolate name. revealed that aac(69)-Ib-cr (19/63) and qnr-type genes (9/63) are the prevalent PMQR genes among ST198 isolates collected in China (12). The emergence of PMQR genes would further largely facilitate the wide dissemination of quinolone resistance among ST198 clones.
The mosaic structure of SGI1 detected in ST198 isolates. A novel SGI1-K variant, carrying a mercuric ion resistance module, was detected in the 18 isolates of 198.2-1. Compared with the prototype of SGI1-K, few deletions were detected in the variant, including S026-resG, tnpR of Tn5393, a bla TEM-1b -bearing Tn2, and DS044 (Fig. 4). The resistance region was completely deleted in the SGI1 carried by the other isolate of 198.2-1 (hn-f5), including the features of SGI1-K, e.g., gene cassettes aacCA5 to aadA7 and the mer module (15). This indicates the occurrence of rapid intraclone evolution. Another SGI1-K variant was identified in three isolates of 198.2-2 (zj-f54, zj-f60, and fj-h1), in which 14 backbone genes (S015 to ;S026, resG, and DS044), tnpR of Tn5393, and IS1133 were deleted (Fig. 4). More deletions were found in the SGI1-K variant carried by three strains, including Tn5393 and Tn2 in gx-h1 and gx-f8 and S011, S012, and Tn5393 in zj-f50. SGI1 was not detected in the two isolates of 198.1. Of note, among SGI-1-positive isolates, zj-f50 was the only one that maintained DS044 and yidY (Fig. 4). This was different from the global isolates reported previously (3), suggesting that Chinese isolates might undergo selection and evolve locally. It has been reported that the structure of SGI1 is highly mosaic in S. Kentucky ST198, mainly caused by IS26 mediating the gain or loss of genes in the island, resulting in numerous variants (15). We suppose that the flexible structure of SGI1 detected in S. Kentucky ST198 may confer an adaptive advantage to this high-risk clone.  Sample  ID  Source Yr  Location KZ CXM CTX CAZ FOX AZM FEP IPM MEM COL SMX TMP CIP TET AZI NAL CHL TGC AMP GEN  zj-f54  Chicken 2017 Zhejiang S I  S  S  R  S  S  S  S  S  R  R  R  R  R  R  R  I  R  R  zj-f60  Chicken 2017 Zhejiang S I  S  S  I  S  S  S  S  S  R  R  R  R  R  R  R  I  R  R  zj-f72 Chicken Chicken 2017 Zhejiang R R R Chicken  Plasmidome of ST198 isolates. Analysis of the plasmidome revealed that 23 strains carried at least one plasmid, covering six different known replicon types (ColRNAI, Col156, Col440I, IncHI2/2A, IncI1, and IncQ1). ColRNAI-type plasmid was the most prevalent one (18/27), followed by Col156 (5/27) and Col440I (4/27). Three different plasmid incompatibility types (IncHI2/2A, IncI1, and IncQ1) were identified in three isolates. We identified that the IncHI2/2A-type plasmid carried by zj-f50 carried numerous AMR genes, including aac(3)-IV, aph(4)-I, sul2, sul3, aadA1, cmlA1, rmtB1, floR, aac (69)-Ib-cr, bla OXA-1 , catB3, arr-3, and dfrA12. It has been known that IncHI2-type plasmids are frequently involved in the acquisition of AMR genes (16)(17)(18). Of note, IncHI2/2Atype plasmids frequently mediate the dissemination of AMR genes across the most prevalent S. enterica serotypes (including Kentucky) (19). This thus raises the concern that the high-risk clone ST198 may become more resistant resulting from the dissemination of such MDR IncHI2/2A-type plasmids in the near future, at least in China.
Conclusion. We here first report the genomic characterization of the S. Kentucky epidemic clone CIP r ST198 with ESC resistance in China. The prevalence ratio of CIP r ST198 is relatively low in human and food isolates; however, the occurrence of interregion and interhost clonal disseminations highlights the necessity of surveillance for the high-risk clone to prevent its further dissemination in China. Of particular concern, the emergence of plasmid-borne AMR genes in ST198 would further worsen the clinical treatment.

MATERIALS AND METHODS
Bacterial isolates collected in this study. A total of 12,379 serotyped S. enterica strains isolated from patients with acute infectious diarrhea were collected by 10 provincial Center for Disease Control and Prevention (CDC) sites during a national surveillance for foodborne pathogens between 2013 and 2017 (Fig. 1), and 3,026 serotyped S. enterica strains isolated from food were collected by these CDC sites during a national surveillance for food contaminants and their virulence factors in the same area and period (Fig. 1). Epidemiological data were recorded for each isolate.
Antibiotic susceptibility testing (AST). The broth dilution method was performed for all S. Kentucky (8:i:z6) strains to determine the MIC values of cefazolin (0.25 to 32 mg/liter), cefuroxime (0.5 to 64 mg/liter), cefotaxime (0.5 to 64 mg/liter), ceftazidime (0.5 to 64 mg/liter), cefoxitin (0.5 to 64 mg/liter), aztreonam (0.25 to 32 mg/liter), cefepime (0.25 to 32 mg/liter), imipenem (0.125 to 16 mg/liter), meropenem (0.125 to 16 mg/liter), colistin (0.25 to 16 mg/liter), trimethoprim-sulfamethoxazole (0.125/2.375 to 16/304 mg/liter), trimethoprim (8 to 1,024 mg/liter), ciprofloxacin (0.03 to 4 mg/liter), tetracycline (1 to 64 mg/liter), azithromycin (0.5 to 64 mg/liter), nalidixic acid (1 to 128 mg/liter), chloramphenicol (1 to 128 mg/liter), tigecycline (0.25 to 32 mg/liter), ampicillin (0.5 to 64 mg/liter), and gentamicin (0.25 to 32 mg/liter). The results were interpreted using the Clinical and Laboratory Standards Institute (CLSI) document M100 breakpoints (20). Strains were defined as high-level ciprofloxacin resistant if the MIC was .4 mg/liter and as high-level resistant to ESCs (i.e., cefotaxime or ceftazidime) if the MIC was $16 mg/liter. All isolates were further tested for ESBL production. Isolates showing a $3 2-fold concentration decrease in an MIC for either cefotaxime (0.5 to 64 mg/liter) or ceftazidime (0.5 to 64 mg/liter) tested in combination with clavulanic acid (4 mg/liter) versus the MIC of the agent when tested alone were considered ESBL producing. Kentucky ST198 isolates collect in this study. The prototype sequence of SGI-K (GenBank accession number AY463797) is shown as a reference. Five variations were detected in our collection, and the number of each variation is shown in parentheses. Black lines indicate the deletion regions corresponding to the reference. Type I to IV variations are supposed to be SGI-K derivatives (in green); type V is not an SGI-K derivative (in purple), since the mercuric ion resistance module was not detected. SGI and chromosome genes (trmE and yidY) are indicated by different colors.