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Genetic characterization of multidrug-resistant Salmonella typhimurium carrying mcr-1 in China 1 2 Running title : Genetic characterization of S . Typhimurium 3 4


 Background: With the rapid emergence of plasmid mediated polymyxin resistance gene mcr-1, multidrug-resistant Salmonella caused great troubles in clinical treatment and attracted extensive attention. Here we report multidrug-resistant Salmonella strains harboring mcr-1 in China, which are resistant to both polymyxin, traditional antibiotics and even clinically wide-used antibiotics. Methods: We screened 1454 strains of Salmonella collected in our laboratory from 2006 to 2018 from 3 provinces or regions for mcr-1 by PCR. Antimicrobial susceptibility testing was determined. Plasmid conjugation assays were carried out to analysis the transferability of polymyxin resistance. Genetic polymorphism analysis of Salmonella was performed using the PFGE, and the plasmid profiles were characterized by S1-PFGE and southern blotting. The plasmids harboring mcr-1 were sequenced and compared. Results: Eleven S. Typhimurium isolates harboring mcr-1 with polymyxin resistance (MICs 4μg/ml) were identified from intestinal infections and foods in China. All S. Typhimurium isolates were multidrug-resistant to traditional antibiotics and even clinically wide-used antibiotics. Three types of plasmids harboring mcr-1 were recovered (IncHI2, IncX4 and IncI2). Compared with the reference plasmid, IncX4 and IncI2 plasmids had extremely similar typical backbone, and contain only mcr-1 resistance gene. However, IncHI2 were the most diverse type of plasmid due to containing a large MDR region, including blaCTX-M, oqxB, sul, aph, aadA and blaTEM. IncHI2 plasmids were observed to contain only one or no insertion sequence ISApl1 around mcr-1, without forming a circular intermediate. Conclusion: With the horizontal transfer of different types of plasmids, mcr-1 is widely spread worldwide. These prevalent plasmids are responsible for resistance to polymyxin, traditional antibiotics and even clinically wide-used antibiotics resulting from transmission of mcr-1 and other resistance genes. Our studying emphasizes the necessity to jointly monitor its international epidemic and preemptive further upgrade.


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The emergence and evolution of multi-drug-resistant (MDR, resistance to three or more classes of 51 reported method [13]. The images were captured by a Gel Doc 2000 system (Bio-Rad), and were 113 imported into the BioNumerics software (v6.0) database for processing and further analysis. 114

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The mcr-1 positive Salmonella strains preserved in our laboratory were sequenced based on the 116 second-generation genome sequencing technique. The DNA was extracted from the overnight cultured 117 strains according to the instructions of the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The 118 mate-pair library was constructed by nucleic acid protein analyzer Qsep100 to obtain DNA fragments 119 (not less than 500bp, not more than 800bp) and sequenced by MiSeq platform sequencer. Sequence 120 reads were assembled into draft continuous sequences (contigs) by Newbler [14] and NxTrim [15], and 121 then splice it through GapFiller, Cytoscape. Complete plasmid genomes were annotated using the 122 online annotation server RAST. Identification of insertion sequence (IS), plasmid replicons and 123 resistance genes is performed by ISfinder (https://www-is.biotoul.fr/search.php), PlasmidFinder 124  (1046), Guangdong (209) and Guangxi (199), we found 11 strains of S. Typhimurium 131 harboring mcr-1 gene. Among them, eight mcr-1 positive S. Typhimurium strains were identified from 132 fecal samples of patients, and three mcr-1 positive S. Typhimurium strains were from pork and pork 133 products. All eleven mcr-1 positive S. Typhimurium strains showed resistance to polymyxin (MICs 134 4μg/ml), and were resistance to multiple antimicrobial agents, including gentamicin, ampicillin, 135 sulfamethoxazole, streptomycin, tetracycline and chloramphenicol. Moreover, six of eight strains 136 isolated from patients were resistant to ceftriaxone and cefotaxime. Among three strains isolated from 137 pork and pork products, one was resistant to ceftriaxone and cefotaxime, and another one was 138 insensitive to azithromycin (Table 1). 139

PFGE, plasmid profiling and southern blotting
140 Eleven mcr-1 positive S. Typhimurium and four mcr-1 negative Salmonella were analyzed by PFGE. 141 Based on the PFGE results of fifteen isolates of Salmonella strains, four clusters were formed among 142 the strains, which were divided according to about 85% homology. In Shanghai, the similarity of five 143 mcr-1 positive Salmonella strains isolated from pork and humans in 2015 was above 95% with the 144 mcr-1 negative Salmonella isolated from humans in 2012. The mcr-1 negative Salmonella strains 145 isolated from pork in 2014 had high homology with mcr-1 positive Salmonella isolated from human in 146 2015 and lower similarity with mcr-1 negative Salmonella isolated from pork in Guangdong. Further 147 analysis of plasmid characteristics, S1-PFGE and southern blotting showed that all isolates carried 148 different plasmids, of which two strains carried two plasmids, others carried one plasmid ( Figure S1). 149 Genetic characterization of plasmids harboring mcr-1 156 We have obtained the plasmid sequence of eleven S. Typhimurium strains. Through the plasmid 157 comparison on NCBI, we downloaded five highly homologous plasmids containing mcr-1 from 158

Plasmid conjugation assays
Salmonella strains as a reference (at above 95% coverage and at above 99% identity). Through the 159 analysis of plasmids, three IncX4, two IncI2 and six IncHI2 plasmids were identified to contain mcr-1 160 resistance genes among 11 strains. Three IncX4 plasmids were 33kb in size, and were extremely 161 similar to pNG14043 from Salmonella in Taiwan. These plasmids were typical IncX4 backbone which 162 varied only by single nucleotide polymorphism (SNP) (at above 99% homology). The insertion 163 sequence IS26 was upstream of mcr-1 in our isolates, and IS15 was on these plasmids but absent 164 around mcr-1. Those plasmids had only mcr-1 genes and no other identifiable resistance genes. (Fig. 2,165 Fig. 5). Two IncI2 plasmids were 50kb, and were similar to pHNSHP45 by E. coli strains from 166 Shanghai in July, 2013. We found that these two IncI2 plasmids have a common plasmid backbone. 167 However, compared with the two IncI2 plasmids, the region around mcr-1 were located in an inverted 168 orientation. The insertion sequence ISApl1 and IS683 were missing in two IncI2 plasmids in this study 169 by comparing with the reference plasmid sequence (Fig. 3, Fig. 5). Unlike the IncX4 and IncI2 types of 170 plasmids, six IncHI2 plasmids were the most diverse type of plasmid due to containing a large MDR 171 region. The MDR region is a region in the IncHI2 plasmid that contains a wide range of resistance 172 genes, integrons and ISs. These IncHI2 plasmids that share a common backbone were 250kb, used 173 pHNSHP45-2 as a reference plasmid with high homology. The MDR region of the reference plasmid 174 contained a variety of resistance genes, including blaCTX- M, oqxA, oqxB, oqxR, sul, aph, aadA, dfrA, 175 floR, aac, fosA, hph, intl. Some drug resistance genes and gene element can be observed in that region 176 of the IncHI2 plasmids in our study, distinguishing them from the reference plasmid sequence (Table  177 1). Compared to the reference plasmid, part of drug-resistant genes in the MDR region has missed in 178 our IncHI2 plasmids (pS49, pS54, pS70), including oqxA, oqxB, oqxR, sul, aadA; The drug-resistant 179 gene dfrA12 is missing in the MDR region of five IncHI2 plasmids except pS51 (Fig. 4). Among six 180 IncHI2 plasmids, five IncHI2 plasmids were observed to contain the insertion sequence ISApl1 which 181 was upstream of mcr-1, whereas another plasmid lacked ISApl1 around mcr-1 (Fig. 5). 182

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Salmonella is a common zoonotic pathogen that can cause bromatoxism and diarrhea of human beings 184 [5,10]. For some special populations, antibiotic therapy is necessary for Salmonella infection. 185 According to the Salmonella monitoring report, the MDR of Salmonella increased 40 percent in the last 186 decade of 20th century [18,19]. Among the many serotypes of Salmonella, the MDR of S. 187 Typhimurium, has drawn global concern in particular. However, the effects of traditional antibiotics 188 and even clinically wide-used antibiotics to S. Typhimurium have been reduced, which include 189 fluoroquinolones, cephalosporins and even azithromycin [20]. In the Salmonella strains, plasmids play a vital role in the acquisition of drug resistance caused by drug 215 resistance genes [26]. The mcr-1 and other resistance genes are usually located on the same or different 216 plasmids. These genes are widely spread in animals, environment and food in many countries and 217 regions around the world through horizontal transfer of plasmids [27]. It is demonstrated that mcr-1 218 and other resistance genes with the spread of Salmonella strains have potential to transfer from food 219 product animal strains to human strains [28,29]. During plasmid transfer, the mcr-1 positive plasmids 220 display significant diversity in terms of antibiotic resistance patterns, incompatibility groups, and 221 genetic contents [3]. IncI2 and IncX4 plasmids which promote Salmonella resistance are the two main 222 types of plasmids spreading globally [14]. In the study, eleven strains of S. Typhimurium found that the 223 plasmids containing mcr-1 were mainly located in IncI2, IncX4 and IncHI2. According to reports in 224 Enterobacteriaceae [30], IncHI2 plasmid (216-280kb), containing a multi-drug resistance region, is the 225 fifth most common plasmid family, and is one of the main plasmid groups carrying mcr-1 gene variants. 226 We observed the coexistence of plasmids containing mcr-1 and multiple drug resistance genes in this 227 work. The reduced sensitivity to multiple antibiotics is attributed to the fact that mcr-1 positive S. 228 Typhimurium also coharbored oqxB, blaTEM and blaCTX resistance genes. These genes are co-located in   pHNSHP45 with GenBank no.KP347127 which was isolated from E. coli strains from Shanghai in July, 419 2013 was used as refrence plasmid (black circle). The outmost circle in red arrows denots the 420 annotations of refrence plasmid. The IS683 and ISApl1 are absent in two IncI2 plasmids in this study. 421 Detailed information of mcr-1 location of plasmids is provided in fugure5. 422

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Fig4. Sequencing alignment of mcr-1 harbouring IncHI2 plasmids. pHNSHP45-2 with GenBank 425 no.KU341381 which was isolated from E. coli strains was used as refrence plasmid (black circle). The 426 outmost circle in red arrows denots the annotations of refrence plasmid. Among the six IncHI2 427 plasmids, pSH51, pS52 and pS53 exhibit sequence with the refrence sequence, others are low sequence 428 homology. An MDR region is exhibieted in the five IncHI2 plasmids. Detailed information of mcr-1 429 location of plasmids is provided in fugure5. 430 431 Fig5. Genetic context of eleven plasmids surrouding the mcr-1 gene. In CDS, red and blue arrows 432 represent mcr-1 and IS respectively, black arrows present plasmid bone. The light blue and orange 433 shaded regions indicate genetic regions that show the direct and reverse nucleotide ideatityhomology 434 between different segments( >99%). 435