Subtyping Salmonella of Pet Dogs With Multilocus Sequence Typing (MLST) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)


 Salmonella, as a zoonotic pathogen, has attracted widespread attention worldwide, especially in the transmission between household pets and humans. Therefore, we investigated the epidemic distribution of dog Salmonella from pet hospitals and breeding base in Xuzhou, Jiangsu Province, China, and used multilocus sequence typing（MLST）and clustered regularly interspaced short palindromic repeats（CRISPRs）to subtype Salmonella isolates. From April 2018 to November 2019, a total of 469 samples were collected from pet hospitals and breeding base, including 339 dog samples and 60 cat samples. S. Kentucky (40.74%) was the most prevalent serotype, but other, such as S. Typhimurium (18.52%) and S. Indiana (18.52%), were also widespread. Eight different sequence type (ST) patterns were Identified by MLST and ST198 was the highest proportion of these isolates. CRISPRs analysis showed that 9 different Kentucky CRISPR types (KCTs) was identified from ST198. 48 spacers including 29 (6 News) for CRISPR1 and 19 (4 News) for CRISPR2 that proved the polymorphic of Salmonella genes in samples from different sources. The analysis demonstrated that the common serotypes were widely present in pet hosts in the same area. This analysis shows that CRISPR genes have better recognition ability in the same serotype, which has a positive effect on the traceability of salmonella and the prevention and treatment of salmonellosis.


Introduction
Salmonella, as a kind of zoonotic bacteria, has caused widespread concern worldwide. In recent years, the number of pets worldwide has increased dramatically, of which cats and dogs account for 80%. The close contact between humans and pets also greatly increases the risk of Salmonella spreading between pets and humans. The positive rate of Salmonella in pet dogs with diarrhea is more than twice that of healthy pet dogs. (Renate Reimschuessel et al. 2017). To make matters worse, healthy pets with no obvious symptoms will also release Salmonella to the outside environment in the form of feces, causing cross-infection between humans and other animals. (Wray and Wray 2000). In southern Ontario, Canada, Murphy tested 188 dogs and 39 cats for Salmonella, and found no positive strains (Murphy et al. 2009). Salmonella can also spread among animal groups (Van Immerseel et al. 2004). There are even individual cases that can prove that pet dogs and cats can spread their own Salmonella to humans (Cherry et al. 2004, Koehler et al. 2006. From 2008 to 2008, in the United States, a study related to human Salmonella infection and contaminated dry dog and cat food showed that Salmonella is more likely to spread between infants and young children (Behravesh et al. 2010). Therefore, it is necessary to pay more attention to Salmonella from pets and Salmonellosis prevention in China which have the largest number of pets.
The genetic characteristics of pet-derived Salmonella have not been extensively studied. At present, molecular detection methods are widely used in the typing of Salmonella strains. Especially MLST, which is more e cient and accurate than pulse eld gel electrophoresis (PFGE), and is more suitable for the identi cation and differentiation of animal Salmonella (Shi et al. 2013). The results of MLST are helpful to analyze the genetic characteristics of different serotypes of Salmonella, and prove the existence of microevolution between different STs. On the other hand, for the same serotype, MLST could not accurately demonstrate the genetic and phenotypic differences of Salmonella (Tennant et al. 2010, Gymoese et al. 2017). Therefore, a new identi cation method was established (Fabre et al. 2012a).
CRISPR has a higher resolution for identical serotypes than PFGE (Thompson et al. 2018). This study investigates the genetic diversity and subtype transmission of pet Salmonella between pets and humans in China. Two molecular typing methods were used. 27 strains of Salmonella from pets were identi ed with different subtypes using MLST; CRISPR was used to analyze the identical serotypes of S. Kentucky and estimate the potential risk of the same subtypes Salmonella spreading between pets and humans.

Multilocus sequence typing (MLST)
The con rmed strains were cultured with LB medium at 37 °C and 120 rpm/min shaking for 18 hours,Then genomic DNA was extracted with the TIANamp Bacteria DNA kit (Tiangen Biotech (Beijing) CO., LTD, China) according to the manufacturer's protocol. Each identi ed strain was characterized by MLST using seven housekeeping (Kidgell et al. 2002). PCR products were delivered to Sangon Biotech (Shanghai) Co., Ltd. for puri cation and sequencing, and the seven pairs of housekeeping gene testing and sequencing results of each strain were uploaded to the MLST database for comparison (http://mlst.warwick.ac.uk/mlst/dbs/ Senterica). This is used to determine the sequence type (ST) of each strain. A minimum spanning tree was generated using BioNumerics software, version 7.5 (Applied Maths, Kortrijk, Belgium) to analyze the distribution of STs from cats, dogs and humans.

Salmonella prevalence and serotypes
Of the total of 467 samples analyzed, 27(5.8%) were positive for Salmonella, of which the isolation rate from dogs was 7.08% (24/339) and the isolation rate from cats was only 2.31% (3/130). Table 1 shows the detailed prevalence of salmonella in pet from pet hospitals and breeding base; 7 different serotypes were identi ed among the 27 positive Salmonella isolates. These serotypes were S. Kentucky, S. Typhimurium, S. Indiana, S. Derby, S. Sandiego, S. London, S. Rissen. The dominant serovar was S. Kentucky followed by S. Typhimurium, S. Indiana and S. Derby. At the same time, both S. Sandiego and S. London found only one sample from a dog, similarly S. Rissen found only one strain in a cat sample. No. of isolates (%) S1 S5 S6 S7 S8 S9 S14 S15 S16 S21 S23 8,20 i; z 6 S. Kentucky 11 (40.74%) S11 S12 S13 S18 S19

Multilocus sequence typing (MLST) analysis
Twenty-seven of the Salmonella isolates were ampli ed and sequenced using seven of housekeeping genes from 399 bp to 501 bp. Table 2 shows in detail the results of MLST analysis of Salmonella,eight different ST patterns were identi ed among these 27 Salmonella isolates. ST198 was the most prevalent STs in the study, represented by 11(40.74%), followed by ST17(18.21%), ST34 (11.11%) and ST39 (11.11%). Interestingly, ST17 and ST39 were detected simultaneously in cat and dog samples. At the same time, most of these Salmonella isolates came from the same pet hospital. Therefore, Salmonella has the risk of cross-infection between pets and can even cause zoonotic diseases. A minimum spanning tree of all STs from the different sources was generated using BioNumerics version 7.6 ( Fig. 1).

Clustered regularly interspaced short palindromic repeats (CRISPRs) analysis
We used CRISPR to investigate the spacer sequences and direct repeat regions of 11 strains of Salmonella Kentucky (

Source Strain (KCTs) ST
A S6(1) S7(2) S8(7) S14(2) S15(3) S9(5) S16 (6)  MonB24 (3) NiaB2 (4) KenB29 (5) ParcB2 (6) KenB30 (7) KenB31 (8) KenB32 (9) CholB4 (10) BloB2 (11) KenB33 ( . In general, Salmonella will pose a great threat to human public health, especially for children and the elderly. For serotypes, the main widespread were Salmonella Kentucky and Salmonella Typhimurium in this study. Therefore, we analyze Salmonella with different molecular methods to determine its clonal structure and determining the diversity among STs in the same serovar. Salmonella Kentucky has a wide range of prevalence and many hosts and through experiments, it has been found that compared with other serotypes, its drug resistance was generally stronger (Zankari et al. 2012). With the rapid development of molecular biology, the typing methods of Salmonella were gradually diversi ed. MLST and CRISPR molecular typing were commonly used typing methods in recent years. Combined with PCR technology, special bacterial gene fragments were ampli ed and sequenced. Through sequence comparison software, we can compare the differences of strains at the genetic level and understand the genetic relationship between strains. The resolution of the two methods were higher than that of traditional serotyping. MLST molecular typing relies on multiple conservative housekeeping gene sequences, with low sequence variation, good experimental reproducibility, and reliable results, which can distinguish the same serotypes (Ho et al. 2017); while CRISPR molecular typing relies on the CRISPR loci of bacteria spacer sequence, the spacer sequence was a short DNA obtained from foreign nucleic acids, such as phage or plasmids, which were inserted into the bacterial chromosomes to prevent them from being infected by plasmids or homologous phages. (Barrangou et al. 2007). Due to the diversity of phages and plasmids pools in the environment, different CRISPRs had emerged. Thus, CRISPRs had higher resolution in differentiate outbreak strains/clones in epidemic clones. (Liu et al. 2011). The deletion and insertion of the spacer sequence form a high degree of polymorphism of the sequence, which makes the CRISPR typing diversi ed. This method had high resolution to distinguish between strains with close relationships.
MLST was usually used to distinguish different Salmonella subtypes, and it can break through the limitations of traditional serotyping. We used MLST to perform rapid and accurate identi cation of serotyping of pet Salmonella from different sources. Each strain has been pro led by different seven alleles. The results showed that a total eight different STs were identi ed in 27 isolates, including ST198, ST17, ST34, ST19, ST39, ST20, ST469 and ST155. Among them, ST198 was the most frequent genotype recovered in this study. According to the 11 strains of Salmonella Kentucky in this study were all ST198, CRISPR molecular typing divides the strains of the same ST type into multiple types. This proved that CRISPR typing is more detailed, not only had the ability to distinguish strains of different serotypes, but also had a certain ability to distinguish strains with high homology, with a higher resolution than MLST typing. However, due to the high variability of the bacterial CRISPR loci sequence and the continuous emergence of new spacer sequences, the CRISPR database had some shortcomings. The experimental data need to be continuously updated and improved by researchers. This was of great signi cance for data sharing in different countries and regions and for the epidemiological analysis of Salmonella.

Declarations
Ethics approval and consent to participate There was no ethical problem in this study, so the samples were feces.

Consent for publication
All authors gave their informed consent prior to their inclusion in the study.
Availability of data and material Not applicable.

Competing of interest
The authors declare that there are con icts of interest.

Funding Information
This work was supported by the Graduate Student Research and Innovation Project of Jiangsu Normal University (2019XKT407, 2020XKT501 and 2020XKT506).
The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Authors' contributions
CY and WFS performed the experiments, interpreted the data and wrote the manuscript; LLW and LXC performed some experiments; AHZ and ZMP participated in experimental design interpreted the data, and supervised the research project. All authors read and approved the nal manuscript.