Serovar and sequence type distribution and phenotypic and genotypic antimicrobial resistance of Salmonella originating from pet animals in Chongqing, China

ABSTRACT A total of 334 Salmonella isolates were recovered from 6,223 pet rectal samples collected at 50 pet clinics, 42 pet shops, 7 residential areas, and 4 plazas. Forty serovars were identified that included all strains except for one isolate that did not cluster via self-agglutination, with Salmonella Typhimurium monophasic variant, Salmonella Kentucky, Salmonella Enteritidis, Salmonella Pomona, and Salmonella Give being the predominant serovars. Fifty-one sequence types were identified among the isolates, and ST198, ST11, ST19, ST451, ST34, and ST155 were the most common. The top four dominant antimicrobials to which isolates were resistant were sulfisoxazole, ampicillin, doxycycline, and tetracycline, and 217 isolates exhibited multidrug resistance. The prevalence of β-lactamase genes in Salmonella isolates was 59.6%, and among these isolates, 185 harbored blaTEM, followed by blaCTX-M (66) and blaOXA (10). Moreover, six PMQR genes, namely, including qnrA (4.8%), qnrB (4.2%), qnrD (0.9%), qnrS (18.9%), aac(6')-Ib-cr (16.5%), and oqxB (1.5%), were detected. QRDR mutations (76.6%) were very common in Salmonella isolates, with the most frequent mutation in parC (T57S) (47.3%). Furthermore, we detected six tetracycline resistance genes in 176 isolates, namely, tet(A) (39.5%), tet(B) (8.1%), tet(M) (7.7%), tet(D) (5.4%), tet(J) (3.3%), and tet(C) (1.8%), and three sulfonamide resistance genes in 303 isolates, namely, sul1 (84.4%), sul2 (31.1%), and sul3 (4.2%). Finally, we found 86 isolates simultaneously harboring four types of resistance genes that cotransferred 2–7 resistance genes to recipient bacteria. The frequent occurrence of antimicrobial resistance, particularly in dogs and cats, suggests that antibiotic misuse may be driving multidrug-resistant Salmonella among pets. IMPORTANCE Pet-associated human salmonellosis has been reported for many years, and antimicrobial resistance in pet-associated Salmonella has become a serious public health problem and has attracted increasing attention. There are no reports of Salmonella from pets and their antimicrobial resistance in Chongqing, China. In this study, we investigated the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella strains isolated from pet fecal samples in Chongqing. In addition, β-lactamase, QRDR, PMQR, tetracycline and sulfonamide resistance genes, and mutations in QRDRs in Salmonella isolates were examined. Our findings demonstrated the diversity of serovars and sequence types of Salmonella isolates. The isolates were widely resistant to antimicrobials, notably with a high proportion of multidrug-resistant strains, which highlights the potential direct or indirect transmission of multidrug-resistant Salmonella from pets to humans. Furthermore, resistance genes were widely prevalent in the isolates, and most of the resistance genes were spread horizontally between strains.

of antimicrobial-resistant Salmonella from pets elsewhere in China and abroad (12-17, 22, 23, 31, 44, 45).Therefore, in this study, we investigated the prevalence, serovar and sequence type (ST) distribution, and antimicrobial susceptibility of Salmonella strains isolated from pets in Chongqing.In addition, we dissected the relationship between phenotypes and genotypes regarding resistance to β-lactams, quinolones, tetracyclines, and sulfonamides and explored the transmission of identified resistance genes in highly resistant isolates.

DISCUSSION
As one of the most important zoonotic pathogens, people often pay attention to Salmonella in food-producing animals or food of animal origin transmission chains (28,46,47) while less attention has been given to Salmonella originating from pets.However, Salmonella colonizing the intestines of pet animals can also be transmitted to humans, leading to the onset of illness that requires attention.For example, one study reported that Salmonella Virchow was transmitted from household dogs to an infant manifesting diarrhea (26).In addition, multistate outbreaks of human Salmonella infections have been linked to small turtles ( 18) and pet hedgehogs (20) in the United States.In practice, this situation may be severely underestimated.
The prevalence of Salmonella among dogs and cats was 4.4% and 3.7%, respectively, in our study, which concurred with previous studies in Xuzhou, China (7.08% in dogs and 2.31% in cats) but was lower than the prevalence in other developing countries, such as Ethiopia (48), Thailand (14,30), and Libya (49).Moreover, in this study, we showed fecal shedding of Salmonella among dogs and cats, in which a significantly greater prevalence was recorded in diarrheal dogs (6.9%) and cats (7.2%) than in apparently healthy dogs and cats (4.0% and 3.1%).This higher prevalence of Salmonella in diarrheal dogs and cats was supported by previous findings in different parts of the globe (13,15,48).In our study, the isolation rate of Salmonella derived from reptiles (turtle: 81.6%; lizard: 66.7%) was not only much greater than that in similar reports from Zhang et al. (turtle: 24.4%) (45) and Wang et al. (turtle: 14.5%) (31) in China but also greater than that in reports from other countries, including Grenada, the West Indies (turtle: 14.7%) (50), Australia (turtle: 20%; lizard: 39.6%; snake: 50%) (51), Portugal (turtle: 20.9%; lizard: 51.9%; snake: 50%) (52), and Korea (turtle: 60%) (29).Eightyfive (78.7%) reptile-derived Salmonella strains were isolated from 108 rectal samples collected at 3 pet clinics and 14 pet shops, indicating the widespread presence of Salmonella in pet reptiles in Chongqing.As some reports (13,48) have stated, the prevalence of Salmonella in pets varies in different regions and countries, probably because of differences in pet sanitary practices, feeding habits, public awareness about pet zoonosis, and the socioeconomic status of the owners.In addition, some factors, such as differences in the season of study, geographical areas, and isolation methods employed, might have also accounted for the observed differences.According to some reports, the proportion of children aged <5 years infected with Salmonella was greater than that of adults (18,31,53), and some outbreaks were associated with pet-associated Salmonella (18).Therefore, it is particu larly important to avoid direct contact between children and reptiles, such as grasping reptiles with bare hands.
A high degree of serovar diversity was observed among Salmonella isolates in this study.Interestingly, the Typhimurium monophasic variant was the most common serovar among the Salmonella isolates identified from both dogs and cats in our study, which was not common in previous studies.The Salmonella Typhimurium monophasic variant is prevalent mainly in humans, food, water, and animals and is strongly associated with the swine food chain (53)(54)(55).However, an outbreak of salmonellosis in a family in central Italy affected two children and involved their three dogs as carriers; phenotypic and genotypic analyses revealed clear transmission of the pathogen identified as a Salmo nella Typhimurium monophasic variant between the human and nonhuman members of the family and the possibility of transmission from a dog to a human (56).Therefore, the prevalence of the Salmonella Typhimurium monophasic variant in pets and the risk of transmission from pets to humans cannot be ignored.The most common STs of the dog-and cat-derived isolates were ST198, ST11, and ST19.Among Salmonella isolates from pet dogs in Xuzhou, China, ST198, ST11, and ST34 were the most prevalent STs (57).However, 26 pet-derived Salmonella isolates were identified as ST10 (number: 22) and ST19 (number: 4) in Hangzhou, China (58).Therefore, the major STs of Salmonella isolated from dogs in Chongqing were roughly the same as those of Salmonella isolated from dogs in Xuzhou but different from those of Salmonella isolated from dogs and cats in Hangzhou.
Local pet hospital veterinarians reported common usage of ampicillin, ceftiofur, cefotaxime, cefovir, meropenem, doxycycline, gentamicin, tilmicosin, tylosin, and azithromycin for pets.However, pet-derived Salmonella isolates exhibit widespread resistance to some antimicrobials that are prohibited or not commonly used in pet clinical practice, such as chloramphenicol, florfenicol, tetracycline, and sulfonamides.For example, sulfonamides are rarely used in pets because the use of sulfonamides in pets sometimes causes the development of urinary stones.It has been reported that Salmonella strains isolated from pets in many countries, including China, are strongly resistant to β-lactams, tetracyclines, and/or sulfonamides (13-15, 30, 48).There might be two major reasons for the severe resistance of these isolates to certain antimicrobials that are not uncommonly used and prohibited in pet clinical practice.First, due to the widespread use of antimicrobials in food-producing animals, drug-resistant Salmonella bacteria from food-producing animals can be disseminated into the external environ ment, and pets can be infected with these drug-resistant bacteria during outdoor activities (59).In addition, pet foods contain a large amount of food-producing animalassociated raw materials, which might be contaminated by drug-resistant Salmonella (12,59).Contaminated foods might transmit drug-resistant Salmonella to pets.Compared with Salmonella from dogs and cats, Salmonella from reptiles was less resistant to antimicrobials and carried fewer resistance genes.This might be due to the limited contact between pet reptiles and the external environment, resulting in fewer opportu nities for acquiring drug-resistant Salmonella.In addition, reptiles are not usually fed with feed.For example, lizards mainly feed on insects than feed, which reduces the chances of lizards being infected with drug-resistant Salmonella in pet food containing raw meat or meat products.
The presence of multidrug-resistant organisms, especially Salmonella isolated from dogs and cats, cannot be ignored.Many different sequence types and serotypes found in dogs and cats in this study were MDR, showing that MDR is not a genotypically limited phenomenon but is indicative of a more widespread phenomenon.Notably, the proportion of multidrug-resistant Salmonella isolates from reptile sources was relatively low.Furthermore, there was no significant difference in the proportion of multidrugresistant bacteria among the serotypes or genotypes for many isolates, indicating that Salmonella's acquisition of resistance was not closely related to their serovars or STs.
Among 199 β-lactamase gene-positive Salmonella isolates, 127 were ESBL-producing strains, of which 52.8% (67/127) carried the bla CTX-M gene.In our previous research on Salmonella originating from food-producing animals, the proportion of ESBL-producing isolates was 24.8% (32/129), and only a small number of strains (6.2%, 8/129) carried the bla CTX-M gene, all of which were isolated from pigs.The proportion of pet-derived Salmonella strains isolated in Chongqing, both those producing ESBLs and those carrying the bla CTX-M gene, was much greater than that of food-producing animal-associated Salmonella strains producing ESBLs and carrying the bla CTX-M gene.This result implied that the source of Salmonella from pets is different from that of Salmonella from food-producing animals in Chongqing.
The coexistence or cotransference of PMQR genes in ESBL-producing strains, especially in bla CTX-M -producing isolates, has been described previously (60)(61)(62)(63).Their coexistence might promote the development of multidrug-resistant isolates under the selective pressure of quinolones and/or cephalosporins (60,61,64).However, among the pet-derived Salmonella isolates, 41.7% (53/127) of the ESBL-producing strains carried PMQR gene(s), but no correlation was found between the cotransfer of the ESBL gene and the PMQR gene.The reason was that the positive rate of the PMQR gene was greater in β-lactamase gene-positive and non-ESBL-producing strains than in ESBL-producing strains, but the difference was not statistically significant.In addition, the PMQR gene exhibited low levels of resistance in Salmonella, as previously reported (65), as only 50% of the PMQR-positive strains were resistant to quinolones.
Tetracycline and sulfonamide resistance genes were prevalent in isolated Salmonella, with positive rates of 52.9% and 90.5%, respectively.Although these two types of resistance genes have rarely been reported in pet-derived Salmonella, they seem to be very common in Salmonella isolates from other sources.For example, the abundances of tet(B) and sul2 were 94.2% and 91.9%, respectively, in 363 Salmonella isolates from Guizhou Province, China (37).Zhu et al. reported that a total of 189 Salmonella isolates were recovered from the cecal contents of broilers, chicken carcasses, chicken meat after the cutting step, and frozen broiler chicken products during the slaughtering process at a slaughterhouse in Sichuan Province, China; the tetracycline resistance genes (tet(A), tet(B), tet(C), and tet(G)) and sulfonamide resistance genes (sul1, sul2, and sul3) were identified in 84 (85.7%) and 89 (97.8%) antibiotic-resistant isolates, respectively (66).Moreover, a study carried out in Bangladesh showed that 81.4% of the Salmonella isolated from broiler farms harbored tet(A), 19.8% harbored tet(B), and 10.47% harbored tet(C), and the prevalence of sul1 was 37.2% (67).The above results indicated that both tetracycline and sulfonamide resistance genes might be prevalent worldwide.The widespread prevalence of these two types of drug-resistance genes in Salmonella from different sources has led to widespread high-level resistance to tetracyclines and sulfonamides.
The resistance rates of the Salmonella isolates to the measured aminoglycosides and amphenicols were 16%-37% and 32%-38%, respectively.Furthermore, we ask the following: what are the resistance genes for aminoglycosides and amphenicols carried by the isolated strains, and how about the prevalence of these resistance genes?In future research, we will continue to detect the resistance genes of both these antimicrobials.
Conjugation experiments demonstrated the cotransference of different types of resistance genes from donor Salmonella to the recipient bacterium E. coli J53 Az R .However, we do not know whether plasmid conjugation can occur between Salmonella isolates.Due to plasmid incompatibility, further research is needed to verify the transfer and frequency of these resistant plasmids between Salmonella isolates.In addition, the structure of plasmids carrying multiple resistance genes deserves further exploration.
Based on the medication status of pets and the antimicrobial resistance and resistance genes of Salmonella isolates obtained from pets in Chongqing, we believe that reducing the use of antimicrobials in food-producing animals might be the key to controlling the development of drug resistance among Salmonella.Fortunately, China launched a campaign to reduce the use of antimicrobials in 2021 (http://www.xmsyj.moa.gov.cn/gzdt/202110/t20211025_6380448.htm).This effort might dramatically reduce the prevalence of drug-resistant Salmonella.In addition, misuse and overuse of antimicrobials in pets might also be important reasons for the emergence of drug-resistant strains.The Chinese government has begun to standardize medication use in pets and has carried out corresponding legisla tive work (http://www.moa.gov.cn/govpublic/xmsyj/202009/t20200914_6352002.htm;http://www.moa.gov.cn/govpublic/xmsyj/202210/t20221012_6413082.htm).It is hoped that with the implementation of the above actions, the resistance of pet-derived Salmonella to antimicrobials may be controlled.
In summary, we first investigated the prevalence, serovar diversity, STs, and antimi crobial resistance of Salmonella strains isolated from pet rectal swabs in Chongqing, China.In addition, β-lactamase, QRDR, PMQR, tetracycline and sulfonamide resistance genes, and mutations in QRDRs among Salmonella isolates were examined.Our findings demonstrated the diversity of serovars and STs among Salmonella isolates.The isolates were widely resistant to antimicrobials, notably with a high proportion of multidrugresistant strains, which highlights the potential direct or indirect transmission of multidrug-resistant Salmonella from pets to humans.Furthermore, resistance genes were widely prevalent in the isolates, and most of the resistance genes were spread horizon tally between species.

Sample collection
From September 2018 to May 2021, a total of 6,223 fresh pet rectal swabs, including 3,638 from dogs, 2,409 from cats, 87 from turtles, 40 from parrots, 21 from lizards, 12 from hedgehogs, 7 from pet rabbits, 6 from pet rats, 2 from foxes, and 1 from a snake, were collected at 50 pet clinics, 42 pet shops, 7 residential areas, and 4 plazas in Chongqing municipality, China (Table S1).All experimental protocols were approved by the Institutional Animal Ethics Committee of Southwest University (approval no.IACUC-20170420-02) and were performed accordingly.
A small sterile cotton-tipped swab (Haoheng, Nanchang, China) was inserted into the anus or cloaca and rotated, gently scraping the mucosa of the lower rectum.The swab was placed into a sterile capped tube containing 2 mL of sterile buffered peptone water (BPW) (Hopebio, Qingdao, China).The sample-containing tube was properly labeled and stored in an ice box maintained at a temperature lower than 8°C.All samples were immediately transported to the laboratory and subjected to microbiological analysis within 6 h.During sample collection, data were collected from staff and/or pet owners using a questionnaire that focused on assessing the possible risk factors for Salmonella infection.

Salmonella isolation and identification
Briefly, rectal swabs in BPW pre-enrichment broth were homogenized using a vortex mixer for 30 s and incubated at 37°C for 16-24 h.A 300 µL pre-enriched suspension was added to 9.7 mL of tetrathionate broth (TTB) (HuanKai Microbial, Guangdong, China) and incubated at 37°C for 18 h.A loop of inoculum from each enrichment culture was streaked onto CHROMagar Salmonella-selective plate (Chromagar, Shanghai, China) at 37°C for 24-48 h.Typical mauve colonies on chromogenic plates were further confirmed by specific polymerase chain reaction (PCR) with Taq PCR Mix (Sangon Biotech, Shanghai, China) targeting the invA gene (28).The confirmed isolates were aliquoted and stored at −80°C in Luria-Bertani (LB) broth supplemented with 50% glycerol until further testing.

Salmonella serotyping and MLST
Serovars of Salmonella isolates were confirmed with the slide agglutination test by determining the types of the O and H antigens with diagnostic sera for Salmonella (Tianrun, Ningbo, China), according to the KauffmannWhite scheme.The antigenic formulae of Grimont and Weill (3) were used to name the serovars.
Primers (Table S14) for the amplification of seven housekeeping genes by PCR were synthesized according to published methods (68).PCR products of amplified genes were sequenced by Sangon Biotech Co., Ltd.The sequencing results of each strain were uploaded to the MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Senterica) to compare the sequence types.

Conjugation experiments
Conjugation experiments were conducted for isolates harboring four or more types of resistance genes by a liquid mating-out assay in LB medium using sodium azide-resistant E. coli J53 Az R as the recipient.The donor bacterium and recipient were co-inoculated in 10 mL of LB media at 37°C for 24-48 h to facilitate plasmid conjugation transferring from the donor to the recipient.Cultured mixtures were inoculated on MacConkey plates supplemented with 100 µg/mL sodium azide, 100 µg/mL tetracycline, or 100 µg/mL ampicillin and incubated at 37°C for another 24-48 h.The presence of resistance genes was detected from the pink colonies as the suspicious transconjugants on sodium azide and tetracycline/ampicillin-containing MacConkey plates by PCR as described.

Statistical analysis
The figure was created with Microsoft Excel version 2013.Statistical analysis was performed using GraphPad Prism (version 8.0).Differences between proportions were calculated using the chi-square test.<i>P values ≤ 0.05, 0.01, or 0.001 were considered to indicate statistical significance.

FIG 1
FIG 1 Multidrug-resistant Salmonella isolated from pet animals in Chongqing.The number of isolates resistant to different categories of antimicrobials is represented on the X axis.The number of isolates is indicated on the Y axis.Colors indicate different pets.Each of the histograms represents the number of isolates of each pet.

TABLE 1
Serovars of Salmonella isolated from pets in Chongqing, China

TABLE 2
Sequence types of Salmonella isolated from pets in Chongqing, China

TABLE 3
Antimicrobial resistance profiles of Salmonella isolated from dogs, cats, turtles, and lizards as determined by the disk diffusion method a a The numbers in the table represent the number of resistant isolates and the percentage of strains isolated from different pets.

TABLE 4
Distribution of multidrug-resistant Salmonella isolates among different serovars

TABLE 5
Distribution of multidrug-resistant Salmonella isolates with different sequence types

TABLE 6
Prevalence of β-lactamase genes in Salmonella isolates originating from different pets a a Genes marked in green are ESBL-producing genes.

TABLE 7
Distribution of ESBL-producing Salmonella isolates among different serovars

TABLE 9
Distribution of QRDR mutations in pet-associated Salmonella isolates

TABLE 10
Prevalence of PMQR genes in Salmonella isolates originating from pets

TABLE 11
Prevalence of tetracycline resistance genes in Salmonella isolates obtained from pets

TABLE 12
Prevalence of sulfonamide resistance genes in Salmonella isolates obtained from pets