Antimicrobial Resistance in Salmonella: Its Mechanisms in Comparison to Other Microbes, and The Reversal Effects of Traditional Chinese Medicine on Its Resistance

Abstract

Salmonella is one of the most notable pathogens leading to the outbreak of foodborne diseases worldwide. Antimicrobial chemotherapy with 3rd-generation cephalosporins or fluoroquinolones is often used for severe infections caused by Salmonella. Therefore, antibiotic or antimicrobial resistance (AMR) of Salmonella is a serious threat to human and animal health in China and worldwide. In order to better understand the current situation and development status of AMR in Salmonella isolates, this chapter will provide an overview of the following: 1. The history and development trend of AMR in Salmonella, and a comparison of its AMR with that of other major pathogenic bacteria in animals. 2. The AMR mechanisms of Salmonella to various antibiotics, with a particular focus on the commonly used antibiotics. 3. The mechanisms of the spread of AMR in Salmonella, including the AMR genes or mobile genetic elements carrying AMR genes among microbes, and among people, animal-derived foods, and the environment. 4. The elimination or reversal of AMR in Salmonella by using traditional Chinese medicine or the active ingredients in traditional Chinese medicine. 5. The development of detection technology for Salmonella serotypes, virulence, and AMR, and the improvement from conventional detection methods to more advanced biological detection methods and bioinformatics technology.

1. Introduction

1.1 Salmonella and Salmonellosis

Salmonella is a spore-free, capsule-free, gram-negative straight bacilli, which widely exists in human and animal intestines. Genus Salmonella currently has two species, Salmonella enterica and Salmonella bongori. The type species, S. enterica, can be further classified into six subspecies with Roman numerals based on their genomic relatedness and biochemical properties, namely, I, S. enterica subsp. enterica; II, S. enterica subsp. salamae; IIIa, S. enterica subsp. arizonae; IIIb, S. enterica subsp. diarizonae; IV, S. enterica subsp. houtenae; and VI, S. enterica subsp. indica [1, 2, 3]. So far, S. bongori (V) has 22 serotypes [4]., and S. enterica has approximately 2600 different serotypes or serovars [2, 5]. Salmonella enterica subsp. enterica (I) is present predominantly in mammals and contributes approximately 99% of Salmonella infections in humans and warm-blooded animals. The other five Salmonella enterica subspecies and S. bongori are mainly found in environment and cold-blooded animals [4, 6]. Among human isolates, S. Enteritidis is the most common serotype, accounting for 65% of all isolates, and Salmonella enterica serovar Typhimurium was reported most frequently among nonhuman isolates, although no serotype predominated [7].

People usually get salmonellosis by eating contaminated foods, particularly foods of animal origin, or by direct contact with infected animals. Salmonella infection causes diarrhea, fever, vomiting and abdominal cramps. Salmonellosis is a common zoonotic disease. It could not only cause serious economic losses in animal production, but also a serious threat to human health [8]. Infection with Salmonella enterica usually results in diarrhea, fever, and abdominal cramps, but some people become asymptomatic or chronic carrier as a source of infection for others.

Salmonella is one of the most notable pathogens leading to the outbreak of foodborne diseases worldwide [8, 9, 10]. In the United States and other developed countries, the annual incidence rate of Salmonella infection is as high as 15.4% [11], and the disease outbreak and hospitalization caused by Salmonella are higher than those caused by other foodborne bacteria [12]. In China, about 300 million people are infected with Salmonella every year [13]. Salmonellosis accounts for 70% - 80% of the total number of foodborne diseases every year in China, and seriously threatens food safety and human health. In one report, 88 Salmonella strains were collected from patients and asymptomatic people in Nantong city of China from 2017 to 2018 [14]. Among these strains, 20 serotypes belonging to 8 serogroups were identified. Salmonella typhimurium remained to be the predominant serotype in strains from both patients and asymptomatic people. Among the 27 strains from patients, S. enteritidis and S. Rissen were shown as the other two major serotypes, while S. London, S. Derby, and S. Meleagridis were demonstrated as the other significant serotypes among the 61 strains from asymptomatic people. AMR testing revealed that 84.1% of strains from both resources were multi-drug resistant. By comparing the characteristics of Salmonella strains from two different kinds of sources, effective strategies would be developed to control Salmonella infection in humans.

Typhoid fever caused by typhoid bacilli is a human acute intestinal infection transmitted between humans. Fowl typhoid is mainly caused by S. typhimurium. Salmonellosis in cattle is mainly caused by S. typhimurium and S. dublin. It mainly occurs in calves aged 10–30 days, and dysentery is the main symptom, so it is also called calf paratyphoid. It is reported that Salmonella spp. are among the most important foodborne pathogens and the third leading cause of human death among diarrheal diseases worldwide [15]. Animals are the primary source of this pathogen, and animal-based foods are the main transmission route to humans. Thus, understanding the global epidemiology of Salmonella serovars is key to controlling and monitoring this bacterium. The study conducted by Rafaela et al. evaluated the prevalence and diversity of Salmonella serovars in animal-based foods (beef, pork, poultry, and seafood) throughout the five continents (Africa, the Americas, Asia, Europe, and Oceania) [15]. The results showed S. typhimurium presented a cosmopolitan distribution in all four assessed matrices and continents. Poultry continues to play a central role in the dissemination of S. enteritidis serovar to humans, and S. Anatum and S. Weltevreden were the most frequently found in beef and seafood, respectively. Careful monitoring of certain serovars and the main vehicles for the transmission of this pathogen will promote the improvement of control programs to reduce the risk of this pathogen reaching humans.

The dominant serotypes of Salmonella from different countries and animals are different. The serotypes of Salmonella from American chickens are mainly from Kentucky [16]. The predominant serotype of Salmonella from cattle in Iran is S. typhimurium [17]. The serotypes of Salmonella from chickens in China are mainly S. Enteriditis, S. Pullorum, and S. typhimurium [18]. In terms of the serotyping of Salmonella, the conventional detection method is to determine the O antigen and H antigen by slide agglutination, and then determine the serotype according to the serum antigen table. Antibiotics have been used in clinical treatment for more than half a century. Antimicrobial therapy of infections based on the antibiotic susceptibility test results and type plays an important role in prevention and treatment of Salmonellosis.

1.2 Antimicrobial-resistance of Salmonella and the effect of traditional Chinese medicines on antibiotic-resistant Salmonella

The overall antibiotic or antimicrobial resistance (AMR) of Salmonella increased significantly from 20% ~ 30% in the early 1990s to 70% at the beginning of this century [8]. Different serotypes show different AMR to antibiotics, and the AMR rate to different antibiotics is also different [9, 10, 11, 12]. In the past three decades, the drug resistance of Salmonella has been significantly enhanced, accompanied by the continuously widened spectrum of multiple AMRs. At present, the antibiotics used for Salmonella are mainly β-lactams, aminoglycosides, sulfonamides, macrolides, phenylpropanols, quinolones, and tetracyclines [19]. With the increasing dosage and abuse of antibiotics, the AMR of Salmonella is becoming more and more prominent. The irrational use of antibiotics has led to a gradual increase in AMR of animal-derived pathogens. From the overall situation of China, China has become one of the countries with the most serious AMR of animal-derived bacteria in the world. The AMR is becoming more and more serious and leads to the effect of clinical treatment decreasing or failing. Multidrug-resistant strains are regionally prevalent and can be transmitted along the food chain, posing risks to food safety and human health.

Different serotypes show different AMR to antibiotics, and the drug resistance rate to different antibiotics is also different [9, 10, 11, 12]. In recent years, Salmonella which has shown resistance to quinolones (ciprofloxacin) and the third-generation cephalosporins (ceftriaxone, cefotaxime) has been reported in China, France, and other countries and regions [20, 21, 22, 23], indicating that with the wide clinical application, the therapeutic effect of ideal antibiotics is also declining. The AMR can be encoded by endogenous AMR genes, or generated by gene mutation or acquisition of exogenous AMR genes carried by mobile genetic elements. Among them, the exogenous AMR genes carried by plasmids, Integron (In), bacteriophages, and Transposon (Tn) can be horizontally transferred through transformation, transduction, and conjugation, which is the main reason for the rapid spread of acquired AMR of bacteria [24].

Different serotypes of Salmonella have different AMR [25], and the rise of AMR levels also brings severe challenges to the prevention and treatment of salmonellosis [26]. Therefore, accurate and rapid serotype identification and AMR detection are of great significance for the prevention and control of salmonellosis [27, 28]. Therefore, how to quickly and efficiently identify the serotype and AMR of Salmonella has become an urgent practical problem, and the introduction of new detection methods is imperative.

Some traditional Chinese medicines have the following properties: anti-bacterial, anti-inflammatory, nourishing and improving immunity, low potential for building tolerance, and low toxicity and side effects. Some studies have shown that traditional Chinese medicine can eliminate AMR plasmids, have a reversal effect on bacterial resistance, and reduce the selection pressure of bacteria [29, 30]. Therefore, as an alternative to antimicrobial agents or a promoter of antimicrobial agents, it has become one of the research hotspots, which has important significance for the prevention and treatment of Salmonella infectious diseases.

1.3 The objective of the chapter

The AMR of Salmonella isolates from humans and animals is becoming more and more serious, which creates great difficulties in the prevention and control of infectious diseases caused by resistant Salmonella isolates. Salmonella with AMR can not only spread widely between animals but also through food to infect humans. Moreover, AMR can also be passed to humans, So it is a huge potential threat to human and animal health in China and worldwide. To make people pay more and more attention to the problem of AMR in Salmonella, this chapter will first review the history and developing trend of AMR in Salmonella. The occurrence and spread mechanisms of the AMR of Salmonella will be clarified, to provide a theoretical basis for searching a new efficacious antibiotic to eliminate and weaken its resistance and control of Salmonellosis caused by resistant Salmonella. In addition, the whole genome sequencing technology has high accuracy in predicting the serotype and AMR of Salmonella. The advanced biological detection methods and bioinformatics technology used in identifying Salmonella serotypes and AMR will be introduced in this chapter. They have broad application prospects in determining salmonella serotype and AMR and the results for prediction will play a very important part in providing strong guidance for the rational use of antibiotics in the clinic.

2. The history and developmental trend of AMR in Salmonella, and a comparison of its AMR with that of other major animal-derived pathogenic bacteria

2.1 Development trend of AMR in Salmonella

Salmonella is one of the most common agents of gastrointestinal disease globally. In the United States, nontyphoidal Salmonella is the second most frequent bacterium causing foodborne illness and the first bacterial pathogen in terms of hospitalizations and deaths. For severe infections, antimicrobial chemotherapy with 3rd-generation cephalosporins or fluoroquinolones is recommended. Therefore, AMR in Salmonella is considered a serious public health threat. 22,102 genomes from public databases were analyzed to track AMR trends in nontyphoidal Salmonella in food animals in the United States. In 2018, genomes deposited in public databases carried genes conferring resistance, on average, to 2.08 antimicrobial classes in poultry, 1.74 in bovines, and 1.28 in swine. There was a decline in AMR of over 70% compared to the levels in 2000 in bovines and swine and an increase of 13% for poultry. Trends in resistance inferred from genomic data showed good agreement with U.S. phenotypic surveillance data. In 2018, resistance to 3rd-generation cephalosporins in bovines, swine, and poultry decreased to 9.97% on average, whereas in quinolones and 4th-generation cephalosporins, resistance increased to 12.53% and 3.87%, respectively.

At present, the antibiotics used for Salmonella are mainly β-lactams, aminoglycosides, sulfonamides, macrolides, phenylpropanols, quinolones, and tetracyclines. The β-lactam mainly includes penicillins (such as ampicillin, carbenicillin, etc.), β-lactam enzyme inhibitors (such as amoxicillin-clavulanic acid, ampicillin-sulbactam, etc.), and cephalosporins (such as ceftriaxone, cefoxitin, etc.). Other antibiotics mainly include aminoglycosides (such as gentamicin, kanamycin, etc.), sulfonamides (such as sulfamethoxazole, trimethoprim-sulfamethoxazole, etc.), macrolides (such as azithromycin, etc.), phenylpropanols (such as chloramphenicol, etc.), quinolones (such as nalidixic acid, ciprofloxacin, etc.) and tetracyclines (such as doxycycline, tetracycline) [18]. With the increasing dosage and abuse of antibiotics, the drug resistance of Salmonella is becoming more and more prominent. Such as the prevention and treatment of the decline, the emergence of new drug resistance genes, and multi-drug resistance (MDR). Salmonella as a zoonosis, the enhancement of AMR is also seriously endangering human health and safety [21]. Salmonella resistance to a single antibiotic first appeared in the 1960s [22]. Subsequently, AMR of Salmonella emerged in different countries and regions of the world, and the isolation rate increased accordingly. In research by Khan et al. [23], the isolation rate of MDR of Salmonella typhi was higher in Asia and Africa. The results showed that the isolation rates in India, Pakistan, and Vietnam were significantly higher than those in Indonesia and China. Fluoroquinolones and cephalosporins are currently the preferred antibiotics for clinical prevention and control of Salmonella infection, but with the irregular use of fluoroquinolones and cephalosporins, the AMR spectrum of Salmonella is wider, and there is a large degree of cross-resistance. Hasan et al. [31] showed among MDR Salmonella, S. paratyphi showed a higher level of resistance to fluoroquinolones. Salmonella strains isolated from animal-derived foods have a high level of resistance to tetracycline. Generally, the resistance rate can reach 80%, and can even reach a high level of 85%. It shows a certain level of resistance to chloramphenicol, penicillin, nalidixic acid, and sulfonamide antibiotics. In addition, the problem of multi-drug resistance is also very serious. The resistance rate to two or more antibiotics can reach 75%, and the resistance rate to five or more antibiotics can reach 30% [24, 32, 33]. The resistance level of Salmonella differs between different studies and regions. Clinically isolated Salmonella strains showed a high level of resistance to nalidixic acid, ampicillin, chloramphenicol, and other antibiotics (65% -90%), and the resistance level to sulfonamides, tetracycline, streptomycin was around 50%, and the resistance to the second and third generation cephalosporins was lower, can reach 10% [34, 35].

At present, the problem of AMR of pathogenic bacteria in veterinary clinics is becoming more and more serious [36]. To promote the growth of livestock and poultry, there will be a large number of antimicrobials used, and many veterinary surgeons in the clinical treatment of antibiotics for the irrational use of non-standard, resulting in a gradual increase in the level of Salmonella resistance, multi-drug resistance is becoming increasingly serious [37]. Changes in the resistance spectrum occur as Salmonella mutates in the natural environment and clinical treatments and are the result of bacterial evolution [38]. Salmonella isolates from clinical specimens have been increasing in recent years, and AMR rates are rising rapidly around the world [39]. With the introduction of new antibiotics into clinical use, the corresponding AMR strains will also be rapidly produced, and single AMR has gradually developed into multidrug resistance. The problem of AMR has become more and more serious, and the problem of bacterial resistance has been paid more and more attention [40, 41]. Salmonella resistance can not only spread widely between animals but also through food to infect humans, causing food poisoning. AMR can also be passed to humans, affecting human health [42]. The increasingly serious AMR of Salmonella has had a great impact on the efficacy of traditional antibiotics, and the increase in the resistance of Salmonella strains to new antibiotics has had a more adverse effect on clinical treatment.

2.1.1 Resistance to tetracyclines

Tetracycline antibiotics are broad-spectrum antibiotics produced by actinomycetes and contain a fused tetraphenyl ring structure [43]. They can be used to treat bacterial diseases caused by Gram-positive and Gram-negative bacteria. Tetracycline antibiotics are mainly divided into two categories: natural and semi-synthetic antibiotics, mainly chlortetracycline, oxytetracycline, methacycline, doxycycline, dimethylaminotetracycline, etc. Due to the characteristics of tetracycline antibiotics, livestock and poultry can only absorb part of them. Most antibiotics will enter the breeding environment in the form of antibiotics themselves or metabolites through the way of livestock and poultry excreta. In addition, livestock and poultry are closely related to human beings. With the continuous development of animal husbandry, bacterial diseases have become increasingly prominent in both intensive farming and free-range farming, and prevention and treatment are facing tremendous pressure. In the prevention or treatment of bacterial diseases, antibiotics are often used. However, when antibiotics are used, there is excessive use, misuse, and abuse, which leads to the specific selection of pathogenic microorganisms by antibiotics and the resistance of pathogenic microorganisms. Among these pathogenic microorganisms, Salmonella is more resistant to tetracycline antibiotics. The resistance of Salmonella to tetracycline antibiotics varies from country to country, which is related to the unreasonable use of tetracycline antibiotics.

Zhang [44] isolated and identified 34 strains of Salmonella from three breeding chicken farms in eastern Liaoning Province. After an antibiotic sensitivity test, 30 of them were resistant to tetracycline. Di et al. [45] found that the resistance rate of swine Salmonella to oxytetracycline was as high as 58.3%. Li et al. [46] found that the resistance rates to doxycycline and oxytetracycline in 247 strains of Salmonella isolated from pigs were as high as 89.77% and 94.88%, respectively. The strains showing resistance to doxycycline and oxytetracycline were as high as 89.3%. This shows that Salmonella is not only resistant to single tetracycline antibiotics but also resistant to two or more tetracycline antibiotics. The continuous emergence of high resistance rates indicates that tetracycline antibiotics are used too much and too frequently in the clinical treatment of avian salmonellosis. The use of tetracycline antibiotics should be appropriately reduced or replaced.

2.1.2 Resistance to quinolone

Quinolone antibiotics, also known as pyruvic acid or pyridine copper acid antibiotics, are a class of synthetic antibiotics with 4-quinolone, which mainly inhibit gram-negative bacteria and mycoplasma. Quinolones have been used to treat human and animal infectious diseases and promote animal growth because of their broad antimicrobial spectrum, strong bactericidal effect, rapid action, lack of cross-resistance with other antibiotics, and few side effects [47]. The common quinolones in clinical treatment are enrofloxacin, ciprofloxacin, ofloxacin, sarafloxacin, difloxacin, and so on.

Yao et al. [48] found that the resistance rate of Salmonella isolated from Shanxi Province, China to the first-generation quinolones was the highest, reaching 56.93%. Zhang et al. [49] found that 2.33% (34 of 1523) of Salmonella enteritidis strains were resistant to ciprofloxacin. Among them, 11 strains had high resistance to ceftriaxone, and all ciprofloxacin-positive strains had resistance to at least 7 antibiotics. From 2013 to 2018, the resistance rate of Salmonella enteritidis to fluoroquinolone enrofloxacin (8.50% -16.30%) showed an increasing trend year by year. In 2012, Li et al. [50] conducted an antibiotic sensitivity test on 62 strains of Salmonella isolated from pigs. The results showed that the resistance rate of fluoroquinolones was 88.7%. As one of the main antibiotics for the treatment of Salmonella, quinolones still have an increasing resistance rate year by year, which has become the hardest hit area of Salmonella resistance.

2.1.3 Resistance to aminoglycosides

There are many kinds of aminoglycoside antibiotics. The earliest aminoglycoside antibiotic is streptomycin, followed by gentamicin, kanamycin, spectinomycin, neomycin, amikacin, netilmicin, and so on. Aminoglycoside antibiotics are mainly divided into two categories: natural and semi-synthetic. Natural aminoglycoside antibiotics include streptomycin, kanamycin, tobramycin, neomycin, spectinomycin, gentamicin, etc. Semi-synthetic aminoglycoside antibiotics include amikacin, netilmicin, etc. [51].

Because of their low price and remarkable effect, aminoglycoside antibiotics are widely used in the treatment and prevention of animal diseases in animal husbandry and aquaculture [52]. However, the use of aminoglycoside antibiotics is abused and abused, resulting in excessive antibiotic residues in animal bodies and AMR. Therefore, the use of aminoglycoside antibiotics has been limited by many countries [53]. Guan et al. [54] conducted an AMR test on 23 isolated and identified Salmonella strains. The results showed that the resistance rate to gentamicin was the highest, which was 66.7%. The resistance rate to spectinomycin was 33.3%, and the resistance rate to kanamycin and tobramycin was 16.7%. The 13 strains of Salmonella isolated by Zhang et al. [55] were tested for AMR to 10 commonly used antibiotics, all of which showed high AMR rates with resistance to more than two antibiotics. Some even achieved resistance to 8 of them, although sensitive to gentamicin and kanamycin. The AMR results varied among the 13 Salmonella isolates, possibly due to the changing breeding environment or AMR. Thus, in recent years, Salmonella resistance to aminoglycoside antibiotics has been very serious, and mostly multi-drug resistance.

2.1.4 Resistance to amide alcohols

Amide alcohol antibiotics are also called chloramphenicol antibiotics. They are a class of antibiotics with broad-spectrum antibacterial amide alcohol substances, which have inhibitory effects on both Gram-positive and negative bacteria. In the field of agriculture in animal husbandry, aquaculture, and chemical industry in the cosmetics industry are widely used, mainly for the treatment of chicken, pig, cattle, and other animals respiratory disease infections. Amide alcohol antibiotics mainly include chloramphenicol, palm chloramphenicol, succinomycin, florfenicol, thiamphenicol, etc.

In 2019, China explicitly banned the continued use of chloramphenicol in foodborne animals. At present, thiamphenicol and florfenicol are widely used as substitutes for chloramphenicol in animal husbandry. With the wide application of amide alcohol antibiotics, the resistance of Salmonella to amide alcohol antibiotics has gradually increased. Huang et al. [56] conducted an antibiotic resistance or AMR test on 61 isolated Salmonella strains. The results showed that the resistance rate to florfenicol accounted for 19.67%. Mondal et al. [57] conducted an AMR test on 9 isolated Salmonella strains. The results showed that 9 Salmonella strains were highly sensitive to ciprofloxacin, kanamycin, nalidixic acid, cotrimoxazole, and ampicillin, but highly resistant to chloramphenicol. Li et al. [58] conducted an AMR test on 215 strains of Salmonella isolated in Henan Province in China. The results showed that the resistance rate to florfenicol was 92.56%, and the AMR was serious. With the extensive use of florfenicol, the number of strains resistant to florfenicol showed an increase. Since February 2022, Salmonella strains resistant to florfenicol mainly belong to S. typhimurium, S. Agona, and S. paratyphi.

2.2 Comparison of AMR in Salmonella with other major animal-derived pathogens

China has become the world’s largest producer and consumer of livestock and poultry products [58]. The production of pork, poultry meat, and eggs has been the world’s first for several consecutive years, and milk production is the third in the world. The rapid growth of China’s aquaculture industry mainly depends on the expansion of the scale of aquaculture and the increase in the number of aquaculture facilities. The large-scale and intensive aquaculture industry continues to develop steadily. Veterinary antibiotics, especially antibiotics, play an important role. However, the irrational use of antibiotics has led to a gradual increase in AMR of animal-derived pathogens. The sensitivity of animal-derived pathogens to quinolones, β-lactams, and other important antibiotics is declining, and the AMR is getting higher and higher. Some clinical isolates of pathogens are resistant to more than 15–20 kinds of antimicrobial agents, leading to livestock and poultry disease prevention and control becoming increasingly close to the embarrassing situation of no antibiotic being available [59]. Streptococcus, Haemophilus parasuis, Pasteurella multocida and other important animal-borne pathogens of amoxicillin, enrofloxacin, and other antimicrobial resistance are becoming more and more serious with clinical treatment, losing effectiveness or failing. In the breeding industry, for a long time, widely through mixing, drinking water to livestock and poultry use of antimicrobial, healthy animal intestinal symbiotic Escherichia coli, Enterococcus resistance to commonly used antibiotics is also increasing year by year [59]. The AMR of Salmonella from livestock and poultry is developing continuously, and the antimicrobial resistance mechanism is becoming more and more complex [60]. Multidrug-resistant strains are regionally prevalent and can be transmitted along the food chain, posing risks to food safety and human health. The emergence and prevalence of five AMR cfr genes have brought great challenges to the clinical treatment of methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus infection [58]. The detection rate of S. aureus clinical strains cfr in developed countries is less than 0.5%. The detection rate of S. aureus clinical strains cfr in China is much higher than that in developed countries by nearly 4%. This gene has even been found in animal-derived Bacillus, Streptococcus, Enterococcus, Escherichia coli, and Proteus, and is mostly located in plasmid DNA that can be horizontally transmitted [58].

Zhao et al. [59] isolated 4 main pathogenic bacteria from 260 cow endometritis samples in Inner Mongolia, including 126 strains of E. coli (48.5%), 84 strains of Streptococcus (32.3%), 53 strains of S. aureus (20.4%) and 21 strains of Salmonella (8.1%). The results of an antimicrobial susceptibility test showed that the resistance rate of E. coli to sulfonamides and benzylaminopyrimidines was more than 98%, and the resistance rate to ceftiofur was 13.7%. The resistance rate of Streptococcus to β-lactams, tetracycline, and kanamycin was more than 80%, and the resistance rate to vancomycin was 26.7%. The resistance rate of S. aureus to β-lactams ranged from 60–85%, to gentamicin and three combinations ranged from 7.5% to 1.2%, and was completely sensitive to vancomycin. The resistance rates of Salmonella to β-lactams, gentamicin, tetracyclines such as oxytetracycline and doxycycline were between 75% and 90%. Salmonella was sensitive to cefotaxime, and the resistance rate was 29%. The resistance rates to aminoglycosides such as tobramycin and amikacin were less than 10%. Four isolates were sensitive to fluoroquinolones and the resistance rates were less than 35%. Zhao et al. [60] isolated pathogenic bacteria from 40 samples of cow endometritis in Xinjiang mainly include E. coli, Staphylococcus, Streptococcus, Bacillus cereus and Salmonella, and the first three pathogens are the main pathogenic bacteria. The results of antibiotic sensitivity test showed that cefotaxime and amoxicillin had obvious antibacterial effect on E. coli, enrofloxacin and kanamycin had obvious antibacterial effect on Staphylococcus, and amoxicillin and ciprofloxacin had obvious antibacterial effects on Streptococcus. Almost all isolated bacteria were resistant to tetracycline and penicillin and were sensitive to quinolones and lactams. Based on the above studies, it seems that Salmonella showed different patterns of AMR to some commonly used antibiotics when compared with several other major animal-derived pathogenic bacterial species. The underlying mechanisms are not clear. They could be due to different bacterial niches, different standards for antibiotic usage and animal breeds. Further research is needed to explore the mechanisms which could be important for designing strategies for migrations of AMR.

3. The AMR mechanisms of Salmonella to various antibiotics, with a particular focus on the commonly used antibiotics

The extensive use of antibiotics has inevitably improved the survival adaptability of pathogenic bacteria and the endogenous flora of humans and animals, and promoted the evolution of their genomes, thus leading to the emergence and spread of AMR strains. At the beginning of this century, the overall AMR of Salmonella increased significantly from 20% ~ 30% in the early 1990s to 70% [29]. Different serotypes show different AMR to antibiotics, and the AMR rate also varies between different antibiotics [30, 61, 62, 63]. In recent years, Salmonella, which has shown resistance to quinolones (ciprofloxacin) and the third generation of cephalosporins (ceftriaxone, cefotaxime) has been reported in China, France, and other countries and regions [64, 65, 66, 67], indicating that with the wide clinical application, the therapeutic effect of ideal antibiotics is also declining. In addition, the emergence and global spread of multi-antibiotic resistant Salmonella make the situation of AMR of Salmonella extremely severe. Therefore, the use of antibiotics should be further standardized and the AMR monitoring of Salmonella should be strengthened in the future.

The biochemical mechanisms of AMR can generally be classified into three categories [68, 69, 70]: 1) Produce inactivating enzymes to destroy antibacterial antibiotics through hydrolysis or modification, so that they can be converted into derivatives without antibacterial activity; 2) Reduce the permeability of the bacterial outer membrane, hinder the entry of antibacterial agents, or strengthen the efflux of active efflux pump to transport antibacterial agents out of the cell to reduce the antibiotic concentration in the cell; 3) To modify the action target of antibiotics or cause target mutation through gene mutation, thereby reducing the affinity of antibiotics to target proteins. The AMR can be encoded by endogenous AMR genes, or generated by gene mutation or acquisition of exogenous AMR genes carried by mobile genetic elements. Among them, the exogenous AMR genes carried by plasmids, Integron (In), bacteriophages, and Transposon (Tn) can be horizontally transferred through transformation, transduction, and conjugation, which is the major reason for the acquired AMR and rapid spread of bacteria [71].

Plasmids are extrachromosomal DNA molecules that can replicate autonomously and can confer host resistance to important antibiotics, including β-Lactamides, aminoglycosaminoamines, tetracyclines, chloramphenicols, sulfonamides, trimethoprims, macrolides and quinolones [72], and conjugated plasmids can transfer AMR to recipient bacteria through conjugation. Plasmids are closely related to the current situation of Salmonella resistance, and heavy metal resistance genes, disinfectant resistance genes, and virulence-related genes carried on plasmids have improved the survival adaptability of Salmonella to the environment [73].

Salmonella has a high level of resistance to quinolones, mainly due to the mutation of gyrA, gyrB, parC and parE genes in the quinolone resistance determining region (QRDR) on the bacterial chromosome, which makes the antibiotics lose their binding sites and efficacy [71]. The quinolone resistance genes qnr, aac (6′) - Ib cr, qepA, and oqxAB carried by plasmids can mediate low levels of quinolone resistance and accelerate the mutation of gyrA, gyrB, parC, and parE genes in QRDR, which is the main reason for the spread of quinolone resistance in Salmonella at present [67, 74].

The tolerance of Salmonella to β-lactam drugs is mainly due to the hydrolysis of antibacterial drugs β-lactamases, and most β-lactamase gene is carried by plasmid. Among them, plasmid-mediated ultra-broad spectrum β-lactamase genes blaCTX-M, blaTEM, and blaSHV, AmpC β-lactamase gene blaCMY and carbapenemase genes blaKPC, blaVIM, blaIMP, and blaOXA are prevalent worldwide [63, 71, 75].

In addition, the plasmid can also achieve the aggregation and transfer of antibiotic-resistant gene clusters by capturing mobile elements such as integrons or transposons. Integron is a natural cloning and expression system found in bacteria in recent years. Although the integron lacks the ability of autonomous movement, it often participates in the transfer as a component of the conjugated plasmid or transposon, thus promoting the diffusion of antibiotic-resistant genes [76]. Vo [77] detected aadA1, aadA2, aadA5, blaPSE-1, blaOXA-30, dfrA1, dfrA12, dfrA17 and sat resistance gene cassettes in the type I integron carried by Salmonella isolates in Vietnam, forming nine different gene box arrays, and most of them are located on conjugative granules, which can transfer resistance to E. coli or S. enteritidis receptor bacteria.

4. The elimination or reversal of AMR in Salmonella by using traditional Chinese medicine or the active ingredients in traditional Chinese medicine

Chinese herbal medicine is natural and has many advantages: low toxicity, and lower residual levels of toxic substances [78]. It plays an active role in modern infection prevention and control. Some traditional Chinese medicines have the following properties: anti-bacterial, anti-inflammatory, nourishing and improving immunity, low potential for building tolerance, and low toxicity and side effects. Some studies have shown that traditional Chinese medicine can eliminate AMR plasmids, have a reversal effect on bacterial resistance, and reduce the selection pressure of bacteria [78, 79]. Therefore, as an alternative to antimicrobial agents or a promoter of antimicrobial agents, it has become a research hotspot, which has important significance for the prevention and treatment of Salmonella infectious diseases.

Some studies have shown that Chinese herbal medicines have a bacteriostatic effect on Salmonella in calves, and the bacteriostatic intensity ranked from strongest to weakest as follows: gallnut, schisandra chinensis, wumei, chebula, Ligustrum lucidum, pomegranate peel, sumu, and scutellaria. Among them, gallnut has the best bacteriostatic effect [79]. Wumei, coptis chinensis, and rhubarb have bacteriostatic effects on the intestinal Salmonella of dairy cows [80], among which, gallnut has good bacteriostatic effects on S. typhimurium and S. cholera-suis isolates from pigs [81]. Ma [82] found that the elimination rates of resistance to amoxicillin and tetracycline were 1% and 5%, respectively, in the resistant Salmonella treated with ebony. Cao [83] found the elimination effects of Galla Chinesis and Scutellaria on Salmonella AMR and with the highest removal rate of resistant strains 23.3%, 15.3% respectively by 20 hours, and 14.7%, 9.9% respectively by 48 hours. The Galla Chinesis and Scutellaria showed resistant plasmid removal rate of 15.6% and 10.8%, respectively.

5. The development of detection technology for Salmonella serotypes, virulence, and AMR, and the change from conventional detection methods to more advanced biological detection methods and bioinformatics technology

Different serotypes of Salmonella have different antimicrobial resistance [25], and the rise of AMR level also brings severe challenges to the prevention and treatment of salmonellosis [26]. Therefore, accurate and rapid serotype identification and AMR detection are of great significance for the prevention and control of salmonellosis [27, 28]. In terms of the serotyping of Salmonella, the conventional detection method is to determine the O antigen and H antigen by slide agglutination, and then determine the serotype according to the serum antigen table. This serotyping technique requires high serum quality, costs a lot, and takes a long time, and some agglutinations are not obvious and difficult to distinguish. In terms of AMR detection of Salmonella, the most commonly used method is the antibiotic sensitivity test recommended by the American Committee for Clinical Laboratory Standardization (CLSI) [84]. However, the accuracy of the experimental results of this method is easily affected by experimental materials, experimental conditions, and personnel operations. In terms of AMR gene detection, common PCR detection techniques cannot identify all AMR genes at once [85]. Therefore, how to quickly and efficiently identify the serotype and AMR of Salmonella has become an urgent practical problem, and the introduction of new detection methods is imperative.

With the increasing maturity of sequencing technology, rapid, low-cost, and cost-effective whole genome sequencing technology (WGS) has been widely used in the research of bacterial epidemiology [86]. At the same time, the development of bioinformatics technology has also promoted the creation of a variety of public databases such as the serological typing of foodborne pathogens and antibiotic-resistant genes, such as the SeqSero serotype database and ResFinder AMR gene database. With the continuous updating and improvement of the databases, the accuracy of automatic data analysis will be higher and higher. Several studies have shown that WGS has broad application prospects in determining Salmonella serotype and AMR genotype, and may replace conventional laboratory methods in the future [87, 88]. At present, there is very limited research in this field in China.

The establishment of serotype databases promotes the application of WGS in Salmonella serotyping. The commonly used serotype databases include SeqSero and SISIR. At present, SeqSero has been updated to SeqSero2, which improves the accuracy of the serotype database. Compared with the SISTR database, SeqSero2 does not need the help of genome-wide multi-site sequence typing research, simplifying the operation process and making the application more convenient [89]. Xu et al. [90] selected 38 Salmonella strains from the American Salmonella surveillance system, and the coincidence rate between the WGS typing results and the original results was 100%. Zhang et al. [91] conducted molecular analysis on 308 known Salmonella serotypes through WGS, among which 304 strains were completely consistent in serotype, with a coincidence rate of 98.7%. Diep et al. [92] collected 100 Salmonella strains from the Netherlands, and the serotypes of 98 Salmonella strains predicted by WGS were consistent with the conventional typing results, with a coincidence rate of 98.0%. Robertson et al. [93] extracted Salmonella WGS data from the SPA public database, and the coincidence rate between the identified serotype and the original results was 95.0%.

In conclusion, WGS typing method has high accuracy in predicting common serotypes. Compared with the conventional serum typing method, WGS typing is faster. For rare serotypes that require different culture media and antisera to determine flagella (H1 and H2), WGS takes only a few minutes, while the conventional serum typing method may take several weeks, sometimes requiring multiple repetitions. Therefore, the typing method based on WGS opens a new door for the identification of Salmonella serotypes, which has great application value in Salmonella serotyping. With the improvement of sequencing technology and the improvement of the databases, WGS typing is expected to become a new standard for Salmonella serotyping [94, 95].

The emergence of AMR is closely related to the existence of AMR genes, and the expression of AMR genes determines bacterial AMR. Research shows that the ResFinder resistance gene database can detect more resistance genes in the prediction of resistance genes, and it is the preferred tool for AMR analysis [96]. Neuert et al. [97] compared the AMR of 3415 Salmonella strains to 15 kinds of antibacterial agents, and their genotypes, and found 97.8% correlation.

Zankari et al. [98] predicted the AMR of 49 strains of S. typhimurium to 17 kinds of antibiotics, which was completely consistent with the results of AMR phenotype. Among 189 Salmonella strains studied by Zhu et al. [99], the coincidence rates of WGS AMR prediction to sulfamethoxazole, ampicillin, and tetracycline with their AMR phenotypes were 97.8%, 94.6% and 85.7%, respectively.

For antibiotics whose AMR genotype is not clear or is still under study, the coincidence rate between the AMR phenotype predicted by WGS and the AMR genotype is relatively low. The resistance mechanism of enrofloxacin and ceftiofur is mainly related to chromosome-mediated mutations. At present, WGS has only detected plasmid-mediated resistance genes, while the resistance genes generated by chromosome mutations have not been detected. This may be due to the low coverage of some regions in the genome sequencing process, preventing the detection of mutation sites, or the emergence of new resistance gene mutations [100].

Overall, the genome-based genotyping method avoids the influence of subjective judgment of conventional serotyping methods and has a high application prospect in serotyping. It is expected to replace conventional serotyping methods. The prediction of AMR by antibiotic resistant genotypes also provides a new perspective and method for clarifying AMR mechanisms and detecting AMR [101]. When new serotypes or AMR genes appear, they can be directly retrieved and analyzed through WGS data, without the need for routine bacterial culture and identification again, which provides a simpler method for the analysis of Salmonella serotypes and AMR. In addition, the application of WGS has also promoted research and development in other directions such as the genetic and variation characteristics of foodborne pathogens, AMR mechanisms [102], and will have an increasing impact on the analysis and research of the molecular biological characteristics of bacteria in different ecosystems and the substitution of traditional methods [103, 104]. With the development of whole gene sequencing technology and the reduction of its cost, rapid screening of antibiotic-resistant genes from genome data by bioinformatics methods has become a research hotspot.

6. Conclusion

The resistance of Salmonella to β-lactams, gentamicin, tetracyclines such as oxytetracycline and doxycycline was serious. However, Salmonella isolates were sensitive to fluoroquinolones, cefotaxime, and aminoglycosides such as tobramycin and amikacin. Salmonella has shown resistance to quinolones (ciprofloxacin) and the third generation cephalosporins (ceftriaxone, cefotaxime) in China, France, and other countries and regions. The resistance of Salmonella from livestock and poultry is developing continuously, and the AMR mechanism is becoming more and more complex. Multidrug-resistant Salmonella is regionally prevalent and can be transmitted along the food chain to human, which make the situation of AMR of Salmonella extremely severe. Therefore, the use of antibiotics should be further standardized and the AMR monitoring of Salmonella should be strengthened in the future.

The increasingly serious AMR of Salmonella has an adverse effect on the clinical treatment of salmonellosis. The biochemical AMR mechanisms of Salmonella are as follows: (1) Produce inactivating enzymes to destroy antibiotics; (2) Reduce the permeability of the bacterial outer membrane; (3) Strengthen the efflux of the active efflux pump to transport antibiotics out of the cell; (4) To modify the action target of antibiotics; (5) Target gene mutation. The serotypes or AMR genes can be retrieved and analyzed through the genome-based genotyping method and WGS data. The development of bioinformatics technology provides a new perspective and method for clarifying AMR mechanisms and detecting AMR.

To a certain degree, the AMR in Salmonella can be eliminated or reversed by traditional Chinese medicine or traditional Chinese medicine active ingredients. Some traditional Chinese medicines have good reversal effects on resistance of Salmonella isolates. By eliminating the resistant plasmids, Chinese herbal medicines can reduce AMR of Salmonella strains and reduce the selection pressure of bacteria. Therefore, some traditional Chinese medicines, as an alternative to antimicrobial agents or a promoter of antimicrobial agents have important significance for the prevention and treatment of Salmonella infectious diseases.

Acknowledgments

The authors would like to acknowledge Wei Mao and Weiguang Zhou, Professors of Veterinary Medicine of Inner Mongolia Agriculture University. They provided a large amount of information on the molecular epidemiology of zoonotic pathogens. In addition, JinShan Cao, the senior vice mayor of Tongliao City in the Inner Mongolia Autonomous Region is acknowledged for proposing the new design ideas before writing the manuscript. During the process of manuscript’s revision, some valuable suggestions were given by Professor Cao. The authors also gratefully acknowledges the support from Inner Mongolia Science and Technology major project (2021ZD0013).