Antibiotic resistance is a global phenomenon resulting in the emergence of pathogens with resistance to clinically important antibiotics, necessitating new treatment strategies [1
]. Antibiotic-resistant bacteria cause life-threatening illness in humans and pose a significant threat to health and well-being. It is estimated that antibiotic-resistant pathogens cause ~2 million illnesses and 23,000 deaths annually in the U.S. These illnesses cause an additional healthcare cost of $
20 billion and a productivity loss of $
35 billion to the U.S. economy. Also, extensive use of antibiotics predisposes individuals to other serious illnesses, such as the Clostridium difficile
infections that result in an estimated 250,000 infections and 14,000 deaths, annually [2
Antibiotic resistance in foodborne pathogens such as Salmonella
is a major concern for public health safety. More focus is required to target them in the animal foods supply [2
is difficult to eliminate from its reservoir hosts, and food animals often serve as reservoirs of the pathogen. Non-typhoidal Salmonella
causes the highest number of illnesses, hospitalizations, and deaths associated with foodborne illness [3
]. It is associated with more than 1,200,000 illnesses annually, and among these at least 100,000 infections are due to antibiotic-resistant Salmonella
, including those that are resistant to clinically-important drugs such as ceftriaxone (36,000 illnesses/year) and ciprofloxacin (33,000 illnesses/year) [2
]. In fact, Salmonella
isolates conferring resistance to ≥5 antibiotics accounted for more than 66,000 illnesses from 2009 to 2011 in the U.S. [2
is a Gram-negative, facultatively anaerobic bacillus belonging to the Enterobacteriaceae
family. The genus Salmonella
is composed of two taxonomical species, Salmonella bongori
, and Salmonella enterica
, with all medically relevant salmonellae a part of the latter. Salmonella enterica
is a diverse species of bacteria consisting of more than 2500 different serovars. The pathogen can be host-adapted, host-restricted, or generalistic, depending on the broad range of hosts that it can infect. The pathogen is ubiquitously present in the human food chain, and is frequently associated with outbreaks of foodborne disease. Outbreak investigations have identified food sources such as vegetables, fresh produce, cereals, cantaloupes, alfalfa sprouts, pistachios, fruit/fruit pulp, ground beef and turkeys, chicken meat and pork, tuna, dried/shredded coconut, and tomatoes as vehicles of Salmonella
-associated foodborne outbreaks in the past decade [4
]. The situation has been aggravated, since antibiotic-resistant clones are frequently implied as the etiological agents in these outbreaks leading to treatment failures, higher risk of bloodstream infections, and increased rate of hospitalizations.
Recently, drug-resistant Salmonella
has been associated with a considerable number of outbreaks in the U.S. A non-typhoidal Salmonella
Urbana, caused disease outbreak through papayas in 2017, and the isolates showed resistance to streptomycin and intermediate resistance to tetracycline [5
]. Another serovar, S.
Poona, caused multistate disease outbreaks through cucumbers in 2015, and the isolates were resistant to either tetracycline or nalidixic acid. In addition, the nalidixic acid-resistant isolate showed a decreased susceptibility to ciprofloxacin, a clinically important drug used in children against Salmonella
]. Multidrug-resistant Salmonella
I 4,,12:i: caused foodborne illness outbreaks through contaminated pork products in 2015, which resulted in severe infection in humans. The isolated strains were resistant to multiple antibiotics, including ampicillin, streptomycin, sulfisoxazole, and tetracycline [7
]. In addition, S.
Enteritidis isolated from raw, frozen, and stuffed chicken entrees associated with multistate disease outbreaks were resistant to ampicillin and tetracycline [8
]. In 2014, S.
Heidelberg was involved in an outbreak through mechanically separated chicken, and 67% of Salmonella
isolates identified were resistant to three or more classes of antibiotics [9
]. In addition, in 2011, multidrug-resistant S.
Heidelberg, and S.
Hadar were associated with disease outbreaks in the U.S. through contaminated ground beef, ground turkeys, and turkey burgers, respectively [10
]. These reports indicate the frequent involvement of antibiotic-resistant Salmonella
in the human food chain, necessitating the exploration of novel non-antibiotic interventions to counteract the pathogen in reservoirs, including food animals.
2. Emergence and Spread of Antibiotic-Resistant Bacteria
Antibiotics are used in food animal production to promote growth and to prevent, (prophylactic), treat (therapeutic), and control (metaphylactic) infectious diseases [13
]. Previous studies indicated that the use of antibiotics for non-therapeutic purposes in poultry, swine, and cattle outweighed what has been used in humans by several-fold with respect to the amount of drugs consumed [13
]. The extensive use of antibiotics in the animal production systems for the purposes mentioned above has also contributed to the development of drug-resistant bacteria. The close association of these bacteria has also been identified in the human food chain. For example, drug-resistant bacteria have been identified from various environmental samples, farms, and retail meat products [13
]. In addition, the non-judicious use of antibiotics has been attributed to foodborne disease outbreaks where the etiological agents have been identified as resistant clones. Although a mandatory withdrawal period is necessary for avoiding the deposition of antibiotic residues in meat, milk, and eggs, lack of proper monitoring could result in residue deposition in the human food chain, resulting in the colonization of resistant bacteria in the human digestive tract [20
A variety of microorganisms are present outside the host, including those found in water, soil, air, and other related environments. These environmental microorganisms are excellent sources of antimicrobial resistance genes (“environmental resistomes”). “Resistome” is a broad term that describes the presence of all antibiotic resistance genes found in free-living organisms in the environment or commensal microbes. The resistome plays a critical role in transferring antimicrobial resistance to pathogenic microorganisms, and directly affects human health by entering the food chain [21
]. Studies have revealed that commensal bacterial species such as the lactic acid bacteria carry resistance genes and might serve as reservoirs of resistant genes for entero-pathogens. For example, tetracycline, vancomycin, and erythromycin resistance genes have been identified from lactic acid bacteria isolated from fermented dairy products, sausages, and raw meat products including poultry, beef, and pork [22
The interaction between the different components in a food chain or the environment further contributes to the spread of antibiotic resistance across species [14
]. Although humans contract infections from farm animals, pets, fresh produce, meat, eggs, and other agricultural and non-agricultural food products, there are multiple entry routes for pathogens to these vehicles [14
]. Foodborne pathogens such as Salmonella
enter a farm from different sources, such as water, litter, personnel, equipment, vehicles, rodents, insects, and pets. In addition, the movement of portable equipment and vehicles can act as a vector for carrying the pathogen to the farm environment or slaughterhouse [23
]. Similarly, antibiotic-resistant bacteria also spread through truck washing systems, lairage, barn floor, barn flush, and holding pens, and potentially end up in animal carcasses during slaughter [24
]. Irrespective of the antibiotic use, antibiotic-resistant pathogens such as S.
Typhimurium have been recovered from swine and poultry housed in antibiotic-free production systems, highlighting the possible role of environmental factors and vectors such as rodents, insects, and birds in spreading resistance [14
The fecal excretion of antibiotic-resistant pathogens such as Salmonella
from livestock and poultry causes the contamination of the farm environment and water systems. Faulty municipal drainage systems could also result in the spread of resistant bacteria from humans to the waterways and the environment [25
]. The use of fecal waste as manure in agricultural lands also contributes to the spread of antibiotic resistance, especially in fresh produce. Antibiotic-resistant foodborne pathogens such as Salmonella
, E. coli
, and Shigella
have frequently been recovered from fresh produce locally grown in the U.S. or imported from other countries [26
]. The application of pesticides, soil contaminated with livestock feces, and the spraying or irrigation of contaminated water cause the spread of resistant bacteria to fruits, vegetables, and fresh produce [27
]. The contamination of waterways also contributes to the pool of resistant bacteria in agriculture and aquaculture. Aquaculture isolates have shown similar resistance patterns to the isolates recovered from terrestrial agriculture, indicating possible contamination of water sources from farmland [25
The development and spread of antibiotic resistance are complicated processes involving different components of the human food chain, and could be a result of the intense use of antibiotics in food animal agriculture, in addition to other contributors. With such severe concerns of antibiotic resistance development in various pathogens, including the emerging multidrug-resistant strains, the Food and Drug Administration (FDA) has recently introduced the Veterinary Feed Directive (VFD) that necessitates the supervision of veterinarians before using clinically-important antibiotics in treating production animals [28
]. The VFD highlights the importance of the judicious use of antibiotics in animal agriculture and demands the development of natural, safe, environmentally-friendly intervention strategies against deadly foodborne pathogens, including Salmonella
4. Mechanisms of Antibiotic Resistance in Salmonella and Public Health Implications
The horizontal transmission of resistance genes plays a vital role in the dissemination of antibiotic resistance in Salmonella enterica
species. These resistance genes can be found in the resistant plasmids or within the chromosome of bacteria. The horizontal transmission of genes mediated by plasmids is the most efficient method of resistance transfer, and is occurring at high frequency involving different resistance genes at a time [79
]. The resistant genes that are acquired by plasmids, integrons, or transposons are capable of transferring resistance to other strains or other species. Transposons are the mobile genetic elements that can carry resistance genes and possess transposase activity providing the recombination of resistance genes with plasmids or the chromosome. Integrons consist of integrase (a recombination enzyme encoded by the intI
gene), a recombination site recognized by integrase, and a promoter which are necessary for the expression of gene cassettes present in the integron [80
]. These arrangements efficiently promote the acquisition of exogenous genes such as antibiotic-resistant genes in the bacterial genome, especially in plasmids. Furthermore, the conjugation events facilitate the spread of resistance genes present in plasmids through transposon or integron to other strains or species [79
The emergence of S.
Typhimurium definitive type (DT)104 as a multidrug-resistant pathogen was a significant issue in animal agriculture. It was first isolated from the United Kingdom, and since then it has been associated with monogastric animals and ruminants, causing foodborne outbreaks through meat and meat products. The chromosomally encoded resistance to ≥5 antibiotics, including ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracyclines, makes this phage type challenging to tackle. The trimethoprim resistance in S.
Typhimurium DT104 has been found to be associated with mobile non-conjugative plasmids [81
The resistance of non-typhoidal Salmonella
to fluoroquinolones is of particular concern since it is the drug of choice to treat invasive salmonellosis in adults. The fluoroquinolone resistance was previously linked to multiple mutations (e.g., amino acid substitutions) in quinolone resistance-determining regions (QRDRs) of the genes that code for gyrase (gyrA
) and topoisomerase IV, which are the targets for fluoroquinolones in bacterial cells. The mutation of these genes results in resistance to fluoroquinolones [81
]. Also, the presence of an active efflux pump was reported in S.
Typhimurium as a mechanism of antibiotic resistance. The overproduction of AcrAB (inner membrane transporter)-TolC (outer membrane transporter)-type efflux pump and associated alterations in outer membrane proteins and lipopolysaccharides synergistically caused less accumulation of ciprofloxacin in S.
Typhimurium, and showed increased resistance in Salmonella.
However, efflux pump blockers significantly increase the susceptibility (16–32 times) of Salmonella
to fluoroquinolones [81
Plasmid-mediated quinolone resistance (PMQR) genes are also involved in resistance build-up in Salmonella.
PMQR genes such as oqxAB
are isolated with high frequency (44% and 89%; oqxAB
, respectively) from ciprofloxacin-resistant clinical isolates of S.
Typhimurium. These PMQR genes along with gyrA
mutations increase the minimum inhibitory concentration of ciprofloxacin by four-fold in S.
Typhimurium. Among other PMQRs, qnr
type genes also bind to DNA gyrase and topoisomerase and prevent the action of fluoroquinolones. Another PMQR gene, qepA
, is associated with efflux pump and excretes fluoroquinolones to the extracellular space. However, further studies are needed to establish their prevalence in non-typhoidal Salmonella
]. As mentioned, the AcrAB-TolC efflux system and its regulatory genes such as marRAB
are found to be involved in fluoroquinolone resistance which increased the minimum inhibitory concentration (MIC) of fluoroquinolones to ≥32 µg/mL in S.
Typhimurium phage type DT204 [85
] and the inactivation of the efflux pump resulted in a 16–32-fold reduction of the MIC of S.
Typhimurium phage type DT204 to ciprofloxacin [86
]. Therefore, the resistance of non-typhoidal Salmonella
to fluoroquinolones is often attributed to a combination of mechanisms [87
spp. showing resistance to extended-spectrum cephalosporins, including ceftriaxone, is a serious concern, since these are the drugs of choice for treating invasive non-typhoidal salmonellosis in children. One of the major mechanisms of developing resistance against β-lactam antibiotics in bacteria is the direct inactivation of antibiotics by enzyme hydrolysis [88
]. The production of extended spectrum β-lactamases (ESBLs) is a major mechanism conferring resistance in most of the Enterobacteriaceae.
Many types of ESBLs are present based on the substrate and inhibitor mechanisms [89
]. The first β-lactamase identified was TEM-1 found in an E. coli
strain isolated from a patient named Temoniera in Greece [90
]. TEM-1 hydrolyzes penicillins and first-generation cephalosporins. A single amino acid substitution to TEM-1 leads to a TEM-2 derivative having the same substrate as that of the TEM-1. The first TEM-type β-lactamase that demonstrated ESBL characteristics was TEM-3 [89
]. The TEM-type β-lactamases are reported in Salmonella
]. Another β-lactamase, SHV (sulphydryl variable) is a plasmid-encoded β-lactamase usually found in Klebsiella pneumoniae
and E. coli
]. The TEM and SHV types of β-lactamases are most common, and are widely distributed in nature with more than 90 types of TEM and more than 25 types of SHV [90
Recently, the emergence of plasmid-mediated ESBLs, namely CTX-M, is of significant concern since it is commonly found in Salmonella
spp. and associated with cefotaxime hydrolysis. The horizontal transfer of CTX-M ESBL genes via conjugation plasmids and transposons are the main process involved in the acquisition of CTX-M ESBLs. The expansion of CTX-M-type β-lactamase has not been explored much, and has been different from TEM- and SHV-type β-lactamases where the amino acid substitutions are common [89
]. However, it has been suggested that serine residue present at position 237 in all CTX-M type enzymes plays a role in displaying extended-spectrum antibiotic resistance [93
]. In Salmonella
, most of the ESBLs (e.g., blaTEM
-1 and blaSHV
-1 gene derivatives), including enzymes conferring resistance to third-generation cephalosporins such as blaCTX
-M and blaSHV
-5, are encoded on transferable plasmids that pose a serious threat to current antibiotic treatment strategies in humans [79
Another class of β-lactamases is the OXA type that confers resistance to ampicillin and cephalolecithin and also possesses strong hydrolytic activity against oxacillin and cloxacillin [94
]. OXA-48 carbapenemase-producing Salmonella
. Kentucky) and OXA-1 encoding poultry isolates have been identified [91
]. PER-type ESBLs (first discovered in Pseudomonas aeruginosa
strains) hydrolyzing penicillins and cephalosporins have also been reported in non-typhoidal Salmonella
]. In addition, intrinsic cephalosporinases such as AmpC-type β-lactamases are also common in non-typhoidal Salmonella
which includes enzyme types such as CMY, DHA, and ACC-1 [81
]. Moreover, Salmonella
serovars exhibiting different β-lactamases such as CMY-7, SHV-9, and OXA-30 were also identified [97
], indicating the possession of a high level of cross-resistance by non-typhoidal Salmonella
Aminoglycoside-modifying enzymes mainly mediate resistance to aminoglycoside antibiotics. The aminoglycoside acetyltransferases modify amino groups in aminoglycoside antibiotics. The genes encoding aminoglycoside acetyltransferases are named as aac
, and are typically located in Salmonella
genomic islands, integrons, and plasmids. These acetyltransferases provide resistance to major antibiotics such as gentamicin and kanamycin. In addition, aminoglycoside hydroxyl group phosphorylating enzymes, namely aminoglycoside phosphotransferases, are involved in resistance development against aminoglycoside antibiotics in Salmonella.
These enzymes are encoded by the genes strA
, respectively) and provide resistance to streptomycin. Some of the aminoglycoside phosphotransferases also provide resistance to kanamycin and neomycin. Nucleotidyltransferases are also hydroxyl group-modifying enzymes present in Salmonella
and are often encoded in aad
genes. Among the different varieties of aminoglycoside adenylyltransferase coding genes, aadA
provides resistance to streptomycin whereas aadB
provides resistance to gentamicin and tobramycin in Salmonella
Tetracycline resistance is mainly developed in Salmonella
due to the acquisition of genes that code for energy-dependent efflux mechanisms [98
]. Mainly tet genes
are involved in efflux mechanisms, and confer resistance to chlortetracycline, doxycycline, oxytetracycline, and tetracycline [99
]. Among these, tet(A)
is common. However, others such as tet(B)
, and tet(H)
have been reported in non-typhoidal Salmonella
from clinical or retail meat isolates [98
]. The tet(A)
genes have been found in plasmids, integrons, and genomic island 1. The tet(B)
are detected in transferable plasmids. The tet(A)
genes are detected in Salmonella
serovars such as S.
Heidelberg, and S.
]. In addition to this, ribosomal protection proteins such as tet(M)
, and tet(32)
prevent the ribosomes from the action of tetracyclines in microorganisms. Some genes encode enzymes such as tet(X)
, and tet (37)
which modify or inactivate the action of tetracyclines. However, the efflux mechanisms are more common [99
The resistance development in microorganisms against phenicol antibiotics including chloramphenicol and florfenicol is mainly by two mechanisms involving efflux pumps or enzymatic inactivation of antibiotics by chloramphenicol O-acetyltransferase. The chloramphenicol O-acetyltransferase enzymes are not capable of inactivating florfenicol since the fluorinated c3 position in florfenicol does not accept acetyl groups. The genes encoding chloramphenicol O-acetyltransferases are referred to as cat
genes and are often associated with plasmids. The cat1
genes have been isolated from non-typhoidal Salmonella
serovars. The cat
genes are associated with plasmids, transposons, or gene cassettes and other mobile genetic elements [101
]. Genes such as cmlA
encode the efflux pumps in Salmonella
isolates. The floR
genes are widely distributed among the Salmonella
serovars and are found to be associated with transferable plasmids and Salmonella
genomic islands [98
The sulfonamide resistance in Salmonella
is due to the presence of the sul
gene, which causes the expression of an insensitive form of dihydropteroate synthetase that cannot be inhibited by sulfonamides. The common sul
genes are sul1
, and sul3
, which have been identified from major Salmonella
serovars, including S.
, Typhimurium, S.
Heidelberg, and S.
Hadar. These genes are present in integrons, Salmonella
genomic islands, or transferrable plasmids [98
]. The dhfr
genes cause the expression of an insensitive form of dihydrofolate reductase (DHFR) that cannot be inhibited by trimethoprim antibiotics and lead to the development of resistance against this class of antibiotics in Salmonella.
These genes are also associated with integrons, plasmids, or Salmonella
genomic islands [98
6. Ongoing Studies with Alternative Interventions against Multidrug-Resistant Salmonella
Heidelberg is emerging as a significant pathogen causing foodborne disease outbreaks in the U.S. through contaminated poultry products. In 2011, the pathogen caused foodborne outbreaks through contaminated ground turkey products that resulted in 136 illnesses in 34 states [10
]. The outbreak isolates were resistant to several commonly prescribed antibiotics, including ampicillin, streptomycin, gentamicin, and tetracycline [10
]. In our lab, we are developing non-antibiotic interventions against this multidrug-resistant Salmonella.
A dairy probiotic bacteria, namely Propionibacterium freudenreichii
, was found effective against Salmonella
spp., including the multidrug-resistant S
. Heidelberg. A study conducted by Nair and Kollanoor-Johny [162
] revealed that probiotic P. freudenreichii
isolated from fermented dairy products was effective against major virulence factors of multidrug-resistant S.
Heidelberg. Compared to the antibiotic-resistant parent strains, probiotic-treated S.
Heidelberg showed reduced multiplication, motility, and adhesion on intestinal epithelial cells. Follow-up in vivo study revealed that supplementation of P. freudenreichii
to 14-day turkey poults resulted in reduced pathogen colonization in the cecum [163
]. Also, P. freudenreichii
in combination with a mannanoligosaccharide prebiotic and a Salmonella
-specific vaccine was found to be effective in reducing cecal colonization of S.
Heidelberg in 7-week and 12-week old turkeys (unpublished data). In another study, we found that multiple combinations of probiotics (Lactobacillus
of turkey-gut origin), prebiotic, and the Salmonella
vaccine were effective in reducing S.
Heidelberg colonization in the cecum of 14-day turkey poults. These treatments also reduced the S.
Heidelberg invasion of the liver and spleen [164
]. In addition, these combinations were also found to be effective in 7-week-old and 12-week-old-turkeys (unpublished data).
We also found plant-derived compounds such as trans
-cinnamaldehyde and pimenta essential oil were effective against multidrug-resistant S.
Heidelberg isolated from ground turkey. Supplementation of trans
-cinnamaldehyde through drinking water resulted in 4.5 log10
CFU/g reduction in cecal colonization and reduced invasion of S.
Heidelberg to the liver and spleen of 14-day old turkey poults [165
]. Pimenta essential oil was effective against S.
Heidelberg attached to the turkey skin. Our studies revealed that pimenta essential oil was effective against the multidrug-resistant S.
Heidelberg attached to the turkey skin and resulted in >2 log10
reduction at simulated chilling or scalding conditions during processing [166
]. More studies are ongoing in the laboratory exploring the potential of antibiotic alternatives against multidrug-resistant enteropathogens.