Mechanisms of Antimicrobial Resistance and Its Diagnostic Techniques in Nontyphoidal Salmonella Infections

Research Article

J Bacteriol Mycol. 2023; 10(2): 1207.

Mechanisms of Antimicrobial Resistance and Its Diagnostic Techniques in Nontyphoidal Salmonella Infections

Hambisa EM*; Umer AA

Animal Health Institute (AHI), PO Box: 04, Sebeta, Ethiopia

*Corresponding author: Hambisa EM Animal Health Institute (AHI), P.O.Box: 04, Sebeta, Ethiopia Tel: +251 932320809 Email: [email protected]

Received: June 01, 2023 Accepted: June 29, 2023 Published: July 06, 2023


Antimicrobial Resistance (AMR) is the decreased sensitivity of microbes to drugs that are capable of causing cell death or inhibition of growth. It is one of the key issues linked with foodborne infections caused by various resistant pathogenic microorganisms. Nontyphoidal Salmonella (NTS) is one of the leading causes of food-borne infections in which its serotypes are usually zoonotic. The source of this resistance is classified as natural and acquired and the mechanism of AMR emergence in NTS can also be divided into two broad categories: biochemical and genetic mechanisms. The biochemical mechanisms are enzymatic inactivation, antimicrobial permeability reduction, active efflux pumps, and others; while the genetics mechanisms include mutation and horizontal and vertical resistant gene transfer. The techniques of AMR diagnosis are conducted by conventional and non-conventional methods. The utilization of antimicrobials for growth promotion, prophylaxis and its misuse in humans or animals, and contamination of the environment are some of the factors that attribute to AMR development. NTS infection is common throughout the world including Ethiopia and developed resistance to different antimicrobials, and the mechanism of AMR development and its diagnosis is not common in the developing countries including our country. The mechanisms by which NTS develop resistance to antimicrobials should be known very well and its dissemination to human, animals, and the environment could be managed, and methods for the diagnosis of AMR in NTS species should be available for the identification of resistant antimicrobials to give proper treatments and other measures. Additionally, proper policies and regulation systems on antimicrobial use and its distribution should be developed and implemented properly.

Keywords: Antimicrobial resistance; Diagnosis; Mechanism; Nontyphoidal salmonella

Abbreviations: ABC: ATP-Binding Cassette; AMR: Antimicrobial Resistance; ARGs: Antimicrobial Resistant Genes; ATP: Adenosine Triphosphate; BLAST: Basic Local Alignment Search Tool; DNA: Deoxyribonucleic Acid; FTIR: Fourier Transform Infrared; HGT: Horizontal Gene Transfer; HT-qPCR: High Throughput Quantitative PCR; iNTS: Invasive Nontyphoidal Salmonellae; LM PCR: Ligation Mediated Polymerase Chain Reaction; LPS: Lipopolysaccharide; MALDI-TOF: Matrix Assisted Laser Desorption/Ionization Time-of-Flight; MDR: Multi-Drug Resistant; MIC: Minimum Inhibitory Concentrations; MLST: Multi Locus Sequence Typing; NGS: Next Generation Sequencing; NTS: Nontyphoidal Salmonella; PCR: Polymerase Chain Reaction; PMQR: Plasmid Mediated Quinolone Resistance; QCM: Quartz Crystal Microbalance; QRDRs: Quinolone Resistance Determining Regions; RNA: Ribonucleic acid; RT-PCR: Reverse Transcriptase Polymerase Chain Reaction; SERS: Surface Enhanced Raman Spectroscopy; SGI1: Salmonella Genomic Island 1; T4SS: Type IV Secretion System; WGS: Whole Genome Sequencing; WHO: World Health Organization; WMS: Whole Metagenome Sequencing


The global increment of foodborne infections linked with antimicrobial-resistant pathogenic microorganisms and the dissemination of Antimicrobial Resistance (AMR) is one of the key issues in developing and developed countries. The emergence of multi-antimicrobial resistant Salmonella strains and the continuous spread of its clones is one related concern for human health in different countries. Incidents of multidrug resistance in Salmonella species, and other bacterial pathogens causing enteric diseases, have been reported in different countries and became a main health concern as they can spread worldwide [1].

Salmonella is a genus of the Enterobacteriaceae family which is characterized as a gram-negative, rod-shaped, non-spore-forming, and facultative anaerobic bacterium and moves using a peritrichous flagellum. Based on the serotype, its genome ranges from 4460 to 4857 Kilobase (kb) [2]. Salmonella can be divided into typhoid and Non-Typhoid Salmonella (NTS) regarding their ability to develop specific pathologies in humans. Typhoid serovars are a subcategory of serovars known as specialists (adapted), capable of infecting and colonizing only a very narrow range of hosts, and include the Typhi, Sendai, and Paratyphi A, B, and C serovars, highly adapted to humans and presenting only higher primates and humans as reservoirs. Generalist serovars are capable of triggering infections in both humans and animals, including NTS Enteritidis and Typhimurium, known mainly for their epidemiological relevance. These serovars are mainly transmitted by animal-based foods, such as beef, pork, poultry, and contaminated raw eggs, fruits, and vegetables can also serve as vehicles. Poor access to improved water supplies and inadequate sanitation facilities, combined with growing urbanization, favor the transmission of NTS serovars [3].

Some NTS serovars demonstrate evident host predilection and usually are associated with a specific animal host, such as Choleraesuis and Dublin, which prefer pigs and cattle, respectively, but may also infrequently cause disease in other mammals, including humans. These NTS can cause invasive infections, and invasive NTS (iNTS) in humans [4].

Nontyphoidal Salmonella is a leading cause of foodborne infection worldwide in which its serotypes are usually zoonotic and have a wide range of animal reservoirs [5]. Some serotypes of Salmonella enterica (S. enterica) are considered emerging zoonotic pathogens, generating outbreaks worldwide in the human population. It is estimated that S. enterica gastroenteritis is responsible for about 93.8 million illnesses and 155,000 deaths worldwide each year, and of these, 80.3 million cases are estimated to be foodborne [6]. It is a facultative intracellular pathogen that is capable of causing different disease syndromes in a wide range of hosts. S. Typhimurium (S. Typhimurium) and Salmonella Enteritidis (S. Enteritidis) are the most frequently isolated serovars throughout the world, leading to severe economic losses [7].

Poultry and other food animals are considered the common reservoirs of S. enterica and undercooked poultry products are the major sources of human infection with NTS. Several studies on Salmonella isolates from poultry products and farms in the past studies were found to be resistant to several antimicrobials. Information on farm-level prevalence and antimicrobial susceptibility status of isolates can explain the level of public health risk associated with poultry products [8]. Antimicrobial Resistance is the decreased sensitivity of microbes to antibiotics that are capable of causing cell death or inhibiting growth [9]. This can be determined through antimicrobial sensitivity testing of Salmonella isolates to determine their susceptibility or resistance to the antibiotics. Resistance in Salmonella is encoded by genes that are present on either chromosome or extra-chromosomal DNA (plasmid) or transferable genetic materials (transposons, integrons), which is determined by genetic or molecular methods. Although resistance may occur due to mutation in key genetic loci in the bacterial genome, most resistance to antimicrobial agents mediated by genes is acquired via mobile genetic elements such as plasmids and transposons [10].

The mechanisms of antimicrobial development to NTS can result from enzymatic inactivation, decreased permeability, and development of efflux pump systems, alteration of target sites, and in most cases in many serovars the overproduction of target sites to overwhelm the used antibiotics. In several cases investigated, antibiotic resistance can be acquired through natural selection or mutation (induced or spontaneous); this however can be chromosomal mutation by the production of chromosomally mediated inducible enzymes or acquisition of plasmid-resistant genes which is the most common genetic basis of antibiotic resistance [11].

The diagnosis of resistance genotype is accomplished through the detection of novel genetic materials and characterization of mutations in specific genes through Polymerase Chain Reaction (PCR), Deoxyribonucleic Acid (DNA) probes, and other amplification techniques [12]. Genotypic analysis of the antibiotic-resistant Salmonella species by use of real-time-polymerase chain reaction and molecular fingerprinting of DNA has been used to good effect. Plasmid gene profile analysis is a quick and relatively easy method to fingerprint strains and has been used in both human and veterinary medicine to study the spread of AMR Salmonella. Phage typing or alternative genetic techniques and full DNA sequencing are increasingly used to study genetic variations in AMR Salmonella species chiefly because of their low-cost automated methods [13].

However, the development of resistance in the responsible pathogens has worsened the situation often with very few resources to investigate and provide reliable susceptibility data on which rational treatments can be based as well as means to optimize the use of antimicrobial agents in most of the developing countries [14]. NTS is one of these resistance-developed microbes having major public health concerns and economic importance throughout the world, and the mechanism of AMR development and its diagnosis is not common in developing countries including Ethiopia.

In light of the above background information and existing facts, this review paper is prepared with the following objectives:

To review the mechanisms by which NTS develops resistance to antimicrobials

To describe the techniques that are used for the diagnosis of AMR in NTS species

Origins of Antimicrobial Resistance

Bacteria as a group or species are not necessarily uniformly susceptible or resistant to any particular antimicrobial agent. Levels of resistance may vary greatly within related bacterial groups. Susceptibility and resistance to antimicrobial agents are often measured as a function of Minimum Inhibitory Concentration (MIC), the minimal concentration of a drug that will inhibit the growth of the bacteria. The susceptibility is a range of the average MICs for any given drug across the same bacterial species. If that average MIC for a species is in the resistant part of the range, the species is considered to have intrinsic resistance to that drug. Bacteria may also acquire resistance genes from other related organisms, and the level of resistance will vary depending on the species and the genes acquired [15].

Natural Resistance

Intrinsic resistance: Intrinsic resistance may be defined as a trait that is shared universally within a bacterial species, is independent of previous antibiotic exposure, and is not related to horizontal gene transfer. The most common intrinsic resistance forms are reduced permeability of the outer membrane (most specifically the Lipopolysaccharide (LPS), in gram-negative bacteria) and the natural activity of efflux pumps [16].

Induced resistance: Induced resistance means that the genes are naturally occurring in the bacteria, but are only expressed to resistance levels after exposure to an antibiotic. Multidrug-efflux pumps are one of the common mechanisms of induced resistance [17].

Acquired Resistance

The genetic material that confers resistance can be acquired through all of the main routes by which bacteria acquire any genetic material: transformation, transposition, and conjugation; all termed Horizontal Gene Transfer (HGT); and, the bacteria may experience mutations to its chromosomal DNA. The acquisition may be temporary or permanent. Plasmid-mediated transmission of resistance genes is the most common route for the acquisition of outside genetic material; bacteriophage-borne transmission is fairly rare. Bacteria can be naturally competent and as a result capable of acquiring genetic material directly from the outside environment. Internally, insertion sequences and integrins may move genetic material around, and stressors (starvation, ultraviolet radiation, chemicals, etc.) are the common causes of genetic mutations (substitutions, deletions, and so on) [18].

Mechanisms of Antimicrobial Resistance In Nontyphoidal Salmonella Infections

Various mechanisms of AMR have been reported and these mechanisms lead to the emergence of multidrug resistance in Salmonella species. These mechanisms can be classified into two broad categories which involve biochemical mechanisms and genetic mechanisms [19].

Biochemical Mechanisms of Antimicrobial Resistance

Enzymatic inactivation: The enzymatic mechanisms of antibiotic resistance include hydrolysis, group transfer, and redox processes [20]. In terms of diversity, evolution, and spread, antibiotic resistance enzymes contribute remarkably to the bacterial ability to overcome antibiotic pressure. The Β-lactamases are the oldest known and the most diverse antibiotic-degrading enzymes that cleave the Β-lactam ring of the penicillin group of antibiotics and render them ineffective. Scientific evidence suggests the existence of Β-lactamases before penicillin was clinically employed, emphasizing that the production of antimicrobial compounds and the mechanisms to endure them occur in parallel in the environment [21].

Antimicrobial permeability reduction: The other mechanism of resistance to antimicrobial agents involves preventing drug permeability and access to the internal milieu of the pathogenic cells [22]. The molecular systems involved in the reduced permeability of antimicrobial agents include resistance mechanisms at the bacterial cell wall. The extensive structural nature of the LPS layer constitutes a formidable barrier to the passage of small molecules, especially those that are growth inhibitory in their properties [23]. Another important molecular mechanism for conferring resistance via permeability reduction involves porins, which are integral outer membrane proteins with water-filled pore-like channels that permit the passage of molecules with definitive sizes and charges. The relationship between bacterial AMR and the outer membrane porins can take one of several ways. A wide-type porin can be highly selective towards the entry of certain nutrients, like sugars, and not permit the passage of many antimicrobial agents. However, for those porins for which no such highly selective properties are a problem, then in such cases, the porin molecules may be depleted from the membrane or functionally disrupted by mutation. In other cases, permissive porins may be regulated by channel blockers or by Ribonucleic Acid (RNA) specific antisense modulators [24].

Active efflux pumps of antimicrobial agents: Efflux pumps are present in all bacteria and are integral parts of bacterial physiology, being involved in diverse functions such as the expulsion of toxic products of metabolism, and the maintenance of homeostasis. However, antibiotics as incidental substrates of efflux pumps have resulted in them being viewed largely as bacterial mechanisms of antimicrobial resistance, and have critical roles in ensuring bacterial survival and evolution into resistant strains. These bacterial multidrug efflux pump systems are energetically driven by Adenosine Triphosphate (ATP) hydrolysis, called primary active transport, and by electrochemical ion gradients or ion motive forces, called secondary active transport [25].

Active transport of antimicrobial agents represents an essential resistance mechanism in bacterial pathogens. As multiple structurally distinct antimicrobial agents with disparate modes of action are exported to the extracellular milieu, their growth inhibitory properties towards bacteria are diminished, if not wholly circumvented. During the primary active transport of antimicrobial agents, bacteria exploit the biological energy stored in the form of intact ATP to export drugs against the drug concentration gradient by performing ATP hydrolysis [26]. During the export of antibacterial agents from bacterial cells, ATP is hydrolyzed to energize the drug translocation through the transporter in an outward direction across the membrane. Thus, as the transporter substrate actively accumulates outside the cell, AMR is conferred upon the bacterial pathogen. One of the best-studied of these primary active drug efflux systems is the ATP-Binding Cassette (ABC) efflux pump family. The ABC transporter represents one of the most abundant protein families known across all taxa of living organisms [27].

Antimicrobial targets alteration: Antimicrobial targets play vital roles in microbial growth or survival and, thus, serve as potentially useful targets for mitigating infection. These targets must differ or be completely absent from humans or the animal species being treated with an antimicrobial to allow for a selective mode of action. A classic example of such a target is peptidoglycan. Peptidoglycan is essential to the growth and survival of many bacterial species and has a chemical structure that is not present in the mammalian hosts they infect. This allows for the targeting of enzymes responsible for the synthesis and assembly of peptidoglycan. The function of proteins associated with these target sites makes it non-viable for a bacterium to evolve resistance by removing these proteins. However, mutations that allow for continued functionality while reducing the ability of an antimicrobial agent to bind them at the target site have been a veritable regularity in the arms race between antimicrobial substances and antimicrobial-resistant bacteria. In addition to peptidoglycan, alteration in target sites has been attributed to ribosomes, nucleic acid enzymes, and LPS [28].

Biofilm formation: A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharides, proteins, and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis and other components of the body's defense system. Biofilm production occurs in many loci, including teeth plaque, water environments, medical catheters, and trauma wounds, and those microorganisms that are found in biofilms are protected from the entry of multiple antimicrobial agents [29]. Thus, biofilms are increasingly becoming a challenge in the human clinical medicine arena when considering potential chemotherapies with antibacterial agents, and this is recognized as one mode of resistance [30].

Antimicrobial targets protection: One of the significant lines of defense against an antimicrobial is the bacterial cell wall. This structure also acts as a physical barrier to encase the cytoplasm and cell membrane from the external world [31]. Prokaryotic cell walls are made up of linear glycan strands cross-linked by small peptides and this peptidoglycan helps to limit which substances can continue towards the cell membrane and ultimately into the cytoplasm. Peptidoglycan also plays an essential role in bacterial growth and proliferation. While the cell wall helps protect cytoplasmic antimicrobial targets, it also ended up being the target for the first natural antibiotic, penicillin, which prevents the complete formation of this barrier by inhibiting peptide cross-linking to occur [32].

With this mechanism of protection compromised due to the advent of Β-lactam antibiotics, prokaryotes began to synthesize another tier of protection: Β-lactamases. These enzymes help to protect the peptidoglycan cell wall from Β-lactam antibiotics, precisely. Β-lactamase enzymes help confer resistant bacterial phenotypes, as their mechanism of action hydrolyzes the Β-lactam ring of such antibiotics, and the resulting chemical structure can no longer hinder bacterial cell wall synthesis [21] (Figure 1) and (Table 1).