Salmonella Infections: An Update, Detection and Control Strategies

Abstract

Salmonella belonging to the Enterobacteriaceae family is commonly divided into Typhoidal and non-typhoidal group. Clinical manifestations can range from gastroenteritis, bacteremia or septicemia without gastrointestinal (GI) upset, enteric fever, chronic carriage to focal infections like osteomyelitis, meningitis, endocarditis etc. Conventional diagnostic tests like blood, stool, food sample culture have a long turnaround time. Chromogenic media modification reduces identification time. Apart from Widal test and ELISA, rapid point of care serological tests like Tubex TF, Typhidot are advantageous in basic set up but limited by modest accuracy in high burden settings. Apart from immunological assays, other diagnostic modalities include PCR, mass spectrophotometry, spectroscopy, optical phenotyping, biosensors etc. Rising drug resistance of Salmonella to first line Ampicillin, Chloramphenicol and Cotrimoxazole with increasing nonsusceptibility to fluoroquinolones and 3rd generation cephalosporin for past few decades is a public threat. Prevention and control measures include basic sanitation, safe water access, safe food handling, public education, physical, chemical, biocontrol methods, vaccination etc. Targeting the menace of antimicrobial resistance in Salmonella species needs a collaborative effort like One Health approach which optimizes the public health, animal and environmental health and reduce the dependency on antibiotics.

1. Introduction

The bacterium Salmonella causes foodborne diseases. It is a gram-negative rod-shaped facultative anaerobic bacterium, predominantly motile with over 2600 serovars [1]. Foodborne diseases is a major public health problem, especially for young children. It is a ubiquitous microorganism which can easily survive in hardy environments. Salmonellosis is one of the major diarrheal diseases worldwide [1]. Apart from diarrhoeal diseases, Typhoidal strains can cause life-threatening enteric fever. In addition to the lack of sanitation and potable water, rising antimicrobial resistance is compounding to the problem [2]. In 1880, Karl Joseph Eberth isolated bacilli presumed as an agent of Typhoid fever from the spleen and mesenteric lymph nodes of patients. It was George Theodor Gaffky, an army surgeon and assistant to Robert Koch who managed to isolate the bacilli in pure culture. The microorganism was named Eberthella Typhi or Eberth-Gaffky bacillus. In 1885, a medical research scientist Theobald Smith in the department headed by a veterinary pathologist, Daniel Elmer Salmon, isolated a microbe called Hog-Cholera Bacillus from porcine intestines affected with swine fever. Joseph Leon Ligneres in the 1900s proposed the name Salmonella for the genus in honor of Daniel E. Salmon [3, 4].

2. Taxonomy and nomenclature

Salmonella is a complex group of bacteria. It belongs to Kingdom Eubacteria, phylum Proteobacteria, class Gamma proteobacteria, order Enterobacterales, family Enterobacteriaceae and Genus Salmonella [5]. The nomenclature of Salmonella is complex, controversial and ever-evolving. It amalgamates genetic, biochemical, and serological classification. Presently, the Centre for Disease Control and Prevention (CDC) uses nomenclature based on recommendations by the World Health Organization (WHO) Collaborating Centre [6]. Based on 16sRNA sequence analysis, this system divides Salmonella into two broad species S. enterica and S. bongori.

S. enterica is again categorized into six subspecies: enterica (I); salamae (II); arizonae (IIIa); diarizonae (IIIb); houtenae (IV); and indica (VI). Roman numerals (I to VI) have been designated to the six subspecies. Subspecies V does not appear in this classification as it was used to describe S.enterica subsp. bongori which has since been promoted to separate Salmonella species [6, 7]. Figure 1 summarizes the taxonomical classification of Salmonella.

Figure 1.

Salmonella taxonomical classification.

Apart from this, further classification of Salmonella subspecies is based on antigenic classification based on the somatic (O) or capsular (K) and flagellar (H) antigen detected by agglutination which was given by Kauffmann-White, now known as Kauffmann-White- Le Minor scheme [7]. Historically, serogroups based on O antigen were designated by alphabets e.g. A, B, C and so on. With the increasing number of serogroups identified, the alphabets were replaced with numbers. In certain Typhoidal serovars, Vi antigen is present superficially over the O antigen layer. An updated scheme with newer serotypes identified by the Word Health Organization Collaborating Centre for Reference and Research on Salmonella at the Pasteur Institute, Paris, France is reported in Research in Microbiology by Popoff et al. yearly, which has reported over 2600 serotypes or serovars [8]. The terminologies serotype and serovar seem to be used interchangeably. The Association of Public Health Laboratories and the World Health Organization prefer to use the term serovar in their documentation [9, 10]. The Rules of the Bacteriological Code also prefers serovar to serotype [10]. The naming of Salmonella strains before 1966 was based on host specificity, geographical origin or clinical features. All older serovars with specific names have been retained to this day with an underlying antigenic formula. Examples of some of the classical serovar names still used to these days are Agama (isolated from the Agama lizard feces), Agona (from cattle in the town Agonas of Ghana in 1952), Choleraesuis (“Swine cholera,” Hog cholera cause), Dublin (isolated from the stool of a patient in Dublin, Ireland in 1929), Heidelberg (isolated from a patient in Heidelberg, Germany in 1933), Infantis (isolated from an infant in Connecticut, USA in 1943), Typhimurium (“mouse typhi,” typhoid fever cause in mice) etc. [3, 6, 7].

Antigenic formula usually begins with subspecies written in Roman letters followed by somatic O antigens, flagellar H antigens phase 1 and phase 2, followed by other results if present. A colon separates major antigens and comma separates components of one antigen. Various symbols appearing in the antigenic formula make it complicated for example, square brackets like [ ] means the O or H antigen in question may be present or absent, ( ) parentheses means antigen is weakly agglutinable, { } curly bracket means antigens are mutually exclusive, hyphen – means particular flagellar phase is absent and underlined O factor means the factor is coded by bacteriophage (lysogenic strain). For example, antigenic formulae for the Salmonella Infantis is: I 6,7,14:r:1,5. Interpretation for the Infantis formula is that it belongs to subspecies enterica I, positive for 6,7,14 O antigen, positive for r H1 antigen and positive for 1 & 5 H2 antigen. A serotype name is not italicized but begins with capital first letter to differentiate from species name when writing a name of a particular named strain. When citing the name first time, “serotype” term should follow the genus name or the word “serotype” can be abbreviated to “ser” e.g. Salmonella serotype Typhi or Salmonella ser Typhi. In subsequent citations in the text, the name is shortened with the genus name followed by the serotype name with capital first letter, e.g., Salmonella Typhi or S. Typhi [8, 9].

3. Disease transmission

Salmonellosis is mostly foodborne. Consumption of contaminated meat, dairy and eggs is the cause as the bacteria mostly resides in the gut of animals and birds, the major sources being pigs, cattle and poultry. Transmission through the consumption of contaminated produce like fruits, vegetables, dried seeds as well as cereals has also been documented [11]. Person-to-person spread occurs in enteric fever via ingestion of food and water contaminated with human feces as Typhoidal fever lacks animal reservoirs. Food handlers especially, asymptomatic carriers of the bacteria with poor hygiene, sanitation and lack of clean water contribute to the spread of the disease [12]. The history of Mary Mallon “Typhoid Mary” is synonymous with the spread of the disease. The Irish-born American cook was a healthy carrier who was found to be responsible for the disease in hundreds of people [13]. The infective dose of bacteria has been reported to be less in non-typhoidal disease to the tune of 10³ whereas approximately 10⁵ bacilli is the infective dose for enteric fever [14, 15]. Various factors exist which increase the susceptibility of the host to Salmonellosis. Children less than 5 years of age, elderly and immunocompromised cases are more susceptible. Hosts with conditions like achlorhydria, gastric surgery, inflammatory bowel disease, hemoglobinopathies, and impaired immune conditions like diabetes mellitus, AIDS, leukemia and lymphomas are more likely to get infected with a lower infective dose [14, 15, 16, 17].

4. Pathogenesis and virulence factors

Salmonella harbors a variety of virulence factors to be fully pathogenic. The establishment of salmonellosis depends on the host cell invasion ability of the particular strain, intracellular proliferation, presence of a complete lipopolysaccharide coat and its toxin secretion ability. The virulence determinants singly or in combination facilitate the entry, attachment, colonization and invasion by overriding host defense mechanisms [18]. Virulence traits include flagella, capsule, plasmids, adhesion systems like adhesins, invasins, fimbriae, hemagglutinins, exotoxins, endotoxins, biofilm formation and type 3 secretion systems (T3SS) encoded on the Salmonella pathogenicity islands (SPI) [18, 19]. The invasion mechanism by Salmonella is partially understood. Following ingestion of the bacteria, the first bottleneck which acts against Salmonella colonization is the host gastric acidity where the pH drops as low as 1.5. Higher pH due to the use of proton pump inhibitors, antacids or achlorhydria along with food particles shields the bacteria and increases the risk of transmission [20].

The bacilli colonize the ileum and colon by attaching to the epithelial cells (M cells) by fimbriae. They enter through its epithelial lining by interacting with apical microvilli. Bacteria-mediated endocytosis occurs when the bacilli trigger the formation of ruffles on the cell membrane of M cells leading to the engulfment of the bacteria in vesicles. These vacuoles containing Salmonella reach the submucosa where they are phagocytosed by macrophages. The bacilli become non-susceptible to the lysosomal enzymes of the macrophages due to the organism’s PhoP/PhoQ regulatory system, following which they spread via lymphatics and blood to other organs of the body [14, 15]. SPIs are gene clusters present on several chromosomal areas which encode for various virulence traits and are plasmid or chromosomally located usually, associated with mobile genetic elements. Several SPIs have been identified but SPI 1–5 have been closely associated with Salmonella serovars especially S. enterica, pathogenic to humans [21]. Host cell invasion including secretion of effector protein responsible for host cell function, encoding for T3SS and macrophage apoptosis induction is mediated by SPI-1 whereas replication within macrophages is done by SPI-2 by maintaining Salmonella-containing vacuole. SPI-2 is also responsible for encoding a second set of T3SS responsible for systemic infection. SPI-3 is reported to be responsible for encoding proteins required for long-term survival of bacilli, SPI-4 and SPI-5 encodes for genes associated with toxin secretion, survival within macrophages and several other sets of T3SS effector proteins [21, 22, 23]. The T3SS encoded by SPIs allows the effector proteins to give rise to a cascade sequence of activation of signal transduction and rearrangement of the cell cytoskeleton leading to ruffling of epithelial cell membrane for pinocytosis [24]. SPIs harbor various genes like spvC concerned with the formation of Salmonella containing vacuole (SCV) following engulfment which can escape host defense mechanism, sopE associated with activation of the signaling pathway for tissue damage and inflammation, suppression of signaling pathway for bacterial multiplication inhibition etc. [25, 26]. SPIs also harbor genes for encoding proteins involved in toxin mediation, virulence enhancement etc. The SCV prevents fusion with lysosomes and allows intracellular multiplication within macrophages. They spread to mesenteric lymph nodes and later reach the reticuloendothelial system (RES) via systemic circulation [24]. The invasion of various organs like gall bladder, liver, bone etc. depends on the interplay of serovars ingested and the effectiveness of the host immune system. Mostly, Salmonellosis causes acute diarrhea as the majority of the extra-intestinal bacilli are killed immediately [16, 27]. Mucosal inflammation causes release of numerous proinflammatory cytokines like IL-1, IL-6, IL-8, TNFα, IFN-γ, IL-12, IL-18 and IL-15 etc. [28]. Acute inflammatory response elicited by the pro-inflammatory cytokines causes intestinal ulceration along with inflammatory symptoms like fever, abdominal pain, diarrhea etc. Salmonella also triggers the activation of intestinal mucosal adenylate cyclase related to local generation of prostaglandin and enterotoxins which increases cyclic AMP, resulting in intestinal secretion. Salmonella escapes from the basal epithelial cells into lamina propria spreading systemically resulting in enteric fever [29].

5. Clinical features

Infections caused by Salmonella can be broadly classified into Typhoidal and non-typhoidal salmonellosis. It can present as gastroenteritis, enteric fever, other extra intestinal manifestations and chronic carriage [30].

5.1 Enteric fever

Enteric fever is caused by S. Typhi and S. Paratyphi A, B and C. Being indistinguishable from each other clinically, Typhoid and Paratyphoid fever are together called Enteric fever. Humans are the only reservoir for enteric fever. The incubation period of Typhoid fever is usually 6–30 days and that of Paratyphoid fever is typically 1–10 days [31]. Disease prodrome includes headache, malaise, loss of appetite, abdominal pain, diarrhea or constipation along with low-grade fever. Slowly, the fever rises from low grade to continuous high grade with the onset of the second week which is known as the step ladder pattern of fever [14, 15]. Pea soup diarrhea is usually seen in patients especially, children but immunosuppressed patients can present with constipation too. Other signs and symptoms include nausea, vomiting, relative bradycardia, rose spot on the trunk (in 30% of cases), hepatosplenomegaly, and myalgia and in some cases, cough and sore throat can also be seen. The dreaded complication occurring in 3% of the cases include gastrointestinal bleeding and intestinal perforation of peyer’s patches at the terminal ileum usually observed in the third week. Other extraintestinal complications include myocarditis, metastasis to bone, joints, urinary tract etc. Gastrointestinal complications can present as pancreatitis, hepatitis and cholecystitis etc. Neurological complications include encephalopathy, meningitis, cerebellar ataxia and neuropsychiatric symptoms etc. [32, 33].

5.2 Non-typhoidal gastroenteritis

Non-typhoidal Salmonella (NTS) are Salmonella strains other than S. Typhi and S. Paratyphi. Primary reservoirs of NTS are usually wild, domesticated animals as well as birds. S. Typhimurium is the most common cause of NTS food poisoning [34]. The incubation period of NTS food poisoning is usually 6–12 hours as compared to days to weeks of Typhoidal incubation time. It is an acute inflammatory gastroenteritis characterized by fever, abdominal pain, diarrhea, nausea, vomiting etc. and is usually self-limiting. This requires no treatment but is indicated in cases with immunosuppressed conditions, children and elderly age groups as it is likely to get complicated [15, 34].

5.3 Invasive non-typhoidal Salmonella (iNTS) infections

The characteristic of iNTS infections is bacteremia by NTS without any focus of infection or gastroenteritis [35]. The most common iNTS strains are Choleraesuis, Dublin, Enteritidis, Typhimurium, Bovismorbificans, Arizona, and Gallinarum etc. Risk factors for bacteremia with other extra-intestinal manifestations include malnutrition, haemolytic anemia (Sickle cell anemia, Thalassemia), T-cell immunosuppression (AIDS, Hodgkin Lymphoma, Steroid Use, Transplantation, Chemotherapy), Schistosomiasis, Chronic granulomatous disease, mutations or acquired auto-immunity compromising IL-12, IFNγ function etc. [36]. The virulence determinants of iNTS are reported to be SPI-1 & SPI-2 responsible for an invasion via T3SS expression, survival of bacteria in macrophages and systemic manifestations; spv (Salmonella plasmid virulence) gene in iNTS is involved in macrophage cytoskeleton disruption, suppression of MAP kinase and NF-κB signaling pathway for prolonged survival of bacteria and host iron homeostasis interference [25, 36, 37]. Manifestations of iNTS infections include most commonly bacteraemia characterized by high-grade fever without formation of rose spots, pneumonia, endocarditis, osteomyelitis, prosthetic joint infections, meningitis, visceral abscess etc. [36].

5.4 Carrier state

Carriers are important from the public health point of view as they are the major cause of outbreaks. Temporary carriers are the cases who continue to shed Typhoid bacteria up to 3 months after disease resolution. It is seen in approximately 10% of cases. In chronic carriers, approximately 2–5% of cases fail to fully clear the infection even after a year of resolution of the disease [38]. Chronic carriers act as a reservoir of infection in the community as they intermittently shed the bacteria in stool or urine. Long-term survival of bacteria in carriers is due to the breach of the epithelial barrier, evasion of the host immune system and the forming of a niche in the host body. Mechanical barrier due to the sphincter of Oddi along with the flushing inability of bacteriostatic bile by the host is associated with the long-term carriage of the pathogen [39]. In contrast to Typhoidal Salmonella, carriage is uncommon, approximately seen in only 0.1% of cases with NT Salmonellosis [34].

6. Epidemiology

World Health Organization reported Typhoid infections affecting approximately 9 million people with over 110,000 deaths as of 2019. Typhoid was mostly reported from the developing nations of Southeast Asia, Africa, the Eastern Mediterranean and the Western Pacific Region [2]. The European countries and USA reported a low number of cases approximately less than 10 per 100,000 population annually with mostly travel-related history to enteric fever endemic countries [40]. Low-incidence countries like Israel noted causative agents of enteric fever to be S. Paratyphi in double the numbers as compared to S. Typhi [41]. Indian subcontinent documented the highest number of cases compared to other regions. The incidence rate for India, Pakistan and Indonesia per 100, 000 was 340, 573 and 148 [42]. Earlier in 2000, African countries reported a low number of Typhoid cases as compared to South Asian countries but in recent years, there are reports of growing numbers of Typhoid fever cases. Poor diagnostic infrastructures and fewer epidemiological studies could be associated with less reporting [42, 43, 44]. A low incidence of 0.38 per 100, 000 of Typhoid Fever was documented in China with cases mostly clustering in children <3 years of age and elderly >60 years of age [45]. Rising incidences of S. Paratyphi infections as compared to S. Typhi infections from Asian countries show the need for detailed epidemiological studies.

Non-typhoidal Salmonella (NTS) affects more than 150 million cases and deaths of approximately 60,000 cases globally, thus becoming one of the leading bacterial causes of diarrhea [46]. The highest incidences were reported from Sub-Saharan African (227/100,000) and European countries (102/100,000). American (23/100,000), South East Asia (21/100,000), Middle East (0.8/100,000) and North Africa (0.8/100,000) documented low incidences of NTS infections [46, 47]. S. Enteritidis was the most common serotype reported globally for causing NTS infections. Second and third most common isolates affecting human were S. Typhimurium and S. Newport respectively [48].

7. Detection

Salmonella can be diagnosed by various techniques starting from conventional methods to newer non-culture methods like molecular-based, immunological-based, molecular-based, mass spectrometry, spectroscopy, optical phenotyping, biosensor methods etc. [49, 50].

7.1 Culture based techniques

Even though culture-based methods are time-consuming, it is still the gold standard in diagnosis. Stools and suspected food products are the most commonly tested clinical samples. Apart from stool, other clinical samples are blood, body fluids, pus, bone marrow, duodenal aspirate, urine, punch biopsy samples of rose spots etc. [51]. The two most commonly done diagnostic tests for enteric fever are culture and Widal test. Bacterial culture depends on the site, age group, antimicrobial intake and stage of illness. Though blood culture is done commonly, the sensitivity is lower and variable (40–80%) as compared to bone marrow culture with a higher sensitivity of more than 80%. Also, blood culture sensitivity is higher in the first week as compared to subsequent weeks of infection and also gets significantly affected with antibiotics intake unlike bone marrow culture [52]. Blood and sterile body fluids can be cultured in blood culture bottles which can be conventional blood culture or automated blood culture bottles (Bactec/BacTAlert) which are further subcultured onto MacConkey agar. Culture methodology is different for contaminated samples like stool or food samples. The fecal samples on day 0 are cultured onto selective media like Xylose Lysine Deoxycholate (XLD) along with an enrichment broth like Selenite F Broth and incubated at 37°C for 18–24 hours. The enrichment broth is further subcultured onto selective media on Day 1. Enrichment broths like Selenite cystine broth, Gram Negative broth, and Tetrathionate broth are required for the selective enrichment of Salmonella over other microflora [53]. FDA’s Bacteriological Analytical Manual (BAM) recommends pre-enriching food samples in non-selective media like buffered peptone water followed by Rappaport Vassialidis soy (RVS) broth and Muller Kauffmann tetrathionate-novobiocin and finally plating onto two selective media [49]. Various selective media for Salmonella apart from XLD agar are Hecktoen Enteric, Deoxycholate Citrate, Salmonella Shigella, Brilliant Green, Bismuth Sulfite agar etc. [54]. The suspicious-looking Salmonella colonies are identified by automated or traditional biochemical methods. The traditional biochemical tests include sugar fermentation, urease, citrate, decarboxylase, indole test, H2S production, MRVP, and nitrate etc. Isolates with presumptive biochemicals are confirmed with serotyping with O and flagellar H antisera and additional biochemical tests may be required in inconclusive results [53, 55]. Automated identification methods which are commonly used include Vitek-2, Microscan Walkaway system, MALDI-TOF etc. Modifications to conventional media like fluorogenic and chromogenic media e.g. SM-ID, BBL CHROMagar Salmonella and Rambach agar etc. decrease the turnover time by 1 day due to direct detection and identification [49, 53].

7.2 Non-culture-based techniques

Immunological assays: The Widal test is a routinely used serological test in high-burden countries. It is an agglutination test quantifying antibodies against lipopolysaccharide O and flagellar H antigen. Though very convenient for low resource settings for its simplicity, the pitfall of this test primarily lies in the need for cautious interpretation of the results, limited sensitivity, specificity and difficulty distinguishing current and past infections [54]. Single unpaired tests showed poor sensitivity and specificity, especially in the early course of infection. Paired serological testing improves diagnostic accuracy but is limited in its clinical utility [51, 54]. Unlike the conventional Widal agglutination test, newer techniques in the immunological assay are based on the principle of Salmonella antigen capture by monoclonal or polyclonal antibodies. Enzyme-linked immunosorbent assays (ELISA) have been used for detection as well as quantification of antibodies targeting O, H, Vi antigens [51]. The diagnostic accuracy of ELISA was found to be better than the Widal test in comparative studies and [56, 57]. However, in the early course of the infection, ELISA done with single unpaired sera suffers from the same limitations of specificity as that of the Widal test [51, 58]. Conventional ELISA in resource-limited settings poses the problem of costly laboratory equipment and the need for trained personnel [59]. Recent advances like manipulation with gold nanoparticles in ELISAs have improved the results by lowering the limit of detection [60]. One of the simplest detection methods is the latex agglutination test where visible agglutination will be seen when antigens coated on Salmonella will bind with antibodies coated on latex particles [49]. Even if outdated, it is still a simple and reliable test for confirmatory analysis of isolates [49, 61]. Commercially available rapid diagnostic kits like Tubex TF immunoassay and Typhidot immunochromatographic test (ICT) have been developed as point-of-care tests for enteric fever [62, 63]. Tubex TF is an inhibition magnetic binding immunoassay which is a semi-quantitative test based on visual interpretation of color development. The principle is based on the inhibition of the reaction of magnetic particles coated with antigens and antibody-coated colored latex particles by IgM antibody against S. Typhi O9 present in the serum. Tubex TF showed a sensitivity of 56–95% and a specificity of 72–95% [62]. Typhidot is a miniaturized dot enzyme immunoassay that detects IgG and IgM antibodies to S. Typhi outer membrane protein (OMP) antigen. Typhidot- M detects recent infection by detecting IgM antibody against S. Typhi after IgG inactivation [63]. It showed moderate sensitivity and specificity of 56–84% and 31–97% respectively [62]. None of them yielded satisfactory results when tested in high-burden countries [64]. Although cheap and simple, clinicians in low-resource endemic settings should cautiously rely on them as early diagnostic tools.

Molecular Assays: Molecular techniques broadly use the principle of DNA/RNA detection of specific pathogens by target or signal amplification or non-amplification methods [49]. PCR is an excellent tool for pathogen detection. The most commonly used PCR targets are flagellum genes like fliC-d for S. Typhi, fliC-a for S. Paratyphi A etc. Other targets are the invasion gene (invA), tetrathionate reductase gene (ttr), Salmonella enterotoxin gene (stn), outer membrane porin F gene (ompF), hyperinvasive locus A (hilA), virulence plasmid gene (spvC) etc. [65]. Isothermal amplification methods are also effective tools for detecting Salmonella like NASBA i.e. nucleic acid sequence-based amplification, RPA i.e. Recombinase polymerase amplification, LAMP i.e. Loop-mediated isothermal amplification etc. [49]. Other examples of molecular diagnostic modalities include DNA Microarray and WGS i.e. Whole Genome Sequencing. Whole genome sequencing has the upper hand over others in its ability to sequence the entire genome of the bacteria. Though having high specificity and sensitivity, high cost and requirement of well-trained personnel is a major drawback [49, 50].

Mass Spectrometry-Based and Spectroscopy Method: MALDI-TOF identifies bacteria after bioinformatics profiling from a library of spectral data from the mass spectral fingerprints which is generated from the isolate. Apart from MALDI-TOF, Liquid chromatography-mass spectrometry (LC-MS) works on a similar principle but is slower than MALDI-TOF [66]. Raman spectroscopy has been increasingly applied to pathogen diagnosis. A Raman spectrum is formed when scattered light from an analyte is analyzed. The Raman spectroscopy has been enhanced by combination with artificial intelligence and in conjunction with nanoparticles like Surface-Enhanced Raman Spectroscopy (SERS). Other rapid spectroscopy techniques include Near Infrared Spectroscopy (NIR), High Spectral Imaging (HSI), and Fournier Transformed Infrared Spectroscopy (FITR) etc. Cross-reactivity in identifying Salmonella serovars can be lessened in spectroscopy-based methods but some disadvantages are fluorescent background, signal saturation, cost and requirement of trained personnel [49, 50, 67, 68].

Optical phenotyping methods like BARDOT (Bacterial Rapid Detection using Optical Scattering Technology) and BISLD (Bacteria Identification System by Light Diffraction) are available. The principle of the light scattering phenomenon is used where scattering images of colonies are compared with reference image libraries [69]. SELA-BARDOT technology uses on plate screening technique with laser which generates individual scatter signatures of multiple pathogens grown on SEL agar and provides results in real time [70]. This optical phenotyping method is non-destructive, has less turnaround time and is economical due to no requirement for any probes or reagents [70]. BISLD (Bacteria Identification System by Light Diffraction) is an automated system for bacterial colony identification. Converging spherical waves on the colonies obtain Fresnel diffraction patterns along with 2-D transmission coefficient maps in order to identify multiple species of bacteria [71].

Biosensors: Biosensor is a compact analytical device which provides information on analytes with the help of a biological recognition element and a transducer [72]. The advantages of biosensors over conventional diagnostic techniques are their higher sensitivity, specificity, portability potential and rapid results [49, 72, 73]. The three main components of a biosensor are a bio receptor, a transducer and an electronic system. The role of the electronic system component in biosensor devices is signal amplification, processing data and displaying the output [49]. Bio receptors are the analytes which recognize the target and the commonly used bioreceptors in Salmonella studies are antibodies, aptamer, antimicrobial peptides, nucleic acid and bacteriophage with their own set of pros and cons [49, 73]. Aptamers are short single-stranded RNA or DNA oligonucleotides which bind the target molecules with high specificity and affinity [74]. Apart from their high specificity and affinity, aptamers have trumped other bio-receptors with various other advantages like their low price, high stability, low limit of detection, ease of production, customization etc. [73, 74]. The only disadvantage of aptamers is that it is nuclease sensitive [49, 74]. Antibody bioreceptors also have good affinity and specificity but it is expensive and unstable [49]. Bacteriophage bioreceptors have the potential to differentiate live from dead bacilli but their capture efficiency is low when dry and also, likely to lyse the bacterial cells in the process [73]. The nucleic acid bioreceptors have high stability but are only limited to genosensors [74]. Antimicrobial peptides demonstrated high affinity and sensitivity but the disadvantage of antimicrobial peptides lies in their poor specificity [49, 73]. The second important component of biosensors is transducers which convert the bioreceptor signals into measurable signals. One of the most common biosensors based on the transducer elements is electrochemical biosensors which can be further classified into amperometric, voltammetric, impedimetric and potentiometric biosensors [73, 75]. Optical biosensors have transducers which convert bioreceptor signals into optical ones and are classified as colourimetric, fluorescent, SERS, and Surface Plasmon Resonance (SPR) biosensors [73, 75, 76]. Other biosensors studied for Salmonella identification in food industries are Piezoelectric, Magnetic Relaxation Switching (MRS), Photothermal Biosensors etc. [49, 50, 72, 77, 78, 79]. Among all the established biosensors, electrochemical biosensors are the most popular and widely studied as they offer major advantages like high specificity, sensitivity, low cost per test, potential on-site testing, portability options etc. A few disadvantages include high initial investment for the instrument and bioreceptor-dependent sample preparation complexity [49, 73, 75]. The transduction principle of the amperometric biosensor is that it measures the current produced due to the redox reaction between two electrodes [80]. Voltammetric biosensors measure the current produced as a function of the varied potential applied in a controlled manner [75]. Impedimetric biosensors measure the electrical impedance generated by the analyte at the electrode surface [75]. The potentiometric transduction principle for biosensors is the measurement of electrical potential between a working electrode and a reference electrode under negligible or zero current flow [80]. Impedimetric biosensors among all the electrochemical biosensors are the most extensively studied due to their label-free quantitative analysis capacity of Salmonella [73]. Emerging onsite and user-friendly biosensors in the pipeline are nanomaterial biosensors, aptamer-based FRET biosensors, microfluidics-based, portable, smartphone-based biosensors and commercial biosensors like Biacore Q100 systems [49, 72, 73, 75, 76, 77, 78, 79]. The appeal of biosensors also lies in their utility in several fields like diagnosis, drug discovery, food safety check, environmental surveillance etc. [49, 73].

8. Antimicrobial treatment and drug resistance

Enteric fever was conventionally treated by first-line antibiotics consisting of ampicillin, chloramphenicol and cotrimoxazole. Strains resistant to these first-line antibiotics were termed as Multi-Drug Resistant (MDR) Salmonella which emerged in the Indian subcontinent, Africa and Southeast Asia in the 1980s causing numerous outbreaks with reports of emerging resistance as early as the 1960s [81, 82]. The shifting of the treatment to fluoroquinolones culminated in MDR strains showing increased resistance to them [83]. It led to a change in prescription to third-generation cephalosporin and azithromycin [84]. Soon, isolated reports of cephalosporin-resistant Typhoid emerged from Bangladesh [85]. The first outbreak of extensively drug-resistant (XDR) typhoid to all three first-line drugs along with fluoroquinolones and cephalosporin emerged from Pakistan in 2016, with reports arising from all endemic countries and even documented in USA [86, 87]. Azithromycin and Carbapenem remain effective against these XDR strains [87]. Though Azithromycin resistance is rare, selective antibiotic pressure eventually leads to the emergence of resistance. Azithromycin-resistant S. Typhi strains have been isolated from Bangladesh [88].

With vaccine coverage, S. Paratyphi infections are doubling in incidence as compared to that of S.Typhi. Though often clubbed together, the resistance profile of S. Paratyphi strains have been found to be different to that of S. Typhi. MDR trait is lower in S. Paratyphi than S. Typhi [89]. Surveillance for Enteric Fever in Asia Project reported MDR S. Paratyphi to be 2%, while none were reported from Nepal or Bangladesh [90]. Even if there is a low incidence of MDR strains, rising minimum inhibitory concentration to fluoroquinolones might point toward not using ciprofloxacin as the drug of choice [91, 92].

AMR is rising in NTS due to the heavy use of antibiotics in animal husbandry. Clinically NTS diarrhea does not need antibiotics unless invasive in nature, elderly, children or immunocompromised case. AMR rates jumped from 20% in the 1990s to 70% in high-income countries by the 2000s according to surveillance data [93]. Of the prevalent serotypes, S. Enteritidis was found to be more susceptible than S.Typhimurium. Ever since the discovery of S.Typhimurium DT104 resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline in the 1990s, such MDR phenotype is on the rise with up to 84% reported in surveillance data from USA [94]. Due to MDR Salmonella strains, cephalosporins and fluoroquinolones have become the drug of choice. NTS cephalosporin resistance has been reported to cause outbreaks with cefotaxime-resistant strains isolated from clinical samples in USA and European countries like Italy, Spain with similar trends emerging from Asian countries like Taiwan [93, 95, 96]. Upto 2.5% resistance to nalidixic acid and a 5% increase in MIC in fluoroquinolones have been reported in the USA [97]. Quinolone-resistant S.Typhimurium DT104 resulted in an outbreak due to its spread from animals to humans [98]. Alarming reports have emerged from Taiwan regarding 98% of a highly invasive NTS S. Cholerasuis found to be resistant to one or more antibiotics. S. Cholerasuis resistant to both ceftriaxone and fluoroquinolones have also been isolated [99, 100].

AMR in Salmonella spp. is either chromosomally mediated or plasmid-mediated. Some of the resistance mechanisms are antibiotic inactivation, alteration of drug targets or efflux pumps mediated by genes via point mutation or transfer by mobile genetic elements, phages or plasmids [101, 102]. Like other gram negative bacilli, TEM, SHV, CTX-M etc. are primary beta-lactamases (ESBL) which are enzymes that cleave beta lactam rings conferring resistance to penicillin and cephalosporin and the gebes transferred by plasmids like IncC and IncI1 [103, 104]. Chloramphenicol resistance genes like Cat1 and Cat2 confer resistance by inactivating the drug by acetyltransferase enzyme [102]. Acquired mutation in quinolone determining region of topoisomerase genes, gyrA, gyrB, parC etc. in chromosomal DNA. Resistance mediated by plasmid is coded by qnr genes that protect the DNA gyrase, aac(6')-ib-cr that decreases binding and qepA, oqxAB genes that code for efflux systems [105, 106]. Tetracycline resistance is conferred by genes tetA, tetB, tetG etc which activates the efflux pump mechanism to drive the drug out of the cell [107]. The resistance genes of sulphonamides and trimethoprim are sul1, sul2, sul3 and dfrA1, dfrA12, dfrV, dhfr1, dhfrV, dhfrA7, dhfr12, dhfr13, dhfr17etc.respectively [107, 108, 109]. The Azithromycin resistance mechanism reported in S.Paratyphi A was due to the R717 mutation in the AcrB protein [88].

9. Prevention and control

Prevention and control of Salmonella infections relies on vaccination, improving food and water sanitation, public education and adopting rational use of antibiotics. Vaccines for Typhoid fever have been available for a long time. Previously used whole-cell inactivated vaccines had numerous adverse reactions and are now no longer in use. One of the licensed vaccines is parenteral Vi capsular polysaccharide (Vi-CPS). Vi antibodies induce complement activation against S. Typhi and Vi-CPS is known to provide a protective antibody response against S. Typhi but there is no evidence of cross-protection against other serovars unless they express Vi. It can be only administered to individuals over 2 years of age [110, 111]. Conjugated Vi antigen with recombinant Pseudomonas aeruginosa Exotoxin A (Vi-rEPA) increases immunogenicity and can be administered in children below 2 years of age unlike Vi- CPS vaccine [110]. Oral live attenuated Ty21a vaccine utilizes S. enterica var. Typhi Ty21a strain which lacks UDP-galactant- 4-epimerase which self destructs after inducing an immune response. Studies show that it offers some cross-protection against S. Paratyphi B but none against S. Paratyphi A [110, 112]. Lack of substantial efficacy data especially below 2 years of age, waning protection post-vaccination, need for boosters and lack of vaccine schedule implementation are some of the factors behind low vaccine uptake in endemic countries [113]. Out of the several typhoid conjugate vaccines (TCV) licensed in India, two have been prequalified by WHO to be used in high-burden countries. TYPBAR TCV uses Ty21 polysaccharide with tetanus toxoid. It’s shown to be 87% efficacious and approved for children and infants less than 2 years of age. The second licensed TCV is TYPHIBEV with similar immunogenicity to TYPBAR TCV. Vi polysaccharide obtained from Citrobacter freundii which is indistinguishable from S. Typhi is conjugated with diphtheria toxin CRM197 as carrier protein [110, 114].

Effective food biosecurity measures to prevent contamination by Salmonella spp. in animals and produces along with isolation and quarantine in animal farms are one of the strategies for prevention and control [115]. Quality control by Hazard analysis and critical control point (HACCP) ensures consumer safety concerning food products and the standards are reviewed every 5 years to ensure the relevance of standards and protocols [116]. National and regional epidemiological surveillance programs are an important tool for detection and rapid awareness to control disease outbreaks [117, 118]. Good sanitation practices involving access to clean water supply, proper food handling practices, hand hygiene practices etc. are keys to curbing Salmonella infections, especially in endemic developing countries [117].

Physical interventions for control of Salmonella with regards to food safety that have been evaluated alone or in combination with other methods include ultrasound, UV, high-pressure processing, irradiation, pulsed light, plasma, thermal processing, high-intensity pulsed electric fields, oscillating magnetic fields etc. [119, 120, 121]. Chemical decontamination methods include the use of biocides like chlorine-based chemicals, chlorine alternatives (e.g. organic acids, peracetic acid), gas treatments (e.g. ozone, chlorine dioxide) etc. [120, 121, 122]. A biocide is a formulation containing an active substance to destroy or control any harmful organism to human or animal health by means other than physical or mechanical action [123]. Biocides for disinfection purposes can be divided into oxidizing (e.g. chlorine, chlorine dioxide, iodine, peroxides, peracetic acid etc.) and non-oxidizing groups (e.g. quaternary ammonium compounds, amphoterics, aldehydes, phenolic compounds, biguanides, and acid anionic agents etc) [122, 124]. However, there are greater implications regarding the widespread use of antimicrobial biocide like the development of biocide resistance, dermal or respiratory exposure effects, environmental threat due to persistence in aquatic systems etc. [122].

One of the novel biocontrol measures includes the use of bacteriophages (phages). Bacteriophages are viruses that infect and replicate in bacterial cells to create new progeny by using the host machinery [125]. The potential of phages against the rise of antibiotic-resistant Salmonella is due to their bacteriolytic activity, stability at a wider range of salt concentrations, pH, temperature, environment-friendly, self-limiting nature, genetic amenability etc. [126]. Phage cocktails with narrow host ranges have been reported to be better at reducing bacterial counts in various foods as they target multiple strains as compared to the use of a single phage [127]. A few examples of phages used against Salmonella in the food industry are SalmonFREE^R, PhageGuardS^TM, SalmoFresh^TM, SalmoLyse^R etc. [117]. Several challenges that exist with phage therapy are the emergence of phage resistance, restricted host range, moderate bacteriolytic activity, lack of safety data and regulatory framework for widespread commercial use etc. [117, 128, 129]. To combat some of the challenges, Genetically engineered phages have been modified to produce depolymerase, quenching enzymes, and hydrolases to act against biofilms, capsules and cell walls to enhance their bacteriolytic power. Also, the species-restricted host range in the engineered phages enables them to target only the pathogenic microbe and protect the commensals [128]. Bacteriophage-derived endolysins and Virion-associated peptidoglycan hydrolases are enzymes which are produced at the end of lytic cycles which hydrolyse the peptidoglycan layer from the outside as well as within the pathogenic bacteria. They are believed to be one of the potential alternative therapies against multidrug-resistant bacteria in multiple fields like food safety, medicine etc. due to good efficiency, rapid action, specificity and low chances of developing resistance [130].

Phytobiotics are plant extracts like herbs and spices which prevent food spoilage from contaminants, increase food shelf life and improve fortified food quality. In animals, phytobiotics owing to their antibacterial effects and microbiota modulation are known to improve carcass quality, decrease pathogenic bacteria proliferation, stimulate the immune system etc. [131, 132]. Bioactive compounds in phytobiotics also alter bacterial cytoplasmic membranes and genetic machinery, disrupt iron uptake pathways etc. [133]. Some examples of herbs and spices used against Salmonella spp. include cinnamon, thyme, oregano, essential oil from Salvia officinalis, lemon, lemongrass etc. [117, 134].

Prebiotics, probiotics, synbiotics and postbiotics help prevent gut colonization by Salmonella. Prebiotics are currently defined as a substrate that is selectively utilized by host microbes conferring a health benefit [135]. The prebiotic foods promote good bacteria to form biofilms on intestinal epithelium which in turn prevent pathogenic bacteria adhesion [136]. Probiotics are living microorganisms that confer a health benefit to the host when administered in the right amount. Probiotics include species of Lactobacillus, Bifidobacterium, Bacillus, Enterococcus and Pediococcus, [137]. Probiotics confer health benefits by competing with pathogens, stimulating the host immune response and inhibiting pathogens by secretion of lactic acid etc. [137]. Synbiotics are a combination of probiotics and prebiotics. The synergistic effects of probiotics and prebiotics inhibit Salmonella or lessen its effect by modulating gut microbiota, decreasing pathogen count, inducing additive effects of the immune system etc. [138]. Another emerging term is the postbiotics. Postbiotic preparations consist of non-viable microbes, their cell fragments and metabolites which are beneficial to the health of the host [139]. Various bacterial metabolites that can be used in postbiotic preparations are enzymes, exopolysaccharides, plasmalogens, organic acids, short-chain fatty acids, peptides etc. [140]. Postbiotics in animals showed reduced Salmonella count as well as increased nutrient absorption [141].

Another alternative method that can target bacteria is nanoparticles. Nanoparticles are ultrafine colloidal particles ranging from 1 to 100 nm in diameter [142]. The antibacterial mechanisms of nanoparticles are still unclear but the current theories are induction of oxidative stress, metal ion release and non-oxidative pathways [142]. Various types of nanoparticles against the control of Salmonella include metal-based, carbon-based, lipid-based and polymeric nanoparticles. Polymeric nanoparticles like chitosan nanoparticles are bigger in size making them efficient for oral uptake. They can withstand stomach acid, increase antigen-presenting cell uptake, stimulate the immune system continuously and act as adjuvants [143]. Silver nanoparticles act as carriers of plant extracts, antibiotics, or vaccines which are delivered on attachment to the cell membrane of Salmonella leading to microbial dysfunction [144].

Tackling drug-resistant Salmonella requires a multi-pronged approach on several fronts. Antibiotic dependency needs to be curbed with alternative non-antibiotic approaches, especially in animal husbandry. Research needs to be done to find newer strategies and further studies are required for the already existing alternative control measures. Since rising drug resistance in Salmonella, affects not only public health but also animal and environmental health, interdisciplinary and international collaborative efforts need to be framed within the One Health approach [145]. One health strategy includes the improved understanding of antimicrobial resistance mechanism through continued education, training and communication; implementation of infection prevention measures, and improved sanitation; antimicrobial stewardship programs for rational use of antibiotics in humans and animals; increased investments for vaccines and non-antimicrobial-based interventions; use of multi-omics (metagenomics, transcriptomics, proteomics etc) as a surveillance tool to identify the origin of antimicrobial resistance genes, its pattern and epidemiology [145, 146].

10. Conclusion

The disease burden caused by Salmonella is substantial and it poses a threat to the public health. Due to its ubiquitous and hardy nature, it persists for weeks in the environment. The multi-drug resistant strains affecting humans having varied virulence factors is a concern on a global level. The emergence of extensively drug-resistant typhoidal Salmonella from southeast Asia as well as reports of resistant non-typhoidal Salmonella is challenging the effective treatment availability. AMR in zoonotically transferable Salmonella is a matter of grave concern because of the extensive use of antibiotics in the husbandry of livestock. One health approach with a view of integrating surveillance and collaboration across human, animal, environmental and other relevant sectors like food industries is one of the key measures in tackling the menace of AMR. Other prevention measures are improving sanitation concerning clean water, safe food handling practices etc. Vaccination for Typhoid has been present for 100 years but suffered from low uptake due to progressively waning efficacy post-vaccination. The pre-qualification of newly licensed TCVs by WHO has been encouraging as the vulnerable age groups of children can be vaccinated. Further research is required for developing vaccines against Paratyphi serovars and non-typhoidal Salmonella, for at-risk groups like HIV and other immunocompromised cases. There is a strong case for developing multivalent vaccines which can target Salmonella serovars simultaneously like trivalent vaccines for invasive NTS/typhoidal strains in Africa and bivalent enteric fever vaccines in Asia or quadrivalent vaccines to provide cross-protection against all major serotypes.

Alternatives to antibiotics are the need of the hour to combat rising antimicrobial resistance in Salmonella. A combination of alternative strategies like biocides, prebiotics, probiotics, bacteriophages etc. can reduce the dependency on antibiotics.

It is of vital importance to commit to research and development so that efficient and advanced methods can quickly detect antibiotic-resistant Salmonella outbreaks for quick response. Besides good prevention and control approaches, continued research in pathogen detection is also crucial for Salmonella mitigation. Conventional culture methods along with non-culture methods like immunological assay, PCR, mass spectrophotometry, spectroscopy, optical phenotyping etc. have their niche in the diagnostic armamentarium. A promising candidate in the Salmonella detection system is biosensors. Apart from medical diagnostics, the appeal of biosensors extends to other fields like environmental surveillance, ensuring food safety, drug discovery etc. Biosensors in the form of lab-on-chip devices which are rapid, highly sensitive, specific, reasonably priced, and miniaturized will be a highly welcome innovation. The commercialization of technology where biosensors are combined with artificial intelligence (AI) will be a scientific breakthrough in the future.

Conflict of interest

The authors declare no conflict of interest.