Literature Review on Salmonella Studies

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In 1884, the first Salmonella species was isolated by veterinary surgeon Daniel E. Salmon from porcine intestines. The isolated species was first referred to as Bacillus choleraesuis, however was renamed Salmonella choleraesuis in 1900 (Ryan). Several years later in 1986, the choleraesuis designation was changed by the Subcommittee of Enterobacteriaceae of the International Committee on Systematic Bacteriology at the XIV International Congress of Microbiology and now the Salmonella type species is recognized as enterica (Ryan).

The genus Salmonella are Gram negative rod-shaped bacteria approximately 2-5 microns in length that belong to the family Enterobacteriaceae. Bacteria belonging to the genus Salmonella are catalase positive, oxidase negative and are not able to form spores (Ryan).  Salmonella are characterized by the possession of peritrichous flagella for mobility. The physiological traits of most of the Salmonella serovars include but are not limited to: the use of citrate as a sole carbon source; fermentation of lactose; production of hydrogen sulfate; and hydrolysis of urea. Salmonella species survival has been reported over a large pH range, from 3.8-9.5 with the optimal growth pH range of 6.5-7.5 (Ryan) (Andino).

Both Salmonella and E. coli are enteric Gram negative bacteria that belong to the family Enterobacteriaceae and possess similar virulence mechanisms such as low pH resistances and the ability to grow on lactose (Windfield). A phylogenetic study conducted by Desai et al. revealed an estimated 140-million-year divergence between Salmonella and E. coli. The divergence of the most recent common ancestor of all Salmonella subspecies resulted in an estimated 657 gene families gained from Escherichia. Of the 657 gene families, 454 gene families were currently present in all of the 29 Salmonella genomes the Desai et al. study sequenced and analyzed. One of the benefits of the genes Salmonella inherently gained from Escherichia allows for Salmonella subspecies to utilize various carbohydrates in anaerobic conditions (Desai).

Approximately 45% of known bacterial genomes have a CRISPR- Cas (clustered regularly interspaced short palindromic repeats and CRISPR associated genes) system to provide immunity protection from invasive genetic elements such as plasmids and bacteriophage. Shariat et al. study performed an extensive sequence analysis on the Salmonella CRISPR-Cas type IE system to acquire a deeper evolutionary perspective of the system. A comparison of CRISPR- Cas sequence data from several different Salmonella enterica serotype isolates (Enteritidis, Typhimurium, Newport and Heidelberg) revealed that leader, operon and array sequences were well conserved among the isolates. CRISPR-Cas systems adapt by obtaining new spacer sequences to combat invading genetic elements however, Salmonella do not acquire new spacers but instead delete or duplicate direct repeat spacer units. Of the isolates sequenced, only 12% of the spacers corresponded with phage and plasmid sequences,  The analysis also determined that Salmonella CRISPR-Cas type IE shared some similarities as well as differences to E. coli CRISPR type I-E. Regulation mechanics of the cas operon is similar in Salmonella and E. coli although the E. coli system has a promoter intergenic region between the cas3 and cse genes while this intergenic region is absent in Salmonella. Collectively, the study concluded that the highly conserved Salmonella CRISPR-Cas system may not be actively involved in immune function but instead may be involved with other processes such as biofilm formation (Shariat).

Salmonella species are typically described as mesophilic organisms but have the ability to thrive at low or high temperatures. The sigma factors of the bacteria sense temperatures changes in the external environment and initiates the activation of rpoH genes to respond to high temperatures. During cold external temperatures Salmonella employs the use of Cold shock proteins to increase the survivability in low temperature environments. Synthesis of these specialized proteins contributes the survivability of Salmonella species in refrigeration conditions (Andino).

The Salmonella genus is categorized into two species: Salmonella enterica and Salmonella bongori (Ryan). Currently, there are six subspecies of Salmonella enterica; of the six, subspecies I is critically recognized as the human disease-causing group while the other subspecies primary reside in cold blooded animals (Desai). A recent phylogenetic study by Desai et al. examined the divergence of subspecies within the species Salmonella enterica using synonymous SNPs. The report concluded that the species separation occurred approximately 27 million years ago and determined that the most recent common ancestor of the subspecies enterica possibly evolved after the emergence of their respective hosts, about 12 million years ago (Desai).

Salmonella bongori is currently recognized by the Judicial Commission of the International Committee for Systematics of Prokaryotes as an entirely separate species from Salmonella enterica. S. bongori is most commonly associated with cold blooded animals such as reptiles, however in extremely rare cases it has been reported to cause disease in humans (Fookes). During the evolutionary divergence of Salmonella from Escherichia, it is proposed that S. bongori lost 63 genes that were present in the most recent common ancestor of Salmonella. Accompanied by the loss of genes, S. bongori does not possess the ability to utilize ethanolamine, catabolize fucose, or perform nitrate and nitrate ammonification (Desai) (Ryan).

During the early 1950’s the serotyping of Salmonella species was conducted by the Kauffman-White scheme using the identification of somatic O and flagellar H surface antigens. The O antigen is found on the outer surface of the lipopolysaccharide of the bacterial membrane, consisting of a heat stable polysaccharide arrangement. The H antigen is located on the filamentous region of the Salmonella flagella which are composed of flagellin subunits (Ryan).  In accordance with the standard identification procedures, the O antigen signifies the group that the Salmonella isolate will be categorized into while the H antigen determines the serovar assignment of the isolate (Ryan). Although horizontal gene transfer events of the O and H antigen genes can cause inaccuracies regarding identification (Desai). Currently, more than 2500 serotypes of Salmonella have been identified with S. enterica Typhimurium , S. enterica Enteritidis , S.enterica Newport, and S. enterica Javiana as the most prevalent isolated serotypes in the United States (Braden).

Salmonella species are enteric organisms that initially invade the host intestinal epithelium by activation of invasion genes (SPI-1). The addition of organic acids was predicted to alter the expression of SPI-1 and as a result interfere with Salmonella virulence. S. Typhimurium exhibited lower epithelial cell invasion when grown in media modified with butyrate and propionate; it was determined that the acids decreased the expression of SPI-1 activators (hilA and invF). Acetate modified media produced a different response; acetate led to an increased expression of hilA and invF activators resulting in greater Salmonella virulence (Van Immerseel).

As Salmonella species takes up residence in a host organism, the host defenses are activated. The pathogens begin their existence intracellularly as they are consumed by macrophage cells and epithelial cells. The pathogens first penetrate the intestinal epithelium by the promotion of early invasion genes (SPI-1); then they can disseminate to other tissues causing gastroenteritis, enteric fever and septicemia (Van Immerseel). Defense mechanisms such as the two- component signal transduction PhoP/PhoQ system comprised of a senor kinase and response regulator is required for Salmonella pathogens to tolerate the harsh acidic environment of phagocytic cells. The PhoP and PhoQ is a highly conserved regulon of Salmonella responsible for the induction of five pag genes that allow for the production of nuclear envelope proteins to improve survival and activate virulence properties (White) (Ren). PhoQ is activated by autophosphorylation in response to external environmental changes such as low concentrations of essential metal ions or changes in pH. The phosphorylated PhoQ then leads to the activation the PhoP which then facilitates the transcription of phoP and other genes contributing to enhanced virulence and production of acid shock proteins. (Ren). PhoP is also involved in the downregulation of the invasion genes SPI-1 master regulator, HilA. During early invasion, the PhoQ/PhoQ system is repressed in order to allow for expression of SPI-1 genes required for initial infection (Ren).

Virulence attributes of Salmonella are also activated by the hilA gene regulated by increased environmental acetic acid concentration (Andino). Overall, the activation of virulence genes is upregulated by external environmental stresses indicating that Salmonella species could exhibit greater infectivity potential and resilience in unfavorable acidic and temperature conditions (Ren, White, Andino).

Salmonellae are facultative anaerobic pathogens whose growth is supported by the environmental conditions of the intestinal tract of humans and animals. Salmonella organisms thriving in the digestive system of a host are often excreted from the host in the feces. Insects and other creatures contact the feces and collect the excreted organisms transmitting Salmonella to other locations such as a water source or food product. Consumption of a contaminated source by a human or animal will introduce Salmonella to the digestive tract, continuing the cycle of Salmonella contamination. The amount of organisms inoculated into the host as well as the host immunity will determine salmonellosis disease progression (Andino).

Salmonella species strategically exploit the host immune system by evading the host’s nutritional immunity. Nutritional immunity of a host is described as a process by which the host restricts the availability of essential transition metals in an effort to prevent the proliferation of pathogenic microbes. Specific genes of the Salmonella species allow the bacterium to acquire the necessary transition metals in the digestive tract of a host organism (Allard) (Hood). A study by Lui et al.  indicated that during a bacterial infection the host upregulates the production of calprotectin, a zinc- and magnesium-dependent antimicrobial protein required to combat microbial growth. Consequently, the presence of the calprotectin provides an ideal environment for S. Typhimurium by the inhibition of the growth of invading microbes while enhancing the growth of S. Typhimurium (Lui).

Beef is one of the top five most common food products susceptible to Salmonella contamination. S. Typhimurium, S. Dublin, and S. Montevideo are three of the most common serotypes infective in cattle causing fever, reduced milk production, and potential miscarriages. In accordance with the 2014 USDA inspection, Salmonella isolates were recovered from 1.6% of the 7,320 ground beef samples. S. Dublin accounted for 12.4 % of the positive samples while S. Montevideo made up 22.4 %. Consumption of contaminated beef products generally results in gastroenteritis (Andino) (USDA/FSIS).

Two of the most medically important serotypes of Salmonella are Salmonella enterica serovars Typhi and Paratyphi, which are the primary causative agents of enteric fevers. These serotypes are distinct from other Salmonella species in that they are strictly human pathogens and do not possess the ability to ferment lactose (Andino) (Braden).

Enteric fever is a progressed salmonellosis disease in humans primarily endemic in Southeast and Central Asian countries and is manifested by typhoidal Salmonella enterica serovars S. enterica Typhi and S. enterica Paratyphi. Symptoms indicative of typhoid fever are fever, rose colored spots on the chest, and bradycardia. Similarly, both typhoidal and non-typhoidal serotypes spread via the fecal oral route. Non-typhoidal serovars (NTS) include S. enterica Typhimurium, S. enterica Enteriditis, S. enterica Newport and S. enterica Heidelberg. NTS are more frequently associated with morbidity and hospitalizations from food contamination, shifting the prevention focus to NTS. Symptoms of NTS salmonellosis are generally self-limiting including nausea, vomiting, fever, chills, abdominal pain, and myalgia (Andino).

S. choleraesuis is one of the most prevalent serotypes (along with S. Typhimurium and S. derby) that infects swine products. Salmonella IV formally referred to as Salmonella marina is associated with reptilian creatures (Braden).

Many Salmonella serovars commonly are found in poultry and poultry processing equipment (Van Immerseel). The most prevalent subspecies of Salmonella involved in food related outbreaks is enterica. The most frequently occurring serovar are S. enterica Enteriditis and S. enterica Typhimurium (Allard). In the United States, S. Enteriditis, is the most frequently reported serovar of Salmonella most commonly associated with poultry products.  In relation to other Salmonella serovars, S. enterica Enteriditis does not produce any symptoms of infection in a chicken host; however if a mother hen is infected with this unique strain then she will transfer bacteria directly to the egg through vertical transmission or horizontal transmission (Andino) (Gantois). Horizontal transmission occurs when Salmonella from contaminated feces or from the colonized gut of the hen infiltrates the egg shell during or after oviposition. Infected reproductive organs of the hen can lead to contamination of the developing egg before oviposition known as a vertical transmission process.  Salmonella contamination inside of eggs is most likely a consequence of infected reproductive organs of the mother hens which results in egg contamination during egg formation (Gantois).

Poultry host specific serovars S. enterica Pullorum and S. enterica Gallinarum cause Pullorum disease and fowl typhoid in poultry but are not usually a risk to humans. Pullorum disease and fowl typhoid are associated with high mortality rates and can affect the national poultry livestock and product supply if untreated. Since 1975 these poultry Salmonella serovars have been eradicated commercially in most of the developing world. The elimination of some species in a niche will reduce the resource competition and allow for a new organism to thrive; it has been proposed that the eradication of these poultry-affecting Salmonella serotypes has allowed for S. Enteriditis to inhabit the vacant niche (Andino).

In 2014, the USDA inspection report revealed that Salmonella was present in 325 broiler chicken carcass samples out of 8816 total sampled. S. kentucky, a serotype not associated with human infection, accounted for 60.8% of the positive samples while S. enterica Enteriditis composed 13.6% of the positive samples (USDA/FSIS).

Over 70% of salmonellosis cases are the result of the consumption of contaminated poultry products such as eggs (Andino). Salmonella species survive with great success in farm environments. Contamination of chickens and eggs is often unnoticed since major pathogenic serotypes such as S. enterica Enteritidis do not produce any signs of disease in the poultry (Braden). Egg shells are considered one of the major sources of contamination. Between 1986 and 1987 65 outbreaks of Salmonella were the cause of 257 hospitalizations and 11 deaths in the United States. Of the 65 cases 77% of 35 of the outbreaks were egg and egg product associated. From 1985-2003 the United States alone reported 997 S. enterica serovar Enteritidis resulting in 33687 illnesses, 3281 hospitalizations and 82 deaths; 75% of the outbreaks were egg and egg product associated. The USDA Agricultural Research Service conducted a study to determine the mode of transmission of Salmonella to egg. Pathogen-free birds were inoculated with S. Enteritidis. After three weeks, S. Enteritidis was recovered from 91% of the bird cloaca, 41% of the egg shells and 71% of the internal egg contents, providing evidence that Salmonella contamination occurs through transovarian transmission (Braden).

Poultry products have had the most Salmonella outbreaks, however this not an exclusive reservoir; many other foods such as vegetable crops, dairy products, and peanut butter are also susceptible to Salmonella contamination. Low moisture foods are often vulnerable to Salmonella contamination depending on the sanitary conditions of the manufacturing and processing facilities (Andino).

In 1993 Germany suffered an outbreak of salmonellosis from contaminated paprika in flavored potato chips, infecting more than 1000 people, with children being the most severely affected. Molecular analysis and serotyping of the contaminated paprika revealed that even small amounts of Salmonella can cause disease. The serovars of Salmonella responsible for the epidemic were S. enterica Saintpaul, S. enterica Javiana, and S. enterica Rubislaw (Lehmacher).

A few years later in 2003 there were several outbreaks of S. Montevideo in New Zealand and Australia from various contaminated sesame seed products. Pulsed Field Gel Electrophoresis analysis of bacterial DNA samples isolated from the contaminated food products and infected patients confirmed that the S. Montevideo serotype was responsible for the outbreaks reported that year (Unicomb).

Many Salmonella serovars commonly are found in poultry and poultry processing equipment. Antimicrobial agents are commonly used to eliminate bacteria as a preventive measure to reduce contamination risk. However, with the emergence of antibiotic resistance of Salmonella species there is great urgency to develop new alternative methods to eliminate Salmonella contamination and infection. Traditionally, many industries use chemical disinfectants, heat treatments, and UV light irradiation to control the possibility of contamination of various pathogenic bacteria including Salmonella (Woolston).

Salmonella contamination is problematic for food manufacturing facilities. Some  strains of Salmonella resist many decontamination applications and have been found to persist in food processing environments for ten years. Resistances to disinfection treatments is possibly linked to the ability for Salmonella to produce a biofilm matrix. A study by Corcoan et al. investigated the properties of  Salmonella biofilm production over time and the efficacy of different disinfecting agents of various common surfaces of food processing equipment. Visible distinctions of Salmonella biofilm formation between 48 hours and 168 hours was apparent by the use of SEM imaging. The study concluded that none of the disinfectants tested on their own were capable of the removal of a 168 hour established Salmonella biofilm. It was mentioned that the context of this study did not correspond with real world applications since there are often abrasive cleaning mechanisms that are also involved in disinfection procedures that were not implemented in the study (Corcoran).

There are other methods besides antimicrobials that are currently utilized to reduce Salmonella contamination. Van Immerseel et al. describes the probiotic and prebiotic application of fermentation acids in water and feed to combat Salmonella growth by decontaminating the food and overall reduction of the amount of Salmonella uptake by the chickens. Van Immerseel et al. mention a study conducted in 1995 of the application of propionic acid to heavily saturated S. Typhimurium infected food and measured a 1000-fold decrease in the pathogen after seven days. After feeding the chickens the acid-supplemented feed they observed a decrease in the number of Salmonella colonies in the chicken caecal from 109 to 102 CFU/g after five days.  MCFA (caproic acid, caprylic acid, capric acid and lauric acid) seem to have decent antimicrobial effect on Salmonella species however this study mentions that large scale studies are necessary to confirm the effects (Van Immerseel).

Another antibiotic alternative approach is the direct use of bacteriophage-encoded enzymes called endolysins. Endolysins are effective in the destruction of bacterial cells by degrading the peptidoglycan layer of the cells. However, endolysin application is limited in effectiveness due to the presence of an outer membrane, which protects Gram negative bacteria (Adhya)(Oliveira). Currently, limited research has been conducted in regard to endolysin activity against Gram negative organisms.

One problematic characteristic of Salmonella species is their innate ability to produce a biofilm matrix. Biofilm production of many Salmonella species is a major consideration to control the probability of contamination of manufacturing equipment and food products. Biofilms are high density polymeric matrices that house and protect microbe communities from environmental conditions such as radiation, pH changes, antimicrobials, and other threats (Paz- Méndez et al.) (Oliveira). Bacteria can exist in several physiological states in a dense biofilm matrix, rendering endolysin and antimicrobial agents ineffective against the bacteria (Oliveira). During biofilm formation, bacteria will bind and divide in exopolysaccharide glycocalyx polymers on a surface creating different microenvironments, contributing to varying susceptibility to antiseptics and disinfectants (Mc Donnell). A recent study conducted by Mendez at el analyzed the biofilm production of Salmonella isolates from poultry houses on several media types on stainless steel and polystyrene. Polystyrene and stainless steel are prominent materials involved in food processing, commonly used as food packaging materials and other surfaces in direct contact with food products. Of the 13 isolates evaluated, all strains with the exception of one were able to initiate biofilm production on all media types on both stainless steel and polystyrene surfaces (Paz-Méndez et al.).

Biofilm production of Salmonella species is of concern especially for fresh food products such as vegetables; some evidence suggests that some types of vegetables secrete products that may enhance and support biofilm production.  A current study organized by Koukkidis et al. found that juices from damaged salad leaves promoted S. enterica attachment, motility and biofilm formation regardless of the established microbiota of the leaves. By using mutant strains of S. enterica, the researchers determined that one of the reasons for enhanced growth of Salmonella in the leaf juice was related to the siderophore-like structures found in the leaves and relative iron availability (Koukkidis). Siderophores are chelating agents designed to uptake iron from the environment and make the iron available to the microbe to perform metabolic processes (Neilands). Siderophore production is essential for the growth and virulence of pathogens such as S. enterica since it allows the pathogen access to iron in transferrin and lactoferrin proteins when iron is scarce in the environment (Neilands) (Hao). Understanding biofilm production mechanisms of Salmonella will ideally provide alternative and effective decontamination methods to prevent the propagation of Salmonella in various food products.

A different study conducted by De Oliveira et al. investigated biofilm production of Salmonella on various surfaces at different temperatures. While the adhesion of Salmonella to various surface types is dependent on the physiochemical properties of the surface, Salmonella species adhere to the surfaces by the production fimbriae, a type of cellular projection such as pilli or curli that allow the cell to adhere to surfaces, and cellulose to compose a biofilm matrix (Althouse) (De Oliveira). The two genes responsible for biofilm creation are agfD, responsible for aggregative fimbriae production, and adrA genes. These genes are co-regulated a by LuxR-type regulator. Cellulose synthesis is dependent on the production of curli fimbriae and can be verified by the observation of red, dry, and rough colony morphology (rdar morphology) of Salmonella at 28°C. All of the Salmonella samples isolated from poultry possessed the agfD and adrA genes and 98.3 % of the tested strains were able to initiate biofilm formation on at least one surface and one of four tested temperatures. Only 55.2% of 96 tested strains and 2.3% of 4 tested strains exhibited the rdar morphology.  They concluded that while there is a strong correlation between the agfD and adrA genes and biofilm formation, the expression of rdar morphology is dependent on several other factors such as pH. Also they concluded that many other genes are likely involved in the formation of a biofilm matrix. Since they discovered that Salmonella biofilm formation can occur on several surfaces at varying temperatures it is crucial that more effective sanitizing practices be implemented to prevent the formation of biofilms on industrial food processing surfaces (De Oliveira).

The over-usage and misuse of antibiotics is not without consequence. Many antimicrobial strains of bacteria have come into existence and are considered one of the greatest threats to human health. Antibiotic resistance is defined as the microbes’ ability to resist the activity of an antimicrobial substance by mean of a structural or functional component such as the absence of the specific antibiotic receptor, the inability to cross the outer membrane of Gram negative bacteria, or the presence of efflux pumps to transport the agents out of the cell (Blair).

A study by Akhtar et al.  proposed that a primary reason antibiotic resistance strains of Salmonella are emerging is because of the limited regulation on antibiotics such as the unnecessary usage of broad spectrum antibiotics. Presently, the only antibiotics available for the treatment of severe S. enterica infections are fluoroquinolone and third generation cephalosporins (Capparelli). Many developing countries such as South Asia have limited restrictions to the access of antimicrobial agents which increases the likelihood of multidrug resistant Salmonella arising. Reduction in the use of broad spectrum antibiotics and more careful use of antibiotics may reduce the amount of resistant strains of Salmonella that are emerging. Vaccine usage has been proposed as a viable option for the treatment of Salmonella infection although it may have limited potential against resistant microbes and have limited effectiveness (Akhtar)(Capparelli).

Many Salmonella serovars are developing resistance to antimicrobial agents such as quinolones, fluoroquinones and some cephalosporins by horizontal gene transfer of the antibiotic resistant genes from other Salmonella strains, other Enterobacteria, and by clonal spread of the antimicrobial resistant serotypes (Andino). A study by Zhang and LeJeune determined that of the twelve different serotypes of Salmonella sampled from bovine, at least one of each serotype had resistance to an antibiotic reagent. Additionally, the transduction of antibiotic resistance occurred in S. enterica Heidelberg to S. enterica Typhimurium was verified in the study. By using PCR with the addition of specific primers they detected ampicillin and tetracycline resistant genes of the bacterial and induced phage genomes. They discovered that the resistant genes were acquired in the phage after propagation and transduction events. The results suggest that phage induction of certain Salmonella strains supports antimicrobial immunity as well as increased virulence via phage transduction transfer (Zhang & LeJeune).

Salmonella serotypes establish antibiotic resistance from the production of specific membrane enzymes that prohibit antimicrobial substances from penetrating the cell and β-lactamase to disrupt the chemistry of the antimicrobials. Transferring drug resistances between Enterobacterial strains involves a resistance transfer factor, a type of plasmid that encodes the multidrug resistance genes. Many different influences contribute to the increase in resistance such as selective environmental pressures and competition for resources (Reis)(Andino)(Zhang).

The most epidemiologically critical S. enterica Enteritidis and S. enterica Typhimurium serovars are believed to have evolved a more extensive resistance to antimicrobials. The Pocurull, Gaines and Mercer, 1971 study primarily focused on the presence and transference of antibiotic resistance of Salmonella isolates from diseased animals from regions of the US. Drug resistance properties were displayed in 935 of the 1,251 of the strains. Of all the isolates analyzed, S.  Typhimurium cultures had the greatest incidence of multiple drug resistance (Pocurull).  During the 1980’s the emergence of a virulent multidrug resilient strain of S. enterica typhimurium was understood to be spread worldwide; distinguished by its chromosomally based resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines (Reis).

Antimicrobial agents are presently used to eliminate bacteria as a preventive measure to reduce contamination risk. However, with the emergence of antibiotic resistance of Salmonella species there is great urgency to develop alternative methods to eliminate Salmonella contamination and infection (Van Immerseel).

In 1896, Ernest Hankin discovered a bactericidal component in the Ganges and Jumna rivers in India effective against Vibrio cholerae. While it is difficult to verify exactly what the antimicrobial agent was, many hypothesize that due to the specificity of the agent it may have been bacteriophage (Abedon) (Hudson).

In 1915, a filtrate solution that was able to degrade a bacterial culture was discovered by British microbiologist Frederick Twork. He identified the transparent filtrate as a bacterial secretion that could not propagate in the absence of bacteria (Wittebole).

It was not until 1917 that bacteriophage manuscripts were published. Felix d’Herelle is famously recognized for his dedication to applying bacteriophage as a therapy.  During his studies he confirmed that bacteriophage propagation occurs in a diseased host. By monitoring the phage titers after infection D’Herrelle recorded an increase in phage titers post infection as well as a stabilization of phage concentration during recovery. Not long after the discovery of bacteriophage, D’Herelle investigated the implications of treating humans with bacteriophage. He used phage to treat patients with bacillary dysentery and cholera, shortly after bacteriophage were utilized to treat wound infections (Cisek).

Current research of bacteriophage applications is seemingly boundless. One of the most promising forms of bacteriophage utility is the development of bacteriophage vaccines; more specifically the use of bacteriophage as a delivery system in conjunction with vaccine agents. Adhya et al. proposes that the use of phage as delivery components in vaccines is advantageous since bacteriophage activate humoral and cell mediated immunity. A phage display vaccine is one of the current methods studied and involves the fusion of foreign antigens along with the normal external phage proteins. Phage DNA vaccines are another type of delivery system characterized by the integration of a foreign antigen gene into the viral genome; the virus then passively transfers the foreign DNA genes to be expressed in the mammalian cell (Adhya).

A study by Van Houten et al. verified that filamentous bacteriophage are ideal synthetic peptide carriers to elicit an antibody response. They first created and purified f1.K/B2.1 phage conjugate complex. Mouse models were immunized with the engineered phage, then by using ELISA they were able to measure antibody titers in response to the synthesized phage. Compared to the control conjugate, ovalbumin/B2.1, the f1.K/B2.1 phage produced a higher antibody titer. The findings of the Van Houten et al. study supports the use of filamentous bacteriophage as immunogenic synthetic peptide carriers to induce a more focused antibody response (Van Houten 2006).

A more recent Van Houten et al. study proposed that low surface complexity of filamentous phage increases the efficacy of the phage as immunologic carriers and will elicit a greater antigen antibody response compared to the standard ovalbumin carrier. To explore the effects of surface complexity on antibody response, the researchers modified phage particles by removing protein elements such the N1 and N2 domains of pIII (Δ3 phage) and altering charges of surface terminal peptide regions of pVIII (Δ8 phage). Mice were then immunized with the phage and antibody titers were detected by ELISA after consecutive immunizations. The Δ3 phage expressed a significantly lower antibody response compared to the control wild type phage and Δ8 phage. The Δ8 phage produced the greatest immunogenicity response compared to the control and Δ3 phage. As a result, surface modification of phage carriers allow for a more focused antibody response to synthetically produced conjugated peptides and not to weakly immunogenic epitopes (Van Houten 2010).

Bacteriophage are not limited to surface antigen display to elicit an antibody response but also have potential in the delivery of antibiotic reagents into a host bacterial cell. Westwater et al. investigated the possible use of a M13 phagemid system to deliver lethal agents to E. coli cells. The Gef and ChpBK lethal plasmid genes were cloned into a vector and ultimately were packaged into phagemid DNA. Mice infected with E. coli were treated with the phagemid; the blood of the mice was collected at one, three, and five-hour intervals to detect the amount of E. coli present in the mouse. The researchers found that the mice treated with the phagemids had reduced amounts of viable bacteria in their systems indicating that the application of phage as lethal agent delivery systems to bacterial cells is plausible (Westwater).

Bacteriophage are microscopic entities of varying complexity composed of nucleic acid protected by a protein coat. Unable to replicate by themselves, they require a host cell to generate new virion progeny. There are two known methods of viral replication, the lytic pathway and the lysogenic pathway. The lytic pathway involves the use of a host cell to create new virions and lysing the host cell as a result (Hudson). The typical lytic cycle duration is approximately 20 to 40 minutes from the initial viral attachment to the release of virion progeny (Abuladze).

T4 and T7 lytic phages have been extensively studied to provide a better understanding of the lytic cycle. The phage genetic material is first injected into the cell. The phage genome then assumes control over the host RNA polymerase to ensure the viral genome is transcribed. The head, tail, and lytic enzymes are all encoded in the viral genome (Davies 2016). The cell machinery is directed to the production of virions by the activation of specific chaperones that assist the appropriate folding of new virion proteins. The host defenses are suppressed by the inactivation of phage restriction processes and inhibition of protease enzymes (Cenens). Lytic phage enzymes referred to as endolysins are synthesized in the host cell near the end of the lytic cycle. Endolysins are peptidoglycan hydrolases that enzymatically destroy the host cell by degrading the peptidoglycan of the cell, ultimately facilitating the release of newly synthesized phage into the external environment (Schmelcher and Loessner).

Phage can also undergo a less destructive lysogenic cycle. This pathway involves the integration of viral genetic elements into the bacterial host genome creating a prophage. Mutations of the bacterial genome are possible during prophage integration considering the enzymes involved in the genome integration processes may place the phage genome in a specific or random location. After genome assimilation, the phage genome is replicated along with the bacterial host genome. When specific sets of conditions are met, such as exposure to UV radiation, antimicrobial agents or other metabolic stresses, repressor genes such as cI are inactivated. Repressor genes control the ability for a phage to become lytic from a lysogenic state. Inactivation of the repressor genes stimulates the lysogenic bacteriophage to become lytic. The transition from lysogenic to lytic is hypothesized to be a survival mechanism of the bacteriophage. When host DNA is damaged, the repressor genes are inactivated leading to the emergence of virulent bacteriophage synthesis, propagating the newly synthesized phage to leave the cell into the environment (Hudson) (Davies).

Redfardt and Rainey used the lysogenic lambda phage, effective against E. coli, to analyze the genetic switches of the induction of prophage. Two host DNA damaging regimes were designed as sensitivity (low dose of mitomycin C) and threshold (high dose of mitomycin C) to analyze the concentration of transmitted lytic phages from the host cell. They propagated lytic phages by exposing the bacterial cells to low and high concentrations of mitomycin C and then calculated the recovered lytic phage titers. Both treatments produced higher phage titers comparative to the control bacteria population, although the high dose of mitomycin C enhanced the induction of prophage, suggesting that increased host DNA damage initiates prophage induction (Redfardt).

Lysogenic phage can provide fitness benefits to the host organism by the expression of phage encoded genes in the host cell that contribute to the host virulence, production of exotoxins, cell attachment and antibiotic resistance. There are many integrated phage-encoded virulence genes for different organisms. S. enterica have a few phage-encoded virulence genes that assist with the survival of S. enterica in macrophages (sodC1, SseI genes from Gifsy-2 phage) and epithelial cell invasion (sopE gene from the φSopE phage) (Davies).

It is possible that the introduction of viral genetic material into a host cell does not integrate with the host genome (lysogenic) nor does the viral genome replicate (lytic). A recent study conducted by Cenes et al. proposed the existence of a phage carrier state and determined that the phage genome is maintained in the host cell without integration into the host chromosome. Using a P22 phage with a  S. Typhimurium bacterial host they were able to express the ORFan gene on the P22 phage. The ORFan gene expression was found in cells that did not display lytic or lysogenic aspects but instead stabilized the viral genome with in the cell.  The pseudo-lysogenic state is unique in that the phage exist in a dormant phase and postpones lytic or lysogenic activation; this adaption may improve phage viability in inhabitable environments by utilizing the host cell to protect the phage genome. In terms of propagation of certain bacteriophage particles dwelling in a pseudo-lysogenic state, it is difficult to detect pseudo-lysogenic phages using traditional plating techniques (Cenens X2).

Phage are often found in the environment in high concentration, considerably in greater concentration comparative to bacterial cells (Cenens). There are approximately 1023 total bacteriophage on the planet, with soil having the greatest concentration of bacteriophage at 109 per gram of soil compared to aquatic environments that range from 104 to 108 phage per milliliter (Wittebole). Some reports claim that the total viral concentration exceeds 1031 virions on earth with archaea and bacterial viruses existing in the greatest abundance (Pietilä). Considering that bacteriophage require a host cell to produce new virion progeny, how is it possible that bacteriophage are in such abundance? One of many possible explanations is the phage chromosomes can exist in a phage carrier state or pseudo-lysogeny, which is a phenomenon characterized by the entry of the phage genome into a host cell where it is maintained and protected from destructive effects (Cenens).

Viruses in general are considered major evolutionary drivers for cellular life since there are no cellular organisms that are invulnerable to viral infections (Pietilä). Since nearly all cellular life has diverse genetic elements such as plasmids or viruses associated with them it is reasonable to suspect the existence of co-evolutionary relationships between the genetic parasites and host organisms. Defense mechanisms against viruses and other genetic invading elements is considered one of the main driving forces in cellular life evolution. Adaptation pressures from viruses or genetic elements is proposed to have a major impact on cell compartmentalization and organization. For example, primordial cells first begin to adapt to invading deleterious genetic elements by creating barriers around microenvironments to form vesicle like structures. The cooperation between hosts and foreign genetic elements is also conserved to have a major impact on evolutionary transitions. Many invading genetic elements provide benefits to the host by the introduction of diversity and protection from superinfection (Koonin).

Bacteriophages are an important component of the gut microbiota and contribute to host intestinal immune responses. Phages can specifically control microbial growth and microbe diversity of the intestinal region by their innate ability to migrate through gut mucosa to lymph and other organs. Due to their innate ability to interact with the glycoproteins on mucus, bacteriophage are found in greater abundance in the mucosa compared to bacteria and defend the epithelial tissue against pathogenic bacterial organisms (Łusiak‑Szelachowsk).

Bacteriophage therapy was first utilized in the 1930’s and 1940’s to successfully treat shigellosis worldwide (Mai).  After the increased development of antibiotics during World War II, the West world transitioned to the use of antimicrobial agents to combat bacterial infection. Antibiotics were relatively easy to produce, stable, and had a broad range spectrum of activity against microbes. The Soviet Union and some Eastern European countries continued the use of bacteriophage to control bacterial infections such as wound infection. Specifically, the Soviet Union used bacteriophage to treat bacillary dysentery in children with high success and were also used to decontaminate hospital rooms (Mai) (Abuladze) (Cisek).

The inadequate research of biological components of bacteriophage and inconsistent usage of phage in therapeutics in vitro lead to ambivalent reports by the American Medical Association, ultimately resulting in the decline of phage usage. Phage therapy was almost exclusively phased out in many regions of the world as the development and use of antibiotic agents such as penicillin dominated (Adhya). It was not until the 1980’s when phage therapy successfully and more efficiently treated Escherichia coli in a mouse model compared to antibiotic agents (Hudson). While phage therapy was abandoned in Western regions between the 1930’s and 1940’s, phage therapy soon was reintroduced in the 1980’s using animal models and later human patients (Wittebole). Less than ten years ago, the US Food Safety and Inspection Service allowed the use of bacteriophage on preprocessed poultry to combat growth of Salmonella (Capparelli). In 2006, the Food and Drug Administration (FDA) allowed for the use of bacteriophage treatment to control Listeria monocytogenes on various foods such as cheeses and other ready-to-eat-food that are often vulnerable to L. monocytogenes contamination (Abuladze).

Bacteriophage used in the therapeutic realm have presented much promise in respect to many different applications. Bacteriophage cocktails have been utilized as a decontaminant in an effort to reduce pathogenic microbe contamination on food processing surfaces and structures (Woolston)(Abduladze). In addition, bacteriophage particles have been implemented directly onto food products such as poultry (Atterbury). In a clinical aspect, phages possess a broad spectrum of application from the treatment of some diseases to vaccine development (Akhtar)(Capparelli). A newly proposed function of bacteriophage therapy is not the direct use of bacteriophage themselves, but the application of the lytic enzymes and this demonstrates extreme potential in many aspects (Oliveira). It is clear that bacteriophage therapy is an innovative approach to various disciplines; further investigation could potentially lead to other phage applications such as bioterrorism agent control (Anderson).

Food safety is one of the fundamental concerns of the entire world. Food products are susceptible to contamination during processing, contamination events reduce the integrity of the manufactured goods and renders them non-consumable. The loss and contamination of the consumable merchandise does not exclusively result in economic cost but also has a major health impact. Understanding methods of which to prevent contamination of food products is critical to decrease the overall impact of compromised food products (Sillankorva) (Goodridge).

One of the major obstacles in food contamination prevention is the development of biofilms in food processing facilities. Some bacteria, such as Salmonella possess the innate ability to form a protective extracellular polymeric matrix that creates a microenvironment for many bacteria to exist in close proximity to one another to exchange compounds such as vital nutrients. These types of structures make it difficult to remove the bacteria from food processing environments. In particular, the seafood, dairy, meat, poultry, and vegetable processing industries have issues with biofilm production of processing equipment and food products. The employment of cleaning and sanitizing agents are implemented although have often been deemed inefficient to reduce biofilm and, in some cases, alter the integrity of the food product. The use of bacteriophage demonstrates much promise against the development of biofilms. Biofilm matrices are a naturally occurring phenomenon in the bacterial world and as a result bacteriophage have evolved adaptive mechanisms to infect cells in a dense polymer environment. Certain bacteriophage have been observed to invade structures by traversing through water channels or by degrading the external matrix by the use of depolymerizing enzymes (Gutierrez).

The bacteriophage of particular interest are those who combat biofilm formation by their ability to penetrate the first external biofilm barrier by the possession of hydrolytic enzymes such as polysaccharide depolymerases that degrade polymers that constitute the biofilm matrix. While some phages do not encode depolymerizing enzymes, phages have been genetically modified to express the enzymes during host infectivity and as a result the phages are able to degrade the biofilm structure and destroy the contained bacterial cells. Phage encoded depolymerase enzyme applications are not limited to the food industry but also have potential in the medical, bioenergetics, and chemical industries (Pires).

An important consideration for the use of bacteriophage for use as a decontaminant is the viability of the phage. Temperature and pH tolerance is fundamental for the use of phage in biocontrol. If the phage are unable to survive in a range of variable temperature and pH ranges, at least within the ranges of most food products, the efficacy of the phage decreases rendering them useless as a biocontrol method (Bao).

Woolston et al. explored the effects of administering two Myoviridae phage cocktails, SalmoFresh™ and SalmoLyse™ to reduce Salmonella on glass and stainless steel contaminated surfaces.  Both of the phage cocktails were tested to determine the host range specificity, each of the cocktails were able to lyse two pathogenic E. coli strains and SalmoLyse™ was effective against a Shigella strain.  The phage cocktails were deemed efficient in reducing the Salmonella serotypes Kentucky S800 and Brandenburg on both stainless-steel surfaces and glass surfaces. Only SalmoLyse™ was able to significantly reduce the growth of Salmonella Paratyphi B on glass surfaces. The use of bacteriophage cocktails on food processing equipment surfaces may induce a selective pressure against the targeted host strain, reducing the ability for the host organisms to inhabit the phage-treated surface environment; however long-term studies are still necessary to verify this claim (Woolston).

Hungaro et al. proposed that bacteriophage as a decontaminant have the potential to replace chemical sanitizing agents. When compared to chemical decontaminant solutions, isolated bacteriophage specimens were found to reduce S. Enteritidis on chicken skin to the same degree. Additionally, the phage were able to remain viable after low temperature storage conditions although phage activity was ideally at 37℃ (Hungaro).

Bacteriophage decontamination practices have also been applied to other food processing surfaces such as glass and gypsum boards. In a study conducted by Aduladze et al., a Mycoviridae bacteriophage cocktail (ECP-100) was used to reduce contamination of Escherichia coli O157:H7 on various surfaces from glass to food products. The ECP-100 phage cocktail was found to significantly reduce E. coli  O157:H7 growth on solid glass and gypsum surfaces. With regards to the food products, the phage cocktail significantly reduced E. coli O157:H7 on the surfaces of broccoli, tomatoes, and spinach in three trials while the E. coli O157:H7 was reduced on red meat in one trial (Aduladze).

The Atterbury et al. study employed the use of three bacteriophage effective in the lysis of Salmonella Enteritidis, Hadar, and Typhimurium. The lytic potential of the bacteriophage isolates was analyzed by introducing the phage into a Salmonella-colonized chicken host in vitro and in vivo. Two of the three phages expressed lytic activity in the chickens, and the first was effective in decreasing the Enteritidis cecal colonies. The second was able to decrease the colony formations of Typhimurium. The last was not effective in the lysis of Hadar serovar. All of the bacteriophage expressed lytic potential in vivo; indicating that even though some phage have lytic potential in vivo this does not mean that they will in vitro. In vivo phage bacterium interactions could be different from in vitro interactions because of the host internal matrix and defenses, which some bacteriophage particles may not be resistant to (Atterbury).

Bardina et al. determined that a cocktail of three Caudovirales bacteriophage were effective against a wide host range as well as showed greater infectivity potential against Salmonella serovars Enteritidis, Typhimurium and others. In vivo application of the phage cocktail did not produce significant results; a potential reason was the ratio between bacteriophage and bacteria did not accurately represent the actual proportions in the environment. They proposed that frequent application of bacteriophage, preferably prior to Salmonella infection, will ideally decrease the overall Salmonella colonization (Bardina).

Bao et al. identified the efficacy of phage treatment on foods featuring Myoviridae phage cocktail directly applied to chicken breast, milk, and Chinese cabbage infected with S.  Enteritidis. Significant reduction of S. Enteritidis was observed in each food category treated with the cocktail. The PC2184 phage and the PA13076 phage in the cocktail individually had different infectivity parameters. PC2184 was more effective in the liquid culture, eliminated the host quickly, and had a greater temperature tolerance. PA13076 phage did not express the same temperature tolerance nor did it have the same infectivity potential as the PC2184 phage, although it was still efficient in terms of reducing the amount of S. Enteritidis. Both of the phage isolates displayed greater stability in the liquid milk medium and at 4°C rather than at 25°C in most of the treated foods (Bao).

Some foods are particularly vulnerable to Salmonella contamination. For example, mung bean sprouts have a susceptibility to Salmonella since the pathogen is found on the seeds and during the bean sprout growth cycle Salmonella can internalize into the developing sprout. The Ye at al study explored the use of both a bacteriophage cocktail in combination with antagonistic bacteria isolated from sprouts to reduce Salmonella on mung bean sprouts. Limited research has been completed to investigate the potential of using both bacteriophage and antagonistic bacteria to reduce a specific target pathogen. The idea of using antagonistic bacteria is to inhibit the growth of specific target pathogens by using bacteria that produce compounds such as lactic acid in addition to prevent the growth of pathogens by increasing the environmental competition. The study determined that antagonistic bacteria in collaboration with bacteriophage cocktails were effective in diminishing the growth of Salmonella on mung bean sprouts (Ye).

It is apparent that dairy products exhibit a certain susceptibility to Salmonella, in the late 1990’s Ontario, Canada fell victim to an outbreak of salmonellosis that affected more than 800 people, primarily children, who ingested contaminated cheese products. Using the exact strain that caused the Ontario outbreak, Modi et al. investigated the ability of phage SJ2 to eradicate the outbreak Salmonella Enteritidis strain from pasteurized and raw cheese products. This study employed the use of bacterial bioluminescent Salmonella Enteritidis to identify the microbial survival in milk used to make cheeses. The inoculated cheeses were then observed using the NightOwl molecular imager to detect the presence of bioluminescent Salmonella Enteritidis. Colony measurements and phage concentration were calculated before the milk was inoculated with the pathogen, after Salmonella inoculation, after phage SJ2 treatment, and several steps during the cheese making process. Salmonella has the innate capability to survive in a raw cheese matrix although the addition of a phage treatment was found to reduce Salmonella counts in cheese. However, it was determined that a phage treatment alone was not able to exclusively prevent Salmonella survival over a 60-day storage period (Modi).

While phage possess a certain efficacy to decrease Salmonella organisms in a food matrix and on the surfaces of processing equipment, other methods may need to be used in tandem to effectively reduce the risk of food borne illness (Toro) Bacteriophage  show much promise to control pathogen propagation in agricultural and other environments; although, considerable research is still essential to determine the practical applications of phage in conjunction with other technologies and the cost associated with the use of phage (Goodridge).

The gut microbiota has been proposed to be a major contributor for many metabolic diseases such as obesity and diabetes. Gut microbiome metagenomic analysis has revealed that bacteriophage are greater in number and diversity compared to eukaryotic viruses (Davies).  Recently, the gut microbiota has become a great research priority with an interest in bacteriophage diversity, abundance and morphology in effort to identify host-phage relationships in the gut.  The colonization of the intestinal microbiome is crucial for healthy development and disease prevention. During the infant life stages, Caudovirales viruses primarily inhabit the gut microbiome of an infant. Further development leads to the formation of single stranded Microviridae in the intestinal microbiome. These among other viruses have been recovered from healthy adult humans in great diversity (Łusiak-Szelachowska).

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