Pathogenic strains of salmonella
1 All Nations University College, Koforidua, Ghana
2 Centre for Bioscience and Nanoscience Research (CBNR, Affiliated to Bharathiar University), Coimbatore, India
3 Jacob Institute of Biotechnology and Bioengineering, Sam Higginbottom University of Agriculture, Technology, and Sciences, Allahabad, U.P, India
The pathogenic strains of Salmonella typhi, paratyphi, and typhimurium are the major cause of typhoid and food poisoning in children and adults in developing countries. According to WHO estimation, 22 million cases of typhoid fever and 200,000 related deaths occur worldwide each year with an additional 6 million cases of paratyphoid fever estimated to occur annually with the highest incidence in children, resulting in a high death rate. The high use of antibiotics has also given rise to drug-resistant strains. Hence, it was of importance to assess the inhibition and quick detection of pathogenic strains of Salmonella. This study aims to investigate the chemically synthesized gold nanoparticles (GNPs) for its antibacterial activity against clinical isolates of S. typhi and S. paratyphi including food sample isolates. The GNPs were characterized using visible color change, UV-Vis spectrophotometry, FTIR, XRD, DLS, FESEM, TEM, and zeta potential. The plasmon peak at 525 nm and 535 nm confirmed the synthesis of gold nanoparticles. The size of the chemically synthesized gold nanoparticles (GNPs) were in the range of 40-60 nm, while FESEM and TEM images revealed that the GNPs were spherical in shape. For antimicrobial activities, five of the Salmonella strains were isolated from fish and egg samples, while the other seven were S. typhi and S. paratyphi from clinical samples. The inhibition factor for GNPs showed higher inhibition against S. paratyphi, while the inhibition factor for S. typhi were found to be higher than Ciprofloxacin-30. This is the first study of the antibacterial efficacy of GNPs against pathogenic strains of Salmonella. The obtained results suggest that nanobioconjugated gold may be of interest in the detection of typhoid and high potential use in areas in biomedicine as an alternative to antibiotics.
The Salmonella spp. are enteric human pathogenic bacteria hazardous to the public health. Salmonella typhi, paratyphi, and typhimurium are the major causes of typhoid and food poisoning, respectively, in children and adults in developing countries. It is therefore relevant to explore the use of new antimicrobial agents for treatment instead of antibiotics as the use of antibiotics has led to the emergence of various drug-resistant strains. Although all noble metals have some potent antimicrobial activity, silver nanoparticles also show significant ability to inhibit the growth of microorganisms and the literature has numerous reports on their therapeutic potentials [1–3]. Furthermore, a comprehensive review article was published highlighting several reports on the antibacterial activity of silver nanoparticles and their therapeutic outcome [2].
Specifically, silver NPs have been reported to have microbial inhibition against Salmonella strains (Salmonella typhi and Salmonella paratyphi) [4]. Also, a green synthesis of silver NPs has been demonstrated to have antibacterial activity against Salmonella typhimurium [5]. It has been further demonstrated that Salmonella growth is greatly inhibited by a synergistic antibacterial activity achieved by combining silver NPs with antibiotics [6]. This study is an attempt to investigate how gold nanoparticles (GNPs) affect these bacteria. Nevertheless, it has been reported that the resistance of various enteric human pathogenic bacteria against many synthetic drugs is being enhanced day by day [7]. According to the earlier published results of the antimicrobial activity of GNPs against human bacterial pathogens, it is outlined that there is no significant reaction for the zone of inhibition for E. coli and S. aureus and only 7 mm and 16 mm were obtained, respectively [8, 9].
Gold nanoparticles (GNPs) have novel properties and applications in nanotechnology and life sciences. It has been used since ancient times to make stained glass, but it was long assumed that the color of the gold suspension was a result of the chemicals used to prepare it. In 1857, Michael Faraday produced the first pure sample of a gold colloid and discovered that its color is due to the size of the gold particles. GNPs have monodispersed nanoparticles with shape- and size-dependent properties, exhibiting the color of ruby/intense red, and a wide range of particle sizes from 5 to 200 nm is often fabricated and used for diagnostic and therapeutic applications. The properties of GNPs and gold ions that make it unique over other metal nanoparticles are found to be its high surface-to-volume ratio for which a large number of surface gold atoms increase the surface chemical reactivity and enhance the chemisorption of molecules (CO and H2O) and its applicability for diagnostic, medical, therapeutic, and biological purposes [10–12]. Its properties such as dielectric function, electrical conductivity, and inertness permit its application to sinter inks, selective coatings, data storage, single electron conductivity, and quantum devices [13–15]. Another unique property is the surface plasmon resonance (SPR), which is a physical concept that describes the collective oscillations of conduction band electrons in the electromagnetic field. This property provides a new platform for the detection of many biological, environmental, and biomolecular targets [16–18]. GNP has a strong affinity for sulfur atoms, and this enhances its ability to have its surface modified through various approaches with S-containing compounds for diagnostic, sensing, and environmental applications [19, 20]. The surface of GNPs can also be modified by many different coating agents having different functionalities to make them useful in biology and medicine [21, 22]. Nowadays, GNPs have been studied and widely exploited in clinical diagnosis [23], biosensors, immunoassays [24], microorganism control [25], genomics [23], and vaccine development [26].
The various methods for the synthesis of GNPs are grouped under the bottom-up and top-down fabrication techniques [27]. They include chemical [28], sonochemical, electrochemical [29, 30], polymer-mediated [31], UV-induced photochemical [32, 33], ultrasound-assisted [34, 35], and laser ablation methods [36], as well as some unconventional (green synthesis or biosynthesis) approaches using microbes and plants. Intracellular and extracellular methods are some of the pathways available for GNP fabrication [37]. The first conventional chemical approach used to synthesized gold (III) derivatives by aqueous citrate was pioneered by Turkevich in 1951 [28]. In this method, the size of the gold nanoparticle obtained are influenced by the ratio of the reducing and stabilizing agents used [38]. Reducing agents such as borohydrate and sodium citrate enabled GNPs to be synthesized in different sizes and shapes (spherical and triangular shapes or in the form of nanorods and nanowires) [39]. However, there are three approaches in the chemical reduction method of synthesis that yield to a size-defined particle. The Turkevich and Frens method is the standard method whereby gold ions are reduced and stabilized by sodium citrate at 100°C. The UV irradiation at room temperature is another approach. Here, the gold solution with an added amount of citrate in a cuvette is placed in front of a UV lamp with a wavelength of 366 nm for two hours and the spectra bands were measured using a spectrometer. The third reduction method is by using ascorbic acid as a reducing and stabilizing agent [29]. The author [33] synthesized GNPs in the presence of ascorbic acid and CTAB to obtain anisotropic GNPs that were applied in semiconductor systems.
Furthermore, the importance of the citrate reduction approach over others denotes that the preparations of GNPs by the chemical method involve two main steps. However, the reduction and the stabilization steps are achieved using sodium citrate in order to avoid aggregation of the particles [40, 41] by achieving a nucleation, growth, and coagulation process. The use of trisodium citrate in fabricating GNPs is a low-cost technology that yields high volumes of GNP and reproducible results in terms of shape and size. This makes it the large-scale production approach chosen by industrial manufacturers and used for commercialization purposes [42].
This work is aimed at fabricating gold nanoparticles (GNP) using a low-cost approach, and its various characterizations were done first by visualization of physical color change and then by UV-Vis, FTIR, XRD, FESEM, TEM, DLS, and zeta potential. Salmonella strains obtained from hospitals and some from fish samples were isolated. All the necessary confirmatory tests were performed using TSI and TCBS media. Finally, the antimicrobial activity of GNPs and ionic gold were evaluated by the standard disk diffusion method and the diameters of the zones of inhibitory concentrations were measured. Also, this work focuses on investigating and evaluating the antimicrobial efficacy of pure GNPs on pathogenic strains of Salmonella which has not really been researched as thoroughly as it deserves.
Gold (III) chloride trihydrate tetrachloroauric (III) acid (HAuCl4·3H2O, M.W. 393.83) was purchased from HiMedia Mumbai, India. Sodium citrate (Na3C6H5O7·2H2O, M.W. 294.10) was purchased from Loba Chemie Pvt. Ltd., Mumbai, India. All solutions were prepared in doubled distilled water (demineralized water) obtained from Medilise Chemicals, Kerala, India. A Ciprofloxacin (CIP-30 mcg) disc was procured from HiMedia Mumbai, India. In this study, nutrient agar, TSI, and TCBS media were procured from HiMedia Mumbai, India. All chemicals used were of analytical grade and used without further purification.
For isolation of Salmonella spp., different clinical samples were collected from three different hospitals in Coimbatore, Tamil Nadu, India. Among the clinical Salmonella strains collected, six were S. typhi and one was S. paratyphi. Also, different types of fishes and eggs were taken from the Ukkadam fish market, Coimbatore (CBE), Tamil Nadu, India. In this study, all other chemicals and reagents used were procured from HiMedia Mumbai, India. All chemicals used were of analytical grade and used without further purification.
The culture methods for the isolation of Salmonellae involve a nonselective preenrichment, followed by selective enrichment, and plating onto selective and differential agars. All samples were aseptically subcultured onto agar slants (HiMedia, Mumbai, India) and incubated at 37°C for 24 hours. After culture growth, the slants were used for further studies [43, 44]. The presumptive Salmonella colonies were then subcultured onto the fresh nutrient broth and incubated for 24 h at 37°C.
The presumptive Salmonella isolates were identified by two confirmatory biochemical tests using the Triple-Sugar-Iron (TSI) agar test and the Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) agar test according to the method in [45, 46], and these were used without modification. The presumptive Salmonella colonies were directly stabbed into the TSI and TCBS agar slants, and the inoculated samples were incubated with a loosened cotton plug for 24 h at 37°C. The TSI agar was checked for alkalinity and the production of hydrogen sulfide gas, while that of the TCBS was checked for the production of black colonies which confirmed that no trace of Vibrio cholerae was falsely isolated. TSI is used to differentiate specific bacteria from the family Enterobacteriaceae based on their ability to ferment glucose, lactose, and sucrose and also with their ability to reduce sulfur to hydrogen sulfide [43]. The TSI slant was used to detect the lactose fermenters and the saccharose and dextrose fermenters. The medium also helped to determine the ability of the organisms to produce H2S. Pinkish slant and yellow butt or black slant and yellow butt were recorded as the positive reaction for Salmonella spp. [43].
All the isolates that were identified and confirmed as Salmonella spp. were used in antimicrobial susceptibility using the Kirby-Bauer disc diffusion method [47]. The isolated organism was subcultured in nutrient broth for 24 hrs. Nutrient agar plates were prepared; after sterilization, the agar was poured on the plate inside the lamina air flow and allowed to solidify. 70 μL of the 24-hour old culture of Salmonella spp. was spread evenly on the agar plate using a sterile cotton swab. After 2 minutes of drying, three different wells (20 mm) were made on the plate using a cork borer and 25 μL and 50 μL of GNP and 10 μL of gold solution were added on it; a standard Ciprofloxacin disc (CIP-30 mcg) was also placed on it and kept for 24 hours of incubation. The diameters of the inhibitory zones were measured in mm; the values obtained were further analyzed.
The gold ion was synthesized using a standard formula formulated by the CBNR laboratory protocol, Coimbatore, India. A 2 mM aqueous solution of gold (III) chloride trihydrate (tetrachloroauric (III) acid) was heated in an ultrasonic bath at 100°C for five minutes and was reduced by 1% sodium citrate in dropwise addition. The solution was boiled further for 5-30 minutes at constant heating till it produced a deep cherry red color [28, 48]. The gold nanoparticle solution was then cooled at room temperature and stored at 4°C till use. It was then subjected to various characterizations like UV-Vis spectra, FTIR, FESEM, TEM, XRD, DLS, and zeta potential.
The microprocessor UV-Vis spectrophotometer (Labtronic, single beam, LT-291, India) analysis was used for the optical analysis of GNPs scanned in the range of 300 nm to 700 nm with the UV-Vis spectrophotometer operating at a resolution of 2 nm. The Fourier transform infrared spectrophotometric (FTIR, Shimadzu, IRTracer-100, Japan) analysis was used to determine infrared intensity against the wavelength of light by identifying the functional group involved in the reduction and formation of the synthesized GNPs. A field emission scanning electron microscope (FESEM, GeminiSEM 500, Carl Zeiss, Germany) and TEM analysis were used for the morphological identification of the size and structure of the synthesized GNPs. X-ray diffraction (XRD, PANalytical X’Pert Pro, Diffractometer, Almelo, Netherlands) analysis was used to confirm the structure. It was scanned in the range of
(10°-80°). Dynamic light scattering (DLS, Nano ZS90, Malvern Instruments Ltd., UK) analysis was used to analyze the particle size and zeta potential to help study the physical property as well as the stability and quality of the synthesized GNPs.
Salmonella was detected in 12 of the total (30) collected samples under study. Seven were from clinical samples and five were isolated from fish and egg food samples. The Salmonella strains were cultured in nutrient media and were ascertained by turbidity and growth of circular, smooth, opaque, and translucent colonies in nutrient agar plates [33, 34].
The Salmonella strains were cultured in nutrient media and were ascertained by turbidity and growth of circular, smooth, opaque, and translucent colonies in nutrient agar plates as reported by the authors [43, 49]. In the TSI slants shown in Figure 1, the organism produced black colonies at the center due to the production of hydrogen sulfide gas. Likewise, in the Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) slants in Figure 2, the organism produced black colonies confirming the absence of Vibrio cholera in the isolates as reported earlier in the literature [50].
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Background
Salmonellae are gram-negative motile, nonsporulating, straight-rod bacteria. The genus Salmonella is named after Daniel E. Salmon, an American veterinarian who first isolated Salmonella choleraesuis from pigs with hog cholera in 1884. [1] .
Salmonellae are intracellular facultative pathogens that may survive in variable conditions. They pose a great threat to the food industry because they are able to adapt to environmental conditions that differ significantly from those in which they normally grow. Pathogenic Salmonella species can move using peritrichal flagellum.
Salmonellae can be isolated in the microbiology laboratory using numerous low-selective media (MacConkey agar, deoxycholate agar), intermediate-selective media (Salmonella-Shigella [SS] agar, Hektoen [HE] agar), and highly selective media (selenite agar with brilliant green). Salmonellae are oxidase-negative and predominantly lactose-negative. Fewer than 1% of nontyphoidal Salmonella (NTS) isolates are lactose-positive (pink on MacConkey agar), but most produce hydrogen sulfide, which is detectable on HE or SS agar. As facultative anaerobes, they grow well both in bottles of standard automated systems for blood cultures and on culture media routinely used for urine, tissue, and respiratory cultures. [2] Individual isolates can then be distinguished with serogrouping, pulsed-field gel electrophoresis, and bacteriophage serotyping techniques.
The nomenclature and classification of Salmonella species have been changed and restructured multiple times. Traditionally, Salmonella species were named in accordance with the Kaufmann-White typing system, defined by different combinations of somatic O, surface Vi, and flagellar H antigens. In 2005, Salmonella enterica finally gained official approval as the type species of the genus Salmonella. The genus Salmonella also contains the species Salmonella bongori and Salmonella subterranean, which was recognized in 2005. [3]
Currently, Salmonella species have the serologically defined names appended as serovars or serotypes. For instance, the current nomenclature of Salmonella typhi is S enterica serovar Typhi. S enterica is preferred over confusing name S choleraesuis, which is also the name of a commonly isolated serotype. [4] To date, more than 2500 serovars of S enterica have been described. Certain serovars are host-restricted, while others have a broad host range. [5]
Pathophysiology
Salmonellosis is caused by all nontyphoid serotypes of the Salmonella genus except for S typhi and Salmonella paratyphi A, B, and C. Salmonellosis-causing serotypes are isolated from humans and animals, including livestock. Serotypes Salmonella Typhimurium, Salmonella enteritidis, Salmonella newport, and Salmonella heidelberg are most often responsible for food poisoning; Salmonella Cholerasuis and Salmonella Dublin also cause diarrheic diseases. [66] Although the infectious dose varies among Salmonella strains, a large inoculum is thought to be necessary to overcome stomach acidity and to compete with normal intestinal flora. Large inocula are also associated with higher rates of illness and shorter incubation periods. In general, about 10 6 bacterial cells are needed to cause infection. Low gastric acidity, which is common in elderly persons and among individuals who use antacids, can decrease the infective dose to 10 3 cells, while prior vaccination can increase the number to 10 9 cells. [16]
The Salmonella infection cycle starts after the ingestion of microbes. Through the stomach, the bacteria reach the small intestine. Infection with salmonellae is characterized by attachment of the bacteria by fimbriae or pili to cells lining the intestinal lumen. Salmonellae selectively attach to specialized epithelial cells (M cells) of the Peyer patches. The bacteria are then internalized by receptor-mediated endocytosis and transported within phagosomes to the lamina propria, where they are released. Once there, salmonellae induce an influx of macrophages (typhoidal strains) or neutrophils (nontyphoidal strains).
The Vi antigen of S typhi is important in preventing antibody-mediated opsonization and complement-mediated lysis. Through the induction of cytokine release and via mononuclear cell migration, S typhi organisms spread through the reticuloendothelial system, mainly to the liver, spleen, and bone marrow. Within 14 days, the bacteria appear in the bloodstream, facilitating secondary metastatic foci (eg, splenic abscess, endocarditis). In some patients, gallbladder infection leads to long-term carriage of S typhi or S paratyphi in bile and secretion to the stool. [17] As a rule, infection with nontyphoidal salmonellae generally precipitates a localized response, while S typhi and other especially virulent strains invade deeper tissues via lymphatics and capillaries and elicit a major immune response.
Virulence factors of salmonellae are complex and encoded both on the organism's chromosome and on large (34-120 kd) plasmids. Some areas of active investigation include the means by which salmonellae attach to and invade the intestine, survive within phagosomes, effect a massive efflux of electrolytes and water into the intestinal lumen, and develop drug resistance. Several Salmonella pathogenicity islands have been identified that mediate uptake of the bacteria into epithelial cells (type III secretion system [TTSS]), nonphagocytic cell invasion (Salmonella pathogenicity-island 1 [SPI-1]), and survival and replication within macrophages (Salmonella pathogenicity-island 2 [SPI-2], phoP/phoQ).
Specific anatomical sites, such as an altered urinary or biliary tract, atherosclerotic aorta, or endovascular devices may facilitate persistent focal Salmonella infection.
A Dangerous Foodborne Pathogen
More than 2,500 serotypes of Salmonella exist. However, only some of these serotypes have been frequently associated with food-borne illnesses. Salmonella is the second most dominant bacterial cause of food-borne gastroenteritis worldwide. Often, most people who suffer from Salmonella infections have temporary gastroenteritis, which usually does not require treatment. However, when infection becom.
More than 2,500 serotypes of Salmonella exist. However, only some of these serotypes have been frequently associated with food-borne illnesses. Salmonella is the second most dominant bacterial cause of food-borne gastroenteritis worldwide. Often, most people who suffer from Salmonella infections have temporary gastroenteritis, which usually does not require treatment. However, when infection becomes invasive, antimicrobial treatment is mandatory. Symptoms generally occur 8 to 72 hours after ingestion of the pathogen and can last 3 to 5 days. Children, the elderly, and immunocompromised individuals are the most susceptible to salmonellosis infections. The annual economic cost due to food-borne Salmonella infections in the United States alone is estimated at $2.4 billion, with an estimated 1.4 million cases of salmonellosis and more than 500 deaths annually. This book contains nineteen chapters which cover a range of different topics, such as the role of foods in Salmonella infections, food-borne outbreaks caused by Salmonella, biofilm formation, antimicrobial drug resistance of Salmonella isolates, methods for controlling Salmonella in food, and Salmonella isolation and identification methods.
eBook (PDF) ISBN: 978-953-51-4378-9
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