Minimum bactericidal concentration of chemically synthesized silver nanoparticles against pathogenic Salmonella and Shigella strains isolated from layer poultry farms

Article history: Received on: 03/04/2017 Accepted on: 08/05/2017 Available online: 30/08/2017 The main objective of the present study is to find an appropriate in-vitro therapy to overcome clinically isolated antibiotic-resistant Salmonella and Shigella species from cases of layer poultry farms suffering from purulent dysentery and diarrhea. The present study demonstrated chemical synthesis of silver nanoparticles (AgNPs) via chemical reduction method and investigation for their antibacterial effect against the isolated bacteria by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Of 32 examined samples, 8 (25%) antibiotic-resistant isolates were isolated and identified including: Shigella flexneri, Salmonella typhimurium (two), Salmonella poona, Shigella boydii, Salmonella montevideo, Shigella sonnei, Salmonella enteritidis. Spherical AgNPs of 10-25 nm in size were synthesized and the AgNPs at a concentration of 16 μgml -1 were found to have both bacteriostatic and bactericidal effects in the case of Salmonella montevideo (layer chicken egg), Shigella sonnei (layer chicken feces), Salmonella enteritidis (layer duck egg) however the AgNPs at a concentration of 8 μgml -1 were found to have both bacteriostatic and bactericidal effects in the case of Salmonella poona (layer chicken feces), Shigella boydii (layer chicken feces), Salmonella typhimurium (layer chicken feces).


INTRODUCTION
Salmonella and Shigella species are Gram-negative rodshaped bacteria that are members of the family Enterobacteriaceae and are considered threatened foodborne pathogens facing food safety and public health (Arena et al., 2017;Pashazadeh et al., 2017).Depending on serology, Salmonella species comprises over 2500 serotypes (Pashazadeh et al., 2017) while Shigella comprises four subspecies: Shigella dysenteriae (serogroup A, 15 serotypes), Shigella flexneri (serogroup B, 14 serotypes), Shigella boydii (serogroup C, 20 serotypes), and Shigella sonnei (serogroup D, a single serotype) (Anderson et al., 2016;Arena et al., 2017).Salmonella is a serious threat facing poultry industries as it has the ability to infect chickens causing diarrhea (Hsu et al., 2016).In the last few years, consumption of contaminated poultry, eggs, and their products become the most common sources of foodborne human salmonellosis (Mahmoud et al., 2015;Park et al., 2015;Kalupahana et al., 2017).On the other hand, Shigella has the ability to infect chickens, which consequently could infect human causing shigellosis (Xu et al., 2004;Shi et al., 2014).The applications of antibiotics for treatment of bacterial infections or as growth promoters have a bad side effect, including: persistence of antibiotics residues in animal products as meat and eggs (Namagirilakshmi et al., 2010;Hsu et al., 2016), another bad side effect is the high incidence of an antibiotic-resistant bacterial species development which is considered one of the public health hazards (Tseng et al., 2014;Hsu et al., 2016).Therefore, it is important to find another efficient treatment for Salmonella and Shigella infection instead of antibiotic.In the last few years, there has been a growing interest in nanotechnology.Indeed, nanoparticles have been gaining importance in recent years and became an effective revolution therapy against pathogenic bacteria due to their bactericidal properties.The nanoparticles size and surface area are significant agents to which their bactericidal mechanism of action attributed to (Devi et al., 2017).Silver nanoparticles (AgNPs) has been gaining importance in recent years due to their broad spectrum bactericidal toxic effects against broad spectrum pathogenic bacteria (Ahmad et al., 2017) even at their minimum concentrations (Devi et al., 2017).Furthermore, the results offered by Raman et al., 2017 suggest that AgNPs can be used as potent bactericidal agents against antibiotic-resistant bacteria.However, to the best of our knowledge, most of the previous studies did not take into account AgNPs bactericidal effect against pathogenic antibiotic-resistant Salmonella and Shigella strains clinically isolated from layer poultry farms.The goal of the present study is to clinically isolate and identify Salmonella and Shigella strains from cases of layer poultry (chickens and ducks) farms suffering from purulent dysentery and diarrhea.The present work was also intended to chemically synthesize AgNPs via chemical reduction method.The synthesized AgNPs will be characterized through high-resolution transmission electron microscopy (HRTEM), ultraviolet-visible (UV-vis) spectroscopy, and Malvern-Zetasizer Nano (ZSP) -UK.The antibacterial effect of the synthesized AgNPs will be investigated through applying of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against the clinically isolated Salmonella and Shigella strains.

Samples collection
A total of 32 samples (15 layer chicken feces, 5 layer chicken eggs, 9 layer duck feces, 3 layer duck eggs) were randomly collected from cases of layer poultry farms suffering from purulent dysentery and diarrhea.The samples were collected aseptically under a sterile condition and transported in an insulated ice box within 4 hours of the collection to the Microbiology and Immunology Department, Veterinary Division, National Research Centre, Cairo, Egypt, where they were analyzed immediately for the presence of pathogenic Salmonella and Shigella.

Salmonella and Shigella Isolation and Identification
For the isolation of Salmonella from chicken or duck feces, a sterile swab of ≈1g fecal sample was collected and added to 50 ml buffered peptone water (Oxoid), blended for 2 min, left for 60 min at room temperature and then incubated at 37°C for 18 hours.After incubation, 0.1 and 1 ml from the previous broth were transferred to 10 ml Rappaport Vassiliadis (RV) (Oxoid) and 10 ml tetrathionate (TT) (Oxoid) broth respectively, followed by 24 h incubation at 42°C and 35°C for RV medium and TT broth respectively.After incubation, a loopful from each broth was streaked onto 2 agar medium (Oxoid) including: Xylose lysine deoxycholate (XLD) and Salmonella-Shigella (SS) plates and then incubated both at 37°C for 24 hours.On the other hand, for the isolation of Shigella from chicken or duck feces, the fecal sample was collected using a sterile swab in sterile tube containing 5 ml buffered peptone water, and then incubated at 37°C for 24 hours and then a loopful from each tube was inoculated onto SS agar medium plates and then incubated at 37°C for 24 hours.Meanwhile, for the isolation of Salmonella and Shigella from chicken or duck eggs, the whole eggs were washed with soap and then rinsed in 70% ethanol for 20 min for surface sterilization.1ml of the separated egg yolk was added to 50 ml lactose broth and 5 ml buffered peptone water for the isolation of Salmonella and Shigella respectively and then completed as mentioned before.After incubation of all cultured petri-dishes, the suspected colonies were then examined through biochemical identification methods followed by API system (Biomerieux).Serological identifications were done using Salmonella and Shigella-specific antisera.All identified isolates were then stored at -20°C in brain heart infusion broth (Oxoid) supplemented with 15% (v/v) sterile glycerol.

Materials used for silver nanoparticles synthesis
All chemicals and reagents used for silver nanoparticles (AgNPs) synthesis were of analytical grades.Silver nitrate (≥ 99.9%), sodium borohydride (≥ 98%), tri-sodium citrate dihydrate (≥ 99%), hydrogen peroxide (50%), and polyvinylpyrrolidone were purchased from Sigma-Aldrich and used as received without further purification.All solutions were prepared using de-ionized water.The borohydride solution was freshly prepared.

Preparation and characterization of silver nanoparticles
AgNPs sample was synthesized by the chemical reduction method.It was prepared by adding 3.5 ml sodium citrate (30 mM), 3.5 ml PVP, and 45 μl H 2 O 2 into the freshly prepared AgNO 3 (50 ml, 0.50 mM in deionized water) under vigorous stirring.After a few minutes, 250 μl of 80 mM NaBH 4 was added to obtain colloidal AgNPs.This mixture was stirred for 3 hours and stored in dark at 4 o C until use (Métraux and Mirkin, 2005).The size and shape of the prepared AgNPs colloidal solution were investigated using HRTEM (a JEOL JEM-1200 EX highresolution transmission electron microscope) operating at 120 kV.UV-visible spectrum of the synthesized AgNPs suspension was obtained on a JASCO UV-VIS spectrophotometer.The particle size distribution of the prepared AgNPs was measured by Malvern-Zetasizer Nano (ZSP) -UK; Size measurement from 0.3 nm (diameter) to 10 microns.

Microorganisms used for evaluation of Antibacterial activities of AgNPs
Stock cultures of the clinically isolated and identified eight pathogenic antibiotic-resistant Salmonella and Shigella strains in the present study including: Shigella flexneri (layer chicken feces), Salmonella typhimurium (layer duck feces), Salmonella poona (layer chicken feces), Shigella boydii (layer chicken feces), Salmonella typhimurium (layer chicken feces), Salmonella montevideo (layer chicken egg), Shigella sonnei (layer chicken feces), Salmonella enteritidis (layer duck egg) were used for evaluation of the antibacterial activities of the synthesized AgNPs.Test organisms were first activated from glycerol by two successive transfers in brain heart infusion broth at 37°C for 24 hours, then streaking on XLD and SS medium for Salmonella and Shigella, respectively.A single pure colony was streaked on brain heart infusion slants then incubated at 37°C for 24 hours, followed by storing at 4°C until the beginning of the experiment.

Bacterium Inoculum preparations for the MIC and MBC experiment
Loopful of culture from each previously inoculated brain heart infusion slants was transferred into brain heart infusion broth.The broth was then incubated with shaking at 37°C for overnight.Then the concentrations of these suspensions were adjusted to be equal to 5×10 5 CFU/ml using sterile saline (Omara, 2017).

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the chemically synthesized AgNPs against the isolated Salmonella and Shigella strains
Antimicrobial activities of the chemically synthesized AgNPs against the clinically isolated antibiotic-resistant Salmonella and Shigella isolates were analyzed through determination of the minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), and MBC/MIC ratio values using a broth microdilution method in the 96-well microtiter plates (Omara, 2017).To each well, 50 μl of the adjusted bacterial inoculum (5 × 10 5 CFU/ml) were added horizontally, while 50 μl of the AgNPs dilution were added vertically.Each plate included growth and sterility control wells; the growth control wells contained MH broth medium with tested bacterial concentrations in order to check the bacterial viability, while the sterility control wells contained only a sterile MH broth in order to check the sterility of the medium used.
The plates were covered slackly with cling film to ensure that the bacteria were not dehydrated and then incubated at 37°C for 18-20 hours.The lowest concentration of each antibacterial agent that inhibited the bacterial growth was then considered as the MIC (CLSI, 2013).After MIC determination, aliquots of 100 μl from each well, which showed no bacterial growth after incubation was seeded in BHI agar plates, which are not supplemented with AgNPs, then were incubated at 37 °C for 20 hours.The lowest concentration of the AgNPs that kills 100% of the initial bacterial population showing no colony on the MH agar after 20 hours of incubation at 37°C was recorded as the MBC (CLSI, 2013;Omara, 2017).

Shigella
Table 1 summarizes the results of antibiotic sensitivity of the isolated Salmonella and Shigella, it can be seen that all the isolated Salmonella and Shigella were multidrug resistant pathogens.

Characteristics of silver nanoparticles
Figure 1 shows high-resolution transmission electron microscopy (HRTEM) image of the synthesized AgNPs.Well homogenous shape of AgNPs is obtained; most of the particles were spherical but some of them having prismatic shape and due to their higher reactivity, the particles tend to agglomerate.Moreover, the size range of the obtained AgNPs are between 10-25 nm.The result of the particle size distribution of the synthesized AgNPs is presented in Fig. 2 which confirms the result obtained from HRTEM analysis.To investigate the structural characteristics of AgNPs, UV-vis spectroscopy technique was used.Fig. 3 depicts Uv-Vis.
spectrum of synthesized AgNPs.The spectrum exhibits two absorption shoulders at wavelength ranging between 350 and 450 nm, indicating the presence of spherical and roughly spherical Ag nanoparticles.

MIC and MBC determination of chemically synthesized AgNPs against the isolated Salmonella and Shigella strains
The MIC (μgml -1 ) and MBC (μgml -1 ) values as well as MBC/MIC ratios of the prepared AgNPs against the eight isolated Salmonella and Shigella strains are presented in Figure 4 and illustrated in Table 2. Table 2: MIC (μgml -1 ) and MBC (μgml -1 ) values as well as the MBC / MIC ratio of AgNPs against the isolated Salmonella and Shigella species.

DISCUSSION
Annually, 155000 death cases were reported due to Salmonella foodborne gastroenteritis (Liu et al., 2013).Recently, a total of 356 persons from 39 US states were infected by S. typhimurium in an outbreak, which occurred due to contact with backyard chicks and ducklings (CDC, 2013;Zheng et al., 2014).In the present study, 8 (25%) Salmonella and Shigella strains were isolated and identified out of the examined 32 samples.Previously, a total of 127 Salmonella isolates were isolated from fecal samples of chicken suffering from diarrhea (Kang et al., 2017).On the other hand, Shigella has been isolated from cases of chicken shigellosis suffered from bloody and purulent dysentery (Xu et al., 2004;Shi et al., 2014).Moreover, Shigella species were detected in 2 (0.25%) of previously examined chicken samples (Ahmed and Shimamoto, 2014).Isolation of Salmonella serovars from ducks has been also recorded from many countries including Egypt (Osman et al., 2014;Tang et al., 2015).In addition, previously examined raw duck meat has been shown to be a reservoir of Salmonella species, especially S. typhimurium (Adzitey et al., 2012).Furthermore, out of total collected 531 duck samples from duck farms, 125/531 (23.5%) were positive for Salmonella infection (Adzitey et al., 2012).On the other hands, Salmonella can reach also the egg yolk by either vertical transmission or horizontal transmission or even through temperature.If Salmonella reaches the egg yolk, it can then grow rapidly, even at room temperature (25°C) (Martelli and Davies, 2012).The vertical transmission (trans-ovarian infection) can be contributed to colonization of Salmonella to the layer reproductive organs (Kalupahana et al., 2017) in which Salmonella can then pass easily to the yolk, yolk membranes, and albumen of the newly formed eggs (Park et al., 2015).While, the horizontal transmission can be contributed to the contact between eggs and anything contaminated with Salmonella as handlers hand, layer fecal matter, pests, and even utensils in which, Salmonella can then pass easily through the shell membrane to the egg contents (Park et al., 2015).Furthermore, the positive differential temperature creates a negative pressure in which, if there is a moist environment at the shell surface, Salmonella can then pass easily to the egg contents (Martelli and Davies, 2012).
Recently, multidrug-resistant (MDR) bacteria have been spread worldwide which represent serious threat public health hazards.In the present study, all the isolated Salmonella and Shigella strains were multidrug resistance pathogens as summarized in Table 1.The administration of antibiotic for treatment of Salmonella or as growth promoters resulted in development of varying degree of Salmonella resistant strains as recorded by Carrique-Mas et al. (2008), Osman et al. (2014);and Tang et al. (2015).A multidrug-resistant (MDR) Salmonella have been previously recorded (Tsai and Hsiang, 2005;Cha et al., 2013;Chang et al., 2014;Tang et al., 2015).
This study presents a pilot study to answer how to overcome antibiotic-resistant Salmonella and Shigella.In the present study, chemically synthesized silver nanoparticles (AgNPs) by reduction method showed efficient antibacterial activities against clinically isolated antibiotic-resistant Salmonella and Shigella strains.In a previous study, a broad spectrum antibacterial activity of AgNPs against various pathogenic strains of bacteria, including Salmonella (Boonkaew et al., 2014;Patil et al., 2015) specially S. typhimurium were reported (Abd-Elnaby et al., 2016) similar to our results.Previously, silver nanoparticles (AgNPs) were reported as an excellent bactericidal agent used for killing bacteria (Raman et al., 2017).The nanoparticles bactericidal properties are attributed mainly to the electrostatic reaction between negative charge of bacterial cell wall and positive charge of nanoparticles metal ions (Prema et al., 2017).The shape, size, surface area, and stability of AgNPs are considered the main factors affecting on AgNPs bactericidal properties (Gliga et al., 2014;Su et al., 2017a).In the present study, the prepared AgNPs are spherical, and a previous study reported that the spherical AgNPs were found to have wider antibacterial activity in Gramnegative organisms (Swarnavalli et al., 2017).In the present study, UV-vis spectroscopy technique used to investigate the structural characteristics of the synthesized AgNPs as presented in Fig. 3, the spectrum exhibits two absorption shoulders at wavelength ranging between 350 and 450 nm, indicating the presence of spherical and roughly spherical Ag nanoparticles.Moreover, when the peak intensity is increased, this means the formation of spherical seed particles.It is important to mention that smaller particles give sharp peaks at shorter wavelengths less than 400 nm as compared to large particles which give broad peaks at longer wavelengths more than 400 nm.Previous study reported that when the absorption peak is ≤400 nm, the size range of nanoparticles would be 10-15 nm and when it is 400-440 nm the corresponding sizes are 20-60nm (Abideen, 2012).The smaller size NPs and larger surface area play a vital role in the antimicrobial performance of nanoparticles (Zafar et al., 2016;Su et al., 2017a).The smaller sized AgNPs (10-25nm) obtained in the present study is considered as an enhancing factor responsible for that observable antibacterial activity against tested Salmonella and Shigella.The results obtained in the present study are consistent with that of other research articles.Indeed, smaller sized AgNPs demonstrated higher bactericidal effect against pathogenic and non-pathogenic bacteria than their larger sized particles (Swarnavalli et al., 2017;Su et al., 2017a).Moreover, the bactericidal properties of AgNPs improved by decreasing AgNPsˊ size (Su et al., 2017a).This phenomenon can be explained by the following; when size of AgNPs decreases, the maximum contact area will increase, and the surface area to volume ratio (SA/ V) for each AgNP will then increase, and this resulting in increasing in relative AgNPs concentration (Agnihotri et al., 2014;Swarnavalli et al., 2017).This can be attributed to higher particle penetration and availability of more area of contact between the bacteria and nanoparticles (Swarnavalli et al., 2017).
Furthermore, the bactericidal activities of AgNPs against Gram-negative bacteria depend on the concentration of AgNPs (Sondi and Salopek-Sondi, 2004;Swarnavalli et al., 2017).Previously, Swarnavalli et al., 2017 reported that, with increasing the concentration of AgNPs, the higher antibacterial effects were observed (as done when the concentration of AgNPs increased from 0.49 to 250 μgml -1 ).In the present study, it is clear that the MIC and MBC values of AgNPs against the tested bacterial isolates were either 8 or 16 μgml -1 .The MBC of AgNPs was observed mainly at a concentration of 16 μgml -1 AgNPs in the case of Shigella flexneri (layer chicken feces), Salmonella typhimurium (layer duck feces), Salmonella montevideo (layer chicken egg), Shigella sonnei (layer chicken feces), Salmonella enteritidis (layer duck egg) while it was 8 μgml -1 AgNPs in the case of Salmonella poona (layer chicken feces), Shigella boydii (layer chicken feces), Salmonella typhimurium (layer chicken feces).
On the other hand, from the MIC and MBC results obtained in the present study, it is clear that, the AgNPs at a concentration of 16 μg ml -1 was found to have both bacteriostatic and bactericidal effects, followed by a concentration of 8 μg ml -1 .In previous studies, the MIC of a chemically synthesized nanoparticles (30-40 nm) against Salmonella were reported at 2.81 μgml -1 .Moreover, the efficient bactericidal activity of AgNPs was obtained at its lowest concentration of 0.35 μgml -1 (Devi et al., 2017).Furthermore, the MBC value of 100 nm sized AgNPs was ≈1 × 104 μgml -1 , while that for 5-10 nm sized AgNPs was 12-25 μgml -1 (Flores et al., 2013).A previous study reported that the calculated MIC of 28 nm sized AgNPs against Salmonella was 25 μgml -1 while it was 12.5 μgml -1 in case of 8 nm sized AgNP (Smekalova et al., 2016).Furthermore, the 3.5 μgml -1 MIC and 6.9 μgml -1 MBC values of AgNPs against Salmonella enterica [ATCC 13076] were previously reported (Singh et al., 2016).Similar to the present study, a chemically synthesized AgNP sized 30-40 nm showed MIC against Salmonella at a concentration of 2.81 μgml -1 (Zafar et al., 2016).
The AgNPs bactericidal properties are attributed mainly to their following abilities: bacterial cell walls and membrane destruction, reactive oxygen species (ROS) generation, vital metabolic enzyme inhibition, and obstruction of DNA replication (Feng et al., 2000;Sondi and Salopek-Sondi, 2004;Kim et al., 2007;Rai et al., 2009;Ahmad et al., 2017).AgNPs have the ability to attach and destruct bacterial cell walls in a dosedependent manner (Cerny and Teuber, 1971;Rhayour et al., 2003;Ahmad et al., 2017) this is confirmed by Electron spin resonance spectroscopy studies which demonstrated that when AgNPs attached to the bacterial cell walls, the free radicals of the AgNPs were then released (Danilcauk et al., 2006;Kim et al., 2007;Swarnavalli et al., 2017).
These released free radical seems to be the main causative agent responsible for final bacterial destruction and death (Devi et al., 2017); as these free radicals begin to make pores in the bacterial cell walls and membranes causing severe permeability of the bacterial cell membrane (Danilcauk et al., 2006;Kim et al., 2007;Rai et al., 2017;Swarnavalli et al., 2017), this is followed by releasing of periplasmic proteins and nucleic acids via cytoplasmic membrane, and so the bacterial cell death will be the final result (Cerny and Teuber, 1971;Rhayour et al., 2003;Ahmad et al., 2017;Rai et al., 2017;Swarnavalli et al., 2017).On the other hand, when AgNPs enter the bacterial cell, they form a low molecular weight region which begin to attack the respiratory chain, so consequently, the cellular signaling pathways will be changed by dephosphorylating assumed key peptide substrates on tyrosine residues (Sondi and Salopek-Sondi, 2004;Mulley et al., 2014;Su et al., 2017b).Moreover, the Ag+ metal ions that released from AgNPs (Feng et al., 2000;Swarnavalli et al., 2017) were found to have obstructing behavior to the bacterial signal transduction pathways (Devi et al., 2017) and moreover they could react with thiol groups present in the bacterial vital enzymes and proteins (Matsumura et al., 2003;Swarnavalli et al., 2017;Su et al., 2017b;Gopinath et al., 2016) and furthermore, they could react with phosphorus-containing compounds like DNA leading to inhibition of bacterial DNA replication, and so the bacterial cell death will be the final result (Jiao et al., 2014;Bao et al., 2015;Su et al., 2017b;Devi et al., 2017).On the other hand, the production of intracellular reactive oxygen species (ROS) by AgNPs occurred via the reaction between the AgNPs with the thiol-containing enzymes of the bacterial respiratory chain (Matsumura et al., 2003;Ahmad et al., 2017).The ROS seems to another causative agent responsible for final bacterial destruction and death (Shrifian-Esfahni et al., 2015;Ahmad et al., 2017) as this ROS is cytotoxic fatal to the bacterial cell in dose and surface charge dependent manner (Ahmad et al., 2017).Indeed, the formed ROS generates an oxidative stress to the bacterial cell resulting in suppression of the bacterial antioxidant defense mechanism, destruction of the bacterial cell membrane as well as DNA and vital enzymes, these circumstances resulting in final bacteria cell death (Kim et al., 2007;Li et al., 2010;Reuter et al., 2010;Ahmad et al., 2017).

CONCLUSION
From the outcome of our investigation, this study demonstrated high level (25%) of antibiotic-resistant Salmonella and Shigella infection, out of 32 examined samples of layer chickens and ducks as well as their eggs.This study suggesting possible applications of chemically synthesized spherical silver nanoparticles (10-25 nm) as an efficient bactericidal agent against clinically isolated antibiotic-resistant Salmonella and Shigella strains.The MBC of AgNPs was best observed mainly at a concentration of 16 μgml-1 followed by 8 μgml-1.In-vivo clinical applications of AgNPs to verify their in-vivo effectiveness will be the next step of our research studies.
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Table 1 :
Antimicrobial susceptibility of the isolated Salmonella and Shigella strains (n = 8).