Biology and Medicine

Salmonellosis is a zoonotic disease causing huge economic losses worldwide, triggering a high rate of morbidity and mortality in humans; it is also one of the most common and aggressive foodborne diseases (ETAS). In Ecuador, 9908 cases of Salmonella were reported in 1990; by 2001 the figure suddenly increased to 18,772; this number has gradually decreased, registering in 2013 only 5972 cases [1]. However, nowadays most of the cases are not recorded because the treatments for controlling disease outbreaks are provided by private physicians (Dr. Rodrigo Paredes personal communication). Salmonellosis is caused by subspecies and serovars of the bacterium Salmonella enterica , which is isolated most frequently from poultry and poultry products. In recent years, according to studies of bird populations made by CONAVE (Corporacion Nacional de Avicultores Del Ecuador “Conave”), from 1990 to 2012 there has been an increase in consumption of chicken of 360%, while egg consumption has had a growth of about 60%. It is evident chicken is one of the main meat products for Ecuadorians.

The genus Salmonella are gram-negative bacilli of the Enterobacteriaceae family, they are none sporulating facultative anaerobes [2]. Subspecies enterica are usually found in warm-blooded animals while subspecies II, IIIa, IIIb, IV and VI are usually found in cold-blooded animals and the environment; nevertheless, all of these can infect humans [3].

An increasingly common feature of the Salmonella strains is that they are more resistant to antibiotics; this is due to the indiscriminate use of them in recent decades. The current spread of antibiotic resistance genes in pathogenic bacteria casts doubt on the effectiveness of antibiotics in the future. In animal production, antibiotics are not only used for therapeutic purposes, but they are also used to stimulate growth. For that reason the overuse of antibiotics should be limited and must urgently seek alternative methods to control bacterial pathogens.

One possible option against antibiotic resistant bacteria is the use of bacteriophages. Bacteriophages have been widely used to treat bacterial diseases in humans, animals and also to reduce the bacterial load in foods from animal and vegetable origin [4,5]. Phages have certain advantages over antibiotics, for example: high specificity, since only pathogenic bacteria of interest, is eliminated [6]. Bacteriophages cause no harm to endogenous individual microflora, no adverse effects on the human or animal immune system [7].

Unfortunately, antibiotics are used uncontrollably everywhere and Ecuador is not an exception. With the implementation of this project, using bacteriophages as a new, safe, and effective biocontrol method for treating infectious diseases caused by resistant pathogenic bacteria, we would introduce safer agricultural practices of food production for Ecuadorian industry.

Salmonellosis is a zoonotic disease causing huge economic losses worldwide, triggering a high rate of morbidity and mortality in humans; it is also one of the most common and aggressive foodborne diseases (ETAS). In Ecuador, 9908 cases of Salmonella were reported in 1990; by 2001 the figure suddenly increased to 18,772; this number has gradually decreased, registering in 2013 only 5972 cases [1]. However, nowadays most of the cases are not recorded because the treatments for controlling disease outbreaks are provided by private physicians (Dr. Rodrigo Paredes personal communication). Salmonellosis is caused by subspecies and serovars of the bacterium Salmonella enterica , which is isolated most frequently from poultry and poultry products. In recent years, according to studies of bird populations made by CONAVE (Corporacion Nacional de Avicultores Del Ecuador “Conave”), from 1990 to 2012 there has been an increase in consumption of chicken of 360%, while egg consumption has had a growth of about 60%. It is evident chicken is one of the main meat products for Ecuadorians.

The genus Salmonella are gram-negative bacilli of the Enterobacteriaceae family, they are none sporulating facultative anaerobes [2]. Subspecies enterica are usually found in warm-blooded animals while subspecies II, IIIa, IIIb, IV and VI are usually found in cold-blooded animals and the environment; nevertheless, all of these can infect humans [3].

An increasingly common feature of the Salmonella strains is that they are more resistant to antibiotics; this is due to the indiscriminate use of them in recent decades. The current spread of antibiotic resistance genes in pathogenic bacteria casts doubt on the effectiveness of antibiotics in the future. In animal production, antibiotics are not only used for therapeutic purposes, but they are also used to stimulate growth. For that reason the overuse of antibiotics should be limited and must urgently seek alternative methods to control bacterial pathogens.

One possible option against antibiotic resistant bacteria is the use of bacteriophages. Bacteriophages have been widely used to treat bacterial diseases in humans, animals and also to reduce the bacterial load in foods from animal and vegetable origin [4,5]. Phages have certain advantages over antibiotics, for example: high specificity, since only pathogenic bacteria of interest, is eliminated [6]. Bacteriophages cause no harm to endogenous individual microflora, no adverse effects on the human or animal immune system [7].

Unfortunately, antibiotics are used uncontrollably everywhere and Ecuador is not an exception. With the implementation of this project, using bacteriophages as a new, safe, and effective biocontrol method for treating infectious diseases caused by resistant pathogenic bacteria, we would introduce safer agricultural practices of food production for Ecuadorian industry.

Bacterial strains

In this study we used two epidemic strains of Salmonella (Salmonella entérica subsp entérica serovar Infantis (SI), and Salmonella entérica subsp entérica serovar Enteritidis (SE)), kindly donated by the Faculty of Veterinary and Animal Science at the Central University of Ecuador. Five other species of bacteria were isolated during this study and used to determine the specificity of Salmonella bacteriophages.

Bacteria were isolated from cloacal swab samples and stool samples collected from eight hens in a craft farm at canton Ruminahui, Pichincha province in Ecuador. Additionally, samples of residual water and different organs: gizzard, intestine, spleen and heart, were randomly taken at selected markets in Quito and Rumiñahui.

Biochemical assays

Standard bacteriological procedures were performed for bacteria management: samples were transported in 10 ml of Buffered Triptone- Water incubated for 24 h at 37°C, pre-enriched in Rappaport Vassiliadis Broth and Tetrathionate Broth Base (Difco Laboratories, United States). A loopful of each sample was streaked onto Xylose lysine deoxycholate agar (XLD) (Difco Laboratories, United States) and Salmonella – Shigella Agar (SS) (Difco Laboratories, United States). Following incubation at 37°C for 24 h, the suspected Salmonella colonies were re-streaked on XLD agar, and then incubated at 37°C for 24 h. A final identification of bacterial colonies was achieved by their biochemical reaction to: oxidase, catalase, MRVP, TSI, SIM, citrate, fermentation of glucose, maltose, sucrose and lactose. The pattern was confirmed using API 20E strips (bioMérieux, Marcy l'Étoile, France).

DNA extraction

DNA from pure bacterial culture was obtained using [8,9] modified protocols. Overnight cultures were centrifuged, pelleted and rinsed twice with TE buffer then pelleted again to re-suspend in 200 µL of TE. Bacteria were exposed to thermal shock by boiling the tubes for five minutes and immediately crushing on icy water for five minutes. After this treatment the samples were centrifuged at 12000 RPM for five minutes. Fifty four microliters of the supernatant were placed in a 200 µL microtube with 6 µL of Proteinase K (10 mg/mL). The mixture was incubated in the thermocycler C1000 Touch de Bio RadPER for 1 hour at 60°C following 15 min at 95°C. The DNA concentration was measured with a nano drop.

PCR detection

Primers with highly conserved regions to detect pathogenic Salmonella were selected from the literature: invA [10], fimC [11], JE0402-1 [12], and histidine operon transport [13]. The cocktail for the PCR reaction contained 4 μl of genomic DNA (100 ng/μl) obtained by heat shock treatment and 16 μl of the following solution: 5% Glycerol, 1.5 Mm MgCl₂, 0.8 Mm dNTPs, 0.2 μM of each primer and 0, 05 U de Platinum Taq- polymerase (Invitrogen, United States). The reactions were performed in a C1000 Touch Thermocycler (Bio Rad) using the following thermal profile: 94°C (5 min) 35 cycles of: 94°C (30 s), 65°C (30 s) this temperature was different according to the primers used, and extension at 72°C (1 min); the program was terminated with a final 72 °C for 5 min. The PCR products were fractionated in 1.5% agarose gel at 100 V for 55 min and stained with 0.03 μl/ml of SYBR® Safe (InvitrogenTM).

Bacteriophages isolation and purification

Bacteriophages were isolated from wastewater of a medium (P) and a small (S) scale poultry processing plants (Table 1) in Pichincha- Ecuador. To increase the number of bacteriophages the samples were enriched twice with: 2.5 mL 0.5X triptose broth (HiMedia) supplemented with 10 mM de MgSO₄ (triptose enriched medium) and 1 mL of overnight Salmonella culture incubated with shaking (170 RPM) at 37°C for 24 h. The samples were then centrifuged at 4000 RPM for 30 minutes at 18°C (1248R – LaboGene Centrifuge). After the second enrichment, 5 mL of the supernatant was filtered using 0.45 and 0.20 μm syringe filters. Then 5 mL of the filtrate was mixed with 5 mL of 3 hour Salmonella growth (in 0.5X triptose enriched broth medium). The mixture was incubated with shaking at 37°C for 24 h. The suspension was centrifuged and filtered. This procedure was repeated six times. The resultant phage suspensions were filtered through a 0.20 μm filter and stored at 4°C. The effect of phage suspensions on Salmonella growth was evaluated by measuring the 3 h cultured Salmonella optical density (OD_o) at 600 nm and the final optical density (OD_f) of the culture after 24 h of incubation with the phage [14].

Table 1: Source of residual water from poultry industries for bacteriophages isolation and bacterial bite.

Bacteriophage titer

Bacteriophage titers were determined using the double layer agar plaque assay. Each phage suspension was serially diluted (10-1 to 10-10) in sterile distilled water. A 100 µL aliquot of each dilution together with 50 µL of cultured Salmonella were mixed with 2.5 mL triptose soft agar (0.4%) tempered to 45°C. The mixture was then spread uniformly through a 1.5% solid triptose agar plate and kept at room temperature for 10 min to solidify. The plaque forming unit (PFU) count was determined after overnight incubation of the plates at 37°C [14].

Host range assays

The host range of each bacteriophage´s cocktail was determined by spotting 5 µL of phage suspensions on a spread lawn of bacteria. After the spots dried, the plates were incubated at 37°C for 24 h and observed for the presence of clear lytic zones over the bacterial lawn. The bacteriophages were challenged with seven different bacteria: SE, SI, Salmonella enterica (unidentified strain), E. agglomerans , C. freundii , P. fluorescens and E. coli . The spot test was performed in triplicate.

Transmission electron microscopy

Negative staining electron microscopy of phages was conducted for morphological characterization. The phage suspensions were stained with 1% phosphotungstic acid (PTA) at pH 7, 2% PTA at pH 5 and 0.5% PTA at pH 4 depending on the origin of the sample. Then the preparations were observed in a transmission electron microscope at 37000 magnifications.

Bacteria isolation and characterization

The genus of each control and isolated bacteria used in this study was established by their biochemic response to pre-established tests (Table 2). Bacteria of the genus Salmonella , Citrobacter , Enterobacter , Pseudomona and Echerichia were identified. To confirm the specie of bacteria, an AP1-20E test was performed with the same samples and controls tested previously (Table 3). The two tests: biochemical reaction and API results coincided with the following species: Salmonella enterica , Citrobacter freundii , Enterobacter agglomerans, Pseudomona fluorescens and Escherichia coli .

Table 2: Biochemical characterization of bacteria isolated from poultry and derivatives. Each result repeated twice. Positive reaction +, Negative reaction -.

Table 3:API-20E test to confirm biochemical characterization of donated and isolated bacteria from poultry industries in Ecuador.

The pathogenicity of the Salmonella species was determined by PCR searching for the presence of pathogenicity genes. Those genes were chosen from reports in the literature. The PCR products obtained from bacterial genomic DNA confirmed the presence of: invA, fimC, histidine transport operon and JEO402-1 that separated Salmonella from the other Enterobacterias and from other none pathogenic Salmonella (Figure 1).

Figure 1: PCR products for Salmonella identification: A. primers US1f /US1r for gene invA, B. primers S212/S500 for gen fimC, C.primers GSAL-F/GSAL-R for histidine transport operón, D. primers ST11-ST15 for gene JEO402-. Where: Se: S. enteritidis , Si:S. infantis , P: S. enterica , En: Enterobacter , Ci: Citrobacter, Ps:Pseudomona, Sg (+): S. gallinarum , Ec: E. coli and B: blank.

Bacteriophages isolation

Four lytic bacteriophage cocktails (PSEA-2, SSEA, PSIA-2 and SSIA) capable of infecting SE and SI were isolated from wastewater of artisanal poultry processing plants.

All isolated cocktails reduced both SE and SI bacterial titer in liquid culture media. After overnight incubation, the OD at 600nm value was decreased in phage treated samples compared with the untreated bacterial controls (Table 4).

Table 4: Effect of phage suspension on Salmonella . Healthy bacterial growth evaluated at three hours, decreased OD at 600 nm after 24 hrs incubation with phage suspension en relation to sterile water control(CSE abd CSI).

The phage cocktails titer was determined by double layer plaque assay: PSEA-2 (1.4x10⁸ UFP/mL), SSEA (1.6×10⁸ UFP/mL), PSIA-2 (9×10⁹ UFP/mL) and SSIA (4×10⁹ UFP/Ml). A mixture of plaques with different diameter were observed: 2, 3, 4 and 5 mm in PSEA-2; 0.5 and 1 mm plaques in SSEA; 2, 3, 4, 5, 7 and 10 mm diameter in PSIA-2 and, 1, 3 and 4 mm plaques in SSIA.

The host range of a bacteriophage is defined by the bacterial genera, species and/or strains it can lysate [15]. The lytic activity of the four bacteriophage cocktails was tested against all seven different bacteria isolated from poultry. PSEA-2 and PSIA-2 cocktails caused total lysis only in SE and SI cultures. These two phage cocktails have great potential for use in biological control of Salmonella due to exhibited specificity in bacterial genera, specie and sub-specie. The cocktails SSEA and SSIA besides having lytic activity against SE and SI, made turbid lytic plaques in P. fluorescens.

The transmission electron microscope (TEM) revealed the presence of tailed phages (Figure 2a, b, c and d) and a polyhedral phage (Figure 2e).

Figure 2: Bacteriophages, transmission electronmicrographies, isolated from waste water from poultry processing plants, using Salmonella enterica as bite.

In this study isolated bacteria from artisanal poultry processed plants were identified, characterized and used to start the development of a control method through the isolation and characterization of specific bacteriophages.

Five genus of bacteria were isolated from poultry and poultry related products gown and processed under artisanal conditions in Ecuador. Those genera were identified and characterized by the biochemical response of each isolate when grown under a set of culture media. The identification was confirmed analyzing the profile of each with the API-20E kit for Enterobacteria identification. Both results coincided identifying Salmonella enterica plus four other bacterial species: Citrobacter freundii , Enterobacter agglomerans , Pseudomona fluorescens and Escherichia coli . Further characterization was done to determine pathogenicity genes in Salmonella enterica isolates. Specific molecular tests, confirmed the presence of genes: invA, fimbC, histidine transport and JEO-4 reported as evidence of plathogenicity in Salmonella species [16,17]. The genes invA and FimC are both essential in the eukaryotic cell invasion [18]. Although fimC in some publications is reported absent from S. gallinarum and S. pollorum [19], in our study fimC amplified S. gallinarum DNA in agreement with others [17,19] who maintain the gene is present but possibly nonfunctional.

The Histidine transport operon and JEO402-1 genes are reported as highly conserved regions in all Salmonella species [12,13]. The presence of these two genes in our study was distinctive of all four strains of Salmonella evaluated.

Our biochemical and molecular results confirm the bacteria used to isolate specific bacteriophages were indeed pathogenic Salmonella enterica . Therefore any phage we could isolate in this study would be applied in the control of the bacteria in further studies.

The literature recognizes as evidence of the lytic activity of bacteriophages, reduction in the optical density of host bacteria cocultivated with lytic phages in relation to bacterial cultures without the phage [20]. In our experiments co-cultivating filtered waste water from artisanal poultry processing plants with SE and SI we saw strong evidence of bacteriophages lytic activity by the constant decrease of bacterial concentration comparing with bacterial cultures without the filtrates. The most efficient phage cocktail isolated was PSIA-2, it decreased the bacteria concentration in 381.82%, followed by PSEA - 2 with 61.11%, SSEA with 49.28% and SSIA with 45.33%.

When measuring the phage concentration of each cocktail, most of them showed a range of plaque sizes, suggesting the presence of different phage species in the samples [21].

The cocktails SSEA and SSIA besides having lytic activity against SE and SI made turbid lytic plaques in P. fluorescens . This is possibly because within the phage cocktail isolated from the wastewater of the small scale poultry processing plant there were bacteriophages capable of recognizing more molecules as receptors. These receptors may be present in bacteria not belonging to Salmonella . The more molecules a phage can recognize as receptors, the wider its host range is [22]. Also, when referring to turbid plaques or media the literature says it is because of the emergence of lysogenic bacteriophages [23]. We would need more experiments to examine those two particular cocktails, whether contains phages able to incorporate in the bacterial genome becoming lysogenic. Because of the clear bacterial specificity at the level of genera, species and subspecies, phages present in PSEA-2 and PSIA-2 have great potential for use in biological control against Salmonella .

Preparations from each of the cocktail of bacteriophages observed at the transmission electron microscope (TEM) revealed the presence of tailed phages and an icosahedral one. Tailed phages are the most abundant group among reported phages and represent 96% of the total; within this group 61% belong to Siphoviridae family, 25% to Myoviridae and 14.5% to Podoviridae [24]. The phages isolated in this study have a size within the range reported for Myoviridae which have icosahedral capsids with diameters larger than 80 nm and their tails are surrounded by a sheath that gives them their contractile nature. Other group of phages isolated in this study are similar to phages from the Syphoviridae having icosahedral heads with diameters ranging between 48 and 75 nm; their tails are long, flexible and noncontractile, their length varies from 110 to 300 nm [25,26] which coincide with our results. Therefore it is clear phages belonging to those families are present in our preparations. We will continue with the molecular characterization of the phages to fully identify them.

In conclusion, we have isolated four cocktails containing lytic phages specific for Salmonella enterica serovars Enteritidis and Infantis present in Ecuador. The cocktails have proven effective at decreasing specific bacterial growth in vitro and to contain bacteriophage particles as observed at the electron microscope. The specificity of the phages was confirmed by challenging them with other Enterobacterias which were not affected using spot test. In such test Salmonella enteritidis did not survive. We have confirmed the efficiency of the phages by two in vitro tests. However we need further studies in order to determine the usefulness of the phages to control the bacteria in vivo , therefore their possible use at the farm level.

Authors are thankful to Army Polytechnic University (ESPE) for providing laboratories facilities and funding the research work, to Central University of Ecuador, Faculty of Veterinary and Zootechnics, for donating the Salmonella strains and to the Center of Electronic Microscopy of the Army Polytechnic University (ESPE) for taking the pictures of phages showed in this paper.