A total of 235 local chicken products (liver, breast, thigh, neck skin, and minced meat) were collected from 25 retail stores and 6 chicken processing plants between January 2018 and October 2021. The products were transported under refrigeration to the National Institute of Health Sciences. Each sample was tested within 24 h after arrival at the laboratory. Twenty-five grams of the sample was mixed in 225 mL of buffered peptone water (Oxoid Ltd., Hampshire, UK) and incubated at 37 °C for 18 h for pre-enrichment. After incubation, 0.1 and 1 mL of the culture was added to 10 mL of Rappaport–Vassiliadis broth (Oxoid) and 10 mL of Hajna tetrathionate broth (Eiken Chemical), respectively, then incubated at 42 °C for 20 h. After incubation, each culture was streaked onto two selective isolation agar plates: xylose-lysine-deoxycholate agar (Oxoid) and CHROMagarTM Salmonella (CHROMagar, Paris, France) then incubated at 37 °C for 24 h. For the selective isolation from minced meat, Mannitol-Lysine-Crystal-Violet-Brilliant-Green agar (Nissui Pharmaceutical, Tokyo, Japan) was used in place of xylose-lysine-deoxycholate agar. Putative Salmonella colonies were biochemically identified as mentioned above. One strain per sample was suspended in 20% glycerol and stored at −80 °C until ready for serotyping and antimicrobial susceptibility testing.
Rappaport vassiliadis broth
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Rappaport-Vassiliadis broth is a microbiological culture medium used for the selective isolation and enrichment of Salmonella species. It is formulated to support the growth of Salmonella while inhibiting the growth of other bacteria.
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Lab products found in correlation
Buffered peptone water (bpw), by Thermo Fisher Scientific (7 mentions)
Xylose lysine deoxycholate agar, by Thermo Fisher Scientific (6 mentions)
Hajna tetrathionate broth, by Eiken Chemical (2 mentions)
Hektoen enteric agar, by Thermo Fisher Scientific (2 mentions)
Salmonella shigella agar, by Thermo Fisher Scientific (2 mentions)
Brilliant green agar, by Thermo Fisher Scientific (2 mentions)
Selenite cystine broth, by Thermo Fisher Scientific (2 mentions)
Vitek 2 compact system, by bioMérieux (2 mentions)
Api 20e strip, by bioMérieux (2 mentions)
Xylose lysine deoxycholate (xld), by Thermo Fisher Scientific (2 mentions)
400 circulator, by Seward Medical (2 mentions)
Sabouraud dextrose agar, by Thermo Fisher Scientific (1 mentions)
Tryptone soya broth (tsb), by Thermo Fisher Scientific (1 mentions)
Macconkey broth, by Thermo Fisher Scientific (1 mentions)
Lactophenol cotton blue stain, by Hardy Diagnostics (1 mentions)
Xylose lysine desoxycholate, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Merck Group (1 mentions)
Brilliance salmonella agar, by Thermo Fisher Scientific (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Xld agar, by Thermo Fisher Scientific (1 mentions)
25 protocols using rappaport vassiliadis broth
1
Salmonella Detection in Chicken Products
2
Salmonella Detection and Serotyping Protocols
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For pre-enrichment, one gram of cecal content sample was mixed with 9 mL buffered peptone water and incubated at 37°C for 18 hr. After incubation, 0.1 and 1 mL of the culture was added to
10 mL Rappaport–Vassiliadis broth (Oxoid) and 10 mL Hajna tetrathionate broth (Eiken Chemical, Tokyo, Japan), respectively. After incubation at 42°C for 20 hr, each culture was streaked onto
xylose-lysine-deoxycholate agar (Oxoid) and CHROMagarTM Salmonella (CHROMagar, Paris, France) selection plates, followed by incubation at 37°C for 24 hr. Two suspect colonies were
biochemically identified, as previously described [21 (link)]. Salmonella isolates were tested by slide agglutination with O antisera (Denka
Co., Tokyo, Japan, and SSI Diagnostica, Copenhagen, Denmark). One of each different O serogroup isolate per flock was tested for flagella antigens by tube agglutination using H antisera
(Denka Co.). Serovars were determined based on the reaction between O and H group antigens according to the Kauffmann–White scheme [9 ]. One of each
different serovar isolate per flock was subjected to antimicrobial susceptibility testing.
10 mL Rappaport–Vassiliadis broth (Oxoid) and 10 mL Hajna tetrathionate broth (Eiken Chemical, Tokyo, Japan), respectively. After incubation at 42°C for 20 hr, each culture was streaked onto
xylose-lysine-deoxycholate agar (Oxoid) and CHROMagarTM Salmonella (CHROMagar, Paris, France) selection plates, followed by incubation at 37°C for 24 hr. Two suspect colonies were
biochemically identified, as previously described [21 (link)]. Salmonella isolates were tested by slide agglutination with O antisera (Denka
Co., Tokyo, Japan, and SSI Diagnostica, Copenhagen, Denmark). One of each different O serogroup isolate per flock was tested for flagella antigens by tube agglutination using H antisera
(Denka Co.). Serovars were determined based on the reaction between O and H group antigens according to the Kauffmann–White scheme [9 ]. One of each
different serovar isolate per flock was subjected to antimicrobial susceptibility testing.
3
Bacterial and Fungal Propagation Protocols
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The bacterial isolates were propagated after Soliman et al. (2021b , 2021c ). Escherichia coli O157: H7 suspension (2.0 × 104 CFU.ml-1) was propagated into Mac-Conkey broth (Oxoid™) at 44°C/24 hours, eosin methylene blue agar (OxoidTM) at 37°C/24 hours, and tryptone soya broth (Oxoid™) providing 1.7 × 1011 CFU. ml-1 suspension. Lyophilized S. Typhimurium (3.4 × 102 CFU) was propagated into Rappaport-Vassiliadis broth (Oxoid™) at 37°C/24 hours, xylose lysine deoxycholate agar (Oxoid™) at 37°C/24 hours, and tryptone soya broth providing 1.5 × 106 CFU.ml-1 suspension.
The fungal isolates were propagated following Soliman et al. (2021b) . Aspergillus niger and C. albicans clinical isolates were propagated into sabouraud dextrose broth (SDB; HIMEDIA®) at 37°C/24–72 hours, sabouraud dextrose agar (SDA; Oxoid™) at 37°C/24 hours, identified with lactophenol cotton blue stain (Hardy Diagnostics®), and resuspended in SDB broth, providing 2.5 × 108 CFU.ml-1 suspensions.
The fungal isolates were propagated following Soliman et al. (2021b) . Aspergillus niger and C. albicans clinical isolates were propagated into sabouraud dextrose broth (SDB; HIMEDIA®) at 37°C/24–72 hours, sabouraud dextrose agar (SDA; Oxoid™) at 37°C/24 hours, identified with lactophenol cotton blue stain (Hardy Diagnostics®), and resuspended in SDB broth, providing 2.5 × 108 CFU.ml-1 suspensions.
4
Isolation and Identification of Salmonella from Various Samples
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Isolation of the microorganism was conducted based on the protocol recommended by the World Organization for Animal Health Terrestrial Manual (Elbediwi et al., 2020b (link), Elbediwi et al., 2021 (link)). Briefly, human (feces, blood, and urine), animal (feces), food or environmental samples were subjected into 10 mL pre-enrichment in buffered peptone water (Oxoid, United Kingdom). Following the initial pre-enrichment in buffered peptone water, 0.1 mL of the pre-enriched samples were added to 10 mL of Rappaport Vassiliadis broth (Oxoid, United Kingdom) and incubated at 42°C for 24 h. The enriched samples were streaked onto Xylose Lysine Desoxycholate (XLD) (Oxoid, United Kingdom). Plates were then incubated at 37°C for 18–24 h. Spherical transparent red or pink colonies with or without typical black centers on XLD, were selected as presumptive Salmonella colonies. The bacterial isolates were then confirmed using polymerase chain reaction (PCR). DNA extraction was done by boiling method. PCR for enterotoxin stn gene for the confirmation of the Salmonella was performed as recommended (Deguenon et al., 2019 (link)).
5
Salmonella Isolation Protocol
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The samples were inoculated in 40 ml of nonselective tryptic soy broth (TSB) (Oxoid Ltd., Basingstoke Hampshire, UK) and incubated for two hours at 37°C with shaking at 100 rpm. Thereafter, 0.1 ml was inoculated into 9.9 ml of Rappaport-Vassiliadis broth (Oxoid Ltd., Basingstoke, UK), a selective enrichment media for Salmonella and incubated at 42°C for 24 hours [17 (link)]. A loopful of the sample was streaked onto Brilliance Salmonella Agar (Oxoid Ltd., Basingstoke Hampshire, England) and incubated at 37°C for 24 hours [18 (link)]. Single presumptive purple/pink colonies typical of Salmonella were picked and subsequently streaked onto Hektoen Enteric Agar (HEA) (Oxoid Ltd., Basingstoke Hampshire, England) and Salmonella Shigella agar (SSA) (Oxoid Ltd., Basingstoke Hampshire, England) plates and incubated at 37°C for 24 hours. Culture plates were examined for the presence of typical colonies based on morphological characteristics, i.e., clear colonies with a black centre on HEA and SSA. Single colonies were subcultured onto nutrient agar and subsequently stored in TSB supplemented with 10% glycerol (Merck, USA) at −60°C until further analysis. Salmonella enterica subsp. enterica serovar Choleraesuis ATCC 10708 (ATCC, Manassas, Virginia, USA) was included as a control strain.
6
Isolation and Detection of Foodborne Pathogens from Eggshells
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The surface of each eggshell was swabbed with a sterile cotton stick. The swabbed cotton sticks were vortexed and suspended in 0.1% (w/v) peptone water. E.coli and Salmonella species were isolated using the standard protocols of the food code established by the Ministry of Food and Drug Safety (MFDS) in Korea. Briefly, EC broth (Oxoid) and MacConkey agar (Oxoid) were used to isolate E. coli. Rappaport Vassiliadis broth (Oxoid) and XLD agar were used to isolate Salmonella species.
Arcobacter species were isolated as described by Lee et al. (2010) (link). Each sample was mixed with Arcobacter-selective broth (Oxoid) including a cefoperazone, amphotericin, and teicoplanin (CAT)-selective supplement (Oxoid) at a 1:10 ratio and incubated in microaerobic conditions at 37℃ for 48 h. The enriched broth was inoculated and incubated in Arcobacter-selective agar (Oxoid) at 37℃ for 48 h. To detect Arcobacter species, species-specific multiplex polymerase chain reaction (PCR) was performed with boiled enriched broth, as described previously (Lee et al., 2010 (link)).
Arcobacter species were isolated as described by Lee et al. (2010) (link). Each sample was mixed with Arcobacter-selective broth (Oxoid) including a cefoperazone, amphotericin, and teicoplanin (CAT)-selective supplement (Oxoid) at a 1:10 ratio and incubated in microaerobic conditions at 37℃ for 48 h. The enriched broth was inoculated and incubated in Arcobacter-selective agar (Oxoid) at 37℃ for 48 h. To detect Arcobacter species, species-specific multiplex polymerase chain reaction (PCR) was performed with boiled enriched broth, as described previously (Lee et al., 2010 (link)).
7
Salmonella Detection Enrichment Protocol
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For detection of Salmonella spp., 25 g of each sample were enriched first at a 1:10 ratio in buffered peptone water (Oxoid; 24 h, 37°C) and subsequently in 10 ml of Rappaport-Vassiliadis broth (Oxoid; 24 h, 41.5°C). The enriched samples were subcultured on xyloselysine-desoxycholate agar (Bio-Rad; 24 h, 37°C) and mannitol lysine crystal violet brilliant green agar (Oxoid; 24 h, 37°C).
8
Isolation and Identification of Salmonella
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Isolation of Salmonella was carried out according to the International Organization for Standardization standard 6579 guidelines20 for isolation and characterisation of Salmonella species. Briefly, pre-enrichment was conducted by transferring each swab into 10 mL tubes containing buffered peptone water (Oxoid, Basingstoke, United Kingdom). Each tube was then thoroughly homogenised by vortexing and incubated at 37 °C for 18–20 h. Subsequently, 0.1 mL of the pre-enrichment broth culture was inoculated into 10 mL of Rappaport Vassiliadis broth (Oxoid, Basingstoke, United Kingdom) for the selective enrichment of Salmonella and incubated at 42 °C for 24 h. A loopful from the overnight enrichment broth was then inoculated onto xylose lysine deoxycholate agar (Oxoid, Basingstoke, United Kingdom) and brilliant green agar (Oxoid, Basingstoke, United Kingdom), and the plates were incubated at 37 °C for 24 h. To obtain pure colonies, the presumptive Salmonella colonies that appeared red with black centres on xylose lysine deoxycholate agar and pinkish-white or red surrounded by a red halo on brilliant green agar were subcultured on xylose lysine deoxycholate agar plates and incubated at 37 °C for 24 h. The pure cultures were subsequently inoculated on nutrient agar slants, incubated at 37 °C for 24 h and stored in the fridge at 4 °C.
9
Salmonella spp. Isolation from Fecal Samples
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Salmonella spp. isolation was executed from each fecal sample following the procedures previously described [21 (link)]. Briefly, about 3 gr of feces was incubated in 10 mL of buffered peptone water at 37 °C for 24 h. One ml of this culture was transferred into 10 mL of Selenite Cystine Broth (Oxoid Ltd., Basingstoke, UK) and 1 mL into 10 mL of Rappaport Vassiliadis Broth (Oxoid Ltd., Basingstoke, UK). The tubes were incubated at 37 °C for 24 h and at 42 °C for 24 h, respectively. One loopful from each broth culture was streaked onto Salmonella-Shigella Agar (Oxoid Ltd., Basingstoke, UK) and Brilliant Green Agar (Oxoid Ltd., Basingstoke, UK) plates. After incubation of the plates at 37 °C for 24 h, suspected colonies were submitted to biochemical characterization.
10
Salmonella Isolation and Identification Protocol
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The procedures of Salmonella isolation and identification were performed, according to standard methods (ISO6579:2002; International Organization for Standardization 2002). Briefly, 25 g from each chicken meat sample were cut into small pieces by sterile scissors. Then add to a stomach bag containing 225 mL sterile Buffered Peptone Water (BPW) (Oxoid, Hampshire, England). For the stool samples, 1 g was added to 9 ml BPW, then all the samples were incubated at 37°C for 18 h. 0.1 mL of the pre-enriched broth was transferred to 10 mL of Rappaport-Vassiliadis broth (Oxoid, Hampshire, England) and incubated at 42°C for 24 h. A loopful of the enriched broth was then streaked on xylose lysine desoxycholate agar and incubated for 24 h at 37°C. Biochemical examination of the suspected Salmonella colonies was performed, according to the (ISO6579:2002; International Organization for Standardization 2002) guidelines [14 ].
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