Isolation of Salmonella from cloacal swabs, poultry drinking water, and feeds was carried out according to the protocols and recommendations of the OIE terrestrial manual (www.oie.int ) and ISO 6579:2002. Briefly, for isolation from cloacal swabs, the swab sticks immersed in 10 mL BPW (Oxoid) were incubated at 37°C for 18 to 24 h. For isolation from flies, trapped flies were processed as previously described (Choo et al., 2011 (link)), immersed in 10 mL BPW (Oxoid), then incubated at 37°C for 18 to 24 h. For isolation from drinking water, 10 mL of the water collected from the farms were each added to 90 mL of BPW and subsequently incubated at 37°C for 18 to 24 h. Similarly, the feeds collected from the farms were first weighed to 10 g using weighing balance, after which the 10 g of feed was inoculated into 90 mL of BPW (Oxoid) and incubated at 37°C for 18 to 24 h. All samples were further processed for Salmonella identification as described below.
Buffered peptone water (bpw)
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, Australia, Italy, Spain, Germany, Canada, Belgium
Buffered peptone water is a general-purpose microbiological culture medium used for the enrichment and recovery of a wide range of microorganisms. It provides a buffered environment and peptone as a source of nutrients to support the growth of microbes.
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Lab products found in correlation
Macconkey agar, by Thermo Fisher Scientific (10 mentions)
Rappaport vassiliadis broth, by Thermo Fisher Scientific (7 mentions)
Brilliant green agar, by Thermo Fisher Scientific (7 mentions)
Nutrient agar, by Thermo Fisher Scientific (6 mentions)
Macconkey agar plate, by Thermo Fisher Scientific (5 mentions)
Xylose lysine deoxycholate agar, by Thermo Fisher Scientific (5 mentions)
Eosin methylene blue agar, by Thermo Fisher Scientific (5 mentions)
Xylose lysine deoxycholate (xld), by Thermo Fisher Scientific (5 mentions)
Xylose lysine deoxycholate xld, by Thermo Fisher Scientific (5 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (4 mentions)
Xylose lysine desoxycholate agar, by Thermo Fisher Scientific (4 mentions)
Xld agar, by Thermo Fisher Scientific (3 mentions)
Rappaport vassiliadis medium, by Thermo Fisher Scientific (3 mentions)
Nutrient broth, by Thermo Fisher Scientific (3 mentions)
Plate count agar, by Thermo Fisher Scientific (3 mentions)
Api 20e system, by bioMérieux (3 mentions)
Glycerol, by Merck Group (3 mentions)
Potato dextrose agar (pda), by Thermo Fisher Scientific (3 mentions)
Nutrient agar na, by Thermo Fisher Scientific (3 mentions)
Modified semisolid rappaport vassiliadis agar plates, by Thermo Fisher Scientific (3 mentions)
203 protocols using buffered peptone water (bpw)
1
Isolation of Salmonella from Poultry Samples
2
Salmonella Isolation from Food Samples
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Pre-enrichment of the samples was conducted in buffered peptone water at 1% (w/v) (BPW, Oxoid Ltd., Basingstoke, UK), which was 25 g of solid sample added to 225 mL of BPW, sponges were added to 90 mL of BPW, and 200 mL of the water samples were centrifuged at 3500× g for 15 min and the pellet was added to 100 mL of BPW incubated at 35 °C for 18 to 24 h. Salmonella isolation was conducted according to the protocol described by USDA [39 ]. Colonies with typical Salmonella characteristics on triple sugar agar and lysine iron agar (Oxoid) were subjected to a serological test by using polyvalent somatic and flagellar antisera (Probac do Brasil, São Paulo, SP, Brazil), and further biochemical characterization by the following tests: urease, indole, methyl red, Voges-Proskauer, citrate, motility, and malonate [39 ]. Isolates identified as Salmonella spp. (n = 210) were serotyped by slide agglutination (Kauffmann–White–Le Minor scheme) using diverse somatic and flagellar antisera in the Bacteriology Section of the Instituto Adolfo Lutz (São Paulo, SP, Brazil) and Enterobacteriaceae Laboratory from the Fundação Oswaldo Cruz (Rio de Janeiro, RJ, Brazil). Isolates were kept stored in trypticase soya broth (TSB, Oxoid) added to glycerol at 10% at −20 °C.
3
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)).
4
Salmonella Quantification in Feces and Organs
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Fecal shedding and organs colonization of STMwt and STMΔznuABC were determined according to the ISO 6579: 2002/Amendment 1:2007 protocol. Briefly, samples were weighed and homogenized in nine parts of Buffered Peptone Water (BPW) (Oxoid Ltd., UK). This initial solution was then used to perform a decimal dilution series carried out by systematically transferring an aliquot of 0.5 ml of each successive dilution in 4.5 ml of BPW. All BPW-diluted samples were incubated at 37°C for 18 ± 3 h. 0.1 ml of cultures were subsequently seeded on Modified Semisolid Rappaport-Vassiliadis (MSRV) agar plates (Oxoid Ltd., UK) and incubated at 41.5°C for 24 h for the selective enrichment of Salmonella. A loopful of growth from each MSRV plate was streaked onto Xylose-Lysine-Desoxycholate Agar (Oxoid Ltd., UK) and Brilliant Green Agar (Oxoid Ltd., UK) plates and hence incubated at 37°C overnight. Suspect Salmonella colonies were subjected to biochemical identification by the BBL Enterotube II (BD Franklin Lakes, USA) and serological identification using Salmonella group-specific antisera (Remel, Lenexa, USA). This is a semi-quantitative approach that allows the quantification of Salmonella in a sample within a tenfold band (detection limit 1 CFU/g feces).
5
Microbiological Analysis of Fish Steaks
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A portion (25 g) of each aseptically removed fish steak (from the upper left side of the fish steak) was placed in 225 mL of sterile, buffered peptone water (Oxoid, Basingstoke, UK), and homogenised in a stomacher apparatus (Stomacher 400 circulator; Seward Ltd, Norfolk, UK) at room temperature for 2 min. Then, buffered peptone water was used to prepare serial decimal dilutions of the homogenates, which were analysed for total viable count (TVC), determined using Plate Count Agar (Oxoid) incubated at 30°C for 48 h (UNI EN ISO 4833:2004; ISO, 2004b ), and Enterobacteriaceae (ISO 21528-2:2004; ISO, 2004a ). After counting, the mean and standard deviation were calculated and data were reported as Log colony forming units per gram of sample (CFU/g).
6
Salmonella Isolation Protocol for Meat and Dairy
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Salmonella isolation was carried out by a standard cultivation method as recommended by ISO 6579-1 [46 ]. Samples (25 g meat or a swab pre-moistened with 25 mL buffered peptone water, Oxoid, UK) were inserted in stomacher bags containing buffered peptone water (225 mL). The homogenization was carried out at 320 rpm for 2 min, followed by incubation at 37 °C overnight. Then, 0.1 mL aliquots were inoculated into tubes containing 10 mL Rappaport Vassiliadis (RV) broth (Oxoid, UK) and then, incubated at 42 °C for 48 h. Then, XLD (xylose lysine deoxycholate) agar (Oxoid, UK) plates were inoculated from each of the RV broths and incubated at 37 °C for 18–24 h. Suspect colonies of Salmonella were biochemically confirmed using the API 20E system (bioMérieux, Marcy-l’Étoile, France). Then, Salmonella isolates were serotyped by using specific Salmonella O and H agglutinating antisera (Difco, Sparks, MD, USA) according to the Kauffman–White serotyping scheme [47 ]. Of note, local S. enterica strains isolated from retail meat and dairy products in Egypt were used as controls for the experiments [17 (link)].
7
Enumeration of Salmonella in Hen Feces
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Fecal samples were aseptically collected from individual hens in Whirl- Pack plastic bags (Thermo Fisher Scientific, Australia) on days 0, 1, 3, 6, 9, and 12 followed by weeks 3, 5, 7, 9, 11, 13, and 15 p.i.
Fecal samples were processed for enumeration of Salmonella by three tube most probable number (MPN) method (Santos et al., 2005 (link); Pavic et al., 2010 (link)). Briefly, 10 g of fecal sample were weighed in sterile Whirl-Pack plastic bag (Thermo scientific, Australia) followed by the addition of 90 ml Buffered peptone water (BPW, (Oxoid, Australia) (1:10); bags were then homogenized for 1 min. From this bag 10 ml of homogenate was placed into three different sterile tubes (100 dilution). Then 1 ml of homogenate sample was transferred to three different tubes containing 9 ml of BPW, and then serially diluted in triplicate tubes of BPW. The tubes were incubated overnight at 37°C. After incubation, 10 μl of BPW from each MPN tube was plated on modified semisolid Rappaport–Vassiliadis (MSRV, Oxoid, Australia) agar plates and incubated at 42°C for 24 h. A loopful of media from the leading edge of white zones from MRSV plate was streaked onto XLD and or Salmonella Brilliance agar plates (Oxoid, Australia) for confirmation of Salmonella.
Fecal samples were processed for enumeration of Salmonella by three tube most probable number (MPN) method (Santos et al., 2005 (link); Pavic et al., 2010 (link)). Briefly, 10 g of fecal sample were weighed in sterile Whirl-Pack plastic bag (Thermo scientific, Australia) followed by the addition of 90 ml Buffered peptone water (BPW, (Oxoid, Australia) (1:10); bags were then homogenized for 1 min. From this bag 10 ml of homogenate was placed into three different sterile tubes (100 dilution). Then 1 ml of homogenate sample was transferred to three different tubes containing 9 ml of BPW, and then serially diluted in triplicate tubes of BPW. The tubes were incubated overnight at 37°C. After incubation, 10 μl of BPW from each MPN tube was plated on modified semisolid Rappaport–Vassiliadis (MSRV, Oxoid, Australia) agar plates and incubated at 42°C for 24 h. A loopful of media from the leading edge of white zones from MRSV plate was streaked onto XLD and or Salmonella Brilliance agar plates (Oxoid, Australia) for confirmation of Salmonella.
8
Isolation and Identification of Foodborne Pathogens
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Twenty-five grams of each raw beef or chicken meat was transferred to a blender bag and homogenized with 225 mL of 0.1% buffered peptone water (BPW; Oxoid, Wesel, Germany). Pre-enrichment was done for 24 h at 37°C. A loop full of the enriched broth was streaked on MacConkey agar plates (Oxoid) and Eosin Methylene Blue Lactose plates (Oxoid) and incubated at 37°C for 24 h. For human samples, hand swabs and stool specimens were directly inserted into sterile tubes containing 10 mL BPW under aseptic conditions and incubated at 37°C for 24 h. Then, the samples were cultivated as described previously.
9
Isolation and characterization of Campylobacter from chicken
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The Campylobacter isolates used in this study were isolated during a separate study investigating the efficacy of sanitizers during chicken meat processing (Chousalkar et al., 2019 (link)). Briefly, chicken meat carcasses (15 birds from each of eight different broiler production sheds) were collected from two separate processing plants prior to the inside-outside wash step. Bacteria were isolated by massaging chicken carcasses (prior to sanitizer exposure) in buffered peptone water (BPW) (Oxoid, Australia). Two hundred microliters of BPW wash was spread plated onto modified charcoal-cefoperazone deoxycholate agar (mCCDA) (Oxoid, Australia) and incubated at 42°C in 10% CO2 for 48 h. Putative, Campylobacter isolates were sub-cultured once to obtain pure cultures, stored at −80°C in 5% glycerol, and further characterized using PCR. A total of 116 Campylobacter isolates were obtained.
In preparation for experiments, bacteria were resuscitated from freezing stocks on to Columbia sheep blood agar (SBA) (Oxoid, Thermo Scientific, Australia) and incubated at 42°C in 10% CO2 for 48 h. The Campylobacter jejuni ATCC 33291 strain was used as a control strain.
In preparation for experiments, bacteria were resuscitated from freezing stocks on to Columbia sheep blood agar (SBA) (Oxoid, Thermo Scientific, Australia) and incubated at 42°C in 10% CO2 for 48 h. The Campylobacter jejuni ATCC 33291 strain was used as a control strain.
10
Isolation of Cronobacter spp. from Dry Powdered Products
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The procedure adopted for the isolation of Cronobacter spp. was based on Iversen (2007) [24 (link)] and the original Enterobacter sakazakii technical specifications (ISO/TS 22964, 2006). Briefly, a 100 g DRP sample was homogenized with 900 mL buffered peptone water (BPW; OXOID, Hampshire, UK) and incubated at 37°C for 18 hr as a pre-enrichment step. Subsequently, 1 mL of the BPW suspension was transferred to 10 mL modified lauryl sulfate tryptose broth (OXOID, Hampshire, UK), and after further incubation at 42°C for 24 hr, the broth was streaked on Druggan Forsythe Iversen (DFI, Oxoid, Hampshire, UK) agar and incubated at 37°C for 24 hr. Greenish/blue colonies on DFI agar were considered presumptive Cronobacter spp. The suspected isolates were subjected to the biochemical galleries by VITEK 2 compact GN (bioMèrieix, France) for identification of Cronobacter, according to the manufacturer's instructions. Two reference strains Cronobacter muytjensii ATCC51329 and Cronobacter sakazakii ATCC 25944 purchased from American Type Culture Collection. Supplemental biochemical differentiation tests were performed according to Farmer (1980) and Iversen (2006) [25 –26 (link)].
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