Buffered peptone water (bpw)

In the course of microbiological testing, 10 g of each sample was blended with 90 mL of Buffered Peptone Water (Oxoid, Basingstoke, UK) for two minutes (BagMixer^® 400 P, Interscience, Saint Nom la Brétèche, France). This initial mixture was labeled 10⁻¹, and a 10⁻² dilution was subsequently prepared using 0.1% peptone water. From both dilutions (10⁻¹ and 10⁻²), 100 µL was plated onto Slanetz–Bartley agar (Oxoid, UK) and incubated for 36–48 h at 41.5 °C [10 (link)]. Following incubation, as many as five colonies per plate that showed typical Enterococcus characteristics were chosen for species confirmation using the Autobio ms1000 MALDI-TOF system (Autobio Diagnostics, Zhengzhou, China). According to the manufacturer’s protocols, identification scores falling above 9.000 were regarded as reliable for species-level classification.

A total number of 285 chicken meat swab samples were gathered from various retail outlets in the Sylhet district. In order to preserve the proper temperature during transportation, swab samples from chicken meat were aseptically collected in disinfected containers containing buffered peptone water (BPW; Oxoid, UK) and brought to the lab in a refrigerated box. At first each sample was inoculated into BPW and underwent incubation at 37 °C for 24 h as pre-enrichment. The increase of turbidity in the BPW media was a sign of bacterial proliferation. The enriched samples were cultured on Eosin Methylene Blue (EMB; Oxoid, UK) agar media following pre-enrichment, and then they were proceeded with aerobic incubation for 18–24 h at 37 °C. After sub culture into MacConkey agar (Oxoid, UK) plates, the suspected colony underwent incubation for 24 h at 37 °C additionally. The initial screening for probable Klebsiella spp. was made easier by the characteristic colony morphology, notably the pink colonies that were indicative of lactose fermentation. Biochemical assays were performed after selection to verify the isolates' identities. The isolates' capacity to ferment lactose was confirmed by the lactose fermentation test conducted on MacConkey agar. Additionally, the ability to hydrolyze urea was also evaluated using a urease test, which showed a pinkening of the medium. The isolates' ability to use citrate as the exclusive carbon source was tested by the citrate utilization test, which again showed a color shift. The methyl red-Voges Proskauer (MR-VP) test helped characterize the isolates by distinguishing between 2,3-butanediol fermentation and mixed acid fermentation, while the indole test assessed tryptophan production, as shown by a color shift with the inclusion of Kovac's reagent. Finally, a motility test was used to assess whether the isolates were motile or not, which is a crucial trait for Klebsiella spp.. All tests were conducted following standard microbiological protocols and the guidelines followed by Liza et al. [3 (link)]. Lastly, PCR was performed for final confirmation on the isolates of Klebsiella spp. that were tentatively positive.

Livers of ill or dead chickens were collected, and approximately 1 cm³ of liver tissue samples were cut in a sterile, ultra-clean room. Samples were enriched in buffered peptone water (Oxoid, Manchester, UK) and incubated at 37 °C for 24 h. The enriched bacterial cultures were streaked on eosin methylene blue (EMB) agar (Oxoid Company, Manchester, UK) using a sterile wire loop and incubated for 24 h at 37 °C. Colonies exhibiting a green metallic sheen were presumptively identified as E. coli. E. coli MG1655 was used as a positive control strain.
Genomic DNA was extracted using the boiling method [14 (link)]. Briefly, 1.5 mL of suspended plaque samples were centrifuged for 15 min (13,523× g, 4 °C). The supernatant was discarded, and the pellet was resuspended in 1 mL of PBS. This washing step was repeated three times. The resuspended samples were incubated at 99 °C for 15 min. After centrifugation (15 min, 13,523× g, 4 °C), the genomic DNA supernatant was immediately cooled to −20 °C for storage.
The genomic DNA supernatant was used as a DNA template. DNA extracted from isolated E. coli strains was amplified using species-specific primers for phoA (F-CGATTCTGGAAATGGCAAAAG, R-CGTGATCAGCGGTGACTATGAC) and E. coli-specific primers for PCR [3 (link)]. DNA from E. coli MG1655 served as the positive control, and PCR reactions without a DNA template served as the negative control. PCR products were analyzed by electrophoresis on 1% agarose gels, with gel images captured and analyzed using the GelDoc© Gel Documentation System (Bio-Rad, Hercules, CA, USA).

In each experiment, 30 chicken carcasses prior to inside outside wash step were collected from a processing plant in large sterile bags. Chickens were weighed and the deep muscle breast temperature was recorded within 20 min of arrival in the laboratory. Individual carcasses were subsequently placed in a whirl pack bag (ThermoFisher Scientific, Scoresby, Australia) and washed with massaging for 2 min in 200 mL buffered peptone water (BPW; ThermoScientific, Oxoid, Scoresby, Australia). Two mL of the BPW wash were spread plated onto five modified charcoal-cefoperazone-deoxycholate agar (mCCDA) (ThermoScientific, Scoresby, Australia) plates (400 µL per plate) and incubated at 42 °C with 10% CO₂ for 48 hours to assess direct Campylobacter spp. counts. From the initial 200 mL BPW wash, 40 mL was incubated overnight at 37 °C. Then, 100 µL of the incubated BPW was transferred into 10 mL Rappaport Vassiliadis soya peptone broth (RVS, ThermoScientific, Oxoid, Scoresby, Australia) and incubated overnight at 42 °C for selective growth of Salmonella enterica serovars. A loop-full of the RVS broth was streaked on to xylose lysine deoxycholate agar (XLD; ThermoScientific, Oxoid, Australia) plates. Suspected Salmonella colonies were subcultured onto Brilliance Salmonella agar (ThermoScientific Oxoid, Scoresby Australia) for confirmation. To quantify the total viable counts (TVC) in the carcass wash, the carcass wash was diluted 10-fold and plated on nutrient agar. The limit of detection for TVC was 0.25 colony forming units (CFU)/cm² of chicken carcass. The plates were incubated overnight at 37 °C and colonies were recorded in colony forming units (CFU). For Campylobacter spp. the limit of detection was 10 CFU/mL of rinsate.
Following the initial BPW wash, 5 carcasses each were placed into six different treatment groups, water and sanitizer wash each at 5 °C, 15 °C, and 22 °C (Table 1). Carcasses were placed into large containers filled with diluted sanitizer and agitated continuously for the entire treatment period (Table 1). Carcasses were then removed and placed in a sterile bag and rinsed as with BPW. The miniaturized Most Probable Number (MPN) method described by [20 (link)], was used to determine the Salmonella load in culture-positive samples. Campylobacter and Salmonella counts as well as the TVC were interpreted per square centimeter of carcass as per the Australian standard [21 ]. Briefly, surface area of a whole chicken carcass in square centimeters was calculated by the following formula, 0.87 m + 635 (m = total mass in grams) and the microorganisms per square centimeter of surface area from the rinse fluid was calculated using the following formula,

Chicken meats of different brands and products were purchased from seven different retail stores in Edmonton, Canada. C. jejuni was isolated as described by Chon et al. (2012) (link) with some modifications. Briefly, the chicken meats were submerged in buffered peptone water (Oxoid, UK) at 37°C overnight and then inoculated in Bolton Campylobacter selective broth (Oxoid) at 42°C for 24 h under microaerobic conditions (5% O₂, 10% CO₂, 85% N₂). Aliquots (100 μl) were serially diluted and spread on Mueller-Hinton (MH) agar plates supplemented with Preston Campylobacter selective supplements (Oxoid). Cultures were incubated at 42°C for 48 h under microaerobic conditions. C. jejuni colonies were confirmed by multiplex PCR as described previously (Wang et al., 2002 (link)). The primer sequences were described in Table 1. All C. jejuni strains were grown on MH media at 42°C under microaerobic conditions. Occasionally, culture media were supplemented with kanamycin (50 μg/ml).

For each sample, minced beef meat was weighed, and 25 g were aseptically placed in a sterile stomacher bag (Fisher Scientific, New Hampshire, USA). The sample was then diluted 1:10 with 225 mL of sterile buffered peptone water (BPW) (Oxoid, Hampshire, UK) and homogenized for 1 min using a stomacher (Thomas Scientific, New Jersey, USA). The suspension was then serially diluted (10-fold) in BPW and 3 dilutions (10⁻¹, 10⁻², 10⁻⁴) were plated on RAPID’E. coli 2 agar plates (BioRad, Hercules, California, USA) in duplicates. The plates were incubated at 44 °C for 18–24 h under aerobic conditions. Colony forming units (CFU) that matched the diagnostic phenotypes (violet to pink E. coli colonies and blue to green colonies for other fecal coliforms) were counted and bacteria densities were determined by averaging the counts from the duplicates. Data were reported as averages of CFU per gram of raw minced beef. BPW without meat was used as control throughout the experiment. E. coli DH5α was used to test the quality of the RAPID’E. coli 2 agar plates. Average CFU counts of E. coli and fecal coliforms were compared using the Student’s t-test. A p < 0.05 was used to identify statistically significant differences.