Macconkey

From January 2019 to September 2021, the isolation and identification of bacterial pathogens in 526 samples from 85 pig farms submitted from various cities in Zhejiang and parts of Anhui were performed. Antibiotic susceptibility paper sheets were purchased from Changde Bikman Biotechnology Co., Ltd. (Hunan, China) (Table 1).
Green Tap Mix enzyme was purchased from Nanjing Novazan Biotechnology Co., Ltd. (Nanjing, China). DL 2000 DNA Marker was purchased from Takara Biomedical Technology (Beijing, China) Co., Ltd. MacConkey, Tryptic Soy Agar (TSA), and Tryptic Soy Broth (TSB) media were purchased from Thermo Fisher Scientific. Calf serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (Huzhou, China). Nicotinamide adenine dinucleotide (NAD) was purchased from Beijing Soleibo Technology Co., Ltd. (Beijing, China).

A 10–15 mL volume of blood was obtained from each rat via puncture in the vena cava inferior. The MLNs of the ileocaecal area were aseptically isolated. After the isolates were ground, 100 μL of homogenized MLNs were cultured on MacConkey (Thermo Fisher Scientific, Waltham, MA), Mueller–Hinton (Thermo Fisher Scientific), and whole blood agar (Bio Merieux, Lyon, France) for 48 hours at 37 °C. BT was defined as the presence of viable organisms in the MLN culture2 (link)29 (link)30 (link). To determine whether bacteraemia was present, 3 mL of blood was drawn from the inferior vena cava and inoculated into aerobic and anaerobic Bactec culture bottles. The cultures were incubated at 35 °C, and the growth value (a measurement of CO₂ production by the bacteria) was continuously monitored for at least 7 days4 (link). For BT monitoring, 10 cirrhotic rats and four control rats were lavaged with 10⁸ RFP-marked E. coli. The small intestine, colon, heart, lung, spleen, MLNs, kidneys, and liver were collected at 2 or 6 hours after lavage. The organs were rinsed in ice-cold PBS twice, and the RFP signal was visualised using a Clairvivo OPT Plus fluorescence microscope (Shimadzu Corporation, Kyoto, Japan) at a wavelength of 583 nm.

Bacterial translocation (BT) is generally considered as the presence of viable organisms in the MLN culture (Teltschik et al. 2012 (link)). Specifically, the MLNs were separated aseptically from the ileocecal zone. After grinding the separation, the homogenized MLNs (100 μL) were put in the MacConkey (Thermo Fisher Scientific), Mueller–Hinton (Thermo Fisher Scientific), and whole blood agar (Bio Merieux, Lyon, France) at 37 °C for approximately 2 days. Ultimately, the number of bacteria per gram was quantified using BT (CFU/g).

The fecal samples from humans and chicken manure were grown directly on Campylobacter Selective Agar (Oxoid, UK) for Campylobacter selection and Xylose-Lysin-Desoxycholat Agar (Oxoid, UK), MacConkey (Oxoid, UK), and Sheep Blood Agar Base (Oxoid, UK) for Salmonella selection. Agar plates were incubated at 37°C and checked for the presence of colonies after 24–48 hours. The chicken meat samples were pre-processed according to the ISO 10272-1:2017 guidelines for Campylobacter and ISO 6579-1:2017 guidelines for Salmonella. From all samples, suspected colonies for each of the species were selected for microbiological confirmation by the Vitek 2 NH ID card for Campylobacter or Vitek 2 GN ID card for Salmonella using the Vitek 2 compact automated system (BioMerieux, France). All isolates which were confirmed as Salmonella or Campylobacter by the Vitek 2 microbial testing system were subjected to further confirmation by PCR. Molecular species identification for Campylobacter spp. was performed using the PCR protocol published by Nayak et al. (29 (link)), while for Salmonella spp., the procedure published by Paião et al. was used (30 (link)).

Bacteriological milk cultures were performed at the University of Milan following published procedures recognized by the NMC (2017). From each sample, 10 μL of milk were spread on blood agar plates (5% defibrinated sheep blood). Plates were incubated aerobically at 37 °C and examined after 24 and 48 h. Colonies were provisionally identified based on size, Gram stain, morphology, and hemolysis pattern. Representative colonies were then subcultured on blood agar plates and incubated again at 37 °C for 24 h to obtain pure cultures. Catalase-negative Gram-positive cocci were identified as Streptococci and species were differentiated by further biochemical tests (growth in 6.5% NaCl broth, esculin hydrolysis, fermentation of ribose, sorbitol, sucrose, and inulin). Coagulase tube test was used to differentiate catalase-positive gram-positive cocci as S. aureus or NAS. Gram-negative isolates were identified using colony morphology, Gram-staining characteristics, oxidase test, indol test, and inoculation in Simmons citrate (Laboratorios Conda, Madrid, Spain), motility indol ornithine, and biochemical reactions on MacConkey (Oxoid, Basingstoke, UK). Microorganisms other than bacteria were confirmed by microscopic appearance. Samples where three or more pathogens grew were considered contaminated.

All samples (358 nasal swab, 408 fecal swab, and 227 milk samples) collected from cattle, buffaloes, sheep, and goats with clinical signs including respiratory manifestations, pneumonia, and diarrhea were placed on peptone water broth (Oxoid, UK) as pre-enrichment for bacterial growth, in accordance with the methods described previously [19 (link)]. A loopful of culture inoculate was streaked on MacConkey (Oxoid) agar. Pink colonies obtained from MacConkey agar that was indole-positive were considered positive for E. coli and maintained at −20°C in 20% glycerol brain heart infusion broth for further confirmation and characterization by polymerase chain reaction (PCR) and antibiotic susceptibility testing, in accordance with a previous study [20 ].