Blood agar base

In accordance with Clinical Laboratory Standards Institute guidelines (CLSI) (10), clinical specimens were inoculated onto the blood agar base (Oxoid, Basingstoke, Hampshire, England) to which 5% sheep blood was added and mannitol salt agar (Oxoid, Basingstoke, Hampshire, England) by using the streaking method. Inoculated plates were incubated at 35–37°C for 18 to 24 hrs aerobically. Bacterial colonies showing typical characteristics of S. aureus (i.e., beta-hemolytic on blood agar and colonies with golden yellow pigmentation on mannitol salt agar) were subjected to subculture onto basic media, Gram stain, catalase, coagulase, and Pastorex staph extract. These catalase, coagulase, Pastorex, and Gram-positive bacteria appearing in grape-like clusters were considered as S. aureus [8 (link), 10 ].

The three isolates of C. jejuni used in the study were 48 (Penner serotype O:19), 75 (serotype O:3), and 111 (serotype O: in 1,44) which originated from the stools of diarrheic patients as stated above. They were found to colonize mouse intestine in previous studies [11 (link), 22 (link)]. Stock cultures were maintained in Brucella broth (Becton & Dickinson, Sparks, MD) with 15% (vol/vol) glycerol at -70°C. Depending upon the purpose, three types of media were used for cultivation. These were: blood agar base (Oxoid, Basingstoke, Hampshire, England) with 5% defibrinated sheep blood; Campylobacter-selective agar with laked horse blood, growth supplement and selective supplement (Oxoid); and a biphasic medium with brain heart infusion agar and broth supplemented with 1% yeast extract [23 (link)]. Cultures were incubated at 42°C for 48 h in a microaerobic atmosphere generated by Campigen (Oxoid). The identity of the bacteria was confirmed by cultural characteristics and molecular methods. The three serotypes generated unique fingerprints by flaA restriction fragment length polymorphism (RFLP) analysis [24 (link)].

Collected purulent material samples were bacteriologically examined following the procedures of Quinn et al. [25 ]. Briefly, each sample was transferred to 10 mL of brain heart infusion broth (BHI; Oxoid, Hampshire, UK), and incubated at 37 °C for 18 h to stimulate the growth of the microorganisms. A loopful of the pre-enriched sample was then plated onto a blood agar base (Oxoid, Hampshire, UK) enriched with 5% sterile defibrinated sheep blood and incubated aerobically at 37 °C for 48 h. The plates were examined for typical growth, morphological features, and hemolytic characteristics. Gram-positive bacilli in small, white, dry, and crumbly colonies were selected for further identification. Biochemical tests including catalase, urease, nitrate reduction, esculin hydrolysis, and fermentation of glucose, lactose, maltose, sucrose, arabinose, fructose, mannose, trehalose, xylose, and salicin sugars were performed [25 ,26 ]. In addition, the suspected colonies were subjected to synergistic hemolysis with Rhodococcus equi and antagonistic hemolysis with Staphylococcus aureus (ATCC 25923), as described by Cowan and Steel [27 ].

Lactobacillus amylovorus, strain P1 (LA), and Lactobacillus mucosae, strain P5 (LB), were isolated from pig feces, characterized by function tests, identified by MALDI-TOF MS and 16S rRNA gene sequencing, and used in the experiments. A probiotic E. coli Nissle 1917 (EcN) is a biologically active compound of a probiotic preparation Mutaflor^® (Ardeypharm, Herdecke, Germany). Salmonella enterica subsp. enterica serovar Typhimurium, strain LT2 (S. Typhimurium, ST) was from a collection of the microorganisms of the Institute of Microbiology of the Czech Academy of Sciences (Novy Hradek, Czech Republic).
Fresh bacterial cultures were prepared for each experiment by cultivation for 16 h at 37 °C. Lactobacilli were cultivated in 10 mL MRS broth (Oxoid). The cells were harvested by centrifugation at 4000 × g for 10 min. The pellet was washed twice with 0.05 M phosphate buffer and resuspended to an approximate density of 8.5 log colony forming units (CFU)/mL. EcN and ST were cultivated overnight on meat-peptone agar slopes (blood agar base; Oxoid), and both resuspended to 8.5 log CFU/mL. The number of CFU was verified by cultivation methods.

The A. butzleri recipient strains used in this work, as well as their origin and characteristics, are shown in Table 1. Two of the donor strains, DQ40A1ΔareB::kan^R and CR1132∆areB::kan^R, were generated by the insertion of a kanamycin resistance cassette (aphA-3) interrupting the areB gene (ABU_RS11090). The aphA-3_cassette was obtained by BamHI and KpnI double digestion of the pUC18-K2 plasmid, followed by binding to the upstream and downstream region of the areB gene by overlap-extension PCR. The purified PCR fragment was used for mutant construction by the agar transformation method, as described elsewhere [49 (link)]. The A. butzleri 851 gyrA mutant strain was generated by transformation of the DQ40A1 strain with a 344 bp PCR fragment of the gyrA gene, carrying the single nucleotide polymorphism (C254T) conferring resistance to ciprofloxacin.
A. butzleri strains were routinely cultured on Blood Agar Base (Oxoid, Basingstoke, England) supplemented with 5% defibrinated horse blood (v/v) (BA—Blood Agar). The bacteria were incubated at 30 °C in a controlled atmosphere (6% O₂, ±7.1% CO₂ and 3.6% H₂) generated by an atmosphere modifier (Anoxomat AN2CTS, Mart Microbiology B.V., Drachten, Netherlands), unless otherwise stated. For transformants selection, strains were transferred to BA plates containing 4 μg/mL of ciprofloxacin, or 30 and 50 μg/mL of kanamycin, or both antibiotics.

The compound (1R, 2 S-(–)2-methylamino-1-phenyl-1-propanol hydrochloride, referred to as MPPH, is also known by its (1R, 2 S)-(–)-ephedrine hydrochloride nomenclature. The substance MPPH was acquired from Cerilliant Corporation, a renowned American corporation specializing in the manufacturing of reference chemical compounds used in toxicology and forensic investigation. The Mueller-Hinton broth (MHB), Mueller-Hinton agar (MHA), nutrient broth (NB), nutrient agar (NA), and blood agar base (BA) were procured from Oxoid Ltd., a company based in Basingstoke, UK. The media utilized in this study were made in strict adherence to the directions provided by the manufacturer.