Microbank tube

C. jejuni M1cam (M1 isolate [35 (link)]) was routinely cultured on Mueller–Hinton (MH) agar (Merck Life Science UK Limited, Dorset, UK) at 42 °C under microaerophilic conditions of 5% O ${}_2$ , 5% CO ${}_2$ , 5% H ${}_2$ , 85% N₂ in an M95 variable atmosphere workstation (Don Whitley Scientific, Bingley, UK). Bacterial stocks were stored at −80 °C in MicroBank tubes (Pro-Lab Diagnostics, Birkenhead, UK). For each experiment and replicate, bacteria were always grown from freezer stocks onto fresh MH agar plates for 48 h and then restreaked and grown for a further 16 h before use.

The performance of the MLSA scheme was further evaluated using field isolates provided by the Bundeswehr Institute of Microbiology (Munich, Germany) and obtained by the Animal and Plant Health Agency (Weybridge, United Kingdom) via brucellosis surveillance activities. In the former case, these represented a diverse panel of Ochrobactrum spp. from a variety of sources and geographical locations, confirmed as Ochrobactrum sp. by both 16S rRNA and recA sequencing (see Scholz et al., 2008a (link) for full details). A panel of 37 isolates was examined (Table 3), representing major clades identified by recA sequence analysis (Scholz et al., 2008a (link)). DNA was prepared as previously described (Scholz et al., 2008a (link)). In the case of Animal and Plant Health Agency (APHA) isolates (n = 13), these were submitted as suspect Brucella sp. of veterinary origin but subsequently identified as non-Brucella members of the Brucellaceae by 16S rRNA sequencing (Wragg et al., 2014 (link)) (Table 3). Pure cultures were stored frozen in either Luria Broth with 15% glycerol or using proprietary MicroBank tubes (ProLab Diagnostics, United Kingdom). Frozen strains were revived by plating onto sheep blood and/or nutrient agar and incubating at 28°C for 24–48 h. Crude thermo-lysates from single colonies were prepared as described above.

Bacterial genus and species were identified using MALDI TOF (Bruker Daltonics Inc.). Initial AST results were determined using the BD Phoenix Automated System (BD Diagnostics). After resistance to traditional β-lactam agents was identified, AST for novel β-lactams was determined using lyophilized Sensititre broth microdilution (BMD) GN7F and MDRGN2F BMD panels (Thermo Fisher Scientific). CLSI interpretive criteria were applied to determine P. aeruginosa susceptibility.⁴ Isolates were stored at −80°C in Microbank tubes (Pro-Lab Diagnostics) until further testing was performed.

The bacterium, designated as strain LAR25 was isolated in Marine Agar (MA) supplemented with mannitol from the inlet water of a commercial gilthead seabream and European seabass hatchery, located in Larymna, Central Greece. LAR25 was identified as Tenacibaculum sp. using molecular methods (PCR amplifying 16S rRNA) and whole genome sequencing. Additionally, five different bacterial strains belonging to four Tenacibaculum species (T. discolor, T. mesophillum, T. soleae, and T. galaicum) kindly offered by Dr. Mikkel Bentzon-Tilia of the Department of Biotechnology and Biomedicine of the Technical University of Denmark were used in the analysis. All the bacterial strains were maintained in Microbank tubes (Pro-lab Diagnostics, Richmond Hill, Canada) at −80°C and were grown in Lysogeny Broth (LB) (10 gL⁻¹ tryptone, 5 gL⁻¹ yeast extract, 10 gL⁻¹ NaCl, 0.75 gL⁻¹ MgSO₄, 1.5 gL⁻¹ KCl, 0.73 gL⁻¹ CaCl₂) at 25°C when used.

Cherry-pit-sized volumes from the stool samples provided were used for broth enrichment in thioglycolate broth (Heipha, Eppelheim, Germany) for 16–24 hours at 37°C. Subsequently, 10 μl preincubated broth was cultured on Brilliance ESBL selective agar (Oxoid, Basingstoke, UK) for the selection of third-generation cephalosporin-resistant bacteria. This agar is made for selective growth of ESBL-positive Enterobacteriaceae. An additional 10 μl for each was incubated on Brilliance VRE agar (Oxoid) for the selection of vancomycin-resistant enterococci (VRE) and on CHROMagar MRSA (CHROMagar, Paris, France) for the selection of methicillin-resistant Staphylococcus aureus (MRSA).
Agar plates were incubated at 37°C for 40–48 hours. All colonies that looked suspicious for Enterobacteriaceae on Brilliance ESBL selective agar (blue, green, brown colonies) were isolated, while suspected Gram-negative nonfermentative rod-shaped bacteria (i.e., yellow or yellowish-brown or greenish-brown colonies) were discarded. Similarly, only colonies that appeared suspicious for MRSA and VRE were selected for further analysis. Suspected enterococci were of blue or violet color; suspected MRSA were of mauve color.
All suspicious isolates were frozen at −80°C in Microbank tubes (Pro-Lab Diagnostics, Bromborough, UK) until further assessment.

Nasal swab samples were streaked onto mannitol salt agar (MSA) (Liofilchem Srl, Teramo, Italy) and incubated aerobically at 37 °C for 24 h, in order to collect bacterial isolates able to grow on this medium. Colonies were firstly identified by standard, rapid screening techniques: colony morphology, presence, or absence of mannitol fermentation on MSA, Gram-staining, catalase reaction, and for staphylococci also the tube coagulase reaction (Oxoid, Ltd., Hampshire, UK) was performed.
Single colonies were subcultured on Columbia Sheep Blood agar (Liofilchem Srl, Teramo, Italy) and after incubation at 37 °C for 24 h, the isolates were identified using the matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) (Bruker Daltonics Inc., Bremen, Germany).
Only staphylococci and enterococci isolates were preserved in 16% v/v glycerol broth and in Microbank tubes (Pro-Lab Diagnostics, Round Rock, TX, USA) at −80 °C for further investigation.