TITLE: Isolation of Klebsiella strains from food samples
AUTHORS: MedVetKlebs consortium

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is intended for isolation of </span><span style = "font-style:italic;">Klebsiella</span><span> strains from different food sources. It is derived from the initial description of the SCAi medium (van Kregten E, Westerdaal, N. A. C., and Willers, J. M. N. New, simple medium for selective recovery of </span><span style = "font-style:italic;">Klebsiella pneumoniae</span><span> and </span><span style = "font-style:italic;">Klebsiella oxytoca </span><span>from human feces. Journal of Clinical Microbiology. 1984;20:936-41) and its validation across a diversity of </span><span style = "font-style:italic;">Klebsiella</span><span> strains (Passet V, Brisse S. 2015. Association of tellurite resistance with hypervirulent clonal groups of</span><span style = "font-style:italic;"> Klebsiella pneumoniae</span><span>. J Clin Microbiol. 53(4):1380-2). </span></div><div class = "text-block">The protocol entails enrichment using Buffered Peptone Water (BPW), and plating on SCAi (Simmons Citrate with Inositol) agar.</div><div class = "text-block">This protocol was optimized by the MedVetKlebs consortium using chicken meat and salad samples. </div></div>

[STEPS]
?. [Pre-treatment of sample]
Cut sample into small slivers to a final weight of 25g.
?. [Pre-treatment of sample]
Dilute the 25g portion in 225ml of Buffered Peptone Water (BPW) broth (1:10 dilution).
?. [Pre-treatment of sample]
Mix the sample using a stomacher for 30 seconds or pulse using a blender.
?. [Pre-treatment of sample]
Incubate the suspension at 37°C ± 1°C for 24 h ± 1 h.
?. [Streak a SCAi medium agar plate]
Following 24h incubation, using a 10μl loop, streak for single colonies onto the surface of a
small petri dish (90 mm) of SCAI medium and incubate at 44°C ± 1°C for 48h ± 1 h.Sometimes, typical colonies (yellow, moist, dome-shaped) can be recognized after 24h culture on plates, but 48 h is much better to discriminate Klebsiella-looking colonies from other ones (E. coli colonies are typically white because they do not use inositol).   It can happen that the medium, which should initially be blue, turns completely yellow, when there are many inositol-fermenting colonies (typically Klebsiella). In these cases, discriminating yellow colonies is less easy. Diluting before streaking could help in these cases.
?. [Purification and identification of suspect Klebsiella colonies]
Typical Klebsiella spp. colonies are yellow on SCAI medium.   Select suspect Klebsiella colonies for subculture and bacterial identification. Streak the selected colonies onto the surface of a non-selective agar medium (e.g., LB or TSA) in a manner which will allow isolated colonies to develop. Incubate plates at 37 °C ± 1°C for 24 h ± 1 h.   Note: If colonies are numerous and close to each other, re-isolate the colony on another SCAi agar plate to control for purity. Incubate for up to 48h.
?. [Purification and identification of suspect Klebsiella colonies]
Determine species of purified suspect K. pneumoniae colonies using MALDI-TOF mass spectrometry and/or species-specific PCR.
?. [Storage of bacterial strains]
Freeze strains confirmed as Klebsiella pneumoniae (or its related species, which also grow on SCAi) at -80°C using CryoBank tubes or equivalent (e.g. BHI + 15% glycerol medium).If several morphotypes are available, you may want to store one colony per morphotype.
?. [Mixed-colony storage for future studies (additional)]
If desired, after picking selected colonies, sweep the remaining SCAi plate content and freeze it at -80°C (e.g., for mixed colonies sequencing) using CryoBank tubes or equivalent (e.g., in house BHI + 15% glycerol medium).