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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is intended for isolation of Klebsiella strains from water samples. </div><div class = "text-block"><span>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. </div></div>

[STEPS]
?. [Pre-treatment of sample]
Water samples are collected in 5L sterile containers and transported to the laboratory. Six samples are collected at each site to give a total of 30 litres per sample.
The water samples are processed using the CapE system by placing the submersible pump into the pooled water samples.NOTE: these first two steps might vary according to the each partner and systems available to collect water samples.
?. [Pre-treatment of sample]
The 0.45μm filter is folded up using sterile forceps and placed in 100mL of Buffered Peptone Water (BPW) for 18-24 hours incubation at 42 ̊C ± 1°C. NOTE: Place all filters used per sample (30L) in the one 100mL BPW container.
?. [Streak a SCAi medium agar plate]
Following 18-24h 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 37°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.
?. [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).
?. [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.