TITLE: Isolation of Klebsiella strains from human or animal fecal material
AUTHORS: Sylvain Brisse, virginie passet, Carla Rodrigues

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is intended for isolation of Klebsiella strains from  human or animal fecal material. 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 Klebsiella pneumoniae and Klebsiella oxytoca from human feces. Journal of Clinical Microbiology. 1984;20:936-41) and its validation across a diversity of Klebsiella strains (Passet V, Brisse S. 2015. Association of tellurite resistance with hypervirulent clonal groups of Klebsiella pneumoniae. J Clin Microbiol. 53(4):1380-2). The protocol entails enrichment using ampicillin-containing broth, and plating on SCAi (Simmons Citrate with Inositol) agar.
 
 
 
 
 
 
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[STEPS]
?. Pre-treatment of sampleEnrich the sample by use of an enrichment broth. We typically use Luria-Bertani broth with 10 mg/L of ampicillin final concentration. Because they carry a constitutively expressed class A beta-lactamase (blaSHV type), the vast majority of Klebsiella isolates will grow/survive, whereas many other bacteria including E. coli (the most abundant potential competitor) are susceptible to ampicillin. Trypto-Casein Soy broth (TSB) can be used instead of LB. Take a swab or a loopful (10 μL) of faecal material, inoculate in 10 mL of LB+Ampicillin broth, mix, and incubate at 37 °C ± 1°C for 24 h± 1 h. 18 hours is also fine  - note that it is best to treat all samples in the same way.
?. Streak a SCAi medium agar plateStreak to isolate single colonies, using a 10 μl loop. Use a Petri dish (90 mm is fine) of SCAi medium. 
 
Incubate at 37°C ± 1°C for 48 h ± 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 of suspect K. pneumoniae colonies 
 Typical Klebsiella spp. colonies are yellow on SCAI medium. 
Select suspect Klebsiella pneumoniae colonies and pick for subculture before bacterial identification.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.
?. Mixed-colony storage for future studiesIf 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).
?. IdentificationStreak 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. 
 
Determine species of purified suspect K. pneumoniae colonies using MALDI-TOF mass spectrometry and/or species-specific PCR.
?. Storage of bacterial strainsFreeze 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.