TITLE: Detection of Klebsiella pneumoniae and closely related species by real-time PCR with the ZKIR system
AUTHORS: Elodie Barbier, Carla Rodrigues, Geraldine Depret, Virginie Passet, Laurent Gal, Pascal Piveteau, Sylvain Brisse

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Klebsiella pneumoniae</span><span> (Kp) is of growing public health concern due to the emergence of multidrug-resistant and virulent strains. Taxonomically, Kp includes seven phylogroups, with Kp1 (</span><span style = "font-style:italic;">K. pneumoniae</span><span style = "font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;">sensu stricto</span><span>) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. </span></div><div class = "text-block"><span>We designed a novel SYBR green real-time PCR assay, named the ZKIR assay, that targets </span><span style = "font-style:italic;">Klebsiella pneumoniae</span><span> and closely related species (phylogroups Kp1 to Kp7). </span></div><div class = "text-block">Based on 48 Kp representing its phylogenetic breadth, and on 88 non-Kp strains, the ZKIR assay detected all Kp and was totally specific for Kp, as no false positive was found.</div><div class = "text-block">We also tested this method on spiked soil microcosms after a 24 h enrichment step (in Lysogeny Broth supplemented with ampicillin 10 mg/l) and a short sample treatment. We showed that this procedure was sensitive enough to detect one single bacterium in 5 g of soil.  </div></div>

[STEPS]
?. qPCR mix preparation (final volume 20 µl)  ABC1Mix reagentsVolume per well (µl)Final concentration2Takyon™ ROX SYBR® MasterMix 2X103Forward (3 µM)2300 nM4Reverse (3 µM)2300 nM5T4 gp32 (optional)0.512.5 µg/ml6PCR grade waterQS 17.5 µl7DNA template2.5
ABC1Mix reagentsVolume per well (µl)Final concentration2Takyon™ ROX SYBR® MasterMix 2X103Forward (3 µM)2300 nM4Reverse (3 µM)2300 nM5T4 gp32 (optional)0.512.5 µg/ml6PCR grade waterQS 17.5 µl7DNA template2.5
?. Thermocycler settings  ABCD1 Temperature (°C)Time (min.) 2Holding stage95.003:00Enzyme activation3Cycling stage95.000:10Denaturation4 60.001:00Annealing (data collection)5Melt Curve stage95.000:15Dissociation stage Program Step and Hold  T° increment +0.3°C6 60.001:007 95.000:15
ABCD1 Temperature (°C)Time (min.) 2Holding stage95.003:00Enzyme activation3Cycling stage95.000:10Denaturation4 60.001:00Annealing (data collection)5Melt Curve stage95.000:15Dissociation stage Program Step and Hold  T° increment +0.3°C6 60.001:007 95.000:15
?. Positive and negative controls:Negative control: PCR grade waterPositive control: Kp1 DNA diluted to 2 ng per µl (Strain ATCC13883T)
?. Melt curve peak established using the ZKIR assay with serial dilutions of K. pneumoniae ATCC13883T. Melting temperature was around 80.2 °C.
?. Sample preparation
?. Sample enrichment step:Soil samples (10 g) are enriched in 90 ml of LB (Lysogeny Broth: 5 g yeast extract, 5 g sodium chloride, 10 g tryptone for 1 liter) supplemented with ampicillin 10 mg/l for 24 h at 30°C.Food samples (25g) such as salad and chicken are enriched in 225 ml of BPW (Buffered Peptone Water) for 24 h at 37°C.
?. Sample treatment step:500 µl of enrichment is centrifuged 5 min at 5 800 g and washed twice with sterile water before boiling for 10 min. Boiled suspensions are then diluted at 1/10 and 1/100 for qPCR. Crude and diluted DNAs are tested with the ZKIR assay.