# The ZKIR assay, a novel real-time PCR method for the detection of Klebsiella pneumoniae and closely related species in environmental Samples

Elodie Barbier, Carla Rodrigues, Géraldine G. Depret, Virginie Passet,
Laurent Gal, Pascal Piveteau, Sylvain Brisse

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Elodie Barbier, Carla Rodrigues, Géraldine G. Depret, Virginie Passet, Laurent Gal, et al.. The ZKIR assay, a novel real-time PCR method for the detection of Klebsiella pneumoniae and closely related species in environmental Samples. Applied and Environmental Microbiology, 2020, 86 (7), pp.1-39.
10.1128/AEM.02711-19. hal-02464159

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https://institut-agro-dijon.hal.science/hal-02464159v1

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1 The ZKIR Assay, a novel Real-Time PCR Method for the Detection of

2 Klebsiella pneumoniae and Closely Related Species

3 in Environmental Samples

4

5

6 Elodie Barbierª#, Carla Rodriguesᵇ, Geraldine Depretª, Virginie Passetᵇ, Laurent Galª, Pascal

7 Piveteauª and Sylvain Brisseᵇ#

8

9 ᵃAgroécologie, AgroSup Dijon, CNRS, INRA, Univ. Bourgogne Franche-Comté, Dijon, France

10 ᵇ Institut Pasteur, Biodiversity and Epidemiology of Bacterial Pathogens, Paris, France

11

12 Running title: Detection of Klebsiella pneumoniae by real-time PCR

13

14 # Corresponding authors

15 Sylvain Brisse:

16 Institut Pasteur, Biodiversity and Epidemiology of Bacterial Pathogens, 28 rue du Docteur Roux,

17 F-75724 Paris, France; E-mail: sylvain.brisse@pasteur.fr; Phone +33 1 45 68 83 34

18 Elodie Barbier:

19 Agroécologie, AgroSup Dijon, CNRS, INRA, Univ. Bourgogne Franche-Comté, 17 rue Sully, F-

20 21000 Dijon, France; E-mail: elodie.barbier@inrae.fr; Phone +33 3 80 69 33 24

21

22 **Keywords:** Klebsiella, phylogroup, soil, detection, screening, ZKIR qPCR, culture method

23

24
25 ABSTRACT

26 Klebsiella pneumoniae (Kp) is of growing public health concern due to the emergence of strains
27 that are multidrug-resistant, virulent, or both. Taxonomically, Kp includes seven phylogroups,
28 with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in
29 environmental sources such as soils and vegetation, which could act as reservoirs of animal and
30 human infections. However, the current lack of screening methods to detect Kp in complex
31 matrices limits research on Kp ecology. Here we analysed 1,001 genome sequences and found
32 that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method,
33 the ZKIR assay, was developed and used to detect Kp in 96 environmental samples. Results were
34 compared to a culture-based method using Simmons citrate agar with 1% inositol (SCAI)
35 medium coupled to MALDI-TOF mass spectrometry identification. Whole-genome sequencing
36 of environmental Kp was performed.

37 The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains
38 were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10⁻¹ CFU g⁻¹
39 after enrichment for 24 h in LB supplemented with ampicillin, and 1.5 × 10³ to 1.5 × 10⁴ CFU g⁻¹
40 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method.
41 Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a
42 predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 MLST
43 sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency
44 (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific and
45 sensitive novel method to detect the presence of Kp in complex matrices, and indicates that Kp
46 isolates from environmental samples differ from clinical isolates.

47

48
49 **IMPORTANCE**

50 The *Klebsiella pneumoniae* species complex (Kp) includes human and animal pathogens, some of 51 which are emerging as hypervirulent and/or antibiotic resistant strains. These pathogens are 52 diverse and classified into seven phylogroups, which may differ in their reservoirs and 53 epidemiology. Proper management of this public health hazard requires a better understanding of 54 Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in 55 complex matrices such as food or the environment has been difficult due to a lack of accurate and 56 sensitive methods. Here, we describe a novel method based on real-time PCR, which enables 57 detection of all Kp phylogroups with high sensitivity and specificity. We used this method to 58 detect Kp isolates from environmental samples, and show based on genomic sequencing that they 59 differ in antimicrobial resistance and virulence gene content, from human clinical Kp isolates.

60 The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will 61 thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, 62 food, animal and human sources.
63 INTRODUCTION

64 Klebsiella pneumoniae (Kp) is one of the leading causes of multidrug resistant (MDR)
65 healthcare-acquired infections, with increasing rates of resistance to carbapenems and other last
66 resort antibiotics being reported (1, 2). Furthermore, Kp is also an important agent of severe
67 community-acquired infections (so-called ‘hypervirulent’ strains) in healthy persons (3), with
68 recent worrisome reports of convergence between hypervirulent and MDR phenotypes (1, 4). Kp
69 is recognized as a colonizer of the throat and the intestinal tract of humans and animals (5–7).

70 The main sources of human exposure to Kp are not well defined. Previous studies
71 highlighted the large distribution of Kp in outdoor environments, including water, sewage, soil
72 and plants (8–13). Animal and human food, particularly retail meat or salad may also be
73 contaminated (6, 14, 15). Many studies suggest that food, water and/or environmental exposure
74 may be associated with virulent and/or antibiotic resistant Kp to humans (13, 14, 16, 17).
75 However, little is known on the relative contribution of these different sources of transmission.
76 Although such information is a prerequisite to control efficiently transmission routes and reduce
77 exposure, the ecology of Kp is currently poorly understood.

78 The systematics of Kp has been refined through recent taxonomic updates, which
79 highlighted the existence of seven phylogroups (Kp1 to Kp7), corresponding to distinct taxa,
80 within Klebsiella pneumoniae sensu lato. The K. pneumoniae species complex includes 5
81 different species: K. pneumoniae sensu stricto (phylogroup Kp1), K. quasipneumoniae [subsp.
82 quasipneumoniae (Kp2) and subsp. similipneumoniae (Kp4)], K. variicola [subsp. variicola
83 (Kp3) and subsp. tropica (Kp5)], ‘K. quasivariicola’ (Kp6) and K. africana (Kp7) (8, 18–20).
84 Most of these taxa are still often misidentified as “K. pneumoniae” or “K. variicola” due to the
85 unsuitability of traditional clinical microbiology methods to distinguish among members of the
86 Kp complex (21). Henceforth, we will use the ‘Kp’ abbreviation to refer collectively to the seven
taxa of the *K. pneumoniae* species complex, and will reserve ‘*K. pneumoniae*’ for *K. pneumoniae* sensu stricto (i.e., phylogroup Kp1). As all Kp are potentially pathogenic for humans and animals and can share acquired resistance and virulence genes, it is important that the seven taxa be considered together when investigating the routes of transmission and ecology of Kp.

Detection of Kp is not well integrated in food or environmental microbiological surveillance programs and there is a general lack of tools and procedures for its detection and quantification.

Culture-based laboratory methods used for the detection of microorganisms in complex matrices are time consuming and have a low throughput. Moreover, Kp culture methods have not been validated so far for food safety screening. Some molecular methods (without need of sequencing) have been proposed over the years for the rapid detection of Kp (22–26). They target the 16S-23S rRNA internal transcribed spacer sequence (ITS) (22), coding sequences of *tyrB* (26), *khe* (25, 27), chromosomal beta-lactamase (*bla*) genes (28, 29) or other molecular targets (23). Some of these targets are described as able to detect the Kp complex (22), whereas others were designed for specific members of the Kp complex, such as Kp1 and Kp3 (23, 24).

Real-time PCR is a powerful approach for the rapid detection and quantification of microorganisms in complex matrices (30). This approach presents multiple advantages including easy standardization and high throughput. The aims of this work were (i) To define the phylogenetic distribution of previously proposed molecular targets for Kp detection, in light of recent taxonomic updates; and (ii) To develop a real-time PCR method for the rapid, specific and sensitive detection of all Kp members. (iii) In addition, we used the novel qPCR method to detect Kp in environmental samples and explored the genomic features (including antibiotic resistance and virulence genes) of the recovered Kp isolates.
109 MATERIALS AND METHODS

110 Bacterial strains and culture conditions

111 A panel of 136 strains from the collections of the Institut Pasteur, Paris (France) and INRA Dijon (France) was used (Table 1). It included the *K. pneumoniae* type strain ATCC13883T and 47 other strains of the Kp complex (Kp1 to Kp7 phylogroups, including type strains) isolated from patients, animals and outdoor environments from multiple geographic locations. Eighty-eight non-Kp bacterial strains from other species of the genus *Klebsiella*, closely related genera (*Raoultella*, *Enterobacter*), and from other species that have similar environmental niches were included for comparison. All strains were regenerated by streaking on tryptone soya agar (TSA, Conda, Spain). After 24h at 37°C, single colonies were transferred to 10 ml tryptone soya broth (TSB, Conda, Spain). Bacterial suspensions were collected after 24 h incubation at 37°C for further experiments.

121

122 Bacterial DNA extraction

123 The bacterial DNA was extracted according to a homemade protocol. One ml of the 24h bacterial culture was centrifuged (7000 g, 10 min, 4°C), the pellet washed with sterile water and suspended in 800 µl Tris (50 mM, pH 8) in which 10 µl EDTA (500 mM, pH 8), 115 µl lysozyme (10 mg/ml, incubation 37°C, 30 min), 57 µl SDS 20% (incubation 65°C, 15 min), 5 µl RNAse (10 mg/ml, incubation 37°C, 30 min) and 23 µl proteinase K (6 mg/ml, incubation 37°C, 1h30) were added. Finally, a 1:1 volume of chloroform/ isoamyl alcohol (25/24/1) was added. The suspension was shaken 3 min at 15 rpm, then centrifuged 3 min at 20 000 g. The supernatant was transferred in a new tube and a 1:1 volume of chloroform/ isoamyl alcohol (24/1) was added.
124
125
126
127
128
129
130
131 After shaking (3 min, 25 rpm) and centrifugation (3 min, 20 000 g), this step was repeated once.
132 After transfer of the supernatant in a new tube, a 1:10 volume of NaCl (5 M) was added (shaking
133 1 min, 15 rpm). Finally, a 1:1 volume of isopropanol (shaking 3 min, 15 rpm) was added.

134 Precipitated DNA filaments were collected with a glass Pasteur pipette and transferred in 400 µl

135 frozen ethanol (70%). After shaking 3 min at 15 rpm, DNA filaments were collected, dried at

136 room temperature and finally dissolved in 100 µl TE (pH 8). DNA concentration was estimated

137 using NanoDrop 2000 Spectrophotometer (ThermoFischer Scientific, USA) and adjusted at 20

138 ng/µl. Purified DNA was stored at -20°C.

139 A simpler protocol was implemented for the PCR specificity assay and validation study. Bacteria

140 grown in TSB or enrichment medium were centrifuged 5 min at 13 000 g. Pellets were suspended

141 in 200 µl of Tris HCl (5 mM pH 8.2) supplemented with 13 µl of Proteinase K (1 mg/ml). After 2

142 h incubation at 55 °C, Proteinase K was inactivated during 10 min boiling. Cell debris were

143 discarded by centrifugation (5 min at 13 000 g) and the supernatant was stored at – 20°C before

144 use for real-time PCR.

145

146 Soil DNA extraction

147 Soil metagenomic DNA was extracted from 2 g aliquots of soil using the modified ISO standard

148 11063 method (31). Crude soil DNA extracts were first purified onto PVPP

149 (polyvinylpolypyrrolidone) minicolumns (BIORAD, France), then with the GeneClean Turbo kit

150 (MP Biomedicals, France) according to the manufacturer's protocol (31). Purified DNA

151 concentrations were determined using a Nanodrop 2000 spectrophotometer.

152

153 *In silico* identification of the target DNA region

154 The phylogenetic distribution of molecular targets that were previously described in the literature

155 for Kp detection, was investigated *in silico*. All available Kp genomes were downloaded from the

156 NCBI database (n=3552, February 2018) and combined with the ones of Institut Pasteur's
157 internal reference collection (n=670) (20, 32, 33), representing a total of 4,222 genomes which
158 included Klebsiella species and closely related Raoultella species. The average nucleotide
159 identity (ANI) metric was used to classify the Kp genomes in each of the phylogroups. To avoid
160 redundancy in the dataset and due to the difficulty of analyzing phylogenetically 4,222 genomes,
161 in the case of the genomes belonging to the Kp complex (which represented the majority of the
162 dataset), we selected unique representatives of each MLST sequence type (ST), as defined using
163 the mlst software tool (https://github.com/tseemann/mlst). This resulted in a final collection of
164 1,001 genomes that were tested for the presence of targets from the literature. Nucleotide BLAST
165 (BLASTN) was used to detect the presence of the target sequences in the 1,001 genomes, with
166 80% nucleotide identity (also with 92% in the case of blaSHV, blaOKP and blaLEN targets) and 80%
167 length coverage as cutoffs. Primer-blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was
168 used (with default parameters) to check the distribution of primer target sites in the genomic
169 sequences. Specificity was defined as the proportion of target organisms among those expected to
170 be amplified by the target assay. Sensitivity was defined as the proportion of target organisms
171 expected to be amplified by the target assay. A phylogenetic tree was constructed (34) and the
172 output was visualized using iTOL (https://itol.embl.de/; Figure 1; Figure S1).

173

174 Design of primers

175 The Kp *tyrB* and *khe* gene sequences (accession numbers: AF074934.1 and AF293352.1) were
176 aligned against the genome sequences of *K. pneumoniae* (CP012744.1, CP012743.1,
177 CP028787.1), *K. variicola* (CP008700.1, CP013985.1, CP017289.1), *K. quasipneumoniae*
178 (CP014071.1, CP023478.1, CP029432.1) and *K. quasivariicola* (CP022823.1) using BLASTN to
179 identify conserved genomic regions within the Kp complex. Sequences of closely related
180 bacterial species *K. oxytoca* (CP027426.1), *Raoultella ornithinolytica* (CP010557.1),
181 *R. planticola* (CP026047.1) and *K. aerogenes* (CP014029.2) were added in the alignments to
182 confirm the specificity of these regions for the Kp complex.

183 Primer sets were designed using Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/ primer3plus.cgi). The specificity of the predicted primer sets and amplicons was
184 checked by applying BLASTN on the GenBank nucleotide collection (nr/nt) from the NCBI
185 database and Multiple primer analyzer (https://www.thermofisher.com) was run to check for
186 dimer formation. The best candidate primer sets defined by this in silico approach were ordered
187 for synthesis at Eurogentec; among these was primer pair ZKIR_F (5'-CTA-AAA-CCG-CCA-TGT-CCG-ATT-TAA- 3') and ZKIR_R (5'-TTC-CGA-AAA-TGA-GAC-ACT-TCA-GA- 3').
188
189
190

191 **Real-time PCR**

192 All real-time PCR assays were performed on an ABI StepOne™ Real-time thermocycler (Fisher
193 Scientific, France) with the following temperature program: 95 °C for 3 min and 40 cycles at
194 95 °C 10 s and 60 °C for 1 min. Melt curves were generated with temperature increments of
195 0.3 °C per cycle from 60 to 95 °C. DNA was amplified in a 20 µl PCR mix containing 10 µl of
196 Takyon Low Rox SYBR MasterMix dTTP Blue (Eurogentec, Belgium), 2 µl of each primer
197 (final concentration 300 nM), 2.5 µl of template DNA and 3.5 µl PCR grade water. For detection
198 of Kp from environmental samples, 0.5 µl of T4 Gene 32 Protein (Sigma Aldrich) was added to
199 the PCR mix. The specificity and cross-reactivity of the ZKIR assay was evaluated with 2 ng of
200 purified DNA of 48 Kp complex strains and 88 non-Kp isolates (Table 1).

201 Two universal primers targeting a 174 base pairs region of the 16S rRNA gene of Eubacteria,
202 341f (5'-CCT-ACG-GGA-GGC-AGC-AG-3') and 515r (5'-ATT-CCG-CGG-CTG-GCA-3'),
203 were used for positive control PCR reactions as described in a previous report (35).

204
205 Standard curve development and sensitivity assessment

206 Aliquots of 2.5 µl adjusted to 7.5 ng, 750 pg, 75 pg, 7.5 pg, 750 fg, 375 fg, 45 fg and 15 fg of
207 genomic DNA of K. pneumoniae ATCC13883T were prepared in triplicate and amplified with the
208 optimized PCR conditions described above. Results were analysed with the StepOne™ Data
209 Analysis software. PCR efficiency was determined. The genome number expressed as logarithm
210 was plotted against Ct values and the correlation coefficient (R²) of the standard curve was
211 calculated.

212

213 Comparison of the ZKIR PCR and culture methods for detection of Kp in environmental
214 samples

215 Ninety-six environmental samples collected between July and September 2018 (23 in July; 39 in
216 August and 34 in September) in Auxonne (Burgundy, France) were analysed for Kp presence
217 using the ZKIR assay and culture methods in parallel. Samples corresponded to bulk soils (n=32),
218 roots (31), leaves (29) and irrigation water (4) and were processed in the lab within 24 hours after
219 sampling. Ten g of soil were weighed in 180 ml pots (Dutscher, France). Plant leaves and roots
220 were properly cut, cleaned with sterile water and transferred in 180 ml pots. Processed samples
221 were suspended in 90 ml of lysogeny broth (LB, 5 g of NaCl, 5 g of yeast extract and 10 g of
222 Tryptone for a 1 l final volume) supplemented with ampicillin (10 mg/l, ampicillin sodium salt,
223 Sigma Aldrich). Five hundred millilitres of water samples were filtered through a 0.25 µm
224 membrane (Millipore, France). The membrane was incubated in 20 ml of LB supplemented with
225 ampicillin as described above. After 24 h of incubation at 30 °C, enrichments were vortexed and
226 500 µl aliquots were centrifuged (5 min at 5800 × g) and washed with sterile water. The pellet
227 was suspended in 500 µl of sterile water and boiled for 10 min. Boiled enrichments were ten-fold
228 diluted (1:10 and 1:100) and the dilutions were used as template for real-time PCR.
In parallel, enrichments were serially diluted (1:10 to 1:10 000) in sterile water before plating on Simmons Citrate Agar enriched with 1% inositol (SCAI medium) (36). Plates were incubated 48 h at 37 °C. Each plate was screened for presumptive colonies of *Klebsiella* (large, yellow, dome-shaped colonies) and ten candidate colonies were purified on SCAI medium and identified using Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry (MALDI Biotyper, Bruker) according to the MALDI Biotyper Compass database version 4.1.80 (Bruker Daltonics, Germany). In addition, whole-genome sequencing (WGS) was performed for environmental Kp isolates (n=31) using the NextSeq-500 sequencing platform (Nextera XT library; 2x150 nt). Genomic assemblies were obtained using SPAdes v3.9. Multilocus sequence typing (MLST) and core-genome MLST (cgMLST) were performed using the BIGSdb-Kp web tool (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html). This resource coupled with Kleborate (https://github.com/katholt/Kleborate) was used to search for antibiotic resistance, virulence and heavy metal tolerance genes, and to predict capsular types. PlasmidFinder was used to look for plasmid replicons (https://cge.cbs.dtu.dk/services/PlasmidFinder/). To construct the tree based on *nif* cluster genes (Figure S2), the DNA region between *nifQ* and *nifJ* genes was extracted from the *nif* carrying strains and from a set of reference strains carrying this cluster (37). Sequences were aligned with MUSCLE, and IQ-TREE (http://iqtree.cibiv.univie.ac.at) was used to reconstruct the maximum likelihood phylogeny using the HKY+I+F+G4 model.

## Determination of the limit of detection of the ZKIR assay in artificially spiked soils

Two soils with contrasted edaphic characteristics (Sandy soil A; clay soil V) and free of indigenous Kp, according to the above procedure, were used. A bacterial suspension of K. pneumoniae ATCC13883T was serially diluted in sterile water and dilutions were enumerated on TSA plates. Five grams aliquots of each soil were spiked with these decreasing Kp dilutions,
resulting in final concentrations from $1.5 \times 10^4$ cfu/g to $1.5 \times 10^{-1}$ cfu/g. Soil samples were

prepared in triplicates with three independently grown inoculums at 4 dilutions (36 spiked

microcosms for each soil). Negative controls were prepared by adding the same volume of sterile

water (3 microcosms for each soil). All 78 spiked and control soil microcosms were enriched for

24 h at 30 °C in 45 ml of LB supplemented with ampicillin (10 mg/l, Sigma Aldrich). Five

hundred µl aliquots of enrichment broth were sampled before and after the 24-h enrichment step,

centrifuged 5 min at 5800 g and washed with sterile water. The pellet was suspended in 500 µl of

sterile water and boiled for 10 min. Boiled enrichments were serially ten-fold diluted (1:10 to

1:100 000) and the diluted suspensions were used for real-time PCR. Moreover, metagenomic

DNA was extracted from spiked soils as described above in ‘Soil DNA extraction’ paragraph.

## Data availability

The detailed ZKIR qPCR operating procedure was made publicly accessible to the scientific

community through the protocols.io platform (https://dx.doi.org/10.17504/protocols.io.7n6hmhe).

Genomic sequences generated in this study were submitted to the European Nucleotide Archive

and are accessible under the BioProject number PRJEB34643.
269 RESULTS

270 Revisiting the phylogenetic distribution of proposed molecular targets for Kp detection

271 Fourteen molecular targets were found in the literature (Figure 1; Table 2) (22–28, 38). Four of them were proposed to detect the Kp complex, while others were designed for specific members of this complex (K. pneumoniae, K. variicola or K. quasipneumoniae; Table 2). Mapping of the presence/absence of the sequence region expected to be amplified by the primers was performed across Klebsiella phylogenetic diversity (Figure 1). Regarding the targets proposed for the entire Kp complex, this in-silico analysis revealed that only khe and tyrB (25, 26) presented both a high sensitivity and a high specificity. We further analysed in silico the primers target sites for these two genes using primer-blast. The results showed that khe primers would be expected to amplify the same region in Raoultella spp. (77 bp, with a sequence identity of the homologous region < 80%, hence not visible on Figure 1), as well as an additional region (348 bp) in these organisms. 272 273 274 275 276 277 278 279 280 281 Furthermore, tyrB primers also appear to be able to amplify the target region in K. aerogenes and Raoultella spp., consistent with the distribution of the target region (Figure 1). Regarding the targets that were proposed to be specific for Kp1, target KpI50233a (24) proved to be the most specific and sensitive one (although with ability to also amplify K. aerogenes isolates), whereas the ones proposed for Kp3 (23) appeared unspecific or to lack sensitivity in our in silico analysis (Figure 1). Regarding the targeted chromosomal class A beta-lactamases genes blaSHV, blaOKP and blaLEN (28), there appeared to be a lack of specificity (Figure 1), which is explained by the high degree of sequence identity between these bla genes, even though they represent distinct targets. To clarify their distribution, we mapped the expected amplicon sequences of blaSHV, blaOKP and blaLEN using a higher threshold (92%, defined based on observed sequence similarity within and between the three gene families) (Figure S1). Using this threshold, we observed a high degree of specificity of the three families for their respective phylogroup/species, as expected 282 283 284 285 286 287 288 289 290 291 292
293 (39). However, it was evident that horizontal gene transfer events implicating the beta-lactamase genes have occurred. In particular, the blaSHV gene was often observed in non-Kp1 phylogroups (particularly Kp2 and Kp4) and even in species outside the Kp complex, such as E. coli or Salmonella spp. This can be attributed to the presence of blaSHV on plasmids and was previously described (21). The gene blaLEN was also observed in a few Kp1 genomes. These observations clearly render the use of the chromosomal beta-lactamase gene targets unreliable as phylogroup/species identification markers (Figure S1).

300

301 ZKIR primers design, PCR assay development and optimization

302 The *tyrB* and *khe* genes were previously proposed as targets for the specific detection of Kp (25, 40). As shown above, *tyrB* and *khe* targets were not totally specific for the Kp complex. We therefore attempted to define primers within the coding sequence of these genes but external to the previously proposed target fragments. However, high identity was observed with non-Kp complex species when blasting the entire coding sequence (data not shown). Consequently, Kp complex-specific primers could not be designed within the coding sequence of these two genes.

308 Interestingly, investigation of sequences upstream of *khe* did reveal a sequence that was highly conserved within the Kp complex. This region located in the intergenic region (IR) upstream of *khe* is partly deleted in other species such as *K. oxytoca* and *Raoultella* spp. As this 249 bp non-coding IR is located between *zur* (zinc uptake regulator) and *khe* (annotated as a putative hemolysin) genes (Figure 2), it was named ZKIR for *zur-khe* intergenic region.

313 A pair of primers (ZKIR_F and ZKIR_R, Figure 2) targeting the ZKIR region was designed and tested in silico. When implemented in the SYBR green PCR assay using reference strain ATCC 700603 (known as *K. pneumoniae*, but belonging in fact to *K. quasipneumoniae* subsp. *similipneumoniae*), these primers successfully amplified a 78 bp sequence, with a melting

316
317 temperature of 80.3 °C. Sequencing of PCR products confirmed amplification of the target sequence.

318 The sensitivity and specificity of the primers were experimentally tested on 2 ng of purified DNA of representatives of Kp phylogroups Kp1 to Kp7 (Table 1) and non-Kp strains. The ZKIR_F and ZKIR_R primers amplified the target sequence in all tested isolates of the Kp complex, with a Threshold Cycle (Ct) value ranging from 13 to 26 and melting temperatures of 80.1 to 80.7 °C. In contrast, when the ZKIR PCR was performed on 88 non-Kp isolates, no amplification was observed, showing that the PCR did not yield false positives. Late, non-specific amplification was recorded with few isolates in the last cycles of the reaction (after cycle 35) and melting temperatures were clearly lower or higher than 80°C, indicating a non-specific amplification. All assays performed on non-Kp samples can therefore be considered negative.

328 **Analytical sensitivity of the ZKIR PCR**

329 PCR reactions were performed with quantities of genomic DNA of *K. pneumoniae* ATCC13883<sup>T</sup> ranging from 7.5 ng (2.5 × 10<sup>6</sup> genomes) to 15 fg (5 genomes) (Figure 3 A and B). Linearity of the assay was evaluated by plotting the Ct values against the log<sub>10</sub> calculated genome number (calculated genome size is about 3.0 fg of DNA per cell, based on a 5.54 Mb *K. pneumoniae* genome). Ct values were closely proportional to the logarithm of the genome number (R<sup>2</sup>=0.99) (Figure 4). The sensitivity of the assay was determined as 15 genomes (45 fg) per reaction mix. No amplification was observed at the lower DNA concentration of 15 fg corresponding to 5 genomes.

338
339 **Analytical sensitivity of the ZKIR assay on soil samples**
In order to assess the performance of the ZKIR assay for detection of Kp directly from soil samples, two soils (A and V) were spiked with bacterial concentrations ranging from $1.5 \times 10^{-1}$ cfu/g to $1.5 \times 10^4$ cfu/g. When soil samples were enriched in LB for 24 h and processed as described above, Kp was detected in all spiked microcosms except in two out of the 3 microcosms of soil A inoculated with the lowest Kp concentration. When the soil/LB suspension was tested prior to incubation, Kp was not detected in any of the spiked microcosms of soil V, whereas in soil A, Kp could only be detected at the highest concentration ($1.5 \times 10^4$ cfu/g).

Finally, when the ZKIR assay was performed using purified metagenomic DNA from soil, positive results were only observed in microcosms spiked with $1.5 \times 10^4$ and $1.5 \times 10^3$ cfu/g in both soils, whereas no positive signal was observed at lower concentrations. The enrichment step thus appeared critical to reach high sensitivity.

### Comparison of the ZKIR real-time PCR and culture-based methods for the detection of Kp in environmental samples

After enrichment and ZKIR real-time PCR, Kp was detected in 41 out of 96 (42.7 %) assayed environmental samples, when the 1:10 dilution was used as template DNA (Table 3). When the 1:100 dilution was used, Kp was detected in a lower number of positive samples (38/96). The Ct values were in the range 18.4 - 36.2. The 96 samples were processed in parallel with the culture-based method. Kp was not detected in any of the ZKIR-negative samples: when presumptive colonies were detected, they were always identified by MALDI-TOF MS as non-Kp, and belonged to closely related species such as *K. oxytoca*, *Raoultella* spp. and *Serratia* spp. In contrast, Kp was isolated in 37 out of the 41 ZKIR positive samples. These isolates were all identified as either *K. pneumoniae* or *K. variicola* by MALDI-TOF MS.
363 Genomic characterization of 31 of these isolates (1 was contaminated and 5 were not yet available) revealed a dominance of Kp1 (n= 20, 65%), followed by Kp3 (n=10, 32%) and Kp4 (n=1, 3%; Table S1). Population diversity analysis based on MLST (41) and cgMLST (24) revealed a high genotypic diversity, with 23 STs and 25 cgMLST types. In five cases, the strain detected in the soil was the same as the one present in the leaves and/or roots (e.g., SB6439 and SB6440; Table S1), showing colonization of several plant sites by the same strain. Only 2 isolates belonging to STs commonly found in the clinical settings were detected (ST37 and ST76).
364
365
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368
369
370 Interestingly, from the predicted O-types, the O3-type represented 50% of Kp population, which contrasts with the nosocomial situation, where types O1 and O2 are dominant (2, 42), but not with human carriage where the O3-type is also dominant (O3-31%, O1-19% and O2-17%; our unpublished results). Also contrasting with the clinical epidemiology of Kp, a low level of antibiotic resistance and virulence genes was observed, with 93.5% of the strains presenting an ancestral 'wild-type' susceptibility genotype (Table S1). The number of strains harboring plasmids detected in these environmental samples was also low (n=9, 29%), as well as the number of plasmid-encoded heavy metal tolerance genes (mainly the silver and copper tolerance clusters). These results contrast with clinical and animal isolates, where plasmids and metal tolerance clusters are common (42, Rodrigues, unpublished results). As expected (1, 37), all Kp3 isolates harbored the *nif* cluster responsible for nitrogen fixation. Interestingly, the *nif* cluster was also present in one Kp1 isolate from soil (SB6181). The phylogenetic analysis (Figure S2) of the *nif* cluster from our environmental isolates compared to a panel of reference strains (37) revealed that strain SB6181 (Kp1) branched within *K. variicola* strains (Kp3), showing that the *nif* cluster in this Kp1 strain was acquired via horizontal gene transfer from a *K. variicola* donor. Kp3 was also the inferred donor of the *nif* gene cluster for Kp5 and Kp6 *nif*-positive strains (Figure S2).
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384
385
386 **Discussion**

387 Routes of transmission of ecologically generalist human pathogens are usually complex and
388 poorly understood. Proper risk management requires a holistic approach which has been theorised
389 as the One Health concept (44). Despite the fact that the number of human infections caused by
390 members of the Kp complex are on the rise and are increasingly resistant to antimicrobial
391 treatment (1, 45), the ecology of Kp remains poorly understood. Identification of the various
392 habitats in which Kp strives, and the routes of transmission to humans, for example through
393 specific types of food, is critical in order to limit exposure. In this prospect, large-scale sampling
394 is needed to define the sources of Kp contamination and such surveys will require sensitive,
395 reliable and cost-efficient detection methods.

396 Several molecular assays for the detection and/or identification of Klebsiella from clinical or food
397 and environmental samples have been previously proposed. These methods allow detecting
398 mainly K. pneumoniae (sensu stricto) and K. variicola (Figure 1). However, the Kp complex
399 currently encompasses 7 taxa. Our in-silico analyses showed that some previously described PCR
400 assays could be useful for the identification of phylogenetic subsets of the Kp complex
401 (Kp150233a for Kp1, blaOKP for Kp2/Kp4 and blaLEN for Kp3/Kp5).

402
403 Given that all members of the Kp complex can cause infections in humans or animals, developing
404 a novel method with the ability to detect the Kp complex by targeting exhaustively all currently
405 known Kp phylogroups would be an important advance. Although several previous targets were
406 designed for the entire Kp complex, they predate recent taxonomic advances and have therefore
407 not been validated on the entire phylogenetic breadth of this bacterial group. Here, we found by
408 in-silico approaches that khe and tyrB assays lack specificity, which may negatively affect Kp
409 detection efforts especially when testing microbiologically complex samples from the
410 environment, such as soil.

411 We therefore aimed to develop a novel real-time PCR assay, and used an intergenic region
412 located adjacent to the previously proposed target gene *khe*. Using well-defined reference strains
413 representative of current taxonomy (20, 32, 33), we show that the novel ZKIR PCR real-time
414 assay allows the accurate detection of all members of the Kp complex. We found complete
415 specificity and sensitivity (i.e., phylogenetic coverage) of this assay based on our reference
416 strains panel. To improve analytical sensitivity, a 24 h enrichment culture followed by an easy,
417 fast and cost-effective sample processing was implemented, upstream of molecular detection
418 using the ZKIR assay. This was necessary for complex matrices such as soil or sewage, where
419 microbial diversity and abundance are high, in which case direct detection of one particular group
420 of organisms, which might be present only at low abundance, is challenging. This procedure
421 turned out to be sensitive enough to detect 1 single Kp bacterium in 5 g of soil. The ZKIR assay
422 also appeared slightly more sensitive than the SCAI culture-based method, which is based on the
423 ability of *Klebsiella* strains to utilize citrate and inositol, itself previously shown to be highly
424 sensitive and to recover most Kp members (36, 46). Therefore, this fast and easy novel molecular
425 method represents a powerful approach for screening large numbers of samples. This will spare
426 the time-consuming handling of numerous presumptive colonies necessary for confirmation of
427 their identification, given that *K. oxytoca*, *Raoultella*, *Serratia* and *Enterobacter* are able to form
428 colonies on SCAI agar, with morphological characteristics similar to Kp (36, 47). As such, the
429 ZKIR protocol was significantly faster than culture, as results were available on average 26 h
430 after sampling (24 h enrichment, sample treatment, PCR) while up to 96 h were necessary when
431 using the culture-based method (24 h enrichment, plate incubation, colony purification, MALDI-
432 TOF identification).
Interestingly, the implementation of the ZKIR PCR real-time assay evidenced a high (43%) detection rate of Kp from environmental samples, which represent niches that are still underexplored for Kp presence and biological characteristics. The high detection rate was largely confirmed by culture, suggesting a low rate of false positive qPCR results. This novel and original sampling strategy allowed us to explore the genomic features of environmental Kp populations. A strong contrast with Kp isolates typically recovered in the clinical setting was observed. First, a high genetic diversity was observed, with no predominant sublineage. This situation contrasts with MDR or hypervirulent Kp populations, from which frequent sublineages (so-called high-risk clones) are recovered. Second, environmental Kp isolates were almost devoid of antibiotic resistance and virulence genes, in conspicuous contrast with clinical samples (48).

Finally, the genomic characteristics of environmental Kp isolates revealed interesting biological features, such as a frequent O3 O-antigen type and the horizontal transfer of nitrogen fixation gene cluster into Kp1, the main phylogroup associated with human infections. These data call for further studies into the biology of soil Kp isolates, which may reveal interesting novel adaptive strategies of this important generalist pathogen. Finally, our findings suggest that environmental Kp populations differ from clinical Kp populations, which implies indirect and possibly complex epidemiological links between environmental and clinical Kp. The ZKIR PCR real-time assay developed herein is expected to enable future large-scale studies into this important question.

In conclusion, the ZKIR assay is a new tool for Kp detection that is highly specific, sensitive, reliable and cost-effective. To accelerate uptake of this method, the corresponding protocol was released publicly on protocols.io (dx.doi.org/10.17504/protocols.io.7n6hmhe). This simple method can easily be implemented in laboratories equipped with real-time PCR thermocyclers (MedVetKlebs consortium, unpublished results). Using the ZKIR method after a short culture
457 enrichment step greatly enhances sensitivity. As this rapid screen of samples allows one to focus

458 only on ZKIR positive samples for more labour-intensive downstream microbiological isolation

459 and characterization, it is our hope that it will contribute to advance knowledge on the biology of

460 environmental Kp and on the reservoirs and transmission routes of this increasingly important

461 group of pathogens.
462 SUPPLEMENTAL MATERIAL

463 SUPPLEMENTAL FILE: 2 PDF files, 1 Excel file

464

465

466 ACKNOWLEDGEMENTS

467 We thank Alexis Criscuolo for access to an early version of the JolyTree software tool, Julien

468 Guglielmini for providing us access to in-house scripts for mapping the target genes, and Juan

469 Sebastian Lopez Fernandes for assistance with genomic analyses.

470 This work is part of the MedVetKlebs project, a component of the One Health European Joint

471 Programme, and has thereby received funding from the European Union's Horizon 2020 research

472 and innovation programme under Grant Agreement No 773830.
473 REFERENCES

474 1. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, Jenney A, Connor TR, Hsu LY, Severin J, Brisse S, Cao H, Wilksch J, Gorrie C, Schultz MB, Edwards DJ, Nguyen KV, Nguyen TV, Dao TT, Mensink M, Minh VL, Nhu NTK, Schultsz C, Kuntaman K, Newton PN, Moore CE, Strugnell RA, Thomson NR. 2015. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in *Klebsiella pneumoniae*, an urgent threat to public health. Proc Natl Acad Sci 112:E3574-E3581.

480 2. David S, Reuter S, Harris SR, Glasner C, Feltwell T, Argimon S, Abudahab K, Goater R, Giani T, Errico G, Aspbury M, Sjunnebo S, Koraqi A, Lacej D, Apfalter P, Hartl R, Glupczynski Y, Huang T-D, Strateva T, Marteva-Proevska Y, Andrasevic AT, Butic I, Pieridou-Bagatzouni D, Maikanti-Charalampous P, Hrabak J, Zemlickova H, Hammerum A, Jakobsen L, Ivanova M, Pavelkovich A, Jalava J, Österblad M, Dortet L, Vaux S, Kaase M, Gatermann SG, Vatopoulos A, Tryfinopoulou K, Tóth Á, Jánvári L, Boo TW, McGrath E, Carmeli Y, Adler A, Pantosti A, Monaco M, Raka L, Kurti A, Balode A, Saule M, Miciuleviciene J, Mierauskaite A, Perrin-Weniger M, Reichert P, Nestorova N, Debattista S, Mijovic G, Lopicic M, Samuelsen Ø, Haldorsen B, Zabicka D, Literacka E, Caniça M, Manageiro V, Kaftandzieva A, Trajkovska-Dokic E, Damian M, Lixandru B, Jelesic Z, Trudic A, Niks M, Schreterova E, Pirs M, Cerar T, Oteo J, Aracil B, Giske C, Sjöström K, Gür D, Cakar A, Woodford N, Hopkins K, Wiuff C, Brown DJ, Feil EJ, Rossolini GM, Aanensen DM, Grundmann H, the EuSCAPE Working Group, the ESGEM Study Group. 2019. Epidemic of carbapenem-resistant Klebsiella pneumoniae in Europe is driven by nosocomial spread. Nat Microbiol 4:1919-1929.

494 3. Ko W-C. 2002. Community-acquired *Klebsiella pneumoniae* bacteremia: global differences in clinical patterns. Emerg Infect Dis 8:160-166.
496 4. Zhang Y, Zeng J, Liu W, Zhao F, Hu Z, Zhao C, Wang Q, Wang X, Chen H, Li H, Zhang F, Li S, Cao B, Wang H. 2015. Emergence of a hypervirulent carbapenem-resistant *Klebsiella pneumoniae* isolate from clinical infections in China. J Infect 71:553–560.

499 5. Gorrie CL, Mirčeta M, Wick RR, Edwards DJ, Thomson NR, Strugnell RA, Pratt NF, Garlick JS, Watson KM, Pilcher DV, McGloughlin SA, Spelman DW, Jenney AWJ, Holt KE. 2017. Gastrointestinal carriage is a major reservoir of *Klebsiella pneumoniae* infection in intensive care patients. Clin Infect Dis 65:208–215.

503 6. Brisse S, Grimont F, Grimont PAD. 2006. The Genus *Klebsiella*, p. 159–196. In Dworkin, M, Falkow, S, Rosenberg, E, Schleifer, K-H, Stackebrandt, E (eds.), The Prokaryotes. Springer New York.

506 7. Munoz MA, Ahlström C, Rauch BJ, Zadoks RN. 2006. Fecal shedding of *Klebsiella pneumoniae* by dairy cows. J Dairy Sci 89:3425–3430.

508 8. Rosenblueth M, Martínez L, Silva J, Martínez-Romero E. 2004. *Klebsiella variicola*, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol 27:27–35.

510 9. Lee D-Y, Shannon K, Beaudette LA. 2006. Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR. J Microbiol Methods 65:453–467.

513 10. Benami M, Gross A, Herzberg M, Orlofsky E, Vonshak A, Gillor O. 2013. Assessment of pathogenic bacteria in treated graywater and irrigated soils. Sci Total Environ 458–460:298–302.

515 11. Barati A, Ghaderpour A, Chew L, Bong C, Thong K, Chong V, Chai L. 2016. Isolation and characterization of aquatic-borne *Klebsiella pneumoniae* from tropical estuaries in Malaysia. Int J Environ Res Public Health 13:426.
518 12. Falomir MP, Gozalbo D, Rico H. 2010. Coliform bacteria in fresh vegetables: from cultivated lands to consumers, volume 2. *In* A. Mendez-Vilas, Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology.

519 to consumers, volume 2. In A. Mendez-Vilas, Current Research, Technology and Education Topics
520 in Applied Microbiology and Microbial Biotechnology.

521 13. Podschun R, Pietsch S, Holler C, Ullmann U. 2001. Incidence of *Klebsiella* species in surface waters
522 and their expression of virulence factors. Appl Environ Microbiol 67:3325-3327.

523 14. Davis GS, Waits K, Nordstrom L, Weaver B, Aziz M, Gauld L, Grande H, Bigler R, Horwinski J,
524 Porter S, Stegger M, Johnson JR, Liu CM, Price LB. 2015. Intermingled *Klebsiella pneumoniae*
525 populations between retail meats and human urinary tract infections. Clin Infect Dis 61:892-899.

526 15. Zadoks RN, Griffiths HM, Munoz MA, Ahlstrom C, Bennett GJ, Thomas E, Schukken YH. 2011.
527 Sources of *Klebsiella* and *Raoultella* species on dairy farms: Be careful where you walk. J Dairy Sci
528 94:1045-1051.

529 16. Runcharoen C, Moradigaravand D, Blane B, Paksanont S, Thammachote J, Anun S, Parkhill J,
530 Chantratita N, Peacock SJ. 2017. Whole genome sequencing reveals high-resolution epidemiological
531 links between clinical and environmental *Klebsiella pneumoniae*. Genome Med 9:6.

532 17. Gregg S. Davis, Lance B. Price. 2016. Recent research examining links among *Klebsiella
533 pneumoniae* from food, food animals, and human extraintestinal infections. Curr Envir Health Rpt
534 3:128-135.

535 18. Brisse S, Passet V, Grimont PAD. 2014. Description of *Klebsiella quasipneumoniae* sp. nov.,
536 isolated from human infections, with two subspecies, *Klebsiella quasipneumoniae* subsp.
537 quasipneumoniae subsp. nov. and *Klebsiella quasipneumoniae* subsp. similipneumoniae subsp. nov.,
538 and demonstration that *Klebsiella Singaporeensis* is a junior heterotypic synonym of *Klebsiella
539 variicola*. Int J Syst Evol Microbiol 64:3146-3152.
540 19. Long SW, Linson SE, Ojeda Saavedra M, Cantu C, Davis JJ, Brettin T, Olsen RJ. 2017. Whole-genome sequencing of a human clinical isolate of the novel species *Klebsiella quasivariicola* sp. nov. Genome Announc 5:e01057-17.

543 20. Rodrigues C, Passet V, Rakotondrasoa A, Diallo TA, Criscuolo A, Brisse S. 2019. Description of *Klebsiella africanensis* sp. nov., *Klebsiella variicola* subsp. *tropicalensis* subsp. nov. and *Klebsiella variicola* subsp. *variicola* subsp. nov. Res Microbiol 170:165-170.

546 21. Long SW, Linson SE, Ojeda Saavedra M, Cantu C, Davis JJ, Brettin T, Olsen RJ. 2017. Whole-Genome Sequencing of Human Clinical *Klebsiella pneumoniae* Isolates Reveals Misidentification and Misunderstandings of *Klebsiella pneumoniae*, *Klebsiella variicola*, and *Klebsiella quasipneumoniae*. mSphere 2:e00290-17.

550 22. Liu Y, Liu C, Zheng W, Zhang X, Yu J, Gao Q, Hou Y, Huang X. 2008. PCR detection of *Klebsiella pneumoniae* in infant formula based on 16S-23S internal transcribed spacer. Int J Food Microbiol 125:230-235.

553 23. Garza-Ramos U, Silva-Sánchez J, Martínez-Romero E, Tinoco P, Pina-Gonzales M, Barrios H, Martínez-Barnetche J, Gómez-Barreto RE, Tellez-Sosa J. 2015. Development of a Multiplex-PCR probe system for the proper identification of *Klebsiella variicola*. BMC Microbiol 15:64.

556 24. Bialek-Davenet S, Criscuolo A, Ailloud F, Passet V, Nicolas-Chanoine M-H, Decre D, Brisse S. 2014. Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2. J Med Microbiol 63:1608-1614.

559 25. Yin-Ching C, Jer-Horng S, Ching-Nan L, Ming-Chung C. 2002. Cloning of a gene encoding a unique haemolysin from *Klebsiella pneumoniae* and its potential use as a species-specific gene probe. Microb Pathog 33:1-6.
562 26. Jeong E-S, Lee K-S, Heo S-H, Seo J-H, Choi Y-K. 2013. Rapid Identification of *Klebsiella pneumoniae*, *Corynebacterium kutscheri*, and *Streptococcus pneumoniae* using triplex Polymerase Chain Reaction in rodents. *Exp Anim* 62:35-40.

565 27. Hartman LJ, Selby EB, Whitehouse CA, Coyne SR, Jaissle JG, Twenhafel NA, Burke RL, Kulesh DA. 2009. Rapid real-time PCR assays for detection of *Klebsiella pneumoniae* with the *rmpA* or *magA* genes associated with the hypermucoviscosity phenotype. *J Mol Diagn* 11:464-471.

568 28. Fonseca EL, Ramos N da V, Andrade BGN, Morais LLCS, Marin MFA, Vicente ACP. 2017. A one-step multiplex PCR to identify *Klebsiella pneumoniae*, *Klebsiella variicola*, and *Klebsiella quasipneumoniae* in the clinical routine. *Diagn Microbiol Infect Dis* 87:315-317.

571 29. Singh K, Mangold KA, Wyant K, Schora DM, Voss B, Kaul KL, Hayden MK, Chundi V, Peterson LR. 2012. Rectal screening for *Klebsiella pneumoniae* carbapenemases: comparison of Real-Time PCR and culture using two selective screening agar plates. *J Clin Microbiol* 50:2596-2600.

574 30. Bilodeau G. 2011. Quantitative polymerase chain reaction for the detection of organisms in soil. *CAB Rev Perspect Agric Vet Sci Nutr Nat Resour* 6.

576 31. Plassart P, Terrat S, Thomson B, Griffiths R, Dequiedt S, Lelievre M, Regnier T, Nowak V, Bailey M, Lemanceau P, Bispo A, Chabbi A, Maron P-A, Mougel C, Ranjard L. 2012. Evaluation of the ISO Standard 11063 DNA extraction procedure for assessing soil microbial abundance and community structure. *PLoS ONE* 7:e44279.

580 32. Rodrigues C, Passet V, Rakotondrasoa A, Brisse S. 2018. Identification of Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola and Related Phylogroups by MALDI-TOF Mass Spectrometry. *Front Microbiol* 9:3000.
583 33. Passet V, Brisse S. 2018. Description of *Klebsiella grimontii* sp. nov. Int J Syst Evol Microbiol 68:377-381.

585 34. Criscuolo A. 2019. A fast alignment-free bioinformatics procedure to infer accurate distance-based phylogenetic trees from genome assemblies. Res Ideas Outcomes 5:e36178.

587 35. López-Gutiérrez et al. - 2004 - Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR. J. Microbiol. Methods 57:399-407.

589 36. Van Kregten E, Westerdaal NAC, Willers JMN. 1984. New, simple medium for selective recovery of *Klebsiella pneumoniae* and *Klebsiella oxytoca* from human feces. J Clin Microbiol 20:6.

591 37. Blin C, Passet V, Touchon M, Rocha EPC, Brisse S. 2017. Metabolic diversity of the emerging pathogenic lineages of *Klebsiella pneumoniae*: Metabolic diversity of *Klebsiella pneumoniae*. Environ Microbiol 19:1881-1898.

594 38. Tian Y, Wang L, Zhang J, Han Q, Xia X, Song Y, Yang G. 2019. CelB is a suitable marker for rapid and specific identification of Klebsiella pneumoniae by the loop-mediated isothermal amplification (LAMP) assay. Braz J Microbiol 50:961-967.

597 39. Haeggman S, Lofdahl S, Paauw A, Verhoef J, Brisse S. 2004. Diversity and Evolution of the Class A Chromosomal Beta-Lactamase Gene in *Klebsiella pneumoniae*. Antimicrob Agents Chemother 48:2400-2408.

600 40. Jeong E-S, Lee K-S, Heo S-H, Seo J-H, Choi Y-K. 2013. Rapid Identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae Using Triplex Polymerase Chain Reaction in Rodents. Exp Anim 62:35-40.

603 41. Diancourt L, Passet V, Verhoef J, Grimont PAD, Brisse S. 2005. Multilocus Sequence Typing of *Klebsiella pneumoniae* Nosocomial Isolates. J Clin Microbiol 43:4178.
605 42. Follador R, Heinz E, Wyres KL, Ellington MJ, Kowarik M, Holt KE, Thomson NR. 2016. The diversity of Klebsiella pneumoniae surface polysaccharides. Microb Genomics 2. 606

607 43. Sütterlin S, Dahlö M, Tellgren-Roth C, Schaal W, Melhus Å. 2017. High frequency of silver resistance genes in invasive isolates of Enterobacter and Klebsiella species. J Hosp Infect 96:256–261. 608 609

610 44. King LJ, Anderson LR, Blackmore CG, Blackwell MJ, Lautner EA, Marcus LC, Meyer TE, Monath TP, Nave JE, Ohle J, Pappaioanou M, Sobota J, Stokes WS, Davis RM, Glasser JH, Mahr RK. 2008. Executive summary of the AVMA One Health Initiative Task Force report. J Am Vet Med Assoc 611 612 613

614 45. Nordmann P, Naas T, Poirel L. 2011. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1798. 615

616 46. Passet V, Brisse S. 2015. Association of Tellurite Resistance with Hypervirulent Clonal Groups of Klebsiella pneumoniae. J Clin Microbiol 53:1380–1382. 617

618 47. Ohtomo R, Saito M. 2003. A New selective medium for detection of Klebsiella from dairy environments. Microbes Environ 18:138–144. 619

620 48. Wyres KL, Holt KE. 2016. Klebsiella pneumoniae Population Genomics and Antimicrobial-Resistant Clones. Trends Microbiol 24:944–956. 621

622

623

624

625 TABLES

626 TABLE 1 Bacterial strains tested to develop the ZKIR PCR assay.
627 **TABLE 2** Molecular target sequences previously proposed for *Klebsiella pneumoniae*
628 identification.

629 **TABLE 3** Comparison between ZKIR PCR and culture results in samples collected in Auxonne,
630 France, between July and September 2018.

631
632 **FIGURES**

633 **FIG 1** Phylogenetic distribution of the molecular targets for *K. pneumoniae* detection described
634 in the literature. The ZKIR target sequence corresponds to target 15; other targets are given in
635 Table 2. The inner circle colored sectors correspond to *K. pneumoniae* phylogroups or other
636 *Klebsiella* species (see color key). Molecular targets were detected in the corresponding genomes
637 using BLASTN with 80% nucleotide identity and 80% length coverage.

638
639 **FIG 2** Genetic context of the ZKIR region on the genome of strain *K. pneumoniae* ATCC 13883<sup>T</sup>
640 (GenBank accession number GCA_000742135.1) and detailed location of ZKIR primers and
641 amplicon within the 249-base pair (bp) region that is specific for the *K. pneumoniae* species
642 complex (boxed area).

643
644 **FIG 3** Amplification curves (A) and melt curve peaks (B) established using real-time PCR
645 targeting the ZKIR region with serial dilutions of *K. pneumoniae* ATCC13883<sup>T</sup> DNA. Triplicate
646 of DNA concentrations of 7.5 ng, 750 pg, 75 pg, 7.5 pg, 750 fg are presented, but lower dilutions
647 are not.

648
649 **FIG 4** Standard curve established using real-time ZKIR PCR with serial dilutions of
650 *K. pneumoniae* ATCC13883<sup>T</sup> DNA from 7.5 ng to 45 fg.
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Phylogenetic distribution of the molecular targets for *K. pneumoniae* detection described in the literature. The ZKIR target sequence corresponds to target 15; other targets are given in Table 2. The inner circle colored sectors correspond to *K. pneumoniae* phylogroups or other *Klebsiella* species (see color key). Molecular targets were detected in the corresponding genomes using BLASTN with 80% nucleotide identity and 80% length coverage.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2** Genetic context of the ZKIR region on the genome of strain *K. pneumoniae* ATCC 13883T (GenBank accession number GCA_000742135.1) and detailed location of ZKIR primers and amplicon within the 249-base pair (bp) region that is specific for the *K. pneumoniae* species complex (boxed area).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 3** Amplification curves (A) and melt curve peaks (B) established using real-time PCR targeting the ZKIR region with serial dilutions of *K. pneumoniae* ATCC13883T DNA. Triplicates of DNA concentrations of 7.5 ng, 750 pg, 75 pg, 7.5 pg and 750 fg are presented, whereas lower dilutions are not.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 4** Standard curve established using real-time ZKIR PCR with serial dilutions of *K. pneumoniae* ATCC13883T DNA from 7.5 ng to 45 fg.</figcaption></figure>
<table><caption>TABLE 1 Bacterial strains tested to develop the ZKIR PCR assay.</caption><thead><tr><th>Organism name (Phylogroup)</th><th>Strain name</th><th>Strain bank ID*</th><th>Country</th><th>Isolation year</th></tr></thead><tbody><tr><td>Klebsiella pneumoniae (Kp1)</td><td>SB4-2</td><td>SB1067</td><td>Netherlands</td><td>2002</td></tr><tr><td>K. pneumoniae (Kp1)</td><td>ATCC 13883<sup>T</sup></td><td>SB132</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. pneumoniae (Kp1)</td><td>MGH 78578</td><td>SB107</td><td>n.a.</td><td>1994</td></tr><tr><td>K. pneumoniae (Kp1)</td><td>none</td><td>SB1139</td><td>Netherlands</td><td>2002</td></tr><tr><td>K. pneumoniae (Kp1)</td><td>5-2</td><td>SB617</td><td>Netherlands</td><td>2000</td></tr><tr><td>K. pneumoniae (Kp1)</td><td>04A025</td><td>SB20</td><td>France</td><td>1997</td></tr><tr><td>K. pneumoniae (Kp1)</td><td>2-3</td><td>SB612</td><td>Netherlands</td><td>2000</td></tr><tr><td>K. pneumoniae (Kp1)</td><td>BJ1-GA</td><td>SB4496</td><td>France</td><td>2011</td></tr><tr><td>K. pneumoniae (Kp1)</td><td>n.a.</td><td>MIAE07651</td><td>France</td><td>2015</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>01A030<sup>T</sup></td><td>SB11</td><td>Austria</td><td>1997</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>none</td><td>SB1124</td><td>Netherlands</td><td>2002</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>U41</td><td>SB2110</td><td>Germany</td><td>1990</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>10A442</td><td>SB224</td><td>Italy</td><td>1998</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>99-1002</td><td>SB2478</td><td>Netherlands</td><td>1999</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>18A451</td><td>SB255</td><td>Spain</td><td>1998</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>11128</td><td>SB3445</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>18A069</td><td>SB59</td><td>Spain</td><td>1997</td></tr><tr><td>K. quasipneumoniae subsp. quasipneumoniae (Kp2)</td><td>Kleb Ali 0320584</td><td>SB98</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>01A065</td><td>SB1</td><td>Austria</td><td>1997</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>07A058</td><td>SB31</td><td>Germany</td><td>1997</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>IPEUC-1516</td><td>SB3278</td><td>France</td><td>1988</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>CIP 53.24</td><td>SB3295</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>Ørskov 1756/51</td><td>SB3301</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>F2R9<sup>T</sup></td><td>SB48</td><td>Mexico</td><td>n.a.</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>6115</td><td>SB489</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>Ørskov 4425/51</td><td>SB497</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. variicola subsp. variicola (Kp3)</td><td>Kp342</td><td>SB579</td><td>USA</td><td>n.a.</td></tr><tr><td>K. quasipneumoniae subsp. similipneumoniae (Kp4)</td><td>09A323</td><td>SB164</td><td>Greece</td><td>1997</td></tr><tr><td>K. quasipneumoniae subsp. similipneumoniae (Kp4)</td><td>12A476</td><td>SB203</td><td>Netherlands</td><td>1998</td></tr><tr><td>K. quasipneumoniae subsp. similipneumoniae (Kp4)</td><td>07A044<sup>T</sup></td><td>SB30</td><td>Germany</td><td>1997</td></tr><tr><td>K. quasipneumoniae subsp. similipneumoniae (Kp4)</td><td>325</td><td>SB3233</td><td>France</td><td>1975</td></tr><tr><td>K. quasipneumoniae subsp. similipneumoniae (Kp4)</td><td>Ørskov 1303/50</td><td>SB3297</td><td>Turkey</td><td>n.a.</td></tr><tr><td>K. quasipneumoniae subsp. similipneumoniae (Kp4)</td><td>Ørskov 4463/52</td><td>SB500</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. quasipneumoniae subsp. similipneumoniae (Kp4)</td><td>CIP 110288</td><td>SB4697</td><td>China</td><td>2010</td></tr><tr><td>K. quasipneumoniae subsp. similipneumoniae (Kp4)</td><td>1-1</td><td>SB610</td><td>Netherlands</td><td>2000</td></tr><tr><td>K. variicola subsp. tropica (Kp5)</td><td>Gal12</td><td>SB824</td><td>Mexico</td><td>n.a.</td></tr><tr><td>K. variicola subsp. tropica (Kp5)</td><td>CDC 4241-71</td><td>SB94</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. variicola subsp. tropica (Kp5)</td><td>885</td><td>SB5439</td><td>Madagascar</td><td>2016</td></tr><tr><td>K. variicola subsp. tropica (Kp5)</td><td>1266<sup>T</sup></td><td>SB5531</td><td>Madagascar</td><td>2016</td></tr><tr><td>K. variicola subsp. tropica (Kp5)</td><td>1283</td><td>SB5544</td><td>Madagascar</td><td>2016</td></tr><tr><td>K. variicola subsp. tropica (Kp5)</td><td>1375</td><td>SB5610</td><td>Madagascar</td><td>2016</td></tr><tr><td>K. variicola subsp. tropica (Kp5)</td><td>814</td><td>SB5387</td><td>Madagascar</td><td>2015</td></tr><tr><td>'K. quasivariicola' (Kp6)</td><td>08A119</td><td>SB33</td><td>Germany</td><td>1997</td></tr><tr><td>'K. quasivariicola' (Kp6)</td><td>10982</td><td>SB6071</td><td>USA</td><td>2005</td></tr><tr><td>'K. quasivariicola' (Kp6)</td><td>01-467-2ECBU</td><td>SB6094</td><td>Madagascar</td><td>2015</td></tr><tr><td>'K. quasivariicola' (Kp6)</td><td>01-310A</td><td>SB6095</td><td>Madagascar</td><td>2013</td></tr></tbody></table>
<table><tr><td>'K. quasivariicola' (Kp6)</td><td>KPN1705<sup>T</sup></td><td>SB6096</td><td>USA</td><td>2014</td></tr><tr><td>K. africana (Kp7)</td><td>200023<sup>T</sup></td><td>SB5857</td><td>Senegal</td><td>2016</td></tr><tr><td>K. michiganensis (Ko1)</td><td>CIP 110787<sup>T</sup></td><td>SB4934</td><td>USA</td><td>2010</td></tr><tr><td>K. michiganensis (Ko1)</td><td>05A071</td><td>SB71</td><td>France</td><td>1997</td></tr><tr><td>K. michiganensis (Ko1)</td><td>09A029</td><td>SB78</td><td>Greece</td><td>1997</td></tr><tr><td>K. grimontii (Ko6)</td><td>07A479</td><td>SB324</td><td>Germany</td><td>1998</td></tr><tr><td>K. grimontii (Ko6)</td><td>06D090</td><td>SB352</td><td>France</td><td>1998</td></tr><tr><td>K. grimontii (Ko6)</td><td>06D021<sup>T</sup></td><td>SB73</td><td>France</td><td>1997</td></tr><tr><td>K. oxytoca (Ko2)</td><td>ATCC 13182<sup>T</sup></td><td>SB175</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. oxytoca (Ko2)</td><td>02A067</td><td>SB131</td><td>Belgium</td><td>1997</td></tr><tr><td>K. oxytoca (Ko2)</td><td>NCTC 49131</td><td>SB136</td><td>n.a.</td><td>n.a.</td></tr><tr><td>K. aerogenes</td><td>none</td><td>MIAE07652</td><td>France</td><td>n.a.</td></tr><tr><td>K. aerogenes</td><td>CIP 60.86<sup>T</sup></td><td>SB3629</td><td>France</td><td>n.a.</td></tr><tr><td>K. aerogenes</td><td>01A089</td><td>SB538</td><td>Austria</td><td>1997</td></tr><tr><td>K. aerogenes</td><td>02A002</td><td>SB539</td><td>Belgium</td><td>1997</td></tr><tr><td>Raoultella terrigena</td><td>ATCC 33257<sup>T</sup></td><td>SB170</td><td>n.a.</td><td>n.a.</td></tr><tr><td>R. terrigena</td><td>17C143</td><td>SB313</td><td>Spain</td><td>1998</td></tr><tr><td>R. terrigena</td><td>V9813596</td><td>SB2796</td><td>Netherlands</td><td>1998</td></tr><tr><td>R. planticola</td><td>01A041</td><td>SB7</td><td>Austria</td><td>1997</td></tr><tr><td>R. planticola</td><td>ATCC 33531<sup>T</sup></td><td>SB174</td><td>n.a.</td><td>n.a.</td></tr><tr><td>R. planticola</td><td>12C169</td><td>SB303</td><td>Netherlands</td><td>1998</td></tr><tr><td>R. ornithinolytica</td><td>ATCC 31898</td><td>SB171</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Acinetobacter calcoaceticus</td><td>ATCC 14987</td><td>none</td><td>USA</td><td>n.a.</td></tr><tr><td>A. lwoffii</td><td>none</td><td>MIAE07654</td><td>France</td><td>1998</td></tr><tr><td>A. johnsonii</td><td>ATCC 17909<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>A. pittii</td><td>none</td><td>MIAE07655</td><td>France</td><td>1998</td></tr><tr><td>Aeromonas caviae</td><td>none</td><td>MIAE07656</td><td>France</td><td>n.a.</td></tr><tr><td>A. hydrophila</td><td>none</td><td>MIAE07657</td><td>France</td><td>n.a.</td></tr><tr><td>Agrobacterium tumefaciens</td><td>C.58</td><td>MIAE07675</td><td>France</td><td>1996</td></tr><tr><td>Alcaligenes faecalis</td><td>none</td><td>MIAE07658</td><td>France</td><td>n.a.</td></tr><tr><td>Bacillus cereus</td><td>ATCC 53522</td><td>none</td><td>USA</td><td>n.a.</td></tr><tr><td>B. cereus</td><td>none</td><td>MIAE07659</td><td>France</td><td>n.a.</td></tr><tr><td>B. circulans</td><td>none</td><td>MIAE07660</td><td>France</td><td>n.a.</td></tr><tr><td>B. megaterium</td><td>none</td><td>MIAE07661</td><td>France</td><td>n.a.</td></tr><tr><td>B.s subtilis</td><td>none</td><td>MIAE07662</td><td>France</td><td>n.a.</td></tr><tr><td>Edwardsiella hoshinae</td><td>DSM 13771<sup>T</sup></td><td>none</td><td>France</td><td>n.a.</td></tr><tr><td>E. tarda</td><td>DSM 30052<sup>T</sup></td><td>none</td><td>USA</td><td>n.a.</td></tr><tr><td>Enterobacter hafnia</td><td>none</td><td>MIAE07653</td><td>France</td><td>n.a.</td></tr><tr><td>Enterococcus casseliflavus</td><td>DSM 20680<sup>T</sup></td><td>none</td><td>n.a.</td><td>1984</td></tr><tr><td>E. faecalis</td><td>DSM 12956</td><td>none</td><td>USA</td><td>n.a.</td></tr><tr><td>E. faecalis</td><td>DSM 20376</td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>E. faecium</td><td>DSM 25389</td><td>none</td><td>Netherlands</td><td>n.a.</td></tr><tr><td>E. faecium</td><td>DSM 6177</td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>E. faecium</td><td>DSM 25644</td><td>none</td><td>Georgia</td><td>n.a.</td></tr><tr><td>E. faecium</td><td>DSM 25697</td><td>none</td><td>South Korea</td><td>n.a.</td></tr><tr><td>E. faecium</td><td>DSM 13590</td><td>none</td><td>Germany</td><td>n.a.</td></tr><tr><td>E. faecium</td><td>DSM 20477<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>E. galinarum</td><td>DSM 20628</td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>E. hirae</td><td>DSM 20160<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>E. raffinosus</td><td>DSM 5633<sup>T</sup></td><td>none</td><td>USA</td><td>1979</td></tr></table>
<table><tr><td>Escherichia coli</td><td>DSM 499</td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>E. coli</td><td>none</td><td>MIAE02388</td><td>France</td><td>2015</td></tr><tr><td>E. coli</td><td>none</td><td>MIAE02376</td><td>France</td><td>2015</td></tr><tr><td>E. coli</td><td>none</td><td>MIAE02510</td><td>France</td><td>2015</td></tr><tr><td>E. coli</td><td>none</td><td>MIAE02381</td><td>France</td><td>2015</td></tr><tr><td>E. coli</td><td>none</td><td>MIAE02198</td><td>France</td><td>2015</td></tr><tr><td>Hafnia alvei</td><td>DSM 30163<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Listeria monocytogenes</td><td>ATCC BAA-679, (EGDe)</td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>L. monocytogenes</td><td>DSM 15675</td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>L. monocytogenes</td><td>DSM 19094</td><td>none</td><td>United Kingdom</td><td>n.a.</td></tr><tr><td>L. innocua</td><td>none</td><td>MIAE07668</td><td>France</td><td>2018</td></tr><tr><td>Morganella morganii</td><td>none</td><td>MIAE07669</td><td>France</td><td>n.a.</td></tr><tr><td>Pantoea agglomerans</td><td>DSM 3493<sup>T</sup></td><td>none</td><td>Zimbabwe</td><td>1956</td></tr><tr><td>Proteus mirabilis</td><td>none</td><td>MIAE07670</td><td>France</td><td>n.a.</td></tr><tr><td>P. vulgaris</td><td>none</td><td>MIAE07671</td><td>France</td><td>n.a.</td></tr><tr><td>Providencia rettgeri</td><td>none</td><td>MIAE07672</td><td>France</td><td>n.a.</td></tr><tr><td>P. stuartii</td><td>none</td><td>MIAE07673</td><td>France</td><td>n.a.</td></tr><tr><td>Pseudomonas brassicacearum</td><td>DSM 13227<sup>T</sup></td><td>none</td><td>France</td><td>n.a.</td></tr><tr><td>P. fluorescens</td><td>DSM 50106</td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>P. gessardii</td><td>CIP 105469<sup>T</sup></td><td>none</td><td>France</td><td>n.a.</td></tr><tr><td>P. jessenii</td><td>CFBP4842</td><td>none</td><td>France</td><td>1995</td></tr><tr><td>P. kilonensis</td><td>DSM 13647<sup>T</sup></td><td>none</td><td>Germany</td><td>n.a.</td></tr><tr><td>P. libanensis</td><td>DSM 17149<sup>T</sup></td><td>none</td><td>Lebanon</td><td>1995</td></tr><tr><td>P. lini</td><td>DSM 16768<sup>T</sup></td><td>none</td><td>France</td><td>n.a.</td></tr><tr><td>P. monteilii</td><td>DSM 14164<sup>T</sup></td><td>none</td><td>France</td><td>1990</td></tr><tr><td>P. putida</td><td>ATCC 12633<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>P. rhodesiae</td><td>DSM 14020<sup>T</sup></td><td>none</td><td>France</td><td>n.a.</td></tr><tr><td>P. thivervalensis</td><td>CFBP5754</td><td>none</td><td>France</td><td>2000</td></tr><tr><td>Salmonella bongori</td><td>DSM 13772<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>S. enterica</td><td>DSM 554</td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>S. enteritidis</td><td>none</td><td>MIAE07674</td><td>France</td><td>2007</td></tr><tr><td>S.subterranea</td><td>DSM 16208<sup>T</sup></td><td>none</td><td>USA</td><td>n.a.</td></tr><tr><td>Serratia fonticola</td><td>DSM 4576<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>S. liquefaciens</td><td>DSM 4487<sup>T</sup></td><td>none</td><td>Ireland</td><td>1997</td></tr><tr><td>Shigella boydii</td><td>DSM 7532<sup>T</sup></td><td>none</td><td>India</td><td>n.a.</td></tr><tr><td>Staphylococcus aureus</td><td>DSM 2569</td><td>none</td><td>South Korea</td><td>n.a.</td></tr><tr><td>S. capitis</td><td>DSM 20326<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>S. epidermidis</td><td>DSM 20044<sup>T</sup></td><td>none</td><td>n.a.</td><td>n.a.</td></tr><tr><td>Stenotrophomonas maltophilia</td><td>DSM 50170<sup>T</sup></td><td>none</td><td>USA</td><td>1961</td></tr><tr><td>Vibrio chagasi</td><td>DSM 17138<sup>T</sup></td><td>none</td><td>Norway</td><td>n.a.</td></tr></table>

n.a. : information not available, T type strain

*SB: Internal strain collection number of the Biodiversity and Epidemiology of Bacterial Pathogens unit, Institut Pasteur ; MIAE : Internal strain collection number of the UMR Agroecologie, INRA
<table><caption>**TABLE 2** Molecular target sequences previously proposed for *Klebsiella pneumoniae* identification.</caption><thead><tr><th>Target</th><th>Annotation</th><th>Amplicon (bp)</th><th>Targeted group</th><th>Reference (PubMed ID)</th></tr></thead><tbody><tr><td>1. 16S-23S rRNA ITS</td><td>tDNA-Ala</td><td>130</td><td>Kp complex</td><td>18579248</td></tr><tr><td>2. 16S-23S rRNA ITS</td><td>tDNA-Ala – 23S rDNA</td><td>260</td><td>Kp complex</td><td>18579248</td></tr><tr><td>3. khe</td><td>hemolysin</td><td>77</td><td>Kp complex</td><td>19644019</td></tr><tr><td>4. tyrB</td><td>tyrosine aminotransferase</td><td>931</td><td>Kp complex</td><td>23357944</td></tr><tr><td>5. KpI50233a</td><td>putative acyltransferase</td><td>484</td><td>Kp1</td><td>25261063</td></tr><tr><td>6. KP878</td><td>transferase</td><td>878</td><td>Kp1</td><td>25886267</td></tr><tr><td>7. KP888</td><td>phosphohydrolase</td><td>888</td><td>Kp1</td><td>25886267</td></tr><tr><td>8. blaSHV</td><td>chromosomal SHV</td><td>995</td><td>Kp1</td><td>28139276</td></tr><tr><td>9. celB</td><td>cellobiose-specific PTS family enzyme IIC component</td><td>180</td><td>Kp1</td><td>31456171</td></tr><tr><td>10. KV770</td><td>phosphoglycerate mutase</td><td>449</td><td>Kp3/5</td><td>25886267</td></tr><tr><td>11. KV1000</td><td>thiopurine S-methyltransferase</td><td>499</td><td>Kp3/5</td><td>25886267</td></tr><tr><td>12. KV1615</td><td>N-acetyltransferase</td><td>438</td><td>Kp3/5</td><td>25886267</td></tr><tr><td>13. blaLEN</td><td>chromosomal LEN</td><td>485</td><td>Kp3/5</td><td>28139276</td></tr><tr><td>14. blaOKP</td><td>chromosomal OKP</td><td>348</td><td>Kp2/4</td><td>28139276</td></tr><tr><td>15. ZKIR</td><td>zur-khe intergenic region</td><td>78</td><td>Kp complex</td><td>This study</td></tr></tbody></table>

ITS, internal transcribed spacer; PTS, phosphotransferase system; bla, beta-lactamase; bp, base pairs.
<table><caption>TABLE 3 Comparison between ZKIR qPCR and culture results obtained using samples collected in Auxonne, France, between July and September 2018.</caption><thead><tr><th>Source</th><th>No. of samples</th><th>qPCR positive samples (%)</th><th>Culture positive samples (%)</th></tr></thead><tbody><tr><td>Bulk soil</td><td>32</td><td>13 (40.6%)</td><td>13 (40.6%)</td></tr><tr><td>Roots</td><td>31</td><td>19 (61.3%)</td><td>16 (51.6%)</td></tr><tr><td>Leaves</td><td>29</td><td>8 (27.6%)</td><td>7 (24.1%)</td></tr><tr><td>Water</td><td>4</td><td>1 (25.0%)</td><td>1 (25.0%)</td></tr><tr><td>Total</td><td>96</td><td>41/96 (42.7%)</td><td>37/96 (38.5%)</td></tr></tbody></table>