Elodie Barbier
email: elodie.barbier@inrae.fr

Carla Rodrigues

Geraldine Depret

Virginie Passet

Laurent Gal

Pascal Piveteau

Sylvain Brisse
email: sylvain.brisse@pasteur.fr

Agroécologie

The ZKIR Assay, a novel Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

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

Klebsiella pneumoniae (Kp) is of growing public health concern due to the emergence of strains that are multidrug-resistant, virulent, or both. Taxonomically, Kp includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) 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. Here we analysed 1,001 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR assay, was developed and used to detect Kp in 96 environmental samples. Results were compared to a culture-based method using Simmons citrate agar with 1% inositol (SCAI) medium coupled to MALDI-TOF mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed.

The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10 -1 CFU g -1 after enrichment for 24 h in LB supplemented with ampicillin, and 1.5 × 10 3 to 1.5 × 10 4 CFU g -1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method.

Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 MLST sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific and sensitive novel method to detect the presence of Kp in complex matrices, and indicates that Kp isolates from environmental samples differ from clinical isolates.

INTRODUCTION

Klebsiella pneumoniae (Kp) is one of the leading causes of multidrug resistant (MDR) healthcare-acquired infections, with increasing rates of resistance to carbapenems and other last resort antibiotics being reported [START_REF] Holt | Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health[END_REF][START_REF] David | Epidemic of carbapenemresistant Klebsiella pneumoniae in Europe is driven by nosocomial spread[END_REF]. Furthermore, Kp is also an important agent of severe community-acquired infections (so-called 'hypervirulent' strains) in healthy persons [START_REF] Ko | Community-acquired Klebsiella pneumoniae bacteremia: global differences in clinical patterns[END_REF], with recent worrisome reports of convergence between hypervirulent and MDR phenotypes [START_REF] Holt | Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health[END_REF][START_REF] Zhang | Emergence of a hypervirulent carbapenem-resistant Klebsiella pneumoniae isolate from clinical infections in China[END_REF]. Kp is recognized as a colonizer of the throat and the intestinal tract of humans and animals [START_REF] Gorrie | Gastrointestinal carriage is a major reservoir of Klebsiella pneumoniae infection in intensive care patients[END_REF][START_REF] Brisse | The Genus Klebsiella[END_REF][START_REF] Munoz | Fecal shedding of Klebsiella pneumoniae by dairy cows[END_REF].

The main sources of human exposure to Kp are not well defined. Previous studies highlighted the large distribution of Kp in outdoor environments, including water, sewage, soil and plants [START_REF] Rosenblueth | Klebsiella variicola, a novel species with clinical and plant-associated isolates[END_REF][START_REF] Lee | Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR[END_REF][START_REF] Benami | Assessment of pathogenic bacteria in treated graywater and irrigated soils[END_REF][START_REF] Barati | Isolation and characterization of aquatic-borne Klebsiella pneumoniae from tropical estuaries in Malaysia[END_REF][START_REF] Falomir | Coliform bacteria in fresh vegetables: from cultivated lands to consumers, volume 2[END_REF][START_REF] Podschun | Incidence of Klebsiella species in surface waters and their expression of virulence factors[END_REF]. Animal and human food, particularly retail meat or salad may also be contaminated [START_REF] Brisse | The Genus Klebsiella[END_REF][START_REF] Davis | Intermingled Klebsiella pneumoniae populations between retail meats and human urinary tract infections[END_REF][START_REF] Zadoks | Sources of Klebsiella and Raoultella species on dairy farms: Be careful where you walk[END_REF]. Many studies suggest that food, water and/or environmental exposure may be associated with virulent and/or antibiotic resistant Kp to humans [START_REF] Podschun | Incidence of Klebsiella species in surface waters and their expression of virulence factors[END_REF][START_REF] Davis | Intermingled Klebsiella pneumoniae populations between retail meats and human urinary tract infections[END_REF][START_REF] Runcharoen | Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae[END_REF][START_REF] Davis | Recent research examining links among Klebsiella pneumoniae from food, food animals, and human extraintestinal infections[END_REF].

However, little is known on the relative contribution of these different sources of transmission.

Although such information is a prerequisite to control efficiently transmission routes and reduce exposure, the ecology of Kp is currently poorly understood.

The systematics of Kp has been refined through recent taxonomic updates, which highlighted the existence of seven phylogroups (Kp1 to Kp7), corresponding to distinct taxa, within Klebsiella pneumoniae sensu lato. The K. pneumoniae species complex includes 5 different species: K. pneumoniae sensu stricto (phylogroup Kp1), K. quasipneumoniae [subsp. quasipneumoniae (Kp2) and subsp. similipneumoniae (Kp4)], K. variicola [subsp. variicola (Kp3) and subsp. tropica (Kp5)], 'K. quasivariicola' (Kp6) and K. africana (Kp7) [START_REF] Rosenblueth | Klebsiella variicola, a novel species with clinical and plant-associated isolates[END_REF][START_REF] Brisse | Description of Klebsiella quasipneumoniae sp. nov., isolated from human infections, with two subspecies, Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a junior heterotypic synonym of Klebsiella variicola[END_REF][START_REF] Long | Wholegenome sequencing of a human clinical isolate of the novel species Klebsiella quasivariicola sp[END_REF][START_REF] Rodrigues | Description of Klebsiella africanensis sp. nov., Klebsiella variicola subsp. tropicalensis subsp. nov. and Klebsiella variicola subsp. variicola subsp. nov[END_REF].

Most of these taxa are still often misidentified as "K. pneumoniae" or "K. variicola" due to the unsuitability of traditional clinical microbiology methods to distinguish among members of the Kp complex [START_REF] Long | Whole-Genome Sequencing of Human Clinical Klebsiella pneumoniae Isolates Reveals Misidentification and Misunderstandings of Klebsiella pneumoniae , Klebsiella variicola , and Klebsiella quasipneumoniae[END_REF]. Henceforth, we will use the 'Kp' abbreviation to refer collectively to the seven on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from 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 [START_REF] Liu | PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer[END_REF][START_REF] Garza-Ramos | Development of a Multiplex-PCR probe system for the proper identification of Klebsiella variicola[END_REF][START_REF] Bialek-Davenet | Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2[END_REF][START_REF] Yin-Ching | Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe[END_REF][START_REF] Jeong | Rapid Identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae using triplex Polymerase Chain Reaction in rodents[END_REF]. They target the 16S-23S rRNA internal transcribed spacer sequence (ITS) [START_REF] Liu | PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer[END_REF], coding sequences of tyrB [START_REF] Jeong | Rapid Identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae using triplex Polymerase Chain Reaction in rodents[END_REF], khe [START_REF] Yin-Ching | Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe[END_REF][START_REF] Hartman | Rapid real-time PCR assays for detection of Klebsiella pneumoniae with the rmpA or magA genes associated with the hypermucoviscosity phenotype[END_REF], chromosomal beta-lactamase (bla) genes [START_REF] Fonseca | A onestep multiplex PCR to identify Klebsiella pneumoniae , Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine[END_REF][START_REF] Singh | Rectal screening for Klebsiella pneumoniae carbapenemases: comparison of Real-Time PCR and culture using two selective screening agar plates[END_REF] or other molecular targets [START_REF] Garza-Ramos | Development of a Multiplex-PCR probe system for the proper identification of Klebsiella variicola[END_REF]. Some of these targets are described as able to detect the Kp complex [START_REF] Liu | PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer[END_REF], whereas others were designed for specific members of the Kp complex, such as Kp1 and Kp3 [START_REF] Garza-Ramos | Development of a Multiplex-PCR probe system for the proper identification of Klebsiella variicola[END_REF][START_REF] Bialek-Davenet | Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2[END_REF].

Real-time PCR is a powerful approach for the rapid detection and quantification of microorganisms in complex matrices [START_REF] Bilodeau | Quantitative polymerase chain reaction for the detection of organisms in soil[END_REF]. 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. on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from

MATERIALS AND METHODS

Bacterial strains and culture conditions

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 ATCC13883 T 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.

Bacterial DNA extraction

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. A simpler protocol was implemented for the PCR specificity assay and validation study. Bacteria grown in TSB or enrichment medium were centrifuged 5 min at 13 000 g. Pellets were suspended in 200 µl of Tris HCl (5 mM pH 8.2) supplemented with 13 µl of Proteinase K (1 mg/ml). After 2 h incubation at 55 °C, Proteinase K was inactivated during 10 min boiling. Cell debris were discarded by centrifugation (5 min at 13 000 g) and the supernatant was stored at -20°C before use for real-time PCR.

Soil DNA extraction

Soil metagenomic DNA was extracted from 2 g aliquots of soil using the modified ISO standard 11063 method [START_REF] Plassart | Evaluation of the ISO Standard 11063 DNA extraction procedure for assessing soil microbial abundance and community structure[END_REF]. Crude soil DNA extracts were first purified onto PVPP (polyvinylpolypyrrolidone) minicolumns (BIORAD, France), then with the GeneClean Turbo kit (MP Biomedicals, France) according to the manufacturer's protocol [START_REF] Plassart | Evaluation of the ISO Standard 11063 DNA extraction procedure for assessing soil microbial abundance and community structure[END_REF]. Purified DNA concentrations were determined using a Nanodrop 2000 spectrophotometer.

In silico identification of the target DNA region

The phylogenetic distribution of molecular targets that were previously described in the literature for Kp detection, was investigated in silico. All available Kp genomes were downloaded from the NCBI database (n=3552, February 2018) and combined with the ones of Institut Pasteur's on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from internal reference collection (n=670) [START_REF] Rodrigues | Description of Klebsiella africanensis sp. nov., Klebsiella variicola subsp. tropicalensis subsp. nov. and Klebsiella variicola subsp. variicola subsp. nov[END_REF][START_REF] Rodrigues | Identification of Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola and Related Phylogroups by MALDI-TOF Mass Spectrometry[END_REF][START_REF] Passet | Description of Klebsiella grimontii sp[END_REF], representing a total of 4,222 genomes which included Klebsiella species and closely related Raoultella species. The average nucleotide identity (ANI) metric was used to classify the Kp genomes in each of the phylogroups. To avoid redundancy in the dataset and due to the difficulty of analyzing phylogenetically 4,222 genomes, in the case of the genomes belonging to the Kp complex (which represented the majority of the dataset), we selected unique representatives of each MLST sequence type (ST), as defined using the mlst software tool (https://github.com/tseemann/mlst). This resulted in a final collection of 1,001 genomes that were tested for the presence of targets from the literature. Nucleotide BLAST (BLASTN) was used to detect the presence of the target sequences in the 1,001 genomes, with 80% nucleotide identity (also with 92% in the case of bla SHV , bla OKP and bla LEN targets) and 80% length coverage as cutoffs. Primer-blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used (with default parameters) to check the distribution of primer target sites in the genomic sequences. Specificity was defined as the proportion of target organisms among those expected to be amplified by the target assay. Sensitivity was defined as the proportion of target organisms expected to be amplified by the target assay. A phylogenetic tree was constructed [START_REF] Criscuolo | A fast alignment-free bioinformatics procedure to infer accurate distance-based phylogenetic trees from genome assemblies[END_REF] and the output was visualized using iTOL (https://itol.embl.de/; Figure 1; Figure S1).

Design of primers

The Kp tyrB and khe gene sequences (accession numbers: AF074934. Primer sets were designed using Primer3Plus (http://www.bioinformatics.nl/cgibin/primer3plus/ primer3plus.cgi). The specificity of the predicted primer sets and amplicons was checked by applying BLASTN on the GenBank nucleotide collection (nr/nt) from the NCBI database and Multiple primer analyzer (https://www.thermofisher.com) was run to check for dimer formation. The best candidate primer sets defined by this in silico approach were ordered 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').

Real-time PCR

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

Two universal primers targeting a 174 base pairs region of the 16S rRNA gene of Eubacteria, 341f (5'-CCT-ACG-GGA-GGC-AGC-AG-3') and 515r (5'-ATT-CCG-CGG-CTG-GCA-3'), were used for positive control PCR reactions as described in a previous report [START_REF] López-Gutiérrez | 2004 -Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR[END_REF].

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

Ninety-six environmental samples collected between July and September 2018 (23 in July; 39 in August and 34 in September) in Auxonne (Burgundy, France) were analysed for Kp presence using the ZKIR assay and culture methods in parallel. Samples corresponded to bulk soils (n=32), roots [START_REF] Plassart | Evaluation of the ISO Standard 11063 DNA extraction procedure for assessing soil microbial abundance and community structure[END_REF], leaves [START_REF] Singh | Rectal screening for Klebsiella pneumoniae carbapenemases: comparison of Real-Time PCR and culture using two selective screening agar plates[END_REF] and irrigation water (4) and were processed in the lab within 24 hours after sampling. Ten g of soil were weighed in 180 ml pots (Dutscher, France). Plant leaves and roots were properly cut, cleaned with sterile water and transferred in 180 ml pots. Processed samples were suspended in 90 ml of lysogeny broth (LB, 5 g of NaCl, 5 g of yeast extract and 10 g of Tryptone for a 1 l final volume) supplemented with ampicillin (10 mg/l, ampicillin sodium salt, Sigma Aldrich). Five hundred millilitres of water samples were filtered through a 0.25 µm membrane (Millipore, France). The membrane was incubated in 20 ml of LB supplemented with ampicillin as described above. After 24 h of incubation at 30 °C, enrichments were vortexed and 500 µl aliquots were 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 ten-fold diluted (1:10 and 1:100) and the dilutions were used as template for real-time PCR. on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/

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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) [START_REF] Van Kregten | New, simple medium for selective recovery of Klebsiella pneumoniae and Klebsiella oxytoca from human feces[END_REF]. Plates were incubated 48 h at 37 °C. Each plate was screened for presumptive colonies of Klebsiella (large, yellow, domeshaped 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; 2×150 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 [START_REF] Blin | Metabolic diversity of the emerging pathogenic lineages of Klebsiella pneumoniae: Metabolic diversity of Klebsiella pneumoniae[END_REF]. 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 ATCC13883 T 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, on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from resulting in final concentrations from 1.5 × 10 4 cfu/g to 1.5 × 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

RESULTS

Revisiting the phylogenetic distribution of proposed molecular targets for Kp detection

Fourteen molecular targets were found in the literature (Figure 1; Table 2) [START_REF] Liu | PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer[END_REF][START_REF] Garza-Ramos | Development of a Multiplex-PCR probe system for the proper identification of Klebsiella variicola[END_REF][START_REF] Bialek-Davenet | Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2[END_REF][START_REF] Yin-Ching | Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe[END_REF][START_REF] Jeong | Rapid Identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae using triplex Polymerase Chain Reaction in rodents[END_REF][START_REF] Hartman | Rapid real-time PCR assays for detection of Klebsiella pneumoniae with the rmpA or magA genes associated with the hypermucoviscosity phenotype[END_REF][START_REF] Fonseca | A onestep multiplex PCR to identify Klebsiella pneumoniae , Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine[END_REF][START_REF] Tian | CelB is a suitable marker for rapid and specific identification of Klebsiella pneumoniae by the loop-mediated isothermal amplification (LAMP) assay[END_REF]. 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 [START_REF] Yin-Ching | Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe[END_REF][START_REF] Jeong | Rapid Identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae using triplex Polymerase Chain Reaction in rodents[END_REF] 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.

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 [START_REF] Bialek-Davenet | Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2[END_REF] proved to be the most specific and sensitive one (although with ability to also amplify K. aerogenes isolates), whereas the ones proposed for Kp3 [START_REF] Garza-Ramos | Development of a Multiplex-PCR probe system for the proper identification of Klebsiella variicola[END_REF] appeared unspecific or to lack sensitivity in our in silico analysis (Figure 1). Regarding the targeted chromosomal class A beta-lactamases genes bla SHV, bla OKP and bla LEN [START_REF] Fonseca | A onestep multiplex PCR to identify Klebsiella pneumoniae , Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine[END_REF], 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 bla SHV , bla OKP and bla LEN 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 on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from [START_REF] Haeggman | Diversity and Evolution of the Class A Chromosomal Beta-Lactamase Gene in Klebsiella pneumoniae[END_REF]. However, it was evident that horizontal gene transfer events implicating the beta-lactamase genes have occurred. In particular, the bla SHV 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 bla SHV on plasmids and was previously described [START_REF] Long | Whole-Genome Sequencing of Human Clinical Klebsiella pneumoniae Isolates Reveals Misidentification and Misunderstandings of Klebsiella pneumoniae , Klebsiella variicola , and Klebsiella quasipneumoniae[END_REF]. The gene bla LEN 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).

ZKIR primers design, PCR assay development and optimization

The tyrB and khe genes were previously proposed as targets for the specific detection of Kp [START_REF] Yin-Ching | Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe[END_REF][START_REF] Jeong | Rapid Identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae Using Triplex Polymerase Chain Reaction in Rodents[END_REF]. 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.

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 noncoding 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.

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. 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.

Analytical sensitivity of the ZKIR PCR

PCR reactions were performed with quantities of genomic DNA of K. pneumoniae ATCC13883 T ranging from 7.5 ng (2.5 × 10 6 genomes) to 15 fg (5 genomes) (Figure 3 A andB). Linearity of the assay was evaluated by plotting the Ct values against the log 10 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 2 =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.

Analytical sensitivity of the ZKIR assay on soil samples

on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/

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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 × 10 -1 cfu/g to 1.5 × 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 × 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 × 10 4 and 1.5 × 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 culturebased 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. on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from Genomic characterization of 31 of these isolates (1 was contaminated and [START_REF] Gorrie | Gastrointestinal carriage is a major reservoir of Klebsiella pneumoniae infection in intensive care patients[END_REF] 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 [START_REF] Diancourt | Multilocus Sequence Typing of Klebsiella pneumoniae Nosocomial Isolates[END_REF] and cgMLST [START_REF] Bialek-Davenet | Development of a multiplex PCR assay for identification of Klebsiella pneumoniae hypervirulent clones of capsular serotype K2[END_REF] 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).

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 [START_REF] David | Epidemic of carbapenemresistant Klebsiella pneumoniae in Europe is driven by nosocomial spread[END_REF][START_REF] Follador | The diversity of Klebsiella pneumoniae surface polysaccharides[END_REF], 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 [START_REF] Holt | Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health[END_REF][START_REF] Blin | Metabolic diversity of the emerging pathogenic lineages of Klebsiella pneumoniae: Metabolic diversity of Klebsiella pneumoniae[END_REF], 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 [START_REF] Blin | Metabolic diversity of the emerging pathogenic lineages of Klebsiella pneumoniae: Metabolic diversity of Klebsiella pneumoniae[END_REF] 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).

Discussion

Routes of transmission of ecologically generalist human pathogens are usually complex and poorly understood. Proper risk management requires a holistic approach which has been theorised as the One Health concept [START_REF] King | Executive summary of the AVMA One Health Initiative Task Force report[END_REF]. Despite the fact that the number of human infections caused by members of the Kp complex are on the rise and are increasingly resistant to antimicrobial treatment [START_REF] Holt | Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health[END_REF][START_REF] Nordmann | Global spread of carbapenemase-producing Enterobacteriaceae[END_REF], the ecology of Kp remains poorly understood. Identification of the various habitats in which Kp strives, and the routes of transmission to humans, for example through specific types of food, is critical in order to limit exposure. In this prospect, large-scale sampling is needed to define the sources of Kp contamination and such surveys will require sensitive, reliable and cost-efficient detection methods.

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

Given that all members of the Kp complex can cause infections in humans or animals, developing a novel method with the ability to detect the Kp complex by targeting exhaustively all currently known Kp phylogroups would be an important advance. Although several previous targets were designed for the entire Kp complex, they predate recent taxonomic advances and have therefore not been validated on the entire phylogenetic breadth of this bacterial group. Here, we found by in-silico approaches that khe and tyrB assays lack specificity, which may negatively affect Kp on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from detection efforts especially when testing microbiologically complex samples from the environment, such as soil.

We therefore aimed to develop a novel real-time PCR assay, and used an intergenic region located adjacent to the previously proposed target gene khe. Using well-defined reference strains representative of current taxonomy [START_REF] Rodrigues | Description of Klebsiella africanensis sp. nov., Klebsiella variicola subsp. tropicalensis subsp. nov. and Klebsiella variicola subsp. variicola subsp. nov[END_REF][START_REF] Rodrigues | Identification of Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola and Related Phylogroups by MALDI-TOF Mass Spectrometry[END_REF][START_REF] Passet | Description of Klebsiella grimontii sp[END_REF], we show that the novel ZKIR PCR real-time assay allows the accurate detection of all members of the Kp complex. We found complete specificity and sensitivity (i.e., phylogenetic coverage) of this assay based on our reference strains panel. To improve analytical sensitivity, a 24 h enrichment culture followed by an easy, fast and cost-effective sample processing was implemented, upstream of molecular detection using the ZKIR assay. This was necessary for complex matrices such as soil or sewage, where microbial diversity and abundance are high, in which case direct detection of one particular group of organisms, which might be present only at low abundance, is challenging. This procedure turned out to be sensitive enough to detect 1 single Kp bacterium in 5 g of soil. The ZKIR assay also appeared slightly more sensitive than the SCAI culture-based method, which is based on the ability of Klebsiella strains to utilize citrate and inositol, itself previously shown to be highly sensitive and to recover most Kp members [START_REF] Van Kregten | New, simple medium for selective recovery of Klebsiella pneumoniae and Klebsiella oxytoca from human feces[END_REF][START_REF] Passet | Association of Tellurite Resistance with Hypervirulent Clonal Groups of Klebsiella pneumoniae[END_REF]. Therefore, this fast and easy novel molecular method represents a powerful approach for screening large numbers of samples. This will spare the time-consuming handling of numerous presumptive colonies necessary for confirmation of their identification, given that K. oxytoca, Raoultella, Serratia and Enterobacter are able to form colonies on SCAI agar, with morphological characteristics similar to Kp [START_REF] Van Kregten | New, simple medium for selective recovery of Klebsiella pneumoniae and Klebsiella oxytoca from human feces[END_REF][START_REF] Ohtomo | A New selective medium for detection of Klebsiella from dairy environments[END_REF]. As such, the ZKIR protocol was significantly faster than culture, as results were available on average 26 h after sampling (24 h enrichment, sample treatment, PCR) while up to 96 h were necessary when using the culture-based method (24 h enrichment, plate incubation, colony purification, MALDI-TOF identification). 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 on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from enrichment step greatly enhances sensitivity. As this rapid screen of samples allows one to focus only on ZKIR positive samples for more labour-intensive downstream microbiological isolation and characterization, it is our hope that it will contribute to advance knowledge on the biology of environmental Kp and on the reservoirs and transmission routes of this increasingly important group of pathogens. Tree scale: 0.01

  After shaking (3 min, 25 rpm) and centrifugation (3 min, 20 000 g), this step was repeated once. After transfer of the supernatant in a new tube, a 1:10 volume of NaCl (5 M) was added (shaking on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from 1 min, 15 rpm). Finally, a 1:1 volume of isopropanol (shaking 3 min, 15 rpm) was added. Precipitated DNA filaments were collected with a glass Pasteur pipette and transferred in 400 µl frozen ethanol (70%). After shaking 3 min at 15 rpm, DNA filaments were collected, dried at room temperature and finally dissolved in 100 µl TE (pH 8). DNA concentration was estimated using NanoDrop 2000 Spectrophotometer (ThermoFischer Scientific, USA) and adjusted at 20 ng/µl. Purified DNA was stored at -20°C.

  1 and AF293352.1) were aligned against the genome sequences of K. pneumoniae (CP012744.1, CP012743.1, CP028787.1), K. variicola (CP008700.1, CP013985.1, CP017289.1), K. quasipneumoniae (CP014071.1, CP023478.1, CP029432.1) and K. quasivariicola (CP022823.1) using BLASTN to identify conserved genomic regions within the Kp complex. Sequences of closely related bacterial species K. oxytoca (CP027426.1), Raoultella ornithinolytica (CP010557.1), on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from R. planticola (CP026047.1) and K. aerogenes (CP014029.2) were added in the alignments to confirm the specificity of these regions for the Kp complex.

on February 7 ,

 7 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from Standard curve development and sensitivity assessment 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 genomic DNA of K. pneumoniae ATCC13883 T were prepared in triplicate and amplified with the optimized PCR conditions described above. Results were analysed with the StepOne™ Data Analysis software. PCR efficiency was determined. The genome number expressed as logarithm was plotted against Ct values and the correlation coefficient (R 2 ) of the standard curve was calculated.

  were submitted to the European Nucleotide Archive and are accessible under the BioProject number PRJEB34643. on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from

  similipneumoniae), these primers successfully amplified a 78 bp sequence, with a melting on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from temperature of 80.3 °C. Sequencing of PCR products confirmed amplification of the target sequence.

on February 7 ,

 7 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from

on February 7 ,

 7 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from 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).

FIG 2

 2 FIG 2Genetic context of the ZKIR region on the genome of strain K. pneumoniae ATCC 13883 T (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).

FIG 3

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

FIG 4 Figure 1 .

 41 FIG 4 Standard curve established using real-time ZKIR PCR with serial dilutions of K. pneumoniae ATCC13883 T DNA from 7.5 ng to 45 fg.

Figure 2

 2 Figure 2Genetic context of the ZKIR region on the genome of strain K. pneumoniae ATCC 13883 T (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).

Figure 3 Figure 4

 34 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 ATCC13883 T 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.A

TABLE 1

 1 Bacterial strains tested to develop the ZKIR PCR assay.

	Downloaded from
	http://aem.asm.org/
	on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque

TABLE 2

 2 Molecular target sequences previously proposed for Klebsiella pneumoniae identification.

TABLE 3

 3 Comparison between ZKIR PCR and culture results in samples collected in Auxonne, 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

	France, between July and September 2018.
	FIGURES
	FIG 1

Table 2 .

 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.

Table 2 .

 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.

	Kp
	1
	0
	02
	85
	395
	5.1
	AS
	M
	285
	39
	5v1

TABLE 2

 2 Molecular target sequences previously proposed for Klebsiella pneumoniae identification.

	Target	Annotation	Amplicon (bp)	Targeted group	Reference (PubMed ID)	Downloaded from
	1. 16S-23S rRNA ITS tDNA-Ala 2. 16S-23S rRNA ITS tDNA-Ala -23S rDNA 3. khe hemolysin 4. tyrB tyrosine aminotransferase 5. KpI50233a putative acyltransferase 6. KP878 transferase	130 260 77 931 484 878	Kp complex 18579248 Kp complex 18579248 Kp complex 19644019 Kp complex 23357944 Kp1 25261063 Kp1 25886267	http://aem.asm.org/
	7. KP888 8. bla SHV 9. celB 10. KV770 11. KV1000 12. KV1615 13. bla LEN 14. bla OKP 15. ZKIR ITS, internal transcribed spacer; PTS, phosphotransferase system; bla, beta-lactamase; bp, base phosphohydrolase 888 Kp1 25886267 chromosomal SHV 995 Kp1 28139276 cellobiose-specific PTS family enzyme IIC component 180 Kp1 31456171 phosphoglycerate mutase 449 Kp3/5 25886267 thiopurine S-methyltransferase 499 Kp3/5 25886267 N-acetyltransferase 438 Kp3/5 25886267 chromosomal LEN 485 Kp3/5 28139276 chromosomal OKP 348 Kp2/4 28139276 zur-khe intergenic region 78 Kp complex This study pairs.	on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque

TABLE 3

 3 Comparison between ZKIR qPCR and culture results obtained using samples collected in Auxonne, France, between July and September 2018.

	Source Bulk soil Roots	No. of samples qPCR positive samples (%) 32 13 (40.6%) 31 19 (61.3%)	Culture positive samples (%) 13 (40.6%) 16 (51.6%)	Downloaded from
	Leaves Water Total	29 4 96	8 (27.6%) 1 (25.0%) 41/96 (42.7 %)	7 (24.1%) 1 (25.0%) 37/96 (38.5%)	http://aem.asm.org/
					on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque

ACKNOWLEDGEMENTS

We thank Alexis Criscuolo for access to an early version of the JolyTree software tool, Julien Guglielmini for providing us access to in-house scripts for mapping the target genes, and Juan Sebastian Lopez Fernandes for assistance with genomic analyses. This work is part of the MedVetKlebs project, a component of the One Health European Joint Programme, and has thereby received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No 773830. on February 7, 2020 at INSTITUT PASTEUR-Bibliotheque http://aem.asm.org/ Downloaded from


D Tournier

P Godignon

J Montserrat

D Planson

J P Chante

F Sarrus

Compatibility of VJFET Technology to MESFET Fabrication and its Interest to System Integration : Fabrication of 6H and 4H-SiC 110V Lateral MESFET

Keywords: MESFET, VJFET, Technology compatibility, system integration

. High energy implantation, RIE etching adjustment and metal contact annealing are the critical steps of the fabrication of this device and will be developed in the process description. Electrical characterization of fabricated MESFET exhibit specific on resistance of 38mΩ.cm² and a transconductance value of 0,4 mS.mm -1 . These results demonstrate VJFET and MESFET fabrication technology compatibility. Keys points to improve for the next generation of devices will be presented as well.

Introduction

Getting both power devices and their associated control circuitry on same die is interesting in terms of efficiency, reducing chips interconnections number subject to both thermal and mechanical stress leading to component failure. A solution to avoid this is to get both power and control on the same wafer. SOI offers solution and lateral power devices have been done with both power and control on the same chip. Unfortunately, those kind of devices are usually designed for low voltage components. Using vertical devices such as VJFET, Thysristor or IGBT allows fabrication of high voltage devices. Getting both high voltage components and its command let us implement intelligent devices such as self protected IGBT against short circuit, or cascode association one same chip. Several solution can be foreseen to realize such components on the same die : connection by bonding, wafer bonding, stack with vertical connection, local SOI zone formation on a vertical component. The challenge consists in the reduction of the number of fabrication steps combined with high efficiency of the completed device.

VJFET and MESFET Fabrication Compatibility.

The fabrication compatibility between a VJFET and a lateral MESFET with a reduced number of mask is presented below. Figures 1 and2 presents cross sections of the VJFET and lateral MESFET and their similarities. Those matches enables the potential realization of both components at same time. The p-type buried layer doesn't have the same role in each device. In the case of the VJFET, it allows current limitation with surface channel definition and a lateral control of the current while applying a bias voltage between gate and source. Regarding the MESFET, the function of the p-type buried layer consist of insulation from the wafer and pinch off of the horizontal channel in conjunction with then Schottky gate action.

MESFET Operation.

Parameters such as channel doping concentration have been fixed by the design of the VJFET.

We have designed the gate of the MESFET considering fabrication capabilities. The source electrode is connected to the p-type buried layer in order to control the gate bias voltage. Without gate bias applied, the MESFET is normally on. Rising the drain bias will cause reverse bias of the pn junction formed between the p-type buried layer and surface channel resulting in a local extension of space charge and the saturation of the current. An ISE-TCAD simulated result view illustrate the mechanism of saturation of the current (figure 3).

Fig. 3 Mesfet current density at pinchoff (V DS = 50V, V GS = 0V).

While applying a gate bias, a restriction of the lateral channel is induced causing reduction of current as in the classical MESFET. This structure allows the control of channel restriction by both JFET (between drain and p-type buried layer) and MESFET effects. passivation

Process Fabrication Description

Both 6H and 4H SiC wafers were used for the device fabrication. N-type epitaxial layers with doping concentration in the range of 5 × 10 15 cm -3 (15µm) were purchase from Cree INC. A high energy Al implantation (2MeV with a dose of 10 14 cm -2 @400ºC) has been performed to form the p-type buried layer. Figure 4 presents simulated profile and SIMS analysis after the thermal annealing step. N + areas were then implanted (Dose of 10 15 cm -2 ) for contact zones of the drain and source. Then an n-type box profile was formed at the surface of the wafer by multiple N implantation (9,8 × 10 12 cm -2 ) to define the surface channel. RIE etching (1,2 µm), adjusted in relation with simulated profile of Al implantation [START_REF] Morvan | Modélisation de l'implantation ionique dans α-SiC et application à la conception de composant de puissance[END_REF], was performed to contact the p-type buried layer. Peripheral protection of the MESFET was realized by a field plate whereas the VJFET is protect by both field plate and JTE. The wafers were finally annealed at 1700 °C / 30 min. Thermal oxidation followed by an oxide deposition was carried out to form the passivation layer. The final step was metal contact formation.

Ni or W layers were deposited and then annealed at the same time for gate and ohmic contacts. Both ohmic and Schottky contacts were simultaneously realized using one mask level for metal patterning. A top picture of the fabricated MESFET is presented in Figure 5. No additional steps are required to achieve VJFET fabrication due to the similarities with MESFET.

Electrical Characterization and Discussion.

The extracted contact resitivity is in the range of 3,57 × 10 -6 Ω.cm² for the Ni 6H-SiC samples (annealing time: 3 min at 900°C) (Figure 6), and 7,7 × 10 -5 Ω.cm² for the 4H-SiC, values in the state of the art [START_REF] Matsunami | Progess in Wide Bandgap Semiconductor SiC for Power devices[END_REF]. The 4H-SiC MESFET specific on resistance is 38 m Ω.cm² and the transconductance is 0,4 mS.mm -1 (higher value : 40mS.mm -1 ). This low value is mainly due to high reverse leakage current of the Schottky contact.

S D G

An on-state current density of 300A/cm² at V DS = 110V has been reached as shown in the Figure 7.

Getting both command and control circuitry on the same chip can cause insulation problems. This is the function of the p-type buried layer. The PN rectifier formed between the p-type buried layer and the epitaxial layer (source/bulk) exhibits a blocking voltage of 950V at 1.10 -3 A/cm², a very low leakage current density compared to saturation current of MESFET. Fig. 7 I DS (V DS ) versus gate to source applied voltage. Dynamic resistance as function of gate to source bias has been investigated and remains constant up to V GS = -15V at a value of 50 kΩ/mm and rises up to 170 kΩ/mm with V GS =-20V. This fact is attribute to gate leakage current. In the case of nickel, a metal anneal has been done. The Schottky gate contact was lost and not connected for this measurement (Figure 8). Good ohmic contacts were formed, leading to a rise of current but inducing a degradation of saturation linearity. Nickel reaction with SiC involving channel geometry modification is probably at the origin of this. Using W instead of Ni should avoid this problem.

Conclusion

Full compatibility of fabrication of SiC-VJFETs and lateral SiC-MESFETs has been demonstrated. High power density has been reached (30kW/cm²). State of art ohmic contact values were obtained. Next batch fabrication challenge will consist of both VJFET and MESFET optimization to realize a self controlled current limiter by a cascode-like association of MESFETs and VJFET . Metallization optimization will remain the main point to improve in the next generation of devices. Therefore, two mask levels for metal deposition and patterning should be envisaged with different metal deposition and annealing steps. This compatibility of fabrication should allow us to increase efficiency of power switches and systems as it enables both power devices and control circuits on the same die.

Fig. 1

 1 Fig. 1 Cross section of the VJFET. Fig. 2 Cross section of the MESFET.

Fig. 4 Simulated

 4 Fig. 4 Simulated Al profile and SIMS analysis results.

Fig. 6 Fig. 5

 65 Fig. 5 Top view of fabricated MESFET.

Fig. 8 .

 8 Fig. 8. Metal annealing effect on electrical characteristics.

Acknowledgment

We would like to thank Ferraz-Shawmut Company for their financial support of this work and the European Community for the Access to Research Infrastructure action of the improving Human Potential Program. High energy implantation was done at the LAMEL (Bologna, Italy).


Tushar Malica

Guillaume Bouchez

Delphine Wolfersberger

Marc Sciamanna

Marc Scia- Manna

Spatiotemporal complexity of chaos in a phase-conjugate feedback laser system

Keywords: Semiconductor lasers, Chaos, Phase conjugation, Information theoretical analysis

teaching and research institutions in France or abroad, or from public or private research centers.

Semiconductor lasers subjected to optical feedback are known to exhibit a diverse range of rich nonlinear effects such as low-frequency fluctuations [START_REF] Mork | Bistability and low-frequency fluctuations in semiconductor lasers with optical feedback: a theoretical analysis[END_REF], self-pulsation [START_REF] Mirasso | Self-pulsating semiconductor lasers: theory and experiment[END_REF], and chaos [START_REF] Cheng | 3d pulsed chaos lidar system[END_REF][START_REF] Uchida | Fast physical random bit generation with chaotic semiconductor lasers[END_REF][START_REF] Goedgebuer | Optical cryptosystem based on synchronization of hyperchaos generated by a delayed feedback tunable laser diode[END_REF][START_REF] Vanwiggeren | Communication with chaotic lasers[END_REF][START_REF] Soriano | Time scales of a chaotic semiconductor laser with optical feedback under the lens of a permutation information analysis[END_REF][START_REF] Sciamanna | Physics and applications of laser diode chaos[END_REF]. Primarily regarding the traditionally undesirable chaotic output, various fields have used it to their advantage and found its application in LIDAR [START_REF] Cheng | 3d pulsed chaos lidar system[END_REF][START_REF] Lin | Chaotic lidar[END_REF], random number generation [START_REF] Uchida | Fast physical random bit generation with chaotic semiconductor lasers[END_REF], optical cryptography [START_REF] Goedgebuer | Optical cryptosystem based on synchronization of hyperchaos generated by a delayed feedback tunable laser diode[END_REF] and secure communication [START_REF] Vanwiggeren | Communication with chaotic lasers[END_REF]. In the context of optical feedback generated chaos, increased spatiotemporal complexity along with high chaos BW provides a truly secure system for communication. The high BW ensures that these applications display consistent high performance in their respective figure of merits. In addition, the desirability for high spatiotemporal complexity reduces the predictability of the system output. In recent years, several feedback configurations have been proposed for enhancing the chaos BW [START_REF] Uchida | Fast physical random bit generation with chaotic semiconductor lasers[END_REF][START_REF] Mercier | High-frequency chaotic dynamics enabled by optical phase-conjugation[END_REF][START_REF] Bouchez | Wideband chaos from a laser diode with phase-conjugate feedback[END_REF][START_REF] Mercier | High-order external cavity modes and restabilization of a laser diode subject to a phase-conjugate feedback[END_REF][START_REF] Someya | Synchronization of bandwidthenhanced chaos in semiconductor lasers with optical feedback and injection[END_REF][START_REF] Sakuraba | Tb/s physical random bit generation with bandwidth-enhanced chaos in three-cascaded semiconductor lasers[END_REF]. Particularly in [START_REF] Mercier | High-frequency chaotic dynamics enabled by optical phase-conjugation[END_REF], the chaos BW was experimentally shown to improve by many orders of magnitude by substituting a conventional mirror with a phase-conjugate mirror to implement a phase-conjugate feedback (PCF) system. In [START_REF] Rontani | Enhanced complexity of optical chaos in a laser diode with phase-conjugate feedback[END_REF], the complexity of the exhibited chaos in a PCF system was theoretically shown to be significantly of superior quality than observed in a conventional optical feedback system (COF).

In this Letter, we analyze experimentally acquired laser output at varying values of PCF [START_REF] Bouchez | Wideband chaos from a laser diode with phase-conjugate feedback[END_REF][START_REF] Mercier | High-order external cavity modes and restabilization of a laser diode subject to a phase-conjugate feedback[END_REF]. System out-puts are analyzed within the framework of Permutation Entropy (PE). We dissect the underlying system characteristic temporal signatures and correlate it with the observed wide-band chaos. PCF in [START_REF] Bouchez | Wideband chaos from a laser diode with phase-conjugate feedback[END_REF] is achieved by employing a self-pumped total-internal-reflection (CAT) configuration using a photorefractive (BaTiO 3 ) crystal. Presented findings show complex outputs with a complexity > 0.85 for feedback values up to 7%. The complexity is also shown for timescales corresponding to various sub-harmonics of the cavity round-trip time and the relaxation oscillation frequency. A limited range of low feedback values (0.9 % -2.22 %) with prominent relaxation oscillation frequency, shows deviation from the typical observations of complexity in the parameter space. This is found to correlate with the chaos BW.

Permutation entropy (PE) is applied for quantification of the complexity. This algorithm, developed by Bandt and Pompe [START_REF] Bandt | Permutation entropy: A natural complexity measure for time series[END_REF], is known to measure the predictability of the recurring temporal patterns in a time series through comparison of the relative magnitude of the data points and calculating the probability distribution of userdefined subsets of the time-series. Its extreme robustness to nonlinearity, along with, simplicity and speed of execution make PE an attractive candidate for complexity analysis [START_REF] Bandt | Permutation entropy: A natural complexity measure for time series[END_REF][START_REF] Riedl | Practical considerations of permutation entropy[END_REF]. Furthermore, it has been employed to a wide range of applications including semiconductor laser systems [START_REF] Soriano | Time scales of a chaotic semiconductor laser with optical feedback under the lens of a permutation information analysis[END_REF][START_REF] Rontani | Enhanced complexity of optical chaos in a laser diode with phase-conjugate feedback[END_REF][START_REF] Yang | Mapping the dynamic complexity and synchronization in unidirectionally coupled external-cavity semiconductor lasers using permutation entropy[END_REF][START_REF] Aragoneses | Unveiling the complex organization of recurrent patterns in spiking dynamical systems[END_REF][START_REF] Toomey | Mapping the dynamic complexity of a semiconductor laser with optical feedback using permutation entropy[END_REF], and the finance sector [START_REF] Zunino | Forbidden patterns, permutation entropy and stock market inefficiency[END_REF]. The algorithm is detailed in [START_REF] Bandt | Permutation entropy: A natural complexity measure for time series[END_REF][START_REF] Riedl | Practical considerations of permutation entropy[END_REF] and summarized here. Consider three user-defined parameters, namely, ordinal pattern length (D), length of the time-series (N ) and the delay (τ ). The ordinal pattern length corresponds to the number of data points extracted from a time-series with each data point τ apart. The delay value is defined as the ratio of the timescale at which one wishes to quantify the complexity to the sampling time (t samp ). The widely accepted conditions for the parameters are 3 ≤ D ≤ 7 and N >> D [START_REF] Riedl | Practical considerations of permutation entropy[END_REF]. The significantly higher length of the time-series, compared to the ordinal pattern length allows the algorithm to construct many subsets of time-series, known as the ordinal pattern sets (∆). A maximum of D! number of ordinal patterns may be constructed. The normalized PE (ρ τ ) for a given probability distribution 'p' associated with 'i' integral number of ordinal patterns and timescale 'τ ' is mathematically given by:

ρ τ = -1 ln(D!) D! i=1 p(∆ i )ln[p(∆ i )] (1)
where 0 ≤ ρ τ ≤ 1 with zero signifying complete predictability while one indicates complete stochasticity. In other words, we visualise a given time series in the form of digitised data points acquired at the sampling time, t samp , storing the amplitude of laser outputs in the complex spatiotemporal domain. The time series follows a helix, wrapped around the helix axis with a constant pitch (β = τ x ) with τ x as the delay at which one wishes to observe the temporal order. The radius of the helix and the number of turns change with the timescale of interest (= τ x .t samp ) for constant length of the helix. An observation axis with length equivalent to the user-defined ordinal pattern length (D) is parallel to the helix axis indicating the data points creating new ordinal patterns. In the presented study, values associated with the timescale corresponding to the external cavity round-trip time (T RT 10.3 ns) referred to as the round-trip delay (τ RT = 824) and its sub-harmonics will be a major timescale of interest. This is because the laser dynamics are known to evolve at timescales proportional to T RT [START_REF] Soriano | Time scales of a chaotic semiconductor laser with optical feedback under the lens of a permutation information analysis[END_REF][START_REF] Sciamanna | Physics and applications of laser diode chaos[END_REF][START_REF] Rontani | Enhanced complexity of optical chaos in a laser diode with phase-conjugate feedback[END_REF][START_REF] Toomey | Mapping the dynamic complexity of a semiconductor laser with optical feedback using permutation entropy[END_REF]. To ensure a high resolution analysis, we choose D = 7, N = 36404, 1τ = t samp =12.5 ps to track changes in the temporal order [START_REF] Riedl | Practical considerations of permutation entropy[END_REF].

The experimental setup shown in Fig. 1 is based on our previous work in [START_REF] Bouchez | Wideband chaos from a laser diode with phase-conjugate feedback[END_REF]. A commercial Fabry-Pérot semiconductor laser (JDS Uniphase DL-SDL-5420) is used with an emission wavelength of 852 nm, the threshold current of 14.9 mA and an operating current range between 20 -130 mA. The experiment was performed at an operating current of 80 mA and the temperature was maintained at 20 • C. The collimating lens is an aspheric objective lens (Newport 5722-B-H) with an effective focal length of 4.5 mm. A beam splitting plate (80:20) redirects the laser emission in three different directions. A part of the transmitted laser beam is coupled into a standard monomode fibre that couples laser emission into a 38 GHz BW photodiode (Newport 1474-A). The free-space optical isolator (Thorlabs IO-3-850-HP) prevents the reflections from the coupler. The system output is acquired as digitized timeseries using an oscilloscope (Teledyne LeCroy 10-Zi-A 36 GHz) at a sample rate of 80 GS/s. The part of the laser beam transmitted through the beam splitting plate is used for PCF generated using a Rh-doped BaTiO3 photorefractive crystal (5 mm × 5 mm × 5 mm). At the entrance of the crystal, the laser beam is known to fan or deviate from the expected straight direction due to the beam coupling between the forward propagating incident waves and its scattering caused by the inhomogeneities in the crystal. The deviated laser beam experiences total internal reflection on the different facets of the bulk crystal. Such reflected beams interact among themselves resulting in a phase conjugated backwards propagating laser beam due to the phenomenon of four-wave mixing [START_REF] Feinberg | Self-pumped, continuous-wave phase conjugator using internal reflection[END_REF]. The remaining laser emission is fed to a power meter for acquiring the feedback value. The external cavity round-trip time (T RT ) is 10. The chaos BW is defined as the upper limit frequency of the RF spectrum containing 80% of the signal energy [START_REF] Bouchez | Wideband chaos from a laser diode with phase-conjugate feedback[END_REF]. It was calculated for every 455.05 ns long time-series for the feedback (η) values between 0 -7 % as shown in Fig. 2. The exhibited dynamical regimes are divided into four regions. Region I is limited to η < 0.9 % with high electronic noise resulting in an artificially created high chaos BW. The system transitions from pure noise to the onset of relaxation oscillations followed by the increase in the magnitude of the relaxation oscillation frequency as one approaches region II. The chaos BW is observed to drop by a magnitude of 10 GHz as the electronic noise becomes a non-contributing factor with increased feedback. Region II is of primary interest in the presented study and encompasses 0.9 % < η < 2.22%. It is further described in Fig. 3. The chaos BW is limited to a varied range of 1.66 GHz before entering region III. This region (2.4 % < η < 4.15 %) is dynamically diverse as the strong feedback drives the system into a combination of external cavity modes and pulsing states. Finally in region IV, the external cavity modes are the sole surviving dynamics for η > 4.3 %. It is known as a virtual spatiotemporal plot in which the timedelay plays a role analog to a virtual spatial coordinate and the dynamics within one time-delay span is followed as time evolves in units of the time-delay [START_REF] Arecchi | Two-dimensional representation of a delayed dynamical system[END_REF]. Such spatio-temporal analogy of the time-delayed laser dynamics has been exploited extensively within the context of the emergence of dissipative spatial solitons [START_REF] Marconi | Vectorial dissipative solitons in vertical-cavity surface-emitting lasers with delays[END_REF] or also the study of collective disordered dynamics in otherwise synchronized networks of spatially extended oscillators -also called chimeras [START_REF] Uy | Optical chimera in light polarization[END_REF][START_REF] Larger | Laser chimeras as a paradigm for multistable patterns in complex systems[END_REF]. Figure 3(a) is produced by dividing the 455.05 ns long time-series into continuous subsets corresponding to the cavity round-trip time.

The horizontal axis represents one round-trip time. Each subsequent subset of the time-series is stacked on top of each other to represent the number of round-trips, i.e., the vertical axis. Each pixel in Fig. 3(a) corresponds to an amplitude of the experimentally acquired data point with the colour scaled in accordance with the colour bar located on the right. Operating conditions in region II exhibit outputs characterized by packets of noise-like signal amplitude spikes when observed over one (or few) cavity round-trips as shown in Fig. 3(b). However, over a few tens of round-trips, some order emerges in the laser dynamics.

Figure 3 (c) shows the typically observed variation in the complexity using PE as a function of delay of the sample time-series shown in Fig. 3 (a) and (b). Recall that a dip in PE value indicates high predictability. A characteristic temporal signature of the laser output is its relaxation oscillation frequency (f RO 5.2 GHz), twice of which is observed at τ = 8 τ RO /2. This is confirmed by the Fourier spectrum analysis of these time series.Time signature corresponding to the relaxation oscillation frequency (τ RO ) is observed for the time-series lying in region I. This signature is observed to disappear for the increased values of feedback. Region II is also observed to exhibit temporal signature at a fractional multiple of the f RO , i.e., (69/2)τ RO . The ordinal pattern distribution characteristic to the timescales associated with τ RO are unlike those at τ RT , regardless of the ordinal pattern length. The τ RT of the experimentally acquired time-series is seen at τ RT = 830 as opposed to the previously estimated value of τ RT = 824. This is found to be the case for all the operating conditions observed over regions I -IV. The associated external cavity round-trip time is equivalent to 10.375 ns (96.39 MHz) as opposed to 10.3 ns (97.09 MHz). This slight mismatch between the statistical calculation and experimental observation is due to the inertia of the laser system and includes uncertainty contributed by the oscilloscope's sampling time. Characteristic temporal signatures are also observed for indicated sub-harmonic multiples of the round-trip time seen in Fig. 3 (c).

Figure 2 shows PE value (ρ RT ) at τ RT as a function of feedback for the entire parameter space region. The complexity is high, upwards of 0.85, for most operating conditions with an exception to the operating conditions exhibiting stationary dynamics or transitioning to the pulsing states. This is expected as a stable train of pulses will have high predictability. However, outside of region III, i.e., operating conditions lying within 3.3 % ≥ η ≥ 1 % and 4.39 % ≤ η ≤ 6.5 % exhibit ρ RT > 0.93 and are hence, highly complex with extremely low predictability. This is highly advantageous for applications concerning secure communication. Generally speaking, ρ RT as a function of feedback strength is observed to follow the pattern of damped oscillations when observed for η < 1% and mostly limits itself between 0.9363 < ρ RT < 0.9869 for η > 1%, excluding the exceptions of stable pulsing states having low complexity. Furthermore, Fig. 2 confirms two things. Firstly, the range of ρ RT values in regions I, II and IV measured experimentally agree with the range of values numerically calculated for the theoretical model in [START_REF] Rontani | Enhanced complexity of optical chaos in a laser diode with phase-conjugate feedback[END_REF]. However, the presented system exhibits consistent high complexity (excluding the pulsing states) as a function of feedback strength, contrary to the trend of monotonic increase followed by a plateau and then a slight decrease predicted by the numerics [START_REF] Rontani | Enhanced complexity of optical chaos in a laser diode with phase-conjugate feedback[END_REF]. Secondly, the experimental implementation of COF as in [START_REF] Toomey | Mapping the dynamic complexity of a semiconductor laser with optical feedback using permutation entropy[END_REF] for such long cavity round-trip length (1.35 m for the case in [START_REF] Toomey | Mapping the dynamic complexity of a semiconductor laser with optical feedback using permutation entropy[END_REF]) has ρ RT ≈ 0.75 for D = 7 at high injection strength and feedback. Additionally, the complexity of the presented system is also observed to be higher compared to that of a COF system calculated numerically in [START_REF] Rontani | Enhanced complexity of optical chaos in a laser diode with phase-conjugate feedback[END_REF]. Hence, we experimentally demonstrate that the PCF system is capable of much higher complex outputs restricted to a much lower variation in complexity over a wide range of feedback strength.

% (a) Spatiotemporal dynamics observed over multiple roundtrips as a function of the round-trip time (ns). The colour bar represents output signal amplitude (a.u.); (b) Sample time-series over one round-trip and, (c) PE (ρτ ) as a function of delay (τ ) showing the general temporal signatures corresponding to the subharmonics of cavity round-trip time (τRT ) and the relaxation oscillation frequency (τRO).

As a general point, we have established that the PCF system exhibits complex dynamics with high chaos BW. Interestingly, a rearrangement of the complexity between the ordinal patterns is observed uniquely to the region II. This is deduced upon observing the correlation between PE values at the characteristic temporal signatures as seen in Fig. 3 (c) and, the chaos BW as we now discuss. higher value than τ RT /5 and lower than τ RT /6 . Outside the window of observation limited to the operating conditions lying in region II, the PE values at τ RT /4 follow the typical general trend of linearly increasing PE values with increasing value of the sub-harmonic round-trip delay. Figure 4 (b) shows the PE value at τ RO /2 steeply increases with an increase in the feedback strength until it saturates before entering region III. Unlike the delay values plotted in Fig. 4 (a), a strong correlation between ρ τ RO /2 , ρ (69/2)τ RO and chaos BW is observed. We emphasise that the subtle complexity reorganization is hidden from isolated analysis of complexity at singular values of delay. Rather, it is revealed upon the comparitive study at specific temporal system signatures and its correlation with chaos BW. The study also highlights (i) such a complexity reorganization of chaos when increasing the feedback rate is obtained while the chaos only shows small variations of its BW and keeps a relatively large BW value; (ii) the chaos BW and the chaos complexity are both associated with peculiar dynamical signatures at T RT and f RO . These features exclusive to region II inform us that the driving dynamics emerge mostly from the undamping of the f RO hence marking a clear transition with the dynamics observe in region III.

Our experimental observations of the presented PCF system shows inherent high spatiotemporal complexity with minimal variation, across a wide range of feedback strengths. From the perspective of complexity, reinforced by the past theoretical analysis, a laser diode subjected to PCF is confirmed to perform better than to COF. Our findings reaffirm the PCF system as an excellent candidate for applications in secure communication providing consistently high chaos BW with high complexity. The identified system characteristic temporal signatures correspond to the sub-harmonics of the cavity round-trip time and the relaxation oscillation frequency. The study presents distinguishing features exclusive to the region of low (region II) feedback strength, namely, (i) evidence of strong correlation between the relaxation oscillation frequency and the chaos BW, (ii) reorganized spatiotemporal complexity and, (iii) lower values of chaos BW relative to the global range of values. These features demarcate a clean transition between the regions of low and high (region III) feedback strengths. Our study deduces the selective dominance of the regulating dynamics in a PCF system. The system dynamics are governed by the relaxation oscillation frequency at low feedback values, while, the external cavity modes control the system dynamics at high feedback strengths. From a broader perspective, we present here a detailed complexity analysis of a spatiotemporal laser dynamics that emerges from a time-delayed laser system. Such analysis benefits from the computation of PE at different time-scales relative to the laser feedback timedelay and laser internal time-scales. The same technique could apply to obtain deeper insight into temporal order and spatial symmetries of complex unstable, transient and turbulent spatio-temporal dynamics that are commonly encountered, for instance, in the transition to chimera states in networks of coupled nonlinear oscillators.

Région Grand-Est, Moselle, Metz Métropole, European Union (FEDER), Ministry of Higher Education and Research (FNADT), AIRBUS-GDI Simulation. Disclosures. The authors declare no conflicts of interest.

  3 ns with corresponding cavity length equivalent to 1.545 m and an external cavity frequency of 97.09 MHz.

Figure 1 :

 1 Figure 1: Laser diode subjected to PCF using a BaTiO3 crystal. BSP: beam splitting plate; PCM: phase-conjugate mirror; ISO: Faraday isolator; PD: photodiode.

Figure 2 :

 2 Figure 2: Dynamics of a PCF system in the context of PE at roundtrip delay (primary vertical axis) and chaos BW (secondary vertical axis) as a function of feedback. Region I (yellow): relaxation oscillation buildup; II (green): low-feedback; III (grey): transition window towards external cavity modes (ECM) with black dots representing pulsed output; IV (red): high-feedback with ECM.Black dots represent pulsing states.

Figure 3 (

 3 Figure3(a) is a 2D representation of the aforementioned helix representation with β = τ RT and the number of turns correspond to the number of round-trips. It is known as a virtual spatiotemporal plot in which the timedelay plays a role analog to a virtual spatial coordinate and the dynamics within one time-delay span is followed as time evolves in units of the time-delay[START_REF] Arecchi | Two-dimensional representation of a delayed dynamical system[END_REF]. Such spatio-temporal analogy of the time-delayed laser dynamics has been exploited extensively within the context of the emergence of dissipative spatial solitons[START_REF] Marconi | Vectorial dissipative solitons in vertical-cavity surface-emitting lasers with delays[END_REF] or also the study of collective disordered dynamics in otherwise synchronized networks of spatially extended oscillators -also called chimeras[START_REF] Uy | Optical chimera in light polarization[END_REF][START_REF] Larger | Laser chimeras as a paradigm for multistable patterns in complex systems[END_REF]. Figure3(a) is produced by dividing the 455.05 ns long time-series into continuous subsets corresponding to the cavity round-trip time.The horizontal axis represents one round-trip time. Each subsequent subset of the time-series is stacked on top of each other to represent the number of round-trips, i.e., the vertical axis. Each pixel in Fig.3(a)corresponds to an amplitude of the experimentally acquired data point with the colour scaled in accordance with the colour bar located on the right. Operating conditions in region II exhibit outputs characterized by packets of noise-like signal amplitude spikes when observed over one (or few) cavity round-trips as shown in Fig.3(b). However, over a few tens of round-trips, some order emerges in the laser dynamics.Figure3(c) shows the typically observed variation in the complexity using PE as a function of delay of the sample time-series shown in Fig.3(a) and (b). Recall that a

Figure 3 :

 3 Figure 3: Typical intracavity dynamics in region II. For η = 1.23 % (a) Spatiotemporal dynamics observed over multiple roundtrips as a function of the round-trip time (ns). The colour bar represents output signal amplitude (a.u.); (b) Sample time-series over one round-trip and, (c) PE (ρτ ) as a function of delay (τ )showing the general temporal signatures corresponding to the subharmonics of cavity round-trip time (τRT ) and the relaxation oscillation frequency (τRO).

Figure 4 (

 4 a) shows the PE values at ρ RT /6 , ρ RT /5 , ρ RT /3 , ρ 2RT /5 , ρ RT /2 and, ρ RT as a function of feedback. The PE values lower as one approaches towards τ RT in ascending order of the delay values. Thus, the correlation and the predictability of the ordinal patterns become progressively stronger as the values corresponding to the sub-harmonic multiple of the round-trip time increases. This trend has an exception at τ = τ RT /4 as indicated using black arrows in Fig.4 (a). The PE values at τ RT /4 are observed to have a

Figure 4 :

 4 Figure 4: PE (primary vertical axis) as a function of feedback for (a) τx indicated in far-right. Black arrows indicate a change in delay branch position for τ = τRT /4. (b) for τ = τRO/2. Secondary vertical axis represents the chaos BW. A similar trend observed at τ = (69/2)τRO.