Definition of an optimized protocol for recovery of KpSC from food samples.

Our initial comparisons of the four different culture protocols (Fig. S2) showed that the most effective method for recovery of KpSC from chicken meat was protocol 1B, which consists of a one-step enrichment in buffered peptone water (BPW). KpSC was recovered from 7 out of 36 (19.4%) samples using this protocol, while only 3 (8.3%) samples were positive using protocol 2 (enrichment in LB broth supplemented with ampicillin) and only 2 (5.6%) using protocol 1C (double enrichment—enrichment in BPW followed by enrichment in LB broth supplemented with ampicillin). No KpSC was isolated using direct plating (protocol 1A).

Further, using BPW enrichment of chicken meat (protocol 1B), a higher recovery of KpSC was observed when streaking a 10-µL loop onto SCAI plates versus spreading 10µL or 100µL of bacterial enrichment. Of the 28 chicken meat samples examined using both methods, KpSC was recovered from 24 (85.7%) samples using the 10-µL loop versus 19 (79.2%) samples after spreading 10µL and 7 (29.2%) samples after spreading 100µL onto SCAI medium. Thus, protocol 1B combined with streaking was used.

Evaluation of the SCAI plate incubation temperature.

We compared two temperatures (37°C and 44°C) for the incubation of SCAI plates (Fig. S3). Collectively, 111 chicken samples were tested, and 67.7% (64/111) of them were positive for KpSC. KpSC was recovered from 59/111 (53%) samples when SCAI medium was incubated at 37°C, as well as when incubated at 44°C, indicating no difference between incubation temperatures (Table S2). However, when data from each individual institution were analyzed, a slightly higher percentage of positive samples was recorded following incubation of SCAI plates at 44°C at two institutions (Agency for Health and Food Safety [AGES] and National University of Ireland Galway [NUIG]), whereas contrasting results were observed at the third institution (Statens Serum Institute [SSI]) (Table S2). As a result, a decision was made to implement incubation of SCAI plates at 44°C±1°C, which was incorporated into our protocol (Fig. 1) (dx.doi.org/10.17504/protocols.io.baxtifnn). All further food samples were tested according to this protocol. Of note, no statistical differences were found in the prevalence of KpSC in free-range and not free-range chicken meat samples (Table S2).

Open in a separate window

FIG 1

Optimized protocol for the recovery and isolation of Klebsiella spp. from food matrices.

Comparison of culture and ZKIR qPCR methods for KpSC detection.

Chicken meat and salad leaf samples were tested for the presence of KpSC using both the ZKIR qPCR assay (13) and the optimized culture protocol (Fig. 1). Regarding the chicken meat samples (n=160), the ZKIR qPCR method detected KpSC in 96 (60.0%) of the samples (Table 1). In comparison, when the culture protocol was used, 83 (51.9%, P=0.177) of the samples were positive for KpSC (82 K. pneumoniae and 1 K. variicola according to matrix-assisted laser desorption ionization–time of flight mass spectrometry [MALDI-TOF MS] results). Regarding salad leaf samples (n=145), 29.7% (43/145) of the samples tested positive for KpSC using the ZKIR qPCR assay, versus 20.7% (30/145; P=0.104; 18 K. pneumoniae and 12 K. variicola according to MALDI-TOF MS results) using the culture method (Table 1). Considering the ZKIR qPCR assay as a reference, the optimized culture protocol showed a sensitivity (true-positive rate) of 84.2%, a specificity (false-positive rate) of 100%, a positive predictive value of 100%, and a negative predictive value of 86.6%.

Comparison of KpSC prevalence in chicken meat and salad samples.

The overall prevalence of KpSC in chicken meat was twice as high as that in salad samples (60.0% versus 29.7%; P<0.00001; Table 1). However, prevalence differed among countries (Fig. 2). The prevalence of KpSC in chicken meat samples ranged between 43.3% and 46.6% for three of the countries (Austria, Italy, and Ireland), whereas in France and Denmark the prevalence was above 70% (72.5% and 90.0%, respectively). Regarding salad samples, the prevalence of KpSC ranged from 3.3% in Ireland to 92% in France, with Austria, Italy, and France being the countries where the highest numbers of samples positive for KpSC were detected.

Open in a separate window

FIG 2

Prevalence of K. pneumoniae species complex by type of sample and by country.

In total, 131 KpSC isolates (92 from chicken meat and 39 from salads) were collected from 113 positive samples using the optimized culture method; in some positive samples, up to 5 different morphotypes were collected (Fig. 2, Table S3).

Antimicrobial susceptibility.

Antimicrobial susceptibility phenotypes were defined for the 131 KpSC isolates using 17 antimicrobial agents representing 11 antimicrobial classes (Table S4); 82% of the isolates presented a wild-type phenotype, being susceptible to all the antibiotics tested except ampicillin, for which constitutive resistance is a characteristic of all KpSC isolates. The highest rates of resistance (detected in>5% of the isolates) were observed for trimethoprim (12% in chicken meat and 13% in salads), trimethoprim-sulfamethoxazole (8% for both), tetracycline (9% in chicken meat and 5% in salads), and piperacillin-tazobactam (7% in chicken meat; Fig. S4, Table S4). Four isolates (2 from each source) were found to be resistant to extended-spectrum cephalosporins, and the presence of extended-spectrum β-lactamase (ESBL) genes in the genomic sequence was confirmed (Table S4). No isolate was found to be resistant to cefoxitin, ertapenem, or netilmicin. Similar levels of resistance were detected among isolates from salads and from chicken meat.

Differences in antimicrobial resistance occurrence rates were observed among countries (Table S4), but the low number of isolates implies precaution in the analysis. For chicken meat, higher resistance rates were detected in Italy (8 to 50%), Austria (7 to 23%), and Ireland (7 to 14%), whereas for salads it was in Italy (33 to 100%), Austria (20%), and France (4 to 12%) (Fig. S4, Table S4).

Nine (6.9%) multidrug-resistant isolates were identified (Table S5), including the four ESBL producers. MDR isolates were recovered in 7 chicken meat and 2 salad samples in Italy (n=3), Austria (n=2), France (n=2), and Ireland (n=2) (Table S5).

Initial evaluation of protocols for recovery of Klebsiella pneumoniae from food.

An initial assessment of four protocols for recovery of KpSC from chicken meat was carried out. In total, 36 chicken meat samples (12 free-range [7 skin-on and 5 skin-off] and 24 non-free range [12 skin-on and 12 skin-off] samples) were collected together with the following metadata: manufacturer, country of origin, and batch number. Each chicken meat sample was cut into small slivers to a final weight of 50 g. This 50-g sample was divided into two portions of 25 g and processed as follows and as illustrated in Fig. S2.

Initial suspension 1. The first 25-g portion was diluted in 225mL (1:10 dilution) of BPW, and the sample was homogenized using a stomacher/blender. Prior to incubation of initial suspension 1, 100µL of the suspension was cultured directly on SCAI medium using a sterile spreader or sterile loop and incubated at 37°C±1°C for 48 h±1 h (Fig. S2 [1A, detection without enrichment]). The remainder of initial suspension 1 was incubated at 37°C±1°C for 24 h±1 h. Following 24 h of incubation of initial suspension 1, 100µL of the suspension was streaked on SCAI medium and incubated at 37°C±1°C for 48 h±1 h (Fig. S2 [1B, detection with BPW enrichment only]). Of the remaining incubated suspension 1, 1mL was inoculated into 9mL of lysogeny broth (LB) with ampicillin (final concentration, 0.01mg/mL) and incubated at 37°C±1°C for 24 h ± 1 h. Following 24 h of incubation, 100µL of the suspension was streaked on SCAI medium and incubated at 37°C±1°C for 48 h±1 h (Fig. S2 [1C, detection with double enrichment]).

Initial suspension 2. The second 25-g portion was diluted in 225mL (1:10 dilution) of LB broth supplemented with ampicillin (final concentration 0.01mg/mL), and the sample was homogenized using a stomacher/blender and incubated at 37°C±1°C for 24 h±1 h. Following 24 h of incubation, 100µL of the suspension was cultured on SCAI medium and incubated at 37°C±1°C for 48 h±1 h (Fig. S2 [2, detection with LB + ampicillin enrichment only]).

Typically, Klebsiella spp. appear yellow on SCAI medium (14). At least five suspected Klebsiella colonies were collected from each SCAI plate and subcultured onto SCAI medium or a nonselective agar for identification using MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). Currently, databases only allow for the identification of K. pneumoniae or K. variicola. Based on the literature and in our test strain set, Kp1, Kp2, and Kp4 strains are identified as K. pneumoniae, whereas Kp3, Kp5, and Kp6 are currently identified as K. variicola (45, 46).

For 28/36 chicken meat samples, the impact on KpSC recovery of culturing using a 10-µL loop versus the spreading of 10µL and 100µL of enrichments onto SCAI medium was also compared.

Evaluation of the optimized protocol and definition of an optimal temperature of incubation.

Following this initial assessment process, the optimized protocol as outlined below and as illustrated in Fig. S3 was evaluated for recovery of KpSC from chicken meat samples using a total of 111 samples of chicken meat (51 free-range [25 skin-on and 26 skin-off] and 60 non-free range [30 skin-on and 30 skin-off]).

Each sample was cut into small slivers to a final weight of 25 g. This was added to 225mL of BPW broth (1:10 dilution), and the sample was mixed using a stomacher or blender. The suspension was then incubated at 37°C±1°C for 24 h±1 h. Following incubation, the suspension was cultured for single colonies on SCAI using a 10-µL loop and incubated at two different temperatures, 37°C±1°C and 44°C±1°C, for 48 h±1 h (Fig. S3) in order to establish an optimal temperature of incubation of SCAI medium for recovery of K. pneumoniae from food matrices.

Comparison of the optimized SCAI culture protocol and ZKIR qPCR.

A comparison of the optimized SCAI culture protocol and the ZKIR qPCR for the detection and isolation of KpSC in food matrices was carried out in 5 European institutions, the Austrian Agency for Health and Food Safety (AGES), French Agency for Food, Environmental and Occupational Health & Safety (ANSES), National University of Ireland Galway (NUIG), Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise Giuseppe Caporale (IZSAM), and the Statens Serum Institute (SSI, Denmark).

Each institution was asked to collect and test, where possible, an additional 30 samples of chicken meat, as well as 30 prepackaged, prewashed salad leaf samples (Table S2). In total, 160 chicken meat samples and 145 salad samples, collected between December 2018 and September 2019 in different supermarkets and representing a variety of suppliers (average of 9 suppliers per country) were tested using the updated optimized protocol (dx.doi.org/10.17504/protocols.io.baxtifnn) for recovery of KpSC from food matrices (Fig. 1). In order to avoid external contamination during the laboratory processing, all samples were analyzed in accordance with laboratory practice as reported in ISO 7218:2007, “Microbiology of Food and Animal Feeding Stuffs—General Requirements and Guidance for Microbiological Examination.”

The ZKIR qPCR was used to detect the presence of KpSC in food samples (13). After enrichment, DNA was prepared for ZKIR qPCR. For 500μL of the enrichment broth was centrifuged (5min at 5,800×g), washed with sterile water, and resuspended in 500μL of sterile water before boiling for 10min. qPCR conditions were as previously described (13) (dx.doi.org/10.17504/protocols.io.7n6hmhe).

Antimicrobial susceptibility testing.

Antimicrobial susceptibility testing was performed on 131 KpSC isolates using either disk diffusion or broth microdilution (GN2F panels [Sensititre; Thermo Fischer Scientific]) methods. Clinical breakpoints were used for interpretation according to EUCAST guidelines (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_9.0_Breakpoint_Tables.pdf), except for kanamycin and streptomycin (47). Escherichia coli ATCC 25922 was used as the control strain. The panel of antimicrobials tested included amoxicillin-clavulanic acid (20/10µg), piperacillin-tazobactam (both using disks of 30/6µg or GN2F plates), cefpodoxime (10µg, GN2F) or cefotaxime (5µg), cefoxitin (30µg, GN2F), aztreonam (30µg), ertapenem (10µg), amikacin (30µg, GN2F), gentamicin (10µg, GN2F), netilmicin (10µg), tobramycin (10µg, GN2F), kanamycin (10µg), streptomycin (30µg), ciprofloxacin (5µg, GN2F) or ofloxacin (5µg), nalidixic acid (30µg), trimethoprim (5µg), trimethoprim-sulfamethoxazole (23.75+1.25µg, GN2F), and tetracycline (30µg). The presence of multidrug-resistant isolates, which are defined as those resistant to at least one agent in three different classes of antimicrobials (48), was analyzed.

Whole-genome sequencing and comparative genomic analyses.

Whole-genome sequencing was performed on the 131 K. pneumoniae isolates. Genomic DNA libraries were prepared using the Nextera XT library prep kit (Illumina, San Diego, USA) following the manufacturer’s protocol. Illumina sequencing was performed at the five partners—AGES and SSI using MiSeq (2×250-bp paired-end sequencing; n=18 and n=31 isolates, respectively), ANSES and NUIG using NovaSeq 6000 (2×150-bp paired-end sequencing; n=52 and n=15, respectively), and IZSAM using NextSeq 500/550 (2×150-bp paired-end sequencing; n=15). Genomic assemblies were obtained using SPAdes v3.9 (49) or Velvet v1.2.10 (50) and were annotated using Prokka v1.12 (51).

Multilocus sequence typing (MLST) (52), core-genome MLST (cgMLST), and cgLIN codes (16) were determined using the BIGSdb-Kp database (https://bigsdb.pasteur.fr/klebsiella/). This web tool and Kleborate (53) (https://github.com/katholt/Kleborate) were used to look for antimicrobial resistance, virulence, and heavy metal tolerance genes and to characterize the capsular synthesis gene cluster. Plasmid replicons were searched using PlasmidFinder (54) (https://cge.cbs.dtu.dk/services/PlasmidFinder/).

For phylogenetic analyses, a core-genome alignment based on the concatenation of 4,125 core genes was obtained with Roary v3.12 (55) using a blastP identity cutoff of 80% and core genes defined as those being present in more than 90% of the isolates. Recombination events were removed using Gubbins v2.2.0 (56), generating a recombination-free alignment comprising 486,290 single-nucleotide variants (SNVs). This recombination-free alignment was used to construct a maximum likelihood phylogenetic tree using IQ-TREE v1.6.11 (model GTR+F+ASC+R5).

Chi square or Fisher exact tests were used to compare the prevalence of KpSC in the different sources and to check the association of the different categorical variables (P values of <0.05 were considered statistically significant).

We thank James Bray and Keith Jolley for technical assistance with genomic assemblies of KpSC isolates from Ireland. We thank the Institut Pasteur teams for the curation and maintenance of BIGSdb-Pasteur databases at http://bigsdb.pasteur.fr/.

This work was supported financially by the MedVetKlebs project, a component of European Joint Program One Health EJP, which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 773830. C.R. was also financed by a Roux-Cantarini grant from Institut Pasteur.

This research was funded, in whole or in part, by Institut Pasteur and by the European Union’s Horizon 2020 research and innovation program. For the purpose of open access, the authors have applied a CC-BY public copyright license to any author manuscript version arising from this submission.

S.B., C.R., D.M., K.H., E.M.N., and F.P. conceptualized the study. G.C., A.C., and F.P. from IZSAM performed the culture media evaluation. K.H., M.L.-M., E.M.N., N.C., and D.M. performed the initial assessments for the establishment of an optimized culture protocol for recovery of KpSC from food matrices. K.H., N.C., M.L.-M., G.B., A.C. and R.G.F. performed the comparison of culture media and the ZKIR qPCR assay. E.B. and P.P. provided laboratory support and validated the analysis of the ZKIR qPCR assay. C.R. and S.B. analyzed all the genomic data, coordinated the study, and wrote the initial draft manuscript. S.B. acquired funding. All authors contributed to writing and editing the manuscript and reviewed the final version.