Nucleic acid lateral flow immunoassay (NALFIA) with integrated DNA probe degradation for the rapid detection of Cronobacter sakazakii and Cronobacter malonaticus in powdered infant formula

Food Control 109 (2020) 106952

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Nucleic acid lateral flow immunoassay (NALFIA) with integrated DNA probe degradation for the rapid detection of Cronobacter sakazakii and Cronobacter malonaticus in powdered infant formula

T

Ömer Akinedena,∗, Tobias Wittwerb, Katrin Geisterb, Madeleine Plötzc,1, Ewald Uslebera a

Chair of Dairy Science, Institute of Veterinary Food Science, Justus-Liebig University Giessen, Ludwigstraße 21, D-35390, Giessen, Germany R-Biopharm AG, An der Neuen Bergstraße 17, D-64297, Darmstadt, Germany c Junior Professorship of Veterinary Food Diagnostics, Institute of Veterinary Food Science, Justus-Liebig University Giessen, Ludwigstraße 21, D-35390, Giessen, Germany b

ARTICLE INFO

ABSTRACT

Keywords: Cronobacter sakazakii Cronobacter malonaticus Enterobacter sakazakii Powdered infant formula Nucleic acid lateral flow immunoassay Rapid detection

A novel nucleic acid lateral flow immunoassay (NALFIA) system for Cronobacter (C.) sakazakii and C. malonaticus is based on amplification of the rpoB gene in the presence of a heterobifunctional, Cronobacter-specific DNA probe. Partial degradation of this probe during amplification is then detected by lateral flow immunoassay. The NALFIA detected all of the 22 C. sakazakii and 8 C. malonaticus isolates under study, but showed no reaction with other Cronobacter species or non-Cronobacter species. The minimal visual and instrumental detection limits were 104 cfu/ ml for both species in buffered peptone water and in reconstituted infant formula. C. sakazakii or C. malonaticus at levels of 100-101 cfu/g in powdered infant formula were reliably detected by NALFI after reconstitution and 24 h incubation at 37 °C. A major advantage of this approach is that the use of an integrated DNA probe provides additional test specificity. Since C. sakazakii and C. malonaticus are by far the most important Cronobacter species occurring in PIF, the novel NALFIA is a promising tool to enhance rapid control of infant formula.

1. Introduction Cronobacter spp. (formerly Enterobacter (E.) sakazakii), members of the Enterobacteriaceae family (Iversen et al., 2008), are opportunistic pathogens which have been associated worldwide with rare but lifethreatening disease (meningitis, septicaemia, necrotizing enterocolitis) in newborn and premature infants. Currently the genus Cronobacter is composed of seven recognized species, but only strains of three species, C. sakazakii, C. malonaticus, and C. turicensis have been associated with clinical infections in infants (Forsythe, 2018). C. sakazakii and C. malonaticus are the most common Cronobacter species associated with human disease (Holý & Forsythe, 2014) and are responsible for the majority of Cronobacter infections in infants (Joseph et al., 2012). C. sakazakii and C. malonaticus are the predominant Cronobacter species in powdered infant formulae (PIF) worldwide (Akineden, Heinrich, Gross, & Usleber, 2017; Yang et al., 2016), including cases in which PIF have been identified as a source of infection for infants (Block et al., 2002; Holý & Forsythe, 2014; Mullane et al., 2007; Van Acker et al., 2001). Although the minimal infectious dose is not known, Cronobacter has

been found at levels less than 1 cfu per 100 g in PIF associated with infections, therefore European Union regulation on microbiological criteria for foodstuffs (European Commission, 2005) requires absence of Cronobacter spp. in 30 × 10 g of PIF intended for special medical purposes for infants under 6 months of age. The reference method for this criterion is ISO/TS 22964:2006, which has recently been updated by ISO 22964:2017 (De Benito et al., 2019). This method is both laborious and time-consuming, and does not allow differentiation between species. Although the reference method is mandatory for all legal purposes, PIF production facilities require suitable additional means for a more rapid presence/absence testing of relevant food safety parameters. In response to the needs of food industry, several complementary techniques have been established, either to obtain results more rapidly, or to enable species differentiation. These include molecular detection methods such as real time PCR (Yan & Fanning, 2015; Zhou et al., 2016; Zimmermann, Schmidt, Loessner, & Weiss, 2014), fluorescence in situ hybridization (Almeida et al., 2009), DNA microarray (Wang et al., 2009), and immunoassays (Blažková, Javůrková, Fukal, & Rauch, 2011; Park et al., 2012; Scharinger, Dietrich, Wittwer, Märtlbauer, & Schauer,

Corresponding author. E-mail address: [email protected] (Ö. Akineden). 1 Present Address: Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173, Hannover, Germany ∗

https://doi.org/10.1016/j.foodcont.2019.106952 Received 9 July 2019; Received in revised form 10 October 2019; Accepted 12 October 2019 Available online 15 October 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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2017; Scharinger, Dietrich, Kleinsteuber, Märtlbauer, & Schauer, 2016; Song, Shukla, Oh, Kim, & Kim, 2015; Xu et al., 2014). A relatively new approach combines PCR amplification with immunochemical detection, most appropriately as lateral flow dipstick tests for rapid detection. These methods combine the power of amplification of the target gene sequence with the sensitivity and ease of use offered by dipstick immunoassay techniques. A promising variant of such methods is NALFIA (Singh, Sharma, & Nara, 2015), which has been successfully applied to the detection of e.g. Cronobacter spp. (Blažková et al., 2011), Listeria monocytogenes (Blažková, Koets, Rauch, & van Amerongen, 2009), methicillin-resistant Staphylococcus aureus (Seidel et al., 2017; Zhang et al., 2017), Mycobacterium tuberculosisComplex (Haridas, Thiruvengadam, & Bishor, 2014) and Toxoplasma gondii infections (Wu et al., 2017). These methods typically use 5′-end labelled primers pairs with different labels for forward and reverse primers. For example, Blažková et al. (2011) used biotin and digoxigenin to label the 5′-end of the forward and reverse primers, respectively, the doubly labelled amplicon was then visualized on a lateral flow system in an anti-digoxigenin carbon-neutravidin sandwich complex. This approach was found convenient and sensitive for detection of Cronobacter at genus level. Here we report evaluation of a novel NALFIA technique which employs a heterobifunctional DNA probe which binds to a specific template DNA locus of C. sakazakii and C. malonaticus framed by the primer pair used for amplification. The degradation products of this probe, produced during the amplification in a singlestep reaction, are then measured by lateral flow immunoassay. Evaluation parameters included sensitivity, specificity and test robustness when applied to detect C. sakazakii and C. malonaticus in PIF.

streptavidin-coated control field. In positive tests, the digoxigenin-labelled fragment of degraded probes, after binding to DIG-mAb, are finally visualized on a test field coated with anti-mAb. Test evaluation can be performed either by visual estimation of color development or by instrumental reading of the reflectance of reaction fields. 2.3. Primer and labelled DNA probe design Primer pairs, DNA probe and strips were prepared and customized as needed for this study in the laboratories of R-Biopharm AG, Germany. The specific PCR primers and the DNA probe, targeting a highly conserved region of rpoB gene of C. sakazakii and C. malonaticus, were designed using PrimerExplorer V4 software (http://primerexplorer.jp/ elamp4.0.0/index.html) according to the sequence data as published in the National Center for Biotechnology (NCBI) GenBank, USA. All primers and probe were blasted against the NCBI nucleotide database to make sure there was no homology with sequences from other organisms. The final set included forward primer designated CsakFmm, reverse primer designated rpoB-RVmm, producing an expected amplicon size of 210 bp. The DNA probe, designated as rpoB-SNDn, was labelled with digoxigenin at the 5′-end, and with biotin at the 3′end (Table 3), to provide a heterobifuntional label. 2.4. PCR amplification and control analysis of PCR products by agarose gel electrophoresis The PCR assay was carried out in a total of 25 μl reaction mixture containing: 1 μl of each primer (1 μmol; CsakFmm and rpoB-RVmm), 1 μl of labelled DNA probe (0.1 μmol; rpoB-SNDn), 12.5 μl of a commercial PCR master mix buffer/reagent solution (SensiFAST Probe NoROX 2x; Bioline, Berlin, Germany), and 9.5 μl sterile distilled water. Finally, 2.5 μl of DNA template was added to each tube. The cycling amplification was conducted on a Bio-Rad T100 Thermocycler (BioRad, Munich, Germany) according to the following steps: initial incubation at 94 °C for 5 min, followed by 35 cycles of 95 °C for 20 s, 65 °C for 45 s. After classical PCR amplification of the target genes, the PCR amplification products were subjected to agarose gel electrophoresis, as control analyses, parallel to the NALFIA procedure. The PCR products (10 μl) were separated on 1.5% agarose gels, followed by ethidium bromide staining and photography under UV light using the GelDoc system (BioRad). DNA molecular size standards (100 bp gene ruler, Fermentas) were included in each agarose gel. For the positive control reaction, plasmid conjugated rpoB sequence was used. Lysis buffer was used as for negative control reaction.

2. Materials and methods 2.1. Bacterial strains The bacterial strains used in this study are listed in Tables 1 and 2 and were all from the institute's strain collection. The selection of Cronobacter strains was based on origin and specific characteristics such as species/subspecies, biotype, and serotype (Akineden et al., 2017). For exclusivity testing, 21 non-Cronobacter strains belonging to different bacterial families and genera were used. 2.2. Test principle and components of NALFIA with integrated DNA probe degradation The test principle of the NALFIA with integrated DNA probe degradation (Fig. 1) is based on amplification of the rpoB gene of C. sakazakii or C. malonaticus in the presence of a heterobifunctional DNA probe labelled with two different haptens, digoxigenin on the 5′ end and biotin on the 3′ end. This probe binds to a specific template DNA locus of C. sakazakii and C. malonaticus, framed by the primer pair used for conventional PCR amplification. The degradation products of this probe, produced during the amplification step, are then measured by lateral flow immunoassay via antibodies against the hapten label. In positive samples, the probe binds to DNA in a region framed by the forward and reverse primers, but is then partially degraded by exonuclease during amplification. The concentration of reagents was selected in such a way that only a part of the probe is degraded during the PCR process, therefore the intact portion of the probe could be used as an internal control. The degradation product is visualized on a lateral flow dipstick format (Fig. 1) which is typically composed of three parts: a sample pad for loading of the sample, a reaction nitrocellulose membrane for formation of the detectable colored lines and finally, an absorbent pad enhancing the capillary driving force and adsorbing non-reacting substances. The membrane has two reaction zones, one for specific detection of degraded probe, one as the control line to identify a valid test. On the NALFIA test strips, intact probe reacts with gold-labelled anti-digoxigenin monoclonal antibodies (DIG-mAb) and is visualized on a

2.5. Lateral flow immunoassay After amplification, 10 μl of the PCR reaction mixture and 90 μl of NALFIA running buffer (45 mmol Bis-Tris, pH 7.5, 100 mmol NaCl; 0.05% Triton X-100; 0.01% sodium azide) were pipetted in a sterile reaction tube and mixed gently using a pipette. Then the test strip was immersed, sample pad first, into the test solution, allowing the liquid to migrate through the dipstick membrane for about 12 min. After this time, the test was completed and a red colored line was discernible for the control field. The color intensities of the control and test lines were evaluated visually by at least two persons. The presence of two, clearly discernible red lines indicated a positive results for C. sakazakii and/or C. malonaticus, while presence of one line (control line) indicated a negative result. In case of invalid test results, neither test nor control lines or only the test line were visible.. After visual evaluation, the NALFIA strips were scanned and the reflectance of the test line quantitatively evaluated using a lateral flow reader (ESEQuant Lateral Flow System, Qiagen, Germany) in colorimetric mode and Lateral Flow Studio software (version 3.03.05, Qiagen). A reflectance signal intensity for the test line of ≥50 mV was set as the cut-off value to identify positive tests. 2

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Table 1 Cronobacter spp. (n = 41) strains used for inclusivity testing of the NALFIA. Species

Strain

Origin

Serotypea

Biotypeb

MLST Sequence typec

C. sakazakii (n = 22)

DSM 4485T DB-372/2k-06 DB-382-3D3-06 DB-376/2e-06 DB-258-1-05 DB-M2 Db-246-3-05 DB-277-1-05 (DB)38a DB-144a-5-05 DB-73a/2-05 DB-264-3-04 DB-141a-1-05 DB-280-2-05 DB-185c/3-05 DB-238/2-05 DB-255/1-05 DB-262/1-05 DB-275/1-05 DB-321/2c-06 DB-338/3j-06 DB-367/1c-05 DSM 18702T DB-160a-6-05 DB-299-6y-06 DB-84a/1-05 DB-271-3-05 IB-37a-2003 DB-143c-7-05 N30 DSM 18703T N35 N160 C32 DSM 21870T N40 DSM 18705T DSM 18706T DSM 18707T LMG 26250T NCTC9529 T

Type strain, clinical isolate, USA PIF (≥4 months)1, Germany PIF (≥4 months), Germany PIF (day 1), Germany PIF (day 1), Germany PIF (≥6 months), Germany PIF (≥6 months), Germany PIF (≥6 months), Germany PIF (≥4 months), Germany PIF (≥4 months), Germany Whey powder, Germany PIF (≥4 months), Germany PIF (≥4 months), Germany PIF (≥6 months), Germany PIF (≥6 months), Germany PIF (≥6 months), Germany PIF (≥4 months), Germany PIF (≥4 months), Germany PIF (≥4 months), Germany PIF (≥4 months), Germany PIF (≥4 months), Germany PIF (≥4 months), Germany Type strain, clinical isolate, USA Milk powder, Germany PIF (≥4 months), Germany PIF (≥8 months), Germany PIF (≥4 months), Germany PIF, Indonesia PIF (≥4 months), Germany Dried pasta, Japan Type strain, clinical isolate (Switzerland) Dried pasta, Germany Dried pasta, Germany Ready to eat salad, Germany Type strain, USA Dried pasta, Germany Type strain, environment, Ireland Type strain, water, Switzerland Type strain, environment, Zimbabwe Type strain, food, Slovakia Type strain, water, United Kingdom

SO1 SO4 SO3 SO1 SO1 SO2 SO1 SO2 SO4 SO2 SO3 SO1 SO7 SO2 SO7 SO4 SO2 SO2 SO3 SO7 SO1 SO3 MaO2 MaO1 MaO2 MaO1 MaO1 – MaO1 – Ctur O1 – – – CmuytO2 – CdubO1 CdubO2 CdubO1 CuniO1 CuniO1

– 1 2 2 1 8 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 9 9 9 5 9 5 5 9 – 16 16 16 – 15 – – – – –

ST8 ST108 ST33 ST108 ST4 ST64 ST1 ST263 ST248 ST20 ST4 ST40 ST83 ST23 – – – – ST20 ST1 ST1 ST1 ST7 ST211 ST7 ST60 ST441 ST138 – ST291 ST19 ST251 ST293 ST252 ST81 – ST106 ST80 ST79 ST98 ST54

C. malonaticus (n = 8)

C. turicensis (n = 4)

C. muytjensii (n = 2) C. C. C. C. C.

dublinensis subsp. dublinensis dublinensis subsp. lausannensis dublinensis subsp. lactaridi condimenti universalis

1

recommended age for feeding. Type strain; ATCC (American Type Culture Collection); DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Leibniz-Institute DSMZ); LMG (Laboratorium voor Microbiologie, Gent, Belgien); NCTC (National Collection of Type Cultures, Culture Collection of Public Health England). a by PCR according to Sun et al. (2011). b according to Farmer (1980) and Iversen, Waddington, Farmer, and Forsythe (2006). c according to Baldwin et al. (2009); -, not defined. T

2.6. Detection limit and specificity of the NALFIA

(ThermoMixer, Eppendorf, Germany). The DNA concentration was determined photometrically (NanoDrop One, Thermo Scientific, Germany), then 5 μl of the mixture were used for PCR. Blank solutions of BPW or reconstituted PIF were used as negative controls. Three independent replicate analyses were performed for each series of experiments. The reactivity pattern of the NALFIA was evaluated against a panel of Cronobacter and non-Cronobacter strains grown from single colony isolates. Inclusivity was studied by testing culture material of 22 C. sakazakii and 8 C. malonaticus strains of various serotypes, biotypes and MLST types (Table 1). The exclusivity was assessed with a panel of 11 strains belonging to other Cronobacter species and 21 other non-Cronobacter bacteria with relevance for PIF (Table 2). All bacterial strains were cultivated on TSA at 37 °C for 24 h. DNA extraction from 2 to 3 colonies of each isolate, suspended with 0.5 ml of lysis buffer in a 2 ml sterile microcentrifuge tube, was performed as described above. At least two independent DNA extractions and analyses were performed for each strain.

The test sensitivity (limit of detection, LOD) of the NALFIA system was assessed using two type strains, C. sakazakii DSM 4485T and C. malonaticus DSM 18702T, employing BPW and PIF as the sample matrix. The absence of Cronobacter spp. in the PIF material was first confirmed by analysis using method ISO/TS 22964:2006. A single colony of each strain from Tryptic Soy Agar (TSA) was suspended in 10 ml of Tryptic Soy Broth (TSB) and cultured at 37 °C for 18 h, the resulting bacteria density typically was around 108 cfu/ml. Then, serial log10 dilutions (10−1 - 10−8) of each culture were prepared in BPW or in reconstituted (10 g PIF plus 80 ml BPW) PIF solution. For colony count, 2 × 100 μl of each dilution were plated in duplicate onto Tryptic Soy Agar (TSA) and incubated overnight at 37 °C. For preparation of artificially contaminated sample matrix, 1 ml each of the 10−1 - 10−8 dilutions were added to test tubes containing BPW (9 ml) or reconstituted PIF (9 ml) under gentle mixing, assuming that this would yield final bacteria numbers of about 107 - 100 cfu/ml. The exact bacterial concentration was obtained retrospectively from colony count data. DNA was extracted from BPW or reconstituted PIF after incubation by mixing 100 μl of sample with 400 μl of lysis buffer (RIDA gene, r-Biopharm, Germany). The mixture was incubated at 95 °C for 5 min

2.7. Detection of C. sakazakii and C. malonaticus in artificially contaminated PIF The applicability of the NALFIA system was evaluated using a 3

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Table 2 Non-Cronobacter (n = 21) strains used for exclusivity testing of the NALFIA. Species

Strain

Origin/Source

Escherichia coli Enterobacter cloacae subsp. cloacae Enterobacter cloacae subsp. dissolvens Enterobacter aerogenes Enterobacter amnigenus Klebsiella pneumoniae subsp. pneumoniae Klebsiella pneumoniae subsp. ozaenae Klebsiella oxytoca Yersinia enterocolitica subsp. palearctica Citrobacter freundii Hafnia alvei Pantoea agglomerans Proteus mirabilis Proteus vulgaris Serratia marcescens Shigela sonneii Salmonella enterica subsp. enterica Pseudomonas aeruginosa Pseudomonas putida Acinetobacter baumannii Staphylococcus aureus

DSM 1103 (ATCC 25922) DSM 30054T (ATCC 13047T) DSM 16657T (ATCC 23373T) DSM 30053T (ATCC 13048T) DSM 4486T (ATCC 33072T) DSM 30104T (ATCC 13883T) DSM 16358T (ATCC 11296T) DSM 5175T (ATCC 13182T) DSM 11502 DSM 30039T (ATCC 8090T) Sal3/2a-99 (2/14) DSM 3493T (ATCC 27155T) DSM 788 (ATCC 14153) DSM 30119 2/53 DSM 5570T (ATCC 29930T) DSM 10062 (ATCC 43845) DSM 939 (ATCC 15442) F91-D DB-207a/3-05 DSM 20372 (ATCC 12598)

Clinic Type strain, clinic Type strain, corn Type strain, clinic Type strain, environment Type strain Type strain, clinic Type strain, clinic Clinic Type strain Mincet meat Type strain, clinic – Faeces Meat Type strain Serovar Senftenberg Environment Cream cheese PIF Clinic, Enterotoxinogenic (seg, sei)

T

Type strain; ATCC (American Type Culture Collection); DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Leibniz-Institute DSMZ); - not defined.

Fig. 1. Schematic representation of the principle of NALFIA with integrated DNA probe degradation. Panel A: Presence of target sequence allows annealing of the primers and binding of the DNA probe labelled with digoxigenin and biotin to the DNA template. Panel B: DNA probe is partially degraded by exonuclease activity of DNA polymerase, Panels C and D: Reactions on the NALFIA lateral flow strip. In negative tests (C) intact DNA probe is captured by gold-labelled anti -digoxigenin monoclonal antibody, this complex is then captured by streptavidin on the control line. For positive tests (D), some digoxigenin is released from the probe and is then captured by gold-labelled anti -digoxigenin monoclonal antibody. This complex passes the control line and is then immobilized at the test line (coated with goat anti mouse-antibody). . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 4

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Table 3 Oligonucleotide primer sequences for amplification of the rpoB gene. Primer/probe

Primer sequence

Tm °C (nearest n)

CsakFmm rpoB-RVmm rpoB-SNDn

5′-ACG CCA AGC CTA TCT CCA CG-3′ 5′-AGA CCG CCC GGG CCA AGA TCA-3′ 5′-DIG-GTGAAAGAGTTCTTCGGTTCAAGCCAGCTTTC-Biotin-3′

60.36 °C 60.71 °C 65.07 °C

commercial product of PIF (recommended use for newborns) purchased in a local German retail market. The absence of Cronobacter spp. in this material was first confirmed by analysis using method ISO/TS 22964:2006. For artificial contamination, test portions of each 10 g of this material were reconstituted with 89 ml of BPW immediately before use. Contamination experiments were done with overnight cultures of four different strains, C. sakazakii DSM 4485T, C. sakazakii DB84a-05, C. malonaticus DSM 18702T, and C. malonaticus DB185-05, all in TSB. Colony count and preparation of log10 dilution series were done as described in 2.6. The 10−7 dilution was used for artificial contamination, based on the assumption that this dilution typically contained about 101 - 102 cfu/ml. One ml of this dilution was added to reconstituted PIF, resulting in an estimated contamination level with Cronobacter cells of 100-101 cfu per gram PIF. PIF (10 g) in blank BPW (90 ml) was used as a negative control. Artificially contaminated samples were incubated at 37 °C for 24 h without agitation. Two portions of each 100 μl of this non-selective enrichment broth were collected for DNA extraction and analysis by NALFIA. Then, 0.1 ml the broth was transferred into 10 ml selective enrichment broth (mLST/vancomycin broth, ISO/TS 22964:2006 (IDF/RM 210: 2006) and incubated again at 37 °C for 18 h. Again, two 100 μl portions of this selective enrichment broth were analysed by NALFIA. To quantitatively estimate the growth of Cronobacter after selective enrichment, log10 serial dilutions (10−4 10−6) from the mLST/vancomycin broth were made with BPW, and 100 μl per dilution plated on Chromogenic Cronobacter Isolation (CCI) agar (Oxoid). For each strain, the spiking experiment was repeated six times.

sakazakii and C. malonaticus, a chromosomal gene with a high level of conservation (Mollet, Drancourt, & Raoult, 1997). The rpoB of both Cronobacter species is suitable for detection with higher sensitivity and specificity than other target genes (Lehner, Fricker-Feer, & Stephan, 2012; Stoop, Lehner, Iversen, Fanning, & Stephan, 2009). Previous studies have shown that the rpoB gene is not suitable to clearly distinguish between both C. sakazakii and C. malonaticus (Akineden et al., 2017). Since C. sakazakii and C. malonaticus are by far the most commonly occurring Cronobacter species in PIF, and to date are the only species related to PIFrelated infections of infants, the NALFIA appears to be primarily suitable for monitoring of infant foods. Although it certainly cannot replace control analyses by the reference method as required by European Commission regulation 2073/2005 (European Commission, 2005), NALFIA could be used as a supplementary rapid test for internal production control purposes in PIF industry. The specificity pattern of the NALFIA described here presents a complementary approach to a previous assay (Blažková et al., 2011) for Cronobacter spp. which is based on the amplification of 16S rRNA gene, in particular since the 16S rRNA gene does not allow the unambiguous identification of Cronobacter spp. (Baldwin et al., 2009). 3.2. Limit of detection of the NALFIA The sensitivity (limit of detection) of the NALFIA was evaluated by using either BPW or Cronobacter-negative, reconstituted PIF, to which C. sakazakii or C. malonaticus type strains had been added at defined cell concentrations (101–107 cfu/ml). For these experiments, PCR amplification was performed on samples after artificial contamination, without prior enrichment. At an instrumental cut-off value of 50 reflectance units, the detection limit for both Cronobacter species both in BPW and in PIF was in a range of 103-104 cfu/ml in BPW (Fig. 3). The lateral flow reader cut-off of ≥50 mV was defined because at this value, visual evaluation of dipsticks also yielded consistently positive samples. Negative samples consistently gave reflectance values of less than 5 mV after instrumental reading. This test sensitivity was found to be highly reproducible, because the relative standard deviation (coefficients of variation of the measurement signal, reflectance mV) were consistently less than 20% for BPW and less than 25% for PIF in each three independent replicate analyses for each strain and matrix. Visual evaluation of the NALFIA test strips yielded identical sensitivity, Cronobacter at cell densities in a range between 103 and 104 cfu/ml were clearly identified as positive in all series of experiments. These results also confirm the test robustness with regard to sample matrix interference. The detection limit of a previous assay (Blažková et al., 2011) for Cronobacter spp. had been determined for extracted DNA only, while in our study a thorough evaluation of the detection limit of the complete analytical procedure was performed based on bacteria numbers. Reconstituted infant formula was found to be an ideal food substrate for NALFIA. Neither sample homogenisation nor extensive sample cleanup was required for analysis. This enabled virtually identical limits of detection for both Cronobacter spp. in BPW and in PIF after 18 h of incubation at 37 °C. However, this may not be the case if other food matrices had to be analysed. As is reflected by results shown in Tables 4 and 5, the strongest factor of variability was not related to assay repeatability but to strain diversity. Depending on the specific isolate, the reflectance readings varied substantially among 22 isolates of C. sakazakii (coefficient of variation 76%) and C. malonaticus (39%), but all isolates were discerned as positive. However, the NALFIA has to be regarded as a qualitative rather than a quantitative assay.

3. Results and discussion 3.1. NALFIA test characterization and test performance Combining the signal amplification power of PCR with the sensitivity and simplicity of lateral flow immunoassays offers promising fields of application in food safety. One major disadvantage of such rapid testing methods is the risk of non-specific amplification, leading to false-positive results. In the present study, a new NALFIA method, which is designed to reduce false-positive results by employing a probe degradation step was developed and applied for the detection of two important pathogens in infant formula, C. sakazakii and C. malonaticus. Examples of lateral flow devices obtained for negative and positive samples is shown in Fig. 2. Instrumental evaluation of the test results by measuring reflectance enabled quantitative results which are more suitable for documentation purposes. Overall, the test performance was found to be straightforward, and the total test time was less than 2 h. NALFIA systems without integrated control via DNA probe degradation have been described for the detection of some bacteria (Blažková et al., 2011; Seidel et al., 2017; Zhang et al., 2017), and for parasites (Wu et al., 2017). To our best knowledge, however, this is the first report that employs a heterobifunctional DNA probe and detection of degradation for pathogens related to food safety, specifically for Cronobacter species. The specificity of the NALFIA was determined using reference and field strains covering all known Cronobacter species, plus a wide range of gramnegative and gram-positive bacteria known to occur in the food environment (Tables 1 and 2). The NALFIA was highly specific for C. sakazakii and C. malonaticus. The cross-reactivity testing of 41 Cronobacter and 21 nonCronobacter isolates revealed neither false positive nor false negative results (Table 4). The NALFIA assay targeted the rpoB sequence specific to C. 5

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Fig. 2. Typical examples of NALFIA lateral flow test devices for positive and negative samples. A, negative result. B1, C. sakazakii DSM 4485T at approx. 106 cfu/ml. B2, C. malonaticus DSM 17802T at approx. 106 cfu/ml. Table 4 Specificity of the NALFIA. Species

Number of strains

Strain diversity

NALFIA result

Serotypesa

Biotypesb

MLST typesc

visual

Reflectance mean ± standard deviation (range, CV)

1, 2, 8

all +

130 ± 99 (75–380, 76%)

all +

280 ± 110 (51–400, 39%)

16 15 n.d. n.d. n.d. n.d. n.d. n.d.

ST1, ST4, ST8, ST20, ST23, ST33, ST40, ST64, ST83, ST108, ST7, ST60, ST138, ST211, ST291, ST441 ST19, ST251, ST252, ST293 ST81 ST106 ST80 ST79 ST98 ST54 –

all all – – – – – all -

all all all all all all all all

C. sakazakii

22

C. malonaticus

8

SO1, SO2, SO3, SO4, SO7 MaO1, MaO2

C. turicensis C. muytjensii C. dublinensis subsp. dublinensis C. dublinensis subsp. lausannensis C. dublinensis subsp. lactaridi C. condimenti C. universalis Other non-Cronobacter species (Table 2)

4 2 1 1 1 1 1 21

CturO1 CmuytO2 CdubO1 CdubO2 CdubO1 CuniO1 CuniO1 –

5, 9

< 50 < 50 < 50 < 50 < 50 < 50 < 50 < 50

a

by PCR according to Sun et al. (2011); baccording to Farmer (1980) and Iversen et al. (2006); caccording to Baldwin et al. (2009); -, negative; +, positive; CV, coefficient of variation.

NALFIA, 1) after non-selective enrichment in BPW and 2) after selective enrichment in mLST/Vancomycin broth, within the ISO/TS 22964:2006 reference method was studied. In each replicate experiment, the presence of Cronobacter was confirmed by completing the analysis by the reference method, to verify that the artificial contamination had been successful. In all six replicate experiments, NALFIA yielded highly positive instrumental reflectance readings for BPW samples (85–440 mV) and for mLST/vancomycin samples (82–480 mV), and all replicate tests were also clearly positive after visual evaluation (Table 5). An inoculation level of approximately 1 cfu/g PIF was close to a realistic contamination scenario of PIF, and still sufficiently high to enable sufficiently accurate quantification. Natural contamination of PIF produce often occurs at very low levels, usually a few cells per 100 g, as estimated for C. sakazakii (Osaili & Forsythe, 2009). Based on re-analyses of colony counts of pre-enrichment BPW broth by microbiological plate count after incubation, the initial spiking level of 100101 cfu/g resulted in final counts of about 107 to 109 cfu/ml after 18 h. Based on these findings, and considering the detection limit of 103104 cfu/g, the NALFIA appears to be capable to detect C. sakazakii and C. malonaticus at initial levels of less than 1 cfu per gram of PIF. This would compare well with previous reports which used cultural detection methods (Muytjens, Roelofswillemse, & Jaspar, 1988; Simmons, Gelfand, Haas, Metts, & Ferguson, 1989), although this range was not studied, because homogeneous artificial contamination of PIF at levels much lower than 1 cfu per gram is difficult to achieve in practice. In any case, the NALFIA is comparable with a similar test described by Blažková et al. (2011), based on the 16S rRNA gene, who claimed a detection limit of less than 10 cells/10 g of PIF. Although these authors

Fig. 3. Characteristic dose-response curves of the NALFIA (instrumental reading, peak height) for serial dilutions of type strains C. sakazakii DSM 4485T and C. malonaticus DSM 17802T in either BPW or reconstituted PIF as the sample matrix. Each data point represents the mean of three independent experiments. The mean coefficients of variation for reflectance (peak height) of replicates at 104, 105, and 106 cfu/g in all series of experiments were 26%, 25%, and 32%, respectively.

3.3. Evaluation of the NALFIA as a means to obtain rapid results within the enrichment procedure of the ISO/TS 22964:2006 reference method In a last series of experiments, detection of C. sakazakii and C. malonaticus in PIF at a low level (approximately 100-101 cfu/g) by 6

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Table 5 Comparison of NALFIA (visual and instrumental evaluation) with reference method ISO/TS 22964:2006 for PIF which was artificially contaminated with Cronobacter strains at a low level (100-101 cfu/g). In each experiment, NALFIA was applied to non-selective enrichment broth (BPW) at 24 h, and to selective enrichment broth (mLST/Vancomycin) at 48 h. For each strain, six independent replicate experiments were performed. Strain

C. C. C. C.

sakazakii DSM 4485T sakazakii DB-185c/3-05 malonaticus DSM 18702T malonaticus DB-84a/1-05

Non-selective pre-enrichment in BPW

Selective pre-enrichment in mLST/Vancomycin broth

Reference method

NALFIA visual

NALFIA instrumental mean ± SD (range)

Reference method

NALFIA visual

NALFIA instrumental mean ± SD (range)

+ + + +

+ + + +

310 229 240 220

+ + + +

+ + + +

270 370 240 270

± ± ± ±

43 (250–380) 57 (120–290) 120 (150–440) 97 (85–370)

± ± ± ±

32 (240–310) 70 (290–480) 79 (160–350) 110 (82–380)

+ positive results in all six replicates.

mentioned that their assay was successfully used to test PIF, experimental data were not provided. In the present study, a comprehensive evaluation of the NALFIA properties has been done, including enhanced specificity controls and rigorous detectability checks with each two strains of C. sakazakii and C. malonaticus, both in BPW and PIF, clearly demonstrating the suitability and robustness of the assay. Furthermore, quantitative measurement of dipsticks by an automated dipstick reader device enabled an objective evaluation of the repeatability of the NALFIA results for both species in real sample material (Table 5).

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4. Conclusion The newly developed NALFIA with integrated DNA probe degradation enabled rapid (90 min) detection of the two most important Cronobacter spp., C. sakazakii and C. malonaticus, in PIF after 18 h nonselective pre-enrichment in BPW. The NALFIA was both sensitive and highly robust towards sample matrix. Reconstituted infant formula was found to be a convenient food matrix for NALFIA, test sensitivity in PIF was equal to that obtained in BPW. Both visual and instrumental evaluation are possible, the latter offering the advantage of an easy documentation of the results. However, the NALFIA presents a significant step towards testing in a laboratory-independent environment. Further work therefore aims at simplifying the PCR amplification step, for example by using battery-operated cycler based on convection, to enable testing at the point of care within the dairy industry. Funding This work was supported in part by the Federal Ministry of Education and Research (BMBF) of Germany (Food supply and analysis (LEVERA), funding code 13N12611). Declaration of competing interest The authors declare no conflict of interest. T. Wittwer and K. Geister are employees of R-Biopharm AG. This manuscript and the experimental work described therein do not involve a commercial product. References Akineden, Ö., Heinrich, V., Gross, M., & Usleber, E. (2017). Reassessment of Cronobacter spp. originally isolated as Enterobacter sakazakii from infant food. Food Microbiology, 65, 44–50. https://doi.org/10.1016/j.fm.2017.01.021. Almeida, C., Azevedo, N. F., Iversen, C., Fanning, S., Keevil, C. W., & Vieira, M. J. (2009). Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter genomospecies (Enterobacter sakazakii) in powdered infant formula. Applied and Environmental Microbiology, 75, 2925–2930. https://doi.org/10. 1128/AEM.02470-08. Baldwin, A., Loughlin, M., Caubilla-Barron, J., Kucerova, E., Manning, G., Dowson, C., et al. (2009). Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes. BMC Microbiology, 9, 223. https://doi.org/10.1186/14712180-9-223. Blažková, M., Javůrková, B., Fukal, L., & Rauch, P. (2011). Immunochromatographic strip test for detection of genus Cronobacter. Biosensors and Bioelectronics, 26, 2828–2834.

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