Membrane Proteins and Proteomics of Cronobacter sakazakii Cells: Reliable Method for Identification and Subcellular Localization.

Members of the genus Cronobacter are responsible for severe infections in infants and immunosuppressed individuals. Although several virulence factors have been described, many proteins involved in the pathogenesis of such infections have not yet been mapped. This study is the first to fractionate Cronobacter sakazakii cells into outer membrane, inner membrane, periplasmic, and cytosolic fractions as the basis for improved proteome mapping. A novel method was designed to prepare the fractionated samples for protein identification. The identification was performed via one-dimensional electrophoresis-liquid chromatography electrospray ionization tandem mass spectrometry. To determine the subcellular localization of the identified proteins, we developed a novel Python-based script (Subcelloc) that combines three web-based tools, PSORTb 3.0.2, CELLO 2.5, and UniProtKB. Applying this approach enabled us to identify 1,243 C. sakazakii proteins, which constitutes 28% of all predicted proteins and 49% of all theoretically expressed outer membrane proteins. These results represent a significant improvement on previous attempts to map the C. sakazakii proteome and could provide a major step forward in the identification of Cronobacter virulence factors. IMPORTANCECronobacter spp. are opportunistic pathogens that can cause rare and, in many cases, life-threatening infections, such as meningitis, necrotizing enterocolitis, and sepsis. Such infections are mainly linked to the consumption of contaminated powdered infant formula, with Cronobacter sakazakii clonal complex 4 considered the most frequent agent of serious neonatal infection. However, the pathogenesis of diseases caused by these bacteria remains unclear; in particular, the proteins involved throughout the process have not yet been mapped. To help address this, we present an improved method for proteome mapping that emphasizes the isolation and identification of membrane proteins. Specific focus was placed on the identification of the outer membrane proteins, which, being exposed to the surface of the bacterium, directly participate in host-pathogen interaction.

Tailor-Made Immunochromatographic Test for the Detection of Multiple 17α-Methylated Anabolics in Dietary Supplements.

In recent years, the undeclared presence of various anabolic androgenic steroids (AAS) in commercial supplements has been confirmed. This fact can be a potential threat to all athletes using these supplements, and therefore, there is of increased interest in the implementation of rapid methods for the detection of AAS. The presented study describes the development of an immunostrip test for the detection of multiple 17α-methylated AAS based on direct and indirect competitive principle using gold nanoparticles as a label. As a capture reagent on test lines conjugated stanazolol to rabbit serum albumin (RSA/ST-3) was used, the intensity of color formed in the test line of the AAS-positive sample was visually distinguishable from that of negative sample within 10 min. The optimized closed direct and indirect format of the test provided a similar visual detection limit (0.7 and 0.9 ng/mL, respectively). The most commonly orally abused AAS (17α-methyltestosterone, methandienone, methyldihydrotestosterone, oxandrolone and oxymetholone) showed a strong cross-reaction. Developed immunostrips were successfully applied to analysis of artificially contaminated dietary supplements with 17α-methylated AASs. The developed immunostrips offer potential as a useful user-friendly method for capturing suspicious dietary supplement samples with different contents of AAS at levels far below the usually used concentrations of AAS.

Sensitive enzyme immunoassay for screening methandienone in dietary supplements.

Methandienone is a synthetic exogenous steroid which, like other anabolic steroids, is strictly regulated in many countries. In recent years, increasing numbers have been detected of illegal additions into dietary supplements of methandienone and other anabolic androgenic steroids (AAS). In this work, a competitive indirect enzyme-linked immunosorbent assay (ELISA) has been constructed for the detection of methandienone using an antiserum against methandienone. Under optimal experimental conditions, the ELISA achieved a limit of detection of 0.04 ± 0.01 µg.g-1. The obtained intra- and inter-day coefficients of variation were less than 8%. The developed ELISA was applied in the analysis of real dietary supplement samples. To minimise the effect of the sample matrix, the sample extracts were simply diluted before addition into the immunoassay. The achieved recovery values were around 100%. Results obtained from the ELISA correlated well, both in terms of accuracy and precision, with those obtained by UHPLC-MS/MS (reference method). The presented ELISA could be successfully applied for the simple screening of dietary supplements.

Lateral flow immunoassay and enzyme linked immunosorbent assay as effective immunomethods for the detection of synthetic cannabinoid JWH-200 based on the newly synthesized hapten.

In recent years, the use of synthetic cannabinoids (SCs) as drugs of abuse has greatly increased. SCs are associated with a risk of severe poisoning or even death. Therefore, more rapid, cost effective and reliable methods are needed, especially for the screening of drivers after traffic accidents and for detailed toxicological analysis in forensic laboratories. In this study, we developed a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of JWH-200 in oral fluids. For this purpose a new hapten was prepared using a ten-step synthetic route. The developed immuno methods are based on antibodies obtained from rabbit immunized with synthesized hapten conjugated to carrier protein. The proposed methods are highly sensitive (LODLFIA = 0.08 ±â€¯0.04 ng mL-1; LODELISA = 0.04 ±â€¯0.02 ng mL-1). They were applied to the quantification of JHW-200 in spiked oral fluids. The recoveries ranged from 82 to 134% for both methods. The results correlated excellently with results obtained using UHPLC-MS/MS (R2LFIA = 0.99; R2ELISA = 0.99). Our developed methods could be an important tool for analyses of JWH-200 in human oral fluids. The one-step LFIA is particularly suitable for roadside and on-site monitoring due to the rapid qualitative results it delivers, while the ELISA is especially useful for laboratory quantitative analyses of positive samples captured by LFIA.

Novel Method for Reliable Identification of Siccibacter and Franconibacter Strains: from "Pseudo-Cronobacter" to New Enterobacteriaceae Genera.

In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method.IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease.

Highly sensitive avidin-biotin ELISA for detection of nandrolone and testosterone in dietary supplements.

Avidin-biotin technology was used for the implementation of an enzyme-linked immunosorbent assay (AB-ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Using click chemistry, novel haptens (linker-optimized biotinylated nandrolone (NT) and testosterone (T) at positions C-3 and C-17, respectively) were designed and synthesized to be then applied as four different immobilized competitors in a proposed set of four indirect competitive AB-ELISAs. Four rabbit polyclonal antibodies of various specificities were prepared using four different immunogens synthesized from C-3 and C-17 carboxymethyloxime and hemisuccinate derivatives of NT and T, respectively. Assembled AB-ELISAs were characterized to establish method parameters such as a half-maximum inhibition concentration (0.18-12.99 ng/mL), limit of detection (0.004-0.032 ng/mL) and linear working range (the best with 0.02-1.38 ng/mL). The stability of the set simulating storage in different conditions was demonstrated. Cross reactivity (CR) was tested for 59 steroids including both endogenous and synthetic analogues in four assembled AB-systems. The focus was placed on the practical use of the method in detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the CR corrected data from AB-ELISA indicated the potential of this method even to quantification of T propionate, NT phenyl propionate, and NT decanoate in such a complex matter. Copyright © 2016 John Wiley & Sons, Ltd.

Novel PCR-RFLP system based on rpoB gene for differentiation of Cronobacter species.

Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.

Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order.

Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.

The potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of biogroups of Cronobacter sakazakii.

RATIONALE: The bacterial genus Cronobacter was established quite recently, in 2008. Therefore, its systematic classification is still in progress as well as the risk assessment of Cronobacter strains. The possibility of rapid identification within the biogroup level has an essential epidemiological significance. We examined the potential of mass spectrometry to accomplish this task on species Cronobacter sakazakii comprising eight different biogroups. METHODS: Members of all Cronobacter sakazakii biogroups were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using intact cells. Analyses were performed on a Biflex IV MALDI-TOF mass spectrometer in the range of 2000 to 20 000 Da in linear mode with an accelerated voltage of 19 kV. RESULTS: Optimal conditions for a proper identification of biogroups, such as suitable cultivation media or growth time of bacteria, were investigated. The biomarker patterns characterizing each of the Cronobacter sakazakii biogroups were obtained. The established identification protocol was applied to ten previously non-identified strains and their biogroups were successfully determined. CONCLUSIONS: The presented work is the first report of successful and rapid bacterial biogroup taxonomy classification using MALDI-TOF-MS that could substitute demanding biochemical testing.

Immunochromatographic strip test for detection of genus Cronobacter.

Members of the genus Cronobacter are opportunistic pathogens formerly known as Enterobacter sakazakii, which induce severe meningitis and sepsis in neonates and infants, with a high fatality rate. In this work, a simple and rapid immunochromatographic strip test for the detection of this pathogen was developed. Following the shortened bacteria cultivation and isolation of DNA, a specific gene sequence targeting 16S rRNA from Cronobacter spp. was amplified by PCR using 5'-end labelled specific primers. The PCR product, amplicon labelled with digoxigenin on one side and biotin on the other side, was directly added to the immunochromatographic strip test, composed of nitrocellulose membrane with bound antibody against digoxigenin in the test line. The visualization was mediated by colloidal carbon conjugated to neutravidin, and the appearance of grey/black line was indicative of the presence of specific amplicon. Colour intensity of the test line in pathogen-positive assay was visually distinguishable from that of negative sample within 10 min. The visual detection limit of PCR product was 8 ng. The specificity of the developed method was confirmed by standard microbiological techniques. Whole detection procedure with the incorporated immunostrip was applied to analysis of infant formulae samples, contaminated with less than 10 cells of Cronobacter spp. per 10 g. The results from immunochromatographic test indicated the absolute agreement with those from standard microbiological methods. Moreover, the developed procedure considerably reduced the total analysis time to 16 h whereas the reference microbiological method needs 6-7 days.

Strip-based immunoassay for rapid detection of thiabendazole.

There is increased interest in the investigation and implementation of rapid screening methods for detection of pesticide residues. This study reports development of an immunostrip test for thiabendazole detection based on indirect competitive principle using carbon particles as a label. Nitrocellulose membrane strip was coated with a thiabendazole-protein conjugate in the defined test zone. In flow of an antibody-carbon complex and thiabendazole along the strip, the intensity of black colour formed in the test line reflected the thiabendazole concentration and semi-quantitative estimation could be carried out visually. The optimized test was accomplished within 10 min and the visual detection limit was achieved 0.25 ng mL(-1) of standard sample. Moreover, immunostrip was evaluated quantitatively using scanning densitometry. Based on standard curve, the detection limit of the proposed test was as low as 0.08+/-0.03 ng mL(-1) with an IC(50) value of 0.60+/-0.08 ng mL(-1) and a linear working range of 0.11-4.13 ng mL(-1). Results of testing precision, stability, and specificity demonstrated that the assay provided a reliable performance. This immunostrip was applied to analysis of spiked fruit juices in range of 0.05-5 mg L(-1). Matrix interferences were avoided by simple dilution of samples. Both visual and instrumental evaluations indicated a good agreement with results obtained by ELISA. Recoveries from juices were from 81.9 to 123.6% and relative standard deviations ranged from 9.9 to 19.3%. The developed strip offers potential as a useful rapid and simple method for screening of thiabendazole in fruit juices at levels far below the maximum residue limits.

Immunochromatographic colloidal carbon-based assay for detection of methiocarb in surface water.

A simple and rapid immunochromatographic assay for a sensitive and inexpensive monitoring of methiocarb in surface water was developed using a binding inhibition format on a membrane strip. In the assay, detection reagent consisted of anti-methiocarb antibody and colloidal carbon-labelled secondary antibody. Methiocarb-ovalbumin conjugate was immobilized in a test line of the strip as a capture reagent. Colour intensity of the test line in methiocarb-positive assay was visually distinguishable from that of negative sample within 10min. The optimized semi-quantitative method provided a visual detection limit of 0.5ngmL(-1). Cross-reactions with other carbamate pesticides were not found (<1%). Only a negligible matrix effect of surface water was recognized. In parallel analyses of spiked water samples, the assay results were in a good agreement with those of ELISA. The stability test indicated the strips could be used at least 2 months without change in performance. All characteristics of the visually evaluated assay mentioned above were verified by instrumental quantification of colour intensity in test lines. The developed immunochromatographic assay offers potential as a useful on-site screening tool for environmental analysis.