Rapid and Specific Ninhydrin-free Chromogenic Assay for Detection of Viable Hippuricase-positive Bacteria and Its Validation in Raw Chicken Meat Samples

Rapid and Specific Ninhydrin-free Chromogenic Assay for Detection of Viable Hippuricase-positive Bacteria and Its Validation in Raw Chicken Meat Samples | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Rapid and Specific Ninhydrin-free Chromogenic Assay for Detection of Viable Hippuricase-positive Bacteria and Its Validation in Raw Chicken Meat Samples Chunyan Yao, Alexandra Schmid, Greta Faccio, Urs Spitz, Vesna Jovanović, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6936616/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Campylobacter infections are behind most pathogenic food contamination cases in humans. Campylobacter jejuni is a pathogen commonly found in the food chain and especially in meat products. Contamination takes place already during the slaughtering process and affects the whole supply chain until the consumer. Campylobacter contamination is still hard to prevent, and this work reports a specific easy-to-read biochemical assay for live Campylobacter with a rapid visual detection from a small bacterial sample. Current PCR- and ninhydrin-based detection assay both present significant limitations in sustainability, applicability, and reliability. This work presents a rapid biochemical easy-to-read assay for live Campylobacter proceeding requiring water-based solutions for an improved safety and sustainability; moreover, it has been validated using chicken meat samples using mass-spectrometry techniques. The assay efficiently allows the discrimination of a microorganism having hippuricase activity, e.g. Campylobacter jejuni , from microorganisms without hippuricase activity as it delivers a bright pink color in positive samples. Health sciences/Diseases/Infectious diseases/Bacterial infection Biological sciences/Microbiology/Pathogens Biological sciences/Microbiology Health sciences/Biomarkers Health sciences/Biomarkers/Diagnostic markers Physical sciences/Chemistry/Biochemistry Physical sciences/Chemistry/Biochemistry/Enzymes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The possibility of identifying the presence of pathogens with a specific, yet easy-to-read, assay is crucial to prevent their spread. At a global scale, Campylobacter infections are behind the majority of diarrheal diseases in humans and can also result in gastroenteritis and be fatal in young children, elderly, and immunosuppressed individuals. Infection is often foodborne, transmitted through contaminated meat like chicken, milk, water, or ice, which are not safely handled or properly heat-treated. In approximately 90% of human Campylobacter infections, the principal pathogen is Campylobacter jejuni ( C. jejuni ) 1 , 2 . Due to frequencies of Campylobacteriosis and the potential sources of Campylobacter spp., their isolation and identification in clinical and food samples is crucial. More recently, C. jejuni has also been identified as a trigger of Guillain-Barré syndrome, a rare immune-mediated acute polyradiculo-neuropathy 3 . The presence of Campylobacter spp. has also been linked to the development of celiac disease 4 , 5 . According to the Genome Taxonomy Database, currently over forty Campylobacter species are recognised 6 , and at least a dozen of them have been implicated in human disease 7 . Members of this genus are typically Gram-negative, non-spore-forming, S-shaped or spiral shaped bacteria, with single polar flagella at one or both ends 8 . Campylobacter jejuni ( C. jejuni ) and Campylobacter coli ( C. coli ) represent the main cause of bacterial diarrhoea in developed countries 9 , 10 , and one of the most important causes of enterocolitis in developing countries 11 . Over 80 % of cases are caused by C. jejuni and 5-10 % of cases are caused by C. coli 12 . Various methods to detect C. jejuni contaminations are based either on its genetic or its metabolic make-up. C. jejuni is one of the few strains producing the enzyme hippuricase (EC 3.5.1.32 hippurate hydrolase), and this has been leveraged to differentiate it from other bacteria such as Campylobacter coli . The hipO gene for hippuricase has been the target for PCR-based tests 13 . With the genetic approach, C. jejuni and C. coli could be indeed differentiated 14 . Campylobacter jejuni is however not the only producer of hippuricase and other Campylobacter spp., e.g., C. avium as well as other species such as Legionella spp. , and Streptococcus agalactiae (known as group B Streptococci) , Gardnerella vaginalis , Listeria monocytogenes , Brachyspira spp., and Staphylococcus aureus produce the enzyme 9,10,11 . However, not all hippuricase-positive bacteria harbor the hipO gene such as C. avium 12 . Although highly sensitive at the gene level, these assays do not provide information on the bacterial viability and require specific equipment and trained personnel). Bessede and colleagues have shown that also mass spectrometry, i.e. MALDI-TOF, can be used to identify Campylobacter isolates rapidly and efficiently at the genus and species level with high accuracy (0.4% of misidentification) 15 . Metabolism-based detection approaches can rely instead on the activity of the hippuricase enzyme itself, that can be considered a biomarker, which is responsible of the hydrolysis, at the peptide linkage site, of hippuric acid to glycine and benzoic acid. Rapid testing currently focusses on the second product of the hippuricase reaction, the glycine amino acid, and provide a visual read-out based on the ninhydrin reaction 13,16 . The amino group of glycine can react with ninhydrin (2,2-dihydroxyindane-1,3-dione) producing a blue coloration. However, the ninhydrin-based reaction is not specific to glycine as it reacts with all molecules carrying alpha-amino groups that are present in the sample limiting its application to complex samples in nutrient-rich culture medium and resulting often in false positives. Moreover, the use of ninhydrin poses health hazards as it can cause irritation on contact, and it is a respiratory tract irritant 14 . Detection methods of benzoic acid, the second product of hippurate hydrolysis, include a color reaction with ferric chloride 17 or a gas-liquid chromatographic method 16 . The former method requires is however time-consuming whereas the latter is equipment-dependent, requires trained personnel and specific sample preparation. This work introduces a rapid, specific, chromogenic assay for detecting hippuricase-positive viable bacteria such as Campylobacter jejuni and it overcomes the limitations of the currently available technologies. The assay is valid for the detection of metabolically active C. jejuni and other hippuricase-positive bacteria, it can be used with different oxidants and outperforms current commercials analogous products. To verify the specificity of this rapid colorimetric test for the detection of the enzymatic hydrolysis of hippurate by hippuricase positive C. jejuni , commercial poultry samples have been analysed and results have been confirmed using nano-liquid chromatography mass spectrometry (nLC-MS/MS) technique. As an additional consideration, being water-based, the assay also presents a higher sustainability level than the current standard ninhydrin-based commercial assays. Results Campylobacter is considered to be the most common bacterial cause of human gastroenteritis in the world. Hippuricase is a rarely found bacterial enzyme that characterizes C. jejuni and a few other bacteria strains that were also tested in this study (Table 1). Hippuricase can thus be considered a key enzyme, a biomarker, for the detection of C. jejuni and its identification in complex samples. Chromogenic assay for the detection of Campylobacter jejuni The assay we report is composed of two main steps based first on the hippuricase enzyme, endogenous of a positive sample, that converts 4-hydroxyhippuric acid (4-HHA) to hydroxybenzoic acid (4-HBA) and glycine (reaction 1, Figure 1), and 4-HBA is subsequently oxidized in the presence of 4-aminoantipyridine (4-AAP) by either a peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ) (approach a, Figure 1) or by sodium periodate as single oxidant (approach c, Figure 1) to a colorful quinoeimine dye with a bright pink color. If the external addition of hydrogen peroxide in the second step is not desired, this can be enzymatically produced in situ using glucose and glucose oxidase (approach b, Figure 1). Positive sample results are indicated by a pink coloration. Detection Campylobacter jejuni using HRP and H 2 O 2 as oxidizing reagent The sample to be tested is prepared by resuspending in buffer a few bacterial colonies picked from an agar plate, i.e., to reach an OD 600 of 0.9. The sample is then incubated with 4-HHA for 70 minutes at 37°C and later added of a 4-AAP, HRP and H 2 O 2 . Increase of the absorbance of the solution at 505 nm is measured in samples comprising C. jejuni (Figure 2B) that develop a pinkish coloration, whereas samples comprising C. coli and Escherichia coli, or without bacteria (sterile control), remain of the initial light yellowish color, upon visual detection (Figure 2A). Determination of the sensitivity level and assessing the interference of endogenous catalase activity To assess the sensitivity of the assay, the bacterial sample was serially diluted with buffer to an OD 600 as low as 0.1 (Figure 2B). Placed in a multi-well plate, samples were then added of 4-HHA and incubated for 1 h at 37°C. After incubation, 4-AAP and HRP were added. The absorbance at 505 nm was monitored immediately. Final reagent concentrations for the assay were 10 mM 4-HHA, 0.5 mM 4-AAP, 4.5 U/mL HRP and 2 mM H 2 O 2 . The sterile control and the samples comprising C. coli remained colorless or slightly yellowish at all the cell concentration tested, whereas the samples comprising Campylobacter jejuni showed a robust red to pink color over a broad range of cell density values, i.e. coloration intensity gradually increased in samples from an OD 600 value of 0.1 to an OD 600 value 0.9. The red/pink color was visible by eye in the sample with an OD 600 value as low as 0.1 (Figure 2B). The results in Figure 2B show that a significant increase of the absorbance is recorded already after a few minutes with a plateau reached eventually, e.g., after about 10 minutes at an OD 600 value of 0.1 demonstrating that the method has a high sensitivity and allows for fast detection of bacteria at low cell density. No background coloration has been observed with the hippuricase-negative bacterial strains. The assay requires hydrogen peroxide as a regent, and the natural production of the enzyme catalase by some microorganisms (Table 1) might cause interference with the assay. We thus tested the formation of the quinoneimine compound produced by oxidative coupling of 4-HBA with 4-AAP and HRP in the presence of the hydrogen-peroxide-using catalase-positive strains. Already all strains tested in Figure 2 A and B are catalase-positive, and results show a significant increase of the absorbance at 505 nm with a plateau reached after about 5 minutes under the conditions tested. Although a concentration of 1 mM was sufficient for signal generation, a final concentration of 10 mM 4-HHA showed the best performance (Figure 2 C). It was however found that hydrogen peroxide should be added freshly after incubating the bacteria with 4-HHA to yield optimal color results. As in Figure 2 D that includes catalase-negative Streptococcus strains, absorbance in the cell-free control sample continued to increase for 15 minutes whereas the reaction stopped after about 5 minutes in the catalase-positive samples ( Campylobacter ). In the catalase-negative samples ( Streptococcus ), the reaction kinetics were similar to the reaction without cell suspension. These results show that catalase most likely degraded the added hydrogen peroxide which caused a stop of the reaction of 4-AAP/HRP with 4-HBA. However, the hippuricase reaction is sufficiently fast and the assay qualifies as a detecting method of hippuricase-producing microorganisms despite the presence of catalase. Detection of Campylobacter jejuni using in-situ -produced H 2 O 2 with glucose/glucose oxidase or using sodium periodate as oxidizing reagent Considering the oxidative properties of hydrogen peroxide and the safety measures that its handling requires, we tested its in situ production using the glucose oxidase action on glucose as an alternative. Campylobacter jejuni DSM 4688 (hippuricase positive) and Campylobacter coli DSM 4689 (hippuricase negative) were cultivated under microaerophilic atmosphere at 37°C for 48 h and later, to assess the sensibility to low cell density, colonies were suspended in buffer and the optical density OD 600 adjusted to 0.5, 0.2 or 0.1. Upon reaction, the samples comprising Campylobacter coli remained colorless or slightly yellowish for all OD 600 values while in the samples comprising Campylobacter jejuni showed a robust red to pink color over a broad range of OD 600 values with gradual increase in intensity from an OD 600 value of 0.1 to an OD 600 value 0.9 as determined by visual inspection (Figure 3A-B). This example confirms that the method is also sensitive and specific with an included enzymatic hydrogen-peroxide-generating system. Aiming at a non-enzymatic version of the essay and at reducing the number of components, sodium periodate was tested as oxidizing agent. The samples were prepared for an OD 600 of 1.1 and then serially diluted with the same buffer to an OD 600 value of 0.5, 0.3, and 0.1. After the incubation phase and the addition of 4-AAP, sodium periodate (NaIO 4 ) was added, and the reaction started. The absorption at 505 nm was recorded visually and with a plate reader at room temperature. The samples comprising Camplyobacter coli or the sterile control remained colorless or slightly yellowish at all OD 600 values while in the samples comprising Campylobacter jejuni showed quickly a red to pink color over the whole range of cellular densities tested which gradual increase in intensity from an OD 600 value of 0.1 to an OD 600 value 1.1 (Figure 3C). The results in Figure 3 show a higher absorbance value at all cell densities (indicated as OD 600 value) for C. jejuni compared to C. coli showing that the method is also specific and sensitive when using NaIO 4 as oxidizing reagent. When comparing the three oxidizing reagents and plotting the endpoint absorbance at 505 nm against the initial cell concentration, a strong correlation was observed. This indicated that the method can be effectively used to estimate the initial cell concentration from a sample (Figure 3D). Assay optimization by tuning oxidative agent concentration A wide range of concentration of each oxidant agent has been tested to ensure the use of an optimal concentration and to ensure alternative reagents are available without compromising the assay’s performance (Figure 4). Cell suspensions ( C. jejuni DSM 4688, OD 600 = 0.5 to 0.6) in sterile 100 mM phosphate buffer pH 7.4 were incubated with 4-HHA at a final concentration of 10 mM for 70 min at 37°C, followed by the addition of 0.5 mM 4-AAP, 4.5 U/mL HRP and H 2 O 2 at a concentration varied between 0 and 16 mM. Concentrations of 1 and 2 mM H 2 O 2 showed the best performance in 15 min with 1 mM giving a faster signal development that reached plateau already after 6 minutes. No coloration was observed, and no signal increase was detected by 505 nm in the cell suspension without oxidants (Figure 4, left panel). Under similar conditions, the enzymatic oxidant-producing system based on glucose oxidase was added using glucose oxidase (GOD) with an activity varied from 0 to 4 U/mL. The enzymatic concentration of 1 U/mL GOD was sufficient for the reaction to develop a sufficient (Figure 4, right panel). No coloration was observed, and no signal increase was detected by 505 nm in the cell suspension without enzyme or oxidative system. The single-oxidant version of the assay was tested with NaIO4and its concentration was varied from 0 to 8 mM. Concentrations of 2 to 4 mM NaIO4 showed the best performance. No coloration was observed, and no signal increase was detected by 505 nm in the cell suspension without NaIO4. Cell suspension without any additives and sterile control were included as negative controls (Figure 4, middle panel). Validation with other hippuricase-positive microorganisms: Streptococcus agalactiae The pathogen C. jejuni enters in contact with humans through contaminated food and water, or companion animals. Aiming at validating the assay with other hippuricase-positive strain, we have tested samples of the Streptococcus agalactiae ATCC®12386 and used Streptococcus pyogenes ATCC®19615, a hippuricase-negative strain, as an additional negative control. Colonies were suspended in buffer for an optical density OD 600 of approx. 0.5. The test was performed using all the three oxidizing systems (Figure 5). The samples with Streptococcus pyogenes and the sterile control remained colorless or slightly yellowish while in the samples comprising Streptococcus agalactiae a vivid pink coloration developed (data not shown). This example confirmed that all the methods presented are also sensitive and specific for distinguishing hippuricase-positive strain Streptococcus agalactiae from hippuricase-negative strain Streptococcus pyogenes, which are important etiological factors in human illnesses. Comparison to commercial ninhydrin-base tests In order to evaluate the competitiveness and ease of adaptation of the proposed biochemical assay, we proceeded to compare its performance with two commercially available alternatives. To ensure comparability, conditions had to be slightly altered but the manufacturer’s instructions were nevertheless followed. The assay performance was compared with two established commercial assays. Biomass of colonies for the here reported assay were dispersed in 0.2 mL sterile 100 mM phosphate buffer pH 7.4 or prepared as instructed by the manual in 0.1 mL sterile water for the Remel assay and in 0.5 mL saline solution for the Millipore assay. To ensure a proper comparability, the optical density OD 600 of all cell suspensions were set to 0.2. In the next step, 25 µL of 100 mM 4-HHA prepared in 100 mM phosphate buffer pH adjusted to 7.4 was added to cell suspensions for the here reported assay. The Hippurate disks and Hippurate strips were dropped in cell suspensions as recommended in the respective manuals. To ensure a proper comparability, all samples were then incubated at 37°C for 1 h. After incubation, the assay was tested in the two variants using the enzymatic systems based on HRP (A) and GOD (B) and a significant pink color was visible by eye in both cases within a few minutes (Figure 6) in sample comprising C ampylobacter jejuni at room temperature; sample comprising Campylobacter coli or sterile control remained slightly yellowish. In the Remel and Millipore assays, the ninhydrin reagent was added to the samples after an incubation of 30 min; a slightly blue color was visible by eye in sample compromising C. jejuni using the Remel Assay, and no color formation can be observed in sample compromising C. jejuni using the Millipore assay. Our method offered a considerable reduction in assay time compared to the Remel Assay, while exhibited a higher sensitivity when compared to the Millipore Assay. Identification of Campylobacter jejuni from chicken meat samples Campylobacter species are widely distributed in most warm-blooded animals, as they are a natural part of the intestinal microbiome of poultry 18 , pigs 19 , cattle 20 dogs and cats 21 . Poultry and poultry products are the main source of the human infections and responsible for between 50 and 80 % of all campylobacter infections 22 . Aiming at validating the biochemical assay with commercial meat samples, a first step of strain enrichment step has been carried out in which Campylobacter spp. were isolated from 65 locally purchased retail chicken meat samples, i.e. using a charcoal-agar-based (mCCDA) selective for Campylobacter . This medium was recommended by the ISO Committee under the specification ISO 10272:1995 for selective isolation and differentiation of Campylobacter species 23 . After 48h incubation at 37°C under microaerobic conditions, agar plates were observed by the naked eye and with a magnifying glass (40x) (Figure 7). Incubation conditions at 37°C rather than 42°C have been indicated to increase the isolation rate of Campylobacter spp. from foods according to the recommendation by the U.K. Ministry of Agriculture, Fisheries and Food 24 . However, C. jejuni is able to survive at temperatures from < 4 °C up to 46 °C and this characteristic of C. jejuni can promote the spreading of infections by meat products especially if they are not properly handled 25 , 26 , 27 . It could be noticed that there were some plates with no bacterial colonies (6 of 65 samples) or with few bacterial colonies (10 of 65 samples), while the other samples presented numerous colonies (Figure S1). In most plates, the characteristic colonies were visibly greyish, flat and moistened, with a metal sheen and tendency to spread. Beside grey colonies, white colonies were also present in some samples (Figure 7, Figure 1S) as well as pink colonies. In a second step, the biochemical assay presented in this article and based on the hydrolysis of hippurate by hippuricase-positive C. jejuni strains has been used to analyze the enriched bacterial samples. After visual assessment of the color development in comparison to a bacteria-free control sample (Figure 8), 11 samples (16.9 %) of the 65 analyzed samples were recognized as positive, i.e. number 3, 4, 7, 36, 38, 41, 46, 47, 55, 61, 62. Among them, sample number 7 gave the strongest and fastest coloration development resulting in a visual intensity comparable to results obtained for bacterial cultures of the optical density (OD 600 ) higher than 0.9 in liquid medium, according to the scale provided. Samples 4 and 36 also gave an intense coloration that was comparable to the one obtained for bacterial cultures in liquid media at an OD 600 between 0.5 and 0.9, while the other eight positive samples had intensity of color below OD 0.2 (Figure 3S). Of the six retail shop used, two of them reported all negative samples, whereas the remaining had at least one positive sample (Table 2). Among individual chicken meat cut types, the highest relative number of positive samples was obtained for liver (30.7 %) and backs (20%), while the lowest number was obtained for legs (7.7 %) and breast (7.7 %). Results validation using LC‐MS/MS‐based peptide sequencing for Campylobacter jejuni identification at protein sequence level Among the tested samples (Figure 8), samples 3, 4, 7, and 41, which gave a recognizable coloration and selected negative samples 30a and b, 31a and b, 45, 17 and 63 (a and b are different parts of plate with unalike colonies) were further analyzed by LC-MS/MS for the presence of Campylobacter -specific proteins. The MS-based analysis of the positive samples revealed the presence of at least seven characteristic of Campylobacter jejuni (Table 3), including a probable histidine-binding protein (UniProt ID: Q46125), Flagellin B (UniProt ID: P56964), major outer membrane protein (UniProt ID: P806729, Large ribosomal subunit protein uL3 (UniProt ID: Q9PLX1), a Fumarate hydratase class II (UniProt ID: O69294), Succinate-CoA ligase [ADP-forming] subunit beta (UniProt ID: A8FKV6), Threonine-tRNA ligase (UniProt ID: A1VXT5), 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (UniProt ID: Q0P823), cysteine synthase B (UniProt ID: P71128) and a catalase (UniProt ID: Q59296). The detection of proteins involved in motility, nutrient transport, and energy metabolism supports the conclusion that C. jejuni was present in the samples tested positive with the assay. The identification of multiple (7 to 9) C. jejuni -specific proteins, according to UniProtKB of reviewed sequences (SwissProt), provides convincing evidence for the presence of C. jejuni in the samples. Homology search identified a few unreviewed protein matches with 100% sequence identity from various Campylobacter species (Table 4). Furthermore, for five of the identified proteins, major outer membrane protein (UniProt ID: P80672), threonine-tRNA ligase (UniProt ID: A1VXT5), cysteine synthase B (UniProt ID: P71128) and catalase (UniProt ID: Q59296), no closely related isoforms with 100% sequence identity from other Campylobacter species were found among unreviewed sequences (Table 4). Eight samples that tested negative in the biochemical assay, i.e. not developing a pink coloration, were also tested by proteomic workflow described above; only one of them, i.e. sample number 30, that tested negative in the enzymatic assay, showed presence of several C. jejuni biomarker proteins in the mass spectrometry analysis. The negative hit obtained with the biochemical assay has been confirmed multiple times sampling different areas of the enrichment plate and no coloration has ever developed (Figure 5S). Discussion The global market for Food Pathogen Testing has been forecasted to surpass USD 9.1 billion by 2028 at a CAGR of 8% 28 . One contributing factor is the increasing attention posed on food safety especially in developing nations. Campylobacteriosis is due to twomajor agents, i.e. Campylobacter jejuni and C. coli, and most assays aim at their specific detection 18 . Current techniques for the detection of Campylobacter rely on PCR-based assays or ninhydrin-based assays, both presenting disadvantages such as the need for a dedicated apparatus or the possibility of false positives. A study has also reported hippuricase-specific antibodies that can result in ELISA-like assays for Campylobacter detection 29 . Here we report a sensitive and specific chromogenic assay exploiting the activity of the Campylobacter endogenous hippuricase enzyme (Figure 4S). The assay proceeds in two enzymatic steps and results in a read-out that can be detected by absorbance measurement but also visually, offering itself to future adaptation to an immobilized form. The assay here presented is functional with a low optical density sample, i.e. OD 600 = 0.1, or of just a few bacterial colonies picked from a culture plate with complex media. This suggests adaptability to the testing of liquid samples and environmental samples. The sensitivity of the popular ninhydrin assay for Campylobacter detection is not clear and prone to provide false positive results. In addition, the assay here presented provides a read-out at a longer wavelength than ninhydrin, e.g. the purple signal at 505 nm vs. the blue signal of ninhydrin. Biochemical chromogenic assays offer the advantages of an easy read-out, often quick, and adaptability to a format that can applied easily in situations of emergency and in low-technology settings. Previously reported chromogenic assays have used a purified hippuricase enzyme; Kasahara and colleagues developed in 1981 an assay targeting the activity of angiotensin-I converting enzyme 30 whereas Saruta and colleagues developed a method for determining the activity of carboxypeptidase A 31 . The Campylobacter assay presented here has multiple competitive advantages such as no need for an extended incubation at 37°C for color development, a significant shorter assay time, and the absence of corrosive organic solutions. The assay can take place in two main steps in which (1) 4-HHA is added, and incubation is carried out, and then (2) 4-AAP and the oxidizing system is added. The read-out is fast, and the signal should be monitored over time. Moreover, it is suitable for samples in complex protein-rich media with no possibility of false positives, in contrast to ninhydrin-based assays. Additionally, this novel assay is water based, making it a more environmentally sustainable alternative to the commercially available ninhydrin-based method. As seen in this work, it also allows easy visual detection of positive samples that were later confirmed by MS analytics. Whereas visual detection might not be a feasible or reliable due to conditions of color-blindness for example, the measuring of UV-Vis absorbance after sample centrifugation, could help in the screening for positive samples. Campylobacter in poultry is a leading foodborne pathogen that causes human gastroenteritis with a risk of development postinfection health issues which can negatively impact the economy 32 . It was found that poultry consumption was the main cause of campylobacteriosis outbreaks between 2007 and 2013 worldwide 33 . In the USA between 2000 and 2016, 28 campylobacteriosis outbreaks were recorded, and the consumption of chicken livers was the main cause 34 . In this study it was found that chicken liver was the most contaminated chicken retail product with 30.7% C. jejuni -positive incidence of the analyzed samples. Poultry farms and processing plants are main sources of chicken contamination by Campylobacter 35 . Colonization of Campylobacter in farm chickens occurs usually due to horizontal transmission from other positive chickens or via drinking water and animal feed. Additionally, farmers, who carry Campylobacter , feces of wild birds colonized by Campylobacter , flies, insects, amoebae, yeasts and molds were found to be also important routes of horizontal transmission of Campylobacter in farms 39 . C. jejuni is a common commensal microorganism in the chicken microbiome 36 , 37 . Besides, other Campylobacter species such as C. lari , C. upsaliensis , C. coli and C. concisus can be also isolated from chicken intestines due to horizontal transmission from different sources to chickens 35 ,38 . Once Campylobacter enters the chicken flock, it spreads rapidly and colonizes the intestinal tracts of most chickens within one week 35 , 39 and can reach the level of 109 cells per g of intestinal tract content 40 . In this study, samples have been sourced from 6 different shops which were supplied by different farms (from farms with different domestic animals in addition to commercial poultry farms), and possibly contaminated with different Campylobacter spp . During the enrichment for Campylobacter spp . from chicken meat samples, different types of colonies were growing on the plates with selective agar suggesting that different species were present. At least three different types of colonies grey, white and pink were identified by color. Many studies confirmed that the number of Campylobacter -positive chicken carcasses significantly increased during poultry processing 41 , 42 , 43 . Leaking of Campylobacter from the gut during evisceration is the most critical point during processing. Bacteria from the gut can contaminate the lower half of the carcasses (breast, neck and wings) more often than the upper half (thighs and drumstick) as the birds are always hanged upside-down by the feet 44 . It has been reported that the number of chicken thighs and breasts which were Campylobacter -positive increased from 0% to 90% after evisceration 45 . This study also confirmed that legs (thighs with drumsticks) have the lowest number of positive samples. Contrary to the other studies 46 , 47 , we found very low contamination with C. jejuni of chicken breast (7.7%). This can be explained by the fact that the breast we purchased in this study were skinless and cut into smaller pieces which were not in contact with bacteria. Heating of poultry carcasses followed by chilling during the processing steps are essential in assisting practices for effective defeathering. During heating, the skin follicles remain open, and this allows bacteria to penetrate the skin and accumulate inside the follicles. This can be the reason why almost the same percentage of positive samples was found in wings and backs samples, but lower in breast. In order to validate the novel biochemical assay, a mass-spectrometry-based analysis of selected positive samples has been carried out and key proteins specific of C. jejuni have been identified confirming the presence of the pathogen. Among the seven negative samples, MS analytics detected C. jejuni proteins in one sample, sample number 30, and this can suggest that the sample has indeed been contaminated but that the strain was probably not metabolically active, especially in regards to hippuricase activity. Such a discrepancy between the biochemical hippuricase-based assay and MS analytics could be explained by the different working principle and by the presence of inactive C. jejuni proteome components in the samples, with belongs to not-metabolically active C. jejuni cells, and thus do not react in the chromogenic assay. Concluding, the novel biochemical chromogenic assay is effective for identifying C. jejuni and other hippuricase-positive bacteria, can be used with various oxidants, and outperforms current commercial products. Its specificity was confirmed by analyzing commercial poultry samples, with results validated using nano-liquid chromatography mass spectrometry. Additionally, the water-based nature of the assay offers a more sustainable alternative to the standard ninhydrin-based commercial assays. Methods Chemicals 4-hydroxyhippuric acid (4-HHA, Bachem 4005059), 4-hydroxybenzoic acid (4-HBA, Merck H20059), 4-aminoantipyridine (4-AAP, Merck 06800), horseradish peroxidase (HRP, Merck 77332) , glucose oxidase (GOD, Merck G2133), sodium periodate (NaIO 4 , Biosynth FS04514), glucose (Merck, G8270). Bacteria cultivation conditions Strains used include Campylobacter jejuni DSM 4688 ( C. jejuni. ), Escherichia coli ( E. coli ) and Campylobacter coli DSM 4689 ( C. coli. ), Streptococcus agalactiae ATCC®12386 ( S. agalactiae ) and Streptococcus pyogenes ATCC®19615 ( S. pyogenes. ), that were cultivated on Columbia Agar with sheep blood (Thermofischer PB5039A) at 37°C for 48 h. Both Campylobacter strains were cultivated under microaerophilic atmosphere (Oxoid, Campylgen CN0025A); to obtain colonies and biomass for testing. Streptococcus agalactiae ATCC®12386 and Streptococcus pyogenes ATCC®19615 were cultivated on Columbia Agar with sheep blood (Thermofischer PB5039A) at 37°C for 48 h. Sample preparation A small sample of a loopful (2-3) colonies have been picked and suspended in sterile 100 mM phosphate buffer pH 7.4 and the optical density OD 600 was set to 0.9, unless otherwise mentioned, and then serially diluted to achieve the desired initial optical density with sterile 100 mM phosphate buffer pH 7.4. Testing of the oxidative agent H 2 O 2 and HRP A bacterial sample with an OD 600 of 0.9 has been serially diluted with buffer to an OD 600 of 0.5, 0.2 or 0.1. Two hundred µL of cell suspensions were then added of 25 µL of 100 mM 4-HHA prepared in 100 mM phosphate buffer with pH 7.4 in a 96-well plate and incubated for 1 h at 37°C. After incubation, 20 µL of the prepared mixture (12.5X) containing 4-AAP and HRP, 5 µL of 100 mM H 2 O 2 were added. The absorbance at 505 nm was recorded using a SpectraMax M5 plate reader (Molecular Devices) at room temperature. Final concentrations for the assay were 10 mM 4-HHA, 0.5 mM 4-AAP, 4.5 U/mL HRP and 2 mM H 2 O 2 . Testing of the oxidative agent H 2 O 2 produced by glucose and glucose oxidase A sample containing Campylobacter jejuni or Campylobacter coli biomass with an OD 600 of 0.9 was serially diluted to an OD 600 of 0.5, 0.2 and 0.1. Two hundred µL of cell suspension were mixed with 25 µL of 100 mM 4-HHA (dissolved in 100 mM phosphate buffer pH 7.4) in microtiter plate and incubated for 1 h at 37°C. After incubation, 10 µL of the prepared mixture (25X) 4-AAP and HRP, 10 µL of 250 mM glucose and 5 µL of GOD 50 U/mL were added. The absorbance at 505 nm was recorded with a SpectraMax M5 plate reader (Molecular Devices). Final concentration of 4-HHA was 10 mM, 4-AAP was 0.5 mM, HRP was 4.5 U/mL, glucose was 10 mM and GOD was 1 U/mL. Testing of the oxidative agent sodium periodate Two hundred µL of prepared sample were mixed with 25 µL of 100 mM 4-hydroxyhippuric acid (prepared in 100 mM phosphate buffer pH adjusted to 7.4) in a microtiter plate and incubated for 1 h at 37°C. After incubation, 5 µL of 100 mM 4-AAP and 10 µL of 50 mM sodium periodate (NaIO 4 ) were added. The absorbance at 505 nm was recorded with a SpectraMax M5 plate reader (Molecular Devices) at room temperature. Final concentrations for the assay are 10 mM 4-HHA, 2 mM 4-AAP and 2 mM NaIO 4. Validation with other hippuricase-positive bacterial strains Similarly to the method for the detection of Campylobacter , biomass of colonies were suspended in sterile 100 mM phosphate buffer pH 7.4 and the optical density OD600 was set to 0.5 to 0.6. Two hundred µL of prepared sample have been mixed with 25 µL of 100 mM 4-HHA prepared in 100 mM phosphate buffer pH adjusted to 7.4 in a microtiter plate and incubated for 80 min at 37°C. In accordance with the present method, the oxidative agents were added after this step. Comparison to ninhydrin-based hippurate tests Tests Hippurate disk (Remel, Thermo Scientific, R21085) and Hippurate strips kit (Millipore 01869) were purchased and used as described by the manufacturer. However, to ensure a proper comparability, the optical density OD 600 of all cell suspensions were set to 0.2 and all samples have been incubated at 37°C for 1 h. Isolation of Campylobacter spp. from chicken meat samples Chicken cuts (breast without skin, and wings, backs and legs with skin) (n=52) and liver (n=13) were purchased from six different retail food stores in Belgrade, Serbia, with each store having its own supplier. All chicken meat samples were fresh. Individual chicken cuts such as breast, legs, liver, wings were kept in the metal vessels in the fridges in the stores. In some stores, a single breast was already cut in smaller pieces, while in others it was kept as a whole. To prevent crosscontamiantion of the samples, these were taken from the container in the shop and placed in a plastic bag (one sample per bag) as the first step at purchasing. The cross-contamination of the samples by bacteria was thus only possible in the store during preparation of chicken cuts or keeping in the containers. During the taking the swabs of the samples, the samples were not taken out of the bag and immediately spread onto the surface of charcoal, cefoperazone, desoxycholate Campylobacter selective agar (mCCDA) base (Millipore®, Sigma-Aldrich, Germany) supplemented with blood free Campylobacter medium selective supplement (Millipore®, Sigma-Aldrich, Germany). Plates (D=55 mm) were incubated at 37 °C for 48 h under microaerophilic conditions (85 % N 2 , 10 % CO 2 , 5% O 2 ; using anaerobic atmosphere generation bags (Sigma-Aldrich, Germany). Enzyme-based colorimetric test for the detection of Campylobacter jejuni A rapid colorimetric test (Biosyth®, United Kingdom) was used for the confirmation of C. jejuni in chicken samples. A newly developed assay was based on detection of the enzymatic hydrolysis of hippurate by hippuricase positive C. jejuni strains. Briefly, 200 μL of component A was inoculated with a loopful of colonies from an overnight culture (48 h, 37°C). After homogenisation by vortexing, 25 μL of component B was added into cell suspension, mixed well and incubated at 37°C for 90 minutes. In the last step, 20 μL of component C was firstly added, mixed well and then 5 μL of component D. After mixing, the development of orange to pink color was observed over the next 5 to 15 minutes at room temperature. Protein identification using nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) Nano-liquid chromatography mass spectrometry (nLC-MS/MS) technique was used for the verification of specificity a rapid colorimetric test for the detection of the enzymatic hydrolysis of hippurate by hippuricase positive C. jejuni strains. Sample preparation Immediately after colorimetric assay, four positive and eight negative samples were taken for further analysis. Briefly, remaining bacteria from the surface of agar were spread onto a new agar plate (D=55 mm), incubated 24 h under the same conditions. After that, individual colonies were transferred onto a new agar plate (D=100 mm) and incubated 48 h at 37°C under microaerobic conditions. Individual colonies or clusters of colonies (2 or 3 per positive sample and 1 per negative) were transferred into 100 μL of 6M urea or 100 μL of buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)(5 times concentrated). The cells transferred in 6M urea were first vigorously mixed and then frozen. The procedure was repeated two times to increase lysis of cells. The cells transferred in the buffer were first vigorously mixed then heated for 10 min at 95°C and frozen. After thawing, the samples were again mixed and heated for 10 min at 95°C prior to SDS-PAGE. SDS-PAGE was performed at 12% gel according to the manufacturer’s recommendations, using a Bio-Rad Mini-Protean electrophoretic unit (Bio-Rad, California, USA). Trypsin digestion Cell lysates were loaded onto a 12% SDS-PAGE gel and run just enough for the proteins to migrate approximately 0.5-1 cm into the separation gel, concentrating them into a single dense band. This step was performed to partially purify the samples and remove small molecule contaminants without full protein size separation. The whole concentrated protein regions were carefully excised and subjected to standard procedure of trypsin digestion (57). Briefly, the gel bands were first washed with 25 mM ammonium bicarbonate and 25 mM ammonium bicarbonate in 50% acetonitrile for 60 and 45 minutes, respectively, followed by reduction with 10 mM dithiothreitol at 57 °C in the dark for 60 minutes and alkylation with 55 mM iodoacetamide at room temperature for 45 minutes, also in the dark. Subsequently, the samples underwent a second series of washes with 25 mM ammonium bicarbonate, 25 mM ammonium bicarbonate in 50% acetonitrile, and 100% acetonitrile for 30, 15, and 5 minutes, respectively. After washing, the proteins were digested with trypsin at a protein-to-enzyme ratio of 30:1, at 37 °C for 18 hours. Digestion was stopped by the addition of 10% formic acid to achieve a final formic acid concentration of 1%. Peptides were then cleaned using Peptide Cleanup Pipette Tips (Agilent Technologies) and concentrated in a SpeedVac vacuum concentrator. The dried peptides were reconstituted in 0.1% formic acid and transferred to autosampler vials for LC-MS/MS analysis. Peptides separation Peptides from in-gel digestion, were chromatographically separated using an UltiMate™ 3000 RSLC nano liquid chromatographic system (Thermo Scientific Inc., Bremen, Germany) and 2-column set up: a trap column C18, 50mm (P/N 160454 Thermo Fisher Scientific) and analytical column PepMap C18, 15 cm × 75 μm, 3 μm particles, and 100 Å pore size (ES800A, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phases were MS-grade (A) water with 0.1 % formic acid and (B) acetonitrile with 0.1 % formic acid. The gradient program was as follows: 0–0.5 min 95 % A, 0.5–10 min 95-66 % A, 10–15 min -66-1 % A, 15–20 min 1% A, 20–23 min 95 % A, with flow rate of 0.25 μL/min. Injection volume was 10 μL. This nLC system was coupled with Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific Inc., Bremen, Germany) equipped with a heated electrospray ionization source. Analysis was performed in positive ion mode. Parameters of the ion source was as follows: spray voltage 1.9 kV, capillary temperature 300 °C, range 300–3000 m/z, resolving power 60 000, 1 micro scan was acquired using Xcalibur (version 4.4) software (Thermo Fisher Scientific) with the precursor mass tolerance of 10 ppm. Peptides identification The identification of proteins was performed by PEAKS Suite 12.5 (Bioinformatics Solutions Inc., Canada). Signature MS/MS spectra were searched using the PEAKS database (DB), and post-translational modification (PTM) algorithms against a database consisting of UniProtKB swiss prot validated sequences and the Max Quant contaminant database. In the PEAKS DB algorithm, the following modifications were considered as variables: oxidation (Met), deamidation (Gln, Asn), and hydroxylation (Pro), while carbamidomethylation (Cys) was set as a fixed modification. Up to two missed trypsin cleavages were allowed per peptide. A semi-specific mode of trypsin cleavage was chosen enabling tryptic and semi-tryptic, peptide detection. Mass tolerances were set to 10 ppm for parent ions and 0.02 Da for fragment ions. Protein filters were set to protein -10 log P≥20, proteins’ unique peptides ≥1, and an ion intensity for confident PTM identification of at least 2%. The peptide filter was set to the false discovery rate <0.1% (35). Trypsin digestion, nLCMS/MS peptides separation and identification Trypsin digestion Cell lysates were loaded onto a 12% SDS-PAGE gel and run just enough for the proteins to migrate approximately 0.5-1 cm into the separation gel, concentrating them into a single dense band. This step was performed to partially purify the samples and remove small molecule contaminants without full protein size separation. The whole concentrated protein regions were carefully excised and subjected to standard procedure of trypsin digestion (57). Briefly, the gel bands were first washed with 25 mM ammonium bicarbonate and 25 mM ammonium bicarbonate in 50% acetonitrile for 60 and 45 minutes, respectively, followed by reduction with 10 mM dithiothreitol at 57 °C in the dark for 60 minutes and alkylation with 55 mM iodoacetamide at room temperature for 45 minutes, also in the dark. Subsequently, the samples underwent a second series of washes with 25 mM ammonium bicarbonate, 25 mM ammonium bicarbonate in 50% acetonitrile, and 100% acetonitrile for 30, 15, and 5 minutes, respectively. After washing, the proteins were digested with trypsin at a protein-to-enzyme ratio of 30:1, at 37 °C for 18 hours. Digestion was stopped by the addition of 10% formic acid to achieve a final formic acid concentration of 1%. Peptides were then cleaned using Peptide Cleanup Pipette Tips (Agilent Technologies) and concentrated in a SpeedVac vacuum concentrator. The dried peptides were reconstituted in 0.1% formic acid and transferred to autosampler vials for LC-MS/MS analysis. Peptides separation Peptides from in-gel digestion, were chromatographically separated using an UltiMate™ 3000 RSLC nano liquid chromatographic system (Thermo Scientific Inc., Bremen, Germany) and 2-column set up: a trap column C18, 50mm (P/N 160454 Thermo Fisher Scientific) and analytical column PepMap C18, 15 cm × 75 μm, 3 μm particles, and 100 Å pore size (ES800A, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phases were MS-grade (A) water with 0.1 % formic acid and (B) acetonitrile with 0.1 % formic acid. The gradient program was as follows: 0–0.5 min 95 % A, 0.5–10 min 95-66 % A, 10–15 min -66-1 % A, 15–20 min 1% A, 20–23 min 95 % A, with flow rate of 0.25 μL/min. Injection volume was 10 μL. This nLC system was coupled with Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific Inc., Bremen, Germany) equipped with a heated electrospray ionization source. Analysis was performed in positive ion mode. Parameters of the ion source was as follows: spray voltage 1.9 kV, capillary temperature 300 °C, range 300–3000 m/z, resolving power 60 000, 1 micro scan was acquired using Xcalibur (version 4.4) software (Thermo Fisher Scientific) with the precursor mass tolerance of 10 ppm. Peptides identification The identification of proteins was performed by PEAKS Suite 12.5 (Bioinformatics Solutions Inc., Canada). Signature MS/MS spectra were searched using the PEAKS database (DB), and post-translational modification (PTM) algorithms against a database consisting of UniProtKB Swiss Prot validated sequences and the Max Quant contaminant database. In the PEAKS DB algorithm, the following modifications were considered as variables: oxidation (Met), deamidation (Gln, Asn), and hydroxylation (Pro), while carbamidomethylation (Cys) was set as a fixed modification. Up to two missed trypsin cleavages were allowed per peptide. A semi-specific mode of trypsin cleavage was chosen enabling tryptic and semi-tryptic, peptide detection. Mass tolerances were set to 10 ppm for parent ions and 0.02 Da for fragment ions. Protein filters were set to protein -10 log P≥20, proteins’ unique peptides ≥1, and an ion intensity for confident PTM identification of at least 2%. The peptide filter was set to the false discovery rate <0.1% (35). Abbreviations 4-hydroxyhippuric acid, 4-HHA; 4-HBA, 4-hydroxybenzoic acid; 4-AAP, 4-aminoantipyridine; GOD, glucose oxidase; HRP, horseradish peroxidase; HRMS, High-resolution mass spectrometry; mCCDA, Modified Charcoal Cefoperazone Deoxycholate Agar; PTM, Post-translational modification. Declarations Competing Interests Y.C., F.G., A.S. and S.U. are employed by Biosynth AG, a corporation that markets most of the enzymes and chemicals used in this protocol. Biosynth AG has provided financial support in the form of authors’ salaries and research materials. The assay presented is commercially available on the company website (www.biosynth.com). The assay subject of this work is the subject of International Patent Application PCT/EP2023/060481. This does not alter our adherence to the journal’s policies on sharing data and materials. No further conflicts of interest are declared. University of Belgrade-Faculty of Chemistry has carried out the testing independently and Biosynth AG partially covered material and shipping costs. Funding No external funding has been received. Author Contribution C.Y. curated the conceptualization, data analysis, investigation, review, and editing; A.S. performed the experimental work and data analysis; G.F. curated the writing, review, and editing; U.S. curated the conceptualization, VJ, TV, and TCV carried out the validation of the assay with chicken samples, mass spectrometry analysis and bioinformatics. Data Availability All data generated or analyzed during this study are included in this published article. References WHO, The Global View of Campylobacterioisis. https://iris.who.int/bitstream/handle/10665/80751/9789241564601_eng.pdf (2012). Center of Disease Control and Prevention. https://www.cdc.gov/campylobacter/technical.html (2019). Finsterer, J. Triggers of Guillain-Barré Syndrome: Campylobacter jejuni Predominates. Int. 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Food Protect. 82, 2126–2134. doi: 10.4315/0362-028X.JFP-19-146 Tables Table 1-Overview of bacterial strains used in this study and info on their reported production of hippuricase or catalase. Strain Catalase Hippuricase Campylobacter jejuni DSM 4688 positive positive Campylobacter coli DSM 4689 positive negative Escherichia coli positive negative Streptococcus agalactiae ATCC®12386™ negative positive Streptococcus pyogenes ATCC®19615™ negative negative Table 2 - Distribution of the positive samples identified with the novel biochemical assay presented in this work among the six retail shops and different meat cuts. Chicken meat samples Total(Positives) Shop Backs Wings Legs Breast Liver 1 5(2) 3(1) 1(0) - 3(0) 2 - 2(0) 1(1) 3(0) 5(2) 3 - 4(0) 0(0) 2(1) 5(2) 4 5(0) 3(0) 2(0) - - 5 - - 7(0) 8(0) - 6 - 4(2) 2(0) - - Total 10(2) 16(3) 13(1) 13(1) 13(4) Abbreviation: - = sample of this type not analyzed (not available). Note: Legs = thigh and drumstick together). Table 3 - Identification of Campylobacter jejuni proteins in positive samples based on LC-MS/MS analysis. *Score (-10logP) represents the identification confidence based on the PEAKS software analysis. C-coverage (%) indicates the proportion of the protein sequence identified by matched peptides. U refers to the number of unique peptides identified per protein. "-" denotes that the protein was not detected in the corresponding sample. Table 4 . C. jejuni biomarker proteins identified by High-resolution mass spectrometry (HRMS) and homologous proteins (100% identity) search results in TrEMBL (TrEMBL release 9.0 | UniProt help | UniProt). C. jejuni biomarker protein Identical proteins retrieved from UniProt TrEMBL Major outer membrane protein (UniProt ID: P80672) Threonine-tRNA ligase (UniProt ID: A1VXT5) Catalase (UniProt ID: Q59296) 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (UniProt ID: Q0P823) Cysteine synthase B (UniProt ID: P71128) Probable histidine-binding protein (UniProt ID: Q46125) Campylobacter coli , Basic amino acid ABC transporter substrate-binding protein (UniProt ID: A0A5T1KBF0) Campylobacter sp. CH185, Transporter substrate-binding domain-containing protein (UniProt ID: A0A4Y8CSE0 Flagellin B (UniProt ID: P56964) Campylobacter coli , Flagellin (uniProt ID: A0A5T1U4W8) Large ribosomal subunit protein uL3 (UniProt ID: Q9PLX1) Campylobacter sp. BCW_8712, 50S ribosomal protein L3 (UniProt ID: A0A2A5M3J8) Fumarate hydratase class II (UniProt ID: O69294) Campylobacter sp. CH185 fumarate hydratase (UniProt ID:A0A4Y8C754) Succinate-CoA ligase [ADP-forming] subunit beta (UniProt ID: A8FKV6) Campylobacter sp. BCW_8712 Succinate--CoA ligase subunit beta (UniProt ID: A0A2A5M3Z4) Additional Declarations Competing interest reported. Y.C., F.G., A.S. and S.U. are employed by Biosynth AG, a corporation that markets most of the enzymes and chemicals used in this protocol. Biosynth AG has provided financial support in the form of authors’ salaries and research materials. The assay presented is commercially available on the company website ( www.biosynth.com ). The assay subject of this work is the subject of International Patent Application PCT/EP2023/060481. This does not alter our adherence to the journal’s policies on sharing data and materials. No further conflicts of interest are declared. University of Belgrade-Faculty of Chemistry has carried out the testing independently and Biosynth AG partially covered material and shipping costs. Supplementary Files 20250619Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Aug, 2025 Reviews received at journal 08 Aug, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviews received at journal 25 Jul, 2025 Reviewers agreed at journal 20 Jul, 2025 Reviewers invited by journal 10 Jul, 2025 Editor assigned by journal 04 Jul, 2025 Editor invited by journal 27 Jun, 2025 Submission checks completed at journal 23 Jun, 2025 First submitted to journal 23 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6936616","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483263008,"identity":"9b39aa0c-984b-450c-9b54-a3bb748f233e","order_by":0,"name":"Chunyan Yao","email":"","orcid":"","institution":"Biosynth AG","correspondingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Yao","suffix":""},{"id":483263009,"identity":"4d1cbe9b-b838-417e-9a0d-6fd500704dd5","order_by":1,"name":"Alexandra Schmid","email":"","orcid":"","institution":"Biosynth AG","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Schmid","suffix":""},{"id":483263011,"identity":"72c6fe25-b6e3-4cdc-82e5-91726b2ea0ff","order_by":2,"name":"Greta Faccio","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYPACOSBmbGD42CAB5jITUA5UymAMphlnQrQwNhOpBWg4bwMDYS3y0YePP/jBYCDP33648bHtDgs5+Rm5xx8XMNjJ6TZg12J4Li2xsYfBwHDGmcRm49wzEsYGN/ISm2cwJBubHcChpYfHsIGH4Q/jBgnGNuncNonEDRI5hs08DAcSt+HUwv+x8Q+DgT1YiyVQy/wZBLTI8/AwAhUYJIK1MAK1NNwgoMWAh81wtoyBQTLIL4a9IL+ceWM4m8cAt1/ke5gffHxTYWDb33784YOfO+rk5NtzDD7zVNjJ4dJiABY3QBYSSEAXQbOlAUOIH4fpo2AUjIJRMGIBABLLWQoqKE4BAAAAAElFTkSuQmCC","orcid":"","institution":"Biosynth AG","correspondingAuthor":true,"prefix":"","firstName":"Greta","middleName":"","lastName":"Faccio","suffix":""},{"id":483263013,"identity":"11674646-0a47-42c2-8ebb-32a1650d00f0","order_by":3,"name":"Urs Spitz","email":"","orcid":"","institution":"Biosynth AG","correspondingAuthor":false,"prefix":"","firstName":"Urs","middleName":"","lastName":"Spitz","suffix":""},{"id":483263014,"identity":"b7b5c1c5-1036-41c5-9dac-3fe21bee1947","order_by":4,"name":"Vesna Jovanović","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Vesna","middleName":"","lastName":"Jovanović","suffix":""},{"id":483263015,"identity":"d4fb1874-891a-4b92-97f5-770969f94e15","order_by":5,"name":"Tamara Vasović","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Tamara","middleName":"","lastName":"Vasović","suffix":""},{"id":483263020,"identity":"9a534776-ec22-41ba-8115-daeda0574067","order_by":6,"name":"Tanja Ćirković Veličković","email":"","orcid":"","institution":"University of Belgrade","correspondingAuthor":false,"prefix":"","firstName":"Tanja","middleName":"Ćirković","lastName":"Veličković","suffix":""}],"badges":[],"createdAt":"2025-06-20 07:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6936616/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6936616/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-24981-x","type":"published","date":"2025-11-20T15:58:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87028557,"identity":"f249d490-00c6-4797-b852-2e28b8377cfe","added_by":"auto","created_at":"2025-07-18 12:33:45","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22188,"visible":true,"origin":"","legend":"\u003cp\u003eBiochemical reaction at the basis of the chromogenic assay for the detection of Campylobacter jejuni and other hippuricase-producing microorganisms. Whereas the first reaction (reaction 1) is catalyzed by the endogenous bacterial hippuricase enzyme, the second reaction (reaction 2) in carried out after addition of 4-aminoantipyrine and an oxidant reagent such as (a) hydrogen peroxide and horseradish peroxidase, (b) hydrogen peroxide produced by glucose oxidase and glucose with peroxidase, or (c) a periodate salt such as sodium periodate.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/2b7714a249771dbca840f05e.jpeg"},{"id":87030200,"identity":"7d01ada3-1660-4423-bd57-672f1a185181","added_by":"auto","created_at":"2025-07-18 12:41:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":343487,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of C. jejuni using the HRP/H2O2 oxidizing system. (A) \u0026nbsp;Intense chromogenic reaction distinguishing C. jejuni from C. coli, E. coli and a cell-free culture medium as negative control. The initial sample optical density is OD\u003csub\u003e600\u003c/sub\u003e = 0.9. (B) Absorbance signal development over time using samples of different initial optical density, i.e. OD\u003csub\u003e600\u003c/sub\u003e =0.1, 0.2, 0.5 or 0.9 with C. jejuni (C. j., red mark) in comparison to C. coli (C. c., blue mark) at similar cell density. Sterile medium is used as control (grey mark). Cell suspensions in sterile 100 mM phosphate buffer pH 7.4 were incubated with 4-hydroxyhippuric acid for 1 h at 37°C, following the addition of 4-aminoantipyrine, horseradish peroxidase and followed with hydrogen peroxide. Inset: image of the reaction mixtures with C. jejuni in compare with C. coli by similar cell density. No coloration is visible with Hippuricase-negative strain C. coli and sterile Control. Strains used are all catalase-producing ones. (C) Absorbance signal development of C. jejuni in comparison to C. coli, and sterile control; final concentrations of 4-hydroxyhippuric acid (4-HHA) were 5 mM, 10 mM, and 20 mM. (D) Absorbance signal development in the chromogenic reaction of 4-hydroxybenzoic acid with 4-aminoantipyrine, horseradish peroxidase and hydrogen peroxide in the presence of bacterial cells with or without catalase, final concentration of 4-hydroxybenzoic acid was 1 mM.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/04e6eda91807b4cd2fb15458.png"},{"id":87028559,"identity":"ef3b0a1e-1e29-4923-8fb1-4a47bbcf77f0","added_by":"auto","created_at":"2025-07-18 12:33:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":417234,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Absorbance signal development over time of C. jejuni (left panel) and C. coli (right panel at similar cell densities. Cell suspensions in sterile 100 mM phosphate buffer pH 7.4 were incubated with 4-HHA for 1 h at 37°C. After incubation, 4-AAP, HRP, glucose and GOD were added. Sterile medium is used as control (crossed marks).\u0026nbsp; (B) Image of the samples after 20 min. Cells were incubated 1 hour at 37°C in the presence of 10 mM 4-HHA and later added of 0.5 mM 4-AAP, 4.5 U/mL HRP, 10 mM glucose and 1 U/mL glucose oxidase. No coloration is seen with hippuricase negative strain C. coli. The assay was carried out as experimental description. Instead of adding H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e directly, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was produced in-situ per Glucose and Glucose Oxidase. (C) Color change obtained from samples containing C. jejuni in compare with C. coli by similar cell density, i.e. 1.1, 0.5, 0.3, and 0.13 obtained using sodium periodate as oxidant in the second reaction of the assay. Endpoint absorbance was taken 10 minutes after all reagents were added. No coloration with hippuricase-negative strain C. coli and the sterile control was visible (image as inset). (D) Linear correlation of absorbance intensity at 505 nm with the optical density measured at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) according to the methods of the present invention using samples with C. jejuni DSM 4688 and different oxidizing reagents. Cell suspensions in sterile 100 mM phosphate buffer pH 7.4 were incubated with 4-HHA for 1h at 37°C, following the addition of 4-aminoantipyrine and different oxidizing reagents: HRP/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (filled square), HRP/Glu/ GOD (filled triangle), NaIO4 (filled circle). Endpoint absorbance after 10 min (absorbance of sterile control subtracted) was evaluated.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/9adb8c7ae3ffc608b3021f76.png"},{"id":87028570,"identity":"b73ff910-c881-4c96-8113-5fbff177c821","added_by":"auto","created_at":"2025-07-18 12:33:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":108425,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of different oxidizing reagents in the assay for detecting hippuricase-positive microorganisms such as C. jejuni DSM 4688. Cell suspensions with an OD\u003csub\u003e600\u003c/sub\u003e = 0.4-0.5 in sterile 100 mM phosphate buffer pH 7.4 were incubated with 4-HHA for 70 min at 37°C, followed by the addition of 4-AAP and various oxidizing reagents: HRP/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (left panel) , NaIO\u003csub\u003e4\u003c/sub\u003e (middle panel), HRP/Glu/GOD (right panel). Cell suspension without any additives and sterile control were included as negative controls. Abbreviations: w/o: without, w: with. Sterile medium is used as control.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/57b4b2ed229e4437b71d12ed.png"},{"id":87028561,"identity":"9311ff20-1c96-477a-a6f4-7bbe37da4e1a","added_by":"auto","created_at":"2025-07-18 12:33:45","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":156272,"visible":true,"origin":"","legend":"\u003cp\u003eTesting additional hippuricase-positive strain Streptococcus agalactiae ATCC®12386 (black bars) and hippuricase-negative Streptococcus pyogenes ATCC®19615 (striped, grey bars) with the three oxidizing agents such as hydrogen peroxide/peroxidase (left), enzymatically produced hydrogen peroxide generating system (middle), and using sodium periodate (right). Endpoint absorbance after 15 min. Sterile medium without additives is used as control (empty bars).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/ce20a7f02f9ec7df4f350b53.jpeg"},{"id":87030206,"identity":"969ee19c-90b7-4f92-bd09-32a0a096712a","added_by":"auto","created_at":"2025-07-18 12:41:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":580930,"visible":true,"origin":"","legend":"\u003cp\u003eVisual comparison of results obtained with the assay described in this work (left panel) and with two commercial ninhydrin-based tests from Remel (middle) and Millipore (right). In left panel, assay ingredients with final concentration of a) 0.5 mM 4-AAP, 4.5 U/mL HRP and 2 mM H2O2; b) 0.5 mM 4-AAP, 4.5 U/mL HRP, 10 mM Glucose and 1 U/mL glucose oxidase were added to the samples.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/2aead70694e070653a5e6ac9.png"},{"id":87028565,"identity":"6073ce23-f57b-4d1d-94b9-560a55866d2f","added_by":"auto","created_at":"2025-07-18 12:33:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":50925,"visible":true,"origin":"","legend":"\u003cp\u003eThe characteristic colonies of grey and white ascribed to Campylobacter spp. on the charcoal, cefoperazone, desoxycholate Campylobacter selective agar after 48 h incubation at 37°C under microaerophilic conditions. According to the specification of the plate manufacturer, the color of the colony of: C. jejuni is grey, C. coli is creamy grey, while C. laridis can be of varying types. \u0026nbsp;The image (left) unedited and (right) at improved contrast for ease of visualization.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/7d45d5f0f2a41c8c77668a40.png"},{"id":87028568,"identity":"f254d9a4-b9e7-422d-877f-7a2282bb0c1e","added_by":"auto","created_at":"2025-07-18 12:33:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":826829,"visible":true,"origin":"","legend":"\u003cp\u003eA selection of the analysed samples and of the reaction samples at completion as assayed with the new method reported in this article showing the range of responses from incolor-to intense red coloration. The pink-reddish coloration is ascribed to the presence of hippuricase enzyme, a biomarker for the presence of bacteria such as Campylobacter jejuni, e.g. in this image samples number 3, 4, 7, 36, 38, 41 were recognized as positive.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/cb672cb2763f9e62ec5d84f8.png"},{"id":96650910,"identity":"e50f81dc-a19d-43c1-8a53-517f27176e52","added_by":"auto","created_at":"2025-11-24 16:12:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3830177,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/3f12348f-a37f-4b62-9102-5aea26b63a5e.pdf"},{"id":87030202,"identity":"dd3f633c-81d7-4045-80ef-28ea86d365d9","added_by":"auto","created_at":"2025-07-18 12:41:45","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6386618,"visible":true,"origin":"","legend":"","description":"","filename":"20250619Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6936616/v1/6eb8a900fd772c966f7d4dba.docx"}],"financialInterests":"Competing interest reported. Y.C., F.G., A.S. and S.U. are employed by Biosynth AG, a corporation that markets most of the enzymes and chemicals used in this protocol. Biosynth AG has provided financial support in the form of authors’ salaries and research materials. The assay presented is commercially available on the company website (www.biosynth.com). The assay subject of this work is the subject of International Patent Application PCT/EP2023/060481. This does not alter our adherence to the journal’s policies on sharing data and materials. No further conflicts of interest are declared. University of Belgrade-Faculty of Chemistry has carried out the testing independently and Biosynth AG partially covered material and shipping costs.","formattedTitle":"\u003cp\u003eRapid and Specific Ninhydrin-free Chromogenic Assay for Detection of Viable Hippuricase-positive Bacteria and Its Validation in Raw Chicken Meat Samples\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe possibility of identifying the presence of pathogens with a specific, yet easy-to-read, assay is crucial to prevent their spread. At a global scale, \u003cem\u003eCampylobacter\u0026nbsp;\u003c/em\u003einfections are behind the majority of diarrheal diseases in humans and can also result in gastroenteritis and be fatal in young children, elderly, and immunosuppressed individuals. Infection is often foodborne, transmitted through contaminated meat like chicken, milk, water, or ice, which are not safely handled or properly heat-treated. In approximately 90% of human \u003cem\u003eCampylobacter\u003c/em\u003e infections, the principal pathogen is \u003cem\u003eCampylobacter jejuni\u003c/em\u003e (\u003cem\u003eC. jejuni\u003c/em\u003e)\u003csup\u003e1\u003c/sup\u003e,\u003csup\u003e2\u003c/sup\u003e. Due to frequencies of Campylobacteriosis and the potential sources of \u003cem\u003eCampylobacter\u003c/em\u003e spp., their isolation and identification in clinical and food samples is crucial. More recently,\u0026nbsp;\u003cem\u003eC. jejuni\u0026nbsp;\u003c/em\u003ehas also been identified as a trigger of Guillain-Barr\u0026eacute; syndrome, a rare immune-mediated acute polyradiculo-neuropathy\u003csup\u003e3\u003c/sup\u003e. The presence of \u003cem\u003eCampylobacter\u003c/em\u003e spp. has also been linked to the development of celiac disease\u003csup\u003e4\u003c/sup\u003e,\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAccording to the Genome Taxonomy Database, currently over forty \u003cem\u003eCampylobacter\u0026nbsp;\u003c/em\u003especies are recognised\u003csup\u003e6\u003c/sup\u003e, and at least a dozen of them have been implicated in human disease\u003csup\u003e7\u003c/sup\u003e. Members of this genus are typically Gram-negative, non-spore-forming, S-shaped or spiral shaped bacteria, with single polar flagella at one or both ends\u003csup\u003e8\u003c/sup\u003e. \u003cem\u003eCampylobacter jejuni\u003c/em\u003e (\u003cem\u003eC. jejuni\u003c/em\u003e) and \u003cem\u003eCampylobacter coli\u0026nbsp;\u003c/em\u003e(\u003cem\u003eC. coli\u003c/em\u003e) represent the main cause of bacterial diarrhoea in developed countries\u003csup\u003e9\u003c/sup\u003e,\u003csup\u003e10\u003c/sup\u003e, and one of the most important causes of enterocolitis in developing countries\u003csup\u003e11\u003c/sup\u003e. Over 80 % of cases are caused by \u003cem\u003eC. jejuni\u003c/em\u003e and 5-10 % of cases are caused by \u003cem\u003eC. coli\u003csup\u003e12\u003c/sup\u003e\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVarious methods to detect \u003cem\u003eC. jejuni\u0026nbsp;\u003c/em\u003econtaminations are based either on its genetic or its metabolic make-up. \u003cem\u003eC. jejuni\u003c/em\u003e is one of the few strains producing the enzyme hippuricase (EC 3.5.1.32 hippurate hydrolase), and this has been leveraged to differentiate it from other bacteria such as \u003cem\u003eCampylobacter coli\u003c/em\u003e. The \u003cem\u003ehipO\u003c/em\u003e gene for hippuricase has been the target for PCR-based tests\u003csup\u003e13\u003c/sup\u003e. \u0026nbsp;With the genetic approach, \u003cem\u003eC. jejuni\u003c/em\u003e and \u003cem\u003eC. coli\u003c/em\u003e could be indeed differentiated\u003csup\u003e14\u003c/sup\u003e. \u003cem\u003eCampylobacter jejuni\u003c/em\u003e is however not the only producer of hippuricase and other \u003cem\u003eCampylobacter\u003c/em\u003e spp., e.g., \u003cem\u003eC. avium\u003c/em\u003e as well as other species such as \u003cem\u003eLegionella spp.\u003c/em\u003e, and \u003cem\u003eStreptococcus agalactiae\u0026nbsp;\u003c/em\u003e(known as\u003cem\u003e\u0026nbsp;\u003c/em\u003egroup B \u003cem\u003eStreptococci)\u003c/em\u003e, \u003cem\u003eGardnerella vaginalis\u003c/em\u003e, \u003cem\u003eListeria monocytogenes\u003c/em\u003e, \u003cem\u003eBrachyspira\u003c/em\u003e spp., and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e produce the enzyme\u003csup\u003e9,10,11\u003c/sup\u003e. However, not all hippuricase-positive bacteria harbor the \u003cem\u003ehipO\u003c/em\u003e gene such as \u003cem\u003eC. avium\u003c/em\u003e\u003csup\u003e12\u003c/sup\u003e. Although highly sensitive at the gene level, these assays do not provide information on the bacterial viability and require specific equipment and trained personnel). Bessede and colleagues have shown that also mass spectrometry, i.e. MALDI-TOF, can be used to identify \u003cem\u003eCampylobacter\u003c/em\u003e isolates rapidly and efficiently at the genus and species level with high accuracy (0.4% of misidentification)\u003csup\u003e15\u003c/sup\u003e. Metabolism-based detection approaches can rely instead on the activity of the hippuricase enzyme itself, that can be considered a biomarker, which is responsible of the hydrolysis, at the peptide linkage site, of hippuric acid to glycine and benzoic acid.\u003c/p\u003e\n\u003cp\u003eRapid testing currently focusses on the second product of the hippuricase reaction, the glycine amino acid, and provide a visual read-out based on the ninhydrin reaction\u003csup\u003e13,16\u003c/sup\u003e. The amino group of glycine can react with ninhydrin (2,2-dihydroxyindane-1,3-dione) producing a blue coloration. However, the ninhydrin-based reaction is not specific to glycine as it reacts with all molecules carrying alpha-amino groups that are present in the sample limiting its application to complex samples in nutrient-rich culture medium and resulting often in false positives. Moreover, the use of ninhydrin poses health hazards as it can cause irritation on contact, and it is a respiratory tract irritant\u003csup\u003e14\u003c/sup\u003e. Detection methods of benzoic acid, the second product of hippurate hydrolysis, include a color reaction with ferric chloride\u003csup\u003e17\u003c/sup\u003e or a gas-liquid chromatographic method\u003csup\u003e16\u003c/sup\u003e. The former method requires is however time-consuming whereas the latter is equipment-dependent, requires trained personnel and specific sample preparation.\u003c/p\u003e\n\u003cp\u003eThis work introduces a rapid, specific, chromogenic assay for detecting hippuricase-positive viable bacteria such as \u003cem\u003eCampylobacter\u003c/em\u003e \u003cem\u003ejejuni and it\u0026nbsp;\u003c/em\u003e overcomes\u0026nbsp;the limitations of the currently available technologies. The assay is valid for the detection of metabolically active \u003cem\u003eC. jejuni\u003c/em\u003e and other hippuricase-positive bacteria, it can be used with different oxidants and outperforms current commercials analogous products. To verify the specificity of this rapid colorimetric test for the detection of the enzymatic hydrolysis of hippurate by hippuricase positive \u003cem\u003eC. jejuni\u003c/em\u003e, commercial poultry samples have been analysed and results have been confirmed using nano-liquid chromatography mass spectrometry (nLC-MS/MS) technique. As an additional consideration, being water-based, the assay also presents a higher sustainability level than the current standard ninhydrin-based commercial assays.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eCampylobacter\u003c/em\u003e is considered to be the most common bacterial cause of human gastroenteritis in the world. Hippuricase is a rarely found bacterial enzyme that characterizes \u003cem\u003eC. jejuni\u003c/em\u003e and a few other bacteria strains that were also tested in this study (Table 1). Hippuricase can thus be considered a key enzyme, a biomarker, for the detection of \u003cem\u003eC. jejuni\u003c/em\u003e and its identification in complex samples.\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\n \u003ch3\u003eChromogenic assay for the detection of \u003cem\u003eCampylobacter jejuni\u0026nbsp;\u003c/em\u003e\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe assay we report is composed of two main steps based first on the hippuricase enzyme, endogenous of a positive sample, that converts \u0026nbsp;4-hydroxyhippuric acid (4-HHA) to hydroxybenzoic acid (4-HBA) and glycine (reaction 1, Figure 1), and 4-HBA is subsequently oxidized in the presence of 4-aminoantipyridine (4-AAP) by either a peroxidase (HRP) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (approach a, Figure 1) or by sodium periodate as single oxidant (approach c, Figure 1) to a colorful quinoeimine dye with a bright pink color. If the external addition of hydrogen peroxide in the second step is not desired, this can be enzymatically produced \u003cem\u003ein situ\u003c/em\u003e using glucose and glucose oxidase (approach b, Figure 1). Positive sample results are indicated by a pink coloration.\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003e\n \u003ch3\u003eDetection \u003cem\u003eCampylobacter jejuni\u0026nbsp;\u003c/em\u003eusing\u003cem\u003e\u0026nbsp;\u003c/em\u003eHRP and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as oxidizing reagent\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe sample to be tested is prepared by resuspending in buffer a few bacterial colonies picked from an agar plate, i.e., to reach an OD\u003csub\u003e600\u003c/sub\u003e of 0.9. The sample is then incubated with 4-HHA for 70 minutes at 37\u0026deg;C and later added of a 4-AAP, HRP and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Increase of the absorbance of the solution at 505 nm is measured in samples comprising \u003cem\u003eC. jejuni\u0026nbsp;\u003c/em\u003e(Figure 2B) that develop a pinkish coloration,\u0026nbsp;whereas samples comprising \u003cem\u003eC. coli\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eEscherichia coli,\u0026nbsp;\u003c/em\u003eor without bacteria (sterile control),\u003cem\u003e\u0026nbsp;\u003c/em\u003eremain of the initial light yellowish color, upon visual detection (Figure 2A).\u003c/p\u003e\n\u003col start=\"3\"\u003e\n \u003cli\u003e\n \u003ch3\u003eDetermination of the sensitivity level and assessing the interference of endogenous catalase activity\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eTo assess the sensitivity of the assay, the bacterial sample was serially diluted with buffer to an OD\u003csub\u003e600\u003c/sub\u003e as low as 0.1 (Figure 2B). Placed in a multi-well plate, samples were then added of 4-HHA and incubated for 1 h at 37\u0026deg;C. After incubation, 4-AAP and HRP were added. The absorbance at 505 nm was monitored immediately. Final reagent concentrations for the assay were 10 mM 4-HHA, 0.5 mM 4-AAP, 4.5 U/mL HRP and 2 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The sterile control and the samples comprising\u003cem\u003e\u0026nbsp;C. coli\u003c/em\u003e remained colorless or slightly yellowish at all the cell concentration tested, whereas the samples comprising \u003cem\u003eCampylobacter jejuni\u003c/em\u003e showed a robust red to pink color over a broad range of cell density values, i.e. coloration intensity gradually increased in samples from an OD\u003csub\u003e600\u003c/sub\u003e value of 0.1 to an OD\u003csub\u003e600\u003c/sub\u003e value 0.9. \u0026nbsp;The red/pink color was visible by eye in the sample with an OD\u003csub\u003e600\u003c/sub\u003e value as low as 0.1 (Figure 2B). The results in Figure 2B show that a significant increase of the absorbance is recorded already after a few minutes with a plateau reached eventually, e.g., after about 10 minutes at an OD\u003csub\u003e600\u003c/sub\u003e value of 0.1 demonstrating that the method has a high sensitivity and allows for fast detection of bacteria at low cell density. No background coloration has been observed with the hippuricase-negative bacterial strains.\u003c/p\u003e\n\u003cp\u003eThe assay requires hydrogen peroxide as a regent, and the natural production of the enzyme catalase by some microorganisms (Table 1) might cause interference with the assay. We thus tested the formation of the quinoneimine compound produced by oxidative coupling of 4-HBA with 4-AAP and HRP in the presence of the hydrogen-peroxide-using catalase-positive strains. Already all strains tested in Figure \u003cem\u003e2\u003c/em\u003eA and B are catalase-positive, and results show a significant increase of the absorbance at 505 nm with a plateau reached after about 5 minutes under the conditions tested. Although a concentration of 1 mM was sufficient for signal generation, a final concentration of 10 mM 4-HHA showed the best performance (Figure \u003cem\u003e2\u003c/em\u003eC). It was however found that hydrogen peroxide should be added freshly after incubating the bacteria with 4-HHA to yield optimal color results. As in Figure \u003cem\u003e2\u003c/em\u003eD that includes catalase-negative \u003cem\u003eStreptococcus\u003c/em\u003e strains, absorbance in the cell-free control sample continued to increase for 15 minutes whereas the reaction stopped after about 5 minutes in the catalase-positive samples (\u003cem\u003eCampylobacter\u003c/em\u003e). In the catalase-negative samples (\u003cem\u003eStreptococcus\u003c/em\u003e), the reaction kinetics were similar to the reaction without cell suspension. These results show that catalase most likely degraded the added hydrogen peroxide which caused a stop of the reaction of 4-AAP/HRP with 4-HBA. However, the hippuricase reaction is sufficiently fast and the assay qualifies as a detecting method of hippuricase-producing microorganisms despite the presence of catalase.\u003c/p\u003e\n\u003col start=\"4\"\u003e\n \u003cli\u003e\n \u003ch3\u003eDetection of \u003cem\u003eCampylobacter jejuni\u0026nbsp;\u003c/em\u003eusing \u003cem\u003ein-situ\u003c/em\u003e-produced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e with glucose/glucose oxidase or using\u003cem\u003e\u0026nbsp;\u003c/em\u003esodium periodate as oxidizing reagent\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eConsidering the oxidative properties of hydrogen peroxide and the safety measures that its handling requires, we tested its \u003cem\u003ein situ\u003c/em\u003e production using the glucose oxidase action on glucose as an alternative.\u003cem\u003e\u0026nbsp;Campylobacter jejuni\u003c/em\u003e DSM 4688 (hippuricase positive) and \u003cem\u003eCampylobacter coli\u003c/em\u003e DSM 4689 (hippuricase negative) were cultivated under microaerophilic atmosphere at 37\u0026deg;C for 48 h and later, to assess the sensibility to low cell density, colonies were suspended in buffer and the optical density OD\u003csub\u003e600\u003c/sub\u003e adjusted to 0.5, 0.2 or 0.1. Upon reaction, the samples comprising \u003cem\u003eCampylobacter coli\u0026nbsp;\u003c/em\u003eremained colorless or slightly yellowish for all OD\u003csub\u003e600\u003c/sub\u003e values while in the samples comprising \u003cem\u003eCampylobacter jejuni\u0026nbsp;\u003c/em\u003eshowed a robust red to pink color over a broad range of OD\u003csub\u003e600\u003c/sub\u003e values with gradual increase in intensity from an OD\u003csub\u003e600\u003c/sub\u003e value of 0.1 to an OD\u003csub\u003e600\u003c/sub\u003e value 0.9 as determined by visual inspection (Figure 3A-B). This example confirms that the method is also sensitive and specific with an included enzymatic hydrogen-peroxide-generating system.\u003c/p\u003e\n\u003cp\u003eAiming at a non-enzymatic version of the essay and at reducing the number of components, sodium periodate was tested as oxidizing agent. The samples were prepared for an OD\u003csub\u003e600\u003c/sub\u003e of 1.1 and then serially diluted with the same buffer to an OD\u003csub\u003e600\u003c/sub\u003e value of 0.5, 0.3, and 0.1. \u0026nbsp;After the incubation phase and the addition of 4-AAP, sodium periodate (NaIO\u003csub\u003e4\u003c/sub\u003e) was added, and the reaction started. The absorption at 505 nm was recorded visually and with a plate reader at room temperature. \u0026nbsp;The samples comprising \u003cem\u003eCamplyobacter coli\u0026nbsp;\u003c/em\u003eor the sterile control remained colorless or slightly yellowish at all OD\u003csub\u003e600\u003c/sub\u003e values while in the samples comprising \u003cem\u003eCampylobacter jejuni\u0026nbsp;\u003c/em\u003eshowed quickly a red to pink color over the whole range of cellular densities tested which gradual increase in intensity from an OD\u003csub\u003e600\u003c/sub\u003e value of 0.1 to an OD\u003csub\u003e600\u003c/sub\u003e value 1.1 (Figure 3C). The results in Figure 3 show a higher absorbance value at all cell densities (indicated as OD\u003csub\u003e600\u003c/sub\u003e value) for \u003cem\u003eC. jejuni\u003c/em\u003e compared to \u003cem\u003eC. coli\u003c/em\u003e showing that the method is also specific and sensitive when using NaIO\u003csub\u003e4\u003c/sub\u003e as oxidizing reagent.\u003c/p\u003e\n\u003cp\u003eWhen comparing the three oxidizing reagents and plotting the endpoint absorbance at 505 nm against the initial cell concentration, a strong correlation was observed. This indicated that the method can be effectively used to estimate the initial cell concentration\u0026nbsp;from a sample (Figure 3D).\u003c/p\u003e\n\u003col start=\"5\"\u003e\n \u003cli\u003e\n \u003ch3\u003eAssay optimization by tuning oxidative agent concentration\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eA wide range of concentration of each oxidant agent has been tested to ensure the use of an optimal concentration and to ensure alternative reagents are available without compromising the assay\u0026rsquo;s performance (Figure 4). Cell suspensions (\u003cem\u003eC. jejuni\u0026nbsp;\u003c/em\u003eDSM 4688, OD\u003csub\u003e600\u003c/sub\u003e = 0.5 to 0.6) in sterile 100 mM phosphate buffer pH 7.4 were incubated with 4-HHA at a final concentration of 10 mM for 70 min at 37\u0026deg;C, followed by the addition of 0.5 mM 4-AAP, 4.5 U/mL HRP and\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eat a concentration varied between 0 and 16 mM. Concentrations of 1 and 2 mM\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e showed the best performance in 15 min with 1 mM giving a faster signal development that reached plateau already after 6 minutes. No coloration was observed, and no signal increase was detected by 505 nm in the cell suspension without oxidants (Figure 4, left panel). Under similar conditions, the enzymatic oxidant-producing system based on glucose oxidase was added using glucose oxidase (GOD) with an activity varied from 0 to 4 U/mL. The enzymatic concentration of 1 U/mL GOD was sufficient for the reaction to develop a sufficient (Figure 4, right panel). No coloration was observed, and no signal increase was detected by 505 nm in the cell suspension without enzyme or oxidative system. The single-oxidant version of the assay was tested with NaIO4and its concentration was varied from 0 to 8 mM. Concentrations of 2 to 4 mM NaIO4 showed the best performance. No coloration was observed, and no signal increase was detected by 505 nm in the cell suspension without NaIO4. Cell suspension without any additives and sterile control were included as negative controls (Figure 4, middle panel).\u003c/p\u003e\n\u003col start=\"6\"\u003e\n \u003cli\u003e\n \u003ch3\u003eValidation with other hippuricase-positive microorganisms: \u003cem\u003eStreptococcus agalactiae\u0026nbsp;\u003c/em\u003e\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe pathogen\u003cem\u003e\u0026nbsp;C. jejuni\u003c/em\u003e enters in contact with humans through contaminated food and water, or companion animals. Aiming at validating the assay with other hippuricase-positive strain, we have tested samples of the \u003cem\u003eStreptococcus agalactiae\u0026nbsp;\u003c/em\u003eATCC\u0026reg;12386 and used \u003cem\u003eStreptococcus pyogenes\u0026nbsp;\u003c/em\u003eATCC\u0026reg;19615, a hippuricase-negative strain,\u003cem\u003e\u0026nbsp;\u003c/em\u003eas an additional negative control.\u003cem\u003e\u0026nbsp;\u003c/em\u003eColonies were suspended in buffer for an optical density OD\u003csub\u003e600\u003c/sub\u003e of approx. 0.5. The test was performed using all the three oxidizing systems (Figure 5). The samples with \u003cem\u003eStreptococcus pyogenes\u0026nbsp;\u003c/em\u003eand the sterile control remained colorless or slightly yellowish while in the samples comprising \u003cem\u003eStreptococcus agalactiae\u0026nbsp;\u003c/em\u003ea vivid pink coloration developed (data not shown).\u003c/p\u003e\n\u003cp\u003eThis example confirmed that all the methods presented are also sensitive and specific for distinguishing hippuricase-positive strain \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e from\u0026nbsp;hippuricase-negative strain \u003cem\u003eStreptococcus pyogenes,\u0026nbsp;\u003c/em\u003ewhich are important etiological factors in human illnesses.\u0026nbsp;\u003c/p\u003e\n\u003col start=\"7\"\u003e\n \u003cli\u003e\n \u003ch3\u003eComparison to commercial ninhydrin-base tests\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eIn order to evaluate the competitiveness and ease of adaptation of the proposed biochemical assay, we proceeded to compare its performance with two commercially available alternatives. To ensure comparability, conditions had to be slightly altered but the manufacturer\u0026rsquo;s instructions were nevertheless followed. The assay performance was compared with two established commercial assays. Biomass of colonies for the here reported assay were dispersed in 0.2 mL sterile 100 mM phosphate buffer pH 7.4 or prepared as instructed by the manual in 0.1 mL sterile water for the Remel assay and in 0.5 mL saline solution for the Millipore assay. To ensure a proper comparability, the optical density OD\u003csub\u003e600\u003c/sub\u003e of all cell suspensions were set to 0.2. In the next step, 25 \u0026micro;L of 100 mM 4-HHA prepared in 100 mM phosphate buffer pH adjusted to 7.4 was added to cell suspensions for the here reported assay. The Hippurate disks and Hippurate strips were dropped in cell suspensions as recommended in the respective manuals. To ensure a proper comparability, all samples were then incubated at 37\u0026deg;C for 1 h. After incubation, the assay was tested in the two variants using the enzymatic systems based on HRP (A) and GOD (B) and a significant pink color was visible by eye in both cases within a few minutes (Figure 6) in sample comprising C\u003cem\u003eampylobacter jejuni\u003c/em\u003e at room temperature; sample comprising \u003cem\u003eCampylobacter coli\u003c/em\u003e or sterile control remained slightly yellowish. In the Remel and Millipore assays, the ninhydrin reagent was added to the samples after an incubation of 30 min; a slightly blue color was visible by eye in sample compromising \u003cem\u003eC. jejuni\u003c/em\u003e using the Remel Assay, and no color formation can be observed in sample compromising \u003cem\u003eC. jejuni\u003c/em\u003e using the Millipore assay.\u003c/p\u003e\n\u003cp\u003eOur method offered a considerable reduction in assay time compared to the Remel Assay, while exhibited a higher sensitivity when compared to the Millipore Assay.\u003c/p\u003e\n\u003col start=\"8\"\u003e\n \u003cli\u003e\n \u003ch3\u003eIdentification of \u003cem\u003eCampylobacter jejuni\u0026nbsp;\u003c/em\u003efrom\u003cem\u003e\u0026nbsp;\u003c/em\u003echicken meat samples\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cem\u003eCampylobacter\u003c/em\u003e species are widely distributed in most warm-blooded animals, as they are a natural part of the intestinal microbiome of poultry\u003csup\u003e18\u003c/sup\u003e, pigs\u003csup\u003e19\u003c/sup\u003e, cattle\u003csup\u003e20\u003c/sup\u003e dogs and cats\u003csup\u003e21\u003c/sup\u003e. Poultry and poultry products are the main source of the human infections and responsible for between 50 and 80 % of all campylobacter infections\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAiming at validating the biochemical assay with commercial meat samples, a first step of strain enrichment step has been carried out in which \u003cem\u003eCampylobacter spp.\u0026nbsp;\u003c/em\u003ewere isolated from 65 locally purchased retail chicken meat samples, i.e. using a charcoal-agar-based (mCCDA) selective for \u003cem\u003eCampylobacter\u003c/em\u003e. This medium was recommended by the ISO Committee under the specification ISO 10272:1995 for selective isolation and differentiation of \u003cem\u003eCampylobacter\u003c/em\u003e species\u003csup\u003e23\u003c/sup\u003e. After 48h incubation at 37\u0026deg;C under microaerobic conditions, agar plates were observed by the naked eye and with a magnifying glass (40x) (Figure 7). Incubation conditions at 37\u0026deg;C rather than 42\u0026deg;C have been indicated to increase the isolation rate of \u003cem\u003eCampylobacter spp.\u003c/em\u003e from foods according to the recommendation by the U.K. Ministry of Agriculture, Fisheries and Food\u003csup\u003e24\u003c/sup\u003e. However, \u003cem\u003eC. jejuni\u003c/em\u003e is able to survive at temperatures from \u0026lt; 4 \u0026deg;C up to 46 \u0026deg;C and this characteristic of \u003cem\u003eC. jejuni\u003c/em\u003e can promote the spreading of infections by meat products especially if they are not properly handled\u003csup\u003e25\u003c/sup\u003e,\u003csup\u003e26\u003c/sup\u003e,\u003csup\u003e27\u003c/sup\u003e. It could be noticed that there were some plates with no bacterial colonies (6 of 65 samples) or with few bacterial colonies (10 of 65 samples), while the other samples presented numerous colonies (Figure S1). In most plates, the characteristic colonies were visibly greyish, flat and moistened, with a metal sheen and tendency to spread. Beside grey colonies, white colonies were also present in some samples (Figure 7, Figure\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e1S) as well as pink colonies.\u003c/p\u003e\n\u003cp\u003eIn a second step, the biochemical assay presented in this article and based on the hydrolysis of hippurate by hippuricase-positive \u003cem\u003eC. jejuni\u003c/em\u003e strains has been used to analyze the enriched bacterial samples. After visual assessment of the color development in comparison to a bacteria-free control sample (Figure 8), 11 samples (16.9 %) of the 65 analyzed samples were recognized as positive, i.e. number 3, 4, 7, 36, 38, 41, 46, 47, 55, 61, 62. Among them, sample number 7 gave the strongest and fastest coloration development resulting in a visual intensity comparable\u0026nbsp;to results obtained for bacterial cultures of the optical density (OD\u003csub\u003e600\u003c/sub\u003e) higher than 0.9 in liquid medium, according to the scale provided. Samples 4 and 36 also gave an intense coloration that was comparable to the one obtained for bacterial cultures in liquid media at an OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003ebetween\u003csub\u003e\u0026nbsp;\u003c/sub\u003e0.5 and 0.9, while the other eight positive samples had intensity of color below OD 0.2 (Figure 3S).\u003c/p\u003e\n\u003cp\u003eOf the six retail shop used, two of them reported all negative samples, whereas the remaining had at least one positive sample (Table 2).\u0026nbsp;Among individual chicken meat cut types, the highest relative number of positive samples was obtained for liver (30.7 %) and backs (20%), while the lowest number was obtained for legs (7.7 %) and breast (7.7 %).\u003c/p\u003e\n\u003col start=\"9\"\u003e\n \u003cli\u003e\n \u003ch3\u003eResults validation using LC‐MS/MS‐based peptide sequencing for \u003cem\u003eCampylobacter jejuni\u0026nbsp;\u003c/em\u003eidentification\u003cem\u003e\u0026nbsp;\u003c/em\u003eat protein sequence level\u003c/h3\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAmong the tested samples (Figure 8), samples 3, 4, 7, and 41, which gave a recognizable coloration and selected negative samples 30a and b, 31a and b, 45, 17 and 63 (a and b are different parts of plate with unalike colonies) were further analyzed by LC-MS/MS for the presence of \u003cem\u003eCampylobacter\u003c/em\u003e-specific proteins. The MS-based analysis of the positive samples revealed the presence of at least seven characteristic of \u003cem\u003eCampylobacter jejuni\u003c/em\u003e (Table 3), including a probable histidine-binding protein (UniProt ID: Q46125), Flagellin B (UniProt ID: P56964), major outer membrane protein (UniProt ID: P806729, Large ribosomal subunit protein uL3 (UniProt ID: Q9PLX1), a Fumarate hydratase class II (UniProt ID: O69294), Succinate-CoA ligase [ADP-forming] subunit beta (UniProt ID: A8FKV6), Threonine-tRNA ligase (UniProt ID: A1VXT5), 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (UniProt ID: Q0P823), cysteine synthase B (UniProt ID: P71128) and a catalase (UniProt ID: Q59296). The detection of proteins involved in motility, nutrient transport, and energy metabolism supports the conclusion that \u003cem\u003eC. jejuni\u003c/em\u003e was present in the samples tested positive with the assay.\u003c/p\u003e\n\u003cp\u003eThe identification of multiple (7 to 9) \u003cem\u003eC. jejuni\u003c/em\u003e-specific proteins, according to UniProtKB of reviewed sequences (SwissProt), provides convincing evidence for the presence of \u003cem\u003eC. jejuni\u003c/em\u003e in the samples. Homology search identified a few unreviewed protein matches with 100% sequence identity from various \u003cem\u003eCampylobacter\u003c/em\u003e species (Table 4). Furthermore, for five of the identified proteins, major outer membrane protein (UniProt ID: P80672), threonine-tRNA ligase (UniProt ID: A1VXT5), cysteine synthase B (UniProt ID: P71128) and catalase (UniProt ID: Q59296), no closely related isoforms with 100% sequence identity from other \u003cem\u003eCampylobacter\u003c/em\u003e species were found among unreviewed sequences (Table 4). \u0026nbsp;Eight samples that tested negative in the biochemical assay, i.e. not developing a pink coloration, were also tested by proteomic workflow described above; only one of them, i.e. sample number 30, that tested negative in the enzymatic assay, showed presence of several \u003cem\u003eC. jejuni\u003c/em\u003e biomarker proteins in the mass spectrometry analysis. The negative hit obtained with the biochemical assay has been confirmed multiple times sampling different areas of the enrichment plate and no coloration has ever developed (Figure 5S).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe global market for Food Pathogen Testing has been forecasted to surpass USD 9.1 billion by 2028 at a CAGR of 8%\u003csup\u003e28\u003c/sup\u003e. One contributing factor is the increasing attention posed on food safety especially in developing nations.\u003c/p\u003e\n\u003cp\u003eCampylobacteriosis is due to twomajor agents, i.e. \u003cem\u003eCampylobacter jejuni\u003c/em\u003e and \u003cem\u003eC. coli,\u003c/em\u003e and most assays aim at their specific detection\u003csup\u003e18\u003c/sup\u003e. Current techniques for the detection of \u003cem\u003eCampylobacter\u003c/em\u003e rely on PCR-based assays or ninhydrin-based assays, both presenting disadvantages such as the need for a dedicated apparatus or the possibility of false positives. A study has also reported hippuricase-specific antibodies that can result in ELISA-like assays for \u003cem\u003eCampylobacter\u003c/em\u003e detection\u003csup\u003e29\u003c/sup\u003e. Here we report a sensitive and specific chromogenic assay exploiting the activity of the \u003cem\u003eCampylobacter\u003c/em\u003e endogenous hippuricase enzyme (Figure 4S). The assay proceeds in two enzymatic steps and results in a read-out that can be detected by absorbance measurement but also visually, offering itself to future adaptation to an immobilized form.\u003c/p\u003e\n\u003cp\u003eThe assay here presented is functional with a low optical density sample, i.e. OD\u003csub\u003e600\u003c/sub\u003e= 0.1, or of just a few bacterial colonies picked from a culture plate with complex media. This suggests adaptability to the testing of liquid samples and environmental samples. The sensitivity of the popular ninhydrin assay for \u003cem\u003eCampylobacter\u003c/em\u003e detection is not clear and prone to provide false positive results. In addition, the assay here presented provides a read-out at a longer wavelength than ninhydrin, e.g. the purple signal at 505 nm vs. the blue signal of ninhydrin.\u003c/p\u003e\n\u003cp\u003eBiochemical chromogenic assays offer the advantages of an easy read-out, often quick, and adaptability to a format that can applied easily in situations of emergency and in low-technology settings. Previously reported chromogenic assays have used a purified hippuricase enzyme; Kasahara and colleagues developed in 1981 an assay targeting the activity of angiotensin-I converting enzyme\u003csup\u003e30\u003c/sup\u003e whereas Saruta and colleagues developed a method for determining the activity of carboxypeptidase A\u003csup\u003e31\u003c/sup\u003e. The Campylobacter assay presented here has multiple competitive advantages such as no need for an extended incubation at 37\u0026deg;C for color development, a significant shorter assay time, and the absence of corrosive organic solutions. The assay can take place in two main steps in which (1) 4-HHA is added, and incubation is carried out, and then (2) 4-AAP and the oxidizing system is added. The read-out is fast, and the signal should be monitored over time. Moreover, it is suitable for samples in complex protein-rich media with no possibility of false positives, in contrast to ninhydrin-based assays. Additionally, this novel assay is water based, making it a more environmentally sustainable alternative to the commercially available ninhydrin-based method. As seen in this work, it also allows easy visual detection of positive samples that were later confirmed by MS analytics. Whereas visual detection might not be a feasible or reliable due to conditions of color-blindness for example, the measuring of UV-Vis absorbance after sample centrifugation, could help in the screening for positive samples.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCampylobacter\u003c/em\u003e in poultry is a leading foodborne pathogen that causes human gastroenteritis with a risk of development postinfection health issues which can negatively impact the economy\u003csup\u003e32\u003c/sup\u003e. It was found that poultry consumption was the main cause of campylobacteriosis outbreaks between 2007 and 2013 worldwide\u003csup\u003e33\u003c/sup\u003e. In the USA between 2000 and 2016, 28 campylobacteriosis outbreaks were recorded, and the consumption of chicken livers was the main cause\u003csup\u003e34\u003c/sup\u003e. In this study it was found that chicken liver was the most contaminated chicken retail product with 30.7% \u003cem\u003eC. jejuni\u003c/em\u003e-positive incidence of the analyzed samples. Poultry farms and processing plants are main sources of chicken contamination by \u003cem\u003eCampylobacter\u003csup\u003e35\u003c/sup\u003e\u003c/em\u003e. Colonization of \u003cem\u003eCampylobacter\u003c/em\u003e in farm chickens occurs usually due to horizontal transmission from other positive chickens or \u003cem\u003evia\u003c/em\u003e drinking water and animal feed. Additionally, farmers, who carry \u003cem\u003eCampylobacter\u003c/em\u003e, feces of wild birds colonized by \u003cem\u003eCampylobacter\u003c/em\u003e, flies, insects, amoebae, yeasts and molds were found to be also important routes of horizontal transmission of \u003cem\u003eCampylobacter\u003c/em\u003e in farms\u003csup\u003e39\u003c/sup\u003e. \u003cem\u003eC. jejuni\u003c/em\u003e is a common commensal microorganism in the chicken microbiome\u003csup\u003e36\u003c/sup\u003e,\u003csup\u003e37\u003c/sup\u003e. Besides, other \u003cem\u003eCampylobacter\u0026nbsp;\u003c/em\u003especies such as \u003cem\u003eC. lari\u003c/em\u003e, \u003cem\u003eC. upsaliensis\u003c/em\u003e, \u003cem\u003eC. coli\u003c/em\u003e and \u003cem\u003eC. concisus\u003c/em\u003e can be also isolated from chicken intestines due to horizontal transmission from different sources to chickens\u003csup\u003e35\u003c/sup\u003e\u003csup\u003e,38\u003c/sup\u003e. Once \u003cem\u003eCampylobacter\u0026nbsp;\u003c/em\u003eenters the chicken flock, it spreads rapidly and colonizes the intestinal tracts of most chickens within one week\u003csup\u003e35\u003c/sup\u003e,\u003csup\u003e39\u003c/sup\u003e\u0026nbsp; and can reach the level of 109 cells per g of intestinal tract content\u003csup\u003e40\u003c/sup\u003e. In this study, samples have been sourced from 6 different shops which were supplied by different farms (from farms with different domestic animals in addition to commercial poultry farms), and possibly contaminated with different \u003cem\u003eCampylobacter\u003c/em\u003e \u003cem\u003espp\u003c/em\u003e. During the enrichment for \u003cem\u003eCampylobacter spp\u003c/em\u003e. from chicken meat samples, different types of colonies were growing on the plates with selective agar suggesting that different species were present. At least three different types of colonies grey, white and pink were identified by color.\u003c/p\u003e\n\u003cp\u003eMany studies confirmed that the number of \u003cem\u003eCampylobacter\u003c/em\u003e-positive chicken carcasses significantly increased during poultry processing\u003csup\u003e41\u003c/sup\u003e,\u003csup\u003e42\u003c/sup\u003e,\u003csup\u003e43\u003c/sup\u003e. Leaking of \u003cem\u003eCampylobacter\u003c/em\u003e from the gut during evisceration is the most critical point during processing. Bacteria from the gut can contaminate the lower half of the carcasses (breast, neck and wings) more often than the upper half (thighs and drumstick) as the birds are always hanged upside-down by the feet\u003csup\u003e44\u003c/sup\u003e. It has been reported that the number of chicken thighs and breasts which were \u003cem\u003eCampylobacter\u003c/em\u003e-positive increased from 0% to 90% after evisceration\u003csup\u003e45\u003c/sup\u003e. This study also confirmed that legs (thighs with drumsticks) have the lowest number of positive samples. Contrary to the other studies\u003csup\u003e46\u003c/sup\u003e,\u003csup\u003e47\u003c/sup\u003e, we found very low contamination with \u003cem\u003eC. jejuni\u003c/em\u003e of chicken breast (7.7%). This can be explained by the fact that the breast we purchased in this study were skinless and cut into smaller pieces which were not in contact with bacteria. Heating of poultry carcasses followed by chilling during the processing steps are essential in assisting practices for effective defeathering. During heating, the skin follicles remain open, and this allows bacteria to penetrate the skin and accumulate inside the follicles. This can be the reason why almost the same percentage of positive samples was found in wings and backs samples, but lower in breast.\u003c/p\u003e\n\u003cp\u003eIn order to validate the novel biochemical assay, a mass-spectrometry-based analysis of selected positive samples has been carried out and key proteins specific of \u003cem\u003eC. jejuni\u003c/em\u003e have been identified confirming the presence of the pathogen. Among the seven negative samples, MS analytics detected \u003cem\u003eC. jejuni\u003c/em\u003e proteins in one sample, sample number 30, and this can suggest that the sample has indeed been contaminated but that the strain was probably not metabolically active, especially in regards to hippuricase activity. Such a discrepancy between the biochemical hippuricase-based assay and MS analytics could be explained by the different working principle and by the presence of inactive C. jejuni proteome components in the samples, with belongs to not-metabolically active \u003cem\u003eC. jejuni\u003c/em\u003e cells, and thus do not react in the chromogenic assay.\u003c/p\u003e\n\u003cp\u003eConcluding, the novel biochemical chromogenic assay is effective for identifying \u003cem\u003eC. jejuni\u003c/em\u003e and other hippuricase-positive bacteria, can be used with various oxidants, and outperforms current commercial products. Its specificity was confirmed by analyzing commercial poultry samples, with results validated using nano-liquid chromatography mass spectrometry. Additionally, the water-based nature of the assay offers a more sustainable alternative to the standard ninhydrin-based commercial assays.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003eChemicals\u003c/h3\u003e\n\u003cp\u003e4-hydroxyhippuric acid (4-HHA, Bachem 4005059), 4-hydroxybenzoic acid (4-HBA, Merck H20059), 4-aminoantipyridine (4-AAP, Merck 06800), horseradish peroxidase (HRP, Merck 77332)\u003cs\u003e,\u003c/s\u003e glucose oxidase (GOD, Merck G2133), sodium periodate (NaIO\u003csub\u003e4\u003c/sub\u003e, Biosynth FS04514), glucose (Merck, G8270).\u003c/p\u003e\n\u003ch3\u003eBacteria cultivation conditions\u003c/h3\u003e\n\u003cp\u003eStrains used include \u003cem\u003eCampylobacter jejuni\u003c/em\u003e DSM 4688 (\u003cem\u003eC. jejuni.\u003c/em\u003e), \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) and \u003cem\u003eCampylobacter coli\u003c/em\u003e DSM 4689 (\u003cem\u003eC. coli.\u003c/em\u003e), \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e ATCC\u0026reg;12386 (\u003cem\u003eS. agalactiae\u003c/em\u003e) and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e ATCC\u0026reg;19615 (\u003cem\u003eS. pyogenes.\u003c/em\u003e), that were cultivated on Columbia Agar with sheep blood (Thermofischer PB5039A) at 37\u0026deg;C for 48 h. Both Campylobacter strains were cultivated under microaerophilic atmosphere (Oxoid, Campylgen CN0025A); to obtain colonies and biomass for testing. \u003cem\u003eStreptococcus agalactiae\u0026nbsp;\u003c/em\u003eATCC\u0026reg;12386 and \u003cem\u003eStreptococcus pyogenes\u0026nbsp;\u003c/em\u003eATCC\u0026reg;19615\u003cem\u003e\u0026nbsp;\u003c/em\u003ewere cultivated on Columbia Agar with sheep blood (Thermofischer PB5039A) at 37\u0026deg;C for 48 h.\u003c/p\u003e\n\u003ch3\u003eSample preparation\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eA small sample of a loopful (2-3) colonies have been picked and\u0026nbsp;suspended in sterile 100 mM phosphate buffer pH 7.4 and the optical density OD\u003csub\u003e600\u003c/sub\u003e was set to 0.9, unless otherwise mentioned, and then serially diluted to achieve the desired initial optical density with sterile 100 mM phosphate buffer pH 7.4.\u003c/p\u003e\n\u003ch3\u003eTesting of the oxidative agent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand HRP\u003c/h3\u003e\n\u003cp\u003eA bacterial sample\u0026nbsp;with an OD\u003csub\u003e600\u003c/sub\u003e of 0.9 has been serially diluted with buffer to an OD\u003csub\u003e600\u003c/sub\u003e of 0.5, 0.2 or 0.1. Two hundred \u0026micro;L of cell suspensions were then added of 25 \u0026micro;L of 100 mM 4-HHA prepared in 100 mM phosphate buffer with pH 7.4 in a 96-well plate and incubated for 1 h at 37\u0026deg;C. After incubation, 20 \u0026micro;L of the prepared mixture (12.5X) containing 4-AAP and HRP, 5 \u0026micro;L of 100 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added. The absorbance at 505 nm was recorded using a SpectraMax M5 plate reader (Molecular Devices) at room temperature. Final concentrations for the assay were 10 mM 4-HHA, 0.5 mM 4-AAP, 4.5 U/mL HRP and 2 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eTesting of the oxidative agent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e produced by glucose and glucose oxidase\u003c/h3\u003e\n\u003cp\u003eA sample containing\u003cem\u003e\u0026nbsp;Campylobacter jejuni\u003c/em\u003e or \u003cem\u003eCampylobacter coli\u003c/em\u003e biomass with an OD\u003csub\u003e600\u003c/sub\u003e of 0.9 was serially diluted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.5, 0.2 and 0.1. Two hundred \u0026micro;L of cell suspension were mixed with 25 \u0026micro;L of 100 mM 4-HHA (dissolved in 100 mM phosphate buffer pH 7.4) in microtiter plate and incubated for 1 h at 37\u0026deg;C. After incubation, 10 \u0026micro;L of the prepared mixture (25X) 4-AAP and HRP, 10 \u0026micro;L of 250 mM glucose and 5 \u0026micro;L of GOD 50 U/mL were added. The absorbance at 505 nm was recorded with a SpectraMax M5 plate reader (Molecular Devices). Final concentration of 4-HHA was 10 mM, 4-AAP was 0.5 mM, HRP was 4.5 U/mL, glucose was 10 mM and GOD was 1 U/mL.\u003c/p\u003e\n\u003ch3\u003eTesting of the oxidative agent sodium periodate\u003c/h3\u003e\n\u003cp\u003eTwo hundred \u0026micro;L of prepared sample were mixed with 25 \u0026micro;L of 100 mM 4-hydroxyhippuric acid (prepared in 100 mM phosphate buffer pH adjusted to 7.4) in a microtiter plate and incubated for 1 h at 37\u0026deg;C. After incubation, 5 \u0026micro;L of 100 mM 4-AAP and 10 \u0026micro;L of 50 mM sodium periodate (NaIO\u003csub\u003e4\u003c/sub\u003e) were added. The absorbance at 505 nm was recorded with a SpectraMax M5 plate reader (Molecular Devices) at room temperature. Final concentrations for the assay are 10 mM 4-HHA, 2 mM 4-AAP and 2 mM NaIO\u003csub\u003e4.\u003c/sub\u003e\u003c/p\u003e\n\u003ch3\u003eValidation with other hippuricase-positive bacterial strains\u003c/h3\u003e\n\u003cp\u003eSimilarly to the method for the detection of \u003cem\u003eCampylobacter\u003c/em\u003e, biomass of colonies were suspended in sterile 100 mM phosphate buffer pH 7.4 and the optical density OD600 was set to 0.5 to 0.6. Two hundred \u0026micro;L of prepared sample have been mixed with 25 \u0026micro;L of 100 mM 4-HHA prepared in 100 mM phosphate buffer pH adjusted to 7.4 in a microtiter plate and incubated for 80 min at 37\u0026deg;C. In accordance with the present method, the oxidative agents were added after this step.\u003c/p\u003e\n\u003ch3\u003eComparison to ninhydrin-based hippurate tests\u003c/h3\u003e\n\u003cp\u003eTests Hippurate disk (Remel, Thermo Scientific, R21085) and Hippurate strips kit (Millipore 01869) were purchased and used as described by the manufacturer. However, to ensure a proper comparability, the optical density OD\u003csub\u003e600\u003c/sub\u003e of all cell suspensions were set to 0.2 and all samples have been incubated at 37\u0026deg;C for 1 h.\u003c/p\u003e\n\u003ch3\u003eIsolation of \u003cem\u003eCampylobacter spp.\u003c/em\u003e from chicken meat samples\u003c/h3\u003e\n\u003cp\u003eChicken cuts (breast without skin, and wings, backs and legs with skin) (n=52) and liver (n=13) were purchased from six different retail food stores in Belgrade, Serbia, with each store having its own supplier. All chicken meat samples were fresh. Individual chicken cuts such as breast, legs, liver, wings were kept in the metal vessels in the fridges in the stores. In some stores, a single breast was already cut in smaller pieces, while in others it was kept as a whole. To prevent crosscontamiantion of the samples, these were taken from the container in the shop and placed in a plastic bag (one sample per bag) as the first step at purchasing. The cross-contamination of the samples by bacteria was thus only possible in the store during preparation of chicken cuts or keeping in the containers. During the taking the swabs of the samples, the samples were not taken out of the bag and immediately spread onto the surface of charcoal, cefoperazone, desoxycholate Campylobacter selective agar (mCCDA) base (Millipore\u0026reg;, Sigma-Aldrich, Germany) supplemented with blood free Campylobacter medium selective supplement (Millipore\u0026reg;, Sigma-Aldrich, Germany). \u0026nbsp;Plates (D=55 mm) were incubated at 37 \u0026deg;C for 48 h under microaerophilic conditions (85 % N\u003csub\u003e2\u003c/sub\u003e, 10 % CO\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e; using anaerobic atmosphere generation bags (Sigma-Aldrich, Germany).\u003c/p\u003e\n\u003ch3\u003eEnzyme-based colorimetric test for the detection of \u003cem\u003eCampylobacter jejuni\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eA rapid colorimetric test (Biosyth\u0026reg;, United Kingdom) was used for the confirmation of \u003cem\u003eC. jejuni\u003c/em\u003e in chicken samples. A newly developed assay was based on detection of the enzymatic hydrolysis of hippurate by hippuricase positive \u003cem\u003eC. jejuni\u003c/em\u003e strains. Briefly, 200 \u0026mu;L of component A was inoculated with a loopful of colonies from an overnight culture (48 h, 37\u0026deg;C). After homogenisation by vortexing, 25 \u0026mu;L of component B was added into cell suspension, mixed well and incubated at 37\u0026deg;C for 90 minutes. In the last step, 20 \u0026mu;L of component C was firstly added, mixed well and then 5 \u0026mu;L of component D. After mixing, \u0026nbsp;the development of orange to pink color was observed over the next 5 to 15 minutes at room temperature.\u003c/p\u003e\n\u003ch3\u003eProtein identification using nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS)\u003c/h3\u003e\n\u003cp\u003eNano-liquid chromatography mass spectrometry (nLC-MS/MS) technique was used for the verification of specificity a rapid colorimetric test for the detection of the enzymatic hydrolysis of hippurate by hippuricase positive \u003cem\u003eC. jejuni\u003c/em\u003e strains.\u003c/p\u003e\n\u003ch4\u003eSample preparation\u003c/h4\u003e\n\u003cp\u003eImmediately after colorimetric assay, four positive and eight negative samples were taken for further analysis. Briefly, remaining bacteria from the surface of agar were spread onto a new agar plate (D=55 mm), incubated 24 h under the same conditions. After that, individual colonies were transferred onto a new agar plate (D=100 mm) and incubated 48 h at 37\u0026deg;C under \u0026nbsp;microaerobic conditions. Individual colonies or clusters of colonies (2 or 3 per positive sample and 1 per negative) were transferred into 100 \u0026mu;L of 6M urea or 100 \u0026mu;L of buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)(5 times concentrated). The cells transferred in 6M urea were first vigorously mixed and then frozen. The procedure was repeated two times to increase lysis of cells. The cells transferred in the buffer were first vigorously mixed then heated for 10 min at 95\u0026deg;C and frozen. After thawing, the samples were again mixed and heated for 10 min at 95\u0026deg;C prior to SDS-PAGE. SDS-PAGE was performed at 12% gel according to the manufacturer\u0026rsquo;s recommendations, using a Bio-Rad Mini-Protean electrophoretic unit (Bio-Rad, California, USA).\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eTrypsin digestion\u003c/h4\u003e\n\u003cp\u003eCell lysates were loaded onto a 12% SDS-PAGE gel and run just enough for the proteins to migrate approximately 0.5-1 cm into the separation gel, concentrating them into a single dense band. This step was performed to partially purify the samples and remove small molecule contaminants without full protein size separation. The whole concentrated protein regions were carefully excised and subjected to standard procedure of trypsin digestion (57). Briefly, the gel bands were first washed with 25 mM ammonium bicarbonate and 25 mM ammonium bicarbonate in 50% acetonitrile for 60 and 45 minutes, respectively, followed by reduction with 10 mM dithiothreitol at 57 \u0026deg;C in the dark for 60 minutes and alkylation with 55 mM iodoacetamide at room temperature for 45 minutes, also in the dark. Subsequently, the samples underwent a second series of washes with 25 mM ammonium bicarbonate, 25 mM ammonium bicarbonate in 50% acetonitrile, and 100% acetonitrile for 30, 15, and 5 minutes, respectively. After washing, the proteins were digested with trypsin at a protein-to-enzyme ratio of 30:1, at 37 \u0026deg;C for 18 hours. Digestion was stopped by the addition of 10% formic acid to achieve a final formic acid concentration of 1%. Peptides were then cleaned using Peptide Cleanup Pipette Tips (Agilent Technologies) and concentrated in a SpeedVac vacuum concentrator. The dried peptides were reconstituted in 0.1% formic acid and transferred to autosampler vials for LC-MS/MS analysis.\u003c/p\u003e\n\u003ch4\u003e\u0026nbsp;Peptides separation\u003c/h4\u003e\n\u003cp\u003ePeptides from in-gel digestion, were chromatographically separated using an UltiMate\u0026trade; 3000 RSLC nano liquid chromatographic system \u0026nbsp; (Thermo Scientific Inc., Bremen, Germany) and \u0026nbsp;2-column set up: a trap column C18, 50mm (P/N 160454 Thermo Fisher Scientific) and analytical column PepMap C18, 15 cm \u0026times; 75 \u0026mu;m, 3 \u0026mu;m particles, and 100 \u0026Aring; pore size (ES800A, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phases were MS-grade (A) water with 0.1 % formic acid and (B) acetonitrile with 0.1 % formic acid. The gradient program was as follows: 0\u0026ndash;0.5 min 95 % A, 0.5\u0026ndash;10 min 95-66 % A, 10\u0026ndash;15 min -66-1 % A, 15\u0026ndash;20 min 1% A, 20\u0026ndash;23 min 95 % A, with flow rate of 0.25 \u0026mu;L/min. Injection volume was 10 \u0026mu;L. This nLC system was coupled with Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific Inc., Bremen, Germany) equipped with a heated electrospray ionization source. Analysis was performed in positive ion mode. Parameters of the ion source was as follows: spray voltage 1.9 kV, capillary temperature 300 \u0026deg;C, range 300\u0026ndash;3000\u0026thinsp;m/z, resolving power 60 000, 1 micro scan was acquired using Xcalibur (version 4.4) software (Thermo Fisher Scientific) with the precursor mass tolerance of 10\u0026thinsp;ppm.\u003c/p\u003e\n\u003ch4\u003ePeptides identification\u003c/h4\u003e\n\u003cp\u003eThe identification of proteins was performed by PEAKS Suite 12.5 (Bioinformatics Solutions Inc., Canada). Signature MS/MS spectra were searched using the PEAKS database (DB), and post-translational modification (PTM) algorithms against a database consisting of UniProtKB swiss prot validated sequences and the Max Quant contaminant database. In the PEAKS DB algorithm, the following modifications were considered as variables: oxidation (Met), deamidation (Gln, Asn), and hydroxylation (Pro), while carbamidomethylation (Cys) was set as a fixed modification. Up to two missed trypsin cleavages were allowed per peptide. A semi-specific mode of trypsin cleavage was chosen enabling tryptic and semi-tryptic, peptide detection. Mass tolerances were set to 10 ppm for parent ions and 0.02 Da for fragment ions. Protein filters were set to protein -10 log P\u0026ge;20, proteins\u0026rsquo; unique peptides \u0026ge;1, and an ion intensity \u0026nbsp;for confident PTM identification of at least 2%. The peptide filter was set to the false discovery rate \u0026lt;0.1% (35).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eTrypsin digestion, nLCMS/MS peptides separation and identification\u003c/h3\u003e\n\u003ch4\u003eTrypsin digestion\u003c/h4\u003e\n\u003cp\u003eCell lysates were loaded onto a 12% SDS-PAGE gel and run just enough for the proteins to migrate approximately 0.5-1 cm into the separation gel, concentrating them into a single dense band. This step was performed to partially purify the samples and remove small molecule contaminants without full protein size separation. The whole concentrated protein regions were carefully excised and subjected to standard procedure of trypsin digestion (57). Briefly, the gel bands were first washed with 25 mM ammonium bicarbonate and 25 mM ammonium bicarbonate in 50% acetonitrile for 60 and 45 minutes, respectively, followed by reduction with 10 mM dithiothreitol at 57 \u0026deg;C in the dark for 60 minutes and alkylation with 55 mM iodoacetamide at room temperature for 45 minutes, also in the dark. Subsequently, the samples underwent a second series of washes with 25 mM ammonium bicarbonate, 25 mM ammonium bicarbonate in 50% acetonitrile, and 100% acetonitrile for 30, 15, and 5 minutes, respectively. After washing, the proteins were digested with trypsin at a protein-to-enzyme ratio of 30:1, at 37 \u0026deg;C for 18 hours. Digestion was stopped by the addition of 10% formic acid to achieve a final formic acid concentration of 1%. Peptides were then cleaned using Peptide Cleanup Pipette Tips (Agilent Technologies) and concentrated in a SpeedVac vacuum concentrator. The dried peptides were reconstituted in 0.1% formic acid and transferred to autosampler vials for LC-MS/MS analysis.\u003c/p\u003e\n\u003ch4\u003e\u0026nbsp;Peptides separation\u003c/h4\u003e\n\u003cp\u003ePeptides from in-gel digestion, were chromatographically separated using an UltiMate\u0026trade; 3000 RSLC nano liquid chromatographic system \u0026nbsp;(Thermo Scientific Inc., Bremen, Germany) and \u0026nbsp;2-column set up: a trap column C18, 50mm (P/N 160454 Thermo Fisher Scientific) and analytical column PepMap C18, 15 cm \u0026times; 75 \u0026mu;m, 3 \u0026mu;m particles, and 100 \u0026Aring; pore size (ES800A, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phases were MS-grade (A) water with 0.1 % formic acid and (B) acetonitrile with 0.1 % formic acid. The gradient program was as follows: 0\u0026ndash;0.5 min 95 % A, 0.5\u0026ndash;10 min 95-66 % A, 10\u0026ndash;15 min -66-1 % A, 15\u0026ndash;20 min 1% A, 20\u0026ndash;23 min 95 % A, with flow rate of 0.25 \u0026mu;L/min. Injection volume was 10 \u0026mu;L. This nLC system was coupled with Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific Inc., Bremen, Germany) equipped with a heated electrospray ionization source. Analysis was performed in positive ion mode. Parameters of the ion source was as follows: spray voltage 1.9 kV, capillary temperature 300 \u0026deg;C, range 300\u0026ndash;3000\u0026thinsp;m/z, resolving power 60 000, 1 micro scan was acquired using Xcalibur (version 4.4) software (Thermo Fisher Scientific) with the precursor mass tolerance of 10\u0026thinsp;ppm.\u003c/p\u003e\n\u003ch4\u003ePeptides identification\u003c/h4\u003e\n\u003cp\u003eThe identification of proteins was performed by PEAKS Suite 12.5 (Bioinformatics Solutions Inc., Canada). Signature MS/MS spectra were searched using the PEAKS database (DB), and post-translational modification (PTM) algorithms against a database consisting of UniProtKB Swiss Prot validated sequences and the Max Quant contaminant database. In the PEAKS DB algorithm, the following modifications were considered as variables: oxidation (Met), deamidation (Gln, Asn), and hydroxylation (Pro), while carbamidomethylation (Cys) was set as a fixed modification. Up to two missed trypsin cleavages were allowed per peptide. A semi-specific mode of trypsin cleavage was chosen enabling tryptic and semi-tryptic, peptide detection. Mass tolerances were set to 10 ppm for parent ions and 0.02 Da for fragment ions. Protein filters were set to protein -10 log P\u0026ge;20, proteins\u0026rsquo; unique peptides \u0026ge;1, and an ion intensity for confident PTM identification of at least 2%. The peptide filter was set to the false discovery rate \u0026lt;0.1% (35).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e4-hydroxyhippuric acid, 4-HHA;\u003cem\u003e\u003cs\u003e\u0026nbsp;\u003c/s\u003e\u003c/em\u003e4-HBA,\u003cem\u003e\u0026nbsp;\u003c/em\u003e4-hydroxybenzoic acid; 4-AAP, 4-aminoantipyridine; GOD, glucose oxidase; HRP, horseradish peroxidase; HRMS, High-resolution mass spectrometry; mCCDA, Modified Charcoal Cefoperazone Deoxycholate Agar; PTM, Post-translational modification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eY.C., F.G., A.S. and S.U. are employed by Biosynth AG, a corporation that markets most of the enzymes and chemicals used in this protocol. Biosynth AG has provided financial support in the form of authors\u0026rsquo; salaries and research materials. The assay presented is commercially available on the company website (www.biosynth.com). The assay subject of this work is the subject of International Patent Application PCT/EP2023/060481. This does not alter our adherence to the journal\u0026rsquo;s policies on sharing data and materials. No further conflicts of interest are declared. University of Belgrade-Faculty of Chemistry has carried out the testing independently and Biosynth AG partially covered material and shipping costs.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNo external funding has been received.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eC.Y. curated the conceptualization, data analysis, investigation, review, and editing; A.S. performed the experimental work and data analysis; G.F. curated the writing, review, and editing; U.S. curated the conceptualization, VJ, TV, and TCV carried out the validation of the assay with chicken samples, mass spectrometry analysis and bioinformatics.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO, The Global View of Campylobacterioisis. https://iris.who.int/bitstream/handle/10665/80751/9789241564601_eng.pdf (2012).\u003c/li\u003e\n\u003cli\u003eCenter of Disease Control and Prevention. https://www.cdc.gov/campylobacter/technical.html (2019).\u003c/li\u003e\n\u003cli\u003eFinsterer, J. 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The interplay between Campylobacter and Helicobacter species and other gastrointestinal microbiota of commercial broiler chickens. Gut Pathog 6, 18 (2014). https://doi.org/10.1186/1757-4749-6-18\u003c/li\u003e\n\u003cli\u003eInternational Organization for Standardization (ISO), (1995). Draft ISO//dIS 10272.\u003c/li\u003e\n\u003cli\u003eMAFF Validated Methods for the Analysis of Foodstuffs: Method for the detection of thermotolerant Campylobacter in Foods (v30) J. Assoc. Publ. Analysts 29. 253-262 (1993)\u003c/li\u003e\n\u003cli\u003eKonkel ME, Kim BJ, Klena JD, Young CR, Ziprin R. Characterization of the thermal stress response of Campylobacter jejuni. Infect Immun. 1998;66:3666\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eRiedel C, F\u0026ouml;rstner KU, P\u0026uuml;ning C, Alter T, Sharma CM, G\u0026ouml;lz G. Differences in the Transcriptomic Response of Campylobacter coli and Campylobacter lari to heat stress. Front Microbiol. 2020;11\u003c/li\u003e\n\u003cli\u003eBhaduri S, Cottrell B. Survival of Cold-stressed Campylobacter jejuni on Ground Chicken and Chicken skin during Frozen Storage. Appl Environ Microbiol. 2004;70:7103\u0026ndash;9\u003c/li\u003e\n\u003cli\u003eGLOBE NEWSWIRE, Facts \u0026amp; Factors, Food Pathogen Testing Industry Trends, Share, Price, Growth, Analysis \u0026amp; Forecast Report https://www.globenewswire.com/news-release/2023/01/17/2590016/0/en/Demand-for-Global-Food-Pathogen-Testing-Market-Size-to-Surpass-USD-9-1-Billion-by-2028-Exhibit-a-CAGR-of-8-Food-Pathogen-Testing-Industry-Trends-Share-Price-Growth-Analysis-Forecas.html (2023). Accessed on 11.01.2024.\u003c/li\u003e\n\u003cli\u003eSteele, M., Gyles, C., Chan, V. L., Odumeru, J. Monoclonal antibodies specific for hippurate hydrolase of Campylobacter jejuni. J. Clin. Microbiol. 40, 1080\u0026ndash;1082; doi: 10.1128/jcm.40.3.1080-1082 (2002).\u003c/li\u003e\n\u003cli\u003eKasahara, Y., Ashihara, Y. Colorimetry of angiotensin-I converting enzyme activity in serum. Clin. Chem. 27, 1922-5 (1981).\u003c/li\u003e\n\u003cli\u003eSaruta, H., Ashihara, Y., Sugiyama, M., Roth, M., Miyagawa, E., Kido, Y., Kasahara, Y. Colorimetric determination of carboxypeptidase A activity in serum. Clin. Chem. 32, 748-51 (1986).\u003c/li\u003e\n\u003cli\u003eBatz, M. B., Hoffmann, S., and Morris Jr., J. G. (2012). Ranking the disease burden of 14 pathogens in food sources in the United States using attribution data from outbreak investigations and expert elicitation. J. Food Protect. 75, 1278\u0026ndash;1291. doi: 10.4315/0362-028X.JFP-11-418.\u003c/li\u003e\n\u003cli\u003eKaakoush, N. O., Casta\u0026ntilde;o-Rodrı́guez, N., Mitchell, H. M., and Man, S. M. (2015). Global epidemiology of Campylobacter infection. Clin. Microbiol. Rev. 28, 687\u0026ndash;720. doi: 10.1128/CMR.00006-15.\u003c/li\u003e\n\u003cli\u003eLanier, W. A., Hale, K. R., Geissler, A. L., and Dewey-Mattia, D. (2018). Chicken liver\u0026ndash;associated outbreaks of campylobacteriosis and salmonellosis, United State\u0026ndash;2016: identifying opportunities for prevention. Foodborne Pathog. Disease 15, 726\u0026ndash;733. doi: 10.1089/fpd.2018.2489.\u003c/li\u003e\n\u003cli\u003eHakeem MJ and Lu X (2021) Survival and Control of Campylobacter in Poultry Production Environment. Front. Cell. Infect. Microbiol. 10:615049.\u003c/li\u003e\n\u003cli\u003eAwad, W. A., Mann, E., Dzieciol, M., Hess, C., Schmitz-Esser, S., Wagner, M., et al. (2016). Age-related differences in the luminal and mucosa-associated gut microbiome of broiler chickens and shifts associated with Campylobacter jejuni infection. Front. Cell. Infect. Microbiol. 6:3389/fcimb.2016.00154:154. doi: 10.3389/fcimb.2016.00154\u003c/li\u003e\n\u003cli\u003eIjaz, U. Z., Sivaloganathan, L., McKenna, A., Richmond, A., Kelly, C., Linton, M., et al. (2018). Comprehensive longitudinal microbiome analysis of the chicken cecum reveals a shift from competitive to environmental drivers and a window of opportunity for Campylobacter. Front. Microbiol. 9:3389/fmicb.2018.02452: 2452. doi: 10.3389/fmicb.2018.02452\u003c/li\u003e\n\u003cli\u003eSahin, O., Morishita, T. Y., and Zhang, Q. (2002). Campylobacter colonization in poultry: sources of infection and modes of transmission. Anim. Health Res. Rev. 3, 95\u0026ndash;105. doi: 10.1079/AHRR200244\u003c/li\u003e\n\u003cli\u003eNewell, D. G., Elvers, K. T., Dopfer, D., Hansson, I., Jones, P., James, S., et al. (2011). Biosecurity-based interventions and strategies to reduce Campylobacter spp. on poultry farms. Appl. Environ. Microbiol. 77, 8605\u0026ndash;8614. doi: 10.1128/ AEM.01090-10.\u003c/li\u003e\n\u003cli\u003eStern, N. J., and Robach, M. C. (2003). Enumeration of Campylobacter spp. in broiler feces and in corresponding processed carcasses. J. Food Protect. 66, 1557\u0026ndash;1563. doi: 10.4315/0362-028X-66.9.1557\u003c/li\u003e\n\u003cli\u003eHakeem MJ and Lu X (2021) Survival and Control of Campylobacter in Poultry Production Environment. Front. Cell. Infect. Microbiol. 10:615049\u003c/li\u003e\n\u003cli\u003eNorthcutt, J. K., Berrang, M. E., Dickens, J. A., Fletcher, D. L., and Cox, N. A. (2003). Effect of broiler age, feed withdrawal, and transportation on levels of coliforms, Campylobacter, Escherichia coli and Salmonella on carcasses before and after immersion chilling. Poultry Sci. 82, 169\u0026ndash;173. doi: 10.1093/ps/82.1.169\u003c/li\u003e\n\u003cli\u003eKeener, K. M., Bashor, M. P., Curtis, P. A., Sheldon, B. W., and Kathariou, S. (2004). Comprehensive review of Campylobacter and poultry processing. Compr. Rev. Food Sci. Food Safety 3, 105\u0026ndash;116. doi: 10.1111/j.1541-4337.2004.tb00060.x\u003c/li\u003e\n\u003cli\u003eKotula, K. L., and Pandya, Y. (1995). Bacterial profile of broiler chickens upon entering the processing plant. J. Food Protect. 58, 1326\u0026ndash;1329. doi: 10.4315/0362-028X-58.12.1326\u003c/li\u003e\n\u003cli\u003eBerrang, M. E., Ladely, S. R., and Buhr, R. J. (2001). Presence and level of Campylobacter, coliforms, Escherichia coli, and total aerobic bacteria recovered from broiler parts with and without skin. J. Food Protect. 64, 184\u0026ndash;188. doi: 10.4315/0362-028x-64.2.184\u003c/li\u003e\n\u003cli\u003eJorgensen, F., Bailey, R., Williams, S., Henderson, P., Wareing, D. R., Bolton, F. J., et al. (2002). Prevalence and numbers of Salmonella and Campylobacter spp. on raw, whole chickens in relation to sampling methods. Int. J. Food Microbiol. 76, 151\u0026ndash;164. doi: 10.1016/S0168-1605(02)00027-2\u003c/li\u003e\n\u003cli\u003eWalker, L. J., Wallace, R. L., Smith, J. J., Graham, T., Saputra, T., Symes, S., et al. (2019). 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Food Protect. 82, 2126\u0026ndash;2134. doi: 10.4315/0362-028X.JFP-19-146\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1-Overview of bacterial strains used in this study and info on their reported production of hippuricase or catalase.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCatalase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHippuricase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter jejuni\u003c/em\u003e DSM 4688\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter coli\u0026nbsp;\u003c/em\u003eDSM 4689\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus agalactiae\u003c/em\u003e ATCC\u0026reg;12386\u0026trade;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus pyogenes\u003c/em\u003e ATCC\u0026reg;19615\u0026trade;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2\u003cstrong\u003e\u0026nbsp;-\u003c/strong\u003e Distribution of the positive samples identified with the novel biochemical assay presented in this work among the six retail shops and different meat cuts.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"349\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99.4269%;\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eChicken meat samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTotal(Positives)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eShop\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacks\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWings\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLegs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBreast\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLiver\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e5(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e5(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e5(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e7(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e8(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e10(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e16(3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e13(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e13(4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 349px;\"\u003e\n \u003cp\u003e\u003csup\u003eAbbreviation: - = sample of this type not analyzed (not available).\u0026nbsp;\u003cbr\u003e\u0026nbsp;Note: Legs = thigh and drumstick together).\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 3\u003cstrong\u003e\u0026nbsp;-\u003c/strong\u003e Identification of Campylobacter jejuni proteins in positive samples based on LC-MS/MS analysis.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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width=\"1045\" height=\"598\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e*Score (-10logP) represents the identification confidence based on the PEAKS software analysis. C-coverage (%) indicates the proportion of the protein sequence identified by matched peptides. U refers to the number of unique peptides identified per protein. \u0026quot;-\u0026quot; denotes that the protein was not detected in the corresponding sample.\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e C. jejuni biomarker proteins identified by High-resolution mass spectrometry (HRMS) and homologous proteins (100% identity) search results in TrEMBL (TrEMBL release 9.0 | UniProt help | UniProt).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. jejuni\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;biomarker protein\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIdentical proteins retrieved from UniProt TrEMBL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003eMajor outer membrane protein (UniProt ID: P80672)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003eThreonine-tRNA ligase (UniProt ID: A1VXT5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003eCatalase (UniProt ID: Q59296)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003e2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (UniProt ID: Q0P823)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003eCysteine synthase B (UniProt ID: P71128)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003eProbable histidine-binding protein (UniProt ID: Q46125)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter coli\u003c/em\u003e, Basic amino acid ABC transporter substrate-binding protein (UniProt ID: A0A5T1KBF0)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter\u003c/em\u003e sp. CH185, Transporter substrate-binding domain-containing protein (UniProt ID: A0A4Y8CSE0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003eFlagellin B (UniProt ID: P56964)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter coli\u003c/em\u003e, Flagellin (uniProt ID: A0A5T1U4W8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003eLarge ribosomal subunit protein uL3 (UniProt ID: Q9PLX1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter\u003c/em\u003e sp. BCW_8712, 50S ribosomal protein L3 (UniProt ID: A0A2A5M3J8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003eFumarate hydratase class II (UniProt ID: O69294)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter\u003c/em\u003e sp. CH185 fumarate hydratase (UniProt ID:A0A4Y8C754)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 283px;\"\u003e\n \u003cp\u003e\u0026nbsp;Succinate-CoA ligase [ADP-forming] subunit beta (UniProt ID: A8FKV6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 318px;\"\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter\u003c/em\u003e sp. BCW_8712 Succinate--CoA ligase subunit beta (UniProt ID: A0A2A5M3Z4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6936616/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6936616/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCampylobacter infections are behind most pathogenic food contamination cases in humans. \u003cem\u003eCampylobacter jejuni\u003c/em\u003e is a pathogen commonly found in the food chain and especially in meat products. Contamination takes place already during the slaughtering process and affects the whole supply chain until the consumer. Campylobacter contamination is still hard to prevent, and this work reports a specific easy-to-read biochemical assay for live \u003cem\u003eCampylobacter\u003c/em\u003e with a rapid visual detection from a small bacterial sample. Current PCR- and ninhydrin-based detection assay both present significant limitations in sustainability, applicability, and reliability. This work presents a rapid biochemical easy-to-read assay for live \u003cem\u003eCampylobacter\u003c/em\u003e proceeding requiring water-based solutions for an improved safety and sustainability; moreover, it has been validated using chicken meat samples using mass-spectrometry techniques. The assay efficiently allows the discrimination of a microorganism having hippuricase activity, e.g. \u003cem\u003eCampylobacter jejuni\u003c/em\u003e, from microorganisms without hippuricase activity as it delivers a bright pink color in positive samples.\u003c/p\u003e","manuscriptTitle":"Rapid and Specific Ninhydrin-free Chromogenic Assay for Detection of Viable Hippuricase-positive Bacteria and Its Validation in Raw Chicken Meat Samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 12:33:40","doi":"10.21203/rs.3.rs-6936616/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-11T15:50:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T20:08:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328356067296261657575202852213707747646","date":"2025-07-31T13:39:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-25T15:56:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121690527323226058622862712876233883595","date":"2025-07-20T08:09:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-10T04:32:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-04T15:17:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-27T08:28:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-23T08:51:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-23T08:48:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2499075-0df3-492f-99bb-cbbb8d8de48a","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51578225,"name":"Health sciences/Diseases/Infectious diseases/Bacterial infection"},{"id":51578226,"name":"Biological sciences/Microbiology/Pathogens"},{"id":51578227,"name":"Biological sciences/Microbiology"},{"id":51578228,"name":"Health sciences/Biomarkers"},{"id":51578229,"name":"Health sciences/Biomarkers/Diagnostic markers"},{"id":51578230,"name":"Physical sciences/Chemistry/Biochemistry"},{"id":51578231,"name":"Physical sciences/Chemistry/Biochemistry/Enzymes"}],"tags":[],"updatedAt":"2025-11-24T16:09:31+00:00","versionOfRecord":{"articleIdentity":"rs-6936616","link":"https://doi.org/10.1038/s41598-025-24981-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-20 15:58:37","publishedOnDateReadable":"November 20th, 2025"},"versionCreatedAt":"2025-07-18 12:33:40","video":"","vorDoi":"10.1038/s41598-025-24981-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-24981-x","workflowStages":[]},"version":"v1","identity":"rs-6936616","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6936616","identity":"rs-6936616","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}