Proteotyping of Campylobacter jejuni by MALDI-TOF MS and Strain Solution Version 2 Software.

Identification of microorganisms by MALDI-TOF MS has become a popular method in the past 20 years. Strain Solution ver. 2 software appended with MALDI-TOF MS enables accurate discrimination of serotypes and strains beyond the genus and species level by creating a theoretical mass-based database. In this study, we constructed a theoretical mass database with the validated biomarkers to proteotype Campylobacter jejuni. Using 10 strains belonging to Campylobacter spp. available from culture collections and 41 Campylobacter jejuni strains isolated from humans and foods, the ribosomal protein subunits L36, L32, S14, L24, L23, L7/L12, and S11 could be selected as the effective biomarkers for the proteotyping of C. jejuni at MALDI-TOF MS. An accurate database of their theoretical mass-based values was constructed by matching these gene DNA sequences and the observed mass peaks. We attempted to automatically classify 41 strains isolated from nature using this database and Strain Solution ver. 2 software, and 38 strains (93%) were correctly classified into the intended group based on the theoretical mass-based values. Thus, the seven biomarkers found in this study and Strain Solution ver. 2 are promising for the proteotyping of C. jejuni by MALDI-TOF MS.

Development and Evaluation of Fluorescence Immunochromatography for Rapid and Sensitive Detection of Thermophilic Campylobacter.

Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are leading causes of foodborne gastroenteritis in Japan. Epidemiological surveillance has provided evidence that poultry meat is one of the main reservoirs for human campylobacteriosis, and therefore, improvement in process hygiene at slaughter is required to reduce the number of human infections. This study thus aimed to develop fluorescent immunochromatography strips for rapid and sensitive detection of thermophilic Campylobacter on poultry carcasses at slaughter. To establish the required detection levels, we first determined the numbers of C. jejuni and C. coli on poultry carcasses at one large-scale poultry slaughterhouse in Japan, resulting in the detection of Campylobacter at 1.97 ± 0.24 log CFU/25 g of neck skin during the post-chilling process by using ISO 10272-2:2017. Our developed Campylobacter fluorescence immunochromatography (FIC) assay exhibited a 50% limit of detection of 3.51 log CFU or 4.34 log CFU for C. jejuni NCTC 11168 or C. coli JCM 2529, respectively. Inclusive and exclusive tests resulted in good agreement. The practical usefulness of this test toward poultry carcasses should be evaluated in future studies, perhaps concentration of the target microorganisms prior to the testing might be helpful to further enhance sensitivity. Nevertheless, our data suggest the potential of FIC for rapid and sensitive detection of thermophilic Campylobacter for monitoring the process hygiene of poultry carcasses at slaughter.

Draft Genome Sequences of Non-H2S-Producing Strains of Salmonella enterica Serovars Infantis, Enteritidis, Berta, and Kiambu in Japan.

We report the draft genome sequences of six strains of Salmonella enterica serovars Berta, Enteritidis, Infantis, and Kiambu, isolated from humans or chicken meats in Osaka, Japan, that were negative for hydrogen sulfide production. Their genome sizes ranged from 4,460,389 to 4,933,483 bp, with 3 to 9 rRNAs and 64 to 73 tRNAs and with coverages of 95× to 159×.

Phylogenetic Diversity and Antimicrobial Resistance of Campylobacter coli from Humans and Animals in Japan.

The phylogenetic diversity and antimicrobial resistance (AMR) of Campylobacter coli from humans and animals in Japan between 2008 and 2014 were investigated. A total of 338 foodborne campylobacterioses were reported in Osaka, and C. coli was isolated from 38 cases (11.2%). In the present study, 119 C. coli strains (42 from humans, 25 each from poultry, cattle, and swine, and 2 from wild mallard) were examined by multilocus sequence typing (MLST). MLST assigned 36 sequence types (STs), including 14 novel STs; all human strains and 91% of animal strains (70/77) were assigned to the ST-828 clonal complex. The predominant human ST was ST-860 (18/42, 43%), followed by ST-1068 (8/42, 19%); these STs were also predominant in poultry (ST-860, 9/25, 36%) and cattle (ST-1068, 18/25, 72%). ST-1562 was only predominant in swine (11/25, 44.0%). Swine strains showed the greatest resistance to erythromycin (EM; 92.0%), while EM resistance was only found in 2 out of the 42 human strains examined (4.8%). All EM-resistant swine strains (n=15) exhibited a common point mutation in the 23S rRNA sequence (A2085G), and the tetO gene was detected in 22 out of the 23 TET-resistant swine strains. A whole genome sequencing analysis of four representative swine ST-1562 strains revealed abundant AMR-associated gene clusters in their genomes, suggesting horizontal gene transfer events during host adaptation. This is the first study to demonstrate the phylogenetic diversity and AMR profiles of C. coli in Japan. The present results suggest that poultry and cattle are major reservoirs, improving our knowledge on the epidemiological and ecological traits of this pathogen.

Development of a rapid immunochromatographic assay to detect contamination of raw oysters with enteropathogenic Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of enteropathogenic Vibrio parahaemolyticus. TDH and TRH are bacterial exotoxins, and their presence in culture medium serves as a specific marker for detecting this significant pathogen. Here, we developed and evaluated an immunochromatographic assay (TDH/TRH-ICA) to simultaneously or individually detect TDH and TRH. The TDH/TRH-ICA detected TDH in all broth cultures of 47 V. parahaemolyticus strains carrying tdh. The genes encoding TRH are classified as variants trh1 and trh2, and TRH was detected in all broth cultures of 25 V. parahaemolyticus strains carrying trh1 and certain proportion (5/31) of broth cultures of V. parahaemolyticus strains carrying trh2. In contrast, TDH and TRH were not detected in broth cultures of 12 non-enteropathogenic V. parahaemolyticus strains without tdh and trh. It was difficult to detect TRH2 using the TDH/TRH-ICA. However, TRH2 may not serve as a suitable marker for detecting enteropathogenic V. parahaemolyticus, and evidence indicates that TRH2 may not contribute to enteropathogenesis. Further, a screening method using a combination of TDH/TRH-ICA and SPP medium supplemented with 1.5% NaCl (modified-SPP medium) detected oyster samples artificially spiked with 1.1-22 colony-forming units of enteropathogenic V. parahaemolyticus per 25g of oysters within approximately 8.5h, including the enrichment culture. The assay may serve as a method that facilitates the rapid and easy detection of raw oysters contaminated with enteropathogenic V. parahaemolyticus.

Production of a novel monoclonal antibody applicable for an immunochromatographic assay for Kudoa septempunctata spores contaminating the raw olive flounder (Paralichthys olivaceus).

Kudoa septempunctata, a myxosporean parasite of the olive flounder (Paralichthys olivaceus), causes foodborne gastroenteritis after ingestion of contaminated raw flounder. Available methods to detect K. septempunctata require expensive equipment, well-trained personnel, and lengthy procedures. Here we generated a novel monoclonal antibody (MAb 15G11) against K. septempunctata and used it to produce a prototype immunochromatographic assay (prototype Kudoa-ICA). Within 15min, the prototype Kudoa-ICA detected ≥1.0×105spores/mL in a spore suspension and ≥2.0×104spores/g of P. olivaceus muscle. The prototype Kudoa-ICA weakly cross-reacted with spores of K. lateolabracis and K. iwatai. cDNA sequence, expression, and western blot analyses revealed that MAb 15G11 detected an approximately 24-kDa protein encoded by a 573bp mRNA. The cDNA nucleotide and predicted amino acid sequences were not significantly similar to any sequence in the GeneBank database. Immunoelectron microscopy revealed that MAb 15G11 reacted with the sporoplasmic cells and mainly with the capsulogenic cells of the K. septempunctata spore. Although the Kudoa-ICA was weakly cross-reactive with two other Kudoa species, it detected >1.0×106spores/g of K. septempunctata in P. olivaceus muscle, which is the criterion used to indicate a violation of the Food Hygiene Law of Japan. We conclude that MAb 15G11 may be suitable for use in an immunochromatographic assay for screening P. olivaceus muscle contaminated with K. septempunctata at food distribution sites such as food wholesalers, grocery stores, and restaurants.

Development of monoclonal antibody-based ELISA for the quantification of orange allergen Cit s 2 in fresh and processed oranges.

The aim of this study was to develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) for the quantification of a major allergen (Cit s 2) in fresh and processed oranges. Purified recombinant Cit s 2 (rCit s 2)-small ubiquitin-like modifier (SUMO) was used for the production of mAbs. In the optimized ELISA, the recovery of rCit s 2 from Navel oranges or orange juice was 107-132%, and the intra- and inter-assay coefficients of variation were 3.1-8.8% and 4.4-11%, respectively. The Cit s 2 content in fresh oranges was determined to be 1,800±430ng/g, while this content was much lower in the processed foods. The developed ELISA demonstrated high reproducibility, sensitivity, and accuracy, and this assay may help individuals with orange allergy by determining Cit s 2 quantities in food products and controlling their Cit s 2 intake.

Evaluation of an immunochromatographic assay for direct identification of thermostable direct hemolysin-producing Vibrio parahaemolyticus colonies on selective agar plates.

We evaluated the utility of an immunochromatographic assay (NH IC TDH) in identifying thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus colonies on selective agar plates. The sensitivity of the NH IC TDH assay was 100% (189 samples) and its specificity was 100% (41 samples) compared with the presence of tdh.

Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus.

To rapidly and simply determine whether or not bacterial colonies growing on agar were Vibrio parahaemolyticus, we developed an immunochromatographic assay (VP-ICA) using two different monoclonal antibodies (designated mAb-VP34 and mAb-VP109) against the delta subunit of V. parahaemolyticus-F0F1 ATP synthase. The epitopes recognized by mAb-VP34 and mAb-VP109 were mapped to sequences of eight ((47)LLTSSFSA(54)) and six amino acid residues ((16)FDFAVD(21)), respectively. An amino acid sequence similarity search of the NCBI database using BLASTP showed that both epitopic amino acid sequences were present together only in V. parahaemolyticus. When 124 V. parahaemolyticus strains and 94 strains of 27 other Vibrio species or 35 non-Vibrio species were tested using the VP-ICA, the VP-ICA identified V. parahaemolyticus with 100% accuracy. The VP-ICA rapidly and simply identified the pathogen directly from a single agar colony within 30 min, indicating that VP-ICA will greatly reduce labor and time required to identify V. parahaemolyticus compared with conventional biochemical tests.

Aflatoxins B and g contamination and aflatoxigenic fungi in nutmeg.

This study examined the distribution of aflatoxigenic fungi in 25 imported Indonesian nutmeg samples contaminated with aflatoxins Bs or Bs and Gs. The incidence of aflatoxigenic fungi in the samples contaminated with high levels of aflatoxin was significantly higher than that in the samples with low levels of the toxins(r=0.752). The aflatoxin production of isolates from the samples in cultures of YES broth was examined by means of TLC and HPLC analyses. The ability of isolates to produce aflatoxins did not necessarily correlate with the contamination levels of aflatoxin in the samples. We isolated aflatoxins B and G-producing fungi from 3 samples contaminated with the high levels of aflatoxins B and G. The aflatoxigenic isolates were identified as Aspergillus nomius and A. bombycis based on morphological characters, growth rates at 37°C and 42°C and also molecular-genetic methods. Our results indicate that these two species are mainly responsible for aflatoxin G contamination in nutmeg products.

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen.

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F(0)F(1) ATP synthase's delta subunit. Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.

Molecular epidemiological investigation of a diffuse outbreak caused by Salmonella enterica serotype Montevideo isolates in Osaka Prefecture, Japan.

In Osaka Prefecture, Japan, three foodborne outbreaks were caused by Salmonella enterica serotype Montevideo in rapid succession between September 2007 and May 2008. Further, Salmonella Montevideo was also isolated from several sporadic diarrhea patients and asymptomatic carriers examined during approximately the identical period. To investigate the relatedness of the isolates, we performed antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) analysis, and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) for 29 Salmonella Montevideo isolates obtained in this region between 1991 and 2008. Although antimicrobial susceptibility tests had low discriminatory power, PFGE patterns revealed 17 unique types with <90% similarity in combined analyses involving XbaI and BlnI. Moreover, we detected three VNTR loci that were useful to genotype Salmonella Montevideo isolates, with our method ultimately classifying the isolates into 11 MLVA types based on differences in repeat unit number in each examined locus. Six isolates obtained from patients of two separate foodborne disease outbreaks, one sporadic patient, and three different carriers between 2007 and 2008 had nearly identical PFGE patterns and were classified into the identical MLVA type; further, the isolates with this PFGE and MLVA pattern appeared only at that time between 1991 and 2008. These data strongly suggest that genetically identical Salmonella Montevideo strains may have caused the 2007 and 2008 outbreaks in Osaka Prefecture. Our results demonstrate that PFGE using XbaI and BlnI is useful for discriminating between Salmonella Montevideo isolates, even within a limited area, and reconfirm that continuous epidemiological surveillance for bacterial intestinal infections such as salmonellosis may be useful to not only monitor changes in the genetic diversity of isolates, but to also detect diffuse outbreaks.

Simultaneous enrichment of shiga toxin-producing Escherichia coli O157 and O26 and Salmonella in food samples using universal preenrichment broth.

Universal preenrichment broth (UPB) was compared with modified Escherichia coli broth with novobiocin (mEC+n) for enrichment of Shiga toxin-producing E. coli O157 and O26, and with buffered peptone water (BPW) for preenrichment of Salmonella enterica. Ten strains each of the three pathogens were inoculated into beef and radish sprouts following thermal, freezing, or no treatment. With regard to O157 and O26, UPB incubated at 42 degrees C recovered significantly more cells from inoculated beef than UPB at 35 degrees C and from radish sprout samples than UPB at 35 degrees C and mEC+n. With regard to Salmonella, UPB incubated at 42 degrees C was as effective as UPB at 35 degrees C and BPW at recovering cells from beef and radish sprout samples. No significant difference was noted between the effectiveness of UPB at 42 degrees C and UPB at 35 degrees C or BPW in the recovery of Salmonella from 205 naturally contaminated poultry samples. By using UPB at 42 degrees C, one O157:H7 strain was isolated from the mixed offal of 53 beef samples, 6 cattle offal samples, and 50 pork samples all contaminated naturally, with no pathogen inoculation. The present study found that UPB incubated at 42 degrees C was as effective as, or better than, mEC+n for enrichment of O157 and O26 and comparable to BPW for preenrichment of Salmonella. These findings suggest that a great deal of labor, time, samples, and space may be saved if O157, O26, and Salmonella are enriched simultaneously with UPB at 42 degrees C.

Comparison of loop-mediated isothermal amplification assay and conventional culture methods for detection of Campylobacter jejuni and Campylobacter coli in naturally contaminated chicken meat samples.

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with Campylobacter jejuni and Campylobacter coli. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto Butzler and modified charcoal cefoperazone deoxycholate agars. Compared with C. jejuni-C. coli isolation using the conventional culture test, the LAMP results showed 98.5% (67/68) and 97.4% (74/76) sensitivity and specificity, respectively, and the positive and negative predictive values were 97.1% (67/69) and 98.7% (74/75), respectively. The conventional culture test required more than 3 to 4 days to isolate and identify C. jejuni and C. coli in the Preston enrichment cultures. In contrast, the LAMP assay was markedly faster, requiring less than 90 min from the beginning of DNA extraction to final detection and differentiation of C. jejuni and C. coli. In total, the LAMP assay required 23.5 to 25.5 h from the beginning of the enrichment culture to final determination. These results suggest that our LAMP assay is a powerful tool for rapid, sensitive, and practical detection of C. jejuni and C. coli which may facilitate surveillance and control of C. jejuni-C. coli contamination in chicken, as well as investigations of food poisoning incidents caused by these organisms. This is the first report of a highly sensitive and specific LAMP assay to detect and differentiate C. jejuni and C. coli in chicken meat samples.

Effect of sample preparation and bacterial concentration on Salmonella enterica detection in poultry meat using culture methods and PCR assaying of preenrichment broths.

We evaluated the sensitivity of a PCR assay in the detection of Salmonella enterica at the broth preenrichment step of poultry meat. A total of 162 retail poultry meat samples, which were prepared by manual massaging, stomacher or no homogenization were compared for Salmonella recovery. Using these homogenization methods, the PCR assay at the broth preenrichment step detected Salmonella in, respectively, 48.9%, 62.2% and 50.0% of meat and giblet samples detected as Salmonella-positive using the culture method. In ground chicken, however, Salmonella was detected in 21.7% of samples treated by stomacher homogenization, compared to 40.7% and 48% of untreated and hand-massaged samples, respectively. These results suggest that stomaching of ground chicken causes excessive effusion of food constituents, which affects PCR results. Using the most probable number (MPN) technique, Salmonella was detected at under 1.0 CFU/g in 12 ground chicken samples and under 10(3)CFU/ml of broth in seven of the 12 broth-enriched samples, which considered the minimum concentration detectable by PCR assay. These results show that Salmonella detection using routine PCR assays is difficult in poultry meat, and in particular ground chicken, due to low amounts of Salmonella and the presence of inhibitors.