Prevalence of Staphylococcus argenteus among food handlers, kitchen utensils, and food samples in Japan.

Staphylococcus argenteus has received increased attention from an aspect of food safety since several food poisoning outbreaks caused by the bacterium were reported in Japan. However, S. argenteus prevalence among food handlers and utensils has not yet been investigated. In this study, we investigated S. argenteus prevalence among a collection of coagulase-positive staphylococci (CPS) that were isolated during food sanitary inspections in Japan. Out of a total of 191 CPS isolates, 14 were identified as S. argenteus. One was isolated from shelled shrimp, nine were isolated from food handlers' hand swabs, and four were isolated from kitchen utensils. Whole-genome sequencing revealed that transmission of S. argenteus from human hands to utensils was possible. Though all 14 isolates were negative for the pvl and tst-1 genes, 6 harbored the seb gene. Only 21.4% of S. argenteus isolates were resistant to antibiotics, while 62.1% of the S. aureus isolates from the same sources were confirmed to be resistant. To the best of our knowledge, this is the first report to demonstrate possible transmission of S. argenteus from food handlers to utensils in food-processing environments.

Analysis of the complete genome sequences of Clostridium perfringens strains harbouring the binary enterotoxin BEC gene and comparative genomics of pCP13-like family plasmids.

BACKGROUND: BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains. RESULTS: In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1-3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their 'accessory regions' can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. CONCLUSION: This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.

Proposal of a novel selective enrichment broth, NCT-mTSB, for isolation of Escherichia albertii from poultry samples.

AIMS: Escherichia albertii is an emerging diarrheagenic pathogen causing food- and water-borne infection in humans. However, no selective enrichment broths for E. albertii have ever been reported. In this study, we tested several basal media, selective supplements and culture conditions which enabled selective enrichment of E. albertii. METHODS AND RESULTS: We developed a selective enrichment broth, novobiocin-cefixime-tellurite supplemented modified tryptic soy broth (NCT-mTSB). NCT-mTSB supported the growth of 22 E. albertii strains, while inhibited growth of other Enterobacteriaceae at 37°C, except for Escherichia coli and Shigella spp. Enrichment of E. albertii was improved further by growth at 44°C, a temperature that suppresses growth of several strains of E. coli/Shigella. Combined use of NCT-mTSB with XR-DH-agar, xylose-rhamnose supplemented deoxycholate hydrogen sulphide agar, enabled isolation of E. albertii when at least 1 CFU of the bacterium was present per gram of chicken meat. This level of enrichment was superior to those obtained using buffered peptone water, modified-EC broth, or mTSB (with novobiocin). CONCLUSIONS: Novobiocin-cefixime-tellurite supplemented modified tryptic soy broth enabled effective enrichment of E. albertii from poultry samples and was helpful for isolation of this bacterium. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this is the first report of selective enrichment of E. albertii from poultry samples.

Multilocus Variable-Number Tandem-Repeat Analysis of Enterohemorrhagic Escherichia coli Serogroups O157, O26, and O111 Based on a De Novo Look-Up Table Constructed by Regression Analysis.

Multilocus variable-number tandem-repeat analysis (MLVA) is a widely accepted molecular typing tool for enterohemorrhagic Escherichia coli (EHEC). However, ensuring the accuracy of MLVA data among multiple laboratories remains difficult. We developed a method of constructing adjusted look-up tables, which are necessary for MLVA profiling, at each laboratory using a regression analysis based on electrophoresis data from 24 in-house reference strains. On performing MLVA against 51 EHEC O157 isolates, the repeat numbers of 46 isolates were determined accurately using the look-up table with a 99% prediction interval, an outcome superior to that when using a 95% prediction interval. For the remaining five isolates, although the electrophoresis size fell outside the look-up table, we were able to predict the repeat number accurately by extrapolation or the nearest values of the look-up table. Our approach provides more accurate results than a nonadjusted conventional look-up table for calibrating MLVA profiles.

Detection of Genetic Elements Carrying vanA in Vancomycin-Resistant Enterococcus saigonensis VE80T Isolated from Retail Chicken Meat.

In this study, we aimed to detect genetic elements carrying vanA in Enterococcus saigonensis VE80T isolated from retail chicken in Vietnam. The structures of vancomycin-resistance determinants and the location of vancomycin-resistance genes were detected by sequencing the vanA gene cluster, Southern hybridization analyses, and whole-genome sequence analyses. The Tn1546-related elements harboring vanA clusters, which exhibited a characteristic structure with five point mutations compared with the prototype Tn1546, were located on the 76-kb plasmid pVE80-1 of VE80T. The vanS sequence of VE80T harboring three point mutations was 100% identical to those of vancomycin-resistant enterococci isolated from poultry in Taiwan and Japan, indicating that the element may be prevalent in poultry production farms in Asia.

Foodborne Outbreak of Group G Streptococcal Pharyngitis in a School Dormitory in Osaka, Japan.

In September 2016, 140 patients with primary symptoms of sore throat and fever were identified in a school dormitory in Osaka, Japan. Epidemiological and laboratory investigations determined that these symptomatic conditions were from a foodborne outbreak of group G streptococcus (GGS), with GGS being isolated from samples from patients, cooks, and foods. The strain of GGS was identified as Streptococcus dysgalactiae subsp. equisimilis of two emm types (stG652.0 and stC36.0). The causative food, a broccoli salad, was contaminated with the two types of S. dysgalactiae subsp. equisimilis, totaling 1.3 × 104 CFU/g. Pulsed-field gel electrophoresis (PFGE) of samples from patients, cooks, and foods produced similar band patterns among samples with the same emm type. This result suggested the possibility of exposure from the contaminated food. The average onset time was 44.9 h and the prevalence rate was 62%. This is the first report to identify the causative food of a foodborne outbreak by Streptococcus dysgalactiae subsp. equisimilis.

Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay.

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

Development and single laboratory validation of an optical biosensor assay for tetrodotoxin detection as a tool to combat emerging risks in European seafood.

Tetrodotoxin (TTX) is a potent neurotoxin emerging in European waters due to increasing ocean temperatures. Its detection in seafood is currently performed as a consequence of using the Association of Analytical Communities (AOAC) mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins, but TTX is not monitored routinely in Europe. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. An AOAC-accredited high-performance liquid chromatography (HPLC) method has now been accepted by the European Union as a first action screening method for PSP toxins to replace the MBA. However, this AOAC HPLC method is not capable of detecting TTX, so this potent toxin would be undetected; thereby, a separate method of analysis is required. Surface plasmon resonance (SPR) optical biosensor technology has been proven as a potential alternative screening method to detect PSP toxins in seafood. The addition of a similar SPR inhibition assay for TTX would complement the PSP assay in removing the MBA. The present report describes the development and single laboratory validation in accordance with AOAC and IUPAC guidelines of an SPR method to be used as a rapid screening tool to detect TTX in the sea snail Charonia lampas lampas, a species which has been implicated in 2008 in the first case of human TTX poisoning in Europe. As no current regulatory limits are set for TTX in Europe, single laboratory validation was undertaken using those for PSP toxins at 800 µg/kg. The decision limit (CCα) was 100 µg/kg, with the detection capability (CCß) found to be ≤200 µg/kg. Repeatability and reproducibility were assessed at 200, 400, and 800 µg/kg and showed relative standard deviations of 8.3, 3.8, and 5.4% and 7.8, 8.3, and 3.7% for both parameters at each level, respectively. At these three respective levels, the recovery of the assay was 112, 98, and 99%.

Real-time PCR assays to detect 10 Shiga toxin subtype (Stx1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) genes.

To develop subtyping methods for Shiga toxin (Stx)1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g genes for epidemiological analyses of Shiga toxin-producing Escherichia coli (STEC), we developed 10 simplex real-time polymerase chain reaction (PCR) assays with reference to 284 valid stx sequences and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates and recombinant plasmids, respectively. Three stx1 and 5 stx2 subtype genes, except for stx2c and stx2d, were detected with high specificity using STEC isolates. However, some stx2a sequences potentially being close to both Stx2a and Stx2d cluster in neighbor-joining cluster analysis were positive for stx2a and stx2d by real-time PCR. For the stx2c assay, the number of real-time PCR cycles was reduced to avoid unnecessary false-positive results. Based on these considerations, the real-time PCR assays developed here might aid epidemiological investigations of infections or outbreaks caused by STEC harboring any of the stx subtype genes.

The quite low cross-reactivity of Kawatsu's anti-tetrodotoxin monoclonal antibody to 5,6,11-trideoxytetrodotoxin, 11-nortetrodotoxin-6(S)-ol, and 11-oxotetrodotoxin, the major tetrodotoxin analogues in pufferfish.

The monoclonal antibody against tetrodotoxin (TTX), prepared by Kawatsu et al. (1997), has been used in several TTX-related studies. Herein, we confirmed the quite low cross-reactivity of this antibody to three major TTX analogues in pufferfish using competitive ELISA: 5,6,11-trideoxyTTX (<2.2%), 11-norTTX-6(S)-ol (<0.3%), and 11-oxoTTX (<1.5%), with reactivity against TTX being 100%. We further confirmed that the presence of these analogues did not cause a marked overestimation of TTX in pufferfish extracts using competitive ELISA.

Detection of paralytic shellfish toxins by a solid-phase inhibition immunoassay using a microsphere-flow cytometry system.

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 µg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Isolation and characterization of Staphylococcus argenteus strains from retail foods and slaughterhouses in Japan.

Staphylococcus argenteus has been recently established as a novel species of Staphylococcus aureus complex. It is known to cause various human diseases, such as skin and soft-tissue infections, sepsis, and staphylococcal food poisoning, although the source of infection has not been clearly described. In food poisoning cases, the source of bacterial contamination in food is unknown. This study examined the prevalence of S. argenteus among retail fresh food and poultry slaughterhouses in Japan. Among 642 food samples examined, successful isolation of S. argenteus was achieved in 21 of 151 (13.9%) chicken samples. No isolations from pork, beef, fish, or vegetables in retail markets were confirmed. Multiple-locus sequence typing revealed that the 21 isolates were classified into four sequence types (ST) that were divided into 14 subtypes using spa-typing. All food isolates were susceptible to methicillin and did not show positivity for the Panton-Valentine leukocidin gene. When bacteria were isolated from two poultry slaughterhouses in the same region, 14 S. argenteus strains were successfully isolated from only one slaughterhouse. Thirteen of 14 strains were isolated from a poultry carcass and slaughterhouse environments during a certain sampling period and were all classified as ST5961 with identical spa-type. Also, the number of single nucleotide variants (SNVs) detected on the core genomes of the same 13 strains were between 0 and 17, suggesting a long-term inhabitation of an S. argenteus strain inside the facility. Furthermore, one isolate from chicken meat was also genetically linked with the same lineage of slaughterhouse isolates, with ≤15 SNVs being detected. Additionally, one slaughterhouse isolate from chiller water and three chicken isolates were classified into the same cluster by phylogenetic analysis, although the number of pairwise SNVs ranged from 62 to 128. To the best of the authors' knowledge, this is the first study that demonstrated S. argenteus in a food processing facility and the possible bacterial contamination on food during food processing.

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.

Evidence of accumulation of tetrodotoxin (TTX) in tissues and body parts of ectoparasitic copepods via their feeding on mucus of TTX-bearing pufferfish.

Adults of the ectoparasitic copepod Caligus fugu found on tetrodotoxin (TTX)-bearing pufferfish such as Takifugu alboplumbeus and Takifugu flavipterus are known to accumulate TTX in body tissues and parts other than the ovaries, oviducts, eggs, and cuticles. This study aimed to demonstrate, using immunoenzymatic staining techniques, that the TTX-free planktonic/infective copepodid stage of C. fugu could accumulate TTX in the tissues after molting into the parasitic stage (chalimus I) and then fed on mucus of host puffers. All the tissues of the planktonic copepodids were completely TTX-free, whereas chalimus I copepods accumulated TTX in parts other than the cuticles, guts, and some muscles. Chalimus IV and adult copepods retained TTX in these body parts but not in the reproductive organs, which were TTX-resistant, indicating that TTX was not vertically transmitted via eggs. Non-cellular TTX-positive contents found in the guts of some chalimi and adults indicated that the copepods potentially accumulated TTX by feeding on host mucus rather than skin tissues and blood. This study revealed that the presence or absence of TTX in some body parts differed among individuals of the parasite.

The First Report of Macrolide-Resistant Bordetella pertussis Isolation in Japan.

We report the first detection of a macrolide-resistant Bordetella pertussis strain in Japan. The isolate was highly resistant to the macrolides (minimum inhibitory concentrations for erythromycin and clarithromycin: > 256 µg/ml, for azithromycin: 32 µg/ml) and A2047G mutation was identified in the 23S rRNA. The Multilocus Sequence Typing and Multilocus Variable Number of Tandem Repeat Analysis genotypes of this isolate were MT195 and ptxP1/ptxA1/prn1/fim3A/fhaB3, respectively, suggesting a relationship with the macrolide-resistant B. pertussis lineage currently found in China. This raises the possibility that macrolide-resistant B. pertussis has already fully spread in Japan. For a better control of B. pertussis infections, the surveillance for macrolide-resistant B. pertussis is essential in not only Japan, but also other Asian countries.

Evaluation of the tetrodotoxin uptake ability of pufferfish Takifugu rubripes tissues according to age using an in vitro tissue slice incubation method.

The tetrodotoxin (TTX) uptake ability of pufferfish Takifugu rubripes tissues and its growth-associated changes were investigated using an in vitro tissue slice incubation method. Tissue slices prepared from the liver, skin, and intestine of a non-toxic cultured adult T. rubripes (20 months old) and incubated with incubation buffer containing 25 µg/mL TTX for 1-48 h showed a time-dependent increase in the TTX content in all tissues. The TTX contents of the skin and intestine slices were comparable to or slightly higher than that of the liver slices, with a similar transition pattern, suggesting similar TTX uptake ability among the skin, intestine, and liver. The TTX uptake ability of the liver and intestine did not differ significantly between young (8 months old) and adult (20 months old) fish, but the skin slices of young fish took up approximately twice as much TTX as that of adult fish, suggesting that the TTX uptake ability of the skin is involved in the growth-dependent changes in the toxin distribution inside the body in T. rubripes. To estimate the TTX uptake pathway in each tissue, an immunohistochemical technique was used to observe temporal changes in the intra-tissue microdistribution of TTX during incubation. The findings suggested that TTX is transferred and accumulates from pancreatic exocrine cells to hepatic parenchymal cells in the liver, from connective tissues to basal cells in the skin, and from villi epithelial cells via the lamina propria to the muscle layer in the intestine.

Isolation and plasmid characterisation of Salmonella enterica serovar Albany harbouring mcr-5 from retail chicken meat in Japan.

The emergence of plasmid-mediated colistin resistance genes (mcr), which is occurring in numerous countries, is a worldwide concern, primarily because colistin is a last-resort antibiotic. Compared to E. coli, prevalence of mcr genes in Salmonella is unclear in Japan. Here we screened for mcr-1-5 genes in our collection of Salmonella strains isolated from retail meat products collected in Japan from 2012 through 2016. We found that Salmonella Albany strain 27A-368 encodes mcr-5 and that mcr genes were undetectable among the remaining 202 isolates. The resistance plasmid p27A-368 was transferred by conjugation to S. Infantis and was stably retained as a transconjugant. Whole-genome sequencing revealed that mcr-5 resided on a 115 kb plasmid (p27A-368). The plasmid backbone of p27A-368 is more similar to that of pCOV27, an ESBL-encoding plasmid recovered from avian pathogenic E. coli, rather than pSE13-SA01718 of S. Paratyphi B that encodes mcr-5. Further, mcr-5 is located on a transposon, and its sequence is similar to that of pSE13-SA01718. A phylogenetic tree based on single nucleotide variants implies a relationship between 27A-368 and S. Albany isolated in Southeast Asian countries.

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.