Development and Effective Utilization of a Rapid Multiplex Real-Time PCR of Diarrheagenic Escherichia coli for Food Poisoning Cases.

Diarrheagenic Escherichia coli (DEC) causes diarrheal symptoms in humans. The comprehensive detection of DEC from feces using SYBR Green real-time PCR assay requires multiple runs. Moreover, PCR screening can have discrepancies related to the conformance between the results from PCR screening and culturing. We aimed to develop a real-time PCR for the comprehensive testing of DEC for diagnostic support that can be used in any general laboratory and proposed its effective utilization. We tested specificity for the designed primer sets using 100 strains. Moreover, screening and isolation of DEC were performed using the proposed multiplex real-time PCR system for 308 fecal samples collected from 37 food poisoning incidences that occurred in Gifu Prefecture, Japan from 2017 to 2019. Furthermore, the factor of discrepant results between PCR screening and culturing was analyzed by quantifying the number of DEC cell and whole E. coli cell using real-time PCR for 47 PCR screening-positive fecal samples. The results obtained from the developed multiplex real-time PCR system were in 99% concordance with those from the conventional techniques. A total of 49 fecal samples were detected with virulence genes for the screening. Of the samples which were positive with virulence genes by PCR screening, 38.3% could not be detected from the strain for bacterial culture. We found that the culturing positive samples were significantly high in numbers for the DEC cells, but no significant difference was noted in the whole E. coli cells with culturing negative samples. The multiplex real-time PCR developed in this study was found to be rapid and practical for DEC testing. The PCR screening for DEC using this method can provide rapid information toward the diagnostic support of DEC infection.

Molecular Typing of Mycobacterium tuberculosis Strains in Gifu Prefecture, Japan, Using Variable Number Tandem Repeat Analysis.

To investigate the molecular epidemiological characteristics of Mycobacterium tuberculosis strains collected from patients in Gifu Prefecture, Japan, 483 M. tuberculosis clinical isolates were analyzed using Japan Anti-Tuberculosis Association (JATA) 18-variable number tandem repeats (VNTR) between 2015 and 2019. To evaluate the lineage of M. tuberculosis strains, JATA18-VNTR profiles were applied to a maximum a posteriori method. The results revealed that the ancient Beijing subfamily, accounting for 57.3% (277/483) of the strains was the most prevalent M. tuberculosis strain. Furthermore, 18 clusters (GC-1-18) were found by minimum spanning tree analysis. The proportion of clustering strains was 9.9% (48/483), and epidemiological links to these clusters were unclear without GC-6 and GC-18. Meanwhile, interestingly, VNTR profiles of GC-7-9 and GC-14 were indistinguishable from the regional epidemic strains of Nagoya City, which has a strong socioeconomic relationship with Gifu Prefecture, but did not match the nationwide epidemic strains. This study suggests that coordinated analyses within the prefectures with strong socioeconomic relationships are important.

Subtype Screening of blaIMP Genes Using Bipartite Primers for DNA Sequencing.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

Single-Tube Multiplex Polymerase Chain Reaction for the Detection of Genes Encoding Enterobacteriaceae Carbapenemase.

A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: blaKPC, blaIMP, blaNDM, blaVIM, blaOXA-48-like, and blaGES. Of 70 blaIMP variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.

Medical clerks in a national university hospital: improving the quality of medical care with a focus on spinal surgery.

In our institution, which is a national university hospital, medical clerks were introduced in 2009 to improve the doctor's working environment. Seventeen clerks were assigned to 9 separate departments and the work content differed greatly among departments, but sufficient professional work was not done efficiently. The purpose of this study is to investigate the effects of the work of medical clerks on improvement of medical quality in recent years. In 2011, we established a central clerk desk on our outpatient floor to improve efficiency and centralize the clerk work. Since 2013, periodic education of clerks on spine disease has been provided by spine doctors, and this has facilitated sharing of information on spinal surgery from diagnosis to surgical treatment. This has allowed medical clerks to ask patients questions, leading to more efficient medical treatment and a potential reduction of doctors' work. In 2016, a revision of the insurance system by the Ministry of Health, Labour and Welfare of Japan increased the amount of medical work that clerks can perform, and it became possible to increase the number of medical clerks. Currently, we have 30 medical clerks, and this has allowed establishment of new clerk desks in other departments to handle patients. A training curriculum will be developed to reduce the burden on doctors further and to improve the quality of medical treatment.

Phenotypic and genetic analyses of Campylobacter jejuni Lior serotype 76 isolated from chicken meat and clinical specimens.

The aim of this study was to examine the link between Campylobacter jejuni isolates obtained from chicken meat (n = 7) and gastroenteritis patients (n = 744). In total, 751 isolates were subjected to Lior serotyping. All the isolates from chicken meats were serotyped as Lior serotype 76 (LIO76). Among 23 of the identified LIO76 strains, 13 strains (6 from chicken meat and 7 from clinical specimens) were indistinguishable by Penner serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis. These strains were isolated in 2 different Japanese prefectures in 2004-2005, suggesting that chicken meat is an etiological agent of Campylobacter gastroenteritis and that a diffuse outbreak occurred during this time. Therefore, a continuous surveillance program should be established in Japan in order to prevent Campylobacter gastroenteritis, especially large-scale food-borne outbreaks.

Mycobacterium species identification--a new approach via dnaJ gene sequencing.

The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.

Use of the novel phylogenetic marker dnaJ and DNA-DNA hybridization to clarify interrelationships within the genus Aeromonas.

The interrelationships of 27 Aeromonas strains were investigated using dnaJ sequences and DNA-DNA hybridization. dnaJ sequence similarities showed a stronger relationship with DNA-DNA relatedness values than did 16S rRNA gene sequence similarities. Additionally, dnaJ sequence analysis, with interspecies divergence over 5.2 % in most cases, gave better resolution than 16S rRNA gene sequences for the differentiation of strains at the species level. Relationships among Aeromonas species were therefore elucidated on the basis of dnaJ sequences and DNA-DNA reassociation. Strains of Aeromonas encheleia and Aeromonas sp. HG11 were unquestionably grouped in the same genetic species, since they shared 98.7 % dnaJ sequence similarity and 82-85 % genomic relatedness. The phylogenetically close relationships obtained from dnaJ sequence analysis (1.7-3.3 % genetic distance) were corroborated by high DNA-DNA relatedness (73-97 %) to support the previous suggestion that Aeromonas culicicola and Aeromonas allosaccharophila are later heterotypic synonyms of Aeromonas veronii. Our findings will contribute to the clarification of controversial relationships in the genus Aeromonas and also demonstrate that analysis of dnaJ sequences can be a powerful tool for interspecies study of the genus.

Phylogeny and species identification of the family Enterobacteriaceae based on dnaJ sequences.

Phylogenetic relations within the family Enterobacteriaceae were analyzed using partial dnaJ sequences of 165 strains belonging to 93 species from 27 enterobacterial genera. The dnaJ phylogeny was in relative agreement with that constructed by 16S rDNA sequences, but more monophyletic groups were obtained from the dnaJ tree than from the 16S rDNA tree. The degree of divergence of the dnaJ gene was approximately 6 times greater than that of 16S rDNA. Also, the dnaJ gene showed the most discriminatory power in comparison with tuf and atpD genes, facilitating clear differentiation of any 2 enterobacterial species by dnaJ sequence analysis. The application of dnaJ sequences to the identification was confirmed by assigning 72 clinical isolates to the correct enterobacterial species. Our data indicate that analysis of the dnaJ gene sequences can be used as a powerful marker for phylogenetic study and identification at the species level of the family Enterobacteriaceae.

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species.

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.

dnaJ gene sequence-based assay for species identification and phylogenetic grouping in the genus Staphylococcus.

In the last few years, many attempts have been made to use conserved gene sequences for identification and for phylogenetic studies of Staphylococcus species. In an effort to identify a more reliable approach, a dnaJ gene sequence-based database was created. In this study, an approximately 883 bp portion of the dnaJ gene sequence from 45 staphylococcal type strains was compared with 16S rRNA and other conserved gene (hsp60, sodA and rpoB) sequences available in public databases. Nucleotide sequence comparisons revealed that the staphylococcal dnaJ gene showed higher discrimination (mean similarity 77.6 %) than the 16S rRNA (mean similarity 97.4 %), rpoB (mean similarity 86 %), hsp60 (mean similarity 82 %) and sodA (mean similarity 81.5 %) genes. Analysis of the dnaJ gene sequence from 20 Staphylococcus isolates representing two clinically important species showed <1 % sequence divergence. Phylogenetic data obtained from the dnaJ gene sequence were in general agreement with those of 16S rRNA gene sequence analysis and DNA-DNA reassociation studies. In conclusion, the dnaJ gene sequence-based assay is an effective alternative to currently used methods, including 16S rRNA gene sequencing, for identification and taxonomical analysis of Staphylococcus species.

Mycobacterium kumamotonense Sp. Nov. recovered from clinical specimen and the first isolation report of Mycobacterium arupense in Japan: Novel slowly growing, nonchromogenic clinical isolates related to Mycobacterium terrae complex.

Three mycobacterium strains isolated from clinical specimens in Japan were provisionally assigned to the genus Mycobacterium based on their phenotypical characteristics. These isolates were further investigated to determine their specific taxonomic statuses. Mycolic acid analysis and 16S rRNA gene, rpoB, and hsp65 sequence data for the isolates showed that they are most similar to M. terrae complex. DNA-DNA hybridization studies indicated that the three strains were of two species and were distinguishable from M. terrae, M. nonchromogenicum, and M. hiberniae. Therefore, these strains represent two novel species within the genus Mycobacterium. However, one potential new species should have been considered as M. arupense with the 16S rRNA gene and hsp65 sequences similarities of 99.8% and 100% respectively; it was isolated from human specimens in the United States and was proposed in June 2006 as a new species. This report describes the first isolation of M. arupense in Japan, suggesting that the organism is clinically relevant. In addition, we propose the novel species designation Mycobacterium kumamotonense sp. nov. The type strain is CST 7247(T) (=GTC 2729(T), =JCM 13453(T), =CCUG 51961(T)).