Association between COVID-19 vaccines and the menstrual cycle in young Japanese women.

INTRODUCTION: Although several studies have investigated the association between coronavirus disease 2019 (COVID-19) vaccines and the menstrual cycle, available data are limited. Therefore, this study investigated the effect of COVID-19 vaccines on the menstrual cycle and the effect of the menstrual cycle phase on the vaccine side effects during vaccine administration in Japan. METHODS: A self-administered questionnaire was used to collect data on the date of vaccination; type of vaccine; type, grade, and duration of the side effects; regularity of menstruation; normal length of the menstrual cycle; and the day one date of menstruation around vaccination. The survey was conducted from October 2021 to March 2022. RESULTS: The difference between the predicted and actual menstrual cycle length was 1.9 ± 3.0, 1.6 ± 2.8 (p = 0.557), and 2.5 ± 3.8 (p = 0.219) days before vaccination and after the first and second dose of the vaccine, respectively. In participants who received vaccinations twice within a single menstrual cycle, this difference was 1.3 ± 3.5 and 3.9 ± 3.3 (p = 0.045) days before and after vaccination, respectively. The grade and proportion of the side effects after the second dose of the vaccine was highest during the menstrual period and lowest during the ovulation period, with a significant effect on headache and chills. CONCLUSION: COVID-19 vaccines tended to prolong the menstrual cycle. The side effects of the COVID-19 vaccine tended to be at a maximum when vaccination occurred during the menstrual period and minimal during the ovulation period.

Detection of Bacillus cereus as a causative agent of emetic food poisoning by an unconventional culture procedure.

Bacillus cereus is known to cause two types of food poisoning: emetic and diarrhoeal. Both diseases are usually self-limiting; however, severe cases have been reported, presenting with acute liver failure and encephalopathy, including rarely fatal cases of vomiting. Clinical laboratories do not routinely test for B. cereus in patients with gastrointestinal disease. Therefore, B. cereus causing food poisoning goes undetected. We report a successful isolation of emetic B. cereus from a patient with food poisoning who presented with severe vomiting, fulminant hepatic failure, and acute encephalopathy, by a non-conventional method. Initially, stool specimens from the patients were routinely cultured to identify the causative organisms of food poisoning. No foodborne pathogens were detected in this study. In contrast, additional clinical and epidemiological information strongly suggested food poisoning by emetic B. cereus. Consequently, we allowed Drigalski agar medium smeared with patient stool specimens to stand at room temperature (approximately 25 °C) for 9 days. After 9 days, mixed bacteria grown on the medium were inoculated onto mannitol egg yolk polymyxin (MYP) agar plates, a selective medium for B. cereus. Typical colonies of B. cereus developed on MYP agar plates. The isolated B. cereus had a cereulide-producing genetic locus (ces) gene encoding the emetic toxin cereulide. The method used in this case study was unique. This method is easy to apply after obtaining an additional clinical and epidemiological information, and this method will improve the diagnostic rate of severe B. cereus food poisoning. This will contribute to the advancement of therapeutics in the future.

Intentions Regarding COVID-19 Vaccination in Females Aged 15-49 Years.

To control the coronavirus disease 2019 (COVID-19) pandemic, the Japanese government is promoting vaccination, which many people are willing to accept; however, some are reluctant to receive vaccinations. The purpose of this study was to analyze the intentions of women aged 15-49 years regarding the COVID-19 vaccination and to identify methods of promoting vaccination. We used secondary data from a web research company of approximately 1020 participants. The data contained the following variables: vaccination status, reasons for not getting vaccinated, and the intentions and reasons related to the third vaccination. We categorized the reasons using text data and evaluated the age-related differences. The proportion of women aged 15-49 years who refused COVID-19 vaccination in Japan was 17.0%, and the rate was not significantly different by age group. The most common reasons were safety and side effect concerns. Of those who received the second vaccination, 32.7% hesitated or refused the third vaccination, and the rate was not significantly different by age group. The reasons were side-effect concerns, a lack of information, and the influence of their surroundings. Addressing the side effects and providing adequate information may help promote vaccination among women aged 15-49 years.

[Detection and Serotyping of Streptococcus pneumoniae Carried in Healthy Adults with a Modified PCR Method].

Detection of Streptococcus pneumoniae colonized in the pharynx of healthy carriers currently relies on conventional culture methods of direct plating with pharyngeal swab specimens. The accurate measurement of the carriage of pneumococci, however, has not been necessarily achieved with these methods due to low density colonization and contamination of numerous oral streptococci that express α-hemolysis. A PCR-based detection method of pneumococci-specific for lytA as well as PCR serotyping of S. pneumoniae was recently developed and their effectiveness was confirmed. We modified the reaction conditions of these methods to improve the detection rate and applied them to the measurement of S. pneumoniae carried in healthy adults. Pharyngeal swab specimens obtained from 110 healthy volunteers over 40 and living in Nagoya were enriched for 5 hours with broth medium supplemented with rabbit serum and the template DNA for PCR was extracted from the mixed enriched culture. Of 110 specimens 36 (32.7%) were lytA-positive, the rate of which was much higher than the results of previous culture-based studies. The DNA template preparations were then used for PCR-based serotyping with primers specific for each of the types included in pneumococcal 23 valent vaccine (PPV23). We found that 28 out of 36 lytA-positive carriers were identified as being positive for the serotypes belonging to PPV23, although serotypes 6A and 6B were indistinguishable with the PCR method. The most frequent serotype was serotype 14, and serotypes 4, 18C, and 6A/B were also frequently identified. Five lytA-positive carriers were previously vaccinated with PPV23, and among them, 4 were positive for serotypes contained in PPV23. We recommend PCR-based identification and serotyping of S. pneumoniae in broth enrichment culture of pharyngeal swab specimens as a reliable method for the surveillance of healthy carriers with low density colonization.

Effect of incubation atmosphere on the production and composition of staphylococcal biofilms.

Staphylococcus aureus and Staphylococcus epidermidis are pathogenic bacteria that often cause invasive infections in humans. In this study, we characterized the composition and growth characteristics of staphylococcal biofilms under various incubation atmospheres. We assessed the effect of incubation atmosphere (aerobic, 5% CO2, anaerobic, and microaerobic) on the biofilm production capabilities of S. aureus strains isolated from healthy volunteers and from patients with catheter-related bloodstream infection. In addition, the composition of S. aureus and S. epidermidis biofilms was determined by assessment of biofilm degradation after treatment with DNase I, proteinase K, and dispersin B. The strains obtained from healthy volunteers and patients showed similar biofilm formation capabilities. Biofilms of S. aureus were rich in proteins when developed under ambient atmospheric conditions, 5% CO2, and microaerobic condition, whereas S. epidermidis biofilms contained large amounts of poly-ß (1, 6)-N-acetyl-D-glucosamine when developed under ambient atmospheric conditions and microaerobic condition. The biofilm-producing capability of S. epidermidis was considerably higher than that of S. aureus under aerobic condition. Staphylococcal isolates obtained from healthy individuals and patients with catheter-related infections have similar biofilm-forming capabilities. Under microaerobic conditions, S. aureus and S. epidermidis form protein-rich and poly-ß (1, 6)-N-acetyl-D-glucosamine-rich biofilms, respectively. These components may play an important role in the development of biofilms inside the body and may be the target molecules to prevent catheter-related infections caused by these organisms.

Draft Genome Sequence of a Legionella pneumophila Serogroup 4 Strain Causing Legionellosis.

Here, we report the draft genome sequence of the Legionella pneumophila Nagoya-1 strain, serogroup 4, which was isolated from a clinical sample from a patient with legionellosis. Several virulence-associated genes, including those encoding the type IV (Dot/Icm) secretion system and effector proteins, were highly conserved.

Detection of methicillin-resistant Staphylococcus aureus using a specific anti-PBP2a chicken IgY antibody.

Immunodetection of methicillin-resistant Staphylococcus aureus (MRSA) by conventional methods employing mammalian immunoglobulins has unknown detection limits, and often yields false-positive results because of the presence of S. aureus protein A, which binds the Fc region of mammalian IgG. In this study, a new PBP2a-specific chicken IgY antibody was developed in inbred and conventional chickens, and used for the detection of MRSA using whole cell lysate samples. Our results showed that this chicken IgY antibody minimized the side effects of protein A. Moreover, enzyme-linked immunosorbent assay and immunochromatography systems were used with a monoclonal and polyclonal anti-PBP2a IgY antibody, clearly differentiating MRSA from methicillin-sensitive S. aureus and other methicillin-sensitive Staphylococcus spp. The detection limit of the immunochromatography was 10(8) colony-forming units; therefore, 1 colony on an agar plate was adequate to distinguish MRSA from non-MRSA. The specificity and sensitivity of this assay were almost similar to that of a commercially available latex agglutination test; however, the procedure used in this study was less complicated. The entire detection procedure, including sample preparation, takes only 20 min and does not require special equipment. Therefore, the use of this IgY antibody as a new tool for the detection of MRSA is highly recommended.

Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products.

Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products.

Complete genome sequence of Bacillus cereus NC7401, which produces high levels of the emetic toxin cereulide.

We report the complete and annotated genome sequence of Bacillus cereus NC7401, a representative of the strain group that causes emetic-type food poisoning. The emetic toxin, cereulide, is produced by a nonribosomal protein synthesis (NRPS) system that is encoded by a gene cluster on a large resident plasmid, pNCcld.

[Capsular types, virulence factors and DNA types of Klebsiella oxytoca strains isolated from blood and bile].

Klebsiella oxytoca is an opportunistic pathogen and is isolated at the second highest frequency among genus Klebsiella from hospitalized patients. According to previous reports, the major virulence factors of K. pneumoniae include capsules and several kinds of pill, whereas the virulence factors of K. oxytoca have not been well investigated. We noticed an increased frequency of K. oxytoca isolates from patients who had undergone a biliary tract operation in a general hospital from May through November, 2009. We then performed a PCR analysis of the virulence factors and an epidemiological analysis with capsular typing (serotyping) and pulsed field gel electrophoresis (PFGE) for K. oxytoca of 11 blood isolates and 10 bile isolates. As a result, serotypes of K9, K15, K26, K31, K43, K47, K55, K70, and K79 were identified in these strains, and K1 and K2 which are frequent serotypes in K. pneumoniae strains were not observed. Two blood isolates of the K55 serotype showed almost the same PFGE pattern, suggesting that these isolates were very closely related and caused cross-infection in a hospital ward. Strains of the K43 serotype were three blood isolates and 1 bile isolate, all of which showed different PFGE patterns. There were no common isolates among the blood and bile isolates. A PCR search revealed that fimH and mrkD genes which are relevant to type 1 and type 2 pili, respectively, were present in all strains, whereas kfuBC, an iron uptake gene, and cf29a were detected in only a few strains. Neither of the mucoid phenotype-related genes magA and rmpA was present in any strains. These results strongly suggest that type 1 and/or type 3 pili would have important roles in the pathogenesis of blood infection and bile infection caused by K. oxytoca.

The cell envelope-associated protein, LytR, regulates the cysteine protease SpeB in Streptococcus pyogenes.

The LytR family of cell envelope-associated transcriptional attenuators in bacteria has been brought into focus of scientific interest on the expression of various virulence factors, as well as bacterial cell envelope maintenance. However, this protein of Streptococcus pyogenes has been only described as cell surface-associated protein, and its function is completely unknown. We created lytR mutant strains from two independent S. pyogenes strains to analyze the function of LytR. The protease assay in culture supernatant showed that lytR mutant had the higher cysteine protease activity than wild-type. Two-dimensional gel electrophoresis and western blotting analysis revealed that the amount of cysteine protease, SpeB in lytR mutant was more compared with that in wild-type. The level of speB mRNA in lytR mutant also increased compared with that of wild-type. The membrane integrity and potential in lytR mutant also were decreased compared with that of wild-type. Murine infection model showed that less survival was detected in mice inoculated with lytR mutant than that with wild-type, and the size of wound lesion of mice with lytR mutant was larger than that with wild-type. Our data suggest that the lytR regulates the expression of SpeB in S. pyogenes with relation to membrane integrity.

A new microarray system to detect Streptococcus pneumoniae serotypes.

Streptococcus pneumoniae, one of the most common gram-positive pathogens to colonize the human upper respiratory tract, is responsible for many severe infections, including meningitis and bacteremia. A 23-valent pneumococcal vaccine is available to protect against the 23 S. pneumoniae serotypes responsible for 90% of reported bacteremic infections. Unfortunately, current S. pneumoniae serotype testing requires a large panel of expensive antisera, assay results may be subjective, and serotype cross-reactions are common. For this study, we designed an oligonucleotide-based DNA microarray to identify glycosyltransferase gene sequences specific to each vaccine-related serotype. Out of 56 isolates representing different serotypes, only one isolate, representing serotype 23A, was not detected correctly as it could not be distinguished from serotype 23F. Our data suggest that the microarray provides a more cost-effective and reliable way of monitoring pneumococcal capsular types.

Structural variation of Tn10 that carries tetB found in fish farm bacteria.

Three variants of the composite transposon Tn10 were extracted from transferable plasmids of fish farm bacteria. These variants were identical in insertions with IS10, but differed in another class I transposon insertion and a region of homologous recombination downstream of tetB.

Variation in M protein production among Streptococcus pyogenes strains according to emm genotype.

M protein is an important virulence determinant in Streptococcus pyogenes, but the amounts of M protein in various strains of the species remain to be elucidated. To assess the amount of M protein in strains of each emm genotype, dot blot analysis was performed on 141 clinically isolated strains. Among the cell membrane-associated proteins, M protein was present in greater quantities in the emm1, 3, and 6 strains than in the other emm strains. In addition three strains, one each of the emm1, 3, and 6 types, showed prolific M protein production (M protein-high producers). These three emm genotypes are frequently isolated in clinical practice. Sequencing of the csrRS gene, one of the two-component signal transduction systems implicated in virulence, was performed on 25 strains bearing different amounts of M protein. CsrS mutations, in contrast to CsrR protein, were detected in 11 strains. The M protein-high producer strain of emm1 type carried two amino acid substitutions, whereas the other three emm1 strains carried only one substitution each. The M protein-high producer expressed its emm gene more strongly than the corresponding M protein-low producer did according to TaqMan RT-PCR. These observations suggest that the accumulation of amino acid substitutions in CsrS protein may contribute, at least in part, to the large amount of M protein production seen in several emm genotypes.

Application of both high-performance liquid chromatography combined with tandem mass spectrometry shotgun and 2-D polyacrylamide gel electrophoresis for streptococcal exoproteins gave reliable proteomic data.

Streptococci secrete a large number of exoproteins including virulence-associated toxins and enzymes. To construct a reliable database of streptococcal exoproteins, we integrated the results that were derived from two approaches: LC-based shotgun proteomic analysis and 2-D PAGE-based proteomic analysis. We identified 74 and 82 proteins by LC-based and gel-based analysis, respectively. Forty-five proteins were identified by both methods. In addition, two proteins, one identified by both methods and the other only by LC-based shotgun analysis, were newly annotated. We therefore found the importance of combinational analysis by the two methods for the construction of a more reliable database.

Prevalence of a streptococcal inhibitor of a complement-mediated cell lysis-like gene (sicG) in Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.

Rapid detoxification of cereulide in Bacillus cereus food poisoning.

Bacillus cereus is recognized as a major pathogenic bacterium that causes food poisoning and produces gastrointestinal diseases of 2 types: emetic and diarrheal. The emetic type, which is often linked to pasta and rice, arises from a preformed toxin, cereulide, in food. Rapid and accurate diagnostic methods for this emetic toxin are important but are limited. Here we describe 3 patients with B cereus food poisoning in which cereulide was detected and measured sequentially. Three family members began to vomit frequently 30 minutes after consuming reheated fried rice. After 6 hours, a 1-year-old brother died of acute encephalopathy. A 2-year-old sister who presented with unconsciousness recovered rapidly after plasma exchange and subsequent hemodialysis. Their mother recovered soon by fluid therapy. From leftover fried rice and the children's stomach contents, B cereus was isolated. Serum cereulide was detected in both children; it decreased to an undetected level in the sister. These cases highlight the importance of measuring the value of cereulide, which would reflect the severity of B cereus emetic food poisoning. The cases also suggest the possible role of blood-purification therapy in severe cases.

Virulence genes, quinolone and fluoroquinolone resistance, and phylogenetic background of uropathogenic Escherichia coli strains isolated in Japan.

A total of 312 uropathogenic Escherichia coli strains were isolated from clinical specimens in 7 hospitals in Aichi Prefecture, Japan. Among them, 113 strains were resistant to quinolone, and 49 strains were resistant to fluoroquinolone. Phylogenetic group B2 was most prevalent in both susceptible strains (148 of 199 strains, 74.4%) and resistant strains (quinolone-resistant strains, 73 of 113 strains, 64.6%; fluoroquinolone-resistant strains, 40 of 49 strains, 81.6%). The resistant strains showed a significantly lower prevalence of virulence genes papA, hlyA, and cnf1 than did the susceptible strains, and this observation was further obvious when compared within B2 group strains. Among the 40 fluoroquinolone-resistant strains belonging to group B2, 37 (92.5%) strains carried PAIusp subtype IIa, 36 strains of which carried E84V mutation in parC, whereas none of the 9 strains belonging to group D carried PAIusp subtype IIa, and only one strain carried the mutation. These observations indicate that the differences of phenotypes including resistance of quinolone and carriage of virulence genes are associated with the complex context of genetic background, including the phylogenetic group and PAIusp subtype.

Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients.

Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in Japan and many other developed countries. Epidemiological studies have revealed that the M1 serotype of Streptococcus pyogenes is the most dominant causative isolate of STSS. Recent characterization of M1 isolates revealed that the mutation of covS, one of the two-component regulatory systems, plays an important role in STSS by altering protein expression. We analyzed the M1 S. pyogenes clinical isolates before or after 1990 in Japan, using two-dimensional gel electrophoresis (2-DE) and pulsed-field gel electrophoresis (PFGE). PFGE profiles were different between the isolates before and after 1990. Markedly different profiles among isolates after 1990 from STSS and pharyngitis patients were detected. Sequence analysis of two-component regulatory systems showed that covS mutations were detected not only in STSS but also in three pharyngitis isolates, in which proteins from the culture supernatant displayed the invasive type. The mutated CovS detected in the pharyngitis isolates had impaired function on the production of streptococcal pyrogenic exotoxin B (SpeB) analyzed by 2-DE. These results suggest that several covS mutations that lead to the malfunction of the CovS protein occurred even in pharyngeal infection.

[Helicobacter pylori infection and autoimmune disease such as immune thrombocytopenic purpura].

Helicobacter pylori infection is implicated in the pathogenesis of extradigestive diseases such as acne rosacea and idiopathic chronic urticaria and autoimmune diseases such as autoimmune gastric atrophy, rheumatoid arthritis, anti phospholipid antibody syndrome, autoimmune thyroiditis, Sjoegren syndrome, Henoch-Schoenlein purpura, and Type B insulin resistance syndrome. H. pylori eradication ameliorated the condition in some, but not all, of those with these autoimmune diseases. Recent studies primarily in Italy and Japan found that H. pylori eradication in those infected with chronic immune thrombocytopenic purpura (ITP) results in a persistent platelet count increase in over half of those treated, suggesting that although pathogenetic mechanisms underlying the relationship between H. pylori infection and autoimmune disease remain unclear, yet-unknown immunological events induced by H. pylori infection almost certainly occur in the development of autoimmune response. A majority of isolated H. pylori strains express human Lewis (Le(x) and/or Le(y) determinants and in some strains, Le(a), Le(b), sialyl-Le(x)), and H determinants in the O-chain of the surface lipopolysaccharide. Previous studies showed that this molecular mimicry helps the bacterium evade host responses while evoking autoantibody responses to Le antigens. The anti-Le(y) autoantibody is also reported to promote H. pylori adhesion to gastric epithelial cells, leading to development of gastric atrophy. Moreover, one can hypothesize that anti-Le autoreactive antibodies induced by H. pylori infection are involved in the development of autoimmune diseases, although no clinical studies showing that anti-Le immune responses are involved in the etiology of these autoimmune diseases have been conducted. Proving this hypothesis would require quantitative and qualitative analysis of autoantibodies and T cell functions to Le antigens. High frequent phase variation of Le structures in the O-polysaccharide of H. pylori may influence the immune response of patients to Le antigens.