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Multilocus variable-number tandem repeat analysis (MLVA) is widely used for genotyping of Bordetella pertussis, the causative bacteria for pertussis. However, MLVA genotyping is losing its discriminate power because prevalence of the epidemic MT27 strain (MLVA-27) is increasing worldwide. To address this, we developed a single nucleotide polymorphism (SNP) genotyping method for MT27 based on multiplexed single-base extension (SBE) assay. A total of 237 MT27 isolates collected in Japan during 1999–2018 were genotyped and classified into ten SNP genotypes (SG1 to SG10) with a Simpson's diversity index (DI) of 0.79 (95% CI 0.76–0.82). Temporal trends showed a marked increase in the genotypic diversity in the 2010s: Simpson's DI was zero in 1999–2004, 0.16 in 2005–2009, 0.83 in 2010–2014, and 0.76 in 2015–2018. This indicates that the SNP genotyping is applicable to the recently circulating MT27 strain. Additionally, almost all outbreak-associated MT27 isolates were classified into the same SNP genotypes for each outbreak. Multiplexed SBE assay allows for rapid and simple genotyping, indicating that the SNP genotyping can potentially be a useful tool for subtyping the B. pertussis MT27 strain in routine surveillance and outbreak investigations. [ABSTRACT FROM AUTHOR]

We evaluated a commercial loop-mediated isothermal amplification (LAMP) assay kit for Bordetella pertussis detection. The LAMP primers were designed to target the ptxP1 allele of the pertussis toxin promoter, but the assay could detect B. pertussis ptxP3 and ptxP8 strains in addition to ptxP1 strains, with high analytical sensitivity. [ABSTRACT FROM AUTHOR]

Abstract: In Japan, the Bordetella pertussis strain Tohama provided by the National Institute of Health, Japan has been used for the production of acellular pertussis (aP) vaccines since 1981. In the present study, in order to verify the genetic consistency of B. pertussis vaccine seed strains, we analyzed the genetic properties of the working seeds obtained from five Japanese vaccine manufacturers, and compared them with those of B. pertussis Tohama reference strains (NIID L-7 and ATCC BAA-589). Genetic analyses with pulsed-field gel electrophoresis and allele typing showed 100% genetic identity among the five seed strains and the Tohama reference strains. In addition, Southern blot analyses revealed the absence of four orthologous genes (BB0537, BB0920, BB1149 and BB4885), which are specifically absent in the strain Tohama, and in the genome of all seed strains tested, suggesting that the regions of difference (RD11–RD14) are absent in their genomes. Consequently, no genetic difference was observed among the working seeds and Tohama reference strains. Our observations indicate that B. pertussis seed strains for Japanese aP vaccine production are genetically comparable with B. pertussis Tohama. [Copyright &y& Elsevier]

Summary: Recently, the incidence of reported pertussis cases of adults has dramatically increased in Japan. In the present study, we analyzed seven Bordetella pertussis isolates recovered from adults in Japan using pulsed-field gel electrophoresis (PFGE) and sequencing of their antigenic and virulence-associated proteins, compared with those from children. PFGE analysis demonstrated that the adult strains were closely related to the child strains (78–100% genetic similarity). On the other hand, the genotyping revealed that 71% (5/7) of the adult strains and 47% (25/53) of the child strains had the same combination of antigenic/virulence-associated allelic variants (ptxS1B/prn1/fim2-1/fim3A/fhaB1/tcfA2) as the Japanese vaccine strain Tohama, respectively. In comparison to the child strains, there was no apparent antigenic and genetic shift in the adult strains. Our result suggests that (i) there is no B. pertussis circulating strain specific to adults and (ii) the antigenic/virulence-associated proteins are unrelated to the rise in adult pertussis incidence in Japan. [Copyright &y& Elsevier]

Abstract: A toxic N-terminal 180-amino-acid fragment (C180) of pertussis toxin S1 subunit has the most potent ability to induce protective immunity against pertussis toxin (PT) following DNA-based immunization [Kamachi K, Arakawa Y. Infect Immun 2004;72:4293–6]. For the development of a safer pertussis DNA vaccine, three plasmids encoding mutant C180 (C180-R9K, C180-E129G and C180-R9K/E129G) were constructed and tested for their protective immunogenicity and cytotoxicity. All of the gene gun delivery of the plasmid, performed by inserting the mutant C180 gene into a mammalian expression vector pcDNA3.1, successfully induced anti-PT IgG antibody production without the loss of immunogenicity in mice. The immunizations of mice with the plasmids significantly inhibited leukocytosis-promoting activity by PT. Among stably transfected Chinese hamster ovary (CHO) cells expressing mutant C180, the expression of C180-R9K and C180-R9K/E129G was non-toxic to the transfectants, confirming that these mutant C180s have no cytotoxicity to mammalian cells. These results indicate that C180-R9K and C180-R9K/E129G genes, especially C180-R9K/E129G, are candidates for safe and effective antigen DNAs in the development of pertussis DNA vaccine. [Copyright &y& Elsevier]

Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis, and detoxified PT is a crucial antigen of acellular pertussis vaccine. Here, plasmid DNA expressing the pertussis toxin S1 subunit (pcDNA/S1) of B. pertussis was evaluated for immunogenicity and for the ability to induce protection against PT challenge or B. pertussis infection in mice. The gene gun delivery of pcDNA/S1, performed by inserting the S1 gene into a mammalian expression vector, successfully induced anti-PT IgG antibody production. Immunization of mice with pcDNA/S1 significantly inhibited leukocytosis-promoting activity caused by PT or B. pertussis. In addition, pcDNA/S1 induced significant protection against intracerebral challenge with a lethal dose of B. pertussis. The results of the present study demonstrated that a DNA vaccine encoding the PT-S1 subunit induced protection against B. pertussis infection in mice. Thus, this vaccine preparation is potentially applicable for the production of novel vaccines against B. pertussis infection. [Copyright &y& Elsevier]

Summary Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori -mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited γ-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-l-norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial γ-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant γ-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that γ-glutamyl transpeptidase plays a significant role in H. pylori -mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial γ-glutamyl transpeptidase function. [ABSTRACT FROM AUTHOR]

We examined the distribution of antibody levels against pertussis toxin (PT) and filamentous hemagglutinin (FHA) among a healthy Japanese population aged from 0 to 77 years. Levels of both antibodies in 1108 serum samples collected in 1994 from nine prefectures were assayed using polystyrene ball ELISA. The ratio of individuals positive (≥10 ELISA U/ml) for anti-PT and anti-FHA antibodies at ages ranging from 0 to 3 years increased rapidly with the increase in the population vaccinated over three times with acellular pertussis (aP) vaccine. However, the ratio of those positive for anti-PT antibody tended to decrease until 6–8 years of age and to increase again from 9 to 19 years among the vaccinated population, although the ratio of individuals positive for anti-FHA antibody remained constant at 80–100% in children and adolescents over 3 years old. Moreover, positivity for anti-PT antibody was high (≥50 ELISA U/ml) in some serum samples collected from adolescents and young adults, suggesting recent symptomatic or asymptomatic infection with circulating Bordetella pertussis. On the other hand, 50–60% of infants below 12 months of age was below the detection limit (1.0 ELISA U/ml) for anti-PT and anti-FHA antibodies, and most early infants were not vaccinated for pertussis. Since intermittent circulation of B. pertussis remains among the Japanese population, complete vaccination with aP vaccine for all infants should be highly recommended to prevent pertussis. [Copyright &y& Elsevier]

The emergence of macrolide-resistant Bordetella pertussis (MRBP) is a significant problem because it reduces treatment options for pertussis and exacerbates the severity and spread of the disease. MRBP has been widely prevalent in mainland China since the 2010s and has been sporadically detected in other Asian countries. In Japan, two MRBP clinical strains were first isolated in Tokyo and Osaka between June and July 2018. The isolates BP616 in Osaka and BP625 in Tokyo harbored the same virulence-associated allelic genes (including ptxP1, ptxA1, prn1, fim3A, and fhaB3) and MT195 genotype and exhibited similar antimicrobial susceptibility profiles. However, despite their simultaneous occurrence, a distinguishable epidemiological link between these isolates could not be established. To gain further insight into the genetic relationship between these isolates in this study, we performed whole-genome analyses. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms revealed that the isolates belonged to one of the three clades of Chinese MRBP isolates, but there were 11 single-nucleotide polymorphism differences between BP616 and BP625. Genome structure analysis revealed two large inversions (202 and 523 kbp) and one small transposition (3.8 kbp) between the genomes. These findings indicate that the two Japanese MRBP isolates are closely related to Chinese MRBP isolates but are genomically distinct, suggesting that they were introduced into Japan from mainland China through different transmission routes. [ABSTRACT FROM AUTHOR]

Autoagglutination (Agg) of Bordetella pertussis is often observed in clinical laboratory. However, its causal factors and frequency in circulating strains are unknown. Repeated single colony isolation enabled us to detect an Agg- mutant in the supernatant of an Agg+ strain of B. pertussis. Whole-genome sequencing and immunoblot analysis disclosed that the Agg- mutant had a single C-deletion in its fim3 promoter region (Pfim3) which abolished Fim3 fimbriae production. A B. pertussis fim3-knock out mutant also lacked the Agg+ phenotype. Agg+ clinical isolates were detected a higher production of Fim3 than Fim3-producing Agg- isolates. B. pertussis is known to harbor multiple Pfim3 poly(C) lengths within a single strain culture and our newly developed PCR/LDR assay revealed that Agg+ isolates harbor the highest Pfim3 poly-14C abundance. We evaluated the frequency of autoagglutination in clinical B. pertussis isolates collected in Japan between 1994 and 2018 (n = 203). Fim3 production was confirmed for 190 isolates and 74.7% of them displayed the Agg+ phenotype. The Agg+ phenotype was strongly associated with Pfim3 poly-14C abundance. Taken together, our findings demonstrated that B. pertussis autoagglutination occurs in response to high Fim3 levels and the Agg+ strain has predominated in Japan over the past two decades. [ABSTRACT FROM AUTHOR]

Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes ( rmtA , rmtB, and armA ), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes ( rmtA , rmtB, and armA ), 10 2 copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA , rmtB , or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae , rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa , and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings. [ABSTRACT FROM AUTHOR]

Pertussis is a human respiratory infection caused by the gram-negative bacterium, Bordetella pertussis. To evaluate the pertussis burden and vaccine efficacy, diagnosis and epidemiological surveillance should be based on accurate and valid diagnostic methods. Recently, the serodiagnostic tests Novagnost Bordetella pertussis IgA and IgM were approved in Japan for pertussis diagnostics. Although the anti-pertussis toxin (PT) IgG assay has been used for pertussis diagnosis worldwide, little is known about the anti-B. pertussis IgA and IgM assays. In this study, serum samples from 460 healthy donors were examined to determine the seroprevalence of anti-B. pertussis IgA and IgM in a Japanese population, and its correlation with donor age. Our data demonstrated that anti-B. pertussis IgA and IgM are positively and negatively correlated with age (r = 0.27, r = -0.37; P < 0.001, respectively). Age-specific analysis revealed high titers of anti-B. pertussis IgA in adults (46–50 years), while anti-B. pertussis IgM titers were high in schoolchildren (6–10, 11–15 years). When applying the arbitrary cut-off values for these ages, 17.6% and 39.5% of healthy donors were interpreted as pertussis-positive or indeterminate with anti-B. pertussis IgA (46–50 years) and IgM (11–15 years) titers, respectively. Overall, our findings indicated that the Novagnost Bordetella pertussis IgA and IgM testing could be greatly affected by subject age, limiting its value for pertussis diagnosis. [ABSTRACT FROM AUTHOR]

Highlights: • Japanese children aged 3–6 years had low-avidity anti-PT IgG with low titers. • Vaccine-induced anti-PT IgG avidity might rapidly wane after ACVs vaccination. • Additional booster vaccination is required for children aged 3–6 years in Japan. Abstract To gain insights into the current Japanese pertussis immunization schedule, we examined the distributions of antibody titers and avidities to pertussis toxin (PT) and filamentous hemagglutinin (FHA) in 460 Japanese healthy subjects (aged 1–60 years) based on age category. Our avidity enzyme-linked immunosorbent assays revealed that young children aged 1–2 years, which corresponded to ages after receiving primary and/or booster pertussis vaccinations, had relatively high-avidity anti-PT IgG (mean avidity index [AI], 40.5%) compared with other age groups (AI, 26.5–31.9%); however, they had relatively low-avidity anti-FHA IgG (AI, 41.8%). In contrast, children aged 3–6 years had both low-avidity anti-PT IgG (AI, 26.5%) and low-avidity anti-FHA IgG (AI, 40.4%). A significant age-related difference in anti-PT IgG avidity was observed between children aged 1–2 years and 3–6 years (P < 0.05); however, the difference in anti-FHA IgG avidity was not significant. The anti-PT IgG avidity was positively correlated with the antibody titer, especially among children aged 1–15 years (r s = 0.508–0.685; P < 0.01), indicating that the avidity of vaccine-induced anti-PT IgG decreases with decreasing IgG antibody titer to PT. Our findings strongly suggest that vaccine-induced anti-PT IgG avidity rapidly wanes after vaccination, but this is not observed for anti-FHA IgG avidity. Because children aged 3–6 years have both low-quantity and low-quality antibodies against PT, an additional booster vaccination with acellular pertussis vaccines is required for such children in Japan. [ABSTRACT FROM AUTHOR]

Macrolide-resistant Bordetella pertussis emerged in Vietnam during 2016-2017. Direct analyses of swab samples from 10 patients with pertussis revealed a macrolide-resistant mutation, A2047G, in the 23S rRNA. We identified the MT104 genotype of macrolide-resistant B. pertussis (which is prevalent in mainland China) and its variants in these patients. [ABSTRACT FROM AUTHOR]

The leukopenic toxicity test (LTT) is used to evaluate the safety and lot-to-lot consistency of influenza hemagglutinin split vaccine (HAv) and is included in the Japanese Minimum Requirements for Biological Products. LTT assesses the reduced leukocyte levels in murine peripheral blood after HAv administration. However, they require large numbers of animals, and therefore it would be beneficial to develop a more accurate and sensitive alternative method. In this study, we selected biomarkers of leukocyte reduction from 18 previously identified marker genes that were associated with an abnormal toxicity test (ATT). Among these 18 genes, the expressions of 15 marker genes were strongly associated with leukocyte reduction levels. A stepwise single addition multiple regression analysis was used to further extract the genes responsible for leukocyte reduction, with significant ( p < 0.25) regression coefficients. The expression of 7 genes significantly predicted the leukocyte reduction. The prediction accuracy of this approach was approximately >90% (mean) for the direct measurement of leukocyte numbers. These results indicate that the expression of these 18 previously identified genes can provide information for both ATT and LTT. [ABSTRACT FROM AUTHOR]

In 2013, national serosurveillance detected a high seroprevalence of antibodies to pertussis toxin (PT) from Bordetella pertussis among Japanese adults. Thus, we aimed to determine the cause(s) of this high seroprevalence, and analyzed the titers of antibodies to PT and filamentous hemagglutinin (FHA) among adults (35–44 years old), young children (4–7 years old), and older children (10–14 years old). Our quantitative analyses revealed that adults had higher seroprevalences of anti-PT IgG and PT-neutralizing antibodies, and similar titers of anti-FHA IgG, compared to the young and older children. Positive correlations were observed between the titers of PT-neutralizing antibodies and anti-PT IgG in all age groups (rs values of 0.326–0.522), although the correlation tended to decrease with age. The ratio of PT-neutralizing antibodies to anti-PT IgG was significantly different when we compared the serum and purified IgG fractions among adults (p = 0.016), although this result was not observed among young and older children. Thus, it appears that some adults had non-IgG immunoglobulins to PT. Our analyses also revealed that adults had high-avidity anti-PT IgG (avidity index: 63.5%, similar results were observed among the children); however, the adults had lower-avidity anti-FHA IgG (37.9%, p < 0.05). It is possible that low-avidity anti-FHA IgG is related to infection with other respiratory pathogens (e.g., Bordetella parapertussis, Haemophilus influenzae, or Mycoplasma pneumoniae), which produces antibodies to FHA-like proteins. Our observations suggest that these adults had been infected with B. pertussis and other pathogen(s) during their adulthood. [ABSTRACT FROM AUTHOR]

Bordetella holmesii causes both invasive and respiratory diseases in humans. Although the number of cases of pertussis-like respiratory illnesses due to B. holmesii infection has increased in the last decade worldwide, little is known about the virulence factors of the organism. Here, we analyzed a B. holmesii isolate that forms large aggregates and precipitates in suspension, and subsequently demonstrated that the autoagglutinating isolate is deficient in Bordetella intermediate protein A (BipA) and that this deletion is caused by a frame-shift mutation in the bipA gene. A BipA-deficient mutant generated by homologous recombination also exhibited the autoagglutination phenotype. Moreover, the BipA mutant adhered poorly to an abiotic surface and failed to form biofilms, as did two other B. holmesii autoagglutinating strains, ATCC 51541 and ATCC 700053, which exhibit transcriptional down-regulation of bipA gene expression, indicating that autoagglutination indirectly inhibits biofilm formation. In a mouse intranasal infection model, the BipA mutant showed significantly lower levels of initial lung colonization than did the parental strain (P < 0.01), suggesting that BipA might be a critical virulence factor in B. holmesii respiratory infection. Together, our findings suggest that BipA production plays an essential role in preventing autoagglutination and indirectly promoting biofilm formation by B. holmesii. [ABSTRACT FROM AUTHOR]

Objectives This study sought to determine the genotypes of circulating Bordetella pertussis , the causative agent of pertussis, in Cambodia by direct molecular typing of clinical specimens. Methods DNA extracts from nasopharyngeal swabs obtained from 82 pertussis patients in 2008–2016 were analyzed by multilocus variable-number tandem repeat analysis (MLVA). B. pertussis virulence-associated allelic genes ( ptxA , prn , and fim3 ) and the pertussis toxin promoter ptxP were also investigated by DNA sequence-based typing. Results Forty-four DNA extracts (54%) yielded a complete MLVA profile, and these were sorted into 8 MLVA types (MT18, MT26, MT27, MT29, MT43, MT72, MT95, and MT200). MT27 and MT29, which are common in developed countries, were the predominant strain types (total 73%). The predominant profile of virulence-associated allelic genes was the combination of ptxP3 / ptxA1 / prn2 / fim3A (48%). MT27 strains were detected during the entire study period, whereas MT29 strains were only found in 2014–2016. Conclusions The B. pertussis population in Cambodia, where a whole-cell pertussis vaccine (WCV) has been continuously used, resembled those observed previously in developed countries where acellular pertussis vaccines are used. Circulating B. pertussis strains in Cambodia were distinct from those in other countries using WCVs. [ABSTRACT FROM AUTHOR]

Prevalence of pertactin-lacking Bordetella pertussis isolates has been observed worldwide. In Japan, however, we found that the frequency of pertactin-deficient isolates in 2014-2016 (8%) was significantly lower than the frequency in 2005-2007 (41%), 2008-2010 (35%), and 2011-2013 (25%). This reduction was closely associated with changes in genotypes. [ABSTRACT FROM AUTHOR]

A large pertussis epidemic occurred between 2008 and 2010 in Japan. To investigate epidemic strains, we analyzed 33 Bordetella pertussis isolates from the epidemic period by sequencing virulence-associated genes (fim3, ptxP, ptxA, and prn) and performing multilocus variable-number tandem repeat analysis (MLVA), and compared these results with those of 101 isolates from non-epidemic, earlier and later time periods. DNA sequencing of the fim3 allele revealed that the frequency of fim3B was 4.3%, 12.8%, 30.3%, and 5.1% within isolates in 2002–2004, 2005–2007, 2008–2010, and 2011–2012, respectively. The isolation rate of the fim3B strain therefore temporarily increased during the epidemic period 2008–2010. In contrast, the frequencies of the virulence-associated allelic variants, ptxP3, ptxA1, and prn2, increased with time during overall study period, indicating that these variants were not directly involved in the occurrence of the 2008–2010 epidemic. MLVA genotyping in combination with analysis of allele types showed that the prevalence of an MT27d strain temporarily increased in the epidemic period, and that this strain carried virulence-associated allelic variants (fim3B, ptxP3, ptxA1, and prn2) also identified in recent epidemic strains of Australia, Europe, and the US. Phenotypic analyses revealed that the serotype Fim3 strain was predominant (≥87%) during all the periods studied, and that the frequency of adhesion pertactin (Prn) non-expressing B. pertussis decreased by half in the epidemic period. All MT27d strains expressed Prn and Fim3 proteins, suggesting that B. pertussis MT27d strains expressing Prn and Fim3B have the potential to cause large epidemics worldwide. [ABSTRACT FROM AUTHOR]

We report a case of a bronchitis caused by Bordetella holmesii in a 2-year-old girl with asthma. The patient had a moderate fever and productive cough, and her condition was initially diagnosed as mycoplasmal bronchitis on the basis of her clinical symptoms and rapid serodiagnosis of mycoplasmal infection. She was treated with a bronchodilator and clarithromycin, which resulted in complete recovery. However, after the initial diagnosis, nucleic acid amplification tests of her sputum showed the absence of both Mycoplasma pneumoniae and Bordetella pertussis infections. Sputum culture showed the presence of a slow-growing, gram-negative bacillus in pure culture on Bordetella agar plates; the bacillus was later identified as B. holmesii. B. holmesii infection is rare in immunocompetent children; however, the organism is a true pathogen that can cause bronchitis in young children with asthma. [ABSTRACT FROM AUTHOR]

We describe the epidemiology of a pertussis outbreak in Japan in 2010-2011 and Bordetella holmesii transmission. Six patients were infected; 4 patients were students and a teacher at the same junior high school. Epidemiologic links were found between 5 patients. B. holmesii may have been transmitted from person to person. [ABSTRACT FROM AUTHOR]

Bordetella pertussis is the etiological agent of whooping cough, a common cause of respiratory illness in both children and adults. In the present study, we investigated the bactericidal activity of four antiseptics-povidone-iodine (PVP-I), benzethonium chloride (BEC), chlorhexidine gluconate (CHG) and benzalkonium chloride (BAC)-against B. pertussis ATCC9797 and clinical isolates. Among the topical antiseptics, PVP-I, BEC, and BAC, PVP-I and BAC in particular, showed high bactericidal activity, whereas CHG had low activity. PVP-I gargle also showed high bactericidal activity, similar to topical PVP-I. However, BEC gargle had low bactericidal activity. Our results indicate that topical PVP-I and BAC, and PVP-I gargle would be useful as effective antiseptics against B. pertussis. [ABSTRACT FROM AUTHOR]

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn-) B. pertussis was observed in Japan. The Prn- isolate was first discovered in 1997, and 33 (27%) Prn- isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn- isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn- isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn- isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn- isolates belong to MLVA-186, and 6 and 3 Prn- isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn- clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn- isolates have a higher growth potential than the Prn+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn- strains have arisen and that Prn expression is not essential for fitness under these conditions. [ABSTRACT FROM AUTHOR]

Abstract: Glutamate dehydrogenase (GDH) is a ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate to glutamate. In Brassica napus, GDH isoenzymes 1 and 7 are hexamers of β and α subunits, respectively and the isoenzyme profile in leaves is known to change on wounding. Here, parallels were sought between the effects of wounding and protoplast isolation because of the possible relevance of changes in GDH activity to the perturbed metabolism in recalcitrant B. napus protoplasts. When leaf protoplasts of B. napus were isolated, GDH7 isoforms predominated. Transcription of GDH2, which encodes the GDH α subunit, was activated and translation of the GDH2 mRNA was also activated to synthesize α subunit polypeptides. When detached leaves absorbed either acidic 5mM jasmonic acid or salicylic acid solutions via petioles, GDH7 isoenzymes were activated and the GDH isoenzyme patterns were similar to those of protoplasts. Salicylic acid β-glycosides were generated soon after treatment with the pectinase–cellulase enzyme solution and peaked at 1h. NMR spectroscopic analysis of protoplasts and unstressed leaves incubated with 5mM 15NH4Cl showed that the change in GDH isoenzyme profile had no effect on ammonium assimilation. Protoplast isolation changed the redox state with NAD(P)H and oxidized glutathione levels increasing, and ascorbate, dehydroascorbate, NAD(P) and glutathione decreasing. ATP content in protoplasts declined to 2.6% of that in leaves, while that in wounded leaves increased by twofold. It is concluded that GDH7 does not support net amination in vivo and it is suggested that the increase in GDH7 activity is a response to oxidative stress during protoplast isolation. [Copyright &y& Elsevier]

Background: Bordetella pertussis is the primary etiologic agent of the disease pertussis. Universal immunization programs have contributed to a significant reduction in morbidity and mortality of pertussis; however, incidence of the disease, especially in adolescents and adults, has increased in several countries despite high vaccination coverage. During the last three decades, strains of Bordetella pertussis in circulation have shifted from the vaccine-type to the nonvaccine-type in many countries. A comparative proteomic analysis of the strains was performed to identify protein(s) involved in the type shift. Methodology/Principal Finding: Proteomic analysis identified one differentially expressed protein in the B. pertussis strains: the type III cytotoxic effector protein BteA, which is responsible for host cell death in Bordetella bronchiseptica infections. Immunoblot analysis confirmed the prominent expression of BteA protein in the nonvaccine-type strains but not in the vaccine-type strains. Sequence analysis of the vaccine-type strains revealed an IS481 insertion in the 59 untranslated region of bteA, 2136 bp upstream of the bteA start codon. A high level of bteA transcripts from the IS481 promoter was detected in the vaccine-type strains, indicating that the transcript might be an untranslatable form. Furthermore, BteA mutant studies demonstrated that BteA expression in the vaccine-type strains is down-regulated by the IS481 insertion. Conclusion/Significance: The cytotoxic effector BteA protein is expressed at higher levels in B. pertussis nonvaccine-type strains than in vaccine-type strains. This type-dependent expression is due to an insertion of IS481 in B. pertussis clinical strains, suggesting that augmented expression of BteA protein might play a key role in the type shift of B. pertussis. [ABSTRACT FROM AUTHOR]

Abstract: The incidence of reported cases with pertussis has increased in young adults in Japan and the lack of additional booster immunizations containing pertussis components is suspected to be one of the causal reasons. Instead of DT immunization at 11–12 years of age, safety and immunogenicity were investigated using 0.2ml and 0.5ml of DTaP. 176 subjects in DTaP 0.5ml, 178 in DTaP 0.2ml, and 197 in DT 0.1ml groups were enrolled in clinical trial. The relative risk of local reactions in the DTaP 0.2ml group compared to the DT 0.1ml group was 1.13 (95% CI: 0.97–1.30), and that of the DTaP 0.5ml to the DT 0.1ml group was 1.34 (95% CI: 1.18–1.53). The relative risks of local pain and heat were 1.62 (95% CI: 1.33–1.98) and 1.59 (95% CI: 1.19–2.13), respectively, in the DTaP 0.5ml group compared to the DT 0.1ml group. Sero-positive rates against PT and FHA were 54% and 82% before immunization and increased to >95% for both after vaccination with no significant difference in GMT. Instead of the scheduled DT program, 0.2ml of DTaP was acceptable and demonstrated efficient immunogenicity. [ABSTRACT FROM AUTHOR]

Abstract: Glutamate dehydrogenase (GDH) is a ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate to glutamine. In Brassica napus, GDH isoenzymes 1 and 7 are hexamers of β and α subunits, respectively. When leaves of B. napus were sliced into narrow strips, seven GDH isoforms appeared with increases in both amination and deamination activity. Isoform profiles ceased changing from 1.5 to 2.5h after wounding. Treatment with jasmonic acid (JA), salicylic acid (SA), ethylene, and oligosaccharides (OGs) changed the profiles. Ammonium ions at the same concentration as the signal substances (5mM) caused only slight induction of the GDH7 isoenzyme. Ethylene was not released until 2h after wounding, whereas JA and OGs were generated soon after wounding and peaked at 30 and 60min after wounding, respectively. Accordingly, the wounding of leaves first releases OGs. Transcription of GDH2 , which encodes the GDH α subunit, was activated by wounding, whereas translation of the GDH2 mRNA was repressed. The GDH7 isoform was activated by the reduction of disulfide bonds in the GDHα subunits, but wounding did not cause synthesis of new α subunits. In leaves, wounding changed the redox state; it decreased NAD(P)H, ascorbic acid, and glutathione levels and increased dehydroascorbic acid. These results indicate that the GDH2 gene is regulated both at the transcriptional and translational levels and that the α subunit proteins are regulated posttranslationally via redox mechanisms. Thus, GDH isoenzyme profiles may reflect the redox state of leaves under oxidative stress. [Copyright &y& Elsevier]

Applicability of the endotoxin test to diphtheria–tetanus-acellular pertussis combined (DTaP) vaccines was examined. We found some DTaP vaccines that strongly interfered with Limulus amoebocyte lysate (LAL) activity of endotoxin without affecting lethal activity of endotoxin in d-galactosamine-treated mice. LAL activity that was interfered in such vaccines increased apparently after the treatment with phosphate buffer at 4 °C for a week. The DTaP vaccines that interfered with the endotoxin test showed no significant effect on endotoxin activity in inducing tumor necrosis factor-alpha (TNF-α) in rabbit peripheral blood. The in vitro TNF-α induction assay was, therefore, suggested to be an appropriate assay method for the quantitative detection of the endotoxin activity in DTaP vaccines. [Copyright &y& Elsevier]

Summary Objectives The present study was designed to determine the genotypes of circulating Bordetella pertussis in the Philippines by direct molecular typing of clinical specimens. Methods Nasopharyngeal swabs (NPSs) were collected from 50 children hospitalized with pertussis in three hospitals during 2012–2014. Multilocus variable-number tandem repeat analysis (MLVA) was performed on the DNA extracts from NPSs. B. pertussis virulence-associated allelic genes ( ptxA , prn , and fim3 ) and the pertussis toxin promoter, ptxP , were also investigated by DNA sequence-based typing. Results Twenty-six DNA extracts yielded a complete MLVA profile, which were sorted into 10 MLVA types. MLVA type 34 (MT34), which is rare in Australia, Europe, Japan, and the USA, was the predominant strain (50%). Seven MTs (MT29, MT32, MT33, and MT283–286, total 42%) were single-locus variants of MT34, while two (MT141 and MT287, total 8%) were double-locus variants of MT34. All MTs had the combination of virulence-associated allelic genes, ptxP1 – ptxA1 – prn1 – fim3A . Conclusions The B. pertussis population in the Philippines comprises genetically related strains. These strains are markedly different from those found in patients from other countries where acellular pertussis vaccines are used. The differences in vaccine types between these other countries and the Philippines, where the whole-cell vaccine is still used, may select for distinct populations of B. pertussis . [ABSTRACT FROM AUTHOR]