A novel vaccine strategy using quick and easy conversion of bacterial pathogens to unnatural amino acid-auxotrophic suicide derivatives.

We propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after the removal of the uAA. The survival time after uAA removal could be controlled by the choice of the uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105 fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates. IMPORTANCE: Live vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of uAAs to control gene expression and to conditionally kill host bacteria as a biological containment system. This report proposes a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system. The balance between safety and immunogenicity can be modulated by the selection of the genetic devices used. Moreover, the uAA-auxotrophy can prevent the vaccine from infecting other individuals or establishing the environment.

Outer membrane protein BamA-based ELISA differentiates Salmonella-vaccinated chickens from naturally infected chickens.

Salmonella often causes subclinical infection in chickens, but antibody tests can find infected individuals and control the spread of infection. In this study, the S. Typhimurium-specific outer membrane, ß-barrel assembly machinery protein A (BamA), was overexpressed in Escherichia coli and purified as a coating antigen to develop a BamA-based enzyme-linked immuno sorbent assay for detecting Salmonella infection. The presence of anti-BamA IgG was detected in the sera of infected BALB/c mice, but not in that of heat-killed Salmonella-vaccinated mice. The assay was validated using White Leghorn chickens and showed similar results. The detection of BamA antibodies in the sera can differentiate infected chickens from vaccinated chickens. This assay will be useful for monitoring Salmonella infection in chickens and possibly in other animals.

Differentiation of Salmonella vaccinated and infected animals by serological detection of antibody to T3SS effector SsaK in an indirect ELISA.

The differentiation of animals that are vaccinated and those that are naturally infected with Salmonella is difficult by conventional serological tests. We have shown here an indirect Enzyme-linked immunosorbent assay for detection of Salmonella infection based on the presence of a Type III secretory effector SsaK in the sera.

Rational Design of Live-Attenuated Vaccines against Genome-Reduced Pathogens.

To develop safe and highly effective live vaccines, rational vaccine design is necessary. Here, we sought a simple approach to rationally develop a safe attenuated vaccine against the genome-reduced pathogen Erysipelothrix rhusiopathiae. We examined the mRNA expression of all conserved amino acid biosynthetic genes remaining in the genome after the reductive evolution of E. rhusiopathiae. Reverse transcription-quantitative PCR (qRT-PCR) analysis revealed that half of the 14 genes examined were upregulated during the infection of murine J774A.1 macrophages. Gene deletion was possible only for three proline biosynthesis genes, proB, proA, and proC, the last of which was upregulated 29-fold during infection. Five mutants bearing an in-frame deletion of one (ΔproB, ΔproA, or ΔproC mutant), two (ΔproBA mutant), or three (ΔproBAC mutant) genes exhibited attenuated growth during J774A.1 infection, and the attenuation and vaccine efficacy of these mutants were confirmed in mice and pigs. Thus, for the rational design of live vaccines against genome-reduced bacteria, the selective targeting of genes that escaped chromosomal deletions during evolution may be a simple approach for identifying genes which are specifically upregulated during infection. IMPORTANCE Identification of bacterial genes that are specifically upregulated during infection can lead to the rational construction of live vaccines. For this purpose, genome-based approaches, including DNA microarray analysis and IVET (in vivo expression technology), have been used so far; however, these methods can become laborious and time-consuming. In this study, we used a simple in silico approach and showed that in genome-reduced bacteria, the genes which evolutionarily remained conserved for metabolic adaptations during infection may be the best targets for the deletion and construction of live vaccines.

Two Akabane virus glycoprotein Gc domains induce neutralizing antibodies in mice.

Akabane virus (AKAV), belonging to the genus Orthobunyavirus and family Peribunyaviridae, causes reproductive and congenital abnormalities in ruminants. Its envelope glycoprotein Gc is a neutralizing antigen, on which at least five distinct antigenic regions have been identified. We attempted to identify the domains using truncated recombinant AKAV Gc proteins expressed in Escherichia coli and monoclonal antibodies (mAbs) with AKAV-neutralizing activity. Dot blot analysis revealed that amino acid positions 1-97 and 189-397 (Gc1-97 and Gc189-397) in the truncated recombinant proteins reacted with the mAbs. Additionally, AKAV was neutralized by sera from mice immunized with these recombinant proteins. The results suggested that the two domains contain neutralizing epitopes and could be potential subunit vaccines against AKAV.

Genetic analysis of an Erysipelothrix rhusiopathiae swine isolate determined to be serovar 2 by a gel double diffusion test but serovar 1a/2 by a serotyping PCR assay.

We previously developed a multiplex PCR assay for the differentiation of serovar 1a, 1b, 2 and 5 strains of Erysipelothrix rhusiopathiae. In this study, we analyzed the serovar-defining chromosomal region of a serovar 2 swine isolate, which was PCR-positive for both serovars 1a and 2 by the multiplex PCR assay. Genetic analysis of the chromosomal region revealed that, as in serovar 1a strains, the ERH_1440 gene, which is usually truncated or missing in serovar 2 strains, was intact in this strain. This paper first shows an E. rhusiopathiae serovar 2 strain possessing an intact ERH_1440 gene and suggests that care may be needed when determining the serovar of such rare strains by PCR assay.

The protective capacity of anti-O4 antigen antibodies against Salmonella infection is influenced by the presence or absence of the O5 antigen.

Anti-O-antigen antibodies, such as anti-O4 antigen IgG, induce protective immunity against Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. S. Typhimurium belongs to the group O4, which can be classified into two serological variants, namely factor O5 antigen positive (O5+) and factor O5 antigen negative (O5-). In this study, we determined the protective immunity induced by anti-O4 antigen IgG against O5+ and O5- S. Typhimurium infection in a mouse model. Unexpectedly, anti-O4 antigen IgG induced protection against O5- of S. Typhimurium, but not against O5+ of S. Typhimurium. We suggest that the affinity of the O4 antigen with anti-O4 antigen IgG is stronger in the O5- S. Typhimurium compared to the O5+ S. Typhimurium. Although anti-O4 antigen IgG has the potential to protect against S. Typhimurium infection, the effects of anti-O4 antigen IgG in protection against Salmonella infection differ depending on the presence or absence of the O5 antigen.

Development of a Multiplex PCR-Based Assay for Rapid Serotyping of Erysipelothrix Species.

The Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping. Among 28 serovars of Erysipelothrix species, specific serovars, namely, 1a, 1b, and 2 of E. rhusiopathiae, are associated mainly with the disease in pigs, poultry, and humans; however, other serovar strains are often simultaneously isolated from diseased and healthy animals, indicating the importance of isolate serotyping for epidemiology. The traditional serotyping protocol, which uses heat-stable peptidoglycan antigens and type-specific rabbit antisera in an agar-gel precipitation test, is time-consuming and labor-intensive. To develop a rapid serotyping scheme, we analyzed sequences of the 12- to 22-kb chromosomal region, which corresponds to the genetic region responsible for virulence of serovar 1a and 2 strains of E. rhusiopathiae, of the 28 serovars of Erysipelothrix species. We confirmed that the serovar 13 strain lacks the genomic region and that some serovar strains possess very similar or the same genetic structure, prohibiting differentiation of the serovars. We created 4 multiplex PCR sets allowing the simultaneous detection and differentiation of the majority of Erysipelothrix serovars. Together with a previously reported multiplex PCR that can differentiate serovars 1a, 1b, 2, and 5, the multiplex PCR-based assay developed in this study covers all but one (serovar 13) of the reported serovars of Erysipelothrix species and should be a valuable tool for etiological as well as epidemiological studies of Erysipelothrix infections.

Successful enrichment of low-abundant comammox Nitrospira from nitrifying granules under ammonia-limited conditions.

In artificial engineered systems, nitrification is a key reaction that accounts for the removal of biological nitrogen. Recently, a single microbe capable of oxidizing ammonia to nitrate, known as a complete ammonia oxidizer (comammox), has been discovered. Although the abundance and diversity of comammox Nitrospira in engineered systems have been identified through molecular-based approaches, the enrichment and isolation of comammox Nitrospira remains a challenge. Therefore, the aim of this study was to enrich comammox Nitrospira from nitrifying granules, which were used to increase the efficiency of biological nitrogen removal in wastewater treatment plants. We sought to accomplish this through the use of a fixed-bed continuous feeding bioreactor. Fluorescence in situ hybridization, 16S rRNA gene amplicon sequencing and qPCR of functional genes were utilized to monitor the growth of nitrifiers including comammox Nitrospira. Cloning of comammox amoA genes identified amoA phylogeny of enriched comammox Nitrospira. This work is an example demonstrating that continuous supply of low ammonium concentrations alongside biomass carriers is effective in cultivating comammox Nitrospira from engineered systems.

Application of stabilized hypobromite for controlling membrane fouling and N-nitrosodimethylamine formation.

Chloramination is a conventional and successful pre-disinfection approach to control biological fouling for reverse osmosis (RO) treatment in water reuse. This study aimed to evaluate the possibility of using a new disinfectant-stabilized hypobromite-in controlling membrane fouling and the formation of a particular carcinogenic disinfection byproduct (DBP)-N-nitrosodimethylamine (NDMA). Our accelerated chemical exposure tests showed that the new disinfectant reduced the permeability of a polyamide RO membrane permeability from 6.7 to 4.1 L/m2hbar; however, its treatment impact was equivalent to that of chloramine. The disinfection efficacy of stabilized hypobromite was greater than that of chloramine when evaluated with intact bacterial counts, which suggests its potential for mitigating membrane biofouling. Additional pilot-scale tests using synthetic wastewater demonstrated that pre-disinfection with the use of stabilized hypobromite inhibits membrane fouling. Among 13 halogenated DBPs evaluated, the formation of bromoform by stabilized hypobromite was higher than that by chloramine at a high dose of 10 mg/L, thus suggesting the need for optimizing chemical doses for achieving sufficient biofouling mitigation. NDMA formation upon stabilized hypobromite treatment in two different types of actual treated wastewaters was found to be negligible and considerably lower than that by chloramine treatment. In addition, NDMA formation potential by stabilized hypobromite was 2-5 orders of magnitude lower than that by chloramine. Our findings suggest the potential of using stabilized hypobromite for controlling NDMA formation and biofouling, which are the keys to successful potable water reuse.

A putative transcription regulator involved in the virulence attenuation of an acriflavine-resistant vaccine strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas.

Acriflavine, an acridine dye that causes frameshift mutations, has been used to attenuate various veterinary pathogens for the development of live vaccines. Erysipelothrix rhusiopathiae Koganei 65-0.15 strain (Koganei) (serovar 1a) is the acriflavine-resistant live vaccine currently used in Japan for the control of swine erysipelas. To investigate the attenuation mechanisms of the Koganei strain, we analyzed the draft genome sequence of the Koganei strain against the reference genome sequence of the E. rhusiopathiae Fujisawa strain (serovar 1a). The sequence analysis revealed a high degree of sequence similarity between the two strains and identified a total of 98 sequence differences within 80 protein-coding sequences. Among them, insertions/deletions (indels) were identified in 9 genes, of which 7 resulted in frameshift and premature termination. To investigate whether these mutations resulted in the attenuation of the Koganei strain, we focused on the indel mutation identified in ERH_0661, an XRE family transcriptional regulator. We introduced the mutation into ERH_0661 of the Fujisawa strain and restored the mutation of the Koganei strain. Animal experiments using the recombinant strains showed that mice survived inoculation with 103 colony forming units (CFUs) (equivalent to approximately 100 50% lethal doses [LD50] of the wild-type Fujisawa) of the recombinant Fujisawa strain, and the mice became ill after inoculation with 108 CFUs of the recombinant Koganei strain. These results suggest that the transcriptional regulator ERH_0661 is involved in the virulence of E. rhusiopathiae and that the ERH_0661 mutation is partially responsible for the attenuation of the Koganei strain.

Genome-Wide Identification of Virulence Genes in Erysipelothrix rhusiopathiae: Use of a Mutant Deficient in a tagF Homolog as a Safe Oral Vaccine against Swine Erysipelas.

Swine erysipelas is caused by the Gram-positive pathogen Erysipelothrix rhusiopathiae The swine erysipelas live vaccine in Japan, the E. rhusiopathiae Koganei 65-0.15 strain (Koganei), has been reported to cause arthritis and endocarditis. To develop a vaccine with increased safety, we used a virulent Fujisawa strain to construct transposon mutants for a total of 651 genes, which covered 38% of the coding sequence of the genome. We screened the mutants for attenuation by inoculating mice with 108 CFU of each mutant and subsequently assessed protective capability by challenging the surviving mice with 103 CFU (102 times the 50% lethal dose) of the Fujisawa strain. Of the 23 attenuated mutants obtained, 6 mutants were selected and evaluated for protective capability in pigs by comparison to that of the Koganei strain. A mutant in the ERH_0432 (tagF) gene encoding a putative CDP-glycerol glycerophosphotransferase was found to be highly attenuated and to induce humoral and cell-mediated immune responses in conventional pigs. An in-frame deletion mutant of the gene, the Δ432 mutant, was constructed, and attenuation was further confirmed in germfree piglets; three of four piglets subcutaneously inoculated with 109 CFU of the Δ432 mutant showed no apparent clinical symptoms, whereas all four of the Koganei-inoculated piglets died 3 days after inoculation. It was confirmed that conventional pigs inoculated orally or subcutaneously with the Δ432 strain were almost completely protected against lethal challenge infection. Thus, the tagF homolog mutant of E. rhusiopathiae represents a safe vaccine candidate that can be administered via the oral and subcutaneous routes.

Wild boars: A potential source of Erysipelothrix rhusiopathiae infection in Japan.

The potential role of wild boars as a source of erysipelas infection was investigated. An ELISA test of wild boar serum samples from 41 prefectures in Japan revealed that proportions of the Erysipelothrix rhusiopathiae-positive samples were very high in all the prefectures, and the mean positive rate was 95.6% (1312/1372). Serovars of E. rhusiopathiae isolates from wild boars were similar to those of previously reported swine isolates, and all serovar isolates tested were found to be pathogenic to mice. These results suggest that wild boars in Japan constitute a reservoir of E. rhusiopathiae and may pose risks to other animals.

Disassociation of Spa type and serovar of an Erysipelothrix rhusiopathiae serovar 6 strain isolated from a diseased pig.

The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae is an important component in protecting pigs against swine erysipelas. The Spa protein has been antigenically divided into 3 types: SpaA, SpaB, and SpaC. Swine erysipelas vaccines are formulated with strains of serovar 1 and/or 2, both of which are SpaA-possessing serovars. The association of Spa type with E. rhusiopathiae serovar has been reported, and therefore, the determination of the Spa type and the serovar of clinical isolates are important to assess vaccine efficacy. An E. rhusiopathiae strain, designated Ireland, was isolated from a diseased pig and identified as serovar 6 by a conventional agar gel precipitation test. Sequence analysis of the chromosomal locus presumably defining the serovar antigenicity of E. rhusiopathiae revealed that the gene content and organization of the chromosomal regions of the Ireland strain were identical to those of the serovar 6 reference strain (Tuzok). Sequence analysis of the spa gene and dot blots using a SpaA-specific monoclonal antibody confirmed that, unlike the Tuzok strain possessing SpaB, the Ireland strain expressed SpaA, indicating that the Spa type is not associated with the serovar in this strain. Thus, further investigation into the association between Spa type and serovar of clinical swine isolates is warranted.

Draft Genome Sequences of Lawsonia intracellularis Swine Strains Causing Proliferative Enteropathy in Japan.

The draft genome sequences of three strains of Lawsonia intracellularis, an obligate intracellular animal pathogen responsible for causing proliferative enteropathy, obtained from swine in different prefectures in Japan revealed the absence of a genomic island previously reported to be linked to host adaptation and to high genomic diversity, despite geographical proximity.

Identification of serovar 1a, 1b, 2, and 5 strains of Erysipelothrix rhusiopathiae by a conventional gel-based PCR.

Among the four species of the genus Erysipelothrix, Erysipelothrix rhusiopathiae is the main species that causes disease in swine and poultry and has also been isolated from human patients. Recently, E. rhusiopathiae infections in domesticated animals have increased in many countries and are also the cause of emerging wildlife disease in arctic and boreal ecosystems. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by their serovars, which are determined based on cell wall antigens. Serotyping of Erysipelothrix is important, as specific E. rhusiopathiae serovars (1a, 1b, and 2) are associated with disease in pigs, poultry, and humans. However, serotyping is laborious and time-consuming and requires a full set of serovar reference strains and strain-specific antiserum. In this study, to develop a conventional gel-based PCR assay that can detect the main disease-associated serovars of E. rhusiopathiae, the draft genome sequences of E. rhusiopathiae strains of serovars 1a, 1b, 2, and 5, the last of which is often isolated from wild animals, were analyzed. Primers were designed based on the serovar-specific sequences of the strains and tested for field strains isolated from extensive origins. Among two hundred and ninety-seven isolates of various serovar strains of E. rhusiopathiae and other Erysipelothrix species, the PCR assay identified serovar 1a, 1b, 2, and 5 strains of E. rhusiopathiae. This conventional gel-based PCR assay should be useful for serovar surveillance of E. rhusiopathiae isolates in domesticated and wild animals as well as in humans.

Identification of the Chromosomal Region Essential for Serovar-Specific Antigen and Virulence of Serovar 1 and 2 Strains of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae causes swine erysipelas, an infection characterized by acute septicemia or chronic endocarditis and polyarthritis. Among 17 E. rhusiopathiae serovars, determined based on heat-stable peptidoglycan antigens, serovars 1 and 2 are most commonly associated with the disease; however, the molecular basis for the association between these serovars and virulence is unknown. To search for the genetic region defining serovar 1a (Fujisawa) strain antigenicity, we examined the 15-kb chromosomal region encompassing a putative pathway for polysaccharide biosynthesis, which was previously identified in the E. rhusiopathiae Fujisawa strain. Six transposon mutants of Fujisawa strain possessing a mutation in this region lost antigenic reactivity with serovar 1a-specific rabbit serum. Sequence analysis of this region in wild-type strains of serovars 1a, 1b, and 2 and serovar N, which lacks serovar-specific antigens, revealed that gene organization was similar among the strains and that serovar 2 strains showed variation. Serovar N strains displayed the same gene organization as the serovar 1a, 1b, or 2 strain and possessed certain mutations in this region. In two of the analyzed serovar N strains, restoration of the mutations via complementation with sequences derived from serovar 1a and 2 strains recovered antigenic reactivity with 1a- and 2-specific rabbit serum, respectively. Several gene mutations in this region resulted in altered capsule expression and attenuation of virulence in mice. These results indicate a functional connection between the biosynthetic pathways for the capsular polysaccharide and peptidoglycan antigens used for serotyping, which may explain variation in virulence among strains of different serovars.

In vivo effect of a TLR5 SNP (C1205T) on Salmonella enterica serovar Typhimurium infection in weaned, specific pathogen-free Landrace piglets.

Toll-like receptor 5 is a pattern-recognition receptor for bacterial flagellin. We previously reported that a single nucleotide polymorphism (SNP) of swine TLR5, C1205T, impairs recognition of Salmonella typhimurium (ST) flagellin and ethanol-killed Salmonella Choleraesuis (SC). In the present study, weaned, specific pathogen-free (SPF) Landrace piglets with CC, CT or TT genotypes were orally infected with ST (L-3569 strain) to determine the effect of this specific SNP on ST infection in vivo. Eighteen ST-infected piglets (six each with CC, CT, or TT) exhibited fever and diarrhea for 1 week after infection. TT piglets had the longest duration of fever. TT piglets had the greatest mean diarrhea score during the experimental period, followed by CT and CC piglets. Fecal ST shedding was greater in CT and TT pigs than CC pigs from 2 days after infection. Serum haptoglobin concentration increased in ST-infected piglets and to greater extents in CT and TT pigs than CC pigs. Daily weight gain was lower in infected pigs, particularly TT piglets, than control pigs. To the best of our knowledge, this study is the first to demonstrate that impairment of TLR recognition affects pig susceptibility to disease in vivo. Thus, piglets with the T allele of swine TLR5 (C1205T) exhibit impaired resistance to ST infection. Furthermore, elimination of the T allele of this SNP from Landrace pigs would lead to enhancement of their resistance to ST infection.

High-rate nitrification of electronic industry wastewater by using nitrifying granules.

Nitrifying granules have a high sedimentation property and an ability to maintain a large amount of nitrifying bacteria in a reaction tank. Our group has examined the formation process of nitrifying granules and achieved high-rate nitrification for an inorganic synthetic wastewater using these granules. In this research, a pilot-scale test plant with an 850-liter reaction tank was assembled in a semiconductor manufacturing factory in order to conduct a continuous water conduction test using real electronics industry wastewater. The aim was to observe the formation of nitrifying granules and determine the maximum ammonia removal rate. The average granule diameter formed during the experiment was 780 µm and the maximum ammonia removal rate was observed to be 1.5 kgN·m-3·day-1 at 20 °C, which is 2.5-5 times faster than traditional activated sludge methods. A fluorescence in situ hybridization analysis showed that ß-proteobacterial ammonia oxidizing bacteria and the Nitrospira-like nitrite-oxidizing bacteria dominate the bacteria population in the granules, and their strong aggregation capacity might confer some benefits to the formation of these nitrifying granules.

Multiplex PCR assay for the simultaneous detection and differentiation of clonal lineages of Erysipelothrix rhusiopathiae serovar 1a strains currently circulating in Japan.

The species Erysipelothrixrhusiopathiae displays genetic heterogeneity; however, E. rhusiopathiae serovar 1a strains currently circulating in Japan exhibit remarkably low levels of genetic diversity and group into clonal sublineages of Lineage IVb (IVb-1 and IVb-2). In the present study, based on whole genome sequencing data, we designed primers for a multiplex PCR assay to simultaneously detect and differentiate the sublineages of E. rhusiopathiae strains. Among the one hundred and twenty-seven isolates of various serovar strains, including isolates from a wide range of hosts and geographic origins, the PCR assay could successfully detect and differentiate the serovar 1a strains belonging to the sublineages.