Quantitative tyramine analysis method for Apis mellifera larvae infected with Melissococcus plutonius, the causative agent of European foulbrood.

Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.

Improvement of the mismatch amplification mutation assay-PCR for discrimination between Streptococcus suis serotypes 2 and 1/2.

A mismatch amplification mutation assay (MAMA)-PCR, which detects a single-nucleotide polymorphism contributed to serological difference between Streptococcus suis serotypes 2 and 1/2, is used to discriminate between these serotypes. The present study reports unusual serotype 1/2 isolates untypable by the MAMA-PCR and improvement of the MAMA-PCR for typing such isolates.

Streptococcus suis surface-antigen recognition by antibodies and bacterial elimination is influenced by capsular polysaccharide structure.

Streptococcus suis is an encapsulated bacterium causing severe diseases in swine. Here, we compared the protective properties of the capsular polysaccharide (CPS) of different S. suis serotypes by using serotype-switched mutants in a mouse model of infection. CPS structure influenced bacterial survival in mice, antibody binding, and antibody-mediated bacterial killing. The CPS of serotypes 3, 4 and 14 allowed more antibody binding and bacterial elimination than the CPS of serotypes 2, 7 and 9. Results suggest that the different CPS structures of S. suis provide varying levels of protection by influencing antigen availability and elimination by the host immune system.

Atypical Melissococcus plutonius strains: their characteristics, virulence, epidemiology, and mysteries.

Melissococcus plutonius is a Gram-positive lanceolate coccus that is the causative agent of European foulbrood, an important bacterial disease of honey bee brood. Although this bacterium was originally described in the early 20th century, a culture method for this bacterium was not established until more than 40 years after its discovery due to its fastidious characteristics, including the requirement for high potassium and anaerobic/microaerophilic conditions. These characteristics were considered to be common to the majority of M. plutonius strains isolated worldwide, and M. plutonius was also thought to be genetically homologous or clonal for years. However, non-fastidious variants of this species (designated as atypical M. plutonius) were very recently identified in Japan. Although the morphology of these unusual strains was similar to that of traditionally well-known M. plutonius strains, atypical strains were genetically very different from most of the M. plutonius strains previously isolated and were highly virulent to individual bee larva. These atypical variants were initially considered to be unique to Japan, but were subsequently found worldwide; however, the frequency of isolation varied from country to country. The background of the discovery of atypical M. plutonius in Japan and current knowledge on atypical strains, including their biochemical and culture characteristics, virulence, detection methods, and global distribution, are described in this review. Remaining mysteries related to atypical M. plutonius and directions for future research are also discussed.

Development of a Streptococcus pluranimalium-specific PCR assay.

Streptococcus pluranimalium, an emerging zoonotic pathogen associated with infections in various animal species and humans, cannot be reliably identified by phenotypic characterization using the commercial kits routinely used in laboratories. We herein developed the first S. pluranimalium-specific PCR assay useful for the easy and reliable identification of this species.

A calf with hind limb paralysis and dysstasia and a genome sequence analysis of an isolated Clostridium perfringens toxinotype E strain.

Clostridium perfringens toxinotype E infections are rare in calves, and the development of intestinal lesions were commonly observed. In 2012, a 6-day-old calf in Japan exhibited swelling with emphysema on the right gluteal region, sudden paralysis of the hind limb and dysstasia. A pathological examination revealed myositis of the gluteal muscle and neuritis of the ischiatic nerve. C. perfringens type E strain CP118 was isolated from the affected muscle. However, the intestinal symptoms and lesions that commonly develop in type E infections in calves were not detected in the present case. Genome analyses revealed that CP118 possessed 16 virulence-related genes, including enterotoxin, and was closely related to other type E and F strains. Particularly, CP118 was more closely related to type E strains from humans, including a food poisoning case, than calf isolates, suggesting its potential to cause food poisoning in humans and, thus, its importance as a potential risk to public health. Since CP118 did not possess the reported toxin genes associated with neuropathy, pyogenic inflammation caused by CP118 and/or other bacteria may have damaged the ischiatic nerve, resulting in neuropathy. Alternatively, unidentified CP118 toxins may have caused the neuropathy. This is the first study to report C. perfringens type E infection with peripheral neuropathy. The distribution of all the reported virulence-related genes in the C. perfringens population as well as the details of this rare case will provide further insights into C. perfringens type E infections.

Disinfectant resistance of Salmonella in in vitro contaminated poultry house models and investigation of efficient disinfection methods using these models.

Salmonellaenterica subsp. enterica (Salmonella) shows disinfectant resistance by forming biofilms on solid surfaces. However, efficient disinfection methods to eliminate Salmonella biofilms from farms have not yet been examined in detail. In this study, more than 80% of Salmonella strains from farms in Yamagata prefecture, Japan, were biofilm producers. Regardless of the extent of their biofilm formation ability, their biofilms were highly resistant to hypochlorous acid on plastic surfaces. To establish efficient disinfection methods in farms, we developed in vitro Salmonella-contaminated poultry house models by depositing dust on ceramic and stainless-steel carriers in poultry houses for one month and culturing a representative Salmonella strain on the carriers. Biofilm-like structures, including Salmonella-like cells, were observed on the models by scanning electron microscopy. Salmonella was not efficiently removed from the models even by cleaning with a surfactant at 25/65°C and disinfection with quaternary ammonium compound or hypochlorous acid at 25°C; on the contrary, viable Salmonella cells increased in some tests under these conditions, suggesting that these models successfully simulate the highly persistent characteristics of Salmonella in farms. However, the persistent bacterial cells were markedly decreased by soaking in 65°C surfactant followed by rinsing with 80°C water, additional cleaning using chlorine dioxide or disinfection with dolomitic lime, suggesting the effectiveness of these methods against Salmonella in farms. Since many different disinfection conditions may be easily tested in laboratories, our models will be useful tools for establishing effective and practical disinfection methods in farms.

Antimicrobial resistance and associated genetic background of Histophilus somni isolated from clinically affected and healthy cattle.

Histophilus somni, a member of the Pasteurellaceae family, causes various diseases, including thrombotic meningoencephalitis and respiratory diseases. Here, 166 isolates recovered from Japanese cattle with various diseases between the late 1970s and the 2010s were subjected to susceptibility testing against 14 antimicrobials (ampicillin, amoxicillin, cefazolin, ceftiofur, kanamycin, streptomycin, nalidixic acid, enrofloxacin, danofloxacin, florfenicol, erythromycin, tylosin, oxytetracycline, and fosfomycin). The proportions of antimicrobial-resistant/intermediate isolates were low in the total isolates, with resistance rates ranging from 0% for ceftiofur and florfenicol to 13.2% for ampicillin. However, relatively high minimum inhibitory concentrations (MICs) and resistance/intermediate rates were observed in the isolates from cattle with respiratory diseases; i.e., 21/53 isolates (39.6%) showed resistance or intermediate to one or more antimicrobials for treatment of respiratory diseases, and the resistance/intermediate rates to oxytetracycline, kanamycin, ampicillin, amoxicillin, nalidixic acid, and danofloxacin were 28.3, 24.5, 24.5, 13.2, 1.9, and 1.9%, respectively. Isolates with high MICs tended to possess antimicrobial resistance genes, which may confer antimicrobial resistance phenotypes. In particular, all isolates with MICs of ampicillin/amoxicillin, kanamycin, and oxytetracycline ≥2 µg/mL, ≥512 µg/mL, and ≥4 µg/mL possessed bla ROB - 1, aphA-1, and tetH/tetR, respectively, whereas isolates whose MICs were lower than the above-mentioned values did not possess these resistance genes. These results suggest that the resistance genes detected in this study are primarily responsible for the reduced susceptibility of H. somni strains to these antimicrobials. As integrative and conjugative element (ICEs)-associated genes were detected only in genetically related isolates possessing antimicrobial resistance genes, ICEs may play an important role in the spread of resistance genes in some genetic groups of H. somni strains.

Whole-Genome Sequences of Bacillus and Paenibacillus sp. Strains Isolated from Honey in Japan.

Knowledge about bacterial species in bee environments is essential for maintaining healthy honeybee colonies. Therefore, we performed whole-genome sequence analysis on bacteria isolated from honey harvested in Japan. This study reports the genomic sequences of the five bacterial strains identified.

Detection of macrolide resistance genes, ermC and ermB, in Japanese honey using real-time PCR assays.

American foulbrood (AFB) is a honeybee disease caused by Paenibacillus larvae, and tylosin is used as the prophylactic in Japan. Honey contains macrolide-resistant bacteria that are a potential source of genes that may confer tylosin resistance to P. larvae. To investigate the potential risk of such genes in Japanese honey, we developed real-time PCR assays for the detection of important macrolide resistance genes, ermC and ermB, and analyzed 116 Japanese honey samples with known contamination status of P. larvae. Consequently, 91.38% of samples contained ermC and/or ermB, and 71.55% of samples contained both ermC and P. larvae, suggesting the possible emergence of tylosin-resistant P. larvae in Japan. Therefore, judicious use of the prophylactic is essential in maintaining its effectiveness.

A novel multiplex PCR assay to detect and distinguish between different types of Paenibacillus larvae and Melissococcus plutonius, and a survey of foulbrood pathogen contamination in Japanese honey.

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.

Suppurative meningoencephalitis and perineuritis caused by Streptococcus gallolyticus in a Japanese Black calf.

A 179-day-old calf, which was weak and stunted, showed neurological signs and was euthanized. Postmortem examination revealed extensive and severe cloudy area in the meninges, and pleural pneumonia. Gram-positive cocci were isolated from systemic organs. Biochemical and 16S rRNA gene sequence analyses identified the isolate as Streptococcus gallolyticus, and its subspecies was suggested to be gallolyticus (SGG). The isolate was classified as a novel sequence type (ST115) by the multilocus sequence typing scheme for SGG and showed susceptibility to penicillin, ampicillin, amoxicillin, florfenicol, sulfamethoxazole-trimethoprim, and chloramphenicol. Histopathologically, suppurative meningoencephalitis and perineuritis were detected. As SGG has been isolated solely from a cow with mastitis in Japan, this is the first SGG infection in a calf with suppurative meningoencephalitis and perineuritis in this country.

Antimicrobial Resistance Genes in Bacteria Isolated From Japanese Honey, and Their Potential for Conferring Macrolide and Lincosamide Resistance in the American Foulbrood Pathogen Paenibacillus larvae.

American foulbrood (AFB) is the most serious bacterial disease of honey bee brood. Spores of the causative agent Paenibacillus larvae are ingested by bee larvae via brood foods and germinated cells proliferate in the larval midgut. In Japan, a macrolide antibiotic, tylosin, is used as the approved prophylactic for AFB. Although tylosin-resistant P. larvae has yet to be found in Japan, it may emerge in the future through the acquisition of macrolide resistance genes from other bacteria, and bacteria latent in brood foods, such as honey, may serve as a source of resistance genes. In this study, to investigate macrolide resistance genes in honey, we attempted to isolate tylosin-resistant bacteria from 53 Japanese honey samples and obtained 209 isolates from 48 samples in the presence of 1 µg/ml of tylosin. All isolates were Gram-positive spore-forming bacteria mainly belonging to genera Bacillus and Paenibacillus, and 94.3% exhibited lower susceptibility to tylosin than Japanese P. larvae isolates. Genome analysis of 50 representative isolates revealed the presence of putative macrolide resistance genes in the isolates, and some of them were located on mobile genetic elements (MGEs). Among the genes on MGEs, ermC on the putative mobilizable plasmid pJ18TS1mac of Oceanobacillus strain J18TS1 conferred tylosin and lincomycin resistance to P. larvae after introducing the cloned gene using the expression vector. Moreover, pJ18TS1mac was retained in the P. larvae population for a long period even under non-selective conditions. This suggests that bacteria in honey is a source of genes for conferring tylosin resistance to P. larvae; therefore, monitoring of bacteria in honey may be helpful to predict the emergence of tylosin-resistant P. larvae and prevent the selection of resistant strains.

Peritrophic matrix-degrading proteins are dispensable virulence factors in a virulent Melissococcus plutonius strain.

European foulbrood (EFB) caused by Melissococcus plutonius is a major bacterial disease of honey bees. Strains of the causative agent exhibit genetic heterogeneity, and the degree of virulence varies among strains. In bee larvae orally infected with the highly virulent strains, ingested bacterial cells colonize the larval midgut and proliferate within the sac of the peritrophic matrix (PM), a barrier lining the midgut epithelium. However, the barrier is degraded during the course of infection, and M. plutonius cells eventually directly interact with the midgut epithelium. As M. plutonius possesses genes encoding putative PM-degrading proteins (enhancin, a chitin-binding domain-containing protein and endo-α-N-acetylgalactosaminidase), we constructed PM-degrading protein gene-knockout mutants from a highly virulent M. plutonius strain and investigated their role in the pathogenesis of EFB. In larvae infected with the triple-knockout mutant, which has no PM-degrading protein genes, M. plutonius that proliferated in the larval midguts was confined to the sac of the PM. However, the midgut epithelial cells degenerated over time, and the mutant killed approximately 70-80% of bee brood, suggesting that although the PM-degrading proteins are involved in the penetration of the PM by M. plutonius, they are not indispensable virulence factors in the highly virulent M. plutonius strain.

Capsular polysaccharide switching in Streptococcus suis modulates host cell interactions and virulence.

The capsular polysaccharide (CPS) of Streptococcus suis defines various serotypes based on its composition and structure. Though serotype switching has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain that express the serotype 3, 4, 7, 8, 9, or 14 CPS. The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of epithelial cells, resistance to phagocytosis by macrophages, killing by whole blood, dendritic cell-derived pro-inflammatory mediator production and virulence using mouse and porcine infection models. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes, although some different mutations other than loci of CPS-related genes were found in each the serotype-switched mutant. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. These findings suggest that serotype switching can drastically alter S. suis virulence and host cell interactions.

Complete Genome Sequences of Two Streptococcus suis Strains Isolated from Asymptomatic Pigs.

Streptococcus suis is an important zoonotic pathogen that causes major economic problems in the pig industry worldwide and serious infections in humans, including meningitis and septicemia. Here, we report the complete genome sequences of two strains isolated from asymptomatic pigs.

Draft Genome Sequence of Brucella ceti Isolated in the Western Pacific Ocean.

In 2018, Brucella ceti was isolated from a bottlenose dolphin from the western Pacific Ocean. Here, we report a draft genome sequence of the isolate BD1442 of sequence type 27, which is the only sequence type known to have been isolated from human clinical cases.

Comparative Study of Immunogenic Properties of Purified Capsular Polysaccharides from Streptococcus suis Serotypes 3, 7, 8, and 9: the Serotype 3 Polysaccharide Induces an Opsonizing IgG Response.

Streptococcus suis is an encapsulated bacterium and one of the most important swine pathogens and a zoonotic agent for which no effective vaccine exists. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. In addition to the previously known serotypes 2 and 14, which are nonimmunogenic, we have recently purified and described the CPS structures for serotypes 1, 1/2, 3, 7, 8, and 9. Here, we aimed to elucidate how these new structurally diverse CPSs interact with the immune system to generate anti-CPS antibody responses. CPS-stimulated dendritic cells produced significant levels of C-C motif chemokine ligand 3 (CCL3), partially via Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88-dependent pathways, and CCL2, via TLR-independent mechanisms. Mice immunized with purified serotype 3 CPS adjuvanted with TiterMax Gold produced an opsonizing IgG response, whereas other CPSs or adjuvants were negative. Mice hyperimmunized with heat-killed S. suis serotypes 3 and 9 both produced anti-CPS type 1 IgGs, whereas serotypes 7 and 8 remained negative. Also, mice infected with sublethal doses of S. suis serotype 3 produced primary anti-CPS IgM and IgG responses, of which only IgM were boosted after a secondary infection. In contrast, mice sublethally infected with S. suis serotype 9 produced weak anti-CPS IgM and IgG responses following a secondary infection. This study provides important information on the divergent evolution of CPS serotypes with highly different structural and/or biochemical properties within S. suis and their interaction with the immune system.

Transcriptional regulator SpxA1a controls the resistance of the honey bee pathogen Melissococcus plutonius to the antimicrobial activity of royal jelly.

Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ-susceptible derivative from a highly RJ-resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10-hydroxy-2-decenoic acid. Moreover, the mutant became susceptible to low-pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild-type and spxA1a mutants revealed that 45 protein-coding genes were commonly upregulated in spxA1a-positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.

Development of a mismatch amplification mutation assay to correctly serotype isolates of Streptococcus suis serotypes 1, 2, 1/2, and 14.

Streptococcus suis is one of the most important bacterial swine pathogens worldwide and is an emerging pathogen in humans. There are 29 serotypes, and serotyping, which is based on the antigenicity of the capsular polysaccharide (CPS) or on its coding genes, is often part of routine identification and provides further information regarding S. suis virulence and zoonotic potential. Serotypes 2 and 14 possess high zoonotic potential, and serotype 1/2 is the serotype most frequently isolated from diseased pigs in North America. PCR has replaced antibody-based techniques to perform serotyping. However, traditional PCR is not able to differentiate serotype 2 from 1/2 and serotype 1 from 14, given that the only difference in the cps loci of those serotype pairs is a nonsynonymous single-nucleotide polymorphism. We developed a mismatch amplification mutation assay (MAMA)-PCR that was able to correctly serotype 148 isolates previously known to be serotypes 1, 2, 1/2, or 14. This technique will be highly useful in animal and human health laboratories performing PCR serotyping of S. suis isolates.