Genome Sequence of Paenibacillus alvei DSM 29, a Secondary Invader during European Foulbrood Outbreaks

Paenibacillus alvei is known as a secondary invader during European foulbrood of honeybees. Here, we announce the 6.83-Mb draft genome sequence of P. alvei type strain DSM 29. Putative genes encoding an antimicrobial peptide, a binary toxin, a mosquitocidal toxin, alveolysin, and different polyketides and nonribosomal peptides were identified.     

The multiplication and spread of sacbrood virus of bees

Sacbrood virus multiplied without causing symptoms in adult bees when it was injected into them or when it was fed to young individuals. More virus accumulated in heads of infected bees than elsewhere in their bodies, and much was in their hypopharyngeal glands. The extract of each infected head contained about 10²medial lethal doses (LD 50s) of sacbrood virus for larvae when given in their food. The infective dose of sacbrood virus by injection for adults was about 10^-4of the LD 50 in food for larvae. The infective dose by mouth for adults was about 10²LD 50s for larvae, but bees older than 4–8 days could not be infected this way. Infection did not spread between adults but is probably transmitted in nature from infected adults to larvae and back to young adults that ingest remains of larvae killed by sacbrood. The youngest adult bees infected with sacbrood ate little pollen and lived only about 3 weeks in the laboratory, as did bees receiving no pollen, whereas bees fed with ample pollen lived 9 weeks.     

Gut flora of Galleria mellonella suppressing ingested bacteria.

Streptococcus faecalis, the only bacterium occurring almost invariably at high populations in guts of Galleria mellonella larvae, suppresses bacteria ingested with food by producing bacteriocin, an antibioticlike substance having a narrow range of bactericidal activity, and by releasing a lysozymelike enzyme, especially in the presence of proteolytic enzymes. The insect intestinal fluid apparently increases the activity of S. faecalis lytic enzyme. Unlike other organisms tested, S. faecalis has shown a strong bactericidal action against various species of unrelated bacteria. Microscopical examination of the sensitive organisms used as indicators has revealed changes resembling formation of protoplasts, gradually leading to destruction of bacterial cells. The insect guts could not be infected, even when the larvae had ingested a high dose of Pseudomonas aeruginosa, Proteus mirabilis, or Bacillus thuringiensis. The mechanism by which S. faecalis could suppress the ingested bacteria is suggested.     

Lysozymelike lytic enzyme of Streptococcus faecalis and its role in the larval development of wax moth, Galleria mellonella

The predominant type of bacteria present in the gut of larval Galleria mellonella were streptococci group D identified as Streptococcus faecalis which showed bacteriolytic activity. Young larvae usually contained mixed populations with a marked dominance of fecal streptococci while normally developed mature larvae most frequently contained large uniform populations of S. faecalis. Pupal stages were found to contain the highest percentage of individuals with pure cultures of fecal streptococci.The author suggests a hypothesis that, owing to its bacteriolytic properties, S. faecalis can be considered as a component of the natural, nonspecific defense mechanism of G. mellonella against bacterial infections. The lytic enzyme released in the exponential growth phase of S. faecalis participates in the selection process stabilizing the microbial flora of wax moth larvae; it limits the population of other forms of bacteria. Larval resistance to bacterial infections to a large extent depends on the magnitude of the populations and thus on S. faecalis muramidase concentration. Bacterial lysozyme inhibited the growth of the ingested organisms and in consequence it prevented the proliferation of undesired bacteria in the digestive tract of Galleria larvae.The lytic enzyme proved to be identical with autolysin, a β-N-acetylmuramide glycanhydrolase (EC 3.2.1.17) which has been isolated from trypsin-speeded wall autolysates of S. faecalis by Shockman and Cheney (1969).     

Survey of six bee viruses using RT-PCR in Northern Thailand

Six honey bee viruses were surveyed using RT-PCR in Northern Thailand where about 80% of Thai apiaries are located. Tested samples were found to be positive for deformed wing virus (DWV), acute bee paralysis virus (ABPV), sacbrood virus (SBV) and Kashmir bee virus (KBV). In the collected samples, neither chronic bee paralysis virus nor black queen cell virus nucleic acids could be detected. It was found that DWV was the most widespread and ABPV was the second most prevalent. Kashmir bee virus was found only in the Lampang province where high infestation of Varroa destructor mite occurred. Tropilaelaps, European foulbrood, and Chalkbrood diseases were found in some apiaries.     

Yeastlike fungi from greater wax moth larvae (Galleria mellonella) fed antibiotics

The effect of the antibiotics oxytetracycline, chloramphenicol, and penicillin on the gut flora of Galleria mellonella larvae was not only to suppress Streptococcus faecalis, a typical gut organism of all stages of the moth, but also simultaneously to cause an increase in the number of yeastlike fungi, Candida guilliermondi, Candida krusei, and Geotrichum candidum. Nystatin prevented or minimized yeast multiplication. Most pupae and adults were sterile or contained only S. faecalis, even when prepupae had contained many fungi. A combination of oxytetracycline-nystatin in a total dosage of 1 and 3 × MIC/cm² of honeycomb surface, respectively, reduced both S. faecalis and fungal counts, so that after a 3-day incubation, most of the larvae were sterile or near sterile.     

Biocidal properties of maltose reduced silver nanoparticles against American foulbrood diseases pathogens

Bee disease caused by spore-forming Paenibacillus larvae and Paenibacillus alvei is a serious problem for honey production. Thus, there is an ongoing effort to find an effective agent that shows broad biocidal activity with minimal environmental hazard. In this study, the biocidal effect of maltose reduced silver nanoparticles (AgNPs) is evaluated against American foulbrood and European foulbrood pathogens. The results demonstrate that the maltose reduced AgNPs are excellent short and long-term biocides against P. larvae isolates. The long-term effect suggests that the Ag⁺ ions are released from the AgNPs with increasing time in a controlled manner.     

Observations on the synthesis and destruction of tyrosine by Streptococcus faecalis R

Streptococcus faecalis R when grown in a double-strength medium rendered the medium deficient in folic acid and tyrosine. The organism was found to be capable of removing approximately ten times as much folic acid from the medium during the 24-hr. incubation period as it requires for maximum growth.Although Streptococcus faecalis R achieved maximum growth in the absence of added tyrosine if pyridoxal was present in the medium, the growth response obtained in the presence of tyrosine was proportional to the amount of tyrosine added at concentrations of tyrosine greater than 10 μ./10 ml. medium.Growth of S. faecalis R in the presence but not in the absence of pyridoxal caused a tyrosine deficiency to develop in the double-strength medium.     

The bacterial flora of the midgut of two Danish populations of healthy fifth instar larvae of the turnip moth, Scotia segetum

Examination of the midgut bacteria of two Danish populations of healthy fifth instar turnip moth larvae, Scotia (=Agrotis) segetum, living on potatoes and celery gave the following results. The total number of living microorganisms in the midgut varied between 1.0 × 10⁴ and 4.0 × 10⁵. Larvae from celery in N. W. Zeeland always contained Streptococcus faecalis and six members of Enterobacteriaceae, viz., Citrobacter freundii, Klebsiella pneumoniae, Hafnia alvei, Proteus mirabilis, P. vulgaris, and Erwinia amylovora. In larvae from potatoes in E. Jutland, the species consistently present were Streptococcus faecalis and four species of Enterobacteriaceae, viz., Escherichia coli, Erwinia amylovora, E. carotovora var. atroseptica, and one other, probably a member of the E. carotovora group. Streptococcus faecalis is supposed to occur as a mutualist in the alimentary tract, suppressing Gram-positive bacteria.     

A PCR Detection Method for Rapid Identification of Melissococcus pluton in Honeybee Larvae

Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.     

Antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases and finding out new strain of Serratia marcescens via DGGE-PCR,as a secondary infection on honeybee larvae

American foulbrood (AFB) and European foulbrood (EFB) are the two major bacterial diseases affecting honeybees, leading to a decrease in viability of the hive, decreasing honey production, and resulting in significant economic losses to beekeepers. Due to the inefficiency and/or low efficacy of some antibiotics, researches with nanotechnology represent, possibly, new therapeutic strategies. Nanostructure drugs have presented some advantagesover the conventional medicines, such as slow, gradual and controlled release, increased bioavailability, and reduced side-effects. In this study, different infected larvae were collected from two apiaries; the combs that had symptoms of American and European foulbrood were isolated. In vitro antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases were characterized using UV–Vis spectrophotometry and scanning electron microscope (SEM) that proves the formation of silver nanoparticles with size range 160–660 nm. The antimicrobial activity of the silver nanoparticles was tested using agar diffusion assay and proved their ability to effectively cease the pathogenic bacterial growth in both AFB and EFB. DGGE-PCR technique has been applied for the identification of un-common bacterial infections honeybees depending on 16S rRNA amplification from their total extracted DNA and has been identified as Serratia marcescens (TES), deposited in GenBank with a new accession number (MT240613). The results were confirmed strain has been detected by DGGE-PCR analysis causing uniquely infected brood that was attacked by the American Foulbrood It could be concluded that greenly synthesized silver nanoparticles is projected to be used as effective treatment for honeybee bacterial diseases. These material need more investigations under field conditions and study the possibility of its residues in honeybee products such as honey, and beeswax.     

Transpuparial transmission of Kashmir bee virus and sacbrood virus in the honey bee (Apis mellifera)

When particles of Kashmir bee virus (KBV) and sacbrood virus (SBV) were fed to larvae of the honey bee, Apis mellifera, in Australian colonies, the resulting pupae became in apparently infected. There was no statistically significant difference in the susceptibility of 1, 2, 3 or 4-day-old larvae for either virus, but 5-day-old larvae were significantly less susceptible to SBV than younger larvae. There was no significant difference in the proportions of pupae that became in apparently infected when, as larvae, they were fed various concentrations of each virus, but significantly more larvae were removed from their cells when fed concentrated preparations of each virus than when fed diluted preparations. Susceptible larvae that became in apparently infected with KBV and SBV developed normally into in apparently infected pupae and later, emerged as in apparently infected worker bees.     

Genetic and Biochemical Diversity among Isolates of Paenibacillus alvei Cultured from Australian Honeybee (Apis mellifera) Colonies

Twenty-five unique CfoI-generated whole-cell DNA profiles were identified in a study of 30 Paenibacillus alvei isolates cultured from honey and diseased larvae collected from honeybee (Apis mellifera) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with DraI, HinfI, CfoI, AluI, FokI, and RsaI. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in the HinfI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using DraI, FokI, and HinfI differentiated P. alvei from the phylogenetically closely related species Paenibacillus macerans and Paenibacillus macquariensis. Alveolysin gene fragments (length, 1,555 bp) were enzymatically amplified from some of the P. alvei isolates (19 of 30 isolates), and RFLP were detected by using the enzymes CfoI, Sau3AI, and RsaI. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%) P. alvei whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28 P. alvei isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and Voges-Proskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity in P. alvei isolates may be a reflection of adaptation to the special habitats in which they originated.     

Presence of Acylated Homoserine Lactones (AHLs) and AHL-Producing Bacteria in Meat and Potential Role of AHL in Spoilage of Meat

Quorum-sensing (QS) signals (N-acyl homoserine lactones [AHLs]) were extracted and detected from five commercially produced vacuum-packed meat samples. Ninety-six AHL-producing bacteria were isolated, and 92 were identified as Enterobacteriaceae. Hafnia alvei was the most commonly identified AHL-producing bacterium. Thin-layer chromatographic profiles of supernatants from six H. alvei isolates and of extracts from spoiling meat revealed that the major AHL species had an R_f value and shape similar to N-3-oxo-hexanoyl homoserine lactone (OHHL). Liquid chromatography-mass spectrometry (MS) (high-resolution MS) analysis confirmed the presence of OHHL in pure cultures of H. alvei. Vacuum-packed meat spoiled at the same rate when inoculated with the H. alvei wild type compared to a corresponding AHL-lacking mutant. Addition of specific QS inhibitors to the AHL-producing H. alvei inoculated in meat or to naturally contaminated meat did not influence the spoilage of vacuum-packed meat. An extracellular protein of approximately 20 kDa produced by the H. alvei wild-type was not produced by the AHL-negative mutant but was restored in the mutant when complemented by OHHL, thus indicating that AHLs do have a regulatory role in H. alvei. Coinoculation of H. alvei wild-type with an AHL-deficient Serratia proteamaculans B5a, in which protease secretion is QS regulated, caused spoilage of liquid milk. By contrast, coinoculation of AHL-negative strains of H. alvei and S. proteamaculans B5a did not cause spoilage. In conclusion, AHL and AHL-producing bacteria are present in vacuum-packed meat during storage and spoilage, but AHL does not appear to influence the spoilage of this particular type of conserved meat. Our data indicate that AHL-producing H. alvei may induce food quality-relevant phenotypes in other bacterial species in the same environment. H. alvei may thus influence spoilage of food products in which Enterobacteriaceae participate in the spoilage process.     

Prevention of Chinese Sacbrood Virus Infection in Apis cerana using RNA Interference

Chinese sacbrood virus (CSBV) is the pathogen of Chinese sacbrood disease, which poses a serious threat to honeybee Apis cerana, and tends to cause bee colony and even the whole apiary collapse. Here we report on prevention of CSBV infection by feeding second instar larvae of A. cerana with specific sequences of CSBV double-stranded RNA (dsRNA). Protection of the bee larvae from CSBV by ingestion of CSBV-derived dsRNA was further demonstrated by quantitative real-time PCR (qRT-PCR) and northern blot analysis. The result provides a potential method to protect A. cerana from CSBV infection.     

Effect of a nuclear polyhedrosis virus on the relationship between Trichoplusia ni (Lepidoptera: Noctuidae) and the parasite, Hyposoter exiguae (hymenoptera: Ichneumonidae)

The effect of a nuclear polyhedrosis virus on the relationship between Trichoplusia ni and the parasite, Hyposoter exiguae, was investigated to determine if the virus could invade and multiply in the tissues of the parasites, if parasites which emerged from virus-infected T. ni larvae had normal emergence, fecundity, and longevity, and if the parasite could serve as a vector for the virus. Light microscopy revealed particles which appeared to be polyhedra within the lumen of the midgut of parasite larvae from virus-infected hosts. Transmission electron microscopy confirmed the presence of polyhedra and free virions within the midgut of the larvae. Polyhedra or free virions were never found within any parasite tissues. Parasite larvae within hosts exposed to virus before parasitization perished when their hosts died of virus infection. Parasite larvae in hosts exposed to virus after parasitization completed their development before their hosts died of virus infection. The proportion of parasites which survived increased as the time between host parasitization and host virus exposure increased. Parasite larvae which developed in hosts exposed to the virus soon after parasitization spent significantly less time in their hosts than did parasites which developed in noninfected hosts. There was no significant difference in time spent in the pupal stage, percent adult emergence, adult longevity with and without food and water, and fecundity of parasites which developed in virus-infected hosts and those which developed in noninfected hosts. Female parasites laid as many eggs in virus-infected hosts as they did in noninfected hosts. Sixty percent of the female parasites which oviposited in virus-infected hosts vectored infective doses of virus to an average of 6% of the healthy hosts subsequently exposed to them. None of the healthy host larvae exposed to male parasites which had been exposed to virus-infected host larvae became infected with the virus. Forty percent of the female parasites which developed in virus-infected hosts transmitted infective doses of the virus to an average of 65% of the healthy host larvae exposed to them. Ninety percent of the male parasites which developed in virus-infected hosts transferred infective doses of the virus to an average of 21% of the healthy host larvae exposed to them.     

The incidence of virus diseases in the honey bee

Chronic bee-paralysis virus and sacbrood virus occur commonly in apparently normal honey-bee colonies in Britain. Most sick adult bees not affected by Nosema apis, Malpighamoeba mellificae or Acarapis woodi have chronic paralysis and most dead larvae not affected by micro-organisms have sacbrood. Both virus diseases are probably limited by hereditary factors, but unknown environmental factors also seem to influence disease. Paralysed bees from Australia, Canada, Eire, Germany and Mexico were found to be infected with chronic paralysis virus.     

Growth of Streptococcus faecalis var. liquefaciens on Plants

The proliferation of Streptococcus faecalis var. liquefaciens on two varieties of beans, and on corn, rye, and cabbage was investigated. Comparisons were made with growth patterns on these same plants exhibited by S. lactis and Lactobacillus plantarum. The ability of each of the bacteria to multiply and to spread to new plant parts as they developed from seed was studied under several environmental conditions. Plants were grown aseptically in glass culture and in sterilized and non-sterilized soil in the greenhouse. Quantitative estimations of increase in bacterial numbers were made. S. faecalis established commensal growth on each of five plants, although selectivity was noted for some plant parts. The organism increased in numbers on the plants equally as well as did the control bacteria, both alone, and in competition with the control bacteria and the microflora of the soil.     

Enterococci in Insects

Enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 10³ to 3 × 10⁷/g of insect. Insects were taken only from nonurban, wild, and cultivated fields and woods. In species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. Enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their role as overwintering agents. They were generally present in species feeding on nectar, succulent plant parts, and on and ir forest litter, but not from insects feeding on less succulent leaves and stems. Streptococcus faecalis was recovered from 32%, Streptococcus faecium from 22.4%, and Streptococcus faecium var. casseliflavus from 43.5% of members of the 37 taxa of insects. S. faecalis and S. faecium var. casseliflavus exhibit a high percent of conformity to the properties published for them. The heterogeneity in properties of S. faecium is similar to that found for the species taken from plants. Many fail to grow in broth at 45 C or in broth containing 6.5% NaCl; 50% of the cultures ferment both melezitose and melibiose, and a few ferment neither sugar. The remainder ferment melibiose only. Failure to reduce methylene blue in milk by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, following the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution.     

蜜蜂幼虫粪肠球菌的分离与鉴定

在对患有欧洲幼虫腐臭病的蜜蜂幼虫进行细菌分离与培养时,获得1株未知菌株。从分离培养特性、形态学、生理生化特性和分子生物学等方面对该菌株进行了鉴定,得知该菌株为欧洲幼虫腐臭病的次生菌——粪肠球菌,属于肠球菌属,并暂定名为Enterococcus faecalis FB102。同时,得知根据该菌的分离培养特性可与欧洲幼虫腐臭病病原区分,并且将其单独接种蜜蜂幼虫后未能使幼虫患病。根据粪肠球菌较欧洲幼虫腐臭病病原易于分离培养的特性,得知通过对粪肠球菌的鉴定可以简化欧洲幼虫腐臭病的诊断过程,而且推测该菌株可能对人体健康有一定的影响。