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.     

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.     

Skin-associated Bacillus, staphylococcal and micrococcal species from the house dust mite, Dermatophagoides pteronyssinus and bacteriolytic enzymes

Dust mites produce bacteriolytic enzymes, one of which belongs to the NlpC/P60 superfamily comprising bacterial and fungal proteins. Whether this enzyme is derived from the mite or from mite-associated microbes is unclear. To this end, the bacteriology of mites per se, and carpet and mattress dust from a group of asthmatic children and their parents was investigated. Dust from parents’ and children’s mattresses yielded significantly more colony forming units compared with dust from their corresponding carpets. Zymography demonstrated some dusts contained bacteriolytic enzymes, and in nine of the twelve dust samples from three of five houses examined, a prominent bacteriolytic band was obtained that corresponded to the mite band, although in one home, other lytic bands were detected. Fifty bacterial isolates were obtained from surface-sterilised, commercially obtained Dermatophagoides pteronyssinus. 16S rRNA, tuf and rpoB gene sequencing of nine Gram-positive isolates identified them as Bacillus cereus, B. licheniformis, Staphylococcus aureus, S. epidermidis, S. capitis and Micrococcus luteus, known human skin commensals. 16S rRNA sequence homologies of four of the nine isolates identified as B. licheniformis formed a distinct phylogenetic cluster. All species secreted lytic enzymes during culture although the lytic profiles obtained differed between the rods and the cocci, and none of the bands detected corresponded to those observed in dust or mites. In conclusion, mites harbour a variety of bacterial species often associated with human skin and house dusts contain bacteriolytic enzymes that may be mite-derived. The identification of a novel cluster of B. licheniformis isolates suggests an ecological adaptation to laboratory-reared D. pteronyssinus. It remains to be determined whether the previously described mite-associated 14 K lytic enzyme is derived from a microbial source.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

Galleria melonella as an experimental in vivo host model for the fish-pathogenic oomycete Saprolegnia parasitica

Oomycetes are eukaryotic pathogens infecting animals and plants. Amongst them Saprolegnia parasitica is a fish pathogenic oomycete causing devastating losses in the aquaculture industry. To secure fish supply, new drugs are in high demand and since fish experiments are time consuming, expensive and involve animal welfare issues the search for adequate model systems is essential. Galleria mellonella serves as a heterologous host model for bacterial and fungal infections. This study extends the use of G. mellonella for studying infections with oomycetes. Saprolegniales are highly pathogenic to the insects while in contrast, the plant pathogen Phytophthora infestans showed no pathogenicity. Melanisation of hyphae below the cuticle allowed direct macroscopic monitoring of disease progression. However, the melanin response is not systemic as for other pathogens but instead is very local. The mortality of the larvae is dose-dependent and can be induced by cysts or regenerating protoplasts as an alternative source of inoculation.     

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.     

Streptococcus faecalis,Bacillus alvei, and sacbrood virus in European foulbrood of the honey bee

Contrary to reports from elsewhere, Streptococcus faecalis or Bacillus alvei did not cause European foulbrood in bee larvae also inoculated with sacbrood virus. The larvae died of sacbrood, by which time S. faecalis had mostly disappeared, although B. alvei multiplied saprophytically, as in European foulbrood, in some of the remains. Larvae that died of sacbrood already contained much sacbrood virus before they were sealed in their cells, when they appeared unaffected by the virus, but when they are most likely to die of European foulbrood, which is caused by Streptococcus pluton, often accompanied by secondary invaders, such as S. faecalis. Therefore, larvae killed by European foulbrood can be expected sometimes to contain much sacbrood virus, particularly as this virus is common.     

The purification and properties of a fibrinolytic neutral metalloendopeptidase from Streptococcus faecalis

A protease has been isolated by affinity chromatography from culture filtrates of a strain of Streptococcus faecalis previously shown to produce a flbrinolytic enzyme. The pH optimum, molecular weight, metal ion chelator sensitivity, and peptidase specificity place this enzyme in the class of bacterial neutral metalloendopeptidase typified by thermolysin and the Bacillus subtilis neutral proteases. Differences with respect to chemical modification and thermal stability exist between the S. faecalis enzyme and the latter proteases. The S. faecalis enzyme (designated EM 19000) renders fibrinogen incoagulable by degradation of the B (β) chains.     

Selective depletion of the plasmatocytes in Galleria mellonella following injection of bacteria

Injection of a suspension of a bacterial pathogen, Bacillus cereus, into larvae of the wax moth, Galleria mellonella, resulted in the disappearance of plasmatocytes from the haemolymph. This depletion effect was dose dependent, and occurred within 5 min of injection of the bacteria. Similar effects, though of lesser intensity, followed injection of a number of other species of both pathogenic and nonpathogenic bacteria. This rapid and specific reaction may play a part in the natural response of insects to the injection of foreign bodies.     

Studies on the in vivo cellular reactions of insects: Clearance of pathogenic and non-pathogenic bacteria in Galleria mellonella larvae

The fate of various doses of bacteria of different pathogenicities injected into Galleria mellonella larvae was monitored over time, from haemocyte and bacterial counts, phagocytic responses and the speed and extent of formation of melanized cell aggregates (nodules). An initial haemocytopenia was recorded in all larvae, probably as a result of wound healing, an increased stickiness of the haemocytes for host tissues and/or cell clump or nodule formation. The results also showed that phagocytosis is the primary cellular defence reaction of this insect for doses of bacteria below ca. 10³ μl^?1 haemolymph while above this level phagocytosis and bacterial clearance are usually rapidly augmented by nodule formation. The extent to which these processes are elicited depends greatly upon the nature of the bacteria injected. In general, the more pathogenic strains produced greater responses than the relatively non-pathogenic forms. This enhanced cellular reactivity was, however, soon overcome by the pathogens which rapidly induced a secondary bacteraemia, a huge drop in haemocyte numbers and death of the larvae. The relative importance of phagocytosis and nodule formation in dealing with various doses of bacteria of differing pathogenicities is discussed.     

The Lysozyme-Induced Peptidoglycan N-Acetylglucosamine Deacetylase PgdA (EF1843) Is Required for Enterococcus faecalis Virulence

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O-acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.     

Pathophysiological influences of the Heterorhabditis bacteriophora complex on seventh-instar larvae of Galleria mellonella: Effect on silk production

Oral introduction of invasive juveniles of Heterorhabditis bacteriophora results in an inhibition of silk production by seventh-instar larvae of Galleria mellonella. The inhibition can be duplicated by intrahemocoelic injection of its bacterial associate alone. The inhibition varies directly with dose and is correlated with a progressive increase in hemolymph bacterial titer.     

Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

The lethal dose (LD)₅₀ values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum.The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues.Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes.The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.     

Bacteriocin Protein BacL1 of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala2-Cross-Bridged Peptidoglycan

Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis and consists of two extracellular proteins, BacL₁ and BacA. We previously reported that BacL₁ protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycan d-isoglutamyl-l-lysine endopeptidase that induces cell lysis of E. faecalis when an accessory factor, BacA, is copresent. However, the target of BacL₁ remains unknown. In this study, we investigated the targeting specificity of BacL₁. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL₁ specifically localized at the cell division-associated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of target E. faecalis cells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of BacL₁. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL₁ and BacA. The static growth state also abolished the binding and targeting of BacL₁ to the cell division-associated site. Furthermore, the targeting of BacL₁ was detectable among Gram-positive bacteria with an l-Ala-l-Ala-cross-bridging peptidoglycan, including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such as Enterococcus faecium, Enterococcus hirae, Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL₁ specifically targets the l-Ala-l-Ala-cross-bridged peptidoglycan and potentially lyses the E. faecalis cells during cell division.     

Level and activities of antioxidants in intestine of larvae Galleria mellonella L. (Lepidoptera, Pyralidae) at peroral infestation by bacteria Bacillus thuringiensis ssp. galleriae

In intestine of larvae Galleria mellonella L. (Lepidoptera, Pyralidae) there were revealed changes of activities of enzymatic antioxidants—superoxide dismutase (SOD), glutathione S-transferase (GT), and catalase and of concentrations of non-enzymatic antioxidants—oxidized and reduced thiols (RSSR/RSH) during development of bacterial infection caused by bacteria Bacillus thuringiensis ssp. galleriae, strain 69-6 (BT). An increase of the activities of SOD and GT as well as of the ratio of oxidized to reduced thiols were shown alongside with a decrease of catalase activity in larvae infestated with BT during the experiment. This seems to be due to that during the pathological process, changes of the ratio of components of antioxidant system take place. It is suggested that changes in the antioxidant system of larvae G. mellonella can occur during the oxidative stress accompanying destructive processes in the intestine at the period of development of bacterial infection.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 18–22.Original Russian Text Copyright © 2005 by Dubovskii, Olifirenko, Glupov.     

Construction of a chimeric lysin Ply187N-V12C with extended lytic activity against staphylococci and streptococci

Developing chimeric lysins with a wide lytic spectrum would be important for treating some infections caused by multiple pathogenic bacteria. In the present work, a novel chimeric lysin (Ply187N-V12C) was constructed by fusing the catalytic domain (Ply187N) of the bacteriophage lysin Ply187 with the cell binding domain (146-314aa, V12C) of the lysin PlyV12. The results showed that the chimeric lysin Ply187N-V12C had not only lytic activity similar to Ply187N against staphylococcal strains but also extended its lytic activity to streptococci and enterococci, such as Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium and Enterococcus faecalis, which Ply187N could not lyse. Our work demonstrated that generating novel chimeric lysins with an extended lytic spectrum was feasible through fusing a catalytic domain with a cell-binding domain from lysins with lytic spectra across multiple genera.     

Characterization of a novel lytic bacteriophage from an industrial <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation process and elimination of virulence using a heterologous CRISPR–Cas9 system

Bacterial–bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.     

Ecology of Bacillus thuringiensis in storage moths

A disease-free stock of Plodia interpunctella was produced by a continuous rearing technique. In dense populations of this stock, 10⁴ or more spores of H serotype V Bacillus thuringiensis applied at one point on the surface of 200 g of food were required to cause epizootics, compared with 10⁷ or more when spread evenly over the surface. In infected populations, spores contaminated the surfaces of all stages of the insect. In diseased larval cadavers there were 5.6–42.2 × 10⁸ spores/g of dry insect (P. interpunctella, Ephestia cautella, Anagasta kuehniella, Ephestia elutella, and Galleria mellonella). Larvae did not cannibalize live larvae while food was present though they sometimes ate cadavers. This is the most potent means of natural spread of the disease. Occurring mainly in protected situations such as food stores, natural infections are usually light, but occasionally spectacular surface accumulations of dead larvae occur, possibly associated with stress, physiological condition of the larvae, serotype of the bacterium, or behavior pattern such as migration. Natural disease may curb infestations in debris, but it attacks too late to prevent excessive damage to stored food. A prophylactic, even admixture of 2 × 10⁹ spores/200 g of food is required for effective insect control.     

Culex pipiens affected by joint infection of a mosquito iridescent virus and Strelkovimermis spiculatus

Dual infections with a mosquito iridescent virus (MIV) and the mermithid nematode, Strelkovimermis spiculatus were recorded in natural Culex pipiens populations around La Plata city, Argentina. S. spiculatus was detected in 82% of samples that were positive for MIV infection. Dissected larvae of Cx. pipiens with patent MIV infection presented 42% infection with S. spiculatus. Larvae of Cx. pipiens exposed to MIV and S. spiculatus under laboratory conditions produced a high joint infection rate (82.5%) while no infection was recorded on larvae exposed to virus suspension only. Field and laboratory results suggest a strong association between S. spiculatus and MIV in natural populations of Cx. pipiens, in which S. spiculatus could be a mode of entry for the virus into the mosquito hemocele.