In vitro growth of bacteria in hemolymph of Galleria mellonella

The in vitro growth patterns of three nonpathogenic species of bacteria in the hemolymph of Galleria mellonella were determined. Similarly, the growth patterns of three pathogenic bacterial species in the hemolymph of normal larvae were compared to patterns in hemolymph from immunized insects. Over the 12-hr observation period, the nonpathogenic species never attained growth in hemolymph that was comparable to that in broth controls. The pathogens in normal hemolymph grew very similarly to controls, whereas, in immune hemolymph the growth of pathogens was greatly inhibited. The growth patterns of both pathogenic and nonpathogenic species of bacteria in normal hemolymph were similar to the in vitro growth patterns previously obtained in whole insect tissue. The possible role of hemolymph in inhibition of growth of bacteria is discussed.     

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.     

Hrp- Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp^- mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an ω-Km interposon within the hrp gene cluster of each strain. The resulting Hrp^- mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp^- mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.     

Location and Survival of Leaf-Associated Bacteria in Relation to Pathogenicity and Potential for Growth within the Leaf

The growth and survival of pathogenic and nonpathogenic Pseudomonas syringae strains and of the nonpathogenic species Pantoea agglomerans, Stenotrophomonas maltophilia, and Methylobacterium organophilum were compared in the phyllosphere of bean. In general, the plant pathogens survived better than the nonpathogens on leaves under environmental stress. The sizes of the total leaf-associated populations of the pathogenic P. syringae strains were greater than the sizes of the total leaf-associated populations of the nonpathogens under dry conditions but not under moist conditions. In these studies the surface sterilants hydrogen peroxide and UV irradiation were used to differentiate cells that were fully exposed on the surface from nonexposed cells that were in “protected sites” that were inaccessible to these agents. In general, the population sizes in protected sites increased with time after inoculation of plants. The proportion of bacteria on leaves that were in protected sites was generally greater for pathogens than for nonpathogens and was greater under dry conditions than under moist conditions. When organisms were vacuum infiltrated into leaves, the sizes of the nonexposed “internal” populations were greater for pathogenic P. syringae strains than for nonpathogenic P. syringae strains. The sizes of the populations of the nonpathogenic species failed to increase or even decreased. The sizes of nonexposed populations following spray inoculation were correlated with the sizes of nonexposed, internal populations which developed after vacuum infiltration and incubation. While the sizes of the populations of the pathogenic P. syringae strains increased on leaves under dry conditions, the sizes of the populations of the nonpathogenic strains of P. syringae, P. agglomerans, and S. maltophilia decreased when the organisms were applied to plants. The sizes of the populations on dry leaves were also correlated with the sizes of the nonexposed populations that developed following vacuum infiltration. Although pathogenicity was not required for growth in the phyllosphere under high-relative-humidity conditions, pathogenicity apparently was involved in the ability to access and/or multiply in certain protected sites in the phyllosphere and in growth on dry leaves.     

Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Stability and Activities of Antibiotics Produced during Infection of the Insect Galleria mellonella by Two Isolates of Xenorhabdus nematophilus

Xenorhabdus nematophilus subsp. dutki, an entomopathogenic bacterium, is vectored by steinernematid nematodes into insects, where it produces broad-spectrum antibiotics. The use of the nematode-bacterium complex against soil-dwelling pest insects could introduce antibiotics into the soil via the dead insect fragments during the emergence phase of the nematodes. Studies on the stability and activities of these antibiotics produced in the insect Galleria mellonella may contribute to assessing the possible impact of antibiotics on soil bacteria. Two isolates of X. nematophilus subsp. dutki (isolates GI and SFU) produced xenocoumacins 1 and 2 in cadavers of G. mellonella larvae in a 1:1 ratio. Total xenocoumacin 1 and 2 production was 800 ng/200 mg (wet weight) of insect tissue for the GI isolate. Antibiotic activity of water extracts from insects that had been infected with X. nematophilus was stable at 60°C for 1 h and after repeated freeze-thaw cycles. The antibiotic titer of extracts held at 27°C declined by day 10. The spectrum of bacterial species killed by antibiotics produced in insect cadavers varied with the isolate of X. nematophilus. Levels of antibiotic activity were greater in vivo than in tryptic soy broth, which may represent a nutrient effect. The bacterial isolate, culture condition, and presence of nematodes influenced the total antibiotic production in vivo. However, the levels of activity were not correlated with bacterial levels in the different growth environments. Insect cadavers with antibiotic activity transiently lowered the numbers of the bacteria in the soil, the extent of decline varying with the strain of X. nematophilus and the time of sampling.     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Discrimination of Food-Borne Microorganisms

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.     

Microbial synergists pathogenic to Lymantria dispar: Chitinolytic and fermentative bacterial interactions

A chitinolytic bacterium isolated from a healthy gypsy moth, Lymantria dispar, larva was shown to be pathogenic to larvae when administered per os after growth on chitin broth. However, the chitin hydrolase produced by this isolate had a pH optimum for activity of 5.5 and the high alkalinity in the mesenteron of L. dispar larvae severely limited chitinolysis of the midgut lining. Fermentative, nonpathogenic, acid-producing bacteria isolated from healthy gypsy moth larvae effectively lowered larval mesenteron pH when administered per os and the combination of fermentative isolates with a crude culture aliquot of the chitinolytic strain produced a synergistic increase in mortality over either dose administered by itself. Increased mortality was also observed for most fermentative strains when they were combined with crude supernatants of centrifuged cultures of the chitinolytic strain, although these combinations proved less effective than when fermenters were added to the whole-culture aliquots of the chitinolytic strain. In vitro studies showed that other bacteria isolated from environments foreign to that of the gypsy moth could ferment carbohydrates with acid production at an alkaline pH; however, in vivo studies demonstrated that these bacteria were incapable both of poising larval midgut pH and of enhancing mortality when added to chitinolytic bacteria.     

Influence of five spiroplasma strains on growth rate and survival of Galleria mellonella (lepidoptera: Pyralidae) larvae

Infection of Galleria mellonella larvae with five spiroplasma strains caused increased mortality and decreased growth rates of larvae. Reductions in growth rate and survival were related to spiroplasma strain and multiplication rate of the spiroplasma in the larvae. Three strains, considered to be ephiphytic on flowers (23-6, SR-3, PPS1), proved highly pathogenic to G. mellonella larvae, whereas strains known to be pathogenic to plants (SC-27) or honeybees (G-1) had a lesser impact.     

Galleria mellonella apolipophorin III – an apolipoprotein with anti-Legionella pneumophila activity

The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III.     

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.     

Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains

Background

Francisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans.

Results

Comparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation.

Conclusion

The chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species.     

Production of Rhamnolipids by Pseudomonas chlororaphis, a Nonpathogenic Bacterium

Rhamnolipids, naturally occurring biosurfactants constructed of rhamnose sugar molecules and β-hydroxyalkanoic acids, have a wide range of potential commercial applications. In the course of a survey of 33 different bacterial isolates, we have identified, using a phenotypic assay for rhamnolipid production, a strain of the nonpathogenic bacterial species Pseudomonas chlororaphis that is capable of producing rhamnolipids. Rhamnolipid production by P. chlororaphis was achieved by growth at room temperature in static cultures of a mineral salts medium containing 2% glucose. We obtained yields of roughly 1 g/liter of rhamnolipids, an amount comparable to the production levels reported in Pseudomonas aeruginosa grown with glucose as the carbon source. The rhamnolipids produced by P. chlororaphis appear to be exclusively the mono-rhamnolipid form. The most prevalent molecular species had one monounsaturated hydroxy fatty acid of 12 carbons and one saturated hydroxy fatty acid of 10 carbons. P. chlororaphis, a nonpathogenic saprophyte of the soil, is currently employed as a biocontrol agent against certain types of plant fungal diseases. The pathogenic nature of all bacteria previously known to produce rhamnolipids has been a major obstacle to commercial production of rhamnolipids. The use of P. chlororaphis therefore greatly simplifies this matter by removing the need for containment systems and stringent separation processes in the production of rhamnolipids.     

Food Microorganisms Influencing the Growth of Staphylococcus aureus

Some 870 cultures of predominating micro-organisms were isolated from market samples of hamburger, fresh pork sausage, fresh fish fillets, stewing beef, frozen chicken pot pie, frozen corn, frozen peas, and pasteurized and raw milk, before and after storage at different temperatures. The isolates were screened for their ability to influence the growth of Staphylococcus aureus strain 196E by means of spot-plate tests on APT and nutrient agars at 25 C. The 438 cultures that influenced the growth of S. aureus were retested on spot plates at 15, 30, and 42 C. After elimination of replicates, the 143 remaining cultures were classified into species, genera, or groups, and 14 different cultures were tested for their influence on the growth of S. aureus in APT broth at 25 C. Over half of the effective cultures inhibited S. aureus and less than half were stimulatory. Pork sausage had the highest proportion of inhibitory cultures, and stewing beef had the lowest. APT agar was better than nutrient agar for screening, and incubation at 15 C gave more effector organisms than at 30 and 42 C. Most of the lactic acid bacteria were inhibitory, but other groups of bacteria contained more stimulatory cultures than inhibitory ones. The three Escherichia coli cultures were stimulatory, but most other Escherichia cultures were inhibitory. Aerobacter and Paracolobactrum isolates were mostly stimulatory. Cultures of other kinds of bacteria were more or less evenly distributed between inhibitory ones and stimulatory ones. Genera containing mostly inhibitory bacteria were Streptococcus, Leuconostoc, and Lactobacillus. Inhibitory species were E. freundii and E. intermedia. Tests with S. aureus in broth indicated that all cultures inhibitory according to spot plates were inhibitory in broth, but stimulation on spot plates did not always indicate the same phenomenon in broth.     

Pathogenesis of Bacillus sphaericus strain SSII-1 infections in Culex pipiens quinquefasciatus ( = C. pipiens fatigans) larvae

Numbers of viable bacteria in second instar Culex pipiens quinquefasciatus larvae were determined following ingestion of pathogenic strain SSII-1 and nonpathogenic Bacillus sphaericus. Numbers of nonpathogenic B. sphaericus recovered from larvae declined rapidly after cessation of feeding, as did numbers of pathogenic SSII-1 cells fed at LD₂₀ dosage. When pathogenic cells were fed at LD₇₀ dosage, the number of B. sphaericus in larvae increased following initial decline. When chloroformtreated SSII-1 cultures, in which all bacteria except spores were dead, were fed at LD₁₀ and LD₉₈ dosages, no viable B. sphaericus were recovered from larvae. In all SSII-1 treatments, other bacterial flora multiplied rapidly in larvae following onset of mortality; the role of this multiplication in the pathogenesis was not determined. It is proposed that toxic material is released when SSII-1 cells are digested and that multiplication of B. sphaericus in the larval gut is not essential in the pathogenesis. There appears to be no difference in the pathogenesis when differing numbers of B. sphaericus. i.e., LD_10–20 or LD_70–98 dosages, are ingested. Possible nature of the toxic material is discussed.     

Interactions between rye (Secale cereale) root border cells (RBCs) and pathogenic and nonpathogenic rhizosphere strains of Fusarium culmorum

Interactions of rye (Secale cereale) root border cells (RBCs), generated during plant growth and surrounding the root cap, with nonpathogenic rhizosphere Fusarium culmorum isolates: DEMFc2 (PGPF) and DEMFc5 (DRMO), and a pathogenic strain DEMFc37 were studied in test tube experiments. The effect of water-suspended RBCs released from the rye root cap on the rate of macroconidia germination and hyphae (mycelial) growth of F. culmorum strains was also examined. It was found that root caps of 3-d-old rye seedlings (with the root length of 20 mm) were surrounded with a layer of RBCs generated in a number specific for this plant species of 1980 ± 30. Introduction of the macroconidia of the tested F. culmorum strains into the root zone of 3-d-old seedlings resulted, after 3 d of incubation, in the formation of mantle-like structures only in the rhizosphere of plants inoculated with the pathogenic DEMFc37 strain. The macroconidia were suspended in (1) water, (2) a water mixture with root caps deprived of RBCs, (3) Martin medium, (4) malt extract broth, and (5) a water mixture with rye RBCs, and their percentage germination was determined during 96-h incubation at 20 °C. Germination of the macroconidia of all the tested F. culmorum strains suspended in the rich growth media (Martin and malt extract broth) and in the mixture with RBCs was significantly speeded up. While only an average of 16.6 % of macroconidia suspended in water germinated after 96-h incubation, more than 90 % of those suspended in the growth media or in the mixture with RBCs germinated after 24 h of incubation. In all the treatments, the highest rate of macroconidia germination was found in suspensions of the pathogenic strain and the lowest in macroconidial suspensions of the PGPF strain. The stimulatory effect of RBCs was not specific to the pathogenic strain. Nevertheless, microscopic observation revealed that it was only in the suspension containing a mixture of rye RBCs and macroconidia of the pathogenic strain that after 48-h incubation compact clusters of hyphae and RBCs, resembling mantle-like structures found in the root zone of plants inoculated only with the pathogenic strain but not inoculated with DRMO and PGPF strain, were formed.     

Ectopic gene conversions in bacterial genomes.

We characterized the gene conversions found between the duplicated genes of 75 bacterial genomes from five species groups (archaea, nonpathogenic and pathogenic firmicutes, and nonpathogenic and pathogenic proteobacteria). The number of gene conversions is positively correlated with the size of multigene families and the size of multigene families is not significantly different between pathogenic and nonpathogenic taxa. However, gene conversions occur twice as frequently in pathogenic species as in nonpathogenic species. Comparisons between closely related species also indicate a trend towards increased gene conversion in pathogenic species. Whereas the length of the conversions is positively correlated with flanking sequence similarity in all five groups, these correlations are smaller for pathogenic firmicutes and proteobacteria than for nonpathogenic firmicutes and proteobacteria. These results are consistent with our previous work on E. coli genomes and suggest that pathogenic bacteria allow recombination between more divergent gene sequences. This higher permissiveness is likely adaptive because it allows them to generate more genetic variability.