Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

Diagnosis of Bacteria In Vitro by Mass Spectrometric Fingerprinting:A Pilot Study

The identification of bacteria by using conventional microbiological techniques can be very time-consuming and circumstantial. In contrast, the headspace screening of bacterial cultures by analyzing their emitted volatile compounds using mass spectrometry might provide a novel approach in diagnostic microbiology. In the present study different strains of Escherichia coli, Klebsiella, Citrobacter, Pseudomonas aeruginosa, Staphylococcus aureus, and Helicobacter pylori were investigated. The volatile compounds emitted by these bacteria in vitro were analyzed using proton-transfer-reaction mass spectrometry, which allows rapid and sensitive measurement. The detected patterns of volatile compounds produced by the investigated bacteria were compared and substantial differences regarding both quantity and quality were observed. In conclusion, the present study is the first to describe headspace screening of bacterial cultures as a potential diagnostic approach in medical microbiology.     

Proferrioxamine profiles of Erwinia herbicola and related bacteria

Two strains of Erwinia herbicola effective in the biocontrol of E. amylovora, the etiological agent of fire blight, were screened for proferrioxamine siderophores by on-line liquid chromatography-electrospray mass spectrometry (LC-MS). Type strains of E. herbicola and Pantoea species were included in this study for taxonomic comparisons. Proferrioxamine profiles similar to that previously described for E. amylovora, including tri- and tetrameric hydroxamates and diaminopropane-containing proferrioxamines, were observed for P. agglomerans, but not for other E. herbicola-like species. Biocontrol activity was not correlated with proferrioxamine synthesis. The results of this study are consistent with the notion that some, but not all, biocontrol strains may inhibit E. amylovora via competition for iron. Further studies into the link between biocontrol of fire blight and siderophores are thus warranted. This study also revealed limitations of standard nutrient utilization and fatty acid profile analyses for the differentiation of P. agglomerans, P. dispersa and other E. herbicola-like species from each other. Given these limitations, LC-MS may become a much needed additional diagnostic tool for the identification of E. herbicola-like strains at the species level.     

In-vitro diagnosis of single and poly microbial species targeted for diabetic foot infection using e-nose technology

Background

Effective management of patients with diabetic foot infection is a crucial concern. A delay in prescribing appropriate antimicrobial agent can lead to amputation or life threatening complications. Thus, this electronic nose (e-nose) technique will provide a diagnostic tool that will allow for rapid and accurate identification of a pathogen.

Results

This study investigates the performance of e-nose technique performing direct measurement of static headspace with algorithm and data interpretations which was validated by Headspace SPME-GC-MS, to determine the causative bacteria responsible for diabetic foot infection. The study was proposed to complement the wound swabbing method for bacterial culture and to serve as a rapid screening tool for bacteria species identification. The investigation focused on both single and poly microbial subjected to different agar media cultures. A multi-class technique was applied including statistical approaches such as Support Vector Machine (SVM), K Nearest Neighbor (KNN), Linear Discriminant Analysis (LDA) as well as neural networks called Probability Neural Network (PNN). Most of classifiers successfully identified poly and single microbial species with up to 90% accuracy.

Conclusions

The results obtained from this study showed that the e-nose was able to identify and differentiate between poly and single microbial species comparable to the conventional clinical technique. It also indicates that even though poly and single bacterial species in different agar solution emit different headspace volatiles, they can still be discriminated and identified using multivariate techniques.     

Headspace-SPME of <Emphasis Type="Italic">in vitro</Emphasis> shoot-cultures and micropropagated plants of <Emphasis Type="Italic">Lavandula viridis</Emphasis>

In this work the volatiles emitted from in vitro shoot-cultures and micropropagated plants of Lavandula viridis L’Hér. were characterized and compared with those obtained from the field-grown mother-plant, using headspace solid phase micro-extraction following by capillary gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS). The headspace composition consisted mainly in oxygenated monoterpenes (66.7–79.2 %), where the major constituents emitted by the mature field-grown mother-plant, in vitro shoot-cultures and micropropagated plants were 1,8-cineole (74.0, 51.9 and 57.8 %) and camphor (2.9, 15.3 and 8.7 %), respectively. The headspace of in vitro shoot-cultures and micropropagated plants showed greater amount of α-pinene, camphene, β-pinene, β-selinene and selina-3,7(11)-diene, when compared with the field-grown mother-plant.     

MALDI-TOF Identification of the Human Gut Microbiome in People with and without Diarrhea in Senegal

Background

In Africa, there are several problems with the specific identification of bacteria. Recently, MALDI-TOF mass spectrometry has become a powerful tool for the routine microbial identification in many clinical laboratories.

Methodology/Principal Findings

This study was conducted using feces from 347 individuals (162 with diarrhea and 185 without diarrhea) sampled in health centers in Dakar, Senegal. Feces were transported from Dakar to Marseille, France, where they were cultured using different culture conditions. The isolated colonies were identified using MALDI-TOF. If a colony was unidentified, 16S rRNA sequencing was performed. Overall, 2,753 isolates were tested, allowing for the identification of 189 bacteria from 5 phyla, including 2 previously unknown species, 11 species not previously reported in the human gut, 10 species not previously reported in humans, and 3 fungi. 2,718 bacterial isolates (98.8%) out of 2,750 yielded an accurate identification using mass spectrometry, as did the 3 Candida albicans isolates. Thirty-two bacterial isolates not identified by MALDI-TOF (1.2%) were identified by sequencing, allowing for the identification of 2 new species. The number of bacterial species per fecal sample was significantly higher among patients without diarrhea (8.6±3) than in those with diarrhea (7.3±3.4; P = 0.0003). A modification of the gut microbiota was observed between the two groups. In individuals with diarrhea, major commensal bacterial species such as E. coli were significantly decreased (85% versus 64%), as were several Enterococcus spp. (E. faecium and E. casseliflavus) and anaerobes, such as Bacteroides spp. (B. uniformis and B. vulgatus) and Clostridium spp. (C. bifermentans, C. orbiscindens, C. perfringens, and C. symbosium). Conversely, several Bacillus spp. (B. licheniformis, B. mojavensis, and B. pumilus) were significantly more frequent among patients with diarrhea.

Conclusions/Significance

MALDI-TOF is a potentially powerful tool for routine bacterial identification in Africa, allowing for a quick identification of bacterial species.     

Hypersensitive response and acyl‐homoserine lactone production of the fire blight antagonists Erwinia tasmaniensis and Erwinia billingiae

Fire blight caused by the Gram‐negative bacterium Erwinia amylovora can be controlled by antagonistic microorganisms. We characterized epiphytic bacteria isolated from healthy apple and pear trees in Australia, named Erwinia tasmaniensis, and the epiphytic bacterium Erwinia billingiae from England for physiological properties, interaction with plants and interference with growth of E. amylovora. They reduced symptom formation by the fire blight pathogen on immature pears and the colonization of apple flowers. In contrast to E. billingiae, E. tasmaniensis strains induced a hypersensitive response in tobacco leaves and synthesized levan in the presence of sucrose. With consensus primers deduced from lsc as well as hrpL, hrcC and hrcR of the hrp region of E. amylovora and of related bacteria, these genes were successfully amplified from E. tasmaniensis DNA and alignment of the encoded proteins to other Erwinia species supported a role for environmental fitness of the epiphytic bacterium. Unlike E. tasmaniensis, the epiphytic bacterium E. billingiae produced an acyl‐homoserine lactone for bacterial cell‐to‐cell communication. Their competition with the growth of E. amylovora may be involved in controlling fire blight.     

Variation in host preferences of malaria mosquitoes is mediated by skin bacterial volatiles

The host preferences of the anthropophilic mosquito species in the Anopheles gambiae complex (Diptera: Culicidae) are mediated by skin bacterial volatiles. However, it is not known whether these mosquitoes respond differentially to skin bacterial volatiles from non‐human host species. In this study, the responses of two malaria mosquito species in the An. gambiae complex, Anopheles gambiae s.s. (hereafter, An. gambiae) and Anopheles arabiensis, with different host preferences, to volatiles released from skin bacteria were tested. Skin bacteria collected from human, cow and chicken skin significantly increased trap catches; traps containing bacteria collected from human skin caught the highest proportions of An. gambiae and An. arabiensis. Traps with bacteria of human origin caught a significantly higher proportion of An. gambiae than of An. arabiensis, whereas bacterial volatiles from the chicken attracted significantly higher numbers of An. arabiensis than of An. gambiae. Additionally, An. gambiae showed a specialized response to volatiles from four specific bacteria, whereas An. arabiensis responded equally to all species of bacteria tested. Skin bacterial volatiles may therefore play important roles in guiding mosquitoes with different host preferences. The identification of these bacterial volatiles can contribute to the development of an odour blend that attracts mosquitoes with different host preferences.     

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster)

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera.The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA.The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines.In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate.     

Olfactory response of a predatory mirid to herbivore induced plant volatiles: multiple herbivory vs. single herbivory

Plants infested with a single herbivore species can attract natural enemies through the emission of herbivore‐induced plant volatiles (HIPVs). However, under natural conditions plants are often attacked by more than one herbivore species. We investigated the olfactory response of a generalist predators Macrolophus caliginosus to pepper infested with two‐spotted spider mites, Tetranychus urticae, or green peach aphid, Myzus persicae, vs. plants infested with both herbivore species in a Y‐tube olfactometer set up. In addition, the constituents of volatile blends from plants exposed to multiple or single herbivory were identified by gas chromatography‐mass spectrometry (GC‐MS). The mirid bugs showed a stronger response to volatiles emitted from plants simultaneously infested with spider mites and aphids than to those emitted from plants infested by just one herbivore, irrespective of the species. Combined with results from previous studies under similar conditions we infer that this was a reaction to herbivore induced plant volatiles. The GC‐MS analysis showed that single herbivory induced the release of 22 additional compounds as compared with the volatiles emitted from clean plants. Quantitative analyses revealed that the amount of volatile blends emitted from pepper infested by both herbivores was significantly higher than that from pepper infested by a single herbivore. Moreover, two unique substances were tentatively identified (with a probability of 94% and 91%, respectively) in volatiles emitted by multiple herbivory damaged plants: α‐zingiberene and dodecyl acetate.     

Identification of Erwinia amylovora, the Fireblight Pathogen, by Colony Hybridization with DNA from Plasmid pEA29

All strains of Erwinia amylovora characterized carry a medium-size plasmid of 29 kilobases (pEA29). We mapped this plasmid with various restriction enzymes, cloned the whole DNA into an Escherichia coli plasmid, and subcloned restriction fragments. These DNA species were used for identification of E. amylovora after handling of strains in the laboratory and also in field isolates. About 70 strains of E. amylovora and 24 strains from nine other species, mainly found in plant habitats, were checked in a colony hybridization test. Virulent and avirulent E. amylovora strains reacted positively, whereas the other species were negative. Apart from the hybridization assay, the positive strains were additionally tested for ooze production on rich agar with 5% sucrose and on immature-pear slices. Unspecific background hybridization of non-E. amylovora strains found for hybridization with the whole E. amylovora plasmid was almost eliminated when a 5-kilobase SalI fragment from pEA29 was used as a probe and when the washes after the hybridization procedure were done with high stringency. Under these conditions, E. amylovora could be readily identified from field isolates.     

Bacteria may contribute to distant species recognition in ant–aphid mutualistic relationships

Mutualistic interactions between ant and aphid species have been the subject of considerable historical and contemporary investigations, the primary benefits being cleaning and protection for the aphids and carbohydrate‐rich honeydew for the ants. Questions remained, however, as to the volatile semiochemical factor influencing this relationship. A recent study highlighted the role of bacterial honeydew volatile compounds in ant attraction. Here, ant's ability to distantly discriminate 2 aphid species was investigated based on bacterial honeydew semiochemicals emissions using a two‐way olfactometer. Both the mutualistic aphid Aphis fabae L. and the nonmyrmecophilous aphid Acyrthosiphon pisum Harris were found to be attractive for the ant Lasius niger L. The level of attraction was similar in both assays (control vs. one of the aphid species). However, when given a choice between these 2 aphid species, ants showed a significant preference for Aphis fabae. Honeydew volatiles, mostly from bacterial origins, are known to be a key element in ant attraction. Using the same olfactometry protocol, the relative attractiveness of volatiles emitted by honeydews collected from each aphid species and by bacteria isolated from each honeydew was investigated. Again, ants significantly preferred volatiles released by Aphis fabae honeydew and bacteria. This information suggests that microbial honeydew volatiles enable ants to distantly discriminate aphid species. These results strengthen the interest of studying the occurrence and potential impact of microorganisms in insect symbioses.     

Plant growth-promoting rhizobacteria do not pose any deleterious effect on cowpea and detectable amounts of ethylene are produced

Summary The use of trap crops such as cowpea could reduce the effects of the root parasitic weed, Striga hermonthica and its subsequent constraints on the growth of cereals. Certain bacteria could augment the trap crop stimulatory effect. We studied the effect of three bacteria introduced to the rhizosphere of three cowpea varieties at planting. Number of days to cowpea flowering was noted and at harvest, data were collected on pod characteristics and biomass. Means of data subjected to ANOVA were compared using Tukey’s Studentized Range Test. We analysed bacterial headspace volatiles for ethylene by gas chromatography and gas chromatography–mass spectrometry. Bacterial type significantly influenced the cowpea varieties with better performance over the non-inoculated control. Average pod weight (g) with bacterial treatment was 37.97 for Enterobacter sakazakii 8MR5, 34.38 for Pseudomonas 44MS8 and 27.46 for Pseudomonas 10M3. Non-inoculated control had an average weight of 20.98 g. Bacteria promoted a significant increase in pod weight (≥30.89%), fresh biomass (≥24.22%), and improved pod number (≥20.54%) and pod wall thickness (≥7.33%) with no deleterious effect on plant health. Ethylene released by the bacteria ranged from trace concentrations in Pseudomonas sp. to 210 nmoles/10⁸ c. f. u./ml in Ent. sakazakii 8MR5.     

Development and evaluation of a real‐time PCR assay targeting chromosomal DNA of Erwinia amylovora

Aims:  To develop and evaluate a new and reliable real‐time PCR detection protocol on chromosomal DNA of the contagious plant pathogenic bacterium Erwinia amylovora, the causal agent of fire blight. Methods and Results:  A Taqman^® minor‐groove‐binder real‐time PCR assay targeting a hypothetical protein coding gene of Erw. amylovora has been developed. Colony PCR of 113 bacterial strains from different taxa was performed to prove specificity. Serial decimal dilutions of Erw. amylovora showed a consistent detection sensitivity of 2 bacterial units per μl. All strains of Erw. amylovora could be identified, and there were no cross‐reactions with matrices or other bacteria also testing naturally contaminated samples. Conclusions:  Rapid, reliable and sensitive detection of Erw. amylovora is important to avoid the spread of the disease within orchards, and the distribution by contaminated plant material or vectors carrying the pathogen. The selected conserved target gene allows relative quantitative detection of Erw. amylovora from different sources and host taxa. The newly developed protocol also enables the detection of recently found natural strains that lack the species‐specific plasmid pEA29, which was so far widely used as target for detection and identification of this plant pathogen by PCR. Significance and Impact of the Study:  This study demonstrates that the newly developed and evaluated real‐time assay can specifically be used for identifying all known strains of the EU quarantine plant pathogen Erw. amylovora. Low concentrations of the bacteria can be detected and relatively quantified using a different target area than other real‐time PCRs designed so far.     

Composition of epiphytic bacterial communities differs on petals and leaves

The epiphytic bacterial communities colonising roots and leaves have been described for many plant species. In contrast, microbiologists have rarely considered flowers of naturally growing plants. We identified bacteria isolated from the surface of petals and leaves of two plant species, Saponaria officinalis (Caryophyllaceae) and Lotus corniculatus (Fabaceae). The bacterial diversity was much lower on petals than on leaves of the same plants. Moreover, the bacterial communities differed strongly in composition: while Pseudomonadaceae and Microbacteriaceae were the most abundant families on leaves, Enterobacteriaceae dominated the floral communities. We hypothesise that antibacterial floral volatiles trigger the low diversity on petals, which is supported by agar diffusion assays using substances emitted by flowers and leaves of S. officinalis. These results suggest that bacteria should be included in the interpretation of floral traits, and possible effects of bacteria on pollination are proposed and discussed.     

Improved recovery of Erwinia amylovora-stressed cells from pome fruit on RESC, a simple, rapid and differential medium

The bacterium Erwinia amylovora causes fire blight, a serious and widespread disease of several pome fruit and ornamental plants. The use of suitable detection tools is essential for preventing its dissemination and, according to the protocol of the European and Mediterranean Plant Protection Organization, the isolation and further identification of E. amylovora is the only conclusive test of its presence. However, bacterial growth on solid media can be hampered when the pathogen is suffering stressful conditions in pome fruit or in other habitats. Since copper is an essential micronutrient that, in E. amylovora, also increases the exopolysaccharide production in rich-nutrient media, we have designed a non-selective differential medium containing 1.5 mM CuSO₄ to improve the recovery of E. amylovora from plants under unfavorable conditions. In this new medium named Recovery Erwinia amylovora-Stressed Cells (RESC), its colonies were easily distinguished by a light yellow color and a high mucus production. The plating recovery of several E. amylovora strains in vitro and from naturally infected samples was significantly improved with respect to other media routinely employed, particularly when the pathogen was suffering stressful conditions. Thus, the recovery of stressed E. amylovora cells (after UV irradiation, nutrient deprivation, or the presence of copper ions in non-copper-complexing media) was significantly enhanced on RESC medium, and their culturability period extended. Therefore, RESC is a useful and valuable medium for the isolation of E. amylovora when adverse conditions in the natural environment are expected.     

Evaluation of bacteria isolated from rice rhizosphere for biological control of charcoal rot of sorghum caused by <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> (Tassi) Goid.

A total of 360 bacteria, isolated from the rhizospheres of a system of rice intensification (SRI) fields, were characterized for the production of siderophore, fluorescence, indole acetic acid (IAA), hydrocyanic acid (HCN) and solubilization of phosphorus. Of them, seven most promising isolates (SRI-156, -158, -178, -211, -229, -305 and -360) were screened for their antagonistic potential against Macrophomina phaseolina (causes charcoal rot in sorghum) by dual culture assay, blotter paper assay and in greenhouse. All the seven isolates inhibited M. phaseolina in dual culture assay, whereas six isolates solubilized phosphorous (except SRI-360), all seven produced siderophore, four produced fluorescence (except SRI-178, -229 and -305), six produced IAA (except SRI-305) and five produced HCN (except SRI-158 and -305). In the blotter paper assay, no charcoal rot infection was observed in SRI-156-treated sorghum roots, indicating complete inhibition of the pathogen, while the roots treated with the other isolates showed 49–76% lesser charcoal rot infection compared to the control. In the antifungal activity test (in green house on sorghum), all the isolates increased shoot dry mass by 15–23% and root dry mass by 15–20% (except SRI-158 and -360), over the control. In order to confirm the plant growth-promoting (PGP) traits of the isolates, the green house experiment was repeated but, in the absence of M. phaseolina. The results further confirmed the PGP traits of the isolates as evidenced by increases in shoot and root dry mass, 22–100% and 5–20%, respectively, over the control. The sequences of 16S rDNA gene of the isolates SRI-156, -158, -178, -211, -229, -305 and -360 were matched with Pseudomonas plecoglossicida, Brevibacterium antiquum, Bacillus altitudinis, Enterobacter ludwigii, E. ludwigii, Acinetobacter tandoii and P. monteilii, respectively in BLAST analysis. This study indicates that the selected bacterial isolates have the potential for PGP and control of charcoal rot disease in sorghum.     

Phytotoxic material from associations betweenErwinia amylovora and pear tissue culture: Possible role in necrotic symptomatology of fireblight disease

Pear cell suspension cultures (PCSC) inoculated with virulent strains of the fireblight bacterium,Erwinia amylovora, exhibited massive necrosis within 7 days, whereas avirulentE. amylovora strains and other enterobacteria generally elicited very slight or no necrotic reactions. These results were generally repeated when pear seedlings (1–2 months old) were inoculated with these same bacterial strains. Fractions derived from massively necrotized PCSC were tested for biological activity against healthy PCSC and pear seedling cuttings. Of these fractions, the dialyzable, 70%-ethanol-soluble material—remaining after precipitation by ethanol of high-molecular-weight polysaccharides (“amylovorin”)—exclusively inhibited growth of pear callus, plasmolyzed and disintegrated pear cells in PCSC, and caused some browning of pear callus. Although cuttings of young pear seedlings placed in solutions containing this dialyzable, ethanol-soluble material generally became massively necrotized within 3 days, some residual methodological problems with this bioassay procedure must be solved. These observations suggest that some relatively small molecule(s) formed in PCSC inoculated with virulentE. amylovora can exert antagonistic biological activity against pear tissue and may play a role in the symptomatology characteristic of the fireblight disease.