Presence Of Other Microorganisms Research Articles

Chikungunya, a new threat propagated by the cosmopolite Aedes albopictus

The recent outbreaks of chikungunya (CHIK) were due to an East-Central-South African genotype harbouring a substitution from an Alanine (E1-226A) to a Valine (E1-226V) at the position 226 in the E1 gene. The new variant E1-226V is very efficiently transmitted by an unusual mosquito vector, Aedes albopictus. We found that (i) Ae. albopictus females ensure a high replication rate of E1-226V up to 109 viral particles per female, (ii) the midgut plays a key role in limiting viral dissemination of E1-226A in the mosquito, (iii) the virus is detectable in the saliva as soon as two days after the infectious blood-meal, and (iv) Ae. albopictus is able to deliver up to 3000 viral particles with its saliva. All these characteristics led to exacerbate CHIK transmission by Ae. albopictus. However, this species is affected by the viral infection. Indeed, CHIK infection reduces sharply the survival of Ae. albopictus, females laying their eggs just before dying. Females did not die from an excess of viral replication but more likely in attempting to mount an immune antiviral response. Moreover, by removing the intracellular bacteria Wolbachia from Ae. albopictus through successive antibiotic treatments, we aimed to determine if Wolbachia interferes with CHIK replication in the mosquito. We found that Ae. albopictus cleaned of Wolbachia was not affected by CHIK infection. Thus, Wolbachia may regulate viral replication in Ae. albopictus with consequences on its survival. So, the response of a vector to a particular pathogen is also closely linked to the presence of other microorganisms. Finally, we showed that Ae. albopictus was able to be orally co-infected with CHIK and dengue (DEN) viruses and to deliver concomitantly infectious particles of both viruses in saliva. This finding is of particular concern as Ae. albopictus is still expanding its geographical range and as both CHIK and DEN viruses can co-circulate in the same geographical regions. Indeed, reports of co-infections in patients with both viruses are increasing.

Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis

In patients afflicted with cystic fibrosis (CF), morbidity and mortality are primarily associated with the adverse consequences of chronic microbial bronchial infections, which are thought to be caused by a few opportunistic pathogens. However, recent evidence suggests the presence of other microorganisms, which may significantly affect the course and outcome of the infection. Using a combination of 16S rRNA gene clone libraries, bacterial culturing and pyrosequencing of barcoded 16S rRNA amplicons, the microbial communities present in CF patient sputum samples were examined. In addition to previously recognized CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, >60 phylogenetically diverse bacterial genera that are not typically associated with CF pathogenesis were also detected. A surprisingly large number of fermenting facultative and obligate anaerobes from multiple bacterial phyla was present in each sample. Many of the bacteria and sequences found were normal residents of the oropharyngeal microflora and with many containing opportunistic pathogens. Our data suggest that these undersampled organisms within the CF lung are part of a much more complex microbial ecosystem than is normally presumed. Characterization of these communities is the first step in elucidating potential roles of diverse bacteria in disease progression and to ultimately facilitate advances in CF therapy.

Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese

Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production.

Influence of Streptococcus mutans on Enterococcus faecalis Biofilm Formation

Introduction An important virulence factor of Enterococcus faecalis is its ability to form biofilms. Most studies on biofilm formation have been carried out by using E. faecalis monocultures. Given the polymicrobial nature of root canal infections, it is important to understand biofilm formation of E. faecalis in the presence of other microorganisms. Methods Eight clinical strains of E. faecalis were tested for biofilm formation on hydroxyapatite disks in the presence and absence of a Streptococcus mutans biofilm. Results Significantly more E. faecalis viable cells were found in biofilms in the presence of S. mutans. This phenomenon was, however, strain-dependent. Of the 8 strains tested, biofilm formation of strains AA-OR34, ER5/1, and V583 was not influenced by S. mutans biofilms. Conclusions The results from this study, especially the strain difference, underline the importance of studying biofilm formation in a more realistic multispecies setting.

PCR-based detection of Phomopsis azadirachtae in die-back affected neem seeds

Phomopsis azadirachtae Sateesh, Bhat & Devaki is the incitant of die-back disease of neem trees. Delayed appearance of conidia and presence of other microorganisms in the neem tissues are the obstacles in the rapid and accurate identification of P. azadirachtae. This work was carried out to develop a methodology for rapid detection of the pathogen in diseased tissues especially in the neem seeds. rDNA sequences of many Phomopsis spp. were retrieved from the database and were subjected for multiple alignment to select a 179 bp conserved sequence. This was used to design Phomopsis specific primer pair (Forward and Reverse) having the potential to produce a 154 bp product in PCR. The primer pair was utilised to detect the presence of P. azadirachtae in diseased neem seeds and other tissues. This is the first report on the PCR-based detection of P. azadirachtae directly in die-back diseased neem tissues. This method can be employed for rapid and reliable detection of P. azadirachtae in die-back affected neem seeds. Hence it will have very good application in seed health testing laboratories.

Microarray of DNA–protein complexes on poly-3-hydroxybutyrate surface for pathogen detection

A novel strategy was developed for the specific immobilization of DNA probes on poly-3-hydroxybutyrate (PHB) surface by using the substrate-binding domain (SBD) of PHB depolymerase as an active binding motif. To demonstrate whether this method can be used for the detection of clinical pathogens, the pathogen-specific biotin-labeled DNA probes were immobilized via core streptavidin (cSA) fused to the SBD. The pathogen-specific 15-mer oligonucleotide probes were designed for four model pathogens, while the target DNAs were prepared by PCR using universal primers. The complex of pathogen-specific probes and cSA-SBD fusion protein was immobilized on the PHB-coated slide by microspotting. This DNA-protein complex microarray was able to successfully diagnose Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, the specific pathogens could be diagnosed in the presence of other microorganisms. Thus, the DNA-protein complex microarray platform technology employing PHB and the SBD reported here can be widely used for the detection of DNA-DNA and DNA-biomolecule interactions without synthetic or chemical modification of biomolecules or solid surface.

The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces

Although Candida containing biofilms contribute to the development of oral candidosis, the characteristics of multi-species Candida biofilms and how oral bacteria modulate these biofilms is poorly understood. The aim of this study was to investigate interactions between Candida albicans and either Candida glabrata or Streptococcus mutans in biofilms grown on various surfaces, with or without saliva. Hydroxyapatite (HA), polymethylmetacrylate (PMMA) and soft denture liner (SL) discs were used as substratum. Counts of viable micro-organisms in the accumulating biofilm layer were determined and converted to colony forming units per unit surface area. Confocal laser scanning microscopy was used to characterize biofilms and to quantitate the number of hyphae in each condition tested. Viable counts of C. albicans and C. glabrata per mm 2 decreased in the order HA > PMMA > SL ( p < 0.05). Biofilms grown on saliva-coated specimens harboured fewer C. glabrata than uncoated specimens ( p < 0.05). Glucose and the presence of S. mutans suppressed C. albicans hyphal formation. Dual Candida species biofilms did not show competitive interaction between the two species. We conclude that Candida biofilms are significantly affected by saliva, substratum type and by the presence of other micro-organisms.

Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.

Perfil etiológico de las infecciones urinarias y patrón de sensibilidad de los uropatógenos

A review on the etiological profile of urinary tract infections in childhood and the sensitivity pattern of urinary pathogens in Spain is presented. Escherichia coli continues to be the main etiological agent of urinary tract infection in childhood. Consequently, its sensitivity pattern will usually determine the choice of empirical therapy. The predominance of E. coli is reduced in certain circumstances, in which the presence of other microorganisms is increased. However, the clinical information available at diagnosis does not allow accurate identification of the etiology; only staining and microscopic urine examination can help in treatment selection. In Spain, E. coli presents a high percentage of resistance to ampicillin and cotrimoxazole, whereas second- and third-generation cephalosporins, fosfomycin, aminoglycosides and amoxicillin-clavulanate maintain high sensitivity. In some areas, amoxicillin-clavulanate and first-generation cephalosporins show high levels of resistance, which can limit their empirical use.

Prokaryotic community composition and ecology of floating macroscopic filaments from an extreme acidic environment, Río Tinto (SW, Spain)

The prokaryotic diversity of macroscopic filaments located at the water surface in an extreme acidic environment, Río Tinto (SW, Spain), has been analysed through denaturing gradient gel electrophoresis (DGGE), cloning of 16S rRNA genes and fluorescence in situ hybridization (FISH). The dominant species present in the macrofilaments were Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans and Acidiphilium spp., which represented the three main bacterial genera found in the water column of the river. However, our results also showed the presence of other microorganisms not previously detected in the Río Tinto. Within the Gammaproteobacteria class, sequences closely related to the iron-oxidizing bacteria WJ2 and DSM 2392 were found, as well as sequences related to the non-acidophilic genera Aeromonas and Acinetobacter. In addition, two other new phylotypes related to Gram-positive species from the genera Desulfosporosinus, Clostridium and Mycobacterium were identified. The presence of these anaerobic microorganisms strongly suggests that the filaments could originate in the deeper parts of the river as a typical acid streamer attached to the rocks or the sediments and be pulled up towards the surface when they reach certain buoyancy.

Simultaneous quantification of pathogenic Campylobacter and Salmonella in chicken rinse fluid by a flotation and real-time multiplex PCR procedure

A procedure for simultaneous quantification of Campylobacter and Salmonella spp. in poultry skin rinse fluids by a flotation and real-time multiplex PCR method is described. Flotation of the target organisms in a discontinuous density gradient separated them from background microflora, particles from poultry skin, dead target cells and PCR inhibitors. Variation of the buoyant density between 1.052 to 1.106 g/ml was measured at different times for various Salmonella strains grown over a period of 4 weeks. This, and the results from earlier studies on the buoyant densities of Campylobacter spp., which were between 1.065 and 1.109 g/ml, led to design of an optimal discontinuous flotation method with three density layers, of 1.048, 1.109 and approximately 1.200 g/ml. This method preceded a real-time multiplex PCR assay using hybridization probes. The specificity of the PCR assay was confirmed on 73 target and non-target strains, and target organisms were detected at the level of one genome per PCR. Results obtained with the combined flotation and real-time multiplex PCR method showed that quantification in rinse fluids was possible down to 3.0 ± 0.3 × 10 3 CFU/ml in the presence of other microorganisms at numbers up to 10 9 CFU/ml.

Use of High-Affinity Cell Wall-Binding Domains of Bacteriophage Endolysins for Immobilization and Separation of Bacterial Cells

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.

Isolation of uncultivated anaerobic thermophiles from compost by supplementing cell extract of Geobacillus toebii in enrichment culture medium

Several researchers have reported that microorganisms can be cultivated only in the presence of other microorganisms. We suggest that a portion of uncultivated microorganisms might be cultivated in the presence of cellular components released from bacteria in their natural environments. In this study, the cell extract of Geobacillus toebii was used to enrich uncultivated thermophiles from compost. In the process of enrichment cultures, cell extract supplementation apparently changed the community composition. This change was monitored by PCR-DGGE targeting 16S rRNA gene. Five novel groups of microorganisms (similarity of 16S rRNA gene to the closest relative <96%) were specifically isolated from enrichment cultures by using cell extract-supplemented culture media. Their growth was found to be dependent on the addition of extract of G. toebii. Putting these findings together, we suggest that the extracts of bacteria could be one of the growth factors in the thermal ecosystem with a possibility of extending other ecological niches.

Bioaugmentation potential of a vinyl chloride-assimilating Mycobacterium sp., isolated from a chloroethene-contaminated aquifer

An aerobic bacterium, Mycobacterium sp. strain TRW-2 that assimilated vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. The strain TRW-2 also degraded cis-dichloroethene ( cis-DCE) in mineral salts medium, but only when VC was present as the primary carbon source. However, no degradation of trans-dichloroethene or trichloroethene occurred in either the presence or absence of added VC. The measured growth yield values were 6.53 and 14.1 g protein/mol of VC and ETH utilized, respectively. Inoculation by strain TRW-2 in microcosms prepared with aquifer samples resulted in rapid degradation of VC, whereas native bacteria degraded negligible amounts of VC within the same time period, thus suggesting bioaugmentation potential of the isolate. Phylogenetic analysis of the 16S rDNA sequence of the isolate revealed 98% sequence similarity to the members of the genus Mycobacterium. In summary, the isolate’s ability to degrade VC, cis-DCE, and ETH and also its ability to survive and degrade VC in the presence of other microorganisms is relevant to the remediation of VC-impacted aquifers.

Isolation and maintenance of symbiotic fungi of ants in the tribe Attini (Hymenoptera: Formicidae)

The isolation and maintenance of symbiotic basidiomycete fungi living in association with ants of the tribe Attini has been hindered by the slow growth rate of these fungi and the presence of other microorganisms on the surface of the material which the ants maintain inside their nests to provide a growth substrate for their symbiont. In this paper we describe a method which increases the efficiency of isolation of these fungal symbionts by over seven fold as compared to traditional isolation procedures. Underground nests of attine ants of the genera Atta, Acromyrmex, Trachymyrmex and Mycetarotes were located, from which samples containing the fungal symbiont and ants were collected and transported to the laboratory where the ants were able to clean the fungal culture and stimulate its growth. As the symbiotic fungus grew, portions of its mycelium were collected and transferred to solid Yeast Nitrogen Base culture medium containing glucose and chloramphenicol. To facilitate the maintenance of the isolates in laboratory cultures, several nutrients were tested to formulate a complex culture medium for fast fungal growth and long-term storage. We successfully applied this methodology to the fungal symbionts of all the ant genera studied, thus producing a useful tool for the creation and maintenance of a comprehensive collection of fungi symbiotic of ants in the tribe Attini.

Simulation Model for Enumeration of Salmonella on Chicken as a Function of PCR Detection Time Score and Sample Size: Implications for Risk Assessment

A data gap commonly identified in risk assessments is the lack of quantitative information on the contamination of food with pathogens. A simulation model that predicts the incidence and distribution of Salmonella contamination on chicken as a function of PCR detection time score and sample size was developed with data from challenge studies with preenrichment samples that were composed of 25 g of chicken and 225 ml of buffered peptone water inoculated with 100.7 to 106Salmonella and incubated at 37°C. At 0, 2, 4, 6, 8, 10, 12, and 24 h of incubation, subsamples were collected and tested for Salmonella by PCR, and a PCR detection time score based on the widths of the bands in the electrophoresis gel was obtained for each preenrichment sample. Standard curves relating PCR detection time score to initial density of Salmonella inoculated were developed for sterile and nonsterile preenrichment samples. Presence of other microorganisms in the preenrichment sample decreased the PCR detection time score at low (<102 per 25 g) but not at high (>102 per 25 g) initial densities of Salmonella and resulted in a nonlinear standard curve rather than the linear standard curve obtained for sterile samples. The predicted incidence and distribution of Salmonella contamination on chicken increased in a nonlinear manner as sample size increased from 25 to 500 g. The new method reduced the time and cost of Salmonella enumeration by eliminating the selective enrichment, selective plating, and confirmation steps of the traditional most-probable-number method. Results are useful for risk assessment because they consider the uncertainty of the standard curve predictions and because they provide distributions of Salmonella contamination for different size samples of chicken that can be directly used in risk assessment.

Biochemical Functions ofBacillus licheniformisin Gnotobiotic Mice

A probiotic bacterial strain, Bacillus licheniformis NCTC 13123, has been mono-associated with germ-free mice. After colonization, the following biochemical microflora-associated characteristics (MACs) were analyzed in the large intestinal contents: excretion of short-chain fatty acids (SCFAs), inactivation of tryptic activity, degradation of β -aspartylglycine, breakdown of mucin, conversion of cholesterol to coprostanol and transformation of bilirubin to urobilins. An additional MAC, b-glucuronidase activity, was tested in vitro. β -Glucuronidase activity was not expressed by the bacterium. The amount of total SCFAs was lower than that previously found in germ-free mice. Utilization of individual SCFAs rather than production by the strain may be assumed. The other MACs remained similar to the basal values in germfree mice. We conclude that B. licheniformis NCTC 13123 is not able to alter any of the microbial functions investigated in vitro or in the gastrointestinal tract of germ-free mice. Whether and to what extent the strain has the capability to interfere with these functions in the presence of other micro-organisms remains to be investigated. Key wordis : Bacillus licheniforms , probiotics, microflora-associated characteristics, germ-free mice.

Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment.

The majority (>99%) of microorganisms from the environment resist cultivation in the laboratory. Ribosomal RNA analysis suggests that uncultivated organisms are found in nearly every prokaryotic group, and several divisions have no known cultivable representatives. We designed a diffusion chamber that allowed the growth of previously uncultivated microorganisms in a simulated natural environment. Colonies of representative marine organisms were isolated in pure culture. These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms. This observation may help explain the nature of microbial uncultivability.

Survival of Salmonella on Tomatoes Stored at High Relative Humidity, in Soil, and on Tomatoes in Contact with Soil

Salmonellosis has been linked to the consumption of several types of raw fruits and vegetables, some of which may have been contaminated with Salmonella before harvesting. The objectives of this study were to investigate water and soil as reservoirs of Salmonella for the contamination of mature green tomato fruits. Salmonella survived for at least 45 days in inoculated moist soil. The population of Salmonella on tomatoes in contact with soil increased by 2.5 log10 CFU per tomato during storage for 4 days at 20°C and remained constant for an additional 10 days. The number of cells inoculated on tomatoes decreased by approximately 4 log10 CFU per tomato during storage for 14 days at 20°C and 70% relative humidity. Fruits in contact with inoculated soil for 1 day at 20°C harbored Salmonella only near or on the skin surface. More Salmonella cells were observed in stem scar and subsurface areas of tomatoes as the time of storage increased. PCR fingerprinting revealed that among five Salmonella serotypes in the inoculum, Salmonella Montevideo was the most persistent on tomatoes in contact with inoculated soil and on spot-inoculated tomatoes, followed by Salmonella Poona and Salmonella Michigan. The results of this study demonstrate that an enhanced green fluorescent protein marker can be used to detect cells and monitor the growth of Salmonella in the presence of other microorganisms. Observations on the infiltration of Salmonella into tomato tissues support the contention that preharvest contact of produce with contaminated water or soil exacerbates problems associated with the postharvest removal of pathogens or their accessibility to treatment with sanitizers.

Toward an understanding of microbial communities through analysis of communication networks.

Bacteria receive signals from diverse members of their biotic environment. They sense their own species through the process of quorum sensing, which detects the density of bacterial cells and regulates functions such as bioluminescence, virulence, and competence. Bacteria also respond to the presence of other microorganisms and eukaryotic hosts. Most studies of microbial communication focus on signaling between the microbe and one other organism for empirical simplicity and because few experimental systems offer the opportunity to study communication among various types of organisms. But in the real biological world, microorganisms must carry on multiple molecular conversations simultaneously between diverse organisms, thereby constructing communication networks. We propose that biocontrol of plant disease, the process of suppressing disease through application of a microorganism, offers a model for the study of communication among multiple organisms. Successful biocontrol requires the sending and receiving of signals between the biocontrol agent and the pathogen, plant host, and microbial community surrounding the host. We are using Bacillus cereus, a biocontrol agent, and the organisms it must interact with, to dissect a communication network. This system offers an excellent starting point for study because its members are defined and well studied. An understanding of signaling in the B. cereus biocontrol system may provide a model for network communication among organisms that share a habitat and provide a new angle of analysis for understanding the interconnections that define communities.