Elias Hakalehto

Fast detection of bacterial growth by using Portable Microbe Enrichment Unit (PMEU) and ChemPro100i® gas sensor

The combination of the Portable Microbe Enrichment Unit (PMEU) cultivation and ChemPro100i((R)) Chemical Detector helps the fast detection of hospital infections. The first alarm of the microbe metabolism and growth in the enrichment culture was achieved in 2-3h when pure cultures of strains isolated from neonatal patients were tested. For the detection of minor concentrations of the bacteria, Bacillus, Staphylococcus, Klebsiella and Escherichia strains were diluted to 0.5-600cells per ml. Their preliminary detection was achieved within 4-5h. The combination of enrichment culture (PMEU) and detection of bacterial products by ion mobility spectrometer is a rapid and sensitive method for diagnosis of bacterial growth. This finding warrants further studies with clinical samples.

Dualistic acidic and neutral glucose fermentation balance in small intestine: Simulation in vitro

Intestinal microbes live in diminished or deprived oxygen conditions. Facultative anaerobic bacteria of the family Enterobacteriaceae ferment glucose in the gut using two main pathways: mixed acid and neutral fermentations. The aim of the present study was to clarify the roles of these fermentations in an in vitro model. Acid-producing Escherichia coli ATCC 25922 and neutral end-products producing Klebsiella mobilis ATCC 13048 were cultured in a Portable Microbe Enrichment Unit (PMEU) which permitted the selection of the gas phase in standardized conditions. In the butanediol production experiments also Klebsiella pneumoniae ssp. pneumoniae IIIa2 E111 strain was also used. Two isomers of 2,3-butanediol were observed in both aerobic and microaerobic Klebsiella cultures. During 7h cultures both E. coli and K. mobilis grew from densities of some million up to some billions per ml. The growth was almost equal in both aerobic and microaerobic conditions. In anaerobic conditions a balance prevailed in mixed E. coli and Klebsiella cultures between the species (final pH was 5.8-6.1), whereas in aerobic conditions the klebsiellas were favoured, and the pH rose up to about 8.5 (in 20h) as in pure Klebsiella cultures. In microaerobic Klebsiella cultures the final pH varied between 6.7 and 7.5. In mixed cultures the growth yields of both bacteria equalled those of the separate pure cultures indicating mutual benefits of the co-existence. Apparently E. coli produced a set of organic acids, which lowered pH. Klebsiellas increased the pH up to 2units probably due to acid conversion in anaerobic conditions to ethanol. We propose that the balance of acid and ethanol as well 2,3-butanediol fermentations contributes to the small bowel pH regulation, which also aids the host in the nutrient uptake in the small intestine.

Composition and Antimicrobial Activity of the Skin Peptidome of Russian Brown Frog Rana temporaria

A nano-HPLC-ESI-OrbiTrap study involving HCD and ETD spectra has been carried out to clarify the composition of the skin peptidome of brown Russian frogs Rana temporaria. This approach allowed determinantion of 76 individual peptides, increasing 3-fold the identified portion of the peptidome in comparison to that obtained earlier with FTICR MS. A search for the new bradykinin related peptides (BRPs) was carried out by reconstructing mass chromatograms based on the ion current of characteristic b- and y-ions. Several peptides were reported in the secretion of R. temporaria for the first time. The overall antibacterial activity of the skin secretion in general and of one individual peptide (Brevinin 1Tb) was determined using PMEU Spectrion (Portable Microbe Enrichment Unit) technology. The inhibitory effects of these peptides on Staphylococcus aureus and Salmonella enterica Serovar typhimutium were equal in scale to that reported for some antibiotics.

Gaseous CO2 signal initiates growth of butyric-acid-producing Clostridium butyricum in both pure culture and mixed cultures with Lactobacillus brevis

Microbial strains produce numerous volatile substances in the anaerobic conditions of the human intestines. The availability of CO(2) is known to be a prerequisite for bacterial growth in general. In experiments with anaerobic Lactobacillus brevis and Clostridium butyricum bacteria in the Portable Microbial Enrichment Unit (PMEU) it was shown that these strains interact; this interaction being mediated by CO(2) emission. CO(2) promoted clostridial growth in pure cultures and mixed cultures with lactobacilli. The growth of C. butyricum in pure cultures was much delayed or did not start at all without CO(2) from outside. Conversely, the onset of growth was provoked by a short (15 min) CO(2) burst. In mixed cultures the presence of lactobacilli in equal numbers speeded up the onset of clostridial growth by 10 h. If C. butyricum cultures designated as PMEU 1, 2, and 3 in cultivation syringes were chained by connecting the gas flow thereby allowing the volatiles of the preceding syringe culture to bubble to the next one, the growth started in 20, 10, or 6 h, respectively. This effect of gaseous emissions from other cultures speeding up the bacterial growth initiation was abolished if the gas was passed through sodium hydroxide to remove the CO(2). The positive contribution of lactobacilli to the growth of butyric-acid-producing clostridia documented in this simulation experiment with PMEU has in vivo implications and indicates molecular communication between the species. CO(2) is a necessary signal for the growth of clostridia, and lactobacilli can promote clostridial growth in mixed cultures where both bacteria grow well with mutual benefit.

Growth and gaseous emissions of pure and mixed small intestinal bacterial cultures: Effects of bile and vancomycin

Simultaneous cultivations in anaerobiosis, aerobiosis and with microaerobic gas mixture were used to clarify the bile (oxgall) effects on the pure and mixed cultures of enterobacterial strains in simulations in Portable Microbe Enrichment Unit (PMEU) linked with ChemPro100i((R)) gas detector. The effects of vancomycin were evaluated in aerobic cultures. Growth and metabolic activity of cultures were also followed by measuring sugar consumption, pH alterations, and colony counts on BD CHROMagar Orientation plates. Results showed that the two fermentatively different strains of facultative anaerobes, Escherichia coli E 17 and Klebsiella mobilis ATCC 13048 grew in balance regardless of oxygen level, bile acid concentration or other components of the mixed cultures, Bacillus cereus or Staphylococcus aureus. When the evaporations of the mixed cultures of E. coli, K. mobilis and S. aureus were compared with the emissions of the corresponding pure cultures by ChemPro100i((R)) gas sensing detector, the pure cultures of bile resistant E. coli and K. mobilis produced more gaseous components than the mixed culture indicating that these organisms cooperate and use the substrate more effectively together than separately. A survey of the aseptic bacterial isolations from the bile tract in a big University Hospital, (Salzburg, Austria) during 3 years, showed that these bacterial groups dominated. Only 13.24% of the 287 patient samples were sterile, and around 180 strains of both E. coli and Klebsiella/Enterobacter groups were found amongst 973 isolates from 249 patients (together 35.57%). Enterococcus sp. accounted for 246 isolates being the largest group of strains (24.25% of all the isolates). In anaerobiosis it was shown that Klebsiella neutralized the acids produced in the mixed acid fermentation of the E. coli. The ethanol produced from both groups evaporated in the gas stream of the PMEU culturing step and its formation also removes excess acidity from the cultures. The synergistic behaviour and symbiotic function between E. coli and Klebsiella/Enterobacter strains is suggested.

Use of portable enrichment unit in rapid characterization of infantile intestinal enterobacterial microbiota

Abstract Objective: The aim of this study was to develop a practical and efficient method for isolation and characterization of the enterobacterial microbiota of infant fecal samples. It could be used for reliable comparisons between individual microflora in different neonates, and in the follow-up of the succession in the floral developments in various nutritional conditions. Methods: A combination of traditional plate culture and different enrichment procedures carried out in a portable microbe enrichment unit (PMEU) was studied. The strains were isolated by plate culture both before and after enrichment. The isolated strains were identified by biochemical tests and the phenotypes of the isolates were compared by the PhenePlate™ technique. To obtain a picture of the formation of intestinal flora during the first year two follow-up samples were collected at the ages of 3 and 12 months from eight Finnish infants (one girl and seven boys). Results: Altogether 21 and 46 enterobacterial isolates were detected in samples taken at the ages of 3 and 12 months, respectively; 7/21 (33%) and 24/46 (52%) isolates were not detected by direct plating but only after enrichment. This material was supposed to demonstrate the differences between the different methodological approaches. Conclusion: There were age-dependent changes in the species proportions of different subpopulations of Enterobacteriaceae detected from the infantile fecal microbiota at different times. The statistically significant difference in the number of isolations with and without enrichment step suggests that the use of enrichment is highly recommendable and the PMEU equipment and method have now proven suitable for that purpose, at least for screening and monitoring the infant fecal flora. The method offers more representative, quicker, and relatively practical tools for obtaining an overall picture of the enterobacterial microbiota of the fecal samples and its variations, thus also giving an idea of the microbial communities in the gastrointestinal tract.

Fast coliform detection in portable microbe enrichment unit (PMEU) with Colilert® medium and bubbling

UNLABELLED Laboratory strains of coliforms Escherichia coli and Klebsiella mobilis were used to artificially contaminate water samples in two different cultivation and detection systems, without and with bubble flow. Samples were collected with an automated system (ASCS). The positive coliform signal caused the color change into yellow (at 550-570nm). This signal could also be transmitted on-line to cell phones. E. coli containing samples emitted UV fluorescence at 480-560nm when activated by UV light. If cultivation was started with inocula varying from 10,000 to 1cfu/ml, the positive detection was obtained between 2 and 18h, respectively, in Colilert medium using Coline PMEU device without gas bubbling. Accordingly, a single K. mobilis cell produced detectable growth in 18h. Various clinical E. coli strains were compared to each other with equal inoculum sizes, and they showed slight variations in the initiation and speed of growth. The gas bubble flow in PMEU Spectrion promoted the mixing and interaction of bacteria and indicator media and speeded the onset of growth. Carbon dioxide also accelerated bacterial growth. In the presence of vancomycin, the onset of E. coli culture growth was speeded up by the volatile outlet flow from previous cultures. In the last cultivation syringe in a series of five, the lag phase disappeared and the growth of the inoculum continued without major interruption. IN CONCLUSION the stimulation of the cultures by the gas flow turned out to be a useful means for improving the detection of indicator bacteria. It could also be used in combination with antibiotic selection in the broth medium.

Volatile fatty acids as an added value from biowaste

The aim of the present work was to provide proof of concept of employing a co-culture of K. mobilis and E. coli for producing short and medium chain volatile fatty acids (VFAs) from kitchen biowaste and potato peels. To this aim, experiments were carried out at pilot-scale installation with a bioreactor of 250L. Different feeding strategies were tested under microaerobic conditions, at pH 6.0-6.5 in order to enhance chain elongation. Acetic acid and ethanol were dominating products in the initial stages of the bioprocess, but in a relatively short time of approx. 20-22h from the process start accumulation of propionic acid took place followed by a chain elongation to butyric and valeric acids. The highest final products yield of 325mg/g TS was achieved for the substrate load of 99.1g TS/L (VS of 91.1g/L) and pH 6.5, with the productivity of 448mg/L/h. However, the highest average VFAs chain length (3.77C) was observed in the process run with the loading of 63.2g TS/L and pH 6.0. In this study, we demonstrated that the existing symbiosis of the co-culture of K. mobilis and E. coli favours formation and chain elongation of VFA, induced most likely by the enhanced ethanol formation. Our finding differs from the previous research which focus mostly on anaerobic conditions of VFAs production. The results provide good basis for further optimisation of VFAs production process.

Bacteriological Indications of Human Activities in the Ecosystems

The central role of microbes in the material cycles in Nature is worth studying for understanding the basis of healthy living on this planet. Human actions have been interfering with the balances in the ecosystems. The microbes compensate these effects by establishing new equilibria. The human impacts on the environment can be monitored by researching the microbial communities in soil, water and air. Wetlands as well as forest ecosystems are important sites for transitions in the natural cycles. Some microbiological follow up via satellite technology, or with climatological point of view, as well as with a focus on species distribution have been included. Any waste outlets from municipal, agricultural or industrial sources contain micro-organisms, whose environmental emission has ecological and health effects. Monitoring these effects is important for improving health, promoting the versatility in Nature, and for all efforts to maintain the functionability of the ecosystems. In environmental microbiology, it is not enough to solely track the intestinal indicator organisms. More holistic views on microbiological phenomena are required. The PMEU (Portable Microbe Enrichment Unit) has been included in this chapter as an example of an approach for implementing fast recovery methods, metabolic monitoring, and automated screening of environmental microbes. These techniques are essential for rooting out many devastating bacteriological diseases, such as cholerae, tuberculosis and salmonellosis. The applications with indicator bacteria could also be broadened into the source tracking of the origins of the pollution.

Enhanced mycobacterial diagnostics in liquid medium by microaerobic bubble flow in Portable Microbe Enrichment Unit

Portable Microbe Enrichment Unit (PMEU) method with microaerobic bubbling speeded up the growth of otherwise slowly starting and propagating Mycobacterium sp. Mycobacterium fortuitum growth was detected after 10-11h and Mycobacterium marinum produced clear growth in 4 days. A mycobacterial environmental isolate was verified in 2 days in the PMEU Spectrion(®) equipped with infrared sensors. In parallel static (without gas bubbling) cultures hardly any growth occurred. In conclusion, PMEU technology provided thus a rapid detection of environmental and clinical mycobacterial isolates. It would also help in the field diagnosis of antibiotic resistant Mycobacterium tuberculosis.