Portable Microbe Enrichment Unit Research Articles

Projected improvement in the fast microbiological analysis of neonatal blood samples

PurposeTo investigate whether Portable Microbe Enrichment Unit (PMEU) Scentrion® (Finnoflag Oy, Kuopio and Siilinjärvi, Finland) can provide a faster, more effective and accurate way of diagnosing sepsis and identifying pathogenic bacteria compared to the standard method in Kuopio University Hospital. Design and samplesBlood samples from 56 diseased neonates were collected and enriched by the PMEU Scentrion® and standard method. After enrichment all detected bacteria were cultured by Eastern Finland Laboratory Centre Joint Authority Enterprise (ISLAB). Main outcome and resultsResults were similar for both methods. Bacterial growth was indicated in 37 samples. Bacterial blood culture was positive in only six blood samples. 26 patients had a clinical diagnosis of sepsis. 20 patients were treated empirically because the bacterium could not be identified. ConclusionsPMEU Scentrion® proved out to be capable of providing early signs of bacterial growth in blood samples with increased efficiency. Development of the PMEU software and use of more specific culture broth could offer means for increased speed, specificity and sensitivity for the analysis of neonatal sepsis. These further improvements could provide tools for verifying more sepsis causing pathogens by rapid methodology.

Enhanced recovery, enrichment and detection of Mycobacterium marinum with the Portable Microbe Enrichment Unit (PMEU)

The use of PMEU significantly accelerated the growth of otherwise slowly growing Mycobacterium sp. Compared to the static reference cultures, M. marinum was detected after 24–48h of cultivation in the PMEU Spectrion® equipped with infrared (IR) sensors. Parallel static cultures did not exhibit or indicate mycobacterial growth within these time limits, and essentially no growth was found in them. The PMEU approach could provide a powerful tool for the rapid diagnosis and determination of environmental and clinical isolates of slow-growing species of mycobacteria. This approach also provides a method for improving diagnostics for M. tuberculosis and other pathogenic mycobacteria, including their antibiotic resistant forms, which represents a significant health problem worldwide and in Africa in particular.

Development of microbiological field methodology for water and food-chain hygiene analysis of Campylobacter spp. and Yersinia spp. in Burkina Faso, West Africa

Field-adaptable research methods for identifying Campylobacter sp., Yersinia sp. and other pathogenic and indicator bacteria were designed in Finland and tested in Burkina Faso. Several bacterial groups were also validated from artificially contaminated samples. Campylobacter strains were cultivated using an innovative gas generation system: The ‘Portable Microbe Enrichment Unit’ (PMEU) which provides microaerobic gas flow into the enrichment broth. This enhanced cultivation system produced rapid growth of several isolates of campylobacteria from water and chicken samples. The latter were obtained from local marketplace samples. No yersinias were found in the field studies, whereas they were readily recovered from the spiked samples, as well as Salmonella sp. and Escherichia coli strains. The PMEU method turned out to be reliable for monitoring of water and food hygiene in remote locations.

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

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.

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.

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.

Minute Microbial Levels Detection in Water Samples by Portable Microbe Enrichment Unit Technology

Water purity requires continuous monitoring. PMEU technology (Portable Microbe Enrichment Unit) provides the means for starting the enrichment and analysis immediately after the sampling. The entire procedure can be automated and microbe levels monitored in real-time from the enrichment broth by using IR, optical, UV or volatiles screening. For example, single cells of Escherichia coli have been verified from water samples in less than 10 hours in an official validation project. Also such pathogens as Salmonella, Campylobacter and Yersinia have been detected rapidly from natural, purified and irrigation waters. The PMEU technology has potential in the continuous monitoring within water departments and distribution systems, as well as in environmental studies. 1. Background In the present manuscript experimental approach and work carried out with the Portable Microbe Enrichment Unit (PMEU) is presented. This device is an ideal tool to investigate micro-organisms life at microscopic and molecular scale. Micro-organisms such as bacteria have a large influence on our lives with impact on economy, various industries, healthcare, agriculture and forestry, as well as in water management. In Finland our team published booklet entitled Vesien Mikrobiaapinen (in English: A Microbiological ABC book of the waters) with the purpose to provide microbiological information to decision makers. As scientists we continuously deal with the public and their representatives: the political decision makers. In order to get funding, we ought to make them understand the importance of environmental protection and conservation of water sources, from the microbiological point of view. One area where micro-organisms have a large influence is the threat and deleterious impact on human and animal health caused by pathogenic micro-organisms. They are potentially distributed via waterways and often disseminated at small concentrations which still pose health hazards. In order to detect these minute concentrations, new tools are needed, also to simulate their biochemical activities. Their detection is not either a single or a simple event but a whole diagnostic process (Figure 1). Focusing on the often neglected step of specimen collection is of crucial importance. Sampling should be carried out considering all the physicochemical parameters, selection of sampling site etc. Usually it is required to transport and store the sample with caution and care. This is the step when most of the microbes present in the sample are often destroyed. They simply cannot survive or do not remain microbiologically viable. In order to avoid these problems, the Portable Microbe Enrichment Unit was developed. Time is saved in the PMEU processing of the samples, since the verification steps could follow sooner compared to traditional microbiology. The basic simple idea is to start the enrichment cultivation right subsequent to sampling. In practice, using the PMEU technology accelerates the growth and metabolism of microbes in a way that as a whole has been designated as enhanced enrichment. It has also opened up new applications in the field of water microbiology, which are briefly introduced in the present article.

Lactic Acid Bacteria Enriched from Human Gastric Biopsies

The purpose of this paper was to check if viable bacteria, in particular lactic acid bacteria (LAB), could be enriched from biopsies obtained from healthy gastroscopy patients. Gastric biopsies were obtained from 13 gastroscopy patients and subjected to an anaerobic or microaerophilic enrichment procedure utilizing the Portable Microbe Enrichment Unit (PMEU). Profuse microbial growth was observed in most cases. Samples plated on MRS showed high numbers of LAB. The most common species characterized were Lactobacillus reuteri, Lact. salivarius, and Streptococcus salivarius. The results demonstrate a continuous presence of viable LAB in healthy stomach. The species are similar to those traditionally used in food applications. The gastric LAB strains could have a potential in developing probiotic foods aimed specially on the upper part of the gastrointestinal tract.

Enterobacterial Microflora in Infancy—A Case Study with Enhanced Enrichment

To validate PMEU (Portable Microbe Enrichment Unit) method for monitoring the composition and development of infantile intestinal enterobacterial microflora. A case study of a boy with neonatal sepsis is presented. During the first 32 months, he was given 19 systemic antibiotic treatments representing seven different antibiotic classes. Seven fecal samples collected at ages from 3.4 to 31.6 months were studied for enterobacterial strains by a combination of enhanced enrichment culture in the PMEU and plate culture. The identification and phenotypic characterization of the isolates was performed by biochemical tests. 37/51 (73%) of the enterobacterial isolates were detected only after the PMEU enrichment. In most samples Escherichia coli strains were predominating, but also several Enterobacter, Klebsiella, Pantoea and Proteus strains could be isolated. It seemed that the antibiotic medications remarkably delayed the development of the intestinal microflora, because first enterobacterial strains were detected only after 6 months of age. The enrichment step turned out to essentially improve the characterization and monitoring of the intestinal enterobacterial microbiota of infants. Compared to plate culture the amount of isolates was 2.6-fold by the PMEU enrichment. This study gives an idea on the development and succession of microbial communities in the gastrointestinal tract and on the variation of the strains due to the intestinal environmental factors.

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 ® 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 ® 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.

Enhanced enrichment and detection of thermotolerant Campylobacter species from water using the Portable Microbe Enrichment Unit and real-time PCR

An enhanced enrichment using the Portable Microbe Enrichment Unit (PMEU) with the microaerobic bubbling of broths was applied for the detection of thermotolerant Campylobacter species from water. This PMEU enrichment was compared with the conventional static enrichment of the international standard ISO 17995:2005. In addition, Campylobacter detection after enrichment using a real-time PCR detection was compared with colony counts. The tests with stressed Campylobacter jejuni cells in drinking water indicated that the PMEU enrichment yielded a significantly higher number of Campylobacter cells in the Bolton broth compared with the conventional static incubation. Application of the real-time PCR technique shortened the Campylobacter detection time. This combination of method modifications can be used for Campylobacter detection from water and adds methodological repertoire for the rapid survey and management of waterborne outbreaks.

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 ® 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 2–3 h 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 0.5–600 cells per ml. Their preliminary detection was achieved within 4–5 h. 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.

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

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.

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 7 h 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 20 h) 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 2 units 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.

Aerobic and anaerobic growth modes and expression of type 1 fimbriae in Salmonella

The aim of this study was to clarify the growth rates of facultatively anaerobic Salmonella enterica serovar Enteritidis strain in aerobic and anaerobic conditions and the expression of type 1 fimbriae in relation to the growth phases. The cultivation was carried out in a Portable Microbe Enrichment Unit (PMEU) where in same conditions one can grow the cells in parallel by modifying, e.g. aerobiosis only. The results obtained show that although the anaerobic metabolism is generally believed to be a slower producer of biomass or metabolites, in these circumstances S. enterica serovar Enteritidis strain gave comparable growth rates in anaerobiosis with nitrogenation as in aerobic cultures with constant aeration. Fimbrial antigens were produced in the beginning of logarithmic phase of the growth cycle both in the aerobic and anaerobic conditions. The fimbria remained in the presence of oxygen. This capability is possibly used for the intrusion of oxygen containing tissues of host body by the invading pathogens. In conclusion S. enterica serovar Enteritidis strain suspensions grow equally well in constant nitrogenation and aeration, and fimbria were produced in both conditions, during the early logarithmic phase but they prevailed in the presence of aeration.