Infection of susceptible horses with Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is associated with commingling. Exposure may occur among horses at equestrian events, sales, or horses moved among different equine stabling environments. Strangles can affect all horses on a farm, leading to the death of up to 10% of cases depending on their immunity status at the time of infection, the development of complications, the success of biosecurity measures, and the use of vaccination. The current retrospective study used ELISAs to measure the exposure of horses to S. equi at a farm that experienced an outbreak of strangles shortly after the introduction of a new horse on the same day that the majority of the 17 resident horses were vaccinated with Strangvac for the first time. One vaccinated horse, which subsequently tested positive for S. equi and EHV-4, developed a cough, elevated body temperature, and nasal discharge 11 days after the first vaccination. Two other horses developed fever for one day at 22 days post-first vaccination, but only one had serological evidence of exposure to S. equi. All vaccinated horses had high antibody titres to vaccine components, whilst 7 of the 17 resident horses, and the new arrival, tested seropositive for exposure to S. equi. Although 3 out of the 17 vaccinated horses developed mild signs of disease before second vaccination, serological data support the effectiveness of vaccination in resident populations of horses to minimise the risk of strangles following the introduction of a new horse.

Infection of horses with Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is associated with the movement of horses from one farm to another, or the attendance of equestrian events and sales. Strangles can affect all horses on a farm, leading to the death of up to 10% of cases depending on the success of biosecurity measures and the use of vaccination. This retrospective study used ELISAs to measure the exposure of horses to S. equi at a farm that experienced an outbreak of strangles shortly after the introduction of a new horse on the same day that the majority of the 17 resident horses were vaccinated with Strangvac for the first time. One vaccinated horse, which subsequently tested positive for S. equi and EHV-4, developed a cough, elevated body temperature and nasal discharge 11 days after first vaccination. Two other horses developed fever for one day at 22 days post-first vaccination, but only one had serological evidence of exposure to S. equi. All vaccinated horses had high antibody titres to vaccine components, whilst seven of the 17 resident horses, and the new arrival, tested seropositive for exposure to S. equi. Although three out of the 17 vaccinated horses developed mild signs of disease before second vaccination, serological data support the effectiveness of vaccination in resident populations of horses to minimise the risk of strangles following the introduction of a new horse.

BACKGROUND:Strangles, caused by Streptococcus equi subspecies equi (S. equi), is a prevalent infectious disease of horses. This is the first report on the use of a new vaccine, Strangvac, in a natural outbreak of strangles. OBJECTIVES:To measure the effects of Strangvac vaccination during an outbreak of strangles at a Swedish farm. STUDY DESIGN:Longitudinal cohort study. METHODS:Healthy horses (n = 17) were vaccinated on day 0, which occurred 23 days after strangles was first confirmed in three unvaccinated horses on the same farm. Blood serum samples were collected on day 0, day 28 and day 489. A combined iELISA was used to measure total antibody titres towards the components of the vaccine. Specific antibody levels in blood sera to S. equi were also quantified using the antigen A/C iELISAs, which can differentiate infected from vaccinated animals. Clinical signs were monitored. RESULTS:All vaccinated horses had increased total antibody titres to CCE, Eq85 and/or IdeE from day 0 (2.50 ± 0.28) to day 28 (3.63 ± 0.31; p < 0.0001). Seropositivity in the antigen A/C iELISA was noted in 8/17 healthy horses on day 0 (prior to vaccination), in 9/16 vaccinates on day 28, and in 3/3 unvaccinated clinical cases on day 28. None of the vaccinated horses developed strangles, but 5/7 of the original vaccinated horses tested seropositive in the A/C iELISA on day 489, which was 377 days after their third vaccination. MAIN LIMITATIONS:Aside from the three unvaccinated clinical cases, no unvaccinated control group was available. CONCLUSIONS:Despite adherence to recommended biosecurity measures, serological evidence of exposure to S. equi was demonstrated in half of the vaccinated horses. Notably, all horses vaccinated during the outbreak responded to the vaccine components and remained healthy, suggesting a protective effect of vaccination of healthy horses with Strangvac in outbreak situations.

Background: Streptococcus equi subspecies equi (S. equi) is the cause of strangles, one of the most prevalent diseases of horses worldwide. The disease is characterised by fever and the formation of abscesses in the lymph nodes of the head and neck, which can restrict the airway. A multicomponent subunit vaccine, Strangvac, has been shown to effectively reduce clinical signs of strangles and to reduce its incidence. Objective: The aim of this study was to determine the immune response against the immunoglobulin-cleaving endopeptidase IdeE, a key protective component within the vaccine and the ability of antibodies to neutralize the proteolytic activity of IdeE. Methods: An in vitro assay was developed to measure the functional inhibition of recombinant IdeE by horse sera pre- and post-vaccination. The IdeE-neutralising titres were compared to the corresponding IdeE-specific antibody titres measured by iELISA (indirect Enzyme-Linked Immunosorbent Assay). Results: A significant IdeE-specific antibody response in blood serum collected from ponies was induced after Strangvac vaccinations. Concomitantly, significant increases in the neutralising activity of IdeE occurred, persisting for at least 12 months post-second vaccination. IdeE-neutralising activity was further increased significantly after a third vaccination, even when the third dose was administered 12 months after the second dose, demonstrating that immunological memory to the vaccine persisted for 12 months. There was a significant correlation between the IdeE-neutralising activity of blood sera and the level of IdeE-specific antibodies. Conclusions: These data provide insights into one potential mechanism by which this vaccine protects Equids against or during S. equi infection.

Streptococcus suis is a severe zoonotic pathogen affecting weaned piglets. No commercial vaccine that provides protection against S. suis is available. A prototype vaccine, tentatively called VASIP (Vaccine Against Streptococcus suis Infection in Pigs), composed of five recombinant fusion proteins, encompassing 23 different protein domains, was used in this study. Pregnant sows were vaccinated on three occasions, at 68, 47 and 19 days prior to farrowing, resulting in high antibody levels, both in sera and in colostrum. Antibodies were transferred to the litter via colostrum. The litters from VASIP-vaccinated and placebo-vaccinated sows were challenged intravenously with S. suis at four or seven weeks of age in two different arms of the study. Body temperature and clinical signs (demeanour, behavioural CNS, and mobility) of infection showed that piglets from vaccinated sows were significantly protected against S. suis infection in the 4-week-old group and that the incidence of severe clinical signs was lower in the 7-week-old group compared with piglets from placebo sows. The study demonstrates the feasibility of vaccinating sows, rather than piglets, using recombinant fusion proteins to maximise protection against S. suis during the period in which they are most at risk of disease.

OBJECTIVES:The increasing frequency of antibiotic-resistant bacterial infections is a major public health challenge, and new antibiotic drugs are urgently needed. A rapid solution to the problem is to repurpose clinically approved compounds with antibacterial properties, such as the nucleoside analogues zidovudine (azidothymidine) or 5-fluoro-2'-deoxyuridine. Here we report the in vitro and in vivo antibacterial properties of double and triple combinations of azidothymidine or 5-fluoro-2'-deoxyuridine with uridine and/or trimethoprim. METHODS:We determined MICs of azidothymidine and 5-fluoro-2'-deoxyuridine, alone or combined with uridine and/or trimethoprim, against a selection of Gram-negative and Gram-positive bacteria. We also measured MICs of a selection of antibiotics of different classes as a function of uridine concentration. The efficacy of azidothymidine and 5-fluoro-2'-deoxyuridine with uridine and/or trimethoprim was measured in a murine peritonitis infection model. RESULTS:The addition of uridine enhanced the in vitro antibacterial activity of azidothymidine and 5-fluoro-2'-deoxyuridine, against Gram-negative and Gram-positive bacteria, respectively. Uridine also enhanced the in vitro antibacterial activity of azidothymidine/trimethoprim and 5-fluoro-2'-deoxyuridine/trimethoprim combinations. Triple combinations containing azidothymidine, trimethoprim and uridine, showed antibacterial synergy against Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) whereas the 5-fluoro-2'-deoxyuridine, trimethoprim and uridine combination showed synergy against the Gram-positive Staphylococcus aureus. The positive effect of uridine on the efficacy of azidothymidine/trimethoprim combination was also observed in vivo in a murine E. coli peritonitis model. CONCLUSIONS:Triple combinations of these clinically approved compounds warrant further investigations as therapies to combat antibiotic-resistant infections.

Abstract Background Digital dermatitis (DD) is a contagious hoof infection affecting cattle worldwide. The disease causes lameness and a reduction in animal welfare, which ultimately leads to major decreases in milk production in dairy cattle. The disease is most likely of polymicrobial origin with Treponema phagedenis and other Treponema spp. playing a key role; however, the etiology is not fully understood. Diagnosis of the disease is based on visual assessment of the feet by trained hoof-trimmers and veterinarians, as a more reliable diagnostic method is lacking. The aim of this study was to evaluate the use of an enzyme-linked immunosorbent assay (ELISA) on bulk tank milk samples testing for the presence of T. phagedenis antibodies as a proxy to assess herd prevalence of DD in Swedish dairy cattle herds. Results Bulk tank milk samples were collected in 2013 from 612 dairy herds spread across Sweden. A nationwide DD apparent prevalence of 11.9% (8.1–14.4% CI95%) was found, with the highest proportion of test-positive herds in the South Swedish regions (31.3%; 19.9–42.4% CI95%). Conclusions This study reveals an underestimation of DD prevalence based on test results compared to hoof trimming data, highlighting the critical need for a reliable and accurate diagnostic method. Such a method is essential for disease monitoring and the development of effective control strategies. The novelty of ELISA-based diagnostic methods for DD, coupled with the disease’s polymicrobial origin, suggests an avenue for improvement. Developing an expanded ELISA, incorporating antigens from various bacterial species implicated in the disease, could enhance diagnostic accuracy. The significance of this study is underscored by the extensive analysis of a substantial sample size (612). Notably, this investigation stands as the largest assessment to date, evaluating the application of ELISA on bulk tank milk for DD diagnosis at the herd level.

Twenty-eight multidrug-resistant bacterial strains closely related or identical to Pedobacter cryoconitis, Pedobacter lusitanus and Pedobacter steynii were isolated from soil samples by selection for multidrug-resistance. Approximately 3–30% of the selected isolates were identified as Pedobacter, whereas isolation without antibiotics did not yield any isolates of this genus. Next generation sequencing data showed Pedobacter to be on 69th place among the bacterial genera (0.32% of bacterial sequences). The Pedobacter isolates produced a wide array of novel compounds when screened by UHPLC-MS/MSMS, and hierarchical cluster analysis resulted in several distinct clusters of compounds produced by specific isolates of Pedobacter, and most of these compounds were found to be peptides. The Pedobacter strain UP508 produced isopedopeptins, whereas another set of strains produced pedopeptins, which both are known cyclic lipodepsipeptides produced by Pedobacter sp. Other Pedobacter strains produced analogous peptides with a sequence variation. Further strains of Pedobacter produced additional novel antibacterial cyclic lipopeptides (ca 800 or 1400 Da in size) and/or linear lipopeptides (ca 700–960 Da in size). A 16S rRNA phylogenetic tree for the Pedobacter isolates revealed several distinct clades and subclades of isolates. One of the subclades comprised isolates producing isopedopeptin analogs, but the isopedopeptin producing isolate UP508 was clearly placed on a separate branch. We suggest that the non-ribosomal peptide synthases producing pedopeptins, isopedopeptins, and the analogous peptides, may derive from a common ancestral non-ribosomal peptide synthase gene cluster, which may have been subjected to a mutation leading to changed specificity in one of the modules and then to a modular rearrangement leading to the changed sequence found in the isopedopeptins produced by isolate UP508.

In the search for new antibiotic compounds, fractionation of Pseudomonas protegens UP46 culture extracts afforded several known Pseudomonas compounds, including 2,4-diacetylphloroglucinol (DAPG), as well as two new antibacterial alkaloids, 6-(pyrrolidin-2-yl)DAPG ( 1 ) and 6-(piperidin-2-yl)DAPG ( 2 ). The structures of 1 and 2 were determined by nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1 and 2 were found to have antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus , with minimal inhibitory concentration (MIC) 2 and 4 μg ml −1 , respectively, for 1 , and 2 μg ml −1 for both pathogens for 2 . The MICs for 1 and 2 , against all tested Gram-negative bacteria, were >32 μg ml −1 . The half maximal inhibitory concentrations against HepG2 cells for compounds 1 and 2 were 11 and 18 μg ml −1 , respectively, which suggested 1 and 2 be too toxic for further evaluation as possible new antibacterial drugs. Stable isotope labelling experiments showed the pyrrolidinyl group of 1 to originate from ornithine and the piperidinyl group of 2 to originate from lysine. The P. protegens acetyl transferase (PpATase) is involved in the biosynthesis of monoacetylphloroglucinol and DAPG. No optical rotation was detected for 1 or 2 , and a possible reason for this was investigated by studying if the PpATase may catalyse a stereo-non-specific introduction of the pyrrolidinyl/piperidinyl group in 1 and 2 , but unless the PpATase can be subjected to major conformational changes, the enzyme cannot be involved in this reaction. The PpATase is, however, likely to catalyse the formation of 2,4,6-triacetylphloroglucinol from DAPG.

Pedobacter cryoconitis strain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A-H (1-8), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1-8 were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B (2), which had the best combination of antibacterial activity, in vitro cytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 μg/mL, respectively. Furthermore, compound 2 had activity against colistin-resistant strains of A. baumannii, E. coli, and Klebsiella pneumoniae, with a MIC down to 8, 2, and 4 μg/mL, respectively. Compound 6 was tested in an E. coli liposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.

Antibiotics are widely used to prevent and treat diseases and promote animal growth in the livestock industry, and therefore antibiotic residues can end up in biogas digestate from processes treating animal manure (AM) and food waste (FW). These digestates represent a potential source of spread of antimicrobial resistance (AMR) when used as fertilisers. This study evaluated AMR risks associated with biogas digestates from two processes, using AM and FW as substrate, by isolation and identification of antibiotic-resistant bacteria (ARB) and testing their susceptibility to different antibiotics. ARB from the digestates were isolated by selective plating. The antibiotic susceptibility profile of isolates was determined using ampicillin, ceftazidime, meropenem, vancomycin, ciprofloxacin, rifampicin, chloramphenicol, clindamycin, erythromycin, tetracycline, gentamicin or sulfamethoxazole/trimethoprim, representing different antibiotic classes with differing mechanisms of action. In total, 30 different bacterial species belonging to seven genera were isolated and classified. Bacillus and closely related genera, including Paenibacillus, Lysinibacillus and Brevibacillus, were the dominant ARB in both digestates. Most of the ARB strains isolated were non-pathogenic and some were even known to be beneficial to plant growth. However, some were potentially pathogenic, such as an isolate identified as Bacillus cereus. Many of the isolated species showed multi resistance and the AM digestate and FW digestate both contain bacterial species resistant to all antibiotics tested here, except gentamicin. A higher level of resistance was displayed by the FW isolates, which may indicate higher antibiotic pressure in FW compared with AM digestate. Overall, the results indicate a risk of AMR spread when these digestates are used as fertiliser. However, most of the ARB identified are species commonly found in soil, where AMR in many cases is abundant already, so the contribution of digestate-based fertiliser to the spread of AMR may still be very limited.

The fungus Aspergillus amoenus Roberg strain UP197 was shown to produce antibacterial tetramic acid based alkaloids. Two new compounds, pyranterreone I and J (1 and 2), were isolated and characterized, in addition to the known compounds cordylactam, 7-hydroxycordylactam, pyranterreone C, D, F and G. Neither the pyranterreones nor the cordylacctams had previously been tested for antimicrobial activity. Thus, all isolated compounds were tested against a panel of clinically important bacteria and fungi. Pyranterreone C was active against Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) between 1 and 8 µg/mL, whereas the MICs for all other compounds were >32 µg/mL. Pyranoterreone C was cytotoxic towards HepG2 cells, and since pyranterreone C reacted rapidly with the nucleophile cysteine, it is likely that the observed antibacterial activity is due to the chemical reactivity rather than enzymatic affinity, making it unsuitable for development as an antibacterial drug.

In the search for new microbial antibacterial secondary metabolites, two new compounds (1 and 2) were isolated from culture broths of Penicillium spathulatum Em19. Structure determination by nuclear magnetic resonance and mass spectrometry identified the compounds as 6,7-dihydroxy-5,10-dihydropyrrolo[1,2-b]isoquinoline-3-carboxylic acid (1, spathullin A) and 5,10-dihydropyrrolo[1,2-b]isoquinoline-6,7-diol (2, spathullin B). The two compounds displayed activity against both Gram-negative and -positive bacteria, including Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus. Compound 2 was more potent than 1 against all tested pathogens, with minimal inhibitory concentrations down to 1 µg/mL (5 µM) against S. aureus, but 2 was also more cytotoxic than 1 (50% inhibitory concentrations 112 and 11 µM for compounds 1 and 2, respectively, towards Huh7 cells). Based on stable isotope labelling experiments and a literature comparison, the biosynthesis of 1 was suggested to proceed from cysteine, tyrosine and methionine via a non-ribosomal peptides synthase like enzyme complex, whereas compound 2 was formed spontaneously from 1 by decarboxylation. Compound 1 was also easily oxidized to the 1,2-benzoquinone 3. Due to the instability of compound 1 and the toxicity of 2, the compounds are of low interest as possible future antibacterial drugs.

Chlorophenols are widespread and of environmental concern due to their toxic and carcinogenic properties. Development of less costly and less technically challenging remediation methods are needed; therefore, we developed a formulation based on micronized vermiculite that, when air-dried, resulted in a granular product containing the 4-chlorophenol (4-CP)-degrading Gram-positive bacterium Arthrobacter chlorophenolicus A6. This formulation and stabilization method yielded survival rates of about 60% that remained stable in storage for at least 3 months at 4 °C. The 4-CP degradation by the formulated and desiccated A. chlorophenolicus A6 cells was compared to that of freshly grown cells in controlled-environment soil microcosms. The stabilized cells degraded 4-CP equally efficient as freshly grown cells in two different set-ups using both hygienized and non-treated soils. The desiccated microbial product was successfully employed in an outdoor pot trial showing its effectiveness under more realistic environmental conditions. No significant phytoremediation effects on 4-CP degradation were observed in the outdoor pot experiment. The 4-CP degradation kinetics from both the microcosms and the outdoor pot trial were used to generate a predictive model of 4-CP biodegradation potentially useful for larger-scale operations, enabling better bioremediation set-ups and saving of resources. This study also opens up the possibility of formulating and stabilizing also other Arthrobacter strains possessing different desirable pollutant-degrading capabilities.

Bioassay-guided fractionation of culture extracts of Serratia plymuthica strain MF371-2 resulted in the isolation of two new antibacterial compounds with potent activity against Gram-positive bacteria, including Staphylococcus aureus LMG 15975 (MRSA). A spectroscopic investigation, in combination with synthesis, enabled the characterization of the compounds as 3-butyryl-4-hydroxy-6-heptyl-5,6-dihydro-2H-pyran-2-one (plymuthipyranone A, 1) and 3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one (plymuthipyranone B, 2). The MIC values for 1 and 2 against S. aureus LMG 15975 were determined to be 1-2 μg mL-1 and 0.8 μg mL-1, respectively. Compound 2 was found to have potent activity against many strains of S. aureus, including several mupirocin-resistant strains, other species of Staphylococcus, and vancomycin-resistant enterococci. Compound 2 was slightly cytotoxic for human cells, with CC50 values between 4.7 and 40 μg mL-1, but the CC50/MIC ratio was ≥10 for many tested combinations of human cells and bacteria, suggesting its possible use as an antibacterial agent. Several analogues were synthesized with different alkyl groups in the 3- and 6-positions (6-13), and their biological properties were evaluated. It was concluded that the activity of the compounds increased with the lengths of the alkyl and acyl substituents.

The urgent need for new antibacterial drugs has led to renewed interest in microorganisms, which historically have been the main source of previously discovered antibiotics. The present study describes the discovery of two new antibacterial oxazolylindole type alkaloids, labradorins 5 (1) and 6 (2), which were isolated and characterized from two isolates of Pseudomonas sp., along with four previously known tryptophane derived alkaloids. The structures of 1 and 2 were determined by NMR spectroscopy and MS, and confirmed by synthesis. During bioassay-guided isolation using several human bacterial pathogens, 1 and 2 displayed activity towards Staphylococcus aureus and Acinetobacter baumannii. The minimal inhibitory concentrations (MIC) of compounds 1 and 2 against S. aureus were 12 μg·mL−1 and 50 μg·mL−1, respectively, whereas the MICs against A. baumannii were >50 μg·mL−1. The CC50 values of compound 1 towards a liver cell line (HEP-G2) and a T-cell line (MT4) were 30 μg·mL−1 and 20 μg·mL−1, respectively, and for compound 2 were >100 μg·mL−1 and 20 μg·mL−1, respectively. Due to the limited potency of compounds 1 and 2, along with their toxicity, the compounds do not warrant further development towards new antibiotics.

In this study we have compared the ability of the organic polymers Ficoll and hydroxyethylcellulose (HEC) and the disaccharides sucrose and trehalose to support cell survival during freeze-drying and subsequent storage of a gram-negative Sphingobium sp. In addition to determination of viability rates, cell integrity was evaluated using lipid peroxidation and RNA quality assays for the different storage conditions and formulation compositions. All formulations resulted in high initial cell survival rates after freeze-drying. However, the disaccharide formulations were superior to the polymer-based formulations in supporting cell survival during storage with the exception of Ficoll that upon storage under vacuum yielded bacterial survival rates equal to that of sucrose. Storage in the presence of both oxygen and moisture was detrimental for bacterial survival in all formulations tested, however, lipid peroxidation or RNA damages were not the controlling mechanisms for cell death in this system. The ability of Ficoll and HEC to support cell survival during freeze-drying show that organic polymers, expected to lack the water replacing capability of e.g. disaccharides, can successfully be used as lyoprotectants. For storage under vacuum conditions we suggest that the intracellular amount of sugars (i.e. trehalose), or other protective native cell components, is sufficient for a basic protection inside the bacteria cell and that the amorphous state is the most important aspect of the formulation excipient. However, when exposed to oxygen and moisture during storage this protection is not sufficient to prevent cell degeneration.

We developed a new approach to separate bacteria from human blood cells based on soft inertial force induced migration with flow defined curved and focused sample flow inside a microfluidic device. This approach relies on a combination of an asymmetrical sheath flow and proper channel geometry to generate a soft inertial force on the sample fluid in the curved and focused sample flow segment to deflect larger particles away while the smaller ones are kept on or near the original flow streamline. The curved and focused sample flow and inertial effect were visualized and verified using a fluorescent dye primed in the device. First the particle behaviour was studied in detail using 9.9 and 1.0 microm particles with a polymer-based prototype. The prototype device is compact with an active size of 3 mm(2). The soft inertial effect and deflection distance were proportional to the fluid Reynolds number (Re) and particle Reynolds number (Re(p)), respectively. We successfully demonstrated separation of bacteria (Escherichia coli) from human red blood cells at high cell concentrations (above 10(8)/mL), using a sample flow rate of up to 18 microL/min. This resulted in at least a 300-fold enrichment of bacteria at a wide range of flow rates with a controlled flow spreading. The separated cells were proven to be viable. Proteins from fractions before and after cell separation were analyzed by gel electrophoresis and staining to verify the removal of red blood cell proteins from the bacterial cell fraction. This novel microfluidic process is robust, reproducible, simple to perform, and has a high throughput compared to other cell sorting systems. Microfluidic systems based on these principles could easily be manufactured for clinical laboratory and biomedical applications.

The human gut microbiota has a substantial impact on human health. Different factors such as disease, diet and drug use can have significant impacts on the gut microbiota. Therefore, it is of interest to have simple, rapid methods for analysis of the composition of the gut microbiota for clinical diagnostic purposes. Since only a minor fraction of the gastrointestinal bacterial community is presently possible to cultivate, molecular approaches are currently the best suited to investigate its composition. However, most of these molecular approaches require technical expertise and expensive equipment to run and they are not routinely available. Ideally, the analyses should be point-of-care options that can be run on a chip. In this study, an existing lab-on-chip (LOC) system for sizing/quantifying DNA was combined with length heterogeneity PCR (LH-PCR), a PCR-based profiling method targeting bacterial 16S rRNA gene sequences, to develop a fast, straightforward, reproducible, and economical method for profiling bacterial communities. The LOC LH-PCR method was first evaluated using a standardized gut cocktail containing genomic DNA from eight different bacterial species representing different genera of relevance for human health. The method was also tested on DNA that was directly extracted from human faecal samples and it was consistently capable of detecting alterations in the bacterial samples before and after antibiotic treatment. Although the resolution of the method needs improvement, this study represents the first step towards development of a diagnostic LOC for profiling gut bacterial communities.

The ability of bacteria to sense their surroundings can be employed to measure the bioavailability and toxicity of pollutants. However, long-term maintenance of both viability and activity of the sensor bacteria is required for the development of cell-based devices for environmental monitoring. To meet these demands, various techniques to conserve such bacteria have been reported, including freeze drying, vacuum drying, continuous cultivation, and immobilisation in biocompatible polymers of organic or inorganic origin. Much effort has been invested in merging these bacterial preservation schemes with the construction of sensor cell arrays on platforms such as biochips or optic fibres, hopefully leading to effective miniaturised whole-cell biosensor systems. These approaches hold much promise for the future. Nevertheless, their eventual implementation in practical devices calls for significant enhancement of current knowledge on formulation of reporter microorganisms.