Neural Immune Communication in the Control of Host-Bacterial Pathogen Interactions in the Gastrointestinal Tract.

The orchestration of host immune responses to enteric bacterial pathogens is a complex process involving the integration of numerous signals, including from the nervous system. Despite the recent progress in understanding the contribution of neuroimmune interactions in the regulation of inflammation, the mechanisms and effects of this communication during enteric bacterial infection are only beginning to be characterized. As part of this neuroimmune communication, neurons specialized to detect painful or otherwise noxious stimuli can respond to bacterial pathogens. Highlighting the complexity of these systems, the immunological consequences of sensory neuron activation can be either host adaptive or maladaptive, depending on the pathogen and organ system. These are but one of many types of neuroimmune circuits, with the vagus nerve and sympathetic innervation of numerous organs now known to modulate immune cell function and therefore dictate immunological outcomes during health and disease. Here, we review the evidence for neuroimmune communication in response to bacterial pathogens, and then discuss the consequences to host morbidity and mortality during infection of the gastrointestinal tract.

In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria.

BACKGROUND: Antimicrobial combinations have been proven as a promising approach in the confrontation with multi-drug resistant bacterial pathogens. In the present study, we identify and characterize a synergistic interaction of broad-spectrum nitroreductase-activated prodrugs 5-nitrofurans, with a secondary bile salt, Sodium Deoxycholate (DOC) in growth inhibition and killing of enterobacteria. RESULTS: Using checkerboard assay, we show that combination of nitrofuran furazolidone (FZ) and DOC generates a profound synergistic effect on growth inhibition in several enterobacterial species including Escherichia coli, Salmonella enterica, Citrobacter gillenii and Klebsiella pneumoniae. The Fractional Inhibitory Concentration Index (FICI) for DOC-FZ synergy ranges from 0.125 to 0.35 that remains unchanged in an ampicillin-resistant E. coli strain containing a ß-lactamase-producing plasmid. Findings from the time-kill assay further highlight the synergy with respect to bacterial killing in E. coli and Salmonella. We further characterize the mechanism of synergy in E. coli K12, showing that disruption of the tolC or acrA genes that encode components of multidrug efflux pumps causes, respectively, a complete or partial loss, of the DOC-FZ synergy. This finding indicates the key role of TolC-associated efflux pumps in the DOC-FZ synergy. Overexpression of Nitric Oxide-detoxifying enzyme Hmp results in a three-fold increase in FICI for DOC-FZ interaction, suggesting a role of nitric oxide in the synergy. We further demonstrate that DOC-FZ synergy is largely independent of NfsA and NfsB, the two major activation enzymes of the nitrofuran prodrugs. CONCLUSIONS: This study is to our knowledge the first report of nitrofuran-deoxycholate synergy against Gram-negative bacteria, offering potential applications in antimicrobial therapeutics. The mechanism of DOC-FZ synergy involves FZ-mediated inhibition of TolC-associated efflux pumps that normally remove DOC from bacterial cells. One possible route contributing to that effect is via FZ-mediated nitric oxide production.

Enhancement of microbial 2,4,6-trinitrotoluene transformation with increased toxicity by exogenous nutrient amendment.

In this study, the bacterial strain Citrobacter youngae strain E4 was isolated from 2,4,6-trinitrotoluene (TNT)-contaminated soil and used to assess the capacity of TNT transformation with/without exogenous nutrient amendments. C. youngae E4 poorly degraded TNT without an exogenous amino nitrogen source, whereas the addition of an amino nitrogen source considerably increased the efficacy of TNT transformation in a dose-dependent manner. The enhanced TNT transformation of C. youngae E4 was mediated by increased cell growth and up-regulation of TNT nitroreductases, including NemA, NfsA and NfsB. This result indicates that the increase in TNT transformation by C. youngae E4 via nitrogen nutrient stimulation is a cometabolism process. Consistently, TNT transformation was effectively enhanced when C. youngae E4 was subjected to a TNT-contaminated soil slurry in the presence of an exogenous amino nitrogen amendment. Thus, effective enhancement of TNT transformation via the coordinated inoculation of the nutrient-responsive C. youngae E4 and an exogenous nitrogen amendment might be applicable for the remediation of TNT-contaminated soil. Although the TNT transformation was significantly enhanced by C. youngae E4 in concert with biostimulation, the 96-h LC50 value of the TNT transformation product mixture on the aquatic invertebrate Tigriopus japonicas was higher than the LC50 value of TNT alone. Our results suggest that exogenous nutrient amendment can enhance microbial TNT transformation; however, additional detoxification processes may be needed due to the increased toxicity after reduced TNT transformation.

Enhanced biohydrogen production from corn stover by the combination of Clostridium cellulolyticum and hydrogen fermentation bacteria.

Hydrogen was produced from steam-exploded corn stover by using a combination of the cellulolytic bacterium Clostridium cellulolyticum and non-cellulolytic hydrogen-producing bacteria. The highest hydrogen yield of the co-culture system with C. cellulolyticum and Citrobacter amalonaticus reached 51.9 L H2/kg total solid (TS). The metabolites from the co-culture system were significantly different from those of the mono-culture systems. Formate, which inhibits the growth of C. cellulolyticum, could be consumed by the hydrogen-evolving bacteria, and transformed into hydrogen. Glucose and xylose were released from corn stover via hydrolysis by C. cellulolyticum and were quickly utilized in dark fermentation with the co-cultured hydrogen-producing bacteria. Because the hydrolysis of corn stover by C. cellulolyticum was much slower than the utilization of glucose and xylose by the hydrogen-evolving bacteria, the sugar concentrations were always maintained at low levels, which favored a high hydrogen molar yield.

High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii.

BACKGROUND: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii ∆dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. RESULTS: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii ∆dhaD resulted in the strain C. werkmanii ∆dhaD∆ldhA∆adhE::ChlFRT which provided the maximum theoretical yield of 1.00 ± 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 ± 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. CONCLUSIONS: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.

PH EFFECT ON ANTAGONISTIC ACTIVITY TOWARDS BACTERIA OF YEASTS ISOLATED FROM HUCUL DAIRY PRODUCTS AND GASTROINTESTINAL TRACT OF HUMAN.

The aim of this work was to study the influence of pH of medium on antagonistic ac- tivity of isolated from authentic Hucul dairy products and gastrointestinal tract (GIT) of Hucul long-livers yeasts towards potentially harmful for humans and animals bacteria. Among 52 tested yeast isolates 14 % yeasts showed considerable antagonistic activity towards Gram-positive bacteria Staphylococcus aureus and only 6 % of them inhibited growth of Gram negative bacteria belonging to genera Escherichia and Citrobacter Most ofyeasts with antagonistic activity (over 70 %) were isolatedfriom long-livers GIT There were identifed two optimal for antagonism areas of pH values of nutrient medium for tested yeasts being around 5.5 and 6.0 for Gram-positive bacteria and around 6.0 and 6.5 for Gram negative bacteria. It appeared that isolated fiom Hucul yogurt Saccharomyces pasterianus yeasts manifested their antagonistic activity in more acidic conditions com- pared to isolates fiom GIT.

1,3-propanediol production with Citrobacter werkmanii DSM17579: effect of a dhaD knock-out.

BACKGROUND: 1,3-propanediol (PDO) is a substantially industrial metabolite used in the polymer industry. Although several natural PDO production hosts exist, e.g. Klebsiella sp., Citrobacter sp. and Clostridium sp., the PDO yield on glycerol is insufficient for an economically viable bio-process. Enhancing this yield via strain improvement can be achieved by disconnecting the production and growth pathways. In the case of PDO formation, this approach results in a microorganism metabolizing glycerol strictly for PDO production, while catabolizing a co-substrate for growth and maintenance. We applied this strategy to improve the PDO production with Citrobacter werkmanii DSM17579. RESULTS: Genetic tools were developed and used to create Citrobacter werkmanii DSM17579 ∆dhaD in which dhaD, encoding for glycerol dehydrogenase, was deleted. Since this strain was unable to grow on glycerol anaerobically, both pathways were disconnected. The knock-out strain was perturbed with 13 different co-substrates for growth and maintenance. Glucose was the most promising, although a competition between NADH-consuming enzymes and 1,3-propanediol dehydrogenase emerged. CONCLUSION: Due to the deletion of dhaD in Citrobacter werkmanii DSM17579, the PDO production and growth pathway were split. As a consequence, the PDO yield on glycerol was improved 1,5 times, strengthening the idea that Citrobacter werkmanii DSM17579 could become an industrially interesting host for PDO production.

Anti-ESBL activity of silver nanoparticles biosynthesized using soil Streptomyces species.

Emergence of antibiotic resistance by bacteria has become a serious threat for public health worldwide. In this study, Streptomyces isolated from fertile soil sample was tested for biosynthesis of silver nanoparticles (AgNps) using cell-free supernatant and synthesized AgNps were screened for anti-ESBL (extended spectrum b-lactamase) activity against multi-drug resistant (MDR) ESBL-producing strain Klebsiella pneumoniae (ATCC 700603) and other medically important pathogens. Synthesis of AgNps was confirmed by change in pale yellow color to dark brown color and characteristic absorption spectra at 420 nm. The XRD spectrum displayed typical peaks of crystalline silver and EDAX analysis showed a major signal for silver. FTIR spectra revealed prominent peaks at 3,294 cm-1 (NH stretching due to amide group), 2,952 cm-1 (aldehydic C­H stretching) 1,658 cm-1 indicating the presence of carbonyl group. AgNps were spherical in shape with size ranging from 20 to 70 nm. The synthesized AgNps showed significant antimicrobial activity against standard ESBL pathogen K. pneumoniae (22 mm), 21 mm against clinical ESBL isolate E. coli and 16 mm against clinical ESBL isolates K. pneumoniae and Citrobacter species, respectively. The results of this study suggest that AgNps synthesized by Streptomyces sp. VITSJK10 can be used as a potential alternative to control MDR ESBL pathogens. The present study aimed for green synthesis of AgNps using Streptomyces species and to explore its anti-ESBL activity.

Bacterial succession and degradative changes by biofilm on plastic medium for wastewater treatment.

Biofilms contain a diverse range of microorganisms and their varying extracellular polysaccharides. The present study has revealed biofilm succession associated with degradative effects on plastic (polypropylene) and contaminants in sludge. The wet weight of biofilm significantly (p < 0.05) increased; from 0.23 ± 0.01 to 0.44 ± 0.01 g. Similarly, the dry weight of the biofilm increased from 0.02 to 0.05 g. Significant reduction in pathogens (E. coli and feacal coliforms) by MPN technique (>80%) and in chemical parameters (decrease in COD, BOD5 of 73.32 and 69.94%) representing diminution of organic pollutants. Energy dispersive X-ray spectroscopy (EDS) of plastic revealed carbon and oxygen contents, further surface analysis of plastic by scanning electron microscopy (SEM) revealed emergence of profound bacterial growth on the surface. Fourier transform infrared (FTIR) spectroscopy conforms its biotransformation under aerobic conditions after 8 weeks. New peaks developed at the region 1050 and 969 cm(-1) indicating CO and CC bond formation. Thus plastic with 6 weeks old aerobic biofilm (free of pathogens, max. weight, and OD, efficient COD & BOD removal ability) is suggested to be maintained in fixed biofilm reactors for wastewater treatment.

Detection of Escherichia coli colonies on confluent plates of chromogenic media used in membrane filtration.

MI agar (MI), Chromocult® Coliform agar ES (Chromocult), and DC with BCIG agar (DC) are chromogenic membrane filtration culture-based methods used to assess microbiological water quality. In this study, their ability to detect Escherichia coli colonies on confluent growth plates was determined by testing water samples containing increasing concentrations of a non-E. coli growing bacterium, Citrobacter youngae. Then, their ability to inhibit the growth of non-coliform bacteria was determined by simultaneously testing 603 well water samples. Results were compared with those obtained with mFC and Colilert® methods. Results showed that the E. coli count was difficult to determine on mFC, Chromocult and DC when non-E. coli colonies reached levels of 10(4)colony forming units (CFU)/100 mL. However, the E. coli count did not interfere with Colilert until non-E. coli colonies reached concentrations of 10(7)CFU/100 mL. No inhibition was observed with MI as E. coli colonies could be easily detected in the presence of at least 10(7)CFU/100 mL of C. youngae. Using well water samples, confluent growth plates were observed for 144, 177, and 185 of the 603 well water samples tested with the MI, Chromocult and DC methods, respectively. Among these confluent growth plates, E. coli colonies were not detected for 10, 20, and 31 water samples. However, they were detected by the mFC and/or Colilert methods. Thus, among the three methods tested, the MI method presented the lowest grow rate of atypic colonies and was the only one that presents no interference in the E. coli count.

Glycerol assimilation and production of 1,3-propanediol by Citrobacter amalonaticus Y19.

Citrobacter amalonaticus Y19 (Y19) was isolated because of its ability for carbon monoxide-dependent hydrogen production (water-gas shift reaction). This paper reports the assimilation of glycerol and the production of 1,3-propanediol (1,3-PDO) by Y19. Genome sequencing revealed that Y19 contained the genes for the utilization of glycerol and 1,2-propanediol (pdu operon) along with those for the synthesis of coenzyme B12 (cob operon). On the other hand, it did not possess the genes for the fermentative metabolism of glycerol of Klebsiella pneumoniae, which consists of both the oxidative (dhaD and dhaK) and reductive (dhaB and dhaT) pathways. In shake-flask cultivation under aerobic conditions, Y19 could grow well with glycerol as the sole carbon source and produced 1,3-PDO. The level of 1,3-PDO production was improved when vitamin B12 was added to the culture medium under aerobic conditions. Under anaerobic conditions, cell growth and 1,3-PDO production on glycerol was also possible, but only when an exogenous electron acceptor, such as nitrate or fumarate, was added. This is the first report of the glycerol metabolism and 1,3-PDO production by C. amalonaticus Y19.

Reduction of NOx in Fe-EDTA and Fe-NTA solutions by an enriched bacterial population.

An enriched biomass was developed from municipal sewage sludge consisting of three dominant bacteria, representing the genera of Enterobacter, Citrobacter and Streptomyces. The biomass was used in a series of batch experiments in order to determine kinetic constants associated with biomass growth and NOx reduction in aqueous Ferrous EDTA/NTA solutions and Ferric EDTA/NTA solutions using ethanol as organic electron donor. The maximum specific reduction rates of NOx in Ferrous EDTA and Ferrous NTA solutions were 0.037 and 0.047mMolesL(-1)d(-1)mg(-1) biomass, respectively while in Ferric EDTA and Ferric NTA solutions were 0.022 and 0.024mMolesL(-1)d(-1)mg(-1) biomass, respectively. In case of Ferric EDTA/NTA solution, the kinetic constants associated with reduction of Ferric EDTA/NTA to Ferrous EDTA/NTA were also evaluated simultaneously. The maximum specific reduction rates of Ferric EDTA and Ferric NTA were 0.0021 and 0.0026mMolesL(-1)d(-1)mg(-1) biomass. The significance of these observations are presented and discussed in this paper.

Effect of surfactant-induced cell surface modifications on electron transport system and catechol 1,2-dioxygenase activities and phenanthrene biodegradation by Citrobacter sp. SA01.

In order to better understand how surfactants affect biodegradation of hydrophobic organic compounds (HOCs), Tween 80 and sodium dodecyl benzene sulfonate (SDBS), were selected to investigate effects on cell surface hydrophobicity (CSH), electron transport system (ETS) activities and phenanthrene biodegradation by Citrobacter sp. SA01. Tween 80 and SDBS increased CSH by 19.8-25.2%, ETS activities by 352.1-376.0µmol/gmin, catechol 1,2-dioxygenase (C12) activities by 50.8-52.7U/L, and phenanthrene biodegradation by 8.9-17.2% separately in the presence of 50mg/L of surfactants as compared to in their absence. Lipopolysaccharide (LPS) release was 334.7µg/mg in the presence of both surfactants whereas in their absence only 8.6-44.4µg/mg of LPS was released. Thus, enhanced LPS release probably increased ETS and C12 activities as well as phenanthrene biodegradation by increasing CSH. The results demonstrate that surfactant-enhanced CSH provides a simple, yet effective strategy for field applications of surfactant-enhanced bioremediation of HOCs.

Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.

Bifidobacteria comprise a significant proportion of the human gut microbiota. Several bifidobacterial strains are currently used as therapeutic interventions, claiming various health benefits by acting as probiotics. However, the precise mechanisms by which they maintain habitation within their host and consequently provide these benefits are not fully understood. Here we show that Bifidobacterium breve UCC2003 produces a cell surface-associated exopolysaccharide (EPS), the biosynthesis of which is directed by either half of a bidirectional gene cluster, thus leading to production of one of two possible EPSs. Alternate transcription of the two opposing halves of this cluster appears to be the result of promoter reorientation. Surface EPS provided stress tolerance and promoted in vivo persistence, but not initial colonization. Marked differences were observed in host immune response: strains producing surface EPS (EPS(+)) failed to elicit a strong immune response compared with EPS-deficient variants. Specifically, EPS production was shown to be linked to the evasion of adaptive B-cell responses. Furthermore, presence of EPS(+) B. breve reduced colonization levels of the gut pathogen Citrobacter rodentium. Our data thus assigns a pivotal and beneficial role for EPS in modulating various aspects of bifidobacterial-host interaction, including the ability of commensal bacteria to remain immunologically silent and in turn provide pathogen protection. This finding enforces the probiotic concept and provides mechanistic insights into health-promoting benefits for both animal and human hosts.

Proteomic analysis on acetate metabolism in Citrobacter sp. BL-4.

Mass production of glucosamine (GlcN) using microbial cells is a worthy approach to increase added values and keep safety problems in GlcN production process. Prior to set up a microbial cellular platform, this study was to assess acetate metabolism in Citrobacter sp. BL-4 (BL-4) which has produced a polyglucosamine PGB-2. The LC-MS analysis was conducted after protein separation on the 1D-PAGE to accomplish the purpose of this study. 280 proteins were totally identified and 188 proteins were separated as acetate-related proteins in BL-4. Acetate was converted to acetyl-CoA by acetyl-CoA synthetase up-regulated in the acetate medium. The glyoxylate bypass in the acetate medium was up-regulated with over-expression of isocitrate lyases and 2D-PAGE confirmed this differential expression. Using (1)H-NMR analysis, the product of isocitrate lyases, succinate, increased about 15 times in the acetate medium. During acetate metabolism proteins involved in the lipid metabolism and hexosamine biosynthesis were over-expressed in the acetate medium, while proteins involved in TCA cycle, pentose phosphate cycle and purine metabolism were down-regulated. Taken together, the results from the proteomic analysis can be applied to improve GlcN production and to develop metabolic engineering in BL-4.

[Effect of heavy metal salts on the growth and pathogenicity factors of opportunistic bacteria].

The paper presents experimental data on the effect of different concentrations of the salts of cadmium, copper, and zinc on the adaptive potentialities of the most common opportunistic microflora in the Volga-Caspian region. The findings suggest that natural microorganisms (Aeromonas and Citrobacter) show a high metal resistance that seems to be associated with steadily increased background levels of these metals in the waters of the Volga-Caspian basin.

Strain-resolved community genomic analysis of gut microbial colonization in a premature infant.

The intestinal microbiome is a critical determinant of human health. Alterations in its composition have been correlated with chronic disorders, such as obesity and inflammatory bowel disease in adults, and may be associated with neonatal necrotizing enterocolitis in premature infants. Increasing evidence suggests that strain-level genomic variation may underpin distinct ecological trajectories within mixed populations, yet there have been few strain-resolved analyses of genotype-phenotype connections in the context of the human ecosystem. Here, we document strain-level genomic divergence during the first 3 wk of life within the fecal microbiota of an infant born at 28-wk gestation. We observed three compositional phases during colonization, and reconstructed and intensively curated population genomic datasets from the third phase. The relative abundance of two Citrobacter strains sharing ~99% nucleotide identity changed significantly over time within a community dominated by a nearly clonal Serratia population and harboring a lower abundance Enterococcus population and multiple plasmids and bacteriophage. Modeling of Citrobacter strain abundance suggests differences in growth rates and host colonization patterns. We identified genotypic variation potentially responsible for divergent strain ecologies, including hotspots of sequence variation in regulatory genes and intergenic regions, and in genes involved in transport, flagellar biosynthesis, substrate metabolism, and host colonization, as well as differences in the complements of these genes. Our results demonstrate that a community genomic approach can elucidate gut microbial colonization at the resolution required to discern medically relevant strain and species population dynamics, and hence improve our ability to diagnose and treat microbial community-mediated disorders.

Resuscitation of eleven-year VBNC Citrobacter.

Citrobacter freundii strain WA1 was stressed by incubation in seawater microcosms for eleven years. After two years of starvation, no culturable strain was observed. Incubation of samples in nutrient-rich broth medium not supplemented with growth factors, however, allowed resuscitation of VBNC cells so that subsequent plating yielded observable colonies for significantly extended periods of time. Recovery of VBNC Citrobacter freundii was obtained by incubation in nutrient broth even after eleven years of starvation. To see whether the samples contained the same strain of Citrobacter freundii inoculated 11 years ago. The complete 16S rRNA gene was PCR amplified and sequenced from initial, stressed and revived strains of Citrobacter freundii strain WA1.The 16S rRNA gene sequences from eleven-year stressed strains were homologous with a high degree of similarity to the GenBank reference strain and were identical to each other.

Decolorization of textile plant effluent by Citrobacter sp. strain KCTC 18061P.

Citrobacter sp. strain KCTC 18061P was found to be able to decolorize textile plant effluent containing different types of reactive dyes. Effects of physico-chemical parameters, such as aeration, nitrogen source, glucose and effluent concentrations on the color removal of real dye effluent by this strain were investigated. The observed changes in the visible spectra indicated color removal by the absorption of dye to cells during incubation with the strain. This strain showed higher decolorization ability under aerobic than static culture conditions. With 1% glucose, this strain removed 70% of effluent color within 5 days. Decolorization was not significantly dependent on the nitrogen sources tested. Chemical oxygen demand (COD) and biological oxygen demand (BOD) were decreased in proportion to incubation times, and their removal rates were about 35% and 50%, respectively, at 7 days of culture.