Anti-Bacterial and Anti-Candidal Activity of Silver Nanoparticles Biosynthesized Using Citrobacter spp. MS5 Culture Supernatant.

The present study described the extracellular synthesis of silver nanoparticles (AgNPs) using environmental bacterial isolate Citrobacter spp. MS5 culture supernatant. To our best knowledge, no previous study reported the biosynthesis of AgNPs using this bacterial isolate. The biosynthesized AgNPs were characterized using different techniques like UV-Vis spectroscopy, fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) equipped with energy dispersive X-ray (EDX). The analysis of UV-Vis spectra revealed absorption maxima at 415 nm due to surface plasmon resonance (SPR) indicated the formation of AgNPs and FTIR spectrum confirmed the participation of proteins molecule in AgNPs synthesis. XRD and EDX spectrum confirmed the metallic and crystalline nature of AgNPs. TEM and SEM showed spherical nanoparticles with a size range of 5-15 nm. The biosynthesized AgNPs showed effective independent as well as enhanced combined antibacterial activity against extended spectrum ß-lactamase (ESBL) producing multidrug resistant Gram-negative bacteria. Further, effective antifungal activity of AgNPs was observed towards pathogenic Candida spp. The present study provides evidence for eco-friendly biosynthesis of well-characterized AgNPs and their potential antibacterial as well as antifungal activity.

Novel repellents for the blood-sucking insects Rhodnius prolixus and Triatoma infestans, vectors of Chagas disease.

BACKGROUND: Studying the behavioral response of blood-sucking disease-vector insects to potentially repellent volatile compounds could shed light on the development of new control strategies. Volatiles released by human facial skin microbiota play different roles in the host-seeking behavior of triatomines. We assessed the repellency effect of such compounds of bacterial origin on Triatoma infestans and Rhodnius prolixus, two important vectors of Chagas disease in Latin America. METHODS: Using an exposure device, insects were presented to human odor alone (control) and in the presence of three individual test compounds (2-mercaptoethanol, dimethyl sulfide and 2-phenylethanol, the latter only tested in R. prolixus) and the gold-standard repellent NN-diethyl-3-methylbenzamide (DEET). We quantified the time the insects spent in the proximity of the host and determined if any of the compounds evaluated affected the behavior of the insects. RESULTS: We found volatiles that significantly reduced the time spent in the proximity of the host. These were 2-phenylethanol and 2-mercaptoethanol for R. prolixus, and dimethyl sulfide and 2-mercaptoethanol for T. infestans. Such an effect was also observed in both species when DEET was presented, although only at the higher doses tested. CONCLUSIONS: The new repellents modulated the behavior of two Chagas disease vectors belonging to two different triatomine tribes, and this was achieved using a dose up to three orders of magnitude lower than that needed to evoke the same effect with DEET. Future efforts in understanding the mechanism of action of repellent compounds such as 2-mercaptoethanol, as well as an assessment of their temporal and spatial repellent properties, could lead to the development of novel control strategies for these insect vectors, refractory to DEET.

The efficacy of 2 phytases on inositol phosphate degradation in different segments of the gastrointestinal tract, calcium and phosphorus digestibility, and bone quality of broilers.

The anti-nutritional effects of dietary inositol phosphates (IP6 through IP3) have been recognized in broiler chickens; however, inositol hexaphosphate (IP6) is more potent than the lower IP esters. The efficacies of 2 commercial phytases, a Buttiauxella sp. phytase (BSP) and a Citrobacter braakii phytase (CBP) at 500 and 1,000 FTU/kg, were studied on IP6-3 concentrations in the crop, proventriculus + gizzard, and distal ileum digesta, and ileal IP6 disappearance in broilers at day 22. Apparent ileal P and Ca digestibility, and bone quality at days 22 and 33 were also measured. Female Ross 308 broilers (n = 1,890; 30 birds × 7 diets × 9 replicates) were fed corn-soy-based crumbled diets. The 7 diets included a primary breeder recommendation-based positive control diet (PC); the PC marginally reduced in available P by 0.146% and Ca by 0.134% of the diet, (NC1) or moderately reduced by 0.174 and 0.159% of the diet, respectively (NC2). Other diets were the NC1 + BSP or CBP at 500 FTU/kg (NC1+500BSP and NC1+500CBP) and the NC2 + BSP or CBP at 1,000 FTU/kg (NC2+1,000BSP and NC2+1,000CBP). Each of the NC1 and NC2 had distal ileum IP6 disappearance similar to that of PC, but each had lower P digestibility and the majority of measured bone quality parameters than the PC. The ileal IP6 levels were decreased by 52.0 and 32.7% for NC1+500BSP and NC1+500CBP, respectively, relative to NC1 and by 73.6 and 50.9% for NC2+1,000BSP and NC2+1,000CBP, respectively, relative to NC2 (P < 0.001), with a similar effect for distal ileum IP6 disappearance. Overall, phytase in the NC diets increased P digestibility, and femur breaking strength and cortical bone mineral density at days 22 and 33. Overall, each of the phytases at each dose degraded IP6-3 across the gastrointestinal tract segments to increase P digestibility and the P and Ca utilization in bone. However, dietary BSP at 1,000 FTU/kg was most effective. Supplemental phytase degrades phytate to decrease the anti-nutritional effects in a dose- and phytase-dependent manner.

Discovery and structure of the antimicrobial lasso peptide citrocin.

We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria Citrobacter pasteurii and Citrobacter braakii We refactored the citrocin gene cluster and heterologously expressed it in Escherichia coli We determined citrocin's NMR structure in water and found that is reminiscent of that of microcin J25 (MccJ25), an RNA polymerase-inhibiting lasso peptide that hijacks the TonB-dependent transporter FhuA to gain entry into cells. Citrocin has moderate antimicrobial activity against E. coli and Citrobacter strains. We then performed an in vitro RNA polymerase (RNAP) inhibition assay using citrocin and microcin J25 against E. coli RNAP. Citrocin has a higher minimal inhibition concentration than microcin J25 does against E. coli but surprisingly is ∼100-fold more potent as an RNAP inhibitor. This suggests that citrocin uptake by E. coli is limited. We found that unlike MccJ25, citrocin's activity against E. coli relied on neither of the two proton motive force-linked systems, Ton and Tol-Pal, for transport across the outer membrane. The structure of citrocin contains a patch of positive charge consisting of Lys-5 and Arg-17. We performed mutagenesis on these residues and found that the R17Y construct was matured into a lasso peptide but no longer had activity, showing the importance of this side chain for antimicrobial activity. In summary, we heterologously expressed and structurally and biochemically characterized an antimicrobial lasso peptide, citrocin. Despite being similar to MccJ25 in sequence, citrocin has an altered activity profile and does not use the same outer-membrane transporter to enter susceptible cells.

Evaluation of a new generation phytase on phytate phosphorus release for egg production and tibia strength in hens fed a corn-soybean meal diet.

To test the effect of several inclusion levels of Citrobacter braakii phytase (CBP), on phytate P release, 420 50-wk-old-Bovans White hens were randomly allocated to 7 treatments with 5 replicates of 12 hens each. The experimental period lasted 12 weeks, first 8 for adaptation and last 4 for data collection. Feed and water were provided ad libitum. Treatments were: (1) a 0.12% basal corn-soybean meal diet deficient only in non-phytate P. Treatments 2 and 3 were added with constant increases of 0.11% inorganic P, to get a linear hen response to P addition. Treatments 4 to 7 were the addition of 300; 600; 1,200; and 1,800 phytase units (FYT)/kg to the basal diet. Variables analyzed were hen productive performance (HPP) and tibia resistance to fracture (TRF), and mineral content. Data were analyzed as a Complete Randomized Design (CRD). The results from treatments 1 to 3 were analyzed by a regression model to test for a significant linear response (P < 0.05). Then for every level of CBP added (treatments 4 to 7), the linear regression equation was solved to find out the equivalent value of released P. Based on hen health and welfare, the response variables that yielded realistic P equivalence values for the CBP levels used in the present trial were the tibia data. Following the significant (P < 0.001) linear response, the equations; TRF, kg (Y = 28.16X + 17.42 R2 = 0.84); Tibia Ca, % (Y = 11.6X + 14.2 R2 = 0.80); Tibia P, % (Y = 11.6X + 6.1 R2 = 0.81); and T ash, % (Y = 33.3X + 38.1 R2 = 0.80). Under the experimental conditions of this trial, the HPP variables were not a sensitive parameter to measure P release; whereas, tibia parameters showed the following average P release values per level of CBP inclusion in the corn- soybean meal diet; 300 FYT/kg = 0.099%, 600 FYT/kg = 0.141%, 1,200 FYT/kg = 0.182%, and 1,800 FYT/kg = 0.198%.

Development of a Rapid and Accurate Identification Method for Citrobacter Species Isolated from Pork Products Using a Matrix-Assisted Laser-Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

Previous detection methods for Citrobacter are considered time consuming and laborious. In this study, we have developed a rapid and accurate detection method for Citrobacter species in pork products, using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). A total of 35 Citrobacter strains were isolated from 30 pork products and identified by both MALDI-TOF MS and 16S rRNA gene sequencing approaches. All isolates were identified to the species level by the MALDI-TOF MS, while 16S rRNA gene sequencing results could not discriminate them clearly. These results confirmed that MALDI-TOF MS is a more accurate and rapid detection method for the identification of Citrobacter species.

Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression.

In a wide variety of bacterial restriction-modification systems, a regulatory `controller' protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Šresolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

Species identification of strains belonging to genus Citrobacter using the biochemical method and MALDI-TOF mass spectrometry.

Strains of genus Citrobacter (152 isolates from 1950 to 1988 deposited in the Czech National Collection of Type Cultures, Prague) were re-classified using biological and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods. One-hundred thirty-six strains (ca. 90 %) were identified to the species level using the biological method with evaluation by Farmer matrix. MALDI-TOF MS exhibited better identification capability, the data being more compact; the method was unambiguously successful in typing 145 (95 %) strains. Comparison of the results of identification by the two methods revealed differences (for 12 samples) in identified species which, considering all biochemical and/or MS characteristics, could be attributed to the natural variability of strains and close relation of the misidentified species (all of them belonged to the Citrobacter freundii complex). Taking into account all the above data, both methods can be considered reliable; however, the MALDI-TOF MS exhibits higher accuracy, efficiency, and rapidity.

Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature.

Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150 kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32 kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87 kDa protein band were identical to that of the large subunit of 95 kDa, indicating that the purified FDHS77 consisted of two subunits; a 95 kDa large subunit and a 32 kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the Vmax of 30.4 U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.

Biohydrogen production by dark fermentation of glycerol using Enterobacter and Citrobacter Sp.

Glycerol is an attractive substrate for biohydrogen production because, in theory, it can produce 3 mol of hydrogen per mol of glycerol. Moreover, glycerol is produced in substantial amounts as a byproduct of producing biodiesel, the demand for which has increased in recent years. Therefore, hydrogen production from glycerol was studied by dark fermentation using three strains of bacteria: namely, Enterobacter spH1, Enterobacter spH2, and Citrobacter freundii H3 and a mixture thereof (1:1:1). It was found that, when an initial concentration of 20 g/L of glycerol was used, all three strains and their mixture produced substantial amounts of hydrogen ranging from 2400 to 3500 mL/L, being highest for C. freundii H3 (3547 mL/L) and Enterobacter spH1 (3506 mL/L). The main nongaseous fermentation products were ethanol and acetate, albeit in different ratios. For Enterobacter spH1, Enterobacter spH2, C. freundii H3, and the mixture (1:1:1), the ethanol yields (in mol EtOH/mol glycerol consumed) were 0.96, 0.67, 0.31, and 0.66, respectively. Compared to the individual strains, the mixture (1:1:1) did not show a significantly higher hydrogen level, indicating that there was no synergistic effect. Enterobacter spH1 was selected for further investigation because of its higher yield of hydrogen and ethanol.

Structural and serological studies on the O-antigen show that Citrobacter youngae PCM1505 must be classified to a new Citrobacter O-serogroup.

The O-polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter youngae PCM1505 was studied by sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopies. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [Formula: see text]. Structural and serological data obtained earlier and in this work show that the strain studied is a candidate to a new Citrobacter O-serogroup.

Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi.

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.

Structure of the O-polysaccharide of Citrobacter youngae PCM 1503.

The O-polysaccharide (O-antigen) was obtained from the lipopolysaccharide of Citrobacter youngae PCM 1503, which is currently classified to the O7 serogroup. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established by sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopy: Structural and serological data of the O-antigen suggest that strain PCM 1503 should be reclassified to a new Citrobacter serogroup.

Purification, characterization, and gene cloning of glucose-1-phosphatase from Citrobacter braakii.

Citrobacter braakii produced an intracellular acid glucose phosphatase (AgpC) which was purified 986 fold to homogeneity with the specific activity of 286 units/mg. AgpC hydrolyzed a wide variety of phosphorylated compounds with high activity for glucose-1-phosphate and glucose-6-phosphate. The optimum pH and temperature for the enzyme activity was pH 5.0 and 45 degrees C, respectively. The Km value for glucose-1-phosphate was 5.12 mM with a Vmax 27.8 U mg(-1). Its molecular weight was 46 kDa by SDS-PAGE gel and the sequence of N-terminal amino acid residues identified was Gln-Thr-Ala-Pro-Glu-Gly-Tyr-Gln-Leu-Gln. The glucose-1-phosphatase gene (agpC) was cloned from the C. braakii genomic library. This gene comprised 1,242 nucleotides and encoded a polypeptide of 413 amino acids. The result of its BLAST search showed a significant similarity with glucose-1-phosphatase from enterobacteria such as E. coli, Enterobacter, Shigella, and Salmonella.

Overexpression, purification and preliminary X-ray diffraction analysis of the controller protein C.Csp231I from Citrobacter sp. RFL231.

Restriction-modification controller proteins play an essential role in regulating the temporal expression of restriction-modification genes. The controller protein C.Csp231I represents a new class of controller proteins. The gene was sublconed to allow overexpression in Escherichia coli. The protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.0 A resolution and belonged to space group P2(1). An electrophoretic mobility-shift assay provided evidence of strong binding of C.Csp231I to a sequence located upstream of the csp231IC start codon.

Conjugal transformation and transposon and chemical mutagenesis of gram-negative selenate-respiring Citrobacter sp. strain JSA.

Conjugal mating between selenate-reducing Citrobacter sp. strain JSA and Escherichia coli S17-1 bearing pSUP2021 allowed transposon mutagenesis and chromosomal transformation. Kanamycin-resistant transconjugants were obtained successfully by this method from a freshwater selenate-respiring Citrobacter sp. strain JSA. The maximum frequency of kanamycin-resistant Tn5 transconjugants was 3.6 x 10(-6) per recipient of this strain. Of these transconjugants, eight strains of selenate reduction-deficient transconjugants living by nitrate reduction were obtained in the strain JSA. Moreover, the same phenotype of deficient mutant was created by chemical mutagenesis with ethylmethanesulfonate. The results strongly indicate that selenate reducing anaerobic respiration was independent of nitrate reduction in the Citrobacter sp. isolate strain JSA.

Synthesis of spacer-equipped di-, tri-, and the tetrasaccharide fragments of the deacetylated O-PS1 of Citrobacter gillenii O9a,9b, and a related pentasaccharide.

The title rhamnooligosaccharides [alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, alpha-D-Rhap4NAc-(1-->2)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, and alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->2)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe] were synthesized in a stepwise fashion from 5-methoxycarbonylpentyl 4-azido-4,6-dideoxy-2-O-benzyl-alpha-D-mannopyranoside and orthogonally protected 1-thioglycoside glycosyl donors. The amorphous, final products were fully characterized as corresponding per-O-acetyl derivatives.

Composition of Acridid gut bacterial communities as revealed by 16S rRNA gene analysis.

The gut bacterial community from four species of feral locusts and grasshoppers was determined by denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rRNA gene fragments. The study revealed an effect of phase polymorphism on gut bacterial diversity in brown locusts from South Africa. A single bacterial phylotype, consistent with Citrobacter sp. dominated the gut microbiota of two sympatric populations of Moroccan and Italian locusts in Spain. There was evidence for Wollbachia sp. in the meadow grasshopper caught locally in the UK. Sequence analysis of DGGE products did not reveal evidence for unculturable bacteria and homologies suggested that bacterial species were principally Gammaproteobacteria from the family Enterobacteriaceae similar to those recorded previously in laboratory reared locusts.

Variation in the nature of organic acid secretion and mineral phosphate solubilization by Citrobacter sp. DHRSS in the presence of different sugars.

A novel phosphate solubilizing bacterium (PSB) was isolated from the rhizosphere of sugarcane and is capable of utilizing sucrose and rock phosphate as the sole carbon and phosphate source, respectively. This PSB exhibited mineral phosphate solubilizing (MPS) phenotype on sugars such as sucrose and fructose, which are not substrates for enzyme glucose dehydrogenase (GDH), along with GDH substrates, viz., glucose, xylose, and maltose, as carbon sources. PCR amplification of the rRNA gene and sequence analysis identified this bacterium as Citrobacter sp. DHRSS. On sucrose and fructose Citrobacter sp. DHRSS liberated 170 and 100 microM free phosphate from rock phosphate and secreted 49 mM (2.94 g/L) and 35 mM (2.1 g/L) acetic acid, respectively. Growth of Citrobacter sp. DHRSS on sucrose is mediated by an intracellular inducible neutral invertase. Interestingly, in the presence of GDH substrates like glucose and maltose, Citrobacter sp. DHRSS produced approximately 20 mM (4.36 g/L) gluconic acid and phosphate released was 520 and 570 microM, respectively. Citrobacter sp. DHRSS GDH activity was found when grown on GDH and non-GDH substrates, indicating that it is constitutive and could act on a wide range of aldose sugars. This study demonstrates the role of different organic acids in mineral phosphate solubilization by rhizobacteria depending on the nature of the available carbon source.