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In this study, we investigated the effects of enzyme-producing probiotic bacteria isolated from the gastrointestinal tract of rainbow trout on the growth performance, feed conversion ratio, and digestive enzyme activity, of fry (Oncorhynchus mykiss). Three isolates (G8/2013, T7/2013 and U5/2013) of candidate bacteria elicited the highest protease, lipase, and amylase activities, respectively. Isolates were identified as Aeromonas sp., Bacillus sp. and Citrobacter braakii by morphological, physiological, biochemical characterizations as well as 16S rRNA gene sequence analysis. The fry basal diet was supplemented with probiotics at varying concentrations; G8 group, Aeromonas sp. 1.72 x 108 CFU/g; U5 group, Bacillus sp. 3.01 x 108 CFU/g; T7 group, C. braakii 2.96 x 108 CFU/g and a mixed group (same bacterial concentrations), and control group (no bacteria). The rainbow trout fry were fed ad libitum in triplicate treatments with supplemented and non-supplemented probiotic diets for 70-days. The total bacterial count in the intestine was significantly higher in the mixed group (30th and 50th days) and U5 group (50th and 70th days) compared with the control group. However, there was no significant difference in weight gain, specific growth rate (SGR), feed conversion ratio (FCR), nutrient digestibility, or digestive enzyme activity among the groups.a

Citrobacter braakii (C. braakii) is widespread in nature, mainly found in human urinary tract. The current study was attempted to investigate the effect of Mr. Trivedi’s biofield treatment on C. braakii in lyophilized as well as revived state for antimicrobial susceptibility pattern, biochemical characteristics, and biotype number. Lyophilized vial of ATCC strain of C. braakii was divided into two parts, Group (Gr.) I: control and Gr. II: treated. Gr. II was further subdivided into two parts, Gr. IIA and Gr. IIB. Gr. IIA was analysed on day 10 while Gr. IIB was stored and analysed on day 159 (Study I). After retreatment on day 159, the sample (Study II) was divided into three separate tubes. First, second and third tube was analysed on day 5, 10 and 15, respectively. All experimental parameters were studied using automated MicroScan Walk-Away® system. The 16S rDNA sequencing of lyophilized treated sample was carried out to correlate the phylogenetic relationship of C. braakii with other bacterial species. The antimicrobial susceptibility and minimum inhibitory concentration showed 39.29% and 15.63% alteration respectively in treated cells of C. braakii as compared to control. Tetracycline showed improved sensitivity pattern, i.e., from resistant to susceptible after biofield treatment, with support of decreased MIC value (>8 to ≤ 4 µg/mL) by two-fold in all the treated samples as compared to the control. Biochemical reactions also showed significant (42.42%) alteration in the treated samples with respect to the control. Biotype numbers with species were substantially changed in Gr. IIA (53131052, Citrobacter freundii complex) on day 10 and in Gr. IIB, Study I (53111052; Citrobacter amalonaticus) on day 159 as compared to the control (77365776; Citrobacter braakii). Moreover, biotype numbers with species were substantially changed in Gr. IIB, Study II after retreatment on day 5 (53111042, Citrobacter amalonaticus) and (53131052; Citrobacter freundii complex) on day 10 and 15 as compared to the control. 16S rDNA analysis showed that the identified microbe as Citrobacter freundii (GenBank Accession Number: DQ517285) with 95% identity. The nearest homolog genus-species of C. braakii was found to be Citrobacter werkmanii (Accession No. AF025373). The results suggested that biofield treatment has a significant impact on C. braakii in lyophilized as well as revived state. https://www.trivedieffect.com/science/phenotyping-and-16s-rdna-analysis-after-biofield-treatment-on-citrobacter-braakii-a-urinary-pathogen https://www.omicsonline.org/open-access/phenotyping-and-16s-rdna-analysis-after-biofield-treatment-on-citrobacter-braakii-a-urinary-pathogen-2332-0672-1000130.php?aid=60954, Trivedi MK, Branton A, Trivedi D, Nayak G, Charan S, et al. (2015) Phenotyping and 16S rDNA Analysis after Biofield Treatment on Citrobacter braakii: A Urinary Pathogen. J Clin Med Genom 3: 129. doi: 10.4172/2472-128X.1000129

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.

Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.

The growth of Listeria monocytogenes during the pathogen specific enrichment of food samples can be limited by the presence of additional microorganisms that are resistant to the selective conditions being applied. If growth is severely limited and minimum post-enrichment threshold levels are not met then the presence of L. monocytogenes may go undetected. Several food products were screened for non-pathogenic commensal or spoilage microorganisms that are capable of growth under the conditions commonly used by regulatory testing laboratories to select for Listeria species. The effect of these potential competitor microorganisms on the ability to detect L. monocytogenes by several common molecular screening assays was then determined. Eight species of bacteria were isolated from foods that demonstrated the ability to grow in buffered Listeria enrichment broth under selective conditions. Growth of these competitor microorganisms during the enrichment incubation resulted in a decrease ranging from 1 to 4 logs in the 48 h population of L. monocytogenes. Three strains of L. monocytogenes representing serotypes 1/2a, 1/2b, and 4b were included in this study but no one serotype appeared to be most or least sensitive to the presence of competitor microorganisms. One additional strain of L. monocytogenes was identified as displaying minimal growth during the enrichment period in the presence of the Citrobacter braakii with the final population only reaching approximately 2.6 log CFU/ml after 48 h which was a 2 log increase over the initial population. This particular strain was subsequently shown to be difficult to detect following enrichment by an automated immunofluorescence assay and an antibody-based lateral flow device assay. In some enrichments, this strain was also difficult to detect by real-time PCR.

The antigenic scheme for the Bethesda-Ballerup group of bacteria established by West and Edwards in 1954 has continued to be applied as a serotyping scheme for Citrobacter freundii. In 1993, however, the classification of the Citrobacter was drastically revised and the species C. freundii redefined by Brenner et al. Accordingly, to judge the propriety to continuously use a single antigenic scheme for the C. freundii complex, the 90 reference strains listed in the antigenic scheme for C. freundii by West and Edwards were characterized phenotypically and specified based on the revised classification. Of these 90 strains, two strains of Hafnia alvei and one of Escherichia coli were found. Among the remaining 87 reference strains, Citrobacter youngae was the predominant species (40 strains), followed by Citrobacter braakii (25 strains), Citrobacter werkmanii (13 strains), and the unnamed Citrobacter genospecies 10 of Brenner et al (six strains). Citrobacter freundii, as redefined, accounted for only three strains and ranked behind the other four species. No overlapping with most of the 42 O-groups and 82 H-antigens was recognized between species with few exceptions. O-groups 1-9 inclusive, which were estimated to represent more than 90% of the former C.freundii strains, occurred in strains of C. youngae and C. braakii; and all nine strains of O-group 29, formerly known as the Ballerup group, were identified as C. braakii. These findings suggest that further study of the serotyping system is needed for all H2S-producing Citrobacter species.

About 100 moribund rainbow trout fry (2-3 g) were brought to the laboratory for examination after an epizootic of citrobacteriosis on a farm in Bursa Province, northwest Turkey, in late March 2012. The dominant bacterium was identified by biochemical, API 20E, and VITEK II rapid tests. Colonies grown on xylose lysine deoxycholate (XLD) agar were yellow with an intensive yellow center and a surrounding zone of yellow precipitation. The fish displayed hyperemia of the mouth, dark color, bilateral exophthalmia, pale liver, and necrotic kidney symptoms. The possible etiologic agent was identified by 16S rRNA gene sequencing as Citrobacter braakii (GenBank accession number JX518488). Histopathological examination of the diseased fish revealed vacuolar degeneration in the liver epithelium, round and limited focal necrotic areas in the liver, desquamation in the intestine epithelium and glands that settled in the propria mucosa, and vacuolar degeneration of the intestinal epithelium. According to ATB VET and antibiogram disc diffusion, the isolated C. braakii strain was sensitive to apramycin, cefoperazone, chloramphenicol, colistin, gentamicin, nitrofurantoin, spectinomycin, and streptomycin but resistant to amoxicillin, amoxicillin/clavulanic acid, cefalotine, cotrimoxazole, enrofloxacin, erythromycin, florfenicol, flumequine, fusidic acid, kanamycin, lincomycin, metronidazole, neomycin, oxacilline, oxolinic acid, oxytetracycline, penicillin, pristinamycin, rifampicin, sulfamethizole, sulfamethoxazole-trimethoprim, tetracycline, and tylosin. We found the bacteria sensitive to 4 mg/l doxycycline in the ATB VET test strip but resistant to 30 mu g/l doxycycline in the antibiogram disc diffusion test.

Cellulitis is usually caused by organisms such as beta-hemolytic streptococci and Staphylococcus aureus. Citrobacter are gram-negative bacilli that can cause opportunistic infections in immunocompromised hosts. They are rarely implicated in skin or soft tissue infections. The genus Citrobacter has been respeciated according to genetic relatedness. Citrobacter braakii refers to the genomospecies 6 of the Citrobacter freundii complex. There are no detailed studies of infections caused by the newly formed specific genetic species. We report a case of C. braakii infection in a renal transplant patient receiving immunosuppressive therapy. The patient's lower extremity cellulitis did not respond to conventional antibiotic therapy. Blood cultures grew C. braakii. Sensitivity studies and treatment with appropriate antibiotics resulted in prompt recovery. Immunosuppressive therapy in renal transplant recipients predisposes to infection by unusual pathogens, and this should be suspected when lack of a clinical response to conventional antibiotics is observed. We believe this is the first reported case of C. braakii cellulitis and bacteremia in a renal transplant recipient.

Phytases (EC 3.1.3) are widely used in animal feed to increase the availability of phosphorus and decrease the anti nutritive effect of myo-inositol hexakisphosphate (InsP₆). The aim of this work was to investigate the stereospecific degradation of InsP₆ in vitro and in vivo by a phytase from Citrobacter braakii (C. braakii), and to study gastric survival of the phytase as well as the site of action in the gastrointestinal tract. The in vitro results showed that the C. braakii phytase belongs to the group of 6-phytases (EC 3.1.3.26). However, in approximately one out of 10 instances the phytase initiated hydrolysis at the D-3 (L-1) position, demonstrating that phytase specificity is not unambiguous. Following the main degradation pathway, InsP₆ was degraded by stepwise removal of the phosphate groups on positions 6/1/5. The stereospecificity was found to be similar under in vitro and in vivo conditions. The phytase was found to be stable in the gastric environment and to be active in the stomach and possibly also in the proximal small intestine. While InsP₄ was accumulated under in vitro conditions this was not the case in vivo, where both InsP₅ and InsP₄ were seen to be hydrolysed in the small intestine, possibly as a combined action of the C. braakii phytase and endogenous phosphatases present in the mucosa. The ability of the C. braakii phytase to focus its activity on degrading InsP₆ to InsP₄ is believed to be a favourable complement to the endogenous phosphatases.

We will examine the use of multi-wavelength UV resonance-Raman signatures to identify the effects of growth phase on different types of bacteria. Gram positive and gram-negative species, Escherichia coli, Bacillus cereus, Citrobacter koseri and Citrobacter braakii were grown to logarithmic and stationary phases in different culture media. Raman spectra of bacteria were obtained by sequential illumination of samples between 220 and 260 nm; a range which encompasses the resonance frequencies of cellular components. In addition to the information contained in the single spectrum, this two-dimensional signature contains information reflecting variations in resonance cross sections with illumination wavelength. Results of our algorithms in identifying the differences between these germs are discussed. Preliminary results indicate that growth affects the Raman signature, but not to an extent that would negate identification of the species.

The aims of the current study were to investigate the prevalence and molecular characteristics of plasmid-mediated quinolone resistance (PMQR) genes from colonizing fecal organisms and to compare the incidence and subtype of these genes according to bacterial species and hospital at five tertiary-care hospitals in Korea. A total of 500 nonduplicated clinical isolates of Enterobacteriaceae were obtained from fecal specimens at five tertiary-care hospitals between March and May 2008. The PMQR genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA) were amplified by PCR and confirmed by direct sequencing of the PCR products. A total of 83 (16.6%) qnr-positive isolates were detected. The prevalence rates of qnrA, qnrB, and qnrS were 1.4%, 13.6%, and 1.6%, respectively. The species distributions of qnrB-positive isolates were Klebsiella pneumoniae (37/109; 33.9%), Citrobacter freundii (10/34; 29.4%), Citrobacter braakii (8/13; 61.5%), and Escherichia coli (8/275; 2.9%). Sixteen subtypes of qnrB were detected, including seven novel variants. The prevalences of aac(6')-Ib-cr and qepA were 15.6% (n=78) and 0.6% (n=3), respectively. The aac(6')-Ib-cr gene was detected in 39 (47.0%) of 83 qnr-positive isolates and 39 (9.4%) of 417 qnr-negative isolates There was one qepA variant containing a novel mutation (Ala231Val). The prevalence of PMQR genes was high in Enterobacteriaceae from stool specimens in Korea, and there was a close relation between qnr and aac(6')-Ib-cr.

Phytases are widely used as feed additives for monogastric animals, which cannot easily utilise the phosphorus bound in phytate (myo-inositol hexakisphosphate). The current study presents a safety evaluation of a 6-phytase produced by an Aspergillus oryzae strain expressing two synthetic genes, both mimicking a phytase gene from a Citrobacter braakii strain. Oral administration of the phytase preparation to rats at a dose level of 0.86 g total organic solids/kg body weight/day for 13 weeks did not cause any adverse effect. The phytase preparation did not exhibit irritative potential when applied locally to the eyes of rabbits or when applied to the skin using the in vitro three-dimensional epidermis model of adult human-derived epidermal keratinocytes. Furthermore, the phytase preparation was found not to represent mutagenic or clastogenic potential in the bacterial reverse mutation assay and in the in vitro micronucleus assays. Based on the toxicological data, the large safety factors calculated under common recommended dose assumptions for broiler chickens and weaned piglets, and the fact that Aspergillus oryzae is considered a safe strain lineage, it is concluded that there are no reasons for safety concerns when using this phytase as a feed additive.

A new biological inspired method to produce nanopalladium is the precipitation of Pd on a bacterium, i.e., bio-Pd. This bio-Pd can be applied as catalyst in dehalogenation reactions. However, large amounts of hydrogen are required as electron donor in these reactions resulting in considerable costs. This study demonstrates how bacteria, cultivated under fermentative conditions, can be used to reductively precipitate bio-Pd catalysts and generate the electron donor hydrogen. In this way, one could avoid the costs coupled to hydrogen supply. The catalytic activities of Pd(0) nanoparticles produced by different strains of bacteria (bio-Pd) cultivated under fermentative conditions were compared in terms of their ability to dehalogenate the recalcitrant aqueous pollutants diatrizoate and trichloroethylene. While all of the fermentative bio-Pd preparations followed first order kinetics in the dehalogenation of diatrizoate, the catalytic activity differed systematically according to hydrogen production and starting Pd(II) concentration in solution. Batch reactors with nanoparticles formed by Citrobacter braakii showed the highest diatrizoate dehalogenation activity with first order constants of 0.45 ± 0.02 h−1 and 5.58 ± 0.6 h−1 in batches with initial concentrations of 10 and 50 mg L−1 Pd, respectively. Nanoparticles on C. braakii, used in a membrane bioreactor treating influent containing 20 mg L−1 diatrizoate, were capable of dehalogenating 22 mg diatrizoate mg−1 Pd over a period of 19 days before bio-Pd catalytic activity was exhausted. This study demonstrates the possibility to use the combination of Pd(II), a carbon source and bacteria under fermentative conditions for the abatement of environmental halogenated contaminants.

DNA relatedness studies (hydroxyapatite method) were done on 112 strains of citrobacteria. By using the recommended definition of a genomospecies 11 genomospecies were identified in the genus Citrobacter. These genomospecies were separable by their biochemical profiles. Citrobacter koseri (Citrobacter diversus) and Citrobacter amalonaticus proved to be homogeneous species, as previously described. C. amalonaticus biogroup 1, as described by Farmer et al. (J. Clin. Microbiol. 21:46-76, 1985), was shown to be a separate homogeneous species, which was named Citrobacter farmeri sp. nov. The Citrobacter freundii complex was quite heterogeneous. C. freundii sensu stricto, as represented by the type strain, contained only 9 of 66 strains in this complex. The remaining 57 strains were members of seven genomospecies. Genomospecies 5, containing 21 strains, was named Citrobacter youngae sp. nov. Genomospecies 6, containing 15 strains, was named Citrobacter braakii sp. nov. Genomospecies 7 and 8, each containing six strains, were named Citrobacter werkmanii sp. nov. and Citrobacter sedlakii sp. nov., respectively. Genomospecies 9, 10, and 11, each containing three strains, were not named.

The O-polysaccharide of Salmonella enterica O59 was studied using sugar analysis and 2D 1 H and 13 C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit was established: →2)-β- d -Gal p -(1→3)-α- d -Glc p NAc-(1→4)-α- l -Rha p -(1→3)-β- d -Glc p NAc-(1→ Accordingly, the O-antigen gene cluster of S. enterica O59 includes all genes necessary for the synthesis of this O-polysaccharide. Earlier, another structure has been reported for the O-polysaccharide of Salmonella arizonae ( S. enterica IIIb) O59, which later was found to be identical to that of Citrobacter ( Citrobacter braakii ) O35 and, in this work, also to the O-polysaccharide of Escherichia coli O15.

This study aims to establish the isolation frequency of species of the family Enterobacteriaceae in fecal samples of South American common sea lion (Otaria flavescens). Thirty fecal samples were collected from the resting ground of an urban sea lions colony in Valdivia, southern Chile. The bacteria species and their isolation frequencies were Edwarsiella tarda (73%), Escherichia coli (70%), Hafnia alvei (33%), Morganella morganii (7%), Proteus mirabilis (7%), Klebsiella pnuemoniae subsp. pneumoniae (3%), Serratia rubidea (3%), Providencia rustigianii (3%) and Citrobacter braakii (3%).

The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: Download : Download full-size image The main d -mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.

An optimized Citrobacter braakii phytase gene, appA-c, was chemically synthesized by oligonucleotides synthesis and over-lap PCR method. The appA-c gene encoding 423 amino acids was cloned into expression vector pPIC9 and transformed into methylotropic yeast Pichia pastoris. From about 2000 transformants, 400 transformants exhibiting phytase activity were obtained. One transformant showing the strongest phytase activity was selected for detailed analyses in 5 L bioreactor. Under control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the transformant was able to secrete 3.85 mg/ml protein to the culture supernatant in about 110 h methanol induction, which comprises of 12,116 U ml(-1) phytase activity. Further characterization of the recombinant phytase was conducted. The optimal pH and temperature for this recombinant phytase was about 4.0 and 50 degrees C, respectively. Fe3+, Zn2+ and Cu2+ could significantly inhibit the recombinant phytase enzyme activity. The specific activity of this recombinant enzyme was 3147 U mg(-1). The K(m) and V(max) values for sodium phytate were determined to be 0.5 mM and 3085 U/mg, respectively. To our knowledge, this is the first report of a chemically synthesized C. braakii appA gene heterologous expression with the highest expression level and highest phytase activity achieved. The novel gene optimization and synthesis method can be applied to other related researches.

The phenomenon of molecular mimicry was found previously for Citrobacter braakii O37, which shared epitopes with human and horse erythrocytes. The aim of this study was to elucidate the basis of the serological cross-reactivity between anti-C. braakii O37 serum and human erythrocytes. The experiments involved analyzing the epitope on the human erythrocyte membrane, that could be recognized by affinity-purified antibodies. The results indicated a specific glycoprotein fraction in immunoblotting, namely band 3, which interacted with the antibodies purified on lipopolysaccharide from C. braakii O37 LPS (LPS O37) and its core affinity columns. Treating the erythrocytes with trypsin, which cleaves glycophorin A, improved the agglutination because band 3 became more available for antibody binding. Isolated band 3 immobilized on an affinity plate could be used to purify antibodies from the anti-C. braakii O37 serum. These antibodies showed a specific reactivity to LPS O37, but not to the related lipopolysaccharide from Salmonella Toucra O48. Furthermore, the inhibition of agglutination with lactose, the diminished interaction of the specific antibodies purified on LPS O37 with endo-beta-galactosidase-treated band 3, and the reactivity of these antibodies to the 40-kDa fragment of band 3 but not to its trypsin-elaborated 60-kDa fragment, all indicated that the epitope is located on the N-glycan of band 3.

The phytase gene appAS was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppAS) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg(-1), respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.

Dear Editor: Citrobacter species, aerobic, gram-negative bacilli in the Enterobacteriaceae family, are normally present in the environment and do not usually cause disease in immunocompetent hosts1. Chronic paronychia is an inflammatory reaction of the nail fold caused by exposure irritants or allergens that can lead to structural changes and increased susceptibility to infection. A 68-year-old woman presented with a 5-day-old, painful grayish abscess (2.0×1.5 cm) on the dorsum of the right thumb (Fig. 1A). Medical history indicated that the patient had contact with a desiccator containing calcium chloride two days before skin eruption. The patient had no current medical issues or surgical history regarding the abscess, and was employed as a housemaid with frequent exposure to water and detergents. She had a 20-year history of chronic paronychia characterized by erythema and mild swelling aggravated by repeated detergent exposure. Topical antibiotics and detergent avoidance temporarily resolved symptoms. A local clinic prescribed 300 mg of roxithromycin daily, which had not resolved the lesion. The patient's lesion was incised and drained, and oral amoxicillin and pivoxyl sulbactam (250 mg each, 3 times daily for 7 days) were prescribed. Culture of lesion identified Citrobacter braakii. By day 7, the abscess had cleared, leaving mild skin excoriation, trachyonychia and Beau's line, which ultimately resolved (Fig. 1B). Fig. 1 (A) A painful grayish 0.8×0.6 inch sized (2.0×1.5 cm) abscess on her right thumb in a 68-year-old women. (B) One week after incision and drainage with antibiotics treatment, the abscess cleared only leaving mild skin excoriation, trachionychia ... Chronic paronychia is an inflammation of the nail fold caused by factors including repetitive trauma and chemical exposure. As a result, the cuticle separates from the nail plate, forming a cleft between the nail fold and plate that predisposes the area to infection2. Upon presentation at our clinic, the patient had a history of chronic paronychia aggravated by detergent exposure. A noticeable cleft was observed in the area of the lesion (Fig. 1B), and we believe that exposure of this cleft to bacteria in a desiccator may have resulted in infection. Citrobacter species are uncommon cutaneous pathogens. Samonis et al.3 reported 5 cases of skin/soft tissue infection among 78 inpatients with confirmed Citrobacter isolates in a study at a Greek hospital. Single cases of folliculitis and skin ulcers caused by Citrobacter species in immunocompetent hosts4 have been reported, but there were no reports of Citrobacter infections manifesting as periungual abscesses. Antibiotic susceptibility differs among Citrobacter subtypes3 and treatment has been based on individual antimicrobial profiles. One study of 22 cases of C. freundii showed 100% resistance to erythromycin, ampicillin, and methicillin5. As was demonstrated in our case study, macrolides including erythromycin and roxithyromycin (often indicated for gram positive/negative cocci infections) were ineffective treatments for C. braakii, a gram-negative bacillus. While oral amoxicillin and pivoxyl sulbactam were prescribed after incision and drainage (I&D) in our study, the Citrobacter isolate was resistant to amoxicillin. Therefore, the I&D procedure was primarily responsible for patient improvement. To our knowledge, this is the first reported case of C. braakii-induced periungual abscess in a patient with chronic paronychia. Additional data on Citrobacter infections are needed to improve comprehension of risk factors, etiology, clinical features, and management of these infections.

Arylsulfatase activity was detected in a bacterial strain, Citrobacter braakii 69-b, isolated from soil by enrichment cultivation using porcine gastric mucin. The production of arylsulfatase was derepressed markedly in a synthetic medium by the addition of tyramine. The purified enzyme hydrolyzed 4-nitrophenyl sulfate, 4-nitrocatechol sulfate, and 3-indoxyl sulfate, and was classified as type I arylsulfatase.

A cost-effective remediation method is needed to remove selenium (Se) from Se-contaminated water. In this study, a selenate [Se(VI)]-reducing bacterium, Citrobacter braakii, that is capable of using molasses as a carbon source to reduce Se(VI) from natural river and drainage waters was isolated. During an 8-day experiment, 87-97% of the added Se(VI) in New River water and White River water, California, was reduced to elemental Se [Se(0)] or transformed to organic Se. In highly saline drainage water, removal of Se(VI) by C. braakii was limited, with 20% Se(VI) removal in a 7-day experiment. Addition of zero-valent iron (ZVI) into these waters along with C. braakii inoculation significantly enhanced the removal of Se(VI) and reduced the formation of organic Se. This study suggests that the combination of a bacterial treatment using inexpensive molasses and ZVI can effectively remove Se from natural river water and agricultural drainage waters.

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppAEc and rAppASc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppASc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppAEc.

Direct and indirect evidence, of unexpected stereoselective reductase-catalysed deoxygenations of sulfoxides, was found. The deoxygenations proceeded simultaneously, with the expected dioxygenase-catalysed asymmetric sulfoxidation of sulfides, during some biotransformations with the aerobic bacterium Pseudomonas putida UV4. Stereoselective reductase-catalysed asymmetric deoxygenation of racemic alkylaryl, dialkyl and phenolic sulfoxides was observed, without evidence of the reverse sulfoxidation reaction, using anaerobic bacterial strains. A purified dimethyl sulfoxide reductase, obtained from the intact cells of the anaerobic bacterium Citrobacter braakii DMSO 11, yielded, from the corresponding racemates, enantiopure alkylaryl sulfoxide and thiosulfinate samples.

The O-specific polysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) was isolated by mild acid hydrolysis of the lipopolysaccharide (LPS) and found to contain d-fucose, l-rhamnose, 4-deoxy-d-arabino-hexose and O-acetyl groups in molar ratios 2 : 1 : 1 : 1. On the basis of methylation analysis and 1H and 13C NMR spectroscopy data, the structure of the branched tetrasaccharide repeating unit of the O-specific polysaccharide was established. Using various serological assays, it was demonstrated that the LPS of strain PCM 1531 is not related serologically to other known 4-deoxy-d-arabino-hexose-containing LPS from Citrobacter PCM 1487 (serogroup O5) or C. youngae PCM 1488 (serogroup O36). Two other strains of Citrobacter, PCM 1504 and PCM 1505, which, together with strain PCM 1531, have been classified in serogroup O6, were shown to be serologically distinct from strain PCM 1531 and should be reclassified into another serogroup.

While searching for Escherichia coli O157 in the aquatic environment of Calcutta using an immunodetection procedure, we fortuitously detected five strains of Citrobacter braakii, which cross-reacted with the commercially available O157 polyvalent antiserum. The five C. braakii isolates gave positive results when a sensitive dot-ELISA was performed with E. coli O157 monoclonal antibody. Further, the O157 monoclonal antibody recognized the bands of proteinase K treated whole cells of lipopolysaccharide of all the C. braakii isolates. Apart from weak reactions with two or three of the DNA probes, all the C. braakii strains did not hybridize with the other probes spanning the minimum region required for O157 O-antigen biosynthesis. These strains did not possess any of the virulence genes that are commonly found in the Shiga toxin-producing E. coli (STEC) specially the serotype O157[ratio ]H7. Therefore, it appears that the serological cross-reaction between C. braakii and E. coli O157 antiserum is based on structural mimicry between the O-polysaccharide of C. braakii and E. coli O157.

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes ( hdc ) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii . In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.

Two 21-day studies were conducted with broilers to evaluate the efficacy of a bacterial 6-phytase from Citrobacter braakii and compare four Pi sources. The four sources were phosphates of monocalcium (MCP), dicalcium (DCP), tricalcium (TCP) with potassium phosphate (KH2PO4) acting as a ‘positive control’ reference. In each study, 336 4-day-old male birds (Ross 708) were blocked based on initial bodyweight (BW) and randomly allotted to one of seven diets with six replicate cages of eight birds each. Access to experimental diets and water from Days 4 to 25 post-hatching was ad libitum. In the first study, the seven diets were: (1) a low-P negative control (NC) corn-soybean meal basal diet formulated to contain crude protein (CP), Ca, total P, and non-phytate P (nPP) at 218, 9.0, 4.5, and 2.0 g/kg, respectively; (2) NC plus 0.75 g Pi from KH2PO4/kg; (3) NC plus 0.75 g Pi from MCP/kg; (4) NC plus 0.75 g Pi from DCP/kg; (5) NC plus 0.75 g Pi from TCP/kg; (6) NC plus phytase at 500 FYT/kg; (7) NC plus phytase at 1000 FYT/kg. Feeding the low-P NC diet reduced (P < 0.01) BW gain (BWG), feed intake (FI), and tibia ash. Supplementing the NC with Pi or phytase linearly improved (P < 0.01) BWG, FI, and tibia ash. Supplementing the NC with 0.75 g/kg Pi from MCP, DCP, or TCP were equipotent in improving BWG and FI; however, percent tibia ash was higher (P < 0.05) in birds fed MCP than either DCP or TCP. The second study was similar to the first study except that Ca, total P, and nPP in the NC were reduced to 7.0, 4.2, and 1.8 g/kg, respectively; and Pi from MCP, DCP, and TCP were reduced to 0.6 g/kg. Similar to observations in the first study, the low-P NC diet reduced (P < 0.01) BWG, FI, and tibia ash; and supplementing the NC with Pi or phytase linearly improved (P < 0.01) BWG, FI, P digestibility and tibia ash. Furthermore, supplementing the NC with 0.75 g Pi from MCP, DCP, or TCP per kg diet were equipotent in improving BWG, FI, and tibia ash though MCP resulted in superior (P < 0.05) retention of P compared with TCP. Results from both studies showed that the phytase was efficacious in releasing phytate-P for growth and bone mineralisation in chickens compensating, at least, the spared Pi, and that Pi sources commonly used in formulating diets of chickens may be different in their potential to supply digestible P.

Citrobacter spp. are gram-negative commensal bacteria that infrequently cause serious nosocomial infections in compromised hosts. They are often resistant to cephalosporins due to overexpression of their chromosomal β-lactamase. During a recent study of multidrug-resistant Enterobacteriaceae (MDRE) in solid-organ transplant patients, we found that almost half of patients colonized with MDRE carried one or more cefpodoxime-resistant Citrobacter freundii , Citrobacter braakii , or Citrobacter amalonaticus strains. Pulsed-field gel electrophoresis showed that 36 unique strains of Citrobacter were present among 32 patients. Genetic and phenotypic analysis of the resistance mechanisms of these bacteria showed that the extended-spectrum β-lactamase (ESBL) SHV-5 or SHV-12 was encoded by 8 strains (26%) and expressed by 7 strains (19%). A number of strains were resistant to other drug classes, including aminoglycosides (28%), trimethoprim-sulfamethoxazole (31%), and fluoroquinolones (8%). PCR and DNA analysis of these multiresistant strains revealed the presence of class I integrons, including the first integrons reported for C. braakii and C. amalonaticus . The integrons encoded aminoglycoside resistance, trimethoprim resistance, or both. Despite the prevalence of MDR Citrobacter spp. in our solid-organ transplant patients, only a single infection with a colonizing strain was recorded over 18 months. Low-virulence Citrobacter spp., which can persist in the host for long periods, could influence pathogen evolution by accumulation of genes encoding resistance to multiple antimicrobial classes.

The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: Download : Download full-size image The main d -mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.

Tributyltin compound (TBT) released into the aquatic environment is generally degraded by bacteria in water and sediment. The isolation of TBT-degrading bacteria from TBT polluted sediment leads to the indication of specific potential TBT degraders. Two new strains of bacteria designated as B2 and B3 were successfully isolated using glycerol medium containing tributyltin chloride (TBTC) at 130 μM from contaminated sediment collected from Bowling Basin in Glasgow. The observed degradation after 14 days of the microcosm from the sediment and the isolated bacteria were investigated at an initial concentration of 1 μM TBTC. It was found that TBT was degraded by the bacterial strains B2 and B3 at 8.3 and 16.9 %, respectively. The results indicate that B2 and B3 are effective as TBT degraders. EC50 of B2 and B3 in water were 88.73 and 112.53 μM TBTC, which were significantly higher than the concentration of TBT measured at the basin, suggesting a low effect of TBT on the growth and activity of bacteria. After identification using API 20E and 16S sequencing, the bacterial isolate strain B2 is Citrobacter braakii and B3 is Enterobacter cloacae. Therefore, this study has discovered two species of high resistance TBT degrader which have never been previously studied or isolated based upon TBT degradation ability.

Bacteria that thrive in extreme conditions and the bacteriophages that infect them are sources of valuable enzymes resistant to denaturation at high temperatures. Many of these heat-stable proteins are useful for biotechnological applications, nevertheless, none have been utilized as antibacterial agents. Here, we demonstrate the bactericidal potential of Ts2631 endolysin from the extremophilic bacteriophage vB_Tsc2631, which infects Thermus scotoductus, against the alarming multidrug-resistant clinical strains of Acinetobacter baumannii, Pseudomonas aeruginosa and pathogens from the Enterobacteriaceae family. A 2&ndash, 3.7 log reduction in the bacterial load was observed in antibacterial tests against A. baumannii and P. aeruginosa after 1.5 h. The Ts2631 activity was further enhanced by ethylenediaminetetraacetic acid (EDTA), a metal ion chelator (4.2 log reduction in carbapenem-resistant A. baumannii) and, to a lesser extent, by malic acid and citric acid (2.9 and 3.3 log reductions, respectively). The EDTA/Ts2631 combination reduced all pathogens of the Enterobacteriaceae family, particularly multidrug-resistant Citrobacter braakii, to levels below the detection limit (&gt, 6 log), these results indicate that Ts2631 endolysin could be useful to combat Gram-negative pathogens. The investigation of A. baumannii cells treated with Ts2631 endolysin variants under transmission electron and fluorescence microscopy demonstrates that the intrinsic antibacterial activity of Ts2631 endolysin is dependent on the presence of its N-terminal tail.