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Citrobacter braakii (C. braakii) is an anaerobic, gram‐negative bacterium that has been isolated from the environment, food, and humans. Infection by C. braakii has been associated with acute mucosal inflammation in the intestine, respiratory tract, and urinary tract. However, the pathogenesis of C. braakii in the gastric mucosa has not yet been clarified. In this study, the bacterium was detected in 35.5% (61/172) of patients with chronic gastritis (CG) and was closely associated with the severity of mucosal inflammation. Citrobacter braakii P1 isolated from a patient with CG exhibited urease activity and acid resistance. It contained multiple secretion systems, including a complete type I secretion system (T1SS), T5aSS and T6SS. We then predicted the potential pilus‐related adhesins. Citrobacter braakii P1 diffusely adhered to AGS cells and significantly increased lactate dehydrogenase (LDH) release; the adhesion rate and LDH release were much lower in HEp‐2 cells. Strain P1 also induced markedly increased mRNA and protein expression of IL‐8 and TNF‐α in AGS cells, and the fold increase was much higher than that in HEp‐2 cells. Our results demonstrate proinflammatory and cytotoxic role of C. braakii in gastric epithelial cells, indicating the bacterium is potentially involved in inducing gastric mucosa inflammation.

Citrobacter braakii (C. braakii) is an anaerobic, gram‐negative bacterium that has been isolated from the environment, food, and humans. Infection by C. braakii has been associated with acute mucosal inflammation in the intestine, respiratory tract, and urinary tract. However, the pathogenesis of C. braakii in the gastric mucosa has not yet been clarified. In this study, the bacterium was detected in 35.5% (61/172) of patients with chronic gastritis (CG) and was closely associated with the severity of mucosal inflammation. Citrobacter braakii P1 isolated from a patient with CG exhibited urease activity and acid resistance. It contained multiple secretion systems, including a complete type I secretion system (T1SS), T5aSS and T6SS. We then predicted the potential pilus‐related adhesins. Citrobacter braakii P1 diffusely adhered to AGS cells and significantly increased lactate dehydrogenase (LDH) release; the adhesion rate and LDH release were much lower in HEp‐2 cells. Strain P1 also induced markedly increased mRNA and protein expression of IL‐8 and TNF‐α in AGS cells, and the fold increase was much higher than that in HEp‐2 cells. Our results demonstrate proinflammatory and cytotoxic role of C. braakii in gastric epithelial cells, indicating the bacterium is potentially involved in inducing gastric mucosa inflammation. [ABSTRACT FROM AUTHOR]

A bacteriophage BD49 specific for Citrobacter braakii was screened out and purified by double-layer plate method. It consists of a polyhedral head of 93.1 ± 1.2 nm long and 72.9 ± 4.2 nm wide, tail fibers, collar, sheath and baseplate. The bacteriophage was identified by morphology observed with transmission electron microscope (TEM), whole genome sequencing carried out by Illumina next generation sequencing (NGS) technique, and gene annotation based on Clusters of Orthologous Groups of proteins (COG) database. It was identified primarily as a member of Caudovirales by morphology and further determined as Caudovirales, Myoviridae, and Citrobacter bacteriophage by alignment of its whole genome sequence with the NCBI database and establishment of phylogenetic tree. The bacteriophage showed good environmental suitability with optimal multiplicity of infection (MOI) of 0.01, proliferation time of 80 min, optimum living temperature of 30–40 °C, and living pH of 5–10. In addition, it exhibited synergistic effect with ciprofloxacin against C. braakii in antibacterial tests. [ABSTRACT FROM AUTHOR]

Huiming Han,1,* Zhi Zhao,2,* Yan Lin,1 Baihui Lin,1 Hao Xu,3 Beiwen Zheng3 1School of Basic Medical Sciences, Beihua University, Jilin, Jilin Province, People’s Republic of China; 2Department of Neonatology, Shaanxi Province People’s Hospital, Xi’an, Shaanxi Province, People’s Republic of China; 3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Beiwen Zheng, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang Province, 310003, People’s Republic of China, Tel +86 571 872 364 23, Fax +86 571 872 364 21, Email zhengbw@zju.edu.cnAbstract: In this study, we describe, for the first time, the co-existence of blaNDM-1and blaOXA-10 in a carbapenem-resistant Citrobacter braakii strain DY2019 isolated from a patient with urinary tract infection in China. We aimed to investigate the genomic context of two β-lactamase-producing plasmids and characterize the transmission mechanism of the carbapenemase-encoding gene. Whole-genome sequencing of strain DY2019 was performed with Nanopore and Illumina platforms, which revealed a chromosome sequence with the length of 4,830,928 bp, an IncC group plasmid pDY2019-OXA (size of 178,134 bp), and a novel IncHI2 group plasmid pDY2019-NDM (length 348,495 bp). A total of 16 antimicrobial resistance genes (ARGs) that confer resistance to nine different antibiotic groups were identified in strain DY2019, and 11 of them were carried by plasmid pDY2019-OXA. These data and analyses suggest that the carbapenem-resistant C. braakii strains may serve as potential reservoir of carbapenemase and highlight the need for further close surveillance of this species in clinical settings.Keywords: Citrobacter braakii, carbapenem-resistant, NDM-1, OXA-10, whole-genome sequencing, urinary tract infection

Citrobacter braakii is an opportunistic pathogen that induces aquatic infections in fish and turtles. In this study, a bacteriophage that infects C. braakii, named vB_CbrM_HP1, was isolated from sewage. This phage belongs to Myoviridae family, Ounavirinae subfamily, Mooglevirus genus. We also used the phage to treat crucian carp infection caused by C. braakii for the first time. vB_CbrM_HP1 was relatively stable at temperatures ranging from 4 to 60°C and pH values ranging from 3 to 11 but float slightly. When the multiplicities of infection (MOI) was 0.0001, the titer reached a maximum of 4.20 × 1010 PFU/ml. As revealed from the results of whole genomic sequence analysis, the total length of vB_CbrM_HP1 was 89335 bp, encoding 135 ORFs, 9 of which were

Abstract A 34‐year‐old woman presented due to progressive painful swelling around the nail of the right index finger. Onychectomy and drainage of the abscess of the affected finger were performed as the inflammation was progressive despite the previous antibiotic therapy. The microbiological culture revealed a ciprofloxacin‐susceptible Citrobacter braakii.

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ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become widespread in hospitals and the environment. Here, we describe a blaKPC-2-carrying plasmid called pCRE3-KPC, which was recovered from a clinical multidrug-resistant Citrobacter braakii CRE3 strain in China. The complete nucleotide sequence of pCRE3-KPC was determined by combining MiSeq and MinION sequencing and then compared with those of three related plasmids. Plasmid conjugal transfer and electroporation tests, modified carbapenem inactivation method, and bacterial antimicrobial susceptibility test were carried out. We compared this plasmid with three related plasmids to verify that the backbone of pCRE3-KPC was composed of the backbones of the IncR plasmid and IncP6 plasmid. Further bioinformatics analysis showed that pCRE3-KPC carried two resistance-related regions (the blaKPC-2 gene cluster and the aacC2-tmrB-related region). The aacC2-tmrB-related region included two novel insertion sequences (ISCfr28 and ISCfr16). IMPORTANCE Reports of human-pathogenic C. braakii strains, especially of strains showing resistance to carbapenems, are rare. To the best of our knowledge, our results represent the first detection of carbapenemase gene blaKPC-2 in C. braakii strains. In addition, we have studied detailed genetic characteristics of the novel IncR/IncP6 hybrid plasmid pCRE3-KPC, which was isolated from a clinical multidrug-resistant Citrobacter braakii CRE3 strain. Our results may provide further insight into the horizontal transfer of multidrug resistance genes in bacteria and into the genomic diversity and molecular evolution of plasmids.

Background: Globally, Salmonella enterica is one of the leading causes of foodborne illness in humans. Food of animal origin is obligatorily tested for the presence of this pathogen. Unfortunately, in meat and meat products, this is often hampered by the presence of background microbiota, which may present as false-positive Salmonella. Methods: For the identification of Salmonella spp. from meat samples of beef, pork, and poultry, the authorized detection method is PN-EN ISO 6579-1:2017-04 with the White–Kauffmann–Le Minor scheme, two biochemical tests: API 20E and VITEK II, and a real-time PCR-based technique. Results: Out of 42 presumptive strains of Salmonella, 83.3% Salmonella enterica spp. enterica, 14.3% Citrobacter braakii, and 12.4% Proteus mirabilis were detected from 180 meat samples. Conclusions: Presumptive strains of Salmonella should be identified based on genotypic properties such as DNA-based methods. The aim of this study was the isolation and identification of Salmonella spp. from miscellaneous meat sorts: beef, pork, and poultry.

La eficacia de dos fitasas (Citrobacter brakii y E. Coli) utilizadas en diferentes dosis (500 y 1,000 FTU) se evaluaron en una dieta deficiente en fósforo (0.15% P disponible) formulada a base de sorgo-soya. Para la estimación de la eficacia en la liberación de P de las fitasas evaluadas se utilizó como referencia fósforo inorgánico suplementado a través de fosfato monodicálcico (FMD), para alcanzar una concentración final de P disponible de 0.23, 0.31 y 0.39 %. Para lo anterior la dieta basal deficiente en P se suplementó como sigue: 1) sin fitasas, sin FMD; 2) 0.08% FMD; 3) 0.16% FMD; 4) 0.24% PMD; 5) citrobacter-500 FTU; 6) coli-500 FTU; 7) citrobacter-1,000 FTU y 8) coli-1,000 FTU. Los tratamientos se aplicaron a 216 pollos de la estirpe Ross (27 pollos/tratamiento) de los 7 a 21 días de edad. La ganancia de peso, eficiencia alimenticia y concentración de ceniza y P en tibias fue mayor en los pollos que recibieron 0.39% PMD y los 1,000 FTU de ambas fitasas, no existiendo diferencias en estos niveles entre los tratamientos FMD y fitasas. La cantidad de fósforo liberado no se afectó (P>0.05) por el tipo de fitasa (promedio 0.142 %), pero sí por el nivel de fitasa utilizado (0.102 vs 0.182 % para 500 y 1,000 FTU respectivamente). Los resultados indican que la suplementación con 1,000 FTU/kg de ambas fitasas en dietas deficientes en P resulta en ganancias y eficiencias alimenticias similares cuando las dietas se suplementan hasta 0.24 % de FMD. Ambas fitasas mejoraron en 78.4 % el aprovechamiento del fósforo fítico en dietas sorgo-soya para pollos en crecimiento.

Abstract Background Polyethylene glycol (PEG) is widely used for bowel cleaning in preparation for colonoscopy because of its safety. Septic shock after PEG preparation is an extremely rare complication. Herein, we describe a case of septic shock that occurred immediately after colonoscopy preparation with PEG. Case presentation A 75-year-old Japanese male who had previously developed diabetes after total pancreatectomy received PEG in preparation for colonoscopy. He had been admitted to the emergency intensive care unit 4 days earlier due to hematochezia presenting with shock. He ingested PEG to prepare for a colonoscopy examination, which was performed to identify the source of his bleeding over a 5-h period, but suddenly exhibited septic shock and markedly elevated procalcitonin levels. A blood culture subsequently revealed Citrobacter braakii. Immediate resuscitation and intensive care with appropriate antibiotics improved his condition. Conclusions Clinicians should be aware of the possibility of deteriorating conditions after bowel preparation with PEG among severely ill patients with recent episodes of hemorrhagic shock.

Introduction: Citrobacter bacteria are gram-negative anaerobic bacilli commonly found in water, soil, food, and the intestinal tracts of animals and humans. Patients at highest risk of these nosocomial infections include neonates and adults who are ≥65 years of age, debilitated, or immunocompromised. Citrobacter braakii (C. braakii), specifically, has been reported to cause sepsis in immunocompromised patients. Herein, we describe a case of an allogeneic stem cell transplant (SCT) adult patient with C. braakii bloodstream infection. Case Report: We report our experience managing a central line-associated bloodstream infection (CLABSI) due to C. braakii in an allogeneic SCT patient. Management and Outcomes: Our patient was initially managed with cefepime. The central venous catheter (CVC) was removed. Blood cultures cleared 24 hours after antibiotic initiation. Therapy was transitioned to oral levofloxacin once susceptibilities resulted. Discussion: The course of this patient highlights the important relationship between an unusual pathogen, C. braakii, in an immunocompromised allogeneic SCT patient. In our case, the source of the bacteremia was most likely a CLABSI given the positive catheter tip cultures. Although this report describes the successful utilization of cefepime and levofloxacin in the treatment of C. braakii infection, caution should be exercised when choosing empiric antimicrobial therapy as AmpC resistance. This clinical scenario can aid health care providers in making informed treatment decisions when faced with patients diagnosed with this relatively uncommon pathogen. Further reports should be published to determine C. braakii bacteremia management in hematopoietic stem cell transplant patients. [ABSTRACT FROM AUTHOR]

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%. [ABSTRACT FROM AUTHOR]

The brackish river prawn (Macrobrachium macrobrachion) is a species of commercial importance in West Africa. However, like other fishery products, it is prone to deterioration due mainly to microbial activities. The present study aimed at evaluating the spoilage characteristics of M. macrobrachion and predicting the growth of the main spoilage bacteria as well as the shelf-life of the product as a function of storage temperature. Freshly caught brackish river prawn samples from Lake Aheme were aerobically stored at 0, 7, 15, and 28 °C and, at pre-determined times during storage, they were taken for microbiological, chemical, and sensory analysis. At sensory rejection times, the spoilage potential of 185 isolates from specific groups of organisms enumerated was assessed in prawn of which the endogenous microbiota was heat inactivated. Isolates capable of producing strong off-odor were identified using 16S rRNA sequencing. Models predicting the maximum growth rate of Pseudomonas spp. and H 2 S-producing bacteria in the brackish river prawn as well as the shelf-life of the product were developed. These models were validated using an independent experiment during which prawn was stored at 0, 4, 10, and 25 °C. Results showed that Pseudomonas spp. at 0 °C, Pseudomonas spp. and H 2 S-producing bacteria at 7 °C, and H 2 S-producing bacteria at 15 °C and 28 °C were the dominant groups of microorganisms during storage. As expected, total volatile basic nitrogen, trimethylamine, and pH with initial values of 21.2 ± 3.0 mg-N/100 g, 4.1 ± 0.8 mg-N/100 g, and 7.46 ± 0.15 increased during storage reaching approximately 35 mg-N/100 g, 10 mg/ 100 g and 8, respectively at sensory rejection times which were 7 h at 28 °C, 1.2 d at 15 °C, 4.6 d at 7 °C, and 11.7 d at 0 °C. The main spoilage organisms were Citrobacter braakii at 28 °C, Citrobacter braakii, Pseudomonas kurunegalensis, and Shewanella bicestrii at 15 °C, Shewanella putrefaciens, Shewanella baltica, and Pseudomonas bubulae at 7 °C, and Pseudomonas versuta at 0 °C. The validation of the developed models showed an adequate agreement between the predicted and observed values. This study highlights the specific spoilage characteristics of the brackish river prawn and reveals that Gram-negative rod bacteria are the main spoilage organisms even at high storage temperatures, contrary to many earlier reports on the spoilage of tropical fishery products. • Spoilage characteristics of Macrobrachium macrobrachion were temperature-dependent. • Potential spoilage organisms were identified by 16S rRNA sequencing. • Even at high storage temperatures, Gram- rods bacteria were surprinsingly the main spoilage organisms • TVBN or TMA can be used as a good chemical spoilage index of M. macrobrachion • An empirical model for prawn shelf-life prediction was developed and validated. [ABSTRACT FROM AUTHOR]

The plasmid-mediated colistin resistance gene mcr-1 has been found worldwide, but the diversity of organisms harbouring this gene is unknown. In this study, 12 colistin-resistant Citrobacter spp. isolates were obtained from diseased or dead chickens in China, and PCR analysis indicated that five were positive for mcr-1 . One Citrobacter braakii strain (SCC4) with a multidrug-resistant phenotype was chosen for further analysis. SCC4 was resistant or intermediate-resistant to ten of the tested antibiotics, and the colistin minimum inhibitory concentration (MIC) was >4 µg/mL. A conjugation assay demonstrated successful transfer of colistin resistance to Escherichia coli strain J53 at a frequency of 10 –7 cells per recipient cell. Whole-genome sequencing revealed that SCC4 contained 13 antibiotic resistance genes in its genome, and the mcr-1 gene resided on a 44-kb self-transmissible IncP-type plasmid of a recently discovered IncP-1 clade. In addition, the mcr-1 gene was part of an insertion element (IS Apl1–mcr-1 – orf– IS Apl1 ) that was excised from the plasmid as a circular intermediate form. This is the first report of mcr-1 -posiitve C. braakii of animal origin and these findings highlight the fact that the mcr-1 gene can be found in normal enteric flora as part of broad-host-range plasmids. [ABSTRACT FROM AUTHOR]

The main objective of this study is to demonstrate the potential of microbial induced calcite precipitation (MICP) as rapid and effective process in the synthesis of fluorescent cadmium sulfide nanoparticles. A urease-producing bacterial isolate AX5, identified as Citrobacter braakii , from calcite rich shells of abalone that could tolerate cadmium at the concentration of 250 mg/l was used. In order to investigate the role of MICP in enhancing the synthesis of CdS nanoparticles, the biomimetic synthesis was carried out by C. braakii in modified nutrient broth without amending any additional organic or toxic surfactants back into the environment. The large amount of monodispersed CdS nanoparticles was harvested and found to bind with biocalcite. XRD analysis confirmed the presence of CdS nanoparticles bound with biocalcite minerals in the precipitation. HRTEM and SEM micrographs showed spherical CdS nanoparticles of particle size in the range of 50–100 nm. The other characteristics of harvested CdS nanoparticles were studied with UV–vis, PL spectra, FTIR, and fluorescence microscopy. The findings of present study have important insights into the application of MICP as a promising mechanism for the biosynthesis of CdS nanoparticles. [ABSTRACT FROM AUTHOR]

Airborne microbes can rapidly spread and cause various infectious diseases worldwide. This necessitates the determination of a fast and highly sensitive detection method. There have been no studies on receptors targeting Citrobacter braakii (C. braakii), a pathogenic bacterium which can exist in the air. In this study, we rapidly isolate an aptamer, a nucleic acid molecule that can specifically bind to C. braakii by centrifugation-based partitioning method (CBPM) reported previously by our groups as omitting the repeated rounds of binding incubation, separation, and amplification that are indispensable for SELEX. The binding affinity and specificity of isolated aptamers are checked using bacteria in liquid culture and recollection solution from aerosolized bacteria. Recollection solutions of the recovered bacteria are obtained by nebulizing, drying, and recapturing with a biosampler. The CB-5 aptamer shows high affinity and specificity for C. braakii (K d : 16.42 in liquid culture and 26.91 nM in recollection from aerosolized sample). Our results indicate the current protocol can be employed for the rapid development of reliable diagnostic receptors targeting airborne bacteria., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

A 34‐year‐old woman presented due to progressive painful swelling around the nail of the right index finger. Onychectomy and drainage of the abscess of the affected finger were performed as the inflammation was progressive despite the previous antibiotic therapy. The microbiological culture revealed a ciprofloxacin‐susceptible Citrobacter braakii. [ABSTRACT FROM AUTHOR]

Abstract: P450cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450cin D241N mutant has been produced and found to be analogous to the P450cam D251N mutant. P450cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31±3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase. [Copyright &y& Elsevier]

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, sulfamethoxazoletrimethoprim, tetracycline, and tylosin. We found the bacteria sensitive to 4 mg/l doxycycline in the ATB VET test strip but resistant to 30 μg/l doxycycline in the antibiogram disc diffusion test. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: A conserved threonine found in the majority of cytochromes P450 (P450s) has been implicated in the activation of dioxygen during the catalytic cycle. P450cin (CYP176A) has been found to be an exception to this paradigm, where the conserved threonine has been replaced with an asparagine. Prior studies with a P450cin N242A mutant established that the Asn-242 was not a functional replacement for the conserved threonine but was essential for the regio- and stereocontrol of the oxidation of cineole. To explore further how P450cin controls the activation of the dioxygen in the absence of the conserved threonine, two concurrent lines of investigation were followed. Modification of P450cin indicated that the Thr-243 was not involved in controlling the protonation of the hydroperoxy species. In addition, the N242T mutant did not enhance the rate and/or efficiency of catalytic turnover of cineole by P450cin. In parallel experiments, the substrate cineole was modified by removing the ethereal oxygen to produce camphane or 2,2-dimethylbicyclo[2.2.2]octane (cinane). An analogous experiment with P450EryF showed that a hydroxyl group on the substrate was vital, and in its absence catalytic turnover was effectively abolished. Catalytic turnover of P450cin with either of these alternative substrates (camphane or cinane) revealed that in the absence of the ethereal oxygen there was still a significant amount of coupling of the NADPH-reducing equivalents to the formation of oxidised product. Again the substrate itself was not found to be important in controlling oxygen activation, in contrast to P450EryF, but was shown to be essential for regio- and stereoselective substrate oxidation. Thus, it still remains unclear how dioxygen activation in the catalytic turnover of cineole by P450cin is controlled. [Copyright &y& Elsevier]

Abstract: The first steps in the biodegradation of 1,8-cineole involve the introduction of an alcohol and its subsequent oxidation to a ketone. In Citrobacter braakii, cytochrome P450cin has previously been demonstrated to perform the first oxidation to produce (1R)-6β-hydroxycineole. In this study, we have cloned cinD from C. braakii and expressed the gene product, which displays significant homology to a number of short-chain alcohol dehydrogenases. It was demonstrated that the gene product of cinD exhibits (1R)-6β-hydroxycineole dehydrogenase activity, the second step in the degradation of 1,8-cineole. All four isomers of 6-hydroxycineole were examined but only (1R)-6β-hydroxycineole was converted to (1R)-6-ketocineole. The (1R)-6β-hydroxycineole dehydrogenase exhibited a strict requirement for NAD(H), with no reaction observed in the presence of NADP(H). The enzyme also catalyses the reverse reaction, reducing (1R)-6-ketocineole to (1R)-6β-hydroxycineole. During this study the N-terminal His-tag used to assist protein purification was found to interfere with NAD(H) binding and lower enzyme activity. This could be recovered by the addition of Ni2+ ions or proteolytic removal of the His-tag. [Copyright &y& Elsevier]

Among the Citrobacter genus, the most commonly isolated bacteria from human specimens are Citrobacter freundii and Citrobacter koseri , and previous cases of infection due to Citrobacter braakii have been rarely reported. We present a case of bacteremia due to C. braakii in a 38-year-old woman with cervical cancer. She was admitted to our hospital with complaints of a fever, chills, and nausea. Blood culture results showed gram-negative bacilli identified as C. braakii via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, although biochemical testing findings were suggestive of C. freundii . Since a rare pathogen was detected in the present case and the results of additional biochemical studies were suggestive of both C. braakii and Citrobacter farmeri , genetic analysis was conducted. Finally, the gram-negative bacilli were confirmed as C. braakii , a member of the C. freundii complex since 1993, by 16S ribosomal RNA gene sequencing analysis. The gastrointestinal tract was considered the portal of entry, because the patient had a rectal fistula and other cultures such as urine and vaginal discharge incubated species other than C. braakii . The patient recovered after receiving treatment with ciprofloxacin for 14 days. The epidemiology and clinical characteristics of C. braakii infection are still unknown because of the limitations in accurate identification by using currently available commercial biochemical testing and previously, only 6 cases of C. braakii infection have been reported. Physicians should focus on this species, because it causes community-acquired infections, although further studies are needed to clarify the clinical characteristics of C. braakii infections. [ABSTRACT FROM AUTHOR]

Abstract: 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 °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. [Copyright &y& Elsevier]

The effect of different physicochemical treatments on the aerobic biodegradability of an industrial wastewater resulting from a cosmetic industry has been investigated. This industrial wastewater contains 11423 and 3148mgL−1 of chemical oxygen demand (COD) and anionic surfactants, respectively. The concentration of COD and anionic surfactants were followed throughout the diverse physicochemical treatments and biodegradation experiments. Different pretreatments of this industrial wastewater using chemical flocculation process with lime and aluminium sulphate (alum), and also advanced oxidation process (electro-coagulation (Fe and Al) and electro-Fenton) led to important COD and anionic surfactants removals. The best results were obtained using electro-Fenton process, exceeding 98 and 80% of anionic surfactants and COD removals, respectively. The biological treatment by an isolated strain Citrobacter braakii of the surfactant wastewater, as well as the pretreated wastewater by the various physicochemical processes used in this study showed that the best results were obtained with electro-Fenton pretreated wastewater. The characterization of the treated surfactant wastewater by the integrated process (electro-coagulation or electro-Fenton)-biological showed that it respects Tunisian discharge standards. [Copyright &y& Elsevier]

The article presents a study on the characterization of arylsulfatase formed by depressed synthesis in Citrobacter braakii. The production of arylsulfatase was derepressed markedly in a synthetic medium by the addition of tyramine. The purified enzyme was classified as type I arylsulfatase. Sulfatases are enzymes that hydrolyze sulfate ester bonds in a wide variety of compounds to produce inorganic sulfates.

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. [ABSTRACT FROM AUTHOR]

Abstract: The treatment of an anionic surfactant rich wastewater by a powerful Citrobacter braakii strain was investigated at large scale. This bacterium was inoculated into a 4.5m3 continuous stirred tank reactor (CSTR) without a membrane. At volumetric loading rates ranged between 0.44 and 0.96gCODl-1d-1, a weak C. braakii population density was observed in the reactor which results in slow anionic surfactants degradation kinetics. The augmented bacterium was diluted and replaced by protozoa and other forms of bacteria after a prolonged experience run. The CSTR was then coupled to a cross-flow ultrafiltration membrane. This allowed complete biomass recycling and partially retention of the surfactants in the reactor. C. braakii became again dominant in the medium. At volumetric loading rates of 0.6–1.5gl-1d-1, the anionic surfactant concentration in the permeate varied from 0 to 40mgl-1 with a mean value of 18mgl-1. After 2months of running, mixed liquor volatile suspended solids (MLVSS) in the reactor increased from 0.71 to 5.92gl-1 and the permeate-flux slightly decreased from 28.75 to 23.75lh-1m-2. The reclaimed water could be reused either for the washing of the material and soil, and to irrigate the parkland of the factory. [Copyright &y& Elsevier]

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 1 H and 13 C 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. [ABSTRACT FROM AUTHOR]

Anionic surfactants, as core components of detergent and cosmetic products formulations, contribute significantly to the pollution profile of sewage and wastewaters of all kinds. In Tunisia more than 100 000 m3 per year of surfactant containing wastewater are released. Treatment by conventional processes such as activated sludge is problematic due to the low kinetics of degradation by unsuitable consortia and to foam production. Bioremediation involves the isolation of suitable microorganisms, adequate process development and in situ biodegradation. The objective of this work was to develop a high performance process for the treatment of anionic surfactant containing wastewater. Screening of bacteria degrading the anionic surfactant sodium lauryl ether sulfate (SLES) was carried out by a conventional enrichment-culture technique. A Citrobacter braakii strain degrading many anionic surfactants was isolated and tested in bioaugmentation for industrial wastewater treatment. It was used for the continuous degradation of highly concentrated SLES synthetic medium and cosmetic industry wastewater. By testing several dilution rates, it was concluded that C. braakii is able to degrade up to 0.065 g l−1 h−1. The hydraulic retention time (HRT) was nearly 20 h. This process was improved by coupling the bioreactor to a mineral microfiltration membrane. This allowed an increase in the SLES elimination rate from 0.065 to 0.15 g l−1 h−1 and to reduce the HRT from 20 to 5 h. Moreover, this integrated system generates microorganism and surfactant free wastewaters able to be rejected without public menace or reused in situ in the industrial process. [Copyright &y& Elsevier]