In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration

Aims: The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla_TEM‐52‐carrying plasmid (lactose‐negative mutant of B1‐54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. Methods and Results: Fresh faeces from a healthy volunteer, negative for cephalosporin‐resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed‐field gel electrophoresis profiles (PFGE). The avian extended‐spectrum β‐lactamase‐positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1‐54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla_TEM‐52‐carrying plasmid. Conclusions: These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. Significance and Impact of the Study: The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.     

Computational fluid dynamics modeling of mass-transfer behavior in a bioreactor for hairy root culture. II. Analysis of ultrasound-intensified process

Recently, cichoric acid production from hairy roots of Echinacea purpurea was significantly improved by ultrasound stimulation in an airlift bioreactor. In this article, the possible mechanism on ultrasound-intensified hairy root culture of E. purpurea in the bioreactor was elucidated with the help of computational fluid dynamics (CFD) simulation, membrane permeability detection, dissolved oxygen concentration detection, confocal laser-scanning microscopy (LSM) observation, and phenylalanine ammonium lyase (PAL) activity analysis. The CFD model developed in Part I was used to simulate the hydrodynamics and oxygen mass transfer in hairy root bioreactor culture stimulated by ultrasound. A dynamic mesh model combined with a changing Schmidt number method was used for the simulation of the ultrasound field. Simulation results and experimental data illustrated that ultrasound intensified oxygen mass transfer in the hairy root clump, which subsequently stimulated root growth and cichoric acid biosynthesis. Ultrasound increased the hairy root membrane permeability, and a high root membrane permeability of 0.359 h(-1) was observed at the bottom region in the bioreactor. LSM observation showed that the change in the membrane permeability recovered to normal in the further culture after ultrasound stimulation. PAL activity in the hairy roots was stimulated by ultrasound increase and was correlated well to cichoric acid accumulation in the hairy roots of E. purpurea.     

Computational fluid dynamics modeling of mass transfer behavior in a bioreactor for hairy root culture. I. Model development and experimental validation

A two-dimensional axisymmetric computational fluid dynamics (CFD) model based on a porous media model and a discrete population balance model was established to investigate the hydrodynamics and mass transfer behavior in an airlift bioreactor for hairy root culture.During the hairy root culture of Echinacea purpurea, liquid and gas velocity, gas holdup, mass transfer rate, as well as oxygen concentration distribution in the airlift bioreactor were simulated by this CFD model. Simulative results indicated that liquid flow and turbulence played a dominant role in oxygen mass transfer in the growth domain of the hairy root culture. The dissolved oxygen concentration in the hairy root clump increased from the bottom to the top of the bioreactor cultured with the hairy roots, which was verified by the experimental detection of dissolved oxygen concentration in the hairy root clump. This methodology provided insight understanding on the complex system of hairy root culture and will help to eventually guide the bioreactor design and process intensification of large-scale hairy root culture.     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

Two-compartment method for determination of the oxygen transfer rate with electrochemical sensors based on sulfite oxidation

The dissolved oxygen concentration is a crucial parameter in aerobic bioprocesses due to the low solubility of oxygen in water. The present study describes a new method for determining the oxygen transfer rate (OTR) in shaken-culture systems based on the sodium sulfite method in combination with an electrochemical oxygen sensor. The method replaces the laborious titration of the remaining sulfite by an on-line detection of the end point of the reaction. This method is a two-step procedure that can be applied in arbitrary flasks that do not allow the insertion of electrodes. The method does not therefore depend on the type of vessel in which the OTR is detected. The concept is demonstrated by determination of the OTR for standard baffled 1-L shake flasks and for opaque Ultra Yield™ flasks. Under typical shaking conditions, k_La values in the standard baffled flasks reached values up to 220 h^-1, whereas the k_La values of the Ultra Yield flasks were significantly higher (up to 422 h^-1).     

Complete mitochondrial genome of Tubulipora flabellaris (Bryozoa: Stenolaemata): the first representative from the class Stenolaemata with unique gene order

Mitochondrial genomes play a significant role in the reconstruction of phylogenetic relationships within metazoans. There are still many controversies concerning the phylogenetic position of the phylum Bryozoa. In this research, we have finished the complete mitochondrial genome of one bryozoan (Tubulipora flabellaris), which is the first representative from the class Stenolaemata. The complete mitochondrial genome of T. flabellaris is 13,763 bp in length and contains 36 genes, which lacks the atp8 gene in contrast to the typical metazoan mitochondrial genomes. Gene arrangement comparisons indicate that the mitochondrial genome of T. flabellaris has unique gene order when compared with other metazoans. The four known bryozoans complete mitochondrial genomes also have very different gene arrangements, indicates that bryozoan mitochondrial genomes have experienced drastic rearrangements. To investigate the phylogenetic relationship of Bryozoa, phylogenetic analyses based on amino acid sequences of 11 protein coding genes (excluding atp6 and atp8) from 26 metazoan complete mitochondrial genomes were made utilizing Maximum Likelihood (ML) and Bayesian methods, respectively. The results indicate the monopoly of Lophotrochozoa and a close relationship between Chaetognatha and Bryozoa. However, more evidences are needed to clarify the relationship between two groups. Lophophorate appeared to be polyphyletic according to our analyses. Meanwhile, neither analysis supports close relationship between Branchiopod and Phoronida. Four bryozoans form a clade and the relationship among them is T. flabellaris + (F. hispida + (B. neritina + W. subtorquata)), which is in coincidence with traditional classification system.     

The complete mitochondrial genome of the Sichuan taimen (Hucho bleekeri): repetitive sequences in the control region and phylogenetic implications for Salmonidae

The complete mitochondrial DNA genome of the Sichuan taimen (Hucho bleekeri) was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequence method. The entire mitochondrial genome of this species is 16,997 bp in length, making it the longest among the completely sequenced Salmonidae mitochondrial genomes. It consists of two ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and one control region (CR). The gene arrangement, nucleotide composition, and codon usage pattern of the mitochondrial genome are similar to those of other teleosts. A T-type mononucleotide microsatellite and an 82 bp tandem repeat were identified in the control region, which were almost identical among the three H. bleekeri individuals examined. Both phylogenetic analyses based on 12 concatenated protein-coding genes of the heavy strand and on just the control region show that H. bleekeri is a basal species in Salmoninae. In addition, Salmo, Salvelinus and Oncorhynchus all represent monophyletic groups, respectively. All freshwater species occupied basal phylogenetic positions, and also possessed various tandem repeats in their mitochondrial control regions. These results support established phylogenetic relationships among genera in Salmonidae based on morphological and molecular analyses, and are consistent with the hypothesis that Salmonidae evolved from freshwater species.