Whole-genome analysis and description of an IMP-8-producing Ochrobactrum anthropi.

OBEJECTIVES: To explore the genomic characterization of an IMP-8-producing Ochrobactrum anthropic and give suggestions for the application of antibiotics. METHODS: In 2021, the infection caused by CRKP was under control after nearly three months of using CAV, however, carbapenem-resistant O. anthropi isolates were collected from a rectal swab sample of a patient with Lumbar Disc Herniation Postoperative Infection. The rectal swab was then enriched in lysogeny broth overnight and inoculated onto China Blue agar plates containing 0.3µg/mL meropenem. And we investigated the characteristics of this carbapenem-resistant O. anthropi by MALDI-TOF MS, Immune colloidal gold technique, conjugation experiment, whole genome sequencing and antimicrobial susceptibility testing. RESULTS: Antimicrobial susceptibility testing showed that the O. anthropi were resistant to imipenem, cefmetazole, ceftazidime, cefotaxime, piperacillin/tazobactam, sulbactam/cefopcrazone, ceftazidime/avibactam, cefepime, ciprofloxacin, aztreonam, and not susceptible to meropenem, ertapenem, polymyxin B, tigecycline, amikacin. Immune colloidal gold technique reflected that this strain produced IMP carbapenemases, and the presence of IMP-8 was verified by WGS, which was located in a 21,442 bp, nonconjugative plasmid. CONCLUSION: Improper antibiotic treatment can cause intestinal flora imbalance and even bacteremia in patients, we should use antibiotics wisely and develop individualized treatment options.

Active-site engineering of ω-transaminase from Ochrobactrum anthropi for preparation of L-2-aminobutyric acid.

BACKGROUND: The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering. RESULTS: Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g. CONCLUSION: This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.

In Vitro Reconstitution of the Pantothenic Acid Degradation Pathway in Ochrobactrum anthropi.

Pantothenic acid is an essential metabolite found throughout all branches of life. Although the enzymes responsible for pantothenic biosynthesis have been characterized, those leading to its biodegradation remain poorly understood. In the study described herein, we showed that use of a "genomic enzymology" strategy enabled identification of four biodegradation pathway genes, which were then confirmed by using kinetic analysis of the purified recombinant enzymes encoded in Ochrobactrum anthropi. The reconstituted pathway converts pantothenic acid to ß-alanine and (R)-pantoate, and then (R)-pantoate to aldopentoate, which is transformed to (R)-3,3-dimethylmalate and hence to α-ketoisovalerate. The pathway genes are common to Proteobacterial genomes in which they are not colocated.

Simultaneous nitrification and aerobic denitrification by a novel isolated Ochrobactrum anthropi HND19.

The study aimed to isolate a novel strain with heterotrophic nitrification and aerobic denitrification ability and evaluate the nitrogen removal characteristics. Results showed that Ochrobactrum anthropi HND19 could remove approximately 98.6% of NH4+-N (104.3 mg·L-1) and 97.6% of NO3--N (98.6 mg·L-1), and the removal rates achieved 4.28 and 4.01 mg-N/(L·h) by heterotrophic nitrification and aerobic denitrification. The optimal incubate conditions of strain HND19 were 120 rpm (shaking speed), 5 ‰ (salinity), 30 °C (temperature), 7.5 (C/N ratio) with sodium acetate as carbon resource. And the removal efficiency of the total nitrogen (TN) realized 73.4% under the optimal conditions. Functional genes (hao, napA, nirK, norB, and nosZ) involved in the nitrogen removal processes were successfully amplified from strain HND19. These findings indicate that the strain HND19 possesses great application feasibility in treating wastewater with high-intensity nitrogen.

A novel global transcriptional perturbation target identified by forward genetics reprograms Vibrio natriegens for improving recombinant protein production.

Vibrio natriegens is known to be the fastest-growing free-living bacterium with the potential to be a novel protein expression system other than Escherichia coli. Seven sampled genes of interest (GOIs) encoding biocatalyst enzymes, including Ochrobactrum anthropi-derived ω-transaminase (OATA), were strongly expressed in E. coli but weakly in V. natriegens using the pET expression system. In this study, we fused the C-terminal of OATA with green fluorescent protein (GFP) and obtained V. natriegens mutants that could increase both protein yield and enzyme activity of OATA as well as the other three GOIs by ultraviolet mutagenesis, fluorescence-activated cell sorting (FACS), and OATA colorimetric assay. Furthermore, next-generation sequencing and strain reconstruction revealed that the Y457 variants in the conserved site of endogenous RNA polymerase (RNAP) ß' subunit rpoC are responsible for the increase in recombinant protein yield. We speculated that the mutation of rpoC Y457 may reprogram V. natriegens's innate gene transcription, thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs. The increase in GOI expression may partly be attributed to the increase in copy number. In conclusion, GOI-GFP fusion combined with FACS is a powerful tool of forward genetics that can be used to obtain a superior expression chassis. If more high-expression-related targets are found for more GOIs, it would make the construction of next-generation protein expression chassis more time-saving.

Genomic and biotechnological insights on stress-linked polyphosphate production induced by chromium(III) in Ochrobactrum anthropi DE2010.

The resistance of microorganisms to heavy metals in polluted environments is mediated by genetically determined mechanisms. One such mechanism includes the intracellular sequestration of heavy metals in polyphosphate (polyP) inclusions. In Cr(III) contaminated mediums, Ochrobactrum anthropi DE2010 is able to bind and sequester Cr(III) in polyP inclusions. In order to further study the relationship between Cr(III) tolerance and polyP production in O. anthropi DE2010, we carried out whole genomic sequencing, analysis of single nucleotide polymorphisms (SNPs), polyP chemical quantification, and determination of the relative abundance and morphometry of polyP inclusions. In the O. anthropi DE2010 genome, six polyP and pyrophosphate (PPi) metabolic genes were found. Furthermore, genomic analysis via SNPs calling revealed that O. anthropi ATCC49188 and DE2010 strains had average variations of 1.51% in their whole genome sequences and 1.35% variation associated with the principal polyP metabolic gene cluster. In addition, the accumulation of polyP in the DE2010 strain and number of polyP inclusions found were directly correlated with the concentration of Cr(III) in contaminated cultures. The results presented in this study may enhance the understanding of polyP production in response to Cr(III) toxicity in the O. anthropi DE2010 strain. This knowledge may facilitate the successful removal of Cr(III) from the natural environment.

Study on biodegradation kinetics of di-2-ethylhexyl phthalate by newly isolated halotolerant Ochrobactrum anthropi strain L1-W.

OBJECTIVE: Di-2-ethylhexyl phthalate (DEHP) pollution is one of the major environmental concerns all over the world. This research aimed at studying the biodegradation kinetics of DEHP by a newly isolated bacterial strain. Water and sediment samples were collected from Wuhan South Lake and potent bacterial isolates were screened for DEHP degradation, characterized by biochemical, physiological, morphological and 16S rDNA gene sequencing, and optimized under suitable pH, temperature, NaCl and DEHP concentrations. DEHP and its metabolites were quantified by High Performance Liquid Chromatography and their degradation kinetics were studied. RESULTS: The newly isolated bacterium was identified as Ochrobactrum anthropi strain L1-W with 99.63% similarity to Ochrobactrum anthropi ATCC 49188. It was capable of utilizing DEHP as the carbon source. The optimum growth temperature, pH, DEHP and NaCl concentration for the strain L1-W were 30 °C, 6, 400 mg/L and 10 g/L respectively. Strain L1-W was capable of degrading almost all (98.7%) of DEHP when the initial concentration was 200 mg/L within a period of 72 h. Besides, it was also found capable of degrading five other phthalates, thus making it a possible candidate for bioremediation of phthalates in the environmental settings.

Microbial associates of the southern mole cricket (Scapteriscus borellii) are highly pathogenic.

We report the isolation and identification of seven bacterial strains and one fungal strain from dead and diseased Scapteriscus borellii mole crickets collected from a golf course in southern California. Using 16S and 18S rRNA gene sequence analysis we identified the microbes as Serratia marcescens (red), S. marcescens (white), S. marcescens (purple), Achromobacter xylosoxidans, Chryseobacterium sp., Ochrobactrum anthropi, Tsukamurella tryosinosolvens, and Beauveria bassiana. We performed a dose response curve for each of these cricket-associated microbial strains (except T. tryosinosolvens) and two other strains of S. marcescens (DB1140 and ATCC 13880). We found that all of these microbes except O. anthropi were highly pathogenic to D. melanogaster compared to the other strains of S. marcescens. Injecting the mole cricket associated strains of Serratia into flies killed all infected flies in ≤24h. For all other strains, the median time to death of injected flies varied in a dose-dependent manner. In vivo growth assessments of these microbes suggested that the host immune system was quickly overcome. We used disease tolerance curves to better understand the host-microbe interactions. Further studies are necessary to understand in mechanistic detail the virulence mechanisms of these mole cricket associated microbes and how this association may have influenced the evolution of mole cricket immunity.

Diversity of Ochrobactrum species in food animals, antibiotic resistance phenotypes and polymorphisms in the blaOCH gene.

Twenty-six lactose non-fermenting, oxidase, urease and citrate-positive Gram-negative rods, isolated from broiler chickens, pigs and cattle at slaughter, were subjected to the matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and 16S rDNA sequencing for identification. Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Ochrobactrum isolates resistant to third-generation cephalosporins were PCR-screened for the presence of the Ochrobactrum anthropi ampC gene (blaOCH). A 547-bp internal segment of blaOCH in the Ochrobactrum spp isolates was amplified with a newly designed primer set, and a phylogenetic reconstruction based on the complete amino acid sequence of blaOCH obtained from nine Ochrobactrum strains in our collection and 20 O. anthropi available in the GenBank was undertaken. All the Ochrobactrum isolates were resistant to the expanded-spectrum beta-lactams and streptomycin. None of the isolates was resistant to imipenem while 41.7% to 50.0% of them were resistant to fluoroquinolones. The blaOCH gene was detected in 16 (66.7%) and 20 (83.3%) of the 24 Ochrobactrum isolates (O. intermedium/O. tritici species), using primers designed for O. anthropi and the newly designed primer set, respectively. Six blaOCH variants grouped into two divergent clusters were identified. This is the first report of the complete nucleotide sequence of the blaOCH gene in non-antropi Ochrobactrum species.

Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation.

BACKGROUND: Whole-cell biocatalysis based on metabolically active baker's yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels. RESULTS: Herein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5'-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using L-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions. CONCLUSIONS: Altogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion.

Utility of Ochrobactrum anthropi YC152 in a Microbial Fuel Cell as an Early Warning Device for Hexavalent Chromium Determination.

Fast hexavalent chromium (Cr(VI)) determination is important for environmental risk and health-related considerations. We used a microbial fuel cell-based biosensor inoculated with a facultatively anaerobic, Cr(VI)-reducing, and exoelectrogenic Ochrobactrum anthropi YC152 to determine the Cr(VI) concentration in water. The results indicated that O. anthropi YC152 exhibited high adaptability to pH, temperature, salinity, and water quality under anaerobic conditions. The stable performance of the microbial fuel cell (MFC)-based biosensor indicated its potential as a reliable biosensor system. The MFC voltage decreased as the Cr(VI) concentration in the MFC increased. Two satisfactory linear relationships were observed between the Cr(VI) concentration and voltage output for various Cr(VI) concentration ranges (0.0125-0.3 mg/L and 0.3-5 mg/L). The MFC biosensor is a simple device that can accurately measure Cr(VI) concentrations in drinking water, groundwater, and electroplating wastewater in 45 min with low deviations (<10%). The use of the biosensor can help in preventing the violation of effluent regulations and the maximum allowable concentration of Cr(VI) in water. Thus, the developed MFC biosensor has potential as an early warning detection device for Cr(VI) determination even if O. anthropi YC152 is a possible opportunistic pathogen.

[Research progress of Ochrobactrum anthropic--A review].

Ochrobactrum anthropi is a gram-negative bacillus, usually known as an opportunistic pathogen. Mostly its infection is related with systemic or local lower immunity, and manifested as bacteremia, meningitis, purulent infection. Recently, along with expanded infection, it has become an important human pathogen. The prevention, clinical diagnose and treatment become complicated because varied clinical symptoms increased antibiotic resistance and cross immune reaction with others pathogens. In this review, we summarized the biological characteristics, differential diagnosis, immunity, resistance and genomic characteristics of Ochrobactrum anthropi, to provide reference for prevention, control and treatment management of this disease.

Development of a loop-mediated isothermal amplification method for the rapid detection of the dioxin-degrading bacterium Ochrobactrum anthropi in soil.

In this study, loop-mediated isothermal amplification (LAMP) and real-time LAMP assays were developed to detect the dioxin-degrading bacterium Ochrobactrum anthropi strain BD-1 in soil. Four primers were designed to use ITS gene amplification for the strain O. anthropi BD-1. The real-time LAMP assay was found to accomplish the reaction by 1 pg of genomic DNA load when used for nucleic acid amplification. This assay was then applied to detect O. anthropi BD-1 in eight soil samples collected from a dioxin-contaminated site. The results demonstrated that these newly developed LAMP and real-time LAMP assays will not only be useful and efficient tools for detecting the target gene, but also be used as molecular tools for monitoring the growth of dioxin-degrading O. anthropi in the soil. This is the first report to demonstrate the use of LAMP assays to monitor the presence of O. anthropi in dioxin-contaminated soil. The application of this method should improve the biomonitoring of dioxin contamination.

Ochrobactrum anthropi bacteremia in a preterm infant with cystic fibrosis.

Ochrobactrum anthropi infection in newborn patients is rare, and the treatment is challenging because of its widespread and unpredictable resistance to antimicrobial agents and discrepancies between in vitro susceptibility and in vivo efficacy. We report the clinical and microbiological characteristics of Ochrobactrum anthropi bacteremia in a preterm patient.

Colonization by endophytic Ochrobactrum anthropi†Mn1 promotes growth of Jerusalem artichoke.

The Ochrobactrum anthropi Mn1 strain, taxonomically identified using 16S ribosomal DNA sequence, was isolated from roots of Jerusalem artichoke. Its endophytic colonization was investigated microscopically using green fluorescent protein introduced by vector pHC60. The strain entered Jerusalem artichoke tissues through the root, and was localized in the roots and stems. The plant growth-promoting (PGP) effects of O. anthropi Mn1 were assessed in greenhouse as well as field trials with different nitrogen supplies. Only under moderate to ample nitrogen supply, could O. anthropi Mn1 promoted growth of host plant. The PGP effects of the strain were symbiotic nitrogen fixation, root morphological optimization and enhanced nutrient uptake. We hypothesize that the symbiotic interspecies interaction might be quorum sensing related.

Ochrobactrum anthropi bacteremia in a preterm infant with cystic fibrosis

Ochrobactrum anthropi infection in newborn patients is rare, and the treatment is challenging because of its widespread and unpredictable resistance to antimicrobial agents and discrepancies between in vitro susceptibility and in vivo efficacy. We report the clinical and microbiological characteristics of Ochrobactrum anthropi bacteremia in a preterm patient.

Typing of Ochrobactrum anthropi clinical isolates using automated repetitive extragenic palindromic-polymerase chain reaction DNA fingerprinting and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

BACKGROUND: Ochrobactrum anthropi (O. anthropi), is a non-fermenting gram-negative bacillus usually found in the environment. Nevertheless, during the past decade it has been identified as pathogenic to immunocompromised patients. In this study, we assessed the usefulness of the automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR-based DiversiLab™ system, bioMèrieux, France) and of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF MS) for typing of twentythree O. anthropi clinical isolates that we found over a four-months period (from April 2011 to August 2011) in bacteriemic patients admitted in the same operative unit of our hospital. Pulsed-field gel electrophoresis (PFGE), commonly accepted as the gold standard technique for typing, was also used. Analysis was carried out using the Pearson correlation coefficient to determine the distance matrice and the unweighted pair group method with arithmetic mean (UPGMA) to generate dendogram. RESULTS: Rep-PCR analysis identified four different patterns: three that clustered together with 97% or more pattern similarity, and one whose members showed < 95% pattern similarity. Interestingly, strains isolated later (from 11/06/2011 to 24/08/2011) displayed a pattern with 99% similarity. MALDI-TOF MS evaluation clustered the twentythree strains of O. anthropi into a single group containing four distinct subgroups, each comprising the majority of strains clustering below 5 distance levels, indicating a high similarity between the isolates. CONCLUSIONS: Our results indicate that these isolates are clonally-related and the methods used afforded a valuable contribution to the epidemiology, prevention and control of the infections caused by this pathogen.

Molecular cloning and characterization of a newly isolated pyrethroid-degrading esterase gene from a genomic library of Ochrobactrum anthropi YZ-1.

A novel pyrethroid-degrading esterase gene pytY was isolated from the genomic library of Ochrobactrum anthropi YZ-1. It possesses an open reading frame (ORF) of 897 bp. Blast search showed that its deduced amino acid sequence shares moderate identities (30% to 46%) with most homologous esterases. Phylogenetic analysis revealed that PytY is a member of the esterase VI family. pytY showed very low sequence similarity compared with reported pyrethroid-degrading genes. PytY was expressed, purified, and characterized. Enzyme assay revealed that PytY is a broad-spectrum degrading enzyme that can degrade various pyrethroids. It is a new pyrethroid-degrading gene and enriches genetic resource. Kinetic constants of Km and Vmax were 2.34 mmol·L(-1) and 56.33 nmol min(-1), respectively, with lambda-cyhalothrin as substrate. PytY displayed good degrading ability and stability over a broad range of temperature and pH. The optimal temperature and pH were of 35°C and 7.5. No cofactors were required for enzyme activity. The results highlighted the potential use of PytY in the elimination of pyrethroid residuals from contaminated environments.