Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles.

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

A novel ß-galactosidase from Klebsiella oxytoca ZJUH1705 for efficient production of galacto-oligosaccharides from lactose.

Galacto-oligosaccharides (GOS), which can be produced by enzymatic transgalactosylation of lactose with ß-galactosidases, have attracted much attention in recent years because of their prebiotic functions and wide uses in infant formula, infant foods, livestock feed, and pet food industries. In this study, a novel ß-galactosidase-producing Klebsiella oxytoca ZJUH1705, identified by its 16S rRNA sequence (GenBank accession no. MH981243), was isolated. Two ß-galactosidase genes, bga 1 encoding a 2058-bp fragment (GenBank accession no. MH986613) and bga 2 encoding a 3108-bp fragment (GenBank accession no. MN182756), were cloned from K. oxytoca ZJUH1705 and expressed in E. coli. The purified ß-gal 1 and ß-gal 2 had the specific activity of 217.56 U mg-1 and 57.9 U mg-1, respectively, at the optimal pH of 7.0. The reaction kinetic parameters Km, Vmax, and Kcat with oNPG as the substrate at 40 °C were 5.62 mM, 167.1 µmol mg-1 min-1, and 218.1 s-1, respectively, for ß-gal 1 and 3.91 mM, 14.6 µmol mg-1 min-1, and 28.9 s-1, respectively, for ß-gal 2. Although ß-gal 1 had a higher enzyme activity for lactose hydrolysis, only ß-gal 2 had a high transgalactosylation capacity. Using ß-gal 2 with the addition ratio of ~ 2.5 U g-1 lactose, a high GOS yield of 45.5 ± 2.3% (w/w-1) was obtained from lactose (40% w/w-1 or 480 g L-1) in a phosphate buffer (100 mM, pH 7.0) at 40 °C in 48 h. Thus, the ß-gal 2 from K. oxytoca ZJUH1705 would be a promising biocatalyst for GOS production from lactose.Key Points• A novel bacterial ß-galactosidase producer was isolated and identified.• ß-Galactosidases were cloned and expressed in heterologous strain and characterized.• Both enzymes have hydrolytic activity but only one have transglycosilation activity.• The developed process with ß-gal 2 could provide an alternative for GOS production.

The Washing Machine as a Reservoir for Transmission of Extended-Spectrum-Beta-Lactamase (CTX-M-15)-Producing Klebsiella oxytoca ST201 to Newborns.

During the period from April 2012 to May 2013, 13 newborns (1 to 4 weeks of age) and 1 child in a pediatric hospital ward in Germany were colonized with Klebsiella oxytoca producing an extended-spectrum beta-lactamase (ESBL) (CTX-M-15). A microbiological source-tracking analysis with human and environmental samples was carried out to identify the source and transmission pathways of the K. oxytoca clone. In addition, different hygienic intervention methods were evaluated. K. oxytoca isolates were detected in the detergent drawer and on the rubber door seal of a domestic washer-extractor machine that was used in the same ward to wash laundry for the newborns, as well as in two sinks. These strains were typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The environmental findings were compared with those for the human strains and the isolates detected on clothing. The results from both techniques showed that the strains were identical (sequence type 201 and PFGE type 00531, a clone specific to this hospital and not previously isolated in Germany), emphasizing the washing machine as a reservoir and fomite for the transmission of these multidrug-resistant bacteria. After the washing machine was taken out of use, no further colonizations were detected during the subsequent 4-year period.IMPORTANCE Washing machines should be further investigated as possible sites for horizontal gene transfer (ESBL genes) and cross-contamination with clinically important Gram-negative strains. Particularly in the health care sector, the knowledge of possible (re-)contamination of laundry (patients' clothes and staff uniforms) with multidrug-resistant Gram-negative bacteria could help to prevent and to control nosocomial infections. This report describes an outbreak with a single strain of a multidrug-resistant bacterium (Klebsiella oxytoca sequence type 201) in a neonatal intensive care unit that was terminated only when the washing machine was removed. In addition, the study implies that changes in washing machine design and processing are required to prevent accumulation of residual water where microbial growth can occur and contaminate clothes.

Characterization of Carbapenemase-Producing Klebsiella oxytoca in Spain, 2016-2017.

There is little information about carbapenemase-producing (CP) Klebsiella oxytoca, an important nosocomial pathogen. We characterized CP K. oxytoca isolates collected from different Spanish hospitals between January 2016 and October 2017. During the study period, 139 nonduplicate CP K. oxytoca isolates were identified; of these, 80 were studied in detail. Carbapenemase and extended-spectrum ß-lactamase genes were identified by PCR and sequencing. Genetic relatedness was studied by pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS), carried out on 12 representative isolates, was used to identify the resistome, to elucidate the phylogeny, and to determine the plasmids harboring carbapenemase genes. Forty-eight (60%) isolates produced VIM-1, 30 (37.5%) produced OXA-48, 3 (3.7%) produced KPC-2, 2 (2.5%) produced KPC-3, and 1 (1.2%) produced NDM-1; 4 isolates coproduced two carbapenemases. By PFGE, 69 patterns were obtained from the 80 CP K. oxytoca isolates, and four well-defined clusters were detected: cluster 1 consisted of 11 OXA-48-producing isolates, and the other three clusters included VIM-1-producing isolates (5, 3, and 3 isolates, respectively). In the 12 sequenced isolates, the average number of acquired resistance genes was significantly higher in VIM-1-producing isolates (10.8) than in OXA-48-producing isolates (2.3). All 12 isolates had chromosomally encoded genes of the blaOXY-2 genotype, and by multilocus sequence typing, most belonged to sequence type 2 (ST2). Carbapenemase genes were carried by IncL, IncHI2, IncFII, IncN, IncC, and IncP6 plasmid types. The emergence of CP K. oxytoca was principally due to the spread of VIM-1- and OXA-48-producing isolates in which VIM-1- and OXA-48 were carried by IncL, IncHI2, IncFII, and IncN plasmids. ST2 and the genotype blaOXY-2 predominated among the 12 sequenced isolates.

Colonization of intestinal microbiota with carbapenemase-producing Enterobacteriaceae in paediatric intensive care units in Cairo, Egypt.

BACKGROUND AND STUDY AIMS: Colonized patients with carbapenamase producing Enterobacteriaceae (CPE) are vulnerable to invasive infections from their endogenous flora. We aimed to assess faecal colonization with (CPE) among children admitted to Cairo University paediatric intensive care units (ICUs). The phenotypic and genotypic characterizations of carbapenemase-producing Enterobacteriaceae were also studied. PATIENTS AND METHODS: A total of 413 Enterobacteriaceae isolates have been isolated from cultured rectal swabs of 100 children. All swabs were inoculated on ChromID™ CARBA agar to screen for carbapenem resistant Enterobacteriaceae (CRE). Disk diffusion method, Modified Hodge test (MHT) and further genotypic detection of carbapenemases genes (blaOXA-48, blaKPC and blaNDM-1, blaVIM and blaIMP) by multiplex PCR were done. RESULTS: Out of 413 Enterobacteriaceae isolates; 100 isolates were defined as CRE. BlaOXA-48 was detected in (33%); Escherichia coli (n = 11), Klebsiella oxytoca (n = 3) and Klebsiella pneumoniae (n = 19), while (27%) carried blaNDM-1Escherichia coli (n = 7), and Klebsiella pneumoniae (n = 20). CONCLUSION: Prevalence of carbapenem resistant Enterobacteriaceae was 24%, various genes of carbapenemases were detected in 80% of carbapenem resistant Enterobacteriaceae with dominance of blaOXA-48. Understanding the colonization status of our patients with strict infection control measures can reduce the risk of horizontal gene transfer of carbapenemases.

Biochemical and structural investigation of sulfoacetaldehyde reductase from Klebsiella oxytoca.

Sulfoacetaldehyde reductase (IsfD) is a member of the short-chain dehydrogenase/reductase (SDR) family, involved in nitrogen assimilation from aminoethylsulfonate (taurine) in certain environmental and human commensal bacteria. IsfD catalyzes the reversible NADPH-dependent reduction of sulfoacetaldehyde, which is generated by transamination of taurine, forming hydroxyethylsulfonate (isethionate) as a waste product. In the present study, the crystal structure of Klebsiella oxytoca IsfD in a ternary complex with NADPH and isethionate was solved at 2.8 Å, revealing residues important for substrate binding. IsfD forms a homotetramer in both crystal and solution states, with the C-terminal tail of each subunit interacting with the C-terminal tail of the diagonally opposite subunit, forming an antiparallel ß sheet that constitutes part of the substrate-binding site. The sulfonate group of isethionate is stabilized by a hydrogen bond network formed by the residues Y148, R195, Q244 and a water molecule. In addition, F249 from the diagonal subunit restrains the conformation of Y148 to further stabilize the orientation of the sulfonate group. Mutation of any of these four residues into alanine resulted in a complete loss of catalytic activity for isethionate oxidation. Biochemical investigations of the substrate scope of IsfD, and bioinformatics analysis of IsfD homologs, suggest that IsfD is related to the promiscuous 3-hydroxyacid dehydrogenases with diverse metabolic functions.

Cloning and characterization of a Flavin-free oxygen-insensitive azoreductase from Klebsiella oxytoca GS-4-08.

OBJECTIVES: This study reports the identification of a new bacterial azoreductase (AzoR) from Klebsiella oxytoca GS-4-08, its heterologous production in Escherichia coli and the decolorization of azo dyes. RESULTS: The AzoR, as a flavin-free, oxygen-insensitive enzyme, has a molecular mass of 22 kDa and a high substrate specificity for methyl red (MR). In the presence of 5 mM of NADH, the enzyme activity for decolorization of 50 mg l-1 of MR reached 5.61 µmol min-1 mg protein-1. Double-reciprocal plots indicated that the NADH and MR reductions proceed by ping-pong mechanism. The calculated Vm was 8.17 µM min-1 mg protein-1, and the AzoR retained over 75% of relative activity under temperature range from 20 to 50 °C. CONCLUSION: This study for the first time identified an AzoR from Klebsiella oxytoca strain, which enabled efficient degradation of azo dyes by strain GS-4-08.

Intestinal persistence of a plasmid harbouring the OXA-48 carbapenemase gene after hospital discharge.

To study intestinal colonization by OXA-48-producing Klebsiella pneumoniae (KpO48) after hospital discharge, stool samples from 22 previously colonized subjects were collected. Time from discharge was 33-611 days, without readmissions. Eight subjects (36%) were identified as blaOXA-48 gene carriers. In all of them the hospital-acquired strain of KpO48 had been lost, and the gene was harboured by other strains of K. pneumoniae, Klebsiella oxytoca and/or Escherichia coli. Our findings show intestinal persistence for several months of a plasmid harbouring the OXA-48 carbapenemase gene in a significant proportion of individuals in the absence of antibiotic treatment.

Carbapenemase-producing Enterobacteriaceae in Mexico: report of seven non-clonal cases in a pediatric hospital.

BACKGROUND: Carbapenemases-producing Enterobacteriaceae (CPE) are a worldwide public health emergency. In Mexico, reports of CPE are limited, particularly in the pediatric population. Here, we describe the clinical, epidemiological, and molecular characteristics of seven consecutive cases in a third-level pediatric hospital in Mexico City over a four-month period during 2016. RESULTS: The Enterobacteriaceae identified were three Escherichia coli strains (producing OXA-232, NDM-1 and KPC-2), two Klebsiella pneumoniae strains (producing KPC-2 and NDM-1), one Klebsiella oxytoca strain producing OXA-48 and one Enterobacter cloacae strain producing NDM-1. The majority of patients had underlying disesases, three were immunocompromised, and three had infections involved the skin and soft tissues. Half patients died as a result of CPE infection. CONCLUSIONS: This study represents the first report of E. coli ST131-O25b clone producing NDM-1 in Latin America. In addition, this study is the first finding of K. oxytoca producing OXA-48 and E. coli producing OXA-232 in Mexican pediatric patients.

Kinetic properties analysis of beta-mannanase from Klebsiella oxytoca KUB-CW2-3 expressed in Escherichia coli.

Endo-1,4-ß-mannanase is an enzyme that can catalyze the random hydrolysis of ß-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and offers many applications in different biotechnology industries. Purification and kinetic properties of the endo-1,4-ß-mannanase from recombinant Escherichia coli strain KMAN-3 were examined. Recombinant ß-mannanase (KMAN-3) was purified 50.5 fold using Ni-NTA Agarose resin and specific activity of 11900 U/mg protein was obtained. Purified KMAN-3 showed a single band on SDS-PAGE with a molecular weight of 43 kDa. Km and Vmax values of KMAN-3 on ivory nut mannan, locust bean gum, defatted copra meal and konjac glucomannan were 243, 3.83 × 105 37 and 2.13 × 106 mg ml-1 and 2940, 61,100, 3930 and 1.56 × 1010 mg-1, respectively. Carboxymethyl cellulose was not digested by KMAN-3.

The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase.

Acireductone dioxygenase (ARD) from the methionine salvage pathway (MSP) is a unique enzyme that exhibits dual chemistry determined solely by the identity of the divalent transition-metal ion (Fe2+ or Ni2+) in the active site. The Fe2+-containing isozyme catalyzes the on-pathway reaction using substrates 1,2-dihydroxy-3-keto-5-methylthiopent-1-ene (acireductone) and dioxygen to generate formate and the ketoacid precursor of methionine, 2-keto-4-methylthiobutyrate, whereas the Ni2+-containing isozyme catalyzes an off-pathway shunt with the same substrates, generating methylthiopropionate, carbon monoxide, and formate. The dual chemistry of ARD was originally discovered in the bacterium Klebsiella oxytoca, but it has recently been shown that mammalian ARD enzymes (mouse and human) are also capable of catalyzing metal-dependent dual chemistry in vitro. This is particularly interesting, since carbon monoxide, one of the products of off-pathway reaction, has been identified as an antiapoptotic molecule in mammals. In addition, several biochemical and genetic studies have indicated an inhibitory role of human ARD in cancer. This comprehensive review describes the biochemical and structural characterization of the ARD family, the proposed experimental and theoretical approaches to establishing mechanisms for the dual chemistry, insights into the mechanism based on comparison with structurally and functionally similar enzymes, and the applications of this research to the field of artificial metalloenzymes and synthetic biology.

VIM-Klebsiella oxytoca outbreak in a Neonatal Intensive Care Unit. This time it wasn't the drain.

OBJECTIVE: We describe an outbreak of VIM-carbapenemase Klebsiella oxytoca (VIM-Kox) in a NICU. MATERIALS AND METHODS: Prospective Epidemiological Surveillance:Systematically (weekly screening cultures) or on admission, if the patient had a history of previous colonization by VIM-Kox.Clinical cultures, done if infection was suspected.Other possible microorganism sources were investigated: their mothers (rectal microbiota), milk packages and preparation apparata in the lactodietary section, echocardiagram transductors, cribs, the sinks (faucets and drains), washing bowls, etc.Molecular typing was performed using the DiversiLab (bioMérieux) system on all VIM-Kox isolated from environment or patients (one by neonate). RESULTS: We identified 20 VIM-Kox cases, the most only presented colonization, but 4 showed infection. Three of the ten sinks (drains) in our NICU, were positive for VIM-Kox. Another four drains harbored P.aeruginosa, S. maltophilia and/or Enterobacter sp. Nevertheless the VIM-Kox bacteria in the sinks (drains) were not the same as those in the patients, who showed three different strains. CONCLUSIONS: A VIM-Kox colonization or infection outbreak in a NICU is described. Rather than environment, not even drains, the source of the outbreak was other patients. The outbreak was relatively brief, as a result of the rapidness with which appropriate measures were taken and followed.

Gut eradication of VIM-1 producing ST9 Klebsiella oxytoca after fecal microbiota transplantation for diarrhea caused by a Clostridium difficile hypervirulent R027 strain.

We report the fecal carriage eradication of a VIM-1-producing ST9 Klebsiella oxytoca strain in a pluripathological 84-year-old woman after fecal microbiota transplantation to control relapsing R027 hypervirulent Clostridium difficile infections. The donor was her son, in which the absence of fecal carbapenemase-producing bacteria was corroborated.

Extended-spectrum ß-lactamase producing Enterobacteriaceae in bulk tank milk from German dairy farms.

Although the dairy farm environment is a known source of extended-spectrum ß-lactamase (ESBL)-producing bacteria, surveillance data on ESBL in the milk production chain are still scarce. This study aimed at estimating the dimensions of the problem for public health and animal welfare by surveying ESBL-producing Enterobacteriaceae in raw bulk tank milk in Germany. Samples from 866 dairy farms, comprising about 1% of the total number of dairy farms in Germany, were first screened for presence of cefotaxime-resistant bacteria by selective enrichment. Suspect colonies were identified phenotypically and further characterized by biochemical and molecular methods, including analysis of resistance genes and clonal diversity in ESBL-producing isolates. Bulk tank milk from 82 (9.5%) farms yielded Enterobacteriaceae with confirmed ESBL-production. The most frequent ESBL-producing species was Escherichia coli (75.6%), followed by Citrobacter spp. (9.6%), Enterobacter cloacae (6.1%), and Klebsiella oxytoca (3.7%), a few isolates belonged to other species within the genera Hafnia, Raoutella and Serratia. The majority of isolates (95.1%) harbored the ß-lactamase blaCTX-M gene, which has gained increased importance among ESBL-producing strains worldwide; the CTX-M group 1 was found to be the dominating (88.4%) phylogenetic group. All ESBL-positive Escherichia coli isolates were clonally heterogeneous, as determined by pulsed-field gel electrophoresis. The results from this survey demonstrate that ESBL-producing bacteria are distributed widely in the dairy farm environment in Germany. Therefore, raw milk is a potential source of exposure for the consumer, which is of increasing importance considering the trend of farmer-to-consumer direct marketing. Furthermore, dairy farm staff have an increased likelihood of exposure to ESBL-producing bacteria. Finally, ESBL-producing bacteria may also be transferred via waste milk to calves, thus further spreading antibiotic resistance in the farm environment.

Molecular characterisation of Klebsiella oxytoca strains isolated from patients with antibiotic-associated diarrhoea.

BACKGROUND AND STUDY AIM: Colitis is a common complication after treatment with antibiotics such as ß-lactams, quinolones, and aminoglycosides. Recently, Klebsiella oxytoca has been implicated in this type of diarrhoea. The prevalence and characterisations of K. oxytoca isolated from patients with antibiotic-associated diarrhoea were investigated. The K. oxytoca isolates were also tested for cytotoxin production. PATIENTS AND METHODS: This study was conducted from May 2011 to Dec 2013. Faecal samples were collected from hospitalised patients receiving antibiotic treatment. Initial cultivation was performed on specific media. The clinical isolates were confirmed by polymerase chain reaction (PCR) using the specific K. oxytoca polygalacturonase (pehX) gene. The double-disc diffusion test was used to detect extended-spectrum beta-lactamase (ESBL)-producing strains. Tracking of ESBL-encoding genes was performed via PCR. The organism was cultured on Hep-2 cell lines for cytotoxin production. RESULTS: Out of 331 samples collected from patients, 40 were confirmed molecularly to be clinical isolates of K. oxytoca. Fourteen (35%) ESBL-producing strains were isolated using the double-disc diffusion method. Among the molecularly confirmed K. oxytoca isolates, seven (17.5%) tested positive for the blaSHV gene, 12 (30%) for blaTEM, 10 (25%) for blaCTX-M, three (7.5%) for blaOXA, nine (22.5%) for blaCTX-M-15, and seven (17.5%) for blaTEM-1. Five (12%) isolates showed cytotoxin activity below 30%, 12 (30%) strains showed moderate cytotoxin activity between 30% and 60%, and 23 (58%) strains showed cytotoxin activity ⩾60%. CONCLUSIONS: The cytotoxin-producing K. oxytoca is found to be one of the causes of antibiotic-induced colitis. Discontinuing treatment and allowing normal intestinal flora to be established or prescribing appropriate medication after antibiogram can help patients with antibiotic-induced haemorrhagic colitis in a timely manner.

Molecular cloning of kman coding for mannanase from Klebsiella oxytoca KUB-CW2-3 and its hybrid mannanase characters.

Gene encoding for ß-mannanase (E.C 3.2.1.78) from Klebsiella oxytoca KUB-CW2-3 was cloned and expressed by an E. coli system resulting in 400 times higher mannanase activities than the wild type. A 3314bp DNA fragment obtained revealed an open reading frame of 1164bp, namely kman-2, which encoded for 387 amino acids with an estimated molecular weight of 43.2kDa. It belonged to the glycosyl hydrolase family 26 (GH26) exhibited low similarity of 50-71% to ß-mannanase produced by other microbial sources. Interestingly, the enzyme had a broad range of substrate specificity of homopolymer of ivory nut mannan (6%), carboxymethyl cellulose (30.6%) and avicel (5%), and heteropolymer of konjac glucomannan (100%), locust bean gum (92.6%) and copra meal (non-defatted 5.3% and defatted 7%) which would be necessary for in vivo feed digestion. The optimum temperature and pH were 30-50°C and 4-6, respectively. The enzyme was still highly active over a low temperature range of 10-40°C and over a wide pH range of 4-10. The hydrolysates of konjac glucomannan (H-KGM), locust bean gum (H-LBG) and defatted copra meal (H-DCM) composed of compounds which were different in their molecular weight range from mannobiose to mannohexaose and unknown oligosaccharides indicating the endo action of mannanase. Both H-DCM and H-LBG enhanced the growth of lactic acid bacteria and some pathogens except Escherichia coli E010 with a specific growth rate of 0.36-0.83h(-1). H-LBG was more specific to 3 species of Weissella confusa JCM 1093, Lactobacillus reuteri KUB-AC5, Lb salivarius KL-D4 and E. coli E010 while both H-KGM and H-DCM were to Lb. reuteri KUB-AC5 and Lb. johnsonii KUNN19-2. Based on the nucleotide sequence of kman-2 containing two open reading frames of 1 and 2at 5' end of the +1 and +43, respectively, removal of the first open reading frame provided the recombinant clone E. coli KMAN-3 resulting in the mature protein of mannanase composing of 345 amino acid residues confirmed by 3D structure analysis and amino acid sequence at N-terminal namely KMAN (GenBank accession number KM100456). It exhibited 10 times higher extracellular and periplasmic total activities of 17,600 and 14,800 units than E. coli KMAN-2. With its low similarity to mannanases previously proposed, wide range of homo- and hetero-polysaccharide specificity, negative effect to E. coli and most importance of high production, it would be proposed as a novel mannanase source for application in the future.

Activity of Ceftazidime-Avibactam against Extended-Spectrum- and AmpC ß-Lactamase-Producing Enterobacteriaceae Collected in the INFORM Global Surveillance Study from 2012 to 2014.

The in vitro activity of ceftazidime-avibactam was evaluated against 34,062 isolates of Enterobacteriaceae from patients with intra-abdominal, urinary tract, skin and soft-tissue, lower respiratory tract, and blood infections collected in the INFORM (International Network For Optimal Resistance Monitoring) global surveillance study (176 medical center laboratories in 39 countries) in 2012 to 2014. Overall, 99.5% of Enterobacteriaceae isolates were susceptible to ceftazidime-avibactam using FDA approved breakpoints (susceptible MIC of ≤8 µg/ml; resistant MIC of ≥16 µg/ml). For individual species of the Enterobacteriaceae, the ceftazidime-avibactam MIC inhibiting ≥90% of isolates (MIC90) ranged from 0.06 µg/ml for Proteus species to 1 µg/ml for Enterobacter spp. and Klebsiella pneumoniae Carbapenem-susceptible isolates of Escherichia coli, K. pneumoniae, Klebsiella oxytoca, and Proteus mirabilis with a confirmed extended-spectrum ß-lactamase (ESBL) phenotype, or a ceftazidime MIC of ≥16 µg/ml if the ESBL phenotype was not confirmed by clavulanic acid inhibition, were characterized further to identify the presence of specific ESBL- and plasmid-mediated AmpC ß-lactamase genes using a microarray-based assay and additional PCR assays. Ceftazidime-avibactam demonstrated potent activity against molecularly confirmed ESBL-producing (n = 5,354; MIC90, 0.5 µg/ml; 99.9% susceptible), plasmid-mediated AmpC-producing (n = 246; MIC90, 0.5 µg/ml; 100% susceptible), and ESBL- and AmpC-producing (n = 152; MIC90, 1 µg/ml; 100% susceptible) isolates of E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis We conclude that ceftazidime-avibactam demonstrates potent in vitro activity against globally collected clinical isolates of Enterobacteriaceae, including isolates producing ESBLs and AmpC ß-lactamases.

[Heterologous expression and characterization of Klebsiella oxytoca lysine decarboxylase].

Cadaverine is a biogenic amine that has the potential to become an important platform chemical for the production of industrial polymers, such as polyamides and polyurethanes. We reported here a lysine decarboxylase from Klebsiella oxytoca. The lysine decarboxylase from Klebsiella oxytoca was cloned to Escherichia coli to get the strain LN18. The specific activity of the crude protein from LN18 reached 30 000 U. The molecular weight was about 80 kDa. The optimum temperature and pH of the crude protein were 55 ℃ and 5.5 respectively. The specific activity could keep over 30% at pH 8.0 compared the one at pH 5.5, much difference from Escherichia coli lysine decarboxylase CadA. Mg²âº was positive to the specific activity, whereas Fe²âº, Zn²âº and Ca²âº were negative.

Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase.

Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.