Increased community-acquired upper urinary tract infections caused by extended-spectrum beta-lactamase-producing Escherichia coli in children and the efficacy of flomoxef and cefmetazole.

BACKGROUND: Urinary tract infections caused by extended-spectrum beta-lactamase-producing bacteria are increasing worldwide. At our hospital, the number of pediatric patients hospitalized because of an upper urinary tract infection has dramatically increased since 2016. In total, 60.5% of urinary tract infections are caused by extended-spectrum beta-lactamase-producing Escherichia coli. Such a high prevalence of extended-spectrum beta-lactamase-producing E. coli has not been detected previously in Japan. Therefore, we evaluated the clinical and bacteriologic characteristics and efficacy of antibiotics against upper urinary tract infections caused by E. coli in children. METHODS: This retrospective study surveyed 152 patients who were hospitalized in the pediatric department of Shimane Prefectural Central Hospital because of upper urinary tract infections caused by E. coli. Medical records were reviewed to examine patient characteristics. O antigens, antibiotic susceptibility, gene typing, and pulse-field gel electrophoresis were studied at the Shimane Prefectural Institute of Public Health and Environmental Science. RESULTS: Urine sample analyses showed extended-spectrum beta-lactamase types such as CTX-M-9 and plural virulence genes. We changed the primary antibiotic treatment to flomoxef or cefmetazole to treat upper urinary tract infections caused by Gram-negative bacilli. After changing treatment, the time to fever alleviation was significantly shortened. CONCLUSION: Extended-spectrum beta-lactamase-producing E. coli should be suspected in community-acquired upper urinary tract infections. Therefore, when treating patients, it is necessary to focus on antibiotic susceptibility and the prevalence of extended-spectrum beta-lactamase-producing bacteria found in each area. Flomoxef and cefmetazole are useful primary treatments for upper urinary tract infections caused by extended-spectrum beta-lactamase-producing E. coli.

Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission.

Antimicrobial resistance through extended-spectrum beta-lactamases (ESBLs) and transferable (plasmid-encoded) cephamycinases (pAmpCs) represents an increasing problem in human and veterinary medicine. The presence of ESBL-/pAmpC-producing commensal enterobacteria in farm animals, such as broiler chickens, is considered one possible source of food contamination and could therefore also be relevant for human colonization. Studies on transmission routes along the broiler production chain showed that 1-day-old hatchlings are already affected. In this study, ESBL-/pAmpC-positive broiler parent flocks and their corresponding eggs, as well as various environmental and air samples from the hatchery, were analyzed. The eggs were investigated concerning ESBL-/pAmpC-producing enterobacteria on the outer eggshell surface (before/after disinfection), the inner eggshell surface, and the egg content. Isolates were analyzed concerning their species, their phylogroup in the case of Escherichia coli strains, the respective resistance genes, and the phenotypical antibiotic resistance. Of the tested eggs, 0.9% (n = 560) were contaminated on their outer shell surface. Further analyses using pulsed-field gel electrophoresis showed a relationship of these strains to those isolated from the corresponding parent flocks, which demonstrates a pseudo-vertical transfer of ESBL-/pAmpC-producing enterobacteria into the hatchery. Resistant enterobacteria were also found in environmental samples from the hatchery, such as dust or surfaces which could pose as a possible contamination source for the hatchlings. All 1-day-old chicks tested negative directly after hatching. The results show a possible entry of ESBL-/pAmpC-producing enterobacteria from the parent flocks into the hatchery; however, the impact of the hatchery on colonization of the hatchlings seems to be low. IMPORTANCE: ESBL-/pAmpC-producing enterobacteria occur frequently in broiler-fattening farms. Recent studies investigated the prevalence and possible transmission route of these bacteria in the broiler production chain. It seemed very likely that the hatcheries play an important role in transmission and/or contamination events. There are only few data on transmission investigations from a grandparent or parent flock to their offspring. However, reliable data on direct or indirect vertical transmission events in the hatchery are not available. Therefore, we conducted our study and intensively investigated the broiler hatching eggs from ESBL-/pAmpC-positive broiler parent flocks as well as the hatchlings and the environment of the hatchery.

Investigation of acid phosphatase variants for the synthesis of phosphate monoesters.

The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;114: 2187-2195. © 2017 Wiley Periodicals, Inc.

Bioanalysis for biocatalysis: multiplexed capillary electrophoresis-mass spectrometry assay for aminotransferase substrate discovery and specificity profiling.

In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections-incubation-separation-enzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion. We used the MINISEP-MS assay to profile the substrate specificity of three aminotransferases (E. coli aspartate aminotransferase, E. coli branched-chain amino acid aminotransferase, and Bacillus sp. YM-1 D-amino acid aminotransferase) for 33 potential amino acid substrates and to measure steady-state kinetics. Using MINISEP-MS, we were able to recapitulate the known substrate specificities and to discover new amino acid substrates for these industrially relevant enzymes. Additionally, we were able to measure the apparent K(M) and k(cat) parameters for amino acid donor substrates of these aminotransferases. Because of its many advantages, the MINISEP-MS assay has the potential of becoming a useful tool for researchers aiming to identify or create novel enzymes for specific biocatalytic applications.

Synthesis and evaluation of malonate-based inhibitors of phosphosugar-metabolizing enzymes: class II fructose-1,6-bis-phosphate aldolases, type I phosphomannose isomerase, and phosphoglucose isomerase.

In the design of inhibitors of phosphosugar metabolizing enzymes and receptors with therapeutic interest, malonate has been reported in a number of cases as a good and hydrolytically-stable surrogate of the phosphate group, since both functions are dianionic at physiological pH and of comparable size. We have investigated a series of malonate-based mimics of the best known phosphate inhibitors of class II (zinc) fructose-1,6-bis-phosphate aldolases (FBAs) (e.g., from Mycobacterium tuberculosis), type I (zinc) phosphomannose isomerase (PMI) from Escherichia coli, and phosphoglucose isomerase (PGI) from yeast. In the case of FBAs, replacement of one phosphate by one malonate on a bis-phosphorylated inhibitor (1) led to a new compound (4) still showing a strong inhibition (K(i) in the nM range) and class II versus class I selectivity (up to 8×10(4)). Replacement of the other phosphate however strongly affected binding efficiency and selectivity. In the case of PGI and PMI, 5-deoxy-5-malonate-D-arabinonohydroxamic acid (8) yielded a strong decrease in binding affinities when compared to its phosphorylated parent compound 5-phospho-D-arabinonohydroxamic acid (2). Analysis of the deposited 3D structures of the kinetically evaluated enzymes complexed to the phosphate-based inhibitors indicate that malonate could be a good phosphate surrogate only if phosphate is not tightly bound at the enzyme active site, such as in position 7 of compound 1 for FBAs. These observations are of importance for further design of inhibitors of phosphorylated-compounds metabolizing enzymes with therapeutic interest.

Computational Elucidation of Phylogenetic, Functional and Structural Features of Methioninase from Pseudomonas, Escherichia, Clostridium and Citrobacter Strains.

BACKGROUND: L-Methioninase (EC 4.4.1.11; MGL) is a pyridoxal phosphate (PLP)-dependent enzyme that is produced by a variety of bacteria, fungi, and plants. L-methioninase, especially from Pseudomonas and Citrobacter sp., is considered as the efficient therapeutic enzyme, particularly in cancers such as glioblastomas, medulloblastoma, and neuroblastoma that are more sensitive to methionine starvation. OBJECTIVE: The low stability is one of the main drawbacks of the enzyme; in this regard, in the current study, different features of the enzyme, including phylogenetic, functional, and structural from Pseudomonas, Escherichia, Clostridium, and Citrobacter strains were evaluated to find the best bacterial L-Methioninase. METHODS: After the initial screening of L-Methioninase sequences from the above-mentioned bacterial strains, the three-dimensional structures of enzymes from Escherichia fergusonii, Pseudomonas fluorescens, and Clostridium homopropionicum were determined through homology modeling via GalaxyTBM server and refined by GalaxyRefine server. RESULTS AND CONCLUSION: Afterwards, PROCHECK, verify 3D, and ERRAT servers were used for verification of the obtained models. Moreover, antigenicity, allergenicity, and physico-chemical analysis of enzymes were also carried out. In order to get insight into the interaction of the enzyme with other proteins, the STRING server was used. The secondary structure of the enzyme is mainly composed of random coils and alpha-helices. However, these outcomes should further be validated by wet-lab investigations.

First description of an extended-spectrum-beta-lactamase-producing multidrug-resistant Escherichia fergusonii strain in a patient with cystitis.

Extended-spectrum-beta-lactamase (ESBL)-producing organisms have captured the attention of clinicians and laboratorians and are agents of nosocomial and community onset infections (J. D. Pitout and K. B. Laupland, Lancet Infect. Dis. 8:159-166, 2008). ESBLs in many enterobacteriaceae and in nonfermenting Gram-negative organisms have been described (K. Bush and G. A. Jacoby, Antimicrob. Agents Chemother. 54:969-976, 2010). We present the first case of a clinical isolate of multidrug-resistant Escherichia fergusonii expressing an extended-spectrum-beta-lactamase (ESBL).

A simple selection strategy for evolving highly efficient enzymes.

Combining tunable transcription with an enzyme-degradation tag affords an effective means to reduce intracellular enzyme concentrations from high to very low levels. Such fine-tuned control allows selection pressure to be systematically increased in directed-evolution experiments. This facilitates identification of mutants with wild-type activity, as shown here for an engineered chorismate mutase. Numerous selection formats and cell-based screening methodologies may benefit from the large dynamic range afforded by this easily implemented strategy.

Asymmetric dihydroxylation of aryl olefins by sequential enantioselective epoxidation and regioselective hydrolysis with tandem biocatalysts.

Chiral aryl vicinal diols were obtained in high ee and yield by asymmetric dihydroxylation of aryl olefins with tandem biocatalysts: one contains an enantioselective styrene monooxygenase, and the other contains a regioselective epoxide hydrolase.

An orthogonal purification strategy for isolating crosslinked domains of modular synthases.

Chemo-enzymatic methods for covalently crosslinking carrier proteins with partner enzymes within modular synthases hold promise for elucidating and engineering metabolic pathways. Our efforts to crystallize the ACP-KS complexes of fatty acid synthases have been complicated by difficulties in the purification of the crosslinked complex from the other proteins in the reaction. Here we present a solution that employs an orthogonal purification strategy to achieve the quantity and level of purity necessary for further studies of this complex.

Production of chitinase from Escherichia fergusonii, chitosanase from Chryseobacterium indologenes, Comamonas koreensis and its application in N-acetylglucosamine production.

The important platform polysaccharide N-acetylglucosamine (GlcNAc) has great potential to be used in the fields of food, cosmetics, agricultural, pharmaceutical, medicine and biotechnology. This GlcNAc is being produced by traditional methods of environment-unfriendly chemical digestion with strong acids. Therefore, researchers have been paying more attention to enzymatic hydrolysis process for the production of GlcNAc. Hence, in this study, we isolated novel chitinase (Escherichia fergusonii) and chitosanase (Chryseobacterium indologenes, Comamonas koreensis) producing strains from Korean native calves feces, and developed the potential of an eco-friendly microbial progression for GlcNAc production from swollen chitin and chitosan by enzymatic degradation. Maximum chitinase (7.24±0.07U/ml) and chitosanase (8.42±0.09, 8.51±0.25U/ml) enzyme activity were reached in submerged fermentation at an optimal pH of 7.0 and 30°C. In this study, sucrose, yeast extract, (NH4)2SO4, and NaCl were found to be the potential enhancers of exo-chitinase activity and glucose, corn flour, yeast extract, soybean flour, (NH4)2SO4, NH4Cl and K2HPO4 were found to be the potential activator for exo-chitosanase activity. Optimum concentrations of the carbon sources for enhanced chitinase activity were 9.91, 3.21, 9.86, 1.66U/ml and chitosanase activity were 1.63, 1.13, 2.28, 3.71, 9.02, 4.93, and 2.14U/ml. These enzymes efficiently hydrolyzed swollen chitin and chitosan to N-acetylglucosamine were characterized by thin layer chromatography and were further confirmed by high-pressure liquid chromatography. From a commercial perspective, we isolated, optimized and characterized exochitinase from Escherichia fergusonii (HANDI 110) and chitosanase from Chryseobacterium indologenes (HANYOO), and Comamonas koreensis (HANWOO) for the large-scale production of GlcNAc facilitating its potential use in industrial applications.

Construction of an Escherichia-Pseudomonas shuttle vector containing an aminoglycoside phosphotransferase gene and a lacZ'' Gene for alpha-complementation.

A new 4.87 kb Escherichia-Pseudomonas shuttle vector has been constructed by inserting a 1.27 kb DNA fragment with a replication origin of a Pseudomonas plasmid pRO1614 into the 3.6 kb E. coli plasmid pBGS18. This vector, designated pJH1, contains an aminoglycoside phosphotransferase gene (aph) from Tn903, a lacZ'' gene for alpha-complementation and a versatile multiple cloning site possessing unique restriction sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, BspMI, PstI, SphI, and HindIII. When pJH1 was transformed into E. coli DH5alpha and into P. putida HK-6, it was episomally and stably maintained in both strains. In addition, the enhanced green fluorescent protein (EGFP) gene which was transcriptionally cloned into pJH1 rendered E. coli cells fluorescence when its transformants were illuminated at 488 nm.

Identifying the methyltransferases for m(5)U747 and m(5)U1939 in 23S rRNA using MALDI mass spectrometry.

There are three sites of m(5)U modification in Escherichia coli stable RNAs: one at the invariant tRNA position U54 and two in 23S rRNA at the phylogenetically conserved positions U747 and U1939. Each of these sites is modified by its own methyltransferase, and the tRNA methyltransferase, TrmA, is well-characterised. Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA, and here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry. A purified recombinant version of YgcA retains its activity and specificity, and methylates U1939 in an RNA transcript in vitro. We were unable to generate a recombinant version of YbjF that retained in vitro activity, so the function of this enzyme was defined in vivo by engineering a ybjF knockout strain. Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification. With this report, the functions of all the E.coli m(5)U RNA methyltransferases are identified, and a more appropriate designation for YbjF would be RumB (RNA uridine methyltransferases B), in line with the recent nomenclature change for YgcA (now RumA).

Tryptophanase of fecal flora as a possible factor in the etiology of colon cancer.

Twenty-three strains of intestinal anaerobes obtained from two laboratories were examined for indole production from tryptophan. Among the 23 isolates tested, three of Bacteroides fragilis thetaiotaomicron and one Citrobacter sp. were indole positive. The tryptophanase of the indole-positive strains of intestinal anaerobes was inducible by tryptophan and was susceptible to glucose repression. The products of tryptophanase activity were formed in stoichiometric amounts by dialyzed, freshly prepared extracts. The tryptophan concentration and tryptophanase activity in feces from rats on an all-meat diet were significantly higher than those in feces from rats on a normal diet. The results indicated that the higher tryptophanase activity in the feces of rats fed an all-meat diet is due to the inducibility of this enzyme by tryptophan and is not due to any inhibitor in the feces of rats on a normal diet. The results also suggested that a population with a diet rich in meat has a greater chance for exposure to possible carcinogens such as indole and other tryptophan metabolites. This agrees with the hypothesis, based on epidemiologic data, that a high intake of meat may be related to the development of colon cancer in man.

A Cell-Cell Communication-Based Screening System for Novel Microbes with Target Enzyme Activities.

The development of synthetic biological devices has increased rapidly in recent years and the practical benefits of such biological devices are becoming increasingly clear. Here, we further improved the design of a previously reported high-throughput genetic enzyme screening system by investigating device-compatible biological components and phenol-mediated cell-cell communication, both of which increased the efficiency and practicality of the screening device without requiring the use of flow cytometry analysis. A sensor cell was designed to detect novel microbes with target enzyme activities on solid media by forming clear, circular colonies with fluorescence around the unknown microbes producing target enzymes. This mechanism of detection was enabled by the combination of pre-effector phenolic substrate treatment in the presence of target enzyme-producing microbes and control of the growth and fluorescence of remote sensor cells via phenol-mediated cell-cell communication. The sensor cells were applied to screen soil bacteria with phosphatase activity using phenyl phosphate as phenolic substrates. The sensor cells facilitated successful visualization of phosphatase activity in unknown microbes, which were identified by 16S rRNA analysis. Enzyme activity assays confirmed that the proposed screening technique was able to find 23 positive clones out of 33 selected colonies. Since many natural enzymatic reactions produce phenolic compounds from phenol-derived substrates, we anticipate that the proposed technique may have broad applications in the assessment and screening of novel microbes with target enzymes of interest. This method also can provide insights into the identification of novel enzymes for which screening assays are not yet available.

The apparent conservation of the internal low efficiency promoter of the tryptophan operons of several species of Enterobacteriaceae.

The specific activities of the tryptophan biosynthetic enzymes were determined for eleven different species of Enterobacteriaceae grown under repressing and non-repressing conditions. In all the bacteria examined the multipliciy of derepression for the first two enzymes of the pathway was at least twofold greater than the multiplicity of derepression for the last three enzymes.

Cloning and expression of a Pseudomonas 3 alpha-hydroxysteroid dehydrogenase-encoding gene in Escherichia coli.

A bacterial strain, B-0831, which produced 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) was isolated and identified as belonging to the genus, Pseudomonas. Molecular weights of the purified 3 alpha HSD, determined by SDS-PAGE and by chromatography on Sephacryl S-200, were about 25 and 50 kDa, respectively. A genomic library of Pseudomonas sp. B-0831, prepared in the plasmid vector pACYC184, was screened with probes based on the amino acid (aa) sequence of the protein to obtain the plasmid, p3 alpha HSD1, identified by hybridization with the probes, that contained a 2.4-kb insert from Pseudomonas DNA. When the 1.4-kb SphI fragment of p3 alpha HSD1 was inserted into the vector, pUC118, and introduced into Escherichia coli DH1 under the control of lacZ promoter in the vector, the transformants produced 200-fold more 3 alpha HSD intracellularly than Pseudomonas sp. B-0831. Sequence analysis of the 3 alpha HSD gene revealed that an ORF encoding 3 alpha HSD consists of 254 aa, with a calculated M(r) of 25,761, suggesting that the enzyme consists of homodimer subunits.

Shigella apyrase--a novel variant of bacterial acid phosphatases?

A virulence-associated ATP diphosphohydrolase activity in the periplasm of Shigella, identified as apyrase, was found to be markedly similar to bacterial non-specific acid phosphatases in primary structure. When the Shigella apyrase sequence was threaded in to the recently published 3D structure of the highly similar (73%) Escherichia blattae acid phosphatase it was found to have a highly overlapping 3D structure. Our analysis, which included assays for phosphatase, haloperoxidase and catalase activities, led us to hypothesize that Shigella apyrase might belong to a new class of pyrophosphatase originating as one more variant in the family of bacterial non-specific acid phosphatases. It revealed interesting structure-function relationships and probable roles relevant to pathogenesis.

Substrate specificity of endo-beta-galactosidases from Flavobacterium keratolyticus and Escherichia freundii is different from that of Pseudomonas sp.

The substrate specificity of endo-beta-galactosidase of Pseudomonas sp. was found to differ from that of Flavobacterium keratolyticus or Escherichia freundii, based on the following experimental results. The endo-beta-galactosidases from these three bacteria released 6-O-sulfo-GlcNAc beta 1-3Gal as one of the major products from keratan sulfates from different sources. In addition to the sulfated disaccharide, Flavobacterium and Escherichia enzymes produced GlcNAc beta 1-3Gal, which is also an integral repeating unit of keratan sulfate, whereas the Pseudomonas enzyme did not release any non-sulfated disaccharide. Tetrasaccharides were prepared from the teleost skin keratan sulfate by digestion with Pseudomonas enzyme followed by gel filtration on Sephadex G-50 chromatography. A part of the tetrasaccharide fraction was hydrolyzed by Flavobacterium enzyme to produce 6-O-sulfo-GlcNAc beta 1-3Gal and GlcNAc beta 1-3Gal, whereas the fraction was completely resistant to retreatment with the Pseudomonas enzyme. Endo-beta-galactosidases from F. keratolyticus and E. freundii hydrolyzed the internal beta-1,4-galactosyl linkage of various neolacto-type glycosphingolipids to produce glucosylceramides. However, these glycosphingolipids were completely resistant to the Pseudomonas enzyme. These findings clearly show that the sulfation on the N-acetylglucosamine adjacent to galactose in the lactosaminoglycans is essential for expression of the Pseudomonas enzyme, but not for that of the Flavobacterium or Escherichia enzyme.

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii, Escherichia fergusonii and Enterobacter cancerogenus.

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.