Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate

Enterobacter cloacae CHE, a clinical strain with overproduced cephalosporinase was found to be highly resistant to the new cephalosporins, cefepime and cefpirome (MICs> or =128 microg ml(-1)). The strain was isolated from a child previously treated with cefepime. The catalytic efficiency of the purified enzyme with the third-generation cephalosporins, cefepime and cefpirome, was 10 times higher than that with the E. cloacae P99 enzyme. This was mostly due to a decrease in K(m) for these beta-lactams. The clinical isolate produced large amounts of the cephalosporinase because introduction of the ampD gene decreased ampC expression and partially restored the wild-type phenotype. Indeed, MICs of cefepime and cefpirome remained 10 times higher than those for a stable derepressed clinical isolate (OUDhyp) transformed with an ampD gene. Sequencing of the ampC gene showed that 18 nucleotides had been deleted, corresponding to the six amino acids SKVALA (residues 289--294). According to the crystal structure of P99 beta-lactamase, this deletion was located in the H-10 helix. The ampR-ampC genes from the clinical isolates CHE and OUDhyp were cloned and expressed in Escherichia coli JM101. The MICs of cefpirome and cefepime of E. coli harboring ampC and ampR genes from CHE were 100--200 times higher than those of E. coli harboring ampC and ampR genes from OUDhyp. This suggests that the deletion, confirmed by sequencing of the ampC gene, is involved in resistance to cefepime and cefpirome. However, the high level of resistance to cefepime and cefpirome observed in the E. cloacae clinical isolate was due to a combination of hyperproduction of the AmpC beta-lactamase and structural modification of the enzyme. This is the first example of an AmpC variant conferring resistance to cefepime and cefpirome, isolated as a clinical strain.     

Imipenem resistance in Enterobacter aerogenes is associated with derepression of chromosomal cephalosporinases and impaired permeability

Enterobacter aerogenes mutants with high-level resistance to imipenem were studied. They were derived from strains characterized by stable over-production of a class-I beta-lactamase. This enzyme (pI = 8.2) exhibited high affinity toward imipenem and hydrolysed the drug slowly. Imipenem-resistant mutants lacked a major 43-kDa outer membrane protein and displayed decreased permeability to cephaloridine. Introduction of a plasmid coding for the regulatory ampD gene abolished beta-lactamase production and rendered the mutants susceptible to imipenem.     

Enterobacter aerogenes OmpX, a cation-selective channel mar- and osmo-regulated

The ompX gene of Enterobacter aerogenes was cloned. Its overexpression induced a decrease in the major porin Omp36 production and consequently a beta-lactam resistance was noted. Purified outer membrane protein X (OmpX) was reconstituted into artificial membranes and formed ion channels with a conductance of 20 pS in 1 M NaCl and a cationic selectivity. Both MarA expression and high osmolarity induced a noticeable increase of the OmpX synthesis in the E. aerogenes ATCC 13048 strain. In addition, OmpX synthesis increased under conditions in which the expression of the E. aerogenes major non-specific porins, Omp36 and Omp35, decreased.     

Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae

BACKGROUND: Porins are channel-forming membrane proteins that confer solute permeability to the outer membrane of Gram-negative bacteria. In Escherichia coli, major nonspecific porins are matrix porin (OmpF) and osmoporin (OmpC), which show high sequence homology. In response to high osmolarity of the medium, OmpC is expressed at the expense of OmpF porin. Here, we study osmoporin of the pathogenic Klebsiella pneumoniae (OmpK36), which shares 87% sequence identity with E. coliOmpC in an attempt to establish why osmoporin is best suited to function at high osmotic pressure. RESULTS: The crystal structure of OmpK36 has been determined to a resolution of 3.2 A by molecular replacement with the model of OmpF. The structure of OmpK36 closely resembles that of the search model. The homotrimeric structure is composed of three hollow 16-stranded antiparallel beta barrels, each delimiting a separate pore. Most insertions and deletions with respect to OmpF are found in the loops that protrude towards the cell exterior. A characteristic ten-residue insertion in loop 4 contributes to the subunit interface. At the pore constriction, the replacement of an alanine by a tyrosine residue does not alter the pore profile of OmpK36 in comparison with OmpF because of the different course of the mainchain. Functionally, as characterized in lipid bilayers and liposomes, OmpK36 resembles OmpC with decreased conductance and increased cation selectivity in comparison with OmpF. CONCLUSIONS: The osmoporin structure suggests that not an altered pore size but an increase in charge density is the basis for the distinct physico-chemical properties of this porin that are relevant for its preferential expression at high osmotic strength.     

Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes

Chloramphenicol has been reported to act as an inducer of the multidrug resistance in Escherichia coli. A resistant variant able to grow on plates containing 64 microg/ml chloramphenicol was obtained from the Enterobacter aerogenes ATCC 13048-type strain. Chloramphenicol resistance was due to an active efflux of this antibiotic and it was associated with resistance to fluoroquinolones and tetracycline, but not to aminoglycoside or beta-lactam antibiotics. MDR in the chloramphenicol-resistant variant is linked to the overexpression of the major AcrAB-TolC efflux system. This overexpression seems unrelated to the global Mar and the local AcrR regulatory pathways.     

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.     

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.     

近三年产气肠杆菌临床感染情况分析

目的 监测嵊州市人民医院近三年产气肠杆菌对常用抗生素耐药情况。方法 对嵊州市人民医院2013年至2015年间收集的产气肠杆菌临床科室分布情况进行统计,并做临床常用抗生素耐药性分析,用WHONET 5.4软件进行统计学分析。结果 分离得到的产气肠杆菌主要来源于神经外科、重症医学科和呼吸内科的痰液和尿液标本。药敏结果显示其对第一、二代头孢类抗生素耐药率普遍较高：对头孢唑啉几乎全耐药,对头孢呋辛耐药率约为55%;第四代的头孢吡肟能明显抑制产气肠杆菌生长,2013年到2015年对头孢吡肟耐药率分别为8.18%、9.14%和10.74%;对氨基糖苷类抗生素庆大霉素和丁胺卡那,以及碳青霉烯类抗生素亚胺培南和美罗培南具有很高的敏感性。结论 嵊州市人民医院院感科近年间通过对产气肠杆菌临床用药的合理监测,及时向临床医生反馈微生物实验室的耐药结果,避免了抗生素滥用,使得其对抗生素耐药率未出现明显提高。氨基糖苷类抗生素和碳青霉烯类抗生素对其仍具有很高的疗效,应继续通过药敏试验加强对其耐药性监测,指导临床合理用药。     

Energy-dependent inactivation of citrate lyase in Enterobacter aerogenes.

Enterobacter aerogenes was grown in continous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation.     

Growth of Enterobacter aerogenes in a chemostat with double nutrient limitations.

The behavior of Enterobacter aerogenes during growth in chemostats limited by single and double nutrient restrictions was examined. On the assumption that different essential nutrients act to limit growth in different ways, we selected pairs of nutrients likely to affect different aspects of metabolism. Results show that macromolecular cell composition can be controlled by using more than one nutrient restriction. The polysaccharide content of the cells is readily manipulated by the ratio of carbon to nitrogen in the inlet nutrients. Also, at low dilution rates, ratios of protein to ribonucleic acid are dependent on the ratio of phosphate to nitrogen in the input nutrients. An examination of both acetic acid and metabolite production (as measured by ultraviolet absorbance of culture filtrates) showed that accumulation of these products was dependent on both dilution rate and type of nutrient limitation(s). These results were examined in terms of the problems of translation of batch to continuous culture processes and the use of selected nutrient limitations to control noncellular product formation.     

Purification and properties of a phosphohydrolase from Enterobacter aerogenes.

A phosphohydrolase from Enterobacter aerogenes which hydrolyzes phosphate mono- and diesters has been purified approximately 50-fold to apparent homoeneity and crystallized. The enzyme is produced when the bacteria utilize phosphate diesters as sole phosphorus source. From sedimentation equilibrium experiments the molecular weight of the native enzyme is 173,000; from sodium dodecyl sulfate polyacrylamide gel electrophoresis the subunit molecular weight is 29,000, indicating that the enzyme is hexameric. The hydrolytic activity of the enzyme using both mono- and diesters is maximal at pH 5; THE Km of the enzyme for bis-p-nitrophenyl phosphate is constant from pH 5 to 8.5 whereas that for p-nitrophenyl phosphate increases about 40-fold as the pH increases over the same range. The phosphodiesterase activity is not inhibited by chelating agents but is inhibited by several divalent metal ions. 31-P NMR spectroscopy was used to identify the hydrolysis products of glycoside cyclic phosphates. The enzyme-catalyzed hydrolysis of methyl beta-D-ribofuranoside cyclic 3:5-phosphate yields exclusively the 5-phosphate whereas that of adenosine 3:5-monophosphate yields a 4:1 mixture of 3- and 5- AMP.     

In vivo modification of porin activity conferring antibiotic resistance to Enterobacter aerogenes

Cephalosporins are widely used in chemotherapy of bacterial infections and resistance mechanisms seriously impair their antibacterial activity. Several resistant strains of Enterobacter aerogenes, a frequently isolated nosocomial pathogen, were analyzed. One isolate exhibited a strong modification of the porin antigenic pattern, especially with an immunological probe directed against an epitope located inside the pore lumen. A strong decrease of cefepime uptake was evidenced for this isolate, similarly to ones observed for porin-deficient strains: these kinetics show a serious alteration of the channel properties which may support cephalosporin resistance. This is the first E. aerogenes isolate using such adaptive response which defines an original enterobacterial answer to cephalosporin.     

产气肠杆菌fhlA基因克隆、表达及重组菌株产氢量

【目的】本研究以产氢细菌产气肠杆菌Enterobacter aerogenes ATCC13408为研究对象,克隆甲酸-氢裂解酶(formate hydrogen lyase,FHL)系统的转录激活蛋白FHL activator(fhlA)基因,构建过表达重组菌株,以提高菌株产氢效率。【方法】利用简并引物和Genome walking技术,克隆fhlA的全长基因,将该基因连接到改造质粒pGEX-4T-2-Cat中,电击转化得到重组菌株,用厌氧发酵方法测定重组细菌的产氢量。【结果】E.aerogenes ATCC13408fhlA ORF全长2073bp,编码一个含690个氨基酸残基的蛋白(GenBank accessionGU188474)。SDS-PAGE和Western blot分析证明fhlA基因在重组菌中得到了融合表达。对重组后菌株的产氢量进行了测定,结果表明:底物产氢潜力由原来的1.23±0.08mol H2/mol葡萄糖提高到了1.48±0.04mol H2/mol葡萄糖,提高了20.36%。【结论】本研究首次克隆了E.aerogenes ATCC13408的fhlA基因,并将该基因在原菌中过量表达。重组后菌株的产氢量得到显著提高,为进一步研究和开发利用E.aerogenes ATCC13408的fhlA基因提供了基础。     

Heterologous expression of a hydrogenase gene in Enterobacter aerogenes to enhance hydrogen gas production

The hydrogenase gene from Enterobacter cloacae (IIT-BT 08) was amplified and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-4T-2-Cat/hydA). The recombinant plasmid was transformed into a hydrogen-producing strain of Enterobacter aerogenes (ATCC13408). SDS–PAGE and western blot analysis confirmed the successful expression of the GST-tagged hydA protein. Anaerobic fermentation for the production of hydrogen from glucose was investigated using E. aerogenes ATCC13408 and the recombinant strain. The results showed that the hydrogen yield markedly increased, from 442.82 ± 22.61 ml/g glucose in the ATCC13408 strain to 864.02 ± 36.8 ml/g glucose in the recombinant. The maximum rate of hydrogen production was found to be 53.49 ± 3.34 ml l⁻¹ h⁻¹ using 1% (w/v) glucose as the substrate at pH 6.0 and a reaction temperature of 37°C.     

Biotransformation of Ferulic Acid to 4-Vinylguaiacol by Enterobacter soli and E. aerogenes

We investigated the conversion of ferulic acid to 4-vinylguaiacol (4-VG), vanillin, vanillyl alcohol, and vanillic acid by five Enterobacter strains. These high-value chemicals are usually synthesized by chemical methods but biological synthesis adds market value. Ferulic acid, a relatively inexpensive component of agricultural crops, is plentiful in corn hulls, cereal bran, and sugar-beet pulp. Two Enterobacter strains, E. soli, and E. aerogenes, accumulated 550–600?ppm amounts of 4-VG when grown in media containing 1,000?ppm ferulic acid; no accumulations were observed with the other strains. Decreasing the amount of ferulic acid present in the media increased the conversion efficiency. When ferulic acid was supplied in 500, 250, or 125?ppm amounts E. aerogenes converted ~72?% of the ferulic acid present to 4-VG while E. soli converted ~100?% of the ferulic acid to 4-VG when supplied with 250 or 125?ppm amounts of ferulic acid. Also, lowering the pH improved the conversion efficiency. At pH 5.0 E. aerogenes converted ~84?% and E. soli converted ~100?% of 1,000?ppm ferulic acid to 4-VG. Only small, 1–5?ppm, accumulations of vanillin, vanillyl alcohol, and vanillic acid were observed. E. soli has a putative phenolic acid decarboxylase (PAD) that is 168 amino acids long and is similar to PADs in other enterobacteriales; this protein is likely involved in the bioconversion of ferulic acid to 4-VG. E. soli or E. aerogenes might be useful as a means of biotransforming ferulic acid to 4-VG.     

丁二酮高产菌株的选育及发酵动力学分析

目的：筛选出稳定的高产丁二酮突变菌株,并对其进行发酵动力学分析。方法：以原养型产气肠杆菌（Enterobacter aero-genes编号CICC 10293）为出发菌株,采用紫外线诱变,并结合亮氨酸平板和丁二酮平板筛选方法,获得耐高浓度底物——葡萄糖的高产丁二酮突变菌株,定名为UV-3,利用气相色谱测定代谢产物量,考察诱变前后菌株代谢途径中碳源的流向。基于Logistic和Luedeking-Piret方程,建立突变株UV-3细胞生长动力学、底物消耗动力学、丁二酮生成动力学模型,确定动力学方程。结果：突变株UV-3的丁二酮产量提高18.7倍,达到1.045g.L-1,乙偶姻产量降低48.4%,乙醇产量降低71.4%,乙酸产量提高34.6%,且遗传性质稳定。用实验数据对各种动力学模型进行了验证,拟合度均为98.5%以上。结论：紫外线是一种操作方便,效果良好的诱变方式。绘制了突变株UV-3的发酵曲线,建立了动力学模型,为优化丁二酮的生产工艺奠定一定的理论基础。     

Purification, characterization and some properties of diacetyl(acetoin) reductase from Enterobacter aerogenes

A new method, faster, milder and more efficient than the one previously described [Bryn, K., Hetland, O. & Stormer, F. C. (1971) Eur. J. Biochem, 18, 116-119], for purification of diacetyl(acetoin) reductase from Enterobacter aerogenes is proposed. The experiments carried out with the electrophoretically pure preparations obtained by this procedure show that the enzyme (a) produces L-glycols from the corresponding L-alpha-hydroxycarbonyls by reversible reduction of their oxo groups and also reduces the oxo group of uncharged alpha-dicarbonyls converting them into L-alpha-hydroxycarbonyls, and (b) is specific for NAD. This is a new enzyme for which we suggest the systematic name of L-glycol: NAD+ oxidoreductase and the recommended name of L-glycol dehydrogenase(NAD). The molecular mass, pI, affinity for substrates and pH profiles of this enzyme are also described.