Epidemiologic and molecular characterization of β-lactamase-producing multidrug-resistant uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients

Uropathogenic Escherichia coli (UPECs) are the predominant cause of asymptomatic bacteriuria (ABU) and symptomatic UTI. In this study, multidrug-resistant (MDR) ABU-UPECs from hospitalized patients of Kolkata, India, were characterized with respect to their ESBL phenotype, acquisition of β-lactamase genes, mobile genetic elements (MGEs), phylotype property, ERIC-PCR profile, sequence types (STs), clonal complexes (CCs) and evolutionary and quantitative relationships and compared to the symptomatic ones to understand their epidemiology and evolutionary origin. Statistically significant incidence of ESBL producers, β-lactamase genes, MGEs and novel phylotype property (NPP) among ABU-UPECs similar to the symptomatic ones indicated the probable incidence of chromosomal plasticity on resistance gene acquisition through MGEs due to indiscriminate drug usage. ERIC-PCR typing and MLST analysis showed clonal heterogeneity and predominance of ST940 (CC448) among asymptomatic isolates akin to symptomatic ones along with the evidence of zoonotic transmissions. Minimum spanning tree analysis showed a close association between ABU-UPEC with known and unidentified STs having NPPs with isolates that belonged to phylogroups clade I, D, and B2. This is the first study that reported the occurrence of MGEs and NPPs among ABU-UPECs with the predominance of ESBL production which displayed the deleterious effect of MDR among this pathogen demanding alternative therapeutic interventions. Moreover, this study for the first time attempted to introduce a new approach to ascertain the phylotype property of unassigned UPECs. Withal, increased recognition, proper understanding and characterization of ABU-UPECs with the implementation of appropriate therapeutic measures against them when necessary are the need of the era which otherwise might lead to serious complications in the vulnerable population.

     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K_m and V_max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert^® (Colilert), Readycult^® Coliforms 100 (Readycult), Chromocult^® Coliform agar ES (Chromocult), and MI agar (MI) are β-galactosidase and β-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect β-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect β-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected β-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected β-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect β-glucuronidase production and MI the weakest to detect β-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.     

Induction of amylomaltase and β-galactosidase in different growth phases of Escherichia coli

The ability to induce amylomaltase and β-galactosidase in E.coli depends on the growth phase of the culture. The optimum for amylomaltase is at the beginning of the stationary phase of growth while that for β-galactosidase is in the logarithmic phase.     

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.     

Pool sizes of metabolic intermediates and their relation to glucose repression of β-galactosidase synthesis in Escherichia coli

1. The intermediary metabolism of two strains of Escherichia coli has been examined. One strain (Q22) exhibits acute transient repression of β-galactosidase synthesis when glucose is supplied to cells growing on glycerol; the other strain (W3110) does not. The two strains do not differ genetically in their lac operons. 2. Strain Q22 uses about twice as much glucose as strain W3110 per unit of cell mass produced. 3. Pentose phosphate-cycle activity in the presence of glucose is much stronger in strain Q22 than in strain W3110. 4. In strain Q22 the pool sizes of glucose 6-phosphate, 6-phosphogluconate, fructose 1,6-diphosphate and NADPH increase when glucose is added to cells growing on glycerol, and β-galactosidase synthesis is severely inhibited. After about 1hr. the synthesis of β-galactosidase is partly resumed, and the pool sizes of the four compounds fall. ATP, NADH and several other phosphorylated compounds show no concentration changes. 5. These concentration changes do not occur in strain W3110, in which β-galactosidase synthesis is only rather weakly repressed by glucose. 6. It is suggested that repression of enzyme synthesis by glucose requires the rapid operation of the pentose phosphate cycle, and is mediated by one of the four substances whose concentration rises and later falls in strain Q22. A definite choice of effector from among these four possibilities cannot at present be made.     

Mechanisms of resistance to cephalosporin and emergence of O25b-ST131 clone harboring CTX-M-27 β-lactamase in extraintestinal pathogenic Escherichia coli from dogs and cats in Japan

Thirty-three cefazolin-resistant extraintestinal pathogenic Escherichia coli strains from companion animals were screened for bla(CMY-1) , bla(CMY-2) , bla(SHV) , bla(TEM) , and bla(CTX-M) genes. Extended-spectrum β-lactamase-producing strains were further characterized by O serotyping and multilocus sequence typing. It was found that 20 and 17 isolates harbored TEM-1 and CMY-2 β-lactamases, respectively, and 13 isolates harbored both β-lactamases. One isolate harbored DHA-1 β-lactamase. Eleven isolates were found to possess CTX-M β-lactamases (CTX-M-27 [n= 6], CTX-M-14 [n= 3], CTX-M-15 [n= 1], and CTX-M-55 [n= 1]). Of 11 CTX-M-positive strains, four strains were O25b-ST131 clones harboring CTX-M-27, and the remaining seven strains belonged to O6-ST127, ONT-ST354, O159-ST539, O1-ST648, O8-ST1642, O25b-ST2042, and ONT-ST2178.     

Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli

Bifidobacterium infantis HL96 produces three -galactosidases (-gal I, II and III). A genomic bank of B. infantis was constructed in E. coli by using pBR322 as a cloning vector. Two E. coli transformants, BIG1 and BIG4, possessing -galactosidase activity, were selected from X-gal plates. They contained two different recombinant plasmids with insert DNA fragments of approx. 4.6 and 4.4 kb, respectively. The restriction maps of pBIG1 and pBIG4 were constructed. -Galactosidases from crude cell-free extracts of B. infantis and of two E. coli recombinants were analyzed by native PAGE and characterized by activity staining. pBIG1 and pBIG4 were shown to carry the genes for -gal I and -gal III, respectively. Optimal pH and temperature for hydrolytic activity of the native enzyme were 7.5 and 40°C, while those for recombinant BIG1 and BIG4 were 7.5, 50°C and 8.0, 40°C, respectively. © Rapid Science Ltd. 1998     

Disulfide bond formation and activation of Escherichia coli β-galactosidase under oxidizing conditions

Escherichia coli β-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version of Escherichia coli β-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation.     

The relationships in biosynthesis of the β-galactosidase- and Pz-proteins in Escherichia coli

E. coli cells possess a protein, Pz, which appears to be synthesized under all conditions of growth. In the presence of a galactoside, the cells also synthesize another protein, Gz, endowed with β-galactosidase properties, and very closely similar to Pz in antigenic structure as well as in solubility properties. The synthesis of Gz interferes very significantly with the net rate of Pz synthesis. Only those species of Enterobacteriaceae which possess the Pz protein are competent to synthesize β-galactosidase.     

Influence of amylose,amylopectin and pullulan on β-glucuronidase activity of starch-degrading Escherichia coli

Synthesis of -glucuronidase in starch-degrading Escherichia coli (S1) was induced by amylose, amylopectin and pullulan supplied in mineral medium as the sole carbon source (1%, w/v). The maximum activity occurred after 4 days when cultures reached the stationary phase of growth, but induction was also evident during log-phase. The effects obtained with amylose, amylopectin and pullulan were higher than that obtained with maize starch.