CTX-M-15 extended-spectrum β-lactamase in a shiga toxin-producing Escherichia coli isolate of serotype O111:H8

We report the discovery of a CTX-M-15-producing Escherichia coli (STEC) of serogroup O111:H8, a major serotype responsible for human enterohemorrhagic Escherichia coli (EHEC) infections. In line with the recent CTX-M-15/O104:H4 E. coli outbreak, these data may reflect an accelerating spread of resistance to expanded-spectrum cephalosporins within the E. coli population, including STEC isolates.     

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.     

Molecular analysis and risk factors for Escherichia coli producing extended-spectrum β-lactamase bloodstream infection in hematological malignancies

Introduction

Patients with hematologic malignancies have greater risk-factors for primary bloodstream infections (BSI).

Methods

From 2004–2009, we analyzed bacteremia caused by extended-spectrum beta-lactamase Escherichia coli (ESBL-EC) (n = 100) and we compared with bacteremia caused by cephalosporin-susceptible E. coli (n = 100) in patients with hematologic malignancies.

Objective

To assess the clinical features, risk factors, and outcome of ESBL-EC BSI in patients with hematologic malignancies, and to study the molecular epidemiology of ESBL-EC isolates.

Results

The main diagnosis was acute leukemia in 115 patients (57.5%). Death-related E. coli infection was significantly increased with ESBL-EC (34% vs. control group, 19%; p = 0.03). Treatment for BSI was considered appropriate in 64 patients with ESBL-EC (mean survival, 245±345 days), and in 45 control patients this was 443±613 (p = 0.03). In patients not receiving appropriate antimicrobial treatment, survival was significantly decreased in cases compared with controls (26±122 vs. 276±442; p = 0.001). Fifty six of the ESBL-EC isolates were characterized by molecular analysis: 47 (84%) expressed CTX-M-15, two (3.6%) SHV, and seven (12.5%) did not correspond to either of these two ESBL enzymes. No TLA-1 enzyme was detected.

Conclusions

Patients who had been previously hospitalized and who received cephalosporins during the previous month, have an increased risk of ESBL-EC bacteremia. Mortality was significantly increased in patients with ESBL-EC BSI. A polyclonal trend was detected, which reflects non-cross transmission of multiresistant E.coli isolates.     

Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

Carboxymethylated beta-galactosidase from Escherichia coli was dissociated at 100 degrees C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore beta-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ;complementable fractions'. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the beta-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented beta-galactosidase can exist.     

Occurrence of extended-spectrum β-lactamases-producing Escherichia coli isolates over gradient pollution in an urban tropical estuary

Bacterial resistance to antimicrobials is a global public health problem that surpasses the human context and can be increased by pollution. However, the lack of systematic monitoring of resistance in some aquatic matrices, such as tropical estuaries, makes it unknown whether its occurrence is associated with anthropogenic pollution in these environments. Therefore, we investigated the occurrence of extended-spectrum beta-lactamases (ESBLs) producing Escherichia coli as a resistance indicator for 12 consecutive months at three representative points of a pollution gradient in Guanabara Bay (GB), Brazil. Sixty-six E. coli strains were selected from 72 samples of GB waters in the presence of ceftriaxone (8 μg mL⁻¹) and identified by MALDI-TOF MS. Of the 66, 55 (83.3%) strains were ESBL producers. They carried beta-lactamase/ESBL genes, with the predominance of bla_CTX-M (54, 98.2%), especially the bla_CTX-M-1,2 allele (49.1%). These strains were detected frequently (81.8%) from the point with the highest pollution levels. Furthermore, the marker for Class 1 integron, intI1 gene, was detected in 54.5% of ESBL producers. These data suggest an association between antimicrobial-resistant E. coli and sewage pollution in aquatic environments raising concerns about the possible risks of human exposure to these waters and fish consumption.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.     

Production of the hybrid protein staphylococcal protein A/Escherichia coli β-galactosidase with E. coli

We have investigated the cultivation of an Escherichia coli strain producing the hybrid protein SpA-βgal. The hybrid protein consists of protein A from Staphylococcus aureus and β-galactosidase from E. coli with retained biological activity of both protein A and β-galactosidase. The expression was controlled by the temperature regulated P_R promoter from phage lambda. By late induction of the product synthesis it was possible to circumvent the problem with plasmid instability. The amount of produced SpA-βgal corresponded to approximately 1256 of the cell dry weight. In shake flask cultures most of the hybrid protein was found in an insoluble form and typical inclusion bodies were observed. However, the major part of the protein could be produced in a soluble and biological active form under controlled conditions in a reactor.     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives

We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.

Methods

ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla _CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla _CTX-M-94 and bla _CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla _CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.

Results

The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla _CTX-M-94 and bla _CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.

Conclusions

This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.     

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.     

Review: Biosafety of E. coli β-glucuronidase (GUS) in plants

The -glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers     

Molecular characterization of β-lactam-resistant Escherichia coli isolated from Fu River, China

The present study aims to demonstrate the β-lactam resistance phenotypes and genotypes of Escherichia coli isolates from the Fu River in Chengdu, southwestern China. We obtained 108 E. coli isolates from nine sampling sites during May and December 2010. The total bacterial count varied from 79 colony-forming units (CFU)/ml to 14,550 CFU/ml, and coliform group number from 13 to 1,435 MPN/ml. Among the 108 isolates, 0-31.48% were resistant to β-lactams and β-lactam inhibitors, 1.85-7.40% to aminoglycoside, 1-20% to fluoroquinolone, and 50% to trimethoprim- sulfamethoxazole. The total bacterial count and antimicrobial resistance of different sites were significantly correlated (P < 0.05). Among the 34 ampicillin-resistant isolates, polymerase chain reaction (PCR) amplification and DNA sequencing showed that bla (TEM), bla (SHV), and bla (CTX-M) were detected in 85.29% (n = 29), 41.18% (n = 14), and 5.88% (n = 2) of the isolates, respectively, whereas bla (KPC) and bla (GES) were not observed in any of the isolates. Enterobacterial repetitive intergenic consensus-PCR patterns revealed that the 34 ampicillin-resistant E. coli isolates belonged to three distinct groups. Plasmid DNAs from the 14 SHV producer isolates yielded one to five bands of ca. 0.15-40 kb. To our knowledge, the current study is the first to describe the phenotypic and genetic characterizations of β-lactam resistance in E. coli isolates of river water origin from the Fu River, Chengdu, southwestern China. Results of the present study suggest that the river water may be considered as a reservoir for antibiotic resistance genes.     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Phylogenetic grouping and virulence potential of extended-spectrum-β-lactamase-producing Escherichia coli strains in cattle

In line with recent reports of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of highly virulent serotypes, such as O104:H4, we investigated the distribution of phylogroups (A, B1, B2, D) and virulence factor (VF)-encoding genes in 204 ESBL-producing E. coli isolates from diarrheic cattle. ESBL genes, VFs, and phylogroups were identified by PCR and a commercial DNA array (Alere, France). ESBL genes belonged mostly to the CTX-M-1 (65.7%) and CTX-M-9 (27.0%) groups, whereas those of the CTX-M-2 and TEM groups were much less represented (3.9% and 3.4%, respectively). One ESBL isolate was stx(1) and eae positive and belonged to a major enterohemorrhagic E. coli (EHEC) serotype (O111:H8). Two other isolates were eae positive but stx negative; one of these had serotype O26:H11. ESBL isolates belonged mainly to phylogroup A (55.4%) and, to lesser extents, to phylogroups D (25.5%) and B1 (15.6%), whereas B2 strains were quasi-absent (1/204). The number of VFs was significantly higher in phylogroup B1 than in phylogroups A (P = 0.04) and D (P = 0.02). Almost all of the VFs detected were found in CTX-M-1 isolates, whereas only 64.3% and 33.3% of them were found in CTX-M-9 and CTX-M-2 isolates, respectively. These results indicated that the widespread dissemination of the bla(CTX-M) genes within the E. coli population from cattle still spared the subpopulation of EHEC/Shiga-toxigenic E. coli (STEC) isolates. In contrast to other reports on non-ESBL-producing isolates from domestic animals, B1 was not the main phylogroup identified. However, B1 was found to be the most virulent phylogroup, suggesting host-specific distribution of virulence determinants among phylogenetic groups.     

Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by “trapping” the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria.     

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.

     

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.