Escherichia coli usually produces only very small amounts of a constitutive AmpC beta-lactamase, but clinical strains overproducing this enzyme have been isolated. Three different ampC promoters of E. coli clinical strains were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene in the pKK232-8 reporter plasmid and their relative strengths were compared by two different methods. The strength of the promoters from AmpC hyperproducers was 70- to 120-fold higher than those from a low-level AmpC producer. One of the strong promoters, which differs from strain K12 at bases -88, -82, -42, -18, -1 and +58, was mutated to abolish the -42 mutation. This change resulted in a 43-fold decrease in CAT concentration. In another promoter, with eight different mutations at positions -88, -82, -32, -18, -1, +5, +24 and +58, the -32T-->A transversion, which created perfect homology with the -35 consensus sequence, was reverted; this led to a 13-fold decrease in CAT concentration. The -42 and -32 mutations play an important role in E. coli resistance to beta-lactams by increasing ampC transcription.

Gastrointestinal disturbances are a side-effect frequently associated with haematological malignancies due to the intensive cytotoxic treatment given in connection with bone marrow transplantation (BMT). However, intestinal microbiota changes during chemotherapy remain poorly described, probably due to the use of culture-based and low-resolution molecular methods in previous studies. The objective of our study was to apply a next generation DNA sequencing technology to analyse chemotherapy-induced changes in faecal microbiota. We included eight patients with non-Hodgkin's lymphoma undergoing one course of BMT conditioning chemotherapy. We collected a prechemotherapy faecal sample, the day before chemotherapy was initiated, and a postchemotherapy sample, collected 1 week after the initiation of chemotherapy. Total DNA was extracted from faecal samples, denaturing high-performance liquid chromatography based on amplification of the V6 to V8 region of the 16S ribosomal RNA (rRNA) gene, and 454-pyrosequencing of the 16 S rRNA gene, using PCR primers targeting the V5 and V6 hypervariable 16S rRNA gene regions were performed. Raw sequence data were screened, trimmed, and filtered using the QIIME pipeline. We observed a steep reduction in alpha diversity and significant differences in the composition of the intestinal microbiota in response to chemotherapy. Chemotherapy was associated with a drastic drop in Faecalibacterium and accompanied by an increase of Escherichia. The chemotherapy-induced shift in the intestinal microbiota could induce severe side effects in immunocompromised cancer patients. Our study is a first step in identifying patients at risk for gastrointestinal disturbances and to promote strategies to prevent this drastic shift in intestinal microbiota.

Escherichia coli strains showing an increased resistance to oxyiminocephalosporins without an extended spectrum Llactamase production have been screened for mutations in their ampC L-lactamase gene promoter. Mutations were found by direct sequencing of seven promoters at positions 342, 332 (box 335), 318, 31, +5, +24 (attenuator), +31 (attenuator) and +58. By using rapid and simple methods, three of these mutations (342, 332 and +24), which could enhance transcription, were searched in 37 resistant and 25 sensitive isolates. The 342 mutation was present in 33 of the 37 promoters from the resistant isolates. The 332 and +24 mutations were present only in three and two promoters, respectively, and they were combined in the most resistant strain of the study. The +24 mutation was detected in another strain associated with a 1-bp insertion between the 335 and 310 conserved sequences. None of these mutations was detected in the ampC L-lactamase from the sensitive isolates. z

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