Characterization of extended-spectrum beta-lactamases in Enterobacteriaceae causing nosocomial infections in a Zagreb University Hospital.

The bacteria producing extended-spectrum beta-lactamases (ESBLs) are increasingly reported. production of ESBLs by Gram-negative bacteria is the major mechanism of resistance to oxymino-cephalosporins and aztreonam. the aim of the present study was to characterize ESBLs produced by Enterobacteriaceae, collected during 2003-2005 in a University Hospital in Zagreb, and to determine the risk factors associated with nosocomial infections due to them. 76 isolates of Enterobacteriaceae were included in the study. Antibiotic susceptibility testing was performed by disk-diffusion and broth microdilution method according to CLSI. beta-lactamases were characterized by PCR and sequencing of bla(ESBL )genes. plasmids were extracted by alkaline lysis method and digested with EcoRI enzyme. Most of the strains displayed CAZ phenotype meaning a higher level of resistance to ceftazidime compared to cefotaxime and ceftriaxone. 50 strains produced SHV-ESBL, 28 tem and 8 CTX-M beta-lactamase. Sequencing of bla(SHV )genes from representative strains revealed SHV-5 beta-lactamase in 6 strains whereas sequencing of bla(CTX-M )genes identified CTX-M-3 beta-lactamase in 3 and CTX-M-15 in 5 strains. Strains were assigned to groups from A to f according to plasmid fingerprinting. The spread of SHV-5-producing strains throughout the hospital units could be due to selective pressure of ceftazidime which is widely prescribed in our hospital thus favoring survival of strains possessing a mutation at the Ambler position 240 responsible for ceftazidime and aztreonam resistance.

Construction and characterization of a Streptomyces rimosus recA mutant: the RecA-deficient strain remains viable.

Although previously reported attempts to construct recA null mutants in Streptomyces spp. have been unsuccessful, we have used the suicide plasmid pErmdeltaRecA to inactivate the recA gene in Streptomyces rimosus by gene disruption. pErmdeltaRecA carries the erythromycin resistance gene ermE and a 451-bp fragment of the S. rimosus recA gene (encoding amino acids 2-151). An erythromycin-resistant clone with single plasmid integration into the recA gene on the chromosome was analyzed in detail. This clone possesses one inactive copy of recA which lacks the entire promoter region and the ATG start codon, and a second, truncated gene that encodes only first 151 amino acids of the RecA protein. This S. rimiosus rec A mutant can therefore be considered a completely RecA-deficient strain. The mutant strain is highly sensitive to UV light. Introduction of a plasmid carrying the wild type S. rimosus recA gene completely restored the UV resistance of the recA mutant to wild-type levels. recA genes encoding RecA proteins with short deletions at the C-terminus (21 and 51 amino acids) could not fully rescue the UV sensitivity of the S. rimosus recA strain, when introduced in the same way.

The recA gene from Streptomyces rimosus R6: sequence and expression in Escherichia coli.

The recA gene from Streptomyces rimusus encodes a 376-amino acids polypeptide (M(r) 39,702) that is one of the largest bacterial RecA proteins observed. Detailed analyses of the Streptomyces RecA proteins showed that all possess an additional and unique C-terminal, rich in lysines and alanines, which can form an additional terminal alpha helix. Expression of the S. rimosus RecA protein in Escherichia coli FR333 (delta recA306) was demonstrated using antibodies raised against E. coli RecA protein; expression was possible only from the S. rimosus promoter. A Streptomyces-E. coli-like promoter sequence (TTGACA-18bp-TCTTAT) was found in the A+ T-rich region 135-165 base pairs upstream from the initiation codon and was related to Bacillus subtilis DNA damage-inducible promoters.

Molecular evidence for the presence of a developmental gene in the lowest animals: identification of a homeobox-like gene in the marine sponge Geodia cydonium.

During the development of higher animals, morphogenetic programs are switched on which are frequently controlled by homeotic genes. Until now these genes have not been identified in the lowest animals, the marine sponges. Since sponges show (i) an antero-posterior and/or dorso-ventral axis during embryogenesis and (ii) a complex differentiation pattern during spicula formation, we hypothesized that in sponges homeotic genes--if present--are also involved in the control of these processes. Therefore, we searched for homeobox or homeobox-like sequences in the marine sponge Geodia cydonium. Here we describe a homeobox-like sequence from these animals; it was isolated from a cDNA library of an adult specimen. The deduced amino acid sequence of the complete homeodomain shares over 70% similarity with other homeodomain sequences, including those from hydra, insects and vertebrates. These data indicate that the sponge homeodomain-like sequence is similar with respect to structure to those of other animals and may suggest that the sponge homeodomain-like sequence(s) might function during developmental processes and/or during spiculogenesis in a similar manner to that known for higher animals.