Promoter Sequences Necessary for High-Level Expression of the Plasmid-Associated ampC β-Lactamase Gene bla(MIR-1)

Little is known about mechanisms involved in high-level expression of plasmid-associated ampC genes. The sequence for bla(MIR-1) has been elucidated, and the gene is not inducible. Although the sequence for the promoter (prA) that drives expression of Enterobacter cloacae chromosomal ampC is present upstream of bla(MIR-1), high-level expression from bla(MIR-1) is directed from a hybrid promoter (prB) located further upstream of prA. The purpose of this study was to determine the influence of each promoter on bla(MIR-1) expression and β-lactam resistance. RNA expression by deletion clones with both promoters was measured and compared to that by clones in which −35 and/or −10 elements of prA and/or prB were altered. Primer extension revealed two start sites for bla(MIR-1) transcription. Expression of bla(MIR-1) in clones with both promoters was 171-fold higher than that in clones carrying only prA. In addition, bla(MIR-1) expression from prA increased 11-fold in the presence of the prB −10 element compared to expression driven from prA alone. Ceftazidime and cefotaxime MICs increased 42- and 64-fold, respectively, for the clone expressing bla(MIR-1) from both promoters compared to expression from prA alone. The upstream promoter prB of bla(MIR-1) is solely responsible for high-level expression required for cefotaxime and ceftazidime resistance. These data suggest that resistance to extended-spectrum cephalosporins mediated by noninducible plasmid-associated ampC genes requires the formation of novel promoter elements that are capable of increasing ampC expression

Rapid Nucleic Acid Isolation Method and Compositions

A method of isolating RNA from a biological specimen is provided, whereby a biological specimen is contacted with an admixture of (i) a mono-phasic solution of phenol and guanidine isothiocyanate and (ii) a lysis buffer under conditions and for a time appropriate to form a homogenate. Next, the homogenate is admixed with a water-immiscible organic solvent under conditions and for a time appropriate to form an aqueous phase and an organic phase. The aqueous phase is then contacted with a C1-C4, lower alcohol under conditions and for a time to form a precipitated RNA. The precipitated RNA is then recovered by centrifugation and decanting of the aqueous phase. The method can also be used to isolate total RNA. In an alternative embodiment, the biological sample is contacted with (i) a lysis buffer, and (ii) a mono-phasic solution of phenol and guanidine isothiocyanate under conditions and for a time appropriate to form a homogenate. The remaining steps of this embodiment are the same as above

Occurrence of Newer β-Lactamases in Klebsiella pneumoniae Isolates from 24 U.S. Hospitals

Despite the discovery of novel β-lactamases such as extended-spectrum β-lactamases (ESBLs), imported AmpC, and carbapenem-hydrolyzing β-lactamases at least a decade ago, there remains a low level of awareness of their importance and how to detect them. There is a need to increase the levels of awareness of clinical laboratories about the detection of newer β-lactamases. Therefore, a study was conducted in 2000 to investigate the occurrence of these β-lactamases in Klebsiella pneumoniae isolates at 24 U.S. medical centers. To enhance the likelihood of detecting imported AmpC and carbapenem-hydrolyzing β-lactamases, participating laboratories were permitted to include archived strains (1996 to 2000) that were intermediate or resistant to either cefoxitin or imipenem. The β-lactamase production of 408 isolates positive by screening of 1,123 isolates was investigated by ESBL phenotypic confirmation tests; and for AmpC and carbapenem-hydrolyzing β-lactamases, three-dimensional tests, isoelectric focusing, β-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular tests were used. ESBL-producing isolates were detected at 18 of the 24 sites (75%), imported AmpC-producing isolates were detected at 10 sites (42%), inducible imported AmpC-producing isolates were detected at 3 sites (12.5%), and a molecular class A carbapenem-hydrolyzing enzyme was detected at 1 site (4%). No class B or D carbapenem-hydrolyzing enzymes were detected. ESBLs and imported AmpC β-lactamases were detected at a significant number of sites, indicating widespread penetration of these enzymes into U.S. medical institutions. Because these enzymes may significantly affect therapeutic outcomes, it is vital that clinical laboratories be aware of them and be able to detect their occurrence

Arterial Catheterization and Infection: Toll-like Receptors in Defense against Microorganisms and Therapeutic Implications

Radial artery catheterization has become a preferred route over femoral artery catheterization, in order to monitor the blood pressure of hemodynamically unstable patients or for repeated sampling of arterial blood gases. While the incidence of catheter‐related infection is lower in the radial artery than the femoral artery, infection remains a major issue that requires attention. In this review of the literature, we discuss infectious complications of radial artery catheterization, with a focus on various risk factors and establishing the most common causative agents. We also critically review the role of the innate immune system involving Toll‐like receptors (TLRs) in host‐defense, with the goal of establishing a common pathway used by the innate immune system via TLRs to combat the pathogens that most commonly cause infection in radial artery catheterization. If this pathway can be therapeutically manipulated to preemptively attack pathogenic agents, immunomodulation may be an option in reducing the incidence of infection in this procedure

Association between Handling of Pet Treats and Infection with Salmonella enterica Serotype Newport Expressing the AmpC β-Lactamase, CMY-2

Resistance to the extended-spectrum cephalosporins can occur in Salmonella species via the production of extended-spectrum and AmpC β-lactamases. We describe human infections with Salmonella enterica serotype Newport phage type 14 strains resistant to ceftazidime (CAZ) and cefoxitin (FOX) related to the handling of pet treats containing dried beef. These strains were isolated from five patients in Calgary, Alberta, Canada, during 2002 and were compared to a strain cultured from a commercial pet treat present at the property of one of the patients. The strains were resistant to FOX, CAZ, cefpodoxime, ampicillin, and chloramphenicol; intermediate resistant to ceftriaxone and cefotaxime; and sensitive to the aminoglycosides, ciprofloxacin, cefepime, and imipenem. Isoelectric focusing, multiplex PCR, and sequencing of the amplicons showed that all strains produced the plasmid-encoded AmpC β-lactamase, CMY-2. Restriction analysis of plasmid DNA following transformation demonstrated that bla(CMY-2) was encoded on an approximately 140-kb plasmid. Pulsed-field gel electrophoresis showed the human and pet treat Salmonella strains to be highly related. This study is the first to implicate the transfer of multidrug-resistant Salmonella species through the handling of commercial pet treats containing animal products. In addition to documenting the first cases of human infection caused by CMY-2-producing S. enterica serotype Newport strains in Canada, this study illustrates the necessity of rapid and accurate laboratory-based surveillance in the identification of novel types of antimicrobial resistance