Plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 in an Enterobacter sp.

A strain of an Enterobacter sp. with reduced susceptibility to imipenem, which produced a plasmid-mediated class A carbapenem-hydrolyzing enzyme, KPC-2 beta-lactamase, was isolated from a patient with sepsis at a Boston hospital. This is the first report of the production of a plasmid-encoded KPC-2 beta-lactamase by an Enterobacter sp.

Modification of the double-disk test for detection of enterobacteriaceae producing extended-spectrum and AmpC beta-lactamases.

Detection of extended-spectrum beta-lactamases (ESBLs) in AmpC-producing Enterobacteriaceae is problematic. A modification of the double-disk test (MDDT) has been developed for successful detection of ESBLs in gram-negative bacilli producing well-characterized beta-lactamases as well as 212 clinical isolates of Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, and Citrobacter freundii. MDDT accurately differentiated between ESBL producers and derepressed chromosomal AmpC mutants. MDDT provides a cost-effective alternative approach for clinical microbiology laboratories for routine susceptibility testing with simultaneous detection of ESBLs in enterobacteriaceae.

Enzymatic characterization of TEM-63, a TEM-type extended spectrum beta-lactamase expressed in three different genera of Enterobacteriaceae from South Africa.

The extended-spectrum beta-lactamase, TEM-63, was identified in three separate genera of South African isolates: Proteus mirabilis, Klebsiella pneumoniae, and Escherichia coli. This paper describes identification of the gene in these isolates and compares relative rates of hyrolysis between TEM-63 and other known ceftazidimases.

Regulation of inducible AmpC beta-lactamase expression among Enterobacteriaceae.

AmpC ss-lactamases are active-site serine enzymes that are primarily cephalosporinases. In many gram negative organisms, including Enterobacter spp.,Citrobacter freundii, Serratia marcescens, Morganella morganii and Pseudomonas aeruginosa, the expression of chromosomal ampC genes is low but inducible in response to ss-lactams and other stimuli. The current working model for AmpC induction requires exposure of bacterial cells to ss-lactam drugs or other stimuli and is linked to the cell wall recycling pathway. Induction of ampC appears to involve several gene products associated with this pathway. These gene products include AmpR, AmpD, and AmpG. In addition, anhydro forms of cell wall precursor muropeptides are believed to act as cofactors for AmpC induction. These cofactors bind to the DNA binding protein, AmpR, and define the role of AmpR as activator. Recent debate has ensued in the literature as to the identification of the precursor muropeptide involved in the activation process. Two candidate muropeptides include 1,6-anhydro-N-acetylmuramic acid L-Ala-D-Glu-meso-diaminopimelic acid (anhydro-MurNAc-tripeptide) and anhydro-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid- D-Ala-D-Ala (pentapeptide). The intent of this review is to address the general mechanism involved in AmpC induction. In doing so, the genes and gene products required for the process of AmpC induction are described. In addition, we review the data addressing cell wall recycling as it relates to AmpC induction.

Molecular characterization of a multiply resistant Klebsiella pneumoniae encoding ESBLs and a plasmid-mediated AmpC.

Organisms encoding multiple antibiotic resistance genes are becoming increasingly prevalent. In this report we describe a multiply resistant Klebsiella pneumoniae which possesses at least five different beta-lactamase genes. Isoelectric focusing, polymerase chain reaction and restriction fragment length polymorphism analysis identified TEM-1, multiple SHVs, OXA-9 and a plasmid-mediated ampC, beta-lactamase. Furthermore, Southern analysis and conjugation experiments established that most of the resistance genes were encoded on one large transferable plasmid. This report demonstrates the complexity of multiply resistant organisms.

beta-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa.

Although resistance to the expanded-spectrum cephalosporins among members of the family Enterobacteriaceae lacking inducible beta-lactamases occurs virtually worldwide, little is known about this problem among isolates recovered in South Africa. Isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis resistant to expanded-spectrum cephalosporins recovered from patients in various parts of South Africa over a 3-month period were investigated for extended-spectrum beta-lactamase production. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Production of extended-spectrum beta-lactamases was evaluated by using the double-disk test, and the beta-lactamases were characterized by spectrophotometric hydrolysis assays and an isoelectric focusing overlay technique which simultaneously determined isoelectric points and general substrate or inhibitor characteristics. DNA amplification and sequencing were performed to confirm the identities of these enzymes. The P. mirabilis and E. coli isolates were found to produce TEM-26-type, SHV-2, and SHV-5 extended-spectrum beta-lactamases. An AmpC-related enzyme which had a pI of 8.0 and which conferred resistance to cefoxitin as well as the expanded-spectrum cephalosporins was found in a strain of K. pneumoniae. This is the first study which has identified organisms producing different extended-spectrum beta-lactamases from South Africa and the first report describing strains of P. mirabilis producing a TEM-26-type enzyme. The variety of extended-spectrum beta-lactamases found among members of the family Enterobacteriaceae isolated from major medical centers in South Africa is troubling and adds to the growing list of countries where these enzymes pose a serious problem for antimicrobial therapy.

Plasmid-mediated resistance to expanded-spectrum cephalosporins among Enterobacter aerogenes strains.

Resistance to expanded-spectrum cephalosporins commonly develops in Enterobacter aerogenes during therapy due to selection of mutants producing high levels of the chromosomal Bush group 1 beta-lactamase. Recently, resistant strains producing plasmid-mediated extended-spectrum beta-lactamases (ESBLs) have been isolated as well. A study was designed to investigate ESBL production among 31 clinical isolates of E. aerogenes from Richmond, Va., with decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk potentiation test. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Beta-lactamases were investigated by an isoelectric focusing overlay technique which simultaneously determined isoelectric points (pIs) and substrate or inhibitor profiles. Decreased susceptibility to cefotaxime, ceftazidime, and aztreonam (MIC range, 1 to 64 microg/ml) was detected and associated with resistance to gentamicin and trimethoprim-sulfamethoxazole. All strains produced an inducible Bush group 1 beta-lactamase (pI 83). Twenty-nine of the 31 isolates also produced an enzyme similar to SHV-4 (pI 7.8), while 1 isolate each produced an enzyme similar to SHV-3 (pI 6.9) and to SHV-5 (pI 8.2). The three different SHV-derived ESBLs were transferred by transconjugation to Escherichia coli C600N and amplified by PCR. Plasmid profiles of the clinical isolates showed a variety of different large plasmids. Because of the linkage of resistance to aminoglycosides and trimethoprim-sulfamethoxazole with ESBL production, it is possible that the usage of these drugs was responsible for selecting plasmid-mediated resistance to extended-spectrum cephalosporins in E. aerogenes. Furthermore, it is important that strains such as these be recognized, because they can be responsible for institutional spread of resistance genes.

The latency-related gene of bovine herpesvirus 1 inhibits the activity of immediate-early transcription unit 1.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory neurons of infected animals. Only one virus-encoded latency-related (LR) gene is expressed during a latent infection. The LR transcript overlaps immediate-early transcription unit 1 (IEtu1) and is anti-sense with respect to IEtu1. The transcriptional start site of the LR RNA was mapped to position 724 of the LR gene, downstream from two putative TATA elements. When LR promoter sequences were deleted from a plasmid containing IEtu1 and the LR gene, the resulting construct trans-activated the HSV-1 thymidine kinase (TK) promoter more efficiently than IEtu1 plus the LR gene. Cotransfection of a plasmid containing the intact LR gene with IEtu1 inhibited the ability of IEtu1 to trans-activate the TK promoter. These results imply that a LR gene product(s) repressed the trans-acting capacity of IEtu1.

Parvovirus NS1 stimulates P4 expression by interaction with the terminal repeats and through DNA amplification.

Parvovirus protein NS1 is required for replication of viral DNA and plays a role in the regulation of viral gene expression. NS1 trans-activates the P38 promoter for capsid protein synthesis and has variable effects on other promoters. In this study, we examined the effects of NS1 on the regulation of its own promoter, P4. A number of plasmid constructions were made with the P4 promoter fused to reporter genes. The effects of NS1 on expression from the P4 promoter differed depending on the construction. Plasmids containing viral sequences which could not replicate showed a decrease in P4 expression on cotransfection with the NS1 gene. However, plasmids having replication-proficient viral sequences showed a three- to fivefold increase in P4 expression dependent on NS1. The effect on NS1 on P4 transcription was also evaluated at the steady-state RNA level. An infectious clone of the LuIII viral genome was modified to an NS1-NS2 null mutant (pLu272) that is competent for viral DNA replication by introducing a frameshift mutation at codon 5 of the NS1 open reading frame. The P4 transcripts of pLu272 are four nucleotides longer than the wild type and can therefore be resolved from the wild type by primer extension analysis. pLu272 allows comparison of the constitutive level of steady-state RNA produced by the pLu272 P4 promoter in the absence or presence of a template replication dependent on NS1 supplied in trans. NS1 increased P4 transcripts about six- to eightfold. Expression of P4 transcripts from clones that could not amplify depended on the presence of an intact inverted terminal repeat sequence at the left end. A clone with an intact viral left end and a defective viral right end gave an NS1-dependent threefold increase in P4 expression. Destruction of terminal hairpins at both ends resulted in no significant increase in P4 expression in the presence of NS1. Thus, the positive effect of NS1 on the steady-state levels of P4 transcripts depends on the amplification of gene copy number and the integrity of the terminal repeats.