Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.

A novel plasmid-mediated quinolone resistance gene, qnrB, has been discovered in a plasmid encoding the CTX-M-15 ␤-lactamase from a Klebsiella pneumoniae strain isolated in South India. It has less than 40% amino acid identity with the original qnr (now qnrA) gene or with the recently described qnrS but, like them, codes for a protein belonging to the pentapeptide repeat family. Strains with qnrB demonstrated low-level resistance to all quinolones tested. The gene has been cloned in an expression vector attaching a polyhistidine tag, which facilitated purification to >95% homogeneity. As little as 5 pM of QnrB-His 6 protected purified DNA gyrase against inhibition by 2 g/ml (6 M) ciprofloxacin. With a PCR assay qnrB has been detected in Citrobacter koseri, Enterobacter cloacae, and Escherichia coli isolates from the United States, linked to SHV-12 ␤-lactamase and coding for a product differing in five amino acids from the Indian (now QnrB1) variety. The qnrB gene has been found near Orf1005 in some, but not all, plasmids and in association with open reading frames matching known chromosomal genes, suggesting that it too was acquired by plasmids from an as-yet-unknown bacterial source.

High-level resistance to ertapenem was produced by ␤-lactamases of groups 1, 2f, and 3 in a strain of Klebsiella pneumoniae deficient in Omp35 and Omp36. From a wild-type strain producing ACT-1 ␤-lactamase, ertapenem-resistant mutants for which the ertapenem MICs were up to 128 g/ml and expression of outer membrane proteins was diminished could be selected.Ertapenem is a potent carbapenem antibiotic for most clinical isolates of Klebsiella pneumoniae, with a typical MIC at which 90% of the isolates tested are inhibited of 0.03 to 0.06 g/ml (6, 9), but occasional strains for which the MICs are Ն16 g/ml have been detected (6, 7). In one such strain resistance was dependent on the presence of the plasmid-mediated extended-spectrum ␤-lactamase (ESBL) SHV-2 and additional host events presumably affecting ertapenem permeativity (7). Further studies were undertaken to elucidate the contribution of ␤-lactamase and host mutation to such exceptional resistance.The K. pneumoniae strain for which the ertapenem MIC was 16 g/ml was treated with ethidium bromide to cure the resident plasmid. The ertapenem MIC for the resulting strain, C2, was still elevated at l g/ml, and the strain was found to be defective in expression of outer membrane porins OmpK35 and OmpK36 (10). To evaluate the influence of different ␤-lactamases on the ertapenem susceptibility of this strain, plasmids were introduced by mating with R ϩ derivatives of Escherichia coli J53 Azi r (met pro; azide resistant) (8), with selection on medium lacking the growth requirements of the donor and containing an antibiotic to which the plasmid provided resistance, if possible a non-␤-lactam so as to avoid inadvertent selection of additional mutations. A few nonconjugative plasmids were introduced by electroporation. MICs were determined by agar dilution on Mueller-Hinton medium with an inoculum of 10 4 organisms per spot according to NCCLS protocols (12). E. coli ATCC 25922 was used for quality control. Antibiotics were obtained from Sigma (St. Louis, Mo.) (cefotaxime) and the pharmaceutical companies AstraZeneca (meropenem), Bristol-Meyers Squibb (cefepime), GlaxoSmithKline (ceftazidime), and Merck & Co. (cefoxitin, ertapenem, and imipenem).In Table 1 the K. pneumoniae C2 derivatives are listed according to the ␤-lactamase classification scheme of Bush et al. (4). The highest ertapenem MICs (Ն128 g/ml) were achieved by ␤-lactamase group 1 enzymes ACT-1, DHA-1, and FOX-1 and by group 2f enzyme KPC-1. KPC-1 is a known carbapenemase (14) and was encoded by a multicopy plasmid, while group 1 enzymes have been reported to express carbapenem resistance in strains lacking outer membrane porins (3, 10). Other group 1 enzymes provided a lesser degree of ertapenem resistance, with FOX-3 and FOX-5 ␤-lactamases conferring MICs of only 8 g/ml. Group 1 enzymes providing ertapenem resistance also increased resistance to imipenem and meropenem but with diminishing effect: the highest imipenem MIC was 64 g/ml, and the highest meropenem MIC was 16 g/ml.With group 2be (ESBL) enzymes, e...

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Many Salmonella typhimurium genes are required for bacterial entry into host cells. P22 transduction analysis has localized several invasion loci near minute 59 on the S. typhimurium chromosome. To further characterize the 59-60 min chromosomal region, we determined the physical and genetic map of 85 kb of S. typhimurium DNA between srl and cysC. It was previously shown that some of the invasion genes from this region are not present in Escherichia coli K-12. We examined whether other S. typhimurium genes on the 85 kb of DNA were similarly absent from E. coli. We found that a contiguous 40 kb fragment of the S. typhimurium chromosome which encodes invasion genes is absent from the corresponding region of the E. coli K-12 chromosome and may represent a 'pathogenicity island'. We speculate that acquisition of the 40 kb region must have significantly advanced the evolution of Salmonella as a pathogen.

Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC 50 s) of 10 M and 12 M, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection.

In the sequence upstream from qnrB (but not qnrA or qnrS) is a LexA binding site. qnrB was shown to be under SOS control by demonstrating that quinolone susceptibility decreased with increasing temperature in a strain with a recA441(Ts) allele, whereas qnrB expression increased in response to ciprofloxacin or mitomycin C in strains with an intact lexA gene.

We have identified 2 novel EBOV inhibitors, MBX2254 and MBX2270, that can serve as starting points for the development of an anti-EBOV therapeutic agent. Our findings also highlight the importance of NPC1-GP interaction in EBOV entry and the attractiveness of NPC1 as an antifiloviral therapeutic target.