bluuebunny/crossref_metadata_2025_split huggingface dataset:
Method for Estimation of Low Outer Membrane Permeability to β-Lactam Antibiotics
Authors: Lakaye Bernard , Dubus Alain , Joris Bernard , Frère Jean-Marie 
Published: October 2002

ABSTRACT

The outer membrane of gram-negative bacteria plays a major role in β-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with β-lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated β-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of β-lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of β-lactamase-deficient strains of
Enterobacter cloacae
and
Enterobacter aerogenes
to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10
−7
cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of
E. cloacae
was 20 to 60 times less permeable than that of
Escherichia coli
, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (
P
= 3.2 × 10
−9
cm/s). Using these values, the MICs for a β-lactamase-overproducing strain of
E. cloacae
were successfully predicted, demonstrating the validity of the method.
Functional Characterization of
Brucella melitensis
NorMI, an Efflux Pump Belonging to the Multidrug and Toxic Compound Extrusion Family
Authors: Braibant Martine , Guilloteau Laurence , Zygmunt Michel S. 
Published: October 2002

ABSTRACT

Two putative proteins (NorMI and NorMII) similar to the multidrug efflux protein NorM of
Vibrio parahaemolyticus
are encoded by the
Brucella melitensis
16 M genome. We show that a drug-hypersusceptible
Escherichia coli
strain overexpressing NorMI displays increased resistance to norfloxacin, ciprofloxacin, gentamicin, tetraphenylphosphonium ion, acriflavine, and berberine. This elevated resistance was proven to be mediated by an energy-dependent efflux mechanism. NorMI belongs to the multidrug and toxic compound extrusion family and is the first multidrug efflux protein identified in
Brucella
spp.
Efficacies of Quinupristin-Dalfopristin Combined with Vancomycin In Vitro and in Experimental Endocarditis Due to Methicillin-Resistant
Staphylococcus aureus
in Relation to Cross-Resistance to Macrolides, Lincosamides, and Streptogramin B- Type Antibiotics
Authors: Pavie Juliette , Lefort Agnès , Zarrouk Virginie , Chau Françoise , Garry Louis , Leclercq Roland , Fantin Bruno 
Published: October 2002

ABSTRACT

A beneficial effect of the combination of quinupristin-dalfopristin and vancomycin was observed against two methicillin-resistant strains of
Staphylococcus aureus
harboring or not harboring the
ermC
gene, which codes for constitutive macrolide, lincosamide, and streptogramin B resistance. The beneficial effect was observed in time-kill studies, in which the drugs were used at inhibitory concentrations, and in a rabbit model of endocarditis, in which the combination was highly bactericidal and more active than monotherapies.
Effect of Lamivudine on Transmission of Human T-Cell Lymphotropic Virus Type 1 to Adult Peripheral Blood Mononuclear Cells In Vitro
Authors: Balestrieri Emanuela , Forte Giancarlo , Matteucci Claudia , Mastino Antonio , Macchi Beatrice 
Published: October 2002

ABSTRACT
The effects of lamivudine (3TC) on in vitro infection of peripheral blood mononuclear cells (PBMC) from healthy donors with human T-cell lymphotropic virus type 1 (HTLV-1) were investigated. Direct measures of viral replication (viral DNA, RNA, and protein) all gave similar, very high 50% inhibitory concentrations in comparison with those previously reported for zidovudine. Nevertheless, 3TC inhibited HTLV-1-driven long-term growth of infected PBMC in vitro at concentrations (6.25 μM) which had poor or no direct antiviral effects, suggesting that another mechanism may be playing a role.
Comparative Evaluation of Disk Diffusion with Microdilution Assay in Susceptibility Testing of Caspofungin against
Aspergillus
and
Fusarium
Isolates
Authors: Arikan Sevtap , Paetznick Victor , Rex John H. 
Published: October 2002

ABSTRACT

We compared the disk diffusion and broth microdilution methods for susceptibility testing of caspofungin against
Aspergillus
(
n
= 78) and
Fusarium
(
n
= 22) isolates. Microdilution testing followed the NCCLS M-38P guidelines but was performed in antibiotic medium 3 supplemented to 2% glucose (AM3). Disk diffusion assays were performed on AM3 agar plates with a 2-μg caspofungin disk. By both methods, caspofungin showed favorable activity against
Aspergillus
isolates and no activity against
Fusarium
isolates. In the disk-based format, intrazonal growth that was not influenced by the drug concentration gradient was consistently observed for all of the
Aspergillus
isolates tested.
Microbial Communities from Methane Hydrate-Bearing Deep Marine Sediments in a Forearc Basin
Authors: Reed David W. , Fujita Yoshiko , Delwiche Mark E. , Blackwelder D. Brad , Sheridan Peter P. , Uchida Takashi , Colwell Frederick S. 
Published: October 2002

ABSTRACTMicrobial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35°C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within theEuryarchaeotaandCrenarchaeotadomains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to theBacteroidetes,Planctomycetes,Actinobacteria,Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.
Microstructure of Anaerobic Granules Bioaugmented with
Desulfitobacterium frappieri
PCP-1
Authors: Lanthier M. , Tartakovsky B. , Villemur R. , DeLuca G. , Guiot S. R. 
Published: October 2002

ABSTRACT

Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with
Desulfitobacterium frappieri
PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for
Eubacteria
and
Archaea
probes showed that
Eubacteria
predominantly colonized the outer layer, while
Archaea
were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day
−1
and a low bacterial diffusion of 10
−7
dm
2
day
−1
. Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.
Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization
Authors: Poppert Sven , Essig Andreas , Marre Reinhard , Wagner Michael , Horn Matthias 
Published: October 2002

ABSTRACT

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly
C. pneumoniae
,
C. trachomatis
,
C. psittaci
, and the recently described chlamydia-like bacteria comprising the novel genera
Neochlamydia
and
Parachlamydia
. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with
C. pneumoniae
and
C. trachomatis
. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.
Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy
Authors: Olsen Katja N. , Budde Birgitte B. , Siegumfeldt Henrik , Rechinger K. Björn , Jakobsen Mogens , Ingmer Hanne 
Published: October 2002

ABSTRACT

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pH
i
) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pH
i
, allowing fast and noninvasive determination of pH
i
that is ideally suited for direct analysis of individual bacterial cells present in complex environments.
virB-Mediated Survival ofBrucella abortusin Mice and Macrophages Is Independent of a Functional Inducible Nitric Oxide Synthase or NADPH Oxidase in Macrophages
Authors: Sun Yao-Hui , den Hartigh Andreas B. , Santos Renato de Lima , Adams L. Garry , Tsolis Renée M. 
Published: October 2002

ABSTRACTTheBrucella abortus virBlocus is required for establishing chronic infection in the mouse. Using in vitro and in vivo models, we investigated whethervirBis involved in evasion of the bactericidal activity of NADPH oxidase and the inducible nitric oxide synthase (iNOS) in macrophages. Elimination of NADPH oxidase or iNOS activity in macrophages in vitro increased recovery of wild-typeB. abortusbut not recovery of avirBmutant. In mice lacking either NADPH oxidase or iNOS, however,B. abortusinfected and persisted to the same extent as it did in congenic C57BL/6 mice up until 60 days postinfection, suggesting that these host defense mechanisms are not critical for limiting bacterial growth in the mouse. AvirBmutant did not exhibit increased survival in either of the knockout mouse strains, indicating that this locus does not contribute to evasion of nitrosative or oxidative killing mechanisms in vivo.
Induction of the Gene Encoding Macrophage Chemoattractant Protein 1 byOrientia tsutsugamushiin Human Endothelial Cells Involves Activation of Transcription Factor Activator Protein 1
Authors: Cho Nam-Hyuk , Seong Seung-Yong , Huh Myung-Sook , Kim Na-Hyun , Choi Myung-sik , Kim Ik-sang 
Published: October 2002

ABSTRACTHuman macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-κB and AP-1.Orientia tsutsugamushicauses vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated thecis-acting andtrans-acting elements involved inO. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-κB activation was observed in HUVEC infected withO. tsutsugamushi, inhibition of NF-κB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position −69 to −63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position −69 to −63−96 to −90 or deletion of NF-κB-binding site at position −69 to −63−88 to −79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-κB binding sites at positions −2640 to −2632, −2612 to −2603 in the enhancer region, or the AP-1 biding site at position −2276 to −2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position −69 to −63 in proximal promoter and TRE at position −2276 to −2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected withO. tsutsugamushi. Although NF-κB activation is not essential per se, the κB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the involvement of the NF-κB may be due to the function of chromatin structures and other transcription cofactors in the regulation of MCP-1 gene expression in response toO. tsutsugamushiinfectioin.
Effect of Vaccination with Carrier Protein on Response to Meningococcal C Conjugate Vaccines and Value of Different Immunoassays as Predictors of Protection
Authors: Burrage Moya , Robinson Andrew , Borrow Ray , Andrews Nick , Southern Joanna , Findlow Jamie , Martin Sarah , Thornton Carol , Goldblatt David , Corbel Michael , Sesardic Dorothea , Cartwight Keith , Richmond Peter , Miller Elizabeth 
Published: October 2002

ABSTRACT

In order to plan for the wide-scale introduction of meningococcal C conjugate (MCC) vaccine for United Kingdom children up to 18 years old, phase II trials were undertaken to investigate whether there was any interaction between MCC vaccines conjugated to tetanus toxoid (TT) or a derivative of diphtheria toxin (CRM
197
) and diphtheria-tetanus vaccines given for boosting at school entry or leaving. Children (
n
= 1,766) received a diphtheria-tetanus booster either 1 month before, 1 month after, or concurrently with one of three MCC vaccines conjugated to CRM
197
or TT. All of the MCC vaccines induced high antibody responses to the serogroup C polysaccharide that were indicative of protection. The immune response to the MCC-TT vaccine was reduced as a result of prior immunization with a tetanus-containing vaccine, but antibody levels were still well above the lower threshold for protection. Prior or simultaneous administration of a diphtheria-containing vaccine did not affect the response to MCC-CRM
197
vaccines. The immune responses to the carrier proteins were similar to those induced by a comparable dose of diphtheria or tetanus vaccine. The results also demonstrate that, for these conjugate vaccines in these age groups, both standard enzyme-linked immunosorbent assays and those that measure high-avidity antibodies to meningococcal C polysaccharide correlated equally well with assays that measure serum bactericidal antibodies, the established serological correlate of protection for MCC vaccines.
Identification and Characterization of aBrucella abortusATP-Binding Cassette Transporter Homolog toRhizobium melilotiExsA and Its Role in Virulence and Protection in Mice
Authors: Rosinha G. M. S. , Freitas Daniela A. , Miyoshi Anderson , Azevedo Vasco , Campos Eleonora , Cravero Silvio L. , Rossetti Osvaldo , Splitter Gary , Oliveira S. C. 
Published: October 2002

ABSTRACTBrucella abortusis a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence ofBrucellaspp. is not fully understood yet. Furthermore, genes that allowBrucellato reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter ofB. abortusstrain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by theexsAgenes ofRhizobium melilotiandMesorhizobium loti, respectively. Additionally,B. abortusExsA, likeR. melilotiandM. lotiExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placedB. abortusExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. InR. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA inBrucellapathogenesis, anexsAdeletion mutant was constructed. Replacement of the wild-typeexsAby recombination was demonstrated by Southern blot analysis ofBrucellagenomic DNA. Decreased survival in mice of theBrucella ΔexsAmutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, theB. abortus exsAdeletion mutant was used as a live vaccine. Challenge experiments revealed that theexsAmutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.
Genomic Analysis ofClostridium perfringensBacteriophage φ3626, Which Integrates intoguaAand Possibly Affects Sporulation
Authors: Zimmer Markus , Scherer Siegfried , Loessner Martin J. 
Published: October 2002

ABSTRACTTwo temperate viruses, φ3626 and φ8533, have been isolated from lysogenicClostridium perfringensstrains. Phage φ3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infectingClostridiumspecies was determined. The virus belongs to theSiphoviridaefamily of the tailed phages, the orderCaudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3′-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of φ3626 with onset of sporulation inC. perfringens. We found that the φ3626 attachment siteattPlies in a noncoding region immediately downstream ofint. Integration of the viral genome occurs into the bacterial attachment siteattB, which is located within the 3′ end of aguaAhomologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence.
MxiE Regulates Intracellular Expression of Factors Secreted by theShigella flexneri2a Type III Secretion System
Authors: Kane Colleen D. , Schuch Raymond , Day William A. , Maurelli Anthony T. 
Published: October 2002

ABSTRACTThemxi-spalocus on the virulence plasmid ofShigella flexneriencodes components of the type III secretion system.mxiE, a gene within this locus, encodes a protein that is homologous to the AraC/XylS family of transcriptional regulators, but currently its role in pathogenesis remains undefined. We characterized the virulence phenotype of a nonpolarmxiEmutant and found that this mutant retained the ability to invade mammalian cells in tissue culture and secrete Ipas (type III effectors required for host cell invasion), although it was less efficient than wild-typeShigellaat cell-to-cell spread. Despite its invasive properties in culture, themxiEmutant was completely avirulent in an animal model. Potential targets for MxiE activation were identified by using promoter-green fluorescent protein fusions, and gene expression was examined under various growth conditions. Six MxiE-regulated genes were discovered:ospB,ospC1,ospE2,ospF,virA, andipaH9.8. Notably, activation of these genes only occurred within the intracellular environment of the host and not during growth at 37°C in liquid culture. Interestingly, all of the MxiE-regulated proteins previously have been shown to be secreted through the type III secretion system and are putative virulence factors. Our findings suggest that some of these Osp proteins may be involved in postinvasion events related to virulence. Since bacterial pathogens adapt to multiple environments during the course of infecting a host, we propose thatShigellaevolved a mechanism to take advantage of a unique intracellular cue, which is mediated through MxiE, to express proteins when the organism reaches the eukaryotic cytosol.
Mutations in the
Bacillus subtilis glnRA
Operon That Cause Nitrogen Source-Dependent Defects in Regulation of TnrA Activity
Authors: Fisher Susan H. , Wray Lewis V. 
Published: October 2002

ABSTRACT

The
Bacillus subtilis
nitrogen transcriptional factor TnrA is inactive in cells grown with excess nitrogen, e.g., glutamine or glutamate plus ammonium, because feedback-inhibited glutamine synthetase (product of
glnA
) binds to TnrA and blocks its DNA-binding activity. Two conditional mutations that allow TnrA-dependent gene expression in cells grown with glutamate plus ammonium, but not in glutamine-grown cells, were characterized. One mutant contained a mutation in the
glnA
ribosome binding site, while the other mutant synthesized a truncated GlnR protein that constitutively repressed
glnRA
expression. The levels of glutamine synthetase were reduced in both mutants. As a result, when these mutants are grown with excess nitrogen in the absence of glutamine, there is insufficient production of the feedback inhibitors necessary to convert glutamine synthetase into its feedback-inhibited form and TnrA-activated genes are expressed at high levels.