bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Bactericidal Activities of Ciprofloxacin, Clinafloxacin, Grepafloxacin, Levofloxacin, Moxifloxacin, and Trovafloxacin against
Streptococcus pneumoniae
in a Dynamic In Vitro Model
Authors: Klepser Michael E. , Ernst Erika J. , Petzold C. Rosemarie , Rhomberg Paul , Doern Gary V.
Published: July 2002

Several new quinolones that exhibit enhanced in vitro activity against
Streptococcus pneumoniae
have been developed. Using a dynamic in vitro model, we generated time-kill data for ciprofloxacin, clinafloxacin, grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin against three isolates of quinolone-susceptible
S. pneumoniae
. Three pharmacokinetic profiles were simulated for each of the study agents (0.1, 1, and 10 times the area under the concentration-time curve [AUC]). Target 24-h AUCs were based upon human pharmacokinetic data resulting from the maximal daily doses of each agent. Ciprofloxacin was the least active agent against all three isolates. With regimens that simulated the human 24-h AUC, ciprofloxacin resulted in an initial, modest decline in the numbers of CFU per milliliter; however, by 48 h the numbers of CFU per milliliter returned to or exceeded the starting inoculum. At the AUC, levofloxacin resulted in variable bacteriostatic and bactericidal activities against the isolates. The remaining agents yielded bactericidal (99.9% reduction) activity by 48 h with regimens that simulated the AUC. At 0.1 time the AUC ciprofloxacin and levofloxacin produced no inhibitory effect, grepafloxacin exhibited bacteriostatic activity, trovafloxacin had mixed static and cidal activities, and clinafloxacin and moxifloxacin caused significant reductions in the numbers of CFU per milliliter by 48 h. All six agents produced cidal activity at 10 times the AUC. In this dynamic in vitro model of infection, the quinolones demonstrated various degrees of activity against
S. pneumoniae
. The rank order of activity, with respect to bactericidal effect, was ciprofloxacin (least active) ≪ levofloxacin < grepafloxacin, trovafloxacin < clinafloxacin and moxifloxacin (most active). The rank order of the agents with respect to the selection of resistance was ciprofloxacin (most likely) > grepafloxacin, moxifloxacin, and trovafloxacin > levofloxacin > clinafloxacin.
Peptide Deformylase as an Antibacterial Drug Target: Assays for Detection of Its Inhibition in
Escherichia coli
Cell Homogenates and Intact Cells
Authors: Apfel Christian M. , Evers Stefan , Hubschwerlen Christian , Pirson Wolfgang , Page Malcolm G. P. , Keck Wolfgang
Published: July 2002

An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [
35
S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.
Activities of the Combination of Quinupristin-Dalfopristin with Rifampin In Vitro and in Experimental Endocarditis Due to
Staphylococcus aureus
Strains with Various Phenotypes of Resistance to Macrolide-Lincosamide-Streptogramin Antibiotics
Authors: Zarrouk Virginie , Bozdogan Bülent , Leclercq Roland , Garry Louis , Feger Celine , Carbon Claude , Fantin Bruno
Published: July 2002

We evaluated the activities of quinupristin-dalfopristin (Q-D), alone or in combination with rifampin, against three strains of
Staphylococcus aureus
susceptible to rifampin (MIC, 0.06 μg/ml) and to Q-D (MICs, 0.5 to 1 μg/ml) but displaying various phenotypes of resistance to macrolide-lincosamide-streptogramin antibiotics:
S. aureus
HM1054 was susceptible to quinupristin and dalfopristin (MICs of 8 and 4 μg/ml, respectively); for
S. aureus
RP13, the MIC of dalfopristin was high (MICs of quinupristin and dalfopristin for strain RP13, 8 and 32 μg/ml, respectively); and
S. aureus
HM1054R was obtained after conjugative transfer of macrolide-lincosamide-streptogramin B constitutive resistance to HM1054, and the MIC of quinupristin for this strain was high (MICs of quinupristin and dalfopristin, 64 and 4 μg/ml, respectively). In vitro time-kill curve studies showed no difference between Q-D and rifampin, at a concentration of four times the MIC, against the three strains. Rabbits with aortic endocarditis were treated 4 days with Q-D, rifampin, or their combination. In vivo, the combination was highly bactericidal and synergistic against strains susceptible to quinupristin (HM1054 and RP13) and sterilized 94% of the animals. In contrast, the combination was neither synergistic nor bactericidal against the quinupristin-resistant strain (HM1054R) and did not prevent the emergence of mutants resistant to rifampin. We conclude that the in vivo synergistic and bactericidal activity of the combination of Q-D and rifampin against
S. aureus
is predicted by the absence of resistance to quinupristin but not by in vitro combination studies.
Vancomycin and Ceftazidime Bioactivities Persist for at Least 2 Weeks in the Lumen in Ports: Simplifying Treatment of Port-Associated Bloodstream Infections by Using the Antibiotic Lock Technique
Authors: Haimi-Cohen Yishai , Husain Neghma , Meenan Jerry , Karayalcin Gungor , Lehrer Michael , Rubin Lorry G.
Published: July 2002

The residual antibiotic concentration of vancomycin (2 mg/ml)- or ceftazidime (2 mg/ml)-heparin solutions instilled in ports in pediatric hematology-oncology patients 1 to 34 days earlier was measured. Antibiotic concentrations of ≥100 μg of either antibiotic per ml persisted for at least 21 days. For treatment of lumenal port infections, antibiotic-heparin dwell times of ≥2 weeks may be appropriate.
Contributions of the 8-Methoxy Group of Gatifloxacin to Resistance Selectivity, Target Preference, and Antibacterial Activity against
Streptococcus pneumoniae
Authors: Fukuda Hideyuki , Kishii Ryuta , Takei Masaya , Hosaka Masaki
Published: July 2002

Gatifloxacin (8-methoxy, 7-piperazinyl-3′-methyl) at the MIC selected mutant strains that possessed
gyrA
mutations at a low frequency (3.7 × 10
−9
) from wild-type strain
Streptococcus pneumoniae
IID553. AM-1147 (8-methoxy, 7-piperazinyl-3′-
H
) at the MIC or higher concentrations selected no mutant strains. On the other hand, the respective 8-H counterparts of these two compounds, AM-1121 (8-
H
, 7-piperazinyl-3′-methyl) and ciprofloxacin (8-
H
, 7-piperazinyl-3′-
H
), at one and two times the MIC selected mutant strains that possessed
parC
mutations at a high frequency (>2.4 × 10
−6
). The MIC of AM-1147 increased for the
gyrA
mutant strains but not for the
parC
mutant strains compared with that for the wild-type strain. These results suggest that fluoroquinolones that harbor 8-methoxy groups select mutant strains less frequently and prefer DNA gyrase, as distinct from their 8-H counterparts. The in vitro activities of gatifloxacin and AM-1147 are twofold higher against the wild-type strain, eight- and twofold higher against the first-step
parC
and
gyrA
mutant strains, respectively, and two- to eightfold higher against the second-step
gyrA
and
parC
double mutant strains than those of their 8-H counterparts. These results indicate that the 8-methoxy group contributes to enhancement of antibacterial activity against target-altered mutant strains as well as the wild-type strain. It is hypothesized that the 8-methoxy group of gatifloxacin increases the level of target inhibition, especially against DNA gyrase, so that it is nearly the same as that for topoisomerase IV inhibition in the bacterial cell, leading to potent antibacterial activity and a low level of resistance selectivity.
Pharmacokinetics and Tissue Penetration of Linezolid following Multiple Oral Doses
Authors: Gee Thekli , Ellis Richard , Marshall Gillian , Andrews Jenny , Ashby Janet , Wise Richard
Published: July 2002

The pharmacokinetics of multiple-dose linezolid were determined following administration of five 600-mg oral doses given every 12 h to each of six healthy male volunteers. Concentrations of the drug were determined in plasma and inflammatory blister fluid using high-pressure liquid chromatography. A mean peak concentration in plasma of 18.3 μg/ml (standard deviation [SD], 6.0) was attained at a mean time of 0.7 h (SD, 0.3) after the final dose. The penetration into the inflammatory fluid was 104% (SD, 20.7). A mean peak concentration of 16.4 μg/ml (SD, 10.6) was attained in the inflammatory fluid at 3 h (SD, 0.6) after the final dose. The elimination half-life from serum and inflammatory fluid was 4.9 (SD, 1.8) and 5.7 (SD, 1.7) h, respectively. The area under the concentration-time curve in plasma and blister fluid was 140.3 (SD, 73.1) and 155.3 (SD, 80.1) μg · h/ml, respectively. These data suggest that linezolid has good tissue penetration, and we can predict that it will be successful in the treatment of a variety of gram-positive infections.
Standardized Method for In Vitro Antifungal Susceptibility Testing of
Candida albicans
Biofilms
Authors: Ramage Gordon , Vande Walle Kacy , Wickes Brian L. , López-Ribot José L.
Published: July 2002

Candida albicans
is implicated in many biomaterial-related infections. Typically, these infections are associated with biofilm formation. Cells in biofilms display phenotypic traits that are dramatically different from those of their free-floating planktonic counterparts and are notoriously resistant to antimicrobial agents. Consequently, biofilm-related infections are inherently difficult to treat and to fully eradicate with normal treatment regimens. Here, we report a rapid and highly reproducible microtiter-based colorimetric assay for the susceptibility testing of fungal biofilms, based on the measurement of metabolic activities of the sessile cells by using a formazan salt reduction assay. The assay was used for in vitro antifungal susceptibility testing of several
C. albicans
strains grown as biofilms against amphotericin B and fluconazole and the increased resistance of
C. albicans
biofilms against these antifungal agents was demonstrated. Because of its simplicity, compatibility with a widely available 96-well microplate platform, high throughput, and automation potential, we believe this assay represents a promising tool for the standardization of in vitro antifungal susceptibility testing of fungal biofilms.
Single-Dose Pharmacokinetics and Penetration of BMS 284756 into an Inflammatory Exudate
Authors: Wise R. , Gee T. , Marshall G. , Andrews J. M.
Published: July 2002

The pharmacokinetics of a single dose of BMS 284756 were determined following oral administration of a 600-mg dose to eight healthy male volunteers. Concentrations of the drug were measured in plasma and a cantharidine-induced inflammatory exudate by a microbiological assay. The mean peak concentration in plasma of 10.4 μg/ml (standard deviation [SD], 1.3 μg/ml) was attained at a mean time of 1.2 h (SD, 0.5 h) after the dose. The penetration into the inflammatory exudate was 82% (SD, 15.7%). A mean peak concentration of 7.2 μg/ml (SD, 2.4 μg/ml) was attained in the inflammatory exudate at 5.3 h (SD, 1.5 h). The elimination half-lives from plasma and inflammatory fluid were 9.8 h (SD, 1.1 h) and 8.5 h (SD, 1.9 h), respectively. The areas under the concentration-time curves for plasma and inflammatory fluid were 96.7 μg · h/ml (SD, 10.3 μg · h/ml) and 77.9 μg · h/ml (SD, 19.2 μg · h/ml), respectively.
Ultrastructural and Biochemical Alterations Induced by 22,26-Azasterol, a Δ
24(25)
-Sterol Methyltransferase Inhibitor, on Promastigote and Amastigote Forms of
Leishmania amazonensis
Authors: Rodrigues Juliany C. F. , Attias Márcia , Rodriguez Carlos , Urbina Julio A. , Souza Wanderley de
Published: July 2002

We report on the antiproliferative effects and the ultrastructural and biochemical alterations induced in vitro by 22,26-azasterol, a sterol Δ
24(25)
-methyltransferase (24-SMT) inhibitor, on
Leishmania amazonensis
. When promastigotes and amastigotes were exposed to 100 nM 22,26-azasterol, complete growth arrest and cell lysis ensued after 72 (promastigotes) or 120 (amastigotes) h. Exposure of parasites to this azasterol led to the complete depletion of parasite endogenous sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (zymosterol, cholesta-5,7,24-trien-3β-ol, and cholesta-7,24-dien-3β-ol), while 14-methyl-zymosterol and 4,14-dimethyl-zymosterol accumulated as a result of simultaneous incubation of the parasites with 22,26-azasterol and ketoconazole, a known inhibitor of the parasite’s sterol C14-demethylase. These results confirmed that 24-SMT is the primary site of action of the azasterol. Profound changes were also observed in the phospholipid compositions of treated cells, in which a twofold reduction in the content of phosphatidylserine was observed; this was accompanied by a concomitant increase in the content of phosphatidylinositol. Transmission electron microscopy showed that 22,26-azasterol induced marked morphological changes, including mitochondrial swelling, invaginations of the inner mitochondrial membrane, and the appearance of large bodies containing concentric membranes. Other modifications included increases in the numbers of acidocalcisomes, megasomes, and lipid inclusions and the appearance of typical autophagic structures and cell body protrusions toward the flagellar pocket. We conclude that the dramatic alteration of the lipid composition of the parasite’s membranes induced by the drug underlies the ultrastructural alterations that lead to the loss of cell viability and that 24-SMT inhibitors could be useful as selective antileishmanial agents.
Broad-Spectrum Antiherpes Activities of 4-Hydroxyquinoline Carboxamides, a Novel Class of Herpesvirus Polymerase Inhibitors
Authors: Oien Nancee L. , Brideau Roger J. , Hopkins Todd A. , Wieber Janet L. , Knechtel Mary L. , Shelly John A. , Anstadt Robert A. , Wells Peter A. , Poorman Roger A. , Huang Audris , Vaillancourt Vallerie A. , Clayton Terrance L. , Tucker John A. , Wathen Michael W.
Published: July 2002

Through broad screening of the compound library at Pharmacia, a naphthalene carboxamide was identified as a nonnucleoside inhibitor of human cytomegalovirus (HCMV) polymerase. Structure-activity relationship studies demonstrated that a quinoline ring could be substituted for naphthalene, resulting in the discovery of a 4-hydroxyquinoline-3-carboxamide (4-HQC) class of antiviral agents with unique biological properties. In vitro assays with the 4-HQCs have demonstrated potent inhibition of HCMV, herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV) polymerases but no inhibition of human α, δ, and γ polymerases. Antiviral cell culture assays have further confirmed that these compounds are active against HCMV, HSV-1, HSV-2, VZV, and many animal herpesviruses. However, these compounds were not active against several nonherpesviruses representing different DNA and RNA virus families. A strong correlation between the viral DNA polymerase and antiviral activity for this class of compounds supports inhibition of the viral polymerase as the mechanism of antiviral activity. Northern blot analysis of immediate-early and late viral transcripts also pointed to a block in the viral life cycle consistent with inhibition of viral DNA replication. In vitro HCMV polymerase assays indicate that the 4-HQCs are competitive inhibitors of nucleoside binding. However, no cross-resistance could be detected with ganciclovir-resistant HCMV or acyclovir-resistant HSV-1 mutants. The unique, broad-spectrum activities of the 4-HQCs may offer new opportunities for treating many of the diseases caused by herpesviruses.
Emergence of Resistance to Protease Inhibitor Amprenavir in Human Immunodeficiency Virus Type 1-Infected Patients: Selection of Four Alternative Viral Protease Genotypes and Influence of Viral Susceptibility to Coadministered Reverse Transcriptase Nucleoside Inhibitors
Authors: Maguire Michael , Shortino Denise , Klein Astrid , Harris Wendy , Manohitharajah Varsha , Tisdale Margaret , Elston Robert , Yeo Jane , Randall Sharon , Xu Fan , Parker Hayley , May Jackie , Snowden Wendy
Published: July 2002

Previous data have indicated that the development of resistance to amprenavir, an inhibitor of the human immunodeficiency virus type 1 protease, is associated with the substitution of valine for isoleucine at residue 50 (I50V) in the viral protease. We present further findings from retrospective genotypic and phenotypic analyses of plasma samples from protease inhibitor-naïve and nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients who experienced virological failure while participating in a clinical trial where they had been randomized to receive either amprenavir or indinavir in combination with NRTIs. Paired baseline and on-therapy isolates from 31 of 48 (65%) amprenavir-treated patients analyzed demonstrated the selection of protease mutations. These mutations fell into four distinct categories, characterized by the presence of either I50V, I54L/I54M, I84V, or V32I+I47V and often included accessory mutations, commonly M46I/L. The I50V and I84V genotypes displayed the greatest reductions in susceptibility to amprenavir, although each of the amprenavir-selected genotypes conferred little or no cross-resistance to other protease inhibitors. There was a significant association, for both amprenavir and indinavir, between preexisting baseline resistance to NRTIs subsequently received during the study and development of protease mutations (
P
= 0.014 and
P
= 0.031, respectively). Our data provide a comprehensive analysis of the mechanisms by which amprenavir resistance develops during clinical use and present evidence that resistance to concomitant agents in the treatment regimen predisposes to the development of mutations associated with protease inhibitor resistance and treatment failure.
Mutations in
ponA
, the Gene Encoding Penicillin-Binding Protein 1, and a Novel Locus,
penC
, Are Required for High-Level Chromosomally Mediated Penicillin Resistance in
Neisseria gonorrhoeae
Authors: Ropp Patricia A. , Hu Mei , Olesky Melanie , Nicholas Robert A.
Published: July 2002

Chromosomally mediated penicillin resistance in
Neisseria gonorrhoeae
occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of
N. gonorrhoeae
to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant
N. gonorrhoeae
(CMRNG) strains for which MICs of penicillin were ≥1 μg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation (
k
2
/
K'
) than wild-type PBP 1 with a variety of β-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered
ponA
gene (
ponA1
) in several CMRNG strains with the wild-type
ponA
gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19
penA4 mtr penB5
) with the
ponA1
gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed
penC
, which was required along with
ponA1
to increase penicillin resistance of PR100 to a high level (MIC = 4 μg/ml). The
penC
locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus,
penC
, is required along with
ponA1
to achieve high-level penicillin resistance.
Identification of a Series of Tricyclic Natural Products as Potent Broad-Spectrum Inhibitors of Metallo-β-Lactamases
Authors: Payne David J. , Hueso-Rodríguez Juan Antonio , Boyd Helen , Concha Néstor O. , Janson Cheryl A. , Gilpin Martin , Bateson John H. , Cheever Christy , Niconovich Nancy L. , Pearson Stewart , Rittenhouse Stephen , Tew David , Díez Emilio , Pérez Paloma , de la Fuente Jesus , Rees Michael , Rivera-Sagredo Alfonso
Published: July 2002

This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-β-lactamase inhibitors. Natural product screening of the
Bacillus cereus
II enzyme identified an extract from a strain of
Chaetomium funicola
with inhibitory activity against metallo-β-lactamases. SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract. The most active of these compounds was SB238569, which possessed

K
i

values of 79, 17, and 3.4 μM for the
Bacillus cereus
II,
Pseudomonas aeruginosa
IMP-1, and
Bacteroides fragilis
CfiA metallo-β-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the
Stenotrophomonas maltophilia
L-1 metallo-β-lactamase (50% inhibitory concentration > 1,000 μM). The lack of activity against angiotensin-converting enzyme and serine β-lactamases demonstrated the selective nature of these compounds. The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 Å. SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove. SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem. Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of
B. fragilis
producing CfiA, making these strains sensitive to meropenem (MIC ≤ 4 μg/ml). Consequently, this series of metallo-β-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-β-lactamases.
Transformation of 2,4,6-Trinitrotoluene by Purified Xenobiotic Reductase B from
Pseudomonas fluorescens
I-C
Authors: Pak Jeong W. , Knoke Kyle L. , Noguera Daniel R. , Fox Brian G. , Chambliss Glenn H.
Published: July 2002

The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from
Pseudomonas fluorescens
I-C, was evaluated by using natural abundance and [U-
14
C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2,6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic
m/z
of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of ∼0.5 mol of nitrite per mol of TNT consumed. The extents of the initial enzymatic reactions were similar in the presence and in the absence of O
2
, but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP
+
. Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed that both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonated dihydride-Meisenheimer complex of TNT were required precursors for the dimerization and nitrite release reactions. The
m/z
376 dimers also reacted with either dansyl chloride or
N
-1-naphthylethylenediamine HCl, providing evidence for an aryl amine functional group. In combination, the experimental results are consistent with assigning the chemical structures of the
m/z
376 species to various isomers of amino-dimethyl-tetranitrobiphenyl. A mechanism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.
Molecular Characterization of Methanotrophic Isolates from Freshwater Lake Sediment
Authors: Auman Ann J. , Stolyar Sergei , Costello Andria M. , Lidstrom Mary E.
Published: July 2002

Profiles of dissolved O
2
and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm and found to be relatively constant at 270 to 350 μmol/liter of sediment/h. Approximately 65% of the methane was oxidized to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by 16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to two copies of
pmoA
and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for
pmoA
genes in each isolate, and in cases where, sequencing was done, the
pmoA
copies were found to be almost identical. PCR primers were developed for
mmoX
which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO) activity. Phylogenetic analysis of the translated PCR products with published
mmoX
sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another containing the orthologs from type II strains. The presence of sMMO-containing
Methylomonas
strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously thought.
Cloning, Sequencing, and Expression of the Pyruvate Carboxylase Gene in
Lactococcus lactis
subsp.
lactis
C2
Authors: Wang H. , O'Sullivan D. J. , Baldwin K. A. , McKay L. L.
Published: July 2002

A functional
pyc
gene was isolated from
Lactococcus lactis
subsp.
lactis
C2 and was found to complement a Pyc defect in
L. lactis
KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The
pyc
gene was also detected in
Lactococcus lactis
subsp.
cremoris
and
L. lactis
subsp.
lactis
bv. diacetylactis strains.
Use of Length Heterogeneity PCR and Fatty Acid Methyl Ester Profiles To Characterize Microbial Communities in Soil
Authors: Ritchie Nancy J. , Schutter Mary E. , Dick Richard P. , Myrold David D.
Published: July 2002

In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5′ domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the β subclass of the class
Proteobacteria
, the
Cytophaga-Flexibacter-Bacteriodes
group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition.
Products from the Incomplete Metabolism of Pyrene by Polycyclic Aromatic Hydrocarbon-Degrading Bacteria
Authors: Kazunga Chikoma , Aitken Michael D.
Published: July 2002

Pyrene is a regulated pollutant at sites contaminated with polycyclic aromatic hydrocarbons (PAH). It is mineralized by some bacteria but is also transformed to nonmineral products by a variety of other PAH-degrading bacteria. We examined the formation of such products by four bacterial strains and identified and further characterized the most apparently significant of these metabolites.
Pseudomonas stutzeri
strain P16 and
Bacillus cereus
strain P21 transformed pyrene primarily to
cis
-4,5-dihydro-4,5-dihydroxypyrene (PYRdHD), the first intermediate in the known pathway for aerobic bacterial mineralization of pyrene.
Sphingomonas yanoikuyae
strain R1 transformed pyrene to PYRdHD and pyrene-4,5-dione (PYRQ). Both strain R1 and
Pseudomonas saccharophila
strain P15 transform PYRdHD to PYRQ nearly stoichiometrically, suggesting that PYRQ is formed by oxidation of PYRdHD to 4,5-dihydroxypyrene and subsequent autoxidation of this metabolite. A pyrene-mineralizing organism,
Mycobacterium
strain PYR-1, also transforms PYRdHD to PYRQ at high initial concentrations of PYRdHD. However, strain PYR-1 is able to use both PYRdHD and PYRQ as growth substrates. PYRdHD strongly inhibited phenanthrene degradation by strains P15 and R1 but had only a minor effect on strains P16 and P21. At their aqueous saturation concentrations, both PYRdHD and PYRQ severely inhibited benzo[
a
]pyrene mineralization by strains P15 and R1. Collectively, these findings suggest that products derived from pyrene transformation have the potential to accumulate in PAH-contaminated systems and that such products can significantly influence the removal of other PAH. However, these products may be susceptible to subsequent degradation by organisms able to metabolize pyrene more extensively if such organisms are present in the system.
Directed Transfer of Large DNA Fragments between
Streptomyces
Species
Authors: Hu Zhihao , Hopwood David A. , Khosla Chaitan
Published: July 2002

The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one
Streptomyces
species to another. The method takes advantage of
tra
gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire ∼22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from
Streptomyces coelicolor
to an engineered derivative of
Streptomyces lividans
.
Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH
Authors: Siegumfeldt Henrik , Björn Rechinger K. , Jakobsen Mogens
Published: July 2002

We describe the dynamics of changes in the intracellular pH (pH
i
) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH
ex
). Strains of
Lactobacillus delbrueckii
subsp.
bulgaricus
,
Streptococcus thermophilus
, and
Lactococcus lactis
were investigated.
Listeria innocua
, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH
i
within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH
ex
from 7.0 to 5.0. Under these conditions, the pH
i
of
L. innocua
remained neutral (between 7 and 8). In contrast, the pH
i
values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH
ex
was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pH
i
at pH
ex
7.0, while different kinetics of pH
i
regulation were observed in different species and also in different strains of
S. thermophilus
.
Biosynthesis of the Lantibiotic Mersacidin: Organization of a Type B Lantibiotic Gene Cluster
Authors: Altena Karsten , Guder André , Cramer Claudia , Bierbaum Gabriele
Published: July 2002

The biosynthetic gene cluster (12.3 kb) of mersacidin, a lanthionine-containing antimicrobial peptide, is located on the chromosome of the producer,
Bacillus
sp. strain HIL Y-85,54728 in a region that corresponds to 348° on the chromosome of
Bacillus subtilis
168. It consists of 10 open reading frames and contains, in addition to the previously described mersacidin structural gene
mrsA
(G. Bierbaum, H. Brötz, K.-P. Koller, and H.-G. Sahl, FEMS Microbiol. Lett. 127:121–126, 1995), two genes,
mrsM
and
mrsD
, coding for enzymes involved in posttranslational modification of the prepeptide; one gene,
mrsT
, coding for a transporter with an associated protease domain; and three genes,
mrsF
,
mrsG
, and
mrsE
, encoding a group B ABC transporter that could be involved in producer self-protection. Additionally, three regulatory genes are part of the gene cluster, i.e.,
mrsR2
and
mrsK2
, which encode a two-component regulatory system which seems to be necessary for the transcription of the
mrsFGE
operon, and
mrsR1
, which encodes a protein with similarity to response regulators. Transcription of
mrsA
sets in at early stationary phase (between 8 and 16 h of culture).
Rapid Detection, Identification, and Enumeration of
Escherichia coli
Cells in Municipal Water by Chemiluminescent In Situ Hybridization
Authors: Stender Henrik , Broomer Adam J. , Oliveira Kenneth , Perry-O'Keefe Heather , Hyldig-Nielsen Jens J. , Sage Andrew , Coull James
Published: July 2002

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable
Escherichia coli
cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of
E. coli
were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in
E. coli
16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of
E. coli
. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of
E. coli
and
Pseudomonas aeruginosa
were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting
P. aeruginosa
16S rRNA. The sensitivity and specificity for the detection of
E. coli
by PNA CISH were then determined using 8
E. coli
strains and 17 other bacterial species, including closely related species. No bacterial strains other than
E. coli
and
Shigella
spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of
E. coli
represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.
Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of
Agrobacterium
Biovars 1 and 2
Authors: Mougel Christophe , Cournoyer Benoit , Nesme Xavier
Published: July 2002

Ecology and biodiversity studies of
Agrobacterium
spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K
2
TeO
3
was analyzed and its potential for isolating
Agrobacterium
spp. directly from soil was evaluated. A K
2
TeO
3
concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K
2
TeO
3
-amended medium. The bona fide agrobacterium colonies growing on media amended with K
2
TeO
3
were confirmed to be
Agrobacterium
colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for
Agrobacterium
biovar 1 and related species (
Agrobacterium rubi
and
Agrobacterium fici
) and for
Agrobacterium
biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the
Agrobacterium
-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10
3
to 10
4
agrobacteria · g of dry soil
−1
in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide
Agrobacterium
biovar 1 strains.
Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1
Authors: Liu Yong , Louie Tai Man , Payne Jason , Bohuslavek Jan , Bolton Harvey , Xun Luying
Published: July 2002

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O
2
. The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent

K
m

for IDA was 4.0 mM with a
k
cat
of 5.3 s
−1
. When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in
Escherichia coli
, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.
In Vivo Regulation of Glutamine Synthetase Activity in the Marine Chlorophyll
b
-Containing Cyanobacterium
Prochlorococcus
sp. Strain PCC 9511 (Oxyphotobacteria)
Authors: El Alaoui Sabah , Diez Jesús , Humanes Lourdes , Toribio Fermı́n , Partensky Frédéric , Garcı́a-Fernández José Manuel
Published: July 2002

The physiological regulation of glutamine synthetase (GS; EC
6.3.1.2
) in the axenic
Prochlorococcus
sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for
Prochlorococcus
growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of
Prochlorococcus
for coping with a light- and nutrient-limited environment.
Study of the Infectivity of Saline-Stored
Campylobacter jejuni
for Day-Old Chicks
Authors: Hald Birthe , Knudsen Katrine , Lind Peter , Madsen Mogens
Published: July 2002

The culturability of three
Campylobacter jejuni
strains and their infectivity for day-old chicks were assessed following storage of the strains in saline. The potential for colonization of chicks was weakened during the storage period and terminated 3 to 4 weeks before the strains became nonculturable. The results from this study suggest that the role of starved and aged but still culturable campylobacters may be diminutive, but even more, that the role of viable but nonculturable stages in campylobacter epidemiology may be negligible. Even high levels of maternally derived anti-campylobacter outer membrane protein serum antibodies in day-old chicks did not protect the chicks from campylobacter colonization.
Analysis of Bacterial Detachment from Substratum Surfaces by the Passage of Air-Liquid Interfaces
Authors: Gómez-Suárez Cristina , Busscher Henk J. , van der Mei Henny C.
Published: July 2002

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (
Streptococcus sobrinus
HG1025,
Streptococcus oralis
J22,
Actinomyces naeslundii
T14V-J1,
Bacteroides fragilis
793E, and
Pseudomonas aeruginosa
974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 × 10
6
cells cm
−2
was reached. For
S. sobrinus
HG1025,
S. oralis
J22, and
A. naeslundii
T14V-J1, the conditioning film consisted of adsorbed salivary components, while for
B. fragilis
793E and
P. aeruginosa
974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with
P. aeruginosa
974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s
−1
), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as “bacterial retention” rather than “bacterial adhesion”.
Survival and Heat Resistance of
Listeria monocytogenes
after Exposure to Alkali and Chlorine
Authors: Taormina P. J. , Beuchat L. R.
Published: July 2002

A strain of
Listeria monocytogenes
isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59°C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21°C. Cells of
L. monocytogenes
incubated at 37°C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56°C than were cells stored in TPB at pH 7.3. The alkaline-stress response in
L. monocytogenes
may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 ± 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log
10
CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log
10
CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56°C than cells treated for 5 min. Contamination of foods by
L. monocytogenes
cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of
L. monocytogenes
against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log
10
-unit reductions is not compromised in these cells.