bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequencing of the
rpoB
Gene in
Legionella pneumophila
and Characterization of Mutations Associated with Rifampin Resistance in the
Legionellaceae
Authors: Nielsen K. , Hindersson P. , Høiby N. , Bangsborg J. M. 
Published: July 2002

ABSTRACT

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the
rpoB
gene are known to cause rifampin resistance in
Escherichia coli
and
Mycobacterium tuberculosis
, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family
Legionellaceae
. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the
Legionella pneumophila rpoB
gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the
rpoB
gene (4,104 bp) and an ORF of 384 bp representing part of the
rpoC
gene was obtained. A 316-bp DNA fragment in the center of the
L. pneumophila rpoB
gene, corresponding to a previously described site for mutations leading to rifampin resistance in
M. tuberculosis
, was sequenced from 18 rifampin-resistant
Legionella
isolates representing four species (
L. bozemanii
,
L. longbeachae
,
L. micdadei
, and
L. pneumophila
), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family
Legionellaceae
.
Anti-Human Immunodeficiency Virus Activities of Nucleosides and Nucleotides: Correlation with Molecular Electrostatic Potential Data
Authors: Mickle Travis , Nair Vasu 
Published: July 2002

ABSTRACT

Examination of the anti-human immunodeficiency virus (HIV) data of some normal and isomeric dideoxynucleosides (ddNs and isoddNs), their three-dimensional (3-D) electron density patterns, their electrostatic potential surfaces (EPS), and their conformational maps reveals some interesting correlations. For example, the EPS of (
S,S
)-isoddA shows regions of high and low electrostatic potential remarkably similar to those of β-
d
-3′-azido-3′-deoxythymidine (β-
d
-AZT), (−)-oxetanocin A, and (−)-carbovir. Such correlations involving EPS data and anti-HIV activity were also found with many other active nucleosides. Conversely, inactive compounds had EPS different from those of compounds in the same series that were active. For example, apio-ddNs, which are inactive against HIV, exhibit clear differences in electrostatic potential and 3-D electron density shape from isoddNs that are active against HIV. Additionally, the inactivity of (
S,S
)-isoddC and (
S,S
)-isoddT can be correlated convincingly with a combination of their EPS data and their conformational energy maps. The electrostatic potential distributions of active nucleoside triphosphates show remarkable correlations. For example, (
S,S
)-isoddATP, AZT triphosphate (AZTTP), and oxetanocin A TP have similar 3-D electron density surface patterns and similar high and low regions of electrostatic potential, which may suggest that these compounds proceed through related mechanisms in their interactions with, and inhibition of, HIV reverse transcriptase (RT). Docking of AZTTP, (
S,S
)-isoddATP, and other active triphosphates into the active site of HIV RT and calculation of the EPS of both the nucleotide and the active site show that there is excellent matching between inhibitor and enzyme binding site EPS data. The structure-activity profile discovered has contributed to the development of a first predictive quantitative structure-activity relationship analysis in the area.
Potent Antipneumococcal Activity of Gemifloxacin Is Associated with Dual Targeting of Gyrase and Topoisomerase IV, an In Vivo Target Preference for Gyrase, and Enhanced Stabilization of Cleavable Complexes In Vitro
Authors: Heaton Victoria J. , Ambler Jane E. , Fisher L. Mark 
Published: July 2002

ABSTRACT

We investigated the roles of DNA gyrase and topoisomerase IV in determining the susceptibility of
Streptococcus pneumoniae
to gemifloxacin, a novel fluoroquinolone which is under development as an antipneumococcal drug. Gemifloxacin displayed potent activity against
S. pneumoniae
7785 (MIC, 0.06 μg/ml) compared with ciprofloxacin (MIC, 1 to 2 μg/ml). Complementary genetic and biochemical approaches revealed the following. (i) The gemifloxacin MICs for isogenic 7785 mutants bearing either
parC
or
gyrA
quinolone resistance mutations were marginally higher than wild type at 0.12 to 0.25 μg/ml, whereas the presence of both mutations increased the MIC to 0.5 to 1 μg/ml. These data suggest that both gyrase and topoisomerase IV contribute significantly as gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-step
gyrA
mutants of
S. pneumoniae
7785 (gemifloxacin MICs, 0.25 μg/ml) encoding Ser-81 to Phe or Tyr, or Glu-85 to Lys mutations. These mutants were cross resistant to sparfloxacin (which targets gyrase) but not to ciprofloxacin (which targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 μg/ml) exhibited an alteration in
parC
resulting in changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10- to 20-fold more effective than ciprofloxacin in stabilizing a cleavable complex (the cytotoxic lesion) with either
S. pneumoniae
gyrase or topoisomerase IV enzyme in vitro. These data suggest that gemifloxacin is an enhanced affinity fluoroquinolone that acts against gyrase and topoisomerase IV in
S. pneumoniae
, with gyrase the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.
Helicobacter pylori
Uptake and Efflux: Basis for Intrinsic Susceptibility to Antibiotics In Vitro
Authors: Bina J. E. , Alm R. A. , Uria-Nickelsen M. , Thomas S. R. , Trust T. J. , Hancock R. E. W. 
Published: July 2002

ABSTRACT

We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567–1572, 1995) that
Helicobacter pylori
has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels.
H. pylori
is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that
H. pylori
is able to take up higher basal levels of the hydrophobic fluorescent probe 1-
N
-phenylnaphthylamine (NPN) than
Pseudomonas aeruginosa
or
Escherichia coli
, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in
H. pylori
(6 to 8 μg/ml) than in
P. aeruginosa
or
E. coli
(0.3 to 0.5 μg/ml), suggesting that
H. pylori
has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic β-lactamase (which
H. pylori
lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in
H. pylori
11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that
H. pylori
contained only these three putative RND efflux systems, named here
hefABC
,
hefDEF
, and
hefGHI
, and that the
hefGHI
system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in
H. pylori
. Independent mutagenesis of the three putative RND efflux operons in the chromosome of
H. pylori
had no effect on the in vitro susceptibility of
H. pylori
to 19 antibiotics. These results, in contrast to what is observed in
E. coli
,
P. aeruginosa
, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of
H. pylori
to antibiotics.
Influence of Shaking on Antifungal Susceptibility Testing of
Cryptococcus neoformans
: a Comparison of the NCCLS Standard M27A Medium, Buffered Yeast Nitrogen Base, and RPMI–2% Glucose
Authors: Rodríguez-Tudela Juan L. , Martín-Díez Francisco , Cuenca-Estrella Manuel , Rodero Laura , Carpintero Yolanda , Gorgojo Begoña 
Published: July 2002

ABSTRACT

Cryptococcus neoformans
is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640–2% glucose, and buffered yeast nitrogen base [BYNB]) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35°C for 48 h were determined. The results indicated that shaking and an inoculum size of 10
5
CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of
C. neoformans
and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI–2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the
C. neoformans
strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for
C. neoformans
.
Prevalence of
gyrA
,
gyrB
,
parC
, and
parE
Mutations in Clinical Isolates of
Streptococcus pneumoniae
with Decreased Susceptibilities to Different Fluoroquinolones and Originating from Worldwide Surveillance Studies during the 1997-1998 Respiratory Season
Authors: Jones Mark E. , Sahm Daniel F. , Martin Nele , Scheuring Sibylle , Heisig Peter , Thornsberry Clyde , Köhrer Karl , Schmitz Franz-Josef 
Published: July 2002

ABSTRACT

From 8,419 worldwide clinical isolates of
Streptococcus pneumoniae
obtained during 1997-1998, 69 isolates with reduced susceptibility or resistance to fluoroquinolones (FQs) were molecularly characterized. For the isolates in this prevalence study, only
parC
(Ser-79→Tyr) and
gyrA
(Ser-81→Phe or Tyr) mutations, especially in combination, were found to contribute significantly to resistance. These mutations influenced the FQ MICs to varying degrees, although the rank order of activity remains independent of mutation type, with ciprofloxacin the least active, followed by levofloxacin, gatifloxacin/grepafloxacin/moxifloxacin/sparfloxacin/trovafloxacin, and clinafloxacin/sitafloxacin. Efflux likely plays a crucial role in reduced susceptibility for new hydrophilic FQs.
Inactivation of the
ampD
Gene in
Pseudomonas aeruginosa
Leads to Moderate-Basal-Level and Hyperinducible AmpC β-Lactamase Expression
Authors: Langaee Taimour Yousef , Gagnon Luc , Huletsky Ann 
Published: July 2002

ABSTRACT

It has been shown in enterobacteria that mutations in
ampD
provoke hyperproduction of chromosomal β-lactamase, which confers to these organisms high levels of resistance to β-lactam antibiotics. In this study, we investigated whether this genetic locus was implicated in the altered AmpC β-lactamase expression of selected clinical isolates and laboratory mutants of
Pseudomonas aeruginosa
. The sequences of the
ampD
genes and promoter regions from these strains were determined and compared to that of wild-type
ampD
from
P. aeruginosa
PAO1. Although we identified numerous nucleotide substitutions, they resulted in few amino acid changes. The phenotypes produced by these mutations were ascertained by complementation analysis. The data revealed that the
ampD
genes of the
P. aeruginosa
mutants transcomplemented
Escherichia coli ampD
mutants to the same levels of β-lactam resistance and β-lactamase expression as wild-type
ampD
. Furthermore, complementation of the
P. aeruginosa
mutants with wild-type
ampD
did not restore the inducibility of β-lactamase to wild-type levels. This shows that the amino acid substitutions identified in AmpD do not cause the altered phenotype of AmpC β-lactamase expression in the
P. aeruginosa
mutants. The effects of AmpD inactivation in
P. aeruginosa
PAO1 were further investigated by gene replacement. This resulted in moderate-basal-level and hyperinducible expression of β-lactamase accompanied by high levels of β-lactam resistance. This differs from the stably derepressed phenotype reported in AmpD-defective enterobacteria and suggests that further change at another unknown genetic locus may be causing total derepressed AmpC production. This genetic locus could also be altered in the
P. aeruginosa
mutants studied in this work.
The Biopesticide
Paenibacillus popilliae
Has a Vancomycin Resistance Gene Cluster Homologous to the Enterococcal VanA Vancomycin Resistance Gene Cluster
Authors: Patel Robin , Piper Kerryl , Cockerill Franklin R. , Steckelberg James M. , Yousten Allan A. 
Published: July 2002

ABSTRACT

We have previously identified, in
Paenibacillus popilliae
, a 708-bp sequence which has homology to the sequence of the enterococcal
vanA
gene. We have performed further studies revealing five genes encoding homologues of VanY, VanZ, VanH, VanA, and VanX in
P. popilliae
. The predicted amino acid sequences are similar to those in VanA vancomycin-resistant enterococci: 61% identity for VanY, 21% for VanZ, 74% for VanH, 77% for VanA, and 79% for VanX. The genes in
P. popilliae
may have been a precursor to or have had ancestral genes in common with vancomycin resistance genes in enterococci. The use of
P. popilliae
biopesticidal preparations in agricultural practice may have an impact on bacterial resistance in human pathogens.
Molecular and Biochemical Heterogeneity of Class B Carbapenem-Hydrolyzing β-Lactamases in
Chryseobacterium meningosepticum
Authors: Bellais Samuel , Aubert Daniel , Naas Thierry , Nordmann Patrice 
Published: July 2002

ABSTRACT

Although the carbapenem-hydrolyzing β-lactamase (CHβL) BlaB-1 is known to be in
Chryseobacterium meningosepticum
NCTC 10585, a second CHβL gene,
bla
GOB-1
, was cloned from another
C. meningosepticum
clinical isolate (PINT). The G+C content of
bla
GOB-1
(36%) indicated the likely chromosomal origin of this gene. Its expression in
Escherichia coli
DH10B yields a mature CHβL with a pI of 8.7 and a relative molecular mass of 28.2 kDa. In
E. coli
, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems. GOB-1 had a broad-spectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam). The catalytic efficiency for meropenem was higher than for imipenem. GOB-1 had low amino acid identity with the class B CHβLs, sharing 18% with the closest, L-1 from
Stenotrophomonas maltophilia
, and only 11% with BlaB-1. Most of the conserved amino acids that may be involved in the active site of CHβLs (His-101, Asp-103, His-162, and His-225) were identified in GOB-1. Sequence heterogeneity was found for GOB-1-like and BlaB-1-like β-lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated
C. meningosepticum
isolates. Each isolate had a GOB-1-like and a BlaB-1-like gene. The same combination of GOB-1-like and BlaB-1-like β-lactamases was not found in two different isolates.
C. meningosepticum
is a bacterial species with two types of unrelated chromosome-borne class B CHβLs that can be expressed in
E. coli
and, thus, may represent a clinical threat if spread in gram-negative aerobes.
Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase
Authors: Iyobe Shizuko , Kusadokoro Haruko , Ozaki Junko , Matsumura Naoki , Minami Shinzaburo , Haruta Shin , Sawai Tetsuo , O'Hara Koji 
Published: July 2002

ABSTRACT

In the course of surveying for the carbapenem-hydrolyzing metallo-β-lactamase gene
bla
IMP
in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-β-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene
bla
IMP-3
was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of
bla
IMP-3
was identical to that of
bla
IMP-1
, except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid
bla
genes were constructed from
bla
IMP-3
and
bla
IMP-1
by recombinant DNA techniques, and β-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the
bla
IMP-3
gene. This indicated that extension of the substrate profile in the metallo-β-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene
bla
IMP-3
is an ancestor of
bla
IMP-1
.
Quinolone Resistance Mutations in
Streptococcus pneumoniae
GyrA and ParC Proteins: Mechanistic Insights into Quinolone Action from Enzymatic Analysis, Intracellular Levels, and Phenotypes of Wild-Type and Mutant Proteins
Authors: Pan Xiao-Su , Yague Genoveva , Fisher L. Mark 
Published: July 2002

ABSTRACT

Mutations in DNA gyrase and/or topoisomerase IV genes are frequently encountered in quinolone-resistant mutants of
Streptococcus pneumoniae
. To investigate the mechanism of their effects at the molecular and cellular levels, we have used an
Escherichia coli
system to overexpress
S
.
pneumoniae
gyrase
gyrA
and topoisomerase IV
parC
genes encoding respective Ser81Phe and Ser79Phe mutations, two changes widely associated with quinolone resistance. Nickel chelate chromatography yielded highly purified mutant His-tagged proteins that, in the presence of the corresponding GyrB and ParE subunits, reconstituted gyrase and topoisomerase IV complexes with wild-type specific activities. In enzyme inhibition or DNA cleavage assays, these mutant enzyme complexes were at least 8- to 16-fold less responsive to both sparfloxacin and ciprofloxacin. The ciprofloxacin-resistant (Cip
r
) phenotype was silent in a sparfloxacin-resistant (Spx
r
)
S
.
pneumoniae gyrA
(Ser81Phe) strain expressing a demonstrably wild-type topoisomerase IV, whereas Spx
r
was silent in a Cip
r
parC
(Ser79Phe) strain. These epistatic effects provide strong support for a model in which quinolones kill
S
.
pneumoniae
by acting not as enzyme inhibitors but as cellular poisons, with sparfloxacin killing preferentially through gyrase and ciprofloxacin through topoisomerase IV. By immunoblotting using subunit-specific antisera, intracellular GyrA/GyrB levels were a modest threefold higher than those of ParC/ParE, most likely insufficient to allow selective drug action by counterbalancing the 20- to 40-fold preference for cleavable-complex formation through topoisomerase IV observed in vitro. To reconcile these results, we suggest that drug-dependent differences in the efficiency by which ternary complexes are formed, processed, or repaired in
S
.
pneumoniae
may be key factors determining the killing pathway.
Streptococcus pneumoniae
Response to Repeated Moxifloxacin or Levofloxacin Exposure in a Rabbit Tissue Cage Model
Authors: Xuan Dawei , Zhong Mingkang , Mattoes Holly , Bui Khanh Q. , McNabb Jocarol , Nicolau David P. , Quintiliani Richard , Nightingale Charles H. 
Published: July 2002

ABSTRACT

The role of moxifloxacin and levofloxacin pharmacokinetics (PK) in antimicrobial efficacy and in the selection of fluoroquinolone-resistant
Streptococcus pneumoniae
strains was investigated using the rabbit tissue cage abscess model. A rabbit tissue cage was created by insertion of sterile Wiffle balls in the dorsal cervical area. Animals orally received a range of moxifloxacin or levofloxacin doses that simulate human PK for 7 days 48 h after the Wiffle balls were inoculated with fluoroquinolone-sensitive
S. pneumoniae
(10
7
CFU). Abscess fluid was collected on a daily basis over 14 days to measure bacterial density and MICs. Moxifloxacin regimens produced a range of area under the concentration-time curve (AUC)/MIC ratios ranging from 9.2 to 444 and peak/MIC ratios ranging from 1.3 to 102. Levofloxacin doses produced AUC/MIC ratios of 5.1 to 85.5 and peak/MIC ratio of 0.9 to 14.8. Moxifloxacin at 6.5, 26, and 42 mg/kg reduced the bacterial log CFU per milliliter in abscess fluid (percentage of that in a sterile animal) by 4.2 ± 2.2 (20%), 5.8 ± 0.4 (100%), and 5.4 ± 0.4 (100%), respectively, over the dosing period. Levofloxacin at 5.5, 22, and 32 mg/kg reduced the log CFU per milliliter in abscess fluid (percentage of that in a sterile animal) by 2.8 ± 0.7 (20%), 5.1 ± 1.3 (80%), and 4.6 ± 1.3 (60%), respectively. Moxifloxacin has a greater bactericidal rate as determined by regression of log CFU versus time data. The AUC/MIC and peak/MIC ratios correlated with the efficacy of both drugs (
P
< 0.05). Resistance to either drug did not develop with any of the doses as assessed by a change in the MIC. In conclusion, data derived from this study show that moxifloxacin and levofloxacin exhibit rapid bactericidal activity against
S. pneumoniae
in vivo, and moxifloxacin exhibits enhanced bactericidal activity compared to levofloxacin, with AUC/MIC and peak/MIC ratios correlated with antimicrobial efficacy for both drugs. The development of fluoroquinolone-resistant
S. pneumoniae
was not observed with either drug in this model.