bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Nutrient Limitation and Stationary-Phase Existence in
Klebsiella pneumoniae
Biofilm Resistance to Ampicillin and Ciprofloxacin
Authors: Anderl Jeff N. , Zahller Jeff , Roe Frank , Stewart Philip S. 
Published: March 2003

ABSTRACT

Biofilms formed by
Klebsiella pneumoniae
resisted killing during prolonged exposure to ampicillin or ciprofloxacin even though these agents have been shown to penetrate bacterial aggregates. Bacteria dispersed from biofilms into medium quickly regained most of their susceptibility. Experiments with free-floating bacteria showed that stationary-phase bacteria were protected from killing by either antibiotic, especially when the test was performed in medium lacking carbon and nitrogen sources. These results suggested that the antibiotic tolerance of biofilm bacteria could be explained by nutrient limitation in the biofilm leading to stationary-phase existence of at least some of the cells in the biofilm. This mechanism was supported by experimental characterization of nutrient availability and growth status in biofilms. The average specific growth rate of bacteria in biofilms was only 0.032 h
−1
compared to the specific growth rate of planktonic bacteria of 0.59 h
−1
measured in the same medium. Glucose did not penetrate all the way through the biofilm, and oxygen was shown to penetrate only into the upper 100 μm. The specific catalase activity was elevated in biofilm bacteria to a level similar to that of stationary-phase planktonic cells. Transmission electron microscopy revealed that bacteria were affected by ampicillin near the periphery of the biofilm but were not affected in the interior. Taken together, these results indicate that
K. pneumoniae
in this system experience nutrient limitation locally within the biofilm, leading to zones in which the bacteria enter stationary phase and are growing slowly or not at all. In these inactive regions, bacteria are less susceptible to killing by antibiotics.
High-Efficiency Generation of Antibiotic-Resistant Strains of
Streptococcus pneumoniae
by PCR and Transformation
Authors: Martín-Galiano Antonio J. , de la Campa Adela G. 
Published: March 2003

ABSTRACT

We designed a method by which to generate antibiotic-resistant strains of
Streptococcus pneumoniae
at frequencies 4 orders of magnitude greater than the spontaneous mutation rate. The method is based on the natural ability of this organism to be genetically transformed with PCR products carrying sequences homologous to its chromosome. The genes encoding the targets of ciprofloxacin (
parC
, encoding the ParC subunit of DNA topoisomerase IV), rifampin (
rpoB
, encoding the β subunit of RNA polymerase), and streptomycin (
rpsL
, encoding the S12 ribosomal protein) from susceptible laboratory strain R6 were amplified by PCR and used to transform the same strain. Resistant mutants were obtained with a frequency of 10
−4
to 10
−5
, depending on the fidelity of the DNA polymerase used for PCR amplifications. Ciprofloxacin-resistant mutants, for which the MICs were four-to eightfold higher than that for R6, carried a single mutation of a residue in the quinolone resistance-determining region: S79 (change to A, F, or Y) or D83 (change to N or V). Rifampin-resistant strains, for which the MICs were at least 133-fold higher than that for R6, contained a single mutation within cluster I of
rpoB
: S482 (change to P), Q486 (change to L), D489 (change to V), or H499 (change to L or Y). Streptomycin-resistant mutants, for which the MICs were at least 64-fold higher than that for R6, carried a mutation at either K56 (change to I, R, or T) or K101 (change to E). PCR products obtained from the mutants were able to transform R6 to resistance with high efficiency (>10
4
). This method could be used to efficiently obtain resistant mutants for any drug whose target is known.
Vancomycin Treatment Failure Associated with Heterogeneous Vancomycin-Intermediate
Staphylococcus aureus
in a Patient with Endocarditis and in the Rabbit Model of Endocarditis
Authors: Moore Matthew R. , Perdreau-Remington Françoise , Chambers Henry F. 
Published: March 2003

ABSTRACT

Heterogeneous resistance to vancomycin is thought to precede emergence of intermediate susceptibility to vancomycin in
Staphylococcus aureus
, but the clinical significance of heterogeneous resistance is unknown. Paired
S. aureus
isolates from a patient with endocarditis who relapsed after vancomycin treatment were tested for heterogeneous resistance to vancomycin. The pretreatment and the relapse clinical isolates (strains SF1 and SF2, respectively) were genotyped by pulsed-field gel electrophoresis. Susceptibility to vancomycin was assessed by the broth dilution method, population analysis, and time-kill studies and in the rabbit model of endocarditis. Strains SF1 and SF2 had similar genotypes, and the vancomycin MICs for the strains were ≤2 μg/ml. SF2 exhibited heterogeneous resistance to vancomycin. Vancomycin eradicated SF1 in the rabbit model of endocarditis, while SF2 persisted at pretreatment levels. Vancomycin treatment failure in this patient with endocarditis was attributable to heterogeneous resistance to vancomycin.
Isolation of Antibiotic Hypersusceptibility Mutants of
Acinetobacter
spp. by Selection for DNA Release
Authors: Lee Hyunwoo , Vázquez-Laslop Nora , Klyachko Katya A. , Neyfakh Alex A. 
Published: March 2003

ABSTRACT

Isolation of bacterial mutants hypersusceptible to antibiotics can reveal novel targets for antibiotic potentiators. However, identification of such mutants is a difficult task which normally requires laborious replica plating of thousands of colonies. The technique proposed here allows for the positive selection of genetic knockout mutants leading to hypersusceptibility. This technique, designated SDR (selection for DNA release), involves introduction of random insertions of a marker gene into the chromosome of a highly transformable bacterial species, followed by treatment of the obtained library with an antibiotic at subinhibitory concentrations. DNA released by lysing bacteria is collected and used to transform fresh bacteria, selecting for insertion of the marker gene. These selection cycles are repeated until variants with a hypersusceptibility phenotype caused by insertion of the marker begin to dominate in the library. This approach allowed for isolation of a number of mutants of the gram-negative opportunistic pathogen
Acinetobacter
sp. susceptible to 4- to 16-times-lower concentrations of ampicillin than wild-type bacteria. The mutations affected proteins involved in peptidoglycan turnover and, surprisingly, proteins involved in exopolysaccharide production. A further modification of the SDR technique is described which allows for selecting mutants hypersensitive to agents that affect bacterial physiology but do not cause cell lysis, e.g., inhibitors of translation. This application of SDR is illustrated here by identification of several mutants of
Acinetobacter
sp. with increased susceptibility (two- to fivefold decrease in the MIC) to erythromycin. The same technique can be used to identify prospective targets for potentiators of many other antibacterial agents.
Flucytosine-Fluconazole Cross-Resistance in Purine-Cytosine Permease-Deficient
Candida lusitaniae
Clinical Isolates: Indirect Evidence of a Fluconazole Uptake Transporter
Authors: Noël Thierry , François Fabienne , Paumard Patrick , Chastin Christiane , Brèthes Daniel , Villard Jean 
Published: March 2003

ABSTRACT

An unusual interaction between flucytosine and fluconazole was observed when a collection of 60
Candida lusitaniae
clinical isolates was screened for cross-resistance. Among eight isolates resistant to flucytosine (MIC ≥ 128 μg/ml) and susceptible to fluconazole (0.5 < MIC < 2 μg/ml), four became flucytosine-fluconazole cross resistant when both antifungals were used simultaneously. Fluconazole resistance occurred only in the presence of high flucytosine concentrations, and the higher the fluconazole concentration used, the greater the flucytosine concentration necessary to trigger the cross-resistance. When the flucytosine- and fluconazole-resistant cells were grown in the presence of fluconazole alone, the cells reversed to fluconazole susceptibility. Genetic analyses of the progeny from crosses between resistant and sensitive isolates showed that resistance to flucytosine was derived from a recessive mutation in a single gene, whereas cross-resistance to fluconazole seemed to vary like a quantitative trait. We further demonstrated that the four clinical isolates were susceptible to 5-fluorouracil and that cytosine deaminase activity was unaffected. Kinetic transport studies with [
14
C]flucytosine showed that flucytosine resistance was due to a defect in the purine-cytosine permease. Our hypothesis was that extracellular flucytosine would subsequently behave as a competitive inhibitor of fluconazole uptake transport. Finally, in vitro selection of spontaneous and induced mutants indicated that such a cross-resistance mechanism could also affect other
Candida
species, including
C. albicans
,
C. tropicalis
, and
C. glabrata
. This is the first report of a putative fluconazole uptake transporter in
Candida
species and of a possible resistance mechanism associated with a deficiency in the uptake of this drug.
Integron-Containing IncU R Plasmids pRAS1 and pAr-32 from the Fish Pathogen
Aeromonas salmonicida
Authors: Sørum Henning , L'Abée-Lund Trine M. , Solberg Asta , Wold Anette 
Published: March 2003

ABSTRACT

A 45-kb R plasmid, pRAS1, that confers resistance to tetracyclines, trimethoprim, and sulfonamides was isolated in 1989 from an atypical strain of the fish pathogen
Aeromonas salmonicida
. This plasmid could be transferred by conjugation to
Escherichia coli
with a high degree of efficiency (frequency, 0.48). The following year pRAS1 was isolated from
A. salmonicida
subsp.
salmonicida
in the same area. Incompatibility group U plasmid pRAS1 contained a drug resistance-determining region of 12 kb consisting of a class 1 integron similar to In4 of Tn
1696
but with a
dfrA16
gene cassette inserted. Close to IS
6100
at the right end of Tn4 was a truncated Tn
1721
. Restriction enzyme analysis showed that R plasmid pAr-32, isolated from
A. salmonicida
in Japan in 1970, had the same backbone structure as pRAS1, while the drug resistance-determining region contained a complex class 1 integron with an
aadA2
cassette; the chloramphenicol resistance gene
catA2
, as in In6 of pSa; and a duplicate of the 3′ conserved segment of the integron.
Engineering Anthracycline Biosynthesis toward Angucyclines
Authors: Metsä-Ketelä Mikko , Palmu Kaisa , Kunnari Tero , Ylihonko Kristiina , Mäntsälä Pekka 
Published: March 2003

ABSTRACT

The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase,
pgaF
, isolated from a silent antibiotic biosynthesis gene cluster. Addition of
pgaF
to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of
pgaF
was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with
aknE2
and
aknF
, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.
Imipenem Resistance in a
Salmonella
Clinical Strain Due to Plasmid-Mediated Class A Carbapenemase KPC-2
Authors: Miriagou Vivi , Tzouvelekis Leonidas S. , Rossiter Shannon , Tzelepi Eva , Angulo Frederick J. , Whichard Jean M. 
Published: March 2003

ABSTRACT

A
Salmonella enterica
serotype Cubana isolate exhibiting resistance to most β-lactam antibiotics, including oxyimino-cephalosporins and imipenem, was isolated from a 4-year-old boy with gastroenteritis in Maryland. β-Lactam resistance was mediated by a conjugative plasmid that encoded KPC-2, a class A carbapenemase previously found in a
Klebsiella pneumoniae
isolate from the Maryland area as well. Sequence analysis of the flanking regions indicated a potential association of
bla
KPC-2
with mobile structures.
Use of Genetically Engineered Phage To Deliver Antimicrobial Agents to Bacteria: an Alternative Therapy for Treatment of Bacterial Infections
Authors: Westwater Caroline , Kasman Laura M. , Schofield David A. , Werner Phillip A. , Dolan Joseph W. , Schmidt Michael G. , Norris James S. 
Published: March 2003

ABSTRACT
The emergence and increasing prevalence of multidrug-resistant bacterial pathogens emphasizes the need for new and innovative antimicrobial strategies. Lytic phages, which kill their host following amplification and release of progeny phage into the environment, may offer an alternative strategy for combating bacterial infections. In this study, however, we describe the use of a nonlytic phage to specifically target and deliver DNA encoding bactericidal proteins to bacteria. To test the concept of using phage as a lethal-agent delivery vehicle, we used the M13 phagemid system and the addiction toxins Gef and ChpBK. Phage delivery of lethal-agent phagemids reduced target bacterial numbers by several orders of magnitude in vitro and in a bacteremic mouse model of infection. Given the powerful genetic engineering tools available and the present knowledge in phage biology, this technology may have potential use in antimicrobial therapies and DNA vaccine development.
Bactericidal Activities of Methoxyfluoroquinolones Gatifloxacin and Moxifloxacin against Aerobic and Anaerobic Respiratory Pathogens in Serum
Authors: Stein Gary E. , Schooley Sharon , Tyrrell Kerin L. , Citron Diane M. , Goldstein Ellie J. C. 
Published: March 2003

ABSTRACT

Gatifloxacin (Bristol-Myers Squibb) and moxifloxacin (Bayer) are new methoxyfluoroquinolones with broad-spectrum activity against aerobic and anaerobic pathogens of the respiratory tract. In this investigation, we analyzed the bactericidal activity in serum over time of these antimicrobials against three aerobic (
Streptococcus pneumoniae, Haemophilus influenzae,
and
Staphylococcus aureus
) and four anaerobic (
Peptostreptococcus micros, Peptostreptococcus magnus, Fusobacterium nucleatum,
and
Prevotella melaninogenica
) bacteria associated with respiratory tract infections. Serum samples were obtained from 11 healthy male subjects following a single 400-mg oral dose of gatifloxacin and moxifloxacin. These samples were collected prior to and at 2, 6, 12, and 24 h after the dose of each drug. Gatifloxacin exhibited bactericidal activity for a median of 12 h against
Streptococcus pneumoniae
(MIC = 0.5 μg/ml),
Peptostreptococcus micros
(MIC = 0.25 μg/ml), and
F. nucleatum
(MIC = 0.5 μg/ml) and 24 h against
H. influenzae
(MIC = 0.03 μg/ml),
Staphylococcus aureus
(MIC = 0.125 μg/ml),
Peptostreptococcus magnus
(MIC = 0.125 μg/ml), and
Prevotella melaninogenica
(MIC = 0.5 μg/ml). Moxifloxacin exhibited bactericidal activity for a median of 24 h against
Streptococcus pneumoniae
(MIC = 0.125 μg/ml),
H. influenzae
(MIC = 0.015 μg/ml),
Staphylococcus aureus
(MIC = 0.06 μg/ml),
F. nucleatum
(MIC = 0.5 μg/ml),
Prevotella melaninogenica
(MIC =0.5 μg/ml),
Peptostreptococcus magnus
(MIC = 0.125 μg/ml), and
Peptostreptococcus micros
(MIC = 0.25 μg/ml). The results from this pharmacodynamic study suggest that these fluoroquinolones would have prolonged killing activity against these organisms in vivo and may have clinical utility in the treatment of mixed aerobic-anaerobic respiratory tract infections.
Rifampin Followed by Ceftriaxone for Experimental Meningitis Decreases Lipoteichoic Acid Concentrations in Cerebrospinal Fluid and Reduces Neuronal Damage in Comparison to Ceftriaxone Alone
Authors: Gerber Joachim , Pohl Karin , Sander Valeska , Bunkowski Stephanie , Nau Roland 
Published: March 2003

ABSTRACT

Rifampin (RIF) releases smaller quantities of lipoteichoic acids (LTAs) from
Streptococcus pneumoniae
than ceftriaxone (CRO). Due to the rapid development of resistance, RIF cannot be used as a single agent for therapy of bacterial meningitis. For this reason, we compared the effect of treatment with RIF followed by treatment with CRO (RIF-CRO) or the effect of treatment with clindamycin (CLI) followed by treatment with CRO (CLI-CRO) to that of CRO alone on the concentrations of LTAs and teichoic acids in vitro. The effects of RIF-CRO on LTA concentrations in cerebrospinal fluid (CSF) and on neuronal injury were investigated in a rabbit model of
S. pneumoniae
meningitis. In vitro, bacterial titers were effectively reduced by CRO, RIF-CRO, and CLI-CRO when each drug was used at 10 μg/ml. The levels of release of LTAs after the initiation of therapy were lower in RIF-CRO- and CLI-CRO-treated cultures than in cultures treated with CRO alone (
P
< 0.05 from 3 to 12 h after initiation of treatment). Similarly, in rabbits, the increase in the amount of LTAs in CSF was lower in RIF-CRO-treated animals than in CRO-treated animals (
P
= 0.02). The density of dentate apoptotic granular cells was lower after RIF-CRO therapy than after CRO therapy (medians, 58.4 and 145.6/mm
2
, respectively; 25th quartiles, 36.3 and 81.7/mm
2
, respectively; 75th quartiles, 100.7 and 152.3/mm
2
, respectively;
P
= 0.03). Therefore, initiation of therapy with a protein synthesis-inhibiting antibacterial and continuation of therapy with a combination that includes a β-lactam may be a strategy to decrease neuronal injury in bacterial meningitis.
Daptomycin Pharmacokinetics and Safety following Administration of Escalating Doses Once Daily to Healthy Subjects
Authors: Dvorchik Barry H. , Brazier David , DeBruin Michael F. , Arbeit Robert D. 
Published: March 2003

ABSTRACT
The purpose of this paper is to establish the pharmacokinetics and safety of escalating, once-daily doses of daptomycin, a novel lipopeptide antibiotic active against gram-positive pathogens, including those resistant to methicillin and vancomycin. This phase 1, multiple-dose, double-blind study involved 24 healthy subjects in three dose cohorts (4, 6, and 8 mg/kg of body weight) who were randomized to receive daptomycin or the control at a 3:1 ratio and administered the study medication by a 30-min intravenous infusion every 24 h for 7 to 14 days. Daptomycin pharmacokinetics was assessed by blood and urine sampling. Safety and tolerability were evaluated by monitoring adverse events (AEs) and laboratory parameters. Daptomycin pharmacokinetics was linear through 6 mg/kg, with a slight (∼20%) nonlinearity in the area under the curve and trough concentration at the highest dose studied (8 mg/kg). The pharmacokinetic parameters measured on the median day of the study period, (day 7) were half-life (∼9 h), volume of distribution (∼0.1 liters/kg), systemic clearance (∼8.2 ml/h/kg), and percentage of the drug excreted intact in urine from 0 to 24 h (∼54%). Daptomycin protein binding (mean amount bound, 91.7%) was independent of the drug concentration. No gender effect was observed. All subjects who received daptomycin completed the study. The frequencies and distributions of treatment-emergent AEs were similar for the subjects who received daptomycin and the control subjects. There were no serious AEs and no pattern of dose-related events. The pharmacokinetics of once-daily administration of daptomycin was linear through 6 mg/kg. For all three doses, plasma daptomycin concentrations were consistent and predictable throughout the dosing interval. Daptomycin was well tolerated when it was administered once daily at a dose as high as 8 mg/kg for 14 days.
Activities of Atazanavir (BMS-232632) against a Large Panel of Human Immunodeficiency Virus Type 1 Clinical Isolates Resistant to One or More Approved Protease Inhibitors
Authors: Colonno Richard J. , Thiry Alexandra , Limoli Kay , Parkin Neil 
Published: March 2003

ABSTRACTTo evaluate the cross-resistance profile of the human immunodeficiency virus type 1 protease inhibitor (PI) atazanavir (BMS-232632), a panel of 551 clinical isolates exhibiting a wide array of PI resistance profiles and a variety of genotypic patterns were assayed for susceptibility to atazanavir and six other PIs: amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir. In general, reductions in atazanavir susceptibility in vitro required several amino acid changes and were relatively modest in degree, and susceptibility was retained among isolates resistant to one or two of the currently approved PIs. There was a clear trend toward loss of susceptibility to atazanavir, as isolates exhibited increasing levels of cross-resistance to multiple PIs. Atazanavir appeared to have a distinct resistance profile relative to each of the other six PIs tested based on susceptibility comparisons against this panel of resistant isolates. Analysis of the genotypic profiles of 943 PI-susceptible and -resistant clinical isolates identified a strong correlation between the presence of amino acid changes at specific residues (10I/V/F, 20R/M/I, 24I, 33I/F/V, 36I/L/V, 46I/L, 48V, 54V/L, 63P, 71V/T/I, 73C/S/T/A, 82A/F/S/T, 84V, and 90M) and decreased susceptibility to atazanavir. While no single substitution or combination of substitutions was predictive of atazanavir resistance (change, >3.0-fold), the presence of at least five of these substitutions correlated strongly with loss of atazanavir susceptibility. Mutations associated with reduced susceptibility to each of the other six PIs were also determined.
Phase I Safety and Pharmacokinetic Trials of 1263W94, a Novel Oral Anti-Human Cytomegalovirus Agent, in Healthy and Human Immunodeficiency Virus-Infected Subjects
Authors: Wang Laurene H. , Peck Richard W. , Yin Yin , Allanson Jane , Wiggs Rebecca , Wire Mary Beth 
Published: March 2003

ABSTRACT

1263W94 [maribavir; 5,6-dichloro-2-(isopropylamino)-1, β-
l
-ribofuranosyl-1-
H
-benzimidazole], a novel benzimidazole compound, has been demonstrated to potently and selectively inhibit human cytomegalovirus replication in vitro and to have favorable safety profiles in animal species. Two phase I trials evaluated the safety and pharmacokinetics of escalating single doses of 1263W94 in 13 healthy subjects (dose, 50 to 1,600 mg) and 17 human immunodeficiency virus (HIV)-infected subjects (dose, 100 to 1,600 mg). No severe safety concerns were observed in the evaluation of adverse events, vital signs, electrocardiograms, and clinical laboratory tests following administration of a single dose of 1263W94. The most frequently reported adverse events in both populations were taste disturbance (80%) and headache (53%). 1263W94 was rapidly absorbed following oral administration, with peak concentrations in plasma (
C
max
) occurring 1 to 3 h after dosing. The increases in the
C
max
of 1263W94 and the area under the concentration-time curve from time zero to infinity (AUC
0-∞
) for 1263W94 were dose dependent;
C
max
increased slightly less than proportionally to the dose, and AUC
0-∞
increased slightly more than proportionally to the dose. 1263W94 was rapidly eliminated, with a mean half-life in plasma of 3 to 5 h; the half-life was independent of the dose level. Less than 2% of the 1263W94 dose administered was eliminated unchanged in urine. The principal metabolite of 1263W94 was 4469W94 (which is derived by N-dealkylation of 1263W94 via CYP3A4), which accounted for 30 to 40% of the dose in urine. Greater than 98% of the 1263W94 in plasma is bound to proteins, and the extent of binding appears to be constant over the dose range of 200 to 1,600 mg. In the trial with HIV-infected subjects, consumption of a high-fat meal decreased the 1263W94 AUC
0-∞
and
C
max
in plasma by ∼30%.