bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow Cytometric Investigation of Filamentation, Membrane Patency, and Membrane Potential in
Escherichia coli
following Ciprofloxacin Exposure
Authors: Wickens H. J. , Pinney R. J. , Mason D. J. , Gant V. A.
Published: July 2002

Ninety-eight percent of the cells in a population of
Escherichia coli
in log-phase growth lost colony-forming ability after being exposed for 3 h to the quinolone antibiotic ciprofloxacin at four times the MIC in nutrient broth, a concentration easily reached in vivo. Flow cytometric analysis, however, demonstrated that only 68% of this bacterial population had lost membrane potential, as judged by the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC
4
(3)], and only 30% could no longer exclude the nucleic acid-binding dye propidium iodide (PI), reflecting lost membrane integrity, efflux mechanisms, or both. Subsequent removal of ciprofloxacin and resuspension in nutrient broth resulted in renewed cell division after 2 h, with a calculated postantibiotic effect (PAE) time of 57 min. The proportion of DiBAC- and PI-fluorescent cells in this recovering population remained stable for more than 4 h after antibiotic removal. Eighty percent of cells present at drug removal were filamentous. Their number subsequently decreased with time, and the increase in particle count seen at the end of the PAE resulted from the division of short cells. Exposure to ciprofloxacin in the presence of the protein synthesis inhibitor chloramphenicol increased colony-forming ability to 60% of starting population numbers. In contrast to ciprofloxacin alone, this antibiotic combination resulted in insignificant filamentation and no dye uptake. Subsequent drug removal and resuspension in nutrient broth resulted in the appearance of filaments within 1 h, with 69% of the population forming filaments at 3 h. Dye uptake was also seen, with 20% of the population fluorescing with either dye after 4 h. We were unable to relate dye uptake to the viable count. Cell division resumed 240 min after removal of both drugs, yielding a PAE calculated at 186 min. Inhibition of protein synthesis with chloramphenicol prevented ciprofloxacin-induced changes in bacterial morphology, cell membrane potential, and ability to exclude nucleic acid-binding dye. These changes persisted beyond the end of the classically defined PAE and were not a definite indicator of cell death as defined by loss of colony formation, which related at least in part to filamentation.
Risk Factors for Recovery of Ampicillin-Sulbactam-Resistant
Escherichia coli
in Hospitalized Patients
Authors: Kaye Keith S. , Harris Anthony D. , Gold Howard , Carmeli Yehuda
Published: July 2002

Ampicillin-sulbactam resistance in
Escherichia coli
is an emerging problem. This study determined risk factors for the recovery of ampicillin-sulbactam-resistant
E. coli
in hospitalized patients. A case-control design was used to compare two groups of case patients with control patients. The first group of case patients consisted of patients from whom nosocomially acquired ampicillin-sulbactam-resistant
E. coli
strains were isolated, and the second group of case patients consisted of patients from whom ampicillin-sulbactam-susceptible
E. coli
strains were isolated. Control patients were a random selection among 5% of all patients admitted during the same time period. Risk factors analyzed included antimicrobial drug exposure, comorbid conditions, and demographics. Univariate and multivariate analyses were performed. Ampicillin-sulbactam-resistant
E. coli
strains were isolated from 175 patients, and ampicillin-sulbactam-susceptible
E. coli
strains were isolated from 577 patients. Nine hundred thirty-four control patients were selected. Exposure to penicillin antibiotics as a class and to ampicillin and ampicillin-sulbactam individually were the only significant, independent risk factors associated with the isolation of ampicillin-sulbactam-resistant
E. coli
(odds ratio [OR] = 2.32 [
P
< 0.001], OR = 3.04 [
P
= 0.02], and OR = 1.72 [
P
= 0.04], respectively), but they were not associated with the isolation of ampicillin-sulbactam-susceptible
E. coli
. Interestingly, exposure to piperacillin-tazobactam tended to protect against the isolation of
E. coli
strains resistant to ampicillin-sulbactam, but this did not reach statistical significance (OR = 0.13;
P
= 0.11).
Non-Target Gene Mutations in the Development of Fluoroquinolone Resistance in
Escherichia coli
Authors: Kern W. V. , Oethinger M. , Jellen-Ritter A. S. , Levy S. B.
Published: July 2002

Mutations in loci other than genes for the target topoisomerases of fluoroquinolones,
gyrA
and
parC
, may play a role in the development of fluoroquinolone resistance in
Escherichia coli
. A series of mutants with increasing resistance to ofloxacin was obtained from an
E. coli
K-12 strain and five clinical isolates. First-step mutants acquired a
gyrA
mutation. Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux. None of the second-step mutants showed additional topoisomerase mutations. All second-step mutants showed constitutive expression of
marA
and/or overexpressed
soxS
. In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations. Attempts to circumvent the second-step Mar mutation by induction of the
mar
locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful. At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the
mar
or
sox
regulons with consequent increased drug efflux.
A between-Species Comparison of Antimicrobial Resistance in Enterobacteria in Fecal Flora
Authors: Österblad Monica , Hakanen Antti , Manninen Raija , Leistevuo Tiina , Peltonen Reijo , Meurman Olli , Huovinen Pentti , Kotilainen Pirkko
Published: July 2002

Enterobacteria in fecal flora are often reported to be highly resistant.
Escherichia coli
is the main species; resistance data on other species are rare. To assess the effect of the host's environment, antimicrobial resistance was determined in fecal species of the family
Enterobacteriaceae
from three populations: healthy people (HP)(
n
= 125) with no exposure to antimicrobials for 3 months preceding sampling, university hospital patients (UP) (
n
= 159) from wards where the antibiotic use was 112 defined daily doses (DDD)/bed/month, and geriatric long-term patients (LTP) (
n
= 74) who used 1.8 DDD/bed/month. The mean length of hospital stay was 5 days for the UP and 22 months for the LTP. The isolates were identified to at least genus level, and MICs of 16 antimicrobials were determined. From the university hospital, resistance data on clinical
Enterobacteriaceae
isolates were also collected. Resistance data for on average two different isolates per sample (range, 1 to 5) were analyzed: 471
E. coli
isolates and 261 other
Enterobacteriaceae
spp. Resistance was mainly found among
E. coli
; even in HP, 18% of
E. coli
isolates were resistant to two or more antimicrobial groups, with MIC patterns indicative of transferable resistance. Other fecal enterobacteria were generally susceptible, with little typically transferable multiresistance. Clinical
Klebsiella
and
Enterobacter
isolates were significantly more resistant than fecal isolates. The resistance patterns at both hospitals mirrored the patterns of antibiotic use, but LTP
E. coli
isolates were significantly more resistant than those from UP. Conditions permitting an efficient spread may have been more important in sustaining high resistance levels in the LTP.
E. coli
was the main carrier of antimicrobial resistance in fecal flora; resistance in other species was rare in the absence of antimicrobial selection.
Combination Antifungal Therapy in Treatment of Murine Pulmonary Mucormycosis: Roles of Quinolones and Azoles
Authors: Sugar Alan M. , Liu Xiu-Ping
Published: July 2002

Amphotericin B is the only recognized antifungal used in the treatment of mucormycosis. In this study, we evaluated various combinations of amphotericin B, fluconazole, and trovafloxacin or ciprofloxacin in the treatment of murine pulmonary mucormycosis. The combination of fluconazole and a quinolone has a marked effect on the outcome of murine pulmonary mucormycosis. Even though we did not optimize therapy with the drugs, these experiments suggest that azoles, especially fluconazole, in combination with either trovafloxacin or ciprofloxacin were effective in the treatment of this aggressive mycosis in the mouse model.
Saccharomicins, Novel Heptadecaglycoside Antibiotics Produced by
Saccharothrix espanaensis
: Antibacterial and Mechanistic Activities
Authors: Singh M. P. , Petersen P. J. , Weiss W. J. , Kong F. , Greenstein M.
Published: July 2002

Saccharomicins A and B, two new heptadecaglycoside antibiotics, were isolated from the fermentation broth of the rare actinomycete
Saccharothrix espanaensis
. They represent a novel class of bactericidal antibiotics that are active both in vitro and in vivo against bacteria and yeast (MICs:
Staphylococcus aureus
, <0.12 to 0.5; vancomycin-resistant enterococci, 0.25 to 16; gram-negative bacteria, 0.25 to >128; and yeast, >128 μg/ml), including multiply resistant strains. Saccharomicins protected mice from lethal challenges by staphylococci (subcutaneous 50% effective dose range of 0.06 to 2.6 mg/kg of body weight, depending on the
S. aureus
strain). The 50% lethal dose by the subcutaneous route was 16 mg/kg. Mechanistic studies with
Escherichia coli imp
and
Bacillus subtilis
suggested complete, nonspecific inhibition of DNA, RNA, and protein biosynthesis within 10 min of drug treatment. Microscopic examination of drug-treated cells also suggested cell lysis. These data are consistent with a strong membrane-disruptive activity. The antibacterial activities of the saccharomicins against gram-positive bacteria were unaffected by the presence of Ca
2+
or Mg
2+
, but activity against gram-negative bacteria was substantially reduced.
Congeners of SMAP29 Kill Ovine Pathogens and Induce Ultrastructural Damage in Bacterial Cells
Authors: Kalfa V. C. , Jia H. P. , Kunkle R. A. , McCray P. B. , Tack B. F. , Brogden K. A.
Published: July 2002

SMAP29, an ovine cathelicidin, was systematically altered to create a family of 23 related peptides for MIC and minimum bactericidal concentration determinations. SMAP28, SMAP29, and a derivative of SMAP29 called ovispirin were all antimicrobial. However, many congeners of SMAP29 and ovispirin were not as active as the parent molecules. With immunoelectron microscopy, SMAP29 was seen on membranes and within the cytoplasm of
Pseudomonas aeruginosa
PAO1.
Antimicrobial Susceptibilities of Group B Streptococci Isolated from Patients with Invasive Disease: 10-Year Perspective
Authors: Murdoch David R. , Reller L. Barth
Published: July 2002

Antimicrobial susceptibility testing of 192 group B streptococcal isolates from patients with invasive disease demonstrated that 31 (16%) were resistant to erythromycin and 17 (9%) were resistant to clindamycin. One isolate demonstrated high-level resistance to streptomycin, but none was highly resistant to gentamicin. Erythromycin and clindamycin are no longer reliable empirical alternatives to penicillin for the treatment and prevention of group B streptococcal infections.
The AbcA Transporter of
Staphylococcus aureus
Affects Cell Autolysis
Authors: Schrader-Fischer Gesine , Berger-Bächi Brigitte
Published: July 2002

Increased production of penicillin-binding protein PBP 4 is known to increase peptidoglycan cross-linking and contributes to methicillin resistance in
Staphylococcus aureus
. The
pbp4
gene shares a 400-nucleotide intercistronic region with the divergently transcribed
abcA
gene, encoding an ATP-binding cassette transporter of unknown function. Our study revealed that methicillin stimulated
abcA
transcription but had no effects on
pbp4
transcription. Analysis of
abcA
expression in mutants defective for global regulators showed that
abcA
is under the control of
agr
. Insertional inactivation of
abcA
by an erythromycin resistance determinant did not influence
pbp4
transcription, nor did it alter resistance to methicillin and other cell wall-directed antibiotics. However,
abcA
mutants showed spontaneous partial lysis on plates containing subinhibitory concentrations of methicillin due to increased spontaneous autolysis. Since the autolytic zymograms of cell extracts were identical in mutants and parental strains, we postulate an indirect role of AbcA in control of autolytic activities and in protection of the cells against methicillin.
OXA-28, an Extended-Spectrum Variant of OXA-10 β-Lactamase fromPseudomonas aeruginosaand Its Plasmid- and Integron-Located Gene
Authors: Poirel Laurent , Girlich Delphine , Naas Thierry , Nordmann Patrice
Published: July 2002

Pseudomonas aeruginosaED-1, isolated from a pulmonary brush of a patient hospitalized in a suburb of Paris, France, was resistant to ceftazidime and of intermediate susceptibility to ureidopenicillins and to cefotaxime. Cloning and expression of the β-lactamase gene content of this isolate inEscherichia coliDH10B identified a novel OXA-10 variant, OXA-28, with a pI value of 8.1 and a molecular mass of 29 kDa. It differed from OXA-10 by 10 amino acid changes and from OXA-13 and OXA-19 by 2 amino acid changes, including a glycine instead of tryptophan at position 164, which is likely involved in its resistance to ceftazidime. Like OXA-11, -14, -16, and -19 and as opposed to OXA-17, OXA-28 predominantly compromised ceftazidime and had only marginal effect on the MICs of aztreonam and cefotaxime inP. aeruginosa. Once expressed inE. coli, OXA-28 raised the MIC of ceftazidime to a much higher level than those of amoxicillin, cephalothin, and cefotaxime (128, 16, 8, and 4 μg/ml, respectively). OXA-28 β-lactamase had a broad spectrum of activity, including ceftazidime. Its activity was partially antagonized by clavulanic acid (50% inhibitory concentration, 10 μM) and NaCl addition. Theoxa28gene cassette was inserted in the variable region of a class 1 integron, In57, immediately downstream of an amino 6′-N-acetyltransferase gene cassette,aac(6′)Ib. The structures of the integrons carrying eitheroxa28,oxa13, oroxa19gene cassettes were almost identical, suggesting that they may have derived from a common ancestor as a result of the common European origin of theP. aeruginosaisolates. In57 was located on a self-transferable plasmid of ca. 150 kb that was transferred fromP. aeruginosatoP. aeruginosa.
An ATP-Binding Cassette Transporter and Two rRNA Methyltransferases Are Involved in Resistance to Avilamycin in the Producer Organism
Streptomyces viridochromogenes
Tü57
Authors: Weitnauer Gabriele , Gaisser Sibylle , Trefzer Axel , Stockert Sigrid , Westrich Lucy , Quiros Luis M. , Mendez Carmen , Salas Jose A. , Bechthold Andreas
Published: July 2002

Three different resistance factors from the avilamycin biosynthetic gene cluster of
Streptomyces viridochromogenes
Tü57, which confer avilamycin resistance when expressed in
Streptomyces lividans
TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of
aviRb
showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in
S. lividans
TK66 of
aviABC1
plus
aviABC2, aviRa
, or
aviRb
conferred different levels of resistance to avilamycin: 5, 10, or 250 μg/ml, respectively. When either
aviRa
plus
aviRb
or
aviRa
plus
aviRb
plus
aviABC1
plus
aviABC2
was coexpressed in
S. lividans
TK66, avilamycin resistance levels reached more than 250 μg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of
S. lividans
TK66(pUWL201) (GWO),
S. lividans
TK66(pUWL201-Ra) (GWRa), or
S. lividans
TK66(pUWL201-Rb) (GWRb), whereas ribosomes of
S. lividans
TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant.
aviRa
and
aviRb
were expressed in
Escherichia coli
, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from
E. coli
as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.
Screening System for Xenosiderophores as Potential Drug Delivery Agents in Mycobacteria
Authors: Schumann Gisbert , Möllmann Ute
Published: July 2002

In order to establish a screening system for xenosiderophores which can be utilized by mycobacteria, we generated a set of mutants of
Mycobacterium smegmatis
that are blocked in different steps of the well-known iron acquisition system. One mutant with a block in mycobactin biosynthesis was generated from strain mc
2
155 by chemical mutagenesis. The exochelin biosynthesis gene
fxbA
and the ferric exochelin uptake gene
fxuA
, previously identified by Fiss et al. (E. H. Fiss, S. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557–559, 1994), were knocked out by gene replacement. Adjacent chromosomal fragments were used for homologous recombination in order to replace wild-type genes by the kanamycin resistance gene from transposon Tn
903
. Gene replacement was confirmed by PCR. The isolated mutants show the expected phenotype:
fxbA
mutants are defective in exochelin biosynthesis, whereas
fxuA
mutants excrete a significantly larger amount of exochelin compared to the amount excreted by the parent strain. This is due to their defectiveness in ferriexochelin uptake, as demonstrated in growth promotion assays. This new set of mutants allows differentiation of siderophores that supply mycobacteria with iron by ligand exchange with exochelin or mycobactin, by the use of separate siderophore uptake routes, or by the use of the exochelin permease. All these types of iron uptake routes were identified with 25 exogenous siderophores as test substances. Siderophores that act without ligand exchange are potential candidates as drug vectors that can be used to overcome permeability-mediated resistance.
Inducible Macrolide Resistance in
Corynebacterium jeikeium
Authors: Rosato Adriana E. , Lee Bonnie S. , Nash Kevin A.
Published: July 2002

Corynebacterium jeikeium
is an opportunistic pathogen primarily of immunocompromised (neutropenic) patients. Broad-spectrum resistance to antimicrobial agents is a common feature of
C. jeikeium
clinical isolates. We studied the profiles of susceptibility of 20 clinical strains of
C. jeikeium
to a range of antimicrobial agents. The strains were separated into two groups depending on the susceptibility to erythromycin (ERY), with one group (17 strains) representing resistant organisms (MIC > 128 μg/ml) and the second group (3 strains) representing susceptible organisms (MIC ≤ 0.25 μg/ml). The ERY resistance crossed to other members of the macrolide-lincosamide-streptogramin B (MLSb) group. Furthermore, this resistance was inducible with MLSb agents but not non-MLSb agents. Expression of ERY resistance was linked to the presence of an allele of the class X
erm
genes,
erm
(X)cj, with >93% identity to other
erm
genes of this class. Our evidence indicates that
erm
(X)cj is integrated within the chromosome, which contrasts with previous reports for the plasmid-associated
erm
(X) genes found in
C. diphtheriae
and
C. xerosis
. In 40% of
C. jeikeium
strains,
erm
(X)cj is present within the transposon, Tn
5432
. However, in the remaining strains, the components of Tn
5432
(i.e., the
erm
and transposase genes) have separated within the chromosome. The rearrangement of Tn
5432
leads to the possibility that the other drug resistance genes have become included in a new composite transposon bound by the IS
1249
elements.
Immunoprecipitation of [
3
H]Dihydroartemisinin Translationally Controlled Tumor Protein (TCTP) Adducts from
Plasmodium falciparum
-Infected Erythrocytes by Using Anti-TCTP Antibodies
Authors: Bhisutthibhan Jamaree , Meshnick Steven R.
Published: July 2002

Artemisinin and its derivatives are endoperoxide-containing antimalarial drugs that appear to form adducts in situ with the
Plasmodium falciparum
translationally controlled tumor protein (TCTP) homolog. Immunoprecipitation with antibody to recombinant TCTP suggests that adducts may form with both monomeric and dimeric TCTP.
New Class of Small Nonpeptidyl Compounds Blocks
Plasmodium falciparum
Development In Vitro by Inhibiting Plasmepsins
Authors: Jiang Suping , Prigge Sean T. , Wei Lan , Gao Yu-e , Hudson Thomas H. , Gerena Lucia , Dame John B. , Kyle Dennis E.
Published: July 2002

Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from
Plasmodium falciparum
and
Plasmodium vivax
have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of
P. falciparum
plasmepsin II and
P. vivax
plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a
K

i

of 1 to 6 μM. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D (
K

i

greater than 280 μM). WR268961 inhibited the growth of
P. falciparum
strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 μg/ml, but was much less toxic to mammalian cells. The Walter Reed chemical database contains over 1,500 compounds with a diphenylurea core structure, 9 of which inhibit the plasmepsins, with
K

i

values ranging from 0.05 to 0.68 μM. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of
P. falciparum
growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.
Countrywide Spread of CTX-M-3 Extended-Spectrum β-Lactamase-Producing Microorganisms of the Family
Enterobacteriaceae
in Poland
Authors: Baraniak Anna , Fiett Janusz , Sulikowska Agnieszka , Hryniewicz Waleria , Gniadkowski Marek
Published: July 2002

Eighty-four clinical isolates of the family
Enterobacteriaceae
, recovered from 1998 to 2000 in 15 hospitals in 10 Polish cities, were analyzed. All the isolates produced β-lactamases with pIs of 8.4 and 5.4, and the pI 8.4 enzymes were demonstrated to hydrolyze cefotaxime but not ceftazidime in the in vitro bioassay. PCR analysis and DNA sequencing have revealed that in all cases the pI 8.4 β-lactamase was probably the CTX-M-3 extended-spectrum β-lactamase (ESBL) variant, which was originally identified in 1996 in Praski Hospital in Warsaw. In the majority of isolates,
bla
CTX-M-3
genes resided within large conjugative plasmids with similar fingerprints, which, in the context of the high degree of diversity of the randomly amplified polymorphic DNA types of the isolates, suggested that horizontal transfer of plasmids was likely the main mechanism of CTX-M-3 spread. The dissemination of plasmids was probably preceded by the center-to-center transmission of several strains, as indicated by the identification by pulsed-field gel electrophoresis of closely related or possibly related
Klebsiella pneumoniae
,
Escherichia coli
, and
Citrobacter freundii
isolates in five different hospitals. CTX-M-3-producing organisms revealed a very high degree of diversity in β-lactam resistance levels and patterns. This was attributed to several factors, such as the production of other β-lactamases including additional ESBLs, possible quantitative variations in CTX-M-3 expression, segregation of AmpC derepressed mutants, and permeability alterations.
bla
VIM-2
Cassette-Containing Novel Integrons in Metallo-β-Lactamase-Producing
Pseudomonas aeruginosa
and
Pseudomonas putida
Isolates Disseminated in a Korean Hospital
Authors: Lee Kyungwon , Lim Jong Back , Yum Jong Hwa , Yong Dongeun , Chong Yunsop , Kim June Myung , Livermore David M.
Published: July 2002

We investigated the phenotypic and genetic properties of metallo-β-lactamase-producing
Pseudomonas
isolates collected at a tertiary-care hospital in Korea since 1995. The prevalence of imipenem resistance among
Pseudomonas aeruginosa
isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 β-lactamase, a class B enzyme previously found only in
P. aeruginosa
isolates from Europe. VIM-2-producing isolates of
Pseudomonas putida
were also detected. Resistance was transferable from both these species to
P. aeruginosa
PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid. Serotyping showed that many of the VIM-2-producing
P. aeruginosa
isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of
Xba
I-digested genomic DNA revealed that many had identical profiles, whereas the
P. putida
isolates were diverse. Sequencing showed that the
bla
VIM-2
genes resided as cassettes in class 1 integrons. In contrast to previous VIM-encoding integrons, the integron sequenced from a
P. aeruginosa
isolate had
bla
VIM
located downstream of a variant of
aacA4. bla
VIM
also lay in a class 1 integron in a representative
P. putida
strain, but the organization of this integron was different from that sequenced from the
P. aeruginosa
strain. In conclusion, the metallo-β-lactamase produced by these imipenem-resistant
Pseudomonas
isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread. Strict measures are required in order to control a further spread of resistance.
Design and Antileishmanial Activity of Amphotericin B-Loaded Stable Ionic Amphiphile Biovector Formulations
Authors: Loiseau P. M. , Imbertie L. , Bories C. , Betbeder D. , De Miguel I.
Published: July 2002

A new delivery system, Ionic Amphiphilic Biovector (ABV), comprised of anionic lipids (dipalmitoyl phosphatidyl glycerol) included in a cationic cross-linked polysaccharide matrix was used as a reservoir for amphotericin B (AmB). Two ABV formulations exhibited an in vitro and in vivo efficacy similar to commercial AmBisome against
Leishmania donovani
-infected mice. The higher stability of these ABV formulations indicates their potential for further development and applications.
Urea Hydrogen Peroxide Reduces the Numbers of Lactobacilli, Nourishes Yeast, and Leaves No Residues in the Ethanol Fermentation
Authors: Narendranath N. V. , Thomas K. C. , Ingledew W. M.
Published: July 2002

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five
Lactobacillus
spp. (
Lactobacillus plantarum
,
L. paracasei
,
Lactobacillus
sp. strain 3,
L. rhamnosus
, and
L. fermentum
) from ∼10
7
to ∼10
2
CFU/ml in a 2-h preincubation at 30°C of normal-gravity wheat mash at ∼21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of
Saccharomyces cerevisiae
in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with
L. paracasei
at ∼10
7
CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of
L. paracasei
at ∼10
7
CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30°C whether the bactericidal agent was added as H
2
O
2
or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H
2
O
2
) increased significantly (
P
= 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H
2
O
2
. H
2
O
2
-resistant mutants were not expected or found when lethal levels of H
2
O
2
or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.
A Rhamnolipid Biosurfactant Reduces Cadmium Toxicity during Naphthalene Biodegradation
Authors: Sandrin Todd R. , Chech Andrea M. , Maier Raina M.
Published: July 2002

A model cocontaminated system was developed to determine whether a metal-complexing biosurfactant, rhamnolipid, could reduce metal toxicity to allow enhanced organic biodegradation by a
Burkholderia
sp. isolated from soil. Rhamnolipid eliminated cadmium toxicity when added at a 10-fold greater concentration than cadmium (890 μM), reduced toxicity when added at an equimolar concentration (89 μM), and had no effect at a 10-fold smaller concentration (8.9 μM). The mechanism by which rhamnolipid reduces metal toxicity may involve a combination of rhamnolipid complexation of cadmium and rhamnolipid interaction with the cell surface to alter cadmium uptake.
Ingested Blood Contributes to the Specificity of the Symbiosis ofAeromonas veroniiBiovar Sobria andHirudo medicinalis, the Medicinal Leech
Authors: Indergand Stefan , Graf Joerg
Published: July 2002

Hirudo medicinalis, the medicinal leech, usually carries in its digestive tract a pure culture ofAeromonas veroniibv. sobria. Such specificity is unusual for digestive tracts that are normally colonized by a complex microbial consortium. Important questions for the symbiotic interaction and for the medical application after microvascular surgery are whether other bacteria can proliferate or at least persist in the digestive tract ofH. medicinalisand what factors contribute to the reported specificity. Using a colonization assay, we were able to compare experimentally the ability of clinical isolates and of a symbiotic strain to colonizeH. medicinalis. The symbioticA. veroniibv. sobria strain proliferated well and persisted for at least 7 days inside the digestive tract. In contrast, the proliferation ofPseudomonas aeruginosaandStaphylococcus aureuswas inhibited inside the animal compared to growth in the in vitro control, indicating that the ingested blood was modified within the digestive tract. However, both strains were able to persist in the digestive tract for at least 7 days. For anEscherichia colistrain, the viable counts decreased approximately 1,000-fold within 42 h. The decrease of viableE. colicould be prevented by interfering with the activation of the membrane-attack complex of the complement system that is present in blood. This suggests that the membrane-attack complex remained active insideH. medicinalisand prevented the proliferation of sensitive bacteria. Thus, antimicrobial properties of the ingested vertebrate blood contribute to the specificity of theA. veronii-H. medicinalissymbiosis, in addition to modifications of the blood inside the digestive tract ofH. medicinalis.
Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts
Authors: Sagt C. M. J. , Kleizen B. , Verwaal R. , de Jong M. D. M. , Müller W. H. , Smits A. , Visser C. , Boonstra J. , Verkleij A. J. , Verrips C. T.
Published: July 2002

Saccharomyces cerevisiae
is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V
HH
antibody fragments by
S. cerevisiae
or
Pichia pastoris
improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.
Sequence Diversity of
rulA
among Natural Isolates of
Pseudomonas syringae
and Effect on Function of
rulAB
-Mediated UV Radiation Tolerance
Authors: Sundin George W. , Jacobs Janette L. , Murillo Jesús
Published: July 2002

The
rulAB
locus confers tolerance to UV radiation and is borne on plasmids of the pPT23A family in
Pseudomonas syringae.
We sequenced 14
rulA
alleles from
P. syringae
strains representing seven pathovars and found sequence differences of 1 to 12% within pathovar syringae, and up to 15% differences between pathovars. Since the sequence variation within
rulA
was similar to that of
P. syringae
chromosomal alleles, we hypothesized that
rulAB
has evolved over a long time period in
P. syringae.
A phylogenetic analysis of the deduced amino acid sequences of
rulA
resulted in seven clusters. Strains from the same plant host grouped together in three cases; however, strains from different pathovars grouped together in two cases. In particular, the
rulA
alleles from
P. syringae
pv. lachrymans and
P. syringae
pv. pisi were grouped but were clearly distinct from the other sequenced alleles, suggesting the possibility of a recent interpathovar transfer. We constructed chimeric
rulAB
expression clones and found that the observed sequence differences resulted in significant differences in UV (wavelength) radiation sensitivity. Our results suggest that specific amino acid changes in RulA could alter UV radiation tolerance and the competitiveness of the
P. syringae
host in the phyllosphere.
Application of a Propionyl Coenzyme A Synthetase for Poly(3-Hydroxypropionate-
co
-3-Hydroxybutyrate) Accumulation in Recombinant
Escherichia coli
Authors: Valentin Henry E. , Mitsky Timothy A. , Mahadeo Debbie A. , Tran Minhtien , Gruys Kenneth J.
Published: July 2002

The genetic operon for propionic acid degradation in
Salmonella enterica
serovar Typhimurium contains an open reading frame designated
prpE
which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381–1388, 1999). In this paper we report the cloning of
prpE
by PCR, its overexpression in
Escherichia coli
, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a

K
m

of 50 μM and a specific activity of 120 μmol · min
−1
· mg
−1
were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate. When
prpE
was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from
Ralstonia eutropha
in recombinant
E. coli
, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with
prpE
, with
acoE
(an acetyl-CoA synthetase gene from
R. eutropha
[H. Priefert and A. Steinbüchel, J. Bacteriol. 174:6590–6599, 1992]), or with
orfZ
(an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from
Clostridium propionicum
[H. E. Valentin, S. Reiser, and K. J. Gruys, Biotechnol. Bioeng. 67:291–299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity.
Diversity of
l
-Methionine Catabolism Pathways in Cheese-Ripening Bacteria
Authors: Bonnarme Pascal , Psoni Lefki , Spinnler Henry E.
Published: July 2002

Enzymatic activities that could be involved in methanethiol generation in five cheese-ripening bacteria were assayed, and the major sulfur compounds produced were identified.
l
-Methionine and α-keto-γ-methyl-thio-butyric acid demethiolating activities were detected in whole cells and cell extracts (CFEs) of all the bacteria tested. No
l
-methionine deaminase activity could be detected in any of the ripening bacteria and
l
-methionine aminotransferase was detected in CFEs of
Brevibacterium linens
,
Micrococcus luteus
, and
Corynebacterium glutamicum
. The results suggest that several pathways for
l
-methionine catabolism probably coexist in these ripening bacteria.
Bacterial Toxin-Antitoxin Gene System as Containment Control in Yeast Cells
Authors: Kristoffersen P. , Jensen G. B. , Gerdes K. , Piškur J.
Published: July 2002

The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. TheEscherichia coli relEandrelBgenes were expressed in the yeastSaccharomyces cerevisiae. Expression of therelEgene was highly toxic to yeast cells. However, expression of therelBgene counteracted the effect ofrelEto some extent, suggesting that toxin-antitoxin interaction also occurs inS. cerevisiae. Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial fermentation processes in which the escape of genetically modified cells would be considered highly risky.
Effect of Flagella on Initial Attachment of
Listeria monocytogenes
to Stainless Steel
Authors: Vatanyoopaisarn Savitri , Nazli Aisha , Dodd Christine E. R. , Rees Catherine E. D. , Waites Will M.
Published: July 2002

At 22°C a flagellin mutant of
Listeria monocytogenes
was found to attach to stainless steel at levels 10-fold lower than wild-type cells, even under conditions preventing active motility. At 37°C, when flagella are not produced, attachment of both strains was identical. Therefore, flagella per se facilitate the early stage of attachment.
Properties of Engineered Poly-3-Hydroxyalkanoates Produced in Recombinant
Escherichia coli
Strains
Authors: Ren Qun , Sierro Nicolas , Kellerhals Michele , Kessler Birgit , Witholt Bernard
Published: July 2002

To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant
Escherichia coli
strains that synthesized PHAs with altered monomer compositions. Experiments with different substrates (fatty acids with different chain lengths) or different
E. coli
hosts failed to produce PHAs with altered physical properties. Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the β-oxidation cycle are accumulated and led to the PHA polymerase precursor
R
-3-hydroxyalkanoates in
E. coli
hosts. By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from
Ralstonia eutropha
and blocking the ketoacyl-CoA degradation step of the β-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor. Introduction of the
phbB
gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point. The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties.
Bacillus thuringiensis
Delta-Endotoxin Cry1C Domain III Can Function as a Specificity Determinant for
Spodoptera exigua
in Different, but Not All, Cry1-Cry1C Hybrids
Authors: de Maagd Ruud A. , Weemen-Hendriks Mieke , Stiekema Willem , Bosch Dirk
Published: July 2002

In order to test our hypothesis that
Bacillus thuringiensis
delta-endotoxin Cry1Ca domain III functions as a determinant of specificity for
Spodoptera exigua
, regardless of the origins of domains I and II, we have constructed by cloning and in vivo recombination a collection of hybrid proteins containing domains I and II of various Cry1 toxins combined with domain III of Cry1Ca. Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ea, and Cry1Fa all become more active against
S. exigua
when their domain III is replaced by (part of) that of Cry1Ca. This result shows that domain III of Cry1Ca is an important and versatile determinant of
S. exigua
specificity. The toxicity of the hybrids varied by a factor of 40, indicating that domain I and/or II modulate the activity as well. Cry1Da-Cry1Ca hybrids were an exception in that they were not significantly active against
S. exigua
or
Manduca sexta
, whereas both parental proteins were highly toxic. Incidentally, in a Cry1Ba-Cry1Ca hybrid, Cry1Ca domain III can also strongly increase toxicity for
M. sexta
.
Cloning, Sequencing, and Expression in
Escherichia coli
of the New Gene Encoding β-1,3-Xylanase from a Marine Bacterium,
Vibrio
sp. Strain XY-214
Authors: Araki Toshiyoshi , Hashikawa Shinnosuke , Morishita Tatsuo
Published: July 2002

The
Vibrio
sp. strain XY-214 β-1,3-xylanase gene cloned in
Escherichia coli
DH5α consisted of an open reading frame of 1,383 nucleotides encoding a protein of 460 amino acids with a molecular mass of 51,323 Da and had a signal peptide of 22 amino acids. The transformant enzyme hydrolyzed β-1,3-xylan to produce several xylooligosaccharides.
Cell Wall Chemical Composition of
Enterococcus faecalis
in the Viable but Nonculturable State
Authors: Signoretto Caterina , del Mar Lleò Maria , Tafi Maria Carla , Canepari Pietro
Published: July 2002

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of
Enterococcus faecalis
in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in
E. faecalis
in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.
Physiological Responses to Starvation in the Marine Oligotrophic Ultramicrobacterium
Sphingomonas
sp. Strain RB2256
Authors: Fegatella Fitri , Cavicchioli Ricardo
Published: July 2002

Sphingomonas
sp. strain RB2256 is representative of the ultramicrobacteria that proliferate in oligotrophic marine waters. While this class of bacteria is well adapted for growth with low concentrations of nutrients, their ability to respond to complete nutrient deprivation has not previously been investigated. In this study, we examined two-dimensional protein profiles for logarithmic and stationary-phase cells and found that protein spot intensity was regulated by up to 70-fold. A total of 72 and 177 spots showed increased or decreased intensity, respectively, by at least twofold during starvation. The large number of protein spots (1,500) relative to the small genome size (ca. 1.5 Mb) indicates that gene expression may involve co- and posttranslational modifications of proteins. Rates of protein and RNA synthesis were examined throughout the growth phase and up to 7 days of starvation and revealed that synthesis was highly regulated. Rates of protein synthesis and cellular protein content were compared to ribosome content, demonstrating that ribosome synthesis was not directly linked to protein synthesis and that the function of ribosomes may not be limited to translation. By comparing the genetic capacity and physiological responses to starvation of RB2256 to those of the copiotrophic marine bacterium
Vibrio angustum
S14 (J. Ostling, L. Holmquist, and S. Kjelleberg, J. Bacteriol. 178:4901–4908, 1996), the characteristics of a distinct starvation response were defined for
Sphingomonas
strain RB2256. The capacity of this ultramicrobacterium to respond to starvation is discussed in terms of the ecological relevance of complete nutrient deprivation in an oligotrophic marine environment. These studies provide the first evidence that marine oligotrophic ultramicrobacteria may be expected to include a starvation response and the capacity for a high degree of gene regulation.
PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant
Pseudomonas fragi
Authors: Fiedler Silke , Steinbüchel Alexander , Rehm Bernd H. A.
Published: July 2002

Recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxy-alkanoate) consisting of medium-chain-length constituents (C
6
to C
14
) (PHA
MCL
), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylase (PhaG), has been identified in
Pseudomonas putida
(B. H. A. Rehm, N. Krüger, and A. Steinbüchel, J. Biol. Chem. 273:24044–24051, 1998). To establish this PHA-biosynthetic pathway in a non-PHA-accumulating bacterium, we functionally coexpressed
phaC1
(encoding PHA synthase 1) from
Pseudomonas aeruginosa
and
phaG
(encoding the transacylase) from
P. putida
in
Pseudomonas fragi
. The recombinant strains of
P. fragi
were cultivated on gluconate as the sole carbon source, and PHA accumulation to about 14% of the total cellular dry weight was achieved. The respective polyester was isolated, and GPC analysis revealed a weight average molar mass of about 130,000 g mol
−1
and a polydispersity of 2.2. The PHA was composed mainly (60 mol%) of 3-hydroxydecanoate. These data strongly suggested that functional expression of
phaC1
and
phaG
established a new pathway for PHA
MCL
biosynthesis from nonrelated carbon sources in
P. fragi
. When fatty acids were used as the carbon source, no PHA accumulation was observed in PHA synthase-expressing
P. fragi
, whereas application of the β-oxidation inhibitor acrylic acid mediated PHA
MCL
accumulation. The substrate for the PHA synthase PhaC1 is therefore presumably directly provided through the enzymatic activity of the transacylase PhaG by the conversion of (
R
)-3-hydroxydecanoyl-ACP to (
R
)-3-hydroxydecanoyl-CoA when the organism is cultivated on gluconate. Here we demonstrate for the first time the establishment of PHA
MCL
synthesis from nonrelated carbon sources in a non-PHA-accumulating bacterium, employing fatty acid de novo biosynthesis and the enzymes PhaG (a transacylase) and PhaC1 (a PHA synthase).