bluuebunny/crossref_metadata_2025 huggingface dataset:
New Carbenicillin-Hydrolyzing β-Lactamase (CARB-7) from
Vibrio cholerae
Non-O1, Non-O139 Strains Encoded by the VCR Region of the
V. cholerae
Genome
Authors: Melano Roberto , Petroni Alejandro , Garutti Alicia , Saka Héctor Alex , Mange Laura , Pasterán Fernando , Rapoport Melina , Rossi Alicia , Galas Marcelo
Published: July 2002

In a previous study, an analysis of 77 ampicillin-nonsusceptible (resistant plus intermediate categories) strains of
Vibrio cholerae
non-O1, non-O139, isolated from aquatic environment and diarrheal stool, showed that all of them produced a β-lactamase with a pI of 5.4. Hybridization or amplification by PCR with a probe for
bla
TEM
or primers for
bla
CARB
gene families was negative. In this work, an environmental ampicillin-resistant strain from this sample, ME11762, isolated from a waterway in the west region of Argentina, was studied. The nucleotide sequence of the structural gene of the β-lactamase was determined by bidirectional sequencing of a
Sau
3AI fragment belonging to this isolate. The gene encodes a new 288-amino-acid protein, designated CARB-7, that shares 88.5% homology with the CARB-6 enzyme; an overall 83.2% homology with PSE-4, PSE-1, CARB-3, and the
Proteus mirabilis
N29 enzymes; and 79% homology with CARB-4 enzyme. The gene for this β-lactamase could not be transferred to
Escherichia coli
by conjugation. The nucleotide sequence of the flanking regions of the
bla
CARB-7
gene showed the occurrence of three 123-bp
V. cholerae
repeated sequences, all of which were found outside the predicted open reading frame. The upstream fragment of the
bla
CARB-7
gene shared 93% identity with a locus situated inside
V. cholerae
's chromosome 2. These results strongly suggest the chromosomal location of the
bla
CARB-7
gene, making this the first communication of a β-lactamase gene located on the VCR island of the
V. cholerae
genome.
Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of
Pseudomonas aeruginosa
Authors: Aires Julio Ramos , Pechère Jean-Claude , Van Delden Christian , Köhler Thilo
Published: July 2002

At least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in
Pseudomonas aeruginosa
. Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system. Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of
Escherichia coli
showed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS). These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge. MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive. Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics. The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol. All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis. It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.
Comparison of Corticosteroids for Treatment of Respiratory Syncytial Virus Bronchiolitis and Pneumonia in Cotton Rats
Authors: Ottolini Martin G. , Curtis Spencer J. , Porter David D. , Mathews Amy , Richardson Joann Y. , Hemming Val G. , Prince Gregory A.
Published: July 2002

Triamcinolone acetonide, methylprednisolone, and dexamethasone were each evaluated in combination with palivizumab (Synagis) for the therapy of established respiratory syncytial virus infection in the cotton rat. Triamcinolone and methylprednisolone proved to be more effective than dexamethasone in reducing lung pathology. No recurrence of viral replication or pulmonary pathology followed the cessation of therapy.
Analysis of Penicillin-Binding Protein Genes of Clinical Isolates of
Streptococcus pneumoniae
with Reduced Susceptibility to Amoxicillin
Authors: du Plessis Mignon , Bingen Edouard , Klugman Keith P.
Published: July 2002

The recent emergence of pneumococcal isolates exhibiting an unusual resistance phenotype of higher amoxicillin MICs in relation to the penicillin MICs prompted an analysis of the
pbp
genes from three such strains isolated in France. For comparison, three amoxicillin-susceptible strains were included in the study. DNA sequence analysis of the
pbp2x
,
pbp2b
, and
pbp1a
genes revealed extensive sequence divergence in all six isolates compared to the sequences of the genes of penicillin-susceptible strain R6. With the exception of
pbp2b
, no amino acid mutations were unique to the resistant isolates. Transformation experiments with cloned
pbp
genes isolated from one of the resistant isolates demonstrated a stepwise development of amoxicillin resistance involving penicillin-binding proteins (PBPs) 2X, 2B, and 1A. Full resistance, equivalent to that of the donor strain, was achieved only when genomic DNA was transformed into R6
2x/2b/1a
mutants, suggesting that full resistance development in this isolate is mediated by a non-PBP determinant. Moreover, the recently identified
murMN
resistance determinant does not appear to have any impact on resistance in this isolate. This determinant (from the French isolate) was, however, able to transform an R6 mutant harboring
pbp2x
,
pbp2b
, and
pbp1a
genes from a Hungarian clone with an extremely high level of penicillin resistance so that it had increased levels of penicillin resistance. These results indicate that the development of high-level β-lactam resistance is a complex process and that the involvement of MurMN in penicillin resistance appears to be dependent on specific mutations in PBPs 2X, 2B, and/or 1A. Furthermore, an additional (as yet unidentified) non-PBP-mediated resistance determinant is required for full resistance development in some pneumococci.
Preclinical and Toxicology Studies of 1263W94, a Potent and Selective Inhibitor of Human Cytomegalovirus Replication
Authors: Koszalka George W. , Johnson Nelson W. , Good Steven S. , Boyd Leslie , Chamberlain Stanley C. , Townsend Leroy B. , Drach John C. , Biron Karen K.
Published: July 2002

1263W94 is a novel benzimidazole compound being developed for treatment of human cytomegalovirus infection. No adverse pharmacological effects were demonstrated in safety pharmacology studies with 1263W94. The minimal-effect dose in a 1-month rat study was 100 mg/kg/day, and the no-effect dose in a 1-month monkey study was 180 mg/kg/day. Toxic effects were limited to increases in liver weights, neutrophils, and monocytes at higher doses in female rats. 1263W94 was not genotoxic in the Ames or micronucleus assays. In the mouse lymphoma assay, 1263W94 was mutagenic in the absence of the rat liver S-9 metabolic activation system, with equivocal results in the presence of the S-9 mix. Mean oral bioavailability of 1263W94 was >90% in rats and ∼50% in monkeys. Clearance in rats and monkeys was primarily by biliary secretion, with evidence of enterohepatic recirculation. In 1-month studies in rats and monkeys, mean peak concentrations and exposures to 1263W94 increased in near proportion to dose. Metabolism of 1263W94 to its primary metabolite, an N-dealkylated analog, appeared to be mediated via the isozyme CYP3A4 in humans. 1263W94 was primarily distributed in the gastrointestinal tract of rats but did not cross the blood-brain barrier. In monkeys, 1263W94 levels in the brain, cerebrospinal fluid, and vitreous humor ranged from 4 to 20%, 1 to 2%, and <1%, of corresponding concentrations in plasma, respectively. The high level of binding by 1263W94 to human plasma proteins (primarily albumin) was readily reversible, with less protein binding seen in the monkey, rat, and mouse. Results of these studies demonstrate a favorable safety profile for 1263W94.
Class 1 Integron Containing a New Gene Cassette,aadA10, Associated with Tn1404from R151
Authors: Partridge Sally R. , Collis Christina M. , Hall Ruth M.
Published: July 2002

The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found onPseudomonas aeruginosaplasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants. Sequencing of the cassette array from the plasmid known as R388::Tn1404revealed two known gene cassettes,oxa10andaadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designatedaadA10, in the orderoxa10-aadB-aadA10.These cassettes replaced thedfrB2-orfAcassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition. The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical). TheaadA10cassette found here has only a simple site containing a 7-bp spacer derived fromattI1in place of a 59-base element and is likely to represent a derivative of the complete cassette. IntI1-mediated deletion of theaadA10cassette was not detected, indicating that this single simple site is either inactive or only weakly active.
Sulfadoxine-Pyrimethamine Resistance in the Rodent Malaria Parasite
Plasmodium chabaudi
Authors: Hayton Karen , Ranford-Cartwright Lisa C. , Walliker David
Published: July 2002

We have studied resistance to sulfadoxine-pyrimethamine (S/P) in the rodent malaria parasite
Plasmodium chabaudi
. A stable S/P-resistant mutant, AS(50S/P), was selected by drug treatment of a clone, AS(PYR), already resistant to pyrimethamine. The sequences of the
P. chabaudi dhfr
and
dhps
genes were obtained and found to be identical in AS(50S/P) and AS(PYR), showing that resistance to S/P in AS(50S/P) was not due to additional mutations in either gene. AS(50S/P) was crossed with a drug-sensitive clone, AJ, and 16 independent recombinant progeny were obtained. These clones were phenotyped for their susceptibility to S/P and to sulfadoxine and pyrimethamine separately. Pyrimethamine resistance was invariably associated with S/P resistance, but no correlation was found between resistance to S/P and resistance to sulfadoxine. Quantitative trait locus analysis of the progeny with 31 chromosome-specific markers showed that mutant
P. chabaudi dhfr,
or one or more genes closely linked to it, was a major determinant of S/P resistance. In addition, the inheritance of genes on chromosomes 5 and 13 from the sensitive parent appeared to contribute to the level of resistance observed. These results demonstrate that the S/P resistance of the AS(50S/P) mutant of
P. chabaudi
does not involve mutation in
dhps
and is not due simply to a combination of two genes determining resistance to pyrimethamine and sulfadoxine separately.
Trends in Antimicrobial Resistance among Urinary Tract Infection Isolates of
Escherichia coli
from Female Outpatients in the United States
Authors: Karlowsky James A. , Kelly Laurie J. , Thornsberry Clyde , Jones Mark E. , Sahm Daniel F.
Published: July 2002

The Infectious Diseases Society of America advocates trimethoprim-sulfamethoxazole (SXT) as initial therapy for females with acute uncomplicated bacterial cystitis in settings where the prevalence of SXT resistance does not exceed 10 to 20%. To determine trends in the activities of SXT, ampicillin, ciprofloxacin, and nitrofurantoin among urine isolates of
Escherichia coli
from female outpatients, susceptibility testing data from The Surveillance Network (TSN) Database-USA (
n
= 286,187) from 1995 to 2001 were analyzed. Resistance rates among
E. coli
isolates to ampicillin (range, 36.0 to 37.4% per year), SXT (range, 14.8 to 17.0%), ciprofloxacin (range, 0.7 to 2.5%), and nitrofurantoin (range, 0.4 to 0.8%) varied only slightly over this 7-year period. Ciprofloxacin was the only agent studied that demonstrated a consistent stepwise increase in resistance from 1995 (0.7%) to 2001 (2.5%). In 2001, SXT resistance among
E. coli
isolates was >10% in all nine U.S. Bureau of the Census regions. At institutions testing ≥100 urinary isolates of
E. coli
(
n
= 126) in 2001, ampicillin (range, 27.3 to 98.8%) and SXT (range, 7.5 to 47.1%) resistance rates varied widely while ciprofloxacin (range, 0 to 12.9%) and nitrofurantoin (range, 0 to 2.8%) resistance rates were more consistent. In 2001, the most frequent coresistant phenotypes were resistance to ampicillin and SXT (12.0% of all isolates; 82.3% of coresistant isolates) and resistance to ampicillin, ciprofloxacin, and SXT (1.4% of all isolates; 9.9% of coresistant isolates). Coresistance less frequently included resistance to nitrofurantoin (3.5% of coresistant isolates) than resistance to ciprofloxacin (15.8%), SXT (95.7%), and ampicillin (98.1%). In conclusion, among urinary isolates of
E. coli
from female outpatients in the United States, national resistance rates to SXT were relatively consistent (14.8 to 17.0%) from 1995 to 2001 but demonstrated considerable regional and institutional variation in 2001. Therapies other than SXT may need to be considered in some locations.
Efficacy of Caspofungin Alone and in Combination with Voriconazole in a Guinea Pig Model of Invasive Aspergillosis
Authors: Kirkpatrick William R. , Perea Sofia , Coco Brent J. , Patterson Thomas F.
Published: July 2002

The antifungal activity of caspofungin acetate (CAS) alone and in combination with voriconazole (VRC) was evaluated in an immunosuppressed transiently neutropenic guinea pig model of invasive aspergillosis. Guinea pigs were immunosuppressed with triamcinolone at 20 mg/kg of body weight/day subcutaneously beginning 4 days prior to lethal intravenous challenge with
Aspergillus fumigatus
and were made temporarily neutropenic with cyclophosphamide administered at 150 mg/kg intraperitoneally (i.p.) 1 day prior to challenge. Therapy with i.p. CAS at 1 and 2.5 mg/kg/day (with and without oral VRC at 5 mg/kg/day), oral VRC at 5 mg/kg/day, or i.p. amphotericin B (AMB) at 1.25 mg/kg/day was begun 24 h after challenge and was continued for 5 days. Mortality occurred in 12 of 12 untreated controls, whereas mortality occurred in 4 of 12 and 6 of 12 guinea pigs treated with CAS at 1 and 2.5 mg/kg/day, respectively, and in 3 of 12 guinea pigs treated with AMB. No mortality occurred among animals treated with CAS at 1 mg/kg/day plus VRC at 5 mg/kg/day, CAS at 2.5 mg/kg/day plus VRC at 5 mg/kg/day, or VRC at 5 mg/kg/day alone. Both CAS regimens increased the survival times and reduced the colony counts in tissue compared with those for the controls. Treatment with VRC and AMB significantly reduced the colony counts in the tissues of selected animals compared with those in the tissues of the controls. Treatment with VRC and AMB also resulted in reductions in colony counts in tissues compared with those in the tissues of animals treated with CAS (the difference was not statistically significant) and improved the survival times but did not sterilize tissues. Combination therapies with CAS plus VRC at either dose reduced colony counts in tissues 1,000-fold over those for the controls and were the only regimens that significantly reduced the numbers of positive cultures. The combinations of CAS plus VRC were highly effective in this model and should be further evaluated for use against invasive aspergillosis.
DNA Gyrase and Topoisomerase IV Mutations Associated with Fluoroquinolone Resistance in
Proteus mirabilis
Authors: Weigel L. M. , Anderson G. J. , Tenover F. C.
Published: July 2002

Mutations associated with fluoroquinolone resistance in clinical isolates of
Proteus mirabilis
were determined by genetic analysis of the quinolone resistance-determining region (QRDR) of
gyrA
,
gyrB
,
parC
, and
parE
. This study included the
P. mirabilis
type strain ATCC 29906 and 29 clinical isolates with reduced susceptibility (MIC, 0.5 to 2 μg/ml) or resistance (MIC, ≥4 μg/ml) to ciprofloxacin. Susceptibility profiles for ciprofloxacin, clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were correlated with amino acid changes in the QRDRs. Decreased susceptibility and resistance were associated with double mutations involving both
gyrA
(S83R or -I) and
parC
(S80R or -I). Among these double mutants, MICs of ciprofloxacin varied from 1 to 16 μg/ml, indicating that additional factors, such as drug efflux or porin changes, also contribute to the level of resistance. For ParE, a single conservative change of V364I was detected in seven strains. An unexpected result was the association of
gyrB
mutations with high-level resistance to fluoroquinolones in 12 of 20 ciprofloxacin-resistant isolates. Changes in GyrB included S464Y (six isolates), S464F (three isolates), and E466D (two isolates). A three-nucleotide insertion, resulting in an additional lysine residue between K455 and A456, was detected in
gyrB
of one strain. Unlike any other bacterial species analyzed to date, mutation of
gyrB
appears to be a frequent event in the acquisition of fluoroquinolone resistance among clinical isolates of
P. mirabilis
.
In Vitro and In Vivo Activities of Tigecycline (GAR-936), Daptomycin, and Comparative Antimicrobial Agents against Glycopeptide-IntermediateStaphylococcus aureusand Other Resistant Gram-Positive Pathogens
Authors: Petersen Peter J. , Bradford Patricia A. , Weiss William J. , Murphy Timothy M. , Sum P. E. , Projan Steven J.
Published: July 2002

Tigecycline (GAR-936) and daptomycin are potent antibacterial compounds in advanced stages of clinical trials. These novel agents target multiply resistant pathogenic bacteria. Daptomycin is principally active against gram-positive bacteria, while tigecycline has broad-spectrum activity. When tested by the standard protocols of the National Committee for Clinical Laboratory Standards in Mueller-Hinton broth II, tigecycline was more active than daptomycin (MICs at which 90% of isolates tested are inhibited, 0.12 to 1 and 0.5 to 16 μg/ml, respectively) against staphylococcal, enterococcal, and streptococcal pathogens. Daptomycin demonstrated a stepwise increase in activity corresponding to an increase in the supplemental concentration of calcium. When tested in base Mueller-Hinton broth supplemented with 50 mg of calcium per liter, daptomycin demonstrated improved activity (MIC90s, 0.015 to 4 μg/ml). The activity of daptomycin, however, equaled that of tigecycline against the glycopeptide-intermediateStaphylococcus aureus(GISA) strains only when the test medium was supplemented with excess calcium (75 mg/liter). Tigecycline and daptomycin demonstrated in vivo efficacies against GISA, methicillin-resistantS. aureus, and methicillin-susceptibleS. aureusstrains in an intraperitoneal systemic murine infection model. These data suggest that tigecycline and daptomycin may offer therapeutic options against clinically relevant resistant pathogens for which current alternatives for treatment are limited.
The AcrAB-TolC Efflux Pump Contributes to Multidrug Resistance in the Nosocomial Pathogen
Enterobacter aerogenes
Authors: Pradel Elizabeth , Pagès Jean-Marie
Published: July 2002

We identified the genes encoding the AcrA-AcrB-TolC efflux pump in
Enterobacter aerogenes
and constructed
acrAB
and
tolC
mutants from a multidrug-resistant isolate. Both derivatives were more susceptible to antibiotics than the parental strain. Sequence analysis and complementation experiments revealed that the multidrug-resistant isolate is an
acrR
mutant.
gyrA
Polymorphism in
Campylobacter jejuni
: Detection of
gyrA
Mutations in 162
C. jejuni
Isolates by Single-Strand Conformation Polymorphism and DNA Sequencing
Authors: Hakanen Antti , Jalava Jari , Kotilainen Pirkko , Jousimies-Somer Hannele , Siitonen Anja , Huovinen Pentti
Published: July 2002

Mutations in the quinolone resistance-determining region of the
gyrA
gene from 138 ciprofloxacin-resistant (MIC, ≥4 μg/ml) and 24 ciprofloxacin-susceptible (MIC, ≤1 μg/ml) clinical
Campylobacter jejuni
isolates were subjected to single-strand conformation polymorphism analysis and sequencing. All of the isolates could be assigned to three genotypic clusters based on silent mutations. All resistant isolates had a point mutation at codon 86.
Novel Complex
sul1
-Type Integron in
Escherichia coli
Carrying
bla
CTX-M-9
Authors: Sabaté Montserrat , Navarro Ferran , Miró Elisenda , Campoy Susana , Mirelis Beatriz , Barbé Jordi , Prats Guillem
Published: July 2002

For the present report, a novel complex class 1 integron, In60, was characterized. Part of this integron includes the
bla
CTX-M-9
gene and its downstream nucleotide sequence, which shares 81% and 78% nucleotide identity with those of
kluA-1
β-lactamase and
orf3
of
K. ascorbata
, respectively. Furthermore, a new insertion sequence, IS
3000
, has been found in In60. PCR analysis indicates that integron In60 is present in 33 of 34 nonclonal enterobacterial isolates carrying the putative β-lactamase CTX-M-9.
Transmission Electron Microscopic Study of Antibiotic Action on
Klebsiella pneumoniae
Biofilm
Authors: Zahller Jeff , Stewart Philip S.
Published: July 2002

The penetration of ampicillin and ciprofloxacin through biofilms formed by
Klebsiella pneumoniae
was confirmed by transmission electron microscopic observation of antibiotic-affected cells at the distal edge of the biofilm. Because the bacteria nevertheless survived antibiotic treatment, some protective mechanism other than inadequate penetration must have been at work in the biofilm.
Differential Ability of Genotypes of 2,4-Diacetylphloroglucinol-Producing
Pseudomonas fluorescens
Strains To Colonize the Roots of Pea Plants
Authors: Landa Blanca B. , Mavrodi Olga V. , Raaijmakers Jos M. , McSpadden Gardener Brian B. , Thomashow Linda S. , Weller David M.
Published: July 2002

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent
Pseudomonas
spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent
Pseudomonas
spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to
Fusarium
wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)
−1
over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene
phlD
were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.
Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes
Authors: Valinsky Lea , Della Vedova Gianluca , Scupham Alexandra J. , Alvey Sam , Figueroa Andres , Yin Bei , Hartin R. Jack , Chrobak Marek , Crowley David E. , Jiang Tao , Borneman James
Published: July 2002

One of the first steps in characterizing an ecosystem is to describe the organisms inhabiting it. For microbial studies, experimental limitations have hindered the ability to depict diverse communities. Here we describe oligonucleotide fingerprinting of rRNA genes (OFRG), a method that permits identification of arrayed rRNA genes (rDNA) through a series of hybridization experiments using small DNA probes. To demonstrate this strategy, we examined the bacteria inhabiting two different soils. Analysis of 1,536 rDNA clones revealed 766 clusters grouped into five major taxa:
Bacillus
,
Actinobacteria
,
Proteobacteria
, and two undefined assemblages. Soil-specific taxa were identified and then independently confirmed through cluster-specific PCR of the original soil DNA. Near-species-level resolution was obtained by this analysis as clones with average sequence identities of 97% were grouped in the same cluster. A comparison of these OFRG results with the results obtained in a denaturing gradient gel electrophoresis analysis of the same two soils demonstrated the significance of this methodological advance. OFRG provides a cost-effective means to extensively analyze microbial communities and should have applications in medicine, biotechnology, and ecosystem studies.
Cloning and Heterologous Expression of an Enantioselective Amidase from
Rhodococcus erythropolis
Strain MP50
Authors: Trott Sandra , Bürger Sibylle , Calaminus Carsten , Stolz Andreas
Published: July 2002

The gene for an enantioselective amidase was cloned from
Rhodococcus erythropolis
MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in
R. erythropolis
MP50. The amidase was heterologously expressed in
Escherichia coli
and shown to hydrolyze 2-phenylpropionamide, α-chlorophenylacetamide, and α-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant
E. coli
strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant
E. coli
strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant
E. coli
strains.
Identification of
glyA
(Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase
l
-Threonine Accumulation by
Corynebacterium glutamicum
Authors: Simic Petra , Willuhn Juliane , Sahm Hermann , Eggeling Lothar
Published: July 2002

l
-Threonine can be made by the amino acid-producing bacterium
Corynebacterium glutamicum
. However, in the course of this process, some of the
l
-threonine is degraded to glycine. We detected an aldole cleavage activity of
l
-threonine in crude extracts with an activity of 2.2 nmol min
−1
(mg of protein)
−1
. In order to discover the molecular reason for this activity, we cloned
glyA
, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged
glyA
, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with
l
-threonine as the substrate was 1.3 μmol min
−1
(mg of protein)
−1
, which was 4% of that with
l
-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential
glyA
gene in the chromosome under the control of P

tac

, making
glyA
expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an
l
-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while
l
-threonine was simultaneously increased by 49%. The intracellular availability of
l
-threonine to aldole cleavage was also reduced by overexpressing the
l
-threonine exporter
thrE
. In
C. glutamicum
DR-17, which overexpresses
thrE
, accumulation of 67 mM instead of 49 mM
l
-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.
Prevalence and Fingerprinting of
Listeria monocytogenes
Strains Isolated from Raw Whole Milk in Farm Bulk Tanks and in Dairy Plant Receiving Tanks
Authors: Waak Elisabet , Tham Wilhelm , Danielsson-Tham Marie-Louise
Published: July 2002

The incidence of
Listeria
species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied.
Listeria monocytogenes
was found in 1.0% and
Listeria innocua
was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of
L. monocytogenes
ml
−1
.
L. monocytogenes
was detected in 19.6% and
L. innocua
was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both
Listeria
species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of
L. monocytogenes
, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No
Listeria
species were found in any other samples from the plant.
Technical-Scale Production of Cyanophycin with Recombinant Strains of
Escherichia coli
Authors: Frey Kay M. , Oppermann-Sanio Fred B. , Schmidt Holger , Steinbüchel Alexander
Published: July 2002

By the use of
Escherichia coli
DH1 harboring
cphA
from
Synechocystis
sp. strain PCC6803, large-scale production of cyanophycin at 30- and 500-liter culture volumes was established. Transcription of
cphA
was controlled by the thermosensitive cI857 repressor, which enabled induction of
cphA
by a simple temperature shift in the culture fluid. Maximum cyanophycin cell content of up to 24% (wt/wt) of cellular dry matter was obtained by induction in the early exponential growth phase and cultivation of the cells in terrific broth complex medium. Synthesis of cyanophycin was found to be strongly dependent on the presence of complex components, and in mineral salts medium the cells synthesized and accumulated cyanophycin only if Casamino Acids were added. Cultivations were done at the 500-liter scale, allowing the provision of cell mass for the preparation of cyanophycin at the kilogram scale. Isolation of cyanophycin was achieved by a new acid extraction procedure which allowed large-scale purification of the polyamide from whole cells.
Expression of a Fungal Hydrophobin in theSaccharomyces cerevisiaeCell Wall: Effect on Cell Surface Properties and Immobilization
Authors: Nakari-Setälä Tiina , Azeredo Joana , Henriques Mariana , Oliveira Rosário , Teixeira José , Linder Markus , Penttilä Merja
Published: July 2002

The aim of this work was to modify the cell surface properties ofSaccharomyces cerevisiaeby expression of the HFBI hydrophobin of the filamentous fungusTrichoderma reeseion the yeast cell surface. The second aim was to study the immobilization capacity of the modified cells. Fusion to the Flo1p flocculin was used to target the HFBI moiety to the cell wall. Determination of cell surface characteristics with contact angle and zeta potential measurements indicated that HFBI-producing cells are more apolar and slightly less negatively charged than the parent cells. Adsorption of the yeast cells to different commercial supports was studied. A twofold increase in the binding affinity of the hydrophobin-producing yeast to hydrophobic silicone-based materials was observed, while no improvement in the interaction with hydrophilic carriers could be seen compared to that of the parent cells. Hydrophobic interactions between the yeast cells and the support are suggested to play a major role in attachment. Also, a slight increase in the initial adsorption rate of the hydrophobin yeast was observed. Furthermore, due to the engineered cell surface, hydrophobin-producing yeast cells were efficiently separated in an aqueous two-phase system by using a nonionic polyoxyethylene detergent, C12-18EO5.
Mucosa-Associated Bacteria in the Human Gastrointestinal Tract Are Uniformly Distributed along the Colon and Differ from the Community Recovered from Feces
Authors: Zoetendal Erwin G. , von Wright Atte , Vilpponen-Salmela Terttu , Ben-Amor Kaouther , Akkermans Antoon D. L. , de Vos Willem M.
Published: July 2002

The human gastrointestinal (GI) tract harbors a complex community of bacterial cells in the mucosa, lumen, and feces. Since most attention has been focused on bacteria present in feces, knowledge about the mucosa-associated bacterial communities in different parts of the colon is limited. In this study, the bacterial communities in feces and biopsy samples from the ascending, transverse, and descending colons of 10 individuals were analyzed by using a 16S rRNA approach. Flow cytometric analysis indicated that 10
5
to 10
6
bacteria were present in the biopsy samples. To visualize the diversity of the predominant and the
Lactobacillus
group community, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was performed. DGGE analysis and similarity index comparisons demonstrated that the predominant mucosa-associated bacterial community was host specific and uniformly distributed along the colon but significantly different from the fecal community (
P
< 0.01). The
Lactobacillus
group-specific profiles were less complex than the profiles reflecting the predominant community. For 6 of the 10 individuals the community of
Lactobacillus
-like bacteria in the biopsy samples was similar to that in the feces. Amplicons having 99% sequence similarity to the 16S ribosomal DNA of
Lactobacillus gasseri
were detected in the biopsy samples of nine individuals. No significant differences were observed between healthy and diseased individuals. The observed host-specific DGGE profiles of the mucosa-associated bacterial community in the colon support the hypothesis that host-related factors are involved in the determination of the GI tract microbial community.
Antibiotic and Biosurfactant Properties of Cyclic Lipopeptides Produced by Fluorescent
Pseudomonas
spp. from the Sugar Beet Rhizosphere
Authors: Nielsen T. H. , Sørensen D. , Tobiasen C. , Andersen J. B. , Christophersen C. , Givskov M. , Sørensen J.
Published: July 2002

Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent
Pseudomonas
spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent
Pseudomonas
spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with
Pseudomonas fluorescens
strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell
P. fluorescens
preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLP-producing
P. fluorescens
strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.
Growth in Coculture Stimulates Metabolism of the Phenylurea Herbicide Isoproturon by
Sphingomonas
sp. Strain SRS2
Authors: Sørensen Sebastian R. , Ronen Zeev , Aamand Jens
Published: July 2002

Metabolism of the phenylurea herbicide isoproturon by
Sphingomonas
sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the β-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that
Sphingomonas
sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for
Sphingomonas
sp. SRS2. This suggests that strain SRS1 supplies amino acids to
Sphingomonas
sp. SRS2, thereby leading to rapid metabolism of
14
C-labeled isoproturon to
14
CO
2
and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by
Sphingomonas
sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by
Sphingomonas
sp. strain SRS2 to a similar extent.
Solid-Phase Contact Assay That Uses alux-MarkedNitrosomonas europaeaReporter Strain To Estimate Toxicity of Bioavailable Linear Alkylbenzene Sulfonate in Soil
Authors: Brandt Kristian K. , Pedersen Anders , Sørensen Jan
Published: July 2002

Information about in situ toxicity of the bioavailable pools of adsorptive soil pollutants is a prerequisite for proper ecological risk assessment in contaminated soils. Such toxicity data may be obtained by assays allowing for direct exposure of introduced test microorganisms to the toxicants, as they appear in solid solution equilibria in the natural soil. We describe a novel sensitive solid-phase contact assay for in situ toxicity testing of soil pollutants based on a recombinant bioluminescent reporter strain ofNitrosomonas europaea. A slurry of the reporter strain and soil sample was shaken for 1 h, after which bioluminescence was measured either directly (soil slurry protocol) or in the supernatant obtained after centrifugation (soil extract protocol). The assay was validated for both protocols by using linear alkylbenzene sulfonate (LAS) as a toxic and adsorptive model compound in the soil samples. Interestingly, LAS showed the same toxicity to the reporter strain with either soil incubation (both protocols) or pure culture, suggesting that adsorbed LAS pools contributed to the observed toxicity. The solid-phase contact assay that used the reporter strain oflux-markedN. europaeawas slightly more sensitive for the detection of LAS toxicity in soil than activity-based assays targeting indigenous nitrifiers and much more sensitive than assays targeting indigenous heterotrophic microbes. We conclude that the new solid-phase contact assay, which is based on direct interaction of the test microorganisms with bioavailable pools of the toxicants in soil, provides a most sensitive and relevant method for evaluating the in situ toxicity and assessing the risks of soil contaminants.
Site-Specific Recombination-Based Genetic System for Reporting Transient or Low-Level Gene Expression
Authors: Casavant N. Carol , Beattie Gwyn A. , Phillips Gregory J. , Halverson Larry J.
Published: July 2002

We report here the construction, characterization, and application of a plasmid-based genetic system that reports the expression of a target promoter by effecting an irreversible, heritable change in a bacterial cell. This system confers strong repression of the reporter gene
gfp
in the absence of target promoter expression and utilizes the site-specific recombination machinery of bacteriophage P22 to trigger high-level reporter gene expression in the original cell and its progeny after target gene induction. We demonstrate the effectiveness of this genetic system by tailoring it to indicate the availability of arabinose to the biological control agent
Enterobacter cloacae
JL1157 in culture and in the barley rhizosphere. The presence of bioavailable arabinose triggered the production of P22 excisionase and integrase from the reporter plasmid pAraLHB in JL1157, and this led to excision of the
c
I repressor gene, which is flanked by
att
sites, and the subsequent irreversible expression of
gfp
in the original cell and in its progeny. In culture, nearly 100% of an
E. cloacae
JL1157(pAraLHB) population expressed
gfp
after exposure to 6.5 to 65 μM arabinose for 3 h. We used this biosensor to demonstrate that arabinose was released from the seeds of several legumes and grass species during germination and from roots of barley seedlings grown hydroponically or in soil. When introduced into microcosms containing barley, the biosensor permitted the localization of arabinose along the roots. Arabinose was present near the root-seed junction and on the seminal roots but was not detected at the root tips. This recombination-based reporter system should be useful for monitoring bacterial exposure to transient or low levels of specific molecules directly in the environment.
Enrichment Double-Antibody Sandwich Indirect Enzyme-Linked Immunosorbent Assay That Uses a Specific Monoclonal Antibody for Sensitive Detection of
Ralstonia solanacearum
in Asymptomatic Potato Tubers
Authors: Caruso Paola , Gorris María Teresa , Cambra Mariano , Palomo José Luis , Collar Jesús , López María M.
Published: July 2002

Sensitive and specific routine detection of
Ralstonia solanacearum
in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical
R. solanacearum
strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29°C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of
R. solanacearum
per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods.
Enzymatic Activities of Bovine Peripheral Blood Leukocytes and Milk Polymorphonuclear Neutrophils during Intramammary Inflammation Caused by Lipopolysaccharide
Authors: Prin-Mathieu C. , Le Roux Y. , Faure G. C. , Laurent F. , Béné M. C. , Moussaoui F.
Published: July 2002

Leukocytes are recruited from peripheral blood into milk as part of the inflammatory response to mastitis. However, excessive accumulation of inflammatory cells alters the quality of milk and the proteases produced by polymorphonuclear neutrophils (PMNs) and macrophages may lead to mammary tissue damage. To investigate PMN recruitment and the kinetics of their intracytoplasmic enzymes in inflammation, we generated mastitis in six cows by intramammary infusion of lipopolysaccharide (LPS). Clinical signs of acute mastitis were observed in all of the cows, and normal status was resumed by 316 h. Intracytoplasmic elastase, collagenase, and cathepsin activities were measured within live cells by flow cytometry in peripheral blood leukocytes and milk PMNs before and during the inflammatory process (at 10 time points between 4 and 316 h). The proportion of immature PMNs was appreciated by CD33 surface labeling measured in flow cytometry. Leukopenia was observed in the peripheral blood 4 h postinfusion, concomitant to an increase in somatic cell counts in milk. CD33+PMNs were preferentially recruited from the peripheral blood to milk. Enzymatic activities were detected in PMNs, lymphocytes, and monocytes at levels depending on the cell type, sample nature, and time of collection. Milk PMNs had lower enzymatic activities than peripheral blood PMNs. This study showed that milk PMNs recruited during LPS-induced experimental mastitis have an immature phenotype and significantly lower enzymatic activities than peripheral blood PMNs. This suggests that CD33, an adhesion molecule, may be involved in the egress from blood to milk and that the enzymatic contents of PMNs are partly used during this process.
Culture of Protozoan Parasites
Authors: Visvesvara Govinda S. , Garcia Lynne S.
Published: July 2002

The in vitro culture of protozoan parasites involves highly complex procedures, which are subject to many variables. These parasites have very complex life cycles and, depending on the life cycle stage, may require different culture parameters. However, in vitro cultivation is important for many reasons, some of which include: diagnosis, antigen and antibody production, assessment of parasite immune modulating capabilities, drug screening, improvements in chemotherapy, differentiation of clinical isolates, determination of strain differences, vaccine production, development of attenuated strains, and the continued supply of viable organisms for studying host-parasite interactions.
Methods for Cultivation of Luminal Parasitic Protists of Clinical Importance
Authors: Clark C. Graham , Diamond Louis S.
Published: July 2002

Cultivation of luminal protistan parasites has a long history. In this review we discuss the methods and media that are most widely used for the establishment and maintenance of the following organisms in culture:Entamoeba histolytica,Giardia intestinalis,Trichomonas vaginalis,Dientamoeba fragilis,Blastocystis hominis, andBalantidium coli. While cultivation is of limited importance in the diagnostic laboratory, it is essential to most research laboratories, and it is toward the latter that this review is primarily aimed.
Vancomycin Resistance in Staphylococci
Authors: Srinivasan Arjun , Dick James D. , Perl Trish M.
Published: July 2002

Vancomycin resistance has been reported in clinical isolates of both coagulase-negative staphylococci and
Staphylococcus aureus
. The emerging threat of widespread vancomycin resistance poses a serious public health concern given the fact that vancomycin has long been the preferred treatment of antibiotic-resistant gram-positive organisms. Though major efforts are now being focused on improving our understanding of vancomycin resistance, there is much that remains unknown at this time. This article reviews the major epidemiologic, microbiologic, and clinical characteristics of vancomycin resistance in both coagulase-negative staphylococci and
S. aureus
. The review begins with a discussion of issues common to both coagulase-negative staphylococci and
S. aureus
, such as definitions, laboratory detection of vancomycin resistance, and infection control issues related to vancomycin-resistant staphylococci. The rest of the article is then devoted to a discussion of issues unique to each organism, including epidemiology, risk factors for infection, mechanisms of resistance, and management options.