bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the Conjugative
mef
Gene in Clinical
Acinetobacter junii
and
Neisseria gonorrhoeae
Isolates
Authors: Luna Vicki A. , Cousin Sydney , Whittington William L. H. , Roberts Marilyn C.
Published: July 2002

The
mef
gene, originally described for gram-positive organisms and coding for an efflux pump, has been identified in clinical isolates of
Acinetobacter junii
and
Neisseria gonorrhoeae
. These strains could transfer the
mef
gene at frequencies ranging from 10
−6
to 10
−9
into one or more of the following recipients: gram-negative
Moraxella catarrhalis
,
Neisseria perflava/sicca
and
Neisseria mucosa
and gram-positive
Enterococcus faecalis
. Three
Streptococcus pneumoniae
strains could transfer the
mef
gene into
Eikenella corrodens
,
Haemophilus influenzae
,
Kingella denitrificans
,
M. catarrhalis
,
Neisseria meningitidis
,
N. perflava/sicca
, and
N. mucosa
at similar frequencies. The
mef
gene can thus be transferred to and expressed in a variety of gram-negative recipients.
Phase I/II Trial of the Pharmacokinetics, Safety, and Antiretroviral Activity of Tenofovir Disoproxil Fumarate in Human Immunodeficiency Virus-Infected Adults
Authors: Barditch-Crovo Patricia , Deeks Steven G. , Collier Ann , Safrin Sharon , Coakley Dion F. , Miller Michael , Kearney Brian P. , Coleman Rebecca L. , Lamy Patrick D. , Kahn James O. , McGowan Ian , Lietman Paul S.
Published: July 2002

Tenofovir DF is an antiviral nucleotide with activity against human immunodeficiency virus type 1 (HIV-1). The pharmacokinetics, safety, and activity of oral tenofovir DF in HIV-1-infected adults were evaluated in a randomized, double-blind, placebo-controlled, escalating-dose study of four doses (75, 150, 300, and 600 mg given once daily). Subjects received a single dose of tenofovir DF or a placebo, followed by a 7-day washout period. Thereafter, subjects received their assigned study drug once daily for 28 days. Pharmacokinetic parameters were dose proportional and demonstrated no change with repeated dosing. Reductions in plasma HIV-1 RNA were dose related at tenofovir DF doses of 75 to 300 mg, but there was no increase in virus suppression between the 300- and 600-mg dose cohorts, despite dose-proportional increases in drug exposure. Grade III or IV adverse events were limited to laboratory abnormalities, including elevated creatine phosphokinase and liver function tests, which resolved with or without drug discontinuation and without sequelae. No patients developed detectable sequence changes in the reverse transcriptase gene.
Molecular Analysis of Tetracycline Resistance in
Pasteurella aerogenes
Authors: Kehrenberg Corinna , Schwarz Stefan
Published: July 2002

Tetracycline-resistant
Pasteurella aerogenes
isolates obtained from the intestinal tract of swine were investigated for their
tet
genes by PCR analysis and hybridization experiments. In contrast to
Pasteurella
isolates from the respiratory tract,
tet
(H) genes were detected in the chromosomal DNA of only 2 of the 24 isolates, one of which also carried two copies of a
tet
(B) gene. All other
P. aerogenes
isolates carried
tet
(B) genes, which are the predominant
tet
genes among
Enterobacteriaceae
. A single isolate harbored a
tet
(B) gene as part of a truncated Tn
10
element on the 4.8-kb plasmid pPAT2. Comparative analysis of the pPAT2 sequence suggested that the Tn
10
relic on plasmid pPAT2 is the result of several illegitimate recombination events. The remaining 21
P. aerogenes
isolates carried one or two copies of the
tet
(B) gene in their chromosomal DNA. In the majority of the cases, these
tet
(B) genes were associated with copies of Tn
10
as confirmed by their
Sfu
I and
Bam
HI hybridization patterns. No correlation between the number of
tet
gene copies and the MICs of tetracycline, doxycyline and minocycline was observed.
Mechanism of Action of the Des-F(6) Quinolone BMS-284756 Measured by Supercoiling Inhibition and Cleavable Complex Assays
Authors: Wu Ping , Lawrence Laura E. , Denbleyker Kenneth L. , Barrett John F.
Published: July 2002

BMS-284756 (T-3811ME), a novel des-F(6) quinolone, was tested in the supercoiling inhibition and cleavable complex assays against
Escherichia coli
DNA gyrase, a target of quinolones. The results suggest that BMS-284756 has the same mechanism of action against DNA gyrase as other quinolones and a similar level of potency.
Germinated and Nongerminated Conidial Suspensions for Testing of Susceptibilities of
Aspergillus
spp. to Amphotericin B, Itraconazole, Posaconazole, Ravuconazole, and Voriconazole
Authors: Espinel-Ingroff A.
Published: July 2002

The effect of germinated and nongerminated conidia of
Aspergillus
spp. on the fungistatic (National Committee for Clinical Laboratory Standards document M38-P) and fungicidal activities (MICs and minimal fungicidal concentrations [MFCs] respectively) of amphotericin B, itraconazole, posaconazole (SCH56592), ravuconazole (BMS-207147), and voriconazole was evaluated. MFCs were the lowest drug dilutions that showed fewer than three colonies (99.9% killing). Overall, the MICs (0.12 to 4 μg/ml) and MFCs (0.5 to >8 μg/ml) of all of the agents tested with both inocula were the same or within 2 dilutions for the 72 isolates. Therefore, MICs and MFCs can be obtained with convenient and standardized nongerminated conidia.
Activities of Linezolid against Rapidly Growing Mycobacteria
Authors: Wallace R. J. , Brown-Elliott B. A. , Ward S. C. , Crist C. J. , Mann L. B. , Wilson R. W.
Published: July 2002

Linezolid is an oxazolidinone available as an oral drug which has activity against most gram-positive bacteria. However, few species of the genusMycobacteriumhave been studied. We tested 249 clinical isolates and 10 reference strains of rapidly growing mycobacteria for susceptibility to linezolid by broth microdilution. Clinical species included theMycobacterium fortuitumgroup (n= 74),M. abscessus(n= 98),M. chelonae(n= 50),M. mucogenicum(n= 10), andM. fortuitumthird biovariant complex (10). The modal MIC forM. mucogenicumwas 1.0 μg/ml, and the MIC at which 90% of the isolates tested are inhibited (MIC90) was 4 μg/ml; the modal MIC for theM. fortuitumgroup was 4 μg/ml, and the MIC90was 16 μg/ml; the modal MIC for theM. fortuitumthird biovariant complex was 4 μg/ml, and the MIC90was 8 μg/ml; the modal MIC forM. chelonaewas 8 μg/ml, and the MIC90was 16 μg/ml; and the modal MIC forM. abscessuswas 32 μg/ml, and the MIC90was 64 μg/ml. Based on peak levels of linezolid in serum of 15 to 20 μg/ml, we propose the following broth MIC breakpoints for these species: susceptible, ≤ 8 μg/ml; moderately susceptible, 16 μg/ml; and resistant, ≥32 μg/ml). These studies demonstrate the excellent potential of linezolid for therapy of rapidly growing mycobacteria.
In Vitro and In Vivo Properties of Ro 63-9141, a Novel Broad-Spectrum Cephalosporin with Activity against Methicillin-Resistant Staphylococci
Authors: Hebeisen Paul , Heinze-Krauss Ingrid , Angehrn Peter , Hohl Peter , Page Malcolm G. P. , Then Rudolf L.
Published: July 2002

Ro 63-9141 is a new member of the pyrrolidinone-3-ylidenemethyl cephem series of cephalosporins. Its antibacterial spectrum was evaluated against significant gram-positive and gram-negative pathogens in comparison with those of reference drugs, including cefotaxime, cefepime, meropenem, and ciprofloxacin. Ro 63-9141 showed high antibacterial in vitro activity against gram-positive bacteria except ampicillin-resistant enterococci, particularly vancomycin-resistant strains of
Enterococcus faecium
. Its MIC at which 90% of the isolates tested were inhibited (MIC
90
) for methicillin-resistant
Staphylococcus aureus
(MRSA) was 4 μg/ml. Ro 63-9141 was bactericidal against MRSA. Development of resistance to the new compound in MRSA was not observed. Ro 63-9141 was more potent than cefotaxime against penicillin-resistant
Streptococcus pneumoniae
(MIC
90
= 2 μg/ml). It was active against ceftazidime-susceptible strains of
Pseudomonas aeruginosa
and against
Enterobacteriaceae
except
Proteus vulgaris
and some isolates producing extended-spectrum β-lactamases. The basis for the antibacterial spectrum of Ro 63-9141 lies in its affinity to essential penicillin-binding proteins, including PBP 2′ of MRSA, and its stability towards β-lactamases. The in vivo findings were in accordance with the in vitro susceptibilities of the pathogens. These data suggest the potential utility of Ro 63-9141 for the therapy of infections caused by susceptible pathogens, including MRSA. Since insufficient solubility of Ro 63-9141 itself precludes parenteral administration in humans, a water-soluble prodrug, Ro 65-5788, is considered for development.
Pharmacodynamics of Amphotericin B in a Neutropenic-Mouse Disseminated-Candidiasis Model
Authors: Andes D. , Stamsted T. , Conklin R.
Published: July 2002

In vivo pharmacodynamic parameters have been described for a variety of antibacterials. These parameters have been studied in correlation with in vivo outcomes in order to determine which dosing parameter is predictive of outcome and the magnitude of that parameter associated with efficacy. Very little is known about pharmacodynamics for antifungal agents. We utilized a neutropenic mouse model of disseminated candidiasis to correlate pharmacodynamic parameters (percent time above MIC [
T
> MIC], area under the concentration time curve [AUC]/MIC ratio, and peak serum level/MIC ratio) for amphotericin B in vivo with efficacy, as measured by organism number in homogenized kidney cultures after 72 h of therapy. Amphotericin B was administered by the intraperitoneal route. Drug kinetics for amphotericin B in infected mice were nonlinear. Serum half-lives ranged from 13 to 27 h. Infection was achieved by intravenous inoculation with 10
6
CFU of yeast cells per ml via the lateral tail vein of neutropenic mice. Groups of mice were treated with fourfold escalating total doses of amphotericin B ranging from 0.08 to 20 mg/kg of body weight divided into 1, 3, or 6 doses over 72 h. Increasing doses produced concentration-dependent killing, ranging from 0 to 2 log
10
CFU/kidney compared to the organism number at the start of therapy. Amphotericin B also produced prolonged dose-dependent suppression of growth after serum levels had fallen below the MIC. Nonlinear regression analysis was used to determine which pharmacodynamic parameter best correlated with efficacy. Peak serum level in relation to the MIC (peak serum level/MIC ratio) was the parameter best predictive of outcome, while the AUC/MIC ratio and
T
> MIC were only slightly less predictive (peak serum level/MIC ratio, coefficient of determination [
R
2
] = 90 to 93%; AUC/MIC ratio,
R
2
= 49 to 69%;
T
> MIC,
R
2
= 67 to 85%). The total amount of drug necessary to achieve various microbiological outcomes over the treatment period was 4.8- to 7.6-fold smaller when the dosing schedule called for large single doses than when the same amount of total drug was administered in 2 to 6 doses. Given the narrow therapeutic window of amphotericin B and frequent treatment failures, these results suggest the need for a reevaluation of current dosing regimens.
Mutations of the
Helicobacter pylori
Genes
rdxA
and
pbp1
Cause Resistance against Metronidazole and Amoxicillin
Authors: Paul Ralf , Postius Stefan , Melchers Klaus , Schäfer Klaus P.
Published: July 2002

To investigate amoxicillin and metronidazole resistance of
Helicobacter pylori
, we compared putative resistance genes between resistant strains obtained in vitro and their sensitive parent strain. All metronidazole-resistant strains had
rdxA
mutations, and an amoxicillin-resistant strain had
pbp1
and
pbp2
mutations. By transforming PCR products of these mutated genes into antibiotic-sensitive strains, we showed that
rdxA
null mutations were sufficient for metronidazole resistance
,
while
pbp1
mutations contributed to amoxicillin resistance of
H. pylori
.
Combination Treatment with Intralesional Cidofovir and Viral-DNA Vaccination Cures Large Cottontail Rabbit Papillomavirus-Induced Papillomas and Reduces Recurrences
Authors: Christensen Neil D. , Han Ricai , Cladel Nancy M. , Pickel Martin D.
Published: July 2002

We used the cottontail rabbit papillomavirus (CRPV) New Zealand White rabbit model to test a combination treatment of large established papillomas with intralesional cidofovir and DNA vaccination to cure sites and reduce recurrences. Intralesional 1% (wt/vol) (0.036 M) cidofovir treatment of rabbit papillomas led to elimination, or “cure,” of the papillomas over a 6- to 8-week treatment period (N. D. Christenson, M. D. Pickel, L. R. Budgeon, and J. W. Kreider, Antivir. Res. 48:131–142, 2000). However, recurrences at periods from 1 to 8 weeks after treatment cessation were observed at approximately 50% of cured sites. DNA vaccinations with CRPV E1, E2, E6, and E7 were initiated either after or at the time of intralesional treatments, and the recurrence rates were observed. When DNA vaccinations were started after intralesional cures, recurrence rates were similar to those of vector-vaccinated rabbits. A small proportion of recurrent sites subsequently regressed (4 out of 10, or 40%) in the vaccinated group versus no regression of recurrences in the vector-immunized group (0 out of 19, or 0%), indicating partial effectiveness. In contrast, when DNA vaccinations were conducted during intralesional treatments, a significant reduction of recurrences (from 10 out of 19, or 53%, of sites in vector-immunized rabbits to 3 out of 20, or 15%, of sites in viral-DNA-immunized rabbits) was observed. DNA vaccination without intralesional treatments had a minimal effect on preexisting papillomas. These data indicated that treatment with a combination of antiviral compounds and specific immune stimulation may lead to long-term cures of lesions without the ensuing problem of papilloma recurrence.
Influence of Human Serum on Antifungal Pharmacodynamics with
Candida albicans
Authors: Zhanel George G. , Saunders David G. , Hoban Daryl J. , Karlowsky James A.
Published: July 2002

Antifungal susceptibilities (NCCLS, approved standard M27-A, 1997) were determined for the reference strain ATCC 90028 and 21 clinical isolates of
Candida albicans
with varying levels of fluconazole susceptibility using RPMI 1640 (RPMI) and 80% fresh human serum–20% RPMI (serum). Sixty-four percent (14 of 22) of the isolates tested demonstrated significant decreases (≥4-fold) in fluconazole MICs in the presence of serum, and the remaining eight isolates exhibited no change. Itraconazole and ketoconazole, two highly protein-bound antifungal agents, had MICs in serum that were increased or unchanged for 46% (10 of 22) and 41% (9 of 22) of the isolates, respectively. All 10 isolates tested against an investigational antifungal agent, LY303366, demonstrated significant increases in the MIC required in serum, while differences in amphotericin B MICs in the two media were not observed. Four of 10 isolates tested demonstrated fourfold higher flucytosine MICs in serum than in RPMI. Postantifungal effects (PAFEs) and 24-h kill curves were determined by standard methods for selected isolates. At the MIC, fluconazole, itraconazole, ketoconazole, flucytosine, and LY303366 kill curves and PAFEs in RPMI were similar to those in serum. Isolates of fluconazole-resistant
C. albicans
required lower MICs in serum than in RPMI, without relative increases in fungal killing or PAFEs. Isolates tested against amphotericin B demonstrated significantly reduced killing and shorter PAFEs in serum than in RPMI without observable changes in MIC. In conclusion, antifungal pharmacodynamics in RPMI did not consistently predict antifungal activity in serum for azoles and amphotericin B. Generally speaking, antifungal agents with high protein binding exhibited some form of reduced activity (MIC, killing, or PAFE) in the presence of serum compared to those with low protein binding.
Antifungal Activities of Posaconazole, Ravuconazole, and Voriconazole Compared to Those of Itraconazole and Amphotericin B against 239 Clinical Isolates of
Aspergillus
spp. and Other Filamentous Fungi: Report from SENTRY Antimicrobial Surveillance Program, 2000
Authors: Pfaller M. A. , Messer S. A. , Hollis R. J. , Jones R. N.
Published: July 2002

Posaconazole, ravuconazole, and voriconazole are new triazole derivatives that possess potent, broad-spectrum antifungal activity. We evaluated the in vitro activity of these investigational triazoles compared with that of itraconazole and amphotericin B against 239 clinical isolates of filamentous fungi from the SENTRY Program, including
Aspergillus
spp. (198 isolates),
Fusarium
spp. (7 isolates),
Penicillium
spp. (19 isolates),
Rhizopus
spp. (4 isolates),
Mucor
spp. (2 isolates), and miscellaneous species (9 isolates). The isolates were obtained from 16 different medical centers in the United States and Canada between January and December 2000. In vitro susceptibility testing was performed using the microdilution broth method outlined in the National Committee for Clinical Laboratory Standards M38-P document. Overall, posaconazole was the most active compound, inhibiting 94% of isolates at a MIC of ≤1 μg/ml, followed by voriconazole (91%), amphotericin B (89%), ravuconazole (88%), and itraconazole (70%). All three new triazoles demonstrated excellent activity (MIC, ≤1 μg/ml) against
Aspergillus
spp. (114
Aspergillus fumigatus
, 22
Aspergillus niger
, 13
Aspergillus flavus
, 9
Aspergillus versicolor
, 8
Aspergillus terreus
, and 32
Aspergillus
spp.): posaconazole (98%), voriconazole (98%), ravuconazole (92%), amphotericin B (89%), and itraconazole (72%). None of the triazoles were active against
Fusarium
spp. (MIC at which 50% of the isolates tested were inhibited [MIC
50
], >8 μg/ml) or
Mucor
spp. (MIC
50
, >8 μg/ml). Posaconazole and ravuconazole were more active than voriconazole against
Rhizopus
spp. (MIC
50
, 1 to 2 μg/ml versus >8 μg/ml, respectively). Based on these results, all three new triazoles exhibited promising activity against
Aspergillus
spp. and other less commonly encountered isolates of filamentous fungi. The clinical value of these in vitro data remains to be seen, and in vitro-in vivo correlation is needed for both new and established antifungal agents. Surveillance efforts should be expanded in order to monitor the spectrum of filamentous fungal pathogens and their in vitro susceptibility as these new antifungal agents are introduced into clinical use.
Efficacies of Fluconazole, Caspofungin, and Amphotericin B inCandida glabrata-Infected p47phox−/−Knockout Mice
Authors: Ju Justina Y. , Polhamus Cynthia , Marr Kieren A. , Holland Steven M. , Bennett John E.
Published: July 2002

Candida glabratais the second leading cause of adult candidemia, resulting in high mortality. Amphotericin B is considered the treatment of choice, while the efficacy of fluconazole is controversial and caspofungin efficacy is unknown. To ascertain drug efficacy in vivo, the utility of a murine model ofC. glabratainfection was investigated.C. glabratawas found to cause progressive, lethal infection when injected intravenously into C57BL/6 mice with reduced oxidative microbicidal capacity due to knockout of the p47phoxgene. Spleen and kidney organ CFU counts were determined in groups of mice 2 days after the mice completed 6 days of daily intraperitoneal drug treatment, which began on the day of infection. Daily injections of fluconazole at 80 mg/kg did not reduce spleen or kidney CFU counts after infection withC. glabratastrains having in vitro fluconazole MICs of 2, 32, or 256 μg/ml compared to saline-treated controls. However, this fluconazole regimen reduced spleen CFU counts in mice infected withCandida albicans, an infection that is known to be responsive to fluconazole. Caspofungin at 5 mg/kg and amphotericin B at 5 mg/kg were both effective in reducing fungal burden in spleens and kidneys ofC. glabrata-infected mice. Ten mice treated for 6 days with caspofungin at 1 mg/kg survived for 15 days, though all 10 saline-injected mice died or were so ill that they had to be sacrificed by 96 h postinfection. This murine model provided evidence of the efficacy of amphotericin B and caspofungin but not of fluconazole againstC. glabratainfection.
New Mg
2+
-Dependent Oxytetracycline Resistance Determinant Tet 34 in
Vibrio
Isolates from Marine Fish Intestinal Contents
Authors: Nonaka Lisa , Suzuki Satoru
Published: July 2002

A new oxytetracycline (OTC) resistance (Otc
r
) determinant, Tet 34, was cloned from chromosomal DNA of
Vibrio
sp. no. 6 isolated from intestinal contents of cultured yellowtail (
Seriola quinqueradiata
). The transformant, containing cloned Tet 34, could grow in broth containing 25 μg of drug per ml with 10 mM MgCl
2
. Tet 34 encoded an open reading frame (ORF) 154 amino acids long. The amino acid sequence of the ORF was homologous to sequences of several bacterial xanthine-guanine phosphoribosyltransferases (XPRTs), which act in purine nucleotide salvage synthesis. Mg
2+
binding site residues and the active site were highly conserved in XPRT and the ORF of Tet 34. The results suggest that Tet 34 encodes a new Mg
2+
-dependent Otc
r
mechanism.
Interaction between Grapefruit Juice and Praziquantel in Humans
Authors: Castro Nelly , Jung Helgi , Medina Roberto , González-Esquivel Dinora , Lopez Mario , Sotelo Julio
Published: July 2002

After a single oral dose of praziquantel with 250 ml of grapefruit juice, the area under the concentration-time curve and the maximum concentration in plasma of praziquantel (
C
max
) were significantly increased (
C
max
for water treatment, 637.71 ± 128.5 ng/ml; and
C
max
for grapefruit juice treatment, 1,037.65 ± 305.7 ng/ml,
P
< 0.05). No statistically significant differences were found in the time to maximum concentration of drug in plasma or elimination half-life.
A Bead-Based Method for Multiplexed Identification and Quantitation of DNA Sequences Using Flow Cytometry
Authors: Spiro Alexander , Lowe Mary , Brown Drew
Published: July 2002

A new multiplexed, bead-based method which utilizes nucleic acid hybridizations on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences is described. The method consists of three elements: beads (5.6-μm diameter) with oligomer capture probes attached to the surface, three fluorophores for multiplexed detection, and flow cytometry instrumentation. Two fluorophores are impregnated within each bead in varying amounts to create different bead types, each associated with a unique probe. The third fluorophore is a reporter. Following capture of fluorescent cDNA sequences from environmental samples, the beads are analyzed by flow cytometric techniques which yield a signal intensity for each capture probe proportional to the amount of target sequences in the analyte. In this study, a direct hybrid capture assay was developed and evaluated with regard to sequence discrimination and quantitation of abundances. The target sequences (628 to 728 bp in length) were obtained from the 16S/23S intergenic spacer region of microorganisms collected from polluted groundwater at the nuclear waste site in Hanford, Wash. A fluorescence standard consisting of beads with a known number of fluorescent DNA molecules on the surface was developed, and the resolution, sensitivity, and lower detection limit for measuring abundances were determined. The results were compared with those of a DNA microarray using the same sequences. The bead method exhibited far superior sequence discrimination and possesses features which facilitate accurate quantitation.
PCR Primers That Amplify Fungal rRNA Genes from Environmental Samples
Authors: Borneman James , Hartin R. Jack
Published: July 2002

Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi:
Ascomycota
,
Basidiomycota
,
Chytridomycota
, and
Zygomycota
. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by
Phythophthora cinnamomi
. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungal community than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses, no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.
Engineering Increased Stability in the Antimicrobial Peptide Pediocin PA-1
Authors: Johnsen Line , Fimland Gunnar , Eijsink Vincent , Nissen-Meyer Jon
Published: July 2002

Pediocin PA-1 is a food grade antimicrobial peptide that has been used as a food preservative. Upon storage at 4°C or room temperature, pediocin PA-1 looses activity, and there is a concomitant 16-Da increase in the molecular mass. It is shown that the loss of activity follows first-order kinetics and that the instability can be prevented by replacing the single methionine residue (Met31) in pediocin PA-1. Replacing Met by Ala, Ile, or Leu protected the peptide from oxidation and had only minor effects on bacteriocin activity (for most indicator strains 100% activity was maintained). Replacement of Met by Asp was highly deleterious for bacteriocin activity.
Colonization of the Stratified Squamous Epithelium of the Nonsecreting Area of Horse Stomach by Lactobacilli
Authors: Yuki Norikatsu , Shimazaki Tomoko , Kushiro Akira , Watanabe Koichi , Uchida Kazumi , Yuyama Teruhiko , Morotomi Masami
Published: July 2002

Selective adhesion to only certain epithelia is particularly common among the bacterial members of the indigenous microflora of mammals. We have found that the stratified squamous epithelium of the nonsecreting area of horse stomach is colonized by gram-positive rods. The microscopic features of a dense layer of these bacteria on the epithelium were found to be similar to those reported in mice, rats, and swine. Adhering microorganisms were isolated and identified asLactobacillus salivarius,L. crispatus,L. reuteri, andL. agilisby DNA-DNA hybridization and 16S rRNA gene sequencing techniques. These lactobacilli associated with the horse, except forL. reuteri, were found to adhere to horse epithelial cells in vitro but not to those of rats. A symbiotic relationship of these lactobacilli with the horse is suggested.
Peptide Nucleic Acid-Mediated PCR Clamping as a Useful Supplement in the Determination of Microbial Diversity
Authors: von Wintzingerode Friedrich , Landt Olfert , Ehrlich Angelika , Göbel Ulf B.
Published: July 2002

Peptide nucleic acid (PNA)-mediated PCR clamping (H. Ørum, P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, and C. Stanley, Nucleic Acids Res. 21:5332–5336, 1993) was introduced as a novel procedure to selectively amplify ribosomal DNAs (rDNAs) which are not frequently found in clone libraries generated by standard PCR from complex microbial consortia. Three different PNA molecules were used; two of these molecules (PNA-ALF and PNA-EUB353) overlapped with one of the amplification primers, whereas PNA-1114F hybridized to the middle of the amplified region. Thus, PCR clamping was achieved either by competitive binding between the PNA molecules and the forward or reverse primers (competitive clamping) or by hindering polymerase readthrough (elongation arrest). Gene libraries generated from mixed rDNA templates by using PCR clamping are enriched for clones that do not contain sequences homologous to the appropriate PNA oligomer. This effect of PCR clamping was exploited in the following two ways: (i) analysis of gene libraries generated by PCR clamping with PNA-ALF together with standard libraries reduced the number of clones which had to be analyzed to detect all of the different sequences present in an artificial rDNA mixture; and (ii) PCR clamping with PNA-EUB353 and PNA-1114F was used to selectively recover rDNA sequences which represented recently described phylogenetic groups (NKB19, TM6, cluster related to green nonsulfur bacteria) from an anaerobic, dechlorinating consortium described previously. We concluded that PCR clamping might be a useful supplement to standard PCR amplification in rDNA-based studies of microbial diversity and could be used to selectively recover members of undescribed phylogenetic clusters from complex microbial communities.
Increased Levels of Markers of Microbial Exposure in Homes with Indoor Storage of Organic Household Waste
Authors: Wouters Inge M. , Douwes Jeroen , Doekes Gert , Thorne Peter S. , Brunekreef Bert , Heederik Dick J. J.
Published: July 2002

As part of environmental management policies in Europe, separate collection of organic household waste and nonorganic household waste has become increasingly common. As waste is often stored indoors, this policy might increase microbial exposure in the home environment. In this study we evaluated the association between indoor storage of organic waste and levels of microbial agents in house dust. The levels of bacterial endotoxins, mold β(1→3)-glucans, and fungal extracullar polysaccharides (EPS) ofAspergillusandPenicilliumspecies were determined in house dust extracts as markers of microbial exposure. House dust samples were collected in 99 homes in The Netherlands selected on the basis of whether separated organic waste was present in the house. In homes in which separated organic waste was stored indoors for 1 week or more the levels of endotoxin, EPS, and glucan were 3.2-, 7.6-, and 4.6-fold higher, respectively (allP< 0.05), on both living room and kitchen floors than the levels in homes in which only nonorganic residual waste was stored indoors. Increased levels of endotoxin and EPS were observed, 2.6- and 2.1-fold (P< 0.1), respectively, when separated organic waste was stored indoors for 1 week or less, whereas storage of nonseparated waste indoors had no effect on microbial agent levels (P> 0.2). The presence of textile floor covering was another major determinant of microbial levels (P< 0.05). Our results indicate that increased microbial contaminant levels in homes are associated with indoor storage of separated organic waste. These increased levels might increase the risk of bioaerosol-related respiratory symptoms in susceptible people.
Inhibition of
Streptococcus mutans
NS Adhesion to Glass with and without a Salivary Conditioning Film by Biosurfactant- Releasing
Streptococcus mitis
Strains
Authors: van Hoogmoed C. G. , van der Kuijl-Booij M. , van der Mei H. C. , Busscher H. J.
Published: July 2002

The release of biosurfactants by adhering microorganisms as a defense mechanism against other colonizing strains on the same substratum surface has been described previously for probiotic bacteria in the urogenital tract, the intestines, and the oropharynx but not for microorganisms in the oral cavity. Two
Streptococcus mitis
strains (BA and BMS) released maximal amounts of biosurfactants when they were grown in the presence of sucrose and were harvested in the early stationary phase. The
S. mitis
biosurfactants reduced the surface tensions of aqueous solutions to about 30 to 40 mJ m
−2
. Biochemical and physicochemical analyses revealed that the biosurfactants released were glycolipids. An acid-precipitated fraction was extremely surfactive and was identified as a rhamnolipidlike compound. In a parallel-plate flow chamber, the number of
Streptococcus mutans
NS cells adhering to glass with and without a salivary conditioning film in the presence of biosurfactant-releasing
S. mitis
BA and BMS (surface coverage, 1 to 4%) was significantly reduced compared with the number of
S. mutans
NS cells adhering to glass in the absence of
S. mitis. S. mutans
NS adhesion in the presence of non-biosurfactant-releasing
S. mitis
BA and BMS was not reduced at all. In addition, preadsorption of isolated
S. mitis
biosurfactants to glass drastically reduced the adhesion of
S. mutans
NS cells and the strength of their bonds to glass, as shown by the increased percentage of
S. mutans
NS cells detached by the passage of air bubbles through the flow chamber. Preadsorption of the acid-precipitated fraction inhibited
S. mutans
adhesion up to 80% in a dose-responsive manner. These observations indicate that
S. mitis
plays a protective role in the oral cavity and protects against colonization of saliva-coated surfaces by cariogenic
S. mutans
.
Development of a Selective Medium for Isolation of
Helicobacter pylori
from Cattle and Beef Samples
Authors: Stevenson Timothy H. , Lucia Lisa M. , Acuff Gary R.
Published: July 2002

Helicobacter pylori
has been isolated from the human stomach with media containing only minimal selective agents. However, current research on the transmission and sources of infection requires more selective media due to the higher numbers of contaminants in environmental, oral, and fecal samples. The objective of this study was to develop and evaluate detection techniques that are sufficiently selective to isolate
H. pylori
from potential animal and food sources. Since
H. pylori
survives in the acidic environment of the stomach, low pH with added urea was studied as a potential selective combination.
H. pylori
grew fairly well on
H. pylori
Special Peptone plating medium supplemented with 10 mM urea at pH 4.5, but this pH did not sufficiently inhibit the growth of contaminants. Various antibiotic combinations were then compared, and a combination consisting of 10 mg of vancomycin per liter, 5 mg of amphotericin B per liter, 10 mg of cefsulodin per liter, 62,000 IU of polymyxin B sulfate per liter, 40 mg of trimethoprim per liter, and 20 mg of sulfamethoxazole per liter proved to be highly selective but still allowed robust colonies of
H. pylori
to grow. This medium was highly selective for recovering
H. pylori
from cattle and beef samples, and it is possible that it could be used to enhance the recovery of this bacterium from human and environmental samples, which may be contaminated with large numbers of competing microorganisms.
Selected Chitinase Genes in Cultured and Uncultured Marine Bacteria in the α- and γ-Subclasses of the Proteobacteria
Authors: Cottrell Matthew T. , Wood Daniel N. , Yu Liying , Kirchman David L.
Published: July 2002

PCR primers were patterned after chitinase genes in four γ-proteobacteria in the families
Alteromonadaceae
and
Enterobacteriaceae
(group I chitinases) and used to explore the occurrence and diversity of these chitinase genes in cultured and uncultured marine bacteria. The PCR results from 104 bacterial strains indicated that this type of chitinase gene occurs in two major groups of marine bacteria, α- and γ-proteobacteria, but not the
Cytophaga-Flavobacter
group. Group I chitinase genes also occur in some viruses infecting arthropods. Phylogenetic analysis indicated that similar group I chitinase genes occur in taxonomically related bacteria. However, the overall phylogeny of chitinase genes did not correspond to the phylogeny of 16S rRNA genes, possibly due to lateral transfer of chitinase genes between groups of bacteria, but other mechanisms, such as gene duplication, cannot be ruled out. Clone libraries of chitinase gene fragments amplified from coastal Pacific Ocean and estuarine Delaware Bay bacterioplankton revealed similarities and differences between cultured and uncultured bacteria. We had hypothesized that cultured and uncultured chitin-degrading bacteria would be very different, but in fact, clones having nucleotide sequences identical to those of chitinase genes of cultured α-proteobacteria dominated both libraries. The other clones were similar but not identical to genes in cultured γ-proteobacteria, including vibrios and alteromonads. Our results suggest that a closer examination of chitin degradation by α-proteobacteria will lead to a better understanding of chitin degradation in the ocean.
Copper Induction of Laccase Isoenzymes in the Ligninolytic Fungus
Pleurotus ostreatus
Authors: Palmieri Gianna , Giardina Paola , Bianco Carmen , Fontanella Bianca , Sannia Giovanni
Published: July 2002

Pleurotus ostreatus
is a white rot basidiomycete that produces several extracellular laccase isoenzymes, including phenol oxidase A1b (POXA1b), POXA2, and POXC. POXC was the most abundant isoenzyme produced under all of the growth conditions examined in this study. Copper was the most efficient inducer of laccase activity among the putative inducers tested. The amounts of all of the previously described laccase isoenzymes increased substantially in copper-supplemented cultures. Under these conditions expression of POX isoenzymes was regulated at the level of gene transcription. It is worth noting that
poxa1b
mRNA was the most abundant induced transcript at all of the growth times analyzed, and the amount of this mRNA increased until day 7. The discrepancy between the
poxa1b
transcript and protein amounts can be explained by the presence of a high level of the protein in
P. ostreatus
cellular extract, which indicated that the POXA1b isoenzyme could be inefficiently secreted and/or that its physiological activity could occur inside the cell or on the cell wall. Moreover, the POXA1b isoenzyme behaved uniquely, as its activity was maximal on the second day of growth and then decreased. An analysis performed with protease inhibitors revealed that the loss of extracellular POXA1b activity could have been due to the presence of specific proteases secreted into the copper-containing culture medium that affected the extracellular POXA1b isoenzyme.
Molecular Analysis of the
pmo
(Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs
Authors: Gilbert Bettina , McDonald Ian R. , Finch Ruth , Stafford Graham P. , Nielsen Allan K. , Murrell J. Colin
Published: July 2002

The particulate methane monooxygenase gene clusters,
pmoCAB
, from two representative type II methanotrophs of the α-
Proteobacteria
,
Methylosinus trichosporium
OB3b and
Methylocystis
sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the
pmo
cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene,
pmoC
, for
Methylocystis
sp. strain M. Immediately upstream of the putative start site, consensus sequences for ς
70
promoters were identified, suggesting that these
pmo
genes are recognized by ς
70
and negatively regulated under low-copper conditions. The
pmo
genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the
Escherichia coli
host. Methanotrophs contain two virtually identical copies of
pmo
genes, and it was necessary to use Southern blotting and probing with
pmo
gene fragments in order to differentiate between the two
pmoCAB
clusters in both methanotrophs. The complete DNA sequence of one copy of
pmo
genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the γ-
Proteobacteria
,
Methylococcus capsulatus
Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of
pmoC
in both
M. trichosporium
OB3b and
Methylocystis
sp. strain M, and not three copies as found in
M. capsulatus
Bath.
Carbon Monoxide Dehydrogenase Activity in
Bradyrhizobium japonicum
Authors: Lorite María J. , Tachil Jörg , Sanjuán Juán , Meyer Ortwin , Bedmar Eulogio J.
Published: July 2002

Bradyrhizobium japonicum
strain 110
spc
4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC
1.2.99.2
) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)
2
subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of
Oligotropha carboxidovorans
. The
B. japonicum
enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the
B. japonicum
CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.
Investigation of the Relationship between Lysogeny and Lysis of
Lactococcus lactis
in Cheese Using Prophage-Targeted PCR
Authors: O'Sullivan David , Ross R. Paul , Fitzgerald Gerald F. , Coffey Aidan
Published: July 2002

The ability of lactococcal strains to lyse (and release intracellular enzymes) during cheese manufacture can be a very desirable trait and has been associated with improvement in flavor and acceleration of cheese ripening. Using a laboratory-scale cheese manufacturing assay, the autolytic behavior of 31 strains of
Lactococcus lactis
was assessed. In general, marked variation was observed between strains with a 20-fold difference between the best and worst lysing strains based on the release of the intracellular enzyme lactate dehydrogenase. In a parallel experiment, the genomes of these strains were examined for the presence of prophage integrase (
int
) sequences by using conserved primer sequences from known lysogenic phage. Results demonstrated that the lytic behavior of lactococcal starter strains significantly correlates with the presence of prophage sequences. These results highlight not only the contribution of prophage to starter cell lysis but also the potential of PCR as a useful initial screen to assess strains for this important industrial trait.
Detection of
Pseudomonas savastanoi
pv. savastanoi in Olive Plants by Enrichment and PCR
Authors: Penyalver Ramón , García Amparo , Ferrer Amparo , Bertolini Edson , López María M.
Published: July 2002

The sequence of the gene
iaaL
of
Pseudomonas savastanoi
EW2009 was used to design primers for PCR amplification. The
iaaL
-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of
P. savastanoi
, whereas genomic DNA from 93 non-
P. savastanoi
isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract.
P. savastanoi
was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover,
P. savastanoi
was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of
P. savastanoi
.
Characterization ofS-Triazine Herbicide Metabolism by aNocardioidessp. Isolated from Agricultural Soils
Authors: Topp Edward , Mulbry Walter M. , Zhu Hong , Nour Sarah M. , Cuppels Diane
Published: July 2002

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Nine gram-positive bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from four farms in central Canada. The strains were divided into two groups based on repetitive extragenic palindromic (rep)-PCR genomic fingerprinting with ERIC and BOXA1R primers. Based on 16S ribosomal DNA sequence analysis, both groups were identified asNocardioidessp. strains. None of the isolates mineralized [ring-U-14C]atrazine. There was no hybridization to genomic DNA from these strains usingatzABCcloned fromPseudomonassp. strain ADP ortrzAcloned fromRhodococcus corallinus. S-Triazine degradation was studied in detail inNocardioidessp. strain C190. Oxygen was not required for atrazine degradation by whole cells or cell extracts. Based on high-pressure liquid chromatography and mass spectrometric analyses of products formed from atrazine in incubations of whole cells with H218O, sequential hydrolytic reactions converted atrazine to hydroxyatrazine and then to the end productN-ethylammelide. Isopropylamine, the putative product of the second hydrolytic reaction, supported growth as the sole carbon and nitrogen source. The triazine hydrolase from strain C190 was isolated and purified and found to have aKmfor atrazine of 25 μM and aVmaxof 31 μmol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 μM EDTA but was inhibited by 100 μM Mg, Cu, Co, or Zn. Whole cells and purified triazine hydrolase converted a range of chlorine or methylthio-substituted herbicides to the corresponding hydroxy derivatives. In summary, an atrazine-metabolizingNocardioidessp. widely distributed in agricultural soils degrades a range ofs-triazine herbicides by means of a novels-triazine hydrolase.
Comparison of 2,4-Dichlorophenoxyacetic Acid Degradation and Plasmid Transfer in Soil Resulting from Bioaugmentation with Two Different pJP4 Donors
Authors: Newby D. T. , Gentry T. J. , Pepper I. L.
Published: July 2002

A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host,
Ralstonia eutropha
JMP134, and a laboratory-generated strain amenable to donor counterselection,
Escherichia coli
D11. The
R. eutropha
strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the
E. coli
strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the
Burkholderia
and
Ralstonia
genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the
E. coli
D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.