bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations at Four Distinct Regions of the
rpoB
Gene Can Reduce the Susceptibility of
Helicobacter pylori
to Rifamycins
Authors: Heep Markus , Odenbreit Stefan , Beck Daniela , Decker Jochen , Prohaska Elke , Rieger Ulrich , Lehn Norbert
Published: July 2002

The transformation of a panel of
rpoB
mutations (codons 525 to 586) from rifampin-resistant donor strains into
Helicobacter pylori
2802A confirmed the MICs associated with the respective mutations.
RpoB
V149X random mutations were generated and induced different levels of resistance, depending on the replacement amino acid. Mutagenesis of the
rpoB
gene at codon position 701 (R701H) induced low-level resistance.
Effect of Oral Administration of Emtricitabine on Woodchuck Hepatitis Virus Replication in Chronically Infected Woodchucks
Authors: Korba Brent E. , Schinazi R. F. , Cote Paul , Tennant Bud C. , Gerin John L.
Published: July 2002

Emtricitabine [(−)FTC] [(−)-β-2′,3′-dideoxy-5-fluoro-3′-thiacytidine] has been shown to be an effective inhibitor of hepatitis B virus (HBV) in cell culture, with a potency and selectivity that are essentially identical to those of lamivudine. The antiviral activity of oral administration of (−)FTC against WHV replication in chronically infected woodchucks, an established and predictive model for antiviral therapy against HBV, was examined in a placebo-controlled study. (−)FTC significantly reduced viremia and intrahepatic WHV replication in a dose-dependent manner that was comparable to the antiviral activity of lamivudine observed in previous studies conducted by our laboratories. No effect on the levels of hepatic WHV RNA or the levels of woodchuck hepatitis surface antigen or anti-woodchuck hepatitis surface and core antibodies in the serum of the treated animals was observed. No evidence of drug-related toxicity was observed in any of the animals treated.
In Vitro Reversion of Amphotericin B Resistance in
Leishmania donovani
by Poloxamer 188
Authors: Espuelas S. , Legrand P. , Loiseau P. M. , Bories C. , Barratt G. , Irache J. M.
Published: July 2002

A micellar formulation of amphotericin B (AmB) solubilized with poloxamer 188 was evaluated against an AmB
Leishmania donovani
-resistant line. A concave isobologram showed a synergistic effect of this association against promastigotes. This result was confirmed with amastigotes since the 50% effective concentration of the new formulation was 100 times less than that of the control AmB formulation.
In Vitro Activities of the Glycylcycline GAR-936 against Gram-Positive Bacteria
Authors: Boucher Helen W. , Wennersten Christine B. , Eliopoulos George M.
Published: July 2002

The in vitro activities of GAR-936, the 9-
t
-butylglycylamido derivative of minocycline, were compared with those of doxycycline, minocycline, and tetracycline against 527 gram-positive clinical isolates. GAR-936 inhibited all strains, including those resistant to other tetracyclines, at concentrations of ≤2 μg/ml, except two strains of JK diphtheroids for which the MIC was 4 μg/ml.
Prophylactic Effect of FK463, a Novel Antifungal Lipopeptide, against
Pneumocystis carinii
Infection in Mice
Authors: Ito Mamoru , Nozu Ryoko , Kuramochi Takashi , Eguchi Natsuko , Suzuki Shuzo , Hioki Kyoji , Itoh Toshio , Ikeda Fumiaki
Published: July 2002

The prophylactic effect of FK463, a new water-soluble echinocandin-like lipopeptide with inhibitory activity against 1,3-β-
d
-glucan synthase, against
Pneumocystis carinii
infection was investigated with the severe combined immunodeficient (SCID) mouse model. Treatment with FK463, pentamidine, and saline only was performed for 6 weeks from the day after the SCID mice were inoculated intranasally with infected lung homogenates. FK463 at 0.2 or 1.0 mg/kg of body weight, pentamidine at 4 mg/kg, or saline was subcutaneously administered daily into the backs of the SCID mice. The effects of the drugs were evaluated by detection of
P. carinii
cysts in mouse lung homogenates by toluidine blue O staining, lung histology, and PCR amplification of a
P. carinii
-specific DNA fragment from the lungs.
P. carinii
cysts were detected in the lungs of all mice administered saline. In contrast, no cysts were detected in mice administered both doses of FK463 and pentamidine. A specific DNA fragment was amplified from all mice administered saline and at least half or more of the mice administered FK463 and pentamidine. These results indicate that FK463 acts on cyst wall formation but not on trophozoite proliferation and is extremely effective in preventing
P. carinii
-associated pneumonia. These results suggest that FK463 is potentially useful as a prophylactic agent against
P. carinii
infection.
Chloramphenicol-Sensitive
Escherichia coli
Strain Expressing the Chloramphenicol Acetyltransferase (
cat
) Gene
Authors: Potrykus Joanna , Wegrzyn Grzegorz
Published: July 2002

An
Escherichia coli
strain (strain CM2555) bearing the chloramphenicol acetyltransferase (
cat
) gene was found to be sensitive to chloramphenicol. We demonstrate that the
cat
gene is efficiently expressed in strain CM2555. Our results suggest that decreased levels of acetyl coenzyme A in
cat
-expressing CM2555 cells in the presence of chloramphenicol may cause the bacterium to be sensitive to this antibiotic.
Bacteriocin Production in Vancomycin-Resistant and Vancomycin-Susceptible
Enterococcus
Isolates of Different Origins
Authors: Del Campo Rosa , Tenorio Carmen , Jiménez-Dı́az Rufino , Rubio Carmen , Gómez-Lus Rafael , Baquero Fernando , Torres Carmen
Published: July 2002

Bacteriocin production was determined for 218
Enterococcus
isolates (
Enterococcus faecalis
[93] and
E. faecium
[125]) obtained from different origins (human clinical samples [87], human fecal samples [78], sewage [28], and chicken samples [25]) and showing different vancomycin susceptibility patterns (vancomycin resistant, all of them
vanA
positive [56], and vancomycin susceptible [162]). All enterococcal isolates were randomly selected except for the vancomycin-resistant ones. A total of 33 isolates of eight different bacterial genera were used as indicators for bacteriocin production. Forty-seven percent of the analyzed enterococcal isolates were bacteriocin producers (80.6% of
E. faecalis
and 21.6% of
E. faecium
isolates). The percentage of bacteriocin producers was higher among human clinical isolates (63.2%, 81.8% of vancomycin-resistant isolates and 60.5% of vancomycin-susceptible ones) than among isolates from the other origins (28 to 39.3%). Only one out of the 15 vancomycin-resistant isolates from human fecal samples was a bacteriocin producer, while 44.4% of fecal vancomycin-susceptible isolates were. The bacteriocin produced by the
vanA
-containing
E. faecium
strain RC714, named bacteriocin RC714, was further characterized. This bacteriocin activity was cotransferred together with the
vanA
genetic determinant to
E. faecalis
strain JH2-2. Bacteriocin RC714 was purified to homogeneity and its primary structure was determined by amino acid sequencing, showing an identity of 88% and a similarity of 92% with the previously described bacteriocin 31 from
E. faecalis
YI717. The presence of five different amino acids in bacteriocin RC714 suggest that this could be a new bacteriocin. The results obtained suggest that the epidemiology of vancomycin resistance may be influenced by different factors, including bacteriocin production.
Combination Effect of Vancomycin and β-Lactams against a
Staphylococcus
aureus
Strain, Mu3, with Heterogeneous Resistance to Vancomycin
Authors: Aritaka Nanae , Hanaki Hideaki , Cui Longzhu , Hiramatsu Keiichi
Published: July 2002

We tested the combined activity of vancomycin and seven β-lactam antibiotics against
Staphylococcus aureus
clinical strain Mu3, which displays heterogeneous resistance to vancomycin. When combined with vancomycin, four of the seven tested β-lactams exhibited an additive effect at or near their MICs, while all showed an antagonistic effect at lower, sub-MIC levels. This study implicated the unpredictable nature of combination therapy of β-lactams and vancomycin against
S. aureus
with reduced susceptibility to vancomycin.
Mechanism and Suppression of Lysostaphin Resistance in Oxacillin-ResistantStaphylococcus aureus
Authors: Climo Michael W. , Ehlert Kerstin , Archer Gordon L.
Published: July 2002

The potential for the development of resistance in oxacillin-resistantStaphylococcus aureus(ORSA) to lysostaphin, a glycylglycine endopeptidase produced byStaphylococcus simulansbiovarstaphylolyticus, was examined in vitro and in an in vivo model of infection. Following in vitro exposure of ORSA to subinhibitory concentrations of lysostaphin, lysostaphin-resistant mutants were idenitifed among all isolates examined. Resistance to lysostaphin was associated with a loss of resistance to β-lactams and a change in the muropeptide interpeptide cross bridge from pentaglycine to a single glycine. Mutations infemA, the gene required for incorporation of the second and third glycines into the cross bridge, were found following PCR amplification and nucleotide sequence analysis. Complementation of lysostaphin-resistant mutants with pBBB31, which encodesfemA, restored the phenotype of oxacillin resistance and lysostaphin susceptibility. Addition of β-lactam antibiotics to lysostaphin in vitro prevented the development of lysostaphin-resistant mutants. In the rabbit model of experimental endocarditis, administration of a low dose of lysostaphin for 3 days led predictably to the appearance of lysostaphin-resistant ORSA mutants in vegetations. Coadministration of nafcillin with lysostaphin prevented the emergence of lysostaphin-resistant mutants and led to a mean reduction in aortic valve vegetation counts of 7.5 log10CFU/g compared to those for untreated controls and eliminated the isolation of lysostaphin-resistant mutants from aortic valve vegetations. Treatment with nafcillin and lysostaphin given alone led to mean reductions of 1.35 and 1.65 log10CFU/g respectively. In ORSA, resistance to lysostaphin was associated with mutations infemA, but resistance could be suppressed by the coadministration of β-lactam antibiotics.
GT160-246, a Toxin Binding Polymer for Treatment of
Clostridium difficile
Colitis
Authors: Kurtz Caroline B. , Cannon E. Pat , Brezzani Alex , Pitruzzello Mary , Dinardo Carol , Rinard Emilie , Acheson David W. K. , Fitzpatrick Richard , Kelly Pamela , Shackett Keith , Papoulis Andrew T. , Goddard Philip J. , Barker Robert H. , Palace Gerard P. , Klinger Jeffrey D.
Published: July 2002

GT160-246, a high-molecular-weight soluble anionic polymer, was tested in vitro and in vivo for neutralization of
Clostridium difficile
toxin A and B activities. Five milligrams of GT160-246 per ml neutralized toxin-mediated inhibition of protein synthesis in Vero cells induced by 5 ng of toxin A per ml or 1.25 ng of toxin B per ml. In ligated rat ileal loops, 1 mg of GT160-246 neutralized fluid accumulation caused by 5 μg of toxin A. At doses as high as 80 mg/loop, cholestyramine provided incomplete neutralization of fluid accumulation caused by 5 μg of toxin A. GT160-246 protected 80% of the hamsters from mortality caused by infection with
C. difficile
, whereas cholestyramine protected only 10% of animals. Treatment of
C. difficile
-infected hamsters with metronidazole initially protected 100% of the hamsters from mortality, but upon removal of treatment, 80% of the hamsters had relapses and died. In contrast, removal of GT160-246 treatment did not result in disease relapse in the hamsters. GT160-246 showed no antimicrobial activity in tests with a panel of 16 aerobic bacteria and yeast and 22 anaerobic bacteria and did not interfere with the in vitro activities of most antibiotics. GT160-246 offers a novel, nonantimicrobial treatment of
C. difficile
disease in humans.
New
erm
Gene in
Staphylococcus aureus
Clinical Isolates
Authors: Matsuoka Mayumi , Inoue Matsuhisa , Nakajima Yoshinori , Endo Yoshihiro
Published: July 2002

We have identified
erm
(Y), a novel gene class that was originally designated
ermGM
, from a
Staphylococcus aureus
strain that has a plasmid that also harbors
msr
(A) and
mph
(C), genes that encode an efflux mechanism and a putative phosphorylase, respectively. The nucleotide and deduced amino acid sequences of
erm
(Y) were 81 and 76% identical to those of
erm
(T), respectively.
Efficacy of TAK-457, a Novel Intravenous Triazole, against Invasive Pulmonary Aspergillosis in Neutropenic Mice
Authors: Hayashi Ryogo , Kitamoto Naomi , Iizawa Yuji , Ichikawa Takashi , Itoh Katsumi , Kitazaki Tomoyuki , Okonogi Kenji
Published: July 2002

TAK-457 is an injectable prodrug of TAK-456, which is a novel oral triazole compound with potent antifungal activity. The in vivo efficacy of TAK-457 was evaluated in two models of invasive pulmonary aspergillosis with CDF1mice and CBA/J mice with transient neutropenia induced by cyclophosphamide. Against the infection in CDF1mice, treatment with 10 mg of TAK-457 and 1 mg of amphotericin B/kg reduced the fungal burden in lungs and rescued all mice. In the infection model with CBA/J mice, TAK-457 at a dose of 10 mg/kg significantly prolonged the survival time of mice, showing significant reduction of lung chitin levels and the plasma β-d-glucan levels. On the other hand, amphotericin B at 1 mg/kg which was a maximum tolerable dose showed slight but not significant prolongation of survival time of mice, although it also reduced the lung chitin levels and the plasma β-d-glucan levels to a lower extent but still significantly. These results suggest that TAK-457 is a promising candidate for development for the treatment of invasive aspergillosis in humans.
Strong Antifungal Activity of SS750, a New Triazole Derivative, Is Based on Its Selective Binding Affinity to Cytochrome P450 of Fungi
Authors: Matsumoto Masaru , Ishida Kazuya , Konagai Akihiro , Maebashi Kazunori , Asaoka Takemitsu
Published: July 2002

SS750 [(
R
)-(−)-2-(2,4-difluorophenyl)-1-(ethylsulfonyl)-1,1-difluoro-3-(1
H
-1,2,4-triazol-1-yl)-2-propanol] is a new triazole, and its potential as an antifungal agent was evaluated by in vitro and in vivo studies. In a comparison of the MICs at which 50% of isolates are inhibited (MIC
50
s) for all strains of
Candida
species and
Cryptococcus neoformans
tested, SS750 was four times or more active than fluconazole and had activity comparable to that of itraconazole. The most important advantage of SS750 was that, when the MIC
90
s were compared, SS750 had 64 and 32 times greater antifungal activities than fluconazole against
Candida krusei
and
Candida glabrata
, respectively, which are intrinsically less susceptible to fluconazole. In cyclophosphamide-immunosuppressed mouse models of systemic and pulmonary candidiasis caused by
C. albicans
, oral SS750 prolonged the number of days of survival of infected animals in a dose-dependent manner and was 4 and ≥64 times more potent than fluconazole and itraconazole, respectively. In a safety profile, SS750, like fluconazole, had less of an affinity for binding to mammalian cytochrome P450 compared with that of ketoconazole, despite its strong affinity for binding to fungal cytochrome P450. The mechanism for the increased in vitro antifungal activity of SS750 against
C. krusei
is partially due to the potent inhibitory activity (3.7 times versus that of fluconazole) of
C. krusei
cytochrome P450 sterol 14α-demethylase; SS750 showed a strong affinity for binding to cytochrome P450 of
C. krusei
, indicating that SS750 acts by inhibiting the cytochrome P450 sterol 14α-demethylase of fungal cells.
In Vitro Activities of BAL9141, a Novel Broad-Spectrum Pyrrolidinone Cephalosporin, against Gram-Negative Nonfermenters
Authors: Zbinden R. , Pünter V. , von Graevenitz A.
Published: July 2002

The activities of BAL9141 (formerly Ro 63-9141), a novel pyrrolidinone-3-ylidenemethyl cephalosporin, against 244 strains of gram-negative nonfermenters were evaluated. The overall MIC at which 50% of isolates are inhibited (MIC
50
) and the overall MIC
90
were 2 and 64 μg/ml, respectively, which are similar to those of imipenem, lower than those of the other cephalosporins tested, amoxicillin, and the ticarcillin-clavulanic acid combination, and much higher than those of ciprofloxacin. BAL9141 shows species-dependent activity in vitro against a variety of gram-negative nonfermentative pathogens.
In Vitro Activities of Peptide Deformylase Inhibitors against Gram-Positive Pathogens
Authors: Wise R. , Andrews J. M. , Ashby J.
Published: July 2002

The activities of six peptide deformylase (PDF) inhibitors against 107 respiratory tract pathogens were studied and compared to those of ciprofloxacin and amoxicillin-clavulanate. Against
Streptococcus
pneumoniae
, BB-83698 and BB-83815 were the most active PDF inhibitors (MIC at which 90% of the organisms tested were inhibited [MIC
90
], 0.25 μg/ml). Five of the agents showed similar activity against
Moraxella
catarrhalis
(MIC
90
, 0.12 μg/ml). All PDF inhibitors were less active against
Haemophilus
influenzae
; BB-3497 was the most active agent (MIC
90
, 2 μg/ml). Five agents were studied against
Chlamydia
spp. and showed activity similar to that of ciprofloxacin (MIC, 0.5 to 4 μg/ml). This study demonstrates that PDF inhibitors have the potential to be developed for the treatment of respiratory tract infections.
Novel Type of Staphylococcal Cassette Chromosome
mec
Identified in Community-Acquired Methicillin-Resistant
Staphylococcus aureus
Strains
Authors: Ma Xiao Xue , Ito Teruyo , Tiensasitorn Chuntima , Jamklang Mantana , Chongtrakool Piriyaporn , Boyle-Vavra Susan , Daum Robert S. , Hiramatsu Keiichi
Published: July 2002

We identified a new type of staphylococcal cassette chromosome
mec
(SCC
mec
) from two community-acquired methicillin-resistant
Staphylococcus aureus
(MRSA) strains. The novel element, designated type IV SCC
mec
, had a unique combination of the class B
mec
gene complex and the type 2
ccr
gene complex and was much smaller in size (21 to 24 kb) than previously identified SCC
mec
elements of hospital-acquired MRSA. Consistent with the strains' susceptibilities to various non-β-lactam antibiotics, the type IV SCC
mec
was devoid of any antibiotic resistance genes other than the
mecA
gene.
In Vitro and In Vivo Activities of a Novel Cephalosporin, BMS-247243, against Methicillin-Resistant and -Susceptible Staphylococci
Authors: Fung-Tomc Joan C. , Clark Junius , Minassian Beatrice , Pucci Michael , Tsai Yuan-Hwang , Gradelski Elizabeth , Lamb Lucinda , Medina Ivette , Huczko Elizabeth , Kolek Benjamin , Chaniewski Susan , Ferraro Cheryl , Washo Thomas , Bonner Daniel P.
Published: July 2002

The recent emergence of methicillin-resistant
Staphylococcus aureus
(MRSA) with decreased susceptibility to vancomycin has intensified the search for alternative therapies for the treatment of infections caused by this organism. One approach has been to identify a β-lactam with improved affinity for PBP 2a, the target enzyme responsible for methicillin resistance in staphylococci. BMS-247243 is such a candidate, with MICs that inhibit 90% of isolates tested (MIC
90
s) of 4, 2, and 8 μg/ml for methicillin-resistant strains of
S. aureus
,
S. epidermidis
, and
S. haemolyticus
, respectively, as determined on plates with Mueller-Hinton agar and 2% NaCl. The BMS-247243 MICs for MRSA were minimally affected by the susceptibility testing conditions (inoculum size, prolonged incubation, addition of salt to the test medium) or by staphylococcal β-lactamases. BMS-247243 MIC
90
s for methicillin-susceptible staphylococcal species ranged from ≤0.25 to 1 μg/ml. The BMS-247243 MIC
90
for β-lactamase-producing
S. aureus
strains was fourfold higher than that for β-lactamase-nonproducing strains. BMS-247243 is hydrolyzed by staphylococccal β-lactamases at 4.5 to 26.2% of the rates measured for cephaloridine. The affinity of BMS-247243 for PBP 2a was >100-fold better than that of methicillin or cefotaxime. BMS-247243 is bactericidal for MRSA, killing the bacteria twice as fast as vancomycin. These in vitro activities of BMS-247243 correlated with its in vivo efficacy against infections in animals, including the neutropenic murine thigh and rabbit endocarditis models involving MRSA strains. In conclusion, BMS-247243 has in vitro and in vivo activities against methicillin-resistant staphylococci and thus may prove to be useful in the treatment of infections caused by these multidrug-resistant organisms.
Purification, Characterization, and Identification of Novel Inhibitors of the β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) from
Staphylococcus aureus
Authors: He Xin , Reynolds Kevin A.
Published: July 2002

Staphylococcus aureus
is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of
S. aureus
requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one
fabH
in the genome of
S. aureus
and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of
Bacillus subtilis
(bFabH1),
Escherichia coli
(ecFabH), and
Mycobacterium tuberculosis
(mtFabH). Additional genomic sequence analysis revealed that this
S. aureus
FabH (saFabH) is not mutated in certain methicillin-resistant
S. aureus
(MRSA) and vancomycin-resistant
S. aureus
(VRSA) strains. saFabH was expressed in
E. coli
with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent

K
m

for malonyl-ACP was 1.76 ± 0.40 μM, and the enzyme was active with acetyl-CoA (
k
cat
, 16.18 min
−1
;

K
m

, 6.18 ± 0.9 μM), butyryl-CoA (
k
cat
, 42.90 min
−1
;

K
m

, 2.32 ± 0.12 μM), and isobutyryl-CoA (
k
cat
, 98.0 min
−1
;

K
m

, 0.32 ± 0.04 μM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC
50
], >100 μM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC
50
, 1.87 ± 0.10 μM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one (IC
50
, 0.775 ± 0.08 μM).
In Vitro and In Vivo Activities of Posaconazole against
Coccidioides immitis
Authors: González Gloria M. , Tijerina Rolando , Najvar Laura K. , Bocanegra Rosie , Rinaldi Michael , Loebenberg David , Graybill John R.
Published: July 2002

Posaconazole (SCH 56592) was tested against 25 strains of
Coccidioides immitis
to determine their in vitro susceptibilities. The geometric mean 48-h MIC of posaconazole (POSA) was 0.5 μg/ml, the MIC range was 0.25 to 1 μg/ml, and the MIC at which 50% of the isolates tested are inhibited (MIC
50
) and the MIC
90
were 0.5 and 1 μg/ml, respectively. The geometric mean 48-h MIC of itraconazole (ITRA) was 0.23 μg/ml, the MIC range was 0.125 to 0.5 μg/ml, and the MIC
50
and MIC
90
were both 0.25 μg/ml. Two strains of
C. immitis
were selected for in vivo studies on the basis of the POSA 48-h MICs for the isolates. POSA orally administered at 0.01, 0.1, 0.5, 1, 5, and 10 mg/kg of body weight/day was compared with ITRA administered at 10 and 30 mg/kg three times a day. The spleens and livers of mice that died or survived to day 50 were removed to measure the fungal burdens. Mice had ≥90% survival when they were treated with ≥0.5 mg of POSA per kg or 30 mg of ITRA per kg. Cultures of whole spleens and livers from mice treated with 10 mg of POSA per kg showed ≥70% sterilization. No sterilization of whole spleens and livers from mice treated with ITRA was seen. POSA displayed potent in vivo activity against the two strains of
C. immitis
tested.
Pharmacokinetics of Piperacillin-Tazobactam in Anuric Intensive Care Patients during Continuous Venovenous Hemodialysis
Authors: Mueller Silke C. , Majcher-Peszynska Jolanta , Hickstein Heiko , Francke Astrid , Pertschy Annette , Schulz Martin , Mundkowski Ralf , Drewelow Bernd
Published: July 2002

The pharmacokinetics of piperacillin-tazobactam were investigated in eight anuric intensive care patients treated by continuous venovenous hemodialysis (CVVHD). The elimination half-life of piperacillin was 4.3 ± 1.2 h, and that of tazobactam was 5.6 ± 1.3 h. The contribution of CVVHD to the overall elimination was relevant (>25%) for both drugs.
In Vitro Activities of Novel Nonfluorinated Quinolones PGE 9262932 and PGE 9509924 against Clinical Isolates of
Staphylococcus aureus
and
Streptococcus pneumoniae
with Defined Mutations in DNA Gyrase and Topoisomerase IV
Authors: Jones Mark E. , Critchley Ian A. , Karlowsky James A. , Blosser-Middleton Renée S. , Schmitz Franz-Josef , Thornsberry Clyde , Sahm Daniel F.
Published: July 2002

Two 8-methoxy nonfluorinated quinolones (NFQs), PGE 9262932 and PGE 9509924, were tested against contemporary clinical isolates of
Staphylococcus aureus
(
n
= 122) and
Streptococcus pneumoniae
(
n
= 69) with genetically defined quinolone resistance-determining regions (QRDRs). For
S. aureus
isolates with wild-type (WT) sequences at the QRDRs, the NFQs demonstrated activities 4- to 32-fold more potent (MICs at which 90% of isolates are inhibited [MIC
90
s], 0.03 μg/ml) than those of moxifloxacin (MIC
90
, 0.12 μg/ml), gatifloxacin (MIC
90
, 0.25 μg/ml), levofloxacin (MIC
90
, 0.25 μg/ml), and ciprofloxacin (MIC
90
, 1 μg/ml). Against
S. pneumoniae
isolates with WT sequences at
gyrA
and
parC
, the NFQs PGE 9262932 (MIC
90
, 0.03 μg/ml) and PGE 9509924 (MIC
90
, 0.12 μg/ml) were 8- to 64-fold and 2- to 16-fold more potent, respectively, than moxifloxacin (MIC
90
, 0.25 μg/ml), gatifloxacin (MIC
90
, 0.5 μg/ml), levofloxacin (MIC
90
, 2 μg/ml), and ciprofloxacin (MIC
90
, 2 μg/ml). The MICs of all agents were elevated for
S. aureus
isolates with alterations in GyrA (Glu88Lys or Ser84Leu) and GrlA (Ser80Phe) and
S. pneumoniae
isolates with alterations in GyrA (Ser81Phe or Ser81Tyr) and ParC (Ser79Phe or Lys137Asn). Fluoroquinolone MICs for
S. aureus
strains with double alterations in GyrA combined with double alterations in GrlA were ≥32 μg/ml, whereas the MICs of the NFQs for strains with these double alterations were 4 to 8 μg/ml. The PGE 9262932 and PGE 9509924 MICs for the
S. pneumoniae
isolates did not exceed 0.5 and 1 μg/ml, respectively, even for isolates with GyrA (Ser81Phe) and ParC (Ser79Phe) alterations, for which levofloxacin MICs were >16 μg/ml. No difference in the frequency of selection of mutations (<10
−8
at four times the MIC) in wild-type or first-step mutant isolates of
S. aureus
or
S. pneumoniae
was detected for the two NFQs. On the basis of their in vitro activities, these NFQ agents show potential for the treatment of infections caused by isolates resistant to currently available fluoroquinolones.
CAU-1, a Subclass B3 Metallo-β-Lactamase of Low Substrate Affinity Encoded by an Ortholog Present in theCaulobacter crescentusChromosome
Authors: Docquier Jean-Denis , Pantanella Fabrizio , Giuliani Francesco , Thaller Maria Cristina , Amicosante Gianfranco , Galleni Moreno , Frère Jean-Marie , Bush Karen , Rossolini Gian Maria
Published: July 2002

The sequenced chromosome ofCaulobacter crescentusCB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned inEscherichia colifromC. crescentustype strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced inE. coliwas purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme ofStenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Kmvalues were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. InC. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, theblaCAUdeterminant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the classProteobacteria.
Activities of Therapeutic Agents and Myristamidopropyl Dimethylamine against
Acanthamoeba
Isolates
Authors: Kilvington Simon , Hughes Reanne , Byas James , Dart John
Published: July 2002

The activities of therapeutic agents and myristamidopropyl dimethylamine (MAPD) against
Acanthamoeba
strains recalcitrant to medical therapy were studied. MAPD minimum cysticidal concentrations were 6.25 to 25 μg/ml; 10 to 30 μg/ml gave at least a 3-log cyst kill after 6 h, and 50 and 100 μg/ml gave at least a 3-log cyst kill within 2 and 1 h, respectively.
Molecular Genetic Manipulation of Truncated Cry1C Protein Synthesis in
Bacillus thuringiensis
To Improve Stability and Yield
Authors: Park Hyun-Woo , Bideshi Dennis K. , Federici Brian A.
Published: July 2002

Cry1 protoxins of
Bacillus thuringiensis
are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by using
cyt1A
promoters to drive expression of the 5′ half of
cry1C
that included in the construct the 5′
cry3A
STAB-SD mRNA stabilizing sequence and the 3′ stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3′ transcriptional termination sequences were important for stabilization of truncated
cry1C
(
cry1C-t
) mRNA. A low level of
cry1C-t
mRNA was present when only the
cyt1A
promoters were used to express
cry1C-t
, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in
cry1C-t
constructs further enhanced synthesis. The Cry1C-t protein was toxic to
Spodoptera exigua
larvae, though the toxicity (50% lethal concentration [LC
50
] = 13.2 μg/ml) was lower than that of full-length Cry1C (LC
50
= 1.8 μg/ml). However, transformation of the HD1 isolate of
B. thuringiensis
subsp.
kurstaki
with the
cry1C-t
construct enhanced its toxicity to
S. exigua
as much as fourfold.
Isolation and Use of a Homologous Histone H4 Promoter and a Ribosomal DNA Region in a Transformation Vector for the Oil-Producing FungusMortierella alpina
Authors: Mackenzie Donald A. , Wongwathanarat Prasert , Carter Andrew T. , Archer David B.
Published: July 2002

Mortierella alpinawas transformed successfully to hygromycin B resistance by using a homologous histone H4 promoter to drive gene expression and a homologous ribosomal DNA region to promote chromosomal integration. This is the first description of transformation in this commercially important oleaginous organism. Two pairs of histone H3 and H4 genes were isolated from this fungus. Each pair consisted of one histone H3 gene and one histone H4 gene, transcribed divergently from an intergenic promoter region. The pairs of encoded histone H3 or H4 proteins were identical in amino acid sequence. At the DNA level, each histone H3 or H4 open reading frame showed 97 to 99% identity to its counterpart but the noncoding regions had little sequence identity. Unlike the histone genes from other filamentous fungi, all fourM. alpinagenes lacked introns. During normal vegetative growth, transcripts from the two histone H4 genes were produced at approximately the same level, indicating that either histone H4 promoter could be used in transformation vectors. The generation of stable, hygromycin B-resistant transformants required the incorporation of a homologous ribosomal DNA region into the transformation vector to promote chromosomal integration.
Decarboxylation of Substituted Cinnamic Acids by Lactic Acid Bacteria Isolated during Malt Whisky Fermentation
Authors: van Beek Sylvie , Priest Fergus G.
Published: July 2002

Seven strains ofLactobacillusisolated from malt whisky fermentations and representingLactobacillus brevis,L. crispatus,L. fermentum,L. hilgardii,L. paracasei,L. pentosus, andL. plantarumcontained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception ofL. hilgardii, these bacteria decarboxylatedp-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition ofp-coumaric acid or ferulic acid to cultures ofL. pentosusin MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture ofL. crispatusandL. pentosusin the presence ofSaccharomyces cerevisiaedecarboxylated addedp-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylatep-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation.
An Improved Spectrophotometric Method To Study the Transport, Attachment, and Breakthrough of Bacteria through Porous Media
Authors: Deshpande P. A. , Shonnard D. R.
Published: July 2002

This study reports an improved spectrophotometric method for studying bacterial (
Pseudomonas fluorescens
UPER-1) transport and attachment in saturated porous media (silica sand). While studying the effect of ionic strength by the traditional packed-column spectrophotometric method, we encountered an artifact. The absorbance of a well-stirred bacterial suspension was found to decrease with time in the presence of high concentrations of sodium and potassium phosphate salts (≥10
−2
M) as the cells continued to age in a resting stage. Our results show that collision efficiency and a bed ripening index will be in error by as much as 20% if breakthrough is measured by the traditional spectrophotometric technique. We present an improved experimental technique that will minimize the artifact and should substantially advance the understanding of bacteria transport in porous media.
Effects of Acid Adaptation of
Escherichia coli
O157:H7 on Efficacy of Acetic Acid Spray Washes To Decontaminate Beef Carcass Tissue
Authors: Berry Elaine D. , Cutter Catherine N.
Published: July 2002

Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of
Escherichia coli
O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of
E. coli
O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of
E. coli
O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three
E. coli
O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted
E. coli
O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4°C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by
E. coli
O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses.
Distribution of Aldoxime Dehydratase in Microorganisms
Authors: Kato Yasuo , Ooi Ryoko , Asano Yasuhisa
Published: July 2002

The distribution of phenylacetaldoxime-degrading and pyridine-3-aldoxime-degrading ability was examined with intact cells of 975 microorganisms, including 45 genera of bacteria, 11 genera of actinomyces, 22 genera of yeasts, and 37 genera of fungi, by monitoring the decrease of the aldoximes by high-pressure liquid chromatography. The abilities were found to be widely distributed in bacteria, actinomyces, fungi, and some yeasts: 98 and 107 strains degraded phenylacetaldoxime and pyridine-3-aldoxime, respectively. All of the active strains exhibited not only the aldoxime-dehydration activity to form nitrile but also nitrile-hydrolyzing activity. On the other hand, all of 19 nitrile-degrading microorganisms (13 species, 7 genera) were found to exhibit aldoxime dehydration activity. It is shown that aldoxime dehydratase and nitrile-hydrolyzing activities are widely distributed among 188 aldoxime and 19 nitrile degraders and that the enzymes were induced by aldoximes or nitriles.
Hydroxylated Metabolites of 2,4-Dichlorophenol Imply a Fenton-Type Reaction in
Gloeophyllum striatum
Authors: Schlosser Dietmar , Fahr Kristina , Karl Wolfgang , Wetzstein Heinz-Georg
Published: July 2002

While degrading 2,4-dichlorophenol, two strains of
Gloeophyllum striatum
, a basidiomycetous fungus causing brown rot decay of wood, simultaneously produced 4-chlorocatechol and 3,5-dichlorocatechol. These metabolites were identified by comparing high-performance liquid chromatography retention times and mass spectral data with those of chemically synthesized standards. Under similar conditions, 3-hydroxyphthalic hydrazide was generated from phthalic hydrazide, a reaction assumed to indicate hydroxyl radical formation. Accordingly, during chemical degradation of 2,4-dichlorophenol by Fenton's reagent, identical metabolites were formed. Both activities, the conversion of 2,4-[U-
14
C]dichlorophenol into
14
CO
2
and the generation of 3-hydroxyphthalic hydrazide, were strongly inhibited by the hydroxyl radical scavenger mannitol and in the absence of iron. These results provide new evidence in favor of a Fenton-type degradation mechanism operative in
Gloeophyllum
.
Chromosomal Integration of
tcb
Chlorocatechol Degradation Pathway Genes as a Means of Expanding the Growth Substrate Range of Bacteria To Include Haloaromatics
Authors: Klemba Michael , Jakobs Barbara , Wittich Rolf-Michael , Pieper Dietmar
Published: July 2002

The
tcbR-tcbCDEF
gene cluster, coding for the chlorocatechol
ortho
-cleavage pathway in
Pseudomonas
sp. strain P51, has been cloned into a Tn
5
-based minitransposon. The minitransposon carrying the
tcb
gene cluster and a kanamycin resistance gene was transferred to
Pseudomonas putida
KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the
tcb
gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the
tcb
gene cluster was apparent. Introduction of two copies of the
tcb
gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the
tcb
chlorocatechol catabolic operon in
P. putida
thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.
Response of Atmospheric Methane Consumption by Maine Forest Soils to Exogenous Aluminum Salts
Authors: Nanba K. , King Gary M.
Published: July 2002

Atmospheric methane consumption by Maine forest soils was inhibited by additions of environmentally relevant levels of aluminum. Aluminum chloride was more inhibitory than nitrate or sulfate salts, but its effect was comparable to that of a chelated form of aluminum. Inhibition could be explained in part by the lower soil pH values which resulted from aluminum addition. However, significantly greater inhibition by aluminum than by mineral acids at equivalent soil pH values indicated that inhibition also resulted from direct effects of aluminum per se. The extent of inhibition by exogenous aluminum increased with increasing methane concentration for soils incubated in vitro. At methane concentrations of >10 ppm, inhibition could be observed when aluminum chloride was added at concentrations as low as 10 nmol g (fresh weight) of soil
−1
. These results suggest that widespread acidification of soils and aluminum mobilization due to acid precipitation may exacerbate inhibition of atmospheric methane consumption due to changes in other parameters and increase the contribution of methane to global warming.
Novel Bacterial and Archaeal Lineages from an In Situ Growth Chamber Deployed at a Mid-Atlantic Ridge Hydrothermal Vent
Authors: Reysenbach Anna-Louise , Longnecker Krista , Kirshtein Julie
Published: July 2002

The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23°22′ N, 44°57′ W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20°C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the
Archaea
or
Bacteria
domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the
Thermococcales
(71%) and
Archaeoglobales
(22%) orders. A sequence belonging to the
Thermoplasmales
confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the
Aquificales
(18%), the ɛ subclass of the
Proteobacteria
(ɛ-
Proteobacteria
) (40%), and the genus
Desulfurobacterium
(25%). Most of the clones (28%) were confined to a monophyletic clade within the ɛ-
Proteobacteria
with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S
0
, SO
4
, thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.
Modeling of Combined Processing Steps for Reducing
Escherichia coli
O157:H7 Populations in Apple Cider
Authors: Uljas Heidi E. , Schaffner Donald W. , Duffy Siobain , Zhao Lihui , Ingham Steven C.
Published: July 2002

Probabilistic models were used as a systematic approach to describe the response of
Escherichia coli
O157:H7 populations to combinations of commonly used preservation methods in unpasteurized apple cider. Using a complete factorial experimental design, the effect of pH (3.1 to 4.3), storage temperature and time (5 to 35°C for 0 to 6 h or 12 h), preservatives (0, 0.05, or 0.1% potassium sorbate or sodium benzoate), and freeze-thaw (F-T; −20°C, 48 h and 4°C, 4 h) treatment combinations (a total of 1,600 treatments) on the probability of achieving a 5-log
10
-unit reduction in a three-strain
E. coli
O157:H7 mixture in cider was determined. Using logistic regression techniques, pH, temperature, time, and concentration were modeled in separate segments of the data set, resulting in prediction equations for: (i) no preservatives, before F-T; (ii) no preservatives, after F-T; (iii) sorbate, before F-T; (iv) sorbate, after F-T; (v) benzoate, before F-T; and (vi) benzoate, after F-T. Statistical analysis revealed a highly significant (
P
< 0.0001) effect of all four variables, with cider pH being the most important, followed by temperature and time, and finally by preservative concentration. All models predicted 92 to 99% of the responses correctly. To ensure safety, use of the models is most appropriate at a 0.9 probability level, where the percentage of false positives, i.e., falsely predicting a 5-log
10
-unit reduction, is the lowest (0 to 4.4%). The present study demonstrates the applicability of logistic regression approaches to describing the effectiveness of multiple treatment combinations in pathogen control in cider making. The resulting models can serve as valuable tools in designing safe apple cider processes.