bluuebunny/crossref_metadata_2025 huggingface dataset:
Candidacidal Activities of Human Lactoferrin Peptides Derived from the N Terminus
Authors: Lupetti Antonella , Paulusma-Annema Akke , Welling Mick M. , Senesi Sonia , van Dissel Jaap T. , Nibbering Peter H.
Published: July 2002

In light of the need for new antifungal agents, the candidacidal activities of human lactoferrin (hLF) and synthetic peptides representing the first, hLF(1-11), and second, hLF(21-31), cationic domains of its N terminus were compared. The results revealed that hLF(1-11) was more effective in killing fluconazole-resistantCandida albicansthan hLF(21-31) and much more effective than lactoferrin, as determined microbiologically and by propidium iodide (PI) staining. By using hLF(1-11) and various derivatives, it was found that the second and third residues of the N terminus of hLF(1-11) were critical for its candidacidal activity. Detailed investigation to elucidate the mechanism of action of hLF(1-11) revealed a dose-dependent release of ATP byCandidaupon exposure to hLF(1-11). Our observations that sodium azide reduced the PI uptake and candidacidal activity of hLF(1-11) and that, upon exposure to hLF(1-11), the fluorescent dye rhodamine 123 first accumulated inside the mitochondria and later was released into the cytoplasm indicate that the peptide triggers the energized mitochondrion. Furthermore, oxidized ATP, which interferes with the interaction of ATP with its extracellular receptors, blocked the candidacidal action of hLF(1-11), as measured microbiologically and by PI staining. Addition of ATP (or analogues) was not a sufficient stimulus to killC. albicansor to act synergistically with suboptimal concentrations of the peptide. The main conclusions are that the first two arginines at the N terminus of hLF are critical in the candidacidal activity of hLF(1-11) and that extracellular ATP is essential but not sufficient for the peptide to exert its candidacidal activity.
Characterization of Spontaneous, In Vitro-Selected, Rifampin-Resistant Mutants of
Mycobacterium tuberculosis
Strain H37Rv
Authors: Morlock Glenn P. , Plikaytis Bonnie B. , Crawford Jack T.
Published: July 2002

Resistance to rifampin in
Mycobacterium tuberculosis
results from mutations in the gene coding for the beta subunit of RNA polymerase (
rpoB
). At least 95% of rifampin-resistant isolates have mutations in
rpoB
, and the mutations are clustered in a small region. About 40 distinct point mutations and in-frame insertions and deletions in
rpoB
have been identified, but point mutations in two codons, those coding for Ser
531
and His
526
, are seen in about 70% of rifampin-resistant clinical isolates, with Ser
531
-to-Leu (TCG-to-TGG) mutations being by far the most common. To explore this phenomenon, we isolated independent, spontaneous, rifampin-resistant mutant versions of well-characterized
M. tuberculosis
laboratory strain H37Rv by plating 100 separate cultures, derived from a single low-density inoculum, onto rifampin-containing medium. Rifampin-resistant mutants were obtained from 64 of these cultures. Although we anticipated that the various point mutations would occur with approximately equal frequencies, sequencing the
rpoB
gene from one colony per plate revealed that 39 (60.9%) were Ser
531
to Leu. We conclude that, for unknown reasons, the associated
rpoB
mutation occurs at a substantially higher rate than other
rpoB
mutations. This higher mutation rate may contribute to the high percentage of this mutation seen in clinical isolates.
Salivary Histatin 5 and Human Neutrophil Defensin 1 Kill
Candida albicans
via Shared Pathways
Authors: Edgerton Mira , Koshlukova Svetlana E. , Araujo Marcelo W. B. , Patel Rashmi C. , Dong Jin , Bruenn Jeremy A.
Published: July 2002

Salivary histatins are a family of basic histidine-rich proteins in which therapeutic potential as drugs against oral candidiasis is apparent, considering their potent in vitro antifungal activity and lack of toxicity to humans. Histatin 5 (Hst 5) kills the fungal pathogen
Candida albicans
via a mechanism that involves binding to specific sites on the yeast cell membrane and subsequent release of cellular ATP in the absence of cytolysis. We explored the killing pathway activated by Hst 5 and compared it to those activated by other antifungal agents. The candidacidal activity of human neutrophil defensin 1 (HNP-1) shared very similar features to Hst 5 cytotoxic action with respect to active concentrations and magnitude of induction of nonlytic ATP efflux, depletion of intracellular ATP pools, and inhibitor profile. Hst 5 and HNP-1 are basic proteins of about 3 kDa; however, they have unique primary sequences and solution structures that cannot explain how these two molecules act so similarly on
C. albicans
to induce cell death. Our finding that HNP-1 prevented Hst 5 binding to the candidal Hst 5 binding protein suggests that the basis for the overlapping actions of these two naturally occurring antimicrobial proteins may involve interactions with shared yeast components.
Nasal Carriage in Vietnamese Children of
Streptococcus pneumoniae
Resistant to Multiple Antimicrobial Agents
Authors: Parry Christopher M. , Diep To Song , Wain John , Hoa Nguyen Thi Tuyet , Gainsborough Mary , Nga Diem , Davies Catrin , Phu Nguyen Hoan , Hien Tran Tinh , White Nicholas J. , Farrar Jeremy J.
Published: July 2002

Resistance to antimicrobial agents in
Streptococcus pneumoniae
is increasing rapidly in many Asian countries. There is little recent information concerning resistance levels in Vietnam. A prospective study of pneumococcal carriage in 911 urban and rural Vietnamese children, of whom 44% were nasal carriers, was performed. Carriage was more common in children <5 years old than in those ≥5 years old (192 of 389 [49.4%] versus 212 of 522 [40.6%];
P
, 0.01). A total of 136 of 399 isolates (34%) had intermediate susceptibility to penicillin (MIC, 0.1 to 1 mg/liter), and 76 of 399 isolates (19%) showed resistance (MIC, >1.0 mg/liter). A total of 54 of 399 isolates (13%) had intermediate susceptibility to ceftriaxone, and 3 of 399 isolates (1%) were resistant. Penicillin resistance was 21.7 (95% confidence interval, 7.0 to 67.6) times more common in urban than in rural children (35 versus 2%;
P
, <0.001). More than 40% of isolates from urban children were also resistant to erythromycin, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline. Penicillin resistance was independently associated with an urban location when the age of the child was controlled for. Multidrug resistance (resistance to three or more antimicrobial agent groups) was present in 32% of isolates overall but in 39% of isolates with intermediate susceptibility to penicillin and 86% of isolates with penicillin resistance. The predominant serotypes of the
S. pneumoniae
isolates were 19, 23, 14, 6, and 18. Almost half of the penicillin-resistant isolates serotyped were serotype 23, and these isolates were often multidrug resistant. This study suggests that resistance to penicillin and other antimicrobial agents is common in carriage isolates of
S. pneumoniae
from children in Vietnam.
A Dose-Response Study of Antibiotic Resistance in
Pseudomonas aeruginosa
Biofilms
Authors: Brooun Alexei , Liu Songhua , Lewis Kim
Published: July 2002

Bacterial biofilms show enormous levels of antibiotic resistance, but little is known about the underlying molecular mechanisms. Multidrug resistance pumps (MDRs) are responsible for the extrusion of chemically unrelated antimicrobials from the bacterial cell. Contribution of the MDR-mediated efflux to antibiotic resistance of
Pseudomonas aeruginosa
biofilms was examined by using strains overexpressing and lacking the MexAB-OprM pump. Resistance of
P. aeruginosa
biofilms to ofloxacin was dependent on the expression of MexAB-OprM but only in the low concentration range. Unexpectedly, biofilm resistance to ciprofloxacin, another substrate of MexAB-OprM, did not depend on the presence of this pump. Dose-dependent killing indicated the presence of a small “superresistant” cell fraction. This fraction was primarily responsible for very high resistance of
P. aeruginosa
biofilms to quinolones. Bacterial cells recovered from a biofilm and tested under nongrowing conditions with tobramycin exhibited higher resistance levels than planktonic cells but lower levels than cells of an intact biofilm.
Characterization of the
fomA
and
fomB
Gene Products from
Streptomyces wedmorensis
, Which Confer Fosfomycin Resistance on
Escherichia coli
Authors: Kobayashi Seiji , Kuzuyama Tomohisa , Seto Haruo
Published: July 2002

Together, the
fomA
and
fomB
genes in the fosfomycin biosynthetic gene cluster of
Streptomyces wedmorensis
confer high-level fosfomycin resistance on
Escherichia coli
. To elucidate their functions, the
fomA
and
fomB
genes were overexpressed in
E. coli
and the gene products were characterized. The recombinant FomA protein converted fosfomycin to fosfomycin monophosphate, which was inactive on
E. coli
, in the presence of a magnesium ion and ATP. On the other hand, the recombinant FomB protein did not inactivate fosfomycin. However, a reaction mixture containing FomA and FomB proteins converted fosfomycin to fosfomycin monophosphate and fosfomycin diphosphate in the presence of ATP and a magnesium ion, indicating that FomA and FomB catalyzed phosphorylations of fosfomycin and fosfomycin monophosphate, respectively. These results suggest that the self-resistance mechanism of the fosfomycin-producing organism
S. wedmorensis
is mono- and diphosphorylation of the phosphonate function of fosfomycin catalyzed by FomA and FomB.
Intracellular Metabolism of β-
l
-2′,3′-Dideoxyadenosine: Relevance to Its Limited Antiviral Activity
Authors: Placidi Laurent , Cretton-Scott Erika , Gosselin Gilles , Pierra Claire , Schinazi Raymond F. , Imbach Jean-Louis , el Kouni Mahmoud H. , Sommadossi Jean-Pierre
Published: July 2002

The intracellular metabolism of the β-
l
- enantiomer of 2′,3′-dideoxyadenosine (β-
l
-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 μM [2′,3′,8-
3
H]-β-
l
-ddA resulted in an increased intracellular concentration of β-
l
-ddA with time, demonstrating that these cells were able to transport β-
l
-ddA. However, it did not result in the phosphorylation of β-
l
-ddA to its pharmacologically active 5′-triphosphate (β-
l
-ddATP). Five other intracellular metabolites were detected and identified as β-
l
-2′,3′-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 ± 0.95, 8.15 ± 2.64, and 15.60 ± 1.74 pmol/10
6
cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a β-glucuronic derivative of β-
l
-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with β-
l
-ddA in the presence of increasing concentrations of 5′-methylthioadenosine resulted in decreased phosphorolysis of β-
l
-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of β-
l
-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of β-
l
-ddA by methylthioadenosine phosphorylase (EC
2.4.2.28
).
Distribution and Content of Class 1 Integrons in Different
Vibrio cholerae
O-Serotype Strains Isolated in Thailand
Authors: Dalsgaard Anders , Forslund Anita , Serichantalergs Oralak , Sandvang Dorthe
Published: July 2002

In this study, 176 clinical and environmental
Vibrio cholerae
strains of different O serotypes isolated in Thailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the
aadB
,
aadA2
,
blaP1
,
dfrA1
, and
dfrA15
gene cassettes, which encode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; β-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the antibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons. However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and 1993 were associated with the emergence of a distinct
V. cholerae
O1 strain which contained the
aadA2
resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982 contained the
aadB
gene cassette. Surprisingly, several strains harbored two integrons containing different cassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among different
V. cholerae
O serotypes of mainly clinical origin in Thailand.
A New Class of Genetic Element, Staphylococcus Cassette Chromosome
mec
, Encodes Methicillin Resistance in
Staphylococcus aureus
Authors: Katayama Y. , Ito T. , Hiramatsu K.
Published: July 2002

We have previously shown that the methicillin-resistance gene
mecA
of
Staphylococcus aureus
strain N315 is localized within a large (52-kb) DNA cassette (designated the staphylococcal cassette chromosome
mec
[SCC
mec
]) inserted in the chromosome. By sequence determination of the entire DNA, we identified two novel genes (designated cassette chromosome recombinase genes [
ccrA
and
ccrB
]) encoding polypeptides having a partial homology to recombinases of the invertase/resolvase family. The open reading frames were found to catalyze precise excision of the SCC
mec
from the methicillin-resistant
S. aureus
chromosome and site-specific as well as orientation-specific integration of the SCC
mec
into the
S. aureus
chromosome when introduced into the cells as a recombinant multicopy plasmid. We propose that SCC
mec
driven by a novel set of recombinases represents a new family of staphylococcal genomic elements.
Ineffectiveness of Echinacea for Prevention of Experimental Rhinovirus Colds
Authors: Turner Ronald B. , Riker Donald K. , Gangemi J. David
Published: July 2002

The purpose of this study was to assess the effectiveness of echinacea for the prevention of experimental rhinovirus colds. Infection occurred in 44 and 57% and illness occurred in 36 and 43% of the echinacea- and placebo-treated subjects, respectively. This preparation of echinacea had no significant effect on either the occurrence of infection or the severity of illness.
Cloning and Sequence of the Gene Encoding a Novel Cefotaxime-Hydrolyzing β-Lactamase (CTX-M-9) from
Escherichia coli
in Spain
Authors: Sabaté Montserrat , Tarragó Raül , Navarro Ferran , Miró Elisenda , Vergés Clara , Barbé Jordi , Prats Guillermo
Published: July 2002

A new CTX-M-type β-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant
Escherichia coli
strain. Despite the close identity that exists between the CTX-M-9 and Toho-2 β-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes. Outside of this region there are only six amino acids substitutions between both proteins.
A Double-Blind Placebo-Controlled Crossover Trial of Intravenous Magnesium Sulfate for Foscarnet-Induced Ionized Hypocalcemia and Hypomagnesemia in Patients with AIDS and Cytomegalovirus Infection
Authors: Huycke Mark M. , Naguib M. Tarek , Stroemmel Mathias M. , Blick Kenneth , Monti Katherine , Martin-Munley Sarah , Kaufman Chris
Published: July 2002

Foscarnet (trisodium phosphonoformate hexahydrate) is an antiviral agent used to treat cytomegalovirus disease in immunocompromised patients. One common side effect is acute ionized hypocalcemia and hypomagnesemia following intravenous administration. Foscarnet-induced ionized hypomagnesemia might contribute to ionized hypocalcemia by impairing excretion of preformed parathyroid hormone (PTH) or by producing target organ resistance. Prevention of ionized hypomagnesemia following foscarnet administration could blunt the development of ionized hypocalcemia. To determine whether intravenous magnesium ameliorates the decline in ionized calcium and/or magnesium following foscarnet infusions, MgSO
4
at doses of 1, 2, and 3 g was administered in a double-blind, placebo-controlled, randomized, crossover trial to 12 patients with AIDS and cytomegalovirus disease. Overall, increasing doses of MgSO
4
reduced or eliminated foscarnet-induced acute ionized hypomagnesemia. Supplementation, however, had no discernible effect on foscarnet-induced ionized hypocalcemia despite significant increases in serum PTH levels. No dose-related, clinically significant adverse events were found, suggesting that intravenous supplementation with up to 3 g of MgSO
4
was safe in this chronically ill population. Since parenteral MgSO
4
did not alter foscarnet-induced ionized hypocalcemia or symptoms associated with foscarnet, routine intravenous supplementation for patients with normal serum magnesium levels is not recommended during treatment with foscarnet.
Human Immunodeficiency Virus Type 1 Mutations Selected in Patients Failing Efavirenz Combination Therapy
Authors: Bacheler Lee T. , Anton Elizabeth D. , Kudish Phil , Baker David , Bunville Julie , Krakowski Karen , Bolling Laura , Aujay Monette , Wang Xin Victoria , Ellis Dawn , Becker Mary F. , Lasut Amy L. , George Henry J. , Spalding Daniel R. , Hollis Greg , Abremski Kenneth
Published: July 2002

Efavirenz is a potent and selective nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Nucleotide sequence analyses of the protease and RT genes (coding region for amino acids 1 to 229) of multiple cloned HIV-1 genomes from virus found in the plasma of patients in phase II clinical studies of efavirenz combination therapy were undertaken in order to identify the spectrum of mutations in plasma-borne HIV-1 associated with virological treatment failure. A K103N substitution was the HIV-1 RT gene mutation most frequently observed among plasma samples from patients for whom combination therapy including efavirenz failed, occurring in at least 90% of cases of efavirenz-indinavir or efavirenz-zidovudine (ZDV)-lamivudine (3TC) treatment failure. V108I and P225H mutations were observed frequently, predominantly in viral genomes that also contained other nonnucleoside RT inhibitor (NNRTI) resistance mutations. L100I, K101E, K101Q, Y188H, Y188L, G190S, G190A, and G190E mutations were also observed. V106A, Y181C, and Y188C mutations, which have been associated with high levels of resistance to other NNRTIs, were rare in the patient samples in this study, both before and after exposure to efavirenz. The spectrum of mutations observed in cases of virological treatment failure was similar for patients initially dosed with efavirenz at 200, 400, or 600 mg once a day and for patients treated with efavirenz in combination with indinavir, stavudine, or ZDV-3TC. The proportion of patients carrying NNRTI resistance mutations, usually K103N, increased dramatically at the time of initial viral load rebound in cases of treatment failure after exposure to efavirenz. Viruses with multiple, linked NNRTI mutations, especially K103N-V108I and K103N-P225H double mutants, accumulated more slowly following the emergence of K103N mutant viruses.
Effect of Xylitol on Growth of
Streptococcus pneumoniae
in the Presence of Fructose and Sorbitol
Authors: Tapiainen Terhi , Kontiokari Tero , Sammalkivi Laura , Ikäheimo Irma , Koskela Markku , Uhari Matti
Published: July 2002

Xylitol is effective in preventing acute otitis media by inhibiting the growth of
Streptococcus pneumoniae
. To clarify this inhibition we used fructose, which is known to block similar growth inhibition observed in
Streptococcus mutans
. In addition, we evaluated the efficacy of sorbitol in inhibiting the growth of pneumococci, as sorbitol is widely used for indications similar to those for which xylitol is used. The addition of 5% xylitol to the growth medium resulted in marked growth inhibition, an effect which was totally eliminated in the presence of 1, 2.5, or 5% fructose but not in the presence of 1 or 5% glucose, 1% galactose, or 1% sucrose. This finding implies that xylitol-induced inhibition of pneumococcal growth is mediated via the fructose phosphotransferase system in a way similar to that in which mutans group streptococcal growth is inhibited. The addition of sorbitol at concentrations of 1, 2.5, or 5% to the growth medium did not affect the growth of pneumococci and neither inhibited nor enhanced the xylitol-induced growth impairment. Thus, it seems that xylitol is the only commercially used sugar substitute proven to have an antimicrobial effect on pneumococci.
Affinities of β-Lactams for Penicillin Binding Proteins of
Chlamydia trachomatis
and Their Antichlamydial Activities
Authors: Storey Christopher , Chopra Ian
Published: July 2002

Binding affinities of β-lactam antibiotics for the three penicillin binding proteins (PBPs) from
Chlamydia trachomatis
were determined in vitro and compared with their antichlamydial activities. Mecillinam selectively inhibited PBP1, with a 50% inhibitory concentration for PBP1 binding (0.2 μg/ml) similar to the MIC (0.1 μg/ml) and minimum bactericidal concentration (0.25 μg/ml). Although the other β-lactams inhibited a wider range of PBPs than mecillinam, their antichlamydial activities were inferior to that of mecillinam.
A MATE Family Multidrug Efflux Transporter Pumps out Fluoroquinolones in
Bacteroides thetaiotaomicron
Authors: Miyamae Shin , Ueda Ohmi , Yoshimura Fuminobu , Hwang Jaiweon , Tanaka Yoshinobu , Nikaido Hiroshi
Published: July 2002

We cloned a gene,
bexA
, that codes for a multidrug efflux transporter from the chromosomal DNA of
Bacteroides thetaiotaomicron
ATCC 29741 by using an
Escherichia coli
Δ
acrAB
Δ
acrEF
mutant as a host. Although the initial recombinant construct contained other open reading frames, the presence of
bexA
alone was sufficient to confer to the
E. coli
host elevated levels of resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Disruption of
bexA
in
B. thetaiotaomicron
made the strain more susceptible to norfloxacin, ciprofloxacin, and ethidium bromide, showing that this gene is expressed in this organism and functions as a multidrug efflux pump. The deduced BexA protein sequence was homologous to the protein sequence of
Vibrio parahaemolyticus
NorM, a multidrug efflux transporter, and thus, BexA belongs to the multidrug and toxic compound extrusion (MATE) family.
Glutathione-Induced Conversion of Pentavalent Antimony to Trivalent Antimony in Meglumine Antimoniate
Authors: Frézard Frédéric , Demicheli Cynthia , Ferreira Claúdio S. , Costa Michelle A. P.
Published: July 2002

The standard treatment of human leishmaniases involves the use of pentavalent antimony [Sb(V)] compounds, including meglumine antimoniate. The mode of action of these compounds has not been fully elucidated. The possibility that Sb(III) is involved has been suggested; however, the biomolecule that may induce the conversion of Sb(V) to Sb(III) has not yet been identified. In the present study, we investigated both the ability of reduced glutathione (GSH) to promote the reduction of Sb(V) into Sb(III) in meglumine antimoniate and the effects of pH and temperature on this transformation. GSH did promote the reduction of Sb(V) into Sb(III) in a dose-dependent manner. When GSH and meglumine antimoniate were incubated together at a GSH/Sb molar ratio superior or equal to 5:1, all antimony was encountered in the reduced form, indicating a stoichiometry of 5:1 between GSH and Sb(V) in the reaction. The reaction between Sb(V) and GSH was favored at an acidic pH (pH 5) and an elevated temperature (37°C), conditions found within the phagolysosome, in which
Leishmania
resides. For instance, about 30% of the Sb(V) (concentration, 2mM) was converted to Sb(III) following incubation for 3 days with 10 mM GSH at pH 5 and 37°C. Our data support the hypothesis that Sb(V) would be converted by GSH, or a related thiol compound, to more toxic Sb(III) in the phagolysosome of macrophages.
High-Dose Intravenous Ribavirin Therapy for Subacute Sclerosing Panencephalitis
Authors: Hosoya Mitsuaki , Shigeta Shiro , Mori Shuichi , Tomoda Akemi , Shiraishi Seiji , Miike Teruhisa , Suzuki Hitoshi
Published: July 2002

Two patients with subacute sclerosing panencephalitis (SSPE) were treated safely and effectively with high doses of intravenous ribavirin combined with intraventricular alpha interferon. The ribavirin concentrations maintained in the serum and cerebrospinal fluid were higher than those which inhibit SSPE virus replication in vitro and in vivo.
Resistance Studies with Daptomycin
Authors: Silverman Jared A. , Oliver Nicole , Andrew Ted , Li Tongchuan
Published: July 2002

We studied the in vitro emergence of resistance to daptomycin using three methods: spontaneous resistance incidence, serial passage in the presence of increasing drug concentrations, and chemical mutagenesis. No spontaneously resistant mutants were obtained for any organism tested (<10
−10
for
Staphylococcus aureus
, <10
−9
for
Staphylococcus epidermidis
, <10
−9
for
Enterococcus faecalis
, <10
−9
for
Enterococcus faecium
, <10
−8
for
Streptococcus pneumoniae
). Population analysis demonstrated that bacterial susceptibility to daptomycin is heterogeneous. Assay results were sensitive to calcium concentration and culture density, both of which can affect apparent resistance rates. Stable
S. aureus
mutants were isolated by both serial passage in liquid media and chemical mutagenesis. The daptomycin MICs for these isolates were 8- to 32-fold higher than for the parental strain. Many mutants with high MICs (>12.5 μg/ml) had significant growth defects but did not display phenotypes typical of
S. aureus
small colony variants. The voltage component (Δψ) of the bacterial membrane potential was increased in three independent resistant isolates. In vivo data showed that some daptomycin-resistant mutants had lost significant virulence. For other mutants, the degree of in vitro resistance was greater than the change in in vivo susceptibility. These results suggest that infection with some daptomycin-resistant organisms may still be easily treatable.
Interaction between DNA Gyrase and Quinolones: Effects of Alanine Mutations at GyrA Subunit Residues Ser
83
and Asp
87
Authors: Barnard Faye M. , Maxwell Anthony
Published: July 2002

DNA gyrase is a target of quinolone antibacterial agents, but the molecular details of the quinolone-gyrase interaction are not clear. Quinolone resistance mutations frequently occur at residues Ser
83
and Asp
87
of the gyrase A subunit, suggesting that these residues are involved in drug binding. Single and double alanine substitutions were created at these positions (Ala
83
, Ala
87
, and Ala
83
Ala
87
), and the mutant proteins were assessed for DNA supercoiling, DNA cleavage, and resistance to a number of quinolone drugs. The Ala
83
mutant was fully active in supercoiling, whereas the Ala
87
and the double mutant were 2.5- and 4- to 5-fold less active, respectively; this loss in activity may be partly due to an increased affinity of these mutant proteins for DNA. Supercoiling inhibition and cleavage assays revealed that the double mutant has a high level of resistance to certain quinolones while the mutants with single alanine substitutions show low-level resistance. Using a drug-binding assay we demonstrated that the double-mutant enzyme-DNA complex has a lower affinity for ciprofloxacin than the wild-type complex. Based on the pattern of resistance to a series of quinolones, an interaction between the C-8 group of the quinolone and the double-mutant gyrase in the region of residues 83 and 87 is proposed.
DNA Gyrase-Mediated Natural Resistance to Fluoroquinolones in
Ehrlichia
spp
Authors: Maurin M. , Abergel C. , Raoult D.
Published: July 2002

Fluoroquinolone susceptibility heterogeneity between various
Ehrlichia
species has been previously demonstrated. In gram-negative bacteria, resistance to fluoroquinolones most often corresponds to specific amino acid variations in a portion of the protein sequence of the A subunit of DNA gyrase (GyrA), referred to as the quinolone resistance-determining region (QRDR). We suspected a similar mechanism to be responsible for natural resistance in some
Ehrlichia
species. To verify this hypothesis, we sequenced the entire
gyrA
gene of the quinolone-susceptible species
Ehrlichia sennetsu
and designed specific primers to amplify and sequence the QRDR of four other
Ehrlichia
species as well as the closely related species
Cowdria ruminantium
. We identified in the fluoroquinolone-resistant species
Ehrlichia chaffeensis
and
Ehrlichia canis
a specific GyrA QRDR amino acid sequence, also present in
C. ruminantium
(whose susceptibility to fluoroquinolones remains unknown). These three species belong to a single phylogenetic cluster referred to as the
E. canis
genogroup. A different GyrA QRDR pattern, shared by the
Ehrlichia
species representatives of the
E. sennetsu
and
Ehrlichia phagocytophila
genogroups, was identified. Three of the four species tested are known to be susceptible to fluoroquinolones. A serine residue in position 83 (
Escherichia coli
numbering) in the susceptible species is replaced by an alanine residue in fluoroquinolone-resistant species. These results are consistent with the current knowledge on fluoroquinolone resistance in other gram-negative bacteria. They are indicative of a natural gyrase-mediated resistance to fluoroquinolones in the
E. canis
genogroup.
Novel Class A β-Lactamase Sed-1 from
Citrobacter sedlakii
: Genetic Diversity of β-Lactamases within the
Citrobacter
Genus
Authors: Petrella Stephanie , Clermont Dominique , Casin Isabelle , Jarlier Vincent , Sougakoff Wladimir
Published: July 2002

Citrobacter sedlakii
2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with β-lactam and quinolone antibiotics. The isolate produced an inducible class A β-lactamase of pI 8.6, named Sed-1, which was purified. Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin. The corresponding gene,
bla
Sed-1
, was cloned and sequenced. Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded β-lactamases from
Citrobacter koseri
(formerly
C. diversus
) (84%),
Klebsiella oxytoca
(74%),
Serratia fonticola
(67%), and
Proteus vulgaris
(63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from
bla
Sed-1
. This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal β-lactamase from
C. koseri
and 63 and 50% identity with the AmpR sequences of
P. vulgaris
and
Enterobacter cloacae
, respectively. By using DNA-DNA hybridization, a
bla
Sed-1
-like gene was identified in two reference strains,
C. sedlakii
(CIP-105037) and
Citrobacter rodentium
(CIP-104675), but not in the 18 strains of
C. koseri
studied. Two DNA fragments were amplified and sequenced from the reference strains of
C. sedlakii
CIP-105037 and
C. rodentium
CIP-104675 using two primers specific for
bla
Sed-1
. They shared 98 and 80% identity with
bla
Sed-1
, respectively, confirming the diversity of the chromosomally encoded class A β-lactamases found in
Citrobacter
.
Once-Daily Oral Gatifloxacin versus Oral Levofloxacin in Treatment of Uncomplicated Skin and Soft Tissue Infections: Double-Blind, Multicenter, Randomized Study
Authors: Tarshis Gary A. , Miskin Barry M. , Jones Terry M. , Champlin John , Wingert Kevin J. , Breen Jeanne D. , Brown Michael J.
Published: July 2002

This was a double-blind, multicenter study in which 410 adults (≥18 years of age) with uncomplicated skin and soft tissue infections (SSTIs) were randomized to receive either 400 mg of gatifloxacin orally once daily or 500 mg of levofloxacin orally once daily for 7 to 10 days. The study protocol called for four assessments—before and during treatment, at the end of treatment, and posttreatment. Efficacy evaluations included clinical response and bacterial eradication rates. Of 407 treated patients, 202 (108 women, 94 men) received gatifloxacin and 205 (111 women, 94 men) received levofloxacin. For clinically evaluable patients, the cure rates were 91% for gatifloxacin and 84% for levofloxacin (95% confidence interval [CI] for the difference, −2.0 to 15.2%). Clinical cure rates for microbiologically evaluable patients were 93% for gatifloxacin and 88% for levofloxacin (95% CI for the difference, −6.5 to 16.8%). The bacterial eradication rate was 92% for each group, with gatifloxacin eradicating 93% of the methicillin-susceptibleStaphylococcus aureusisolates and levofloxacin eradicating 91% of them. Both drugs were well tolerated. Most of the adverse events were mild to moderate, and nausea was the most common adverse event in each treatment arm. Once-daily oral gatifloxacin (400 mg) is clinically efficacious and well tolerated compared with once-daily levofloxacin (500 mg) for the treatment of patients with uncomplicated SSTIs.
Specific Inhibition of Human Immunodeficiency Virus Type 1 (HIV-1) Integration in Cell Culture: Putative Inhibitors of HIV-1 Integrase
Authors: Vandegraaff Nick , Kumar Raman , Hocking Helen , Burke Terrence R. , Mills John , Rhodes David , Burrell Christopher J. , Li Peng
Published: July 2002

To study the effect of potential human immunodeficiency virus type 1 (HIV-1) integrase inhibitors during virus replication in cell culture, we used a modified nested Alu-PCR assay to quantify integrated HIV DNA in combination with the quantitative analysis of extrachromosomal HIV DNA. The two diketo acid integrase inhibitors (L-708,906 and L-731,988) blocked the accumulation of integrated HIV-1 DNA in T cells following infection but did not alter levels of newly synthesized extrachromosomal HIV DNA. In contrast, we demonstrated that L17 (a member of the bisaroyl hydrazine family of integrase inhibitors) and AR177 (an oligonucleotide inhibitor) blocked the HIV replication cycle at, or prior to, reverse transcription, although both drugs inhibited integrase activity in cell-free assays. Quercetin dihydrate (a flavone) was shown to not have any antiviral activity in our system despite reported anti-integration properties in cell-free assays. This refined Alu-PCR assay for HIV provirus is a useful tool for screening anti-integration compounds identified in biochemical assays for their ability to inhibit the accumulation of integrated HIV DNA in cell culture, and it may be useful for studying the effects of these inhibitors in clinical trials.
Molecular Characterization of Multidrug-Resistant Isolates of
Mycobacterium tuberculosis
from Patients in North India
Authors: Siddiqi Noman , Shamim Mohammed , Hussain Seema , Choudhary Rakesh Kumar , Ahmed Niyaz , Prachee  , Banerjee Sharmistha , Savithri G. R. , Alam Mahfooz , Pathak Niteen , Amin Amol , Hanief Mohammed , Katoch V. M. , Sharma S. K. , Hasnain Seyed E.
Published: July 2002

The World Health Organization has identified India as a major hot-spot region for
Mycobacterium tuberculosis
infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of
M. tuberculosis
from India to identify the respective mutations. The loci selected were
rpoB
(rifampin),
katG
and the ribosomal binding site of
inhA
(isoniazid),
gyrA
and
gyrB
(ofloxacin), and
rpsL
and
rrs
(streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the
rpoB
locus could be correlated with the drug resistance levels exhibited by the
M. tuberculosis
isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the
katG
locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the
inhA
gene. A Ser95Thr substitution in the
gyrA
locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant
M. tuberculosis
strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.
Topoisomerase II and IV QuinoloneResistance-Determining Regions in
Stenotrophomonas maltophilia
Clinical Isolates with Different Levels of QuinoloneSusceptibility
Authors: Valdezate Sylvia , Vindel Ana , Echeita Aurora , Baquero Fernando , Cantó Rafael
Published: July 2002

The quinolone resistance-determining regions
(QRDRs) of topoisomerase II and IV genes from
Stenotrophomonas
maltophilia
ATCC 13637 were sequenced and compared with the
corresponding regions of 32 unrelated
S. maltophilia
clinical
strains for which ciprofloxacin MICs ranged from 0.1 to 64 μg/ml. GyrA
(Leu-55 to Gln-155,
Escherichia coli
numbering), GyrB (Met-391 to
Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments
from strain ATCC 13637 showed high degrees of identity to the corresponding
regions from the phytopathogen
Xylella fastidiosa
, with the
degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity
to the corresponding regions from
Pseudomonas aeruginosa
(70.9 to
88.6%) and
E. coli
(73.0 to 88.6%) were observed. Amino acid
changes were present in GyrA fragments from 9 of the 32 strains at
positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no
consistent relation to higher ciprofloxacin MICs. The absence of changes at
positions 83 and 87, commonly involved in quinolone resistance in
gram-negative bacteria, was unexpected. The GyrB sequences were identical
in all strains, and only one strain (ciprofloxacin MIC, 16 μg/ml)
showed a ParC amino acid change (Ser-80→Arg). In contrast, a high
frequency (16 of 32 strains) of amino acid replacements was present in
ParE. The frequencies of alterations at positions 437, 465, 477, and 485
were higher (
P
< 0.05) in strains from cystic fibrosis
patients, but these changes were not linked with high ciprofloxacin MICs.
An efflux phenotype, screened by the detection of decreases of at least
twofold doubling dilutions of the ciprofloxacin MIC in the presence of
carbonyl cyanide
m
-chlorophenylhydrazone (0.5 μg/ml) or
reserpine (10 μg/ml), was suspected in seven strains. These results
suggest that topoisomerases II and IV may not be the primary targets
involved in quinolone resistance in
S.
maltophilia.
Pharmacokinetics, Excretion, and Mass Balance of Liposomal Amphotericin B (AmBisome) and Amphotericin B Deoxycholate in Humans
Authors: Bekersky Ihor , Fielding Robert M. , Dressler Dawna E. , Lee Jean W. , Buell Donald N. , Walsh Thomas J.
Published: July 2002

The pharmacokinetics, excretion, and mass balance of liposomal amphotericin B (AmBisome) (liposomal AMB) and the conventional formulation, AMB deoxycholate (AMB-DOC), were compared in a phase IV, open-label, parallel study in healthy volunteers. After a single 2-h infusion of 2 mg of liposomal AMB/kg of body weight or 0.6 mg of AMB-DOC/kg, plasma, urine, and feces were collected for 168 h. The concentrations of AMB were determined by liquid chromatography tandem mass spectrometry (plasma, urine, feces) or high-performance liquid chromatography (HPLC) (plasma). Infusion-related side effects similar to those reported in patients, including nausea and back pain, were observed in both groups. Both formulations had triphasic plasma profiles with long terminal half-lives (liposomal AMB, 152 ± 116 h; AMB-DOC, 127 ± 30 h), but plasma concentrations were higher (
P
< 0.01) after administration of liposomal AMB (maximum concentration of drug in serum [
C
max
], 22.9 ± 10 μg/ml) than those of AMB-DOC (
C
max
, 1.4 ± 0.2 μg/ml). Liposomal AMB had a central compartment volume close to that of plasma (50 ± 19 ml/kg) and a volume of distribution at steady state (
V
ss
) (774 ± 550 ml/kg) smaller than the
V
ss
of AMB-DOC (1,807 ± 239 ml/kg) (
P
< 0.01). Total clearances were similar (approximately 10 ml hr
−1
kg
−1
), but renal and fecal clearances of liposomal AMB were 10-fold lower than those of AMB-DOC (
P
< 0.01). Two-thirds of the AMB-DOC was excreted unchanged in the urine (20.6%) and feces (42.5%) with >90% accounted for in mass balance calculations at 1 week, suggesting that metabolism plays at most a minor role in AMB elimination. In contrast, <10% of the liposomal AMB was excreted unchanged. No metabolites were observed by HPLC or mass spectrometry. In comparison to AMB-DOC, liposomal AMB produced higher plasma exposures and lower volumes of distribution and markedly decreased the excretion of unchanged drug in urine and feces. Thus, liposomal AMB significantly alters the excretion and mass balance of AMB. The ability of liposomes to sequester drugs in circulating liposomes and within deep tissue compartments may account for these differences.
Cloning and Expression of Class A β-Lactamase Gene
blaA
BPS
in
Burkholderia pseudomallei
Authors: Cheung Terence K. M. , Ho P. L. , Woo Patrick C. Y. , Yuen K. Y. , Chau P. Y.
Published: July 2002

The β-lactamase gene
blaA
BPS
in
Burkholderia pseudomallei
was cloned and expressed in
Escherichia coli
. BPS-1 is a cephalosporinase with an isoelectric point of 7.7. Sequence analysis of BPS-1 revealed conserved motifs typical of class A β-lactamases and a relationship to the PenA (in
B. cepacia
) and BlaI (in
Yersinia enterocolitica
) lineages.
Interactions between Biocide Cationic Agents and Bacterial Biofilms
Authors: Campanac C. , Pineau L. , Payard A. , Baziard-Mouysset G. , Roques C.
Published: July 2002

The resistance of bacterial biofilms to physical and chemical agents is attributed in the literature to various interconnected processes. The limitation of mass transfer alters the growth rate, and physiological changes in the bacteria in the film also appear. The present work describes an approach to determination of the mechanisms involved in the resistance of bacteria to quaternary ammonium compounds (benzalkonium chloride) according to the C-chain lengths of those compounds. For
Pseudomonas aeruginosa
CIP A 22, the level of resistance of the bacteria in the biofilm relative to that of planktonic bacteria increased with the C-chain length. For cells within the biofilm, the exopolysaccharide induced a characteristic increase in surface hydrophilicity. However, this hydrophilicity was eliminated by simple resuspension and washing. The sensitivity to quaternary ammonium compounds was restored to over 90%.
Staphylococcus aureus
CIP 53 154 had a very high level of resistance when it was in the biofilm form. A characteristic of bacteria from the biofilm was a reduction in the percent hydrophobicity, but the essential point is that this hydrophobicity was retained after the biofilm bacteria were resuspended and washed. The recovery of sensitivity was thus only partial. These results indicate that the factors involved in biofilm resistance to quaternary ammonium compounds vary according to the bacterial modifications induced by the formation of a biofilm. In the case of
P. aeruginosa
, we have underlined the involvement of the exopolysaccharide and particularly the three-dimensional structure (water channels). In the case of
S. aureus
, the role of the three-dimensional structure is limited and drastic physiological changes in the biofilm cells are more highly implicated in resistance.
Cytological Effects of Cellulases in the Parasitism of
Phytophthora parasitica
by
Pythium oligandrum
Authors: Picard Karine , Tirilly Yves , Benhamou Nicole
Published: July 2002

The ubiquitous oomycete
Pythium oligandrum
is a potential biocontrol agent for use against a wide range of pathogenic fungi and an inducer of plant disease resistance. The ability of
P. oligandrum
to compete with root pathogens for saprophytic colonization of substrates may be critical for pathogen increase in soil, but other mechanisms, including antibiosis and enzyme production, also may play a role in the antagonistic process. We used transmission electron microscopy and gold cytochemistry to analyze the intercellular interaction between
P. oligandrum
and
Phytophthora parasitica
. Growth of
P. oligandrum
towards
Phytophthora
cells correlated with changes in the host, including retraction of the plasma membrane and cytoplasmic disorganization. These changes were associated with the deposition onto the inner host cell surface of a cellulose-enriched material.
P. oligandrum
hyphae could penetrate the thickened host cell wall and the cellulose-enriched material, suggesting that large amounts of cellulolytic enzymes were produced. Labeling of cellulose with gold-complexed exoglucanase showed that the integrity of the cellulose was greatly affected both along the channel of fungal penetration and also at a distance from it. We measured cellulolytic activity of
P. oligandrum
in substrate-free liquid medium. The enzymes present were almost as effective as those from
Trichoderma viride
in degrading both carboxymethyl cellulose and
Phytophthora
wall-bound cellulose.
P. oligandrum
and its cellulolytic enzymes may be useful for biological control of oomycete pathogens, including
Phytophthora
and
Pythium
spp., which are frequently encountered in field and greenhouse production.
Oxygen-Dependent Growth of the Sulfate-Reducing Bacterium
Desulfovibrio oxyclinae
in Coculture with
Marinobacter
sp. Strain MB in an Aerated Sulfate-Depleted Chemostat
Authors: Sigalevich Pavel , Cohen Yehuda
Published: July 2002

A chemostat coculture of the sulfate-reducing bacterium
Desulfovibrio oxyclinae
and the facultatively aerobic heterotroph
Marinobacter
sp. strain MB was grown for 1 week under anaerobic conditions at a dilution rate of 0.05 h
−1
. It was then exposed to an oxygen flux of 223 μmol min
−1
by gassing the growth vessel with 5% O
2
. Sulfate reduction persisted under these conditions, though the amount of sulfate reduced decreased by 45% compared to the amount reduced during the initial anaerobic mode. After 1 week of growth under these conditions, sulfate was excluded from the incoming medium. The sulfate concentration in the growth vessel decreased exponentially from 4.1 mM to 2.5 μM. The coculture consumed oxygen effectively, and no residual oxygen was detected during either growth mode in which oxygen was supplied. The proportion of
D. oxyclinae
cells in the coculture as determined by in situ hybridization decreased from 86% under anaerobic conditions to 70% in the microaerobic sulfate-reducing mode and 34% in the microaerobic sulfate-depleted mode. As determined by the most-probable-number (MPN) method, the numbers of viable
D. oxyclinae
cells during the two microaerobic growth modes decreased compared to the numbers during the anaerobic growth mode. However, there was no significant difference between the MPN values for the two modes when oxygen was supplied. The patterns of consumption of electron donors and acceptors suggested that when oxygen was supplied in the absence of sulfate and thiosulfate,
D. oxyclinae
performed incomplete aerobic oxidation of lactate to acetate. This is the first observation of oxygen-dependent growth of a sulfate-reducing bacterium in the absence of either sulfate or thiosulfate. Cells harvested during the microaerobic sulfate-depleted stage and exposed to sulfate and thiosulfate in a respiration chamber were capable of anaerobic sulfate and thiosulfate reduction.