bluuebunny/crossref_metadata_2025_split huggingface dataset:
Screening and Characterization of Mutations in Isoniazid-Resistant
Mycobacterium tuberculosis
Isolates Obtained in Brazil
Authors: Cardoso Rosilene Fressatti , Cooksey Robert C. , Morlock Glenn P. , Barco Patricia , Cecon Leticia , Forestiero Francisco , Leite Clarice Q. F. , Sato Daisy N. , Shikama Maria de Lourdes , Mamizuka Elsa M. , Hirata Rosario D. C. , Hirata Mario H. 
Published: August 2004

ABSTRACT

We investigated mutations in the genes
katG
,
inhA
(regulatory and structural regions), and
kasA
and the
oxyR-ahpC
intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible
Mycobacterium tuberculosis
isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of
katG
, the regulatory region and codons 16 and 94 of
inhA
,
kasA
, and the
oxyR-ahpC
intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in
katG
were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported
katG
mutations. Mutations in the
inhA
promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had
inhA
structural gene mutations, and 10.3% had mutations in the
oxyR-ahpC
intergenic region (one, nucleotide −48, previously unreported). Polymorphisms in the
kasA
gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G
269
A substitution. Although KatG

315

substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.
Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria
Authors: Szynol A. , de Soet J. J. , Sieben-van Tuyl E. , Bos J. W. , Frenken L. G. 
Published: August 2004

ABSTRACTEnzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products. Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies. Variable domains (VHH) derived from llama heavy-chain antibodies are particularly suited for such an approach. The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies. In this study, a fusion protein consisting of VHHand GOx was constructed and expressed bySaccharomyces cerevisiae. A llama was immunized withStreptococcus mutansstrain HG982. Subsequently, B lymphocytes were isolated and cDNA fragments encoding the VHHfragments were obtained by reverse transcription-PCR. After construction of a VHHlibrary inEscherichia coliand screening of the library against mutans group streptococci andStreptococcus sanguinisstrains, we found two VHHfragments with high specificities forS. mutansstrains. A GOx gene was linked to the two VHHgenes and cloned intoS. cerevisiaeyeasts. The yeasts expressed and secreted the recombinant proteins into the growth medium. The test of binding of fusion proteins to oral bacteria through their VHHfragments showed thatS. mutanshad been specifically targeted by GOx-S120, one of the fusion protein constructs. A low concentration of the fusion protein was also able to selectively killS. mutanswithin 20 min in the presence of lactoperoxidase and potassium iodide. These findings demonstrate that the fusion protein GOx-VHHis potentially valuable in the selective killing of target bacteria such asS. mutans.
A Mucoadhesive Polymer Extracted from Tamarind Seed Improves the Intraocular Penetration and Efficacy of Rufloxacin in Topical Treatment of Experimental Bacterial Keratitis
Authors: Ghelardi Emilia , Tavanti Arianna , Davini Paola , Celandroni Francesco , Salvetti Sara , Parisio Eva , Boldrini Enrico , Senesi Sonia , Campa Mario 
Published: August 2004

ABSTRACT

Bacterial keratitis is a serious infectious ocular disease requiring prompt treatment to prevent frequent and severe visual disabilities. Standard treatment of bacterial keratitis includes topical administration of concentrated antibiotic solutions repeated at frequent intervals in order to reach sufficiently high drug levels in the corneal tissue to inhibit bacterial growth. However, this regimen has been associated with toxicity to the corneal epithelium and requires patient hospitalization. In the present study, a mucoadhesive polymer extracted from tamarind seeds was used for ocular delivery of 0.3% rufloxacin in the treatment of experimental
Pseudomonas aeruginosa
and
Staphylococcus aureus
keratitis in rabbits. The polysaccharide significantly increased the intra-aqueous penetration of rufloxacin in both infected and uninfected eyes. Rufloxacin delivered by the polysaccharide reduced
P. aeruginosa
and
S. aureus
in the cornea at a higher rate than that obtained by rufloxacin alone. In particular, use of the polysaccharide allowed a substantial reduction of
S. aureus
in the cornea to be achieved even when the time interval between drug administrations was extended. These results suggest that the tamarind seed polysaccharide prolongs the precorneal residence times of antibiotics and enhances drug accumulation in the cornea, probably by reducing the washout of topically administered drugs. The tamarind seed polysaccharide appears to be a promising candidate as a vehicle for the topical treatment of bacterial keratitis.
Sequence-Selective Recognition of Extended-Spectrum β-Lactamase GES-2 by a Competitive, Peptide Nucleic Acid-Based Multiplex PCR Assay
Authors: Weldhagen Gerhard F. 
Published: August 2004

ABSTRACT

Extended-spectrum β-lactamases (ESBLs) in
Pseudomonas aeruginosa
, such as GES-2, which compromises the efficacy of imipenem, tend to be geographically restricted. The CC-to-AA base pair substitution at positions 493 and 494 of the
bla
GES-2
-coding region distinguishes this ESBL from
bla
GES-1
and the
bla
IBC
-type genes, making it an ideal target for the development of a novel sequence-specific, peptide nucleic acid (PNA)-based multiplex PCR detection method. By using two primer pairs in conjunction with a PNA probe, this method provided an accurate means of identification of
bla
GES-2
compared to standard PCR and gene sequencing techniques when it was used to test 100
P. aeruginosa
clinical isolates as well as previously published, well-described control strains encompassing all presently known genes in the
bla
GES-IBC
ESBL family. This novel method has the potential to be used in large-scale, cost-effective screening programs for specific or geographically restricted ESBLs.
Genes Required for Intrinsic Multidrug Resistance in
Mycobacterium avium
Authors: Philalay Julie S. , Palermo Christine O. , Hauge Kirsten A. , Rustad Tige R. , Cangelosi Gerard A. 
Published: August 2004

ABSTRACT

Genes required for intrinsic multidrug resistance by
Mycobacterium avium
were identified by screening a library of transposon insertion mutants for the inability to grow in the presence of ciprofloxacin, clarithromycin, and penicillin at subinhibitory concentrations. Two genes,
pks12
and Maa2520, were disrupted in multiple drug-susceptible mutants. The
pks12
gene (Maa1979), which may be cotranscribed with a downstream gene (Maa1980), is widely conserved in the actinomycetes. Its ortholog in
Mycobacterium tuberculosis
is a polyketide synthase required for the synthesis of dimycocerosyl phthiocerol, a major cell wall lipid. Mutants of
M. avium
with insertions into
pks12
exhibited altered colony morphology and were drug susceptible, but they grew as well as the wild type did in vitro and intracellularly within THP-1 cells. A
pks12
mutant of
M. tuberculosis
was moderately more susceptible to clarithromycin than was its parent strain; however, susceptibility to ciprofloxacin and penicillin was not altered.
M. avium
complex (MAC) and
M. tuberculosis
appear to have different genetic mechanisms for resisting the effects of these antibiotics, with
pks12
playing a relatively more significant role in MAC. The second genetic locus identified in this study, Maa2520, is a conserved hypothetical gene with orthologs in
M. tuberculosis
and
Mycobacterium leprae
. It is immediately upstream of Maa2521, which may code for an exported protein. Mutants with insertions at this locus were susceptible to multiple antibiotics and slow growing in vitro and were unable to survive intracellularly within THP-1 cells. Like
pks12
mutants, they exhibited increased Congo red binding, an indirect indication of cell wall modifications. Maa2520 and
pks12
are the first genes to be linked by mutation to intrinsic drug resistance in MAC.
Effect of Ertapenem Protein Binding on Killing of Bacteria
Authors: Nix David E. , Matthias Kathryn R. , Ferguson Emily C. 
Published: August 2004

ABSTRACT

The effect of protein binding on the antimicrobial activity of ertapenem was evaluated using the bacterial kill rate and concentration-response studies. Various proportions of human serum were utilized to determine the total and free-drug concentrations using a validated high-performance liquid chromatography assay. The MICs and kill curves were determined for test isolates of
Enterobacter cloacae
and
Staphylococcus aureus
at various percentages of human serum. The killing of bacteria was analyzed in relation to the free and total concentrations of ertapenem at various proportions of human serum. It was determined that unbound ertapenem was responsible for the antimicrobial activity against the test isolates.
Disruption of the
Candida albicans CYB5
Gene Results in Increased Azole Sensitivity
Authors: Rogers K. M. , Pierson C. A. , Culbertson N. T. , Mo C. , Sturm A. M. , Eckstein J. , Barbuch R. , Lees N. D. , Bard M. 
Published: August 2004

ABSTRACT

Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochrome-mediated reactions, aerobically synthesized heme. Cytochrome
b
5
is required for sterol C5-6 desaturation and the encoding gene,
CYB5
, is nonessential in
Saccharomyces cerevisiae
. Cyb5p and Ncp1p (cytochrome P-450 reductase) appear to have overlapping functions in this organism, with disruptions of each alone being viable. The cytochrome P-450 reductase phenotype has also been shown to demonstrate increased sensitivity to azole antifungals. Based on this phenotype, the
CYB5
gene in the human pathogen
Candida albicans
was investigated to determine whether the
cyb5
genotype was viable and would also demonstrate azole sensitivity. Sequential disruption of the
CYB5
alleles by direct transformation resulted in viability, presumably conferred by the presence of a third copy of the
CYB5
gene. Subsequent disruption procedures with a
pMAL2-CYB5
rescue cassette and a
CYB5-URA3
blaster cassette resulted in viable
cyb5
strains with no third copy. The
C. albicans CYB5
gene is concluded to be nonessential. Thus, the essentiality of this gene and whether we observed two or three alleles was dependent upon the gene disruption protocol. The
C. albicans cyb5
strains produced a sterol profile containing low ergosterol levels and sterol intermediates similar to that reported for the
S. cerevisiae cyb5
. The
C. albicans cyb5
shows increased sensitivity to azoles and terbinafine, an inhibitor of squalene epoxidase, and, unexpectedly, increased resistance to morpholines, which inhibit the
ERG2
and
ERG24
gene products. These results indicate that an inhibitor of Cyb5p would not be lethal but would make the cell significantly more sensitive to azole treatment.
In Vivo Imaging of Bioluminescent
Escherichia coli
in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy
Authors: Jawhara Samir , Mordon Serge 
Published: August 2004

ABSTRACT

A rapid, continuous method for noninvasively monitoring the effectiveness of several antibacterial agents in real time by using a model of wound infection was developed. This study was divided into three steps: (i) construction of a plasmid to transform
Escherichia coli
into a bioluminescent variant, (ii) study of the bioluminescent
E. coli
in vitro as a function of temperature and pH, and (iii) determination of the MIC and the minimal bactericidal concentration of sulfamethoxazole-trimethoprim (SMX-TMP). Finally, the efficacy of SMX-TMP was monitored in vivo in a cutaneous wound model (hairless rat) infected with this bioluminescent bacterium by using a bioluminescence imaging system.
E. coli
was transformed by electroporation with a shuttle vector (pRB474) containing the firefly (
Photinus pyralis
) luciferase gene, resulting in a bioluminescent phenotype. It was found that pH 5.0 was optimal for incorporation of the susbstrate
d
-luciferin for the luciferase reaction. In vitro, when the agar dilution method, standard turbidity assays, and the bioluminescence imaging system were used,
E. coli
(pRB474) proved to be susceptible to SMX-TMP. In vivo, at 4 h, SMX-TMP treatment was already efficient compared to no treatment (
P
= 0.034). At 48 h, no bioluminescence was detected in the wound, demonstrating the susceptibility of
E. coli
to SMX-TMP. In conclusion, this study points out the advantage of using bioluminescence imaging to evaluate the effects of antibiotics for the treatment of acute infections in vivo in a nondestructive and noninvasive manner.
The
mef
(A) Gene Predominates among Seven Macrolide Resistance Genes Identified in Gram-Negative Strains Representing 13 Genera, Isolated from Healthy Portuguese Children
Authors: Ojo K. K. , Ulep C. , Van Kirk N. , Luis H. , Bernardo M. , Leitao J. , Roberts M. C. 
Published: August 2004

ABSTRACT

Of the 176 randomly selected, commensal, gram-negative bacteria isolated from healthy children with low exposure to antibiotics, 138 (78%) carried one or more of the seven macrolide resistance genes tested in this study. These isolates included 79 (91%) isolates from the oral cavity and 59 (66%) isolates from urine samples. The
mef
(A) gene, coding for an efflux protein, was found in 73 isolates (41%) and was the most frequently carried gene. The
mef
(A) gene could be transferred from the donors into a gram-positive
E. faecalis
recipient and a gram-negative
Escherichia coli
recipient. The
erm
(B) gene transferred and was maintained in the
E. coli
transconjugants but was found in 0 to 100% of the
E. faecalis
transconjugants tested, while the other five genes could be transferred only into the
E. coli
recipient. The individual macrolide resistance genes were identified in 3 to 12 new genera. Eight (10%) of the oral isolates and 30 (34%) of the urine isolates for which the MICs were 2 to >500 μg of erythromycin per ml did not hybridize with any of the seven genes and may carry novel macrolide resistance genes.
Azithromycin Inhibits MUC5AC Production Induced by thePseudomonas aeruginosaAutoinducerN-(3-Oxododecanoyl) Homoserine Lactone in NCI-H292 Cells
Authors: Imamura Yoshifumi , Yanagihara Katsunori , Mizuta Yohei , Seki Masafumi , Ohno Hideaki , Higashiyama Yasuhito , Miyazaki Yoshitsugu , Tsukamoto Kazuhiro , Hirakata Yoichi , Tomono Kazunori , Kadota Jun-ichi , Kohno Shigeru 
Published: August 2004

ABSTRACTThe features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus.Pseudomonas aeruginosais one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease. The quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL)] not only regulates bacterial virulence but also is associated with the immune response. In this study, we investigated whether 3O-C12-HSL could stimulate the production of a major mucin core protein, MUC5AC. The effect of a macrolide on MUC5AC production was also studied. 3O-C12-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners. A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C12-HSL. 3O-C12-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-κB phosphorylation in cells, and this induction was suppressed by azithromycin. 3O-C12-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059. Our findings suggest that theP. aeruginosaautoinducer 3O-C12-HSL contributes to excessive mucin production in chronic bacterial infection. Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction.
Molecular Basis of Resistance to Macrolides and Other Antibiotics in Commensal Viridans Group Streptococci and
Gemella
spp. and Transfer of Resistance Genes to
Streptococcus pneumoniae
Authors: Cerdá Zolezzi Paula , Laplana Leticia Millán , Calvo Carmen Rubio , Cepero Pilar Goñi , Erazo Melisa Canales , Gómez-Lus Rafael 
Published: August 2004

ABSTRACT

We assessed the mechanisms of resistance to macrolide-lincosamide-streptogramin B (MLS
B
) antibiotics and related antibiotics in erythromycin-resistant viridans group streptococci (
n
= 164) and
Gemella
spp. (
n
= 28). The macrolide resistance phenotype was predominant (59.38%); all isolates with this phenotype carried the
mef
(A) or
mef
(E) gene, with
mef
(E) being predominant (95.36%). The
erm
(B) gene was always detected in strains with constitutive and inducible MLS
B
resistance and was combined with the
mef
(A/E) gene in 47.44% of isolates. None of the isolates carried the
erm
(A) subclass
erm
(TR),
erm
(A), or
erm
(C) genes. The
mel
gene was detected in all but four strains carrying the
mef
(A/E) gene. The
tet
(M) gene was found in 86.90% of tetracycline-resistant isolates and was strongly associated with the presence of the
erm
(B) gene. The

cat
pC194

gene was detected in seven chloramphenicol-resistant
Streptococcus mitis
isolates, and the
aph
(
3′
)-
III
gene was detected in four viridans group streptococcal isolates with high-level kanamycin resistance. The
intTn
gene was found in all isolates with the
erm
(B),
tet
(M),
aph
(
3′
)
-III
, and

cat
pC194

gene. The
mef
(E) and
mel
genes were successfully transferred from both groups of bacteria to
Streptococcus pneumoniae
R6 by transformation. Viridans group streptococci and
Gemella
spp. seem to be important reservoirs of resistance genes.
Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “
Streptomyces carzinostaticus
”
Authors: Otsuka Miyuki , Ichinose Koji , Fujii Isao , Ebizuka Yutaka 
Published: August 2004

ABSTRACT

Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by “
Streptomyces carzinostaticus
” var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in
Streptomyces coelicolor
CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH
2
-dependent halogenases involved in the formation of halometabolites. The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite.
Increased Serum Resistance in
Klebsiella pneumoniae
Strains Producing Extended-Spectrum β-Lactamases
Authors: Sahly H. , Aucken H. , Benedí V. J. , Forestier C. , Fussing V. , Hansen D. S. , Ofek I. , Podschun R. , Sirot D. , Tomás J. M. , Sandvang D. , Ullmann U. 
Published: August 2004

ABSTRACT

The aim of this study was to determine whether there is an association between serum resistance, O serotypes, and the production of extended-spectrum β-lactamases (ESBLs) in
Klebsiella pneumoniae
. Ninety ESBL-producing and 178 non-ESBL-producing
K. pneumoniae
isolates gathered in five European countries were O serotyped and tested for sensitivity to the serum's bactericidal effect. The frequency of serum-resistant isolates was higher among ESBL-producing strains (30%; 27/90 isolates) than among non-ESBL-producing strains (17.9%; 32/178 isolates) (
P
= 0.037; odds ratio [OR] = 1.96; 95% confidence interval [95% CI] = 1.08 to 3.53). Although O1 was the most common O serotype in both
Klebsiella
groups, its frequency among ESBL-producing strains was significantly higher (59%; 53/90 isolates) than among non-ESBL producers (36%; 64/178 isolates) (
P
= 0.0006; OR = 2.5; 95% CI = 1.52 to 4.29). Furthermore, the prevalence of the O1 serotype was higher among serum-resistant strains of both ESBL-producing (74%; 20/27isolates) and non-ESBL producers (75%; 24/32 isolates) than among serum-sensitive ESBL producers (52.4%; 33/63 isolates) and non-ESBL producers (27.4%; 40/146 isolates). Serum resistance among ESBL-producing strains (36%; 17/47 isolates) versus non-ESBL-producing strains (16%; 27/166 isolates) was also significantly higher after the exclusion of clonal strains (
P
= 0.0056; OR = 2.9; 95% CI = 1.41 to 6.01). Sixteen ESBL types were detected, among which the frequency of serum resistance was significantly lower among the SHV-producing strains (9/48 isolates) than among the TEM producers (16/35 isolates) (
P
= 0.016; OR = 3.65; CI = 1.3 to 9.7). Curing ESBL-coding plasmids did not influence the serum resistance of the bacteria; all six plasmid-cured derivatives maintained serum resistance. The present findings suggest that ESBL-producing strains have a greater pathogenic potential than non-ESBL-producing strains, but the linkage between O serotypes, serum resistance, and ESBL production remains unclear at this stage.