bluuebunny/crossref_metadata_2025 huggingface dataset:
Activities of Temporin Family Peptides against the Chytrid Fungus (Batrachochytrium dendrobatidis) Associated with Global Amphibian Declines
Authors: Rollins-Smith Louise A. , Carey Cynthia , Conlon J. Michael , Reinert Laura K. , Doersam Jennifer K. , Bergman Tomas , Silberring Jerzy , Lankinen Hilkka , Wade David
Published: February 2003

Temporin A and structurally related peptides produced in amphibian dermal granular glands and in wasp venom were tested for growth inhibition ofBatrachochytrium dendrobatidis, a pathogen associated with global amphibian declines. Two natural amphibian temporins, a wasp temporin, and six synthetic analogs effectively inhibited growth. Differences in potency due to amino acid substitution suggest that ability to penetrate membranes and form an α-helical structure is important for their effectiveness against this pathogen.
In Vitro Effects of Ciprofloxacin and Roxithromycin on Apoptosis of Jurkat T Lymphocytes
Authors: Jun Yong-Taek , Kim Hee-Jung , Song Min-Jin , Lim Ji-Hyang , Lee Dong-Gun , Han Kyung-Ja , Choi Su-Mi , Yoo Jin-Hong , Shin Wan-Shik , Choi Jung-Hyun
Published: February 2003

Ciprofloxacin (CPFX) and roxithromycin (RXM) induced apoptosis of activated Jurkat T cells in vitro. CPFX showed concentration-dependent acceleration of apoptosis of activated Jurkat T cells by enhancing the expression of FasL and activities of caspase-3 and -8. RXM accelerated cell death, enhanced expression of FasL and caspase-3 but not caspase-8, and did not show the concentration dependency.
Imipenem Resistance in
Salmonella enterica
Serovar Wien Related to Porin Loss and CMY-4 β-Lactamase Production
Authors: Armand-Lefèvre L. , Leflon-Guibout V. , Bredin J. , Barguellil F. , Amor A. , Pagès J. M. , Nicolas-Chanoine M.-H.
Published: February 2003

Two multidrug-resistant
Salmonella enterica
serovar Wien strains (SW468 and SW1107) were isolated in 2001 in Tunis. Both strains produced the β-lactamases TEM-1, SHV-2a, and CMY-4, whereas strain SW1107 also produced the β-lactamase CTX-M-3. The imipenem-resistant strain (SW468) was totally devoid of the OmpF-immunorelated porin. Imipenem resistance was shown as being related to porin loss and CMY-4 β-lactamase production.
A New Sulfonamide Resistance Gene (
sul3
) in
Escherichia coli
Is Widespread in the Pig Population of Switzerland
Authors: Perreten Vincent , Boerlin Patrick
Published: February 2003

A new gene,
sul3
, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from
Escherichia coli
was characterized. Expression of the cloned
sul3
gene conferred resistance to sulfamethoxazole on
E. coli
. Two copies of the insertion element IS
15
Δ
/26
flanked the region containing
sul3
. The
sul3
gene was detected in one-third of the sulfonamide-resistant pathogenic
E. coli
isolates from pigs in Switzerland.
Relationships between Respiration and Susceptibility to Azole Antifungals in
Candida glabrata
Authors: Brun Sophie , Aubry Christophe , Lima Osana , Filmon Robert , Bergès Thierry , Chabasse Dominique , Bouchara Jean-Philippe
Published: February 2003

Over the past two decades, the incidence of infections due to
Candida glabrata
, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly. Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species. In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of
C. glabrata
were evaluated. Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide. Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles. Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed. This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar. This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy. Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency. Finally, this mutant exhibited cross-resistance to all the azoles tested. In conclusion, blockage of respiration in
C. glabrata
induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA. This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain.
Quinolone-DNA Interaction: Sequence-Dependent Binding to Single-Stranded DNA Reflects the Interaction within the Gyrase-DNA Complex
Authors: Noble Christian G. , Barnard Faye M. , Maxwell Anthony
Published: February 2003

We have investigated the interaction of quinolones with DNA by a number of methods to establish whether a particular binding mode correlates with quinolone potency. The specificities of the quinolone-mediated DNA cleavage reaction of DNA gyrase were compared for a number of quinolones. Two patterns that depended on the potency of the quinolone were identified. Binding to plasmid DNA was examined by measuring the unwinding of pBR322 by quinolones; no correlation with quinolone potency was observed. Quinolone binding to short DNA oligonucleotides was measured by surface plasmon resonance. The quinolones bound to both single- and double-stranded oligonucleotides in an Mg
2+
-dependent manner. Quinolones bound to single-stranded DNA with a higher affinity, and the binding exhibited sequence dependence; binding to double-stranded DNA was sequence independent. The variations in binding in the presence of metal ions showed that Mg
2+
promoted tighter, more specific binding to single-stranded DNA than softer metal ions (Mn
2+
and Cd
2+
). Single-stranded DNA binding by quinolones correlated with the in vitro quinolone potency, indicating that this mode of interaction may reflect the interaction of the quinolone with DNA in the context of the gyrase-DNA complex.
Low-Level Resistance to Rifampin in
Streptococcus pneumoniae
Authors: Meier Patricia Stutzmann , Utz Silvia , Aebi Suzanne , Mühlemann Kathrin
Published: February 2003

Rifampin is recommended for combination therapy of meningitis due to β-lactam-resistant
Streptococcus pneumoniae
. High-level rifampin resistance (MIC, ≥4 mg/liter) has been mapped to point mutations in clusters I and III of
rpoB
of the pneumococcus. The molecular basis of low-level resistance (MICs, ≥0.5 and <4 mg/liter) was analyzed. Spontaneous mutants of clinical pneumococcal isolates were selected on Columbia sheep blood agar plates containing rifampin at 0.5, 4, 10, or 50 mg/liter. Low-level resistance could be assigned to mutations in cluster II (I
545
N, I
545
L). Sensitive (MIC, <0.048 mg/liter) wild-type strains acquired low-level resistance at a rate approximately 10 times higher than that at which they acquired high-level resistance (average mutation frequencies, 2.4 × 10
−7
for low-level resistance versus 2.9 × 10
−8
for high-level resistance [
P
< 0.0001]). In second-step experiments, the frequencies of mutations from low- to high-level resistance were over 10 times higher than the frequencies of mutations from susceptibility to high-level resistance (average mutation frequencies, 7.2 × 10
−7
versus 5.0 × 10
−8
[
P
< 0.001]). Mutants with low-level resistance were stable upon passage. Sequencing of a clinical isolate with low-level resistance (MIC, 0.5 mg/liter) revealed a Q
150
R mutation upstream of cluster I. The frequencies of mutations to high-level resistance for this strain were even higher than the rates observed for the in vitro mutants. Therefore, a resistance-mediating mutation located outside clusters I, II, and III has been described for the first time in the pneumococcus. In vitro low-level rifampin resistance in
S. pneumoniae
could be mapped to cluster II of
rpoB
. Mutants of pneumococcus with low-level resistance may be selected in vivo during therapy in tissue compartments with low antibiotic concentrations and play a role in the development of resistance.
Resistance Genes of Aminocoumarin Producers: Two Type II Topoisomerase Genes Confer Resistance against Coumermycin A1and Clorobiocin
Authors: Schmutz Elisabeth , Mühlenweg Agnes , Li Shu-Ming , Heide Lutz
Published: February 2003

The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A1, and clorobiocin were investigated. All three clusters contained agyrBRresistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A1clusters were found to contain an additional, similar gene, namedparYR. Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression ofgyrBRand likewise ofparYRinStreptomyces lividansTK24 resulted in resistance against novobiocin and coumermycin A1, suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and ofStreptomyces coelicolorcontained two additional genes which hybridized with eithergyrBRorparYRand which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes,novAandcouR5, were found in the novobiocin and the coumermycin A1cluster, respectively. Expression of these genes inS. lividansTK24 resulted in moderate levels of resistance against novobiocin and coumermycin A1, suggesting that these genes may be involved in antibiotic transport.
Prevalence of Tetracycline Resistance Genes in Oral Bacteria
Authors: Villedieu A. , Diaz-Torres M. L. , Hunt N. , McNab R. , Spratt D. A. , Wilson M. , Mullany P.
Published: February 2003

Tetracycline is a broad-spectrum antibiotic used in humans, animals, and aquaculture; therefore, many bacteria from different ecosystems are exposed to this antibiotic. In order to determine the genetic basis for resistance to tetracycline in bacteria from the oral cavity, saliva and dental plaque samples were obtained from 20 healthy adults who had not taken antibiotics during the previous 3 months. The samples were screened for the presence of bacteria resistant to tetracycline, and the tetracycline resistance genes in these isolates were identified by multiplex PCR and DNA sequencing. Tetracycline-resistant bacteria constituted an average of 11% of the total cultivable oral microflora. A representative 105 tetracycline-resistant isolates from the 20 samples were investigated; most of the isolates carried tetracycline resistance genes encoding a ribosomal protection protein. The most common
tet
gene identified was
tet
(M), which was found in 79% of all the isolates. The second most common gene identified was
tet
(W), which was found in 21% of all the isolates, followed by
tet
(O) and
tet
(Q) (10.5 and 9.5% of the isolates, respectively) and then
tet
(S) (2.8% of the isolates). Tetracycline resistance genes encoding an efflux protein were detected in 4.8% of all the tetracycline-resistant isolates; 2.8% of the isolates had
tet
(L) and 1% carried
tet
(A) and
tet
(K) each. The results have shown that a variety of tetracycline resistance genes are present in the oral microflora of healthy adults. This is the first report of
tet
(W) in oral bacteria and the first report to show that
tet
(O),
tet
(Q),
tet
(A), and
tet
(S) can be found in some oral species.
Diversity of Tetracycline Resistance Genes in Bacteria from Chilean Salmon Farms
Authors: Miranda Claudio D. , Kehrenberg Corinna , Ulep Catherine , Schwarz Stefan , Roberts Marilyn C.
Published: February 2003

Twenty-five distinct tetracycline-resistant gram-negative bacteria recovered from four Chilean fish farms with no history of recent antibiotic use were examined for the presence of tetracycline resistance (
tet
) genes. Sixty percent of the isolates carried 1 of the 22 known
tet
genes examined. The distribution was as follows. The
tet
(A) gene was found in six isolates. The
tet
(B) gene was found in two isolates, including the first description in the genus
Brevundimonas
. Two isolates carried the
tet
(34) and
tet
(B) genes, including the first description of the
tet
(34) gene in
Pseudomonas
and
Serratia
and the first description of the
tet
(B) gene in
Pseudomonas
. The
tet
(H) gene was found in two isolates, which includes the first description in the genera
Moraxella
and
Acinetobacter
. One isolate carried
tet
(E), and one isolate carried
tet
(35), the first description of the gene in the genus
Stenotrophomonas
. Finally, one isolate carried
tet
(L), found for the first time in the genus
Morganella
. By DNA sequence analysis, the two
tet
(H) genes were indistinguishable from the previously sequenced
tet
(H) gene from Tn
5706
found in
Pasteurella multocida
. The
Acinetobacter radioresistens
isolate also harbored the Tn
5706
-associated 1,063-bp IS element IS
1597
, while the
Moraxella
isolate carried a 1,026-bp IS-like element whose 293-amino-acid transposase protein exhibited 69% identity and 84% similarity to the transposase protein of IS
1597
, suggesting the presence of a novel IS element. The distribution of
tet
genes from the Chilean freshwater ponds was different than those that have previously been described from other geographical locations, with 40% of the isolates carrying unidentified tetracycline resistance genes.
New Anti-Human Immunodeficiency Virus Type 1 6-Aminoquinolones: Mechanism of Action
Authors: Parolin Cristina , Gatto Barbara , Del Vecchio Claudia , Pecere Teresa , Tramontano Enzo , Cecchetti Violetta , Fravolini Arnaldo , Masiero Sara , Palumbo Manlio , Palù Giorgio
Published: February 2003

A 6-aminoquinolone derivative, WM5, which bears a methyl substituent at the N-1 position and a 4-(2-pyridyl)-1-piperazine moiety at position 7 of the bicyclic quinolone ring system, was previously shown to exhibit potent activity against replication of human immunodeficiency virus type 1 (HIV-1) in de novo-infected human lymphoblastoid cells (V. Cecchetti et al., J. Med. Chem. 43:3799-3802, 2000). In this report, we further investigated WM5's mechanism of antiviral activity. WM5 inhibited HIV-1 replication in acutely infected cells as well as in chronically infected cells. The 50% inhibitory concentrations were 0.60 ± 0.06 and 0.85 ± 0.05 μM, respectively. When the effects of WM5 on different steps of the virus life cycle were analyzed, the reverse transcriptase activity and the integrase and protease activities were not impaired. By using a transienttrans-complementation assay to examine the activity of WM5 on the replicative potential of HIV-1 in a single round of infection, a sustained inhibition of Tat-mediated long terminal repeat (LTR)-driven transcription (>80% of controls) was obtained in the presence of 5 μM WM5. Interestingly, the aminoquinolone was found to efficiently complex TAR RNA, with a dissociation constant in the nanomolar range (19 ± 0.6 nM). These data indicate that WM5 is a promising lead compound for the development of a new class of HIV-1 transcription inhibitors characterized by recognition of viral RNA target(s).
Cefpodoxime-Proxetil versus Trimethoprim-Sulfamethoxazole for Short-Term Therapy of Uncomplicated Acute Cystitis in Women
Authors: Kavatha Demetra , Giamarellou Helen , Alexiou Zoe , Vlachogiannis Nickolas , Pentea Stavroula , Gozadinos Thomas , Poulakou Garyphalia , Hatzipapas Agissilaos , Koratzanis George
Published: February 2003

One hundred sixty-three women with uncomplicated acute lower urinary tract infections were included in a multicenter randomized study comparing cefpodoxime-proxetil (one 100-mg tablet twice daily) with trimethoprim-sulfamethoxazole (one double-strength tablet [160/800 mg] twice daily) for 3 days. A total of 30 women in both arms were excluded from the study for various reasons. At 4 to 7 days after the discontinuation of therapy, 62 of 63 (98.4%) cefpodoxime-proxetil recipients and 70 of 70 (100%) trimethoprim-sulfamethoxazole patients were clinically cured and demonstrated bacteriological eradication, respectively. At 28 days after treatment, 48 of 55 (87.3%) and 43 of 50 (86%) cefpodoxime-proxetil recipients as well as 51 of 60 (85%) and 42 of 50 (84%) trimethoprim-sulfamethoxazole recipients were clinically cured and demonstrated bacteriological eradication, respectively. Independently of the prescribed regimen, a significant difference (
P
< 0.001) in failure rates was observed only for patients with a previous history of three or more episodes of acute cystitis per year. With the exception of one patient in the trimethoprim-sulfamethoxazole arm who discontinued therapy because of gastrointestinal pain, both antimicrobials were well tolerated. In conclusion, cefpodoxime-proxetil treatment for 3 days was as safe and effective as trimethoprim-sulfamethoxazole for 3 days for the treatment of uncomplicated acute cystitis in women.
Short-Course Artesunate Treatment of Uncomplicated
Plasmodium falciparum
Malaria in Gabon
Authors: Borrmann Steffen , Adegnika Ayola A. , Missinou Michel A. , Binder Ronald K. , Issifou Saadou , Schindler Andreas , Matsiegui Pierre-Blaise , Kun Jürgen F. J. , Krishna Sanjeev , Lell Bertrand , Kremsner Peter G.
Published: February 2003

Artesunate is one of the most important antimalarial agents available, since it is effective against parasites that have developed resistance to conventional antimalarials in sub-Saharan Africa. Antimalarial combination chemotherapies with artesunate (4 mg/kg of body weight once daily for 3 days) as one partner have been proposed. However, the efficacy of a 3-day course of artesunate alone has never been evaluated in individuals in Africa (which has 90% of the worldwide malaria burden) living in regions of hyperendemicity, where a considerable degree of immunity might substantially enhance the efficacy of short courses of artesunate compared to those in regions where the levels of endemicity are low. This lack of information does not permit a systematic assessment of the value of artesunate-based combination chemotherapies in Africa. Therefore, we studied the efficacy and safety of a 3-day course of artesunate (4 mg/kg of body weight, orally, once daily) for the treatment of uncomplicated
Plasmodium falciparum
malaria in Gabonese patients aged 4 to 15 years (
n
= 50). Artesunate was well tolerated, and no severe adverse event was reported. Parasite elimination was rapid and was achieved in all patients within ≤72 h (geometric mean time to elimination, 34 h). The PCR-corrected cure rate by day 14 was 92% (46 of 50 patients), but it dropped to 72% (36 of 50 patients) by day 28. We conclude that a 3-day course of artesunate fails to achieve sufficiently high cure rates for uncomplicated falciparum malaria in Gabonese children.
Synergistic Activity of Colistin and Ceftazidime against Multiantibiotic-Resistant
Pseudomonas aeruginosa
in an In Vitro Pharmacodynamic Model
Authors: Gunderson Brent W. , Ibrahim Khalid H. , Hovde Laurie B. , Fromm Timothy L. , Reed Michael D. , Rotschafer John C.
Published: February 2003

Despite the marketing of a series of new antibiotics for antibiotic-resistant gram-positive bacteria, no new agents for multiple-antibiotic-resistant gram-negative infections will be available for quite some time. Clinicians will need to find more effective ways to utilize available agents. Colistin is an older but novel antibiotic that fell into disfavor with clinicians some time ago yet still retains a very favorable antibacterial spectrum, especially for
Pseudomonas
and
Acinetobacter
spp. Time-kill curves for two strains of multiantibiotic-resistant
Pseudomonas aeruginosa
were generated after exposure to colistin alone or in combination with ceftazidime or ciprofloxacin in an in vitro pharmacodynamic model. MICs of colistin, ceftazidime, ciprofloxacin, piperacillin-tazobactam, imipenem, and tobramycin were 0.125, ≥32, >4, >128/4, 16, and >16 mg/liter, respectively. Colistin showed rapid, apparently concentration-dependent bactericidal activity at concentrations between 3 and 200 mg/liter. We were unable to detect increased colistin activity at concentrations above 18 mg/liter due to extremely rapid killing. The combination of colistin and ceftazidime was synergistic (defined as at least a 2-log
10
drop in CFU per milliliter from the count obtained with the more active agent) at 24 h. Adding ciprofloxacin to colistin did not enhance antibiotic activity. These data suggest that the antibacterial effect of colistin combined with ceftazidime can be maximized at a peak concentration of ≤18 mg/liter.
Activities of Garenoxacin (BMS-284756) and Other Agents against Anaerobic Clinical Isolates
Authors: Hecht David W. , Osmolski J. R.
Published: February 2003

A total of 590 clinical isolates consisting of 33 species of both gram-positive and gram-negative anaerobes were collected from nine centers in the Chicago area in 1998-1999. The largest number of isolates (330 isolates, 56%) belonged to the
Bacteroides
group. Isolates were tested by agar dilution against garenoxacin (BMS-284756, T-3811 ME), trovafloxacin, moxifloxacin, clindamycin, imipenem, piperacillin-tazobactam, and cefoxitin. All but one species (2% of
Bacteroides vulgatus
isolates) were fully susceptible to piperacillin-tazobactam and imipenem. A number of species were resistant to clindamycin. Among the fluoroquinolones, garenoxacin and trovafloxacin had an MIC at which 90% of the isolates tested were inhibited of <4 μg/ml for all but two species (
Fusobacterium mortiferum
/
varium
and
Peptostreptococcus anaerobius
).
Clinical Concentrations of Thioridazine Kill Intracellular Multidrug-Resistant
Mycobacterium tuberculosis
Authors: Ordway Diane , Viveiros Miguel , Leandro Clara , Bettencourt Rosário , Almeida Josefina , Martins Marta , Kristiansen Jette E. , Molnar Joseph , Amaral Leonard
Published: February 2003

The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant
Mycobacterium tuberculosis.
These compounds have not been used as anti-
M. tuberculosis
agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular
M. tuberculosis
at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills
M. tuberculosis
organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant
M. tuberculosis
organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant
M. tuberculosis
isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent.
In Vitro Activity of S-3578, a New Broad-Spectrum Cephalosporin Active against Methicillin-Resistant Staphylococci
Authors: Fujimura Takaji , Yamano Yoshinori , Yoshida Isamu , Shimada Jingoro , Kuwahara Shogo
Published: February 2003

The in vitro antibacterial activity of S-3578, a new parenteral cephalosporin, against clinical isolates was evaluated. The MICs of the drug at which 90% of the isolates were inhibited were 4 μg/ml for methicillin-resistant
Staphylococcus aureus
(MRSA) and 2 μg/ml for methicillin-resistant
Staphylococcus epidermidis
, which were fourfold higher than and equal to those of vancomycin, respectively. The anti-MRSA activity of S-3578 was considered to be due to its high affinity for penicillin-binding protein 2a (50% inhibitory concentration, 4.5 μg/ml). In time-kill studies with 10 strains each of MRSA and methicillin-susceptible
S. aureus
, S-3578 caused more than a 4-log
10
decrease of viable cells on the average at twice the MIC after 24 h of exposure, indicating that it had potent bactericidal activity. Furthermore, in population analysis of MRSA strains with heterogeneous or homogeneous resistance to imipenem, no colonies emerged from about 10
9
cells on agar plates containing twice the MIC of S-3578, suggesting the low frequency of emergence of S-3578-resistant strains from MRSA. S-3578 was also highly active against penicillin-resistant
Streptococcus pneumoniae
(PRSP), with a MIC
90
of 1 μg/ml, which was comparable to that of ceftriaxone. S-3578 also had antibacterial activity against a variety of gram-negative bacteria including
Pseudomonas aeruginosa
, though its activity was not superior to that of cefepime. In conclusion, S-3578 exhibited a broad antibacterial spectrum and, particularly, had excellent activity against gram-positive bacteria including methicillin-resistant staphylococci and PRSP. Thus, S-3578 was considered to be worthy of further evaluation.
In Vivo Killing of
Porphyromonas gingivalis
by Toluidine Blue-Mediated Photosensitization in an Animal Model
Authors: Kömerik N. , Nakanishi H. , MacRobert A. J. , Henderson B. , Speight P. , Wilson M.
Published: February 2003

Porphyromonas gingivalis
is one of the major causative organisms of periodontitis and has been shown to be susceptible to toluidine blue-mediated photosensitization in vitro. The aims of the present study were to determine whether this technique could be used to kill the organism in the oral cavities of rats and whether this would result in a reduction in the alveolar bone loss characteristic of periodontitis. The maxillary molars of rats were inoculated with
P. gingivalis
and exposed to up to 48 J of 630-nm laser light in the presence of toluidine blue. The number of surviving bacteria was then determined, and the periodontal structures were examined for evidence of any damage. When toluidine blue was used together with laser light there was a significant reduction in the number of viable
P. gingivalis
organisms. No viable bacteria could be detected when 1 mg of toluidine blue per ml was used in conjunction with all light doses used. On histological examination, no adverse effect of photosensitization on the adjacent tissues was observed. In a further group of animals, after time was allowed for the disease to develop in controls, the rats were killed and the level of maxillary molar alveolar bone was assessed. The bone loss in the animals treated with light and toluidine blue was found to be significantly less than that in the control groups. The results of this study show that toluidine blue-mediated lethal photosensitization of
P. gingivalis
is possible in vivo and that this results in decreased bone loss. These findings suggest that photodynamic therapy may be useful as an alternative approach for the antimicrobial treatment of periodontitis.
Alteration of
Escherichia coli
Topoisomerase IV to Novobiocin Resistance
Authors: Hardy Christine D. , Cozzarelli Nicholas R.
Published: February 2003

DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of
Escherichia coli
. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This
parE
mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.
Proteomic Approach to Understanding Antibiotic Action
Authors: Bandow Julia Elisabeth , Brötz Heike , Leichert Lars Ingo Ole , Labischinski Harald , Hecker Michael
Published: February 2003

We have used proteomic technology to elucidate the complex cellular responses of
Bacillus subtilis
to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.
Ergosterol Biosynthesis Inhibitors Become Fungicidal when Combined with Calcineurin Inhibitors against
Candida albicans
,
Candida glabrata
, and
Candida krusei
Authors: Onyewu Chiatogu , Blankenship Jill R. , Del Poeta Maurizio , Heitman Joseph
Published: February 2003

Azoles target the ergosterol biosynthetic enzyme lanosterol 14α-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of
Candida albicans
during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type
C. albicans
when used in conjunction with CsA and FK506. Similarly,
C. albicans
mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type
C. albicans
. These drug combinations also exert synergistic effects against two other
Candida
species,
C. glabrata
and
C. krusei
, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance.
Modulation of the Activity of Secretory Phospholipase A
2
by Antimicrobial Peptides
Authors: Zhao Hongxia , Kinnunen Paavo K. J.
Published: February 2003

The antimicrobial peptides magainin 2, indolicidin, and temporins B and L were found to modulate the hydrolytic activity of secretory phospholipase A
2
(sPLA
2
) from bee venom and in human lacrimal fluid. More specifically, hydrolysis of phosphatidylcholine (PC) liposomes by bee venom sPLA
2
at 10 μM Ca
2+
was attenuated by these peptides while augmented product formation was observed in the presence of 5 mM Ca
2+
. The activity of sPLA
2
towards anionic liposomes was significantly enhanced by the antimicrobial peptides at low [Ca
2+
] and was further enhanced in the presence of 5 mM Ca
2+
. Similarly, with 5 mM Ca
2+
the hydrolysis of anionic liposomes was enhanced significantly by human lacrimal fluid sPLA
2
, while that of PC liposomes was attenuated. These results indicate that concerted action of antimicrobial peptides and sPLA
2
could improve the efficiency of the innate response to infections. Interestingly, inclusion of a cationic gemini surfactant in the vesicles showed an essentially similar pattern on sPLA
2
activity, suggesting that the modulation of the enzyme activity by the antimicrobial peptides may involve also charge properties of the substrate surface.
Efflux-Mediated Resistance to Tigecycline (GAR-936) in
Pseudomonas aeruginosa
PAO1
Authors: Dean Charles R. , Visalli Melissa A. , Projan Steven J. , Sum Phaik-Eng , Bradford Patricia A.
Published: February 2003

Pseudomonas aeruginosa
strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 μg/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline,
P. aeruginosa
PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 μg/ml) was specifically associated with loss of MexXY. Transcription of
mexX
and
mexY
was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 μg/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The
mexAB-oprM
repressor gene,
mexR
, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The
mexCD-OprJ
repressor gene,
nfxB
, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from
P. aeruginosa
, are generally inferior substrates for
P. aeruginosa
efflux pumps than are narrower-spectrum tetracyclines.
New Insertion Sequence Elements in the Upstream Region of
cfiA
in Imipenem-Resistant
Bacteroides fragilis
Strains
Authors: Kato Naoki , Yamazoe Kikuo , Han Chang-Gyun , Ohtsubo Eiichi
Published: February 2003

The 747-bp
cfiA
gene, which encodes a metallo-β-lactamase, and the regions flanking
cfiA
in six imipenem-resistant and four imipenem-susceptible
Bacteroides fragilis
strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the
cfiA
genes (designated

cfiA
1

to

cfiA
10

) of all 10 strains tested varied from that of the standard
cfiA
gene from
B. fragilis
TAL2480. However, putative proteins encoded by the
cfiA
variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that
cfiA
variants have the capability of producing metallo-β-lactamases with full catalytic activities. PCR assay indicated that six metallo-β-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of
cfiA
. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of
cfiA
revealed that there were five new kinds of insertion sequence (IS) elements (designated IS
612
, IS
613
, IS
614
, IS
615
, and IS
616
, with a size range of 1,594 to 1,691 bp), of which only IS
616
was found to be almost identical to IS
1188
, one of the IS elements previously identified in the upstream region of
cfiA
. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of
cfiA
. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS
612
, IS
613
, IS
614
, and IS
615
belong to the IS
4
family, which includes IS
942
, previously found in some drug-resistant
B. fragilis
strains, but that IS
616
belongs to the IS
1380
family. All the IS elements appear to have putative promoter motif sequences (the −7 region's TAnnTTTG motif and the −33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of
cfiA
upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the
cfiA
gene.
High Variability of Plasma Drug Concentrations in Dual Protease Inhibitor Regimens
Authors: Guiard-Schmid Jean-Baptiste , Poirier Jean-Marie , Meynard Jean-Luc , Bonnard Philippe , Gbadoe Ayi Hola , Amiel Corinne , Calligaris Frédérique , Abraham Bruno , Pialoux Gilles , Girard Pierre-Marie , Jaillon Patrice , Rozenbaum Willy
Published: February 2003

Ritonavir (RTV) strongly increases the concentrations of protease inhibitors (PIs) in plasma in patients given a combination of RTV and another PI. This pharmacological interaction is complex and poorly characterized and shows marked inter- and intraindividual variations. In addition, RTV interacts differently with saquinavir (SQV), indinavir (IDV), amprenavir (APV), and lopinavir (LPV). In this retrospective study on 542 human immunodeficiency virus-infected patients, we compared inter- and intraindividual variability of plasma PI concentrations and correlations between the
C
min
(minimum concentration of drug in plasma) values for RTV and the coadministered PI
C
min
values. Mean RTV
C
min
s are significantly lower in patients receiving combinations containing APV or LPV than in combinations with SQV or IDV. With the most common PI dose regimens (600 mg of IDV twice a day [BID], 800 mg of SQV BID, and 400 mg of LPV BID), the interindividual
C
min
variability of patients treated with a PI and RTV seemed to be lower with APV and LPV than with IDV and SQV. As regards intraindividual variability, APV also differed from the other PIs, exhibiting lower
C
min
variability than with the other combinations. Significant positive correlations between RTV
C
min
and boosted PI
C
min
were observed with IDV, SQV, and LPV, but not with APV. Individual dose adjustments must take into account the specificity the pharmacological interaction of each RTV/PI combination and the large inter- and intraindividual variability of plasma PI levels to avoid suboptimal plasma drug concentrations which may lead to treatment failure and too high concentrations which may induce toxicity and therefore reduce patient compliance.
Pharmacokinetics of Intravenous Acyclovir, Zidovudine, and Acyclovir-Zidovudine in Pregnant Rats
Authors: Brown Stacy D. , Bartlett Michael G. , White Catherine A.
Published: February 2003

The pharmacokinetics and placental transfer of acyclovir and zidovudine monotherapies and acyclovir-zidovudine combination therapy were compared in the pregnant rat. Timed-pregnancy Sprague-Dawley rats were used for the study. Doses of 60 mg of each drug/kg of body weight in monotherapy and in combination therapy were given by intravenous bolus, and samples of maternal plasma, amniotic fluid, fetal tissue, and placental tissue were collected over a period of 8 h postdose. Concentrations of each drug in the various matrices were measured by high-performance liquid chromatography. All data were analyzed by using WinNonlin. A one-compartment model with first-order elimination was used to fit the AZT plasma data from the combination therapy rats, but the plasma data from the other groups were fit to a two-compartment model. Tissue data were analyzed by noncompartmental analysis to generate area-under-the-concentration-time-curve values. Implementation of the combination therapy altered the pharmacokinetics of each drug compared to its monotherapy pharmacokinetics. The combination of these two drugs may potentiate fetal and amniotic fluid exposures to each drug.
Comparing Pharmacokinetics of Amoxicillin Given Twice or Three Times per Day to Children Older than 3 Months with Pneumonia
Authors: Fonseca Walter , Hoppu Kalle , Rey Luís C. , Amaral João , Qazi Shamim
Published: February 2003

For children with ambulatory pneumonia, the World Health Organization (WHO) recommends oral amoxicillin (15 mg/kg of body weight/dose) thrice daily (t.i.d.) or oral cotrimoxazole (4 mg of trimethoprim/kg/dose) twice daily (b.i.d.). The more frequent amoxicillin dosing may lead to compliance problems. To compare the pharmacokinetics and levels of amoxicillin in plasma in the current WHO acute respiratory infection recommendations with the 25-mg/kg/dose b.i.d. regimen, we performed a two-group parallel study of 66 children ages 3 to 59 months with pneumonia. Amoxicillin was given orally at 25 mg/kg/dose b.i.d. or 15 mg/kg/dose t.i.d. Amoxicillin concentrations were determined by high-performance liquid chromatography after the first dose on days 1 and 3. After the first dose on day 1, the mean area under the concentration-time curve (AUC) for amoxicillin after the 25-mg/kg dose was 54.7 versus 24.9 μg · h/ml after the 15-mg/kg dose. After the first dose on day 3, the mean AUC was 44.1 versus 28.5 μg · h/ml. All but two children had plasma amoxicillin concentrations above 0.5 μg/ml for >50% of the dose interval on both days. Six children on day 1 and five children on day 3 had concentrations above 1.0 μg/ml for <50% of the dose interval. On day 1, 16 of 27 children in the b.i.d. group and 11 of 26 children in the t.i.d. group had concentrations that were above 2.0 μg/ml for <50% of the dose interval, and on day 3, 18 of 31 children in the b.i.d. group and 8 of 31 children in the t.i.d. group had concentrations that were above 2.0 μg/ml for <50% of the dose interval. Amoxicillin b.i.d. is a feasible alternative for t.i.d. dosing. To lengthen the time above the MIC at higher concentration levels, a 30- to 40-mg/kg/dose b.i.d. should be considered instead of the 25 mg/kg/dose used in this study.
Marine Bacteria Cause False-Positive Results in the Colilert-18 Rapid Identification Test for
Escherichia coli
in Florida Waters
Authors: Pisciotta John M. , Rath Damon F. , Stanek Paul A. , Flanery D. Michael , Harwood Valerie J.
Published: February 2003

The Colilert-18 system for enumeration of total coliforms and
Escherichia coli
is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of
E. coli
numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including
E. coli
, could be cultured from 100% of yellow fluorescent wells (denoting
E. coli
-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the “positive” wells inoculated with marine samples.
Ortho
-nitrophenyl-β-
d
-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-β-
d
-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of
Vibrio cholerae
(ONPG positive) and
Providencia
sp. (MUG positive) ml
−1
inoculated into Colilert-18 caused false-positive
E. coli
results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml
−1
in seawater diluted 1:10 with freshwater to ≈5,000 CFU ml
−1
in seawater diluted 1:20 with freshwater. Estimated
E. coli
numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU·100 ml
−1
, while
E. coli
numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU·100 ml
−1
. The lower estimates of
E. coli
numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of
E. coli
in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.