bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Efflux System in Inducibly Erythromycin-Resistant Strains of
Streptococcus pyogenes
Authors: Giovanetti Eleonora , Brenciani Andrea , Burioni Roberto , Varaldo Pietro Emanuele
Published: November 2002

Streptococcus pyogenes
strains inducibly resistant (iMLS phenotype) to macrolide, lincosamide, and streptogramin B (MLS) antibiotics can be subdivided into three phenotypes: iMLS-A, iMLS-B, and iMLS-C. This study focused on inducibly erythromycin-resistant
S. pyogenes
strains of the iMLS-B and iMLS-C types, which are very similar and virtually indistinguishable in a number of phenotypic and genotypic features but differ clearly in their degree of resistance to MLS antibiotics (high in the iMLS-B type and low in the iMLS-C type). As expected, the iMLS-B and iMLS-C test strains had the
erm
(A) methylase gene; the iMLS-A and the constitutively resistant (cMLS) isolates had the
erm
(B) methylase gene; and a control M isolate had the
mef
(A) efflux gene.
mre
(A) and
msr
(A), i.e., other macrolide efflux genes described in gram-positive cocci, were not detected in any test strain. With a radiolabeled erythromycin method for determination of the intracellular accumulation of the drug in the absence or presence of an efflux pump inhibitor, active efflux of erythromycin was observed in the iMLS-B isolates as well as in the M isolate, whereas no efflux was demonstrated in the iMLS-C isolates. By the triple-disk (erythromycin plus clindamycin and josamycin) test, performed both in normal test medium and in the same medium supplemented with the efflux pump inhibitor, under the latter conditions iMLS-B and iMLS-C strains were no longer distinguishable, all exhibiting an iMLS-C phenotype. In conjugation experiments with an iMLS-B isolate as the donor and a Rif
r
Fus
r
derivative of an iMLS-C isolate as the recipient, transconjugants which shared the iMLS-B type of the donor under all respects, including the presence of an efflux pump, were obtained. These results indicate the existence of a novel, transferable efflux system, not associated with
mef
(A) or with other known macrolide efflux genes, that is peculiar to iMLS-B strains. Whereas the low-level resistance of iMLS-C strains to MLS antibiotics is apparently due to
erm
(A)-encoded methylase activity, the high-level resistance of iMLS-B strains appears to depend on the same methylase activity plus the new efflux system.
Genes Involved in Bacitracin Resistance in
Streptococcus mutans
Authors: Tsuda Hiromasa , Yamashita Yoshihisa , Shibata Yukie , Nakano Yoshio , Koga Toshihiko
Published: November 2002

Streptococcus mutans
is resistant to bacitracin, which is a peptide antibiotic produced by certain species of
Bacillus
. The purpose of this study was to clarify the bacitracin resistance mechanism of
S. mutans
. We cloned and sequenced two
S. mutans
loci that are involved in bacitracin resistance. The
rgp
locus, which is located downstream from
rmlD
, contains six
rgp
genes (
rgpA
to
rgpF
) that are involved in rhamnose-glucose polysaccharide (RGP) synthesis in
S. mutans.
The inactivation of RGP synthesis in
S. mutans
resulted in an approximately fivefold-higher sensitivity to bacitracin relative to that observed for the wild-type strain Xc. The second bacitracin resistance locus comprised four
mbr
genes (
mbrA
,
mbrB
,
mbrC
, and
mbrD
) and was located immediately downstream from
gtfC
, which encodes the water-insoluble glucan-synthesizing enzyme. Although the bacitracin sensitivities of mutants that had defects in flanking genes were similar to that of the parental strain Xc, mutants that were defective in
mbrA
,
mbrB
,
mbrC
, or
mbrD
were about 100 to 120 times more sensitive to bacitracin than strain Xc. In addition, a mutant that was defective in all of the
mbrABCD
genes and
rgpA
was more sensitive to bacitracin than either the RGP or Mbr mutants. We conclude that RGP synthesis is related to bacitracin resistance in
S. mutans
and that the
mbr
genes modulate resistance to bacitracin via an unknown mechanism that is independent of RGP synthesis.
New Site of Modification of 23S rRNA Associated with Clarithromycin Resistance of
Helicobacter pylori
Clinical Isolates
Authors: Fontana Carla , Favaro Marco , Minelli Silvia , Criscuolo Anna Angela , Pietroiusti Antonio , Galante Alberto , Favalli Cartesio
Published: November 2002

Resistance of
Helicobacter pylori
to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve
H. pylori
eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from
H. pylori
isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for
H. pylori
isolation. Eighty-six gastric biopsy specimens yielded
H. pylori
-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 μg/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an
Hha
I restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates.
Low Frequencies of Resistance among
Staphylococcus
and
Enterococcus
Species to the Bactericidal DNA Polymerase Inhibitor
N
3
-Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil
Authors: Butler Michelle M. , Skow Donna J. , Stephenson Ryan O. , Lyden Patrick T. , LaMarr William A. , Foster Kimberly A.
Published: November 2002

The 6-anilinouracils (AUs) constitute a new class of bactericidal antibiotics selective against gram-positive (Gr
+
) organisms. The AU family of compounds specifically inhibits a novel target, replicative DNA polymerase Pol IIIC. Like other antibiotics, AUs can be expected to engender the development of resistant bacteria. We have used a representative AU and clinically relevant strains of
Staphylococcus aureu
s and
Enterococcus
to determine the frequency and mechanism(s) of resistance development. The frequency of resistance was determined by using
N
3
-hydroxybutyl 6-(3′-ethyl-4′-methylanilino) uracil (HBEMAU) and commercially available antibiotics at eight times the MICs. For all five Gr
+
organisms tested, the frequency of resistance to HBEMAU ranged from 1 × 10
−8
to 3 × 10
−10
. The frequencies of resistance to the antibiotics tested, including rifampin, gentamicin, and ciprofloxacin, were either greater than or equal to those for HBEMAU. In order to understand the mechanism of resistance, HBEMAU-resistant organisms were isolated. MIC assays showed that the organisms had increased resistance to AU inhibitors but not to other families of antibiotics. Inhibition studies with DNA polymerases from HBEMAU-sensitive and -resistant strains demonstrated that the resistance was associated with Pol IIIC. DNA sequence analysis of the entire
polC
genes from both wild-type and resistant organisms revealed that the resistant organisms had a sequence change that mapped to a single amino acid codon in all strains examined.
Pharmacokinetics and Efficacies of Liposomal and Conventional Formulations of Tobramycin after Intratracheal Administration in Rats with PulmonaryBurkholderia cepaciaInfection
Authors: Marier Jean-Francois , Lavigne Jean , Ducharme Murray P.
Published: November 2002

The objective of the present study was to determine the pharmacokinetics and efficacies of liposomal and conventional formulations of tobramycin againstBurkholderia cepaciain a model of chronic lung infection. Male Sprague-Dawley rats were inoculated intratracheally with 106CFU of a very resistant strain ofB. cepacia(strain BC 1368; MIC, 128 μg/ml) to establish lung infection. A 1,200-μg dose of tobramycin was administered intratracheally as a liposomal formulation and as a conventional formulation. Rats were anesthetized and exsanguinated by cardiac puncture at different times over 24 h to assess pulmonary tobramycin concentrations and the number of residual CFU. Pharmacokinetic parameters were calculated by using a two-compartment model with NONMEM. The mean half-life at the β phase (t1/2β) and the pulmonary exposure (the area under the concentration-time curve [AUC]) of liposomal tobramycin were 19.7 h (coefficient of variation [CV], 24.2%) and 6,811 μg · h/lungs (CV, 19.7%), respectively. The pharmacokinetics of conventional tobramycin were statistically different, with at1/2βand AUC of 12.9 h (CV, 31.4%) and 821 μg · h/lungs (CV, 15.0%), respectively. Pearson chi-square analyses were performed on residual CFU data distributed in the following categories: <103, 103to 105, and >105. Differences in CFU data between formulations showed a statistical trend (P< 0.10) when data from all time points were used, and statistically significant differences were found after 12 h (P< 0.05), with greater eradication achieved with the liposomal formulation. In conclusion, intratracheal administration of tobramycin in liposomes was associated with marked changes in the pharmacokinetics of the drug in the lung and an apparent trend for a prolonged efficacy againstB. cepacia. These results support the hypothesis that inhalation of liposomal tobramycin may improve the management of chronic pulmonary infections caused by resistant bacteria in patients with cystic fibrosis.
Ex Vivo Pharmacodynamics of Amoxicillin-Clavulanate against β-Lactamase-Producing
Escherichia coli
in a Yucatan Miniature Pig Model That Mimics Human Pharmacokinetics
Authors: Bronner Stéphane , Murbach Valérie , Peter Jean-Daniel , Levêque Dominique , Elkhaïli Hassan , Salmon Yves , Dhoyen Nathalie , Monteil Henri , Woodnutt Gary , Jehl François
Published: November 2002

The objective of the present study was to investigate the potential bactericidal activity of amoxicillin-clavulanate against β-lactamase-producing
Escherichia coli
strains and to elucidate the extent to which enzyme production affects the activity. Six adult Yucatan miniature pigs received a single intravenous dose of 1.1 g of amoxicillin-clavulanate as an intravenous infusion over 30 min. The pharmacokinetic parameters were determined for the serum samples and compared to the published data for humans (2.2-g intravenous dose). The parameters were comparable for the two species, and therefore, the miniature pig constitutes a good model for pharmacodynamic study of amoxicillin-clavulanate. Therefore, the model was used in an ex vivo pharmacodynamic study of amoxicillin-clavulanate against four strains of
Escherichia coli
producing β-lactamases at different levels. The
E. coli
strains were cultured with serial dilutions (1:2 to 1:256) of the serum samples from the pharmacokinetic study, and the number of surviving bacteria was determined after 1, 3, and 6 h of exposure. Amoxicillin-clavulanate at concentrations less than the MIC and the minimal bactericidal concentration had marked bactericidal potency against the strain that produced low levels of penicillinase. For high-level or intermediate-level β-lactamase-producing strains, the existence of a clavulanate concentration threshold of 1.5 to 2 μg/ml, below which there was no bactericidal activity, was demonstrated. The index of surviving bacteria showed the existence of mixed concentration- and time-dependent actions of amoxicillin (in the presence of clavulanate) which varied as a function of the magnitude of β-lactamase production by the test strains. This study shows the effectiveness of amoxicillin-clavulanate against low- and intermediate-level penicillinase-producing strains of
E. coli
. These findings are to be confirmed in a miniature pig experimental infection model.
Benznidazole Treatment following AcuteTrypanosoma cruziInfection Triggers CD8+T-Cell Expansion and Promotes Resistance to Reinfection
Authors: Olivieri Bianca Perdigão , Cotta-de-Almeida Vinícius , Araújo-Jorge Tania
Published: November 2002

Many studies have shed light on the mechanisms underlying both immunoprotection and immune dysregulation arising afterTrypanosoma cruziinfection. However, little is known about the impact of benznidazole (N-benzyl-2-nitroimidazole acetamide), the drug available for clinical treatment of the infection, on the immune system in the infected host. In the present study we investigated the effect of benznidazole therapy on the lymphoid compartment during the course of experimentalT. cruziinfection. Although amelioration of a variety of clinical and parasitological signs was observed in treated mice, amelioration of splenocyte expansion was not detected. Interestingly, this sustained splenomegaly observed in benznidazole-treated mice showed a preferential expansion of CD8+T lymphocytes. Moreover, although benznidazole treatment blocked the expansion of recently activated CD4+and CD8+T cells seen in infected hosts, benznidazole treatment led to a selective expansion of effector and memory CD8+T lymphocytes in association with a lower rate of apoptosis. In addition, the surviving treated animals were protected from reinfection. Together, these data suggest that, in addition to its well-known direct role in blocking parasite replication in vivo, benznidazole appears to directly affect immune regulation inT. cruzi-infected hosts.
Therapeutic Efficacy of Topically Applied KP-103 against Experimental Tinea Unguium in Guinea Pigs in Comparison with Amorolfine and Terbinafine
Authors: Tatsumi Yoshiyuki , Yokoo Mamoru , Senda Hisato , Kakehi Kazuaki
Published: November 2002

The therapeutic efficacy of KP-103, a novel topical triazole, in a guinea pig tinea unguium model was investigated. Experimental tinea unguium and tinea pedis were produced by inoculation ofTrichophyton mentagrophytesSM-110 between the toes of the hind paw of guinea pigs. One percent solution (0.1 ml) of KP-103, amorolfine, or terbinafine was topically applied to the nails and whole sole of an infected foot once daily for 30 consecutive days, and terbinafine was also orally administered at a daily dose of 40 mg/kg of body weight for 30 consecutive days, starting on day 60 postinfection. The fungal burdens of nails and plantar skin were assessed using a new method, which makes it possible to recover infecting fungi by removing a carryover of the drug remaining in the treated tissues into the culture medium. Topically applied KP-103 inhibited the development of nail collapse, significantly reduced the fungal burden of the nails, and sterilized the infected plantar skin. On the other hand, topical amorolfine and topical or oral terbinafine were ineffective for tinea unguium, although these drugs eradicated or reduced the fungal burden of plantar skin. The in vitro activities of amorolfine and terbinafine againstT. mentagrophytesSM-110 were 8- and 32-fold, respectively, decreased by the addition of 5% keratin to Sabouraud dextrose broth medium. In contrast, the activity of KP-103 was not affected by keratin because its keratin affinity is lower than those of the reference drugs, suggesting that KP-103 largely exists in the nails as an active form that was not bound to keratin and diffuses in the nail without being trapped by keratin. The effectiveness of KP-103 against tinea unguium is probably due to its favorable pharmacokinetic properties in the nails together with its potent antifungal activity.
Effect of Auristatin PHE on Microtubule Integrity and Nuclear Localization in
Cryptococcus neoformans
Authors: Woyke Tanja , Roberson Robert W. , Pettit George R. , Winkelmann Günther , Pettit Robin K.
Published: November 2002

The mechanism of action of the fungicidal peptide auristatin PHE was investigated in
Cryptococcus neoformans
. Since auristatin PHE causes budding arrest in
C. neoformans
(T. Woyke, G. R. Pettit, G. Winkelmann, and R. K. Pettit, Antimicrob. Agents Chemother.
45:
3580-3584, 2001), microtubule integrity and nuclear localization in auristatin PHE-treated cells were examined. Iterative deconvolution in conjunction with an optimized
C. neoformans
microtubule immunolabeling procedure enabled detailed visualization of the microtubule cytoskeleton in auristatin PHE-treated
C. neoformans
. The effect of auristatin PHE on
C. neoformans
microtubule organization was compared with that of the tubulin-binding agent nocodazole. Both drugs produced complete disruption first of cytoplasmic and then of spindle microtubules in a time- and concentration-dependent manner. Sub-MICs of auristatin PHE caused complete microtubule disruption within 4.5 h, while 1.5 times the nocodazole MIC was required for the same effect. For both drugs, disruption of microtubules was accompanied by blockage of nuclear migration and of nuclear and cellular division, resulting in cells arrested in a uninucleate, large-budded stage. Nocodazole and the linear peptide auristatin PHE are remarkably different in structure and spectrum of activity, yet on the cellular level, they have similar effects.
Mechanism by Which Mutations at His274 Alter Sensitivity of Influenza A Virus N1 Neuraminidase to Oseltamivir Carboxylate and Zanamivir
Authors: Wang Michael Z. , Tai Chun Y. , Mendel Dirk B.
Published: November 2002

Oseltamivir carboxylate is a potent and specific inhibitor of influenza neuraminidase (NA). An influenza A/H1N1 variant selected in vitro with reduced susceptibility to oseltamivir carboxylate contains a His274Tyr mutation. To understand the mechanism by which a His274Tyr mutation gives rise to drug resistance, we studied a series of NA variant proteins containing various substitutions at position 274. Replacement of His274 with larger side chain residues (Tyr or Phe) reduced the NA sensitivity to oseltamivir carboxylate. In contrast, replacement of His274 with smaller side chain residues (Gly, Asn, Ser, and Gln) resulted in enhanced or unchanged sensitivity to oseltamivir carboxylate. Previous studies have suggested that the slow-binding inhibition of NA by oseltamivir carboxylate is a result of the reorientation of Glu276. Loss of this slow-binding inhibition in the His274Tyr and His274Phe mutant NA but not in His274Asn, His274Gly, His274Ser, or His274Gln supports the conclusion that the conformational change of Glu276 is restricted in the His274Tyr and His274Phe mutant NA upon oseltamivir carboxylate binding. Interestingly, His274Asn, as well as His274Gly, His274Ser, and His274Gln, also displayed reduced sensitivity to zanamivir and its analogue, 4-amino-Neu5Ac2en. Substitution of His274 with Tyr in influenza A/Tokyo/3/67 (H3N2) recombinant NA did not affect the susceptibility to oseltamivir carboxylate. These data indicate that the volume occupied by the amino acid side chain at position 274 can influence the sensitivities of influenza N1 NA but not of N2 NA to both oseltamivir carboxylate and zanamivir.
Carbapenem Resistance in a Clinical Isolate of
Enterobacter aerogenes
Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs
Authors: Yigit Hesna , Anderson Gregory J. , Biddle James W. , Steward Christine D. , Rasheed J. Kamile , Valera Lourdes L. , McGowan John E. , Tenover Fred C.
Published: November 2002

We investigated the mechanism of imipenem resistance in
Enterobacter aerogenes
strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 μg/ml and the meropenem MIC was 8 μg/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was ≤1 μg/ml and the meropenem MIC was ≤0.25 μg/ml. Cefepime MICs also decreased from 8 to 1 μg/ml; however, the MICs of ceftazidime (≥128 μg/ml), cefoxitin (≥32 μg/ml), and cefotaxime (≥64 μg/ml) remained the same. The β-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain
E. aerogenes
ATCC 13048 were determined. Strains 810 and 810-REV each produced two β-lactamases with pIs of 8.2 and 5.4. The β-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to
Escherichia coli
porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of
Klebsiella pneumoniae
(anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data suggest that imipenem resistance in
E. aerogenes
810 is primarily associated with the lack of expression of the analogs of the OmpC (42-kDa) and OmpF (39-kDa) outer membrane proteins, which also results in decreased susceptibility to meropenem and cefepime.
Identification of
vat
(E) in
Enterococcus faecalis
Isolates from Retail Poultry and Its Transferability to
Enterococcus faecium
Authors: Simjee S. , White D. G. , Wagner D. D. , Meng J. , Qaiyumi S. , Zhao S. , McDermott P. F.
Published: November 2002

Sixteen isolates of
Enterococcus faecalis
were recovered from retail poultry samples (seven chickens and nine turkeys) purchased from grocery stores in the greater Washington, D.C., area. PCR for known streptogramin resistance genes identified
vat
(E) in five
E. faecalis
isolates (three isolates from chickens and two isolates from turkeys). The
vat
(E) gene was transmissible on a ca. 70-kb plasmid, along with resistance to erythromycin, tetracycline, and streptomycin, by conjugation to
E. faecalis
and
Enterococcus faecium
recipient strains. DNA sequencing showed little variation between
E. faecalis vat
(E) genes from the chicken samples; however, one
E. faecalis vat
(E) gene from a turkey sample possessed 5 nucleotide changes that resulted in four amino acid substitutions. None of these substitutions in the
vat
(E) allele have previously been described. This is the first report of
vat
(E) in
E. faecalis
and its transferability to
E. faecium
, which indicates that
E. faecalis
can act as a reservoir for the dissemination of
vat
(E)-mediated streptogramin resistance to
E. faecium
.
Mechanisms of Decreased Susceptibility to Cefpodoxime in
Escherichia coli
Authors: Oliver Antonio , Weigel Linda M. , Rasheed J. Kamile , McGowan John E. , Raney Patti , Tenover Fred C.
Published: November 2002

Cefpodoxime is one of five antimicrobial agents recommended by the National Committee for Clinical Laboratory Standards for screening isolates of
Klebsiella
spp. and
Escherichia coli
for extended-spectrum β-lactamase (ESBL) production. In a prior study, we noted that among 131
E. coli
isolates for which the MIC of at least one extended-spectrum cephalosporin (ESC) or aztreonam was ≥2 μg/ml (suggesting the presence of ESBL production), there were 59 isolates (45.0%) for which the MIC of cefpodoxime was 2 to 4 μg/ml (i.e., a positive ESBL screening test), but the MICs of ceftazidime, cefotaxime, and ceftriaxone were ≤1 μg/ml (below the ESBL screening breakpoint). Thus, the results appeared to be false-positive ESBL screening tests. These 59 isolates were divided into five phenotypic groups based on the susceptibility patterns of the organisms to a variety of β-lactam agents and further characterized. The first group (32 isolates) all produced a TEM-1 β-lactamase, and changes in the major outer membrane proteins were detected in representative strains. The second group (18 isolates) lacked
bla
TEM
but showed a number of porin changes; some also showed a modest elevation in production of the AmpC chromosomal β-lactamase. In the third phenotypic group (seven isolates) all expressed an OXA-30 β-lactamase. Some also harbored altered porins. The two remaining phenotypes each had a distinct pattern of porin changes with or without β-lactamase production. These data indicate that several factors are associated with decreased susceptibility to cefpodoxime in
E. coli
, but none of the mechanisms are related to ESBL production. Current screening methods produced false-positive ESBL results for these isolates. Such isolates should not be classified as containing ESBLs, nor should interpretations of ESCs or aztreonam susceptibility be changed to resistant on test reports for these isolates.
Occurrence of Newer β-Lactamases in
Klebsiella pneumoniae
Isolates from 24 U.S. Hospitals
Authors: Moland Ellen Smith , Black Jennifer A. , Ourada Jason , Reisbig Mark D. , Hanson Nancy D. , Thomson Kenneth S.
Published: November 2002

Despite the discovery of novel β-lactamases such as extended-spectrum β-lactamases (ESBLs), imported AmpC, and carbapenem-hydrolyzing β-lactamases at least a decade ago, there remains a low level of awareness of their importance and how to detect them. There is a need to increase the levels of awareness of clinical laboratories about the detection of newer β-lactamases. Therefore, a study was conducted in 2000 to investigate the occurrence of these β-lactamases in
Klebsiella pneumoniae
isolates at 24 U.S. medical centers. To enhance the likelihood of detecting imported AmpC and carbapenem-hydrolyzing β-lactamases, participating laboratories were permitted to include archived strains (1996 to 2000) that were intermediate or resistant to either cefoxitin or imipenem. The β-lactamase production of 408 isolates positive by screening of 1,123 isolates was investigated by ESBL phenotypic confirmation tests; and for AmpC and carbapenem-hydrolyzing β-lactamases, three-dimensional tests, isoelectric focusing, β-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular tests were used. ESBL-producing isolates were detected at 18 of the 24 sites (75%), imported AmpC-producing isolates were detected at 10 sites (42%), inducible imported AmpC-producing isolates were detected at 3 sites (12.5%), and a molecular class A carbapenem-hydrolyzing enzyme was detected at 1 site (4%). No class B or D carbapenem-hydrolyzing enzymes were detected. ESBLs and imported AmpC β-lactamases were detected at a significant number of sites, indicating widespread penetration of these enzymes into U.S. medical institutions. Because these enzymes may significantly affect therapeutic outcomes, it is vital that clinical laboratories be aware of them and be able to detect their occurrence.
Antimicrobial Susceptibilities of Diverse
Bacillus anthracis
Isolates
Authors: Coker Pamala R. , Smith Kimothy L. , Hugh-Jones Martin E.
Published: November 2002

A test of 25 genetically diverse isolates of
Bacillus anthracis
was conducted to determine their susceptibility to seven clinically relevant antimicrobial agents. Etest strips (AB BIODISK, Solna, Sweden) were used to measure the MICs for the isolates. Using the National Committee for Clinical Laboratory Standards MIC breakpoints for staphylococci, three isolates were found to be resistant to penicillin and five were found to be resistant to cefuroxime. The penicillin-resistant isolates were negative for β-lactamase production. Continued surveillance of
B. anthracis
field isolates is recommended to monitor antimicrobial susceptibility.
In Vitro Activity of Micafungin (FK-463) against
Candida
spp.: Microdilution, Time-Kill, and Postantifungal-Effect Studies
Authors: Ernst Erika J. , Roling Ellen E. , Petzold C. Rosemarie , Keele Douglas J. , Klepser Michael E.
Published: November 2002

We evaluated the in vitro activity of the new echinocandin antifungal micafungin against
Candida
spp. using microdilution and time-kill methods. Additionally, we examined the postantifungal effect (PAFE) of micafungin. Finally, we evaluated the effect of the addition of serum and plasma on the MIC of micafungin. Four
Candida albicans
isolates and two isolates of each
Candida glabrata
,
Candida krusei
, and
Candida tropicalis
were selected for testing. The MICs of micafungin were determined in RPMI 1640 medium buffered with morpholinepropanesulfonic acid alone and with the addition of 10, 20, and 50% human serum and plasma. MICs were determined by using two endpoints: a prominent reduction in growth (the MIC at which 80% of isolates are inhibited [MIC
80
]) and complete visual inhibition of growth (MIC
100
). The minimum fungicidal concentration (MFC) of micafungin for each isolate was also determined. Time-kill curves were determined for each isolate in RPMI 1640 medium with micafungin at concentrations ranging from 0.125 to 16 times the MIC
80
to assess the correlation between MIC
80
and fungicidal activity. PAFE studies were conducted with each isolate by using concentrations ranging between 0.25 and 4 times the MIC
80
. The MIC
80
s for the test isolates ranged from 0.0039 to 0.25 μg/ml. Overall, the addition of serum or plasma increased the MIC 6 to 7 doubling dilutions for
C. albicans
and 3 to 4 doubling dilutions for
C. krusei
and
C. tropicalis
. Micafungin time-kill studies demonstrated fungicidal activity at concentrations ranging from 4 to 16 times the MIC
80
. Micafungin is very potent agent against a variety of
Candida
spp., producing fungicidal activity against 7 of 10 isolates tested. A PAFE was observed against all isolates. The PAFE was influenced by the drug concentration, with the highest concentration resulting in the longest observed PAFE in each case. The highest concentration tested, four times the MIC, resulted in a PAFE of more than 9.8 h for 5 of 10 isolates tested (range, 0.9 to ≥20.1 h).
Antiviral Activities and Cellular Toxicities of Modified 2′,3′-Dideoxy-2′,3′-Didehydrocytidine Analogues
Authors: Stuyver Lieven J. , Lostia Stefania , Adams Marjorie , Mathew Judy S. , Pai Balakrishna S. , Grier Jason , Tharnish Phillip M. , Choi Yongseok , Chong Youhoon , Choo Hyunah , Chu Chung K. , Otto Michael J. , Schinazi Raymond F.
Published: November 2002

The antiviral efficacies and cytotoxicities of 2′,3′- and 4′-substituted 2′,3′-didehydro-2′,3′-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC
90
s], 0.14 to 5.2 μM), but marked increases in EC
90
s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC
90
s, 53 to >100 μM). The β-
l
-enantiomers of both classes of compounds were more potent than the corresponding β-
d
-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC
90
, 0.25 μM). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 μM. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4′-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of β-
d
-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC
90
, 0.08 μM) and lamivudine-resistant HIV-1 (EC
90
= 0.15 μM) is markedly reduced by introduction of a 3′-fluorine in the sugar (EC
90
s of compound 2a, 37.5 and 494 μM, respectively).
Mutagenically Separated PCR Assay for Rapid Detection of M41L and K70R Zidovudine Resistance Mutations in CRF01_AE (Subtype E) Human Immunodeficiency Virus Type 1
Authors: Myint Lay , Ariyoshi Koya , Yan Hua , Frater Alexander J. , Auwanit Wattana , Pathipvanith Panita , Yamada Kaneo , Matsuda Masakazu , Chiba Tomoko , Fujita Kazunori , McClure Myra , Weber Jonathan N. , Sugiura Wataru
Published: November 2002

A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01_AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries.
Synthetic Analogues of β-1,2 Oligomannosides Prevent Intestinal Colonization by the Pathogenic Yeast
Candida albicans
Authors: Dromer Françoise , Chevalier Reynald , Sendid Boualem , Improvisi Luce , Jouault Thierry , Robert Raymond , Mallet Jean Maurice , Poulain Daniel
Published: November 2002

The pathogenic yeast
Candida albicans
displays at its cell surface β-1,2 oligomannosides (β-1,2-Mans). In contrast to the ubiquitous α-Mans, β-1,2-Mans bind to galectin-3, a major endogenous lectin expressed on epithelial cells. The specific role of β-1,2-Mans in colonization of the gut by
C. albicans
was assessed in a mouse model. A selected virulent strain of
C. albicans
(expressing more β-1,2-Man epitopes) induced more intense and sustained colonization than an avirulent strain (expressing less β-1,2-Man epitopes). Synthetic (Σ) β-and α-linked tetramannosides with antigenicities that mimicked the antigenicities of
C. albicans
-derived oligomannosides were then constructed. Oral administration of Σβ-1,2-Man (30 mg/kg of body weight) prior to inoculation with the virulent strain resulted in almost complete eradication of yeasts from stool samples, whereas administration of Σα-Man at the same dose did not. As most cases of human systemic candidiasis are endogenous in origin, this first demonstration that a synthetic analogue of a yeast adhesin can prevent yeast colonization in the gut opens the possibility of new prophylactic strategies.
Coadministration of Indinavir and Nelfinavir in Human Immunodeficiency Virus Type 1-Infected Adults: Safety, Pharmacokinetics, and Antiretroviral Activity
Authors: Riddler Sharon A. , Havlir Diane , Squires Kathleen E. , Kerr Brad , Lewis Ronald H. , Yeh Kuang , Wynne Linda Hawe , Zhong Ling , Peng Yahong , Deutsch Paul , Saah Alfred
Published: November 2002

Combinations of protease inhibitors (PIs) can have potentially beneficial pharmacokinetic interactions, resulting in higher drug levels and less frequent dose administration. Indinavir (IDV) and nelfinavir (NFV) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) protease and are commonly prescribed antiretroviral agents. Pilot pharmacokinetic data suggested a bidirectional enhancing interaction between IDV and NFV. A phase II study was conducted to evaluate the safety, pharmacokinetics, and antiviral activity of IDV plus NFV given in a combination every 12 h in HIV-1-infected subjects. IDV plus NFV was given as a twice-daily regimen to 20 HIV-1-infected subjects who were PI naive (11 of 20 were antiretroviral naive). After week 18, nucleoside reverse transcriptase inhibitors were added to the treatment regimen in seven subjects. The enrolled subjects had a geometric mean baseline plasma HIV-1 RNA of 63,095 copies/ml and a mean CD4
+
cell count of 266 cells/mm
3
. Pharmacokinetic evaluations were performed at the following doses: IDV at 1,000 mg every 12 h (q12h) plus NFV at 750 mg q12h, IDV at 1,000 mg q12h plus NFV at 1,000 mg q12h, and IDV at 1,200 mg q12h plus NFV at 1,250 mg q12h. The coadministration of IDV plus NFV resulted in a modest inhibition of IDV elimination, resulting in a plasma profile of IDV 1200 mg q12h (with NFV at 1,250 mg q12h) that was comparable to the standard IDV dose of 800 mg q8h. In contrast, IDV had no apparent effect on the pharmacokinetic profile of NFV. The combination of IDV and NFV was generally well tolerated and resulted in sustained virologic suppression with 45% of the subjects having an HIV-1 RNA level in plasma of <400 copies/ml at week 72 (intent-to-treat).
Synthetic Peptides That Exert Antimicrobial Activities in Whole Blood and Blood-Derived Matrices
Authors: Yeaman Michael R. , Gank Kimberly D. , Bayer Arnold S. , Brass Eric P.
Published: November 2002

Peptides that exert antimicrobial activity in artificial media may lack activity within blood or other complex biological matrices. To facilitate the evaluation of antimicrobial peptides for possible therapeutic utility, an ex vivo assay was developed to assess the extent and durability of peptide antimicrobial activities in complex fluid biomatrices of whole blood, plasma, and serum compared with those in conventional media. Novel antimicrobial peptides (RP-1 and RP-11) were designed based in part on platelet microbicidal proteins. RP-1, RP-11, or gentamicin was introduced into biomatrices either coincident with, or 2 h prior to, inoculation with anEscherichia colitarget organism. Antimicrobial activities of peptides were assessed by quantitative culture 2 h after bacterial inoculation and compared to those of peptide-free and gentamicin controls. In whole blood and homologous plasma or serum, introduction of RP-1 or RP-11 coincident withE. coliwas associated with a significant reduction in CFU per milliliter versus the respective peptide-free controls. Moreover, substantial antimicrobial activity remained when RP-1 or RP-11 was placed into whole blood or plasma 2 h prior toE. coliinoculation. These results suggest that the peptides were not rapidly inactivated within these biomatrices. Peptide antimicrobial activities were negatively affected by preincubation in serum or in heat-inactivated serum, compared with those of the respective controls. Peptides RP-1 and RP-11 were consistently effective at lower concentrations in biomatrices than in artificial media, indicating favorable antimicrobial interactions with components of blood or blood fractions. Collectively, these findings support the concept that synthetic peptides can be designed to exert potent antimicrobial activities in relevant and complex biological matrices.
Analysis of an 8.1-kb DNA Fragment Contiguous with the Erythromycin Gene Cluster of
Saccharopolyspora erythraea
in the
eryCI
-Flanking Region
Authors: Reeves Andrew R. , Weber Gerhard , Cernota William H. , Weber J. Mark
Published: November 2002

An 8.1-kb region of the
Saccharopolyspora erythraea
genome, significant for its contiguity to the known genes of the erythromycin biosynthetic gene cluster, was mutationally analyzed and its DNA sequence was determined. The region lies immediately adjacent to
eryCI
. The newly characterized region is notable for a large, 3.0-kb segment, predicted not to be translated, followed by four probable genes: an acetyltransferase gene, a protease inhibitor gene, a methyltransferase gene, and a transposase gene. Because the probable functions of the genes in this region are not required for erythromycin biosynthesis or resistance and because a deletion of a 6.0-kb portion of this region had no effect on erythromycin biosynthesis, this region marks the outside boundary of the erythromycin gene cluster. Therefore,
eryCI
represents the end of the cluster. These results complete the analysis of the erythromycin gene cluster and eliminate the possibility that additional sought-after pathway-specific structural or regulatory genes might be found within or adjacent to the cluster.
ABC Transporters and Azole Susceptibility in Laboratory Strains of the Wheat Pathogen
Mycosphaerella graminicola
Authors: Zwiers Lute-Harm , Stergiopoulos Ioannis , Van Nistelrooy Johannes G. M. , De Waard Maarten A.
Published: November 2002

Laboratory strains of
Mycosphaerella graminicola
with decreased susceptibilities to the azole antifungal agent cyproconazole showed a multidrug resistance phenotype by exhibiting cross-resistance to an unrelated chemical, cycloheximide or rhodamine 6G, or both. Decreased azole susceptibility was found to be associated with either decreased or increased levels of accumulation of cyproconazole. No specific relationship could be observed between azole susceptibility and the expression of ATP-binding cassette (ABC) transporter genes
MgAtr1
to
MgAtr5
and the sterol P450 14α-demethylase gene,
CYP51
. ABC transporter MgAtr1 was identified as a determinant in azole susceptibility since heterologous expression of the protein reduced the azole susceptibility of
Saccharomyces cerevisiae
and disruption of
MgAtr1
in one specific
M. graminicola
laboratory strain with constitutive
MgAtr1
overexpression restored the level of susceptibility to cyproconazole to wild-type levels. However, the level of accumulation in the mutant with an
MgAtr1
disruption did not revert to the wild-type level. We propose that variations in azole susceptibility in laboratory strains of
M. graminicola
are mediated by multiple mechanisms.
Virtual Inhibitory Quotient Predicts Response to Ritonavir Boosting of Indinavir-Based Therapy in Human Immunodeficiency Virus-Infected Patients with Ongoing Viremia
Authors: Shulman Nancy , Zolopa Andrew , Havlir Diane , Hsu Ann , Renz Cheryl , Boller Sheila , Jiang Ping , Rode Richard , Gallant Joel , Race Elizabeth , Kempf Dale J. , Sun Eugene
Published: November 2002

Depending on the degree of underlying resistance present, optimization of the pharmacokinetics of protease inhibitors may result in improved virologic suppression. Thirty-seven human immunodeficiency virus (HIV)-infected subjects who had chronic detectable viremia and who were receiving 800 mg of indinavir three times a day (TID) were switched to 400 mg of indinavir BID with 400 mg of ritonavir two times a day (BID) for 48 weeks. Full pharmacokinetic evaluations were obtained for 12 subjects before the switch and 3 weeks after the switch. Combination therapy increased the indinavir predose concentrations in plasma by 6.47-fold, increased the minimum concentration in serum by 3.41-fold, and reduced the maximum concentration in serum by 57% without significantly changing the area under the plasma concentration-time curve at 24 h. At week 3, 58% (21 of 36) of the subjects for whom postbaseline measurements were available achieved a viral load in plasma of <50 copies/ml or a reduction from the baseline load of ≥0.5 log10copies/ml. Of these subjects, 82% (14 of 17) whose viruses had three or fewer protease inhibitor mutations and 88% (14 of 16) whose viruses had an indinavir virtual phenotypic susceptibility test of more than sixfold less than that for the baseline isolate were considered virologic responders. The indinavir virtual inhibitory quotient, which is a function of baseline indinavir phenotypic resistance (estimated by virtual phenotype) and the indinavir predose concentration in plasma achieved with indinavir-ritonavir combination therapy, was the best predictor of a viral load reduction. Sixteen subjects discontinued the study by week 48 due to adverse events, predominantly related to hyperlipidemia. Pharmacokinetic intensification of indinavir-based therapy with ritonavir reduced the viral loads in subjects but added toxicity. The virtual inhibitory quotient, which incorporates both baseline viral resistance and the level of drug exposure in plasma, was superior to either baseline resistance or drug exposure alone in predicting the virologic response.
Inactivation of Human Immunodeficiency Virus Type 1 by Porphyrins
Authors: Vzorov Andrei N. , Dixon Dabney W. , Trommel Jenna S. , Marzilli Luigi G. , Compans Richard W.
Published: November 2002

We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-β-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 μg/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-
meso
-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.