bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant Bactericidal/Permeability-Increasing Protein (rBPI21) in Combination with Sulfadiazine Is Active againstToxoplasma gondii
Authors: Khan Anis A. , Lambert Lewis H. , Remington Jack S. , Araujo Fausto G.
Published: October 2018

The activity of recombinant bactericidal/permeability-increasing protein (rBPI21), alone or in combination with sulfadiazine, on the intracellular replication ofToxoplasma gondiiwas assessed in vitro and in mice with acute toxoplasmosis. rBPI21markedly inhibited the intracellular growth ofT. gondiiin human foreskin fibroblasts (HFFs). Following 72 h of exposure, the 50% inhibitory concentration of rBPI21forT. gondiiwas 2.6 μg/ml, whereas only slight cytotoxicity for HFF cells was observed at the concentrations tested. Subsequent mathematical analyses revealed that the combination of rBPI21with sulfadiazine yielded slight to moderate synergistic effects againstT. gondiiin vitro. Infection of mice orally with C56 cysts or intraperitoneally (i.p.) with RH tachyzoites resulted in 100% mortality, whereas prolongation of the time to death or significant survival (P= 0.002) was noted for those animals treated with 5 to 20 mg of rBPI21per kg of body weight per day. Treatment with rBPI21in combination with sulfadiazine resulted in significant (P= 0.0001) survival of mice infected i.p. with tachyzoites but not of mice infected orally withT. gondiicysts. These results indicate that rBPI21is active in vitro and in vivo againstT. gondiiand that its activity is significantly enhanced when it is used in combination with sulfadiazine. To our knowledge, this is the first report of the activity of rBPI21against a protozoan parasite.
Nitrite Reductase from
Pseudomonas aeruginosa
Released by Antimicrobial Agents and Complement Induces Interleukin-8 Production in Bronchial Epithelial Cells
Authors: Sar Borann , Oishi Kazunori , Wada Akihiro , Hirayama Toshiya , Matsushima Kouji , Nagatake Tsuyoshi
Published: October 2018

We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of
Pseudomonas aeruginosa
, could induce interleukin-8 (IL-8) generation in a variety of respiratory cells, including bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65:2648–2655, 1997). In this report, we examined the mode of nitrite reductase (PNR) release from a serum-sensitive strain of live
P. aeruginosa
cells during in vitro treatment with four different antimicrobial agents or human complement. Bacterial killing of
P. aeruginosa
by antimicrobial agents induced PNR release and mediated IL-8 production in human bronchial epithelial (BET-1A) cells. Among these agents, imipenem demonstrated rapid killing of
P. aeruginosa
as well as rapid release of PNR and resulted in the highest IL-8 production. Complement-mediated killing of
P. aeruginosa
was also associated with PNR release and enhanced IL-8 production. The immunoprecipitates of the aliquots of bacterial culture containing imipenem or complement with anti-PNR immunoglobulin G (IgG) induced a twofold-higher IL-8 production than did the immunoprecipitates of the aliquots of bacterial culture with a control IgG. These pieces of evidence confirmed that PNR released in the aliquots of bacterial culture was responsible for IL-8 production in the BET-1A cells. Furthermore, the culture supernatants of the BET-1A cells stimulated with aliquots of bacterial culture containing antimicrobial agents or complement similarly mediated neutrophil migration in vitro. These data support the possibility that a potent inducer of IL-8, PNR, could be released from
P. aeruginosa
after exposure to antimicrobial agents or complement and contributes to neutrophil migration in the airways during bronchopulmonary infections with
P. aeruginosa.
Molecular Characterization of an Antibiotic Resistance Gene Cluster of
Salmonella typhimurium
DT104
Authors: Briggs Connie E. , Fratamico Pina M.
Published: October 2018

Salmonella typhimurium
phage type DT104 has become an important emerging pathogen. Isolates of this phage type often possess resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT resistance). The mechanism by which DT104 has accumulated resistance genes is of interest, since these genes interfere with treatment of DT104 infections and might be horizontally transferred to other bacteria, even to unrelated organisms. Previously, several laboratories have shown that the antibiotic resistance genes of DT104 are chromosomally encoded and involve integrons. The antibiotic resistance genes conferring the ACSSuT-resistant phenotype have been cloned and sequenced. These genes are grouped within two district integrons and intervening plasmid-derived sequences. This sequence is potentially useful for detection of multiresistant DT104.
Structure of In31, a
bla
IMP
-Containing
Pseudomonas aeruginosa
Integron Phyletically Related to In5, Which Carries an Unusual Array of Gene Cassettes
Authors: Laraki Nezha , Galleni Moreno , Thamm Iris , Riccio Maria Letizia , Amicosante Gianfranco , Frère Jean-Marie , Rossolini Gian Maria
Published: October 2018

The location and environment of the acquired
bla
IMP
gene, which encodes the IMP-1 metallo-β-lactamase, were investigated in a Japanese
Pseudomonas aeruginosa
clinical isolate (isolate 101/1477) that produced the enzyme. In this isolate,
bla
IMP
was carried on a 36-kb plasmid, and similar to the identical alleles found in
Serratia marcescens
and
Klebsiella pneumoniae
clinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn
402
-like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the
bla
IMP
cassette is the first of an array of five gene cassettes that also includes an
aacA4
cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the
catB
family, (ii) a
qac
allele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents.
Biochemical Characterization of the
Pseudomonas aeruginosa
101/1477 Metallo-β-Lactamase IMP-1 Produced by
Escherichia coli
Authors: Laraki Nezha , Franceschini Nicola , Rossolini Gian Maria , Santucci Pasqualino , Meunier Cécile , de Pauw Edwin , Amicosante Gianfranco , Frère Jean Marie , Galleni Moreno
Published: October 2018

The
bla
IMP
gene coding for the IMP-1 metallo-β-lactamase produced by a
Pseudomonas aeruginosa
clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in
Escherichia coli
BL21(DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from
E. coli
were identical to those of the enzyme produced by
P. aeruginosa
. The IMP-1 metallo-β-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-
o
-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the
P. aeruginosa
101/1477 metallo-β-lactamase was not pH dependent.
In Vitro Activities of Two Ketolides, HMR 3647 and HMR 3004, against Gram-Positive Bacteria
Authors: Malathum Kumthorn , Coque Teresa M. , Singh Kavindra V. , Murray Barbara E.
Published: October 2018

The in vitro activities of two new ketolides, HMR 3647 and HMR 3004, were tested by the agar dilution method against 280 strains of gram-positive bacteria with different antibiotic susceptibility profiles, including
Staphylococcus aureus
,
Enterococcus faecalis
,
Enterococcus faecium
,
Streptococcus
spp. (group A streptococci, group B streptococci,
Streptococcus pneumoniae
, and alpha-hemolytic streptococci). Seventeen erythromycin-susceptible (Em
s
), methicillin-susceptible
S. aureus
strains were found to have HMR 3647 and HMR 3004 MICs 4- to 16-fold lower than those of erythromycin (MIC at which 50% of isolates were inhibited [MIC
50
] [HMR 3647 and HMR 3004], 0.03 μg/ml; range, 0.03 to 0.06 μg/ml; MIC
50
[erythromycin], 0.25 μg/ml; range, 0.25 to 0.5 μg/ml). All methicillin-resistant
S. aureus
strains tested were resistant to erythromycin and had HMR 3647 and HMR 3004 MICs of >64 μg/ml. The ketolides were slightly more active against
E. faecalis
than against
E. faecium
, and MICs for individual strains varied with erythromycin susceptibility. The MIC
50
s of HMR 3647 and HMR 3004 against Em
s
enterococci (MIC ≤ 0.5 μg/ml) and those enterococcal isolates with erythromycin MICs of 1 to 16 μg/ml were 0.015 μg/ml.
E. faecalis
strains that had erythromycin MICs of 128 to >512 μg/ml showed HMR 3647 MICs in the range of 0.03 to 16 μg/ml and HMR 3004 MICs in the range of 0.03 to 64 μg/ml. In the group of
E. faecium
strains for which MICs of erythromycin were ≥512 μg/ml, MICs of both ketolides were in the range of 1 to 64 μg/ml, with almost all isolates showing ketolide MICs of ≤16 μg/ml. The ketolides were also more active than erythromycin against group A streptococci, group B streptococci,
S. pneumoniae
, rhodococci, leuconostocs, pediococci, lactobacilli, and diphtheroids. Time-kill studies showed bactericidal activity against one strain of
S. aureus
among the four strains tested. The increased activity of ketolides against gram-positive bacteria suggests that further study of these agents for possible efficacy against infections caused by these bacteria is warranted.
Presence of
mefA
and
mefE
Genes in
Streptococcus agalactiae
Authors: Arpin C. , Daube H. , Tessier F. , Quentin C.
Published: October 2018

Eighteen unrelated clinical isolates of
Streptococcus agalactiae
with the M phenotype harbored an
mef
gene. DNA sequencing showed that one of nine strains contained
mefA
(producing one amino acid substitution), whereas the remaining eight carried
mefE
(identity, 100%). Restriction analysis of PCR products indicated that the nine other strains also contained
mefE
.
Alterations in GyrA and ParC Associated with Fluoroquinolone Resistance in
Enterococcus faecium
Authors: el Amin Nagwa , Jalal Shah , Wretlind Bengt
Published: October 2018

High-level quinolone resistance in
Enterococcus faecium
was associated with mutations in both
gyrA
and
parC
genes in 10 of 11 resistant strains. One low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as
Enterococcus faecalis
.
Trovafloxacin in Combination with Vancomycin against Penicillin-Resistant Pneumococci in the Rabbit Meningitis Model
Authors: Rodoni Dina , Hänni Fränzi , Gerber Cynthia M. , Cottagnoud Marianne , Neftel Klaus , Täuber Martin G. , Cottagnoud Philippe
Published: October 2018

Trovafloxacin, a new fluoroquinolone, produced bactericidal activity (−0.33 ± 0.13 Δlog
10
CFU/ml · h; intravenously [i.v.] administered dose, 15 mg/kg) comparable to that of vancomycin (−0.39 ± 0.18 Δlog
10
CFU/ml · h; i.v. administered dose, 20 mg/kg) in the treatment of experimental meningitis in rabbits due to a pneumococcal strain highly resistant to penicillin (MIC of penicillin G, 4 μg/ml). The combination of both drugs significantly increased (
P
 < 0.05) the killing rate (−0.60 ± 0.23 Δlog
10
CFU/ml · h) compared to that produced by either monotherapy. These results were also confirmed in vitro.
Effect of 3-Hydroxyphthaloyl-β-Lactoglobulin on Vaginal Transmission of Simian Immunodeficiency Virus in Rhesus Monkeys
Authors: Wyand Michael S. , Manson Kelledy H. , Miller Christopher J. , Neurath A. Robert
Published: October 2018

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is the major cause of the ongoing AIDS epidemic. Application of chemical barrier methods is expected to contribute to the worldwide control of this epidemic. Bovine β-lactoglobulin modified by 3-hydroxyphthalic anhydride (3-hydroxyphthaloyl-β-lactoglobulin [3HP-β-LG]) was shown to inhibit HIV-1, HIV-2, simian immunodeficiency virus (SIV), herpes simplex virus type 1 and 2, and
Chlamydia trachomatis
infection in vitro. Here, we show that 3HP-β-LG not formulated into any vehicle protected three of six rhesus monkeys against vaginal infection by SIV. Incorporation of the compound into an appropriate vehicle is expected to increase the degree of protection. 3HP-β-LG may be effective as a vaginal inhibitor of HIV-1 infection in humans.
Growth in the Presence of Salicylate Increases Fluoroquinolone Resistance in
Staphylococcus aureus
Authors: Gustafson John E. , Candelaria Pierre V. , Fisher Scott A. , Goodridge Jodie P. , Lichocik Tanya M. , McWilliams Tracy M. , Price Christopher T. D. , O’Brien Frances G. , Grubb Warren B.
Published: October 2018

Salicylate and acetylsalicylate slightly increased fluoroquinolone resistance in ciprofloxacin-susceptible and -resistant
Staphylococcus aureus
. Salicylate allowed a greater number of cells from ciprofloxacin-susceptible and -resistant strains to survive on high fluoroquinolone concentrations. Salicylate also increased the frequency with which a susceptible strain mutated to become more resistant to ciprofloxacin.
Thiolated Recombinant Human Tumor Necrosis Factor-Alpha Protects againstPlasmodium bergheiK173-Induced Experimental Cerebral Malaria in Mice
Authors: Postma Nancy S. , Hermsen Rob C. , Crommelin Daan J. A. , Eling Wijnand M. C. , Zuidema Jan
Published: October 2018

The introduction of reactive thiol groups in recombinant human tumor necrosis factor (TNF) alpha (rhTNF-α) by the reagent succinimidyl-S-acetylthioacetate resulted in the formation of a chemically stabilized rhTNF-α trimer (rhTNFα-AT; as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis). rhTNFα-AT showed a substantially enhanced protective efficacy against the development of experimental murine cerebral malaria (ECM) after intravenous injection compared to the protective efficacy of nonmodified rhTNF-α. Administration of thiolated rhTNF-α with protected thiol groups (rhTNFα-ATA; no stabilized trimers in vitro) exhibited the same protective efficacy against ECM, while in vitro bioactivity was reduced. Parasitemia was significantly suppressed in rhTNF-treated mice that were protected against ECM but not in treated mice that developed ECM. Protection against ECM was not related to increased concentrations in plasma of soluble TNF receptor 1 and 2 directly after injection or at the moment of development of ECM in nontreated mice. The results indicate that thiolation of rhTNF-α leads to the formation of stable trimers with increased potential in vivo.
Distribution of Genes Encoding Resistance to Macrolides, Lincosamides, and Streptogramins among Staphylococci
Authors: Lina Gerard , Quaglia Alain , Reverdy Marie-Elisabeth , Leclercq Roland , Vandenesch François , Etienne Jerome
Published: October 2018

The relative frequency of 10 determinants of resistance to macrolides, lincosamides, and streptogramins was investigated by PCR in a series of 294 macrolide-, lincosamide-, and/or streptogramin-resistant clinical isolates of
Staphylococcus aureus
and coagulase-negative staphylococci isolated in 1995 from 32 French hospitals. Resistance was mainly due to the presence of
ermA
or
ermC
genes, which were detected in 259 strains (88%), in particular those resistant to methicillin (78% of the strains). Macrolide resistance due to
msrA
was more prevalent in coagulase-negative staphylococci (14.6%) than in
S. aureus
(2.1%). Genes related to
linA/linA′
and conferring resistance to lincomycin were detected in one strain of
S. aureus
and seven strains of coagulase-negative staphylococci. Resistance to pristinamycin and quinupristin-dalfopristin was phenotypically detected in 10 strains of
S. aureus
and in three strains of coagulase-negative staphylococci; it was always associated with resistance to type A streptogramins encoded by
vat
or
vatB
genes and occurred in association with
erm
genes. The
vga
gene conferring decreased susceptibility to type A streptogramins was present alone in three strains of coagulase-negative staphylococci and in combination with
erm
genes in 10 strains of coagulase-negative staphylococci. A combination of
vga-vgb-vat
and
ermA
genes was found in a single strain of
S. epidermidis
.
Pharmacokinetics of Gatifloxacin and Interaction with an Antacid Containing Aluminum and Magnesium
Authors: Lober Silke , Ziege Susanne , Rau Margot , Schreiber Gabriele , Mignot Alain , Koeppe Peter , Lode Hartmut
Published: October 2018

The pharmacokinetics of gatifloxacin (400 mg orally) and the influence of the antacid aluminum magnesium hydroxide (20 ml of Maalox 70) on the bioavailability of gatifloxacin in 24 healthy volunteers were assessed. In an open, randomized, six-period crossover study, the volunteers received either gatifloxacin alone (treatments A and D); aluminum magnesium hydroxide concomitant with gatifloxacin (treatment C); or aluminum magnesium hydroxide 2 h before (treatment B), 2 h after (treatment E), or 4 h after gatifloxacin administration (treatment F). Gatifloxacin concentrations were measured by a validated bioassay and high-performance liquid chromatography. Pharmacokinetics of a single 400-mg dose of gatifloxacin alone were characterized as follows (mean ± standard deviation): peak concentration (
C
max
), 3.8 ± 0.5 (treatment A) and 3.4 ± 0.9 (treatment D) μg/ml; time to
C
max
, 1.4 ± 0.8 (treatment A) and 1.7 ± 0.7 (treatment D) h; area under the curve from time zero to infinity (AUC
0–∞
), 33.5 ± 5.9 (treatment A) and 31.4 ± 3.4 (treatment D) μg · h/ml; urine recovery, (83 ± 6)% (treatment A) and (84 ± 8)% (treatment D). Comparison of the results obtained by bioassay showed a good correlation. Aluminum magnesium hydroxide administration 2 h before (treatment B) or concomitant with (treatment C) gatifloxacin decreased the
C
max
by 45% (2.1 ± 1.2 μg/ml) or even 68% (1.2 ± 0.4 μg/ml) highly significantly (
P
< 0.01). AUC
0–∞
was significantly reduced from 33.5 ± 5.9 to 19.4 ± 6.9 μg · h/ml (by 42%) or even to 11.9 ± 3.3 μg · h/ml (by 64%) (
P
< 0.01). If aluminum magnesium hydroxide was given 2 h after gatifloxacin (treatment E), there was no significant reduction of concentration in serum but AUC
0–∞
was significantly reduced from 31.4 ± 3.4 to 25.9 ± 5.3 μg · h/ml (18%) (
P
< 0.01). Aluminum magnesium hydroxide given 4 h after gatifloxacin (treatment F) showed no influence on the gatifloxacin pharmacokinetics. Therefore, the optimal time between gatifloxacin application and the intake of an aluminum-containing antacid should be 4 h.
From
vanA Enterococcus hirae
to
vanA Enterococcus faecium
: a Study of Feed Supplementation with Avoparcin and Tylosin in Young Chickens
Authors: Robredo Beatriz , Singh Kavindra V. , Baquero Fernando , Murray Barbara E. , Torres Carmen
Published: October 2018

Fifteen newborn chickens were isolated in separate cages after 1 month of living together, divided into three groups, and challenged for 5 weeks with seed food which either was supplemented with avoparcin (10 mg/kg of animal food) or tylosin (40 mg/kg) or was nonsupplemented. At 9 weeks of age and after the 5-week challenge, all chickens received nonsupplemented feed for 4 additional weeks. At 4, 9, and 13 weeks of life, feces were collected and inoculated on M-
Enterococcus
agar plates with and without vancomycin (4 μg/ml).
vanA
-containing
Enterococcus hirae
was isolated from 11 of 15 chickens before antibiotic challenge, without detection of vancomycin-resistant
Enterococcus faecium
. At 9 weeks of age and after the 5-week avoparcin challenge,
vanA E. hirae
strains were no longer detected, but five of five chickens now had
vanA E. faecium
. At a lower frequency,
vanA E. faecium
had also displaced
vanA E. hirae
in both the tylosin group (one of four chickens) and the control group (two of five chickens). One month after avoparcin discontinuation, the number of chickens colonized with
vanA E. faecium
decreased from five to one. All
vanA
-containing
E. hirae
strains detected in the first month of life and most of the
vanA
-containing
E. faecium
strains detected in the second month of life showed identical
Apa
I and
Sma
I restriction patterns, respectively, when analyzed by pulsed-field gel electrophoresis. All
vanA E. hirae
isolates transferred glycopeptide and macrolide resistance to
Enterococcus faecalis
JH2-2 in vitro; the level of glycopeptide resistance was higher in the transconjugants than in the donor
E. hirae
strains. These data suggest that
E. hirae
may be a significant source of
vanA
determinants with the potential of transfer to other enterococcal species from humans or animals.
Lack of Effect of Zafirlukast on the Pharmacokinetics of Azithromycin, Clarithromycin, and 14-Hydroxyclarithromycin in Healthy Volunteers
Authors: Garey Kevin W. , Peloquin Charles A. , Godo Paul G. , Nafziger Anne N. , Amsden Guy W.
Published: October 2018

This randomized, open-label, crossover study was conducted to investigate whether the coadministration of zafirlukast would affect the pharmacokinetics of azithromycin, clarithromycin, or 14-hydroxyclarithromycin (14-OHC). Twelve healthy subjects (six males and six females) received single 500-mg doses of azithromycin and clarithromycin with and without zafirlukast given to a steady-state concentration. Blood was collected prior to all macrolide doses and for 3 and 10 days after each clarithromycin and azithromycin dose, respectively. Serum was assayed for azithromycin, clarithromycin, and 14-OHC concentrations by validated high-performance liquid chromatography assay systems. Data analyses were done by noncompartmental and nonparametric methods. Analysis of the patients indicated that the addition of steady-state concentrations of zafirlukast did not significantly alter the pharmacokinetic parameters of or overall exposure (based on the area under the concentration-time curve) to azithromycin, clarithromycin, and 14-OHC. While zafirlukast is a known inhibitor of CYP3A4, it does not appear to exert a clinically or statistically significant pharmacokinetic effect on azithromycin, clarithromycin, or 14-OHC.
In Vitro Activities of the Potent, Broad-Spectrum Carbapenem MK-0826 (L-749,345) against Broad-Spectrum β-Lactamase-and Extended-Spectrum β-Lactamase-Producing
Klebsiella pneumoniae
and
Escherichia coli
Clinical Isolates
Authors: Kohler Joyce , Dorso Karen L. , Young Katherine , Hammond Gail G. , Rosen Hugh , Kropp Helmut , Silver Lynn L.
Published: October 2018

An important mechanism of bacterial resistance to β-lactam antibiotics is inactivation by β-lactam-hydrolyzing enzymes (β-lactamases). The evolution of the extended-spectrum β-lactamases (ESBLs) is associated with extensive use of β-lactam antibiotics, particularly cephalosporins, and is a serious threat to therapeutic efficacy. ESBLs and broad-spectrum β-lactamases (BDSBLs) are plasmid-mediated class A enzymes produced by gram-negative pathogens, principally
Escherichia coli
and
Klebsiella pneumoniae
. MK-0826 was highly potent against all ESBL- and BDSBL-producing
K. pneumoniae
and
E. coli
clinical isolates tested (MIC range, 0.008 to 0.12 μg/ml). In
E. coli
, this activity was associated with high-affinity binding to penicillin-binding proteins 2 and 3. When the inoculum level was increased 10-fold, increasing the amount of β-lactamase present, the MK-0826 MIC range increased to 0.008 to 1 μg/ml. By comparison, similar observations were made with meropenem while imipenem MICs were usually less affected. Not surprisingly, MIC increases with noncarbapenem β-lactams were generally substantially greater, resulting in resistance in many cases.
E. coli
strains that produce chromosomal (Bush group 1) β-lactamase served as controls. All three carbapenems were subject to an inoculum effect with the majority of the BDSBL- and ESBL-producers but not the Bush group 1 strains, implying some effect of the plasmid-borne enzymes on potency. Importantly, MK-0826 MICs remained at or below 1 μg/ml under all test conditions.
Diversity of Substitutions within or Adjacent to Conserved Amino Acid Motifs of Penicillin-Binding Protein 2X in Cephalosporin-Resistant
Streptococcus pneumoniae
Isolates
Authors: Asahi Yasuko , Takeuchi Yasuo , Ubukata Kimiko
Published: October 2018

The sequence of an approximately 1.1-kb DNA fragment of the
pbp2x
gene, which encodes the transpeptidase domain, was determined for 35 clinical isolates of
Streptococcus pneumoniae
for which the cefotaxime (CTX) MICs varied. Strains with substitutions within a conserved amino acid motif changing STMK to SAFK and a Leu-to-Val change just before the KSG motif were highly resistant to CTX (MIC, ≧2 μg/ml). Strains with substitutions adjacent to SSN or KSG motifs had low-level resistance. The amino acid substitutions were plotted on the three-dimensional crystallographic structure of the transpeptidase domain of PBP2X. Transformants containing
pbp2x
from strains with high-level CTX resistance increased the CTX MIC from 0.016 μg/ml to 0.5 to 1.0 μg/ml.
Improved Derivatives of Bactenecin, a Cyclic Dodecameric Antimicrobial Cationic Peptide
Authors: Wu Manhong , Hancock Robert E. W.
Published: October 2018

Both linear and cyclic derivatives of the cyclic 12-amino-acid antimicrobial peptide bactenecin were designed based on optimization of amphipathicity and charge location. In general, increasing the number of positive charges at the N and C termini and adding an extra tryptophan residue in the loop not only increased the activities against both gram-positive and gram-negative bacteria but also broadened the antimicrobial spectrum.
In Vitro Activities of Antibiotics Alone and in Combination against
Brucella melitensis
at Neutral and Acidic pHs
Authors: Akova Murat , Gür Deniz , Livermore David M. , Kocagöz Tanil , Akalin H. Erdal
Published: October 2018

Brucellae survive acidic pHs in phagolysosomes. Azithromycin, streptomycin, and quinolones were active against
Brucella melitensis
at pH 7.0 but not at pH 5.0; rifampin and doxycycline retained activity at pH 5.0. Regardless of pH, azithromycin-rifampin and ofloxacin-rifampin showed less synergy than established streptomycin-doxycycline and rifampin-doxycycline combinations.
The Trailing End Point Phenotype in Antifungal Susceptibility Testing Is pH Dependent
Authors: Marr Kieren A. , Rustad Tige R. , Rex John H. , White Theodore C.
Published: October 2018

The interpretation of end points in azole antifungal drug susceptibility testing is problematic, in part due to incomplete growth inhibition of
Candida
species. Such trailing growth can cause the MICs of fluconazole for some isolates to be low (<1 μg/ml) after 24 h of growth but much higher (>64 μg/ml) after 48 h. Isolates having this type of growth have been described as having a low-high phenotype. Although these isolates would be considered resistant by current National Committee of Clinical Laboratory Standards definitions, growing evidence suggests that they are susceptible in vivo. To further characterize these isolates in vitro, microdilution susceptibility testing comparing the complex defined medium RPMI 1640 to a defined minimal medium (yeast nitrogen broth) was performed. Isolates having trailing growth in MOPS (morpholinepropanesulfonic acid)-buffered RPMI 1640 (pH 7.0) were found to have clear end points in the minimal medium at its native pH of 4.5. The pH of the medium influenced the low-high phenotype, as these same isolates trailed in minimal medium adjusted to a pH of ≥6.0 but did not trail in RPMI 1640 adjusted to a pH of ≤5.0. This pH effect was independent of the medium buffering capacity, as trailing was decreased in both minimal medium and RPMI 1640 (pH 4.5) buffered in citrate. Adjustment in the pH of MOPS-buffered RPMI 1640 reduced trailing in multiple strains of
Candida albicans
without affecting the MICs for isolates having known susceptible (low-low) and resistant (high-high) phenotypes. Adjustment of the medium pH could be considered to eliminate trailing in azole drug susceptibility testing.
Activities of β-Lactams and Macrolides against
Helicobacter pylori
Authors: Hassan Ibrahim J. , Stark Roger M. , Greenman John , Millar Michael R.
Published: October 2018

A continuous-culture system (chemostat) was used to study the activities of β-lactam antimicrobial agents, clarithromycin, and 14-OH-clarithromycin against slowly growing
Helicobacter pylori
NCTC 11637.
H. pylori
was grown to steady state before exposure to these antimicrobial agents at ×8 the MIC. The bactericidal actions of combinations of amoxicillin and clarithromycin were also studied. Viable counts (numbers of CFU per milliliter) were determined at 2-h intervals for 12 h and at 20 h after the addition of antibiotics. The effects of pH changes (6.5 to 7.4) on the activities of amoxicillin, clarithromycin, and the combination of these against
H. pylori
NCTC 11637 were also studied. Viable counts following exposure to ampicillin, cefixime, ceftazidime, cefuroxime, cefotaxime, azlocillin, and piperacillin at 20 h showed bacteriostatic activity. Imipenem, meropenem, amoxicillin, clarithromycin, and 14-OH-clarithromycin reduced the viable counts by 3 log
10
CFU/ml (≥99.9% killing). Imipenem was the most rapidly bactericidal against
H. pylori
NCTC 11637. Results of the pH experiments showed that amoxicillin was bactericidal at pHs 6.5 to 7.4. Clarithromycin was bactericidal at pH 7.0 to 7.4 but was bacteriostatic at pH 6.5. The combination of amoxicillin and clarithromycin was bactericidal at pHs 6.5 and 7.0. A batch culture (flask system) was also used to investigate 12 strains of
H. pylori
for their susceptibilities to β-lactams, clarithromycin, and/or 14-OH-clarithromycin in order to determine whether results from the chemostat model can be reproduced with batch cultures. Results of the chemostat time-kill kinetic study were reproducible in our batch culture flask system. The role of carbapenems in the eradication of
H. pylori
should be investigated.
Use of Microdilution Panels with and without β-Lactamase Inhibitors as a Phenotypic Test for β-Lactamase Production among
Escherichia coli
,
Klebsiella
spp.,
Enterobacter
spp.,
Citrobacter freundii
, and
Serratia marcescens
Authors: Thomson Kenneth S. , Sanders Christine C. , Moland Ellen Smith
Published: October 2018

Over the past decade, a number of new β-lactamases have appeared in clinical isolates of
Enterobacteriaceae
that, unlike their predecessors, do not confer β-lactam resistance that is readily detected in routine antibiotic susceptibility tests. Because optimal methodologies are needed to detect these important new β-lactamases, a study was designed to evaluate the ability of a panel of various β-lactam antibiotics tested alone and in combination with β-lactamase inhibitors to discriminate between the production of extended-spectrum β-lactamases, AmpC β-lactamases, high levels of K1 β-lactamase, and other β-lactamases in 141 isolates of
Escherichia coli
,
Klebsiella pneumoniae
,
Klebsiella oxytoca
,
Enterobacter cloacae
,
Enterobacter aerogenes
,
Citrobacter freundii
, and
Serratia marcescens
possessing well-characterized β-lactamases. The microdilution panels studied contained aztreonam, cefpodoxime, ceftazidime, cefotaxime, and ceftriaxone, with and without 1, 2, and 4 μg of clavulanate per ml or 8 μg of sulbactam per ml and cefoxitin and cefotetan with and without 8 μg of sulbactam per ml. The results indicated that a minimum panel of five tests would provide maximum separation of extended-spectrum β-lactamase high AmpC, high K1, and other β-lactamase production in
Enterobacteriaceae
. These included cefpodoxime, cefpodoxime plus 4 μg of clavulanate per ml, ceftazidime, ceftriaxone, and ceftriaxone plus 8 μg of sulbactam per ml. Ceftriaxone plus 2 μg of clavulanate per ml could be substituted for cefpodoxime plus 4 μg of clavulanate per ml without altering the accuracy of the tests. This study indicated that tests with key β-lactam drugs, alone and in combination with β-lactamase inhibitors, could provide a convenient approach to the detection of a variety of β-lactamases in members of the family
Enterobacteriaceae.
Species Differences in the Proportion of Plasma Lipoprotein Lipid Carried by High-Density Lipoproteins Influence the Distribution of Free and Liposomal Nystatin in Human, Dog, and Rat Plasma
Authors: Ramaswamy Manisha , Wallace Thomas L. , Cossum Paul A. , Wasan Kishor M.
Published: October 2018

The objective of this study was an interspecies comparison of free nystatin (NYS) and liposomal NYS (Nyotran) distribution in plasma. NYS and liposomal NYS at concentrations of 5, 10, and 20 μg of NYS/ml were incubated in human, dog, and rat plasma for 5, 60, and 180 min at 37°C. Following these incubations, plasma samples were separated into their high-density lipoprotein (HDL), triglyceride-rich lipoprotein, low-density lipoprotein, and lipoprotein-deficient plasma (LPDP) fractions by density-gradient ultracentrifugation, and each fraction was assayed for NYS by high-pressure liquid chromatography. Total plasma and lipoprotein cholesterol, triglyceride, and protein concentrations in each human, dog, or rat plasma sample were determined by enzymatic assays. When NYS and liposomal NYS were incubated in human, dog, or rat plasma, the majority of the NYS was recovered in the LPDP fraction. For the 5- and 60-min incubation times for all plasmas measured, a significantly greater percentage of NYS was recovered in the lipoprotein fraction (primarily HDL) following the incubation of liposomal NYS than following the incubation of NYS. There was a significant correlation between the lipoprotein lipid and protein profiles in human, dog, and rat plasmas and the distribution of NYS and liposomal NYS in plasma. In particular, differences in the proportion of plasma lipoprotein cholesterol, triglyceride, and apolar lipids (cholesteryl ester and triglycerides) carried by HDL influenced the distribution of NYS and liposomal NYS within plasmas of different species. These findings suggest that the distribution of NYS among plasma lipoproteins of different species is defined by the proportion of lipid carried by HDL, and this is possibly an important consideration when evaluating the pharmacokinetics, toxicities, and activities of these compounds following administration to different animal species.
Emergence in France of Multiple Clones of Clinical
Streptococcus pneumoniae
Isolates with High-Level Resistance to Amoxicillin
Authors: Doit Catherine , Loukil Chawki , Fitoussi Frederic , Geslin Pierre , Bingen Edouard
Published: October 2018

The genetic relatedness of French isolates of
Streptococcus pneumoniae
highly resistant to amoxicillin (MIC, ≥4 μg/ml, equal to or exceeding those of penicillin) was investigated by molecular fingerprinting. The results suggest that high-level resistance to amoxicillin has emerged within preexisting penicillin-resistant clones.
Rifampin and Rifabutin Resistance Mechanism in
Helicobacter pylori
Authors: Heep Markus , Beck Daniela , Bayerdörffer Ekkehard , Lehn Norbert
Published: October 2018

Eighty-one clinical isolates of
Helicobacter pylori
showed no resistance to rifampin (MIC range, 0.032 to 2 μg/ml; MIC at which 50% of isolates are inhibited [MIC
50
], 0.25 μg/ml). The MIC
50
of rifabutin was 0.008 μg/ml (
n
= 16). All resistant laboratory mutants of
H. pylori
ATCC 43504 showed amino acid exchanges in codons 524 to 545 or codon 585 of the
rpoB
gene, corresponding to the gene sequences from
Mycobacterium tuberculosis
and
Escherichia coli.
Pharmacokinetics and Inflammatory-Fluid Penetration of Moxifloxacin following Oral or Intravenous Administration
Authors: Wise Richard , Andrews Jennifer M. , Marshall Gill , Hartman Giles
Published: October 2018

A single 400-mg oral or intravenous (i.v.) dose of moxifloxacin was given to each of eight healthy male volunteers, and 6 weeks later the dose was administered by the other route. The concentrations of the drug in plasma, cantharidin-induced inflammatory fluid, and urine were measured over the subsequent 24 h. The mean maximum concentrations observed in plasma were 4.98 μg/ml after oral dosing and 5.09 μg/ml after i.v. dosing. The mean maximum concentrations attained in the inflammatory fluid were 2.62 and 3.23 μg/ml, respectively. The mean elimination half-lives from plasma were 8.32 and 8.17 h, respectively. The overall penetration into the inflammatory fluid was 103.4 and 104.2%. Over 24 h 15% of the drug was recovered in the urine when administered by either route.
In Vitro Antifungal Susceptibilities of
Scopulariopsis
Isolates
Authors: Aguilar C. , Pujol I. , Guarro J.
Published: October 2018

MICs and minimum fungicidal concentrations of amphotericin B, miconazole, itraconazole, ketoconazole, fluconazole, and flucytosine against 17 isolates of
Scopulariopsis
spp. were determined by a broth microdilution method. All the isolates were resistant to itraconazole, fluconazole, and flucytosine, and amphotericin B, miconazole, and ketoconazole MICs were low for only a few.
Identification and Analysis of the Balhimycin Biosynthetic Gene Cluster and Its Use for Manipulating Glycopeptide Biosynthesis in
Amycolatopsis mediterranei
DSM5908
Authors: Pelzer S. , Süßmuth R. , Heckmann D. , Recktenwald J. , Huber P. , Jung G. , Wohlleben W.
Published: October 2018

Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer,
Amycolatopsis mediterranei
DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (
oxyA
to -
C
) showed significant sequence similarities to cytochrome P450 monooxygenases and one (
bhaA
) showed similarities to halogenase, and the genes
bgtfA
to -
C
showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene
bgtfB
, produced at least four different compounds which were not glycosylated but still antibiotically active.
DNA Cleavage Activities of
Staphylococcus aureus
Gyrase and Topoisomerase IV Stimulated by Quinolones and 2-Pyridones
Authors: Saiki Anne Y. C. , Shen Linus L. , Chen Chih-Ming , Baranowski John , Lerner Claude G.
Published: October 2018

We have cloned
Staphylococcus aureus
DNA gyrase and topoisomerase IV and expressed them in
Escherichia coli
as polyhistidine-tagged proteins to facilitate purification and eliminate contamination by host enzymes. The enzyme preparations had specific activities similar to previously reported values. Potassium glutamate (K-Glu) stimulated the drug-induced DNA cleavage activity and was optimal between 100 and 200 mM for gyrase and peaked at 100 mM for topoisomerase IV. Higher concentrations of K-Glu inhibited the cleavage activities of both enzymes. Using a common buffer system containing 100 mM K-Glu, we tested the enzyme-mediated DNA cleavage activities of both gyrase and topoisomerase IV with oxolinic acid, norfloxacin, ciprofloxacin, trovafloxacin, clinafloxacin, and the 2-pyridone ABT-719. As expected, all drugs tested demonstrated greater potency against topoisomerase IV than against gyrase. In addition, cleavage activity was found to correlate well with antibacterial activity.
Topoisomerase Sequences of Coagulase-Negative Staphylococcal Isolates Resistant to Ciprofloxacin or Trovafloxacin
Authors: Dubin Donald T. , Fitzgibbon Joseph E. , Nahvi Massoumeh D. , John Joseph F.
Published: October 2018

Coagulase-negative staphylococcal isolates (
n
= 188) were screened for susceptibility to oxacillin, ciprofloxacin, and trovafloxacin, a new fluoroquinolone. At an oxacillin concentration of ≥4 μg/ml, 43% were methicillin resistant; of these, 70% were ciprofloxacin resistant (MIC, ≥4 μg/ml). Of the methicillin-resistant, ciprofloxacin-resistant isolates, 46% were susceptible to ≤2 μg of trovafloxacin per ml and 32% were susceptible to ≤1 μg of trovafloxacin per ml. Sixteen isolates, including twelve that expressed fluoroquinolone resistance, were chosen for detailed analysis. Identification of species by rRNA sequencing revealed a preponderance of
Staphylococcus haemolyticus
and
S. hominis
among fluoroquinolone-resistant strains. Segments of genes (
gyrA
and
grlA
) encoding DNA gyrase and DNA topoisomerase IV were sequenced. Considerable interspecies variation was noted, mainly involving noncoding nucleotide changes. Intraspecies variation consisted of coding changes associated with fluoroquinolone resistance. As for
S. aureus
, ciprofloxacin resistance (MIC, ≥8 μg/ml) and increased trovafloxacin MICs (0.25 to 2 μg/ml) could be conferred by the combined presence of single mutations in each
gyrA
and
grlA
gene. Trovafloxacin MICs of ≥8 μg/ml also occurred, but these required an additional mutation in
grlA
.
Multiple Mechanisms of Action for Inhibitors of Histidine Protein Kinases from Bacterial Two-Component Systems
Authors: Hilliard Jamese J. , Goldschmidt Raul M. , Licata Lisa , Baum Ellen Z. , Bush Karen
Published: October 2018

Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NR
II
-NR
I
, with 50% inhibitory concentrations (IC
50
s) ranging from 1.9 to >500 μM and MICs ranging from 0.5 to >16 μg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4× MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC
50
s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 μg/ml). Fifteen of 24 compounds also caused hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.
Activities of Sordarins in Murine Histoplasmosis
Authors: Graybill John R. , Najvar Laura , Fothergill Annette , Bocanegra Rosie , de las Heras Federico Gomez
Published: October 2018

Sordarins are new antifungals which inhibit fungal protein synthesis by blocking elongation factor 2. Three compounds were evaluated in a murine model of histoplasmosis. Immune-competent mice were infected intravenously with 10
6
to 10
8
CFU of
Histoplasma capsulatum
yeast cells. Mice were treated either orally with sordarins or fluconazole from day 2 through 8 after infection or intraperitoneally with amphotericin B during the same period. Protection was measured by increased rates of survival for 30 days after infection or reduction of lung or kidney tissue counts 9 days after infection. All three of the antifungal drugs tested were protective compared with controls. Sordarins were effective at doses as low as 2 mg/kg of body weight/day. This novel class of drugs compared favorably with amphotericin B and fluconazole for the treatment of histoplasmosis.
Class C β-Lactamases Operate at the Diffusion Limit for Turnover of Their Preferred Cephalosporin Substrates
Authors: Bulychev Alexey , Mobashery Shahriar
Published: October 2018

It has been suggested that class C β-lactamases have evolved to carry out a metabolic reaction other than hydrolysis of β-lactam antibiotics. It is demonstrated in the present study that the class C β-lactamase from
Enterobacter cloacae
P99 has reached the diffusion limit in its ability to hydrolyze its preferred cephalosporin substrates. The increase in the solution viscosity by addition of a microviscogen (sucrose) caused the decline in the parameter
k
cat
/

K
m

for hydrolysis of cephaloridine and cephalosporin C (approximately 2.5-fold at a relative viscosity of 2.9). A similar increase in viscosity has no effect on the turnover rate of the poorer substrates cefepime and penicillin G. Addition of a macroviscogen (polyethylene glycol) to the reaction mixture did not change the rate of turnover for any of the substrates tested because in this case the viscogen would not interfere with the motion of small molecules, as was expected. Therefore, it would appear that the driving force behind the evolution of this class C β-lactamase and, in principle, other enzymes of this class is indeed the functional reaction of this enzyme as a drug resistance factor.
Effect of Fluoroquinolone Concentration on Selection of Resistant Mutants of
Mycobacterium bovis
BCG and
Staphylococcus aureus
Authors: Dong Yuzhi , Zhao Xilin , Domagala John , Drlica Karl
Published: October 2018

When
Mycobacterium bovis
BCG and
Staphylococcus aureus
were plated on agar containing increasing concentrations of fluoroquinolone, colony numbers exhibited a sharp drop, followed by a plateau and a second sharp drop. The plateau region correlated with the presence of first-step resistant mutants. Mutants were not recovered at concentrations above those required for the second sharp drop, thereby defining a mutant prevention concentration (MPC). A C-8-methoxy group lowered the MPC for an
N
-1-cyclopropyl fluoroquinolone.
Sequence of the MIR-1 β-Lactamase Gene
Authors: Jacoby George A. , Tran John
Published: October 2018

The complete nucleotide sequence of the plasmid-mediated MIR-1 β-lactamase gene confirms its relationship to chromosomally located
ampC
genes of
Enterobacter cloacae
.
bla
MIR-1
is not part of a typical gene cassette but does lie near an element that could be involved in its capture on a plasmid.
pncA
Mutations in Pyrazinamide-Resistant
Mycobacterium tuberculosis
Isolates from Northwestern Russia
Authors: Marttila Harri J. , Marjamäki Merja , Vyshnevskaya Elena , Vyshnevskiy Boris I. , Otten Tatjana F. , Vasilyef Alexandr V. , Viljanen Matti K.
Published: October 2018

Thirty-six pyrazinamide-resistant and eight pyrazinamide-susceptible
Mycobacterium tuberculosis
isolates from Russia were analyzed for their
pncA
mutations. Thirty-one (86.1%) of the resistant isolates had a mutation either in
pncA
or upstream of the gene. Twenty of the 23 different mutations found in this study had not been described earlier.
pncA
genotype correlated well with pyrazinamidase activity and BACTEC 460 susceptibility test results.