bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of the Emergence of Ciprofloxacin Resistance in
Streptococcus pneumoniae
by the Multidrug Efflux Inhibitor Reserpine
Authors: Markham Penelope N.
Published: October 2018

Recent evidence supports the contribution of a multidrug efflux mechanism to fluoroquinolone resistance in
Streptococcus pneumoniae
. In this paper I show that reserpine, an inhibitor of multidrug transporters in gram-positive bacteria, dramatically suppresses the in vitro emergence of ciprofloxacin-resistant variants of
S. pneumoniae
, suggesting that the combination of a fluoroquinolone with an inhibitor of multidrug transport may help preserve the efficacy of this class of antibiotics.
Heterogeneity in the
vanB
Gene Cluster of Genomically Diverse Clinical Strains of Vancomycin-Resistant Enterococci
Authors: Dahl Kristin Hegstad , Simonsen Gunnar Skov , Olsvik Ørjan , Sundsfjord Arnfinn
Published: October 2018

Molecular analysis of 17 genomically unrelated clinical VanB-type vancomycin-resistant enterococcus isolates from hospital patients in Germany, Norway, Sweden, the United Kingdom and the United States revealed three subtypes of the
vanB
gene cluster—
vanB1
,
vanB2
, and
vanB3
—which was in accordance with previous subtyping of the ligase gene sequence. There was no correlation between
vanB
subtype and levels of vancomycin resistance. All strains studied carried a structurally conserved
vanB
gene cluster as shown by long-range PCR (long PCR) covering 5,959 bp of the published sequence in
vanB1
strain V583. Restriction analysis of long PCR amplicons displayed one unique
vanB1
pattern and a second
vanB2
- and
vanB3
-specific pattern. The

vanS
B
-vanY
B

intergenic sequences with flanking coding regions were identical within each
vanB
subtype with one exception. A U.S.
vanB2
isolate had a 789-bp enlargement of this region containing a putative open reading frame (ORF) with substantial homology to an ORF in the
Clostridium perfringens
IS
1469
insertion element. The molecular heterogeneity within the
vanB
gene cluster has implications for the selection of PCR primers, as the primers must ensure detection of all
vanB
subtypes, and is of importance when considering reservoirs and dissemination of
vanB
resistance. The molecular identity within the
vanB1
and the
vanB2
subtype indicates horizontal transmission of both gene clusters between isolates in different geographical areas. Restriction analysis of long PCR
vanB
amplicons may reveal specific varieties that can be used as epidemiological markers for mobile determinants conferring VanB-type resistance. The finding of three distinct
vanB
gene clusters should encourage a search for different environmental reservoirs of
vanB
resistance determinants.
In Vitro Antibacterial Activities of Platelet Microbicidal Protein and Neutrophil Defensin against
Staphylococcus aureus
Are Influenced by Antibiotics Differing in Mechanism of Action
Authors: Xiong Yan-Qiong , Yeaman Michael R. , Bayer Arnold S.
Published: October 2018

Thrombin-induced platelet microbicidal protein-1 (tPMP-1) and human neutrophil defensin-1 (HNP-1) are small, cationic antimicrobial peptides. These peptides exert potent in vitro microbicidal activity against a broad spectrum of human pathogens, including
Staphylococcus aureus
. Evidence suggests that tPMP-1 and HNP-1 target and disrupt the bacterial membrane. However, it is not yet clear whether membrane disruption itself is sufficient to kill the bacterium or whether subsequent, presumably intracellular, events are also involved in killing. We investigated the staphylocidal activities of tPMP-1 and HNP-1 in the presence or absence of pretreatment with antibiotics that differ in their mechanisms of action. The staphylocidal effects of tPMP-1 and HNP-1 on control cells (no antibiotic pretreatment) were rapid and concentration dependent. Pretreatment of
S. aureus
with either penicillin or vancomycin (bacterial cell wall synthesis inhibitors) significantly enhanced the anti-
S. aureus
effects of tPMP-1 compared with the effects against the respective control cells over the entire tPMP-1 concentration range tested (
P
< 0.05). Similarly,
S. aureus
cells pretreated with these antibiotics were more susceptible to HNP-1 than control cells, although the difference in the effects against cells that received penicillin pretreatment did not reach statistical significance (
P
< 0.05 for cells that received vancomycin pretreatment versus effects against control cells). Studies with isogenic pairs of strains with normal or deficient autolytic enzyme activities demonstrated that enhancement of
S. aureus
killing by cationic peptides and cell wall-active agents could not be ascribed to a predominant role of autolytic enzyme activation. Pretreatment of
S. aureus
cells with tetracycline, a 30S ribosomal subunit inhibitor, significantly decreased the staphylocidal effect of tPMP-1 over a wide peptide concentration range (0.16 to 1.25 μg/ml) (
P
< 0.05). Furthermore, pretreatment with novobiocin (an inhibitor of bacterial DNA gyrase subunit B) and with azithromycin, quinupristin, or dalfopristin (50S ribosomal subunit protein synthesis inhibitors) essentially blocked the
S. aureus
killing resulting from exposure to tPMP-1 or HNP-1 at most concentrations compared with the effects against the respective control cells (
P
< 0.05 for a tPMP-1 concentration range of 0.31 to 1.25 μg/ml and for an HNP-1 concentration range of 6.25 to 50 μg/ml). These findings suggest that tPMP-1 and HNP-1 exert anti-
S. aureus
activities through mechanisms involving both the cell membrane and intracellular targets.
Pharmacodynamics of Trovafloxacin, Ofloxacin, and Ciprofloxacin against
Streptococcus pneumoniae
in an In Vitro Pharmacokinetic Model
Authors: Lister Philip D. , Sanders Christine C.
Published: October 2018

An in vitro pharmacokinetic model was used to simulate the pharmacokinetics of trovafloxacin, ofloxacin, and ciprofloxacin in human serum and to compare their pharmacodynamics against eight
Streptococcus pneumoniae
strains. The MICs of ofloxacin and ciprofloxacin ranged from 1 to 2 μg/ml. Trovafloxacin was 8- to 32-fold more potent, with MICs of 0.06 to 0.12 μg/ml. Logarithmic-phase cultures were exposed to peak concentrations of trovafloxacin, ofloxacin, or ciprofloxacin achieved in human serum after 200-, 400-, and 750-mg oral doses, respectively. Trovafloxacin was dosed at 0 and 24 h, and ofloxacin and ciprofloxacin were dosed at 0, 12, and 24 h. Human elimination pharmacokinetics were simulated, and viable bacterial counts were measured at 0, 2, 4, 6, 8, 12, 24, and 36 h. Trovafloxacin was rapidly and significantly bactericidal against all eight strains evaluated, with viable bacterial counts decreasing at least 5 logs to undetectable levels. Times to 99.9% killing were only 1 to 3 h. Although the rate of killing with ofloxacin was substantially slower than that with trovafloxacin, ofloxacin was also able to eradicate all eight strains from the model, despite a simulated area under the inhibitory curve/MIC ratio (AUC/MIC) of only 49. In contrast, ciprofloxacin eradicated only five strains (AUC/MIC = 44) from the model. Against the other three strains (AUC/MIC = 22), the antibacterial activity of ciprofloxacin was substantially diminished. These data corroborate clinical data and suggest that trovafloxacin has a pharmacodynamic advantage over ciprofloxacin and ofloxacin against
S. pneumoniae
in relation to its enhanced antipneumococcal activity.
BOCILLIN FL, a Sensitive and Commercially Available Reagent for Detection of Penicillin-Binding Proteins
Authors: Zhao Genshi , Meier Timothy I. , Kahl Steven D. , Gee Kyle R. , Blaszczak Larry C.
Published: October 2018

We describe a new, sensitive, rapid, and nonradioactive method involving the use of the commercially available BOCILLIN FL, a fluorescent penicillin, as a labeling reagent for the detection and study of penicillin-binding proteins (PBPs). This method allowed rapid detection of 30 ng of a purified PBP protein under UV light and of 2 to 4 ng of the protein with the aid of a FluorImager. This method also allowed rapid determination of the PBP profiles of
Escherichia coli
,
Pseudomonas aeruginosa
, and
Streptococcus pneumoniae
. The PBP profiles obtained are virtually identical to those reported previously with
3
H-,
14
C-, or
125
I-labeled penicillin. Using this method enabled us to determine the 50% inhibitory concentrations of the penicillin-sensitive and -resistant PBP2x proteins of
S. pneumoniae
for penicillin G, thereby allowing a direct evaluation of their relative affinities for penicillin G. Finally, this method also allowed us to compare relative affinities of a PBP2x protein for different β-lactam antibiotics with the aid of fluorescence polarization technology and to monitor a PBP2x protein during purification.
Sequence Analysis of the
gyrA
and
parC
Homologues of a Wild-Type Strain of
Vibrio parahaemolyticus
and Its Fluoroquinolone-Resistant Mutants
Authors: Okuda Jun , Hayakawa Eriko , Nishibuchi Mitsuaki , Nishino Takeshi
Published: October 2018

Vibrio parahaemolyticus
causes seafood-borne gastroenteritis in humans. It is particularly important in Japan, where raw seafood is frequently consumed. Fluoroquinolone is one of the current drugs of choice for treating patients infected by
V. parahaemolyticus
because resistant strains are rarely found. To study a possible fluoroquinolone resistance mechanism in this organism, nucleotide sequences that are homologous to known
gyrA
and
parC
genes have been cloned from
V. parahaemolyticus
AQ3815 and sequenced by amplification with degenerate primers of the quinolone resistance-determining region (QRDR), followed by cassette ligation-mediated PCR. Open reading frames encoding polypeptides of 878 and 761 amino acid residues were detected in the
gyrA
and
parC
homologues, respectively. The
V. parahaemolyticus
GyrA and ParC sequences were most closely related to
Erwinia carotovora
GyrA (76% identity) and
Escherichia coli
ParC (69% identity) sequences, respectively. Ciprofloxacin-resistant mutants of AQ3815 were obtained on an agar medium by multistep selection with increasing levels of the quinolone. One point mutation only in the
gyrA
QRDR was detected among mutants with low- to intermediate-level resistance, while point mutations in both the
gyrA
and
parC
QRDRs were detected only in strains with high-level resistance. These results strongly suggest that, as in other gram-negative bacteria, GyrA and ParC are the primary and secondary targets, respectively, of ciprofloxacin in
V. parahaemolyticus
.
In Vitro Development of Resistance to Five Quinolones and Amoxicillin-Clavulanate in
Streptococcus pneumoniae
Authors: Davies Todd A. , Pankuch Glenn A. , Dewasse Bonifacio E. , Jacobs Michael R. , Appelbaum Peter C.
Published: October 2018

The ability of 50 sequential subcultures in subinhibitory concentrations of ciprofloxacin, levofloxacin, grepafloxacin, sparfloxacin, trovafloxacin, and amoxicillin-clavulanate to select for resistance was studied for six penicillin-susceptible and four penicillin-intermediate pneumococci. Subculturing in ciprofloxacin, grepafloxacin, levofloxacin, and sparfloxacin led to selection of mutants requiring increased MICs for all 10 strains, with MICs rising from (i) 0.5 to 4.0 to (ii) 4.0 to 32.0 μg/ml after 7 to 12 passages for ciprofloxacin, from (i) 0.06 to 0.25 to (ii) 0.5 to 8.0 μg/ml after 5 to 23 passages for grepafloxacin, from (i) 0.5 to 1.0 to (ii) 4.0 to 64 μg/ml after 14 to 49 passages for levofloxacin, and from (i) 0.125 to 0.25 to (ii) 1.0 to 16.0 μg/ml after 8 to 26 passages for sparfloxacin. Subculturing in trovafloxacin led to increased MICs for eight strains, with MICs rising from (i) 0.06 to 0.125 to (ii) 0.5 to 8.0 μg/ml after 6 to 28 passages. Subculturing in amoxicillin-clavulanate led to raised MICs for only one strain, with the MIC rising from 0.015 to 0.125 μg/ml after 24 passages. Double mutations in both ParC and GyrA led to high-level quinolone resistance when ParC mutations were at S79. Trovafloxacin MICs were 1 to 2 μg/ml in double mutants with ParC mutations at positions other than S79 (e.g., D83). Mutations in ParE (at D435, R447, and E474) and GyrB (at S405, D406, and D435) were found in four and six mutants, respectively. In the presence of reserpine, 29 mutants had lower ciprofloxacin MICs (2 to 16 times lower), 8 mutants had lower levofloxacin MICs (2 times), and one mutant had a lower trovafloxacin MIC (2 times), suggesting the involvement of an efflux mechanism. In contrast to the case for quinolones, subculturing in the presence of amoxicillin-clavulanate did not select for resistance to this drug.
Pharmacokinetics of Rifapentine in Subjects Seropositive for the Human Immunodeficiency Virus: a Phase I Study
Authors: Keung Anther C.-F. , Owens Robert C. , Eller Mark G. , Weir Scott J. , Nicolau David P. , Nightingale Charles H.
Published: October 2018

Rifapentine is undergoing development for the treatment of pulmonary tuberculosis. This study was conducted to characterize the single-dose pharmacokinetics of rifapentine and its 25-desacetyl metabolite and to assess the effect of food on the rate and extent of absorption in participants infected with human immunodeficiency virus (HIV). Twelve men and four women, mean age, 38.6 ± 6.9 years, received a single 600-mg oral dose of rifapentine in an open-label, randomized two-way, complete crossover study. Each volunteer received rifapentine following a high-fat breakfast or during a fasting period. Serial blood samples were collected for 72 h and both rifapentine and its metabolite were assayed by a validated high-performance liquid chromatography method. Pharmacokinetics of rifapentine and 25-desacetylrifapentine were determined by noncompartmental methods. Mean (± the standard deviation) maximum concentrations of rifapentine in serum and areas under the curve from time zero to infinity following a high-fat breakfast were 14.09 ± 2.81 and 373.63 ± 78.19 μg/ml, respectively, and following a fasting period they were 9.42 ± 2.67 and 256.10 ± 86.39 μg · h/ml, respectively. Pharmacokinetic data from a previously published healthy volunteer study were used for comparison. Administration of rifapentine with a high-fat breakfast resulted in a 51% increase in rifapentine bioavailability, an effect also observed in healthy volunteers. Although food increased the exposure of these patients to rifapentine, the infrequent dosing schedule for the treatment of tuberculosis (e.g., once- or twice-weekly dosing) would be unlikely to lead to accumulation. Additionally, autoinduction has been previously studied and has not been demonstrated with this compound, unlike with rifabutin and rifampin. Rifapentine was well tolerated by HIV-infected study participants. The results of our study suggest that no dosage adjustments may be required for rifapentine in HIV-infected patients (Centers for Disease Control and Prevention classification A1, A2, B1, or B2) undergoing treatment for tuberculosis.
β-Lactamase Production and Antimicrobial Susceptibility of Oral Heterogeneous
Fusobacterium nucleatum
Populations in Young Children
Authors: Könönen E. , Kanervo A. , Salminen K. , Jousimies-Somer H.
Published: October 2018

Oral
Fusobacterium nucleatum
populations from 20 young, healthy children were examined for β-lactamase production. Ten children (50%) harbored, altogether, 25 β-lactamase-positive
F. nucleatum
isolates that were identified as
F. nucleatum
subsp.
polymorphum
,
F. nucleatum
subsp.
nucleatum
, and
F. nucleatum
subsp.
vincentii
(J. L. Dzink, M. T. Sheenan, and S. S. Socransky, Int. J. Syst. Bacteriol. 40:74–78, 1990). In vitro susceptibility of these β-lactamase-producing and 26 non-β-lactamase-producing
F. nucleatum
isolates was tested with penicillin G, amoxicillin-clavulanic acid, tetracycline hydrochloride, metronidazole, trovafloxacin, and azithromycin. Except for penicillin G, the antimicrobials exhibited good activity against all
F. nucleatum
isolates.
Pharmacokinetics of a Clarithromycin Suspension Administered via Nasogastric Tube to Seriously Ill Patients
Authors: Fish Douglas N. , Abraham Edward
Published: October 2018

The pharmacokinetics of clarithromycin and its 14-(
R
)-hydroxylated metabolite were studied on two separate occasions after nasogastric administration of 500 mg of a clarithromycin suspension to 16 seriously ill adults in an intensive care unit. The clarithromycin suspension appeared to be adequately absorbed, and the pharmacokinetics of neither clarithromycin nor 14-(
R
)-hydroxyclarithromycin differed significantly between the two dosing periods. No substantial differences in pharmacokinetics were observed compared to previously published studies of other adult populations. Minimal intrapatient variability of pharmacokinetic parameters was observed in these seriously ill patients.
Correlation between In Vitro Susceptibility Determined by E Test and Response to Therapy with Amphotericin B: Results from a Multicenter Prospective Study of Candidemia
Authors: Clancy Cornelius J. , Nguyen M. Hong
Published: October 2018

Ninety-nine
Candida
bloodstream isolates underwent testing for susceptibility to amphotericin B by the E test, and the results were correlated with patients’ responses to amphotericin B. The MICs for isolates that were associated with therapeutic failure were significantly higher than the MICs for those associated with therapeutic success. A MIC of ≥0.38 μg/ml identified isolates likely to be associated with therapeutic failure.
Distribution and Antimicrobial Activity of Ciprofloxacin in Human Soft Tissues
Authors: Brunner Martin , Hollenstein Ursula , Delacher Simon , Jäger Dorothea , Schmid Rainer , Lackner Edith , Georgopoulos Apostoulos , Eichler Hans Georg , Müller Markus
Published: October 2018

Interstitial ciprofloxacin concentrations in soft tissues were measured by microdialysis following intravenous administration of 200 mg to each of eight healthy volunteers. Interstitial ciprofloxacin concentrations were significantly lower than corresponding total serum drug concentrations; the interstitium-to-serum concentration ratios ranged from 0.55 to 0.73. An in vitro simulation based on interstitial pharmacokinetics showed a substantially lower antimicrobial activity than did the simulation based on serum pharmacokinetics. Thus, ciprofloxacin concentrations at the site of effect may be subinhibitory although effective concentrations are attained in serum.
Intrapulmonary Concentrations of Pyrazinamide
Authors: Conte John E. , Golden Jeffrey A. , Duncan Sheila , McKenna Elaine , Zurlinden Elisabeth
Published: October 2018

The objective of this study was to compare the steady-state plasma and intrapulmonary concentrations of orally administered pyrazinamide in normal volunteers and subjects with AIDS. Pyrazinamide was administered at 1 g once daily for 5 days to 40 adult volunteers (10 men with AIDS, 10 normal men, 10 women with AIDS, and 10 normal women). Subjects with AIDS and with more than four stools per day were excluded. Blood was obtained prior to administration of the first dose, 2 h after the last dose, and at the completion of bronchoscopy and bronchoalveolar lavage, which were performed 4 h after the last dose. Standardized bronchoscopy was performed without systemic sedation. The volume of epithelial lining fluid (ELF) recovered was calculated by the urea dilution method. The total number of alveolar cells (AC) was counted in a hemocytometer, and differential cell counting was performed after cytocentrifugation. Pyrazinamide was measured by high-performance liquid chromatography. The presence of AIDS or gender had no significant effect on the concentrations of pyrazinamide in plasma. The concentrations of pyrazinamide in ELF and AC were lower in the subjects with AIDS than in the subjects without AIDS, but the difference was not significant. The concentrations in plasma (mean ± standard deviation) were 25.1 ± 7.6 and 21.1 ± 6.8 μg/ml at 2 and 4 h after the last dose, respectively, and were not significantly different from the concentration (17.4 ± 16.9 μg/ml) in AC. The concentration of pyrazinamide in ELF was high (431 ± 220 μg/ml) and was approximately 4 to 40 times the reported MIC for pyrazinamide-susceptible strains ofMycobacterium tuberculosis. The high concentration of pyrazinamide in ELF may contribute in part to the effectiveness of the drug in treating pulmonary tuberculosis.
ACTG 260: a Randomized, Phase I-II, Dose-Ranging Trial of the Anti-Human Immunodeficiency Virus Activity of Delavirdine Monotherapy
Authors: Para Michael F. , Meehan Patricia , Holden-Wiltse Jeanne , Fischl Margaret , Morse Gene , Shafer Robert , Demeter Lisa M. , Wood Kenneth , Nevin Tom , Virani-Ketter Nzeera , Freimuth William W.
Published: October 2018

ACTG 260 was an open-label, four-arm trial designed to study the safety and anti-human immunodeficiency virus (anti-HIV) activity of delavirdine monotherapy at three ranges of concentrations in plasma compared to those of control therapy with zidovudine or didanosine. Delavirdine doses were adjusted weekly until subjects were within their target trough concentration range (3 to 10, 11 to 30, or 31 to 50 μM). A total of 113 subjects were analyzed. At week 2, the mean HIV type 1 (HIV-1) RNA level declines among the subjects in the three delavirdine arms were similar (0.87, 1.08, and 1.02 log
10
for the low, middle, and high target arms, respectively), but by week 8, the subjects in the pooled delavirdine arms showed only a 0.10 log
10
reduction. In the subjects in the nucleoside arm, mean HIV-1 RNA level reductions at weeks 2 and 8 were 0.67 and 0.55 log
10
, respectively. Because viral suppression by delavirdine was not maintained, the trial was stopped early. Rash, which was usually self-limited, developed in 36% of subjects who received delavirdine. Delavirdine monotherapy has potent anti-HIV activity at 2 weeks, but its activity is time limited due to the rapid emergence of drug resistance.
Activity of Gatifloxacin Compared to Those of Five Other Quinolones versus Aerobic and Anaerobic Isolates from Skin and Soft Tissue Samples of Human and Animal Bite Wound Infections
Authors: Goldstein Ellie J. C. , Citron Diane M. , Merriam C. Vreni , TyRrell Kerin , Warren Yumi
Published: October 2018

The activity of gatifloxacin against 308 aerobes and 112 anaerobes isolated from bite wound infections was studied. Gatifloxacin was active at ≤0.016 μg/ml against all 148
Pasteurella
isolates (eight species and three subspecies) tested and all other aerobes tested, including
Actinobacillus-Haemophilus
spp.,
Eikenella corrodens
,
Neisseria weaveri
,
Weeksella zoohelcum
, staphylococci, and streptococci. Fusobacteria were sometimes resistant. Gatifloxacin MICs at which 90% of the isolates were inhibited were 0.125 μg/ml against
Bacteroides tectum
and
Prevotella
spp., 0.25 μg/ml against
Porphyromonas
spp., and 0.5 μg/ml against peptostreptococci.
Pharmacokinetics of Azithromycin Administered Alone and with Atovaquone in Human Immunodeficiency Virus-Infected Children
Authors: Ngo Leock Y. , Yogev Ram , Dankner Wayne M. , Hughes Walter T. , Burchett Sandra , Xu Jing , Sadler Brian , Unadkat Jashvant D.
Published: October 2018

To evaluate if atovaquone (ATQ) interacts pharmacokinetically with azithromycin (AZ) in human immunodeficiency virus-infected children, 10 subjects (ages, 4 to 13 years) were randomized in a crossover study to receive AZ (5 mg/kg/day) alone (ALONE) or AZ (5 mg/kg/day) and ATQ (30 mg/kg/day) simultaneously (SIM) prior to receiving AZ and ATQ staggered by 12 h. Despite a lack of significant difference in the mean AZ pharmacokinetic parameters, the steady-state values of AZ’s area under the concentration-time curve from 0 to 24 h and maximum concentration in serum were consistently lower (n= 7 of 7) for the SIM regimen than they were for the ALONE regimen. A larger study will be required to determine if ATQ affects AZ pharmacokinetics and efficacy in a clinically significant manner.
In Vitro Activities of Pyronaridine, Alone and in Combination with Other Antimalarial Drugs, against
Plasmodium falciparum
Authors: Ringwald Pascal , Eboumbou Else Carole Moukoko , Bickii Jean , Basco Leonardo K.
Published: October 2018

The in vitro activities of pyronaridine, alone and in combination with established antimalarial drugs, were assessed by isotopic microtest. Pyronaridine was highly active against all Cameroonian isolates. A positive correlation was observed between the response to pyronaridine and that to chloroquine. Drug combination studies showed synergy between pyronaridine and primaquine, additive effects with 4-aminoquinolines, and weak antagonism with dihydroartemisinin, antifolates, or amino alcohols.
Prospective Evaluation of the Effect of an Aminoglycoside Dosing Regimen on Rates of Observed Nephrotoxicity and Ototoxicity
Authors: Rybak Michael J. , Abate Betty J. , Kang S. Lena , Ruffing Michael J. , Lerner Stephen A. , Drusano George L.
Published: October 2018

The nephrotoxicity and ototoxicity associated with once-daily versus twice-daily administration of aminoglycosides was assessed in patients with suspected or proven gram-negative bacterial infections in a randomized, double-blind clinical trial. Patients who received therapy for ≥72 h were evaluated for toxicity. Patients also received concomitant antibiotics as deemed necessary for treatment of their infection. Plasma aminoglycoside concentrations, prospective aminoglycoside dosage adjustment, and serial audiologic and renal status evaluations were performed. The probability of occurrence of a nephrotoxic event and its relationship to doses and daily aminoglycoside exposure served as the main outcome measurement. One hundred twenty-three patients were enrolled in the study, with 83 patients receiving therapy for at least 72 h. For 74 patients plasma aminoglycoside concentrations were available for analysis, and the patients formed the group evaluable for toxicity. The primary infectious diagnosis for the patients who were enrolled in the study were bacteremia or sepsis, respiratory infections, skin and soft tissue infections, or urosepsis or pyelonephritis. Of the 74 patients evaluable for toxicity, 39 received doses twice daily and 35 received doses once daily and a placebo 12 h later. Nephrotoxicity occurred in 6 of 39 (15.4%) patients who received aminoglycosides twice daily and 0 of 35 patients who received aminoglycosides once daily. The schedule of aminoglycoside administration, concomitant use of vancomycin, and daily area under the plasma concentration-time curve (AUC) for the aminoglycosides were found to be significant predictors of nephrotoxicity by multivariate logistic regression analysis (
P
≤ 0.001). The time to a nephrotoxic event was significantly influenced by vancomycin use and the schedule of administration, as assessed by Cox proportional hazards modeling (
P
≤ 0.002). The results of the multivariate logistic regression analysis and the Cox proportional hazards modeling demonstrate that both the probability of occurrence and the time to occurrence of aminoglycoside nephrotoxicity are influenced by the schedule on which the aminoglycoside is administered as well as by the concomitant use of vancomycin. Furthermore, this risk of occurrence is modulated by the daily AUC for aminoglycoside exposure. These data suggest that once-daily administration of aminoglycosides has a predictably lower probability of causing nephrotoxicity than twice-daily administration.
Roles of β-Lactamases and Porins in Activities of Carbapenems and Cephalosporins against
Klebsiella pneumoniae
Authors: Martínez-Martínez Luis , Pascual Alvaro , Hernández-Allés Santiago , Alvarez-Díaz Dolores , Suárez Ana Isabel , Tran John , Benedí Vicente Javier , Jacoby George A.
Published: October 2018

Two clinical isolates of extended-spectrum β-lactamase (ESBL)-producing
Klebsiella pneumoniae
were noted to be less susceptible than expected to imipenem. Both were missing outer membrane proteins that serve as channels for antibiotic entry. The role of β-lactamase in resistance was investigated by eliminating the original ESBL and introducing plasmids encoding various ESBLs and AmpC β-lactamase types, by studying the effect of an increased inoculum, and by evaluating interactions with β-lactamase inhibitors. The contribution of porin deficiency was investigated by restoring a functional
ompK36
gene on a plasmid. Plasmids encoding AmpC-type β-lactamases provided resistance to imipenem (up to 64 μg/ml) and meropenem (up to 16 μg/ml) in strains deficient in porins. Carbapenem resistance showed little inoculum effect, was not affected by clavulanate but was blocked by BRL 42715, and was diminished if OmpK36 porin was restored. Plasmids encoding TEM- and SHV-type ESBLs conferred resistance to cefepime and cefpirome, as well as to earlier oxyimino-β-lactams. This resistance was magnified with an increased inoculum, was blocked by clavulanate, and was also lowered by OmpK36 porin restoration. In addition, SHV-2 β-lactamase had a small effect on carbapenem resistance (imipenem MIC, 4 μg/ml, increasing to 16 μg/ml with a higher inoculum) when porins were absent. In
K. pneumoniae
porin loss can thus augment resistance provided either by TEM- or SHV-type ESBLs or by plasmid-mediated AmpC enzymes to include the latest oxyimino-β-lactams and carbapenems.
Reversal of Tetracycline Resistance Mediated by Different Bacterial Tetracycline Resistance Determinants by an Inhibitor of the Tet(B) Antiport Protein
Authors: Nelson Mark L. , Levy Stuart B.
Published: October 2018

Active efflux is a useful strategy by which bacteria evade growth inhibition by antibiotics. Certain semisynthetic tetracycline (TC) analogs, substituted at the 13th carbon at C-6 on ring C of the TC molecule, blocked TC efflux as revealed in everted membrane vesicles from class B TC-resistant (Tc
r
)
Escherichia coli
(M. L. Nelson, B. H. Park, J. S. Andrews, V. A. Georgian, R. C. Thomas, and S. B. Levy, J. Med. Chem. 36:370–377, 1993). A representative C-13-substituted analog, 13-cyclopentylthio-5-OH-TC (13-CPTC), was shown to competitively inhibit TC translocation by the Tet(B) protein, blocking the uptake of TC into vesicles and therefore the efflux of TC from whole cells. Against Tc
r
E. coli
, 13-CPTC, when used in combination with doxycycline, produced synergistic inhibition of growth. 13-CPTC was shown to increase the uptake of [
3
H]TC into the resistant cells. 13-CPTC alone was a potent growth inhibitor against TC-susceptible (Tc
s
) and Tc
r
Staphylococcus aureus
and enterococci specifying class K or class L efflux-dependent TC resistance mechanisms or, unexpectedly, the class M ribosomal protection mechanism. These findings indicate that derivatives of TC, identified by their ability to block the Tet(B) efflux protein, can restore TC activity against Tc
r
bacteria bearing either of the two known resistance mechanisms. Blocking drug efflux and increasing intracellular drug concentrations constitute an effective approach to reversing TC resistance and may be generally applicable to other antibiotics rendered ineffective by efflux proteins.
Mefloquine Is Active In Vitro and In Vivo againstMycobacterium aviumComplex
Authors: Bermudez Luiz E. , Kolonoski Peter , Wu Martin , Aralar Priscilla A. , Inderlied Clark B. , Young Lowell S.
Published: October 2018

Despite the development of several agents, new classes of antimicrobials with activity against theMycobacterium aviumcomplex (MAC) are needed. Based on a broad screening of compounds, we found that mefloquine has MICs of 8 to 16 μg/ml by the BACTEC system and 16 μg/ml by broth microdilution for five MAC strains tested. An expansion of the screening with broth microdilution to 24 macrolide-susceptible strains and 6 macrolide-resistant strains determined that the MIC for all strains was 16 μg/ml. To determine the intracellular activity of mefloquine, U937 macrophage monolayers infected with MAC strain 101, 100, or 109 (serovars 1, 8, and 4) were treated with mefloquine daily, and the number of intracellular bacteria was quantitated after 4 days. Significant growth inhibition against the three MAC strains at concentrations greater than or equal to 10 μg/ml (P< 0.05) was obtained. Due to the encouraging anti-MAC activity, in vivo efficacy in beige mice infected with MAC 101 was evaluated. Animals were treated with 5, 10, 20, or 40 mg/kg of body weight daily, three times a week, twice a week, or once a week for 4 weeks, and bacteria were quantitated in blood, liver, and spleen. No toxicity was observed with any of the treatment regimens. Mefloquine had borderline bactericidal activity at a dosage of 40 mg/kg daily (100% inhibition compared with a 1-week control), and significant inhibition was obtained at dosages of 40 mg/kg three times a week, as well as 20 mg/kg daily. Mefloquine had no significant effect on bacteremia. A combination of mefloquine and ethambutol showed significantly more activity than did either drug alone in liver, spleen, and blood; the combination was also bactericidal againstM. avium. Although safety is a potential concern, mefloquine and related compounds deserve further investigation as anti-MAC therapies.
In Vitro and In Vivo Activities of Syn2190, a Novel β-Lactamase Inhibitor
Authors: Nishida Kouichi , Kunugita Chieko , Uji Tatsuya , Higashitani Fusahiro , Hyodo Akio , Unemi Norio , Maiti Samarendra N. , Phillips Oludotun A. , Spevak Paul , Atchison Kevin P. , Salama Sameeh M. , Atwal Harninder , Micetich Ronald G.
Published: October 2018

Syn2190, a monobactam derivative containing 1,5-dihydroxy-4-pyridone as the C-3 side chain, is a potent inhibitor of group 1 β-lactamase. The concentrations of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% for Syn2190 against these enzymes were in the range of 0.002 to 0.01 μM. These values were 220- to 850-fold lower than those of tazobactam. Syn2190 showed in vitro synergy with ceftazidime and cefpirome. This synergy was dependent on the concentration of the inhibitor against group 1 β-lactamase-producing strains, such as
Pseudomonas aeruginosa
,
Enterobacter cloacae
,
Citrobacter freundii
, and
Morganella morganii
. However, against β-lactamase-derepressed mutants of
P. aeruginosa
, the MICs of ceftazidime plus Syn2190 were not affected by the amount of β-lactamase, and the values were the same for the parent strains. The MICs at which 50% of isolates are inhibited (MIC
50
s) of ceftazidime plus Syn2190 were 2- to 16-fold lower than those of ceftazidime alone for ceftazidime-resistant, clinically isolated gram-negative bacteria. Similarly, the MIC
50
s of cefpirome plus Syn2190 were two- to eightfold lower for cefpirome-resistant clinical isolates. The synergies of Syn2190 plus ceftazidime or cefpirome observed in vitro were also reflected in vivo. Syn2190 improved the efficacies of both cephalosporins in both a murine systemic infection model with cephalosporin-resistant rods and urinary tract infection models with cephalosporin-resistant
P. aeruginosa
.
Unusual Antimicrotubule Activity of the Antifungal Agent Spongistatin 1
Authors: Ovechkina Yulia Y. , Pettit Robin K. , Cichacz Zbigniew A. , Pettit George R. , Oakley Berl R.
Published: October 2018

Spongistatin 1, a macrocyclic lactone from the marine sponge
Hyrtios erecta
, has broad-spectrum antifungal activity. Since this compound is a potent antimicrotubule agent in mammalian cells, we examined its effects on the filamentous fungus
Aspergillus nidulans
to determine if its antifungal effects are due to antimicrotubule activity. At 25 μg/ml (twice the MIC), spongistatin 1 caused a greater-than-twofold elevation of the chromosome and spindle mitotic indices. Immunofluorescence microscopy revealed that mitotic spindles were smaller and shorter than in control germlings. However, late-anaphase and telophase nuclei were seen occasionally, and this suggests that the spindles are capable of segregating chromosomes. Spongistatin 1 had more dramatic effects on cytoplasmic microtubules. At 30 min after initiation of treatment, 83% of germlings contained fragmented microtubules and after 2 h of treatment, microtubules had disappeared completely from 82% of germlings. In contrast, microtubules disappeared rapidly and completely from germlings treated with benomyl. We conclude that spongistatin 1 has antimicrotubule activity in
A. nidulans
and that its mechanism of action may involve a novel microtubule-severing activity.
Identification of Functional Amino Acids in the Macrolide 2′-Phosphotransferase II
Authors: Taniguchi Kazuo , Nakamura Akio , Tsurubuchi Kazue , Ishii Aki , O’Hara Koji , Sawai Tetsuo
Published: October 2018

Macrolide 2′-phosphotransferase [MPH(2′)] transfers the γ phosphate of ATP to the 2′-OH group of macrolide antibiotics. The role of aspartic acids in the putative ATP-binding site of MPH(2′)II was investigated through the substitution of alanine for aspartate by site-directed mutagenesis. D200A, D209A, D219A, and D231A mutant strains were unable to inactivate the substrate oleandomycin, while a D227A mutant retained 7% of the activity of the original enzyme.
Contribution of
kasA
Analysis to Detection of Isoniazid-Resistant
Mycobacterium tuberculosis
in Singapore
Authors: Lee Ann S. G. , Lim Irene H. K. , Tang Lynn L. H. , Telenti Amalio , Wong Sin Yew
Published: October 2018

Genotypic analysis of resistance to isoniazid (INH) in
Mycobacterium tuberculosis
is complex due to the various genes potentially involved. Mutations in ketoacyl acyl carrier protein synthase (encoded by
kasA
) were present in 16 of 160 (10%) INH-resistant isolates (R121K [
n
= 1], G269S [
n
= 3], G312S [
n
= 11], G387D [
n
= 1]). However, G312S was also present in 6 of 32 (19%) susceptible strains.
kasA
analysis contributed marginally to the performance of INH genotypic testing in Singapore. The significance of
kasA
polymorphisms in INH resistance should be carefully established.
In Vitro Activities of Polycationic Peptides Alone and in Combination with Clinically Used Antimicrobial Agents against
Rhodococcus equi
Authors: Giacometti A. , Cirioni O. , Ancarani F. , Del Prete M. S. , Fortuna M. , Scalise G.
Published: October 2018

The in vitro activities of magainin II, nisin, and ranalexin alone and in combination with other antimicrobial agents against six clinical isolates of
Rhodococcus equi
were investigated by MIC and time-kill studies. All isolates were more susceptible to nisin. A positive interaction was observed when the peptides were combined with ampicillin, ceftriaxone, rifabutin, rifampin, azithromycin, clarithromycin, and vancomycin.
Characterization of
fmtA
, a Gene That Modulates the Expression of Methicillin Resistance in
Staphylococcus aureus
Authors: Komatsuzawa Hitoshi , Ohta Kouji , Labischinski Harald , Sugai Motoyuki , Suginaka Hidekazu
Published: October 2018

FmtA is a factor which affects the methicillin resistance level in methicillin-resistant
Staphylococcus aureus
. Since FmtA has two of three conserved motifs which are typically found in penicillin-binding proteins (PBPs) and β-lactamases, we investigated the penicillin-binding activity of recombinant FmtA and found no such activity. Immunoblotting analysis revealed that FmtA localizes in the membrane fraction. To investigate the function of FmtA, high-pressure liquid chromatography analysis of cell wall muropeptides was performed with an
fmtA
-inactivated mutant and its parent. The mutant showed a reduced cross-linking and partially reduced amidation of glutamate residues in the peptidoglycan of the mutant. The transcription of
fmtA
was dose dependently increased by the addition of β-lactam antibiotics, fosfomycin, and bacitracin, while its transcription was not changed by the addition of vancomycin or tetracycline. These results reveal that Fmt is a membrane-located, non-penicillin-binding protein and that mutation of
fmtA
affects the cell wall structure, although its precise function is still unknown.
Fluoroquinolone Resistance in
Bacteroides fragilis
following Sparfloxacin Exposure
Authors: Peterson M. L. , Hovde L. B. , Wright D. H. , Hoang A. D. , Raddatz J. K. , Boysen P. J. , Rotschafer J. C.
Published: October 2018

In vitro pharmacodynamic studies investigating the antimicrobial properties of five fluoroquinolones, (trovafloxacin, sparfloxacin, clinafloxacin, levofloxacin, and ciprofloxacin) against
Bacteroides fragilis
ATCC 23745 were conducted. The times required to reduce the viable counts by 3 log units were as follows: clinafloxacin, 2.9 h; levofloxacin, 4.6 h; trovafloxacin, 6 h; and sparfloxacin, 10 h. Exposure to ciprofloxacin did not achieve a 3-log decrease in viable counts. The susceptibility of
B. fragilis
was determined both prior to exposure and following 24 h of exposure to each of the five fluoroquinolones tested. The MICs of clinafloxacin, levofloxacin, trovafloxacin, sparfloxacin, ciprofloxacin, metronidazole, cefoxitin, chloramphenicol, and clindamycin were determined by the broth microdilution method. The MICs for
B. fragilis
preexposure were as follows: clinafloxacin, 0.25 μg/ml; trovafloxacin, 0.5 μg/ml; sparfloxacin, 2 μg/ml; levofloxacin, 2 μg/ml; and ciprofloxacin, 8 μg/ml. Similar pre- and postexposure MICs were obtained for cultures exposed to trovafloxacin, clinafloxacin, levofloxacin, and ciprofloxacin. However, following 24 h of exposure to sparfloxacin, a fluoroquinolone-resistant strain emerged. The MICs for this strain were as follows: clinafloxacin, 1 μg/ml; trovafloxacin, 4 μg/ml; sparfloxacin, 16 μg/ml; levofloxacin, 16 μg/ml; and ciprofloxacin, 32 μg/ml. No changes in the susceptibility of
B. fragilis
pre- and postexposure to sparfloxacin were noted for metronidazole (MIC, 1 μg/ml), cefoxitin (MIC, 4 μg/ml), chloramphenicol (MIC, 4 μg/ml), and clindamycin (MIC, 0.06 μg/ml). Resistance remained stable as the organism was passaged on antibiotic-free agar for 10 consecutive days. Mutant
B. fragilis
strains with decreased susceptibility to clinafloxacin, trovafloxacin, sparfloxacin, levofloxacin, and ciprofloxacin were selected on brucella blood agar containing 8× the MIC of levofloxacin at a frequencies of 6.4 × 10
−9
, 4× the MICs of trovafloxacin and sparfloxacin at frequencies of 2.2 × 10
−9
and 3.3 × 10
−10
, respectively, and 2× the MIC of clinafloxacin at a frequency of 5.5 × 10
−11
; no mutants were selected with ciprofloxacin. The susceptibilities of strains to trovafloxacin, levofloxacin, clinafloxacin, sparfloxacin, and ciprofloxacin before and after exposure to sparfloxacin were modestly affected by the presence of reserpine (20 μg/ml), an inhibitor of antibiotic efflux. The mechanism of fluoroquinolone resistance is being explored, but it is unlikely to be efflux due to a lack of cross-resistance to unrelated antimicrobial agents and to the fact that the MICs for strains before and after exposure to sparfloxacin are minimally affected by reserpine.
Inhibition ofEscherichia coli-Induced Meningitis by Carboxyfullerence
Authors: Tsao Nina , Kanakamma Puthuparampil P. , Luh Tien-Yau , Chou Chen-Kung , Lei Huan-Yao
Published: October 2018

The effect of a water-soluble malonic acid derivative of carboxyfullerence (C60) againstEscherichia coli-induced meningitis was tested. C60 can protect the mice fromE. coli-induced death in a dose-dependent manner. C60 administered intraperitoneally as late as 9 h afterE. coliinjection was still protective. The C60-treated mice had less tumor necrosis factor alpha and interleukin-1β production by staining of brain tissue compared to the levels of production for nontreated mice. TheE. coli-induced increases in blood-brain barrier permeability and inflammatory neutrophilic infiltration were also inhibited. These data suggest that C60 is a potentially therapeutic agent for bacterial meningitis.
Mononuclear and Polymorphonuclear Leukocyte Dispositions of Clarithromycin and Azithromycin in AIDS Patients Requiring
Mycobacterium avium
Complex Prophylaxis
Authors: Bui Khanh Q. , McNabb JoCarol , Li Chonghua , Nightingale Charles H. , Nicolau David P.
Published: October 2018

The intracellular dispositions of clarithromycin and azithromycin in AIDS patients requiring
Mycobacterium avium
complex (MAC) prophylaxis were studied. The dispositions of both drugs in mononuclear and polymorphonuclear leukocytes were markedly different. Our data support the proven efficacy of these agents for MAC prophylaxis since clarithromycin and azithromycin displayed sustained intracellular concentrations which exceeded their MICs for MAC throughout the dosing periods.
Improvement of ST0452
N
-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering
Authors: Honda Yuki , Nakano Shogo , Ito Sohei , Dadashipour Mohammad , Zhang Zilian , Kawarabayasi Yutaka
Published: October 2018

We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes.
Limited Marginal Utility of Deep Sequencing for HIV Drug Resistance Testing in the Age of Integrase Inhibitors
Authors: Dalmat Ronit R. , Makhsous Negar , Pepper Gregory G. , Magaret Amalia , Jerome Keith R. , Wald Anna , Greninger Alexander L.
Published: October 2018

HIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect low-frequency alleles.
Towards universal health coverage: including undocumented migrants
Authors: Onarheim Kristine Husøy , Melberg Andrea , Meier Benjamin Mason , Miljeteig Ingrid
Published: October 2018

As countries throughout the world move towards universal health coverage, the obligation to realise the right to health for undocumented migrants has often been overlooked. With unprecedented millions on the move – including refugees, asylum seekers, internally displaced persons, and returnees – undocumented migrants represent a uniquely vulnerable subgroup, experiencing particular barriers to health related to their background as well as insecure living and working conditions. Their legal status under national law often restricts access to, and affordability of, healthcare services. While striving to ensure health for all, national governments face challenging priority setting dilemmas in deciding: who to include, which services to provide, and how to cover out-of-pocket expenses. Building on comparative experiences in Norway, Thailand and the United States – which reflect varied approaches to achieving universal health coverage – we assess whether these national approaches provide rights-based access to affordable essential healthcare services for undocumented migrants. To meet the shared Sustainable Development Goal on universal health coverage, the right to health must be realised for all persons – including undocumented migrants. To ensure universal health coverage in accordance with the right to health, governments must evaluate laws, regulations, policies and practices to evaluate: whether undocumented migrants are included, to which services they have access, and if these services are affordable. Achieving universal health coverage for everyone will require rights-based support for undocumented migrants.