bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Different Factors on the Probability of Clinical Response in Tigecycline-Treated Patients with Intra-Abdominal Infections
Authors: Bhavnani S. M. , Rubino C. M. , Ambrose P. G. , Babinchak T. J. , Korth-Bradley J. M. , Drusano G. L.
Published: December 2009

Patients with intra-abdominal infections differ with regard to the type of infection and the severity of illness. However, the impact of these factors, together with differences in drug exposure, on clinical response is not well understood. Using phase 2 and 3 data for patients with complicated intra-abdominal infections, the relative importance of tigecycline exposure, host factors, and disease factors, alone or in combination, for the probability of clinical response was examined. Patients with complicated intra-abdominal infections who received tigecycline intravenously as a 100-mg loading dose followed by 50 mg every 12 h for 5 to 14 days and who had adequate clinical, pharmacokinetic, and response data were evaluated. Multivariable logistic regression was used to identify factors associated with clinical response. A final multivariable logistic regression model demonstrated six factors based on 123 patients to be predictive of clinical success: a weight of <94 kg (
P
= 0.026), the absence of
Pseudomonas aeruginosa
in baseline cultures (
P
= 0.021), an APACHE II score of <13 (
P
= 0.029), non-Hispanic race (
P
= 0.005), complicated appendicitis or cholecystitis (
P
= 0.004), and a ratio of the area under the concentration-time curve (AUC) to the MIC (AUC/MIC ratio) of ≥3.1 (
P
= 0.003). The average model-predicted probability of clinical success when one unfavorable factor was present was 0.940. This probability was lower (0.855) when the AUC/MIC ratio was <3.1 and the remaining five factors were set to the favorable condition. The average model-predicted probability of clinical success in the presence of two unfavorable factors was 0.594. These findings demonstrated the impact of individual and multiple factors on clinical response in the context of drug exposure.
Pharmacokinetics of Antituberculosis Drugs in Pulmonary Tuberculosis Patients with Type 2 Diabetes
Authors: Ruslami Rovina , Nijland Hanneke M. J. , Adhiarta I. Gusti N. , Kariadi Sri H. K. S. , Alisjahbana Bachti , Aarnoutse Rob E. , van Crevel Reinout
Published: December 2009

Altered pharmacokinetics of antituberculosis drugs may contribute to an increased risk of tuberculosis treatment failure for diabetic patients. We previously found that rifampin exposure was 2-fold lower in diabetic than in nondiabetic tuberculosis patients during the continuation phase of treatment. We now examined the influence of diabetes on the pharmacokinetics of antituberculosis drugs in the intensive phase of tuberculosis treatment, and we evaluated the effect of glycemic control. For this purpose, 18 diabetic and 18 gender- and body weight-matched nondiabetic tuberculosis patients were included in an Indonesian setting. Intensive pharmacokinetic sampling was performed for rifampin, pyrazinamide, and ethambutol at steady state. The bioavailability of rifampin was determined by comparing rifampin exposure after oral versus intravenous administration. Pharmacokinetic assessments were repeated for 10 diabetic tuberculosis patients after glycemic control. No differences in the areas under the concentration-time curves of the drugs in plasma from 0 to 24 h postdose (AUC
0-24
), the maximum concentrations of the drugs in plasma (
C
max
), the times to
C
max
(
T
max
), and the half-lives of rifampin, pyrazinamide, and ethambutol were found between diabetic and nondiabetic tuberculosis patients in the intensive phase of tuberculosis treatment. For rifampin, oral bioavailability and metabolism were similar in diabetic and nondiabetic patients. The pharmacokinetic parameters of antituberculosis drugs were not correlated with blood glucose levels or glucose control. We conclude that diabetes does not alter the pharmacokinetics of antituberculosis drugs during the intensive phase of tuberculosis treatment. The reduced exposure to rifampin of diabetic patients in the continuation phase may be due to increased body weight and possible differences in hepatic induction. Further research is needed to determine the cause of increased tuberculosis treatment failure among diabetic patients.
Pharmacokinetics of Acyclovir and Its Metabolites in Cerebrospinal Fluid and Systemic Circulation after Administration of High-Dose Valacyclovir in Subjects with Normal and Impaired Renal Function
Authors: Smith James P. , Weller Stephen , Johnson Benjamin , Nicotera Janet , Luther James M. , Haas David W.
Published: December 2009

Valacyclovir, the
l
-valyl ester prodrug of acyclovir (ACV), is widely prescribed to treat infections caused by varicella-zoster virus or herpes simplex virus. Rarely, treatment is complicated by reversible neuropsychiatric symptoms. By mechanisms not fully understood, this occurs more frequently in the setting of renal impairment. We characterized the steady-state pharmacokinetics of ACV and its metabolites 9-[(carboxymethoxy)methyl]guanine (CMMG) and 8-hydroxy-acyclovir (8-OH-ACV) in cerebrospinal fluid (CSF) and the systemic circulation. We administered multiple doses of high-dose valacyclovir to 6 subjects with normal renal function and 3 subjects with chronic renal impairment (creatinine clearance [CrCl], ∼15 to 30 ml/min). Dosages were 2,000 mg every 6 h and 1,500 mg every 12 h, respectively. Indwelling intrathecal catheters allowed serial CSF sampling throughout the dosing interval. The average steady-state concentrations of acyclovir, CMMG, and 8-OH-ACV were greater in both the systemic circulation and the CSF among subjects with impaired renal function than among subjects with normal renal function. However, the CSF penetration of each analyte, reflected by the CSF-to-plasma area under the concentration-time curve over the 6- or 12-h dosing interval (AUC
τ
) ratio, did not differ based on renal function. Renal impairment does not alter the propensity for ACV or its metabolites to distribute to the CSF, but the higher concentrations in the systemic circulation, as a result of reduced elimination, are associated with proportionally higher concentrations in CSF.
Evidence of Selective Sweeps in Genes Conferring Resistance to Chloroquine and Pyrimethamine in
Plasmodium falciparum
Isolates in India
Authors: Mixson-Hayden Tonya , Jain Vidhan , McCollum Andrea M. , Poe Amanda , Nagpal Avinash C. , Dash Aditya P. , Stiles Jonathan K. , Udhayakumar Venkatachalam , Singh Neeru
Published: December 2009

Treatment of
Plasmodium falciparum
is complicated by the emergence and spread of parasite resistance to many of the first-line drugs used to treat malaria. Antimalarial drug resistance has been associated with specific point mutations in several genes, suggesting that these single nucleotide polymorphisms can be useful in tracking the emergence of drug resistance. In India,
P. falciparum
infection can manifest itself as asymptomatic, mild, or severe malaria, with or without cerebral involvement. We tested whether chloroquine- and antifolate drug-resistant genotypes would be more commonly associated with cases of cerebral malaria than with cases of mild malaria in the province of Jabalpur, India, by genotyping the
dhps
,
dhfr
,
pfmdr-1
, and
pfcrt
genes using pyrosequencing, direct sequencing, and real-time PCR. Further, we used microsatellites surrounding the genes to determine the origins and spread of the drug-resistant genotypes in this area. Resistance to chloroquine was essentially fixed, with 95% of the isolates harboring the
pfcrt
K76T mutation. Resistant genotypes of
dhfr
,
dhps
, and
pfmdr-1
were found in 94%, 17%, and 77% of the isolates, respectively. Drug-resistant genotypes were equally likely to be associated with cerebral malaria as with mild malaria. We found evidence of a selective sweep in
pfcrt
and, to a lesser degree, in
dhfr
, indicating high levels of resistance to chloroquine and evolving resistance to pyrimethamine. Microsatellites surrounding
pfcrt
indicate that the resistant genotypes (SVMNT) were most similar to those found in Papua New Guinea.
Detection of KPC in
Acinetobacter
spp. in Puerto Rico
Authors: Robledo Iraida E. , Aquino Edna E. , Santé María I. , Santana Jorge L. , Otero Diana M. , León Carlos F. , Vázquez Guillermo J.
Published: December 2009

During an island-wide PCR-based surveillance study of beta-lactam resistance in multidrug-resistant (MDR)
Escherichia coli
,
Klebsiella pneumoniae
,
Pseudomonas aeruginosa
, and
Acinetobacter calcoaceticus-baumannii
complex isolates obtained from 17 different hospitals, 10 KPC-positive
Acinetobacter
isolates were identified. DNA sequencing of the
bla
KPC
gene identified KPC-2, -3, and -4 and a novel variant, KPC-10. This is the first report of a KPC-type beta-lactamase identified in
Acinetobacter
species.
Single Nucleotide Polymorphism Analysis of the Major Tripartite Multidrug Efflux Pump of
Escherichia coli
: Functional Conservation in Disparate Animal Reservoirs despite Exposure to Antimicrobial Chemotherapy
Authors: Elkins Christopher A. , Mullis Lisa B. , Lacher David W. , Jung Carina M.
Published: December 2009

AcrAB-TolC imparts a strong intrinsic resistance phenotype to many clinically significant molecules in
Escherichia coli
. This complex is composed of a pump, AcrB, and a periplasmic protein, AcrA, that exports substrates through a common outer membrane porin, TolC. A sequence survey of the pump-specific components,
acrA
and
acrB
, was conducted on three discrete animal reservoirs: rodents, bovines, and catfish. Although two of the reservoirs (bovine and catfish) were agrarian, and antibiotic use (ceftiofur and oxytetracycline/Romet 30, respectively) was reported for them, the vast majority of structural polymorphisms were silent except for T104A (AcrA) and Q733R (AcrB), found in certain bovine-derived strains. Overall, the genes were well conserved, with high ratios of synonymous to nonsynonymous substitutions (

d
S

/

d
N

ratios), consistent with or, in the case of
acrB
, better than those of standard multilocus sequence typing (MLST) loci. Furthermore, predicted recombination points from single nucleotide polymorphism (SNP) patterns in
acrB
support a modular evolution of transporter proteins, consistent with an ancient origin. However, functional studies with clones representing the major silent SNPs and the nonsilent mutation in
acrB
failed to generate significant differences in resistance to a range of common efflux pump substrates. Interestingly, a comparison between log-phase
acrA
and
acrB
expression profiles yielded inconsistent trends, with
acrB
expression increasing modestly (<1.8-fold) in many strains from the antibiotic-enriched pools. Our results suggest that structural polymorphisms in this major efflux pump system may not contribute significantly to adaptive resistance by altering function or substrate specificity but may have a potential use in improving phylogenetic relationships and/or source tracking.
Acquisition of Flucytosine, Azole, and Caspofungin Resistance in
Candida glabrata
Bloodstream Isolates Serially Obtained from a Hematopoietic Stem Cell Transplant Recipient
Authors: Chapeland-Leclerc Florence , Hennequin Christophe , Papon Nicolas , Noël Thierry , Girard Aurélie , Socié Gérard , Ribaud Patricia , Lacroix Claire
Published: December 2009

We describe the acquisition of flucytosine, azole, and caspofungin resistance in sequential
Candida glabrata
bloodstream isolates collected from a bone marrow transplant patient with clinical failure. Point mutations in
C. glabrata FUR1
(
CgFUR1
) and
CgFKS2
and overexpression of
CgCDR1
and
CgCDR2
were observed in resistant isolates.
Lack of Pharmacokinetic Interaction between Amdoxovir and Reduced- and Standard-Dose Zidovudine in HIV-1-Infected Individuals
Authors: Hurwitz Selwyn J. , Asif Ghazia , Fromentin Emilie , Tharnish Phillip M. , Schinazi Raymond F.
Published: December 2009

Amdoxovir (AMDX) inhibits HIV-1 containing the M184V/I mutation and is rapidly absorbed and deaminated to its active metabolite, β-d-dioxolane guanosine (DXG). DXG is synergistic with zidovudine (ZDV) in HIV-1-infected primary human lymphocytes. A recentin silicopharmacokinetic (PK)/enzyme kinetic study suggested that ZDV at 200 mg twice a day (b.i.d.) may reduce toxicity without compromising efficacy relative to the standard 300-mg b.i.d. dose. Therefore, an intense PK clinical study was conducted using AMDX/placebo, with or without ZDV, in 24 subjects randomized to receive oral AMDX at 500 mg b.i.d., AMDX at 500 mg plus ZDV at 200 or 300 mg b.i.d., or ZDV at 200 or 300 mg b.i.d. for 10 days. Full plasma PK profiles were collected on days 1 and 10, and complete urine sampling was performed on day 9. Plasma and urine concentrations of AMDX, DXG, ZDV, and ZDV-5′-O-glucuronide (GZDV) were measured using a validated liquid chromatography-tandem mass spectrometry method. Data were analyzed using noncompartmental methods, and multiple comparisons were performed on the log-transformed parameters, at steady state. Coadministration of AMDX with ZDV did not significantly change either of the plasma PK parameters or percent recovery in the urine of AMDX, DXG, or ZDV/GZDV. Larger studies with AMDX/ZDV, with a longer duration, are warranted.
Comparative Pharmacodynamics of Amphotericin B Lipid Complex and Liposomal Amphotericin B in a Murine Model of Pulmonary Mucormycosis
Authors: Lewis Russell E. , Albert Nathan D. , Liao Guangling , Hou Jingguo , Prince Randall A. , Kontoyiannis Dimitrios P.
Published: December 2009

We compared the kinetics of amphotericin B (AMB) lung accumulation and fungal clearance by liposomal amphotericin B (L-AMB) and amphotericin B lipid complex (ABLC) in a neutropenic murine model of invasive pulmonary mucormycosis (IPM). Immunosuppressed BALB/c mice were inoculated with 1 × 10
6
Rhizopus oryzae
spores and administered L-AMB or ABLC at daily intravenous doses of 1, 5, or 10 mg/kg of body weight for 5 days starting 12 h after infection. At a dose of 10 mg/kg/day, both L-AMB and ABLC were effective at reducing the
R. oryzae
lung fungal burden and achieved lung tissue concentrations exceeding the isolate mean fungicidal concentration (MFC) of 8 μg/ml by 72 h. When ABLC was dosed at 5 mg/kg/day, the ABLC-treated animals had significantly higher AMB lung concentrations than the L-AMB treated animals at 24 h (6.64 and 1.44 μg/g, respectively;
P =
0.013) and 72 h (7.49 and 1.03 μg/g, respectively;
P
= 0.005), and these higher concentrations were associated with improved fungal clearance, as determined by quantitative real-time PCR (mean conidial equivalent of
R. oryzae
DNA per lung, 4.44 ± 0.44 and 6.57 ± 0.74 log
10
, respectively;
P
< 0.001). Analysis of the AMB tissue concentration-response relationships revealed that the suppression of
R. oryzae
growth in the lung required tissue concentrations that approached the MFC for the infecting isolate (50% effective concentration, 8.19 μg/g [95% confidence interval, 2.81 to 18.1 μg/g]). The rates of survival were similar in the animals treated with L-AMB and ABLC at 10 mg/kg/day. These data suggest that higher initial doses may be required during L-AMB treatment than during ABLC treatment of experimental IPM.
Early Curative Applications of the Aminoglycoside WR279396 on an ExperimentalLeishmania major-Loaded Cutaneous Site Do Not Impair the Acquisition of Immunity
Authors: Lecoeur Hervé , Buffet Pierre A. , Milon Geneviève , Lang Thierry
Published: December 2009

Topical therapy is an attractive approach for the treatment ofLeishmaniamajorcutaneous leishmaniasis (CL). WR279396, an expanded-spectrum aminoglycoside ointment, is now in phase 3 trials. Because the application of a cream is easier than the injection of pentavalent antimony, many patients with CL will likely be treated with WR279396 soon after the onset of a lesion. However, this new therapeutic approach may impair the acquisition of immunity. We evaluated the impact of early topical therapy on acquired immunity in an optimized mouse model ofL. major-induced CL. The efficacy of the WR279396 ointment in this model has been established previously. Acquired immunity was defined as the absence of lesions upon reinoculation of the same parasite isolate at a different skin site. Bioluminescence-based follow-up of luciferase-expressingL. majorloads was also performed. In this model, the control ofL. majorloads at the initial inoculation site and the acquisition of immunity are simultaneous (day 22 postinoculation). The clinical and parasitological efficacies of WR279396 applied as early as day 11 postinoculation, i.e., during theL. majormultiplication phase, did not impair the acquisition of immunity to a secondL. majorchallenge. This is reassuring from the perspective of the wide deployment of WR279396-based therapy in foci whereL. majoris endemic.
In Vitro
Activity of CEM-101, a New Fluoroketolide Antibiotic, against
Chlamydia trachomatis
and
Chlamydia
(
Chlamydophila
)
pneumoniae
Authors: Roblin Patricia M. , Kohlhoff Stephan A. , Parker Charles , Hammerschlag Margaret R.
Published: December 2009

The
in vitro
activities of CEM-101, telithromycin, azithromycin, clarithromycin, and doxycycline against 10 isolates each of
Chlamydia trachomatis
and
Chlamydia
(
Chlamydophila
)
pneumoniae
were tested. The MIC at which 90% of the isolates of both
C. trachomatis
and
C. pneumoniae
were inhibited and the minimal bactericidal concentration at which 90% of the isolates were killed by CEM-101 were 0.25 μg/ml (ranges, 0.125 to 0.5 μg/ml for
C. trachomatis
and 0.25 to 1.0 μg/ml for
C. pneumoniae
).
Stage-Dependent Inhibition of HIV-1 Replication by Antiretroviral Drugs in Cell Culture
Authors: Donahue Daniel A. , Sloan Richard D. , Kuhl Björn D. , Bar-Magen Tamara , Schader Susan M. , Wainberg Mark A.
Published: December 2009

Recent clinical trials have shown that the use of the HIV-1 integrase (IN) inhibitor raltegravir (RAL) results in drops in the viral load that are more rapid than those achieved by use of the reverse transcriptase (RT) inhibitor efavirenz. Previously, mathematical modeling of viral load decay that takes into account the stage of viral replication targeted by a drug has yielded data that closely approximate the clinical trial results. This model predicts greater inhibition of viral replication by drugs that act later in the viral replication cycle. In the present study, we have added drugs that target entry, reverse transcription, integration, or proteolytic processing to acutely infected cells and have shown modest viral inhibition by entry inhibitors, intermediate levels of inhibition by RT and IN inhibitors, and high levels of inhibition by protease inhibitors relative to the levels of growth for the no-drug controls. When dual or triple combinations of these drugs were added to acutely infected cells, we found that the levels of inhibition achieved by any given combination were comparable to those achieved by the latest-acting drug in the combination. In single-round infections in which the kinetics of reverse transcription and integration had been determined by quantitative PCR, addition of IN inhibitors at various times postinfection resulted in levels of inhibition equal to or greater than those achieved by addition of RT inhibitors. Collectively, our data providein vitroevidence of the stage-dependent inhibition of HIV-1 by clinically relevant drugs. We discuss how stage-dependent inhibition helps to explain the unique viral load decay dynamics observed clinically with RAL.
pilF
Polymorphism-Based PCR To Distinguish
Vibrio vulnificus
Strains Potentially Dangerous to Public Health
Authors: Roig Francisco J. , Sanjuán Eva , Llorens Amparo , Amaro Carmen
Published: December 2009

Vibrio vulnificus
is a heterogeneous species that comprises strains virulent and avirulent for humans and fish, and it is grouped into three biotypes. In this report, we describe a PCR-based methodology that allows both the species identification and discrimination of those isolates that could be considered dangerous to public health. Discrimination is based on the amplification of a variable region located within the gene
pilF
, which seems to be associated with potential human pathogenicity, regardless of the biotype of the strain.
Genome-Wide Screening and Identification of Factors Affecting the Biosynthesis of Prodigiosin by
Hahella chejuensis
, Using
Escherichia coli
as a Surrogate Host
Authors: Kwon Soon-Kyeong , Park Yon-Kyoung , Kim Jihyun F.
Published: December 2009

A marine bacterium,
Hahella chejuensis
, recently has attracted attention due to its lytic activity against a red-tide dinoflagellate. The algicidal function originates from its red pigment, prodigiosin, which also exhibits immunosuppressive or anticancer activity. Genome sequencing and functional analysis revealed a gene set contained in the
hap
gene cluster that is responsible for the biosynthesis of prodigiosin. To screen for the factors affecting the prodigiosin biosynthesis, we constructed a plasmid library of the
H. chejuensis
genomic DNA, introduced it into
Escherichia coli
strains harboring the
hap
cluster, and observed changes in production of the red pigment. Among the screened clones,
hapXY
genes whose products constitute a two-component signal transduction system were elucidated as positive regulators of the pigment production. In addition, an Hfq-dependent, noncoding region located at one end of the
hap
cluster was confirmed to play roles in regulation. Identification of factors involved in the regulation of prodigiosin biosynthesis should help in understanding how the prodigiosin-biosynthetic pathway is organized and controlled and also aid in modulating the overexpression of prodigiosin in a heterologous host, such as
E. coli
, or in the natural producer,
H. chejuensis
.
β-(1,3)-Glucan Exposure Assessment by Passive Airborne Dust Sampling and New Sensitive Immunoassays
Authors: Noss Ilka , Wouters Inge M. , Bezemer Gillina , Metwali Nervana , Sander Ingrid , Raulf-Heimsoth Monika , Heederik Dick J. J. , Thorne Peter S. , Doekes Gert
Published: December 2009

Associations between house dust-associated β-(1,3)-glucan exposure and airway inflammatory reactions have been reported, while such exposures in early childhood have been suggested to protect against asthma and wheezing. Most epidemiological studies have used reservoir dust samples and an inhibition enzyme immunoassay (EIA) for β-(1,3)-glucan exposure assessment. The objective of this study was to develop inexpensive but highly sensitive enzyme immunoassays to measure airborne β-(1,3)-glucans in low-exposure environments, like homes. Specificities of available anti-β-(1,3)-glucan antibodies were defined by direct and inhibition experiments. Three suitable antibody combinations were selected for sandwich EIAs. β-(1,3)-Glucans in passive airborne dust collected with an electrostatic dust fall collector (EDC) and floor dust from seven homes were measured with the three EIAs. Floor dust samples were additionally analyzed in the inhibition EIA. The sandwich EIAs were sensitive enough for airborne glucan measurement and showed different specificities for commercial glucans, while the β-(1,3)-glucan levels in house dust samples correlated strongly. The feasibility of measuring glucans in airborne dust with the recently introduced EDC method was further investigated by selecting the most suitable of the three EIAs to measure and compare β-(1,3)-glucan levels in the EDC and in floor and actively collected airborne dust samples of the previously performed EDC validation study. The EDC β-(1,3)-glucan levels correlated moderately with β-(1,3)-glucans in actively collected airborne dust and floor dust samples, while the glucan levels in the airborne dust and floor dust samples did not correlate. The combination of the newly developed β-(1,3)-glucan sandwich EIA with EDC sampling now allows assessment in large-scale population studies of exposure to airborne β-(1,3)-glucans in homes or other low-exposure environments.
Selection of a
Bacillus pumilus
Strain Highly Active against
Ceratitis capitata
(Wiedemann) Larvae
Authors: Molina C. Alfonso , Caña-Roca Juan F. , Osuna Antonio , Vilchez Susana
Published: December 2009

Ceratitis capitata
(Wiedemann) (Diptera: Tephritidae), the Mediterranean fruit fly (medfly), is one of the most important fruit pests worldwide. The medfly is a polyphagous species that causes losses in many crops, which leads to huge economic losses. Entomopathogenic bacteria belonging to the genus
Bacillus
have been proven to be safe, environmentally friendly, and cost-effective tools to control pest populations. As no control method for
C. capitata
based on these bacteria has been developed, isolation of novel strains is needed. Here, we report the isolation of 115 bacterial strains and the results of toxicity screening with adults and larvae of
C. capitata
. As a result of this analysis, we obtained a novel
Bacillus pumilus
strain, strain 15.1, that is highly toxic to
C. capitata
larvae. The toxicity of this strain for
C. capitata
was related to the sporulation process and was observed only when cultures were incubated at low temperatures before they were used in a bioassay. The mortality rate for
C. capitata
larvae ranged from 68 to 94% depending on the conditions under which the culture was kept before the bioassay. Toxicity was proven to be a special characteristic of the newly isolated strain, since other
B. pumilus
strains did not have a toxic effect on
C. capitata
larvae. The results of the present study suggest that
B. pumilus
15.1 could be considered a strong candidate for developing strategies for biological control of
C. capitata
.
Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of
Escherichia coli
O157:H7 and
Campylobacter jejuni
Authors: Rapp Delphine , Waller John , Brightwell Gale , Muirhead Richard W.
Published: December 2009

Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both
Escherichia coli
O157:H7 and
Campylobacter jejuni
bacteria present in feces at concentrations ranging between 2 log
10
and 6 log
10
CFU g
−
1
.
Evaluation of
Lactobacillus sobrius/L. amylovorus
as a New Microbial Marker of Pig Manure
Authors: Marti Romain , Dabert Patrick , Ziebal Christine , Pourcher Anne-Marie
Published: December 2009

Based on a comparison of the dominant microbial populations in 17 pig manure samples and using a molecular typing method, we identified a species,
Lactobacillus sobrius
and
Lactobacillus amylovorus
(which now are considered a single species and are designated
L. sobrius
/
amylovorus
here), that was consistently found in manure. The aim of the present study was to confirm by real-time PCR the relevance of this species as a marker of pig fecal contamination. The specificity of
L. sobrius
/
amylovorus
was evaluated in human and animal DNA extracted from feces. The real-time PCR assay then was applied to water samples, including effluents from urban wastewater treatment plants, runoff water, and rivers.
L. sobrius
/
amylovorus
was consistently present in all samples of swine origin: 48 fecal samples, 18 from raw manure and 10 from biologically treated manure at mean concentrations of 7.2, 5.9, and 5.0 log
10
cells/g, respectively. The species was not detected in any of the other livestock feces (38 samples from cattle and 16 from sheep), in the 27 human fecal samples, or in the 13 effluent samples from urban wastewater treatment plants. Finally,
L. sobrius
/
amylovorus
was not detected in runoff water contaminated by cattle slurry, but it was quantified at concentrations ranging from 3.7 to 6.5 log
10
cells/100 ml in runoff water collected after pig manure was spread on soil. Among the stream water samples in which cultured
Escherichia coli
was detected, 23% tested positive for
L. sobrius
/
amylovorus
. The results of this study indicate that the quantification of
L. sobrius
/
amylovorus
using real-time PCR will be useful for identifying pig fecal contamination in surface waters.
Characterization of Ozone Disinfection of Murine Norovirus
Authors: Lim Mi Young , Kim Ju-Mi , Lee Jung Eun , Ko GwangPyo
Published: December 2009

Despite the importance of human noroviruses (NoVs) in public health, little information concerning the effectiveness of ozone against NoVs is available. We determined the efficacy of ozone disinfection using murine norovirus (MNV) as a surrogate of human NoV. MNV in ozone demand-free buffer was exposed to a predetermined dose of ozone at two different pHs and temperatures. The virus remaining in the solution was analyzed by plaque assay, real-time TaqMan reverse transcriptase PCR (RT-PCR) (short template), and long-template conventional RT-PCR. Under all conditions, more than 99% of the MNV was inactivated by ozone at 1 mg/liter within 2 min. Both RT-PCR assays significantly underestimated the inactivation of MNV, compared with that measured by plaque assay. Our results indicate that NoV may be more resistant to ozone than has been previously reported. Nevertheless, proper ozone disinfection practices can be used to easily control its transmission in water.
NoxE NADH Oxidase and the Electron Transport Chain Are Responsible for the Ability of
Lactococcus lactis
To Decrease the Redox Potential of Milk
Authors: Tachon Sybille , Brandsma Johannes Bernhard , Yvon Mireille
Published: December 2009

The redox potential plays a major role in the microbial and sensorial quality of fermented dairy products. The redox potential of milk (around 400 mV) is mainly due to the presence of oxygen and many other oxidizing compounds.
Lactococcus lactis
has a strong ability to decrease the redox potential of milk to a negative value (−220 mV), but the molecular mechanisms of milk reduction have never been addressed. In this study, we investigated the impact of inactivation of genes encoding NADH oxidases (
noxE
and
ahpF
) and components of the electron transport chain (ETC) (
menC
and
noxAB
) on the ability of
L. lactis
to decrease the redox potential of ultrahigh-temperature (UHT) skim milk during growth under aerobic and anaerobic conditions. Our results revealed that elimination of oxygen is required for milk reduction and that NoxE is mainly responsible for the rapid removal of oxygen from milk before the exponential growth phase. The ETC also contributes slightly to oxygen consumption, especially during the stationary growth phase. We also demonstrated that the ETC is responsible for the decrease in the milk redox potential from 300 mV to −220 mV when the oxygen concentration reaches zero or under anaerobic conditions. This suggests that the ETC is responsible for the reduction of oxidizing compounds other than oxygen. Moreover, we found great diversity in the reducing activities of natural
L. lactis
strains originating from the dairy environment. This diversity allows selection of specific strains that can be used to modulate the redox potential of fermented dairy products to optimize their microbial and sensorial qualities.
Effects of Carbon Dioxide on Growth of ProteolyticClostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome
Authors: Artin Ingrid , Mason David R. , Pin Carmen , Schelin Jenny , Peck Michael W. , Holst Elisabet , Rådström Peter , Carter Andrew T.
Published: December 2009

The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO2(10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolyticClostridium botulinumtype A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolyticC. botulinumtypes B and E, CO2had little effect on any of these parameters. At all CO2concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.
Methanotrophic Communities in Brazilian Ferralsols from Naturally Forested, Afforested, and Agricultural Sites
Authors: Dörr Nicole , Glaser Bruno , Kolb Steffen
Published: December 2009

Conversion of forests to farmland permanently lowers atmospheric methane consumption due to unresolved reasons. Alphaproteobacterial methanotrophs were predominant in forested soils and gammaproteobacterial species were predominant in farmland soils of subtropical ferralsols in Brazil. The capability of atmospheric methane consumption was obliterated in farmland soils, suggesting a shift from oligotrophic to copiotrophic species.
Distribution and Diversity of
Escherichia coli
Populations in the South Nation River Drainage Basin, Eastern Ontario, Canada
Authors: Lyautey Emilie , Lu Zexun , Lapen David R. , Wilkes Graham , Scott Andrew , Berkers Tanya , Edge Thomas A. , Topp Edward
Published: December 2009

We investigated the prevalence and diversity of
Escherichia coli
strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada. The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface water samples were collected from 24 sampling sites.
E. coli
densities ranged from undetectable to 1.64 × 10
5
CFU 100 ml
−1
and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307
E. coli
isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from 0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use, discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection of
Campylobacter
species was associated with statistically lower Simpson indices (greater diversity;
P
< 0.05). Waterborne
E. coli
isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall, our data indicated that the densities and distribution of
E. coli
in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne
E. coli
populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized
E. coli
strains.
Genes Involved in Long-Chain Alkene Biosynthesis in
Micrococcus luteus
Authors: Beller Harry R. , Goh Ee-Been , Keasling Jay D.
Published: December 2009

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in
Micrococcus luteus
ATCC 4698, a close relative of
Sarcina lutea
(now
Kocuria rhizophila
), which 4 decades ago was reported to biosynthesize
iso
- and
anteiso
-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from
M
.
luteus
(Mlut_13230-13250) in a fatty acid-overproducing
Escherichia coli
strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C＝C bonds). Heterologous expression of Mlut_13230 (
oleA
) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and
in vitro
studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C
27
monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the
M
.
luteus
genes were responsible for production of these metabolites. Studies with wild-type
M
.
luteus
showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (β-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during fatty acid biosynthesis.
Long-Term Persistence and Leaching ofEscherichia coliin Temperate Maritime Soils
Authors: Brennan Fiona P. , O'Flaherty Vincent , Kramers Gaelene , Grant Jim , Richards Karl G.
Published: December 2009

Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, usingEscherichia colias a marker, in four Irish soils (n= 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on theE. colithat leached from the soil was observed. Leaching ofE. coliwas observed predominantly from one soil type (average, 1.11 ± 0.77 CFU ml−1), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism.E. coliwas found to have persisted in control soils for more than 9 years, indicating that autochthonousE. colipopulations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use ofE. colias an indicator of fecal pollution of waters in these regions.
Biogenic Silver for Disinfection of Water Contaminated with Viruses
Authors: De Gusseme Bart , Sintubin Liesje , Baert Leen , Thibo Ellen , Hennebel Tom , Vermeulen Griet , Uyttendaele Mieke , Verstraete Willy , Boon Nico
Published: December 2009

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study,
Lactobacillus fermentum
served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag
0
. The antiviral action of biogenic Ag
0
was examined in water spiked with an
Enterobacter aerogenes
-infecting bacteriophage (UZ1). Addition of 5.4 mg liter
−1
biogenic Ag
0
caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (
n
Ag
0
) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter
−1
ionic Ag
+
resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag
0
were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag
0
was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag
0
m
−2
on the filter (135 mg biogenic Ag
0
kg
−1
filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag
0
and its promising opportunities for continuous water disinfection.
Identification of a Secreted Lipolytic Esterase in
Propionibacterium freudenreichii
, a Ripening Process Bacterium Involved in Emmental Cheese Lipolysis
Authors: Dherbécourt J. , Falentin H. , Jardin J. , Maillard M.-B. , Baglinière F. , Barloy-Hubler F. , Thierry A.
Published: December 2009

Lipolysis plays an important role in the formation of cheese flavor. In Emmental cheese, the main part of lipolysis has been associated with the presence of
Propionibacterium freudenreichii
, a species used as a ripening culture. Our aim was to identify the most probable lipolytic esterase(s) involved in cheese lipolysis by
P. freudenreichii
. Since cheese lipolysis mainly occurs during
P. freudenreichii
growth, we hypothesized that
P. freudenreichii
possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of
P. freudenreichii
CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted
in silico
. The esterase activity in extracellular and intracellular extracts of
P. freudenreichii
was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed
P. freudenreichii
CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining
in silico
, biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in
P. freudenreichii
and is active on milk fat. Therefore, it is likely a key component in cheese lipolysis by
P. freudenreichii
.
Identification of a Two-Component Regulatory Pathway Essential for Mn(II) Oxidation in
Pseudomonas putida
GB-1
Authors: Geszvain Kati , Tebo Bradley M.
Published: December 2009

Bacterial manganese(II) oxidation has a profound impact on the biogeochemical cycling of Mn and the availability of the trace metals adsorbed to the surfaces of solid Mn(III, IV) oxides. The Mn(II) oxidase enzyme was tentatively identified in
Pseudomonas putida
GB-1 via transposon mutagenesis: the mutant strain GB-1-007, which fails to oxidize Mn(II), harbors a transposon insertion in the gene
cumA
.
cumA
encodes a putative multicopper oxidase (MCO), a class of enzymes implicated in Mn(II) oxidation in other bacterial species. However, we show here that an in-frame deletion of
cumA
did not affect Mn(II) oxidation. Through complementation analysis of the oxidation defect in GB-1-007 with a cosmid library and subsequent sequencing of candidate genes we show the causative mutation to be a frameshift within the
mnxS1
gene that encodes a putative sensor histidine kinase. The frameshift mutation results in a truncated protein lacking the kinase domain. Multicopy expression of
mnxS1
restored Mn(II) oxidation to GB-1-007 and in-frame deletion of
mnxS1
resulted in a loss of oxidation in the wild-type strain. These results clearly demonstrated that the oxidation defect of GB-1-007 is due to disruption of
mnxS1
, not
cumA
::Tn
5
, and that CumA is not the Mn(II) oxidase.
mnxS1
is located upstream of a second sensor histidine kinase gene,
mnxS2
, and a response regulator gene,
mnxR
. In-frame deletions of each of these genes also led to the loss of Mn(II) oxidation. Therefore, we conclude that the MnxS1/MnxS2/MnxR two-component regulatory pathway is essential for Mn(II) oxidation in
P. putida
GB-1.
Involvement of a Novel ABC Transporter and Monoalkyl Phthalate Ester Hydrolase in Phthalate Ester Catabolism by
Rhodococcus jostii
RHA1
Authors: Hara Hirofumi , Stewart Gordon R. , Mohn William W.
Published: December 2009

Phthalate esters (PEs) are important environmental pollutants. While the biodegradation of the parent compound, phthalate (PTH), is well characterized, the biodegradation of PEs is not well understood. In particular, prior to this study, genes involved in the uptake and hydrolysis of these compounds were not conclusively identified. We found that
Rhodococcus jostii
RHA1 could grow on a variety of monoalkyl PEs, including methyl, butyl, hexyl, and 2-ethylhexyl PTHs. Strain RHA1 could not grow on most dialkyl PEs, but suspensions of cells grown on PTH transformed dimethyl, diethyl, dipropyl, dibutyl, dihexyl and di-(2-ethylhexyl) PTHs. The major products of these dialkyl PEs were PTH and the corresponding monoalkyl PEs, and minor products resulted from the shortening of the alkyl side chains. RHA1 exhibited an inducible, ATP-dependent uptake system for PTH with a

K
m

of 22 μM. The deletion and complementation of the
patB
gene demonstrated that the ATP-binding cassette (ABC) transporter encoded by
patDABC
is required for the uptake of PTH and monoalkyl PEs by RHA1. The hydrolase encoded by
patE
of RHA1 was expressed in
Escherichia coli
. PatE specifically hydrolyzed monoalkyl PEs to PTH but did not transform dialkyl PEs or other aromatic esters. This investigation of RHA1 elucidates key processes that are consistent with the environmental fate of PEs.
Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences
Authors: Ramsook Caleen B. , Tan Cho , Garcia Melissa C. , Fung Raymond , Soybelman Gregory , Henry Ryan , Litewka Anna , O'Meally Shanique , Otoo Henry N. , Khalaf Roy A. , Dranginis Anne M. , Gaur Nand K. , Klotz Stephen A. , Rauceo Jason M. , Jue Chong K. , Lipke Peter N.
Published: December 2009

The occurrence of highly conserved amyloid-forming sequences in
Candida albicans
Als proteins (H. N. Otoo et al., Eukaryot. Cell
7:
776–782, 2008) led us to search for similar sequences in other adhesins from
C. albicans
and
Saccharomyces cerevisiae
. The β-aggregation predictor TANGO found highly β-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were β-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the
S. cerevisiae
proteome. High β-aggregation potential peptides from
S. cerevisiae
Flo1p and
C. albicans
Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of
C. albicans
Als5p and
S. cerevisiae
Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation
in vivo
: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.