bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro activity of roxithromycin against the Mycobacterium tuberculosis complex
Authors: Rastogi N , Goh K S , Ruiz P , Casal M
Published: June 2012

Roxithromycin has recently been shown to possess significant in vitro activity against a variety of atypical mycobacteria such as the M. avium complex, M. scrofulaceum, M. szulgai, M. malmoense, M. xenopi, M. marinum, and M. kansasii and rare pathogens like M. chelonei and M. fortuitum. In the present investigation, screening of its in vitro activity was further extended by testing it against 34 strains belonging to the M. tuberculosis complex (including M. tuberculosis, M. africanum, M. bovis, and M. bovis BCG). The MICs were determined by the radiometric BACTEC 460-TB methodology at pHs of both 6.8 and 7.4, as well as with 7H10 agar medium by the 1% proportion method. With the exception of M. bovis BCG (MIC ranges, 0.5 to 4 micrograms/ml at pH 6.8 and 0.25 to 2 micrograms/ml at pH 7.4), MICs for all of the isolates were significantly greater (MIC ranges, 32 to > 64 micrograms/ml at pH 6.8 and 16 to > 32 micrograms/ml at pH 7.4) than those reported previously for atypical mycobacteria. Roxithromycin MICs of 64 or > 64 micrograms/ml for all of the M. tuberculosis isolates screened were found by the 7H10 agar medium method. Roxithromycin, however, showed a pH-dependent bactericidal effect against M. tuberculosis because the drug was relatively more active when it was used at pH 7.4 than when it was used at pH 6.8. We conclude that roxithromycin per se is not a drug of choice for the treatment of M. tuberculosis infection or disease; however, considering its pharmacokinetics, eventual anti-tubercle bacillus activity in an in vivo system cannot yet be excluded. We suggest that the use of roxithromycin in chemoprophylactic regimens for the prevention of opportunistic infections (including M. avium complex infections) in patients with AIDS should be carefully monitored, and patients should be enrolled in such a regimen only after it has been excluded that the patient das an underlying infection of disease caused by M. tuberculosis.
Results of German multicenter study of antimicrobial susceptibilities of Trichophyton rubrum and Trichophyton mentagrophytes strains causing tinea unguium. German Collaborative Dermatophyte Drug Susceptibility Study Group
Authors: Korting H C , Ollert M , Abeck D
Published: June 2012

Thirty-two strains of Trichophyton rubrum and 16 strains of Trichophyton mentagrophytes isolated from patients with tinea unguium in various parts of Germany were subjected to a microdilution test with six systemic or topical antimycotic agents. Apart from griseofulvin, there were no species-specific differences between the two species. Terbinafine was the most active antimycotic agent, with a MIC not exceeding 0.05 micrograms/ml.
The PETT series, a new class of potent nonnucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase
Authors: Ahgren C , Backro K , Bell F W , Cantrell A S , Clemens M , Colacino J M , Deeter J B , Engelhardt J A , Hogberg M , Jaskunas S R
Published: June 2012

To identify the minimal structural elements necessary for biological activity, the rigid tricyclic nucleus of the known human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor tetrahydroimidazobenzodiazepinthione was subjected to systematic bond disconnection to obtain simpler structures. A rational selection and testing of modeled analogs containing these potential pharmacophoric moieties led to the discovery of a new series of nonnucleoside inhibitors of RT. The lead compound of this new PETT series of nonnucleoside RT inhibitors, N-(2-phenylethyl)-N'-(2-thiazolyl)thiourea (LY73497), was found to inhibit HIV-1 but not HIV-2 or simian immunodeficiency virus in cell culture at micromolar concentrations. This derivative was also found to inhibit HIV-1 RT. Through an integrated effort involving synthesis and molecular modeling, compounds with nanomolar potency against HIV-1 in cell culture were developed. In these studies, LY300046-HCl was identified as a potent nonnucleoside inhibitor of HIV-1 RT possessing favorable pharmacokinetic properties.
Drug interactions with zidovudine phosphorylation in vitro
Authors: Hoggard P G , Veal G J , Wild M J , Barry M G , Back D J
Published: June 2012

We have investigated the effect of a range of drugs (some commonly coadministered with zidovudine [ZDV] to human immunodeficiency virus-positive patients) on intracellular phosphorylation of ZDV by stimulated peripheral blood mononuclear cells, Molt 4 cells, and U937 cells in vitro. Of the drugs tested (azoles, antiviral agents, antibiotics, and anticancer agents), only doxorubicin and ribavirin caused inhibition of anabolite formation as measured by high-performance liquid chromatography. This in vitro approach may provide important leads to potential interactions at the phosphorylation level in patients with human immunodeficiency virus disease. It is reassuring that so many commonly administered drugs do not alter ZDV phosphorylation.
Antiviral effect in human cytomegalovirus-infected cells, pharmacokinetics, and intravitreal toxicology in rabbits of acyclovir diphosphate dimyristoylglycerol
Authors: Shakiba S , Freeman W R , Flores-Aguilar M , Munguia D , Tatebayashi M , Besen G , Amani R , Wiley C A , Vuong C , Aldern K A
Published: June 2012

Acyclovir diphosphate dimyristoylglycerol (ACVDP-DG) is a lipid prodrug which is active against ACV-resistant strains of herpes simplex virus because of its intracellular metabolism to ACV monophosphate. In human cytomegalovirus (HCMV)-infected MRC-5 cells, ACVDP-DG was ninefold more active than ACV. When liposomal [8-3H]ACVDP-DG was injected intravitreally at the maximum nontoxic dose of 1 mumol in rabbits, the drug remained above its estimated 90% HCMV-inhibitory concentration for 18 days. Intravitreal ganciclovir persists above its 90% inhibitory concentration for only 1 to 2 days. ACVDP-DG may be useful as a local treatment for HCMV retinitis.
Synergistic activities of clarithromycin and antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis
Authors: Cavalieri S J , Biehle J R , Sanders W E
Published: June 2012

The rise of multidrug-resistant Mycobacterium tuberculosis has complicated therapy for tuberculosis and led us to search for a potentially active combination of drugs against these strains. The susceptibilities of 12 strains of multidrug-resistant M. tuberculosis to standard antituberculous drugs (isoniazid, rifampin, ethambutol, and pyrazinamide), clarithromycin, and its metabolite, 14-hydroxyclarithromycin, were determined by use of the BACTEC radiometric method. All strains were resistant to at least two of the antituberculous drugs. Clarithromycin and 14-hydroxyclarithromycin MICs were in the range indicating resistance at > or = 8.0 micrograms/ml for all strains. Combination testing by the BACTEC method was performed with various concentrations of isoniazid, rifampin, and ethambutol, and with clarithromycin/14-hydroxyclarithromycin at fixed concentrations of 2.0/0.5 micrograms/ml, respectively. Addition of clarithromycin/14-hydroxyclarithromycin to these antituberculous drug mixtures resulted in a 4- to 32-fold reduction in MICs of isoniazid, rifampin, and ethambutol and made resistant strains susceptible. Fractional inhibitory concentrations ranged from 0.23 to 0.50 for all strains, suggesting a synergistic interaction between standard antituberculous drugs and clarithromycin/14-hydroxyclarithromycin. The ability of clarithromycin/14-hydroxyclarithromycin to enhance the activities of isoniazid, ethambutol, and rifampin in vitro suggests that this combination may be efficacious in the treatment of multidrug-resistant M. tuberculosis infections.
Susceptibilities of several drug-resistant herpes simplex virus type 1 strains to alternative antiviral compounds
Authors: Andrei G , Snoeck R , De Clercq E
Published: June 2012

Resistant herpes simplex virus type 1 strains were obtained under the selective pressure of acyclovir, ganciclovir, bromovinyldeoxyuridine, foscarnet, 2-phosphonylmethoxyehtyl (PME) derivatives of adenine and 2,6-diaminopurine, 3-hydroxy-2-phosphonylmethoxypropyl derivatives of adenine and cytosine, and 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S2242). The drug susceptibility profiles of resistant strains point to differences in the modes of action of PME and 3-hydroxy-2-phosphonylmethoxypropyl derivatives and common mechanisms of action of foscarnet, S2242, and PME derivatives against herpes simplex virus type 1 replication.
In vitro evaluation of combination of fluconazole and flucytosine against Cryptococcus neoformans var. neoformans
Authors: Nguyen M H , Barchiesi F , McGough D A , Yu V L , Rinaldi M G
Published: June 2012

Amphotericin B and fluconazole are current acceptable therapies for cryptococcal meningitis; however, their effect remains suboptimal. The combination of fluconazole and flucytosine has yielded encouraging clinical results in human immunodeficiency virus patients with cryptococcal meningitis. To investigate the biological basis of this finding, we performed in vitro combination testing of fluconazole and flucytosine against 50 clinical strains of Cryptococcus neoformans var. neoformans. Synergy (fractional inhibitory concentration index of < 1.0) was observed in 62% of cases, while antagonism (fractional inhibitory concentration index of > 2.0) was not observed. For cases in which synergy was not achieved (autonomous or additive effects), the beneficial effect of the combination was still seen (i.e., there was still a decrease, although not as dramatic, in the MIC of one or both drugs when used in combination). The in vitro inhibitory action of flucytosine was greatly enhanced by the addition of fluconazole; the flucytosine MICs for Cryptococcus isolates were markedly decreased to concentrations which were severalfold lower than the achievable cerebrospinal fluid flucytosine concentration. On the other hand, the addition of flucytosine did not greatly enhance the in vitro activity of fluconazole if the initial fluconazole MIC for the isolate was > or = 8 micrograms/ml. Controlled clinical studies are warranted to further elucidate the potential utility of fluconazole-flucytosine combination therapy.
Cloning and characterization of a 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ib, from Pseudomonas aeruginosa
Authors: Schwocho L R , Schaffner C P , Miller G H , Hare R S , Shaw K J
Published: June 2012

A novel gene encoding an aminoglycoside 3-N-acetyltransferase, which confers resistance to gentamicin, astromicin, and sisomicin, was cloned from Pseudomonas aeruginosa Stone 130. Its sequence was determined and found to show considerable similarity to an aac(3)-I gene previously cloned from R plasmids from Enterobacter, Pseudomonas, and Serratia spp. We have designated the genes from the R plasmids and this work aac(3)-Ia and aac(3)-Ib, respectively. The two aac(3)-I genes share 74% nucleotide identity, and their deduced protein products are 88% similar. These data suggest that the genes derive from a common ancestor. Homology between the flanking sequences of both aac(3)-I genes and other resistance determinants known to reside in integron environments was also observed. Intragenic probes specific for either aac(3)-Ia or aac(3)-Ib were used in hybridization studies with a series of gentamicin-, astromicin-, and sisomicin-resistant clinical isolates. Of 59 clinical isolates tested, no isolates hybridized with both probes, 30 (51%) hybridized with the aac(3)-Ia probe, 12 (20%) hybridized with the aac(3)-Ib probe, and 17 (29%) did not hybridize with either probe. These data suggest the existence of at least one other aac(3)-I gene.
In vitro activities of PS-15, a new dihydrofolate reductase inhibitor, and its cyclic metabolite against Mycobacterium avium complex
Authors: Meyer S C , Majumder S K , Cynamon M H
Published: June 2012

PS-15, a new dihydrofolate reductase inhibitor, and its cyclic metabolite were evaluated for in vitro activity against 31 clinical Mycobacterium avium complex isolates. Broth dilution MICs of PS-15 ranged from 16 to 64 micrograms/ml. The cyclic metabolite was three to five times more active than the parent compound. Further evaluation of these compounds in an M. avium-infected murine test system will be of interest.
Cloning and characterization of the Pseudomonas aeruginosa pbpB gene encoding penicillin-binding protein 3
Authors: Liao X , Hancock R E
Published: June 2012

Clones containing the pbpB gene which encodes penicillin-binding protein (PBP) 3 of Pseudomonas aeruginosa were detected by hybridization by PCR amplification with primers based on the conserved sequences of high-molecular-weight PBPs. The translated amino acid sequence demonstrated 45% identity and had a total of 66% conserved amino acids relative to the Escherichia coli PBP3. The pbpB gene was located upstream of a gene homologous to the E. coli murE gene, which encodes uridine diphosphate-N-acetyl muramic acid-tripeptide synthetase. The overexpressed pbpB gene product reacted with 3H-penicillin G and had an apparent molecular weight of 60,000.
Bactericidal properties of Campylobacter jejuni-specific immunoglobulin M antibodies in commercial immunoglobulin preparations
Authors: Autenrieth I B , Schwarzkopf A , Ewald J H , Karch H , Lissner R
Published: June 2012

Campylobacter jejuni is one of the most common enterocolitis-causing microorganisms worldwide. It is of particular importance in immunodeficient patients, who frequently are prone to develop extraintestinal manifestations. Since these cases respond poorly to antibiotic treatment, a supportive immunomodulating therapy including the administration of C. jejuni-specific immunoglobulins would be desirable. In the present study, nine commercial immunoglobulin preparations for intravenous use were tested for the presence of C. jejuni lipopolysaccharide (LPS)- and outer membrane protein (OMP)-specific antibodies by using immunoblot and enzyme-linked immunosorbent assay techniques. The immunoglobulin G (IgG) antibody reactivities against these antigens were comparable in eight of nine tested immunoglobulin preparations. Only in one preparation were C. jejuni OMP- and LPS-specific IgM antibodies found. In this preparation the immunoblot test revealed a strong reactivity against both flagellin and a major OMP. Moreover, all immunoglobulin preparations recognized OMPs of C. jejuni serotypes Lior 4, 9, 11, and 29 equally strongly, while the reactivity to an anti-Lior 36 isolate was less marked. Furthermore, the bactericidal properties of three immunoglobulin preparations were tested by means of chemiluminescence signaling in and bacterial killing by human polymorphonuclear leukocytes (PMNL). The results show that the IgM preparation enhanced Campylobacter-triggered chemiluminescence signaling in PMNL as well as killing of C. jejuni by PMNL, while the other immunoglobulin preparations did not do so. These results suggest that the administration of immunoglobulin preparations containing C. jejuni-specific IgM antibodies would be beneficial for patients with severe C. jejuni infections.
Anti-human immunodeficiency virus (HIV) activities of halogenated gomisin J derivatives, new nonnucleoside inhibitors of HIV type 1 reverse transcriptase
Authors: Fujihashi T , Hara H , Sakata T , Mori K , Higuchi H , Tanaka A , Kaji H , Kaji A
Published: June 2012

Halogenated gomisin J (a derivative of lignan compound), represented by the bromine derivative 1506 [(6R, 7S, S-biar)-4,9-dibromo-3,10-dihydroxy-1,2,11,12-tetramethoxy-6, 7-dimethyl-5,6,7,8- tetrahydrodibenzo[a,c]cyclo-octene], was found to be a potent inhibitor of the cytopathic effects of human immunodeficiency virus type 1 (HIV-1) on MT-4 human T cells (50% effective dose, 0.1 to 0.5 microM). Gomisin J derivatives were active in preventing p24 production from acutely HIV-1-infected H9 cells. The selective indices (toxic dose/effective dose) of these compounds were as high as > 300 in some systems. 1506 was active against 3'-azido-3'-deoxythymidine-resistant HIV-1 and acted synergistically with AZT and 2',3'-ddC. 1506 inhibited HIV-1 reverse transcriptase (RT) in vitro but not HIV-1 protease. From the time-of-addition experiment, 1506 was found to inhibit the early phase of the HIV life cycle. A 1506-resistant HIV mutant was selected and shown to possess a mutation within the RT-coding region (at position 188 [Tyr to Leu]). The mutant RT expressed in Escherichia coli was resistant to 1506 in the in vitro RT assay. Some of the HIV strains resistant to other nonnucleoside HIV-1 RT inhibitors were also resistant to 1506. Comparison of various gomisin J derivatives with gomisin J showed that iodine, bromine, and chlorine in the fourth and ninth positions increased RT inhibitory activity as well as cytoprotective activity.
Diversity among the gram-positive acetyltransferases inactivating streptogramin A and structurally related compounds and characterization of a new staphylococcal determinant, vatB
Authors: Allignet J , el Solh N
Published: June 2012

A gene encoding an acetyltransferase inactivating streptogramin A (SgA) and structurally similar compounds was isolated from a staphylococcal plasmid and sequenced. This gene, designated vatB, potentially encodes a 212-amino-acid protein, VatB, of 23,320 Da with 47.4 and 58.4% amino acid identities with two other enzymes with the same activity, Vat and SatA, respectively, which are encoded by a staphylococcal plasmid and an enterococcal plasmid, respectively. The C-terminal parts of these three enzymes share significant homology with the C-terminal parts of 10 other acetyltransferases modifying various substrates. A pair of degenerate primers representing the conserved motifs shared by VatB, Vat, and SatA was designed to detect the three genes encoding these SgA acetyltransferases. Five of 12 clinical SgAr Staphylococcus aureus isolates tested carried neither these genes nor the gene vga, which confers resistance to SgA by a different mechanism, suggesting that another gene(s) and possibly another mechanism of resistance to SgA in staphylococci remains to be characterized.
In vitro antimycobacterial activities of pyrazinamide analogs
Authors: Yamamoto S , Toida I , Watanabe N , Ura T
Published: June 2012

We synthesized various pyrazine derivatives and pyrazinamide analogs and assayed their antimycobacterial activities in vitro in order to find new drugs which are more active against Mycobacterium tuberculosis than pyrazinamide and also active against Mycobacterium avium and Mycobacterium intracellulare. Of the drugs synthesized, four drugs, namely, pyrazine thiocarboxamide, N-hydroxymethyl pyrazine thiocarboxamide, pyrazinoic acid n-octyl ester, and pyrazinoic acid pivaloyloxymethyl ester, were not only bacteriostatic but also bacteriocidal against these three species of mycobacteria in vitro under conditions in which pyrazinamide showed no or little activity. In conclusion, these four drugs are possible candidates for new antimycobacterial agents, and animal experiments are now under way.
Two Boundaries Separate Borrelia burgdorferi Populations in North America
Authors: Margos Gabriele , Tsao Jean I. , Castillo-Ramírez Santiago , Girard Yvette A. , Hamer Sarah A. , Hoen Anne Gatewood , Lane Robert S. , Raper Steve L. , Ogden Nicholas H.
Published: June 2012

Understanding the spread of infectious diseases is crucial for implementing effective control measures. For this, it is important to obtain information on the contemporary population structure of a disease agent and to infer the evolutionary processes that may have shaped it. Here, we investigate on a continental scale the population structure ofBorrelia burgdorferi, the causative agent of Lyme borreliosis (LB), a tick-borne disease, in North America. We test the hypothesis that the observed population structure is congruent with recent population expansions and that these were preceded by bottlenecks mostly likely caused by the near extirpation in the 1900s of hosts required for sustaining tick populations. Multilocus sequence typing and complementary population analytical tools were used to evaluateB. burgdorferisamples collected in the Northeastern, Upper Midwestern, and Far-Western United States and Canada. The spatial distribution of sequence types (STs) and inferred population boundaries suggest that the current populations are geographically separated. One major population boundary separated westernB. burgdorferipopulations transmitted byIxodes pacificusin California from Eastern populations transmitted byI. scapularis; the other divided Midwestern and Northeastern populations. However, populations from all three regions were genetically closely related. Together, our findings suggest that although the contemporary populations of North AmericanB. burgdorferinow comprise three geographically separated subpopulations with no or limited gene flow among them, they arose from a common ancestral population. A comparative analysis of theB. burgdorferiouter surface protein C (ospC) gene revealed novel linkages and provides additional insights into the genetic characteristics of strains.
Accumulation of Intracellular Glycogen and Trehalose by Propionibacterium freudenreichii under Conditions Mimicking Cheese Ripening in the Cold
Authors: Dalmasso Marion , Aubert Julie , Even Sergine , Falentin Hélène , Maillard Marie-Bernadette , Parayre Sandrine , Loux Valentin , Tanskanen Jarna , Thierry Anne
Published: June 2012

Seven
Propionibacterium freudenreichii
strains exhibited similar responses when placed at 4°C. They slowed down cell machinery, displayed cold stress responses, and rerouted their carbon metabolism toward trehalose and glycogen synthesis, both accumulated in cells. These results highlight the molecular basis of long-term survival of
P. freudenreichii
in the cold.
Virulence and Metabolic Characteristics of Salmonella enterica Serovar Enteritidis Strains with Different
sefD
Variants in Hens
Authors: Morales Cesar A. , Guard Jean , Sanchez-Ingunza Roxana , Shah Devendra H. , Harrison Mark
Published: June 2012

Salmonella enterica
serovar Enteritidis is one of a few
Salmonella enterica
serotypes that has SEF14 fimbriae encoded by the
sef
operon, which consists of 4 cotranscribed genes,
sefABCD
, regulated by
sefR
. A parental strain was used to construct a
sefD
mutant and its complement, and all 3 strains were compared for gene expression, metabolic properties, and virulence characteristics in hens. Transcription of
sefD
by wild type was suppressed at 42°C and absent for the mutant under conditions where the complemented mutant had 10
3
times higher transcription. Growth of the complemented mutant was restricted in comparison to that of the mutant and wild type. Hens infected with the wild type and mutant showed decreased blood calcium and egg production, but infection with the complemented mutant did not. Thus, the absence of
sefD
correlated with increased metabolic capacity and enhanced virulence of the pathogen. These results suggest that any contribution that
sefD
makes to egg contamination is either unknown or would be limited to early transmission from the environment to the host. Absence of
sefD
, either through mutation or by suppression of transcription at the body temperature of the host, may contribute to the virulence of
Salmonella enterica
by facilitating growth on a wide range of metabolites.
Differential
In Vivo
Gene Expression of Major Leptospira Proteins in Resistant or Susceptible Animal Models
Authors: Matsui Mariko , Soupé Marie-Estelle , Becam Jérôme , Goarant Cyrille
Published: June 2012

Transcripts of
Leptospira
16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns
in vivo
compared to
in vitro
cultures. LipL32 expression was downregulated
in vivo
and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated
in vivo
. Thus, we demonstrated that a virulent strain of
Leptospira
differentially adapts its gene expression in the blood of infected animals.
Metagenomic Analyses of Drinking Water Receiving Different Disinfection Treatments
Authors: Gomez-Alvarez Vicente , Revetta Randy P. , Santo Domingo Jorge W.
Published: June 2012

A metagenome-based approach was used to assess the taxonomic affiliation and function potential of microbial populations in free-chlorine-treated (CHL) and monochloramine-treated (CHM) drinking water (DW). In all, 362,640 (averaging 544 bp) and 155,593 (averaging 554 bp) pyrosequencing reads were analyzed for the CHL and CHM samples, respectively. Most annotated proteins were found to be of bacterial origin, although eukaryotic, archaeal, and viral proteins were also identified. Differences in community structure and function were noted. Most notably,Legionella-like genes were more abundant in the CHL samples while mycobacterial genes were more abundant in CHM samples. Genes associated with multiple disinfectant mechanisms were identified in both communities. Moreover, sequences linked to virulence factors, such as antibiotic resistance mechanisms, were observed in both microbial communities. This study provides new insights into the genetic network and potential biological processes associated with the molecular microbial ecology of DW microbial communities.
Internalization and Dissemination of Human Norovirus and Animal Caliciviruses in Hydroponically Grown Romaine Lettuce
Authors: DiCaprio Erin , Ma Yuanmei , Purgianto Anastasia , Hughes John , Li Jianrong
Published: June 2012

Fresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both pre- and postharvest stages. However, the ecology of human NoV in fresh produce is poorly understood. In this study, we determined whether human NoV and its surrogates can be internalized via roots and disseminated to edible portions of the plant. The roots of romaine lettuce growing in hydroponic feed water were inoculated with 1 × 10
6
RNA copies/ml of a human NoV genogroup II genotype 4 (GII.4) strain or 1 × 10
6
to 2 × 10
6
PFU/ml of animal caliciviruses (Tulane virus [TV] and murine norovirus [MNV-1]), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were homogenized, and viral titers and/or RNA copies were determined by plaque assay and/or real-time reverse transcription (RT)-PCR. For human NoV, high levels of viral-genome RNA (10
5
to 10
6
RNA copies/g) were detected in leaves, shoots, and roots at day 1 postinoculation and remained stable over the 14-day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (10
1
to 10
3
PFU/g) were detected in leaves and shoots at days 1 and 2 postinoculation, but virus reached a peak titer (10
5
to 10
6
PFU/g) at day 3 or 7 postinoculation. In addition, human NoV had a rate of internalization comparable with that of TV as determined by real-time RT-PCR, whereas TV was more efficiently internalized than MNV-1 as determined by plaque assay. Taken together, these results demonstrated that human NoV and animal caliciviruses became internalized via roots and efficiently disseminated to the shoots and leaves of the lettuce.
Firing Range Soils Yield a Diverse Array of Fungal Isolates Capable of Organic Acid Production and Pb Mineral Solubilization
Authors: Sullivan Tarah S. , Gottel Neil R. , Basta Nicholas , Jardine Philip M. , Schadt Christopher W.
Published: June 2012

Anthropogenic sources of lead contamination in soils include mining and smelting activities, effluents and wastes, agricultural pesticides, domestic garbage dumps, and shooting ranges. While Pb is typically considered relatively insoluble in the soil environment, some fungi may potentially contribute to mobilization of heavy metal cations by means of secretion of low-molecular-weight organic acids (LMWOAs). We sought to better understand the potential for metal mobilization within an indigenous fungal community at an abandoned shooting range in Oak Ridge, TN, where soil Pb contamination levels ranged from 24 to >2,700 mg Pb kg dry soil
−1
. We utilized culture-based assays to determine organic acid secretion and Pb-carbonate dissolution of a diverse collection of soil fungal isolates derived from the site and verified isolate distribution patterns within the community by 28S rRNA gene analysis of whole soils. The fungal isolates examined included both ascomycetes and basidiomycetes that excreted high levels (up to 27 mM) of a mixture of LMWOAs, including oxalic and citric acids, and several isolates demonstrated a marked ability to dissolve Pb-carbonate at high concentrations up to 10.5 g liter
−1
(18.5 mM) in laboratory assays. Fungi within the indigenous community of these highly Pb-contaminated soils are capable of LMWOA secretion at levels greater than those of well-studied model organisms, such as
Aspergillus niger
. Additionally, these organisms were found in high relative abundance (>1%) in some of the most heavily contaminated soils. Our data highlight the need to understand more about autochthonous fungal communities at Pb-contaminated sites and how they may impact Pb biogeochemistry, solubility, and bioavailability, thus consequently potentially impacting human and ecosystem health.
Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2
Authors: Furuya Toshiki , Arai Yuka , Kino Kuniki
Published: June 2012

Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with
p
-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore,
Escherichia coli
cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for
p
-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.
Chemotaxis to Furan Compounds by Furan-Degrading Pseudomonas Strains
Authors: Nichols Nancy N. , Lunde Tristan A. , Graden Kevin C. , Hallock Kate A. , Kowalchyk Cara K. , Southern Rebecca M. , Soskin Ellen J. , Ditty Jayna L.
Published: June 2012

Two
Pseudomonas
strains known to utilize furan derivatives were shown to respond chemotactically to furfural, 5-hydroxymethylfurfural, furfuryl alcohol, and 2-furoic acid. In addition, a LysR-family regulatory protein known to regulate furan metabolic genes was found to be involved in regulating the chemotactic response.
Enumerating Viruses in Coral Mucus
Authors: Leruste Amandine , Bouvier Thierry , Bettarel Yvan
Published: June 2012

The distribution of viruses inhabiting the coral mucus remains undetermined, as there is no suitable standardized procedure for their separation from this organic matrix, principally owing to its viscosity and autofluorescence. Seven protocols were tested, and the most efficient separations were obtained from a chemical treatment requiring potassium citrate.
The Physiological Opportunism of Desulfitobacterium hafniense Strain TCE1 towards Organohalide Respiration with Tetrachloroethene
Authors: Duret Aurélie , Holliger Christof , Maillard Julien
Published: June 2012

Desulfitobacterium hafniense
strain TCE1 is capable of metabolically reducing tetra- and trichloroethenes by organohalide respiration. A previous study revealed that the
pce
gene cluster responsible for this process is located on an active composite transposon, Tn-
Dha1
. In the present work, we investigated the effects on the stability of the transposon during successive subcultivations of strain TCE1 in a medium depleted of tetrachloroethene. At the physiological level, an increased fitness of the population was observed after 9 successive transfers and was correlated with a decrease in the level of production of the PceA enzyme. The latter observation was a result of the gradual loss of the
pce
genes in the population of strain TCE1 and not of a regulation mechanism, as was postulated previously for a similar phenomenon described for
Sulfurospirillum multivorans
. A detailed molecular analysis of genetic rearrangements occurring around Tn-
Dha1
showed two independent but concomitant events, namely, the transposition of the first insertion sequence, IS
Dha1
-a, and homologous recombination across identical copies of IS
Dha1
flanking the transposon. A new model is proposed for the genetic heterogeneity around Tn-
Dha1
in
D. hafniense
strain TCE1, along with some considerations for the cleavage mechanism mediated by the transposase TnpA1 encoded by IS
Dha1
.
The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain
Authors: Rodríguez-Rubio Lorena , Gutiérrez Dolores , Martínez Beatriz , Rodríguez Ana , Götz Friedrich , García Pilar
Published: June 2012

Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from
Staphylococcus aureus
bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits
in vitro
lytic activity against live
S. aureus
cells, an activity that could eventually find use in the treatment of infections.
Alternative Approach To Modeling Bacterial Lag Time, Using Logistic Regression as a Function of Time, Temperature, pH, and Sodium Chloride Concentration
Authors: Koseki Shige , Nonaka Junko
Published: June 2012

The objective of this study was to develop a probabilistic model to predict the end of lag time (λ) during the growth of
Bacillus cereus
vegetative cells as a function of temperature, pH, and salt concentration using logistic regression. The developed λ model was subsequently combined with a logistic differential equation to simulate bacterial numbers over time. To develop a novel model for λ, we determined whether bacterial growth had begun, i.e., whether λ had ended, at each time point during the growth kinetics. The growth of
B. cereus
was evaluated by optical density (OD) measurements in culture media for various pHs (5.5 ∼ 7.0) and salt concentrations (0.5 ∼ 2.0%) at static temperatures (10 ∼ 20°C). The probability of the end of λ was modeled using dichotomous judgments obtained at each OD measurement point concerning whether a significant increase had been observed. The probability of the end of λ was described as a function of time, temperature, pH, and salt concentration and showed a high goodness of fit. The λ model was validated with independent data sets of
B. cereus
growth in culture media and foods, indicating acceptable performance. Furthermore, the λ model, in combination with a logistic differential equation, enabled a simulation of the population of
B. cereus
in various foods over time at static and/or fluctuating temperatures with high accuracy. Thus, this newly developed modeling procedure enables the description of λ using observable environmental parameters without any conceptual assumptions and the simulation of bacterial numbers over time with the use of a logistic differential equation.
Evaluation of Gene Modification Strategies for the Development of Low-Alcohol-Wine Yeasts
Authors: Varela C. , Kutyna D. R. , Solomon M. R. , Black C. A. , Borneman A. , Henschke P. A. , Pretorius I. S. , Chambers P. J.
Published: June 2012

Saccharomyces cerevisiaehas evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO2. Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene,GPD1, was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites.
Pyrosequencing-Derived Bacterial, Archaeal, and Fungal Diversity of Spacecraft Hardware Destined for Mars
Authors: La Duc Myron T. , Vaishampayan Parag , Nilsson Henrik R. , Torok Tamas , Venkateswaran Kasthuri
Published: June 2012

Spacecraft hardware and assembly cleanroom surfaces (233 m
2
in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m
2
) than colocated spacecraft hardware (187 OTU; 162 m
2
). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space.
Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation
Authors: Sygmund Christoph , Kracher Daniel , Scheiblbrandner Stefan , Zahma Kawah , Felice Alfons K. G. , Harreither Wolfgang , Kittl Roman , Ludwig Roland
Published: June 2012

The genome of
Neurospora crassa
encodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome of
N. crassa
and preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced in
Pichia pastoris
. With the cytochrome domain-dependent one-electron acceptor cytochrome
c
, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (
k
cat
and

K
m

values) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the heme
b
cofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) from
N. crassa
was expressed in
P. pastoris
. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposed
in vivo
function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.
MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria
Authors: Xu Ying , Gao Xiaoli , Wang Song-He , Liu Hong , Williams Peter A. , Zhou Ning-Yi
Published: June 2012

Klebsiella pneumoniae
M5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene cluster
mhbRTDHIM
conferred the ability to grow on 3-hydroxybenzoate to
Escherichia coli
and
Pseudomonas putida
PaW340. Four of the six genes (
mhbDHIM
) encode enzymes converting 3-hydroxybenzoate to pyruvate and fumarate via gentisate. MhbR is a gene activator, and MhbT is a hypothetical protein belonging to the transporter of the aromatic acid/H
+
symporter family. Since a transporter for 3-hydrxybenzoate uptake has not been characterized to date, we investigated whether MhbT is responsible for the uptake of 3-hydroxybenzoate, its metabolic intermediate gentisate, or both. The MhbT-green fluorescent protein (GFP) fusion protein was located on the cytoplasmic membrane.
P. putida
PaW340 containing
mhbR
Δ
TDHIM
could not grow on 3-hydroxybenzoate; however, supplying
mhbT
in
trans
allowed the bacterium to grow on the substrate.
K. pneumoniae
M5a1 and
P. putida
PaW340 containing recombinant MhbT transported
14
C-labeled 3-hydroxybenzoate but not
14
C-labeled gentisate and benzoate into the cells. Site-directed mutagenesis of two conserved amino acid residues (Asp-82 and Asp-314) and a less-conserved residue (Val-311) among the members of the symporter family in the hydrophilic cytoplasmic loops resulted in the loss of 3-hydroxybenzoate uptake by
P. putida
PaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.
Randomized, Controlled Trial of a 13-Valent Pneumococcal Conjugate Vaccine Administered Concomitantly with an Influenza Vaccine in Healthy Adults
Authors: Frenck Robert W. , Gurtman Alejandra , Rubino John , Smith William , van Cleeff Martin , Jayawardene Deepthi , Giardina Peter C. , Emini Emilio A. , Gruber William C. , Scott Daniel A. , Schmöle-Thoma Beate
Published: June 2012

A randomized, double-blind, phase 3 trial evaluated the immunogenicity, safety, and tolerability of a 13-valent pneumococcal conjugate vaccine (PCV13) coadministered with trivalent inactivated influenza vaccine (TIV) in pneumococcal vaccine-naive adults. Participants ages 50 to 59 years (n= 1,116) received TIV with PCV13 (group 1) or placebo (group 2) (1:1 randomization); 1 month later, group 1 received placebo and group 2 received PCV13. A hemagglutination inhibition (HAI) assay for TIV and a standardized enzyme-linked immunosorbent assay for pneumococcal serotype-specific immunoglobulin G (IgG) were performed and opsonophagocytic activity (OPA) titers (assessedpost hoc) were measured at baseline and 1 and 2 months postvaccination. The rises in HAI assay geometric mean titer (GMT) and percentage of participants in groups 1 and 2 with ≥4-fold increases in HAI responses (A/H1N1, 84.0% and 81.2%, respectively; A/H3N2, 71.1% and 69.5%, respectively; and B, 60.6% and 60.3%, respectively) were similar. In group 1, all serotypes met the predefined IgG geometric mean concentration (GMC) ratio noninferiority criterion relative to group 2, but GMCs were lower in group 1 than group 2. When comparing group 1 with group 2, 5 serotypes did not meet the OPA GMT ratio noninferiority criterion, and OPA GMTs were significantly lower for 10 serotypes. PCV13 injection site reactions were similar and mostly mild in both groups. Systemic events were more frequent in group 1 (86.2%) than group 2 (76.7%;P< 0.001); no vaccine-related serious adverse events occurred. Coadministration of PCV13 and TIV was well tolerated but associated with lower PCV13 antibody responses and is of unknown clinical significance. Given the positive immunologic attributes of PCV13, concomitant administration with TIV should be dictated by clinical circumstances.
Draft Genome Sequence of Salt-Tolerant Yeast Debaryomyces hansenii var. hansenii MTCC 234
Authors: Kumar Shailesh , Randhawa Anmoldeep , Ganesan Kaliannan , Raghava Gajendra Pal Singh , Mondal Alok K.
Published: June 2012

Debaryomyces hansenii
is one of the most halotolerant species of yeast, and the genome sequence of
D. hansenii
strain CBS767 is already available. Here we report the 11.46-Mb draft genome of
D. hansenii
strain MTCC 234, which is even more halotolerant than strain CBS767. Comparative analysis of these sequences would definitely provide further insight into the halotolerance of this yeast.
Progress in Antifungal Susceptibility Testing of Candida spp. by Use of Clinical and Laboratory Standards Institute Broth Microdilution Methods, 2010 to 2012
Authors: Pfaller M. A. , Diekema D. J.
Published: June 2012

Antifungal susceptibility testing of
Candida
has been standardized and refined and now may play a useful role in managing
Candida
infections. Important new developments include validation of 24-h reading times for all antifungal agents and the establishment of species-specific epidemiological cutoff values (ECVs) for the systemically active antifungal agents and both common and uncommon species of
Candida
. The clinical breakpoints (CBPs) for fluconazole, voriconazole, and the echinocandins have been revised to provide species-specific interpretive criteria for the six most common species. The revised CBPs not only are predictive of clinical outcome but also provide a more sensitive means of identifying those strains with acquired or mutational resistance mechanisms. This brief review serves as an update on the new developments in the antifungal susceptibility testing of
Candida
spp. using Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) methods.