bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multicenter Evaluation of a Sequence-Based Protocol for Subtyping Shiga Toxins and Standardizing Stx Nomenclature
Authors: Scheutz Flemming , Teel Louise D. , Beutin Lothar , Piérard Denis , Buvens Glenn , Karch Helge , Mellmann Alexander , Caprioli Alfredo , Tozzoli Rosangela , Morabito Stefano , Strockbine Nancy A. , Melton-Celsa Angela R. , Sanchez Maria , Persson Søren , O'Brien Alison D. 
Published: July 2012

ABSTRACT

When Shiga toxin-producing
Escherichia coli
(STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by
Shigella dysenteriae
type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and
stx
genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.
Phylogenetic Distribution of CTX-M- and Non-Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates: Group B2 Isolates, Except Clone ST131, Rarely Produce CTX-M Enzymes
Authors: Brisse Sylvain , Diancourt Laure , Laouénan Cédric , Vigan Marie , Caro Valérie , Arlet Guillaume , Drieux Laurence , Leflon-Guibout Véronique , Mentré France , Jarlier Vincent , Nicolas-Chanoine Marie-Hélène ,   the Coli β Study Group
Published: July 2012

ABSTRACT

Escherichia coli
is the species most frequently associated with clinical infections by extended-spectrum-β-lactamase (ESBL)-producing isolates, with the CTX-M ESBL enzymes being predominant and found in genetically diverse
E. coli
isolates. The main objective of this study was to compare, on the basis of a case-control design, the phylogenetic diversity of 152 CTX-M-producing and 152 non-ESBL-producing clinical
E. coli
isolates. Multilocus sequence typing revealed that even though CTX-M enzymes were largely disseminated across the diversity of
E. coli
isolates, phylogenetic group B2 showed a particularly heterogeneous situation. First, clone ST131 of group B2 was strongly associated with CTX-M production (55 [79%] of 70 isolates), with CTX-M-15 being predominant. Second, the remaining members of group B2 were significantly less frequently associated with CTX-M production (9 [12%] of 75) than
E. coli
phylogenetic groups A, B1, and D (88 [55%] of 159). CTX-M-producing ST131
E. coli
isolates were significantly more frequent in patients hospitalized in geriatric wards or long-term care facilities. Besides, the non-ESBL ST131 isolates significantly more frequently showed resistance to penicillins than the non-ESBL, non-ST131 isolates did. In conclusion, the present study emphasizes the particular antimicrobial resistance and epidemiologic characteristics of clone ST131 within group B2, which could result from the higher antibiotic exposure of this clone, as it is the predominant clone of group B2 carried in the human gut.
Detection of Kudoa septempunctata 18S Ribosomal DNA in Patient Fecal Samples from Novel Food-Borne Outbreaks Caused by Consumption of Raw Olive Flounder (Paralichthys olivaceus)
Authors: Harada Tetsuya , Kawai Takao , Jinnai Michio , Ohnishi Takahiro , Sugita-Konishi Yoshiko , Kumeda Yuko 
Published: July 2012

ABSTRACT

Kudoa septempunctata
is a newly identified myxosporean parasite of olive flounder (
Paralichthys olivaceus
) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of
K. septempunctata
18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect
K. septempunctata
in feces. Simultaneously, we compared the relative extraction efficacy of
K. septempunctata
DNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting
K. septempunctata
DNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 10
1
spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of
K. septempunctata
DNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by
K. septempunctata
in laboratory investigations.
Hepatitis C Virus-Mediated Inhibition of Cathepsin S Increases Invariant-Chain Expression on Hepatocyte Surface
Authors: Kim Hangeun , Mazumdar Budhaditya , Bose Sandip K. , Meyer Keith , Di Bisceglie Adrian M. , Hoft Daniel F. , Ray Ranjit 
Published: July 2012

ABSTRACTHepatocytes are the main source of hepatitis C virus (HCV) replication and contain the maximum viral load in an infected person. Chronic HCV infection is characterized by weak cellular immune responses to viral proteins. Cathepsin S is a lysosomal cysteine protease and controls HLA-DR–antigen complex presentation through the degradation of the invariant chain. In this study, we examined the effect of HCV proteins on cathepsin S expression and found it to be markedly decreased in dendritic cells (DCs) exposed to HCV or in hepatocytes expressing HCV proteins. The downregulation of cathepsin S was mediated by HCV core and NS5A proteins involving inhibition of the transcription factors interferon regulatory factor 1 (IRF-1) and upstream stimulatory factor 1 (USF-1) in gamma interferon (IFN-γ)-treated hepatocytes. Inhibition of cathepsin S by HCV proteins increased cell surface expression of the invariant chain. In addition, hepatocytes stably transfected with HCV core or NS5A inhibited HLA-DR expression. Together, these results suggested that HCV has an inhibitory role on cathepsin S-mediated major histocompatibility complex (MHC) class II maturation, which may contribute to weak immunogenicity of viral antigens in chronically infected humans.
Hepatitis E Virus Quasispecies and the Outcome of Acute Hepatitis E in Solid-Organ Transplant Patients
Authors: Lhomme Sebastien , Abravanel Florence , Dubois Martine , Sandres-Saune Karine , Rostaing Lionel , Kamar Nassim , Izopet Jacques 
Published: July 2012

ABSTRACTHepatitis E virus (HEV) infections are responsible for chronic hepatitis in immunocompromised patients, and this can evolve to cirrhosis. Like all RNA viruses, HEV exists as a mixture of heterogeneous viruses defining quasispecies. The relationship between the genetic heterogeneity described as a quasispecies, cytokine secretion, and the outcome of acute hepatitis in immunocompromised patients remains to be elucidated. We cloned and sequenced the region encoding the M and P capsid domains of HEV from eight solid-organ transplant (SOT) patients with acute HEV infection who subsequently cleared the virus and from eight SOT patients whose infection became chronic. We analyzed the cytokines and chemokines in the sera of these SOT patients by multianalyte profiling. The nucleotide sequence entropy and genetic distances were greater in patients whose infections became chronic. A lowerKa/Ksratio was associated with the persistence of HEV. The patients who developed chronic infection had lower serum concentrations of interleukin-1 (IL-1) receptor antagonist and soluble IL-2 receptor. Increased concentrations of the chemokines implicated in leukocyte recruitment to the liver were associated with persistent infection. Those patients with chronic HEV infection and progressing liver fibrosis had less quasispecies diversification during the first year than patients without liver fibrosis progression. Great quasispecies heterogeneity, a weak inflammatory response, and high serum concentrations of the chemokines involved in leukocyte recruitment to the liver in the acute phase were associated with persistent HEV infection. Slow quasispecies diversification during the first year was associated with rapidly developing liver fibrosis.
Combination of Immune and Viral Factors Distinguishes Low-Risk versus High-Risk HIV-1 Disease Progression in HLA-B*5701 Subjects
Authors: Norström Melissa M. , Buggert Marcus , Tauriainen Johanna , Hartogensis Wendy , Prosperi Mattia C. , Wallet Mark A. , Hecht Frederick M. , Salemi Marco , Karlsson Annika C. 
Published: July 2012

ABSTRACT

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1
in vivo
evolution and epitope-specific CD8
+
T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4
+
T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8
+
T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8
+
T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.
Binding of Cellular Export Factor REF/Aly by Kaposi's Sarcoma-Associated Herpesvirus (KSHV) ORF57 Protein Is Not Required for Efficient KSHV Lytic Replication
Authors: Li Da-Jiang , Verma Dinesh , Swaminathan Sankar 
Published: July 2012

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is expressed early during lytic KSHV replication, enhances expression of many KSHV genes, and is essential for virus production. ORF57 is a member of a family of proteins conserved among all human and many animal herpesviruses that are multifunctional regulators of gene expression and act posttranscriptionally to increase accumulation of their target mRNAs. The mechanism of ORF57 action is complex and may involve effects on mRNA transcription, stability, and export. ORF57 directly binds to REF/Aly, a cellular RNA-binding protein component of the TREX complex that mediates RNA transcription and export. We analyzed the effects of an ORF57 mutation known to abrogate REF/Aly binding and demonstrate that the REF-binding mutant is impaired in activation of viral mRNAs and noncoding RNAs confined to the nucleus. Although the inability to bind REF leads to decreased ORF57 activity in enhancing gene expression, there is no demonstrable effect on nuclear export of viral mRNA or the ability of ORF57 to support KSHV replication and virus production. These data indicate that REF/Aly-ORF57 interaction is not essential for KSHV lytic replication but may contribute to target RNA stability independent of effects on RNA export, suggesting a novel role for REF/Aly in viral RNA metabolism.
N-Linked Glycosylation of GP5 of Porcine Reproductive and Respiratory Syndrome Virus Is Critically Important for Virus Replication
In Vivo
Authors: Wei Zuzhang , Lin Tao , Sun Lichang , Li Yanhua , Wang Xiaoming , Gao Fei , Liu Runxia , Chen Chunyan , Tong Guangzhi , Yuan Shishan 
Published: July 2012

ABSTRACT

It has been proposed that the N-linked glycan addition at certain sites in GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) is important for production of infectious viruses and viral infectivity. However, such specific N-linked glycosylation sites do not exist in some field PRRSV isolates. This implies that the existence of GP5-associated glycan
per se
is not vital to the virus life cycle. In this study, we found that mutation of individual glycosylation sites at N30, N35, N44, and N51 in GP5 did not affect virus infectivity in cultured cells. However, the mutants carrying multiple mutations at N-linked glycosylation sites in GP5 had significantly reduced virus yields compared with the wild-type (wt) virus. As a result, no viremia and antibody response were detected in piglets that were injected with a mutant without all N-linked glycans in GP5. These results suggest that the N-linked glycosylation of GP5 is critically important for virus replication
in vivo
. The study also showed that removal of N44-linked glycan from GP5 increased the sensitivity of mutant virus to convalescent-phase serum samples but did not elicit a high-level neutralizing antibody response to wt PRRSV. The results obtained from the present study have made significant contributions to better understanding the importance of glycosylation of GP5 in the biology of PRRSV.
The Role of Clonal Interference in the Evolutionary Dynamics of Plasmid-Host Adaptation
Authors: Hughes Julie M. , Lohman Brian K. , Deckert Gail E. , Nichols Eric P. , Settles Matt , Abdo Zaid , Top Eva M. 
Published: July 2012

ABSTRACT

Promiscuous plasmids replicate in a wide range of bacteria and therefore play a key role in the dissemination of various host-beneficial traits, including antibiotic resistance. Despite the medical relevance, little is known about the evolutionary dynamics through which drug resistance plasmids adapt to new hosts and thereby persist in the absence of antibiotics. We previously showed that the incompatibility group P-1 (IncP-1) minireplicon pMS0506 drastically improved its stability in novel host
Shewanella oneidensis
MR-1 after 1,000 generations under antibiotic selection for the plasmid. The only mutations found were those affecting the N terminus of the plasmid replication initiation protein TrfA1. Our aim in this study was to gain insight into the dynamics of plasmid evolution. Changes in stability and genotype frequencies of pMS0506 were monitored in evolving populations of MR-1 (pMS0506). Genotypes were determined by sequencing
trfA1
amplicons from individual clones and by 454 pyrosequencing of whole plasmids from entire populations. Stability of pMS0506 drastically improved by generation 200. Many evolved plasmid genotypes with point mutations as well as in-frame and frameshift deletions and duplications in
trfA1
were observed in all lineages with both sequencing methods. Strikingly, multiple genotypes were simultaneously present at high frequencies (>10%) in each population. Their relative abundances changed over time, but after 1,000 generations only one or two genotypes dominated the populations. This suggests that hosts with different plasmid genotypes were competing with each other, thus affecting the evolutionary trajectory. Plasmids can thus rapidly improve their stability, and clonal interference plays a significant role in plasmid-host adaptation dynamics.

IMPORTANCE
Promiscuous plasmids play an important role in the spread of antibiotic resistance and many other traits between closely and distantly related bacteria. However, little is known about the dynamics by which these broad-host-range antibiotic resistance plasmids adapt to novel bacteria and thereby become more persistent, even in the absence of antibiotics. In this study, we show that after no more than 200 generations of growth in the presence of antibiotics, a plasmid that was initially poorly maintained in a novel bacterial host evolved to become drastically more persistent in the absence of antibiotics. In each of the evolving populations, an unexpectedly large number of bacterial variants arose with distinct mutations in the plasmid’s replication initiation protein. Our results suggest that clonal interference, characterized by competition between variant clones in a population, plays a major role in the evolution of the persistence of drug resistance.
Modified Lipooligosaccharide Structure Protects Nontypeable Haemophilus influenzae from IgM-Mediated Complement Killing in Experimental Otitis Media
Authors: Langereis Jeroen D. , Stol Kim , Schweda Elke K. , Twelkmeyer Brigitte , Bootsma Hester J. , de Vries Stefan P. W. , Burghout Peter , Diavatopoulos Dimitri A. , Hermans Peter W. M. 
Published: July 2012

ABSTRACT

Nontypeable
Haemophilus influenzae
(NTHi) is a Gram-negative, human-restricted pathogen. Although this bacterium typically colonizes the nasopharynx in the absence of clinical symptoms, it is also one of the major pathogens causing otitis media (OM) in children. Complement represents an important aspect of the host defense against NTHi. In general, NTHi is efficiently killed by complement-mediated killing; however, various resistance mechanisms have also evolved. We measured the complement resistance of NTHi isolates isolated from the nasopharynx and the middle ear fluids of OM patients. Furthermore, we determined the molecular mechanism of NTHi complement resistance. Complement resistance was strongly increased in isolates from the middle ear, which correlated with decreased binding of IgM. We identified a crucial role for the R2866_0112 gene in complement resistance. Deletion of this gene altered the lipooligosaccharide (LOS) composition of the bacterium, which increased IgM binding and complement-mediated lysis. In a novel mouse model of coinfection with influenza virus, we demonstrate decreased virulence for the R2866_0112 deletion mutant. These findings identify a mechanism by which NTHi modifies its LOS structure to prevent recognition by IgM and activation of complement. Importantly, this mechanism plays a crucial role in the ability of NTHi to cause OM.

IMPORTANCE
Nontypeable
Haemophilus influenzae
(NTHi) colonizes the nasopharynx of especially young children without any obvious symptoms. However, NTHi is also a major pathogen in otitis media (OM), one of the most common childhood infections. Although this pathogen is often associated with OM, the mechanism by which this bacterium is able to cause OM is largely unknown. Our study addresses a key biological question that is highly relevant for child health: what is the molecular mechanism that enables NTHi to cause OM? We show that isolates collected from the middle ear fluid exhibit increased complement resistance and that the lipooligosaccharide (LOS) structure determines IgM binding and complement activation. Modification of the LOS structure decreased NTHi virulence in a novel NTHi-influenza A virus coinfection OM mouse model. Our findings may also have important implications for other Gram-negative pathogens harboring LOS, such as
Neisseria meningitidis
,
Moraxella catarrhalis
, and
Bordetella pertussis
.
External Validity in Randomised Controlled Trials of Acupuncture for Osteoarthritis Knee Pain
Authors: Purepong Nithima , Jitvimonrat Anusorn , Sitthipornvorakul Ekalak , Eksakulkla Sukanya , Janwantanakul Prawit 
Published: July 2012

Objectives To assess two aspects of the external validity of acupuncture research for osteoarthritis knee pain and determine the common acupoints and treatment parameters used. Methods The external validity of 16 randomised controlled trials (RCTs) was investigated using a scale consisting of two aspects: reporting and performance. The reporting aspect included acupuncturist's background, study location, treatment detailed, patient characteristics, positive trial results, adverse effects and between-group statistical differences, whereas treatment appropriateness, appropriate controls and outcomes were classified as the performance aspect. Acupuncture treatment in RCTs was compared with common practice according to the literature sources and survey of acupuncturists working in different parts of Thailand. Results The levels of external validity for the reporting and performance aspects were in the range of 31.3% to 100%. Statistic values such as mean difference and confidence interval were reported by the minority of trials (43.8%). Patient satisfaction and quality of life were seldom used (31.3%). There were minor differences between research and practice in terms of the points used (25.0%), number of treatment sessions (6.3%) and frequency (12.5%). The most frequently used points were ST34, ST35, ST36, SP6, SP9, SP10, GB34, Xiyan and ah shi points, and the commonly used treatment parameters were 20 minutes, 10–15 sessions and two treatments weekly. Conclusions Reporting of the external validity of acupuncture RCTs for knee pain was notably inadequate in terms of trial setting, treatment provider and statistical reporting. The majority of studies involved appropriate controls and outcomes and applied acupuncture treatments in line with practice.