Prevalence and risk factors for colonization by Clostridium difficile and extended-spectrum ß-lactamase-producing Enterobacteriaceae in rehabilitation clinics in Germany.

BACKGROUND: Rehabilitation clinics may vary widely in terms of type of care provided, duration of hospital stay, and case severity. Few data are available on prevalence of Clostridium difficile or extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) colonization in rehabilitation clinics in Germany. AIM: This study investigated the frequency of intestinal colonization by these pathogens among patients in rehabilitation clinics of different specialization. METHODS: In the scope of a point prevalence study, faecal samples and demographic and clinical data were collected in five rehabilitation clinics. Samples were screened for C. difficile and ESBL-E by culture. Isolates were characterized by polymerase chain reaction for C. difficile toxins A and B, for ß-lactamase genes, and by molecular typing including pulsed-field gel electrophoresis and PCR-based ribotyping. FINDINGS: Of 305 patients screened, 11.1% were colonized by toxigenic C. difficile and 7.5% by ESBL-E. Colonization rates differed markedly between facilities, ranging from 1.6% to 26.3% for C. difficile and from zero to 23.7% for ESBL-E. Prevalence of colonization by C. difficile and ESBL-E was higher in neurological rehabilitation clinics than in clinics with other specialties (P<0.001). Molecular typing revealed six patients from one neurological rehabilitation clinic harbouring a unique C. difficile strain (ribotype 017). CTX-M-15 was the most prevalent ESBL type. We detected several indistinguishable pairs of ESBL-E isolates within some facilities. CONCLUSION: Significant differences were found in the prevalence of C. difficile and ESBL-E between rehabilitation clinics. Facilities providing specialized medical care for critically ill patients had higher prevalence rates. These results may help to delineate the requirements for infection prevention and control in rehabilitation clinics.

Degradation kinetics of chlorinated aliphatic hydrocarbons by methane oxidizers naturally-associated with wetland plant roots.

Chlorinated aliphatic hydrocarbons (CAHs) are common groundwater contaminants that can be removed from the environment by natural attenuation processes. CAH biodegradation can occur in wetland environments by reductive dechlorination as well as oxidation pathways. In particular, CAH oxidation may occur in vegetated wetlands, by microorganisms that are naturally associated with the roots of wetland plants. The main objective of this study was to evaluate the cometabolic degradation kinetics of the CAHs, cis-1,2-dichloroethene (cisDCE), trichloroethene (TCE), and 1,1,1-trichloroethane (1,1,1TCA), by methane-oxidizing bacteria associated with the roots of a typical wetland plant in soil-free system. Laboratory microcosms with washed live roots investigated aerobic, cometabolic degradation of CAHs by the root-associated methane-oxidizing bacteria at initial aqueous [CH4] ~1.9mgL(-1), and initial aqueous [CAH] ~150µgL(-1); cisDCE and TCE (in the presence of 1,1,1TCA) degraded significantly, with a removal efficiency of approximately 90% and 46%, respectively. 1,1,1TCA degradation was not observed in the presence of active methane oxidizers. The pseudo first-order degradation rate-constants of TCE and cisDCE were 0.12±0.01 and 0.59±0.07d(-1), respectively, which are comparable to published values. However, their biomass-normalized degradation rate constants obtained in this study were significantly smaller than pure-culture studies, yet they were comparable to values reported for biofilm systems. The study suggests that CAH removal in wetland plant roots may be comparable to processes within biofilms. This has led us to speculate that the active biomass may be on the root surface as a biofilm. The cisDCE and TCE mass losses due to methane oxidizers in this study offer insight into the role of shallow, vegetated wetlands as an environmental sink for such xenobiotic compounds.

The porcine lymphotropic herpesvirus 1 encodes functional regulators of gene expression.

The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1(h), ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta, (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1 of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1(h) and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation.

The core 2 beta-1,6-N-acetylglucosaminyltransferase-mucin encoded by bovine herpesvirus 4 was acquired from an ancestor of the African buffalo.

The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only viral gene known to date that encodes a homologue of the cellular core 2 beta-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M). To investigate the origin and evolution of the Bo17 gene, we analyzed its distribution among BoHV-4 strains and determined the sequences of Bo17 from nine representative strains and of the C2GnT-M gene from six species of ruminants expected to encompass the group within which the gene acquisition occurred. Of 34 strains of BoHV-4, isolated from four different continents, all were found to contain the Bo17 gene. Phylogenetic analyses indicated that Bo17 was acquired from a recent ancestor of the African buffalo, implying that cattle subsequently acquired BoHV-4 by cross-species transmission. The rate of synonymous nucleotide substitution in Bo17 was estimated at 5 x 10(-8) to 6 x 10(-8) substitutions/site/year, consistent with previous estimates made under the assumption that herpesviruses have cospeciated with their hosts. The Bo17 gene acquisition was dated to around 1.5 million years ago. Bo17 sequences from BoHV-4 strains from African buffalo and from cattle formed two separate clades, estimated to have split about 700,000 years ago. Analysis of the ratio of nonsynonymous to synonymous nucleotide substitutions revealed a burst of amino acid replacements subsequent to the transfer of the cellular gene to the viral genome, followed by a return to a strong constraint on nonsynonymous changes during the divergence of contemporary BoHV-4 strains. The Bo17 gene represents the most recent of the known herpesvirus gene acquisitions and provides the best opportunity for learning more about this important process of viral evolution.

Transport issues and bioremediation modeling for the in situ aerobic co-metabolism of chlorinated solvents.

For aerobic co-metabolism of chlorinated solvents to occur, it is necessary that oxygen, a primary substrate, and the chlorinated compound all be available to an appropriate microorganism--that is, a microorganism capable of producing the nonspecific enzyme that will promote degradation of the contaminant while the primary substrate is aerobically metabolized. Thus, the transport processes that serve to mix the reactants are crucial in determining the rate and extent of biodegradation, particularly when considering in situ biodegradation. These transport processes intersect, at a range of scales, with the biochemical reactions. This paper reviews how the important processes contributing to aerobic co-metabolism of chlorinated solvents at different scales can be integrated into mathematical models. The application of these models to field-scale bioremediation is critically examined. It is demonstrated that modeling can be a useful tool in gaining insight into the physical, chemical, and biological processes relevant to aerobic co-metabolism, designing aerobic co-metabolic bioremediation systems, and predicting system performance. Research needs are identified that primarily relate to gaps in our current knowledge of inter-scale interactions.

A biomechanical evaluation of mandibular angle fracture plating techniques.

PURPOSE: The purpose of this investigation was evaluate the biomechanical behavior of a vast array of fixation philosophies and techniques that address mandibular angle fractures. MATERIALS AND METHODS: A total of 150 polyurethane synthetic mandible replicas (Synbone, Laudquart, Switzerland,) were used in this investigation. Five controls and 5 each of 14 different fixation philosophies and techniques were subjected to vertical loading at the incisal edge and then repeated for contralateral loading in the molar region by an Instron 1331 (Instron, Canton, MA) servohydraulic mechanical testing unit. The fixation philosophies and techniques evaluated were the lag screw technique, monocortical superior border plating techniques with varying sizes of plates and screws, monocortical 2-plate techniques with varying forms of fixation, monocortical tension band systems with associated bicortical stabilization plates of various types, and various forms of reconstruction plates. Load/displacement data within a 0 to 200 N range were recorded. Yield load, yield displacement, and stiffness were determined. Mean and standard deviations were calculated, and statistically significant differences within and among categories were determined using an analysis of variance (P <.05). Second-order polynomial best-fit curves were also created for each group to further evaluate and compare the mechanical behavior. RESULTS: For incisal edge loading, statistically significant differences (P <.05) were found for stiffness between some of the monocortical superior border fixation techniques, as well as for yield displacement between several forms of monocortical 2-plate fixation techniques. No other differences were found within categories or among the groups that best represented their categories. For contralateral molar loading, statistically significant differences existed within and among categories. CONCLUSIONS: Under the conditions of this experiment, all systems met or exceeded currently identified postoperative functional requirements for incisal edge loading, but failed to meet them for contralateral molar loading.

Sorption and biodegradation of vapor-phase organic compounds with wastewater sludge and food waste compost.

To test the possible use of composted food waste and wastewater sludge as biofilters to treat gas-phase volatile organic compounds (VOCs), batch experiments were conducted with an isolated strain that could degrade aromatic compounds under aerobic conditions. A benzene and trichloroethylene (TCE) mixture was used as the gas-phase pollutant in experiments with composted food waste, sludge, and soil. Under aerobic conditions, benzene was degraded as a primary substrate and TCE was degraded cometabolically, with water contents varying from 6 to 60% (volume of water added/volume of solid). Optimal water content for VOC removal was 12% for the soil, 36% for the composted food waste, and 48% for the sludge. The extent of VOC sorption and biodegradation at the optimal water content was different for each material. With the same initial VOC concentration, more VOCs were removed by sorption onto the composted food waste and the sludge, while less VOCs were biodegraded in comparison with the results using soil. The reason the biodegradation in the soil was greater may be partly attributed to the fact that, due to less sorption, the aqueous-phase concentration of VOCs, which microorganisms could utilize as a carbon source or cometabolize, was higher. We also speculate that the distribution of microorganisms in each medium affects the rate of biodegradation. A large number of microorganisms were attached to the composted food waste and sludge. Mass transfer of VOCs and oxygen to these microorganisms, which appear to have been heterogeneously distributed in clusters, may have been limited, resulting in hindered biodegradation.

Detection and multigenic characterization of a novel gammaherpesvirus in goats.

Evidence for the existence of a caprine gammaherpesvirus was obtained by analysis of peripheral blood leucocytes of goats with PCR assays that target the herpesvirus genes encoding the glycoprotein B (gB), the DNA polymerase (DPOL) and the terminase (TERM) with degenerate and deoxyinosine-substituted primers. A contiguous 3.6 kbp sequence extending from the gB to the DPOL gene was then determined with specific primers. All sequences (gB, DPOL and TERM) showed a close relationship with the corresponding genes of the Gammaherpesvirinae. Alignment of amino acid sequences revealed a particularly high percentage of identity with the ovine herpesvirus type 2 (>83%), followed by the alcelaphine herpesvirus 1 (>76%) and the bovine lymphotropic herpesvirus (>61%). Phylogenetic analyses confirmed these relationships. The putative novel goat herpesvirus from which these sequences originate was tentatively designated caprine herpesvirus 2. This virus is the first gammaherpesvirus recognized in goats.

Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus.

A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.

Genome sequence of bovine herpesvirus 4, a bovine Rhadinovirus, and identification of an origin of DNA replication.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus of cattle. The complete long unique coding region (LUR) of BoHV-4 strain 66-p-347 was determined by a shotgun approach. Together with the previously published noncoding terminal repeats, the entire genome sequence of BoHV-4 is now available. The LUR consists of 108,873 bp with an overall G+C content of 41.4%. At least 79 open reading frames (ORFs) are present in this coding region, 17 of them unique to BoHV-4. In contrast to herpesvirus saimiri and human herpesvirus 8, BoHV-4 has a reduced set of ORFs homologous to cellular genes. Gene arrangement as well as phylogenetic analysis confirmed that BoHV-4 is a member of the genus Rhadinovirus. In addition, an origin of replication (ori) in the genome of BoHV-4 was identified by DpnI assays. A minimum of 1.69 kbp located between ORFs 69 and 71 was sufficient to act as a cis signal for replication.

Identification and sequence analysis of the glycoprotein B gene of porcine cytomegalovirus.

Porcine cytomegalovirus (PCMV) is one of the pathogens that should be eliminated from pigs intended for use as organ donors in xenotransplantation. For this purpose, reliable diagnostic test systems are needed. To provide a basis for this goal and to analyse the evolutionary relationships of PCMV within the herpesvirus family, the putative glycoprotein B (gB) gene of PCMV was identified by assuming gene colinearity and a relative conservation of nucleotide sequences in comparison with closely related herpesviruses. Using this approach the complete nucleotide sequence of the PCMV gB gene was determined. A protein of 860 amino acids was deduced and a putative cleavage site, conserved cysteine residues, as well as potential N-terminal glycosylation motifs were identified. In a comparison of PCMV gB with the corresponding region of other herpesviruses, the highest identities were found with human herpesviruses 6 and 7 (HHV-6 and 7; 43.4% and 42.6%, respectively). Also in phylogenetic analysis, the PCMV gB clustered with HHV-6 and HHV-7. Between the complete gB sequences of five different PCMV strains and isolates from the United Kingdom, Germany, Spain, Japan and Sweden, differences of 3.4% were found, indicating a considerable intra-species variation. The characterisation of the protein deduced from the identified gene provides further evidence that this is indeed the gB gene of PCMV and provides important taxonomical information regarding PCMV. The identification of the gB gene should facilitate the development of sensitive and robust diagnostic methods for the PCMV screening of pigs.

Characterization of the DNA polymerase loci of porcine cytomegaloviruses from diverse geographic origins.

Porcine cytomegalovirus (PCMV) is an undesired pathogen in pigs intended for use as organ donors in xenotransplantation. In the present work, we characterized the first set of genes of PCMV. From a German isolate, the DNA polymerase (DPOL) locus was amplified and two complete open reading frames (ORF) as well as two partial ORFs including the complete DPOL gene and the 3'-end of the glycoprotein gB gene were sequenced. The deduced amino acid sequences showed the highest identities with the respective proteins of the betaherpesviruses, in particular those (ORFs 36-39) of the human herpesviruses 6 and 7 (HHV-6 and -7). In phylogenetic analysis, PCMV clustered also with HHV-6 and HHV-7. On this basis, PCMV could be firmly classified to the Betaherpesvirinae and tentatively assigned to the genus Roseolovirus. In addition to the German isolate, the DPOL gene was analysed from a British and a Japanese strain as well as a Spanish isolate. Differences of 0.4 to 1% were found on the nucleotide and the amino acid level. On the basis of the conserved regions, primer pairs were selected for PCR which detected PCMV in blood and tissue samples from four European countries. Therefore, these are the first nucleic acid-based test systems which were shown to universally detect PCMV. The application of these assays to organs of domestic pigs from Germany revealed a PCMV prevalence of > 50%.

Genetics of atopy in a mouse model: polymorphism of the IL-5 receptor alpha chain.

To study the genetics of atopy systematically we established a mouse model that provides the general phenotype of atopy: the early response characteristic of IgE-dependent eczema or atopic dermatitis, and the diagnostic test of atopy, the skin-prick test. Using an immediate cutaneous hypersensitivity test (ICHS) against birch pollen extract we could classify A/J and C57BL/6 (B6) inbred mouse strains respectively as high responder and low responders. The F1 hybrids were found to be high responders with incomplete penetrance. Backcrossing F1 mice to the low responder B6 strain yielded three classes of responders, high, intermediate, and low. A genome-wide microsatellite screen of the backcross progeny disclosed suggestive linkage to a microsatellite marker on chromosome 6 close to the locus of the IL-5 receptor alpha chain. Its allelic variation in A/J and B6 strains was investigated and two major differences were detected. Firstly, a nucleotide exchange in the 5' untranslated region of B6 mRNA resulted in increased transcription/translation of a reporter construct. Higher expression of the receptor on the cell surface would be expected to favor an allergic immune response. Secondly, the two alleles are differentially spliced so as to yield two soluble isoforms in A/J mice versus one in B6 mice. Higher expression of soluble IL-5R would be expected to reduce the level of allergy through capture of IL-5. Thus both findings conform to the expectation based on susceptibility to atopy and thus identify the IL-5R alpha chain as a likely contributor to the genetics of atopy.

Early aldosterone effect in distal colon by transcriptional regulation of ENaC subunits.

Aldosterone-induced sodium absorption is mediated by the epithelial Na(+) channel (ENaC). It is thought that the "early effect" is not based on genomic regulation of ENaC expression, because ENaC subunit transcription was reported to start later than Na(+) transport. We investigated electrogenic Na(+) absorption (J(Na)) and, in identical tissues, mRNA expression of ENaC subunits in early (EDC) and late (LDC) distal colon of the rat. In both segments, 8-h in vitro incubation with 3 nM aldosterone enhanced expression of beta- and gamma-ENaC mRNA and induced J(Na). J(Na) was 10 times higher in LDC than in EDC. alpha-ENaC mRNA was unchanged in EDC, whereas it decreased in LDC. In LDC, beta- and gamma-ENaC mRNA was induced 1 h after aldosterone addition, whereas J(Na) became apparent >1 h later. Downregulation of alpha-ENaC mRNA did not take part in acute regulation because it started after a lag time of 3 h. Time correlation of beta- and gamma-ENaC induction and J(Na) stimulation suggests that the early aldosterone effect on Na(+) absorption in distal colon is caused by transcriptional upregulation of beta- and gamma-ENaC expression.

Bovine herpesvirus type 2 is closely related to the primate alphaherpesviruses.

Bovine herpesvirus type 2 (BoHV-2), also known as bovine mammillitis virus, is classified in the Family Herpesviridae, Subfamily Alphaherpesvirinae, and Genus Simplexvirus along with herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) and other primate simplexviruses on the basis of similarities in 4 genes within the 15 kb U(L) 23-29 cluster. This could be explained either by a global similarity or a recombination event that brought primate herpesviral sequences into a bovine virus. Our sequences for DNA polymerase (U(L)30), a large gene adjacent to the previously identified conserved cluster, and glycoprotein G (U(S)4), a gene as distant from the cluster as possible on the circularized genome, confirm the close relationship between BoHV-2 and the primate simplexviruses, and argue for a global similarity and probably a close evolutionary relationship. Thus one can speculate that BoHV-2 may represent a greater hazard to humans than has been appreciated previously.

Genetic analysis of the bovine herpesvirus type 4 gene locus for the putative terminase.

The complete DNA sequence of the 10-45 kbp HindIII B fragment of bovine herpesvirus type 4 (BoHV-4) was determined. This fragment contains nine complete and two incomplete open reading frames (ORFs), all of which are homologous to herpesvirus saimiri (HVS), Kaposi's Sarcoma-associated herpesvirus (HHV-8) and Epstein-Barr virus (EBV). Particularly, the arrangement of the gene for the terminase-related protein with the two coding exons 29a/29b is conserved among all herpesviruses sequenced to date. The intron carries the ORFs 30 to 33 in the opposite direction. Analysis by reverse transcription and polymerase chain reaction (PCR) of the transcript across the proposed splice junction of the ORF 29a/29b and subsequent sequence determination of the amplified product revealed the precise structure of the splice junction. Furthermore, the phylogenetic analysis of the 29a/29b protein and its counterparts in other herpesviruses revealed that BoHV-4 clustered in the genus Rhadinovirus of the subfamily Gammaherpesvirinae.

In vitro and in vivo effects of genistein on murine alveolar macrophage TNF alpha production.

The present study was performed to determine whether genistein could inhibit in vivo LPS-induced alveolar macrophage TNFalpha production and thus reduce the alveolar neutrophil influx following LPS. In vitro incubation with genistein completely inhibited LPS-induced TNFalpha production by alveolar macrophages (AM) from BALB/c mice. Subsequently mice were pretreated with intraperitoneal genistein or vehicle, then received nasal LPS to induce an alveolitis. Genistein was then administered every eight hours for five days following LPS. At 24 hours after LPS, the bronchoalveolar lavage (BAL) TNFalpha and ex vivo TNFalpha production from AM, were lower in the genistein treated animals. As well, total BAL white blood cell (WBC) count was reduced in the genistein as compared to the vehicle-only group. The percent neutrophils and the resolution of neutrophils were similar between genistein and vehicle groups. Therefore, genistein was able to decrease AM TNFalpha production, and was associated with a decrease in BAL WBC count post-LPS.