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.

A silicon sensor-based filtration immunoassay using biotin-mediated capture.

A sensitive sandwich immunoassay for human chorionic gonadotropin (hCG) was developed with biotin-mediated filtration capture and silicon sensor detection. A high density of biotin on the membrane assured efficient capture of complexes containing streptavidin and analyte. Capture efficiency was not affected over a wide range of filtration flow rates or biotin concentrations. The assay utilized the pH sensing ability of the light addressable potentiometric sensor (LAPS) for the detection of urease-antibody conjugates. A LAPS reader was constructed which allowed the enzyme conjugate to be detected in approximately 1 microliter volumes. Effects from variations in detection volume were studied. 10 pg of hCG could be detected in an assay time of 20 min with four standard deviations separation from background. Comparison to a commercial RIA was made.

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.

Biological and molecular aspects of bovine herpesvirus 4 (BHV-4).

This review summarizes most recent information on the bovine cytomegalovirus BHV-4. The virus is not associated with clearly defined clinical entities in cattle. It can easily be isolated in tissue culture and has a broad host range. BHV-4 strains are rather similar in restriction enzyme analysis of their DNAs, the size of the pr DNAs, however, differs. The genome represents a gamma-herpesvirus. Because of its uniqueness BHV-4 is discussed as an appropriate vector.

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.

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.

Genome organization of the herpesviruses: minireview.

Fundamentally, the members of the herpesvirus family can be divided into six genome structure types designated by the letters A-F. Most of the viruses relevant to animal diseases belong to the D or E type. Whereas the biological function of the different DNA structures is so far unknown, studies on HSV genome structures indicate a rolling-circle mechanism as a mode of virus replication. Furthermore, the terminal repetitive segments seem to be involved in cleavage/packaging mechanisms and probably in the integration process of virus into the host genome. In the well-studied D and E type viruses a great number of genes are colinearly arranged and are homologous regarding their sequences. Thus genes encoding alpha-proteins map at least in part within the reiterated sequences, whereas beta- and gamma-polypeptide genes are largely located within the unique sequences of the long and short components.

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.

Detection of two novel porcine herpesviruses with high similarity to gammaherpesviruses.

Evidence for the existence of porcine gammaherpesviruses was obtained by PCR and sequence analysis. Initially, samples of peripheral blood mononuclear cells (PBMC), spleens, lungs, kidneys and livers of pigs from Germany and Spain were tested with a PCR assay which targets conserved regions of the herpesvirus DNA polymerase gene with degenerate and deoxyinosine-substituted primers. Amplicons of identical sequence were obtained from one spleen and two PBMC samples. This sequence showed a high percentage of identity with the DNA polymerase genes of herpesviruses of the oncogenic subfamily Gammaherpesvirinae. Alignment of amino acid sequences showed the highest identity values with bovine gammaherpesviruses, namely alcelaphine herpesvirus type 1 (68%), ovine herpesvirus type 2 (68%) and bovine lymphotropic herpesvirus (67%). Comparison with pseudorabies virus and porcine cytomegalovirus, which are the only porcine herpesvirus species presently known, showed values of only 41%. PCR analysis of PBMC (n = 39) and spleen (n = 19) samples from German pigs, using primers specific for the novel sequence, revealed a prevalence of 87 and 95%, respectively. In this analysis, three out of eight spleen samples from Spanish pigs were also positive. Subsequent sequencing of the amplicons revealed the presence of two closely related gammaherpesvirus sequences, differing from each other by 8% at the amino acid level. The putative novel porcine herpesviruses, from which these sequences originated, were tentatively designated porcine lymphotropic herpesvirus type 1 and type 2 (PLHV-1 and PLHV-2). When using pig organs for xenotransplantation, the presence of these viruses has to be considered.

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.

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.

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.

Structure and function of the prDNA and the genomic termini of the gamma2-herpesvirus bovine herpesvirus type 4.

The linear virion DNA of bovine herpesvirus type 4 (BHV-4) is flanked by tandem repeats designated polyrepetitive DNA (prDNA). To investigate the structure and functional role of the prDNA for cleavage/packaging of progeny viral DNA, the complete nucleotide sequence (2267 bp) of a cloned prDNA unit of BHV-4 was determined. Moreover, the terminal fragments of the genome and the junctions between prDNA and the central unique DNA were analysed. In order to characterize the function of the prDNA of BHV-4, a transient packaging assay was developed. The prDNA has a G+C content of 71.1%. Its structure is composed of numerous internal repeats and every unit contains the conserved sequence of the cleavage/packaging signal. A fragment of 443 bp comprising the cleavage/packaging signal was found to be sufficient for cleavage and encapsidation of replicated concatemeric viral DNA. These results suggest that prDNA is a functionally important region of the genome of BHV-4.

A bovine herpesvirus (BHV-4) as passenger virus in ethmoidal tumours in Indian cattle.

A herpesvirus was isolated from tumours of the ethmoidal mucosa in two of three head of cattle in the State of Kerala, India. The virus designated M40 was cytopathic for a variety of cultured bovine and porcine cells and it did not kill suckling mice or chicken embryos. Sera from tumour-bearing cattle and goats reacted with the M40 virus. Immunofluorescence tests with FITC-conjugated IgG from a bovine monospecific antiserum to bovine herpesvirus 4 (BHV-4) stained the M40 virus specific antigen in infected cells. Experimental infection of goats with the M40 virus did not result in development of tumours. This virus is therefore considered to represent a "passenger" virus. A great similarity was found between restriction patterns of DNAs extracted from M40 virus and the strain 66-P-347, a reference strain of the BHV-4 group.

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%.

Distribution and relevance of equine herpesvirus type 2 (EHV-2) infections.

Equine herpesvirus type 2 (EHV-2) is a slow-growing, cytopathogenic gammaherpesvirus, which is suggested to be ubiquitous in the equine population. However, its precise role as a pathogen and its tissue tropism remains uncertain. To estimate the prevalence of EHV-2 in Germany and to investigate the possible pathogenicity of the virus, peripheral blood leucocytes (PBL) from 172 horses were examined for EHV-2 DNA by a sensitive and specific nested PCR based on the EcoRI-N genomic fragment and by classical cocultivation. PBL samples from 51% of the horses were positive by PCR and virus was isolated from 31% of the horses by cocultivation. However, almost all animals were seropositive for EHV-2. This may indicate that PBL do not harbour EHV-2 indefinitely after infection. Furthermore, a correlation between clinical signs and EHV-2 as a causative agent could not be determined. Nevertheless, the prevalence of virus was high among horses with upper respiratory tract disease, abortion and severe ataxia. The products of the second round of the PCR reactions showed size polymorphism. Sequencing of the products revealed that these size differences were due to repetition of the motif (AGACAGGGGCCATGCTGGC) between 9-16 times depending on the isolate, suggesting that the nested PCR might be a useful tool for the differentiation of EHV-2 isolates.