Extended Survival and Persistence of Campylobacter spp. in Water and Aquatic Biofilms and Their Detection by Immunofluorescent-Antibody and -rRNA Staining

In water microcosm experiments, the survival times of Campylobacter isolates differed by up to twofold, as determined by culturing; this difference increased to fourfold when particular combinations of temperature and oxygenation were used. The mean survival times were much longer at 4 and 10°C (202 and 176 h, respectively) than at 22 and 37°C (43 and 22 h, respectively). The influence of anaerobiosis on survival time was less dramatic and differed considerably between isolates. In a two-stage water distribution model preparation containing a biofilm consisting of standardized autochthonous water microflora, Campylobacter isolates continued to differ in survival time. However, the survival times of cultures were considerably longer in the presence of the autochthonous water microflora (strains CH1 and 9752 survived 700 and 360 h, respectively, at 4°C) than in the sterile microcosms (strains CH1 and 9752 survived 230 and 157 h, respectively). Although increased temperature and oxygenation were generally detrimental to culturability, the interaction of these two factors influenced the two strains examined differently. When the organisms were grown aerobically at 30°C, the survival of the two strains was reversed; aerobiosis decreased the survival time of strain CH1 by 30%, but unexpectedly improved the persistence time of strain 9752 by more than threefold. Persistence times within biofilms were much longer when they were determined by detection methods not involving culturing. Immunofluorescent-antibody staining demonstrated that the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4°C, respectively. The specificity of detection within intact biofilms was reduced because of high background fluorescence. However, preliminary studies with a Campylobacter-specific rRNA probe revealed the same extended persistence of the pathogen within the biofilms.     

Molecular Analysis of Hypervirulent Somatic Hybrids of the Entomopathogenic Fungi Beauveria bassiana and Beauveria sulfurescens

Protoplast fusion of diauxotrophic mutants of a Beauveria bassiana entomopathogenic strain (Bb28) and a Beauveria sulfurescens toxinogenic strain (Bs2) produced hybrids which were significantly different from the parents in pathogenicity. Some of the hybrids were hypervirulent and killed insects more quickly than the Bb28 strain, probably because these hybrids had acquired the toxic activity of the Bs2 strain. By using six nuclear genes and a telomeric fingerprint probe, the molecular structures of the hybrids were studied. The results demonstrated the occurrence of parasexual events. Hybrids appeared to be diploid or aneuploid, with portions of the genome being heterozygous. A mitochondrial molecular marker indicated homoplasmy of the hybrids and inheritance of mitochondria from strain Bs2 or Bb28. The pathogenicities and the ploidies of the hybrids remained stable after passage through the host insect, showing that somatic hybridization provides an attractive method for the genetic improvement of biocontrol efficiency in the genus Beauveria.     

Pattern and Performance of Understory Bamboos (Chusquea spp.) under Different Canopy Closures in Old-Growth Oak Forests in Costa Rica1

The effect of canopy closure on spatial and morphological patterns of three understory Chusquea species was studied in pristine upper montane oak forests. Plots with either Chusquea talamancenis Widmer & L.G. Clark or C. tomentosa Widmer & L.G. Clark were set out in gaps, under intermediate tree canopy and under closed canopy. The third species Chusquea foliosa L.G. Clark was only found in gap and intermediate canopy sites in the study area. Clumps of all bamboo species tend to be few and large under open conditions and numerous and small under more shady conditions. The larger size of clumps in gaps is reflected in the number of culms per clump, the diameter of the culms and, as a consequence, clump basal area, clump crown area, and biomass (index of plant volume) compared to intermediate and closed canopy. The smaller clump density in gaps compared to closed canopy implies that there is more intraspecific competition and density dependent mortality (self-thinning effect) when a bamboo species is dominant under favourable light conditions. Parameters of performance like culm length, number of nodes per culm, number and length of primary branches, and number of branch nodes seem not to be affected by the light regime, unlike total number of branches, total branch length, and the branching pattern. Species differ in their response to the light environment: C. tomentosa and C. foliosa have a higher degree of morphological plasticity than C. talamancensis, which in turn, appears to be more shade tolerant. No difference between species has been found regarding their contribution to vegetation, parameters reflecting abundance per unit area (site clump area, site crown area and site index of clump volume) were similar for all three species.     

A single red-light pulse leads to a block of greening in the high-pigment-1 mutant of tomato

A single pulse of red light (R) given to 4-d-old etiolated high-pigment-1 (hp-1) mutant tomato (Solanum lycopersicum L.) seedlings followed by a 3-d dark period is demonstrated to result in a block of greening in subsequent white light. Wild-type seedlings green normally under this regime. The block of greening in the hp-1 mutant depends on the length of the dark period before and after the R pulse and operates via the low-fluence-response mode of phytochrome action. This block of greening takes place in hp-1 double mutants lacking either phytochrome A or phytochrome B1, but is absent in the hp-1 triple mutant lacking both phytochromes A and B1. These observations enable a screen to be devised for new phytochrome B1 mutants either within the photoreceptor or mutants defective in phytochrome B1-signalling steps which result in loss of capacity to green, by mutagenising the phytochrome A-deficient hp-1, fri double mutant. Received: 20 February 1998 / Accepted: 18 June 1998     

The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus . We report the discovery of four additional pil genes: pilD , a homologue of type IV prepilin leader peptidases; and pilG , pilH and pilI , which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH . Null mutants of pilG , pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.     

Validation of a NAT-based Mycoplasma assay according European Pharmacopoiea

Eucaryotic expression systems are widely used to produce biologicals for human use, e.g. vaccines, recombinant proteins and monoclonal antibodies. As part of the safety testing the current U.S. Food and Drug Administration (FDA) regulatory guidelines as well as several European Pharmacopoiea monographs requests the demonstration of the absence of Mycoplasma in the cell culture in the bioreactors prior to harvest and further downstream processing. In recent years progress has been made in the development of a sensitive NAT-based method for the detection of Mycoplasma species in CHO cells, e.g. Eldering et al. This method is based on a nucleic acid amplification technique using a very sensitive touch-down PCR-profile. The presence of mollicutes DNA in the test specimens is determined by an approx. 450 bp target sequence which is amplified and this amplicon is finally detected by polyacrylamide gel electrophoresis. Based on this method a ready-to-use test kit was developed. In this report the validation of both method variants according the European Pharmacopoiea monograph 2.6.7 “Mycoplasmas” is described. The validation demonstrated the robustness and precision as well as a sufficient specificity of both assay formats. The validated sensitivity fulfills the requirements of the European Pharmacopoiea for a PCR-based method proposed as an alternative to the time consuming indicator cell culture and the culture method for the detection of Mollicutes (requested sensitivity of at least 10 colony-forming-units/mL).     

Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G₀/G₁ arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin.     

The Prognostic Value of the Work Ability Index for Sickness Absence among Office Workers

BackgroundThe work ability index (WAI) is a frequently used tool in occupational health to identify workers at risk for a reduced work performance and for work-related disability. However, information about the prognostic value of the WAI to identify workers at risk for sickness absence is scarce.ObjectivesTo investigate the prognostic value of the WAI for sickness absence, and whether the discriminative ability differs across demographic subgroups.MethodsAt baseline, the WAI (score 7-49) was assessed among 1,331 office workers from a Dutch financial service company. Sickness absence was registered during 12-months follow-up and categorised as 0 days, 0<days<5, 5≤days<15, and ≥15 days in one year. Associations between WAI and sickness absence were estimated by multinomial regression analyses. Discriminative ability of the WAI was assessed by the Area Under the Curve (AUC) and Ordinal c-index (ORC). Test characteristics were determined for dichotomised outcomes. Additional analyses were performed for separate WAI dimensions, and subgroup analyses for demographic groups.ResultsA lower WAI was associated with sickness absence (≥15 days vs. 0 days: per point lower WAI score OR=1.27; 95%CI 1.21-1.33). The WAI showed reasonable ability to discriminate between categories of sickness absence (ORC=0.65; 95%CI 0.63-0.68). Highest discrimination was found for comparing workers with ≥15 sick days with 0 sick days (AUC=0.77) or with 1-5 sick days (AUC=0.69). At the cut-off for poor work ability (WAI≤27) the sensitivity to identify workers at risk for ≥15 sick days was 7.5%, the specificity 99.6%, and the positive predictive value 82%. The performance was similar across demographic subgroups.ConclusionsThe WAI could be used to identify workers at high risk for prolonged sickness absence. However, due to low sensitivity many workers will be missed. Hence, additional factors are required to better identify workers at highest risk.     

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.