[Climate changes and emerging diseases. What new infectious diseases and health problem can be expected?]. / Die Auswirkungen des Klimawandels. Welche neuen Infektionskrankheiten und gesundheitlichen Probleme sind zu erwarten?

Increasing temperatures, but also other climatic factors, will have an impact on human health. Apart from the direct consequences of extreme weather conditions (e.g., heat-related fatalities), indirect health consequences in the long-term are also of great importance. In addition to a likely increase in allergic diseases and additional complications in the course of cardiovascular and respiratory diseases, infectious diseases are of particular interest. In Germany, endemic pathogens, such as hantavirus (with its reservoir in small rodents), tick-borne pathogens (Borrelia burgdorferi, tick-borne encephalitis virus), and certain food- and water-borne pathogens, are of concern. Mild winters favor rodent populations and may result in hantavirus epidemics in the subsequent summer period. Statistical analyses show a significant association between temperature and campylobacter incidence in Germany. An outbreak of rodent-borne leptospirosis among strawberry harvesters enhanced by heavy rainfalls illustrates how weather conditions may influence disease occurrence. Pathogens that are non-endemic in Germany but are imported by humans, vectors, and reservoir animals pose an additional risk to the population. Increasing temperatures improve the conditions for establishment of new vectors and for autochthonous transmission of some pathogens (e.g., chikungunya, dengue, West Nile virus, malaria, or leishmaniasis). Climatic and ecologic conditions in Germany currently do not favor autochthonous outbreaks for most of these pathogens. However, if temperatures increase, as expected, such outbreaks will become more likely. Germany should enhance its research in public health activities in the field of climate change and infectious diseases.

Characterization of vibrio cholerae O1 antigen as the bacteriophage K139 receptor and identification of IS1004 insertions aborting O1 antigen biosynthesis.

Bacteriophage K139 was recently characterized as a temperate phage of O1 Vibrio cholerae. In this study we have determined the phage adsorption site on the bacterial cell surface. Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor. In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139. Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide. By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection. Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfb region). Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004 into the rfb gene cluster. Treating one wbeW::IS1004 serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance. Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation. This strain had a significant reduction in its ability to colonize the mouse small intestine.

Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella.

The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. In this study, we infected single-cell stages of D. discoideum with different Legionella species. Intracellular growth of Legionella in this new host system was compared with their growth in the natural host Acanthamoeba castellanii. Transmission electron microscopy of infected D. discoideum cells revealed that legionellae reside within the phagosome. Using confocal microscopy, it was observed that replicating, intracellular, green fluorescent protein (GFP)-tagged legionellae rarely co-localized with fluorescent antibodies directed against the lysosomal protein DdLIMP of D. discoideum. This indicates that the bacteria inhibit the fusion of phagosomes and lysosomes in this particular host system. In addition, Legionella infection of D. discoideum inhibited the differentiation of the host into the multicellular fruiting stage. Co-culture studies with profilin-minus D. discoideum mutants and Legionella resulted in higher rates of infection when compared with infections of wild-type amoebae. Because the amoebae are amenable to genetic manipulation as a result of their haploid genome and because a number of cellular markers are available, we show for the first time that D. discoideum is a valuable model system for studying intracellular pathogenesis of microbial pathogens.

Detection of Staphylococcus aureus and Staphylococcus epidermidis in clinical samples by 16S rRNA-directed in situ hybridization.

Staphylococcus epidermidis and Staphylococcus aureus are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates of S. aureus and S. epidermidis adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of S. aureus and S. epidermidis by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of S. epidermidis in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of S. aureus, and the differentiation of S. epidermidis from other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms.

Specific detection of Legionella pneumophila: construction of a new 16S rRNA-targeted oligonucleotide probe.

Based on comparative sequence analysis, we have designed an oligonucleotide probe complementary to a region of 16S rRNA of Legionella pneumophila which allows the differentiation of L. pneumophila from other Legionella species without cultivation. The specificity of the new probe, LEGPNE1, was tested by in situ hybridization to a total of four serogroups of six strains of L. pneumophila, five different Legionella spp. and three nonlegionella species as reference strains. Furthermore, L. pneumophila cells could be easily distinguished from Legionella micdadei and Pseudomonas aeruginosa cells by using in situ hybridization with probes LEGPNE1, LEG705, and EUB338 after infection of the protozoan Acanthamoeba castellanii.

Looking at Christmas trees in the nucleolus.

We describe novel nucleolar structures, observed by thin section electron microscopy in oocyte nuclei of the grasshopper Locusta migratoria, which we interpret, based on morphological and compositional criteria, as rDNA transcription units. Morphologically they resemble the condensed and foreshortened "Christmas trees" seen in Miller spreads of nucleolar chromatin prepared from the same biological material. They contain DNA and rRNA as shown by immunocytochemistry and in situ hybridization and are concentrated in several intranucleolar cavities. The presumptive rDNA transcription units extend throughout the interior of these nucleolar pockets or are selectively enriched at their outermost zones in close contact with the surrounding fibrillarin-positive dense component. We suggest that the nucleolar pockets of Locusta oocytes are equivalent to the fibrillar centers of somatic nucleoli and discuss possible implications for the current understanding of the functional organization of nucleoli.

Specific detection of Candida albicans and Candida tropicalis by fluorescent in situ hybridization with an 18S rRNA-targeted oligonucleotide probe.

In situ hybridization of whole cells with rRNA-targeted, fluorescently labelled oligonucleotide probes is a powerful method to specifically detect microorganisms in their natural habitat without cultivation and subsequent identification by phenotypic characterization. To examine the use of this method for the specific detection of pathogenic Candida species, we have designed an oligonucleotide probe which binds to the 18S rRNA of C. albicans and C. tropicalis, the two most important pathogenic Candida species, and differentiates them from other clinically relevant species. After establishing suitable hybridization conditions, we confirmed the specificity of our probe O20 in RNA dot blot hybridizations with a series of reference strains and clinical isolates of medically important Candida species. All C. albicans and C. tropicalis strains hybridized with the probe, whereas all strains of C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. kefyr and C. lusitaniae did not. When we used the fluorescently labelled probe O20 to specifically detect single cells of the two target species by in situ hybridization, both C. albicans and C. tropicalis reacted strongly with the probe and could be clearly differentiated from C. krusei and C. parapsilosis, although the latter organism contains only two nucleotide mismatches in the probe target region. This discrimination capacity was also seen when mixed suspensions of C. albicans and C. parapsilosis were hybridized with the probe. After infection of a human endothelial cell line with C. albicans and C. krusei, C. albicans cells adhering to the endothelial cells were easily distinguishable from the C. krusei cells by fluorescent in situ hybridization with probe O20. In addition, germ tubes and hyphae of C. albicans were also efficiently labelled. The application of fluorescently labelled rRNA-targeted oligonucleotide probes therefore appears to be a valuable tool for the specific detection and identification of different members of the genus Candida, which does not require any cultivation.

Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells.

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.

Immunocytochemical localization of DNA in synaptonemal complexes of rat and mouse spermatocytes, and of chick oocytes.

The distribution of DNA in synaptonemal complexes of rat and mouse spermatocytes, and of chick oocytes was investigated by immunogold electron microscopy. Except for a few specific sites, DNA was not immunolocalized in the space between lateral elements of the complex. Some labeled fibrils connecting the lateral elements with the central element were observed associated with recombination nodules or near them. However, other labeled fibrils in the space between lateral elements did not appear to present any relationship to recombination nodules. The immunocytochemical approaches used here confirmed the presence of significant amounts of DNA in the lateral elements as previously indicated by preferential DNA staining methods. Furthermore, our findings support the view that recombination nodules are the site of chiasma formation.

Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin.

Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin LIII did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.