Relatedness of wildlife and livestock avian isolates of the nosocomial pathogen Acinetobacter baumannii to lineages spread in hospitals worldwide.

The natural habitats and potential reservoirs of the nosocomial pathogen Acinetobacter baumannii are poorly defined. Here, we put forth and tested the hypothesis of avian reservoirs of A. baumannii. We screened tracheal and rectal swab samples from livestock (chicken, geese) and wild birds (white stork nestlings) and isolated A. baumannii from 3% of sampled chicken (n = 220), 8% of geese (n = 40) and 25% of white stork nestlings (n = 661). Virulence of selected avian A. baumannii isolates was comparable to that of clinical isolates in the Galleria mellonella infection model. Whole genome sequencing revealed the close relationship of an antibiotic-susceptible chicken isolate from Germany with a multidrug-resistant human clinical isolate from China and additional linkages between livestock isolates and human clinical isolates related to international clonal lineages. Moreover, we identified stork isolates related to human clinical isolates from the United States. Multilocus sequence typing disclosed further kinship between avian and human isolates. Avian isolates do not form a distinct clade within the phylogeny of A. baumannii, instead they diverge into different lineages. Further, we provide evidence that A. baumannii is constantly present in the habitats occupied by storks. Collectively, our study suggests A. baumannii could be a zoonotic organism that may disseminate into livestock.

Hatchery workers' IgG antibody profiles to airborne bacteria.

Occupational exposure to high concentrations of airborne bacteria in poultry production is related to an increased risk of respiratory disorders. However, etiology and in particular microorganisms' potential role in pathogenesis still needs to be elucidated. Thus, detection of specific antibodies against occupational microbial antigens may lead to identification of potentially harmful species. For the purpose of IgG titer determination, indirect immunofluorescence on various bacterial isolates from duck hatchery air was combined with image-based quantification of fluorescence intensity. Moreover, in addition to established assays with pure bacterial cultures, a new approach utilized complex bioaerosol samples for detection of anti-microbial antibodies in human sera by determination of percentages of antibody-bound cells in different serum dilutions. Mean titers in sera from hatchery workers and a non-exposed control group did not display significant differences for most tested isolates and application of comprehensive cluster analysis to entire titer data revealed no structure reflecting workers and controls group. Furthermore, determination of immunoreactivity to the complete microbial community in workplace air displayed similar proportions of antibody-bound cells in both groups. Although no general differences in immunoreaction patterns were observed, mean titers to a Proteus mirabilis isolate and to 3 of 4 distinct Acinetobacter baumannii isolates were higher in the group of hatchery workers than in the reference group indicating a potential applicability as exposure markers. We conclude, despite long term bioaerosol exposure, hatchery workers' IgG antibody profiles to tested antigens did not differ substantially from those of the control group. However, increased workers' titers to A. baumannii and clinical relevance of this species should lead to further investigations regarding potential involvement in pathogenesis of occupational respiratory disorders.

Eggshells as a source for occupational exposure to airborne bacteria in hatcheries.

Occupational exposure to high concentrations of airborne bacteria in poultry production is related to an increased risk of respiratory disorders. However, potential sources and formation of hatchery bioaerosols are rarely characterized. In this study, bacterial multiplication on fresh shell fragments from turkey hatching eggs under conditions present in a hatcher incubator was investigated. A 105-fold amplification was observed both by colony count and total cell count gaining 4 × 107 cfu/cells per gram eggshell within 30 hr of incubation. Furthermore, the bacterial community present on eggshells was analyzed by generation of 16S rRNA gene clone libraries and identification of eight isolates. RFLP analysis revealed no shift in community composition during incubation and Enterococcus faecalis and Enterococcus gallinarum were found as the predominant species on turkey eggshells, both have been classified as risk group 2 microorganisms (German TRBA 466). Since Enterococcus spp. were found as predominant species on turkey eggshells, contribution of this genus to bioaerosol formation was demonstrated. During different work activities with poult and eggshell handling concentrations of airborne enterococci up to 1.3 × 104 cfu m-3 were detected. In contrast, no enterococci were identified at a day without poult or eggshell processing. In conclusion, turkey hatching eggs carry a viable specific microflora from breeder flocks to hatcheries. After hatching of turkey poults, hatcher incubators and eggshell fragments provide appropriate conditions for excessive bacterial growth. Thus, high bacterial loads on eggshell fragments are a source of potential harmful bioaersols caused by air flows, poult activity, and handling of equipment.

Automated Image Analysis for Determination of Antibody Titers Against Occupational Bacterial Antigens Using Indirect Immunofluorescence.

Employees who are exposed to high concentrations of microorganisms in bioaerosols frequently suffer from respiratory disorders. However, etiology and in particular potential roles of microorganisms in pathogenesis still need to be elucidated. Thus, determination of employees' antibody titers against specific occupational microbial antigens may lead to identification of potentially harmful species. Since indirect immunofluorescence (IIF) is easy to implement, we used this technique to analyze immunoreactions in human sera. In order to address disadvantageous inter-observer variations as well as the absence of quantifiable fluorescence data in conventional titer determination by eye, we specifically developed a software tool for automated image analysis. The 'Fluorolyzer' software is able to reliably quantify fluorescence intensities of antibody-bound bacterial cells on digital images. Subsequently, fluorescence values of single cells have been used to calculate non-discrete IgG titers. We tested this approach on multiple bacterial workplace isolates and determined titers in sera from 20 volunteers. Furthermore, we compared image-based results with the conventional manual readout and found significant correlation as well as statistically confirmed reproducibility. In conclusion, we successfully employed 'Fluorolyzer' for determination of titers against various bacterial species and demonstrated its applicability as a useful tool for reliable and efficient analysis of immune response toward occupational exposure to bioaerosols.

STR typing of formalin-fixed paraffin embedded (FFPE) aborted foetal tissue in criminal paternity cases.

Sexual assault or rape cases occasionally result in unwanted pregnancies. In almost all such cases the foetus is aborted. A forensic laboratory may receive the foetus, the placenta, or paraffin embedded abortion material for paternity testing. Obtaining a foetal profile DNA from a foetus or placenta may not be successful due to the age or condition of the tissue. Moreover, maternal contamination of placental material will invariably result in a mixed DNA profile. However, the use of properly screened abortion material from paraffin blocks will almost always result in obtaining a foetal DNA profile. Furthermore, foetal tissue fixed in paraffin blocks does not require special conditions for submission and storage as required to preserve fresh foetal or placental tissue. As hospitals routinely prepare foetal tissue in paraffin blocks, which should be readily obtainable by forensic laboratories, these samples would appear to be the preferred choice for paternity testing.

Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei.

The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1ß, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.

The use of adhesive tape for recovery of DNA from crime scene items.

The selection of the appropriate method of collection of biological material from crime scene items can be crucial to obtaining a DNA profile. The three techniques commonly used for sampling items are: cutting, swabbing, and taping. The tape sampling technique offers an advantage, in that it enables the collection of a potentially highly informative source of DNA, shed epithelial cells, from selected areas on crime scene items (the inside fingers of a glove, for instance). Furthermore, surface collection of biological material by taping reduces co-sampling of known PCR inhibitors such as clothing dyes. The correct choice of tape for crime scene item sampling is important. Not all tapes are suitable for biological trace evidence collection as well as DNA extraction. We report on one tape that met both these criteria. Three different cases are presented which demonstrate the usefulness of adhesive tape sampling of crime items. Finally, the advantages of the tape collection technique are discussed and guidelines for preferred areas of tape sampling on various casework items are presented.

Erroneous gender identification by the amelogenin sex test.

Human gender identification, based on the amelogenin gene, has important applications in forensic casework, prenatal diagnosis, DNA databasing, and blood sample storage. However, we report on the first known case, in the Israeli population, of an amelogenin sex test failure on a phenotypically normal male. He was typed as a female by both the AmpFlSTR SGM plus and GenePrint kits. Subsequent, karyotyping of the soldier's blood sample showed no abnormalities. These results suggest that the determination of sex, based on the amelogenin test, should be interpreted cautiously.