Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms--which stain is suitable?

BACKGROUND There is confusion over the definition of the term "viability state(s)" of microorganisms. "Viability staining" or "vital staining techniques" are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data. DISCUSSION Many terms describe "vitality states" of microorganisms, however, several of them are misleading. Authors define "viable" as "capable to grow". Accordingly, staining methods are substitutes, since no staining can prove viability.The reliability of a commercial "viability" staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the "viability" kit are dependent on the stains' concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique.To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research.Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported. SUMMARY - The nomenclature regarding "viability" and "vitality" should be used carefully.- The manual of the commercial "viability" kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an array of literature.- Results obtained with various stains are influenced by the relationship between bacterial counts and the amount of stain used in the test. Corresponding vitality data are prone to artificial shifting.- As microbiological parameter the Plating Efficiency should be used for comparison.- Ethidium Bromide is mutagenic. Researchers should be aware that alternative staining compounds may also be or even are mutagenic.

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses

Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.

Epitheliocystis and the importance of Chlamydia-related bacteria on fish health

This book offers unique coverage of all presently known amoeba-resistant microorganisms and their significance in the study of infectious diseases. It highlights the role of free-living amoebae as a widespread evolutionary crib for the development of virulence traits in resistant microbes, including the ability of intracellular bacteria to survive to other phagocytic cells such as human macrophages. The emphasis is on public health risks associated with the presence in drinking water of intra-amoebal bacteria as well as the ecology and pathogenic role of amoebae-resisting bacteria as new emerging human pathogens

Penetrability of the inner mucus layer: who is out there?

Large numbers of microorganisms colonise the skin and mucous membranes of animals, with their highest density in the lower gastrointestinal tract. The impact of these microbes on the host can be demonstrated by comparing animals (usually mice) housed under germ-free conditions, or colonised with different compositions of microbes. Inbreeding and embryo manipulation programs have generated a wide variety of mouse strains with a fixed germ-line (isogenic) and hygiene comparisons robustly show remarkably strong interactions between the microbiota and the host, which can be summarised in three axioms. (I) Live microbes are largely confined to their spaces at body surfaces, provided the animal is not suffering from an infection. (II) There is promiscuous molecular exchange throughout the host and its microbiota in both directions [1]. (III) Every host organ system is profoundly shaped by the presence of body surface microbes. It follows that one must draw a line between live microbial and host “spaces” (I) to understand the crosstalk (II and III) at this interesting interface of the host-microbial superorganism. Of course, since microbes can adapt to very different niches, there has to be more than one line. In this issue of EMBO Reports, Johansson and colleagues have studied mucus, which is the main physical frontier for most microbes in the intestinal tract: they report how different non-pathogenic microbiota compositions affect its permeability and the functional protection of the epithelial surface [2].

Colonization with resistant microorganisms in patients transferred from abroad: who needs to be screened?

BACKGROUND While multi-drug resistant organisms (MDRO) are a global phenomenon, there are significant regional differences in terms of prevalence. Traveling to countries with a high MDRO prevalence increases the risk of acquiring such an organism. In this study we determined risk factors for MDRO colonization among patients who returned from a healthcare system in a high-prevalence area (so-called transfer patients). Factors predicting colonization could serve as screening criteria to better target those at highest risk. METHODS This screening study included adult patients who had been exposed to a healthcare system abroad or in a high-prevalence region in Switzerland over the past six months and presented to our 950-bed tertiary care hospital between January 1, 2012 and December 31, 2013, a 24-month period. Laboratory screening tests focused on Gram-negative MDROs and methicillin-resistant Staphylococcus aureus (MRSA). RESULTS A total of 235 transfer patients were screened and analyzed, of which 43 (18 %) were positive for an MDRO. Most of them yielded Gram-negative bacteria (42; 98 %), with only a single screening revealing MRSA (2 %); three screenings showed a combination of Gram-negative bacteria and MRSA. For the risk factor analysis we focused on the 42 Gram-negative MDROs. Most of them were ESBL-producing Escherichia coli and Klebsiella pneumoniae while only two were carbapenemase producers. In univariate analysis, factors associated with screening positivity were hospitalization outside of Europe (p < 0.001), surgical procedure in a hospital abroad (p = 0.007), and - on admission to our hospital - active infection (p = 0.002), antibiotic treatment (p = 0.014) and presence of skin lesions (p = 0.001). Only hospitalization outside of Europe (Odds Ratio, OR 3.2 (95 % CI 1.5- 6.8)) and active infection on admission (OR 2.7 (95 % CI 1.07- 6.6)) remained as independent predictors of Gram-negative MDRO colonization. CONCLUSION Our data suggest that a large proportion of patients (i.e., 82 %) transferred to Switzerland from hospitals in high MDRO prevalence areas are unnecessarily screened for MDRO colonization. Basing our screening strategy on certain criteria (such as presence of skin lesions, active infection, antibiotic treatment, history of a surgical procedure abroad and hospitalization outside of Europe) promises to be a better targeted and more cost-effective strategy.

A comprehensive assessment of environmental ultraviolet radiation induced DNA damage in marine microorganisms

There is evidence that ultraviolet radiation (UVR) is increasing over certain locations on the Earth's surface. Of primary concern is the annual pattern of ozone depletion over Antarctica and the Southern Ocean. Reduction of ozone concentration selectively limits absorption of solar UV-B (290–320 nm), resulting in higher irradiance at the Earth's surface. The effects of ozone depletion on the human population and natural ecosystems, particularly the marine environment, are a matter of considerable concern. Indeed, marine plankton may serve as sensitive indicators of ozone depletion and UV-B fluctuations. Direct biological effects of UVR result from absorption of UV-B by DNA. Once absorbed, energy is dissipated by a variety of pathways, including covalent chemical reactions leading to the formation of photoproducts. The major types of photoproduct formed are cyclobutyl pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone dimer [(6-4)PD]. Marine plankton repair these photoproducts using light-dependent photoenzymatic repair or nucleotide excision repair. The studies here show that fluctuations in CPD concentrations in the marine environment at Palmer Station, Antarctica correlate well with ozone concentration and UV-B irradiance at the Earth's surface. A comparison of photoproduct levels in marine plankton and DNA dosimeters show that bacterioplankton display higher resistance to solar UVR than phytoplankton in an ozone depleted environment. DNA damage in marine microorganisms was investigated during two separate latitudinal transects which covered a total range of 140°. We observed the same pattern of change in DNA damage levels in dosimeters and marine plankton as measured using two distinct quantitative techniques. Results from the transects show that differences in photosensitivity exist in marine plankton collected under varying UVR environments. Laboratory studies of Antarctic bacterial isolates confirm that marine bacterioplankton possess differences in survival, DNA damage induction, and repair following exposure to UVR. Results from DNA damage measurements during ozone season, along a latitudinal gradient, and in marine bacterial isolates suggest that changes in environmental UVR correlate with changes in UV-B induced DNA damage in marine microorganisms. Differences in the ability to tolerate UVR stress under different environmental conditions may determine the composition of the microbial communities inhabiting those environments. ^

Antibody chemical reactivity: Beneficial and pathogenic roles

Antibodies (Abs) to autoantigens and foreign antigens (Ags) mediate, respectively, various pathogenic and beneficial effects. Abs express enzyme-like nucleophiles that react covalently with electrophiles. A subpopulation of nucleophilic Abs expresses proteolytic activity, which can inactivate the Ag permanently. This thesis shows how the nucleophilicity can be exploited to inhibit harmful Abs or potentially protect against a virus. ^ Inactivation of pathogenic Abs from Hemophilia A (HA) patients by means of nucleophile-electrophile pairing was studied. Deficient factor VIII (FVIII) in HA subjects impairs blood coagulation. FVIII replacement therapy fails in 20-30% of HA patients due to production of anti-FVIII Abs. FVIII analogs containing electrophilic phosphonate group (E-FVIII and E-C2) were hypothesized to inactivate the Abs by reacting specifically and covalently with nucleophilic sites. Anti-FVIII IgGs from HA patients formed immune complexes with E-FVIII and E-C2 that remained irreversibly associated under conditions that disrupt noncovalent Ab-Ag complexes. The reaction induced irreversible loss of Ab anti-coagulant activity. E-FVIII alone displayed limited interference with coagulation. E-FVIII is a prototype reagent suitable for further development as a selective inactivator of pathogenic anti-FVIII Abs. ^ The beneficial function of Abs to human immunodeficiency virus type 1 (HIV-1) was analyzed. HIV-1 eludes the immune system by rapidly changing its coat protein structure. IgAs from noninfected subjects hydrolyzed gp120 and neutralized HIV-1 with modest potency by recognizing the gp120 421-433 epitope, a conserved B cell superantigenic region that is also essential for HIV-1 attachment to host cell CD4 receptors. An adaptive immune response to superantigens is generally prohibited due to their ability to downregulate B cells. IgAs from subjects with prolonged HIV-1 infection displayed improved catalytic hydrolysis of gp120 and exceptionally potent and broad neutralization of diverse CCR5-dependent primary HIV isolates attributable to recognition of the 421-433 epitope. This indicates that slow immunological bypass of the superantigenic character of gp120 is possible, opening the path to effective HIV vaccination. ^ My research reveals a novel route to inactivate pathogenic nucleophilic Abs using electrophilic antigens. Conversely, naturally occurring nucleophilic Abs may help impede HIV infection, and the Abs could be developed for passive immunotherapy of HIV infected subjects. ^

PRODUCTION, DEGRADATION AND ACCUMULATION OF ORGANICS BY WASTE STABILIZATION POND MICROORGANISMS

The objectives of this research were: to determine the contribution of algae to commonly run water quality variables, to evaluate waste pond micoorganisms' capacity to degrade and accumulate ten EPA priority pollutants, and to determine the environmental fate of those compounds in a laboratory

Table 1 Microorganisms, identified with DOGE analysis. 16S rDNA sequences, obtained from sequencing of DOGE bands, were assigned to the corresponding microorganisms

Microorganisms play an important role in the transformation of material within the earth's crust. The storage of CO2 could affect the composition of inorganic and organic components in the reservoir, consequently influencing microbial activities. To study the microbial induced processes together with geochemical, petrophysical and mineralogical changes, occurring during CO2 storage, long-term laboratory experiments under simulated reservoir P-T conditions were carried out. Clean inner core sections, obtained from the reservoir region at the CO2 storage site in Ketzin (Germany) from a depth of about 650 m, were incubated in high pressure vessels together with sterile synthetic formation brine under in situ P-T conditions of 5.5 MPa and 40°C. A 16S rDNA based fingerprinting method was used to identify the dominant species in DNA extracts of pristine sandstone samples. Members of the alpha- and beta-subdivisions of Proteobacteria and the Actinobacteria were identified. So far sequences belonging to facultative anaerobic, chemoheterotrophic bacteria (Burkholderia fungorum, Agrobacterium tumefaciens) gaining their energy from the oxidation of organic molecules and a genus also capable of chemolithoautotrophic growth (Hydrogenophaga) was identified. During CO2 incubation minor changes in the microbial community composition were observed. The majority of microbes were able to adapt to the changed conditions. During CO2 exposure increased concentrations of Ca**2+, K**+, Mg**2+ and SO4**2- were observed. Partially, concentration rises are (i) due to equilibration between rock pore water and synthetic brine, and (ii) between rock and brine, and are thus independent on CO2 exposure. However, observed concentrations of Ca**2+, K**+, Mg**2+ are even higher than in the original reservoir fluid and therefore indicate mineral dissolution due to CO2 exposure.