Microbiology Spectrum

Supplementary Information 2. Representative movies of the phase contrast and epifluorescence (reporting sfGFP(Sp) expressed from the TnJM1 transposon inserted into the indicated genes) channels of reconstructed mutants grown on MOPS agarose pads. Due to differences in fluorescence expression among the isolated clones, image brightness was adjusted for each mutant separately and fluorescence intensity can therefore not be compared between videos.

The upload contains raw data files for the article "Interplay between Porphyromonas gingivalis hemophore-like protein HmuY and Kgp/RgpA gingipains plays a superior role in heme supply" which shows the production of the HmuY protein in various P. gingivalis mutant strains. These data are not present in the supplementary data of publication. Article abstract To acquire heme as a source of iron and protoporphyrin IX, Porphyromonas gingivalis uses gingipains, Hmu, and Hus systems. The aim of this study was to assess the correlation between the production and function of the most important virulence factors of P. gingivalis involved in heme supply, namely, hemophore-like proteins (HmuY and HusA) and gingipains. Respective mutant strains were used, and the expression of genes at the transcript and protein levels, as well as the importance of these genes’ products for virulence potential, was examined. We found that HmuY and Kgp/RgpA gingipains are among the main P. gingivalis virulence factors synergistically engaged in heme supply. Their expression is related mainly when P. gingivalis grows in conditions rich in iron and heme sources, resembling those found in severe periodontitis. We confirmed that HmuY production is strictly dependent on the availability of heme and iron in the external environment, whereas we did not observe such dependence in the production of HusA. Moreover, we found that the HmuY protein can easily sequester heme from the HusA protein. The only correlation in the production of HmuY and HusA hemophore-like proteins could occur in P. gingivalis grown in conditions rich in iron and heme sources, mimicking an environment typical for severe periodontitis. Based on our observations, we suggest that HmuY is the major heme-binding protein produced by P. gingivalis, especially in iron- and heme-depleted conditions, typical for healthy periodontium and the initial stages of infection. The HusA protein could play a supporting role in P. gingivalis heme uptake. The Word document (Data description.docx) contains a description of files.

This is the supplemented material to the publication "Mycobacteriosis in Various Pet and Wild Birds from Germany: Pathological Findings, Coinfections, and Characterization of Causative Mycobacteria". The causative agents and confounding factors of mycobacteriosis in a set of pet (n=45) and some wild birds (n=5) from Germany were examined in this study. Not only Mycobacterium genavense (Mg), but also M. avium subsp. avium (Maa) and M. avium subsp. hominissuis (Mah), contributed to mycobacteriosis in these birds. The isolates were characterized by a combination of different typing methods. The genetic diversity of isolates belonging to Mg, Maa and Mah differed. Various coinfections by viruses, endoparasites, fungi and other bacterial species did not affect the manifestation of mycobacteriosis. Cross pathological fidings were more often seen in mycobacteriosis caused by Ma compared to Mg suggesting a different pathogenicity of the two species. New genotypes of Mah were identified in these birds that is important for epidemiological studies and for understanding the zoonotic role of this pathogen, as the subsp. hominissuis represents an increasing public health concern. The study provides some evidence of correlation between individual Maa genotypes and virulence which will have to be confirmed by broader studies.

Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens like Salmonella enterica and Listeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detection of S. enterica and L. monocytogenes by qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n = 1,990) was analyzed by multiplex qPCR specific for S. enterica and L. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection of S. enterica and L. monocytogenes were 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter for S. enterica; for L. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for both S. enterica and L. monocytogenes, indicating that water type may influence the agreement of these results.

Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens like Salmonella enterica and Listeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detection of S. enterica and L. monocytogenes by qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n = 1,990) was analyzed by multiplex qPCR specific for S. enterica and L. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection of S. enterica and L. monocytogenes were 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter for S. enterica; for L. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for both S. enterica and L. monocytogenes, indicating that water type may influence the agreement of these results.

Traditional indigo dyeing through anaerobic fermentation has recently gained worldwide attention in efforts to address concerns regarding the sustainability of industrial indigo dyeing and the impact of toxic reducing agents such as sodium dithionite (Na2S2O4) on human health and the ecological environment. Intriguingly, changes in the microbiota during indigo fermentation are known to potently affect the onset of indigo reduction, and thus elucidation of the microbial community transitions could help develop methods to control the initiation of indigo reduction. Here, we investigated the microbiota associated with the traditional indigo dyeing practiced in Hunan, China. Specifically, we identified the bacterial and fungal components of the microbiota at distinct stages in the indigo fermentation process by analyzing 16S rRNA gene and internal transcribed spacer sequences. Our analyses revealed two substantial changes in the microbiota during the traditional indigo fermentation process. The first change, which was probably caused by the introduction of Chinese liquor (featuring a high alcohol concentration), resulted in decreased bacterial diversity and increased proportions of Pseudomonas, Stenotrophomonas, and Bacillaceae family members. The second change, which could be attributed to the addition of specific plant species, led to an increase in the abundance of Alkalibacterium, Amphibacillus, the obligate anaerobe Turicibacter, the facultative anaerobe Enterococcus, and ZOR0006, as well as to a decrease in the pH and redox potential values. Our results indicate that the specific plant mixture included in the procedure here could be used as an effective additive to accelerate the initiation of indigo reduction during the fermentation process. To the best of our knowledge, this is the first report revealing the fungal diversity during the indigo fermentation process and, furthermore, showing that the fungal diversity has remained in transition despite the relatively stable bacterial diversity in the proper indigo fermentation process. Although traditional indigo fermentation in China is challenging to manage, we can benefit from local knowledge of the fermentation process, and understanding the scientific bases of traditional indigo fermentation will facilitate the development of environmentally friendly procedures.