100 Doors X Level 9
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100 Doors X Level 9
100 Doors X Level 9
100 Doors X Level 9 Walkthrough, Solutions, Cheats, Answers for iPhone, iPad, Android, Kindle, iPod Touch and other device by MPI Soft.
- “Door Color?” is the clue for this level.
- There are 5 different colors.
- Dark Red, Lime, Cyan, Red and Orange.
- Use first alphabet of those colors to match the clue.
- Answers in the picture.
- Tap by following the answers and open door.
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To the best of our knowledge, this study represents the first comprehensive, culture–independent characterization and visualization of the bacterial kitchen sponge microbiome. Previously, Flores and coworkers (2013)30 analyzed a single kitchen sponge sample from Boulder in Colorado (USA), and detected 123 OTUs (of which about 20% were singletons) with a phylogenetic diversity metric (PD) of 7.88. Our dataset was based on 14 sponges separated into top and bottom parts, each, and yielded 362 OTUs in total, with an average of 31 OTUs (8–62) and an average PD of 4.28 (1.92–7.46) per sample, not including singleton OTUs. If singleton OTUs were included, the total number of OTUs would increase to 1823 and our dataset would yield an average of 96 OTUs (42–199) and an average PD of 10.06 (5.08–18.50) per sponge sample. Thus, the data from the study of Flores and coworkers (2013)30 and our data were quite similar regarding species richness and diversity. However, we preferred to remove singletons, because of the well–known issue of pyrosequencing–generated artifacts32, 33. Moreover, by manual screening of the singleton OTUs, no further taxa, including well-known pathogens, were retrieved. Therefore, we decided to exclude singletons to avoid inflation of the diversity indices, although a few members of the rare microbiota might have been excluded from the analysis by doing so.
Flores and co-workers30 showed a relative dominance (40.39%) of the family Moraxellaceae in their single sponge sample, which is very similar to our value obtained for this family on the basis of 28 samples (36.04%). The relative abundance of other taxa, including Pseudomonadaceae, Rhizobiaceae, Flavobacteriaceae and Staphylococcaceae was also similar; notable differences, however, also appeared for certain taxa, including Xanthomonadaceae, Enterobacteriaceae, Sphingomonadaceae, Acetobacteraceae and especially [Weeksellaceae] (Supplementary Table S3). These differences might simply result from the different sample numbers, but might also be due to geographical and/or cultural (food, cleaning habits etc.) differences between the underlying countries. While the similarities suggest a kitchen sponge core-microbiome, the more variable fractions might differ from region to region. Clearly, more data from different regions are needed to support this hypothesis, but apparently bacteria affiliated with the Moraxellaceae seem “typical” for kitchen sponges. Interestingly, Moraxellaceae have been consistently detected on sink surfaces, faucets, refrigerator doors and stoves30, i.e. surfaces that might be regularly cleaned with kitchen sponges, suggesting them as source for these surface contaminations. However, Moraxellaceae also represent typical human skin bacteria, suggesting also other sources for the contamination of kitchen surfaces. In turn, human skin (hands) might represent a source for the contamination of the sponges with Moraxellceae during use. Recently it has been shown that in particular Moraxellaceae get significantly enriched on cotton laundry during a domestic washing process34.
Notably, Enterobacteriaceae were of low relative abundance (1.18%) and were only partly related to genera including pathogenic species, such as Escherichia sp. We also screened all excluded singletons and detected only 53 singleton OTUs additionally affiliated with Enterobacteriaceae, representing an additional 0.18% of relative abundance of the non-rarified dataset. Also other typical enteropathogenic genera were not detected here, such as Campylobacter sp.
The scarce retrieval of bacterial taxa targeted in previous studies of kitchen sponges, such as coliforms, can be explained with the very high abundance of total bacteria in the sponges, which locally exceeded 1010 bacterial cells per cm3, based on our FISH analyses. CFU counts of total heterotrophic bacteria indicated kitchen sponges as the most densely colonized bacterial reservoir of the BE, with average counts of ~107–109 CFUs per sponge13, 14, 21, 29, which is consistent with our observation, assuming that only 1–3% of the environmental bacteria are cultivable on the currently available media35. Rossi and coworkers (2012)19 reported a retrieval or 2.5 * 108 CFUs/sponge (so, about 107 CFUs per cm3) of fecal coliforms, while Josephson et al. (1997)23 reported 7.8 * 108 per swab area. Averaging these two values (3.9 * 108) and assuming the same amount of Enterobacteriaceae in our sponge samples, then a relative abundance of 0.975% should be expected in our samples for the observed bacterial abundance (~4 * 1010); our pyrosequencing–detected relative abundance of Enterobacteriaceae was about 1.18%, which is very close. Moreover, a bacterial abundance of 4 * 1010 was not an average value per sponge, but represented heavily contaminated local sponge sites. Therefore, considering a lower average abundance of bacteria in the sponges, the fraction of Enterobacteriaceae would be expected to be even greater than 0.975%, thus getting even closer to the observed value of 1.18%.
We found 5 out of the 10 most abundant OTUs to be closely related to RG2-species from the genera Acinetobacter, Moraxella and Chryseobacterium. Clearly, relatedness based on partial 16S rRNA gene sequences is only a weak indicator for the pathogenic potential of the identified bacteria, and we are not aware of any case, in which an infection with these bacteria was explicitly reported from a domestic environment. Nevertheless, kitchen surfaces are generally regarded as vehicles for transmission of infections36 and metagenome reconstruction recently retrieved pathogenicity genes–harboring Acinetobacter baumanni genomes from kitchen counters10. Therefore, we believe that in view of the high bacterial load of kitchen sponges, the dominance of bacteria closely related to species that clearly can cause infections in humans37, 38, warrants attention and underlines the need for appropriate hygiene measures, particularly in BE environments with many immunocompromised persons, such as hospitals, nurseries, schools and houses of home–handled patients. Kitchen sponges are likely to collect, incubate and spread bacteria from and back onto kitchen surfaces, from where they might eventually find their way into the human body, e.g. via the human hands or contaminated food. In addition, direct contact of a sponge with food and/or the human hands might transfer bacteria in and onto the human body, where they might cause infections, depending on their pathogenic potential and the environmental conditions.
Interestingly, in addition to being a RG2-species, Moraxella osloensis, is also known for generating malodor in laundry39. The abundant occurrence of this bacterium might be responsible for bad smelling kitchen sponges, too. As “special cleaning” measures even increased the relative abundance of Moraxella, cleaned sponges might paradoxically smell more often. However, corresponding studies are still missing.
Sanitation by boiling or microwave treatment has been shown to significantly reduce the bacterial load of kitchen sponges19, 21 and can therefore be regarded as a reasonable hygiene measure. However, our data showed that regularly sanitized sponges (as indicated by their users) did not contain less bacteria than uncleaned ones. Moreover, “special cleaning” even increased the relative abundance of both the Moraxella– and Chryseobacterium–affiliated OTUs (Fig. 3B). Presumably, resistant bacteria survive the sanitation process and rapidly re–colonize the released niches until reaching a similar abundance as before the treatment (Fig. 6A). This effect resembles the effect of an antibiotic therapy on the gut microbiota40, 41, and might promote the establishment of higher shares of RG2-related species in the kitchen sponges. Although further analyses, including controlled sanitation experiments, are needed to substantiate these findings, our data allow careful speculation that a prolonged application of sanitation measures of kitchen sponges is not advisable.
Analysis of co–occurrence patterns are useful to identify recurrent associations of relevant organisms. We aimed to find out whether non–pathogenic bacteria could act as inhibitors of RG2-related species. However, our data argue against such a protection, instead, associations of RG2-related OTUs were found more often, which suggests a synergy between these species. Whether this has any consequences in terms of clinical relevance remains to be demonstrated.
FISH coupled with confocal microscopy is a robust complementary approach that can be used to validate and support the results of next generation sequencing in microbial ecology studies42,43,44. In this work, FISH–CLSM confirmed the relative abundance of Gammaproteobacteria in selected samples. So far, only a single study has directly visualized the bacterial colonization of a kitchen sponge before, however, by electron microscopy45, which does not reveal the activity of the inspected cells as the rRNA–based FISH method does. Nevertheless, those images are consistent with our observations, showing dense, biofilm–like clusters of bacterial cells over the sponge surface. Although we calculated the bacterial density of localized, heavy colonized sponge sites (Supplementary Video File S1), a statistically averaged bacterial abundance over whole sponges remains to be assessed. Nevertheless, our work revealed an amazing bacterial colonization of kitchen sponges, and visualized its extent for this common BE–microbial hot spot for the first time. We are convinced that in particular our FISH data (including the Supplementary Video File S1) are very suitable for hygiene education and will help to create even more awareness for kitchen sponges as hygienically relevant microbial incubators.