Dishwasher Microbiome: Microbial diversity and putative opportunistic pathogens in dishwasher biofilm communities
“Home is a place where we feel save, and so do the microorganisms”.
Extreme habitats are not only present in nature; they exist also within our modern homes. Indoor extreme environments are represented by household appliances, such as dishwashers, and are generally considered clean and abiotic. However, conditions within dishwashers, such as fluctuating low and high temperatures, pH, NaCl concentrations, presence of detergents and shear forces from water ejectors, that inhibit most microorganisms, enable enrichment of the sturdy and polyextremotolerant ones. These selected microorganisms may represent a so far overlooked hidden danger.
Research on dishwasher microbiome started in 2011 with the discovery that black yeasts, especially the opportunistic black yeast Exophiala dermatitidis (Fig 1), colonize rubber seals of domestic dishwashers. This global phenomenon attracted even more attention with the isolation of additional fungal human opportunistic pathogens from dishwashers, such as Candida parapsilosis, Exophiala phaeomuriformis, Aureobasidium melanogenum, Fusarium dimerum, Magnusiomyces capitatus and Saprochaete clavata. Due to the emphasis on fungi, the bacterial community of dishwasher remained initially unexplored.
Figure 1: Pure culture (A) of black yeast Exophiala dermatitidis, rarely isolated from natural environments, but frequently isolated from human made environments, such as dishwashers (B), kitchen facilities (C), saunas, …
To address this issue, our research focused on identifying bacterial and fungal diversity from bioﬁlms grown on one cm2 surface of rubber seals of 24 different household dishwashers. On rubber seals microorganisms form solid and persistent biofilm structures (Fig 2), that were scraped from the surface of the seals with a sterile scalpel and analysed for inter- and intrakingdom interactions, important for the shaping of the microbial community. DNA extractions were performed followed by high throughput DNA sequencing. Further we studied how specific conditions of the dishwashers, such as years of use, frequency of usage, temperature of washing and incoming tap water hardness impact the abundance and diversity of microorganisms.
Figure 2: Biofilm, formed on rubber seals in household dishwashers. The red square represents the sampling site of 1 cm2.
Our analysis included 24 randomly selected domestic dishwashers. Each dishwasher owner filled out a questionnaire with information about the usage of the investigated dishwasher. After scraping the sampling site of 1 cm2 size from the rubber seal, we isolated the total DNA of the scraped biofilm, using a molecular kit, and followed by amplification of 16S rRNA for bacteria and ITS rRNA for fungi. In the final step we used Ilumina MiSeq sequencer to obtain the amplicons of each dishwasher. Several data analyses were performed to evaluate the obtained results.
Figure 3: Bacterial and fungal genera that were represented in the number of dishwashers samples in the study.
Dishwasher biofilms were composed of diverse fungal and bacterial phyla (Fig 3). Microbiome resulted in bacterial genera such as Pseudomonas, Escherichia, and Acinetobacter, known to include opportunistic pathogens, that were represented in most samples. The most frequently encountered fungal genera belong to Candida, Cryptococcus, and Rhodotorula, also known to include opportunistic pathogenic representatives. The age, usage frequency, and hardness of incoming tap water of dishwashers had the most signiﬁcant impact on bacterial and fungal community compositions.
Figure 4: Bacterial community changed over the years of use and frequency of use of the dishwashers.
With the aging of the machine, the microbial community that lives on the seals, changed. In the initial phase (roughly in the first four years), we found mainly bacterial species from the phylum Proteobacteria (Fig 4), while other bacterial species were represented in smaller numbers. Amongst fungi Ascomycetes (92%) (Fig 5) prevailed, represented by genera Candida, Saccharomyces and Debaryomyces.
In the older dishwashers, age of 5-7 years, Proteobacteria decreased significantly and were replaced with bacterial species from the phyla Actinobacteria and Firmucutes. In the fungal part of the community we observed a similar phenomenon. Absolute initial dominance of the ascomycetes was half diminished and replaced with basidiomycetous fungi, in particular from the genera Cryptococcus and Rhodotorula.
Figure 5: Fungal community shifted with dishwasher’s years of use, the frequency of usage and the incoming tap water hardness.
The frequency of machine usage use also had an impact on the bacterial community. In dishwashers that were used more often (1-2x per day), different species of bacteria were observed than in less used (1-3 x per week) dishwashers. Representatives of bacterial pathogenic species appeared more frequently at lower washing frequencies. At higher washing frequencies, more than 95% of fungi were ascomycetous yeasts from the genera Candida, Saccharomyces and Debaryomyces.
The hardness of the tap water to which the dishwasher was connected had a significant impact on the fungal population. The fungi were more often isolated from water samples with a higher content of calcium ions and with a moderate presence of magnesium. From dishwashers that were connected to hard tap water we identified different genera of fungi than from dishwashers connected to softer water. In particular, the presence of calcium ions influenced the colonization by the black yeast Exophiala dermatitidis.
Candida spp., found at the highest prevalence (100%) in all dishwashers, show that this genus was very well adapted to grown in man-made systems like dishwashers. In mixed bacterial-fungal bioﬁlms, the early adhesion, species contact and interactions are vital in the process of bioﬁlm formation. Mixed complexes of bacteria and fungi could provide a preliminary biogenic structure for the establishment of bioﬁlms. This study confirms that household appliances like dishwashers support growth of microbial biofilms, colonized by poly-extremotolerant bacteria and fungi, could present a potential source of domestically sourced infections.
The signiﬁcance of our research is in identifying the microbial composition of bioﬁlms formed in a broadly used household appliance, dishwashers, in describing how diverse abiotic conditions affect the composition of mixed fungal bacterial microbiota and which key members were present in early colonization processes.
Our article entitled “Microbiomes in Dishwashers: Analysis of the microbial diversity and putative opportunistic pathogens in dishwasher biofilm communities” was published in Applied and Environmental Microbiology, 10.1128/AEM.02755-17.
Written by: Jerneja Zupančič, Prem K. Raghupathi and Nina Gunde-Cimerman