Distinct Resistomes and Microbial Communities of Soils, Wastewater Treatment Plants and Households Suggest Development of Antibiotic Resistances Due to Distinct Environmental Conditions in Each Environment.

The use of antibiotics in humans and animals results in a release of excess antibiotic residues into the environment through wastewaters and insufficient removal in wastewater treatment plants (WWTP), leading to increasing numbers of bacteria enriched in antibiotic resistance genes (ARG). However, the potential transfer of ARG and their host bacteria between different environments remains largely unexplored. Since many factors need to be fulfilled for a transfer between different environments, we hypothesized that antibiotic resistance (ABR) is less frequently transferred between environments in the same geographical region but rather develops and clusters in each distinct environment, leading to characteristic metagenome patterns in samples of different environments. We sampled agricultural soils, a WWTP and private households and performed metagenomic analyses to evaluate differences and potential overlaps in bacterial communities and resistomes of different environments. Wastewater revealed significantly higher richness of ARG (n = 40) and mobile genetic elements (n = 52) than soil and household samples. Bacterial communities differed between the environments and antibiotic resistance factors clustered distinctly. Overall, only few overlaps of ARG between the environments were observed, leading to the conclusion that ABR predominantly develops in individual environments as caused by environmental filtering for ARG, while a transfer between different environments is less likely.

The Toothbrush Microbiome: Impact of User Age, Period of Use and Bristle Material on the Microbial Communities of Toothbrushes.

Toothbrushes play a central role in oral hygiene and must be considered one of the most common articles of daily use. We analysed the bacterial colonization of used toothbrushes by next generation sequencing (NGS) and by cultivation on different media. Furthermore, we determined the occurrence of antibiotic resistance genes (ARGs) and the impact of different bristle materials on microbial growth and survival. NGS data revealed that Enterobacteriaceae, Micrococcaceae, Actinomycetaceae, and Streptococcaceae comprise major parts of the toothbrush microbiome. The composition of the microbiome differed depending on the period of use or user age. While higher fractions of Actinomycetales, Lactobacillales, and Enterobacterales were found after shorter periods, Micrococcales dominated on both toothbrushes used for more than four weeks and on toothbrushes of older users, while in-vitro tests revealed increasing counts of Micrococcus on all bristle materials as well. Compared to other environments, we found a rather low frequency of ARGs. We determined bacterial counts between 1.42 × 106 and 1.19 × 107 cfu/toothbrush on used toothbrushes and no significant effect of different bristles materials on bacterial survival or growth. Our study illustrates that toothbrushes harbor various microorganisms and that both period of use and user age might affect the microbial composition.

The Household Resistome: Frequency of ß-Lactamases, Class 1 Integrons, and Antibiotic-Resistant Bacteria in the Domestic Environment and Their Reduction during Automated Dishwashing and Laundering.

Households provide a habitat for bacteria originating from humans, animals, foods, contaminated clothes, or other sources. Thus, bacteria carrying antibiotic resistance genes (ARGs) may be introduced via household members, animals, or the water supply from external habitats into private households and vice versa. Since data on antibiotic resistance (ABR) in the domestic environment are limited, this study aimed to determine the abundance of ß-lactamase, mobile colistin resistance, and class 1 integron genes and the correlation of their presence and to characterize phenotypically resistant strains in 54 private households in Germany. Additionally, the persistence of antibiotic-resistant bacteria during automated dishwashing compared to that during laundering was assessed. Shower drains, washing machines, and dishwashers were sampled and analyzed using quantitative real-time PCR. Resistant strains were isolated, followed by identification and antibiotic susceptibility testing using a Vitek 2 system. The results showed a significantly higher relative ARG abundance of 0.2367 ARG copies/16S rRNA gene copies in shower drains than in dishwashers (0.1329 ARG copies/16S rRNA gene copies) and washing machines (0.0006 ARG copies/16S rRNA gene copies). blaCMY-2, blaACT/MIR, and blaOXA-48 were the most prevalent ARG, and intI1 occurred in 96.3% of the households, while no mcr genes were detected. Several ß-lactamase genes co-occurred, and the resistance of bacterial isolates correlated positively with genotypic resistance, with carbapenemase genes dominating across isolates. Antibiotic-resistant bacteria were significantly reduced during automated dishwashing as well as laundering tests and did not differ from susceptible strains. Overall, the domestic environment may represent a potential reservoir of ß-lactamase genes and ß-lactam-resistant bacteria, with shower drains being the dominant source of ABR.IMPORTANCE The abundance of antibiotic-resistant bacteria and ARGs is steadily increasing and has been comprehensively analyzed in natural environments, animals, foods, and wastewater treatment plants. In this respect, ß-lactams and colistin are of particular interest due to the emergence of multidrug-resistant Gram-negative bacteria. Despite the connection of private households to these environments, only a few studies have focused on the domestic environment so far. Therefore, the present study further investigated the occurrence of ARGs and antibiotic-resistant bacteria in shower drains, washing machines, and dishwashers. The analysis of the domestic environment as a potential reservoir of resistant bacteria is crucial to determine whether households contribute to the spread of ABR or may be a habitat where resistant bacteria from the natural environment, humans, food, or water are selected due to the use of detergents, antimicrobial products, and antibiotics. Furthermore, ABR could limit the options for the treatment of infections arising in the domestic environment.

Winter is coming - Impact of temperature on the variation of beta-lactamase and mcr genes in a wastewater treatment plant.

Wastewater treatment plants (WWTP) play a key role in the dissemination of antibiotic resistance and analyzing the abundance of antibiotic resistance genes (ARGs) and resistant bacteria is necessary to evaluate the risk of proliferation caused by WWTPs. Since few studies investigated the seasonal variation of antibiotic resistance, this study aimed to determine the abundance of beta-lactamase and mcr genes and to characterize phenotypic resistant strains in a WWTP in Germany over the seasons. Wastewater, sewage sludge and effluent samples were collected over a one year period and analyzed using quantitative real-time PCR. Resistant strains were isolated, followed by identification and antibiotic susceptibility testing using VITEK 2. The results show a significantly higher occurrence of nearly all investigated ARGs in the wastewater compared to sewage sludge and effluent. ARG abundance and temperature showed a negative correlation in wastewater and significant differences between ARG abundance during warmer and colder seasons were determined, indicating a seasonal effect. Co-occurrence of mcr-1 and carbapenemase genes in a multi-drug resistant Enterobacter cloacae and Escherichia coli producing extended-spectrum beta-lactamase (ESBL) was determined. To the best of our knowledge, this is the first detection of mcr-1, blaVIM and blaOXA-48 in an ESBL-producing E. coli. Although wastewater treatment reduced the abundance of ARGs and resistant strains, a dissemination into the river might be possible because carbapenemase-, CTX-M- and mcr-1-gene harboring strains were still present in the effluent.