Metagenomic Insights Into the Changes of Antibiotic Resistance and Pathogenicity Factor Pools Upon Thermophilic Composting of Human Excreta

Abstract

In times of climate change, practicing a form of sustainable, climate-resilient and productive agriculture is of primordial importance. Compost could be one form of sustainable fertilizer, which is increasing humus, water holding capacity, and nutrient contents of soils. It could thereby strengthen agriculture toward the adverse effects of climate change, especially when additionally combined with biochar. To get access to sufficient amounts of suitable materials for composting, resources, which are currently treated as waste, such as human excreta, could be a promising option. However, the safety of the produced compost regarding human pathogens, pharmaceuticals (like antibiotics) and related resistance genes must be considered. In this context, we have investigated the effect of 140- and 154-days of thermophilic composting on the hygienization of human excreta and saw dust from dry toilets together with straw and green cuttings with and without addition of biochar. Compost samples were taken at the beginning and end of the composting process and metagenomic analysis was conducted to assess the fate of antibiotic resistance genes (ARGs) and pathogenicity factors of the microbial community over composting. Potential ARGs conferring resistance to major classes of antibiotics, such as beta-lactam antibiotics, vancomycin, the MLSB group, aminoglycosides, tetracyclines and quinolones were detected in all samples. However, relative abundance of ARGs decreased from the beginning to the end of composting. This trend was also found for genes encoding type III, type IV, and type VI secretion systems, that are involved in pathogenicity, protein effector transport into eukaryotic cells and horizontal gene transfer between bacteria, respectively. The results suggest that the occurrence of potentially pathogenic microorganisms harboring ARGs declines during thermophilic composting. Nevertheless, ARG levels did not decline below the detection limit of quantitative PCR (qPCR). Thresholds for the usage of compost regarding acceptable resistance gene levels are yet to be evaluated and defined.

Fate of Horizontal-Gene-Transfer Markers and Beta-Lactamase Genes during Thermophilic Composting of Human Excreta

“…The present study aimed to analyze the fate of beta-lactamase genes, as well as marker genes for HGT during thermophilic composting of human excreta. These results complement a previous study on the same samples analyzing antibiotic resistance and virulence genes [ 62 ].…”

“…This leads to higher variation in replicate measurements [ 68 ]. The bla gene abundances were also low in comparison with other ARGs analyzed before [ 62 ]. This is interesting, since metagenomic data on the same samples revealed the highest abundance for the group of beta-lactam ARGs, with significant decreases during composting [ 62 ].…”

“…The study aimed to assess the fate of three beta-lactam ARGs of high clinical relevance, as well as the class 1 integron-integrase gene intI1 and korB marker gene for IncP-1 plasmids as indicators for HGT during thermophilic composting of human excreta amended with green cuttings and straw, with and without biochar. The trial and results of cultivation-based and molecular analyses on the bacterial community and ARGs for different antibiotic classes have been published previously [ 61 , 62 ].…”

“…The qPCR analyses were conducted as stated in [ 62 ] on a LightCycler ® 480 II instrument (Roche Diagnostics Ltd.). Primer sequences, PCR amplicon sizes, and references are listed in Table 1 .…”

“…We previously assessed the bacterial community in the same samples using 16S rRNA gene amplicon sequencing, as well as cultivation-based approaches, to analyze potential human pathogens in the mature compost [ 61 ]. Moreover, we performed metagenomic sequencing and qPCR, analyzing virulence factors and ARGs conferring resistance to different antibiotic classes [ 62 ]. This study complements our previous publications by examining the fate of environmentally relevant beta-lactamase genes and the impact of thermophilic composting on the horizontal spread of resistance genes.…”