Antibiotic resistance crisis and the influence of land use practices

Nowadays, we are in an antibiotic resistance crisis as antibiotic-resistant bacteria (ARBs) have become a medical priority problem. Infections that were treatable in the past cause acute problems today and, in increasing numbers, even death. In fact, it is estimated that ARBs are responsible for 670,000 infections in Europe per year, whereof 33,000 lead to the patient’s death (Cassini et al. 2019).

Furthermore, they are the cause for 870,000 years under disability adjusted conditions and lead in

7 Goossens 2019). Bacterial pathogens are often not only drug resistant but can contain MGEs stocked with several different resistance mechanisms, rendering them multidrug resistant (MDR) (Partridge et al. 2018). Examples for bacteria that are frequently MDR are the ESKAPEE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli (Santajit and Indrawattana 2016;

Partridge et al. 2018). Another name that should be mentioned in this context is MDR Clostridium difficile which can cause infections of the gastro-intestinal tract as a consequence of an imbalance in the intestinal microflora, due to antibiotic treatment (Spigaglia, Mastrantonio, and Barbanti 2018). The ESKAPEE pathogens as well as C. difficile are notorious with respect to globally occurring nosocomial (hospital acquired) infections which are extremely difficult to treat and therefore a serious threat to human health.

Even though the highest density of ARGs is prevalent in bacteria from clinical settings (Surette and Wright 2017), the true origin of the respective genes is in most cases still unclear. As outlined above, the soil bacterial resistome comprises an inconceivably large variety of resistance genes which is very likely the origin of many pathogen encoded ARGs. This theory is supported by the mentioned fact that intrinsic resistance mechanisms can be mobilized and acquisition of ARGs through HGT is by far more efficient than the invention of novel resistance mechanisms via mutational changes. In fact, it has been discovered that specific ARGs which are encoded by known human pathogens, such as K. pneumoniae or Salmonella typhimurium, show 100% identity to genes of soil bacteria (Forsberg et al. 2012). Furthermore, evidence suggests that ARGs can be transferred from harmless soil bacteria to hazardous pathogens via HGT events (Forsberg et al. 2012; Pärnänen et al. 2016). Therefore, it can be concluded that ARGs and ARBs can spread to humans through direct or indirect contact with the soil microbial community (EMA 2018; Forsberg et al. 2012; Canteón 2009). These circumstances underline the importance of the in depth study of the soil resistome in order to identify unknown resistance mechanisms that may become problematic in the future.

The occurrence of MDR pathogens is closely linked to human use of antibiotics since the middle of the 20^th century (Surette and Wright 2017). An accumulation of ARGs in MGEs and their efficient spread over species borders probably occurs much more frequently nowadays because of the selection

8 pressure, established through anthropogenic antibiotic pollution (Bengtsson-Palme, Kristiansson, and Larsson 2018). Particularly relevant in this regard is the treatment of livestock in agriculture, a practice that is quite common, due to the prevalent factory farming and the associated higher infection risk of farm animals. A major fraction of all human diseases develops in animals (van Doorn 2014) that are potentially colonized by bacteria which have evolved resistance mechanisms as a result of continuous antibiotic exposure. Humans can pick up these antibiotic resistant pathogens via the food chain and fall sick with hard-to-treat infections. An example for such a food-borne infection is campylobacteriosis, a gastro-intestinal disease that is caused by Campylobacter species which are very frequently resistant to fluoroquinolones (EFSA and ECDC 2019) (bacteriocidal antibiotics that inhibit DNA replication).

Another reason why antibiotic treatment of livestock is problematic is the large proportion of antibiotics that are excreted functionally by the treated animals. Consequently, manure is often enriched with the active compounds as well as with bacteria that have developed resistance against these harmful substances (Berendsen et al. 2015). When manure is applied as organic fertilizer, an increase in the abundance of medically relevant ARGs and MGEs within the soil microbial community can occur (Graham et al. 2016; Jechalke et al. 2014; Binh et al. 2007). Additionally, antibiotics, ARBs and ARGs can disseminate throughout the environment via surface water run offs, dust and migrating wild animals (Allen et al. 2010). This leads to a circulation of ARGs between soil, human and livestock, driven by the evolutionary pressure established through antibiotic application or pollution (Figure 2).

9 Figure 2. The role of the environment in the recruitment of antibiotic resistance genes (ARGs) to human pathogens. This takes place in four major steps: (1) emergence of novel resistance factors in the environment, (2) mobilization onto mobile genetic elements, (3) transfer of ARGs to human pathogens, and (4) dissemination of ARGs into the human microbiome. The arrow widths approximately mirror the estimated frequency of each event.

Adapted from Bengtsson-Palme, Kristiansson, and Larsson 2018.

To find ways to counteract the dissemination of ARGs, it is necessary to consider the contribution of a variety of parameters, many of which are presently still elusive. For example, detailed information on the overall contribution of land use types and intensities that influence the development and transmission of ARGs in soil microbiomes need to be evaluated. Currently, most studies that address this issue focus on a small number of study plots, or set up microcosms from just one or two

10 soils, and simulate land use by e.g. spiking manure with antibiotics. However, data from various soil sites over a large spatial scale with a variety of realistic land use histories is still lacking. This would allow a deeper understanding of the effect of different land use types and intensities on the abundance and transmission of ARGs and MGEs throughout soil ecosystems which could be, together with data on other environmental resistomes, useful for the development of approaches to overcome the antibiotic resistance crisis.