**1. Introduction**

The emergence of antibiotic resistances is a worldwide rising phenomenon. It is not restricted to clinical settings and it reaches environmental settings and their associated ecological habitats. In particular, surface waters such as rivers, lakes or coastal waters act as reservoirs for resistant bacteria owing to anthropogenic activities and influences such as industrial or urban sewage [1–5]. The discharge of resistant bacteria in combination with antibiotics and/or other chemical compounds into the water bodies is likely to select for antibiotic resistances within microbial communities [6–8]. Therefore, effluents or insufficient water management promotes the distribution of resistant bacteria and facilitates the spread of resistance genes [9]. This trend of emerging antibiotic-resistant bacteria speeds up by the overuse of antibiotics in human and veterinary medicine, and a subsequent release of these substances into the environment [10].

The massive health problem that arises from the current situation concerns (opportunistic) pathogens that gained multidrug resistance (MDR) to a broad spectrum of antibiotics. ESBL-(extended-spectrum b-lactamase) or carbapenemase-producing *Enterobacteriaceae*, notably *Klebsiella pneumoniae*, are described not only in clinical but also in different aquatic settings all around the world, including Austria [2–4,11–13].

Last-resort antibiotics act as ultimate force to overcome those multiresistant strains. Tigecycline is such an antibiotic and is often the last or the penultimate choice (besides colistin) to treat infections caused by those pathogens [14,15]. Hence, occurrence of tigecycline resistance is a major threat to every medical institution. Cases of tigecycline non-susceptible *Klebsiella pneumoniae* in clinical settings are reported worldwide [16,17] but are rather rare regarding environmental settings.

There are different mechanisms that can lead to an acquired tigecycline resistance, most of them based on chromosomal mutations. Gene network of the efflux pump, AcrAB-TolC is associated with tigecycline non-susceptibility and its regulators has been analyzed with regard to tigecycline non-susceptibility in prior studies. In particular, mutation in the repressors RamR, MarR and SoxR of the regulators (RamA, MarA and SoxS) of the efflux pump were found to be responsible [18–22].

An additional mutation in the ribosomal RPS10 protein, which is located close to the ribosomal binding site of tigecycline is likely to influence the binding properties between the ribosome and tigecycline [21].

The aim of this study was to elucidate the resistance mechanism that causes tigecycline non-susceptibility and to question whether this mechanism is plasmid or chromosomally mediated. In order to detect a potential plasmid-encoded resistance mechanism, transformation experiments were performed.

#### **2. Material and Methods**

## *2.1. Sample Collection*

Water samples were taken for microbiological investigations during a survey from the river Mur in the center of Graz (47◦4 38" N; 15◦25 60" E); each sample in two sterile 500 mL glass flasks, 30 cm below the river surface, 50 cm apart from the river bank.
