*3.4. Determination of a Chromosomally-Encoded Resistance Mechanism*

All alleles of *soxR*, *marR* and *rpsJ* were identical with sequences from tigecycline susceptible *Klebsiella pneumoniae* strains previously described (GenBank accession numbers: CP000647.1 [22], CP009461.1 [28], CP003999.1 [29], KC843636.1 [21]). Even though *marR* of MurTR-KL002 harbored a silent mutation (C270A), no other mutations within these genes could be observed.

With regard to the reference strain *Klebsiella pneumoniae* subsp. *pneumoniae* MGH 78578 (GenBank accession number: CP000647.1 [22]), mutations in both isolates could be observed within the *ramR* allele. *RamR* of MurTR-KL001 primarily harbored a four base pair deletion (Δ 518–521CCCG) resulting in a frameshift. Secondarily, a silent point mutation was on position 291 with a G to A mutation. *RamR* of MurTR-KL002 harbored a point mutation (152A > C), which resulted in an amino acid substitution (K51T) (Table 1).

#### **4. Discussion**

Tigecycline non-susceptible *Klebsiella pneumoniae* were recently isolated from heavily polluted coastal waters in Brazil [30] and less recently from hospital sewage in Saudi Arabia [31]. Other resistant *Enterobacteriaceae* could be recovered from drinking water samples in India [32].

However, such reports from more "decent" aquatic settings are rare in the current literature. Recent cases of tigecycline-resistant *Klebsiella pneumoniae* were reported from urban surface waters in Brazil: from a river downstream of a wastewater treatment plant, in Curitiba [1], and from an urban lake and reservoir in the city of Sao Paulo [4].

Even though the two isolates belong to two newly described and distinctly different MLST types, they seem to share the same resistance mechanism (even though owing two different mutations), which could indicate a common selective pressure. They were susceptible to all other tested antibiotics. Taking also into account that both isolates belong to two new MLST types it is very unlikely that these isolates are a contamination from a clinical source. Although low concentrations of antibiotics can cause an ecological shift towards less susceptible bacteria, it is rather unlikely that the river Mur was contaminated with tigecycline [33].

A recent study demonstrated cross-resistance to antibiotics, including tetracycline, in association with the resistance to linalool, a component of basil oil that is used as a natural preservative. The increased resistance to linalool was accompanied by the overexpression of the AcrAB efflux pump suggesting linalool as potential substrate [34]. A similar cross-resistance to antibiotics was observed in association with the resistance to pine oil and the tolerance to solvents; in both cases resistance correlated with the activity of the AcrAB efflux pump [35]. Decreased susceptibility to triclosan, a biocide, was also reported in the course of *acrAB* overexpression. Moreover, the AcrAB efflux pump extrudes dyes and detergents, and appears to play a more crucial role, as it is embedded in fundamentally physiological functions; for instance, in cell-to-cell communication and in virulence. It appears plausible that a cross-resistance to an antibiotic could easily fall within a more fundamentally microbial purpose as long as the overexpression of the efflux pump is favored within an ecological and physiological setting. In that manner, higher concentrations of any potential substrate could select for, i.e., a tigecycline resistance [36–38].

Nikaido et al. and Baucheron et al. proposed a mechanism of induction for the AcrAB locus. They suggested that indole and bile bind to RamR, thereby inhibiting its repressing effect on *ramA* transcription, and therefore promoting the induction of the *ramR* and *acrAB* locus. Yamasaki et al. further reported that different substrates can bind to RamR due to a flexible binding pocket and upon binding the DNA binding affinity of RamR decreases. Therefore, substrates could act as extracellular signals that force subsequent induction of *ramA* and *acrAB* expression, whenever the efflux system is overloaded. However, a mutation within RamR can also lead to the induction of the efflux pump resembling a permanent sensing signal. That arrangement may endure in a suitable ecological or physiological condition [39,40].

RamR represents a genetic hotspot for mutations as far as clinical Klebsiella pneumoniae isolates are concerned [18–21]. None of the reported mutations are identical, the closest mutation to the MurTR-KL001 isolate was described by Rosenblum et al. [41]. Nevertheless, reports of aquatic isolates harboring such mutations are absent in the current literature.

#### **5. Conclusions**

The presence of two genetically different isolates suggests that river water may bear substances that favor mutations that can lead to this efflux pump-driven resistance. The origin of these substances (e.g., triclosan or heavy metals) may be waste water or surface run-off after rainfall. Therefore, the occurrence and impact on human health of such mutations in bacteria in surface waters must be further investigated.

**Author Contributions:** Gernot Zarfel conceived and designed the study; Alexander Hladicz, Gernot Zarfel performed the experiments; Alexander Hladicz, Clemens Kittinger and Gernot Zarfel analyzed the data; Alexander Hladicz wrote the manuscript. Clemens Kittinger and Gernot Zarfel edited the manuscript.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.
