*3.3. VRE*

VRE could be detected in four of eleven (36%) activated sludge samples represented by one Enterococcus isolate each. All four isolates were identified as *Enterococcus faecium* and harboured the *vanA* gene. All isolates showed highly similar resistance patterns. They were all resistant to ampicillin, teicoplanin, and vancomycin; three isolates showed additional resistance to trimethoprim/sulfamethoxazole (Table 1).

#### **4. Discussion**

The omnipresence of ESBL in environmental population of Enterobacteriaceae is widely demonstrated. The findings of this study go in full concordance with prior results. This includes also the isolated species (mostly *E. coli*) and the detected genes (CTX-M family) being dominant [8,11,29,30].

Other studies concerning *E. coli* from sewage sludge also reported tetracycline, ampicillin/clavulanic acid and trimethoprim/sulfamethoxazole as antibiotics with the highest nonsusceptibility rate. These antibiotics showed the highest non-susceptibility in ESBL *E. coli* from Austrian sewage sludge as well [31,32]. Regarding co-resistance, the isolates did not show a reduced occurrence as can be observed in ESBL isolates from surface waters, without direct wastewater influence. Resistance to quinolones was very common and most of the isolates could be classified as multiresistant (resistance to three or more tested antibiotic classes). Environment and residence time in the WWTP seem not to favour a potential adaptation process in the ESBL population. The permanent entry of ARB from different sources in the activated sludge basin and the horizontal gene transfer are the dominant factors for the composition of resistant bacteria. Selection pressure due to different substances, does not seem to have enough time in this environment to contribute to resistance development [9,10,30,33–37]. Therefore, the isolates of this study reflect rather the situation of clinical ESBL isolates where this kind of co-resistance and multiresistance is dominant. Interestingly the majority of the ESBL Enterobacteriaceae isolates remained susceptible to the tested 4th generation cephalosporin (cefepim).

The isolation of a PER-1 producing *Aeromonas* spp. is more remarkable. There are reports of PER-1 based ESBL (also in *Aeromonas*) in European surface waters, nevertheless clinical isolates with this enzyme are reported rarely. In Austria, this is the first PER-1 producer documented so far [38,39].

The MRSA isolates from the sewage sludge can be linked to hospital settings. A multiresistant phenotype including the aminoglycoside gentamicin is a typical characteristic of hospital acquired (HA)-MRSA. T032 is a common *spa* type of the ST22-MRSA-IV (Barnim epidemic MRSA strain). It is the most prevalent HA-MRSA in Europe and has spread in Austria since the beginning of this decade. The second gentamicin resistant (t067) isolate can be linked to the so called paediatric clone. The resistance pattern of this MRSA isolate, with the exotic *spa* type t6613, showed similarity with CA-MRSA, but did not harbour the genes for the PVL toxin [40,41].

In general MRSA isolates from surface water are rather rare, with only low number of analysed isolates. Therefore an estimation which of the three MRSA types is more dominant in water environment is difficult to make [15,16,42,43].

VRE isolates showed nearly identical features in terms of species, gene and resistance pattern. Likewise MRSA, VRE isolates were only investigated and isolated in few studies compared to studies with ESBL isolates. This is remarkable because in contrast to Staphylococci, Enterococci have a

much better ability to survive in surface water and they are indicator bacteria for water quality assessment [44–46]. Therefore, the exclusivity of *vanA* isolates is more likely to be based on the low number of sludge isolates. Furthermore other environmental VRE isolates from Austria revealed also *vanB* [44–49].

However, there is much evidence that confirms the presence of diverse and plentiful ARB in fertilizer produced from livestock animals [50,51]. There appears to be significant variability on wastewater management across different industrialized countries. In high income countries sewer connectivity is generally high, whereas in many middle and low income countries sewer connectivity is low and untreated sewage is discharged mainly to surface water bodies [52,53].
