*3.1. ESBL Gram Negative Bacilli Isolates*

In total, 117 Enterobacteriaceae were screened for multidrug resistance phenotypically. Genetic analysis revealed 32 different positive isolates consisting of 21 *E. coli*, seven *Klebsiella pneumoniae*, three *Enterobacter* sp. and one *Raoultella ornithinolytica* (Table 1). Members of the CTX-M gene family were the most predominant ESBL genes.

The most detected ESBL gene was *bla*CTX-M-15, which was present in twelve (28.6%) of the 32 isolates followed by *bla*CTX-M-1, which was found in six (14.3%) isolates. In addition, five (11.9%) of the isolates harboured the *bla*CTX-M-14, three (7.1%) *bla*CTX-M-3, and one (2.4%) the *bla*CTX-M-38 gene. The non-CTX-M ESBL genes *bla*SHV-2 and *bla*SHV-12 were detected in four isolates from activated sludge.

Bacteria with ESBL phenotypes frequently carry additional antibiotic resistances. For the purpose of phenotypic differentiation, all ESBL *E. coli* isolates were tested for their susceptibility to 19 antibiotics. The antibiotic resistances of each of the investigated isolates are listed in Table 1.

No ESBL-producing Enterobacteriaceae showed resistance to tigecycline, amikacin and the carbapenems imipenem and meropenem. Penicillin-inhibitor combinations such as amoxicillin/clavulanic acid (53.1%, 17 of 32) and piperacillin/tazobactam (9.4%, 3 of 32) showed reduced efficacy against the ESBL producing Enterobacteriaceae. The cephamycin cefoxitin revealed resistance to eight (25%) isolates.

The most common co-resistance rates among the ESBL producing Enterobacteriaceae isolates to non-beta lactam antibiotics were detected for the quinolones, nalidixic acid 75% (24 of 32), ciprofloxacin 56.3% (18 of 32) and moxifloxacin 53.1% (17 of 32). The co-resistance for tetracycline was as high as 53.1% (17 of 32) and for the drug combination trimethoprim/sulfamethoxazole 50% (16 of 32). Co-resistance rates to aminoglycoside compounds were low with 34.4% (11 of 32) for gentamicin and 0% for amikacin.

Two ESBL-producing isolates were resistant to three antibiotics and 26 of the isolates were resistant to more than three antibiotic classes, which lead to a number of 28 ESBL isolates that could be assigned as multidrug resistant (Table 1).

Additional 25 Pseudomonadales were isolated from the ESBL screening plates but genetic analysing showed no positive confirmation for ESBL genes. Only one *Aeromonas* spp. isolate was tested positive for the ESBL gene *bla*PER-1. This isolate revealed resistance to ceftazidime and meropenem.


 **1.** Detected resistance genes and resistance pattern of all isolates. Non *E. coli* Enterobacteriaceae are automatically set resistant to AM according EUCAST.

**Table**



MEM, meropenem; CIP, ciprofloxacin; MXF, moxifloxacin; GM, gentamicin; SXT, trimethoprim/sulfamethoxazole; TE, tetracycline; NA, nalidixic acid; C, chloramphenicol; TEC, teicoplanin; VA, vancomycin; P, penicillin; E, erythromycin; NOR, norfloxacin. b Resistance genes *bla*TEM-1, *bla*SHV-1 and *bla*SHV-11 encoding non-extended-spectrum-β-lactamases.
