Spread and persistence of antibiotic resistance pose a severe threat to human health, yet there is still lack of knowledge about reservoirs of antibiotic resistant bacteria in the environment. We took the opportunity of the Joint Danube Survey 3 (JDS3), the world's biggest river research expedition of its kind in 2013, to analyse samples originating from different sampling points along the whole length of the river. Due to its high clinical relevance, we concentrated on the characterization of Pseudomonas spp. and evaluated the resistance profiles of Pseudomonas spp. which were isolated from eight sampling points. In total, 520 Pseudomonas isolates were found, 344 (66.0%) isolates were identified as Pseudomonas putida, and 141 (27.1%) as Pseudomonas fluorescens, all other Pseudomonas species were represented by less than five isolates, among those two P. aeruginosa isolates. Thirty seven percent (37%) of all isolated Pseudomonas species showed resistance to at least one out of eleven tested antibiotics. The most common resistance was against meropenem (30.4% / 158 isolates) piperacillin/tazobactam (10.6% / 55 isolates) and ceftazidime (4.2% / 22 isolates). 16 isolates (3.1% / 16 isolates) were multi-resistant. For each tested antibiotic at least one resistant isolate could be detected. Sampling points from the upper stretch of the River Danube showed more resistant isolates than downriver. Our results suggest that antibiotic resistance can be acquired by and persists even in Pseudomonas species that are normally not in direct contact with humans. A possible scenario is that these bacteria provide a reservoir of antibiotic resistance genes that can spread to related human pathogens by horizontal gene transfer.

In a clinical setting it seems to be normal these days that a relevant proportion or even the majority of different bacterial species has already one or more acquired antibiotic resistances. Unfortunately, the overuse of antibiotics for livestock breeding and medicine has also altered the wild-type resistance profiles of many bacterial species in different environmental settings. As a matter of fact, getting in contact with resistant bacteria is no longer restricted to hospitals. Beside food and food production, the aquatic environment might also play an important role as reservoir and carrier. The aim of this study was the assessment of the resistance patterns of Escherichia coli and Klebsiella spp. out of surface water without prior enrichment and under non-selective culture conditions (for antibiotic resistance). In addition, the presence of clinically important extended spectrum beta lactamase (ESBL) and carbapenmase harboring Enterobacteriaceae should be investigated. During Joint Danube Survey 3 (2013), water samples were taken over the total course of the River Danube. Resistance testing was performed for 21 different antibiotics. Samples were additionally screened for ESBL or carbapenmase harboring Enterobacteriaceae. 39% of all isolated Escherichia coli and 15% of all Klebsiella spp. from the river Danube had at least one acquired resistance. Resistance was found against all tested antibiotics except tigecycline. Taking a look on the whole stretch of the River Danube the proportion of multiresistances did not differ significantly. In total, 35 ESBL harboring Enterobacteriaceae, 17 Escherichia coli, 13 Klebsiella pneumoniae and five Enterobacter spp. were isolated. One Klebsiella pneumoniae harboring NMD-1 carbapenmases and two Enterobacteriaceae with KPC-2 could be identified. Human generated antibiotic resistance is very common in E. coli and Klebsiella spp. in the River Danube. Even isolates with resistance patterns normally associated with intensive care units are present.

Antibiotic resistant bacteria (ARB) in the aquatic environment are reported from all over the world and their presence in the environment has become quite common. The current most prominent example is the presence of beta-lactamases harboring Enterobacteriaceae. The aim of this study was to investigate the presence and diversity (on the genetic and phenotypic levels) of extended spectrum beta-lactamases (ESBL) and carbapenemases harboring Enterobacteriaceae from the River Mur in the center of Graz, Austria's second largest city. Thus over a period of four months water samples were taken, filtrated and screened for these bacteria. All samples revealed ESBL harboring Enterobacteriaceae, of which all with only one exception were Escherichia coli. Dominant ESBL gene family was CTX-M, represented by subgroups CTX-M-1 group, CTX-M-2 group and CTX-M-9 group. Surprisingly co-resistances to non-beta-lactam antibiotics were low, only resistance to trimethoprim was detected in 50% of all (70) isolates. One Klebsiella oxytoca with GES-1 was isolated. To date, GES ESBL has never been reported from Austria before and only rarely from other European countries. Screening for carbapenemase harboring Enterobacteriaceae revealed one Enterobacter cloacae with the gene for VIM-1. Members sharing the same multi-locus-sequence-type (MLST) as well as members of the same rep PCR clusters occurred at different sampling time points. ESBL-harboring Enterobacteriaceae are common in Austrian river water, is dominated by Escherichia coli and CTX-M enzymes. Furthermore, some of the isolates could be linked to different origins.

Acinetobacter spp. occur naturally in many different habitats, including food, soil, and surface waters. In clinical settings, Acinetobacter poses an increasing health problem, causing infections with limited to no antibiotic therapeutic options left. The presence of human generated multidrug resistant strains is well documented but the extent to how widely they are distributed within the Acinetobacter population is unknown. In this study, Acinetobacter spp. were isolated from water samples at 14 sites of the whole course of the river Danube. Susceptibility testing was carried out for 14 clinically relevant antibiotics from six different antibiotic classes. Isolates showing a carbapenem resistance phenotype were screened with PCR and sequencing for the underlying resistance mechanism of carbapenem resistance. From the Danube river water, 262 Acinetobacter were isolated, the most common species was Acinetobacter baumannii with 135 isolates. Carbapenem and multiresistant isolates were rare but one isolate could be found which was only susceptible to colistin. The genetic background of carbapenem resistance was mostly based on typical Acinetobacter OXA enzymes but also on VIM-2. The population of Acinetobacter (baumannii and non-baumannii) revealed a significant proportion of human-generated antibiotic resistance and multiresistance, but the majority of the isolates stayed susceptible to most of the tested antibiotics.

Wastewater contains different kinds of contaminants, including antibiotics and bacterial isolates with human-generated antibiotic resistances. In industrialized countries most of the wastewater is processed in wastewater treatment plants which do not only include commercial wastewater, but also wastewater from hospitals. Three multiresistant pathogens—extended spectrum β-lactamase (ESBL)-harbouring Enterobacteriaceae (Gram negative bacilli), methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE)—were chosen for screening in a state of the art wastewater treatment plant in Austria. Over an investigation period of six months all three multiresistant pathogens could be isolated from activated sludge. ESBL was the most common resistance mechanism, which was found in different species of Enterobacteriaceae, and in one Aeromonas spp. Sequencing of ESBL genes revealed the dominance of genes encoding members of CTX-M β-lactamases family and a gene encoding for PER-1 ESBL was detected for the first time in Austria. MRSA and VRE could be isolated sporadically, including one EMRSA-15 isolate. Whereas ESBL is well documented as a surface water contaminant, reports of MRSA and VRE are rare. The results of this study show that these three multiresistant phenotypes were present in activated sludge, as well as species and genes which were not reported before in the region. The ESBL-harbouring Gram negative bacilli were most common.