Nowadays, antimicrobial resistance (AMR) is a major health threat, and according to the World Health Organization (WHO), we are in severe danger of entering a post-antibiotic era, where simple bacterial infections will become untreatable (WHO, 2017). AMR is a phenomenon that has probably existed since microorganisms exist. Microorganisms produce antimicrobials to outcom-pete other microorganisms in their struggle for limited resources. The discovery of antimicrobials by man, their use, and overuse in our fight against infectious disease and the resulting environmental pollution caused by diverse anthropogenic activities (human medicine, veterinary medicine, agriculture) are factors that have been facilitating the development and spread of AMR. The development of AMR and the emergence of resistant microorganisms are linked to the use of antimicrobials (Goossens et al., 2005). The dramatic health threat caused by AMR is illustrated by the development of a common “One Health” global action plan on AMR by the WHO (WHO, 2015), by the World Organization for Animal Health (OIE, 2016), and the Food and Agriculture Organization (FAO, 2016) with the aim to minimize the impact of AMR. The focus areas of this global action plan include implementing AMR surveillance and antimicrobial residue monitoring in the environment (FAO, 2016). There is an increasing number of reports on the occurrence of clinically relevant multidrug-resistant (MDR) pathogens in the aquatic environment (Zurfluh et al., 2013; Mahon et al., 2017; Zarfel et al., 2017; Khan et al., 2018; Lepuschitz et al., 2017, 2018, 2019). Water is one of the most important habitats for bacteria. It is also a major medium for bacteria to disseminate and potentially to exchange among different environmental compartments, such as waste, surface, and drinking water (Vaz-Moreira et al., 2014). Studies increasingly emphasize the importance of aquatic systems as antibiotic resistance reservoirs, including antibiotic residues, antibiotic-resistant bacteria, and antibiotic-resistant genes, which can be exchanged between pathogenic and nonpathogenic bacteria (Baquero et al., 2008; Zhang et al., 2009; Rizzo et al., 2013; Manaia et al., 2016). However, at present, it is not clear to what extent environmental bacteria are a source for novel resistance mechanisms or which circumstances force them to spread antibiotic resistance. Therefore, the question how antibiotic resistance in the water environment affects human health still needs to be investigated and discussed (Vaz-Moreira et al., 2014). The aim of our study was to assess the burden of AMR caused by
In July and August 2017, 27 water samples were collected (according to ÖNORM M 6230, 2015) from 27 bathing sites, all of which fulfilled the criteria set by the EU Bathing Water Directive. Three sites were arbitrarily chosen per state. Water samples were collected in a sterile 500-ml glass flask, 30 cm below the river/lake surface, 2 m from the bank. A 100-ml water sample aliquot was filtered using 0.45-μm pore-sized membranes (Microfil® S device; Merck, Vienna, Austria), and the filtrate was incubated in thioglycollate broth (Becton Dickinson, Franklin Lakes, NJ, USA) at 37°C overnight. To detect vancomycin resistance and screen for carbapenemase-producing and extended-spectrum beta-lactamase (ESBL)-producing isolates, 50 μl of overnight cultures was subcultivated on selective chromogenic media (chromID™ VRE, chromID™ CARBA, and chromID™ ESBL (bioMérieux, Marcy-l’Étoile, France). For the detection of methicillin-resistant staphylococci (MRSA), the overnight cultures were cultivated on BBL™ CHROMagar™ MRSA II (Becton Dickinson, Vienna, Austria). Subcultivated single colonies were identified at species level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) mass spectrometry using an MALDI Biotyper (Bruker, Billerica, MA, USA).
From subcultivated isolates, the DNA was extracted using the MagAttract High-Molecular-Weight DNA Kit (Qiagen, Hilden, Germany) and quantified fluorometrically on a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using a target-specific Qubit assay for double stranded deoxyribonucleic acid (dsDNA BR Assay Kit, Thermo Fisher Scientific). The Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) was used to prepare libraries for whole genome sequencing (WGS) according to manufacturer’s protocol. Paired-end sequencing (2 × 300 bp) of genomic libraries was performed using the Illumina Miseq instrument. Sequencing coverage calculator (www.
The screening for antimicrobial-resistant bacteria yielded negative results in 23 of the 27 samples (Figure 1). Four water sample subcultures yielded growth on one chromogenic medium each: chromID™ CARBA (water sample K3) and chromID™ ESBL (water samples B1, NOE2, V2). No growth was observed on chromID™ VRE and on BBL™ CHROMagar™ MRSA II plates. Primary species identification using MALDI-TOF-MS identified one bacterial species in each of the four water samples (Table 1).
Antibiotic-resistant bacteria detected at Austrian bathing sites
Tabelle 1. Vorkommen von antibiotikaresistenten Bakterien in österreichischen Badegewässern
MALDI-TOF-MS | Water sample ID | Agar plate yielding isolate | Collection date | Federal state | Bathing site |
---|---|---|---|---|---|
B1 | chromID™ ESBL | 11.07.2017 | Burgenland | Stausee Forchtenstein | |
K3 | chromID™ CARBA | 28.08.2017 | Carinthia | Ossiachersee Bodensdorf | |
NOE2 | chromID™ ESBL | 10.07.2017 | Lower Austria | Donaualtarm Greifenstein | |
V2 | chromID™ ESBL | 05.09.2017 | Vorarlberg | Bregenz Wocherhafen |
The
In Table 2, the
Antimicrobial susceptibility testing results of four resistant water isolates
Tabelle 2. Ergebnisse der Antibiotika-Resistenztestung der vier resistenten Wasserisolate
Sample ID | AM | AMC | PIP-TAZ | CXM-AX | FOX | CTX | CAZ | FEP | ATM | IPM | MEM | AN | GM | CIP | MXF | TGC | FOS | SXT |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
N0E2 ( | ≥32 | ≥32 | ≥128 | NA | NA | ≥64 | ≥64 | ≤1 | 16 | ≤0.25 | ≤0.25 | ≤2 | ≤1 | ≤0.25 | ≤0.25 | 1 | 64 | ≤20 |
B1 ( | - | - | 8 | - | - | - | 4 | 2 | 16 | 1 | ≤0.25 | ≤2 | ≤1 | ≤0.25 | - | - | - | - |
K3 ( | ≥32 | ≥32 | ≤4 | NA | NA | ≤1 | ≤1 | ≤1 | ≤1 | ≥16 | ≥16 | ≤2 | ≤1 | ≤0.25 | 0.5 | ≤0.5 | ≤16 | ≤20 |
V2 ( | ≥32 | 8 | ≤4 | ≥64 | ≤4 | ≥64 | ≤1 | 2 | 4 | ≤0.25 | ≤0.25 | ≤2 | ≤1 | ≤0.25 | ≤0.25 | ≤0.5 | ≤16 | ≤20 |
Interpretation of MIC breakpoints (mg/L) according to the EUCAST criteria (red = resistant, orange = intermediate, green = sensitive); AM = ampicillin, AMC = amoxicillin/clavulanic acid, PIP-TAZ = piperacillin/tazobactam, CXM-AX = cefuroxime axetil, FOX = cefoxitin, CTX = cefotaxime, CAZ = ceftazidime, FEP = cefepime, ATM = aztreonam, IPM= imipenem, MEM = meropenem, AN = amikacin, GM = gentamicin, CIP = ciprofloxacin, MXF = moxifloxacin, TGC = tigecycline, FOS = fosfomycin, SXT= trimethoprim/sulfamethoxazole; NA = no defined breakpoints available
In our study, the screening for antimicrobial-resistant bacteria was negative in 23 of the 27 samples. Resistant bacteria were detected from 4 of the 27 bathing sites. Two of the four isolates carried plasmids:
The detection of MRSA was negative in all investigated isolates, and there are only a few studies describing the cultivation of MRSA from water samples (Tolba et al., 2008; Boopathy 2017; Lepuschitz et al., 2017, 2018). Up to date there are no official guidelines for the detection of MRSA from water samples, which might lead to the underestimation of MRSA in the environment. According to the definition by Magiorakos et al. (2012), three of the four bacteria isolated in our study on Austrian bathing sites were non-susceptible to at least one agent in three or more antimicrobial categories and therefore categorized as MDR bacteria (Magiorakos et al., 2012). Only the
We consider the public health risk at Austrian bathing sites authorized under the EU Bathing Water Directive to be low despite the occurrence of MDR bacteria. However, our results confirm the existing risk for dissemination of MDR bacteria via the aquatic environment.