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 Enterobacteriales, enterococci, methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas spp. (organisms commonly considered microbial indicators for water contamination (WHO, 2003)) in Austrian bathing sites, an aqueous environment supposedly less prone to anthropogenic influence than regular surface waters.
2 Materials and methods
2.1 Sampling, enrichment, and cultivation of resistant bacteria
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).
2.2 Antimicrobial susceptibility testing
In vitro susceptibility testing was performed with VITEK 2 Compact System (bioMérieux, Marcy-l’Étoile, France) using VITEK® 2 AST196 and AST-P586 cards interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria for Enterobacteriaceae, Pseudomonas spp., and Enterococcus spp. (European Committee on Antimicrobial Susceptibility Testing, EUCAST Clinical Breakpoint Tables v. 8.0, valid from January 01, 2018).
2.3 DNA isolation and whole genome sequencing
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. illumina.com/CoverageCalculator) was used to calculate for a desired mean coverage of at least 100-fold. De novo assembly of raw reads was performed using SPAdes (version 3.9.0) (Bankevich et al., 2012) and next generation sequencing (NGS) data interpretation was carried out using SeqSphere+ software (Ridom, Münster, Germany). Primary species confirmation was performed using rMLST (ribosomal multilocus sequence typing) (Jolley et al., 2012). For further phylogenetic analysis, the classical MLST (multi-locus sequence type) (Jolley et al., 2004) was extracted from the WGS data. PlasmidFinder 1.3 (Carattoli et al., 2014), Serotype-Finder 2.0 (Joensen et al., 2015), and VirulenceFinder 2.0 (Joensen et al., 2014) available from the Center for Genomic Epidemiology web server (http://www.genomi-cepidemiology.org) and the Comprehensive Antibiotic Resistance Database (CARD) (Jia et al., 2017) were used to identify Escherichia coli serotypes and to search for the presence of plasmids, virulence genes, or genes conferring antibiotic resistance.
3.1 Strain isolation and primary species identification
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|
|Pseudomonas aeruginosa||B1||chromID™ ESBL||11.07.2017||Burgenland||Stausee Forchtenstein|
|Enterobacter mori||K3||chromID™ CARBA||28.08.2017||Carinthia||Ossiachersee Bodensdorf|
|Enterobacter cloacae||NOE2||chromID™ ESBL||10.07.2017||Lower Austria||Donaualtarm Greifenstein|
|Escherichia coli||V2||chromID™ ESBL||05.09.2017||Vorarlberg||Bregenz Wocherhafen|
3.2 Whole genome sequencing analysis
The Pseudomonas aeruginosa isolate was assigned to classical ST2698; no plasmids were detected. The Enterobacter mori isolate was assigned to classical ST1009; no plasmids were identified. WGS analysis of the Enterobacter cloacae isolate was assigned to classical ST102 and the presence of three plasmids (IncHI2A (accession no. BX664015), pSL483 (CP001137), IncFII (CP001919)) was identified. Subtyping of the E. coli isolate was assigned to classical ST10 (Warwick scheme), serotype H40, and the presence of the plasmid IncI1 (AP005147) was identified. The analysis of the E. coli isolate with VirulenceFinder revealed the presence of one virulence gene iss (increased serum survival, CP001509) and the absence of Shiga-toxin genes.
3.3 In vitro and in silico antimicrobial resistance analysis
In Table 2, the in vitro susceptibility testing results of the four resistant water isolates (B1, K3, NOE2, and V2) using VITEK 2 Compact System method are summarized. The P. aeruginosa isolate harbored 51 AMR gene loci in total, of which 42 were associated with antibiotic efflux, 4 with target alteration, and 5 with antibiotic inactivation (FosA, OXA-50, PDC-3, APH(3’)-IIb, and catB7). The isolate was susceptible to piperacillin/tazobactam, ceftazidime, cefepime, imipenem, meropenem, amikacin, gentamicin, and ciprofloxacin and intermediately resistant to aztreonam (MIC 16 mg/L). The E. mori isolate was resistant to imipenem and meropenem. Additionally, it was resistant to ampicillin, amoxicillin/clavulanic acid, and moxifloxacin. It harbored 23 AMR gene loci in total, of which 15 were associated with antibiotic efflux, 4 with target alteration, and 4 with antibiotic inactivation (NmcR, FosA2, ACT-29, and IMI-2). The E. cloacae isolate was an AmpC producer. It was resistant to ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, cefotaxime, ceftazidime, aztreonam, and fosfomycin. It harbored 25 AMR gene loci in total, of which 14 were associated with antibiotic efflux, 7 with target alteration, 1 with target replacement (sul1), and 3 with antibiotic inactivation (FosA2, aadA2, and ACT-24). The E. coli isolate produced ESBL and showed resistance to ampicillin, cefuroxime axetil, and cefotaxime and intermediate resistance to cefepime (minimum inhibitory concentration (MIC) = 2 mg/L) and aztreonam (MIC 4 mg/L). This isolate V2 harbored 53 AMR gene loci in total, of which 40 were associated with antibiotic efflux, 10 with target alteration, 2 with antibiotic inactivation (AmpC and CTX-M-1), and 1 with target protection (mfpA).
Antimicrobial susceptibility testing results of four resistant water isolates
Tabelle 2. Ergebnisse der Antibiotika-Resistenztestung der vier resistenten Wasserisolate
|N0E2 (E. cloacae)||≥32||≥32||≥128||NA||NA||≥64||≥64||≤1||16||≤0.25||≤0.25||≤2||≤1||≤0.25||≤0.25||1||64||≤20|
|B1 (P. aeruginosa)||-||-||8||-||-||-||4||2||16||1||≤0.25||≤2||≤1||≤0.25||-||-||-||-|
|K3 (E. mori)||≥32||≥32||≤4||NA||NA||≤1||≤1||≤1||≤1||≥16||≥16||≤2||≤1||≤0.25||0.5||≤0.5||≤16||≤20|
|V2 (E. coli)||≥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: E. cloacae yielded three plasmids and the E. coli isolate one plasmid. These findings indicate that antibiotic resistance genes were acquired by multiple separate acquisition events mediated by plasmids (Villa et al., 2012; Voulgari et al., 2014). The occurrence of plasmids raises concerns about the possibility of the detected strains contributing to the dissemination of resistance genes among bacterial species in the water environment (Cloutier and McLellan, 2017; Rothenheber and Jones, 2018; Schang et al., 2016). Vancomycin-resistant enterococci were not detected in any of the water samples. In Austrian hospitals, these grampositive bacteria never reached the high clinical importance observed in the United States (BMG, 2017). In 2017, the ratio of vancomycin resistance among blood culture isolates from Austria was 0% for Enterococcus faecalis and only 3.2% for Enterococcus faecium (BMG, 2017).
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 P. aeruginosa isolate must not be considered multidrug resistant. Non-fermenting bacteria, such as P. aeruginosa, are not only found in the clinical setting but also occur naturally in the aquatic environment (Kittinger et al., 2017). Our findings are in accordance with those of Suzuki et al., who in 2013 postulated that in advanced nations where the majority of the population is urban and where medical services are widespread, antibiotic-resistant bacteria such as P. aeruginosa are likely to be widely distributed, even in apparently pristine rivers (Suzuki et al., 2013). In 2017, the ratio of antibiotic resistance among P. aeruginosa blood culture isolates from Austria was 5% for aminoglycosides, 8.7% for ceftazidime, 13.5% for piperacillin/tazobactam, 12.3% for fluoroquinolones, and 13.9% for carbapenems (BMG, 2017). Pseudomonads can carry multiple intrinsic and acquired resistance genes and mobile genetic elements, exchange them with other Enterobacteriaceae, and are known to be the origin of several carbapenemase families (Pfeifer et al., 2010; Farinas and Martinez, 2013). The risk of intrinsic resistances found in environmental microorganisms being transferred to pathogens is an international concern (Forsberg et al., 2012; Cox and Wright, 2013; Singer et al., 2016). Two of the four antibiotic-resistant bacteria isolated in our study belonged to the genus Enterobacter. Although not surveyed under the European Antimicrobial Resistance Surveillance Network (EARS-Net), Enterobacter spp. have great relevance as invasive clinical pathogens. In 2017, the WHO published it’s first-ever list of antibiotic-resistant “priority pathogens”—a catalog of 12 families of bacteria that pose the greatest threat to human health. The most critical group of all includes Acinetobacter, Pseudomonas, and Enterobacteriaceae. Enterobacter spp. have become resistant to a large number of antibiotics, including carbapenems and third-generation cephalosporins—the main antibiotics for treating infections caused by MDR bacteria. The detection of a carbapenem-resistant E. mori isolate at a Carinthian bathing site clearly underlines this resistance threat. Recently, the first clinical carbapenemase-carrying E. mori isolate was described in Austria (Hartl et al., 2019). This isolate was obtained from a 59-year-old patient suffering from acute otitis externa after visiting a thermal bath, which indicates water as the source of infection with antibioticresistant bacteria (Hartl et al., 2019). Also the finding of an ESBL-producing E. coli mirrors the situation in clinical microbiology. According to the “Der Österreichische Resistenzbericht” (AURES) report 2017, 49.5% of invasive E. coli isolates were resistant to aminopenicillins, 20.5% resistant to fluoroquinolones, 9.6% to third-generation cephalosporins, and 7.7% to aminoglycosides (BMG, 2017). Rivers and lakes are considered relevant reservoirs for MDR bacteria, because they pool materials from different origins, such as wastewater plants, water of urban or industrial effluents, agricultural activities, or rain (Lupo et al., 2012; Zurfluh et al., 2013; Kittinger et al., 2017). Our findings reveal that even ecologically pristine waters used for recreational activities can harbor resistant isolates. Exner et al. (2018) who evaluated potential health risks of water bodies contaminated with antibiotic-resistant pathogens reported that bathers without increased vulnerability and bathers with increased vulnerability should be differentiated. Ingestion of bathing water can theoretically lead to colonization of the gastrointestinal tract, but this is considered unlikely, given the low levels of swallowed bathing water or concentrations of antibiotic-resistant pathogens in open water. With regard to bathing in water authorized under the EU Bathing Water Directive, possible exposure to antibiotic-resistant bacteria according to the current knowledge poses no increased health risk for bathers without increased vulnerability; this assumes that the criteria of the EU Bathing Water Directive are met. They include intact skin of the bather and no antibiotics taken, observing the general rules of hygiene. For bathers with increased vulnerability or predisposition, risk of infection cannot be ruled out under the following conditions: open, extensive wounds; extensive skin disease; or prolonged intake of antibiotics. For these reasons, these persons should generally not bathe in open and untreated water irrespective of the presence of antibiotic-resistant pathogens (Exner et al., 2018).
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.
Bankevich A. Nurk S. Antipov D. Gurevich A.A. Dvorkin M. Kulikov A.S. Lesin V.M. Nikolenko S.I. Pham S. Prjibelski A.D. Pyshkin A.V. Sirotkin A.V. Vyahhi N. Tesler G. Alekseyev M.A. and P.A. Pevzner (2012): SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology 19 455–477.
- Export Citation
Bankevich, A., Nurk, S., Antipov, D., Gurevich, A.A., Dvorkin, M., Kulikov, A.S., Lesin, V.M., Nikolenko, S.I., Pham, S., Prjibelski, A.D., Pyshkin, A.V., Sirotkin, A.V., Vyahhi, N., Tesler, G., Alekseyev, M.A. and P.A. Pevzner (2012): SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology 19, 455–477.)| false 10.1089/cmb.2012.0021
Baquero F. Martınez J.L. and R. Canton (2008): Antibiotics and antibiotic resistance in water environments. Current Opinion in Biotechnology 19 260–265.
Boopathy R. (2017): Presence of methicillin resistant Staphylococcus aureus (MRSA) in sewage treatment plant. Bioresource Technology 240 144–148.
Bundesministerium für Gesundheit (BMG) (2017): Resistenzbericht Österreich AURES 2017. Antibiotikaresistenz und Verbrauch antimikrobieller Substanzen in Österreich. 2018. Eigenverlag Bundesministerium für Gesundheit Wien.
Carattoli A. Zankari E. García-Fernández A. Voldby Larsen M. Lund O. Villa L. Aarestrup F.M. and H. Hasman (2014): In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrobial Agents and Chemotherapy 58 3895–3903.
- Export Citation
Carattoli, A., Zankari, E., García-Fernández, A., Voldby Larsen, M., Lund, O., Villa, L., Aarestrup, F.M. and H. Hasman (2014): In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrobial Agents and Chemotherapy 58, 3895–3903.)| false 10.1128/AAC.02412-14 24777092
Cloutier D.D. and S.L. McLellan (2017): Distribution and differential survival of traditional and alternative indicators of fecal pollution at freshwater beaches. Applied and Environmental Microbiology 83 e02881-16.
Cox G. and G.D. Wright (2013): Intrinsic antibiotic resistance: mechanisms origins challenges and solutions. International Journal of Medical Microbiology 303 287–292.
Exner M. Schmithausen R. Schreiber C. Bierbaum G. Parcina M. Engelhart S. Kistemann T. Sib E. Walger P. and T. Schwartz (2018): Zum Vorkommen und zur vorläufigen hygienisch-medizinischen Bewertung von Antibiotika-resistenten Bakterien mit humanmedizinischer Bedeutung in Gewässern Abwässern Badegewässern sowie zu möglichen Konsequenzen für die Trinkwasserversorgung. Hygiene und Medizin 43 D46–D54.
Fariñas M.C. and L. Martínez-Martínez (2013): Multiresistant Gram-negative bacterial infections: Enterobacteria Pseudomonas aeruginosa Acinetobacter baumannii and other non-fermenting Gram-negative bacilli. Enfermedades Infecciosas y Microbiologia Clinica 31 402–409.
- Export Citation
Fariñas, M.C. and L. Martínez-Martínez (2013): Multiresistant Gram-negative bacterial infections: Enterobacteria,)| false Pseudomonas aeruginosa Acinetobacter baumanniiand other non-fermenting Gram-negative bacilli. Enfermedades Infecciosas y Microbiologia Clinica, 31, 402–409. 10.1016/j.eimc.2013.03.016 23684390
Forsberg K.J. Reyes A. Wang B. Selleck E.M. Sommer M.O. and G. Dantas (2012): The shared antibiotic resistome of soil bacteria and human pathogens. Science 337 1107–1111.
FAO (2016): The FAO action plan on antimicrobial resistance 2016-2020. 2016. Food and Agriculture Organization of the United Nations Rome.
Goossens H. Ferech M. Vander Stichele R. Elseviers M. and the ESAC Project Group (2005): Outpatient antibiotic use in Europe and association with resistance: a cross-national database study. Lancet 365 579–587.
Hartl R. Kerschner H. Gattringer R. Lepuschitz S. Allerberger F. Sorschag S. Ruppitsch W. and P. Apfalter (2019): Whole-genome analysis of a human Enterobacter mori isolate carrying a blaIMI-2 carbapenemase in Austria. Microbial Drug Resistance 25 94–96.
- Export Citation
Hartl, R., Kerschner, H., Gattringer, R., Lepuschitz, S., Allerberger, F., Sorschag, S., Ruppitsch, W. and P. Apfalter (2019): Whole-genome analysis of a human)| false Enterobacter moriisolate carrying a blaIMI-2 carbapenemase in Austria. Microbial Drug Resistance 25, 94–96. 10.1089/mdr.2018.0098
Jia B. Raphenya A.R. Alcock B. Waglechner N. Guo P. Tsang K.K. Lago B.A. Dave B.M. Pereira S. Sharma A.N. Doshi S. Courtot M. Lo R. Williams L.E. Frye J.G. Elsayegh T. Sardar D. Westman E. L. Pawlowski A.C. Johnson T.A. Brinkman F.S. Wright G.D. and A.G. McArthur (2017): CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Research 45 D566–D573.
- Export Citation
Jia, B., Raphenya, A.R., Alcock, B., Waglechner, N., Guo, P., Tsang, K.K., Lago, B.A., Dave, B.M., Pereira, S., Sharma, A.N., Doshi, S., Courtot M., Lo, R., Williams, L.E., Frye, J.G., Elsayegh, T., Sardar, D., Westman, E. L., Pawlowski, A.C., Johnson, T.A., Brinkman, F.S., Wright, G.D. and A.G. McArthur (2017): CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Research 45, D566–D573.)| false 10.1093/nar/gkw1004 27789705
Joensen K.G. Scheutz F. Lund O. Hasman H. Kaas R.S. Nielsen E.M. and F.M. Aarestrup (2014): Real-time whole-genome sequencing for routine typing surveillance and outbreak detection of verotoxigenic Escherichia coli Journal of clinical microbiology 52 1501–1510. Joensen K.G. Tetzschner A.M. Iguchi A. Aarestrup F.M. and F. Scheutz (2015): Rapid and easy in silico serotyping of Escherichia coli using whole genome sequencing (WGS) data. Journal of Clinical Microbiology 53 2410–2426.
- Export Citation
Joensen, K.G., Scheutz, F., Lund, O., Hasman, H., Kaas, R.S., Nielsen, E.M. and F.M. Aarestrup (2014): Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic)| false Escherichia coliJournal of clinical microbiology 52, 1501–1510. Joensen, K.G., Tetzschner A.M., Iguchi A., Aarestrup F.M. and F. Scheutz (2015): Rapid and easy in silicoserotyping of Escherichia coliusing whole genome sequencing (WGS) data. Journal of Clinical Microbiology 53, 2410–2426. 10.1128/JCM.03617-13 24574290
Jolley K.A. Chan M.S. and M.C. Maiden (2004): mlstdbNet–distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics 5 86.
Jolley K.A. Bliss C.M. Bennett J.S. Bratcher H.B. Brehony C. Colles F.M. Wimalarathna H. Harrison O.B. Sheppard S.K. Cody A.J. and M.C. Maiden (2012): Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain. Microbiology 158 1005–1015.
- Export Citation
Jolley, K.A., Bliss, C.M., Bennett, J.S., Bratcher, H.B., Brehony, C., Colles, F.M., Wimalarathna, H., Harrison, O.B., Sheppard, S.K., Cody, A.J. and M.C. Maiden (2012): Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain. Microbiology 158, 1005–1015.)| false 10.1099/mic.0.055459-0 22282518
Khan F.A. Hellmark B. Ehricht R. Söderquist B. and J. Jass (2018): Related carbapenemase-producing Klebsiella isolates detected in both a hospital and associated aquatic environment in Sweden. European Journal of Clinical Microbiology & Infectious Diseases 37 2241– 2251.
Kittinger C. Lipp M. Baumert R. Folli B. Koraimann G. Toplitsch D. Liebmann A. Grisold A.J. Farnleitner A.H. Kirschner A. and G. Zarfel (2016): Antibiotic resistance patterns of Pseudomonas spp. isolated from the river Danube. Frontiers in Microbiology 7 586.
- Export Citation
Kittinger, C., Lipp, M., Baumert, R., Folli, B., Koraimann, G., Toplitsch, D., Liebmann A., Grisold A.J., Farnleitner A.H., Kirschner A. and G. Zarfel (2016): Antibiotic resistance patterns of)| false Pseudomonasspp. isolated from the river Danube. Frontiers in Microbiology 7, 586. 27199920
Lepuschitz S. Mach R. Springer B. Allerberger F. and W. Ruppitsch (2017): Draft Genome Sequence of a Community-Acquired Methicillin-Resistant Staphylococcus aureus USA300 Isolate from a River Sample. Genome Announcements 5 e01166-17.
Lepuschitz S. Huhulescu S. Hyden P. Springer B. Rattei T. Allerberger F. Mach R.L. and W. Ruppitsch (2018): Characterization of a community-acquiredMRSA USA300 isolate from a river sample in Austria and whole genome sequence based comparison to a diverse collection of USA300 isolates. Scientific Reports 8 9467.
- Export Citation
Lepuschitz, S., Huhulescu, S., Hyden, P., Springer, B., Rattei, T., Allerberger, F., Mach R.L. and W. Ruppitsch (2018): Characterization of a community-acquiredMRSA USA300 isolate from a river sample in Austria and whole genome sequence based comparison to a diverse collection of USA300 isolates. Scientific Reports 8, 9467.)| false 10.1038/s41598-018-27781-8 29930324
Lepuschitz S. Schill S. Stoeger A. Pekard-Amenitsch S. Huhulescu S. Inreiter N. Hartl R. Kerschner H. Sorschag S. Springer B. Brisse S. Allerberger F. Mach R.L. and W. Ruppitsch (2019): Whole genome sequencing reveals resemblance between ESBL-producing and carbapenem resistant Klebsiella pneumoniae isolates from Austrian rivers and clinical isolates from hospitals. Science of the Total Environment 662 227–235.
- Export Citation
Lepuschitz, S., Schill, S., Stoeger, A., Pekard-Amenitsch, S., Huhulescu, S., Inreiter, N., Hartl, R., Kerschner, H., Sorschag, S., Springer, B., Brisse, S., Allerberger, F., Mach, R.L. and W. Ruppitsch (2019): Whole genome sequencing reveals resemblance between ESBL-producing and carbapenem resistant)| false Klebsiella pneumoniaeisolates from Austrian rivers and clinical isolates from hospitals. Science of the Total Environment 662, 227–235. 10.1016/j.scitotenv.2019.01.179
Lupo A. Coyne S. and T.U. Berendonk (2012): Origin and evolution of antibiotic resistance: the common mechanisms of emergence and spread in water bodies. Frontiers in Microbiology 18 1–13.
Magiorakos A.P. Srinivasan A. Carey R.B. Carmeli Y. Falagas M.E Giske C.G. Harbarth S. Hindler J.F. Kahlmeter G. Olsson-Liljequist B. Paterson D.L. Rice L.B. Stelling J. Struelens M.J. Vatopoulos A. Weber J.T. and D.L. Monnet (2012): Multidrug-resistant extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection 18 268–281.
- Export Citation
Magiorakos, A.P., Srinivasan, A., Carey, R.B., Carmeli, Y., Falagas, M.E, Giske, C.G., Harbarth, S., Hindler, J.F., Kahlmeter, G., Olsson-Liljequist, B., Paterson, D.L., Rice, L.B., Stelling, J., Struelens, M.J., Vatopoulos, A., Weber, J.T. and D.L. Monnet (2012): Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection 18, 268–281.)| false 10.1111/j.1469-0691.2011.03570.x
Mahon B. M. Brehony C. McGrath E. Killeen J. Cormican M. Hickey P. Keane S. Hanahoe B. Dolan A. and D. Morris (2017): Indistinguishable NDMproducing Escherichia coli isolated from recreational waters sewage and a clinical specimen in Ireland 2016 to 2017. Euro Surveillance 22 pii=30513.
Manaia C.M. Macedo G. Fatta-Kassinos D. and O.C. Nunes (2016): Antibiotic resistance in urban aquatic environments: can it be controlled? Applied microbiology and biotechnology 100 1543–1557.
ÖNORM M 6230 (2015): Bathing waters – Requirements for water quality analysis and assessment. Austrian Standards Vienna.
OIE (2016): The OIE Strategy on Antimicrobial Resistance and the Prudent Use of Antimicrobials World. 2016. World Organization for Animal Health Paris http://www.oie.int/fileadmin/Home/eng/Media_Center/docs/pdf/PortailAMR/EN_OIE-AMRstrategy.pdf.Accessed on 6 May 2019.
Pfeifer Y. Cullik A. and W. Witte (2010): Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens. International Journal of Medical Microbiology 300 371–379.
Rizzo L. Manaia C. Merlin C. Schwartz T. Dagot C. Ploy M.C. Michael I. and D. Fatta-Kassinos (2013): Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. Science of the Total Environment 447 345–360.
- Export Citation
Rizzo, L., Manaia, C., Merlin, C., Schwartz, T., Dagot, C., Ploy, M.C., Michael, I. and D. Fatta-Kassinos (2013): Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. Science of the Total Environment 447, 345–360.)| false 10.1016/j.scitotenv.2013.01.032
Rothenheber D. and S. Jones (2018): Enterococcal concentrations in a coastal ecosystem are a function of fecal source input environmental conditions and environmental sources. Applied and Environmental Microbiology 84 e01038-18.
Schang C. Henry R. Kolotelo P.A. Prosser T. Crosbie N. Grant T. Cottam D. O’Brien P. Coutts S. Deletic A. and D.T. McCarthy (2016): Evaluation of techniques for measuring microbial hazards in bathing waters: A comparative study. PLOS ONE 11 e0155848.
- Export Citation
Schang, C., Henry, R., Kolotelo, P.A., Prosser, T., Crosbie, N., Grant, T., Cottam D., O’Brien, P., Coutts, S., Deletic, A. and D.T. McCarthy (2016): Evaluation of techniques for measuring microbial hazards in bathing waters: A comparative study. PLOS ONE 11, e0155848.)| false 10.1371/journal.pone.0155848
Singer A.C. Shaw H. Rhodes V. and A. Hart (2016): Review of antimicrobial resistance in the environment and its relevance to environmental regulators. Frontiers in Microbiology 7 1728.
Suzuki Y. Kajii S. Nishiyama M. and A. Iguchi (2013): Susceptibility of Pseudomonas aeruginosa isolates collected from river water in Japan to antipseudomonal agents. Science of the Total Environment 450 148–154.
Tolba O. Loughrey A. Goldsmith C.E. Millar B.C. Rooney P.J. and J.E. Moore (2008): Survival of epidemic strains of healthcare (HA-MRSA) and communityassociated (CA-MRSA) methicillin-resistant Staphylococcus aureus (MRSA) in river- sea-and swimming pool water. International Journal of Hygiene and Environmental Health 211 398–402.
- Export Citation
Tolba, O., Loughrey, A., Goldsmith, C.E., Millar, B.C., Rooney, P.J. and J.E. Moore (2008): Survival of epidemic strains of healthcare (HA-MRSA) and communityassociated (CA-MRSA) methicillin-resistant)| false Staphylococcus aureus(MRSA) in river-, sea-and swimming pool water. International Journal of Hygiene and Environmental Health 211, 398–402. 10.1016/j.ijheh.2007.06.003
Vaz-Moreira I. Nunes O.C. and C.M. Manaia C. Bacterial diversity and antibiotic resistance in water habitats: searching the links with the human microbiome. FEMS Microbiology Reviews 38 761–778.
Villa L. Poirel L. Nordmann P. Carta C. and A. Carattoli (2012): Complete sequencing of an IncH plasmid carrying the bla(NDM-1) bla(CTX-M-15) and qnrB1 genes. Journal of Antimicrobial Chemotherapy 67 1645–1650.
Voulgari E. Gartzonika C. Vrioni G. Politi L. Priavali E. Levidiotou-Stefanou S. and A. Tsakris (2014): The Balkan region: NDM-1-producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece. Journal of Antimicrobial Chemotherapy 69 2091–2097.
- Export Citation
Voulgari, E., Gartzonika, C., Vrioni, G., Politi, L., Priavali, E., Levidiotou-Stefanou, S. and A. Tsakris (2014): The Balkan region: NDM-1-producing)| false Klebsiella pneumoniaeST11 clonal strain causing outbreaks in Greece. Journal of Antimicrobial Chemotherapy 69, 2091–2097. 10.1093/jac/dku105
WHO (2003): Guidelines for safe recreational water environments. Volume 1 Coastal and fresh waters. World Health Organization Geneva.
WHO (2015): Global action plan on antimicrobial resistance. 2015. Geneva World Health Organization http://www.wpro.who.int/entity/drug_resistance/resources/global_action_plan_eng.pdf Accessed on 6 May 2019.
WHO (2017): Fact sheet 2018- Antibiotic resistance. 2017. Geneva World Health Organization https://www.who.int/en/news-room/fact-sheets/detail/antibiotic-resistance Accessed on 6 May 2019.
Zarfel G. Lipp M. Gürtl E. Folli B. Baumert R. and C. Kittinger (2017): Troubled water under the bridge: Screening of River Mur water reveals dominance of CTX-M harboring Escherichia coli and for the first time an environmental VIM-1 producer in Austria. Science of the Total Environment 593 399–405.
Zhang X.X. Zhang T. and H.H. Fang (2009): Antibiotic resistance genes in water environment. Applied Microbiology and Biotechnology 82 397–414.
Zurfluh K. Hächler H. Nüesch-Inderbinen M. and R. Stephan (2013): Characteristics of extended-spectrum β-lactamase- and carbapenemase-producing Enterobacteriaceae isolates from rivers and lakes in Switzerland. Applied and Environmental Microbiology 79 3021–3026.
- Export Citation
Zurfluh, K., Hächler, H., Nüesch-Inderbinen, M. and R. Stephan (2013): Characteristics of extended-spectrum β-lactamase- and carbapenemase-producing)| false Enterobacteriaceaeisolates from rivers and lakes in Switzerland. Applied and Environmental Microbiology 79, 3021–3026. 23455339 10.1128/AEM.00054-13