The\u00a0detection of\u00a0high numbers of\u00a0resistant or multi-resistant E.\u00a0coli<\/em> strains in\u00a0and downstream of\u00a0treated effluents indicates the\u00a0importance of\u00a0the\u00a0aquatic environment in\u00a0the\u00a0spread of\u00a0AMR, which may be a\u00a0consequence of\u00a0the\u00a0suggested transfer of\u00a0resistance between bacterial strains in\u00a0WWTP. The\u00a0screening findings point to the\u00a0need for detailed study of\u00a0AMR in\u00a0the\u00a0environment, which is essential for success in\u00a0efforts to reduce the\u00a0current health threats posed by ATB resistance in\u00a0the\u00a0Czech Republic and worldwide.<\/p>\n Antimicrobial resistance (AMR) is the\u00a0ability of\u00a0bacteria to resist the\u00a0effect of\u00a0antibiotics (ATBs), i.e. substances that can kill them or stop their growth. The\u00a0natural property of\u00a0every organism, including bacteria, is to survive and reproduce. If contact with ATB prevents them from doing so, they will try to find a\u00a0way to avoid their negative effect. This is how mutations and genetic transfers occur, which cause the\u00a0initially sensitive bacteria to become partially or completely resistant to the\u00a0action of\u00a0ATB. The\u00a0main\u00a0cause is excessive contact of\u00a0bacteria with ATB caused by their incorrect or inappropriate use and the\u00a0occurrence of\u00a0ATB in\u00a0the\u00a0environment. The\u00a0consequence is the\u00a0fact that there are dangerous resistant bacteria in\u00a0the\u00a0world today, for which neither standard nor reserve ATBs work. Currently, we can only defend against them with higher doses or other types of\u00a0ATB which, however, are in\u00a0limited quantities; this can mean a\u00a0greater burden on the\u00a0body and more side effects for patients. At the\u00a0same time, there is a\u00a0possibility that the\u00a0bacterium will find a\u00a0way to defeat ATB; over 35,000 people are reported to die in\u00a0the\u00a0EU each year related to AMR\u00a0[1, 2].<\/p>\n Contributing to the\u00a0spread of\u00a0AMR is the\u00a0excretion of\u00a0ATB into wastewater (up to 80\u00a0%) and the\u00a0overuse of\u00a0ATB in\u00a0the\u00a0agricultural sector, where, until 2006, preventive administration of\u00a0ATB to farm animals to promote growth was practiced (and in\u00a0some non-EU countries it is still being practiced). The\u00a0emergence of\u00a0resistant bacteria in\u00a0the\u00a0ATB-contaminated environment is a\u00a0source of\u00a0AMR that has not yet been fully explored. In\u00a0the\u00a0clinical sector, the\u00a0use of\u00a0broad-spectrum ATBs that act against a\u00a0wide spectrum of\u00a0bacteria, underdosing of\u00a0recommended therapeutic doses that cause bacteria to adapt to low ATB levels, and inconsistent diagnosis of\u00a0the\u00a0causative agent (e.g. viral infections treated by ATB) contribute to AMR.<\/p>\n ATB effect of\u00a0is also complicated by the\u00a0ability of\u00a0some bacteria, including E.\u00a0coli<\/em>, to produce extended spectrum beta-lactamases (ESBL), which hydrolyse frequently used ATB (including penicillins and cephalosporins).<\/p>\n Clinical and veterinary medicine at the\u00a0European and global level has been studying AMR and the\u00a0effects of\u00a0its spread intensively. In\u00a02019, the\u00a0WHO ranked AMR among the\u00a0ten most significant health threats; in\u00a02022, the\u00a0European Commission, together with EU member states, designated AMR as one of\u00a0the\u00a0three priority health threats\u00a0[3]. Adopted in\u00a0June 2023, the\u00a0European Council\u2019s\u00a0recommendation on strengthening EU measures to combat antimicrobial resistance within\u00a0the\u00a0framework of\u00a0the\u00a0\u201cOne Health\u201d approach\u00a0[4] now contains specific goals that each member state should achieve by 2030. For the\u00a0Czech Republic, the\u00a0goals are listed in\u00a0the\u00a0Strategy of\u00a0the\u00a0National Antibiotic Programme of\u00a0the\u00a0Czech Republic for 2024\u20132030:<\/p>\n AMR in\u00a0the\u00a0environment was not a\u00a0priority concern until recently. Findings demonstrating its importance were accepted in\u00a02017 in\u00a0the\u00a0UN Frontiers 2017 study\u00a0[5\u20137]. Professor W. Gaze pointed out that ATB release is an overlooked problem, but one that could be key to the\u00a0development of\u00a0resistant strains, and sparked a\u00a0commitment to tackle AMR across sectors, resulting in\u00a0the\u00a0\u201cOne Health\u201d initiative. The\u00a0risk lies in\u00a0the\u00a0fact that the\u00a0majority of\u00a0ATB in\u00a0non-metabolized form together with resistant bacteria (ARB) gets into water and soil, where it meets environmental bacteria and creates conditions for the\u00a0mutual exchange of\u00a0genetic information. Environmental conditions and other contaminants (heavy metals, disinfectants, etc.) also contribute to the\u00a0transmission, which can increase selection pressure and thus the\u00a0potential for the\u00a0emergence of\u00a0a\u00a0large number of\u00a0new resistances. Pathogenic bacteria with clinically relevant genes originating from the\u00a0environment have been found\u00a0[7]. So far, both resistant and multi-resistant bacteria (i.e. those that carry resistance to more than three ATB groups) have been found in\u00a0all types of\u00a0water, including groundwater. Contamination with resistant bacteria or resistance genes is risky for sources of\u00a0drinking water and surface water used for bathing, where it can be transmitted to the\u00a0human body via the\u00a0faecal-oral route. Food chain\u00a0AMR contamination can occur with irrigation water, aquaculture, and the\u00a0application of\u00a0sewage sludge and farmyard manure to agricultural land\u00a0[8]. The\u00a0mechanisms of\u00a0possible aquatic environment contamination by AMR are shown in\u00a0Fig.\u00a01<\/em>.<\/p>\n <\/p>\n The\u00a0aquatic environment is contaminated with resistant bacteria primarily through wastewater treatment plants (WWTPs), which are considered hot spots for the\u00a0spread of\u00a0AMR in\u00a0the\u00a0aquatic environment. Together with ATB, ARBs enter WWTP from human digestive and excretory systems and are present here in\u00a0varying degrees of\u00a0metabolism, depending on their stability in\u00a0the\u00a0aquatic environment. Despite the\u00a0high efficiency of\u00a0existing treatment technologies, which reach values of\u00a0around 99\u00a0% when removing microbial pollution, a\u00a0large amount of\u00a0ARB and ARG is released into the\u00a0recipient. A\u00a0large amount of\u00a0ATB which cannot be broken down by current technologies is also present in\u00a0treated municipal wastewater and wastewater from the\u00a0production of\u00a0pharmaceuticals discharged into rivers, together with little-known products of\u00a0their decomposition. To supplement information on the\u00a0AMR occurrence in\u00a0the\u00a0population connected to individual WWTPs\u00a0[4], data is also used which is obtained during monitoring of\u00a0raw wastewater based on WES principle (Wastewater and Environmental Surveillance).<\/span><\/p>\n Knowledge of\u00a0the\u00a0current state of\u00a0AMR occurrence in\u00a0the\u00a0Czech Republic is at a\u00a0very low level; following the\u00a0activities of\u00a0other EU countries, it is necessary to contribute to its expansion in\u00a0order to obtain\u00a0data for effective protection of\u00a0human health and the\u00a0environment.<\/span><\/p>\n In\u00a0the\u00a0Czech Republic, there is currently no systematic monitoring of\u00a0watercourses with regard to AMR. Information on the\u00a0status can only be derived from the\u00a0research activities of\u00a0several scientific teams that deal with this issue from different perspectives (e.g. University of\u00a0Chemistry and Technology, Prague; Pardubice University; Veterinary University, Brno; National Institute of\u00a0Public Health, Prague). Interest in\u00a0the\u00a0AMR issue is supported by the\u00a0revised Urban Wastewater Treatment Directive 271\/91\/EEC, which will enter into force at the\u00a0end of\u00a02024. Within\u00a0this Directive, a\u00a0number of\u00a0changes are expected to help improve the\u00a0quality of\u00a0surface water and reduce the\u00a0health risks associated with its use. The\u00a0monitoring of\u00a0substances that can affect human health should be gradually introduced, including, in\u00a0addition to AMR, the\u00a0direct monitoring of\u00a0viruses, PFAS (perfluorinated and polyfluorinated alkyl substances) and microplastics. In\u00a0the\u00a0future, the\u00a0issue should also be included in\u00a0the\u00a0Water Framework Directive 2000\/60\/EC<\/span><\/em>.<\/span><\/p>\n Our goal was to obtain\u00a0initial information about AMR occurrence in\u00a0surface and wastewater in\u00a0the\u00a0Czech Republic. The\u00a0screening was aimed at detecting the\u00a0occurrence of\u00a0antibiotic resistance to selected ATBs in\u00a0the\u00a0indicator bacterium E.\u00a0coli<\/span><\/em>, isolated from surface and wastewater at WWTP influent and effluent using the\u00a0disk diffusion method.<\/span><\/p>\n To compare the WWTP influence, the locations of surface water from main watercourses above and below the effluent of municipal wastewater from large urban areas with uniform sewage, and surface water samples from smaller watercourses flowing into the Vltava were selected. At the same time, wastewater samples were analysed at the influent and effluent of these WWTPs. The samples were taken continuously in 2022\u20132024 and were classified into the categories ABOVE (13 samples from watercourses above large municipal WWTPs), BELOW (53 samples from watercourses below the effluent of treated wastewater from large municipal WWTPs at a distance of 500 m to 10 km), INFLUENT (19 samples from influents to the WWTP after rough mechanical pretreatment), EFFLUENT (26 samples of treated wastewater on effluent from WWTPs with different treatment technologies), and STREAM (20 samples from the Vltava tributaries of different water bearing, into which smaller WWTPs and other effluents are discharged). A total of 131 samples were included in the study. Samples were taken in the standard sampling method for microbiological analysis.<\/span><\/p>\n In the samples, E. coli<\/em> bacteria were determined by cultivation on mFC agar [9]. From each sample, in the optimal case, four different E. coli<\/em> strains were selected and isolated, for which AMR was determined by the disk diffusion method. A pure bacterial culture grown overnight on a solid non-selective\u00a0medium (Trypton Yeast Extract Agar) was suspended in\u00a0physiological solution to a\u00a0turbidity level of\u00a00.5 \u00b1 0.1 according to the\u00a0McFarland turbidity scale, i.e.\u00a01\u20132\u00a0\u00d7\u00a0108 cells\/ml. The\u00a0suspension was spread evenly on plates with Mueller-Hinton agar, on which disks containing ATB of\u00a0different concentrations were subsequently placed using an applicator (Tab. 1<\/em>). After 18 \u00b1 2 hours of\u00a0incubation at 36 \u00b1 2 \u00b0C, the\u00a0inhibition zones of\u00a0individual ATBs were read (breakpoint means of\u00a0inhibition zones were adopted from the\u00a0EUCAST tables\u00a0[10]), see Fig. 2<\/em>. ATBs and their concentrations were selected based on information on the\u00a0occurrence of\u00a0resistance in\u00a0clinical areas, the\u00a0use of\u00a0ATB in\u00a0the\u00a0Czech Republic, and the\u00a0properties of\u00a0ATB in\u00a0the\u00a0aquatic environment so as to cover as many ATB groups as possible (source: NRL for ATB SZ\u00da, EUCAST\u00a0[10]).<\/p>\n <\/p>\n The\u00a0determination of\u00a0E.\u00a0coli<\/em> resistance to selected ATBs was complemented by the\u00a0detection of\u00a0the\u00a0production of\u00a0extended-spectrum beta-lactamases (ESBL).<\/p>\n A\u00a0selected sample volume (usually 1\u2013100 ml) was filtered through a\u00a0sterile nitrocellulose membrane filter with a\u00a0porosity of\u00a00.45 \u00b5m, which was then placed on a\u00a0TBX agar (Tryptone Bile\u00a0\u00d7\u00a0Glucuronide agar) plate supplemented with cefotaxime (4 \u00b5g\/ml). TBX agar without ATB was used to determine the\u00a0total number of\u00a0E.\u00a0coli<\/span><\/em> in\u00a0the\u00a0water sample. Cultivation took place in\u00a0an incubator at a\u00a0temperature of\u00a036 \u00b1 1 \u00b0C for 21 \u00b1 3 hours. From each sample, four presumptive colonies of\u00a0ESBL-positive E.\u00a0coli<\/span><\/em> strains were subjected to two tests\u00a0\u2013 CDT (Combination Disk Diffusion Test) and DDST (Double Disk Synergy Test) according to the\u00a0procedure for performing and interpreting the\u00a0results\u00a0[11], see Fig.\u00a03<\/span><\/em>. ESBL detection uses inhibition of ATB hydrolysis by clavulanic acid.\u00a0<\/span>For\u00a0CDT, cephalosporin\u00a0disks containing cefotaxime and ceftazidime and combined cefotaxime\/clavulanic acid and ceftazidime\/clavulanic acid disks are used. Four disks (two cephalosporins and two combination disks) are used per isolate. The\u00a0interpretation of\u00a0the\u00a0CDT test results (Fig.\u00a03<\/span><\/em>) is based on the\u00a0reading of\u00a0the\u00a0size of\u00a0the\u00a0inhibition zones of\u00a0each cephalosporin\u00a0separately compared to the\u00a0combination of\u00a0the\u00a0cephalosporin\u00a0and clavulanic acid. For DDST, cephalosporin\u00a0disks and a\u00a0clavulanic disk are used. The\u00a0principle is to use cephalosporin\u00a0disks next to a\u00a0clavulanic disk with a\u00a0distance of\u00a020 mm from the\u00a0centre. After incubation, the\u00a0interaction between individual cephalosporins and clavulanic acid is monitored (Fig.\u00a03<\/span><\/em>).<\/span><\/p>\n The\u00a0samples were divided into five categories to evaluate the\u00a0indicative occurrence of\u00a0AMR in\u00a0surface and wastewater. Samples taken at influents (INFLUENT) and effluents (EFFLUENT) from large WWTPs, in\u00a0watercourses above (ABOVE) and below (BELOW) the\u00a0effluent of\u00a0treated wastewater from WWTPs and in\u00a0smaller watercourses (STREAM) where smaller WWTPs are located were compared. The\u00a0obtained results were evaluated within\u00a0individual categories and processed into a\u00a0graph. The\u00a0assessment was made for the\u00a0\u201crelative percentage of\u00a0strains with proven resistance\u201d to individual ATBs. This was obtained by adding the\u00a0actually tested proportion of\u00a0strains to the\u00a0total number of\u00a0E.\u00a0coli<\/span><\/em> in\u00a0the\u00a0sample.<\/span><\/p>\n The\u00a0accuracy of\u00a0the\u00a0results is affected by the\u00a0relatively low proportion of\u00a0tested strains (0.00004\u20130.19\u00a0%) due to high microbial load of\u00a0surface and wastewater samples. The\u00a0proportion of\u00a0ESBL-positive and multi-resistant strains was evaluated separately (i.e. strains with simultaneous resistance to at least three groups of\u00a0ATBs, with 3rd and 4th generation cephalosporins considered as one group).<\/span><\/p>\n During 2022\u20132024, 131 water samples from five categories were tested. The\u00a0numbers of\u00a0samples in\u00a0individual categories are shown in\u00a0Tabs. 2<\/span><\/em> and 3<\/span><\/em>. Tab. 2<\/span> <\/em>also shows the\u00a0relative percentage shares of\u00a0E.\u00a0coli<\/em> <\/span>strains with proven antibiotic resistance to the\u00a0tested ATB in\u00a0individual categories; Tab. 3<\/span><\/em> shows the\u00a0number of\u00a0samples with proven antibiotic resistance to the\u00a0tested ATB in\u00a0individual categories. The\u00a0results are shown in\u00a0Fig.\u00a04<\/span><\/em>.<\/span><\/p>\n <\/p>\n <\/p>\n Most samples were tested from the\u00a0BELOW category, which included wastewater recipients at different distances (500 m to 10 km) from their effluents. In\u00a0this category, 201 E.\u00a0coli<\/span><\/em> strains were tested out of\u00a0a\u00a0total of\u00a0more than 400,000\u00a0detected. Resistance to all tested ATBs was found; compared to\u00a0the\u00a0other categories, there was a\u00a0high proportion of\u00a0fosfomycin-resistant strains (22\u00a0%), in\u00a0a\u00a0similar proportion to the\u00a0INFLUENT and EFFLUENT category (17\u201324\u00a0%). The\u00a0most common was resistance to gentamicin\u00a0(55\u00a0%), the\u00a0least common, as in\u00a0the\u00a0other categories, was resistance to meropenem (1\u00a0%).<\/span><\/p>\n Lower proportions of\u00a0antibiotic-resistant strains were found in\u00a0the\u00a0ABOVE category, where the\u00a0profiles of\u00a0larger watercourses above the\u00a0WWTP effluent were included. This category served as a\u00a0control comparison of\u00a0the\u00a0status above versus the\u00a0status below the\u00a0effluent of\u00a0large WWTPs, which are considered significant AMR sources. However, even in\u00a0this \u201ccontrol\u201d category, E.\u00a0coli<\/span><\/em> with resistance to six of\u00a0the\u00a0eight tested ATBs were found. However, no sample showed resistance to nitrofurantoin\u00a0and meropenem.<\/span><\/p>\n Other significant categories for comparison were INFUNENT and EFFLUENT from the\u00a0WWTP. Despite a\u00a0significant reduction in\u00a0the\u00a0number of\u00a0E.\u00a0coli<\/span><\/em> in\u00a0the\u00a0effluents due to good treatment efficiency, resistance to all tested ATBs was found in\u00a0both categories. The\u00a0share of\u00a0both resistant strains and positive samples was unexpectedly higher in\u00a0the\u00a0EFFLUENT category. The\u00a0exception was resistance to gentamicin, which was similarly high in\u00a0both categories (50\u201355\u00a0%). In\u00a0the\u00a0STREAM category, where samples of\u00a0different Vltava tributaries were included, into which smaller WWTPs are discharged, resistance to all tested ATBs was also demonstrated. The\u00a0proportions of\u00a0resistant E.\u00a0coli<\/span><\/em> and samples were lower, similarly to the\u00a0ABOVE category.<\/span><\/p>\n The\u00a0largest proportion of\u00a0resistant E.\u00a0coli<\/span><\/em> and samples was clearly identified for gentamicin\u00a0(46\u201355\u00a0% of\u00a0strains), while the\u00a0least represented was resistance to meropenem and nitrofurantoin\u00a0(0\u20138\u00a0% of\u00a0strains). Resistance to second generation cephalosporins was detected in\u00a09\u201326\u00a0% of\u00a0E.\u00a0coli<\/span><\/em> strains; it was also significant in third and fourth generation cephalosporins (3\u201323 % of strains), see Fig. 5<\/em>.<\/span><\/p>\n <\/p>\n Many E.\u00a0coli<\/span><\/em> strains showed multiple resistance (Fig.\u00a06<\/span><\/em>). The\u00a0occurrence of\u00a0resistance to three to five ATB groups was most common in\u00a0the\u00a0BELOW and EFFLUENT category. In\u00a0the\u00a0EFFLUENT category, resistance to six and seven ATB groups was also detected.<\/span><\/p>\n <\/p>\nINTRODUCTION<\/h2>\n
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\n<\/li>\n![\"\"](\"https:\/\/www.vtei.cz\/wp-content\/uploads\/2024\/12\/Zverinova-obr-1-1.jpg\")
<\/a>\nFig. 1. Mechanisms of potential spread of AMR in the water environment<\/h6>\n
METHODS<\/h2>\n
Sampling<\/span><\/h3>\n
Procedure for E.\u00a0coli<\/em> isolation and determination of\u00a0sensitivity to antibiotics by disk diffusion method<\/h3>\n
Tab.\u00a01. List of antibiotics used and their concentration in the discs<\/h5>\n
<\/a>\n<\/a><\/h6>\nFig. 2. Inhibition zones of the tested E. coli<\/em> strain, example of a sensitive (obvious inhibition zone around the antibiotics disc) and resistant strain (small or no inhibition zone around the antibiotics disc); the size of inhibition zones is given in EUCAST<\/h6>\n
E. coli<\/em> determination with production of extended-spectrum beta-lactamases<\/h3>\n
<\/a>\nFig. 3. Confirmation of ESBL on the E. coli<\/em> isolates by CDT and DDST tests (above:\u00a0CDT test, below: DDST test)<\/h6>\n
Evaluation of\u00a0results<\/h3>\n
RESULTS<\/h2>\n
Tab.\u00a02. Relative proportion of E. coli<\/em> strains with proven AMR in each category<\/h5>\n
<\/a>\nTab. 3. Numbers of samples in each category with demonstrated AMR in E. coli<\/em><\/h5>\n
<\/a>\n<\/h6>\n
<\/a>Fig. 4.\u00a0\u00a0Relative proportion of E. coli<\/em> strains with proven AMR in each category<\/h6>\n
\nThe\u00a0most significant increase in\u00a0the\u00a0proportion of\u00a0ARBs occurred with cefepime, nitrofurantoin, and meropenem.<\/span><\/p>\n<\/a><\/h6>\nFig. 5. Relative proportion of occurred resistance to individual antibiotics (incl.\u00a0Multiple\u00a0resistance; in %)<\/h6>\n
<\/a>\nFig. 6. Number of E. coli<\/em> strains with proven resistance to 3\u20137 groups of antibiotics<\/h6>\n