INTRODUCTION<\/h2>\n
The\u00a0detection of\u00a0intestinal enterococci may appear straightforward at first glance, but it nevertheless has its pitfalls. On the\u00a0one hand, intestinal enterococci are, in\u00a0the\u00a0new drinking water legislation, one of\u00a0the\u00a0two key microbiological indicators of\u00a0faecal contamination, with the\u00a0highest parametric value. On the\u00a0other hand, alternative methods based on the\u00a0detection of\u00a0enterococci through \u03b2-D-glucosidase activity are gaining ground, extending the\u00a0target group to all enterococci, i.e. Enterococcus<\/span> spp. However, relatively little is known about the\u00a0ecology of\u00a0enterococci, particularly in\u00a0drinking water, which complicates the\u00a0interpretation of\u00a0the\u00a0results obtained. This study was therefore conducted, and the\u00a0results were presented at the\u00a0Vod\u00e1rensk\u00e1 biologie 2025<\/em> conference<\/span>\u00a0[1]. A\u00a0revised and extended version is presented here.<\/span><\/p>\n Intestinal (faecal) enterococci are Gram-positive spherical or ovoid bacteria arranged in\u00a0pairs or chains and belong to the\u00a0genus Enterococcus<\/span><\/em> (order Lactobacillales<\/em>, phylum Firmicutes<\/em>). Advances in\u00a0molecular genetic methods in\u00a0taxonomy have led to a\u00a0continuous increase in\u00a0the\u00a0number of\u00a0described enterococcal species; for example, only 19 species were known in\u00a01995, whereas at present 60 species have been validly described\u00a0[2]. With the\u00a0now widely used MALDI-TOF method, individual species can\u00a0be identified, allowing a\u00a0more detailed interpretation of\u00a0the\u00a0results obtained. For example, among 101 isolates from various surface, technological, and drinking waters, the\u00a0following species were identified: E. faecalis<\/span><\/em> (26.7\u00a0%), E. hirae<\/span><\/em> (20.8\u00a0%), E. faecium<\/span><\/em> (18.8\u00a0%), E.\u00a0casseliflavus<\/span><\/em> (15.8\u00a0%), E. durans<\/span><\/em> (11.8\u00a0%), and E. mundtii<\/span><\/em> and E. moraviensis<\/span><\/em> (both 2.3\u00a0%)\u00a0[3].<\/span><\/p>\n Intestinal enterococci are regarded as indicators of\u00a0faecal contamination, and their importance has increased in\u00a0recent years \u2013 under the\u00a0new legislation\u00a0[4, 5], together with Escherichia coli<\/span><\/em>, they represent a\u00a0key indicator and must be determined in\u00a0all types of\u00a0analysis (both reduced and full). This is associated not only with an\u00a0increased number of\u00a0samples analysed, but also with a\u00a0higher number of\u00a0positive detections that need to be properly interpreted.<\/span><\/p>\n The\u00a0frequency of\u00a0detection in\u00a0drinking water over the\u00a0past five years in\u00a0the\u00a0Czech Republic, based on data from the\u00a0National Institute of\u00a0Public Health (Drinking Water Quality Report)\u00a0[6], is presented in\u00a0Tab.\u00a01<\/span><\/em>. At present, enterococci are the\u00a0core parameter and its detection is included in\u00a0all drinking water samples; consequently, the\u00a0number of\u00a0analyses, and thus the\u00a0number of\u00a0positive detections, is increasing (in\u00a0Tab.\u00a01<\/span><\/em>, a\u00a0marked difference can\u00a0be seen particularly between 2023 and 2024). The\u00a0higher numbers of\u00a0detected enterococci are mainly associated with the\u00a0increased number of\u00a0tests performed. However, as the\u00a0figures are still relatively low, it will be important to continue monitoring this situation.<\/span><\/p>\n There is a\u00a0substantial body of\u00a0scientific literature on intestinal enterococci; however, it predominantly consists of\u00a0studies describing the\u00a0sources from which particular species have been isolated or the\u00a0description and characterisation of\u00a0new species. Where environmental studies do exist, they are primarily focused on surface waters and bathing waters, as well as on sludge and sediments. Studies dealing with a\u00a0more in-depth investigation of\u00a0enterococci isolated from drinking water are lacking. Thus, although numerous studies\u00a0[7,\u00a08] address the\u00a0question of\u00a0\u201cwho isolated which enterococcus, when, and from where\u201d, relatively little is known about their actual ecology, particularly in\u00a0relation to drinking water, drinking water treatment, and their survival and growth in\u00a0biofilms.<\/span><\/p>\n Although all commonly identified enterococcal species occur in\u00a0the\u00a0intestines of\u00a0humans or warm-blooded animals, a\u00a0distinction is made between species that are typically faecal (E. faecium<\/span><\/em>, E. faecalis<\/span><\/em>, E. hirae<\/span><\/em>, E. durans<\/span><\/em>) and those\u00a0<\/span>associated with possible proliferation on plant material (E. mundtii<\/span><\/em>, E. casseliflavus<\/span><\/em>)\u00a0[3]. Enterococci are also frequently used as indicators in\u00a0microbial source tracking (MST), and various methods for their elimination from the\u00a0aquatic environment have been described. They are also considered significant carriers of\u00a0antibiotic resistance (e.g. to vancomycin\u00a0and ampicillin). In\u00a0the\u00a0environment, they often occur in\u00a0a\u00a0non-virulent VBNC (Viable But Non Culturable) state.<\/span><\/p>\n For the\u00a0determination of\u00a0intestinal enterococci in\u00a0drinking water, the\u00a0long-established method according to \u010cSN EN ISO 7899-2\u00a0[9] is used. This method includes membrane filtration of\u00a0samples, incubation for 48 hours at\u00a036\u00a0\u00b0C on Slanetz and Bartley agar, and confirmation for 2 hours at 44 \u00b0C on bile esculin\u00a0azide agar (hereafter BEA). Intestinal enterococci are defined as red to maroon colonies (Fig.\u00a01<\/span><\/em>) that, after subculture on the\u00a0confirmation medium, show blackening of\u00a0the\u00a0medium beneath the\u00a0colony. This method is intended to detect predominantly enterococcal species of\u00a0faecal origin\u00a0(E. faecalis<\/span><\/em>, E. faecium<\/span><\/em>, E. durans<\/span><\/em>, and E. hirae<\/span><\/em>). This is also related to the\u00a0reduction of\u00a0the\u00a0confirmation time from four to two hours in\u00a02001, based on the\u00a0assumption that typical faecal enterococci exhibit faster and more intense BEA activity. More recent methods, which aim to serve as alternatives, are often based on the\u00a0activity of\u00a0the\u00a0enzyme \u03b2-D-glucosidase, which enables the\u00a0detection of\u00a0all enterococcal species (Enterococcus<\/span><\/em> spp.)\u00a0[10]. This does not appear to be appropriate, particularly because this parameter is not merely an\u00a0indicator but a\u00a0key parameter with a\u00a0limit value (unsurpassable parametric value). On the\u00a0contrary, a\u00a0more appropriate approach for improving the\u00a0interpretation of\u00a0enterococci results would be to move in\u00a0the\u00a0opposite direction, namely towards the\u00a0identification of\u00a0individual species.<\/span><\/p>\n This study included strains isolated from treated drinking water (but not from wells), obtained over a\u00a0two-year period from operational hydroanalytical laboratories. The\u00a0strains were purified, identified using the\u00a0MALDI-TOF method (with the\u00a0application of\u00a0formic acid), and the\u00a0confirmation test was repeated, with results recorded after 2, 4, and 24 hours. In\u00a0addition, the\u00a0strains were tested for \u03b2-D-glucosidase activity in\u00a0a\u00a0selective medium (Enterolert DW, IDEXX; more recently also according to ISO 7899-3\u00a0[11]).<\/span><\/p>\n Finally, representatives of\u00a0the\u00a0most frequently isolated species were tested for sensitivity to free chlorine using a\u00a0method modified according to Annex\u00a04 of\u00a0Decree No. 409\/2005 Coll.\u00a0[12]. A\u00a0solution of\u00a0sodium hypochlorite was added to a\u00a0measured volume (1,000\u00a0ml) of\u00a0settled tap water at laboratory temperature to achieve a\u00a0free chlorine concentration in\u00a0the\u00a0range of\u00a00.15\u20130.17\u00a0mg\/L. The\u00a0solution was then artificially inoculated with the\u00a0tested strains of\u00a0the\u00a0genus Enterococcus<\/span><\/em>. The\u00a0initial concentration of\u00a0the\u00a0test strain\u00a0was approximately 10\u2075\u00a0CFU\/mL. Before the\u00a0test, the\u00a0contaminated water was thoroughly mixed (e.g. by shaking) to ensure uniform distribution of\u00a0microorganisms. At test intervals of\u00a01, 5, and 30\u00a0minutes, 0.5\u00a0ml of\u00a0the\u00a0prepared solution was inoculated onto the\u00a0surface of\u00a0a\u00a0solid culture medium, and after incubation for 48\u00a0hours at (36\u00a0\u00b1\u00a02) \u00b0C, the\u00a0colonies grown on the\u00a0surface were counted. During the\u00a0test period, the\u00a0test solution in\u00a0the\u00a0flask was continuously mixed. Simultaneously, the\u00a0original suspension was inoculated to determine the\u00a0initial number of\u00a0enterococci. All specified time intervals were tested; however, only the\u00a0results after 1\u00a0minute were evaluated, as the\u00a0results after 5 and 30\u00a0minutes were mostly negative. For each species, a\u00a0strain\u00a0isolated from treated drinking water and a\u00a0strain\u00a0isolated from the\u00a0natural environment (bathing water) were tested in\u00a0parallel, and all assays were performed in\u00a0duplicate. After incubation, the\u00a0relative reduction of\u00a0the\u00a0tested species (strain) after 1\u00a0minute of\u00a0exposure to free chlorine was calculated in\u00a0comparison with the\u00a0control number.<\/span><\/p>\n A\u00a0total of\u00a0227 strains isolated in\u00a0five water management laboratories from drinking (treated) water were processed; of\u00a0these, 134 were subsequently identified as species belonging to the\u00a0genus Enterococcus<\/span><\/em> (a\u00a0total of\u00a010 species), while 93\u00a0belonged to other genera, with a\u00a0clear predominance of\u00a0Aerococcus viridans<\/span><\/em>.<\/span><\/p>\n The\u00a0species composition of\u00a0enterococci and their relative distribution are shown in\u00a0Fig.\u00a02<\/span><\/em>. The\u00a0most frequently identified species was E. casseliflavus<\/span><\/em> (31\u00a0%), followed by E. faecium<\/span><\/em> (25\u00a0%), E. hirae<\/span><\/em> (13\u00a0%), E. faecalis<\/span><\/em> (10\u00a0%), and E.\u00a0mundtii<\/span><\/em> (9\u00a0%). Enterococcal species considered to be of\u00a0faecal origin\u00a0according to \u010cSN\u00a0EN\u00a0ISO\u00a07899-2\u00a0[9] (faecalis<\/span>, faecium<\/span>, hirae<\/span>, durans<\/span>) accounted for only 54\u00a0%. For comparison, our earlier unpublished results from the\u00a0identification of\u00a0612\u00a0enterococci from bathing waters showed that the\u00a0most frequently identified species was E.\u00a0faecium<\/span><\/em> (25.2\u00a0%), followed by E. faecalis<\/span><\/em> (21.1\u00a0%), E. durans<\/span><\/em> (17.3\u00a0%), and E. casseliflavus<\/span><\/em> (14.4\u00a0%). Such datasets can, of\u00a0course, be compared only to a\u00a0limited extent; nevertheless, it is evident that different matrices yield different results (unfortunately, the\u00a0literature cited in\u00a0the\u00a0Introduction\u00a0[3] analysed enterococci from a\u00a0\u201cmixture of\u00a0matrices\u201d). That bathing water represents a\u00a0completely different matrix is also apparent from the\u00a0composition of\u00a0the\u00a0accompanying microflora. According to our previous results, as well as the\u00a0cited literature\u00a0[3], the\u00a0species most frequently interfering with the\u00a0determination of\u00a0intestinal enterococci in\u00a0bathing waters was Lactobacillus plantarum<\/span><\/em>, whereas in\u00a0drinking water the\u00a0most prevalent species of\u00a0background microflora was Aerococcus viridans<\/span><\/em>.<\/span><\/p>\n The\u00a0high occurrence of\u00a0E. casseliflavus<\/span><\/em> in\u00a0drinking water cannot yet be reliably interpreted; however, it should be noted that this was not a\u00a0case of\u00a0\u201cmany strains from a\u00a0single sample\u201d, as is often observed in\u00a0bathing waters, but rather a\u00a0more continuous occurrence. In\u00a0previously cited studies, this species has often been associated with possible proliferation on plant material. In\u00a0drinking water, the\u00a0key question is how it behaves, for example, in\u00a0biofilms or on sand filters, which is not yet known. In\u00a0contrast to bathing water samples, where this species may proliferate, for example, on reeds and the\u00a0membrane filter may then be covered with these (rather small) colonies, in\u00a0this case overgrown filters were generally not observed. When membrane filters were covered with small colonies, these were exclusively A. viridans<\/span><\/em>.<\/span><\/p>\n The\u00a0results of\u00a0the\u00a0confirmation and supplementary tests for individual species are presented in\u00a0Tab.\u00a02<\/span><\/em>. In\u00a0addition to the\u00a0absolute number of\u00a0positive reactions for strains of\u00a0individual species, the\u00a0table also shows the\u00a0relative proportion of\u00a0false-positive results (in\u00a0background microflora) and false-negative results (in\u00a0enterococci).<\/span><\/p>\n According to the\u00a0current version of\u00a0\u010cSN EN ISO 7899-2\u00a0[9], the\u00a0duration of\u00a0the\u00a0BEA test is 2 hours, which was taken as the\u00a0reference time. Additional times (the\u00a0previously used 4 hours and 24 hours) were tested for the\u00a0purpose of\u00a0further discussion of\u00a0the\u00a0results. However, the\u00a0difference between 2 and 4\u00a0hours was minimal. A\u00a0false-negative BEA test (i.e. after 2 hours of\u00a0incubation) was recorded in\u00a010\u00a0% of\u00a0enterococci, most frequently in\u00a0E. gallinarum<\/span><\/em>, E. casseliflavus<\/span><\/em>, and E. durans<\/span><\/em>. A\u00a0false-negative result in\u00a0the\u00a0\u03b2-D-glucosidase test was observed in\u00a0only 1\u00a0% of\u00a0enterococcal strains, but a\u00a0further seven strains (5.3\u00a0%) showed a\u00a0weak reaction. Unfortunately, it is not precisely known how a\u00a0positive GLD test should appear, as no comparator is available for the\u00a0Enterolert DW test. A\u00a0false-positive BEA test (after 2 hours of\u00a0incubation) was recorded in\u00a08\u00a0% of\u00a0strains of\u00a0the\u00a0accompanying microflora; in\u00a0some strains of\u00a0A. viridans<\/span><\/em>, the\u00a0positive reaction faded during the\u00a0subsequent 20 hours. A\u00a0false-positive \u03b2-D-glucosidase test was observed in\u00a014\u00a0% of\u00a0strains.<\/span><\/p>\n The\u00a0most frequently isolated enterococcal species and the\u00a0most common background species, A. viridans<\/span>, were selected for testing their sensitivity to\u00a0free chlorine. It is generally accepted that enterococci are less sensitive to\u00a0the\u00a0effects of\u00a0free chlorine than, for example, coliform bacteria, and this is also confirmed by the\u00a0results of\u00a0our operational testing of\u00a0disinfectants (commercial, unpublished data). For each species, two strains were tested (three strains in\u00a0the\u00a0case of\u00a0E. faecalis<\/span><\/em>), with one strain always isolated from treated drinking water (and<\/span><\/p>\n therefore potentially exposed to free chlorine) and the\u00a0other from a\u00a0typical natural environment (bathing water). According to legislative requirements\u00a0[12], enterococci counts must be reduced by at least three orders of\u00a0magnitude at a\u00a0free chlorine concentration of\u00a00.3\u00a0mg\/L. The\u00a0strains investigated in\u00a0this study largely met this requirement even at half the\u00a0concentration of\u00a0free chlorine (0.15\u00a0mg\/L). The\u00a0results are illustrated in\u00a0Fig.\u00a03<\/span><\/em>. The\u00a0least sensitive species were E.\u00a0hirae<\/span><\/em>, A. viridans<\/span><\/em>, and a\u00a0strain\u00a0of\u00a0E. mundtii<\/span><\/em> isolated from drinking water (rather than\u00a0from bathing water), whereas the\u00a0most sensitive species was E.\u00a0durans<\/span><\/em>. In\u00a025\u00a0% of\u00a0E. durans<\/span> strains, a\u00a0delayed BEA test reaction was observed (Tab.\u00a02<\/span><\/em>), which may be attributed to stress in\u00a0strains originating from treated water. A\u00a0certain\u00a0degree of\u00a0resistance to free chlorine in\u00a0E. hirae<\/span> may explain\u00a0why this species ranked third among the\u00a0identified strains, and a\u00a0similar resistance in\u00a0A. viridans<\/span><\/em> may account for its frequent occurrence as accompanying microflora. The\u00a0most resistant strain, E. hirae<\/span><\/em>, is also prescribed for testing the\u00a0bactericidal properties of\u00a0disinfectants\u00a0[13]. Until the\u00a0final experiment, it appeared that strains from natural environments were more sensitive to the\u00a0effects of\u00a0free chlorine (possibly due to the\u00a0absence of\u00a0stress?) than\u00a0strains isolated from drinking water; however, this was clearly not the\u00a0case for E. mundtii<\/span><\/em>, where the\u00a0opposite was observed.<\/span><\/p>\n Although the\u00a0\u201ccollection of\u00a0enterococci\u201d lasted at least two years, the\u00a0number of\u00a0strains obtained was not particularly high (enterococci = 134 + accompanying microflora = 93). Nevertheless, valuable data were obtained. The\u00a0most frequently isolated species from drinking water was E. casseliflavus<\/span><\/em>, which is usually less abundant and occurs rather sporadically in\u00a0natural waters. This occurrence cannot yet be fully interpreted; one possible explanation is its ability to survive in\u00a0biofilms (?). This species has also been associated with the\u00a0potential for proliferation on plant material. Among the\u00a0accompanying microflora, the\u00a0most frequently identified species was A. viridans<\/span>, which, together with E.\u00a0hirae<\/span>, showed the\u00a0lowest sensitivity to free chlorine. The\u00a0method according to \u010cSN\u00a0EN\u00a0ISO\u00a07899-2 is fully suitable for the\u00a0determination of\u00a0intestinal enterococci in\u00a0drinking water. The\u00a0use of\u00a0alternative methods based on the\u00a0determination of\u00a0\u03b2-D-glucosidase (GLD) activity is not entirely appropriate, as it expands the\u00a0group of\u00a0\u201cintestinal enterococci\u201d to include Enterococcus<\/span>\u00a0spp., thereby extending detection to species with an\u00a0uncertain\u00a0faecal origin. Moreover, the\u00a0interpretation of\u00a0enterococci results would benefit from the\u00a0opposite approach, namely the\u00a0identification of\u00a0strains and interpretation of\u00a0their occurrence in\u00a0the\u00a0environment. In\u00a0addition, the\u00a0most frequently detected species of\u00a0accompanying microflora, A. viridans<\/span>, shows false-positive GLD results in\u00a015\u00a0% of\u00a0cases. Given that this is a\u00a0key parameter with the\u00a0highest parametric value, such an\u00a0expansion of\u00a0the\u00a0\u201cgroup\u201d could lead to complications.<\/span><\/p>\n This publication was supported by the\u00a0Ministry of\u00a0Health of\u00a0the\u00a0Czech Republic\u00a0\u2013 Institutional Support (National Institute of\u00a0Public Health \u2013 SZ\u00da, No. 75010330). Special thanks are extended to the\u00a0staff of\u00a0laboratories of\u00a0water supply operators who provided strains for this study.<\/span><\/span><\/em><\/p>\n The\u00a0Czech version of\u00a0this article was peer-reviewed, the\u00a0English version was translated from the\u00a0Czech original by Environmental Translation Ltd.<\/span><\/p>\n","protected":false},"excerpt":{"rendered":" Intestinal enterococci are one of the two core microbiological parameters in the new drinking water legislation, used as indicators of faecal contamination. They must be detected in all analyses of drinking water. A total of 134 strains of enterococci (Enterococcus spp.) and 93 strains identified as background microflora, or as potentially false-positive strains, were examined from operational samples of treated (i.e. drinking) water.<\/p>\n","protected":false},"author":8,"featured_media":39085,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[94,87,92],"tags":[290,4147,4149,4148],"coauthors":[231,4134],"class_list":["post-38958","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-current-issue","category-hydrochemistry-radioecology-microbiology","category-main","tag-drinking-water","tag-intestinal-enterococci","tag-sensitivity-to-free-chlorine","tag-species-composition"],"acf":[],"_links":{"self":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/38958","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/users\/8"}],"replies":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/comments?post=38958"}],"version-history":[{"count":6,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/38958\/revisions"}],"predecessor-version":[{"id":39192,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/38958\/revisions\/39192"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/media\/39085"}],"wp:attachment":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/media?parent=38958"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/categories?post=38958"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/tags?post=38958"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/coauthors?post=38958"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}OVERVIEW OF THE ISSUE<\/h2>\n
Tab. 1. Detection of intestinal enterococci in drinking water in the Czech Republic (categories: > 5,000 population supplied, < 5,000 population supplied, total number of samples analysed, number of positive samples, arithmetic mean and maximum value) in 2020\u20132024 (CFU = colony forming units)<\/h5>\n
![\"\"](\"https:\/\/www.vtei.cz\/wp-content\/uploads\/2026\/06\/Baudisova-tab-1-3.jpg\")
<\/a>\n<\/a>\nFig. 1\u00a0a, b. Left: Aerococcus viridans, forming very small (mostly non-blackening) colonies; right: detection of\u00a0intestinal enterococci<\/h6>\n
METHODOLOGY<\/h2>\n
RESULTS AND DISCUSSION<\/h2>\n
Identification of\u00a0species<\/h3>\n
<\/a>\nFig. 2. Occurrence of\u00a0individual species of\u00a0intestinal enterococci isolated from\u00a0drinking\u00a0water<\/h6>\n
Confirmation and supplementary tests<\/h3>\n
Sensitivity of\u00a0enterococci to free chlorine<\/h3>\n
Tab. 2. Results of additional tests for strains of individual species; the number of strains examined (n), positive results of the BEA test after 2, 4 and 24 hours, positive results of\u00a0the\u00a0\u03b2-D-glucosidase (GLD)\u00a0test. False results (false positives for background microflora or false negatives for enterococci) for BEA\u00a0tests after 2 hours and GLD\u00a0 are given separately [%]<\/h5>\n
<\/a>\nCONCLUSION<\/h2>\n
Acknowledgements<\/h3>\n