Agricultural and Municipal Waste Water as a Source of Antibiotic-Resistant Enterococci

Cupáková, ·. , J . Luká‰ová: Agricultural and Municipal Waste Water as a Source of Antibiotic-Resistant Enterococci. Acta Vet. Brno 2003, 72: 123-129. The occurrence of enterococci in different waste waters from two cattle farms in south and north Moravia and sewage treatment plant of the University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic was investigated. Resistance and/or sensitivity of selected isolates was tested to various antibiotics. The samples were collected in the course of two years (1999-2000). The total counts of enterococci varied from 103 to 105 colony-forming units (CFU) per ml. Among 100 isolated strains 60 strains were identified as Enterococcus faecalis, 10 as Ent. durans as well as Ent. hirae, 8 strains were allotted to Ent. faecium, 5 to Ent. mundtii, 3 to Ent. gallinarum, 2 to Ent. casseliflavus, one strain was identified as Enterococcus spp. and one strain was not specified as enterococcus. The susceptibility to antibiotics was tested by both, the agar disk diffusion method and the microdilution method. The majority of enterococci (95%) was resistant to more than one antibiotic tested, especially to clindamycin, penicillin, cephalotin, ofloxacin and tetracycline. No vancomycin-resistant strain was found. Our results confirm that agricultural and municipal waste waters might be an important source of antibiotic-resistant enterococci. Enterococci, waste water, silage, liquid manure, cattle, antibiotic-resistance Enterococci are a part of the common microflora in the gastrointestinal tract of warmblooded animals and birds. In addition they can occur in soil, surface or waste waters, on plants and vegetables (Franz et al. 1999). Enterococci are now considered as the third most common pathogens causing nosocomial infections and “superinfections” in hospitalized patients, accounting for nearly 12% of these infections (Linden et al. 1999). The most common enterococci-associated infections are infections of the urinary tract, bacteremia, endocarditis, intra-abdominal and pelvic infections. The very consequential ones are neonate meningitis and bacteremia and central nervous system infections in adults (Murray 1990; Franz et al. 1999). Vancomycin-resistant enterococci (VRE) represent a serious problem at present. They are usually isolated from environment, waste waters, animals, foods of animal origin. They still represent the worldwide problem for hospitalized patients, where the glycopeptide antibiotics vancomycin and teicoplanin are used in the therapy of several infections caused by Gram-positive bacteria (Klare et al. 1993; Borgen et al. 2001). The emergence of VRE in Europe is attached to the long-time use of the glycopeptide antibiotic avoparcin as a growth promoter for farm animals with the possibility of inducing cross-resistance to vancomycin. In 1997 it was banned in EU (Grosso et al. 2000). Many studies indicate food animals as reservoirs of resistant enterococci which might be transmitted to humans through the food chain and represent a potential risk for the consumers (Bates et al. 1994; Bager et al. 1997; Klein et al. 1998; Grosso et al. 2000). The aim of this study was to investigate the occurrence of enterococci in different kinds of agricultural and municipal waste waters and to test their sensitivity to selected antimicrobial agents. ACTA VET. BRNO 2003, 72: 123–129 Address for correspondence: MVDr. ·árka Cupáková Department of Milk Hygiene and Technology University of Veterinary and Pharmaceutical Sciences Brno Palackého 1-3, 612 42 Brno, Czech Republic Phone: +420 5 4156 2593 E-mail: cupakovas@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm

Enterococci are a part of the common microflora in the gastrointestinal tract of warmblooded animals and birds.In addition they can occur in soil, surface or waste waters, on plants and vegetables (Franz et al. 1999).
Enterococci are now considered as the third most common pathogens causing nosocomial infections and "superinfections" in hospitalized patients, accounting for nearly 12% of these infections (Linden et al. 1999).The most common enterococci-associated infections are infections of the urinary tract, bacteremia, endocarditis, intra-abdominal and pelvic infections.The very consequential ones are neonate meningitis and bacteremia and central nervous system infections in adults (Murray 1990;Franz et al. 1999).
Vancomycin-resistant enterococci (VRE) represent a serious problem at present.They are usually isolated from environment, waste waters, animals, foods of animal origin.They still represent the worldwide problem for hospitalized patients, where the glycopeptide antibiotics vancomycin and teicoplanin are used in the therapy of several infections caused by Gram-positive bacteria (Klare et al. 1993;Borgen et al. 2001).The emergence of VRE in Europe is attached to the long-time use of the glycopeptide antibiotic avoparcin as a growth promoter for farm animals with the possibility of inducing cross-resistance to vancomycin.In 1997 it was banned in EU (Grosso et al. 2000).Many studies indicate food animals as reservoirs of resistant enterococci which might be transmitted to humans through the food chain and represent a potential risk for the consumers (B ates et al. 1994;B ager et al. 1997;Klein et al. 1998;Grosso et al. 2000).
The aim of this study was to investigate the occurrence of enterococci in different kinds of agricultural and municipal waste waters and to test their sensitivity to selected antimicrobial agents.

Samples
The occurrence of enterococci was studied in different waste waters during the years 1999-2000.The following samples were collected: -12 samples of mixed waste waters from litter bed and milking parlor and 5 samples of silage waste waters from a cattle farm in the district of Blansko, south Moravia; -6 samples of cattle liquid manure from a cattle farm in the district of Nov˘ Jiãín, north Moravia; -10 samples of waste waters affluent to the sewage treatment plant of the Veterinary university Brno, Czech Republic.
Bacterial strains A total of 100 bacterial strains were isolated during the microbiological analysis of agricultural and municipal waste water.The samples were diluted according to âSN ISO 7218.The total numbers of enterococci were determined by inoculation of 0.1 ml of the respectived dilution of the sample onto Slanetz-Bartley agar (HiMedia, Mumbai, India), which contains NaN 3 as a selective agent for enterococci.The plates were incubated at 37 C for 24 h.Three colonies with different but "enterococcal" colony morphology from all tested samples were picked up.The purity of the isolated strains was checked by the streak plate technique on blood agar (Blood agar base No. 2, HiMedia, and sheep blood, Bioveta, Ivanovice na Hané, CZ) and Slanetz-Bartley agar (HiMedia).

Identification of isolated strains
The following tests were used for the identification of isolated strains: Gram staining, appearance of colonies on blood agar, catalase production, growth at 45 °C, in broth (tryptone glucose yeast extract broth, HiMedia) with 6.5% NaCl and at pH 9.6 (Havlová et al. 1993), growth on bile-esculin agar (Oxoid, Basingstoke, Hampshire, England).Phenotyping of selected isolates was done using the Api Rapid ID 32 Strep system (BioMerieux, L'Etoile, France) and completed by production of pyrrolidonyl arylamidase (PYRAtest, Pliva-Lachema, a.s., Brno, Czech Republic), motility and production of yellow pigment.

Tests of sensitivity to antimicrobial agents
The susceptibility to antibiotics was tested by the agar disc diffusion method (DD) and the microdilution method (MD) to show the minimal inhibition concentration of antibiotic used (MIC) as well.
Resistance to antibiotics of isolated enterococci performed by agar disc diffusion method is shown in Table 2.The majority of tested isolates were resistant to CLI (98.99%) and OFL (90.91%).More than a half of the isolates was resistant to CLT and PEN (66.67%).No strain was resistant to vancomycin and teicoplanin; only 32 strains shown VAN-intermediate resistance.The strains were resistant at least to one and not more than to seven antibiotics tested.The majority of strains was simultaneously resistant to four (37.37%) or five (26.26%) drugs.The most frequently occurring resistant phenotypes were CLI-CLT-OFL-PEN (27 strains) and CLI-CLT-OFL-PEN-TTC (21 strains).
In terms of the MIC for the individual anti-microbial drugs, resistance to CLT, PEN and CLI was demonstrated in more than 95% of enterococcal strains and more than a half of them   (1996) isolated 45 strains of enterococci from municipal waste waters; 32 of them were Enterococcus faecalis, 10 isolates belonged to Ent. faecium and three to Ent. hirae.Lauková et al. (1997) studied the occurrence of enterococci in municipal sewage from different sewage treatment plants in the Eastern Slovakia.They analyzed 2 000 isolates and documented the predominant occurrence of Ent.faecium (50%), followed by Ent.gallinarum (25.5%) and Ent.casseliflavus (10.1%).These presented studies suggest a higher incidence of Ent.faecium in municipal sewage and waste waters.Our results show an expressive prevalence of Enterococcus faecalis strains in different types of agricultural waste waters, which could be explained by the fact that the gastrointestinal tract of farm animals is probably more colonized by this species.Most enterococci are naturally or inherently resistant to various drugs, including cephalosporins, oxacillin and to clinically achievable concentrations of clindamycin and aminoglycosides, a great part is relatively resistant to penicillin and ampicillin (Murray 1998;Urbá‰ková 1999).Important is their resistance to tetracyclines, macrolides and chloramphenicol (Gray et al. 1991;F ranz et al. 1999). K lare et al. (1993) showed that glycopeptide-resistant E. faecium can be found not only in clinical samples but also in environmental samples as well as in waste waters or sewage treatment plants.Our results are in agreement with these data.Schlegelová et al. (2002) described in their study resistance to TTC (MIC 90 >32 mg l -1 ) and to ERY (MIC 90 >32 mg l -1 ) among Enterococcus faecalis strains isolated from bulk milk samples.Compared to our results, MIC 90 16 mg l -1 for TTC and MIC 90 2 mg l -1 for ERY, a lower prevalence of resistant strains of Ent.faecalis was demonstrated.Bates et al. (1994) isolated vancomycin-resistant E. faecium from a duck, a chick, a turkey, a dog, a pony and from pigs, but VRE were not isolated from cattle or sheep.Devriese et al. (1996) isolated VanA-resistant Ent.faecium strains from the intestines and faeces of horses, dogs, chickens and pigs, VanA-positive strains identified as Ent.durans from gallinaceous birds, Ent.faecalis in a horse and Ent.gallinarum in a pheasant.Furthermore, a prevalence of vancomycin-resistant Ent.faecium was studied in faecal samples from pigs in Spain.There, 43 from 240 pig farms represented in the sampling had  (Herrero et al. 2000).Koláfi et al. (2000) described for the first time the occurrence of VRE in animals in the Czech Republic.The VRE were isolated from 2.5% of examined hens and 75% of them was diagnosed as Ent.faecium phenotype VanA.In our study noVRE strain from agricultural waste water samples was detected.Multidrug-resistant enterococci are commonly isolated from humans, sewage, aquatic habitats, agriculture and animal sources.It indicates their ability to enter the human food chain.Lauková et al. (1997) found that enterococci isolated from waste waters were resistant at least to one (except vancomycin) and not more than to six of the examined antibiotics, most of them being biresistant.Similarly, Koláfi et al. (2000) reported about multiresistant enterococci isolated from hens.These findings are in accordance with our results.In this study 95% multiresistant strains of Enterococcus spp. in waste water samples were determined.
Finally, a large spectrum of enterococci was isolated and identified from various agricultural waste waters.Ent.faecalis predominated among them.The majority of enterococci were resistant to more than one antibiotics tested, above all to CLI, PEN, CLT, OFL and TTC.But no VRE strain was demonstrated.Our results suggest that agricultural waste waters might be an important source of resistant enterococci, which could be taken into account in their further application in agriculture.
range of antimicrobial agents for enterococcal species sults were obtained in other studies, which showed distribution of enterococci in sewage or waste waters, e.g.V aldivia et al.

Table 1
Numbers of strains of Enterococcus spp.isolated from different agricultural waste waters

Table 3
Numbers of antibiotic-resistant strains of Enterococcus spp.isolated from different sources (microdilution method)