Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd
?? 200584667674Original
Article
Antibiotic-resistant enterococciA. Manero, X. Vilanova, M.
Cerdà-Cuéllar and A. R. Blanch
Environmental Microbiology (2006) 8(4), 667–674
doi:10.1111/j.1462-2920.2005.00945.x
Vancomycin- and erythromycin-resistant enterococci in a pig farm and its environment Albert Manero,1 Xavier Vilanova,2 Marta Cerdà-Cuéllar3 and Anicet R. Blanch2* 1 Laboratori d’Anàlisi Mediambiental, Secció Microbiologia, Aigües de Terrassa, S.A., Carrer del Nord, 88, E 08221 Terrassa (Barcelona), Catalonia, Spain. 2 Departament de Microbiologia, Universitat de Barcelona, Diagonal 645, E 08028 Barcelona, Catalonia, Spain. 3 Centre de Recerca en Sanitat Animal, Campus Universitat Autònoma de Barcelona, E 08193 Bellaterra (Barcelona), Catalonia, Spain. Summary A high prevalence of vancomycin- and erythromycinresistant enterococci (VRE and ERE respectively) in a pig farm and its environment was observed. A similar structure and composition of enterococcal populations was detected between urban sewage and those associated with the pig environment. Enterococcus faecium was the most predominant species among VRE isolates from both animal and human origin. The high population similarity index (Sp) obtained comparing VRE and ERE isolates from urban sewage and pig slurry suggests that there are certain strains circulating through the food chain from farms to humans. Erythromycin resistance was present in a wider variety of clones and species of enterococci in both pigs and humans than vancomycin resistance. Introduction The enterococci are members of the normal intestinal flora in humans and other animals (Murray, 1990; Devriese et al., 1991; 1992; 1994). They are released into the environment by animal and human faecal materials. Moreover, some enterococci are ubiquitous and can be found free-living in soil or on plants (Devriese et al., 1991; Leclerc et al., 1996; Flahaud et al., 1997; Domig et al., 2003). Enterococci are commonly used as faecal indicators because of their abundance in faeces and their long survival in the environment (Flahaud et al., 1997). Although enterococci are not regarded as primary pathogens, they are major nosocomial pathogens due to Received 20 June, 2005; accepted 22 September, *For correspondence. E-mail
[email protected]; Tel. 93 4029012; Fax (+34) 93 4039047.
2005. (+34)
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd
their ability to acquire high-level resistance to antimicrobial agents (Murray, 1990; 1998; Jett et al., 1994; Flahaud et al., 1997). Several molecular and clinical studies have reported strains that are resistant to antibiotics and the risk that they might be transmitted from farm animals to humans through the food chain (Klein et al., 1998; Woodford et al., 1998; Lemcke and Bülte, 2000; Robredo et al., 2000; Torell et al., 2003; Kühn et al., 2005). In the past few years, the appearance of enterococci resistant to the glycopeptide vancomycin (vancomycin-resistant enterococci, VRE) has become an important public health concern because of the use of vancomycin in treating enterococcal infections (Huycke et al., 1998). Vancomycin-resistant enterococci are becoming increasingly common in hospitals and healthy humans, and have also been isolated from sewage waters, faeces from different animals and from food of animal origin (Klare et al., 1993; 1995; Aarestrup, 1995; Borgen et al., 2000; Blanch et al., 2003; Garcia-Migura et al., 2005). Some authors suggested that the extensive use in animal production of glycopeptides as avoparcin has created an animal reservoir of resistant enterococci (Robredo et al., 1999). On the other hand, epidemiological studies in the USA suggested the spread of resistant strains from the hospital environment to the food chain (Coque et al., 1996). Erythromycinresistant enterococci (ERE) have been also related to the use of the 16C-macrolide tylosin as a growth promoter (Van den Bogaard et al., 1997). VRE are also frequently resistant to the 14C-macrolide erythromycin (Robredo et al., 1999). In this study, the isolation of VRE and ERE and the biochemical fingerprints of enterococcal populations in a pig farm environment were used to analyse the ecological and epidemiological role of enterococci in a first stage of the food chain. The study was focused on pig farms because they are animals that are most likely to produce slurry that is used as natural fertilizer. Moreover, pig husbandry used avoparcin and tylosin as growth promoters for many years. In order to compare human enterococcal populations with those associated with the pig environment, urban sewage isolates were also studied. Results Prevalence of enterococci A total of 285 samples were collected: 68 from pig faeces,
668 A. Manero, X. Vilanova, M. Cerdà-Cuéllar and A. R. Blanch 47 from pig slurry, 35 from pig feed, 46 from soil receiving pig slurry, 13 from crops receiving pig slurry, 33 from soil receiving no animal fertilizer, 33 from crop receiving no animal fertilizer and 10 from urban sewage. Most of the samples (215 of 285) showed growth on m-Enterococcus Agar (MEA) and Bile Esculin Agar (BEA), consequently presenting enterococcal strains (ENT). Enterococci were isolated in all samples of urban sewage, pig faeces and pig slurry. Prevalence of enterococci was also high for pig feed (94%). However, enterococci were found in about 50% of soils fertilized with pig slurry and crops from the corresponding farmland. Enterococci were isolated in less than 50% of soil and crop samples receiving no animal fertilizer (Table 1). Pig slurry and urban sewage showed the highest enterococci concentrations, whereas soil and crops, with or without pig slurry, showed the lowest levels (Table 1). Prevalence of vancomycin-resistant enterococci Enterococci resistant to 8 mg l−1 (VRE8) and 20 mg l−1 (VRE20) vancomycin were found in 16% and 13%, respectively, of samples positive for enterococci. The VRE20 were mostly found when samples were preenriched in EBV8. Only in about 25% of samples that showed a presence of VRE20 after enrichment they were also isolated on direct plating. The highest prevalence of VRE20 was obtained in urban sewage water (100%), followed by pig slurry and pig faeces (34% and 16% respectively) (Table 1). No VRE20 were isolated from pig feed, soil, or crops with or without slurry. Prevalences of VRE8 isolates are not shown because only isolates growing in 20 mg l−1 vancomycin (VRE20) were considered as vancomycin-resistant enterococci. Enumeration of VRE8 and VRE20 isolates are not shown because they occurred in very low numbers and were mostly found when enrichment was performed.
Prevalence of erythromycin-resistant enterococci Erythromycin-resistant enterococci to 8 mg l−1 (ERE8) were isolated in 58% of samples positive for enterococci. A 100% prevalence was obtained in urban sewage, pig faeces and pig slurry. Prevalence was 40–50% for pig feed and crops and soil receiving pig slurry as fertilizer. On the other hand, very low prevalence was obtained in soil and crops receiving artificial fertilizer (6% and 0% respectively) (Table 1).
Phenotypic diversity of enterococcal populations A total of 2593 Enterococcus strains isolated from MEA were phenotyped: 662 from pig faeces, 717 from pig slurry, 460 from pig feed, 269 from soil with pig slurry, 84 from crops with pig slurry, 25 from soil without natural fertilizer, 149 from crops without natural fertilizer and 227 from urban sewage. The numbers of phenotyped VRE20 were 19, 29 and 49 from pig faeces, pig slurry and urban sewage respectively. A total of 562 ERE8 were also phenotyped: 206 from pig faeces, 208 from pig slurry, 26 from pig feed, 64 from soil with pig slurry, 19 from crops with pig slurry, 3 from soil without natural fertilizer and 36 from urban sewage. Table 1 shows the diversity of the bacterial populations (Di) of enterococcal populations (ENT, VRE20 and ERE8) from different origins. The highest diversity indexes for ENT isolates were obtained in urban sewage, pig feed, pig slurry and soil receiving pig slurry samples, indicating the presence of a large variety of enterococcal phenotypes (Table 1). Pig faeces showed lower diversity than pig slurry and soil fertilized with pig slurry. Diversity was also calculated for VRE20 and ERE8 isolates. The Di for VRE20 was higher in urban sewage than in pig faeces and slurry. The highest diversity indexes for ERE8 were obtained in urban sewage, pig slurry and pig
Table 1. Prevalence (%), enumeration [colony-forming units (cfu) g−1, average value in positive samples] and diversity index values (Di) of enterococci in different samples types. cfu g−1
Prevalence
PF (n = 68) PS (n = 47) PD (n = 35) SS (n = 46) CS (n = 13) SA (n = 33) CA (n = 33) US (n = 10)
Di
ENT
ERE8
VRE20
ENT
ERE8
VRE20
ENT
ERE8
VRE20
100 100 94 48 62 36 45 100
100 100 40 39 54 6 0 100
16.1 34 0 0 0 0 0 100
nd 1.0E+05 2.0E+03 6.0E+02 2.0E+02 9.0E+00 2.0E+02 8.0E+03
nd 1.0E+05 2.0E+02 1.0E+02 2.0E+02 0.4 0.0E+00 1.0E+03
nd nd nd nd nd nd nd nd
0.871 0.946 0.954 0.944 0.818 0.923 0.932 0.949
0.842 0.903 0.923 0.896 0.865 0.667 nd 0.946
0.719 0.631 nd nd nd nd nd 0.861
ENT, enterococci on MEA; ERE8, erythromycin-resistant enterococci on MEAE8; VRE20, vancomycin-resistant enterococci on MEAV20; PF, pig faeces; PS, pig slurry; PD, pig feed; SS, soil receiving pig slurry; CS, crops receiving pig slurry; SA, soil not receiving pig slurry; CA, crops not receiving pig slurry; US, urban sewage; nd, not determined. © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 667–674
Antibiotic-resistant enterococci 669 Table 2. Population similarity indexes (Sp) between enterococcal populations from different sample types.
CS US SA SS PD PF PS
CA
CS
US
SA
SS
PD
PF
0.15/nd/nd 0.29/nd/nd 0.24/nd/nd 0.31/nd/nd 0.16/nd/nd 0.12/nd/nd 0.25/nd/nd
– 0.21/0.21/nd 0.22/0.12/nd 0.26/0.25/nd 0.26/0.20/nd 0.26/0.20/nd 0.22/0.34/nd
– – 0.22/0.09/nd 0.32/0.18/nd 0.30/0.22/nd 0.29/0.12/0.04 0.42/0.30/0.31
– – – 0.34/0.11/nd 0.36/0.19/nd 0.26/0.46/nd 0.18/0.06/nd
– – – – 0.33/0.25/nd 0.32/0.28/nd 0.40/0.29/nd
– – – – – 0.27/0.25/nd 0.31/0.22/nd
– – – – – – 0.35/0.29/0.03
Values presented as ENT/ERE8/VRE20. ENT, enterococci on MEA; ERE8, erythromycin-resistant enterococci on MEAE8; VRE20, vancomycin-resistant enterococci on MEAV20; CA, crops not receiving pig slurry; CS, crops receiving pig slurry; US, urban sewage; SA, soil not receiving pig slurry; SS, soil receiving pig slurry; PD, pig feed; PF, pig faeces; PS, pig slurry; nd, not determined.
feed. The diversity of erythromycin-resistant isolates was again lower in pig faeces than in pig slurry (Table 1). Phenotypic similarities between enterococcal populations from different sample types The population similarity indexes (Sp) between enterococcal populations are presented in Table 2. The highest Sp value for ENT isolates was obtained comparing pig slurry with urban sewage water samples. A high similarity was obtained comparing VRE20 isolates from urban sewage and pig slurry, whereas the VRE20 isolates from pig faeces were very different (Table 2). A similar situation was observed for ERE8 isolates: there was more similarity between urban sewage and pig slurry than between pig faeces and pig slurry (Table 2). Species distribution Species distribution among ENT isolates showed some differences between the eight kinds of analysed samples. However, the same predominant species were always present: Enterococcus faecium, E. faecalis and E. hirae (Table 3). Different proportions of E. faecalis and E. faecium were observed between pig faeces and slurry samples (Table 3). Soil and crops that had received pig slurry showed a decrease in E. faecalis and an increase in other enterococcal species respect to pig slurry (Table 3). Distribution of different species among the VRE20 showed a high predominance of E. faecium (Table 3). A similar species distribution was obtained for ERE8 with respect to ENT isolates (Table 3), although higher percentage of E. faecalis and E. faecium was detected on crops receiving pig slurry. Discussion For infection control purposes, it is imperative to differentiate VRE from other intrinsically vancomycin-resistant enterococci presenting non-transferable low-level resis-
tance as E. casseliflavus, E. flavescens and E. gallinarum species [minimum inhibitory concentration (MIC) values of 4–16 mg l−1] (Quednau et al., 1998; Willey et al., 1999; Grohs et al., 2000). The isolation on MEA8 allowed the recovery of all the VRE facilitating the recovery of any stressed environmental strain. Later, isolates were selected by their capability to growth in 20 mg l−1. Thus, in this study only isolates growing in 20 mg l−1 vancomycin (VRE20) were considered as vancomycin-resistant enterococci. These species with intrinsically low-level resistance to vancomycin seldom cause infection and are rarely associated with transmission and hospital outbreaks (Madani et al., 1999; Ratanasuwan et al., 1999). However, E. gallinarum strains possessing transferable high-level vancomycin resistance have been described (Chen et al., 2000). Despite the rarity of these isolates, in our study two vancomycin-high-resistant isolates from pig slurry were classified to the E. casseliflavus, E. flavescens and E. gallinarum species group. Enterococci resistant to 20 mg l−1 vancomycin were detected in 13% of samples with enterococci present. They were mostly found when overnight enrichment was performed. These findings suggest that VRE20 occurred in very low numbers, indicating that they make up only a small part of the enterococcal populations in most samples. This fact was true especially in pig farm environment. Therefore, the use of enrichment procedures could be essential for detection of VRE20 in certain environmental samples. All the urban sewage samples, 16% and 34% of pig faeces and slurry samples, respectively, presented VRE20 isolates. These prevalences are still high considering that avoparcin has been banned in all European Union countries since 1997. These results obtained several years after their ban (no differences of VRE20 occurrence was observed over the investigation period) suggest that the use of avoparcin may not have been the exclusive source for the development of vancomycin resistance in animals and the environment. Alternatively, the extensive use of feed additives such as tylosin may have created co-linkage between the vancomycin resistance genes and resistance determi-
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 667–674
ENT, enterococci on MEA; ERE8, erythromycin-resistant enterococci on MEAE8; VRE20, vancomycin-resistant enterococci on MEAV20; PF, pig faeces; PS, pig slurry; PD, pig feed; CS, crops receiving pig slurry; SS, soil receiving pig slurry; CA, crops not receiving pig slurry; SA, soil not receiving pig slurry; US, urban sewage.
0 4 – – – – – 0 3 5 4 0 4 – 0 6 3 5 4 5 12 1 8 6 0 0 – – – – – 0 0 0 0 0 0 – 0 3 0 0 9 3 3 0 4 0 0 7 – – – – – 0 4 1 23 5 23 – 0 0 2 3 5 8 10 10 0 2 0 0 – – – – – 2 24 18 23 5 23 – 0 3 21 23 11 47 21 15 0 18 100 89 – – – – – 88 61 23 42 48 42 – 100 42 68 24 56 32 43 42 83 42 0 0 – – – – – 10 8 53 8 42 8 – 0 46 6 45 16 5 11 32 4 32 PF PS PD CS SS CA SA US
ERE8 ERE8 VRE20 ERE8 ENT VRE20 ERE8 ENT
ERE8
VRE20
ENT
ERE8
VRE20
ENT
E. hirae E. faecium E. faecalis
Table 3. Percentage of Enterococcus species distribution of ENT, ERE8 and VRE20 isolates.
E. cas-gal-flav. group
ENT
E. mundtii
VRE20
ENT
Other
VRE20
670 A. Manero, X. Vilanova, M. Cerdà-Cuéllar and A. R. Blanch nants to other antibiotics used as growth promoters (Aarestrup, 2000). Moreover, other authors suggest the existence of selection pressures other than antibiotic usage (Bahirathan et al., 1998; Klein et al., 1998). The high prevalence of VRE20 in samples from human origin (urban sewage) indicates a potential risk of nosocomial infections caused by these bacteria. Moreover, this high prevalence suggests the possible spread of VRE by contamination of comestible vegetables if inadequate disposal of slurries or treated sewage is performed as it has been proposed in previous studies (Robredo et al., 2000). Results of pig feed analysis were in agreement with other studies where it is observed that the origin of the vancomycin-resistant strains in pigs can not be due to feed contamination with enterococci (Robredo et al., 1999). A high predominance of E. faecium species was observed for VRE20 from both animal (pig faeces and slurries) and human origin (urban sewage). This fact is in agreement with other authors that described E. faecium as the predominant vancomycin-resistant species from non-hospitalized citizens, farmers, farm animals, meat products and from sewage treatment plants (Klare et al., 1993; 1995; Aarestrup, 1995; Borgen et al., 2000). The high Sp indexes obtained comparing VRE20 isolates from urban sewage and pig slurry suggests the possible circulation of certain strains through the food chain from farms to humans. This observation is in line with molecular observations by others authors that report identical genotypes in VRE strains from humans and pigs (Jensen, 1998; Willems et al., 2000). Vancomycin-resistant enterococci isolates of this study were included in an international comparative study on vancomycin resistance spreading (Kühn et al., 2005). In this study, MIC values and molecular screening for van genes was performed. It was suggested that the spread of the vanA gene cluster from animal VRE to human strains may contribute to the dissemination of VRE, although clonal spread of VRE between animals and humans could not be demonstrated. It was observed that when the same PhP type was found in a human and an animal isolate no known epidemiological relations existed. So, the relation between animal and human VRE is rather due to spread of the vanA gene cluster between different enterococcal strains as it has already been reported in previous studies (Willems et al., 1999; Klare et al., 2003). Pig slurry and soil fertilized with pig slurry showed an increment of diversity with respect to pig faeces. In soil receiving pig slurry this fact can be explained by the presence of ubiquitous enterococci species as E. mundtii (Table 3) that can be found free-living in soil or on plants (it was also observed in pig feed and soil and crops not receiving pig slurry). However, the increment of diversity in pig slurry compared with pig faeces is surprising. It was expected that certain enterococci would survive better
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 667–674
Antibiotic-resistant enterococci 671 outside the intestine, and this should have resulted in a decreased diversity in these types of samples. This increment of diversity, the different proportion of E. faecalis and E. faecium species between pig faeces and pig slurry, and the low similarity between VRE20 from pig faeces and pig slurry indicate important ecological changes during the accumulation of slurry. The high similarity populations obtained when comparing VRE20 from human origin (urban sewage) with those of pig slurry suggests that the use of pig slurry as a fertilizer without any pre-treatment could act as possible vehicle for VRE20 spreading. However, we were unable to detect VRE20 from soil and crops receiving pig slurry. The European Union has banned the use of tylosin as a growth promoter since 1999. Erythromycin resistance in enterococci is related to the use of tylosin as described above. A 100% prevalence of ERE was obtained in samples from pig faeces, pig slurry and urban sewage. Significant prevalences were also obtained in samples from pig feed and from soil and crops receiving pig slurry as fertilizer. The proportion of ERE8 isolates per sample was very high (around 100%) in pig slurry and crops receiving pig slurry, and around 10% in other samples related with pig production (pig feed and soil receiving pig slurry) as well as in sewage (data from pig faeces not analysed). However, very low prevalences and charges of ERE8 isolates were obtained in soil and crop samples not receiving pig slurry. Thus, the presence of high proportions of ERE8 isolates in the environment could indicate a possible use of pig slurry in farmland as a natural fertilizer. The high diversity indexes of ERE8 and their similar species distribution obtained with respect to the total enterococci suggests that erythromycin resistance is present in a wide variety of clones and species of enterococci such as pigs as humans. The high similarity between ERE8 isolates from samples of the pig production environment and human sewage also suggests the strains circulation through the food chain from farms to humans. Despite the similarity indexes between both ERE8 and VRE20 isolates from human and pig origin suggests this circulation, a common source and their simply presence in both environments can not be discarded.
Experimental procedures Samples and sampling treatment Three different kinds of samples were collected from a pig farm in the northeast of Spain: faeces, slurry and pig feed. Samples from soil and crops destined for human consumption that receive pig slurry from this farm as a natural fertilizer were also collected. For comparison, samples from soil and crops receiving only artificial fertilizers were also taken. Additionally, sewage samples were collected from a water treat-
ment plant receiving human sewage from an urban zone. The sampled pig farm and water treatment plant are located on Barcelona province, northeast of Spain. Soil surface samples were collected from four areas of one square metre each with a distance of at least 10 m between them. In each sampling square, five subsamples were collected and pooled together in a plastic bag and carefully mixed. Crop samples were collected from the same field as soil samples. A crop sample was defined as five plants from a square metre of field. The plants were pooled together and homogenized. Pig feed samples were collected from the feeding boxes. Fresh faecal samples were collected from the floors of the pig litters. Pig slurry from a collecting tank and raw urban sewage from the ingress of the treatment plant were collected according to standard methods (APHA, 1998). These strains were isolated and maintained on MEA medium (Difco, Detroit, USA). Environmental samples were taken in sterile bottles and stored at 4°C during transportation. They were then processed within 2 h.
Isolation and enumeration of enterococci Aliquots of 20 g of samples from soil, crop and pig feed were transferred to a flask and diluted with 180 ml of phosphatebuffered saline pH 7.4 (PBS). Samples were stirred at room temperature for 20 min, and settling was allowed for 5 min. Pig faeces (1 g) and pig slurry samples (1 ml) were diluted 1/10 with PBS and vortexed. Sewage samples were treated according to the standard methods (APHA, 1998). Samples with presumed high counts of enterococci were subject to serial dilutions in PBS. Enterococci were counted by membrane filtration using MEA (Difco), with a previous 2 h preincubation of membranes at 37°C on Brain–Heart Infusion Agar (BHIA) (Difco) (APHA, 1998) for sewage, soil, crop, pig feed and pig slurry samples. For faeces samples 10 µl of the dilution in PBS was streaked on MEA plates. Inoculated plates were incubated at 37°C for 48 h. Enterococci were confirmed by transferring the membrane (by peaking for faeces isolates) on to BEA (Difco) and incubating at 45°C for 1 h (Anonymous, 1984; Figueras et al., 1998). Dilutions of sewage, soil, crop, pig feed and pig slurry samples were also cultivated by membrane filtration on MEA with a supplement of 8 mg l−1 vancomycin (MEAV8) and on MEA supplemented with 8 mg l−1 erythromycin (MEAE8). Isolates from MEAV8 plates were also tested for resistance to 20 mg l−1 by streaking on MEAV20 plates (MEA with 20 mg l−1 vancomycin). Pig faeces isolates were tested for vancomycin resistance to 8 mg l−1 and 20 mg l−1 and for erythromycin resistance to 8 mg l−1 by streaking these isolates on MEAV8, MEAV20 and MEAE8 respectively. In order to detect low numbers of VRE, all samples were also grown in Enterococcosel broth enrichment medium (Beckton and Dickinson) supplemented with 8 mg l−1 vancomycin (EBV8). The homogenized and diluted samples (10 ml) were mixed with equal volumes of 2× EBV8. After incubation for 24 h at 37°C, 10 µl of the enrichment was streaked on MEAV8. Plates were incubated for 48 h at 37°C. Later, isolates were also tested for resistance to 20 mg l−1 by streaking on MEAV20 plates.
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 667–674
672 A. Manero, X. Vilanova, M. Cerdà-Cuéllar and A. R. Blanch Biochemical fingerprinting
Species identification
To measure the bacterial diversity, 24 colonies of each sample of sewage, soil, crop, pig feed and pig slurry were isolated from MEA plates as previously described (Bianchi and Bianchi, 1982). Because eight faecal samples were collected from litters of the same unit at the farm, eight colonies were picked from each of these samples. Additionally, colonies growing on MEAV8 and MEAE8 plates were also isolated for biochemical fingerprinting. Pure cultures were obtained on BHIA by incubation overnight at 37°C. Isolates were phenotyped using the PhP-RF microplates of the Phene-Plate System (PhPlate Microplates Techniques AB, Sweden), following the manufacturer’s instructions and previous studies (Kühn et al., 1991). Biochemical fingerprinting is based on the kinetics of several discriminating biochemical reactions, performed in microtitre plates. The Phene-Plate System for enterococci consists of 96-well microplates with dehydrated substrates. Tests used in the PhP-RF plates are acidification from Larabinose, lactose, melibiose, melezitose, raffinose, inositol, sorbitol, mannose, D-galacturonic acid gamma lactone, amygdaline and gluconate. To obtain a biochemical fingerprint for each isolate, the rate of each acidification reaction is measured by the absorbance reading at 620 nm. Several readings during the incubation period at 37°C are used to evaluate the kinetics for the fermentation of each substrate. Absorbance is measured at 16 h, 40 h and 64 h after inoculation. The average of absorbance values for each well provides the biochemical PhP phenotype for each strain (Kühn et al., 1991). The Di in each sample was calculated from Simpson’s index (Hunter and Gaston, 1988; Kühn et al., 1991). The Di measures the distribution of isolates into different phenotypic types. It is high (maximum value 1) if the isolates are distributed evenly into many different types, and is low (minimum value 0) if certain types are of the dominant bacterial population. The structure and composition of enterococcal populations were analysed for each sample by cluster analysis on the basis of the biochemical PhP phenotypes of the isolates using the unweighted pair group method analysis (UPGMA). The Sp, a similarity coefficient that measures the proportion of isolates that are identical in two compared samples (Kühn et al., 1991), were calculated. On the basis of the results of the biochemical tests, isolates for each sampling site were pooled to determine the main biochemical PhP phenotypes (main clusters) of bacterial populations. Clusters were defined by isolates showing a similarity index equal to or higher than 0.975. Isolates showing the highest minimum and the highest mean similarity to all other isolates belonging to the same PhP phenotype were selected as representative strains (Kühn et al., 1991). It has been demonstrated that phenotyping of enterococci using the Phene-Plate System is providing identical clustering results than DNA fingerprinting using pulsed field gel electrophoresis (PFGE) (Kühn et al., 1995; Iversen et al., 2002; 2004; Torell et al., 2003). It supports the use of this phenotyping method for characterization of enterococci populations in this study. Additionally, all isolated VRE strains were supplied for further MIC characterization and molecular screening of van genes for epidemiological analyses in an international study on vancomycin resistance spreading (Kühn et al., 2005).
Representative strains were identified at the species level following the procedures and the biochemical key for Enterococcus spp., as previously described (Manero and Blanch, 1999). The PhP biochemical fingerprints of these identified representative strains were used to make up a reference database relating PhP phenotypes with Enterococcus species. The PhP phenotypes of all isolates subjected to biochemical fingerprinting were then compared with those on the database, and a presumed species identification was obtained for each isolate based on a correlation coefficient higher than 0.90. Statistical analyses were performed using the software PhPWin® (PhPlate Microplates Techniques AB, Sweden). Enterococcus gallinarum, E. casseliflavus and E. flavescens were treated as a group following the suggestions by other authors that they comprise in a single group (Dutka-Malen et al., 1995; Descheemaeker et al., 1997; Teixeira et al., 1997; Patel et al., 1998; Quednau et al., 1998; Baele et al., 2000).
Acknowledgements This research was supported by the European Project FAIR5CT97-3709 and by the Project 2001SGR00099 from the Catalan Government. A. Manero received a fellowship from the Ministerio de Educación y Cultura of the Spanish Government (AP97 44007540).
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