Enterococci Isolated from Japanese Quails Exposed to Microgravity Conditions and Stability of their Properties

Enterococci isolated from the crop and caecum of Japanese quails exposed to 7 day conditions of microgravity were re-vitalized after their dry-freezing long storage. Originally, the strains were isolated from Japanese quails after their landing from flight onboard the orbital station Mir during the experiment in August 1990. Because taxonomy as well as the studies concerning the bacteriocins, especially those produced by enterococci, have been continually developed for years, the aim of this study was to confirm species identification, stability of the properties of enterococci as well as to test new properties after their long storage. Genotyping allotted the strains to the species E. faecium. Lactic acid production was detected in similar amounts in the strains before and after their long-storage in dry-frozen form. The strains were vancomycinsensitive and kanamycin-resistant before as well as after their long-time storage. Variability in sensitivity to different antibiotics was found among the strains tested even before and after longtime storage. Each of the strains possessed at least one structural enterocin gene. The structural genes for enterocin A, P, B, L50B were detected in E. faecium EP7. E. faecium EP2, EEP4 have the genes for ent A, B, L50B. The gene for ent P was detected only in the strain EP7. The most often detected was ent A gene followed by ent genes B, L50B. All strains inhibited growth of at least 4 out of 15 indicators. The stability of the enterococcal properties determined before as well as after their dry-freezing was not influenced during their long-term storage; moreover, new properties were determined. Enterococci, storage, enterocin gene, properties, stability Japanese quails represent suitable animal laboratory model for several reasons: their small size, low husbandry costs, short generation interval and adaptability to a wide range of husbandry conditions. Therefore, in past they were an object of the scientific space research programme (Boďa 1979) called Interkozmos that was finished in 1990s. In this programme many research issues were investigated and solved; e.g. the effect of microgravity on endocrine functions and adaptation processes (Juráni et al. 1988), embryonic development (Boďa et al. 1992) and/or morphological changes in the small intestine under space-flight conditions (Cigánková et al. 2000). Moreover, in the framework of the Interkozmos programme-Incubator 2, firstly also microflora of Japanese quails exposed to microgravity conditions was analysed in comparison to conventional Japanese quails with an impact on lactic acid bacteria such as lactobacilli, enterococci and staphylococci (Lauková et al. 1991, 1993a, 1995). The most studied were enterococci that were also found to produce antimicrobial substances bacteriocins (Lauková et al. 1993bc). The studied isolates were dry-frozen and stored for a long period for other tests. Because taxonomy as well as the studies concerning bacteriocins, especially those produced by enterococci, were continually widely developed for years and enriched with new knowledge (Aymerich et al. 1996; Casaus et al. 1997; Cintas et al. 1998, 2000; De Vuyst et al. 2003; Mareková et al. 2007; Franz et al. 2007), the aim of this study was to confirm the species identification, stability and properties of enterococci after their long-time storage as well as to genotype them and to test them for the previously not determined properties. ACTA VET. BRNO 2009, 78: 253–258; doi:10.2754/avb200978020253 Address for correspondence: MVDr. Andrea Lauková, PhD. IAP SAS, Šoltésovej 4-6 040 01 Košice Slovakia Phone:+421-55-633 0283, 7922 964 Fax:+421-55-7287842, E-mail:laukova@saske.sk http://www.vfu.cz/acta-vet/actavet.htm Materials and Methods Six strains of enterococci isolated from the crop (EEP4, EP2) and caecum (EFP7, EP7, EFP4, EFP5) of Japanese quails exposed to 7 days of microgravity conditions were re-vitalized after their long-time dry-freezing storage (18 years). Originally, the strains were isolated from Japanese quails after their flight onboard the orbital station Mir during the experiment carried out in August 1990. The samples were treated as described previously (Lauková et al. 1991). Dry-frozen strains were cultivated in the Brian Heart Infusion (Becton & Dickinson, Cockeysville, USA) for 18 h at 37 oC. Then they were plated onto M-Enterococcus agar as well as Brian Heart agar supplemented with defibrinated sheep blood to check their purity. In the past, the strains were phenotyped by the API 20 Strep identification kit (BioMeriux, l’Etoile, France) and analysed to produce lactic acid (Pryce 1969) have bacteriocin-like activity (Skalka et al. 1983) and tested for sensitivity/resistance to antibiotics (NCCLS 2002). After their re-vitalization, they were genotyped using primer sequences and PCR method (Woodford et al. 1997) to confirm their taxonomy. Beside the above-mentioned tests, they were tested for possession of structural genes for enterocin production, and their antimicrobial activity was tested against additional indicator organisms (Table 2). To detect the structural genes for enterocin production, 2 μl of template was added to 8.75 μl of the reagent mixture which contained 0.5 μl each of the primers, 1 μl of (10 mM) dNTPs, 1.5 μl of (5 mM) MgCl2, 5 μl of 10 × reaction buffer, 0.25 μl of 1 U Taq polymerase (Invitrogen) and water to the total volume 50 μl. The sequences of the primer pairs used for PCR-amplification of the structural enterocin genes (Ent) A, P, L50B, and B are listed in Table 1. The reaction conditions for EntA detection included 5 min denaturation at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 58° C, 30 s at 72 °C; then by 5 min at 72 °C and a cool down to 4 °C. For EntP, L50B and B, the temperature of 56 °C instead of 58 °C was used as the annealing temperature. PCR products were visualised by 2% agarose electrophoresis, containing 1 μg of ethidium bromide. Positive control strains for PCR reactions were as follows: E. faecium EK13 (CCM 7419) strain for EntA (Mareková et al. 2003), E. faecium AL41 strain for EntP (Lauková et al. 2003), E. faecium L50 strain for EntL50B and B (Cintas et al. 1998; Casaus et al. 1997). DNA extraction from each strain was obtained by a simple procedure: 1 loopful of bacterial colony (10 μl) was resuspended in 30 μl of sterile injection water and vortexed for 10 min. Then the supernatant aliquots were used as template DNA. The antimicrobial activity of the strains was originally tested for 4 indicators such as Streptococcus bovis Sb 36 (Dr. Jonecová, Faculty of Medicine, University of PJ Šafárik, Košice, Slovakia); Enterococcus faecium EF26/42 own isolate (from rumen contents), Staphylococcus aureus CB40 (University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic), Corynebacterium renale CCM 5740 (Czech Culture Collection of Microorganisms in Brno, Czech Republic). Additionally, the following 11 indicator strains were used: E. avium EA5, L. innocua LMG 13568 (Collection of Microorganisms in Ghent, Belgium), E. faecium H14a, Enterococcus sp. H2, H4, LT2, H12, H14b, Staphyloccocus sp. SH14, SH9 (isolates from chickens, Salmonella enterica serovar Enteritidis PT4 (Dr. Šišák, Brno, Czech Republic). Activity was confirmed by the occurrence of inhibitory zone around indicator organisms.


Materials and Methods
Six strains of enterococci isolated from the crop (EEP4, EP2) and caecum (EFP7, EP7, EFP4, EFP5) of Japanese quails exposed to 7 days of microgravity conditions were re-vitalized after their long-time dry-freezing storage (18 years).Originally, the strains were isolated from Japanese quails after their flight onboard the orbital station Mir during the experiment carried out in August 1990.The samples were treated as described previously (Lauková et al. 1991).Dry-frozen strains were cultivated in the Brian Heart Infusion (Becton & Dickinson, Cockeysville, USA) for 18 h at 37 ºC.Then they were plated onto M-Enterococcus agar as well as Brian Heart agar supplemented with defibrinated sheep blood to check their purity.In the past, the strains were phenotyped by the API 20 Strep identification kit (BioMeriux, l'Etoile, France) and analysed to produce lactic acid (Pryce 1969) have bacteriocin-like activity (Skalka et al. 1983) and tested for sensitivity/resistance to antibiotics (NCCLS 2002).After their re-vitalization, they were genotyped using primer sequences and PCR method (Woodford et al. 1997) to confirm their taxonomy.Beside the above-mentioned tests, they were tested for possession of structural genes for enterocin production, and their antimicrobial activity was tested against additional indicator organisms (Table 2).
To detect the structural genes for enterocin production, 2 μl of template was added to 8.75 μl of the reagent mixture which contained 0.5 μl each of the primers, 1 μl of (10 mM) dNTPs, 1.5 μl of (5 mM) MgCl 2, 5 μl of 10 × reaction buffer, 0.25 μl of 1 U Taq polymerase (Invitrogen) and water to the total volume 50 μl.The sequences of the primer pairs used for PCR-amplification of the structural enterocin genes (Ent) A, P, L50B, and B are listed in Table 1.The reaction conditions for EntA detection included 5 min denaturation at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 58° C, 30 s at 72 °C; then by 5 min at 72 °C and a cool down to 4 °C.For EntP, L50B and B, the temperature of 56 °C instead of 58 °C was used as the annealing temperature.PCR products were visualised by 2% agarose electrophoresis, containing 1 μg of ethidium bromide.Positive control strains for PCR reactions were as follows: E. faecium EK13 (CCM 7419) strain for EntA (Mareková et al. 2003), E. faecium AL41 strain for EntP (Lauková et al. 2003), E. faecium L50 strain for EntL50B and B (Cintas et al. 1998;Casaus et al. 1997).DNA extraction from each strain was obtained by a simple procedure: 1 loopful of bacterial colony (10 μl) was resuspended in 30 μl of sterile injection water and vortexed for 10 min.Then the supernatant aliquots were used as template DNA.
Lactic acid production was detected in the same or very similar amounts in the strains before as well as after dry-freezing in the range values from 0.54 ± 0.07 up to 2.0 ± 0.10 mmo/l (Table 2).The strains were vancomycin-sensitive and kanamycin-resistant before as well as after dry-freezing (Table 2).Variability in sensitivity to gentamicin, tetracycline, streptomycin, ampicillin, rifampicin, penicillin and chloramphenicol was found among the strains tested even when sensitivity or resistance were compared before and after dry-freezing.The strains were also sensitive to erythromycin; only EEP4 strain was originally resistant, and it was found to be polyresistant.
Each of the six strains possesses at least one structural enterocin gene among those tested (Table 3).The structural genes for ent A, P, B and L50B production was detected in E. faecium EP7 strain.E. faecium EP2 and EEP4 have the genes for ent A, B, L50B production.The gene for ent P was detected only in the strain EP7.The most detected was ent A gene (in 5 isolates) followed by the gene for ent B production (in 4 isolates) and L50B (in 3 isolates).

Table 1 .
The sequences of the primer pairs used for PCR-amplification of the structural enterocin genes