Scholarly article on topic 'Enterococci Isolated from Japanese Quails Exposed to Microgravity Conditions and Stability of their Properties'

Enterococci Isolated from Japanese Quails Exposed to Microgravity Conditions and Stability of their Properties Academic research paper on "Biological sciences"

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Academic research paper on topic "Enterococci Isolated from Japanese Quails Exposed to Microgravity Conditions and Stability of their Properties"

ACTA VET. BRNO 2009, 78: 253-258; doi:10.2754/avb200978020253

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 vancomycin-sensitive 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 (Bod'a 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 (Jurani et al. 1988), embryonic development (Bod'a et al. 1992) and/or morphological changes in the small intestine under space-flight conditions (Cigankova 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 (Laukova et al. 1991, 1993a, 1995). The most studied were enterococci that were also found to produce antimicrobial substances - bacteriocins (Laukova 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; Marekova 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.

Andrea Laukova, Gabriela Michlovicova

Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovakia

Received July 1, 2008 Accepted February 9, 2009


Address for correspondence:

MVDr. Andrea Laukova, PhD. IAP SAS, Soltesovej 4-6 040 01 Kosice Slovakia

Phone:+421-55-633 0283, 7922 964 Fax:+421-55-7287842,

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 (Laukova et al. 1991). Dry-frozen strains were cultivated in the Brian Heart Infusion (Becton & Dickinson, Cockeysville, USA) for 18 h at 37 °C. 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 of template was added to 8.75 of the reagent mixture which contained 0.5 each of the primers, 1 |il of (10 mM) dNTPs, 1.5 |il of (5 mM) MgCl2 5 |il of 10 x reaction buffer, 0.25 of 1 U Taq polymerase (Invitrogen) and water to the total volume 50 |il. 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 ^g of ethidium bromide. Positive control strains for PCR reactions were as follows: E. faecium EK13 (CCM 7419) strain for EntA (Marekova et al. 2003), E. faecium AL41 strain for EntP (Laukova 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 ^1) was resuspended in 30 of sterile injection water and vortexed for 10 min. Then the supernatant aliquots were used as template DNA.

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


R 5' - CCC TGG AAT TGC TCC ACC TAA - 3' (Aymerichetal. 1996)


R 5' -TCC TGC AAT ATT CTC TTT AGC - 3' (Cintas et al. 1997)


R 5' - TAG CCA TTT TTC AAT TTG ATC - 3' (Cintas et al. 1998)


R 5' -AGA GTA TAC ATT TGC TAA CCC - 3' (Casaus et al. 1997)

The antimicrobial activity of the strains was originally tested for 4 indicators such as Streptococcus bovis -

Sb 36 (Dr. Jonecova, Faculty of Medicine, University of PJ Safarik, Kosice, 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 H Enterococcus sp. H2, H4, LT2, H12, H , Staphyloccocus sp. SH14, SH9 (isolates from chickens, Salmonella enterica serovar Enteritidis PT4 (Dr. Sisak, Brno, Czech Republic). Activity was confirmed by the occurrence of inhibitory zone around indicator organisms.


Before dry-freezing the tested strains were allotted by phenotyping to the species

Enterococcus casseliflavus (EC7/EFP7), Enterococcus sp. EP2, EFP5, E. avium EP7 and E. faecium, EEP4, EFP4. However, genotyping using PCR method allotted all six strains to the species E. faecium.

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/1 (Table 2).

Table 2. Lactic acid production and antibiotic sensitivity or resistance of enterococci, isolated from Japanese quails under microgravity conditions before and after 18 years dry-freezing storage

Strains LA Van Kan Gen Tct Stm Ery Rif Amp Ch Pn

EC71 0.90 ± 0.05 S R R R R S S R nd R

EFP7* 0.93 ± 0.01 S R S S S S S S S S

EP72 0.93 ± 0.0 s R R S R S R S S R

EP7* 0.93 ± 0.02 s R R S R s R S s R

EFP43 nd s R R R S s R R R nd

EFP4* 0.86 ± 0.0 s R S S S s S S S S

EFP5" 0.54 ± 0.07 s R S s S s S s s S

EFP5* 1.05 ± 0.02 s R S s s s s s s S

EP25 0.98 ± 0.05 s R s s s s s s s s

EP2* 1.24 ± 0.02 s R s s s s s s s s

EEP46 2.00 ± 0.10 s R R R R R R R R R

EEP4* 1.50 ± 0.07 s R R R R S R S S S

LA-lactic acid expressed in mmol/1; Van-vancomycin (30 ^g); Kan-kanamycin (30 ^g); Gen-gentamicin (10 ^g); Tct-tetracycline (30 ^g); Stm-streptomycine (30 ^g); Ery-erythromycin (15 ^g); Rif-rifampicin (30 ^g); Amp-ampicillin (10 ^g); Ch-chloramphenicol (30 ^g); Pn-penicilline (10 IU); S-sensitive; R-resistant; nd-not determined; 1-6Enterococcus faecium-beiore dry-freezing; *the strains after re-vitalizing after 18 years

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.

Table 3. The presence of structural genes for enterocins P, A, B, L50B and inhibitory activity of tested enterococci against the additional indicator microorganisms

Strains Ent P A B L50B Sb1 Sa2 EF3 EA54 Hl25 Li6 Cr7

EFP7 - - + - + + + - + - +

EP7 + + + + + + + - + - -

EFP4 - + - - + + + - - - +

EFP5 - + - - nd nd nd - - - -

EP2 - + + + + + nd - + - +

EEP4 - + + + + + nd - + - +

+: the presence of gene or inhibition of the indicator organism; -: absence of the structural gene or inhibition of the indicator; 1Sb-Streptococcus bovis 36 (Dr. Jonecova, Faculty of Medicine, University of PJ Safarik, Kosice, Slovakia); 2Staphylococcus aureus CB40-Czech Culture Collection of Microorganisms, CCM, Brno; 3Enterococcus faecium EF26/42, 4E. avium EA5 (Dr. Laukova, IAP SAS, Slovakia);5Enterococcus sp. H12-(Dr. Michlovicova, IAP SAS); 6L. innocua LMG 13568-Ghent, Belgium; Cf-Corynebacterium renale CCM 5740; Enterococcus sp. H2, H4, LT2, H14b, E. faecium H14a Staphylococcus sp. SH14, SH9 (Dr. Michlovicova, IAP SAS, Kosice) S. enterica serovar Enteritidis PT4 were not inhibited by the tested strains; besides E. faecium EEP4 strain which inhibited the strains indicated in the table as well as enterococci and staphylococci from chickens (together 13 strains among 15 tested).

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).

All strains inhibited the growth of at least 4 out of 15 indicators (Table 3). The principal (most sensitive) indicators generally used in our study, E. avium EA5 strain as well as

L. innocua LMG 13568, were resistant to the strains tested. The growth of S. enterica PT4 was not inhibited by the substances produced by the tested enterococci from Japanese quails. The growth of S. aureus CB40 as well as C. renale CCM 5740 was inhibited (before dry-freezing).


It seems (also from our previous studies) that the phenotyping of E. faecium is in harmony with its genotyping (unpublished data). However, there were differences found in phenotyping and genotyping of the species E. casseliflavus or E. avium (Kirschner et al. 2001). E. faecium is a frequently determined species from the genus Enterococcus among poultry faecal samples (Strompfová et al. 2003, 2007; Tejedor-Junco et al. 2005; Lauková et al. 2008). There are suggestions that especially plasmid-encoded properties such as antibiotic resistance or bacteriocin production could be missing or changed by subsequent passaging of the strains when they are re-vitalized after storage (Lauková and Kuncová 1991). Under our conditions, unchanged LA amounts were detected in enterococci after their long-term storage. This stability could be probably supported by the chromosomal encoding of LA as has been found e.g. for rifampicin resistance in enterococcci (Lauková and Kuncová 1991; Rice et al. 1991). Detection of the structural enterocin genes by PCR is a method that was not available years ago. Therefore it was interesting to determine it when the strains tested were found to produce an antimicrobial substance. There are differences in the ent genes occurrence among E. faecium species isolated from different ecosystems. Strompfová and Lauková (2007) reported the presence of only ent A and P structural genes among 5 bacteriocin producing E. faecium strains isolated from the caecum, ileum and crop of chickens. Moreover, contrary to the results presented here, ent P gene was the most frequently occurring gene among 234 enterococcal strains from different sources (Strompfová et al. 2008). Among rabbit enterococci, ent A, P, L50B genes were determined but not ent B (Simonová and Lauková 2007). On the other hand, ent B gene was found in enterococci isolated from horses (Lauková et al. 2008). Taking into account bacteriocin production and structural genes detection, EEP4 strain was the most active; although it could be probably discussed that it produces one among enterocins tested and/or a new substance; it will be researched in more detail in our further studies. Interestingly, S. aurues CB40 as well as C. renale CCM 5740 were mostly inhibited before dry-freezing. That is, bacteriocin production as well as antibiotic resistance are plasmid-encoded properties which as formerly mentioned could be lost by subsequent manipulation (Lauková and Kuncová 1991). In the case of our strains this phenomenon could be hypothetically supposed, although in preliminary studies no plasmids were determined (unpublished data).

Changes in antibiotic sensitivity or resistance in the strains after strain re-vitalization could be explained by different ways; e.g. by the molecular way - the structural gene transport (Lauková et al. 1990), or pragmatically by better quality of modern disks. It is also interesting that polyresistant EEP4 strain showed the best antimicrobial activity; it could be due to bacteriocin but also due to LA production (the highest value among 6 strains tested). The knowledge on bacteriocin production among the isolates presented give us opportunity to search for the probable new substance, to spread information in association with space research as well as to select the strain for other e.g. probiotic testing. It can be concluded that the tested enterococci maintained stability of the properties determined before as well as after their long-term storage by dry-freezing; moreover, additional properties were determined.

Enterokoky z japonskych prepelíc vystavenych podmienkam mikrogravitácie

a stabilita ich vlastností

Enterokoky izolované z hrvol'a a céka japonskych prepelíc (vystavenych po dobu 7 dni podmienkam mikrogravitácie) boli lyofilizované pre dlhodobé uskladnenie. Kmene boli

izolované z japonskych prepelíc po ich návrate z letu na palube orbitálnej stanice Mir pocas experimenta v auguste roku 1991v rámci vyskumného vesmirneho programu Interkozmos-Inkubátor 2. Pretoze taxonomía ako aj poznatky o bakteriocinoch, najmä o tych, ktoré sú produkované enterokokmi sa rokmi vyvijali, ciel'om predkladanej práce bolo geno-typizáciou overit' taxonomickú príslusnost' uskladnenych kmeüov, prehodnotit' ich stabilitu a vlastnosti po dlhodobom uskladneni ako aj zistit' u nich prítomnost' génov pre produkciu niektorych doposial' známych enterocínov. Vyselektovanych 6 kmeüov bolo genotypizáciou priradenych к druhu Enterococcus faecium. Intenzita produkcie kyseliny mliecnej ostala nezmenená po dlhodobom skladovaní testovanych kmeüov. Enterokoky boli citlivé na vankomycin a rezistentné na kanamycín. Variabilita v citlivosti ci rezisten-cii к testovanym antibiotikám bola zistená pred i po ich skladovaní lyofilizáciou. Kazdy testovany kmeü obsahoval aspoü jeden strukturálny gén pre produkciu testovaného enterocínu. Strukturálne gény pre produkciu enterocínov A, P, B, L50B boli detegované u kmeüa E. faecium EP7. Kmene EP2, EEP4 obsahovali gény pre enterocíny A, B, L50B. Gén pre produkciu enterocínu P bol zisteny len u kmeüa EP7. Najcastejsie bol detegovany ent A gén, potom gény pre produkciu enterocínov В a L50B. Vsetky kmene inhibovali rast najmenej 4 z 15 indikátorovych mikroorganizmov. Enterokoky si zachovali svoje vlastnosti i po dlhodobom skladovaní v lyofilizovanej forme. Naviac, boli u nich detegované i d'alsie vlastnosti.


This study is dedicated to the memory of Professor Koloman Bod'a who founded the Institute of Animal Physiology in Kosice and who worked with great enthusiasm for many years in the space scientific programme Interkozmos. We also thank Mrs. Margita Bodnárová for her excellent technical assistance. A part of the study was supported by the Vega Project 2/0008/08.


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