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International Journal of Food Microbiology 81 (2002) 1 – 10 Identification and antibiotic susceptibility of bacterial isolates R. Temmermana,*, B. Pot a, G. Huys a, J. Swings a,b aLaboratorium voor Microbiologie, Universiteit Gent, K.L. Ledeganckstr. 35, B-9000 Ghent, Belgium bBCCMTM/LMG Bacteria Collection, K.L. Ledeganckstr. 35, B-9000 Ghent, Belgium Received 20 May 2001; received in revised form 27 December 2001; accepted 16 April 2002 In the present study, a total of 55 European probiotic products were evaluated with regard to the identity and the antibiotic resistance of the bacterial isolates recovered from these products. Bacterial isolation from 30 dried food supplements and 25dairy products, yielded a total of 268 bacterial isolates selected from several selective media. Counts of food supplementsshowed bacterial recovery in 19 (63%) of the dried food supplements ranging from 103 to 106 CFU/g, whereas all dairyproducts yielded growth in the range of 105 – 109 CFU/ml. After identification of the isolates using whole-cell protein profiling,mislabeling was noted in 47% of the food supplements and 40% of the dairy products. In six food supplements, Enterococcusfaecium was isolated whereas only two of those products claim this species on their label. Using the disc diffusion method,antibiotic resistance among 187 isolates was detected against kanamycin (79% of the isolates), vancomycin (65%), tetracycline(26%), penicillinG (23%), erythromycin (16%) and chloramphenicol (11%). Overall, 68.4% of the isolates showed resistanceagainst multiple antibiotics including intrinsic resistances. Initially, 38% of the isolated enterococci was classified asvancomycin resistant using the disc diffusion method, whereas additional broth dilution and PCR assays clearly showed that allE. faecium isolates were in fact vancomycin susceptible.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Probiotics; Label correctness; Identification; Antibiotic susceptibility testing promoting properties of probiotic lactic acid bacteria(LAB) as reviewed by including The past 5 years have witnessed a strong expansion stabilisation of the intestinal microflora by competi- of the probiotic market and, in parallel, a rise in the number of research projects addressing fundamental of lactose intolerance (de Vrese et al., 2001), preven- and applied aspects of probiotics. New research tech- nologies have supported earlier suggestions of health prevention of colon cancer and stimulation of the immune system (Isolauri et al.,2001). Bringing a probiotic to the market involves a * Corresponding author. Tel.: +32-9-264-52-49; fax: 32-9-264- step-wise process that needs to be carefully monitored in order to obtain a correctly labeled, functional, and E-mail address: 0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 1 6 2 - 9 R. Temmerman et al. / International Journal of Food Microbiology 81 (2002) 1–10 Saarela et al., 2000). If a product is not labeled expiry date. All products were examined using a set correctly, safety and functionality cannot be guaran- of four isolation media under standardized cultivation teed due to lack of documentation of the product conditions. For the isolation of Lactobacillus and components. However, as many manufacturers rely Lactococcus strains, De Man Rogosa and Sharpe Agar on the widely acknowledged but occasionally debated (MRSA) medium (CM361, Oxoid, Basingstoke, UK) GRAS (‘generally regarded as safe’) status of lacto- was used, whereas streptococci and enterococci were isolated on M17 medium (CM785, Oxoid) and on characterization of probiotic LAB strains with regard Kanamycine Aesculine Azide Agar Base (KAAAB) to taxonomic status, antibiotic resistance, and viru- (CM591, Oxoid), respectively. For the isolation of bifidobacteria, Transgalacto-Oligosaccharides (TOS) Microbial analyses of probiotic dairy products have demonstrated that the identity and the number following composition: 10 g Trypticase Soy Broth of recovered species do not always correspond to the (81-1768-0, Becton Dickinson, Sparks, USA), 1 g Yeast Extract (L21, Oxoid), 3 g KH2PO4 (1627, Vel, Holzapfel et al., 1998; Hamilton-Miller et al., 1999).
Leuven, Belgium), 4.8 g K2HPO4 (1628, Vel), 3 g However, it should be noted, that each of the cited (NH4)2SO4 (1.01217.1000, Merck, Darmstadt, Ger- studies was rather limited in number and type of many), 0.2 g MgSO4Á7 H2O (1433, Vel), 0.5 g L- products or was mainly restricted to national products.
cystein hydrochloride (C4820, Sigma, Bornem, Bel- Various opinions exist as to whether it might be gium), 15 g Na-propionate (P1880, Sigma), 10 g desirable that some probiotic strains show resistance Transgalacto-OligoSaccharides (TOS, Honsha, to specific antibiotics that are, for instance, involved Tokyo, Japan) and 15 g agar (L11, Oxoid) dissolved in 1000 ml of distilled water. Products were sampled On the other hand, the commercial introduction of by preparing 10-fold dilutions of 100 Al of the dairy probiotics containing antibiotic resistant strains may products or 100 mg of the food supplements in 10 ml also have negative consequences, for example, when pepton-physiological solution [PPS, 0.1% (w/v) Pep- resistance is transferred to intestinal pathogens ton (Oxoid, L37) and 0.85% (w/v) NaCl in distilled water]. A total of 50 Al of each dilution was plated in In the current paper, an extensive study is pre- triplicate on all media, using the Whitley Automatic sented to verify the label correctness of a range of Spiral Plater (WASPk; Led Techno, Eksel, Belgium).
European probiotic food supplements and dairy prod- All plates were incubated at 37 jC under aerobic ucts, together with the antibiotic susceptibility testing conditions, except for TOS plates that were incubated of the product isolates. For each of these products, the anaerobically (80% N2, 10% H2 and 10% CO2) using label information was checked through taxonomic an anaerobic chamber. After incubation for 48 h, characterisation of the recoverable bacterial strains colony counts were performed and three to five using whole-cell protein profiling. In addition, indi- colonies were picked based on different colony mor- vidual susceptibilities were determined for a selection phologies. Selected colonies were further purified on MRSA medium except that those recovered fromTOS medium were cultured on Modified ColumbiaAgar (MCA) comprising 23 g special pepton (L72, Oxoid), 1 g soluble starch (1.01252.0250, Merck), 5 gNaCl (1.06404.1000, Merck), 0.3 g cystein – HClÁH2O, 5 g glucose (500520-887, Vel) and 15 g agar dis-solved in 1000 ml of distilled water. The latter A total of 55 probiotic products, collected in eight medium was also used in a second screening round European countries, comprised 30 dried food supple- of products that claimed bifidobacteria on their labels, but did not produce any Bifidobacterium strain on the Dairy products were collected using a refrigerated TOS medium during the first isolation round. Prod- box. None of the 55 products had exceeded their ucts that did not yield any isolates were again R. Temmerman et al. / International Journal of Food Microbiology 81 (2002) 1–10 Table 1Probiotic food supplements: comparison of label claims with identification results of isolates from the products Lb. acidophilus, Lb. rhamnosus, Lb. bifidumb Lb. bulgaricus, B. infantis, S. thermophilus Lb. acidophilus, Lb. bulgaricus, B. bifidum Lb. = Lactobacillus, B. = Bifidobacterium, S. = Streptococcus, E. = Enterococcus, Lc. = Lactococcus, P. = Pediococcus.
Lb. bulgaricus = corresponds to Lactobacillus delbrueckii ssp. bulgaricus.
a Type of product: P = Powder, C = Capsule, T = Tablet, PC = Powder as Coating.
b Indistinct or invalid name.
R. Temmerman et al. / International Journal of Food Microbiology 81 (2002) 1–10 Table 2Probiotic dairy products: comparison of label claims with identification results of isolates from the products Lb. acidophilus, S. thermophilus,Lb. paracasei ssp. paracasei S. thermophilus, Lc. lactis ssp.
lactis Lb. acidophilus, B. longum, S. thermophilus Lb. acidophilus Gilliland, living yoghurt Lb. acidophilus, Lb. johnsonii,S. thermophilus Lb. casei GGb, Lb. acidophilus, B. bifidum All dairy products were fermented drinks or yoghurt based products.
Lb. = Lactobacillus, B. = Bifidobacterium, S. = Streptococcus, Lc. = Lactococcus.
Lb. bulgaricus = corresponds to Lactobacillus delbreuckii ssp. bulgaricus.
a Indicated on the product label as ‘active bifidus’.
b Indistinct or invalid name.
screened, now using anaerobic and micro-aerophilic 2.2. Identification of recovered isolates (3,5% CO2, 5% O2, 7,5% H2, 84% N2) cultivationconditions. In addition, these products were subjected Isolates were identified by Sodium Dodecyl Sul- to an enrichment step in MRS broth (CM359, Oxoid) phate – Polyacrylamide Gelelectrophoresis (SDS – using the same aerobic and anaerobic incubation PAGE) analysis of whole-cell proteins, using stand- ardized cultivation conditions for comparison with the R. Temmerman et al. / International Journal of Food Microbiology 81 (2002) 1–10 available protein pattern database of lactic acid bac- vancomycin resistance in enterococci. First, by grow- ing the enterococci in a series of Trypticase Soy Broth proteins was performed according to the method (L21, Oxoid) Yeast Extract (211768, Becton Dick- inson) (TSYE) tubes containing different concentra- bacteria. Extracts were separated using SDS – PAGE tions of vancomycin or teicoplanin, the Minimal with a 5% total acrylamide stacking gel (12 mm long) Inhibitory Concentration (MIC) for these antibiotics and a 12% total acrylamide separation gel (126 mm was determined according the protocol as described long). Gels were stained using Coomassie Blue R- 250. The patterns were then densitometrically digi- the MIC value obtained for both antibiotics is consid- tized using an LKB 2202 Ultrascan Laser Densitom- ered indicative for the preliminary classification as to eter (LKB, Bromma, Sweden). Subsequently, these what extent a strain is vancomycin resistance. In a digital protein patterns were normalized using Gel- second approach, the presumptive presence of vanco- Compar software (Applied Maths, Sint-Martens- mycin resistance was assessed, using a PCR protocol Latem, Belgium) so it became possible to identify the isolates by comparison of their protein patterns pairs (A1, A2 and B1, B2) specific for the vancomy- with the SDS – PAGE protein pattern database avail- able at the laboratory. Upon repeated analyses, theinter-gel and intra-gel reproducibility was found to be90.3% and 97.1%, respectively.
3.1. Bacterial isolation from probiotic products At least one isolate per identified species recovered Depending on the medium used, colony counts of from a given product was included for antibiotic the 25 investigated dairy products ranged from 105 to susceptibility testing, resulting in 187 isolates screened 109 CFU/ml. Among the 30 food supplements tested for possible resistance against a selection of six anti- in this study, counts varied from below 1 to 106 CFU/ biotics, including kanamycin (30 Ag), vancomycin (30 g. During a first isolation round, we were unable to Ag), erythromycin (10 Ag), tetracycline (30 Ag), chlor- isolate viable bacteria out of 12 (i.e. 40%) of the food amphenicol (30 Ag) and penicillinG (10 Ag) using a supplements. These products were subjected to a slightly modified version of the agar disc diffusion second isolation round including an enrichment step in MRS broth and applying anaerobic as well as MRS broth (Oxoid, CM 359) (MC Broth for bifido- micro-aerophilic conditions. Only 1 of these 12 prod- bacteria) for 48 h at 37 jC. Following the preparation ucts displayed bacterial growth in MRS broth but of a 10-fold dilution in PPS, freshly poured MRSA again not on MRS agar plates. At the end of the two plates (MCA for bifidobacteria) were equally inocu- isolation rounds, a total of 323 isolates were obtained.
lated with this dilution. Antibiotic discs (Oxoid) were All isolated genera with exception of Bifidobacterium placed on the inoculated plates using the Oxoid Disc grew on all media used. However, it was noted that Dispenser. Following a 24-h incubation at 37 jC, lactobacilli and enterococci grew best on MRS agar inhibition zones around the discs were measured using and KAAAB, streptococci grew best on M17 and a digital callipers (Mauser, Switzerland). Results were TOS, and bifidobacteria only grew on TOS.
interpreted according to the cut-off levels proposed bywith strains considered resistant 3.2. Identification of recovered isolates if inhibition zone diameters were equal to or smallerthan 19 mm for penicillinG, 14 mm for vancomycin and tetracycline, and 13 mm for kanamycin, chloram- 2. From a total of 323 isolates, 268 bacteria could be identified at the species level. The remaining 55 In addition to the agar disc-diffusion method, two isolates were classified as yeasts after microscopical other methods were used to confirm the presence of investigation or were lost during purification on MRS R. Temmerman et al. / International Journal of Food Microbiology 81 (2002) 1–10 frequently recovered species among the food supple- Summary of isolation and identification results ments was Enterococcus faecium followed by Lacto- bacillus rhamnosus. Of the 6 products in which E.
faecium was found, only two actually claimed this species on their label. Lactobacillus acidophilus, which was claimed on the label of 22 food supple- ments, could only be isolated out of 2 of these products. Although all 13 food supplements claiming bifidobacteria were screened twice using two different media for the selective isolation of Bifidobacterium (TOS and MCA), only 3 of these 13 products pro- duced a bifidobacterial strain. Among the 25 dairy products, Lb. acidophilus was claimed, as well as isolated most frequently. As it was the case with the food supplements, only a poor retrieval of bifidobac- teria was possible among the dairy products, despite the use of two different media. Only 2 out of 14 dairyproducts claiming bifidobacteria actually produced a agar. Only six products yielded all species indicated Bifidobacterium strain during isolation.
on the product label. However, when disregarding thepresence of the yoghurt cultures Streptococcus ther- mophilus and Lactobacillus delbrueckii subsp. bul-garicus, this number of products rises to 13. In 19 Of the 268 identified isolates, 187 strains were products, the isolated species were entirely different subjected to antibiotic susceptibility testing using the from those mentioned on the product label, even after agar disc diffusion method of which the results are a second isolation round using a new batch of the presented in It was found that 79% and 65% same products. In a brief summary is given of of the isolates were resistant to kanamycin and van- the isolation and identification results. The most comycin, respectively. Furthermore, 23% and 21% of Table 4Percent of isolates resistant against six tested antibioticsa using the disc diffusion method Lactobacillus delbreuckii ssp. bulgaricus (3) a K = Kanamycin, TE = Tetracycline, E = Erythromycin, P = PenicillinG, C = Chloramphenicol, VA = Vancomycin.
b Total percentage of resistance calculated as the number of isolates (from a total of 187 isolates) resistant against the respective antibiotic.
R. Temmerman et al. / International Journal of Food Microbiology 81 (2002) 1–10 the isolates were grouped as resistant or intermedi- because of a too-long shelf-life period although the ately resistant, respectively, to penicillin. Concerning products investigated had not yet reached the expiry the other antibiotics, the intermediate resistant fraction date at the moment of the isolation procedure. Some was never larger than 6.5%. It was also found that immunological activities have been assigned to dead 38% of the isolated enterococci were vancomycin resistant according to the disc diffusion method. These promoting properties, e.g. competitive exclusion of resistant enterococci originated from four dried food pathogens, nutrient supplementation for the host, and supplements. However, when using the dilution anti-tumor effects can only be exerted by living method and a PCR assay for confirmation, none of the presumptively vancomycin resistant Enterococcus the present findings, it is therefore more likely for strains were found to be resistant against vancomycin dairy products to exert these probiotic properties than (MIC < 2 Ag/ml (results not shown)).
Identification of 268 isolates using protein profil- ing revealed that E. faecium was the most frequently recovered species out of the food supplements. Thistaxon was found in 6 out of the 19 food supplements Considering the significant rise in the annual (32%) containing living bacteria. With the exception consumption of probiotic products worldwide, it is of one product, E. faecium was the only species important that such products are correctly labeled and that the probiotic strains are well-documented Because of the high isolation numbers (104 – 105 CFU/g), it is unlikely that E. faecium entered the in’t Veld, 1999). Hitherto, in Europe, there are no production process via contamination. The second widely acknowledged regulations concerning the most frequently recovered species in food supple- labeling issues and claims that can be made by the ments was Lb. rhamnosus. Lb. acidophilus was claimed to be present in 22/30 (73%) products but O’Donnell, 1998; Przyrembel, 2001). Our findings clearly indicate the need for such regulations. Counts growth was observed for Lb. acidophilus on MRSA of viable bacteria were subtantially lower among the medium compared to Lb. rhamnosus, Lb. casei and E.
30 food supplements compared to the 25 dairy faecium, the relatively low recovery rate cannot be products. Possibly, higher isolation numbers could clearly explained. Likewise, the poor retrieval of have been obtained when food supplements were bifidobacteria could not readily be explained because analysed using anaerobic isolation conditions. How- isolation results were comparable after testing various ever, it can be speculated that the significant differ- isolation parameters, e.g. atmosphere, temperature and ence in relative numbers between the two main types duration of incubation (data not shown). More likely, of products will not be affected by incubation under it is possible that the nutritional content of the TOS aerobic or anaerobic conditions. Therefore, it is and MCA medium used in this study did not meet the possible that a number of the investigated food specific growth requirements of a number of probiotic supplements may comprise a bacterial concentration bifidobacterial strains. Therefore, it can be speculated below the minimum value required for any probiotic that more products claiming bifidobacteria may have strain to affect the gastro-intestinal tract, and thus, to produced these organisms during isolation, when a be able to promote a significant health effect. Using series of well-defined strain-specific media were used.
our protocol, a total of 11 food supplements (37%) The need for broad-spectrum isolation media for did not yield any viable bacteria on the four isolation bifidobacteria is clearly demonstrated by this study media. It can be speculated that the absence of living bacteria in a dried food supplement is due to damage the tested food supplements, a total of nine products of the culture caused by the drying and capsulation contained species other than those stated on the process, the possibility that some of these food product label. This mislabeling has also been reported supplements were sterilised for safety reasons or R. Temmerman et al. / International Journal of Food Microbiology 81 (2002) 1–10 al. (1998). Strikingly, 38% of the E. faecium isolates (1999) for 20 out of 29 tested food supplements.
also showed to be resistant against vancomycin Since S. thermophilus and Lb. delbrueckii subsp.
according to the disc diffusion method. However, bulgaricus are the main starter cultures of yoghurt, it these findings could not be confirmed by the dilution could be expected that these two species were among the most frequently isolated ones from the dairy products. However, Lb. delbrueckii subsp. bulgaricus 1995). Collectively, these findings indicate that all was only found once possibly because this species is enterococci isolated from probiotic products were rapidly overgrown by other lactobacilli in the dairy susceptible to vancomycin, which again highlights products. The fact that Lb. acidophilus was more the limited reliability of the disc diffusion method to easily isolated from dairy products than from food determine the occurrence of vancomycin resistance supplements, could be related to (1) the supporting matrix of the product in which the strains have to frequencies of vancomycin resistance found among survive for the complete shelf-life, (2) the ambient other lactic acid bacterial genera do not pose a temperature at which the different products are problem as this type of vancomycin resistance is usually maintained, (3) the total shelf-life (average different from the inducible, transferable mechanism of 30 days for dairy products, average of 24 months for food supplements) or (4) to the individual strain et al., 2000). The lactobacilli in the present study differences with respect to survival in the stationary comprised strains resistant to tetracycline (29.5%), phase at the given temperature. Fourteen dairy prod- chloramphenicol (8.5%), and erythromycin (12%) ucts also claim to contain bifidobacteria, whereas in and overall, more than 68% of our isolates exhibited only two instances, Bifidobacterium lactis was recov- resistance to two or more antibiotics (data not ered instead of the claimed B. longum. As outlined shown). With regard to general concerns on biosaf- above, the low recovery of bifidobacteria might be ety of probiotics, further research should focus on due to the lack of optimal isolation media for specific the location and potential transferability of these Bifidobacterium strains. Although to a lesser extent than with food supplements, our results suggest that In conclusion, it can be stated that quite a number also quite a number of dairy products suffer from of dried food supplements and—to a lesser extent— mislabeling, which underscores similar findings of dairy products are incorrectly or inadequately labeled with regard to the correct identity of the incorporated probiotic strains. Despite earlier reports concerning Using the disc diffusion method, high frequencies of resistance were detected for kanamycin (79%) and Holzapfel et al., 1998; Hamilton-Miller et al., vancomycin (65%). Most of the kanamycin resistant 1999), the new data indicate that this situation has isolates belonged to the genera Lactobacillus and not significantly improved. Although specific anti- Enterococcus. The latter genus is intrinsically resist- biotic resistance traits among probiotic strains may be finding that 81% of the isolated lactobacilli was also tetracycline, chloramphenicol and erythromycin resistant against kanamycin somewhat counteracts resistance among the investigated probiotic isolates the specificity of the Enterococcus-specific KAAAB indicates that continuous attention should be paid to medium. Likewise, the relatively high percentage of the selection of probiotic strains free of transferable vancomycin resistance observed among the entire antibiotic resistance. It is of paramount importance collection of isolates is due to the fact that the that at a time when consumers become more aware of majority of the lactobacilli are intrinsically resistant the importance of good nutrition and health, probiotic products designed especially for their health promot- intraspecies variations were found among the Lb.
ing purposes are safe and well-documented in order johnsonii and Lb. acidophilus isolates, which is in to provide consumers with the full benefits of the R. Temmerman et al. / International Journal of Food Microbiology 81 (2002) 1–10 Holzapfel, W.H., Haberer, P., Snel, J., Schillinger, U., Huis in’t Veld, J.H.J., 1998. Overview of gut flora and probiotics. Interna-tional Journal of Food Microbiology 41, 85 – 101.
This research was financially supported by a PhD Isolauri, E., Su¨tas, Y., Kankaanpa¨a¨, P., Arvilommi, H., Salminen, S., grant from the Flemish Institute for the Promotion of 2001. Probiotics: effects of immunity. American Journal of Clin- Innovation by Science and Technology (IWT—Vlaan- ical Nutrition 73, S444 – S450 (suppl.).
deren, Brussels, Belgium). Part of this research was Kirby, W.M.M., Bauer, A.W., Sherris, J.C., Turck, M., 1966. Anti- supported by Research Programme 3G030901 of the biotic susceptibility testing by a standardized single disc meth-od. American Journal of Clinical Pathology 45, 493 – 496.
Fund for Scientific Research—Flanders (Belgium) Klein, G., Hallmann, C., Casas, I.A., Abad, J., Louwers, J., Reuter, (F.W.O.—Vlaanderen). G.H. is a postdoctoral fellow G., 2000. Exclusion of vanA, vanB and vanC type glycopeptide of the Fund for Scientific Research—Flanders (Bel- resistance in strains of Lactobacillus reuteri and Lactobacillus gium) (F.W.O.—Vlaanderen). Also, we would like to rhamnosus used as probiotics by Polymerase Chain Reaction thank Dirk Dewettinck, Dirk Gevers and Geert Kindt and hybridization methods. Journal of Applied Microbiology89, 815 – 824.
for their contributions to this research.
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