Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococcus faecium strains of dairy, animal and clinical origin

Enterococci are part of the dominant microbiota of several dairy products. They are also present in the gut of humans and animals. Their presence in traditional raw milk cheeses is probably due to faecal contamination of milk during milking. Due to their importance as a cause of nosocomial infections, enterococci are acquiring increased significance. Such infections are becoming more and more difficult to treat as resistance to antibiotics increases. The aim of this investigation was to compare the potential virulence of Enterococcus faecium isolated from different ecological habitats and to establish if strains isolated from dairy products should really be considered as potential pathogens. In the present work, the antibiotic resistance pattern of 40 E. faecium strains isolated from dairy products, 26 E. faecium isolated from ewes' faeces and 28 clinical isolates of the same species was studied, and checks were made to see if known virulence determinants were present. Resistance to 12 different antibiotics commonly used in the treatment of human infections was tested using the broth microdilution method as described by the NCCLS. In addition, polymerase chain reaction (PCR) tests were carried out to see if genes for vancomycin resistance were present. The presence of the aggregation substance (AS) gene, the surface protein gene esp, the accessory colonisation factor ace, the Enterococcus faecalis endocarditis antigen efaA and the gelatinase gelE gene, which are involved in the virulence of enterococci, were also tested by PCR. The results of this study clearly indicate that E. faecium strains isolated from both cheese and sheep faeces are less pathogenic than those isolated from clinical samples. A similar pattern of resistance to antibiotics was observed in both dairy and animal strains. It was also found that there was difference in the kind of virulence determinants present in dairy and clinical isolates, while no virulence traits were found in sheep faeces strains. The results of this study suggest that E. faecium from traditional Sardinian raw milk cheeses should not be considered to be the main source of untreatable nosocomial enterococcal infections in humans in the island of Sardinia.     

Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins

The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria. No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria, Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA gene only occurred among E. faecium strains. The incidence of beta-hemolysis was not restricted to E. faecalis strains, but among the E. faecium strains the structural genes of cytolysin were not detected. beta-Hemolysis occurred in strains both from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or probiotics.     

Intestinal survival and persistence of probiotic Lactobacillus and Bifidobacterium strains administered in triple-strain yoghurt

The aim of the present study was to investigate the intestinal survival and persistence of probiotic strains Lactobacillus F19, Lactobacillus acidophilus NCFB 1748, and Bifidobacterium animalis subsp. lactis Bb-12 consumed in a yoghurt (ABC product), and also their effect on the intestinal microbiota. Based on the results of culture studies and strain-level analysis by randomly amplified polymorphic DNA (RAPD) fingerprinting Lactobacillus F19 and B. animalis subsp. lactis Bb-12 survived well through the human gastrointestinal tract; they were detected in reasonable numbers in the faeces of 100% and 79% of the study subjects, respectively. Ingestion of the probiotic yoghurt increased transiently the numbers of bifidobacteria and lactobacilli. For lactobacilli the increase was due to the detection of the ingested probiotic strains in faeces, while in bifidobacteria the increase was likely caused by the increase of indigenous bifidobacteria since the ingested Bifidobacterium strain did not comprise the predominant part of bifidobacterial population during the intervention. Probiotic strains were infrequently detected in mucosal biopsy samples. The present study indicates that developing probiotic food products with multiple probiotic strains is feasible.     

Survival of potentially probiotic enterococci in dairy matrices and in the human gastrointestinal tract

The aim of this study was to investigate the stability of Enterococcus strains isolated from a traditional Portuguese cheese and previously proved to be safe, in dairy matrices, and to assess survival of the best strains in the human gastrointestinal (GI) tract. Enterococcus faecium 32 and Enterococcus durans 37 were added to yoghurt that was ingested by 4 healthy adults. Detection of the enterococcal strains was performed with RAPD-PCR. The intervention trial showed transient colonisation with both strains, via presence in faeces during the ingestion period and disappearance by 10 d post-ingestion. Viable numbers of enterococci increased during the consumption period by 1.8–4.4 log-values, and returned to baseline level during the follow-up period. Based on data of the dairy matrix stability trials and human intervention study involving yoghurt ingestion, E. faecium 32 survived well both in the food matrix and in the human GI tract, thus showing probiotic potential.     

Species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in Germany

Presently, enterococci take the third place of bacterial pathogens associated with nosocomial infections, after staphylococci and Escherichia coli. Especially, the resistances of enterococci to several available antibiotics are threatening. We attempted to determine which species of enterococci could be found in food of animal origin and their significance according to their antibiotic resistances for human beings. From November 2000 to May 2002 we investigated 155 samples of food of animal origin bought in retail outlets in Germany: 27 samples of sausages, 19 of ham, 83 of minced meat, 26 of cheese. From these food samples we isolated 416 enterococcal strains. The most frequent species was Enterococcus faecalis (299 strains); furthermore, we found Enterococcus faecium (54 strains), Enterococcus durans together with Enterococcus hirae (24 strains), Enterococcus casseliflavus (22 strains), Enterococcus avium (9 strains) and Enterococcus gallinarum (8 strains). We focused on the resistance patterns of 118 selected E. faecium and E. faecalis strains to 13 antimicrobial active agents (ampicillin, amoxicillin/clavulanic acid, avilamycin, chloramphenicol, enrofloxacin, erythromycin, flavomycin, gentamicin, penicillin, quinupristin/dalfopristin, teicoplanin, tetracycline and vancomycin). From the clinical point of view, the situation of antibiotic resistance to the examined antimicrobial agents seemed to be favourable. The investigated strains were sensitive to ampicillin and amoxicillin/clavulanic acid. These antibiotics are, in combination with an aminoglycoside, for example gentamicin, agents of choice for the treatment of enterococcal infections in human medicine. Only one E. faecium strain was resistant to penicillin, while all strains were sensitive to the glycopeptide antibiotics, vancomycin and teicoplanin. Resistances found against the antibiotics, tetracycline, quinupristin/dalfopristin and erythromycin, are causes for concern.     

Evaluation of cheddar cheese as a food carrier for delivery of a probiotic strain to the gastrointestinal tract.

Cheddar cheese was evaluated as a food carrier for the delivery of viable microorganisms of Enterococcus faecium (Fargo 688; Quest Int., Naarden, The Netherlands) to the gastrointestinal tract. This strain had previously been shown to possess properties required of a probiotic microorganism including the ability to relieve irritable bowel syndrome. The strain was found to survive to high numbers in Cheddar cheese during ripening at 8 degrees C for 15 mo (4 x 10(8) cfu/g) and in yogurt during storage at 4 degrees C for 21 d (4 x 10(7) cfu/g). In an in vitro model system, Cheddar cheese was found to have a greater protective effect than yogurt upon exposure of the probiotic culture to porcine gastric juice at pH 2. Subsequently, a feeding trial involving 8 pigs per group was performed in which a rifampicin-resistant variant of the probiotic strain was fed for 21 d at a mean daily intake of 1.3 x 10(10) cfu/d from Cheddar cheese or 3.7 x 10(9) cfu/d from yogurt. During the feeding period, Cheddar cheese yielded a significantly higher mean fecal probiotic count (2 x 10(6) cfu/g of feces) than did yogurt (5.2 x 10(5) cfu/g of feces). These data indicate that mature Cheddar cheese compares very favorably with fresh yogurt as a delivery system for viable probiotic microorganisms to the gastrointestinal tract.     

Enterococci in foods--a conundrum for food safety

Enterococci form part of the lactic acid bacteria (LAB) of importance in foods. They can spoil processed meats but they are on the other hand important for ripening and aroma development of certain traditional cheeses and sausages, especially those produced in the Mediterranean area. Enterococci are also used as human probiotics. However, they are important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics, but antibiotic resistance alone cannot explain the virulence of some of these bacteria. Virulence factors such as adhesins, invasins and haemolysin have been described. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some harbour virulence traits and generally, Enterococcus faecalis harbours more of them than Enterococcus faecium. Regulations in Europe stipulate that safety of probiotic or starter strains is the responsibility of the producer; therefore, each strain intended for such use should be carefully evaluated. For numerous questions, immediate answers are not fully available. It is therefore suggested that when considering an Enterococcus strain for use as a starter or probiotic culture, it is imperative that each particular strain should be carefully evaluated for the presence of all known virulence factors. Ideally, such strains should harbour no virulence determinants and should be sensitive to clinically relevant antibiotics. In general, E. faecium appears to pose a lower risk for use in foods, because these strains generally harbour fewer recognised virulence determinants than E. faecalis. Generally, the incidence of such virulence determinants among E. faecium strains is low, as compared to E. faecalis strains, probably as a result of the presence of pheromone-responsive plasmids.     

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.     

Vancomycin-resistant enterococci from animal sources in Korea

Enterococci for which the minimum inhibitory concentration (MIC) of vancomycin was >/=8 mg/l were isolated from meat, feces, and raw milk samples collected in Korea from March to November 2003. Among the 243 vancomycin-resistant enterococci (VRE) that were identified the vanA vancomycin resistance gene was carried by 51 Enterococcus faecium and one Enterococcus sp., vanC1 was carried by 151 Enterococcus gallinarum, vanC2 was carried by 39 Enterococcus casseliflavus, and one Enterococcus sp. carried no van genes. Of the isolated enterococci carrying vanA, 4% were found to be highly resistant to gentamicin and 11% were resistant to ampicillin. Further genotyping of the E. faecium isolates carrying vanA using pulsed-field gel electrophoresis (PFGE) revealed extensive heterogeneity. The vancomycin resistance transferability test revealed that only two of the 52 enterococci carrying the vanA gene were able to transfer vancomycin resistance to other enterococci. The VRE were recovered from various animal sources with a particularly high prevalence of E. faecium carrying the vanA gene being found in poultry meat.     

Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut

A total of seventy lactic acid bacteria (LAB) were isolated from the faeces of healthy humans and their identities were confirmed by sequencing of their 16S rDNA genes. Of these only 5 isolates were found to resist bile salts and indicated survival in the simulated in vitro digestion assay which reproduces the stomach and intestinal digestion indicating their tolerance to gastric enzymes and the low pH conditions. Species that showed the best resistance to these conditions were: Lactobacillus casei, Lactobacillus sp., uncultured bifidobacteria, Enterococcus faecalis and Streptococcus anginosus. These strains were investigated further to study their capacity to adhere to human intestinal Caco-2 cells. E. faecalis was the most adherent strain. Examination of the virulence determinants for this strain indicated that it was positive for efaAfs, gelE, agg, cpd, cob, ccf and cad, a profile that is similar to that of many E. faecalis isolates from food sources. The cytolysin biosynthetic genes cylA, cylB and cylM that are more associated with the clinical isolates of E. faecium were not detected in this strain. The antibiotic susceptibility tests indicated that the strain was sensitive to vancomycin, tetracycline, rifampicin and erythromycin but resistant only to kanamycin and chloramphenicol. These data suggest that the strain E. faecalis CP58 may be tested further for beneficial properties and developed as a new probiotic.     

Survival and in vivo adhesion of human isolates Lactobacillus gasseri LF221 and K7 in weaned piglets and their effects on coliforms, clostridia and lactobacilli viable counts in faeces and mucosa

In in vivo study on 24 weaned piglets (8 per group), the survival rates of human isolates Lactobacillus gasseri K7 and LF221 were quantified by selective enumeration on MRS agar with rifampicin, and the presence of both strains in intestinal mucosa was examined. Faeces from individual animals were analysed for the number (cfu/g) of coliforms, lactobacilli, clostridia and both of the two probiotic strains during 2-weeks probiotic application period (5 x 10(10) cfu of individual strain/day) and 1 week after the probiotic treatment was ceased. Samples of duodenum, jejunum and ileum of sacrificed animals (5th or 20th day) were also examined microbiologically. A great variability in the microflora of faeces and mucosa was observed even between equally treated animals. The survival of both Lb. gasseri strains was established by their detection in the faeces (2.5 x 10(5) to 3.3 x 10(5) cfu of K7 strain/g faeces; 4.5 x 10(5) to 5 x 10(5) cfu of LF221 strain/g). In two animals, the LF221 or K7 viable cells were found in the faeces 6 d after ceasing probiotic application. In both animals from the group fed with Lb. gasseri K7 that were sacrificed 5 d after weaning, the presence of K7 strain was found either in the mucosa of duodenum (140 cfu/10 cm2) and jejunum (170 cfu/10 cm2) or in the ileum (1600 cfu/10 cm2). LF221 cells were detected in the ileal mucosa of one piglet (820 cfu/10 cm2). The results demonstrated the capability of both tested strains of in vivo adhesion to intestinal mucosa and of temporary colonisation of the piglets' intestine.     

Antibiotic resistance and incidence of Enterococcus species in Turkish white cheese

Thirty samples of Turkish white cheese were analysed for the presence of Enterococcus spp., and presumptive isolates were identified by morphological, cultural and biochemical tests and confirmed by the API 20 Strep System. Among the 101 isolates of Enterococcus spp., 62 were E. faecalis , 25 were E. faecium , seven were E. durans , five were E. mundtii and two were E. hirae/dispar . The resistance of the isolates to 13 different antibiotics was determined by the Kirby–Bauer disc diffusion test. Resistance to streptomycin, erythromycin, oxacillin and vancomycin was frequently found in these enterococci, and resistance to vancomycin was found in 96.8% of E. faecalis isolates and 76% E. faecium . The most effective antimicrobials were ampicillin (69.3% of isolates inhibited) and imipenem (76.3% of isolates inhibited). This examination confirmed the presence of enterococci, especially vancomycin-resistant strains, in Turkish white cheese, indicating poor sanitary conditions during production and processing and a significant health risk for consumers.     

Species identification and detection of vancomycin resistance genes in enterococci of animal origin by multiplex PCR

A multiplex PCR assay for the differentiation of species and detection of vancomycin (van) resistance genes in enterococci was established. Three hundred sixty-seven enterococcal strains isolated from food were selected from a sample collection and were examined with regards to the existence of four vancomycin resistance genes (vanA, vanB, vanC1 and vanC2) and to species differentiation (E. faecalis, E. faecium, E. casseliflavus and E. gallinarum) by multiplex PCR. Apart from unambiguous results for the majority of strains, the E. faecium specific primers used here, showed weak points in the specificity and sensitivity, respectively. Despite these rare instances, we succeeded in optimising the multiplex PCR assay by variation of annealing temperature and primer combination. The assay presented here, with its optimised parameters, is therefore suitable for routine use and because of time and material saving, it is an alternative and rapid method in comparison to conventional tests.     

In vitro assessment of the gastrointestinal transit tolerance of taxonomic reference strains from human origin and probiotic product isolates of Bifidobacterium

Next to health promoting effects, the functional aspect of probiotic strains also involves their capacity to reach the colon as viable metabolically active cells. The present study aimed to assess the potential of 24 probiotic product isolates and 42 human reference strains of Bifidobacterium to survive gastrointestinal transit under in vitro conditions. The survival capacity of exponential and stationary phase cultures upon exposure to gastric and small intestinal juices was determined using a recently developed microplate-based assay in combination with the LIVE/DEAD BacLight Bacterial Viability kit. All 66 strains tested displayed a considerable loss in viability during exposure to an acidic pepsin containing solution (pH 2.0). Among the 10 taxa tested, cultures of B. animalis ssp. lactis appeared to be most capable to survive gastric transit. Although to a lesser extent, the presence of bile salts also affected the viability of most of the strains tested. Except for 3 strains, all 66 strains showed bile salt hydrolase activity using an agar-based assay. In contrast, the bifidobacterial strains used in this study appeared to possess a natural ability to survive the presence of pancreatin (pH 8.0). Although the effect was not significant, a slightly enhanced tolerance to gastrointestinal transit was observed when cells were in the stationary phase, especially when exposed to acid, compared with cells being in the exponential phase. Survival in the gastrointestinal tract appeared to be largely strain-dependent and hence implies that different strains will likely display a different behavior in functionality. The assay used in this study allows an initial assessment of strains for use as probiotic cultures prior to selecting potential candidate strains for further investigation in vivo.     

Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract

Apart from genotypic identification methods, there is a need for reliable conventional phenotypic identification schemes for simple and rapid determination of enterococcal species in food or in the gastro-intestinal tract (GIT). Only a limited number of enterococcal species is of importance for the ecology of the GIT or the food microflora, including E. faecalis, E. faecium, E. durans/hirae, E. gallinarum and E. casseliflavus. After genus identification the differentiation within these species can include, e.g. mannitol and arabinose fermentation and growth at 50 degrees C. Widely used commercial identification systems may fail to precisely identify rare species. Ecological aspects should also be taken into account. In the human GIT E. faecium is the most common species whereas in most animal species E. faecalis is at least present in the same amount. Especially in foods of animal origin (cheese, pork meat, beef, poultry meat) also E. faecalis is very frequent. This is of special interest as glycopeptide resistance is most often found in human clinical E. faecium strains as well as in E. faecium from the environment or animal samples and less frequent in E. faecalis strains. EU experts propose as safety criteria for probiotics in feed additives the exclusion of resistances or the lack of transferability. This proposal can also be applied to enterococci in foods. Specific resistances must be excluded, but transferability or acquisition of resistance (e.g. vancomycin) cannot be excluded per se. However, technologically used strains should differ from clinical strains concerning their resistance patterns and transfer rates.     

Assessment of safety of enterococci isolated throughout traditional Terrincho cheesemaking: virulence factors and antibiotic susceptibility

Enterococci account for an important fraction of the adventitious microflora of traditional cheeses manufactured in Mediterranean countries from small ruminants' raw milk and play an important role in the development of suitable organoleptic characteristics of the final product. It has been suggested that animals used for food or animals that supply edible products are a reservoir of antibiotic-resistant enterococci. The main purpose of this research effort was thus to identify, to the species level, a total of 73 enterococci with high tolerance to acidic pH and bile salts (as prevailing environmental conditions in the first portion of the gastrointestinal tract), which were previously isolated from the milk feedstock to the final product of Terrincho cheesemaking, and to determine their profiles of antibiotic susceptibility, coupled with the occurrence of specific virulence factors (especially in those that might eventually be claimed to exhibit suitable probiotic and technological performances). Isolates, identified by both API 20 STREP and PCR methods, were found to belong to the following Enterococcus species: E. casseliflavus, E. durans, E. faecalis, E. faecium, and E. gallinarum. Susceptibility of those isolates was observed to most antibiotics tested, whereas none harbored aminoglycoside resistance genes. PCR screenings for cytolysin genes (cylL(L), cylL(s), cylM, cylB, and cylA), surface adhesin genes (efaA(fs), efaA(fm), and esp), the aggregation protein gene (agg), and the extracellular metalloendopeptidase gene (gelE) were performed. All isolates proved negative for cylL(L), cylM, cylB, and agg genes. Both E. faecalis strains were positive for the cell wall-associated protein Esp and the cell wall adhesin efaA(fs), whereas the cell wall adhesin efaA(fm) was detected in 11 of the 12 E. faecium strains. Only one strain possessed the cylL(s) determinant, and another possessed the cylA gene. Incidence of virulence determinants was thus very low; hence, the enterococcal adventitious microflora tested is essentially safe.     

The survival and persistence in the human gastrointestinal tract of five potential probiotic lactobacilli consumed as freeze-dried cultures or as probiotic sausage

Among five lactobacilli (L. plantarum MF1291, MF1298, DC13, L. pentosus MF1300 and L. salivarius DC5) which were administrated as freeze-dried cultures for 17 volunteers, MF1298 and DC13 were the most frequently reisolated strains in faeces demonstrating the human gastric survival of these strains. Furthermore, MF1298 and DC13 persisted in the same volunteer after ended intake, suggesting host-specific persistence behaviour. When MF1298 was administrated as sausage fermented with this strain, the number of volunteers harbouring MF1298 increased from 4 to 10 indicating that the sausage matrix protects the survival through the gastrointestinal tract (GIT).     

Effect of Enterococcus faecium on microbiological,physicochemical and sensory characteristics of Greek Feta cheese

Greek Feta cheese was prepared using as adjunct starter cultures Enterococcus faecium FAIR-E 198, E. faecium FAIR-E 243, and their combination. Numbers of enterococci in the control and in the batches containing E. faecium strains as adjunct starters rapidly increased until day 15 of ripening, and then remained constant. Both E. faecium strains positively affected the counts of non-starter lactic acid bacteria (NSLAB), micrococci and coliforms, while thermophilic cocci were not influenced. Moreover. E. faecium FAIR-E 243 enhanced the growth of mesophilic cocci and thermophilic bacilli. Physicochemical characteristics, such as pH, moisture, ash, salt in moisture and fat in dry matter (FDM) were not influenced by the addition of the E. faecium strains. The most pronounced effect was observed in the case of proteolysis. Both E. faecium strains, either as sole adjunct starter or in combination, increased the proteolytic index and the free amino groups concentration, and enhanced degradation of alpha(s1)- and beta-caseins in comparison to the control. Furthermore, the reverse-phase (RP)-HPLC peptide profiles of the water-soluble nitrogen (WSN) fractions were significantly affected by the addition of enterococci. The main volatile compounds produced were ethanol, acetate, acetone, acetaldehyde, acetoin and diacetyl, with highest amounts determined for ethanol, followed by acetate. Both E. faecium strains positively affected taste, aroma, colour and structure of the full-ripened cheeses, as well as the overall sensory profile. The present work emphasizes the technological significance of E. faecium strains and supports their use as adjunct cultures in the manufacture of Feta cheese.     

Enterococcus faecalis and Enterococcus faecium isolates from milk,beef, and chicken and their antibiotic resistance

The occurrence and antibiotic resistance of enterococci, especially Enterococcus faecalis and Enterococcus faecium, in milk, beef, and chicken in Gaborone, Botswana, were studied. Enterococci were isolated from these sources with the use of bile esculin agar and identified with API 20 Strep kits. Antibiotic resistance was determined by the disk diffusion method. The antibiotics tested were vancomycin, teicoplanin, ampicillin, tetracycline, and cephalothin. Among the 1,467 enterococci isolated from the samples, E. faecalis (46.1%) and E. faecium (29.0%) were found to be the predominant species. Other enterococcal species made up 25% of the isolates. More than 96 and 97% of the E. faecalis and E. faecium isolates, respectively, were found to be resistant to ampicillin. Almost 34, 27.3, and 22.4% of the E. faecalis isolates from milk, beef, and chicken, respectively, were also resistant to cephalothin. The percentages of E. faecium isolates that were found to be resistant to cephalothin were 32.8, 16.9, and 17.3% for milk, beef, and chicken, respectively. Resistance to vancomycin was widespread. It was found that 18.8, 7.8. and 13.1% of the E. faecalis isolates from milk, beef, and chicken samples, respectively, were resistant to vancomycin. In contrast, 32.8, 24.7, and 30.7% of the E. faecium isolates from milk, beef, and chicken samples, respectively, were resistant to vancomycin. Isolates that were resistant to multiple drugs were found in relatively large numbers.     

Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: selective isolation from faeces and identification using monoclonal antibodies.

Lactobacillus casei strain Shirota (LCS) is a probiotic bacterium used in the production of fermented milk products and lactic acid bacteria preparations. To investigate the survival of LCS in the gastrointestinal tract, we have developed a selective medium and specific monoclonal antibodies to isolate and identify this strain. Selective LLV agar medium was prepared by modifying LBS medium, a selective medium for lactobacilli, through the replacement of glucose with lactitol as a carbon source and vancomycin as a selective antibiotic. Culture in LLV agar medium followed by ELISA using monoclonal antibodies specific for LCS was able to detect the organism in faeces. Using this method, we studied the faecal recovery of LCS in individuals who drank 125 ml of fermented milk which contained 10(10) live LCS for 3 days. The mean recovery was about 10(7) live bacteria per gram of faeces, indicating that LCS survived transit through the gastrointestinal tract after ingestion of the fermented milk.