Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population

Kumis is a traditional fermented cow milk produced and consumed in South West Colombia. The main objective of this research was to studied the enterococcal population, present in 13 kumis samples traditionally manufactured, for their role as beneficial organisms or opportunistic pathogens. The molecular identification of 72 isolates evidenced that Enterococcus faecalis and E. faecium were the dominant species. The genes gelE, esp, asa1, cyl and hyl, all associated with virulence factors in enterococci, were detected in 30 isolates, while 42 were free of virulence determinants. Skim milk media were fermented by all the different isolates and further tested for proteolysis (free NH₃ groups), Angiotensin-I Converting Enzyme (ACE) inhibitory activity and biogenic amines production. Nine E. faecalis and two E. faecium strains produced fermented milk with ACE-inhibitory activity values ranging from 39.7% to 84.35% .The digestion of fermented milk samples by pepsin and pancreatin evidenced an increase in ACE inhibitory activity, with E. faecalis KE09 as the best producer (IC50 = 14.25 μg ml⁻¹). Moreover, the strains showed a very low tyrosine decarboxylase activity and did not produce histamine during 48 h fermentation in milk. This study underlines the that Colombian kumis is a good source of not virulent enterococci able to produce fermented milks with ACE-inhibitory activity.     

Properties and safety aspects of Enterococcus faecium strains isolated from Chungkukjang, a fermented soy product

Enterococcus faecium isolated from Chungkukjang, a Korean traditional fermented soybean food was studied for their functional characteristics as potential new starter culture and safety. Microbiological analysis of ripened Chungkukjang revealed the presence of an enterococcal population in numbers of up to 6 log CFU per g. Seven isolates with higher activity were selected for further study and the strains were identified as E. faecium. The E. faecium strains showed resistance against simulated gastrointestinal conditions such as acidic environment and the presence of bile salts. These strains also showed bile salt hydrolase activity but neither hemolytic activity nor virulence determinant such as gelE and efaAfm. All strains were susceptible to glycopeptides and lacked potential as vancomycin-resistant enterococci (VRE). Two strains, S2C10 and S2C11, showed inhibited the viability of Listeria monocytogenes in vitro. The ability was probably due to the production of bacteriocin. The lipase activity influenced the stability, while either acidic condition or high temperature did not play a significant role in the activity of the antimicrobial substances. The strains also produced thermostable listericidal antimicrobial substance. For this reason, the strains could be used as selected starters or protective cultures in soybean fermented food production.     

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,characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey

The aim of the present work is to provide information about Enterococcus strains isolated from traditional Turkish cheese samples in Ankara (Turkey), focusing on their prevalence, phenotypic and genotypic characteristics, and antibiotic resistance. A total of 213 probable enterococcal isolates isolated from 215 samples were identified by phenotypic and genotypic methods. As a result of 16S rDNA sequence analysis, 88 of the 213 enterococci strains were identified as Enterococcus faecium and 125 as Enterococcus faecalis. The E. faecalis strains (58.7%) were identified as the dominant species isolated from cheese samples in Turkey. The 213 Enterococcus strains were tested for susceptibility to 12 different antimicrobial agents. The resistance phenotype were as follow: nalidixic acid (100%), kanamycin (98.6%), rifampicin (78.4%), ampicillin (48.8%), ciprofloxacin (45.5%), erythromycin (18.8%), tetracycline (11.7%), penicillin G (5.6%), chloramphenicol (4.2%), gentamycin (3.8%) and streptomycin (1.4%). None of the strains was resistant to vancomycin. E. faecium strains showed more resistant phenotypes than E. faecalis strains as shown by the antibiotic resistance levels. It was also observed that the resistance of E. faecium and E. faecalis strains against the antibiotics was statistically significant (p ? 0.05). In total, 100% of E. faecium and 88.8% of E. faecalis strains were resistant to multiple drugs.     

Safety aspects of enterococci from the medical point of view

Enterococci occur in a remarkable array of environments. They can be found in soil, food, and water, and make up a significant portion of the normal gut flora of humans (10(5)-10(7)/g of stool) and animals. As other bacteria of the gut flora, enterococci can also cause infectious diseases. Most clinical isolates are Enterocococus faecalis, which account for 80-90% of clinical strains. Enterocococus faecium accounts for 5-10% of such isolates. Typical enterococcal infections occur in hospitalised patients with underlying conditions representing a wide spectrum of severity of illness and immune modulation. Enterococci today rank second to third in frequency among bacteria isolated from hospitalised patients. They are isolated from urinary tract infections, intra-abdominal and pelvic infections, bacteremias, wound and tissue infections, and endocarditis--often as part of a polymicrobial flora. Surprisingly, little is known about the factors that contribute to the ability of enterococci to cause infections. Many strains of E. faecalis produce a cytolysin (haemolysin) exhibiting tissue-damaging capacity. Further extracellular products often observed in clinical isolates are a proteinase (gelatinase), hyaluronidase, and extracellular superoxide. Furthermore, many of the clinical isolates possess the aggregation substance on the surface and an extracellular surface protein, both contributing to the adherence to eucaryotic cells. Some strains of E. faecalis, and many E. faecium strains are resistant to multiple antimicrobials. The ultimate role of all these factors in enterococcal pathogenicity remains to be determined. It was previously thought that enterococcal infections were endogenously acquired from the patient's own gut flora. A rather new concept that has emerged is that enterococcal disease is a two-stage process. There is an initial colonisation of the gastrointestinal tract by enterococcal strains possessing virulence traits and/or antibiotic resistance. Subsequently, this population spreads, often facilitated by antibiotic elimination of competitors. For a selected number of patients, there is subsequent tissue invasion from the gastrointestinal tract reservoir. From this concept, it can be deduced that enterococcal strains without virulence traits and antibiotic resistances exogenously transferred into the human gut via food products or probiotics will not represent any risk for immunocompetent individuals. In very severely immunocompromised patients, however, a risk for enterococcal disease by such strains cannot completely be excluded.     

Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses

Prevalence of enterococci and antibiotic resistance profiles of Enterococcus faecalis was analyzed in 126 French cheeses from retail stores. Forty-four percent of pasteurized or thermised-milk cheeses, and up to 92% of raw-milk cheeses contained detectable enterococci. A total of 337 antibiotic resistant enterococci were isolated in 29% and 60% of pasteurized-milk and raw-milk cheeses, respectively. E. faecalis was the predominant antibiotic resistant species recovered (81%), followed by Enterococcus faecium (13%), and Enterococcus durans (6%). The most prevalent antibiotic resistances were tetracycline (Tet) and minocycline (Min), followed by erythromycin (Ery), kanamycin (Kan) and chloramphenicol (Cm). The most common multiple antibiotic resistance phenotype was Cm Ery Kan Min Tet. The occurrence of antibiotic genes, as searched by PCR, was 100 % for aph3′IIIa, 96 % for ermB, 90 % for tetM and 80 % for catA in isolates resistant to Kan, Ery, Tet or Cm, respectively. MLST analysis of 30 multidrug resistant E. faecalis revealed that ST19, CC21, CC25 and CC55 isolates were the most common in cheeses. In conclusion, as in many other European countries, French cheeses do contain enterococci with multiple antibiotics resistances. However, low occurrence of high-level gentamicin resistant or sulfamethoxazole/trimethoprim-resistant enterococci and absence of vancomycin- or ampicillin- resistant enterococci indicate that cheeses cannot be considered as a major reservoir for nosocomial multi-drug resistant enterococci.     

In vitro growth of enterococci of bovine origin in bovine mammary secretions from various stages of lactation

In vitro growth responses of Enterococcus faecium and Enterococcus faecalis were tested in cell-free, fat-free bovine mammary secretions. Mammary secretions were collected during the dry period, and during early, late, and extended lactation. Sixty-three enterococcal isolates from aseptically collected bovine quarter milk samples and bedding samples from a commercial dairy herd were tested. Isolates from bovine quarter milk samples originated from mammary glands with clinical mastitis, cows with composite somatic cell score >4, postpartum milk samples, or from routine milk samples submitted to a mastitis diagnostic laboratory. Source of enterococcal isolates and the species significantly contribute to the ability of organisms to multiply in mammary secretions from various stages of lactation. Isolates collected from milk samples of the commercial herd and isolates from milk submitted to a mastitis diagnostic lab did not display enhanced growth in mammary secretions compared with isolates from bedding. Growth responses of E. faecalis were greater than those for E. faecium in secretions collected during the dry period, late lactation, and extended lactation. Bacterial growth did not differ between enterococcal species in mammary secretion collected from cows in early lactation. Differences in bacterial growth between E. faecalis and E. faecium in mammary secretions may indicate differences between species in susceptibility of mammary glands during the lactation cycle.     

Characterization of antibiotic resistant enterococci isolated from untreated waters for human consumption in Portugal

Untreated drinking water is frequently overlooked as a source of antibiotic resistance in developed countries. To gain further insight on this topic, we isolated the indicator bacteria Enterococcus spp. from water samples collected in wells, fountains and natural springs supplying different communities across Portugal, and characterized their antibiotic resistance profile with both phenotypic and genetic approaches. We found various rates of resistance to seven antibiotic families. Over 50% of the isolates were resistant to at least ciprofloxacin, tetracyclines or quinupristin-dalfopristin and 57% were multidrug resistant to ≥ 3 antibiotics from different families. Multiple enterococcal species (E. faecalis, E. faecium, E. hirae, E. casseliflavus and other Enterococcus spp) from different water samples harbored genes encoding resistance to tetracyclines, erythromycin or gentamicin [tet(M)-46%, tet(L)-14%, tet(S)-5%, erm(B)-22%, aac(6´)-Ie-aph(2″)-12%] and putative virulence factors [gel-28%, asa1-16%]. The present study positions untreated drinking water within the spectrum of ecological niches that may be reservoirs of or vehicles for antibiotic resistant enterococci/genes. These findings are worthy of attention as spread of antibiotic resistant enterococci to humans and animals through water ingestion cannot be dismissed.     

Probiotic and Technological Properties of Enterococci Isolates from Infants and Cheese

Abstract

In the present study 16 enterococcal strains isolated from infant feces and/or Feta cheese were tested for their ability to metabolize in milk and resist specific conditions of the gastrointestinal tract, in order to finally select interesting strains for further studies, for their evaluation as dietary adjuncts. The strains were characterized by phenotypic criteria (API ID 32 STREP) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Whole-cell protein analysis by SDS-PAGE confirmed the taxonomic allocation of 14 strains suggested by phenotypic criteria as E. durans (4 strains), E. faecalis (3 strains), and E. faecium (7 strains). Two strains biochemically characterized as E. faecalis were assigned to other species by SDS-PAGE. All strains grew well in bile concentrations 0–1%. The strains were also able to withstand low pH (3.0) values for 4?h, eventhough for some strains a reduction by ～1–2 log₁₀ cfu/mL was observed. Most of the strains produced tyramine and all of them were susceptible to vancomycin. In addition, six out of seven E. faecium strains from either feces or cheese were β-haemolytic. The strains exhibited antagonistic activities towards enterococci, lactic acid bacteria, and Cl. sporogenes. A considerable phenotypic diversity was found among the test strains concerning their acidifying and proteolytic activities. Results of this study give useful information for the selection of appropriate strains with interesting probiotic and technological properties.     

Is the production of the biogenic amines tyramine and putrescine a species-level trait in enterococci?

Biogenic amines (BA) are toxic nitrogenous compounds that can be accumulated in foods via the microbial decarboxylation of certain amino acids. Lactic acid bacteria (LAB) strains belonging to different species and genera have been described as BA producers and are mainly responsible for their synthesis in fermented foods. It is generally accepted that the capacity to produced BAs is strain-dependent. However, the large number of enterococci identified as BA producers suggests that the aminogenic trait may be a species-level characteristic. Enterococcus faecalis, Enterococcus faecium and Enterococcus durans strains of different origin were analysed to determine their capacity to produce tyramine and putrescine. The presence of the genes responsible for this and the identity of their flanking regions were checked by PCR. The results suggest that tyramine biosynthesis is a species-level characteristic in E. faecalis, E. faecium and E. durans. Putrescine synthesis was found to be a species-level trait of E. faecalis, with production occurring via the agmatine deamination pathway. Some E. faecium strains of human origin also produced putrescine; this trait was probably acquired via horizontal gene transfer.     

Enterococcus populations in artisanal Manchego cheese: biodiversity, technological and safety aspects

Enterococci represent a considerable proportion of the microbiota in Manchego cheeses. In this study, a total of 132 enterococci isolated from good quality Manchego cheeses from two dairies at different ripening times were genotypically characterized and identified using molecular techniques. Representative isolates from the clusters obtained after genotyping were assayed for some enzymatic activities considered to have a potential role in cheese ripening, and for 2,3-butanedione and acetoin production, evaluation of odor intensity and appearance in milk and safety evaluation. Enterococcus faecalis was the predominant specie, accounting for 81.8% of the total isolates, while Enterococcus faecium, Enterococcus hirae and Enterococcus avium were present in low proportions. The number of genotypes involved at each ripening time varied both between dairies and with the ripening times; genotype E. faecalis Q1 being present in almost all the samples from both dairies. Eight isolates showed a higher proteolytic activity and 3 isolates produced high quantities of acetoin-diacetyl, for which reason they are interesting from a technological standpoint. A low antibiotic resistance was found and almost all the strains were susceptible to clinically important antibiotics. On the contrary, only four isolates (E. faecalis C4W1 and N0W5, and E. faecium N32W1 and C16W2) did not harbor some of the virulence genes assayed.     

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.     

Diversity,distribution and antibiotic resistance of Enterococcus spp. recovered from tomatoes,leaves, water and soil on U.S. Mid-Atlantic farms

Antibiotic-resistant enterococci are important opportunistic pathogens and have been recovered from retail tomatoes. However, it is unclear where and how tomatoes are contaminated along the farm-to-fork continuum. Specifically, the degree of pre-harvest contamination with enterococci is unknown. We evaluated the prevalence, diversity and antimicrobial susceptibilities of enterococci collected from tomato farms in the Mid-Atlantic United States. Tomatoes, leaves, groundwater, pond water, irrigation ditch water, and soil were sampled and tested for enterococci using standard methods. Antimicrobial susceptibility testing was performed using the Sensititre microbroth dilution system. Enterococcus faecalis isolates were characterized using amplified fragment length polymorphism to assess dispersal potential. Enterococci (n = 307) occurred in all habitats and colonization of tomatoes was common. Seven species were identified: Enterococcus casseliflavus, E. faecalis, Enterococcus gallinarum, Enterococcus faecium, Enterococcus avis, Enterococcus hirae and Enterococcus raffinosus. E. casseliflavus predominated in soil and on tomatoes and leaves, and E. faecalis predominated in pond water. On plants, distance from the ground influenced presence of enterococci. E. faecalis from samples within a farm were more closely related than those from samples between farms. Resistance to rifampicin, quinupristin/dalfopristin, ciprofloxacin and levofloxacin was prevalent. Consumption of raw tomatoes as a potential exposure risk for antibiotic-resistant Enterococcus spp. deserves further attention.     

Prevalence of enterococci in frozen dairy products and their pathogenicity

Enterococci were present in all the samples of frozen dairy products examined in the present study. The total bacterial counts as well as enterococcal counts were generally very high in Kulfi (a frozen product similar to ice cream) and Kulfi mix. Among 161 enterococcal isolates recovered, the predominant type was Streptococcus faecalis var. faecalis, the majority of isolates coming from Kulfi and Kulfi mix samples. Although 58 cultures produced deoxyribonuclease, only two of them exhibited thermonuclease (TNase) production. Hyaluronidase activity was shown by three strains. However, 31 strains were alpha haemolytic, while five showed beta haemolysis on rabbit blood agar. Out of these haemolytic strains tested, 21 produced lethal toxicity in mice and 20 strains showed culture virulence, postmortem studies revealed congestion of heart, liver, kidney and spleen. The two TNase positive strains of Streptococcus faecium produced fluid accumulation in ligated ileal loops of rabbits and distension in the gastrointestinal tracts of infant mice.     

Characterization of enterococci isolated from homemade Bulgarian cheeses and katuk

A collection of 107 lactic acid bacteria (LAB) isolates was obtained from traditional Bulgarian dairy products??homemade cheeses and katuk samples, produced from heat-treated cow, goat, ewe and buffalo milk without the addition of any bacterial starter culture. The samples were collected from mountain region of Rhodope (south part of Bulgaria), Tracian valley and mountain region of Stara Planina (west part of Bulgaria). These LAB produced bacteriocin-like inhibitory substances (BLIS) and proteinases. Preliminary strain determination was performed according to their fermentation ability using API 50CHL and API 20 Strep. Most of the characterized strains (58) belong to genus Enterococcus; 21, 20 and 11 strains were identified as Lactobacillus sp., Streptococcus sp. and Lactococcus sp., respectively. Isolated enterococcal strains were characterized using phenotypic features as well as by DNA typing. The strains were identified as Enterococcus faecium (34), Enterococcus durans (22) and Enterococcus faecalis (2). The proteolytic activity varied from 0.094 to 0.455?mM/L Gly into the group of E. faecium and from 0.109 to 0.487?mM/L Gly into the group of E. durans strains, while both E. faecalis strains showed relatively high proteolytic activity. The samples obtained after 3?h of hydrolysis of ??-casein by E. faecalis B1 strain were further used for mass spectrometry analysis, and 31 peptides were identified.     

2-Phenylethylamine formation by enterococci in vitro

The ability of enterococci to form PEA in vitro was investigated on a test strain Fs 202 of species Ec. faecalis from cheese. 22 enterococcal strains were isolated from foodstuff: nine strains from cheese, three strains from minced pork, seven strains from carcasses of pigs, two strains from chicken carcasses and one strain from cattle carcasses. The enterococci were examined in a test medium with additives of L-phenylalanine, L-tyrosine and dextrose. The amount of formed PEA was measured in periods of 24 h in test strain Fs 202 using RP-HPLC. Simultaneously, the pH-value and the number of microbes were determined at start and 72 h. The tested strains formed maximal or significant PEA amounts in test medium at 30 °C incubation temperature in a period of 72 h. In the same period strains without PEA formation were detected. A regular and intensive PEA formation was found by six Ec. faecalis, five Ec. hirae and two Ec. faecium strains. Ec. faecium could form only moderate PEA amounts. Amine traces or no PEA at all were detected for one Ec. faecium strain, three Ec. durans strains, two Ec. gallinarum strains and one Ec. casseliflavus strain. Potential starter organisms or probiotic strains should be tested in vitro at 30 °C for 72 hours for ability to form PEA. For toxicological reasons, it is important that those strains not have any ability to form PEA.     

The occurrence of virulence traits among high-level aminoglycosides resistant Enterococcus isolates obtained from feces of humans, animals, and birds in South Korea

Enterococcus isolates (1500) obtained from the feces of 48 humans, 209 domesticated food animals, and 155 wild geese in South Korea were characterized with respect to species status by PCR analyses and resistance to antibiotics. Of the 1500 strains examined, the majority (n = 577) were Enterococcus faecalis from 224 (54.4%) of the samples feces, while 299 were of E. faecium from 125 of the samples (30.3%), 224 were E. hirae from 101 (24.5%) of the samples, 94 were E. casseliflavus from 43 (10.4%) of the samples, and one was E. gallinarum. While 305 isolated from 125 (30.3%) of the samples were unidentified species. Approximately 15, 60, 50, 55, 3, and 40% of samples obtained from beef cattle, chickens, ducks, swine, wild geese, and humans, respectively, yielded Enterococcus isolates that were resistant to high-levels of aminoglycosides (i.e., of gentamicin, kanamycin, and streptomycin, minimum inhibitory concentrations were > 1000 mg/l). The 180 Enterococcus isolates that showed high levels of resistance to aminoglycoside antibiotics (HLAR) were screened for virulence genes encoding for aggregation substance (agg), cytolysin activator (cylA), gelatinase (gelE) and surface protein (esp). Of those, the gelE gene was found most frequently in chickens and ducks of the HLAR isolates, while 56 E. faecalis and 13 E. faecium HLAR were gelatinase positive and showed hemolysin activity. Multiple antibiotic resistant Enterococcus isolates carrying virulence genes were most frequently isolated from poultry and swine, and were mostly E. faecalis or E. faecium. These findings suggest that restriction of the use of antibiotics in food animal operations in South Korea, especially those involved in poultry and swine production would be desirable.     

Dissemination of Enterococcus faecalis and Enterococcus faecium in a Ricotta Processing Plant and Evaluation of Pathogenic and Antibiotic Resistance Profiles

In this work, the sources of contamination by Enterococcus spp. in a ricotta processing line were evaluated. The isolated strains were tested for virulence genes (gelE, cylA,B, M, esp, agg, ace, efaA, vanB), expression of virulence factors (hemolysin and gelatinase), and the resistance to 10 different antibiotics. Enterococcus faecium and Enterococcus faecalis were subjected to discriminatory identification by intergenic spacer region (ITS)‐polymerase chain reaction and sequencing of the ITS region. The results showed that Enterococcus spp. was detected in the raw materials, environment samples and the final product. None of the 107 Enterococcus isolates were completely free from all virulence genes considered. A fraction of 21.5% of the isolates containing all of the genes of the cylA, B, M operon also expressed β‐hemolysis. Most of the isolates showed the gelE gene, but only 9.3% were able to hydrolyze gelatin. In addition, 23.5% of the observed Enterococcus isolates had the vanB gene but were susceptible to vancomycin in vitro. The dissemination of antibiotic‐resistant enterococci was revealed in this study: 19.3% of the E. faecium samples and 78.0% of the E. faecalis samples were resistant to at least one of the antibiotics tested. Sequencing of region discriminated 5 and 7 distinct groups among E. faecalis and E. faecium, respectively. Although some similarity was observed among some of the isolates, all E. faecalis and E. faecium isolates had genetic differences both in the ITS region and in the virulence profile, which makes them different from each other.     

Screening of strains of Lactococci isolated from Egyptian dairy products for their proteolytic activity

A collection of cocci isolates (293) obtained from traditional Egyptian dairy products collected from four Egyptian regions yielded 151 lactic acid bacteria (LAB) cocci isolates. Among them, 24 isolates were characterised as highly proteolytic. SDS–PAGE showed that 6 isolates were the most proteolytically active, which were classified into Enterococcus faecalis HH22 (4 isolates) and Enterococcus faecium DO623 (2 isolates). The proteolytic activity of E. faecalis was higher than that of E. faecium (particularly on β-casein). The maximal degradation of milk proteins was achieved at pH 6.5–7.2 (E. faecalis) or pH 6.5 (E. faecium) and at 42 °C for both strains. The proteolytic activities of the two strains were inhibited mostly by the presence of EDTA, showing that their proteases belong mainly to metalloproteases. A slight inhibition of proteolysis by PMSF in the case of E. faecalis HH22 suggests a limited inclusion of serine proteases in its protease system.     

Species distribution,antibiotic resistance and virulence traits in enterococci from meat in Tunisia

Antimicrobial resistance and the mechanisms implicated were studied in 119 enterococci from 105 meat samples from Tunisian markets. Almost 24.5% of recovered enterococci showed resistance against four or more antimicrobial agents and these isolates were identified to the species level. Enterococcus faecalis was the most prevalent species (41%). High percentages of erythromycin and tetracycline resistances were found among our isolates, and lower percentages were identified to aminoglycosides, ciprofloxacin and chloramphenicol. All tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in 78.5% of erythromycin-resistant isolates, ant(6)-Ia gene in 58.8% of streptomycin-resistant isolates, and cat(A) gene in one chloramphenicol-resistant isolate. Forty-eight isolates carried the gelE gene and exhibited gelatinase activity. The hyl and esp genes were detected in one and three Enterococcus faecium isolates, respectively. Streptomycin-resistant isolates showed a high genetic diversity by PFGE and MLST. Meat might play a role in the spread through the food chain of enterococci with these virulence and resistance characteristics to humans.