Charles M. A. P. Franz

Enterococci at the crossroads of food safety

Enterococci are gram-positive bacteria and fit within the general definition of lactic acid bacteria. Modern classification techniques resulted in the transfer of some members of the genus Streptococcus, notably some of the Lancefields group D streptococci, to the new genus Enterococcus. Enterococci can be used as indicators of faecal contamination. They have been implicated in outbreaks of foodborne illness, and they have been ascribed a beneficial or detrimental role in foods. In processed meats, enterococci may survive heat processing and cause spoilage, though in certain cheeses the growth of enterococci contributes to ripening and development of product flavour. Some enterococci of food origin produce bacteriocins that exert anti-Listeria activity. Enterococci are used as probiotics to improve the microbial balance of the intestine, or as a treatment for gastroenteritis in humans and animals. On the other hand, enterococci have become recognised as serious nosocomial pathogens causing bacteraemia, endocarditis, urinary tract and other infections. This is in part explained by the resistance of some of these bacteria to most antibiotics that are currently in use. Resistance is acquired by gene transfer systems, such as conjugative or nonconjugative plasmids or transposons. Virulence of enterococci is not well understood but adhesins, haemolysin, hyaluronidase, aggregation substance and gelatinase are putative virulence factors. It appears that foods could be a source of vancomycin-resistant enterococci. This review addresses the issue of the health risk of foods containing enterococci.

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.

Incidence of Virulence Factors and Antibiotic Resistance among Enterococci Isolated from Food

ABSTRACT The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.

Enterococci as probiotics and their implications in food safety.

Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. 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 can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis.

Diversity and applications of Bacillus bacteriocins

Members of the genus Bacillus are known to produce a wide arsenal of antimicrobial substances, including peptide and lipopeptide antibiotics, and bacteriocins. Many of the Bacillus bacteriocins belong to the lantibiotics, a category of post-translationally modified peptides widely disseminated among different bacterial clades. Lantibiotics are among the best-characterized antimicrobial peptides at the levels of peptide structure, genetic determinants and biosynthesis mechanisms. Members of the genus Bacillus also produce many other nonmodified bacteriocins, some of which resemble the pediocin-like bacteriocins of the lactic acid bacteria (LAB), while others show completely novel peptide sequences. Bacillus bacteriocins are increasingly becoming more important due to their sometimes broader spectra of inhibition (as compared with most LAB bacteriocins), which may include Gram-negative bacteria, yeasts or fungi, in addition to Gram-positive species, some of which are known to be pathogenic to humans and/or animals. The present review provides a general overview of Bacillus bacteriocins, including primary structure, biochemical and genetic characterization, classification and potential applications in food preservation as natural preservatives and in human and animal health as alternatives to conventional antibiotics. Furthermore, it addresses their environmental applications, such as bioprotection against the pre- and post-harvest decay of vegetables, or as plant growth promoters.

Antibiotic Resistances of Starter and Probiotic Strains of Lactic Acid Bacteria

ABSTRACT The antibiotic resistances of 45 lactic acid bacteria strains belonging to the genera Lactobacillus, Streptococcus, Lactococcus, Pediococcus, and Leuconostoc were investigated. The objective was to determine antibiotic resistances and to verify these at the genetic level, as is currently suggested by the European “qualified presumption of safety” safety evaluation system for industrial starter strains. In addition, we sought to pinpoint possible problems in resistance determinations. Primers were used to PCR amplify genes involved in β-lactam antibiotic, chloramphenicol, tetracycline, and erythromycin resistance. The presence of ribosomal protection protein genes and the ermB gene was also determined by using a gene probe. Generally, the incidences of erythromycin, chloramphenicol, tetracycline, or β-lactam resistances in this study were low (<7%). In contrast, aminoglycoside (gentamicin and streptomycin) and ciprofloxacin resistances were higher than 70%, indicating that these may constitute intrinsic resistances. The genetic basis for ciprofloxacin resistance could not be verified, since no mutations typical of quinolone resistances were detected in the quinolone determining regions of the parC and gyrA genes. Some starter strains showed low-level ampicillin, penicillin, chloramphenicol, and tetracycline resistances, but no known resistance genes could be detected. Although some strains possessed the cat gene, none of these were phenotypically resistant to chloramphenicol. Using reverse transcription-PCR, these cat genes were shown to be silent under both inducing and noninducing conditions. Only Lactobacillus salivarius BFE 7441 possessed an ermB gene, which was encoded on the chromosome and which could not be transferred in filter-mating experiments. This study clearly demonstrates problems encountered with resistance testing, in that the breakpoint values are often inadequately identified, resistance genes may be present but silent, and the genetic basis and associated resistance mechanisms toward some antibiotics are still unknown.

Functional and Safety Aspects of Enterococci Isolated from Different Spanish Foods

The incidence and diversity of enterococci in retail food samples of meat, dairy and vegetable origin was investigated. Enterococci were present, at concentrations of 10(1) to 10(4) CFU/g. Fifty selected isolates from food samples grouped in two separate clusters by RAPD analysis. Cluster G1 (72% of the isolates) contained the E. faecium CECT 410T type strain, and also showed a high degree of genetic diversity. Cluster G2 (28% of the isolates) contained the E. faecalis CECT 481T type strain and was genetically more homogeneous. Virulence traits (haemolysin, gelatinase or DNAse activities, or the presence of structural genes cylL, ace, asal and esp) were not detected. All isolates were sensitive to the antibiotics ampicillin, penicillin, gentamicin, streptomycin and chloramphenicol. A high pecentage of isolates were resistant to erythromycin and rifampicin. Many isolates showed intermediate sensitivity to several antibiotics (tetracycline, ciprofloxacin, levofloxacin, or quinupristin/dalfopristin). Vancomycin and teicoplanin resistance was detected in one strain, but vanA, vanB, vanC1, vanC2 or vanC3 genes were not detected. Many of the isolates showed functional properties of food or health relevance. Production of antimicrobial substances was detected in 17 of the isolates, and 14 of them carried structural genes for enterocins A, B and/or P.

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces.

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by α‐chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS‐PAGE, was approximately 3·4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.

Production and characterization of enterocin 900, a bacteriocin produced by Enterococcus faecium BFE 900 from black olives.

Enterococcus faecium BFE 900 isolated from black olives produced a bacteriocin termed enterocin 900, which was antagonistic towards Lactobacillus sake, Clostridium butyricum, enterococci as well as Listeria spp. including Listeria monocytogenes. Enterocin 900 was inactivated by pepsin, alpha-chymotrypsin, proteinase K and trypsin but not by catalase, alpha-amylase, or other non-proteolytic enzymes tested. The bacteriocin was heat stable, retaining activity after heating at 121 degrees C for 15 min. Enterocin 900 was active at pH values ranging from 2.0-10.0, with highest activity at pH 6.0. Bacteriocin production occurred in the late logarithmic growth phase when culture density was ca. log 8.0 CFU ml-1. Enterocin 900 was produced in media with initial pH ranging from 6.0-10.0, but not in media with a pH lower than 6.0. Medium composition, especially the concentrations of peptone and yeast extract influenced bacteriocin production, with no bacteriocin being produced in the absence of either of these compounds. No plasmids could be isolated from Enterococcus faecium BFE 900, indicating that the gene for bacteriocin activity is located on the chromosome.

Microbiological and biochemical profile of cv. Conservolea naturally black olives during controlled fermentation with selected strains of lactic acid bacteria

The effect of controlled fermentation processes on the microbial association and biochemical profile of cv. Conservolea naturally black olives processed by the traditional anaerobic method was studied. The different treatments included (a) inoculation with a commercial starter culture of Lactobacillus pentosus, (b) inoculation with a strain of Lactobacillus plantarum isolated from a fermented cassava product and (c) uninoculated spontaneous process. Microbial growth, pH, titratable acidity, organic acids and volatile compounds were monitored throughout the fermentation. The initial microbiota consisted of Gram-negative bacteria, lactic acid bacteria and yeasts. Inhibition of Gram-negative bacteria was evident in all processes. Both starter cultures were effective in establishing an accelerated fermentation process and reduced the survival period of Gram-negative bacteria by 5 days compared with the spontaneous process, minimizing thus the likelihood of spoilage. Higher acidification of the brines was observed in inoculated processes without any significant difference between the two selected starter cultures (113.5 and 117.6mM for L. plantarum and L. pentosus, respectively). L. pentosus was also determined as the major species present during the whole process of spontaneous olive fermentation. It is characteristic that lactic acid fermentation was also initiated rapidly in the spontaneous process, as the conditions of fermentation, mainly the low salt level (6%, w/v) favored the dominance of lactic acid bacteria over yeasts. Lactic, acetic and propionic were the organic acids detected by HPLC in considerable amounts, whereas citric and malic acids were also present at low levels and degraded completely during the processes. Ethanol, methanol, acetaldehyde, ethyl acetate were the major volatile compounds identified by GC. Their concentrations varied among the different treatments, reflecting varying degrees of microbial activity in the brines. The results obtained from this study could help the Greek table olive industry to improve the existing processing schemes in order to increase product consistency and quality expanding the international market for naturally black olives.