Wilhelm H. Holzapfel

Lactic acid bacteria of foods and their current taxonomy

Application of molecular genetic techniques to determine the relatedness of food-associated lactic acid bacteria has resulted in significant changes in their taxonomic classification. During the 1980s the genus Streptococcus was separated into the three genera Enterococcus, Lactococcus and Streptococcus. The lactic acid bacteria associated with foods now include species of the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. The genus Lactobacillus remains heterogeneous with over 60 species (ymol% G+C content ranging from 33 to 55), of which about one-third are strictly heterofermentative. However, many changes have been made and reorganization of the genus along lines that do not follow previous morphological or phenotypic differentiation from Leuconostoc and Pediococcus is being studied. Phylogenetically belonging to the Actinomyces branch of the bacteria, Lactobacillus bifidus has been moved to the genus Bifidobacterium also on account of its greater than 50 mol% G+C content. It is therefore no longer considered one of the lactic acid bacteria senso strictu, which form part of the Clostridium branch of the bacteria. The new genus Weissella has been established to include one member of the genus Leuconostoc (Leuc, paramesenteroides) and heterofermentative lactobacilli with unusual interpeptide bridges in the peptidoglycan. Contrary to the clear-cut division of the streptococci, morphological and physiological features of Weissella do not directly support this grouping which now incorporates species that produce D(-)- as well as DL-lactate. The new genus Carnobacterium is morphologically similar to the lactobacilli, but it shares some physiological similarities (e.g. growth at pH 9.5) and a common phylogenetic branch with the genus Enterococcus. The review includes information on the taxonomic changes and the relationship of the bacteria of food fermentation and spoilage.

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.

Improved screening procedure for biogenic amine production by lactic acid bacteria

An improved screening plate method for the detection of amino acid decarboxylase-positive microorganisms (especially lactic acid bacteria) was developed. The suitability and detection level of the designed medium were quantitatively evaluated by confirmation of amine-forming capacity using an HPLC procedure. The potential to produce the biogenic amines (BA) tyramine, histamine, putrescine, and cadaverine, was investigated in a wide number of lactic acid bacteria (LAB) of different origin, including starter cultures, protective cultures, type strains and strains isolated from different food products. Also, several strains of Enterobacteriaceae were examined. Modifications to previously described methods included lowering glucose and sodium chloride concentrations, and increasing the buffer effect with calcium carbonate and potassium phosphate. In addition, pyridoxal-5-phosphate was included as a codecarboxylase factor for its enhancing effect on the amino acid decarboxylase activity. The screening plate method showed a good correlation with the chemical analysis and due to its simplicity it is presented as a suitable and sensitive method to investigate the capacity of biogenic amine production by LAB. Tyramine was the main amine formed by the LAB strains investigated. Enterococci, carnobacteria and some strains of lactobacilli, particularly of Lb. curvatus. Lb. brevis and Lb. buchneri, were the most intensive tyramine formers. Several strains of lactobacilli, Leuconostoc spp., Weissella spp. and pediococci did not show any potential to produce amines. Enterobacteriaceae were associated with cadaverine and putrescine formation. No significant histamine production could be detected for any of the strains tested.

The genera of lactic acid bacteria.

Introduction. Phylogenetic Relationships of Lactic Acid Bacteria. The Genus Lactobacillus. The Genus Streptococcus. The Genus Pediococcus. The Genus Lactococcus. The Genus Leuconostoc. The Genus Bifidobacterium. The Genus Carnobacterium. The Genus Enterococcus. Spore-Forming, Lactic Acid Producing Bacteria of the Genera Bacillus and Sporolactobacillus. References. Index

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.

Taxonomy and important features of probiotic microorganisms in food and nutrition

Lactic acid bacteria are among the most important probiotic microorganisms typically associated with the human gastrointestinal tract. Traditionally, lactic acid bacteria have been classified on the basis of phenotypic properties, eg, morphology, mode of glucose fermentation, growth at different temperatures, lactic acid configuration, and fermentation of various carbohydrates. Studies based on comparative 16S ribosomal RNA sequencing analysis, however, showed that some taxa generated on the basis of phenotypic features do not correspond with the suggested phylogenetic relations. Thus, some species are not readily distinguishable by phenotypic characteristics. This is especially true for the so-called Lactobacillus acidophilus group, the Lactobacillus casei and Lactobacillus paracasei group, and some bifidobacteria, strains of which have been introduced in many probiotic foods, eg, the novel yogurt-like commodities. Consequently, modern molecular techniques, including polymerase chain reaction-based and other genotyping methods, have become increasingly important for species identification or for the differentiation of probiotic strains. Probiotic strains are selected for potential application on the basis of particular physiologic and functional properties, some of which may be determined in vitro. The classification and identification of a probiotic strain may give a strong indication of its typical habitat and origin. The species, or even genus name, may also indicate the strains safety and technical applicability for use in probiotic products. Molecular typing methods such as pulsed-field gel electrophoresis, repetitive polymerase chain reaction, and restriction fragment length polymorphism are extremely valuable for specific characterization and detection of such strains selected for application as probiotics.

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.

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.

Use of starter cultures in fermentation on a household scale

Abstract An overview on the role and potential of microorganisms in traditional food fermentations is given. Prospects for the implementation of starter cultures for homescale food fermentations are discussed against the background of the present situation, socio-economic and rural/logistic constraints, and the potential for improvement of traditional processes. Special attention is given to the African situation and the well-accepted lactic fermented, traditional foods. Perspectives are given on the improvement of the nutritional value and the overall product safety, with reference to strain selection on the basis of particular phenotypic and technical features.