Gerhard Reuter

Taxonomy and physiology of probiotic lactic acid bacteria

The current taxonomy of probiotic lactic acid bacteria is reviewed with special focus on the genera Lactobacillus, Bifidobacterium and Enterococcus. The physiology and taxonomic position of species and strains of these genera were investigated by phenotypic and genomic methods. In total, 176 strains, including the type strains, have been included. Phenotypic methods applied were based on biochemical, enzymatical and physiological characteristics, including growth temperatures, cell wall analysis and analysis of the total soluble cytoplasmatic proteins. Genomic methods used were pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA-PCR (RAPD-PCR) and DNA-DNA hybridization for bifidobacteria. In the genus Lactobacillus the following species of importance as probiotics were investigated: L. acidophilus group, L. casei group and L. reuteri/L. fermentum group. Most strains referred to as L. acidophilus in probiotic products could be identified either as L. gasseri or as L. johnsonii, both members of the L. acidophilus group. A similar situation could be shown in the L. casei group, where most of the strains named L. casei belonged to L. paracasei subspp. A recent proposal to reject the species L. paracasei and to include this species in the restored species L. casei with a neotype strain was supported by protein analysis. Bifidobacterium spp. strains have been reported to be used for production of fermented dairy and recently of probiotic products. According to phenotypic features and confirmed by DNA-DNA hybridization most of the bifidobacteria strains from dairy origin belonged to B. animalis, although they were often declared as B. longum by the manufacturer. From the genus Enterococcus, probiotic Ec. faecium strains were investigated with regard to the vanA-mediated resistance against glycopeptides. These unwanted resistances could be ruled out by analysis of the 39 kDa resistance protein. In conclusion, the taxonomy and physiology of probiotic lactic acid bacteria can only be understood by using polyphasic taxonomy combining morphological, biochemical and physiological characteristics with molecular-based phenotypic and genomic techniques.

Elective and selective media for lactic acid bacteria

Abstract Lactic acid bacteria comprise a heterogeneous group of microorganisms. Isolates from food include representatives of the genera Lactobacillus, Streptococcus, Pediococcus and Leuconostoc. As no universal selective medium exists for the cultivation of all lactic acid bacteria the well-known media for lactobacilli may be modified by changing the pH value, the concentrations of inhibitory agents and the incubation time and temperature. It may be necessary to select media for special fields of application, e.g. milk and milk products, meat and meat products. Surface plating under microaerophilic or anaerobic conditions is generally recommended. Generally suitable as selective media for lactic acid bacteria are MRS-medium, adapted to a lower pH (e.g. 5.7) with the addition of an inhibitor (e.g. sorbic acid) or a modified Rogosamedium with increased pH (e.g. 6.2). The LaS (lactobacilli-sorbic acid) medium (pH 5.0) incubated for at least 48 h is also suitable but fails to recover enterococci. However, this can be compensated by a special enterococcus medium used in parallel. Elective media for defined groups of lactic acid bacteria such as lactic streptococci or yoghurt bacteria have recently been used successfully. One of those is the M 17 medium for lactic streptococci which must be used strictly according to the recommendations of the authors. Good growth can also be obtained with the BRIGGS or RCM medium, the latter supplemented with 1% dextrose or lactose. For the differentiation of the yoghurt lactic acid bacteria the TPPY medium proved suitable as well as the LSD medium which was introduced recently. No special medium can be recommended at present for the selective cultivation of leuconostocs or pediococci. Selection of media has to be determined by the microflora of the habitat to be investigated considering the individual experience of the investigator. It seems reasonable in many cases to use two media at the same time. Quality assurance of media must be carried out regularly using test strains to check selectivity and productivity. The easiest way is a semi-quantitative loop streaking technique which may be used in different modifications. The confirmation of testing can be done by a quantitative drop-plating procedure.

Identification of probiotic cultures in food samples

Probiotic cultures are used increasingly in dairy products. Confusion occurred due to false declarations and the uncritical selection of strains. Efficacy of probiotic cultures depends on special properties of single strains, however. Over the last 30 years, own experience was made with strains of the genera Lactobacillus and Bifidobacterium, especially out of the groups of Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus reuteri/fermentum which were collected from commercial probiotic food samples as well as from pharmaceuticals and nutrients for animals. They were compared with reference strains from culture collections. Phenotypic criteria revealed a grouping of isolates at genus level and, in most cases, a correct identification of species, too. False declarations of cultures used world-wide as probiotics could thus be detected. In order to confirm questionable species or to characterise specific strains within a species, molecular-based methods had to be applied. Good results were obtained by (1) SDS-PAGE of soluble proteins of cells, (2) RAPD-PCR, (3) PFGE-technique and (4) dot blot hybridisation methods using gene probes. Costs and workload differed considerably. SDS-PAGE and RAPD-PCR required data bases, dot blot hybridisation worked faster and more productively. For confirmations, at least one molecular-based method has to be applied.

Emended Descriptions of Lactobacillus sake (Katagiri, Kitahara, and Fukami) and Lactobacillus curvatus (Abo-Elnaga and Kandler): Numerical Classification Revealed by Protein Figerprinting and Identification Based on Biochemical Patterns and DNA-DNA Hybridizations

A former subgenus of the genus Lactobacillus, “Streptobacterium,” comprises a wide range of species, including the so-called “atypical streptobacteria,” which includes the Lactobacillus sake-Lactobacillus curvatus group. Various identification systems and differentiation criteria for L. sake and L. curvatus have been described previously and are well established. The phenotypic diversity within these two species was the reason for comparing phenotypic variations with DNA homology data. Previously described biotypes of L. sake (Katagiri, Kitahara, and Fukami) and L. curvatus (Abo-Elnaga and Kandler) were the basis for selecting strains. Strains of all known biotypes of these species were examined to determine their biochemical reactions, their physiological growth characteristics, their total soluble protein contents as determined by a pattern analysis in which native polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with colloidal Coomassie blue and silver diamine stainine were used, and their levels of DNA homology as determined by DNA-DNA hybridization experiments. All of the phenotypic analyses revealed a diversity within the taxa, whereas the DNA-DNA hybridization analysis revealed that the level of genomic homogeneity within each species was relatively high. The phenotypic diversity and genomic homogeneity which we observed allowed us to describe subgroups of L. sake and L. curvatus. The descriptions of L. sake and L. curvatus are emended accordingly. These subgroups which we describe may be the basis for defining subspecies within these two species.

Culture media for enterococci and group D-streptococci.

Lancefield group D-streptococci are contaminants of various food commodities, especially those of animal origin. They encompass the new genus Enterococcus comprising 13 known species and some species of streptococci which have their habitat in the intestine of animals, e.g. Streptococcus bovis, suis and equinus. The serologically based grouping may no longer constitute the best definition for streptococci from the food chain. Food hygiene monitoring systems using enterococci as indicators need reliable methods for selective cultivation and identification of marker strains. Up to now more than 100 modifications of selective media have been described for isolating streptococci or enterococci from various specimens. The selection of a medium requires either experience or consultation. It depends on the kind of specimen, the method of cultivation (plate count or membrane filter) and whether or not the habitat is heavily contaminated with other organisms. The choice of media is made more difficult as commercial versions of the same culture medium may vary in recipe and/or performance from producer to producer. Therefore, reviewing the literature may help in the choice of medium and confirmation tests. The selectivity and productivity of some commonly used or cited media are reported here, partly based on our own experience: citrate azide tween carbonate agar (CATC), kanamycin aesculin azide agar (KAA) and M-enterococcus agar (ME) including earlier results with aesculin bile azide agar (ABA), and thallous acetate tetrazolium glucose agar (TITG). No medium was completely selective for all group D-streptococci or for all enterococci but some media were highly selective for a single Enterococcus species, e.g., for E. faecalis which serves as indicator of human pollution. Confirmatory tests must be carried out when experience in the evaluation procedure is limited. Selective media for enterococci should be used only after or while checking in parallel their selectivity and productivity against appropriate test organisms.

Impedance measurement as a rapid method for the determination of the microbial contamination of meat surfaces, testing two different instruments

Abstract The suitability of the impedance measurement method was considered as a rapid method for use in the routine estimation of the microbial contamination of meat surfaces. Two instruments which have been developed recently were available, namely the Bactometer 32 and the 8-Channel Malthus-Meter. A total of 256 destructive and 82 rinsed samples were taken from beef carcasses and examined chilling temperatures for 3–7 days. Preliminary experiments using both instruments with 49 destructive samples yielded the correlation of r = −0.912 with s y · x = 0.39 (Malthus-Meter) and of r = −0.906 with s y · x = 0.42 (Bactometer) compared with cfu-values obtained by the drop plating method. 22 rinsed samples produced values of r = −0.812 with s y · x = 0.57 (Malthus-Meter) and r = −0.714 with s y · x = 0.83 (Bactometer). The Bactometer was manually more convenient. The interpretation of the results was easier with the Malthus-Meter. Some technical difficulties occuring with the Malthus-Meter may have been caused by the prototype nature of the instrument available. Further investigations with the Bactometer 32 yielded faster and better results using a Stomacher instead of an Ultra Turrax for homogenisation and brain heart infusion broth enriched with 0.1% yeast extract instead of tryptone soya broth with yeast extract. Correlation of r = −0.928 with s y · x = 0.17 could be achieved with destructive samples and of r = −0.896 with s y · x = 0.3 with rinsed samples. Microbial combination of 10 7 cells and above, per cm 2 meat surface, could be accurately detected within 2 h ( α = 0.05) including the preparation procedure. The impedance reactions were influenced to a large degree by the Enterobacteriaceae. This group correlates very well with the total count of meat surface flora. Impedance measurement can therefore be recommended as a routine method for the determination of the microbial load of this biotope of food microflora.

Selective media for group D streptococci

Abstract Group D streptococci are contaminants of different food commodities, especially those of animal origin. They encompass the physiological subgroups of enterococci, non-enterococci and enterococcus-like organisms. The first and the last subgroups may be included in the proposed new genus Enterococcus in the future. The serologically base grouping may be no longer constitute the best definition of streptococci which are important in food hygiene. Up to the present day more than 100 modifications of selective media have been described for group D streptococci or enterococci. Therefore it is impossible to recommend a universal medium. The choice of a medium depends on whether group D streptococci in total or enterococci are to be detected and whether or not the habitat is highly contaminated. The selectivity and productivity of the following media were compared using a set of test strains: citrate-azide-Tween-carbonate-agar (CATC), kanamycin-aesculin-azide-agar (KAA) and M-enterococcus-agar (ME). Earlier results [Reuter (1978) Arch. Lebensmittelhyg. 29, 84–91] with these and some other media, e.g. aesculin-bile-azide-agar, thallous-acetate-agar, crystal violet-azide-agar, streptococci-selective-agar were considered as well. No medium was completely selective for group D streptococci or for enterococci. Either growth of non-wanted strains occurred or growth rate of single wanted strains was reduced. The parallel use of two media, one highly, the other moderately selective is a reasonable way to obtain best results from a food habitat. Choice of media must be a result of personal experience on particular habitats to be investigated. Confirmatory tests have to be performed in many cases. Commercially prepared media may be used after checking their selectivity and productivity against appropriate test organisms.

The bioluminescence technique as a rapid method for the determination of the microflora of meat

Abstract The adenosine triphosphate (ATP) content of bacterial cells was determined by means of the bioluminescence technique. Primarily 9 bacteria species which had been selected from meat microflora were tested. Beef meat surfaces were examined using a destructive method (237 samples) and a rinse method (85 samples). The ATP content of the cells of pure cultures varied according to the type of bacteria and the physiological state of the culture. During the lag- and log-phases of growth the ATP level was higher than in the stationary and decline phases. Differences of up to 80% were observed. Rod shaped bacteria yielded higher percentage differences than cocci. However, a good correlation could be observed between ATP values and the total number of viable cells, viz. r = 0.92 with s y · x = 0.41 (drop plating method), r = 0.91 with s y · x = 0.42 (surface plating method) and r = 0.94 with s y · x = 0.37 (an epifluorescent filter technique). A very close relationship of r = 0.96 with s y · x = 0.28 was detected in the case of Enterobacteriaceae and Pseudomonadaceae. With heavily contaminated meat samples correlations of r = 0.66 to 0.87 were achieved. Deviations were caused by the sampling procedure, the methods of preparation and the chemical treatment. Treatment with a nucleotide-releasing agent for somatic cells and arsenate-malate buffer was found to be the most suitable way to eliminate the somatic ATP within the sample. The lowest reliable detectable number of microorganisms/ml homogenate was about 5 × 10 6 (= about 10 5 per test) cells corresponding to 1 × 10 −8 mol ATP/l. An average conversion factor of 1.5 mamol/cell ( =11.5 × 10 −18 mol) was found to be justifiable for a prognostic estimation of viable cells from meat samples. About 100 samples per day can be assayed using a manual technique involving one person. The running costs are moderate (about 4.- DM/sample). Microprocessor computerized bioluminescence equipment can analyse 70 to 80 samples/h at an even lower cost level. The bioluminescence method has therefore been proved to be a convenient, feasible, rapid method for the determination of microbial contamination of meat samples.

A selective medium for the detection and enumeration of mesophilic sulphite-reducing clostridia in food monitoring programs

Abstract Experiments with 54 strains of 12 clostridia species and 51 ‘representative microorganisms’ from food microflora were carried out. Selectivity, significance of sulphite reduction and growth rates were tested with a recently developed sul-phite-cycloserine-azide medium (SCA) and five well-known selective media for clostridia (sulphite-polymyxin-sulfadiazin agar [SPS], tryptone-sulphite-neomycin agar [TSN], oleandomycin-polymyxin-sulfadiazin-perfringens-selective agar [OPSP], Shahidi- Ferguson-perfringens-selective agar [SFP], tryptose-sul-phite-cycloserine-selective agar [SC]). As elective medium Brewer agar was used. Pour plating and cultivation in anaerobic jars were done. The SCA medium showed the best results with regard to selectivity and colony count rates (productivity) compared with the other selective media. A clear differentiation of the mesophilic sulphite-reducing clostridia from any accompanying lactic acid bacteria or enterococci was possible by the way of identification of the blackening of the colonies (sulphite reduction). A confirmation that the blackened colonies were clostridia and not any exceptionally growing sulphite-reducing Enterobacteriaceae could be easily achieved by means of Gram-staining.

Intra-species characterization of clinical isolates and biotechnologically used strains of Lactobacillus rhamnosus by analysis of the total soluble cytoplasmatic proteins with silver staining☆

Lactobacillus rhamnosus is often used in milk products because of its technological properties, however it is supposed to be related to human infections as well. Therefore, 23 L. rhamnosus strains, including the type strain, were investigated. Nine strains resulted from biotechnological sources, 14 isolates had a clinical background, 13 came from a swedish culture collection. Biochemical and physiological properties were proved by classical tests. Analysis of the total soluble cytoplasmatic protein patterns was performed with diamine silver staining, a technique not previously applied to lactobacilli. Classical tests were able to confirm all strains as L. rhamnosus. Analysis of protein patterns allowed to differentiate between three clusters. Cluster I contained the type strain and biotechnologically used strains and one clinical strain isolated in Berlin. The second consisted of both, technological and clinical strains. The third cluster contained clinical isolates alone. SDS-PAGE of proteins together with diamine silver staining seems to be helpful to detect intra-species differences. It was stated, that technologically used strains of L. rhamnosus could clearly be differentiated from each other and also from clinical isolates.