Lactococcus piscium: a psychrotrophic lactic acid bacterium with bioprotective or spoilage activity in food-a review.

The genus Lactococcus comprises 12 species, some known for decades and others more recently described. Lactococcus piscium, isolated in 1990 from rainbow trout, is a psychrotrophic lactic acid bacterium, probably disregarded because most of the strains are unable to grow at 30°C. During the last 10 years, this species has been isolated from a large variety of food: meat, seafood and vegetables, mostly packed under vacuum (VP) or modified atmosphere (MAP) and stored at chilled temperature. Recently, culture-independent techniques used for characterization of microbial ecosystems have highlighted the importance of Lc. piscium in food. Its role in food spoilage varies according to the strain and the food matrix. However, most studies have indicated that Lc. piscium spoils meat, whereas it does not degrade the sensory properties of seafood. Lactococcus piscium strains have a large antimicrobial spectrum, including Gram-positive and negative bacteria. In various seafoods, some strains have a protective effect against spoilage and can extend the sensory shelf-life of the products. They can also inhibit the growth of Listeria monocytogenes, by a cell-to-cell contact-dependent. This article reviews the physiological and genomic characteristics of Lc. piscium and discusses its spoilage or protective activities in food.

Characterization of European Yersinia enterocolitica 1A strains using restriction fragment length polymorphism and multilocus sequence analysis.

UNLABELLED: Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2-5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2-4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2-4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2-5. SIGNIFICANCE AND IMPACT OF THE STUDY: Yersinia enterocolitica strains are currently divided into six biotypes and two subspecies. Strains of biotype 1A, which are phenotypically and genotypically very heterogeneous, are classified as subspecies palearctica. In this study, European Y. enterocolitica 1A strains isolated from both human and nonhuman sources were characterized using restriction fragment length polymorphism and multilocus sequence analysis. The European biotype 1A strains formed a separate group, which differed from strains belonging to subspecies enterocolitica and palearctica. This may indicate that the current division between the two subspecies is not sufficient considering the strain diversity within Y. enterocolitica.

Packaging gas selects lactic acid bacterial communities on raw pork.

AIMS: To study the effect of different CO2-rich packaging atmospheres on the composition of lactic acid bacterial communities proliferating on raw pork. METHODS AND RESULTS: Raw pork loin was inoculated with a mixture of 14 lactic acid bacteria (LAB) strains previously associated with meat and packaged with four gas atmospheres: (i) 100% CO2 (ii) 80% N2 20% CO2 (iii) 80% N2, 20% CO2, 0·4% CO and (iv) 80% O2, 20% CO2. The colony counts of LAB, pH and composition of packaging gas were monitored every other day during the storage of 14 days at +6°C. The compositions of lactic acid bacterial communities on pork were evaluated after 7 days of storage with culture-independent, terminal restriction fragment length polymorphism analysis of 16S rRNA gene fragments. After 14 days of storage, the compositions of lactic acid bacterial communities were evaluated using identification of plate-grown LAB isolates by numerical ribopattern analysis. The results showed that (i) high concentration of CO2 in packaging atmosphere favoured Lactobacillus sp. (ii) high concentration of O2 favoured Leuconostoc spp. (iii) atmosphere with 80% N2, 20% CO2 favoured Lactococcus sp. CONCLUSIONS: The composition of modified packaging atmosphere is a major factor selecting lactic acid bacterial communities proliferating on raw meat. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides an explanation for the compositions of lactic bacterial communities on modified atmosphere packaged raw meat observed in other studies. The results should be considered when attempting to manipulate LAB communities in raw meat, e.g. by protective cultures.

Characteristics of Yersinia enterocolitica biotype 1A strains isolated from patients and asymptomatic carriers.

Yersinia enterocolitica biotype 1A strains are frequently isolated from the environment, foods, and animals, and also from humans with yersiniosis. There are controversial reports on the pathogenicity of biotype 1A strains. In this study, 811 fecal samples from asymptomatic humans from Switzerland were studied for the presence of Y. enterocolitica. Nine (1.1%) of the 811 samples were positive for Y. enterocolitica 1A. These strains were compared with 12 Y. enterocolitica 1A strains from Swiss patients with diarrhea isolated in the same year. Almost all (20/21) Y. enterocolitica 1A strains carried the ystB gene, seven strains carried the hreP gene, and none carried the ail, ystA, myfA, yadA, or virF genes. Most (17/21) Y. enterocolitica 1A strains belonged to two major clusters, A and B, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Strains of cluster B were only isolated from humans with diarrhea; however, ystB and hreP genes were detected in strains from both clinical and non-clinical samples and from strains of clusters A and B. Using ribotyping, six restriction patterns among biotype 1A strains were obtained with HindIII enzyme. The most common ribotype (RT I) was found in strains isolated from humans with and without diarrhea. All biotype 1A strains had a unique NotI profile by pulsed-field gel electrophoresis (PFGE), showing a very high genetic diversity. In this study, Y. enterocolitica 1A strains from clinical and non-clinical samples could not be clearly differentiated from each other. More research is needed in order to prove that biotype 1A strains are a primary cause for human yersiniosis and not only a secondary finding.

Identification and antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from bovine milk samples.

The conventional identification of Streptococcus uberis/parauberis group (n = 137) in clinical and subclinical bovine mastitis samples originating from 111 different farms was compared with identification based on 16 and 23S rRNA gene HindIII RFLP patterns used as operational taxonomic units in numerical analyses. On the basis of ribopattern analysis only 2 isolates belonged to S. parauberis, which is thus not a frequent cause of bovine intramammary infections in Finland. According to in vitro antimicrobial susceptibility testing, Streptococcus uberis is susceptible to beta-lactam antibiotics. The prevalence of erythromycin (15.6%) and oxytetracycline (40.6%) resistance of clinical S. uberis isolates was higher than reported previously among subclinical isolates. The 2 subclinical S. parauberis isolates were susceptible to all the antimicrobials tested.

Bovine intramammary infections caused by coagulase-negative staphylococci may persist throughout lactation according to amplified fragment length polymorphism-based analysis.

Persistence of coagulase-negative staphylococci (CNS) in intramammary infections during lactation was studied in a research dairy herd of University of Helsinki. Milk samples from 328 udder quarters of 82 dairy cows (30 primiparous, 52 multiparous) were collected 2 wk before calving, at calving, and every 4 wk thereafter until the end of lactation or until the cow left the herd. The CNS isolated from the milk samples were analyzed with the API Staph ID 32 (bioMérieux, Marcy l'Etoile, France) test (API) and genotyped using amplified fragment length polymorphism (AFLP) analysis. The AFLP patterns were used for similarity analysis between CNS isolates and for species identification. For the latter, AFLP patterns of CNS isolates and staphylococcal type strains were used as operational taxonomic units in numerical analysis. In addition, the somatic cell count (SCC) of the milk samples was measured during lactation. A CNS infection was considered persistent when isolates originating from the same quarter had identical AFLP patterns on at least 3 consecutive samplings. In total, 63 CNS infections were detected during lactation in 30 and 33 quarters in the first and later lactations, respectively. Twenty-nine of these infections persisted and 34 were transient. Most of the persistent infections lasted until the end of lactation. In 57 quarters, CNS infection was detected before calving, at calving, or both, but only half of these quarters were infected by CNS during subsequent lactation. The geometric mean of SCC in quarters during persistent CNS infection was 657,600 cells/mL, and the mean of SCC in quarters with transient CNS infection was 619,100 cells/mL. The median of SCC in quarters during persistent CNS infection was 355,400 cells/mL, and the median of SCC in quarters with transient CNS infection was 133,500 cells/mL. According to both the API test and AFLP results, Staphylococcus chromogenes and Staphylococcus simulans were the CNS species isolated most often. Identification results for API and AFLP corresponded in 71.9% of the isolates.

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 strain's 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.

Comparative molecular field analysis of some clodronic acid esters.

Comparative molecular field analysis (CoMFA) has been used as a three-dimensional quantitative structure-activity relationship (QSAR) method to correlate three different types of biological activity data with physicochemical properties of some clodronate ester analogues, which act as bone-resorption regulators in cell cultures and rats. The QSAR studies show the importance of the steric properties of these new bisphosphonate derivatives for the inhibition of bone resorption in bone cell cultures and for their bioavailability in rats. This information will be used in predicting the structure of new more potent bisphosphonic compounds.

Ropy slime-producing Lactobacillus sake strains possess a strong competitive ability against a commercial biopreservative.

Aseptically handled Frankfurters were treated with a commercial Lactobacillus alimentarius biopreservative and inoculated with different cell concentrations of four ropy slime-producing Lactobacillus sake strains. The packages were vacuum sealed and kept at 6 degrees C for 28 days, after which the production of ropy slime was evaluated. The inoculation test was controlled by sealing the different control packages containing either aseptically manufactured sausages without any bacterial inoculation, packages containing biopreservative only or packages inoculated only with the four different ropy slime-producing strains. Authenticity of the biopreservative strain after the cold storage period was ascertained by performing EcoRI restriction endonuclease analysis of 30 randomly selected isolates originating from the biopreservative control packages. All patterns were identical to the pattern of the original L. alimentarius biopreservative strain. The biopreservative was found to be ineffective against the four ropy slime-producing L. sake strains. The strongest slime producers inoculated with approximately 1 colony forming units (CFU)/cm2 could compete efficiently with the L. alimentarius inoculated at a level of 10(7) CFU/cm2 on sausage surfaces. This commercial biopreservative failed to occupy the vital niche of the four ropy slime-producing L. sake strains leading to spoilage in almost all packages.

rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime.

The rRNA gene restriction patterns (ribotypes) of 69 ropy slime producing Lactobacillus sake strains isolated mainly from vacuum-packaged meat products of ten meat plants were determined. Ribotypes were compared to the corresponding patterns of non-ropy L. sake strains, and also to other species of the genus Lactobacillus, Carnobacterium and Weissella associated with meat products. Ropy slime-producing L. sake strains were divided into four characteristic groups corresponding to the phenotypic carbohydrate grouping. No association between certain ribotypes and individual plants was detected. Ribotyping was unable to distinguish slime producing and non-ropy strains of L. sake group sharing the same carbohydrate pattern. Otherwise, ribotyping distinguished the ropy slime producing strains from the non-ropy L. sake reference strains and all L. sake strains from other species of the genus Lactobacillus, Carnobacterium and Weissella. These results suggest that ribotyping is a suitable method for detection and surveillance of the contamination of ropy slime-producing L. sake strains but the patterns alone cannot be used as markers of slime production capability.

Characterization of Lactobacillus sake strains associating with production of ropy slime by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) patterns.

Randomly amplified polymorphic DNA (RAPD) patterns and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of ropy slime-producing Lactobacillus sake strains. The two most revealing commercially available primers (OPJ 12 and OPJ 16, Operon Inc. Alameda, USA) and two rare-cutting enzymes (AvrII and SmaI) were chosen from a pretested lot for the typing of 69 ropy slime-producing strains, 7 non-ropy isolates and 4 non-ropy reference strains. Both RAPD and PFGE patterns confirmed the group division established in previous studies and provided new information concerning ropy slime-producing strains. PFGE patterns were found to have the greatest discriminatory power, revealing the genetic variation of the main group of ropy slime-producing L. sake strains and distinguishing all non-ropy strains from slime-producers.

Characterisation of lactic acid bacteria from spoiled, vacuum-packaged, cold-smoked rainbow trout using ribotyping.

A total of 405 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged, salted, sodium nitrite- or potassium nitrate-treated, cold-smoked rainbow trout stored at 4 degrees C or 8 degrees C were characterised and identified using a molecular method. The isolates were initially classified according to their restriction endonuclease profiles using HindIII and EcoRI restriction endonucleases and further characterised by rRNA gene restriction patterns (ribotypes). Numerical analysis of these ribopatterns was performed together with 19 reference LAB strain patterns in order to identify the isolates to species level. The strains were divided with HindIII and EcoRI ribopatterns into ten and nine clusters at the similarity level of 65% and 50%, respectively. The Leuconostoc-clusters and the Lb. sakei/Lb. curvatus-clusters formed the two main groups. Only one isolate was identified as Lactobacillus plantarum and no Carnobacterium strains were discovered. For both enzymes, the 35 isolates possessing six individual ribotypes and forming five clusters could not be identified further with the reference strains used. The relative proportion of Leuconostoc mesenteroides subsp. mesenteroides was higher in all samples stored at 4 degrees C. Most of the Leuconostoc citreum were found in the samples stored at 8 degrees C, and particularly in the nitrite-treated samples.

The spoilage flora of vacuum-packaged, sodium nitrite or potassium nitrate treated, cold-smoked rainbow trout stored at 4 degrees C or 8 degrees C.

The spoilage flora of vacuum-packaged, salted, cold-smoked rainbow trout fillets, with or without the addition of nitrate or nitrite, stored at 4 degrees C and 8 degrees C, was studied. Of 620 isolates, lactic acid bacteria were the major fraction (76%), predominating in all samples of spoiled product. However, the phenotypical tests used were insufficient to identify the lactic acid bacteria to the species level. Gram-positive, catalase-positive cocci, gram-negative, oxidase-negative rods and gram-negative, oxidase-positive rods were found in 6%, 16% and 2% of the samples, respectively. Of 39 gram-positive, catalase-positive cocci, 29 were identified as staphylococci and 10 as micrococci. Eighty-five isolates were found to belong to the family Enterobacteriaceae, with 45 of those being Serratia plymuthica. Eleven isolates from the nitrate treated samples stored at 8 degrees C were identified as Pseudomonas aeruginosa. The occurrence of P. aeruginosa and staphylococci in the nitrate-containing samples, stored at 8 degrees C, may cause problems with respect to the safety of the product. The types of lactic acid and other bacteria in the spoilage flora were generally reduced by the addition of nitrate or nitrite to fillets.

Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR.

Random amplified polymorphic DNA analysis (RAPD) and repetitive element sequence-based PCR (rep-PCR) were evaluated with respect to their applicability to characterise Clostridium botulinum group I and II strains, the species causing human botulism. Fifteen group I and 21 group II strains of various geographical and temporal origins were characterised with four single arbitrary RAPD primers at low stringency amplification conditions and with a degenerate REP primer pair at moderately stringent conditions. Ready-To-Go RAPD Analysis Beads and Ready-To-Go PCR Beads were used for PCR reactions with RAPD and rep-PCR, respectively. Arbitrary primer OPJ 6 yielded the most discriminating patterns, and distinguished group II C. botulinum serotypes at the strain level. Group I strains were mainly discriminated at the serotype level. The discriminatory power of rep-PCR was found to be inferior to that of RAPD. The REP1R-Dt and REP2R-Dt primer pair generated group I- and II-specific fragments and arbitrary primer OPJ 13 produced a serotype E-specific fragment. The use of pre-dispensed and pre-optimised beads attributed to highly reproducible results. As compared to more time-consuming typing methods, such as pulsed-field gel electrophoresis (PFGE), both RAPD and rep-PCR were characterised by rapid performance and a typeability of 100%.

Ribotyping as an identification tool for Clostridium botulinum strains causing human botulism.

Ribotyping was used for characterisation of 68 Clostridium botulinum strains and five related Clostridium species to determine the applicability of this method for identification of species causing human botulism. Thirteen restriction enzymes were initially tested for suitability for ribotyping of C. botulinum, of which EcoRI and HindIII were selected. Both enzymes clearly differentiated between proteolytic (group I) and a nonproteolytic (group II) strains of C. botulinum, and can be recommended for Group/species identification. Using a commercial software package (GelCompar), a numerical analysis of the discriminatory abilities of EcoRI and HindIII ribotyping within and between the two C. botulinum groups was performed. EcoRI had the higher discriminatory index (0.982), but the ribopatterns generated with group II strains were partly muddled and difficult to interpret. All HindIII ribopatterns were easy to analyse and the discriminatory index for all strains was almost equally high (0.954), whereas this enzyme did not discriminate well between group I isolates. The Clostridium strains diverged at 35+/-13% (mean+/-standard deviation) Dice similarity in dendrograms based on cluster analysis of the ribotyping results. These findings are in good agreement with taxonomical ribotyping studies with other bacterial genera, indicating that ribotyping is a highly suitable method for C. botulinum species identification.

Lactobacillus alimentarius: a specific spoilage organism in marinated herring.

Spoilage characterised by bulging of lids and gas formation affected various product lots of different marinated herring types. Microbiological analyses resulted in growth on MRS and Rogosa SL agar. Altogether, 206 randomly selected colonies from two unspoiled and ten spoiled samples were characterised using phenotypical key tests and a 16 + 23S rRNA gene-based RFLP identification database. L. alimentarius was found to be the specific spoilage organism in all samples. All isolates obtained from the different product types were of the same clonal type. The slight rise in pH value together with marked gas production suggested a rare lactic acid bacteria spoilage type called 'protein swell'. L. alimentarius has not been previously associated with herring spoilage.

Persistence in bovine mastitis of Staphylococcus aureus clones as assessed by random amplified polymorphic DNA analysis, ribotyping and biotyping.

Staphylococcus aureus isolates (N = 40) from bovine mastitis were characterized by random amplified polymorphic DNA-PCR (RAPD-PCR), ribotyping and biotyping. The isolates were collected in the veterinary surveillance area of the Ambulatory Clinic, Faculty of Veterinary Medicine, University of Helsinki from 20 quarters during the acute phase of infection and from the same quarters 3 weeks after cessation of therapy. The aim of the study was to compare the S. aureus isolates taken from the same quarter at different times to verify persistence of virulent strains in infected quarters and to compare the discriminatory power of the diagnostic methods. Using all methods (except for a commercial diagnostic test), the paired isolates of S. aureus were identical. Results suggest that the chronic nature of S. aureus infections was due to the persistence of the original infective strain. More laborious ribotyping and the more convenient RAPD-PCR method produced identical results. The molecular methods differentiated the 40 isolates into 6 distinct genotypes. Biotyping produced partially identical results to RAPD and ribotyping. A commercial diagnostic test system identified only 3 S. aureus biotypes.

Hydrophobicity and central nervous system agents: on the principle of minimal hydrophobicity in drug design.

The problem of getting drugs across the so-called blood-brain barrier (BBB) has long been under extensive investigation; however, the other side of the problem, that of keeping drugs out of the central nervous system (CNS), has not been studied so intently. As we strive to make more and more refined drugs with fewer side effects, the problem of keeping drugs out of the CNS has possibly become more important than getting them in. The role of lipophilicity has long been recognized as being important in CNS penetration by chemicals, but we believe that not enough attention has been devoted to just exactly what is meant when it is said that "a lipophilic drug is needed for CNS penetration." How lipophilic? Can hydrophilic properties keep drugs out of the CNS? How hydrophilic should they be? There are other reasons for making drugs hydrophilic. Hydrophobic drugs, other factors being equal, are more inhibitory of biochemical systems than hydrophilic congeners. Evidence is beginning to show that cytochrome P450 is induced in direct proportion to hydrophobicity by a variety of compounds, and cytochrome P450 may produce modifications in lipophilic molecules in the body. Hydrophobic drugs are more slowly eliminated. This report discusses these problems in terms of the octanol-water (log P) hydrophobic scale. The principle is proposed that, without convincing evidence to the contrary, drugs should be made as hydrophilic as possible without loss of efficacy. Antihistamines are discussed in terms of what kind of hydrophobic-hydrophilic balance is best to avoid CNS-related problems.

Prevalence of the enterotoxin gene and clonality of Clostridium perfringens strains associated with food-poisoning outbreaks.

The prevalence of the enterotoxin gene in a well-characterized collection of 71 Clostridium perfringens strains from 36 separate food-poisoning cases or outbreaks was analyzed with the polymerase chain reaction (PCR). The clonality of 39 strains originating from 14 outbreaks where at least two isolates were available was studied with pulsed-field gel electrophoresis (PFGE) using SmaI and ApaI restriction endonucleases. The cpe gene PCR assay was found to correlate well with Clostridium perfringens enterotoxin (CPE) production in vitro with reverse passive latex agglutination. Of the C. perfringens food and clinical food-poisoning isolates 24 (86%) and 38 (88%) were cpe-positive, respectively. Different PFGE patterns indicated that multiple cpe-positive clones are frequently present within one outbreak. The existence of cpe-positive and negative isolates with identical or nearly identical PFGE patterns in a single outbreak suggests that the cpe gene may be in a movable genetic element.

The effect of marination on lactic acid bacteria communities in raw broiler fillet strips.

Marination with marinade containing salt, sugar, and acetic acid is commonly used in Finland to enhance the value of raw broiler meat. In this study, we investigated the effect of marination, marinade components and storage time on composition of bacterial communities in modified atmosphere-packaged (MAP) broiler fillet strips. The communities were characterized using two culture-independent methods: 16S rRNA gene fragment sequencing and terminal restriction fragment length polymorphism. In unmarinated broiler fillet strips, Lactococcus spp. and Carnobacterium spp. predominated at the early storage phase but were partially replaced by Lactobacillus spp. and Leuconostoc spp. when the chilled storage time was extended. In the marinated fillet strips, Lactobacillus spp. and Leuconostoc spp. predominated independent from the storage time. By mixing the different marinade components with broiler meat, we showed that marination changed the community composition and favored Leuconostoc spp. and Lactobacillus spp. by the combined effect of carbohydrates and acetic acid in marinade. Marination increased the maximum level of lactic acid bacteria in broiler meat and enhanced CO(2) production and acidification of meat during the chilled storage. Accumulation of CO(2) in package head-space due to the enhanced growth of Leuconostoc spp. in marinated meat may lead to bulging of packages, which is a spoilage defect frequently associated with marinated and MAP raw broiler preparations in Finland.