Pavel Švec

Enterococcus haemoperoxidus sp. nov. and Enterococcus moraviensis sp. nov., isolated from water

A polyphasic taxonomic approach was used to study atypical enterococci isolated from surface waters. All strains were characterized by physiological and biochemical tests as well as by genotyping. The results of biochemical tests and tRNA intergenic length polymorphism analysis (tDNA-PCR) divided all studied strains uniformly into two groups. Because these groups were clearly separated from all enterococcal species described to date, 16S rDNA sequence analysis, DNA base composition analysis and DNA-DNA hybridization of representative strains were done to elucidate the taxonomic position of the analysed groups. On the basis of the results obtained, the names Enterococcus haemoperoxidus (type strain CCM 4851T = LMG 19487T) and Enterococcus moraviensis (type strain CCM 4856T = LMG 19486T) are proposed for the two hitherto undescribed species. The type strains and reference cultures have been deposited in the Czech Collection of Microorganisms (CCM), Masaryk University, Brno, Czech Republic, and in the BCCM/LMG Culture Collection, Ghent University, Belgium.

Antibiotic resistance in faecal bacteria (Escherichia coli, Enterococcus spp.) in feral pigeons.

Aims:  To determine the presence of antibiotic‐resistant faecal Escherichia coli and Enterococcus spp. in feral pigeons (Columba livia forma domestica) in the Czech Republic.

The effect of ripening and storage conditions on the distribution of tyramine, putrescine and cadaverine in Edam-cheese.

The aim of the work was to describe the development of selected biogenic amines (histamine, tyramine, putrescine and cadaverine) in 4 layers of Dutch-type cheese (Edam-cheese) depending on 3 ripening/storage regimes during a 98-day period. Biogenic amines were analysed by means of ion-exchange chromatography. A further goal was to identify microbial sources of biogenic amines in the material analysed. Phenotype characterization and repetitive sequence-based PCR fingerprinting were used to identify the isolated bacteria. The highest content of tyramine, putrescine and cadaverine was determined in cheeses stored in a ripening cellar at a temperature of 10 degrees C during the whole observation period. Lower biogenic amines content was determined in samples which were moved into a cold storage device (5 degrees C) after 38 days of storage in a ripening cellar (10 degrees C). The lowest concentrations of biogenic amines were detected in cheeses which were moved into a cold storage device (5 degrees C) after 23 days of storage in a ripening cellar (10 degrees C). During the 98-day period, histamine was not detected in any of the regimes. Within the cheeses analysed, non-starter lactic acid bacteria Lactobacillus curvatus, Lactobacillus casei/paracasei and Lactobacillus plantarum were detected as the main producers of the biogenic amines tested. In starter bacteria Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris the decarboxylase activity tested was not detected.

Occurrence of Enterococcus spp. in waters

We studied 630 bacterial strains isolated from surface waters and determined as enterococci on the basis of their growth on Slanetz-Bartley agar in typical colonies. The strains were tested and characterized by several key conventional tests for basic differentiation of enterococci and by commercial test kits. We identified 135 strains ofE. fœcium (21%), 115E. fœcalis (18%), 30E. mundtii (5%), 27E. hirae (4%), 22E. casseliflavus (3%), 21E. gallinarum (3%), 17E. durans-E. hirae complex (3%), 5E. durans (1%), and 1 strain ofE. avium. 150 strains were classified only asEnterococcus sp. (25%) and 107 strains (17%) isolated from Slanetz-Bartley agar were not enterococci. We found that the non-enterococcal group consisted of other Gram-positive cocci and Gram-positive and Gram-negative rods. Based on the identification we tried to find a relation between taxonomic position of isolated strains and their colony morphology on Slanetz-Bartley agar. Out of the total of 523 identified enterococci, 345 strains (66%) formed purple colonies, 136 red colonies (26%), 37 pink colonies (7%) and 5 cream colored colonies (1%). There was no correlation among the color, size or colony morphology and the taxonomic characterization of enterococcal strains.

Lactobacillus apis sp. nov., from the stomach of honeybees (Apis mellifera), having an in vitro inhibitory effect on the causative agents of American and European foulbrood

A taxonomic study was performed on Gram-stain-positive, catalase-negative and regular rod-shaped bacterial strains R4B(T) and R4C, isolated from the stomachs of honeybees. 16S rRNA gene sequence analysis revealed that the phylogenetic position of the novel strains was within the genus Lactobacillus; the highest sequence similarity to R4B(T) was shown by Lactobacillus acidophilus BCRC 10695(T) (93.6 %). Lower sequence similarities were found to other obligately homofermentative lactobacilli. A PCR-DGGE method could detect the sequence of the 16S rRNA gene of strain R4B(T) at different developmental stages of honeybees occurring in two different locations in the Czech Republic. The distinctiveness of the strains from other lactobacilli was also confirmed by analysis of sequences of other phylogenetic markers applicable to the taxonomy of the genus Lactobacillus, ribotyping and rep-PCR analysis. The DNA G+C content of strain R4B(T) was 41.3 mol%. The predominant cellular fatty acids of strain R4B(T) were C18 : 1ω9c, summed C19 : 1ω6c/C19 : 0 cyclo ω10c, C16 : 0, summed C18 : 1ω7c/C18 : 1ω6c and summed C16 : 1ω7c/C16 : 1ω6c. The major polar lipids of strain R4B(T) were glycolipids, lipids and phospholipids. Phenotypic and phylogenetic characteristics also confirmed the independent status of the strains at the species level. Interestingly, strain R4B(T) was able to inhibit growth in vitro of Paenibacillus larvae subsp. larvae (causal agent of American foulbrood in honeybees) and Melissococcus plutonius (causal agent of European foulbrood). The name Lactobacillus apis sp. nov. is proposed for this novel taxon; the type strain is R4B(T) ( = CCM 8403(T) = LMG 26964(T)).

16S rRNA gene-based identification of cultured bacterial flora from host-seeking Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks, vectors of vertebrate pathogens

A total of 151 bacterial isolates were recovered from different developmental stages (larvae, nymphs and adults) of field-collected ticks (67 strains from Ixodes ricinus, 38 from Dermacentor reticulatus, 46 from Haemaphysalis concinna). Microorganisms were identified by means of 16S rRNA gene sequencing. Almost 87 % of the strains belonged to G+ bacteria with predominantly occurring genera Bacillus and Paenibacillus. Other G+ strains included Arthrobacter, Corynebacterium, Frigoribacterium, Kocuria, Microbacterium, Micrococcus, Plantibacter, Rhodococcus, Rothia, and Staphylococcus. G− strains occurred less frequently, comprising genera Advenella, Pseudomonas, Rahnella, Stenotrophomonas, and Xanthomonas. Several strains of medical importance were found, namely Advenella incenata, Corynebacterium aurimucosum, Microbacterium oxydans, M. schleiferi, Staphylococcus spp., and Stenotrophomonas maltophilia. Data on cultivable microbial diversity in Eurasian tick species D. reticulatus and H. concinna are given, along with the extension of present knowledge concerning bacterial flora of I. ricinus.

Ribotyping of Lactobacillus casei group strains isolated from dairy products

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping withEcoRI andHindIII restriction enzymes. Biotyping assigned 14 strains asLactobacillus casei, 6 strains asLactobacillus paracasei subsp.paracasei and 12 asLactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding toL. rhamnosus andL. casei/L. paracasei subsp.paracasei. TheHindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast,EcoRI ribotyping revealed high intraspecies variability. All ribotypes ofL. casei andL. paracasei subsp.paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strainsL. casei CCM 7088T (= ATCC 393T) andLactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile ofL. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy ofL. casei andL. paracasei species revealed by other studies as well as reclassification of the type strainL. casei CCM 7088T asL. zeae and designation ofL. casei CCM 7089 as the neotype strain.

Identification of Staphylococcus spp. using (GTG)5-PCR fingerprinting

A group of 212 type and reference strains deposited in the Czech Collection of Microorganisms (Brno, Czech Republic) and covering 41 Staphylococcus species comprising 21 subspecies was characterised using rep-PCR fingerprinting with the (GTG)₅ primer in order to evaluate this method for identification of staphylococci. All strains were typeable using the (GTG)₅ primer and generated PCR products ranging from 200 to 4500 bp. Numerical analysis of the obtained fingerprints revealed (sub)species-specific clustering corresponding with the taxonomic position of analysed strains. Taxonomic position of selected strains representing the (sub)species that were distributed over multiple rep-PCR clusters was verified and confirmed by the partial rpoB gene sequencing. Staphylococcus caprae, Staphylococcus equorum, Staphylococcus sciuri, Staphylococcus piscifermentans, Staphylococcus xylosus, and Staphylococcus saprophyticus revealed heterogeneous fingerprints and each (sub)species was distributed over several clusters. However, representatives of the remaining Staphylococcus spp. were clearly separated in single (sub)species-specific clusters. These results showed rep-PCR with the (GTG)₅ primer as a fast and reliable method applicable for differentiation and straightforward identification of majority of Staphylococcus spp.

Characterization of yellow-pigmented and motile enterococci isolated from intestines of the garden snail Helix aspersa

Aims: Enterococci associated with garden snails (Helix aspersa) were studied in order to obtain reliable species identification and characterization.

Lactobacillus spp. associated with early childhood caries

A group of 69 lactobacilli was isolated from caries lesions and root canals of early childhood caries (ECC) affected children treated in the Department of Pedodontics (Children’s Teaching Hospital, Brno, Czech Republic). Biochemical and physiological properties of all strains were characterized by API 50 CH kit and conventional tube tests. The rep-PCR fingerprinting with the (GTG)5 primer was used for genotypic grouping of the isolates. The (GTG)5-PCR fingerprinting grouped all analyzed strains into a few clusters in nearly full agreement with phenotype identification results and clarified the taxonomic position of 13 biochemically unidentified strains. In total, 20 strains of Lactobacillus fermentum, 17 L. rhamnosus, 14 L. casei/paracasei, 7 L. gasseri, 7 L. salivarius and 4 L. plantarum were identified. Mixtures of two or even three Lactobacillus spp. were isolated from a few root canal content samples. Results obtained by biotyping and (GTG)5-PCR were generally comparable except for L. gasseri strains that were not biochemically identified. The (GTG)5-PCR fingerprinting was shown to be quicker, easier to perform and more reliable than biotyping. Our results imply this molecular method as a good tool for screening and identification of Lactobacillus spp. inhabiting dental plaque.