Bifidobacterium apri sp. nov., a thermophilic actinobacterium isolated from the digestive tract of wild pigs (Sus scrofa).

Fresh samples of intestinal contents of three wild pigs originating from the Central Bohemia region were examined for the presence of bifidobacterial strains. During the study, we isolated many fructose-6-phosphate phosphoketolase-positive, strictly anaerobic, irregular rod-shaped bacterial isolates. Three of them were preliminarily identified as representing a novel species of the genus Bifidobacterium because their 16S rRNA gene sequence similarity with the closest relatives of thermophilic bifidobacteria (Bifidobacterium boum DSM 20432T, Bifidobacterium thermophilum DSM 20210T, Bifidobacterium thermacidophilumsubsp. porcinum LMG 21689T, Bifidobacterium thermacidophilumsubsp. thermacidophilum DSM 15837T) was in the range of 97.9 - 98.4 %. All three bacterial isolates had identical 16S rRNA, dnaJ1, fusA, gyrB and rplB gene sequences. Isolate RP115T was chosen as a representative of the bacterial group and DNA G+C content (mol%) determination, biochemical tests and analyses of physiological and morphological characteristics, habitat and chemotaxonomic traits (peptidoglycan structure, cellular fatty acids and polar lipids profile) were performed. The DNA-DNA hybridization analyses of RP115T and species representing the group of thermophilic bifidobacteria revealed values in the range from 33 to 53 %. This fact, together with relatively low sequence similarities of particular phylogenetic markers among examined bacterial strains and the phenotyping and chemotaxonomy results obtained, indicated that the evaluated bacterial isolate should be classified as representing a separate taxon within the specific group of thermophilic bifidobacteria. The name Bifidobacterium apri (of boar) sp. nov. has been proposed for the representative strain RP115T (=CCM 8605T=DSM 100238T=LMG 28779T).

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)).

Vagococcus entomophilus sp. nov., from the digestive tract of a wasp (Vespula vulgaris).

Three unknown Gram-stain-positive, catalase-negative, facultatively anaerobic and coccus-shaped strains of bacteria were isolated from the digestive tracts of wasps (Vespula vulgaris). Analysis of 16S rRNA gene sequences revealed that these strains had identical sequences and showed that Vagococcus salmoninarum, with 96.2% sequence similarity, was the closest phylogenetic neighbour. Further analyses based on hsp60 and pheS gene sequences of representatives of the family Enteroccocaceae and genotypic and phenotypic characterization using (GTG)5-PCR fingerprintings, EcoRI ribotyping, DNA G+C content, whole-cell protein profiling, cellular fatty acid profiles analysis and extensive biotyping confirmed that the investigated strains were representatives of a novel bacterial species within the genus Vagoccocus for which the name Vagoccocus entomophilus sp. nov. is proposed. The type strain is VOSTP2(T) ( = DSM 24756(T) = CCM 7946(T)).

Reclassification of Bifidobacterium stercoris Kim et al. 2010 as a later heterotypic synonym of Bifidobacterium adolescentis.

The taxonomic position of Bifidobacterium stercoris Eg1(T) ( = JCM 15918(T)) based on comparative 16S rRNA gene and hsp60 sequence analyses was found to be controversial, as the strain showed high similarity to the type strain of Bifidobacterium adolescentis, CCUG 18363(T). Therefore, the relationship between the two species was investigated by a taxonomic study that included, in addition to re-evaluation of the 16S rRNA gene sequence, determination of DNA-DNA binding and multilocus sequence analysis (MLSA) of housekeeping genes encoding the DNA-directed RNA polymerase B subunit (rpoC), putative xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp), elongation factor EF-G (fusA), 50S ribosomal protein L2 (rplB) and DNA gyrase B subunit (gyrB). Comparative 16S rRNA gene sequence analysis showed relatively high similarity (98.9 %) between B. stercoris KCTC 5756(T) and B. adolescentis ATCC 15703(T). MLSA revealed close relatedness between B. stercoris KCTC 5756(T) and B. adolescentis CCUG 18363(T), with 99.3-100 % similarity between the rpoC, xfp, fusA, rplB and gyrB gene sequences. In addition, relatively high dnaJ1 gene sequence similarity of 97.7 % was found between the strains. Similar phenotypes and a high DNA-DNA binding value (78.9 %) confirmed that B. stercoris and B. adolescentis are synonymous. Based on these results, it is proposed that the species Bifidobacterium stercoris Kim et al. 2010 should be reclassified as a later heterotypic synonym of Bifidobacterium adolescentis Reuter 1963 (Approved Lists 1980).

[Nocardia farcinica as the causative agent of a brain abscess in a patient with interstitial lung disease]. / Nocardia farcinica jako puvodce abscesu mozku u pacienta s intersticiálním onemocnením plic.

This case report describes a two-step protocol for the identification of the causative agent of nocardiosis in a patient with brain abscess, antibiotic susceptibility testing and etiological treatment after neurosurgery. The patient treated with corticosteroids for pulmonary fibrosis and presenting with multiple neurological manifestations was admitted to a neurosurgery clinic. CT and contrast MRI revealed an expansive multilocular lesion 45 x 35 mm in size in the left parietal lobe, differentially diagnosed as malignant glioma. The lesion was biopsied and the histology showed a brain abscess containing white blood cells and dead tissue. The aspirated pus culture yielded bacteria of the genus Nocardia that were further identified, in the first step, by phenotypic methods (Gram positivity, partial acidoresistance, airborne mycelium detection, growth at 45 degrees C, lysozyme resistance and antibiotic resistance phenotype) as belonging to resistance phenotype V., v.s. N. farcinica (resistance to aminoglycosides except amikacin and to third-generation cephalosporins). In the second step of the polyphasic identification, rDNA was isolated and a 1000 bp part of the 16S rRNA gene was sequenced. Sequence comparison with the GenBank database using BLAST software identified the agent as N. farcinica (100%). The isolate was tested for susceptibility by the NCCLS /CLSI dilution method and showed good susceptibility to co-trimoxazole, amikacin and imipenem. The patient was treated with long-term intravenous cotrimoxazole acid in combination with amikacin and his clinical condition and laboratory parameters of inflammation improved. N. farcinica is among the three most frequently isolated Nocardia species in Europe as well as in the Czech Republic where it was repeatedly recovered from the lungs and respiratory tract of immunocompromised patients with systemic nocardiosis.

[The use of molecular genetics techniques in clinical microbiology--final report from the workshop of the Molecular Microbiology Working Group TIDE]. / Aplikace metod molekulární genetiky v klinické mikrobiologii: zpráva z jednání Pracovní skupiny molekulárni mikrobiologie--TIDE.

In the last decade, there has been a rapid development in the use of molecular genetics methods in clinical microbiology. Novel technologies bring new knowledge and approaches to various disciplines of microbiology--taxonomy, identification of microbes, clinical diagnosis, epidemiology of infectious diseases and antibiotic resistance. This article summarizes the conclusions from the workshop of the Molecular Microbiology Working Group TIDE held during the Second Annual Meeting of the Society for Medical Microbiology of the J. E. Purkyne Czech Medical Association.

Characterization of Aeromonas encheleia strains isolated from aquatic environments in the Czech Republic.

AIMS: Characterization and identification of Aeromonas strains isolated from surface and underground waters using phenotypic and genotyping methods. METHODS AND RESULTS: Biotyping using the ENTEROtest 24 kit and conventional biochemical and physiological tests assigned four strains to Aeromonas encheleia, whereas three isolates were identified as ambiguous Aeromonas bestiarum/Aeromonas caviae and one strain as Aeromonas eucrenophila/Aeromonas encheleia. Further characterization grouped the analysed strains together with Aer. encheleia CCM 4582(T) and assigned the analysed group as members of Aer. encheleia species using ribotyping, whole-cell protein analysis and ERIC-PCR fingerprinting. The results obtained were verified by DNA gyrase A subunit gene sequencing. All analysed isolates showed unique molecular patterns, except for isolates P 1769 and CCM 7407, which revealed the same EcoRI ribotype profile and proved to be identical strains. CONCLUSIONS: Our results imply that Aer. encheleia strains occur in unpolluted surface as well as in underground waters and demonstrate applied methods as suitable for their identification. SIGNIFICANCE AND IMPACT OF THE STUDY: To our best knowledge, this is the first report of the isolation and identification of Aer. encheleia in the Czech Republic.

Characterization of Lactococcus lactis subsp. lactis isolated from surface waters.

A group of nine presumptive enterococci was isolated on enterococcal selective media Slanetz-Bartley agar and/or kanamycin-esculin-azide agar during a screening of Enterococcus spp. in surface waters. All strains formed a homogeneous cluster separated from all enterococcal species using rep-PCR fingerprinting with the (GTG)5 primer but they matched fingerprints revealed by Lactococcus lactis subsp. lactis representatives. Further identification using extensive biotyping and automated ribotyping with EcoRI (RiboPrinter microbial characterization system) confirmed all strains as L. lactis subsp. lactis in full correspondence with the (GTG)5-PCR. We demonstrated that L. lactis subsp. lactis strains occur in different surface waters and can be confused with enterococci due to their positive growth on selective enterococcal media as well as positive results in tests commonly used for identification of the genus Enterococcus (esculin hydrolysis, acetoin and pyrrolidonyl arylamidase production, growth at 10 degrees C and in 6.5% NaCl). The (GTG)5-PCR fingerprinting was revealed as a reliable and fast method for the identification of L. lactis subsp lactis while automated ribotyping with EcoRI proved to be a good tool for intrasubspecies typing purposes.

First data on uranium uptake in three nototheniid fishes from Antarctica (James Ross Island).

Recent studies have confirmed historic atmospheric deposition of uranium in Antarctica, with a steep and significant increase in levels deposited since the 1980s in Antarctic Peninsula ice core samples. To date, however, there has been little or no attention paid to uranium in the Antarctic food web. Here, we present results for uranium content in scales of three common nototheniid species (Trematomus bernacchii, Gobionotothen gibberifrons, Notothenia coriiceps) from coastal waters off James Ross Island (Antarctic Peninsula). While mean total uranium levels (mean ±â€¯SD) were low and similar between species (N. coriiceps 0.08 µg g-1 ± 0.01, T. bernacchii 0.17 µg g-1 ± 0.10; G. gibberifrons 0.11 µg g-1 ± 0.04), linear regressions against standard length indicated bioaccumulation in T. bernacchii (ANOVA, F = 7.8349, P = 0.0076). We suggest this may be the result of dietary specialisation on prey with calcareous shells that accumulate uranium. To the best of our knowledge, this paper provides the first quantitative baseline data on uranium levels in coastal Antarctic nototheniids. While the low levels recorded are unlikely to represent a threat within the food chain, we suggest that further long-term trophic studies (including stable isotope analysis) are needed, recognising that the feeding ecology of individual species (and even individuals) can have a strong effect on overall trends.

Brucellosis of the common vole (Microtus arvalis).

A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.

Ribotyping and biotyping of Staphylococcus epidermidis isolated from hospital environment.

The Staphylococcus strains acquired from scrapings from hospital environments were identified to the species level based on their biochemical properties. From the monitored sample the Staphylococcus epidermidis strains were selected for more accurate typing and tested on their virulence factor and ribotyped. The biotyping of S. epidermidis did not show any considerable intraspecific variation of these isolates and there were no atypical reactions, with the exception of three strains (out of 33). In contrast, the results of ribotyping showed greater heterogeneity of strains and unequivocally demonstrated the relation between the ribotype and the place of sample drawing. In addition to this fact, the found ribotypes repeat in the same environment in the long-term which suggests the occurrence and persistence of the same strains of conditionally pathogenic bacteria in hospital environment. We showed that ribotyping is a suitable method for precise and reliable detection of some coagulase-negative staphylococci.

Properties of the strains Enterococcus haemoperoxidus and E. moraviensis, new species among enterococci.

Antibiotic susceptibility or resistance, urease activity, detection of the structural genes for bacteriocin production, bacteriocin activity as well as sensitivity of the isolates to enterocins (Ent) A and M were determined in 23 isolates of new species Enterococcus haemoperoxidus and E. moraviensis. The majority of the strains were antibiotic sensitive and exhibited low urease activity (< 10 nkat/mL). The most frequently detected genes for Ent were entA and entP. However, only the strain 466 of E. haemoperoxidus produced an antibacterial substance with inhibitory activity against 21 G+ indicators. It was partially purified reaching an activity of up to 12 800 AU/mL. This bacteriocin active strain also possessed the genes for EntA and EntP. The other strains did not inhibit the indicator strains. The substance produced by the 466 strain was active even after a 5-months storage at +4 and -20 degrees C. This substance has proteolytic and hydrophilic character, pH optimum of bacteriocin production by this strain being between 4 and 7. While E. moraviensis strains showed sensitivity to EntA (produced by E. faecium EK13) and to EntM (produced by E. faecium AL41), E. haemoperoxidus strains were sensitive to EntA (except strain 382) but less sensitive to the treatment by EntM.

[Aeromonas spp. as the causative agent of acute diarrhoea in children under 1 year of age]. / Aeromonas spp. jako puvodce akutních prujmu detí do jednoho roku.

STUDY OBJECTIVE: To establish whether there is a link between cases of acute watery diarrhoea and a specific Aeromonas species. MATERIALS AND METHODS: Eight strains studied were identified as aeromonads and were further characterized by biochemical tests, fatty acid analysis and ribotyping. RESULTS: Aeromonads were isolated repeatedly from stool specimens of four children under one year of age with acute diarrhoea, two of whom were admitted to hospital. Of eight isolated aeromonads strains six were identified as A. caviae, one was classified into A. veronii bv. sobria and one could not be identified to the species level. Only two A. caviae strains from one patient were found to be identical by ribotyping while the Aeromonas species (strains) isolated from the other cases differed from one another. Contaminated fresh water, contaminated food and contact with travellers with imported diarrhoea were identified as probable sources of infection. CONCLUSION: Four cases of acute gastroenteritis in small children document that aeromonads are not rare and can cause serious health problems. However, epidemiological links remain unclear. We did not prove correlation between the four serious cases of acute diarrhoea and specific Aeromonas species but the results suggest the predominant role of A. caviae.

Classification of Aeromonas spp. isolated from water and clinical sources and distribution of virulence genes.

In this work, 84 isolates of aeromonads were isolated from water and clinical samples, identified, and characterized. Identification was based on routine phenotyping combined with multiplex PCR. In this study, multiplex PCR was retested and reevaluated and its identification key was enhanced by 17 newly described species and five subspecies. Identification score increased from 36 % (only phenotyping) to 90 % when supported with multiplex PCR. Further description of isolates included detection of eight virulence genes. These genes were overall present in 46 % (act), 2.4 % (ast), 80 % (alt), 40 % (ahh1), 20 % (asa1), 69 % (pla/lip/lipH3/alp-1), 69 % (ser), and 81 % (fla), and no significant differences between water and clinical isolates were found. Results of this work show that the proper combination of different approaches is necessary for final identification of Aeromonas spp. at the species level. Multiplex PCR was shown to have limits in final identification, specifically inability to distinguish four species pairs and one triplet as their gene profiles are identical. However, it seems to be rapid and easy to do method able to support routine biochemical identification in laboratories. Moreover, our results supported previous proposal of reclassification of "Aeromonas hydrophila subsp. dhakensis" and "Aeromonas aquariorum" as identical species.

Ribotyping of Lactobacillus casei group strains isolated from dairy products.

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

Identification of Staphylococcus piscifermentans from dog feces.

The AD 2 strain isolated from feces of a healthy dog in Slovakia was characterized phenotypically by the conventional tests and commercial identification kits API Staph and ID32 Staph. Results of biochemical tests identified the strain as S. piscifermentans, fully corresponding with the species description. Further characterization by whole-cell protein profile analysis (SDS-PAGE) confirmed the identification based on biochemical tests and showed that the AD 2 strain is S. piscifermentans; lactic acid production, urease activity, bacteriocin production and the antibiotic susceptibility of it were also determined. S. piscifermentans AD 2 isolated first from an animal source was deposited in the Czech Collection of Microorganisms as Staphylococcus piscifermentans CCM 7165.

Evaluation of ribotyping for characterization and identification of Enterococcus haemoperoxidus and Enterococcus moraviensis strains.

Seven Enterococcus moraviensis and 16 Enterococcus haemoperoxidus as well as nine reference cultures of other enterococcal species obtained from the Czech Collection of Microorganisms were characterized using ribotyping with EcoRI and HindIII in the present work. The ribopatterns obtained by both restriction enzymes clearly distinguished all E. moraviensis and E. haemoperoxidus strains from the other enterococci (E. faecalis, E. faecium, E. avium, E. raffinosus, E. pseudoavium, E. malodoratus) and they differentiated both species from each other as well. Although all strains were isolated from different sampling sites, many strains shared the same band patterns. E. moraviensis formed four ribogroups using EcoRI and two ribogroups using HindIII restriction enzyme. E. haemoperoxidus gave six different patterns with EcoRI and five using the HindIII restriction enzyme.

Identification of Staphylococcus and Micrococcus species with the STAPHYtest system.

A collection of 216 well-characterized strains of Staphylococcus, Micrococcus and Stomatococcus was examined by a commercially available STAPHYtest system (Lachema, Brno, Czechoslovakia). The results of STAPHYtest agreed with those of conventional tests. The STAPHYtest permitted a clear-cut separation of Staphylococcus from Micrococcus and Stomatococcus strains and correctly identified 104 of 145 (72%) Staphylococcus strains after 24 h of incubation. However, it allowed the identification only of 19 of 29 validly published Staphylococcus species. The STAPHYtest proved to be a simple and rapid system for the separation of staphylococci from micrococci and for the identification of most frequent clinically significant staphylococci.

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 of E. foecium (21%), 115 E. faecalis (18%), 30 E. mundtii (5%), 27 E. hirae (4%), 22 E. casseliflavus (3%), 21 E. gallinarum (3%), 17 E. durans-E. hirae complex (3%), 5 E. durans (1%), and 1 strain of E. avium. 150 strains were classified only as Enterococcus 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. Our 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.

Characterization of esculin-positive Pseudomonas fluorescens strains isolated from an underground brook.

A group of sixteen esculin-positive fluorescent pseudomonads isolated from an underground brook flowing through a cave complex was characterized by biotyping, multiple enzyme restriction fragment length polymorphism analysis of 16S rDNA (MERFLP), ribotyping and whole-cell fatty-acid methyl-esters analysis (FAME). All strains were phenotypically close to Pseudomonas fluorescens, but they revealed high biochemical variability as well as some reactions atypical for P. fluorescens species. Because identification of pseudomonads by of biochemical testing is often unclear, further techniques were employed. Fingerprints obtained by MERFLP clearly showed that all strains represent P. fluorescens species. Ribotyping separated the strains analyzed into four groups corresponding almost completely (with the exception of one strain) to the clustering based on biochemical profiles. FAME analysis grouped all the strains into one cluster together with the P. putida (biotype A, B), P. chlororaphis and P. fluorescens biotype F representatives, but differentiated them from other FAME profiles of all pseudomonads included in the standard library TSBA 40 provided by MIDI, Inc.