Hana Salmonová

A new medium containing mupirocin, acetic acid, and norfloxacin for the selective cultivation of bifidobacteria.

Various culture media have been proposed for the isolation and selective enumeration of bifidobacteria. Mupirocin is widely used as a selective factor along with glacial acetic acid. TOS (transgalactosylated oligosaccharides) medium supplemented with mupirocin is recommended by the International Dairy Federation for the detection of bifidobacteria in fermented milk products. Mupirocin media with acetic acid are also reliable for intestinal samples in which bifidobacteria predominate. However, for complex samples containing more diverse microbiota, the selectivity of mupirocin media is limited. Resistance to mupirocin has been demonstrated by many anaerobic bacteria, especially clostridia. The objective was to identify an antibiotic that inhibits the growth of clostridia and allows the growth of bifidobacteria, and to use the identified substance to develop a selective cultivation medium for bifidobacteria. The susceptibility of bifidobacteria and clostridia to 12 antibiotics was tested on agar using the disk diffusion method. Only norfloxacin inhibited the growth of clostridia and did not affect the growth of bifidobacteria. Using both pure cultures and faecal samples from infants, adults, calves, lambs, and piglets, the optimal concentration of norfloxacin in solid cultivation media was determined to be 200xa0mg/L. Our results showed that solid medium containing norfloxacin (200xa0mg/L) in combination with mupirocin (100xa0mg/L) and glacial acetic acid (1xa0mL/L) is suitable for the enumeration and isolation of bifidobacteria from faecal samples of different origins.

Galliscardovia ingluviei gen. nov., sp. nov., a thermophilic bacterium of the family Bifidobacteriaceae isolated from the crop of a laying hen (Gallus gallus f. domestica)

Bacteria with potential probiotic applications are not yet sufficiently explored, even for animals with economic importance. Therefore, we decided to isolate and identify representatives of the family Bifidobacteriaceae, which inhabit the crop of laying hens. During the study, a fructose-6-phosphate phosphoketolase-positive strain, RP51T, with a regular/slightly irregular and sometimes an S-shaped slightly curved rod-like shape, was isolated from the crop of a 13u2009-month-old Hisex Brown hybrid laying hen. The best growth of the Gram-stain-positive bacterium, which was isolated using Bifidobacterium-selective mTPY agar, was found out to be under strictly anaerobic conditions, however an ability to grow under microaerophilic and aerobic conditions was also observed. Sequencing of the almost complete 16S rRNA gene (1444u2009bp) showed Alloscardovia omnicolens CCUG 31649T and Bombiscardovia coagulans BLAPIII/AGVT to be the most closely related species with similarities of 93.4 and 93.1u200a%, respectively. Lower sequence similarities were determined with other scardovial genera and other representatives of the genus Bifidobacterium. Taxonomic relationships with A. omnicolens and other members of the family Bifidobacteriaceaewere also demonstrated, based on the sequences of dnaK, fusA, hsp60 and rplB gene fragments. Low sequence similarities of phylogenetic markers to related scardovial genera and bifidobacteria along with unique features of the bacterial strain investigated within the family Bifidobacteriaceae(including the lowest DNA G+C value (44.3u2009mol%), a unique spectrum of cellular fatty acids and polar lipids, cellular morphology, the wide temperature range for growth (15-49u2009°C) and habitat) clearly indicate that strain RP51T is a representative of a novel genus within the family Bifidobacteriaceae for which the name Galliscardovia ingluviei gen. nov., sp. nov. (RP51T=DSM 100235T=LMG 28778T=CCM 8606T) is proposed.

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).

Anticlostridial agent 8-hydroxyquinoline improves the isolation of faecal bifidobacteria on modified Wilkins-Chalgren agar with mupirocin.

The need for suitable selective cultivation media for the isolation of Bifidobacterium spp. continues to be a real concern in the field of intestinal microbiology. Isolation of bifidobacteria from human and animal faecal samples using selective agar plating may be problematic especially in samples with increased clostridial counts than bifidobacterial counts. Due to the absence of anticlostridial agents in existing selective media, clostridia can displace bifidobacteria resulting in incorrect estimation of their counts. Therefore, we supplemented the existing selective medium ‘modified Wilkins Chalgren agar with mupirocin’ (MWM) with 90 mg l−1 of 8‐hydroxyquinoline (8HQ), which was recently proved to act selectively against clostridia. The newly composed ‘modified Wilkins–Chalgren agar with 8HQ’ (MWMQ) was tested on pure bifidobacterial and clostridial strains, their mixtures, and using faecal samples of mammalian origin; its selectivity was evaluated by genus‐specific identification of isolates. The results demonstrated that the presence of 8HQ in this agar eliminated the growth of nonbifidobacterial strains on MWMQ compared to that on MWM, whereas the recovery of bifidobacterial counts was at satisfactory levels. In conclusion, MWMQ could be recommended for bifidobacterial isolation from human and animal faeces especially when bifidobacteria are not numerically dominant and there are chances of clostridial contamination.

Identification of microbiota associated with Pectinatella magnifica in South Bohemia

Abstract The bacterial diversity of Pectinatella magnifica colonies sampled from pounds in South Bohemia during the summer of 2012 was investigated. The bacterial counts determined after cultivation on modified yeast extract-tryptone agar (Oxoid) supplemented with glucose (1 g L−1) varied from 4.22 to 6.61 and from 1.30 to 6.85 log CFU/g for aerobes and anaerobes, respectively. Higher counts were found in the superficial structures of Pectinatella colonies than in the inner gelled mass. Neither a trend in bacterial numbers at the individual site during the season, nor correlations between bacterial counts in P. magnifica and the surrounding water were observed. Fifty-four isolates were identified by sequencing the 16S rRNA gene and through MALDI-TOF MS analysis. Species of Aeromonas and Aquitalea were the predominantly isolated bacteria, but members of Chryseobacterium, Herbaspirillum, Enterobacter, Lactococcus, Leuconostoc, Pseudomonas and Sphingomonas were also found. As listed genera are wildly distributed in different water, soil, and plant samples, we conclude thatPectinatella colonies are inhabited by environmental bacteria. Nevertheless, a symbiotic relationship of these bacteria with P. magnifica cannot be excluded.

Alloscardovia venturai sp. nov., a fructose 6-phosphate phosphoketolase-positive species isolated from the oral cavity of a guinea-pig (Cavia aperea f. porcellus)

A slightly irregular, short rod-shaped bacterial strain, MOZIV/2T, showing activity of fructose 6-phosphate phosphoketolase was isolated from the oral cavity of a home-bred guinea-pig. Based on comparative 16S rRNA gene sequence analyses, its closest relatives were Alloscardovia omnicolens DSM 21503T and Alloscardovia criceti DSM 17774T with 96.0 and 95.6u200a% pairwise similarities, respectively. Completeness of the compared sequences was 97.3 and 96.9u200a%, respectively. Growth was found only under anaerobic conditions. Activities of α- and β-gluco(galacto)sidases were detected in strain MOZIV/2T, which is characteristic for almost all members of the family Bifidobacteriaceae. Sequencing of other molecular markers (fusA, gyrB and xfp) revealed low gene sequence similarities to A. omnicolens DSM 21503T ranging from 72.7 to 87.5u200a%. Strain MOZIV/2T differed from other species within the genus Alloscardovia by the presence of C18u200a:u200a1ω9t. In addition, much higher proportions of C8u200a:u200a0, C11u200a:u200a0, C12u200a:u200a0, C14u200a:u200a1, C16u200a:u200a1 and C17u200a:u200a0 fatty acids were found in cells of strain MOZIV/2T. The peptidoglycan structure was of type A4α [l-Lys(l-Orn)-d-Asp], which is consistent with its classification within the genus Alloscardovia. The DNA G+C content (45.8u2009mol%) was lower than those found in other alloscardovia. Phylogenetic studies and evaluation of phenotypic characteristics including the results of biochemical, physiological and chemotaxonomic analyses confirmed the novel species status for strain MOZIV/2T, for which the name Alloscardovia venturai sp. nov. is proposed. The type strain is MOZIV/2T (=DSM 100237T=CCM 8604T=LMG 28781T).

Lactobacillus caviae sp nov., an obligately heterofermentative bacterium isolated from the oral cavity of a guinea pig (Cavia aperea f. porcellus)

A Gram-stain-positive, facultatively anaerobic, and catalase- and oxidase-negative bacterial strain designated MOZM2T, having 98.4u200a% 16S rRNA gene sequence identity with Lactobacillus reuteri DSM 20016T, was isolated from a swab of the oral cavity of a home-bred guinea pig. Comparative analyses based on the hsp60, pheS and tuf genes confirmed L. reuteri as its closest relative species, with calculated sequence similarities of 92.8, 88.8 and 96.9u200a%, respectively. DNA-DNA hybridisation revealed a 42u200a% degree of genetic similarity between the novel strain and L. reuteri DSM 20016T. Strain MOZM2T degrades carbohydrates via the 6-phosphogluconate/phosphoketolase pathway, evidenced by its production of gas from glucose and the end products of hexose catabolism. Comparative analysis of the cellular fatty acid profiles determined significant differences between MOZM2T and L. reuteri DSM 20016T in their proportions of C8u200a:u200a0, C14u200a:u200a1, C17u200a:u200a0, C18u200a:u200a2ω6t and C20u200a:u200a0 fatty acids. Results of genotypic analyses also demonstrated differences between these two strains. They also differed in DNA G+C content, and some biochemical and physiological characteristics. We therefore believe that the examined bacterial isolate should be considered as a new taxon within the group of obligately heterofermentative lactobacilli. The species name Lactobacillus caviae sp. nov. is proposed, of which the type strain is MOZM2T (=CCM 8609T=DSM 100239T=LMG 28780T).

Assessment of Chemical Impact of Invasive Bryozoan Pectinatella magnifica on the Environment: Cytotoxicity and Antimicrobial Activity of P. magnifica Extracts

Pectinatella magnifica, an invasive bryozoan, might significantly affect ecosystem balance due to its massive occurrence in many areas in Europe and other parts of the world. Biological and chemical analyses are needed to get complete information about the impact of the animal on the environment. In this paper, we aimed to evaluate in vitro cytotoxic effects of five extracts prepared from P. magnifica using LDH assay on THP-1 cell line. Antimicrobial activities of extracts against 22 different bacterial strains were tested by microdilution method. Our study showed that all extracts tested, except aqueous portion, demonstrated LD50 values below 100 μg/mL, which indicates potential toxicity. The water extract of P. magnifica with LD50 value of 250 μg/mL also shows potentially harmful effects. Also, an environmental risk resulting from the presence and increasing biomass of potentially toxic benthic cyanobacteria in old colonies should not be underestimated. Toxicity of Pectinatella extracts could be partially caused by presence of Aeromonas species in material, since we found members of these genera as most abundant bacteria associated with P. magnifica. Furthermore, P. magnifica seems to be a promising source of certain antimicrobial agents. Its methanolic extract, hexane, and chloroform fractions possessed selective inhibitory effect on some potential pathogens and food spoiling bacteria in the range of MIC 0.5–10 mg/mL. Future effort should be made to isolate and characterize the content compounds derived from P. magnifica, which could help to identify the substance(s) responsible for the toxic effects of P. magnifica extracts.

Cultivable bacteria from Pectinatella magnifica and the surrounding water in South Bohemia indicate potential new Gammaproteobacterial, Betaproteobacterial and Firmicutes taxa

Pectinatella magnifica is a freshwater bryozoan, which has become a subject of scientific interest because of its invasive expansion worldwide. To obtain a comprehensive overview of its influence on environments, information on associated bacteria is needed. In this study, cultivable bacteria associated with P. magnifica were investigated. In total, 253 isolates were selected for preliminary identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and clustered based on repetitive extragenic palindromic-PCR profiles. Among these, 169 strains were selected and identified using 16S rRNA gene comparative analyses. The sequences were grouped into 76 phylotypes and affiliated with 67 species. The majority of isolated bacteria belonged to Gammaproteobacteria, followed by Betaproteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Most strains within the Betaproteobacteria were isolated exclusively from bryozoan colonies. Aeromonas was the genus predominantly isolated from both P. magnifica and the water samples. Based on 16S rDNA similarity values, 15 putative new species belonging to the genera Aeromonas, Aquitalea, Clostridium, Herbaspirillum, Chromobacterium, Chryseobacterium, Morganella, Paludibacterium, Pectobacterium, Rahnella, Rhodoferax and Serratia, and putative new genera belonging to families Clostridiaceae and Sporomusaceae were revealed. The majority of the detected bacteria were species widely distributed in the environments; nevertheless, a possible symbiotic association of two new putative species with P. magnifica cannot be excluded.