Accurate enumeration of Paenibacillus mucilaginosus (formerly Bacillus mucilaginosus) bacterium from environmental samples on solid medium is challenging owing to its extensive extracellular polysaccharides (EPS) excretion. In the present study, P. mucilaginosus enumeration has been facilitated by a simple modification: addition of triphenyl tetrazolium chloride (TTC) to growth medium and application of a second soft agar layer. Results show distinctively better and accurate colonies' count. This method can be applied to all bacterial species that produce excessive EPS that may interfere with direct count.

• MeSH
• agar chemie   MeSH
• barvicí látky chemie   metabolismus   MeSH
• kultivační média chemie   MeSH
• Paenibacillus růst a vývoj   MeSH
• počet mikrobiálních kolonií metody   MeSH
• polysacharidy metabolismus   MeSH
• tetrazoliové soli chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• technické zprávy MeSH
• Názvy látek
• agar MeSH
• barvicí látky MeSH
• kultivační média MeSH
• polysacharidy MeSH
• tetrazoliové soli MeSH
• triphenyltetrazolium MeSH Prohlížeč

AIM: Raman spectroscopy is an analytical method with a broad range of applications across multiple scientific fields. We report on a possibility to differentiate between two important Gram-positive species commonly found in clinical material - Staphylococcus aureus and Staphylococcus epidermidis - using this rapid noninvasive technique. MATERIALS & METHODS: For this, we tested 87 strains, 41 of S. aureus and 46 of S. epidermidis, directly from colonies grown on a Mueller-Hinton agar plate using Raman spectroscopy. DISCUSSION & CONCLUSION: The method paves a way for separation of these two species even on high number of samples and therefore, it can be potentially used in clinical diagnostics.

• Klíčová slova
• Raman spectroscopy, Staphylococcus epidermidis, Staphyococcus aureus, rapid diagnostics,
• MeSH
• bakteriologické techniky MeSH
• lidé MeSH
• počet mikrobiálních kolonií metody   MeSH
• Ramanova spektroskopie metody   MeSH
• stafylokokové infekce diagnóza   MeSH
• Staphylococcus aureus izolace a purifikace   patogenita   MeSH
• Staphylococcus epidermidis izolace a purifikace   patogenita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

This study provides information on the occurrence of Arcobacter in several types of water and food products of animal origin in the Czech Republic. We processed 190 samples using the modified method, and the occurrence of Arcobacter spp. was confirmed in 36.8 % of these. This total incidence consisted of Arcobacter butzleri (27.3 %), Arcobacter cryaerophilus (8.4 %) and Arcobacter skirrowii (1.1 %). We newly described the common presence of Arcobacter spp. in sewage water in the Czech Republic that is released into waterways after processing in water treatment plants (86.7 %). All the acquired isolates were subject to detailed confirmation with subsequent species classification using multiplex PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, we used a modification of a method using passive filtration of an enriched sample, which could be suitable for the isolation of Arcobacter, especially in combination with Campylobacter selective agar chromogenic medium. Our studies have shown this agar to be quite suited to the isolation of Arcobacter and that it can be an appropriate instrument for accelerating culture diagnostics.

• MeSH
• Arcobacter genetika   růst a vývoj   izolace a purifikace   metabolismus   MeSH
• kultivační média metabolismus   MeSH
• odpadní vody mikrobiologie   MeSH
• počet mikrobiálních kolonií přístrojové vybavení   metody   MeSH
• potravinářská mikrobiologie * MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• kultivační média MeSH
• odpadní vody MeSH

An international standard already exists for the selective enumeration of bifidobacteria in milk products. This standard uses Transgalactosylated oligosaccharides (TOS) propionate agar supplemented with mupirocin. However, no such standard method has been described for the selective enumeration of bifidobacteria in probiotic supplements, where the presence of bifidobacteria is much more variable than in milk products. Therefore, we enumerated bifidobacteria by colony count technique in 13 probiotic supplements using three media supplemented with mupirocin (Mup; 100mg/l): TOS, Bifidobacteria selective medium (BSM) and modified Wilkins-Chalgren anaerobe agar with soya peptone (WSP). Moreover, the potential growth of bifidobacterial strains often used in probiotic products was performed in these media. All 13 products contained members of the genus Bifidobacterium, and tested mupirocin media were found to be fully selective for bifidobacteria. However, the type strain Bifidobacterium bifidum DSM 20456 and collection strain B. bifidum DSM 20239 showed statistically significant lower counts on TOS Mup media, compared to BSM Mup and WSP Mup media. Therefore, the TOS Mup medium recommended by the ISO standard cannot be regarded as a fully selective and suitable medium for the genus Bifidobacterium. In contrast, the BSM Mup and WSP Mup media supported the growth of all bifidobacterial species.

• Klíčová slova
• Bifidobacteria, Enumeration, Mupirocin, Probiotic supplements, Selective media,
• MeSH
• antibakteriální látky metabolismus   MeSH
• bakteriální nálož metody   MeSH
• Bifidobacterium účinky léků   růst a vývoj   MeSH
• kultivační média chemie   MeSH
• mupirocin metabolismus   MeSH
• počet mikrobiálních kolonií metody   MeSH
• probiotika analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antibakteriální látky MeSH
• kultivační média MeSH
• mupirocin MeSH

An agar selective enumeration of necrotoxigenic Escherichia coli O55 (NTEC2) and probiotic E. coli Nissle 1917, using modified MacConkey agar, was developed to study bacterial interference between these E. coli strains in a gnotobiotic piglet model. Replacement of lactose with saccharose in the agar enables the direct visual enumeration of red colonies of E. coli O55 and yellow colonies of E. coli Nissle 1917 that are co-cultured in the same Petri dish. A total of 336 colonies (168 for each color) were subjected to strain-specific PCR identification with LNA probes. Sensitivity, specificity, and positive and negative predictive values were 96.43%, 95.83%, 95.86% and 96.41% respectively in E. coli O55, and 98.21%, 97.02%, 97.06% and 98.19% respectively in E. coli Nissle 1917. Color-based enumeration of both E. coli strains in colonic contents and mesenteric lymph nodes homogenates of gnotobiotic piglets demonstrated the applicability of this method for the gnotobiotic piglet model of enteric diseases.

• Klíčová slova
• Cytotoxic necrotizing factor, E. coli Nissle 1917, E. coli O55, LNA probes, MacConkey agar, NTEC,
• MeSH
• agar chemie   MeSH
• barva MeSH
• Escherichia coli klasifikace   genetika   růst a vývoj   metabolismus   MeSH
• infekce vyvolané Escherichia coli farmakoterapie   mikrobiologie   MeSH
• kultivační média chemie   metabolismus   MeSH
• lidé MeSH
• počet mikrobiálních kolonií přístrojové vybavení   metody   MeSH
• prasata MeSH
• probiotika aplikace a dávkování   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• agar MeSH
• kultivační média MeSH

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of paratuberculosis in ruminants, has a lipid-rich cell wall which facilitates its survival and persistence in the environment. This property of the organism is exploited when it is cultured as decontaminating agents and antibiotics are used to suppress the growth of contaminating microflora, but such treatments can also negatively affect the isolation of MAP itself. The objective of this study was to assess the effect of the 'VAN' antibiotics (vancomycin, amphotericin B and nalidixic acid) on the viability of MAP using a propidium monoazide real time quantitative PCR (PMA qPCR) and culture. Long-term (5 week) treatment with VAN antibiotics resulted in a larger decrease in bacterial numbers compared to short-term (3 day) exposure. The PMA qPCR assay indicated that 50 μg/mL of vancomycin, 50 μg/mL of nalidixic acid, and 200 μg/mL of amphotericin B were 'threshold' concentrations, respectively, above which the decline in the viability of MAP was statistically significant. Using culture, these threshold concentrations were 100 μg/mL of vancomycin, 50-100 μg/mL of nalidixic acid, and 100 μg/mL of amphotericin B, respectively. Given that the two methods were found to be comparable, the PMA qPCR is a potentially more convenient and effective alternative to culture in detecting MAP.

• MeSH
• amfotericin B farmakologie   MeSH
• antibakteriální látky farmakologie   MeSH
• azidy metabolismus   MeSH
• časové faktory MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• kyselina nalidixová farmakologie   MeSH
• Mycobacterium avium subsp. paratuberculosis účinky léků   růst a vývoj   izolace a purifikace   metabolismus   MeSH
• paratuberkulóza diagnóza   mikrobiologie   MeSH
• počet mikrobiálních kolonií metody   MeSH
• přežvýkavci mikrobiologie   MeSH
• propidium analogy a deriváty   metabolismus   MeSH
• vankomycin farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amfotericin B MeSH
• antibakteriální látky MeSH
• azidy MeSH
• kyselina nalidixová MeSH
• propidium monoazide MeSH Prohlížeč
• propidium MeSH
• vankomycin MeSH

Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

• MeSH
• DNA fungální chemie   genetika   MeSH
• Glomeromycota genetika   růst a vývoj   MeSH
• kořeny rostlin mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• Medicago sativa mikrobiologie   MeSH
• mikrobiální interakce MeSH
• mitochondriální DNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• počet mikrobiálních kolonií metody   MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA fungální MeSH
• mitochondriální DNA MeSH

We developed and assessed the diagnostic value of a novel quantitative nested real-time (QNRT) PCR assay targeting the internal transcribed spacer region of ribosomal DNA (rDNA) in a guinea pig model of invasive pulmonary aspergillosis. Groups of 5 immunosuppressed animals that were infected using an aerosol chamber with Aspergillus fumigatus conidia were humanely terminated 1 h postinoculation and at days 3, 5, 7, and 11 postchallenge, and lung tissue, bronchoalveolar lavage (BAL) fluid, whole blood, and serum samples were collected. The QNRT PCR results obtained with the serum and BAL fluid were compared to those achieved with galactomannan and (1→3)-β-d-glucan assays. High fungal burden levels were detected by QNRT PCR in both lung tissue and BAL fluid in all infected animals at each time point, and the sensitivity of each assay in BAL fluid was 100% by day 3 and remained so through the remainder of the study. The sensitivity of detection of fungi in whole blood and serum samples was significantly lower, and some samples remained negative by all three assays despite the advanced stage of the infection. From these data, we can conclude that this novel QNRT PCR method was highly sensitive for the detection of A. fumigatus from different types of samples in this model. In addition, BAL fluid samples appeared to be the most suitable for the early diagnosis of invasive pulmonary aspergillosis. When testing serum, the use of a combination of available assays may increase the possibility of early detection of this opportunistic mycosis.

• MeSH
• Aspergillus fumigatus izolace a purifikace   MeSH
• beta-glukany analýza   MeSH
• bronchoalveolární lavážní tekutina mikrobiologie   MeSH
• DNA fungální genetika   MeSH
• galaktosa analogy a deriváty   MeSH
• invazivní plicní aspergilóza mikrobiologie   patologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mannany analýza   MeSH
• mezerníky ribozomální DNA genetika   MeSH
• modely nemocí na zvířatech MeSH
• morčata MeSH
• mykologie metody   MeSH
• plíce mikrobiologie   MeSH
• počet mikrobiálních kolonií metody   MeSH
• polymerázová řetězová reakce metody   MeSH
• proteoglykany MeSH
• senzitivita a specificita MeSH
• zvířata MeSH
• Check Tag
• morčata MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• beta-glukany MeSH
• DNA fungální MeSH
• galactomannan MeSH Prohlížeč
• galaktosa MeSH
• mannany MeSH
• mezerníky ribozomální DNA MeSH
• polysaccharide-K MeSH Prohlížeč
• proteoglykany MeSH

The aim of this study was to assess the diagnostic properties of the two selective plating media and a chromogenic medium for identification of Bacillus cereus. The 324 isolates were B. cereus (37%), Bacillus weihenstephanensis (45%) or Bacillus thuringiensis (18%), as identified by a new combination of techniques. All isolates were growing on mannitol-egg yolk-polymyxin agar (MYP), and they did not form acid from mannitol. However, a significant lower number of B. thuringiensis isolates did not show lecithinase activity. All isolates were also growing on polymyxin-egg yolk-mannitol-bromothymol blue agar (PEMBA); however, 11% isolates indicated that they did produce acid from mannitol, and 15% isolates did not show any lecithinase activity. Five of the isolates did not grow at all on the chromogenic agar, and 14 of the growing isolates were β-glucosidase negative. It is concluded that the two recommended selective plating media MYP and PEMBA for detection of B. cereus group bacteria both have their limitations for identification of some B. cereus, B. weihenstephanensis or B. thuringiensis. However, MYP is preferable compared to PEMBA. The chromogenic medium has its own advantages and limitations, and some of the limitations seem to be solved by incubation at 30°C instead of the recommended 37°C.

• MeSH
• Bacillus cereus klasifikace   růst a vývoj   izolace a purifikace   metabolismus   MeSH
• Bacillus thuringiensis klasifikace   růst a vývoj   izolace a purifikace   metabolismus   MeSH
• Bacillus klasifikace   růst a vývoj   izolace a purifikace   metabolismus   MeSH
• členovci mikrobiologie   MeSH
• grampozitivní bakteriální infekce mikrobiologie   MeSH
• kultivační média metabolismus   MeSH
• lidé MeSH
• počet mikrobiálních kolonií metody   MeSH
• potravinářská mikrobiologie MeSH
• půdní mikrobiologie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• kultivační média MeSH

Organic acids can be used as feed supplements or for treatment of poultry carcasses in processing plants. The antimicrobial activity of nineteen organic acids and two monoacylglycerols in cultures of Campylobacter jejuni CCM 6214(T) (ATCC 33560) was determined using a SYBR Green-based real-time PCR assay. The IC(50) was a concentration at which only 50 % of a bacteria specific DNA sequence was amplified. Caprylic, capric and lauric acids were the most efficient antimicrobials among the compounds tested (IC(50) < or = 0.1 mg/mL). In a weakly acidic environment (pH 5.5), the antimicrobial activity was more pronounced than at pH 6.5. At pH 5.5, oleic and fumaric acid also had clear antimicrobial activity, as did monocaprylin. The antimicrobial activity of acetic, butyric, stearic and succinic acid was low. In cells treated with fumaric acid, the potential of potassium and tetraphenylphosphonium ion-selective electrodes changed, indicating an increase in cytoplasmic and outer membrane permeability, respectively. No changes in membrane permeability were observed in cells treated with capric acid or monocaprin. Transmission electron microscopy revealed separation of the inner and outer membrane in cells treated with capric and fumaric acid, as well as cytoplasmic disorganization in cells exposed to capric acid.

• MeSH
• antibakteriální látky farmakologie   MeSH
• barvení a značení metody   MeSH
• benzothiazoly MeSH
• buněčná membrána ultrastruktura   MeSH
• Campylobacter jejuni účinky léků   fyziologie   ultrastruktura   MeSH
• chinoliny MeSH
• diaminy MeSH
• inhibiční koncentrace 50 MeSH
• kyseliny karboxylové farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• monoglyceridy farmakologie   MeSH
• organické látky metabolismus   MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• počet mikrobiálních kolonií metody   MeSH
• polymerázová řetězová reakce MeSH
• transmisní elektronová mikroskopie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• benzothiazoly MeSH
• chinoliny MeSH
• diaminy MeSH
• kyseliny karboxylové MeSH
• monoglyceridy MeSH
• organické látky MeSH
• SYBR Green I MeSH Prohlížeč